Cloning, characterization, and molecular manipulation of plant terpene synthases

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dc.contributor.advisorMatsuda, Seiichi P. T.en_US
dc.creatorSchepmann, Hala Gobranen_US
dc.date.accessioned2009-06-04T06:37:34Zen_US
dc.date.available2009-06-04T06:37:34Zen_US
dc.date.issued2002en_US
dc.description.abstractGinkgo biloba produces the ginkgolides, a structurally novel class of diterpenes. Part I of this thesis describes the cloning and functional characterization of levopimaradiene synthase, which catalyzes the initial cyclization step in ginkgolide biosynthesis. A G. biloba cDNA library was prepared from seedling roots and a probe was amplified using primers corresponding to conserved gymnosperm terpene synthase sequences. Colony hybridization and rapid amplification of cDNA ends yielded a full-length clone encoding a predicted protein (873 amino acids, 100,289 Da) similar to known gymnosperm diterpene synthases. The sequence includes a putative N-terminal plastid transit peptide and three aspartate-rich regions. The full-length protein expressed in Escherichia coli cyclized geranylgeranyl pyrophosphate to levopimaradiene, which was identical to a synthetic standard by GC/MS analysis. Removing 60 or 79 N-terminal residues increased levopimaradiene production, but a 128-residue N-terminal deletion lacked detectable activity. This is the first cloned ginkgolide biosynthetic gene and the first in vitro observation of an isolated ginkgolide biosynthetic enzyme. Additionally, production of abietatriene, the immediate hydrocarbon precursor of the ginkgolides, was achieved. Expression of G. biloba levopimaradiene synthase in Saccharomyces cerevisiae , metabolically engineered for geranylgeranyl pyrophosphate over-production, yielded levopimaradiene, abietadiene, abietatriene, and (+)-copalol. Part II of this thesis describes molecular manipulation of Arabidopsis thaliana cycloartenol synthase. Residues critical to enzymatic activity were identified, altered, and functionally characterized. Furthermore, cycloartenol synthase double point mutants were studied to determine the effects on catalytic function.en_US
dc.format.extent137 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoTHESIS BIOCHEM. 2002 SCHEPMANNen_US
dc.identifier.citationSchepmann, Hala Gobran. "Cloning, characterization, and molecular manipulation of plant terpene synthases." (2002) Diss., Rice University. <a href="https://hdl.handle.net/1911/18130">https://hdl.handle.net/1911/18130</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/18130en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectBiochemistryen_US
dc.subjectOrganic chemistryen_US
dc.titleCloning, characterization, and molecular manipulation of plant terpene synthasesen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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