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Metabolic engineering of Clostridium cellulovorans for selective n-butanol production from cellulose Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Teng Bao, M.S. Graduate Program in Chemical Engineering The Ohio State University 2019 Dissertation Committee Shang-Tian Yang, Advisor Charles E. Bell Jeffrey Chalmers Eduardo Reátegui 1 Copyrighted by Teng Bao 2019 2 Abstract n-Butanol as a promising alternative biofuel has gained high interest. Especially, compared with methanol and ethanol, n-butanol presents superior fuel properties and can be used as an ideal substitute of gasoline because of its high energy density, low water solubility, and low vapor pressure. Unfortunately, bio-butanol production through the conventional acetone-butanol-ethanol (ABE) fermentation process is not economically feasible because of the co-production of other metabolites and the use of expensive food- based feedstock. Lignocellulosic biomass, an abundant, cheap, and renewable source, is a desired feedstock for biofuels production. Conventional biorefinery of lignocellulosic biomass requires different operations, which is not cost-effective due to the complex process and high equipment capital requirement. Currently, several process strategies have been developed for cellulosic butanol production. Among them, consolidated bioprocessing (CBP) combines enzyme production, biomass hydrolysis, and sugar fermentation into one step, which greatly simplifies the process and dramatically reduces the equipment investment. Clostridium cellulovorans is a strictly anaerobic, cellulolytic bacterium among the most interesting candidates for CBP of lignocellulosic biomass. Recently, a bifunctional aldehyde/alcohol dehydrogenase gene adhE2 from Clostridium acetobutylicum has been overexpressed in C. cellulovorans for n-butanol production from cellulose. However, this ii recombinant strain produced n-butanol from microcrystalline cellulose at a low titer and yield insufficient for industrial application. In addition, there are two type II restriction modification (RM) systems in C. cellulovorans and currently available plasmids would be digested by C. cellulovorans cell extract even after in vivo methylation, making it difficult to transfer and express genes in C. cellulovorans for further metabolic engineering. Therefore, efficient transformation for developing better recombinant C. cellulovorans for butanol production is urgently required. In this study, the RM system of C. cellulovorans was analyzed and a new Clostridium shuttle plasmid pYL001 without Cce743I and Cce743II restriction sites was constructed to overcome the transformation barrier in C. cellulovorans. The new plasmid with improved post-electroporation procedure significantly improved the transformation efficiency and allowed the use of unmethylated DNA for metabolic engineering of C. cellulovorans. Then a synthetic butanol formation pathway was introduced in C. cellulovorans by overexpressing three different aldehyde/alcohol dehydrogenases gene combinations (acidogenesis: adhE2-bdhB, solventogenesis: adhE1-bdhB, and alcohologenesis: adhE2) from C. acetobutylicum. Due to different substrate and cofactor specificities, overexpressing different ADH and BDH in C. cellulovorans resulted in different amounts of alcohols production. Among them, C. cellulovorans adhE2 with the highest aldehyde/alcohol dehydrogenase activities produced the highest n-butanol titer of ~4 g/L with a high yield of ~0.22 g/g. Finally, modular metabolic engineering strategies, including strengthening the butyryl-CoA biosynthesis and improving intracellular NADH availability, were applied to redistribute the carbon flux towards butanol production in C. iii cellulovorans. Heterologous thiolase (thlA) and 3-hydroxybutyryl-CoA dehydrogenase (hbd) genes from C. acetobutylicum and Clostridium tyrobutyricum, respectively, were overexpressed in C. cellulovorans adhE2 to increase the flux from C2 to C4 metabolites. In addition, ferredoxin-NAD(P)+ oxidoreductase (fnr) from C. acetobutylicum, which can regenerate the intracellular NAD(P)H and thus increase alcohols formation, was also overexpressed. In batch fermentation with adding methyl viologen (MV) as an electron carrier, the recombinant C. cellulovorans adhE2-fnrCA-thlACA-hbdCT was able to direct the carbon flux towards n-butanol biosynthesis, leading to a high n-butanol titer of 5.56 g/L with a high yield of 0.34 g/g cellulose, which were the highest ever obtained for butanol production from cellulose in a mono-culture fermentation. These results indicated that C. cellulovorans is a promising CBP platform host for bio-butanol production from lignocellulosic biomass. iv Dedication This document is dedicated to my family and friends. v Acknowledgments I would like to express my deep and sincere gratitude to my advisor, Dr. Shang- Tian Yang for his continuous support of my Ph.D. study and research, for his insight, enthusiasm, and immense knowledge. He not only guided me how to solve the problems in studies, but also taught me how to face the difficulties in my life. His aggressive upward style also affected me a lot, from which I will benefit a lot in the future. I am also very thankful to the rest of my committee members, Prof. Jeffrey Chalmers and Prof. Eduardo Reátegui for their encouragement, insightful comments, and challenging questions. My sincere thanks also go to Dr. Mei Shao, Dr. Siguang Sui, Dr. Hungwei Chien, Mrs. Bianca Olson and Mr. David Ramey, for offering me summer internship opportunities to work on diverse exciting projects