H , . lg”? d" it. . n w gx‘ . A. 2‘ . t w , . :. It Lmuflfizs. “.1. .' in. ' x I. £3313. . .‘ Ltfiuu... xi.-qg£.§. . I» . 9.31114 ft 11.9“" 22.! 21.3! no... . r l. .7543; an .2. 4 .Iu...z.‘-... n (”can .2 § .1 3.....1. , . .2. . » ,6 I... . i 1r.) 1. ; . S (-935 LIBRARY ‘7 \ MIChIQu a State University This is to certify that the thesis entitled STRATEGIES FOR IMPROVING SYNTHESIS OF 3-DEHYDROSHIKIMIC ACID AND SHIKIMIC ACID FROM D-GLUCOSE presented by Justas Jancauskas has been accepted towards fulfillment of the requirements for the MS. degree in Chemistry "g. FVW‘Vfiar/ / Major Professor’s Signature 04-24-2006 Date MSU is an Affirmative Action/Equal Opportunity Institution A. ~n-x------ 1---.-I-.--—I—I-.—.-l--.---.-.--.---—‘-VA 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. DATEDUE DAIEDUE DAIEDUE 6/07 p:/CIRC/DateDue.indd-p.1 STRATEGIES FOR IMPROVING SYNTHESIS OF 3-DEHYDROSHIKIMIC ACID AND SHIKIMIC ACID FROM D-GLUCOSE 'By Justas Jancauskas A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 2006 Copyright by J ustas Jancauskas 2006 ABSTRACT STRATEGIES FOR IMPROVING SYNTHESIS OF 3-DEHYDROSHIKIMIC ACID AND SHIKIMIC ACID FROM D-GLUCOSE By Justas Jancauskas Shikimate pathway product yields in microbial synthesis are ultimately limited by phosphoenolpyruvate and D-erythrose 4-phosphate availability, which are derived from glycolysis, and by the amount of carbon that is converted to the cell biomass and C02. A 21% increase in synthesized 3—dehydroshikimic acid under fed-batch conditions was observed with adenylate kinase overexpression. Mutants possessing AathH mutation showed a decrease in 3-dehydroshikimic acid titer, yield and biomass formation due to an inactivated oxidative phosphorylation pathway. Deletion of the sucA gene resulted in reduced CO2 formation by E. coli. E. coli Shikimate dehydrogenase (AroE) catalyzes the reduction of 3- dehydroshikimic acid and is feedback inhibited by shikimic acid, which leads to 3- dehydroshikimic acid accumulation during shikimic acid biosynthesis. A second E. coli Shikimate dehydrogenase, YdiB, showed no inhibition with shikimic acid. Bioinformatics tools were used to identify a K. pneumoniae quinate dehydrogenase encoding gene qad. The identified gene was overexpressed in E. coli, and purified enzyme showed 70% inhibition in the presence of 20 mM of shikimic acid, while AroE and B. subtilis Shikimate dehydrogenase were 90% inhibited. E. coli AroE mutant library was generated and screened for a feedback-insensitive Shikimate dehydrogenase. To my mother, niece and family For their constant love and support. iv ACKNOWLEDGMENTS First and foremost, I would like to thank my supervisor Prof. John W. Frost for allowing me to take part in his research. His passion about scientific research, aggressiveness and strong work ethic has certainly been inspiring. I am indebted to him for his guidance and encouragements throughout my graduate studies. I would also like to thank the members of my graduate committee, Prof. Babak Borhan, Prof. Robert E. Maleczka, Jr. and Prof. David P. Weliky for their input during the preparation of thesis. I am grateful to Dr. Karen M. Draths for her friendship, patient guidance, invaluable suggestions and endless support in my research, and for proofreading my thesis. I am also thankful to Carolyn Wemple for help in all the encountered bureaucratic issues during my stay at MSU. I wish to thank Dr. Ningqing Ran and Dr. Jain Yi for their discussions and guidance in experimental techniques. My gratitude is given to the Frost group members, past and present, including Kin Sing Stephen Lee, Dr. Dongming Xie, Dr. Stephen Cheley, Dr. Jiantao Guo, Dr. Wei Niu, Dr. Wensheng Li, , Dr. Heather Stuben and Adam Bush. I am especially grateful to Dr. Mapitso N. Molefe, Man Kit Lau, Dr. Jihane Achkar, Xiaofei .Iia, Jinsong Yang, and Brad Cox for their much needed support throughout my time in the group and whose friendships I will treasure forever. Finally, I owe the sinceriest of thanks to my friends Kostas, Thalia, Nineta, Kyoungsoo and his family, Diana and David, Algirdas, Arunas, Chrysoula, and Fei for their understanding, help and support. This thesis is dedicated to my parents, Alberta Jancauskiene and Zigmantas Jancauskas, my brother, Mindaugas Jancauskas, and his family. TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... ix LIST OF FIGURES ........................................................................................................ xi LIST OF ABBREVIATIONS.........................................‘ .............................................. xiii CHAPTER ONE ............................................................................................................. 1 Introduction ..................................................................................................... 1 The Shikimate pathway ................................................................................. 2 3-Dehydroshikimic acid ............................................................................... 5 Shikimic acid ............................................................................................... 6 Increasing of carbon flow into the Shikimate pathway .................................. 8 Directed evolution ...................................................................................... 11 References ..................................................................................................................... 14 CHAPTER TWO .......................................................................................................... 20 Modifying central metabolism of Escherichia coli ......................................... 20 Introduction ................................................................................................ 20 Glycolytic flux and ADP availability .......................................................... 23 Biomass formation and CO2 generation ...................................................... 26 Biocatalyst design ....................................................................................... 27 Fermentation conditions ............................................................................. 32 Effect of Aath H mutation on 3-dehydroshikimic acid and the Shikimate pathway byproducts synthesized by E. coli ................................................. 33 Effect of adenylate kinase (adk) expression on 3-dehydroshikimic acid and the Shikimate pathway byproducts synthesized by E. coli ............................ 36 Effect of AsucA mutation on 3-dehydroshikimic acid and the Shikimate pathway byproducts synthesized by E. coli ................................................. 39 Discussion .................................................................................................. 40 References ..................................................................................................................... 44 CHAPTER THREE ....................................................................................................... 48 A search for feedback-insensitive Shikimate dehydrogenase ........................... 48 Introduction ............................................................................................... 48 Purification of E. coli Shikimate dehydrogenases YdiB and AroE ............... 50 Enzymatic evaluation of Shikimate dehydrogenases AroE and YdiB .......... 53 Evaluation of YdiB under fermentor—controlled conditions ......................... 57 Quinate dehydrogenase (Qad) from Klebsiella pneumoniae ........................ 60 Shikimate dehydrogenase (AroD) from Bacillus subtilis ............................ 69 Directed evolution of E. coli Shikimate dehydrogenase (AroE) ................... 74 Discussion .................................................................................................. 80 References ..................................................................................................................... 83 vi CHAPTER FOUR ......................................................................................................... 86 Experimental .................................................................................................. 86 General methods ............................................................................................ 86 Spectroscopic measurements ...................................................................... 86 Bacteria strains and plasmids ......................................................................... 86 Storage of bacterial strains and plasmids .................................................... 88 Culture medium ......................................................................................... 88 Fed—batch fermentation (general) ................................................................ 89 Glucose-rich fermentor conditions ............................................................. 90 Glucose-limited fermentor conditions ......................................................... 91 Analysis of fermentation broths .................................................................. 91 Genetic manipulations .................................................................................... 92 General .......................................................................................................... 92 Large scale purification of plasmid DNA ................................................... 94 Small scale purification of plasmid DNA ................................................... 94 Determination of DNA concentration ......................................................... 95 DNA precipitation ...................................................................................... 95 Restriction enzyme digestion of DNA ........................................................ 95 Agarose gel electrophoresis ........................................................................ 96 Isolation of DNA from agarose .................................................................. 96 Treatment of vector DNA with calf intestinal alkaline phosphatase (CIAP) 97 Treatment of DNA with Klenow fragment ................................................. 97 Ligation of DNA ........................................................................................ 98 Preparation and transformation of competent cells ..................................... 98 Purification of genomic DNA ..................................................................... 99 Pl-mediated transduction ......................................................................... 100 Enzyme assays ......................................................................................... 102 Adenylate kinase assay ............................................................................. 102 DAHP synthase assay .............................................................................. 102 Phosphoenolpyruvate synthase assay ........................................................ 103 Transketolase assay .................................................................................. 104 Shikimate dehydrogenase assay in forward direction ................................ 105 Shikimate dehydrogenase assay in reverse direction ................................. 105 Quinate dehydrogenase assay ................................................................... 106 Chapter two ................................................................................................. 106 E. coli KL3AathH::FRT-tet-FRT ........................................................... 106 E. coli KL3AsucA::FRT-tet-FRT .............................................................. 107 Plasmid pJJ 1 .224A ................................................................................... 107 Plasmid pJJ 1 .262A ................................................................................... 107 Plasmid pJJ1.266A ................................................................................... 108 Chapter three ............................................................................................... 108 Purification of GST-tagged proteins ......................................................... 108 Protein gel (SDS-PAGE) .......................................................................... 109 Purification of K. pneumoniae quinate dehydrogenase .............................. 111 Km and Vm measurment of E. coli Shikimate dehydrogenases AroE and YdiB ........................................................................................ 112 vii Plasmid p112.134A ................................................................................... 112 Plasmid pJJ2.297A ................................................................................... 112 Plasmid pJJ3.041A ................................................................................... 113 Plasmid pJJ3.247A... ................................................................................ 113 Plasmid pJJ3.289AP ................................................................................. 113 Plasmid pJJ4.024A ................................................................................... 114 Plasmid pJJ4.025A ................................................................................... 114 Plasmid pJJ4.118A ................................................................................... 114 Plasmid pJJ4.150A ................................................................................... 115 Plasmid pJJ4. 171A ................................................................................... 115 PCR mutagenesis of aroE gene ................................................................ 115 Preparation of electrocompetent E. coli cells ............................................ 116 High-throughput screening of AroE mutant library ................................... 117 References ................................................................................................................... 1 19 viii Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Table 9. LIST OF TABLES KL3Aath H::F RT-tet-F RT characterization on minimal-salt plates. ................ 28 Concentrations and yields of 3-dehydroshikimic acid and byproducts synthesized by E. coli KL3 and KL3Aath H::F RT-tet-F RT bearing pKL5.17A and pJY1.216A plasmids. .............................................................. 34 Concentrations and yields of 3-dehydroshikimic acid and byproducts synthesized by E. coli KL3/pJJ 1 .266A. ........................................................... 37 PEP synthase and adenylate kinase specific activities for E. coli KL3/pJJ 1 .266A cultured under glucose-rich conditions. .................................. 38 Concentrations and yields of 3-dehydroshikimic acid and byproducts synthesized by E. coli KL3/pKL5.17A and KL3AsucA::FRT-tet-FRT/pKL5.17A under glucose-rich conditions ................ 39 3-Dehydroshikimic acid yield as a function of biomass. ................................... 42 Shikimic acid and 3-dehydroshikimic acid ratios, Shikimic acid yield and total hydroaromatic yield produced by recombinant E. coli under fermentor- controlled, glucose-rich conditions. ................................................................. 49 Shikimate dehydrogenase AroE and YdiB kinetic parameters for 3-dehydroshikimic acid. .................................................................................. 55 Shikimate dehydrogenase YdiB complementation assay. ................................. 56 Table 10. Concentrations and yield of Shikimic acid and byproducts synthesized by E. coli SP1.1/pKD12.138 and SP1.1/pJJ4.l71A under glucose-rich conditions. 60 Table 11. E. coli AB2834 complementation assay with plasmid pTW8090A localized quinate dehydrogenase. ................................................................................... 61 Table 12. Purification of K. pneumoniae Qad from E. coli DHSa/pTW8090A. ............. 62 Table 13. ClustalW alignment results of quinate dehydrogenases. ................................. 65 Table 14. Quinate dehydrogenase specific activity ......................................................... 65 Table 15A. Microorganzims with sequence similarities to Qad and AroE. ..................... 69 Table ISB. Microorganzims with sequence similarities to Qad and AroE. ..................... 70 Table 16. Mutant candidates chosen for second round of screening. .............................. 78 ix Table 17. Candidates after second round screening. ....................................................... 79 Table 18. Bacterial strains and plasmids. ....................................................................... 87 LIST OF FIGURES Figure 1. The Shikimate pathway and biosynthesis of quinic acid and gallic acid. ........... 3 Figure 2. Value-added chemicals synthesized from glucose via 3-dehydroshikimic acid intermediacy. ............................................................. 5 Figure 3. Use of Shikimic acid in synthetic chemistry. .................................................... 7 Figure 1. Glycolysis and biogenesis of D-erythrose 4-phosphate and phosphoenolpyruvic acid. .............................................................................. 21 Figure 2. E. coli futile cycle employing Ppc and Pck. ................................................... 23 Figure 6. ATP synthase assembly to the cytoplasmic membrane. .................................. 24 Figure 7. Futile cycle for ADP generation inside the cell. ............................................. 25 Figure 8A. Construction of plasmid pJJ 1.224A ............................................................. 29 Figure 9. E. coli KL3/pKL5.17A (A) and KL3AathH::FRT-tet-FRT/pKLS.17A (B) Figure 10. Figure 1 1. cultured under glucose-rich conditions ........................................................... 35 CO2 produced per dry cell weight. Legend: KL3/pKL5. 17A (solid line ), KL3AsucA::FRT-tet-FRT/pKL5.17A (dashed line). ..................................... 40 Proposed pathway for acetate formation during KL3AathH::FRT—tet- F RT/pKLS. 17A and KL3Aath H : :F RT-tet-F RT/pJ Y1 .216A fermentation. .................................. 42 Figure 12. Construction of plasmids pJJ2.134A and pJJ2.297A. ................................... 51 Figure 13. SDS-PAGE of E. coli YdiB purification. ..................................................... 52 Figure 14. SDS-PAGE of E. coli AroE purification. ..................................................... 53 Figure 15. Specific activity of YdiB as a function of pH. .............................................. 54 Figure 16. Inhibition of AroE and YdiB by Shikimic acid. ............................................ 55 Figure 17A. Construction of plasmid pJJ4.150A ............................................................ 58 Figure 18. E. coli SP1.1/pKD12.138 (A) and SP1.1/pJJ4.17lA (B) cultured under glucose-rich conditions. ............................................................................... 60 Figure 19. SDS-PAGE of K. pneumoniae Qad. ............................................................. 62 xi Figure 20. Inhibition profiles for Qad and AroE. .......................................................... 63 Figure 21. Klebsiella pneumoniae MGH78578 quinate dehydrogenase qad sequence. ..64 Figure 22. Construction of plasmid pJJ3.289AP. .......................................................... 66 Figure 23A. Construction of plasmid pJJ4.024A. .......................................................... 67 Figure 24. Construction of the plasmid pJJ4.118A. ....................................................... 72 Figure 25. SDS—PAGE of B. subtilis AroD purification. ............................................... 73 Figure 26. B. subtilis Shikimate dehydrogenase inhibition with Shikimic acid. .............. 74 Figure 27. Construction of plasmid pJJ3.247A .............................................................. 76 Figure 28. Shikimate dehydrogenase inhibition profile of JJ4679 F2, H5, A7, H7 mutants after shake-flask experiment ...................................................................... 79 xii Ac ADP ATP Ap ApR CIAP Cm CmR DAHP DCU DHQ DHS DO DTT E4P EMP FBR GA IPTG LIST OF ABBREVIATIONS acetyl adenosine diphosphate adenosine triphosphate ampicillin ampicillin resistance gene base pair calf intestinal alkaline phosphatase chloramphenicol chloramphenicol resistance gene 3-deoxy-D-arabino-heptulosonic acid 7-phosphate digital control unit 3-dehydroquinic acid 3-dehydroshikimic acid dissolved oxygen dithiothreitol D-erythrose 4-phosphate Embden-Meyerhof pathway feedback resistant gallic acid hour isopropyl B-D-thiogalactopyranoside xiii Kan KanR kb (‘0! kg LB M9 min mL mM pM NAD NADH NADP N ADPH OD ORF PEP PID kanamycin kanamycin resistance gene kilobase turnover number kilogram Michaelis constant Luria broth molar minimal salts minute milliliter microliter millimolar rnicromolar 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 optical density open reading frame phosphoenolpyruvic acid proportional-integral-derivative xiv PCR Phe psi PTS QA SA SDS Tc tet TCA TSP Tyr UV polymerase chain reaction L-phenylalanine pounds per square inch phosphotransferase system quinic acid rotations per minute Shikimic acid sodium dodecyl sulfate tetracycline tetracycline resistance gene tricarboxylic acid L-tryptophan sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 L-tyrosine ultraviolet XV CHAPTER ONE Introduction Much of the chemical industry is based on technologies where starting materials are derived from fossil fuels. As a nonrenewable natural resource, petroleum has several environmental and geopolitical problems associated with its use. For example, benzene is a potent carcinogen.l Interest in using renewable starting materials has grown with the increasing demand and cost of petroleum.2 In addition to the actual cost of petroleum, both the health costs‘ and the petroleum feedstocks have to be considered?3 Carbohydrates such as glucose, xylose and arabinose are abundant and can be used in biocatalytic processes to obtain products that are currently manufactured by the chemical industry using traditional technology.4 In the future, microbial synthesis alone or combined with chemical synthesis will grow and replace many chemical syntheses currently employedx" D-Glucose being renewable and the most abundant carbohydrate holds great potential as a starting material for microbial synthesis. Today, the majority of glucose used in microbial synthesis is derived from corn starch processed by wet milling operations. Another major source of glucose is cellulose. However, better technology for cellulose depolymerization must be developed in order to for cellulose to find use as a glucose source.6 To compete with chemical synthesis, microbial synthesis must produce value- added chemicals in a high yield and a high titer. There are two general methods to increase the yield and titer of desired products: direct more carbon flow into biosynthetic pathway and eliminate byproduct accumulation during biosynthesis. Chapter 2 of this thesis will focus on increasing the yield and titer of 3-dehydroshikimic acid (DHS) synthesized by an Escherichia coli strain. Strategies used for increasing 3- dehydroshikimic acid yield could also be applied for Shikimic acid and other molecules derived from 3-dehydroshikimic acid. Previous work in the Frost group explored microbial synthesis of 3-dehydroshikimic acid by genetically engineered E. coli strain KL3/pJY1.216A, which synthesized 69 g/L of 3-dehydroshikimic acid in 35% yield from glucose.7 Expression of plasmid-localized AroFFBR, which encodes feedback insensitive 3- deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase directed more carbon flow into the Shikimate pathway. Overexpression of plasimd-localized tktA, which encodes transketolase, and plasmid-localized ppsA, which encodes phosphoenolpyruvate synthase, increased the in vivo availability of D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP), which are the substrates for AroF‘q’R. Chapter 2 investigates whether an increase in glycolytic flux and reduction of CO2 and biomass formation has a positive effect on the 3-dehdyroshikimic acid concentration and yield. In chapter 3, a slightly different approach is taken. Previously in the Frost group, it was determined that Shikimic acid is a mixed type inhibitor of E. coli Shikimate dehydrogenase (AroE).8 A search for a feedback insensitive Shikimate dehydrogenase will be described. Two strategies were followed to find a Shikimate dehydrogenase that was less feedback inhibited by Shikimic acid than the wild-type E. coli AroE. The Shikimate pathway The Shikimate pathway exists in plants, bacteria and fungi, and is involved in the transformation of carbohydrate into aromatics including amino acids L-tyrosine, L- 2 phenylalanine and L-tryptophan, and vitamin precursors such as 2, 3-dihydroxybenzoate, p-hydroxybenzoate and p-aminobenzoate.9 Mammals are incapable of de novo biosynthesis of aromatics and must acquire these metabolites from their diet. The first step of the Shikimate pathway is the irreversible condensation of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to form 3-deoxy-D- arabino-heptulosonic acid 7-phosphate (DAHP), which is catalyzed by DAHP synthase (Figure 1). E. coli has three DAHP isozymes AroF, AroG and AroH, which are feedback inhibited, respectively, by L-tyrosine, L-phenylalanine and L-tryptophan.l0 OH O HZOSPOMH OH HO HQ HQ D-erythrose 4-phosphate ’- COZH ‘ COZH ' COZH AroF O AroB fl ‘—"—’ ——’ <— ‘. AroG ; OH O s OH HO‘ 2 OH 0P03H2 Am“ H203PO OH 0H 0H 3-dehydroquinic - - H . umlc 002 3-deoxy-o-arabino-heptulosonic acnd qacid phosphoenolpyruvic acid acid 7-phosphate 'AroD 002H C02H COzH COzH AroE ? AIOK a ' HO OH H203POW ; OH AI'OL HO‘ i OH O 5- OH OH OH OH OH shikimate 3-phOSphate shikimic 3-dehydroshikimic gallic acid acid acrd JAroA COZH C02H , L-phenylalanlne AroC ' L-tryptophan \v JL JL L-tyrosine H203HPO i O COzH i O COzH fofic acid OH OH 5-enolpyruvylshikimate chorismic acid 3-phosphate Figure 1. The Shikimate pathway and biosynthesis of quinic acid and gallic acid. Enzymes: DAHP synthase (AroF, AroG, AroH); 3-dehydroquinate synthase (AroB); 3- dehydroquinate dehydratase (AroD); Shikimate dehydrogenase (AroE); Shikimate kinase (AroK, AroL); 5-enolpyruvoylshikimate 3-phosphate synthase (AroA); chorismate synthase (AroC); unknown(?). DAHP is further converted to 3-dehydroquininc acid (DHQ) by aroB-encoded DHQ synthase.” A syn elimination of water from DHQ is catalyzed by 3-dehydroquinate dehydrogenase (AroD) and produces 3-dehydroshikimic acid.