P-coumaric acid (p-CA), a pant metabolite with antioxidant and anti-inflammatory activity, is extensively utilized in biomedicine, food, and cosmetics industry. In this study, a synthetic pathway (PAL) for p-CA was designed, integrating three enzymes (AtPAL2, AtC4H, AtATR2) into a higher l-phenylalanine-producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of AtC4H in the PAL pathway was a bottleneck for increasing p-CA titers. To overcome this limitation, the soluble expression of AtC4H was enhanced through N-terminal modifications. And an optimal mutant, AtC4H^L373T/G211H, which exhibited a 4.3-fold higher k_cat/K_m value compared to the wild type, was developed. In addition, metabolic engineering strategies were employed to increase the intracellular NADPH pool. Overexpression of ppnk in engineered E. coli PHCA20 led to a 13.9-folds, 1.3-folds, and 29.1% in NADPH content, the NADPH/NADP⁺ ratio and p-CA titer, respectively. These optimizations significantly enhance p-CA production, in a 5-L fermenter using fed-batch fermentation, the p-CA titer, yield and productivity of engineered strain E. coli PHCA20 were 3.09 g/L, 20.01 mg/g glucose, and 49.05 mg/L/h, respectively. The results presented here provide a novel way to efficiently produce the plant metabolites using an industrial strain.

中文翻译：

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用于从葡萄糖高效生产对香豆酸的系统工程大肠杆菌


对香豆酸（p-CA）是一种具有抗氧化和抗炎活性的植物代谢物，广泛应用于生物医学、食品和化妆品行业。在这项研究中，设计了p-CA的合成途径（PAL），将三种酶（AtPAL2、AtC4H、AtATR2）整合到高产L-苯丙氨酸的菌株大肠杆菌PHE05中。然而，PAL 途径中 AtC4H 的可溶性表达和活性较低是增加 p-CA 滴度的瓶颈。为了克服这一限制，通过 N 端修饰增强了 AtC4H 的可溶性表达。并开发出最佳突变体AtC4H ^L373T/G211H ，其k _cat /K _m 值比野生型高4.3倍。此外，还采用代谢工程策略来增加细胞内 NADPH 池。在工程化大肠杆菌 PHCA20 中过度表达 ppnk 导致 NADPH 含量、NADPH/NADP ⁺ 比率和 p-CA 滴度分别提高 13.9 倍、1.3 倍和 29.1%。这些优化显着提高了 p-CA 产量，在采用分批补料发酵的 5 L 发酵罐中，工程菌株 E. coli PHCA20 的 p-CA 滴度、产量和生产率分别为 3.09 g/L、20.01 mg/g 葡萄糖和分别为 49.05 毫克/升/小时。这里提出的结果提供了一种利用工业菌株有效生产植物代谢物的新方法。