| Marine actinomycetes have the potential to synthesize new natural bioactive products different from terrestrial actinomycetes due to their special marine habitats.However,under the current culture conditions,most of the secondary metabolic gene clusters of marine actinomycetes are silent.New techniques are needed to activate these silent gene clusters so as to develop the potential resource of marine actinomycetes.In this paper,we hope to develop a genome-based cluster activation strategy suitable for marine actinomycetes,which includes optimization of genetic manipulation,utilization of promoters and application of CRISPR/Cas9 gene editing technology.First,in order to test whether the CRISPR/Cas9 system previously constructed in our laboratory can be applied to actinomycetes,we applied Streptomyces venezuelae ATCC 15439 and Streptomyces laurentii ATCC 31255 as the experimental objects.Experimental conditions such as the ratio of donor and recipient,heat shock temperature,conjugation time and antibiotic dosage were optimized.The effects of CRISPR related components Cas9 and sgRNA on the growth of the strain were also tested.We improved the low conjugation efficiency of non-pattern strains and accumulated experience for the genetic manipulation of Marine actinomycetes.Secondly,this study designed and synthesized a series of promoters,which include constitutive type and inductive type.We used the strain Streptomyces coelicolor M512 as the host,the positive regulatory gene redD of natural red pigment synthesis as the report gene.We used the free plasmid vector pCas9intoMyces-0 to transfer the promoter and report gene into the host.The contents of red pigment in fermentation products was detected.Constitutive promoters P5/P6 with overexpression ability and promoters P9/P10 with induction effect were screened out.These promoters can be used for subsequent activation of silencing gene clusters in marine actinomycetes.Then we carried out the gene editing of marine actinomycetes.23 secondary metabolite gene clusters were located by antiSMASH in the genome of marine actinomycete Streptomyces sp.HNS054.We successfully knocked out 10 of these gene clusters.And the constitutive promoter P5 was inserted into the upstream of the regulatory gene(lasR2)of gene cluster BGC1.1.Finally a total of 11 modified strains with good genetic stability were obtained.Phenotypic analysis was performed on the genomic modified strains.By detecting the antibacterial activity of the fermentation broth of these strains,we found that the antibacterial activity of the HNS054 against Bacillus subtilis disappeared after three gene clusters(including BGC1.1)were knocked out seperately.We preliminarily judged that these three gene clusters were related to the biosynthesis of antimicrobial active substances secreted by HNS054.However,the modified strain that inserted promoter P5 to increase the expression level of BGC1.1 gene cluster did not increase its antibacterial activity.We detected the fermentation extracts of the above strains by LC-MS and observed the changes in the expression of secondary metabolites in the modified strains.In conclusion,this paper successfully realized CRISPR/Cas9 gene editing of marine actinomycetes Streptomyces sp.HNS054.A series of genetically modified strains were obtained and a gene-editing protocol for marine actinomycetes was established from genome prediction,plasmid vector construction,conjugation transfer to genotype and phenotype verification.This paper will lay a foundation for the future research on natural active products of marine actinomycetes. |
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