The Regulatory Mechanisim Of Multiple Secondary Metabolism Processes In Streptomyces Chattanoogensis

The genus Streptomyces are particularly abundant source of natural products. It is well studied that there are numerous silent secondary metabolite biosynthetic gene clusters in the industrial producing strain of streptomycetes, besides the target metabolite production. How to activate these cryptic gene clusters has shown to be a problem in the utilization of the Streptomyces genome resource. The biosynthetic process of secondary metabolism is controlled by complex and multi-level regulatory networks, among which the y-butyrolactone (GBL) autoregulatory signaling cascades have been shown to be an important one in Streptomyces. In this study, we focused on the Streptomyces chattanoogensis L10, which is the industrial producing strain of natamycin. Further, we studied both the coordinate regulatory mechanism of GBL receptors and the molecular mechanism of activation of the silent angucycline biosynthetic gene cluster.In our previous study, the GBL receptor ScgR and the GBL synthease ScgA made up a global regulatory system. According to the genome wide transcriptome analysis of the △scgA, ScgA was involved in primary metabolism process including glycolysis and TCA cycle. Meanwhile, the transcription of some regulator and some secondary metabolism gene clusters were also changed in AscgA according to the gene array data. The protein SprA is an important homologure to ScgR. Like deletion of scgA, the disruption of sprA resulted in decreased production of the antibiotic natamycin in liquid culture and retarded morphological differentiation on solid agar. Electrophoretic mobility shift assays and DNase I footprinting assays showed that SprA bound to two specific autoregulatory element (ARE) sequences located upstream of the sprA gene, indicating that its expression is self-regulated. According to the gene array data, we found that the expression of sprA was shown to be positively regulated by ScgA. While, SprA was involved in biosynthesis of GBL by repressing the expression of scgA. An Escherichia coli-based luciferase report system demonstrated that SprA directly repressed the expression of scgR. This work indicates that SprA acts as a pleiotropic regulator of both morphogenesis and the production of natamycin through regulating the transcription of scgA and scgR.Genomic sequencing of S. chattanoogensis L10 has revealed the presence of thirty-four secondary metabolite biosynthetic gene clusters (including natamycin gene cluster), while most of them are cryptic under our laboratory conditions. Bioinformatics analysis of these gene clusters revealed a silent angucycline biosynthetic gene cluster (termed chattamycin gene cluster). The over-expression of a pathway specific activator gene under the constitutive ermE* promoter successfully triggered the expression of the angucycline biosynthetic genes. Two novel members of angucycline antibiotic family chattamycin A and chattamycin B were further isolated and elucidated. Cell viability assays demonstrated that chattamycin B possesses a promising antitumor activities against human cancer cell lines. Further study revealed that the production of chattamycins were coordinately controlled by ScgR, SprA and ScgA. The results presented here probably provide a feasible method to active silent angucycline biosynthetic gene clusters to discover potential new drug leads.