Shewanella oneidensis MR-1 is a potent hydrogen producer in the deficiency of exogenous electron acceptors. The electron transfer pathway for hydrogen production remains unclear, although enzymes for hydrogen production have been identified in S. oneidensis MR-1. In this study, we investigated the electron transfer pathway from formate to hydrogen, given that formate is commonly a key chemical for bacterial hydrogen production. We revealed that two formate dehydrogenases FdhA1B1C1 and FdhA2B2C2, rather than FdnGHI, played a dominant role in formate-driven hydrogen production. Menaquinone was indispensable for the electron transfer from formate to hydrogen, which excluded the presence of formate hydrogen-lyase in S. oneidensis MR-1. A previously proposed formate dehydrogenase subunit HydC was identified as a menaquinone-binding subunit of [FeFe] hydrogenase HydAB, and the hydABC operon is conserved in bacteria living in diverse environments. A formate exporter FocA and transcriptional regulator FhlA were identified for their effect on formate metabolism and hydrogen production. FhlA positively affected the metabolism of formate and hydrogen by regulating the expression of fdhA2B2C2, fdnGHI, focA, and dld-II. Overall, the electron transfer pathway deciphered in this work will facilitate the improvement of biohydrogen production by S. oneidensis MR-1.Key Points• The electron transfer pathway from formate to hydrogen in MR-1 is deciphered.• Menaquinone is indispensable for hydrogen production.• A cytochrome b subunit transfers electrons from menaquinone to [FeFe] hydrogenase.
Keywords: Electron transfer; Formate dehydrogenase; Hydrogen; Hydrogenase; Shewanella.