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(£23.11. 1.1.115! y f . 1». ....-.‘.‘.«5e .5. «I Ii... 3 1.. r » I..v¢\o } $33.4. .. .3..xp!v.. l Q)..|us.. 3. ”1!- A. an; anfiafii , A: as; J . ikvuhilfi at! .I . 1 I u!!- chulnfl.’ 3 _ I .1“ .50. 1.. .1 Enos". \..,~ 3.3.0....- .....5. . p This is to certify that the dissertation entitled CHEMO-ENZYMATIC SYNTHESIS OF CATECHOL AND INVESTIGATION OF BIOSYNTHETIC NITRATION FOR SYNTHESIS OF ARYLNITRO MOLECULES presented by Wensheng Li has been accepted towards fulfillment of the requirements for the PhD degree in Chemistry Qa/a aka/W2 Major Professor’ s Signature 3 //<’/@ (I: Date MSU is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University 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 2/05 p:/ClRC/DateDue.indd-p.1 CHEMO-ENZYMATIC SYNTHESIS OF CATECHOL AND INVESTIGATION OF BIOSYNTHETIC NITRATION FOR SYNTHESIS OF ARYLNITRO MOLECULES By Wensheng Li A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 2005 ABSTRACT CHEMO-ENZYMATIC SYNTHESIS OF CATECHOL AND INVESTIGATION OF BIOSYNTHETIC NITRATION FOR SYNTHESIS OF ARYLNITRO MOLECULES By Wensheng L1 Sustainable development is a worldwide movement of the chemical industry and requires environmentally benign chemical manufacturing and minimizing the use of non— renewable resources. Biocatalytic synthesis, in the forms of enzymatic synthesis and microbial synthesis, is attractive for industrial organic synthesis. However, organic synthesis using biocatalysis to meet the requirements of sustainable development encounters two challenges. One is inhibition or even toxicity of molecules involved in the biosynthetic reactions towards enzymes and/or living cells. The other is the limited number of enzymes available for chemical conversion. As a case study of the challenges presented by the microbial toxicity of many aromatic chemicals, the synthesis of catechol from D-glucose is examined. One route, which led to an increase of catechol productivity, is to reduce the concentration of catechol synthesized in culture medium by resin-based extractive fermentation and hence minimizing the catechol toxicity towards E. coli used as biocatalyst. Other routes include hybrid microbial synthesis and chemical synthesis to completely avoid interfacing catechol and microbes. E. coli strains were constructed to accumulate non-toxic shikimate pathway intermediates, 3-dehydroquinic acid and 3—dehydroshikimic acid, which were chemically converted to catechol by heating in near critical H20. The less- toxic protocatechuic acid, which was also converted to catechol in near critical H20, was synthesized either chemically from 3-dehydroquinic acid and 3-dehydroshikimic acid, or microbially from glucose via regular or resin-based extractive fed-batch fermentation. By comparison, catechol was synthesized from glucose with the highest yield 43% (mol/mol) via intermediacy of protocatechuic acid obtained from extractive fermentation. Elucidation of biosynthetic pathways may lead to the discovery of enzymes with new functionalities. Enzymatic routes can then be established for organic synthesis. Arylnitro-containing antibiotics, pyrrolomycin A was isolated and characterized from Actinosporogum vitaminophilus and dioxapyrrolomycin from Streptomycses Sp. UpJohn UC11065. Experiments were designed to investigate the biosynthetic pathway of pyrrolomycin A, with emphasis on the mechanism of biosynthetic nitration. One of the hypothesized mechanisms, direct nitration, was found unlikely involved in the biosynthesis of pyrrolomycin A. However, evidence from an indirect method suggested bacterial nitric oxide synthase (NOS) was most likely responsible for biosynthetic nitration in the synthesis of pyrrolomycin A. Further genetic information is required to full understand bacterial biosynthetic nitration. Copyright by Wensheng Li 2005 To: My Son My Wife My Mother My whole family ACKNOWLEDGMENTS I would like to thank all the people who helped me. First and foremost, 1 want to thank my research advisor Prof. John Frost for his guidance and encouragement throughout the 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. Jame E. Jackson and Prof. James H. Gieger for their intellectual input and encouragement during the preparation of this dissertation. I am grateful to Dr. Karen M. Draths for her wealth of knowledge that she freely shared with me. I also wish to thank all of the Frost group members, both past and present, Dr. Chad Hansen, Dr. Padmesh Venkitasubramanian, Dr. Jian Yi, Dr. Jiantao Guo, Dr. Wei Niu, Dr. Ningqing Ran, Dr. Dongming Xie, Dr. Jihane Achkar, Dr. Mo Xian, Dr.. Mapitso Molefe, Ms. Heather Stueben, Mr. Xiaofei Jia, Mr. Justas Jancauskas, Mr. Man—Kit Lau, Mr. Jingsong Yang and Mr. Brad Cox for the happies we had together during all these years. Last but not least I would like to thank my mother Gengying Huang, my wife Dr. Chunfeng Zhang, my son Kevin Y. Li, my parents-in-law Yuping Zhang and Shuhua Dong and all my family members for their love, constant encouragement and support through out my life. This thesis is dedicated to them. vi TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... x LIST OF FIGURES ......................................................................................................... xi LIST OF ABBREVIATIONS ......................................................................................... xv CHAPTER ONE .............................................................................................................. 1 INTRODUCTION: CHEMO-ENZYMATIC SYNTHESIS AND ELUCIDATION BIOSYNTHETIC PATHWAY FOR ORGANIC SYNTHESIS ........................................ 1 Overview ...................................................................................................................... 1 Chemical Synthesis in Conjunction with Microbial Synthesis ....................................... 3 Microbial Synthesis in Conjunction with Chemical Synthesis ....................................... 6 s-Caprolactam. ......................................................................................................... 7 Shikimate pathway and its manipulation for organic synthesis. ................................. 8 Chemo-enzymatic synthesis based on Shikimate pathway. ...................................... l6 Elucidation of Biosynthetic Pathway for Organic Synthesis ........................................ 21 The DXP pathway .................................................................................................. 22 Polyketide synthesis and nonribosomal peptide synthesis ........................................ 24 Reference ................................................................................................................... 30 CHAPTER TWO ........................................................................................................... 36 CHEMO-ENZYMATIC SYNTHESIS BASED ON SHIKIMATE PATHWAY: BENZENE-FREE SYNTHESIS OF CATECHOL ......................................................... 36 Introduction ................................................................................................................ 36 Catechol as organic building block. ........................................................................ 36 Early synthetic efforts towards catechol. ................................................................. 38 The toxicity of catechol towards organisms ............................................................. 43 One-Step Microbial Synthesis of Catechol Based on Shikmate pathway ..................... 45 E. Coli construct design. ......................................................................................... 45 Extractive fermentation ........................................................................................... 49 Synthesis of catechol under fermentor conditions .................................................... 52 Chemo-enzymatic Synthesis of Catechol Based on Shikimate Pathway ...................... 55 Synthesis of 3-dehydroquinate (DHQ). ................................................................... 55 Synthesis of 3-dehydroshikimate (DHS). ................................................................ 58 Synthesis of PCA. ................................................................................................... 61 Chemical conversion of DHQ, DHS and PCA to catechol in near critical H20. ....... 69 Overall conversion from glucose to catechol. .......................................................... 72 Discussion .................................................................................................................. 75 Sustainable development consideration for catechol synthesis ................................. 75 Non-benzene Based Synthesis of Catechol Impeded by molecular Toxicity. ........... 77 Near Critical Reaction is the Best Option to Synthesize Catechol ............................ 78 Conclusion .............................................................................................................. 81 Reference ................................................................................................................... 83 vii CHAPTER THREE ........................................................................................................ 89 investigation of BlOsynthetc NITRATION PATHWAY FOR nitroaromatics SYNTHESIS .................................................................................................................. 89 Introduction ................................................................................................................ 89 Overview ................................................................................................................ 89 Hypothesis of pyrrolomycin A biosynthetic pathway. ............................................. 92 Synthesis of Pyrrolomycin A and Dioxapyrrolomycin ................................................ 98 Purification of Actinosporangium vitaminophilus (ATCC 31673). .......................... 98 Chemical synthesis of pyrrolomycin A .................................................................. 101 Microbial Synthesis of Pyrrolomycin A. ............................................................... 103 Microbial Synthesis of dioxapyrrolomycin by Streptomyces SP. UpJohn UC11065.105 Hypothesis of Direct Bionitration Mechanism .......................................................... 105 Isotope labeling experiments ................................................................................. 106 In vitro Reactions with SNAC as PCP Analogues. ................................................ 107 In vitro Reactions with Ketone as Post-Modification Intermediates ....................... 111 Hypothesis of NOS-mediated Bionitration Mechanism ............................................. 115 Genetic approach to probe nitric oxide synthase .................................................... 118 An Alternative Way to Understand Bacterial Nitration Reaction ............................... 120 Cloning and heteroexpression of deiNOS .............................................................. 120 Synthesis of nitrotryptophan. ................................................................................ 123 Nitric oxide production assay activity. .................................................................. 123 Nitric oxide synthase nitration activity. ................................................................. 125 Disccussion .............................................................................................................. 127 Is direct nitration the mechanism? ......................................................................... 127 Is NOS-mediated nitration the mechanism? .......................................................... 130 Does NOS mediate bacterial nitration? ................................................................. 131 Reference ................................................................................................................. 141 CHAPTER FOUR ........................................................................................................ 146 EXPERIMENTAL ....................................................................................................... 146 General Chemistry .................................................................................................... 146 Chromatography. .................................................................................................. 146 Spectroscopic Measurements. ............................................................................... 147 Bacterial Strains and Plasmids. ............................................................................. 149 Storage of Bacterial Strains and Plasmids. ............................................................ 149 Culture Medium .................................................................................................... 150 General Fed-Batch Ferrnentor Conditions. ............................................................ 153 Analysis of Fermentation Broth. ........................................................................... 154 Genetic Manipulations .......................................................................................... 155 Enzyme Assays ..................................................................................................... 165 Chapter Two ............................................................................................................. 167 Plasmid construction ............................................................................................. 167 Microbial synthesis of catechol. ............................................................................ 167 Microbial synthesis of protocatechuic acid. ........................................................... 168 Microbial synthesis of catechol and PCA with in situ fermentation. ...................... 168 Microbial synthesis of 3-dehydroshikimic acid. .................................................... 171 viii Microbial synthesis of 3-dehydroquinic acid. ........................................................ 172 Chemical synthesis of protocatechuic acid. ........................................................... 173 Chemical synthesis of catechol. ............................................................................ 174 Chapter Three ........................................................................................................... 176 Purification of Actinosporangium vitaminophilus (ATCC 31673). ........................ 176 Culturing A. vitaminophilum and Streptomyces sp. UpJohn UC11065. .................. 178 Chemical synthesis of pyrrolomycin A .................................................................. 179 Microbial synthesis of pyrrolomycin A by A. vitaminophilum. .............................. 180 Dioxapyrrolomycin synthesized by UpJohn Streptomyces UC11065. .................... 183 Synthesis of pyrrole-2-acyl thioester (SNAC). ...................................................... 183 Synthesis of pyrrole—phenol ketone intermediates. ................................................ 185 Synthesis of nitrotryptophan. ................................................................................ 187 In vitro activity with SNAC and possible post-modification intermediates. ........... 189 Plasmid construction and deiNOS heteroexpression. ............................................. 189 NO production assay ............................................................................................. 190 Biosynthetic nitration assay. ................................................................................. 191 Reference ................................................................................................................. 193 Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. LIST OF TABLES Adsorption of procatehuic axid and catechol on anion exchange resins. ........... 51 Adsorption of catechol on adsorbent resins. ..................................................... 51 Identification of Actinosporangium vitaminophilus (ATCC 31673). ............... 101 Chemical synthesis of pyrrolomycin A ........................................................... 103 Pyrrolomycin A production in minimal medium. ........................................... 104 Nitration activity of nitric oxide synthase. ...................................................... 126 All Bacterial strains and plasmids. ................................................................. 149 LIST OF FIGURES Figure l. Chemo-enzymatic synthesis of L-ascorbic acid (vitamin C). ............................. 6 Figure 2. Chemo—enzymatic synthesis of e—caprolactam ................................................... 8 Figure 3. The Shikimate pathway in Escherichia coli. .................................................... 10 Figure 4. Typical chemicals derived from Shikimate pathway metabolites. .................... 12 Figure 5. In vivo synthesis of E4P in E. coli. ................................................................. 14 Figure 6. PEP availability and variant carbon channel into Shikimate pathway ............... 15 Figure 7. Microbial synthesis in conjunction with chemical synthesis based on Shikimate pathway ........................................................................................................ 18 Figure 8. Hypothesis microbial synthesis of hydroquinone from D-glucose. .................. 20 Figure 9. DXP pathway and its application in organic synthesis ..................................... 24 Figure 10. Polyketide synthesis and nonribosomal peptide synthesis and natural products .......................................................................................................... 26 Figure 11. PKS/NRPS hybrid synthesis of pyoluteorin .................................................. 27 Figure 12. Synthesis of ansamitocin from proansamitocin by post modification of PKS. 28 Figure 13. Synthesis of acyl-ACP (PKS) and aminoacyl-PCP (NRPS) protein and their N-acetylcysteamine (NAC) thioester (SNAC) mimic ..................................... 29 Figure 14. N-acetylcysteamine (NAC) thioester (SNAC) small molecules serve as PKS substrates for unnatural products synthesis derived from 6- deoxyerythronolide B. ................................................................................... 29 Figure 15. Application of catechol in agriculture: two—step synthesis of carbofuran. ..... 36 Figure 16. Application of catechol in perfumery: 4—step synthesis of vanillin. ............... 37 Figure 17. Application of catechol in pharmaceutical: chemical and microbial synthesis of L-Dopa from catechol. .............................................................................. 38 Figure 18. Chemical synthesis of catechol from petroleum based starting material. ....... 39 Figure 19. Metabolism of aromatic compounds through catechol intermediates. (. ........ 40 Figure 20. Microbial synthesis of catechol from D-glucose ............................................ 42 xi 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. Figure 40. Figure 41. Molecular modes of catechol toxic action in cells ......................................... 45 Construction of plasmid pWL1.284A ............................................................ 47 Construction of plasmid pWL1.290A ............................................................ 48 Resin-based extractive fermentation of protocatechuic acid and catechol ...... 51 Catechol synthesized by E. coli WNl/pWLl.290A was cultured under glucose-rich condition ................................................................................ 54 Construction of pJY1.216A. ......................................................................... 56 DHQ synthesized by E. coli QPI.1/pJY1.216A under glucose-rich condition.58 Reactivity of DHS and value-added compounds synthesized from D—glucose via 3-dehydroshikimic acid intermediacy ................................................... 59 DHS synthesized by E. coli KL3/pJY1.216A under glucose-rich condition... 60 DHS reactivity at pH 7.0 at different phosphate concentration. ..................... 62 DHS reactivity at pH 2.5 (top) and pH 2.2 (bottom). ..................................... 63 Construction of plasmid pWL2.46B .............................................................. 67 PCA synthesized by E. coli KL3/pWL2.46B was under regular fermentor conditions ................................................................................................... 68 PCA synthesized by E. coli KL3/pWL2.46B under resin (AG1X8) based fermentor conditions .................................................................................. 68 Reactivity of 3-dehydroquinate in near critical H20. ..................................... 71 Reactivity of 3-dehydroshikimate in near critical H20. ................................. 72 Reactivity of protocatechuate in water. ......................................................... 72 Interfacing of microbial and chemical synthesis of catechol from D-glucose via intermediacy of DHQ and DHS .............................................................. 73 Interfacing of microbial and chemical synthesis of catechol from D-glucose via intermediacy of chemically synthesized PCA. ........................................ 74 Interfacing of microbial and chemical synthesis of catechol from D-glucose via intermediacy of microbially synthesized of PCA from regular and extractive fermentor conditions .................................................................... 75 DHQ degradation competed against in high temperature H20. ...................... 81 xii Figure 42. Natural products containing aromatic nitro group. ........................................ 89 Figure 43. Biosynthesis of phloroglucinol ...................................................................... 90 Figure 44. Chemical synthesis of triaminotrinitrobenzene (TATB). ............................... 91 Figure 45. Hypothetic enzymatic synthesis of triaminotrinitrobenzene (TATB) ............. 92 Figure 46. Pyoteorin biosynthetic mechanism pathway and hypothesis of pyrrolomycin A biosynthetic pathway ............................................................................... 93 Figure 47. In vitro tyrosine nitration mechanisms with different ways to producing nitrogen dioxide radical. .............................................................................. 94 Figure 48. Biosynthetic pathway of pyrrolnitrin ............................................................. 96 Figure 49. Proposed biosynthetic pathway from PABA to PNBA in the biosynthetic pathway of aureothin ................................................................................... 97 Figure 50. Proposed mechanism of the biosynthesis of chloramphenicol. ...................... 97 Figure 51. Chemical synthesis of pyrrolomycin A. ...................................................... 103 Figure 52. Hypothesis of direct biosynthetic nitration with pyrrolyl-2-acyl-peptidyl carrier protein (PCP) or pyrrolyl-2-acyl-N-acetylcysteamine thioester (SNAC) as chlorination and nitration reaction ............................................ 108 Figure 53. Synthesis of the unsubstituted and 2, and 3—chloro substituted pyrrole-SNAC.110 Figure 54. Synthesis of DlF-I by post PKS modification reactions from THPH ........... 112 Figure 55. Hypothesis mechanism of biosynthetic nitration by post PKS modification reactions. ................................................................................................... I 12 Figure 56. Friedel-Craft reaction to couple pyrrolyl-acyl chlorinde and 2, 4- dichlorophenol/anisole ............................................................................... 1 13 Figure 57. Synthesis of possible ketone intermediates .................................................. 114 Figure 58. Incorporation of 15N into NO from isotope labeled L-arginine-guanido-‘SN2 by nitric oxide synthase. ................................................................................. 115 Figure 59a. Production of pyrrolomycin A in the presence of NOS inhibitor, NAME. . 116 Figure 60. Griess reagent assay (top) and hemoglobin assay (bottom) for nitric oxide synthase activity. ....................................................................................... 117 Figure 61. Designing degenerate primers for consensus PCR based on the conserved domains (highlighted by underline) amino acid sequences. ........................ 119 xiii Figure 62. Inverse PCR. .............................................................................................. 120 Figure 63. Cloning of deiNOS under different promotors. ........................................... 122 Figure 64. Synthesis of nitrotryptophan. ...................................................................... 123 Figure 65. Nitric oxide synthase activity. ..................................................................... 124 Figure 66. Glucose oxidase catalyzes the oxidation of glucose by air 02 and simutanuously generates H202. ............................................................... 126 Figure 67. Possible washout of incorporated '80 from the nitro group .......................... 128 Figure 68. Mass spectra of dioxapyrrolomycin ............................................................ 134 Figure 69. Mass spetra of pyrrolomycin A synthesized by A. vitaminophilus (ATCC 31673). ................................................................................................... 137 Figure 70. Mass spetra of pyrrolomycin B synthesized by A. vitaminophilus (ATCC 31673) .................................................................................................... 140 xiv Ac ADP ATP AP ApR bp CA CIAP Cm CmR DAHP DCU DEAD DEAE DHQ DHS D.O. DTT E4P EPSP FBR GA Hb HPLC LIST OF ABBREVIATIONS acetyl adenosine diphosphate adenosine triphosphate ampicillin ampicillin resistance gene base pair chorismic acid calf intestinal alkaline phosphatase chloramphenicol chloramphenicol resistance gene 3-deoxy-D-arabin0-heptulosonic acid 7-phosphate digital control unit diethyl azodicarboxylate diethylaminoethyl 3-dehydroquinic acid 3-dehydroshikimic acid dissolved oxygen dithiothreitol D-erythrose 4-phosphate 5-enolpyruvoylshikimate 3-phosphate feedback resistant gallic acid hour hemoglobin high pressure liquid chromatography XV IPTG Kan Kan” kb m LB M9 metHb min mL 14L mM uM NAD NADH NADP NADPH NMR NO NOS NRPS ORF PCA PEP isopropyl fl-D-thiogalactopyranoside kanamycin kanamycin resistance gene kilobase kilogram Michaelis constant Luria-Bertani molar minimal salts methemoglobin minute milliliter microliter millimolar micromolar 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 spectroscopy nitric oxide nitric oxide synthesis nonribosomal peptide synthesis open reading frame protocatechuic acid phosphoenolpyruvic acid xvi PCR Phe PKS Tyr Trp psi PTS QA rpm SA SDS S3P Tc TCA TSP UV oxyHb polymerase chain reaction L-phenylalanine polyketide synthesis pyruvate L-tyrosine L-tryptophan pounds per square inch phosphotransferase system quinic acid rotations per minute shikimic acid sodium dodecyl sulfate shikimate 3-phosphate tetracycline tricarboxylic acid sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 ultraviolet oxyhemoglobin xvii CHAPTER ONE ROD TIO ° HEMO-E ZYMATI Y THEI A DEL IDT I FBI YNTHETI PA H AY F R R ANI YNTHEI Overview "Sustainable development is development that meets the needs of the present without compromising the ability of future generations to meet their own needs." —the World Commission on Environment and Development (the Brundtland Commission), 1987. Sustainable development is a worldwide movement of the chemical industry and requires environmentally benign chemical manufacturing and minimizing the use of non- renewable resources. While only 100 years ago, renewable resources were the primary source of fuel and major raw materials for industrial chemical synthesis, the past half- century saw an upsurge of the petroleum chemistry, which channeled the primary dependence of energy and industrial feedstock into fossil fuel.1 The petroleum chemistry is currently meeting the present needs. People all over the world are enjoying the products from petroleum chemistry. However, it is time to return to the old days with the increasing concerns upon petroleum oil availability and environmental pollution. It is believed that petroleum oil is a finite resource, because its formation from biomass needs millions of years, while its consumption is increasing rapidly with the development of modern society.2 The technologies of industrial chemical processes had been improved to minimize the effect of petroleum chemistry on the environment, especially the use of chemical catalysts such as the Zigiel-Natta catalyst used for polymerization of ethylene and propylene. However, the pollution resulted from petroleum chemistry won't be diminished as both starting materials and products are not environmentally benign. Compared to chemical catalysis, biocatalysis in the forms of enzyme or microbe is not different, just better.3 However, the facts that biocatalysts function mostly in mild conditions of temperature and pH and preferentially in aqueous media make biocatalytic synthesis a good candidate for environmentally benign manufacturing processes. The unsurpassed regio- and enatioselectivity of enzyme reactions is also attractive for synthetic organic chemistry, especially in the pharmaceutical industry. Furthermore, the starting materials of enzyme reactions are always derived from renewable resources such as carbohydrate, lignin, animal and plant waste.4 The tendency of biocatalytic synthesis to partially, if not completely, replace the petroleum-based chemical process is unavoidable.4 Biocatalysis thus represents the movement of industrial chemistry towards sustainable development. However, the application of biocatalytic synthesis encounters two challenges. One is the inhibition or even toxicity of molecules involved in the reactions towards enzymes or tmicrobes used as biocatalyst. This is especially true for the biosynthesis of aromatic compounds. The other is the limited number of enzymes available for desired chemical conversions, although enzymes can catalyze a large variety of chemical reactions from hydralation to Diels-Alder reactions.4 One answer to enzyme inhibition is protein engineering to find inhibition-resistant genes by introducing mutations to the wild type gene, or shuffling the naturally related genes.5 In the case of highly toxic molecules, it is important to reduce or completely avoid interfacing toxic molecules with biocatalytic live cells. The strategies had been employed include in situ removal of toxic products by extractive fermentation and chemical conversion of microbially synthesized nontoxic intermediates into toxic molecules. The answer to the second challenge will be mainly based on the discovery of enzymes with new function. Since enzymes are always converting a certain intermediate to another in a biosynthetic pathway, elucidation of new biosynthetic pathways may result in discovery of enzymes with new functionality. In addition, elucidation of biosynthetic pathways also helps the discovery of rate—limiting steps and therefore, helpd finding a reasonable strategy to overcome it. Elucidation of new biosynthetic pathways is thus always attractive for organic synthesis. This dissertation will report our efforts addressing these two challenges. The first chapter is compromised by brief introduction of the applications of chemo—enzymatic synthesis and how organic synthesis benefits from the discovery of biosynthetic pathways. In the second chapter, chemo-enzymatic routes based on shikimate pathway were developed for catechol synthesis, a pseudocommodity chemical that is toxic towards microbes. By interfacing microbial and chemical synthesis, E. coli strains were engineered to convert D-glucose into intermediates with non or reduced microbial toxicity, followed by chemical conversion of the nontoxic intermediates into the aromatic product, catechol. In chaper 3, our attempts to elucidating the biosynthetic pathway of arylnitro-containing antibiotics, pyrrolomycin A, especially the mechanism of biosynthetic nitration reaction will be described. By doing this, we hoped to establish a general route for biosynthetic nitration reaction, which can be used for the enzymatic synthesis of arylnitro molecules such as the therrnoenergetic molecule, triaminotrinitrobenzene. Chemical Synthesis in Conjunction with Microbial Synthesis Although dramatic progresses had been made, biocatalysis is still not competitive with the petroleum chemistry from the economic point in most cases. Up to date, chemical synthesis is the dominant method in both industry and laboratory synthesis. However, in certain steps, chemical conversions may have to be under very harsh conditions and lack the required high selectivity. The advantages of biocatalysis aforementioned make it a promising supplement, or even replacement of chemical synthesis in industrial chemistry, especially, pharmaceutical chemistry, due to the pressure of synthetic efficiency, environmental and resource concerns. L-Ascorbic acid is the first pharmaceutical molecule, which was synthesized in a chemo-enzymatic way (Figure 1).‘5 Since no industrially useful microbes are available for L-ascorbic acid synthesis, the 6-step Reichstein-Griissner synthesis, which includes 5 chemical steps and 1 enzymatic step, contributes 50% of the $500 million/year L-ascorbic acid market.7 The synthesis started with the reduction of D-glucose to D-sorbitol, followed by selective enzymatic oxidation of D-sorbitol to L-sorbose. The protected L-sorbose is again oxidized to diacetone-2-keto-L-gluconic acid, which was deprotected to obtain the common intermeidate, 2-keto-L-gluconic acid. 2-Keto—L-gluconic acid was then cyclized to L-ascorbic acid (Figure 1, a-f).4 Further chemo-enzymatic routes toward L-ascorbic acid are based on enzymatic synthesis of the intermediates on the Reichstein-Griissner route via intermediacy of 2-keto-L-gluconic acid. In the D-sorbitol pathway (Figure 1, 1- 4 and 0, genetic engineered Gluconobacter oxydans was able to convert D-sorbitol to 2- keto-L-gluconic acid in a single step biosynthesis, which reduced the chemical steps in the Reichstein-Grfissner synthesis from 5 to 2 (step I and f).8 A recombinant Erwinia sp was further genetically engineered for converting D-glucose to 2-keto-L-gluconic acid dirctly, which left the Reichstein-Grijssner synthesis to only one chemical step (step f).9 The ultimate goal of microbially synthesizing L-ascorbic acid directly from glucose depends on enzymatic conversion of 2-keto-L-gluconic acid to L-ascorbic acid, which will elimilate all chemical steps. A gene is now obtained in Candida blankii and Cryptococcus dimmnae7a and expressed for this conversion. According to the above description, although the Reichstein-Griissner synthesis had already been optimized with glucose as starting material and use less organic chemicals and solvents, the increasing steps of enzymatic conversions still relieve the L-ascorbic acid synthesis from the tedious chemical protection and deprotection, chemical reaction working up, and the use of any harsh chemicals and organic solvents. The efficiency of the overall synthesis is also dramatically increased by the enzymatic conversions used in the overall synthesis. OH OH <—I—- Ho“' . OH HO“ 5H HO 5H 5H D-gluconic acid D-glucose D-sorbitol L-sorbose l“ 1' /OH 10 0 OH “0 I OH 0 o — 0 0H - o ; OH 5 O“ 0 5H HO OH OH /I \‘< 2—keto-D- - - - - diacetone-L- gluconic acid L ascorbic acnd L sorbosone sorbose O OH OH HO 1 0 iv H020 \OH 6 H020 O —-> = O‘X—Z‘o O & OH O 5 OH 0‘“ O OH OH 4 \‘< 2, 5-diketo-D 2-keto-L- diacetone—Z-keto- -gluconic acid gulonic acid L-gulonic acid Figure 1. Totally 4 synthetic routes towards L-ascorbic acid (vitamin C). Reichstein- Griissner synthesis (a-f): (a) H2, Pt/C; (b) D—sorbitol dehydrogenase; (c) acetone, H+; (d) KMnO4, OH'; (e) H3O+; (f) MeOH, H+. D-Sorbitol pathway (1-4, f): (1) H2, WC; (2) D-sorbitol dehydrogenase; (3) L-sorbose dehydrogenase; (4) L-sorbosone dehydrogenase. 