文献类型：期刊文献

英文题名：Multilevel cytochrome P450 engineering integrating protein language model-guided evolution enables de novo biosynthesis of amentoflavone in Escherichia coli

作者：Dai, Xuehui[1,2,3];Zhu, Jiangming[1,2,3];Wu, Zheyu[2,3];Wang, Guangyi[2,3];Zhang, Yanchen[2,3];Tian, Chenfei[2];Ren, Yuhong[1];Liu, Haili[1,2];Wang, Yong[1,2,3]

机构：[1]East China Univ Sci & Technol, Sch Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Inst Plant Physiol & Ecol, CAS Ctr Excellence Mol Plant Sci, Lab Synthet Biol,State Key Lab Plant Trait Design, Shanghai 200032, Peoples R China;[3]Univ Chinese Acad Sci, Beijing 100039, Peoples R China

年份：2026

卷号：456

外文期刊名：BIORESOURCE TECHNOLOGY

收录：;EI(收录号：20262120777945);WOS:【SCI-EXPANDED(收录号:WOS:001781585500001)】；

基金：This work is financially supported by the National Key R&D Program of China (No. 2024YFA0919900), the National Natural Science Foundation of China (No. 32571708), the State Key Laboratory of Plant Trait Design, the Key Laboratory of Plant Carbon Capture, Chinese Academy of Sciences, and the Shanghai Municipal Science and Technology Major Project.

语种：英文

外文关键词：Amentoflavone; Cytochrome P450; Protein language model; Metabolic engineering; Biflavonoid biosynthesis

摘要：Amentoflavone-type biflavonoids exhibit potent neuroprotective activities but are limited by scarce natural abundance and inefficient chemical synthesis routes. Here, we establish a stable Escherichia coli platform for de novo amentoflavone biosynthesis through multilevel engineering of a cytochrome P450-dependent oxidative coupling module. Systematic optimization of redox partner pairing, N-terminal expression regulation, and intrinsic catalytic activity substantially enhances the performance of the key P450 enzyme GbCYP90J6. Protein language model-guided directed evolution identifies high-activity variants that, when combined with an optimized expression tag, markedly improve in vivo catalysis. Integration of precursor flux balancing, expression optimization, and enzyme evolution results in an overall 8.34-fold increase in de novo amentoflavone production, reaching 6.59 mg/L in shake-flask cultivation. Fed-batch fermentation in a 5-L bioreactor increases the amentoflavone titer to 22.01 mg/L, demonstrating pathway scalability while revealing cellular tolerance as a remaining bottleneck. Together, this study establishes a framework for engineering P450-dependent intermolecular C-C coupling and highlights the necessity of balancing catalytic efficiency with cellular robustness in microbial cell factories.