Cloning And Characterizations Of Two Key Enzyme Genes Involved In The Biosynthesis Pathway Of Taxol Precursors.

Taxol,isolated from Taxus species,is one of the best anti-tumor drugs.However,Taxus species is nationwide-protected with slow growth rate and low taxol content.To solve the problem of limited taxol availability,genetic engineering technology offers a brand new way.Therefore,The molecular biology research of taxol biosynthesis pathways is a prerequisite work for it's engineering.Taxol is originated from recently unveiled MEP pathway.In order to map the biosynthesis of taxol precursors at the level of molecular genetics,The full-length cDNAs of two key genes involved in the MEP pathway were cloned through RACE method,characterized and functionally identified,which was respectively TmMECT(the gene encoding 2-C-Methyl-D-erythritol 4-phosphate cytidyltransferase from Taxus media),TmMECPS(the gene encoding 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase from Taxus media).2-C-Methyl-D-erythritol 4-phosphate cytidyltransferase(MECT,EC:2.7.7.60)is the third enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate(MEP)Taxol biosynthesis.The full-length MECT cDNA sequence(designated TmMECT)was cloned and characterized for the first time from Taxus media,using Rapid Amplification of cDNA Ends(RACE)technique.The full-length cDNA of TmMECT was 1301-bp containing 5'and 3' untranslated regions,poly A tail and a 939 bp open reading frame(ORF)encoding a peptide of 312 amino acids with a calculated molecular mass of 34.33 kDa and an isoelectric point of 8.96.Tissue expression pattern analysis indicated that TmMECT expressed constitutively in all tissues including leaves,roots,barks and stems,with higher expression in leaves and barks.The color complemetion assay demonstrated that TmMECT could accelerate the accumulation ofβ-carotene.The cloning and characterization of TmMECT will be helpful to understand more about the role of MECT involved in the Taxol biosynthesis at the molecular level. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase(MECPS,EC:4.6.1.12)is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate(MEP)pathway of Taxol biosynthesis.The full-length cDNA sequence(designated as TmMECPS)was cloned and characterized for the first time from Taxus media,using RACE technique.According to the bioinformations analysis,the full-length cDNA of TmMECPS was 899 bp containing a 726-bp ORF encoding a peptide of 241 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.22.Comparative and bioinformatic analyses revealed that TmMECPS had extensive homology with MECPSs from other species and contained conserved residues owned by the MECPS protein family.Phylogenetic analysis indicated that TmMECPS was more ancient than other plant MECPSs.Tissue expression pattern analysis indicated that TmMECPS expressed in all tested tissues including roots,stems, barks and leaves but at different levels,with higher expression levels in tender barks and leaves than roots and stems.The color complementation assay demonstrated that TmMECPS could promoted the accumulation ofβ-carotene.The cloning and characterization of TmMECPS will be helpful to understand more about the role of MECPS involved in the Taxol biosynthesis at the molecular level.