A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.

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We have developed efficient biocatalytic processes for the preparation of chiral alcohols, such as (R)-1,3-butanediol, ethyl (S)-4-chloro-3-hyroxybutanoate, ethyl (R)-4-chloro-3-hyroxybutanoate, (S)-5-chloro-2-pentanol, (R)-5-chloro-2-pentanol, and (S)-cyclopropylethanol by stereospecific enzymatic oxidoreduction on a practical level. These chiral alcohols are very important synthons for the synthesis of various pharmaceutical intermediates that lead to antibiotics and inhibitors of HMGCoA reductase. Here, we present practical applications on biocatalysis using novel recombinant whole-cell biocatalysts that catalyzed enantioselective oxidation and asymmetric reduction with a coenzyme regeneration system.

This paper is a report on the successful application of bioinformatics to enzyme screening. The synthesis of ethyl ( S)-4-chloro-3-hydroxybutanoate (ECHB) by asymmetric reduction of ethyl 4-chloroacetoacetate (ECAA) using fabG-homologues was studied. beta-Ketoacyl-acyl carrier protein reductases from both Escherichia coli and Bacillus subtilis, which are components of type II fatty acid synthase, could reduce ECAA to ( S)-ECHB with 94-98% ee. Furthermore, acetoacetyl-CoA reductases (ARs) from both Ralstonia eutropha and Zoogloea ramigera, whose genes are significantly similar to fabG genes and play a physiological role in the biosynthesis of poly-beta-3-hydroxybutyrate, could also catalyze the asymmetric reduction of ECAA to ( S)-ECHB with >99% ee. ( S)-ECHB was synthesized to 48.7 g/l with an optical purity of 99.8% ee, using recombinant E. coli cells coexpressing AR from R. eutropha and glucose dehydrogenase from B. subtilis for the regeneration of NADPH.