文献类型：期刊文献

中文题名：Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin

作者：Bing-Yao Sun[1];Hua-Lu Sui[1];Zi-Wei Liu[1];Xin-Yi Tao[1];Bei Gao[1];Ming Zhao[1];Yu-Shu Ma[1];Jian Zhao[1];Min Liu[1];Feng-Qing Wang[1];Dong-Zhi Wei[1]

机构：[1]State Key Lab of Bioreactor Engineering,Newworld Institute of Biotechnology,East China University of Science and Technology,Shanghai 200237,China

年份：2022

卷号：9

期号：1

起止页码：811

中文期刊名：Bioresources and Bioprocessing

外文期刊名：生物资源与生物加工(英文)

基金：supported by National Key Research and Development Program of China(No.SQ2020YFC210061);the National Natural Science Foundation of China(No.31500043);the Natural Science Foundation of Shanghai(No.21ZR1417200);the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

语种：英文

中文关键词：Flavin-containing monooxygenase;Indirubin;Loop region;Substrate tunnel;Structure-guided enzyme engineering;Microbial synthesis

摘要：Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia.Here,we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase(bFMO)for the efficient production of indirubin in Escherichia coli.A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity,resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5-and 2.0-fold,respectively.A combined modification at the two regions(K223R/D317S)achieved a 6.6-fold improvement in catalytic efficiency(k_(cat)/K_(m))due to enhancing π-π stacking interactions stabilization.Finally,an engineered E.colistrain was constructed by metabolic engineering,which could produce 860.7 mg/L(18 mg/L/h)indirubin,the highest yield ever reported.This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin.