Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2011 Isobutene formation from 3-hydroxy-3-methylbutyrate (3-HMB) by the Saccharomyces cerevisiae diphosphomevalonate decarboxylase (ScMDD) and directed enzyme evolution to improve enzyme function David Gogerty Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Gogerty, David, "Isobutene formation from 3-hydroxy-3-methylbutyrate (3-HMB) by the Saccharomyces cerevisiae diphosphomevalonate decarboxylase (ScMDD) and directed enzyme evolution to improve enzyme function" (2011). Graduate Theses and Dissertations. 10353. https://lib.dr.iastate.edu/etd/10353 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Isobutene formation from 3-hydroxy-3-methylbutyrate (3-HMB) by the Saccharomyces cerevisiae diphosphomevalonate decarboxylase (Sc MDD) and directed enzyme evolution to improve enzyme function by David Gogerty A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee: Thomas A. Bobik, Major Professor Alan DiSpirito Basil Nikolau Donald Beitz Robert Brown Iowa State University Ames, Iowa 2011 Copyright © David Gogerty, 2011. All rights reserved. ii TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES viii ACKNOWLEDGEMENTS ix ABSTRACT xi CHAPTER 1. General Introduction 1 I. Dependence on fossil fuels 1 II. Reducing our dependence through biofuels 3 III. Alternative biofuels 6 CHAPTER 2. Literature Review 8 Introduction 8 Alternative biofuels production 9 I. Butanol 9 II. Microbial hydrocarbon biosynthesis 17 III. Fatty acid biosynthesis 17 IV. Alkane biosynthesis 20 V. Ethylene 21 VI. Isobutene 22 Optimizing enzyme function 25 I. Rational design 25 II. Directed enzyme evolution 26 Mevalonate Decarboxylase 35 I. Mevalonate diphosphate decarboxylase (MDD) 35 II. Putative mevalonate monophosphate decarboxylase (MPD) 41 iii CHAPTER 3. Formation of Isobutene from 3-hydroxy-3-methylbutyrate by 44 Diphosphomevalonate Decarboxylase Abstract 44 Introduction 46 Materials and methods 47 Results 52 Discussion 60 CHAPTER 4. Mevalonate Growth Selection 66 Abstract 66 Introduction 67 Materials and methods 73 Results 77 Discussion 93 CHAPTER 5. Development of an Isobutene Biosensor 99 Abstract 99 Introduction 99 Materials and methods 102 Results 106 Discussion 114 iv CHAPTER 6. Synthetic Gene Shuffling to Expand Mevalonate Diphosphate 120 Decarboxylase Sequence Diversity Abstract 120 Introduction 120 Materials and methods 123 Results 124 Discussion 130 CHAPTER 7. Conclusion and Future Work 133 I. 3-HMB Synthesis 133 II. Search for mevalonate monophosphate decarboxylase 137 III. Additional increases in enzyme activity (biosensor) 139 IV. Directed evolution by mevalonate selection 141 ABBREVIATIONS 144 REFERENCES 145 v LIST OF FIGURES Figure 1. Native Clostridium butanol biosynthetic pathway. 12 Figure 2. Ehrlich pathway for alternative alcohol production. 15 Figure 3. Fatty acid biosynthetic pathway. 19 Figure 4. Ethylene biosynthetic pathways. 22 Figure 5. Isobutene biosynthesis from Rhodotorula minuta . 24 Figure 6. Mevalonate and methylerythritol phosphate pathways. 36 Figure 7. Mevalonate diphosphate decarboxylase reaction mechanism. 39 Figure 8. Reactions catalyzed by diphosphomevalonate decarboxylase (MDD). 52 Figure 9. GC-MS of isobutene produced by whole cells of Sc MDD variant R74H. 55 Figure 10. MS spectrum for isobutene produced by Sc MDD versus library standards. 56 Figure 11. SDS-PAGE analysis of His 6-Sc MDD purification. 57 Figure 12. Structural mapping of Sc MDD variants. 60 Figure 13. Possible pathway for the production of 3-HMB from glucose. 63 Figure 14. A step-wise approach to directed enzyme evolution. 70 Figure 15. Proposed isopentenol pathway. 71 Figure 16. Proposed isopentenol pathway with MEP and MVA pathways. 72 Figure 17. Isopentenol growth assay for isopentenyl phosphate kinase after 24 h. 79 vi Figure 18. Isopentenol growth assay for isopentenyl phosphate kinase after 103 h. 80 Figure 19. Chromosomal map of the lytB area of E. coli . 81 Figure 20. PCR confirmation of lytB knockout. 82 Figure 21. PCR confirmation of lytB knockout with IPK insertion. 84 Figure 22. GC-FID analysis of isopentenol production by ten mevalonate isolates. 88 Figure 23. Isopentenol-producing activity of MS-Sc-1 variant versus wild-type 89 (DG157). Figure 24. Detailed growth inhibition studies of fosmidomycin on revertant 91 phenotype. Figure 25. Broad study of growth inhibition by fosmidomycin on multiple revertants. 92 Figure 26. pTS biosensor fluorescence. 107 Figure 27. Fluorescence assay to determine the isobutene sensitivity of 107 the pTS biosensor. Figure 28. Luminescence assay to determine the E. coli -based biosensor’s 109 sensitivity to isobutene. Figure 29. pPROBE luminometer test. 109 Figure 30. β-galactosidase assay of an E. coli based pPROBE vector containing 111 the biosensor cassette (DG94). vii Figure 31. β-galactosidase assay of reengineered biosensor construct (DG112a). 