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Genomic Analyses of Paenibacillus Polymyxa CR1, a Bacterium with Potential Applications in Biomass Degradation and Biofuel Production

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Genomic Analyses of Paenibacillus Polymyxa CR1, a Bacterium with Potential Applications in Biomass Degradation and Biofuel Production Western University Scholarship@Western Electronic Thesis and Dissertation Repository 5-26-2015 12:00 AM Genomic analyses of Paenibacillus polymyxa CR1, a bacterium with potential applications in biomass degradation and biofuel production Alexander W. Eastman The University of Western Ontario Supervisor Dr. Ze-Chun Yuan The University of Western Ontario Graduate Program in Microbiology and Immunology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Alexander W. Eastman 2015 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Other Microbiology Commons Recommended Citation Eastman, Alexander W., "Genomic analyses of Paenibacillus polymyxa CR1, a bacterium with potential applications in biomass degradation and biofuel production" (2015). Electronic Thesis and Dissertation Repository. 2873. https://ir.lib.uwo.ca/etd/2873 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Genomic analyses of Paenibacillus polymyxa CR1, a bacterium with potential applications in biomass degradation and biofuel production (Thesis format: Monograph) by Alexander William Eastman Graduate Program in Microbiology and Immunology A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Alexander William Eastman 2015 i Abstract Lignin is a polyphenolic heteropolymer constituting between 18 to 35% of lignocellulose and is recognized as preventative of cellulosic biofuel commercialization. Paenibacillus polymyxa CR1 was isolated from naturally degrading corn stover and shown to produce alcohols using lignin as a sole carbon source. Genome sequencing and comparative genomics of P. polymyxa CR1 identified two homologs, a Dyp-type peroxidase and a laccase, which have previously been implicated in lignin metabolism in other bacteria. Knockout mutants of the identified genes displayed no growth deficiency and P. polymyxa CR1 is incapable of metabolizing common aromatic intermediates of lignin, suggesting the bacterium employs a novel catabolic pathway. To identify genes involved in lignin metabolism, a transposon library was generated and screened for abnormal lignin growth phenotypes. The results contained within will help elucidate the genetic basis of known functions helping delineate regulatory pathways and metabolic versatility in P. polymyxa relevant to lignin metabolism. Keywords Sequencing, genomics, genetics, comparative genomics, lignocellulose, lignin, metabolism, biofuels, renewable energy, finishing, transposon mutagenesis, ribosomal RNA ii Co-Authorship Statements Complete Genome Sequence of Paenibacillus polymyxa CR1, a Plant Growth- Promoting Bacterium Isolated from the Corn Rhizosphere Exhibiting Potential for Biocontrol, Biomass Degradation, and Biofuel Production. Alexander W. Eastman 1,2, Brian Weselowski 1, Naeem Nathoo 1,3, and Ze-Chun Yuan1,2 1 Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, Government of Canada, London, Ontario, Canada N5V 4T3 2 Western University, Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, London, Ontario, Canada N6A 5C1 3Western University, Department of Biology, London, Ontario, Canada N6A 5B7 Portions of this work have been published in Genome Announcements 2014, 2(1): e01218-13 Brian Weselowski aided in performing experiments. Naeem Nathoo aided in analysis of scaffolding and in silico alignments. Dr. Yuan contributed to editing of the manuscript. Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing. Alexander W. Eastman 1,2, and Ze-Chun Yuan 1,2 1 Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, Government of Canada, London, Ontario, Canada N5V 4T3 2 Western University, Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, London, Ontario, Canada N6A 5C1 Portions of this work has been published in Frontiers in Microbiology 2015, 7:769 Dr. Yuan contributed to the design of the experiments and editing of the manuscript. Comparative and genetic analysis of the four sequenced Paenibacillus polymyxa genomes reveals a diverse metabolism and conservation of genes relevant to plant- growth promotion and competitiveness. Alexander W. Eastman 1,2, David E Heinrichs 2, and Ze-Chun Yuan 1,2 1 Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, Government of Canada, London, Ontario, Canada N5V 4T3 2 Western University, Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, London, Ontario, Canada N6A 5C1 iii Portions of this work have been published in BMC Genomics 2014, 15:851 Dr. Yuan contributed to the conceptualization of the research and the writing and editing of the manuscript. Dr. Heinrichs supervised and edited the writing of the manuscript. iv Acknowledgments I would like to express my gratitude to my co-supervisors Dr. Ze-Chun Yuan and Dr. David Heinrichs who have always been available for guidance and support over the course of my research. I would also like to thank the members of my committee Dr. Martin McGavin and Dr. Yuhai Cui for their invaluable insights into limitations and areas in need of clarification and focus in my work, as well their time and efforts put into reading and editing my thesis. A special thanks goes out to Alex Molnar for his work on figures. I would like to thank members of my immediate lab group, Naeem Nathoo and Brian Weselowski who have helped maintain my sanity and dedication at various times and made the long hours at the lab seem like mere minutes. Finally I would like to thank my friends and family, with expressive thanks to Shilpa Goel, for enduring my endless ranting and incessant science both when things we running smoothly and when experiments were inevitably failing. v List of Abbreviations ABC transporter- ATP binding cassette transporter ATP- adenosine triphosphate BLAST- basic local alignment search tool Bp- base pairs CDS- coding sequences CO2- carbon dioxide COG- cluster of orthologous groups Contig – contiguous DNA sequence DNA – deoxyribonucleic acid DyP- dye-decolourizing peroxidase GC- gas chromatography GH- glycoside hydrolase GHG- greenhouse gas Kan- kanamycin sulphate KO- KEGG orthology LB- lysogeny broth LCB- local collinear block Lig- Kraft lignin LiP- lignin peroxidase MFS- major facilitator superfamily MM- minimal media Mn- manganese NCBI- National Centre for Bioinformatics PCR- polymerase chain reaction vi PTS- phosphor transferase system rDNA- DNA region encoding ribosomal subunit genes RNA- ribonucleic acid rRNA- mature ribosomal subunit RNA TAE- tris base, acetic acid ethylenediaminetetraacetic acid Tet- tetracycline Tn- transposon tRNA- transfer RNA US- United States of America VP-versatile peroxidase G- giga (109) M- mega (106) k-kilo (103) m- milli (10-3) n- nano (10-6) p- pico (10-9) Pa- Pascal M- molar (mol/L) g- gram L- litre vii Table of Contents Abstract ............................................................................................................................... i Co-Authorship Statements ............................................................................................... ii Acknowledgments ............................................................................................................ iv Table of Contents ............................................................................................................ vii List of Tables ..................................................................................................................... 2 List of Figures .................................................................................................................... 3 List of Appendices ............................................................................................................. 5 Chapter 1 – Introduction .................................................................................................. 6 Biofuels History .......................................................................................................................... 6 First-Generation Biofuels .......................................................................................................... 7 Cellulosic Biofuels ...................................................................................................................... 9 Lignocellulose ........................................................................................................................... 12 Lignin Structure and Synthesis ............................................................................................. 16 Current Limitations of Lignin on Cellulosic Biofuel ............................................................ 19 Chemical and Industrial Delignification Processes .............................................................. 20 Acid or Alkali Hydrolysis ..................................................................................................... 20 Ionic Liquids ........................................................................................................................
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