Accurate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants.

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents.

Improving peppermint essential oil yield and composition by metabolic engineering.

Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost.

A systems biology approach identifies the biochemical mechanisms regulating monoterpenoid essential oil composition in peppermint.

The integration of mathematical modeling and experimental testing is emerging as a powerful approach for improving our understanding of the regulation of metabolic pathways. In this study, we report on the development of a kinetic mathematical model that accurately simulates the developmental patterns of monoterpenoid essential oil accumulation in peppermint (Mentha x piperita). This model was then used to evaluate the biochemical processes underlying experimentally determined changes in the monoterpene pathway under low ambient-light intensities, which led to an accumulation of the branchpoint intermediate (+)-pulegone and the side product (+)-menthofuran. Our simulations indicated that the environmentally regulated changes in monoterpene profiles could only be explained when, in addition to effects on biosynthetic enzyme activities, as yet unidentified inhibitory effects of (+)-menthofuran on the branchpoint enzyme pulegone reductase (PR) were assumed. Subsequent in vitro analyses with recombinant protein confirmed that (+)-menthofuran acts as a weak competitive inhibitor of PR (K(i) = 300 muM). To evaluate whether the intracellular concentration of (+)-menthofuran was high enough for PR inhibition in vivo, we isolated essential oil-synthesizing secretory cells from peppermint leaves and subjected them to steam distillations. When peppermint plants were grown under low-light conditions, (+)-menthofuran was selectively retained in secretory cells and accumulated to very high levels (up to 20 mM), whereas under regular growth conditions, (+)-menthofuran levels remained very low (<400 muM). These results illustrate the utility of iterative cycles of mathematical modeling and experimental testing to elucidate the mechanisms controlling flux through metabolic pathways.

Menthofuran regulates essential oil biosynthesis in peppermint by controlling a downstream monoterpene reductase.

(+)-Pulegone is a central intermediate in the biosynthesis of (-)-menthol, the most significant component of peppermint essential oil. Depending on environmental conditions, this branch point metabolite may be reduced to (-)-menthone en route to menthol, by pulegone reductase (PR), or oxidized to (+)-menthofuran, by menthofuran synthase (MFS). To elucidate regulation of pulegone metabolism, we modified the expression of mfs under control of the CaMV 35S promoter in transformed peppermint plants. Overexpression and cosuppression of mfs resulted in the respective increase or decrease in the production of menthofuran, indicating that the control of MFS resides primarily at the level of transcription. Significantly, in both WT peppermint as well as in all transformed plants, the flux of (+)-pulegone through PR correlated negatively with the essential oil content of menthofuran, such that menthofuran, and pulegone increased, or decreased, in concert. These results suggested that menthofuran itself might influence the reduction of pulegone. Although (+)-menthofuran did not inhibit (+)-PR activity, stem feeding with menthofuran selectively decreased pr transcript levels in immature leaves, thereby accounting for decreased reductase activity and increased pulegone content. These data demonstrate that the metabolic fate of (+)-pulegone is controlled through transcriptional regulation of mfs and that menthofuran, either directly or indirectly, influences this process by down-regulating transcription from pr and/or decreasing pr message stability. The ability to reduce both menthofuran and pulegone levels is of commercial significance in improving essential oil quality; however, the physiological rationale for such complex regulation is presently unclear.

Abietadiene synthase catalysis: mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement.

Abietadiene synthase catalyzes the committed step in resin acid biosynthesis, forming a mixture of abietadiene double-bond isomers by two sequential, mechanistically distinct cyclizations at separate active sites. The first reaction, protonation-initiated cyclization, converts the universal diterpene precursor geranylgeranyl diphosphate to the stable bicyclic intermediate copalyl diphosphate. In the second, magnesium ion-dependent reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone and is coupled, by intramolecular proton transfer within a transient pimarenyl intermediate, to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane structure. Alternative deprotonations of the terminal abietenyl carbocation provide a mixture of abietadiene, levopimaradiene, and neoabietadiene, and this product profile varies as a function of pH. Mutational analysis of amino acids at the active site of a modeled structure has identified residues critical for catalysis, as well as several that play roles in specifying product formation, apparently by ligation of a magnesium ion cofactor. These results strongly suggest that choice between alternatives for deprotonation of the abietenyl intermediate depends more on the positioning effects of the carbocation-diphosphate anion reaction partners than on the pKa of multiple participating bases. In one extreme case, mutant N765A is unable to mediate the intramolecular proton transfer and aborts the reaction, without catalyzing 1,2-methyl migration, to produce only sandaracopimaradiene, thereby providing supporting evidence for the corresponding stereochemistry of the cryptic pimarenyl intermediate of the reaction pathway.