Rodney Croteau

Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase.

Peppermint (Mentha × piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition.

Sesquiterpene synthases from grand fir (Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of delta-selinene synthase and gamma-humulene synthase.

Grand fir (Abies grandis) has been developed as a model system for the study of oleoresin production in response to stem wounding and insect attack. The turpentine fraction of the oleoresin was shown to contain at least 38 sesquiterpenes that represent 12.5% of the turpentine, with the monoterpenes comprising the remainder. Assays of cell-free extracts from grand fir stem with farnesyl diphosphate as substrate indicated that the constitutive sesquiterpene synthases produced the same sesquiterpenes found in the oleoresin and that, in response to wounding, only two new products were synthesized, δ-cadinene and (E)-α-bisabolene. A similarity based cloning strategy yielded two new cDNA species from a stem cDNA library that, when expressed in Escherichia coli and the gene products subsequently assayed, yielded a remarkable number of sesquiterpene products. The encoded enzymes have been named δ-selinene synthase and γ-humulene synthase based on the principal products formed; however, each enzyme synthesizes three major products and produces 34 and 52 total sesquiterpenes, respectively, thereby accounting for many of the sesquiterpenes of the oleoresin. The deduced amino acid sequence of the δ-selinene synthase cDNA open reading frame encodes a protein of 581 residues (at 67.6 kDa), whereas that of the γ-humulene synthase cDNA encodes a protein of 593 residues (at 67.9 kDa). The two amino acid sequences are 83% similar and 65% identical to each other and range in similarity from 65 to 67% and in identity from 43 to 46% when compared with the known sequences of monoterpene and diterpene synthases from grand fir. Although the two sesquiterpene synthases from this gymnosperm do not very closely resemble terpene synthases from angiosperm species (52–56% similarity and 26–30% identity), there are clustered regions of significant apparent homology between the enzymes of these two plant classes. The multi-step, multi-product reactions catalyzed by the sesquiterpene synthases from grand fir are among the most complex of any terpenoid cyclase thus far described.

Cyclization Enzymes in the Biosynthesis of Monoterpenes, Sesquiterpenes, and Diterpenes

Terpene synthases catalyze the first committed steps in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes. An overview is presented of the enzymology and mechanism of these terpene synthases, and their molecular cloning, expression, and sequence analysis. Detailed structural and functional evaluation of four representative monoterpene, sesquiterpene, and diterpene synthases is also presented.

Strategies for transgenic manipulation of monoterpene biosynthesis in plants

Monoterpenes, the C(10) isoprenoids, are a large family of natural products that are best known as constituents of the essential oils and defensive oleoresins of aromatic plants. In addition to ecological roles in pollinator attraction, allelopathy and plant defense, monoterpenes are used extensively in the food, cosmetic and pharmaceutical industries. The importance of these plant products has prompted the definition of many monoterpene biosynthetic pathways, the cloning of the relevant genes and the development of genetic transformation techniques for agronomically significant monoterpene-producing plants. Metabolic engineering of monoterpene biosynthesis in the model plant peppermint has resulted in yield increase and compositional improvement of the essential oil, and also provided strategies for manipulating flavor and fragrance production, and plant defense.

Monoterpene Synthases from Grand Fir (Abies grandis) cDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF MYRCENE SYNTHASE, (−)-(4S)-LIMONENE SYNTHASE, AND (−)-(1S,5S)-PINENE SYNTHASE

Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (−)-limonene and (−)-α- and (−)-β-pinene are prominent components; (−)-limonene and (−)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression inEscherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (−)-limonene synthase, a myrcene synthase, and a (−)-pinene synthase that produces both α-pinene and β-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA blot hybridization using probes derived from the three monoterpene synthase cDNAs. The availability of cDNA species encoding these monoterpene synthases will allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects. These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic olefin products.

Bornyl diphosphate synthase: Structure and strategy for carbocation manipulation by a terpenoid cyclase

The x-ray crystal structure of dimeric (+)-bornyl diphosphate synthase, a metal-requiring monoterpene cyclase from Salvia officinalis, is reported at 2.0-Å resolution. Each monomer contains two α-helical domains: the C-terminal domain catalyzes the cyclization of geranyl diphosphate, orienting and stabilizing multiple reactive carbocation intermediates; the N-terminal domain has no clearly defined function, although its N terminus caps the active site in the C-terminal domain during catalysis. Structures of complexes with aza analogues of substrate and carbocation intermediates, as well as complexes with pyrophosphate and bornyl diphosphate, provide “snapshots” of the terpene cyclization cascade.

Menthol biosynthesis and molecular genetics

Abstract(−)-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.

Monoterpene Synthases from Common Sage (Salvia officinalis) cDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF (+)-SABINENE SYNTHASE, 1,8-CINEOLE SYNTHASE, AND (+)-BORNYL DIPHOSPHATE SYNTHASE

Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of thep-menthane (1,8-cineole), pinane (α- and β-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli. The heterologously expressed synthases produce (+)-bornyl diphosphate, 1,8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-α-pinene, (+)-camphene, and (±)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (−)-α-pinene, (+)- and (−)-β-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of γ-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes.

Floral Scent Production in Clarkia (Onagraceae) (I. Localization and Developmental Modulation of Monoterpene Emission and Linalool Synthase Activity)

The flowers of many plants emit volatile compounds as a means of attracting pollinators. We have previously shown that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of approximately 8 to 12 volatile compounds[mdash]three monoterpenes and nine benzoate derivatives (R.A. Raguso and E. Pichersky [1994] Plant Syst Evol [in press]). Here we report that the monoterpene alcohol linalool is synthesized and emitted mostly by petals but to a lesser extent also by the pistil and stamens. Two linalool oxides are produced and emitted almost exclusively by the pistil. These three monoterpenes are first discernible in mature unopened buds, and their tissue levels are highest during the first 2 to 3 d after anthesis. Levels of emission by the different floral parts throughout the life span of the flower were correlated with levels of these monoterpenes in the respective tissues, suggesting that these monoterpenes are emitted soon after their synthesis. Activity of linalool synthase, an enzyme that converts the ubiquitous C10 isoprenoid intermediate geranyl pyrophosphate to linalool, was highest in petals, the organ that emits most of the linalool. However, linalool synthase activity on a fresh weight basis was highest in stigma and style (i.e. the pistil). Most of the linalool produced in the pistil is apparently converted into linalool oxides. Lower levels (0.1%) of monoterpene emission and linalool synthase activity are found in the stigma of Clarkia concinna, a nonscented relative of C. breweri, suggesting that monoterpenes may have other functions in the flower in addition to attracting pollinators.

Isolation of Secretory Cells from Plant Glandular Trichomes and Their Use in Biosynthetic Studies of Monoterpenes and Other Gland Products

The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes.