Preliminary Study On The Mechanism Of A Regulator Involved In The Biosynthesis Of An Aromatic Polyketide Antibiotic Medermycin

Medermycin, produced by Streptomyces sp. AM-7161, is a polyketide antibiotic and possesses antitumor and antibacterial activity. med-ORF10, a small gene located in medermycin biosynthetic gene cluster, has an undesignated function. Functional analysis in vivo has supported that med-ORF10could play a regulatory role in medermycin biosynthesis. The present research intended to investigate preliminaryly the mechanism of this gene involved in medermycin biosynthesis:1. Transcriptional analysis of a target gene regulated by med-ORF10using qRT-PCRBy Half-quatitive RT-PCR it was found previously that med-ORF10certainly regulates the target gene med-ORF12which encodes a ketoreductase. In order to further quantify the effect of its regulation, firstly we extracted total RNAs respectively from two strains including CH999/pIK340("heterologous-expression wild-type strain" S. coelicolor) and CH999/pAYT64("heterologous-expression wed-ORF10-deficient mutant strain" S. coelicolor), then reverse-transcribed them into cDNA. Secondly, we performed qRT-PCR using these total DNAs as templates and23S rRNA gene as control. Our data showed that at the level of transcription, the expression of med-ORF12in "heterologous mutant strains" was downregulated about5folds, compared to that in "heterologous wild-type strain". This result was consistent with the speculation that med-ORF10is a positive regulatory gene.2. Expression and purification of Med-ORF10med-ORF10was revelealed to play an important regulatory role in medermycin biosynthesis. In order to further study the regulatory mechanism, we expressed and purified Med-ORF10protein as well as preparing polyclonal antibodies against Med-ORF10.Firstly, med-ORF10was amplified from a medermycin-gene-cluster-containing plasmid pIK340by PCR. Then med-ORF10was subcloned into an expression vector pET28a(+), yielding a recombinant expression plasmid pHSL79. pHSL79was introduced into E. coli BL21(DE3) competent cells to establish the prokaryotic expression system BL21(DE3)/pHSL79. Highly efficient expression of Med-ORF10was achieved after optimization of induction conditions. After being purified, Med-ORF10was used to immunize the rabbit and mouse and polyclonal antibodies against Med-ORF10were prepared. Western blot showed the resultant polyclonal antibodies had high specificity against Med-ORF10. 3. The interaction between Med-ORF10and the promoter of the target genePreliminary studies have supported that med-ORF10is a positive regulatory gene by controlling expression of some target genes, inlcuding med-ORF12. The present research intended to investigate the relationship between Med-ORF10and the182-bp promoter (Pmed-ORF12) of med-ORF12by EMSA.Firstly, Pmed-ORF12was labeled by biotin. Then, the labeled Pmde-oRF12was incubated with pufied Med-ORF10protein. Finally, the mixtures were analyzed by electrophoresis in the native polyacrylamide gel. The EMSA showed that there was no obvious binding between Med-ORF10and Pmed-ORF12under the current conditions.