文献类型：期刊文献

英文题名：Engineering Bacillus subtilis Isoleucine Dioxygenase for Efficient Synthesis of (2S,3R,4S)-4-Hydroxyisoleucine

作者：Du, Ping[1];Yan, Shuai[2];Qian, Xiao-Long[1];Pan, Jiang[1];Zhang, Zhi-Jun[1];Yu, Hui-Lei[1];Xu, Jian-He[1]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, Sch Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai Jiao Tong Univ, Dept Endocrinol & Metab, Shanghai Gen Hosp, Shanghai 200080, Peoples R China

年份：2020

卷号：68

期号：49

起止页码：14555

外文期刊名：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

收录：;EI(收录号：20205109663215);WOS:【SCI-EXPANDED(收录号:WOS:000599505900026)】；

基金：This work was financially supported by the National Key Research and Development Program of China (2019YFA0905000), the National Natural Science Foundation of China (32071475, 21922804, 21672063, and 21536004), the Natural Science Foundation of Shanghai (no. 18ZR1408400), and the Fundamental Research Funds for the Central Universities (22221818014).

语种：英文

外文关键词：biocatalysis; isoleucine dioxygenase; directed evolution; (2S,3R,4S)-4-hydroxyisoleucine; space-time yield

摘要：Isoleucine dioxygenase (IDO)-catalyzed hydroxylation of isoleucine is a promising method for the synthesis of the diabetic drug (2S,3R,4S)-4-hydroxyisoleucine [(2S,3R,4S)-4-HIL]. However, the low activity of IDO significantly limits its practical application. In this work, a high-throughput screening method was developed and directed evolution was performed on the IDO from Bacillus subtilis, resulting in a double mutant with improvements in specific activity, protein expression level, and fermentation titer of 3.2-, 2.8-, and 9.4-fold, respectively. L-Isoleucine (228 mM) was completely converted to (2S,3R,4S)-4-HIL by the best variant with a space-time yield of up to 80.8 g L-1 d(-1), which is the highest record reported so far. With a further increase of the substrate loading to 1 M, a high conversion of 91% could also be achieved. At last, enzymatic synthesis of (2S,3R,4S)-4-HIL was successfully carried out on a 3 L scale, indicating tremendous potential of the IDO variant I162T/T182N for green and efficient production of (2S,3R,4S)-4-HIL.