Functional Study On A Few Regulatory And Structural Genes Located In Medermycin Biosynthetic Gene Cluster

Medermycin,a polyketide antibiotic,is produced by Streptomyces sp.AM-7161,and can be active against Gram-positive bacteria and many types of tumors.A few proposed regulatory genes including med-ORF11 and med-ORF10 located in medermycin biosynthetic gene cluster has yet to be investigated,while med-ORF9 in the same cluster was proposed to encode an enoyl reductase involved in pyran ring formation.The present research intends to investigate the function of these genes in medermycin biosynthesis in a wild-type producer Streptomyces sp.AM-7161 as follows:(1)Overexpression of med-ORF11 in Streptomyces sp.AM-7161Similarity analysis showed that Med-ORF11 belongs to Streptomyces antibiotic regulatory protein(SARP)family which are activators and widely distributed in antibiotics biosynthetic pathways.So med-ORF11 was supposed to regulate positively the medermycin biosynthesis.In the present research,med-ORF11 amplified from the genomic DNA of medermycin wild producer Streptomyces sp.AM-7161,was cloned into the expesssion vector pIJ8600 which could intrgrate onto the chromosome of many Streptomycete hosts,to generate an expression plasmind named as pHSL49.After pHSL49 was introduced into Streptomyces sp.AM-7161 by conjugation,the resultant exconjugant AM-7161/pHSL49 were confirmed by antitiotic selection and PCR amplification.AM-7161/pHSL49 was incubated on R4 agar plates and shows stronger redbrown color indicating medermycin production than AM-7161,impling med-ORF11 might promote production of medermycin.(2)Gene knockout of med-ORF 11 in the wild type producer AM-7161Since overexpression of med-ORF11 was observed to promote medermycin production,next we hope to study whether medermycin production decreases if the gene med-ORF11 was knockout from the genome of AM-7161.The 2-kb left region(left arm)in the upstream of med-ORF11 and the 2-kb right arm in the downstream of med-ORF11)were amplified respectively,and ligated together,then cloned into the vector pYH7 with sagregational instability to generate pHSL47 to be used for/med-ORF11 knockout.Next,pHSL47 was introduced into the Streptomyces sp.AM-7161 by conjugation,med-ORF11 deficiency mutant AM-7161-WW-1 was selected from the exconjugants and confirmed by PCR amplification.AM-7161-WW-1 was incubated on R4 agar plates and showed a significant reduction of medermycin production than AM-7161,based on the observation of lighter redbrown pigmentation.HPLC analysis of the broth of AM-7161-WW-1 liquid cultivation showed the medermycin production in AM-7161-WW-1 was decreased sharply comparing with that in AM-7161,further suggestiong that med-ORF11 product acted as an activator in medermycin biosynthesis.(3)Interaction between of Med-ORF 10 and target promoter Pmed-ORF 12med-ORF10 was found previously to function involved in the regulation for medermycin biosynthesis with unknown mechasim.Transcriptional analysis and similarity analysis deduced that expression of a structural enzyme in medermycin pathway,Med-ORF 12 should be regulated by Med-ORF 10.but it still remains obscure whether Med-ORF 10 will operate on the promoter of med-ORF12 in a direct or indirect way.Firstly,we wanted to test whether the 182-bp proposed promoter region(Pmed-ORF12)of med-ORF12 could be active in E.coli:we introduced pHSL33 carrying(Pmed-ORF12)located upstream of a reporter gene GFP into heterologous host E.coli BL21(DE3).Western blot showed GFP expression under the existance of Pmed-ORF12in the recombinant strain BL21(DE3)/pHSL33,implying the region of Pmed-ORF12)is active in E.coli.Then,we intended to introduce the med-ORF 10-expression plamid pHSL79 into BL21(DE3)/pHSL33 to test whether Med-ORF10 could promoter GFP expression when these two plasmids coexist in BL21(DE3).Theoretically,they could be coexisted in one cell under the selection with two antibiotics(Kanamycin and Apramycin),because pHSL79 and pHSL33 carry kanamycin-and apramycin-resistance genes respectively.However,we found that these two plasmids can not coexist and were recombinated together in two-plasmid system,cofired by enzyme digestion and PCR amplification.(4)Anlysis of med-ORF9 involved in the pyran ring formation of medermycinmed-ORF9 was deduced to be an enoyl reductase gene,and proposed to be involved in pyran ring formation the the medermycin biosynthetic pathway.But we found that in AM-7161,this gene was proposed to be lower efficiently functional or disfunctional,because bioinformatic analysis demostrated that the N-terminal of med-ORF9 shows significantly difference from its homologous gene actVI-ORF2,which was proved previously to participate in the pyran formation of actinorhodin.So we amiplified gene actVI-ORF2 firstly from the actinrhodin gene cluster,then constructed an expression plasmid pHSL48,The introduction of pHSL48 in AM7161 was under the way to check whether if medermycin production will be increaded if med-ORF9 could be restored by the actVI-ORF2.