The Study Of The Regulation Of Natamycin Thesis In Streptomyces Chattanoogensis

Natamycin is a polyene macrolide compound produced by Streptomyces. It is a highly efficient, broad-spectrum and secure antifungal agent, which has been widely used as food preserative as well as drugs. This study focus on understanding the regulation of natamycin biosynthesis in an industrial natamycin-producer Streptomyces chattanoogensis L10. These research will be paralleled for enhanced production of NTM in industry and hence of both academic value and commercial interest.First, the complete natamycin biosynthetic gene cluster of S. chattanoogensis L10was cloned and confirmed by the disruption of the two pathway-specific activator genes, scnRⅠ and scnRⅡ. The transcriptional orgnization of the scn cluster was determined by RT-PCR and5'RACE. By using both in vitro and in vivo assays, ScnRII was shown to directly bind to the promoter region of operons, and activate their transcription, while ScnRⅠ appears to influence the expression of scn cluster indirectly. Comparative cluster analysis with its counterpart pim cluster in S. natalensis revealed different cluster architecture between these two clusters. The evolutionary change of nucleotide sequence in the intergenic regions caused different transcriptional orgnization between these two clusters and resulted in altered gene regulation. These results provided new insight into the evolution of antibiotic biosynthetic gene clusters.In addition, we also cloned a pleitropic regulator adpA-Ch and the gamma-butyrolactone regulatory system (scgA, scgX and scgR) in S. chattanoogensis, both of which were found to be involved in morphological differentiation and secondary metabolism. The adpA-Ch deletion mutant ZJUD5showed a conditionally sparse aerial mycelium formation phenotype and defects in sporulation; it also loses the ability to produce natamycin and a diffusible yellow pigment. Quantative transcriptional analysis showed that the expression level of the natamycin regulatory gene scnRⅠ decreased20-fold in ZJUD5, while the transcription of the other activator gene scnRⅡ was not significantly affected. EMS A showed that AdpA-Ch binds to its own promoter but fails to bind to the promoter region of scnRⅠ, indicating the control of scnRI by AdpA-Ch is exerted in an indirect way.γ-butyrolactones (GBLs) produced by several Streptomyces species have been shown to serve as quorum sensing signalling molecules for activating antibiotics production. The GBL system (CHB system) of Streptomyces chattanoogensis L10consists of three genetically linked genes, scgA, scgX and scgR. scgR encode a GBL receptor, while both scgA and scgX contribute to GBL production. Both in vitro and in vivo assays showed that ScgR could bind to the promoter region of scgA. This binding activity can be abolished by addition of a GBL-fraction extract from the cluture of this strain. CHB mutant strains had a growth defect in liquid YEME medium and delayed or abolished the morphological differentiation and secondary metabolites productions on solid medium depending on medium composition. An additional direct ScgR-target gene gbdA was identified by genomic SELEX. Comparative proteomic analysis revealed that CHB system affected the expression of more than50proteins, including enzymes involved in carbon uptake, primary metabolism and stress response. This study revealed a novel GBL system involving nutrient utilization, triggering adaptive responses, and finally distating the switch from primary to secondary metabolism.