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Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
PLoS One. 2013; 8(12):e83586.Plos

Abstract

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.

Authors+Show Affiliations

Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi, P. R. China ; National Key Laboratory for Food Science, Jiangnan University, Wuxi, P. R. China.Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi, P. R. China ; Tianjin Institute of Industrial Biotechnology, The Chinese Academy of Sciences, Tianjin, P. R. China.Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi, P. R. China ; National Key Laboratory for Food Science, Jiangnan University, Wuxi, P. R. China.Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi, P. R. China ; National Key Laboratory for Food Science, Jiangnan University, Wuxi, P. R. China.Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi, P. R. China ; National Key Laboratory for Food Science, Jiangnan University, Wuxi, P. R. China.Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi, P. R. China ; National Key Laboratory for Food Science, Jiangnan University, Wuxi, P. R. China.Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, United States of America.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

24358299

Citation

Zhang, Rongzhen, et al. "Efficicent (R)-phenylethanol Production With Enantioselectivity-alerted (S)-carbonyl Reductase II and NADPH Regeneration." PloS One, vol. 8, no. 12, 2013, pp. e83586.
Zhang R, Zhang B, Xu Y, et al. Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration. PLoS ONE. 2013;8(12):e83586.
Zhang, R., Zhang, B., Xu, Y., Li, Y., Li, M., Liang, H., & Xiao, R. (2013). Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration. PloS One, 8(12), e83586. https://doi.org/10.1371/journal.pone.0083586
Zhang R, et al. Efficicent (R)-phenylethanol Production With Enantioselectivity-alerted (S)-carbonyl Reductase II and NADPH Regeneration. PLoS ONE. 2013;8(12):e83586. PubMed PMID: 24358299.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration. AU - Zhang,Rongzhen, AU - Zhang,Botao, AU - Xu,Yan, AU - Li,Yaohui, AU - Li,Ming, AU - Liang,Hongbo, AU - Xiao,Rong, Y1 - 2013/12/17/ PY - 2013/09/16/received PY - 2013/11/13/accepted PY - 2013/12/21/entrez PY - 2013/12/21/pubmed PY - 2014/10/22/medline SP - e83586 EP - e83586 JF - PloS one JO - PLoS ONE VL - 8 IS - 12 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/24358299/Efficicent__R__phenylethanol_production_with_enantioselectivity_alerted__S__carbonyl_reductase_II_and_NADPH_regeneration_ L2 - http://dx.plos.org/10.1371/journal.pone.0083586 DB - PRIME DP - Unbound Medicine ER -