| Actinomycetes have the characteristics of wide distribution,strong adaptability and diverse metabolites,and are important biological resources of antibiotics and anti-tumor and anti-plant diseases.Under the conventional laboratory culture conditions,most natural product biosynthetic gene clusters are in a state of no expression or low expression.Therefore,the establishment of genetic manipulation system of actinomycetes with potential for mining secondary metabolites is helpful to explore active natural products and analyze the biosynthetic pathway of target active molecules.Two Streptomyces strains CPCC 202372 and NEAU-GS3 with good antibacterial and antitumor activities were obtained by fermentation and metabolic spectrum analysis of the existing strains in the laboratory.Through the analysis and identification of their main secondary metabolites,it was determined that the main secondary metabolites of strain CPCC 202372 was polyketmycin,and the main secondary metabolites of NEAU-GS3 were GTRI-02 and SEK-4b.Only two kinds of compounds were found in the above research,suggesting that these two strains have the potential for further exploration.In addition,as an important resource,Amycolatopsis orientalis has always had difficulties in genetic manipulation.Therefore,this study intends to evaluate the potential of three strains by genome-wide sequencing and bioinformatics analysis,and establish efficient and stable genetic manipulation systems for three strains of actinomycetes by using CRISPR-cBEST technology.Then,the established system is used to construct the mutant inactivated strain of the target gene,locate the biosynthetic gene cluster of the main active compounds and infer the function of the inactivated gene based on metabolic spectrum analysis.The main results are as follows:（1）The whole genome analysis of Streptomyces CPCC 202372 showed that the genome size was 11.2 Mb and the DNA G+C content was 70.12%.Bioinformatics analysis showed that there were 32 gene clusters encoding secondary metabolites in the genome,and the similarity between Region 3 and the reported gene cluster of polyketomycin biosynthesis reached 95%,suggesting that Rregion 3 may contain the gene cluster responsible for polyketmycin biosynthesis.In addition,the genome size of NEAU-GS3 is 8.3 Mb,and the content of DNA G+C is 72.64%.Bioinformatics analysis shows that there are 27 biosynthetic gene clusters encoding secondary metabolites in the genome,among which Region 29.1 contains the gene cluster for the synthesis of polyketide II.It is speculated that Region 29.1 may contain the biosynthetic gene clusters of GTRI-02 and SEK-4b.（2）The genetic manipulation systems of two non-model Streptomyces strains,CPCC 202372and NEAU-GS3,was established for the first time by conjugation transfer and CRISPR-cBEST technology.The specific conditions of CPCC 202372 are as follows:adding 75 mmol/L Mg Cl2to the culture medium;the spores were thermally stimulated at 45℃for 10 min and pre-germinated for 2 h;the donor-recipient ratio is 1:1;and covered with antibiotic solution after 18 h of culture.The specific conditions of NEAU-GS3 are as follows:adding 100 mmol/L Ca Cl2to the culture medium;the spores were thermally stimulated at 50℃for 10 min and pre-germinated for 6 h;the donor-recipient ratio is 10:1;and covered with antibiotic solution after 16 h of culture.（3）The genetic manipulation system of rare actinomycete Amycolatopsis orientalis CPCC203345 was successfully established for the first time.The traditional PEG-mediated protoplast transformation method and CRISPR-cBEST technology were used to explore and optimize the conditions for establishing the genetic manipulation system.The details are as follows:GY liquid medium was used as the optimum mycelium growth medium,after shaking culture at 28℃for 32 h,2%glycine was added to continue culture for 24 h,the bacteria were collected and incubated with1 mg/m L lysozyme at 37℃for 45 min,and R2YE medium was used as protoplast regeneration medium for subsequent culture.（4）The inactivation of key genes in the biosynthesis gene cluster of potential secondary metabolites of three actinomyces was verified.Based on the established genetic manipulation system,five gene-inactivated strains were successfully constructed,namely:Δorf788、Δorf794、Δorf811、Δorf6107 andΔorf6108.The LC-MS analysis of mutant inactivated strain CPCC 202372showed that polyketomycin inΔorf788 was significantly reduced,and a new peak was found,whose molecular weight was 14 Da less than polyketomycin.Considering that gene orf 788encoded methyltransferase,it was speculated that it might participate in a methylation reaction in polyketomycin biosynthesis.In the metabolic spectrum ofΔorf811,polyketomycin was hardly produced,and a new peak with molecular weight of 572 Da was found.Considering that gene orf811 encodes a glycosyltransferase,it is speculated that it may participate in the second glycosyl reaction in the synthesis of polyketomycin.The yield of polyketomycin inΔorf794 decreased greatly,and no new peak was found.The gene orf 794 encoded acyl-Co A,which suggested that it may be a positive regulator of polyketomycin yield.By analyzing the secondary metabolites of the mutant NEAU-GS3,it was found that the yield of GTRI-02 and SEK-4b in the metabolic spectrum of the mutantsΔorf6107 andΔorf6108 decreased obviously,which indicated that the gene cluster in Region 29.1 was responsible for the biosynthesis of GTRI-02 and SEK-4b.To sum up,this study successfully established an efficient and stable genetic manipulation system for non-model Streptomyces CPCC 202372 and NEAU-GS3 by using CRISPR-cBEST technology for the first time.The gene cluster of polyketomycin biosynthesis was located by the plasmid-mediated gene inactivation technology,and the functions of key genes were preliminarily predicted,which is of great significance to the study of the biosynthesis pathway of polyketomycin and actinobacine.On this basis,CRISPR-cBEST technology was innovatively combined with PEG-mediated protoplast transformation technology,and an efficient genetic manipulation system of Amycolatopsis orientalis CPCC 203345 was established for the first time,which confirmed that CRISPR-cBEST technology could be applied to the establishment of genetic manipulation system of Amycolatopsis orientalis.It provides effective technical support for mining active natural products of rare actinomycetes and promotes the efficient discovery of microbial drug source molecules. |
