Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli

Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD⁺/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD⁺/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H₂ production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H₂ production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme.