Engineering Cupriavidus necator and Escherichia coli for the sustainable manufacturing of 3-hydroxypropionic acid

The platform chemical 3-hydroxypropionic acid (3HP) has been identified by the US Government as a key target for biological production, with a global market projected to reach $1 billion by 2030. Current production methods are expensive, limiting the potential of 3HP as a precursor to valuable compounds including acrylic acid. This thesis presents a novel technique for biosynthesis of 3HP using the bacterium Cupriavidus necator, a versatile biomanufacturing host that can utilise a range of carbon sources, including CO2.

The enzymes malonyl-CoA reductase (MCR) and malonate semialdehyde reductase (MSR) from archaea were expressed in C. necator to enable the conversion of malonyl-CoA to 3HP. Kinetic analysis of MCR from Sulfolobus tokodaii and MSR from Metallosphaera sedula was performed in vitro. The synthesis of 3HP was then developed in vivo and fed-batch fermentation produced a final titre of 1.34 ± 0.13 g/L 3HP, with a yield of 0.028 ± 0.004 g 3HP / g sodium gluconate and a productivity of 0.014 ± 0.002 g/L/h. This is the highest reported 3HP titre from C. necator, outlining the potential of using MCR and MSR. However, production was limited by low malonyl-CoA availability.

A novel adaptive laboratory evolution method was developed for increasing the intracellular concentration of malonyl-CoA, and the method was assessed in Escherichia coli. A plasmid (S1-tet) was constructed that linked higher malonyl-CoA levels to increased resistance to the antibiotic tetracycline. To drive cells to accumulate more malonyl-CoA, E. coli DH5α with S1-tet was cultivated in increasing tetracycline concentrations. Evolved strains were isolated and their production of 3HP via malonyl-CoA was measured, achieving a titre of 10.9 ± 0.92 g/L from glucose. This is one of the highest reported flask 3HP titres, and a 12-fold increase compared to unevolved E. coli DH5α, demonstrating the capacity of the method for raising malonyl-CoA availability.