신규 면역억제제 토토마이세틴 고생산을 위한 토토마이세틴 생합성 조절 네트워크 규명

Title
신규 면역억제제 토토마이세틴 고생산을 위한 토토마이세틴 생합성 조절 네트워크 규명
Authors
나지혜
Keywords
신규면역억제제토토마이세틴고생산을위한토토마이세틴생합성조절네트워크규명
Issue Date
2012
Publisher
인하대학교
Abstract
Tautomycetin (TMC) is an unusual linear polyketide compound esterificated with a cyclic anhydride, which exhibits a novel activated T cell-specific immunosuppressant and anti-cancer activities. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster including a TMC pathway-specific gene, tmcN from Streptomyces sp. CK4412, whose over-expression led to significantly increased TMC productivity. In addition, we also reported that wblA acted as a global down-regulator of antibiotic biosynthesis through pathway-specific regulator Streptomyces species. Here, we confirm that tmcT is another TMC pathway-specific regulatory gene present within the TMC biosynthetic cluster, because tmcT deletion resulted in the complete loss of TMC production and complementation by a tmcT-carrying integrative plasmid significantly restored TMC biosynthesis. We also identified a 0.39 kb wblA ortholog (named wblAtmc) via genomic DNA library screening from Streptomyces sp. CK4412, showing 96% amino acid identity compared to a previously known S. coelicolor wblA. A targeted gene disruption of wblAtmc from the Streptomyces sp. CK4412 exhibited approximately 2-fold higher TMC productivity than that from the wild-type strain. Moreover, transcript analyses of the TMC biosynthetic and regulatory genes revealed that the expression of tmcT is strongly down-regulated by wblAtmc. These results imply that TMC biosynthetic regulation network is controlled by two pathway-specific positive regulatory genes, wblAtmc-independent tmcN as well as wblAtmc-dependent in Streptomyces sp. CK4412.
Description
Abstract I Contents Ⅱ List of Figures IV List of Tables VI 1. Introduction 1 1.1 Streptomyces 1 1.2 Antibiotics biosynthesis regulatory systems in Streptomyces 1 1.3 Tautomycetin 2 2. Experimental section 5 2.1 Bacterial strains and culture condition 5 2.2 Screening and sequence analysis of wblAtmc 5 2.3 Construction of ΔwblAtmc and ΔtmcT mutants via PCR-targeted gene disruption 6 2.4 Construction of integrative plasmid for complementation of ΔwblAtmc or ΔtmcT 9 2.5 HPLC quantification and antifungal bioassay for TMC 11 2.6 Isolation of total RNA and gene expression analysis by RT-PCR 11 3. Results and Discussion 15 3.1 In silico anaylsis of pathway-specific regulatory gene, tmcT from Streptomyces sp. CK4412 15 3.2 Cloning and sequence analysis of wblA ortholog from Streptomyces sp. CK4412 15 3.3 TMC productivity stimulation via wblAtmc disruption from Streptomyces sp. CK4412 18 3.4 Loss of TMC production via tmcT disruption from Streptomyces sp. CK4412 19 3.5 TMC biosynthetic regulatory cascade of global regulatory wblAtmc and two pathway-specific regulatory tmcN and tmcT 23 4. Conclusion 26 5. References 27 Figure 1 4 Figure 2 7 Figure 3 17 Figure 4 21 Figure 5 22 Figure 6 25 Table 1 10 Table 2 13
URI
http://dspace.inha.ac.kr/handle/10505/23632
Appears in Collections:
College of Natural Science(자연과학대학) > Ocean Sciences (해양과학) > Theses(해양과학 석박사 학위논문)
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