‘2 Reduction of 3- dehydroshikimic acid by NADPH affords Shikimic acid. The reaction is catalyzed by aroE—encoded Shikimate dehydrogenase,l3 which is feedback inhibited by Shikimic acid.8 A second putative Shikimate/quinate dehydrogenase isozyme YdiB was recently identified in E. coli.'4 The next step requires ATP and is catalyzed by two Shikimate kinase isozymes, AroK and AroL.l5 Phosphorylated Shikimic acid is a substrate for 5- enolpyruvoylshikimate 3-phosphate synthase encoded by the aroA gene, which catalyzes enolpyruvoyl addition to the C-5 hydroxy using PEP as a substrate.” The last enzyme of the Shikimate pathway is chorismate synthase (AroC).l7 It catalyzes 1,4-trans elimination of phosphate to form chorismic acid. Chorismic acid is the branching point for biosynthesis of aromatics. All three aromatic amino acids and aromatic vitamins are synthesized from this branching point. Regulation of the Shikimate pathway in E. coli occurs at the protein level via feedback inhibition and at the transcriptional level. The transcription of L-tyrosine- sensitive AroF and L-phenyla1anine-sensitive AroG isozymes is regulated by a transcriptional repressor encoded by the tyrR gene.l0 Transcription of L-tryptophan- sensitive AroH is regulated by the trpR gene product.10 The same transcription regulators control Shikimate kinase aroL transcription, while aroK is constitutively expressed in E. coli. '0 3-Dehydroshikimic acid 3-Dehydroshikimic acid is an intermediate in the Shikimate pathway and can be viewed as a branch point from which natural products can be biosynthesized without intermediacy of chorismic acid (Figure 2). The aromatization of 3-dehydroshikimic acid to protocatechuic acid is crucial to the value of 3-dehydroshikimic acid as an intermediate for the synthesis of several commodity and fine chemicals. Protocatechuic acid is a branch point in the biosynthesis of catechol's, cis, cis-muconic acid”, vanillinzo, gallic acid21 and pyrogallol.21 Hydrogenation of cis-cis-muconic acid affords adipic acid.'9 0 H OH 0 AOH i OH We" HO OH OH vanillin D-glulcose COZH COzH ‘— fl HOzc\/\/\COZH HO O s OH OH OH adipic acid protocatechuic acid DHS I I \I COZH CCOZH \ COZH HO _. O OH HO OH HO OH cis, cis-muconic catechol OH OH acid gallic acid PYFOQaIIOI Figure 2. Value-added chemicals synthesized from glucose via 3-dehydroshikimic acid intermediacy. The Frost group developed E. coli KL3/pJYl.216A as a construct capable of converting glucose into 3-dehydroshikimic acid.7 This 3-dehydroshikimic acid- synthesizing biocatalyst was constructed by disruption of the genomic aroE locus (Figure 1) in E. coli, which resulted in accumulation of 3-dehydroshikimic acid in the culture medium. Integration of a second aroB copy in the genome and plasmid-localized expression of feedback-insensitive, aroFFBR-encoded DAHP synthase, tktA-encoded transketolase and ppsA-encoded PEP synthase, increased the flow of carbon into the Shikimate pathway.7 Shikimic acid It has been demonstrated that Shikimic acid is a useful chiral compound. It is a six- membered carboxylic ring with three stereogenic centers, and an endocyclic olefin. Schreiber and coworkers have exploited it as the core scaffold for a large combinatorial library synthesis (Figure 3).22 Frost and coworkers reported that Shikimic acid also serves as an environmentally-friendly precursor for phenol23 and p-hydroxybenzoic acid (Figure 3).24 The importance of Shikimic acid increased with discovery of Tamiflu” (Figure 3), which is a potent inhibitor for Influenza A and Influenza B neuraminidases.25 Tamiflu was approved by the FDA and is currently marketed by Roche as an orally administered antiinfluenza drug. Use of Shikimic acid in Tamiflu synthesis and other synthetic applications was previously restricted by its price and availability. Before a microbial synthesis of Shikimic acid was developed by the Frost group the only source for Shikimic acid was isolation from the pericarps of Chinese star anise (lllicium sp. plants).26 cozn OH p-hydroxybenzoic acid 0 O O\/ R O §—N 0 ll cozH H”. N. : ‘— 6‘ 4— ‘— _’ O .” 04 :o:i:‘ H0“ i OH ’NH2 0“. H '0 “0 0 0“ j “”1.” O shikimic acid Tamiflu I Q OH phenol Figure 3. Use of shikimic acid in synthetic chemistry. Genetically engineered E. coli SP1.1/pKD12.138 was constructed for microbial synthesis of shikimic acid.” This biocatalyst was created by disrupting the Shikimate kinase-encoded aroK and aroL genomic loci in E. coli. Also integration of a second aroB copy in the genome and overexpression of plasmid-localized feedback insensitive aroFFBR- ‘ encoded DAHP synthase and tktA-encoded transketolase, permitted an increase of carbon flow into the Shikimate pathway, which ultimately translated into 52 g/L titer of shikimic acid synthesized in 18% yield from glucose. The SP1.1/pKD12.138 fermentation process has been scaled up and is licensed by Roche to provide an alternative source of shikimic acid for the manufacture of Tamiflu. Increasing of carbon flow into the Shikimate pathway Modifications of central metabolism in E. coli to channel more carbon flow into the Shikimate pathway has been the focus of considerable research activity.28 The first strategy employed was overexpression of feedback resistant AroFFBR 29 which helped to overcome feedback inhibition of DAHP synthase caused by aromatic amino acids.30 Insensitivity to feedback inhibition by aromatic amino acids increases the in vivo catalytic activity of DAHP synthase. Because of successfully increased carbon flow into the Shikimate pathway, resulting from increased DAHP activity, DAH accumulation together with increased 3-dehydroshikimic acid level was observed during E. coli A82834 cultivation.31 The absence of catalytically-active Shikimate dehydrogenase, which catalyzes conversion of 3-dehydroshikimic acid into the shikimic acid (Figure 1), in E. coli ABZ834 resulted in the accumulation of 3-dehydroshikimic acid in the culture medium. Accumulation of DAH, which results from DAHP dephosphorylation, lowers 3- dehydroshikimic acid and shikimic acid titer and yield. An approximately twofold increase in DHQ synthase specific activity is required8 to eliminate DAH accumulation which can be accomplished by introducing a second copy of aroB into the genome of E. coli.32 Several different routes were pursued for circumventing transcriptional repression of aroF.33 One strategy utilized two plasmid-localized copies of aroFmR, and it resulted in accumulation of 39 g/L of 3-dehydroshikimic acid synthesized in 16% yield, while one aroFFBR copy afforded 20 g/L of 3-dehydroshikimic acid synthesized in 17% yield.33 A further improvement in 3-dehydroshikimic acid synthesis was achieved once aroFFBR was overexpressed together with unmodified native promoter PamF, which presumably helped Lek!» to titrate away the cellular supply of TyrR protein. Accumulation of 41 g/L of synthesized 3-dehydroshikimic acid in 18% yield was observed.33 These results indicated that the best configuration was to include in the plasmid one copy of Pam; and aroFFBR under its native promoter. The same study also demonstrated that higher DAHP synthase activity did not necessarily translate into higher yields or titers of Shikimate pathway products. This indicated that the substrates for DAHP synthase were limiting factors in increasing carbon flow directed into the Shikimate pathway. Frost and coworkers published the first work suggesting that D-erythrose 4—phosphate (E4P) availability was an important factor limiting in viva DAHP synthase activity.34 The reason for low E4P availability might be due to its tendency to polymerize in solution.35 Dissociation back to the monomeric form of E4P is quite slow. This is probably the reason why E. coli cells keeps low steady-state concentrations of E4P. To increase the intracellular concentration of E4P, tktA-encoded transketolase was overexpressed, which led to an increased titer and yield of 3- dehydroshikimic acid28f and shikimic acid.27 With increased in viva E4P availability, availability of phosphoenolpyruvate (PEP) became a limiting factor. A number of different cellular processes and enzymes compete with DAHP synthase for PEP including pyruvate kinase (Figure 4), PEP carboxylase (Figure 5), and the PEP-dependent carbohydrate:phosphotransferase system (PTS) for transport of glucose and structurally related sugars inside the cell. Efforts to improve 28c,36 and intracellular PEP availability began with genomic inactivation of PEP carboxylase pyruvate kinase,37 which did not lead to significantly improved bioconversions of aromatic amino acids. A better strategy was reported by Liao and coworkers where they achieved an increase in DAH synthesized from glucose.“ 28" Liao used an E. coli aroB mutant (inactive 3-dehydroquinate synthase, Figure 1) with plasmid-localized aroGFBR-encoded feedback insensitive DAHP synthase, transketolase and PEP synthase. This strategy was based on recycling pyruvic acid, which is generated by PTS during glucose transport, back to PEP. The construction of E. coli strain KL3/pJY1.216A in the Frost group afforded the best 3-dehydroshikimic acid producer built so far.7 Plasmid pJY1.216A carried aroFFBR- encoded feedback-insensitive DAHP isozyme, tktA-encoded transketolase, ppsA-encoded PEP synthase and the PM. -encoded promoter region of DAHP synthase. The same approach was taken to create a better shikimic acid-producing biocatalyst in SP1.1/pKD15.071B.38 Another strategy was to use a non-PTS mechanism for glucose transport into the cell, thereby eliminating consumption of a mole of PEP for each mole of glucose transported into the cell. It was shown, that heterologous expression of Zymomonas mobilis plasmid-localized glf (glucose facilitator protein) and glk (glucose kinase) in a PTS deficient E. coli strain would reconstitute glucose transport and phosphorylation.39 A decrease of 3-dehydroshikimic acid was obtained in 3-dehydroshikimic acid-producing strain when compared to KL3/pJYl.216A results."'0 A slight increase in titer and yields of shikimic acid for shikimic acid-producing strain was observed when compared to SP1.1/pKD15.07lB results.38 Chapter 2 will focus on other methods for increasing the yield of 3- dehydroshikimic acid synthesized by E. coli. DAHP synthase substrates PEP and E4P are derived from glycolysis, so an increase in glycolytic flux would provide more starting material for DAHP synthase. On the other hand, carbon flow to biomass and CO2 formation contributes to a reduction in yields for biosynthesized products. The effect of 10 reducing CO2 and biomass formation and alternatively, increasing glycolytic flux will be examined for 3-dehydroshikimic acid-producing strains under fermentor-controlled conditions. Directed evolution Natural selection has created a large variety of enzymes superbly adapted to catalyze an array of chemical reactions. However, there are still too few enzymes available for catalyzing reactions of interest and many of these do not have optimal properties for use in biotechnological processes. There are two ways to obtain enzymes with desired properties. One traditional approach entails the screening of microorganisms from the strain collections or environmental isolates for desired enzyme activities. Due to the vast number of microorganisms on Earth, one might ultimately identify an enzyme with the desired properties. The biggest drawback with this method is the need to screen large numbers of microorganisms and enzymes. Bioinformatics is a complimentary strategy for searching and identification of new enzymes. Bioinformatic tools can narrow down possibilities in the search for new enzymes. However, the biggest limitation now is genomics and proteomics data availability. Even though bioinforrnatics can guide in the identification of new enzymes, wild-type enzymes might not have specificity for a particular substrate or will be kinetically slow and thus not useful in biocatalysis. Directed evolution“ has become a powerful technique for improving the properties of enzymes. Natural evolution took billions of years. In contrast, directed evolution is a relatively fast process and typically generates mutant libraries of 104 — 106 variants.42 The challenge of this tool is identification of mutant enzymes in the library, which possess the sought after properties. Directed evolution begins with generation of a mutant library of 11 the gene of interest. Genes with random mutations are inserted into an expression vector and the resulting plasmid is transformed into the microorganism. The second step, which is more labor intensive, entails the selection or screening of the library for the enzyme possessing the desired characteristics. Identified mutants with improved properties can be selected for the next round of directed evolution. Beyond random mutagenesis, there is DNA shuffling. This technique is based on fragmentation of parental genes with DNaseI and reassembly of the fragments to a full length gene by repeated cycles of overlap extension reactions.“b'° Another commonly used directed evolution method is error-prone polymerase chain reaction (EP-PCR) to introduce random point mutations across the gene.” PCR mutagenesis utilizes a low- fidelity polymerase such as Taq (isolated from Thermus aquaticus) in combination with reaction conditions (discussed in chapter 3) that promote point mutations. Identifying a desired mutant is the most challenging step of directed evolution. Selection might be used when an enzyme is essential for growth of the host organism.43 High-throughput selection is limited only by the size of the library. Screening, on the other hand, is limited not by the size of the library but by the throughput associated with evaluation of the mutants. During the screening process, each member of the library is assayed either by directly measuring enzyme activities or by indirectly measuring product formation or substrate depletion. Common analytical techniques used include calorimetry, high-performance liquid chromatography (HPLC) and gas chromatography. The screening is usually carried out in a 96-well or 384-well format microtiter plate and robotic instruments are employed to facilitate the process. Whole cells, cell lysates or partially purified enzymes can be used for the screening. The advantage of the screening 12 is that assays can be specifically designed for a desired enzyme, although such screens require more time to execute relative to employment of selection techniques. Over the past few years, directed evolution has proven to be a powerful tool. Protein engineering of P450 monooxygenases used rational evolution (protein design combined with directed evolution) and directed evolution to alter the substrate specificity and characteristics of the enzyme.44 Hiratke and coworkers reported increased amide- hydrolyzing activity of Pseudomonas aeruginosa lipase after a single round of error-prone mutagenesis.45 Significant results where obtained from directed evolution by the Romesberg and Schultz groups. DNA polymerase is a highly specific enzyme, which recognizes only four natural nucleotide bases. Romesberg and coworkers expanded this specificity and successfully incorporated 2’-0-methyl ribonucleoside in the DNA strand.46 Schultz and coworkers were the first to synthesize in vivo proteins containing unnatural amino acids473 after they evolved orthogonal aminoacyl-tRNA synthase and t-RNA pair.47b This demonstrates that directed evolution combined with an efficient selection method can be a very powerful tool. The attempts to identify and create feedback-insensitive Shikimate dehydrogenase will be discussed in Chapter 3. The inhibition with shikimic acid of the second E. coli Shikimate dehydrogenase YdiB, K. pneumoniae quinate dehydrogenase Qad and B. subtilis Shikimate dehydrogenase AroD was measured. Generation of an E. coli Shikimate dehydrogenase AroE mutant library by error-prone PCR and high-throughput screening of the mutant library will also be discussed in chapter 3. 13 REFEREN ES 1 (a) Wong, 0.; Raabe, G. K. Critical Review of Cancer Epidemiology in Petroleum Industry Employees, with a Quantitative Meta-Analysis by Cancer Site. Am. J. Ind. Med. 1989, 15, 283-310. (b) Lewis, R. J. Carcinogenically Active Chemicals, Van Nostrand Reinhold, New York, 1991, p. 68. (c) O’Connor, S. R.; Farmer, P. B.; Lauder, I. Benzene and Non-Hodgkin’s Lymphoma. J. Pathol. 1999, 189, 448-453. ((1) Lan Q.; Zhang, L.; Li, G.; Vermeulen, R.; Weinberg, R. S.; Dosemeci, M.; Rappaport, S. M.; Shen, M.; Alter, B. P.; Wu, Y.; Kopp, K.; Waidyanatha, S.; Rabkin, Ch.; Guo, W.; Chanock, S.; Hayes, R. B.; Linet, M.; Kim, S.; Yin, S.; Rothman, N.; Smith, M. T. Hematotoxicity in Workers Exposed to Low Levels of Benzene. Science, 2004, 306, 1774-1776. 2 (a) Tullo, A. Benzene Costs Hurt Customers. Chem. Eng. News 2004, 82, 15-17. (b) http://www.eia.doe.gov/oiaf/aeo/index.html. Annual Energy Outlook 2005 with Projections to 2025. 3 (a) Yoshida, J .; Inomata, M. Trends in Developments of Aromatics Production Technologies. Aramatikkusu 2002, 54, 123-135. (b) Tullo, A. H. A New Source. Chem. Eng. News 2003, 81, 16-17. 4 (a) Frost, J. W.; Lievense, J. Prospects for Biocatalytic Synthesis of Aromatics in the let- Century. New J. Chem. 1994, I8, 341. (b) Bongaerts, J .; Kramer, M.; Muller, U.; Raeven, L.; Wubbolts, M. Metabolic Engineering for Microbial Production of Aromatic Amino Acids and Derived Compounds. Metabal. Eng. 2001, 3, 289-300. 5 http://\V\\«'\v.eere.energygov/industry/chemicals/Visions biocatalysishtmI. Chemical Industry of the Future. 6 (a) Bozell, J. J.; Landucci, R. Alternate Feedstocks Program Technical and Economic Assessment; U. S. Department of Energy, Office of Industrial Technologies, 1993. (b) Lynd, L. R.; Cushman, J. H.; Nichols, R. J.; Wyman, C. E. Fuel Ethanol from Cellulose Biomass. Science 1991, 251, 1318-1323. (c) Zaldivar, J .; Nielson, J .; Olsson, L. Fuel Ethanol Production from Lignocellulose: A Challenge for Metabolic Engineering and Process Integration. Appl. Microbiol. Biotechnol. 2001, 56, 17-34. 7 Yi, J.; Li, K.; Draths, K. M.; Frost, J. W. Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli. Biatechnal. Prag. 2002, 18, 1141-1148. 8 Dell, K. A.; Frost, J. W. Idnetification and Removal of Impediments to Biocatalytic Synthesis of Aromatics from D-Glucose: Rate-Limiting Enzymes in the 14 Common Pathway of Aromatic Amino Acid Biosynthesis. J. Am. Chem. Soc. 1993, 115, 11581-11589. 9 (a) Haslam, E. In Shikimic Acid: Metabolism and Metabolites; Wiley: New York, 1993. (b) Bentley, R. The Shikimate Pathway - a Metabolic Tree with Many Branches. Crit. Rev. Biochem. Mol. Biol. 1990, 25, 307-384. (c) Herrmann, K. M. In Amino Acids: Biosynthesis and Genetic Regulation; Herrmann, K. M., Somerville, R. L., Ed.; Addison- Wesley: Reading, 1983: p. 301. 10 Pittard, A. J. Biosynthesis of the Aromatic Amino Acids. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. Neidhardt, F. C. ed. ASM Press (Washington, DC: American Society for Microbiology) 1996, p. 458-484. 11 (a) Frost, J. W.; Bender, J. L.; Kadonaga, J. T.; Knowles, J. R. Dehydroquinate Synthase from Escherichia coli: Purification, Cloning, and Construction of Overproducers of the Enzyme. Biochemistry 1984, 23, 4470-4475. (b) Carpenter, E. P.; Hawkins, A. R.; Frost, J. W.; Brown, K. A. Structure of Dehydroquinate Synthase Reveals an Active Site Capable of Multistep Catalysis. Nature 1998, 394, 299-302. 12 (a) Smith, B. W.; Turner, M. J .; Haslam, E. Shikimate Pathway. 4. Stereochemistry of 3-Dehydroquinate Dehydratase Reaction and Observations on 3-Dehydroquinate Synthetase. J. Chem. Sac., Perkins Trans. 1 1975, I, 52-55. (b) Duncan, K.; Chaudhuri, S.; Campbell, M. S.; Coggins, J. R. The Overexpression and Complete Amino Acid Sequence of Escherichia coli 3-Dehydroquinase. Biochem. J. 1986, 238, 475-483. 13 (a) Anton, I. A.; Coggins, J. R. Sequencing and Overexpression of the Escherichia coli araE Gene Encoding Shikimate Dehydrogenase. Biochem. J. 1988, 249, 319-326. (b) Chaudhuri, S.; Coggins, J. R. The Purification of Shikimate Dehydrogenase from Escherichia coli. Biochem. J. 1985, 226, 217—223. 14 (a) Michel, G.; Roszak, A. W.; Sauve, V.; Maclean, J.; Matte, A.; Coggins, J. R.; Cygler, M.; Lapthom, A. J. Structures of Shikimate Dehydrogenase AroE and Its Paralog YdiB. J. Biol. Chem. 2003, 278, 19463-19472. (b) Benach, J.; Lee, I.; Edstorrn, W.; Kuzin, A. P.; Chiang, Y.; Acton, T. 13.; Montelione, G. T.; Hunt, J. F. The 2.3-}. Crystal Structure of the Shikimate 5-Dehydrogenase Orthologue YdiB from Escherichia coli Suggests a Novel Catalytic Enviroment for an NAD-dependent Dehydrogenase. J. Biol. Chem. 2003, 278, 19176-19182. 15 (a) DeFeyter, R. C.; Pittard, J. Genetic and Molecular analysis of AroL, the Gene for Shikimate Kinase-II in Escherichia coli K-12. J. Bacterial. 1986, 165, 226-232. (b) DeFeyter, R. C.; Pittard, J. Purification and Properties of Shikimate Kinase-II From Escherichia coli K-12. J. Bacteriol. 1986, 165, 331-333. (c) Laner-Olesen, A.; Marinus, 15 M. G. Identification of the Gene (AroK) Encoding Shikimic Acid Kinase-I of Escherichia coli. J. Bacteriol. 1992, I 74, 525-529. 16 Duncan, K.; Coggins, J. R. The SerC-AroA Operon of Escherichia coli: A Mixed Function Operon Encoding Enzymes from 2 Different Amino Acid Biosynthetic Pathways. Biochem. J. 1986, 234, 49-57. (b) Duncan, K.; Lewendon, A.; Coggins. J. R. The Purification of 5-Enolpyruvylshikimate 3-Phosphate Synthase from an Overproducing Strain of Escherichia coli. F EBS Lett. 1984, 165, 121-127. 17 White, P. J .; Millar, G.; Coggins, J. The Overexpression, Purification and Complete Amino-Acid Sequence of Chorismate Synthase from Escherichia coli K12 and its Comparison with the Enzyme from Neuraspara crassa. Biochem. J. 1988, 251, 313- 322. 18 (a) Draths, K. M.; Frost, J. W. Environmentally Compatible Synthesis of Catechol from D-Glucose. J. Am. Chem. Soc. 1995, 117, 2395-2400. (b) Li, W.; Xie, D.; Frost, J. W.. Benzene-Free Synthesis of Catechol: Interfacing Microbial and Chemical Catalysis. J. Am. Chem. Soc. 2005, 127, 2874-2882. 19 (a) Draths, K. M.; Frost, J. W. Environmentally Compatible Synthesis of Adipic acid from D-Glucose. J. Am. Chem. Soc. 1994, 116, 399-400. (b) Niu, W.; Draths, K. M.; Frost, J. W. Benzene-Free Synthesis of Adipic Acid. Biatechnal. Prag. 2002, 18, 201-211. 20 Li, K.; Frost, J. W. Synthesis of Vanillin from Glucose. J. Am. Chem. Soc. 1998, 120, 10545-10546. 21 Kambourakis, S.; Draths, K. M.; Frost, J. W. Synthesis of Gallic Acid and Pyrogallol from Glucose: Replacing Natural Product Isolation with Microbial Catalysis. J. Am. Chem. Soc. 2000, 122, 9042-9043. 22 (a) Tan, D. S.; Foley, M. A.; Shair, M. D.; Schreiber, S. L. Stereoselective Synthesis of over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assays. J. Am. Chem. Soc. 1998, 120, 8565-8566. (b) Tan, D. S.; Foley, M. A.; Stockwell, B. R.; Shair, M. D.; Schreiber, S. L. Synthesis and Preliminary Evaluation of a Library of Polycyclic Small Molecules for Use in Chemical Genetic Assays. J. Am. Chem. Soc. 1999, 121, 90073- 9087. 23 Gibson, J. M.; Thomas, P. S.; Thomas, J. D.; Barker, J. L.; Chandran, S. S.; Harrup, M. K.; Draths, K. M.; Frost, J. W. Benzene-Free Synthesis of Phenol. Angew. Chem., Intl. Ed. 2001, 40, 1945-1948. 16 24 Barker, J. L.; Frost, J. W. Microbial Synthesis of p-Hydroxybenzoic Acid from Glucose. Biotechnol. Bioeng. 2001, 76, 376-390. 25 Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan, S.; Bischofberger, N .; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, W. G.; Stevens, R. C. Influenza Neuraminidase Inhibitors Possessing a Novel Hydrophobic Interaction in the Enzyme Active Site: Design, Synthesis, and Structural Analysis of Carbocyclic Sialic Acid Analogues With Potent Anti-influenza Activity. J. Am. Chem. Soc. 1997, 119, 681- 690. 26 Weber, W. F. Hoffmann-La Roche, Ltd., personal communication. 27 Knop, D. R.; Draths, K. M.; Chandran, S. S.; Barker, J. L.; von Daeniken, R.; Weber, W.; Frost, J. W. Hydroaromatic Equilibration During Biosynthesis of Shikimic Acid. J. Am. Chem. Soc. 2001, 123, 10173-10182. 28 (a) Draths, K. M.; Pompliano, D. L.; Conley, D. L.; Frost, J. W.; Berry, A.; Disbrow, G. L.; Staversky, R. J .; Lievense, J. C. Biocatalytic Synthesis of Aromatics from D-Glucose: The Role of Transketolase. J. Am. Chem. Soc. 1992, 114, 3956-3962. (b) Gubler, M., Jetten, M., Lee, S. H., Sinskey, A. J. Cloning of the Pyruvate-Kinase Gene (Pyk) of Corynebacterium glutamicum and Site-Specific Inactivation of Pyk in a Lysine- Producing Corynebacterium lactafermentum Strain. Appl. Env. Microbiol. 1994, 60, 2494- 2500. (c) Miller, J. E.; Backman, K.C.; O'Conner, M. J.; Hatch, R. T. Production of Phenylalanine and Organic-Acids by Phosphoenolpyruvate Carboxylase-Deficient Mutants of Escherichia coli. J. Ind. Microbial. 1987, 2, 143-149. ((1) Patnaik, 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. (e) Patnaik, R.; Spitzer, R. G.; Liao, J. C. Pathway Engineering for Production of Aromatics in Escherichia coli: Confirmation of Stoichiometric Analysis by Independent Modulation of AroG, TktA, and Pps Activities. Biotechnol. Bioeng. 1995, 46, 361-370. (f) Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R. M.; Frost, J. W. Fed-Batch Ferrnentor Synthesis of 3-Dehydroshikimic Acid Using Recombinant Escherichia coli. Biotechnol. Bioeng. 