., . . .. ..., . 5‘53: «.121. . . 11L. 4 - I l. u z: 3.. .7; . . _ . .134 . I 1 SI. 1 ~. .2. .9 .KA . I i at.— .6 . v... u‘ U... . 4%. «#7 ”qtvdulukiilw a up I! §. _ ‘ _ agfiwmmakufi #00 L1 This is to certify that the dissertation entitled BIOSYNTHESIS OF PRECURSORS TO THE AMINOSHIKIMATE PATHWAY AND MICROBIAL SYNTHESIS OF 5-AMINO-5-DEOXYSHIKIMATE presented by 0 I'd >- £9 E 3‘) g Jlantao Guo 99 i.- 'E _.l .9 D 2 has been accepted towards fulfillment of the requirements for the Ph. D. degree in Chemistry 2L W Major Professor’s Signature J/M/osl Date MSU is an Affirmative Action/Equal Opportunity Institution .- _.-.-.-—-o- - _.‘---¢—n--—..—-.—a_-.-. _- PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/07 p:/CIRC/DateDue.indd-p.1 BIOSYNTHESIS OF PRECURSORS TO THE AMINOSHIKIMATE PATHWAY AND MICROBIAL SYNTHESIS OF S-AMINO—S-DEOXYSHIKIMATE By J iantao Guo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 2004 ABSTRACT BIOSYNTHESIS OF PRECURSORS TO THE AMINOSHIKIMATE PATHWAY AND MICROBIAL SYNTHESIS OF 5-AMINO-5-DEOXYSHIKIMATE By J iantao Guo A variety of biologically active natural products are derived from 3-amino-5- hydroxybenzoate including rifamycins, ansamitocins, and mitomycins. It has been proposed that the biosynthesis of 3-amino-5-hydroxybenzoate proceeds via a novel variant of the shikimate pathway, which is called the aminoshikimate pathway. Although enzyme-catalyzed condensation of iminoE4P with phosphoenolpyruvate to form 4- amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP) has been proposed to be the first committed step in the aminoshikimate pathway, this enzyme activity has never been detected in vitro. This thesis establishes'the details of iminoE4P biosynthesis and its relationship with kanosamine biosynthesis. The result is the first demonstration of in vitro enzyme activity leading to formation of aminoDAHP. 3-Amino-3-deoxy-D-fructose 6-phosphate is identified as the biosynthetic precursor to iminoE4P in the cell-free lysate of Amycolatopsis mediterranei (ATCC 21789). Transketolase-catalyzed transfer of a glycoaldehyde from 3-amino-3-deoxy-D- fructose 6-phosphate to appropriate aldehyde receptors generates iminoE4P. Condensation of the resulting iminoE4P with phosphoenolpyruvate affords aminoDAHP. Orf 15 and RifH from A. mediterranei are assigned, respectively, as the dedicated transketolase and aminoDAHP synthase. 3-Amino-3-deoxy-D-fructose 6-phosphate biogenesis is subsequently traced to kanosamine. Biosynthesis of kanosamine and, ultimately, glutamine are thus established as the source of the aminoshikimate pathway’s nitrogen atom. To gain additional insights into the aminoDAHP formation in vivo, complexation between 0rf15-encoded transketolase and rifH—encoded aminoDAHP synthase is examined. The relationship between kanosamine biosynthesis, iminoE4P biosynthesis, and the aminoshikimate pathway is ultimately exploited to synthesize S-amino-S- deoxyshikimate. S-Amino-S-deoxyshikimate is also demonstrated to form in A. mediterranei fermentations when rifI-encoded aminoshikimate dehydrogenase is overexpressed. A two-step synthesis of S-amino-S-deoxyshikimate from glucose is then detailed involving synthesis of kanosamine from glucose using Bacillus pumilus (ATCC21143) and subsequent conversion of kanosamine into S-amino-S-deoxyshikimate catalyzed by E. coli SPl.1/pJG9.24OA. Bacillus pumilus synthesizes 25 g/L of kanosamine in 28% (mol/mol) yield from glucose. E. coli _SP1.1/pJG9.240A then synthesizes 1.2 g/L of S-amino-S-deoxyshikimate in 19% (mol/mol) yield from kanosamine. Copyright by Jiantao Guo 2004 To my family and friends For their love and encouragement ACKNOWLEDGMENTS The first person I would like to thank is Prof. John Frost for his patience, guidance, and encouragement throughout the course of my graduate career. His dedication and demand for high quality research will be a standard I will try to maintain throughout my career. In addition, I would like to thank the members of my graduate committee, Prof. Babak Borhan, Prof. David P. Weliky, and Prof. James H. Gieger for their input during the preparation of this thesis. I am grateful to Dr. Karen M. Draths for her wealth of knowledge that she freely shared with me. I thank her from the bottom of my heart for the arduous task of proof reading my thesis and being critical about it. I would like to extend my gratitude towards the past members of the group including Dr. Sunil Chandran, Dr. Chad Hansen, Dr. Dave Knop, and Dr. Padmesh Venkitasubramanian who helped me in various ways during the early stages of my graduate career. I would also like to thank the present group members Wei Niu, Jian Yi, Ningqing Ran, Mapitso Molefe, Heather Stueben, Xiaofie Jia, Wensheng Li, Man-Kit Lao, Justas Jancauskas, Jinsong Yang, Lee Kin Sing, and Dr. Jihane Achkar for their assistance and their friendship. Last but not least I would like to thank my parents for their love, constant encouragement and support through out my life. This thesis is dedicated to them. vi TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................... XII LIST OF TABLES .................................................................................................... XVII LIST OF ABBREVIATIONS ..................................................................................... XIX CHAPTER ONE ............................................................................................................. 1 INTRODUCTION ........................................................................................................... 1 Study of Biosynthetic Pathways .............................................................................. 4 Biocatalytic synthesis of natural products .......................................................... 4 Biosynthesis of unnatrual products .................................................................... 7 Biosynthesis of biosynthetic pathway intermediates .......................................... 8 Biosynthesis of Ansamycins ................................................................................... 9 Ansamycins ...................................................................................................... 9 Biosynthesis of ansamycins ............................................................................ 12 CHAPTER TWO .......................................................................................................... 23 IN VITRO ELABORATION OF THE PRECURSORS TO THE AMINOSHIKIMATE PATHWAY .................................................................................................................. 23 Introduction .......................................................................................................... 23 IminoE4P and AminoDAHP Biosynthesis ............................................................ 26 Overview ........................................................................................................ 26 Synthesis of 3-amino-3-deoxy-D-fructose 6-phosphate (aminoF6P) ................ 30 Experimental designs for examination of aminoF6P as the biosynthetic precursor to iminoE4P ....................................................... 32 Enzyme purifications and A. mediterranei cell-free lysate preparation ............ 33 Reaction of aminoF6P in the presence of E. coli transketolase (TktA) and DAHP synthase (AroFFBR) ............................................................ 35 Reaction of aminoF6P in the presence of E. coli transketolase (TktA) and the hypothetical aminoDAHP synthase (RifH) from A. mediterranei ........ 38 Reaction of aminoF6P in A. mediterranei cell-free lysate ................................ 39 RifH and other possible aminoDAHP synthases in A. mediterranei ................. 41 3-Amino-3—deoxy-D-fructose 6-phosphate Biosynthesis ........................................ 43 Overview ........................................................................................................ 43 Synthesis of kanosamine 6-phosphate ............................................................. 45 Experimental designs for examination of kanosamine 6-phosphate as a biosynthetic precursor to 3-amino-3-deoxy-D-fructose 6-phosphate ......... 46 Reaction of kanosamine 6-phosphate in the presence of yeast phosphoglucose isomerase, E. coli transketolase (TktA), and DAHP synthase (AroFFBR) ........................................................................ 47 Reaction of kanosamine 6-phosphate in the presence of yeast vii phosphoglucose isomerase, E. coli transketolase (TktA), and A. mediterranei aminoDAHP synthase (RifH) .......................................... 48 Reaction of kanosamine 6-phosphate in A. mediterranei cell-free lysate .......... 49 Complexation of RifH—encoded AminoDAHP Synthase with 0rf15-encoded Transketolase ............................ , ................................................... 50 Overview ........................................................................................................ 50 Glycerol effects on aminoDAHP synthesis from aminoF6P ............................ 51 Examination of the interaction between rifl-I-encoded aminoDAHP synthase and 0rf15—encoded transketolase using a bacterial two-hybrid system ............. 53 Kanosamine and Kanosamine 6-phosphate Biosynthesis ....................................... 59 Overview ........................................................................................................ 59 Synthesis of UDP-D-6,6-[2H2]-glucose ............................................................ 61 Synthesis of UDP-3-keto-D-glucose ................................................................ 63 Isolation of kanosamine biosynthetic genes from B. pumilus ........................... 64 Kanosamine biosynthesis in A. mediterranei ................................................... 75 Kanosamine 6-phosphate biosynthesis ............................................................ 77 The Source of the Nitrogen Atom Incorporated into the Aminoshikimate Pathway ................................................................................................................ 8 1 Discussion ............................................................................................................ 83 CHAPTER THREE ..................................................................................................... 104 IN VIVO ELABORATION OF THE AMINOSHIKIMATE PATHWAY AND BIOCATALYTIC SYNTHESIS OF 5-AMINO-5-DEOXYSHIKIMATE .................. 104 Introduction ........................................................................................................ 104 Biocatalytic Synthesis of 5-Amino-5-deoxyshikimate by Recombinant A. medzterranet .............................. 107 Overview ...................................................................................................... 107 Plasmid construction ..................................................................................... 110 Transformation (electroporation) of A. mediterranei with pJG8.219A ........... 1 1 1 Batch fermentaor conditions ......................................................................... l 12 Overexpression of Rifl in A. mediterranei ..................................................... 113 Biocatalytic Synthesis of Aminoshikimate by Recombinant E. coli ..................... 115 Overview ...................................................................................................... l 15 Biocatalytic synthesis of aminoshikimate from glucose ................................. 116 Overview ................................................................................................ 116 Host strain ............................................................................................... 117 Plasmid construction ............................................................................... 1 18 Fed-batch fermentor conditions ............................................................... 127 Fermentation of E. coli SP1. l/JG7.071A ................................................. 128 Biocatalytic synthesis of aminoshikimate from glucose via intermediacy of kanosamine .......................................................................... 131 Overview ................................................................................................ 13 1 Biosynthesis of kanOsamine by B. pumilus .............................................. 133 Fed-batch fermentor conditions ............................................................... 135 Biosynthesis of aminoshikimate by E. coli SP1.l/pKD12.138 ................. 136 Biosynthesis of aminoshikimate by E. coli SP1.l/pJGS.166A ................. 137 viii Biosynthesis of aminoshikimate by E. coli SP1. 1/pJG6.071A ................. 147 Biosynthesis of aminoshikimate by SP1.l/pJG6.18lB and SP1.l/pJG6.223B .................................................................................... 148 Biosynthesis of aminoshikimate by SP1.l/pJG6.238A and SP1.l/pJG9.240A ................................... y ................................................. 155 Discussion .......................................................................................................... 165 CHAPTER FOUR ....................................................................................................... 172 EXPERIMENTALS .................................................................................................... 172 General Methods ................................................................................................ 172 General Chemistry .............................................................................................. 172 Chromatography ................................................................................................. 172 Spectroscopic Measurements .............................................................................. 174 Chemical Assays ................................................................................................ 175 Thiobarbituric acid (TBA) assay ................................................................... 175 Organic and inorganic phosphate assay ......................................................... 176 Ninhydrin assay ............................................................................................ 177 Bacterial Strains and Plasmids ............................................................................ 178 Storage of Bacterial Strains and Plasmids ........................................................... 178 Culture Medium ................................................................................................. 179 Fermentation Conditions ..................................................................................... 182 General ......................................................................................................... 182 Fed-batch fermentations of E. coli ................................................................. 183 Batch fermentations of A. mediterranei ......................................................... 184 Fed-batch fermentations of B. pumilus .......................................................... 185 Analysis of Culture Supernatant ........................................... . .............................. 186 Product Purification from Fermentation Broth .................................................... 187 Purification of kanosamine from fermentation broth of B. pumilus ................ 187 Purification of aminoshikimate from fermentation broth of A. mediterranei .. 188 Purification of aminoshikimate from fermentation broth of E. coli ................ 188 Genetic Manipulations ........................................................................................ 189 General procedures ....................................................................................... 189 Determination of DNA concentration ............................................................ 190 Large scale purification of plasmid DNA ...................................................... 190 Small scale purification of plasmid DNA ...................................................... 192 Restriction enzyme digestion of DNA ........................................................... 193 Agarose gel electrophoresis ........................................................................... 194 Isolation of DNA from agarose ..................................................................... 194 Treatment of DNA with Klenow fragment .................................................... 195 Treatment of DNA with Mung bean nuclease ................................................ 195 Treatment of vector DNA with calf intestinal alkaline phosphatase (CIAP)... 196 Ligation of DNA ........................................................................................... 196 Purification of E. coli genomic DNA ............................................................ 196 Purification of B. pumilus genomic DNA ...................................................... 198 Purification of A. mediterranei genomic DNA .............................................. 199 Preparation and transformation of E. coli competent cells ............................. 200 ix Preparation and electroporation of A. mediterranei eletrocompetent cells ...... 201 General Enzymology ........................................................................................... 203 General information ...................................................................................... 203 DAHP synthase (AroFFBR) and aminoDAHP (RifH) synthase assay .............. 203 Transketolase (TktA or Orf 15) assay ............................................................. 204 Shikimate dehydrogenase (AroE) assay ........................................................ 205 Aminoshikimate dehydrogenase (RifI) assay ................................................. 205 Kanosamine kinase (le and RifN) assay ..................................................... 206 UDP-3-keto-D-glucose dehydrogenase assay ................................................ 206 Protein gel (SDS-PAGE) ............................................................................... 206 CHAPTER TWO ................................................................................................ 208 Synthetic Procedures .......................................................................................... 208 Synthesis of 3-amino-3-deoxy-[3-D-fructofuranose 6-phosphate .................... 208 Synthesis of kanosamine 6-phosphate ........................................................... 212 Synthesis of UDP—6,6-[2H2]-D-glucose .......................................................... 216 Genetic Manipulations ........................................................................................ 218 Plasmid pJG6. 128A ...................................................................................... 218 Plasmid pJG7.241 ......................................................................................... 218 Plasmid pJG7.246 ......................................................................................... 219 Plasmid pJG9.150 ......................................................................................... 219 Plasmid pJG9. 154 ......................................................................................... 219 Plasmid pJG9.213 ......................................................................................... 219 Plasmid pJG9.251 ......................................................................................... 220 Enzyme Purifications .......................................................................................... 220 E. coli tktA-encoded transketolase................................... .............................. 220 E. coli aroFFBR-encoded DAHP synthase ...................................................... 222 A. mediterranei rifl-l-encoded aminoDAHP synthase .................................... 223 A. mediterranei rifN-encoded kanosamine kinase .......................................... 224 B. pumilus UDP-3-keto-D-glucose dehydrogenase ........................................ 225 Recombinant UDP-3-keto—D-glucose dehydrogenase .................................... 227 Cell-free Lysate Preparations .............................................................................. 228 Cell-free lysate of A. mediterranei ................................................................ 228 Cell-free lysate of B. pumilus ........................................................................ 229 In Vitro Enzymatic Reactions ............................................................................. 229 Biosynthesis of aminoDAHP from aminoF6P ............................................... 229 Biosynthesis of aminoDAHP from kanosamine 6-phosphate ......................... 231 Biosynthesis of kanosamine from UDP-glucose ............................................ 232 Biosynthesis of 6,6-[2H2]-kanosamine ........................................................... 233 Biosynthesis of 7,7-[2H2]-aminoDAHP ......................................................... 233 le or RifN-catalyzed phosphorylation of kanosamine ................................. 235 BacterioMatch Two-Hybrid System .................................................................... 236 Inverse PCR ....................................................................................................... 238 CHAPTER THREE ............................................................................................ 238 Strain Purification ............................................................................................... 238 Genetic Manipulations ........................................................................................ 241 Plasmid pJGS. 165A ...................................................................................... 241 Plasmid pJGS. 166A ...................................................................................... 241 Plasmid pJG6.07OA ...................................................................................... 241 Plasmid pJG6.071A ...................................................................................... 242 Plasmid pJG6.128A ...................................................................................... 242 Plasmid pJG6. 154A ..................................... , ................................................. 243 Plasmid pJG6.1558 ...................................................................................... 243 Plasmid pJG6.180A ...................................................................................... 243 Plasmid pJG6.181B ...................................................................................... 244 Plasmid pJG6.222A ...................................................................................... 244 Plasmid pJG6.223B ...................................................................................... 245 Plasmid pJG6.238A ...................................................................................... 24S Plasmid pJG7.032A ...................................................................................... 246 Plasmid pJG7.039A ...................................................................................... 246 Plasmid pJG7.046A ...................................................................................... 246 Plasmid pJG7.056A ...................................................................................... 247 Plasmid pJG7.071A ...................................................................................... 247 Plasmid pJG8.155 ......................................................................................... 248 Plasmid pJG8.219A ...................................................................................... 248 Plasmid pJG9.240A ...................................................................................... 249 REFERENCES ............................................................................................................ 250 xi LIST OF FIGURES Figure 1. Structures of rifamycins, rifampicin, geldanamycin, and ansamitosins .............. 2 Figure 2. Synthetic applications of 5-amino-5-deoxyshikimate ........................................ 3 Figure 3. The biosynthetic building units of rifamycin S ................................................ 12 Figure 4. [1-'3C]-3-amino-5-hydroxybenzoate incorporation experiments ..................... 13 Figure 5. The shikimate pathway ................................................................................... 14 Figure 6. Labeling patterns of shikimate, 3-amino-5-hydroxybenzoate, and rifamycin from 13 C-labeled glucose and glycerate .......................................................... 15 Figure 7. Labeling pattern of mitomycin C from labeled D-erythrose and pyruvic acid ............................................................................................ 17 Figure 8. Rifamycin biosynthetic gene cluster of A. mediterranei S699 and proposed enzyme functions ..................................................................... 18 Figure 9. The proposed aminoshikimate pathway .......................................................... 21 Figure 10. Biosynthesis of iminoE4P and aminoDAHP ................ . ................................ 23 Figure 1 1. RifK and RifL homology .............................................................................. 27 Figure 12. Biosynthesis of E4P and hypothetical biosyntheses of iminoE4P .................. 28 Figure 13. Synthesis of 3-amino-3-deoxy—D-fructose 6-phosphate (aminoF6P) .............. 31 Figure 14. Examination of 3-amino-3-deoxy-D-fructose 6-phosphate as the biosynthetic precursor to iminoE4P .................................................................................. 33 Figure 15. TktA-catalyzed fragmentation of aminoF6P .................................................. 38 Figure 16. Naturally occurring 3-aminosugars ............................................................... 43 Figure 17. Hypothetical aminoF6P biosynthesis ............................................................ 44 Figure 18. Synthesis of kanosamine 6-phosphate ........................................................... 45 Figure 19. Examination of kanosamine 6-phosphate as a biosynthetic precursor to aminoF 6P ................................................................................................. 47 xii Figure 20. Figure 21. Figure 22. Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. Figure 29. Figure 30. Figure 31. Figure 32. Figure 33. Figure 34. Figure 35. Figure 36. Figure 37. Figure 38. Figure 39. Examine protein-protein interaction using a bacterial two-hybrid system ...... 54 B-Galactosidase activity assay ...................................................................... 57 Antibiotics containing kanosamine or kanosamine-like structure .................. 59 Hypothetical kanosamine and kanosamine 6-phosphate biosynthesis ............ 60 Synthesis of UDP-D-6,6-[2H2]-glucose ......................................................... 62 Synthesis of UDP-3-keto-D-glucose .............................................................. 63 Enzyme encoding genes associated with 3-amino-5-hydroxybenzoate biosynthesis and hypothetical kanosamine and kanosamine 6—phosphate biosynthesis in A. mediterranei .................................................................... 65 Kanosamine biosynthesis with fractioned B. pumilus cell-free lysate ............ 66 Enzyme assay designs for UDP-3-keto-D-glucose dehydrogenase ................. 67 Absorption spectra of UDP-3-keto-D-glucose dehydrogenase ....................... 68 N-terminal sequences of UDP-3-keto-D-glucose dehydrogense ..................... 70 Inverse PCR ................................................................ . ................................ 71 The sequence of B. pumilus UDP-3-keto-D-glucose dehydrogenase .............. 72 Modified kanosamine biosynthetic pathway in B. pumilus ............................ 73 Phosphorylation of kanosamine .................................................................... 78 Kanosamine 6-phosphate biosynthesis .......................................................... 81 The biosynthesis of precursors to the aminoshikimate pathway in A. mediterranei ................................................................................................. 84 'H NMR of chemically synthesized 3—amino-3-deoxy-D-fructose 6—phosphate .................................................................................................. 88 13C NMR of chemically synthesized 3-amino-3-deoxy-D-fructose 6-phosphate .................................................................................................. 89 COSY of chemically synthesized 3-amino-3-deoxy-D-fructose 6-phosphate .................................................................................................. 90 xiii Figure 40. Figure 41. Figure 42. Figure 43. Figure 44. Figure 45. Figure 46. Figure 47. Figure 48. Figure 49. Figure 50. Figure 51. Figure 52. Figure 53. Figure 54. Figure 55. Figure 56. Figure 57. Figure 58. Figure 59. HMQC of chemically synthesized 3-amino-3-deoxy-D-fructose 6-phosphate .................................................................................................. 91 'H NMR of chemically synthesized kanosamine 6-phosphate ....................... 92 13C NMR of chemically synthesized kanosamine 6-phosphate ...................... 93 COSY of chemically synthesized kanosamine 6-phosphate ........................... 94 HMQC of chemically synthesized kanosamine 6-phosphate ......................... 95 lH NMR of 4-amino-3,4-dideoxy-D-arabin0-heptulosonic acid 7-phosphate (aminoDAHP) .......................................................................... 96 13C NMR of 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP) .......................................................................... 97 COSY of 4-amino-3,4-dideoxy-D-arabin0-heptulosonic acid 7-phosphate (aminoDAHP) .......................................................................... 98 HMQC of 4-amino-3,4-dideoxy-D-arabin0-heptulosonic acid 7-phosphate (aminoDAHP) .......................................................................... 99 lH NMR of kanosamine l-phosphate .......................................................... 100 13C NMR of kanosamine l-phosphate ......................................................... 101 1H NMR of kanosamine .............................................................................. 102 '3C NMR of kanosamine ............................................................................. 103 The aminoshikimate pathway and biocatalytic synthesis of 5-amino-5-deoxyshikimate .................................................................... 105 Synthesis of Tamiflu ................................................................................... 106 Preparation of Plasmid pJG8.155 ................................................................ 108 Preparation of Plasmid pJG8.219A ............................................................. 109 Biocatalytic synthesis of aminoshikimate in E. coli ..................................... 116 Preparation of Plasmid pJG6.154A ............................................................. 119 Preparation of Plasmid pJG6.ISSB ............................................................. 120 xiv Figure 60. Preparation of Plasmid pJG7.032A ............................................................. 121 Figure 61. Preparation of Plasmid pJG7.039A ............................................................. 122 Figure 62. Preparation of Plasmid pJG7.046A ............ y ................................................. 123 Figure 63. Preparation of Plasmid pJG7.056A ............................................................. 124 Figure 64. Preparation of Plasmid pJG7.07lA ............................................................. 125 Figure 65. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG7.071A under glucose-limited conditions ......................................................................... 129 Figure 66. Two-step biosynthesis of aminoshikimate from glucose .............................. 131 Figure 67. Purification and identification of B. pumilus (ATCC 21143) ....................... 132 Figure 68. Biosynthesis of aminoshikimate by E. coli SP1.l/pKD12.138 under glucose-limited conditions ......................................................................... 137 Figure 69. Preparation of Plasmid pJG5.165A ............................................................. 140 Figure 70. Preparation of Plasmid pJG5.166A ............................................................. 141 Figure 71. Biosynthesis of aminoshikimate by E. coli SP1. 1/pJG5. 166A under Figure 72. Figure 73. Figure 74. Figure 75. Figure 76. Figure 77. Figure 78. Figure 79. glucose- limited conditions ......................................................................... 142 Biosynthesis of aminoshikimate by E. coli SP1.l/pJG5.166A under glucose-limited conditions without kanosamine supplementation ............... 143 Biosynthesis of aminoshikimate by E. coli SP1.l/pJG5.l66A under glucose-rich conditions .............................................................................. 144 Preparation of Plasmid pJG6.070A ............................................................. 145 Preparation of Plasmid pJG6.07lA ............................................................. 146 Biosynthesis of aminoshikimate by E. coli SP1 .1/pJG6.071A under glucose-rich conditions .............................................................................. 148 Preparation of Plasmid pJG6.180A ............................................................. 149 Preparation of Plasmid pJG6.181B ............................................................. 150 Biosynthesis of aminoshikimate by E. coli SP1.l/pJG6.1818 under glucose-rich conditions .............................................................................. 151 XV Figure 80. Figure 81. Figure 82. Figure 83. Figure 84. Figure 85. Figure 86. Figure 87. Figure 88. Figure 89. Figure 90. Preparation of Plasmid pJG6.222A ............................................................. 152 Preparation of Plasmid pJG6.223B ............................................................. 153 Biosynthesis of aminoshikimate by E. coliISP1.1/pJG6.223B under glucose-rich conditions .............................................................................. 154 Preparation of Plasmid pJG6.128A ............................................................. 157 Preparation of Plasmid pJG6.238A ............................................................. 158 Biosynthesis of aminoshikimate by E. coli SP1 . 1/pJG6.23 8A under glucose-rich conditions .............................................................................. 159 Biosynthesis of aminoshikimate by E. coli SP1.l/pJG9.24OA under glucose-rich conditions ............................................................................. 159 Preparation of Plasmid pJG9.240A ............................................................. 160 1H NMR of the culture supernatant of SP1.l/pJG9.240 after 48 h ............... 169 1H N MR of 5-amino-5-deoxyshikimate purified from fermentation broth 170 13C NMR of 5-amino-5-deoxyshikimate purified from fermentation broth .. 171 xvi Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Table 9. Table 10. Table 1 1. Table 12. Table 13. Table 14. Table 15. Table 16. Table 17. Table 18. LIST OF TABLES Origin and biological activities of various natural ansamycins ...................... 10 Proposed functions of rifbiosynthetic gene products .................................... 19 Plate selection for characterization of orfl 5 gene product ............................. 29 Restriction enzyme maps of plasmids pJG4.150 and pSK4.172 .................... 30 Restriction enzyme maps of plasmids pSK4. 172, pKL4.71, MMBOIZ, EDAHPB, and PEI ....................................................................................... 34 Reaction of aminoF 6P in the presence of transketolase, DAHP synthase, and aminoDAHP synthase ............................................................................ 36 Reaction of aminoF6P in the presence of different DAHP synthases. ........... 42 Reaction of kanosamine 6-phosphate (K6P) in the presence of yeast phosphoglucose isomerase, transketolase, DAHP synthase, and aminoDAHP synthase .................................................................................. 49 Glycerol effects on DAHP synthase activities and aminoDAHP synthesis from aminoF6P in A. mediterranei cell-free lysates ....................... 52 Restriction enzyme maps of plasmids using in the bacteria two-hybrid experiments .................................................................................................. 55 Study of protein-protein interactions by bacterial two-hybrid system ............ 56 Purification of B. pumilus UDP-3-keto-D-glucose dehydrogenase ................ 69 Restriction enzyme maps of plasmids pJG9. 197 and pJG9.251 .................... 70 Genetic organization around UDP-3-keto-D-glucose dehydrogenase ............ 74 Kanosamine biosynthesis in A. mediterranei ................................................ 75 Restriction enzyme map of plasmid pRM070 ............................................... 78 Biosynthesis of kanosamine from UDP-glucose in A. mediterranei cell-free lysates ............................................................................................ 79 15 . . . . _ , N enrichments 1n kanosamine produced in A. medzterranez xvii cell-free lysates with different '5 N labeled nitrogen sources ......................... 82 Table 19. B. pumilus (ATCC 21143) strain identification and isolation ...................... 133 Table 20. Improving kanosamine synthesis by B. pumilus fermentation ..................... 135 Table 21. Product concentrations and yields achieved by catalysts grown under different culture conditions. .............................................................. 1.39 Table 22. DAHP synthase specific activities measured during fermentation runs ....... 162 Table 23. Transketolase specific activities measured during fermentation runs .......... 163 Table 24. Kanosamine kinase specific activities measured during fermentation runs.. 164 xviii Ac ADP AHBA LIST OF ABBREVIATIONS acetyl adenosine diphosphate 3-amino-5-hydroxybenzoate aminoDAHP 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate aminoDHQ aminoDHS aminoF6P K6P aminoS7P aminoSA AMP Ca CIAP Cm DAHP DCIP DEAE DHAP 5—amino-5-deoxy-3-dehydroquinic acid; aminoDHS 5-amino-5—deoxy-3-dehydroshikimate 3-amino-3-deoxy-D-fructose 6-phosphate kanosamine 6-phosphate 4-amino-4-deoxy-sedoheptulose 7-phosphate 5-amino-5-deoxyshikimate adenosine monophosphate ampicillin adenosine triphosphate base pair carbenicillin calf intestinal alkaline phosphatase chloramphenicol 3-deoxy-D-arabino-heptulosonic acid 7- phosphate dichloroindophenol diethylaminoethyl dihydroxyacetone l-phosphate xix DHQ DHS DNA DO DTT E4P F6P FAB FBR FPLC G6P GAP Glu h His HPLC HRMS iminoE4P IPTG Kan k kg 3-dehydroquinate 3-dehydroshikimate 3-deoxyribonucleic acid dissolved oxygen dithiothreitol D-erythrose 4-phosphate erythromycin D-frctose 6-phophate fast atom bombardment feed back resistant fast protein liquid chromatography D-glucose 6-phosphate D-glyceraldehydes 3-phosphate L- glutamate hour L-histidine high pressure liquid chromatography high resolution mass spectrometry l -imino- 1 —deoxy-D-erythrose 4—phosphate isopropyl B-D-thiogalactopyranoside kanamycin rate constant kilogram XX LB mg 11L mM MS min NAD NADH NADP N ADPH N MR OD PEG PEP PCR PMS PPm psi R5P RNA rpm luria broth molar milligram milliliter microliter millimolar mass spectrometry minute nicotinamide adenine dinucleotide, oxidized form nicotinamide adenine dinucleotide, reduced form nicotinamide adenine dinucleotide phosphate, oxidized form nicotinamide adenine dinucleotide phosphate, reduced form nuclear magnetic resonance optical density polyethylene glycol phosphoenolpyruvate polymerase chain reaction phenazine methosulfate parts per million pounds per square inch D-ribose 5-phosphate ribonucleic acid revolutions per minute xxi S7P SA SDS Tc THF TLC TMS TSP UTP UV room temperature D-sedoheptulose 7-phosphate shikimate sodium dodecyl sulfate tetracycline tetrahydrofuran thin-layer chromatography trimethylsilyl sodium 3-(trimethylsilyl)propionic-2,2,3,3-d4 uridine 5’-triphosphate ultraviolet xxii CHAPTER ONE INTRODUCTION Natural products have served as major sources of medicinal agents for centuries. However, natural or semisynthetic products derived from microorganisms and used in the pharmaceutical industry have a much shorter history. Their impact on medicine has only dated back about 60 years to the introduction of penicillin.1 Microbes, marine sponges, and plants have become major sources of medically important natural products. The ansamycins2 constitute a noteworthy class of bioactive natural products, which are obtained from microorganisms. Rifamycins and ansamitosins (Figure 1) are examples of ansamycin natural products that display, respectively, antibacterial and antitumor activities. Given their important medicinal applications, understanding the way ansamycins are put together by living organisms has been vigorously pursued by scientists.3 The work carried out in completion of this thesis focused on elaborating the precursors to the aminoshikimate pathway, which results in the formation of 3-amino-5- hydroxybenzoate, a biosynthetic precursor to the ansamycins4 and the mitomycins.5 Beyond answering fundamental biosynthetic questions associated with the aminoshikimate pathway, developing an understanding of the molecular aspects of this pathway could have many important practical consequences. For example, elaboration of the aminoshikimate pathway could lead to development of genetically modified strains capable of synthesizing these medically important products or analogs in higher concentrations and yields. Additionally, unnatural starter units could be biosynthesized by manipulating the 3-amino-5-hydroxybenzoate biosynthetic pathway. These unnatural starter molecules could then be utilized by polyketide synthases to biosynthesize unnatural polyketides, which might have novel bioactivities. 5-Amino-5- deoxyshikimate, which could be made by genetic modification of the aminoshikimate pathway, is a hydroaromatic compound. Similar to the application of shikimate,6 the carbocyclic array of stereogenic centers and functional groups make 5-amino-5- deoxyshikimate an interesting core scaffold for combinatorial library construction (Figure 2). 5-Amino-5-deoxyshikimate might also be useful as a starting material for the synthesis of drugs such as Tamiflu (Figure 2). Shikimate is currently used as the starting material for the synthesis of this neuraminidase inhibitor produced and marketed by .. 7 Roche as an antunfluenza agent. / . CH3 H305 5 H H Ft = COCH3 ansamitocin P-1 Fl = COCHZCHa ansamitocin P-2 R = COCH(CH3)2 ansamitocin P-3 n = OCHQCOOH. n' = H rifamycin B , , R = COCHchcha ansamitocin P-4 n = OCOCH20H, n' = H rifamycin L X = 0 "lama.” G n = OH, R' = H ,_, rifamycin sv X = CO “'3'“de 3 Fl = OH, R' = CH=N-N‘_’N-CH3 ritampicin Me Me OMe OyNHz OYN O {O o o y R ’- OMe CH30 OH H30 N 2(N-R' H3C N :(N-CH3 O O R = CH30. R' = H mitomycin A . R = NH2, Fi' = H mitomycin C geldanamycm Fl = NH2, Fi' = CH3 portiromycin mitomycin B Figure 1. Structures of rifamycins, rifampicin, geldanamycin, and ansamitosins. H OO\/ 0 N-. cozH combinatorial ‘ ............ —>_. \I library . ‘ be 0‘“ "N112 OH 5H WY 0 5-amino—5-deoxyshikimate _ Tamlflu Figure 2. Synthetic applications of S-amino-S-deoxyshikimate. Chapter One will present an overview of ansamycin biosynthesis and the origin of the aromatic starter unit, which is derived from the aminoshikimate pathway. Chapter Two details research that elaborates the biosynthetic precursors to the aminoshikimate pathway and provides mechanistic details of the biosynthesis of these precursors. Two classical methods used in the study of natural product biosynthesis are applied in this chapter. The first method comprises synthesizing the putative biosynthetic precursors and incubating them in Amycolatopsis mediterranei (ATCC21789) cell-free lysate, which is then analyzed for the formation of the aminoshikimate pathway intermediates. The second method involves isolating and characterizing enzymes associated with this pathway. In Chapter Three of this dissertation, biocatalytic synthesis of 5-amino—5- deoxyshikimate will be discussed. Although this hydroaromatic alkaloid is the namesake of the aminoshikimate pathway, it is not known to be a metabolite formed during biosynthesis of 3-amino-5-hydroxybenzoate. The formation of 5-amino-5- deoxyshikimate from kanosamine in E. coli provides another evidence for the elucidated mechanism of the iminoE4P biosynthesis and the aminoshikimate pathway. By combining the results of in vitro (Chapter Two) and in vivo (Chapter Three) studies, 1- imino-l-deoxy-D-erythrose 4-phosphate (iminoE4P), 3-amino-3-deoxy-D-fructose 6- phosphate (aminoF6P), kanosamine 6-phosphate, and kanosamine are confirmed as biosynthetic precursors to the aminoshikimate pathway. For the first time, in vitro biosynthesis of aminoDAHP is demonstrated. Study of Biosynthetic Pathways Natural products represent a valuable source of metabolites that are essential for the production of many bioactive goods, including numerous commercial antibiotics and anticancer drugs. By understanding how these natural products are created in nature, scientists can gain knowledge on how to manipulate or modify biosynthetic pathways to improve or alter biocatalytic syntheses of medically important natural products. Unraveling the precursors, intermediates, enzymes, and encoding genes associated with biosynthetic pathways also allows scientists to manipulate the pathways to produce products that are not found in nature and may possess unique biological properties. In addition, biocatalytic synthesis of intermediates in biosynthetic pathways can be exploited to gain access to chiral synthons as well as other starting materials and intermediates of use to chemical synthesis. Biocatalytic synthesis of natural products Between 1981 and 2002, about 78% of antibiotics and 60% of anticancer drugs introduced were natural products or derived from natural products.8 Some well-known antibiotics that are either natural products or synthesized from natural products include the B-lactams penicillin G1 (natural product) and cephalosporins9 (semisynthetic), the aminoglycosides neomycin10 (natural product) and gentamicinll (natural product), the macrolides erythromycin12 (natural product), azithromycinl3 (semisynthetic). and clarithromycin14 (semisynthetic), the polypeptide polymixin B15 (natural product), the aminocyclitol spectinomycin16 (natural product), the glycopeptide vancomycinl7 (natural product), and tetracycline (natural product).18 Some notable anticancer drugs currently in clinical use or clinical trials include the plant products taxol19 and vincristine,20 the marine products dolastatin21 and bryostatin,22 and the microbial products bleomycin23 and mitomycin C. 24 An obstacle encountered with using natural products directly as drugs or for the synthesis of drugs is that the quantities available from natural sources are often limited. One way to overcome this limitation entails de novo chemical synthesis. Many medicinally important natural products have been produced via total synthesis. However, these syntheses often require multiple steps, prohibitive numbers of man-hours, large quantities of starting materials, and generate considerable volumes of harmful waste for the synthesis of limited amounts of the target molecule. Although creative and intellectually rewarding, total synthesis can be an inefficient means to produce large quantities of natural products. Biosynthetic and semisynthetic approaches are therefore employed as the alternative method to obtain significant amounts of natural products or . . . . . . 