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, AtC4HL373T/G211H, which exhibited a 4.3‐fold higher kcat/Km 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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利用葡萄糖高效生产对香豆酸的系统工程大肠杆菌

磷‐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，合成途径（PAL）p‐CA 的设计整合了三种酶（在PAL2，在C4H,在ATR2) 进入更高我苯丙氨酸生产菌株大肠杆菌PHE05。然而，可溶性表达和活性较低在PAL途径中的C4H是增加的瓶颈p‐CA 滴度。为了克服这个限制，可溶性表达在C4H 通过 N 端修饰得到增强。和一个最佳突变体，在C4HL373T/G211H，表现出 4.3 倍的高k猫/K米与野生型相比的价值，被开发出来。此外，还采用代谢工程策略来增加细胞内 NADPH 池。过度表达彭克在工程中大肠杆菌PHCA20 导致 NADPH 含量（NADPH/NADP）分别增加 13.9 倍、1.3 倍和 29.1%+比率和p分别为-CA滴度。这些优化显着增强了p‐CA 生产，在 5 L 发酵罐中采用补料分批发酵，p‐工程菌株的CA滴度、产量和生产力大肠杆菌PHCA20 分别为 3.09 g/L、20.01 mg/g 葡萄糖和 49.05 mg/L/h。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。