Identification Of The Secondary Metabolite In Streptomyces Pactum Act12 And Functional Analysis Of A LuxR Family Regulator SPA5613

Streptomyces pactum Act12 is multi-active actinobacteria which has excellent bio-control activity, such as growth promoting for strawberry, and a higher production for tanshinone, however, there is no abuntant of secondary metabolites in previous study and massive biosynthetic gene cluster, which indicates the most of the clusters in it is silent or has a low expression level. To study its promoting mechanism, it is necessary to know its second metabolitess in laboratory conditions. We seek the optimum fermention condition and then we obtain the fermentation extract with bulk fermentation. we get three compounds by multistep separation and purification. The purified compounds were analyzed and by MS and NMR and the structures were under identification.Most of the clusters in Act12 is silent or has a low expression level in previous study which proving gene resourses for active compounds. We have a functional analysis and characterization of a halogenase-containing cluster with a LuxR family regulator SPA5613 in Streptomyces pactum Act12. The gene spa5613 was inactivated by homologous recombination, then the changes in metaboblites was analyzed in the mutant strain to explore the function of SPA5613. The constructed recombinant plasmid pKCJ17(pKC1139-spa5913::kan)which contains two homologous fragments of the gene spa5613 and a kanamycin marker gene between them was conjugally transformed from E.coli into the Streptomyces pactum Act12, and then a gene deficiency strain was obtained. The metabolites of the gene deficiency strain in different medium were extracted and the fermentation components were subjected to the antagonistic experiment, HPLC analysis. The results indicate that a new metabolic product arises and a stronger bioactivity appears in the deficiency strain than the wild type. Conclusion: Gene inactivation and genetic complementation of spa5613 revealed that the SPA5613 is a negative regulator. This work provides new insights into Lux R family transactional regulator and the feasibility of activating of a silent gene cluster by the manipulation of candidate negative regulator in biocontrol strain Streptomyces pactum Act12.