Efficicent (R)-Phenylethanol Production with Enantioselectivity-Alerted (S)-Carbonyl Reductase II and NADPH Regeneration

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	Efficicent (R)-Phenylethanol Production with Enantioselectivity-Alerted (S)-Carbonyl Reductase II and NADPH Regeneration
	Zhang, Rongzhen 1,2,3; Zhang, Botao 1,2,4; Xu, Yan 1,2,3; Li, Yaohui 1,2,3; Li, Ming 1,2,3; Liang, Hongbo 1,2,3; Xiao, Rong 5
刊名	PLOS ONE
	2013-12-17
卷号	8 期号:12
英文摘要	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.
WOS标题词	Science & Technology
类目[WOS]	Multidisciplinary Sciences
研究领域[WOS]	Science & Technology - Other Topics
关键词[WOS]	ANTI-PRELOG STEREOSPECIFICITY ; GLUCOSE-DEHYDROGENASE GENES ; ESCHERICHIA-COLI ; CARBONYL REDUCTASE ; CANDIDA-PARAPSILOSIS ; ETHYL (S)-4-CHLORO-3-HYDROXYBUTANOATE ; TRANSLATION INITIATION ; COFACTOR REGENERATION ; CYTOCHROME-P450 2D6 ; EXPRESSION
收录类别	SCI
语种	英语
WOS记录号	WOS:000328737700063
内容类型	期刊论文
源URL	[http://124.16.173.210/handle/834782/1277]
专题	天津工业生物技术研究所_基因工程与微生物应用技术研究组宋恢_期刊论文
作者单位	1.Jiangnan Univ, Minist Educ, Key Lab Ind Biotechnol, Wuxi, Peoples R China 2.Jiangnan Univ, Sch Biotechnol, Wuxi, Peoples R China 3.Jiangnan Univ, Natl Key Lab Food Sci, Wuxi, Peoples R China 4.Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Tianjin, Peoples R China 5.Rutgers State Univ, Ctr Adv Biotechnol & Med, Piscataway, NJ 08854 USA
推荐引用方式 GB/T 7714	Zhang, Rongzhen,Zhang, Botao,Xu, Yan,et al. Efficicent (R)-Phenylethanol Production with Enantioselectivity-Alerted (S)-Carbonyl Reductase II and NADPH Regeneration[J]. PLOS ONE,2013,8(12).
APA	Zhang, Rongzhen.,Zhang, Botao.,Xu, Yan.,Li, Yaohui.,Li, Ming.,...&Xiao, Rong.(2013).Efficicent (R)-Phenylethanol Production with Enantioselectivity-Alerted (S)-Carbonyl Reductase II and NADPH Regeneration.PLOS ONE,8(12).
MLA	Zhang, Rongzhen,et al."Efficicent (R)-Phenylethanol Production with Enantioselectivity-Alerted (S)-Carbonyl Reductase II and NADPH Regeneration".PLOS ONE 8.12(2013).