taxane and methyl-jasmonate

We report the production of taxadiene by transformation of N. benthamiana with a taxadiene synthase gene. The production was significantly increased by an elicitor treatment or metabolic pathway shunting. Paclitaxel (Taxol(®)) was first isolated from the bark of the pacific yew tree as an anticancer agent and has been used extensively to treat various types of cancer. Taxadiene, the first committed product of paclitaxel synthesis is cyclized from geranylgeranyl diphosphate (GGPP), and further complex hydroxylation and acylation processes of the unique taxane core skeleton produce paclitaxel. To accomplish de novo production of taxadiene, we transformed Nicotiana benthamiana with a taxadiene synthase (TS) gene. The introduced TS gene under the transcriptional control of the CaMV 35S promoter was constitutively expressed in N. benthamiana, and the de novo production of taxadiene was confirmed by mass spectroscopy profiling. Transformed N. benthamiana homozygous lines produced 11-27 μg taxadiene/g of dry weight. The highest taxadiene production line TSS-8 was further treated with an elicitor, methyl jasmonate, and metabolic pathway shunting by suppression of the phytoene synthase gene expression which resulted in accumulation of increased taxadiene accumulation by 1.4- or 1.9-fold, respectively. In summary, we report that the production of taxadiene in N. benthamiana was possible by the ectopic expression of the TS gene, and higher accumulation of taxadiene could be achieved by elicitor treatment or metabolic pathway shunting of the terpenoid pathway.

Topics: Acetates; Alkenes; Antineoplastic Agents, Phytogenic; Bridged-Ring Compounds; Cyclopentanes; Diterpenes; Gene Silencing; Humans; Isomerases; Metabolic Engineering; Metabolic Networks and Pathways; Nicotiana; Oxylipins; Paclitaxel; Plant Growth Regulators; Polyisoprenyl Phosphates; Taxoids; Taxus

Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant cell cultures. Cyclodextrins, which are cyclic oligosaccharides, can form inclusion complexes with nonhydrophilic secondary products, thus increasing their excretion from the producer cells to the culture medium. In the present work, using a selected Taxus x media cell line cultured in a two-stage system, the relationship between taxane production and the transcript profiles of several genes involved in taxol metabolism was studied to gain more insight into the mechanism by which these two elicitors regulate the biosynthesis and excretion of taxol and related taxanes. Gene expression was not clearly enhanced by the presence of cyclodextrins in the culture medium and variably induced by methyl jasmonate, but when the culture was supplemented with both elicitors, a synergistic effect on transcript accumulation was observed. The BAPT and DBTNBT genes, which encode the last two transferases involved in the taxol pathway, appeared to control limiting biosynthetic steps. In the cell cultures treated with both elicitors, the produced taxanes were found mainly in the culture medium, which limited retroinhibition processes and taxane toxicity for the producer cells. The expression level of a putative ABC gene was found to have increased, suggesting it played a role in the taxane excretion. Taxol biosynthesis was clearly increased by the joint action of methyl jasmonate and cyclodextrins, reaching production levels 55 times higher than in nonelicited cultures.

Topics: Acetates; Biosynthetic Pathways; Bridged-Ring Compounds; Cells, Cultured; Cyclodextrins; Cyclopentanes; Drug Synergism; Gene Expression Regulation, Plant; Oxylipins; Plant Proteins; Taxoids; Taxus

Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures. In the current study, cultures with different aggregation profiles were established to create predictable differences in paclitaxel accumulation upon MeJA elicitation. Expression of known paclitaxel biosynthetic genes in MeJA-elicited cultures exhibiting both substantial (15-fold) and moderate (2-fold) differences in paclitaxel accumulation was analyzed using quantitative reverse transcriptase PCR. Each population exhibited the characteristic large increase in paclitaxel pathway gene expression following MeJA elicitation; however, differences in expression between populations were minor, and only observed for the cultures with the 15-fold variation in paclitaxel content. These data suggest that although upregulation of biosynthetic pathway gene expression contributes to observed increases in paclitaxel synthesis upon elicitation with MeJA, there are additional factors that need to be uncovered before paclitaxel productivity can be fully optimized.

Topics: Acetates; Bridged-Ring Compounds; Cell Culture Techniques; Cyclopentanes; Gene Expression Regulation, Plant; Oxylipins; Paclitaxel; Plant Cells; Plant Proteins; Taxoids; Taxus