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|Engineering of geranyl diphospate C-methyltransferase for the development of new diterpenoid precursors
|Yeni diterpen öncülleri geliştirmek amacıyla geranil difosfat C-metiltransferaz enzimin değiştirilmesi
|Izmir Institute of Technology
|Terpenoids constitute the most diverse family of natural products. They are involved in several biological functions and are used in medical and industrial applications. The key to their diverse biological activities is their structural diversity. Terpenoids are synthesized in three stages, all of which contribute to generation of structural diversity. In the terpenoid biosynthetic pathways, terpene synthases generate larger linear terpenoid precursors from smaller units via condensation reactions, terpene cyclases transform precursors via cyclization reactions, and then tailoring enzymes modify terpenoid products via addition of functional groups. Recently discovered geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is able to modify a linear monoterpenoid precursor, geranyl diphoshate (GPP), to produce a non-canonical terpenoid precursor, 2-methylgeranyl diphosphate. Modification of GPP by GPPMT is the first example of modification of a canonical linear isoprenoid precursor in nature. This study aims to achieve enzymatic synthesis of novel methylated non-canonical diterpenoid precursors, such as 2-methylgeranylgeranyl diphosphate (2MGGPP) by engineering GPPMT. The novel non-canonical precursors may later be utilized by cyclases to enhance the diversity of the terpenome. For example, taxadiene synthase could utilize 2MGGPP to generate variants of taxadiene, the precursor of the leading anti-cancer drug paclitaxel (Taxol®). Candidate mutants predicted to use GGPP as substrate were selected via in silico analysis of GPPMT structure. These mutations were introduced using the Quick-change site-directed mutagenesis. Mutant genes were expressed in E.coli strains. Mutant proteins were purified by Fast Protein Liquid Chromatography. Catalytic activities of mutants against canonical terpenoid precursors were determined by SAM methyltransferase assay.
|Includes bibliographical references (leaves: 27-30)
Text in English; Abstract: Turkish and English
x, 30 leaves
Thesis (Master)--Izmir Institute of Technology, Molecular Biology and Genetics, Izmir, 2014
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|Master Degree / Yüksek Lisans Tezleri
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