Metabolic engineering of Cupriavidus necator H16 for the production of high value chemicals

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Gas fermentation technology based on microbial conversion of carbon monoxide (CO), carbon dioxide (CO2), and methane (CH4) into chemicals and fuels has the potential to replace the existing fossil fuel based technologies and could provide a desirable alternative for the production of low carbon fuels for transportation, green chemicals or other valuable monomers that can be further processed into biopolymers and other high value chemicals. These processes would greatly contribute to reduction of Green House Gas (GHG) emissions by converting waste gasses from steel manufacturing, oil refining, coal and natural/shale gas into valuable products. Cupriavidus necator H16 (formerly known as Ralstonia eutropha) is a Gram-negative, non-spore forming, facultatively chemolithoautotrophic bacterium able to grow on organic substrates or H2 and CO2 under aerobic conditions. Its ability to grow on CO2 as sole carbon source makes it an attractive chassis organism for the sustainable production of high value platform chemicals from waste gasses. The aim of this project is to metabolically engineer Cupriavidus necator H16 to produce a valuable platform chemical, 3-hydroxypropanoic acid or 3HP. This intermediate product can be converted into acrylic acid, biodegradable polyesters, superabsorbent polymers and acrylic acids among other highly valued industrial products. It can be synthesised via glycerol, lactate, malonyl-CoA or beta-alanine intermediates via at least seven different biosynthetic pathways (Kumar, 2013). Three of the proposed pathways are thermodynamically favourable, and the most favourable pathway, proceeding via beta-alanine will be tested during this PhD for 3HP production in C. necator.The synthetic pathway for 3-HP production from beta-alanine has been previously described in E. coli and yeast. In yeast, the intermediate beta-alanine was converted into malonic semialdehyde either by the action of beta-alanine-pyruvate aminotransferase (BAPAT) or y-amino butyrate transaminase (GABT), and further reduced into 3-HP by the action of either 3-hydroxypropionate dehydrogenase (HPDH) or 3-hydroxyisobutyrate dehydrogenase (HIBADH). Homologues of all four candidate genes have been identified in the genome of C. necator H16 and will be subjected to enzyme characterisation. In addition, several synthetic operons will be built and tested for efficient production of beta-alanine and conversion of beta-alanine to 3HP