Sunday, July 26, 2009
P29

Production of ethyl (S)-4-chloro-3-hydroxybutanoate by Saccharomyces cerevisiae reductase coupled with regeneration of NADPH

Jihye Jung, Yun Hee Choi, and Hyung Kwoun Kim. Department of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon, South Korea

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is used as building block for the synthesis of Atorvastatin, a chiral drug for hypercholesterolemia acting as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. In this study, 13 different microbial reductases have been cloned and expressed in Escherichia coli. Then their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed using a spectrophotometer. Amidst them, No. 2, 3, and 8 reductase (Baker’s yeast YDL124W, YOR120W, and YOL151W, respectively) showed the highest activity for ECOB. They displayed higher activity with NADPH than NADH. Among them, only No. 2 reductase produced (S)-ECHB exclusively with an ee value of 100%, whereas No. 3 and 8 reductases made (R)-form alcohol with an ee value of 100%. The recombinant YDL124W reductase was then purified using Ni-NTA column chromatography and desalting column chromatography. The purified reductase displayed optimum temperature and pH of 45°C and pH 6.5, respectively. It was rather stable up to 25°C and pH 5-9 for 30 min. To perform continuously the reduction of the substrate, a glucose dehydrogenase-coupling reaction was introduced as a NADPH regeneration system. In addition, the effects of substrate/product inhibition, solution’s pH change, and organic solvent concentration on the coupling reaction were studied to optimize the reaction conditions. These results demonstrated that in conjunction with a glucose dehydrogenase-coupling reaction the recombinant yeast YDL124W reductase could be used to produce (S)-ECHB.

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