Structurally Guided Reprogramming of Valerenadiene Synthase.

Valerena-1,10-diene synthase (VDS) catalyzes the conversion of the universal precursor farnesyl diphosphate into the unusual sesquiterpene valerena-1,10-diene (VLD), which possesses a unique isobutenyl substituent group. In planta, one of VLD's isobutenyl terminal methyl groups becomes oxidized to a carboxylic acid forming valerenic acid (VA), an allosteric modulator of the GABAA receptor. Because a structure-activity relationship study of VA for its modulatory activity is desired, we sought to manipulate the VDS enzyme for the biosynthesis of structurally diverse scaffolds that could ultimately lead to the generation of VA analogues. Using three-dimensional structural homology models, phylogenetic sequence comparisons to well-characterized sesquiterpene synthases, and a substrate-active site contact mapping approach, the contributions of specific amino acid residues within or near the VDS active site to possible catalytic cascades for VLD and other sesquiterpene products were assessed. An essential role of Tyr535 in a germacrenyl route to VLD was demonstrated, while its contribution to a family of other sesquiterpenes derived from a humulyl route was not. No role for Cys415 or Cys452 serving as a proton donor to reaction intermediates in VLD biosynthesis was observed. However, a gatekeeper role for Asn455 in directing farnesyl carbocations down all-trans catalytic cascades (humulyl and germacrenyl routes) versus a cisoid cascade (nerolidyl route) was demonstrated. Altogether, these results have mapped residues that establish a context for the catalytic cascades operating in VDS and future manipulations for generating more structurally constrained scaffolds.

Mouse lipogenic proteins promote the co-accumulation of triacylglycerols and sesquiterpenes in plant cells.

MAIN CONCLUSION: Mouse FIT2 protein redirects the cytoplasmic terpene biosynthetic machinery to lipid-droplet-forming domains in the ER and this relocalization supports the efficient compartmentalization and accumulation of sesquiterpenes in plant cells. Mouse (Mus musculus) fat storage-inducing transmembrane protein 2 (MmFIT2), an endoplasmic reticulum (ER)-resident protein with an important role in lipid droplet (LD) biogenesis in mammals, can function in plant cells to promote neutral lipid compartmentalization. Surprisingly, in affinity capture experiments, the Nicotiana benthamiana 5-epi-aristolochene synthase (NbEAS), a soluble cytoplasm-localized sesquiterpene synthase, was one of the most abundant proteins that co-precipitated with GFP-tagged MmFIT2 in transient expression assays in N. benthamiana leaves. Consistent with results of pull-down experiments, the subcellular location of mCherry-tagged NbEAS was changed from the cytoplasm to the LD-forming domains in the ER, only when co-expressed with MmFIT2. Ectopic co-expression of NbEAS and MmFIT2 together with mouse diacylglycerol:acyl-CoA acyltransferase 2 (MmDGAT2) in N. benthamiana leaves substantially increased the numbers of cytoplasmic LDs and supported the accumulation of the sesquiterpenes, 5-epi-aristolochene and capsidiol, up to tenfold over levels elicited by Agrobacterium infection alone. Taken together, our results suggest that MmFIT2 recruits sesquiterpene synthetic machinery to ER subdomains involved in LD formation and that this process can enhance the efficiency of sesquiterpene biosynthesis and compartmentalization in plant cells. Further, MmFIT2 and MmDGAT2 represent cross-kingdom lipogenic protein factors that may be used to engineer terpene accumulation more broadly in the cytoplasm of plant vegetative tissues.

Engineering triterpene metabolism in the oilseed of Arabidopsis thaliana.

