Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources. We metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture. We for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

中文翻译：

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代谢工程大肠杆菌高效生产艾卡里苷 D2

Icariside D2 是一种植物来源的天然糖苷，具有抑制血管紧张素转换酶和杀死白血病癌细胞的药理活性。通过植物提取和化学合成生产淫羊藿苷 D2 效率低且对环境不友好。微生物细胞工厂为利用可再生和可持续的生物资源经济地生产淫羊藿苷 D2 提供了一条有吸引力的途径。我们在工程大肠杆菌中代谢构建了艾卡里苷 D2 的生物合成途径。我们筛选了尿苷二磷酸糖基转移酶 (UGT) 并获得了一个活性 RrUGT3，该 RrUGT3 可在酚类位置对酪醇进行区域特异性糖基化，以专门合成淫羊藿苷 D2。我们将异源基因放入大肠杆菌细胞中，用于从头合成伊卡里苷 D2。通过微调启动子和拷贝数以及平衡基因表达模式以减少代谢负担，构建了BMD10单一培养物。同时，为了平衡通路强度，我们通过将淫羊藿苷 D2 生物合成基因分配到两个大肠杆菌菌株 BMT23 和 BMD12 来建立 BMT23-BMD12 共培养，分别负责从优先木糖和葡萄糖中的淫羊藿苷 D2 生物合成。在补料分批摇瓶发酵的最佳条件下，使用葡萄糖作为唯一碳源的 BMD10 单一培养产生 3.80 g/L 的淫羊藿苷 D2，而使用葡萄糖-木糖混合物的 BMT23-BMD12 共培养产生 2.92 g/L 的淫羊藿苷 D2 . 我们首次报道了用于从头高效生产具有克滴度的伊卡里苷 D2 的工程化大肠杆菌。这将是利用可再生碳源微生物生产淫羊藿苷 D2 的有效且可持续的方法。大肠杆菌-E。大肠杆菌共培养方法不仅限于糖苷生产，还可以应用于其他生物产品。