Cloning of terpene synthases from plants

As part of a program to clone Artemisia annua sesquiterpene biosynthetic genes, a cDNA encoding epi-cedrol synthase was isolated from an A. annua leaf cDNA library by a homology-based strategy. The cDNA was functionally expressed in Escherichia coli and the encoded protein shown to produce (-) epicedrol and cedrol in a 96:4 ratio. The structure of epicedrol was determined by GC-MS and NMR and cedrol was identified by GC-MS and GC co-elution with the authentic sample. Neither cedrol nor epicedrol was detected in an extract of the plant from which the cDNA library was made. In addition, a sesquiterpene synthase like gene was cloned from A. thaliana ecotype Columbia by RT-PCR and the cDNA was expressed in E. coli. The second part of this thesis, describes the study of triterpene biosynthesis. A yeast strain with squalene synthase (erg9) and lanosterol synthase (erg7) double mutation was made by homologous recombination. This strain has been used by other group members to express A. thaliana cycloartenol synthase mutants and S. cerevisiae lanosterol synthase mutants. More accurate enzyme product ratios were obtained for the mutants expressed in this erg9 and erg7 double mutant yeast strain compared to the erg7 single mutant SMY8. In order to study the mechanism of oxidosqualene cyclases, A. thaliana was chosen as the model plant for cloning oxidosqualene cyclases. A triterpene synthase was isolated from the A. thaliana ecotype Landsberg young seedling cDNA library and the cloned gene had the second exon missing compared to cycloartenol synthase and lupeol synthase cloned from same organism. The cloned gene was expressed in yeast and no oxidosqualene cyclase activity was detected. An oxidosqualene cyclase like open reading frame ORF3, which is adjacent to lupeol synthase on the genomic DNA was cloned from A. thaliana ecotype Landsberg cDNA library. An open reading frame ORF1 found in the A. thaliana sequencing database, that has 71.4% identity to lupeol synthase was cloned from A. thaliana ecotype Columbia total RNA by RT-PCR. The cDNA of these two ORFs were expressed in yeast and no oxidosqualene cyclase activity was detected