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 environmentfriendly 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 P yhcN . This system enabled us to obtain a mutant strain exhibiting a 47% increase in the half-inhibitory concentration (IC 50 ) 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 ecofriendly approach.