Cloning Of Phenazine Biosynthesis Gene Cluster And Improvement The Phenazines Producivity In Pseudomonas Chlororaphis GP72

Pseudomonas chlororaphis GP72, isolated from the green pepper rhizosphere, is an effective root-colonizing bio-control agent against soil-borne pathogenic fungi on crop plants. This capability is primarily depending on its ability to produce two different types of antibiotics, phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). However, the molecular mechanism of the production of these two chemical compounds is still unknow.Since previous reports have shown that 2-OH-PHZ exhibited a stronger anti-fungal activity than PCA alone, and its production in the wild-type GP72 was relatively low, it was essential to study the conversion mechansim of 2-OH-PHZ as well as the regulation network in GP72 so as to promote its production.In this study, the phenazine biosynthesis gene cluster was cloned and analyzed by PCR and random PCR gene walking, providing the solid basis for further study on antibiotics in this strain. About 12418 bp sequences were got and the map of the phenazine biosynthesis gene cluster was shown.Gene rpeA and phzO were insertionally mutated to construct GP72AN, GP72ON respectively and GP72ANON collectively. The inactivation of rpeA resulted in a 5-fold increase of the production of PCA, as well as 2-OH-PHZ.An intermediate was found and identified as 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) in the culture of GP72AN and it could not be found in the culture of GP72ANON, which confirmed the hypothesis of Thomashow about the biosynthsis pathway of 2-OH-PHZ in P. chlororaphis strain 30-84, which indicated that 2-OH-PHZ was derived from PCA, involving only one enzyme PhzO that catalyzed the conversion of PCA to 2-OH-PCA, and then 2-OH-PCA spontaneously turned to 2-OH-PHZAt last, baffled shake flasks were adopted in the fermentation test of GP72AN and GP72ANON, and additional PCA was added into the broth culture to dertermine the conversion efficiency of PCA to 2-OH-PHZ under current culture conditions. Results showed that the adoption of baffled shake flasks greatly improved the production of phenazines and PCA exerted a positive feedback regulation on itself accumulation and thus enhance the production of 2-OH-PHZ. The production of PCA was eventually enhanced to 930 ppm compared with 30 ppm in the wild-type, and 2-OH-PHZ to 170 ppm compared with 5 ppm, respectively.