Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase

Biotechnology is a promising approach for the generation of hydrogen, but is not yet commercially viable. Metabolic engineering is a potential solution, but has largely been limited to native pathway optimisation. To widen opportunities for use of non-native NiFe] hydrogenases for improved hydrogen production, we introduced a cyanobacterial hydrogen production pathway and associated maturation factors into Escherichia coli. Hydrogen production is observed in vivo in a hydrogenase null host, demonstrating coupling to host electron transfer systems. Hydrogenase activity is also detected in vitro. Hydrogen output is increased when formate production is abolished, showing that the new pathway is distinct from the native formate dependent pathway and supporting the conclusion that it couples cellular NADH and NADPH pools to molecular hydrogen. This work demonstrates non-native hydrogen production in E. coli, showing the wide portability of NiFe] hydrogenase pathways and the potential for metabolic engineering to improve hydrogen yields.