2, 5-diketo-D-gluconic acid pathway (i-iv, f) (i) glucose dehydrogenase; (ii) gluconic acid dehydrogenase; (iii) 2-keto-D-gluconic acid dehydrogenase; (iv) 2, 5- diketo-D-gluconic acid reductase. One step biosynthesis of L-ascorbic acid from D- glucose (1). Microbial Synthesis in Conjunction with Chemical Synthesis Although biocatalytic synthesis is our ultimate goal for organic synthesis, it won’t do everything in some cases. Inhibition and toxicity of molecules towards enzymes and living cells as well as the limit number of reactions catalyzed by enzymes are the two main barriers. In such extreme cases, chemical synthesis can be a good supplement to enzymatic synthesis. With the renewable resources to be the starting materials of the overall synthesis, the goal to minimize the environmental impact of the necessitated chemical steps could be achieved by carefully selected reaction conditions. The synthesis of oxygenated aromatic compounds and their derivatives are typical examples. About 98% of this kind of chemicals is derived from petroleum chemistry via intermediacy of benzene, a volatile, carcinogen molecule.I0 Environmentally benign synthesis with carbohydrate as starting materials is thus highly desirable. e-Caprolactam. e-Caprolactam is used as the monomer for nylon-6 fibers and plastics production and one of chemical intermediates produced mostly in industry.11 All commercial processes for the manufacture of caprolactam are based on benzene, which is oxidized to phenol followed by hydrogenation to afford cyclohexanone. By reacting with hydroxylamine, cyclohexanone is converted to cyclohexanone oxime, which undergoes molecular rearrangement to the seven-membered ring, caprolactam (Figure 2).” By comparison of the structural similarity of e-caprolactam and L-lysine, which is now successfully synthesized from D-glucose by fermentation, a biobased synthesis of £- caprolactam is proposed. Up to now, enzymatic conversion of L-lysine to e-caprolactam is not available. However, a hybrid of chemical/microbial synthesis will permit the overall e-caprolactam synthesis from renewable source and minimizes its environmental impacts. This strategy is now under development in the Frost group (Figure 2).12 OH O NOH ©==©=>©i>© lb OH O OH + o " r3113 C NH —>—’ + W — ——> .=. OH H3N C02 HO OH — i D-glucose L-lysine caprolactam Figure 2. Chemo-enzymatic synthesis of e-caprolactam. (a) (N HZOH)2H2SO4, then NH3. (b) H2504'SO3, then N H3. (c) cyclization and hydrogen deamination. Shikimate pathway and its manipulation for organic synthesis. The shikimate pathway is the primary route to aromatic amino acids (tyrosine, phenylalanine and tryptophan) and aromatic vitamins.l3 It provides alternative biosynthetic intermediates for a variety of aromatic products and products derived from aromatic compounds, which are currently synthesized from benzene. Starting with the condensation of phosphoenolpyruvic acid (PEP) and D-erythrose 4-phosphate (E4P) to afford 3-deoxy-D-arabin0-heptulosonic acid 7-phosphate (DAHP) catalyzed by DAHP synthase (AroF, AroG, AroH), the shikimate pathway goes through seven biosynthetic reactions and ends with chorismic acid. The amazing enzyme, 3-dehydroquinic acid (DHQ) synthase (AroB) then converts DAHP to DHQ through 5 continuous steps including oxidation, elimination, reduction, ring opening, and intramolecular aldol condensation. Syn-elimination of H20 from DHQ catalyzed by aroD-encoded DHQ dehydratase affords 3-dehydroshikimate (DHS), which is reduced to shikimic acid (SA) in the 4‘h step catalyzed by an NADP-dependent shikimate dehydrogenase, encoded by aroE. Shikimate kinase (AroA, AroL) then transfers a phosphoryl group from ATP to 8 shikimic acid to afford shikimate 3-phosphate (S3P). The more reactive species, S3P is further condensed with phosphoenolpyruvate to yield 5—enolpyruvylshikimate 3- phosphate (EPSP) catalyzed by aroA-encoded EPSP synthase, releasing inorganic phosphate simultaneously. This step, however, is reversible.14 The second elimination reaction in the shikimate pathway is the chorismate synthase (AroC) catalyzed trans 1,4- elimination of phosphate from EPSP to yield the final product, chorismic acid. Chorismic acid then serves as the substrate for the synthesis of the three aromatic amino acids, L-tryptophan, L-tyrosine, and L-phenylalanine, via three terminal pathways.13 Chorismic acid is also converted into p-hydroxybenzoic acid, p-aminobenzoic acid, and 2,3-dihydroxybenzoic acid, which are the precursors for the biosynthesis of ubiquinone, folic acid, and enterobactin respectively. Ubiquinone (coenzyme Q) functions in the respiratory chain as an electron transporter, folic acid is a carrier of one-carbon units in biosynthetic reactions, and enterobactin is a chelating agent in bacterial iron uptake.15 The discovery of the shikimate pathway in plants and microorganisms including both bacteria and fungi is of great importance. It provides target enzymes for screening new nontoxic herbicides and antibiotics due to the absence of the shikimate pathway in animals.16 A broad—spectrum herbicide, glyphosate (N-phosphonomethylglycine) has been successfully marketed to inhibit EPSP synthase by competitively binding against phosphoenolpyruvate at this enzyme’s active site.17 OH O .\OH L-Tyr, L-Phe, L-Trp s. OH HO OH I T T D-glucose l C02H OPO H 1 0H 0 0 JL 3 2 + H203POM : O COZH COzH 5 OH pEp E4P OH chorismic acid H01: lg COZH 002H H203PO _ OJLCOZH H203PO OH OH DAHP EPSP lb if HQ, ECOZH CO?“ 0 a OH H203PO a OH OH OH DHQ SSP 1° Te COQH COzH Kl O a OH HO s OH OH DHS SA Figure 3. The shikimate pathway in Escherichia coli. Enzymes (encoding genes) (a) DAHP synthase (aroF, aroG, aroH); (b) DHQ synthase (aroB); (c) DHQ dehydratase (aroD); (d) shikimate dehydrogenase (aroE); (e) shikimate kinase (aroL, aroK); (f) EPSP synthase (aroA); (g) chorismate synthase (aroC). Abbreviations: phosphoenolpyruvate (PEP), D-erythrose 4—phosphate (E4P), 3~deoxy-D-arabin0-heptulosonic acid 7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS), shikimic acid (SA), shikimate 3-phosphate (S3P), 5-enolpyruvylshikimate 3-phosphate (EPSP). 10 The other application of the discovery of the shikimate pathway is that it provides important synthetic intermediates towards industrial chemicals and pharmaceutical chemicals (Figure 4). Metabolites in the shikimate pathway can be chemically or enzymatically converted to chemicals including quinic acid, gallic acid, pyrrogal and Tamiflu. Quinic acid is a highly functionalized, six—membered carbocyclic ring with multiple asymmetric centers and was used as an organic chiral synthon for combinatorial synthesis18 and natural products synthesis such as the neutral sphongomyelinase inhibitor, (+)-eutypoxide B”. Gallic acid and its esters are used in industry as food and cosmetics antioxidants and also used as synthetic blocks for the synthesis of antibiotic trimethoprim.20 Pyrogallol is the thermal decarboxylation product of gallic acid and is used in the production of dyes and photographic developers.20 The most famous use of the shikimate pathway metabolites may be the synthesis of neuraminidase inhibitor GS- 4104, an anti-influenza drug sold under the name TamifluTM form shikimic acid. 2‘ The Roche licensed drug is also found to be effective for bird flu currently spreads in south Asian. It is therefore urgent to channel carbon flux into shikimate pathway to improve the synthesis of shikimate pathway metabolites. The main challenge is that shikimate pathway in prokaryotes such as E. coli is very well regulated by both feedback inhibition and transcriptional control. Based on the knowledge of mechanism of shikimate pathway, the first effort will be searching inhibition-resistant genes to circumvent these barriers. Other challenges include the availability of the two starting materials of the shikimate pathway, phosphoenolpyruvate (PEP), D-erythrose 4-phosphate (E4P), which are derived for glycolysis. 11 ”033 © © Ccoou H 0 COOH catechol HO” COOH \ T 1 COgEt .[ l <— shikimate athwa metabolite/Isl —> Ho“ i OH p y \go‘ “V: ’NHz-H3P04 p-hydroxybezoic acid hydroqumone adipic acid 0” NHAc quinic acid ©/ Tamiflu COzH HO OH OH H300 phenol pyrogallol vanillin gallic acid Figure 4. Typical chemicals derived from shikimate pathway metabolites. DAHP synthase and inhibition impediments. Common to most metabolic pathways, the first committed step is always rate limiting. In the shikimate pathway, the first step is catalyzed by 3 isoenzymes (AroF, AroG, AroH) whose in vivo activities are subjected to both feedback inhibition and transcriptional control.13 The DAHP synthase is feedback inhibited by the 3 terminal aromatic amino acids, phenylalanine (AroG), tyrosine (AroF) and tryptophan (AroH). In E. coli, the aroG and aroF-encoded two isoenzymes contribute the major DAHP synthase activity, and their expression subjects to regulation of the TyrR repressor protein.22 In the presence of tyrosine and/or tryptophan, TyrR binds to specific sequences upstream of the aroG and aroF known as tyrR boxes, which repress the transcription of the two genes. When constructing E. coli strains for the biosynthesis of shikimate pathway intermediates, strategies were then developed to circumvent the two impediments based on the knowledge of the mechanism of DAHP 12 synthase. A mutation P148L was introduced into AroF by UV radiation, which led to the enzyme feedback insensitive (aroFFBR).23 Theoretically, providing more binding sites for the limited numbers of activated TyrR repressor protein in vivo can depress the transcriptional control. One choice is expressing extra copies of plamid localized arol‘i‘mm.23 Another choice is inserting of an extra copy of promoter Pam; into the plasmid, which provides 3 binding sites for activated TyrR repressor protein and allows aroFm being expressed from its native promoter. As a result, the DAHP synthase activity was maintained at a decent level to channel carbon flux into the shikimate pathway.23 The 5-step conversion of DAHP to DHQ catalyzed by DHQ synthase encoded by aroB is another rate-limiting step for the synthesis of shikimate pathway intermediates. Inserting an extra copy of aroB in the E. coli genome was demonstrated to be effective to overcome this rate-limiting step as no DAHP dephosphated and exported product DAH was detected in the culture supernatant from the fermentations of DHS and shikimic acid production strains.23'24 An E. coli strain KL3/pI(D11.29123 (encoding serA, aroFFBR and Pump) was constructed to synthesize DHS with aroFFBR and Pam].— expressed from plasmid to overcomet the feedback inhibition and transcriptional repression. DHS was accumulated 41 g/L with a total yield of 21% from D-glucose due to the lack of SA dehydrogenase (AroE) activity and an extara copy of aroB inserting in the genomic serA locus. TktAand thailam Drath et al demonstrated that after increasing DAHP synthase catalytic activity to a certain level, no improvements of aromatic amino acids biosynthesis could be achieved.25 This suggested the limit availability of the starting 13 materials. E4P availability is the one that was first studied. In wild-type E. coli, E4P is synthesized through three enzymatic reactions, which are catalyzed by transketolase (encoded by tktA or tktB) and transaldolase (encoded by talA or talB) (Figure 5). Overexpression of either transketolase or transaldolase was effective for increasing carbon flux into shikimate pathway,26 and thansketolase encoded by tktA was mostly used in the biosynthesis of shikimate pathway metabolites. Cloning tktA in the plasmid pKDl 1.291A obtained E. coli KL3/pKL5.17A, which accumulated 58 g/L DHS with a total yield of 27% from D—glucose when cultured under fermentor controlled conditions.23 Compared to E. coli KL3/PKD11.291A, which lacks the overpression of tktA, E. coli KL3/pKL5.17A increases of DHS productivity in both titer and yield is very obvious. H203PO OH 0 o H203PO OH H203PO OH OH 5 + H203PO a H —> I\/'\n’ + g OH OH OH OH 5H 0 OH 0 D-fructose D-glyceraldehyde D-xylulose 6-phosphate 3-phosphate 5-phosphate H203PO OH 0 H203PO OH 0 H203P0 OH H203PO OH 9H OH a I : + : ; H ‘— WH + i 5 OH OH OH OH OH OH O OH on o D-fructose D-ribose D-sedoheptulose 6—phosphate 5-phosphate 7-phosphate H203PO OH OH OH O H203PO OH H203PO OH 0 ° + V15 b H .5. i. H203PO i H —_>l i + F. OH OH O OH OH O OH OH OH D-sedoheptulose D-glyceraldehyde D-erythrose D-tructose 7-phosphate 3-phosphate 4-phosphate 6-phosphate Figure 5. In vivo synthesis of E4P in E. coli. Enzymes (encoding genes) (a) transketolase (tktA, or tktB); (b) transaldolase (talA, or talB). PTS system and PEP availability. Not all phosphoenolpyruvate (PEP) generated from glycolysis is able to condense with E4P to afford DAHP because of its multiple functions in vivo. One main reason that limits PEP availability for shikimate pathway is 14 that PEP loses its phosphate group to D—glucose in the phosphoenolpyruvatezcarbohydrate phosphotransferase system (PTS) (Figure 6) for glucose transportation into cells. The product, pyruvate is then oxidized through TCA cycle to C02, and as a result, the maximum theoretical yield of shikimate pathway products is 43% based on a stoichiometric analysis.27 Increasing the second substrate availability therefore depended on recycling pyruvate or avoiding the loss of PEP. D-glucose al BPTS PEP ——> TCA cycle pyruvate _ b 0P03H2 OH CO H ,\0H 2 0 _. PEP C HQ. 002H s OH OH 0 O HZOSPO OH H203P0\}\/u\ 5 OH D-glucose OH d H203PO OH E4P DAHP O /U‘cozH pyruvate Figure 6. PEP availability and variant carbon channel into shikimate pathway. Enzymes (genes): (a) glucose facilitator protein (glF), glucose kinase (glK); (b) PEP synthase (ppsA); (c) DAHP synthase (aroFFBR); (d) 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase (dgoA). Abbreviations: PEP, pyruvate phosphate; PTS, phosphoenolpyruvate:carbohydrate phosphotransferase system; E4P, D-erythrose 4- phosphate; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate. Re—phosphate pyruvate is a straightforward route to recycle PEP. This was achieved by overexpression of PEP synthase encoded ppsA (Figure 6).28 E. coli strain KL3/pJY1.2l6A, which differents from KL3/pKL5.17A by a plasmid located ppsA expressed under the control of IPT G, synthesized 69 g/L DHS with a total yield 51% from D-glucose. The second route is to replace PTS-mediated glucose transport system by the Zymomanas mabilis glf—encoded glucose facilitator protein. The intracellular 15 glucose is then phosphated by Zymamonas mobilis glK-encoded glucose kinase (Figure 6).29 E. coli strain JY 1/pJY2. 183A was constructed and differed from KL3/pKL5.17A by a plasmid located glfglk cassette expressed under Pm promoter and a PTS knock-out in the host strain.29 DHS was accumulated when culturing JYl/pJY2.l83A under fermentor conditions in 60 g/L with a total yield of 41% from glucose.29 While the titers and yields of DHS synthesized by KL3/pJY1.216A and JYl/pJY2.183A are comparable, the increase is obvious when compared with KL3/pKL5.17A. The dramatic increasing of the total yield from the above two PEP recycling system reflects the 2 fold theoretical yield increment of DHS from glucose.23 Alternatively, when pyruvate, instead of pyruvate phosphate, was used as one of the substrate for shikimate pathway, the consumption of PEP by the PTS will not be a concern. Directed evolution of dgoA encoding 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase creates a pyruvate based shikimate pathway (Figure 6).30 In this pathway, DAHP is not a product of the condensation of E4P and PEP, but E4P and pyruvate. E. coli NR7/pEC03-lserA was constructed, which differed from KL3/PKL5.17A by the genomic inactivation of all DAHP synthase isozymes and localization of the evolved dgaA, instead of aroFFBR in plasmid. Culturing NR7/pEC03- lserA under fermentor conditions synthesized 12 g/L DHS in 5% yield from glucose.30 Although DHS productivity is not comparable with KL3/pKL5.17A, this route can be a supplement to shikimate pathway for organic synthesis. Chemo-enzymatic synthesis based on shikimate pathway. The above efforts to direct carbon flux into shikimate pathway enhanced the ability for microbial synthesis of shikimate pathway intermediates and hence the value 16 added industrial chemicals such as phenol, hydroquinone, adipic acid and p- hydroxybenzoic acid (Figure 4). These huge volume marketed molecules are all currently synthesized from the volatile, carcinogen molecule, benzene, which is ultimately derived from the non-renewable source petroleum oil (Figure 7). Sustainable development requires renewable resources and environmentally benign processes for industrial synthesis. However, because of their high toxicity to live cells or the lack of enzymatic conversion of certain steps, genetically engineered E. coli strains are either incapable of producing these chemicals in high yields and titers, or even unable to produce them at all. Chemo-enzymatic routes thus provide alternatives to synthesize such chemicals as hydroquinone (high toxicity), p-hydroxybenzoic acid (toxicity), adipic acid (lack of enzymatic conversion), and phenol (toxicity and lack of enzymatic conversion). 17 HO COO COZH C02H £500: £5. £5H-—+» JL OH OH Ho“ OH COOH DHQ DHS shikimoicH acid chrismic acid lAroE 11 l c V 1 UciC HO, COOH CCOOH COOH q CCOOH Q “' cis, cis- HO OH muconic acid OH OH quinic: HaCid l b phenol p-hydroxybenzoic acid 1 a CCOOH I / OH COOH : adipic acid \ 3 OH_ benzene hydroqumone Figure 7. Microbial synthesis in conjunction with chemical synthesis based on the shikimate pathway. Routes toward chemo-enzymatic synthesis: a. (i) E. coli QPI.1/pKD12.138, (ii) NaOCl; b. (i) E. coli WNl/pWN2.248, (ii) H2, 50 psi., 10% Pt/C; c. (i) E. coli SP1.1/pKD12.138, (ii) 350 °C, H20; (1. (i) E. coli SP1.1/pKD12.138, (ii) 1 M H2504 in AcOH. PM is used for synthetic resins, dyes, pharmaceuticals, pesticides, perfumes, lubricating oils and solvents.31 The dominant synthetic method of phenol with a 5 x 109 kg/year market consumes 20% of the global benzene production. The Fridel-Craft reaction of benzene under harsh conditions affords cumene, followed by Hock oxidation to produce phenol (Figure 7).32 No intermediates in the shikimic pathway can be enzymatically converted to phenol, and therefore, one-step microbial synthesis is not available. E. coli SP1.1/p1(D12.13833 was constructed for microbial synthesis of shikimic acid from glucose, and a further modified construct SP1.1pts/pSC6.090 accumulated 87 18 g/L shikimic acid in a 36% (mol/mol) yield.34 Aromatization and decarboxylation of microbially synthesized shikimic acid in near-critical water afforded phenol in an overall 14% yield from D-glucose (Figure 7).35 Synthesis of phenol via this method is not yet economically competitive with the current industrial synthesis. However, it created a right track of benzene-free synthesis of phenol and represents the industry movements towards sustainable development. p-Hydroxybenzoic acid itself is used in liquid crystal polymer and several of its esters are used as food preservatives, known as parabens.36 Unlike phenol, the microbial synthesis of p-hydroxybenzoic acid is well established by taking advantage of p- hydroxybezoic acid’s intermediacy in the biosynthesis of coenzyme Q in microbes. E. coli JBl61/pJB2.274 accumulated p-hydroxybezoic acid under fermentor conditions in modest tier (12 g/L) and yield (13%) by expressing chroismate lyase encoding ubiC from plasmid (Figure 7).37 One obvious impediment of the not so satisfied productivity is the toxicity of p-hydroxybezoic acid towards E. coli.38 Although in situ removal of p- hydroxybezoic acid from fermentation could partially repress the toxic effect,39 further improvement of p-hydroxybezoic acid productivity was also impeded by the negative effect of overexpressing ubiC to the DAHP synthase activity in E. coli.37 Further barriers encountered by E. coli JBl61/pJB2.274 include the slow 1 sec'1 turnover of UbiC40 and the enzyme feedback inhibitions (AroF and AroE) and rate-limiting steps (AroF/G/H, AroB, AroE, AroL/K, AroC) and transcriptional controls (AroF/G/H and AroL/K) in the shikimate pathway (Figure 3).24 Insersion of a PmcaroAaroLaraCaroBkanR cassette into the E. coli genome required by JBl61 may not contend enough with all the impediments.37 Chemo-enzymatic synthesis of p—hydroxybenzoic acid takes advantage of 19 the well established shikimic acid synthesis strategy to accumulate shikimic acid in 87 g/L titer and convert it to p-Hydroxybenzoic acid by refluxing shikimic acid in acetic acid containing] M sulfuric acid in an overall 15% yield from D-glucose.41 Hydroguinone is a pseudocommodity chemical used in photographic developer and in the synthesis of polymerization inhibitors and rubber antioxidants.42 Theoritically, the establishment of microbial synthesis of p-hydroxybenzoic acid provides microbial synthesis of hydroquinone direct from glucose. Conversion of p-hydroxybenzoic acid into hydroquinone can be catalyzed by p-hydroxybenzoate l-hydroxylase (Figure 8), an enzyme found in Candida parapsilosis.43 Isolation and expression of the C. parapsilosis gene encoding p-hydroxybenzoate l-hydroxylase into p-hydroxybenzoic acid synthesis strain JBl61/pJBZ.27437 would achieve a single microbe capable of catalyzing the conversion of glucose into hydroquinone. However, except for all the aforementioned impediments encountered in the microbial synthesis of p-hydroxybenzoic acid, hydroquinone itself is highly toxic towards E. coli for its inhibition of sugar catabolism and damage to plasma membrane.44 OH E. coli COZH p-hydroxybenzoate OH 0 A0“ JB161/pJ82.274 1-hydroxyiase g HO OH OH NADPH NADP+ OH 02 D-glucose phydroxygenzoic hydroquinone acn Figure 8. Hypothesis microbial synthesis of hydroquinone from D-glucose. The unavailability of microbial synthesis of hydroquinone from glucose led to establishing a chemo-enzymatic synthesis by dehydration and oxidative decarboxylation of quinic acid.45 Shikimate pathway diverted at DHQ results in the biosynthesis of quinic 20 acid from glucose (Figure 7). E.coli strain QPI.1/pKD12.138 accumulated 49 g/L quinic acid in 20% yield from glucose under fermentor controlled conditions.45 Oxidative chemical decarboxylation of quinic acid in fermentation broth was achieved under different conditions in very decent yield (around 90% mol/mol). House hold bleach (NaOCl), halide free oxidant ((NH4)2Ce(SO4)3 and V205) and catalytic oxidant (Ag3PO4 with K28208 as cooxidant) were all capable of converting quinic acid to hydroquinone.4S Apidic acid is a building block of nylon 6,6 with an annual market 2.2 x 109 kg.46 Carbon flux channeled into shikimate pathway was diverted at DHS and ended up to cis, cis-muconic acid by heteroexpression of K. pneumoniae aroZ encoding DHS dehydratase, aroY encoding protochatechuate decarboxylase and A. calcoaceticus catA encoding catechol 1, 2-dioxygenase (Figure 7).47 E. coli WNl/pWN2.24847b was constructed, in which, enomic located tktA, aroB and lasmid located aroFm and P g P aroF directed carbon flux into shikimate pathway, while genomic located aroZ and plasmid located catX, aroY diverted shikimate pathway at the DHS point. Culturing WNl/pWN2.248 under fermentor controlled conditions synthesized 37 g/L cis, cis- muconic acid with 22% yield from glucose.47b Benzen-free synthesis of adipic acid from glucosewas then achieved by catalytic hydrogenation of cis, cis-muconic acid in fermentation broth into adipic acid in 97% (mol/mol) yield (Figure 7).47b Elucidation of Biosynthetic Pathway for Organic Synthesis Tremendous efforts exerted on elucidating the shikimate pathway had led to the synthesis of the pathway metabolites and molecules with added value (Figure 4, Figure 7). To meet the requirement of sustainable development, a long-term goal of microbial 21 synthesis to replace chemical synthesis will also be based on discovering enzymes with new functionality. Except for the environmental and resource concerns, modern therapy requires synthesis of new antibiotics and anticancer drugs. In the last two decades, about 78% of antibiotics and 60% of anticancer drugs introduced were natural products or derived from natural products (unnatural products).48 Although creative and intellectually rewarding, chemical synthesis is not always capable of producing such natural or unnatural products, while microbial synthesis provides promising alternative for the synthesis or semisynthesis of these molecules. Elucidation of biosynthetic pathway is thus of great importance in a sense not only for direct synthesis of natural products, but it provides genetic bases for manipulation of biosynthetic pathway for the synthesis of unnatural products. The elucidation of DXP pathway, polyketide synthesis (PKS) and nonribosomal peptide synthesis (NRPS) are typical examples. The DXP pathway It was well established that the long acyclic isoprenic chains of quinones and isoprenoids including sterols, carotenoids and hopanoids are derived from simple building blocks, isopentenyl diphosphate (IPP) and dimethylally diphosphate (DMAPP).49 Early isotope labeling experiments for the biosynthesis of cholesterol in liver tissues and ergosterols in yeast suggested that acetate was the unique starting material of IPP and DMAPP and a so-called MVA pathway for IPP and DMAPP synthesis was elucidated.50 However, the isotope-labeling pattern of hopanoids synthesized by bacteria suggested the addition of a C2 unit derived from pyruvate, instead of acetate.51 This led to the hypothesis of an alternative carbon source and hence an alternative biosynthetic pathway of IPP and DMAPP.52 D-Glyceraldehyde 3-phosphate 22 and pyruvate were further identified as direct precursors of IPP and DMAPP,53 and this pathway is named after its first committed metabolite as deoxy-D-xylulose 5-phosphate (DXP) pathway.” 5“ Unlike the well-characterized MVP pathway with acetyl coenzyme A as the precursor, DXP pathway starts with the thiamine dependent condensation of pyruvate and glycealdehyde 3-phosphate to yield DXP catalyzed by dxs-encoded DXP synthase (DXS). DXP reductoisomerase encoded by dxr then catalyzes the rearrangement and reduction of DXP to afford 2-methylerythritol 4-phosphate (MEP), which undergoes 3 more steps to synthesize IPP and DMAPP (Figure 9).52‘54 By tracking the changes of various isoprenoid end products such as carotenoid and ubiquinone with overexpression or subexpression of dxs, the first enzyme in the DXP pathway is found to be rate-limiting for isoprenoid biosynthesis in both bacteria55 and plants.56 Organic synthesis application of this discovery includes the synthesis of 1—deoxy-D-xylulose. Overexpression of dxs, which encodes DXP synthase, in Escherichia coli SP1.1/pPV4.230 accumulated 18 g/L l-deoxy-D-xylulose when cultured under fermentor-controlled conditions?”7 The molecule l-deoxy-D-xylulose is one of the pentose precursors in Maillard reactions to synthesize the key flavor components, 4- hydroxyfuranones.SB The modest productivity of l—deoxy-D-xylulose compared to the shikimate pathway products led to further mechanism studies of DXP pathway. As common to most biosynthetic pathways, the first step might be a rate-limiting step because of possible enzyme inhibitions. Further increasing the productivity of 1-deoxy- D-xylulose will depend on evolving the rate-limiting enzyme, DXS, to obtain inhibition insensitive gene. The discovery of the potential antimalarial drug,59 fosmidomycin’s inhibition towards DXR by competitively binding against its substrate DXP provides 23 selection of directed evolution. Theoretically, the introduction of mutagenesis, which enables E. coli SP1.1/pPV4.230 resistance to fosmidomycin inhibition, to dxs encoding DXP synthase, will correspondently increases DXS activity. As a result, l-deoxy-D- xylulose accumulation will be increased. After two rounds of error-prone PCR mutagenesis, E. coli SP1.1/pPV4.230 gained 50 times higher resistance to fosmidomycin.60 Although no increase of l-deoxy-D-xylulose productivity was obtained, this results opened the avenue for further directed evolution of enzyme DXS for organic synthesis. 0 Jim 0P206H3 pyruvate DXS O DXR H04 IPP HopoaHz—_> OPO3H2 —-> OH OH JV IIJH/‘opoan2 DXP MEP 0P206H3 dephosphate DMAPP GSP and export 0 Ho... a H0 / b H8/ H8, HO/ OH \ O + \ S + S ”O o o 1-deoxy- , D-xylulose 4 hydroxyfuranones Figure 9. DXP pathway and its application in organic synthesis. Synthesis of 4- hydroxyfuranones and additional flavor compounds from l-deoxy-D-xylulose. Key: (a) H, 02; (b) H2S, phosphate buffer, pH 4.5-6.5. Abbreviations: DXP, l-deoxy-D- xylulose S-phosphate; MEP, 2-methylerythritol 4—phosphate; DXS, DXP synthase; DXR, DXP reductoisomerase. Polyketide synthesis and nonribosomal peptide synthesis. Polyketide synthesis (PKS) and nonribosomal peptide synthesis (NRPS) are well “programmed” synthesis catalyzed by organized multi-enzyme complexes of remarkable size. The complexes, known as polyketide synthase and nonribosomal peptide synthase, 24 are organized into repeated functional modules for different stage synthesis.61 In each module, there are 3 core domains, which are acyl tranferase (AT-domain), acyl carrier protein (ACP—domain) and keto synthase (KS-domain) for PKS and aminoacyl adenylation domain (A—domain), peptidyl carrier protein (PCP) and condensation-domain (C~domain) for NRPS. During PKS, organic acids (such as malonylic acid) in their CoA thioester active forms were selectively loaded onto AT-domain, and then transferred onto ACP-domain to form acyl-ACP protein. Elongation was then obtained on KS-domain by the condensation of different acyl-ACP protein. Similarly, amino acids were selectively activated as aminoacyl adenylates and loaded onto A-domain during N RPS. The aminoacyl adenylates is transferred onto PCP domain to form aminoacyl-PCP proteins, which were condensed to form peptide bonds in the C-domain. In both cases, elongation might undergo as many as l to 11 steps until the synthesized polyketides or polypeptides are terminated by cyclization or hydrolation (Figure 10).61 The 3 core domains, together with variable numbers of auxiliary domains responsible for modification of the growing polyketides and polypeptides, established the main stream to synthesize a large variety of natural products (Figure 10) with antibiotic, imminosuppression, antitumor, antifungi and antiparasite activities?" 62 Although PKS and NRPS differ in several aspects including different starting materials, starting materials activation, elongation reaction types and termination models, the two parallel synthesis convergent to form hybrid synthesis by domain and module shuffling in nature.62 25 s’ polyketide synthesis 0 SH A o c n' H o HZNYILOAMP + pal" —» HQNW/lLSmCPn —>"Rco.NJ\WN\HLS,PCPn a a H o 9 A1 ATP 0 ll 0 A, PCPn-i . "RCO S noanbosomal H2N \HLOH Fl' polypeptide synthesis R OH 0 N02 H O 0' HO CI 7 / N / \ I N 0' N HO OH N I If. OH H 0 HO CI H o pyoluteorin differentiation-inducing thaxtomin factor (DlF-1) OH CI \ 0 9 ° ~H .fl‘“ HO OH O . / , NJ=o phloroglucmol MeO“.H5 H erythromycin A ansamitocin Figure 10. Polyketide synthesis, nonribosomal peptide synthesis and natural products. Abbreviations: PKS: AT, acytransferase; ACP, acyl carrier protein; KS, keto synthase. NRPS: A, aminoacyl adenylation domain; PCP, peptidyl carrier protein; C, condensation domain. Pyoluteorin is an antifungal natural product synthesized by Pseudomonas fluorescens Pf-5 through a PKS/NRPS hybrid gene cluster pltLABCDEFGMR.63 NRPS is responsible for the synthesis of pyrrole ring, while PKS involves in the synthesis of the 26 resorcinol (Figure 11). The carrier protein encoded by pltL possesses the conserved sequence of both PCP and ACP proteins, which acts as the interfacing of PKS and NRPS.63a This knowledge provides the basis for genetic manipulation and combinatorial biosynthesis for synthesizing unnatural polyketides and polypeptides by interchanging modules,64 mixing genes from different types of PKS and NRPS,65 and employing unnatural starter units.“5 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.67 - CI Cl HO OH NRPS PKS / \ N 30' / \ S‘PIIL —">. Cl H O H O u 0 HO L-pyrrole pyoluteorin Figure 11. PKS/NRPS hybrid synthesis of pyoluteorin. PltL, carrier protein; NRPS, nonribosomal peptide synthesis; PKS, polypeptide synthesis. Along with the polyketide and polypeptide assembly line, modifications of the backbones including reduction, oxidation, methylation and halogenation could occur simultaneously.62 However, the diversity of polyketides and polypeptides was also contributed from the tailoring reactions after the PKS and NRPS assembling line. Glycosylation and halogenation, which are very important for the bioactivity of mature products, are among the mostly happened post-modification reaction of PKS and N RPS.62 Although all domains in PKS synthase are highly substrates specific, in the biosynthesis of ansamitocin, the substrate specificity of most post-PKS gene products are not as high as the PKS assembly lines.“ In the biosynthesis of ansamitocin by Actinosynnema pretiosum, proansamitocin released from the PKS complex will undergo chlorination, 0- methylation, N-methylation, epoxidation, acylation, and carbamoyl transferforamtion 27 until the mature product was obtained (Figure l2).“’8 And the priority of different post- PKS steps is variable (Figure 12).68 This finding Opened the way of selective organic synthesis of PKS and N RPS derivatives with designed functionality. M35 / \s' é NAD MeO H0 H proansamitocin ansamitocin Figure 12. Synthesis of ansamitocin from proansamitocin by post modification of PKS. Reactions includes chlorination, O-methylation, N-methylation, epoxidation, acylation, and carbamoyl transferforamtion as highlighted as bold. Another discovery that helps organic synthesis of “designed” natural products is the involvement of phosphopantetheinyl transferases. In PKS and NRPS, ACP and PCP proteins are first expressed in inactive forms (ape-ACP and apo-PCP) before a 4’- phosphopantetheinyl group is transferred from CoASH to the conserved serine residue of ACP and PCP proteins (Figure 13).69 The posttranslational modifications then activate the carrier proteins (halo-ACP and halo-PCP). Because of the structural similarity between 4’-phosphopantetheinyl and N—acetylcysteamine (NAC) (Figure 13), acyl- and aminoacyl-N-acetylcysteamine (NAC) thioester (SNAC) could serve as mimic substrates of acyl-ACP and aminoacyl-PCP proteins in the PKS and NRPS.70 The chemically synthesized SNAC, thus facilitated the elucidation of the complicated PKS and NRPS pathway70 as well as synthesizing unnatural polyketides.7| An example of SNAC application is the modification of 6-deoxyerythronolide B (6-dEB) core structure. Genetic blocks of the PKS pathway that leads to the synthesis of 6-dEB resulted in the 28 inability of microbes for 6~dEB, the macrocyclic precursor of natural product, erythromycin.71 Exogenous addition of small molecule SNAC, however, recovered the 6- deoxyerythronolide B synthase (DEBS) functionality and unnatural polyketides were obtained from different SNAC precursors (Figure 14).71 Hit 0 no Han/KAMP R 7 R aminoacyl-SNAC 0:1tll/E"). H H 0H 0 HSNN NWO-P-O aminoacyl- -PCP protein 0 o c') o NAC 6‘: N RXSH JOL n n O” o H o R s’V Wolf—o ACP/PCP protein 0 O o acyl-SNAC Wild/Efra- , H acyl-ACP protein 0 Figure 13. Synthesis of acyl-ACP (PKS) and aminoacyl-PCP (NRPS) protein and their N-acetylcysteamine (NAC) thioester (SNAC) mimic. 0“ 0 mutated DEBS RKKHLSNAC > R2 R1=Et R2=Me 6-deoxyerythronolide B R1=BU R2=Me R1=CH2Ph R2=Me R1=vinyl R2=Me Figure 14. N-acetylcysteamine (NAC) thioester (SNAC) small molecules serve as PKS substrates for unnatural products synthesis derived from 6-deoxyerythronolide B. 29 Reference ' Witcoff, H. A.; Reuben, B. G. Industrial Organic Chemistry, Wiley, New York, 1996. 2 (a) Campbell, C. J.; Laherrere, J. H. The End of Cheap Oil. Sci. Am. 1998, 73-83. (b) Kerr, R. A. The Next Oil Crisis Looms Large-and Perhaps Close. Science 1998, 281,73-83. 3 Knowles, J. R. Enzyme Catalysis: not different, just better. Nature, 1991, 350, 121- 124. 4 Bommarius, A. S.; Riebel, B. R. 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S.; Zuber, P.; LaCelle, M.; Marahiel, M. A.; Reid, R.; Khosla, C.; Walsh, C. T. A New Enzyme Superfamily-the PhosphopantetheinylTransferases. Chem. Biol. 1996, 3, 923-936. 7° Ehmann, D. E.; Trauger, J. W.; Stachelhaus, T.; Walsh, C. Aminoacyl-SNACS as Small-molecule Substrates for the Condesation Domains of Nonribosomal Peptide Synthetases. Chem. Biol. 2000, 7, 765-772. 7‘ (a) Jacobsen, J. R.; Hutchinson, C. R.; Cane, D. E.; Khosla, C. Precursor-Directed Biosynthesis of Erythromycin Analogs by an Engineered Polyketide Synthase. Science 1997, 277, 367-369. (b) Kinoshita, K.; Williard, P. G.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2001, 123, 2495-2502. (c) Kinoshita, K.; Pfeifer, B. A.; Khosla, C.; Cane, D. E. Biaarg. Med. Chem. Lett. 2003, 13, 3701-3704. 35 CHAP’T ER TWO HEM -ENZYMATI Y THE I BA ED HIKIMATE AT A ' BENZE E-FREE YNTHE I F ATE H L Introduction Catechol as organic building block. Catechol, with an annual market capacity over 2.5 x 107 kg, is widely used as building block for organic synthesis in industry including food, medicine, and agriculture.‘ Of the global production of catechol, it is assumed that 50 % is used as starting material for insecticides. Carbofuran (trade name Furadan) and propoxur (trade name Baygon) are two important carbamate insecticides that are produced from catechol.lb The main challenge for the two-step synthesis of carbofuran is the regioselectivity in the first step to form 7-benzofuranol. The obtained 7-benzofuranol then reacts with methyl isocynate to afford carbofuran (Figure 15).2 @OHJK/ CISiO2/WO4 —>&X‘LN. O O + OH heat Et3N/benzene O N’ catechol 7-benzofuranol carbofuran Figure 15. Application of catechol in agriculture: two-step synthesis of carbofuran. The second largest consumes of catechol are perfumery and drug industries. 