Squalene and botryococcene are linear, hydrocarbon triterpenes that have industrial and medicinal values. While natural sources for these compounds exist, there is a pressing need for robust, renewable production platforms. Oilseeds are an excellent target for heterologous production because of their roles as natural storage repositories and their capacity to produce precursors from photosynthetically-derived carbon. We generated transgenic Arabidopsis thaliana plants using a variety of engineering strategies (subcellular targeting and gene stacking) to assess the potential for oilseeds to produce these two compounds. Constructs used seed-specific promoters and evaluated expression of a triterpene synthase alone and in conjunction with a farnesyl diphosphate synthase (FPS) plus 1-deoxyxylulose 5-phosphate synthase (DXS). Constructs directing biosynthesis to the cytosol to harness isoprenoid precursors from the mevalonic acid (MVA) pathway were compared to those directing biosynthesis to the plastid compartment diverting precursors from the methylerythritol phosphate (MEP) pathway. On average, the highest accumulation for both compounds was achieved by targeting the triterpene synthase, FPS and DXS to the plastid (526.84 µg/g seed for botryococcene and 227.30 µg/g seed for squalene). Interestingly, a higher level accumulation of botryococcene (a non-native compound) was observed when the biosynthetic enzymes were targeted to the cytosol (>1000 µg/g seed in one line), but not squalene (natively produced in the cytosol). Not only do these results indicate the potential of engineering triterpene accumulation in oilseeds, but they also uncover some the unique regulatory mechanisms controlling triterpene metabolism in different cellular compartments of seeds.

Engineering linear, branched-chain triterpene metabolism in monocots.

Triterpenes are thirty-carbon compounds derived from the universal five-carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large-scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T0 mature plants) were obtained using the cytosolic-targeting strategy. Plastid-targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid-targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

Agronomic and chemical performance of field-grown tobacco engineered for triterpene and methylated triterpene metabolism.

Squalene is a linear intermediate to nearly all classes of triterpenes and sterols and is itself highly valued for its use in wide range of industrial applications. Another unique linear triterpene is botryococcene and its methylated derivatives generated by the alga Botryococcus braunii race B, which are progenitors to fossil fuel deposits. Production of these linear triterpenes was previously engineered into transgenic tobacco by introducing the key steps of triterpene metabolism into the particular subcellular compartments. In this study, the agronomic characteristics (height, biomass accumulation, leaf area), the photosynthetic capacity (photosynthesis rate, conductance, internal CO2 levels) and triterpene content of select lines grown under field conditions were evaluated for three consecutive growing seasons. We observed that transgenic lines targeting enzymes to the chloroplasts accumulated 50-150 times more squalene than the lines targeting the enzymes to the cytoplasm, without compromising growth or photosynthesis. We also found that the transgenic lines directing botryococcene metabolism to the chloroplast accumulated 10- to 33-fold greater levels than the lines where the same enzymes were targeted to in the cytoplasm. However, growth of these high botryococcene accumulators was highly compromised, yet their photosynthesis rates remained unaffected. In addition, in the transgenic lines targeting a triterpene methyltransferase (TMT) to the chloroplasts of high squalene accumulators, 55%-65% of total squalene was methylated, whereas in the lines expressing a TMT in the cytoplasm, only 6%-13% of squalene was methylated. The growth of these methylated triterpene-accumulating lines was more compromised than that of nonmethylated squalene lines.

Mapping a kingdom-specific functional domain of squalene synthase.

Squalene synthase catalyzes the first committed step in sterol biosynthesis and consists of both an amino-terminal catalytic domain and a carboxy-terminal domain tethering the enzyme to the ER membrane. While the overall architecture of this enzyme is identical in eukaryotes, it was previously shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implied a unique component of the fungal carboxy-terminal domain was responsible for the complementation phenotype. To identify this motif, we used Saccharomyces cerevisiae with a squalene synthase knockout mutation, and expressed intact and chimeric squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. However, when highly expressed, non-fungal squalene synthases could not complement the yeast mutation and instead led to the accumulation of a toxic intermediate(s) as defined by mutations of genes downstream in the ergosterol pathway. Restoration of the complete complementation phenotype was mapped to a 26-amino acid hinge region linking the catalytic and membrane-spanning domains specific to fungal squalene synthases. Over-expression of the C-terminal domain containing a hinge domain from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Because this hinge region is unique to and highly conserved within each kingdom of life, the data suggests that the hinge domain plays an essential functional role, such as assembly of ergosterol multi-enzyme complexes in fungi.

