Study On The Elimination Methods Of Linear Mega-plasmid In Streptomyces Cattleya 46488

Compared with other prokaryotes,Streptomyces has a special genomic structure,which is characterized by a large genome,generally in Mb base pairs,high GC contect in genome,and is a rare species of bacteria containing linear chromosomes and linear plasmids.Streptomyces cattleya is one of the few microorganisms in nature that can synthesize fluorochemicals.Our team have performed a genome-wide sequencing of the Streptomyces cattleya DSM 46488,and the sequencing results were consistenet with the results of pulsed-field gel electrophoresis.It was found that the genome of this strain contains two replicons,which are 6.3 Mb linear chromosomes and 1.8 Mb linear megaplasmid respectively.The analysis of this mega-plasmid revealed that it did not encode any rRNA or tRNA genes associated with primary metabolism and did not contain conserved genes from other sequenced Streptomyces chromosomes.What's more,a replication initiation region of 2 kb in size and capable of conferring circular replication to the plasmid was identified and proved in the vicinity of the parAB gene.The plasmid in Streptomyces is linear and often has a low copy number,so it is more stable than the circular plasmid.However,it has been reported that linear plasmid in Streptomyces can be eliminated by knocking out the essential genes for plasmid replication process.Since the mega-plasmid of S.cattleya DSM 46488 does not contain any essential genes,this paper explores its stability in the genome by trying to eliminate it.First,we attempt to eliminate the plasmid by SDS with high temperature mutagenesis,but it has not been successful.After that,the parAB gene in the mega-plasmid was knocked out by method in molecular genetics.After several rounds of relaxation culture,it was found that the mega-plasmid was not eliminated.Therefore,a fragment of 8 kb including the minimal replication region and its upstream and downstream was knocked out,but after several rounds of relaxation culture,the mega-plasmid was still not be eliminated.What's more,we also attempt to eliminating the mega-plasmid by using the CRISPR/Cas9-CodA?sm?system which is codon-optimized and adapted for Streptomyces gene editing by Sun Yuhui.The CRISPR system is an immune defense system existing in prokaryotes.In order to explore the characteristics and unique features of the CRISPR system in Streptomyces cattleya,and achieving gene editing by using this system,we used bioinformatics method to compare and analyze 182 CRISPR loci in 46 strains of Streptomyces.The analysis including sequence alignment and similarity analysis of repeat sequence DRs,spacers;recognition of homologous sequences of spacers on foreign plasmids or phage;prediction and classification of cas gene clusters.Finally,two CRISPR loci were predicted in S.cattleya DSM 46488,and the CRISPR_IDs were NC₀16111₁,NC₀17586₁,respectively.In addition,there was no cas gene cluster be predicted near these two loci.Furthermore,the plasmid containing gDNA could not enter the S.cattleya DSM 46488 when we editing the mega-plasmid using CRISPR/Cas9-CodA?sm?.In conclusion,the linear mega-plasmid was tried to be eliminated by different methods,but no mutant strains can obtained,which confirmed that the 1.8 Mb linear mega-plasmid with a large proportion of the genome in the S.cattleya DSM 46488 is very stable.Besides,the analysis of 182 CRISPR loci in 46 strains of Streptomyces will help to carry out the genome evolution study of Streptomyces in the future.