文献类型：期刊文献

英文题名：Multi-dimensional metabolic engineering and enzyme engineering in Escherichia coli for highly efficient biosynthesis of 2,5-furandicarboxylic acid

作者：Cui, Yanan[1,2];Zhang, Peiyi[1,2];Miao, Renjie[1,2];Zhao, Mengqi[1,2];Fan, Liqiang[3];Li, Xu[1,2];Qiu, Yongjun[1,2];Deng, Chen[1,2];Zhao, Liming[1,2,3]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg Technol SCI, Shanghai 200237, Peoples R China;[3]East China Univ Sci & Technol, Shanghai Frontiers Sci Ctr Optogenet Tech Cell Met, Sch Pharm, 130 Mei Long Rd, Shanghai 200237, Peoples R China

年份：2026

卷号：457

外文期刊名：BIORESOURCE TECHNOLOGY

收录：;EI(收录号：20262320828771);WOS:【SCI-EXPANDED(收录号:WOS:001785568100001)】；

基金：This work was financially supported by the National Key Research and Development Program of China (2022YFC2104500), the National Natural Science Foundation of China (32301214, 32327801), Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China (JYB2025XDXM509), the Shanghai Commission of Science and Technology (24HC2820700), ECUST-Jingbo Joint Research Institute of Applied Technology Exploratory Research Projects (JJHP2020029-12).

语种：英文

外文关键词：5-Hydroxymethylfurfural; 5-Furandicarboxylic acid; Metabolic engineering; Cofactors engineering; Transporter engineering; Fermentation

摘要：2,5-Furandicarboxylic acid (FDCA) is a key monomer widely used in the plastic, dye, pharmaceutical, pesticide and resin industries. Presently, its biosynthesis via microbial fermentation is severely limited by the cytotoxicity of substrate 5-hydroxymethylfurfural (HMF). In this study, using Escherichia coli as a host, efficient biosynthesis of FDCA was achieved through multi-step metabolic engineering and protein engineering. The novel oxidative pathway for converting HMF to FDCA was constructed and optimized by screening candidate genes and optimizing gene combination, linkage strategies, copy number, and translation intensity. Subsequently, a comprehensive deep mutation screening of aldehyde dehydrogenase EcALDH was performed using the VenusFactory platform, and the mechanisms were elucidated through kinetic analysis and molecular dynamics (MD) simulations. The H263A mutant increased the catalytic efficiency toward the key intermediates 5-hydroxymethyl-2-fur-ancarboxylic acid (HMFCA) and 5-formyl-2-furancarboxylic acid (FFCA) by 828.4% and 340.3%, respectively. Further, FDCA production was enhanced by increasing the availability of the cofactors nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), while reducing extracellular leakage of the intermediate HMFCA. Transcriptomic analysis identified 26 significantly upregulated candidate genes potentially associated with HMFCA transport. Notably, inactivation of aromatic amino acid transporter (AroP) increased the FDCA concentration to 77.73 mM. Finally, fed-batch fermentation in a 5-L bioreactor produced 170.72 mM (26.65 g/L) FDCA with a molar yield of 94.8% relative to HMF added. These results demonstrate that the engineered E. coli strain constructed in this study can serve as a promising platform for efficient and sustainable production of FDCA, laying a solid foundation for industrial biomanufacturing.