| Phenzine-1-carboxamide is a secondary metabolite produced by some strains of Pseudomonas and a kind of biologically active antifungal agents. The PCA biosynthesis is mainly by Shikimate pathway. It was commonly accepted that the phenazine biosynthetic gene cluster in Pseudomonas contains seven genes, but until now it has been unknown exactly which one functions the most significantly in the pathway. The evidenced result was that phzA/B/C cannot be the key one. PhzA and PhzB were not surposed necessary and the level of chorismic acid that is on the down-stream of PhzC cannot influence PCA directly. In this study we selected phzD, phzE, phzF and phzG as candidates to approach and analyze the relative significances of phz genes through the methods of over-expression and antisense RNA. Pseudomonas. sp. M18 used in this study was found by our laboratory and can produce two different kinds of antifungal agents, phenazine-1-caboxylic acid (PCA) and pyoluteorin (Plt) at one time, which was first time reported.We constructed the over-expression functional plusmids of phzD, phzE, phzF and phzG, and transformed them into Psedomonas sp. M18 for fermentation of PCA. The fermentation results indicated that in the four genes phzD had the most significant effect for the PCA biosynthesis for enhancing the PCA product by more than 30% by phzD over-expression strain compared to the control of strain bearing void plasmid. This illustrated that phzD played most important role in the pathway if only single gene was considered. The experiments of inoculation period and availability of oxygen indicated that the over-expression strains and wild type of Pseudomonas M18 had different requirements in cultural conditions. Using the ristiction of antisense RNA of phzD gene, the level of PCA product was greatly decreased in the fermentation experiments of Pseudomonas M18. |
