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

Abstract:

Abstract: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．［Objective and Methods］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．［Results］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．［Conclusion］These data indicated that Med-ORF12 expressing efficiently in the secondary metabolism could be involved in the biosynthesis of medermycin in the medermycin-producers．