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As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids.

Assuming biofuels generated via the fermentation of sugars derived from cellulosic and non-cellulosic constituents of biofuels crops is providing a substantial contribution to our future energy needs, augmenting and amending the productivity of these biofuel crops is now a major research thrust worldwide. One way of enhancing these biofuels crops will be to engineer them for additional value-added components such as oils, that can be used for efficient fuel production and the manufacturing of other high-value products currently derived from petroleum oils. Towards this end, we are engineering optimized production of long, branched-chain hydrocarbon biosynthesis into plants suitable as biofuels crops. Branched chain hydrocarbons, like methylated triterpenes, are readily cracked into paraffins and naphthenes that can either be distilled to combustible fuels (gasoline, jet fuel and diesel), or can be used directly for the synthesis of plastics, nylons, paints and other oil-derived products manufactured by di...

Isoprenoids are a class of compounds derived from the five carbon precursors, dimethylallyl diphosphate, and isopentenyl diphosphate. These molecules present incredible natural chemical diversity, which can be valuable for humans in many aspects such as cosmetics, agriculture, and medicine. However, many terpenoids are only produced in small quantities by their natural hosts and can be difficult to generate synthetically. Therefore, much interest and effort has been directed toward capturing the genetic blueprint for their biochemistry and engineering it into alternative hosts such as plants and algae. These autotrophic organisms are attractive when compared to traditional microbial platforms because of their ability to utilize atmospheric CO2 as a carbon substrate instead of supplied carbon sources like glucose. This chapter will summarize important techniques and strategies for engineering the accumulation of isoprenoid metabolites into higher plants and algae by choosing the correct host, avoiding endogenous regulatory mechanisms, and optimizing potential flux into the target compound. Future endeavors will build on these efforts by fine-tuning product accumulation levels via the vast amount of available "-omic" data and devising metabolic engineering schemes that integrate this into a whole-organism approach. With the development of high-throughput transformation protocols and synthetic biology molecular tools, we have only begun to harness the power and utility of plant and algae metabolic engineering.

Plant cytochrome P450 (CYP) enzymes are involved in the biosynthesis of many primary and secondary metabolites including phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates. However, while hundreds of CYP genes have been identified in plant genomes, relatively few have been functionally characterized. We report here the cloning and characterization of a CYP enzyme from tobacco (Nicotiana tabacum L.) that demethylates nicotine to form nornicotine, a precursor to the nitrosamine N'-nitrosonornicotine (NNN). Microsomal demethylase activity was first shown to be induced in leaves treated with the growth regulator ethylene, required molecular oxygen and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for full activity, and exhibited a K m of 3.9 μΜ for nicotine and a maximum turnover rate (V max ) of 8.9 pkat mg protein. Equally important, microsomal activity was reduced greatly by several inhibitors of CYP-mediated reactions. Using a polymerase chain reaction based strategy with degenerate primers designed to conserve P450 motifs and mRNA from ethylene-treated leaves, putative gene fragments representing 32 different P450 gene families were isolated and numerous full-length genes were cloned. Employing Genechip r (Affymetrix Inc., Santa Clara, CA) microarray hybridizations, the steady-state mRNA level of two highly related full-length cDNAs and one cDNA fragment were demonstrated to be highly expressed in ethylene-treated vs. control plant material. Microsomes from yeast over-expressing the two full-length cDNAs along with two other highly homologous P450 genes demonstrated that only one cDNA, D121-AA8 (GenBank accession no. DQ205656) encoded for demethylation activity comparable to that found in planta. Molecular modeling was used to identify putative active site residues and for comparisons to other putative demethylase cDNAs.

Tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases (TEAS and HPS) catalyze the cyclizations and rearrangements of (E,E)-farnesyl diphosphate (FPP) to the corresponding bicyclic sesquiterpene hydrocarbons. The complex mechanism proceeds through a tightly bound (R)-germacrene A intermediate and involves partitioning of a common eudesm-5-yl carbocation either by angular methyl migration, or by C-9 methylene rearrangement, to form the respective eremophilane and spirovetivane structures. In this work, the stereochemistry and timing of the proton addition and elimination steps in the mechanism were investigated by synthesis of substrates bearing deuterium labels in one or both terminal methyl groups, and in the pro-S and pro-R methylene hydrogens at C-8. Incubations of the labeled FPPs with recombinant TEAS and HPS, and with the chimeric CH4 hybrid cyclase having catalytic activities of both TEAS and HPS, and of unlabeled FPP in D2O, together with gas chromatography-mass spectrometry (GC-MS) and/or NMR analyses of the labeled products gave the following results: (1) stereospecific CH3-->CH2 eliminations at the cis-terminal methyl in all cases; (2) similar primary kinetic isotope effects (KIE) of 4.25-4.64 for the CH3-->CH2 eliminations; (3) a significant intermolecular KIE (1.33+/-0.03) in competitive cyclizations of unlabeled FPP and FPP-d6 to premnaspirodiene by HPS; (4) stereoselective incorporation of label from D2O into the 1beta position of epiaristolochene; (5) stereoselective eliminations of the 1beta and 9beta protons in formation of epiaristolochene and its delta(1(10)) isomer epieremophilene by TEAS and CH4; and (6) predominant loss of the 1alpha proton in forming the cyclohexene double bond of premnaspirodiene by HPS and CH4. The results are explained by consideration of the conformations of individual intermediates, and by imposing the requirement of stereoelectronically favorable proton additions and eliminations.

A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing β-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for α-barbatene, thujopsene, and β-chamigrene biosynthesis.

A series of eremophilane sesquiterpene alcohols and hydrocarbons was prepared from the phytoalexin capsidiol (1) for mechanistic studies with epiaristolochene synthase and epiaristolochene dihydroxylase. Among them, 3-deoxycapsidiol (10) was obtained through selective derivatization and reductive cleavage of the equatorial 3 alpha hydroxyl group. Two novel isomers of aristolochene and eremophilene were accessed from the 1- and 3-deoxycapsidiol isomers. 4-Epieremophilene (17) was obtained by conjugate reduction of epiaristolochen-1-one tosylhydrazone with catecholborane followed by sulfinate elimination and diimide rearrangement. Epimerization of epiaristolochen-3-one (27a) at the C4 methyl followed by reductions led to the previously unknown aristolochene isomer, eremophila-9(10),11(12)-diene (30). Optical rotations and characteristic (1)H NMR data for the related eremophilenols and dienes are collected in Tables 1 and 2. Finally, bioassays were used to assess the antifungal potencies of capsidiol and its synthetic derivatives. The minimum inhibitory concentration for capsidiol (3-10 ng) was at least 1 order of magnitude lower than that of any of the derivatives and considerably lower than those previously reported for ketoconazole, nystatin, and propiconazole.

Plants have the capacity to produce a wide array of terpene compounds – compounds that provide ecological and physiological benefit to plants. Terpenes also have important practical applications such as flavors, fragrances and medicines. Liat Sharon-Asa and colleagues have recently isolated and characterized a sesquiterpene synthase from Citrus that is providing a new window into the biochemical mechanics of these enzymatic wizards and hints for the future metabolic engineering of terpene biosynthesis.

5-epi-Aristolochene dihydroxylase (EAH) catalyzes unique stereo- and regiospecific hydroxylations of a bicyclic sesquiterpene hydrocarbon to generate capsidiol. To define functional and mechanistic features of the EAH enzyme, the utility of a coupled assay using readily available sesquiterpene synthases and microsomes from yeast overexpressing the EAH enzyme was determined. Capsidiol and deoxycapsidiol biosyntheses were readily measured in coupled assays consisting of 5-epi-aristolochene synthase and EAH as determined by the incorporation of radiolabeled farnesyl diphosphate into thin-layer chromatography-isolated products and verified by gas chromatography-mass spectrometry analysis. The assays were dependent on the amounts of synthase and hydroxylase protein added, the incubation times, and the presence of nicotinamide adenine dinucleotide phosphate. The utility of this coupled assay was extended by examining the relative efficiency of the EAH enzyme to catalyze hydroxylations of different sesquiterpene skeletons generated by other terpene synthases.

Squalene synthase (SS) represents a putative branch point in the isoprenoid biosynthetic pathway capable of diverting carbon flow specifically to the biosynthesis of sterols and, hence, is considered a potential regulatory point for sterol metabolism. For example, when plant cells grown in suspension culture are challenged with fungal elicitors, suppression of sterol biosynthesis has been correlated with a reduction in SS enzyme activity. The current study sought to correlate changes in SS enzyme activity with changes in the level of the corresponding protein and mRNA. Using an SS-specific antibody, the initial suppression of SS enzyme activity in elicitor-challenged cells was not reflected by changes in the absolute level of the corresponding polypeptide, implicating a post-translational control mechanism for this enzyme activity. In comparison, the absolute level of the SS mRNA did decrease approximately 5-fold in the elicitor-treated cells, which is suggestive of decreased transcription of the SS gene. Study of SS in intact plants was also initiated by measuring the level of SS enzyme activity, the level of the corresponding protein, and the expression of SS gene promoter-reporter gene constructs in transgenic plants. SS enzyme activity, polypeptide level, and gene expression were all localized predominately to the shoot apical meristem, with much lower levels observed in leaves and roots. These later results suggest that sterol biosynthesis is localized to the apical meristems and that apical meristems may be a source of sterols for other plant tissues.

