BIOCHEMICAL AND STRUCTURAL STUDIES OF MICROBIAL ENZYMES FOR THE BIOSYNTHESIS OF 1,3-BUTANEDIOL by Taeho Kim A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto © Copyright by Taeho Kim Biochemical and Structural Studies of Microbial Enzymes for the Biosynthesis of 1,3-Butanediol Taeho Kim Doctor of Philosophy Department of Chemical Engineering and Applied Chemistry University of Toronto 2019 Abstract Protein design approach for systems metabolic engineering entails discovery of novel enzymes and protein engineering for improving biocatalysts activity. It allows us to design and optimize cellular metabolism for the generation of novel metabolic pathways, aiming at the production of chemicals from renewable biomass. This thesis is aimed at two topics: the enzyme discovery and engineering for optimization of the biosynthetic pathway for 1,3-butanediol (1,3BDO), and the in vivo demonstration of the protein design in E. coli and cyanobacteria. The 1,3BDO pathway consists of three steps: pyruvate, a natural metabolite, is converted into acetaldehyde by pyruvate decarboxylase (PDC); it is followed by aldol condensation using 2-deoxyribose-5- phosphate aldolase (DERA), yielding 3-hydroxybutanal (3-HB); an aldo-keto reductase (AKR) catalyzes the reduction of 3-HB to produce 1,3BDO. Thus, we specifically investigated novel DERA and AKR in this thesis. First, PA1127 from Pseudomonas aeruginosa was identified based on the activity on 3-HB. The AKR was biochemically and structurally studied, providing insights into further applications with a remarkable substrate promiscuity. Next, BH1352 from Bacillus halodurans was identified with a high activity of aldol condensation. As the DERA-catalyzed condensation was the rate-limiting step, we rationally designed site-directed mutagenesis for enhancing 1,3BDO synthesis from DERA-AKR coupled reaction; two of the mutations showed 2.6 times higher in vitro activity compared to BH1352 wildtype. The two mutations were then ii introduced to E. coli fermentation for 1,3BDO biosynthesis from glucose. The Phe160Tyr single mutation exhibited a 5-fold increase in 1,3BDO titer, and the Phe160Tyr/Met173Ile double mutation displayed a 6-fold increase in 1,3BDO production with a 7-fold improvement in glucose yield compared to the wildtype. Finally, the biosynthetic pathway was introduced into Synechococcus elongatus PCC 7942 for the photosynthetic conversion of CO2 into 1,3BDO. Although we were not able to obtain the strain of cyanobacterial 1,3BDO production yet, the cyanobacterial engineering system was established, and the heterologous expression of the pathway genes was demonstrated via genetic engineering of Synechococcus elongatus PCC 7942. iii Acknowledgement First, I would like to dedicate this thesis to my parents. They could not have been more supportive during my entire life. I am also very grateful for my sister for so many advices and supports that helped me through the Ph.D. I sincerely appreciate the guidance and teaching from my supervisors, Dr. Yakunin and Dr. Mahadevan. Dr. Yakunin has always made himself available for me to discuss research subjects, and always provided me with directions and answers. He not just made me a researcher with a doctoral degree but transformed me into an analytical thinker. Dr. Mahadevan has always been supportive that he inspired me with many creative ideas and helped me challenging a variety of projects. I am also very grateful for my friends and colleagues in Enzyme and Genomic Lab and Systems Metabolic Engineering Lab. Anna could not have been more kind and helpful to me for the last 5 years, and Sofia had to take too many responsibilities to manage the lab and help everyone doing their jobs. I cannot thank you two enough for that. Kayla has been a good friend as well as an inspiring colleague, helping me so much of my Ph. D thesis work. I also feel grateful to Mahbod, Kevin, Tommy, Vik, Naveen, Mabel, Andy, and Susie for welcoming me when I joined Biozone and being supportive friends throughout my Ph.D. Last, but not least, I would like to express my genuine gratitude to Jeong for getting me an opportunity to join Biozone at U of Toronto and having always been a mentor to me. iv Table of Contents ABSTRACT ....................................................................................................................................... II ACKNOWLEDGEMENT ................................................................................................................... IV LIST OF FIGURES ........................................................................................................................ VIII LIST OF TABLES ............................................................................................................................ XII LIST OF APPENDICES .................................................................................................................. XIII LIST OF ABBREVIATIONS .............................................................................................................. XV CHAPTER 1. THESIS BACKGROUND, MOTIVATION, AND OUTLINE ........................................................... 1 1.1. Background ........................................................................................................................................ 1 1.1.1. Biocatalysis and biocatalysts ...................................................................................................... 1 1.1.2. Systems metabolic engineering .................................................................................................. 3 1.2. Motivation and objective of this Ph. D study .................................................................................... 5 1.2.1. Motivation .................................................................................................................................. 5 1.2.2. Main objective ............................................................................................................................ 6 1.3. Thesis outline ..................................................................................................................................... 9 CHAPTER 2. LITERATURE REVIEW AND GENERAL INTRODUCTION ...................................................... 11 2.1. Aldo-keto Reductases ...................................................................................................................... 11 2.1.1. Introduction .............................................................................................................................. 11 2.1.2. Microbial AKRs ....................................................................................................................... 13 2.2. Aldol Reaction and Aldolases ......................................................................................................... 15 2.2.1. Aldol reaction ........................................................................................................................... 15 2.2.2. Aldolases .................................................................................................................................. 16 2.2.3. 2-deoxyribose-5-phsophate aldolase (DERA) .......................................................................... 18 2.3. 1,3-Butanediol production technologies and applications ............................................................... 23 2.3.1. Introduction .............................................................................................................................. 23 2.3.2. Pathway for bioconversion of glucose into 1,3BDO ................................................................ 26 2.3.3. Aldolase-based biosynthetic pathway for 1,3BDO .................................................................. 26 2.4. Cyanobacteria engineering .............................................................................................................. 28 v 2.4.1. Motivation of cyanobacteria engineering study ....................................................................... 28 2.4.2. Cyanobacteria ........................................................................................................................... 29 2.4.3. Notes in cyanobacterial engineering ........................................................................................ 33 2.5. Hypotheses and research objectives ................................................................................................ 35 2.6. Publication status and contribution .................................................................................................. 38 CHAPTER 3. NOVEL ALDO-KETO REDUCTASES FOR THE BIOCATALYTIC CONVERSION OF 3- HYDROXYBUTANAL TO 1,3-BUTANEDIOL: STRUCTURAL AND BIOCHEMICAL STUDIES ...................... 41 3.1. Abstract ............................................................................................................................................ 41 3.2. Introduction ..................................................................................................................................... 42 3.3. Materials and Methods .................................................................................................................... 45 3.4. Results and Discussion ...................................................................................................................