The Activtion Of Silent Gene Clusters And Products Identification In Streptomyces Act12

Streptomyces pactum Act12 is a multifunctional actinomycete strain isolated from an extreme environment on the Qinghai-Tibet Plateau,China.Previous study indicated that the strain has some excellent biological activity,for example,the strain can suppress the growth of plant fungal pathogens and promote the growth of strawberry,melon and cotton and other crops.Through the study of the whole genome sequencing of the strain and the on-line analysis by using antiSMASH we found that the genome of Act12 may contain abundant secondary metabolites biosynthesis gene cluster,however,when we detected the fermentation extract of Act12 under laboratory conditions,we found that the content and types of secondary metabolites contained in the strain are fewer,which indicates that the vast majority of the gene cluster in Act12 is silent or low expression.In order to activate the silence gene cluster and full excavate the active compound resources in Act12,as well as to further explore the relevant activity mechanism,in this study,by the chromosome gene inactivation technique based on homologous recombination with linearized DNA fragments,we successfully gained four mutant strains,we have respectively inactivated a LuxR family transcriptional regulator SPA4754,a TetR family transcriptional regulator SPA0520,the global regulatory gene nsdA and the global regulatory gene bldA,and we respectively obtained the mutant strains Δspa4754,Δspa0520,ΔnsdA and ΔbldA.We also detected the changes of pathogenic microorganism antagonism activity and compare metabolic spectrum analysis about the four mutant strains.Comparison with the experimental result showed that when spa0520(GenBank Accession No.KY368145)was inactivated in Act12,and by the fungal cake method,the mutant strainΔspa0520 had more significant inhibiting effect on several fungal pathogens than its parent strain Act12.The comparison of metabolic patterns analysis based on the high performance liquid chromatography(HPLC)showed that the fermented product of the mutant strainsΔspa0520 has a more significant differences peak than the wild type strains Act12.we obtained the fermentation extract of the mutant strainsΔspa0520 with bulk fermentation,and we purified the corresponding substance of the significant differences peak.By high resolution mass spectrometry analysis methods,the structure of the substance was preliminary analyzed and identified.The result of the final analysis for the substance predicted that the compound may be the oligomycin D.At the same time,we obtain the corresponding genetic complement strain Δspa0520C by the genetic complement experiments,and we also detected the changes of pathogenic microorganism antagonism activity and compare metabolic spectrum analysis about the genetic complement strain Δspa0520C.Comparison with the experimental result showed that the inhibiting effect on several fungal pathogens and the metabolic patterns analysis based on HPLC about the genetic complement strain Δspa0520C was same as its parent strain Act12.Through gene deletion and complementation experiments about spa0520,we identified spa0520 negatively regulating oligomycin D biosynthesis in Act12,and it may also have multiple effect regulation effect.The experiment result provided the help for the research about the regulation mechanism of other secondary metabolites in Act12,and at the same time,by adopting the method of knocking out the negative control gene provided the beneficial reference for the activation to the silence gene clusters.