文献类型：期刊文献

英文题名：Rational Design of Taxadiene Hydroxylase by Ancestral Enzyme Construction and the Elucidation of Key Amino Acids

作者：Zhang, Zihan[1];Huang, Longhao[1];Zhang, Zhi-Jun[1];Xu, Jian-He[1];Yu, Hui-Lei[1]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2023

卷号：62

期号：22

起止页码：3214

外文期刊名：BIOCHEMISTRY

收录：;EI(收录号：20234815110435);WOS:【SCI-EXPANDED(收录号:WOS:001096870800001)】；

基金：The work was financially supported by the National Natural Science Foundation of China (no. 32271540), the National Key Research and Development Program of China (grant 2019YFA0905000), Program of Shanghai Academic Research Leader (21XD1400800), and the Fundamental Research Funds for the Central Universities (grant no. 22221818014).

语种：英文

外文关键词：Amino acids - Biochemistry - Enzymes

摘要：Cytochrome P450 monooxygenases (CYP450s) play an important role in the biosynthesis of natural products by activating inert C-H bonds and inserting hydroxyl groups. However, the activities of most plant-derived CYP450s are extremely low, limiting the heterologous biosynthesis of natural products. Traditional enzyme engineering methods, either rational or screening-based, are not suitable for CYP450s because of the lack of crystal structures and high-throughput screening methods for this class of enzymes. CYP725A4 is the first hydroxylase involved in the biosynthesis pathway of Taxol. Its low activity, promiscuity, and multispecificity make it a bottleneck in Taxol biosynthesis. Here, we identified key amino acids that affect the in vivo activity of CYP725A4 by constructing the ancestral enzymes of CYP725A4. We obtained positive mutants that showed an improved yield of hydroxylated products based on the key amino acids identified, providing guidance for the modification of other CYP450s involved in the biosynthesis of natural products.