'b The two most successful syntheses based on catechol in this area are the syntheses of vanillin and L-Dopa. Vanillin is an everyday flavoring agent used in food and cosmetics.I Its synthesis (Figure 16)lb started with the condensation of partially protected catechol, guaiacol with glyoxylic acid to afford mandelic acid, which is air oxidized to 36 phenylglyoxylic acid. Vanillin is then obtained by acidification and decarboxylation of 4—hydroxyl-3-methoxyphenyl glyoxylic acid. HJKgOH HO COQH COQH ©__> (CH30)2SO4 QC N OH HO OH ”3‘30 8 H300 H300 H3CO catechol guaiacol mandelic acid phenyel‘gcliyljoxyllc vanillin Figure 16. Application of catechol in perfumery: 4-step synthesis of vanillin. L-Dopa was first used in 1938 as an antiparkinson drug, which was successfully synthesized from catechol using both chemical and microbial methods (Figure 17).3 Asymmetric chemical synthesis of L-Dopa was first developed by Nobel Prize laureate, William S. Knowels. He applied Rh complex catalyzed asymmetric hydrogenation to the synthesis of L-Dopa and obtained 95% enatioselectivity more than 30 years ago.3 Microbial synthesis of L-Dopa takes advantage of the tyrosine degradation pathway. The enzyme tyrosine phenol-lysae (TPL), isolated from Erwinia herbicala4 and Synbiabatecterium sp SC-l,5 catalyzes the degradation of tyrosine to phenol, pyruvate and ammonia. The reaction is reversible and if phenol is replaced by catechol, microbial synthesis of L-Dopa can be achieved. The synthetic application of catechol in perfumery and drug industries accounts for 35 — 40 % of global catechol.lb However, this number is being changed as the synthesis of vanillin is changed from the process using lignin in pulp waste liquor to the synthesis using catechol as starting material, which will then drive up the demand for catechol.lb 37 Catechol is also used as an organic sython for polymerization inhibitors and directly used as photographic developer, analytical reagent, and antioxidant. All these account for the remaining 10 —15% world consumption of catechol.lb o H 1)AcHNACozH/ACQO \ 002H —> : QOH OCHs 2) ”20 AcomHAc OH OH OCH3 cate°h°' vanillin 1) H2/Rh(DiPAMP) 2) H30+ O tyrosine phenol-Iysae C02H + NOH + NH4+, > NHAC OH ‘ H O OH O OH catechol L-Dopa tyrosine phenol-lysae (TPL): or: o . , , DiPAM : producd by Emma herblcola or Symbiobacterium sp. SC-1 GP :Q H3CO Figure 17. Application of catechol in pharmaceutical industry: chemical and microbial synthesis of L-Dopa from catechol. Early synthetic efforts towards catechol. The huge market for catechol requires efficient synthesis of catechol itself. Although both chemical and microbial syntheses are available, chemical synthesis dominates the catechol productivity. Chemical synthesis of catechol. Catechol and its derivatives exist in nature in different forms such as tannin and lignin and the very early source of catechol is low- temperature carbonization of coal or dry distillation from catechin.1 Catechol was also obtained from the hydrolysis of chlorophenol with aqueous NaOH in the presence of CuSO4 or CuzO at 190 °C (Figure 18).lb The application of this method is limited by the 38 availability of starting materials and simultaneous production of chlorine salt even though the yield from 2-chlorophenol to catechol is pretty high. A more efficient synthesis of catechol is based on a two-step oxidation of cumene with O2 and H202 (Figure 18).1 Cumene is the Fridel-Craft reaction product of benzene. Hock oxidation of cumene leads to the formation of phenol and acetone. Further oxidation of phenol with 70% hydrogen peroxide affords a mixture of hydroquinone and catechol that can be separated by successive distillation. The above routes include harsh reaction conditions and produce large amount byproduct salts. They also create environmental problems, as well as using non-renewable source,6 petroleum-based starting materials. hydroquinone o 654601—60 benzene cumene phenol catechol 2-chlorophenol Figure 18. Chemical synthesis of catechol from petroleum based starting material. Conditions: (a) propylene, solid H3PO4, 200~260 °C, 400~600 psi; (b) Oz, 80~130 °C, then S02, 60~100 °C; (c) 70% H202, EDTA, Fe2+ or C0“, 70~80 °C; (d). NaOH, CuSO4 or CuzO, 190 °C, 3 h. As the long-term goal of sustainable development, microbial synthesis is always prior to chemical synthesis. Based on different biosynthetic pathway, two different microbial synthetic routes were developed. One is based on the aromatic compound degradation pathway and the other on the shikimate pathway. Benzene-based microbial synthesis. The first route for microbial synthesis of catechol was based on the aerobic catabolism pathway of aromatic compounds. Natural evolution creates catabolism pathway to degrade the toxic aromatic compounds and 39 minimizes their effect on organisms. Although the initial conversions of aromatic compounds in this pathway were carried out by a large variety of enzymes, the central intermediates of aromatic compounds catabolism are limited to a few dihydroxyaromatic molecules such as protocatechuic acic and catechol (Figure 19).7 The aromatic diols were then diverged into either artha cleavage pathway characterized by catechol l, 2- oxygenase or meta cleavage pathway characterized by catechol 2, 3-oxygenase.8 The artha cleavage pathway is also known as the ketoadipate pathway named after the common intermediate B-ketoadipate. Both pathways end up with central metabolic routes, such as tricarboxylic acid cycle7. HNAD+ NADH C 6 20° L- or: OH OH benzene catechol 2-hydroxymuconic cis-dihydrodiol l 6-semialdehyde b / O R =H, -OH, -NH2, CI, -coz- .. . £388: 3 Egg: :1,» TCA cycle cis, cis-muconic acid B-ketoadipic acid Figure 19. Metabolism of aromatic compounds through catechol intermediates. (a) benzene cis-dihydrogenase, (b) catechol l, 2-oxygenase, (c) catechol 2, 3-oxygenase. Microbes screened for the synthesis of catechol were therefore capable of catabolizing aromatic compounds to catechol, but are defective in catechol catablism. According to this, microbes were able to accumulate catechol after the two genes encoding 1,2-oxygenase and catechol 2, 3- -oxyg enase were knocked out. A wide variety of starting materials including benzene,9 phenol,lo anilinell and bezoate'2 were up-taken by different bacterial strains to synthesize catechol. Although most of the starting materials were converted to catechol in a decent yield, the use of this kind of microbial 40 synthesis was limited by factors including the insolubility of starting materials} ‘0' “ harsh conditions,10 the labile mutants?“ “ and the toxicity of both product and starting materials.9' '0' ” As a result, the titers of catechol accumulated were not higher than 5 g/L. The above microbial syntheses minimized the use of organic chemicals and inorganic compounds, which lead to minimize their impact on environment. However, they share the same non-renewable starting materials with chemical synthesis. Long- term reliance on petroleum for organic synthesis must and will face the declining availability of petroleum oil6 and geopolitical issues.” The sustainable development movement for chemical industry prefers renewable starting materials for organic synthesis. D-Glucose-based microbial synthesis. Plant-derived starch, hemicellulose, and cellulose can serve as the renewable feeding stocks from which glucose is derived. Microbial synthesis of catechol with D-glucose as starting material was based on the shikimate pathway (Figure 4). In conjunction with the araZ—encoded 3-dehydroshikimate dehydratase and aro Y-encoded protocatechuate decarboxylase, carbon flux directed into the shikimate pathway is diverted at 3-dehydroshikimate into synthesis of catechol (Figure 20).14 To achieve this goal, a two—plasmid E. coli strain AB2834/pKD136/pKD9.069A was constructed, in which, the resistance to ampicillin (ApR) encoded by pKDl36 and the resistance to chlomophenicol (CmR) encoded by pKD9.069A facilitated stable plasmid maintenance in medium containing both antibiotics.l4 Host strain ABZ83415 was selected for its mutation in its araE locus that rendered the shikimate dehydrogenase catalytically inactive and DHS was available for conversion to catechol (Figure 20). Plasmid pKDl36l6 carries tktA encoding 41 transketolase, araF encoding DAHP synthase, and aroB encoding 3-dehydroquinic acid synthase for smooth direction of carbon flux into the shikimate pathway as described in chapter one. Plasmid pKD9.069A” carries araZ encoding 3-dehydroshikimic acid dehydratase and araY encoding protocatechuate decarboxylase (Figure 20) for converting DHS to catechol. The two non-shikimate pathway genes, araZ and araY were isolated from Klebsiella pneumaniae. Assembly and screening a genomic library of Klebsiella pneumaniae resulted in localization of a 3.5 kb fragment (araY) and a 2.3 kb fragement (araZ), which were heterogeneously expressed in E. coli for catechol synthesis.l4 COQH COQH L- h alan'ne H.xOH pKD136 araE p enyl I —’ L-tyrosine OH HO OH OH L-tryptophan 3-dehydroshikimic acid shikimic acid D-glucose laroZ 002H OH araY <1 —> OH HO OH protocatechic acid catechol Figure 20. Microbial synthesis of catechol from D-glucose. Enzymes (genes): pKD136 encoding transketolase (tktA), 3-deoxy-D-arabina-heptulosonic acid (DAH) 7-phosphate (DAHP) synthase (aroF), and 3-dehydroquinic acid synthase (aroB); shikimate dehydrogenase (aroE); 3-dehydroshikimic acid dehydratase (araZ); protocatechuic acid decarboxylase (ara Y). E. coli AB2834/pKD136/pKD9.069A accumulated 2.0 g/L when cultured in minimal medium under shake flask conditions with D-glucose as sole carbon source.14 Catechol toxicity toward E. coli constructed for synthesis is apparently the impetement to further increase its yield from glucose.[4 In this charter, two distinct strategies have been employed to circumvent this challenge. One is the extractive fermentation, which 42 m reduced interfacing of the toxic molecule with E. coli cells by in situ removing catechol with resin. The other is the combination of microbial and chemical synthesis, in which E. coli strains were constructed to convert D-glucose into intermediates with non or reduced microbial toxicity, followed by chemical conversion of the intermediates into the aromatic product, catechol by heating near critical H20. The intermediates employed are 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS), and protocatechate (PCA). The toxicity of catechol towards organisms Catechol is an aromatic diol, which makes it chemically active. Its chemical reactions include chelation with heavy metals, redox cycling and production of reactive oxygen species in the presence of heavy metals and oxygen (Figure 21).17 Based on the chemical reactions, the molecular modes of toxic action in cells were well summarized.17 The first toxicity comes from catechol-evolved redox reactions. Although under physiological conditions, catechol is not auto-oxidized, it will react with O2 in the presence of metal ions (e.g. Cu“, Fe“) to produce reactive oxygen species, such as hydrogen peroxide, superoxide, and hydroxyl radicals. DNA oxidative damage was observed under these conditions.18 One or two electrons were transferred to Cu (II) to form semiquinone radical or ortho-benzoquinone (Figure 21), and the resulted Cu (1) will catalyze the reduction of molecular O2 to yield reactive oxygen species. A DNA-Cu-oxo complex is formed, which breaks DNA strains by splitting hydroxyl groups in the vicinity of the DNA.19 The second catchol-invovled redox reaction in vivo is the oxidation of catechol by oxidative enzymes (peroxisases, tyrosinase, prostaglandin synthase) to generate two reactive species, semiquinone radical or ortho-benzoquinone.20 The two reactive species produced from catechol are toxic to organisms. The semiquinone radical 43 will oxidize the thiol groups of glutathione and cysteine residues in proteins and generate thiyl radicals. The oxidative stress in cells is increased because of the consumption of the important reductant glutathione21 and protein cross-links are induced because of the coupling reactions between thiyl radicals (Figure 21).20 The artha-benzoquinone can also bind to proteins by reacting with thiol groups in the cysteine residues22 or the e—amino group of lysine residues, and as a result, protein crosslinks are induced (Figure 21).23 The other aspect of catechol’s toxicity toward organisms comes from its chelation with heavy metals. Catechol can form stable complexes with di- and trivalent metal ions at wide range of pH.24 At physiological conditions, the chelation of catechol with metals such as iron (11 or III) will affect cell growth, as well as inhibit or even inactivate enzymes. Aromatic amino acid hydroxylase could be inhibited by catechol because of the stable complex formed between catechol and iron at its active sites.25 Chelation of catechol to Fe (III) induces the reduction of Fe (III) to Fe (II), which inactives Iipoxygenase, an enzyme that catalyzes the oxidation of an unsaturated fatty acid by atmospheric oxygen.”5 However, it is also interesting to note that therapeutic chelating agents are under development based on the catechol to remove over loaded iron for patients with thalassemia.27 H proein-N e 0 d S-protein ._ (I _. HO O OH OH OH lie affect membrane b OH a 0“: enzyme inhibition potential OH 0' e ' and inactivation lie, 0 . ' ' f . - cross linkage and DNggélgggve 9 [:E _> GS/proein S _’ increase cell OH GS/proein'S oxidative stress Figure 21. Molecular modes of catechol toxic action in cells: a. chelation with iron; b. bind to membrane; c. Cu (11), 02 or oxidative enzymes; d. inactivation of protein by binding to thiol groups of cysteine residues; e. inactivation of protein by binding to 8- amino groups of lysine residues; f. reacts with thiol group in cysteine residue or glutathione (GSH); g. oxidative damage of DNA. Of all the molecular modes, it was not clear which toxic mode plays the important role of the toxicity of catechol towards E. coli. However, catechol’s impact on E. coli growing was quite clear as at concentration of 2.5 mM, catechol completely prevented cell growth in solid medium.14 And in fermentation, the synthesis of 3-dehydroshikimic acid by E. coli A82834/pKD136 was decreased by 80% in the presence of 25 mM catechol.l4 In this research, in situ removal of catechol with extractive fermentation and chemo-enzymatic routes were developed to resolve this issue. One-Step Microbial Synthesis of Catechol Based on Shikimate pathway E. Cali construct design. Instead of using the two-plasmid E. coli strain ABZ834/pKD136/pKD9.069A,14 E. coli strain WNl/pWL1.290A was constructed for the biocatalytic synthesis of catechol. Carbon flux was directed into the shikimate pathway by overexpression of the 45 rate-limiting enzymes (araF’iBR and aroB) and substrate-producing enzyme (tktA). Among them, feedback-insensitive allele of 3-deoxy-D-arabino-heptulosonic acid 7- phosphate (DAHP) synthase encoded by aro)“FBR 28' 29 was expressed from plasmid pWLl.290A (Figure 22, Figure 23), together with promoter (PamF) to repress transcription control and allow araF‘cBR expressed from its native promoter.29 A copy of aroB encoding 3-dehydroquinic acid synthase was inserted into the genomic serA locus in the form of araBaraZ cassette. Transketolase (tktA), as a tktAaraZ cassette, was inserted into the lacZ locus of KL7,31 which was derived from AB283415 to increase D- erythrose 4-phosphate (E4P) availability.30 As a result, host strain WNl already possesses two copies of 3-dehydroshikimic acid dehydratase (araZ) to direct the shikimate pathway carbon flux diverted at 3-dehydroshikimic acid (DHS) to synthesize protocatechuic acid (PCA) (Figure 20). Overall conversion of glucose to catechol is then achieved by a plasmid located protocatechuate decarboxylase (araY) expressed from its Pmc promoter (Figure 20), which decarboxylates PCA to catechol. Host strain WNl was designed with two copies of araZ in the genome to minimize foreign gene expression from the plasmid.31 32 Another feature of WNl is the insertion of the araBaraZ cassette into the 3-phosphoglycerate dehygenase (serA) locus, which requires a copy of plasmid- located serA gene (Figure 23) to synthesize L-serine for stable plasmid maintenance. pRC1.558 Smal digestion Smal Smal serA 8: § 1) Smal digestion 2) CIAP treatment pWL1.284A 7.55 kb 59 ‘ Figure 22. Construction of plasmid pWL1.284A. 47 pMF63A 1) PCR 2) Xbal digestion Xbal pamF Xbal Figure 23. Construction of plasmid pWL1.290A. 48 Exit tilt i501 In! in an rec til hig pit 21$ 2’ a ad C0 Extractive fermentation. The development of biocatalyts in conjunction with fermentation technology has been a major step towards the incorporation of renewable raw materials into mainstream chemical manufacture. However, product toxicity/inhibition to microbes and products isolation from fermentation broth are the two challenges to conventional fermentation.33 Integration of fermentation and product recovery was obtained by extractive fermentation, which reduces the product in the fermentor to a microbial tolerable level and hence increases the productivity. Low boiling point products such as ethanol were recovered by CO2 stripping}4 and vacuum fermentation.” A hollow-fiber membrane extract fermentation was developed for microbial production of propionic acid, which has higher boiling point.” Resin based extraction approaches were employed for the production of molecules in their solid forms including L-phenylalanine,37 lactic acid38 and p-hydroxybenzoic acid.39 The productivity increased by extractive fermentation could be as high as 80%. The high toxicity of catechol towards E. coli requires us to use extractive fermentation to increase its productivity. Another molecule in this research, ptotocatechuic acid (PCA), has no impact on cell growth, but has downstream effects on 3-dehydroshikic acid production. This suggested that PCA is mildly toxic to E. coli.14 Thus, extractive fermentation was used for the microbial synthesis of catechol as well as microbial synthesis of PCA. Proper ion-exchange resins were first screened for in situ adsorption of catechol and PCA from fermentation broths. A series of preliminary comparative experiments were carried out with anion-exchange resins, AG 1-X8, Dowex 1X4, Dowex 1X8 and Amberlite IRA-400. AG 1-X8 is a strong anion-exchange resin 49 with quaternary ammonium functional groups attached to the styrene divinylbenzene copolymer lattice. Dowex 1X4 and 1X8 are polystyrene resins with trimethylbenzene ammonium functional groups. And Amberlite IRA-400 is a polystyrene resin with quaternary ammonium functional groups. A 50 mL solution containing 15.6 g/L protocatechuic acid and 5.6 g/L catechol was incubated with 10 g wet resin in 250 mL shaking flasks to compare their adsorption efficiencies (Table 1). AG 1-X8 was selected to be effective for both PCA and catechol adsorption. The hydrophobic adsorbent resins were also screened for catechol adsorption by similar experiments with 5 g polystyrene resins including Sepabeads SP850, 5 types of Amberlite XAD resin (XAD-2, -4, -16, - 16HP, and ~1180), 3 types of Diaion resins (Diaion SP207, HP20SS and HP2MG) and polymethacrylic resin MCI GEL CHP20P. Sepabeads SP850 was found to be the most effective one to adsorb catechol (Table 2). The resins selected were applied to extractive fermentation and both AG1X8 and Sepabeads SP850 were found non-toxic to E. coli cells and suitable for in situ extractive fermentation. Although AG l-X8 is selected for efficient catechol adsorption, Sepabeads SP850 is more effective in catechol fermentation. A 20% higher titer of catechol was achieved with Sepabeads SP850 than AG 1X8, while the yield was almost the same. The possible reason is that adsorption of acetate accumulated in high concentration (up to 30 g/L) during catechol fermentation would affect the efficiency of catechol adsorption to AG 1-X8 resin. Besides that, using the Sepabeads SP850 is more convenient for catechol recovery as well as resin preparation and regeneration. Therefore, AG 1-X8 was chosen for extractive PCA fermentation and Sepabeads SP850 for extractive catechol fermentation. 50 Table 1. Adsorption of procatehuic axid and catechol on anion exchange resins. Resin 0 AG l-X8 Dowex 1X8 Dowex 1X4 IRA-400 PCA adsorption b-Cvd (%) 91 75 87 88 catechol adsorption C-d (%) 86 79 73 80 ‘1 Ten grams resin was added into 50 mL solution, the mixture was incubated at 28 °C for 4 h. b PCA: procatechuic acid. C The initial concentrations of protocatechuic acid and catechol were 15.5 g/L and 5.5 g/L respectively. d The data shown are percentage (mol/mol) of PCA or catechol bound to resin. Table 2. Adsorption of catechol on adsorbent resins. Resin a XAD-2 XAD-4 XAD-16H? XAD-1180 XAD-l6 catechol adsorption 5'6 (%) 5 33 1 l 9 42 Resin SP850 HP2083 HPZMG CHPZOP SP207 catechol adsorption bfi (%) 60 24 36 29 44 ‘1 Five grams resin was added into 50 mL solution, the mixture was incubated at 28 °C for 4 h. bThe initial concentration of catechol was 9 g/L (73 mM), respectively. C The data shown are percentage (mol/mol) of catechol bound to resin. t——I l——‘—_[ ——I 2 _1 r \ $ Ell-(:1 y B 3 a M- am 3,: g K DLDLi. [It \ /J 1 Figure 24. Resin-based extractive fermentation of protocatechuic acid and catechol. keys: 1 fermentor, 2 peristaltic pump, 3 resin column. An in situ extraction and product recovery system was then designed for the extractive fermentation of catechol and PCA (Figure 24). Fermentation broth was 51 pumped out from the fermentor and flowed through the resin-packed columns. After a certain amount of products were adsorbed on one column, the broth flow was switched to another column and the previous column was eluted with solvent to recover products and regenerate resin. Productivities of both catechol and PCA were were almost doubled with extractive fermentation. Synthesis of catechol under fermentor conditions. E. coli WNl/pWL1.290A was cultured under glucose-rich condition for 36 h at pH 7.0 with the dissolved oxygen (D0) to be controlled at a set point 20% air saturation. Plasmid maintenance relied on nutritional pressure as opposed to resistance to antibiotics. Glucose was added 30 g/L initially and was maintained at 10-20 g/L during the fermentation process. A 10 mL broth was sampled every 6 h to analyze the fermentation products started from the 12 h time point. After 36 h fermentation, catechol was synthesized at a titer of 4.8 g/L in a 6% (mol/mol) yield, along with 30 g/L DHS and 27 g/L acetate. The high level of DHS and acetate suggests the inhibition of catechol towards E. coli strain (Figure 25). Extractive fermentation was then employed with columns packed by Sepabead SP850 resin for in situ removal and recovery of catechol during the fermentation process. The broth was pumped out to a flow rate high enough to create a fluidized bed to avoid any possible dead comer in the columns and also increase cells viability by keeping the concentration of catechol in broth below 18 mM (~2 g/L) (Figure 26). Multiple parallel columns were used alternatively for the in situ regeneration of resin. After 36 h, catechol was accumulated at 8.5 g/L in a 9% (mol/mol) yield from D-glucose, which was much higher than the regular fermentation without resin (4.8 g, yield 6% (mol/mol)) (Figure 25, Figure 26). 52 Culturing WN l/pWL1.290A under fermentor controlled conditions accumulated catechol only in low titer and yield. Although the increases are obvious, the titer and yield of catechol accumulated under extractive fermentation is far from satisfactory because of catechol’s high toxicity towards E. coli. Our effort was then turned to alternative chemo-enzymatic routes, in which, interface of cells with the toxic molecules was completely avoided. 53 00 U1 ................................................ MN _5_L 03 0010010010 ............................................. .......................................... catechol, DHS, acetate and cell mass (g/l.) 0 12 18 24 30 36 time (h) Figure 25. Catechol synthesized by Escherichia coli WNl/pWLl.290A under regular fermentor conditions. Abbreviations: DHS, 3-dehydroshikimic acid. Legend: black bars, catechol; open bars, DHS; grey bars, acetate; solid circles, dry cell mass. 35 30- N 01 l .5 U1 1 and cell mass (g/L) 8 8 catechol, DHS, acetate 01 time (h) Figure 26. Catechol synthesized by E. coli WNl/pWL1.290A under resin (Sepabead SP850)-based extractive fermentor conditions. Abbreviations: DHS, 3-dehydroshikimic acid. Legend: stapled bars, catechol in fermentation broth; black bars, total catechol including catechol in broth and on column; open bars, DHS; grey bars, acetate; solid circles, dry cell mass. 54 Chemo-enzymatic Synthesis of Catechol Based on Shikimate Pathway Unlike the previous one-step synthesis, catechol was synthesized through multiple steps, which completely avoid the interface of microbes with the toxic molecule catechol. Non-toxic intermediates, 3-dehydroquinic acid and 3-dehydroshikimic acid were microbially synthesized under fermentor-controlled conditions. The isolated intermediates were then dehydrated and decarboxylated to catechol in near critical H20. Alternatively, the less toxic intermediate, protocatechuic acid was either synthesized by microbes direct from glucose or chemically converted from 3-dehydroquinic acid and 3- dehydroshikimic acid in fermentation broth. Protocatechuic acid was then purified and decarboxylated to catechol in near critical H20. Synthesis of 3-dehydroquinate (DHQ). 3-Dehydroquinic acid (DHQ) is the first non-toxic intermediate investigated for chemo-enzymatic synthesis of catechol. While chemical synthesis of DHQ was achieved from quinic acid,“0 microbial synthesis is obviously preferred with elucidation of the shikimate pathway. E.coli construct design. E. coli strain QP1.1/pJY1.2l6A was designed for microbial synthesis of DHQ based on the shikimate pathway. E. coli AB2848 ‘5 contains a mutation in its genomic araD-encoded 3-dehydroquinic acid dehydratase locus (Figure 3). Site—specific insertion of an additional aroB encoded DHQ synthase into its serA locus yielded E. coli QPl.1.‘" The host strain thus will accumulate DHQ because of its lacking AroD activity for DHQ dehydration to DHS. The inactivity of SerA requires a serA gene in plasmid pJY 1.216A (Figure 27) for stable plasmid maintenance as described before. As previously descriped in constructing WNl/pWL1.290A, the rate-limiting step 55 was overcome by the plasmid-carried araFBR-encoded feedback inhibition insensitive DAHP synthase and an additional copy of the Pam}.- promoter. The availability of the two substrates, E4P and PEP, was increased by plasmid pJY1.216A located tktA-encoded transketolase3O and an open reading frame of ppsA-encoded phosphoenolpyruvate 42. 43 synthase. While tktA was expressed from its native promoter, ppsA was located behind a toe promoter (Pm) in plasmid. The ppsA expression was then controlled by lac repressor-encoding lale and addition of isa-propyl B-D-thiogalactopyranoside (IPT G) to the culture medium. Figure 27. Construction of pJY1.216A. Synthesis of DHO under fermentor conditions. E. coli QPl.l/pJY1.216A was cultured under fed-batch fermentor conditions in minimal medium (M9) for 42 h to accumulate DHQ. Aromatic amino acids and aromatic vitamins were supplemented to the medium due to the interuption of the shikimate pathway, through which, aromatic compounds are synthesized. Through out the whole 42 h fermentation, the dissolved oxygen was maintained at 20% air saturation by varying the impeller speed and 56 temperature maintained at 36 0C and pH 7.0. The concentration of starting material glucose was maintained at 55-170 mM by manually adjusting the glucose feeding rate. To induce the expression of PEP synthase, a certain amount of IPTG was added to a final concentration of 0.5 mM at 18, 24, 30, and 36 h after inoculation of the culture medium. Aliquots of the culture medium at certain time points were removed and concentrations of DHQ and DAH were measured by NMR. After 42 h, QPl.l/pJY1.216A synthesized 58 g/L of 3-dehydroquinic acid in 28% yield (mol/mol) and 13 g/L DAH (Figure 28). Fermentation broth was then centrifuged at (14000 g, 20 min) to remove cells, followed by acidification to pH 2.5 with concentrated HZSO4 (3~5 mL) to allow protein precipitation. The precipitated protein was then removed by a second centrifugation at 4 0C (14000 g, 20 min). The resulting broth is therefore cell and protein free. DHQ could be extracted from the fermentation broth with EtOAc by liquid-liquid continuous extraction with 67% (mol/mol) recovery yield. After removing EtOAc, DHQ was again dissolved in H20 and dried with lyophilization. The obtained off-white powder DHQ was dehydrated and decarboxylated to catechol in near critical HZO. However, the challenge of DHQ isolation was not only the low recovery and tedious work of continuous extraction, but also the instability of DHQ as it could be decomposed by retro-aldol reactions. To circumvent this challenge, DHQ was first converted to PCA by refluxing DHQ fermentation broth under N2 atmosphere. The resulted PCA was easily extracted from the aqueous solution and catechol synthesis was achieved by heating PCA in near critical H20. 57 ----------- ....................................... O) O ...................................................... cell mass (g/L) A O DHQ, DAH, acetate, N o O 12 18 24 3O 36 42 time (h) Figure 28. DHQ synthesized by E. coli QPl.l/pJY 1.216A under glucose-rich condition. Abbreviations: DHQ, 3-dehydroquinic acid, DAH, 3-deoxy-D-arabina-heptulosonic acid. Legend: open bars black bars, DHQ; grey bars, DAH; black bars, acetate; solid circles, dry cell mass. Synthesis of 3-dehydroshikimate (DHS). Overview. The hydroaromatic metabolite, 3—dehydroshikimic acid (DHS) is situated midway through the shikimic acid pathway. Although it is a potential antioxidant due to its high oxygen containing structure, 44 the relatively stable shikimate pathway metabolite is mostly served as divergent for aromatic oxygenated chemicals (Figure 29), which were either originated from petroleum chemistry (catechol, adipic acid, vanillin) or isolated from scarce natural sources (gallic acid, pyrogallol). Industrially important molecules microbially synthesized from glucose via DHS l,45 protocatechuic acid, catechol,46 and cis, intermediacy included gallic acid,45 pyrogallo cis-muconic acid.47 Chemical routes were also employed for the synthesis of molecules with added values via DHS intermediacy. Direct dehydration of DHS afforded protocatechuic acid48 and direct oxidation produced gallic acid.49 Microbial synthesized 58 cis,cis-muconic acid was also converted to adipic acid by a simple and environmentally benign chemical reduction.47 COZH 002H COQH COQH £1 —> QC, 001 10,, ‘— O 5 OH Ho/éz‘OHT—L-> HO/QOHZ OH 3-dehydroshikimic enediol enediol dihydrogallic diketone acnd acid _ ii 13 19 OH COZH COZH CHO COZH O -‘OH b c ——> —> 5 OH HO HSCO H300 HO OH HO OH OH OH OH OH D-glucose protocatechuic acid vanillic acid vanillin gallic acid it i“ e CCOQH f CCOZH HO \ COQH ' 002H HO OH OH OH catechol cis, cis-muconic acid adipic acid pyrogallol Figure 29. Reactivity of DHS and value-added compounds synthesized from D-glucose via 3-dehydroshikimic acid intermediacy. Keys: (a) araZ, 3-dehydroshikimate dehydratase or heating; (b) C OMT, catechol-0-methyltransferase; (c) aryl aldehyde dehydrogenase; (d) araY, protocatechuate decarboxylase; (e) catA, catechol 1, 2- dioxygenase; (f) 10% Pt/C, H2, 3400 kPa; (g) pabA *, mutated p-hydroxybenzoate hydroxylasecatechol or Oz, Cuz”, an“, AcOH; (h) ara Y, PCA decarboxylase. E. coli construct design. Construction of E. coli KL3/pJYl.216A was described in a previous report to synthesize 3-dehydroshikimic acid.50 The plasmid pJY1.216A (Figure 27) used for the biosynthesis of DHS was the same that used for biosynthesis of DHQ. Host strain KL329 was derived from AB2834l5 by inserting an additional araB encoding DHQ synthase into the genomic serA locus. Unlike the host strain used for 59 synthesis of DHQ, the genomic araD-encoded 3-dehydroquinic acid dehydratase was not disrupted, which facilitates the dehydration of DHQ to DHS. on O 80 O) O l ............................................ A O .................................... DHS DHQ, DAH, dry cell weight (g/l.) 8 it f ‘5' l :J 0 12 18 24 time (h) Figure 30. DHS synthesized by E. coli KL3/pJY1.216A under glucose-rich condition. Abbreviations: DHQ, 3-dehydroquinic acid, DAH, 3-deoxy-D-arabina-heptulosonic acid; DHS, 3-dehydroshikimic acid. Legend: open bars black bars, DHS; grey bars, DAH; black bars, DHQ; solid circles, dry cell mass. gmthesis of DHS under fermentor conditions. As E. coli KL3/pJY1.2l6A and QPl.l/pJY1.26A share similar characterization, microbial synthesis of DNS under fermentor controlled conditions will have the same profile. Culturing E. coli KL3/pJYl.216A accumulated 61 g/L of 3-dehydroshikimic acid with a yield of 36% (mol/mol) under fed-batch fermentation conditions (36 °C, pH 7.0) for 42h with IPTG added for ppsA expresion (Figure 30). In addition to DHS, E. coli KL3/pJY1.216A also accumulated 7 g/L DHQ and 19 g/L DAH. Trace amount of acetate (data not shown) suggested the non-toxicity of DHS towards E. coil strains. After the removal of cells and proteins as described, the fermentation broth was used for the chemical conversion of DHS to PCA or isolated by liquid-liquid continuous extraction49 in an 80% (mol/mol) 60 recovery yield. After dried with lyophilization, the off-white powder 3-dehydroshikimic acid was then used for the chemical conversion to catechol in near critical H20. Synthesis of PCA. Up to now, the lack of industrial scale synthesis of protocatechuic acid doesn’t necessarily suggest that PCA has no potential applications. As described above, PCA is the necessary intermediate for the synthesis of catechol, adipic acid and vanillin. It was also demonstrated recently that PCA isolated from Hibiscus sabdarifi‘a could induce human leukaemia cell apoptosis.“ PCA also inhibits the carcinogenic action of various chemicals in different tissues of rat,52 including diethlnitrosamine in the liver, 4- nitroquinoline-l-oxide in the oral cavity, azoxymethane in the colon, N-methyl-N— nitrosourea in glandular stomach tissue and N-butyl-N-(4-hydroxybutyl)nitrosamine in the bladder. PCA is the continuous dehydration product of DHQ and DHS, which provides both microbial and chemical synthesis routes to PCA used as an intermediate for catechol synthesis Chemical Synthesis of PCA from DHO and DHS. Enzymatic conversion of DHQ to DHS and DHS to PCA were achieved by araD-encoded DHQ dehydratase and aro Y- encoded DHS dehydratase. Similarly, both isolated 3-dehydroshikimic acid and 3- dehydroquinic acid are readily aromatized to protocatechuic acid in acidic condition? 5“ although either the yield was low53 or the reactions needed to be run at high temperature.54 In the study of DHS oxidation, inorganic phosphate was found to catalyze the oxidation of DHS to gallic acid,44 and protocatechuic acid was detected as byproduct with a 12% (mol/mol) yield (Figure 29),44 which provoked us to develop conditions to prevent DHS oxidation and increase DHS dehydration in fermentation broth. The first 61 effort was to optimize chemical dehydration of DHS in fermentation broth under N2 atmosphere to prevent DHS oxidation. DHS was then dissolved in synthetic fermentation broth to mimic DHS microbial synthesis conditions and heated to reflux at different pH values and different inorganic phosphate concentrations since inorganic phosphate catalyzed DHS oxidation.44 Reactions under these mimic conditions suggest, under N2 atmosphere, the concentration of inorganic phosphate was not important for DHS aromatization, while the pH value is critical for the dehydration of DHS to PCA. 100 2 I311-: <0 gen. 80. ................................................................ «LL'. 29. 5‘5 so- ................................................................ 2%; (U %% 40- ............................................................. SE SE", 20.. .- ............................................. 0—0) E [1 Ji '1 L 0 f I I fl T I 0 0.5 1 15 2 2.5 3 time(h) 2100 E ES,- 80- ..... .. ............................................................... 0v (UII BE 6‘: 50..... .- ......................................... . ..................... D 22 (“0 BE 404. ..................................................... 030 SE 53% 20. ............................................ “-0) ES 0 I I I r O 1 1.5 2 2.5 3 time(h) Figure 31. DHS reactivity at pH 7.0 at different phosphate concentrations. Phosphate concentrations: 8.6 mM (top); 43.0 mM (bottom) Abbreviations: DHS, 3- dehydroshikimic acid; PCA, protocatechuic acid. Legend: solid square, yield of PCA; solid circle, unreacted DHS. 62 At a relatively higher pH condition (pH 7.0), protocatechuic acid was obtained in only 22% (mol/mol) yield after 3 h (Figure 31). On the other hand, at a lower pH condition (pH 2.5), the dehydration was slow, but the yield of protocatechuic acid was dramatically increased (80% mol/mol) (Figure 32, top). Since phosphate concentration had no effect on DHS dehydration, the fermentation broth phosphate concentration was chosen for chemical synthesis of PCA from DHS. 100 80- ...... so. ...... -_ 4o. ...... PCA yield and unreacted DHS (mol/mol) at pH 2 5 20+ ----- 100 80- 60---(- 4o- 20 .. PCA yield and unreacted DHS (mol/mol) at pH 2 2 O 3 6 9 12 15 18 21 24 27 time (h) Figure 32. DHS reactivity at pH 2.5 (top) and pH 2.2 (bottom). Abbreviations: DHS, 3- dehydroshikimic acid; PCA, protocatechuic acid. Legend: solid square, yieldof PCA; solid circle, unreacted DHS. 155 Dowex-50 (H+ form) was used as catalyst for the dehydration of alcoho and to remove aromatic impurities in the fermentation broth. An alternative way to acidify the 63 3-dehydroshikimic acid-containing fermentation broth was passing the broth through ion exchange resin Dowex-50 (H+ form). Refluxing the resulting broth for 27 hours afforded protocatechuic acid with an 88% (mol/mol) yield (Figure 32, bottom). According to the control experiments, dehydration of DHS to PCA was obtained by passing the DHS-containing fermentation broth through Dowex-50 (H” form) and refluxing for 27 h under N2 atmosphere. Dowex-50 (H+ form) acidification also facilitates future PCA purification by retaining any contaminating aromatic compounds. The developed dehydration conditions were applied to the chemical synthesis of PCA from DHS and DHQ in fermentation broth. Cell and protein free fermentation broth containing DHS obtained by culturing E. coli KL3/pJY1.2l6A was eluted through Dowex-50 (H+ form) and refluxed under N2 atmosphere for 27 h to afford protocatechuic acid in a 94% (mol/mol) yield. DHQ is readily dehydrated to the more stable DHS even at room temperature. Dehydration conditions for DHS to PCA can be applied to continuous dehydration of DHQ. Therefore, refluxing fermentation broth containing DHQ obtained by culturing QPl.l/pJY1.216A produced protocatechuic acid quantitatively after refluxing for 27 h. Compared to the tedious liquid-liquid continuous extraction, PCA obtained in this way was easily and quantitatively hand extracted from fermentation broth. After pump dry overnight, the obtained brown PCA was heated in near-critical water to afford catechol (yield: 89% and 87% (mol/mol)). Microbial Synthesis of PCA under regulaiand extractive fermentor conditions. Microbial synthesis of protocatechuic acid (PCA) is highly possible based on the two facts that enzymatic dehydration of DHS to PCA is achievd by araZ-encoded DHS dehydratase (Figure 29) and PCA is mildly toxic towards E. coli.M An E. coli strain was constructed to synthesize PCA based on the shikimate pathway (Figure 29). KL3 was constructed as described above with genomic araE- encoded shikimic acid dehydrogenase disrupted, which will prevent the conversion of DHS to shikimic acid and channel carbon flux into microbial synthesis of PCA. Plasmid, pWL2.46B encodes Ptac, ppsA, lale, araFFBR, Pump, tktA, oral, and serA (Figure 33). It was constructed by inserting the cassette tktAaraZ, obtained by partial digestion of pWN1.200A3' with Xbal and EcaRl, into the HindIII site of pJY1.211A.50 Plasmid pWL2.46B differs from pJY1.216A by an additional copy of DHS dehydratase encoding araZ, which was expressed from its native promoter, to catalyze the dehydration of DHS to PCA.” 5" E. coli KL3/pWL2.46B accumulated 41 g/L PCA in 26% (mol/mol) yield when cultured under D-glucose rich fermentation conditions for 48 h, instead of 42 h (Figure 34). PCA was then quantitatively extracted with EtOAc from cell-free and protein—free fermentation broth and pumped dry overnight. The obtained PCA was decarboxylated by heating in near-critical water to afford catechol. To further increase protocatechuic acid yield and titer, E. coli KL3/pWL2.46B was cultured under resin-based extractive fermentation conditions as described before (Figure 24). Four columns with 120 mL AG 1-X8 resin for each were used respectively at 18-30 h, 30-36 h, 36-42 h, and 42-48 h to remove PCA in situ from the broth. The concentrations of PCA in the broth were kept below 15 g/L during the extractive fermentation (Figure 35). As expected, both PCA titer and yield was significantly increased. The effective concentration of PCA at 48 h ((PCA eluted off from all four columns + PCA in the broth)/the volume of cell-free fermentation broth at 48 h) was 72 g/L with a yield 49 % (mol/mol) based on glucose consumed. They are respectively 76% 65 and 88% higher than the titer (41 g/L) and yield (26%, mol/mol) achieved at 48 h in regular fermentation without resin extraction. PCA obtained in this route was then purified and decarboxylated to give a 43% (mol/mol) overall yield of catechol from glucose, which is the highest of all approaches. 66 pWN1 .200A 1) Xbal digestion 2) EcoFll partial digestion 3) Klenow treatment (Xbal) (EcoRl) l tktAaraZ (4.4 kb) 1) Hindlll digestion 2) Klenow treatment 3) CIAP treatment ligation Figure 33. Construction of plasmid pWL2.46B. 67 80 604 ---------------------------------- ............ cell mass (g/l.) PCA, DAH and dry 0 12 18 24 30 36 42 48 time (it) Figure 34. PCA synthesized by E. coli KL3/pWL2.46B was under regular fermentor conditions. Abbreviations: DAH, 3-deoxy-D-arabino-heptulosonic acid; PCA, protocatechuic acid. Legend: open bars, protocatechuic acid (PCA); black bars, 3- deoxy-D-arabino-heptulosonic acid (DAH); solid circle, dry cell weight. 80 ................................................. O) O J PCA, DAH and dry cell mass (g/L) A O N O L time (b) Figure 35. PCA synthesized by E. coli KL3/pWL2.46B under resin (AG1X8) based fermentor conditions. Abbreviations: DAH, 3-deoxy-D-arabino-heptulosonic acid; PCA, protocatechuic acid. Legend: stapled bars, protocatechuic acid (PCA) in fermentation broth; open bars, total PCA including PCA in fermentation broth and on column; black bars, 3-deoxy-D-arabino-heptulosonic acid (DAH); solid circle, dry cell weight. 68 Chemical conversion of DHQ, DHS and PCA to catechol in near critical H20. For the industrial level synthesis of catechol, sustainable development requires renewable starting materials and environmentally benign reactions. With the necessary intermediates synthesized from glucose, chemo-enzymatic synthesis of catechol can be achieved by careful selection of reaction conditions to minimize its impact on the environment. H20 is always considered to be environmentally benign compared to organic solvent. Based on this, near critical HZO conditions were developed for catechol synthesis. Near critical HzO as acid and base catalyst. Acids or bases can be catalysts for textbook organic reactions. Dehydration of alcohol is achieved easily in aqueous mineral acid or alkali base solutions. Decarboxylation, however, is always achieved in organic solvent quinoline with copper as catalysts7 In ambient HZO, acid catalyzed decarboxylation occurs only to a-keto acids, while base catalyzed decarboxylation always leads to anhydration. The dramatic physic-chemical change of H20 when heated to high temperature, high pressure incurs its applications as an environmentally benign medium for organic reactions.58 At ambient temperature, most organic compounds can’t be dissolved in HZO. When H20 is heated to near-critical or supercritical point, the decrease of dielectric constant makes it a good solvent for organic compounds.58 On the other hand, an increase in the dissociation constant by 3 orders of magnitude allows water to act as both acidic and basic catalysts. Dehydration and decarboxylation are then facilitated in high temperature H20. For example, decarboxylation of aromatic carboxylic acid was achieved when heated to about 255 °C in HzO without adding copper catalyst.59 The mechanism of decarboxylation was believed to be catalyzed by the conjugated acid- 69 base formed in near critical H2058 A previous report60 indicated that dehydration and decarboxylation of shikimic acid affored phenol in near-critical water with high yield. This observation raised the possibility that a synthetic route to catechol could be achieved by employing the same strategy with different starting materials. Two important intermediates in the common pathway of aromatic amino acid biosynthesis (Figure 1),“ DHQ and DHS, had been obtained with high titers and yields as described above from E. coli fermentations. Heated in near-critical water, 3-dehydroquinic acid and 3- dehydroshikimic acid would undergo dehydration and decarboxylation to produce catechol. Protocatechuic acid, the immediate precursor of catechol, which was obtained by both microbial and chemical synthesis from glucose, would undergo one-step decarboxylation to produce catechol in near-critical water. Reactivity of DHO. DHS and PCA in near critiaal H20. Since H20 in near critical conditions can act as both acid and base, we expected that DHQ, DHS and PCA could be smoothly converted to catechol in high temperature H20. Thus, the first effort is to heat the 3 intermediates in near-critical water and optimize temperatures for the chemical conversions (Figure 36-Figure 38). Temperatures applied to near critical reaction were 190 °C, 210 °C, 230 °C, 250 °C, 270 °C, 290 °C, 310 °C, 330 °C, 350 °C and 374 °C. Under all 3 reactions, PCA was detected at temperatures below 290°C and the yield of catechol decreased above 290°C (Figure 36). The detection of PCA at temperatures below 290 °C is understandable as decarboxylation is more difficult than dehydration. And the decrease in yield can be accounted for the degradation of catechol under high temperature. A little bit unexpected result is the detection of DHS at 190 °C and 210 °C when heating DHS in near critical HZO (Figure 37), while the incomplete dehydration of 70 DHQ was not observed at these two temperatures (Figure 36). Another observation is the mass balance was well maintained in the DHS and PCA cases and not in the DHQ case. Explanation for this depends on the possible retro-aldol reaction occurred when heating DHQ in high temperature H20. According to the reaction profiles, the yields of catechol increased form 190 °C to 290 °C, followed by decreasing afterward, and 290 °C was identified as the optimal temperature for the conversion. The isolated yields of catechol from 3-dehydroquinic acid and 3-dehydroshikimic acid and protocatechuic acid were 54%, 90% and 94% (mol/mol), respectively (Figure 36-Figure 38). 100 30. ..................................................... 60- - --------------------------------------------------- 40-» 204 o .-..I,i 190 210 230 250 270 290 310 330 350 374 temperature (°C) catechol and PCA (% mol/mol) Figure 36. Reactivity of 3-dehydroquinate in near critical HZO. Abbreviations: PCA, protocatechuate; DHQ, 3-dehydroquinate. Yield, (mol product/mol DHQ)x100%. Legends: open bars, PCA; solid bars, catechol. 71 .5 O O ............... (D O a l 60» 1 40» 20 oil-11L i 190 210 230 250 270 290 310 330 350 374 temperature (°C) catechol. PCA and DHS (% mol/mol) Figure 37. Reactivity of 3-dehydroshikimate in near critical H20. Abbreviations: PCA, protocatechuate; DHS, 3-dehydroshikimate. Yield, (mol product/moi DHS)x100%. Legends: grey bars, DHS; open bars, PCA; black bars, catechol. 100 sow-w- —————————— 50. .-. ..... 404 ... ..... 20«»«~--~ catechol and PCA (% mol/mol) 190 210 230 250 270 290 310 330 350 374 temperature (°C) Figure 38. Reactivity of protocatechuate in water. Abbreviations: PCA, protocatechuate. Yield, (mol product/mo] PCA)x100%. Legends: open bars, PCA; black bars, catechol. Overall conversion from glucose to catechol. In the above description, DHS , DHQ and PCA were synthesized from D-glucose through both chemical and microbial routes based on the shikimate pathway. The obtained intermediates were then chemically converted to catechol in near critical water 72 through the method developed in this version. These compromised 6 routes of chemo- enzymatic synthesis of the toxic molecule, catechol. Microbial Synthesized DHO and DHSjS intermediaLea. The first 2 efforts include microbial synthesis of DHQ and DHS when culturing E. coli QPl.l/pJY1.216A and KL3/pJY1.216A under fermentor controlled conditions in yields of 28% and 36% (mol/mol). The extraction of DHQ and DHS from fermentation broth, however, is a tedious and low recovery yields (67% and 80%, mol/mol) process. The yields of reactions in near critical HZO for DHS are excellent, while that of DHQ is only mild. These afford the overall yields of catechol from glucose to be 10% and 24% (mol/mol) from D-glucose (Figure 39). 0,, COZH COZH COHQ‘fi HOD overall 10% OH 67% HO OH D- -g|ucose DHQO (broth) DHQO (isolated) catechol OH COZH 002H 0 overall 26% HO OH D-glucose DHSO (broth) DHS (isolated) catechol Figure 39. Interfacing of microbial and chemical synthesis of catechol from D-glucose via intermediacy of DHQ and DHS. Conditions: a. E. coli QPl.l/pJYl.2l6A; b. E. coli KL3/pJY1.216A; c. liquid-liquid continuous extraction with EtOAc; d. H20, 290 °C. Chemical 3 nthesized PCA as intermediate. To avoid the tedious and low recovery of continuous extraction of DHQ and DHS from fermentation broth, microbially synthesized DHQ and DHS were first converted to PCA in pretty good yields (100% and 94%). The overall synthesis of catechol in these routes was also facilitated by the hand extraction of PCA from fermentation broth quantitatively. PCA obtained was then 73 converted to catechol in 87% and 89% (mol/moi) yields. Although there is one extra step in these routes, the high yields in each step increased the overall yields of catechol to 25% and 30% (mol/mol) from D-glucose respectively (Figure 40). H o,, COQH C02 overall 25% OH 28% OH 100% H0 87% HO OH D-gluoose DHQ (broth) PCA (isolated) catechol OH 0021-1 COZH O .\OH a c (1 HO —> ——> —> overall 30% = OH 36% o i OH 94% H0 39% HO HO OH OH OH D't'llucose DHS (broth) PCA (isolated) catechol Figure 40. Interfacing of microbial and chemical synthesis of catechol from D-glucose via intermediacy of chemically synthesized PCA. Conditions: a. E. coli QPl.l/pJY 1.216A; b. E. coli KL3/pJY1.216A; c. refluxing at pH 2.2 and extracted with EtOAc; d. H20, 290 °C. Microbiaflgynthesized PCA as intermedifl A more direct route to obtain PCA was microbial synthesis of PCA from D-glucose by E. coli KL3/pWL2.46B under fed- batch regular fermentor conditions and extractive conditions with very decent yields (26% and 49%, mol/mol). The quantitatively extracted PCA from fermentation broth was heated in near critical H20 in yields of 91% and 87% (mol/mol). These two routes saved the tedious and low recovery liquid-liquid continuous extraction as well as the one extra step for converting DHQ and DHS into PCA. As a result, the overall conversion of catechol from glucose reached the highest 43% (mol/mol) yield (Figure 41). 74 002H 002H HOD overall 24% OH 26% HQ 100% HQ 91% HO HO OH D-glucose PCA (broth) PCA (isolated) catechol COQH COQH ‘ TU overall 43% 3 OH 49% H0 100% H0 87% HO HO OH D-glucose PCA (broth) PCA (isotaled) catechol Figure 41. Interfacing of microbial and chemical synthesis of catechol from D-glucose via intermediacy of microbially synthesized of PCA from regular and extractive fermentor conditions. Conditions: a. E. coli KL3/pWL2.46B, regular fermentation; b. E. coli KL3/pWL2.46B, extractive fermentation; c. hand extracted with EtOAc or recovery from resin; (1. H20, 290 °C. Discussion Sustainable development consideration for catechol synthesis. Sustainable development represents the modern development of the chemical industry, which has two concerns, renewable source and environmental impact. The very early catechol production, distilling from coal tar, is not efficient, based on nonrenewable resource and an environment disaster. Current industrial synthesis of catechol moved from hydroxylation of 2-chlorophenol to oxidation of phenol and the environmental impact is minimized, though not completely avoided (Figure 18). However, the petroleum-based procedure with benzene as the starting material leaves much space for improvement in contend with sustainable development. It is believed that petroleum oil is a finite resource, because its formation from biomass requires millions of years and its consumption is increasing rapidly with the development of modern society.62 The 75 starting material benzene is a volatile, carcinogenic molecule and any leaking of the reaction system will be a disaster for human health. It is highly desirable to develop more environmentally benign alternatives using renewable source for the synthesis of chemicals with huge market such as catechol. The biodegradation pathway of aromatic compounds provides possible biocatalytic syntheses of catechol. Bioconversions of readily available starting materials such as benzene, aniline, benzoate, phenol, and toluene to catechol have been reported.“12 Catechol productivity is comparable with glucose-based microbial synthesis. Under both conditions, catechol could be produced about 5 g/L and the yields are even better when using aromatic molecules as starting materials. However, increasing yields using aromatic molecules as starting materials to industry level can’t be achieved due to the toxicity of both reactants and products and the insolubility of starting materials in HZO, which is the preferred medium for enzymatic reactions in most cases. Research efforts on this route, however, are not completely unnecessary. Catechol is one of the central intermediatea in aromatics degradation pathways. Microbes screened for the synthesis of catechol from these starting materials will help to degrade the toxic aromatic molecules discarded from the modern chemical industry. Another concern of this synthesis is that all these starting materials are derived from the petroleum chemistry. The long-tenn goal for sustainable development of the chemical industry is expected to be based on starting materials derived from renewable resources. In a word, this biosynthetic route is not practical for industrial synthesis, and doesn’t meet the sustainable development movement for industry production. Plant-derived starch, hemicellulose, and cellulose can serve as the renewable feeding stocks from which glucose is derived. This makes 76 microbial synthesis of catechol with glucose as starting material very necessary and highly possible. Non-benzene Based Synthesis of Catechol Impeded by molecular Toxicity. Microbe-catalyzed synthesis with D-glucose as starting material had been exploited to synthesize an extensive range of chemicals with industrial importance. Successfully syntheses include ethanol, L-ascorbic acid, L-lysine, L-lactic acid and l, 3- propaneldiol. However, developing a one-step biocatalytic synthesis of aromatic molecules from D-glucose was frequently complicated by their toxicity to microbes used as catalysts. Because of the toxicity of p-hydroxybenzoate, extractive fermentation had been employed to increase the biosynthetic yield of p-hydroxybenzoate. And because of the toxicity problems, chemo—enzymatic methods were employed for biosynthesis of hydroquinone and phenol. In catechol’s case, the toxicity problem is significant. When culturing E. coli WNl/pWL1.290A under fermentor-controlled conditions, catechol only accumulated in 4.8 g/L with poor yield. Toxicity is the problem that must be solved for the microbial synthesis of catechol with glucose as starting material. E.coli WNl/pWL1.290A synthesized 4.1 g/L catechol and as much as 30 g/L DHS (Figure 25A). In this case, the accumulation of DHS doesn’t mean that the downstream enzyme, araZ-encoded DHS dehydratase is rate-limiting. Instead, DHS accumulation is because of catechol’s toxicity, which affected microbial metabolism. This conclusion is based on the following observations. The first observation is, during the fermentation, except for DHS, acetate accumulation is also significant (27 g/L). On the contrary, acetate accumulation is not important when culturing E. coli strains to synthesize non-toxic molecules DHQ (Figure 28) and DHS (Figure 30), even during the 77 synthesis of the somewhat toxic molecule PCA (Figure 34). This strongly suggested that cell growth was affected by toxic molecules during catechol fermentation. The second observation is, during the microbial synthesis of PCA, DHS accumulation is not significant, which suggested the activity of araZ-encoded DHS dehydratase is sufficient to direct the carbon flux diverted at DHS. Draths et al’s previous research14 also demonstrated that, at the concentration of 2.5 mM, catechol would totally inhibit the growth of E. coli A82834/pKD136. Also in this research, the synthesis of DHS by E. coli ABZ834/pKDl36 rest cells was reduced 15% with the addition of 10 mM catechol and 80% with 25 mM catechol. Therefore, it is the toxicity of catechol that impedes the microbial synthesis of catechol from glucose. It is therefore necessary to develop efficient synthetic routes to circumvent the toxicity barrier based on the shikimate pathway with glucose as starting material. Resin-based extractive fermentation is a choice. Employing resin-based extractive fermentation increased both titer and yield of catechol accumulated in E. coli WNl/pWKl.290A fermentation, and cells also survived from resins used to recover the product. It is still extremely difficult to reduce the concentration of catechol in culture medium to a low level (for example, at least as low as 2.5 mM), at which, microbe growth is not severely prevented and the metabolism in microbes is not affected. Chemo- enzymatic synthesis therefore is the only choice. Near Critical Reaction is the Best Option to Synthesize Catechol. Near critical H20 condition for organic synthesis is environmentally benign. However, it is also energy consuming. The successfully developed chemo-enzymatic Synthesis makes near critical reaction the best option to synthesize catechol from glucose. 78 Microbial synthesis of non-toxic or less toxic intermediates, followed by chemical conversion of these intermediates to catechol could avoid the interface of catechol with microbes used as biocatalysts and hence circumvent the toxicity barrier. A related strategy had been reported to achieve catechol from glucose with overall 22% yield.63 Phosphorylation of glucose catalyzed by hexokinase affored glucose 6—phosphate, which underwent cyclization catalyzed by 2-deoxy-scylla-inosose synthase. The 2-deoxy- scyllo-inosose obtained from the two-step in vitro enzymatic reactions was then converted into catechol upon reaction with HI and HOAc. However, synthesis of 2- deoxy-scylla-inosose from glucose using an intact microbe remains to be established. Different E. coli strains had been reported to accumulate the two intermediates DHQ and DHS in the shikimate pathway.” 50“ Chemical conversions of DHQ and DHS to catechol were not developed as far as we know. Mechanism study shows that the conversions could be achieved by dehydration and decarboxylation reactions in strong acidic conditions. However, PCA was detected as the only product with yields of 90% (mol/mol) and 99% (mol/mol) respectively when DHS was refluxed in 12 M HCl and acetic acid (containing 1 M HZSOH) for 24 h. When DHS was heated to reflux in 8 M H2804 for 24 h, catechol was detected as product in 14% (mol/mol) yield. A strong acidic resin, nafion was used as acid catalysis for dehydration with benzene as the reaction medium. The chemical conversion of DHQ, DHS and PCA then depended on the strong acid-base system provided by water at high temperature. The attempt of heating DHQ and DHS fermentation broths to synthesize catechol turned out to be unsuccessful as no product was detected. It is then necessary to extract DHQ and DHS from fermentation broths for chemical conversions in near-critical water. 79 The low recovery yield of DHQ and DHS from the tedious liquid-liquid continuous extraction leads to the development of protocatechuic acid as synthetic precursor, which can be easily and quantitatively extracted from fermentation broth. However, due to the oxidation of 3-dehydroshikimic acid to gallic acid,45 PCA was not obtained in high yield from heating 3-dehydroshikimic acid containing fermentation broth at pH 7.0 even under N2 atmosphere (Figure 31). On the other hand, in acidic conditions, oxidation of 3-dehydroshikimic acid is not important and protocatechuic acid was obtained in a high yield (Figure 32). The in situ conversions of DHQ and DHS in fermentation broth to protocatechuic acid were efficient after the broths were acidified with Dowex-50 (H+ form). One-step synthesis of PCA from D- glucose under fed-batch fermentation conditions with E. coli constructs KL3/pWL2.46B was studied. When cultured under fermentor-controlled conditions, KL3/pWL2.46B synthesized 41 g/L PCA with 26% yield from glucose. PCA is less toxic than catechol as 25 mM (~4 g/L) PCA resulted in 10% reduction in DHS production, compared with the 80% reduction at the same concentration of catechol.46 Resin-beased extractive fermentation is then expected to improve its titer and yield in culture medium. And the fact is that PCA was accumulated 72 g/L in 49% yield from glucose (Figure 35). Catechol synthesis via PCA intermediacy achieved the highest yield 43% from glucose. The reactivity of DHQ at high temperature is quite different from those of DHS and PCA. Compared to the more than 90% yields of catechol from DHS and PCA heated in HZO, the 54% yield obtained from DHQ is not vey impressive. An unexpected observation is the mass balance was well maintained in the DHS and PCA cases and not in the DHQ case. Explanation for this depends on the competitive retro-aldol ring 80 opening reaction against the dehydration reaction facilitated by the acid-base presented in high temperature H20 (Figure 42). The retro-aldol reaction products were then degraded through further reto-aldol or decraboxylation reactions to small molecules or even charcoal, which accounts for the mass lost. DHS won’t undergo retro-aldol ring opening reaction as it forms enediol, which was further dehydrated to the stable aromatic ring product (Figure 42). HO— HQ 002H 0 OH O ——> WOO H—T degradation 5 2 OH O 2 OH OH DHQ H+H COZH COZH COZH HHC ’11—»,{EL-4 O OH OH DHQ DHS enediol PCA Figure 42. DHQ degradation competed against in high temperature HZO. Conclusion. Tremendous efforts had been exerted to improve the titers and yields of shikimate pathway products and byproducts by fed-batch E. coli fermentation. While direct biosynthesis of catechol from D-glucose was hindered by the product toxicity, productivity of catechol was improved by in situ recovery of product from fermentation broth with resin and by chemo-enzymatic reactions through non-toxic intermediates in the shikimate pathway or less-toxic byproducts of the shikimate pathway. H20 in near- critical conditions was exploited as the chemical reaction medium to convert precursors to catechol effectively. The highest yield, 43% (mol/mol), of catechol from D-glucose was improved by resin-based extractive E. coli fermentation to synthesize protocatechuic 81 acid as synthetic precursor of catechol. Therefore, the central finding of this research is not only comparing different synthetic routes for catechol synthesis based on the shikimate pathway. 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M.; Frost, J. W. Benzene-Free Synthesis of Hydroquinone. J. Am. Chem. Soc. 2001, 123, 10927-10934. 42 Niersbach, M.; Kreuzaler, F.; Geerse, R. H.; Postma, P. W.; Hirsch, H. J. Cloning and Nucleotide Sequence of the Escherichia coli K-12 PpsA Gene Encoding PEP Synthase. Mal. Gen. Genet. 1992, 231, 332-336. “3 Pataik, R.; Liao, J. C. Engineering of Escherichia coli Central Metabolism for Aromatic Metabolite Production with Near Theoretical Yield. Appl. Environ. Microbial. 1994, 60, 3903-3908. 4" (a) Richman, J. E.; Chang, Y.-C.; Kambourakis, S.; Draths, K. M.; Almy, E.; Snell, K. D.; Strasburg, G. M.; Frost, J. W. Reaction of 3-dehydroshikimic acid with molecular oxygen and hydrogen peroxide: products, mechanism, and associated antioxidant activity. J. Am. Chem. Soc. 1996, 118, 11587-11591. (b) Kambourakis, 8.; Frost, J. W. Synthesis of Gallic Acid: Cu21-Mediated Oxidation of 3-Dehydroshikimic Acid. J. Org. Chem. 2000, 65 , 6904—6909. 45 (a) Kambourakis, S.; Draths, K. M.; Frost, J. W. J. Am. Chem. Soc. 2000, 122, 9042. 4" Draths, K. M.; Frost, J. W. J. Am. Chem. Soc. 1995, 117, 2395. 47 (a) Niu, W.; Draths, K. M.; Frost, J. W. Biotechnol. Prog. 2002, I 8, 201. (b) Draths, K. M.; Frost, .1. W. J. Am. Chem. SOC. 1994, 116, 399. 48 Kambourakis, S. Ph. D Dissertation; Michigan State University: 2000. 49 Kambourakis, 8.; Frost, J. W. Synthesis of Gallic Acid: Cu2+-Mediated Oxidation of 3—dehydroshikimic acid. J. Org. Chem. 2000, 65, 6904-6909. 86 5° Yi, J.; Li, K.; Draths, K. M.; Frost, J. W. Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in Escherichia coli. Biotechnol. Prog. 2002, I8, 1141-1148. 5’ Tseng, T. H.; Kao, T. W.; Chu, C. Y.; Chou, F. P.; Lin, W. L.; Wang, C. J. Induction of apoptosis by Hibiscus protocatechuic acid in human leukemia cells via reduction of retinoblastoma (RB) phosphorylation and Bel-2 expression. Biochem. Pharmacol. 2000, 60, 307-315. 52 Ali, B. H.; Wabel, N. A.; Blunden, G. 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Reactivity of organic Compounds in Superheated Water: General Background. Chem. Rev. 2001, 101, 837-892. (b) Akiya, N.; Savage, P. E. Roles of Water for Chemical Reactions in High-Temperature Water. Chem. Rev. 2002, 102, 2725-2750. (c) Patrick, H. R.; Griffith, K.; Liotta, C. L.; Eckert, C. A. Near-Critical Water: A Benign Medium for catalytic Reactions. Ind. Eng. Chem. Res. 2001, 40, 6063-6067. 59 (a) An, J.; Bagnell, L.; Cablewski, T.; Strauss, C. R.; Trainor, R. W. Applications of High-Temperature Aqueous Media for Synthetic Organic Reactions. J. Org. Chem. 1997, 62, 2505-2511. (b) Kaatritzky, A. R.; Lapucha, A. R.; Murugan, R.; Luxem, F. J. Aqueous Hi gh-Temperature Chemistry of Carbo- and Heterocycles. 1. Introduction and Reaction of 3-Pyridylmethanol, Pyridine-3-carboxaldehyde, and Pyridine-3-carboxylic Acid. Energy Fuels 1990, 4, 493-498. 6° Gibson, J. M.; Thomas, P. S.; Thomas, J. D.; Barker, J. L.; Chandran, S. S.; Harrup, M. K.; Drath, K. M.; Frost, J. W. Benzene-Free Synthesis of Phenol. Angew. Chem. Int. Ed. 2001, 40, 1945-1948. 87 6’ Pittard, A. J. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed.; Neidhardt, F. C., Ed.; ASM Press: Washington, DC, 1996; Chart 58. 62 (a) Campbell, C. J.; Laherrere, J. H. The End of Cheap Oil. Sci. Am. 1998, 73-83. (b) Kerr, R. A. The Next Oil Crisis Looms Large-and Perhaps Close. Science 1998, 281, 73-83. (’3 Kakinuma, K.; Nango, E.; Kudo, F.; Matsushima, Y.; Eguchi, T. An Expeditions Chemo-enzymatic Route from Glucose to Catechol by the Use of 2-Deoxy-scylla-inosose Synthase. Tetrahedron Lett. 2000, 41, 1935. 6“ Kambourakis, S. Ph. D Dissertation; Michigan State University: 2000. 88 ' ‘ CHAPTER THREE 1 E T1 FBI Y THET RA I NPATH Y NITR AR MATI Y T E Introduction Overview Biosynthetic nitration may be one of the few reactions, which are not completely understood up to now. Molecules bearing an aromatic nitro group are not widely occurred in nature, but many of these compounds have antibiotic, antifugal and phytoxic activities such as pyrrolomycins, chloramphenicol, pyrronitrin and thaxtomin (Figure 43). Elucidating the mechanism of the biosynthetic nitration reaction is therefore of great interest for expanding our scientific knowledge and for organic synthesis. Cl N020H C' , Cl NO2 / \ C. \ NH Cl N / \ H Cl N CI N02 H OH CI CI pyrrolomycin B pyrrolomycin E pyrrolnitrin Cl CI No2 CI N02 OH “TI/Ca ’Z/ \3 Cl / \ i O 0 Cl N CI \ H H oVo OZN 0“ pyrrolomycin A dioxapyrrolomycinNO chloramphenicol /OCH3 H W0” aureothin thaxtomin Figure 43. Natural products containing an aromatic nitro group. 89 Microbial synthesis of the thermal stable energetic compound 1, 3, S-triamino-Z, 4, 6-trinitrobenzene (TATB) is a good target for elucidating biosynthetic nitration for organic synthesis. TATB possesses high nitrogen content in the form of arylamino and arylnitro groups. Currently, the TATB synthesis started from the nitration of 1, 3, 5- trichlorobezene with nitric acid, followed by the nucleophilic substitution of chlorine with amino groups at high temperature and high pressure.‘ The harsh reaction conditions and chlorine waste produced during the two-step synthesis provoke milder and cleaner procedures. Phloroglucinol is unique in its ability to react with nucleophiles, electrophiles and radicals. Synthesis of TATB from phloroglucinol can be divided into two steps, synthesis of the aromatic core structure and the functionalization of the aromatic ring. Microbial synthesis of phloroglucinol was achieved recently by recombination of the E. coli strain W3110(DE3)/pJA3.l3lA, which accumulates phloroglucinol in a 15 g/L titer under extractive fermentor conditions.2 Polyketide synthase (PKS) gene cluster PhlABCD found in Pseudomonas fluorescens Pf-S is encoded for the biosynthesis of acetylphloroglucinol.3 Examination of the PKS cluster led to the discovery of the condensation of 3 malonyl-CoA to afford phloroglucinol when pth was expressed in E. coli (Figure 44).2 The next issue is to find methods for introducing the 3 nitro groups into the aromatic ring and substitution of the hydroxyl groups. OH OH OH 0 o o -‘ Pth "’ 3HOMSC0A 5 OH HO OH HO OH glucose malonyl-CoA phloroglucinol Figure 44. Biosynthesis of phloroglucinol. Condensation of 3 malonyl-COA followed by atomization affored phloroglucinol. 9O A 3-step TATB synthesis from phloroglucinol includes nitration in mild conditions, alkylation with tripropyl orthoformate and amination in liquid ammonia with an overall yield of 87% (Figure 45).4 Though very efficient, this route still includes harsh reaction conditions. TATB is finding application to replace the unstable energetic molecule trinitrotoluene (TNT). As a long-term goal of synthesis, enzymatic nitration is CI : 8 N02 b 02N CI CI preferred. . N02N trIchlorobenzene T ATB 6 0“ OCH3 O C HN02 d OZN NC2 HO OH H3CO OCH3 N02 phloroglucinol Figure 45. Chemical synthesis of triaminotrinitrobenzene (TATB). Keys: (a) HNO3, oleum, 150 °C; (b) NH3/O.4 MPa, toluene, 150 °C; (c) NZOS/H2804, -8-10 °C; (d) tripropyl orthoformate, 110-120 °C; (e) NH3 (liquid). Microbial synthesis in its simplest form is the use of enzymes to catalyze chemical reactions and these reactions occur mostly in mild conditions and environmentally benign. The reactivity of phloroglucinol and existence possible arylamino oxidase and arylnitro synthase responsible for synthesizing arylamino and arylnitro compounds in nature provides possibility of the enzymatic functionalization of phloroglucinol (Figure 46). 91 NH2 No2 /HN : NH —>ON :1 NO OH 2 2 2 2 How/(”CL NH2 Phloroglucinol\ O:NJ¢[:OZ H]? N02 TATB Figure 46. Hypothetic enzymatic synthesis of triaminotrinitrobenzene (TATB). Keys: (a) arylamino oxidase; (b) arylnitro synthase. Hypothesis of pyrrolomycin A biosynthetic pathway. Pyrrolomycin A (Figure 43) is a natural product isolated from Actinosporangium vitaminophilus (ATCC 31673), also known as Streptomyce vitaminophilus, whose biosynthetic pathway remains unknown.5 The core structure of pyrrolomycin A is an electron rich aromatic ring, which, similar to phloroglucinol, will be readily attacked by nucleophilic, electrophilic and radical substitution. Studying the mechanism of the biosynthetic nitration of pyrrolomycin A maybe helpful to achieve microbial synthesis of TATB from phloroglucinol. Natural products sharing the same pyrrole core structure include arylnitro molecules (pyrrolomycin B, pyrrolomycin E, and dioxapyrrolomycin“) and non-arylnitro molecules (pyrrolomycin C, pyrrolomycin D, pyoluteorin and a series of coumermycin). Among these molecules, the biosynthesis of pyoluteorin, which was synthesized by Pseudomonas fluorescens Pf-S, was mostly studied. It is accepted that polyketide synthesis (PKS) is responsible for resorcinol ring synthesis and nonribosomal peptide Synthesis (NRPS) for pyrrole ring synthesis. 7 L-Proline was first activated and loaded onto NRPS enzyme complex. Oxidation then affords pyrrolyl-Z-carboxylate peptidyl 92 carrier protein (PCP), which will be chlorinated8 and interfaced with PKS to synthesize the final product (Figure 47). The structural similarity between pyoluteorin and the pyrrolomycin A (Figure 47) suggests they share similar mechanism in the biosynthesis of the pyrrole core structure and the chlorination of the pyrrole ring (Figure 47). Since the nitration reaction is another modification of the pyrrole ring, it may be occurred along with chlorination reactions. The biodecarboxylation can take place after the core moiety is relieved from the NRPS assembly line by hydration. Other molecules sharing the pyrrole core structure may undergo the same biosynthetic pathway. Our attention is then focused on the biosynthetic nitration step. Up to now, 3 different bacterial nitration reaction models had been proposed, which are nitric oxide synthase (NOS)—mediated nitration, arylamino oxidase nitration and direct nitration. NRPS CI PKS Cl HO OH / \ S~ a / [h _. (H PCP_>C|/Z/—N_\>\«S-PCP —> ("W 0 O H o H 0 HO Cl pyoluteorin NR ”OH—ES WS‘PCP a MST“, —' CI N o O H o LproIIne lb lb N02 CI No2 0' N02 . . m /N\ S~PCP—>C|/Z/—';\Sfi <—> l <—> —> . ° N02 R R R R T R peroxidase N02 + H202 ——> ONOO—‘—_ .NO + 0; Figure 48. In vitro tyrosine nitration mechanisms with different ways to producing nitrogen dioxide radical. 