Highly stereoselective reagents for beta-keto ester reductions by genetic engineering of baker's yeast

While whole cells of baker's yeast (Saccharomyces cerevisiae) are a conveni ent biocatalytic reducing agent for a wide variety of carbonyl compounds, m ixtures of stereoisomeric alcohols are often observed since the organism co ntains a large number of reductase enzymes with overlapping substrate speci ficities but differing stereoselectivities. We sought to improve the perfor mance of baker's yeast for beta -keto ester reductions by using recombinant DNA techniques to alter the levels of three enzymes known to play importan t roles in these reactions (fatty acid synthase, Fasp; aldo-keto reductase, Ypr1p; alpha -acetoxy ketone reductase, Gre2p). A complete set of "first-g eneration" yeast strains that either lack or overexpress each of these thre e enzymes was created and tested for improvements in stereoselective reduct ions of a series of beta -keto esters. On the basis of these results, multi ply modified ("second-generation") strains were created that combined gene knockout and overexpression in single strains. In some cases, these additio nal modifications further improved the stereoselectivities of beta -keto es ter reductions, thereby making several beta -hydroxy ester building blocks readily available by reactions that can be performed by nonspecialists. Thi s work also revealed that additional yeast proteins participate in reducing beta -keto esters, and further progress using this strategy will require e ither additional genetic manipulations or the expression of yeast reductase s in hosts that lack enzymes with overlapping substrate specificity.