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(-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint (Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general "allylic oxidation-conjugate reduction" scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1R, 3R, 4S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3-and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (2)-trans-isopiperitenol to (2)-isopiperitenone in peppermint and (2)-trans-carveol to (2)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (2)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (2)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (2)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5#-truncated cDNA encoding the spearmint homolog, (2)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5#-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The fulllength spearmint dehydrogenase shares .99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (2)-trans-isopiperitenol and (2)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/ reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene reductases of mint involved in (2)-menthol biosynthesis. The isolation of the genes specifying redox enzymes of monoterpene biosynthesis in mint indicates that these genes arose from different ancestors and not by simple duplication and differentiation of a common progenitor, as might have been anticipated based on the common reaction chemistry and structural similarity of the substrate monoterpenes.Monoterpenes, the C 10 members of the terpenoid family of secondary metabolites, are largely responsible for the fragrance and flavor characteristics of plants and are well known as components of plant essential oils (Parry, 1969). The essential oils of both peppermint (Mentha x piperita), a sterile cross between Mentha spicata and Mentha aquatica (Murray et al., 1972;Harley and Brighton, 1977), and spearmint (Mentha spicata) consist primarily of monoterpenes (Lawrence, 1981) that are distinguished by the position of oxygenation on the p-menthane ring conferred by two distinct regiospecific cytochrome P450 limonene hydroxylases (Fig. 1). Thus, following cycliz...

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Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (−)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed. monoterpene synthase | enzyme catalysis | mechanism | carbocation | structure-function relationship T erpenoids are a structurally diverse group of metabolites with functions in both primary and secondary (or specialized) metabolism. Primary metabolites derived from terpenoid pathway intermediates in plants include sterols, carotenoids, and the side chains of chlorophylls, tocopherols, and quinones of electron transport systems. Many plant hormones are also products of terpenoid metabolism, including abscisic acid, cytokinins, brassinosteroids, and strigolactones (1). Secondary plant metabolites of terpenoid origin can play critical defense-related roles (e.g., sesquiterpene lactones and triterpene saponins serve as antifeedants) and are dominant constituents of essential oils and resins (mono-, sesqui-, and diterpenes) (2). Terpene synthases (TPSs) convert a prenyl diphosphate of a specific chain length to the first pathway-specific (often cyclic) intermediate in the biosynthesis of each class of terpenoids. Whereas some terpene synthases are remarkably specific and only generate one product from a prenyl diphosphate precursor, others release a larger number of products from a common substrate, thus contributing to terpenoid chemical diversity (3). The genomes of plants may only contain one TPS gene [e.g., ent-kaurene (diterpene) synthase in the moss Physcomitrella patens (Hedw.) Bruch & Schimp.], but often harbor sizable families of TPS genes with more than 20 members, which is another source of terpenoid structural variety (4).All monoterpene synthases (MTSs) use either geranyl diphosphate (GPP) or its 2Z-isomer neryl diphosphate as substrate, but the sequence conservatio...

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.

(−)-Menthone is the predominant monoterpene produced in the essential oil of maturing peppermint (Mentha x piperita) leaves during the filling of epidermal oil glands. This early biosynthetic process is followed by a second, later oil maturation program (approximately coincident with flower initiation) in which the C3-carbonyl of menthone is reduced to yield (−)-(3R)-menthol and (+)-(3S)-neomenthol by two distinct NADPH-dependent ketoreductases. An activity-based in situ screen, by expression in Escherichia coli of 23 putative redox enzymes from an immature peppermint oil gland expressed sequence tag library, was used to isolate a cDNA encoding the latter menthone:(+)-(3S)-neomenthol reductase. Reverse transcription-PCR amplification and RACE were used to acquire the former menthone:(−)-(3R)-menthol reductase directly from mRNA isolated from the oil gland secretory cells of mature leaves. The deduced amino acid sequences of these two reductases share 73% identity, provide no apparent subcellular targeting information, and predict inclusion in the short-chain dehydrogenase/reductase family of enzymes. The menthone:(+)-(3S)-neomenthol reductase cDNA encodes a 35,722-D protein, and the recombinant enzyme yields 94% (+)-(3S)-neomenthol and 6% (−)-(3R)-menthol from (−)-menthone as substrate, and 86% (+)-(3S)-isomenthol and 14% (+)-(3R)-neoisomenthol from (+)-isomenthone as substrate, has a pH optimum of 9.3, and K  m values of 674 μ  m, > 1 mm, and 10 μ  m for menthone, isomenthone, and NADPH, respectively, with a k  cat of 0.06 s−1. The recombinant menthone:(−)-(3R)-menthol reductase has a deduced size of 34,070 D and converts (−)-menthone to 95% (−)-(3R)-menthol and 5% (+)-(3S)-neomenthol, and (+)-isomenthone to 87% (+)-(3R)-neoisomenthol and 13% (+)-(3S)-isomenthol, displays optimum activity at neutral pH, and has K  m values of 3.0 μ  m, 41 μ  m, and 0.12 μ  m for menthone, isomenthone, and NADPH, respectively, with a k  cat of 0.6 s−1. The respective activities of these menthone reductases account for all of the menthol isomers found in the essential oil of peppermint. Biotechnological exploitation of these genes could lead to improved production yields of (−)-menthol, the principal and characteristic flavor component of peppermint.

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