title: In situ expression of (R)-carbonyl reductase rebalancing an asymmetric pathway improves stereoconversion efficiency of racemic mixture to (S)-phenyl-1,2-ethanediol in Candida parapsilosis CCTCC M203011.
abstract: BACKGROUND
  Candida parapsilosis (R)-carbonyl reductase (RCR) and (S)-carbonyl reductase (SCR) are involved in the stereoconversion of racemic (R,S)-1-phenyl-1,2-ethanediol (PED) to its (S)-isomer. RCR catalyzes (R)-PED to 2-hydroxyacetophenone (2-HAP), and SCR catalyzes 2-HAP to (S)-PED. However, the stereoconversion efficiency of racemic mixture to (S)-PED is not high because of an activity imbalance between RCR and SCR, with RCR performing at a lower rate than SCR. To realize the efficient preparation of racemic mixture to (S)-PED, an in situ expression of RCR and a two-stage control strategy were introduced to rebalance the RCR- and SCR-mediated pathways.
  
  
  RESULTS
  An in situ expression plasmid pCP was designed and RCR was successfully expressed in C. parapsilosis. With respect to wild-type, recombinant C. parapsilosis/pCP-RCR exhibited over four-fold higher activity for catalyzing racemic (R,S)-PED to 2-HAP, while maintained the activity for catalyzing 2-HAP to (S)-PED. The ratio of k cat /K M for SCR catalyzing (R)-PED and RCR catalyzing 2-HAP was about 1.0, showing the good balance between the functions of SCR and RCR. Based on pH and temperature preferences of RCR and SCR, a two-stage control strategy was devised, where pH and temperature were initially set at 5.0 and 30 °C for RCR rapidly catalyzing racemic PED to 2-HAP, and then adjusted to 4.5 and 35 °C for SCR transforming 2-HAP to (S)-PED. Using these strategies, the recombinant C. parapsilosis/pCP-RCR catalyzed racemic PED to its (S)-isomer with an optical purity of 98.8 % and a yield of 48.4 %. Most notably, the biotransformation duration was reduced from 48 to 13 h.
  
  
  CONCLUSIONS
  We established an in situ expression system for RCR in C. parapsilosis to rebalance the functions between RCR and SCR. Then we designed a two-stage control strategy based on pH and temperature preferences of RCR and SCR, better rebalancing RCR and SCR-mediated chiral biosynthesis pathways. This work demonstrates a method to improve chiral biosyntheses via in situ expression of rate-limiting enzyme and a multi-stage control strategy to rebalance asymmetric pathways.
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China. rzzhang@jiangnan.edu.cn.|National Key Laboratory for Food Science, Jiangnan University, Wuxi, 214122, People's Republic of China. rzzhang@jiangnan.edu.cn.|School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, People's Republic of China. rzzhang@jiangnan.edu.cn.
authors.forename: Rongzhen
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China. yxu@jiangnan.edu.cn.|National Key Laboratory for Food Science, Jiangnan University, Wuxi, 214122, People's Republic of China. yxu@jiangnan.edu.cn.|School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, People's Republic of China. yxu@jiangnan.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Liang
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Yaohui
authors.initials: Y
authors.lastname: Li
authors.affiliation: Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ, 08854, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People's Republic of China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Ni
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002175:Candida / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019855:Ethylene Glycol / Q000378:metabolism; D005026:Ethylene Glycols / Q000737:chemistry* / Q000378:metabolism*; D015870:Gene Expression; D053858:Metabolic Networks and Pathways / Q000235:genetics
chemical_list: D005026:Ethylene Glycols; C031474:styrene glycol; D000429:Alcohol Oxidoreductases; D000449:Aldehyde Reductase; D019855:Ethylene Glycol
keywords: (R)-carbonyl reductase; (S)-1-phenyl-1,2-ethanediol; Candida parapsilosis; In situ expression; Racemic 1-phenyl-1,2-ethanediol; Stereoconversion