The Polyphasic Taxonomy Of Two Novel Actinomycetes And Establishment Of Genetic Manipulation System Of Streptomyces Alarensis TRM 155-22

The special extremely saline-alkali environment of Xinjiang,may contain abundant resources of new actinomycetes species;The preliminary study of our group found that TRM 155-22,the new species of actinomycetes,can produce effective inhibitory oxanthenoxin on the cotton wilt.And biosynthesis gene cluster of oxanthenoxin has not been reported.In this study,two strains of actinomycetes from typical saline environment in Xinjiang were confirmed belonging to the new species of genus actinomycetes through polyphasic taxonomy.The genetic manipulation system of Streptomyces alarensis TRM 155-22 was constructed and the recombinant plasmids of oxy 1060 and oxy 1063 genes were constructed based on the analysis of TRM 155-22 genome sequencing.Key genes of Biosynthetic gene cluster of Streptomyces alarensis are about to be verificated to explore the synthetic pathway of oxanthenoxin and to establish the foundation for directional biosynthesis.1.Polyphasic taxonomy of new species TRM 46250 and TRM 41337By identifying polyphasic taxonomy,including genotype characteristics,morphological characteristics,physiological and biochemical characteristics and chemical classification characteristics of the strains TRM46250 and TRM 41337,and comparative analysis on similar strains confirmed these two strains belonging to the new species of genus actinomycetes,and they are named Nocardiopsis akesuensis and Streptomyces salilacus.2.Establishment of genetic manipulation system of Streptomyces alarensis TRM 155-22The genetic manipulation system of the anthrone antibiotic oxanthroxin-producing strain Streptomyces alarensis TRM 155-22 was established in order to knockout the oxanthrone-related biosynthetic gene.Firstly,the protoplasts of Streptomyces alarensis TRM 155-22 were successfully prepared,Then,the integrated plasmid pKC1139 was used as exogenous DNA to investigate and optimize the Methods for transformation and protoplast transformation of Streptomyces alarensis TRM 155-22,and the plasmid pKC1139 was successfully transformed into Streptomyces alarensis.At last,comparison of these two methods showed that the transformation method of Escherichia coli-Streptomyces was more simple,and the conversion efficiency was 84.2% in MS medium.so,the best way to transfer into the exogenous gene was determidend to be conjugative transfer.3.Construction of Recombinant Plasmid of oxy 1060 and oxy 1063 of Streptomyces alarensis TRM155-22After whole genome sequencing of Streptomyces alarensis,we analyzed the genomes using antiSMASH,and found the two key genes,oxy 1060 and oxy 1063,in synthesis process.Homology analysis showed that oxy 1060 has 85% identity with actinorhodin polyketide synthase acyl carrier gene and oxy 1063 has 92% identity with actinorhodin polyketide putative beta-ketoacyl gene.So,we suggested that it was probaly involved in starting and elongation of polyketide chains.We designed the primers to amplify the upstream and downstream homologous arms of the two key genes and the Apr resistance gene.Then this five fragments were ligated into the pSK plasmid for sequencing.The sequencing results showed that the amplified fragment was 100% matched with the designed gene fragment without mutation.Then,we ligated the upstream and downstream homologous arms of oxy 1060 and oxy 1063 and the Apr resistance gene into pJTU1278 plasmid,the recombinant plasmid of oxy 1060 and oxy 1063 of Streptomyces alarensis TRM 155-22 was successfully established,aiming at knocking out and analyzing the oxy 1060 and oxy 1063 gene.The identification of two strains of actinomycetes enriched the resource library of the actinomycetes species.Establishment of genetic manipulation system and Construction of Recombinant Plasmid of oxy1060 and oxy 1063 provided research foundation to analize the oxanthenoxin of Biosynthetic gene cluster.