Molecular Biology Of The Biosynthetic Pathways Of Taxol Precursors And Metabolic Engineering Of Anti-Tumor Terpenoid Indole Alkaloids

Taxol, one of the most potent and efficient anti-tumor agents, is originated from the classic MVA pathway and recently unveiled DXP pathway, In order to map the biosynthesis of taxol precursors at the level of molecular genetics, 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 three key genes involved in the MVA pathway were cloned through RACE method, characterized and functionally identified, which was respectively tmhmgr(the gene encoding 3-Hydroxy-3-methylglutaryl-CoA reductase from Taxus media), tmfps (the gene encoding Farnesyl diphosphate synthase from Taxus media) and tmipi (the gene encoding Isopentenyl diphosphate isomerase from Taxus media). Two key genes encoding the committed-step enzymes involved in the DXP 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), and the geranylgeranyl diphosphate synthase gene from Taxus media and its correlative genomic sequence were also cloned and characterized. 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, whichresulted in pushing forward the metabolic flux to the downstream of the DXP pathway. Southern blot analysis revealed that tmhmgr, tmjps, tmipi or tmdxs was not a single-gene but belonged to its gene family in Taxus media. Northern blot analysis showed that tmhmgr and tmjps 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, tmjps expressed constitutively in roots, stems and needles, tmggpps was an intron-free gene with the low level of constitutive expression; when Taxus media 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. 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. When Taxus media cells were treated with UV and MeJA, RT-PCR of tmdxs revealed that UV and MeJA could up regulate its expression. The responses of tmggpps and tmdxs to MeJA might give reasons that MeJA induced over production of taxol. 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.Camptothecin, vinblastine and vincristine were potent anti-tumor agents, all belonging to TIAs but with very low contents in planta. Therefore, metabolic engineering of these TIAs would be a good alternative way to solve the shortage problem of these agents. The three key genes, including tdc (the gene encoding tryptophan decarboxylase from Catharanthus roseus), str (the gene encoding strictosidine synthase from C. roseus) and g10h (the gene encoding geraniol 10-hydroxylase from C. roseus), were isolated by RT-PCR and used for constructing plant expression vectors. ORCA3, a transcription regulator involved in the TIAs biosynthetic pathway in C. roseus, was also cloned and used for constructing plant expression vector. After a series of molecular operation, four plant expression vectors were obtained: p1304++tdc+str, p1304++g10h, p1304++orca3, p1304++g10h+orca3, and the resulting four vectors were introduced into Agrobacterium tumejaciens strain LBA4404 and disarmed strain...