Uncovering the Role of PhzC as DAHP Synthase in Shikimate Pathway of Pseudomonas chlororaphis HT66

SIMPLE SUMMARY: This study investigated PhzC, one essential 3-Deoxy-D-arabino-heptulosnate-7-phosphate (DAHP) synthase that catalyzes the first step of the shikimate pathway in Pseudomonas chlororaphis. We identified and characterized phzC, which is different from the reported DAHP synthase encoding genes aroF, aroG and aroH in E. coli. PhzC accounts for approximately 90% of the total DAHP synthase activities in P. chlororaphis and it plays the most critical role in four DAHP synthases in the shikimate pathway. Moreover, the results showed that phzC in P. chlororaphis HT66 is not sensitive to feedback inhibition. This study demonstrated that PhzC is essential for phenazine-1-carboxamide (PCN) biosynthesis without inhibition in feedback by PCN production. It highlighted the importance of PhzC and applying P. chlororaphis for shikimate pathway-derived high-value biological production. ABSTRACT: DAHP synthase catalyzes the first step in the shikimate pathway, deriving the biosynthesis of aromatic amino acids (Trp, Phe and Tyr), phenazine-1-carboxamide, folic acid, and ubiquinone in Pseudomonas chlororaphis. In this study, we identified and characterized one DAHP synthase encoding gene phzC, which differs from the reported DAHP synthase encoding genes aroF, aroG and aroH in E. coli. PhzC accounts for approximately 90% of the total DAHP synthase activities in P. chlororaphis HT66 and plays the most critical role in four DAHP synthases in the shikimate pathway. Inactivation of phzC resulted in the reduction of PCN production by more than 90%, while the absence of genes aroF, aroG and aroH reduced PCN yield by less than 15%, and the production of PCN was restored after the complementation of gene phzC. Moreover, the results showed that phzC in P. chlororaphis HT66 is not sensitive to feedback inhibition. This study demonstrated that gene phzC is essential for PCN biosynthesis. The expression level of both phzC and phzE genes are not inhibited in feedback by PCN production due to the absence of a loop region required for allosteric control reaction. This study highlighted the importance of PhzC and applying P. chlororaphis for shikimate pathway-derived high-value biological production.