Purification and Molcular Cloning of Sinefungin synthetase fom Streptomyces incarnatus NRRL8089.

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Sinefungin is a nucleoside antibiotic, in which a molecule of L-omithine is linked to the 5'-end of adenosine through a CC bond. The antibiotic, isolated from the culture broth of Streptomyces incarnatus NRRL 8089, has a strong inhibitory effect on various fungi and trypanosome, and is expected to be useful as an anti-malaria drug as well. It has been reported that the cell-free extract prepared from a variant of incarnatus produced sinefungin from L-arginine and ATP in the presence of pyridoxal-5'-phosphate, but the enzymes involved in sinefungin-production have not been characterized due to the instability and low expression in the producer strain. The present study was undertaken to characterize the enzymes responsible for sinefungin production.In the present research, we have first established DNA methylse inhibition assay for detecting sinefungin. Then, the sinefungin producer strain underwent mutagenesis by ultraviolet light irradiation on the protoplast, and a high-producer stra … More in was selected among the mutants that acquired rifampicin-resistance. Because the enzyme activity of sinefungin synthetase was still not detected in the crude cell extract, we attempted to clone the genes involved in sinefungin production. The genome DNA was partially digested, ligated to a vector plasmid pKU110, and transformed in Streptomyces lividans TK24. Three transformants (#19, #20, and #21) were screened from over 200 of transformants as the candidate strain that produces sinefungin. The transformants were cultivated in 2 L of the medium, and sinefungin was isolated from the culture broth by means of charcoal column chromatography, cation-exchange column chromatography, and anion-exchange column chromatography. The strain #19 produced sinefungin more than the strains #20 and #21. We attempted to isolate the recombinant plasmids from these sinefungin-producing transformants, but the conventional isolation protocol did not yield plasmid. We examined the presence of the thiostrepton-resistance marker sequence, which is located in the vector plasmid, by polymerase chain reaction, but the corresponding DNA fragment was not amplified. It was concluded that the genes of sinefungin production from S. incarnatus were integrated to the genome DNA of S. lividans. Two references were published in the present research. Less