P164: Enhancement of H2 and CO2 Uptake for the Production of Biodiesel in Ralstonia eutropha

Utilization of renewable CO₂ and H₂ as feedstocks for the production of biodiesel electrofuels compatible with existing infrastructures has immense potential. Ralstonia eutropha can grow autotrophically, using energy (ATP) and reductant (NAD(P)H) obtained from the oxidation of H₂ via a membrane-bound hydrogenase (MBH) and a soluble hydrogenase (SH) to fix CO₂ via the Calvin-Benson-Bassham (CBB) cycle. Our research addresses the enhancement of H₂ and CO₂ substrate utilization to improve product yields by focusing on two strategies: (1) increased expression of CBB cycle genes to enhance CO₂ fixation rates; and (2) increased expression of the MBH and SH operons and their maturation machinery to enhance oxidation of H₂ and subsequent generation of NAD(P)H and ATP. Increased expression of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for assimilation of CO₂ into the CBB cycle, resulted in 6-fold improvement in CO₂ fixation in vitro. Over-expression of the MBH operon alone (includes all necessary maturation factors), the SH structural genes plus maturation factors (from the MBH operon), or both enhanced in vitro activity up to 10-fold. Ongoing research involves analysis of the relative contribution of the two endogenous cbb operons, genome incorporation of genetic changes for optimal expression of both CBB cycle enzymes and MBH/SH, and determination of activity enhancement, both in vitro and in vivo, and effects on cellular cofactor ratios and downstream carbon sinks in these genetically altered strains. Results from these studies will improve our understanding of autotrophic metabolism in this organism and will provide tools necessary for the enhancement of carbon fixation, energy, and reductant levels necessary for improved production yields.