Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis.

Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides.

Taxol biosynthesis: differential transformations of taxadien-5 alpha-ol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cells.

The biosynthesis of the diterpenoid antineoplastic drug Taxol in Taxus species involves the cyclization of the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation (with allylic rearrangement) of this olefin precursor to taxa-4(20),11(12)-dien-5 alpha-ol, and further oxygenation and acylation reactions. Based on the abundances of naturally occurring taxoids, the subsequent order of oxygenation of the taxane core is considered to occur at C10, then C2 and C9, followed by C13, and finally C7 and C1. Circumstantial evidence suggests that the acetylation of taxadien-5 alpha-ol may constitute the third specific step of Taxol biosynthesis. To determine whether taxadienol or the corresponding acetate ester serves as the direct precursor of subsequent oxygenation reactions, microsomal preparations isolated from induced Taxus cells and optimized for cytochrome P450 catalysis were incubated with each potential substrate. Both taxadienol and taxadienyl acetate were oxygenated to the level of a diol and to higher polyols at comparable rates by cytochrome P450 enzymes of the microsomal preparation. Preparative-scale incubation allowed the isolation of sufficient quantities of the diol derived from taxadienol to permit the NMR-based structural elucidation of this metabolite as taxa-4(20),11(12)-dien-5 alpha,13 alpha-diol, which may represent an alternate route of taxoid metabolism in induced cells. GC-MS-based structural definition of the diol monoacetate derived in microsomes from taxadienyl acetate confirmed this metabolite as taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol, thereby indicating that acetylation at C5 of taxadienol precedes the cytochrome P450-mediated insertion of the C10-beta-hydroxyl group of Taxol.

Isolation of labeled 9-dihydrobaccatin III and related taxoids from cell cultures of taxuscell cultures of taxus canadensis elicited with m.

Cell suspension cultures of Taxus canadensis rapidly produced paclitaxel (1) and other taxoids in response to elicitation with methyl jasmonate. Three of these taxoids, of potential value in the synthesis of taxoid analogues, have been isolated from cell cultures of Taxus canadensis and identified as 13-acetyl-9-dihydrobaccatin III (2), baccatin VI (3), and 9-dihydrobaccatin III (4). Of these metabolites, 9-dihydrobaccatin III (4) has not been isolated from any Taxus species, whereas 13-acetyl-9-dihydrobaccatin III (2) and baccatin VI (3) have been isolated from a number of natural sources. 2D NMR techniques, mass spectrometry, and partial synthesis were used to rigorously elucidate the structure and stereochemistry of these natural products.

Partial purification and characterization of acetyl coenzyme A: taxa-4(20),11(12)-dien-5alpha-ol O-acetyl transferase that catalyzes the first acylation step of taxol biosynthesis.

The acetylation of taxa-4(20),11(12)-dien-5alpha-ol is considered to be the third specific step of Taxol biosynthesis that precedes further hydroxylation of the taxane nucleus. An operationally soluble acetyl CoA:taxadienol-O-acetyl transferase was demonstrated in extracts of Taxus canadensis and Taxus cuspidata cells induced with methyl jasmonate to produce Taxol. The reaction was dependent on both cosubstrates and active enzyme, and the product of this acetyl transferase was identified by radiochromatographic and GC-MS analysis. Following determination of the time course of acetyl transferase appearance in induced cell cultures, the operationally soluble enzyme was partially purified by a combination of anion exchange, hydrophobic interaction, and affinity chromatography on immobilized coenzyme A resin. This acetyl transferase has a pI and pH optimum of 4.7 and 9.0, respectively, and a molecular weight of about 50,000 as determined by gel permeation chromatography. The enzyme shows high selectivity and high affinity for both cosubstrates, with Km values of 4.2 and 5.5 microM for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced Taxol precursors, 10-deacetylbaccatin III or baccatin III. This acetyl transferase is insensitive to monovalent and divalent metal ions, is only weakly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and coenzyme A, and resembles in general properties the few other O-acetyl transferases of higher plant origin that have been examined.

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate.

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.

Cloning and functional expression of a cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production.

Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an induced T. canadensis cell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and [4-14C]isopentenyl diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90-100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative "mature proteins" from which the transit peptide was deleted, were transformed into a yeast mutant defective for the beta-subunit of type II geranylgeranyl transferase. Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant. However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl diphosphate as the cosubstrate for the geranylgeranyl diphosphate synthase reaction. In vitro activity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS-PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain. Results from the analysis of geranylgeranyl diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in induced T. canadensis suspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction.

Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures.

The cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene represents the first committed, and a slow, step in the complex biosynthetic pathway leading to the anticancer drug Taxol. The cyclization enzyme, taxadiene synthase, has been previously purified from Pacific yew (Taxus brevifolia) stem and characterized, and the corresponding cDNA has been isolated. To better assess the role of taxadiene synthase in the control of pathway flux in Canadian yew (T. canadensis) cells, a reliable system for production of Taxol in suspension culture, the enzyme from this source was isolated and shown to be chromatographically, electrophoretically, and kinetically identical to that of T. brevifolia stem. Results from the analysis of enzyme activity levels during the time course of Taxol accumulation in developing cell cultures of T. canadensis indicate that rate-limiting transformations lay farther down the pathway than the cyclization step in this system.

Cytochrome P450-catalyzed hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol: the first oxygenation step in taxol biosynthesis.

BACKGROUND: The structural complexity of taxol dictates continued reliance on biological production methods, which may be improved by a detailed understanding of taxol biosynthesis, especially the rate-limiting steps. The biosynthesis of taxol involves the cyclization of the common isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5), 11(2)-diene followed by extensive, largely oxidative, modification of this diterpene olefin. We set out to define the first oxygenation step in taxol biosynthesis. RESULTS: Microsomal enzymes from Taxus stem and cultured cells were used to define the first hydroxylation of taxadiene. We confirmed the structure of the reaction product (taxa-4(20), 11(12)-dien-5alpha-ol) by synthesizing this compound. The responsible biological catalyst was characterized as a cytochrome P450 (heme thiolate protein). In vivo studies confirmed that taxadienol is a biosynthetic intermediate and indicated that the hydroxylation step that produces this product is slow relative to subsequent metabolic transformations. CONCLUSIONS: The structure of the first oxygenated intermediate on the taxol pathway establishes that the hydroxylation reaction proceeds with an unusual double bond migration that limits the mechanistic possibilities for subsequent elaboration of the oxetane moiety of taxol. The reaction is catalyzed by a cytochrome P450, suggesting that the seven remaining oxygenation steps in taxol biosynthesis may involve similar catalysts. Because the first oxygenation step is slow relative to subsequent metabolic transformations, it may be possible to speed taxol biosynthesis by isolating and manipulating the gene for the taxadiene-5-hydroxylase that catalyzes this reaction.

Increased efficacy of Bacillus thuringiensis subsp. kurstaki in combination with tannic acid.

We identified tannic acid as an inexpensive additive that increased the efficacy of sublethal concentrations of Bacillus thuringiensis subsp. kurstaki (Berliner). Tannic acid mimicked the active constituents contained in an aqueous, tannin-rich extract of Taxus baccata (L.) bark that retarded development of Heliothis virescens (F.) larvae at 10,000 ppm; most larvae remained in first and second stage when treated with 250-10,000 ppm of tannic acid. Instar development of Trichoplusia ni (Hübner) larvae was affected in a concentration-dependent manner by 2.5-500 ppm of tannic acid. In subsequent bioassays, tannic acid at 25-500 ppm in combination with B. thuringiensis (1.63 micrograms [AI]/ml diet) yielded mean mortalities of 57-75%, whereas treatments with B. thuringiensis alone produced 10% mortality. Mean mortalities in the 3.0, 4.5, and 6.75 micrograms (AI) B. thuringiensis per milliliter of diet treatments (5.5; 8.0, and 30%, respectively) were significantly higher in the presence of 250 and 2,500 ppm tannic acid; in these treatments we observed 78-94% mortality. Addition of tannic acid increased the activity of concentrations of 3-4.5 micrograms (AI) B. thuringiensis per milliliter of diet to approximately that of a concentration of 13 micrograms (AI) B. thuringiensis per milliliter of diet alone (85-95% mortality). Although deaths caused by a formulation of B. thuringiensis + tannic acid occurred more slowly than with high rates of B. thuringiensis alone, such formulations would have the advantages of arresting development, minimizing foliar damage, and decreasing the concentration of B. thuringiensis used.

Initiation and growth of cell lines of Taxus brevifolia (Pacific yew).

Conditions have been established for the induction and maintenance of callus cultures of Taxus brevifolia (Pacific yew) from bark, stem, and needle tissues. Cultures were established on a modified Gamborg's B5 medium, 1% sucrose, 0.2% casamino acids and 1 mg/L 2,4-D. There was no apparent inhibition of callus induction as a result of taxol concentration in the explant material. Cell lines derived from explants of individual trees were used to investigate growth characteristics. Although none of the cell lines contained taxol, some contained low levels of related taxanes. Variability was observed with each cell line in response to light, and auxin type and concentration. Growth index was most affected by cell line, followed by auxin type and concentration. These culturing methods may be useful for the goal of developing a highproducing cell line applicable for large-scale taxol production.