多拉菌素产生菌aveD基因缺失突变株的构建

Deletion Analysis of aveD Gene from a Streptomyces avermitilis Mutant Producing Doramectin

阿维链霉菌(Streptomyces avermitilis)bkd76-3在发酵过程中添加环己羧酸(CHC)可产生抗寄生虫药物多拉菌素(doramectin,阿维菌素衍生物CHC-B1),但同时还产生其它三种无效组分CHC-B2、CHC-A1、CHC-A2。利用基因缺失载体pXJ04(pKC1139∷△aveD1+△aveD2)对该菌株的aveD基因进行缺失,获得的aveD缺失突变株经摇瓶发酵和HPLC检测,发现只存在2种产物,经LC/MS分析验证,这两种产物分别为CHC-B1和CHC-B2,表明该突变株完全丧失了合成CHC-A1和CHC-A2的能力。缺失突变株的CHC-B1产量较出发菌株提高了78.19%,CHC-B2的产量提高了602.3%,发酵产物中有效组分多拉菌素的比例增加了93.16%。该缺失突变是在染色体上通过同源双交换完成的,不会发生进一步的重组,因此突变株具有良好的遗传稳定性,在工业生产上具有应用价值。

The avermectin analog doramectin（CHC-B1）,sold commercially as DectomexTM,was co-produced with the undesired analog CHC-B2,CHC-A1 and CHC-A2 by Streptomyces avermitilis bkd76-3 with the supplementation of cyclohexanecarboxylic acid（CHC） during fermentation.Gene deletion vector pXJ04（pKC1139∷aveD1＋aveD2） was used to delete aveD gene in S.avermitilis bkd76-3.The aveD gene was displaced by deletion allele on the plasmid via double crossover.Shaking flask experiments and HPLC analysis showed that the aveD deletion mutant no longer produced the undesired analogue CHC-A1 and CHC-A2,and only made two components which were confirmed as CHC-B1 and CHC-B2 by LC/MS analysis.The yield of CHC-B1 improved 78.19%,and B2 improved 602.3%,the ratio of effective component had a 93.16% increase.The deletion mutant was proved to be genetically stable,and thus might be promising strain in industrial production of doramectin.