Cloning, characterization, and molecular manipulation of plant terpene synthases.

摘要

Ginkgo 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.