title: [Expression detection of med-ORF12 encoding a stereochemical ketoreductase possibly involved in medermycin biosynthesis].
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
  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.
  
  
  CONCLUSIONS
  These data indicated that Med-ORF12 expressing efficiently in the secondary metabolism could be involved in the biosynthesis of medermycin in the medermycin-producers.
authors.affiliation: College of Life Sciences, Central China Normal University, Wuhan 430079, China. rui.sun.ips@gmail.com
authors.forename: Rui
authors.initials: R
authors.lastname: Sun
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.forename: Caixia
authors.initials: C
authors.lastname: Gong
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Aibing
authors.initials: A
authors.lastname: Zeng
authors.forename: Aiying
authors.initials: A
authors.lastname: Li
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis*; D005798:Genes, Bacterial; D007106:Immune Sera / Q000276:immunology; D005810:Multigene Family; D009285:Naphthoquinones / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids; D011817:Rabbits; D000095622:Benzoisochromanequinones
chemical_list: D000903:Antibiotics, Antineoplastic; D007106:Immune Sera; D009285:Naphthoquinones; C013104:medermycin; D000095622:Benzoisochromanequinones