Catharanthus roseus (L.) G. Don hairy roots harboring hamster 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) (EC 1.1.1.88) cDNA without membrane-binding domain were evaluated by quantifying the levels of sterols and some indol-alkaloids. Clone 236, with the highest hybridization signal, had the lowest soluble and microsomal HMGR activity and produced more ajmalicine and catharanthine than the control but had reduced campesterol concentration. Clone 19, with low hybridization signal, had high soluble HMGR activity and produced high levels of campesterol and five to seven times more serpentine than the control but a low level of ajmalicine and no accumulation of catharanthine. These results suggest a possible role for HMGR in indole alkaloid biosynthesis and a possible cosuppression of both the endogenous and foreign HMGR genes in clone 236.

Squalene and botryococcene are branched-chain, triterpene compounds that arise from the head-to-head condensation of two molecules of farnesyl diphosphate to yield 1'-1 and 1'-3 linkages, respectively. The enzymes that catalyze their formation have attracted considerable interest from the medical field as potential drug targets and the renewable energy sector for metabolic engineering efforts. Recently, the enzymes responsible for botryococcene and squalene biosynthesis in the green alga Botryococcus braunii race B were characterized. To better understand how the specificity for the 1'-1 and 1'-3 linkages was controlled, we attempted to identify the functional residues and/or domains responsible for this step in the catalytic cascade. Existing crystal structures for the mammalian squalene synthase and Staphylococcus dehydrosqualene synthase enzymes were exploited to develop molecular models for the B. braunii botryococcene and squalene synthase enzymes. Residues within the active sites that could mediate catalytic specificity were identified, and reciprocal mutants were created in an attempt to interconvert the reaction product specificity of the enzymes. We report here the identification of several amino acid positions contributing to the rearrangement of the cyclopropyl intermediate to squalene, but these same positions do not appear to be sufficient to account for the cyclopropyl rearrangement to give botryococcene.

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 μmax. 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. Biotechnol. Bioeng. 2015;112: 1523–1532. © 2015 Wiley Periodicals, Inc.

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.

Phytoalexins play an important role in the inducible defense responses of plants against diseases caused by fungi. Some enzymes, involved in the respective biosynthetic pathways, catalyze key steps. Farnesyl pyrophosphate (FPP) is a key intermediate in the biosynthesis of terpenes. It may be converted into several cyclic and some acyclic sesquiterpenic derivatives, sterol precursors, or to geranylgeranyl pyrophosphate according to the requirements of the plant cells. Specific cyclization reactions of FPP are catalyzed by particular sesquiterpene cyclases. The 5-epi-aristolochene synthase (EAS) enzyme of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) produce the 5-epi-aristolochene, which is the immediate precursor of the bicyclic phytoalexin capsidiol. Oligonucleotides homologous to 3′-end specific regions of tobacco and Hyoscyamus muticus inducible sesquiterpene cyclase genes were used with the system for rapid amplification of cDNA ends (3′-RACE) technique to prepare pepper EAS-cDNA fragments (PEAS-cDNA). Three specific PEAS-cDNAs were isolated (PEAS1, PEAS18, and PEAS55). Northern blots of total RNA samples from pepper plant tissues challenged with cellulase or Phytophthora capsici showed different expression levels of the respective transcripts. PEAS1 was used to identify the corresponding elicitor-stimulated gene (gPEAS1). The nucleotide sequence of the proposed gPEAS1 promoter showed putative stimuli- and tissue-specific responsive elements. A pepper 5-epi-aristolochene synthase gene family of 5–8 members was demonstrated by Southern blot.

Jasmonates are well documented for their ability to modulate the expression of plant genes and to influence specific aspects of disease/pest resistance traits. We and others have been studying the synthesis of sesquiterpene phytoalexins in elicitor/pathogen-challenged plants and have sought to determine if methyl jasmonate (MeJA) could substitute for fungal elicitors in the induction of capsidiol accumulation by tobacco cell cultures. The current results demonstrate that MeJA does in fact induce phytoalexin accumulation, but with a much more delayed induction time course than elicitor. While elicitor treatment induced strong but transient changes in key enzymes of sesquiterpene biosynthesis, sesquiterpene cyclase, and aristolochene/deoxy-capsidiol hydroxylase, MeJA did not. Instead, MeJA caused a protracted induction of cyclase activity and only a low level of hydroxylase activity. MeJA induced the expression of at least two sesquiterpene cyclase genes, including one that had not been observed previously in elicitor-induced mRNA populations. Only a small portion of the total sesquiterpene cyclase mRNA induced by MeJA was associated with polysomal RNA, suggesting that the MeJA treatment imposed both transcriptional and posttranscriptional regulation in tobacco cells. These results are not consistent with MeJA playing a role in orchestrating defense responses in elicitor-treated tobacco cells, but do provide evidence that MeJA induces a subset of genes coding for the biosynthesis of sesquiterpene phytoalexins.