1999, 64, 61-73. 29 (a) Weaver, L. M.; Herrmann, K. M. Cloning of an AroF Allele Encoding a Tyrosine-Insensitive 3-Deoxy-D-arabina-Heptulosonate 7-Phosphate Synthase. J. Bacterial. 1990, 172, 6581. (b) Mikola, M. R.; Widman, M. T.; Worden, R. M. In Situ Mutagenesis and Chemotactic Selection of Microorganisms in a Diffusion Gradient Chamber. Appl. Biochem. Biotechnol. 1998, 70- 72, 905-918. 30 Ogino, T.; Garner, C.; Markley, J. L.; Herrmann, K. M. Biosynthesis of Aromatic- Compounds - l3C NMR-Spectroscopy of Whole Escherichia coli Cells. Prac. Natl. Acad. Sci. USA 1982, 79, 5828-5832. 17 31 Draths, K. M.; Frost, J. W. Genomic Direction of Synthesis During Plasmid-Based Biocatalysis. J. Am. Chem. Soc. 1990, 112, 9630-9632. 32 Snell, K. D.; Draths, K. M.; Frost, J. W. Synthetic Modification of the Escherichia coli Chromosome: Enhancing the Biocatalytic Conversion of Glucose Into Aromatic Chemicals. J. Am. Chem. Soc. 1996, 118, 5605-5614. 33 Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R. M.; Frost, J. W. Fed-Batch Fermentor Synthesis of 3-Dehydroshikimic Acid Using Recombinant Escherichia coli. Biotechnol. Bioeng. 1999, 64, 61-73. 34 (a) Draths, K. M.; Frost, J. W. Synthesis Using Plasmid-Based Biocatalysis: Plasmid Assembly and 3-Deoxy-D-Arabina-Heptulosonate Production. J. Am. Chem. Soc. 1990, 112, 1657-1659. (b) Frost, J. W. US. Patent 5,168,056, 1992. 35 Williams, J. F.; Blackmore, P. F.; Duke, C. C. MacLeod, J. K. Fact, Uncertainty and Speculation Concerning the Biochemistry of D-Erythrose-4-Phosphate and its Metabolic Roles. Int. J. Biochem. 1980, 12, 339-344. 36 Backman, K.C. US. Patent 5,169,768, 1992. 37 Mori, M.; Yokota, A. ; Sugitomo, S.; Kawamura, K. Patent JP 62,205,782, 1987. 38 Chandran, S. S.; Yi, J.; Draths, K. M.; von Daeniken, R.; Weber, W.; Frost, J. W. Phosphoenolpyruvate Availability and the Biosynthesis of Shikimic Acid. Biotechnol. Prag. 2003, 19, 808-814. 39 (a) Parker, C.; Barnell, W. 0.; Snoep, J. L.; Ingram, L. O.; Conway, T. Characterization of the Zymomanas mobilis Glucose Facilitator Gene Product (ng) in Recombinant Escherichia coli: Examination of Transport Mechanism, Kinetics and the role of Glucokinase in Glucose Transport. Mal. Microbial. 1995, 15, 795-802. (b) Snoep, J. L.; Arfman, N.; Yomano, L.- P.; Fliege, R. K.; Conway, T.; Ingram, L. 0. Reconstruction of Glucose Uptake and Phosphorylation in a Glucose-Negative Mutant of Escherichia coli by Using Zymomanas mabilis Genes Encoding the Glucose Facilitator Protein and Glucokinase. J. Bacterial. 1994, I 76, 2133-2135. (c) Weisser, P.; Kramer, R.; Sahm, H.; Sprenger, G. A. Functional Expression of the Glucose Transporter of Zymomanas mabilis Leads to Restoration of Glucose and Fructose Uptake in Escherichia coli Mutants and Provides Evidence for Its Facilitator Action. J. Bacteriol. 1995, 177, 3551-3554. 40 Yi, J .; Draths, K. M.; Li, K.; Frost, J. W. Altered Glucose Transport and Shikimate Pathway product Yields in E. coli. Biotechnol. Prag. 2003, 19, 1450-1459. 18 41 (a) Cadwell, R. C.; Joyce, G. F. Randomization of Genes by PCR Mutagenesis. PCR Methods Appl. 1992, 2, 28-33. (b) Stemmer, W. P. C. DNA Shuffling by Random Fragmentation and Reassembly: in vitra Recombination for Molecular Evolution. Prac. Natl. Acad. Sci. USA 1994, 91, 10747-10751. (c) Stemmer, W. P. C. Rapid Evolution of a Protein in vitra by DNA Shuffling. Nature 1994, 370, 389-391. 42 Schmidt-Dannert, C. Directed Evolution of Single Proteins, Metabolic Pathways and Viruses. Biochemistry 2001, 44, 13125-13136. 43 Fastrez, J. In Viva Versus In Vitra Screening or Selection for Catalytic Activity in Enzymes and Abzymes. Mal. Biotechnol. 1997, 7, 37-55. 44 Urlacher, V. B.; Lutz-Wahl, S.; Schmid, R. D. Microbial P450 Enzymes in Biotechnology. App. Microbial. Biotechnol. 2004, 64, 317-325. 45 Fujii, R; Nakagawa, Y.; Hiratake, J .; Sogabe, A.; Sakata, K. Directed Evolution of Pseudamanas aeruginasa Lipase for Improved Amide-hydrolyzing Activity. Protein Eng. Des. Sel. 2005, 18, 93-101. 46 Fa, M.; Redeghieri, A.; Henry, A. A.; Romesberg, F. E. Expanding the Substrate Repertoire of a DNA Polymerase by Directed Evolution. J. Am. Chem. Soc. 2004, 126, 1748-1754. 47 (a) Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G. Generation of Bacterium with a 21 Amino Acid Genetic Code. J. Am. Chem. Soc. 2003, 125, 935—939. (b) Liu, D. R.; Schultz, P. G. Progress Toward the Evolution of an Organism with an Expanded genetic Code. Prac. Nat. Acad. Sci. USA 1999, 96, 4780-4785. 19 CHAPTER TWO Modifying central metabolism of Escherichia coli Introduction A variety of strategies have been employed for improving the yields of Shikimate pathway metabolites synthesized by E. cali."2'3 These strategies focused on expression of feedback-insensitive DAHP synthase as well as increasing the availability of D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP). E4P and PEP are the substrates of DAHP synthase, which is the first enzyme in the Shikimate pathway and catalyzes an irreversible condensation to form 3-deoxy-D-arabina-heptulosonic acid 7-phosphate (DAHP) (Figure l). E4P is derived from the pentose phosphate pathway and PEP is formed in the Embden-Meyerhof pathway (glycolysis) as shown in Figure 4. Initial attempts to increase carbon flow into the Shikimate pathway examined overexpression of feedback-insensitive DAHP synthase isozymes.4 Frost and co-workers used a 3- dehydroshikimic acid-synthesizing strain to demonstrated that the increases in overexpression of DAHP synthase leads to increased accumulation of 3-dehydroshikimic acid and byproducts in the broth.lf However, they also successfully demonstrated that additional increases in DAHP synthase overexpression failed to have a positive impact, i.e. increased in synthesized 3-dehydroshikimic acid accumulating in the culture medium.1f Therefore, either availability of E4P or PEP had to be a limiting factor, since both molecules are used as the substrates for DAHP synthase. Overexpression of plasmid-localized tktA-encoding transketolase resulted in higher DAH production, which was interpreted as increase in E4P availability.’ A 3-dehydroshikirnic acid titer of 41 g/L 20 synthesized in 18% yield increased to 58 g/L synthesized in 24% yield once transketolase was overexpressed together with feedback-insensitive DAHP synthase in E. coli KL3/pKL5.17A.” 0 CPD H OH 3 2 OH /u\ll’ /‘cozH OH , OH 0 (Ox/1‘ ‘><‘PTS 0 "OH 0 am OH 5 0“ ADP ATP OH 2K“, H203PO OH i 0 HO OH pyruvic acid glucose 6-phosphate glucose PykA ATP Pgi Pku ADP ——>TktA H203PO\/'\)L H203”) 0“ a H CO H OH OH 2 OH h phosphoenolpyruvic acid D-e rose fructose 6—phosphate 4-pIr1Il3tsphate Eno ATP PkaADP 092 H203PO 0P03H2 ROWOH 0 0 OH OH R1=H; R2=P03H2 2—phosphoglycerate OH Gpm< =P H ;R -H 3- hos h I oerate fructose 1,6-diphosphate R1 03 2 2" p p ogy {ATP Fba Fba P k / \ g ADP o - OH Tpi OH Worm/"Cori +——- H203P0\/'\IrH fag. H203PO\/'\rOP03H2 N D NADH o dihydroxyacetone 0 phosphate 9gfig;%%g¥ge Pr 1 ,3-diphosphoglycerate Figure 4. Glycolysis and biogenesis of D-erythrose 4-phosphate and phosphoenolpyruvic acid. Enzymes: phosphoenolpyruvate:carbohydrate phosphotransferase (PTS); glucokinase (le); phosphoglucose isomerase (Pgi); phosphofructose kinase (Pfk); fructose 1,6-diphosphate aldolase (Fba); glyceraldehyde 3- phosphate dehydrogenase (Gap); phosphoglycerate kinase (ng); enolase (Eno); pyruvate kinase (PykA, Pku); transketolase (TktA). In order to eliminate PEP limitation, a couple of strategies have been employed.3'6""'° Liao and co-workers demonstrated that a two fold increase in DAHP 21 production can be achieved by overexpression of phosphoenolpyruvate synthase, an enzyme that converts pyruvate to PEP with concomitant hydrolysis of ATP.Id The combined positive effect on synthesized DAHP was observed once Liao and coworkers overexpressed transketolase and phosphoenolpyruvate synthase in the presence of feedback-insensitive DAHP synthase.“ Frost and co-workers demonstrated the combined effect of transketolase and phosphoenolpyruvate synthase overexpression by using E. coli KL3/pJY1.216A, which resulted in the best 3-dehydroshikimic acid-producing strain so far with accumulation of 69 g/L of 3-dehydroshikimic acid under glucose-rich conditions in 35% yield.3 Another strategy for increasing PEP availability employed a non phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) to phosphorylate and transport glucose across the cytoplasmic membrane. This strategy was based on the fact that during PTS, which is the system that E. coli employs for glucose transport, one molecule of PEP is used to phosphorylate and transport one molecule of sugar, therefore limiting PEP in viva availability. PEP used during PTS-mediated transport of glucose is converted to pyruvate, which is ultimately channeled to the tricarboxylic acid cycle (TCA). Therefore, E. coli lacking PTS-mediated transport and phosphorylation of glucose was used to increase in viva PEP availability. Frost and coworkers showed that E. coli pts‘ host strain JYl with plasmid pJY2.183A-localized transketolase and feedback-insensitive DAHP synthase overexpression synthesized 60 g/L of 3- dehydroshikimic acid in 34% yield.6 This showed an increase in 3-dehydroshikimic acid titer and yield when compared to KL3/pKL5.17A, which uses PTS for glucose transport (previously described). However, the increase was not as significant as for 22 KL3/pJYl.216A (previously described), which employs phosphoenolpyruvate synthase for PEP recycling during PTS transport of glucose. Glycolytic flux and ADP availability The availability of E4P and PEP is ultimately governed by glycolysis (Figure 4). Therefore, an increase in glycolytic flux should increase intracellular availability of E4P and PEP, which will translate into increased 3-dehydroshikimic acid titer and yield. Liao and coworkers have previously demonstrated that a twofold increase in glycolytic flux could be achieved by expressing plasmid-localized genes in E. coli that create futile cycles to consume ATP.7 He employed phosphoenolpyruvate carboxylase (Ppc) and phosphoenolpyruvate carboxykinase (Pck) to create a futile cycle between PEP and oxaloacetate which resulted in the net reaction in hydrolysis of ATP (Figure 5).7b Observed increase in oxygen and glucose consumption and excretion of fermentation products such as pyruvate and acetate, indicated an increase in glycolysis. CO2 PI fl HON/fir COZH Pck o o phosphfiriicqlpyruvic @213, ATP oxaloacetic acid 2 Figure 5. E. coli futile cycle employing Ppc and Pck. Jensen and coworkers tested the hypothesis as to whether ATP consumption by cellular processes determines the steady-state flux throughout glycolysis.8 He employed a PI subunit of H*ATPase that simply hydrolyzed ATP to ADP and Pi, without coupling to any metabolic pathways. The (FIF0)H*ATP synthase complex consists of two parts, the membrane integral part, F0, which forms a proton channel, and the cytoplasmic part, 23 F,, which interconverts proton motive force and free-energy in the form of ATP (Figure 6). His study showed that the [ATP]/ [ADP] ratio declined gradually as the expression of ATPase increased. The added ATPase activity resulted in up to a 70% increase in the rate of glycolysis.811 These results indicate that the majority of glycolytic flux control resides outside glycolysis, i.e. in enzymes that consume ATP. Ingram and coworkers developed a different approach. Instead of expressing a plasmid-localized F, subunit of ATPase they introduced a Aath H mutation in athBEFHAGDC Operon.9 Proteins F and H of the atp Operon are responsible for F, subunit attachment to the F0 subunit (Figure 6). Deletion of ath H prevents this attachment and releases the F, subunit into the cytoplasm. This ruptures oxidative ADP conversion to ATP, while the F, subunit is still active and catalyzes ATP hydrolysis to ADP and inorganic phosphate.9 H+ +‘/202 + e' —> HzO periplasm : L j cytoplasmic Electron Transport I:o membrane System cytoplasm ( W N DH AD+ ADP ATP i H. AAthH AtplBEFHAGDC Figure 6. ATP synthase assembly to the cytoplasmic membrane. 24 A different approach can be used for creating a futile cycle during PTS transport of glucose (Figure 7). Glucose is transported inside the cell using PTS and for each mole of glucose transported, one mole of phosphoenolpyruvate (PEP) is consumed, and one mole of pyruvate is produced (Equation 1, Figure 7). Previously a strategy employing plasmid-localized phosphoenolpyruvate synthase (ppsA) expression was used for PEP regeneration from pyruvate and ATP.3 Therefore, phosphoenolpyruvate synthase can also be interpreted as an ATPase (Equation 2, Figure 7). Adenylate kinase catalyzes the interconversion of AMP and ATP to two moles of ADP (Equation 3, Figure 7). The overall stoichiometry for the PTS system, phosphoenolpyruvate synthase, and adenylate kinase operating together is the conversion of 2 ATP to 2 ADP for every molecule of glucose transported inside the cell (Equation 4, Figure 7). PTS - glucose + pyruvic (1) glucose + phosphggircijolpyruwc ——> 6-phosphate acid . P sA (2) pyruvrc + ATP _p_> phosphoenolpyruvic + AMP + P, acid acid Adk (3) AMP + ATP _. 2 ADP (4) g|ucose + 2 ATP _, glucose 6-phosphate + 2ADP + P, Figure 7. Futile cycle for ADP generation inside the cell. Enzymes: phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS); phosphoenolpyruvate synthase (PpsA); adenylate kinase (Adk). As postulated before, the increase in glycolytic flux should ultimately translate into higher 3-dehydroshikimic acid titer and yield. Therefore, two strategies were employed to increase the rate of glycolysis. The first strategy introduced a Aath H mutation in 3-dehydroshikimic acid-producing E. coli host KL3. The second strategy was to express plasmid-localized adenylate kinase. The impact of strategies employed to increase the glycolytic flux was evaluated, based on the yield of the 3-dehydroshikimic 25 acid and Shikimate pathways byproduct mixture synthesized by E. coli constructs cultivated under fermentor-controlled conditions. Biomass formation and C02 generation Product yield during the microbial synthesis is limited not only by enzyme activity but also by the amount of the carbon source that is converted to cell biomass and catabolized to C02. Cell biomass formation is directly proportional to the number of ATP molecules synthesized per molecule of catabolized glucose as starting material.” This is the reason for the high yields and low biomass formation associated with anaerobic metabolism (4 ATP/glucose) versus aerobic fermentation (36 ATP/glucose). Under aerobic fermentative conditions glucose can be converted to high levels of cell biomass and C02.” This results in significant reduction of synthesized product yields during aerobic growth. During cultivation of E. coli under anaerobic conditions, CO2 as well as NADH formation are reduced by repression of 2-ketoglutarate dehydrogenase (SucAB).ll Ingram and coworkers have achieved similar levels of CO2 reductions and NADH formation during the aerobic growth of E. coli by introducing a mutation in the sucA gene.9 The same strategy in reduction of CO2 formation by introducing a AsucA mutation in the 3-dehydroshikimic acid-producing strain was evaluated and will be presented later in this chapter. Strains carrying the Aath H mutation should generate less biomass due to the inactivated oxidative phosphorylation pathway and a strain with the AsucA mutation should generate less CO2 due to inactivated 2-ketoglutarate. The effect of these mutations on synthesized 3-dehydroshikimic acid titer and yield was separately evaluated under fermentor-controlled conditions. 26 Biocatalyst design E. coli host strains KL3Aath H ::F RT-tet-F RT and KL3AsucA : :F RT-tet-F RT were constructed to evaluate the impact of Aath H and AsucA mutation on synthesized 3- dehydroshikimic acid titer and yield. E. coli KL3l2 carried a mutation in the Shikimate dehydrogenase gene araE, which results in 3-dehydroshikimic acid accumulation in fermentation broth (Figure 1). In addition, a second copy of 3-dehydroquinate synthase (aroB) was inserted into the serA locus that encode 3-phosphoglycerate dehydrogenase. Accumulation of DAH was previously observed with increased carbon flow into the Shikimate pathway.l3 Two genomic copies of aroB eliminated DAH accumulation and directed more carbon flow into the Shikimate pathway.l2 A second useful feature of aroB insertion in serA is that the strain is no longer capable of synthesizing L-serine. This serves as nutritional pressure on the strain to maintain a serA-encoding plasmid when the strain is cultured in minimal salts medium. KL3AathH::FRT—tet-FRT was made from E. coli KL3 by P1 phage mediated transduction using E. coli TC21 (W3110, AathH::FRT-tet-FRT) as a donor strain.9 E. coli strain KL3 bearing AathH::FRT-tet-FRT mutation was selected on LB/Tc plates. Integration of the AathH::FRT-tet-FRT mutation was confirmed by PCR analysis. Genomic DNA of control strain KL3 and the putative mutant KL3AathH::FRT-tet-FRT was isolated.[4 Primers corresponding to 5’ atpE (ATGGAAAACTGAATATG) and 5’ atpH (TTAAGACTGCAAGACGTC) were used in PCR analysis and afforded a 2.3 kb fragment for the mutant and 1.3 kb fragment for the control strain.9 Additionally, the mutant was characterized by the growth pattern on selective plates. KL3Aath H: :F RT- tet-F RT failed to grow on succinate-minimal plates in the absence of glucose (Table 1).9 27 This demonstrates that the oxidative phosphorylation pathway was not operational since cells could not grow in the absence of fermentable carbon source, i.e. employing substrate level phosphorylation that is used for ATP generation during anaerobic cultivation. ‘5 Table 1. KL3AathH::FRT-tet-FRT characterization on minimal-salt plates. Strain M9/glucose/aros/ser‘ M9/succinate/aros/ser KL3 + + KL3AathH::FRT-tet-FRT + _ ”Abbreviations: aromatic amino acids (phenylalanine, tyrosine and tryptophan) and aromatic vitamins (2,3-dihydroxybenzoic acid, p-aminobenzoic acid and p- hydroxybenzoic acid) (aros), serine (ser). KL3AsucA::FRT-tet-FRT was made from E. coli KL3 by P1 phage mediated transduction using E. coli T C25 (W3110, AsucA::FRT-tet-FRT)9 as a donor strain to inactivate 2-ketoglutarate dehydrogenase. E. coli strain KL3 bearing the AsucA::FRT-tet- F RT mutation was selected on LB/Tc plates. The successful integration of the AsucA::FRT-tet-FRT mutation was confirmed by PCR analysis. Genomic DNA of control strain KL3 and putative mutant KL3AsucA::F RT-tet-F RT was isolated.” Primers corresponding to 5’ sucA (ATGCAGAACAGCGCTTTG) and 5’ (T'I‘TI‘CGACGTTCAGCGC) were used in PCR analysis and afforded 3.3 kb for KL3AsucA::F RT-tet-F RT mutant and 2.8 kb for the control strain.9 The inactivation of 2- ketoglutarate dehydrogenase (AsucA) resulted in an undesirable auxotrophic requirement for succinate during growth on glucose-minimal medium. A serial dilution method was used to isolate the KL3AsucA::FRT-tet-FRT (Succ+) mutant. Cells were grown in minimal medium containing glucose and decreasing amounts of succinate (4 mM to 0.2 mM), as described previously.9 Each time after cells were transferred to the glucose- minimal medium lacking succinate, no growth was observed. Therefore, in subsequent 28 experiments succinate was supplemented at 2 g/L concentration when glucose-minimal medium was used for cell growth. BamHI Smal Sail . EcoRl HIndIII P180 PCR adktrom RB791 genomic DNA 11) Smal and Sail digest Smal Sail 0.9 kb \waufifij/ adk 1) Smal and Sail digest 2) CIAP treatment T4 Ligase Sail Hinolll Smal adk EooFIl Ptac pJJ1.224A 6.2 kb Iale Figure 8A. Construction of plasmid pJJ 1.224A. Plasmid pJJ1.266A (Figure 8C) was constructed to evaluate the impact of adenylate kinase (adk) overexpression. As discussed earlier, adenylate kinase overexpression was expected to create a futile cycle for ATP hydrolysis during PTS-mediated glucose transport inside the cell (Figure 7). Plasmid construction began with amplification of a 0.9 kb adk” locus from RB791 genomic DNA that was 29 subsequently inserted into the pJF118EHl7 vector to afford plasmid pJJ1.224A in which adk is transcribed from the vector-encoded Pm sequence (Figure 8A). Confirmation that adenylate kinase was expressed from PM. promoter of pJJ1.224A relied on assay of adenylate kinase. Adenylate kinase specific activity for DH5a/pJJ 1.224A strain was 6.4 U/mg while E. coli DHSa/pJFl 18EH specific activity was 0.6 U/mg, therefore indicating a tenfold achieved overexpression of adenylate kinase. BamHl Sail Hindl I | PCR Pmadk from pJJ1.224A 11) San digest Sail Smal Sail I I 1.2 kb I Pm adk 1) Sail digest 2) CIAP treatment T4 Ligase Sail BamHl p 881‘ BamHl ”0F pJJ1 .262A 12.3 kb A y. // , w” EcoHI Figure 8B. Construction of plasmid pJJ1.262A. 30 Amplification of the 1.2 kb Pmadk fragment from pJJ 1.224A plasmid and subsequent insertion into pJY1.211A3 resulted in plasmid pJJ 1.262A (Figure SB). The final plasmid pJJ 1.266A was constructed by insertion of tktA, which was liberated from pMFS 1A'8 and ligated into plasmid pJJ 1.262A (Figure 8C). Sail pMF51A 1) BamHl digest 2) Klenow treatment (BamHl) 2.4 kb ECORI 353333Ifiiiirirjjzgzgziizflgz 1) Hindlll digest WI 2) Klenow treatment 3) CIAP treatment T4 Ligase BamHI San BamHI (Hindlll) (Smal) (Smal) . ' R Eco” 14.7 kb AP 1,: ppsA // EcoFil Figure 8C. Construction of plasmid pJJ 1.266A. 31 The requirement for plasmid-localized serA locus was described earlier. In addition, plasmid pJJ 1.266A contains phosphoenolpyruvate (PEP) synthase encoded by ppsA ORF, which is directly inserted behind a Pm promoter and ribosomal binding site. Overexpression of PpsA was required because it recycled pyruvic acid generated during PTS-mediated glucose transport, back to PEP and it was essential for futile cycle creation, as previously described (Equation 2, Figure 7). Because the final plasmid has lacIQ-encoded lac repressor, the expression of adenylate kinase and PEP synthase was controlled by the concentration of isopropyl B-D-thioglucopyranoside (IPTG) in the culture medium. E. coli host strain KL3 and its derivatives required supplementation with aromatic amino acids and aromatic vitamins, due to inactive Shikimate dehydrogenase, when cultured in glucose-minimal medium. To overcome feedback inhibition of DAHP synthase caused by the aromatic amino acid supplements, plasmid- localized feedback-insensitive DAHP synthase encoded by aral'v"FBR was required. An additional copy of plasmid-localized PmF was shown to increase expression levels of DAHP synthase,If presumably by titrating the cellular supply of TyrR repressor protein.” It also has been shown that increased expression of plasmid-localized tktA resulted in higher 3-dehydroshikimic acid titer and yield during the fermentation5 due to increased availability of D-erythrose 4-phosphate inside the cell (Figure 4). Fermentation conditions The impact of E. coli genetic modifications on the yields and concentrations of synthesized 3-dehydroshikimic acid and Shikimate pathway byproducts was evaluated under fed-batch controlled conditions. Fermentations were run under glucose-rich and glucose-limited conditions in a 2.0 L working volume fermentor. A concentration range 32 of 55-170 mM glucose in the fermentation medium was maintained by manually adjusting the rate of glucose addition under glucose-rich conditions while a steady state concentration of approximately 0.2 mM glucose was maintained under glucose-limited conditions. During cultivation under glucose-limited conditions, glucose addition was controlled automatically by maintaining a steady concentration of dissolved oxygen. Glucose-rich conditions can lead to excessive generation of acetic acid, which is toxic to E. coli and many other microbes. 2° Glucose-limited conditions minimize generation of acetic acid but can lead to excessive CO2 generation resulting in lower product yields.20 Thus both glucose-rich and glucose-limited conditions were evaluated for 3- dehydroshikimic acid and Shikimate pathway byproducts synthesis. Temperature was maintained at 36 °C and pH was maintained at 7.0. Dissolved oxygen concentration was maintained at 20% of air saturation under both glucose-rich and glucose-limited conditions. All ferrnentations were run in duplicate and reported results represent the average of two runs. Effect of AathH mutation on 3-dehydroshikimic acid and the Shikimate pathway byproducts synthesized by E. coli E. coli host KL3AathH::FRT-tet-FRT bearing pKL5.17A or pJY1.216A plasmids were tested under glucose-rich conditions. Dissolved oxygen concentration was maintained at 20% air saturation by varying impeller speed. A fermentation run was terminated when the amount of 3-dehydroshikimic acid synthesized in a six h time period was less than 5%. This resulted in fermentation times that varied between 42 and 60 hours, depending on the host and plasmid that were used. Host strains bearing pJY1.216A required addition of IPTG for expression of plasmid localized PEP synthase. 33 An optimal concentration of 12 mg/L of IPTG was previously determined3 and this quantity was initially added at the 18 hour mark and then every 6 hours until the end of the fermentation. Starting at the 12 hour mark, aliquots of the culture medium were removed every 6 hours and concentrations of 3-dehydroshikimic acid, 3-dehydroquinic acid, gallic acid, 3-deoxy-D-arabina-heptulosonic acid (DAH), and acetic acid were determined by 1H NMR using response factors determined with authentic samples of all molecules (Table 2). Table 2. Concentrations and yields of 3-dehydroshikimic acid and byproducts synthesized by E. coli KL3 and KL3AathH ::FRT-tet-F RT bearing pKL5.17A and pJY1.216A plasmids. Entry Strain Time [DHsr DHS [DAH] [DHQ] [GA] Total 33:13:? [acetate] (h) (g/L) Yield” (g/L) (g/L) (g/L) Yield‘ L‘L/L) (g/L) 1“I KL3/pKL5.17A 42 46 26% 6.9 6.5 7.5 37% 19 0.6 2" KL3AathH::FF?T-tet-FRT/ 54 15 12% 0 2.3 2.5 15% 14 3.3 pKL5.17A 3" KL3/pJY1.216A 48 61 34% 7.5 6.9 5.2 46% 22 0.1 4” KL3AathH::FRT-tet-FFIT/ 60 11 13% O 0 0 13% 13 0.6 pJY1.216A “Glucose-rich conditions. ”Glucose-limited conditions. CAbbreviations: 3-dehydroshikimic acid (DHS), 3-deoxy-D-arabina-heptulosonic acid (DAH), 3-dehydroquinic acid (DHQ), gallic acid (GA). d (mol DHS)/(mol glucose consumed). ‘ (mol DHS + mol DAH + mol DHQ + mol GA)/(mol glucose consumed). 