Isolation and functional analysis of a cDNA encoding a myrcene synthase from holm oak (Quercus ilex L.).

An 859-bp cDNA segment of a terpene synthase gene was amplified by PCR from the evergreen sclerophyllous holm oak (Quercus ilex L.) using heterologous primers for conserved regions of terpene synthase genes (TPS) in dicotyledonous plants. Based on the sequence of this segment, homologous primers were designed for amplification by RACE-PCR of a cDNA segment carrying the monoterpene synthase gene myrS. The gene encodes a protein of 597 amino acids including an N-terminal putative plastid transit peptide. The gene without the segment encoding the transit peptide was cloned by PCR into a bacterial expression vector. Expression in Escherichia coli yielded an active monoterpene synthase, which converted geranyl diphosphate (GDP) predominantly into the acyclic monoterpene myrcene and to a very small extent into cyclic monoterpenes. Sequence comparison with previously cloned monoterpene synthases revealed that the myrcene synthase from Q. ilex belongs to the TPSb subfamily.     

Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-beta-ocimene synthase

The Arabidopsis genome project has recently reported sequences with similarity to members of the terpene synthase (TPS) gene family of higher plants. Surprisingly, several Arabidopsis terpene synthase-like sequences (AtTPS) share the most identity with TPS genes that participate in secondary metabolism in terpenoid-accumulating plant species. Expression of a putative Arabidopsis terpene synthase gene, designated AtTPS03, was demonstrated by amplification of a 392-bp cDNA fragment using primers designed to conserved regions of plant terpene synthases. Using the AtTPS03 fragment as a hybridization probe, a second AtTPS cDNA, designated AtTPS10, was isolated from a jasmonate-induced cDNA library. The partial AtTPS10 cDNA clone contained an open reading frame of 1665 bp encoding a protein of 555 amino acids. Functional expression of AtTPS10 in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate (C(10)) into the acyclic monoterpenes beta-myrcene and (E)-beta-ocimene and small amounts of cyclic monoterpenes. Based on sequence relatedness, AtTPS10 was classified as a member of the TPSb subfamily of angiosperm monoterpene synthases. Sequence comparison of AtTPS10 with previously cloned monoterpene synthases suggests independent events of functional specialization of terpene synthases during the evolution of terpenoid secondary metabolism in gymnosperms and angiosperms. Functional characterization of the AtTPS10 gene was prompted by the availability of Arabidopsis genome sequences. Although Arabidoposis has not been reported to form terpenoid secondary metabolites, the unexpected expression of TPS genes belonging to the TPSb subfamily in this species strongly suggests that terpenoid secondary metabolism is active in the model system Arabidopsis.     

Characterization of geraniol synthase from the peltate glands of sweet basil

The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 microM were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.     

Biosynthesis of marine natural products: isolation and characterization of a myrcene synthase from cultured tissues of the marine red alga Ochtodes secundiramea

The acyclic monoterpene myrcene is the likely progenitor of the unusual cytotoxic halogenated monoterpenes that are found in marine algae and that function as feeding deterrents to herbivores. Myrcene synthase was isolated from suspension cultures of the marine red alga Ochtodes secundiramea, representing the first enzyme of this type from a marine organism. The algal myrcene synthase produces exclusively myrcene from the natural substrate geranyl diphosphate (GDP), utilizes Mg(+2) as the required divalent metal ion cofactor, has a molecular mass of about 69 kDa, and exhibits a pH optimum near 7.2. These features are similar to those of monoterpene synthases from terrestrial organisms. When incubated with neryl diphosphate (the cis-isomer of GDP), the O. secundiramea myrcene synthase produces the cyclic monoterpene limonene, whereas incubation with (+/-)linalyl diphosphate (the tertiary allylic isomer of geranyl diphosphate) yields both acyclic and cyclic monoterpenes. These results suggest that the enzyme is incapable of isomerizing geranyl diphosphate to linalyl diphosphate, a feature common to all monoterpene cyclases from terrestrial sources. The limited catalytic capability of the myrcene synthase may reflect the ancient evolutionary origin of the producing organism. The ability to assay this enzyme in cultured algae, grown under strictly defined conditions, provides an unparalleled opportunity to delineate factors eliciting the biosynthesis of this class of secondary metabolites, to investigate the metabolic pathway leading to the halogenated monoterpenes, and to determine their role in the chemical ecology of marine algae.     

Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers

Terpenoids emitted from snapdragon flowers include three monoterpenes derived from geranyl diphosphate (GPP), myrcene, ( E )-β-ocimene and linalool, and a sesquiterpene, nerolidol, derived from farnesyl diphosphate (FPP). Using a functional genomics approach, we have isolated and biochemically characterized two nearly identical nerolidol/linalool synthases, AmNES/LIS-1 and AmNES/LIS-2, two enzymes responsible for the terpenoid profile of snapdragon scent remaining to be characterized. The AmNES/LIS-2 protein has an additional 30 amino acids in the N-terminus, and shares 95% amino acid sequence identity with AmNES/LIS-1, with only 23 amino acid substitutions distributed across the homologous regions of the proteins. Although these two terpene synthases have very similar catalytic properties, and synthesize linalool and nerolidol as specific products from GPP and FPP, respectively, they are compartmentally segregated. GFP localization studies and analysis of enzyme activities in purified leucoplasts, together with our previous feeding experiments, revealed that AmNES/LIS-1 is localized in cytosol, and is responsible for nerolidol biosynthesis, whereas AmNES/LIS-2 is located in plastids, and accounts for linalool formation. Our results show that subcellular localization of bifunctional enzymes, in addition to the availability of substrate, controls the type of product formed. By directing nearly identical bifunctional enzymes to more than one cellular compartment, plants extend the range of available substrates for enzyme utilization, thus increasing the diversity of the metabolites produced.     

Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua

A cDNA encoding farnesyl diphosphate (FPP) synthase (FPPS) has been cloned from a cDNA library of Artemisia annua. The sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with a calculated molecular weight of 39 420 kDa. The deduced aa sequence of the cDNA was highly similar to FPPS from other plants, yeast and mammals, and contained the two conserved domains found in polyprenyl synthases including FPPS, geranylgeranyl diphosphate synthases and hexaprenyl diphosphate synthases. The expression of the cDNA in Escherichia coli showed enzyme activity for FPPS in vitro.     

Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate.

Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.     

Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate

Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including β-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism.     

Molecular cloning and characterization of a new linalool synthase

Mentha citrata Ehrh. (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (-)-3R-linalool and its acetate ester. A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the (--)-3R-linalool synthase. The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceae) family (62-72% identity), but differs substantially from that of 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain). Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a K(m) value for the natural substrate, geranyl diphosphate, of 56 microM with k(cat) of 0.83 s(-1). These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-linalool synthase from Clarkia. Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is not an efficient precursor of linalool with the M. citrata synthase. Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3R-linalool.     

Structure and evolution of linalool synthase

Plant terpene synthases constitute a group of evolutionarily related enzymes. Within this group, however, enzymes that employ two different catalytic mechanisms, and their associated unique domains, are known. We investigated the structure of the gene encoding linalool synthase (LIS), an enzyme that uses geranyl pyrophosphate as a substrate and catalyzes the formation of linalool, an acyclic monoterpene found in the floral scents of many plants. Although LIS employs one catalytic mechanism (exemplified by limonene synthase [LMS]), it has sequence motifs indicative of both LMS-type synthases and the terpene synthases employing a different mechanism (exemplified by copalyl diphosphate synthase [CPS]). Here, we report that LIS genes analyzed from several species encode proteins that have overall 40%-96% identity to each other and have 11 introns in identical positions. Only the region encoding roughly the last half of the LIS gene (exons 9-12) has a gene structure similar to that of the LMS-type genes. On the other hand, in the first part of the LIS gene (exons 1-8), LIS gene structure is essentially identical to that found in the first half of the gene encoding CPS. In addition, the level of similarity in the coding information of this region between the LIS and CPS genes is also significant, whereas the second half of the LIS protein is most similar to LMS-type synthases. Thus, LIS appears to be a composite gene which might have evolved from a recombination event between two different types of terpene synthases. The combined evolutionary mechanisms of duplication followed by divergence and/or "domain swapping" may explain the extraordinarily large diversity of proteins found in the plant terpene synthase family.     

