链霉菌负调控因子突变筛选系统的构建

Construction of screening system for mutation of negative regulatory genes in Streptomyces

【目的】构建用于阻遏链霉菌隐性次级代谢基因簇表达的负调控因子筛选的报告系统。【方法】通过"REDIRECT(Rapid Efficient Directed Recombination Time Saving)"技术结合链霉菌温和噬菌体BT1整合酶的体内位点特异性重组技术,对链霉菌中多基因进行无痕敲除。以链霉菌隐性次级代谢基因簇中受阻遏的启动子驱动链霉菌中保守的inoA构建报告质粒,针对阻遏次级代谢基因簇表达的负调控基因的突变进行检测,以验证报告系统的可行性。【结果】本研究首先通过对天蓝色链霉菌的肌醇从头合成途径关键酶基因inoA,及合成黄色聚酮类隐性抗生素(yellow cryptic polyketide,yCPK)的途径特异性负调控基因scbR2依次进行了无痕敲除,以构建进一步筛选所用的受体菌,再以scbR2阻遏的cpkO启动子控制inoA的表达构建了报告质粒pIJ8660::P cpkO::inoA。结果显示沉默的cpkO启动子在突变的受体菌中被激活并使inoA得到了表达,可以使inoA的光秃型突变表型在不添加肌醇的培养基上恢复到产孢的野生型表型。【结论】inoA可以作为新的链霉菌普遍适用的报告基因,可方便地通过表型变化的观察进行筛选,同时可针对性对负调控基因的突变进行检测,可应用于链霉菌隐性抗生素激活的研究。

[ Objective] We aimed to create a novel report system for screening the mutation of the negative regulatory genes, especially for those repressing the expression of cryptic antibiotics clusters. [ Metbods]We used marker-free gene disruption strategy, which combines with the ＂ REDIRECT （Rapid Efficient Directed Recombination Time Saving）＂ technology and in vivo site-specific recombination by Streptomyces phage ФBT1 integrase, to construct a scbR2/inoA double mutant strain of S. coelicolor M145. This strain was used as the host of the report system. For the construction of the reporter plasmid, the ScbR2 repressed promoter of cpkO from CPK （ cryptic polyketide） cluster was used to drive the expression of a promoterless conserved gene inoA of S. coelicolor. Then the reporter plasmid was introduced into the host strain described above to test the availability of inoA as a reporter gene in this system. [ Results] The scbR2/inoA double mutant strain gave rise to a bald pheno type on MM medium in the absence of inositol, and produced yellow pigmented secondary metabolite by the disruption of scbR2 to release the repression of cpkO, a pathway specific activator gene situated in CPK cluster. After introducing the reporter plasmid into this test stain, the resulting strain recovered the phenotype as wild-type strain, indicating that the promoter of cpkO can drive the expression of inoA in scbR2 mutant and consequently restore the biosynthesis of inositol. [ Conclusion] Our results indicated that inoA can be used as a novel reporter gene for Streptomyces, especially for detecting the activation of the ＂ silent＂ promoter. This report system might be available for screening the mutation of the negative regulatory genes for the cryptic secondary metabolic gene clusters.