The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [ 3 H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [ 3 H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [ 3 H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [ 3 H]F-OH or [ 3 H]geranylgeraniol ([ 3 H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [ 3 H]F-P and [ 3 H]F-P-P were synthesized when exogenous [ 3 H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor–product relationship between [ 3 H]F-P and [ 3 H]F-P-P. In agreement with this kinetic pattern of labeling, [ 32 P]F-P and [ 32 P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [γ- 32 P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [ 3 H]GG-OH to [ 3 H]geranylgeranyl monophosphate and [ 3 H]geranylgeranyl pyrophosphate ([ 3 H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [ 3 H]CTP was formed when microsomes were incubated with [ 3 H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation.

Summary The first unique step in the synthesis of the tobacco phytoalexin capsidiol is cyclization of farnesyl pyrophosphate catalyzed by 5-epi-aristolochene synthase (EAS), a sesquiterpene cyclase. Earlier work demonstrated that the elicitor-inducibility of this enzyme activity corresponded to the transcriptional activation of at least one gene, EAS4, of a rather complex gene family consisting of > 10 members. To investigate the mechanism(s) controlling expression of this gene, fragments of the EAS4 promoter were examined for binding by proteins in nuclear and whole-cell extracts. A strong protein binding site (TAC box; ACTCTACAGTACTC) was identified between ‐245 and ‐232 by the electrophoretic mobility shift assay, and DNAase I and methylation interference footprinting. Several distinctly migrating bands representing protein‐ TAC box complexes were also observed in the mobility shift assays, and the relative abundance of these bands varied in extracts from cells at different stages of EAS induction. The TAC box binding factor (TacBBF) was purified > 450-fold from crude whole-cell extracts by a combination of DNA affinity and cation exchange chromatography. The purified fractions were enriched for polypeptides of 17 and 19 kDa and the DNA binding properties of these preparations were characterized. Mutation of 2 bp in the TAC box prevented protein binding in vitro and

International audience; The induction of sesquiterpene cyclase, a key phytoalexin biosynthetic enzyme, and the accumulation of phytoalexins in relation to the induction of a hypersensitive response (HR) and cell necrosis in tobac- co (Nicotiana tabacum L.) were investigated. When tobacco leaves were inoculated with virulent or avirulent isolates of Ralstonia solanacearum, steady-state levels of mRNA complementary to cDNA of the sensitivity- related (sts) gene str319 were dramatically induced. This cDNA clone is greater than 90% homologous with a gene coding for 5-epi-aristolochene synthase (EAS), previously described as a branch-point enzyme regulat- ing the synthesis of capsidiol, the major sesquiterpenoid phytoalexin found in tobacco. Accumulation of EAS transcripts in leaves after inoculation with virulent and avirulent strains of R. solanacearum, or after treatment with necrotizing or non-necrotizing elicitins was rapid but transient, and restricted to the site of infiltration. Two highly similar sesquiterpene cyclase activities, 5-epi- aristolochene synthase and a vetispiradiene synthase-like activity, were found in extracts of elicitin-challenged and R. solanacearum-inoculated tobacco. Under all condi- tions tested, the induction of cyclase activity was closely correlated with induction of the cyclase mRNA level. In contrast, high levels of capsidiol were found only after treatment with the necrosis-inducing elicitin cryptogein, or after infiltration with HR-inducing bacterial strains. Low levels of capsidiol did accumulate after application of capsicein, an elicitin that induces little or no necrosis on tobacco, or after infection with a virulent bacterium. Hence, capsidiol accumulation, not 5-epi-aristolochene synthase gene expression or total sesquiterpene cyclase enzyme activity, appears to be a good marker for the HR of tobacco.

Summary With the aim of studying the production of chloroplast isoprenoid derivatives, chimeric gene constructs comprising partial and full-length forms of the Arabidopsis thaliana hmg1 gene, encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in sense and antisense forms were fused to a chloroplast transit peptide (CTP) sequence from a pea ribulose bisphosphate carboxylase/oxygenase (Rubisco) small subunit gene. These plasmids were placed downstream from the constitutive CaMV 35S promoter and introduced into tobacco ( Nicotiana tabacum cv. Xanthi) by Agrobacterium -mediated transformation. Southern and northern blot analysis confirmed the transformation and transcription of the respective constructs within transgenic tobacco plants. In addition total and chloroplast specific HMGR enzyme activities, and the levels of chlorophyll, carotenoids and total sterols were analyzed. Results of these analyses suggest that neither partial nor full-length sense constructs had any effect on chloroplast isoprenoid production. However, the corresponding antisense constructs decreased general isoprenoid levels. The significance of these results is discussed.

