Molecular Cloning, Characterization And Functional Analysis Of MCT And MCS Genes Of Taxus Chinensis

Taxol, originally isolated from Taxus species, is a very promising anticancer drug used in the treatment of some certain cancers. It is not a successive way to extracting taxol from natural taxus species, due to the slow growth of taxus species and very low content of taxol in plantlets. Other more efficient means or renewable resources for taxol production need to be developed to meet the market demands, such as microbial, chemical synthesis of taxol. Due to the low productivity and high effective cost of other means, genetic engineering for manipulation of the taxol biosynthetic pathway is an attractive strategy to increase taxol production. Although many genes involved in taxol biosynthesis have been isolated successfully, the properties of these genes are still limited.There are two up stream pathways for synthesis taxol precursors, in the cytosol via the mevalonate pathway and recently unveiled methylerythritol phosphate (MEP) pathway. To research the molecular mechanism in biosynthesis of taxol precursors and provide new target genes for metabolic engineering of MEP pathway, we cloned and characterized the MCT, MCS genes involved in the biosynthetic pathway of taxol.2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MCT), the third-step enzyme of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, catalyzes MEP and CTP yielding 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. The full-length cDNA of TcMCT was isolated using Rapid Amplification of cDNA Ends (RACE) technique and characterized for the first timee, the new cDNA was named as TcMCT. The full-length of TcMCT was 1293bp containing a 942 bp coding region encoding a 313 amino acids peptide with a calculated molecular mass of 34.33KD and an isoelectric point of 8.96. Comparative and bioinformatic analyses revealed that TcMCT had extensive homology with MCTs from other species, especially had high similarities with MCTs from other plant species. Phylogenetic analysis suggested that MCTs divided into two groups including planta and bacterial. TcMCT belongs to plant MCTs’family. Tissue expression pattern analysis showed that TcMCT expressed constitutively in tissues of taxuschinensis, with the highest expression level in the bark. In the present work, TcMCT gene was expressed in E coli M15 strain. With N-terminal fused a histidine-tagged sequence, TcMCT could be purified by one-step nickel affinity chromatography. SDS-PAGE indicated that the protein purified form the fraction was 35kDa. Enzyme activity assay suggested that the fusion protein has activity. Circular dichroism spectra indicated that the protein secondary structures for a-helix,β-sheet,β-turn and random coil were 70.9%,0%,0%and 29.1%. The color complemetion assay indicated that TcMCT could result in the accumulation ofβ-carotene. 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), the fifth enzyme of 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, catalyzes CDP-MEP to form ME-cPP. The full-length cDNA sequence of TcMCS was cloned via RACE technology and characterized. The new cDNA was designated as TcMCS. The full-length cDNA of TcMCS was 1081-bp containing a 744 bp open reading frame (ORF) encoding a peptide of 247 amino acids with a calculated molecular mass of 26.1KDa and an isoelectric point of 8.97. Comparative and bioinformatics analysis showed TcMCS was highly homology with MCSs from other plant species and containing a 57 aa plastid transport peptides. Phylogenetic analysis revealed that MCSs divided into two groups including plant and bacterial, TcMCS belonged to plant MCSs’family. The secondary structure of TcMCS by SOPMA analysis showed that the putative TcMCS protein contained 26% alpha helix,23% extended strand,7%beta turn, and 44% random coil.3D structural modeling showed that these highly conserved activity centre residues are Asp98, His100, and His132 correspondingly, suggesting that TcMCS has a similar biological function with other MCSs. The color complemetion assay indicated that TcMCS could resulted in the accumulation ofβ-carotene.The cloning, characterization and functional analysis of TcMCT and TcMCS will be helpful to understand more about their important roles involved in Taxol biosynthesis in T.chinensis at the molecular level and provides candidate genes for metabolic engineering of Taxol.