3-DEHYDROSHIKIMIC ACID titer and yield for KL3AathH::FRT—tet- FRT/pKL5.17A construct decreased to 15 g/L and 12% (entry 2, Table 2) while control strain KL3/pKL5.17A produced 46 g/L of 3-dehydroshikimic acid in a 26% yield (entry 1, Table 2). New constructs produced less biomass (14 g/L) (entry 2, Table 2), however the yield of 3-dehydroshikimic acid was not improved. Acetate accumulation in the broth increased 6-fold and at the end of KL3AathH::FRT-tet-FRT/pKLS.17A fermentation was 3.3 g/L of acetate, while the control strain (entry 1, Table 2) had only 0.6 g/L. An increase in acetate production under aerobic condition usually indicates that cells are not 34 operating at optimal efficiency due to an imbalance between glucose uptake and glucose ' The rate of cell growth and 3- consumed by biosynthesis and energy production.2 dehydroshikimic acid formation was slower than during the control experiment (Figure 9) and they required an additional 12 h of growth to reach the best possible titer of 15 g/L. Accumulation of byproducts 3-dehydroquinic acid and gallic acid decreased proportionally to 3-dehydroshikimic acid accumulation (3-fold) and was 2.3 g/L and 2.5 g/L respectively at the end of fermentation (entry 2, Table 2). No accumulation of DAH was observed while during KL3/pKL5.17A cultivation, 6.9 g/L of DAH accumulated. A. B 50* e l - s E 401 __________________ —.__. ...- E .9 1 .9 s : — s 78 303 -------------- II-m ---- -—- E E —- E '2): 20: ————————— 1L——i r——I 0——1‘I-—- '2): .‘E . £9. 8 I 8 <. 10: ------ °--- -—-1 t"‘ ---- --- <. (D . (D I I l :l: D 011.1- C 12 18 24 30 36 42 12 18 24 30 36 42 48 54 Time (h) Time 00 Figure 9. E. coli KL3/pKL5.17A (A) and KL3AathH::FRT-tet-FRTIpKL5.17A (B) cultured under glucose-rich conditions. Legend: 3-dehydroshikirnic acid (open bars), acetic acid (black bars), dry cell weight (black circles). Glucose-limited conditions were employed to eliminate acetate production. Acetate levels decreased from 3.3 g/L (entry 2, Table 2) to 0.6 g/L (entry 3, Table 2) but it was still a little bit higher than the 0.1 g/L obtained during KL3/pJYl.216A fermentation (entry 4, Table 2). KL3Aath H:.°F RT-tet—F RT/pJY1.216A strain showed a 35 sixfold decrease in 3-dehydroshikimic acid concentration with its synthesis of 11 g/L (entry 4, Table 2) as compared to the control experiment where 61 g/L (entry 3, Table 2) was synthesized by E. coli. Even with decreased acetate levels, 3-dehydroshikimic acid was synthesized in only 13% yield (entry 4, Table 2) while control strain KL3/pJYl.216A synthesized 3-dehydroshikimic acid in 34% yield (entry 3, Table 2). A similar decrease in biomass formation (13 g/L) was observed under glucose-limited conditions as compared to cultivation under glucose-rich conditions (14 g/L). Fermentation also required an additional 12 h and the total time required to achieve the highest 3-dehydroshikimic acid concentration was 60 h. None of the fermentation byproducts such as 3-dehydroquinic acid, gallic acid and DAH, was observed during KL3AathH::FRT-tet-FRT/pJYl.216A cultivation under glucose-limited conditions (entry 4, Table 2). Effect of adenylate kinase (adk) expression on 3-dehydroshikimic acid and the Shikimate pathway byproducts synthesized by E. coli Glucose-rich and glucose-limited culture conditions were explored to evaluate the effect of adenylate kinase overexpression on 3-dehydroshikimic acid synthesis (Table 3). The titer of 3-dehydroshikimic acid synthesized by E. coli KL3/pJJ 1.266A under glucose-rich conditions was 64 g/L (entry 2, Table 3), and was comparable to the 62 g/L produced by control strain KL3/pJY1.216A (entry 1, Table 3). No significant effect was observed for the formation of byproducts with DAH, 3-dehydroquinic acid and gallic acid accumulating at 12 g/L, 8.2 g/L and 4 g/L, respectively (entry 2, Table 3). Biomass formation (24 g/L) and acetate accumulation (0.2 g/L) did not change significantly and cultivation time remained at 42 h (entry 2, Table 3) as compared to the control 36 experiment (entry 1, Table 3). Synthesized 3-dehydroshikimic acid yield (35 %) and total yield (49%) was similar to the control experiment (entry 1, Table 3) 33% and 47%, respectively. An increase in the synthesized titer of 3-dehydroshikimic acid was observed under glucose-limited conditions, where KL3/pJJ 1.266A synthesized 74 g/L of 3- dehydroshikimic acid in 36% yield (entry 4, Table 3) as compared to the control strain KL3/pJYl.216A 61 g/L and 34%, respectively (entry 3, Table 3). E. coli KL3/pJJ 1.266A showed slightly elevated levels of DAH (10 g/L) and 3-dehydroquinic acid (9.8 g/L) accumulation in the broth, while gallic acid and acetate were produced to the same extent (entry 4, Table 3). The cultivation time of 48 h was the same for KL3/pJY1.216A and KL3/pJJl.266A under glucose-limited conditions (entry 3 and 4, Table 3) and was 6 h longer than under glucose-rich conditions (entry 1 and 2, Table 3). Even though an increase in 3-dehydroshikimic acid and byproduct titers was observed, the total synthesized yield of 3-dehydroshikimic acid and byproducts remained almost the same (47%) as during KL3/pJY1.216A cultivation (46%). Table 3. Concentrations and yields of 3-dehydroshikimic acid and byproducts synthesized by E. caliKL3/pJJ1.266A. Dry cell . Time [DHS]° DHs [DAH] [DHQ] [GA] Total . [acetate] Em” S"""" (n) (Q/L) Yield" (g/L) (g/L) (GIL) Yield‘ “(g/‘3',“ (g/L) 1" KL3/pJY1.216A 42 62 33% 11 7.5 6.2 47% 22 0.3 2‘ KL3/pJJ1.266A 42 64 35% 12 8.2 4.0 49% 24 0.2 3" KL3/pJY1.216A 48 61 34% 7.5 6.9 5.2 46% 22 0.1 4" KL3/pJJ1.266A 48 74 36% 10 9.8 5.3 47% 25 0.1 “Glucose-rich conditions. ”Glucose-limited conditions. ‘Abbreviations: 3—dehydroshikimic acid (DHS), 3-deoxy-D-arabina-heptulosonic acid (DAH), 3-dehydroquinic acid (DHQ), gallic acid (GA). 4 (mol DHS)/(mol glucose consumed). ‘ (mol DHS + mol DAH + mol DHQ + mol GA)/(mol glucose consumed). 37 Plasmids pJY1.216A and pJJ 1.266A required IPTG addition to the fermentation medium. The optimal concentration of 12 mg/L of IPT G for pJY1.216A was determined before and this led to a slight decline (0.07 — 0.05 U/mg) in the specific activity of phosphoenolpyruvate synthase during the course of the microbial synthesis.3 In contrast to pJY1.216A, plasmid pJJ 1.266A has two Pm promoters while pJY1.216A has only one. Initial experiments showed that KL3/pJJ 1.266A fermentation with 12 mg/L IPT G had a stable 0.03 U/mg of phosphoenolpyruvate synthase specific activity during the course of fermentation (entry 1, Table 4). A decision was made to increase the concentration of IPTG to 24 mg/L every six h for all KL3/pJJ 1.266A fermentations. Phosphoenolpyruvate synthase showed similar declining specific activity from 0.08 U/mg to 0.02 U/mg as previously reported3 for 3-dehydroshikimic acid production during KL3/pJYl.216A fermentation under glucose-rich conditions (0.07 - 0.05 U/mg). The increase in IPTG concentration resulted in increased adenylate kinase specific activity that was relatively stable at approximately 7 U/mg over the course of the fermentation once IPTG addition started after 18 h from the beginning of fermentation. Adenylate kinase overexpression of almost tenfold was achieved after addition of 24 mg/L of IPTG (42 h time point, entry 2, Table 4) when compared to wild-type activity (18 h time point before was taken before IPTG addtion). Table 4. PEP synthase and adenylate kinase specific activities for E. coli KL3/pJJ 1.266A cultured under glucose-rich conditions. Ent IPTG PEP synthase (U/mg)” Adenylate kinase (U/mg)“ ry addn-’ 18 h 30 h 42 h 18 h 30 h 42 h 1 12 0.010 0.03 0.03 0.81 2.8 4.5 2 24 0.010 0.08 0.02 0.81 6.9 7.4 " Amount of IPT G (mg) added at 18, 24, 30, and 36 hours. b One unit (U) of PEP synthase corresponds to consumption of 1 umole of pyruvate per min at 30 °C. " One unit (U) of adenylate kinase corresponds to formation of 1 umole of ATP per min at 25 °C.22 38 Effect of AsucA mutation on 3-dehydroshikimic acid and the Shikimate pathway byproducts synthesized by E. coli E. coli KL3AsucA::FRT-tet-FRT/pKLS.17A was used to evaluate the effect on 3- dehydroshikimic acid titer and yield by the deletion of the sucA-encoded subunit of 2- oxoglutarate dehydrogenase. 3-Dehydroshikimic acid was synthesized at 7.8 g/L concentration and 6% yield (entry 2, Table 5) and showed a significant decrease in the synthesized yield and titer of 3-dehydroshikimic acid relative to the 46 g/L synthesized by KL3/pKL5.17A in 26% yield (entry 1, Table 5). A notable increase to 9.6 g/L of acetate production during KL3AsucA::FRT-tet-FRT/pKL5.17A fermentation as well as decrease from 19 g/L (entry 1, Table 5) to 12 g/L in biomass was observed (entry 2, Table 5). Fermentation time was increased to 48 h in order to achieve the highest 3- dehydroshikimic acid titer. No accumulation of DAH or 3-dehydroquinic acid and a decrease in gallic acid (0.8 g/L) was observed during KL3AsucA::FRT-tet- F RT/pKLS. 17A cultivation (entry 2, Table 5) while KL3/pKL5.17A synthesized DAH, 3- dehydroquinic acid and gallic acid in titer of 6.9, 6.5 and 7.5 g/L respectively (entry 1, Table 5). Table 5. Concentrations and yields of 3-dehydroshikimic acid and byproducts synthesized by E. coli KL3/pKL5.17A and KL3AsucA::FRT-tet-FRT/pKL5.17A under glucose-rich conditions. Dry cell . Time [DHS]‘ DHS [DAH] [DHQ] [GA] Total . [acetate] entry 3“” (h) (g/L) Yield" (g/L) (g/L) (g/L) Yield“ "$3? (g/L) 1 KL3/pKL5.17A 42 46 26% 6.9 6.5 7.5 37% 19 0.6 2 KL3AsucA::FRT-tet-FRT/ 48 7.8 6% o o 0.8 6% 12 9.6 pKL5.17A “Abbreviations: 3-dehydroshikimic acid (DHS), 3-deoxy-D-arabina-heptulosonic acid (DAH), 3-dehydroquinic acid (DHQ), gallic acid (GA). ” (mol DHS)/(mol glucose + mol sodium succinate consumed). ‘(mol DHS + mol DAH + mol DHQ + mol GA)/( mol glucose + mol sodium succinate consumed). 39 As was described earlier, deletion of the sucA gene encoding 2-ketoglutarate dehydrogenase should reduce CO2 formation. In order to verify this hypothesis, the fermentor exhaust line was connected to Sartorius/BBI Systems Gas Analyzer, which permitted online measuring of CO2 concentration in the exhaust gas. As shown in Figure 10, CO2 produced per dry cell weight by KL3AsucA::FRT-tet-FRT/pKL5.17A strain (dashed line) was decreased as compared to control KL3/pKL5.17A strain (solid line). 25 20 002 / Dry Cell O T l I I I Y I 0 6 12 18 24 30 36 42 48 Time (h) Figure 10. CO2 produced per dry cell weight. Legend: KL3/pKL5.17A (solid line ), KL3AsucA : :F RT-tet-F RT /pKL5. 17A (dashed line). Discussion Both gene deletions (athH and sucA) resulted in opposite results for what had been anticipated. Mutants possessing the sucA deletion required succinate supplementation when cultivated under minimal salt conditions in the presence of glucose as a carbon source. A significant reduction in CO2 formation was observed for KL3AsucA::FRT-tet-FRT/pKL5.17A strain as had been expected, although inactivation of sucA resulted in the synthesis of a significantly lower yield of 3-dehydroshikimic acid. 40 Mutants bearing a Aath H mutation showed also decreased formation of 3- dehydroshikimic acid, DAH, 3-dehydroquinic acid and gallic acid as compared to the control strains cultivated on both glucose-rich and glucose-limited conditions. Reduction in biomass was observed for both Aath H and AsucA mutants. E. coli strain KL3AathH::FRT-tet-FRT/pKLS.17A as well as KL3AsucA::FRT-tet-FRT/pKL5.17A exhibited increased acetate formation when cultivated under glucose-rich conditions. This increase in acetate levels could be explained by the pathway described in Figure 11. The majority of ATP produced in E. coli under aerobic conditions is from oxidative phosphorylation (Figure 6).15 Deletion of athH genes disrupts the oxidative phosphorylation pathway inside E. coli (Figure 6) thus leading to ATP starvation. Formation of ATP is directly proportional to the biomass formation” and the introduced AathH mutation led to less biomass accumulation during the fermentation process (Table 2). On the other hand, E. coli with the AathH mutation lies somewhere in the middle of anaerobic and aerobic metabolism, i.e. it is not able to regenerate ATP using the oxidative phosphorylation pathway, but it is able to use oxygen as an external electron acceptor for reoxidation of NADH to NAD*. It is well known that the major route for ATP generation under anaerobic conditions is from phosphate acetyltransferase and the acetate kinase pathway.23 Pyruvate formate-lyase is involved in NADH regeneration during anaerobic growth of E. coli.24 The low 3-dehydroshikimic acid titer and yields under glucose-rich and glucose- limiting conditions can now be explained. First of all, carbon flow is directed into formation of acetate and CO2 (Figure 11). Secondly, pyruvate is used in ATP and NADH regeneration. 41 o )ROH P" A Pta )0L 0 Ack i CoA " o 7 S: 7 S: o-P-OH 7 S OH NAD' NADH. Pi CoA 0” ADP ATP PYFUVIC acet l-CoA acet l hos hate acetic acid y y p p acid Figure 11. Proposed pathway for acetate formation during KL3AathH::FRT-tet- FRT/pKL5.17A and KL3AathH::FRT-tet-FRT/pJY1.216A fermentation. Enzymes: pyruvate formate-lyase (Pfl); phOSphate acetyltransferase (Pta); acetate kinase (Ack). Previously, plasmid pJY 1.216A was used to recycle pyruvate back to PEP due to expression of a plasmid-localized phosphoenolpyruvate synthase (ppsA) .3 KL3Aath H : :F RT-tet-F RT/pJYl.216A fermentation was run under glucose-limited conditions in order to recycle pyruvate back to PEP and to eliminate acetate accumulation in the broth. However, pyruvate formate-lyase (Figure 11) competes with phosphoenolpyruvate synthase expression and employing plasmid pJYl.216A did not increase 3-dehydroshikimic acid titer and yield (entry 4, Table 2). The effects on reduced biomass and CO2 generation are compared in Table 6. All newly constructed mutants had much smaller cell catalytic efficiency (entries 2, 4 and 5) as compared to the control strains (entries 1 and 2). Mutants possessing AsucA mutation produced least 3-dehydroshikimic acid (0.7 g) per each gram of biomass, while KL3/pJYl.216 produced four times more 3-dehydroshikimic acid (2.7 g) per each gram of biomass. Table 6. 3-Dehydroshikimic acid yield as a function of biomass. Entry Strain Cell catalytic efficiencyc 1‘ KL3/pKL5.17A 2.4 2" KL3AathH::FFiT-tet-FFIT/pKLS.17A 1 .1 3" KL3/pJY1.216A 2.7 4" KL3AathH::FRT-tet-FHT/pKL5.17A 0.9 5" KL3AsucA::FRT-tet-FH T/pKL5.1 7A 0.7 “Glucose-rich conditions. bGlucose-limited conditions. “(gram DHS)/(gram dry cell). 42 More promising results were obtained from expression of plasmid-localized adenylate kinase (adk). With a successful tenfold overexpression level of adenylate kinase (entry 2, Table 4) an increase in 21% of 3-dehydroshikimic acid titer was obtained under glucose-limited conditions, while there were no effects during fermentation under glucose-rich conditions. Glucose is the limiting factor, thus an increase in glycolytic flux under glucose-limiting conditions has a higher impact than under glucose-rich conditions. This suggests that glycolytic flux is not a limiting factor for higher 3-dehydroshikimic acid production under glucose-rich conditions. Adenylate kinase overexpression did not have any effect on total yield of Shikimate pathway byproducts. 43 REFEREN E 1 (a) Draths, K. M.; Pompliano, D. L.; Conley, D. L.; Frost, J. W.; Berry, A.; Disbrow, G. L.; Staversky, R. J.; Lievense, J. C. Biocatalytic Synthesis of Aromatics from D-Glucose: The Role of Transketolase. J. Am. Chem. Soc. 1992, 114, 3956-3962. (b) Gubler, M.; Jetten, M.; Lee, S. H.; Sinskey, A. J. Cloning of the Pyruvate-Kinase Gene (Pyk) of Corynebacterium glutamicum and Site-Specific Inactivation of Pyk in a Lysine- Producing Corynebacterium lactafermentum Strain. Appl. Env. Microbiol. 1994, 60, 2494-2500. (c) Miller, J. E.; Backman, K. C.; O’Conner, M. J.; Hatch, R. T. Production of Phenylalanine and Organic-Acids by Phosphoenolpyruvate Carboxylase- Deficient Mutants of Escherichia coli. J. Ind. Microbiol. 1987, 2, 143-149. ((1) Patnaik, 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. (e) Patnaik, R.; Spitzer, R. G.; Liao, J. C. Pathway Engineering for Production of Aromatics in Escherichia coli: Confirmation of Stoichiometric Analysis by Independent Modulation of AroG, TktA, and PpsA Activities. Biotechnol. Bioeng. 1995, 46, 361-370. (1') Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R. M.; Frost, J. W. Fed- Batch Fermentor Synthesis of 3-Dehydroshikimic Acid Using Recombinant Escherichia coli. Biotechnol. Bioeng. 1999, 64, 61-73. 2 (a) Flores, N.; Xiao, J .; Berry, A.; Bolivar, F.; Valle, F. Pathway Engineering for the Production of Aromatic Compounds in Escherichia coli. Nat. Biotechnol. 1996, 14, 620-623. (b) Chen, R.; Yap, W. M. G. J.; Postma, P. W.; Bailey, J. E. Comparative Studies of Escherichia coli Strains Using Different Glucose Uptake Systems: Metabolism and Energetics. Biotechnol. Bioeng. 1997 , 56, 583-590. (c) Chen, R.; Hatzimanikatis, V.; Yap, W. M. G. J .; Postma, P. W.; Bailey, J. E. Metabolic Consequences of Phosphotransferase (PTS) Mutation in a Phenylalanine-Producing Recombinant Escherichia coli. Biotechnol. Prag. 1997, I 3, 768-775. ((1) Baez, J. L.; Bolivar, F.; Gosset, G. Determination of 3-Deoxy-D-Arabina-Heptulosonate 7-Phosphate Productivity and Yield from Glucose in Escherichia coli Devoid of the Glucose Phosphotransferase Transport System. Biotechnol. Bioeng. 2001, 73, 530-535. (e) Flores, S.; Gosset, G.; Flores, N.; De Graaf, A. A; Bolivar, F. Analysis of Carbon Metabolism in Escherichia coli Strains with an Inactive Phosphotransferase System by l3C Labeling and NMR Spectroscopy. Metabal. Eng. 2002, 4, 124-137. 3 Yi, J.; Li, K.; Draths, K. M.; Frost, J. W. Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli. Biotechnol. Prag. 2002, 18, 1141-1148. 4 (a) Ogino, T.; Garner, C.; Markley, J. L.; Herrmann, K. M. Biosynthesis of Aromatic Compounds: l3C NMR Spectroscopy of Whole Escherichia coli Cells. Prac. Natl. Acad. Sci. USA 1982, 79, 5828-5832. (b) Weaver, L. M.; Herrmann, K. M. Cloning of an AroF Allele Encoding a Tyrosine-Insensitive 3-Deoxy-D-Arabina-Heptulosonate 7- Phosphate Synthase. J. Bacterial.l990, 172, 6581-6584. 5 Draths, K. M.; Pompliano, D. L.; Conley, D. L.; Frost, J. W.; Berry, A.; Disbrow, G. L.; Staversky, R. J .; Lievense, J. Biocatalytic Synthesis of Aromatics from D-Glucose: the Role of Transketolase. J. Am. Chem. Soc. 1992, 114, 3956-3962. 6 Yi, J.; Draths, K. M.; Li, K.; Frost, J. W. Altered Glucose Transport and Shikimate Pathway Product Yields in E. coli. Biotechnol. Prag. 2003, 19, 1450-1459. 7 (a) Pantiak, R.; Roof, W. D.; Young, R. F.; Liao, J. C. Stimulation of Glucose Catabolism in Escherichia coli by a Potential Futile Cycle. J. Bacterial. 1992, 174, 7527- 7532. (b) Chau, Y.-P.; Liao, J. C. Metabolic Responses to Substrate Futile Cycling in Escherichia coli. J. Biol. Chem. 1994, 269, 5122-5126. 8 (a) Koebmann, B. J.; Westerhoff, H. V.; Snoep, J. L.; Nilsson, D.; Jensen, P. R. The Glycolytic Flux in Escherichia coli Is Controlled by the Demand for ATP. J. Bacterial. 2002, 184 , 3909-3916. (b) Koebmann, B. J.; Westerhoff, H. V.; Snoep, J. L.; Solem, C.; Pedersen, M. B.; Nilsson, D.; Michelsen, 0.; Jensen, P. R. The Extent to which ATP Demand Controls the Glycolytic Flux Depends Strongly on the Organism and Conditions for Growth. Mal. Biol. Rep. 2002, 29, 41-45. 9 Causey, T. 8.; Zhou, S.; Shanmugam, K. T.; Ingram, L. 0. Engineering the Metabolism of Escherichia coli W3110 for the Conversion of Sugar to Redox-Neutral and Oxidized Products: Homoacetate Production. Prac. Natl. Acad. Sci. U.S.A. 2003, 100, 852-832. 10 Gottschalk, G. Bacterial Metabolism, 2nd Ed.; Springer-Verlag: New York, 1986, Chapter 3. 11 (a) Park, S.-J.; Chao, G.; Gunsalus, R. P. Aerobic Regulation of the SucABCD Genes of Escherichia coli, Which Encode a-Ketoglutarate Dehydrogenase and Succinyl Coenzyme a Synthase: Roles of ArcA, Fnr, and the Upstream sdhCDAB Promoter. J. Bacterial. 1997, 179, 4138-4142. (b) Cunningham, L.; Guest, J. R. Transcription and Transcript Processing in the sdhCDAB-sucABC D Operon of Escherichia coli. Microbiology 1998, 144, 2113-2123. 12 Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R. M.; Frost, J. W. Fed-Batch Fermentor Synthesis of 3-Dehydroshikimic Acid Using Recombinant Escherichia coli. Biotechnol. Bioeng. 1999, 64, 61-73. 45 13 Snell, K. D.; Draths, K. M.; Frost, J. W. Synthetic Modification of the Escherichia coli Chromosome: Enhancing the Biocatalytic Conversion of Glucose Into Aromatic Chemicals. J. Am. Chem. Soc. 1996, 118, 5605-5614. 14 Pitcher, D. G.; Saunders, N. A.; Owen, R. J. Rapid Extraction of Bacterial Genomic DNA with Guanidium Thiocyanate. Letters in Applied Microbiology 1989, 8, 151-156. 15 Neijssel, O. M.; De Matos, M. J. T.; Tempest, D. W. Growth Yield and Energy Distribution. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed.; Neidhardt, F. C., Ed.; ASM Press: Washington, DC, 1996; Pp 1683-1692. 16 Brune, M.; Schumann R.; Wittinghofer. Cloning and Sequencing of Adenylate Kinase Gene (Adk) of Escherichia coli. Nucleic Acids Res. 1985, 13, 7139-7151. 17 Furste, J. P.; Pansegrau, W.; Frank, R.; Blocker, H.; Scholz, P.; Bagdasarian, M.; Lanka, E. Molecular Cloning of the Plasmid Rp4 Primase Region in a Multi-Host-Range Tacp Expression Vector. Gene 1986, 48, 119-131. 18 Farabaugh, M. A. Biocatalytic Production of Aromatics from D-Glucose. MS. Thesis, Michigan State University, 1996. 19 Cobbett, C. S.; Delbridge, M. L. Regulatory Mutants of the AroF-TyrA Operon of Escherichia coli K-12. J. Bacterial. 1987, 169, 2500-2506. 20 (a) Konstantinov, K. B.; Nishio, N.; Yoshida, T. Glucose Feeding Strategy Accounting for the Decreasing Oxidative Capacity of Recombinant Escherichia coli in Fed-Batch Cultivation for Phenylalanine Production. J. Ferment. Bioeng. 1990, 70, 253- 260. (b) Konstantinov, K. B.; Nishio, N.; Seki, T.; Yoshida, T. Physiologically Motivated Strategies for Control of the Fed-Batch Cultivation of Recombinant Escherichia coli for Phenylalanine Production. J. Ferment. Bioeng. 1991, 71, 350-355. (c) Kleman, G. L.; Strohl, W. R. Acetate Metabolism by Escherichia coli in High-Cell-Density Fermentation. Appl. Environ. Microbial. 1994, 60, 3952-3958. 21 El-Mansi, E. M. T.; Holms, W. H. Control of Carbon Flux to Acetate Excretion During Growth of E. coli in Batch and Continuous Culture. J. Gen. Microbial. 1989, 135, 2875-2883. 22 Tanizawa, Y.; Kishi, F.; Kaneko, T.; Nakazawa, A. High Level Expression of Chicken Muscle Adenylate Kinase in Escherichia coli. J. Biochem., 1987, 101, 1289- 1296. 46 23 Bbck, A.; Sawers, G. Fermentation. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed.; Neidhardt, F. C., Ed.; ASM Press: Washington, DC, 1996; Pp 262-282. 24 Kessler, D.; Knappe, J. Anaerobic Dissimilation of Pyruvate. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed.; Neidhardt, F. C., Ed.; ASM Press: Washington, DC, 1996; Pp 199-205. 47 CHAPTER THREE A search for feedback-insensitive Shikimate dehydrogenase Introduction Many strategies have been developed to improve titers and yields of shikimic acid synthesized by E. coli. An increase in the intracellular concentration of phosphoenolpyruvate showed that more carbon flow was directed into the Shikimate pathway when feedback insensitive DAHP synthase and transketolase were overexpressed.l The benchmark strain for shikimic acid production, SP1.1/pKD12.138, had overexpressed araE-encoded Shikimate dehydrogenase, tktA-encoded transketolase and araFFBR-encoded DAHP synthase and synthesized 52 g/L of shikimic acid from glucose in 18% yield and 24% total combined yield of shikimic acid, 3-dehydroshikimic acid and quinic acid.2 Two strategies were employed to increase PEP availability. E. coli SP1.1/pKD15.071B with expression of plasmid-localized ppsA-encoded phosphoenolpyruvate synthase in addition to AroFFBR, TktA and AroE, synthesized 66 g/L of shikimic acid in 23% yield and a combined 29% yield of shikimic acid and byproducts.la This strategy relied on the fact that pyruvate, which is generated from PEP during PTS glucose transport, was recycled back to PEP by phosphoenolpyruvate synthase. The second strategy employed Glf-mediated glucose transport by facilitated diffusion in a PTS inactive E. coli strain SP1.1pts/pSC6.090B. Shikimic acid was synthesized at 71 g/L in a 27% yield and combined 34% yield was achieved in this case. The strategies examined so far for improving shikimic acid biosynthesis were based on increasing carbon flow into the Shikimate pathway. As described earlier they 48 employed increasing specific activities of DAHP synthase and transketolase, and increasing intracellular phosphoenolpyruvate levels. With increased carbon flow into the Shikimate pathway, synthesized shikimic acid yield was increased from 12% to 23% and the total hydroaromatic yield was increased from 15% to 29% (Table 7).” 