Yifu Gong

Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast

In plants, the first committed step in the pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34). Here we report for the first time the cloning of a full-length cDNA encoding HMGR (Tm-HMGR) from a taxol-producing gymnosperm, Taxus media Rehder. The full-length cDNA of Tm-HMGR (GenBank accession number: AY277740) was 2307 base pairs (bp), with a 1791-bp open reading frame (ORF) encoding a 596-amino-acid polypeptide. Bioinformatic analysis revealed that Tm-HMGR contained two trans-membrane domains and a catalytic domain, and showed high homology to other plant HMGRs. Phylogenetic analysis indicated that Tm-HMGR was more ancient than other plant HMGRs. The structural modelling showed that Tm-HMGR had the typical spatial structure of HMGRs whose catalytic domains could be folded and divided into three spatial domains, L-domain, N-domain and S-domain. Southern blot analysis revealed that Tm-HMGR belonged to a small HMGR gene family. Northern blot analysis showed that Tm-HMGR was expressed in roots, stems and needles, with higher expression in stems and needles than in roots. Functional complementation of Tm-HMGR in a HMGR-deficient mutant yeast demonstrated that Tm-HMGR mediated the biosynthesis of mevalonate and provided the general precursor for taxol biosynthesis.

[An intron-free methyl jasmonate inducible geranylgeranyl diphosphate synthase gene from Taxus media and its functional identification in yeast].

Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes and, in particular, Taxol, one of the most potent antitumor drugs. In order to investigate the role of GGPP synthase in Taxol biosynthesis, we cloned, characterized, and functionally expressed the GGPPS gene from Taxus media. Using the genome walking strategy, a 3743-bp genomic sequence of T. media was isolated which contained a 1182-bp open reading frame (ORF) encoding a 393-amino acid polypeptide that showed a close similarity to other plant GGPPSs. Subsequently, the full-length cDNA of the GGPPS gene of T. media (designated TmGGPPS) was amplified by RACE. Bioinformatic analysis showed that TmGGPPS was an intron-free gene, and its deduced polypeptide contained all five conserved domains and functional aspartate-rich motifs of the prenyltransferases. By constructing the phylogenetic tree of plant GGPPSs, it was found that plant-derived GGPPSs could be divided into two classes, those of angiosperms and gymnosperms, which might have evolved in parallel from the same ancestor. To our knowledge, this was the first report that the geranylgeranyl diphosphate synthase genes were free of introns and evolved in parallel in both angiosperms and gymnosperms. The coding sequence of TmGGPPS was expressed through functional complementation in a yeast mutant lacking GGPPS activity (SFNY368), and the transgenic yeast was shown to have this activity. This was also the first time SFNY368 was used to identify the function of plant-derived GGPPSs. Furthermore, investigation of the effect of methyl jasmonate (MeJA) on the expression of TmGGPPS showed that MeJA-treated T. media cultured cells had much higher expression of TmGGPPS than untreated cells.

[Isolation and characterization of a 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene from Taxus media].

Abstract2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) synthase (MECS, EC: 4.6.1.12) is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and further Taxol biosynthesis. The full-length MECS cDNA sequence (GenBank accession number DQ286391) was cloned and characterized for the first time from Taxus media, using the Rapid Amplification of cDNA Ends (RACE) technique. The full-length cDNA of Tmmecs was 1081 bp containing a 741 bp open reading frame (ORF) encoding a peptide of 247 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.97. Comparative and bioinformatic analyses revealed that TmMECS had extensive homology with MECSs from other plant species. Phylogenetic analysis indicated that TmMECS was more ancient than other plant MECSs. Southern blot analysis revealed that Tmmecs belonged to a small gene family. Tissue expression pattern analysis indicated that Tmmecs expressed constitutively in all tissues including roots, stems and leaves. The cloning and characterization of Tmmecs will be helpful to understand more about the role of MECS involved in the Taxol biosynthesis at the molecular level.

Characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from Camptotheca acuminata

Abstract5-enolpyruvylshikimate 3-phosphate synthase (EPSPS; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a critical enzyme in the shikimate pathway. The full-length EPSPS cDNA sequence (CaEPSPS, GenBank accession number: AY639815) was cloned and characterized for the first time from woody plant, Camptotheca acuminata, using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of CaEPSPS was 1778 bp containing a 1557 bp ORF (open reading frame) encoding a polypeptide of 519 amino acids with a calculated molecular mass of 55.6 kDa and an isoelectric point of 8.22. Comparative and bioinformatic analyses revealed that CaEPSPS showed extensive homology with EPSPSs from other plant species. CaEPSPS contained two highly conserved motifs owned by plant and most bacteria EPSPSs in its N-terminal region. Phylogenetic analysis revealed that CaEPSPS belonged to dicotyledonous plant EPSPS group. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in leaves, stems and roots, with the lower expression being found in roots. The coding sequence of CaEPSPS gene was successfully subcloned in a plasmid-Escherichia coli system (pET-32a), and the cells containing the plasmid carrying the CaEPSPS gene exhibited enhanced tolerance to herbicide glyphosate, compared to the control.

Molecular cloning and heterologous expression of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus x media

A full-length cDNA encoding 10-deacetylbaccatin III-10-O-acetyl transferase (designated as TmDBAT), which catalyzes the acetylation of the C-10 hydroxyl group of the advanced metabolite 10-deacetylbaccatin III (10-DAB) to yield baccatin III, the immediate diterpenoid precursor of Taxol, was isolated from Taxus x media. Heterologous expression of TmDBAT in E. coli demonstrated that TmDBAT was a functional gene. Tissue expression pattern analysis revealed that TmDBAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, implying that TmDBAT was tissue-specific. Expression profiling analysis of TmDBAT under different elicitor treatments including silver nitrate, ammonium ceric sulphate and methyl jasmonate indicated that TmDBAT was an elicitor-responsive gene. Southern blot analysis suggested that TmDBAT belonged to a small multigene family.