94 As mammalian NOS-mediated nitration is widely accepted, the discovery of nitric oxide synthase in bacteria provokes the research on the possible involvement of nitric oxide synthase in microbial biosynthetic nitration. Nitric oxide synthases from bacteria were found to be homologue to the N-terminal of mammalian NOS oxidase domain, but lack the C-terminal reductase domain and the N-terminal zinc-binding domain.18 Evidence of NO production from B. sublitis was also observed,18b which suggested that bacterial NOS might be involved in the biosynthetic nitration. Genetic and isotope labeling experiments suggested that Streptomyces NOS participated in thaxtomin nitration.‘9 Thaxtomin is a dipeptide cytotoxin.19 Its production in Streptomyces turgidiscabies was largely reduced by NOS inhibitors, nitro-L-arginine methyl ester (NAME),19 and NOS gene knockout.‘9 However, the NOS gene knockout didn’t completely eliminate thaxtomin production, which suggested that there were alternative NO sources in the bacteria.l9b A direct evidence of the involvement of bacterial NOS in the biosynthetic nitration reaction was the in vitro nitration activity observed with purified NOS isolated from Deinococcus readioduram (ATCC 31673).20 It was reported that this NOS could nitrate both L-tyrosine and L-tryptophan in the presence of hydrogen peroxide.20 Although the mechanism remains unclear, the bacterial NOS-mediated nitration seems not completely non-enzymatic compared to their mammalian counter partner. Mmino oxidase-mediated biosynthetic nitration. Oxidation of an arylamino group to an arynitro group compromises the second category of bacterial nitration reaction. Pyrrolnitrin, an antibiotic with antifungal activity is produced by many Pseudomonas strains.2i A gene cluster prnApranrnCprnD was cloned from P. 95 fluorescens and expressed into E. coli and was further identified to be responsible for the pyrrolnitrin biosynthesis with tryptophan as starting material (Figure 49).22 In this cluster, prnA and prnC genes encode halogenases, and prnB encodes an enzyme that catalyzes the rearrangement of 7—chloro-tryptophan.22 The fourth enzyme encoded by prnD has high similarity with dioxygenase, which catalyzed the conversion of amino- rrolnitrin to rrolnitrin.22 This conversion indicates PmD is an a lamine oxidase. W W ry + NH3 + .NH2 CO; PrnA CO; PrnB \ _, \ _. N N H CI H 7-chlorotryptophan I C ,- NH Cl Cl \ ’ ’ —> —> NH Cl 2 NH2 No2 Cl Cl monodechloro- . _ . . . aminopyrrolnitrin amInopyrrolnItrIn pyrrolnItrIn tryptophan Figure 49. Biosynthetic pathway of pyrrolnitrin. Enzymes (genes): halogenase (prnA); isomerase (prnB); halogenase (prnC); aryliamino oxidase (prnD). The second piece of evidence for the oxidation of arylamino group to arylnitro group came from the biosynthesis of aureothin synthesized by Stretpomyces thioluteus, which exhibits antitumor, antifungal and insecticidal activity.23 An enzyme, AurF was discovered that catalyzed the oxidation of a shikimic intermediate, p-aminobenzoate to p- nitrobenzoate, which was loaded onto a polyketide synthase as primer and eventually built up to aureothin.23a AurF is an N-oxygenase that catalyzes the step-by-step oxidation through the p-hydroxylaminobenzoate and p-nitrosobenzoate intermediates (Figure 3).23b Another shikimate pathway derived antibiotic, chloramphenicol shares similar conversion 96 of p-aminobenzyl to p-nitrobenzyl group24 and might be also achieved by a N-oxygenase (Figure 51). COOH COOH COOH COOH AurF AurF AurF —> —> —> NH2 HN ‘OH N :0 N02 PABA PHABA PNSBA PNBA n 11 PKS O shikimate Wm. / / apthway OQN 0 O . OCH3 aureothin Figure 50. Proposed biosynthetic pathway from PABA to PNBA in the biosynthetic pathway of aureothin. Abbreviation: AurF, N-dioxygenase; PABA, p-aminobenzoate; PHABA, p-hydroxyaminobenzoate; PNSBA, p-nitrosobenzoate; PNBA, p- nitrobenzoate; PKS, polyketide synthesis. C02H OH H CI arylamine OH H CI Q 0 _ N CI oxidase ; ‘n/kCl JL —-> i O —> ;\ O o 002H HQN \OH OZN OH OH d' hl l ' chorismic acid '0 gfi’ggfigye}?:emmo' chloramphenicol Figure 51. Proposed mechanism of the biosynthesis of chloramphenicol. Direct biosynthetic nitration. Unlike genetic probing of the above categories of biosynthetic nitration, the hypothesis of the third category was based on isotope-labeling experiments in the biosynthesis of dioxapyrrolomycin synthesized by Streptomyces fumanus}s When culturing this S. fumanus strain, dual labeled K‘SN‘BO3 was fed in the medium and products were collected. It was interesting that the arylnitro group on the pyrrole ring was also dual labeled, which suggested that the nitro group was intactly derived from NO3‘. Based on this observation, it was hypothesized that the nitro group 97 was introduced to the aromatic ring by a direct nitration reaction.25 If this is the case, the biosynthetic nitration of pyrrolomycin products (pyrrolomycin A. B and E) that share similar core structure and substitution patterns (Figure 43), might include a direct biosynthetic nitration. The reaction can be a nucleophilic, electrophilic or radical process. This chapter presents our efforts to probe the biosynthetic nitration mechanism in the synthetic pathway of pyrrolomycin A. Searching for the arylnitro synthase will then start with culturing A. vitaminophilu to confirm pyrrolomycin A production. A. vitaminophilu obtained from ATCC was purified and an authentic sample of pyrrolomycin A was synthesized. As a supplemental effort, dioxapyrrolomycin synthesized by Streptomyces SP. (UpJohn UC11065) was also confirmed. The direct biosynthetic nitration reaction mechanism was tested by isotope-labeling experiment, in vitro reaction with mimic intermediate analogues and in vitro reactions with possible putative intermediates. Another mechanism, a NOS-mediated biosynthetic reaction mechanism was also examined with isotope labeling, inhibition experiments and genetic approach. As the inability to obtain biosynthetic evidences, an alternative way to understand NOS-mediated biosynthetic nitration was employed by an in vitro nitration reaction with NOS isolated from Deinococcus radioduran (ATCC 17939). Synthesis of Pyrrolomycin A and Dioxapyrrolomycin Purification of Actinosporangium vitaminophilus (ATCC 31673). The strain A. vitaminophilus (ATCC 31673) was a Streptomyces related bacterial, which froms pseudosporangia when growing in glucose-citrate medium for 14 days.26 98 However, culturing A. vitaminophilus (ATCC 31673) according to literature5 could not always produce pyrrolomycin A, which suggested that the strain purchased from ATCC was contaminated. Consequently, a six-step identification and isolation procedure was carried out.26 It included hydrolysis of starch, liquefaction of gelatin, reduction of nitrate, a growth test in 1.5% NaCl, peptonization and coagulation of skim milk, and formation of melanoid pigment. Hydrolysis of starch.27 The extracellular enzymes (Jr-amylase and oligo-l,6- glucosidase are able to break starch into glucose. As a result, when starch is used as the only carbon source in solid medium, A. vitaminophilus, which produces such enzymes, will consume starch around their environment. Positive results will be observed when applied to iodine reaction. Gelatin liquefaction.27 Gelatin is composed of protein and is solid at room temperature. A. vitaminophilus, which produces hydrolytic exoenzymes known as gelatinases, would digest and liquefy gelatin and produce a positive result. Reduction of nitrate.27 A. vitaminophilus could reduce nitrate to nitrite and some other nitrogenous compounds, such as molecular nitrogen, ammonia or nitric oxide, using the enzyme nitrate reductase. Culturing this strain in the presence of KNO3 will result in the production of nitrite, which could be detected by reacting with Griess reagents, sulfanilic acid and a-naphthylamine, to produce a positive result, pink color. Peptonization and coagulation of skim milk.27 Skim milk provides lactose as carbohydrate source and casein as primary protein source for cell growth, and is supplemented with pH indicator, azolitmin. B-Galactosidase, produced by microbes, could hydrolyze lactose to galactose and glucose, and glucose may the be oxidized to acid, which decreases the pH value. Accumulated acid can also cause precipitation of casein and form acid clots. This is a positive result. On the other hand, casein could be partially digested and NH3 would be released to raise the pH value and no peptonization will be observed, which is a negative results. Proteolytic enzymes such as rennin, pepsin and chymotrypsin could digest casein but coagulate the milk, which will disturb the results. 28 Most Streptomyces contain a gene encoding tyrosinase. Melanoid pigment. Expression of this gene in the presence of tyrosine would lead to the formation of a dark brown melanin pigment. A. vitaminophilus is a Streptomyces-related strain, but not Streptomyces and will produce a negative result. In the experiment, 20 single colonies were streaked out to obtain the 2"d generation colonies, which were selected to run all the tests. According to the literature,26 starch hydrolysis, gelatin liquefaction, and reduction of nitrate are supposed to be positive, while peptonization and coagulation of skim milk and the melanoid pigment test are supposed to be negative. And cells grow in 1.5% NaCl, not in 3% NaCl. After growing cells for 14 to 21 days, however, only the NaCl test and skim milk test showed some differentia, and all colonies produce negative result in gelatin liquefaction (Table 3). According to the result, 9 colonies, no. 2, 7, 8,10, 14, 15, 18, 19, 20 were selected for further screening by culturing the strain and detecting the production of pyrrolomycin A by HPLC. As a result, colonies no. 2, 14, 15 and 18, which consistantly produce pyrrolomycin A were selected for further experiments. These 4 colonies were grown in ISPII medium and stored at —80 °C in 10% glycerol. 100 Table 3. Identification of Actinosporangium vitaminophilus (ATCC 31673). l starch nitrate gelatin melanoid litmus NaCl tgéerance co ony hydrolysis reduction liquefaction pigment milk 0 (1 5) 3 0 blank _ a - _ _ P /H b _ _ _ 1 + + - - L/C + - - 2 + + - - L/C + + - 3 + + - - P/H + + - 4 + + - - P/H + - - 5 + + - - L/C + - - 6 + + - - P/C - - _ 7 + + - - L/C + + - 8 + + - - L/C + + - 9 + + - - P/H + + - 10 + + - - P/H + + - I l + + - - UP + _ _ 12 + + ‘ - L/H + + - 13 + + — - P/C + - - 14 + + - - P/H + + _ 15 + + - - P/H + + - 16 + + - - B/H + + - 17 + + - - P/C + + - 18 + + — - P/H + + - 19 + + - - P/H + + - 20 + + - - P/H + + - W31 100 - — - - P/C atcc6051d + + + _ B /H “ “-“ means the result is negative; “+” means the result is positive. bAbbreviations: P, the color is purple; L, the color is lavender; B, the color is blue; H, the medium is homologous; C, coagulation formed. CE. coli w3110 was used as negative control for hydrolysis of starch, liquefaction of gelatin, reduction of nitrate. dBacillus subtilis (ATCC6051) was used as positive control for hydrolysis of starch, liquefaction of gelatin, reduction of nitrate. Chemical synthesis of pyrrolomycin A. The first step to elaborate the biosynthesis of pyrrolomycin A was to chemically synthesize pyrrolomycin A as an authentic sample. In the early 19808, Koyama et al reported the first synthesis.29 Pyrrolomycin A was produced at room temperature in 13% yield by direct chlorination of 3-nitropyrrole with 2 eq sufuryl chloride (SOzClz) in acetic 101 acid solution. However, no pyrrolomycin A was obtained when we tried to repeat the synthesis, as no aromatic H in 1H NMR indicating any mono- and di-chlorinated products. The product here is probably the trichlorinated nitro-pyrrole. Efforts to prevent the 3rd chlorination by decreasing the temperature to 0 °C turned out to be ineffective (Table 4, entry 1 and 2). Except for Lewis acid catalyzed chlorination,30 Lewis base, ethyl ether would stabilize the electrophile, Cl+ produced by sufuryl chloride (50202),“ which can chlorinate the aromatic ring. Inert solvent CH2C12 was used to dissolve the starting material and reduce the amount of ether needed (Figure 52). Although no dichloro- products was detected at room temperature even after 16 h, bringing the reaction temperature below 0 °C was effective to obtain dichloro- products (Table 4). When exactly 2 eq of sufuryl chloride (SOZCIZ) was used to control the chlorination extension, mono- and tri-chlorinated product couldn’t be completely avoided. Furthermore, the a-position of the pyrrole ring is the most electron-rich and easily attacked. As a result, 2, 5-dichloro-3-nitropyrrole, instead of pyrrolomycin A, was the dominant species. The chlorination conditions were then optimized (Table 4) at —15 °C for 2 h to obtain the highest yields of pyrrolomycin A. The chlorinated mixture was then separated by flash column to afford 2, 5-dichloro-3-nitropyrrole (53% mol/mol), and pyrrolomycin A (15% mol/mol), 5-chloro-3-nitropyrrole (trace) and 2, 4, 5-trichloro-3- nitropyrrole (23% mol/mol) (Figure 52). Pyrrolomycin A was further crystallized from EtOAc and hexane to afford a yellow solid, which has the same spectroscopic characterization as reported.29 102 N02 N02 CI N02 N02 CI N02 $02C12/Et20 _ / \ o a / \ + / \ + / \ + / \ N -15C.r-t-/CH20I2 CI N CI N CI N CI CI N CI H H H H H 2-chloro-4- pyrrolomycin A 2,5-dichloro-4- 2,4,5-trichloro-3— 3-nitropyrrole nitropyrrole nitropyrrole nitropyrrole 1 (trace) 2 (15%) 3 (53%) 4 (23%) Figure 52. Chemical synthesis of pyrrolomycin A. Table 4. Chemical synthesis of pyrrolomycin A. l t T o T' h product so ven emp ( C) Ime ( ) 1 2 3 4C HOAC RT. 1 -a - - + HOAC 0 1 - - - + CHzClz/EtQO _ RT. 2 +1? - + + CHZClz/EtzO R.T. l6 - trace + + CHzClz/EtzO 0 1 + trace + + CHzClz/EtzO —5 1 + 6 + + CHZCIZ/EtzO -15 2 + 15 + + ‘1 — not detected by lHNMR; b + detected by lHNMR; C structure not fully Characterized. Microbial Synthesis of Pyrrolomycin A. A. vitaminophilus (ATCC 31673) was first isolated from a soil sample in Nagano, Japan.5a A series of antibiotics, pyrrolomycin A, B, C, D and E were also isolated from the same fermentation broth by culturing A. vitaminophilus? Of the 5 antibiotics, pyrrolomycin A, B and E contain a nitro group attached to the pyrrole core structure. Pyrrolomycin production in rich medium. Our first effort was to culture the strain according to literature5 to verify that pyrrolomycin A was synthesized by this strain. A single colony grown on SYE (starch, Yeast extract) or ISP2 plate was inoculated in 5 mL seed medium for 3 days, which was then transferred to 100 mL seed culture for 2 days and then 1000 mL production medium for 5 days. All the cultures were incubated at 28 °C in a 250—rpm shaker. The resulting fermentation broth obtained was centrifuged to separate the supernatant and mycelia. The supernatant was extracted with EtOAc 103 directly, while the mycelia were first stirred with acetone to break cells and then extracted with EtOAc. After purification with flash column and recrystallized from EtOAc and hexane, a total 25 mg pyrrolomycin A was isolated from the two extractions of every liter culture medium, among which, about 20 mg pyrrolomycin A was found in the supernatant. HPLC was used to detect pyrrolomycin A by comparing with the synthetic sample. Aside from pyrrolomycin A, significant amount of pyrrolomycin B (about 2 mg/L) and pyrrolomycin D (less than 1 mg/L) were also detected from the culture medium. No significant amounts of other pyrrolomycin products were detected by LC-MS. Pyrrolomycin production in minimal medium. Except for the rich medium used, defined medium based on ISP432 with a different nitrogen source were used to synthesize pyrrolomycin A (Table 5). A. vitaminophilus was inoculated in rich medium as seed culture. 100 mL was then transferred to 1000 mL defined medium and incubated for 5 days to accumulate pyrrolomycin A. No obvious differentiation of pyrrolomycin A production was observed with different N sources, which suggested that A. vitaminophilum could grow on a variety of N sources. Table 5. Pyrrolomycin A production in minimal medium. nitrogen source pyrrolomycin A (mg/L) rich medium (control) 20.0 KNO3 63 (NH4)ZSO4 9.0 L-arginine 8.0 L~proline 5.2 KNO3 and (NH,,)ZSO4 7.6 KNO3 and L-arginine 7.2 KNO3 and L-proline 6.6 (NH4)ZSO4 and L—arginine 7.6 (NH4)ZSO4 and L-proline 6.9 L-arginine and L-proline 7.6 carbon source: starch and D-glucose. 104 Microbial synthesis of dioxapyrrolomycin by Streptomyces sp. UpJohn UC11065. The biosynthetic nitration reactions are rare in bacteria. Another example is the biosynthesis of dioxapyrrolomycin, in which the arylnitro group was believed to be an intact conversion from nitrate.25 Streptomyces SP. UpJohn UC11065 was isolated from the soil in Kalamazoo, Michigan, USA. The spore growing strain could produce 5 mg/L diaoxapyrronycin. 33 Streptomyces sp. UpJohn UC11065 was cultured in the same way as A. vitaminophilus (ATCC31673). A 1000 mL production medium obtained was then centrifuged to separate the supernatant and mycelia. While the supernatant was directly extracted with EtOAc, the mycelia were stirred with acetone for 2 h to export any dioxapyrrolomycin from the mycelia into solution that can be extracted with EtOAc. Dioxapyrrolomycin was purified with flash column and an Rf=0.3 fraction was collected when the column was eluted with EtOAc and hexane (1:5). Further recrystallization afford 1.5 mg yellow needle solid, most of which was extracted from the mycelia. After characterization by 1H NMR, 13C NMR and Mass spetrum., this fraction was confirmed to be dioxapyrrolomycin according to literature.6 Hypothesis of Direct Bionitration Mechanism The intact incorporation of dual isotope labeled 1(‘5N‘803 into the nitro group in the dioxapyrrolomycin observed by Carter25 suggested a direct biosynthetic nitration mechanism in the synthesis of this molecule. As the substitution patterns of pyrrolomycin A and dioxapyrrolomycin are the same, we assumed that a similar mechanism might be involved in the synthesis of pyrrolomycin A. Isotope labeling experiments and in vitro reactions were set up to test this hypothesis. 105 Isotope labeling experiments. In the study of dioxapyrrolomycin biosynthesis, double labeled K‘SN'SO3 was fed into the culture of Streptomyces fumanus, and the product was collected.” It was interesting that the nitro group in the pyrrole ring was also double labeled, which suggested that the arylnitro group was intact from N031 Based on this observation, the Carter group claimed a direct nitration mechanism in the biosynthesis of dioxapyrrolomycin.25 Considering the structural similarity of pyrrolomycin A and dioxapyrrolomycin, an isotope—labeling experiment was designed to test whether the nitro group was intact from NO3'. Our first experiment used the single labeled K'SNO3 to make sure this experiment would work, as K‘SNO3 is much cheaper than K'SN‘BO3. A. vitaminophilus (ATCC31673) was cultured as described with 100 mg K‘SNO3 added to the production medium. Pyrrolomycin A was then purified and 15N was incorporated into pyrrolomycin A as detected by Mass Spectrum. The next step was to determine whether the nitro group was introduced to the pyrrole ring through a direct bionitration step. Thus, dual labeled K'SN'BO3 was supplemented to the accumulation medium and cells were cultured in the same way as before. To our surprise, only l5N, not 180, was incorporated into pyrrolomycin A. According to the mass spectra, 15N was specifically incorporated into the nitro group, not the N in the pyrrole ring. Losing NO2 from the molecular ion leads to losing a fragment of 47 (‘5N02), not 46 (”NOZ). The same conclusion was reached based on the other arylnitro bearing molecule, pyrrolomycin B, which was co-purified from the same medium. 15N was also specifically incorporated into the nitro group, not the N in the pyrrole ring. It is thus safe to say the 15N was incorporated into the nitro group, not in the pyrrole ring in both pyrrolomycin A and B. 106 It is unlikely that the nitro groups in these two molecules were intact from N0; if no washout of '80 occurred from the nitro group when the molecules were incubate in HZ‘SO. However, in both NOS-mediated and arylamino oxidase mechanisms, the single isotope incorporation is highly possible because the O in both situations comes from air 02. Further isotope labeling experiments were conducted in defined medium with KNO3 as the only N source supplemented with 15% K‘SN 180. However, both 15N and 180 were not incorporated into the isolated pyrrolomycin products, pyrrolomycin A, B and dioxapyrrolomycin. This is not explainable as at least 15N was incorporated into the pyrrolomycin in rich medium. In vitro Reactions with SNAC as PCP Analogues. The isotope labeling experiments failed to tell us that a direct nitration reaction was involved in the biosynthesis of pyrrolomycin A, pyrrolomycin B and dioxapyrrolomycin. It is not clear whether there was no direct nitration at all or the '80 was washout during the incubation. By comparing with the biosynthesis of pyoluteorin, a hybrid PKS and NRPS may also be responsible for pyrrolomycin A synthesis. During pyoluteorin biosynthesis, L-proline was first activated and loaded onto NRPS as a peptidyl carrier protein (PCP) intermediate, which undergoes further oxidation and chlorination to form the pyrrole intermediate (Figure 47).7' 8 The PCP protein also serves as the interfacing point of polyketide/nonribozomal polypeptide synthase modules, which are loaded onto the polyketide synthase module to synthesize the benzene ring.8 Specifically, the chlorination reaction is an eletrophilic substitution catalyzed by a FADHz-dependent haloganase, whose substrate is pyrrolyl-2-acyl-peptidyl carrier protein (PCP) (Figure 47).8 We assume that the PCP protein is also the substrates of the similar 107 substituting nitration reaction. Based on this observation, one possible nitration mechanism could be substitution of either pyrrolyl-Z-acyl-PCP or dichloro pyrrolyl-2- acyl-PCP (Figure 53). Although it is not clear whether the substitution is a radical or electrophilic reaction. 34 Another possible mechanism could be the nucleophilic substitution of C1 by a NO; group (Figure 53). The modified pyrrolyl-2-acyl-PCP will then be hydrolyzed and decarboxylated to afford pyrrolomycin A. N02 CI No2 ms I R —*. N02 ms ‘ —> M N CI H u NO + O 2 O {:11 CI CI N02 C, N02 INS? £3 111139 —> ,Z/—\S 9' N NO + C' N —’CI N H O 2 H O H o N CI CI CI 2 (5' C. N02 N02" / \ S~I=I —> ’ S~n —’__. Z/ \S CI N CI N C, N H O H O H R = peptidyl carrier protein (PCP) or . . pyrrolomycin A N-acetylcysteamme thIoester (SNAC) Figure 53. Hypothesis of direct biosynthetic nitration with pyrrolyl-2-acyl-peptidyl carrier protein (PCP) or pyrrolyl-2-acyl-N-acetylcysteamine thioester (SNAC) as substrates of chlorination and nitration reaction. On the other hand, acyl- and aminoacle-acetylcysteamine thioester (SNAC) could in vivo and in vitro serve as mimic substrates of acyl-ACP and aminoacyl-PCP in PKS and NRPS (Figure 13),35 the chemically synthesized pyrrolyl-2-acyl-SNACs may also serve as the substrates of the chlorination and nitration reactions (Figure 53). Based on these two observations, incubating crude lysate prepared from the pyrrolomycin A producer with pyrrolyl—2—acyl-SNACs was expected to show in vitro nitration activity. Since pyrrolomycin B was detected together with pyrrolomycin A from the A. 108 vitaminophilus culture medium, any detection of the two molecules will suggest in vitro nitration activity. We then synthesized possible peptidyl carrier protein (PCP) analogues and prepared crude lysate for in vitro reactions to test this hypothesis. Preparation of crude lysate of A. vitaminophilum. Culturing A. vitaminophilum in rich medium with CaCO3 and soybean meal brought problems for reparation of crude lysate with French press, as the two insoluble components would block the passage of the French press. This problem was resolved by either breaking the cell with sonicator or culturing the cells in media comprised of all soluble components. Thus, CaCO3 was not used and soybean meal was replaced with a H20 soluble enzyme-digesting product of soybean meal. A. vitaminophilum was also cultured in ISP2 medium and defined medium without any insoluble components. Pyrrolomycin A production was confirmed in all culture broths before mycelia were collected to prepare crude lysate. Synthesis of SNAC thioeste as PCP anw The syntheses of biomimic small molecules (SNAC thioesters) of PCP proteins were achieved by BBC coupling of N- acetylcysteamine with the corresponding pyrrole-Z-carboxylic acid (Figure S4). The synthesis of the non-chlorine substituted pyrrole SNAC was begun with the commercially available pyrrolyl-Z-carboxylic acid. The syntheses of di-, and tri-chloropyrrole-SANC was started with a commercially available compound, 2-(trichloroacetyl)pyrrole. Chlorination of 2-(trichloroacetyl)pyrrole with two equilibriums of sufuryl chloride (SOZCIZ) in acetic acid would afford 4, 5-dichloropyrrole-2-yl-trichloromethyl ketone and 3, 4, S-trichloropyrrole-Z-yl- trichloromethyl ketone simutanously. After separation by flash column, the two ketones were hydrolyzed in 0.5 M NaOH, followed by acidification to obtain the corresponding di- and tri-chloroacid, which coupled with N- 109 acetylcysteamine to get the final products. The main challenge of the synthesis is the isolation of the coupled products from the starting material, N—acetylcysteamine. Although silica gel treated with CuSO4 could provide better separation, further purification of the products was achieved by repeated recrystallization. This is the main reason that the yields are relatively low. 0 /\ OH 3 N (BOVSWNJOK HSNAC: HSwNJK H 800/0 H CI CI CI 0 / \ CCI c / \ OH 8 / \ 0% S—wlpfir _.c. N SWNJK H o 85% H o 30% H o H / \ CCI3 _b, 53 % N H O ClmCClac ——-> (fizz—R0 Hi’CITZ—‘Sfifsw 650/° 20°/o 33% Figure 54. Synthesis of the unsubstituted and 2, and 3-chloro substituted pyrrole-SNAC. Conditions: 3. HSNAC, EDC°HCI, DMAP, DMF;36 b. SOzClz/HOAC;37 c. NaOH, then BC].37 In vitro reaction with SNAC thioester. The synthesized SNAC analogues were then used to investigate the biosynthesis of pyrrolomycin A in vitro with crude cell lysate. A 1 mL cell lysate was incubated with 0.3 mM SNAC analogues, 5 mM NaCl, supplemented with either 5 mM KNO2 or KNO3 and possible cofactors such as ATP, NADH or FADHZ. The reaction was then incubated at 28 °C and loaded onto HPLC to detect the production of pyrrolomycin A and pyrrolomycin B. After up to 48 h, both unchlorinated and 2-chlorinated pyrrole SNAC disappeared completely and a new peak showed up in HPLC chromatography. In the reaction with 3-chlorinated pyrrole SNAC, an unknow product as well as the substrate was also detected by HPLC. Although all the 110 3 unknow components could not be identified and the reactions couldn’t be duplicated, the inability to detect pyrrolomycin A and B suggested that either the SNAC mimic molecules were not accepted by the PKS/N RPS modules or they are not the intermediates of nitration reaction in the biosynthesis of pyrrolomycin A. In vitro Reactions with Ketone as Post-Modification Intermediates. Although the modification of polyketides and polypeptides mostly occurr in the polyketide synthase and nonribosomal peptide synthase modules, post modification was also possible.38 In the biosynthesis of differentiation-inducing factor (DIF—l), the key intermediate, (2, 4, 6-trihydroxyphenyl)-l-hehax-l-one (THPH) was chlorinated and methylated in vitro after being released from the polyketide synthase complex (Figure 55).”3 Another evidence of post modification of polyketide synthesis is the biosynthesis of ansamitocin as we described in chapter one. Proansamitocin released from the PKS complex will undergo chlorination, O-methylation, N-methylation, epoxidation, acylation, and carbamoyl transferforamtion until the mature product was obtained (Figure 12).38b Accoding to the biosynthesis of DIF-l and ansamitocin, a mechanism was proposed for the chlorination and nitration of the pyrrole ring by a post PKS/NRPS synthesis modification in the biosynthesis of pyrrolomycin A (Figure 5). In this mechanism, L- proline was activated, oxidized and loaded onto the NRPS complex as pyrrolyl-Z-acyl- PCP protein, which was further loaded onto a PKS complex.7' 8 The polyketide intermediate was then elongated by 3 molecules of malonyl CoA, which in turn was reduced and terminated as phenyl pyrrolyl ketone and released from the PKS assembly line. The ketone substrates could then be post-modified by any of the chlorination, nitration, hydroxylation and decarboxylation reactions to achieve the pyrrolomycin A, B, 111 C and D . Chlorination, however, more likely occurred on the NRPS complex8 instead of post PKS modification. We then synthesized possible pyrrole phenol ketone intermediates and in vitro reactions were set up with the crude lysate of A. vitaminophilum to test this hypothesis. 0 OHO Cl Polyketide _. WWW b synthesis OH CH3O OH CI (2 4 5 tnhydroxyphenyl) differentiation-inducin - 1-hehax-1-one (THPH) dlCthfO-THPH factor (DIP-1) g 5: a. H202/Cl7chloroperoxidase b. methyltransierase I Figure 55. Synthesis of DIF—l by post PKS modification reactions from THPH. I}- O O P PKS 0HN_..R S SPCP—> /\ __.a /\ N OH—> N H malonleoA H 0 0 O OH L-prolin: CI b CI No2 CI 1 OOH pyrrolomycin A l \ pyrrolomycin C / \ CI No2 Cl C' N 0' CI CI CI / u 0 OH CI H l \ /f/ O OH R / \ C CI N C. CI N CI H OH H 0 OH pyrrolomycin B pyrrolomycin D Figure S6. Hypothesis mechanism of biosynthetic nitration by post PKS modification reactions. Possible enzymatic reactions included: (a) Claisen condensation and products released from PKS; (b) benzene ring chlorination; (c) pyrrole ring chlorination; (d) nitration; (e) hydralation, and decarboxylation; (f) ketone reduction. Synthesis of pyrrole phenol ketones. Synthesis efforts towards the pyrrole phenol ketone started with the coupling of pyrrole and phenol species by Friedel-Craft reaction with AlCl3 or SnCl4 as catalyst (Figure 3). The 4, 5-chloropyrrole-2-carboxylic acid synthesized as described above was reduced to acyl chloride with SOCI2 and coupled 112 with 2, 4~dichlorophenol by a Friedel-Craft reaction. However, the Friedel-Craft reaction product in CHzCl2 catalyzed by AlCl3 and SnCl4 turned out to be an ester, which was not further rearranged to the desired ketone (Figure 57). The ester product was also obtained even with 2, 4-dichloroanisole as the starting material because the demethylation occurred first in the presence of AlCl3.39 The Fries rearrangement40 couldn’t complete by increasing the reaction temperature to 80 °C by using 1, 2-dichloroethane as solvent. Further increasing the reaction temperature to 100 °C and 120 0C with nitromethane and nitrobenzene as solvents didn’t help the rearrangement, instead, the products could end up with polymer.“ / \ CI + a ,Zl—Mo b Cl’mfif <10|_’ CI N X’ C. / \ CI H o H o N CH CI H o OCH3 R=HorCH3 Figure 57. Friedel-Craft reaction to couple pyrrolyl-acyl chlorinde and 2, 4- dichlorophenol/anisole. Conditions of step (a): (1) AlCl3/CZH2CIZ, rt; (2) AlCl3/ClCH2CH2Cl, reflux (82 °C); (3) AlCl3/CH3.NO2 (101°C); (4) AlCl3/nitrobenzene (120 °C). Conditions of step d: (l) AlCl3/CH3NO2 (101°C); (2) AlCl3/nitrobenzene (120 °C). As the Fridel-Craft reaction failed to couple the two aromatic rings, we turned to link metalized pyrrole species with acyl chloride. Protection and partial deprotection of 3, S-dichlorosalicylic acid (or salicylic acid to synthesize 2-hydroxybenzoylpyrrole) afforded the protected salicylic acid, which will be coupled with N-pyrrolylmagnesium bromide. This coupled ketone could be deprotected to yield 2-(3, 5- dichlorosalicyloyl)pyrrole or 2-(hydroxybenzoyl)pyrrole. The coupled ketone could also be further chlorinated to afford the protected pyrrolomycin C and D, which were then deprotected to obtain pyrrolomycin C and D (Figure 58). 113 fl CH3CH2MgBr> m N N H MgBr N-pyrrolylmagnesium bromide (not isolated) a / \ HO R 88°/ CI R ii CH 0 O R=H,CI O OH O OCH3 2-(2- Hydroxybenzoyl) bl 45% pyrrole dC1OO°/o CI CI CI / \ R /N\ CI N C|53°/o/ 27% Cl Cl H CCH3 0 OCHs o OCHa 1OOC°I/o 110070 /H\:1C|100% H 0 OH 0 OH 0 OH pyrrolomycin C 2- -,(3 :-dichlorosalicyloyl) pyrrolomycin D pyrrole Figure 58. Synthesis of possible ketone intermediates. a. (i) CH3I, K2C03, DMF, 50 °C; (ii). NaOH, then HCl; (iii) SOCIZ/DMF/CHZCIZ; b. N-pyrrolylmagnesium bromide/toluene/T HF, 0 °C to rt; c. SOzClz/EtzO/CHzClz, -78 °C to rt; d. BBr3, CHZCIZ. With the proposed intermediates for the post modification synthesized, in vitro reactions were set up to monitor the production of pyrrolomycin A or B by HPLC. Cell lysate was incubated with 2-(2-hydroxybenzoyl)pyrrole, 2-(3, 5- dichlorosalicyloyl)pyrrole, pyrrolomycin C or pyrrolomycin D, supplemented with KNO2 or KNO3. However, no new peaks showed up from HPLC after incubating the substrates for 48 h, and only starting materials were recoveried from reactions with all the 4 intermediates. which suggests either no post modification to the ketones at all or the enzyme activity in crude lysate is very low. 114 Hypothesis of N OS-mediated Bionitration Mechanism In Vitro nitric oxide synthase assay. In the study of the biosysnthesis of thaxtomin, Loria19 claimed that nitric oxide synthase was involved in the biosynthetic nitration reaction based on 3 experiments. One is the incorporation of 15N into the NO2 group from L—Arg-guanido-‘SNZ. The second is the inhibition of thaxtomin production by a typical NOS inhibitor N-nitro-L-arginine methyl ester (NAME). The final one is the detection of NOS activity in the crude lysate. Similar experiments were designed to test whether the NOS was involved in the nitration reaction of pyrrolomyicn A. Our first effort was culturing A. vitaminophilus (ATCC31673) in rich medium supplemented with 50 mg and 100 mg L-Arginine-guanido-‘SNZ. If the nitro group in pyrrolomycin A and B came from the NO produced by a nitric oxidase synthase, it would be 15N labeled (Figure 59).42 Pyrrolomycin A and B obtained in this broth was isolated by a flash column and detected by HPLC and MS. However, no 15N was incorporated into both pyrrolomycin A and B. H2‘5N H2‘5N OH H2‘5N O H2‘5N ):15NH ):15N HN>;15N H)=O HN HN NADPH NADPH + 1 5N0 02 + + HaN'“ H3N"' H3N"' H3N"' O O ‘0 “O ”O L-arginine N-hydroxy-L-arginine L-citruline Figure 59. Incorporation of 15N into NO from isotope labeled L-arginine-guanido-‘SN2 by nitric oxide synthase. The second effort was to measure pyrrolomycin A production in the presence of NOS inhibitor, NAME. 5 mL of the 100 mL seed culture 2 as described above was 115 transferred to a 100 mL culture medium supplemented with NAME at different concentrations and kept on growing at 28 °C. A 5 mL sample was taken out every 24 h and pyrrolomycin A production was detected by HPLC. This inhibitor didn’t affect the cell growth, and no apparent inhibition of pyrrolomycin A production was observed at all inhibitor concentrations (Figure 60). 4O 0 o :7 l I! E 30 . ...... . ............................................................ g C) 3 .< D E 20 . ................................................................... 3. E .9 9 '5. 1o . ..................................................................... Q B 0 9 fi I T I O 24 48 72 96 120 time (h) Figure 60a. Production of pyrrolomycin A in the presence of NOS inhibitor, NAME. Concentration of NAME: solid square, 0 FM; solid circle, 15 pM; open circle, 30 yM; open square, 60 pM. 40 I O U A I "E‘ 30 .......................... 0 ............ B. ............ C1 .......... \U) 0 § . < E 20 .................................................................... g. E .9 9 ; 10 ..................................................................... Q. B 0 g I f I I 0 24 48 72 96 120 time (b) Figure 60b. Production of pyrrolomycin A in the presence of NOS inhibitor, NAME. Concentration of NAME: solid square, 0 yM; solid circle, 120 ”M; open circle, 200 yM; open square, 500 FM. 116 The third experiment was to detect NOS activity with crude cell lysate. Nitric oxide synthase catalyzes the oxidation of L-arginine to citrulline and NO was produced (Figure 59). If an NOS exists, incubating L-arginine, H202 and possible cofactors with crude lysate would allow the detection of NO production by Griess reagent assay or hemoglobin assay. 