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

Abstract

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 ؋ 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 M). 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 M). These results illustrate the utility of iterative cycles of mathematical modeling and experimental testing to elucidate the mechanisms controlling flux through metabolic pathways.kinetic modeling ͉ monoterpene biosynthesis ͉ isoprenoid ͉ menthofuran ͉ pulegone reductase

Mathematical Modeling-Guided Evaluation of Biochemical, Developmental, Environmental, and Genotypic Determinants of Essential Oil Composition and Yield in Peppermint Leaves

“…All modeling assumptions were inferred directly from experimental observations, thus providing us with a unique opportunity to evaluate the depth of our understanding of this pathway. Here, we will provide a general overview of modifications to our first generation model (described in Rios-Estepa et al, 2008). A description of all modeling assumptions is given in Supplemental Data S1.…”

“…1). Follow-up biochemical studies established that this prediction was correct (Rios-Estepa et al, 2008), thus illustrating the utility of an approach that integrates mathematical modeling with experimental testing.…”

“…For dynamic systems, kinetic modeling is regarded as the generally most suitable method (McNeil et al, 2000;Poolman et al, 2004;Bruggeman and Westerhoff, 2006;Rios-Estepa and Lange, 2007;Mendes et al, 2009). Building on the rich body of published data on the enzymology and physiology of the peppermint monoterpene pathway (for review, see Croteau et al, 2005), we recently developed a first generation kinetic model to simulate the dynamics of peppermint monoterpene composition (Rios-Estepa et al, 2008). Modeling indicated that the monoterpene profiles observed in leaves of plants grown under lowlight conditions could be explained if one assumed that (+)-menthofuran, a dead-end side product, acted as a heretofore unknown competitive inhibitor against (+)-pulegone, the primary substrate of the branch point enzyme (+)-pulegone reductase (PR; Fig.…”

The application of synthetic biology to elucidation of plant mono-, sesqui- and diterpenoid metabolism

“…Enabled by the localization of methol production to protruding glandular trichomes that could be physically isolated (Gershenzon et al, 1992), this work began with enzymatic assays with plant cellfree extracts, progressed to molecular genetic identification of the relevant biosynthetic enzymes (for both on-and off-pathway reactions), leading to genetic/metabolic engineering in peppermint (Mentha x piperita) plants (Croteau et al, 2005). The full elucidation of the relevant pathway and (relatively few) branch points enabled a systems biology approach to understanding of biochemical regulation (Rios-Estepa et al, 2008), and comprehensive mathematical modeling guided evaluation of the pathway (Rios-Estepa et al, 2010). Notably, in part based on these advances, mint plants have been engineered for higher and more reliable yield of menthol, as demonstrated in multi-year field trials (Lange et al, 2011).…”

Essential oil research: past, present and future

“…[20] Systems biology approaches seem to be the next steps to identifying still unknown mechanisms in the EO components biosynthesis. [21] As a result of these eff orts, genetically or phytochemically tailored plants are to be expected in the future.…”