112 Figure 32. β-galactosidase assay to determine substrate specificity of the 113 TbuT biosensor. Figure 33. ClustalW analysis of six homologs for synthetic gene shuffling. 125 Figure 34. Sc MDD amino acid sequence with residue labels. 126 Figure 35. Gel confirmation of the reconstitution of MDD from synthetic 129 gene shuffling. Figure 36. Potential 3-HMB biosynthetic pathways. 135 Figure 37. The two plasmid system for an E. coli based biosensor. 141 viii LIST OF TABLES Table 1. Select fuel molecules and their specs. 10 Table 2. Bacterial strains and plasmids used in chapter 3. 48 Table 3. Isobutene production by whole cells producing Sc MDD and variants. 54 Table 4. Isobutene formation by purified His 6-Sc MDD and His 6-Sc MDD2. 58 Table 5. Growth phenotype chart of lytB knockout mutants. 83 Table 6. Bacterial strains and plasmids used in chapter 4. 96 Table 7. Induction ratios for the E. coli based biosensors DG94 and DG112. 113 Table 8. Bacterial strains and plasmids used in chapter 5. 118 Table 9. Oligonucleotides designed for synthetic gene shuffling of Sc MDD. 128 ix ACKNOWLEDGEMENT I would like to thank my major professor Dr. Thomas Bobik, for the patient advice he has provided throughout my graduate work. His knowledge of the scientific tools needed to investigate our system as well as his keen interest in finding a solution to the problems we face was a constant source of help and encouragement thoughout. I look forward to applying the lessons that he guided me through in my future endeavors. I also would like to give a special thank you to all of my committee members who have given advice and guidance during my graduate work. Dr. Alan Dispirito, Dr. Basil Nikolau, Dr. Donald Bietz, and Dr. Robert Brown are all leaders of their fields and I feel very fortunate to have them as my committee members. Several colleagues have also given advice and support during my graduate work. Dr. Chenguang Fan, Dr. Shouqiang Cheng, Dr. Sharmistha Sinha, Dr. Huilin Zhu, and especially Christian Bartholomay have all been readily available for scientific conversations and advice. I would especially like to thank Stephanie Cline for her hard work on these projects, which would have been hard to complete to the extent that we did without her help. I would also like to acknowledge others who have worked in the lab, including Dr. Tracie Bierwagen, Flora Liu, Netra Agarkar, Casey Volker and Ji-An Chooi. Finally, I would like to say a big thank you to my family. My wife Jessica and son James have worked hard to give me the support to complete this work and I love x them very much. I would also like to thank my parents, Dan and Lana, as well as Jessica’s parents, Dan and Cheryl, for their indispensable help and support. xi ABSTRACT Dependence on petroleum for energy and petrochemical products has led to high energy costs, a polluted environment, the depletion of a finite resource (oil), and the reliance on hostile nations for our energy security. Biofuels promise to alleviate some or all of these issues but remain costly and inefficient to produce, and difficult to integrate into our existing transportation infrastructure. A renewable fuel molecule capable of replacing petroleum for our fuel and chemical industries is desired. Isobutene is an important commercial chemical used for the synthesis of butyl rubber, terephthalic acid, specialty chemicals, and a gasoline performance additive known as alkylate. Currently, isobutene is produced from petroleum and hence is nonrenewable. Here we report that the Saccharomyces cerevisiae mevalonate diphosphate decarboxylase ( Sc MDD) can convert 3-hydroxy-3-methylbutyrate (3-HMB) to isobutene. Whole cells of Escherichia coli producing Sc MDD formed isobutene from 3-HMB at a rate of 154 pmol h -1 g cells -1. His 6-Sc MDD was purified by nickel affinity chromatography and shown to produce isobutene from 3-HMB at a rate of 1.33 pmol min -1 mg -1 protein. In contrast, no isobutene was detected from control cells lacking Sc MDD, and controls showed that both His 6-Sc MDD and 3-HMB were required for detectable isobutene formation in enzyme assays. Sc MDD was subjected to error-prone PCR and 2 improved variants were characterized, Sc MDD1 (I145F) and Sc MDD2 (R74H). Whole cells of E. coli producing Sc MDD1 and Sc MDD2 produced isobutene from 3-HMB at rates of 3000 and 5888 pmol -1 -1 h g cells which are 19- and 38-fold increases compared to cells producing His 6- Sc MDD. Although Sc MDD was shown to be amenable to manipulation in order to xii increase its activity on 3-HMB, we estimate that a 10 6 fold increase in activity is needed for commercial application.