25 . . . . their derivatives. One example 18 Vitamin B12, which was originally discovered as an antipemicious anemia factor. Total chemical synthesis of vitamin B12 was achieved by Woodward and Eschenmoser in the 1960’s and 1970’s.26 It took more than 100 scientists and a period of 11 years to accomplish this feat in synthetic organic chemistry.27 Although this synthesis had a pervasive impact on organic chemistry, its 70-step synthesis makes any industrial production of vitamin B12 by chemical synthesis too expensive to be commercially viable. In contrast, a liter of Pseudomonas denitrificans 7 can make 60 mg of vitamin 8,2 from sucrose at 30°C in 3 days.2 ' 28 This is the reason that fermentations, instead of chemical synthesis, are used exclusively for the commercial production of vitamin B”. Like synthetic methods continuously being modified and discovered to make synthesis more efficient, microbial strains can also be developed to produce higher concentrations and yields of natural products. Historically, random mutagenesis has been used to increase the yields of microbe-synthesized natural products. This approach has had many successes, as exemplified by the 4000-fold improvement in penicillin production achieved by random mutagenesis of parental strains.29 However, random mutagenesis has several major drawbacks. Mutated strains often acquire detrimental mutations that impair their growth and metabolic vigor. The time required to obtain improved strains by random mutagenesis is often long and not predictable. The random mutations leading to high concentrations and yields of biosynthesized product are not readily identified, which prevents analogous mutations from being introduced into other strains that biosynthesize the same or related natural products. These drawbacks inspired scientists to find alternative ways to increase the yields of natural products synthesized by microbes. The cumulative elaboration of biosynthetic pathways and the development of new genomic technologies have resulted in a revolution in biocatalytic synthesis of natural products. Amplified expression of biosynthetic gene clusters and genetic modification of biosynthetic pathways have become widely employed alternatives for improving microbial synthesis of natural products.30 For example, an increase of 176% in the penicillin productivity (g/L) was achieved when the whole penicillin gene cluster was amplified in Penicillium chrysogenum Wis 54-1255.31 This increase in productivity of penicillin corresponds to about 5 years (1972—1977) of strain improvement using . 32 random mutageneSis. Biosynthesis of unnatural products With microbes developing resistance to almost every antibiotic in clinical use and people’s quest for effective anticancer agents, efforts have been continuously made to identify new classes of drugs. Unnatural product biosynthesis, which entails the microbial synthesis of drugs not found in nature, has become a new approach for the discovery of candidates for drug development. Biosynthesis of unnatural agents was pioneered by Hopwood in the 1970’s with initial attempts to produce hybrid antibiotics using protoplast fusion techniques in Streptomyces spp.33 Currently, manipulation of polyketide biosynthetic pathways represent one of the major areas of research in unnatural product biosynthesis.34 Polyketides are complex natural products that are produced by microorganisms and plants. They are biosynthesized by polyketide synthases, which are composed of many component enzymes or modules with various . . .. 3 . . , . . . . . catalytic act1vrties. 6 Starting from Collie 3 pioneering work in the biosyntheSIS of 35.36 polyphenols in 1907, enormous progress has been made in understanding biosynthetic pathways of polyketides during the past century.36 This knowledge provides the basis for producing unnatural polyketides by interchanging modules in different type I polyketide synthases (PKSs),37 mixing genes from different types (type I and II) of polyketide systems,38 and employing unnatural starter units.39 For example, a library of 61 erythromycin analogs was generated by genetic manipulation of 6-deoxyerythronolide B synthase (DEBS), a polyketide synthase that produces the macrolide ring of erythromycin.37 These unnatural erythromycin analogs would be impractical to produce by chemical methods. Unnatural product biosynthesis is a relatively young area of research. No clinically useful drug has been made via this approach. However, it is expected to be useful in the pharmaceutical industry and complements total synthesis, semisynthesis, and combinatorial synthesis.4O When integrated with combinatorial and chemical synthesis, it has the potential of leading to totally new and unique structural classes and activity types of drugs that would not have emerged exclusively from a synthetic program or a biosynthetic program. Biosynthesis of Biosynthetic pathway intermediates Biosynthesis offers many benefits when integrated with traditional synthetic chemistry. It provides efficient and economical access to some important pharmaceutical intermediates. It also provides access to molecules that are difficult to make using traditional organic synthesis. Regio- and stereoselective transformations, reaction conditions employing ambient temperature and pressure, and less organic waste are the other attractions of biosynthesis. Microbial synthesis of biosynthetic pathway intermediates can be achieved by using mutant or genetically modified strains. Mutant strains are usually obtained by random mutagenesis. To genetically modify wild type strains requires the knowledge of the intermediates, enzymes, and encoding genes in a given biosynthetic pathway. Although the database required for genetic manipulation of a biosynthetic pathway in a microbe is substantial, it does provide more options and better chances for success. For example, shikimate is a seven-carbon carbocyclic intermediate in the common pathway of aromatic amino acid biosynthesis.4| The stereogenic centers and functional groups in shikimate make it an attractive chiral synthon for use in various synthetic schemes.42 It is originally isolated from the fruits of the Illicium plants,43 which complicated its commercial use as a starting material for the synthesis of the neuraminidase inhibitor Tamiflu.44 Based on the biosynthetic database available for the shikimate pathway, recombinant E. coli biocatalysts have been designed, which are capable of producing high concentrations of shikimate from glucose in good yields.45 This microbial synthesis enables shikimate to be a viable starting material in the commercial synthesis of Tamiflu (Figure 2). Biosynthesis of Ansamycins Ansamycins The ansamycins46 are a class of medically important natural products (Figure 1). The term ansamycin refers to chemical structures having an aromatic chromophore joined at two nonadjacent positions by an aliphatic ansa polyketide chain.47 The ansa chain is linked as an amide to the amino group of the chromophore. The aromatic chromophore in most of the known ansamycins is capable of forming a quinone-hydroquinone structure (Figure 1). To date, about 120 naturally-occurring ansamycins have been discovered.48 9 The ansamycins are typically divided into two groups according to their chromophores: the naphthalenoid ansamycins and the benzenoid ansamycins.46 The well-known naphthalenoid ansamycins include actamycin, ansathiazin, awamycin, halomicin, naphthomycin, rifamycin, streptovaricin, and tolypomycin (Table 1). The notable benzenoid ansamycins include ansamitocin, ansatrienin, geldanamycin, and maytansines (Table 1). Table 1. Origin and biological activities of various natural ansamycins. antibiotic producer biological activity actamycin Streptomyces sp. E784 antibacterial ansamitocin Actinosynnema pretiosum antitumor ansathiazin Streptomyces albolongus antibacterial ansatrienin Streptomyces collinus antibacterial, antifungal awamycin Streptomyces sp. antitumor geldanamycin Streptomyces hygroscopicus antitumor halomicin Micromonospora halophytica antibacterial maytansines Celastraceae, Rhamnaceae antitumor naphthomycin Streptomyces collinus antibacterial, antifungal rifamycin Amycolatopsis mediterranei antibacterial streptovaricin Streptomyces spectabilis antibacterial tolypomycin Streptomyces tolypophorus antibacterial Rifamycins, which are produced by Amycolatopsis mediterranei, were first isolated in Lepetit Research Laboratories49 as a complex of at least five components denoted rifamycins B, G, L, S, and SV (Figure 1). These were the first antibiotics found to selectively inhibit RNA synthesis by binding to bacterial RNA polymerase.50 10 Rifamycin B (Figure 1), although less active than other analogs,51 was chosen for development due to its stability, ease of isolation, solubility at physiological pH, and spontaneous transformation into more active rifamycin S in aqueous solution. It has been used clinically in a synthetically modified form called rifampicin52 (Figure 1) since the 1960’s, and it is still one of the first-line therapies for the treatment of tuberculosis and other mycobacterial infections.53 Geldanamycin (Figure 1) was isolated from Streptomyces hygroscopicus var. geldanus by DeBore and co-workers while screening for substances inhibiting the growth and multiplication of protozoa.S4 It belongs to the benzenoid ansamycins and possesses a shorter ansa chain than that of rifamycins. Geldanamycin is a potent antitumor antibiotic,55 which is active at nanomolar concentration against many (about 60) cell lines. It binds specifically to the heat shock protein Hsp90 and to its endoplasmic reticulum homologue GP96, and thus interferes with the cellular stress response and the conformational maturation of proteins.56 Ansamitosins (Figure l) are a class of benzenoid ansamycins produced by Actinomycete Actinosynnema pretiosum ssp. pretiosum and a mutant strain . . . 57 . . . . Actmosynnema pretzosum ssp. auranncum. They were first isolated by Higashide and co-workers in 1977.58 The structures and antitumor activities of ansamitocins are similar to those of the maytansinoids, which are extraordinarily potent antitumor agents that were originally isolated from plant families Celastraceae (Maytenus spp.), Rhamnaceae (Colubrina californica and Colubrina texensis), and Euphorbiaceae (Trewia nudzflora and Mallotus anomalus Meer et Chan).59 Ansamitosins are reported to interfere with the 11 dynamic instability of microtubules. As a result, mitotic cells are arrested in the M-phase of the cell division cycle, which leads to apoptotic cell death.60 Biosynthesis of ansamycins _/ propionate rifamycin S — acetate Figure 3. The biosynthetic building units of rifamycin S. The carbon skeleton of the ansamycins closely resembles that of macrolide antibiotics of the erythromycin type.61 In the ansa chain, the methyl groups alternate with hydroxyl groups (Figure 3). This feature suggests that the ansa chain originates from a polyketide chain formed by condensation of methylmalonate units with the insertion of several malonate units. The biosynthesis of ansamycins was first studied in the formation of rifamycin S by incorporation experiments with 1“C- and 3H-labeled precursors}52 Analysis of the radioactivity of different fragments of the molecule, obtained by chemical degradation, established that propionate and acetate was incorporated head to tail (Figure 3), which is in agreement with the general pattern of a polyketide chain. Using l3C enriched precursors and with the help of 13’C NMR, the 12 biosynthetic origin of the carbon atoms of the ansa chain of rifamycin S was fully established (Figure 3).63 The other ansamycins that have been used to study the ansa . . . . 64 . 65 . 66 chain formation include geldanamycm, naphthomycm, actamycrn, and . . 67 streptovaricm. *cozu H0 NHZ 3-amino-5-hydroxybenzoate O y NH2 0 O pMe N N ' CH3 0 porfiromycin ansamitocin P-3 Figure 4. [133C]-3-amino-S-hydroxybenzoate incorporation experiments. According to the labeling studies on rifamycin S biosynthesis,62‘ 63 only seven carbon atoms (01 to C-7, Figure 3) of rifamycin S were not derived from propionate and acetate. These seven carbon atoms formed a substituted aromatic chromophore, which appeared to constitute the initiator molecule for polyketide synthases in ansa chain . . 68 . . . . . . biosyntheSIS. This aromatic unit also serves as the biosynthetic precursor in the mitomycin family, as exemplified by porfiromycin in Figure 4. Its structure resembles a meta-substituted aminobenzoate, which is unusual. The previously known natural aminobenzoates are ortho or para substituted, as exemplified by anthranilic acid and p- 13 aminobenzoic acid, respectively. The biosynthetic precursor of this aromatic . . 69 chromophore was studied by several research groups. Based on the results of genetic and specific feeding experiments,70 the biosynthetic precursor of the aromatic chromophore was identified as 3-amino-5-hydroxybenzoate (Figure 4). 3-Amino-5- hydroxybenzoate was demonstrated to restore rifamycin B production in an A. mediterranei mutant deficient in rifamycin B production.69 In the labeling experiments, [1-'3C]-3-amino-5-hydroxybenzoate was shown to specifically label the quinone methide carbon of streptovaricin (Figure 4).703 It was also reported that the ‘3C NMR of the benzyl carbon of ansamitocin was greatly enhanced by feeding [1-‘3C]-3-amino-5- hydroxybenzoate (Figure 4).70b In addition, the incorporation of [1-‘3C]-3-amino-5- hydroxybenzoate into porfiromycin, a mitomycin group antibiotic, was observed (Figure 701: 4). COZH HZOSPO Pi HO]. COzH Pi H04 C02H H20 C02H COZH “E." 1.. o .2. 5:. _. (1 01" a s O” b 0 s O“ c o , OH d 140‘“ , OH Hzoapomo HZOaPO OH OH 5H OH OH DAHP DHQ DHS shikimate E4P Figure 5. The shikimate pathway. (a) DAHP synthase; (b) 3-dehydroquinate synthase; (c) 3-dehydroquinate dehydratase; (d) shikimate dehydrogenase. Abbreviations: PEP, phosphoenolpyruvate; E4P, D- erythrose 4-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate; DHQ, 3-dehydroquinate; DHS 3-dehydroshikimate. With the identification of 3-amino-5-hydroxybenzoate as the biosynthetic precursor to ansamycin and mitomycin aromatic chromophores, attention subsequently l4 turned to delineation of the biosynthesis of 3-amino-5-hydroxybenzoate. The shikimate pathway (Figure 5) origin of 3-amino-5-hydroxybenzoate was established by feeding experiments with labeled precursors (Figure 6).71 Both [1-‘3C]-D-glucose and [6-‘3C]-D- glucose specifically labeled C—1 and C-10 of rifamycin (Figure 6). [1-‘3C]-D-Glycerate labeled C-3 and C-8 of rifamycin (Figure 6). These data were consistent with a shikimate pathway origin of the chromophore. In the shikimate pathway, [1-‘3C]-D-glucose and [6- l3C]-D-glucose label C-3 of phosphoenolpyruvate and C-4 of D-erythrose 4-phosphate, and [1-‘3C]-D-glycerate labels C-1 of phosphoenolpyruvate and C-2 of E4P (Figure 6). COZH COZH 0 b0” OH : HONOH ' NH2 HO NH2 0‘ H06 0” AHBA AHBA glycerate glucose / \ OH 50 H OH H203PO\/'\/§O 002“ [\2 H203POMO o s ’\ o H 0 PO 5 H 0 PO 2 3 OH 2 3 OH E4P PEP v PEP l E4P Me Me Me Me ° COzH 7 2' 002H '6: Ho“' 3 OH Ho“' 5 OH OH OH shikimate shikimate rifamycin 8 Figure 6. Labeling patterns of shikimate, 3-amino-5-hydroxybenzoate, and rifamycin from l3C-labeled glucose and glycerate. Abbreviations: iminoE4P, l-imino- l-deoxy-D-erythrose 4-phosphate; PEP, phosphoenolpyruvate; AHBA, 3-amino-5- hydroxybenzoate. Although a shikimate pathway origin for 3-amino-5-hydroxybenzoate was . . . . . . 7la-c. 72 . 73 verified, no incorporation was observed when labeled shikimate, qurnate, or 3- 15 dehydroquinate 71d were tested as precursors. Based on these results, it was speculated that 3-amino-5-hydroxybenzoate biosynthesis branched off from the shikimate pathway at a step earlier than the formation of 3-dehydroquinate. Experiments with auxotrophic mutants blocked in different steps of aromatic amino acid biosynthesis were subsequently investigated.74 A. mediterranei A8, which is a transketolase mutant, produced no aromatic amino acids and much less rifamycin B relative to the parent strain. Since transketolase was necessary for the production of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP), which is an essential intermediate of the shikimate pathway, Ghisalba and co-workers postulated that the branch point for the formation of 3-amino-5- hydroxybenzoate from shikimate pathway was at or after the formation of DAHP.74a Another mutant, A. mediterranei A10, was deficient in 3-dehydroquinate synthase activity. Rifamycin production was not affected in this mutant. This indicated that biosynthesis of 3-amino-5-hydroxybenzoate must diverge from the shikimate pathway at a step prior to the formation of 3-dehydroquinate.74C An important discovery in 3-amino-5-hydroxybenzoate biosynthesis came from work by Hornemann and coworkers on the biosynthesis of mitomycin C (Figure 7).75 The carbon carrying the amino group in 3-amino-5-hydroxybenzoate could be equivalent to either C-3 or OS of shikimate pathway intermediates (Figure 5). Since the C-3 carbon atoms of 3-dehydroquinate and 3-dehydroshikimate are present as keto groups, a plausible hypothesis was that the nitrogen might be introduced at C—3 of a shikimate pathway intermediate by a transamination reaction.71a However, the analysis of the labeling of mitomycin C derived from feeding D-[4-‘4C]-erythrose and pyruvic acid 16 revealed l“C labeling at C—2 of mitomycin C (Figure 7).75 This result indicated that nitrogen was attached to the C-4 carbon atom of DAHP to afford 4-amino-3,4-dideoxy-D- arabino-heptulosonic acid 7-phosphate (aminoDAHP) before the formation of 5-amino- 5-deoxy—3—dehydroquinate (aminoDHQ, Figure 7). If nitrogen was introduced by transamination at the C-3 of 3-dehydroquinate to form 5-amino-5-deoxy-3- dehydroquinate, D-[4-‘4C]-erythrose should label C-4 carbon of mitomycin C. The results led to a hypothesis that aminoDAHP or a closely related molecule was an early precursor in the biosynthesis of 3-amino-5-hydroxybenzoate. 0“ “203'300 Ho, COOH HO/h . 4 >‘H'IOI, + OTCOOH _____ COZH _____ HO\\' 1 H >WV HNH2 O D-erythrose pyruvic aicd aminoDAHP aminoDHQ AHBA mitomycin C Figure 7. Labeling pattern of mitomycin C from labeled D-erythrose and pyruvic acid. Abbreviations: aminoDAHP, 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7- phosphate; aminoDHQ, 5-amino-5-deoxy-3-dehydroquinate; AHBA, 3-amino-5- hydroxybezoate. A major step forward in the delineation of the pathway came with the proposal by Floss that the nitrogen atom in 3-amino-5-hydroxybenzoate is derived from aminoDAHP.76 Floss synthesized aminoDAHP, 5-amino-5-deoxy-3-dehydroquinate (aminoDHQ), and 5-amino-5-deoxy-3-dehydroshikimate (aminoDHS) and demonstrated 17 that each of these substrates could be converted into 3-amino-5—hydroxybenzoate in cell- free lysate of the rifamycin B producer A. mediterranei.76a He subsequently purified 3- amino-S-hydroxybenzoate synthase and cloned the encoding gene rifK.77 Based on the observation that the genes encoding for a given antibiotic biosynthesis are clustered together, the rifK gene was subsequently used to identify the rif biosynthetic gene cluster required for biosynthesis of 3-amino-5-hydroxybenzoate in A. mediterranei S699. A region of 95-kb of DNA surrounding the rifK gene was isolated and sequenced (Figure 8).78 The genes that are related to 3-amino-5-hydroxybenzoate biosynthesis in this gene cluster are shown in Figure 8. 95 kb g, A —'l )L )P V ' ' V :1 rifA rifB rifC rifD rifE rifF: r ‘\ rifG rifH rifl rifK rifL rifM rifN orf9 orf15 rifJ : l—<:l—l—l:>-l:>- ‘I ' l '71— 35.6 kb Figure 8. Rifamycin biosynthetic gene cluster of A. mediterranei 8699 and proposed enzyme functions. Proposed functions (encoding gene): modular type I polyketide synthase (rifA to rifE); amide synthase (rifF); S-amino-S-deoxy-3-dehydroquinate synthase (rifG); aminoDAHP synthase (rifH); aminoshikimate dehydrogenase (rifl); 3- amino-S-hydroxybenzoate synthase (rifK); oxidoreductase (rifL); phosphatase (rifM); glucokinase (rifN); transaminase (0rf9), transketolase (0rf15); 5-amino-5-deoxy-3- dehydroquinate dehydratase (rifJ). 18 Table 2. Proposed functions of nf biosynthetic gene products.“ en 2:3?313522‘: (3p...iiiqi’Srliil‘g’fiiilmyoz.) Proposedfunction 1 RifG AroB (E. coli, 49/33) aminoDHQ synthase 2 RifH AroG (L. esculentum, 54/34) aminoDAHP synthase 3 R111 AroE (Synechocystis Sp. 56/29) 3333:2323? 4 RifJ AroD (A. pleuropneumoniae, 63/41) $332236 5 RifK AHBAS (S. collinus, 86/70) AHBA synthase 6 RifL Pur10 (S. alboniger, 55/29) oxidoreductase 7 RifM CbbzP (R. eutropha, 55/32) phosphatase 8 RifN Xle (Synechocystis sp. 52/29) kinase 9 Orf9 YokM (B. subtilis, 58/30) transaminase 10 Orf 15 TktA (E. coli, 58/32) transketolase 3 Abbreviations: aminoshikimate, 5-amino-5-deoxyshikimate; aminoDHQ, 5-amino-5- deoxy-3-dehydroquinate; aminoDAHP, 4-amino-3,4-dideoxy-D-arabin0-heptulosonic acid 7-phosphate; AHBA, 3-amino-5-hydroxybenzoate. Located immediately upstream from rifK are three genes showing high sequence homology to shikimate pathway genes (Figure 8).78 They are rifG, rifH, and rifl, which encode homologues to 3-dehydroquinate synthase (entry 1, Table 2), plant-like DAHP synthase (entry 2, Table 2), and shikimate dehydrogenase (entry 3, Table 2), respectively. However, no 3-dehydroquinate dehydratase homology was found in the nearby region. The closest gene with high sequence homology to a type II 3-dehydroquinate dehydratase (entry 4, Table 2) is rifJ, which is located about 30-kb downstream of the 3-amino-5- 1.9 hydroxybenzoate synthase gene (rsz).78 Inactivation of rifJ reduces rifamycin B formation to 10% of wild type levels and rifamycin B synthesis can be fully restored in an A. mediterranei rifJ mutant by 3-amino-S-hydroxybenzoate supplementation.79 Inactivation experiments also showed that rifH-encoded DAHP synthase and rifG- . . . . . . 79 encoded 3-dehydroqu1nate synthase are essential for r1famyc1n B biosyntheSis. However, inactivation of rzfl has no notable effect on r1famyc1n B production. 9 Located immediately downstream from the rsz gene is rifL (Figure 8),78 which was annotated as an oxidoreductase (entry 6, Table 2). It is translationally coupled to rsz and essential for 3-amino-S-hydroxybenzoate biosynthesis.79 In the same . . . . . . . . . . . 78 transcription unit With rsz and rsz are two additional genes, rifM and rsz (Figure 8), which were annotated as phosphatase (entry 7, Table 2) and glucokinase (entry 8, Table 2), respectively. Both rifM and rifN gene products are absolutely necessary for rifamycin B production since inactivation of rifM or rifN yield rifamycin B non-producing mutants and these mutants can be complemented with 3-amino-5-hydroxybenzoate to restore rifamycin B biosynthesis.79 Heterologous coexpression of rifG, rifH, rifK, rifL, rifM, rifN, and rifJ in Streptomyces coelicolor YU105 resulted in production of 3—amino-5- hydroxybenzoate.79 It indicates that the products of these seven genes are necessary for 3-amino-5-hydroxybenzoate production. Based on the results described above and on a report from the laboratory of J iao80 that the amide nitrogen of glutamine is the best source of nitrogen in rifamycin biosynthesis, Floss and co-workers proposed the 3-amino-5-hydroxybenzoate biosynthetic pathway shown in Figure 9.76 Condensation of iminoE4P with 20 phosphoenolpyruvate gives aminoDAHP and is catalyzed by the rifH gene product. AminoDAHP is then cyclized to aminoDHQ, which is catalyzed by rifC-encoded aminoDHQ synthase. A rifJ-encoded aminoDHQ. dehydratase-catalyzed dehydration reaction gives aminoDHS, which is converted into 3-amino-5-hydroxybenzoate by dehydration and enolization. COZH ”203m pi Ho, COZH pi H,O COZH H o 0H2 H2 0 C02H PEP + J» Jag + OH 3 NH." O HdNHz HO NH2 H203PO\/KANH H203PO 5H aminoDAHP aminoDHQ aminoDHS AHBA iminoE4P Figure 9. The proposed aminoshikimate pathway. Enzymes (encoding genes): (a) aminoDAHP synthase, rifH; (b) 5-amino-5-deoxy-3- dehydroquinate synthase, rifG; (c) 5-amino-5-deoxy-3-dehydroquinate dehydratase, rifJ; (d) 3-amino-5-hydroxybenzoate synthase, rsz. Abbreviations: iminoE4P, l-imino-l- deoxy-D-erythrose 4-phosphate; PEP, phosphoenolpyruvate; aminoDAHP, 4-amino-3,4- dideoxy-D—arabino-heptulosonic acid 7-phosphate; aminoDHQ, 5-amino-5-deoxy-3- dehydroquinate; aminoDHS, 5-amino-5-deoxy-3-dehydroshikimate; AHBA, 3-amino—5- hydroxybenzoate. When expressed in E. coli, rifH-encoded DAHP synthase can catalyze the synthesis of DAHP from E4P and phosphoenolpyruvate. However, no aminoDAHP was formed when D-erythrose 4-phosphate, phosphoenolpyruvate, and nitrogen sources were incubated with rifH-encoded DAHP synthase.“ Floss suggested that rifH-encoded DAHP synthase must combine with another protein in order to synthesize aminoDAHP. The heterologously expressed RifG from S. lividans 1326 could utilize both DAHP and aminoDAHP as substrates.82 The rifK gene product has been shown to possess 3-amino- 21 5-hydroxybenzoate synthase activity by enzyme assay.77 The enzymatic activity of RifJ has also been demonstrated:33 The other gene products that are absolutely necessary for 3-amino-5- hydroxybenzoate biosynthesis are rifL-encoded oxidoreductase, rifM-encoded phosphatase, and riflV—encoded kinase activity. The functions of these gene products are based on sequence homology with the genetic sequences encoding known enzyme activities.78 Their actual enzymatic activities have not been experimentally established with enzyme assays. Since they are not required for 3-amino-5-hydroxybenzoate biosynthesis after the formation of aminoDAHP according to the proposed aminoshikimate pathway (Figure 9), the enzymes encoded by these genes were suggested to be associated with the formation of aminoDAHP by Floss.79 However, the role of RifL, RifM, and RifN in aminoDAHP biosynthesis had not been demonstrated prior to the initiation of research discussed in this thesis. 22 CHAPTER TWO IN VITRO ELABORATION OF THE PRECURSORS TO THE AMINOSHIKIMAT E PATHWAY Introduction The aminoshikimate pathway (Figure 9, Chapter 1) was first discovered and studied in rifamycin B producer Amycolatopsis mediterranei. Its end product, 3-amino- 5-hydroxybenzoate, serves as an initiator for polyketide synthases in the biosynthesis of ansamycins.84 Floss and coworkers proposed that the key step of the aminoshikimate pathway was catalyzed by a modified DAHP synthase (Figure 10), which might carry an additional protein subunit or domain that would generate a molecule of ammonia from glutamine in the active site of this enzyme. The ammonia subsequently could form a Schiff’s base with E4P to yield iminoE4P. Condensation with phosphoenolpyruvate would yield 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP, Figure 10). 002H lutamate g H203P0’j\ H04 COZH H203P0\)\/\O H203P0\/K/\ PEP > O NH N ; NH2 H203PO OH glutamine Pi iminoE4P aminoDAHP Figure 10. Biosynthesis of iminoE4P and aminoDAHP. Abbreviations: E4P, D-erythrose 4—phosphate; iminoE4P, l-imino—1-deoxy-D-erythrose 4-phosphate; aminoDAHP, 4-amino-3,4-dideoxy—D-arabino-heptulosonic acid 7- phosphate. Conversions (enzymes): (a) ammonia generation subunit or domain of aminoDAHP synthase; (b) aminoDAHP synthase. 23 Floss and coworkers have demonstrated that a small amount of [l-‘4C]- aminoDAHP could be detected by TLC and autoradiography when [l-“C]- phosphoenolpyruvate and E4P were incubated with A. mediterranei S699 cell-free lysate.76a Although this result might be consistent with biosynthesis of iminiE4P from E4P via a transamination reaction (Figure 9), the possibility remained that [1-‘4C]- aminoDAHP was formed from [l-”C]-phosphoenolpyruvate and a small amount of an endogenous source of iminoE4P (or precursor to iminoE4P) in the undialyzed lysate of A. mediterranei S699. Addition of L-glutamine to undialyzed A. mediterranei cell-free lysate did not have an impact on the amount of [1-‘4C]-aminoDAHP synthesized from [1- '4C]-phosphoenolpyruvate and FARMl This observation indicated that either E4P was not the biosynthetic precursor to iminoE4P or L-glutamine was not the source of nitrogen for the hypothesized transamination. Further experiments showed that 15N enrichments in 3-amino-5—hydroxybenzoate were only 2.7% and 2.4%, respectively, when [amide-LN]- L-glutamine or 'SNH4CI was incubated with phosphoenolpyruvate and E4P in cell-free lysate of A. mediterranei S699.76b Based on these observations, the role of E4P as a biosynthetic precursor to iminoE4P and the role of L-glutamine as the nitrogen source for the aminoshikimate pathway were called into question. To elaborate the biosynthesis of iminoE4P and the source of the nitrogen atom incorporated into the aminoshikimate pathway, the Floss group turned to identification of a transaminase that catalyzed transamination of E4P to form iminoE4P. Following inspection of the rif biosynthetic gene cluster, the orf9 gene was discovered to have a high sequence homology to genes that encode dNTP-hexose aminotransferases.78 Floss therefore suggested that orf9 possessed the necessary enzymatic activity for introducing 24 the nitrogen atom into the aminoshikimate pathway. Unfortunately, inactivation of orf9 showed no impact on rifamycin B production.79 With 0rf9 out of consideration, the search fora dedicated transaminase continued. Previous experiments showed that heterologous coexpression of rifG, rifH, rifK, rifL, rifM, rifN, and rifJ resulted in production of 3-amino-5-hydroxybenzoate in Streptomyces coelicolor YU105.79 Based on this observation, Floss and coworkers proposed that the transaminase activity must reside on one of these seven genes. The rifK gene product, which had been identified as 3-amino-5-hydroxybenzoate synthase, also showed high sequence homology to transaminases in deoxysugar biosynthesis. More importantly, the rifK gene product binds pyridoxamine 5-phosphate as well as pyridoxal 5-phosphate,77 which is a widely used cofactor in transamination reactions. Based on these observations, Floss proposed that the rifK gene product carries two functions: transaminase and 3—amino-5-hydroxybenzoate synthase. The transaminase activity of the rifK gene product was proposed to either catalyze transamination of E4P or to introduce a nitrogen atom into another biosynthetic precursor of the aminoshikimate pathway in conjugation with the rifL, rifM, or rifN gene products. However, no experimental evidence was provided to support this hypothesis and the enzymatic activities of the rifK (transaminase), rifL, rifM, and rifN gene products were not addressed. In this chapter, a different formulation for the biosynthesis of iminoE4P was tested. Putative precursors to iminoE4P were synthesized and incubated in A. mediterranei cell-free lysate followed by analysis for the formation of the aminoshikimate pathway intermediate aminoDAHP and the aminoshikimate pathway product 3-amino-5-hydroxybenzoate. In the process, enzymes involved in the 25 biosynthesis of iminoE4P and aminoDAHP were isolated and characterized. 3-Amino-3- deoxy-D-fructose 6-phosphate, kanosamine 6-phosphate, and kanosamine were identified as precursors to iminoE4P. Because of its role in the biogenesis of iminoE4P, the long— ignored biosynthesis of kanosamine was also investigated. This led to the identification of glutamine as the source of the nitrogen atom incorporated into kanosamine and the aminoshikimate pathway. IminoE4P and AminoDAHP Biosynthesis Overview The focus of our hypothesis is the formulation that an aminosugar is the molecular source from which the aminoshikimate pathway derives its nitrogen atom. The hypothesis is based on a possible aminosugar biosynthetic pathWay that resides in the rif biosynthetic gene cluster of A. mediterranei S699. The rifK-encoded protein is closely related to a class of transaminases in the biosynthesis of some secondary metabolites, which include StsC (S. griseus) in streptomycin biosynthesis,85 Tle (S. fradz'ae) in tylosin biosynthesis,86 and Pur4 (S. alboniger) in puromycin biosynthesis (Figure 11).87 The rifl. gene product is a possible oxidoreductase, which has sequence homology to PurlO in the biosynthesis of puromycin (Figure 11).87 In addition to being immediately 79 adjacent to each other, rifK and rifL also share the same transcription unit.78‘ A plausible scenario would be that RifL oxidizes a sugar’s hydroxyl group to yield a ketone, which can be converted to an amino group by a RifK-catalyzed transamination 26 reaction to yield an aminosugar. This aminosugar then serves as a biosynthetic precursor to iminoE4P, whose condensation with phosphoenolpyruvate would yield aminoDAHP. HO OH HO pH > StsC l Hon-QC —-——> HOI--§:> streptomycin HO .’OH HO ’OH Me Me O Tle 0 HOW -'IOTDP—> Hou- .uo'rop '——’» tylosin O 'OH H2N ’OH H409P3O O R H409P30 O R H203PO O R H203PO O R ———p - s -, Pur10 -, Pur7 k»): Pur4 ,g—l, ' pummyc'" HO 0H 0 OH 0 OH H2N OH NH2 N \ N R: u I ‘) N N Figure 11. RifK and RifL homology. The formulation of an aminosugar, as opposed to E4P, as the biosynthetic precursor to iminoE4P was also based on the chemical properties of E4P and iminoE4P. As an aldose phosphate that cannot intramolecularly cyclize and thereby has a tendency to dimerize, E4P can only exist as a monomer in dilute solutions under a very limited range of conditions.88 Nature apparently deals with the problematic solution chemistry of E4P by balancing the rate of biosynthetic formation and utilization of E4P so that the steady-state concentration of E4P remains low in the cell. It is also possible that nature may take advantage of metabolite channeling to directly transfer E4P from the active site of one enzyme (transketolase) to the active site of the next enzyme (DAHP synthase). 27 E4P may thus not be free in solution in a microbe’s cytoplasm. IminoE4P possesses a similar structure to that of E4P and possibly displays the same problematic solution chemistry as E4P in addition to being prone to hydrolytic loss of its nitrogen atom. Instead of dealing with the troublesome solution chemistry of both E4P and iminoE4P at the same time, nature might possibly utilize a stable aminosugar as the biogenic source of iminoE4P. With this initial assumption, efforts were made to identify this hypothetical aminosugar. H203PO O OH O a CH 0 9H fill/OH + HVOPO3H2 ——> H203PO\}\AR + H()\/u\/’\/Qp03H2 HO“. H OH 6H 6H R = OH F6P R = o E4P R = NH2 aminoF6P GAP R = NH iminoE4P XSP H 0 PO 2 3 ,.. 0 OH O b OH ”203% 0 OH HO "’//OH + HJ|Y\OP03H2 —’ H203PONR + "’I/OH Re‘ OH OH 6H H6 OH R = NH2 aminoS7P GAP R = NH iminoE4P F6P Figure 12. Biosynthesis of E4P and hypothetical biosynthesis of iminoE4P. Enzymes: (a) transketolase; (b) transaldolase. Abbreviations: F6P, D-fructose 6- phosphate; aminoF6P, 3-amino-3-deoxy-D-fructose 6-phosphate; GAP, D-glyceraldehyde 3-phosphate; E4P, D-erythrose 4-phosphate; iminoE4P, l-imino-l-deoxy-D-erythrose 4- phosphate; XSP, D-xylulose S-phosphate; S7P, D-sedoheptulose 7-phosphate; aminoS7P, 4-amino-4-deoxy-sedoheptulose 7-phosphate. Consideration of how E4P is biosynthesized provided clues to formulate how iminoE4P might be biosynthesized (Figure 12). E4P is generated via the pentose phosphate pathway from two different precursors, D-fructose 6-phosphate (F6P), and D- sedoheptulose 7-phosphate (S7P, Figure 12). Transketolase catalyzes the transfer of a glycoaldehyde group from D-fructose 6-phosphate to an aldehyde receptor such as D- 28 glyceraldehyde 3-phosphate (GAP) to form E4P and D-xylulose 5-phosphate (X5P). Transaldolase catalyzes the transfer of a dihydroxyacetone group from D-sedoheptulose 7-phosphate to D-glyceraldehyde 3-phosphate to form E4P and D-fructose 6-phosphate. Similar to the formation of E4P, iminoE4P could potentially be biosynthesized from 3- amino-3-deoxy-D-fructose 6-phosphate (aminoF6P) and/or 4-amino-4-deoxy-D- sedoheptolose 7-phosphate (aminoS7P). Transketolase-catalyzed reaction of 3-amino-3- deoxy-D-fructose 6-phosphate would result in transfer of a glycoaldehyde unit to an aldehyde receptor such as D-glyceraldehyde 3—phosphate and generation of iminoE4P (Figure 12). Alternatively, transaldolase-catalyzed reaction of 4-amino-4-deoxy-D- sedoheptolose 7-phosphate would result in transfer of a dihydroxyacetone unit to D- glyceraldehyde 3-phosphate and generation of iminoE4P (Figure 12). Table 3. Plate selection for characterization of 01115 gene product. lug/glucose M9/glucose M9/glucose entry strain (40 h) 0.5 mM IPTG" 0.5 mM IPTG“ (20h) (40 h) 1 1315020353) + - + 2 BJ502(DE3)/pJG4. 150 + +++ +++++ (T7, 0rf15, AmpR) 3 BJ502(DE3)/pSK4.172 +++++ +++ +++++ (PM, tktA, Cm“) a [PT G, isopropyl B-D-thiogalactopyranoside. In the sequenced rif biosynthetic gene cluster89 of A. mediterranei S699 (Figure 8, Page 18), the 0rf15 gene product has sequence homology to loci that encode transketolase in other organisms (cbbT of Xanthobater flavus90 and tktA in Synechocystis sp.9l). To confirm that the 0rf15 encoded transketolase, the open reading frame of 0rf15 was PCR 29 amplified and cloned into vector pT7-7 to yield (Table 4) plasmid pJG4.150 (T7, 0rf15, Amp“). E. coli BJ502(DE3), which carried a leaky transketolase mutation, only formed tiny colonies after 40 hour of incubation on M9/glucose medium at 37 °C (entry 1, Table 3). After transformation, the growth (entry 2, Table 3) of E. coli BJ502(DE3)/pJG4.150 (T7, 0rf15, Amp“) on M9/glucose/O.5 mM IPT G solid medium was similar to the growth (entry 3, Table 3) of E. coli B1502(DE3)/pSK4.172 (PM. tktA, Cm“) on the same medium. Without IPT G supplementation, the overexpression of 0rf15 was not induced and E. coli BJ502(DE3)/pJG4.150 (T7, 0rf15, Amp“) only formed tiny colonies after 40 h of incubation (entry 2, Table 3). These observations confirmed that the 0rf15 gene product was a transketolase. On the other hand, no transaldolase—encoding gene was found in the rifbiosynthetic gene cluster based on sequence homology. This suggested that 3-amino- 3-deoxy-D-fructose 6-phosphate and not 4-amino-4-dideoxy-D-sedoheptolose 7- phosphate was more likely to be the biosynthetic precursor of iminoE4P. Table 4. Restriction enzyme maps of plasmids pJG4.150 and pSK4.172. Plasmid Plasmid Map“ N B 89 pJG4.150 I . . I ——> ——-> <— T7 orf15 Am," B B pSK4.172 l l ' I —> ——> —> Plac tktA Cm“ “ B = BamHI, Bg = BglII, N = NdeI. Synthesis of 3-amino-3-deoxy-D-fructose 6-phosphate (aminoF6P) To determine whether 3-amino-3-deoxy-D-fructose 6-phosphate was the 30 biosynthetic precursor to iminoE4P, it was synthesized by hexokinase-catalyzed phosphorylation of 3-amino-3-deoxy-D-fructose (aminoF) derived from chemical synthesis from D-fructose (Figure 13). The enzymatic phosphorylation was employed because of its regiospecific phosphorylation of the C-6 hydroxyl group. Chemical phosphorylation was a less attractive synthetic option as this approach would require additional protection and deprotection steps. HO 0H0 _| “QM Q0“— —»” ~Q°o° 0|: ”/0 0" 'II/0 HO OH #0 OH 7‘6“ 0 D-fructose 2 (L R0 0 0+ d O 0 e O OH (DD-<12, 0 On- ”,0 \LZ'WIOH ’I/ 7%) NO OH 7K0 NH2 Ho“ NH2 aminoF 3 4 f CR: R: :03H2 aminOFOP Figure 13. Synthesis of 3-amino-3-deoxy-D-fructose 6-phosphate (aminoF6P). (a) H2804, acetone, 52%; (b) RuCl3, NaIO4, KZCO3, Et3BnNC1, CHC13/I-I20 (1:1), reflux, 99%; (c) HZNOH-HCI, NaOAc, CH3CN/HZO (1:1), 91%; (d) LiAlH4, THF, reflux, 11%; (e) 2 N HCl, 25°C, quant.; (0 ATP, MgClz, hexokinase, citric acid, pH 8, 87%. Starting from D-fructose, hydroxyl groups on C-1, C-2, C-4, and C-5 were selectively protected to give 1 in 52% yield. The remaining C-3 hydroxyl group of compound 1 was subsequently oxidized to afford ketosugar 2 in a yield of 99%. Treatment of ketosugar 2 with HzNOH-HCI and NaOAc in CH3CN/HzO (1:1) gave oxime 3 in 91% yield. Reduction by LiAlH4 afforded a mixture of amines in 70% de favoring the epimer of amine 4. Although efforts were made to improve the stereoselectivity of the oxime reduction, significant improvements were not achieved. 31 Treatment of amine 4 with 2 N HCl afforded 3-amino-3—deoxy-D-fructose (aminoF) in a quantitative yield. Conversion of 3-amino-3-deoxy-D-fructose to 3-amino-3-deoxy-D- fructose 6-phosphate employed hexokinase-catalyzed phosphoryl group transfer from ATP. Since 3-amino-3-deoxy-D-fi'uctose is not a native substrate of hexokinase, citric acid was added in the reaction as an activator.92 Addition of citric acid increased the yield of 3-amino-3-deoxy-D-fructose 6-phosphate from 40% to 87% and decreased the amount of hexokinase required from 10,000 units to 500 units. The final product was purified by anion exchange column (AGl-X8) and characterized by 1H NMR (Figure 37), '3 C NMR (Figure 38), 31P NMR, two-dimensional NMR (Figure 39 and Figure 40), and high resolution electrospray mass spectrometry. Neither 3-amino-3-deoxy-D-fructose nor its phosphate monoester had been previously reported in the literature. Experimental designs for examination of aminoF6P as the biosynthetic precursor to iminoE4P ' Direct isolation of iminoE4P in proposed transketolase-catalyzed reaction of 3- amino-3-deoxy-D-fructose 6-phosphate (aminoF6P) was not attempted due to the predicted instability of iminoE4P. Instead, an in situ generation and trapping strategy was employed. Trapping of iminoE4P generated from transketolase-catalyzed glycoaldehyde transfer from 3-amino-3-deoxy-D-fructose 6-phosphate to D-ribose 5- phosphate (RSP, Figure 14) was attempted using its condensation with phosphoenolpyruvate catalyzed by DAHP (or aminoDAHP) synthase. Isolation of aminoDAHP would implicate formation of iminoE4P. In this experimental design, D- ribose 5-phosphate was used as an aldehyde receptor instead of glyceraldehyde 3- phosphate since it is cheaper and easier to handle. 32 0 0P03H2 R H203PO 0 OH H RSP OH OH HO,_ COZH K(___Z'~,,OH 50H I H203PO\/'\-ANH O #03 e a = : HO NH2 0 9H iminoE4P 0” '3 Pi HOPO 5H NHz HONR . 2 3 aminoF6P S7P 5H Jo aminoDAHP 0P03H2 OH 0” H04, COZH H P 203 O i \O O O 0H d 3'. , OH E4P P' H203PO OH DAHP Figure 14. Examination of 3-amino-3-deoxy-D-fructose 6-phosphate as the biosynthetic precursor to iminoE4P. (a) transketolase; (b) DAHP synthase or aminoDAHP synthase; (0) hydrolysis; (d) DAHP synthase or aminoDAHP synthase. Abbreviations: aminoF6P, 3-amino-3-deoxy-D-fructose 6-phosphate; R5P, D-ribose 5- phosphate; S7P, D-sedoheptulose 7-phosphate; iminoE4P, l-imino-l-deoxy-D-erythrose 4-phosphate; aminoDAHP, 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7- phosphate; E4P, D-erythrose 4-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate. Enzyme purifications and A. mediterranei cell-free lysate preparation To conduct the iminoE4P in situ generation/trapping experiments, E. coli tktA- encoded transketolase and. aroFFB“-encoded DAHP synthase were partially purified. Three DAHP synthase isozymes (RifH, Dahpsl, and DahpsII) from Amycolatopsis mediterranei S699 were partially purified as recombinant 6-His tagged proteins. Cell- free lysate was also prepared from Amycolatopsis mediterranei (ATCC 21789), which contains all the enzymes of the aminoshikimate pathway. E. coli tktA-encoded transketolase was purified from E. coli BL21(DE3)/pSK4.172 (PW, tktA, Cm“)187 following literature procedures.93 E. coli aroFF““-encoded DAHP synthase was purified94 from E. coli BL21(DE3)/pKL4.71 (P 1m" 33 aroF’JB“, lac/Q, Amp“). The purification was performed using a DEAE-cellulose column after protamine sulfate treatment and (NH,,)2SO4 precipitation. Table 5. Restriction enzyme maps of plasmids pSK4.172, pKL4.71, MMB012, EDAHPB, and PEI. plasmid Plasmid Map8 a e pSK4.172 I I J ' —-> ——> ——> Plac tktA CmR N E E N pKL4.71 ‘ ' ‘ J +— —> Ar > [80F P lac aroFFBR AmpR A A MMBO12 ‘ ‘ —> ———> ——> T7, lacO, His, n'fH AmpR A A EDAHPB L ' —>.