Promoting the Synthesis of Salicylic Acid in Escherichia coli Based on Metabolic Engineering and Multi-enzyme Cascade Strategy

Abstract:

[Objective] The production of the aromatic compound salicylic acid (SA) is limited by its inhibitory effects on enzymes related to cellular energy metabolism. This study aims to develop a strategy for avoiding the impact of SA on enzymes related to cellular energy metabolism and increase the yield of SA.[Method] A highly efficient Escherichia coli strain was designed to improve SA production by optimizing metabolic pathways and enzyme systems.[Result] The genome-scale metabolic model GEMS1515 was employed to predict the key bottle necks in the phosphoenolpyruvic acid (PEP) pathway and SA biosynthesis pathways. The SA yield was successfully increased from 834.82 mg/L to 1 143.56 mg/L by targeted knockout of fbaA and dcuA genes. Furthermore, the enzyme fusion strategy, combining flexible and rigid peptide linkers with TRAP scaffold proteins (TRAP1 and TRAP3), further improved the catalytic efficiency. The optimized strain achieved a maximum SA yield of 2 100.00 mg/L, which represented an increase of 151% over that of the initial strain.[Conclusion] Integrating metabolic engineering, multi-enzyme cascade strategy, and machine learning demonstrate great application potential in optimizing the biosynthesis of high-value compounds.