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

Medermycin is an antibiotic produced by many streptomycetes, including Streptomyces sp AM-7161. Medermycin possesses antitumor and antibacterial activity as a strong inhibitor against signal transduction pathways in many types of tumor cells. In medermycin biosynthetic gene cluster, med-ORF10 was a gene with undesignated function. Because its homologies are found in many other antibiotic biosynthetic gene clusters, we speculated that med-ORF10 and its homologous genes are essential in these antibiotics biosynthetic pathways, and they may regulate the biosynthesis of these antibiotics.In order to investigate the function and mechanism of a proposed transcription regulator gene (med-ORF10) in the medermycin biosynthetic gene cluster, we utilized genetics, molecular biology, analytical chemistry methods to determine med-ORF10 function in vivo, and performed a preliminary study on its action mode:1. Genetic experiments show that med-ORF10 possesses a regulatory functionFirstly, we introduced a med-ORF10-containing plasmid pHSL98, derived from a streptomyces auto-replicating plasmid pWHM4*, into a medermycin-producing strain, Streptomyces sp. AM-7161, by protoplast transformation for overexpression of med-ORF10; Secondly, we determined the pigmentation level of over-expression strain AM-7161/pHSL98. Our data showed that the over-expression strain could accumulate obviouly stronger pigmentation than the wild type strain AM-7161 both on solid and liquid media, implying the over-expression of med-ORF10 can promote the production of medermycin; Thirdly, we measured the fermentation broth of AM-7161/pHSL98 by LC/MS, and found that the production of medermycin in the over-expression strain AM-7161/pHSL98 was promoted by about 2.3-fold as well as the production of an intermediate increased by about 6-fold. These data show that med-ORF10 may be a regulatory gene as an activator.2. Target genes located in the medermycin gene cluster and regulated by med-ORF10 were determined using RT-PCRFirstly, we extracted total RNAs respectively from two strains ("heterologous-expression mutant strain Streptomyces coelicolor CH999/pAYT64" and "heterologous-expression wild-type strain Streptomyces coelicolor CH999/pIK340); Secondly, we performed half-quantiative RT-PCR using these total RNAs as templates and 23 S rRNA as control. Our data showed that the transcription of some genes including med-ORF12, med-ORF1 and med-ORF11 in the mutant CH999/pAYT64 was promoted at different levels, comparing to that in CH999/pIK340, suggesting that the expression of these genes might be regulated by med-ORF10 in a direct or indirect way.3. Preliminary study on the mechanism of med-ORF10 regulating target genesFirstly, we constructed a positive control system for promoter-detection in the wild-type Streptomyces sp AM-7161 using a proved constitutive promoter Pactâ…¢which was cloned onto a streptomyces-promoter-probe vector pIJ8660 (the resultant plasmid was named as pHSL32). We introduced pHSL32 into AM-7161 by protoplast transformation, and could detect green fluorescence from GFP expression in AM-7161/pHSL32 under the fluorescence microscopy. Secondly, we analyzed the proposed promoter regions on the medermycin gene cluster and cloned the proposed promoter (Pmed-ORF12) of a main target gene med-ORF12 onto the same promoter-probe vector pIJ8660, and obtained a new plasmid pHSL33. The transformation of pHSL33 into AM-7161 was under the way.