The number for organic syntheses in which enzymes are used as catalysts is increasing rapidly, due to their superior stereo-and regio-specificity under mild pH and temperature conditions ¹.  Coupling cascading enzyme reactions for the conversion of low value renewable feedstocks into high value products represents a keystone of renewable green chemistry. However one of the main limitations to the application of such enzyme systems to energy-intensive redox reactions is the cost of providing a continuous supply of diffusible cofactors or cosubstrates ². Thus there is an emerging requirement to develop successful multi-enzyme bioredox cascades using heterogenous enzyme catalysts coupled with cell-free cofactor regeneration ³.

To this end, we identified twenty two enzymes that could, in principle, be used in a multi-enzyme bio-redox cascade to produce dihydroxyacetone phosphate (DHAP) from glycerol via an intermediate (glycerol-3-phosphate) and at the expense of the cofactors ATP and NAD(P), which are converted to ADP and NAD(P)H, respectively. The enzymes were expressed in E. coli to assess ease of production, and on this criterion twelve were selected for further characterization. From the kinetic properties of the twelve ‘short-listed’ enzymes two were identified that were suitable for each of the following functions: ATP-dependent production of glycerol-3-phosphate from glycerol, NAD(P)-dependent production of DHAP from glycerol-3-phosphate, oxygen-dependent recycling of NAD(P) from NAD(P)H, and acetyl phosphate-dependent recycling of ATP from ADP. Cascades of these enzymes that produced DHAP from glycerol were demonstrated in batch reactors. In addition, we illustrated that stereospecific aldolase enzymes could be coupled with the DHAP produced from the multi-enzyme bioredox casacade to produce a variety of chiral sugars with potential as feedstocks for fine chemical and pharmaceutical industry applications.