[Efficient biosynthesis of (S)-1-phenyl-1,2-ethanediol catalyzed by (S)-carbonyl reductase Ⅱ and glucose dehydrogenase].

OBJECTIVE

To realize efficient biosynthesis of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol, we designed a co-expression system containing Candida parapsilosis CCTCC M203011 (S)-carbonyl reductase Ⅱ (SCRⅡ) and Bacillus sp. YX-1 glucose dehydrogenase (GDH) in Escherichia coli BL21(DE3), based on the optimal ratio between the specific activities of the two enzymes.

METHODS

The enzymes SCRⅡ and GDH were purified from their corresponding recombinant E. coli strains. When the purified SCRⅡ and GDH were used for the reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol, the optimal ratio between their specific activities, the optimal temperature and pH were determined. Based on above results, a co-expression system E. coli BL21(DE3)/S-SD-AS-G harboring SCRⅡ and GDH was constructed.

RESULTS

SCRⅡ and GDH exhibited specific activities of 1.3 U/mg and 13.5 U/mg. When the total enzyme activity was 1 U, the optimal ratio of their activities is between 1:1 and 5:1, and the optimal temperature and pH are 30℃ and 7.0, respectively. So we designed a co-expression system E. coli BL21/S-SD-AS-G, in which the ratio of the SCRⅡ and GDH genes is 1:1. The specific activities of SCRⅡ and GDH are 0.76 U/mg and 0.73 U/mg in the cell-free extracts of E. coli BL21(DE3)/S-SD-AS-G, respectively. The ratio between SCRⅡ and GDH activity is 1:1. Under the optimal conditions, the system showed excellent performance to produce (S)-1-phenyl-1,2-ethanediol with an optical purity and a yield both over 99% during the reduction of 2-hydroxyacetophenone. With respect to the recombinant E. coli BL21(DE3)/pET-SCRⅡ, the co-expression system obviously improved the yield of (S)-1-phenyl-1,2-ethanediol and reduced biotransformation time from 24 h to 13 h.

CONCLUSION

This work provides the research foundation on the construction of a co-expression system containing a target chiral catalyst and a cofactor-regeneration enzyme for efficient chiral biosynthesis based on the optimal ratio of SCRⅡ and GDH activities.