A new 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene from Taxus media: Cloning, characterization and functional complementation

Abstract

2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECPS, EC: 4.6.1.12) is the fifth enzyme of the methylerythritol phosphate (MEP) pathway of Taxol biosynthesis. The full-length cDNA sequence (designated as TmMECPS) was cloned and characterized from Taxus media. The full-length cDNA of TmMECPSwas 899 bp containing a 723 bp open reading frame (ORF) encoding a polypeptide of 241 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.22. Comparative analysis indicated that TmMECPS was similar with other plant MECPSs with the conserved Zn2+ ligands and CDP-binding residues, the subcellular prediction showed that TmMECPS owned a 60 amimo-acid plastidial transit peptide at it N-terminus. In the phylogenetic analysis, plant MECPSs were divided into 2 groups including angiosperm MECPSs and gymnosperm MECPSs. Tissue expression pattern analysis indicated thatTmMECPS expressed in all tested tissues including tender barks, leaves, roots and stems but at different levels, TmMECPS had higher expression levels in tender barks and leaves than that in roots and stems. The genetic complementation assay demonstrated that TmMECPS did encode the enzyme that had the MECPS activity like Arabidopsis MECPS. The cloning and characterization of TmMECPS will be helpful to understand more about the role of MECPS involved in the taxol precursor biosynthesis at the molecular level. Key words: MECPS gene, cloning, color complementation assay, Taxus media, expression.