————> ——> T7, IacO, H155 dahpsl Amp“ A A PEI ' , ' —II> ————> ——n> T7, [300, His; dahpsll AmpR " B = BamHI, N = Nrul, E =Ec0RI, A = AflIII. The A. mediterranei rifH gene product was suggested to be an aminoDAHP synthase.95 It was purified as a 6-His tagged protein from E. coli BL21(DE3)/MMB012 (T7, lacO, Hisé, rifH, Amp“)96 by chromatography using a Ni-NTA Agarose column following the manufacture’s protocol. The dahpsl—encoded DAHP synthase was purified as a 6-His tagged protein from E. coli BL21(DE3)/EDAHPB (T7, lacO, His6, dahpsl, Amp“)97 using a Ni-NTA Agarose column. The dahpsll-encoded DAHP synthase was 34 purified as a 6-His tagged protein from E. coli BL21(DE3)/PEI (T7, lacO, His6, dahpsll, Amp“)98 by Ni-NTA Agarose chromatography. A. mediterranei (ATCC 21789) cell-free lysate preparation followed literature procedure76a with slight modifications. The harvested mycelia was washed with 50 mM Tris-HCl buffer (pH 7.5) and reisolated by centrifugation. The mycelia were then resuspended in Tris-HCl buffer and disrupted by two passages through a French Press. The cell debris was removed by centrifugation and the supernatant was directly used in most of the cell-free reactions. In cases where removing small molecule were required, diafiltrations were carried out after centrifugation. Diafiltration was performed by using Millipore PM-lO membranes and an Aminco stirred cell (300 mL). Dilution to 250 mL followed by concentration to approximately 25 mL was repeated 3 to 6 times. Reaction of aminoF6P in the presence of E. coli transketolase (TktA) and DAHP synthase (AroF“““) With the literature showing that the E. coli shikimate pathway enzymes can bind aminoshikimate pathway substrates76a and our proposal that iminoE4P biosynthesis is similar to E4P biosynthesis, the combined action of E. coli tktA-encoded transketolase and aroFFBR-encoded DAHP synthase was tested. IminoE4P generation from 3-amino-3- deoxy-D-fructose 6-phosphate could be implicated by the formation of aminoDAHP (Figure 14). To conduct the test, 3-amino-3-deoxy-D-fructose 6-phosphate, D-ribose 5- phosphate, and phosphoenolpyruvate were incubated with partially purified E. coli tktA- encoded transketolase and E. coli aroFFm-encoded DAHP synthase (entry 1, Table 6). 35 Table 6. Reaction of aminoF6P in the presence of transketolase, DAHP synthase, and aminoDAHP synthase.a entry reaction condition products (% yield) aminoF6P, R5P, PEP ; l E. coli TktA transketolase (9 units), E. coli DAHP (53) AroFFB“ DAHP synthase (660 units), pH 7.3 aminoF6P, R5P, PEP; E. coli TktA transketolase (9 units), aminoDAHP (2); 2 . . . . b A. medzterranez rzfl-I gene product (64 units ), DAHP (35) pH7.3 aminoF6P, R5P, PEP; aminoDAHP (7); 3 A. mediterranei cell-free lysate (DAHP synthase DAHP (l9); AHBA (3) activity of 0.2 units), pH 7.3 F6P, R5P, PEP, glutamine, (NH4)ZSO4; 4 A. mediterranei cell—free lysate (DAHP synthase activity of 0.2 units), pH 7.3 E4P, PEP, glutamine, (NH4)2SO4; 5 A. mediterranei cell-free lysate (DAHP synthase activity of 0.2 units), pH 7.3 aminoF6P, R5P, PEP, glutamine, (NH4)2SO4; 6 A. mediterranei cell-free lysate (DAHP synthase activity of 0.2 units), pH 7.3 Tyr (5), Phe (5) DAHP (29) aminoDAHP (trace) aminoDAHP (7); DAHP (l8); AHBA (3) Tyr (5), Phe (5) 3 Abbreviations: aminoF6P, 3-amino-3-deoxy-D-fructose 6—phosphate; R5P, D-ribose 5- phosphate; PEP, phosphoenolpyruvate; aminoDAHP, 4-amino-3,4-dideoxy-D-arabino- heptulosonic acid 7-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7- phosphate; AHBA, 3-amino-5-hydroxybenzoate; Tyr, tyrosine; Phe, phenylalanine; F6P, D-fructose 6-phosphate. b RifH-encoded protein was assayed as DAHP synthase activity according to reference 93. After incubation of the reaction mixture at 28°C for 6 h, protein was removed by ultrafiltration. The crude, protein-free reaction mixture was applied to a Dowex 50 (H*) strong cation exchange column. The column was washed with H20 and eluted with a linear gradient of AcOH. The only fraction that tested positive towards the thiobarbiturate colorimetric assay99 (TBA) was in the void. Based on ‘H and 13C NMR 36 analysis, DAHP was the product formed in a yield of 53%. Formation of aminoDAHP was not observed. Since 3-amino-3-deoxy-D-fructose 6-phos‘phate, D-ribose 5-phosphate, and phosphoenolpyruvate were the only carbon sources added in the reaction, DAHP must be derived from these three substrates. A plausible explanation for DAHP formation entails conversion of 3-amino-3-deoxy-D-fructose 6-phosphate into iminoE4P catalyzed by tktA- encoded transketolase. However, TktA- generated iminoE4P was either not a substrate or a very poor substrate of aroFFB“-encoded DAHP synthase and subsequently hydrolyzed to afford E4P. DAHP formation is then the result of DAHP synthase-catalyzed condensation of iminoE4P-derived E4P with phosphoenolpyruvate (Figure 14). The fragmentation of 3-amino-3-deoxy-D-fructose 6-phosphate in the presence of tkIA-encoded transketolase was further examined by a coupled enzyme assay shown in Figure 15. The TktA-catalyzed reaction of 3-amino-3-deoxy-D-fructose 6-phosphate resulted in transfer of a glycoaldehyde unit to E4P and generation of iminoE4P and fructose 6-phosphate. The resulting fructose 6-phosphate was subsequently isomerized to glucose 6-phosphate with catalysis of yeast phosphoglucose isomerase. Oxidation of glucose 6-phosphate catalyzed by glucose 6-phosphate dehydrogenase generated phosphogluconate and NADH, whose formation was monitored by following the increase in absorbance at 340 nm. The specific activity of TktA was 0.02 unit/mg when 3-amino- 3-deoxy-D-fructose 6-phosphate and E4P were used as substrates. The control experiment showed no increase in absorbance at 340 nm in the absence of transketolase when 3-amino-3-deoxy-D-fructose 6-phosphate was incubated with yeast phosphoglucose isomerase, glucose 6-phosphate dehydrogenase, and NAD. The results confirmed that 37 iminoE4P could be synthesized from 3-amino-3-deoxy-D-fructose 6-phosphate using ketol transfer catalyzed by E. coli tktA-encoded transketolase. H203PO 0 OH H203PO 0H 8 0 OH OH ."’/OH + HZOSPOMO—fi ."”OH + HZOSPONNH H0“ NHz 5H Ho“ OH 6H aminoF6P E4P fructose 6-phosphate iminoE4P b H203PO NADPH NADP H203PO O O Hobo = \c/ HobOH HO ’OH HO 6H phosphogluconate glucose 6-phosphate Figure 15. TktA-catalyzed fragmentation of aminoF6P. Enzymes: (a) E. coli tktA-encoded transketolase; (b) yeast phosphoglucose isomerase; (c) glucose 6-phosphate dehydrogenase. Abbreviations: aminoF6P, 3-amino-3-deoxy-D- fructose 6-phosphate; E4P, D-erythrose 4—phosphate; iminoE4P, l-imino-l-deoxy-D- erythrose 4-phosphate. Reaction of aminoF6P in the presence of E. coli transketolase (TktA) and the hypothetical aminoDAHP synthase (RifH) from A. mediterranei With aroFFB“-encoded DAHP synthase apparently incapable of catalyzing the condensation of iminoE4P with phosphoenolpyruvate, The TktA-catalyzed reaction of 3- amino-3-deoxy-D-fructose 6-phosphate was then examined in the presence of the A. mediterranei rifH gene product, which was purified from E. coli BL21(DE3)/MMB012. The rifH gene product had been suggested to be an aminoDAHP synthase based on sequence homology and location in the rif biosynthetic gene cluster. However, its ability to catalyze the formation of aminoDAHP from iminoE4P and phosphoenolpyruvate had not been demonstrated. After incubation of 3-amino-3-deoxy-D-fructose 6-phosphate, D-ribose 5- phosphate, and phosphoenolpyruvate in the presence of tktA-encoded transketolase and 38 rifH-encoded enzyme at 28°C for 6 h, proteins were removed by ultrafiltration. The crude product mixture was fractionated using a Dowex 50 (H*) strong cation exchange column. A fraction that did not bind to Dowex 50 (H*) and tested positive to thiobarbiturate colorimetric assay99 was confirmed by 1H and 13C NMR to contain DAHP. Elution of the Dowex 50 (H*) column with acetic acid solution (1 M) produced another fraction that tested positive to the thiobarbiturate colorimetric assay. This fraction was further purified by HPLC on an AXpak WA-624 weak anion exchange column followed by a sugar KS-801 strong cation exchange column. Fractions containing product that tested positive to the thiobarbiturate colorimetric assay and ninhydrin assay'm were pooled and concentrated. Although the 'H NMR (Figure 45) and [3C NMR (Figure 46) of this compound did not totally agree with the NMR data of aminoDAHP published in the literature,'01 the two-dimentional NMRs (Figure 47, Figure 48) and mass spectrometry confirmed that this fraction of product contained aminoDAHP. To obtain higher yields of aminoDAHP after purification, a modified purification method was subsequently developed (see Chapter 4). By using this modified method, a 2% yield of aminoDAHP was obtained (entry 2, Table 6). Reaction of aminoF6P in A. mediterranei cell-free lysate The successful generation of aminoDAHP supported our hypothesis that iminoE4P was generated from 3-amino-3-deoxy-D-fructose 6-phosphate. A new question was also raised. What would happen if Orf 15 was used instead of TktA? To answer this question, A. mediterranei (ATCC 21789) cell-free lysate, which should contain both native rifH-encoded enzyme and native 0rf15-encoded enzyme, was employed to conduct 39 the biosynthesis of aminoDAHP from 3-amino-3-deoxy-D-fructose 6-phosphate. Incubation of 3-amino-3-deoxy-D-fructose 6-phosphate, D-ribose 5-phosphate, and phosphoenolpyruvate in crude A. mediterranei cell—free lysate yielded aminoDAHP in 7% yield. DAHP, 3-amino-5-hydroxybenzoate, tyrosine, and phenylalanine were also formed, respectively, in 19%, 3%, 5%, and 5% yield (entry 3, Table 6). Formation of 3- amino—S-hydroxybenzoate reflected the fact that all of the aminoshikimate pathway enzymes were present in the wild type A. mediterranei cell-free lysate. DAHP formation indicated that iminoE4P hydrolysis with resulting formation of E4P was occurring in the cell-free lysate. To further confirm that 3-amino-3-deoxy-D-fructose 6-phosphate was the nitrogen source of iminoE4P, several control experiments were conducted. In the first control experiment, D-fructose 6-phosphate, D-ribose 5-phosphate, phosphoenolpyruvate, and glutamine/(NH4)2SO4 as possible sources of nitrogen were incubated in crude A. mediterranei cell-free lysate (entry 4, Table 6). No aminoDAHP formation was detected. In a second control experiment (entry 5, Table 6), E4P and phosphoenolpyruvate were incubated with glutamine and (NH4)2SO4 in crude A. mediterranei cell-free lysate. A trace amount of aminoDAHP could be detected by the thiobarbiturate colorimetric assay when the reaction products were fractioned using Dowex 50 (H*). However, the amount of aminoDAHP formed in the reaction was too small to be detected by 500 MHz 1H NMR. In the last control experiment, addition of glutamine and (NH4)ZSO4 to the reaction of 3-amino-3-deoxy-D-fructose 6-phosphate, D-ribose 5-phosphate, and phosphoenolpyruvate in A. mediterranei cell-free lysate (entry 6, Table 6) did not give a higher yield of aminoDAHP than that formed in the same reaction solution lacking 40 glutamine and (NH4)ZSO4. These observations supported the hypothesis that iminoE4P was derived from 3-amino-3—deoxy-D-fructose 6-phosphate. Derivation of iminoE4P from 3-amino-3-deoxy-D-fructose 6-phosphate requires that all of the carbon and nitrogen atoms of iminoE4P should be derived from 3-amino-3- deoxy-D-fructose 6-phosphate. Giver that Floss and coworkers proposed that iminoE4P was produced from E4P by a transamination reaction, the possibility remained that 3- amino-3-deoxy-D-fructose 6-phosphate might be just the source of the nitrogen atom but not the source of the carbon atoms of iminoE4P. To address this issue, 3-['5N]-amino-3- deoxy-6,6-[2H2]-D-fructose 6-phosphate was synthesized from 6,6-[2H2]-D-fructose and 15NHQOH-HCI via the same synthetic route specified in Figure 13. Considering the amount of this precious compound, 3-['SN]-amino-3-deoxy-6,6-[2H2]-D-fructose 6- phosphate was diluted with unlabeled 3-amino-3-deoxy-D-fructose 6-phosphate to afford material that gave M+3, M+2, and M+l ions with relative intensities of 10.97%, 0.24%, and —0.6%, respectively. Incubation of 3-['SN]-amino-3-deoxy-6,6-[2H2]-D-fructose 6- phosphate, phosphoenolpyruvate, and D-ribose 5-phosphate in A. mediterranei cell-free lysate led to the formation of aminoDAHP that gave M+3, M+2, and M+1 ions with relative intensities of 10.24%, 0.5%, and 0.42%. Based on the retention of both '5N and 2H labels, 3-amino-3-deoxy-D-fructose 6-phosphate was confirmed to be the biosynthetic precursor to all of the carbon and nitrogen atoms of iminoE4P. RifH and other possible aminoDAHP synthases in A. mediterranei A higher yield of aminoDAHP was obtained when A. mediterranei cell-free lysate was used instead of heterologously expressed RifH. The presence in A. mediterranei of 41 aminoDAHP synthase isozymes in addition to RifH might explain this observation. Two additional DAHP synthases encoded by loci dahpsI and dahpsll have been identified in A. mediterranei.'02 To examine whether dahpsI-encoded DAHP synthase and dahpsll- encoded DAHP synthase from A. mediterranei possess aminoDAHP synthase activity, these two enzymes were purified from E. coli BL21(DE3)/EDAHPB (T7, lacO, Hisé, dahpsl, Amp“) and BL21(DE3)/PEI (T7, lacO, His6, dahpsll, Amp“), respectively, and tested by in vitro enzyme reactions. Table 7. Reaction of aminoF6P in the presence of different DAHP synthases.a entry reaction condition products (% yield) aminoF6P, R5P, PEP; _ . 1 E. coli TktA transketolase (9 units), 32111313385?) (2)’ A. mediterranei RifH (64 units“), pH7.3 aminoF6P, R5P, PEP ; 2 E. coli TktA transketolase (9 units), DAHP (41) A. mediterranei Dahpsl (64 units“), pH7.3 aminoF6P, R5P, PEP; 3 E. coli TktA transketolase (9 units), DAHP (39) A. mediterranei DahpsII (64 units“), pH7.3 3 Abbreviations: aminoF6P, 3-amino-3—deoxy-D-fructose 6-phosphate; R5P, D-ribose 5- phosphate; PEP, phosphoenolpyruvate; aminoDAHP, 4-amino-3,4-dideoxy-D-arabin0- heptulosonic acid 7-phosphate; DAHP, 3-deoxy-D-arabin0-heptulosonic acid 7- phosphate; AHBA, 3-amino-5-hydroxybenzoate; F6P, D-fructose 6-phosphate. b RifH, Dahpsl, and DahpsII were assayed as DAHP synthase activity according to reference 93. Incubation of 3-amino-3-deoxy-D-fructose 6-phosphate, D-ribose 5-phosphate, and phosphoenolpyruvate in the presence of tktA-encoded transketolase and dahpsl- encoded DAHP synthase (entry 2, Table 7) or dahpsll-encoded DAHP synthase (entry 3, Table 7) did not lead to aminoDAHP formation. Instead, DAHP was obtained as the sole 42 product in 41% and 39% yields, respectively, when dahpsI-encoded DAHP synthase (entry 2, Table 7) and dahpsll-encoded DAHP synthase (entry 3, Table 7) were used. These results suggested that dahpsI-encoded DAHP synthase and dahpsll-encoded DAHP synthase did not possess aminoDAHP synthase activity. 3-Amino-3-deoxy-D-fructose 6-phosphate Biosynthesis Overview With identification of 3-amino-3-deoxy-D-fructose 6-phosphate as a biosynthetic precursor to iminoE4P, attention turned to delineation of the source of this amino carbohydrate. Aminosugars can be found in a wide variety of natural products such as 03 glycolipids, glycoproteins, and secondary metabolites.1 Naturally occurring 3- aminosugars (Figure 16) include 3-amino-3-deoxy-D-glucose (kanosamine),104 3-amino- 3,6-dideoxyhexose (mycaminose)!OS and desosamine.106 These aminosugars have been reported to be biosynthesized from their corresponding ketosugars via pyridoxal 5’- phosphate (PLP)-dependent transamination reactions. Similarly, 3-amino-3-deoxy-D- fructose 6-phosphate could be synthesized either from D-fructose 6-phosphate via 3-keto- D-fructose 6-phosphate or from D-fructose via 3-keto-D-fructose followed by phosphorylation (Figure 17). HO H3C H3C o o 0 HOW OH HO» OH OH H2N ’OH (CH3)2N ’OH (CH3)2N ’OH kanosamine mycaminose desosamine Figure 16. Naturally occurring 3-aminosugars. 43 HO 0 OH Ho 0 OH H203PO 0 OH H203PO 0 0H \LZCWH a five“ \szon c \LZzon Ho“ OH Ho“ 0 HO“ 0 Ho“ OH fructose 3-keto-fmctose 3-keto-F6P Fep \ y H203PO 0 OH "II/OH Ho“ NH2 7 aminoF6P \ O H203PO H203Po\)K,NH2 0 HObOH AHAP + OH " H203PO\/'\n,H o HZN OH K6P GAP Figure 17. Hypothetical aminoF6P biosyntheses. Enzymes: (a) oxidoreductase; (b) i) transaminase; ii) kinase; (c) oxidoreductase; (d) transaminase; (e) isomerase; (f) i) aldolase; ii) phosphatase. Abbreviations: F6P, fructose 6-phosphate; 3-ketoF6P, 3-keto-D-fructose 6-phosphate; aminoF6P, 3-amino-3-deoxy-D- fructose 6-phosphate; GAP, glyceraldehyde 3-phosphate; AHAP, 3-amino-l- hydroxyacetone 1~phosphate. Other possible biosynthetic precursors to 3-amino-3-deoxy-D-fructose 6- phosphate include 3-amino-1-hydroxyacetone or kanosamine 6-phosphate (Figure 17). Aldolase-catalyzed condensation of 3-amino-1-hydroxyacetone with D-glyceraldehyde 3- phosphate would afford 3-amino-3-deoxy-D-fructose 1, 6-bisphosphate, which could be subsequently converted to 3-amino—3-deoxy-D-fructose 6-phosphate by enzymatic hydrolysis of the phosphate group on O] (Figure 17). 3-Amino-3-deoxy-D-fructose 6- phosphate could also derive from kanosamine 6-phosphate by an isomerization reaction as shown in Figure 17. 3-Amino-1-hydroxyacetone was not known as a natural product. On the other hand, kanosamine 6-phosphate could be biosynthesized by phosphorylation 104, 107 of kanosamine, which is a natural product as well as an intermediate en route to 44 kanamycin biosynthesis.108 Based on the foregoing analysis, we proposed that A. mediterranei might utilize kanosamine 6-phosphate as a biosynthetic intermediate to incorporate the nitrogen atom into the aminoshikimate pathway. Kanosamine 6- phosphate was therefore synthesized and tested as a precursor to 3-amino-3-deoxy-D- fructose 6-phosphate and an intermediate of the aminoshikimate pathway. Synthesis of kanosamine 6-phosphate HO O O O O a O O O Hobw —>>‘o153-‘3< $740131"; —°—~ $019"; HO 6H HO “’0 O “’0 HO“. >0 6 D-glucose 5 7 H2N 9 g (R = H kanosamine R = P03H2 kanosamine 6-phosphate O 0 R0 L 74%"0 —e—> 7kdkgbg< —'—> HobOH N3 ’0)< H2” ’0 A >014 8 Figure 18. Synthesis of kanosamine 6-phosphate. (a) acetone, ZnClz, H3PO4, 68%; (b) PDC, (CH3CO)20, CHzClz, reflux, 91%; (c) NaBH4, EtOH/Hzo (9:1), 0 °C, 90%; (d) (i) (CF3SOZ)20, pyridine, CHZCIZ, -20 °C, quantitative, (ii) NaN3, DMF, 50 °C, 92%; (e) LiAlH4, Hp, 94%; (f) 2 N HCl, 25 °C, quantitative; (g) ATP, MgClz, hexokinase, citric acid, pH 8, 90%. Kanosamine 6-phosphate was synthesized by hexokinase-catalyzed phosphorylation of kanosamine chemically synthesized from D-glucosem'184 (Figure 18). Starting from D-glucose, hydroxyl groups on 01, C-2, C-5, and C-6 were selectively protected to give 5 in 68% yield. The O3 hydroxyl group of 5 was subsequently oxidized by refluxing with pyridinium dichromate (PDC) and (CH3CO)ZO in CHZCI2 to 45 afford 1,2:5,6-di-0-isopropylidene-3-keto-a-D-glucofuranose 6 in 91% yield. Subsequent diastereoselective (> 99% de) reduction of 6 provided 1,225,6-di-0- isopropylidene-a-D-allofuranose 7 for an overall net inversion of configuration at G3 in 90% yield. Activation of the C-3 hydroxyl group as a triflate ester followed by nucleophilic displacement with NaN3 introduced the requisite nitrogen atom at C-3. Reduction with LiAll-l4 afforded 1,2:5,6-di-0-isopropylidene-kanosamine 9 in a yield of 94%. Treatment of amine 9 with 2 N HCl gave kanosamine in a quantitative yield. Hexokinase-catalyzed phosphorylation of kanosamine employed citric acid as an activator and afforded kanosamine 6—phosphate in 90% yield (Figure 41 to Figure 44). The overall yield of kanosamine 6-phosphate was 43% from starting D-glucose. Experimental designs for examination of kanosamine 6-phosphate as a biosynthetic precursor to 3-amino-3-deoxy-D-fructose 6-phosphate Direct investigation of 3-amino-3-deoxy-D-fructose 6-phosphate formation from kanosamine 6-phosphate in A. mediterranei cell-free lysate or in the presence of yeast phosphoglucose isomerase was first attempted. However, formation of 3-amino-3- deoxy-D-fructose 6—phosphate and consumption of kanosamine 6-phosphate could not be detected by lH NMR after 6 h of incubation. One explanation for the absence of 3- amino-3-deoxy-D-fructose 6-phosphate is an equilibrium favoring kanosamine 6- phosphate for the enzyme-catalyzed isomerization reaction (Figure 19). A driving force was therefore needed to push the equilibrium towards the formation of 3-amino-3-deoxy- D-fructose 6-phosphate. Since 3-amino-3-deoxy-D-fructose 6-phosphate had been demonstrated to be a biosynthetic precursor to aminoDAHP, the isomerization reaction might be driven forward by converting small, steady-state concentration of 3-amino-3- 46 deoxy-D-fructose 6-phosphate into aminoDAHP. Formation of aminoDAHP would not only indicate that kanosamine 6-phosphate could be isomerized to 3-amino-3-deoxy-D- fructose 6-phosphate, but would also implicate that kanosamine 6-phosphate as a biosynthetic precursor to iminoE4P and aminoDAHP (Figure 19). 0 0P03H2 R H203PO OH HJKS’H asp Arm Ho, cozn OH O c‘ a .- HO NH2 0 OH iminoE4P OH b NH2 Pi HZOSPO 6H ' F6P HO R amino 3. ' S7P OH . aminoDAHP hydrolysns 1L isomerization 0P03H2 OH 0“ H04, 002H H203PO H203P0\/KAO 0 o O 3 LT HObOH 0“ c P, i OH ; ' H203P0 OH H2N 0“ E4P DAHP kanosamine 6-phosphate Figure 19. Examination of kanosamine 6-phosphate as a biosynthetic precursor to aminoF6P. (a) transketolase; (b) aminoDAHP synthase; (c) DAHP synthase. Abbreviations: aminoF6P, 3-amino-3-deoxy-D-fructose 6-phosphate; R5P, D—ribose 5- phosphate; S7P, D-sedoheptulose 7-phosphate; iminoE4P, l-imino-l-deoxy-D-erythrose 4-phosphate; aminoDAHP, 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7- phosphate; E4P, D-erythrose 4-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate. Reaction of kanosamine 6-phosphate in the presence of yeast phosphoglucose isomerase, E. coli transketolase (TktA), and DAHP synthase (AroFm‘) The role of kanosamine 6-phosphate as a precursor to iminoE4P, as implicated by formation of aminoDAHP was first examined by employing an assembled in vitro enzymatic system. Kanosamine 6-phosphate, D-ribose 5-phosphate, and phosphoenolpyruvate were incubated with yeast phosphoglucose isomerase, Escherichia coli tktA-encoded transketolase, and E. coli aroFFBR-encoded DAHP synthase (entry 1, 47 Table 8). After the completion of the reaction, proteins were removed by ultrafiltration. The protein-free reaction mixture was then applied to AG-l X8 anion exchange resin (acetate form). The column was washed with H20. and eluted with a linear gradient of AcOH. Fractions that tested positive towards thiobarbiturate colorimetric assay and ninhydrin assay were collected. Based on 1H and l3C NMR analysis, aminoDAHP was not formed. Nonetheless, formation of DAHP did indicate that E4P was generated. 3- Amino-3-deoxy-D-fructose 6-phosphate must therefore have been an intermediate as hydrolysis of transketolase-generated iminoE4P was the only source of E4P. This, in turn, demonstrated that kanosamine 6-phosphate could be enzymatically isomerized to 3- amino-3-deoxy-D-fructose 6-phosphate. Reaction of kanosamine 6-phosphate in the presence of yeast phosphoglucose isomerase, E. coli transketolase (TktA), and A. meditenanei aminoDAHP synthase (RifH) Incubation of kanosamine 6-phosphate, D-ribose S-phosphate, and phosphoenolpyruvate in the presence of yeast phosphoglucose isomerase, Escherichia coli tktA-encoded transketolase, and rifH-encoded aminoDAHP synthase was examined (entry 2, Table 8). After completion of the reaction, proteins were removed by ultrafiltration. The products of the reaction were purified by an open AG-l X8 anion exchange column, an HPLC AXpak WA-624 weak anion exchange column, and an HPLC sugar KS-801 strong cation exchange column. Thiobarbiturate colorimetric assay and ninhydrin assay were used to identify fractions containing DAHP or aminoDAHP. Formation of aminoDAHP was observed in a 2% yield along with formation of DAHP in a 30% yield (entry 2, Table 8). 48 Table 8. Reaction of kanosamine 6-phosphate (K6P) in the presence of yeast phosphoglucose isomerase, transketolase, DAHP synthase, and aminoDAHP synthase. entry reaction condition A products (% yield) K6P, R5P, PEP; yeast phosphoglucose isomerase (60 units), E. coli TktA transketolase (9 units), E. coli AroFFBR DAHP synthase (660 units), pH 7.3 K6P, R5P, PEP; yeast phosphoglucose isomerase (60 units), E. coli aminoDAHP (2); DAHP (39) 2 TktA transketolase (9 units), A. mediterranei RifH DAHP (30) aminoDAHP synthase (64 unitsb), pH7.3 K6P, R5P, PEP; aminoDAHP (6); 3 A. mediterranei cell-free lysate (DAHP synthase DAHP (20); AHBA (2) activity of 0.2 units), pH 7.3 Tyr (3), Phe (2) G6P, R5P, PEP, glutamine, (NH4)ZSO4; 4 A. mediterranei cell-free lysate (DAHP synthase DAHP (21) activity of 0.2 units), pH 7.3 Abbreviations: K6P, kanosamine 6-phosphate; R5P, D-ribose S-phosphate; PEP, phosphoenolpyruvate; aminoDAHP, 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate; DAHP, 3-deoxy-D-arabin0-heptulosonic acid 7-phosphate; AHBA, 3- amino-S-hydroxybenzoate; Tyr, tyrosine, Phe, phenylalanine; G6P, D-glucose 6- phosphate. b AminoDAHP synthase was assayed as DAHP synthase activity according to reference 93. Reaction of kanosamine 6-phosphate in A. mediterranei cell-free lysate It had been demonstrated previously that incubation of 3-amino-3-deoxy-D- fructose 6-phosphate with cell-free lysate of A. mediterranei (ATCC 21789) gave higher yields of aminoDAHP.“) Similarly, a higher yield of aminoDAHP was obtained when kanosamine 6-phosphate, D-ribose S-phosphate, and phosphoenolpyruvate were incubated in A. mediterranei cell-free lysate (entry 3, Table 8). The yield of aminoDAHP 49 from kanosamine 6-phosphate was 6 % and was virtually the same as that employing 3- amino-3-deoxy-D-fructose 6-phosphate as starting material. The formation of DAHP was observed once again (entry 3, Table 8) and its formation would result from the hydrolysis of iminoE4P following the previously discussed mechanism (Figure 19). No aminoDAHP formation was observed when D-glucose 6-phosphate, D-ribose 5- phosphate, and phosphoenolpyruvate were incubated in A. mediterranei cell-free lysate with glutamine and (NH4)2SO4 as possible sources of nitrogen (entry 4, Table 8). Com lexation of Ri -enc ded Amin DAHP S nthase with 0rf15-encoded Transketolase Overview In the previously described cell-free reactions, incubation of 3-amino-3-deoxy-D- fructose 6-phosphate or kanosamine 6-phosphate with D-ribose 5-phosphate and phosphoenolpyruvate in A. mediterranei cell-free lysate led to formation of aminoDAHP and DAHP. It would seem odd for nature to go through the trouble of biosynthesizing iminoE4P only to have it partition between enzyme-catalyzed condensation with phosphoenolpyruvate and hydrolysis to E4P. One possible explanation is that hydrolysis of iminoE4P is prevented by channeling of this intermediate in intact A. mediterranei. Metabolite channeling is a process by which a product from one enzymatic reaction is a substrate for a second enzymatic reaction without diffusion into the surrounding solution. In vivo, complexation between 0rf15-encoded transketolase and rifH-encoded aminoDAHP synthase might minimize hydrolysis of iminoE4P after its generation by Orf15—catalyzed ketol transfer from 3-amino-3-deoxy-D-fructose 6-phosphate and prior to its RifH-catalyzed condensation with phosphoenolpyruvate. 50 Channelingl ‘0 provides a solution to a number of different challenges encountered in cellular metabolism. For example, a product of one enzyme could be a substrate for a second, diffusion-limited enzyme. Channeling could reduce the transit time for the movement of the first enzyme’s product to the second enzyme’s active site. Alternatively, a metabolic intermediate might also be labile. Channeling could prevent a labile intermediate from coming into contact with the external reactive environment thereby preserving the chemical integrity of the intermediate. A metabolic intermediate may also be a substrate for more than one enzyme. With channeling, a metabolic intermediate can be delivered selectively between complexing enzymes and thus segregate the intermediate from competing enzymatic transformations. In the case of iminoE4P, channeling may protect this intermediate from hydrolysis. Support for this possibility followed from the higher yields of aminoDAHP synthesized in A. mediterranei cell-free lysate, which contained both RifH and Orf15 relative to that of the assembled system where TktA was used instead of Orf15. Another observation possibly supportive of channeling followed from the reduced hydrolysis of iminoE4P when glycerol was added to A. mediterranei cell-free lysates. To explore the possible channeling of iminoE4P between 0rf15-encoded transketolase and rifH-encoded aminoDAHP synthase, this section describes the results of an investigation employing a bacterial two-hybrid system. Glycerol effects on aminoDAHP synthesis from aminoF6P AminoDAHP as well as DAHP formation was observed in the cell-free lysate of A. mediterranei. The formation of DAHP apparently resulted from the hydrolysis of 51 iminoE4P to E4P during the bioconversion. This suggested that the rate of iminoE4P utilization was significantly lower than that of its hydrolysis. An effectively higher aminoDAHP synthase activity was needed in order to trap more iminoE4P and improve the yields of aminoDAHP synthesized from 3-amino-3-deoxy-D-fructose 6-phosphate or kanosamine 6-phosphate in the cell-free lysate of A. mediterranei. Table 9. The effect of glycerol on the biosynthesis of aminoDAHP from aminoF6P in A. mediterranei cell-free lysates.‘I entry glycerol aminoF6P aminoDAHP DAHP aminoDAHP synthase (%) remaining (%)b (%)b (%)b activityd (units) 1 0 0 5 15 0.2 2 10 68 6 8 0.2 3 20 64 13 tracec 0.3 4 30 69 15 trace° 0.3 3 Reaction conditions: aminoF6P, ribose 5-phosphate, phosphoenolpyruvate; A. mediterranei cell-free lysates, pH 7.3; b Yields are relative to the amount of aminoF 6P consumed in the reaction; ° non-quantifiable concentration by IH NMR; d AminoDAHP synthase was assayed as DAHP synthase activity according to ref 93. Polyols, and glycerol in particular, can help suppress conformational flexibility and stabilize proteins against denaturation.m To examine the effects of glycerol on aminoDAHP biosynthesis from 3-amino-3-deoxy-D-fructose 6-phosphate, a series of reactions were run with different amounts of glycerol added to A. mediterranei cell lysate buffer (Table 6). Without glycerol addition, all 3-amino—3-deoxy-D-fructose 6—phosphate was consumed in the reaction. DAHP and aminoDAHP were formed in yields of 15% and 5%, respectively (entry 1, Table 9). More than 60% of the initially added 3-amino-3- deoxy-D-fructose 6-phosphate remained in the reaction mixture and the ratio of aminoDAHP to the DAHP formation changed when glycerol was included in A. 52 mediterranei cell lysate buffer. In the presence of 10% glycerol, more aminoDAHP (6%) and less DAHP (8%) were formed in the reaction (entry 2, Table 9). When glycerol concentration was increased to 20% and 30%, only trace amounts of DAHP was detected (entry 3, 4, Table 9). The yield of DAHP decreased from 8% to a trace amount, and the yield of aminoDAHP increased from 6% to 15% (entry 2-4, Table 9). Examination of the interaction between rifH-encoded aminoDAHP synthase and orfl5-encoded transketolase using a bacterial two-hybrid system The two-hybrid system is a molecular genetic tool to study protein-protein interactions. The BacterioMatch Two-Hybrid system (Stratagene) is based on transcriptional activation.1 '2 A protein of interest, referred as the bait, is fused to the full- length bacteriophage A repressor protein (AC1), which can bind specifically to the A. operator sequence with its amino—terminal DNA-binding domain (Figure 20). The corresponding target protein is fused to the a-subunit of RNA polymerase (RNAP-a, Figure 20). The reporter strain contains a reporter gene cassette that is incorporated on an F’ episome (Figure 20). The activatable promoter in the reporter gene cassette is a modified lac promoter that contains a single A operator centered at position -62, replacing the CAP-binding site originally associated with the lac promoter. When the reporter strain is co-transformed with bait and target plasmids, the bait protein can be tethered to the A. operator sequence upstream of the reporter promoter through the DNA- binding domain of AC1 (Figure 20). If the bait and target protein interact, they would recruit and stabilize the binding of RNA polymerase at the promoter and activate the transcription of the reporter gene, the AmpR gene (Figure 20), which enable the reporter strain to grow in the presence of carbenicilline (or ampicillin). A second reporter gene, 53 B-galactosidase, is expressed from the same activatable promoter, providing an additional mechanism to validate the bait and target interaction (Figure 20). While the yeast two-hybrid system has been widely and successfully exploited,l '3 the E. coli two-hybrid system has several advantages: E. coli grows much faster than yeast, it is transformed with higher efficiency, and isolating plasmid DNA from E. coli is easier than that of yeast. There are also some disadvantages associated with use of a bacterial two-hybrid system. Some heterologous proteins could be toxic to an E. coli host. E. coli is also unable to catalyze a number of posttranslational modifications mediated by S. cerevisiae. ColR1 ori HNAP-a recombinant pBT plasmid recombinant target pTRG plasmid p15A ori fl/ M... I Pucz PF“ 1. operator amp' m m- PInez .E- nNAp ....,.... l E——— Figure 20. Examination of protein-protein interactions using a bacterial two-hybrid system. 54 Table 10. Restriction enzyme maps of plasmids using in the bacteria two-hybrid experiments. Plasmid Plasmid Map” 8 B pJG9.150 ‘ ' ‘ ’ —> —> —_>. <-—— PIac-Uvs AC1 01115 CmR B B pJG9.154 ‘ ' ' ‘ —> ; 4 <——'—— Plpp/lac-UVS RNAP-a rifH Ten 8 B 9.169.213 L ‘ ‘ ' —> —> _—.> <—— PIac-UVS AC1 tktA CmFl B B pBT-LGF2 ‘ ' ‘ ' —-—--> ——> ———-—-> +— PIac-UV5 AC1 LGFZ CrnR B B pTRG-Galll ‘ ' 4 ’ ——-> > > ‘——— PIppflac-UVS HNAP-a Gall! TCR a B = BamHI. To detect the possible interaction between 0rf15-encoded transketolase and rifH- encoded aminoDAHP synthase, 0rf15 and rifH genes were inserted into plasmid pBT and pTRG, respectively, to give plasmid pJG9.150 (P,m.,UV5, AC1, 0rf15, Cm“) and plasmid pJG9.154 (PWNWUW, RNAP-a, rrfli, Tc“). Co-transformation of the bait plasmid pJG9.150 (P,a,._UV5, AC1, 0rf15, Cm“) and target plasmid pJG9.154 (P,pp,,m.,uv5, RNAP-a, rifl-I, Tc“) into the BacterioMatch Two-hybrid reporter strain was followed by selection for positive interactions using medium containing 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin. The selection plates were incubated at 30°C for 24 h. The use of carbenicillin as the selective antibiotic instead of ampicillin was because the reporter strain possessed a background level of ampicillin resistance due to basal transcription of the reporter cassette. The use of high concentration of carbenicillin can minimize background growth of the reporter strain. 55 Table 11. Study of protein-protein interactions by bacterial two-hybrid system. LB/CaTchKan B-galactosidase en strainsa . . . . try selectionb act1v1ty (unit/mg) RS/pBT-LGFZ/pTRG-Galll 1 (P,,,_UV,, AC1, LGF2, Cm“)/(P,,,p,,m._uv5, . +++ 0.19 RNAP-a, GalII, Tc“) RS/pBT/pTRG 2 (P,,,_U,,,, AC1, CmR)/(P,,,,,,,,,._W,, RNAP-a, 0.015 Tc“) RS/pJG9.150/pJG9.154 3 (P,,,,U,,,, AC1, 01f15,lac1",Cm“)/(P,pp,,ac, +++ 0.23 W5, RNAP-a, rifH, Tc“) RS/pJG9.150/pTRG 4 (PlaC-UV5’ AC1, 0,1159 laClq9 cmR)/(Plpp/lac- W5, RNAP-a, Tc“) RS/pBT/pJG9.154 5 (PlaC-UVS’ AC1, cmR)/(Plpp/lac-UV5’ RNAP'G, riflI, Tc“) RS/pJG9.213/pJG9.154 6 (PlaC-UVS’ AC1, tktA! cmR)/(PlppllaC-UV5’ (+) 0018 RNAP-a, nfl-I, Tc“) RS/pJG9.2l3/pTRG 7 (PlaC-UVS' AC1, tktAa cmR)/(Plpp/IaC-UV5’ RNAP-a, Tc“) a RS is the reporter strain for the BacterioMatch Two-Hybrid system. b Ca, carbenicillin; Tc, tetracycline; Cm, chloramphenicol; Kan, kanamycin. Several control experiments were carried out simultaneously (Table 11). The dimerization domain (LGF2) of the yeast transcriptional activator Ga14 and GalllP protein had been shown to interact in E. coli cells. These polypeptides are expressed by the pBT-LGF2 and the pTRG-Gall 1P control plasmids respectively and are used as positive control. When plasmid pBT-LGF2 (1’10.-szle I , LGF2, Cm“) and pTRG-GalII (P,,,,,,,ac_UV5RNAP-a, Galll Tc“), were co-transformed (entry 1, Table 11) into the BacterioMatch two-hybrid system reporter strain competent cells, a positive interaction 56 would be indicated by the growth of colonies on LB agar plates supplemented with 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin. As negative controls, co-transformation of the BacterioMatch two-hybrid system reporter strain using recombinant bait plasmid pJG9.150 and non—recombinant target plasmid pTRG (entry 4, Table 11), or non—recombinant bait plasmid pBT and recombinant target plasmid pJG9.154 (entry 5, Table 11), or non—recombinant bait plasmid pBT and non—recombinant target plasmid pTRG (entry 2, Table 11), respectively, should not produce colonies on LB agar plates supplemented with 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin. HO OZN HO N02 0 B-galactosidase 0 __ HO O —= HO OH + O HO ’OH HO "OH 2-nitrophenyl-B-D-galactopyranoside B-D-galactopyranose 2-nitrophenol Figure 21. B-Galactosidase activity assay. Colonies appeared after 24 h of incubation when BacterioMatch two-hybrid system reporter strain was either cotransformed with pBT-LGF2 (Plac-UVS’ AC1, LGF2, Cm“) and pTRG-GalII (P,pp,,m._uv5, RNAP-a, Gall] Tc“) or cotransformed with pJG9.150 (Edam, AC1, 0rf15, Cm“) and pJG9.154 (P,pp,,ac,uv5, RNAP-a, rifH, Tc“). There were no colonies produced in negative control plates (Table 11). Colonies from the above screening were subsequently screened for the presence of B-galactosidase activities. Both RS/pBT-LGF2/pTRG-GalII and RS/pJG9.150/pJG9.154 colonies showed blue color when grown on 5-bromo-4-chloro-3-indolyl—B-D-galactopyranoside (X—Gal) indicator 57 plates. Based on enzyme assay (Figure 21) that monitors the formation of 2-nitrophnol, greatly enhanced B-galactosidase activities were observed for both RS/pBT-LGF2/pTRG- GalII (0.19 unit/mg; entry 1, Table 11) and RS/pJG9.150/pJG9.154 (0.23 unit/mg; entry 3, Table 7)when comparing to the activity of control (0.015 unit/mg; entry 2, Table 11). The results suggested that 0rf15-encoded transketolase and rifH-encoded aminoDAHP synthase complexed with one another when co-expressed in E. coli. Using the same method, the interaction between TktA and RifH was studied by co-expression of the bait plasmid pJG9.213 (Fwd/V5, AC1, tkrA, Cm“) and target plasmid pJG9.154 (P,p,,,,a,_Uv5, RNAP-a, rifH, Tc“) in the BacterioMatch two-hybrid reporter strain followed by selection for positive interactions using medium containing 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin. Only a very few colonies appeared on the plate after 30 h of incubation at 30°C. When subsequently screened for the presence of B-galactosidase activity, only very faint blue color was formed. Based on enzyme assay, no apparent enhancement (entry 6, Table 11) of B-galactosidase activity was observed relative to the control (entry 2, Table 11). The results indicated that rkrA-encoded transketolase and rifH-encoded aminoDAHP synthase did not complex, or had a very weak interaction when co- expressed in E. coli. The two-hybrid results were consistent with our previous observations. More aminoDAHP was synthesized in the presence of RifH and Orf15. A lower yield of aminoDAHP was obtained when RifH and TktA were used together. Further evidence of metabolite channeling between 0rf15-encoded transketolase and rifH-encoded aminoDAHP synthase will be discussed in Chapter 3. 58 Kanosamine and Kanosamine 6-phosphate Biosynthesis Overview With identification of 3-amino-3-deoxy-D-fructose 6-phosphate and kanosamine 6-phosphate as defined biosynthetic precursors of the aminoshikimate pathway, attention turned to delineation of the biosynthesis of kanosamine 6-phosphate. As discussed in the previous section, kanosamine is likely to serve as a biosynthetic precursor to kanosamine 6-phosphate. The riflV gene product, which is part of the rif gene cluster and necessary for 3-amino-S-hydroxybenzoate biosynthesis in A. mediterranei, has high sequence homology to glucokinase in various microorganisms (47% identity to ansB of Streptomyces collinus and 42% identity to gdnK of Streptomyces hygroscopicus). This suggested that kanosamine 6-phosphate is biosynthesized from kanosamine with the phosphorylation catalyzed by the rifN-encoded glucokinase in A. mediterranei. H2N H2N H2N +10% Hofig Hofl HO HO HZN H0 H2 NO H2N H2N H2N ggmo, NH, wk”, 35mm OH NH2 0H0 NH2 OH NH2 PQOH 0%; \—|§,0H O O kanamycin OH amikacin tobramycin 0“ natamycin oleandomycin R = H daunomycin R = OH adriamycin Figure 22. Antibiotics containing kanosamine or kanosamine-like structure. 59 0 HO HO HO NH 0 ATP ADP o UTP PP o 9 Q I ,g HOI- OH >4+ 1—1o:-----0P03H2 A4» HOI- «no-P-o-P-o o" 0 > -, OH OH 17 HO OH HO OH HO OH HO OH D-glucose glucose 1-phosphate - UDP-glucose 0 NAD HO NH NADH I NH 0 0 9 Q J§ %* HO'" "'O'P'O'P'O HO" "'O P 0' P' 0 ON 0 NADH 0 -,OH OH OH Elke 6H 6H OH h HO OH HO OH UDP-3-keto-D-glucose UDP-kanosamine HO H203PO 0 ATP ADP o 'T’ HO" OH 34> HO"' OH UMP P H2N H2N ’OH kanosamine kanosamine 6-phosphate Figure 23. Hypothetical kanosamine and kanosamine 6-phosphate biosynthesis. (a) i) glucokinase; ii) phosphoglucomutase; (b) UDP-glucose pyrophosphorylase; (c) UDP- 3-keto-D-glucose dehydrogenase; (d) UDP-3-keto-D-glucose transaminase; (e) UDP— kanosamine phosphatase; (f) kanosamine kinase. Abbreviations: AT(D)P, adenosine 5’- tri(di)phosphate; UD(M)P, uridine 5’-tri(mono)phosphate; lPPi, inorganic pyrophosphate; Pi, inorganic phosphate. Many naturally occurring antibiotics contain kanosamine or kanosamine-like moiety (Figure 22). Therefore, the importance of elaboration of kanosamine biosynthesis is not limited to the study of the biosynthetic precursors to the aminoshikimate pathway. Kanosamine biosynthesis was first observed and studied in Bacillus pumilus (formerly Bacillus aminoglucosidicus) in the 1960’s.104 Incubation of [U-‘4C]-glucose, ATP, UTP, NAD,g1utamine, and Mg+2 in dialyzed cell- free lysate of B. pumilus led to the formation of [HQ-kanosamine.1040 Likewise, incubation of UDP-[U-‘4C]-glucose in B. pumilus cell 1 04c lysate with NAD and glutamine led to the formation of [U-‘4C]-kanosamine. The distribution of radioactivity in kanosamine produced from [1-‘4C]-glucose and [6—‘4C]- 60 glucose was measured. Kanosamine synthesized from [l-‘4C]-glucose contained 73% of the total radioactivity in C-1 and kanosamine synthesized from [6-‘4C]-glucose contained 104C 58% of the radioactivity in C-6. These results suggest that kanosamine is biosynthesized from glucose via the intermediacy of UDP-glucose and the whole carbon . . . . . 4 skeleton of glucose 13 incorporated into kanosamme. The prev10usly proposed route10 C for kanosamine biosynthesis from glucose is summarized in Figure 23. D-Glucose is first converted into UDP-glucose by way of D-glucose l-phosphate. The requirement of NAD suggests that UDP-glucose is oxidized to UDP-3-keto-D-glucose, which undergoes transamination with glutamine to afford UDP—kanosamine. Subsequent hydrolysis of UDP-kanosamine generates kanosamine. The route (Figure 23) for kanosamine biosynthesis was proposed 40 years ago. . . . . . 104~ However, no intermediates in th1s proposed pathway have ever been isolated. L Recently, kanosamine biosynthesis in B. pumilus was investigated using cell-free reactions in the Frost group.”4 UDP-glucose and UDP-3-keto-D-glucose have been confirmed as biosynthetic precursors to kanosamine in B. pumilus. In this section, isolation and characterization of kanosamine biosynthetic enzymes in B. pumilus will be discussed. Based on the properties of the isolated UDP-3-keto—D-glucose dehydrogenase, a modified kanosamine biosynthetic pathway is proposed. Characterization of the kanosamine biosynthetic pathway in A. mediterranei is also discussed in this section. Synthesis of UDP-6,6-[2H2]-D-glucose UDP—6,6-[2H2]-D-glucose was synthesized from 6,6-[2HZJ-D-glucose by in vitro enzyme-catalyzed conversion summarized in Figure 24. In this one-pot enzymatic 61 conversion, 6,6-[2Hz]-D-glucose was first phosphorylated by UTP in the presence of hexokinase to afford 6,6- [2H2]-D- glucose 6-phosphate, which was subsequently converted to 6,6-[2H2]-a-D-glucose l-phosphate by phosphoglucomutase. The UDP generated in the phosphorylation reaction was recycled back to UTP by pyruvate kinase-catalyzed phosphate transfer from phosphoenolpyruvate to UDP. UTP then reacted with 6,6-[2H2]- a-D-glucose l-phosphate in the presence of UDP-glucose pyrophosphorylase to give UDP-6,6-[2H2]-D-glucose and PP,. H203POD Hm OH ——’ HO'“ O'”OP03H2 HO OH HO OH 6, 6- [2H2J- D- -g|ucose 6, 6- [:HOZJ- 0- glucose 6.6- [2H2]- D glucose 6-phosph ate 1—phosphate PEP Pyruvate o 0 HO D NH 0 Q 0 fig UTP HOI- «o P- o P- o o” 0 A 9] -, 6H 6H ' HO OH { 6 HO OH PP: —’ Pi UDP-6,6-[2HZJ-D-glucose Figure 24. Synthesis of UDP-D-6,6-[2H2]-glucose. (a) hexokinase; (b) phosphoglucomutase; (c) pyruvate kinase; ((1) UDP-glucose pyrophosphorylase; (e) inorganic pyrophosphatase. Abbreviations: UD(M)P, uridine 5’- tri(mono)phosphate; PEP, phosphoenolpyruvate; PPi, inorganic pyrophosphate; Pi, inorganic phosphate. The limiting step of the bioconversion was the formation of 6,6-[2H2]-a-D-glucose l-phosphate catalyzed by phosphoglucomutase. The equilibrium might disfavor formation of 6,6-[2H2]-a-D-glucose 1-phosphate.“5 However, addition of inorganic pyrophosphatase allowed the overall reaction proceed in the direction of UDP-6,6-[2H2J- 62 D-glucose by converting inorganic pyrophosphate to inorganic phosphate. a-D—Glucose 1,6-diphosphate was used as an activator of phosphoglucomutase. Since high concentrations of UTP can inhibit UDP-glucose pyrophosphorylase,ll6 UTP was maintained at a low concentration throughout the reaction by adding 6,6-[2H2]-D-glucose, UTP, and phosphoenolpyruvate in portions. Product UDP-6,6-[2H2]-D-glucose was purified using Dowex-1X2-200 anion exchange resin and obtained as a white power after concentration and lyophilization. The final yield of UDP-6,6-[2H2]-D-glucose was 80% relative to D-6,6-[2H2]-glucose. Synthesis of UDP-3-keto-D-glucose O O HO O O D (“1:4 Agmbacterium HO O O O [Kr HOW -uo-P-o-P-o o N 0 '"me 80'9"“ : H01» -uo-P-o-P-o o N O ', ' ' standing cells in .’ ' ' HO OH OH OH 5 mM Tris-HCI. pH 8 O OH OH OH h HO OH HO OH UDP-glucose 3-keto-UDP-gluoose Figure 25. Synthesis of UDP-3-keto-D-glucose. UDP-3-keto-D-glucose was synthesized from UDP-glucose by a whole cell- catalyzed bioconversion (Figure 25). A strain called Agrobacterium tumefaciens IAM- 1525 (NCPPB 396) contains 3-keto-D-glucose dehydrogenase activity. It was originally reported that this strain could oxidize sucrose to 3-keto-sucrose.117 It was also reported that this strain could oxidize UDP-glucose to UDP-3-keto-D-glucose.118 A. tumefaciens was grown at 30°C with sucrose supplementation. After the completion of the growth, cells were washed with Tris-HCl buffer and resuspended in 5 mM Tris-HCl. UDP- glucose was added and the reaction mixture was shaken at 30°C until no UDP-glucose 63 was detected by lH N MR. After removing cells by centrifugation, the crude UDP-3-keto- D-glucose was purified by passing through a Dowex 50 (H1) column. Concentration to a small volume followed by lyophilization afforded UDP-3-keto-D-glucose as a white powder in a yield of 69% relative to UDP-glucose. Isolation of kanosamine biosynthetic genes from B. pumilus Kanosamine biosynthetic pathway brought to our attention because kanosamine 6-phosphate has been directly implicated as one of the biosynthetic intermediates of the aminoshikimate pathway and this aminosugar was likely biosynthesized from kanosamine. Kanosamine was indeed formed when UDP-glucose, glutamine, and NAD were incubated in cell-free lysate of A. mediterranei (details later in this chapter). Following inspection of the sequenced rif biosynthetic gene cluster of A. mediterranei and proposed kanosamine biosynthesis in B. pumilus, we found that all the genes and encoded enzymes required for biosynthesis of kanoSamine 6-phosphate might reside in the cluster (Figure 26). RifL-catalyzed oxidation of UDP-glucose would yield UDP-3-keto-D-glucose, which could undergo a transamination reaction catalyzed by rifK- encoded transaminase. Hydrolysis of the resulting UDP-kanosamine catalyzed by rifM- encoded UDP-kanosamine phosphatase would give kanosamine. Phosphorylation of kanosamine catalyzed by riflV—encoded kanosamine kinase would afford kanosamine 6- phosphate. Biosynthesis of kanosamine might thus follow the same pathway in A. medirerranei as in B. pumilus. However, UDP-3-keto-D-glucose formation was not observed when UDP-glucose and NAD were incubated with heterologously expressed RifL. Similarly, UDP-kanosamine formation was not observed when UDP-3-keto-D- 64 glucose was incubated with glutamine, NADH, and heterologously expressed RifK. The failure to observe enzyme activities for enzymes encoded by genes putatively involved in kanosamine biosynthesis in A. mediterranei prompted efforts to identify the enzymes and encoding genes involved in biosynthesis of kanosamine in B. pumilus. (1) me rifH rifl rifK rifL rifM rifN 0rf15 rifJ (ll) 0 O o o [“120 Q Q N121) OI“ 'IO-PHOP-O —a—> HOI' 'I-OPO-P-O O —> DH H H OH OH o 6 U o 0.. U OOH HO OH UDP- D- -glucosHeO UDP-3-keto-D-glucose O (“NH R0 COzH I A c 0 9 Ohio P- o P- 0 ON 0 —+ H01» OH —__>_. DH OH OH -, HO NH2 H2N OH HO OH - UDP-kanosamine kanosamine AHBA d(R= R: H203P kanosamine 6- -phosphate Figure 26. Enzymes and the encoding genes associated with 3-amino-5- hydroxybenzoate biosynthesis and hypothetical kanosamine and kanosamine 6- phosphate biosynthesis in A. mediterranei. (I) Enzymes (encoding gene): aminodehydroquinate synthase (rifG); aminoDAHP synthase (rifH); aminoshikimate dehydrogenase (rifl); 3-amino-S-hydroxybenzoate synthase/transaminase (rifK); UDP-3- keto-D-glucose dehydrogenase (rifL); UDP-kanosamine phosphatase (rijM); kanosamine kinase (rifN); transketolase (0rf15); aminodehydroquinate dehydratase (rifJ). (II) (a) rifL-encoded UDP-3-keto-D-glucose dehydrogenase; (b) rifK-encoded UDP-3-keto-D- glucose transaminase; (c) rifM-encoded UDP-kanosamine phosphatase; ((1) riflV— encoded kanosamine kinase; (e) the aminoshikimate pathway. As the genomic DNA sequence of B. pumilus was not available and molecular biological manipulation of B. pumilus is still in its infancy, classical enzyme purification was employed as a first step towards ultimate isolation of kanosamine biosynthetic genes. 65 The crude lysate of B. pumilus was first partitioned into four fractions by passing through a Sephadex G-100 column (Pharmacia, Figure 27). After UDP-glucose, NAD, and L- glutamine were incubated with each fraction at 30°C for 6 h, no kanosamine formation was detected. Kanosamine formation could only be. observed when fraction I and II were combined. The results indicated that at least two individual enzymes (or enzyme complexes) were needed for kanosamine biosynthesis from UDP- glucose in B. pumilus. O HO 0 o ([31: HO 0 .. .. enzyme 0 HOW 'IIO-P-O-P-O ON 0 t Ho» OH . ' ' NAD, glutamine . , OH OH , HO OH H2N OH HO OH kanosamine enzyme production crude lysate of B. pumilus l' I" III, or IV Iandll + Sephadex G-100 | and m ’ land IV I l l 1 fractions: | u m IV II and III II and IV II and IV Figure 27 . Kanosamine biosynthesis with fractioned B. pumilus cell-free lysate. When B. pumilus cell-free lysate was further purified by FPLC on a HiTrap DEAE column, UDP-3-keto-D-glucose dehydrogenase activity was detected by enzyme assay as shown in Figure 28 (Method I). The specific activity of UDP-3-keto-D-glucose dehydrogenase was 0.38 units/mg after consecutive FPLC purifications using HiTrap DEAE and RESOURCE Q columns. However, the UDP-3-keto-D—glucose dehydrogenase activity was lost after an additional purification step using FPLC Superdex 200 prep column or a Phenyl HP column. 66 Method I. O O HO 6”” UDP-glucose HO (‘LNH O 9 Q I A 3-dehydrogenase O 9 O I «g Ho» «uo-P-o-P-o o” 0 w HOI" --uo-P-o-P-o o” 0 ', OH OH , OH OH HO OH 1.? NAD NADH O H h HO OH ' HO OH UDP-glucose 3-keto-UDP-glucose Method ll. 0 O HO (‘LNH UDP-glucose HO NH O 9 9 | A 3-dehydrogenase O 9 9 I * HOI" 'IIO-P-O-P-O o” 0 HOW "IO-E-O-P-O ON 0 ', > OH OH HO OH OH OH 1? PMsox PMSred 0 OH 17 HO OH HO OH UDP-glucose DCIPre d DCIPOX 3-keto-UDP-glucose (colorless) (blue) Me Cl PF (1: 1:} WQQ N Cl PMS, phenazine methosulfate DCIP, dichloroindophenol Figure 28. Enzyme assay designs for UDP-3-keto-D-glucose dehydrogenase. One possible explanation for the loss of enzyme activity during purification included loss of cofactor or removal of a redox-active protein. Two 3-keto-glucoside dehydrogenses had previously been reported in the literature. Both of them utilize flavine adenine dinucleotide (FAD) as their co-factor.l '9 To test whether UDP-3-keto-D-glucose dehydrogenase from B. pumilus similarly requires FAD as its co—factor, a new enzyme assay method was employed (Method 11, Figure 28). The UDP—3-keto-D-glucose dehydrogenase was assayed using reduction of 2,6-dichloroindophenol (DCIP) as the electron acceptor and phenazine methosulfate (PMS) as the intermediate electron carrier.120 This method is widely used in the assay for flavoproteins.121 UDP-3-keto—D- glucose dehydrogenase was successfully assayed using DCIP oxidation with PMS 67 serving as the electro carrier. The specific activity of UDP-3-keto-D-glucose dehydrogenase was 10 unit/mg after purification using FPLC Superdex 200 column. 0.025 002 ~- . A d I‘. 0.015 .4- o 300 350 400 450 500 550 Wave Length (nm) Absorbanoe < 1 / l Figure 29. Absorption spectra of UDP-3-keto-D-glucose dehydrogenase. a (solid line), UDP-3-keto-D-glucose dehydrogenase; b (dashed line), UDP-3-keto-D- glucose dehydrogenase with UDP-glucose. The absorption spectrum of the enzyme showed maxima near 380 nm and 455 nm (Figure 29), which is a characteristic of flavoproteins. On addition of 1.0 mM UDP- glucose, the chromophore was bleached. A plot of this substrate-induced difference spectrum (Figure 29) confirmed the presence of a flavine chromophore. To further confirm that the purified enzyme did possess UDP-3-keto-D-glucose dehydrogenase activity, UDP-glucose was incubated with 2,6-dichloroindophenol (DCIP) and phenazine methosulfate (PMS) in the presence of the purified enzyme at 30°C for 6 h. Based on 1H and ”C NMR, UDP-3-keto-D-glucose was formed in 60% yield. The finalized enzyme purification was performed on FPLC using HiTrap DEAE column, RESOURCE Q column, and Superdex 200 prep column followed by a Phenyl HP column. Yields and specific activities for each step of the purification are summarized in 68 Table 12. After purification, the N-terminal sequence of UDP-3-keto—D-glucose dehydrogenase was obtained (Figure 30). Based on the protein sequence, degenerate oligonucleotides were to be designed and used as PCR primers for isolation and cloning of the gene encoding UDP-3-keto-D-glucose dehydrogenase from B. pumilus genomic DNA. Table 12. Purification of B. pumilus UDP-3-keto-D-glucose dehydrogenase. total protein total activity specific activitya yield purification (mg) (units) (unit/mg) (%) f01d lysate 360 21.6 0.06 100 l HiTrap DEAE 14 19.6. 1.4 91 23 RESOURCE Q 4.2 17.2 4.1 80 68 Superdex 200 1.0 9.6 9.6 43 160 Phenyl HP 0.5 9.3 18.6 43 310 a One unit of enzyme activity: 1 ymol of 2,6-dichloroindophenol (DCIP) reduced per min at 25°C and pH 7.0 However, an alternative strategy for isolation of the encoding gene was successfully employed before a protocol was finalized for the purification of UDP-3- keto-D-glucose dehydrogenase to homogeneity. Halomonas sp. contains a sequenced gene encoding 3-keto—glucoside 3dehydrogenase, which can oxidize a-methyl-D- glucoside to or-methyl-3-l t > IacF Pm PCR product AmpR X B B X pJGQ.251 . H ' 4 __. —> <—— T5, His6 UDPGDH AmpR a N = NruI, S = Smal, X = XhoI, B = BamHI. 7O restriction enzyme digestion =21 > :2: + I 2:: genomic DNA intramolecular l ligation PCR ‘ primers C 1 Figure 31. Inverse PCR. Legend: filled area, known DNA sequence. To solve this problem, inverse PCR122 (Figure 31) was employed. Standard PCR amplifies segments of DNA that lie between two inward-pointing primers. By contrast, inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available. Since the gene sequence in the middle of UDP-3-keto-D-glucose dehydrogenase gene should be correct, primers were designed based on this part of the gene. To conduct inverse PCR, B. pumilus genomic DNA was first digested with the restriction enzyme PstI. The individual restriction fragments were subsequently converted into circles by intramolecular ligation. After purification, the circularized DNAs were then used as templates in PCR. The unknown sequence was amplified by two primers that bind specifically to the known sequence of UDP-3-keto-D-glucose dehydrogenase gene and point in opposite directions. Based on the results of inverse PCR, the actual DNA sequence of the 5’- and 3’-end of UDP-3- keto—D-glucose dehydrogenase gene were indeed different from the DNA sequence amplified using primers based on the sequence encoding 3-keto-glucose dehydrogenase in Halomonas sp. When the DNA sequence was translated into protein sequence, no change in N-terminal sequence and two amino acids difference in the C-terminal of the 71 protein were observed (Figure 26 and 27). The DNA and protein sequence of UDP-3- keto-D-glucose dehydrogenase from B. pumilus was shown in Figure 32. 1 ATG GCA GAC AAT CAT TAT GAT GCG ATT GTT GTC GGT TCA GGC ATA AGT GGA GGC TGG GCT GCA AAG 1’ M A D N H Y D A I V V G S G I S G G W A A K 67 GAA CTA ACC GAA AAA GGC CTG AAG GTT CTG CTG CTG GAG CGC GGC AGA AAC ATC GAG CAC ATT AAG 23’ E L T E K G L K V L L L E R G R N I E H I K 133 GAT TAT CAG AAT GCC GAC AAG GAA GCC TGG GAC TAT CCC CGC AAC CGC GCC ACG CAG GAG ATG AAG 45’ D Y Q N A D K E A W D Y P R N R A T Q E M K 199 GCG AAG TAT CCG GTG CTC AGC CGC GAC TAT CTG CTG GAA GAG GCC ACG CTC GGC ATG TGG GCC GAC 67’ A K Y P V L S R D Y L L E E A T L G M W A D 265 GAG CAG GAA ACG CCC TAT GTC GAA GAA AAG CGT TTC GAC TGG TTC CGT GGT TAT CAC GTC GGC GGC 89’ E Q E T P Y V E E K R F D W F R G Y H V G G 331 CGT TCG CTG CTG TGG TGG TCG CAG ACC GAT TTC GAG GCC AAT GCC AAA GAA GGG ATC GCC GTT GAC 111’ R S L L W W S Q T D F E A N A K E G I A V D 397 TGG CCG ATC CGT TAT CAG GAC ATG GCT CCC TGG TAT GAT TAT GTC GAG CGT TTC GCC GGC ATT TCC 133’ W P I R Y Q D M A P W Y D Y V E R F A G I S 463 GGC AGC CGC GAG GGG CTG GAC ATT CTT CCT GAT GGT GAA TTC CTG CCG CCT ATT CCG CTG AAT TGC 155’ G S R E G L D I L P D G E F L P P I P L N C 529 GTT GAA GAG GAT GTG GCA CGC CGG TTG AAA ACT GCC TTT AAA GGC ACG CGA CAC CTC ATC AAT TCG 177’ V E E D V A R R L K T A F K G T R H L I N S 595 CAG TGC GCC AAC ATC ACC CAG GAA CTT CCC GAT CGC ACG CGC TGC CAG TTC CGC AAC TGC TGC CGG 199’ Q C A N I T Q E L P D R T R C Q F R N C C R 661 AAT TGC CAC TAT CGA AAT AAA TGC TTT CTG CGT TGC CCT TAT GGT GCC TAT TTC AGC CCC CAG TCC 221’ N C H Y R N K C F L R C P Y G A Y F S P Q S 727 GGA AAT CTT ACG CTC CGG CCT TTC TCC ATC GTC AAG GAA ATC CTT TAC GAC AAG GAC AAG AAG AAG 243’ G N L T L R P F S l V K E I L Y D K D K K K 793 GCG CGC GGT GTC GAA ATC ATC GAT GCC GAA ACG AAC CTG ACC TAT GAA TAC ACC GCC GAT GTG ATC 265’ A R G V E | I D A E T N L T Y E Y T A D V I 859 TTC CTC AAC GCT TCG ACG CTC AAC TCG ACC TGG GTT CTG ATG AAC TCG GCC ACA GAC GTG TGG GAG 287’ F L N A S T L N S T W V L M N S A T D V W E 925 GGA GGG CTC GGC AGC AGC TCG GGC GAA CTC GGC CAC AAT GTG ATG GAC CAC CAG GAG CAT TTC CGC 309’ G G L G S S S G E L G H N V M D H Q E H F R 991 ATG GGC GCC ACC GGC GAG GTG GAA GGT TTC GAA GAG TTT TAT TTC AAG GGC CGC CGC CCG GCG GGA 331’ M G A T G E V E G F E E F Y F K G R R P A G 1057 TTC TAT ATT CCC CGT TTC CGC AAC ACC GGC GAC GAC AAG CGG AAC TAT CTG CGC GGT TTC GGT TAT 353’ F Y I P R F R N T G D D K R N Y L R G F G Y 1123 CAG GGA TCC GCC AGC CGT TCG CGC TGG GAG CGG GAA ATC GCC GAA CTC AAC ATC GGA GCC GAC TAC 375’ Q G S A S R S R W E R E I A E L N I G A D Y 1189 AAG GAA GCG CTG ACC CAG CCG GGC GCC TGG ACT ATC GGC ATG ACT GCC TTC GGT GAA ATG CTG CCT 397’ K E A L T Q P G A W T | G M T A F G E M L P 1255 TAT CAC GAG AAC CGC ATG CTG GTG AAG CTG GAC CAC GAC AAG AAG GAC AAA TGG GGC CTG CCA GTC 419’ Y H E N R M L V K L D H D K K D K W G L P V 1321 CTG TCG ATG AAT GTC GAG ATG AAG CTC GAT ATG CGC GAA GAC ATG GTC AAT GAC GCA GTC GAG ATG 441’ L S M N V E M K L D M R E D M V N D A V E M 1387 TTC GAA GCG GTC GGC ATC AAG AAC GTC AAA CCG TCG AGG GGC AGC TAT GCG CCG GGC ATG GGC ATC 463’ F E A V G I K N V K P S R G S Y A P G M G I 1453 CAT GAA ATG GGT ACG GCC CGC ATG GGA CGC GAC CCC AAA ACT TCC GTG CTT AAT GGC AAC AAC CAG 485’ H E M G T A R M G R D P K T S V L N G N N Q 1519 GTC TGG GAT GCG CCG AAC GTC TTT GTC ACT GAC GGT GCC TGC ATG ACT TCG GCC TCC TGC GTC AAC 507’ V W D A P N V F V T D G A C M T S A S C V N 1585 CCG TCG CTC ACC TAC ATG GCG CTG ACG GCG CGT GCC GCC GAA TTT GCC GTT TCC GAA CGT AAG CAG 529’ P S L T Y M A L T A R A A E F A V S E R K Q 1651 GGG AAC TTG GCA TGA 551’ G N L A 0 Figure 32. The sequence of B. pumilus UDP-3-keto-D-glucose dehydrogenase. 72 Using the modified primers, the UDP-3-keto-D-glucose dehydrogenase gene was amplified from B. pumilus genomic DNA by PCR. The PCR product was subsequently cloned into pJG7.246 (T5, Hisé, lacIQ, Amp“) to yield (Table 13) pJG9.251 (T5, His6, UDPGDH, lacIQ, Amp“). After transformation, growth, and induction, UDP-3-keto-D- glucose dehydrogenase was purified from the crude lysate of E. coli XL-l Blue/pJG9.251 using a nickel-nitrilotriacetic acid (Ni-NTA) column. The specific activity of UDP-3- keto-D-glucose dehydrogenase was 2.3 unit/mg protein. Incubation of UDP-glucose, 2,6- dichloroindophenol (DCIP), and phenazine methosulfate (PMS) with purified enzyme at 30°C for 6 h led to a quantitative yield of UDP-3-keto-D-glucose. The results confirmed that the isolated gene indeed encoded UDP-3-keto-D-glucose dehydrogenase. Based on the redox requirement of this enzyme, a modified kanosamine biosynthetic pathway in B. pumilus was proposed (Figure 33). O O HO NH FAD HO NH 0 9 P I x a o P 9 I 1 HOW -IIO-P-O-P-O ON 0 ATP HOI» «IO-P-O-P-O ON 0 ', OH OH > OH OH HO OH b FADH2 0 OH h HO OH HO OH UDP-glucose 3-keto-UDP-glucose O HO (“NH HO b O 9 9 K c O ———> Hoh- «no-P-o-P-o o" 0 T HOI-- OH ‘, OH OH ‘, H2N OH h UMP, Pi H2N OH HO OH . UDP-kanosamine kanosamine Figure 33. Modified kanosamine biosynthetic pathway in B. pumilus. (a) UDP-3- keto-D-glucose dehydrogenase; (b) transaminase; (c) phosphatase. After the isolation of UDP-3-keto-D-glucose dehydrogenase and its encoding gene were completed, attention turned to identification of UDP-3-keto-D-glucose 73 transaminase and UDP-kanosamine phosphatase. With the hope that kanosamine biosynthetic genes are clustered together, neighboring genes of UDP-3-keto-D-glucose dehydrogenase were elucidated by chromosome walking using previously described inverse PCR technique. Five additional open reading frames were identified and their possible functions were assigned based on sequence homology (Table 14). Unfortunately, no transaminase or phosphatase activities were identified. Table 14. Genetic organization around UDP-3-keto-D-glucose dehydrogenase. entry gene proposed function 1 orfA transcription regulator 2 0113 gluconate dehydrogenase 3 orfC UDP-3-keto-D-glucose dehydrogenase 4 orfD sugar phosphate isomerase/epimerase 5 0rf15 oxidoreductase 6 orfF sugar transport protein With the success of using homologous PCR during identification of UDP-3-keto- D-glucose dehydrogenase gene in B. pumilus, the similar strategy was again employed to identify the UDP-3-keto-D-glucose transaminase-encoding gene. Two open reading frames can be amplified from B. pumilus genomic DNA by using two sets of primers based on deoxysugar aminotransferase sequence from Oceanobacillus 5pm However, no UDP-3-keto-D-glucose transaminase activity was detected when they were heterologously expressed in E. coli. Both genes encode unknown enzyme activity based on sequence homology. Enzyme purification will be attempted to identify genes that encode UDP-3-keto-D—glucose transaminase and UDP-kanosamine phosphatase. 74 Kanosamine biosynthesis in A. mediterranei With kanosamine 6-phosphate established as a precursor to iminoE4P, kanosamine became an obvious candidate as a source of the nitrogen atom for the aminoshikimate pathway. In vitro bioconversion of kanosamine into aminoDAHP once again employed A. mediterranei cell-free lysate. Incubation of kanosamine with ATP, D- ribose 5-phosphate, and phosphoenolpyruvate in A. mediterranei cell-free lysate with 20% glycerol led to the formation of aminoDAHP in 1% yield (entry 1, Table 15). No aminoDAHP was detected when ATP, D-ribose 5—phosphate, and phosphoenolpyruvate were incubated in A. mediterranei cell-free lysate (entry 2, Table 15). The results establish kanosamine as a biosynthetic precursor to iminoE4P and the aminoshikimate pathway. Table 15. Kanosamine biosynthesis in A. mediterranei.a entry reaction condition . products (% yield) kanosamine, ATP, R5P, and PEP; A. mediterranei 1 cell-free lysate, 20% glycerol, pH 6.8 am1noDAHP(1) 2 ATP, R5P, and PEP; A. mediterranei cell-free --- lysate, 20% glycerol, pH 6.8 3 UDP-6,6-[2H2]-D-glucose, NAD, glutamine; 6,6-[2H2]-kanosamine A. mediterranei cell-free lysate, pH 6.8 (5) 4 UDP-D-glucose, NAD, glutamine, ATP, R5P, and ___ PEP; A. mediterranei cell-free lysate, pH 6.8 UDP-6,6-[2H2]-D-glucose, NAD, glutamine, ATP, 5 R5P, and PEP; A. mediterranei cell-free lysate, 20% glycerol, pH 6.8 7,7-[2H2]—aminoDAHP (0.9) UDP-3-keto-D-glucose, N ADH, and glutamine; A. mediterranei cell-free lysate, pH 6.8 kanosamine (6) 3 Abbreviations: R5P, D-ribose S-phosphate; PEP, phosphoenolpyruvate; aminoDAHP, 4- amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate. 75 Attention then turned to elaboration of kanosamine biosynthesis in A. mediterranei. Based on the supposition that A. mediterranei and B. pumilus might share a similar kanosamine biosynthetic pathway, UDP-glucose was first examined as the biosynthetic precursor to kanosamine. As expected, incubation of UDP-6,6-[2H2]-D- glucose with NAD and glutamine in A. mediterranei cell-free lysate led to the formation of 6,6-[2H2]-kanosamine in 5% yield (entry 3, Table 15). Based on positive electrospray mass spectrometry, UDP-6,6-[2H2]-D-glucose and 6,6-[2H2]-kanosamine have M + 2 ions with relative intensities of 98.08% and 97.83%, respectively. However, incubation of UDP-glucose with B-NAD, L-glutamine, ATP, D-ribose 5-phosphate, and phosphoenolpyruvate in A. mediterranei cell-free lysate did not produce quantifiable concentrations of aminoDAHP (entry 4, Table 15). Given the importance of glycerol on the biosynthesis of aminoDAHP from the corresponding aminosugar precursors, incubation of UDP-6,6-[2H2]-D-glucose with NAD, glutamine, ATP, D-ribose 5- phosphate, and phosphoenolpyruvate in A. mediterranei cell-free lysate was subsequently examined in the presence of 20% glycerol. 7,7-[2H2]-AminoDAHP was formed in 0.9% yield (entry 5, Table 15). At the same time, UDP-3-keto-D-glucose was also confirmed as a biosynthetic precursor to kanosamine in A. mediterranei by another group member (entry 6, Table 15).]24 The observations confirmed that kanosamine was biosynthesized from UDP-glucose via UDP-3-keto-D-glucose in A. mediterranei and kanosamine was a precursor to iminoE4P biosynthesis and the source of nitrogen for the aminoshikimate pathway. Unlike B. pumilus, the DNA sequences of rzf biosynthetic genes had been delineated (Figure 8, Chapter 1). The rifL gene product, which is necessary for 3-amino- 76 5-hydroxybenzoate biosynthesis, has high sequence homology to a class of oxidoreductases. It is immediately downstream from the rifK gene, which displays high sequence homologies to aminotransferases. RifM, the other gene immediately adjacent to rz’fL, is assigned as a phosphatase. This suggeststhat the joint action of rifK-encoded transaminase and rifL-encoded oxidoreductase converts UDP-glucose to UDP- kanosamine, which is subsequently hydrolyzed by rifM—encoded kanosamine phosphatase to yield kanosamine. Recently, the enzymatic activities of rifK—encoded UDP-3-keto-D- glucose transaminase and rifL-encoded UDP-3—keto-D-glucose dehydrogenase were confirmed by another group member.‘25 Incubation of UDP-glucose, dichloroindophenol (DCIP), and phenazine methosulfate (PMS) with rifL—encoded oxidoreductase led to the formation of UDP-3-keto-D- glucose in 69% yield. Similarly, incubation of UDP-3-keto- D-glucose, glutamine, and PLP with rifK-encoded transaminase led to the formation of UDP-kanosamine in a yield of 25%. UDP-kanosamine has also been chemically synthesized in our lab.l26 It will be used to address the enzymatic activity of the rifM gene product. Kanosamine 6-phosphate biosynthesis Kanosamine and kanosamine 6-phosphate have been identified as precursors to iminoE4P biosynthesis. An enzyme-catalyzed phosphoryl group transfer reaction is clearly required for phosphorylation of kanosamine. The rifN gene product, which is part of the rif biosynthetic gene cluster and necessary for 3-amino-5-hydroxybenzoate biosynthesis in A. mediterranei, has high sequence homology to glucokinase in various 77 I'll-II IIIIIIIIIII Illllll’lllll'lilllr 1111' lilil llllill‘lll‘lll‘ll I? 1|" I‘lll‘ll ll: microorganisms. This suggested that kanosamine was phosphorylated by a riflV—encoded glucokinase to form kanosamine 6-phosphate (Figure 34). HO H203PO 0 ATP ADP O HO"' OH i—INZ. H0... OH . l HZN OH H2N ’OH kanosamine kanosamine 6—phosphate Figure 34. Phosphorylation of kanosamine. The riflV gene product was partially purified from BL21(DE3)/pRM070 (77, Hisé, rzflV, Amp“) as a 6-His tagged protein using a Ni-NTA Agarose column to purify the enzyme. Kanosamine, ATP, MgClz, and citric acid were incubated with partially purified RifN at room temperature. The pH of the reaction mixture was maintained between 7.5 and pH 8.0 by addition of 1 N aqueous NaOH. After 24 h of incubation, the crude reaction mixture was purified using AG-l X8 anion exchange resin (acetate form). Kanosamine 6-phosphate, was isolated as a white solid in 50% yield. This confirmed that RifN was a kinase and could catalyze the phosphorylation of kanosamine. At the same time, Professor Floss reported the kinetic evaluation of RifN and confirmed that RifN . . . 127 was a spec1fic kanosamine kinase. Table 16. Restriction enzyme map of plasmid pRM070. Plasmid Plasmid Map“ A A pRM070 I I —> ———> -——> T7, [300, Hiss rifN AmpR I A = AflIII 78 Table 17. lysates.ail Biosynthesis of kanosamine from UDP-glucose in A. mediterranei cell-free O (‘1: NH; HO H N 7 OH O 9 9 ‘ 2 m + HOli- “no-3-0-3-0 ON 0 > H H HO OH . HO OH L-glutamine UDP-glucose A. mediterranai cell-tree lysate O HO NH HO 0 Q 9 I k 0 Ho» «Io-P-o-P-o o” 0 + HOI-- -IIP03H2 + ', OH OH -, H2N OH HZN OH HO OH UDP-galactose HO O HO" OH + H0 kanosamine l-phosphate + B-NAD Ho 0 Hoh- -IIP03H2 HO “OH glucose 1-phosphate NH2 0 o ' OH + HzNMrOH + HOMOH o o O o o o H2N “’OH kanosamine L-glutamic acid u-ketoglutaramic acid a-ketoglutaric acid entry products yield (%)b 1 L-glutamine 52° 2 L-glutamic acid 29 ° 3 a-ketoglutaramic acid 2C 4 a-ketoglutaric acid 1 c 5 UDP- glucose 10d 6 UDP-galactose 2 d 7 kanosamine 5 d 8 kanosamine l-phosphate 3 d 9 glucose l-phosphate 1 1 d “ Reaction conditions: UDP-glucose, B-NAD, L-glutamine; A. mediterranei cell-free lysates, pH 6.8; b Yields are based on 1H NMR integration relative to an internal standard of the sodium salt of 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP, 6:0.00 ppm); c Yields are relative to initially added L-glutamine; (d) Yields are relative to initially added UDP- glucose. 79 During the study of kanosamine biosynthesis in A. mediterranei, incubation of UDP-glucose, NAD, and L-glutamine in cell-free lysate led to the formation of a number of other products in addition to kanosamine. The major components in the reaction mixture were partially separated and identified (Table 17). L-Glutamic acid, or- ketoglutaric acid, a-ketoglutaramic acid, and unreacted L-glutamine were all found in the reaction mixture. The yields of the four compounds were quantified by 'H NMR (entry 1-4, Table 17). Unreacted UDP-glucose along with a small quantity of UDP-galactose was also found in the reaction mixture (entry 5, 6, Table 17). Interestingly, an unknown compound, which was tentatively identified as kanosamine l-phosphate based on 1H NMR (Figure 49) and high resolution mass spectrometry (entry 8, Table 17), was isolated from the reaction mixture. To fully characterize this compound, an authentic sample of . . . . 6 kanosamine 1-phosphate was synthesrzed from kanosamine by another group member.l2 By comparing ]H and 13C NMR with the authentic sample, the unknown compound was confirmed to be kanosamine l-phosphate. I The formation of kanosamine l-phosphate might be a side reaction similar to the reaction leading to formation of glucose 1-phosphate. An unknown UDP-glucose diphosphatase or UDP-glucose pyrophosphorylase in the cell-free lysate might be responsible for both glucose 1-phosphate and kanosamine l-phosphate formation. Conversion of kanosamine l-phosphate to kanosamine 6-phosphate by mutase might constitute a pathway to biosynthesis of this precursor to iminoE4P in A. mediterranei (Figure 35). It could be synthesized by kinase-catalyzed phosphorylation of kanosamine or derived from mutase-catalyzed phosphoryl group migration in kanosamine l- 80 phosphate. Both kanosamine and kanosamine l-phosphate would be derived from UDP- kanosamine. 0 1H HO 0 0 (“1H 0 .. .. O __a_. Hob..io-P-o-P-Q N O HO O 9 9 HOII' «no-P-o-P-o 0 -, OH OH . OH OH H2N OH :1: O O I I?- Z O HO OH HO OH UDP-glucose UDP-kanosamine HO O —-b—> HOII' OH -—-L H2N OH H203PO kanosamine O —— '-—> HOII- OH H2N ’OH HO c kanosamine 6-phosphate ——> Ho" "'PO3H2 C! H2N ’OH kanosamine 1-phosphate Figure 35. Kanosamine 6-phosphate biosynthesis. (a) (i) UDP—3-keto-D-glucose dehydrogenase; (ii) UDP-3-keto-D-glucose transaminase; (b) phosphatase; (c) UDP-sugar diphosphatase or pyrophosphorylase; (d) kanosamine kinase (rifN); (e) mutase. The Source of the Nitrogen Atom Incorporated into the Aminoshikimate Pathway Once iminoE4P was demonstrated to be derived via kanosamine biosynthesis, attention turned to elaboration of the source of the aminoshikimate pathway’s nitrogen atom. L-Glutamine, which served as the source of the nitrogen atom in kanosamine biosynthesis, was likely the source of the aminoshikimate pathway’s nitrogen atom. The 81 question was which nitrogen atom in glutamine was incorporated into the aminoshikimate pathway (Table 18). To answer this question, 15N enrichments in kanosamine were analyzed when [amine-'SN]-L-glutamine and [amide-”NLL-glutamine were used as nitrogen source, respectively. Table 18. 15N enrichments in kanosamine produced‘ in A. mediterranei cell-free lysates with different 15N labeled nitrogen sources.a 5:1?“ HO NAD 0 :11pr no- P- o- P 04w 0 . > HObOH > OH OH nitrogen source .’ H2N OH UDP -Hglucose OOH [ISM-kanosamine NH2 ‘5qu [amine-‘5N1-L-glutamine [amide-‘5N]-L-glutamine entry l5N labeled glutamine dialysis cycles :::::::l:e(l;i): l [amine-‘5N]-L-glutamine 3 10 2 [amine-”N] -L-glutamine 4 13 3 [amine-'SN] -L-glutamine 6 1 2 4 [an1ide-‘5N]-L-glutamine 3 69 5 [amide-‘5N]-L—glutamine 4 85 6 [amide-'SN]-L-glutamine 6 89 a Reaction conditions: UDP—glucose, B-NAD, L-glutamine-amide-‘SN (or L-glutamine- amine-'SN); A. mediterranei cell-free lysates, pH 6.8; b l5N enrichments were determined by electrospray mass spectrometry. Incubation of UDP-glucose, NAD, and [amine-‘5N]-L-glutamine or [amide-UN]- L-glutamine in A. mediterranei cell-free lysate led to the formation of kanosamine with 15N enrichments of 9.5% and 69.4%, respectively (entry 1, 4, Table 18). The [amide- 82 ISN]-L-glutamine labelings were consistent with the presence of an alternative source of nitrogen that was difficult to remove by dialysis. In addition, 15N label exchange appeared to occur between [an1ine-‘5N]-L-glutamine and this alternative nitrogen source. When more thorough dialysis was carried out, 15Nlenrichments in kanosamine increased from 69.4% to 89.1% (entry 4, 5, 6, Table 18) with [amide-”NLL-glutamine being used as sole nitrogen source. The results suggested that the amide nitrogen of L—glutamine was the preferred source of the nitrogen of kanosamine biosynthesis in A. mediterranei cell- free lysates. It was consistent with the result that a significant amount of glutamic acid as well as only a small amount of a-ketoglutaramic acid was formed when UDP-glucose was incubated with [S-NAD and glutamic acid in A. mediterranei cell-free lysate (entry 2, 3, Table 17). Discussion In this chapter, iminoE4P, 3-amino-3-deoxy-D-fructose 6-phosphate, kanosamine 6-phosphate, and kanosamine have been identified as biosynthetic precursors to the aminoshikimate pathway. In turn, the biosyntheses of these precursors to the aminoshikimate pathway were also investigated. This demonstrates the intersection of kanosamine biosynthesis, iminoE4P biosynthesis, and the aminoshikimate pathway (Figure 36). Both kanosamine 6-phophate and 3-amino-3-deoxy-D-fructose 6-phosphate can be converted into aminoDAHP with virtually the same yields in A. mediterranei cell-free lysate. This suggested that kanosamine is isomerized to 3-amino-3-deoxy-D-fructose 6- 83 phosphate. Although no candidate gene encoding an isomerase has been found in the rif biosynthetic gene cluster, a “housekeeping” isomerase may be capable of converting kanosamine 6-phophate into 3-amino-3-deoxy-D-fructose 6-phosphate. O O HO (“NH HO [‘LNH O 9 9 ,k a 0 Q 9 ,Is b Hon. dig-[P-o-P-o ON 0 —> Hon. -IIO-?-O-?-O ON 0 —> ', OH OH ', OH OH HO OH O OH HO OH HO OH UDP-glucose 3-keto-UDP-glucose HO UDP 0 ATP ADP c . d H N ’OH HO NH 2 H203PO o O O I lg kanosamine 0 9 Ho... .iio-P-o-P-o ON O —— i_.. HOII- OH —» ', OH OH " HZN OH 17 HO HzN OH HO OH 9 O f kanosamine 6-phosphate VD HO'" "'P03H2— UMP HZN ’OH kanosamine 1-phosphate H203PO O R 0P03H2 HJY /\ H203PO . HO Hoi. 0 OH 0 OH ’, H203P0\/'\/c LY Ho--- -“ _. 002H H N :. 0 9H OH I COZH 2 ‘OH HO 'R P, HZN HzN aminoF6P S7P OH iminoE4P aminoDAHP AHBA Figure 36. The biosynthesis of precursors to the aminoshikimate pathway in A. mediterranei. Enzyme (encoding gene): (a) UDP-3-keto-D-glucose dehydrogenase (rifL); (b) UDP-3-keto-D-glucose transaminase (rifK); (c) UDP-kanosamine phosphatase (rifM); (d) kanosamine kinase (rifN); (e) UDP-kanosamine diphosphatase; (f) phosphokanosamine mutase; (g) phosphokanosamine isomerase; (h) transketolase (0rf15); (i) aminoDAHP synthase (rifl-I); (j) the aminoshikimate pathway. Abbreviations: aminoF6P, 3-amino-3-deoxy-D-fructose 6-phosphate; R5P, D-ribose 5-phosphate; S7P, D-sedoheptulose 7-phosphate; iminoE4P, l-imino-l—deoxy-D-erythrose 4-phosphate; aminoDAHP, 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate; AHBA, 3- amino-S-hydroxybenzoate. 84 Two pathways are possible for the biosynthesis of iminoE4P from UDP- kanosamine in A. mediterranei (Figure 36). Kanosamine 6-phosphate could be synthesized by kinase-catalyzed phosphorylation of kanosamine or derived from mutase- catalyzed phosphoryl group migration in kanosamine l-phosphate. Identification of Rifl\l as a specific kanosamine kinase adds credence to the first hypothetical pathway. There is currently no phosphokanosamine mutase candidate. Considering the ubiquitous presence of pyrophosphorylase and phosphatase activities, kanosamine 1-phosphate formation could be an artifact. However, it also could serve as an alternative route to kanosamine 6-phosphate. Incubation of 3-amino-3-deoxy-D-fructose 6-phosphate with D-ribose 5-phosphate in A. mediterranei cell-free lysate yields iminoE4P, which partly condenses with phosphoenolpyruvate to give aminoDAHP and is partly hydrolyzed to E4P to afford 3- deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP). In the presence of at least 20% glycerol, aminoDAHP becomes the sole product of the reaction without any detectable amount of DAHP formation. Complexation between Orf15 and RifH is ' identified by the bacterial two-hybrid experiments. The attendant channeling should result in a more efficient utilization of iminoE4P and minimize its hydrolysis. The fate of 3-amino-3-deoxy-D -fructose 6-phosphate in the in vitro bioconversions en route to aminoDAHP synthesis is an interesting question. Since the total recovered mass from the in vitro reaction is far less that 100% and 3-amino-3- deoxy-D—fructose 6-phosphate is considered as a stable compound, 3-amino-3-deoxy-D- fructose 6-phosphate must be converted into some unidentified material other than aminoDAHP and DAHP. Although the low mass balance is expected when A. 85 mediterranei cell-free lysate is used as it contains many other enzymes that might catalyze side reactions of 3-amino-3-deoxy-D-fructose 6-phosphate, less than 50% mass recovery can be accounted for when purified transketolase and DAHP (or aminoDAHP) synthase (entry 1, 2, Table 6) are employed. The total consumption of 3-amino-3-deoxy- D-fructose 6—phosphate indicates that it must be completely converted into iminoE4P. Based on the unstable nature of iminoE4P, it is likely that most of the mass is lost right after the formation of iminoE4P. Part of the iminoE4P condenses with phosphoenolpyruvate to form aminoDAHP. Part of the iminoE4P is hydrolyzed to E4P, which condense with phosphoenolpyruvate to yield DAHP. The rest of the iminoE4P may polymerize since iminoE4P cannot intramolecularly cyclize. Similarly, the E4P, derived from hydrolysis of iminoE4P, is also capable of polymerization. Preliminary experimental results show that kanosamine is biosynthesized from UDP-glucose via the intermediacy of UDP-3-keto-D—glucose and UDP-kanosamine. The kanosamine biosynthetic pathway is similar in A. mediterranéi as that in B. pumilus. In A. mediterranei, RifL and RifK were identified as UDP-3-keto-D-glucose dehydrogenase and UDP-3-keto-D-glucose transaminase, respectively. In B. pumilus, UDP-3-keto-D- glucose dehydrogenase activity was isolated and characterized. In vitro, the FAD- dependent UDP-3-keto-D-glucose dehydrogenase could use dichloroindophenol, phenazine methosulfate, or other artificial electron acceptors. In vivo, however, it requires a natural electron acceptor to recycle FAD. During the purification, an NAD- dependent enzyme assay could identify the activity of UDP-3-keto-D-glucose dehydrogenase at an early stage of the purification but this NAD-dependent activity was lost upon further purification. It is likely that UDP-3-keto-D-glucose dehydrogenase has 86 a close relationship with an NAD-dependent enzyme, which could serve as an electron acceptor. The other possible electron acceptor could be a certain cytochrome. In A. tumefaciens, 3-keto-glucoside dehydrogenase transfers electrons from its prosthetic FAD to cytochrome C552 exclusively.128 To examine whether UDP-3-keto-D- glucose dehydrogenase similarly utilizes cytochrome as its electron acceptor, cytochromes in B. pumilus could be isolated and tested for their ability to be reduced by direct acceptance of electrons from UDP-3-keto-D-glucose dehydrogenase. A bacterial two-hybrid system would be another choice to identify the electron acceptor, which, in theory, should form a complex with UDP-3—keto-D-glucose dehydrogenase. In conclusion, we have identified seven biosynthetic precursors to the aminoshikimate pathway including UDP-glucose, UDP-3-keto-D-glucose, UDP- kanosamine, kanosamine, kanosamine 6-phosphate, 3-amino-3-deoxy-D-fructose 6- phosphate, and iminoE4P and characterized four pathway enzymes including rifH- encoded aminoDAHP synthase, rifN-encoded kanosamine kinase, rifL-encoded UDP-3- keto-D-glucose dehydrogenase, and rifK-encoded transaminase. The long-standing puzzle associated with the absence of assayable aminoDAHP synthase activity and the source of the nitrogen atom for the aminoshikimate pathway has been solved. 87 JC I IiT -JL I 'r-‘TT f7 I T Figure 37. 1H NMR of chemically synthesized 3-amino-3-deoxy-D-fructose 6- phosphate. 88 40 r In TTT‘T'm-TT'T—l’] ‘r‘l Hfi‘TTT‘T-r‘l I‘T‘I ] TI ‘0 IIYH‘T‘TT‘T—T‘TTT'II IIITI 100 r r T—fiFW 130 I T 140 160 1.0 TITTITT—I—V'T—T, TWIITT Figure 38. 13C NMR of chemically synthesized 3-amino-3-deoxy-D-fructose 6- phosphate. 89 2."! EM ; is. in g a» @o E.— w 00 :0. —o :n Ii 1 .. Ito . E -w- 3% it in. .. :'t a P_ In i": 5:: g to M Euro RP DO 57% TITTTuTIITITTTTIVTIIIIIIT‘l—ITTIIIIITIT[IrIWI—ITITTITITTITII'IT l‘ O O O F. N M V“ I'D D O O O O O O O O l O N M n M V’ V’ V t V Q h v fi—TVP Figure 39. COSY of chemically synthesized 3-amino-3-deoxy-D-fructose 6- phosphate. 90 40 140 120 100 1'1 (m) 160 100 TTWTTTTTT‘IYTTTTTIIV[—TYT‘TTTTTIITYTIT—ITT—ITIIIIIIIIllIIYII‘ITIITTITITYYTITTTTITYIITT—IjTrrT—VTITITTTITITITTTT IIIIR‘YTTUITIITIIITUIIITIIITTTITTIT‘TTfT]TIIIITIT H 1' I! U B C O N “v Figure 40. HMQC of chemically synthesized 3-amino-3-deoxy-D-fructose 6- phosphate. 91 T l r—‘I I T I I 7 I fi—r'r‘rr‘fi—fr 1 Figure 41. 1H NMR of chemically synthesized kanosamine 6-phosphate. 92 20 {ITTYFTTTleTIrITTTTI 40 V‘TWTTrrTIIIT—TITTTTIITIIITTTTITITTITTYIYTYYX 1‘0 140 120 100 iTTr1TTTFTITTTIrTTIITITTTITTTTIY 1.0 TIYTTTI er, I Figure 42. 13C NMR of chemically synthesized kanosamine 6-phosphate. 93 ——+ “*— m . II'.‘ "It TITIITTIITTTIITTWTITITIIITITIITTTITIlIllfiITTTTTIr IIITIIIIIIT‘IIITIIYITTIIIIIIITIIIIT‘IIIIVTITTITITII Ed on in wt in u r~ o a U 0! II. Figure 43. COSY of chemically synthesized kanosamine 6-phosphate. 94 71 (PM) _ _ _ ~—o )- p- P I r- b— ‘ D- ’- LO cu . c _ _ _ h- h- 0 T? h h V I— F- O — - Ls .. E . >- ' T o. _ b— ‘ ”O _ i-. b v- _ __ A h o .- ICE 9 —g~v _ H I I! P __ II b p— P ,9 >—N ’- dd . r F. p- p to 73 p ._ _ _ p- .0 :0 .H _ P- y— - ,. _ to Ho ,. pd h II .. p- i- I- '8 fl IjjrlIIIIIIIITTITIYIITTII[TITTIYVTITTTTTITT’TWTYT N M Q‘ I! ’0 F O O A Figure 44. HMQC of chemically synthesized kanosamine 6-phosphate. 95 . -fi— Y . A AJLM _ -1441; 4 4 j I 7 T I I 1,—7— ‘Tuur- T'I'“ Y 4 T j r j I I I j 1 r j Figure 45. 1H NMR of 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7- phosphate (aminoDAHP). 96 40 100 120 140 160 1.0 inr‘rYIITTIIIIIIVIIIIIIITITTIIIIIYITYIYIIleIYIIIIIIIIIrYIIIIIIIYFIIIIIVIIYWYIFUIIIIIIIIT‘IIIIIITIIIII] Figure 46. 13C NMR of 4-aminc-3,4-dideoxy-D-arabino-heptulosonic acid 7- phosphate (aminoDAHP). 97 4 F1 (M) h YIIIYTfiIYIII‘IIIIIIIIII[VIII]IIIIIIYTIIIYIIITITTII IIIIIIYYTYIYIY‘IITIIFTYY—[jTjTlIIIIIIIIIIIYF V In D 'I‘ O G Trflu EH N n N h v FT Figure 47. COSY of 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP). 98 p- r I J, Lo I 1 Z I; L- » L' i ,_g { I i I r f p. _____j : O F w J, b- r L y- 1 P— L b- 1 O F’ o P'— r - U .___i . : f f —-——-4 o b 2—8 1 '- A 1 r— _4 : L— ’_ 8 v y.— C H "' E II. 1 D- » :O 1 ~53 p- ] i L _ P C o . _‘_ _ W p H *— It '_'_ 1 S f _ O r E.“ _ v-I )- 1 r _1 h E o I :0 r ,_ H y- ‘t f 4 t _ O ’ III!IIIITfiIIIYYIIITI‘TITIrIITYTTYITIYTTYTYYYIITY a Ennnvnwhom V N h Figure 48. HMQC of 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP). 99 Figure 49. 1H NMR of kanosamine l-phosphate. 100 LALMMM L a Y Tfi—V'Tfi 1 T 1 I T _ THY" ‘7"_T‘TTT7I“ Figure 50. 1”C NMR of kanosamine l-phosphate. 101 Yty'ti1lvvvr] irYTTYI VV—TTVUIIUrIrIII—YIIIYWITTTTI]Vrli‘fiTTTf‘TIIIYTIWIYYTYYIYj'WITrrfiTTf [7YTYIYYIYITVT1’T7Y 40 100 120 140 160 1.0 Ll. Figure 51. 1H NMR of kanosamine. 102 Figure 52. 13C NMR of kanosamine. 103 IleIU'ITTIWTIIITTYIYIIIIYrTTIIIlI'TIYITII'Y‘TIIIIUYITIUUTYIITTIIW'YIIIUITIIIIIIIIIIUIIIIIITIIYII‘IIIIIII] 40 100 120 140 160 100 CHAPTER THREE IN VIVO ELABORATION OF THE AMINOSHIKIMATE PATHWAY AND BIOCATALYTIC SYNTHESIS OF S-AMINO-S-DEOXYSHIKIMATE Introduction l-Imino—l-deoxy-D-erythrose 4-phosphate (iminoE4P), 4-amino-3,4-dideoxy-D- arabino-heptulosonic acid 7-phosphate (aminoDAHP), 5-amino-5-deoxy-3- dehydroquinic acid (aminoDHQ), and 5-amino-5-deoxy-3-dehydroshikimate (aminoDHS) have been proposed as intermediates of the aminoshikimate pathway.76 Floss synthesized and demonstrated that aminoDAHP, aminoDHQ, and aminoDHS could be converted into 3-amino-5-hydroxybenzoate (AHBA) in cell-free lysate of rifamycin B producer A. mediterranei.763 In the previous chapter, kanosamine, kanosamine 6- phosphate, 3-amino-3-deoxy-D-fructose 6-phosphate (aminoF6P), and iminoE4P were similarly confirmed as precursors to the aminoshikimate pathway. With respect to the aminoshikimate pathway enzymes, the rifH gene product was suggested to be an aminoDAHP synthase with the ability to catalyze transamination of E4P as a small amount of aminoDAHP could be detected when E4P and 76a phosphoenolpyruvate were incubated with glutamine in presence of RifH. Our observations (see Chapter 2) confirm that RifH is an aminoDAHP synthase. However, it did not possess aminotransferase activity. IminoE4P, one of the substrates of rin- encoded aminoDAHP synthase, was biosynthesized via transketolase-catalyzed ketol transfer from aminoF6P.129 Recently, the catalytic activities of some other enzymes that 104 catalyze the formation of precursors to the aminoshikimate pathway were investigated by both the Floss127 and the Frost groups.130 Based on observations obtained from in vitro bioconversions, RifL, RifK, and RifN have been confirmed to be UDP-3-keto-D-glucose dehydrogenase, UDP-3-keto-D-glucose transaminase (also a 3-amino-5-hydroxybenzoate synthase), and kanosamine kinase, respectively. COZH A pl HO,“ COZH p. H04 COZH H2O PEP _2:_, 0 _A_. _L. OH a i NH2 b 0 g NH2 C H203PO\/'\_ANH H203PO OH OH 6H aminoDAHP aminoDHQ iminoE4P COZH CO H d —_’__, rifamycinB 2 HO NH2 AHBA o ; NH2 aminoDHS 8 HOW ; NH2 OH aminoSA Figure 53. The aminoshikimate pathway and biocatalytic synthesis of S-amino-S- deoxyshikimate. (a) rifH—encoded aminoDAHP synthase; (b) rifC-encoded S-amino-S- deoxy-3-dehydroquinate synthase or aroB-encoded 3-dehydroquinate synthase; (c) rifJ- encoded 5-amino-5-deoxy-3-dehydroquinate dehydratase or aroD-encoded 3- dehydroquinate dehydratase; (d) rifK-encoded 3-amino-5-hydroxybenzoate synthase; (e) rifl-encoded aminoshikimate dehydrogenase or aroE-encoded shikimate dehydrogenase. Abbreviations: iminoE4P, l-imino-l-deoxy-D-erythrose 4-phosphate; PEP, phosphoenolpyruvate; aminoDAHP, 4-amino-3,4-dideoxy-D-arabino-heptulosonic acid 7-phosphate; aminoDHQ, 5-amino-5-deoxy-3-dehydroquinate; aminoDHS, S-amino-S- deoxy-3-dehydroshikimate; aminoSA, 5-amino-5-deoxyshikimate. As stated in Chapter 1, our efforts towards elaboration of the aminoshikimate pathway focused on both elucidation of the biosynthetic pathway and exploiting the 105 pathway for biocatalytic synthesis of intermediates or derivatives of this pathway, which could be used as chiral sythons or applied in the discovery and practical synthesis of potent drugs. Development of a process to make 5-amino-5-deoxyshikimate (Figure 53 and Figure 54), a derivative of the aminoshikimate pathway, will be discussed in this chapter. It could potentially be used as starting material. to achieve an azide-free synthesis of Tamiflu (Figure 54), an antiinflunza drug marketed by Roche. Biocatalytic synthesis of S-amino-S-deoxyshikimate was examined by using genetically modified A. mediterranei and E. coli. COZH co?_CH3 COZH COZCHa a <5 6 f ————> ——-> ——-> Ho“ . OH HO“ ,: M0M0“ . N3 Mon/Io“ .,; 6H 0 5,; NH . . . . R = H R = H shlklmuc acnd c( R = MOM e( R = Ms ll 0 O COZH . ‘/ Glucose ———g——> a ”n?.* i \I \v 7 Ho“‘ . NH2 0 NH2 OH Han/ 5-amino-5-deoxyshikimate , Tamiflu o Figure 54. Synthesis of Tamiflu. Key: (a) MeOH, H“; (b) Ph3P, CH302CN=NC02CH3, THF, 0°C to LL; (0) MeOCHZCl, DIPEA, CHZClz; (d) NaN3, NH4C1, MeOH/HZO; (e) MeSOZCI, Et3N, CH2C12; (f) PH3P, THF then Et3N/H20; (g) microbial synthesis. 106 Biocatalytic Synthesis of 5-Amino-5-deoxyshikimate by Recombinant A. mediterranei Overview Conversion of 5-amino-5-deoxy-3-dehydroshikimate (aminoDHS), which is the last step in the aminoshikimate pathway, into 3-amino-5-hydroxybenzoate catalyzed by rsz-encoded 3-amino-5-hydroxybenzoate synthase. If an appropriate dehydrogenase was expressed in A. mediterranei, aminoDHS could be captured and converted into 5- amino-S-deoxyshikimate (Figure 53). Interestingly, a gene in the rif biosynthetic gene cluster, designated rifl, encodes an enzyme possessing homology with a shikimate dehydrogenase. This rifI gene product was proposed to catalyze the reduction of aminoDHS to S-amino-S-deoxyshikimate and was therefore designated as an aminoshikimate dehydrogenase.131 The functional role of RifI during rifamycin biosynthesis was not clear since its disruption had no effect on rifamycin B synthesis.79 Considering its ability to convert aminoDHS to 5-amino-5-deoxyshikimate, Rifl was suggested to serve as a regulator to prevent uncontrolled accumulation of 3-amino-5- hydroxybenzoate in the cell.79 Although no 5-amino-5-deoxyshikimate has ever been detected in the cultures of wild type A. mediterranez', overexpression of rifl-encoded aminoshikimate dehydrogenase in A. mediterranei might compete with rifK-encoded 3- amino-5-hydroxybenzoate synthase and convert a significant amount of aminoDHS into S-amino-S-deoxyshikimate. 