Deciphering and Expending Clostridium formicoaceticum Metabolism Based on Whole Genome Sequencing THESIS Presented in Partial Fulfillment of Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Teng Bao Graduate Program in Chemical Engineering The Ohio State University 2016 Master’s Examination Committee: Shang-Tian Yang, Advisor Jeffrey Chalmers Copyrighted by Teng Bao 2016 Abstract Clostridium formicoaceticum is a Gram-negative homoacetogenic bacterium for acetic acid production and is also considered as a promising heterologous host for biofuel production. Here, its genome has been completely sequenced and consists of a 4.5-Mbp chromosome without containing any plasmid. The results of sequence analysis indicate that C. formicoaceticum is able to grow on several sugars and other organic substrates. In addition, it also exhibits a highly conserved Wood-Ljungdahl pathway gene cluster, which shows an identical arrangement as that in other autotrophic acetogens, such as Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. Differently from these homoacetogens, C. formicoaceticum contains a Na+- translocating ATPase and Rnf system for energy conversion. Such ATP generation system has also been sufficiently elucidated in Acetobacterium woodii. However, no growth defects were observed when C. formicoaceticum was grown on the heterotrophic and autotrophic medium under low sodium ion concentration, respectively. Otherwise, unlike Moorella thermoacetica and C. aceticum, neither completely cytochromes nor quinones synthesis genes are identified in the genome. Therefore, like C. ljungdahlii, C. formicoaceticum generates proton gradient via Rnf system and belongs to a H+-type homoacetogen. Moreover, ethanol formation may proceed by acetaldehyde dehydrogenases, alcohol dehydrogenases, and reversible ii aldehyde oxidoreductases under excessive reducing equivalent condition. Such information will be valuable in developing strategies for fermentation and metabolic engineering to produce bio-based chemicals and fuels in C. formicoaceticum. iii Dedication This document is dedicated to my family and friends. iv Vita 2008….B.S. Biological Engineering, Zhejiang University of Science & Technology 2012………………….………….M.S. Biological Engineering, Jiangnan University 2015 to present…………Graduate, Chemical Engieering, The Ohio State University Field of Study Major Field: Chemical Engineering v Table of Contents Abstract ......................................................................................................................... ii Dedication .................................................................................................................... iv Vita ................................................................................................................................ v List of Tables .............................................................................................................. viii List of Figures .............................................................................................................. ix Chapter 1: Introduction ................................................................................................ 1 1.1 Biofuels ........................................................................................................................... 1 1.2 Clostridial Fermentation ................................................................................................. 3 1.3 The First-generation and the Second-generation Biofuels .............................................. 4 1.4 Clostridium formicoaceticum .......................................................................................... 6 Chapter 2: Material and Methods ................................................................................. 8 2.1 Strains and Plasmids ....................................................................................................... 8 2.2 Cultural Media and Growth Condition ............................................................................ 8 2.2 Analytical Methods ....................................................................................................... 10 2.3 Sequencing, Gene Prediction, and Annotation .............................................................. 10 2.4 Characterization of the Restriction Modification System by Restriction Assay ........... 11 2.5 Nucleotide Sequence Accession Number ...................................................................... 12 Chapter 3: Deciphering and Expending C. formicoaceticum Metabolism Based on Whole Genome Sequencing ....................................................................................... 