2 However, the molar ratio between shikimic acid and 3-dehydroshikimic acid declined from 5.9 to 4.1 (Table 7). Previous research determined that accumulation of 3-dehydroshikimic acid and quinic acid is caused by hydroaromatic equilibration where shikimic acid is transported back inside the cell and converted to quinic acid through intermediacy of 3- dehydroshikimic acid and 3-dehydroquinic acid? It was also shown that glucose-rich conditions successfully inhibited quinic acid formation and afforded shikimic acid in higher yield and titer? Previous research has also determined that Shikimate dehydrogenase AroE inhibition by shikimic acid is a linear mixed-type inhibition with an inhibition constant (K ,) 0.16 mM.3 Therefore, accumulation of 3-dehydroshikimic acid (decrease in SA/DHS) during hydroaromatic equilibration can be caused by shikirrric acid feedback inhibition of Arc. Table 7. Shikimic acid and 3-dehydroshikimic acid ratios, shikimic acid yield and total hydroaromatic yield produced by recombinant E. coli under fermentor- controlled, glucose-rich conditions. Entry Strain :ielevant characteristics Ratio‘ “86:1,, giggle 1 SP1.1/pKD12.1 12 serA, aroF‘B“, PmcaroE 5.9 12% 15% 2 SP1.1/pKD12.138 serA, araFFB", PmcaroE, tktA 4.7 18% 24% 3 SP1.1pts/pSC6.090A pts‘lserA, era/58“, PmcaroE, tktA, Pm glf glk 4.7 21 % 28% 4 SP1.1/pKD15.071B serA, araFFBR, anaraE, tktA, ppsA 4.1 23% 29% “(produced shikimic acid)/(produced 3-dehydroshikimic acid). b(mol shikimic acid)/(mol glucose consumed). ‘(mol shikimic acid + mol 3-dehydroshikimic acid + mol quinic acid)/(mol glucose consumed). Removing shikimic acid feedback inhibition of Shikimate dehydrogenase might decrease 3-dehydroshikimic acid accumulation and therefore increase the titer and yield 49 synthesized shikimic acid. Two strategies were developed to obtain a feedback insensitive Shikimate dehydrogenase. One strategy called for identification of a non-E. coli Shikimate dehydrogenase that was insensitive to shikimic acid inhibition, followed by heterologous expression of the enzyme in an E. coli shikimic acid-producing strain. The biggest obstacle to this strategy was to identify a Shikimate dehydrogenase that was insensitive to shikimic acid inhibition from all of the Shikimate dehydrogenases found in nature. The screening of each Shikimate dehydrogenase would be time consuming and labor intensive. The second strategy for obtaining a feedback insensitive Shikimate dehydrogenase utilized directed evolution of E. coli wild-type Shikimate dehydrogenase. It was previously shown that use of feedback insensitive DAHP synthase led to increased carbon flow into the Shikimate pathway.4 The screening of a library of Shikimate dehydrogenase mutants was thus pursued. The advantage of this method is that screening protocols were automated and performed faster than manual screening. Purification of E. coli Shikimate dehydrogenases YdiB and AroE Previously in the literature, E. coli YdiB was named a putative Shikimate dehydrogenase, because it showed activity towards shikimic acid5 and the E. coli ydiB gene is in the same operon as another Shikimate pathway enzyme araD-encoded 3- dehydroquinate dehydratase. Crystal structures of YdiB and AroE were found to be very similar, even though ydiB share only 25% nucleotide sequence identity with araE.5a It was also demonstrated that YdiB catalyzes conversion of shikimic acid into 3- dehydroshikimic acid and conversion quinic acid into 3-dehdyroquinic acid in the presence of NADP“ and NAD*.5“ However, no inhibition or in viva studies were 50 performed with YdiB. Therefore, enzymatic evaluation of E. coli wild-type YdiB seemed reasonable and began with purification of the enzyme. Amplification of the ydiB and araE ORF’s from wild-type E. coli W3110 genomic DNA with subsequent insertion into EcoRI site of pGEX-4T-1 vector afforded plasmids pJJ2.134A and pJJ2.297A, respectively (Figure 12). The glutathione transferase (GST) encoding gene sequence was linked to the ydiB and araE ORF through the thrombin cleavage site recognition sequence (Figure 12). Salt Smal Xhol PCR ydiB from W3110 genomic DNA 50°F" N0" PCR araE from W3110 genomic DNA BamHl Thrombin 1) EcoRl digest GST P EcoFII E0081 “3" 0.9 kb ydiB 500m EcaFil 0.9 kb araE 1) E0081 digest 2) CIAP treatment T4 Ligase San Smal Xhol EcoFil N011 EcaFil , BamHl “”8 Thrombin \.‘ '\\ pJJ2.134A 5.8 kb Figure 12. Construction of plasmids pJ12.134A and pJJ2.297A. 51 Overexpression of YdiB was carried out in E. coli BL21/pJJ2.134A. Transcription of the ydiB sequence with an N-terrninus GST tag was performed from Pm promoter after addition of IPTG to the culture medium. Cell lysate was applied to a Glutathione Sepharose column and unbound proteins were washed out. Thrombin was then applied to the column and YdiB was cleaved from column bound GST. YdiB protein solution (Figure 13) was obtained after elution of YdiB through a benzamidine column that helped to capture thrombin due to benzamidine’s high affinity binding to serine protease. An identical procedure was used to purify AroE using construct BL21/pJJ2.297A (Figure 14). ma 1 2 3 4 5 6 7 8 . _ 10 kDa 205 I ' . ‘7 m l. 1 - i" , ' .‘ 205 116 1 116 97 97 66 66 45 I 45 29 29 __ , . I 1,,- r, . ., .. ”$211334, 4‘ Figure 13. SDS-PAGE of E. coli YdiB purification. Legend: molecular weight markers (1, 10), crude lysate (2), flow-through after Glutathione Sepharose column (3), last wash of Glutathione Sepharose column (4), l — 5 fractions collected after benzamidine column (5 - 9). Molecular weight: YdiB (32 kDa), GST (29 kDa), Thrombin (37 kDa). 52 kDa 205 116 97 66 45 29 Figure 14. SDS-PAGE of E. coli AroE purification. Legend: molecular weight markers (1), crude lysate (2), flow-through after Glutathione Sepharose column (3), l - 3 fractions collected after benzamidine column (4 — 6). Molecular weight: AroE (30 kDa), GST (29 kDa), Thrombin (37 kDa). Enzymatic evaluation of Shikimate dehydrogenases AroE and YdiB Shikimate dehydrogenase activity is routinely measured in the reverse direction by monitoring the reduction of NADP" during shikimic acid conversion to 3- dehydroshikimic acid.‘5'5 In this study Shikimate dehydrogenase activity had to be measured in forward direction since shikimic acid was used as an inhibitor and not as a substrate. Conversion of 3-dehydroshikimic acid to shikimic acid with subsequent oxidations of NADPH was monitored at 340 nm.6c One unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 umol of NADPH per minute in the presence of 3-dehydroshikimic acid. There was no reported literature precedent for YdiB assay in the forward direction. The pH optimum of YdiB was determined by measuring the enzyme specific activity at varying pH (Figure 15). The optimal pH was determined to be 5.3. YdiB assays were subsequently carried out at pH 5.3, and AroE assays were performed at pH 7.0 as described by Coggins.6c 53 100 Specific activity (U/mg) Figure 15. Specific activity of YdiB as a function of pH. For enzymatic evaluation of AroE and YdiB, Km, and kcan were determined (Table 8). The K, for 3-dehydroshikimic acid was determined under the following conditions: NADPH was held constant at 0.2 mM (K m = 0.03)3 and 3-dehydroshikimic acid concentrations were 0.03, 0.05, 0.06, 0.1, 0.2 mM. A Km = 0.1 mM was measured for AroE, which is in good agreement with the literature reported value of 0.072 mM.3 As Table 8 indicates, the Km value for 3-dehydroshikimic acid is 100 times lower for AroE (0.1 mM) relative to YdiB (10 mM). In addition, the turnover number km, for AroE (361 s") is significantly higher than for YdiB (83 3"). These findings lead to the conclusion that the primary Shikimate dehydrogenase in E. coli is AroE. YdiB assays were subsequently carried out at pH 5.3 in the presence of 20 mM of 3-dehydroshikirnic acid and AroE assays were performed at pH 7.0 with a 0.2 mM concentration of 3— dehydroshikimic acid . 54 Table 8. Shikimate dehydrogenase AroE and YdiB kinetic parameters for 3-dehydroshikimic acid. Enzyme Km (mM) km, s‘1 AroE 0.10 361 YdiB 10 83 Inhibition of YdiB and AroE by shikimic acid was measured at range 0 - 20 mM of shikimic acid (Figure 16). The results indicate that YdiB shows no inhibition at the 20 mM concentration of shikimic acid while AroE is 90% inhibited at the same concentration. This suggests that YdiB might be used instead of AroE in the shikimic acid-producing strain. 100 0 0 ., o o so:———————————3—+ ————————— o 0 R? 2.. so —-—-~o- ------------------ C .9 E 40 no? ————————————————————— E .. - O 20 31,0- —————————————————————— 0 0 :H—v—r—r-rfi—r—I—I—Q V—Ifi 111111 0 5 10 15 20 Shikimate (mM) Figure 16. Inhibition of AroE and YdiB by shikimic acid. Legend: AroE (diamonds), YdiB (circles). To evaluate YdiB activity in viva, complementation assays were performed. E. coli strain AB2834 lacks Shikimate dehydrogenase activity due to mutation in the araE gene.7 AB2834 is not capable of growing on glucose as the exclusive source of carbon in minimal salt medium without aromatic amino acid and aromatic vitamin supplementation or alternatively in the absence of shikimic acid supplementation? Plasmid pJY1.43 55 (CmR, PmaraE in pSU18) was used as the control and plasmid pJJ3.041A (ApR, PmydiB in pKK223-3) was used in the complementation experiment. Growth of E. coli AB2834/pJY1.43A and AB2834/pJJ3.041A were tested on a series of glucose minimal salts conditions as shown in Table 9. AB2834 was capable to grow only on glucose minimal salts medium supplemented with all aromatic amino acids and aromatic vitamins. AB2834/pJJ 1.043A showed regular size of colonies at 24 h on glucose minimal salts medium (Table 9). These control experiment results are the same as previously reported by Pittard and Wallace.7 E. coli AB2834 carrying plasmid encoded- ydiB showed colonies at 24 h only when culturing on glucose minimal salts medium supplemented with all aromatic amino acid and aromatic vitamins. It took 120 h for AB2834/pJJ3.041A colonies to appear on M9/glucose plate. The same results were obtained when complementation experiments were repeated on glucose minimal salts medium in the presence of chloramphenicol for AB2834/pJY1.43A and ampicillin for AB2834/pJJ3.041A. Antibiotics were added to ensure plasmid maintenance inside the cell during the 120 h cultivation. Table 9. Shikimate dehydrogenase YdiB complementation assay. . ,, A82834 AB2834/pJY1 .043A AB2834/@3041 A Selection Plate 24h 24h 24h 120h M9/glucose - + - + M9/glucose/T yr - + - + M9/glucose/Phe - + - + M9/glucose/Tyr/Phe - + - + M9/glucose/Aros + + + + LB/Ap - + + LB/Cm + - - “Abbreviations: phenylalanine (Phe), tyrosine (Tyr), aromatic amino acids (phenylalanine, tyrosine and tryptophan) and aromatic vitamins (2,3-dihydroxybenzoic acid, p-aminobenzoic acid and p-hydroxybenzoic acid) (Aros), ampicillin (Ap), chloramphenicol (Cm). 56 Complementation experiments showed YdiB is a significantly poorer enzyme in viva for catalyzed conversion of 3-dehydroshikimic acid to shikimic acid relative to AroE. However, YdiB was not inhibited by shikimic acid. Therefore, in order to evaluate YdiB as a Shikimate dehydrogenase in a shikimic acid-producing strain, it has to be overexpressed. Evaluation of YdiB under fermentor-controlled conditions Strain SP1.1/pKD12.138 is a benchmark strain in the Frost group for shikimic acid production? A new plasmid identical to pKD12.138 bearing a ydiB insert in place of araE was designed for shikimic acid production (Figure 17). Plasmid pKD12.112 was the precursor for pKD12.138, and therefore, the construction of plasmid pJJ4.150A started with digestion of plasmid pKD12.112 with KpnI and BamHI restriction endonucleases followed by treatment with Klenow fragment. This lead to loss of the PmaraE serA fragment. A PCR amplified PmydiB fragment from pJJ3.041A was inserted into pretreated pKD12.112 yielding plasmid pJJ4.150A (Figure 17A). Digestion of plasmid pNR8.146A with XbaI restriction endonuclease liberated an intact tktA serA fragment, which was cloned into pJJ4.150A and afforded target plasmid pJJ4.171A (Figure 17B). The impact of plasmid-expressed ydiB on the yields and concentrations of shikimic acid and Shikimate pathway byproducts was evaluated under fed-batch controlled conditions. Fermentations were run under glucose-rich conditions in a 2.0 L working volume fermentor. A concentration range of 55-170 mM glucose was maintained. Temperature was maintained at 36 °C, pH was maintained at 7.0 and 57 dissolved oxygen was maintained at 20% air saturation. All the fermentations were run in duplicate and reported results represent an average of two runs. ”M- // \\ ,I/ \ Xbal IPA. PKg172-k1b12A BamH, PCR P,,,,ydieirom pJJ3.041A // (Smal) .v,/,'//'/serA YI1) Kpnl and BamHI digest Kpnl BamHl Smal Kpnl ( ) 1.2 kb 1) Kpnl and BamHI digest PtacydiB 2) CIAP treatment I T4 Ligase I I \ Xb l I pJJ4.150A . a \ 5.7 kb 77) ,N BamHl \ \P‘Iac / / ; / PtaeYdIB Figure 17A. Construction of plasmid pJJ4.150A. 58 pNR8.146A 6.7 kB II pJJ4.150A I 5.7 kb Pstl Xbal BamHl l1) Xbal digest Xbal Xbal 3.8 kb :3:323:3:3:f:j:i:f:i:f:§7i:§:if]. 1) Xbal digest serA 2) CIAP treatment tktA I T4 Ligase 9.5 kb pJJ4.171A \\ Figure 17B. Construction of plasmid pJJ4.171A Shikimic acid was produced at 16 g/L in a 12% yield from glucose (entry 2, Table 10). No accumulation of acetate was observed. Accumulation of 3-dehydroshikimic acid 59 as well as quinic acid was observed during the course of the fermentation, which resulted in a SA/DHS ratio equal to one. Table 10. Concentrations and yield of shikimic acid and byproducts synthesized by E. coli SP1.1/pKD12.138 and SP1.1/pJJ4.171A under glucose-rich conditions. [SA]‘ SA [DHS] [QA] Total Entry St'a'" (g/L) Yield" (g/L) (g/L) Yieldc 1 SP1.1/pKD12.138 62 33% 1 1 7.5 47% 2 SP1.1/pJJ4.171A 16 12% 16 12 31% ”Abbreviations: shikimic acid (SA), 3-dehydroshikimic acid (DHS), quinic acid (QA). ”(mol SA)/(mol glucose consumed). “(mol SA + mol DHS + mol QA)/(mol glucose consumed). A. B. Q 60 E, 3’ E E 50 .Q’ .91 11;: (1) 3 = E 40 3 2‘ g 30 3 ‘5 :l: :5 20 3 5 10 O <' <' 12 18 24 36 42 48 60 12 18 24 36 42 48 60 Time (h) Time (h) Figure 18. E. coli SP1.1/pKD12.138 (A) and SP1.1/pJJ4.171A (B) cultured under glucose-rich conditions. Legend: shikimic acid (open bars), 3-dehydroshikimic acid (grey bars), quinic acid (black bars), dry cell weight (black circles). Quinate dehydrogenase (Qad) from Klebsiella pneumoniae It has been shown that E. coli Shikimate dehydrogenase AroE is involved in hydroaromatic equilibration and accumulation of quinic acid during the shikimic acid microbial synthesis by reducing 3-dehydroquinic acid to quinic acid (Figure 1).2 The first 60 reported microbial synthesis of quinic acid relied on heterologous expression in E. coli of the Klebsiella pneumoniae qad gene that encodes quinate dehydrogenase.8 Qad catalyzed the reduction of DHQ to quinic acid. In K. pneumoniae, Qad oxidizes quinic acid to DHQ in the presence of N AD“, which is the first step in quinic acid catabolism.9 Since E. coli AroE Shikimate dehydrogenase catalyzes reduction of 3-dehydroquinic and 3- dehydroshikimic acids, the question arose to whether K. pneumoniae Qad quinate dehydrogenase also catalyzed 3-dehydroshikimic acid reduction and whether shikimic acid inhibits this conversion. E. coli AB2834 complementation was first appraised using plasmid-localized, qad-encoded quinate dehydrogenase. Plasmid pTW8090A8'” (CmR, qad) was used in complementation experiment. E. coli AB2834/pTW8090A grew on glucose-minimal plates in 18 hours (Table 11). Results clearly indicate that Qad can convert 3- dehydroshikimic acid to shikimic acid. Table 11. E. coli AB2834 complementation assay with plasmid pTW8090A containing a qad insert. Medium A32834 AB2834/pTW8090A M9/Glucose - + LB/Cm - + The purification of K. pneumoniae Qad from DHSa/pTW8090A was performed as previously described (Table 12).” Additionally, a Resource-Q column was used in the final purification step in order to remove protein impurities obtained after the RedA column step (Lane 4, Figure 19). This resulted in a more highly purified Qad (Lane 5-7, Figure 19). 61 Table 12. Purification of K. pneumoniae Qad from E. coli DHSa/pTW8090A. Purification Total protein Total Units Specific activity“ Purification Yield step (m9) (U1 (U/mgl (fold) 1%) crude extract 520 4100 7.9 1.0 (NH.)2SO. (35-65%) 260 2900 1 1 1.4 71 FtedA 81 1400 17 2.2 34 Resource-Q 28 930 33 4.2 23 " One unit (U) of quinate dehydrogenase corresponds to the formation of 1 umole of NADH in the presence of quinic acid per min at 25 °C. kDa twee-1m""'....;flx.2 . » kDa 205 m :11: ‘" 205 116 ‘m , 116 97 u _ 97 45 “an 45 29 29 u ”'1'" » . -~-J-m- mamas... .e .1 .r—Ifih“. Figure 19. SDS-PAGE of K. pneumoniae Qad. Legend: olecular weight markers (1, 8), crude lysate (2), after (NH4)2SO4 precipitation (3), after RedA column (4), 1 - 3 fractions collected after ResourceQ column (5 — 7). Molecular weight: Qad (32 kDa). . v, Partially purified Qad was used in the inhibition assays. Inhibition of K. pneumoniae Qad and E. coli AroE with quinic and shikimic acid was measured (Figure 20). Shikimate dehydrogenase assays were performed in the forward direction with the reduction of 3-dehydroshikimic acid to shikimic acid and concomitant oxidation of NADPH to NADP‘ followed at 340 nm.6 Quinate dehydrogenase activity was measured in the reverse direction with the oxidation of quinic acid to 3-dehydroquinic acid and concomitant reduction of NAD+ to NADH measured at 340 nm. 62 Results indicated that AroE is not inhibited by quinic acid. On the other hand, Qad is approximately 20% less feedback inhibited by shikimic acid relative to AroE. To determine if Shikimate dehydrogenase activity less sensitive to feedback inhibition by shikimic acid would increase shikimic acid production under fermentor-controlled conditions, a Shikimate producer carrying plasmid-expressed qad in place of araE was constructed. 100 A ‘I A A 80 ——————————— E—e —————————— A A it 23 ___ ___________ I __o__'__. c 60 T I 0 ° a 40 _-__: ________________ . E o 20 —————————————————————— 0 g 0 5 10 15 20 mmumumM) Figure 20. Inhibition profiles for Qad and AroE. Legend: Qad inhibition with shikimic acid (diamonds), Qad inhibition with quinic acid (squares), AroE inhibition with shikirrric acid (triangles), AroE inhibition with quinic acid (circles). Plasmid pTW8090A had a 3 kb K. pneumoniae genomic DNA insert that encoded qad,” but it was necessary to reduce the size of DNA fragment to a minimum. A search for the qad ORF began using the ERGOll database. Most of the quinate dehydrogenases are PQQ-dependent enzymes (EC number 1.1.99.25). Only four quinate dehydrogenases were published in the ERGO database as NAD(P)H dependent (EC number 1.1.1.24). Those quinate dehydrogenases are found in Debaromyces hansenii, Emiricella nidulans, Magnaparthe grisea and Neuraspora crassa. Protein sequences of all four quinate 63 1" T 7"“ . {TS-fl dehydrogenases were used to BLAST12 against protein sequence of K. pneumoniae MGH 78578, which is available from Washington University at St. Louis.13 Four hits were obtained which had up to 25% amino acid identity with each quinate dehydrogenase protein sequence. The nucleotide sequence of each hit was then translated into all six protein frames on an ExPASy server. '4 Protein sequences from newly obtained ORFs were used to BLAST against the K. pneumoniae protein sequence. Only one sequence showed 100% amino acid identity and the corresponding ORF that was 864 nucleotides in length was designated as qad (Figure 21). 1 ATGGCAGAAC GTATTACTGG ACACACTGAG CTGATTGGCC TGATCGCCAC CCCGA'ITCGT 61 CACAGCATGT CGCCCACCAT GCACAACGAG GC'I'I'TCGCCC ATCTCGGGCT GGACTACGTC 121 TATCTCGC'I'I' 'ITGAAGTCGG TAACCAGGAG CTAAAAGACG TGGTGCAGGG C'I'I'CCGGGCG 181 ATGAAGCTGC GCGGTITI'AA CGTCTCTATG CCGAACAAAA CCGAGATTI'G CCAGTATCTC ‘ 241 GATAAACTGT CGCCGGCGGC ACAGCTGGTC GGGGCGGTCA ATACCGTGGT CAACGACGAC 301 GGCGTACTGA CCGGACATAT CACCGATGGC ACCGGCTACA TGCGCGCCCT TAGCGAAGCC 361 GGTATCGATA TCATCGGCAA GAAGATGACC GTACTCGGCG CCGGCGGCGC GGCAACGGCG 421 CTCTGCGTCC AGGCAGCACT GGATGGCGTG AAGGCGATCT CCATCTTCAA CCGCCGCGAT 481 AAATTCTI'CG CCAACGCAGA AGAAACCGTG GCCAAGATCC GCCACAACAC CGACTGCGAG 541 ATCCATCTGT TCGATCTCGA CGATCATGAC AAGCTGCGCG CCGAAAT'I'GA CAGCAGCGTG 601 ATCCTGACCA ATGCCACCGG GGTCGGCATG AAGCCG'ITCG AAGGCCAGAT GCTGCTACCT 661 GACGACAGCT TCCTGCGTCC GGACCTGATC GTCTCCGACG TCGTCTACAA CCCGCGCAAA 721 ACCCACCTGC TGGAAGTGGC CGAGAAAAAA GGCTGCCGCA CGCTGAACGG CCTGGGGATG 781 ATGCTGTGGC AAGGCGCGCG CGCG'ITCGAA ATCTGGACGG GCAAACAGAT GCCGGTTGAT 841 TACATCAAGA GCATTCTG'I'I' CTAA Figure 21. Klebsiella pneumoniae MGH78578 quinate dehydrogenase qad sequence. Quinate dehydrogenase Qad sequence was used in a BLAST against the entire ERGO database (604 genomes at that time). Only one sequence showed up after BLAST and that was the sequence of E. coli YdiB. It showed 50% amino acid and 57% nucleotide identity with K. pneumoniae Qad. The protein and nucleotide sequences of K. pneumoniae quinate dehydrogenase Qad, E. coli Shikimate dehydrogenase YdiB, and four previously described quinate dehydrogenases were aligned using a ClustalWl5 algorithm (Table 13). Only K. pneumoniae Qad and E. coli AroE showed high protein and gene sequence identity (entry 5, Table 13). Other quinate dehydrogenases did not 64 show high identity to either K. pneumoniae Qad or E. coli AroE at the protein or nucleotide levels. However, they showed some identities with each other (Table 13). Table 13. ClustalW alignment results of quinate dehydrogenases. Entry . . Length . . Length . Identity (°/o) organ's'“ Am'."° Nucleotide organ's'“ Am'."° Nucleotide Am'."° Nucleotide acrd aCld ac1d 1 K. pneumoniae 287 864 D. hansenii 317 954 18 1 2 K. pneumoniae 287 864 E. nidulans 329 990 19 5 3 K. pneumoniae 287 864 M. grisea 326 984 22 7 4 K. pneumoniae 287 864 N. crassa 321 966 21 7 5 K. pneumoniae 287 864 E. cali' 288 867 50 57 6 D. hansenii 317 954 E. nidulans 329 990 35 20 7 D. hansenii 317 954 M. grisea 326 984 40 26 8 D. hansenii 317 954 N. crassa 321 966 35 37 9 D. hansenii 317 954 E. coli 288 867 22 16 10 E. nidulans 329 990 M. grisea 326 984 42 55 11 E. nidulans 329 990 N. crassa 321 966 39 43 12 E. nidulans 329 990 E. coli 288 867 23 6 13 M. grisea 326 984 N. crassa 321 966 58 58 14 M. grisea 326 984 E. coli 288 867 22 2 1 5 N. crassa 321 966 E. coli 288 867 1 8 2 " E. coli Shikimate dehydrogenase protein YdiB and ORF ydiB sequences. To construct a shikimic acid producer where E. coli araE is replaced by K. pneumoniae qad, the same strategy as described for construction of pJJ4.171A was used. Ampification by PCR of qad from pTW8090A resulted in a ~09 kb size band on agarose gel. Insertion of qad ORF into the EcoRI site of pKK233-3 afforded plasmid pJJ3.289AP (Figure 22), which was transformed into DHSa host strain. The resulting construct showed fivefold overexpression of quinate dehydrogenase (Table 14). Plasmid pJJ3.289AP was used for P qad amplification and subsequent construction of plasmid lac pJJ4.025A (Figure 23). Table 14. Quinate dehydrogenase specific activity. Entry Strain Specific activity (U/mg)__ 1 DHSa/pKK223-3 0 2 DH5a/pJJ3.289AP 4.8 65 Smal Pstl EcaFII Hindlll PCR qad from pTW8090A l1) EcoRl digest pKK223-3 Ec 0R1 E coFil 4.6 kb 0.9 kb qad 1) 500111 digest 2) CIAP treatment T4 Ligase Smal Pstl Figure 22. Construction of plasmid pJJ3.289AP. 66 Xbal i/ I’D/ac PKDIZ-“ZA 33ml... PCR quad from pJJ3.289AP (Smal) 7.7 kb p\7/ ////serA 1) Kpnl and BamHI digest ECOF" K I 8 HI Smal pn am Kpnl ( ) 1.2 kb ' Ptac qad 1) Kpnl and BamHI digest 2) CIAP treatment I T4 Ligase II pJJ4.024A Xbal BamHI EcoRl EcoFt’l Kpnl Figure 23A. Construction of plasmid pJJ4.024A. 67 1%, P311 Xbal BamHl ‘ \ l1) Xbal digest 50°F" EcoRl Kpn' Xba' 3.8 kb Xba' 1) Xbal digest 2) CIAP treatment serA I tktA T4 Ligase Xbal tktA ft?! x" y _~ APR Xbal .4 pJJ4.025A ’i‘ 9.5 kb BamHl EcoFIl Figure 23B. Construction of plasmid pJJ4.025A. After inserting the tktA serA fragment in the last step (Figure 23B), quinate dehydrogenase specific activity decreased ten times. The final construct SP1.1/pJJ4.025A showed only a 0.3 U/mg specific activity for quinate dehydrogenase 68 when it was cultivated under glucose-limited conditions. No evaluation of shikimic acid production was performed with such a low quinate dehydrogenase specific activity. Shikimate dehydrogenase (AroD) from Bacillus subtilis K. pneumoniae Qad showed 20% less inhibition by shikimic acid relative to E. coli AroE, as described previously (Figure 20). On the other hand, E. coli Shikimate dehydrogenase AroE has a high km, and a low K,,,. The search for other Shikimate dehydrogenases that would have similar properties of Qad and AroE was conducted. BLAST with the Qad sequence against the entire ERGO database resulted in 184 sequences. A similar number of sequences (175) was obtained after AroE BLAST against the ERGO database. Sequence identities from microorganisms, which showed protein sequence similarity to Qad and AroE are summarized in Table 15. Bacillus subtilis 168 Shikimate dehydrogenase AroD” was chosen for further evaluation as a potential Shikimate dehydrogenase for a shikimic acid-producing strain. Selection of this B. subtilis AroD was influenced by a couple of reasons. First of all, it was well known that heterologous expression of B. subtilis genes in E. coli can be achieved without any complications.17 Secondly, B. subtilis Shikimate dehydrogenase had the same protein sequence identity to Qad and AroE (entry 47, Table ISB). 69 .. '7 N: o 5 mwm or w om vw m ”_> 596me 6:23. wwwoo<< 25:33 «3430:3203 wN Fooxos. m 2 we www vww or :V 5 0: vac; 63:08:63 «3880396 wwwoozam we 3 www wwm or :4 5 0: wm omEoEamEq 2.886665% wwwwomdwm m. we mww vww or 5‘ mm o: “.8 omEoana «3880396 nwmooudwm I 3‘ onw mwm w mm 5 mm 23 6:889:95 w Pwooswwm 9 en onw mww m t on 0: mm 33 ESE: muooooofiobm. mepozim NF .8 Fww www v R _.w m: 83 .= 3m 8.:ng 380883 was Foxjm S we 5w wwm or ww em 9. «.800: .628 3:630:63 www 5<04m or wv 2w «mm or mm wm mm :ww ms. 666993 maoooooaqobw Rmoowdwm m we mew mom or ww ww mm Fiénwuw 8:06on 6:889:85 wwwoohwm w we mnw «mm or Aw ww mm mpww<02 ms. 8:393 8880396 vwmwoxowm n we mnw «mm or wm wm Nm ms. 93:69.3 3880395 wwio>dwm w on NNw mum m w 5 ow Nome 3:0 maoo::.: 3:26: wmm 523m w E wa mum we 8. ww wm 0-00m moSSoXQSoE «:66: $300.24”. v aw omw mww we mm ww om odOw 8:62.889: Emu: wwwmoOEDm m we wrw NR cor w 09 em or 52, «TX :8 «Ectocomm wwwwo>>0mm m ww vww nwm w 09 em 00. wnwwnIOE omEoanE 3:96:62 P oh 596. £96. 62 02 3.: 80 Esme}. @L :60 3828 6:60 529: o. 328. 9 5:8. 9 >552 9 228. 2259885 9 “E0 ram 00 onzoopoaz mch< U: 7O ww wow wow mm mm 3. wm vaO 8.9092909 9299mm wwwwosme Fw ww 5w wwm w wv mm mm 829088 202820008 88005. ow mm 03 mam Z 2 mm 8 08. 