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Expanding Molecular Tools for the Metabolic Engineering of Ralstonia Eutropha H16 Abayomi Oluwanbe Johnson

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Expanding Molecular Tools for the Metabolic Engineering of Ralstonia Eutropha H16 Abayomi Oluwanbe Johnson EXPANDING MOLECULAR TOOLS FOR THE METABOLIC ENGINEERING OF RALSTONIA EUTROPHA H16 By: ABAYOMI OLUWANBE JOHNSON A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Engineering Department of Chemical and Biological Engineering July 2018 (This page has been left blank intentionally) i DECLARATION This thesis is a product of original research conducted by me as a PhD student at the Department of Chemical and Biological Engineering, University of Sheffield. All sources of information herein have been duly referenced. This thesis, in its entirety, has never been previously submitted at this University or any other institution. This thesis is written in conformance to the rules of Alternative Format Thesis (Code Of Practice For Research Degree Program 2017-18) of the University of Sheffield. Some parts of this thesis have been previously published/submitted as peer-reviewed scientific publications. i ASSOCIATED PUBLICATIONS Johnson, A. O., Gonzalez Villanueva, M., & Wong, T. S. (2015). Engineering Ralstonia eutropha for chemical production. In Industrial Processes and Nanotechnology (Vol. 10). USA: Studium Press LLC. • Johnson, A.O., Gonzalez, V.M., Wong, L., Steinbüchel, A., Tee, K.L., Xu, P., Wong, T.S., 2017. Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories. Metab. Eng. Johnson, A. O., Gonzalez-Villanueva, M., Tee, K. L. & Wong, T. S. 2018. An Engineered Constitutive Promoter Set with Broad Activity Range for Cupriavidus necator H16. ACS Synthetic Biology. ii ACKNOWLEDGEMENTS I would like to thank my esteemed supervisor – Dr. Tuck Seng Wong for his invaluable supervision, support and tutelage during the course of my PhD degree. My gratitude extends to the Faculty of Engineering for the funding opportunity to undertake my studies at the Department of Chemical and Biological Engineering, University of Sheffield. Additionally, I would like to express gratitude to Dr. Kang Lan Tee for her treasured support which was really influential in shaping my experiment methods and critiquing my results. I also thank Dr. David Gonzalez, Dr. Pawel Jajeniak, Dr. Amir Zaki Abdullah Zubir for their mentorship. I would like to thanks my friends, lab mates, colleagues – Abdulrahman Alessa, Miriam Gonzalez-Villanuva, Inas Al-nuaemi, Jose Avalos, Valeriane Keita, Melvin Mikuzi, Robert Berchtold for a cherished time spent together in the lab, and in social settings. My appreciation also goes out to my family and friends for their encouragement and support all through my studies. iii SUMMARY Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a non-pathogenic chemolithoautotrophic soil bacterium. It has increasingly gained biotechnological interest for its use as a microbial cell factory for the production of several valuable bio-based chemicals. However the absence of a large repertoire of molecular tools to engineer this organism remains a critical limiting factor to exploiting its full biotechnological potential. Also, adopting established molecular tools applicable to the more notable microbial hosts such as E. coli and Saccharomyces cerevesiae is severely hampered by chassis- incompatibility and functional variability of essential biological parts. The work detailed in this thesis focuses on the development of key molecular tools crucial to improving the biosynthesis of malonyl-CoA - a precursor metabolite required for the biosynthesis of fatty acids and potentially several valuable bio-products in Ralstonia eutropha H16. All molecular tools developed were based on the broad host range (BHR) plasmid vector backbone of pBBR1MCS1 – a R. eutropha H16-compatible vector. Firstly, to facilitate heterologous pathway optimization, a combination of pre-existing and novel methods of genetic modifications were applied to engineer a collection of 42 promoters. Promoter strengths were characterized using a fluorescence-based assay and benchmarked to the dose-dependent activity of an L-arabinose-inducible PBAD promoter. Next, to detect intracellular accumulation of malonyl-CoA, transcriptional factor-based malonyl-CoA- sensing genetic circuits were developed via careful selection from the promoter collection. Thirdly, BHR L-arabinose-inducible -Red plasmid vectors were developed for mediating -Red-based genome editing. These were first tested in E. coli BW25113 to confirm their functionality and then subsequently tested in R. eutropha H16. Overall, the collection of engineered promoters yielded a 137-fold range of promoter activity and the malonyl-CoA biosensors responded to changing malonyl-CoA concentrations. The BHR -Red