13 3.1 General Features of the C. formicoaceticum Genome ................................................... 13 3.2 Wood-Ljungdahl Pathway ............................................................................................. 16 3.3 Acetate, Ethanol, and Butanol Production and Utilization ............................................ 20 3.4 Substrate Utilization ...................................................................................................... 23 vi 3.5 Energy Conservation ..................................................................................................... 27 3.6 Intermediary Metabolism .............................................................................................. 31 3.7 Restriction Modification System Analysis .................................................................... 32 Chapter 4: Discussion ................................................................................................. 38 Chapter 5: Conclusions and Future Study .................................................................. 47 5.1 Constructing Transformation System in C. formicoaceticum ....................................... 47 5.2 Expression of Ethanol or n-Butanol Biosynthesis Pathway in C. formicoaceticum ...... 48 5.3 Increasing the Intracellular NADH Availability in C. formicoaceticum ....................... 49 Reference ................................................................................................................... 51 vii List of Tables Table 1. Chemical structural of butanol……….……….……….……………....…….2 Table 2. The application of butanol in different industries areas……….…………….2 Table 3. The bacteria and plasmids used in this study. …………….……..…………8 Table 4. General features of the Clostridium formicoaceticum ATCC 27076 genome…………………………………………………………………………….....15 Table 5. COG categories of C. formicoaceticum ATCC 27076. ……………...……..16 Table 6. Genes attributable to ethanol production in C. formicoaceticum ATCC 27076…........................................................................................................................21 Table 7. Genomic analysis of restriction modification systems in C. formicoaceticum……………………………………………………………………..33 Table 8. Number of predicted restriction sites in PMTL80000 plasmids susceptible to digestion by C. formicoaceticum...……..………..……..……..………..……..….…37 Table 9. Number of sites on important components of PMTL80000 plasmids digested by C. formicoaceticum. ……..……..………..…..……..………..……..……...…..…37 viii List of Figures Figure 1. ABE fermentation pathways of C. acetobutylicum…………………………4 Figure 2. Genomic map representation of the C. formicoaceticum chromosome…...14 Figure 3. KEGG categories of C. formicoaceticum ATCC 27076. ……….…………15 Figure 4. The Wood-Ljungdahl pathway in C. formicoaceticum……..……………..19 Figure 5. The comparison of the Wood-Ljungdahl pathway gene cluster of C. formicoaceticum with other sequenced acetogens……...……………………………19 Figure 6. Acetate and ethanol metabolic pathway in C. formicoaceticum..………….20 Figure 7. Butanol metabolic pathway in C. formicoaceticum ……………….………22 Figure 8. The EMP pathway and relevant enzymes in C. formicoacetium……..……24 Figure 9. The PPP pathway and relevant enzymes in C. formicoacetium……………24 Figure 10. The glycerol metabolic pathway in C. formicoacetium……...……….…..26 Figure 11. Energy conservation types in Homoacetogens…...……….……….……..28 Figure 12. Cytochrome/menaquinone system in C. formicoaceticum…...……….…29 Figure 13. F1F0 ATPase in C. formicoaceticum………….……….……..……….…30 Figure 14. Alignment of the deduced amino acid sequence of the proteolipids of C. formicoaceticum with proteolipids of other organisms…………………………….30 Figure 15. Rnf system in C. formicoaceticum…………………………….……….31 ix Figure 16. Digestion of several plasmids by crude cell extract of C. formicoaceticum at 12 h.. …………………………..………………………………………..…….…35 Figure 17. Digestion of several plasmids by crude cell extract of C. formicoaceticum at 24 h. ……………………………………………………………………..………36 Figure 18. Phylogenetic tree of C. formicoaceticum ATCC 27076. ……….………40 Figure 19. The Wood-Ljungdahl pathway in A. woodii. …………………..………41 Figure 20. The Wood-Ljungdahl pathway in C. formicoaceticum..………………..41 Figure 21. The reductive glycine pathway in C. formicoaceticum..………………..41 Figure 22. Batch fermentations of C. formicoaceticum with fructose (A), formate (B), and glycerol (C) as substrate in serum bottles…………………………………...….44 Figure 23. Batch fermentations of C. formicoaceticum with ethanol (A) and butanol (B) as substrate in serum bottles.………………………………...……………….…45 Figure 24. Gas fermentations of C. formicoaceticum at the optimum growth pH 7.6 in serum bottles..………………………………..….…………………………….……45 Figure 25. Constructing ethanol or