Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China.
Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China; School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China.
Metab Eng. 2022 Nov;74:206-219. doi: 10.1016/j.ymben.2022.10.015. Epub 2022 Nov 4.
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.
染料木苷是豆类中发现的生物活性异黄酮糖苷之一，具有巨大的营养和药用价值。目前市售的异黄酮是通过直接从植物中提取得到的。然而，由于其在植物中的含量低且结构复杂，通过植物提取或化学合成获得这种植物药受到了限制。在这里，构建了大肠杆菌细胞工厂，以可持续地从甘油生产染料木苷。首先，我们在大肠杆菌中重建了前体染料木黄酮生物合成途径，然后通过鉴定限速步骤并应用人工蛋白支架系统，将其产量提高了 668%。然后通过功能筛选和引入糖基转移酶、UDP-葡萄糖途径和特定的染料木苷外排泵，从甘油从头生产染料木苷，获得了 48.1mg/L 的染料木苷。进一步的工程大肠杆菌菌株配备了改进的丙二酰辅酶 A 途径、替代的甘油利用途径、乙酰辅酶 A 羧化酶 (ACC) 和 CRISPR 干扰 (CRISPRi) 介导的调控，在摇瓶培养中可生产高达 137.8mg/L 的染料木苷。最后，通过在 3-L 生物反应器中进行分批补料发酵，可获得 202.7mg/L 的染料木苷。本研究首次实现了从甘油从头生产染料木苷，为低成本微生物生产糖苷异黄酮奠定了基础。此外，多相工作流程还可为在其他微生物宿主中工程化生物合成途径提供参考，从而实现复杂天然产物的绿色制造。
