生防菌密旋链霉菌Act12中SPA7074缺失突变株的构建及其次级代谢产物鉴定

Construction of SPA7074-deficient mutant of biocontrol strain Streptomyces pactum Act12 and characterization of its secondary metabolites

摘要:

[目的] 通过基因工程手段构建生防菌Act12转录调控因子SPA7074缺失突变株，并挖掘其中活性次级代谢产物资源和探讨其活性机理。[方法] 利用同源重组方法敲除Act12基因组中可能的TetR家族转录调控因子编码基因spa7074（accession number：KU955325），平板实验检测缺失突变株发酵液抑菌活性的变化，并通过HPLC比较代谢图谱，然后通过质谱及核磁共振对差异峰对应化合物进行结构鉴定。[结果] SPA7074缺失突变株对几种病原真菌的拮抗活性显著增强，比较代谢图谱表明出现数个差异峰，将最显著差异峰所对应化合物进行分离纯化鉴定，结果为寡霉素D。[结论] 本研究通过基因工程手段敲除生防菌株Act12中的负转录调控转录因子，使得突变菌株抑菌活性显著增强，并获得了产量达野生型菌株7倍的寡霉素D高产菌株Δspa7074。

Abstract:

[Objective] To disrupt spa7074, which encodes a member of the TetR family transcriptional factors, in biocontrol strain Act12 and characterize the secondary metabolites in the mutant strain. [Methods] We disrupted the gene spa7074 by homologous recombination. The secondary metabolites of the mutant strain Δspa7074 and Act12 were detected by HPLC. The structure was analyzed by MS and NMR. [Results] Compared to the wild-type strain, the production of some unknown compounds in the mutant strain Δspa7074 increased obviously. We purified one of the compounds and identified as oligomycin D by MS and NMR analysis. [Conclusion] An oligomycin D-producing strain Δspa7074 was derived via genetic engineering.