自溶链霉菌遗传操作研究

Genetic manipulation in Streptomyces autolyticus

放线菌新种自溶链霉菌能够产生一种称为自溶霉素的抗肿瘤活性产物.虽然自溶霉素具有成为抗肿瘤药物的良好前景,但是自溶链霉菌遗传操作方法的缺乏严重阻碍了对自溶霉素生物合成的研究.本工作对自溶链霉菌中各种遗传操作方法进行了探索.优化了PCR扩增的反应体系和程序,建立了外源DNA导入的方法,确立了构建基因敲除突变株的最佳方案.此外,也摸索了自溶霉素发酵和HPLC检测的适宜条件.利用所建立的方法对自溶链霉菌基因almRⅠ和almRⅡ的功能进行了初步研究,结果表明它们在自溶霉素生物合成过程中起正调控作用.自溶链霉菌遗传操作方法的建立为进一步开发利用放线菌资源奠定了基础.

A new species of actinomycetes,Streptomyces autolyticus,produces a bioactive compound called autolytimycin that possesses potent anti-tumor activity.Although autolytimycin is promising to be developed as an anticancer drug,the lack of genetic manipulation tools in S.autolyticus severely hampered the study on autolytimycin biosynthesis.In this work,a series of genetic manipulation methods in S.autolyticus were explored.The PCR amplification system was optimized,the most efficient way to introduce foreign DNA into S.autolyticus was established,and the best procedure for constructing knockout mutants was determined.In addition,the optimum conditions for fermentation and HPLC analysis of autolytimycin were also explored.The set of procedures was used to study the function of almRⅠ and almRⅡ genes of S.autolyticus,and it was found that they played positive regulatory roles in the biosynthesis of autolytimycin.The methods established for genetic manipulation in S.autolyticus set up a platform for the further studying and utilizing actinomycetes resources.