Systems engineering Escherichia coli for efficient production p‐coumaric acid from glucose

Abstract 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 ( At PAL2, At C4H, At ATR2) into a higher l ‐phenylalanine‐producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of At C4H in the PAL pathway was a bottleneck for increasing p ‐CA titers. To overcome this limitation, the soluble expression of At C4H was enhanced through N‐terminal modifications. And an optimal mutant, At C4H L373T/G211H , which exhibited a 4.3‐fold higher k cat / K m 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.