Engineering Triterpene and Methylated Triterpene Production in Plants Provides Biochemical and Physiological Insights into Terpene Metabolism.

Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31-C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g(-1) fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene.

Spatial and temporal regulation of sterol biosynthesis in Nicotiana benthamiana.

Nicotiana benthamiana was used as a model to investigate the spatial and developmental relationship between sterol synthesis rates and sterol content in plants. Stigmasterol levels were approximately twice the level in roots as that found in aerial tissues, while its progenitor sterol sitosterol was the inverse. When incorporation of radiolabeled precursors into sterols was used as measure of in vivo synthesis rates, acetate incorporation was similar across all tissue types, but approximately twofold greater in roots than any other tissue. In contrast, mevalonate incorporation exhibited the greatest differential with the rate of incorporation in roots approximately one-tenth that in apical shoots. Similar to acetate, incorporation of farnesol was higher in roots but remained fairly constant in aerial tissues, suggesting less regulation of the downstream sterol biosynthetic steps. Consistent with the precursor incorporation data, analysis of gene transcript and measurements of putative rate-limiting enzyme activities for 3-hydroxy-3-methylglutaryl-coenzyme A synthase (EC 2.3.3.10) and reductase (EC 1.1.1.34) showed the greatest modulation of levels, while the activity levels for isopentenyl diphosphate isomerase (EC 5.3.3.2) and prenyltransferases (EC 2.5.1.10 and EC 2.5.1.1) also exhibited a strong but moderate correlation with the development age of the aerial tissues of the plants. Overall, the data suggest a multitude of means from transcriptional to posttranslational control affecting sterol biosynthesis and accumulation across an entire plant, and point to some particular control points that might be manipulated using molecular genetic approaches to better probe the role of sterols in plant growth and development.

Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

Building terpene production platforms in yeast.

Plants and microbes commonly make terpenes and terpenoids in small amounts and as complex mixtures, and their chemical synthesis is often costly and inefficient. Hence, there are many efforts to create robust and efficient biological production platforms for this interesting class of molecules. In this study, our effort was directed towards building a yeast production platform using an unbiased genetic selection approach. Yeast strain BY4741 was subjected to EMS mutagenesis, followed by selection for growth in the presence of nystatin, squalestatin, and exogenous cholesterol. This unbiased screen selected for mutant yeast lines having a dispensable mevalonate pathway and containing uncharacterized SUE (sterol uptake enhancement) mutations supporting aerobic uptake of exogenous sterol. These mutants were next screened for high level accumulation of farnesol (FOH), an indicator for high level accumulation of the key intermediate FPP, farnesyl diphosphate. To further improve the FPP pool in these mutants, insertional mutations into the ERG9 gene (coding for squalene synthase) were introduced into those lines capable of accumulating ≥50 mg farnesol/L. This generated another series of lines that accumulated farnesol levels over 70 mg/L in small-scale shake cultures. To evaluate the utility of these lines as a general production platform for specific terpenes, select SUE/erg9 lines were transformed with a vector harboring the Hyoscyamus muticus premnaspirodiene synthase (HPS) gene encoding for a sesquiterpene synthase. The new yeast line ZX178-08 accumulated the highest level of premnaspirodiene, up to 116 mg/L, with FOH levels of 23.6 mg/L. In comparison, the parental line BY4741 accumulated 10 times less premnaspirodiene, 10.94 mg/L, with no farnesol detectable. Co-expression of the HPS gene with an amino-terminal truncated, catalytic form of the hamster HMGR gene, tHMGR, increased premnaspirodiene accumulation to 170.23 ± 30.44 mg/L, almost a 50% increase. Further utility of this yeast line was demonstrated for triterpene production. When engineered for the production of a non-native triterpene, Zx178-08 accumulated upwards of 60 mg/L of botryococcene. To engineer more native triterpene accumulation, additional insertion mutants into the ERG1 gene (coding for squalene epoxidase) were evaluated. Insertion of a simple selection marker followed by over-expression of a heterologous squalene synthase gene resulted in greater than 85 mg/L of squalene. However, when the ERG1 insertional mutant included chromosomal insertion of a truncated, heterologous HMGR gene, squalene production was more than tripled to 270 mg/L. These results are discussed in comparison to other recently developed terpene production platforms.

