Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.

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 caus...

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Autores principales:	Rongzhen Zhang, Botao Zhang, Yan Xu, Yaohui Li, Ming Li, Hongbo Liang, Rong Xiao
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/35cf5e245ba14e7393292cf3bc9e3e9d
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spelling	oai:doaj.org-article:35cf5e245ba14e7393292cf3bc9e3e9d2021-11-18T08:41:34ZEfficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.1932-620310.1371/journal.pone.0083586https://doaj.org/article/35cf5e245ba14e7393292cf3bc9e3e9d2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24358299/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Rongzhen ZhangBotao ZhangYan XuYaohui LiMing LiHongbo LiangRong XiaoPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 12, p e83586 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rongzhen Zhang Botao Zhang Yan Xu Yaohui Li Ming Li Hongbo Liang Rong Xiao Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
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.
format	article
author	Rongzhen Zhang Botao Zhang Yan Xu Yaohui Li Ming Li Hongbo Liang Rong Xiao
author_facet	Rongzhen Zhang Botao Zhang Yan Xu Yaohui Li Ming Li Hongbo Liang Rong Xiao
author_sort	Rongzhen Zhang
title	Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
title_short	Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
title_full	Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
title_fullStr	Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
title_full_unstemmed	Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
title_sort	efficicent (r)-phenylethanol production with enantioselectivity-alerted (s)-carbonyl reductase ii and nadph regeneration.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/35cf5e245ba14e7393292cf3bc9e3e9d
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Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.

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