43 In the Griess reagent assay, NO was oxidized by H202 to NOZ', which was further oxidized by Griess reagent one to azide. The obtained azide then coupled with Griess reagent two to afford the red azo product, which can be monitor at ODS.40 nm (Figure 61). H202 _ NO —> N02 4. NEN NH2 H+ N02- '1’ —'> SOZNHZ SOQNHQ sulfanilamide ,1] N (Griess reagent 1) HN/\‘ H2N + NH2 00 _. H2NOZS-Q-N=N O SOZNHZ Q N-(1-naphthyl)ethylenediamine azo product (Griess reagent 2) (red 00'0') NO/o2 Oxyhemoglobin (Fez*) —-—> Methemoglobin (Fe3*) Figure 61. Griess reagent assay (top) and hemoglobin assay (bottom) for nitric oxide synthase activity. In the hemoglobin assay (Figure 61), iron (11) in oxyhemoglobin is stoichiometrically oxidized to Fe (III) in methemoglobin by NO, which can be continuously monitored at OD“), nm. And because this redox reaction is 26 times faster than the reaction between molecular oxygen and NO,43 the hemoglobin assay can be assayed in the presence of air. Reactions were set up according to literature.43 However, 117 no activity was observed from both assays. This suggested that, in solution, no free NO was produced.18 All in vitro NOS assay suggested that no NOS exists in A. vitaminophilus. However, exclusion of the existence of NOS may not be made until further experiments. The complex of arginine metabolism in microbes could be misleading to the isotope labeling reaction and the mammalian NOS inhibitor, NAME might not be an inhibitor for bacterial NOS, although it is an inhibitor for mammalian NOS. Genetic approach to probe nitric oxide synthase. To solve the in vitro NOS problem, a genetic approach was employed to probe NOS. Amplifying a target gene by DNA polymerase chain reaction (PCR) enables cloning of the target gene and overexpressing it for in vivo and in vitro reactions. This requires two short sequences known as primers based on the target gene sequence. However, the lack of any gene sequence information of both A. vitaminophilus and Streptomyces sp. UpJohn UC11065 makes a regular PCR impossible. Consensus PCR is a technology used to probe target genes without sequence information from organisms according to the conserved amino acid sequences that are shared by enzymes from closely related organisms.44 Degenerate primers were designed according to the alignment of NOS from bacteria (Figure 2). Amplification of the inner fragment of the target gene between the two-conserved domains will then provide part of the target gene sequence information. This information is then the basis for designing further primers to amplify the whole target gene by inverse PCR. 118 SO ---------- QSDQPVPLARRLEQVRAAIDATGTYRBTTAELVYGARVAWRNSSRCIGRL 110 Sac ---------- QSDQPVPLARRLBOVRAAIDATGTYRHTTAELVYGARVAHRRSSRCIGRL 110 St ---------- QSDQAVPLTRRLDOVRAAIDATGTYRHTTAELVFGARVAWRNSSRCIGRL 110 83v AATAFLTLHHTBBRLGDPARRIAAAHA!IABTGTYRHTTEELVIGARVAWRNANRCIGRL 300 B. ---------- HXDRLAD ------- IRS!IDLTGSYVHTKBELBBGAKHAWRNSNRCIGRL 43 Dr ---------- tflBBHGEPG——LPARLRAVBEAGLWWPTSAELTWGARVAHRNSTRCVGRL 67 g. 3 3. g t. .0 w:.ifigtit SI YWNSLRVLDRRDATAPDEIKRBLCTBLRQATNGGRIRPVISVPAPDSPGRPGPOVHNEQL 170 Ste YWNSLRVLDRRDATAPDIIBRRLCTKLROATNGGRIRPVISVPAPDSPGRPGPQVWNBQL 170 St YWNSLRVLDRRDTTAPEVIHRHLCTHLRQATRGGBIRPVISVPAPDAPSRPGPRVWNBQL 170 Sav YHBSLCVRDRRDVRDAKDVABASADHLRBATRDGRIRALITVPAPDAPGRPGPRIWNBQL 360 B. FWNSLNVIDRRDVRTKEEVRDALtflHIITATNNGKIRPTITIPPPEEKGEXOVEIHNHQL 103 Dr YVEALSVRDLRELNTAQAVYBALLQHLDDAPCGGflIRPVISVFGP ------ GVRLBNPQL 121 :..30 0 O O: . 3 O: Q ‘0'... 03'. O .3 O 0. SI IRYAGYRRDDGTVLGDPRTADLTEAILRLGVQGCPOGPPDVLPLVIDTPDD-KPRYPBLP 229 Sac IRYAGYRRDDGTVLGDPRTADLTBAILRLGHQGCPOGPFDVLPLVIDTPDD—KPRFFBLP 229 St VRYAGBRRDDGTVLGDPRSADLTEAIRGLGWOGGROGPPDVLPLVIDAHDD-KPRPPBLP 229 Snv IRYAGYARPGGAVTGDPRNAGLTALARRLGW?GGPGSPPDVLPLIVQSAGD-RPRHPTLP 419 as IRYAGYB-SDGERIGDPASCSLTAACEILGHRG-BRTDFDLLPLIPRHRGDBQPVHYELP 161 Dr IRYA ---------- DDPINADFVDKLRRPGWQP-RGBRIEVLPLLIBVNGR--ABLF8LP 168 3". ." ...3. x" '3:"‘s. . . x *' So RBLVLEVPITHPDVPRLABLGLRWBAVPVISNHRLRIGGHDYPLAPFNGWYHGTBIGARN 289 Sac RBLVLEVPITHPDVPRLAELGLRHHAVPVISNHRLRIGGHDYPLAPFNGWYHGTBIGARN 289 St RZVVLEVPITHPDVPRLABLCLRHKAVPVISNHRLBIGGYDYPLAPFNGWYHGTEIGVRN 289 Buv EDAVLBVALTHPEYPHWRSLGLRWEAVPALAGHCLESGGICYPAAPINGWYHGTBIGARN ‘79 BI RSLVIBVPITHPDIZAFSDLELKHYGVPIISDMXLBVGGIBINAAPPNGWYNGTEIGARN 221 Dr POAVQEVAITHPVCLGIGBLGLRWHALPVISDHBLDIGGLHLPCA-ISGHYVQTEIAAkD 227 . 0 fit.‘...‘ .0 O‘.;.:I 3“. C it; 9&0.- O.....: S. LVDBDRYNNLPAVAACLQLDTTSBSTLWRDRALVELNVAVLHSPBAAGVRI8DRBZBSRR 349 Ste LVDEDRYNHLPAVAACLQLDTTSBSTLWRDRALVBLNVAVLBSISAAGVRI508833888 369 St LVDZARYNLLPAVAACLQLDTTSESTLWRDRALVELNVAVLHSPAAAGVRISDHHEBSRR 349 Sav LADADRYDLLPHLADRLGLDTRSDRSLWKDRALVBLNRSVLRSFDRAGVTVTDHHTBSLR 539 B. LADBKRYDKLKKVASVIG1AADYNTDLHKDQALVBLNXAVLHS!KRQGVSIVDHHTAASQ 281 Dr LADVGRYDOLPAVAflALGLDTSEERTLWRDRALVBLNVAVLBSFDAAGVKLADHHTVTAB 281 0.. it: 0 :0 3 3 g 3 .0‘03000900 30...; it 3 .0. g g S. FLAHLAKEBRQGRTVSADWSNIVPPLSGGITPVFHRYYDNVDORPNFYPHQ --------- 400 Sac FLAHLAREERQGRTVSADWSHIVPPLSGGITPVPHRYYDNVDQRPNPYPHQ --------- 400 St PLABLTKEERQGRTVPADWSWIVPPLSSGITPV?HRYYDNADQRPNPYPHQ --------- 400 Sav PLTflLDRBERXGRRVGADHSHIVPPISGSATPVFHRTYBTVBRHPAYVHHPZALARARGB 599 Bi IKRPBBOEEEAGRKLTGDHTWLIPPISPAATHIFHRSYDRSIVRPNYPYODRPYB ----- 336 Dr flVRFBEREARAGRBVRGKWSHLVPPLSPATTPLHSRRYRARBESPRFVRARCPIHTPTVR 347 . . a. . 'fi : ..':'::"z' . ' s: ' ' . 3 III III-I-I-II Figure 62. Designing degenerate primers for consensus PCR based on the conserved domains (highlighted by underline) amino acid sequences. 53, S. scabies; Sac, S. acidiscabies; St, S. turgidiscabies; Sav, S. avermitilis; BS, B. subtilis; Dr, D. radiodurans. Unlike standard PCR, which amplifies segments of DNA that lie between two inward-pointing primers, inverse PCR45 is used to amplify and clone unknown DNA based on outward-pointing primers (Figure 63). Genomic DNA that contains the target gene is digested into small linear fragments and re-ligated into circular DNA. Inverse PCR then amplifies the circular DNA based on primers designed according to the partial 119 SI ---------- QSDQPVPLARRLBQVRAAIDATGTYRHTTABLVYGARVAWRNSSRCIGRL 110 Sec ---------- QSDOPVPLARRLEOVRAAIDATGTYRHTTAfiLVYGARVAWRNSSRCIGRL 110 St ---------- QSDOAVPLTRRLDQVRAAIDATGTYRHTTAELVPGARVAWRNSSRCIGRL 110 53V ‘ AATAFLTLBHTEERLGDPARRIAAAHABIABTGTYRHTTEELVIGARVAWRNBNRCIGRL 300 B. ---------- HXDRLAD ------- IRS!IDLTGSYVBTKIBLBHGAKHAWRNSNRCIGRL ‘3 Dr ---------- PHBEHGBPG--LPARLRAVBEAGLWWPTSAELTWGAKVAWRNSTRCVGRL ‘7 g. g g. g 0. I. mtg...x..0 SI YWNSLRVLDRRDATAPDEIHRHLCTHLRQATNGGRIRPVISVTAPDSPGRPGPQVWNBQL 170 SIC YHNSLRVLDRRDATAPDBIHRHLCTHLRQATNGGRIRPVISVYAPDSPGRPGPQVWNBQL 170 St YWNSLRVLDRRDTTAPEVIHRHLCTHLRQATNGGRIRPVISVPAPDAPSRPGPRVWNBQL 170 88V YWHSLCVRDRRDVRDAKDVAEASADHLREATRDGRIRALITVFAPDAPGRPGPRIWNEQL 350 Bl PWNSLNVIDRRDVRTKZEVRDALPHHISTATNNGKIRPTITIFPPEEKGEKQVBIWNHQL 103 Dr YWEALSVRDLRZLNTAQAVYBALLOHLDDAPCGGHIRPVISVFGP ------ GVRLHNPQL 121 ‘o_,o c o a, . g o; o .03... .;.o t .3 I 0. SI IRYAGYRRDDGTVLGDPRTADLTBAILRLGWOGCPQGPFDVLPLVIDTPDD-KPRPPEL? 229 Sac IRYAGYRRDDGTVLGDPRTADLTBAILRLGWOGCPQGPFDVLPLVIDTPDD-KPRFFBLP 229 St VRYAGHRRDDGTVLGDPRSADLTBAIRGLGWQGGROGPVDVLPLVIDAHDD-KPRPFZL? 229 SDV IRYAGYARPGGAVTGDPRHAGLTALARRLGWPGGPGSPPDVLPLIVQSAGD-RPRWPTLP ‘19 BI IRYAGYB-$DGBRIGDPASCSLTAACEBLGWRG-BRTDFDLLPLIPRHRGDZQPVHYELP 151 Dr IRYA ---------- DDPINADPVDKLRRFGWQP~RGBR’EVLPLLIBVNGR--RBLP8LP 168 3". ." ...:. :" 'zrfififis. . . z 0' S. RBLVLEVPITHPDVPRLABLGLRWBAV?VISHIRLRIGGHDYPLAPFNGWYHGTDIGARN 289 Sac RZLVLEVPITHPDVPRLAELGLRWHAVPVISHIRLRIGGHDYPLAPFNGWYHGTZIGARN 289 St RIVVLEVPITHPDVPRLAELCLRWKAVPVISNHRLRIGGYDYPLAPFNGWYHGTEIGVRN 289 BIV EDAVLEVALTBPBYPWWRSLGLRWHAVPALAGHCLESGGICYPAAPFNGWYHGTEIGARN ‘79 BI RSLVIBV?ITBPDIEAPSDLELKWYGVPIISDHKLIVGGIHYNAAPPNGWYKGTBIGARN 221 Dr PQAVQEVAITHPVCLGIGELGLRWKALPVISDHBLDIGGLHLPCA-PSGWYVQTBIAARD 227 . a c..'ooa . .. .3i;.;. 3". a II: . ;‘;;:‘ 090...: S. LVDEDRYNHLPAVAACLQLDTTSBSTLWRDRALVELNVAVLBSPEAAGVRISDHHEBSRR 349 8.0 LVDEDRYNMLPRVAACLQLDTTSBSTLWRDRALVELNVAVLHSPIAAGVRISDSKIBSR‘ 389 St LVDEARYNLLPAVAACLOLDTTSESTLWRDRALVBLNVAVLHSFAAAGVRISDRHEBSRR 349 88V LADADRYDLLPHLADRLGLDTRSDRSLWXDRALVBLNRSVLHSPDRAGVTVTDHHTBSLR 539 BI LADBKRYDKLRKVASVIGIAADYNTDLWKDQALVBLNXAVLHSYKKQGVSIVDHHTAASQ 281 Dr LADVGRYDQLPAVARALGLDTSRERTLWRDRALVILNVAVLBSFDAAGVRLADHHTVTAI 287 ¢_c no; a g. l 3 3 3 .0:.:...... 3.90.; at ' 0.. ' 3 SI FLAHLAKBBRQGRTVSADWSWIVPPLSGGITPVPHRYYDNVDQRPNFYPBQ --------- ‘00 SIC PLAHLAKBZRQGRTVSADWSWIVPPLSGGITPVPHRYYDNVDQRPNPYPBQ --------- ‘00 St PLABLTXEERQGRTVPADWSWIVPPLSSGITPVFHRYYDNADQRPNPYPHQ --------- ‘00 88V FLTHLDREBRXGRRVGADHSHIVPPISGSATPVFHRTYETVERHPAYVHBPEALARARGB 599 BI FRRFBEOEZEAGRXLTGDWTWLIPPISPAATHIFHRSYDNSIVKPNYFYODXPYB ----- 335 Dr HVRFEEREARAGREVRGKWSWLVPPLSPATTPLWSRRYRAREBSPRPVRARCPFHTPTVH 387 o o 8‘ . .. I ...8.88"8. o * 88 . ' ' I III III-III..- Figure 62. Designing degenerate primers for consensus PCR based on the conserved domains (highlighted by underline) amino acid sequences. Ss, S. scabies; Sac, S. acidiscabies; St, S. turgidiscabies; Sav, S. avermitilis; BS, B. subtilis; Dr, D. radiodurans. Unlike standard PCR, which amplifies segments of DNA that lie between two inward-pointing primers, inverse PCR45 is used to amplify and clone unknown DNA based on outward-pointing primers (Figure 63). Genomic DNA that contains the target gene is digested into small linear fragments and re-ligated into circular DNA. Inverse PCR then amplifies the circular DNA based on primers designed according to the partial 119 sequences obtained from consensus PCR. Therefore, the whole target gene sequences will be available to pull out the whole gene from genomic DNA. restriction enzyme digestion :21 = :2: + I :2 genomic DNA intramolecular l ligation PCR primers ‘2. <—— J ‘____ Figure 63. Inverse PCR. Ligend: filled area, gene sequence obtained from consensus PCR. Genomic DNA of pyrrolomycin A and dioxapyrrolomycin producers, A. vitaminophilum and Streptomyces sp. UpJohn UC11065 were prepared according to the Kirby mix procedure.‘“5 The PCR products were then cloned into TOPO 10 vector for sequencing. However, after several PCR reactions, no gene with homologue to NOS was pulled out from the genome of pyrrolomycin A and dioxapyrrolomycin producers, A. vitaminophilum and Streptomyces sp. UpJohn UC11065. In stead, DNA fragments, which have high homology to B-galactosidase (EC 3.2.1.23), 2-oxoisovalerate dehydrogenase a subunit, 2-oxoacid ferredoxin oxidoreductase B subunit, or transporter (membrane protein), were identified. These results, however didn’t exclude the existence of NOS in the genome of these two bacteria due to the complex factors that may affect the consensus PCR results. An Alternative Way to Understand Bacterial N itration Reaction Cloning and heterogenous expression of deiNOS. The previous efforts to test the direct nitration reaction and NOS-mediated 120 nitration reaction didn’t provide positive results. One possibility can be the total non- involvement of these two reactions in the synthesis of pyrrolomycin A by A. vitaminophilus. However, the enzymatic turnovers of both polyketide synthase and non- ribosomal peptide synthase are normally pretty low, which could lead to an inability to detect any in vitro activity. In this case, the best way to detect any in vitro activity will depend on the overexpressed gene. PKS and NRPS are most likely responsible for the biosynthesis of pyrrolomycin A. With no gene information available from A. vitaminophilus, we designed an alternative way to understand the bacterial nitration mechanism. The strong evidences of the evolvement of NOS in the biosynthetic nitration of thaxtomnl9 and the regioseletive nitration of tryptophan20 by NOS invoke us to put more work on the NOS-mediated nitration reaction. Heterogenous expression of deiNOS-encoded nitric oxide synthase isolated from Deinococcus radiodurans (ATCC 13939) in E. coli was believed to catalyze the in vitro nitration of L-tryptophan.20 Our primary goal would be to measure the in vitro nitration activity of NOS. Thus, the 1.1 kb fragment of NOS was PCRed out from the genomic DNA of Deinococcus radiodurans (ATCC 13939)47 and cloned into pJF118EH, pQE3O and pETle for heteroexpression. The gene would then be expressed under Pm, T5 and T7 promotors (Figure 64) in E. coli. All plasmids contain a lac/Q gene, which allows the expression of NOS under control of IPTG, In addition, deiNOS was also put after a 6 x His-tag in the vector of pQE3O and pEI‘lS for convenient purification. 121 NB ll pWL4.106 (6.8 kb) _. ‘— Ap T7 N05 lacl ? P l pWL4.134 (6.5 kb) ._ ., Ap Ptac N05 lacl B P I | pWL4.135 (4.7 kb) ._ _, Ap i5 N05 lacl Figure 64. Cloning of deiNOS under different promotors. Restriction enzyme sites are abbreviated as follows: N=Ndel, B=BamHI, P=Pstdl. Plasmid pWL4.134 and pWL4.135 were transformed into E. coli DHSa for large- scale production of NOS. However, no overexpression obtained at temperatures of 22 °C, 30 °C and. 37 °C and IPTG concentration from 0.1 mM to 1.0 mM, and this may be due to the expression burden of rare codens of deiNOS for E. coli. To solve this problem, the NOS gene was cloned under T7 and E. coli BL21(DE3)/pWL4.106 was constructed. Effort was then focused on over expressing NOS under a T7 promotor from E. coli BL21(DE3)/pWL4.106. The NOS gene was over expressed under these conditions, although it forms inclusion body and no NOS activity was detected. Expression conditions were then optimized. BL21(DE3)/pWL4.lO6 was incubated at 37°C until OD600=0.4-O.6 and was completely cooled down on ice H20. [PT G was added at a concentration of 0.5 mM and the cells were grown at 22 oC overnight. The NOS gene was over expressed under such a condition with detected enzyme activiry. The critical point for over expression of the deiNOS gene is, therefore cooling the culture to 0 °C before induction with IPF G. The protein was then purified with Ni-NTA argose resin and in vitro reactions were set up for nitration activity. L-Tyrosine and L-tryptophan were then selected as substrates as described by literature.20 122 Synthesis of nitrotryptophans. For detection of in vitro nitration activity of deiNOS with L-tryptophan as substrate, we first synthesized the four nitrotryptophan isomers as they were all possible nitration products. Started with different isomers of nitroindole, Mannich reaction of nitroindole with formaldehyde and dimethyl amine in HOAc produced the gramine, which was condensed with diethyl acetamidomalonate. The condensation product was then hydrolyzed in concentrated HCl to afford the nitrotryptophan in the salt formation with HCl.48 The yields for each steps are from 70% to 90% (mol/mol). o / HNJK HOAC, HCHO (37%), N\ FOWOI O \ (CH3)2NH (40%). \ O . \ _ _._ \ Ong '— 02N . / / N 90-95°C, 4h, 75% N Toluene/NaOH, H H reflux, 10 h, 70% nitroindole nitrogramine HCI, reflux, 1811, 90% nitrotryptophan Figure 65. Synthesis of nitrotryptophan. Nitric oxide production assay activity. With the deiNOS purified, it is possible to assay its activity. Our first effort was to assay the production of NO with Griess reagent (Figure 61).43 In this assay, L- arginine, deiNOS protein, and H202 were incubated at room temperature. A aliquot was taken out every 3 min and heated at 100 °C to quench the reaction. After 10 min for complete oxidation of NO with H202, half volume of Griess reagents one and two were added continuously and allowed 10 min to develop the red color, which suggested the 123 production of NO as positive result. The red azo product was also measured at OD540 nm and NO amount was calculated based on standard curves. Specific activity of the NOS enzyme is 0.002 U/mg (Figure 66). 60 y = 2.3467x + 28.56 01 O A O .................................................... A L v I I N 0 concentration of nitrite (uM) (D O _L O ...................................................... 0 time (min) Figure 66. Nitric oxide synthase activity. Reaction (1 mL): deiNOS 25 14M, H202 10 mM, L-Arginine 10 mM. A 100 A. sample was taken out every 3 min and quenched by heating at 100 °C for 3 min. After allowing the oxidation for 15 min, 50 A Griess reagent R1 and R2 were added consequently. The pink color was developed for 10 min and nitrite concentration was determined by measuring OD540. The Griess reagent NOS activity is based on the detection of NO derivatives. A direct detection of NO could be based on hemoglobin assay.43 The reactions were set up according to literature, however, no activity was observed. This suggested that, in solution, no free NO was produced.18 A possible reason is that NO produced by NOS is bound to the enzyme, instead of released into solution. In hemoglobin assay, the heme protein is too bulky to approach the bound NO and no activity observed, while in the Griess reagent assay, small molecule, H202 can easily approach the NO and activity was detected. 124 Nitric oxide synthase nitration activity. The NO production activity encouraged us to further investigate the nitration activity of deiNOS. It was reported that, in the presence of H202, deiNOS could catalyze the nitration of L-tryptophan specifically at the 4 position.20 In this reaction, what can be excluded is that NO produced in this reaction is not oxidized to NC; by H202 as is the case in the Griess reagent assay reaction. What is the reactive nitrogen species responsible for the nitration remains unclear. Another observation is that, in mammalian tissues and fluids, the nitration of protein tyrosine residues is a NOS-mediated, non- enzymatic catalyzed reaction.9 Theoretically, any active aromatic molecules will also be nitrated by reactive nitrogen species derived from NOS. This hypothesis invokes us to investigate the deiNOS nitration activity with phenol, L-tyrosine, L-tryptophan and phloroglucinol as substrates (Table 6). On the other hand, peroxynitrite, which is decomposed to the reactive species NOZ+, is widely believed to be a nitration reagent for in vitro nitration of aromatic molecules, although the yields obtained were pretty low. As an alternative, in situ generation of peroxynitrite by oxidation of NO with [(02 could also nitrate aromatic substrates. And the nitration of the selected substrates by commercially available peroxynitrite served as our control reaction (Table 6). After incubating the deiNOS with aromatic substrates, L-arginine and oxidants (H202 or K02) for 4 h, weak nitration activity was observed when L-tyrosine and L-trptophan as substrates, while no activity was detected with phenol and phloroglucinol as substrates. This in vitro nitration activity mediated by NOS is based on the extra addition of oxidant. As a long-term goal to establish a microbial synthesis of arylnitro chemicals, in situ enzymatic generation of oxidants will pave the way for this purpose. Glucose oxidase oxidizes glucose to D- 125 V glucono-l, S-lactone by O2 and generates H202 simultaneously (Figure 67). L-Tyrosine and L-trptophan were nitrated when they were incubated with deiNOS, L-arginine, glucose and glucose oxidase all together (Table 6). OH 0 .xOH glucose oxidase .\OH 02 + O > O + H202 . OH EC 1 .1 .3.4 . OH HO OH HO OH D-glucose D-glucono—l, 5-Iactone Figure 67. Glucose oxidase catalyzes the oxidation of glucose by air O2 and simutanuously generates H202. The bacterial NOS-mediated nitration therefore, is not completely non-enzymatic since it has substrate selectivity. As no free NO was detected in solution by the hemoglobin assay, the NO produced was not released. Nitration substrates may bind to NOS and most likely to the same site of pterine (H4B) and serves as the electron acceptor as the role of H4B, which inhibits the tryptophan nitration by deiNOS.20 This provides the possibility to evolve bacterial NOS to mediate the nitration of aromatic molecules such as phloroglucinol. The in situ enzymatic H202 generation for nitration also provides the possibility to heteroexpress genes encoding glucose oxidase or any other H202- generating enzyme such as pyranose oxidase in E. coli for biosynthetic nitration. Table 6. Nitration activity of nitric oxide synthase. ONOO'“ H202 NOSb/H202 K02 NOS/K02 NOS/GODC phenol 1%‘1 - - - - - L-Tyr 1% - 1% - 2% 1% L-Trp 1% - 1% - 3% 5% phloroglucinol 0.2%e - - - - - ‘1 all substrates were incubated with L-arginine and the nitration reagents. 1’ NOS, nitric oxide synthase. C glucose oxidase. d ortho- and para-nitrophenol. ‘3 mononitrophloroglucinol. 126 fi- 1 - -- I 2‘ Disccussion During our efforts to discover enzymes with new functions for organic synthesis, our interests turned to elucidating the biosynthetic nitration mechanism. This can be achieved by studying the biosynthesis of molecules bearing arylnitro groups. At present, 3 mechanisms were proposed for the bacterial aromatic nitration reactions, NOS- mediated nitration, oxidation of arylamino groups and direct nitration. Oxidation of arylamino group to arylnitro group mechanism has been established and genes encoding for N-oxidation were identified in the biosynthesis of aureothin23 and pyrrolnitrin.21 The involvement of bacterial NOS in the synthesis of an arylnitro molecule, thaxtomin A, was confirmed by gene knockout and enzyme inhibition experiments.‘9 However, the so called direct nitration hypothesis was just based on the observation that feeding dual labeled nitrate in the culture medium of the dioxapyrrolomycin producer led to dual labeled nitro group in dioxapyrrolomycin.25 Our effort is then to determine which is the nitration mechanism involved in the biosynthesis of pyrrolomycin A. Is direct nitration the mechanism? Pyrrolomycin A shares a similar core structure and substitution pattern with dioxapyrrolomycin. Our research efforts to elucidate the bacterial nitration mechanism and use it for organic synthesis started with the direct nitration hypothesis. By feeding dual labeled K‘SN'803 in the culture medium of A. vitaminophilus, a dual labeled nitro group in pyrrolomycin A was highly expected as is the case of dioxapyrrolomycin. However, according to the mass spectrum, a l6—fragment loss accounts for the loss of O, while a 47-fragment cleavage can be explained with the loss of 15N02. After the NO2 was cleaved from the molecule, the fragment pattern is the same as the non-labeled 127 pyrrolomycin A obtained from both chemical and microbial synthesis. This clearly suggested that only 15N, not 180 was incorporated into the nitro group of pyrrolomycin A (Figure 70) and the N in the pyrrole ring was not labeled. According to this, the direct nitration mechanism is not likely involved in the biosynthesis of pyrrolomycin A. From the point view of chemistry, O exchange between an aromatic nitro group and H20 is not favored. However, final conclusion won’t be made before incubating pyrrolomycin A in Hz'gO to exclude the possible 180 washout during the incubation. The mass spectrum of pyrrolomycin B, which was isolated together with pyrrolomycin A, also indicated that ”N, not '80 was incorporated into the nitro group, not the N in the pyrrole ring. 5 16 18 N u H2 0 H pyrrolomycin A pyrrolomycin A Figure 68. Possible washout of incorporated 180 from the nitro group. The other two nitration mechanisms, arylamino oxidation and NOS-mediated nitration, provide a theoretical base for the observation of the single 15N incorporation into the nitro group from the dual labeled feeding experiments. In the arylanimo oxidation mechanism, K'SN 1803 could be converted to a nucleophilic amino group, which was oxidized to a nitro group (Figure 50). As a result, the N incorporated into the pyrrolomycin A was l"N labeled, while the O was acquired from the molecule Oz, and thus not 18O labeled. In the NOS-mediated nitration mechanism, in vivo metabolism of K"’N"’O3 leads to the synthesis of 15N labeled L—arginine, from which, NO was produced. Nitration resulting from NOS would incorporate 15N into the nitro group, not 180 because the O source in NO is derived from the molecular O2 (Figure 59). Except for the isotope labeling experiment, in vitro reactions with crude lysate 128 were set up to test the direct nitration mechanism. It is believed that a hybrid PKS and NRPS is responsible for the biosynthesis of pyoluteorin, and the electrophilic chlorination occurs on the pyrrolyl-2-acyl-PCP (Figure 47). The structural similarity between pyrrolomycn A and pyoluteorin invoked us to assume that the two molecules share the similar biosynthetic mechanism that the pyrrolyl-2—acyl-PCP serves as the substrates of electrophilic chlorination and nitration. Therefore, pyrrolyl-2-acyl-SNACs were synthesized as the mimic substrates of pyrrolyl-2-acyl-PCPs (Figure 53) and used to detect in vitro nitration activity. During our research, no in vitro activity was observed, which also suggested the non-involvement of the direct nitration mechanism. However, these experiments could also be misleading because of the following reasons. One is that SNAC thioesters do not always serve as PKS/NRPS substrates. And even if SNACs could be the substrates, the activity of PKS/NRPS with SNAC substrates could be much lower than the already low activity of PKS/NRPS with their native substrates. The third misleading reason is that, in the synthesis of pyrrolomycin A from the pyrrolyl-2-acyl- PCP, other enzymatic steps including hydration and decarboxylation are also involved. The slow turnover of any of these enzymes would affect the detection of the desired products. Post modification of the core structure released from the PKS/NRPS assembly line also occurrs in natural products synthesis. Substrates of the tailoring enzymes were believed not as specific as those of the PKS/NRPS modules. If the direct nitration occurs in the post modification steps, possible intermediates could be screened for the in vitro nitration activity. The negative results again, only suggested the non-evolvement of a direct nitration mechanism. Misleading reasons for these experiments including the slow 129 turnover of PKS/NRPS enzymes and the limited number of substrates screened. Evidences confirming the involvement of the direct nitration mechanism will then come from genetic approaches including cloning and expression gene for in vitro activity and gene knockout for in vivo activity. This depends on the identification of the gene cluster for the biosynthesis of pyrrolomycin A and is now uongoing in Dr. Parry’s lab in Rice university. Is NOS-mediated nitration the mechanism? According to the first isotope labeling experiments, NOS-mediated nitration is likely responsible for pyrrolomycin A synthesis. The second isotope labeling experiment was then employed to confirm this hypothesis with L—arginine-guanido—”N2 as the substrate for NO production. If the nitration mechanism is NOS-mediated, ”N will be incorporated into NO produced by NOS (Figure 59) and further incorporated into the nitro group of pyrrolomycin A. No incorporation of IN from L-arginine-guanido-‘SN2 was observed. The risk of the in vivo isotope labeling experiment includes the complexity of L-arginine metabolism pathways in microbes."9 In addition to the production of NO, arginine can be catabolized by arginase and end up into the urea cycle, which is the general pool of nitrogen source. At the same time, the inhibition of pyrrolomycin A production by A. vitaminophilus in the presence of NAME was not observed, either. NAME, which was a competitive inhibitor of mammalian NOS,’0 couldn’t affect thaxtomin A production at concentrations from 16 to 64 pM.'9" We are not sure whether the nitration is not mediated by NOS or NAME is not an inhibitor of bacterial NOSs. The genetic approach didn’t provide any clues of the involvement of NOS in the biosynthetic nitration of pyrrolomycin A synthesis. 130 Does NOS mediate bacterial nitration? Although not completely eliminated, the thaxtomin A production was dramatically decreased by NOS gene knockout in several Streptomyces bacteria.‘9b This suggested the existence of alternate nitrogen soureces for nitration. On the other hand, both expression of NOS and exogenous addition of NO sources recovered the thaxtomin A production by mutated Streptomyces strains.19 According to our own experiments, phenol and phloroglucinol were not nitrated by purified deiNOS, while the nitration L- Trp and L—Tyr are very obvious, although the yields are pretty low (Table 6). One interesting observation is the substrate selectivity during the NOS-mediated nitration reactions. It suggests that the nitration reaction most likely occurred after the substrates binds to the enzyme. And the binding site may be the cofactor H4B site because H4B inhibits the amino acid nitration in vitro.20 All the bacterial NO production was assayed by an indirect method with Griess reagents to measure the production of nitrite obtained by H202 oxidation of NO.18 No free NO was detected in the solution by the hemoglobin assay.18 A possible reason is NO binds to the enzyme. During the reaction process, transient intermediate Fe(II)O2 is formed to activate 02 for rodex reactions. An intermediate of Fe(IlI)NO was detected by rapid-scanning stopped-flow spectroscopy during the decay of Fe(II)OZ, which clearly suggested the production of NO by Bacillus subtilis.”" The two enzyme bound species, substrate and NO might approach each other for nitration reaction. It is also interesting to notice that no cofactors were necessary in both the NO production assay and the nitration assay with deiNOS (Figure 66, Table 6). In mammalian, all 3 isoforms of NOSs contain an oxygenase domain (NOSoxy), which 131 catalyzes the S-electron transfer oxidation reaction (Figure 59), and a reductase domain, which is responsible for electron donation.42 How the bacterial NOS produces NO without the electron donation domain remains unclear. One unusual tryptophanyl tRNA synthetase in D. radiodurans was believed to serve as an electron donor based on its copurification with NOS and its enhancement of NO productivity.51 Based on the genomic information, an electron donating protein, sulfite reductase flavoprotein (SiR- FP) was identified in Bacillus. subtilis and displays a domain organization (FMN, FAD 52 and NADPH domains) similar to that of mammalian NOSred' Zemojtel et a1 hypothesized that SiR—FP might be involved in the electron transfer for NOS although it also catalyzes the electron transfer from NADPH to reduce sulfite to sufide.52 However, in the present of H202, the tryptophanyl tRNA synthetase only increased the NOS activity, instead of creating the NOS activity.51 This suggests that H202 serves as the electron donor and is important for the bacterial NOS activity, and the tryptophanyl tRNA synthetase only enhances it. Both our nitration assay and literature nitration assay20 indicate, in the presence of H202, NOS activity was detected. However, this observation is not surprising. H202 could reduce heme iron (III) to iron (II) and assisted the mammalian oxydase domain (NOSOXy) alone to oxidize L-argine to L-citruline and NO.53 It also oxidize NO to NOZ', which can be detected by Griess reagents. The bacterial NOS-mediated nitration, therefore, is not completely non- enzymatic based on its substrate selectivity. The successful in situ enzymatic H202 generation for nitration makes the nitration reaction to be completely enzymatic. Although in vivo nitration used this method will have to deal with the toxicity of H202 towards microbes, we hope this provides some clues for studying the mechanism of 132 biosynthetic nitration. ‘ 308.0 25- 73.0 273.0 109.0 “ 2°‘5..° - I, 122'“ 13"0 l 2".0 . ‘ 35‘.“ 33 38!.0 111- 1 1151.0 ,1 252:0 300.0 ‘ 366.0 . ( l l 1 s- 1:, l 1 1‘ ‘ M l \ 231. o\' ll . 336. o l so too 1511 zoo zoo . also ' noo all 133 .P’I‘TflflSUI‘IaifiETIUZIS-56' er 2 . cq: - — : 7080 816 unmet Bplaz308 391586136 TIC:5938494 Flagsflmm. Pile Textmcan 50- 50 100! 3l3 -5.985 95- .5.635 90- ~ .‘5 . 335 85 -5 . 035 Figure 69. Mass spectra of dioxapyrrolomycin. top, copied from literature and bottom, obtained from that synthesized by Streptomyces sp. UpJohn UC11065. 134 10 0"- 95 90 85 80" '75 70 65 60 55 50 45 40 35 30 25 20 15 10 '8 37 40 '72 62 99 l l.il I 60 80 10 1:25 135 10 I: 95 90 85 80 75 '70 65 60 55 50 40 35 30 25 20 15 10 37 4O '72 62 60 80 99 100 H- 136 134 126 0'53: 1 a O 150 ”ii 1.6 O l..,...., 1 O 2 O 1.00 107 1'31 95 90 85 80 75 7O 65 60. 55 '8 50 45 40 72 35 30 25 20 15 37 62 99 10 S. 1314 125 150 165 o i ll .f g. '1 H! . 40 0 so 1 o' 1 o 10 10 1.0 20 Figure 70. Mass spetra of pyrrolomycin A synthesized by A. vitaminophilus (ATCC 31673). Pyrrolomycin isolated from isotope labeling experiments: top, KNO3; middle, K”NO3; bottom, K”N'803. 137 100111 95 90 85 80 '75 7O 65 60 55 50 45 40 35 30 25 20 15 10 s o! 59 62 (I 'l 87 l i' ‘1’“ 1‘} ll k: 10 p i Ill-i v I 0 147 138 274 308 339 1' I l; . 3 1o-' qu 9'.5 so as so 15 '10 65 60. g 55 ' so 45 40 35 30 25 308 20 147 233 ’l 87. 174 l 274 Y 356 l l H, l l 11| 2:? I ‘ l~ ‘, l ll1‘1 I "I'll 1;.11 :1 '{ ll: i1 1' ill .11 ' . 1 l7’! . 1' .: 1 ‘ 1'0 1 O O 139 10v 291 95 - 90 as so 75 70 65 60 55 274 357 50 l 45 4o 35 30 25 308 20 1‘9 174 238 340 l 15 73 , , , 111 . 10 5- .. l. 50 Hull: Ii ' 10 . ‘ 1r. _ . ' , lll' , . ., w' " _T ,,;:.l (:3 ‘1 ° 300 3 O Figure 71. Mass spetra of pyrrolomycin B synthesized by A. vitaminophilus (ATCC 31673). 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USA 2004, 101, 15881-15886. 52 Zemojtel, T.; Wade, R. C.; Dandekar, T. In Search of the Prototype of Nitric Oxide Synthase. FEBS Lett. 2003, 554, 1-5. 53 (a) Adhikari, 5.; Ray, 8.; Gachhui, R. Catalase Activity of Oxygenase Domain of Rat Neuronal Nitric Oxide Synthase. Evidence for Product Formation from L-Arginine. (b) Pufahl, R. A.; Wishnok, J. S.; Marietta, M. A. Hydrogen Peroxide-supported Oxidation of N’"-Hydroxy-L-arginine by Nitric Oxide Synthase. Biochemistry 1995, 34, 1930-1941. 145 CHAPTER FOUR EXPERIMENTAL General Chemistry All reactions sensitive to air and moisture were carried out in oven and/or flame dried glassware under positive argon pressure. Air or moisture sensitive reagents and solvents were transferred to reaction flasks fitted with rubber septa via syringes. Solvents were removed using either a Biichi rotary evaporator at water aspirator pressure or under high vacuum (0.5 mm Hg). Tetrahydrofuran and diethyl ether were distilled under nitrogen from sodium/benzophenone. Methylene chloride, benzene, triethylamine and pyridine were distilled over calcium hydride before use. Organic solutions of products were dried over NaZSO4. Most chemicals were purchased from Aldrich. Sodium salt of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid (TSP) was purchased from Lancaster Synthesis Inc. Distilled deionized water was used for all purposes. Charcoal (Darco® G-60 ~100 mesh) was used for decolorization at a final concentration of 0.1 g/mL. Chromatography. Silica gel 60 (40—63 am, E. Merck) was used for flash chromatography. Analytical thin-layer chromatography (TLC) utilized precoated plates of silica gel 60 F- 254 (0.25 mm, Whatman). TLC plates were visualized with UV or immersing in different stain solution such as phosphomolybdic acid stain (7% phosphomolybdic acid in ethanol) and potassium permanganate stain (1% KMnO4, 6.7% KZCO3 and 0.08% NaOH in H20). 146 HPLC chromatography was performed on an Agilent 1100 instrument installed with ChemStation acquisition software (Rev. A.08.03). Column used for all the HPLC purpose was a Zorbax SB-C18 reverse phase column (250 mm x 4.6 mm) and the detector was a VWD UV detector. Solvents were routinely filtered through 0.25-um membranes (Pall corporation) prior to use. Dowex 50 (H’) was purchased from Aldrich-Sigma, which was cleaned by treatment with bromine. An aqueous suspension of resin was adjusted to pH 14 by addition of solid KOH. Bromine was added to the solution until the suspension turned a golden yellow color. Additional bromine was added (1-2 mL) to obtain a saturated solution. The mixture was left to stand at room temperature overnight, and the Dowex 50 resin was collected by filtration and washed exhaustively with water followed by 6 N HCI. In an extreme situation that large amount of bromine was used to clean Dowex 50 resin, ethanol was firstly used to remove most of the bromine, followed by H20 and HCI washing. Ni-NTA resin was purchased from Qiagen. Spectroscopic Measurements. IH NMR and ‘3C NMR spectra were recorded on either a Varian VX-300 or a Varian VXR-SOO FT -NMR spectrometer. Chemical shifts for 1H NMR spectra are reported (in parts per million) relative to internal tetramethylsilane (Me4Si, 6 = 0.0 ppm) with CDCl3, d 6-acetone, d6-DMSO as solvent and to internal sodium 3- (trimethylsilyl)propionate-2,2,3,3-d4 (TSP, (5 0.0 ppm) with D20 as solvent. Chemical shifts for 13C NMR spectra are reported (in parts per million) relative to internal tetramethylsilane (Me4Si, 6 = 0.0 ppm) with CDCl3, d6-acetone, d6-DMSO as solvents I47 and to internal acetonitrile (CH3CN, (5 3.69 ppm) with DZO as 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). 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. 148 Bacterial Strains and Plasmids. Table 7. Selected bacterial strains and plasmids. Strain/ Plasmid Relevant Characteristics Source Strain 31210353) E. coli B F‘ dcm ampT hst(rB' mB') gal A Novagen (DE3) DHS“ F’ endAI hstl7(r'Km+K) supE44 thi-I recAI Inventrogen gyrA relAI ¢80lacZDM15 A(lacZYA-argF)U,69 Actinosparagum Pyrrolomycin A producer ATCC vitaminophilus KL3 AB2834 serA::aroB Lab QP1.1 AB2848 serA::araB Lab WN l AB2834 serA::araBaraZ, Laz::tktAaraZ Lab Actinosparagum Pyrrolomycin A producer ATCC vitaminophilus Streptomyces sp Dioxapyrrolomycin producer UpJohn UC11065 Plasmid pJF118EH ApR, Ptac lale lab PQE30 ApR, lale, T5 Qiagen PET15b ApR, lac/Q, T7 Inventrogen pMF63A ApR, araFFBR Lab pRC1.55B serA Lab pWN1.079A CmR, araFFBR, araY Lab pWN 1.200A tktAaraZ Lab pJY1.21 1A ApR, Ptac ppsA, serA, araFFBR, Pomp, lac/Q Lab pJY1.216A ApR, Pm ppsA, serA, araFFBR, Pamp, tktA, lac/Q Lab PWL1.284A pWN1.079A, serA Chapter 2 PWL1.290A PWL1.284A, Pam; Chapter 2 PWL2.46B PJY1.211A, tktAaraZ Chapter 2 PWL4.106 PET15b, deiNOS Chapter 3 PWL4.134 pJ F1 18EH, deiNOS Chapter 3 PWL4.135 pQE30, deiNOS Chapter 3 Storage of Bacterial Strains and Plasmids. All bacterial strains including Escherichia coli, Actinosporagium vitaminophilum, Streptomyces UpJohn UC11065, were stored at -78 °C in glycerol. 