Preparation and characterization of sinefungin synthetase

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Sinefungin is a nucleoside antibiotic, in which a molecule of L-methionine is linked to the 5'-end of adenosine through a C-C bond. The antibiotic, isolated from the culture broth of Streptomyces incarnates NRRL8089, 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 S. incarnates produced sinefungin from L-arginine and ATP in the presence of pyridoxal-5'-phosphate, but the enzyme(s) involved in sinefungin-production have not been characterized clue to the instability and low expression of the responsible enzymes in the producer strain. In the present study we have constructed a new expression-vector of pTRA502, which derives from pKU110 shuttle vector. The present study was undertaken to identify genes involved in the sinefungin biosynthesis. We have constructed an original cloning vector of pTRA502 by replacing thiostrepton resistant gene with that of neomycin resistant gene of pKU110. Limited digestion of genome DNA was carried out with Sau3AI restrictions enzyme to obtain genetic fragments of about 5-6 kb. Optimal conditions were examined by restriction enzyme concentrations, enzyme concentration and incubation time. There was a problem that the collection of DNA was low in the process to extract DNA. The amount of collection and purity was improved by using the kit of glass beads. Shot-gun cloning is now being carried out with pTRA502 vector. The transformants were examined for the sinefungin producing ability by a capillary electrophoresis.