文献类型：期刊文献

英文题名：Development of an Engineered Ketoreductase with Simultaneously Improved Thermostability and Activity for Making a Bulky Atorvastatin Precursor

作者：Gong, Xu-Min[1,2];Qin, Zhen[1,3];Li, Fu-Long[1,2];Zeng, Bu-Bing[4];Zheng, Gao-Wei[1,2];Xu, Jian-He[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, Shanghai 200237, Peoples R China;[3]East China Univ Sci & Technol, R&D Ctr Separat & Extract Technol Fermentat Ind, Shanghai 200237, Peoples R China;[4]East China Univ Sci & Technol, Shanghai Key Lab New Drug Design, Shanghai 200237, Peoples R China

年份：2019

卷号：9

期号：1

起止页码：147

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20190206361877);WOS:【SCI-EXPANDED(收录号:WOS:000455286600015)】；

基金：This work was financially supported by the National Natural Science Foundation of China (grant nos. 21472045, 21878085, and 21536004) and the Fundamental Research Funds for the Central Universities (22221818014).

语种：英文

外文关键词：biocatalysis; asymmetric reduction; ketoreductase; protein engineering; thermostabilization; atorvastatin precursor

摘要：Protein engineering is a powerful strategy for enhancing the properties of enzymes for industrial applications. However, thermostabilizing an enzyme via this strategy while simultaneously improving its activity is challenging due to the well-known stability-activity trade-off. Herein, using native ketoreductase LbCR, thermostability and activity were evolved separately by directed evolution, generating mutations V198I and M154I/A155D with increased thermostability and mutations A201D/A202L with increased enzymatic activity. On the basis of additivity and cooperative mutational effects, variants LbCR(M6) (M154I/A155D/A201D/A202L) and LbCR(M8) (M154I/A155D/V198I/A201D/A202L) with simultaneously improved thermostability and activity were subsequently constructed by combining mutations. Analysis of variant structures demonstrated that increased thermostability was largely attributed to rigidification of flexible loops around the active site through the formation of additional hydrogen bonds and hydrophobic interactions. The best variant LbCR(M8) displayed a 1944-fold increase in half-life at 40 degrees C and a 3.2-fold improvement in catalytic efficiency compared with the wide-type enzyme. Using only 1 g L-1 of lyophilized E. coli cells coexpressing this LbCR(M8) and glucose dehydrogenase BmGDH as a catalyst, t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate up to 300 g L-1 loading was completely reduced within 6 h at 40 degrees C, yielding the corresponding t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate (ATS-7) with >99.5% de and a space-time yield of up to 1.05 kg L-1 day(-1). These results demonstrated that LbCR(M8) is an attractive biocatalyst for the synthesis of ATS-7, an advanced chiral intermediate for the production of the cholesterol-lowering drug atorvastatin.