Genetic engineering of Cupriavidus necator’s autotrophic metabolism for enhanced CO2 capture and polyhydroxybutyrate (PHB) synthesis

Cupriavidus necator is a facultative chemolithoautotrophic bacterium capable of converting CO2 into polyhydroxybutyrate (PHB), a biodegradable polymer with bioplastic properties. The bacterium has been widely studied as a platform for PHB production due to its flexible metabolism and ability to accumulate high intracellular levels of the polymer. However, the process is not yet competitive with the production costs of traditional plastics, requiring further optimisation. In this work, the enhancement of the CO2 uptake rate and PHB yield of C. necator is being investigated both by bioprocess optimisation and via genetic engineering. C. necator DSM545 was grown in a fed-batch mode using 10% CO2, 10% O2 and 60% H2, achieving a maximum PHB yield of 0.69 g/L in 4 days under nitrogen limitation, with an average CO2 uptake rate of 1.14 mmol CO2 L -1 h -1 and 65% efficiency of CO2 consumption. In parallel, mutant strains were created which overexpress key genes or regulatory elements of the autotrophic metabolism. These include bifunctional fructose-1,6-bisphosphatase sedoheptulose-1,7-bisphosphatase (F/SBPase) of the Calvin-Benson-Bassham (CBB) cycle, HoxA hydrogenase regulator, Cytochrome c. reductase, and a recombinant soluble transhydrogenase. Growth tests showed that these genetic modifications impacted upon gas consumption patterns, with implications on strategies to maximise the bioconversion efficiency of C. necator. Finally, the system was applied to capture CO2 derived from fermentation during wine production, exemplifying its application in an industrial process.