Efficient Synthesis of Shikimic Acid in Escherichia coli through Self-Feedback Regulation and Accelerated Evolution

Shikimic acid (SA), a critical intermediate in the synthesis of numerous high-value aromatic compounds, is extensively used in the food and pharmaceutical industries. In this study, efficient SA synthesis in Escherichia coli was achieved through dynamic and static regulatory strategies. Initial optimization of key enzymes in the SA pathways AroGfbr, AroB, AroD, and AroE and the growth-regulating gene pykF enabled the accumulation of 7.4 g/L SA. Subsequently, the expression of gapN and zwF was adjusted to ensure adequate NADPH supply. The SA sensor module was then optimized to achieve a response range up to 25 g/L SA, and a negative feedback regulation system was constructed and characterized to balance cellular growth with maximal SA production. An accelerated evolution strategy enhanced the strain's tolerance and identified the regulatory factor StpA that confers tolerance through transcriptome analysis. Finally, the tolerant engineered strain SA35 was fermented in a 5 L bioreactor, and the SA titer was 1.3 times higher than that of the control strain SA33, reaching 97.3 g/L, with a glucose conversion rate of 0.32 g/g. Therefore, these methods provide insights for the efficient synthesis of SA and its derivatives.