Genetic manipulation system and genomic library of Streptomyces luteosporeus NRRL 2401

Abstract:

[Objective] To clone the biosynthetic gene clusters for secondary metabolites, we developed the genetic modification system and constructed a genomic library of Streptomyces luteosporeus NRRL 2401. [Methods] The genetic modification system was developed by using conjugal transfer vectors pSET152, pPM927 and pJTU1278 which were transferred from Escherichia coli ET12567/pUZ8002 to S. luteosporeus. The genomic library of S. luteosporeus NRRL 2401 was constructed by the fosmid vector pCClFOS^TM, with E. coli EPl300TM-T1R as the host strain. A PCR-based method was then developed for screening the biosynthetic gene clusters of secondary metabolites in the constructed genomic library. [Results] Vectors pSET152, pPM927 and pJTU1278 were successfully transferred into S. luteosporeus for genetic modification, with pSET152 presenting the highest transformation efficiency. The constructed genomic library of S. luteosporeus NRRL 2401 contained 2880 clones with an average -35 kb inserted DNA fragment in each clone, indicating the 99.99% coverage of the genome in the library. In this genomic library, we detected 9 clones containing possible indolmycin biosynthesis genes by the PCR-based screening method. [Conclusion] A stable, efficient genetic modification system and high-quality genomic library could be used for discovery of the biosynthetic gene clusters for secondary metabolites in S. luteosporeus NRRL 2401.