Dataset Information

A novel carbonyl reductase with anti-Prelog stereospecificity for the production of t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate.

ABSTRACT: A novel gene (crc1) from Candida boidinii was cloned and then overexpressed in a recombinant strain BL21(DE3)/pET30a-crc1 of Escherichia coli. The resulting carbonyl reductase was prepared through fermentations using the recombinant strain. The purified enzyme showed an NADPH-dependent activity and specific activity was 4.65 U/mg using t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (ATS-6) as substrate. The enzyme was optimally active at 35 °C and pH 7, respectively. The apparent K m and V max of the enzyme for ATS-6 are 1.5 mM and 21.1 ?mol/min mg, respectively, indicating excellent anti-Prelog stereospecificity. Under the optimum condition, t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate (ATS-7) was prepared with the enzyme with high d.e. value (99.9%) and good conversion (94%) in 4 h, indicating high stereoselectivity and conversion efficiency in biotransformation of ATS-6 to ATS-7.

SUBMITTER: Jin Q

PROVIDER: S-EPMC6497681 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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A novel carbonyl reductase with anti-Prelog stereospecificity for the production of t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate.

Jin Qingchao Q Wu Zhige Z Dou Yanping Y Yang Yu Y Xia Jingjing J Jin Zhihua Z

3 Biotech 20190502 5

A novel gene (crc1) from Candida boidinii was cloned and then overexpressed in a recombinant strain BL21(DE3)/pET30a-crc1 of Escherichia coli. The resulting carbonyl reductase was prepared through fermentations using the recombinant strain. The purified enzyme showed an NADPH-dependent activity and specific activity was 4.65 U/mg using t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (ATS-6) as substrate. The enzyme was optimally active at 35 °C and pH 7, respective ...[more]

PMID: 31065494

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Project description:The NADPH-dependent (S)-carbonyl reductaseII from Candida parapsilosis catalyzes acetophenone to chiral phenylethanol in a very low yield of 3.2%. Site-directed mutagenesis was used to design two mutants Ala220Asp and Glu228Ser, inside or adjacent to the substrate-binding pocket. Both mutations caused a significant enantioselectivity shift toward (R)-phenylethanol in the reduction of acetophenone. The variant E228S produced (R)-phenylethanol with an optical purity above 99%, in 80.2% yield. The E228S mutation resulted in a 4.6-fold decrease in the K M value, but nearly 5-fold and 21-fold increases in the k cat and k cat/K M values with respect to the wild type. For NADPH regeneration, Bacillus sp. YX-1 glucose dehydrogenase was introduced into the (R)-phenylethanol pathway. A coexpression system containing E228S and glucose dehydrogenase was constructed. The system was optimized by altering the coding gene order on the plasmid and using the Shine-Dalgarno sequence and the aligned spacing sequence as a linker between them. The presence of glucose dehydrogenase increased the NADPH concentration slightly and decreased NADP(+) pool 2- to 4-fold; the NADPH/NADP(+) ratio was improved 2- to 5-fold. The recombinant Escherichia coli/pET-MS-SD-AS-G, with E228S located upstream and glucose dehydrogenase downstream, showed excellent performance, giving (R)-phenylethanol of an optical purity of 99.5 % in 92.2% yield in 12 h in the absence of an external cofactor. When 0.06 mM NADP(+) was added at the beginning of the reaction, the reaction duration was reduced to 1 h. Optimization of the coexpression system stimulated an over 30-fold increase in the yield of (R)-phenylethanol, and simultaneously reduced the reaction time 48-fold compared with the wild-type enzyme. This report describes possible mechanisms for alteration of the enantiopreferences of carbonyl reductases by site mutation, and cofactor rebalancing pathways for efficient chiral alcohols production.

Dataset Information

A novel carbonyl reductase with anti-Prelog stereospecificity for the production of t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate.

Publications

A novel carbonyl reductase with anti-Prelog stereospecificity for the production of <i>t</i>-butyl 6-cyano-(3<i>R</i>, 5<i>R</i>)-dihydroxyhexanoate.

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