Study On The Mechanism Of Genes Involved In The Biosynthesis Of An Aromatic Polyketide Antibiotic Medermycin

Medermycin,a polyketide antibiotic,is produced by Streptomyces sp AM-7161.It belongs to a benzoisochromanequinone antibiotic family with strong antibacterial and anti-tumor activity.It is featured with two opposite chiral centers at C3 and C15(C13S/C15R).The present research aimed to characterize the function of a tailoring structural gene med-ORF9 and a regulatory gene med-OrF10 located in medermycin biosynthetic cluster:(1)Characterization of med-ORF9 proposed to be a tailoring gene in medermycin pathway.Blast analysis of med-ORF9 sequence released in GenBank proposed it to be an enoyl-reductase-encoding gene,probably involved in the formation of C15R configuration of medermycin.However,the sequence of med-ORF9 amplified from the wild type strain was proved to be different from that in GenBank,and proposed its enoyl-reductase-activity to be lost due to the point mutation(namely as med-ORF9*).Firstly,function correction using the med-ORF9 sequence from GenBank was carried out via DNA synthesis,PCR and subcloning.The correct med-ORF9 was cloned onto an integrative plasmid pIJ8600,generating an expression plasmid pHSL108.After introducing this plasmid into the wild type strain,LC/MS analysis of the resultant recombinant strain liquid culture indicated obviously.increased yield of medermcin accumulation,in an obvious contrast to that in the wild type strain.Meanwhile,under the current analytic conditions,two compounds related to medermycin were detected accumulated,probably representing two different chiral structures of medermycin.Next,we performed knockout of med-ORF9*in the wild-type strain.The resultant med-ORF9*-deficient mutant still could accumulate medermycin and its proposed chiral structure,implying that this gene(med-ORF9*)with point mutation became inessential for medermycin biosynthesis,while the correct med-ORF9 still plays an important role in controlling the stereospecificity of medermycin.In order to perform enzymatic assay in vitro,we established prokaryotic expression systems of med-ORF9:using pRSET(A)and pET28a as vectors,both correct med-ORF9 and mutant med-ORF9*were cloned onto these vectors respectively,and then introduced into E.coli BL21(DE3)for inducible expression.We could detect expression of the correct med-ORF9 in E.coli,but the mutant med-ORF9*could not express in E.coli.(2)Mechanistically study on a regulatory gene med-ORF10med-ORF10 was a regulatory gene located in the biosynthetic cluster of medermycin.Previous qRT-PCR assay suggested that it controlled the expression of med-ORF12,a structural gene in the tailoring step of medermycin biosynthesis.Here,we used qRT-PCR to detect whether any other med genes were controlled by med-ORF10.Our data showed the transcription levels of many genes besides med-ORF12 were regulated significantly by Med-ORF10,including med-ORF11,med-ORF1.Interestingly,the expression of med-ORF11(another regulatory gene located in the medermycin cluster)showed remarkable difference by 70 fold between the wild type strain and med-ORF10-deficient strain.In order to rule out the possibility that the expression of these structural genes were directly controlled by med-ORF11(i.e..via an indirect controlling mode by med-ORF10),we detected the expression levels of these genes in the med-ORF11-overexpressing strain and found that the increase of med-ORF11 expression would not promote the expression of these several structural genes.Using polyclonal antibody of med-ORF10 prepared in previous experiments,we could not detect the expected-sized protein band indicating med-ORF10 expression in the wild-type strain.Here,we fused the sequence of Flag tag to med-ORF10 and constructed a fusion protein-encoding gene(Flag-med-ORF10).After it was cloned onto an expression vector and introduced into the wild type strain,we could detect the expected-sized protein band,indicating that med-ORF10 was expressed in the wild-type strain.Previous experiments revealed that Med-ORF10 could not bind the promoter regions of its target genes.To further investigate if Med-ORF10 plays its regulatory role via protein interaction,we selected the fusion-gene(Flag-med-ORF10)-containing strain for co-immunoprecipitation experiments,using a med-ORF10-deficient strain as negative control.After centrifugation for the protein complex composed of argarose-bead-proteinA/G-antibody-Flag-Med-ORF10 and possible target protein,we performed immuno-detection for the interaction between Med-ORF10 and its possible target protein using polyclonal antibodies against two proteins in medermycin pathway(glycosyltransferase Med-ORF8 and ketoreductase Med-ORF12).However,under the current analytic conditions,we could not detect any interaction between Med-ORF10 and these two structural proteins,though both them were controlled by Med-ORF10 at transcription and translational levels,confirmed previously.