107 pRL-GO 1) PCR 2) Sphl/Xbal digest i Sphl Xbal Pamy 0.3-kb Xbal 0.8-kb FKN108 1) PCR 2) Xbal/EcoRl digest EcoRl rifl 1) Sphl/EcoRl digest 2) CIAP treatment ligation Sphl Xbal EcoRl Smal BamHl Pstl pJGB 155 Hindlll 5.2-kb pBR§22 Figure 55. Preparation of Plasmid pJG8.155. 108 pJG8.155 1) PCR 2) Mlul digest J, Mlul Mlul Pamy, rifl 1.1-kb 1) Mlul digest 2) CIAP treatment ligation Figure 56. Preparation of Plasmid pJG8.219A. 109 Plasmid construction Although A. mediterranei belongs to the same order (Actinomycetes) to which Streptomyces belongs, molecular biological manipulation of A. mediterranei is still in its infancy.132 This is mainly due to the lack of a suitable plasmid with a selection marker gene for vector development in A. mediterranei. Attempts have been made to develop . . . . 133 surtable cloning vectors for A. medzterranel. However, only a few of them have ever met with any success. For this study, cloning vector pRL-60 (Figure 56)132 was employed. Plasmid pRL-60 is a shuttle vector that contains two origins of replication. One origin of replication is derived from pA387sz-rep, which functions in A. mediterranei. The other origin of replication is derived from pBR3221pBR-ori and functions in E. coli. Plasmid pRL-6O also carries three marker genes encoding resistance to kanamycin and neomycin (kanR/NeoR), resistance to erythromycin (ermE), and the a-amylase gene (amy). Resistance to kanamycin and neomycin functions effectively in E. coli. The remaining two markers encoding resistance to erythromycin and a-amylase work in A. mediterranei. The overexpression of a-amylase enables A. mediterranei to hydrolyze starch. A major disadvantage of using pRL-6O as a protein expression vector is that it does not provide multiple cloning sites, promoter, or a ribosomal binding site. In order to use this vector for protein expression, an A. mediterranei recognizable ribosomal binding site and promoter are required for transcription and translation. Since a-amylase is already expressed in A. mediterranei, its ribosomal binding site and promoter are not of concern. 110 The plasmid preparation was initiated with PCR amplification (Figure 55) of the (at-amylase promoter and ribosomal binding site (PM) from plasmid pRL60. The open reading frame of rifl gene was also PCR-amplified (Figure 55) from cosmid FKN108,134 which contains part of the rz'f biosynthetic gene cluster. These two fragments were then ligated and inserted (Figure 55) into vector pKK223-3 to give a new plasmid pJG8.155 (Pmrifl, Amp“). The resulting Pamgifl was subsequently PCR-amplified and ligated (Figure 56) into Mlul site of vector pRL-6O to yield plasmid pJGS.219A (Pamyrifl, ermE, amy, KanR/NeoR), which would be suitable for Rifl overexpression in A. mediterranei. Transformation (electroporation) of A. mediterranei with pJG8.219A With an expression vector for rifl-encoded aminoshikimate dehydrogenase in hand, the next step was to develop a transformation method. Standard heat shock and conjugation procedures used in Streptomyces sp. are not applicable to A. mediterranei.135 Electroporation has been reported to be the only general transformation method for A. mediterranei.‘32 However, electroporation conditions needed to be optimized for each strain of A. mediterranei in order to obtain reasonable transformation efficiencies. The best conditions developed in our lab for A. mediterranei (ATCC 21789) were: field strength of 7.5 kV/cm, resistance of 600 S2, and capacitance of 25 HF. Under such electroporation conditions, a transformation efficiency of 1x103 transformants/ug DNA was routinely obtained. The preparation of electro-competent cells followed literature procedures‘32 with a few modifications. After being harvested by centrifugation, the mycelia were washed with sterilized water (pre-chilled on ice) and collected by centrifugation. After removing 111 the supernatant, the mycelia were resuspended in 10% glycerol containing 4 ug/mL lysozyme and incubated for 20 min at 25 °C. The mycelia were then harvested by centrifugation. Collected cells were washed with 10 mL 10% glycerol, harvested, and resuspended in 4 mL of 10% glycerol. This cell suspension was then electroporated (BioRad Gene Pulser) with plasmid pJGS.219 at field strength of 7.5 kV/cm, resistance of 600 Q, and capacitance of 25 uF. Electroporated cells were plated on YM medium and incubated overnight. The YM agar plates were then overlaid with 2.5 ml of YM soft agar containing erythromycin and followed by incubation at 28°C until colonies appeared. The erythromycin-resistance colonies from the initial selection were subjected to a second round of screen by using on- amylase as the marker. Batch fermentor conditions Fermentations were conducted in a B. Braun M2 culture vessel with a 2 L working capacity. Environmental conditions were supplied by a B. Braun Biostat MD controlled by a DCU. Data was acquired on a Dell Optiplex GX200 personal computer utilizing B. Braun MFCS/Win software. PID control loops were used to control temperature, pH, and glucose addition. The temperature was maintained at 28 °C and the pH was maintained at 7.0 by addition of NH4OH or 2 N H2304. Dissolved oxygen (D.O.) was monitored using a Mettler-Toledo 12 mm sterilizable O2 sensor fitted with and lngold A-type O2 permeable membrane. D.O. was maintained at 10% air saturation throughout the course of the fermentations. Antifoam (Sigma 204) was manually pumped into the vessel as needed. 112 Fermentation inoculants were initiated by introduction of a single colony into 100 mL of YMG medium and grown at 28°C with agitation for 72 to 168 h until an appropriate OD600 was reached (1.2-2.0). The 100 mL culture was then transferred to the fermentation vessel. The DC. levels were controlled at 10% air saturation during the course of the run. With the airflow at an initial setting of 0.06 L/L/min, the DD. concentration was maintained by increasing the impeller speed from its initial set point of 200 rpm to its preset maximum of 1100 rpm. With the impeller rate constant at 1100 rpm, the mass flow controller then maintained the DD. concentration by increasing the airflow rate from 0.06 L/L/min until a maximum airflow rate was reached (usually less than 0.2 L/L/min). Afterwards, airflow was maintained at 0.2 L/L/min, and the impeller was allowed to vary in order to maintain the DO. concentration at 10% air saturation. The impeller speed typically varied from 200 to 1000 rpm during the remainder of the run. Overexpression of RifI in A. mediterranei After transformation, the aminoshikimate synthesizing capability of A. mediterranei/pJG8219A was examined under batch fermentor conditions at 28°C and pH 7.0. After 12 days of cultivation, A. mediterranei/pJG8219A synthesized 0.2 g/L of aminoshikimate and 1.4 g/L of rifamycin B. The concentration of aminoshikimate was determined by 1H NMR and the concentration of rifamycin B was determined by spectrophotometry.136 The average specific activity of aminoshikimate dehydrogenase during A. mediterranei/pJGS219A fermentation was 0.2 unit/mg. A. mediterranei/pRL- 60 was examined under identical batch fermentor conditions. Accumulations of 1.5 g/L 113 of rifamycin B was observed but no aminoshikimate accumulation could be detected. The average specific activity of aminoshikimate dehydrogenase during A. mediterranei/pRL-60 fermentation was 0.08 unit/mg. The low aminoshikimate synthesis was disappointing relative to our goal of biocatalytic synthesis of aminoshikimate. It might result from the competition for aminoDHS between rifK-encoded 3-amino-5-hydroxybenzoate synthase and rifl-encoded aminoshikimate dehydrogenase. Under an ideal situation, overexpression of rifl-encoded aminoshikimate dehydrogenase in a RifK mutant strain would give a higher yield of aminoshikimate. However, the rifK gene product has been proposed to catalyze transamination of UDP-3-keto-D-glucose during kanosamine biosynthesis. A problematic exercise would be anticipated involving elimination of the RifK 3-amino—5- hydroxybenzoate synthase activity while leaving RifK transaminase activity. The best scenario for us would be if RifK was not the only transaminase in A. mediterranei that possessed UDP-3—keto-D-glucose transaminase activity. To examine this possibility, A. mediterranei RifK mutant (HGF014)137 was cultured under batch fermentor conditions and the fermentation products were subsequently analyzed. AminoDHS is converted to 3-amino-5-hydroxybenzoate in the last step of the aminoshikimate pathway, in a reaction catalyzed by rifK-encoded 3- amino-S-hydroxybenzoate synthase (Figure 53). If RifK is not the only transaminase, aminoDHS or other aminoshikimate pathway intermediates would be accumulate in the culture supernatant of A. mediterranei HGF014. If RifK is the only transaminase, the aminoshikimate pathway will be robbed of iminoE4P due to the absence of kanosamine biosynthesis attendant with loss of UDP-kanosamine formation. Therefore, no 114 aminoshikimate pathway intermediates or rifamycin B would be expected to accumulate in the culture supernatant. After 7 days of cultivation, A. mediterranei HGF014 did not synthesize any detectable amount of aminoshikimate pathway intermediates or rifamycin B. The only product detected in the culture supernatant was gluconic acid. This result was consistent with RifK being the only transaminase for conversion of UDP-3-keto-D- glucose into UDP-kanosamine. Biocatalytic Synthesis of Aminoshikimate By Recombinant E. coli. Overview In contrast to A. mediterranei, E. coli does not possess the aminoshikimate pathway. However, UDP-glucose is a biosynthetic metabolite in E. coli. It is an essential 138 intermediate in the biosynthesis of membrane-derived oligosaccharides of E. coli. It also serves as biosynthetic precursor to trehalose, which has been proposed to increase both the heat and the osmotic stress tolerance of E. coli cells.139 The presence of UDP- glucose in E. coli raises the possibility that aminoshikimate can be synthesized by E. coli if the aminoshikimate pathway enzymes are heterologously overexpressed in E. coli (Figure 57). The advantage of using E. coli as the host to synthesize aminoshikimate lies in the relative ease with which this microbe can be cultured and genetically manipulated. The disadvantage of using E. coli as host to construct an aminoshikimate producer is associated with heterologous protein expression. Since E. coli is a gram-negative 115 bacteria and A. mediterranei is a GC-rich gram-positive bacteria, A. mediterranei proteins are typically not expressed well in E. coli. HO HO HO HO 0 a o b o C o HOI-- «0H —’—» HOI" "IOUDP —> HOI" "IOUDP —> HOn- «ouop ’OH HO ’OH 0 ’OH ' HZN ’OH HO glucose UDP-glucose 3-keto-UDP-glucose UDP-kanosamine H 0 PO HO H203PO 2 3 d O O f HOII. f HObOH —e—> HobOHa OOH 4* H203P0\/'\/\NH UDP HZN ’OH HZN ’OH H2“ 1 OH kanosamine K6P aminoF6P iminoE4P 5: £1 £1 COW—l2. gum, NH2 Ho‘ NH; NADP aminoDAHP aminoDHQ aminoDHS aminoshikimic acid Figure 57. Biocatalytic synthesis of aminoshikimate in E. coli. (a) native E. coli UDP-glucose biosynthesis; (b) rifL-encoded UDP-3-keto-D-glucose dehydrgenase; (c) rifK-encoded UDP-3-keto-D-glucose transaminase; (d) rifM-encoded UDP-kanosamine phosphatase or native E. coli phosphatase; (e) rifN-encoded kanosamine kinase or native E. coli phosphoglucokinase; (f) isomerase; (g) 0rf15- encoded transketolase or tktA-encoded transketolase; (h) rifH-encoded aminoDAHP synthase; (i) rifC-encoded aminoDHQ synthase or aroB-encoded 3-dehydroquinate synthase; (j) rifl-encoded aminoDHQ dehydratase or aroD-encoded 3-dehydroquinate dehydratase; (k) rifl-encoded aminoshikimate dehydrogenase or aroE-encoded shikimate dehydrogenase. Biocatalytic synthesis of aminoshikimate from glucose Overview Biocatalytic synthesis of aminoshikimate from glucose in E. coli requires heterologous expression of rifl-encoded UDP-3-keto-D-glucose dehydrgenase, rifK- encoded UDP-3-keto-D-glucos transaminase, rifM-encoded UDP-kanosamine phosphatase, rifN-encoded kanosamine kinase, rifH-encoded aminoDAHP synthase, rifG- 116 encoded aminoDHQ synthase, rifl-encoded aminoDHQ dehydratase, and 0rf15-encoded transketolase (Figure 57). Given that E. coli enzymes tktA-encoded transketolase, aroB- encoded 3-dehydroquinate synthase, and aroD-encoded 3-dehydroquinate dehydratase can catalyze reactions on the amino counterparts (aminoF6P, aminoDAHP, and aminoDHQ) of their native substrates (F6P, DAHP and 3-dehydroquinate), heterologous overexpression of 0rf15-encodedtransketolase, rifC—encoded aminoDHQ synthase, and rifJ-encoded aminoDHQ dehydratase in E. coli are therefore not necessary for aminoshikimate synthesis. Host Strain E. coli SPl.1 was used as host strain for aminoshikimate synthesis. The SP1.1 host strain was derived from RB791 and lacks catalytically active shikimate kinase. It had been previously prepared in the laboratory and used for synthesis of shikimate.45 The absence of shikimate kinase activity in SP1.1 precludes the possibility of phosphorylation 45a, b of aminoshikimate. Construction of SP1.1 began with the homologous recombination of the aroB gene into the serA locus of E. coli RB791 resulting in RB791 serAzzaroB. RB791 serA::aroB was then subjected to two successive P1 phage-mediated transductions to transfer the aroL478zzTn10 and aroKzszR loci of AL0807140 onto the genome and eliminate shikimate kinase activity. Since inactivation of the shikimate kinases precluded de novo biosynthesis of aromatic amino acids and aromatic vitamins, growth of all constructs required supplementation with L-phenylalanine, L-tyrosine, L- tryptophan, p-hydroxybenzoic acid, p-aminobenzoic acid, and 2,3-dihydroxybenzoic acid. 117 Plasmid construction In order to synthesize aminoshikimate in E. coli, plasmid pJG7.071A (Figure 64) was constructed. It carried a rifKrifL cassette, a rierifN cassette, rifl-1, and aroE each under the control of a tac promoter as well as serA and tktA under the control of their native promoters. The combination of rifL-encoded UDP-3-keto-D-glucose dehydrogenase and rifK-encoded UDP-3-keto-D-glucose transaminase was to catalyze the conversion of UDP-glucose into UDP-kanosamine. Tandem activities of rifM—encoded phosphatase and rifN-encoded kanosamine kinase were to mediate the synthesis of kanosamine 6-phosphate from UDP-kanosamine. Our anticipation was that the native E. coli phosphoglucose isomerase would convert kanosamine 6-phosphate into aminoF6P followed by fragmentation of aminoF6P to form imino4P catalyzed by native tktA- encoded transketolase. Condensation of this iminoE4P with phosphoenolpyruvate to form aminoDAHP was to be catalyzed by rifl-I-encoded aminoDAHP synthase. The native E. coli shikimate pathway enzymes AroB, AroD, and AroE (Figure 57) were then to convert aminoDAHP into aminoshikimate. Localization of native tktA-encoded transketolase and aroE-encoded shikimate dehydrogenase on both the genome and the plasmid was deemed necessary to ensure the elevated specific activities required for conversion of aminoF6P into iminoE4P and for conversion of aminoDHS into aminoshikimate. The serA gene encodes 3-phosphoglycerate dehydrogenase, which is necessary for biosynthesis of L-serine in E. coli. Host strain SPl.1 possesses a mutated genomic copy of serA and is therefore incapable of growth in minimal salts medium lacking L- serine supplementation. However, inclusion of a functional serA gene on each plasmid 118 restores the ability of the cell to synthesize L-serine, allowing growth of the catalyst in minimal salts medium only if the plasmid was successfully maintained by the cell. This . . . . 141 strategy has been used extenswely as an effective means for plasmid maintenance. FKN108 1) PCR 2) EcoFlI digest pKK223-3 4.6-kb Ii Scal Scal pBR322 rifK rifL or, ,. 2.3-Rb 1) 1'5le digest 2) Klenow treatment 3) CIAP treatment ligation (EOORI) (EcoRl) Smal BamHl Pstl Hindlll pJGG.154A 6.9-kb pBR322 . ori h Figure 58. Preparation of Plasmid pJG6.154A. 119 (Bglll) pJG6.154A NCO] pJG5.166A Sphl/Hindlll digest ' 1 1,5-kb Ncol Sphl Hindlll 1) Ncol digest Mung bean nuclease 2) Klenow treatment treatment 3) CIAP treatment ligation (Bglll) pJG6.1553 13.2-kb Figure 59. Preparation of Plasmid pJG6.155B. 120 (Bglll) pRM30 1) Xbal/Hindlll ' pJGG.155B digest __ 13.2-kb v thaal Hindlll rifL 1 .5-kb 1) Xhol digest Klenow treatment 2) Klenow treatment 3) CIAP treatment ligation (Bglll) pJG7.032A 14.1-kb Figure 60. Preparation of Plasmid pJG7.032A. 121 FKN108 1) PCR 2 Ec RI d' est ) ° '9 pKK223-3 v 4.6-kb EcoRI E RI 0° penszz rifM 0.8-kb 1) EcoRl digest 2) CIAP treatment ligation EcoRl Smal BamHI ' Pstl Hindlll pJG7.039A 5.4-kb pBR322 .. ori Figure 61. Preparation of Plasmid pJG7.039A. 122 FKN108 1) PCR 2) 15le digest EcoRl EcoRl rifN 0.9-kb 1) EcoRl digest 2) CIAP treatment ligation pJG7.046A 5.5-kb pBR322 ori Figure 62. Preparation of Plasmid pJ G7 .046A. 123 EcoRl pJG7.046 Smal BamHl Pstl Ss ldi est ' p g pJG7.039A Hmdm 5.4-kb Sspl Sspl rifN 1.5-kb 1) Smal digest 2) CIAP treatment ligation pJG7.056A 6.9-kb Hindlll pBFl322 I“ 011 Figure 63. Preparation of Plasmid pJG7.056A. 124 (Bglll) pJG7.056 Sphl/Hindll pJG7.032A digest ‘ _ 14.1 -kb it Sphl Hindlll Pm rifM rifN 2.3-kb 1) Ncol digest Mung bean nuclease 2) Klenow treatment treatment 3) CIAP treatment ligation (Bglll) pJG7.071A 16.4-kb Figure 64. Preparation of Plasmid pJG7.071A. 125 The construction of pJG7.07 1A was initiated by the preparation of plasmid pJG6.155B (Figure 59). The rifK and rifL loci were PCR-amplified from cosmid FKN108]34 and inserted (Figure 58) into vector pKK223-3 to give plasmid pJG6.154A (PmrifKrifL, Amp“). The resulting PmrifKrifL DNA fragment was then excised from plasmid pJG6.154A and inserted (Figure 59) into pJG5.166A (Pmrifli, PmaroE, serA, tktA, Amp“; see later part of this chapter for the detailed construction of pJG5.166A), to afford plasmid pJG6.155B (P,m.rz'j‘H, P,m.ar0E, serA, tktA, P,anfKrz'fL). At this stage, a communication from the Floss group revealed that E. coli could not recognize A. mediterranei ribosomal binding sites. Native E. coli ribosomal binging sites were therefore required for overexpression of RifK and RifL. In plasmid pJG6.155B, rifK gene was cloned behind native E. coli ribosomal binding site and rifL was still behind A. mediterranei ribosomal binding site. To solve this problem, the rifL gene with an E. coli 142 and ribosomal binding site was excised out from plasmid pRM030 (pT7, rifL, Amp“) cloned (Figure 60) into plasmid pJG6.155B to give a new plasmid pJG7.032A (Pmrer, PmaroE, serA, tktA, PmcrifKrifL). The next construction step was to add rifM and rifN genes into plasmid pJG7.032A. Following the construction of the PmrifKrifL cassette, rifM and rifN were attached to E. coli recognizable ribosomal binding sites, respectively, before their insertions into plasmid pJG7.032A. The preparations began with PCR amplifications of rifM and rifN genes from cosmid FKN108. The rifM (Figure 61) and rifN (Figure 62) were then cloned into pKK223-3 respectively to afford pJG7.039A (PmrifM, Amp“) and pJG7.046A (PmriflV, Amp“). The rifN gene with E. coli recognizable ribosomal binding site was subsequently excised out of plasmid pJG7.046A and inserted (Figure 63) into 126 pJG7.039A to afford plasmid pJG7.056A (PmrierifiV, Amp“). The rifN locus with E. coli ribosomal binding site was oriented such that transcription was from the same tac promoter, which was placed in front of rsz locus. The resulting PmrierifN cassette was then excised out of plasmid pJG7.056A and ligated into pJG7.032A to yield (Figure 64) the desired plasmid pJG7.07lA (PmrifH, PmaroE, serA, tktA, P ,acrszrifL, P,m.rierifN). The orientation of the P,m.rierifN cassette was in the opposite direction as that of P,m.rifKrij‘L cassette. Fed-batch fermentor conditions The fed-batch fermentor equipment was the same as that described in the early section of this Chapter (page 104). Fermentations were run at 33 °C, pH 7.0, and the dissolved oxygen (D.O.) level was maintained at 10%. The initial glucose concentration was 25 g/L and pH maintained at 7.0 by addition of NH4OH or 2 N H2804. Antifoam (Sigma 204) was manually pumped into the vessel as needed. Fermentation inoculants were initiated by introduction of a single colony into 5 ml. of M9 medium and grown at 37 °C with agitation for 20 to 24 h until the culture was turbid. This starter culture was subsequently transferred to 100 mL of M9 medium and grown for an additional 10 to 12 h at 37 °C and 250 rpm. After an appropriate ODwO was reached (2.0-3.0), the inoculant was transferred to the fermentation vessel. The fed-batch fermentation carried out in this section of study was run under glucose-limited conditions. The initial glucose concentration in the fermentation medium was 25 g/L and a steady state concentration of less than 1 g/L of glucose was maintained after the preset maximum of air flow (1.0 L/L/min) and impeller speed (1 100 rpm) were reached. The 127 entire fermentation can be divided into three stages, each of which corresponds to a different procedure for controlling the DO. level at 10%. In the first stage, D.O. concentration was maintained by increasing the impeller speed from its initial set point of 50 rpm to its preset maximum of 1100 rpm while the airflow was maintained at an initial setting of 0.06 L/L/min. In the second stage, the mass flow controller maintained D.O. levels by increasing the airflow rate from 0.06 L/L/min to a preset maximum of 1.0 L/L/min with the impeller constant at 1100 rpm. In the third stage, D.O. levels were finally maintained at 10% air saturation for the remainder of the fermentation by oxygen sensor-controlled glucose feeding under constant impeller speed and constant airflow rate. Fermentation of E. coliSP1.1/pJG7.071A After transformation, E. coli SP1.l/pJG7.07lA was examined under glucose- limited fed-batch fermentor conditions at 33°C, pH 7.0, with dissolved oxygen maintained at a set point of 10% air saturation. L-Phenylalanine, L-tyrosine, and L- tryptophan were added to the culture medium at the beginning of the fermentor run as required for cell growth and at 16 and 30 h to inhibit native E. coli DAHP synthase activity. After 48 h of cultivation, no detectable amount of aminoshikimate and 1.8 g/L of shikimate were synthesized (Figure 65). The results were disappointing and might be explained if native E. coli phosphoglucose isomerase was incapable of catalyzing the isomerization between aminoF6P and kanosamine 6-phosphate. However, this possibility can be eliminated since experiments have shown that kanosamine can be converted into aminoshikimate in 128 E. coli without overexpression of a dedicated A. mediterranei phosphokanosamine isomerase (see the results presented in the later part of this Chapter). As an alternative explanation, iminoE4P may indeed be formed in the fermentation but subsequently hydrolyzed to E4P. Conversion into DAHP might then be catalyzed by incompletely inhibited native E. coli DAHP synthases or rifl-I-encoded aminoDAHP synthase, which also possessed DAHP synthase activity.81 This would explain the lack of aminoshikimate and the presence of shikimate in the fermentation. 4 40 < 3 "303 ‘é’A TE» gee ' . ' - if; ' “'5’ 1« . l’l . --10 g 0"—'.T n l r“i I 0 30 36 In In I 0 12 18 24 time (h) 42 48 Figure 65. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG7.071A under glucose-limited conditions. Legend: shikimate (SA), open columns; dry cell weight, closed circles. In a recent paper,I43 Khosla and coworkers demonstrated the formation of 3- amino-S-hydroxybenzoate (3.1 mg/L) in E. coli when a hybrid aminoshikimate pathway was assembled from five A. mediterranei genes and two Actinosynnema pretiosum genes. Genes from A. mediterranei included rifH, rifK, rifL, rifM, and rifN. Genes from A. pretiosum included asm4 7 and asm23. Asm47 and Asm23, which are homologous to RifG and RifJ respectively, were used simply because RifG and RifJ were poorly 129 expressed in E. coli. Our aminoshikimate biosynthetic genes contained rifH, rifK, rifL, rifM, rifN, tktA, and aroE. AroE-encoded shikimate dehydrogenase was designed to convert aminoDHS to aminoshikimate in a competition with rifK—encoded 3-amino-5- hydroxybenzoate synthase. Its overexpression should not be responsible for the absence of both aminoshikimate and 3-amino-S-hydroxybenzoate. TktA-encoded transketolase catalyzed the fragmentation of aminoF6P to form iminoE4P. Its overexpression could not account for the formation of 3-amino-5-hydroxybenzoate in Khosla’s construct and the absence of aminoshikimate in our construct as well. RifG (or Asm47) and RifJ (or Asm23) were not overexpressed in our construct since native E. coli AroB and AroD could be substituted for RifG (or Asm47) and RifJ (or Asm23), respectively.76a‘ '44 Although AroB and AroD are not overexpressed, the basal expression of AroB and AroD in E. coli was expected to lead to enough enzyme activities to convert aminoDAHP into aminoDHS. Carefully studying Khosla’s paper revealed that they used a very unique culture condition for 3-amino-5—hydroxybenzoate synthesis in E. coli. Cultivation at a very low temperature (13°C) was required to adequately express all of the aminoshikimate 43 . . . . . . . . pathway enzymes.l Thls lS conSIStent With our speculation that aminoshiklmate pathway enzymes were not adequately expressed in E. coli at 33°C. Due to our inability to run our fermentor at 13°C, we have not examined aminoshikimate synthesis by E. coli SP1 . l/pJG7.07 l A at low temperatures. 130 Biocatalytic synthesis of aminoshikimate from glucose via intermediacy of kanosamine Overview While the ultimate goal is to produce aminoshikimate from glucose and ammonium ion by a one-step microbial synthesis, a useful intermediate goal entails synthesis of aminoshikimate by a two-step biosynthesis via an intermediacy of a readily accessible material (Figure 66). Kanosamine, which can be obtained in a substantial quantity by culturing B. pumilus (ATCC 21143), was chosen as intermediate to bridge the two-step biosynthesis (Figure 66). The advantage of the two—step biosynthesis lies in that it could reduce the total number of A. mediterranei proteins required for aminoshikimate synthesis in E. coli. HO V HO 1 COzH O I 0 11 HO!" "IOH —> HOI" OH —’ . .’ '/ HO“ ; NHZ HO OH H2N OH 6H glucose [ kanosamine aminoshikimate H203PO H203PO O 0 OH HO". OH HO'“ ,\\ .’ COzH HZN OH HzN K6P aminoDAHP b dIPEP 6 OH HOn. O —> H203PONNH 3 OH OH H2N "OH aminoF6P iminoE4P Figure 66. Two-step biosynthesis of aminoshikimate from glucose. Key: (1) B. pumilus; (II) recombinant E. coli; (a) kinase; (b) isomerase; (c) transketolase; (d) aminoDAHP synthase; (e) native E. coli shikimate pathway. 131 In this section, biosynthesis of kanosamine by B. pumilus fermentation will be discussed. Several stains of E. coli were developed and examined for their ability to synthesize aminoshikimate from kanosamine under fed-batch fermentor conditions. Based on fermentation results, a few questions associated with the aminoshikimate pathway will be discussed. sample to beidenfified catalase test + _ other strains Voges-Proskauer test — + other strains IGrowth in anaerobic agar l | + _ other strains lGrowth in 7% NaCl — + other strains , I HydrolySIs of starch — + B. pumilus other strains I Citrate uptake test — + other 8. pumilus | Sugar uptake test — + Other 8. pumilus B pumilus ATCC 21143 Figure 67. Purification and identification of B. pumilus (ATCC 21143). 132 Biosynthesis of kanosamine by B. pumilus A strain of Bacillus pumilus (ATCC 21143, formerly Bacillus aminoglucosidicus), which produces kanosamine as a secondary metabolite, was isolated by a Japanese group from a soil sample collected on the shores of Lake Haruna.104a According to the literature, this strain was capable of producing 1 to 4 g/L of kanosamine from glucose when grown on soybean meal as the nitrogen source. Table 19. B. pumilus (ATCC 21143) strain identification and isolation. f number of entry test expected number 0 colonies testing phenotype colonies tested _ , a posrtive 1 catalase test + 50 50 2 Voges-Proskauer test + 20 20 3 growth test in anaerobic agar - 20 20 4 growth test in 7% NaCl + 20 19 5 hydrolysis of starch - 15 14 6 citrate uptake test + 10 _ 10 7 I sugar uptake test + 10 10 a Positive result denotes expected result for B. pumilus (ATCC 21143). However, no detectable amount of kanosamine was synthesized when B. pumilus (ATCC 21143) was cultured under conditions described in the literature.104C One 1 04c abnormal character of the bacteria was its growth rate. According to the literature, B. pumilus (ATCC 21143) should be grown on solid nutrient agar at 37°C for 17 to 20 h. In our case, however, large colonies were observed after 6 h at 37°C. It suggested that the strain we purchased from ATCC was either not B. pumilus (ATCC 21143), or it was contaminated with some fast-growing microorganism. Consequently, a seven-step 133 145, 10421 identification and isolation procedure was carried out (Figure 67). It included catalase test, Voges-Proskauer test, growth test in anaerobic agar, growth test in 7% NaCl, starch hydrolysis test, citrate uptake test, and sugar uptake test. All the characteristics of B. pumilus (ATCC 21143) relative to these tests were shown in Figure 67. Contaminants were found during the starch hydrolysis test and growth test in medium containing 7% NaCl (entry 4, 5, Table 19). B. pumilus cultured under a variety of conditions accumulated kanosamine. For example, soybean meal, peanut meal, and cornsteap liquor were each adequate sources of nitrogen while glucose and sucrose were acceptable carbon sources, as had been preViously mentioned in the literature.10 C Mannitol and 11108110] were other suitable . . 4 . . carbon sources for kanosamine syntheSIS.l 6 The titer was about 1 g/L when B. pumilus . . . . 4 . was fermented under culture conditions reported in the literature.10 C The syntheSized concentration of kanosamine was at the same level as reported in the literature. However, the titer was not high enough for us to make a significant amount of kanosamine. Fermentation conditions needed to be modified to improve the titer of kanosamine synthesis by B. pumilus. Since secondary metabolism, such as rifamycin B, increases at lower culture temperatures,147 this was the first parameter examined for improved kanosamine synthesis. When the temperature was decreased from 34 °C to 30 °C, a two-fold increase in kanosamine synthesis was observed (entry 2, Table 20). The second modification was the nitrogen source. When soy flour was used instead of soytone (digested soybean meal), 4 g/L of kanosamine was synthesized (entry 3, Table 20). The third change was to use fed-batch fermentation conditions (in terms of glucose feeding) instead of shake flask 134 culture conditions. The D.O. concentration of fermentation was maintained at 10% air and pH was set at 7. Under such conditions, 20 g/L kanosamine was synthesized (entry 4, Table 20). When the soy flour added to the medium was increased from 20 g/L to 30 g/L, kanosamine synthesis increased to 25 g/L (entry 5, Table 20). Table 20. Improving kanosamine synthesis by B. pumilus fermentation. entry teomp. nitrogen amount of nitrogen fermentation titer“ ( C) source source (g/L) type (g/L) l 34 soytone 20 batchc 1 a 2 30 soytone 20 batchc 2 a 3 30 soy flour 20 batchc 4 b 4 30 soy flour 20 fed-batchd 20 b 5 30 soy flour 30 fed—batchd 25 b a single runs; b duplicated runs; C cultured in shake flask without pH and dissolved oxygen control; d cultured in fermentor with pH (7.0) and dissolved oxygen (10%) control; c titer was calculated based on NMR. Fed-Batch fermentor conditions The fed-batch fermentor equipment was the same as those described in previous section of this Chapter (page 104). Fermentations were run at 33 °C, pH 7.0, and the dissolved oxygen (D.O.) level was maintained at 10%. Two different conditions that differed in the steady state concentration of D-glucose in the culture medium were employed. A steady state concentration of less than 1 g/L of glucose was maintained in glucose-limited conditions and a concentration of 15-25 g/L of glucose was employed for glucose-rich conditions. The initial glucose concentration in the fermentation medium was 25 g/L for glucose-limited conditions and 30 g/L for glucose-rich conditions. 135 The same method was used to control D.O. concentration at 10% of air saturation under glucose-limited conditions. For fermentations that employed glucose-rich conditions, a stainless steel baffle cage consisting of four 1/2" x 5" baffles was placed in the fermentation vessel and three different staged methods were used to maintain the DO. concentration at 10% of air saturation. In the first stage, the DO. concentration was maintained by increasing the impeller speed from its initial set point of 50 rpm to a preset maximum of 1100 rpm while the airflow was maintained at an initial setting of 0.06 L/L/min. In the second stage, the mass flow controller maintained the DO. concentration by increasing the airflow rate from 0.06 L/L/min to a preset maximum of 1.0 L/L/min with the impeller rate constant at 1100 rpm. These two stages take anywhere from 12 h to 18 h for completion depending on'the strain under investigation. Until this stage, except for the initial 30 g/L, no additional glucose was added. As soon as the air flow reached 1.0 L/L/min at a constant impeller speed of 1100 rpm, the glucose pump was switched on and hereafter the DO. level was maintained steady at 10% by varying the stirrer from 750 rpm to 1600 rpm. The concentration of the glucose solution added was 65% (w/v) and the rate of glucose addition was controlled so as to maintain a glucose concentration of approximately 15-25 g/L in the fermentation at any given time. Biosynthesis of aminoshikimate by E. coli SP1.l/pKD12.138 Shikimate synthesizing E. coli SP1.l/pKDl2.138 (aroFFBR, P aroE, tktA, serA, lac Amp“), which had been previously constructed in the laboratory,mb‘ C was first examined for its ability to synthesize aminoshikimate from kanosamine. E. coli SP1.l/pKDl2.138 was cultured under glucose-limited fed-batch fermentation conditions at 33°C, pH 7.0, 136 with dissolved oxygen maintained at a set point of 10% air saturation. L-Phenylalanine, L-tyrosine, and L-tryptophan were added to the culture medium at the beginning of the fermentor run as required for cell growth. Kanosamine (2.5 g each time) was added to the culture medium at 18, 24, and 30 h. After 48 h of cultivation, E. coli SP1.l/pKD12.l38 consumed 53% of the initially added kanosamine and synthesized 40.1 g/L of shikimate. No aminoshikimate was detected in the fermentation broth (entry 1, Table 21). Although the observations indicated that kanosamine could be metabolized by E. coli, the fate of kanosamine and how E. coli utilized the kanosamine were not known. 45 40 40“ 1735 A . :3" f 8A 25 -- . . fl l “L25 E: g3 20.. ' "20 E g 15-- 1.5 8 x 10“ . H 710 s .. -5 (5)-H. ”.J-L -. Tl i L110 0 12 18 24 30 36 42 48 time(h) Figure 68. Biosynthesis of aminoshikimate by E. coli SP1.l/pKD12.138 under glucose-limited conditions. Legend: shikimate (SA), open columns; kanosamine, filled columns; dry cell weight, closed circles. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG5.166A In order to use E. coli as host to synthesize aminoshikimate from kanosamine, kanosamine kinase, phosphokanosamine isomerase, transketolase, aminoDAHP synthase, 137 aminoDHQ synthase, aminoDHQ dehydratase, and aminoshikimate dehydrogenase were required. Since native E. coli tktA-encoded transketolase, aroB-encoded 3- dehydroquinate synthase, aroD-encoded 3-dehydroquinate dehydratase, and aroE- encoded shikimate dehydrogenase are able to catalyze reactions on the amino counterparts (aminoF6P, aminoDAHP, aminoDHQ, and aminoDHS) of their native substrates (F6P, DAHP, 3-dehydroquinate, and 3-dehydroshikimate), only rifN-encoded kanosamine kinase, phosphokanosamine isomerase (unidentified), and rer-encoded aminoDAHP synthase were necessary (Figure 66). Given that kanosamine could be metabolized by E. coli and it had a similar structure to that of glucose, it was suspected that the native E. coli glucokinase or phosphoenolpyruvate:carbohydrate phosphotransferase (PTS) system was capable of phosphorylating kanosamine. Phosphokanosamine isomerase had not been identified. Native E. coli phosphoglucose isomerase might be capable of catalyzing the isomerization between kanosamine 6- phosphate and aminoF6P. Based on these initial assumptions, rifH—encoded aminoDAHP synthase was overexpressed in our first construct as the only aminoshikimate pathway enzyme. The plasmid construction was initiated by PCR amplification of rifH from cosmid FKN108. After digestion, the PCR product was cloned into vector pKK223-3 to yield (Figure 69) plasmid pJG5.165A (PmrifH, Amp“). The resulting PmrifH fragment was subsequently excised out of plasmid pJG5.165A and inserted into a unique BglII site in plasmid pKD12.138 (tktA, aroFFB“, serA, aroE, Amp“) to afford (Figure 70) plasmid pJG5.166A (PmrifH, PmaroE, serA, tktA, Amp“) in which aroFfii“ was simultaneously deactivated. 138 damn—«8:3 88:88 :0 women 863 86:: 2E. 6 ”88 86333.6 6 Am m5 :osficoEoEmsm 88305.: 53, £82280 5880033 .U Eocfififioamsm 05:38:83 BEE? 8:256:00 Uganomooim .m ”Aw DD cosficoEoEmsm 2:858:83: 5:3 .mcoEcaoo 8638:6863m .< a ms. 2 N: E 0 5:665: #65 .Ea 866$ flaws...“ $3.83: . am as 6 3o 8 o 58$ was is: 83.36: ”mum“ £8.83:.:m pm 4 . Ed 46 o isf as: as... 862$ Ease mambefieam as 2 2 3 0 seem as: is... 8&6..an Excess: m _ m _ .83: . Em 96 o o I. o «as... .38: is... .885 43:85 38.83:.Em am 6: :3 2 o ”:95. as .ssa. 88.20: 8:83: $2.83: . am a. e 6 _ .o 3 < $5.. is: .36.... .885 East 82 .83: . Em pm o o 6.: m “82 as .Ea. 86...»: Essen: <66 : .8 a: . :5 N o o o4 < Mai .36.. is .835 .8166 fiafiaxeaam : ”Mow—MM «WNW—wee AWWV 66:00 8558820 2883 39:88 .055 8:35:89 9:518 «53%? .56.:— Eseuu mama—3.3 .3 69632“ 8.3.» can 88355259 335.5 AN 93.; 139 FKN103 - Hindlll 1)PCR 2) EcoRl digest pKK223-3 4.6-kb EcoRl EcoRl rifH 1.3-kb pBR322 ori 1) EcoRl digest 2) CIAP treatment ligation BamHI EcoRl EcoRl Smal BamHl Pstl Hindlll pJG5.165A 5.9-kb pBR322 . ori Figure 69. Preparation of Plasmid pJG5.165A. 140 pJG5.165A BamHl digest AmpR pKD12.138 ii BamHl BamHl Ptac rifH 1 .6-kb 1) Bglll digest 2) CIAP treatment ligation (Bglll) Figure 70. Preparation of Plasmid pJG5.166A. 141 < ‘3 a E 6 -- c» E v w. o E if)" 4 o 3 E 3 2 “i o l l :2; O C 3 | 0 4i—C % . : - -‘ O 12 18 24 30 time (h) Figure 71. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG5.166A under glucose-limited conditions. Legend: kanosamine, filled columns; shikimate (SA), open columns; aminoshikimate (aminoSA), dashed columns; dry cell weight, closed circles. After transformation, E. coli SP1.l/pJG5.l66A was cultured under glucose- limited, fed-batch fermentation conditions. L-Phenylalanine, L-tyrosine, and L- tryptophan were added to the culture medium at the beginning of the fermentor run as required for growth and at 16 and 30 h to inhibit native E. coli DAHP synthase activity. Kanosamine (2.5 g each time) was added at 18, 24, and 30 h. Synthesis of aminoshikimate along with shikimate was observed (entry 3, Table 21, Figure 71). Aminoshikimate formation was confirmed by 1H NMR (Figure 89), ”C NMR (Figure 90), and high resolution FAB mass spectrometry. E. coli SP1.l/pJGS.166A synthesized 0.16 g/L aminoshikimate and 5.1 g/L of shikimate over a 48 h of cultivation. The synthesis of aminoshikimate from kanosamine validated our speculations that native E. coli kinase (or PTS system) and isomerase were able to catalyze reactions on the amino counterparts (kanosamine and kanosamine 6-phosphate) of their native substrates 142 (glucose and glucose 6-phosphate). However, they might not work very well, which might explain the low titer. When E. coli SP1.l/pJG5.l66A were cultured under glucose—limited conditions without kanosamine supplementation, it synthesized no aminoshikimate and 1.6 g/L of shikimate (Figure 72, entry 2, Table 21). The results implicated that kanosamine was essential for the synthesis of aminoshikimate in E. coli and therefore proven to be a biosynthetic intermediate of the aminoshikimate pathway. 8 40 § 8 ._ 35 2 g 6-u- “L30 U, 3 . :7 3' O . . . 25 .c 650 0 .g 5%) 4- O “20 3 E» --15 " a. g. D 2"” "" 10 '0 0 (D C > 11 ii H H” 0"—. l _I n‘f n! l ’T ’ 1 ' 0 0 12 18 24 3O 36 42 48 time (h) Figure 72. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG5.l66A under glucose-limited conditions without kanosamine supplementation. Legend: shikimate (SA), open columns; dry cell weight, closed circles. In the above two fermentations, more shikimate (5.1 g vs 1.6 g) was synthesized when kanosamine was added to the culture medium. It suggested that E. coli SP1.l/pJGS.166A could synthesize shikimate both from glucose through the E. coli common pathway and from kanosamine via the assembled aminoshikimate biosynthetic 143 pathway. The hydrolysis of iminoE4P and formation of E4P was likely the explanation for shikimate synthesis from kanosamine. To identify better culture conditions for aminoshikimate synthesis in E. coli, glucose-rich culture conditions was examined. Under such conditions, E. coli SP1.l/pJGS.166A was cultured with glucose concentrations being maintained at 15 to 25 g/L after phase change and with the other conditions remaining the same as those employed during glucose-limited cultivation. More aminoshikimate was synthesized by E. coli SP1. 1/pJG5. 166A when cultivated under glucose-rich conditions (Figure 73; entry 4, Table 21) relative to that of glucose-limited conditions. Under glucose-rich conditions, E. coli SP1.l/pJG5.166A synthesized 0.81 g/L of aminoshikimate and 3.7 g/L of shikimate. This observation was consistent with enzyme assay results, which indicated that higher DAHP synthase activities (entry 4, Table 22) and transketolase activities (entry 4, Table 23) were achieved under glucose-rich conditions. kanosamine, SA, aminoSA (9/L) dry cell weight (g/L) time (h) Figure 73. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG5.l66A under glucose-rich conditions. Legend: kanosamine, filled columns; shikimate (SA), open columns; aminoshikimate (aminoSA), dashed columns; dry cell weight, closed circles. 144 EDAHPB . Hindlll 1)PCR 2) EcoRl digest pKK223-3 lLGkb EcoRl EcoRl pBR322 dahpsl ori 1 .3-kb 1) EcoRl digest 2) CIAP treatment ligation Ba\mHl EcoRl EooRl Smal BamHl Pstl Hindlll pJGG.O70A 5.9-kb Figure 74. Preparation of Plasmid pJG6.070A. 145 pJG6.070A Ncol BamHl digest v BamHl BamHl NCOI Pm dahpsl 1 .6-kb 1) Bglll digest 2) CIAP treatment ligation (Bglll) Figure 75. Preparation of Plasmid pJG6.071A. 146 Biosynthesis of aminoshikimate by E. coli SP1.l/pJ G6.07 1A There are two more DAHP synthase isozymes besides RifH and the encoding genes have been cloned from A. mediterranei.144 One of the deduced peptide sequences (DahpsI) was closely related to the product of the rifH gene and to type II DAHP synthases148 from higher plants. Another one (Dahpsll) was annotated as the housekeeping activity to support aromatic amino acids biosynthesis. To examine whether dahpsl-encoded DAHP synthase from A. mediterranei possesses in vivo aminoDAHP synthase activity, E. coli SP1.l/pJG6.071A (P dahpsl, P aroE, serA, tktA, AmpR) was (ac lac constructed (Figure 75) and evaluated for aminoshikimate synthesis. The construction of plasmid pJG6.071A was initiated by PCR amplification of dahpsl from plasmid EDAHPB.97 The PCR product was then cloned (Figure 74) into vector pKK223-3 to yield plasmid pJG6.07OA (Pmdahpsl, Amp“). The resulting Pmdahpsl fragment was subsequently excised out of plasmid pJG6.070A and inserted (Figure 75) into a unique BglII site in plasmid pKD12.138 (tktA, aroFFBR, serA, aroE, Amp“) to yield pJG6.071A (P dahpsl, P aroE, serA, tktA, Amp“) in which aroFFBR was (ac tat simultaneously inactivated. After transformation, the resulting E. coli SPl.l/pJG6.071A was cultured under glucose-rich conditions identical to those employed to study aminoshikimate synthesis in E. coli SP1.l/pJG5.166A. No aminoshikimate and 4.1 g/L of shikimate were synthesized after 48 h of cultivation (entry 5, Table 21, Figure 76). The results suggested that dahpsl- encoded DAHP synthase did not possess in vivo aminoDAHP synthase activity but in vivo DAHP synthase activity. It was consistent with our in vitro result (entry 2, Table 7) that no aminoDAHP formation was observed when dahpsI-encoded DAHP synthase was 147 used to catalyze the condensation between iminoE4P and phosphoenolpyruvate. The low shikimate synthesis might result from poor expression of dahpsl-encoded DAHP synthase or its partial inhibition by aromatic amino acids, which were known to feedback inhibit native E. coli DAHP synthases. 8 40 3‘3 435 A g 6 . 0 o . -3o 3 “i -- 25 E 332 o '5’ 053’ 4“ "20 3 c -- = E 15 8 9, 2“ --10 P: o p 'o :5 ' I «5 x ' ' lo 48 time (h) Figure 76. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG6.071A under glucose-rich conditions. Legend: kanosamine, filled columns; shikimate (SA), open columns; dry cell weight, closed circles. Biosynthesis of aminoshikimate by SP1.l/pJG6.1818 and SP1.l/pJG6.223B Although E. coli SP1.l/pJGS.l66A synthesized aminoshikimate from kanosamine, the titer of aminoshikimate was low. An increase in kanosamine kinase activity might help convert more kanosamine into kanosamine 6-phosphate and lead to a higher titer of aminoshikimate. Based on in vitro enzyme assays and bioconversions, . . . 