文献类型：期刊文献

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

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

机构：[1]East China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2022

卷号：9

期号：1

外文期刊名：BIORESOURCES AND BIOPROCESSING

收录：;EI(收录号：20224513084501);WOS:【SCI-EXPANDED(收录号:WOS:000817300600001)】；

基金：This study was 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), and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

语种：英文

外文关键词：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 pi-pi stacking interactions stabilization. Finally, an engineered E. coli strain 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.