Terpene cyclases catalyze the synthesis of cyclic terpenes with 10-, 15-, and 20-carbon acyclic isoprenoid diphosphates as substrates. Plants have been a source of these natural products by providing a homologous set of terpene synthases. The crystal structures of 5-epi-aristolochene synthase, a sesquiterpene cyclase from tobacco, alone and complexed separately with two farnesyl diphosphate analogs were analyzed. These structures reveal an unexpected enzymatic mechanism for the synthesis of the bicyclic product, 5-epi-aristolochene, and provide a basis for understanding the stereochemical selectivity displayed by other cyclases in the biosynthesis of pharmacologically important cyclic terpenes. As such, these structures provide templates for the engineering of novel terpene cyclases.

Treatment of tobacco (Nicotiana tabacum) cell-suspension cultures with cryptogein, an elicitin protein from Phytophthora cryptogea, resulted in the release of a factor(s) that diffused through a 1000-D cutoff dialysis membrane and was capable of inducing sesquiterpene cyclase enzyme activity (a key phytoalexin biosynthetic enzyme in solanaceous plants) when added to fresh cell-suspension cultures. The diffusible factor(s) was released from cells over a 20-h period and induced a more rapid induction of cyclase enzyme activity than did direct treatment of the cultures with pure elicitin protein. The diffusible factor also induced a more rapid accumulation of transcripts encoding for sesquiterpene cyclase, acidic and basic chitinase, and hsr203 (a putative hypersensitive response gene) than did elicitin treatment. The diffusible factor(s) was resistant to protease, pectinase, Dnase, and RNase treatments, was not extractable into organic solvents, and was not immunoprecipitable when challenged with polyclonal antibodies prepared against elicitin protein. The diffusible factor(s) could not induce the release of more factor, suggesting that it was a terminal signal. These results are consistent with the notion that cells directly challenged or stimulated by pathogen-derived elicitors release diffusible secondary signal molecules that orchestrate the induction of complementary defense responses in neighboring cells.

Cyclic terpenes and terpenoids are found throughout nature. They comprise an especially important class of compounds from plants that mediate plant- environment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities. Molecular comparisons of several terpene cyclases, the key enzymes responsible for the multistep cyclization of C10, C15, and C20 allylic diphosphate substrates, have revealed a striking level of sequence similarity and conservation of exon position and size within the genes. Functional domains responsible for a terminal enzymatic step were identified by swapping regions approximating exons between a Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) gene and a Hyoscyamus muticus vetispiradiene synthase (HVS) gene and by characterization of the resulting chimeric enzymes expressed in bacteria. While exon 4 of the TEAS gene conferred specificity for the predominant reaction products of the tobacco enzyme, exon 6 of the HVS gene conferred specificity for the predominant reaction products of the Hyoscyamus enzyme. Combining these two functional domains of the TEAS and HVS genes resulted in a novel enzyme capable of synthesizing reaction products reflective of both parent enzymes. The relative ratio of the TEAS and HVS reaction products was also influenced by the source of exon 5 present in the new chimeric enzymes. The association of catalytic activities with conserved but separate exonic domains suggests a general means for generating additional novel terpene cyclases.

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 [13C]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. Background: Therapeutic values of Valeriana officinalis have been associated with sesquiterpenes whose biosynthetic origins have remained enigmatic. Results: A cyclobutenyl intermediate in the catalytic cascade of valerena-1,10-diene synthase is reported. Conclusion: A new class of sesquiterpene synthases for the biosynthesis of sesquiterpenes harboring isobutenyl functional groups is proposed. Significance: Similar catalytic mechanisms from evolutionarily diverse organisms are proposed and portend sources for sesquiterpene diversity.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The rate-limiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMGR activity could be limiting in plants, we introduced a constitutively expressing hamster HMGR gene into tabacco (Nicotiana tabaccum L.) plants to obtain unregulated HMGR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMGR gene led to a 3- to 6-fold increase in the total HMGR enzyme activity. Total sterol accumulation was consequently increased 3- to 10-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. The level of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMGR activity, these results indicate that the activity of one or more later enzyme(s) in the pathway must also be involved in determining the relative accumulation of end-product sterols. The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.

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.

This review first summarizes the diverse nature of isoprenoids found in plants, emphasizing the wide range of physiological functions these compounds serve. The biosynthetic origins of isoprenoids have occupied chemists and biochemists for decades, and the second section of this review recaps some of the conceptual models used to rationalize key biosynthetic reactions in the isoprenoid pathway. The third section describes briefly some of the recently developed experimental systems that have helped researchers uncover much of the biochemistry and molecular biology of isoprenoids. The fourth section compares the deduced amino acid sequences of enzymes with similar catalytic functions and attempts to correlate these sequences with the known enzymology. The fifth and final section focuses on our limited understanding of how isoprenoid biosynthesis is regulated in plants.

