l-Phenylalanine (l-Phe) is widely used in the food and pharmaceutical industries. However, the biosynthesis of l-Phe using Escherichia coli remains challenging due to its lower tolerance to high concentration of l-Phe. In this study, to efficiently synthesize l-Phe, the l-Phe biosynthetic pathway was reconstructed by expressing the heterologous genes aroK1, aroL1, and pheA1, along with the native genes aroA, aroC, and tyrB in the shikimate-producing strain E. coli SA09, resulting in the engineered strain E. coli PHE03. Subsequently, adaptive evolution was conducted on E. coli PHE03 to enhance its tolerance to high concentrations of l-Phe, resulting in the strain E. coli PHE04, which reduced the cell mortality to 36.2% after 48 h of fermentation. To elucidate the potential mechanisms, transcriptional profiling was conducted, revealing MarA, a DNA-binding transcriptional dual regulator, as playing a crucial role in enhancing cell membrane integrity and fluidity for improving cell tolerance to high concentrations of l-Phe. Finally, the titer, yield, and productivity of l-Phe with E. coli PHE05 overexpressing marA were increased to 80.48 g/L, 0.27 g/g glucose, and 1.68 g/L/h in a 5-L fed-batch fermentation, respectively.

中文翻译：

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用于高水平生产 L-苯丙氨酸的大肠杆菌代谢工程

l-苯丙氨酸（l -Phe）广泛应用于食品和制药行业。然而，由于大肠杆菌对高浓度l -Phe的耐受性较低，因此利用大肠杆菌生物合成l -Phe仍然具有挑战性。在本研究中，为了有效合成l -Phe，通过在莽草酸生产菌株E中表达异源基因aroK1、aroL1和pheA1以及天然基因aroA、aroC和tyrB来重建l -Phe生物合成途径。大肠杆菌SA09，产生工程菌株大肠杆菌PHE03。随后，对大肠杆菌PHE03进行适应性进化，增强其对高浓度l -Phe的耐受性，产生菌株大肠杆菌PHE04，发酵48小时后，细胞死亡率降低至36.2%。为了阐明潜在的机制，进行了转录分析，揭示了 MarA（一种 DNA 结合转录双重调节因子）在增强细胞膜完整性和流动性以提高细胞对高浓度l -Phe 的耐受性方面发挥着至关重要的作用。最后，在 5 L 补料分批发酵中，大肠杆菌PHE05 过表达marA的l -Phe滴度、产量和生产率提高至 80.48 g/L、0.27 g/g 葡萄糖和 1.68 g/L/h ， 分别。