149 Plasmids were transformed into E. coli DHSa for long-term storage. Preparation of bacteria glycerol freeze samples started from introduction of a single colony of the desired strain picked from an agar plate into 5 mL medium. E. coli strains were cultured in LB medium containing appropriate amount of antibiotics at 37 °C with agitation for 12 h. Actinosporagium vitaminophilum (ATCC31673) and Streptomyces sp. UpJohn UC11065 were cultured in ISP2 medium at 28 °C with agitation for 120 h. Glycerol freeze samples were prepared by addition 0.75 mL of bacteria culture to 0.25 mL of sterile 80% (v/v) aqueous glycerol solution. The solutions were mixed, allowed to stand at room temperature for 2 h, and then stored at -7 8 °C. Culture Medium. Bacto’tryptone, Bacto yeast extract, Bacto polypeptone, malt extract, casamino acids, and agar were purchased from Difco. Meat extract and com distiller’s soluble was obtained from Sigma. Soybean soil was purchased from ICN Biomedicals Inc. Maltose syrup and pharmemedia were gifts from Strader-Ferris international and Traders Protein respectively. All culture solutions were prepared in distilled, deionized water. Mediums used for growing E. coli strains. LB medium1 (1 L): Bacto tryptone (10 g), Bacto yeast extract (5 g), and NaCl (10 g). M9 saltsl (1 L): NazHPO4 (6 g), KHZPO4 (3 g), NH4C| (l g), and NaCl (0.5 g). M9 minimal medium (1 L): D-glucose (10 g), MgSO4 (0.12 g), and thiamine hydrochloride (0.001 g) in 1 L of M9 salts. M9 medium (1 L) was supplemented where appropriate with L-phenylalanine (0.040 g), L-tyrosine (0.040 g), L-tryptophan (0.040 g), p-hydroxybenzoic acid (0.010 g), potassium p- aminobenzoate (0.010 g), and 2,3-dihydroxybenzoic acid (0.010 g). 150 Antibiotics were added where appropriate to the following final concentrations: ampicillin (Ap), 50 ug/mL; chloramphenicol (Cm), 20 ug/mL. Stock solution of ampicillin (Ap) was prepared in water and chloramphenicol solution was prepared in 95% ethanol. Aqueous stock solutions of isopropyl-B-D-thiogalactopyranoside (IPTG) were prepared at various concentrations. Solutions of LB medium, M9 inorganic salts, MgSO4, and D-glucose were autoclaved individually and then mixed. Solutions of aromatic amino acids, aromatic vitamins, L-serine, thiamine hydrochloride, antibiotics, and IPTG were sterilized through 0.22-um membranes. Other solid media were prepared by addition of Difco agar to a final concentration of 1.5% (w/v) to the liquid medium. E. coli fermentation medium (1 L) contained KZHPO4 (7.5 g), ammonium iron (III) citrate (0.3 g), citric acid monohydrate (2.1 g), L-phenylalanine (0.7 g), L-tyrosine (0.7 g), L- tryptophan (0.35 g), and concentrated H2304 (1.2 mL). Fermentation medium was adjusted to pH 7.0 by addition of concentrated NH4OH before autoclaving. The following supplements were added immediately prior to initiation of the fermentation: D- glucose, MgSO4 (0.24 g), p-hydroxybenzoic acid (0.010 g), potassium p-aminobenzoate (0.010 g), 2,3-dihydroxybenzoic acid (0.010 g), and trace minerals including (NH4)6(Mo7OZ4)-4HZO (0.0037 g), ZnSO4-7HZO (0.0029 g), H3BO3 (0.0247 g), CuSO4-5HZO (0.0025 g), and MnC12-4HZO (0.0158 g). IPTG stock solution was added as necessary to the indicated final concentration. Carbon source D-glucose feed solution, and MgSO4 (l M) solution were autoclaved separately. Glucose feed solution (650 g/L) was prepared by combining 300 g of glucose and 280 mL of H20. Solutions of aromatic vitamins, trace minerals, and IPTG were sterilized through 0.22-um membranes. Antifoam (Sigma 204) was added to the fermentation broth as needed. 151 Media used for growing A. vitaminajahilum (ATCC31673) and Streptomyces UC11065. ISPl medium (1 L): Bacto tryptone (5 g), Bacto yeast extract (5 g), pH 7.0. ISP2 medium (1 L): Bacto Malt extract (10 g), Bacto yeast extract (5 g) and D- glucose (4 g), pH 7.2. ISP4 medium (1 L): Glucose (10 g), starch (10 g), KZHPO4 (1 g), MgSO4 (1 g), NaCl (1 g), (NH4)ZSO4 (2 g), CaCO3 (2 g), FeSO4°7HzO (1 mg), MnC12'7HzO (1 mg), ZnSO4'7HZO (1 mg), pH 7.2. Nitrogen source (NH,,)ZSO4 was also replaced by KNO3 (3.3 g), L-arginine (2 g), L-proline (2 g). ISP7 medium (1 L): Glycerol (15 g), L-tyrosine (0.5 g), L-asparagine (1.0 g), KZHPO4 (0.5 g), MgSO4'7HzO (0.5 g), NaCl (0.5 g), FeSO4'7HzO (1 mg), MnC12'7HzO (1 mg), ZnSO4'7HzO (1 mg), pH 7.2. SYE medium (1 L): soluble starch (15.0 g), yeast extract (4.0 g), KHZPO4 (1.0 g), MgSO4°7HzO (0.5 g), pH 7.2. Seed medium (1 L): soluble starch (10 g), glucose (10 g), polypepton (5 g), meat extract (2 g), yeast extract (3 g), soybean meal (2 g), CaCO3 (2 g). pH 7.0. Production medium (1 L): maltose syrup (20 g), soybean meal (10 g), pharmemedia (5 g), corn distiller’s soluble (2.5 g), CaCO3 (1.0 g), supplemented by 100 yL stock solution of 50 mg FeSO4°7HzO, 5 mg NiCl2°6HzO, and 5 mg CoC12'6HzO. Solid ISP2 and SYE medium were prepared by addition of Difco agar to a final concentration of 2% (w/v) to the liquid medium. 152 General Fed-Batch Fermentor Conditions. Fermentations employed a 2.0 L working capacity B. Braun M2 culture vessel. Utilities were supplied by a B. Braun Biostat MD controlled by a DCU-3. Data acquisition utilized a Dell Optiplex Gs+ 5166M personal computer (PC) equipped with B. Braun MFCS/Win software (v1.1) or a Dell Optiplex GX200 personal computer (PC) equipped with B. Braun MFCS/Win software (v2.0). Temperature, pH, and carbon source feeding were controlled with PID control loops. pH was maintained at 7.0 by addition of concentrated NH4OH or 2 N H2804. Dissolved oxygen (D.O.) was measured using a Mettler-Toledo 12 mm sterilizable O2 sensor fitted with an Ingold A-type Oz permeable membrane. Inoculants were started by introduction of a single colony picked from an agar plate into 5 mL of M9 medium. Cultures were grown at 37 °C with agitation at 250 rpm until they were turbid and subsequently transferred to 100 mL of M9 medium. Cultures were grown at 37 °C and 250 rpm for an additional 10 h. The inoculant (OD600 = 1.0-3.0) was then transferred into the fermentation vessel and the batch fermentation was initiated (t = 0 h). Three staged methods were used to maintain D.O. concentrations at desired air saturation during the fermentations. 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 rate. With the impeller speed constant, 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. At constant impeller speed and constant airflow rate, the DO. concentration was finally maintained at the desired air saturation for the remainder of the fermentation by oxygen sensor-controlled carbon 153 source feeding. At the beginning of this stage, the DO. concentration fell below the desired air saturation due to residual initial carbon source in the medium. This lasted for approximately 10 min to 30 min before carbon source feeding commenced. The carbon source feed PID control parameters were set to 0.0 s (off) for the derivative control (1:0) and 999.9 3 (minimum control action) for the integral control (171). XP was set to 950% to achieve a KC of 0.1. Analysis of Fermentation Broth. Samples (5-10 mL) of fermentation broth were removed at the indicated timed intervals. Cell densities were determined by dilution of fermentation broth with water (1:100) followed by measurement of absorption at 600 nm (OD600). Dry cell weight of E. coli cells (g/L) was calculated using a conversion coefficient of 0.43 g/L/OD6OO. The remaining fermentation broth was centrifuged to obtain cell-free broth. For the biosynthesis of 3-dehydroshikimate, cis, cis-muconic acid, D—xylonate, and L-arabinonate, solute concentrations in the cell-free broth were quantified by 1H NMR. A portion (0.5-2.0 mL) of the cell-free broth was concentrated to dryness under reduced pressure, concentrated to dryness one additional time from D20, and then redissolved in DZO containing a known concentration of the sodium salt of 3- (trimethylsilyl)propionic—2,2,3,3—d4 acid (TSP, Lancaster Synthesis Inc.). Concentrations were determined by comparison of integrals corresponding to each compound with the integral corresponding to TSP (6 = 0.00 ppm) and were converted by response factors determined using authentic materials. Compounds were quantified using the following resonances: 3-deoxy-D-arabina-heptulosonic acid (DAH, 6 1.81, t, 1 H); 3- dehydroquinate (DHQ, 6 4.38, d, l H); 3-dehydroshikimate (DHS, 6 4.28, d, l H); 154 cis,cis-muconic acid (6 6.02, d, 2 H); catechol (6 6.90, m, 4 H); gallic acid (GA, 6 7.02, s, 2 H); D-xylonic acid (6 4.08, d, 1 H); L-arabinonic acid (6 4.24, dd, 1 H); and L-arabina- 1,4-lactone (6 4.64, d, l H). A standard concentration curve was determined for metabolites using solutions of authentic samples. Concentrations were calculated by application of the following response factor: 3-dehydroshikimate, 0.95; 3- dehydroquinate, 0.89; 3-deoxy-D-arabina-heptulosonic acid, 1.22; gallic acid, 1.36; cis, cis-muconic acid, 0.96; D-xylonic acid, 0.85; L-arabinonic acid, 0.88. For the biosynthesis of 1,2,4-butanetriol, the concentration 1,2,4-butanetriol in cell-free broth was quantified by GC analysis. A portion of the fermentation broth (0.5-1.0 mL) was concentrated to dryness under reduced pressure, and the residue was redissolved in pyridine (0.9 mL). To this pyridine solution, dodecane (0.1 mL) and bis(trimethylsilyl)trifluoroacetamide (BSTFA, 2 mL, 7.53 mmol) were sequentially added. Silyation of 1,2,4—butanetriol was carried out at room temperature with stirring for 10 h. Samples were then analyzed using gas chromatography. Genetic Manipulations GeneLaL Recombinant DNA manipulations generally followed procedures described by Sambrook.2 Restriction enzymes were purchased from Gibco BRL or New England Biolabs. T4 DNA ligase, large fragment of DNA polymerase I (Klenow fragment) and dNTP’s were purchased from Invitrogen. Calf intestinal alkaline phosphatase was purchased from New England Biolabs. Fast-Link DNA ligase was purchased from Epicentre Technologies. Agrose (electrophoresis grade) was purchased from Invitrogen. 155 Phenol was prepared by addition of 0.1% (w/v) 8-hydroxyquinoline into distilled phenol. Two extractions of phenol with an equal volume of 1 M Tris—HCI (pH 8.0) were followed by extraction with 0.1 M Tris-HCI (pH 8.0) until the pH of the aqueous layer was greater than 7.6. Phenol was stored under an equal volume of 0.1 M Tris-HCI (pH 8.0) at 4 °C. SEVAG was a mixture of chloroform and isoamyl alcohol (24:1, v/v). TE buffer contained 10 mM Tris-HCI (pH 8.0) and 1 mM NazEDTA (pH 8.0). Endostop solution (10X concentrated) contained 50% glycerol (v/v), 0.1 M NazEDTA (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, 1 mL of 10X Endostop was mixed with 0.12 mL of DNase-free RNase. DNase-free RNase was prepared by dissolving 10 mg RNase in 1 mL of 10 mM Tris-HCI (pH 7.5) and 15 mM NaCl. Following inactivation of the DNase activity by heating at 100 °C for 15 min, the solution was stored at —20 °C. PCR (Polmerase Chain Reaction). Regular PCR amplifications were carried out as described by Sambrook.2 Each reaction (0.1 mL) contained 10 mM KCl, 20 mM Tris- HCl (pH 8.8), 10 mM (NH4)2SO4, 2 mM MgSO,,, 0.1% Triton X-100, dATP (0.2 mM), dCT P (0.2 mM), dGTP (0.2 mM), dTTP (0.2 mM), template DNA (0.02 ug-l ug), 0.5 uM of each primer, and 2 units of Vent polymerase. For onsensus PCR, gene alignment was conducted using web-based tool, San Diego Supercomputer Center (SDSC) Biology Workbench (http://workbench.sdsc.edu) and degenerate primers were designed using web-based tool Consensus Degenerate Hybrid Oligonucleotide Primers (CODEHOP, http://lfioinformatics.weizmann.ac.il/blocks/codehop.html). Gene blast uses Integrated 156 Genomics bioinformation system (ERGO, http://ergo.integratedgenomics.com/ERGO). PCR was conducted as above. PCR was conducted with Taq polymerase and amplified DNA fragment was cloned and sequenced using Top 10 TOPO TA Cloning ®Kits Primers were synthesized by the Macromolecular Structure Facility at Michigan State University. Determination of DNA concentration. In order to determine the concentration of DNA, an aliquot (10 uL) of sample was diluted to 1 mL in TE and the absorbance at 260 nm was measured relative to TE. The DNA concentration was calculated based on the fact that the absorbance at 260 nm of a 50 ug mL-1 of plasmid DNA is 1.0. Large Scale Purification of Plasmid DNA. Routine purification of plasmid DNA on a large scale followed a modified alkaline lysis procedure described by Sambrook.2 A single colony of a strain containing the desired plasmid was inoculated into a 2 L Erlenmeyer flask containing LB (500 mL) and the appropriate antibiotics. Following incubation in a gyratory shaker (250 rpm) at 37 °C for 12-14 h, cells were harvested by centrifugation (4 000g, 5 min, 4 °C) and then resuspended in 10 mL of cold GETL solution [50 mM glucose, 20 mM Tris-HCI (pH 8.0), 10 mM NazEDTA (pH 8.0)] into which lysozyme (5 mg mL") had been added immediately prior to use. The suspension was kept at room temperature for 5 min. Addition of 20 mL of 1% sodium dodecyl sulfate (w/v) in 0.2 N NaOH was followed by gentle mixing and storage on ice for 15 min. A 15 mL aliquot of ice-cold solution containing 3 M KOAc (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 of the mixture resulted in formation of a white precipitate. Following storage on ice for 10 min, the sample was centrifuged (48 000g, 20 min, 4 °C) to remove 157 cellular debris. The resulting supernatant was transferred equally to two centrifuge tubes, and then mixed with isopropanol (0.6 volume) to precipitate DNA. After the samples were stored at room temperature for 15 min, the DNA was recovered by centrifugation (20 000g, 20 min, 4 oC). The DNA pellet was then rinsed with 70% ethanol and dried. The isolated DNA was dissolved in 3 mL TE and transferred to a 15 mL Corex tube. The solution was thoroughly mixed with 3 mL of cold 5 M LiCl, and then centrifuged (12 000g, 10 min, 4 °C) to remove high molecular weight RNA. The clear supernatant was transferred to a Corex tube, treated with an equal volume of isopropanol (6 mL) and was gently mixed. The precipitated DNA was collected by centrifugation (12 000g, 10 min, 4 °C), rinsed with 70% ethanol and dried. After redissolving the DNA pellet in 0.5 mL of TE containing DNase-free RNase (20 ug/mL), the solution was transferred to a 1.5 mL microcentrifuge tube and stored at room temperature for 30 min. Following addition of 0.5 mL of 1.6 M NaCl containing 13% PEG-8000 (w/v), the solution was mixed and centrifuged (microcentrifuge, 5 min, 4 °C) to recover the precipitated DNA. The supernatant was discarded and the pellet was dissolved in 0.4 mL of TE. The sample was sequentially extracted with phenol (0.4 mL), phenol and SEVAG (0.4 mL each) and finally SEVAG (0.4 mL). 10 M NH4OAc (10 mL) 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 room temperature for 5 min and then centrifuged (microcentrifuge, 5 min, 4 °C). The resulting DNA pellet was rinsed with 70% ethanol, dried, and dissolved in 0.2-0.5 mL of TE. For the purpose of DNA sequencing, purification of plasmid DNA employed a Qiagen Plasmid Maxi Kit purchased from Qiagen. The manufacture’s procedure was followed 158 during the experiment, and the resulting DNA was dissolved in sterile water to facilitate DNA sequencing. Small Scale Purification of Plasmid DNA. A single colony of a strain containing the desired plasmid was inoculated into LB (5 mL) containing the appropriate antibiotics. Following incubation at 37 °C with agitation (250 rpm) overnight, cells were harvested from 3 mL of culture in a 1.5 mL microcentrifuge tube by centrifugation. The cell pellet was resuspended in 0.1 mL of cold GETL solution into which lysozyme (5 mg mL“) was added immediately before use. The sample was kept on ice for 10 min. Addition of 0.2 mL of 1% sodium dodecyl sulfate (w/v) in 0.2 N NaOH was followed by gentle mixing and storage on ice for 5-10 min. To the resulting sample was added 0.15 mL of cold 3 M KOAc solution. The mixture was shaken vigorously and then stored on ice for 5 min. Following removal of precipitated cellular debris by centrifugation (microcentrifuge, 20 min, 4 °C), the supernatant was transferred to a fresh microcentrifuge tube and extracted with phenol and SEVAG (0.2 mL each). The aqueous DNA solution was transferred to a fresh microfuge tube and mixed well with 1 mL of 95% ethanol. After storage at room temperature for 5 min, DNA was precipitated by centrifugation (15 min, room temperature). The DNA pellet was rinsed with 70% ethanol, dried, and redissolved in 50-100 uL of TE. DNA isolated using this method was used for restriction enzyme analysis. Restriction enzyme digestion of DNA. Restriction enzyme digests were performed in buffers provided by the enzyme suppliers. A typical restriction enzyme digest contained approximately 1 ug of DNA (in 10 uL of TE), 2 uL of restriction enzyme buffer (10X concentration), 1 uL of bovine serum albumin (BSA) (2 mg mL“), 1 159 [1L of restriction enzyme and 6 uL TE. After incubation at 37 °C for 1-2 h, the sample was mixed with 2 ML of Endostop (10 X concentrated) and analyzed by agarose gel electrophoresis. When DNA was required for cloning experiments, restriction digestion was terminated by addition of l uL of 0.5 M NazEDTA (pH 8.0). Following extraction with phenol and SEVAG (0.1 mL each), DNA was thoroughly mixed with 0.1 volume of 3 M NaOAc (pH 5.2) and precipitated by addition of 3 volumes of 95% ethanol. After storage at -78 °C for 3 h, precipitated DNA was recovered by centrifugation (15 min, 4 °C), rinsed with 0.1 mL of 70% ethanol and centrifuged (15 min, 4 °C). DNA was dried and redissolved in TE. Alternatively DNA was isolated from the restriction digestion mixture utilizing Zymoclean DNA Clean and Concentrate Kit (Zymo Research) following the protocol recommended by the manufacturer. 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. DNA fragments smaller than 1 kb were resolved in 2% agarose. Addition of ethidium bromide (0.5 ug mL") to the agarose allowed for visualization of DNA fragments under ultraviolet exposure. Two sets of DNA size markers were employed to estimate the size of DNA fragments between 0.5 kb and 23 kb: it DNA digested with Hindlll resulted in bands of 23.1 kb, 9.4 kb, 6.6 kb, 4.4 kb, 2.3 kb, 2.0 kb, and 0.6 kb, and )\. DNA digested with EcoRI and Hindlll resulted in bands of 21.2 kb, 5.] kb, 5.0 kb, 4.3 kb, 3.5 kb, 2.0 kb, 1.9 kb, 1.6 kb, 1.4 kb, 0.9 kb, 0.8 kb, and 0.6 kb. For DNA fragments smaller than 1 kb, a 100 bp DNA ladder purchased from Invitrogen was utilized as a DNA size marker. 160 Isolation of DNA from Agarose. The band of agarose containing the DNA of interest was excised from argrose gel using a razor blade under long wavelength UV light (365 nm) to avoid damages to DNA. The excised agarose was transferred into a 1.5 mL microfuge tube. Two methods were used to isolate the DNA from the agarose. The first method involved chopping the agarose plug thoroughly with a razor blade and transfering it to a 0.5 mL microfuge tube, which was packed tightly with glass wool and had an 18 gauge hole at the bottom. While centrifuging for 5 min using a Beckman microfuge, the aqueous solution was collected in a 1.5 mL microfuge tube. The DNA was precipitated from the aqueous soltion by addition of 3 M NaOAc (pH 5.2, 0.1 vol) and 95% ethanol (2-3 vol) as previously described. DNA was subsequently redissolved in TE. In a second method, the DNA was isolated from the agarose plug using Zymoclean Gel DNA Recovery Kit (Zymo Research) by following manufacturer recommended procedure. Treatment of Vector DNA with Calf Intestinal Alkaline Phosphatase. Plasmids digested with a single restriction enzyme were dephosphorylated to prevent self-ligation. Digested vector DNA was dissolved in TE or sterile H20 (88 uL). To this sample 10 [LL of dephosphorylation buffer (10 X concentration) and 2 uL of calf intestinal alkaline phosphatase (2 units) were added. The reaction was incubated at 37 °C for 1 h. The phosphatase was inactivated by addition of 1 ML 0.5 M EDTA (pH 8.0) followed by heat treatment (65 °C, 20 min). After sequential extraction with phenol and SEVAG (100 uL each) to remove protein, the DNA was precipitated as previously described and redissolved in TE. Treatment of DNA with Klenow Fragmeat. DNA fragments with recessed 3' termini were modified to blunt-ended fragments by treatment with the Klenow fragment 161 of E. coli DNA polymerase I. Since the Klenow fragment works well in common restriction enzyme buffers, there was no need to purify the DNA after restriction digestion and prior to filling recessed 3' termini. To a 20 uL of digested DNA sample (0.8-2 pg), 2 pL of a solution containing 25 mM of each of the four dNT P’s and 2.5 units of Klenow fragment were added. After thorough mixing, the reaction was allowed to stand at room temperature for 20 min. The Klenow reaction was quenched either by sequential extraction with equal volume of phenol and SEVAG or by addition of Endostop (10 X concentrated, 2 uL). DNA isolation from the resulting aqueous solution employed either DNA precipitation or agarose gel electrophoresis as previously described. Ligation of DNA. DNA ligations using T4 DNA ligase were designed to result in a molar ratio of 1:3 between vectors and insert DNAs. A typical ligation reaction contained 0.1 ug vector DNA, 0.05 to 0.2 ug insert DNA, 2 uL of T4 ligation buffer (5 X concentration), 1 uL of T4 DNA ligase (2 units), and TE to a final volume of 10 uL. The reaction was carried out at 16 °C for at least 4 h. In an alternative method, the Fast-link DNA Ligation Kit (Epicentre Technologies) was employed according to the procedures recommended by the manufacturer. Ligation mixture was used to transform chemically competent cells without purification. Inorganic salts and protein was removed from the ligation reactions using Zymoclean DNA Clean and Concentrate Kit prior to transforming electrocompetent cells. Preparzflm and Transformation of Competent Cells. Chemically competent and electrocompetent cells were prepared using procedures modified from Sambrook.2 Preparation of chemically competent cells started with introduction of a single colony I62 into 5 mL LB containing appropriate antibiotics. Following incubation at 37 °C with agitation overnight, 1 mL of the culture was transferred to a 500 mL Erlenmeyer flask containing 100 mL LB and appropriate antibiotics. The cells were cultured in a gyratory shaker (250 rpm, 37 °C) until they reached the mid-log phase of growth (OD600 = 0.4- 0.6). The culture was transferred to a centrifuge bottle that was previously sterilized with bleach and rinsed with sterile water. The cells were harvested by centrifugation (4 000g, 5 min, 4 °C) and the supernatant was discarded. All manipulations were carried out on ice during the remaining part of the procedure. Cells were resuspended in 100 mL of ice- cold 0.9% NaCl (w/v), harvested by centrifugation, and resuspended in 50 mL of ice-cold 100 mM CaClz. The suspension was stored on ice for a minimum of 30 min and centrifuged (4 000g, 5 min, 4 °C). The resulting cell pellet was resuspended in 4 mL of ice-cold 100 mM CaCl2 (v/v) containing 15% glycerol (v/v). Aliquots (0.25 mL) were dispensed into ice-cold 1.5 mL sterile microfuge tubes and immediately frozen in liquid nitrogen. Competent cells were stored at -78 °C without significant loss of transformation efficiency over a period of six months. Prior to transformation, frozen chemically competent cells were thawed on ice for about 5 min. An aliquot of plasmid (1 to 10 uL) or DNA ligation mixture was added to thawed competent cells (0.1 mL). After gentle mixing, the solution was kept on ice for 30 min. The cells were then heat shocked at 42 °C for 2 min and subsequently stored on ice for 1 min. LB (0.5 mL) was added to the cells, and the sample was incubated at 37 °C for 1 h without agitation. Cells were harvested using microcentrifugation (30 s), resuspended in 0.1 mL of LB and plated onto an LB plate containing the appropriate antibiotics. If the transformation was to be plated onto minimal medium plates, the cells 163 W6 531 in In were washed once with an aliquot of M9 salts (0.5 mL). After resuspension in fresh M9 salts (0.1 mL), the cells were spread onto the plates. Competent cells that were not transformed with any DNA, were subjected to the same transformation procedure. Such treated cells were plated onto LB plates to check the viability of the competent cells, and were also plated onto selection medium to verify the absence of contaminations. Preparation of electrocompetent cells followed the same procedure as described above for cell growth and harvest. Harvested cells were then washed with two portions (250 mL each) of ice-cold sterile double distilled water. After the second treatment with water, cells were collected by centrifugation (4 000g, 5 min, 4 °C) and resuspended in 50 mL of aqueous 10% glycerol (v/v) solution. The cell suspension was centrifuged (4 000g, 5 min, 4 °C). The resulting cell pellet was gently resuspended in 5 mL of ice-cold aqueous 10% glycerol. Aliquots (0.25 mL) of cells were dispensed into ice-cold sterile microfuge tubes, frozen in liquid nitrogen, and stored at —78 °C. Electroporation was performed in Bio-Rad Gene Pulser cuvettes with an electrode gap of 0.2 cm. The cuvettes were chilled on ice for 5 min prior to use. Plasmid DNA (dissolved in sterile water, 1-5 uL) or purified DNA ligation reaction was mixed with 0.1 mL of electrocompetent cells. After storage on ice for 5 min, the solution was transferred to a chilled cuvette. Moisture on the outer surface of the cuvette was removed before it was placed in the sample chamber of Bio-Rad Gene Pulser. The instrument was set at 2.5 kvolts, 25 uF, and 200 Ohms. A single pulse was applied to the sample which typically resulted in a time constant of 4—5 ms. The cuvette was removed, and 0.5 mL of LB was added into it. Contents of the cuvette were transferred to a 1.5 mL microfuge tube, incubated at 37 °C for 1 h, and plated on the appropriate selective medium. 164 Enzyme Assays Ogre—r211, Cells were collected by centrifugation at 4 000g and 4 °C for 5 min. Harvested cells were resuspended in the appropriate buffer and subsequently disrupted by two passages through a French press (16,000 psi, SLM Aminco). Cellular debris was removed by centrifugation (48 000g, 20 min, 4 °C). Protein concentrations were determined using the Bradford dye-binding method.3 A standard curve was prepared using bovine serum albumin. Protein assay solution was purchased from Bio-Rad. Protein SDS-PAGE Analysis. Protein SDS-PAGE analysis followed the procedure described by Harris.4 Preparation of a 10% separating gel started from mixing 3.33 mL of 30% (w/v) aqueous acrylamide stock solution containing N,N’-methylene- bisacrylamide (0.8% (w/v)), 2.5 mL of 1.5 M Tris-HCI (pH 8.8), and 4 mL of distilled deionized water. After degassing the solution using a water aspirator for 30 min, 0.1 mL of 10% (w/v) aqueous ammonium persulfate solution, 0.1 mL 10% (w/v) aqueous SDS solution, and 0.005 mL of N, N, N’, N’-tetramethylethylenediamine (TEMED) were added. The mixture was mixed thoroughly and poured into a 0.1 cm-width gel cassette to about 1.5 cm below the top of the gel cassette. t-Amyl alcohol was overlaid on 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 30% acrylamide stock solution containing N,N’-methylene- bisacrylamide (0.8% (w/v)), 2.5 mL Tris-HCI solution (0.5 M, pH 6.8), and 5.55 mL of distilled deionized water. After degassing for 30 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 thoroughly. 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 165 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 glycine (192 mM), Tris base (25 mM), and 0.1% SDS (w/v). Following dilution with Laemmli sample buffer (10 uL, Sigma S-3401) consisting of 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and Tris-HCI (125 mM, pH 6.8), each protein sample (10 uL) was heated at 100 °C for 10 min. Samples and markers (MW-SDS-200, Sigma) were then loaded into the sample wells and the gel was run under constant current at 30 mA until the blue tracking dye (bromophenol blue) reached the interface of stacking gel and separating gel. The protein gel was then run at a higher current (50 mA). When the blue tracking dye reaches the bottom of the gel, electrophoresis was terminated. The protein gel was subsequently removed from the cassette and submerged in 10% (w/v) aqueous trichloroacetic acid solution with constant shaking for 30 min. The protein gel was then transferred into a solution containing 0.1% (w/v) Comassie Brilliant Blue R, 45% (v/v) MeOH, 10% (v/v) HOAC in H20 and stained with constant shaking for 4 h. Destaining of the protein gel was carried out in a solution containing 45% (v/v) MeOH, 10% (v/v) HOAC in H20 for 2-3 h. For long-term storage, SDS-PAGE gels were sealed in plastic bags containing 10% glycerol. 166 Chapter Two Plasmid construction. pWLl.290A. This 7.7-kb plasmid is a pSU18-derived plasmid that encodes ara Y, PamF, araFFBR, serA, and C m”. A 1.6-kb fragment encoding 3-phosphoglycerate dehygenase (serA) was obtained by digestion of pRC 1.55B with Smal and was inserted into the Smal site of pWN1.079A to afford pWLl.284A. A 0.15-kb sequence encoding the three operator binding sites associated with the araFFBR gene and designated Pamp was amplified from pMF63A5 using the following primers: 5’- GCTCTAGAGAATTCAAAGGGAGTGTA and 5’- GCTCTAGACCTCAGCGAGGATGACGT. Localization of the P F fragment of 070 DNA into the Xbal site of pWLl.284A afforded pWLl.290A. pWL2.46B. Plasmid pWL2.46B is a pJF118EH-derived plasmid that encodes lale, PmC, ppsA, serA, Ramp, araFmR, tktA, araZ and ApR. Plasmid pJYl.211A was digested with Hindlll and treated with Klenow fragment. Partial digestion of pWNl.200A with EcaRI afforded a 4.5-kb fragment of DNA cassette tktAaraZ. After treatment with Klenow fragment, the tktAaraZ cassette was inserted into the Hindlll site of pJY 1.21 1A to afford the 15.5 kb plasmid pWL2.46B. Microbial synthesis of catechol. E. coli WNl/PWL1.290 was cultured under glucose rich condition as described above with the addition of 0.5 mL IPTG every 6 h started at 18 h (2 h after the phase change) to 30 h. In this condition, catechol was synthesized in a yield of 5.9% (mol/mol) with a titer 4.2 g/L. Fermentation broth (1000 mL each) was centrifuged at 4 °C (14000 g, 20 min) to remove cells, and the resulting culture supernatant was further acidified to 167 pH 2.5 with concentrated H2804 (3~5 mL) to allow the precipitation of protein. The precipitated protein was then removed by another centrifugation at 4 °C (14000g, 20 min). The resulting fermentation broth containing catechol was then extracted with EtOAc (3 x 600 mL). The organic layers were combined, washed with saturated NaCl solution, then dried over NazSO4 and concentrated under vacuum to afford a brown residue. Kugelrohr distillation of the residue under reduced pressure afforded catechol as white crystals with a 75% (mol/mol) recovery yield. Microbial synthesis of protocatechuic acid. E. coli KL3/pWL2.46B was cultured under glucose rich condition as described above with the addition of 0.5 mL IPTG every 6 h started at 24 h (2 h after the phase change) to 42 h. In this condition, protocatechuic acid was accumulated at a titer of 41 g/L in 26% (mol/mol) yield. Fermentation broth (1000 mL each) was centrifuged at 4 °C (14000 g, 20 min) to remove cells, and the resulting culture supernatant was further acidified to pH 2.5 with concentrated HZSO4 (3-5 mL) to allow the precipitation of protein. The precipitated protein was then removed by another centrifugation at 4 °C (14000g, 20 min). Protocatechuic acid was isolated from this broth simply by hand extraction with ethyl acetate (3 x 600 mL). The extract was dried over anhydrous NaZSO4 and solvent was removed under reduced pressure to afford protocatechuic acid as brown powder in a 100% (mol/mol) recovery yield. Microbial synthesis of catechol and PCA with in situ fermentation. Selection of resin for extractive fermentation. The anion-exchange resin AG 1- X8 (chloride form, 50-100 mesh) was obtained from Bio-Rad. Dowex 1X4 (chloride form, 50-100 mesh), Dowex 1X8 (chloride form, 50-100 mesh) and Amberlite IRA-400 168 (chloride form, 20-50 mesh) were obtained from Supelco. All the anion-exchange resins were converted to phosphate form by elution with 10 bed volumes of l M KHZPO4 prior to use. The polymeric adsorbent resins Sepabeads SP850 (20-60 mesh), Amberlite XAD- 2, XAD-4, XAD-16, XAD-16HP, XAD-1180 (20-60 mesh), Diaion SP207 (20-60 mesh), Diaion HP2088 (75-150 am), Diaion HP2MG (25-50 mesh), and MCI GEL CHP20P (75-150 ,um) were obtained from Supelco. The anion-exchange resins were used for adsorption of both protocatechuic acid and catechol while the polymeric hydrophobic resins were used only for catechol adsorption. To select out the best anion-exchange resin for adsorption of protocatechuic acid and catechol, procatechuic acid (3.1 g, 20 mmol) and catechol (1.1 g, 10 mmol) were dissolved in 200 mL distilled, deionized water to make a solution containing 15.5 g/L protocatechuic acid and 5.5 g/L catechol. The resulted solution was equally divided into four 250 mL flasks with each flask containing 50 mL solution. Ten grams (wet weight) of AG 1-X8, Dowex 1X4, Dowex 1X8, and IRA-400 resin were added separately to the four flask and incubated with shaking at 250 rpm, 28 °C for 4 h. To select out the best adsorbent resin for adsorption of catechol, catechol (4.5 g, 41 mmol) was dissolved in 500 mL distilled, deionized water. The resulted solution was equally divided into ten 250 mL flasks with each flask containing 50 mL solution. Five grams of Sepabeads SP850, Amberlite XAD-2, XAD-4, XAD-16, XAD-16HP, XAD-1180, Diaion SP207, HP2088, HP2MG, and MCI GEL CHP20P were added separately to the ten flasks and incubated with shaking at 250 rpm, 28 °C for 4 h. Adsorption efficiency of resin was determined by checking the decrease in concentrations of protocatechuic acid and/or catechol in the solution after addition of resin. All the resins used for extractive fermentation were 169 sterilized prior to use by elution with 2 bed volumes of ethanol followed by elution with 5 bed volumes of autoclaved distilled, deionized water. Unless specified, AG 1-X8 was used for extractive protocatechuic acid fermentation and SP850 for extractive catechol fermentation in all reported results. In situ fermention of catechtLand protocatechuic acid. All extractive fermentation experiments were performed in a 2-L fermentor (B.Braun) with 1-1.5 L working volume and an external extraction cycle driven by a peristaltic pump after a column extraction unit (Figure 5). Generally, the extraction was initiated at the beginning of the third phase of dissolved oxygen control (i. e. 12-18 h). For extractive fermentation of protocatechuic acid, four Bio-Rad Econo columns (O25x300 mm) packed with 120 mL (bed volume) AG l-X8 resin for each were prepared and used for in situ extraction at 18-30 h, 30-36 h, 36-42 h, 42-48 h, respectively. The circulation flow rate for the extraction of protocatechuic acid was 5-7 mL/min. For the extractive catechol fermentation, three Bio-Rad Econo columns (Q25x200 mm) packed with 80 mL (bed volume) Sepabeads SP850 resin for each were prepared and used for in situ extraction at 12-24 h, 24-30 h, 30-36 h, respectively. The circulation flow rate for catechol extraction was 10-12 mL/min. All the columns were operated in a fluidized-bed mode during the in situ extraction process. The columns after using for protocatechuic acid or catechol extraction were further disposed for product recovery and regeneration of resin in the following process. To recover protocatechuic acid adsorbed on the AGl-X8 resin, the column was rinsed with 5 bed volumes of distilled, deionized water to remove the residual cells, followed by 15 bed volumes of acidic ethanol (1 M trifluoroacetic acid in 95% ethanol) 170 to elute protocatechuic acid. The eluted solution was collected and used for analysis and purification of protocatechuic acid. The resin was regenerated by further elution with ten bed volumes of KHZPO4 (1M) and washed with 5 bed volumes of distilled, deionized water. The regenerated AG 1-X8 resin could be reused for extractive fermentation by sterilization with 2 bed volumes of ethanol and washed with 5 bed volumes of autoclaved distilled, deionized water, respectively. By elution with 10 bed volumes of distilled, deionized water, almost all cells and more than 60% of catechol were eluted off from the Sepabeads SP850 column. The residual catechol on the resin was recovered by a further elution with 10 bed volumes of ethanol. The resin could be reused for extractive catechol fermentation after a final rinse with 5 bed volumes of autoclaved distilled, deionized water. Microbial synthesis of 3-dehydroshikimic acid. E. coli KL3/pJYl.216A was cultured under glucose rich condition as described above with the addition of 0.5 mL IPTG every 6 h started at 18 h (2 h after the phase change) to 36 h. In this condition, 3-dehydroshikimic acid was synthesized in a yield of 36% (mol/mol) with a titer 65 g/L. Fermentation broth (1000 mL each) was centrifuged at 4 °C (14000 g, 20 min) to remove cells, and the resulting culture supernatant was further acidified to pH 2.5 with concentrated H2504 (3-5 mL) to allow the precipitation of protein. The precipitated protein was then removed by another centrifugation at 4 °C (14000g, 20 min). And 3-dehydroshikimic acid was isolated by continuous liquid-liquid extraction from the resulting cell-free and protein-free fermentation broth. A 1.5 L continuous liquid-liquid extraction apparatus was loaded with 500 mL fermentation broth and filled with EtOAC. The mixture was then stirred at a rate to create a translucent 171 colloidal suspension of ethyl acetate in the extraction cylinder. The organic solution containing 3-dehydroquinic acid was replaced with fresh ethyl acetate (400 mL) every 12 h. A combined total of 1.2 L ethyl acetate part was dried over NazSO4 and filtered through 20-30 g of Darco G-60 (100 mesh) activated charcoal by a fritted glass funnel. The filtrate was concentrated under vacuum until the first crystal of 3-dehydroquinic acid came out. This DHS saturated solution was then kept at 4 °C for 4 h to allow the complete crystallization of DHS, which was then collected by filtration and pumped overnight with a 80% (mol/mol) recovery yield. Microbial synthesis of 3-dehydroquinic acid. E. coli QPl. l/pJY1.216A was cultured under glucose rich condition as described above with the addition of 0.5 mL IPT G every 6 h started at 18h (2 h after the phase change) to 36 h. In this condition, 3-dehydroquinic acid was synthesized in a yield of 28% (mol/mol) with a titer 58 g/L. Fermentation broth (1000 mL each) was centrifuged at 4 °C (14000 g, 20 min) to remove cells, and the resulting culture supernatant was further acidified to pH 2.5 with concentrated HZSO4 (3~5 mL) to allow the precipitation of protein. The precipitated protein was then removed by another centrifugation at 4 °C (14000g, 20 min). Continuous extraction of cell-free and protein-free fermentation broth containing 3-dehydroquinic acid was conducted as follows. The cell—free and protein- free broth was first hand extracted with ethyl acetate (1 x 100 mL), and then mixed with 110 mL of ethanol. The mixture was loaded into a continuous liquid-liquid extraction apparatus, which was filled with EtOAc. The mixture was then stirred in at a rate to create a translucent colloidal suspension of ethyl acetate in the extraction cylinder. The organic solution containing 3-dehydroquinic acid was replaced with fresh ethyl acetate 172 (400 mL) and 110 mL fresh ethanol was added to the extraction cylinder every 24 h. A combined total of 1.2 L EtOAc solution was collected and dried over NaZSO4, followed by filtration through 20~30 g of Darco G-60 (100 mesh) activated charcoal with a fritted glass funnel. The filtrate was concentrated under vacuum and the residue was redissolved in 50 mL water, which was filtered through 5~8 g of Darco G-60 (100 mesh) activated charcoal. After removing water by lyophilization, 3-dehydroquinic acid was obtained as an off-white powder with a 64% (mol/mol) recovery yield. Chemical synthesis of protocatechuic acid. A 40g/L 3-dehydroshikimic acid solution was prepared by dissolving 2 g of 3- dehydroshikimic acid in 50 mL synthetic fermentation broth and was adjusted to required pH 2.5 or pH 7.0 with concentrated HZSO4 or NH3'HZO. The 3-dehydroshikimic acid solution at pH 2.5 was also eluted through a 10 g Dowex-50 (H+ form). The three different solutions were then heated to reflux under N2 atmosphere. Samples were taken out to monitor the production of protocatechui acid by NMR Spectrometer every 3 h (pH 2.5) or 0.5 h (pH 7.0). Reactions were stopped when either 3-dehydroshikimic acid was used up or the yield of protocatechuic acid didn’t increase any more. The cell-free and protein-free fermentation broth (1000 mL) obtained as described above containing 3-dehydroquinic acid or 3-dehydroshikimic acid was eluted through 200 g Dowex-50 (H+ form) and then heated to reflux under N2 atmosphere for 24 h. The product, protocatechuic acid, was extracted with ethyl acetate (3 x 600 mL). The combined organic part was dried over NaZSO4 and solvent was removed under reduced pressure to afford protocatechuic acid as brown powder. 