Metabolic Engineering of Escherichia coli Nissle 1917 for Efficient Production of p -Coumaric Acid

p-Coumaric acid is a valuable phytochemical with significant roles in anticancer cell proliferation, antianxiety, and neuroprotection and as a key precursor for various flavonoids. However, the production of p-coumaric acid in microorganisms is often limited by enzyme compatibility and its antimicrobial effects. In this study, a p-coumaric acid producing Escherichia coli strain was constructed. First, the cryptic plasmids pMUT1 and pMUT2 were eliminated from E. coli Nissle 1917 by using the CRISPR/Cas9 method to mitigate their interference with heterologous gene expression, and the resulting strain WEN01 was used to screen for the genes encoding for tyrosine ammonia-lyase with superior host compatibility. Next, the gene tyrR encoding a global regulator was knocked out to alleviate the repression of l-tyrosine production. The key genes pheL and pheA involved in phenylalanine biosynthesis were knocked out to reduce byproduct formation, resulting in the strain WEN06. Finally, the quorum sensing system was used to overexpress the key genes aroGfbr and tyrAfbr in the l-tyrosine biosynthetic pathway, and the resulting strain WEN06/pWT101-AT, pWT104F could produce 462.6 mg/L p-coumaric acid in shake flask fermentation. In fed-batch fermentation, the engineered strain WEN06/pWT101-AT, pWT104F could produce 10.3 g/L p-coumaric acid with a glucose conversion yield of 0.13 g/g and a productivity of 0.14 g/L/h. This work provides a novel strategy for the efficient production of p-coumaric acid and lays a foundation for the efficient production of antimicrobial natural products in bacteria.