[Tn4560 transposition in Streptomyces ansochromogenes and its application for cloning of genes involved in nikkomycin biosynthesis].

No transformant was obtained when pUC1169(pMT660::Tn4556::vph) from S. coelicolor J1501 was used to transform the protoplast of S. ansochromogenes by general transformation for many times. Transformants were obtained when the protoplast of S. ansochromogenes was prepared at 50 degrees C for 30 min by heat attenuation of restriction. But the transformation frequency is very low, only 0.4 transformant per microgram DNA. Transformation frequency increased 10(3)-10(4) times when the protoplast of S. ansochromogenes was transformed by pUC1169 from itself. With Tn4560 transposition, 8 mutants lost ability to produce nikkomycin were screened from 4068 colonies with VioR-ThioS. Southern blotting suggested that Tn4560 was inserted in at least 4 positions of chromosomal DNA in S. ansochromogenes. By using the cloned 30 kb DNA fragment involved in nikkomycin biosynthesis as a probe, southern hybridization was carried out with 8 mutant total DNA digested with different enzymes. Only total DNA from Nik5 displayed signals, which were the same as those of the wild type. With HPLC analysis, 8 mutants could be classified into two types for nikkomycin production, one is Nik5, the other is Nik1, 2, 3, 4, 6, 7 and Nik8. As deduced preliminarily, the deletion of large chromosomal DNA fragment involved in nikkomycin biosynthesis took place in the 7 mutants. The partial gene library of Nik5 was constructed, the 4.2 kb and the 0.6 kb chromosomal DNA fragment flanking Tn4560 were cloned. Sequence analysis showed that the 4.8 kb DNA fragment (GenBank accession number:AF349618) contains two complete ORFs and one partial ORF.