Metabolic engineering of Pseudomonas putida KT2440 for high-yield production of protocatechuic acid.

Protocatechuic acid (PCA) has been widely utilized in conventional pharmaceutical, cosmetic and functional food industries. Currently, chemical synthesis and solvent extraction are the main methods for commercial production, indicating several disadvantages. In this study, we developed a method for the biosynthesis of PCA in Pseudomonas putida KT2440 in high yield. First, we developed constitutive promoters with different expression intensities for fine-tuned gene expression. Second, we improved the biosynthesis of "natural" PCA in P. putida KT2440 via multilevel metabolic engineering strategies: overexpression of rate-limiting enzymes, removal of negative regulators, attenuation of pathway competition, and enhancement of precursor supply. Finally, by further bioprocess engineering efforts, the best-producing strain reached a titer of 12.5 g/L PCA from glucose at 72 h in a shake flask and 21.7 g/L in fed-batch fermentation without antibiotic pressure. This was the highest PCA titer from glucose using metabolically engineered microbial cell factories reported to date.