Triterpene hydrocarbon production engineered into a metabolically versatile host--Rhodobacter capsulatus.

Triterpene hydrocarbon biosynthesis of the ancient algae Botryococcus braunii was installed into Rhodobacter capsulatus to explore the production of C30 hydrocarbon in a host capable of diverse growth habits-utilizing carbohydrate, sunlight or hydrogen (with CO2 fixation) as alternative energy feedstocks. Engineering an enhanced MEP pathway was also used to augment triterpene accumulation. Despite dramatically different sources of carbon and reducing power, nearly the same level of botryococcene or squalene (∼5 mg oil/g-dry-weight [gDW]) was achieved in small-scale aerobic heterotrophic, anaerobic photoheterotrophic, and aerobic chemoautotrophic growth conditions. A glucose fed-batch bioreactor reached 40 mg botryococcene/L (∼12 mg/gDW), while autotrophic bioreactor performance with CO2 , H2 , and O2 reached 110 mg/L (16.7 mg/gDW) during batch and 60 mg/L (23 mg/gDW) during continuous operation at a dilution rate corresponding to about 10% of µ(max). Batch and continuous autotrophic specific productivity was found to reach 0.5 and 0.32 mg triterpene/g DW/h, comparable to prior reports for terpene production driven by heterotrophic growth conditions. This demonstrates the feasibility of alternative feedstocks and trophic modes to provide comparable routes to biochemicals that do not rely on sugar.

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

Hydrocarbon production in high density Botryococcus braunii race B continuous culture.

Continuous cultures of Botryococcus braunii race B were maintained at photosynthetic cell densities as high as 20 g dry weight per liter for up to 3 months. Growth associated triterpene hydrocarbon accumulation was nearly constant at 22.5% of dry weight for a range of growth rates maintained by daily replacement of 5-15% of the respective cultures. The ability to achieve high cell concentrations and oil levels of roughly 5 g triterpene oil/L resulted from a combination of high light (∼ 1/4 full sun for 15 h/day) and replenishing stoichiometrically balanced growth medium. Due to light-limited growth conditions, cell concentration dropped nearly linearly with increased dilution rate. This reduction in cell number resulted in increased productivity per cell at higher dilution rates and was accompanied by a dramatic increase in algae colony size from 0.09 to 0.343 mm at high dilution rate. This change in colony size resulted in an equally dramatic change in optical density (OD) per gram dry weight, which precluded use of simple correlations of OD and cell concentration. A trickle-film photobioreactor was also demonstrated as a scalable approach to achieving these ultra-high cell concentrations. Additional media analysis revealed a steady increase in photobioreactor conductivity suggesting an accumulation of ions may be the reason for rapid culture crash and washout observed at all dilution rates after several months of continuous operation. The volumetric productivity of 22.5 mg oil/L/photo-h reported here is more than an order of magnitude higher than previous reports for B. braunii race B, reflecting the high cell densities used in this work and substantiating a higher metabolic rate for B. braunii race B than previously surmised from its relatively long doubling times.

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii.

Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.

Functional identification of triterpene methyltransferases from Botryococcus braunii race B.

Botryococcus braunii race B is a colony-forming, green algae that accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by dimethylated to tetramethylated forms of botryococcene and squalene. Although unusual mechanisms for the biosynthesis of botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the S-adenosyl methionine-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono- and dimethylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, whereas TMT-3 showed a striking preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast overexpressing the respective genes, which encode for membrane-associated enzymes. Structural examination of the in vivo yeast generated mono- and dimethylated products by NMR identified terminal carbons, C-3 and C-22/C-20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites are identical to those previously reported for the triterpenes extracted from the algae. The availability of closely related triterpene methyltransferases exhibiting distinct substrate selectivity and successive catalytic activities provides important tools for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.

