Slow-Starter Enzymes: Role of Active-Site Architecture in the Catalytic Control of the Biosynthesis of Taxadiene by Taxadiene Synthase.

Taxadiene synthase (TXS) catalyzes the formation of natural product taxa-4(5),11(12)-diene (henceforth taxadiene). Taxadiene is the precursor in the formation of Taxol, which is an important natural anticancer agent. In the current study, we present a detailed mechanistic view of the biosynthesis of taxadiene by TXS using a hybrid quantum mechanics-molecular mechanics potential in conjunction with free energy simulation methods. The obtained free-energy landscape displays initial endergonic steps followed by a stepwise downhill profile, which is an emerging free-energy fingerprint for type I terpene synthases. We identify an active-site Trp residue (W753) as a key feature of the TXS active-site architecture and propose that this residue stabilized intermediate cations via π-cation interactions. To validate our proposed active TXS model, we examine a previously reported W753H mutation, which leads to the exclusive formation of side product cembrene A. The simulations of the W753H mutant show that, in the mutant structure, the His side chain is in the perfect position to deprotonate the cembrenyl cation en route to cembrene formation and that this abortive deprotonation is an energetically facile process. On the basis of the current model, we propose that an analogous mutation of Y841 to His could possibly lead to verticillane. The current simulations stress the importance of the precise positioning of key active-site residues in stabilizing intermediate carbocations. In view of the great pharmaceutical importance of taxadiene, a detailed understanding of the TXS mechanism can provide important clues toward a synthetic strategy for Taxol manufacturing.

Impact of drought stress on specialised metabolism: Biosynthesis and the expression of monoterpene synthases in sage (Salvia officinalis).

In previous experiments, we demonstrated that the amount of monoterpenes in sage is increased massively by drought stress. Our current study is aimed to elucidate whether this increase is due, at least in part, to elevated activity of the monoterpene synthases responsible for the biosynthesis of essential oils in sage. Accordingly, the transcription rates of the monoterpene synthases were analyzed. Salvia officinalis plants were cultivated under moderate drought stress. The concentrations of monoterpenes as well as the expression of the monoterpene synthases were analyzed. The amount of monoterpenes massively increased in response to drought stress; it doubled after just two days of drought stress. The observed changes in monoterpene content mostly match with the patterns of monoterpene synthase expressions. The expression of bornyl diphosphate synthase was strongly up-regulated; its maximum level was reached after two days. Sabinene synthase increased gradually and reached a maximum after two weeks. In contrast, the transcript level of cineole synthase continuously declined. This study revealed that the stress related increase of biosynthesis is not only due to a "passive" shift caused by the stress related over-reduced status, but also is due - at least in part-to an "active" up-regulation of the enzymes involved.

Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221.

Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and ß-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/ß-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively).

Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids.

BACKGROUND: Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. RESULTS: S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production. CONCLUSIONS: Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.

Challenges posed to bornyl diphosphate synthase: diverging reaction mechanisms in monoterpenes.

The simplest form of terpenoid chemistry is found for the monoterpenes, which give plants fragrance, flavor, and medicinal properties. Monoterpene synthases employ geranyl diphosphate as a substrate to generate an assortment of cyclic products. In the current study we present a detailed analysis of the multiple gas-phase reaction pathways in the synthesis of bornyl cation from geranyl diphosphate. Additionally, the fate of the proposed bornyl cation intermediate in the bornyl diphosphate synthase reaction is investigated by molecular dynamics simulations. We employ accurate density functional theory (DFT) methods after careful validation against high-level ab initio data for a set of model carbocations. The gas-phase results for the monoterpene reactions indicate a diverging reaction mechanism with multiple products in the absence of enzymatic control. This complex potential energy surface includes several possible bifurcation points due to the presence of secondary cations. Additionally, the suggested bornyl cation intermediate in the bornyl diphosphate synthase reaction is studied by molecular dynamics simulations employing a hybrid quantum mechanics (DFT)-molecular mechanics potential energy function. The simulations suggest that the bornyl cation is a transient species as in the gas phase and that electrostatic steering directs the formation of the final product, bornyl diphosphate.

Syn stereochemistry of cyclic ether formation in 1,8-cineole biosynthesis catalyzed by recombinant synthase from Salvia officinalis.

Regiospecifically labeled geranyl diphosphates ((2E,6E)-[1,1,8,8,8-(2)H(5)]- and (2E,6Z)-[1,1,9,9,9-(2)H(5)]-GDP) and D(2)O incorporation, in concert with NMR spectrometry, were employed to demonstrate a unique intramolecular syn-facial protonation-cyclization mechanism of action of 1,8-cineole synthase.