Isolation and partial characterisation of a putative monoterpene synthase from Melaleuca alternifolia.

Melaleuca alternifolia (Cheel) is an Australia native tree harvested for its monoterpene-rich, essential oil. Monoterpene synthases (E.C. 4.2.3.20) were partially purified from the flush growth of the commercially important, high terpinen-4-ol chemotype of M. alternifolia. The purified fractions produced an acyclic monoterpene, linalool that is not present in the essential oil. To further characterise the monoterpene synthase, a cDNA library was constructed and 500 expressed sequence tags (ESTs) were sequenced to isolate putative terpene synthases. A single clone with similarity to the TspB gene sub-family of angiosperm monoterpene and isoprene synthases was isolated but was truncated at the 5' end. This single clone was used to design a probe for a cDNA library and was applied to isolate a full-length clone. This gene encoded a polypeptide 583 amino acids in length (67 kDa) including a putative transit peptide. Heterologous expression of the gene in Escherichia coli and subsequent assay of the recombinant enzyme did not result in the production of terpinen-4-ol, the major constituent of tea tree oil, or of its precursor sabinene hydrate. Significant quantities of linalool were observed in these assays, and in the assays of monoterpene synthase activity of a native enzyme in vitro, but the racemic nature of the linalool means that it may have a non-enzymatic origin.     

A cDNA clone for beta-caryophyllene synthase from Artemisia annua

An homology-based cloning strategy yielded a full-length cDNA from Artemisia annua that encoded a protein of 60.3 kDa which resembled a sesquiterpene synthase in sequence. Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of farnesyl diphosphate to beta-caryophyllene, a sesquiterpene olefin found in the essential oil of A. annua. In reaction parameters and kinetic properties, beta-caryophyllene synthase resembles other sesquiterpene synthases of angiosperms. The beta-caryophyllene synthase gene is expressed in most plant tissues during early development, and is induced in mature tissue in response to fungal elicitor thus suggesting a role for beta-caryophyllene in plant defense.     

Evolution of the isoprene biosynthetic pathway in kudzu

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.     

Identification, functional characterization, and regulation of the enzyme responsible for floral (E)-nerolidol biosynthesis in kiwifruit (Actinidia chinensis)

Flowers of the kiwifruit species Actinidia chinensis produce a mixture of sesquiterpenes derived from farnesyl diphosphate (FDP) and monoterpenes derived from geranyl diphosphate (GDP). The tertiary sesquiterpene alcohol (E)-nerolidol was the major emitted volatile detected by headspace analysis. Contrastingly, in solvent extracts of the flowers, unusually high amounts of (E,E)-farnesol were observed, as well as lesser amounts of (E)-nerolidol, various farnesol and farnesal isomers, and linalool. Using a genomics-based approach, a single gene (AcNES1) was identified in an A. chinensis expressed sequence tag library that had significant homology to known floral terpene synthase enzymes. In vitro characterization of recombinant AcNES1 revealed it was an enzyme that could catalyse the conversion of FDP and GDP to the respective (E)-nerolidol and linalool terpene alcohols. Enantiomeric analysis of both AcNES1 products in vitro and floral terpenes in planta showed that (S)-(E)-nerolidol was the predominant enantiomer. Real-time PCR analysis indicated peak expression of AcNES1 correlated with peak (E)-nerolidol, but not linalool accumulation in flowers. This result, together with subcellular protein localization to the cytoplasm, indicated that AcNES1 was acting as a (S)-(E)-nerolidol synthase in A. chinensis flowers. The synthesis of high (E,E)-farnesol levels appears to compete for the available pool of FDP utilized by AcNES1 for sesquiterpene biosynthesis and hence strongly influences the accumulation and emission of (E)-nerolidol in A. chinensis flowers.     

Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species

The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as alpha-pinene and beta-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of alpha-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of alpha-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species.     

cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis).

Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)) and geranylgeranyl diphosphate (C(20)), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C(15)) synthases and diterpene (C(20)) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction.     

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula: ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL cis-PRENYL DIPHOSPHATE SYNTHASE GENE,LAVANDULYL DIPHOSPHATE SYNTHASE*

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent K_m and k_cat values of 208 ± 12 μm and 0.1 s⁻¹, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.