title: Efficicent (R)-phenylethanol production with enantioselectivity-alerted (S)-carbonyl reductase II and NADPH regeneration.
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.affiliation: 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.
authors.forename: Rongzhen
authors.initials: R
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Botao
authors.initials: B
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: 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.
authors.forename: Yaohui
authors.initials: Y
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Liang
authors.affiliation: Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, United States of America.
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D019943:Amino Acid Substitution; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D010626:Phenylethyl Alcohol / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary / Q000235:genetics; D013237:Stereoisomerism
chemical_list: D009249:NADP; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D010626:Phenylethyl Alcohol