Addition of formate dehydrogenase increases the production of renewable alkane from an engineered metabolic pathway

An engineered metabolic pathway consisting of reactions that convert fatty acids to aldehydes and eventually alkanes would provide a means to produce biofuels from renewable energy sources. The enzyme aldehyde-deformylating oxygenase (ADO) catalyzes the conversion of aldehydes and oxygen to alkanes and formic acid and uses oxygen and a cellular reductant such as ferredoxin (Fd) as co-substrates. In this report, we aimed to increase ADO-mediated alkane production by converting an unused by-product, formate, to a reductant that can be used by ADO. We achieved this by including the gene (fdh), encoding formate dehydrogenase from Xanthobacter sp. 91 (XaFDH), into a metabolic pathway expressed in Escherichia coli Using this approach, we could increase bacterial alkane production, resulting in a conversion yield of ∼50%, the highest yield reported to date. Measuring intracellular nicotinamide concentrations, we found that E. coli cells harboring XaFDH have a significantly higher concentration of NADH and a higher NADH/NAD⁺ ratio than E. coli cells lacking XaFDH. In vitro analysis disclosed that ferredoxin (flavodoxin):NADP⁺ oxidoreductase could use NADH to reduce Fd and thus facilitate ADO-mediated alkane production. As formic acid can decrease the cellular pH, the addition of formate dehydrogenase could also maintain the cellular pH in the neutral range, which is more suitable for alkane production. We conclude that this simple, dual-pronged approach of increasing NAD(P)H and removing extra formic acid is efficient for increasing the production of renewable alkanes via synthetic biology-based approaches.