This study shows that the organic cofactor riboflavin provides a cheap and atom-efficient source of reducing equivalents to sustain biocatalytic alkene reductions by ene-reductase enzymes, when coupled with a H2-driven or electrochemical recycling system. We employ the robust NiFe hydrogenase, Escherichia coli Hyd1, for H2-driven riboflavin reduction and show the feasibility of unmediated electrochemical recycling of reduced riboflavin at a simple carbon electrode. We show that H2-driven reduction of riboflavin can be extended to continuous flow with a packed bed reactor comprising of Hyd1 immobilized on a carbon support. These findings demonstrate that there is scope for replacing the expensive nicotinamide cofactors—NADH or NADPH with riboflavin, for applications of ene-reductases in biotechnology in either batch or continuous flow and in electrosynthesis.
