Identification Of A Medermycin-producing Streptomycete And Functional Study On The Med-ORF10 Involved In Medermycin Biosynthetic Regulation

The strain AM-7161(AM-7161) produces an antibiotic medermycin showing strong anticancer activity. However, as a producer of an important antibiotic, this strain has not been classified systematically so far and was temporally referenced as Streptomyces sp. AM-7161. med-ORF10 gene located in the medermycin biosynthetic cluster was proposed to be a novel transcriptional regulatory gene. In the present study, the strain AM-7161 was firstly identified by classical classification and 16S rRNA-sequence analysis; and then functional analysis of med-ORF10 was conducted by bioinformatics, gene complementation and gene knock-out. The main results are summarized as follows:The strain AM-7161 showed a typical morphological characteristics of Streptomycetes by Gram-staining and SEM observation as well as growth curve on different cultural conditions, but grew much faster than the common streptomycetes. Phylogenetic tree analysis of AM-7161 on the basis of 16S rRNA-sequence indicated that the Streptomyces sp. AM-7161 and Streptomyces nashvillensis strain xsd08147 share 99%homo logy at the nucleotide sequence, therefore, we suggest that the AM-7161 will be classified into a subspecies of Streptomyces nashvillensis.Bioinformatics analysis for the primary, secondary and tertiary structure of Med-ORF10 shows that Med-ORF10 is a small and hydrophobic protein containing threeα-helices, fourβ-sheets and a SH3 (src homolog 3) motif as well as a casein kinase 2 motif. Only one conserved NTF2 domain (Nuclear Transporter Factor 2) was found in Med-ORF10. Homologous genes of med-ORF10 are found to exist widely in the biosynthetic gene clusters of many aromatic polyketide antibiotics, implying that it is an essential gene for biosynthesis of these metabolites. med-ORF10 shares the highest homology with the act VI-ORFA gene which is a novel and positive transcriptional regulatory gene in the biosynthesis of actinorhodin. Therefore, med-ORF 10 is considered to have the similar function to act VI-ORFA.Molecular genetic analysis of med-ORF10 in vivo showed that:both the reduced production of spores and accumulation of medermycin were observed by the knock-out of med-ORF 10 and its function was restored by complementation with med-ORF10, indicating that med-ORF10 is a positive regulatory gene in the pathway of medermycin biosynthesis and might have an impact on the differentiation of Streptomyces sp. AM-7161.The present study provides us with useful information for the further research of the biosynthetic pathway of medermycin.