Biosynthesis of three benzoic acid derivatives in Escherichia coli

Benzoic acid, an aromatic compound conventionally obtained from coal tar, can also be produced via microbial biotransformation. The shikimate pathway of Escherichia coli provides a route for the biosynthesis of aromatic acids, with its intermediates serving as valuable starting materials for the synthesis of benzoic acid derivatives. Here, we report the E. coli-based synthesis of three benzoic acid derivatives: 3,4-dihydroxybenzoic acid (protocatechuic acid, 3,4-DHBA), gallic acid (GA), and beta-glucogallin. The enzyme QuiC was used to catalyze the conversion of 3-dehydroshikimate to 3,4-DHBA. For GA production, a screening of four pobA mutants was conducted to identify the most efficient mutant. A grape-derived uridine diphosphate-dependent glucosyltransferase (UDP-GT) was utilized for the glucosylation of GA to beta-glucogallin. To improve the production titers of 3,4-DHBA and GA, a shikimate pathway gene module and a specifically engineered E. coli mutant were employed, resulting in the accumulation of 451.3 mg/L 3,4-DHBA and 123.4 mg/L GA. beta-Glucogallin synthesis was achieved through a stepwise process, wherein one E. coli strain produced GA, and its clarified culture medium was subsequently used by a second E. coli strain for beta-glucogallin formation. Optimization of the ratio between the GA-containing supernatant and the second cell culture led to a beta-glucogallin yield of 118.5 mg/L.