A convenient in vitro transcription system using monocot and dicot whole cell extracts and circular DNA templates has been developed. The system consists of incubating template and whole cell extract to generate initiation complexes, followed by addition of nucleotide triphosphates to support elongation, and primer extension assay to detect authentic transcripts. This in vitro transcription system required circularized templates and was essentially inactive with linearized templates. Accurate in vitro transcription of a rice phenylalanine ammonia-lyase (PAL) promoter-beta-glucuronidase (GUS) gene fusion and a tobacco sesquiterpene cyclase promoter-GUS gene fusion was examined in their homologous whole cell extracts, and the optimal concentrations for several reaction components, including DNA template, whole cell extract, monovalent and divalent cations, were determined for specific initiation from the in vivo start site. Transcription was inhibited by low concentrations of alpha-amanitin, demonstrating that the reaction was mediated by RNA polymerase II. Accurate transcription initiation was dependent on the TATA-box motif within the respective promoters. Based on the effect of delayed addition of sarkosyl at a concentration sufficient to inhibit transcription initiation but not elongation, three to four rounds of transcription were initiated in standard assays.

Specialized compounds from photosynthetic organisms serve as rich resources for drug development. From aspirin to atropine, plant-derived natural products have had a profound impact on human health. Technological advances provide new opportunities to access these natural products in a metabolic context. Here, we describe a database and platform for storing, visualizing and statistically analyzing metabolomics data from fourteen medicinal plant species. The metabolomes and associated transcriptomes (RNAseq) for each plant species, gathered from up to twenty tissue/organ samples that have experienced varied growth conditions and developmental histories, were analyzed in parallel. Three case studies illustrate different ways that the data can be integrally used to generate testable hypotheses concerning the biochemistry, phylogeny and natural product diversity of medicinal plants. Deep metabolomics analysis of Camptotheca acuminata exemplifies how such data can be used to inform metabolic understanding of natural product chemical diversity and begin to formulate hypotheses about their biogenesis. Metabolomics data from Prunella vulgaris, a species that contains a wide range of antioxidant, antiviral, tumoricidal and anti-inflammatory constituents, provide a case study of obtaining biosystematic and developmental fingerprint information from metabolite accumulation data in a little studied species. Digitalis purpurea, well known as a source of cardiac glycosides, is used to illustrate how integrating metabolomics and transcriptomics data can lead to identification of candidate genes encoding biosynthetic enzymes in the cardiac glycoside pathway. Medicinal Plant Metabolomics Resource (MPM) [1] provides a framework for generating experimentally testable hypotheses about the metabolic networks that lead to the generation of specialized compounds, identifying genes that control their biosynthesis and establishing a basis for modeling metabolism in less studied species. The database is publicly available and can be used by researchers in medicine and plant biology.

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.

Development of next-generation sequencing, coupled with the advancement of computational methods, has allowed researchers to access the transcriptomes of recalcitrant genomes such as those of medicinal plant species. Through the sequencing of even a few cDNA libraries, a broad representation of the transcriptome of any medicinal plant species can be obtained, providing a robust resource for gene discovery and downstream biochemical pathway discovery. When coupled to estimation of expression abundances in specific tissues from a developmental series, biotic stress, abiotic stress, or elicitor challenge, informative coexpression and differential expression estimates on a whole transcriptome level can be obtained to identify candidates for function discovery.

5-Epi-aristolochene synthase is a sesquiterpene cyclase activity found in pathogen-challenged tobacco cells, but not in nonchallenged tissues, and appears to be encoded by a complex gene family. As a prerequisite to assessing the functional significance of these multiple genes, bacterial expression systems were examined for their capacity to express a tobacco sesquiterpene cyclase cDNA. Insertion of full-length 5-epi-aristolochene synthase cDNA into two commonly used expression vectors, pET-11d and pGBT-T19, resulted in high level expression of the cyclase activity. The highest level of expression occurred 3 h after induction with low concentrations (0.1-0.5 mM) of IPTG, incubation at 27 degrees C instead of 37 degrees C, and in the bacterial host strain BL21(DE3). Under these conditions, the cyclase protein constituted 5 to 8% of the soluble and 35% of the total Escherichia coli proteins. Enzyme reaction products of the native tobacco and recombinant enzyme were identical, based on argentation-thin layer chromatography. Deletion mutants of the cyclase gene corresponding to the amino and carboxy termini of the enzyme were prepared. The cyclase proteins resulting from bacterial expression of these mutant constructs were found largely in the insoluble protein fraction and no soluble enzyme activity was detected.

