High-Yielding Protocatechuic Acid Synthesis from l-Tyrosine in Escherichia coli

Protocatechuic acid (PCA) is an important drug intermediate with antibacterial and antioxidant properties. In this study, the whole-cell bioconversion of l-tyrosine into PCA was explored using artificial enzymatic cascades engineered in Escherichia coli (E. coli). In particular, the first biocatalytic route comprises l-amino acid deaminase (LAAD) from Proteus mirabilis, hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis, two-component flavin-dependent monooxygenase (HpaBC) from E. coli, hydroxymandelate oxidase (HMO) from Streptomyces coelicolor, benzoylformate decarboxylase (BFD) from Pseudomonas putida (P. putida), and aldehyde dehydrogenase (ALDH) from Saccharomyces cerevisiae. Combining LAAD–HmaS–HpaBC resulted in efficient synthesis of 3,4-dihydroxymandelate (DHMA), which could be further converted to PCA by HMO–BFD–ALDH to a final conversion of 64.4%. The second route utilizes 4-hydroxybenzoate (HBA) hydroxylase (PobA from P. putida) to convert HBA into PCA. As the recombinant E. coli produced >99% conversion of l-tyrosine into HBA within 12 h, further incorporation of PobA resulted in complete conversion of HBA into PCA, reaching >99% conversion. In summary, the developed biocatalytic pathway has great potential to produce various high-valued fine chemicals such as 4-hydroxymandelate (HMA), DHMA, HBA, and PCA.