Systems metabolic engineering of Escherichia coli for high-yield production of Para-hydroxybenzoic acid

Para-hydroxybenzoic acid (PHBA) is extensively used as an additive in the food and cosmetics industries, significantly enhancing product shelf life and stability. While microbial fermentation offers an environment-friendly and sustainable method for producing PHBA, the titer and productivity are limited due to product toxicity and complex metabolic flux distributions. Here, we initially redesigned a L-phenylalanine-producing Escherichia coli by employing rational metabolic engineering strategies, resulting in the production of PHBA reached the highest reported level of 14.17 g/L. Subsequently, a novel accelerated evolution system was devised comprising deaminase, the alpha subunit of RNA polymerase, an uracil-DNA glycosylase inhibitor, and the PHBA-responsive promoter PyhcN. This system enabled us to obtain a mutant strain exhibiting a 47% increase in the half-inhibitory concentration (IC50) for PHBA within 15 days. Finally, the evolved strain achieved a production of 21.35 g/L PHBA in a 5-L fermenter, with a yield of 0.19 g/g glucose and a productivity rate of 0.44 g/L/h. This engineered strain emerges as a promising candidate for industrial production of PHBA through an eco-friendly approach.