Sustainable production of genistin from glycerol by constructing and optimizing Escherichia coli

• Published in: Metabolic engineering Vol. 74; pp. 206 - 219
• Main Authors: Wang, Zhe, Li, Xiaonan, Dai, Yiqiang, Yin, Liqing, Azi, Fidelis, Zhou, Jianzhong, Dong, Mingsheng, Xia, Xiudong
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.11.2022
• Subjects: Escherichia coli; Genistin; Isoflavone; Isoflavone synthase; Metabolic engineering; Isoflavone; Metabolic engineering; Genistin; Escherichia coli; Isoflavone synthase
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2022.10.015

Genistin is one of the bioactive isoflavone glucosides found in legumes, which have great nutraceutical and pharmaceutical significance. The market available isoflavones are currently produced by direct plant extraction. However, its low abundance in plant and structural complexity hinders access to this phytopharmaceutical via plant extraction or chemical synthesis. Here, the E. coli cell factory for sustainable production of genistin from glycerol was constructed. First, we rebuilt the precursor genistein biosynthesis pathway in E. coli, and its titer was then increased by 668% by identifying rate-limiting steps and applying an artificial protein scaffold system. Then de novo production of genistin from glycerol was achieved by functional screening and introduction of glycosyl-transferases, UDP-glucose pathway and specific genistin efflux pumps, and 48.1 mg/L of genistin was obtained. A further engineered E. coli strain equipped with an improved malonyl-CoA pathway, alternative glycerol-utilization pathways, acetyl-CoA carboxylase (ACC), and CRISPR interference (CRISPRi) mediated regulation produced up to 137.8 mg/L of genistin in shake flask cultures. Finally, 202.7 mg/L genistin was achieved through fed-batch fermentation in a 3-L bioreactor. This study represents the de novo genistin production from glycerol for the first time and will lay the foundation for low-cost microbial production of glucoside isoflavones. In addition, the multiphase workflow may provide a reference for engineering the biosynthetic pathways in other microbial hosts as well, for green manufacturing of complex natural products. [Display omitted] •Biosynthesis of genistin from glycerol was achieved by E. coli for the first time.•Glycerol was more favorable to isoflavone synthase expression in E. coli.•Glycosylation of genistein alleviated product inhibition of IFS in E. coli.•The multiphase optimization strategy further improved genistin production in E. coli.•The optimized strain produces up to 202.7 mg/L of genistin in fed-batch culture.