阿维菌素B1a组分高产菌株的高通量选育

Breeding and high throughput screening overproducing avermectin B1a strains

为筛选阿维菌素高产菌株,建立了阿维菌素高产菌株的高通量初筛及复合诱变技术选育高产菌株的方法。初筛首先采用24孔板培养突变株,发酵培养5 d,甲醇直接浸提固体培养物,最后利用96孔板-酶标仪检测浸提液在紫外波段245 nm吸收。以阿维链霉菌(Streptromyces avermitilis)编号为AV-X-95的阿维菌素产生菌作为出发菌株,通过紫外线(UV)照射45 s和亚硝基胍(NTG)处理60 min的组合诱变方法对出发菌株的孢子悬液进行了连续的诱变处理。研究结果表明,1 728株突变子的经高通量初筛后得到8株高产菌株,经摇瓶复筛和稳定性考察得到高产菌株AW4-329,其B1a产量较出发菌株提高了15.1%。

To increase production of avermectins and the most effective fractions B1a,a more rapid and sensitive high throughput screening method for large-scale mutations was constructed.The mutants were miniaturized in 24 deep-well microplate,the culture was extracted with methanol after 5 days fermentation,and avermectin producing monitor was with 96 deep-well and microplate-reader using UV absorbance at 245 nm.The original strain（numbered AV-X-95）was treated with repeated multiple mutation with ultraviolet radiation（UV） 45 s and nitrosoguanidine（NTG） 60 min treatment.Eight high producers were obtained by the high throughput screening method.The B1a production of AW4-329 increased by 15.1%,and had stable inheritance after verification in the shake flask experiments.