Rigoberto Rios-Estepa1, Glenn W. Turner2, James M. Lee3, and B. M. Lange2. (1) School of Chemical Engineering and Bioengineering, Washingtn State University, P.O. BOX 643710, Pullman, WA 99163, (2) Institute of Biological Chemistry, Washington State University, P.O. BOX 646340, Pullman, WA 99163, (3) School of Chemical Engineeering and Bioengineering, Washington State University, P.O. BOX 643710, Pullman, WA 99163
Essential oils derived from peppermint (Mentha x piperita) have been valued for thousands of years. These oils, the major components of which are the monoterpenes menthone (M-one) and menthol (M-ol), are synthesized in specialized anatomical structures on the leaf surface termed peltate glandular trichomes. Under adverse environmental conditions (low ambient light, low water, high night temperatures) peppermint oil may contain elevated levels of the monoterpene pathway intermediate pulegone (Pul) and the pathway byproduct menthofuran (MF), thus rendering an oil of poor quality. We developed a kinetic mathematical model of monoterpene biosynthesis in peppermint oil gland secretory cells, which allowed us to simulate the effects of varying environmental conditions on essential oil composition and yield. Interestingly, this model predicted that a concomitant increase in Pul and MF could only be accounted for when an inhibition of pulegone reductase (PulR), the enzyme that converts the intermediate Pul into M-one, by MF was assumed. Thus, the gene encoding peppermint PulR was expressed in E. coli, the recombinant protein purified and the effect of MF on recombinant PulR activity tested. Our kinetic analyses clearly established that MF acts as a competitive inhibitor (Ki = 300 µM), competing with Pul for the substrate binding site in PulR. We then isolated secretory cells and subjected them to steam distillations. MF turned out to accumulate at high levels (20 mM) in secretory cells obtained from peppermint plants grown under stress conditions but not in controls maintained under regular growth conditions. In transgenic plants expressing an antisense version of the menthofuran synthase (MFS) gene, which encodes the enzyme responsible for the synthesis of MF, low levels of MFS transcripts correlated with decreased amounts of Pul and MF under regular growth and stress conditions, indicating that the inhibition of PulR by MF was alleviated. These results demonstrate the utility of using mathematical modeling for guiding targeted metabolic engineering approaches in crop plants.