Klymyshyn D. Gene cloning system for nogalamycin producer Streptomyces nogalater IMET43360

Gene cloning system in nogalamycin producer strain S. nogalater IMET43360 was developed using intergeneric conjugation from E. coli ET 12567 (pUB307). Integrative conjugative plasmids pVWB and pRT801 are stably inherited in the chromosome of S. nogalater strain after several passages under non selective conditions. Transconjugants of these strains lost replicative conjugative plasmids pSOK101, pCHZ101, pKC1139, pKC1218 with high frequency after several passages without selective antibiotics. Dynamic of conjugative transfer from E. coli to S. nogalater IMET43360 was investigated and transconjugants starting from the twelve hour of mating were obtained. Plasmid pVWB integrates into one site of S. nogalater IMET43360 chromosome and pRT801 - into two sites. This is potentially very attractive for development of stable recombinant strains. Transconjugants S. nogalater containing pVWB plasmid has antibiotic production inhibited. Replicative conjugative plasmids and integrative pRT801 do not influent on the level of nogalamycin production. We discovered that the presence of all plasmids used in this study had no effect on cultural and morphological properties in S. nogalater IMET43360. The simplicity and universality of the intergeneric conjugation from E. coli ET 12567 makes it a very promising and powerful tool. Some features of nogalamycin biosynthesis regulation in S. nogalater IMET43360 were studied. Gene snorA was cloned from S. nogalater chromosome and showed to be involved in nogalamycin biosynthesis control at the level of activation of structural genes expression. This gene was disrupted within the S. nogalater IMET43360 chromosome. In this case mutants were characterized by the complete fail of nogalamycin production. The obtained strain, with disrupted snorA gene was called S. nogalater A1. This mutant strain was complemented via introduction of snorA gene in replicative plasmid. The level of nogalamycin biosynthesis in the obtained strain was restored. Such effect is typical for mutations in regulatory genes. Overexpression of snorA gene in the wild type strain leads to the increasing of antibiotic biosynthesis up to 5 times. The O-methyltransferase genes snogM, snogL and snogY were cloned from the nogalamycin biosynthesis cluster. Heterologous expression of these genes under control of constitutive promoter of pKC1218E plasmid in aranciamycin and doxorubicin producer strains S. echinatus DSM40730 and S. рeucetius subsp. сaesius was performed. It was found that S. еchinatus DSM40730 strain, containing copies of snogM gene in pKC1218E plasmid, produced a novel compound. This fact is a result of the aranciamycin molecule modification by the expression of this gene in S. еchinatus.