Isolation And Identification Of Streptomyces And Establishment Of Crispr/Cas9 Gene Editing Method

Streptomyces is the largest genus of actinomycetes,it produces a large number secondary metabolites with high application values,including various water-soluble and fat-soluble pigments as well as natural active substances which possess antibacterial activity,antifungal activity,antiviral activity and enzyme inhibitory activity,which can be applied in food and medicine industries.Soil is the main source of Streptomyces,has important research and application value to their isolation and identification.Streptomyces is an excellent host with clear genetic background for expression of exogenous gene.However,the final transformation efficiency is low due to the relatively complicated expression of foreign genes and the existence of a restriction modification system in Streptomyces.It is necessary to optimize the expression system in Streptomyces.Editing the genome of Streptomyces with traditional homologous recombination is a heavy workload and takes long time.In recent years,genome editing technology based on CRISPR/Cas9 system has the advantages of simple operation and high editing efficiency,and it is allowed to target and repair multiple genes simultaneously.Edit the genome and optimize the expression system of Streptomyces with this technique can contribute to establishing a more complete genetic manipulation system of Streptomyces and exploring more valuable secondary metabolites.In this study,Streptomyces were isolated and identified from soil by morphological analysis and molecular biology techniques.An efficient gene editing method for Streptomyces was established by using CRISPR/Cas9 system.The main results are listed as follows:1.Isolation and identification of Streptomyces.Ten strains were isolated and purified from the soil by three-zone plate scribing method,numbered LS-1 to LS-10,and the 16S rDNA region was amplified and sequenced respectively.The sequence of strain were compared to that of the database by EzBioCloud analysis.Downloading the model strains that have higher sequence homology,and then construct the phylogenetic tree using the Neighbor-Joining method in the phylogenetic tree construction software MEGA 6.0.Combining with morphological observation of 10 actinomycetes strains,they are identified as S.deccanensis,S.canus,S.collimus,S.melanogenes,S.albogriseolus,S.ossamyceticus,S.pseudovenezuelae,S.racemochromogenes,S.Xanthocidicus,S.albidoflavus.2.Construction of a Streptomyces gene editing plasmid.In this study,the PKS-NRPS hybrid gene sshg₀5713 in LS-10(S.albidoflavus)was selected for targeted knockout.Using pCRISPomyces-2 as prototype plasmid,the designed gRNA(24 bp)was inserted into pCRISPomyces-2 by Golden Gate Assembly.The white positive clones were screened by phenotypic blue-white spot,and the correct plasmid pCm-2-20nt inserted into gRNA(24 bp)was obtained by sequencing.The left and right 1 KB homologous repair templates were amplified by PCR from LS-10(S.albidoflavus).The modified plasmid pCm-2-2kb for LS-10(S.albidoflavus)was successfully constructed by inserting it into pCm-2-20nt by enzyme digestion.3.Establishment and optimization of gene editing methods for Streptomyces.E.coli S17-1/lamp pir and ET12567/pUZ8002 were used to conjugate and transfer.E.coli S17-1/lamp pir/pCm-2-2kb,ET12567/pUZ8002/pCm-2-2kb were introduced into LS-10(S.albidoflavus),respectively.The heterozygous gene sshg₀5713(67bp)was successfully knocked out.The editing efficiency of both strains was 75%.However,the editing time of E.coli S17-1/lamp pir as assistant bacteria is as long as 10 days,while that of ET12567/pUZ8002 is only 3 days,which is more than three times shorter.In this study,10 strains of actinomycetes were isolated and identified from soil,which provided resources for exploiting new secondary metabolites.An efficient gene editing method for Streptomyces was established based on type II CRISPR/Cas9 system.The PKS-NRPS hybrid gene sshg₀5713(67bp)in LS-10(S.albidoflavus)was successfully knocked out.This method can be applied not only to LS-10(S.albidoflavus),but also to other streptomyces.