Study On The Biosynthesis Mechnism Of 2-hydroxy-phenazine And The Two-component System RpeA/RpeB In Pseudomonas Chlororaphis GP72

The phenazine derivative 2-hydroxyphenazine?2-OH-PHZ?plays an important role in the biocontrol of plant diseases,and exhibits stronger bacteriostatic and fungistatic activity than phenazine-1-carboxylic acid?PCA?toward some pathogens.However,the yield of 2-OH-PHZ in fermentation culture is quite low and enhancement in our understanding of the reaction kinetics may contribute to improvements in large-scale,high-yield production of 2-OH-PHZ for biological control and other applications.Three mechanisms have been proposed to account for the conversion of PCA to 2-OH-PHZ,although the mechanism responsible has not been clearly defined.To identify the mechanism,PhzO,the key enzyme involved in the conversion of PCA to 2-OH-PHZ,was subcloned from Pseudomonas chlororaphis GP72 and heterologously expressed in Escherichia coli BL21.In this study we confirmed that free PCA is converted to2-hydroxy-phenazine-1-carboxylic acid?2-OH-PCA?by the action of a single enzyme PhzO,and particularly demonstrate that this reaction is dependent on NADP?H?and Fe³⁺.Fe³⁺enhanced the conversion from PCA to 2-OH-PHZ and 28?was a optimum temperature for the conversion.However,PCA added in excess to the culture inhibited the production of 2-OH-PHZ.PhzO has been shown to be responsible for the conversion of PCA to2-OH-PHZ,however the kinetics of the reaction have not been systematically studied.In this study,2-OH-PCA was extracted and purified from the broth,and it was found that the decarboxylation of 2-OH-PCA could occur without the involvement of any enzyme.A kinetic analysis of the conversion of2-OH-PCA to 2-OH-PHZ in the absence of enzyme was examined under different temperatures and pHs in vitro,and was found to follow first-order reaction kinetics.In the fermentation,the concentration of 2-OH-PCA increased to about 90 mg/L within a red precipitate fraction,as compared to37mg/L within the supernatant.The results of this study elucidate the reaction kinetics involved in the biosynthesis of 2-OH-PHZ and provide insights into in vitro methods to enhance yields of 2-OH-PHZ.RpeA/RpeB is a putative two component system that regulates phenazines biosynthesis in Pseudomonas chlororaphis GP72.RpeA is a repressor for Phenazine biosynthesis in P.chlororaphis GP72.In this work,the cognate response regulator gene rpeB is first cloned by PCR,and is studied to understand its regulation of phenazine biosynthesis and the function of putative two-component regulatory system rpeA/rpeB.In contract to rpeA mutant,the rpeB mutant produces fewer phenazines than the wild type,suggesting that RpeB has a positive regulation on phenazines biosynthetic genes.Also,the rpeA/rpeB double mutant produced a lower amount of phenzines than wild type.Transcriptional levels of phenazines biosynthetic gene phzE and quorum sensing gene phzR which controlling phenazines production positively in GP72 wild type,rpeA mutant and the rpeB mutant were measured by quantitative real-time PCR,and the results suggested that RpeB positively controls phzR expression and AHL production.Complementation assays showed that the production of phenazines can be restored by complement rpeB gene in the rpeB mutant,while overexpression of rpeB gene in GP72 wild type doesn't enhance the phenazines biosynthesis remarkably.Additionally,the rpeA and the rpeB expression level is decreased in minimal culture or at high temperature while the phenazines production is also decreased consistant with the conculsion that RpeA/RpeB belong to the same two component system.The results suggest that RpeA/RpeB regulatory system conbines the positive regulator and repressor for Phenazine biosynthesis.Futher,we investigate RpeA/RpeB regulatory role in low-phosphate medium.The results demonstrated that RpeA positively regulates the phenazines'production by phosphorylate RpeB leading to the increased production of phenazines in MOPS medium.Finally,we investigate on the regulatory roles that RND efflux transporter RndB plays on the production of phenazines in GP72.We construct the rndB mutant GP72RN.The results showed that rndB regulates the phenazines biosynthesis in different way in different environment.Though rndB didn't affect the phenazines biosynthesis in KB medium,it positively regulated the phenazines biosynthesis in M9 medium.