20292 520099028 83228 9. vw wnw owm w wv Nw wm warn. 029920289020 8209002220m 5985wa w\. an mww mam v m 5 mm 08050... am 0292.50 9202028 8083me t. ww mww wow w w 5 wm 729801 .20 098202 9282028 woooozxwm wn mm «8 8m 9 or mm mm 885 $850st @88sz ms nw oow wow m mv vw ow 80808.22 .29 990929082 880045;. K mw www wwm E 3 mm 8 w—wo 82280820 82089an vwmoooom Q. ww wow 5w 5 mm mm mm 2962002095 88:: 022302300 9809.39”. Nu nw nww wnm 3 ww ow mm Tm: 250200290289 992220090282 wownoszim I. 9 «8 8m 9 mm mm 8 2:700? Ease 59228822 «98:8 9. on ovw mum w vw Po 5 fivd 8998200 29002000222 wvwwoawmm mw mm Em {N v m mm on 9 0289.228 5202390 82880: 8 mm nww wwm w— w 5 mm 07... 808298 8829920 wvwvoaNm Fw ow 9w mum mm m vw mm 89$ 00.: 0.2922098 8000003288 Nwm—oamwm ow ww 5w wmm mw op mw vm wwwwhx 0.328022 8228202800 kwwSEOm mm 8 5w wmm mm m 5 mm 038-00.: 02890029 82200092293 wwvrowum ww mm onw www rm 3 no ww .29 829.209 92085 wmwwomzwm um mm mww vwm R w ww wm own? 93 8202.289 988$ Foomoawwm wm wm onw mwm E E. R wm 82020 920005 mwmmo>w¢ mm 8 now www NF op ow mm wnv Exam >>Nm 8 5 now wwm «F or on mm owns. 898 8000099285 mmw82528. 9 32:00. 9 32200. 9 >5ch 9920909022 0. “EC 95 00 0028.032 2.29 $0.32 020 an 3 8:20:56 00:0: 8 2:? 25025 00032 .mmn 0.2m 71 Plasmid pJJ4.118A was constructed to facilitate B. subtilis Shikimate dehydrogenase AroD purification. The construction of the plasmid started from amplification of the aroD ORF from B. subtilis genomic DNA with subsequent insertion into vector pGEX-4T-1 EcoRI site (Figure 24). GST PCR aroD from B. subtilis168 genomic DNA Ptac pGEX-4T-1 1) EooRl digest 4 . 9 kb ‘5‘- t>q\\ If? ‘: ECOR | ECOR' it x , ~ aroD 1) EcoRl digest 2) CIAP treatment T4 Ligase Figure 24. Construction of the plasmid pJJ4.118A. 72 Purification of B. subtilis Shikimate dehydrogenase was performed using the same conditions as previously described for the purification of E. coli YdiB and AroE. Enzyme assays were performed in the forward direction and reduction of 3- dehydroshikimic acid with subsequent oxidation of NADPH was followed at 340 nm. Enzyme purified to homogeneity (Figure 25) showed 96 U/mg specific activity. Inhibition levels with shikimic acid were measured (Figure 26) and showed more rapid and higher inhibition than for E. coli Shikimate dehydrogenase AroE (Figure 16). 1 2 3 4 5 6 7 8 kDa 205 116 97 29 Figure 25. SDS-PAGE of B. subtilis AroD purification. Legend: molecular weight markers (1), crude lysate (2), flow-through after Glutathione Sepharose column (3), 1 mL 1 — 5 fractions collected after benzamidine column (4 — 8). Molecular weight: AroD (31 kDa), GST (29 kDa), Thrombin (37 kDa). 73 100 _ . . i ao-_————' ————————— .————'————— . I . ’o‘ . ' 22 so.—'————._ __________________ c . g § 404—1 ————————————————————— E . O 20 —————————————————————— 0 ................... O 5 10 15 20 Shikimate (mM) Figure 26. B. subtilis AroD and E. coli AroE Shikimate dehydrogenases inhibition by shikimic acid. Legend: AroD (squares), AroE (circles). Further evaluation of B. subtilis Shikimate dehydrogenase AroD in the shikimic acid- producing strain, seemed unnecessary, since it was more sensitive to feedback inhibition by shikimic acid relative to E. coli AroE Shikimate dehydrogenase. Directed evolution of E. coli Shikimate dehydrogenase (AroE) DAHP synthase activity is critical in regulating the carbon flow into the common pathway of aromatic amino acid biosythesis.‘ All three DAHP synthase isozymes AroF, AroG, AroH are inhibited by aromatic amino acids. Use of a feedback resistant mutant isozyme of DAHP synthase was the first step needed to direct more carbon flow into the Shikimate pathway.‘ Accumulation of byproducts such as 3-dehydroshikimic acid and quinic acid was attributed, respectively, to feedback inhibition of AroE by shikimic acid and to hydroaromatic equilibration.2 Previously described attempts to identify feedback 74 resistant Shikimate dehydrogenase were unsuccessful. Therefore, directed evolution was chosen to create a feedback resistant AroE mutant. Error-prone PCR18 was used to create a mutant library. The goal of mutagenic PCR is to introduce random mutations along the gene (rather than achieve a high level of gene amplification). The protocol used for error-prone PCR is derived from the standard PCR protocol and the following changes were made to enhance the mutation rate: the MgCl2 concentration was increased from 1.5 mM to 7 mM to stabilize noncomplementary pairs; 0.2 mM MnCl2 was added to the reaction mixture to reduce template specificity; the concentrations of dCTP and dTTP were increased from 0.2 mM to 1 mM to promote misincorporation; the amount of Taq polymerase was increased to 5 units to promote chain extension beyond positions of base mismatches; the annealing temperature was lowered to 45 °C to decrease the fidelity of polymerase. E. coli araE ORF from plasmid pJY1.43A was amplified by mutagenic PCR and the resulting DNA was ligated into pKK223-3 to afford a family of plasmids designated as pJJ3.247A carrying mutant aroE (Figure 27). The mutation rate (average 2 nucleotides/gene) was determined by sequencing twenty-four mutants at the Genomic Technology Support Facility at Michigan State University. The resulting ligation mixture was transformed into E. coli AB2834, which is deficient in Shikimate dehydrogenase activity due to a mutation in the aroE gene.7 The transformation mixture was plated on selective M9/Glucose/Ap plates. This selection method relied on the fact that only colonies carrying a functional mutant aroE would grow. For the first 2000 mutants, serial dilutions (“pics”) on M9/Glucose/Ap plates were used to isolate single colonies. Isolated single colonies from 75 “pies” were screened for feedback insensitive Shikimate dehydrogenase. For the rest of the 8000 mutants no serial dilutions were performed and a screening was performed using robotics for colony inoculation and liquid handling. Robotic instruments were available at Genomic Technology Support Facility at Michigan State University. Smal Pstl EcoRl Hindlll aroE mutagenic PCR from pJY1.43A $7 // ' l1) EcoRI and Pstl digest ; pKK223-3 {K 4.6 kb £001?! 0 8 kb Psti \\\ ' aroE \\ \W’ 1) EcoRI and Pstl digest 2) CIAP treatment T4 Ligase Psn Hindlll Figure 27 . Construction of plasmid pJJ3.247A. The screening method relied on the Shikimate dehydrogenase assay6 where enzyme activity was measured in the forward direction in the presence of 30 mM and in 76 1.1. the absence of shikimic acid in the reaction mixture. The slope ratio (“Slope with SA”/“Slope without SA”) was calculated and compared to the wild-type AroE slope ratio obtained under the same conditions. The mutant was considered as a potential candidate if the following two conditions are met: the slope ratio was greater or equal to the wild- type AroE slope ratio and the mutant “Slope without SA” was at least 50 % of wild-type AroE slope under the same conditions. The first condition filtered out mutants that have higher feedback sensitivity on shikimic acid relative to wild-type AroE. The second condition filtered out partially inactivated enzymes. If a mutant enzyme is almost inactive, the slope with and without shikimic acid will be very similar in magnitude and the slope ratio will be close to 1, therefore indicating a false positive result. In order to trust the screening results, additional tests were performed. In the first test, the 92 of the best mutants were re-screened using the same method. Table 16 summarizes colonies that were chosen for the second round screening. Results that passed the second round screening are shown in Table 17. Of the 92 colonies that passed both conditions in the first round of screening, only 28 mutants passed when they were re-screened for the second time. This indicated that results from the first screen are only preliminary and contain many false positive results. The four best candidates (JJ4679 F2, H5, A7, H7) were selected after second round of screening and were tested for Shikimate dehydrogenase feedback inhibition with shikimic acid after colonies were grown under shake flask conditions instead of 96-well plates as before. Shikimate dehydrogenase inhibition levels were measured with increasing shikimic acid concentration (Figure 28) and it indicated that none of the mutant Shikimate dehydrogenase showed increased 77 feedback insensitivity to shikimic acid as compared to wild-type E. coli AroE Shikimate dehydrogenase. Table 16. Mutant candidates chosen for second round of screening. Wens Plate 10‘ Flati021b natioc" (12:21:; 02:, Wells Plate ID Rati021 RatioC (3:31:22 ('3‘de 06 0073 0.67 0.29 0.36 F1 E7 JJ78 0.27 0.17 0.10 A6 G11 004657 0.63 0.17 0.46 E1 66 0074 0.26 0.16 0.06 F6 06 0075 0.61 0.29 0.32 0.1 64 0074 0.26 0.18 0.08 GS F6 0075 0.60 0.29 0.32 H1 01 1 0070 0.26 0.16 0.10 G5 F12 0071 0.51 0.32 0.19 E2 F4 0076 0.25 0.17 0.06 E6 A3 0075 0.50 0.29 0.21 62 H2 JJ67 0.25 0.13 0.13 H4 F2 0073 0.49 0.29 0.20 02 H9 004657 0.25 0.17 0.08 A7 69 0074 0.46 0.16 0.27 A2 ca 0072 0.25 0.17 0.07 67 C11 0075 0.45 0.29 0.17 63 D4 0076 0.25 0.17 0.06 H6 07 0073 0.45 0.29 0.16 03 B8 004657 0.24 0.17 0.07 G7 E3 0073 0.42 0.29 0.14 B4 09 0076 0.24 0.17 0.07 C7 66 JJ76 0.42 0.27 0.16 E3 06 004657 0.24 0.17 0.07 H7 A3 0073 0.41 0.29 0.13 A5 611 0072 0.24 0.17 0.06 C8 A5 0073 0.40 0.29 0.11 05 E11 004657 0.24 0.17 0.06 06 E1 .1060 0.40 0.32 0.06 D6 D7 0072 0.23 0.17 0.06 G8 F1 0074 0.36 0.18 0.20 02 F6 004657 0.23 0.17 0.05 A9 G4 0076 0.36 0.27 0.11 F5 E1 0074 0.23 0.16 0.04 E10 F11 JJ4657 0.37 0.17 0.19 F2 A4 JJ4657 0.23 0.17 0.05 E9 E5 0073 0.36 0.29 0.07 D7 65 JJ4657 0.23 0.17 0.05 F9 E6 0075 0.36 0.29 0.07 F7 011 0067 0.22 0.13 0.10 H5 G10 0071 0.35 0.32 0.03 H11 E10 004657 0.22 0.17 0.04 A11 69 0076 0.35 0.17 0.16 H2 H10 0076 0.22 0.17 0.05 610 E7 0072 0.35 0.17 0.17 A3 06 0076 0.21 0.17 0.04 611 G7 0060 0.35 0.32 0.03 E12 F10 004657 0.21 0.17 0.03 D11 61 0073 0.34 0.29 0.05 09 F3 JJ62 0.21 0.14 0.06 66 F4 0076 0.34 0.27 0.07 E7 A3 JJ4657 0.20 0.17 0.03 612 F4 004657 0.34 0.17 0.16 C3 E4 0062 0.20 0.14 0.05 69 611 0.173 0.33 0.29 0.04 C10 A6 0070 0.19 0.16 0.03 E11 G6 004657 0.33 0.17 0.15 F3 H3 0070 0.19 0.16 0.03 A12 E3 0079 0.33 0.16 0.14 (33 H6 JJ67 0.16 0.13 0.05 H6 F5 0079 0.32 0.16 0.14 H3 GB 0067 0.18 0.13 0.05 09 E6 0074 0.32 0.16 0.14 A4 A6 JJ67 0.16 0.13 0.05 (39 Ge JJ75 0.32 0.29 0.03 F11 63 0063 0.17 0.14 0.03 C11 E12 0074 0.32 0.16 0.13 F4 C7 0066 0.17 0.13 0.05 A10 E7 JJ4657 0.31 0.17 0.14 04 A5 JJ67 0.17 0.13 0.04 G10 03 0076 0.31 0.27 0.04 H10 H6 0069 0.16 0.13 0.03 611 H9 0067 0.31 0.13 0.18 G2 65 0061 0.16 0.10 0.06 E6 F6 0076 0.31 0.17 0.14 C4 F7 0065 0.16 0.10 0.07 A6 02 0076 0.31 0.17 0.13 E4 E6 JJ66 0.15 0.09 0.06 F6 05 0072 0.30 0.17 0.13 64 F6 0065 0.14 0.10 0.05 H9 H6 0079 0.30 0.16 0.12 65 H10 0065 0.14 0.10 0.04 010 G8 0076 0.30 0.27 0.03 F12 H9 0065 0.14 0.10 0.04 F10 G7 0074 0.26 0.16 0.09 66 06 0061 0.13 0.10 0.03 612 D8 0074 0.28 0.18 0.09 C6 F3 0065 0.13 0.10 0.03 H12 69 .1070 0.27 0.16 0.11 05 F7 JJ66 0.12 0.09 0.03 012 A6 0070 0.27 0.16 0.11 E5 011 0.166 0.12 0.09 0 03 012 “plate ID from the first round screening. ”Mutant slope ratio (“Slope with SA”/“Slope without SA”). cControl slope ratio (“Slope with SA”/“Slope without SA”). dRatio2l-RatioC. ‘New assigned location in the plate JJ4679 for the second round screening. 78 Table 17. Candidates after second round screening. First screening Second screening Wells Plate ID“ Ratioz1" RatioC° 6:122:22" Wells Plate ID Flati021 RatioC 33:31:: H3 JJ70 0.19 0.16 0.03 A12 JJ4679 0.25 0.21 0.04 E6 JJ74 0.32 0.18 0.14 A4 JJ4679 0.22 0.21 0.01 H9 JJ4657 0.25 0.17 0.08 A7 JJ4679 0.35 0.21 0.15 F7 0065 0.16 0.10 0.07 A8 JJ4679 0.24 0.21 0.04 H10 JJ78 0.22 0.17 0.05 B10 JJ4679 0.23 0.21 0.03 06 J061 0.13 0.10 0.03 812 JJ4679 0.25 0.21 0.05 F4 JJ4657 0.34 0.17 0.16 C3 JJ4679 0.28 0.21 0.07 89 JJ70 0.27 0.16 0.11 CS JJ4679 0.23 0.21 0.03 G11 JJ72 0.24 0.17 0.06 C8 JJ4679 0.21 0.21 0.01 H10 JJ65 0.14 0.10 0.04 D10 JJ4679 0.28 0.21 0.08 C1 1 .1066 0.12 0.09 0.03 D12 JJ4679 0.27 0.21 0.07 E1 JJ74 0.23 0.18 0.04 E10 JJ4679 0.22 0.21 0.01 A8 JJ70 0.19 0.16 0.03 E11 JJ4679 0.34 0.21 0.13 02 JJ78 0.31 0.17 0.13 E4 JJ4679 0.29 0.21 0.09 85 JJ61 0.16 0.10 0.06 E8 JJ4679 0.24 0.21 0.03 A4 JJ4657 0.23 0.17 0.05 E9 JJ4679 0.23 0.21 0.02 G8 JJ76 0.30 0.27 0.03 F12 JJ4679 0.29 0.21 0.08 F1 1 JJ4657 0.37 0.17 0.19 F2 JJ4679 0.38 0.21 0.17 E12 JJ74 0.32 0.18 0.13 F4 JJ4679 0.26 0.21 0.05 G4 JJ76 0.38 0.27 0.1 1 F5 JJ4679 0.28 0.21 0.07 85 JJ4657 0.23 0.17 0.05 F9 JJ4679 0.22 0.21 0.02 A3 JJ4657 0.20 0.17 0.03 G12 JJ4679 0.24 0.21 0.03 H9 JJ67 0.31 0.13 0.18 G2 JJ4679 0.27 0.21 0.07 05 0072 0.30 0.17 0.13 G4 JJ4679 0.22 0.21 0.01 B4 JJ74 0.26 0.18 0.08 G6 JJ4679 0.23 0.21 0.03 03 JJ76 0.31 0.27 0.04 H10 JJ4679 0.27 0.21 0.06 C1 1 0067 0.22 0.13 0.10 H5 JJ4679 0.36 0.21 0.15 D8 JJ4657 0.24 0.17 0.07 H7 JJ4679 0.34 0.21 0.13 “plate ID from the first round screening. bMutant slope ratio (“Slope with SA”/”Slope without SA”). CControl slope ratio (“Slope with SA”/”Slope without SA”). dRatioZl-RatioC. 100 l - I 80- ——————— e —————————————— 4 3? . v 60-l———-.— —————————————————— c . g . 1:3 40¢ —————————————————————— - E “a 201:1- ______________________ og ....... . . ”.mfim .. 0 10 20 30 Shikimate (mM) Figure 28. Shikimate dehydrogenase inhibition profile of JJ 4679 F2, H5, A7, H7 mutants after shake-flask experiment. Legend: F2 (squares), H5 (triangles), A7 (circles), H7 (pluses), control (diamonds). 79 Discussion Three Shikimate dehydrogenase activities were screened for inhibition with shikimic acid. E. coli Shikimate dehydrogenase YdiB showed no inhibition with shikimic acid. B. subtilis AroD was more sensitive to shikimic acid feedback inhibition than E. coli AroE. K. pneumoniae Qad was somewhere in the middle in terms of its sensitivity towards feedback inhibition by shikimic acid relative AroE and Qad. Even though YdiB was insensitive to shikimic acid, E. coli strain SP1.1/pJJ4.171A synthesize less shikimic acid than the benchmark strain SP1.1/pKD12.138 (Table 10). This failure can be attributed to slow enzyme kinetic characteristics and the operational pH Optimum. As shown previously, YdiB is more sluggish than AroE with the km, that is four times lower (Table 8). Also the optimal pH value of 5.3 for an E. coli enzyme is surprising, since E. coli is typically cultured from pH 7 to pH 8. YdiB might be actually enzymatically inactive in wild-type E. coli given its pH optimum. However, expression of plasmid- localized ydiB, was sufficient to restore the growth of E. coli AB2834 on glucose minimal salts medium (Table 11). Accumulation of 3-dehydroshikimic acid and quinic acid was also observed during SP1.1/pJJ4. 171A fermentation (Table 10). The final ratio of SA:DHS:QA was almost 1:1:1. Accumulation of 3-dehydroshikimic acid likely was a result of small YdiB activity (0.1 U/mg) during the course of the fermentation. Quinic acid accumulation during the fermentation process might be due to hydroaromatic equilibration. YdiB may discriminate between 3-dehydroshikimic acid and 3- dehydroquininc acid to an even lesser extent relative to E. coli AroE, therefore leading to 1:1 DSH:QA ratios. If that is the case, maybe quinic acid accumulation during SP1.1/pKDl2.138 fermentation can be not only due to AroE activity2 but also due to 80 YdiB activity. Therefore, a new experiment should be done by knocking out the ydiB gene in the SP1.1 host strain. Comparison of the accumulated products in the broth between SP1.1/pKD12.138 and SP1.1AydiB/pKD12.138 would help to evaluate this possibility. Directed evolution of Shikimate dehydrogenase AroE combined with high- throughput screening did not afford feedback insensitive Shikimate dehydrogenase. A couple of reasons might be responsible for this failure. First of all, only 10,000 mutants were screened from the library, while usually up to 10‘5 mutants are screened.'9 Second reason might be a difference in protein concentration from one sample to the next. It was measured (results are not shown) that cell growth inside the deep-well varied depending on the well position within a growth plate (outer wells grew better than inner), therefore different amounts of proteins where obtained for screening. As one can observe in Table 16, values in the “RatioC” column vary from 0.32 to 0.03, indicating that even control slope ratio was not consistent from one screening plate to the next. Probably the most important problem in this screening was the lack of uniform cell. None of the previously described strategies helped to increase the concentration and/or yield of microbe-synthesized shikimic acid. Another strategy would be to identify the export system for shikimic acid. If such a system exists in E. coli, overexpression of efflux proteins would lower in vivo concentration of shikimic acid. This would drive the equilibrium towards shikimic acid formation from 3-dehydroshikimic acid (Figure 1) and would result in higher titers and yields of shikimic acid as well as higher SA/DHS ratios. Transcription analysis using gene-chips resulted in identification of the p-hydroxybenzoic acid efflux system after wild-type E. coli W3110 was treated with p-hydroxybenzoic 81 acid.20 Identical experiments could be performed with shikimic acid. The control strain could use E. coli wild-type W3110. Addition of shikimic acid to the W3110 culture should trigger the shikimic acid export system. Transcriptional analysis of E. coli treated with shikimic acid could reveal the proteins responsible for shikimic acid export out of the cytoplasm. 82 REFEREN ES 1 (a) Chandran, S. S.; Yi, J.; Draths, K. M.; von Daeniken, R.; Weber, W.; Frost, J. W. Phosphoenolpyruvate Availability and the Biosynthesis of Shikimic Acid. Biotechnol. Prog. 2003, 19, 808-814. (b) Yi, J.; Li, K.; Draths, K. M.; Frost, J. W. Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli. Biotechnol. Prog. 2002, 18, 1141-1148. 2 Knop, D. R.; Draths, K. M.; Chandran, S. S.; Barker, J. L.; von Daeniken, R.; Weber, W.; Frost, J .W. Hydroaromatic Equilibration During Biosynthesis of Shikimic Acid. J. Am. Chem. Soc. 2001, 123, 10173-10182. 3 Dell, K. A.; Frost, J. W. Identification and Removal of Impediments to Biocatalytic Synthesis of Aromatics from D-Glucose: Rate-Limiting Enzymes in the Common Pathway of Aromatic Amino Acid Biosynthesis. J. Am. Chem. Soc. 1993, 115, 11581-11589. 4 (a) Weaver, L. M.; Herrmann, K. M. Cloning of an Arof Allele Encoding a Tyrosine-Insensitive 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase. J. Bacterial.l990, I 72, 6581-6584. (b) Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R. M.; Frost, J. W. Fed-Batch Fermentor Synthesis of 3-Dehydroshikimic Acid Using Recombinant Escherichia coli. Biotechnol. Bioeng. 1999, 64, 61-73. 5 (a) Michel, G.; Roszak, A. W.; Sauve, V.; Maclean, J .; Matte, A.; Coggins, J. R.; Cygler, M.; Lapthom, A. J. Structures of Shikimate Dehydrogenase AroE and Its Paralog YdiB. J. Biol. Chem. 2003, 278, 19463-19472. (b) Benach, J .; Lee, 1.; Edstorm, W.; Kuzin, A. P.; Chiang, Y.; Acton, T. B.; Montelione, G. T.; Hunt, J. F. The 2.3-A Crystal Structure of the Shikimate 5-Dehydrogenase Orthologue YdiB from Escherichia coli Suggests a Novel Catalytic Enviroment for an NAD-dependent Dehydrogenase. J. Biol. Chem. 2003, 278, 19176-19182. 6 (a) Chaudhuri, 8.; Anton, I. A.; Coggins, J. R. Shikimate Dehydrogenase from Escherichia coli. Meth. Enzymol. 1987, 142, 315-320. (b) Lumsden, J .; Coggins, J. R. The Subunit Structure of the arom Multienzyme Complex of Neurospora crassa. A Possible Pentafunctional Polypeptide Chain. Biochem. J. 1977, 161, 599-607. (c) Coggins, J. R.; Boocock, M. R.; Chaudhuri, S.; Lambert, J. M.; Lumsden, J.; Nimmo, G. A.; Smith, D. D. S. The arom Multifunctional Enzyme from Neurospora crassa. Meth. Enzymol. 1987, 142, 325-341. 7 Pittard, J .; Wallace, B. J. Distribution and Function of Genes Concerned with Aromatic Biosynthesis in Escherichia coli. J. Bacteriol. 1966, 91 , 1494-1508. 83 8 Draths, K. M.; Ward, T. L.; Frost, J. W. Biocatalysis and Nineteenth Century Organic Chemistry: Conversion of D-Glucose into Quinoid Organics. J. Am. Chem. Soc. 1992, 114, 9725-9726. 9 (a) Mitsuhashi, S.; Davis, B. D. Aromatic Biosynthesis XIII. Conversion of Quinic Acid to 5-Dehydroquinic Acid by Quinic Dehydrogenase. Biochim. Biophys. Acta 1954, 15, 268-280. (b) Davis, B. D.; Gilvarg, C.; Mitsuhashi, S. Enzymes of Aromatic Biosynthesis: Quinic Dehydrogenase from Aerobacter aerogenes. Methods Enzymol. 1955, 2, 307-311. B?- 10 Ward, T. L. Biocatalytic Synthesis of Quinic Acid and Application to the Production of Commercial Quinoid Organic Products. M.S. Thesis 1993. Purdue University. ll http://ergo.illlcgl‘atcdgenomics.com/ERGO/ J 12 Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, 2.; Miller W.; Lipman, D. J. Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs. Nucleic Acid Res. 1997 , 25, 3389-3402. 13 http://gcnomcgld.wusjl.cdlfipljgiggs/bzlctcri:1,l_/'_kpncumon incl 14 htlp://ca.expalsyorg/ 15 ht.11121211:unsgbjrac._lechLlst_zll_L1 16 Nasser, L.; Nester, E. W. Aromatic Amino Acid Biosynthesis: Gene-Enzyme Relationships in Bacillus subtilis. J. Bacteriol. 1967, 94, 1706-1714. 17 Vellanoweth, R. L. Translation and Its Regulation. In Bacillus subtilis and Other Gram—Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. Sonenshein, A. L., Ed.; ASM Press: Washington, DC, 1993; pp 699-711. 18 Cadwell, R. C.; Joyce, G. F. Mutagenic PCR. PCR Methods Applic. 1994, 4, 136- 139. 19 Schweinhost, A. Advanced Screening Strategies for Biocatalyst Discovery. In Directed Molecular Evolution of Proteins or How to Improve Enzymes for Biocatalysis. Brakmann, S.; Johnsson K., Ed.; Wiley-VCH Verlag GmbH, Weinheim, 2002; pp 159- 176. 84 20 Van Dyk, T. K.; Templeton, L. J .; Cantera, K. A.; Sharpe, P. L.; Sariaslami, F. S. Characterization of the Escherichia coli AaeAB Efflux Pump: a Metabolic Relief Valve? J. Bacteriol. 2004, 186, 7196-7204. -P_J_ 85 CHAPTER FQQR Experimental General methods Spectroscopic measurements 1H NMR spectra were recorded on a Varian 300 MHz VX-300 FT -NMR spectrometer. Chemical shifts were reported in parts per million (ppm) downfield from internal sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP, 6 = 0.00) with D20 as solvent. TSP was purchased from Lancaster. 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. Bacteria strains and plasmids All the strains and plasmids used are shown in Table 18. E. coli K-12 strain RB791 was obtained from the American Type Culture Collection (ATCC strain 53622). E. coli AB2834,‘ were obtained from the E. coli Genetic Stock Center at Yale University. E. coli KL3,2 SP1.1,3 were constructed in the lab previously. E. coli TC214 and TC254 were provided by Professor L. 0. Ingram (University of Florida). Plasmid constructions were carried out in E. coli DHSa, which is available from Invitrogen. Plasmid pJF118EH5 was provided by Professor M. Bagdasarian (Michigan State University). Plasmids pSU18,6 were obtained previously by this lab. Plasmids pKL5.17A,2 pKD12.112,3 pKD12.138,3 pNR8.146,7 pTW8090A,8 pJY1.43A, pJYl.211A” and pJY1.216A,'l pMFS 1A9 were constructed in the lab previously. 86 t. in Table 18. Bacterial strains and plasmids. Strain/Plasmid Relevant Characteristics Source Strain RB791 W31 10 lacL81" ATCC AB2834 tsx-352 gan42 A’ aroE353 malT352 CGSC KL3 AB2834 serA::aroB Ref. 2 SP1. 1 RB791 serA.°.°aroB aroL4 78: :TnI 0 aroKI 7: :C mR Ref. 3 RB79lserA::aroB RB791 serA::aroB Ref. 3 DHSa F ¢801acZAMI5 A(lacZYA-argF) U169 recAI endAIInvitrogen hstI 7(rk_, mk+) phoA A’ supE44 thi-I gyrA96 relAI TC21 W31 10 Aath szF RT-tet-F RT Ref. 4 TC25 W31 10 AsucAzzF RT-tet-F RT Ref. 4 KL3AathH:: AB2834 serA::aroB Aath H::F RT-tet-F RT Chapter 2 F RT-tet-F RT KL3AsucA2: FR T- AB2834 serA::aroB AsucAzzF RT—tet-F RT Chapter 2 let-F RT Plasmid pKK223-3 ApR, PM. Amersham Bioscience pJF118EH ApR, Pm lacl" in pKK223-3 Ref. 5 pGEX-4T-1 ApR, Pm GST, lacI" Amersham Bioscience pSU18 Cm“, PmlacZ’, p 15A replicon Ref. 6 pMF51A ApR, tktA Lab pKD12.112 ApR, aroFFBR, PmaroE, serA in pSU18 Ref. 3 pKD12. 138 ApR, aroFmR, tktA, PmaroE, serA in pKD12.112 Ref. 3 pKL5.17A Cm“, tktA in pKDII.29IA Ref. 2 pNR8.146 serAtktA in p34e Ref. 7 pTW8090A Cm“, qad in pSUl9 Ref. 6 pJY1.43 Cm“, PmaroE, in pSU18 Lab pJYl.211A ApR, serA, aroFFBR, PmF, PmppsA Ref. 11 pJYl.216A ApR, serA, aroFFBR, Pomp tktA, PmppsA Ref. 11 pJJl.224A Pmadk in pJFl 18EH Chapter 2 pJJ 1.262A ApR, serA, aroFFBR, PamF, PmppsA, Pmadk Chapter 2 pJJ 1.266A ApR, serA, aroFFBR, PmF, tktA, P, psA, Pmadk Chapter 2 p112. 