Construction Of Pseudomonas Sp. M18GQ And Its Fermentation Conditions Optimization Phenazing-1-carboxylic Acid

Pseudomonas sp. M18 is a root-colonizing biocontrol agent that was isolated from the rhizosphere of sweet melon in Shanghai suburb of China. This strain produces two different antibiotics, PCA and pyoluteorin, and has many unusual regulatory features on the production of secondary metabolites. As wide type M18 is not suitable for large scale commercial application, there is an urgent requirement to improve PCA production using strategies of both genetic modification of pseudomonad strains and culture medium optimization. Both the qscR gene and gacA gene are active in quorum sensing system and signal transduction pathway that negatively control phz gene expression. In our initial pilot studies, the genetically modified mutant M18G and M18Q, were constructed by the chromosomally inactivating the negative regulators gacA or qscR to promote phz gene cluster expression and PCA production.Statistics based experimental designs were applied to optimize the culture medium components for enhancing phenazine-1-carboxylic acid (PCA) production by Pseudomonas sp. strain M18GQ, a gacA and qscR gene double chromosomally inactivated mutant of the wild type Pseudomonas sp. strain M18. We primarily screened out the proper age of seed, liquid volume (ml), inoculum (%), temperature, pH and rotary speed using one-factor-at-a-time screening. The medium components, including soybean meal, glucose, and corn steep liquor, had significant effects on PCA production based on a 25-1 fractional factorial design. The concentrations of these three significant factors were subsequently optimized using a central composite design. An optimum concentration of soybean meal 73.3 g/L, corn steep liquor 18.1 g/L, glucose 17.9 g/L, and ethanol 18.0 ml/L was obtained by response surface analysis. The predicted maximum PCA yield was as high as 4011.5 mg/L, and was further verified by validation experiments. The double strain M18GQ produced 4032.2 mg/L PCA, which was almost 2 to 3.3-fold that produced by either single mutant M18G and M18Q.