A mutant Y3 blocked in the thienamycin biosynthetic pathway was obtained from thienamycin producing strain Streptomyces cattleya by NTG treatment. Preliminary cloning system has been established on the basis of studies on the conditions for protoplast formation, regeneration, as well as DNA transformation for Y3 mutant strain. The shotgun cloning was carried out from S. cattleya using pIJ680 as a vector and the Y3 mutant as a host. The transformant No. 12 produced a thienamycin-like substance identified by paper chromatography and HPLC analysis. A recombinant plasmid p6BC12, which has molecular size of 9.8kb and an insert of 4.5kb, could be recovered. Southern hybridization confirmed that the transformant No. 12 harbors the recombinant plasmid p6BC12. The intermediate accumulated by Y3 mutant was identified as a polypeptide. The product of transformant containing p6BC12 could turn this polypeptide to a bioactivity substance in vitro. This gene can hybridizate with S. lipmanii IPNS gene. We presume that a cyclase gene from S. cattleya was cloned according to the function of the expression product.