127, . . both rifN-encoded kanosamine kinase from A. medzterranez and glk-encoded glucokinaseI49 from Zymomonas mobilis showed activity towards phosphorylation of 148 kanosamine (Table 23). Aminoshikimate synthesis from kanosamine was therefore examined in the presence of RifN and le respectively. EcoFtl Smal BamHl BamHl T0325 PS" p - Hindlll 1) PCR 2) EcoRl digest pKK223-3 4.6-kb EcoRl EcoRl glk pBFl322 1.1-kb 0ri .- 1) EcoRl digest 2) CIAP treatment ligation EcoRl ' Smal BamHl Pstl Hindlll pJGG.180A 5.7-kb Figure 77. Preparation of Plasmid pJG6.180A. 149 (Bglll) pJG6.180A BamHl digest BamHI BamHl Ptac g/k 1 .4-kb 1) Neal digest Klenow treatment 2) Klenow treatment 3) CIAP treatment ligation (Bglll) pJGG.181 B Figure 78. Preparation of Plasmid pJG6.1818. 150 le-encoded glucokinase from Z. mobilis was examined first. The open reading frame of glk was PCR amplified from plasmid pTC325150 and cloned (Figure 77) into vector pKK223-3 to afford plasmid pJG6.180A (nglk, Amp“). The resulting nglk was subsequently excised out of plasmid pJG5.18OA and ligated (Figure 78) with linearized plasmid pJGS.l66A (PmrifH, PmaroE, serA, tktA, Amp“) to yield plasmid pJG6.181B (Pmrifl-I, PmcaroE, serA, tktA, Pmcglk). ' ’ ._ 30 a) O O -- 20 r 15 N dry cell weight (g/L) kanosamine, SA, aminoSA (Q/L) A time (h) Figure 79. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG6.181B under glucose-rich conditions. Legend: kanosamine, filled columns; shikimate (SA), open columns; aminoshikimate (aminoSA), dashed columns; dry cell weight, closed circles. After transformation, synthesis of aminoshikimate using E. coli SP1.l/pJG6.181B (PmcrifH, PmaroE, serA, tktA, nglk) was examined by glucose-rich fed-batch fermentation. It synthesized 1.1 g/L of aminoshikimate and consumed 80% of the initially added kanosamine (Figure 79, entry 6, Table 21). Compared with E. coli SP1.l/pJGS.l66A (entry 4, Table 21), E. coli SP1.l/pJG6.l8lB (entry 6, Table 21) synthesized 36% more aminoshikimate and consumed 13% more kanosamine after 48 h 151 of cultivation. The yield of aminoshikimate synthesized from kanosamine was also increased about 20%. The results were consistent with our speculation that higher kanosamine kinase activity would result in increased kanosamine consumption and aminoshikimate synthesis. EcoRl Smal BamHl BamHI FKN108 Pstl Hindlll 1) PCR 2) EcoRl digest pKK223-3 4.6-kb EcoRl EcoRl rifN 1 .O-kb ori pBR322 1) EcoFtl digest 2) CIAP treatment ligation EooRl Smal BamHl Pstl Hindlll pJGG.222A 5.6-kb pBR322 . ori Figure 80. Preparation of Plasmid pJG6.222A. 152 (Bglll) pJGS.222A BamHl digest 11 BamHl BamHl Pm rifN 1 .3-kb 1) Ncol digest Klenow treatment 2) Klenow treatment 3) CIAP treatment ligation (Bglll) pJG6.223B Figure 81. Preparation of Plasmid pJG6.223B. 153 With the promising results obtained with glk-encoded glucokinase overexpression, aminoshikimate synthesis was subsequently examined in the presence of rifN-encoded kanosamine kinase. To conduct the investigation, plasmid pJG6.223B (Pmrifl-I, PmaroE, serA, tktA, PmriflV) was prepared (Figure 81) by localizing PmrifN onto plasmid pJG5.166A. The construction began with PCR amplification of rifN gene from cosmid FKN108. The resulting PCR product was then cloned (Figure 80) into vector pKK223-3 to yield pJG6.222A (Pmrzflv, Amp“). The PmrifN fragment was excised out of plasmid pJG6.222A and then ligated to linearized plasmid pJGS.166A to afford pJG6.223B (Figure 81). kanosamine, SA, aminoSA (g/L) A dry cell weight (glL) 1218 24 30 36 42 48 time(h) Figure 82. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG6.223B under glucose-rich conditions. Legend: kanosamine, filled columns; shikimate (SA), open columns; aminoshikimate (aminoSA), dashed columns; dry cell weight, closed circles. After transformation, the aminoshikimate producing capability of E. coli SP1.l/p pJG6.223B was determined under glucose-rich conditions identical to those employed to study E. coli construct SP1.l/p pJG6.181B. Accumulation of only 0.24 g/L of aminoshikimate was observed after 48 h of cultivation (Figure 82, entry 7, Table 21) 154 although more kanosamine was consumed to E. coli SP1.l/pJG6.181B (entry 6, Table 21). E. coli SP1.l/p pJG6.223B also synthesized 6.4 g/L of shikimate (Figure 82, entry 7, Table 21). It was quite surprising that lower aminoshikimate. synthesis was observed when glk-encoded glucokinase was substituted with rifN-encoded kanosamine kinase. This might indicate that glk-encoded glucokinase was a better kanosamine kinase than the rifN gene product. However, enzyme assays (entry 2, 3, table 24) showed that kanosamine kinase activity of RifN was two to three times higher than that of le in the respective aminoshikimate-synthesizing microbes. The observations in enzyme assay were consistent with the fact that more kanosamine was consumed in the presence of RifN. The lower aminoshikimate synthesis with RifN overexpression could be explained as follows. RifN-encoded kanosamine kinase was indeed a better kanosamine kinase. Its overexpression resulted in a faster formation of kanosamine 6-phosphate, which, in turn, resulted in a higher intracellular concentration of iminoE4P. When iminoE4P concentration was higher than desired, the likelihood of its hydrolysis increased. The hydrolyzed iminoE4P was eventually converted into shikimate. The fermentation results did show higher concentrations of synthesized shikimate when rifN-encoded kanosamine kinase was overexpressed (entry 7, Table 21) instead of glk-encoded glucokinase (entry 6, Table 21). Biosynthesis of aminoshikimate by SP1.l/pJG6.238A and SP1.l/pJG9.240A Based on fermentation results obtained above, a key impediment to higher aminoshikimate synthesis from kanosamine in E. coli appeared to be the hydrolysis of 155 iminoE4P. If this problem could be overcome, an improved aminoshikimate synthesis was likely to be achieved. 0rf15-encoded transketolase and rifH-encoded aminoDAHP synthase were demonstrated to form a complex in vivo (see Chapter 2), which might facilitate channeling of iminoE4P from its formation to its condensation with phosphoenolpyruvate and therefore reduce hydrolysis. To examine the effects of metabolite channeling on aminoshikimate synthesis, E. coli aminoshikimate producer (Figure 84) SP1.l/pJG6.238A (PmrifH, PmaroE, serA, lac)", Pmorf15, PmrifN) and (Figure 87) SP1.l/pJG9.240A (Pmcriffl, PmaroE, serA, lacl", P 0rf15, nglk) were constructed. Plasmid pJG6.238A was created by inserting lalePmorfIS and PmrifN into pJGS.l66A. The open reading frame of 0rf15 was PCR-amplified from cosmid FKN108 and inserted (Figure 83) into vector pJF118EH to give plasmid pJG6.128A (PmorfIS, lacl“, Amp“). The resulting lachPmorfI5 was excised out of pJG6.128A and the PmrifN fragment was excised out of plasmid pJG6.222A (Figure 80). These two fragments were then ligated (Figure 84) with linearized plasmid pJGS.166A (Rama, PmaroE, serA, tktA, Amp“) to generate the desired plasmid pJG6.238A (Pmcrifl-I, P aroE, serA, lacI", 10C Pm.-0rf15- PmJiflVl After 48 h of cultivation, E. coli SP1.l/pJG6.238A (Pmrifl-I, P aroE, serA, lacI", Pmorf15, P,a,rz'j‘N) synthesized 0.4 g/L of aminoshikimate and 6.3 g/L of shikimate (Figure 85, entry 8, Table 21). When compared to construct E. coli SP1.l/pJG6.223B (entry 7, Table 21) culturing under the same conditions, E. coli SP1.l/pJG6.238A (entry 8, Table 21, Figure 85) synthesized more aminoshikimate (0.40 g vs 0.24 g) with an increased yield (6% vs 4%). 156 FKN108 1)PCR 2) EcoRl digest pJF1 18EH v 5.3-kb EcoRl EcoRl pBR322 0rf15 ° 1 .7-kb 1) EcoRl digest 2) CIAP treatment ligation EcoRl Smal BamHl Pstl pJGS.128A Hindlll 7.0-kb penaza Figure 83. Preparation of Plasmid pJG6.128A. 157 (Bglll) pJG6.128A pJG6.222A Nrul/Hindlll Alflll/Scal NCO' digest digest Nrul Hindlll Alflll Scal . Ncol IacP, Pm 0rf15 Pia.- rIfN 3.4-kb 3.1-kb Hindlll 1) Ncol/Hindlll digest 2) CIAP treatment figafion (Bglll) pJGG.238B 13.9-kb Hindlll Figure 84. Preparation of Plasmid pJG6.238A. 158 CD 4o -- 35 -- 30 H -- 25 -- 20 O) 1 dry cell weight (g/L) N 1 kanosamine, SA, aminoSA (9/L) .b time (h) Figure 85. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG6.238A under glucose-rich conditions. Legend: kanosamine, filled columns; shikimate (SA), open columns; aminoshikimate (aminoSA), dashed columns; dry cell weight, closed circles. 8 4o (<8 -35 A .E o . E 6 2 H 30 i, m E <'»~ .9 (De 4 g “:59 = r. E. s 2“ v C (U x 0.. time (h) Figure 86. Biosynthesis of aminoshikimate by E. coli SP1.l/pJG9.240A under glucose-rich conditions. Legend: kanosamine, filled columns; shikimate (SA), open columns; aminoshikimate (aminoSA), dashed columns; dry cell weight, closed circles. 159 pJGB.128A Nrul/Seal 11 .9-kb Nrul Scal lac/0, Pm 0rf15 4.1 -kb Xbal 1) Xbal digest 2) Klenow treatment 3) CIAP treatment ligation pJGQ.240A 12.9-kb (Xbal) Figure 87. Preparation of Plasmid pJG9.240A. 160 Plasmid pJG9.24OA (PmrifH, P .aroE, serA, lacl", P lac morf15, nglk) was created (Figure 87) by inserting a lacIQPmorfIS fragment into plasmid pJG6.181B. After transformation, the synthesis of aminoshikimate by E. coli SP1.1/ pJG9.24OA was examined under glucose-rich fermentation conditions. It synthesized 1.2 g/L of aminoshikimate and 7 g/L of shikimate (Figure 86, entry 9, Table 21). Comparing to construct E. coli SP1.l/pJG6.181B (Pmrlfl-I, PmaroE, serA, tktA, nglk) cultivated under the same conditions, E. coli SP1.l/pJG9.240A synthesized more aminoshikimate (1.2 g vs 1.1 g) with no increase in yield. It was the current best E. coli aminoshikimate producer. The slightly increase in titers of aminoshikimate, however, did not provide strong arguments to support our metabolite channeling hypothesis, which was implicated by the results of bacterial two-hybrid experiments. One possible explaination was that 0rf15—encoded transketolase was not expressed well in E. coli. The number of the active portein complexs of 0rf15-encoded transketolase with rifH-encoded aminoDAHP synthase was too small to have a signofocant impact on preventing the hydrolysis of iminoE4P. 161 .couflcoEo—nasm 0553288— 53, .202250 notewooaw .U ”cosflcofioamsm 2:830:3— :55; 5:066:00 BEE—emooiw .m ”souficoEoEmsm 2:830wa 53 accuse-So BEE—-8833 .< a 86 NS 2 .o o 3%....» fiber; #82 is. .885 ESE <8~doazém a . . . 23$ . a . 8 o S o 2 o u .283 was .83 £243.24 £25 — _ - _ . _c .v r. b )— _. _ )- p- Lo _0 l— - b _. _ _ h F- -9 Fa _ _ _ _ _ - _ ~o —0 run h _ _ _ D b— [o —« EH , b— . _ )— L3 l. _H h b p— - _ _ -o ~40 "H r F _ _ h h ~o »—o ”r. _ p— p- h- p _ _ ~o —o _ _N l- h p— F L- _ ~o —« “N b h h b )— p- Figure 90. 13C NMR of S-amino-S-deoxyshikimate purified from fermentation broth. 171 CHAPTER FOUR EXPERIMENTAL General Methods General Chemistry ' All reactions sensitive to air and moisture were carried out in flame or oven-dried glassware under a positive atmosphere of argon. Air or moisture sensitive reagents and solvents were transferred to reaction flasks fitted with rubber septa via oven-dried syringes or cannula. Unless otherwise specified, all reactions were carried out at room temperature. Solvents were removed using either a Biichi rotary evaporator at water aspirator pressure or under high vacuum (0.5 mm Hg). Acetone was dried over anhydrous ZnClz. CHzClz, Et3N, and pyridine were distilled from calcium hydride under nitrogen. Tetrahydrofuran and diethyl ether were distilled under nitrogen from sodium/benzophenone. Water used in synthesis was glass- distilled and deionized. All other reagents and solvents were used as available from commercial sources. Organic solutions of products were dried over anhydrous N aZSO4 or MgSO4. The sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP) was purchased from Lancaster Synthesis Inc. [Amine-'SN]-L-glutamine, [amide-'SNJ-L- glutamine, D-6,6-[2H2]~glucose, D-6,6-[2H2]-fructose, and 'SNHZOH-HCI were obtained from Cambridge Isotope Laboratories, Inc. Chromatography HPLC analysis was preformed on an Agilent 1100 series HPLC with ChemStation acquisition software (Rev. A.08.03). Columns used include Agilent 172 ZORBAX C-18 reverse phase analytical column (4.6 mm x 150mm), Alltech C-18 reverse phase semi—prep column (22 mm x 250 mm), AXpak WA-624 weak anion exchange column (Showa Denko, 6 mm x 150 mm), and sugar KS-801 strong cation exchange column (Showa Denko, 8 mm x 300 mm). Solvents were routinely filtered through 0.45-um membranes (Gelman Science). Analytes were detected at 250 nm, 254 nm, or 260 nm as specified. Protein purification utilized an AKTA Purifier FPLC system with Unicorn acquisition software 4.10 (Amersham Biosciences). Columns used include HiPrep DEAE FF column (16 mm x 100 mm, 16 mL), Resource Q column (6.4 mm x 30 mm, 1 mL and 16 mm x 30 mm, 6 mL), HiLoad Superdex 200 (prep grade) column (16 mm x 600 mm), and HiTrap Phenyl HP column (16 mm x 25mm, 5 mL). All the columns were purchased from Amersham Biosciences. Solvents were routinely filtered through 0.45- pm membranes (Gelman Science) and degassed under reduced pressure for 30 min prior to use. Proteins were detected at 260 nm. AGl-X8 (acetate form and chloride form) was purchased from Bio-Rad. Sephadex G-100 was purchased from Pharmacia. Diethylaminoethyl cellulose (DEAE) was purchased from Whatman. Ni-NTA resin was purchased from Qiagen. Dye matrix selection kit was obtained from Amicon. Dowex 1 (200-400 mesh, chloride form) and Dowex 50 (ZOO-400 mesh, H+) were purchased from Sigma-Aldrich. Radial chromatography was performed on a Harrison Associates Chromatotron (model 7924), using 1, 2 or 4 mm layers of silica gel 60 PF254 containing gypsum (E. Merck). Silica gel 60 (40-63 um, E. Merck) was used for flash chromatography. Analytical thin-layer chromatography (TLC) utilized precoated glass plates of silica gel 173 60 F-254 (0.25 mm, Whatman). TLC plates were visualized by UV or by immersion in anisaldehyde stain (by volume: 93% ethanol, 3.5% sulfuric acid, 1% acetic acid, and 2.5% anisaldehyde), vanillin stain (by volume: 98% ethanol, 1% sulfuric acid, and 1% vanillin), or phosphomolybdic acid stain (7% phosphomolybdic acid in ethanol, w/v) followed by heating. Spectroscopic Measurements 1H NMR and 13C NMR spectra were recorded on a Varian VX-300 FT -NMR or a Varian VX-SOO FT-NMR spectrometer. Chemical shifts for 1H NMR spectra are reported (in parts per million) relative to internal tetramethylsilane (Me4Si, a = 0.0 ppm) with CDCl3 as solvent and to sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP, a = 0.0 ppm) when D20 was the solvent. The following abbreviations are used to describe spin multiplicity: s (singlet), d (doublet), t (triplet), q (quartet), m (unresolved multiplet), dd (doublet of doublets), b (broad). l3C NMR spectra were recorded at 125 MHz. Chemical shifts for I3C NMR spectra are reported (in parts per million) relative to internal tetramethylsilane (Me4Si, a = 0.0 ppm) with CDCl3 as solvent and to sodium 3- (trimethylsilyl)propionate-2,2,3,3-d4 (TSP, 6 = 0.0 ppm) when D20 was the solvent. 31P NMR spectra were recorded on a 121 MHz Varian spectrometer and chemical shifts are reported (in parts per million) relative to external 85% phosphoric acid (0.0 ppm). UV and visible measurements were recorded on a Perkin-Elmer Lambda 3b UV-Vis spectrophotometer or on a Hewlett Packard 8452A Diode Array Spectrophotometer equipped with HP 89532A UV-Visible Operating Software. Fast atom bombardment (FAB) mass spectra were obtained on a double focusing Kratos MSSO mass spectrometer at Michigan State University and electrospray ionization (ES) mass spectra were obtained 174 on a direct infusion electrospray mass spectrometer at Department of Chemistry and Biochemistry at University of South Carolina. Concentrations of fermentation and cell-free reaction products were determined by comparison of the integrals corresponding to each compound with the integral corresponding to TSP (6:0.00 ppm) in the 1H NMR. Compounds were quantified using the following resonances: shikimate (6 4.40, t, l H), aminoshikimate (6 6.56, m, 1 H), kanosamine (6 5.27, d, 0.45 H), aminoDAHP (6 1.85, dd, 1 H), and DAHP (6 1.80, dd, 1 H). Concentrations of above compounds derived from their respective 1H NMR integral values tended to be overestimated and their precise concentrations were calculated by application of the following formulas: [SA (mM)]wu,, = 0.67 x [SA (mM)]NMR + 0.27; [aminoSA (mM)],cma, = 0.71 x [aminoSA (mM)]NMR + 0.14; [kanosamine (mM)]actual = 0.78 x [kanosamine (mM)]NMR + 0.15; and [DAHP (mM)],cm,, = 0.65 x [DAHP (mM)]NMR + 0.21. The concentration of aminoDAHP was calculated using the formula obtained for DAHP. These equations were obtained as follows: A known quantity of each compound was dissolved in 10 mL of D20 to obtain a stock solution. Various known volumes of the stock solution of each compound were concentrated under reduced pressure and redissolved in 1 mL of DZO containing 10 mM TSP and their 1H NMR spectra were recorded. The solute concentration in each sample that was estimated for 1H NMR was plotted against the calculated concentration for that sample resulting in the calibration curve. Chemical Assays Thiobarbituric acid (TBA) assay 175 The reagents used in the thiobarbituric acid (TBA) assay99 include solution A: 0.2 M NaIO4 in 8.2 M H3PO4; solution B: 0.8 M NaAsO2 and 0.5 M Na2S04 in 0.1 M H2804; and solution C: 0.04 M thiobarbituric acid in 0.5 M NaZSO4 (pH 7.0). An aliquot (0.1 mL) of the DAHP (or aminoDAHP) containing sample was reacted with 0.1 mL of solution A at 37 °C for 5 min. The reaction was quenched by addition of solution B (0.5 mL) and vortexed until a dark brown color disappeared. Upon addition of 3 mL of solution C, the sample was heated at 100 °C for 15 min. Samples were cooled briefly and the pink (or orange) chromophore was extracted into 4 mL of cyclohexanone. The aqueous and organic layers were separated by centrifugation (2000g, 15 min). The absorbance of the organic layer was recorded at 549 nm (e = 68000 L mol—l cm'l). Organic and inorganic phosphate assay Reagents used to quantify both organic phosphate and inorganic phosphate'80 include 10 % Mg(NO_,)2 (w/v, dissolved in ethanol), 0.5 M HCl, 10% ascorbic acid (w/v, dissolved in H20), and 0.42% (NH,,)2MoO4 (w/v, dissolved in 1 M H2304). The assay solution (freshly mixed) consists of one volume of 10% ascorbic acid and six volumes of 0.42% (NH4)2MoO4. To assay for organic phosphate, 100 uL of 10% Mg(NO;.,)2 was added to a test tube (1.3 mm x 100 mm) containing 100 uL of sample. The resulting mixture was then evaporated to dryness over a flame, leaving a white solid. To this test tube was added 600 uL of 0.5 M HCl. After the white solid was dissolved, the solution was heated at 100 °C for 15 min in a boiling water bath. Assay solution (1400 uL, described above) was 176 added to the cooled sample and the resulting mixture was kept at 45 °C for 20 min. If the original sample contains either inorganic phosphate or any organic phosphate, a blue color will develop. To assay for inorganic phosphate, 600 uL of 0.5 M HCl and 1400 uL of assay solution were directly added to a test tube containing 100 uL of sample. The resulting reaction mixture was then heated at 45 °C for 20 min. Blue color is developed if sample contains inorganic phosphate. The phosphate concentration of a sample was quantified by comparing the absorbance at 820 nm of the sample to a standard curve that is prepared using a phosphorous standard solution (0.65 mM in 0.05 M HCl, Sigma 661-9). Ninhydrin assay The ninhydrin reagent100 contains NaOAc (15%, w/v), sulfolane (40%, v/v), ninhydrin (2%, w/v), and hydrindantin (0.36%, w/v) in H20. ThepH of the reagent was adjusted to 2.5 with glacial acetic acid and the reagent was then filtered through filter paper. An aliquot (50 uL) of the sample was added to a test tube (13 mm x 100 mm) containing 500 uL of ninhydrin reagent. The resulting mixture was heated at 100 °C for 5 min. A purple color develops if the sample contains free amino group. The concentration of amino group containing compound in a sample was quantified by comparing the absorbance at 570 nm of the sample to a standard curve that is prepared using glycine as the standard. 177 Bacterial Strains and Plasmids E. coli DHSa [F’ endAI hstI7(r'Km+K) supE44 thi-I recAI gyrA relAI ¢801acZAM15 A(lacZYA-argF)U,69], JWFl (RB791 serA),lS3 JM109 [e14-(McrA-) recAI endAI gyrA96 thi-I hst17(r'Km+K) supE44 relAI A(lac-pr0AB) [F’ traD36 proAB lacIQDM15]], and RB791 (W3110 lacL8IQ) were obtained previously by this laboratory. E. coli XLl-Blue MRF’ Kan [A(mcrA)183 A(mchB-hstMR-mrr)173 endAI supE44 thi- IrecAI gyrA96 relAI lac [F’ proAB lacIQZAMI5 Tn5 (KanR)] was purchased from Stratagene. E. coli SP1 . l45b (RB791 serA::aroB ar0L478.'.°Tn10 ar0K17::CmR) was used as the host strain for all the E. coli fermentations. Amycolatopsis mediterranei (ATCC 21789) and B. pumilus (ATCC 21143) were purchased from the American Type Culture 134 Collection (ATCC). A. mediterranei HGF014 mum”4 and cosmid FKN108, plasmid EDAHP,97 PEI,98 MMBOIZ,96 and pRM030142 were generously provided by Professor Heinz G. Floss (University of Washington). Shuttle vector pRL-60132 was generously provided by Professor Rup Lal (University of Delhi). Plasmid pTC325150 was obtained from Professor L. Ingram (University of Florida). Plasmid pKD12.13845 was previously constructed in this laboratory. Plasmid pQE30 was obtained from Qiagen. Plasmids pBT, pTRG, pBT-LGF2, and pTRG-GalII were purchased from Stratagene. Plasmid pKK223-3,155 pJF118EH,156 and pT7-7157 were obtained previously by this laboratory. Storage of Bacterial Strains and Plasmids All bacterial strains were stored at -78 °C in glycerol. Plasmids were transformed into E. coli DHSa, JWFl, JM109, or XL-l Blue MRF’ Kan for long-term storage. 178 Glycerol samples were prepared by adding 0.75 mL of an overnight culture to a sterile vial containing 0.25 mL of 80% (v/v) glycerol. The solution was mixed, left at room temperature for 2 h, and then stored at ~78 °C. Culture Medium Bacto tryptone, Bacto tryptone peptone, Bacto yeast extract, Bacto malt extract, agar, and soytone were purchased from Difco. Casein enzyme hydrolysate was obtained from Sigma. Meat extract was obtained from Fluka. Soy flour and peanut meal were purchased from ICN Bioscience. Nutrient agar was purchased from Oxoid. 158 All solutions were prepared in distilled, deionized water. LB medium (1 L) contained Bacto tryptone (10 g), Bacto yeast extract (5 g), and NaCl (10 g). M9 salts159 (l L) contained NazHPO4 (6 g), KHZPO4 (3 g), NH4C1 (l g), and NaCl (0.5 g). M9 minimal medium159 contained D-glucose (10 g), MgSO4 (0.12 g), and thiamine hydrochloride (0.001 g) in l L of M9 salts. M9 medium (1 L) was supplemented where appropriate with L-phenylalanine, L-tyrosine, L-tryptophan, and L-serine to a final concentration of 40 ug/mL and with p—hydroxybenzoic acid, potassium p-aminobenzoate, and 2,3-dihydroxybenzoic acid to a final concentration of 10 ug/mL. Antibiotics were added where required to the following final concentrations: chloramphenicol (Cm), 34 ug/mL; ampicillin (Ap), 50 ug/mL; carbenicillin (Ca), 200 ug/mL; tetracycline (Tc), 12.5 ug/mL; kanamycin (Kan), 50 ug/mL; and erythromycin (Ery), 200 or 500 ug/mL, as specified. Isopropyl [3-D-thiogalactopyranoside (IPTG) was added to the culture medium of strains containing inducible promoters including Pm, P77, or P75. Inorganic salts, D- glucose, and MgSO4 solutions were autoclaved separately while thiamine hydrochloride, 179 amino acids, aromatic vitamins, antibiotics, and IPT G were sterilized by passage through a 0.22-um membrane. Solid medium was prepared by the addition of 1.5% (w/v) agar to the liquid medium. Soft agar (100 mL) contained Bacto tryptone (1 g), Bacto yeast extract (0.5 g), and agar (0.55 g). A. mediterranei was grown in either YMG, vegetative, TYN, or production medium. YMG medium132 (1 L) contained Bacto yeast extract (4 g), Bacto malt extract (10 g), and glucose (4 g). Vegetative medium160 (1 L) contained meat extract (5 g), Bacto tryptone peptone (5 g), Bacto yeast extract (5 g), casein enzyme hydrolysate (2.5 g), glucose (20 g), and NaCl (1.5 g). TYN medium!32 (1 L) contained tryptone (10 g), yeast extract (2.5 g), and NaCl (5 g). Production medium!“ (1 L) contained glucose (115 g), peanut meal (25 g), (NH4)2SO4 (9.6 g), CaCO3 (9.5 g), and 1 mL of trace element solution consisting (1 L) of MgSO4-7 H20 (1 g), CuSO4-5 H20 (1 g), FeSO4-7 H20 (1 g), MnSO4-HZO (1 g), and Co(NO3)2-6 H20 (1 g). D-Glucose (60%, w/v) was autoclaved separately while trace element solution was sterilized by passage through a 0.22-um membrane. Solid YMG and TYN medium were prepared by the addition of 1.5% (w/v) agar to the liquid medium. YMG soft agar was prepared by the addition of 0.55% (w/v) agar to the liquid medium. 8. pumilus was grown either on solid nutrient agar or in liquid SSNG or SNG medium. Nutrient agar plates were prepared by the addition of 2r.8% (w/v) nutrient agar to H20. SSNG medium162 (1 L) contained soy flour (15 g), soytone (1 g). NaCl (6 g), and glucose (10 g). SNG mediummc contained (1 L) soytone (15 g), NaCl (3 g), and glucose (10 g). D-Glucose (20%, w/v) was autoclaved separately. 180 The standard E. coli fermentation medium45b (1 L) contained K2HP04 (7.5 g), citric acid monohydrate (2.1 g), ammonium iron (III) citrate (0.3 g), concentrated H2804 (1.2 mL), L-phenylalanine (0.7 g), L-tyrosine (0.7 g), and L-tryptophan (0.35 g). The pH of the medium was adjusted to 7.0 with concentrated NH4OH prior to autoclaving. The following supplements were added to the medium immediately prior to initiation of the fermentation: D-glucose (25 g or 30 g), MgSO4 (0.24 g), p-hydroxybenzoic acid (0.01 g), 2,3-dihydroxybenzoic acid (0.01 g), potassium p-aminobenzoate (0.01 g), and trace minerals including (NH4)6(Mo7Oz4)-4 H20 (0.0037 g), ZnSO4-7 H20 (0.0029 g), H3BO3 (0.0247 g), CuSO4-5 H20 (0.0025 g), and MnC12-4 H20 (0.0158 g). Solutions of D- glucose and MgSO4 were autoclaved separately while aromatic vitamins and trace minerals solutions were sterilized by passage through a 0.22 pm membrane. Aromatic amino acids were added to the culture medium at the beginning of the fermentor run as required for cell growth and at 16 h and 30 h to inhibit native E. coli DAHP synthases. Aqueous kanosamine solutions (25%, w/v), which were sterilized by passage through a 0.22 pm membrane, were added at 18, 24, and 30 h (10 mL per time point). Antifoam (Sigma 204) was added as needed. Under glucose-limited conditions, addition of D- g‘lucose (65% aqueous solution, w/v) was controlled by the oxygen sensor in order to maintain 10% air saturation. Under glucose-rich conditions, D-glucose (65% aqueous solution, w/v) was added to the vessel at a rate sufficient to maintain a glucose concentration of 15-30 g/L. Glucose concentration was determined using the Glucose Diagnostic Kit from Sigma. The standard A. mediterranei fermentation medium161 (1 L) contained soy flour (32 g) and (NH4)2SO4 (7.5 g). The pH of the medium was adjusted to 7.0 with 181 concentrated NH4OH prior to autoclaving. D-Glucose (126 g, 65% w/v) solution was added following sterilization. The following supplements were added to the medium immediately prior to initiation of the fermentation: MgSO4 (l g) and trace minerals including (NH4)6(Mo7Oz4)-4 HZO (0.0001 g), ZnSO4-7 H20 (0.005 g), CoC12° 6 H20 (0.0002 g), CuSO4-5 H20 (0.00033 g), Fe2(SO.,)3-7 H20 (0.001 g), and MnSO4-HZO (0.0004 g). Solution of D-glucose (65%, w/v) and MgSO4 (1 M) were autoclaved separately while trace mineral solution was sterilized by passage through a 0.22 am membrane. Antifoam (Sigma 204) was added as needed. The standard B. pumilus fermentation medium162 (1 L) contained soy flour (30 g), soytone (1 g), and NaCl (9 g). The pH of the medium was adjusted to 7.0 with 1 N NaOH prior to autoclaving. D-Glucose (30 g, 50% w/v) solution, which was autoclaved separately, was added to the medium immediately prior to initiation of the fermentation. One more batch of soy flour (10 g, autoclaved in 50 mL H20) was added at 48 h. Additional D-glucose (65%, w/v) was added to the fermentor vessel during a fermentation run to maintain a glucose concentration of 15-50 g/L. Glucose concentration was determined using the Glucose Diagnostic Kit from Sigma. Fermentation Conditions General Fermentations were conducted in a B. Braun M2 culture vessel with a 2 L working capacity. The vessel was modified using a stainless steel baffle cage containing four 1/4" x 4" baffles for all E. coli fermentations run under glucose-rich conditions. Environmental conditions were supplied by a B. Braun Biostat MD controlled by a DCU. 182 Data was acquired on a Dell Optiplex GX200 personal computer utilizing B. Braun MFCS/Win software. PID control loops were used to control temperature, pH, and glucose addition. The temperature was maintained at 33 °C, 30 °C, or 28 °C as specified, and the pH was maintained at 7.0 by addition of concentrated NH4OH or 2 N H2804. Glucose was added as a 65% (w/v) solution if required. Dissolved oxygen (D.O.) was monitored using a Mettler-Toledo 12 mm sterilizable O2 sensor fitted with an lngold A- type 02 permeable membrane. D.O. was maintained at 10% air saturation throughout the course of the fermentations. Antifoam (Sigma 204) was added manually as needed. Fed-batch fermentations of E. coli Growth of an inoculant was initiated by introduction of a single colony into a culture tube (18 mm x 150 mm) containing 5 mL of M9 medium and grown at 37 °C with agitation for 20 to 24 h. The 5 mL culture was then transferred to a 500 mL flask containing 100 mL of M9 medium and grown for an additional 10 to 12 h at 37 °C and 250 rpm. After an appropriate OD600 was reached (1.0-3.0), the inoculant was transferred to the fermentation vessel. The initial glucose concentration in the fermentation medium was 20 g/L for glucose-limited conditions and 30 g/L for glucose-rich conditions. For glucose-limited conditions, three staged methods were used to maintain D.O. concentrations at 10% air saturation during the fermentation runs. With the airflow at an initial setting of 0.06 L/L/min, the DO. concentration was maintained by increasing the impeller speed from its initial set point of 50 rpm to its preset maximum of 1100 rpm. With the impeller rate constant at 1100 rpm, the mass flow controller then maintained the DO. concentration by increasing the airflow rate from 0.06 L/L/min to a preset 183 maximum of 1.0 L/L/min. At constant impeller speed and constant airflow rate, the DO. concentration was finally maintained at 10% air saturation for the remainder of the fermentation by oxygen sensor-controlled glucose feeding. PID control parameters were set to 0.0 (off) for the derivative control (15D), and 999.9 3 (minimum control action) for integral control (n). Xp was set to 950.0% to achieve a KC of 0.1. For fermentations that employed glucose-rich conditions, the following three staged methods were used to maintain the DO. concentration at 10% air. With the airflow at an initial setting of 0.06 L/L/min, the DO. concentration was maintained by increasing the impeller speed from its initial set point of 50 rpm to a preset maximum of 1100 rpm. With the impeller rate constant at 1100 rpm, the mass flow controller then maintained the DO. concentration by increasing the airflow rate from 0.06 L/L/min to a preset maximum of 1.0 L/L/min. After the preset maxima of 1100 rpm and 1.0 LIL/min were reached, the third stage of the fermentation was initiated in which glucose (65% w/v) was added to the vessel at a rate sufficient to maintain a glucose concentration of 15-30 gfL for the remainder of the run. Airflow was maintained at 1.0 L/L/min, and the impeller was allowed to vary in order to maintain the DO. concentration at 10% air saturation. The impeller speed typically varied from 750 to 1600 rpm during the remainder of the run. Batch fermentations of A. mediterranei Growth of an inoculant was initiated by introduction of a single colony from a YMG plate into 100 mL of YMG medium in a 500 mL flask with baffles and grown at 28 °C with agitation for 72 to 168 h until significant growth was achieved (0D,,00 1.2-2.0). 184 The 100 mL culture was then transferred to the fermentation vessel. The D.O. levels were controlled at 10% air saturation during the course of the run. With the airflow at an initial setting of 0.06 L/L/min, the DO. concentration was maintained by increasing the impeller speed from its initial set point of 200 rpm to its preset maximum of 1100 rpm. With the impeller rate constant at 1100 rpm, the mass flow controller then maintained the DO. concentration by increasing the airflow rate from 0.06 LIL/min until a maximum airflow rate was reached (usually less than 0.2 L/L/min). Afterwards, airflow was maintained at 0.2 L/L/min, and the impeller was allowed to vary in order to maintain the DO. concentration at 10% air saturation. The impeller speed typically varied between 500 and 1000 rpm during the remainder of the run. Fed-batch fermentations of B. pumilus Growth of an inoculant was initiated by introduction of a single colony from a nutrient agar plate into 100 mL of SSNG medium in a 500 mL flask with baffles and grown at 30 °C with agitation. Since SSNG medium was a heterogeneous medium (a suspension of soy flour), cell growth could not be monitored by following OD600. The pH of the culture supernatant was used to monitor the stage of the cell growth. The pH of the culture supernatant decreased from 7.0 to approximately 5.0 during the first 24 h of growth and increased from 5.0 to 8.5163 thereafter due to the formation of kanosaminemc When the pH of the culture supernatant reached 7.5 (30-48 h), the 100 mL culture was then transferred to the fermentation vessel. Three staged methods were used to maintain D.O. concentrations at 10% air saturation during the fermentation run. With the airflow at an initial setting of 0.06 L/L/min, the DO. concentration was 185 maintained by increasing the impeller speed from its initial set point of 200 rpm to its preset maximum of 1100 rpm. With the impeller rate constant at 1100 rpm, the mass flow controller then maintained the DO. concentration by increasing the airflow rate from 0.06 L/L/min until a maximum airflow rate was reached (usually less than 0.5 L/L/min). Afterwards, airflow was maintained at 0.5 LIL/min, and the impeller was allowed to vary in order to maintain the DO. concentration at 10% air saturation. The impeller speed typically varied between 500 and 1100 rpm during the remainder of the run. Additional D-glucose (65% w/v) was added to the fermentor vessel during the fermentation run to maintain a glucose concentration of 15-50 g/L. Glucose concentration was determined using the Glucose Diagnostic Kit from Sigma. Analysis of Culture Supernatant For strains being evaluated in shake flasks, samples (4 mL) of the culture were taken at timed intervals and the cells were removed by microcentrifugation. For strains being evaluated in fermentors, samples (5 mL) of broth were taken at indicated intervals, and cell densities of E. coli were determined by dilution of fermentation broth with water (1:100) followed by measurement of absorption at 600 nm (ODwO). Dry cell weight for E. coli (g/L) was obtained using a conversion coefficient of 0.43 g/L/OD600. The remaining fermentation broth was microcentrifuged to obtain cell-free broth. Cell densities of A. mediterranei and B. pumilus were not measured if heterogeneous fermentation medium was used. Cell-free samples of A. mediterranei and B. pumilus were obtained by microcentrifugation of the corresponding fermentation broth. 186 Solute concentrations in the cell-free culture supernatant were determined by 1H NMR. Solutions were concentrated to dryness under reduced pressure, concentrated to dryness on additional time from D20, and then redissolved in D20 containing a known concentration of the sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP). Concentrations were determined by comparison of the integrals corresponding to each compound with the integral corresponding to TSP (6:0.00 ppm) in the 1H NMR. Compounds were quantified using the following resonances: shikimate (6 4.40, t, 1 H), aminoshikimate (6 6.56, m, l H), and kanosamine (6 5.27, d, 0.45 H). The concentrations of shikimate, aminoshikimate , and kanosamine were calculated by application of the previously described calibration formulae. Product Purification from Fermentation Broth Purification of kanosamine from fermentation broth of B. pumilus After the completion of the fermentation, the fermentation broth was passed through four layers of cheesecloth and the cells in the filtrate were removed by centrifugation (6400g, 4°C, 10 min). Activated charcoal (10 g/L) was then added to the supernatant and the mixture was shaken at 37°C and 250 rpm for 1 h. The charcoal was removed by filtration through Celite and the protein in the filtrate was removed by ultrafiltration. The resulting protein free solution was concentrated to about 200 — 300 mL and applied to Dowex 50 (H*, 2.5 cm x 51 cm, 250 mL) resin. The column was washed with H20 (600 mL) and eluted with a linear gradient (600 mL + 600 mL, 0 to 0.5 M) of HCl. Fractions testing positive by the ninhydrin assay were collected and evaporated under reduced pressure to give kanosamine as a slightly brown solid (very hygroscopic). 1H NMR (500 MHz, DZO, TSP as reference): 6 5.27 (d, J = 3.5 Hz, 0.4 H), 187 4.72 (d, J = 8 Hz, 0.6 H), 4.39 (m, l H), 3.74-4.38 (m, 2 H), 3.67 (ddd, J = 10, 10, 4.5 Hz, 1 H), 3.57 (ddd, J = 7.5, 5.5, 2 Hz, 0.5 H), 3.45 (dd, J = 10, 8 Hz, 0.5 H), 3.41 (dd, J = 10, 10 Hz, 0.5 H), 3.25 (dd, J = 10, 10 Hz, 0.5 H). 13C NMR (125 MHz, D20, TSP as reference): 6 99.0, 94.1, 79.7, 74.2, 73.4, 71.0, 68.9, 68.8, 63.2, 63.0, 60.8, 58.1. Purification of aminoshikimate from fermentation broth of A. mediterranei After the completion of the fermentation, the fermentation broth was passed through four layers of cheesecloth and the cells in the filtrate were removed by centrifugation (6400g, 4°C, 10 min). Activated charcoal (10 g/L) was then added to the supernatant and the mixture was shaken at 37°C and 250 rpm for 1 h. The charcoal was removed by filtration through Celite and the protein in the filtrate was removed by ultrafiltration. A portion of the protein free solution (100 mL) was applied to Dowex 50 (H+, 2.5 cm x 10 cm, 50 mL) resin. The column was washed with H20 (100 mL) and eluted with a linear gradient (150 mL + 150 mL, 0-0.5 M) of HCl. Ninhydrin positive fractions were collected and the solvent evaporated under reduced pressure to give aminoshikimate as a pale yellow solid. 1H NMR (500 MHz, D20, TSP as reference): 6 6.56 (m, 1H, H-2), 4.39 (dd, J = 5, 5 Hz, 1 H, H-3), 3.85 (dd, J = 11, 5 Hz, 1 H, H-4), 3.51 (ddd, J = 11, 11, 6 Hz, 1 H, H-S), 2.96 (dd, J = 17, 6 Hz, 1 H, H-6a), 2.37 (m, 1 H, H-6b), 13C NMR (125 MHz, D20, TSP as reference): 6 177.0, 138.4, 133.3, 72.4, 67.9, 50.7, 32.8. HRFABMS: calcd for C7H12NO4 (M + H“): 174.0720. Found: 174.0760. Purification of aminoshikimate from fermentation broth of E. coli After the completion of the fermentation, the cells were removed by centrifugation (6400g, 4°C, 10 min). The protein in the supernatant was removed by 188 ultrafiltration. A portion of the protein free solution (100 mL) was applied to Dowex 50 (H”, 2.5 cm x 10 cm, 50 mL) resin. The column was washed with H20 (100 mL) and eluted with a linear gradient (150 mL + 150 mL, 0-0.5 M) of HCl. Ninhydrin positive fractions were collected and the solvent evaporated. under reduced pressure to give aminoshikimate as a pale yellow solid. IH NMR (500 MHz, D20, TSP as reference): 6 6.56 (m, 1H, H-2), 4.39 (dd, J = 5, 5 Hz, 1 H, H-3), 3.85 (dd, J = 11, 5 Hz, 1 H, H-4), 3.51 (ddd, J = 11, 11, 6 Hz, 1 H, H-S), 2.96 (dd, J = 17, 6 Hz, 1 H, H—6a), 2.37 (m, 1 H, H-6b), 13C NMR (125 MHz, D20, TSP as reference): 6 177.0, 138.4, 133.3, 72.4, 67.9, 50.7, 32.8. HRFABMS: calcd for C7H12NO4 (M + H”): 174.0720. Found: 174.0752. Genetic Manipulations General procedures Standard protocols were used for construction, purification, and analysis of plasmid DNA.‘59 E. coli DHSa, JM109, XL-l Blue MRF’ Kan, and JWFl were used as the host strain for plasmid manipulations. T4 DNA Ligase, Large Fragment of DNA polymerase I (Klenow fragment), dNTP’s, and agarose (electrophoresis grade) were purchased from Invitrogen. Fastlink DNA ligase was purchased from Epicentre. Restriction enzymes were purchased from Invitrogen or New England Biolabs. Calf intestinal alkaline phosphatase was purchased from New England Biolabs. The BacterioMatch Two-Hybrid System Kit was obtained from Stratagene. PCR amplifications were performed as described by Sambrook.159 Each reaction (0.1 mL) contained 10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 2 mM MgSO,,, 189 0.1% Triton X-100, dATP (0.2 mM), dCTP (0.2 mM), dGTP (0.2 mM), d'ITP (0.2 mM), template DNA (0.02 tag or 1 pg), 0.5 1.1M of each primer, and 2 units of Vent polymerase. Primers were synthesized by the Macromolecular Structure Facility at Michigan State University. SEVAG is a mixture of chloroform and isoamyl alcohol (24:1 v/v). TE buffer contained 10 mM Tris-HCl (pH 8.0) and 1 mM disodium EDTA (pH 8.0). Endostop solution (10X concentration) contained 50% glycerol (v/v), 0.1 M disodium EDTA (pH 7.5), 1% sodium dodecyl sulfate (SDS) (w/v), 0.1% bromophenol blue (w/v), and 0.1% xylene cyanole FF (w/v) and was stored at 4 °C. Prior to use, 0.12 mL of DNase-free RNase was added to 1 mL of 10X Endostop solution. Determination of DNA concentration The concentration of DNA in a sample was determined as follows: an aliquot (10 uL) of the DNA solution was diluted to 1 mL in TE and the absorbance at 260 nm was measured relative to the absorbance of TE. An absorbance of 1.0 at 260 nm corresponds to 50 ug/mL of double stranded DNA. Large scale purification of plasmid DNA Plasmid DNA was purified on a large scale using a modified lysis method as 9 described by Sambrook et al.15 In a 2 L Erlenmeyer flask, 500 mL of LB medium containing the appropriate antibiotic was inoculated from a single colony, and the culture was incubated at 37 °C (E. coli strains harboring plasmids used for two-hybrid experiments were grown at 30 °C) for approximately 12 h (24 h if culturing at 30 °C) 190 with agitation at 250 rpm. In cases where the plasmid did not possess an antibiotic marker, 500 mL of M9 minimal medium was used and the culture was shaken (250 rpm) at 37°C for 24 h. Cells were harvested by centrifugation (4000g, 5 min, 4 °C) and then resuspended in 10 mL of cold solution 1 (50 mM glucose, 20 M Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0) into which lysozyme (5 mg/mL) had been added immediately before use. The suspension was stored at room temperature for 5 min. Addition of 20 mL of solution 2 (1% SDS (w/v) in 0.2 N NaOH) was followed by gentle mixing and storage on ice for 15 min. Fifteen milliliters of ice cold solution 3 (prepared by mixing 60 mL of 5 M KOAc, 11.5 mL of glacial acetic acid, and 28.5 mL of water) was added. Vigorous shaking resulted in formation of a white precipitate. After the suspension was stored on ice for 10 min, the cellular debris was removed by centrifugation (48000g, 20 min, 4 °C). The supernatant was transferred to two centrifuge bottles and isopropanol (0.6 volumes) was added to precipitate the DNA. After the samples were left at room temperature for 15 min, the DNA was recovered by centrifugation (20000g, 20 min, 4 °C). The DNA pellet was rinsed with 70% ethanol and dried. The isolated DNA was dissolved in TE (3 mL) and transferred to a Corex tube. Cold 5 M LiCl (3 mL) was added and the solution was gently mixed. The sample was centrifuged (12000g, 10 min, 4 °C) to remove high molecular weight RNA. The clear supernatant was transferred to a Corex tube and isopropanol (6 mL) was added followed by gentle mixing. The precipitated DNA was collected by centrifugation (120003, 10 min, 4°C). The DNA was then rinsed with 70% ethanol and dried. After redissolving the DNA in 0.5 mL of TE containing 20 ug/mL of RN ase, the solution was transferred to a 1.5 mL microcentrifuge tube and stored at rt for 30 min. DNA was precipitated from the 191 solution upon addition of 500 ML of 1.6 M NaCl containing 13% PEG-8000 (w/v). The solution was mixed and microcentrifuged (10 min, 4 °C) to recover the precipitated DNA. The supernatant was removed and the DNA was then redissolved in 400 [L of TE. The sample was extracted sequentially with phenol (400 uL), phenol and SEVAG (400 uL each), and finally SEVAG (400 uL). Ammonium acetate (10 M, 100 uL) was added to the aqueous DNA solution. After thorough mixing, 95% ethanol (1 mL) was added to precipitate the DNA. The sample was left at rt for 5 min and then microcentrifuged for 5 min at 4 °C. The DNA was rinsed with 70% ethanol, dried, and then redissolved in 250-500 uL of TE. Large scale purifications of plasmid DNA were also performed by using the Maxi Kit (Qiagen) following the protocol supplied by the manufacturer. Small scale purification of plasmid DNA An overnight culture (5 mL) of the plasmid-containing strain was grown in LB medium containing the appropriate antibiotics. In cases where nutritional pressure was used to maintain plasmid, 5 mL of M9 minimal medium was used and the culture grown for 24 h. Cells from 3 mL of the culture were collected in a 1.5 mL microcentrifuge tube by microcentrifugation. The harvested cells were resuspended in 0.1 mL of cold solution 1 into which lysozyme (5 mg mL") was added immediately before use. The suspension was incubated on ice for 10 min and then treated with solution 2. The mixture was shaken gently and kept on ice for 5-10 min. To this sample was added 0.15 mL of cold solution 3, and the mixture was shaken vigorously, resulting in formation of a thick white precipitate. The sample was stored on ice for 5 min, after which the precipitate was 192 removed by microcentrifugation (15 min, 4°C). The supernatant was transferred to another microcentrifuge tube and extracted with equal volumes of phenol and SEVAG (0.2 mL each). The aqueous phase (approximately 0.5 mL) was transferred to a microfuge tube and the DNA was precipitated by addition of 95% ethanol (1 mL). The sample was left at room temperature for 5 min before microcentrifugation (15 min, rt) to isolate the DNA. The DNA pellet was rinsed with 70% ethanol, dried, and redissolved in 50-100 uL TE. DNA isolated from this method was used for restriction enzyme analysis. Restriction enzyme digestion of DNA A typical digest (10 uL) contained approximately 7.5 uL of DNA (0.1 ug/uL in TE), 1 uL of restriction enzyme buffer (10X concentration), 1 ML of restriction enzyme, and 0.5 uL of bovine serum albumin (BSA, 2 mg/mL). Reactions were incubated at 37 °C for l h. Digests were terminated by addition of 1 uL of Endostop solution (10X concentration) and subsequently analyzed by agarose gel electrophoresis. When DNA was required for subsequent cloning, restriction digests were terminated by addition of l uL of 0.5 M EDTA (pH 8.0) followed by extraction of the DNA with equal volumes of phenol and SEVAG and precipitation of the DNA. DNA was precipitated by addition of 0.] volume of 3 M NaOAc (pH 5.2) followed by thorough mixing and the addition of 3 volumes of 95% ethanol. Samples were stored for at least 1 h at -78 °C. Precipitated DNA was recovered by microcentrifugation (15 min, 4 °C). Ethanol (70%) was added and the sample was microcentrifuged for 10 min at 4°C. Ethanol was then discarded, and the DNA was air dried and redissolved in TB. 193 Alternatively, the DNA was isolated from the reaction mixture using Zymoclean DNA Clean and Concentrate Kit (Zymo Research). Agarose gel electrophoresis Agarose gels were run in TAE buffer containing 40 mM Tris-acetate and 2 mM EDTA (pH 8.0). Gels typically contained 0.7% agarose (w/v) in TAE buffer. Lower concentrations of agarose (0.3%) were used to resolve genomic DNA and electrophoresis was conducted at 4 °C instead of rt. Ethidium bromide (0.5 tag/ml) was added to the agarose to allow visualization of DNA fragments under a UV lamp. The size of the DNA fragments were determined by comparison to DNA fragments contained in the following: A. DNA digested with HindIII (23.1-kb, 9.4-kb, 6.6-kb, 4.4-kb, 2.3-Rb, 2.0-kb, and 0.6- kb) and 7t DNA digested with EcoRI and Hindlll (21.2-kb, 5.1-kb, 5.0-kb, 4.3-kb, 3.5-Rb, 2.0-kb, 1.9-kb, 1.6-kb, 1.4-kb, 0.9-kb, 0.8-kb, and 0.6-kb). Isolation of DNA from agarose Zymoclean DNA Isolation Kit (Zymo Research) was used to isolate DNA from agarose. The band of agarose containing the DNA of interest was excised from the gel with a razor and transferred to a 1.5 mL microfuge tube. Three volumes of agarose dissolving buffer was added to each volume of agarose gel and the resulting suspension was incubated at 42°C with intermittent vortexing until the gel was completely dissolved. DNA in the dissolved agarose solution was subsequently purified using a Zymo-Spin Column. 