阿维链霉菌中aveD基因插入失活产生的异常组分(英文)

Unusual metabolites produced by recombinant Streptomyces avermitilis after insertional inactivation into aveD gene

目的探索阿维链霉菌中aveD基因插入失活后对发酵产物组分的影响。方法采用将抗生素(安普霉素)抗性基因插入到aveD基因的方法,构建了重组质粒pID03;利用接合转移的方法将重组质粒导入到阿维链霉菌S-2(Streptomyces avermitilis S-2)菌株中,并通过抗性标记筛选双交换的菌株。结果得到了aveD基因插入失活的菌株AveD24。该菌株不再产生4个A组分,只产生4个B组分,同时还产生2个异常的组分,经HPLC和质谱分析,初步确定异常组分D24-1为寡霉素A,另一异常组分D24-2为5-酮avermectin la。

Objective To construct a Streptomyces avermitilis strain producing only “B” components by the replacement of aveD gene with a resistance cassette. Methods A DNA fragment carrying aveD gene amplified by PCR were interrupted by apr （apramycin resistance gene） inserted to a restriction site of NruI, the constructed recombinant plasmid pID03 was transferred by conjugation via E. coli ET12567（pUZ8002） into a wild type strain, and S. avermitilis S-2 obtained a mutant strain AveD24. Conclusions Analysis of the metabolites of AveD24 by HPLC re- vealed that the disappearance of component “A” with still remaining of component B, as expected. But two unexpected compounds were also identified as being oligomycin A and 5-oxoavermectin la by HPLC and LC-MS.