Dissertation > Industrial Technology > Chemical Industry > Pharmaceutical chemical industry > Antibiotics manufacture

The Study of the Pathway-specific Regulation of Natamycin Biosynthesis in Streptomyces Chattanoogensis

Author SunZhiHao
Tutor LiYongQuan
School Zhejiang University
Course Biochemistry and Molecular Biology
Keywords Streptomyces chattanoogensis pathway-specific regulator hierarchical control ATPase activity cholesterol oxidase
Type Master's thesis
Year 2013
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Antibiotics are usually biosynthesized by a large gene cluster in gram-positive Streptomycetes. The control of antibiotic production is a complex process involving multiple levels of regulation. The pathway-specific regulation which lies in the lowest level affect only a single antibiotic biosynthesis pathway.The natamycin biosynthesis gene cluster was recently cloned from Streptomyces chattanoogensis L10. Previous disruption of scnRⅠ and scnRⅡ respectively revealed two positive pathway-specific regulatory genes (scnRⅠ,scnRⅡ) within natamycin cluster. The full-length scnRⅡ was overexpressed in Escherichia coli as GST-fused protein. ScnRⅡ-GST binds directly to promoters of senⅠ, scnS2, scnJ, scnK, scnA, scnE, scnSl, scnD, as demonstrated by electrophoretic mobility shift assays. Therefore, ScnRII could activates the transcriptional of genes within natamycin cluster by directly binding to promoters of seven genes responsible for biosynthesis, tailoring and transportation.The transcription of scnRⅡ, a pathway-specific regulatory gene, was controlled by ScnRI. EMSA and Real-time PCR revealed that ScnRI bind directly to the intergenic region between scnRⅡ and scnRⅠ. In addition, ScnRI contains an AAA domain in the middle of the protein as characterized by highly conserved Walker A and Walker B motif. As measured in vitro, ScnRI exhibited ATPase activity. Point mutation of Wakler A motif in ScnRI highly decreased its ATPase activity and failed to complement the natamycin production in scnR1deletion mutant. Therefore, ScnRⅠ directly increase the transcription of scnRⅡ in natamycin biosynthesis and the ATPase activity is indispensable for the regulatory function of ScnRI.Searching from the previously sequenced natamycin gene cluster, we found a putative cholesterol oxidase encoding gene, scnE, which locates in the center of the cluster. Its unknown function and central location prompted us to evaluate its role in natamycin production. Therefore, the scnE deletion mutant was constructed via PCR targeting method and the natamycin production of the mutant decreased by about30%compared with wild type. In addition, the SignalP predicted its signal peptide in its N-terminal. Given that no obvious role for a cholesterol oxidase could be predicted in natamycin biosynthesis, we hypothesize that ScnE functions extracellularly to produce small signal molecules via oxidation, thus to exert "regulatory" role in natamycin production.In this study, a hierarchical regulation of two pathway-specific regulators was clarified in natamycin biosynthesis:ScnRI, a transcriptional regulator with ATPase activity, directly enhances the transcription of scnRⅡ in natamycin biosynthesis in Streptomyces chattanoogensis L10thus to increase the natamycin production. In addition, we also found a potential "regulatory" gene, scnE, in natamycin production.

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