Gallic acid fermentation by metabolically engineered Escherichia coli producing p-hydroxybenzoate hydroxylase from Hylemonella gracilis NS1.

Plant-derived phenolic gallic acid (GA) is an important raw material for antioxidants and food additives. Efforts to ferment GA using microbial processes have aimed at minimizing production costs and environmental load using enzymes that hydroxylate p-hydroxybenzoate and protocatechuate (PCA). Here, we found a p-hydroxybenzoate hydroxylase (PobA) in the bacterium Hylemonella gracilis NS1 (HgPobA) with 1.5-fold more hydroxylation activity than that from Pseudomonas aeruginosa PAO1 and thus converted PCA to GA more efficiently. The PCA hydroxylation activity of HgPobA was improved by introducing the amino acid substitutions L207V/Y393F or T302A/Y393F. These mutants had 2.9- and 3.7-fold lower K_m^app for PCA than wild-type HgPobA. An Escherichia coli strain that reinforces shikimate pathway metabolism and produces HgPobA when cultured for 60 h generated 0.27 g L^-1 of GA. This is the first report of fermenting glucose to generate GA using a natural enzyme from the PobA family. The E. coli strain harboring the HgPobA L207V/Y393F mutant increased GA production to 0.56 g L^-1. During the early stages of culture, GA was fermented at a 10-fold higher rate by a strain producing either HgPobA L207V/Y393F or T302A/Y393F compared with wild-type HgPobA, which agreed with the high k_cat^app/K_m^app PCA values of this mutant. We enhanced a PobA isozyme and its PCA hydroxylating function to efficiently and cost-effectively ferment GA.