Geranyl diphosphate synthase from Abies grandis: cDNA isolation,functional expression,and characterization

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.     

Alternative termination chemistries utilized by monoterpene cyclases: chimeric analysis of bornyl diphosphate, 1,8-cineole,and sabinene synthases

Monoterpene cyclization reactions are initiated by ionization and isomerization of geranyl diphosphate, and proceed, via cyclization of bound linalyl diphosphate, through a series of carbocation intermediates with ultimate termination of the multistep cascade by deprotonation or nucleophile capture. Three structurally and mechanistically related monoterpene cyclases from Salvia officinalis, (+)-sabinene synthase (deprotonation to olefin), 1,8-cineole synthase (water capture), and (+)-bornyl diphosphate synthase (diphosphate capture), were employed to explore the structural determinants of these alternative termination chemistries. Results with chimeric recombinant enzymes, constructed by reciprocally substituting regions of sabinene synthase with the corresponding sequences from bornyl diphosphate synthase or 1,8-cineole synthase, demonstrated that exchange of the C-terminal catalytic domain is sufficient to completely switch the resulting product profile. Exchange of smaller sequence elements identified a region of roughly 70 residues from 1,8-cineole synthase that, when substituted into sabinene synthase, conferred the ability to produce 1,8-cineole. A similar strategy identified a small region of bornyl diphosphate synthase important in conducting the anti-Markovnikov addition to the bornane skeleton. Observations made with these chimeric monoterpene cyclases are discussed in the context of the recently determined crystal structure for bornyl diphosphate synthase.     

Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.     

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.     

Unusual features of a recombinant apple alpha-farnesene synthase

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.     

Purification of 4S-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita) and spearmint (Mentha spicata).

The p-menthane monoterpenes of the Mentha species are biosynthesized from geranyl pyrophosphate via the monocyclic olefin 4S-limonene. A monoterpene cyclase was isolated from both Mentha x piperita (peppermint) and Mentha spicata (spearmint) that catalyzes the cyclization of geranyl pyrophosphate to 4S-limonene. This enzyme, 4S-limonene synthase, was purified to apparent homogeneity by dye ligand, anion exchange, and hydrophobic interaction chromatography. Since the monoterpenes of Mentha are synthesized and secreted in modified epidermal hairs called glandular trichomes, an extract of isolated glandular trichome cells was used as the source of this enzyme. A combination of gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that purified 4S-limonene synthase had a native molecular weight of 56,000 and was monomeric. The principal product of the enzyme was enantiomerically pure (-)-4S-limonene, and a catalytic constant of 0.3/s was determined. The basic properties of 4S-limonene synthase from both M. x piperita and M. spicata are identical and, in general, are similar to those of other monoterpene, sesquiterpene, and diterpene cyclases isolated from microorganisms and higher plants.     

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.     

Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-beta-D-glucopyranoside

Petunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in the various tissues is not present as free linalool, but was efficiently converted to non-volatile S-linalyl-beta-D-glucopyranoside by the action of endogenous glucosyltransferase. The results presented demonstrate that monoterpene production can be altered by genetic modification, and that the compounds produced can be converted by endogenous enzymatic activity.     

Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita).

(4S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha x piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (greater than 94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg2+ or Mn2+, with Mn2+ preferred. The Km value measured for geranyl pyrophosphate was 1.8 microM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence.     

Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.     

Antigenic cross-reactivity among monoterpene cyclases from grand fir and induction of these enzymes upon stem wounding.

A major wound response in grand fir (Abies grandis) sapling stems is the rapid increase in monoterpene production at the site of injury. Monoterpene cyclases (synthases) catalyze the formation of monoterpenes from geranyl pyrophosphate, and total cyclase activity increases markedly on wounding. At least six distinct cyclases, producing different monoterpene products, have been isolated from wounded grand fir saplings and characterized. The predominant wound-inducible cyclase produces both alpha- and beta-pinene. This pinene cyclase was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein. The antibody preparation was found to cross-react by Western blotting with other grand fir monoterpene cyclases that produce different olefinic products, but not with monoterpene cyclases from related conifer species (Pinus contorta and P. ponderosa) or from angiosperms (Mentha piperita and M. spicata). The increase in monoterpene cyclase activity after wounding was closely correlated with the appearance of new cyclase protein as determined by immunoblotting. These results indicate that the wound-dependent increase in monoterpene cyclase activity is a consequence of de novo synthesis of cyclase protein.     

Characterization of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin)

Gamma-terpinene is a monoterpene and a major component of essential oils made from citrus fruits and shows strong antioxidant activity in various assay systems. Plant gamma-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be gamma-terpinene by GC/MS.     