Laboratory assays were conducted to determine the sensitivity of tomato and tobacco cell suspension cultures and tomato and pepper cotyledons to clomazone. A comparison of fresh weight and carotenoid content indicated up to a three-fold difference between the clomazone-tolerant to- bacco and clomazone-susceptible tomato cell suspension cul- tures. In contrast, an approximate 60-fold difference between the tolerant pepper and susceptible tomato cotyledons was observed when total chlorophyll and carotenoid contents were measured. The effect of clomazone and its possible metabolites on in vivo and in vitro extractable IPPisomerase (EC 53.3.2) and prenyl transferase (EC 2.5.1.29) activity was investigated. There was no clear inhibitory effect of cloma- zone or possible clomazone metabolites upon enzyme activity in tomato or tobacco cell suspension cultures or on light or dark grown tomato or pepper cotyledons. No specific enzy- matic target site of clomazone was identified in correlation with the reduction in total chlorophyll or carotenoid content. Nomenclature: Clomazone, 2-((2-chlorophenyl)methyl)-4,4- dimethyl-3-isoxazolidinone; pepper, Capsicum annum L. cv Yolo Wonder L; tobacco, Nicotiana tabacum L. 'KY-14'; tomato, Lycopersicon esculentum Mill. 'Niagara.'

Most people are familiar with terpenes whether they know it or not. These are the compounds that give many of our foods wonderful aromas and tastes and many of our household cleaning agents their fresh scents. But these are just some of the obvious ways we have taken advantage of these compounds. Biologically, terpenes are immensely important compounds in plants, fungi, bacteria, and insects that provide unique means for these organisms to sense and interact with their environment as well as serve as hormones that orchestrate developmental programs. For example, plants emit a wide range of terpenes that attract beneficial insects (1). Some plant families are notable also for their ability to synthesize and secrete antimicrobial terpenes as a defense compound against fungal and bacterial challenges (2). Still other plants that occupy resource-limited habitats such as deserts are known to constitutively secrete terpenes into their rhizosphere as a means of inhibiting the growth of competitor plant species (3). Fungi and bacteria likewise are known to synthesize a plethora of terpenes (4). Gibberella fujikuroi produces the well characterized plant growth regulator gibberellic acid, a diterpene, when it infects rice (5). This infection process results in unusually tall rice plants that can lodge easily and plants having other developmental abnormalities. Fusarium species and other relatives probably represent an even greater agricultural problem, because these fungi produce particularly noxious terpenes known as mycotoxins when they infect wheat and other stored grains (6).

(+)-Germacrene A, an important intermediate in sesquiterpene biosynthesis, was isolated in pure form from a genetically engineered yeast and was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR, 1 H NMR, and 13 C NMR data. Variable-temperature 500 MHz 1 H NMR spectra in CDCl 3 showed that this flexible cyclodecadiene ring exists as three NMR-distinguishable conformational isomers in a ratio of about 5:3:2 at or below ordinary probe temperature (25 °C). The conformer structures were assigned by 1 H NMR data comparisons, NOE experiments, and vicinal couplings as follows: 1a (52%, UU), 1b (29% UD), and 1c (19%, DU).

Terpene cyclases catalyze the synthesis of cyclic terpenes with 10-, 15-, and 20-carbon acyclic isoprenoid diphosphates as substrates. Plants have been a source of these natural products by providing a homologous set of terpene synthases. The crystal structures of 5-epi-aristolochene synthase, a sesquiterpene cyclase from tobacco, alone and complexed separately with two farnesyl diphosphate analogs were analyzed. These structures reveal an unexpected enzymatic mechanism for the synthesis of the bicyclic product, 5-epi-aristolochene, and provide a basis for understanding the stereochemical selectivity displayed by other cyclases in the biosynthesis of pharmacologically important cyclic terpenes. As such, these structures provide templates for the engineering of novel terpene cyclases.

The initial step in the conversion of the isoprenoid intermediate farnesyl diphosphate to the sesquiterpenoid phytoalexin capsidiol in elicitor-treated tobacco tissues is catalyzed by an inducible sesquiterpene cyclase [5-epi-aristolochene synthase (EAS)]. Two independent cDNA clones (cEAS1 and cEAS2) encoding EAS were isolated from an elicitor-induced tobacco cDNA library by differential hybridization and subsequently were characterized by hybrid selection--in vitro translation. Insertion of cEAS1, a partial cDNA clone encoding 175 C-terminal amino acids, into an Escherichia coli expression vector resulted in accumulation of a fusion protein immunodetectable with EAS-specific polyclonal antibodies. The cDNA clones were used to isolate two full-length EAS genes that mapped 5 kilobases (kb) apart on one 15-kb genomic clone. The nucleotide sequences of the structural gene components were identical from 388 base pairs (bp) upstream of the transcription initiation site to 40 bp downstream of the translation termination codon, suggesting a relatively recent duplication event. The genes consist of 1479-bp open reading frames, each containing five introns and specifying 56,828-Da proteins. The N-terminal amino acid sequence deduced from the genomic clones was identical to the first 16 amino acids of the EAS protein identifiable by Edman degradation. RNA blot hybridization with cEAS1 demonstrated a mRNA induction time course consistent with the induction of the EAS mRNA translational activity with maximum levels 4-6 h after elicitation. EAS mRNA was not detected in control cells. DNA blot-hybridization analysis of genomic DNA revealed a copy number of approximately 12-15 for EAS-like genes in the tetraploid tobacco genome. The conservation of a putative allelic prenyl diphosphate binding motif is also discussed.

