EXPANDING MOLECULAR TOOLS FOR THE METABOLIC ENGINEERING OF RALSTONIA EUTROPHA H16

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 λ-Red plasmid vectors were developed for mediating λ-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 λ-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.