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 convenient biocatalytic reducing agent for a wide variety of carbonyl compounds, mixtures of stereoisomeric alcohols are often observed since the organism contains a large number of reductase enzymes with overlapping substrate specificities but differing stereoselectivities. We sought to improve the performance of baker's yeast for beta-keto ester reductions by using recombinant DNA techniques to alter the levels of three enzymes known to play important roles in these reactions (fatty acid synthase, Fasp; aldo-keto reductase, Ypr1p; alpha-acetoxy ketone reductase, Gre2p). A complete set of "first-generation" yeast strains that either lack or overexpress each of these three enzymes was created and tested for improvements in stereoselective reductions of a series of beta-keto esters. On the basis of these results, multiply modified ("second-generation") strains were created that combined gene knockout and overexpression in single strains. In some cases, these additional modifications further improved the stereoselectivities of beta-keto ester reductions, thereby making several beta-hydroxy ester building blocks readily available by reactions that can be performed by nonspecialists. This work also revealed that additional yeast proteins participate in reducing beta-keto esters, and further progress using this strategy will require either additional genetic manipulations or the expression of yeast reductases in hosts that lack enzymes with overlapping substrate specificity.
Rodríguez
S
S
Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
Kayser
M M
MM
Stewart
J D
JD
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D029721:Drosophila Proteins; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000502:physiology
D029721:Drosophila Proteins; D009124:Muscle Proteins; C109229:ko protein, Drosophila; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D064429:Fatty Acid Synthases