Molecular Cloning Of Genes Coding Enzymes In The Upstream Biosynthesis Pathway Of Taxol

The upstream biosynthesis pathway of Taxol is metabolic pathway before GGPP. As the common biosynthesis pathway of biterpenoids, it includs MVA pathway, DXP pathway and the part from IPP to GGPP. In order to clone genes coding the important enzymes in this upstream biosynthesis pathway of taxol, an easy and efficient protocol was developed for isolating good-quality total RNA from various mature tissues including fruits, leaves, stems and roots of Taxus plant. On the basis of this protocol, the full-length cDNAs of six key genes were cloned through RACE method, characterized, which were respectively tmhmgr(the gene encoding 3-Hydroxy-3-methylglutaryl-CoA reductase from Taxus media), tmipi (the gene encoding Isopentenyl diphosphate isomerase from Taxus media) involved in the MVA pathway, tmdxs (the gene encoding 1-Deoxy-D-xylulose 5-phosphate synthase from Taxus media) and tmdxr (the gene encoding 1-Deoxy-D-xylulose 5-phosphate reductoisomerase from Taxus media) involved in DXP pathway, tmfps (the gene encoding Farnesyl diphosphate synthase from Taxus media), tmggpps (the geranylgeranyl diphosphate synthase gene from Taxus media) from the biosynthesis pathway from IPP to GGPP. The different mutant yeast strains were applied to respectively identify the function of tmhmgr, tmfps and tmggpps by functionally genetic complementation; the function of tmipi was identified by over expressing in E. coli strain XL1-Blue with the carotenoid-producing plasmid pAC-BETA, which resulted in pushing forward the metabolic flux to the downstream of the DXP pathway. Southern blot analysis revealed that tmhmgr, tmfps, tmipi or tmdxs was not a single-gene but belonged to its gene family in Taxus media. Northern blot analysis showed that tmhmgr and tmfps expressed in the roots, stems and needles of Taxus media, but with higher level of expression for tmhmgr in the needles and stems, that was coincident with the fact that stems and needles were used for extracting taxol and its derivatives. tmfps expressed constitutively in roots, stems and needles. when Taxus cuspidata cells were treated with MeJA, the expression of tmggpps increased greatly and maintained high expression level in a relative long time that was helpful to the accumulation of taxol.The genomic sequence of tmggpps was also cloned and characterized. it was found that tmggpps was an intron-free gene with the low level of constitutive expression. A G-box was found in the upstream sequence of tmggpps, which was the same as the MeJA-responsive G-box of the strictosidine synthase gene in C. roseus, and this might explain that tmggpps was up-regulated by MeJA. It was important to clone and characterize the genes described before, which was very helpful to map the taxol biosynthetic pathways at the level of molecular genetics and biochemistry and also provided more potential targets for metabolic engineering of taxol.