RICE UNIVERSITY Engineering the Production of Itaconic Acid in Escherichia coli by Tao Lin A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE Master of Science APPROVED, THESIS COMMITTEE Ka-Yiu San, E. D. Butcher Professor in Bioengineering and Professor of Chemical Engin~ 7t!-:: Michael C. Gustin, Professor, Biochemistry and Cell Biology Ronald Parry, Professor of Chemistry and Professor of Biochemistry and Cell Biology HOUSTON, TEXAS Jan 2011 Abstract Engineering the Production of Itaconic Acid in Escherichia coli by Tao Lin Itaconic acid (IA) has many versatile applications in science and medicine. Engineering Escherichia coli can provide a reliable route for IA production, where cis-aconitate decarboxylase (CAD) is one essential enzyme in the process. The synthetic cad gene has been cloned into various vectors to improve the expression rate and folding activities. The strain bearing plasmid pTrc99a-cad displays a significant amount of IA production, while the CAD protein expressed from the same plasmid displays the highest enzymatic activity in vitro. To improve the reliability of forming the cis-aconitate intermediate, the aconitase genes from Aspergillus terreus have been cloned into the pTrc99a vector. The expression of aconitate hydratase leads to a peak at 10.3 min in the HPLC spectra after 4 hours incubation. The peak will be analyzed to determine whether it is cis-aconitate. The obtained data will provide data on possible metabolic pathways of IA production in Aspergillus terreus. Acknowledgments First of all, I would like to sincerely thank my advisor, Dr. George Bennett, for his steady support, guidance, and advice all the time. His wisdom and encouragement kept motivating me through the good and tough times during the research. Secondly, I would like to thank my committee members, Dr. Kathleen Beckingham, Dr. Michael Gustin, and Dr. Ka-Yiu San for their advice and suggestions during the research progress. Their brilliant suggestions ease the difficulties of the research and contributed significantly to my research. Additionally, I need to thank Dr. Ronald Parry to serve in my thesis committee. Thirdly, I have to thank my colleagues in Drs. Bennett and San labs for their help and advice: Xianpeng, Chandresh, Yipeng, Huasong, Mary, Jiangfeng, Mai, Xiuzhun, and Haijun, Apita, and Grant. Xianpeng helped me initiate my project. The rest of my colleagues taught me many useful molecular biology techniques and guide me through many obstacles during the research. Additionally, I need to thank specially to Mary Harrison and Dr. Zhang. Mary is always ready to help me and comfort me during the research. Dr. Zhang helped me a lot in cloning the genes from Aspergillus terre us. Fourthly, I would like to present my gratitude to Dr. Greg May and his lab members in M.D. Anderson Cancer Center. Their expertise in Aspergillus fungi has made great contribution to my research. I really appreciate their help. Finally, I need to thank my parents and my brother for their full support during the research. Their encouragement motivated me during my research. The project is supported by Shell Sustainability Grant. ii Table of Contents Abstract ........................................................................................................................................... i Acknowledgments ......................................................................................................................... ii Table of Contents ......................................................................................................................... iii List of Figures .............................................................................................................................. vii List of Tables ................................................................................................................................ ix List of Abbreviation ...................................................................................................................... x CHAPTER 1 Introduction ........................................................................................................... 1 1.1 Overview............. ................ .......................................................................................... 1 1.2 Project Objectives......................................................................................................... 1 1.3 Background ................................................................................................................... 3 1.3.1 Characteristics and applications of IA ........................................................... 3 1.3.2 Industrial production ofIA and IA producing strains .................................... 4 1.3.3 Metabolic pathway of IA production in A. terreus ........................................ 5 1.3.4 Characteristics of cis-aconitate decarboxylase (CAD) .................................. 6 1.3.5 General information about aconitases (Acn) ................................................. 7 1.3.6 Aconitases in E. coli .................................................................................... 10 1.3.7 The 2-Methylcitrate dehydratase from Shewanella oneidensis (S. oneidensis) ............................................................................................................................... 10 iii 1.3.8 Debate on the existence of citrate dehydratase ............................................ 12 1.3.9 Sequence analysis of potential aconitases from A. terre us .......................... 13 CHAPTER 2. Materials and Methods ...................................................................................... 15 2.1 Strains and plasmids ....................... ... ......................................................................... 15 2.2 Media .......................................................................................................................... 16 2.2.1 LB medium and Aspergillus medium .......................................................... 16 2.2.2 Storage Medium ........................................................................................... 16 2.2.2.1 Glycerol stocks .................................................................................... 16 2.2.2.2 Agar plates .......................................................................................... 17 2.3 Cell Cultivation ........................................................................................................... 17 2.3.1 Overnight aerobic tubes ............................................................................... 17 2.3.2 Collect the spores from Aspergillus species ................................................ 17 2.3.3 Aerobic Shake Flask .................................................................................... 18 2.4 Analytical Techniques ................................................................................................ 18 2.4.1 Cell Density Determination (OD) ................................................................ 18 2.4.2 Extracellular metabolites analysis - HPLC .................................................. 19 2.4.3 Enzyme Assays of CAD .............................................................................. 19 2.4.3.1 Crude Extract ...................................................................................... 19 2.4.3.2 CAD enzyme assay ............................................................................. 20 2.4.3.3 Aconitase assay ................................................................................... 20 2.5 Genetic Manipulation.................................................................................................. 21 2.5.1 Chromosomal DNA Purification and Plasmid Isolation .............................. 21 iv 2.5.2 DNA Digestion and Ligation ....................................................................... 21 2.5.3 Agarose Gel Electrophoresis ........................................................................ 22 2.5.4 PCR .............................................................................................................. 22 2.5.5 Cell Transformation .................... , ................................................................ 23 2.5.6 Gene Cloning and Expression .................................................................................. 23 CHAPTER 3. Expression of cad gene in various vectors in E. coli ........................................ 24 3.1 CAD protein expressed from pTrc99a-cad plasmid is soluble and functional in the cytoplasm .......................................................................................................................... 24 3.2 CAD protein expressed from pTrcHis-cad vector was functional and visualized through Western Blot........................................................................................................ 28 3.3 Expression of cad gene in other high-copy plasmids was not successfuL ................ 30 3.4 Comparison of expression of constructed plasmids in various strains using different carbon sources ................................................................................................................... 33 CHAPTER 4. Exploring the potential aconitase gene from Aspergillus species and Shewanella oneidensis ................................................................................................................. 39 4.1 No "citrate dehydratase"