plasmids showed high recombination efficiency in E. coli BW25113. The molecular tools developed from this work will further facilitate rapid control and regulation of gene expression in R. eutropha, particularly for malonyl-CoA engineering. iv TABLE OF CONTENT DECLARATION ................................................................................................................... I ASSOCIATED PUBLICATIONS ..................................................................................... II ACKNOWLEDGEMENTS ............................................................................................... III SUMMARY ........................................................................................................................ IV TABLE OF CONTENT ....................................................................................................... V TABLE OF FIGURES ......................................................................................................... X TABLE OF TABLES ...................................................................................................... XIV LIST OF ABBREVIATIONS ....................................................................................... XVII CHAPTER ONE .................................................................................................................. 1 LITERATURE REVIEW .................................................................................................... 1 1 LITERATURE REVIEW .............................................................................................. 2 1.1 ENGINEERING MICROBIAL CELL FACTORIES FOR BIO-MANUFACTURING .............. ERROR! BOOKMARK NOT DEFINED. 1.2 R. EUTROPHA H16 – AN INDUSTRIAL WORK-HORSE FOR CHEMICAL PRODUCTION ERROR! BOOKMARK NOT DEFINED. 1.3 ENGINEERING STRATEGIES FOR CHEMICAL PRODUCTION IN R. EUTROPHA ........... ERROR! BOOKMARK NOT DEFINED. 1.4 METABOLIC ENGINEERING IN R. EUTROPHA .............ERROR! BOOKMARK NOT DEFINED. 1.4.1 EVOLUTIONARY ENGINEERING OR ADAPTIVE EVOLUTIONERROR! BOOKMARK NOT DEFINED. 1.4.2 RECOMBINANT STRAIN ENGINEERING ..................ERROR! BOOKMARK NOT DEFINED. 1.4.3 GENOME EDITING .................................................ERROR! BOOKMARK NOT DEFINED. 1.5 EXPANDING CARBON SUBSTRATE RANGE OF R. EUTROPHA ...... ERROR! BOOKMARK NOT DEFINED. 1.5.1 GLUCOSE..............................................................ERROR! BOOKMARK NOT DEFINED. 1.5.2 GLYCEROL ...........................................................ERROR! BOOKMARK NOT DEFINED. 1.5.3 SUGARS DERIVED FROM LIGNOCELLULOSIC BIOMASSERROR! BOOKMARK NOT DEFINED. 1.5.4 CARBON DIOXIDE .................................................ERROR! BOOKMARK NOT DEFINED. 1.6 ENGINEERING STRATEGIES FOR BIOPRODUCT SYNTHESIS IN R. EUTROPHA ........... ERROR! BOOKMARK NOT DEFINED. 1.6.1 PHA PRODUCTION................................................ERROR! BOOKMARK NOT DEFINED. 1.6.2 BIOFUEL PRODUCTION ..........................................ERROR! BOOKMARK NOT DEFINED. 1.6.3 METHYL CITRATE .................................................ERROR! BOOKMARK NOT DEFINED. 1.6.4 FERULIC ACID.......................................................ERROR! BOOKMARK NOT DEFINED. 1.6.5 CHIRAL SYNTHONS ...............................................ERROR! BOOKMARK NOT DEFINED. 1.6.6 BIOHYDROCARBONS ............................................ERROR! BOOKMARK NOT DEFINED. v 1.6.7 OTHER BIOPOLYMERS ..........................................ERROR! BOOKMARK NOT DEFINED. 1.7 EMERGING STRATEGIES TO EXPAND CHEMICAL RANGE ........... ERROR! BOOKMARK NOT DEFINED. 1.7.1 IN SILICO METABOLIC PATHWAY ANALYSIS AND DESIGNERROR! BOOKMARK NOT DEFINED. 1.7.2 PROTEIN ENGINEERING .........................................ERROR! BOOKMARK NOT DEFINED. 1.7.3 SYNTHETIC BIOLOGY ............................................ERROR! BOOKMARK NOT DEFINED. 1.8 BIOPROCESS ENGINEERING STRATEGIES FOR INDUSTRIAL APPLICATIONS OF R. EUTROPHA ERROR! BOOKMARK NOT DEFINED. 1.8.1 FEEDSTOCK ..........................................................ERROR! BOOKMARK NOT DEFINED. 1.8.2 BIOREACTOR AND FERMENTATION STRATEGY......ERROR! BOOKMARK NOT DEFINED. 1.9 FUTURE OUTLOOK ...................................................ERROR! BOOKMARK NOT DEFINED. 1.10 SCOPE OF RESEARCH .................................................................................................. 39 1.11 WHY MALONYL-COA? .............................................................................................. 40 1.12 STRATEGIES FOR MALONYL-COA ENGINEERING IN MODEL MICROBIAL FACTORIES ... 41 1.13 DEVELOPMENT OF CHASSIS-COMPATIBLE EXPRESSION VECTORS ............................... 48 1.14 PROMOTER ENGINEERING ........................................................................................... 49 1.15 METABOLITE SENSING GENETIC
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