A simplified procedure for cell-free biocatalytic reductions of conjugated C=C double bonds using old yellow enzymes (OYEs) is reported. Instead of indirectly regenerating YqjM (an OYE homologue from B. subtilis) or NemA (N-ethylmale-A C H T U N G T R E N N U N G imide reductase from E. coli) via regeneration of reduced nicotinamide cofactors, we demonstrate that direct regeneration of catalytically active reduced flavins is an efficient and convenient approach. Reducing equivalents are provided from simple sacrificial electron donors such as ethylenediaminetetra-A C H T U N G T R E N N U N G acetate (EDTA), formate, or phosphite via photocatalytic oxidation. This novel photoenzymatic reaction scheme was characterized. Up to 65% rates of the NADH-driven reaction were obtained while preserving enantioselectivity. The chemoselectivity of the novel approach was exclusive. Even when using crude cell extracts as biocatalyst preparations, only C=C bond reduction was observed while ketone and aldehyde groups remained unaltered. Overall, a simple and practical approach for photobiocatalytic reductions is presented.
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