Engineering triterpene metabolism in tobacco.

Terpenes comprise a distinct class of natural products that serve a diverse range of physiological functions, provide for interactions between plants and their environment and represent a resource for many kinds of practical applications. To better appreciate the importance of terpenes to overall growth and development, and to create a production capacity for specific terpenes of industrial interest, we have pioneered the development of strategies for diverting carbon flow from the native terpene biosynthetic pathways operating in the cytosol and plastid compartments of tobacco for the generation of specific classes of terpenes. In the current work, we demonstrate how difficult it is to divert the 5-carbon intermediates DMAPP and IPP from the mevalonate pathway operating in the cytoplasm for triterpene biosynthesis, yet diversion of the same intermediates from the methylerythritol phosphate pathway operating in the plastid compartment leads to the accumulation of very high levels of the triterpene squalene. This was assessed by the co-expression of an avian farnesyl diphosphate synthase and yeast squalene synthase genes targeting metabolism in the cytoplasm or chloroplast. We also evaluated the possibility of directing this metabolism to the secretory trichomes of tobacco by comparing the effects of trichome-specific gene promoters to strong, constitutive viral promoters. Surprisingly, when transgene expression was directed to trichomes, high-level squalene accumulation was observed, but overall plant growth and physiology were reduced up to 80 % of the non-transgenic controls. Our results support the notion that the biosynthesis of a desired terpene can be dramatically improved by directing that metabolism to a non-native cellular compartment, thus avoiding regulatory mechanisms that might attenuate carbon flux within an engineered pathway.

Doubly deuterium-labeled patchouli alcohol from cyclization of singly labeled [2-(2)H(1)]farnesyl diphosphate catalyzed by recombinant patchoulol synthase.

Incubations of isotopically pure [2-(2)H(1)](E,E)-farnesyl diphosphate with recombinant patchoulol synthase (PTS) from Pogostemon cablin afforded a 65:35 mixture of monodeuterated and dideuterated patchoulols as well as numerous sesquiterpene hydrocarbons. Extensive NMR analyses ((1)H and (13)C NMR, (1)H homodecoupling NMR, HMQC, and (2)H NMR) of the labeled patchoulol mixture and comparisons of the spectra with those of unlabeled alcohol led to the conclusion that the deuterium label was located at positions (patchoulol numbering system) C5 (both isotopomers, ca. 100%) and C12 (minor isotopomer, 30-35%), that is, an approximately 2:1 mixture of [5-(2)H(1)]- and [5,12-(2)H(2)]-patchoulols. Low-resolution FIMS analyses and isotope ratio calculations further corroborated the composition of the mixture as mainly one singly deuterated and one doubly deuterated patchoulol. From a mechanistic point of view, the formation of [5,12-(2)H(2)]patchoulol is rationalized through the intermediacy of an unknown exocyclic [7,10:1,5]patchoul-4(12)-ene (15-d(1)), which could incorporate a deuteron at the C-12 position on the pathway to doubly labeled patchoulol. The corresponding depletion of deuterium content observed in the hydrocarbon coproducts, beta-patchoulene and alpha-guaiene (55% d(0)), identified the source of the excess label found in patchoulol-d(2). Comparison of the PTS amino acid sequence with those of other sesquiterpene synthases, and examination of an active site model, suggested that re-orientation of leucine 410 side chain in PTS might facilitate the creation of a 2-pocket active site where the observed deuteron transfers could occur. The retention of deuterium at C5 in the labeled patchoulol and its absence at C4 rule out an alternative mechanism involving two consecutive 1,2-hydride shifts and appears to confirm the previously proposed occurrence of a 1,3-hydride shift across the 5-membered ring. A new, semisystematic nomenclature is presented for the purpose of distinguishing the three different skeletal structures of the patchoulane sesquiterpenes.