High-yield production of medium-chain fatty acids (MCFAs) via engineered Escherichia coli metabolic pathways for flavor enhancement of dried-fish

Medium-chain fatty acids (MCFAs) are high-value flavor enhancers critical for improving the sensory quality of processed foods like dried-fish. Traditional plant-derived MCFAs production faces sustainability limitations, necessitating efficient microbial alternatives. Here, we engineered Escherichia coli ( E. coli ) for high-yield MCFAs synthesis via targeted metabolic rewiring. In this study, we optimized a platform based on de novo fatty acid synthesis to increase the production of MCFAs. Using the pTargetF and pCas dual plasmid knockout system, single gene knockout was sequentially performed on E . coli , they were IdhA and poxB genes, which consume pyruvate, as well as the pta gene, which consumes acetyl CoA, and the aas gene that can reduce the toxicity of MCFAs to the host, followed by multi gene combination knockout. All combination knockout strains express thioesterase gene (CpFatB 1 ). The best-performing strain, strain MG9 (MG1655△ aas △ pta [pTrc99a-CpFatB 1 ]), was selected for further optimization. After 144h of cultivation with D-fructose, 680 mg/L of C8 fatty acid was obtained. We explored purification methods for C8 fatty acid, and purified C8 fatty acid reached 99.75% purity with 82.15% recovery yield. Crucially, applying these biosynthesized C8 fatty acid to industrial fish drying elevated the kinds of flavor compounds similarity to traditional products from 38.57% to 68.57%. This work establishes a sustainable platform for high-purity MCFAs production and demonstrates their direct utility in enhancing premium food flavors, offering a scalable solution for the food industry.