Identification of key enzymes and tolerance mechanisms for protocatechuic acid synthesis in Escherichia coli via systematic metabolic engineering.

Protocatechuic acid (PCA), a phenolic acid with strong antioxidant, antibacterial, and anti-aging properties, is widely used in medicine, food, and cosmetics. This study achieved efficient PCA synthesis in Escherichia coli through a systematic metabolic engineering approach. The 3-dehydroshikimic acid pathway was identified as optimal, and aroE expression was regulated by the growth-dependent promoter PrrnA P1 to balance cell growth and production. A high-potential 3-dehydroshikimic dehydratase (Qa-4) was screened, and a W169F/F311Y mutant was engineered via substrate channel optimization, increasing PCA titer to 4.38 g/L. Accelerated evolution enhanced strain tolerance to PCA, and transcriptome analysis revealed proX as a key resistance factor. Its integration into the W169F/F311Y/A27M strain further increased the titer to 5.04 g/L. The NOG pathway was introduced to improve carbon efficiency and redirect carbon flux. This raised PCA production to 5.6 g/L. Finally, in a 5 L bioreactor maintained at pH 7.0, PCA titer reached 26.7 g/L, successfully achieving the efficient synthesis of PCA. Our strategies provide valuable technical guidance for developing microbial cell factories for PCA and its derivatives.