Aims We aimed to explore Yarrowia lipolytica carbonyl reductases as effective biocatalysts and to develop efficient asymmetric reduction systems for chiral alcohol synthesis. Methods and Results Yarrowia lipolytica carbonyl reductase genes were obtained via homologous sequence amplification strategy. Two carbonyl reductases, YaCRI and YaCRII, were identified and characterized, and used to catalyse the conversion of 2-hydroxyacetophenone (2-HAP) to optically pure (S)-1-phenyl-1,2-ethanediol. Enzymatic assays revealed that YaCRI and YaCRII exhibited specific activities of 6 center dot 96 U mg(-1) (99 center dot 8% e.e.) and 7 center dot 85 U mg(-1) (99 center dot 9% e.e.), respectively, and showed moderate heat resistance at 40-50 degrees C and acid tolerance at pH 5 center dot 0-6 center dot 0. An efficient whole-cell two-phase system was established using reductase-expressing recombinant Escherichia coli. The conversion of 2-HAP (20 center dot 0 g l(-1)) conversion with the solvent of dibutyl phthalate was approximately 70-fold higher than in water. Furthermore, the two recombinant E. coli displayed biocatalyst activity and enantioselectivity towards several different carbonyl compounds, and E. coli BL21 (DE3)/pET-28a-yacrII showed a broad substrate spectrum. Conclusions A new whole-cell recombinant E. coli-based bioreduction system for enantiopure alcohol synthesis with high enantioselectivity at high substrate concentrations was developed. Significance and Impact of the Study We proposed a promising approach for the efficient preparation of enantiopure chiral alcohols.