in their groups. I also want to thank all current and previous lab mates, including Dr. Jingbo Zhao, Dr. Meng Lin, Dr. Jin Huang, Dr. Xian Zhang, Dr. Xin Xin, Dr. Chi Cheng, Mrs. Li Lu, Ms. Fengli Zhang, Ms. You Li, and Ms. Zhen Qin for useful discussions. Especially, I would like to thank Dr. Zhao, who helped me with many experimental details, and taught me genetic engineering design when I first joined the project. In addition, I also would like to thank all my friends met at the Ohio State University. vi Financial supports from US Department of Energy EERE (DE-EE0007005) and SBIR (DE-FOA-0001771) program and ENGIE-Axium scholarship during the course of this research are acknowledged. Last but not the least, I would like to thank my girlfriend, Xuyao Liu, for her support and encouragement during my graduate study. I also want to deeply thank my parents, Jinhua Bao and Ji Guan, for giving me their selfless love and forever support. vii Vita November 1990……….……………………………………….Born-Jiashan, P.R. China 2012.…B.S. Bioengineering, Zhejiang University of Science & Technology, P.R. China 2015……………………………..M.S. Bioengineering, Jiangnan University, P.R. China 2015-2019…………..Graduate Teaching/Research Associate, Department of Chemical and Biomolecular Engineering, The Ohio State University Publications 1. Teng Bao, Jingbo Zhao, Jing Li, Xin Liu, and Shang-Tian Yang. 2019. n-Butanol and ethanol production from cellulose by Clostridium cellulovorans overexpressing heterologous aldehyde/alcohol dehydrogenases. Bioresource Technology, 3:121316. 2. Teng Bao, Jingbo Zhao, Qianxia Zhang, and Shang-Tian Yang. 2019. Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans. Applied Microbiology and Biotechnology, 103:5391-5400. 3. Teng Bao, Chi Cheng, Xin Xin, Jufang Wang, Minqi Wang, and Shang-Tian Yang. 2019. Deciphering mixotrophic Clostridium formicoaceticum metabolism and energy conservation: Genomic analysis and experimental studies. Genomics, 111:1687-94. 4. Chi Cheng, Teng Bao, and Shang-Tian Yang. 2019. Engineering Clostridium for improved solvent production: Recent progress and perspective. Applied Microbiology and Biotechnology. 103:5549-5566. 5. Jianfa Ou, Teng Bao, Patrick Ernst, Yingnan Si, Sumanth Prabhu, Hui Wu, Jianyi viii Zhang, Lufang Zhou, Shang-Tian Yang, Xiaoguang Liu. 2019. Intracellular metabolism analysis of Clostridium cellulovorans via modeling integrating proteomics, metabolomics and fermentation. Process Biochemistry. In press. 6. Jing Li, Yinming Du, Teng Bao, Jie Dong, Meng Lin, Hojae Shim, Shang-Tian Yang. 2019. n-Butanol production from lignocellulosic biomass hydrolysates without detoxification by Clostridium tyrobutyricum Δack-adhE2 in a fibrous-bed bioreactor. Bioresource Technology. 289:121749. (Co-first author) 7. Jin Huang, Yinming Du, Teng Bao, Meng Lin, Jufang Wang, Shang-Tian Yang. 2019. Production of n-butanol from cassava bagasse hydrolysate by engineered Clostridium tyrobutyricum overexpressing adhE2: Kinetics and cost analysis. Bioresource Technology. 292:121969. (Co-first author) 8. Xian Zhang, Rumeng Han, Teng Bao, Xiaojing Zhao, Xiangfei Li, Manchi Zhu, Taowei Yang, Meijuan Xu, Minglong Shao, Youxi Zhao, Zhiming Rao. 2019. Synthetic engineering of Corynebacterium crenatum to selectively produce acetoin or 2,3-butanediol by one step bioconversion method. Microbial Cell Factories. 18:128. 9. Zhiping Xiao, Chu Cheng, Teng Bao, Lujie Liu, Bin Wang, Wenjing Tao, Xun Pei, Shang-Tian Yang, and Minqi Wang. 2018. Production of butyric acid from acid hydrolysate of corn husk in fermentation by Clostridium tyrobutyricum: kinetics and process economic analysis. Biotechnology for Biofuels, 11(1), 164. 10. Niyomukiza Samuel, Teng Bao, Xian Zhang, Taowei Yang, Meijuan Xu, Xin Li, Irene Komera, Tuyishime Philibert, and Zhiming
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    ADVANCING TOWARDS BIOMASS-DERIVED SYNGAS FERMENTATION EVALUATION OF PROCESS PARAMETERS AND GAS COMPOSITION EFFECTS Zur Erlangung des akademischen Grades einer DOKTORIN DER INGENIEURWISSENSCHAFTEN (DR.-ING.) von der KIT-Fakultät für Chemieingenieurwesen und Verfahrenstechnik des Karlsruher Instituts für Technologie (KIT) genehmigte DISSERTATION von M. Sc Alba Infantes-López aus Mataró (Barcelona), Spain Tag der mündlichen Prüfung: 14.08.2020 Erstgutachter: Prof. Dr. Christoph Syldatk Zweitgutachter: Prof. Dr. Nicolaus Dahmen Zweitgutachterin: Dr. Anke Neumann This document is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License (CC BY-SA 4.0): https://creativecommons.org/licenses/by-sa/4.0/deed.en “Le rêve c’est tout – la technique, ça s’apprend.” Dreams are everything, technique can be learned. Jean Tinguely PREAMBLE Parts of this thesis have been published as peer reviewed research articles. They describe the main findings of my work. At the end of any applicable paragraph a reference note states the publication in which the content has been previously published. The text of these paragraphs is partially identical to the content of the publications. Layout, citation style, figures and formatting have been modified and adjusted to the style of this dissertation. Chapters that contain contents of previously published work are as follows: Chapters with the heading “General procedures for STR experiments”, “Impact of medium components and process parameters”, the discussion and the conclusion include content of the publication Infantes, A., Kugel, M., and Neumann, A. (2020). Evaluation of media components and process parameters in a sensitive and robust fed-batch syngas fermentation system with Clostridium ljungdahlii. Fermentation. 6(2), 61.