[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.%[目的]绿针假单胞菌GP72是一种植物根围促生细菌,其分泌的次级代谢产物2-羟基-吩嗪(2-OH-PHZ)具有广谱抗真菌活性,但其产量较低,不能满足农业生产的应用需求,因此需对GP72进行改造,从而提高产量.[方法]从GP72的野生株出发,首次将2-OH-PHZ合成途径的限制性因子PhzO用绿色荧光蛋白(GFP)替换,以一种新型的常压室温等离子体技术(Atmospheric and room temperature plasma,ARTP)进行诱变,通过酶标仪测定96孔板中突变株的荧光强度进行高通量筛选;最后将荧光强度高的菌株中绿色荧光蛋白(Green fluorescent protein,GFP)替换为PhzO以获得2-OH-PHZ高产突变株.[结果]经过五轮诱变后,获得一株荧光强度增加1.62倍的突变株,用phzO基因回替后,该突变株在KB培养基中摇瓶培养时2-OH-PHZ的产量为野生型的4.62倍.[结论]基于安全、高效ARTP诱变技术,并以GFP替换限制性因子作为标记进行高通量筛选,可以快速获得高产2-OH-PHZ的GP72突变株,克服了传统诱变育种方法筛选难度大、费时费力的不足,为其它微生物的育种提供了参考.
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