Construction Of A Genetically Modified Strain Of Pseudomonas Sp. M18GQ Harboring PME6032Phz And Optimization Of Phenazine-1-carboxylic Acid Prodcution Using Response Surface Methodology

Pseudomonas sp. stain M18, a new kind of plant growth-promoting rhizobacteria isolated from the melon roots in the suburb of Shanghai, would be a promising strain as a powerful and effective bio-control agent due to its secondary metabolite phenazine-1-carboxylic acid (PCA), which has a broad-spectrum antifungal activity. PCA production was enhanced in Pseudomonas sp. M18 wild strain and its mutants carrying recombinant pME6032Phz for phz gene cluster overexpression, among which Pseudomonas sp. strain M18GQ/pME6032Phz, a gacA and qscR double gene chromosomally inactivated mutant harboring pME6032Phz, showed the highest PCA yield. The conditions for fermentation and isopropyl-β-D-1-thio- galactopyranoside (IPTG) induction were optimized for strain M18GQ/pME6032Phz in shake flask experiments. A one-factor-at-a-time approach, followed by a fractional factorial design (FFD) identified soybean meal, corn steep liquor, and ethanol as statistically significant factors. Optimal concentrations and mutual interactions of the factors were then determined by the method of steepest ascent and by response surface methodology (RSM) based on the center composite design (CCD). The predicted PCA production was 6335.2 mg/l after 60 hour fermentation in the optimal medium of 65.02 g soybean meal, 15.36 g corn steep liquor, 12 g glucose, 21.70 ml ethanol, and 1g MgSO4 per liter in the flask fermentations, with induction of 1.0 mmol/l IPTG 24 hours after inoculation. In an experimental validation under these conditions, the maximum PCA production was 6365.0 mg/l. This represents a ~60% increase over production by strain M18GQ in optimal conditions, and a 60-fold as much as that of strain M18 in basal culture medium.