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

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Clavulanic acid (CA) is produced by Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. clavuligerus.

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