[Expression detection of med-ORF12 encoding a stereochemical ketoreductase possibly involved in medermycin...

Medermycin, an aromatic polyketide antibiotic produced by streptomyces, possesses a stereochemical-pyran-ring lactone critical for its strong anticancer activity. The med-ORF12 located in the biosynthetic gene cluster of medermycin encodes a stereochemical ketoreductase. Based on many indirect data, we proposed it to be involved in the enantioselective reduction at C-3 in the formation of the pyran ring of medermycin. The direct genetic evidence about the function of med-ORF12 in the medermycin-producing strain has yet to be obtained. Enzymatic features, expression and regulation pattern of Med-ORF12 in the medermycin-producer still remain obscure.

The present study aimed to investigate the expression profiles of med-ORF12 and relationship between Med-ORF12 and medermycin accumulation in medermycin-producers using prokaryotic expression, protein purification, polyclonal antiserum preparation, western blot.

First, we established a prokaryotic expression system of med-ORF12 using a pET vector and optimized the induction conditions to accumulate the soluble Med-ORF12. Subsequently, we acquired the polyclonal antiserum against Med-ORF12 by immunizing the New Zealand rabbit with the purified protein. Finally, we detected the expression pattern of med-ORF12 in the medermycin producers with the obtained polyclonal antiserum, and found that med-ORF12 could express with a fairly high amount during the late stationary phase of the medermycin-producers, consistent with the accumulation of medermycin as a secondary metabolite.

These data indicated that Med-ORF12 expressing efficiently in the secondary metabolism could be involved in the biosynthesis of medermycin in the medermycin-producers.