194 Treatment of DNA with Klenow fragment DNA fragments with recessed 3’ termini were modified to DNA fragments with blunt ends by treatment with the Klenow fragment of E. coli DNA polymerase 1. After restriction enzyme digestion (20 uL) of the DNA (1.5 pg) was complete, a solution (1 uL) containing each of the four dNTP’s (25 mM) was added. Addition of l to 2 units of Klenow fragment was followed by incubation at rt for 30 min. Since the Klenow fragment works well in the common restriction enzyme buffers, there was no need to purify the DNA after restriction digestion and prior to filling recessed 3’ termini. Klenow reactions were quenched by extraction with equal volumes of phenol and SEVAG. DNA was recovered by precipitation as described previously and redissolved in TE. Alternatively, the DNA was isolated from the reaction mixture by using Zymoclean DNA Clean and Concentrator Kit (Zymo Research). Treatment of DNA with mung bean nuclease DNA fragments with 3’ overhang were modified to DNA fragments with blunt ends by treatment with mung bean nuclease. After restriction digestion (120 uL) of the DNA (9 u g) was complete, DNA was purified by Zymoclean DNA clean and concentration Kit (Zymo Research). A typical reaction (160 uL) contained approximately 9 ug DNA, 16 uL of mung bean nuclease buffer (10X concentration), 8 uL of mung bean nuclease, and 136 uL of TE. Reactions were incubated at 30 °C for 30 min. After the completion of the reaction, DNA was purified by using Zymoclean DNA Clean and Concentrator Kit. 195 Treatment of vector DNA with calf intestinal alkaline phosphatase (CIAP) Following restriction enzyme digestion, vectors were dephosphorylated to prevent self-ligation. Digested vector DNA was dissolved in TE (88 uL). To this sample was added 10 uL of dephosphorylation buffer (10X concentration) and 2 uL of calf intestinal alkaline phosphatase (2 units). The reaction was incubated at 37 °C for 1 h. Phosphatase was inactivated by the addition of 1 uL of 0.5 M EDTA (pH 8.0) followed by heat treatment (65 °C, 20 min). The sample was extracted with phenol and SEVAG (100 uL each) to remove the protein, and the DNA was precipitated as previously described and redissolved in TE. Alternatively, the DNA was isolated from the reaction mixture by using Zymoclean DNA Clean and Concentrator Kit. Ligation of DNA DNA ligations were designed so that the molar ratio of insert DNA to vector DNA was 3 to l. A typical ligation reaction contained 0.03 to 0.1 ug of vector DNA and 0.05 to 0.2 ug of insert DNA in a combined volume of 7 uL. To this sample, 2 uL of ligation buffer (5X concentration) and 1 uL of T4 DNA ligase (2 units) were added. The reaction was incubated at 16 °C for at least 4 h and then was used to transform competent cells. In an alternative method, the Fast-link DNA Ligation Kit (Epicentre) was used according to the manufacturer’s protocol. Purification of E. coli genomic DNA Genomic DNA was purified using a modified method described by Silhavy.164 After being cultured in LB at 30 °C with shaking at 250 rpm for approximately 12 h, the 196 cells were harvested by centrifugation (4000g, 5 min, 4 °C). The harvested cells were resuspended in 9.5 mL of TE and transferred to a small centrifuge bottle. To the cell suspension, 0.5 mL of 10% SDS and 0.05 mL of freshly prepared proteinase K (20 mg/mL) were added. After being incubated at 37 °C for 1 h, NaCl (5 M, 1.8 mL) solution was added. The sample was mixed thoroughly and 1.5 mL of CTAB/NaCl solution (aqueous solution containing 0.1 g/mL of hexadecyltrimethylammonium bromide and 0.041 g/mL of NaCl) was added. After mixing thoroughly, the sample was incubated at 65 °C for 20 min. The sample was transferred to two Corex tubes and extracted with an equal volume of SEVAG. After centrifugation (6000g, 10 min, 4 °C), the aqueous portion was transferred to fresh Corex tubes and the DNA was precipitated by the addition of 0.6 volumes of isopropanol to each Corex tube. All transfers of aqueous layers were carried out using large bore pipette tips to minimize shearing of the genomic DNA. After storage for 2 h at rt, threads of DNA were spooled onto a flame- sealed Pasteur pipette and transferred to a single Corex tube containing 70 % ethanol. The ethanol was removed, the DNA was dried, and resuspended in 1 mL of TE. To this solution, 10 uL of RNase (10 mg/mL) was added and the solution was stored at rt for 4 h. After a combined extraction with phenol (1 mL) and SEVAG (1 mL), 0.1 mL of 3 M NaOAc (pH 5.2) was added to the aqueous layer and mixed thoroughly. DNA was precipitated by the addition of 3 mL of 95% ethanol. After storage at rt for 1.5 h, the DNA threads were spooled onto a flame-sealed Pasteur pipette and dipped into 1 mL of 70% ethanol. The spooled DNA was then transferred to a 1.5 mL microfuge tube, dried, and redissolved in 0.5 mL of TE. DNA was quantified as described in the early part of this Chapter. 197 Purification of B. pumilus genomic DNA 1453 Genomic DNA was purified following a literature method. B. pumilus was grown in 100 mL of SNG medium at 30 °C with shaking at 250 rpm for approximately 36 h. The cells were harvested by centrifugation (4400g, 5 min, 4 °C) and the culture medium was discarded. Harvested cells were resuspended in 20 mL of lysis buffer containing 0.15 M NaCl, 0.1 M EDTA, and 0.5 mg/mL lysozyme (pH 8.0) and incubated at 37 °C for 1 h (swirling occasionally). The following solutions were consecutively added in the indicated order: 1 mL of 10% SDS, 4 mL of 5 M NaClO4, and 40 mL of SEVAG. The solution was thoroughly, but gently mixed follow each addition. The final mixture was then shaken gently at rt for 15 min. After centrifugation (17000g, 20 rrrin, 4 °C), the aqueous portion was transferred to a 100 mL beaker. Cold 95% ethanol (2 volume) was added and the solution was mixed well. DNA was spooled out of the solution with a flame-sealed Pasteur pipette and the DNA was transferred to another beaker containing 20 mL of cold 70% ethanol. After washing, the DNA was spooled onto a flame-sealed Pasteur pipette and transferred to a microfuge tube, air dried, and dissolved in 5 mL TEN buffer consisting of 10 mM Tris-HCl (pH 7.6), 1 mM EDTA, and 10 mM NaCl. To this solution, 20 uL of DNase-free RNase (10 mg/mL) was added and the solution was stored at 37 °C for l h. After a combined extraction with phenol and SEVAG (1:1, 1 volume), DNA was precipitated by the addition of 10 mL of 95% ethanol, spooled onto a flame-sealed Pasteur pipette, washed with cold 70% ethanol, dried, and redissolved in 1 mL of TE. DNA was quantified as described earlier in this Chapter. 198 Purification of A. mediterranei genomic DNA .165 . . A. medzterranez Genomic DNA was purified following a literature method. was grown in 100 mL of vegetative medium at 28 °C with shaking at 250 rpm for approximately 72 h. The mycelia were harvested by centrifugation (17700g, 5 min, 4 °C) and the culture medium was discarded. The harvested mycelia were washed with 10 mM EDTA (pH 8.0, 50 mL). After centrifugation (17700g, 5 min, 4 °C), the cells were resuspended in 3 mL of TEZSS buffer (25 mM Tris-HCI, pH 8.0, 25mM EDTA, 300 mM sucrose, and 2 mg/mL of lysozyme) and incubated at 37 °C for 10 min. Four milliliter of 2xKirby mixI66 consisting of 2% SDS (w/v), 6% phenol (v/v), and 12% sodium 4- aminosalicylate (w/v) was added and the resulting mixture was gently agitated for 1 min on vortex mixer. SEVAG (8 mL) was added, and the sample was gently agitated for 15 s on vortex mixer. After centrifugation (22003, 10 min, 4 °C), the aqueous portion was transferred to a fresh tube containing 3 mL of SEVAG and 0.6 mL of 3 M NaOAc (unbuffered), gently agitated on vortex mixer, and centrifuged at 2200g and 4°C for 10 min. The aqueous portion was transferred to a fresh tube and 0.6 volumes of isopropanol was added. After mixing, the DNA was spooled out of the solution with a flame-sealed Pasteur pipette and washed with cold 70% ethanol. The DNA was then redissolved in 0.05 mL of T E. To this solution, RN ase (10 mg/mL) was added to a final concentration of 40 ug/mL and the solution was stored at 37 °C for 30 min. After a combined extraction with phenol and SEVAG (1:1, 1 volume), DNA was precipitated by the addition of 0.6 volumes of isopropanol, spooled onto a flame-sealed Pasteur pipette, washed with cold 70% ethanol, dried, and redissolved in 1 mL of TE at 55 °C. DNA was quantified as described earlier in this Chapter. 199 Preparation of and transformation of E. coli competent cells Competent cells were prepared using a procedure modified from Sambrook et 159 al A single colony was inoculated into 5 mL‘of LB containing the necessary antibiotics. After overnight growth, an aliquot (1 mL) of the culture was transferred to a 500 mL Erlenmeyer flask containing 100 mL of LB containing the necessary antibiotics. The cells were cultured at 37 °C with shaking at 250 rpm until an OD600 of 0.4-0.6 was reached. The entire culture was transferred to a centrifuge bottle that was sterilized with bleach and rinsed exhaustively with sterile water. The cells were harvested by centrifugation (4000g, 4 °C, 5 min) and the culture medium was discarded. All manipulations were carried out on ice during the remainder of the procedure. Harvested cells were resuspended in 100 mL of ice cold 0.9% N aCl. After centrifugation at 4000g and 4 °C for 5 min, the cells were resuspended in ice cold 100 mM CaCl2 (50 mL) and stored on ice for 30 min. The cells were then collected by centrifugation (4000g, 5 min, 4 °C) and resuspended in 4 mL of ice cold 100 mM CaCl2 containing 15% glycerol (v/v). Aliquots (0.25 mL) of competent cells were transferred to 1.5 mL microfuge tubes, frozen in liquid nitrogen, and stored at -78 °C. In order to perform a transformation, frozen competent cells were thawed on ice for 5 min prior to use. A small aliquot (l to 10 uL) of plasmid DNA or a ligation reaction was added to the thawed competent cells (0.1 mL). The solution was gently mixed and stored on ice for 30 min. The cells were then heat shocked at 42 °C for 2 min and then placed on ice for 2 min. LB (0.5 mL) was added to the cells, and the sample was incubated at 37 °C for 1 h (or 30 °C for 1.5 h with agitation). After incubation, cells 200 were collected by microcentrifugation. If the transformation was to be plated onto LB plates, 0.5 mL of the culture supernatant was removed. The cells was then resuspended in the remaining 0.1 mL of LB and spread onto LB plates containing the appropriate antibiotics. If the transformation was to be plated onto minimal medium plates, all the culture supernatant was removed. The cells were washed with 0.5 mL of M9 salts and collected by microcentrifugation. After removal of all the supernatant, the cells were resuspended in 0.1 mL of M9 salts and spread onto the appropriate plates. A sample of competent cells with no DNA added was also carried through the transformation procedure as a control. These cells were used to check the viability of the competent cells and to verify the absence of growth on selective medium. Preparation of and electroporation of A. mediterranei electrocompetent cells Electrocompetent A. mediterranei were prepared using a procedure modified from literature procedures.132 A single colony of A. mediterranei was transferred from a YMG plate to a 500 mL flask (with baffles) containing 50 mL of TYN medium and the resulting culture was incubated at 28 °C with agitation. An aliquot (1 mL) from this fully grown culture (OD600 2.0-2.5) was used to inoculate 100 mL of TYN medium. A. mediterranei was then cultured at 28 °C with shaking at 250 rpm until the late log phase was reached (OD600 1.2). The entire culture was transferred to a centrifuge bottle that was sterilized with bleach and rinsed exhaustively with sterile water. The mycelia were harvested by centrifugation (11000g, 10 min, 4 °C) and the culture medium was discarded. The harvested mycelia were washed with 100 mL of ice cold sterilized water, and the mycelia were collected by centrifugation (11000g, 10 min, 4 °C). The harvested 201 mycelia were resuspended in 10% glycerol containing 4 ug/mL lysozyme and incubated for 20 minutes at 25 °C. The mycelia were then harvested by centrifugation (4400g, 5 min) and washed with ice cold 10% glycerol (10 mL). After centrifugation (4400g, 5 min, 4 °C), the pelleted mycelia were resuspended in 2 mL of ice cold 10% glycerol. Aliquots (0.2 mL) of competent cells in 1.5 mL microfuge tubes were frozen in liquid nitrogen and stored at -78 °C. In order to perform a transformation, frozen electrocompetent cells were thawed on ice for 5 min. T hawed cells (200 uL) were transferred to an electroporation cuvette (0.2 cm, pre-chilled) and 2.0 ug of DNA was added. The electroporations (Bio-Rad Gene Pulser) were performed at field strength of 7.5 kV/cm, resistance of 600 S2, and capacitance of 25 uF. Electroporated cells were then plated onto YMG medium and incubated overnight. The YMG agar plates were then overlaid with 2.5 mL of YMG soft agar containing 500 ug/mL of erythromycin and continued to incubate at 28°C until colonies appeared. Under these transformation parameters, a transformation efficiency of 1x103 transformants/ug DNA was routinely obtained. The erythromycin-resistant colonies were then subjected to a second screen using (it-amylase as a morphological marker.132 Erythromycin-resistant colonies were replicated onto YMG plates containing 1% starch (w/v) and 200 ug/mL of erythromycin. The starch was autoclaved together with YMG medium while erythromycin solution was sterilized by passage through a 0.22 gm membrane. After incubation at 28 °C for 4 days, the colonies were exposed to iodine vapours and the activity of (at-amylase was detected by the appearance of a colorless halo around colonies harboring plasmid. 202 General Enzymology General information E. coli and B. pumilus cells were harvested at 40003 for 5 min at 4°C and A. mediterranei were harvested at 110003 for 5 min at 4°C. Cells lysis was achieved by two passages through a French pressure cell (SLM Aminco) at 16,000 psi. Cellular debris was removed from the lysate by centrifugation (300003, 30 min, 4 °C). Protein solutions were concentrated by ultrafiltration using either Millipore PM-lO membranes (10,000 MWCO) or Centricon concentrators (Amicon). Protein concentrations were determined using the Bradford dye—binding procedure167 using protein assay solution purchased from Bio-Rad. The assay solution was prepared by diluting 20 mL of the Bio-Rad concentrate to 100 mL with water followed by gravity filtration of the resulting solution. Assay solution (5 mL) was added to an aliquot of protein containing solution (diluted to 0.1 mL) and the sample was vortexed. After allowing the color to develop for 5 min, the absorbance at 595 nm of the solution was measured. Protein concentrations were determined by comparison to a standard curve prepared using bovine serum albumin. DAHP synthase (AroFFBR) and aminoDAHP (RifH) synthase assay DAHP synthase and aminoDAHP synthase were assayed according to the procedure described by Schoner.I68 D-Erythrose 4-phosphate169 (E4P) and phosphoenolpyruvateI70 (PEP) required for the assay of DAHP synthase activity were synthesized in this laboratory according to literature procedures. A dilute solution of E4P was first concentrated to 12 mM by rotary evaporation and neutralized with 5 N KOH. 203 Two different solutions were prepared and incubated separately at 37 °C for 5 min. The first solution (1 mL) contained E4P (6 mM), phosphoenolpyruvate (12 mM), ovalbumin (1 mg/mL), and potassium phosphate (25 mM, pH 7.0). The second solution (0.5 mL) contained an appropriate amount of enzyme that had been diluted with buffer consisting of 50 mM potassium phosphate (pH 7.0), 0.5 mM phosphoenolpyruvate, and 250 mM 1,3-propanediol. After the two solutions were mixed (time = 0), aliquots (0.15 mL) were removed at timed intervals and quenched with 0.1 mL of 10% trichloroacetic acid (w/v). Precipitated protein was removed by microcentrifugation and the DAHP in each sample was quantified using the thiobarbituric acid assay99 as described before. One unit of DAHP synthase activity was defined as the formation of 1 umol of DAHP per min per mg of protein at 37 °C. Transketolase (TktA or Orf15) assay Transketolase activity was assayed using the method of Paoletti.171 A dilute solution of E4P169 was concentrated to 10 mM, and the pH was adjusted to 7.0 with KOH. The assay mixture (1 mL) contained 200 mM triethanolamine (pH 7.6), 5 mM MgC12, 0.1 mM thiamine pyrophosphate, 0.4 mM NADP, 0.4 mM B-hydroxypyruvate, 0.2 mM E4P, glucose-6-phosphate dehydrogenase (8 units), and phosphoglucose isomerase (4 units). The solution was allowed to equilibrate at rt for 5 min and the absorbance at 340 nm was monitored. After all the unreacted D-glucose 6-phosphate from the E4P synthesis had reacted, an appropriate amount of transketolase containing solution was added and the reaction was monitored at 340 nm for 3 to 5 min. One unit of 204 transketolase activity was defined as the formation of 1 umol of NADPH (e = 6220 L -1 -r . . mol cm ) per min per mg of protein. Shikimate dehydrogenase (AroE) assay AroE-encoded shikimate dehydrogenase activity was measured in the reverse direction.172 The reaction (1 mL) contained 780 uL of reverse assay buffer (100 mM Tris, pH 9), 100 ptL of shikimate (40 mM, dissolved in reverse assay buffer), and 20 uL of diluted AroE solution. The reaction was initiated by the addition of 100 uL of NADP+ (2 mM). Formation of NADPH was monitored by following the increase in absorbance at 340 nm. One unit of shikimate dehydrogenase activity was defined as the formation of l umol of NADPH (a = 6220 L mol'] cm") per min per mg of protein. Aminoshikimate dehydrogenase (RifI) assay Aminoshikimate dehydrogenase activity was measured. in the reverse direction following the procedure designed for shikimate dehydrogenase assay.172 The reaction (1 mL) contained 780 uL of reverse assay buffer (100 mM Tris, pH 9), 100 uL of aminoshikimate (purified from fermentation broth, 40 mM, dissolved in reverse assay buffer), and 20 ML of diluted Rifl solution. The reaction was initiated by the addition of 100 uL of NADP+ (2 mM). Formation of NADPH was monitored by following the increase in absorbance at 340 nm. One unit of aminoshikimate dehydrogenase activity was defined as the formation of 1 umol of NADPH (e = 6220 L mol'l cm'l) per min per mg of protein. 205 Kanosamine kinase (le and RifN) assay Kanosamine kinase activity was measured in a coupled enzyme system using pyruvate kinase and lactate dehydrogenase.173 The assay mixture (1 mL) contained 50 mM HEPES (pH7.6), 10 mM MgC12, 2 mM ATP, 0.2 mM NADH, 2 mM phosphoenolpyruvate, 2 mM kanosamine, pyruvate kinase (10 units), L-lactate dehydrogenase (10 units), and 20 uL of diluted le or RifN kinase. The absorbance at 340 nm was monitored for several minutes. One unit of le or RifN kinase activity was defined as the consumption of 1 umol of NADH (a = 6220 L mol'l cm'l) per min per mg of protein at rt and pH 7.5. UDP-3-keto-D-glucose dehydrogenase assay UDP-3-keto-D-glucose dehydrogenase activity was measured at rt by monitoring the reduction of 2,6-dichloroindophenol (DCIP) at 600 nm in the presence of phenazine methosulfate (PMS).174 A typical reaction mixture contained 10 mM phosphate buffer (pH 7.0), 60 uM 2,6-dichloroindophenol, 1 mM phenazine methosulfate, 1.5 mM UDP- glucose, and an appropriate amount of enzyme. The final assay volume was 1 mL. The decrease in absorbance at 340 nm was monitored for several minutes. One unit of enzyme activity was defined as the amount necessary to reduce l umol of 2,6- dichloroindophenol per min at 25°C and pH 7.0. A molar extinction coefficient for 2,6- dichloroindophenol of 21,000 L mol'I cm'1 was used for activity calculations.I75 Protein gel (SDS-PAGE) SDS-PAGE analysis was followed the procedure described by Harris et al.176 The separating gel was prepared by mixing 3.33 mL of 30% acrylamide stock solution 206 (w/v in H20), 2.5 mL of 1.5 M Tris-HCl (pH 8.8), and 4 mL of distilled deionized water. After degassing the solution using a water aspirator for 20 min, 0.1 mL of 10% ammonium persulfate (w/v in H20), 0.1 mL 10% SDS (w/v in H20), and 0.005 mL of N, N, N’, N’-tetramethylethylenediamine (TEMED) was added. After mixing gently, the separating gel was poured into the gel cassette to about 1.5 cm below the top of the gel cassette. t—Amyl alcohol was overlaid on the top of the solution and the gel was allowed to polymerize for 1 h at rt. The stacking gel was prepared by mixing 1.7 mL of 30% acrylamide stock solution, 2.5 mL of 0.5 M Tris-HCl (pH 6.8), and 5.55 mL of distilled deionized water. After degassing for 20 min, 0.1 mL of 10% ammonium persulfate, 0.1 mL 10% SDS, and 0.01 mL of TEMED was added, and the solution was mixed gently. t- Amyl alcohol was removed from the top of the gel cassette, which was subsequently rinsed with water and wiped dry. After insertion of the comb, the gel cassette was filled with stacking gel solution, and the stacking gel was allowed to polymerize for 1 h at rt. After removal of the comb, the gel cassette was installed into the electrophoresis apparatus. The electrode chamber was then filled with electrophoresis buffer containing 192 mM glycine, 25 mM Tris base, and 0.1% SDS. Each protein sample (10 uL) was diluted with Laemmli sample buffer”7 (10 uL, Sigma 3-3401) consisting of 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 125 mM Tris- HCl (pH 6.8). Samples and markers (MW-SDS-200, Sigma) were loaded into the sample well and the gel was run under constant current at 30 mA when the blue tracking dye (bromophenol blue) was within stacking gel. After the blue tracking dye reached the separating gel, a higher current (50 mA) was applied to the gel. At the completion of electrophoresis (blue tracking dye reaches the bottom of the gel), the gels were removed 207 from the cassettes and fixed in a solution of 10% (w/v) aqueous trichloroacetic acid for 30 min. After staining in a solution containing 0.1% (w/v) Coomassie Brilliant Blue R, 45% (v/v) MeOH, 10% (v/v) HOAc in H20 for 3 h, the protein gels were destained in a solution of 45% (v/v) MeOH, 10% (v/v) HOAc in H20. The gels were then sealed in a sheet protector. CHAPTER TWO Synthetic Procedures Synthesis of 3-amino-3-deoxy-B-D-fructofuranose 6-phosphate 1,2:4,5-Di-0-isopropylidene-fi-D-fructopyranose (1).178 A suspension of D- fructose (36 g, 200 mmol) in dry acetone (700 mL) and concentrated HZSO4 (3.5 mL) was stirred at rt under Ar until the sugar had dissolved (about 1.5 h). An ice-cold solution of 11 g (275 mmol) of NaOH in 100 mL of H20 was gradually. added over 3 min with stirring. The acetone was subsequently removed under reduced pressure, and the residue was extracted with CHzCl2 (3x). The extracts were combined and washed with water (2x). Drying and concentration gave a white solid, which was recrystallized by dissolving in boiling ether (5 mL/g), adding hexane, and cooling to give 1,2:4,5-di-0- isopropylidene-B-D-fructopyranose (27 g, 52%). lH NMR (CDCl3): 6 4.21 (ddd, J = 6, 3, 1 Hz, 1 H), 4.18 (d, J: 9 Hz, 1 H), 4.14 (dd, J = 7, 7 Hz, 1 H), 4.12 (dd, J=14, 3 Hz,1 H), 4.01 (d, J = 14 Hz, 1 H), 3.98 (d, J = 9 Hz, 1 H), 3.67 (dd, J = 8, 7 Hz 1H), ), 2.15 (d, J: 8 Hz, 1 H), 1.53 (s, 3H), 1.51 (s, 3H), 1.44 (s, 3H), 1.37 (s, 3H). 13C NMR (CDC13): 6 111.9, 109.4, 104.6, 77.3, 73.4, 72.4, 70.4, 60.8, 27.9, 26.4, 26.3, 26.0. 208 1,2:4,5-Di-0-isopropylidene—D-erythro-2,3-hexodiuro-2,6-pyranose (2).179 To a mechanically stirred mixture of 1,224,5-di-0-isopropylidene-B-D-fructopyranose 1 (21 g, 81 mmol), PhCH2(Et3)NCl (0.92 g, 4 mmol), NaIO4 (25.6 g, 120 mmol), and KZCO3 (1.7 g, 12.4 mmol) in CHCl3-HZO (1:1, v/v, 140 mL) was added RuCl3-HZO (0.6 g, 2.9 mmol). After 12 h of stirring at rt, 2-propanol (24 mL) was added. The mixture was stirred for an additional hour and then filtered through Celite. The organic layer was separated and the water layer was extracted with CHZCI2 (2x). The combined organic layers were washed with saturated NazSO3, H20, and then brine. Drying and concentration gave a white solid. Purification by flash chromatography (EtOAc/hexane, 1:4, v/v) afforded the ketone as a white solid (20.6 g, 99%). 1H NMR (CDC13): 6 4.72 (d, J = 6 Hz, 1 H), 4.61 (d, J = 10 Hz, 1 H), 4.55 (m, 1 H), 4.38 (dd, J = 14, 3 Hz, 1 H), 4.12 (d, J = 14 Hz, 1 H), 4.00 (d, J = 10 Hz, 1 H), 1.55 (s, 3H), 1.46 (s, 3H), 1.40 (s, 6H). 13C NMR (CDCl3): 6 196.9, 113.8, 110.7, 104.2, 77.9, 75.9, 70.1, 60.1, 27.2, 26.5, 26.1, 26.0. 1,2:4,5-Di-0-isopropylidene-B-deoxy-3-oxime-B-D-fructopyranose (3). To a solution of NHzOH-HCl (6.0 g, 86 mmol) and NaOAc (7.8 g, 95 mmol) in 100 mL of H20 was added l,224,5-di-0-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose 2 (20 g, 77.5 mmol), dissolved in 100 mL of CH3CN. The mixture was stirred at rt under Ar overnight. After concentration in vacuo, the residue was extracted with CHzCl2 (3x). The combined organic layers were washed with water and brine. Drying and concentration gave a white solid. Purification by flash chromatography (EtOAc/hexane, 1:2, v/v) afforded the oxime as a white solid (19.3 g, 91%). 1H NMR (CDC13): 6 4.80 (d, J = 8 Hz, 1 H), 4.42 (d, J = 9 Hz, 2 H), 4.08 (d, J = 9 Hz, 1 H), 3.84-3.88 (m 1 H), 3.78 209 (d, J = 14 Hz, 1 H), 1.55 (s, 3 H), 1.53 (s, 3 H), 1.50 (s, 3 H), 1.36 (s, 3 H). 13C NMR (CDC13): 6 149.1, 112.9, 110.4, 102.2, 73.7, 73.6, 73.5, 63.8, 27.2, 26.4, 26.2, 25.2. HRFABMS: calcd for C,2H,.,NO6 (M + H”): 274.1291. Found: 274.1293. 1,2:4,5-Di-0-isopropylidene-3-amino-3-deoxy-fi-D-fructopyranose (4). To a cold suspension of LiAlH4 (11.1 g, 292 mmol) in dry THF (900 mL) under Ar was added l,224,5-di-0-isopropylidene-3-deoxy-3-oxime-B—D-fructopyranose 3 (20 g, 73.3 mmol). The reaction mixture was refluxed under Ar for 6 h, with stirring. To the cooled reaction mixture, 11.1 mL of H20 was added dropwise followed by addition of 22.2 mL of 15% aqueous NaOH solution and finally 11.1 mL of H20. The solution was filtered through Celite and concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc/hexane, 5:1, v/v) to give 1,2:4,5-di-O-isopropylidene-3-amino- 3-deoxy-[3-D-fructopyranose as a white solid (2.09 g, 11%). lH NMR (CDC13): 6 4.26 (d, J = 9 Hz, 1 H), 4.04 — 4.15 (m, 3 H), 3.96 (d, J = 9 Hz, 1 H), 3.89 (dd, J = 8, 6 Hz, 1 H), 2.79 (d, J = 9 Hz, 1 H), 1.52 (s, 3 H), 1.50 (s, 3 H), 1.43 (s, 3 H), 1.37 (s, 3 H). 13C NMR (CDC13): 6 111.8, 109.1, 105.8, 79.2, 73.1, 72.4, 60.4, 53.9, 28.4, 26.4, 26.3, 26.2. HRFABMS: calcd for C12H21NO5 (M + H’): 260.1489. Found: 260.1504. 3-Amino-3-deoxy-D-fructose. A solution of 1,2:4,5-di-0-isopropylidene-3- amino-3-deoxy-B-D-fructopyranose 4 (1.0 g, 38.6 mmol) in 2 N HCl (15 mL) was stirred at rt overnight. Evaporation of solvent afforded a quantitative yield of 0.83 g of 3-arnino- 3-deoxy-D-fructose (both or and [3 forms), which was isolated as a slightly yellow solid. 1H NMR (D20): 6 4.38 (dd, J = 9, 9 Hz, 0.2 H), 4.23 (m, 0.1 H), 4.05-4.09 (m, 1.5 H), 210 3.99 (m, 0.7 H), 3.96 (m, 0.2 H), 3.67-3.85 (m, 3.6 H), 3.52 (d, J = 11 Hz, 0.7 H). 13C NMR (D20): 6 103.9, 97.9, 85.3, 75.1, 70.7, 69.9, 68.8, 66.2, 64.4, 61.0, 55.5. HRFABMS: calcd for C6HI3NOS (M + H’): 180.0872. Found: 180.0867. 3-Amino-3—deoxy-D-fructose 6-phosphate. In a 250 mL Erlenmeyer flask equipped with a stir bar were combined 3-amino-3-deoxy-D-fructose (0.44 g, 2.04 mmol), ATP (sodium salt, 2.2 g, 4.0 mmol), MgClZ'HzO (1.0 g, 4.9 mmol), citric acid-H20 (0.4 g, 1.9 mmol), and 140 mL of deionized water. The pH of the solution was adjusted to 8.0 with aqueous NaOH, and the solution was deoxygenated with Ar for 5 minutes. The reaction was initiated by the addition of hexokinase (500 units). The reaction mixture was stirred slowly at rt for 24 h and the pH was maintained between 7.5 and 8.0 by the addition of 1 N aqueous NaOH. The pH was adjusted every hour for six hours and then monitored every six hours until the end of the reaction. The crude reaction mixture was applied to AG-l X8 anion exchange resin (2.5 cm x 16 cm, 80 mL, acetate form). The column was washed with distilled water (160 mL) and eluted with a linear gradient (200 mL + 200 mL, 0-2.0 M) of HOAc. Column fractions containing inorganic phosphate and phosphate esters were identified by colorimetric assay.180 Fractions containing phosphate esters were combined and concentrated to a small volume (about 5 mL) and then lyophilized to dryness. The product, 3-amino-3-deoxy-D-fructose 6-phosphate, was a white solid (0.46 g, 87%). 1H NMR (D20): 6 4.48 (dd, J = 8, 8 Hz 1 H, H-4), 4.08 (m, 1 H, H-6'), 4.07 (m, 1 H, H-5), 4.00 (m, l H, H-6), 3.82 (d, J = 9 Hz 1 H, H-3), 3.67 (s, 2 H, H-1, 1'). 13C NMR (D20): 6 104.0 (C-2), 83.9 (JPOCC = 8.5 Hz, C- 5), 75.0 (C-4), 67.4 (JPOC = 4.8 Hz, C-6), 66.2 (C-l), 60.9 (C-3). 31P NMR (D20): 6 1.09. 211 gDQCOSY (D20): H-3 => H-4; H-4 => H-5, H-5 => H-6, 6'. gHMQC (D20): H-1, 1' => C-l; H-3 => C-3; H-4 => C-4; H-5 => C-5; H-6, 6' => C-6. HRFABMS: calcd for c,H,,No,P (M + 14*): 260.0535. Found: 260.0538. Synthesis of kanosamine 6-phosphate 1,2:5,6-Di-0-isopropylidene—a-D-glucofuranose (5).181 To a suspension of D- glucose (20 g, 111 mmol) in dry acetone (200 mL) was added anhydrous ZnCl2 (16 g, 117 mmol), followed by 85% H3PO4 (0.6 mL). The reaction mixture was stirred at rt for 36 h. The mixture was filtered through Celite and neutralized to pH 7 by the addition of 10% aqueous NaOH solution. The acetone was removed under reduced pressure, and the residue was extracted with CH2C12 (3x). The extracts were combined and washed with water (2x). Drying and concentration gave a white solid, which was recrystallized to afford 1,2:5,6-di-O-isopropylidene-or-D-glucofuranose (19.6 g, 68%). 'H NMR (CDCl3): 6 5.90 (d, J = 3.5 Hz, 1 H), 4.49 (d, J = 3.5 Hz, 1 H), 4.29 (m,2 H), 4.13 (dd, J = 8.5, 6 Hz, 1 H), 4.03 (dd, J = 8, 3 Hz, 1 H), 3.95 (dd, J = 8.5, 5 Hz, 1 H), 2.57(d, J = 4 Hz, 1 H), 1.46 (s, 3 H), 1.41 (s, 3 H), 1.34 (s, 3 H), 1.29 (s, 3 H). ”C NMR (CDC13): 6 111.9, 109.7, 105.3, 85.1, 81.2, 75.3, 73.5 67.7, 26.9, 26.8, 26.2, 25.2. 182 . Acetic 1,2:5,6-Di-0-isopropylidene-3-keto-a-D-glucofuranose (6). anhydride (21.9 mL, 232 mmol) was added to pyridinium dichromate (27.8 g, 73.9 mmol) in dry CHzCl2 (210 mL). 1,2:5,6—Di-0-isopropylidene-or-D-glucofuranose 5 (19 g, 73.0 mmol) was added in 10 mL of CHzCl2 and the mixture was heated in an oil bath (75°C) for 4 h. After the completion of the reaction, the mixture was diluted with EtOAc 212 (300 mL) and the precipitate was filtered. After evaporation of the solvent, diethyl ether was added (200 mL) and the mixture was filtered again. Drying and concentration gave a yellow oil. Purification by flash chromatography (EtOAc/hexane, 1:4, v/v) afforded the ketone as a colorless oil (17.2 g, 91%). 1H NMR (CDC13): 6 6.14 (d, J = 4.5 Hz, 1 H), 4.39 (d, J = 4.5 Hz, 1 H), 4.35-4.38 (m, 2 H), 4.01-4.03 (m, 2 H), 1.46 (s, 3 H), 1.43 (s, 3 H), 1.34 (s, 6 H). ”C NMR (CDC13): 6 208.8, 114.3, 110.4, 103.1, 79.0, 77.3, 76.4, 64.3, 27.6, 27.2, 26.0, 25.3. 1,2:5,6-Di-0-isopropylidene-a-D-allofuranose (7).183 The 1,2:5,6-di-0- isopropylidene-3-keto-or-D-glucofuranose 6 (16 g, 62.0 mmol) was dissolved in 100 mL of 90% aqueous EtOH and cooled to 0°C. NaBH4 (6 g, 160 mmol) was added and the mixture was stirred at 0°C for 1 h. After destroying unreacted borohydride by addition of an excess of NH4Cl, the resulting mixture was concentrated and the residue was extracted with CHzCl2 (3x). The extracts were combined and washed with water (2x). Drying and concentration gave a yellow oil. Purification by flash chromatography (EtOAc/hexane, 1:2, v/v) afforded the 1,2:5,6-di-O-isopropylidene-a-D-allofuranose as a white solid (14.5 g, 90%). 1H NMR (CDC13): 6 5.82 (d, J = 4 Hz, 1 H), 4.61 (dd, J = 5, 4 Hz, 1 H), 4.3 (m, l H), 4.00-4.10 (m, 3 H), 3.82 (dd, J = 6.5, 5 Hz, 1 H), 2.56 (d, J = 8 Hz, 1 H), 1.58 (s, 3 H), 1.47 (s, 3 H), 1.39 (s, 3 H), 1.37 (s, 3 H). ”C NMR (CDC13): 6 112.9, 109.9, 104.0, 79.9, 79.0, 75.7, 72.6, 66.0, 26.6, 26.5, 26.3, 25.3. 1 ,2:5,6-Di-0-isopropylidene-3-azido-3-deoxy-a-D-glucofuranose (8). I 83 1 ,2: 5 , 6-Di-0-isopropylidene-a-D-allofuranose 7 (14 g, 53.8 mmol) was dissolved in dry 213 CH2C12 (160 mL) containing pyridine (8.8 mL, 108.8 mmol) and the solution was cooled to —30°C under an atmosphere of Ar. Trifluoromethanesulphonic anhydride (10.8 mL, 64.2 mmol) was added dropwise over 15 min and the suspension was stirred at the same temperature for a further 30 min. The reaction was quenched by the addition of CH3OH (6.5 mL, 160 mmol) and the clear solution obtained was allowed to warm to rt. The solution was washed with water (2x), dried and evaporated to a pale yellow syrup of crude 1,2:5,6-di-0-isopropylidene-3-0-trifluorometanesulphonyl-a-D-allofuranose. The unpurified product was dissolved in dimethylformamide (110 mL) and stirred at 50°C overnight with NaN3 (14 g, 215 mmol). After cooling to rt, the solvent was evaporated and the residue partitioned between CHCl3 (100 mL) and water (100 mL). The organic layer was dried and concentrated to a yellow syrup. Purification by flash chromatography (EtOAc/hexane, 1:5, v/v) afforded the l,2:5,6-di-0-isopropylidene-3- azido-3-deoxy-(1-D-glucofuranose as a colorless oil (14.1 g, 92%). 1H NMR (CDC13): 6 5.84 (d, J = 3.5 Hz, 1 H), 4.60 (d, J = 3.5 Hz, 1 H), 4.23 (m, 1 H), 4.12 (dd, J: 8.5, 6 Hz, 1 H), 4.08 (m, 2 H), 3.96 (dd, J = 8.5, 5 Hz, 1 H), 1.50 (s, 3 H), 1.42 (s, 3 H), 1.35 (s, 3 H), 1.31 (s, 3 H). ”C NMR (CDC13): 6 112.3, 109.6, 105.1, 83.5, 80.6, 73.1, 67.7, 66.4, 26.9, 26.7, 26.2, 25.2. 1,2:5,6-Di-0-isopropylidene-kanosamine (9).184 To a cold suspension of LiAlH4 (7.43 g, 196 mmol) in dry ethyl ether (800 mL) under an Ar atmosphere was added 1,2:5,6-di-0-isopropylidene-3-azido-3-deoxy-a-D-glucofuranose 8 (14 g, 49 mmol). The reaction mixture was stirred at rt for 2 h. To the cooled reaction mixture, 7.5 mL of water was added dropwise followed by addition of 15 mL of 15% aqueous NaOH 214 and finally 7.5 mL of water. The solution was filtered through Celite and concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc/hexane, 6:1, v/v) to give 1,2:5,6-di-0-isopropylidene—kanosamine as a colorless oil (11.9 g, 94%). lH NMR (CDCl_.,): 6 5.89 (d, J =4 Hz, 1 H), 4.39 (d, J = 4 Hz, 1 H), 4.14—4.22 (m, 2 H), 4.03 (dd, J = 8.5, 4 Hz, 1 H), 3.99 (dd, J = 8.5, 5 Hz, 1 H), 3.56 (d, J = 4 Hz, 1 H), 1.51 (s, 3 H), 1.42 (s, 3 H), 1.35 (s, 3 H), 1.31 (s, 3 H). ”C NMR (CDC13): 6 111.6, 109.4, 105.1, 86.6, 81.5, 73.0, 68.2, 57.4, 26.9, 26.8, 26.2, 25.3. Kanosamine. A solution of 1,2:5,6-di-0-isopropylidene-3-amino-3-deoxy-0t-D- glucofuranose (1 g, 38.6 mmol) in 2 N HCl ( 15 mL) was stirred at rt overnight. Evaporation of solvent afforded a quantitative yield of 0.83 g of kanosamine, which was isolated as a white solid. lH NMR (D20): 6 5.27 (d, J = 3.5 Hz, 0.4 H), 4.72 (d, J = 8 Hz, 0.6 H), 4.39 (m, 1 H), 3.74-3.87 (m, 2 H), 3.67 (ddd, J = 10, 10, 4.5 Hz, 1 H), 3.57 (ddd, J = 7.5, 5.5, 2 Hz, 0.5 H), 3.45 (dd, J = 10, 8 Hz, 0.5 H), 3.41 (dd, J = 10, 10 Hz, 0.5 H), 3.25 (dd, J = 10, 10 Hz, 0.5 H). ”C NMR (D20): 6 99.0, 94.1, 79.7, 74.2, 73.4, 71.0, 68.9, 68.8, 63.2, 63.0, 60.8, 58.1. HRFABMS: calcd for CGHUNO5 (M + H”): 180.0872. Found: 180.0868. Kanosamine 6-phosphate. In a 250 mL Erlenmeyer flask equipped with a stir bar were combined kanosamine (0.44 g, 2.04 mmol), ATP (sodium salt, 2.2 g, 4.0 mmol), MgC12°6HZO (1.0 g, 4.9 mmol), citric acid°HzO (0.4 g, 1.9 mmol), and 140 mL of deionized water. The pH of the solution was adjusted to 8.0 with aqueous NaOH, and the solution was deoxygenated with Ar for 5 minutes. Hexokinase (500 units) was added. The reaction mixture was stirred slowly at rt for 24 h and the mixture was maintained 215 between pH 7.5 and pH 8.0 by the addition of 1 N aqueous NaOH. The pH was adjusted every hour for six hours and then monitored every six hours until the end of the reaction. The crude reaction mixture was applied to AG-l X8 anion exchange resin (2.5 cm x 16 cm, 80 mL, acetate form). The column was washed with distilled water (160 mL) and eluted with a linear gradient (200 mL + 200 mL, 0-2 M) of acetic acid. Column fractions containing inorganic phosphate and phosphate esters were identified by colorimetric assay.180 Fractions containing phosphate esters were combined and concentrated to a small volume (about 5 mL) and then lyophilized overnight to dryness. Kanosamine 6- phosphate was isolated as a white solid (0.50 g, 90%). llH NMR (D20): 6 5.27 (d, J = 3.5 Hz, 0.5 H, H-l), 4.74 (d, J = 7.5 Hz, 0.5 H, H-l), 4.02-4.17 (m, 2 H, H-6, 6’), 3.99 (br d, J = 9.5 Hz, 0.5 H, H-S), 3.8 (m, 1.5 H, H-4, H-2), 3.67 (br (1, J = 9.5 Hz, 0.5 H, H-5), 3.48 (dd, J = 10.5, 7.5 Hz, 0.5 H, H-2), 3.43 (dd, J = 10.5, 10.5 Hz, 0.5 H, H-3), 3.25 (dd, J = 10.5, 10.5 Hz, 0.5 H, H-3). ”C NMR (D20): 6 99.1 (C-l), 94.2 (C-l), 78.5 (JPOCC = 7.6 Hz, C-5), 73.3 (C-2), 73.2 ((JPOCC = 7.6 Hz, C-5), 70.9 (C-2), 68.4 (C-4), 68.3 (C—4), 66.2 (JPOC = 3.8 Hz, C-6), 60.5 (C-3), 57.7 (C-3). ). 3'P NMR (D20): 6 1.60. gDQCOSY (D20): H-l => H-2; H-2 => H-3; H-3 => H-4; H-4 => H-5; H-5 => H-6. gHMQC (D20): H-l => C-l; H-2 :5 C-2; H-3 => C-3; H-4 => C-4; H-5 => C-5; H-6, 6’ => C-6. HRFABMS: calcd for C6H,5NOSP (M + H’): 260.0535. Found: 260.0542. Synthesis of UDP-6,6-[2H2]-D-glucose A 60 mL solution of 100 mM triethanoamine buffer (pH 8.0) containing 6,6- [2H2]-D-glucose (20 mg, 0.11 mmol), UTP (sodium salt, 55 mg, 0.11 mmol), MgC12-6HZO (4 mM), [3 -mercaptoethanol (10 mM), a-D-glucose 1,6-diphosphate cyclohexylammonium salt (33 uM), and phosphoenolpyruvate (25 mg, 0.12 mmol) was 216 deoxygenated with Ar. Hexokinase (100 units), phosphoglucose mutase (100 units), UDP-D-glucose pyrophosphorylase (100 units), pyruvate kinase (200 units), and inorganic pyrophosphatase (200 units) were added. The reaction mixture was slowly stirred at rt. The synthesis was monitored by. TLC (solvent system: 85:15:01 EtOH:HZO:85% H3PO4, v/v). When the reaction was nearly complete, 1 mL of fresh substrate solution containing 6,6- [2H2]-D- glucose (20 mg, 0.11 mmol), UTP (sodium salt, 55 mg, 0.11 mmol), and phosphoenolpyruvate (25 mg, 0.12 mmol) was added to the reaction mixture. The same substrate solution was added 7 more times. After completion of the reaction, the protein was removed by ultrafiltration. The clear reaction mixture was then applied to Dowex anion exchange resin (200-400 mesh, bicarbonate form, 2.5 cm x 20 cm, 100 mL). The column was washed with water (200 mL) and eluted with a linear gradient (250 mL + 250 mL, 0-1 M) of NaHCO3. Fractions containing UDP-6,6- [2H2]-D-glucose were identified by organic phosphate assay180 and NMR, pooled, adjusted to pH 4 by addition of Dowex 50 (200-400 mesh, Hf) resin, and stirred for 30 min. After removal of the resin by filtration, the filtrate was neutralized to pH 7. The desired UDP-6,6—[2H2]-D-glucose was obtained as a white powder after concentration and lyophilization. The final yield of UDP-6,6-[2H2]-D-glucose was 80%. 1H NMR (D20): 6 7.96 (d, J = 8 Hz, 1 H, H-6"), 6 6.00 (d, 1 H, H-l’), 6 5.98 (d, 1 1H, H-S”), 6 5.59 (dd, J = 7.5, 4 Hz, 1 H, H-l), 6 4.41 (m, 2 H, H-2', 3’), 6 4.30 (m, 1 H, H-4'), 6 4.25 (m, 1 H, H- 5’), 6 3.90 (d, J = 10 Hz, 1 H, H-5), 6 3.81 (dd, J = 10, 9 Hz, 1 H, H-4), 6 3.56 (ddd, J = 11, 4, 4 Hz, 1 H, H-2), 6 3.46 (dd, J = 10, 9 Hz, 1 H, H-3). ”C NMR (D20): 6 169.1, 154.8, 144.5, 105.6, 98.6 (d, J = 7.8 Hz), 91.1, 86.2 (d, J = 8.5 Hz), 76.6, 76.3, 76.2, 74.8, (d, J = 5.6 Hz), 72.6, 72.3, 67.9 (d, J = 4.8 Hz), 63.1 (m). 31P NMR (D20): 6 -10.5 (d, J = 217 20.8 Hz), 6 -12.1 (d, J = 20.8 Hz). ESMS: (M - H’): 567, 96.4% of di-deuterium enrichment. Genetic Manipulations Plasmid pJG6.128A This 7.0-kb plasmid was created by inserting the 0rf15 locus of A. mediterranei into plasmid pJF118EH. A 1.7-kb DNA fragment containing the open reading frame of 0rf15 was PCR amplified from cosmid FKN108I34 employing the following primers containing EcoRI restriction sequences: 5’-AAGAATTCAGAGGCATCACGTGTGAT TGA and 5’-AAGAATTCATGCAGATGACCGAAGAGAAC. Digestion of the PCR product with EcoRI followed by insertion into the plasmid pJF118EH, which was linearized by EcoRI digestion, resulted in plasmid pJG6.128A. The 0rf15 locus was oriented such that transcription was from the tac promoter and in the opposite direction as lac/Q. Plasmid pJG7.241l A 1.7-kb DNA fragment containing the open reading frame of 0rf15 was excised out of plasmid pJG6.128A (Pm, 0rf15, 1acIQ, Amp“) by digestion with EcoRI and then digested with mung bean nuclease. Blunt end ligation of this 1.7-kb fragment into plasmid pJG7.246 (PT5, Iale, Amp“), which had been linearized by BamHl digestion followed by treatment with Klenow fragment, afforded the 7.2-kb plasmid pJG7.24l (P75, 0rf15, Iale, Amp“). The 0rf15 locus oriented in the same direction as the T5 promoter. 218 Plasmid pJG7.246 Plasmid pPV4.222 (PT5, dxr, IacIQ, Amp“)185 was digested with BamHI and the 5.3-kb DNA fragment was gel purified. Self-ligation of this 5.3-kb DNA fragment afforded the 5.3-kb plasmid pJG7.246 (P75, I acIQ, Amp“). Plasmid pJG9.150 The DNA fragment of 0rf15 was excised out of plasmid pJG7.241 (P75, 0rf15, IacIQ, Amp“) by digestion with BamHI. Ligation to the BamHI site of plasmid pBT (Stratagene) afforded plasmid pJG9.150 (P,,,,._UV5ACI, 0rf15, Cm“). The 07f15 locus was oriented in the same direction as the lac- U V5 promoter. Plasmid pJG9.154 The rifH locus was amplified by PCR from plasmid pJG5.165A (PmrifH, Amp“; see later part of this Chapter for detail) using the following primers containing BamHl terminal recognition sequences: 5’-TAGGATCCATGAAGCGGCAGCCGGAC'I'I‘ C and 5’-TAGGATCCCGACTAGCG CAGCATCTTCG. The amplified 1.3-kb PCR fragment was digested with BamHI and ligated into the BamHI site of plasmid pTRG to yield plasmid pJG9.154 (P,p,,,,ac_UV5RNAP-a, rifH, Tc“) in which the rifH gene is oriented in the same orientation as the lpp/lac-U V5 promoter. Plasmid pJG9.213 The tktA fragment was amplified by PCR from plasmid pKD12.13845 using the following primers containing BamHl terminal recognition sequences: 5’-CGGGATCCA 219 TGTCCTCACGTAAAGAGCTT and 5’-CGGGATCCTCAAGGTAATAAAAAAG GTCG. The amplified 2.0-kb PCR fragment was digested with BamHI and ligated into the BamHI site of plasmid pBT to create plasmid pJG9.213 (P,aC_UV5AC1, tktA, Cm“) in which the tktA gene is oriented in the same orientation as lac-U V5 promoter. Plasmid pJG9.251 The UDP-3-keto-D-glucose dehydrogenase gene (UDPGDH) was amplified by PCR from B. pumilus genomic DNA using the following primers containing Bglll terminal recognition sequences: 5’-GAAGATCTATGGCAGACAATCATTATGAT and 5’-GAAGATCT TGGTTCTCGGTTCAGGTCGTG. The resulting 2.3-kb amplified fragment was digested with Bglll and ligated into the BamHI site of plasmid pJG7.246 to create plasmid pJG9.251 (PT5, UDPGDH, lale, Amp“) in which the UDP-3-keto-D- glucose dehydrogenase gene is oriented in the same orientation as T5 promoter. Enzyme Purifications E. coli tktA-encoded transketolase E. coli tktA-encoded transketolase was purified186 from E. coli BL21(DE3)/pSK4.172 (Pl... tktA, Cm“).'87 Buffers used in the purification included buffer A: K2HP04 (50 mM), MgCl2 (1 mM), and dithiothreitol (1 mM), pH 7.35; buffer B: KZHPO4 (250 mM), MgCl2 (1 mM), and dithiothreitol (1 mM), pH 7.35; buffer C: 220 KzHPO4 (10 mM) and MgCl2 (1 mM), pH 7.35; and buffer D: KZHPO4 (10 mM), MgCl2 (1 mM), and KCl (500 mM), pH 7.3. Single colonies of E. coli BL21(DE3)/pSK4.172 were used to inoculate 50 mL LB (2x) containing chloramphenicol. After 12 hours of growth, 2 mL culture (7x) was transferred to 1 L LB (7x) containing chloramphenicol and grown at 37°C for 12 hours. Cells (43 g) were collected from the above cultures by centrifugation at 44003 for 5 min at 4 °C. Cells were resuspended in 90 mL of buffer A and lysed by two passages through a French press at 16000 psi. Cellular debris was removed by centrifugation at 480003 and 4 °C for 25 min. To the clarified cellular lysate containing approximately 3800 mg of protein was added 50 mL hydroxyapatite in a slurry of 70 mL of buffer A. After centrifugation at 176003 for 20 min at 4 °C, the pellet was resuspended in 60 mL of buffer A and centrifuged at 176003 and 4 °C for 10 min. The combined supernatant (188 mL) was loaded on a DEAE-cellulose column (2.5 cm x 31 cm, 150 mL), which had been equilibrated with buffer A. The column was washed with buffer A (300 mL) and eluted with a linear gradient (500 mL + 500 mL) of buffer A and buffer B. Fractions that tested positive towards transketolase assay were combined, concentrated to approximately 13 mL, and dialyzed against buffer C overnight. The protein was then applied to a HPLC Resource Q (16 mm x 30 mm, 6 mL) column, which had been equilibrated with buffer C. The column was washed with buffer C (18 mL) and eluted with a linear gradient (30 mL + 30 mL) of buffer C and buffer D at a flow rate of 1 mL/min. Fractions containing transketolase were combined, concentrated, dialyzed against buffer A, quick frozen in liquid nitrogen, and stored at —80°C. The specific activity of purified transketolase was 5.6 unit/mg and the total activity obtained in the purification was 174 units. 