产环脂肽Bacilus velezensis G-1高效诱变育种及对链格孢霉的抑菌效果

Efficient Breeding of Cyclic Lipopeptide Producing Bacillus velezensis G-1 by Mutagenesis and Its Antifungal Activity Against Alternaria alternata

摘要:

为提高产环脂肽野生菌贝莱斯芽孢杆菌(Bacillus velezensis)G-1的环脂肽产量和降低其生产成本,以链格孢霉菌为靶标,以廉价菜粕为氮源,以抑菌活性为评价指标,探究了紫外诱变、微波诱变、ARTP(常压室温等离子体)诱变、NTG(亚硝基胍)诱变、DES(硫酸二乙酯)诱变和EMS(甲基磺酸乙酯)诱变6种单一和复合诱变方式对B.velezensis G-1环脂肽合成的影响,旨在建立B.velezensis G-1高产环脂肽突变体的高效诱变方法和筛选出以菜粕为氮源的高产脂肽突变体。结果表明,6种单一诱变方式中化学诱变的正向突变率明显高于物理诱变,且化学诱变中NTG和EMS正向突变率较高,分别为59.97%和59.40%；物理诱变中ARTP正向突变率较高,为31.37%；同单一诱变方式相比,NTG 150μg/mL、EMS 0.6mol/L和ARTP 160 s的复合诱变可显著提高B.velezensis G-1的正向突变率,从复合诱变菌株中筛选出1株高产环脂肽突变体,可使环脂肽产量提升2.33倍,离体和活体抑菌活性分别提升52.20%和96.55%。研究结果表明,NTG+EMS+ARTP的复合诱变可成功获取高产环脂肽的B.velezensis G-1突变体,并进一步筛选到了可利用菜粕高产环脂肽且对链格孢霉具有良好抑菌效果的突变体。研究旨在为高产环脂肽B.velezensis G-1突变体筛选及其环脂肽在防治果蔬采后链格孢霉菌侵染的产业化应用奠定理论基础。

Abstract:

In order to improve the cyclic lipopeptide production of wild-type Bacillus velezensis G-1 and reduce the production cost, Alternaria alternata was used as the target, cheap rapeseed meal was used as the nitrogen source, and antifungal activity was used as the evaluation index. The effects on the synthesis of cyclic lipopeptides in B.velezensis G-1 was studied by single and compound mutagenesis methods of ultraviolet, microwave, ARTP(atmospheric room temperature plasma), NTG (nitroso guanidine), DES (diethyl sulfate) and EMS (ethyl methylsulfonate) mutagenesis. The aim was to establish an efficient mutagenesis method for high-yield cyclic lipopeptide mutants of B.velezensis G-1 and to select high-yield strain with rapeseed meal as nitrogen source. The results showed that the forward mutation rates of chemical mutagenesis were significantly higher than those of physical mutagenesis in six kinds of single mutagenesis methods. The forward mutation rate of chemical mutagenesis was higher for NTG and EMS, which were 59.97% and 59.40%, respectively. In physical mutagenesis methods, ARTP had a higher forward mutation rate of 31.37%. The combined treatment of NTG 150μg/mL, EMS 0.6mol/L, and ARTP 160s significantly increased the forward mutation rate of B.velezensis G-1. A high-yield cyclic lipopeptide mutant selected from the compound mutagenic strain could increase the production of cyclic lipopeptide by 2.33 times. The antifungal activity in vitro and in vivo increased by 52.20% and 96.55%, respectively. In this study, the combined mutagenesis method of NTG+EMS+ARTP could successfully obtain the B.velezensis G-1 mutant with high-yield cyclic lipopeptide, and further selected out mutants with high-yield cyclic lipopeptide by rapeseed meal. This study laid a theoretical foundation for the industrial application of the high-yielding B.velezensis G-1 mutants and its cyclic lipopeptides in the prevention of A. alternata infection after fruit and vegetable harvest.