Exploring ethanol utilization for NADH regeneration in whole cell biocatalysis

Cofactor regeneration is an established technology for enabling the recycling of cofactors such as NADH and NADPH needed by oxidoreductase performing biocatalytic reactions. Without cofactor regeneration, stoichiometric amounts of cofactors would need to be provided which hampers the widespread adoption of biocatalysis to perform enantioselective reactions for the pharmaceutical and fine chemical industry. In the area of NADH regeneration, glucose dehydrogenase is the dominant method, but it suffers from relative lack of efficiency and poor NADH yield per glucose molecule. This presentation sought to highlight the potential of an alternative NADH regeneration system built upon the ethanol/ethanol utilization pathway system that, theoretically, could regenerate two NADH per ethanol molecule. Preliminary results showed that the ethanol utilization pathway could engender growth in Escherichia coli; thereby, validating its functional status in channelling flux from ethanol to acetyl-CoA that connects to central carbon metabolism which helps power cell growth. Performance assessment with an acetophenone to 1-phenylethanol transformation in a two-phase system suggests that ethanol utilization pathway could be usefully coupled to a biotransformation reaction for cofactor regeneration. Considering that successful coupling of ethanol utilization with biotransformation requires both reactions to be similar in speed, the results bring confidence that ethanol utilization pathway may find a useful niche in biocatalysis. Finally, lack of substrate toxicity effect from acetophenone in the coupled biotransformation system with cofactor regeneration validates the utility of the second organic phase in storing hydrophobic acetophenone and protecting E. coli whole cells. Collectively, ethanol utilization pathway holds potential as a high efficiency NADH regeneration system for whole-cell biocatalysis. This is validated by experimental results indicating successful coupling of ethanol utilization pathway with biotransformation reaction in a two-phase setup.