The Polyphasic Taxonomy Of Three Novel Actinomycetes And Construction Of ActD-24 Gene Recombinant Plasmid Of Streptomyces Mutabilis TRM45540

Southern Xinjiang sufficient duration of sunshine, less rainfall, dry climate, forming a typical extreme salinity environment, which is rich in actinomycetes new species. In this study, three new species of actinomycetes from southern saline environment were multiphase taxonomic identification and to determine its classification status; at the same time, we got a high yield actinomycin D of Streptomyces mutabilis TRM45540 from Lop Nur, bioinformatics to predict the strain actinomycin D biosynthetic gene cluster, presumably Act D-24 gene is a membrane transporter function and regulats actinomycin D synthesis, but not yet seen some relevant reports about it. This study constructed a Act D-24 gene recombinant plasmid of actinomycin D gene cluster of streptomyces mutabilis TRM45540, in order to verify the Act D-24 gene function and its function in the biosynthetic pathways of actinomycin D. The main research results as follows:1. The strain TRM46794-61, TRM49032 and TRM49642 through polyphasic taxonomy, including genotype characteristics, morphological characteristics, physiological and biochemical characteristics, chemical classification characteristics, and similar strains comparative analysis confirmed the three strains belonging to the new species of genus Streptomyces, followed by the name Streptomyces canalis, type strain TRM46794-61T; Streptomyces gobiensis, type strain TRM49032T; Streptomyces ruoqiangensis, type strain TRM49642 T.2. CD-Search of NCBI database predicted the function of Act D-24 gene has a conserved domain of membrane transport proteins; while BLAST alignments showed the sequence of Act D-24 gene has the most similar with the putative membrane protein of streptomyces lividans TK24 at 87% similarity, therefore presumed Act D-24 gene has a membrane transporter function. Constructing p KC1139-T1 Kan T2 mutation carriers to verify the function of Act D-24 gene. The specific process of mutation carriers build: design of homologous upstream fragment length of 411 bp, downstream fragment length of 573 bp, a marker gene(kan) fragment of 1491 bp, and the three fragment was connected with the shuttle vector p KC1139 by enzyme-linked way, obtain mutation carrier p KC1139-T1 Kan T2, about 9 kb. The mutation carriers into E.coli ET12567, and complete recombinant plasmid.In this study, the identification of three new species of actinomycetes enriched the actinomycetes species repository, and the construction of Act D-24 recombinant plasmids provided the research foundation of the regulation of actinomycin D.