Enhancement of CO2 and H2 Uptake for the Production of Biodiesel in Cupriavidus Necator

Cupriavidus necator fixes CO{sub 2} through the Calvin-Benson-Bassham (CBB) cycle using electrons and energy obtained from the oxidation of H{sub 2}. Producing biodiesel-equivalent electrofuel from renewable CO{sub 2} and H{sub 2} has immense potential, especially if the fuel is compatible with the existing fuel infrastructure. This research addressed enhanced substrate utilization by focusing on two strategies: (1) optimizing transcriptional regulations to afford over-expression of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for assimilation of CO{sub 2} into the CBB cycle; and (2) hydrogenase over-expression by introduction of additional copies of genes encoding a membrane-bound hydrogenase (MBH), a soluble hydrogenase (SH), and their maturation machinery to enhance oxidation of H{sub 2} to generate NAD(P)H and ATP required for CO{sub 2} fixation. Incorporation of these strategies into a single production strain resulted in 6-fold CO{sub 2} and 3-fold H{sub 2} uptake improvement, in vitro, with the overarching goal of providing abundant reducing equivalents towards the economic production of biodiesel in C. necator.