Molecular Breeding And Its Classification Of Isthromycin Producing Strain

Bitespiramycin(BT) is a fermentation product of a genetically engineered strain which was constructed by integrating the4"-isovaleryltransferase gene(ist) from streptomyces thermotolerans through homologous recombination into the chromosome of spiramyc in-producing strain streptomyces spiramyceticus F21. BT had completed the clinical trial and now is in the application process of obtaining new drug certificate. Bitespiramycin is a multi-component antibiotic consisted mainly of4"-isovalerylspiramycin(isomycin) Ⅰ, Ⅱ and Ⅲ. Study showed no significant difference in antibacterial activity among BT and between isomycin Ⅰ,Ⅱ,Ⅲ. So single component of isomycin may exhibit similar antibacterial activity as multi-component of BT.Obtaining single component of isomycin producing strain would greatly facilitated to improve the drug quality control and preparation process in commercial production. In previous study, a isomycin-Ⅰ producing strain WSJ-2was constructed by inactivation of the3-0-AT gene (sspA) in WSJ-1which was responsible for the acylation of SP I to SP Ⅱ and Ⅲ.But the low productivity of isomycin-Ⅰ by WSJ-2strain restricted its practical application at large scale.AcyB2, a novel transcriptional activator, neighbour with acyBl(ist) gene in carbomycin (16-membered macrolide antibiotic) producer Streptomyces thermotolerans, was proved to be essential for the expression of ist in Streptomyces. The ist gene could be expressed in Streptomyces lividans only in the presence of acyB2gene,but so far, increasing fermentation production and proportion of the single component of antibiotics by heterologously introducing the two genes(ist and acyB2) into antibiotic producer strains had not been reported. In this work, the construction of genetically engineered strain with high proportion and high production of isomycin Ⅰ,a single component of biterspiramycin,through co-expression of linked ist-acyB2genes in Streptomyces spiramyceticus WSJ-2was presented.A recombinant plasmid pSET152-ia, carrying4"-isovaleryltransferase gene(ist) with positive regulatory gene acyB2in Streptomyces thermotolerans, was constructed and introduced into Streptomyces spiramyceticus WSJ-2by protoplast transformation. The recombinant plasmid pSET152-ia was integrated into the chromosome of WSJ-2by attp attachment site. A new isomycin I producing strain, Streptomyces spiramyceticus WSJ-IA, was generated. Under appropriated conditions the fermentation titre of Streptomyces spiramyceticus WSJ-IA was4.14times higher than its original strain WSJ-2and the proportion of isomycin I was3.47times higher than WSJ-2. Experimental data proved that introduction of linked ist-acyB2genes into Streptomyces spiramyceticus improved the proportion of the isomycin single component and its fermentation titre significantly in WSJ-2. These results will facilitate to develop bitespiramycin single component as an effective new antibiotic with WSJ-IA strain and promote further studies on the regulatory mechanism of heterologous expression of ist gene in Streptomyces spiramyceticus. Bitespiramycin (BT) is a group of4"-acylated spiramycins with4"-isovalerylspiramycin Ⅰ, Ⅱ and Ⅲ as the major component. Isovalerylspiramycin (isomycin) Ⅰ is a single component of BT generated from a gene diruptant of BT bioengineerd strain.A Streptomyces spiramyceticus WSJ-IA strain (herein designated as an isomycin I producer) with high proportion and high production of isovalerylspiramycin I was obtained by cloning and expression of acyB2(Regulatory gene) and ist(Isovaleryl transferase gene) into an isomycin I producting strain. As a single component of BT isomycin Ⅰ has the potential in developing of a commercial drug. Since WSJ-IA and BT producer both originated from Streptomyces spiramyceticus F21have been passed through sequencial manipulations by gene engineering and multiiple mutation breeding, it is essencial to carryout taxonomic studies to define the engineered WSJ-IA and its original parent strain Streptomyces spiramyceticus F21for inspecting the genetic stability, meanwhile, which could be served as a genetic markers in protecting the production rights in the future.Streptomyces taxonomy has become current study hot spot, the molecular taxonomy is developing quickly, which is based on the analysis of the highly conserved sequences of streptomyces16S rRNA genes, but it is proved that insufficient resolution in many species relationship whithin streptomyces remain. Multilocus sequence analysis provides a valuable tool that could help to reine the syntematic localization of this genus.Taxonomic studies were carried out to define the engineered isomycin I producer and its originated strain Streptomyces spiramyceticus F21.16S rRNA gene sequencing and five housekeeping genes(atpD、gyrB、rpoB、recA and trpB)coding proteins sequencing combining the traditional taxonomic studies, such as morphological, cultural, physiological and biochemical characteristics were performed for both strains. The Streptomyces spiramyceticus WSJ-IA and Streptomyces spiramyceticus F21share highly similar phenotypes and16S rRNA gene sequences as well as in the phenogenetic analysis of the five housekeeping gene protein sequences. While their16S rRNA gene sequences and the five housekeeping gene protein sequences phylogenetically were located in different clades with all other strains so far described. And the closely related strains with each gene coding protein were also different, none of them was reported as spiramycin producer. Our results suggested that Streptomyces spiramyceticus F21was possible a new Streptomyces sp. of spiramycin producer.16S rRNA sequence and five housekeeping genes (atpD、gyrB、rpoB、recA and trpB) coding proteins sequences could be served as genetic markers in identifying the engineered strain’s stability in long term of the commercial production. This study implied the significance of using these genetic markers in the inspecting the genetic stability of genetic streptomyces strains in the long term of large-scale production.