173 Chemical synthesis of catechol. Conversion of DHS. DHQ and PCA to catechol in mr critical water. Aqueous solutions of 3-dehydroquinic acid, 3-dehydroshikimic acid or protocatechuic acid were heated to 190 °C, 230 °C, 250 °C, 270 °C, 290 °C, 310°C, 330 °C, 350 °C and 374 °C in a Parr (Model No. 4742) hi gh-pressure, stainless steel reaction vessel with working volume of 21 mL. A sand bath was used to heat the reactor with a steady heating rate provided by heating mantle controlled by a TEMP-O-Trol thermo-control unit (Model TOT- VOVC). In temperature optimization experiments, 3-dehydroquinic acid and 3- dehydroshikimic acid were isolated from fermentation broth by continuous liquid-liquid extraction, and protocatechuic acid was purchased from Aldrich. Distilled, deionized water was used for all reactions. Ethyl acetate was used to extract organic compounds and the resulting solutions were dried over anhydrous NazSO4. A certain amount of 3-dehydroquinic acid (2.11 g, 11 mmol), 3-dehydroshikimic acid (1.89 g, 11 mmol) or protocatechuic acid (1.69 g, 11mmol) was dissolved in 6 mL of water, which was degassed by a subsurface feed of Ar for 15 min followed by a subsurface feed of CO2 for another 15 min. After flushing the headspace with Ar, the high-pressure reaction vessel was sealed according to the manufacture’s specifications and submerged in a sand bath. An average heating rate of 1.5 oC/min was maintained until the desired final temperature was reached. After the final temperature was maintained (+/- 4 °C) for 30 min, the vessel was removed from the sand bath and cooled to room temperature by placing under a cooling fan. The reaction mixture was then extracted with Ethyl acetate (5 x 20 mL). The vessel interior was also thoroughly rinsed with ethyl acetate. The organic layers were combined, washed with saturated NaCl 174 solution, then dried over NaZSO4 and concentrated in vacuum to give a brown residue. Kugelrohr distillation of the residue under reduced pressure afforded catechol as white crystals. Conversion of DHS to catechol in strong acid conditions. A portion of 0.5 g 3- dehydroshikimic acid was refluxed in 5 mL 12 M HCI or acetic acid (containing 1 M H2504) for 24 h. After cooling to room temperature, the reaction solutions were then poured into 50 mL ice H20 and extracted with EtOAc (3 x 30 mL). The extract was rinsed with H20 (2 x 100 mL), and concentrated to dry to afford protocatechuic acid in yields of 90% (mol/mol) and 99% (mol/mol) respectively. 3-Dehydoshikimic acid (0.5 g) was also heated to reflux in 5 mL 8 M HZSO4 for 24 h and followed the same working up procedure. Catechol was detected as product in 14% (mol/mol) yield after Kugelrohr distillation. 175 Chapter Three Purification of Actinosporangium vitaminophilus (ATCC 31673). A. vitaminophilum (ATCC31673) was first grow on ISP2 solid medium and 20 single colonies were further streaked out on ISP2 medium to obtain single colonies the second generation, which were selected to run all the tests. Bacillus subtilis (ATCC 6051, single colonies obtained by growing on ATCC medium 265) and wild type E. coli W31 10 (single colonies obtained by growing on LB medium) were selected as control. Hydrolysis of starch.6 Single colonies of A. vitaminophilum, B. subtilis and E. coli W3110 were replicated on an ISP4 based solid medium with starch as the only carbon source. After incubating at 28 °C for 3 days, the plate was exposed to iodine and the hydrolysis of starch was observed after 10 min. The results of A. vitaminophilum and B. subtilis were positive, while the results of E. coli were negative. Gelatin liquefaction. Single colonies of A. vitaminophilum, B. subtilis and E. coli W31 10 were stab inoculated in culture tubes in 5 mL ISP2 based medium supplemented with 120 g/L gelatin. After incubated at 28 °C for two weeks, the tubes were kept at 4 °C for 10 min to allow the solidification of unliquefied gelitan. The results of A. vitaminophilum, B. subtilis were positive, while the results of E. coli were negative. Reduction of nitrate.27 Single colonies of A. vitaminophilum, B. subtilis and E. coli W3110 were inoculated in 5 mL ISP2 based liquid medium supplemented with 0.1% KNO3. Cells were allowed to grow at 28 0C up to 3 weeks. One drop of solution A (0.8% sulfanilic acid in 5.0 M HOAC) and solution B (0.5% naphthylamine in 5.0 M HOAC) was added to 1 mL culture samples consequently. The culture of A. 176 vitaminophilum and B. subtilis produced pink or brown color, which suggested positive results. The culture of E. coli produced pink or brown color only after the addition of Zn powder, which suggested negative results. Peptonization and coagulation of skim milk.27 Single colonies of A . vitaminophilum, B. subtilis and E. coli W3110 were inoculated in 5 mL litmus milk medium (100 g litmus milk in 1000 mL, pH 6.8 after autoclave). Skim milk provides lactose as carbohydrate source and casein as primary protein source, and supplemented with pH indicator, azolitmin. B-Galactosidase could hydrolyze lactose to galactose and glucose, and glucose may then end up with acid, which lows the pH value. Accumulating acid may also cause precipitation of casein and form acid clot. On the other hand, casein could be partially digested and NH3 would be released to raise the pH value. Proteolytic enzymes such as rennin, pepsin and chymotrypsin could digest casein and coagulate the milk. Melanoid pigment.7 Single colonies of A. vitaminophilum, B. subtilis and E. coli W3110 were inoculated in 5 mL ISPl or ISP7 medium. After growing at 28 °C for 2 weeks, 2 mL culture was mixed with 1 mL 0.4% L-tyrosine or L-dopa in 2 mL phophate buffer (100 mM, pH 5.9) and incubated at 37 °C for 30 min. No dark brown melanin pigment formation suggested the negative results from all the tests. NaCl tolerance. Single colonies of A. vitaminophilum were replicated on solid medium (1 L medium contained 1% yeast extract, 2% soluble starch, 1.5% agar and different concentrations of NaCl: 0, 0.75%, 1.5%, 3%, 5%). Colonies, which grew on 1.5% NaCl and not grew on 3% NaCl after 2 to 3 weeks were selected out. 177 After all the purification, 9 out of 20 colonies were selected out and cultured as described above to assay the production of pyrrolomycin A. Of the 9 colonioes, 4 colonies were confirmed to synthesize pyrrolomycin A with a titer of 20 mg/L. These 4 colonies were grown in ISPII medium and stored at -80 °C in 10% glycerol. Culturing A. vitaminophilum and Streptomyces sp. UpJohn UC11065. A single colony from SYE (starch, Yeast extract) plate or ISP2 plate was incubated in 5 mL seed culture medium-l at 28 °C with shaking at 250 rpm for 3 days. The 3-day old seed culture-2 was transferred to a 50 mL seed medium-2 for 2 days. The second seed culture was then transferred to 1000 mL production medium and grew in shake flask for 5 days. Preparation of crude lysate of A. vitaminophilum (ATCC31673) and Strethamyces sp. UpJohn UC11065. After incubating the strain in production medium (both rich and minimal) for 5 days, cell lysate was prepared in the following way. A 5 mL portion of fermentation broth was first taken out and extracted with 10 mL EtOAc to detect the production of pyrrolomycin A by HPLC. A 1000 mL pyrrolomycin A production culture was then centrifuged at 4 °C (2 700 g, 10 min) to collect the mycelium, which was further suspended in 30 mL phosphate buffer (pH 7.3) and centrifuged at 4 °C (2 700 g, 10 min) to wash the mycelium. The resulting mycelium was again suspended in 10 mL phophate buffer and cell lysate was obtained by two passages of french press at 16 000 psi, or by sonication followed by centrifugation at 4 °C (47 000 g, 20 min) to remove the cell debris. The resulting cell lysate was then ready for in vitro reactions. Preparation of genomic DNA from A. vitaminophilum (ATCC31673) and Streptomyces sp. UpJohn UC11065. Genomic DNA preparation was conducted 178 according to Kirby mix procedure.8 Solutions specific for this procedure are prepared as the following method. TEZSS buffer: 25 mM Tris-HCI, pH 8; 25 mM EDTA pH 8; 0.3 M sucrose. 2 x Kirby mix: 2 g TPNS (sodium tri-isopropylnaphthalene sulphate, SDS can be used instead), 12 g sodium 4-aminosalicilate (BDH), 5 mL 2M Tris-HCI pH 8, 6 mL equilibrated phenol pH 8.0, make up to 100 mL HzO. Other solutions were prepared as described in the general part of this chapter. A. vitaminophilum (ATCC31673) and Streptomyces sp. UpJohn UC11065 were cultured in ISP2 medium as described. Mycelia was harvested from 500 mL antibiotics production medium was resuspended in 3 mL TEZSS buffer with addition of 100 aM lysozyme solution. After incubating at 37 °C for 10 min, add 4 mL 2 x Kirby mix and gently agitate for 1 min on a vortex mixer. Add 8 mL phenol/chloroform/isoamyl alcohol and agitate for 15 s as above and centrifuged 10 min (1500 g) at room temperature. The upper aqueous phase was then transferred to a fresh tube containing 3 mL phenol/ch]oroform/isoamyl alcohol and agitated for 1 min and cetrifuged again as described above. To this mixture, 0.6 vol isopropanol was added to precitate the DNA and spool DNA onto a sealed Pasteur pipette. Following a brief rinse with 5 mL 70% ethanol, the DNA was air dried and resuspended in 1 mL of TE for future use. Chemical synthesis of pyrrolomycin A. A well-stirred solution of sufuryl chloride (0.9 mL, 6.6 mmol, Aldrich) in 8 mL diethyl ether was added dropwise to a solution of 3-nitropyrrole (0.336 g, 3mmol, TCI) in 30 mL CHzClz, which was cooled to —15 °C before hand. After being stirred at —15 °C for 2 h, the reaction mixture was concentrated under reduced pressure to obtain a yellow 179 residue. This residue was then separated by flash column to afford 2,5-dichloro-3- nitropyrrole (53% mol/mol), and pyrrolomycin A (15% mol/mol), 5-chloro-3-nitropyrrole (trace) and 2, 4, 5-trichloro-3-nitropyrrole (23% mol/mol) (Scheme 7). The 2, 5- dichloro-3-nitropyrrole was crystallized from EtOAc and hexane to afford a yellow solid. 1H NMR (d6-acetone, ppm): 6 6.75 (s, 1H), 12.5 (broad, 1 H). 13C NMR (d6-acetone, ppm): 6 103, 117, 120, 138. MS (EI, relative intensity, %): 180 (M‘, 100), 182 (M + 2, 76), 184 (M + 4, 20), 164 (10), 150 (28), 134 (50), 107 (93), 62 (44). HRMS (El) calculated for C,,HC12NZO2 (M+) 179.9493. Found: 179.9499. m. p. 148-150 °C. Pyrrolomycin A was crystallized from EtOAc and hexane to afford a yellow solid. 1H NMR (d6-acetone): 6 7.96 (s, 1 H), 12.5 (broad, 1 H). l3C NMR (d6-acetone): 6 104, 116, 121, 133. MS (EI, relative intensity, %): 180 (M‘, 98), 182 (M + 2, 69), 184 (M + 4, 13), 164 (7), 150 (10), 134 (30), 107 (100), 28 (58) (Figure 1a). HRMS (EI) calculated for C4HC12N202 (M*) 179.9493. Found: 179.9489. m. p. 210-212 °C. The mono- and trichlorinated products were not characterized. Microbial synthesis of pyrrolomycin A by A. vitaminophilum. General. The broth was then centrifuged at 4 °C (13500 g, 10 min) to separate supernatant from the mycelium. The supernatant (1000 mL) was extracted with ethyl acetate (3 x 600 mL). The mycelium was suspended in 500 mL 50% acetone aqueous solution and stirred for 2 h. The stirred mixture was then filtered through celite to remove solid residue and the acetone in the resulting solution was distilled away. The aqueous solution was then extracted with EtOAc (3 x 100 mL). The two organic parts were combined and dried over NaZSO4, followed by being concentrated under vacuum to afford a yellow oil residue. The yellow oil was load onto HPLC column to detect 180 pyrrolomycin A by comparing with the synthetic sample. The oil residue was also loaded on silica flash column and eluted with EtOAc/hexane to obtain 25 mg pyrrolomycin A, which was further recrystallized from EtOAc and hexane. The 1H NMR, 13C NMR and MS of the purified pyrrolomycin A correspond to the synthetic sample. Microbial synthesis of pyrrolomycin A by supplementing isotope labeled substrates. Actinosporangium vitaminophilus (ATCC31673) was cultured as described above with 100 mg K‘SNO3, K‘5N 1803 or L-Arginine-guanido-‘SN2 fed in the production medium. Pyrrolomycin A was then purified as described above as a mixture of pyrrolomycin A and pyrrolomycin B. 1H NMR (d6-acetone, ppm): pyrrolomycin A, 6 7.96 (s, 1H); Pyrrolomycin B, 6 7.35 (d, J=2 Hz, 1 H), 7.19 (d, J=2 Hz, 1 H), 4.41 (s, 2 H). MS of pyrrolomycin A and B isolated from A. vitaminophilum culture broth without supplementing KNO3 (EI, relative intensity, %): pyrrolomycin A, 180 (M+, 75), 182 (M++2, 50), 184 (M++4, 10), 150 (23), 107 (100); pyrrolomycin B 354 (M+, 63), 356 (M++2, 100), 358 (M++4, 41), 360 (M++6, 7), 337 (19), 308 (30) (Figurelb). MS of pyrrolomycin A and B isolated from A. vitaminophilum culture broth supplementing KNO3 (El, relative intensity, %): pyrrolomycin A 180 (M+, 94), 182 (M++2, 52), 184 (M++4, 11), 150 (10), 107 (100); pyrrolomycin B 354 (M+, 76), 356 (M++2, 100), 358 (M++4, 44), 360 (M++6, 9), 337 (18), 308 (29). (Figurelc). MS of pyrrolomycin A and B isolated from A. vitaminophilum culture broth supplementing K'SNO3 (El, relative intensity, %):. Pyrrolomycin A, 181 (M+, 78), 183 181 (M++2, 55), 185 (M++4, 25), 150 (21), 107 (100); pyrrolomycin B, 355 (M+, 80), 357 (M++2, 100), 359 (M++4, 52), 361 (M++6, 14), 338 (21), 308 (41). MS of pyrrolomycin A and B isolated from A. vitaminophilum culture broth supplementing K'5N1803 (El, relative intensity, %):. Pyrrolomycin A, 181 (M+, 78), 183 (M*+2, 55), 185 (M"+4, 25), 150 (21), 107 (100); pyrrolomycin B, 355 (M+, 80), 357 (M*+2, 100), 359 (M*+4, 52), 361 (M*+6, 14), 338 (21), 308 (41). MS of pyrrolomycin A and B isolated from A. vitaminophilum culture broth supplementing L-Arginine-guanido-‘SN2 (EI, relative intensity, %):. Pyrrolomycin A, 181 (M+, 78), 183 (M++2, 55), 185 (M++4, 25), 150 (21), 107 (100); pyrrolomycin B, 355 (M+, 80), 357 (M++2, 100), 359 (M++4, 52), 361 (M++6, 14), 338 (21), 308 (41). Microbial synthesis of pyrrolomycin A in defined fldium. A 20 mL seed culture (rich medium) obtained by inoculating a single colony for 3 days was transferred to 100 mL seed culture (rich medium) for 2 more days. An aliquot of 5 mL this medium was transferred to 100 mL ISP4 minimal mediums with different N sources. After 5 days, 5 mL of each broth was extracted with EtOAC and production of pyrrolomycin A was detected by HPLC. Microbial synthesis of pyrrolomycin A at the presence of NOS inhibitor. A 5 mL of the second stage seed culture as described above was transferred to a 100 mL culture medium supplemented with the NOS inhibitor, N-nitro-L-arginine methyl ester (NAME) at different concentrations of 0, 15, 30, 60, 120, 200 500 uM and kept on growing at 28 °C. A 5 mL sample was taken out every 24 h and pyrrolomycin A production was detected by HPLC. No apparent inhibition was observed at all inhibitor concentrations. 182 Dioxapyrrolomycin synthesized by UpJohn Streptomyces UC11065. UpJohn Streptomyces UC11065 was cultured in the same way as A. vitaminophilus (ATCC31673).9 A 1000 mL production medium was then centrifuged at 4°C (13500 g, 15 min). The mycelium was stirred with 200 mL acetone for 2 h and the mycelium residue was removed by filtration. Acetone was then distilled away under vacuum and the resulting aqueous part was extracted with 3 x 100 mL EtOAc. The supernatant was directly extracted with 3 x 600 mL EtOAc. The two extracts were then combined and dried over Nast4. After being concentrated to dry, the residue was loaded on a flash column and a Rf=0.3 fraction with the ratio of EtOAc and hexane to be 1 to 5 was collected and further recrystallized to afford 1.5 mg yellow needle solid. After characterization by 1H NMR, 13C NMR and MS, this fraction was confirmed to be dioxapyrrolomycin. 1H NMR (d6—acetone, ppm) 6 11.50 (broad, 1H), 7.44 (d, J=2.0, 1 H), 7.16 (dd, J=2.5, 2.0, 1 H), 6.89 (s, 1 H), 5.54 (d, J=2.5 2H). ”C NMR (d6-acetone, ppm) 6 148.9, 130.9, 130.1, 126.6, 126.5, 126.4, 124.7, 123.1, 106.1, 91.3, 69.3. MS (EI, relative intensity, %): 382 (M+, 10), 384 (M+ + 2, 12), 386 (M+ + 4, 6), 388 (M+ + 8), 352 (M+-NO, 20), 306 (M+-NOZ-CHzO, 77), 308 (306 + 2, 100) (Figure 6). HRMS: calculate 381.9081, found 381.9087. Synthesis of pyrrole-Z-acyl thioester (SNAC). The synthesis of the non-chloro substituted pyrrole SNAC was started with the commercially available. To a solution of pyrrolyl-2-carboxylic acid (111 mg, 1 mmol), 1 mmol HSNAC (0.31 mL, 1mmol), and 0.75 mmol DMAP (93 mg, 0.75 mmol) in 10 mL DMF was added a premixed solution of EDC'HCI (216 mg, 1.1 mmol) and DMAP (136 mg, 1.1 mmol) in 10 mL of dry DMF dropwise. The resulting mixture was allowed to 183 stir at room temperature for 3 h, and was diluted with 300 mL EtOAC, washed with H20 (1 X 100 mL), and brine (2 X 100 mL). The product extracted with EtOAC was purified by flash column and further crystallized from EtOAc and hexane in an 80% (mol/mol) yield. 1H NMR (dé-acetone, ppm) 6 7.30 (broad, 1H), 7.15 (m, 1 H), 6.96 (m, l H), 6.23 (s, 1 H), 3.38 (m, 2H), 3.10 (t, J=6.6 Hz, 2H), 2.84 (broad), 1.85 (s, 3H). 13C NMR (d6- acetone, ppm) 6 180, 170, 131, 125, 116, 111,40, 28,23. The synthesis of 2 and 3 chlorinated pyrrole-SANC started with 2- (trichloroacetyl) pyrrole. Two equilibriums of sufuryl chloride (SOZCIZ) (1.1 mL, 11 mmol) in acetic acid (25 mL) was slowly added to the solution of 2- (trichloroacetyl)pyrrole (1.06 g, 5 mmol) in acetic acid (25 mL). After stirring overnight, the solution was concentrated to dry first under water aspirator pressure then under high vacuum (0.5 mm Hg). The resulting residue was redissolved in 50 mL diethyl ether and rinsed with 50 mL saturated NaHCO3 and 2 x 50 mL brine. The ether solution was then dried over NazSO4 and shaked with 2 g charcoal for 2 min. A pale yellow solid was obtained after filtration through celite and concentrated to dry. The 2 chlorinated products, 4, 5-dichloropyrrole-2-yl-trichloromethyl ketone (yield, 53% mol/mol) and 3, 4, S-trichloropyrrole-Z-yl- trichloromethyl ketone (yield, 33% mol/mol) were separated by flash column. The 2 chlorinated products, 4, 5-dichloropyrrole—2-yl-trichloromethyl ketone (281 mg, 1 mmol) or 3, 4, 5-trichloropyrrole-2-yl-trichloromethyl ketone (316 mg, 1 mmol) was suspended in 10 mL HZO. NaOH (1.0 M, 1.2 equilibrium) was dropped slowly until all solid was dissolved. The solution was then acidified with concentrated HCI to pH 1.0 and the resulting acid was extracted with diethyl ether. The extract was then dried over 184 NazSO4 and concentrated to dry to afford 2 and 3 chlorinated pyrrole-2-carboxylic acid in the yields of 85% (mol/mol) and 65% (mol/mol). The two acids were pumped dry and used for the next step directly without further purification. The two acids were then coupled with HSNAC as described above to afford the corresponding SNAC analogues in relatively low yields, 30% (mol/mol) and 20% (mol/mol). Dichlorinated product 1H NMR (d6-acetone, ppm) 6 7.30 (broad, 1H), 6.95 (s, l H), 3.37 (m, 2H), 3.10 (t, J=6.6 Hz, 2H), 2.60 (broad), 1.85 (s, 3H). 13C NMR (d6- acetone, ppm) 6 180, 170, 128, 120, 115, 111, 40, 28, 23. Trichlorinated product: 1H NMR (d6-acetone, ppm) 6 7.38 (broad, 1H), 3.38 (m, 2H), 3.20 (t, J=6.6 Hz, 2H), 2.80 (broad, 1 H), 1.85 (s, 3H). 13C NMR (d6-acetone, ppm) 6 180, 170, 128, 121, 114, 111, 40, 28, 23. Synthesis of pyrrole-phenol ketone intermediates. Salicylic acid chloride and 3, 5-dichlorosalicylic acid chloride. Salicylic acid (6.9 g, 50 mmol) or 3, 5-dichlorosalicylic acid (10.35 g, 50 mmol) was dissolved in 100 mL DMF and heated to 50 °C. To the above solution, KZCO3 (15.2 g, 120 mmol) was added to form a yellow solution and stirred at 50 0C for 30 min, followed by the addition of CH3I (8 mL, 128 mmol). After stirring at this temperature for 3 h, the reaction solution was poured into 500 mL H20 and extracted with diethyl ether (3 X 300 mL). The extract was then dried over NaZSO4 and concentrated under vacuum to afford the completely protected salicylic acid. The double protected salicylic acid was the refluxed in 250 mL, 10% NaOH for 2 h. The solution was cooled to room temperature and extracted once with diethyl ether to remove any double protected starting material. The aqueous solution was further acidified with concentrated HCI to pH 2.0 and stirred 185 overnight to allow the complete precipitation of the partially protected methyl salicylic acid (70% mol/mol) and 3,5-dichloromethy1 salicylic acid (88% mol/mol). Methyl salicylic acid. 1H NMR (d6-acetone, ppm) 6 7.90 (l H, dd, J=), 7.58 (1H, ddd, J=), 7.22 (1 H, d, J=), 7.07 (1 H, ddd, =), (3 H, s); 13C NMR (d6-acetone, ppm) 6 166, 159, 135, 132, 121, 120, 113, 20. The partially protected acid (10 mmol) was suspended in 60 mL CHzCl2 and a solution of SOCl2 (2.1 mL, 30 mL)/DMF (20 drops) in 30 mL CHzCl2 was added in slowly. The reaction mixture was stirred at room temperature for 1 h, followed by refluxing for 30 min until the solution became clear. The completion of the reaction was also monitored with IR by the disappearance of absorption at 1668 cm'1 and the appearance of 1780 cm". The resulting solution was then concentrated under vacuum and pumped dry overnight for the next coupling reaction. 2-(2-hydroxylbenzoyl)pyrrole and 2-(2-hydroxyl 3.S-dichlorobenzopryrrole. Freshly distilled pyrrole (0.75 mL, 11 mmol) was dissolved in 30 mL toluene and allowed to cool down to 0 °C. To this solution, ethyl magnesium bromide (1. 0 M in THF, 11 mL) was dropped in cold and the solution was stirred at room temperature for 30 min to allow the completely formation of pyrrole magnesium bromide. To this solution, 10 mmol salicylic acid chloride or 3, 5-dichlorosalicylic acid chloride in 30 mL THF was added in slowly and stirred at room temperature for 3 h. The reaction was then quenched with 200 mL ice H20 and extracted with EtOAC (3 X 150 mL). The extract was dired over Nast4 and concentrated to dry under vacuum and the coupling ketone product was purified with flash column. The purified product was then dissolved in CHZCI2 in an inert atmosphere and cooled to —78 °C. BBr3 (1 M in CHZCIZ, 1.1 eq.) was added and the 186 reaction solution was stirred at —78 °C for 1 h. After carefully being brought to room temperature, the solution was diluted and quenched with EtOAc (20 X) and ice H20 (20 X). The aqueous part was separated from the organic part and extracted with EtOAc. The combined EtOAc was then dried over NazSO4 and concentrated to dry under vacuum to afford 2-(2-hydroxylbenzoyl)pyrrole (50% mol/mol) and 2-(2-hydroxyl 3,5- dichlorobenzoyl)pyrrole (45% mol/mol). Pyrrolomycin C and pyrrolomycin D. The synthesized 3,5-dichloromethyl salicylic acid (1.35 g, 5 mmol) was dissolved in 100 mL CHZCI2 and cooled to —78 °C. To this solution, well-stirred SOzCl2 in 30 mL diethyl ether was added slowly. The solution was stirred at -78 °C for 10 min and immediately concentrated to dry under vacuum. The 2 and 3 chlorined products were separated by flash column and deprotected as described above to afford two yellow solid, pyrrolomycin C (53% mol/mol) and pyrrolomycin D (27% mol/mol). Synthesis of nitrotryptophan. Formaldehyde (37% in H20, 0.18 mL, 2.4 mmol), dimethyl amine (40% in H20, 0.38 mL, 3 mmol) and nitroindole (324 mg, 2 mmol) were added consequently into 5 mL ice cold HOAc. The temperature was then increased to 90-95 °C and the reaction solution was stirred at this temperature for 10 h. The solution was then cooled to room temperature and poured into 35 mL ice H20. A yellow precipitate was formed after adjusting pH to 10 and stirring in ice HZO for 2 h. The product gramine was collected by filtration and washed with H20 until pH 7.0 and pumped dry overnight with a yield around 75% (mol/mol). The obtained gramine (219 mg, 1 mmol) was suspended in 5 mL toluene with diethyl acetamidomalonate (260 mg, 1.2 mmol) and NaOH (30 mg, 0.75 187 mmol). After refluxing for 10 h, the reaction mixture was cooled to room temperature and poured into 10 mL ice HZO to allowed the precipitation of the condensation product. The product was collected by filtration and rinsed with diluted HOAc in a yield from 67 to 73% (mol/mol). These condensation products was refluxed in 5 mL concentration HCI for 18 h to afford nitrotryptophan HCl salts (yields were from 51 to 90%, mol/mol), which were precipitated after the solution was cooled to room temperature. Nitrotryptophans could be further purified with Dowex 50 (H+ form) from their HCl salt forms. 4-nitrotryptophan. 1H NMR (d6-DMSO, ppm) 6 13.85 (1 H, b), 12.19 (1 H, s), 8.28 (2 H, b), 7.87 (1 H, d, J=8.0), 7.84 (1 H, d, J=8.0), 7.65 (l H, d, J=2.5), 7.27 (1 H, t, J=8.0), 3.93 (1 H, not split well), 3.50 (1 H, dd, J=15.0, 5.5), 3.25 (1 H, dd, J=15.0, 8.5); ”C NMR 6 162.85, 134.07, 132.01, 122.60, 112.22, 110.43, 110.27, 109.61, 98.70, 46.04, 20.90. 5-nitrotryptophan (d6-DMSO, ppm) 'H NMR 6 13.85 (1 H, b), 11.96 (1 H, s), 8.62 (1 H, d, J=2.0), 8.43 (2 H, b), 7.98 (1 H, dd, J=8.5, 2.5), 7.54 (1 H, d, J=8.5), 7.53 (1 H, d, J=2.0), 4.19 (1 H, d, 5.0), 3.60 (2 H, dd, 5.0) : ”C NMR 6 170.69, 140.50, 139.41, 129.10, 126.54, 116.54, 115.90, 111.98, 109.90, 52.57, 25.46. 6-nitrotryptophan (d6-DMSO, ppm) 1H NMR 6 13.85 (1 H, b), 12.00 (1 H, s), 8.47 (2 H, b), 8.35 (1 H, d, J=2.5), 7.87 (1 H, dd, J=9.0, 2.0), 7.78 (1 H, d, J=9.0), 7.70 (l H, d, J=2.5), 4.1 (1 H, d, b), 3.60 (2 H, d, 6.5) : ”C NMR 6 170.54, 141.91, 134.59, 132.12, 131.95, 118.69, 113.71, 108.41, 108.29, 52.49, 25.54. 7-nitrotryptophan. (dé-DMSO, ppm) 1H NMR 6 13.80 (1 H, b), 11.88 (1 H, s), , 8.49 (2 H, b), 8.15 (1 H, d, J=8.0) 8.10 (1 H, d, J=8.0), 7.48 (l H, d, J=2.5), 7.24 (1 H, t, 188 J=8.0), 4.18 (l H, d, b), 3.39 (2 H, d, 5.0) : 13C NMR 6 170.53, 132.47, 131.63, 128.50, 128.41, 127.24, 118.62, 118.44, 109.14, 52.40, 25.30. In vitro activity with SN AC and possible post-modification intermediates. A. vitaminophilus (ATCC31673) was cultured as described to synthesize pyrrolomycin A. Cells were harvested at 4300 g for 10 min at 4°C from 500 mL culture medium, and washed with 30 mL phosphate buffer (pH 7.0) twice to remove as most of pyrrolomycin A as possible. The resulting cells were then suspended in 10 mL phosphate with buffer supplemented with] mM PMSF and 1 mM DTT and broken by two passages of french press (16000 psi). The cell debris was then discarded after centrifuged at 48000 g for 30 min at 4 °C. The crude lysate was then used for the in vitro reaction. A 5 mL in vitro reaction solution include 0.3 mM SNAC analogue, 5 mM NaCl, supplemented with either 5 mM KNO2 or KNO3. The reaction was then incubated at 28°C and monitored by TLC and then loaded onto HPLC to detect the synthesis of pyrrolomycin A. An aliquot of 0.5 mL samples were taken out for to monitor pyrrolomycin A and pyrrolomycin B production at 1, 2, 4, 8,12, 24 and 48 h by HPLC. In vitro reactions with ketone intermediates pyrrolomycin C, pyrolomycin D, 2- (2-hydroxy1benzoyl)pyrrole and 2-(2-hydroxyl 3,5-dichlorobenzoyl)pyrrole were set up as described about. The only difference is, the four intermediates were dissolved in DMSO (5% v/v of final volume) before adding to the reaction solution. Plasmid construction and deiN OS heteroexpression. Plasmid construction. NOS gene from the genomic DNA of D. radioduran (ATCC 13939D) was amplified with primers: (upper) ATGAGTTGCCCCGCFGCC and (down) l'l 'l'ATCGTGGGGTTACC. To clone gene deiNOS into BamHI and PstI sites of 189 pJFl 18EH and pQE30, short sequences CGGGATCC (BamHI site) and AACTGCAT (PstI site) were added to the corresponding upper and down stream primers. And plasmid pWL4.134 and pWL4.135 was constructed. To clone gene deiNOS into NdeI and BamHI sites of pET-15B, short sequences GGAATTCCATATG (NdetI site) and CGGGATCC (BamHI site) were added to the corresponding upper and down stream primers. And plamid pWL4.106 was constrycted. Gene expression. Plasmid pWL4.134 and pWL4.135 were transformed into E. coli DHSa. A single colony of E. coli strain DHSa/pWL4.l34 or DHSa/pWL4.135 was inoculated in 5 mL LB medium and grew overnight at 37°C. The 5 mL culture was then transferred to 500 mL LB medium at different temperatures, 22°C, 30°C and 37°C until ODw0=0.4~0.6. And the expression of NOS gene was induced by adding different concentrations of IPT G (0.2 mM. 0.5 mM and 1.0 mM) and grew 4-8 h before cells were harvested. Plasmid pWL4.106 was transformed into BL21(DE3) to obtain E. coli BL21(DE3)/pWL4.106. A single colony of E. coli strain, BL21(DE3)/pWL4.106 was then inoculated in 5 mL LB medium and grew overnight at 37°C. The 5 mL culture was further transferred to 500 mL LB medium and incubated at 37°C until OD600=0.4~0.6. At this point, the culture was put in ice H20 until it was completely cooled down. IPTG was added at a concentration of 0.5 mM and the cells were grown at 22°C overnight. The NOS gene was over expressed under such a condition. The protein was then purified with Ni-NTA argose resin. NO production assay. Griess reagent assay NO; response factor for Griess reagent assay was determined with two different ranges of KNO2 concentrations (0, 1, 2, 3, 4, 5 mM and 0, 190 5, 10, 15, 20, 25 mM). For a 1 mL reaction, 0.5 mL Griess reagent 1 and 2 were added continuously and allowed 15 min for color development at room temperature. Azo product formation was monitored at ODS”. Enzyme purified as described was used for this assay. Reaction (1 mL) was set up as: deiNOS 25 uM, H202 5 mM, L-Arginine 5 mM. A 100 A sample was taken out every 3 min and quenched by heating at 100 °C for 3 min. After allowing the oxidation for 15 min, 50 7» Griess reagent R1 and R2 were added consequently. The pink color was developed for 10 min and nitrite concentration was determined by measuring ODS“). Hemoglobin assay. Hemoglobin (Hb) purchased from Sigma contains a certain amount of heme in ferric (Fe3*, metHb) was dissolved in H20 and solid sodium dithionite was added to in a 2-3 fold molar excess over heme in order to ensure complete reduction of methemoglobin (MetHb). Assay reaction was then set up as Phosphate buffer (50 mM, pH 7.5), L-Arginine (5 mM) Oxyhemoglobin (8 11M), NADPH (0.1 mM), Mg(OAc)2 (1 mM), Calmodulin (1 11M), CaC12(1 mM), FAD (1 11M) and H4B (10 11M). deiNOS enzyme (25 uM) was added to start assay. The activity was monitored at OD401 nm. Biosynthetic nitration assay. For nitration reaction with peroxynitrite, it concentration was determined right before the reaction according to it absorption at 302 nm (8302:1670 M'1cm" in 0.1 M NaOH). The NO production activity of protein deiNOS purified used for nitration assay was first determined by Griess reagent assay. Reactions (1 mL) were sep up in as deiNOS (25 uM), L-Arginine (5 mM), substrates (5 mM) and H202/KO2 (5 mM) phosphate buffer (50 mM, pH 7.5). After stirring at 37 °C for 4 h, reactions were 191 quenched with 0.5 mL methanol (1% HOAC) and loaded onto HPLC to detect nitration activity. For in situ generation of H202, 10 mM glucose and 500 units of glucose oxidase, instead of HzOzwas added to reactions. 192 Reference ‘ Miller, J. H. Experiments in Molecular Genetics; Cold Spring Harbor Laboratory: Plainview, NY, 1972. 2 Sambrook, J .; Russell, D. W. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 2001. 3 Bradford, M. M. Anal. Biochem. 1976, 72, 248. 4 Harris, E. L. V.; Angal, S. In Protein Purification Methods: A Practical Approach; Oxford University Express: Oxford, New York, Tokyo, 1989. 5 Farabaugh, M. A. Biocatalytic Production of Aromatics from D-Glucose. MS. Thesis, Michigan State University, 1996. 6 Jeboffe, M. J .; Pierce, B. E. A Photograph Atlas for the Microbiology Laboratory; Morton Publishing Company: Englewood, Colorado, 1999. 7 (a) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A. Practical Streptomyces Genetics. John Innes: Norwich, 2000; pp 342-343. (b) Kom-Wendisch, F.; Kutzner, H. J. In the Prakaryates; Springer-Verlag: New York, 1992; pp 921-995. 8 Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A. Practical Stryptamyces Genetics; The John Innes Foundation, Norwich, England. 2000, pp 161- 171. 9 (8) Carter, G. T.; Nietsche, J. A.; Goodman, J. J.; Torrer, M. J.; Dunne, T. S.; Borders, D. B.; Testa, R. T. LL-F42248-alpha, A Novel Chlorinated Pyrrole Antibiotics. J. Antibiot. 1987, 40, 233-236. (b) Conder, G. A.; Zielinski, R. J.; Johnson, S. S.; Kuo, M. ST.; Cox, D. L.; Marshall, V. P.; Haber, C. L; Diroma, P. J.; Nelson, S. J.; Conklin, R. D.; Lee, B. L.; Geary, T. G.; Rothwell, J. T.; Sangster, N. C. Anthelmintic activity of Dioxapyrrolomycin. J. Antibiot. 1992, 45, 977-983. 193 ‘11:. 1' ll- 3.8.451 ,L. t. 1... Erna er. 1 I! 371.3)! .I‘p ‘ &» ‘31....23. to... 2 Sun 1 w. «Waxy . ‘fl 1 Eu .. s an», .. 15:3. . ..1r1_z . L r. Niki .. 4x.» 5% :2... F, 42 3 . $11....mi. .. .. s 3 .P... 95cmkun . .. , . J} v.» 4v t. . .Eufirl