Enhanced production of 2-hydroxyphenazine in Pseudomonas chlororaphis GP72

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from the green pepper rhizosphere that synthesizes two phenazine derivatives: phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). The 2-OH-PHZ derivative shows somewhat stronger broad-spectrum antifungal activity than PCA, but its conversion mechanism has not yet been clearly revealed. The aim of this study was to clone and analyze the phenazine biosynthesis gene cluster in this newly found strain and to improve the production of 2-OH-PHZ by gene disruption and precursor addition. The conserved phenazine biosynthesis core operon in GP72 was cloned by PCR, and the unknown sequences located upstream and downstream of the core operon were detected by random PCR gene walking. This led to a complete isolation of the phenazine biosynthesis gene cluster phzIRABCDEFG and phzO in GP72. Gene rpeA and phzO were insertionally mutated to construct GP72AN and GP72ON, respectively, and GP72ANON collectively. The inactivation of rpeA resulted in a fivefold increase in the production of PCA, as well as 2-OH-PHZ. The addition of exogenous precursor PCA to the broth culture, to determine the conversion efficiency of PCA to 2-OH-PHZ under current culture conditions, revealed that PCA had a positive feedback effect on its own accumulation, leading to enhanced synthesis of both PCA and 2-OH-PHZ. The production of 2-OH-PHZ by GP72AN increased to about 170 μg ml(-1), compared with just 5 μg ml(-1) for the wild type. The hypothesis of biosynthetic pathway for 2-OH-PHZ from PCA was confirmed by identification of 2-hydroxyphenazine-1-carboxylic acid as an intermediate in the culture medium of the high-phenazine producing GP72AN mutant.