View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Waterloo's Institutional Repository Development of genetic tools for metabolic engineering of Clostridium pasteurianum by Michael Pyne A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Chemical Engineering Waterloo, Ontario, Canada, 2014 © Michael Pyne 2014 Author’s declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract Reducing the production cost of industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to a complete lack of genetic tools and techniques available for the manipulation of this promising bacterium. This thesis encompasses the development of fundamental genetic tools and techniques that will permit extensive genetic and metabolic engineering of C. pasteurianum. We initiated our genetic work with the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency up to 7.5 × 104 transformants µg-1 DNA, an increase of approximately three orders of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Following development of a gene transfer methodology, we next aimed to sequence the entire genome of C. pasteurianum. Using a hybrid approach involving 454 pyrosequencing, Illumina dye sequencing, and single molecule real-time sequencing platforms, we obtained a iii near-complete genome sequence comprised of 12 contigs, 4,420,100 bp, and 4,056 candidate protein-coding genes with a GC content of 30.0%. No extrachromosomal elements were detected. We provide an overview of the genes and pathways involved in the organism’s central fermentative metabolism. We used our developed electrotransformation procedure to investigate the use of established clostridial group II intron biology for constructing chromosomal gene knockout mutants of C. pasteurianum. Through methylome analysis of C. pasteurianum genome sequencing data and transformation assays of various vector deletion constructs, we identified a new Type I restriction-modification system that inhibits transfer of vectors harboring group II intron gene knockout machinery. We designated the new restriction system CpaAII and proposed a recognition sequence of 5’-AAGNNNNNCTCC-3’. Overcoming restriction by CpaAII, in addition to low intron retrohoming efficiency, allowed the isolation of a gene knockout mutant of C. pasteurianum with a disrupted CpaAI Type II restriction system. The resulting mutant strain should be efficienty transformed with plasmid DNA lacking M.FnuDII methylation. Lastly, we investigated the use of plasmid-based gene overexpression and chromosomal gene downregulation to alter gene expression in C. pasteurianum. Using a β-galactosidase reporter gene, we characterized promoters corresponding to the ferredoxin and thiolase genes of C. pasteurianum and show that both promoters permitted high-level, constitutive gene expression. The thiolase promoter was then utilized to drive transcription of an antisense RNA molecule possessing complementarity to mRNA of our β-galactosidase reporter gene. Our antisense RNA system demonstrated 52-58% downregulation of plasmid encoded β- galactosidase activity throughout the duration of growth. In an attempt to perturb the central fermentative metabolism of C. pasteurianum and enhance butanol titers, we prepared several antisense RNA constructs for downregulation of 1,3-propanediol, butyrate, and hydrogen production pathways. The resulting downregulation strains are expected to exhibit drastically altered central fermentative metabolism and product distribution. Taken together, we have demonstrated that C. pasteurianum is amendable to genetic manipulation through the development of methods for plasmid DNA transfer and gene overexpression, knockdown, and knockout. Further, our genome sequence should provide valuable nucleotide sequence information for the application of our genetic tools. Thus, the genome sequence, electrotransformation method, and associated genetic tools and techniques iv reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium. Keywords: Antisense RNA, biofuels, butanol, Clostridium, genetic engineering, glycerol, knockout, metabolic engineering, overexpression, restriction, transformation v Acknowledgements I am infinitely grateful to have Professor C. Perry Chou, Professor Duane Chung, and Professor Murray Moo-Young as my PhD supervisors. They have pushed me immensely over the past 6 years, but I learned very early on that they want nothing more than for their students to succeed. It was both refreshing and valuable to have supervisors with such different scientific approaches and outlooks. Professor Chung – from you I learned that curiosity and skepticism are just as important as intellect. I have benefitted greatly from your entrepreneurial viewpoint. Professor Chou – you demonstrated that work ethic, attention to detail, and modesty are essential to a successful career in academia. If I inherited even a fraction of these traits from you, I will surely succeed in my career. Professor Moo-Young – you showed me first-hand of what hard work and ambition can achieve in science. I am honoured to have been your student. I am greatly indebted to my committee members – Professor Frank Gu, Professor Bill Anderson, and the late Professor Jeno Scharer from the Department of Chemical Engineering and Professor Trevor Charles from the Department of Biology – for attending my comprehensive examination and defense, and providing valuable suggestions and comments. I am also honoured to have had Professor E. Terry Papoutsakis from the University of Delaware serve as the external examiner and share his expertise at my oral defense. I owe sincere thanks to our collaborators, Professor Steven Brown and Sagar Utturkar at Oak Ridge National Laboratories in Oak Ridge, Tennessee, for their genome sequencing contributions. I would also like to thank Dr. Jiri Perutka (University of Texas at Austin), Scott Binford (Pacific Biosciences), and Dr. Bill Jack and Dr. Geoffrey Wilson (New England Biolabs) for their valuable input and assistance. This work would also not have been possible without plasmid and strain donations from Professor E. Terry Papoutsakis (University of Delaware), Professor Nigel Minton (University of Nottingham), Professor Sheng Yang (Shanghai Institutes for Biological Sciences), Professor George Bennett (Rice University), and New England Biolabs. I acknowledge financial support from Natural Sciences and Engineering Research Council (NSERC) of Canada and the Canada Research Chair program. I would also like to acknowledge the hard-working staff and faculty members of the Department of Chemical Engineering, especially Rose Guderian, Judy Caron, Liz Bevan, Ingrid Sherrer, Camille Graham, Rick Hecktus, and Bert Habicher. vi I cannot think of anyone better than Karan Sukhija to have started grad school with 6 years ago. This work would not have been possible without the help of Karan and all of my other past and present lab mates – Niju, Shreyas, Victoria, Weifang, Val, Kajan, Fred, Steve, Daryoush, Rana, Lu, Saideh, Bahram, Adam, Lamees, Yasmin, Will, Ziggy, Ian, Mark, Aaqil, and Nimhani. These people made the lab a fun, helpful, and cooperative environment. I also cannot envision a successful laboratory without the dedication of co-op students. Although there have been too many over the years to list, I am grateful for the hard work provided by all of our co-op students, especially