Phenazine-1-carboxylic acid production in a chromosomally non-scar triple-deleted mutant Pseudomonas aeruginosa using statistical experimental designs to optimize yield
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We constructed a non-scar triple-deleted mutant Pseudomonas aeruginosa to improve phenazine-1-carboxylic acid (PCA) yield and then optimized the culture conditions for PCA production. Using a non-scar deletion strategy, the 5′-untranslated region of the phz1 gene cluster and two genes, phzM and phzS, were knocked out of the P. aeruginosa strain M18 genome. The potential ability for high-yield PCA production in this triple-deleted mutant M18MSU1 was successfully realized by using statistical experimental designs. A 25–1 fractional factorial design was used to show that the three culture components of soybean meal, corn steep liquor and ethanol had the most significant effect on PCA production. Using a central composite design, the concentration of the three components was optimized. The maximum PCA production was predicted to be 4,725.1 mg/L. With the optimal medium containing soybean meal 74.25 g/L, corn steep liquor 13.01 g/L and ethanol 21.84 ml/L, a PCA production of 4,771.2 mg/L was obtained in the validation experiments, which was nearly twofold of that before optimization and tenfold of that in the wild-type strain. This non-scar triple-deleted mutant M18MSU1 may be a suitable strain for industrial production of this biologically synthesized fungicide due to its high PCA production, presumed safety, thermal adaptability and cost-effectiveness.
KeywordsPseudomonas aeruginosa M18MSU1 Non-scar deletion Phenazine-1-carboxylic acid Culture medium optimization
This study was supported by the National Key Basic Research Program (973 Program, No. 2009CB118906) and the Shanghai Science and Technology Program (No. 08391911900). We are very grateful to Dr. Ya-Wen He for his technical assistance, the referees and editor for their helpful and valuable suggestions and revisions.
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