Application Of Small Genome Technology In The Transformation Of Enramycin Producing Strains

Enramycin is a secondary metabolite produced by Streptomyces fungicidicus,an actinomycete isolated from soil.It has a good inhibitory effect on Clostridium capsulatum and Streptococcus in animals.Enramycin has many advantages over other antibiotics,such as good stability,low residue and resistance to drug.It has been widely used in poultry industry to add to animal feed to promote animal growth.In order to further simplify the genetic operation process of actinomycetes and shorten the screening cycle of the recombinant strains,a counter selectable marker gene--uracil phosphoribosyl transferase gene(upp)was introduced into the genetic operation of Streptomyces fungicidicus.By deletion of the upp gene in the recipient strain and construction of a suicide vector that carries the upp gene expression cassette,a inframe deletion system was developed.The introduction of upp gene shortened the average screening cycle of Streptomyces recombinant strains by about 2 weeks,and further reduced the probability of false positive recombinant strains.The simplicity of this system should make it adaptable for continuous markerless gene deletion in other actinomycetes.It is worth to be popularized.In this study,the genome of Streptomyces fungicides was optimized and simplified by using small genome technology on the basis of complete genome sequencing.The whole genome sequencing results showed that the genome consisted of 6740768 bp chromosomes.Bioinformatics analysis revealed that there were 6991 genes and 925644 bp linear plasmid in Streptomyces fungicidicus genome.In addition to the gene cluster related to enramycin biosynthesis,there were four large fragments of non-ribosomal polypeptide synthase(NRPS)gene and three larger polyketide synthase genes(PKS)in the genome.The total length of these seven gene clusters was 162788 bp.About 2.41%of the total genome.In order to simplify genome of Streptomyces fungicidicus to increase enramycin production and bacterial growth rate,the strategy of combining temperature-sensitive plasmids with tsr and upp forward and backward screening markers was adopted.Firstly,seven secondary metabolic gene clusters identified by genome data mining were knocked out separately.The inability of nrps1 and nrps4 to be knocked out may be related to cell wall synthesis.After nrps2 and nrps3 genes were knocked out,the production levels of strains under shake flask fermentation increased by 42.8 and 40.6%,respectively.The deletion of gene clusters other than pks3 after gene knockout of polyketides had a significant effect on mycelial growth,spore formation and enramycin production.Among them,the growth rate and the production level of pks1 gene knockout decreased significantly,and the spore color changed from original gray to gray-white after pks2 gene knockout.Then,in this study,nrps2 and nrps3 blocking strains with high production capacity were continuously and tracelessly knocked out.The increase of production level of double blocking strains did not exceed that of single blocking strains.It is concluded that enramycin production by Streptomyces fungicides is regulated by complex factors,not by the interaction of secondary metabolic flows of the same kind,which forms a complex network relationship.The production level of enramycin can not be calculated accumulatively by simple addition and subtraction.