Purification and characterization of the monoterpene cyclase gamma-terpinene synthase from Thymus vulgaris

The monoterpene cyclase, gamma-terpinene synthase, from Thymus vulgaris (thyme) leaves was purified to apparent homogeneity by isoelectric focusing and dye-ligand, anion-exchange, hydrophobic interaction, and gel permeation chromatography. The enzyme has a native molecular weight of 96,000 as determined by gel permeation chromatography, and exhibited a specific activity of 538 nmol/h.mg protein (turnover number of approximately 0.01/s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two apparently identical subunits of Mr approximately 55,000. The protein was very hydrophobic, and possessed a pI value of 4.85 as determined by isoelectric focusing. Maximum activity was observed at pH 6.8 in the presence of 20 mM Mg2+; 5 mM Mn2+ could support catalysis, albeit at a much lower rate. The Km value for the substrate, geranyl pyrophosphate, was 2.6 microM. Cyclase activity was inhibited by cysteine- and histidine-directed reagents. Purified gamma-terpinene synthase also possessed the ability to cyclize geranyl pyrophosphate to small amounts of alpha-thujene and to lesser quantities of myrcene, alpha-terpinene, limonene, linalool, terpinen-4-ol, and alpha-terpineol, all of which appear to be coproducts of the reaction sequence leading to gamma-terpinene. In general properties, the gamma-terpinene synthase from thyme leaves resembles other monoterpene cyclases as well as sesquiterpene and diterpene cyclases.     

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.     

Mechanism of monoterpene cyclization: stereochemical aspects of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which ultimately cyclizes to the various monoterpene skeletons. Previous experimental approaches using the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and racemic methanogeranyl diphosphate, in attempts to dissect the cryptic isomerization step from the normally coupled reaction sequence, were thwarted by the limited product available from native monoterpene synthases and by the inability to resolve chiral monoterpene products at the microscale. These approaches were revisited using three recombinant monoterpene synthases and chiral phase capillary gas chromatographic methods to separate antipodal products of the substrate analogs. The recombinant monoterpene olefin synthases, (-)-limonene synthase from spearmint and (-)-pinene synthase from grand fir, yielded essentially only achiral, olefin products (corresponding to the respective analogs and homologs of myrcene, trans-ocimene and cis-ocimene) from 6,7-dihydrogeranyl diphosphate and (2S,3R)-methanogeranyl diphosphate; no significant amounts of terpenols or homoterpenols were formed, nor was direct evidence obtained for the formation of the anticipated analog and homolog of the tertiary intermediate linalyl diphosphate (i.e., 6,7-dihydrolinalyl diphosphate and homolinalyl diphosphate, respectively). In the case of recombinant (+)-bornyl diphosphate synthase from common sage, the achiral olefins were generated, as before, from 6,7-dihydrogeranyl diphosphate and (2R,3S)-methanogeranyl diphosphate, but 6,7-dihydrolinalool and homolinalool also comprised significant components of the respective product mixtures, indicating greater access of water to the active site of this enzyme compared to the olefin synthases; again, no direct evidence for the production of 6,7-dihydrolinalyl diphosphate or homolinalyl diphosphate was obtained. Resolution of the terpenol products of (+)-bornyl diphosphate synthase, by chiral phase separation, revealed the predominant formation of (3R)-dihydrolinalool from dihydrogeranyl diphosphate and of (4S)-homolinalool from (2R,3S)-methanogeranyl diphosphate. The opposite stereochemistries of these products indicates water trapping from opposite faces of the corresponding tertiary carbocationic intermediates of the respective reactions, a phenomenon that appears to result from the binding conformations of these substrate analogs. Although these experiments failed to provide direct evidence for the tertiary intermediate of the tightly coupled isomerization-cyclization sequence, they did reveal a mechanistic difference between the olefin synthases and bornyl diphosphate synthase involving access of water as a participant in the reaction.     

Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.     

A cDNA clone for 3-carene synthase from Salvia stenophylla

The essential oil of Salvia stenophylla contains (+)-3-carene as the principal monoterpene component. Using an enriched cDNA library prepared from mRNA isolated from S. stenophylla peltate glandular trichomes, and a homology-based cloning strategy, a full-length cDNA was isolated that encoded a preprotein of 69.7 kDa which resembled a monoterpene 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 geranyl diphosphate to (+)-3-carene and to lesser amounts of limonene, myrcene, 4-carene and beta-phellandrene. This multiple-product synthase is responsible for the production of all of the essential oil monoterpenes of S. stenophylla.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

Mono and diterpene production in Escherichia coli

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.