221 E. coli aroFFB“-encoded DAHP synthase DAHP synthase was purified168 from E. coli BL21(DE3)/pKL4.71 (Pm, aroFuB“, IacIQ, Amp“).188 Buffers used in the purification included buffer A: KZHPO4 (50 mM), phosphoenolpyruvate (10 mM), and CoSO4-7HZO (0.5 mM), pH 6.5; buffer B: KZHPO4 (100 mM), phosphoenolpyruvate (0.5 mM), and 1,3-propandiol (250 mM), pH 6.5; and buffer C: KzHPO4 (500 mM), phosphoenolpyruvate (0.5 mM), and 1,3-propandiol (250 mM), pH 6.5 Single colonies of E. coli BL21(DE3)/pKL4.7l were used to inoculate 50 mL LB (2x) containing ampicillin. After 12 hours of growth, 4 mL of culture (7x) was transferred to 1 L LB (7x) containing ampicillin and grown at 37°C until OD600 reached 0.5. IPTG was added to the culture to a final concentration of 1 mM. After an additional 4 hr growth at 37°C, the cells (33 g) were harvested by centrifugation at 44003 for 5 min at 4 °C. Cells were resuspended in 132 mL of buffer A and lysed by two passages through a French press at 16000 psi. Cellular debris was removed by centrifugation at 480003 and 4 °C for 25 min. To the clarified cellular lysate containing approximate 2700 mg of protein, 27.5 mL of 2 % (w/v) protamine sulfate solution was added dropwise to give a final concentration of 0.2 mg protarrrine sulfate per mg of protein. The resulting suspension was stirred at 4 °C for 30 min and the precipitate was removed by centrifugation (480003, 4 °C, 30 min). To the supernatant was added 0.02 g of CoSO4-7H20 followed by 42 g of (NH4)2SO4 to a final concentration of 50% (w/v). The resulting precipitate was removed by centrifugation (270003, 20 min, and 4 °C) and (NH4)._,SO4 was added to the supernatant to achieve a 65% (w/v) concentration of 222 (NH4)ZSO,. After stirring for an additional 30 min, the precipitated was collected by centrifugation (270003, 30 min, and 4 °C) and redissolved in 30 mL of cold buffer B. The resulting solution was dialyzed against buffer B overnight and then loaded on a DEAE-cellulose column (2.5 cm x 20 cm, 100 mL), which had been equilibrated with buffer B. The column was washed with buffer B (200 mL) and eluted with a linear gradient (500 mL + 500 mL) of buffer B and buffer C. Fractions that containing DAHP synthase were combined, concentrated, dialyzed against buffer C, quick frozen in liquid nitrogen, and stored at —80°C. The specific activity of purified DAHP synthase was 38 unit/mg and the total activity obtained in the purification was 2660 units. A. mediterranei rifH-encoded aminoDAHP synthase AminoDAHP synthase was purified‘89 from BL21(DE3)/MMB012 (T7, [(100, His,, rifH, Amp“)96 on a Ni-NTA Agarose column (Qiagen). Buffers used in the purification included buffer A: KZHPO4 (50 mM), . imidazole (10 mM), phosphoenolpyruvate (0.5 mM), pH 8.0; buffer B: KZHPO4 (50 mM), imidazole (20 mM), NaCl (300 mM), pH 8.0; buffer C: KZHPO4 (50 mM), imidazole (250 mM), NaCl (300 mM), pH 8.0; and buffer D: KZHPO4 (50 mM), phosphoenolpyruvate (0.5 mM), pH 7.0. A single colony of E. coli BL21(DE3)/MMB012 was used to inoculate 5 mL LB containing ampicillin. After 12 hours of growth, the 5 mL culture was transferred to 1 L LB containing ampicillin and grown at 28°C until the OD600 reached 0.5. IPTG was added to the culture to a final concentration of 0.1 mM. After an additional 8 hr growth at 28°C, the cells (5 g) were harvested by centrifugation (44003 for 5 min at 4 °C), resuspended in Buffer A (15 mL), and lysed by two passages through a French press at 223 16000 psi. Cellular debris was removed by centrifugation at 480003 and 4 °C for 25 min. To the supernatant was added a 50% slurry (w/v) of Ni-NTA agarose resin (1 mL resin per 4 mL of crude lysate), and the mixture was stirred at 4 °C for an hour. The lysate resin slurry was then transferred to a polypropylene column (Qiagen) and the column was washed with Buffer B (2 x 4 mL per mL of Ni-NTA agarose resin). The 6-His tagged protein was eluted from the column by washing with buffer C (2 x 1 mL per mL of Ni- NTA agarose resin). The eluted protein was concentrated, dialyzed against buffer D, quick frozen in liquid nitrogen, and stored at —80°C. The specific activity of purified aminoDAHP synthase (assayed as DAHP synthase activity)93 was 0.3 unit/mg and the total activity obtained in the purification was 8 units. A. mediterranei rifN-encoded kanosamine kinase Kanosamine kinase was purified from BL21(DE3)/pRM070 (T7, lacO, His6, rifN, Amp“)190 by Ni-NTA Agarose column (Qiagen). Buffers, used in the purification included buffer A: Tris-HCl (25 mM), imidazole (10 mM), MgCl2 ( 10 mM), pH 8.0; buffer B: Tris-HCl (25 mM), imidazole (20 mM), NaCl (300 mM), pH 8.0; buffer C: Tris-HCl (25 mM), imidazole (250 mM), NaCl (300 mM), pH 8.0; buffer D: Tris-HCl (25 mM), MgCl2 (10 mM), pH 7.0. A single colony of E. coli BL21(DE3)/pRM070 was used to inoculate 5 mL LB containing ampicillin. After 12 hours of growth, the 5 mL culture was transferred to 1 L LB containing ampicillin and grown at 28°C until OD,>00 reached 0.5. IPT G was added to the culture to a final concentration of 0.1 mM. After an additional 8 hr growth at 28°C, the cells (6 g) were harvested by centrifugation (44003 for 5 min at 4 °C), resuspended in 224 Buffer A (18 mL), and lysed by two passages through a French press at 16000 psi. Cellular debris was removed by centrifugation at 480003 and 4 °C for 25 min. To the supernatant, a 50% slurry (w/v) of Ni-NT A agarose resin was added (1 mL resin per 4 mL of crude lysate), and the mixture was stirred at 4 °C for an hour. The lysate resin slurry was transferred into a polypropylene column (Qiagen) and washed with Buffer B (2 x 4 mL per mL of Ni-NTA agarose resin). The 6-His tagged protein was eluted from the column by washing with a solution of buffer C (2 x 1 mL per mL of Ni-NTA agarose resin). The eluted protein was concentrated, dialyzed against buffer D, quick frozen in liquid nitrogen, and stored at —80°C. The specific activity of purified kanosamine kinase was 0.4 unit/mg and the total activity obtained in the purification was 11 units. B. pumilus UDP-3-keto-D-glucose dehydrogenase UDP-3-keto-D-glucose dehydrogenase was purified from B. pumilus. Buffers used in the purification included buffer A: 20 mM Tris-HCl, 0.5 mM DTT, and 0.5 mM PMSF, pH 7.3; buffer B: 20 mM Tris-HCl, pH 7.3; buffer C: 20 mM Tris-HCl, 600 mM NaCl, pH 7.3; buffer D: 20 mM Tris-HCl, 150 mM NaCl; and buffer B: 20 mM Tris-HCl, 1 M (NH4)2SO4, pH 7.3. Growth of an inoculant was initiated by introduction of a single colony of B. pumilus from a nutrient agar plate into 100 mL of SSNG medium in a 500 mL flask with baffles and grown at 30 °C with agitation. After 30 h of growth, the 100 mL culture was then transferred to the fermentation vessel and the fermentation was run for 48 h. Cells were collected by first passing through four layers of cheesecloth followed by centrifugation (64003, 4°C, 10 min). The cells (27) were resuspended in buffer A (55 225 mL) and lysed by two passages through a French press at 16000 psi, and the cellular debris was removed by centrifugation (480003, 4 °C, 25 min). The supernatant was applied in 10 mL aliquots to a HiT rap DEAE column (1.6 x 10 cm) equilibrated with buffer B. The column was washed with 3 column volumes of buffer B followed by elution with a linear gradient of buffer B and buffer C (20 column volume) at a flow rate of 3 mL/min. Five milliliter fractions were collected. UDP-3-keto-D-glucose dehydrogenase containing fractions were identified by enzyme assay, combined, and concentrated to 20 mL. After dialysis against buffer B (3x), the solution was loaded in 5 mL aliquots onto a Resource Q column (16 mm x 30 mm, 6 mL) equilibrated with buffer B. The column was washed with 3 column volumes of buffer B followed by elution with a linear gradient (20 column volume) of buffer B and buffer C at a flow rate of 4.0 mL/min. Fractions (2 mL each) containing UDP-3-keto-D-glucose dehydrogenase were pooled and concentrated to 10 mL. After dialysis against buffer D (3x) and concentration (1.5 mL), the whole protein solution was loaded onto a Superdex 200 (prep grade, 16 mm x 600 mm) equilibrated with buffer D. The column was eluted with 2 column volumes of buffer D at a flow rate of 1.0 mL/min. Fractions (2 mL per tube) containing UDP-3-keto- D-glucose dehydrogenase were combined and concentrated to 2 mL. After dialysis against buffer B (3x), the protein was loaded onto a Phenyl HP column (16 mm x 25mm, 5 mL). The column was washed with 3 column volumes of buffer B followed by elution with a linear gradient (20 column volume) of buffer B and buffer B at a flow rate of 3.5 mL/min. Fractions (1 mL per tube) containing UDP-3-keto-D-glucose dehydrogenase were combined and concentrated to 2 mL. After dialysis against buffer B (3x), the enzyme was quick frozen in liquid nitrogen and stored at -80°C. The specific activity of 226 purified UDP-3-keto-D-glucose dehydrogenase was 18.6 unit/mg and the total activity obtained in the purification was 9.3 units. Recombinant UDP-3-keto-D-glucose dehydrogenase Recombinant UDP-3-keto-D-glucose dehydrogenase was purified from XL-l Blue/pJG9.251 by Ni-NTA Agarose column (Qiagen). Buffers used in the purification included buffer A: KZHPO4 (50 mM), imidazole (10 mM), phosphoenolpyruvate (0.5 mM), pH 8.0; buffer B: KZHPO4 (50 mM), imidazole (20 mM), NaCl (300 mM), pH 8.0; buffer C: KZHPO4 (50 mM), imidazole (250 mM), NaCl (300 mM), pH 8.0; and buffer D: KZHPO4 (50 mM), phosphoenolpyruvate (0.5 mM), pH 7.0. A single colony of E. coli XL-l Blue/pJG9.251 was used to inoculate 5 mL LB containing ampicillin. After 12 hours of growth, the 5 mL culture was transferred to 1 L LB containing ampicillin and grown at 30°C until an OD600 reached 0.5. IPT G was added to the culture to a final concentration of 1 mM. After an additional 8 hr growth at 30°C, the cells (5 g) were harvested by centrifugation (44003 for 5 min at 4 °C), resuspended in Buffer A (15 mL), and lysed by two passages through a French press at 16000 psi. Cellular debris was removed by centrifugation at 480003 and 4 °C for 25 min. To the supernatant, a 50% slurry (w/v) of Ni-NTA agarose resin was added (1 mL resin per 4 mL of crude lysate), and the mixture was stirred at 4 °C for an hour. The lysate resin slurry was then filled into a polypropylene column (Qiagen) and the column was washed with Buffer B (2 x 4 mL per mL of Ni-NTA agarose resin). The 6—His tagged protein was eluted from the column by washing with buffer C (2 x 1 mL per mL of Ni-NTA agarose resin). The eluted protein was concentrated, dialyzed against buffer D, quick 227 frozen in liquid nitrogen, and stored at —80°C. The specific activity of purified recombinant UDP-3—keto-D-glucose dehydrogenase was 2.3 unit/mg and the total activity obtained in the purification was 30 units. Cell-free Lysate Preparations Cell-free lysate of A. mediterranei A. mediterranei was first grown76a on a YNG plate at 28°C for 4 days. This plate culture of A. mediterranei was then used to inoculate vegetative medium. A 50 mL vegetative culture was grown in a 500 mL flask with baffles for two days at 28°C and 250 rpm and then a portion of this vegetative culture (5 mL) was used to inoculate 50 mL of production medium in 500 mL flask with baffles. The synthesis of rifamycin B was checked by spectrophotometryI91 after 4 days of incubation at 28°C and 280 rpm. The culture that showed the highest synthesis of rifamycin B was used to inoculate 500 mL YMG medium. After 3 days of incubation at 28°C and 300 rpm, the mycelia were harvested by centrifugation (86003, 4°C, 5 min). After washing the mycelia with 50 mM Tris-HCl buffer (pH 7.5), the cells were harvested by centrifugation (110003, 4°C, 5 min). The mycelia were resuspended in Tris-HCl buffer (pH 7.5) containing 1 mM PMSF (5 mL/g of wet cells). Cells were then disrupted by two passages through a French press (16000 psi). The cell debris was removed by centrifugation (480003, 4 °C, 25 min) and the supernatant was used directly in most of the cell-free reactions. In cases that removing micro solute were required, dialysis were carried out after centrifugation. Dialysis was performed using a Millipore PM-lO membrane and an Aminco stirred cell (300 mL). The cell lysate (about 30 mL) was first diluted to 300 mL followed by 228 concentration (to about 25 mL). More buffer (225 mL) was added to the concentrator. The sample was concentrated to 25 mL again and the process was repeated until the concentration of the micro solute was sufficiently reduced. Typically, 3 to 6 cycles were performed to remove most of initial salt content. Finally, the lysate was concentrated to about 30 mL. Cell-free lysate of B. pumilus B. pumilus was first grow on a nutrient agar plate at 37 °C for 18-24 h. A single colony from the plate was used to inoculate SSNG medium. A 100 mL culture was grown in a 500 mL flask with baffles for 1.5 to 2 days at 30°C and 250 rpm. The whole 100 mL culture was then transferred to a 4 L flask with baffles containing 1 L of the same medium. After 2 days of incubation at 30°C and 250 rpm, cells were collected by first passing through four layers of cheesecloth followed by centrifugation (64003, 4°C, 10 min) of the filtrate. After washing the cells with 50 mM Tris-HCl buffer (pH 7.5), the , cells were harvested by centrifugation (64003, 4°C, 10 min). The cells were resuspended in 2 mL of 50 mM phosphate buffer (pH 7.0) per 1 g of wet cells and were disrupted by two passages through a French press (16000 psi). The cell debris was removed by centrifugation (480003, 4°C, 25 min). In Vitro Enzymatic Reactions Biosynthesis of aminoDAHP from aminoF6P 3-Amino-3-deoxy-D-fructose 6-phosphate (aminoF6P, 0.025 g, 0.096 mmol), D- ribose 5-phosphate (0.027 g, 0.099 mmol), and phosphoenolpyruvate (0.042 g, 0.20 mmol) were incubated with A. mediterranei cell-free lysate containing 0.2 units of DAHP 229 synthase activity, or purified transketolase (9 units) and aminoDAHP synthase (64 units) at 28°C for 6 h. Protein was subsequently removed by ultrafiltration. Crude, protein-free reaction mixtures were applied to an AG-l X8 anion exchange resin (2.5 cm x 6 cm, 30 mL, acetate form). The column was washed with H20 (60 mL) and eluted with a linear gradient (120 mL + 120 mL, 0-3.0 M) of AcOH. Fractions that tested positive by thiobarbiturate colorimetric assay (TBA)99 were collected and further purified by HPLC using an AXpak WA-624 weak anion exchange column (6 mm x 150 mm), which had been equilibrated with distilled water. The column was eluted with AcOH (0.5 M, pH 4). Fractions containing aminoDAHP were identified by TBA assay and combined. The solvent was evaporated and then azeotroped with water to remove AcOH. The residue was purified using a sugar KS-801 strong cation exchange column (8 mm x 300 mm), which had been equilibrated with distilled water. The column was eluted with a linear gradient (0-20 mM, 40 column volume) of NH4HCO3 (pH 7.3). Fractions containing aminoDAHP were identified by TBA assay, and the NH4HCO3 was removed by passing through a Dowex 50 (H’, 2.5 cm x 6 cm, 30 mL) column. AminoDAHP was eluted from the column by a linear gradient (100 mL + 100 mL, 0-2.0 M) of AcOH. Fractions testing positive towards the TBA assay were pooled. The solvent was evaporated, azeotroped with water to remove AcOH and then concentrated to dryness. The final yield of aminoDAHP was determined by 1H NMR. 1H NMR (D20): 6 4.15 (m, 2 H, H-7, 7’), 3.95 (m, 1 H, H-6), 3.61 (ddd, J = 13, 10, 5 Hz, 1 H, H-4), 3.52 (dd, J = 9, 9 Hz, 1 H, H- 5), 2.30 (dd, J = 13, 5 Hz, 1 H, H-3eq), 1.85 (dd, J = 13, 13 Hz, 1 H, H-3ax). 13C NMR (D20): 6 175.0 (C-l), 95.5 (C-2), 74.1 (JPOC = 6 Hz, 07), 67.6 (JPOCC = 4 Hz, C-6), 65.2 (C-5), 57.3 (C-4), 38.2 (C-3). 31P NMR (D20): 6 1.46. COSY (D20): H-3ax => H-3eq; 230 H-3ax, H-3eq => H-4; H-4 => H-5; H-5 => H-6; H-6 => H-7, 7'. HMQC (D20): H-3eq; H-3ax => 03; H-4 => C-4; H-S => C-S; H-6 => C-6; H-7, 7' => C-7. HRFABMS: calcd for C7HI3N09P (M - H1): 286.0328. Found: 286.0312. Biosynthesis of aminoDAHP from kanosamine 6-phosphate Kanosamine 6-phosphate (0.025 g, 0.096 mmol), D-ribose 5-phosphate (0.027 g, 0.099 mmol), and phosphoenolpyruvate (0.042 g, 0.20 mmol) were incubated with A. mediterranei cell-free lysate containing 0.2 units of DAHP synthase activity, or purified transketolase (9 units), aminoDAHP synthase (64 units), and commercial yeast phosphoglucose isomerase (60 units) at 28°C for 6 h. Protein was subsequently removed by ultrafiltration. The crude, protein-free reaction mixture was applied to an AG-l X8 anion exchange resin (2.5 cm x 6 cm, 30 mL, acetate form). The column was washed with water (60 mL) and eluted with a linear gradient (120 mL + 120 mL, 0-3.0 M) of AcOH. Fractions that tested positive by thiobarbiturate colorimetric assay99 (TBA) were collected and further purified by HPLC using an AXpak WA-624 weak anion exchange column (6 mm x 150 mm), which had been equilibrated with distilled water. The column was eluted with AcOH (0.5 M, pH 4). Fractions containing aminoDAHP were identified by TBA assay and combined. The solvent was evaporated and then azeotroped with water to remove AcOH. The residue was then purified using a sugar KS-801 strong cation exchange column (8 mm x 300 mm) which was equilibrated with distilled water. The column was eluted with a linear gradient (0-20 mM, 40 column volume) of NH4HCO3 (pH 7.3). Fractions containing aminoDAHP were identified by TBA assay, and the NH4HCO3 was removed by passing through a Dowex 50 (W, 2.5 cm x 6 cm, 30 231 mL) column. AminoDAHP was eluted from the column by a linear gradient (100 mL + 100 mL, 0-2.0 M) of AcOH. Fractions testing positive towards the TBA assay were pooled. The solvent was evaporated, azeotroped with water to remove AcOH and then concentrated to dryness. The final yield of aminoDAHP was determined by 1H NMR. 1H NMR (D20, TSP as reference): 6 4.15 (m, 2 H, H-7, 7’), 3.95 (m, 1 H, H-6), 3.61 (ddd, J = 13, 10, 5 Hz, 1 H, H-4), 3.52 (dd, J: 9, 9 Hz, 1 H, H-5), 2.30 (dd, J: 13, 5 Hz, 1 H, H- 3eq), 1.85 (dd, J = 13, 13 Hz, 1 H, H-3ax). 13C NMR (D20): 6 175.0 (C-l), 95.5 (C-2), 74.1 (JPOC = 6 Hz, C-7), 67.6 (JPOCC = 4 Hz, C-6), 65.2 (C-5), 57.3 (C-4), 38.2 (G3). 3'P NMR (D20): 6 1.46. COSY (D20): H-3ax =9 H-3eq; H-3ax, H-3eq => H-4; H-4 => H-5; H-5 => H-6; H-6 => H-7, 7'. HMQC (D20): H-3eq; H-3ax => 03; H-4 => C-4; H-5 = C- 5; H-6 => C-6; H—7, 7' => C-7. HRFABMS: calcd for C7HI3N09P (M - H’): 286.0328. Found: 286.0323. Biosynthesis of kanosamine from UDP-glucose UDP-D-glucose (0.025 g, 0.096 mmol), L-glutamine (0.027 g, 0.099 mmol), and B-NAD (0.042 g, 0.20 mmol) were incubated with A. mediterranei cell-free lysate (60 mg of protein, 28°C) or B. pumilus cell-free lysate (50 mg of protein, 30°C) for 6 h. Protein was subsequently removed by ultrafiltration. The crude, protein-free reaction mixture was applied to Dowex 50 (H+, 2.5 cm x 3 cm, 15 mL). The column was washed with water (60 mL) and eluted with a linear gradient (120 mL + 120 mL, 0-0.5 M) of HCl. Fractions containing kanosamine were identified by the ninhydrin assay, pooled, and concentrated to dryness. The final yield of kanosamine was determined by 1H NMR. 1H NMR (13,0): 6 5.27 (d, J = 3.5 Hz, 0.4 H), 4.72 (d, J = 8 Hz, 0.6 H), 4.39 (m, 1 H), 3.74- 232 3.87 (m, 2 H), 3.67 (ddd, J = 10, 10, 4.5 Hz, 1 H), 3.57 (ddd, J = 7.5, 5.5, 2 Hz, 0.5 H), 3.45 (dd, J = 10, 8 Hz, 0.5 H), 3.41 (dd, J = 10, 10 Hz, 0.5 H), 3.25 (dd, J = 10, 10 Hz, 0.5 H). ”C NMR (D20): 6 99.0, 94.1, 79.7, 74.2, 73.4, 71.0, 68.9, 68.8, 63.2, 63.0, 60.8, 58.1. Biosynthesis of 6,6-[2H2]-kanosamine UDP-6,6-[2H2]-D-glucose (0.025 g, 0.096 mmol), L-glutamine (0.027 g, 0.099 mmol), and B-NAD (0.042 g, 0.20 mmol) were incubated with A. mediterranei cell-free lysate (pH 6.8, 60 mg protein) at 28°C for 6 h. Protein was subsequently removed by ultrafiltration. The crude, protein—free reaction mixture was applied to Dowex 50 (H’, 2.5 cm x 3 cm, 15 mL). The column was washed with water (60 mL) and eluted with a linear gradient (120 mL + 120 mL, 0-0.5 M) of HCl. Fractions that tested positive towards ninhydrin colorimetric assay were collected. The yield of D-6,6-[2H2]-kanosamine was 5 %. 1H NMR (D20): 6 5.27 (d, J = 3.5 Hz, 0.4 H), 4.72 (d, J = 8 Hz, 0.6 H), 3.90 (d, J = 11 Hz, 0.5 H), 3.79 (dd, J = 11, 3.5 Hz, 0.5 H), 3.67 (ddd, J = 10, 10, 4.5 Hz, 1 H), 3.57 ((1,! = 11 Hz, 0.5 H), 3.45 (dd, J = 10, 8 Hz, 0.5 H), 3.41 (dd, J = 10, 10 Hz, 0.5 H), 3.25 (dd, J = 10, 10 Hz, 0.5 H). ”C NMR (D20): 6 99.0, 94.1, 79.7, 74.2, 73.4, 71.0, 69.0, 68-3, 62.8 (m), 60.9, 58.1. ESMS: (M + Na’): 204, 97.8% of di-deuterium enrichment. Biosynthesis of 7,7-[2H2]-aminoDAHP UDP-6,6-[2Hzl-D-glucose (210 mg, 0.34 mmol), B-NAD (315 mg, 0.47 mmol), L- glutamine (250 mg, 1.7 mmol), ATP (300 mg, 0.5 mmol), D-ribose 5-phosphate (100 mg, 0-36 mmol), and phosphoenolpyruvate (100 mg, 0.48 mmol) were incubated with 233 dialyzed A. mediterranei cell-free lysate (with 20% glycerol, 60 mg protein) at 28°C for 6 h. Protein was subsequently removed by ultrafiltration. The crude, protein-free reaction mixture was applied to an AG-l X8 anion exchange column (2.5 cm x 2 cm, 10 mL, acetate form). The column was washed with water (30 mL) and eluted with a linear gradient (100 mL + 100 mL, 0-3.0 M) of AcOH. Fractions that tested positive by TBA assay were collected and further purified by HPLC using an AXpak WA-624 weak anion exchange column (6 mm x 150 mm), which had been equilibrated with distilled water. The column was eluted with AcOH (0.5 M, pH 4). Fractions containing aminoDAHP were identified by TBA assay and combined. The solvent was evaporated and then azeotroped with water to remove AcOH. The residue was purified on a sugar KS-801 strong cation exchange column (8 mm x 300 mm), which had been equilibrated with distilled water. The column was eluted with a linear gradient (0-20 mM, 40 column volume) of NH4HCO3 (pH 7.3). Fractions containing aminoDAHP were identified by TBA assay, and the was NH4HCO3 removed by passing through a Dowex 50 (H", 2.5 cm x 6 cm, 30 mL) column. AminoDAHP was eluted from the column by a linear gradient (100 mL + 100 mL, 0-2.0 M) of AcOH. Fractions testing positive towards the TBA assay were pooled. The solvent was evaporated, azeotroped with water to remove AcOH and then concentrated to dryness. The final yield of 7,7-[2H2]-aminoDAHP was 0.9%. 1H NMR (500 MHz, D20, TSP as reference): 6 3.94 (m, 1 H, H-6), 3.61 (ddd, J = 13, 10, 5 Hz, 1 H, H-4), 3.52 (dd, J = 9, 9 Hz, 1 H, H-5), 2.29 (dd, J = 13, 5 Hz, 1 H, H-3eq), 1.85 (dd, J = 13, 13 Hz, 1 H, H-3ax). ”C NMR (D20): 6 175.0 (C-l), 95.5 (C-2), 67.6 (C-6), 65.2 (C-5), 57.6 (C-4), 38.2 (G3). 31P NMR (D20): 6 1.46. ESMS: (M - H’): 288, 95.3% of di-deuterium enrichment. 234 le or RifN-catalyzed phosphorylation of kanosamine Kanosamine (0.1 g, 0.6 mmol), ATP (sodium salt, 0.5 g, 0.9 mmol), MgClz-6HZO (0.2 g, 1 mmol), and citric acid'HzO (0.1 g, 0.48 mmol) were dissolved in deionized water. The pH of the solution was adjusted to 8.0 with aqueous NaOH, and the solution was deoxygenated with Ar for 5 rrrinutes. After the addition of le (15 mg protein) or RifN (15 mg protein), the reaction mixture was stirred slowly at rt for 24 h and the mixture was maintained between pH 7.5 and pH 8.0 by the addition of 1 N aqueous NaOH. The pH was adjusted every hour for six hours and then monitored every six hours until the end of the reaction. The crude reaction mixture was then applied to AG-l X8 anion exchange resin (2.5 cm x 16 cm, 80 mL, acetate form). The column was washed with distilled deionized water (160 mL) and eluted with a linear gradient (200 mL + 200 mL, 0-2 M) of AcOH. Column fractions containing inorganic phosphate and phosphate esters were identified by colorimetric assay.180 Fractions containing phosphate V esters were combined and concentrated to dryness. The products, kanosamine 6- phosphate, were isolated as a white solid in 20% and 50% yields when le and RifN were used, respectively. 1H NMR (D20): 6 5.27 (d, J = 3.5 Hz, 0.5 H, H-l), 4.74 (d, J = 7.5 Hz, 0.5 H, H-l), 4.02-4.17 (m, 2 H, H-6, 6’), 3.99 (br (1, J = 9.5 Hz, 0.5 H, H-S), 3.8 (m, 1.5 H, H-4, H-2), 3.67 (br d, J = 9.5 Hz, 0.5 H, H-5), 3.48 (dd, J = 10.5, 7.5 Hz, 0.5 H, H-2), 3.43 (dd, J = 10.5, 10.5 Hz, 0.5 H, H-3), 3.25 (dd, J = 10.5, 10.5 Hz, 0.5 H, H- 3). ”C NMR (D20): 6 99.1 (C-l), 94.2 (C-l), 78.5 (JPOCC = 7.6 Hz, C-5), 73.3 (C-2), 73.2 ((JPOCC = 7.6 Hz, C-5), 70.9 (C-2), 68.4 (C-4), 68.3 (C-4), 66.2 (JPOC = 3.8 Hz, 06), 60.5 (C-3), 57.7 (C-3). ). 31P NMR (D20): 6 1.60. gDQCOSY (D20): H-l => H-2; H-2 => H- 235 3; H-3 => H-4; H-4 = H-5; H-5 => H-6. gHMQC (D20): H-l => C-l; H-2 => C-2; H-3 => C-3; H-4 => C-4; H-S => C-5; H-6, 6’ => C-6. BacterioMatch Two-Hybrid System To assay for protein-protein interactions, BacterioMatch reporter competent cells were cotransformed with recombinant plasmid pairs encoding the genes of the two proteins of interest. The resulting transformants were plated onto LB agar plates supplemented with 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin. The plates were incubated at 30°C for approximately 24 hours. Colonies grow only if the two proteins have an interaction. Several control experiments were carried out simultaneously. The interaction between the dimerization domain (LGF2) of the yeast transcriptional activator Gal4 and Gall lP protein were used as positive control. The positive interaction was indicated by the growth of colonies on LB agar plates supplemented with 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin when plasmid pBT-LGF2 (Pmm, AC1, LGF2, Cm“) and pTRG-Galll (PW/mm, RNAP-a, Galll Tc“) were co-transformed into the BacterioMatch two-hybrid reporter strain competent cells. As negative controls, recombinant bait plasmid and non—recombinant target plasmid, or non—recombinant bait plasmid and recombinant target plasmid, or non—recombinant bait plasmid and non-recombinant target plasmid, respectively, were used to co-transform the BacterioMatch two-hybrid system reporter strain. No colony should be observed when these negative control trnasforments were grown on LB agar plates supplemented with 236 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin. After the initial screen by growing on LB agar plates supplemented with 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin, the appeared colonies were subjected to a second screen. Single putative positive colonies in the first round of selection were replicated onto a LB plate supplemented with 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, 50 ug/mL kanamycin, and 80 ug/mL 5-bromo-4-chloro-3-indolyl-B-D- galactopyranoside (X-gal). The plate was incubated at 37 °C for 12 to 30 h. The appearance of blue color implicated that the two proteins have an interaction. B-Galactosidase activities were also measured as described by the manufacturer’s protocol. A single colony was used to inoculate 100 mL of LB containing 250 ug/mL carbenicillin, 12.5 ug/mL tetracycline, 34 ug/mL chloramphenicol, and 50 ug/mL kanamycin. After 24 hours of growth at 37 °C, the cells wereharvested by centrifugation at 44003 for 5 min at 4 °C. Cells were resuspended in 250 mM Tis buffer (pH 8.0, 2 mL per g of cell) and lysed by two passages through a French press at 16000 psi. Cellular debris was removed by centrifugation at 480003 and 4 °C for 25 min. The assay reaction (1 mL) contained 200 uL of cleavage buffer (0.6 M NaZHPO4, 0.4 M NaHzPO4, 0.1 M KCl, and 0.01 M MgSO4, pH 7), 70 uL of ortho-nitrophenyl-B-D-galactopyranoside (ONPG, dissolved in sterilized water), cell lysate (1, 5, or 10 uL), and water (29, 25, or 20 uL). The total volume was 300 uL. The enzyme assay mixture was incubated at 37°C for 30 minutes. A faint yellow color was developed if B-galactosidase was present. The reaction was stopped by the addition of 500 uL stop buffer (1 M NazCO3). The final 237 volume was 800 uL. The absorbance at 420 nm was recorded against a blank containing ONPG and cleavage buffer without lysate. One unit of B-galactosidase activity was defined as the formation of 1 umol of ortho—nitrophenol per min at 37 °C. Inverse PCR Inverse PCR was conducted using a procedure described by Sambrook et al.159 Genomic DNA of B. pumilus was digested with appropriate restriction enzyme (Pstl, Mlul, Sphl, Xbal, or Xhol). A series of ligation reaction containing the cleaved and purified genomic DNA at a concentration ranging from 0.1 to 1 ug /mL were set up. A typical ligation (100 uL) reaction contained cleaved genomic DNA (10 ng, 50 ng, and 100 ng), 10 uL of ligation buffer (10X concentration), 4 HL of bacteriophage T4 DNA ligase, 10 uL of ATP (10 mM), and H20 (make up to 100 uL). Reactions were incubated at 16 °C for 12 h. After purification, the circulized DNA fragments were used as templates in PCR amplification. CHAPTER THREE Strain Purification A seven-step identification and isolation procedure was carried out to purify B. pumilus ATCC 21143.”'45 The initial purification step was a test for catalase activity known to be expressed by B. pumilus. Cell cultures grown for 24 h on nutrient agar plate were flooded with 0.5 238 mL of 10% hydrogen peroxide. A rapid bubbling of gas indicates the presence of catalase activity. The Voges-Proskauer test was based on the detection of acetylmethylcarbinol (acetoin) arising from metabolism of glucose. Bacteria metabolizes glucose to pyruvic acid, which is further metabolized to acetoin. After being grown in Voges-Proskauer medium containing (1 L) proteose peptone (7 g), glucose (5 g), and NaCl (5 g) at 35 °C for 24 h, 2.5 mL broth was transferred to a test tube. To this test tube was added 0.6 mL of 5% or-naphthol solution followed by 0.2 mL of 40% KOH solution. or-Naphthol catalyzes the conversion of acetoin to diacetyl in the presence of oxygen. KOH absorbs carbon dioxide and acts as an oxidizing agent to complete the conversion from acetoin to diacetyl. Diacetyl then reacted with guanidine-containing compounds such as arginine contained in the Voges-Proskauer medium. The formation of a pinkish-red color indicated the presence of acetoin. For the rapid Voges-Proskauer test, creatine was also added as an additional source of guanidine nucleus. Once added, creatine reacted with diacetyl to promote color development. 2 B. pumilus is unable to grow anaerobically. In this test, anaerobic agar was used as growth medium. It contained (1 L) trypticase (20 g), glucose (10 g), NaCl (5 g), sodium thioglycolate (2 g), sodium formaldehyde sulfoxylate (1 g), methylene blue (0.002), and agar (15 g). Thioglycolate and sodium formaldehyde sulfoxylate act as reducing agents to remove oxygen from the broth. Methylene blue was used as a chemical indicator in the broth. The absence of blue color indicates anaerobic conditions. Tubes of anaerobic agar were inoculated with colonies from nutrient agar plate by 239 stabbing to the bottom of the culture tubes. The state of cell growth was recorded at 3 and 7 days of incubation at 30 °C. B. pumilus is capable of growth in the presence of elevated NaCl (7%, w/v) concentration. Twenty tubes of nutrient broth containing 7% NaCl were inoculated and incubated at 30 °C with agitation at 250 rpm. The state of cell growth was recorded after 5 days of incubation. B. pumilus is unable to hydrolyze extracellular starch. Single colonies were replicated on nutrient agar plate containing 1% potato starch and incubated at 30°C for 48 h. Two methods were used to visualize the hydolysis of starch. The first one was iodine treatment. Iodine reacted with starch to produce a blue color. Hydrolysis was indicated by the development of a colorless zone surrounding the growth. The second method was ethanol treatment. The unchanged starch turned white and opaque. A clear zone underneath and around the growth was an indicator of hydrolysis. B. pumilus is able to use citrate as a sole carbon source. Phenol red plate was used to perform this test. It contained (1 L) sodium citrate (2 g) as a sole carbon source, NH4H2PO4 (1 g) as a sole nitrogen source, phenol red (0.008 g) as a pH indicator, KZHPO4 (1 g), NaCl (5 g), MgSO4 (0.5 g), and agar (15 g). The utilization of citrate was indicated by the formation of a red color underneath and around the growth. B. pumilus can utilize glucose, arabinose, sucrose, mannitol, and inositol as a sole carbon source. It is unable to utilize sorbitol, maltose, and lactose. The test medium contained (1 L) carbohydrate (5 g), yeast extract (0.2 g), (NH,,)2HPO4 (1 g), KCl (0.2 g), MgSO4 (0.2 g), and bromocresol blue (0.006). The utilization of a certain carbohydrate was indicated by the formation of a blue color underneath and around the growth. 240 Genetic Manipulations Plasmid pJG5.165A This 5.9-kb plasmid was created by inserting rifH into plasmid pKK223-3. The construction started with PCR amplification of a 1.3-kb rifH gene from cosmid F KN108I34 utilizing the following primers containing EcoRI restriction sequences: 5’- AAGAATTCCGACTAGCGCAGCATCTTCG and 5’-AAGAATTCGTGAAGCGGCA GCCGGACTT. Digestion of the resulting PCR product with EcoRI followed by ligation into the EcoRI site of plasmid pKK223-3 afforded plasmid pJG6.165A. The orientation of the rifl-I was in the same direction as the tac promoter. Plasmid pJG5.166A This 11.5—kb plasmid was constructed by inserting locus PmrifH into the Bglll site of plasmid pKD12.138.45 The construction started with digestion of plasmid pJG5.165A with BamHI. The resulting 1.6-kb PmCri/H DNA fragment was gel purified. Plasmid pKD12.138 was digested with Bglll. Ligation of these two purified fragments yielded plasmid pJG5.166A. The orientation of Pmrin was such that transcription was in the same direction as PmaroE. Plasmid pJG6.070A This 5.9-kb plasmid was created by inserting the dahpsI locus into plasmid pKK223-3. The construction started with PCR amplification of a 1.3-kb dahpsI gene from plasmid EDAHPB97 utilizing the following primers containing EcoRI restriction 241 sequences: 5’-AAGAATTCATGACGCTCTCCGCCGGACCG and 5’-AAGAATTC TTCCATGGTACCAGCTGCAGA. The amplified PCR product was digested with EcoRI and inserted into plasmid pKK223-3, which was linearized by digestion with EcoRI, to give plasmid pJG6.070A. The orientation of the dahpsl was in the same direction as the tac promoter. Plasmid pJG6.071A This 11.5-kb plasmid was constructed by inserting locus Pmdahpsl into the Bglll site of plasmid pKD12.138.45 The construction started with digestion of plasmid pJG6.070A with BamHI. The resulting 1.6-kb Pmdahpsl DNA fragment was gel purified. Plasmid pKD12.138 was digested with Bglll. Ligation of these two purified fragments yielded plasmid pJG6.071A. The orientation of PmdahpsI was such that transcription was in the same direction as PmaroE. Plasmid pJG6.128A This 7.0—kb plasmid was created by inserting the 0rf15 locus into plasmid pJF118EH. A 1.7-kb DNA fragment containing the open reading frame of 0rf15 was PCR amplified from cosmid FKN108134 employing the following primers containing EcoRI restriction sequences: 5’-AAGAATTCAGAGGCATCACGTG TGATTGA and 5’-AAGAATTCATGCAGATGACCGAAGAGAAC. Digestion of the PCR product with EcoRI followed by insertion into the plasmid pJF118EH, which was linearized by EcoRI digestion, resulted in plasmid pJG6.128A. The orfl5 locus was oriented such that transcription was from the tac promoter and in the opposite direction as lacIQ. 242 Plasmid pJG6.154A This 6.9-kb plasmid was created by inserting the rifKrifL cassette into plasmid pKK223-3. A 2.3-kb rle and rifl. DNA fragment was PCR amplified from cosmid FKN108134 employing the following primers containing ScaI restriction sequences: 5’- AAAAGTACTATGAACGCGCGAAAGGCACCGGAA and 5’-AAAAGTACTGGAG CGCTCCTTCATCACTGGTAC. The amplified PCR product was digested with Seal and inserted into plasmid pKK223-3, which was linearized by digestion with EcoRI followed by treatment with Klenow fragment, to give plasmid pJG6.154A. The orientation of the rifKrifL was in the same direction as the tac promoter. Plasmid pJG6.155B This 13.2-kb plasmid was created by inserting the PmrifKrifL cassette into plasmid pJG5.166A. The construction started with digestion of plasmid pJG6.154A with Sphl and Hindlll to liberate the 2.7-kb PmrifKrifL cassette. After treatment with mung bean nuclease, the DNA fragment of PmrifKrifL was gel purified. Blunt end ligation of this 2.7-kb fragment into plasmid pJG5.166A, which was linearized by removing the [3- lac gene with Ncol digestion followed by treatment with Klenow fragment, afforded the 13.2-kb plasmid pJG6.155B. The orientation of the PmrifKrifl. cassette was in the opposite direction as that of Pmrifl-I. Plasmid pJG6.180A This 5.7-kb plasmid was created by inserting glk locus into plasmid pKK223-3. The construction started with PCR amplification of a 1.1-kb glk containing DNA 243 fragment from plasmid pTC325 utilizing the following primers containing EcoRI restriction sequences: 5’-ACGAATTCATGGAAATTGTTGCGATTGAC and 5’-AC GAATTCGAAGGCAGCCTCTTAAAT TCA. The amplified PCR product was digested with EcoRI and inserted into plasmid pKK223-3, which was linearized by digestion with EcoRI, to yield plasmid pJG6.180A. The orientation of the glk locus was in the same direction as the lac promoter. Plasmid pJG6.181B This 11.1-kb plasmid was created by replacing the 1.0-kb B—lac gene of plasmid pJG5.166A with the 1.1-kb nglk fragment. The construction started with digestion of plasmid pJG6.180A with BamHI followed by treatment with Klenow fragment and gel purification. Plasmid pJG5.166A was digested with Neal and the resulting 8.9-kb fragment was subsequently treated with Klenow fragment and gel purified. The two purified fragments were ligated to generate plasmid pJG6.181B. The orientation of Pmcglk was such that transcription was in the opposite direction as that of PtacrifH. Plasmid pJG6.222A This 5.6-kb plasmid was created by inserting rifN locus into plasmid pKK223-3. The construction started with PCR amplification of rifN locus from cosmid FKN108134 employing the following primers containing EcoRI restriction sequences: 5’- AtGAATTCGTGAGCAGTGCTGCGAGGCCG and S’-ATGAATTCGTGGGCACC CCGTACCACCTC. The amplified PCR product was digested with EcoRI and subsequently inserted into the EcoRI site of plasmid pKK223-3 to give plasmid 244 pJG6.222A. The rl]N locus was oriented such that transcription was from the tac promoter and in the same direction as the genetic marker bla conferring resistance to Ap. Plasmid pJG6.223B This 11.8-kb plasmid was created by inserting the PmrifiV fragment into plasmid pJG5.166A. The construction started with digestion of plasmid pJG6.222A with BamHI to liberate the 1.3-kb PmriflV. After treatment with Klenow fragment, the DNA fragment of PmrifN was gel purified. Plasmid pJG5.166A was digested with Neal and subsequently treated with Klenow fragment followed by gel purification. The two purified fragments were ligated to generate plasmid pJG6.223B. The PmriflV locus was in the opposite orientation as that of PmrifH. Plasmid pJG6.238A This 13.9-kb plasmid was created by inserting the lacIQPmorfI5 cassette and PmrifN locus into plasmid pJG5.166A. The construction started with digestion of plasmid pJG6.128A with Nrul and Hindlll. The DNA fragment containing lalePmorfI5 locus was gel purified. Plasmid pJG6.222A was digested with Seal and AflIII to liberate the 1.3-kb PmrifN locus, which was subsequently gel purified. Plasmid pJGS.l66A was digested with Ncol and HindIII followed by gel purification. The three purified fragments were ligated to generate plasmid pJG6.238A. The Pmorfl5 and Pmrsz loci were placed such that transcriptions were in the opposite direction as that of PmrifH and laClQ. 245 Plasmid pJ G7 .032A This 14.1-kb plasmid was created by inserting the rifL locus with E. coli ribosomal binding site into plasmid pJG6.155B. The construction started with digestion 142 of plasmid pRM030 with X bal and HindIII .to liberate the 1.5-kb DNA fragment containing the locus of rifL cloned behind E. coli ribosomal binding site. Blunt end ligation of this 1.5-kb fragment into plasmid pJG6.ISSB, which had been linearized by partially removing the rifL gene with Xhol digestion followed by treatment with Klenow fragment, afforded the 14.1-kb plasmid pJG7.032A. The rifL locus with E. coli ribosomal binding site was oriented such that transcription was from the same tac promoter, which was placed in front of rifK locus. Plasmid pJG7.039A This 5.4-kb plasmid was created by ligation of the rifM locus into plasmid 134 pKK223-3. A 0.8-kb rifM gene was PCR amplified from cosmid FKN108 employing the following primers containing EcoRI restriction sequences: 5’- AAGAATTCATGACGCTCTCCGCCGGACCG and 5’-AAGAATTC’ITCCATGGT ACCAGCTGCAGA. The amplified PCR product was digested with EcoRI and subsequently inserted into the EcoRI site of pKK223-3 to give plasmid pJG7.039A. The orientation of the rifM locus was in the same direction as that of tac promoter. Plasmid pJG7.046A This 5.5-kb plasmid was created by ligation of the rifN DNA fragment into plasmid pKK223-3. The construction started with PCR amplification of the 0.9-kb riflV 246 locus from cosmid FKN108134 employing the following primers containing EcoRI restriction sequences: 5’-ATGAATTCGTGAGCAGTGCTGCGAGGCCG and 5’-AT GAATTCGTGGGCACCCCGTACCACCTC. The amplified PCR product was digested with EcoRI and subsequently inserted into the EcoRI site of pKK223-3 to give plasmid pJG7.046A. The orientation of the rifN locus was in the same direction as that of tac promoter. Plasmid pJG7.056A This 6.9-kb plasmid was created by inserting the rifN with E. coli ribosomal binding site into pJG7.039A. The construction started with digestion of plasmid pJG7.046A with Sspl to liberate a 1.5-kb DNA fragment containing the riflV locus with E. coli ribosomal binding site. Blunt end ligation of this 1.5-kb DNA fragment into plasmid pJG7.039A, which had been linearized by Smal digestion, afforded the 6.9-kb plasmid pJG7.056A. The rifN locus with E. coli ribosomal binding site was oriented such that transcription was from the same tac promoter, which was placed in front of rifM locus. Plasmid pJ G7 .07 1A This 16.4-kb plasmid was created by inserting the Pmrl'erifN cassette into plasmid pJG7.032A. The 2.3-kb PmrszriflV cassette was excised out of plasmid pJG7.056A by digestion with Sphl and Hindlll followed by treatment with Mung bean nuclease. Blunt end ligation of this 2.3-kb fragment into plasmid pJG7.032, which had been linearized by Ncol digestion followed by treatment with Klenow fragment, afforded 247 the 16.4-kb plasmid pJG7.071A. The orientation of the PmrieriflV cassette was in the opposite direction as that of PmrifKrifL. Plasmid pJG8.155 This 5.2-kb plasmid was created by inserting the amylase promoter and ribosomal binding site Pam, and rifl locus into plasmid pKK223-3. A 0.8-kb rifl gene was PCR amplified from cosmid FKN108134 employing the following primers containing Xbal and EcoRI restriction sequences: 5’-A'ITCTAGAATGCCCATCAGCGGT ACCACC and 5’- ATGAATTCTCGTCAACCCAGGCCGAAGAA. The resulting rifl DNA fragment was then digested with Xbal and EcoRI. A 0.32-kb Pam, DNA fragment was PCR amplified from plasmid pRL-60132 employing the fowling primers containing Sphl and Xbal restriction sequences: 5’-ATCGCATGCGATCGCCCACCAGACC GTGCA and 5’- A'I'TCTAGAGTGGTGCCTCCTGATCGGGGG. The PCR product was subsequently digested with Sphl and Xbal. Plasmid pKK223-3 was digested with Sphl and EcoRI. The three purified fragments were ligated to yield plasmid pJG8.155. The orientation of the rifl locus was in the same direction as that of amy promoter. Plasmid pJG8.219A This l-1.3-kb plasmid was created by inserting the Pamyrifl locus into plasmid pRL- 132 60. The construction started with PCR amplification of Pmrifl locus from plasmid pJG8.155 employing the following primers containing Mlul restriction sequences: 5’- TAGACGCGTCCCACCAGACCGTGCAGCGCA and 5’-AGTACGCGTGTCAACCC AGGCCGAAGAAAT. The resulting 1.1-kb DNA fragment was digested with Mlul and 248 inserted into the Mlul site of plasmid pRL-60 to generate plasmid pJG8.219A. The orientation of Pamyrlfl was such that transcription was in the opposite direction as that of amy. Plasmid pJG9.240A This 12.9-kb plasmid was created by replacing the 3.1—kb fragment containing tktA locus with the 4.1-kb lacIQPmorfIS cassette. The construction started with digestion of plasmid pJG6.128A with Nrul and ScaI to liberate a 4.1-kb DNA fragment containing the lalePmorf15 cassette. 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