Integration of enzyme, strain and reaction engineering to overcome limitations of baker's yeast in the asymmetric reduction of alpha-keto esters.
We report on the development of a whole-cell biocatalytic system based on the popular host Saccharomyces cerevisiae that shows programmable performance and good atom economy in the reduction of alpha-keto ester substrates. The NADPH-dependent yeast reductase background was suppressed through the combined effects of overexpression of a biosynthetic NADH-active reductase (xylose reductase from Candida tenuis) to the highest possible level and the use of anaerobic reaction conditions in the presence of an ethanol co-substrate where mainly NADH is recycled. The presented multi-level engineering approach leads to significant improvements in product optical purity along with increases in the efficiency of alpha-keto ester reduction and co-substrate yield (molar ratio of formed alpha-hydroxy ester to consumed ethanol). The corresponding alpha-hydroxy esters were obtained in useful yields (>50%) with purities of > or =99.4% enantiomeric excess. The obtained co-substrate yield reached values of greater than 1.0 with acetate as the only by-product formed.
Kratzer
Regina
R
Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria.
Egger
Sigrid
S
Nidetzky
Bernd
B
D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism; D000693:Anaerobiosis; D002384:Catalysis; D004952:Esters / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D024861:Genetic Enhancement / Q000379:methods*; D007651:Keto Acids / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D004952:Esters; D007651:Keto Acids; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D000431:Ethanol; D000449:Aldehyde Reductase