Functional Analyses Of The Sviscsn Gene Cluster In Streptomyces Virginiae IBL14

CRISPR system is known as clustered,interspaced short palindromic repeats(CRISPR)in bacteria and archaea,which serves as an accessory immune function in prokaryotes.The exogenous genetic material(viral DNA or plasmid)fragment was cloned into the repeat interval of CRISPR to become spacer.When the exogenous genetic material reintroduces into the bacteria,CRISPR identified and degraded it by specific identification,so as to achieve the role of immunity.Streptomyces virginiae IBL14 is a laboratory strain which can grow on a variety of steroids as the only carbon source,and it was found that the strain can biocatalytic diosgenin(Diosgenin)F-ring(C-25 hydroxylated)microorganism as so far.The results showed that there were 18 CRISPR loci in chromosomes,among which there was a Cas protein group between the two CRISPR loci,and the order was cas7,cas5,cas3,cas4,cas1,Cas2,it was named as IB-svi type CRISPR-Cas system,it has been confirmed to be self-chromosome gene editing.cytochrome p450 gene(svu001),RmLC-like gene(svicup02),cytochrome p450 gene(svu015)and nitric oxide synthase gene(svinos01)were found in the chromosome of the strain.The transcript was named as sviscsn gene cluster.The aim of this study was to research the structure and function of cytochrome P450 in Streptomyces virginiae IBL14 and knock out the gene from four genes of sviscsn gene cluster through its own I B-svi type CRISPR-Cas system.The corresponding mutant strains were used to analyze the function of the enzyme gene in combination with biotransformation,physiological morphology and bioinformatics.The recombinant strain was constructed with Escherichia coli as the host,and the biotransformation of arginine was carried out by recombinant bacteria.product separation and identification,to determine the enzyme gene function of transformation pathway.The results of this study are as follows:1)There was a sviscsn gene cluster in the genome IBL14 by the bioinformatics software analysis;2)Five mutant strains IBL14?svu001,IBL14?svicup02,IBL14?svu015 IBL14?svinos01,IBL14?sviscsn were obtained from the knockout of I B-svi type CRISPR-Cas system 3)It is interesting that the sequence of knockout of sviscsn gene cluster resulted in a change of colony colour,but the colony colour of all knockout mutant was recoverd found that the five mutant strains grew in the medium;4)The four genes were cloned into Escherichia coli to achieve the corresponding recombinants,then to induce the expression and the addition of arginine.It was found that Svu001 and Svu015 could introduce N-atoms on L-arginine with regional and A-?-hydroxy.