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Metabolic Engineering of Clostridium Tyrobutyricum for Enhanced N-Butanol Production and Sugar Utilization DISSERTATION Presente

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Metabolic Engineering of Clostridium Tyrobutyricum for Enhanced N-Butanol Production and Sugar Utilization DISSERTATION Presente Metabolic Engineering of Clostridium tyrobutyricum for Enhanced n-Butanol Production and Sugar Utilization DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Le Yu, B.S. Graduate Program in The Ohio State Biochemistry Program The Ohio State University 2015 Dissertation Committee: Professor Shang-Tian Yang, Advisor Professor Jeffrey Chalmers Professor Andre Palmer Professor Hua Wang Copyright by Le Yu 2015 Abstract With diminished availability of fossil fuels and volatility of petroleum prices, concerns over the reliability in fossil-based energy supply and stability of society and economy are on the rise. The development of bio-based energy resources is becoming more and more important, as the demand for renewable and green energy sources is rapidly expanding. n-Butanol, a four-carbon alcohol, has been widely considered as a superior substitute for current gasoline additive ethanol and a prospective alternative for traditional transportation fuel. Therefore in recent years, increasing attentions have been paid to butanol production from biological approach, and extensive studies have been carried out to better transform biobutanol technology into viable large-scale industrial application. Clostridium tyrobutyricum ATCC 25755 is characterized as a native acidogen, which only produces acetate and butyrate as its main metabolites. Compared to other solventogenic Clostridium, simpler metabolic pathways and less complex regulatory mechanism that C. tyrobutyricum processes make it easier to manipulate and control. With advances in metabolic engineering techniques of Clostridium, establishment of a butanol producing pathway in C. tyrobutyricum was achieved previously. However, less than 10 g/L of butanol was produced in batch fermentation with a large amount of acid accumulated, indicating that further genetic manipulation is required to improve butanol ii production and reduce by-product accumulation. In order to solve the problem of low butanol production brought by acid accumulation, the effect of CoA transferase (encoded by ctfAB) on butanol production was investigated in this study. The CoA transferase from solventogenic clostridia can catalyze the following reaction: acetate/butyrate + acetoacetyl-CoA → acetyl/butyryl-CoA + acetoacetate, and can thus decrease acetate and butyrate levels and increase butanol production. The plasmid pMTL007 was used to co-express adhE2 (aldehyde /alcohol dehydrogenase) and ctfAB from C. acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from C. beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (Δack, adhE2). Compared to the control C. tyrobutyricum (Δack, adhE2), all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22-0.26 g/g (vs. 0.10-0.13 g/g) and productivity to 0.35 g/L·h (vs. 0.13 g/L·h). Meanwhile, acetate and butyrate decreased 54% and 75%, respectively. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation. An exclusive Pta-Buk reverse pathway for butyrate re-uptake by C. tyrobutyricum was also proposed in this study. Another problem of C. tyrobutyricum is that the native strain cannot catabolize maltose and soluble starch as its substrates. Therefore in this study, C. tyrobutyricum was further optimized by introducing two extracellular α-glucosidases encoded by agluI and agluII from C. acetobutylicum ATCC 824. The mutants showed active hydrolytic capability of maltose and soluble starch. Significant increase in butanol production by using these two substrates was also observed. Compared to the parental strain C. iii tyrobutyricum (Δack, adhE2) grown on glucose, mutants expressing agluI can direct more butanol production (17.2 vs. 9.5 g/L) with a higher butanol yield (0.20 vs. 0.10 g/g) and productivity (0.29 vs. 0.16 g/L·h). In comparison with C. acetobutylicum ATCC 824, the mutant produced more butanol from maltose (17.2 vs. 11.2 g/L) and soluble starch (16.2 vs. 8.8 g/L) in batch fermentations. The mutant strain was also stable without antibiotics, reaching a high butanol productivity of 0.40 g/L·h. Similar to other Clostridium, glucose-mediated carbon catabolite repression (CCR) impedes efficient utilization of xylose in C. tyrobutyricum when the preferred sugar source glucose is present, which would lead to inefficient utilization of xylose in lignocellulosic biomass and low butanol production. To eliminate the bottleneck, three rate-limiting enzymes in xylose catabolism encoded by xylT, xylA, and xylB were overexpressed in