Abstract: 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.