134A ydiB in pGEX-4T-l Chapter 3 pJJ2.297A aroE in pGEX—4T-1 Chapter 3 pJ13.041A ApR, PmydiB in pKK223-3 Chapter 3 pJJ3.247A Ap“, PmaroE in pKK223-3 Chapter 3 pJJ3.289AP Ap“, quad in pKK223-3 Chapter 3 pJJ4.024A ApR, aroFFBR, quad Chapter 3 pJJ4.025A ApR, aroFFBR, quad, tktA, serA Chapter 3 pJJ4.118A aroD in pGEX—4T—l Chapter 3 pJJ4.150A ApR, aroFFBR, PmydiB Chapter 3 pJJ4.171A ApR, aroFmR, PmydiB, tktA, serA Chapter 3 87 Storage of bacterial strains and plasmids All bacterial strains were stored at -78 °C in glycerol. Plasmids were transformed into DH501 for long-term storage. Glycerol samples were prepared by adding 0.75 mL of an overnight culture to a sterile vial containing 0.25 mL of 80% (v/v) glycerol. The solution was mixed, left at room temperature for 2 h and then stored at -78 °C. Culture medium All solutions were prepared in distilled, deionized water. LB mediumIO (1 L) contained Bacto tryptone (10 g), Bacto yeast extract (5 g), and NaCl (10 g). L-Brothlo (l L) contained Bacto tryptone (10 g), Bacto yeast extract (5 g), NaCl (5 g), glucose (1 g) and CaCl2 (2.5 mM). Soft agarl0 (100 mL) contained Bacto tryptone (1 g), Difco agar (0.55 g), and NaCl (0.5 g). M9 saltsl0 (1 L) contained NazHPO4 (6 g), KHZPO4 (3 g), NH4C1 (1 g), and NaCl (0.5 g). M9 medium contained carbon sources (D-glucose, D- xylose, D-maltose or D-mannitol, 10 g), MgSO4 (0.12 g), and thiamine (0.001 g) in 1 L of M9 salts. Solutions of inorganic salts, magnesium salts, and carbon sources were autoclaved separately and then mixed. Antibiotics were added where appropriate to the following final concentrations unless noted otherwise: chloramphenicol, 20 ug/mL; ampicillin, 50 ug/mL; tetracycline, 12.5 ug/mL. Stock solution of antibiotics were prepared in water with the exception of chloramphenicol which was prepared in 95% ethanol and tetracycline which was prepared in 50% aqueous ethanol. L-Phenylalanine, L-tyrosine, L-tryptophan, and L-serine were added to M9 medium where indicated to a final concentration of 0.04 g/L. Antibiotics, isopropyl B-D-thioglucopyranoside (IPTG), thiamine, and amino acid supplementations were sterilized through 0.22-um membranes prior to addition to M9 medium. Solid medium was prepared by addition of 1.5% (w/v) 88 Difco agar to the medium. Fermentation medium (1 L) contained K2HPO4 (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), and L-tryptophan (0.35 g), and concentrated H2S04 (1.2 mL). The culture medium was adjusted to pH 7.0 by addition of concentrated NH40H before autoclaving. The following supplementations were added immediately prior to initiation of the fermentation: glucose (19-24 g under glucose-limited conditions or 30 g under glucose-rich conditions), MgSO4 (0.24 g), aromatic vitamins p-aminobenzoic acid (0.01 g), 2,3-dihydroxybenzoic acid (0.01 g), and p-hydroxybenzoic acid (0.01 g), and trace minerals (NH4)6(M07024)-4H20 (0.0037 g), ZnSO4~7HzO (0.0029 g), H3BO3 (0.0247 g), CuSO4'5HzO (0.0025 g), and MnC12'4HzO (0.0158 g). D-Glucose and MgSO4 were autoclaved separately while aromatic vitamins and trace minerals were sterilized through 0.22-um membranes prior to addition to the medium. Fed-batch fermentation (general) Fermentations'l employed a 2.0 L working capacity B. Braun M2 culture vessel fitted with a stainless steel baffle cage consisting of four 1/2” x 5” baffles. Utilities were supplied by a B. Braun Biostat MD controlled by a DCU-1 or DCU-3. Data acquisition utilized a Dell Optiplex Gst 5166M personal computer (PC) equipped with B. Braun MFCS/Win software (v2.0). Temperature, pH, and dissolved oxygen (D.O.) were controlled with PID control loops. Temperature was maintained at 36 °C, and pH was maintained at 7.0 by addition of concentrated NH4OH or 2 N H2804. Dissolved oxygen was measured using a Mettler-Toledo 12 mm sterilizable O2 sensor fitted with an Ingold A-type O2 permeable membrane. D.O. was maintained at 20% air saturation. Exhaust 89 CO2 was measured using gas analyzer purchased from Sartorius/BBI. Antifoam (Sigma 204) was added as needed. Inoculants were prepared by introduction of a single colony into 5 mL of M9 medium. The culture was grown at 37 °C with agitation at 250 rpm until they were turbid (~18-30 h) and subsequently transferred to 100 ml. of M9 medium. Cultures were grown at 37 °C for an additional 12 h. The inoculant (OD600 = 1.0-2.0) was then transferred into the fermentor vessel and the batch fermentation was initiated (t = 0 h). Glucose-rich fermentor conditions The initial glucose concentration in the fermentation medium was 30 g/L. Three staged methods were used to maintain D.O. levels at 20% air saturation during the course of the fermentations. With the airflow at an initial setting of 0.06 LIL/min, D.O. concentration was maintained by increasing the impeller speed from its initial set point of 50 rpm to a preset maximum of 750 rpm. With the impeller rate constant at 750 rpm, the mass flow controller then maintained D.O. levels by increasing the airflow rate from 0.06 L/L/min to a preset maximum of 1.0 LIL/min. After the preset maxima of 750 rpm and 1.0 L/L/min were reached, the third stage of the fermentation was initiated in which glucose (65% w/v) was added to the vessel at a rate sufficient to maintain a glucose concentration in the range of 5 to 30 g/L for the remainder of the run. Airflow was maintained at 1.0 LIL/min, and the impeller was allowed to vary in order to maintain the DO. concentration at 20% air saturation. The impeller speed typically varied from 750 rpm to 1400 rpm during the remainder of the run. A solution of IPTG (100 mM; 0, 0.25, 0.50, 0.75, 1.0, and 2.0 mL) was added at timed intervals after initiation of the run to 90 achieve reported IPTG concentrations of 0, 6.0, 12, 18, 24, and 48 mg/L, respectively in the fermentation medium. Glucose-limited fermentor conditions The initial glucose concentration in the fermentation medium was 19-24 g/L, depending on the strain being examined. Three staged methods were used to maintain D.O. levels at 20% air saturation, with the first two stages identical to those described for the glucose-rich conditions. After the preset maxima of 750 rpm and 1.0 LIL/min of airflow were reached, the third stage of the fermentation was initiated in which the DO. concentration was maintained at 20% air saturation for the remainder of the run by oxygen sensor-controlled glucose feeding. At the beginning of this stage, the DO. concentration initially fell below 20% air saturation due to residual glucose in the medium. This lasted for up to 30 min before glucose (65% w/v) feeding commenced. The glucose feed PID control parameters were set to 0.0 5 (off) for the derivative control (1:0) and 999.9 3 (minimum control action) for the integral control (1,). XP was set to 950% to achieve a Kc of 0.1. A solution of IPTG (100 mM; 0, 0.25, 0.50, 0.75, 1.0, and 2.0 mL) was added at timed intervals after initiation of the run to achieve reported IPT G concentrations of 0, 6.0, 12, 18, 24, and 48 mg/L, respectively in the fermentation medium. Analysis of fermentation broths Samples (5 mL) of fermentation broth were taken 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 (ODwO). Dry cell weight (g/L) was 91 calculated using a conversion coefficient of 0.43 g/L/OD600. The remaining fermentation broth was centrifuged to obtain cell-free broth. Glucose concentrations in cell-free broth were measured using the Glucose Diagnostic Kit purchased from Sigma. Solute concentrations in the cell-free broth were quantified by 'H NMR. Solutions were concentrated to dryness under reduced pressure, concentrated to dryness one additional time from D20, and then redissolved in D20 containing a known concentration of the sodium salt of 3-(trimethylsilyl)propionic- 2,2,3,3-d4 acid (TSP). lH NMR spectra were recorded and concentrations were determined by comparison of integrals corresponding to each compound with the integral corresponding to TSP (0 = 0.00 ppm). A standard concentration curve was determined for each metabolite using solutions of authentic, purified metabolites. The following resonances were used to quantify each compound: shikimic acid (0 4.45, d, J = 3.7 Hz, 1 H); 3-dehydroshikimic acid (0 4.28, d, J = 11.5 Hz, 1 H); 3-dehydroquinic acid (0 4.38, d, J = 9.3 Hz, 1 H); quinic acid (0 4.16, m, 1 H); 3-deoxy-D-arabino-heptulosonic acid (0 1.81, dd, J = 12.4, 12.4 Hz, 1 H); and gallic acid (0 7.02, s, 2 H). The following response factor was used for each molecule: shikimic acid, 0.70; 3-dehydroshikimic acid, 0.95; 3- dehydroquinic acid, 0.89; quinic acid 0.75; 3-deoxy-D-arabino-heptulosonic acid, 1.22; gallic acid, 1.36. Genetic manipulations General Recombinant DNA manipulations generally followed methods described by Sambrook.12 Restriction enzymes were purchased from Invitrogen or New England 92 Biolabs. FasteLinkTM DNA Ligation Kit was obtained from Epicentre. Zymoclean Gel DNA Recovery Kit and DNA Clean & Concentrator Kit was obtained from Zymo Research Company. Maxi, Midi and Mini Plasmid Purification Kits were obtained from Qiagen. Calf intestinal alkaline phosphatase was obtained from New England Biolabs. Agarose (electrophoresis grade) was obtained from Invitrogen. Phenol was prepared by addition of 0.1 % (w/v) 8-hydroxyquinoline to distilled, liquefied phenol. Extraction with an equal volume of 1 M Tris-HCl (pH 8.0) two times was followed by extraction with 0.1 M Tris-HCl (pH 8.0) until the pH of the aqueous layer was greater than 7.6. Phenol was stored at 4 °C under an equal volume of 0.1 M Tris-HCl (pH 8.0). SEVAG was a mixture of chloroform and isoamyl alcohol (24:1 v/v). TE buffer contained 10 mM Tris-HCl (pH 8.0) and 1 mM NazEDTA (pH 8.0). Endostop solution (10X concentration) 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, 0.12 mL of DNase-free RNase was added to 1 mL of 10X Endostop solution. DNase-free RNase was purchased from Roche or (10 mg mL‘l) was prepared by dissolving RNase in 10 mM Tris-Cl (pH 7.5) and 15 mM NaCl. DNase activity was inactivated by heating the solution at 100 °C for 15 min. Aliquots were stored at -20 °C. PCR amplifications were carried out as described by Sambrook.‘2 Each reaction (0.1 mL) contained 10 mM KCl, 20 mM Tris-Cl (pH 8.8), 10 mM (NH4)ZSO4, 2 mM MgSO4, 0.1% Triton X-100, dATP (0.2 mM), dCTP (0.2 mM), dGTP (0.2 mM), dTTP (0.2 mM), template DNA, 0.5 11M of each primer, and 2 units of Platinum Taq HiFi or Pfu polymerase also have been used for PCR reaction with the reaction buffers provided. Initial template concentrations varied from 0.02 ug to 1.0 pg. 93 Large scale purification of plasmid DNA In a 2 L Erlenmeyer flask, LB (500 mL) containing the appropriate antibiotics was inoculated from a single colony, and the culture was incubated in a gyratory shaker (250 rpm) for 14 h at 37 °C. DNA was purified using a Qiagen Maxi Kit or Midi Kit as described by the manufacturer. The purity of DNA isolated by this method was adequate for DNA sequencing. Small scale purification of plasmid DNA An overnight culture (5 mL) of the plasmid-containing strain was grown in LB containing the appropriate antibiotics.” Cells from 3 mL of the culture were collected in a 1.5 mL microcentrifuge tube by centrifugation. The resulting cell pellet was liquefied by vortexing (30 sec) and then resuspended in 0.1 mL of cold GETL solution into which lysozyme (5 mg mL‘l) had been added immediately before use. The solution was stored 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 sample was added 0.15 mL of cold KOAc solution. The solution was shaken vigorously and stored on ice for 5 min before centrifugation (15 min, 4 °C). The supernatant was transferred to another microcentrifuge tube and extracted with equal volumes of phenol and SEVAG (0.2 mL). The aqueous phase (approximately 0.5 mL) was transferred to a fresh microfuge tube, and DNA was precipitated by the addition of 95% ethanol (1 mL). The sample was left at room temperature for 5 min before centrifugation (15 min, room temperature) to collect the DNA. The DNA pellet was rinsed with 70% ethanol, dried, and redissolved in 50 -100 uL TE. DNA isolated from this method was used for 94 restriction enzyme analysis, and the concentration was not determined by spectroscopic methods. Determination of DNA concentration The concentration of DNA in the sample was determined as follows. An aliquot (10 uL) of the DNA was diluted to 1 mL in TE and the absorbance at 260 nm was measured relative to the absorbance of TE. The DNA concentration was calculated based on the fact that the absorbance at 260 nm of a 50 pg mL'] of plasmid DNA is 1.0. DNA precipitation DNA was precipitated by addition of 0.1 volume of 3 M NaOAc (pH 5.2) followed by thorough mixing and addition of 3 volumes of 95% ethanol. Samples were stored for at least 2 h at -78 °C. Precipitated DNA was recovered by centrifugation (15 min, 4 °C). To the DNA pellet was added 70% ethanol (100 uL), and the sample was centrifuged again (15 min, 4 °C). DNA was dried and redissolved in TE. Restriction enzyme digestion of DNA Restriction enzyme digests were performed using restriction enzyme buffers supplied by Invitrogen or New England Biolabs. A typical digest contained approximately 0.8 ug of DNA in 8 uL TE, 2 uL of restriction enzyme buffer (10X concentration), 1 11L of restriction enzyme, and TE to a final volume of 20 1.1L. Reactions were incubated at 37 °C for 1 h. Digests were terminated by addition of 2.2 uL of Endostop solution (10X concentration) and subsequently analyzed by agarose gel electrophoresis. When DNA was required for subsequent cloning, restriction digests 95 were terminated by addition of 1 uL of 0.5 M NazEDTA (pH 8.0) followed by extraction of the DNA with equal volumes of phenol and SEVAG and precipitation of the DNA. 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. Higher concentrations of agarose (1%-2%) were used to resolve DNA fragments smaller than 1 kb. Lower concentrations of agarose (0.35%) were used to resolve DNA fragments larger than 10 kb. Ethidium bromide (0.5 ug mL'l) was added to the agarose to allow visualization of DNA fragments over a UV lamp. The size of the DNA fragments were determined by using two sets of DNA standards: A. DNA digested with Hindlll (23.1-kb, 9.4-kb, 6.6-kb, 4.4-kb, 2.3-kb, 2.0-kb, and 0.6-kb) and A DNA digested with EcoRI and Hindlll (21.2-kb, 5.1-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). Also 100 bp DNA Ladder (Invitrogen) was used to determine the size of small DNA fragements. The ladder consists of 15 blunt-ended fragments ranging in length from 100 to 1500 bp, at 100 bp increments, and an additional fragment at 2,072 bp. Isolation of DNA from agarose The band of agarose containing DNA of interest was excised from the gel while visualized with high wavelength UV and chopped thoroughly with a razor in a plastic weighing tray. The agarose was then transferred to a spin column consisting of a 500 11L microfuge tube packed tightly with glass wool and with an 18 gauge hole in its bottom. The spin column was then centrifuged for 5 min using a microcentrifuge to separate the 96 DNA solution from the agarose. The DNA-containing aqueous phase collected after centrifugation were mixed with 3 M NaOAc and 95% ethanol. The DNA was precipitated as described previously and redissolved in TE. Alternatively, the band of agarose containing DNA of interest was excised from the gel while visualized with high wavelength UV. Zymoclean Gel DNA Recovery Kit was used to isolate DNA from the agarose gel according to the protocol provided by the Zymo Research Company. Treatment of vector DNA with calf intestinal alkaline phosphatase (CIAP) Plasmid vectors digested with a single restriction enzyme were dephosphorylated to prevent self-ligation. Vector DNA after digestion was immediately combined in a total volume of 60 uL. To this sample was added 7 [AL of dephosphorylation buffer (10X concentration, provided by enzyme supplier) and 3 [AL of calf intestinal alkaline phosphatase (3 units). The reaction was incubated at 37 °C for 1 h. The phosphatase was inactivated by addition of 1 uL of 0.5 M NazEDTA (pH 8.0) followed by heat treatment (65 °C, 20 min). The sample was extracted with phenol and SEVAG (100 11L each) to remove protein, and the DNA was precipitated as previously described and redissolved in TE. Treatment of DNA with Klenow fragment DNA with recessed 3' termini was modified to blunt-ended fragment by treatment with the Klenow fragment of E. coli DNA polymerase 1. After the DNA (0.8-2 ug) restriction digestion was completed in a 20 11L reaction, a solution (1 11L) containing each of the desired dNTPs was added to provide a final concentration of 1 mM for each dNTP. 97 Addition of 1-2 units of the Klenow fragment to the reaction was followed by incubation of the mixture at room temperature for 20-30 min. Since the Klenow fragment works well in the common buffers used for restriction digestion of DNA, there was no need to purify the DNA after restriction digestion and prior to filling recessed 3' termini. Klenow reactions were quenched by extraction with equal volumes of phenol and SEVAG. DNA was recovered by DNA precipitation. Ligation of DNA Alternatively, Fast-Link DNA Ligation Kit (Epicentre) was used for ligation of insert DNA with cohesive or blunt ends into vectors with compatible cohevsive ends according to the protocol provided by the manufacturer. Preparation and transformation of competent cells Competent cells were prepared using a procedure modified from Sambrook.12 An aliquot (1 mL) from an overnight culture (5 mL) was used to inoculate 100 mL of LB (500 mL Erlenmeyer flask) containing the appropriate antibiotics. The cells were cultured in a gyratory shaker (37 °C, 250 rpm) until they reached the mid-log phase of growth (judged from the absorbance at 600 nm reaching 0.4-0.6). The culture was poured into a large centrifuge bottle that had been previously sterilized with bleach and rinsed with sterile water. The cells were collected by centrifugation (4 000g, 5 min, 4 °C) and the culture medium was discarded. All manipulations were canied out on ice during the remaining portion of the procedure. The cell pellet was washed with 100 mL of cold 0.9% NaCl (w/v) and then resuspended in 50 mL of cold 100 mM CaClz. The suspension was stored on ice for a minimum of 30 min and then centrifuged (4 000g, 5 min, 4 °C). 98 The cell pellet was resuspended in 4 mL of cold 100 mM CaClz containing 15% glycerol (v/v). Aliquots (0.25 mL) were dispensed into 1.5 mL microcentrifuge tubes and immediately frozen in liquid nitrogen. Competent cells were stored at -78 °C with no significant decrease in transformation efficiency over a period of six months. Frozen competent cells were thawed on ice for 5 min before transformation. A small aliquot (1 to 10 uL) of plasmid DNA or a ligation reaction was added to the thawed competent cells (0.1 mL). The solution was gently mixed and stored on ice for 30 min. The cells were then heat shocked at 42 °C for 2 min and placed on ice briefly (1 min). LB (0.5 mL, no antibiotics) was added to the cells, and the sample was incubated at 37 °C (no agitation) for 1 h. Cells were collected in a microcentrifuge (30 s). If the transformation was to be plated onto LB plates, the cells were resuspended in a small volume of LB medium (0.1 mL), and then spread onto plates containing the appropriate antibiotics. If the transformation was to be plated onto minimal medium plates, the cells was washed once with the same minimal medium. After resuspension in fresh minimal medium (0.1 mL), the cells was spread onto the plates. A sample of competent cells with no DNA added was also carried through the transformation procedure as a control. These cells were used to check the viability of the competent cells and to verify the absence of growth on selective medium. Purification of genomic DNA Genomic DNA was purified using a method described by Pitcher.‘3 Broth cultures (20 mL) were harvested at the end of the exponential growth phase by centrifugation (1 000g, 15 min, room temperature). A small cell pellet was obtained. The cells of Gram-positive species were resuspended in 100 uL of fresh lysozyme (50 99 mg/mL) in TE buffer and incubated at 37°C for 30 min. The Gram-negative species were resuspended in 100 11.1 of TE buffer without enzyme treatment and incubated at 37°C for 30 min. Cells were lysed with 0.5 mL 5 M guanidium thiocyanate (Sigma), 100 mM EDTA and 0.5% v/v sarkosyl (GES reagent), which was prepared as follows. Guanidium thiocyanate (60 g), 0.5 M EDTA at pH 8 (20 mL) and deionized water (20 mL) were heated at 65°C with mixing until dissolved. After cooling, 5 mL of 10% v/v sarkosyl were added, the solution was made up to 100 mL with deionized water, filtered through a 0.22-um membrane and stored at room temperature. Cell suspensions were vortexed briefly and checked for lysis (clear solution) after 5-10 min. The lysates were cooled on ice and 0.25 mL cold 7.5 M ammonium acetate was added with mixing on ice for 10 min. To this sample, 0.5 mL SEVAG was added, and the solution was mixed thoroughly. After centrifugation in a 1.5 mL Eppendorf tube (25 000g, 10 min, room temperature), supernatant fluids were transferred to Eppendorf tubes and 0.54 volumes of cold 2-propanol was added. The tubes were inverted for 1 min to mix the solutions and the fibrous DNA precipitate was deposited by centrifugation (6 5003, 20 6, room temperature). Pellets of DNA were washed five times in 70% ethanol and dried at room temperature for 20 min. Genomic DNA was redissolved in 100 11L TE. Pl-mediated transduction Transduction with P1 phage was carried out using a method modified from Miller.l4 P1 phage lysate was prepared by propagation of phage in the donor strain using the following procedure. Serial dilutions of P1 phage stock (0.1 mL, 10'1 to 10’s) in LB were prepared in sterile test tubes (13 x 100 mm). An aliquot (0.1 mL, approximately 5 x 108 cells) of an overnight culture of the donor strain was added to each tube. Sterile, 100 molten soft agar (45 °C) was added to each tube. The contents of each tube were mixed and poured immediately onto a pre-warrned (37 °C) L plate, swirling gently to achieve uniform coverage of the plate. After the agar had solidified, the plates were incubated at 37 °C until confluent lysis had occurred (approximately 8 h). Because the multiplicity of infection is critical to phage generation, confluent lysis occurred on only one or two of the plates. L-Broth (4 mL) was added to these plates, which were then stored overnight at 4 °C to allow the phage particles to diffuse into the broth. The L-broth was collected from the plate and vortexed with several milliliters of CHCl3 to make certain that all of the cells had lysed. The solution was centrifuged (2 000g, 5 min, room temperature) to separate the layers. Aqueous phage lysate was stored in 1.5 mL microfuge tubes over several drops of CHCl3 at 4 °C. Infection of the recipient strain with phage lysate proceeded as follows. Overnight culture (2 mL) of the recipient strain was centrifuged (microfuge, 30 s, 4 °C) and the growth medium discarded. The cells were resuspended in 1 mL of 5 mM CaCl2 and 100 mM MgSO4 and shaken (200 rpm) at 37 °C for 15 min to promote aeration of the cells. In the meantime, 0.1 mL serial dilutions (10° to 103) of phage lysate in LB were prepared in sterile microfuge tubes. An aliquot (0.1 mL) of aerated recipient cells was added to each of the phage dilutions, the samples were gently mixed and then incubated at 37 °C for 20 min without shaking. Sodium citrate (1 M, 0.2 mL) was added to each sample, and the cells were harvested (microfuge, 30$, room temperature) and resuspended in 0.2 mL of LB containing 100 mM sodium citrate. After incubation at 30 °C for 30 min, cells were again harvested (microfuge, 30 s, room temperature), resuspended in 0.1 mL of growth medium, and plated out onto appropriate agar plates. 101 Enzyme assays After collected and resuspended in proper resuspension buffer, the cells were disrupted by two passages through a French pressure cell (SLM Aminco) at 16000 psi. Cellular debris was removed from the lysate by centrifugation (48 0003, 20 min, 4 °C). Protein was quantified using the Bradford dye-binding procedure.15 A standard curve was prepared using bovine serum albumin. The protein assay solution was purchased from Bio-Rad. Adenylate kinase assay Adenylate kinase was assayed according to the procedure described by Tanizawa.l6 Harvested cells were resuspended in 100 mM Tris-HCl buffer (pH 7.4) and disrupted using a French press as described above. Cell lysates where diluted in Tris-HCl (pH 7.4) buffer prior to the assay. The assay (1 mL) contained Tris-HCl (100 mM, pH 7.4) buffer, KCl (100 mM), MgCl2 (2 mM), glucose (10 mM), NADP+ (0.5 mM), ADP (4 mM), hexokinase (7 units) and glucose 6-phosphate dehydrogenase (9 units). Assay mixture was incubated for 2 min, and addition of cell lysate started the reaction. Formation of NADPH was monitored at 340 nm for 120 seconds. One unit of adenylate kinase corresponds to formation of 1 mole of ATP per min at room temperature. DAHP synthase assay DAHP synthase was assayed according to the procedure described by Schoner.” Harvested cells were resuspended in 50 mM potassium phosphate (pH 6.5) that contained 10 mM PEP and 0.05 mM CoClz. The cells were disrupted using a French press as described above. Cellular lysate was diluted in a solution of potassium phosphate (50 102 mM), PEP (0.5 mM), and 1,3-propanediol (250 mM), pH 7.0. A dilute solution of E4P was first concentrated to 12 mM by rotary evaporation and neutralized with 5 N KOH. Two different solutions were prepared and incubated separately at 37 °C for 5 min. The first solution (1 mL) contained E4P (6 mM), PEP (12 mM), ovalbumin (1 mg/mL), and potassium phosphate (25 mM), pH 7.0. The second solution (0.5 mL) consisted of the diluted lysate. After the two solutions were mixed (time = 0), aliquots (0.15 mL) were removed at timed intervals and quenched with 0.1 ml. of 10% trichloroacetic acid (w/v). Precipitated protein was removed by centrifugation, and the DAHP in each sample was quantified using thiobarbituric acid assay18 as described below. An aliquot (0.1 mL) of DAHP containing sample was reacted with 0.1 mL of 0.2 M NaIO4 in 8.2 M H3PO4 at 37 °C for 5 min. The reaction was quenched by addition of 0.8 M NaAst in 0.5 M Na2SO4 and 0.1 M H2S04 (0.5 mL) and vortexed until a dark brown color disappeared. Upon addition of 3 mL of 0.04 M thiobarbituric acid in 0.5 M NaZSO4 (pH 7), the sample was heated at 100 °C for 15 min. Samples were cooled (2 min), and the pink chromophore was then extracted into distilled cyclohexanone (4 mL). The aqueous and organic layers were separated by centrifugation (2 000g, 15 min, room temperature). The absorbance of the organic layer was recorded at 549 nm (e = 68000 L mol“l cm“). One unit of DAHP synthase activity was defined as the formation of 1 umol of DAHP per min at 37 °C. Phosphoenolpyruvate synthase assay PEP synthase activity was assayed at 30 °C according to the procedure described by Cooper.'9 The reaction (1 mL) contained 100 mM Tris-HCl (pH 8.0), 10 mM MgC12, 10 mM ATP, 1.25 mM sodium pyruvate and was initiated by addition of cell-free lysate. 