Breeding of Pseudomonas chlororaphis GP72 based on ARTP mutagenesis and high throughput screening

Abstract:

[Objective] Pseudomonas chlororaphis GP72 is a plant growth-promoting rhizobacterium. Its secondary metabolite 2-hydroxy-phenazine (2-OH-PHZ) has broad-spectrum antimicrobial activity, although its yield is too low to apply in agricultural pest management. [Methods] The limiting factor PhzO in 2-OH-PHZ biosynthesis pathway was replaced by green fluorescent protein (GFP), and a novel atmospheric and room temperature plasma (ARTP) technology was used to mutagenize the strain. High-throughput screening of the mutants in a 96-well plate was done by using a microplate reader. Finally, GFP was replaced reversibly by PhzO in the strain with high fluorescence intensity to obtain the 2-OH-PHZ high-yield mutant. [Results] The yield of 2-OH-PHZ was 4.62 times higher than that of the wild-type strain in KB medium in shake flask culture. [Conclusion] The high yield of 2-hydroxy-phenazine GP72 mutants could be obtained rapidly and safely by high-throughput screening based on ARTP mutagenesis using GFP substitution of the restriction factor as the marker. This method overcomes the shortcomings of traditional breeding methods. This method could be used as a reference for other microbial breeding.