C. tyrobutyricum (Δack, adhE2). The resulted mutant realized efficient co-utilization of glucose and xylose with a similar consumption rate. Moreover, compared to the control, the mutant produced more butanol (12.0 vs. 3.2 g/L) with higher butanol yield (0.12 vs. 0.07 g/g) and productivity (0.17 vs. 0.07 g/L·h) from the co-utilization of glucose and xylose. The fermentation with different glucose-to-xylose ratios demonstrated that efficient and simultaneous glucose and xylose utilization could be achieved even at low xylose levels. Efficient and complete co-utilization of glucose and xylose within soybean hull hydrolysate by the mutant further confirmed its exceptional capability of using cheap and abundant lignocellulosic biomass as feedstock. In summary, two major problems in butanol-producing C. tyrobutyricum including acid accumulation and poor sugar utilization have been addressed. Through genetic engineering, the mutants developed in this study have realized enhanced butanol iv production with more robust sugar utilization capability. With the ability to produce high levels of butanol from low-cost soluble starch and lignocellulose, the engineered C. tyrobutyricum strains developed in this study should have good potential for application in industrial biobutanol production. v Dedication Dedicated to my parents and grandparents vi Acknowledgements First of all, I would like to express my sincere gratitude to my advisor, Dr. Shang-Tian Yang, for his enormous support and guidance during my 5-year graduate study. His in-depth insight and comprehension of this project has been a true blessing, and his inspiring suggestions and continuous encouragements have been a great motivation to me whenever I encountered difficulties. I am also very grateful to have the opportunity to study in Dr. Yang’s lab. The knowledge and techniques I learned will be a priceless treasure for my future career. I also want to give my special thanks to Dr. Jeffrey Chalmers, Dr. Andre Palmer, and Dr. Hua Wang as my committee members. Their kind and valuable suggestions also contributed a lot to the completion of this project. I want to express my particular thanks to my colleague Dr. Jingbo Zhao, who taught me every basic operation in metabolic engineering, genetic modifications, and anaerobic culture when I first came to this group. His concentration and devotion to this field also set a great example to me. I also want to thank my colleague Dr. Mengmeng Xu for the collaboration and assistance to my work. Great appreciation is given to all my previous and current colleagues in Dr. Yang’s lab, including Dr. Mingrui Yu, Dr. Xiaoguang Liu, Dr. Baohua Zhang, Dr. Zhongqiang Wang, Dr. Yinming Du, Dr. Yipin Zhou, Dr. Chih-Chin Chen, Dr. Jie Dong, Dr. Fangfang Liu, Dr. Xiaorui Yang, Dr. Wenyan Jiang and Dr. Meng Lin. Their instruction and assistance are indispensable to my progress in vii both professional expertise and teamwork ability. Financial supports from the Ohio Department of Development Third Frontier Advanced Energy Program and National Science Foundation STTR Program are deeply appreciated. Finally, I would like to thank my parents, Mr. Junping Yu and Mrs. Xiaoyu Liu, and my grandparents Mr. Dezhong Yu and Mrs. Yueying You for giving me their great support and love in the pursuit of my dream. viii Vita June 2006 ……………………………………………………Tianjin No.1 Senior High 2006-2010……………………………………………………B.S. Biological Sciences, Nankai University 2010 to present……………………………………………Graduate Research Associate, The Ohio State University Publications Yu L, Zhao JB, Xu M, Dong J, Varghese S, Yu MR, Tang IC, Yang ST (2015) Metabolic engineering of Clostridium tyrobutyricum for n-butanol production: Effects of CoA transferase. Appl Microbiol Biotechnol. DOI: 10.1007/s00253-015-6566-5. Xu M, Zhao J, Yu L, Tang IC, Xue C, Yang ST. (2015) Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production. Appl. Microbiol. Biotechnol. 99:1011-22. Yu L, Xu M, Tang IC, Yang ST (2015) Metabolic engineering of Clostridium tyrobutyricum for n-butanol production through co-utilization of glucose and xylose. Biotechnol Bioeng. Accepted. Fields of Study Major Field: Biochemistry Specialty: Biochemical Engineering ix Table of Contents Abstract ............................................................................................................................... ii Dedication ...........................................................................................................................vi Acknowledgements ........................................................................................................... vii Vita ......................................................................................................................................ix
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