103 Aliquots (100 111) of reaction mixture were removed at 1 min intervals and immediately added to a microcentrifuge tube containing 0.33 mL of a 0.1% aqueous solution of 2, 4- dinitrophenylhydrazine and 0.9 mL H20. The resulting mixture was incubated at 30 °C for 10 min. Following addition of 1.67 mL of 10% (w/v) NaOH, the mixture was further incubated at 30 °C for 10 min. The disappearance of pyruvate was quantified by measuring the absorbance at 445 nm. A molar extinction coefficient of 18,000 L mol'l cm'l was used to quantify pyruvate. One unit of PEP synthase activity was defined as the amount of enzyme that catalyzed the consumption of 1 umol of pyruvate per min at 30 °C. Transketolase assay Transketolase was assayed using a coupled enzyme procedure described by Paoletti.20 The assay solution (1 mL) contained triethanolamine buffer (150 mM, pH 7.6), MgCl2 (5 mM), thiamine pyrophosphate (0.1 mM), NADP (0.4 mM), 0- hydroxypyruvate (0.4 mM), D-erythrose 4-phosphate (0.1 mM), glucose 6-phosphate dehydrogenase (3 units), and phosphoglucose isomerase (10 units). The solution was incubated at room temperature and the absorbance at 340 nm was monitored for several minutes. After all the unreacted D-glucose-6-phosphate from the D-erythrose 4- phosphate synthesis had reacted, an aliquot of transketolase solution was added and the reaction monitored at 340 nm for 10-20 min. One unit of transketolase activity was defined as the formation of 1 mol of NADPH (e = 6220 L mol'l cm") per min. 104 Shikimate dehydrogenase assay in forward direction Shikimate dehydrogenase was assayed using 3-dehydroshikimic acid as the substrate according to the procedure described by Coggins.” Lysate was prepared and protein concentrations were determined as previously mentioned. Cells were harvested and resuspended in 100 mM potassium phosphate buffer (pH 7.4), NazEDTA (1 mM) and diethiothreitol (0.4 mM). Cellular lysate was diluted in 100 mM potassium phosphate buffer (pH 7.4). Assays (1 mL) contained potassium phosphate (100 mM, pH 7.0) buffer, 3-dehydroshikimic acid (2 mM), and fi-NADPH (0.2 mM) sodium salt. Potassium phosphate, 3-dehydroshikimic acid, and [El-NADPH solutions were mixed, and the spectrophotometer was zeroed. Addition of diluted lysate initiated the assay. The depletion of NADPH was monitored at 340 nm (e = 6,220 M'l cm") for 60 seconds. One unit of Shikimate dehydrogenase activity was defined as the formation of l umol of N ADP per minute at room temperature. Shikimate dehydrogenase assay in reverse direction Shikimate dehydrogenase was assayed using shikimic acid as the substrate according to the procedure described by Chaudhuri et a1.22 Lysate was prepared and protein concentrations were determined as previously mentioned. Cells were harvested and resuspended in a buffer containing Tris-HCl (100 mM, pH 7.5), NaQEDTA (1 mM) and diethiothreitol (0.4 mM). Cellular lysate was diluted in 100 mM Tris-HCl (pH 9.0). Assays (1 mL) contained Tris-HCl (100 mM, pH 9.0), shikimic acid (4 mM), and )3- NADP (2 mM) sodium salt. Tris-HCl, shikimic acid, and diluted lysate solutions were mixed, and the spectrophotometer was zeroed. Addition of fi-NADP initiated the assay. The formation of NADPH was monitored at 340 nm (e = 6,220 M“l cm") for 60 seconds. 105 One unit of Shikimate dehydrogenase activity was defined as the formation of 1 umol of N ADPH per minute at room temperature. Quinate dehydrogenase assay Quinate dehydrogenase was assayed using quinic acid according to the procedure described by Davis.23 Lysate was prepared and protein concentrations were determined as previously mentioned. Cells were harvested and resuspended in a buffer containing 100 mM potassium phosphate (pH 7.6). Cellular lysate or purified enzyme was diluted in 100 mM potassium phosphate (pH 7.6). Diluted enzyme solution was treated with 1M KCN (1.0% vol/vol) and incubated at 4 °C for at least 10 min prior the continuous assay. Assay (1 mL) contained potassium bicarbonate (32 mM, pH 9.4), NAD+ (0.32 mM) and clarified lysate. Addition of quinic acid (3.2 mM) initiated the assay. The formation of NADPH was monitored at 340 nm (e = 6,220 M’l cm") for 60 seconds. One unit of quinate dehydrogenase activity was defined as the formation of 1 umol of NADH per minute at room temperature. Chapter two E. coli KL3AathH::FRT-tet-FRT E. coli KL3AathH::FRT-tet-F RT was made from E. coli KL3 by P1 transduction using E. coli TC21 as the donor strain. Colonies were plated on selective LB/O.5 Tc/ plates containing 3% glucose. The resulting colonies were screened for AathH::FRT- tet-F RT mutation by checking the growth on the following plates: no growth LB/Ap and LB/Kan plates; no growth on M9 succinate plates ; growth on M9 glucose plates with aromatic and serine supplementation. Additionally it was characterized by PCR analysis. 106 5’-atpE (ATGGAAAACTGAATATG) and 5’-atpH (TTAAGACTGCAAGACGTC) primers were used in PCR analysis and afforded 2.3 kb fragment for mutant and 1.3 kb for control strain E. coli KL3. E. coli KL3AsucA::FR T -tet-FRT E. coli KL3AsucA::FRT—tet-FRT was made from E. coli KL3 by P1 transduction using E. coli TC25 as the donor strain. Colonies were plated on selective LB/0.5 Tc/ plates containing 3% glucose. The resulting colonies with AsucA::F RT-tet-F RT mutation were characterized by PCR analysis. 5’-sucA (ATGCAGAACAGCGCTTTG) and 5’- sucA ('I'I'I‘TCGACG'I'I‘CAGCGC) primers were used in PCR analysis and afforded 3.3 kb fragment for mutant and 2.8 kb for control strain E. coli W3110. Plasmid pJJ 1.224A The adk gene was amplified from RB791 genomic DNA using the following primers: 5’-TCCCQCGGGCGCTTTTTCAAAAAATTCG and 5’- ACGCGTCGACGCAAC'ITGTTGATAA'ITGT. Smal and SalI restriction sequences (underlined nucleotides) were included to facilitate cloning, respectively. Localization of the resulting 0.9-kb adk fragment into vector ppJFl 18Eh, which has previously been incubated with Smal and SalI followed by CIAP treatment, afforded pJJ 1.224A. Transcription of the adk gene in pJJ 1.224A proceeds in the same orientation relative to the vector-encoded Pm sequence. Plasmid pJJ 1.262A The Pmadk fragment was amplified from pJJ 1.224A plasmid using the following primers: 5’-ACGCGTQGACGGAGCTTATCGACTGCACG and 5 ’- 107 ACGCGTCGACGCAAC’ITGTI‘GATAATI‘GT. SalI restriction sequences (underlined nucleotides) were included to facilitate cloning. Localization of the Pmadk fragment into the Soil site of pJYl.211A afforded pJJ 1.262A, where Pmadk is in opposite direction relative to the aroFFBR. Plasmid pJJl.266A Following digestion of pMF51A with BamHI, the resulting 2.4-kb tktA fragment was treated with Klenow fragment and ligated to pJYl.211A, which had previously been incubated with HindIII followed by Klenow fragment. The 14.7-kb plasmid pJJ 1.266A was isolated in which transcription of the tktA gene proceeds in the same orientation as transcription from the Pmadk. Chapter three Purification of GST-tagged proteins A11 protein manipulations were carried at 4 °C. GST purification kit was purchased from Amersham Biosciences to facilitate GST tagged protein purification. Buffers used in purification included PBS buffer: NaCl (0.14 M), KCl (2.7 mM), NazHPO4 (10.1 mM), KHZPO4 (1.8 mM), pH 7.3; resuspension buffer: PBS buffer, dithiotreitol-DTI‘ (0.4 mM); binding buffer: sodium phosphate buffer (20 mM, pH 7.5), NaCl (0.15 M), high salt wash buffer: sodium phosphate buffer (20 mM, pH 7.5), NaCl (1 M); buffer B: Tris-HCl (50 mM, pH 7.5), DTI‘ (0.4 mM), KCl (50 mM), benzamidine HCl (1 mM), glycerol (50%). 108 A single colony of E. coli was used to inoculate 5 mL 2xYTA (tryptone (l6g/L), yeast extract (10 g/L), NaCl (5 g/L)) containing ampicillin. After 12 hours of growth, the 5 mL culture was transferred to 2 L 2xYTA medium containing ampicillin and grown at 37 °C until OD600 reached 0.6. IPTG was added to the culture to a final concentration of 0.5 mM. After an additional 6 h growth at 37 °C, the cells were harvested by centrifugation (6000g for 10 min at 4 °C), resuspended in 0.9% NaCl solution and harvested by centrifugation. Resulted cell pellet was resuspended in resuspension buffer, and lysed by two passages through a French press at 16000 psi. Cellular debris was removed by centrifugation at 20000g and 4 °C for 30 min. Resuspension buffer was added to clarified cell lysate (50 mL final volume) and resulted solution was applied to 1 mL GST-Sepharose column, earlier prepared by manufactures protocol. Column and bounded protein were washed with 30 bed volumes (6 x 5 mL) of resuspension buffer. A thrombin solution was added to column bounded GST tagged protein (50 U/2L culture) and column was incubated at room temperature for 12 hrs. Protein cleaved from GST tag was eluted together with thrombin from the column by washing with a high salt buffer (4 x 1 mL per mL of GST-Sepharose resin). The eluted protein was concentrated, dialyzed against buffer B, liquated and quick frozen in liquid nitrogen, and stored at -80 °C. Protein gel (SDS-PAGE) SDS-PAGE analysis was followed the procedure described by Harris.24 The separating gel was prepared by mixing 3.33 mL of 30% acrylamide stock solution (w/v in HZO), 2.5 mL of 1.5 M Tris-HCl (pH 8.8), and 4 mL of distilled deionized water. After degassing the solution using a water aspirator for 20 min, 0.1 mL of 10% ammonium persulfate (w/v in H20), 0.1 mL 10% SDS (w/v in H20), and 0.005 mL of N, N, N’, N’- 109 tetramethylethylenediamine (TEMED) was added. After mixing gently, the separating gel was poured into the gel cassette to about 1.5 cm below the top of the gel cassette. t- Amyl alcohol was overlaid on the top of the solution and the gel was allowed to polymerize for 1 h at rt. The stacking gel was prepared by mixing 1.7 mL of 30% acrylamide stock solution, 2.5 mL of 0.5 M Tris-HCl (pH 6.8), and 5.55 mL of distilled deionized water. After degassing for 20 min, 0.1 mL of 10% ammonium persulfate, 0.1 mL 10% SDS, and 0.01 mL of TEMED was added, and the solution was mixed gently. t- Amyl alcohol was removed from the top of the gel cassette, which was subsequently rinsed with water and wiped dry. After insertion of the comb, the gel cassette was filled with stacking gel solution, and the stacking gel was allowed to polymerize for 1 h at rt. After removal of the comb, the gel cassette was installed into the electrophoresis apparatus. The electrode chamber was then filled with electrophoresis buffer containing 192 mM glycine, 25 mM Tris base, and 0.1% SDS. Each protein sample (10 11L) was diluted with Laermnli sample buffer” (10 1.1L, Sigma S-3401) consisting of 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 125 mM Tris-HCl (pH 6.8). Samples and markers (MW-SDS-200, Sigma) were loaded into the sample well and the gel was run under constant current at 30 mA when the blue tracking dye (bromophenol blue) was within stacking gel. After the blue tracking dye reached the separating gel, a higher current (50 mA) was applied to the gel. At the completion of electrophoresis (blue tracking dye reaches the bottom of the gel), the gels were removed from the cassettes and fixed in a solution of 10% (w/v) aqueous trichloroacetic acid for 30 min. After staining in a solution containing 0.1% (w/v) Coomassie Brilliant Blue R, 45% (v/v) MeOH, 10% (v/v) HOAc in H20 for 3 h, the protein gels were destained in a 110 solution of 45% (v/v) MeOH, 10% (v/v) HOAc in H20. The gels were then sealed in a sheet protector. Purification of K. pneumoniae quinate dehydrogenase Amicon MatrexTm Red Gel A was used during the purification. Buffers used in the purification included: buffer A: Tris-HCl (50 mM, pH 7.5); and buffer B: Tris-HCl (50 mM, pH 7.5) and KC] (400 mM); buffer C: Tris-HCI (50 mM, pH 7.5) and KCI (600 mM). All protein manipulations were carried out at 4 °C. Protein solutions were concentrated by ultrafiltration (YM- 10 membranes from Millipore-Amicon). Single colony of E. coli DHSor/pTW8090A was used to inoculate 1 L of LB/Cm medium with 0.1 mM IPTG. Culture was grown overnight (~11 hours) at 37 °C and harvested by centrifugation (6000g for 10 min at 4 °C). Cell paste (6g) was suspended in 12 mL of buffer A and the cells were lysed by two passes through a French press at 16000 psi. Cell debris was removed by centrifugation at 18000 g for 30 min. The 35- 60% ammonium sulfate fraction was precipitated, redissolved in buffer A, and dialyzed against buffer A. The resulting supernatant was applied to a column of Matrex'rm Red Gel A (30 mg/lOO mL media) that was previously equilibrated with buffer A. Elution was completed with a linear gradient (buffer A-buffer B). Active fractions were concentrated, dialyzed against buffer A, quick frozen, and stored at -80 °C. A small portion (27 mg) of partially purified quinate dehydrogenase was applied to 6 mL Resource Q (Amersham Biosciences) column and eluted with linear gradient (buffer A-buffer C). Active fractions were collected, dialyzed against buffer A, aliquoted in 50 mL, quick frozen and stored at -80 °C. 111 K... and Vm measurment of E. coli Shikimate dehydrogenases AroE and YdiB The K, of Shikimate dehydrogenase for 3-dehydroshikimic acid was measured under the following conditions: NADPH was held constant at 0.2 mM, 3- dehydroshikimic acid concentrations were 0.03, 0.05, 0.06, 0.1, 0.2, 0.4 mM. The Km and Vm was calculated using Prism software. Plasmid pJJ2.134A This 5.8-Rb plasmid was constructed by ligation the open reading frame (ORF) of ydiB into the EcoRI site of pGEX-4T-1. The ydiB ORF was amplified from E. coli W3110 genomic DNA using the following primers: 5’- CGGAATTCATGGATGTTACCGCAAAATAC and 5’- CGGAATTCTCAGGCACCGAACCCCAT. Localization of the resulting 0.9-kb fragment into pGEX-4T-1, which had been previously treated with EcoRI and CIAP afforded pJJ2.134A, where ydiB ORF is transcribed from Pm. promoter. Plasmid pJJ2.297A This 5.7-kb plasmid was constructed by ligation the open reading frame (ORF) of aroE into the EcoRI site of pGEX-4T-1. The aroE ORF was amplified from E. coli W3110 genomic DNA using the following primers: 5’- GGAATTCAT GGAAACCT ATGCT GT and 5 ’-GGAA'ITQTCACGCGGACAA'ITCCT. Localization of the resulting 0.8-kb fragment into pGEX-4T-1, which had been previously treated with EcoRI and CIAP afforded pJJ2.297A, where ydiB ORF is transcribed from Pm promoter. 112 Plasmid pJJ3.041A This 5.5-kb plasmid was constructed by ligation the open reading frame (ORF) of ydiB into the EcoRI site of pKK223-3. The ydiB ORF was amplified from E. coli W3110 genomic DNA using the following primers: 5’- CGGAATTCATGGATGTTACCGCAAAATAC and 5’- CGGAATTCTCAGGCACCGAACCCCAT. Localization of the resulting 0.9-kb fragment into pKK223-3, which had been previously treated with EcoRI and CIAP afforded pJJ2.134A, where ydiB ORF is transcribed from P promoter. lac Plasmid pJJ3.247A The aroE ORF from plasmid pJY1.43A was subject to PCR mutagenesis using the following primers: 5’-GGAATTCATGGAAACCTATGCTGT and 5’- AACTGCAGTCACGCGGACAA'ITCCTCC. The PCR product was purified and digested with EcoRI/Pstl. The resulting DNA was ligated to pKK223-3, which has been previously digested with EcoRI/PstI and treated with CIAP. The resulting ligation mixture was introduced by transformation into BA2834 electrocompetent cells and the resulting transformants were selected at 37 °C on the minimal selection plate M9/Glucose/Ap. The resulting colonies were subject to high-throughput screening. Plasmid pJJ3.289AP This 5.5-kb plasmid was constructed by ligation the Open reading frame (ORF) of qad into the EcoRI site of pKK223-3. The qad ORF was amplified from K. pneumoniae MGH78678 genomic DNA using the following primers: 5 ’- CGGAATTCATGGCAGAACGTATTACTGGAC and 5’- 113 CGGAATTC'ITAGAACAGAATGCTC’I'I‘GATG. Localization of the resulting 0.9-kb fragment into pKK223-3, which had been previously treated with EcoRI and CIAP afforded pJJ3.289A, where qad ORF is transcribed from P (0C promoter. Plasmid pJJ4.024A The construction of 5.7-kb plasmid pJJ4.024A started from amplification of quad fragment from pJJ3.289AP plasmid using the following primers: 5’- CGGGTACCGGAGCTTATCGACTGCACG and 5’- CGGGATCCTI‘AGAACAGAATGCTCTTGATG. Plasmid pKD12.112 was digested with Kpnl/BamHI and treated with CIAP and liberated fragment PmaroE, serA was separated from the plasmid. Resulted plasmid was ligated with 1.1. -kb PCR product, which had been previously digested with Kpnl/BamHI. Plasmid pJJ4.025A Construction of pJ4.025A began with pNR8.146A. A 3.8-kb tktA, serA-encoding fragment was liberated from pNR8.146A by digestion with Xbal. Insertion of the tktA, serA fragment into Xbal site of pJJ4.024A yielded a 9.5-kb plasmid pJJ4.025A. Plasmid pJJ4.] 18A This 5.8-kb plasmid was constructed by ligation the open reading frame (ORF) of B. subtilis aroD into the EcoRI site of pGEX-4T-1. The aroD ORF was amplified from B. subtilis 168 genomic DNA using the following primers: 5 ’- CGGAATTCATGAAAAAGCTGTACGGGGTT and 5’- CGGAA'ITC'ITAACATTCTGTI‘CCTCCTAATT. Localization of the resulting 0.9-kb 114 fragment into pGEX-4T-1, which had been previously treated with EcoRI and CIAP afforded pJJ4.118A, where B. subtilis aroD ORF is transcribed from P promoter. lac Plasmid pJJ4.150A The construction of 5.7-kb plasmid pJJ4.150A started from amplification of PmydiB fragment from pJJ3.041A plasmid using the following primers: 5’- CGGGTACCGGAGCTTATCGACTGCACG and 5’- CGGAATTCTCAGGCACCGAACCCCAT. Plasmid pKD12.112 was digested with Kpnl/BamHI and treated with CIAP and liberated fragment PmaroE, serA was separated from the plasmid. Resulted plasmid was ligated with 1.1-kb PCR product, which had been previously digested with Kpnl/BamHI and afforded plasmid pJJ4. 150A. Plasmid pJJ4.171A Construction of pJ4.171A began with pNR8. 146A. A 3.8-kb tktA, serA-encoding fragment was liberated from pNR8.146A by digestion with Xbal. Insertion of the tktA, serA fragment into Xbal site of pJJ4.150A yielded a 9.5-kb plasmid pJJ4.025A. PCR mutagenesis of araE gene The protocol for mutagenic PCR is derived from standard PCR conditions as described by Cadwell.“ Mutagenic PCR buffer (10X) contained 70 mM MgC12, 500 mM KCl, 100 mM Tris (pH 8.3), and 0.1% (w/v) gelatin. Deoxynucleoside triphosphate mix (10X) contained 2 mM dGTP, 2 mM dATP, 10 mM dCTP, and 10 mM dTTP. The mutagenic PCR reaction was prepared by mixing 10 11L 10X mutagenic PCR buffer, 10 uL 10X dNTP mix prepared above, 14 11L 50 mM MgC12, 30 pmoles of each primer, 20 ng of input DNA, and an amount of water that brought the total volume to 88 ul. To this 115 sample was added 4 11L 5 mM MnCl2 (make sure a precipitate was not formed) and 2 it] Taq polymerase (5 Unit), bringing the final volume to 100 11L. The PCR reaction was performed in a thermal cycler (Eppendorf, Mastercycler Gradient). The sample was incubated for 30 cycles of 94°C for 45 s, 45°C for 1 min, and 72°C for 45 s. Preparation of electrocompetent E. coli cells E. coli AB2834 was prepared for electroporation with the following procedure. E. coli strain AB2834 was initially grown from a single colony in 5 mL LB/Cm and shaken for 16 h at 37°C . From the 5 mL culture, 2 mL was added to 500 mL 2 x YT medium (Bacto-tryptone 16 g, yeast extract 5 g, NaCl 5 g per liter) which was grown until reaching an absorbance of 0.6 - 0.8 at 600 nm. The cells were chilled to 4°C then harvested by centrifugation (3 000g, 5 min, 4 °C). The supernatant was discarded, and the cell pellet was resuspended in 500 mL ice cold water. The cells were harvested by centrifugation (3 000g, 10 min, 4 °C) then resuspended in 250 mL ices cold water. Following harvesting by the same conditions, the cells were resuspended in 100 mL ice cold 10% glycerol solution (Sterile filtered, not autoclaved). The cells were once again harvested by the same conditions and resuspended in 1.5 mL ice cold 10% glycerol solution. 0.1 mL aliquots were fast frozen in liquid nitrogen and store at -80 °C. For transformation, 40 uL of cells were combined with plasmid DNA followed by incubation for 2 min at 0 °C. The cells were then transferred to a pre-chilled electroporation cuvette with a 0.2 cm gap width. The cells were electroporated at 2.5 kV, 25 11F, and 200 Ohms using Gene Pulser II (Bio-rad). Following electroporation, 1 mL room temperature SOC medium was immediately added to the cuvette and incubated at 37 °C for 1 h. The entire cell solution was plated with the appropriate selection plate. 116 The SOC medium used for transformation of electrocompetent cells was prepared as the following. Tryptone (2 g), yeast extract (0.5 g), NaCl (1 mL 1 M NaCl), KCl (0.25 mL 1 M KCl) was added to 97 mL of distilled water. After dissolving and autoclaving. 1 mL 1 M MgCl2 and 1 mL 2 M glucose was added to the solution. The complete medium was filtered through a 0.22 pm filter. High-throughput screening of AroE mutant library All screening manipulations were performed in a 96-well plates. Inoculation was performed using a Mantis (GeneMachine) robot, liquid transfers were performed using 8- and 96-chanell Biomek 2000 and Biomek FX (Beckman Coulter) stations, respectively, available at Genomic Technology Support Facility at Michigan State University. Solutions used in screening were used as follow: dilution buffer: potassium phosphate buffer (100 mM, pH 7.4), 0.4 mM DTT; lysis buffer: potassium phosphate buffer (100 mM, pH 7.4), 0.4 mM DTT, leugBuster‘D (Novagene). A single colonies from mutant library of E. coli AB2834/pJJ3.247A from selected plates (described above) were used to inoculate 96-deepwell plates. Well Al was left empty as negative control, wells Bl, Cl and D1 were inoculated with AB2834/pJJ3.247A harboring wild-type aroE, and rest of wells were inoculated with library mutants. Colonies were grown overnight at 37 °C with agitation at 250 rpm. Glycerol freeze plate was made by transferring 75 11L of overnight culture to the 25 11L 80% glycerol and was stored at -80 °C. Remaining culture was harvested by centrifugation (1800 g, 10 min, 4 °C), cell pellet was resuspended in 100 11L of lysis buffer and was incubated with agitation for 30 min. Addition of 100 mL of dilution buffer was followed by centrifugation (3800 g, 30 min, 4 °C) to remove cell debris. 117 Cell lysate was diluted 10 and 20 times for Shikimate dehydrogenase activity measurements (as described above) in the presence and absence of shikimic acid, respectively. Depletion of NADPH was followed at 340 nm using SpectraMax 384 Plus (Molecular Devices) plate reader. Ratio of slope in the presence and absence of shikimic acid was calculated for each mutant and was compared to the ratio of control wells. The mutant was considered as potential candidate if slope in the absence of shikimic acid was greater or equal to 50% of wild-type enzyme slope and mutant slope ratio was greater than slope ratio of wild-type enzyme (average of B 1, C1, D1 well results). 118 REFERENCES l Pittard, J.; Wallace, B. J. Distribution and Function of Genes Concerned with Aromatic Biosynthesis in Escherichia coli. J. Bacteriol. 1966, 91 , 1494-1508. 2 Li, K.; Mikola, M. R.; Draths, K. M.; Worden, R. M.; Frost, J. W. Fed-Batch Fermentor Synthesis of 3-Dehydroshikimic Acid Using Recombinant Escherichia coli. Biotechnol. Bioeng. 1999, 64, 61-73. 3 Knop, D. R.; Draths, K. M.; Chandran, S. S.; Barker, J. L.; von Daeniken, R.; Weber, W.; Frost, J. W. Hydroaromatic Equilibration During Biosynthesis of Shikimic Acid. J. Am. Chem. Soc. 2001, 123, 10173-10182. 4 Causey, T. 3.; Zhou, S.; Shanmugam, K. T.; Ingram, L. 0. Engineering the Metabolism of Escherichia coli W3110 for the Conversion of Sugar to Redox-Neutral and Oxidized Products: Homoacetate Production. Proc. Natl. Acad. Sci. U.S.a. 2003, 100, 852-832. 5 Furste, J. P.; Pansegrau, W.; Frank, R.; Blocker, H.; Scholz, P.; Bagdasarian, M.; Lanka, E. Molecular Cloning of the Plasmid Rp4 Primase Region in a Multi-Host-Range tacP Expression Vector. Gene 1986, 48, 119-131. 6 Bartolome, B.; Jubete, Y.; Martinez, E.; de la Cruz, F. Construction and Properties of a Family of pACYC184-derived Cloning Vectors Compatible with pBR322 and its Derivatives. Gene, 1991, 102, 75-78. 7 Ran, N. Synthesis of Aromatics and Hydroaromatics from D-Glucose Via a Native and a Variant of the Shikimate pathway. PhD Disertation, Michigan State University, 2004. 8 Ward, T. L. Biocatalytic Synthesis of Quinic Acid and Application to the Production of Coimercial Quinoid Organic Products. MS Thesis, Purdue University, 1993. 9 Farabaugh, M. A. Biocatalytic Production of Aromatics from D-Glucose. M.S. Thesis, Michigan State University, 1996. 10 Miller, J. H. Experiments in Molecular Genetics; Cold Spring Harbor Laboratory: Plainview, NY, 1972. 119 11 Yi, J.; Li, K.; Draths, K. M.; Frost, J. W. Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli. Biotechnol. Prog. 2002, 18, 1141-1148. 12 Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Plainview, NY, 1990. 1.3 Pitcher, D. G.; Saunders, N. A.; Owen, R. J. Rapid Extraction of Bacterial Genomic DNA with Guanidium Thiocyanate. Lett. Appl. Microbiol. 1989, 8, 151-156. 14 Miller, J. H. A short course in bacterial genetics; Cold Spring Harbor Laboratory: Plainview, NY, 1992. 15 Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248-254. 16 Tanizawa, Y.; Kishi, F.; Kaneko, T.; Nakazawa, A. High Level Expression of Chicken Muscle Adenylate Kinase in Escherichia coli. J. Biochem., 1987, 101, 1289- 1296. 17 Schoner, R.; Herrmann, K. M. 3-Deoxy-D-arabinose-heptulosonate 7-phosphate synthase. J. Biol. Chem. 1976, 251, 5440. 18 Gollub, E.; Zalkin, H.; Sprinson, D. B. Assay for 3-deoxy-D-arabinose- heptulosonate acid 7-phosphate synthase. Meth. Enzymol. 1971, 17A, 349. 19 Cooper, R. A. Phosphoenolpyruvate Synthetase. Methods Enzymol. 1969, I3, 309— 314. 20 Paoletti, F.; Williams, J. F.; Horecker, B. L. An enyzmic method for the analysis of D-erythrose 4-phosphate. Anal. Biochem. 1979, 95, 250-253. 21 Coggins, J. R.; Boocock, M. R.; Chaudhuri, S.; Lambert, J. M.; Lumsden, J.; Nimmo, G. A.; Smith, D. D. S. The arom Multifunctional Enzyme from Neurospora crassa. Meth. Enzymol. 1987, 142, 325-341. 22 Chaudhuri, 8.; Anton, I. A.; Coggins, J. R. Shikimate dehydrogenase from Escherichia coli. Methods Enzymol. 1987, I 42, 315-319. 120 23 Davis, B. D.; Gilvarg, C.; Mitsuhashi, S. Methods Enzymol. Enzymes of Aromatic Biosynthesis: Quinic Dehydrogenase from Aerobacter aerogenes. 1955, 2, 307- 311. 24 Harris, E. L. V.; Angal, S. In Protein Purification Methods: A Practical Approch; Oxford University Express: Oxford, New York, Tokyo, 1989. 25 Laemmli, U. K. Nature 1970, 227, 680-685. 26 Cadwell, R. C.; Joyce, G. F. Mutagenic PCR. PCR Methods Applic. 1994, 4, 8136-8139. 121 111111111111111111111Willi