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [(35)S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.

The olefin reaction products of the potato and tobacco sesquiterpene cyclases were partially separated from each other by argenation chromatography. Arachidonic acid-elicited potato tuber discs or cellulase-elicited tobacco cell suspension cultures metabolized the olefin reaction products of potato and tobacco sesquiterpene cyclases into different sets of compounds. These results provide evidence that the two olefin cyclase products are chemically different. The Km of the potato sesquiterpene cyclase for farnesyl diphosphate was 7μM and 1.0 mM for magnesium chloride. Inorganic diphosphate inhibited enzyme activity with an ID50 of approximately 4 mM.

Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-β-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a Km for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.

The specificity of mevinolin as an inhibitor of sterol and sesquiterpene metabolism in tobacco cell suspension cultures was examined. Exogenous mevinolin inhibited [14C]acetate, but not [3H]mevalonate incorporation into free sterols. In contrast, mevinolin inhibited the incorporation of both [14C]acetate and [3H]mevalonate into capsidiol, an extracellular sesquiterpene. Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase was inhibited greater than 90% by 3 μ m mevinolin, while squalene synthetase was insensitive to even 600 μ m mevinolin. Sesquiterpene cyclase, the first branch point enzyme specific for sesquiterpene biosynthesis, was inhibited in a dose-dependent manner by mevinolin with a 50% reduction in activity at 100 μ m . Kinetic analysis indicated that the mechanism for inhibition was complex with mevinolin acting as both a competitive and noncompetitive inhibitor. The results suggest that the mevinolin inhibition of [3H]mevalonate incorporation into extracellular sesquiterpenes can, in part, be attributed to a secondary, but specific, site of inhibition, the sesquiterpene cyclase.

Magnolia grandiflora (Southern Magnolia) is a primitive evergreen tree that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young leaves were isolated, and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted farnesyl diphosphate (C15) predominantly to β-cubebene, while Mg17 converted geranyl diphosphate (C5) to α-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all three genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 messenger RNAs in stamens. A putative amino-terminal signal peptide of Mg17 targeted the reporter green fluorescent protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, the intron-exon organizations for the three Magnolia TPS genes were different from one another and from other well-characterized TPS gene sets. The Mg17 gene consists of six introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only four introns, and Mg25 has only a single intron located near the 5′ terminus of the gene. Our results suggest that the structural diversity observed in the Magnolia TPS genes could have occurred either by a rapid loss of introns from a common ancestor TPS gene or by a gain of introns into an intron-deficient progenote TPS gene.

Plant natural products play essential roles in plant survivability and many of them are used as nutrients, colorants, flavors, fragrances, and medicines. Genetic engineering of plants for natural products can help alleviate the demands for limited natural resources. Successes in enhancing production capacities have included manipulating blocks of genes coding for segments of pathways, over-expression of putative rate-limiting steps in pathways, expression of transcription factors regulating the entire metabolic pathways, and the construction of novel branch pathways capable of diverting carbon to the biosynthesis of unique metabolites in unexpected intracellular compartments. Further enhancements are likely if more efficient pathways can be constructed, providing for the efficient channeling of intermediates to final products, and if the means for sequestering natural products in planta can be accomplished.

An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a Km for farnesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly- and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitor-treated cells resulting from either the expression of multiple genes or multiple post-translational processing events.

Solavetivone, a potent antifungal phytoalexin, is derived from a vetispirane-type sesquiterpene, premnaspirodiene, by a putative regio- and stereo-specific hydroxylation, followed by a second oxidation to yield the alpha,beta-unsaturated ketone. Mechanistically, these reactions could occur via a single, multifunctional cytochrome P450 or some combination of cytochrome P450s and a dehydrogenase. We report here the characterization of a single cytochrome P450 enzyme, Hyoscyamus muticus premnaspirodiene oxygenase (HPO), that catalyzes these successive reactions at carbon 2 (C-2) of the spirane substrate. HPO also catalyzes the equivalent regio-specific (C-2) hydroxylation of several eremophilane-type (decalin ring system) sesquiterpenes, such as with 5-epi-aristolochene. Moreover, HPO displays interesting comparisons to other sesquiterpene hydroxylases. 5-Epi-aristolochene di-hydroxylase (EAH) differs catalytically from HPO by introducing hydroxyl groups first at C-1, then C-3 of 5-epi-aristolochene. HPO and EAH also differ from one another by 91-amino acid differences, with four of these differences mapping to putative substrate recognition regions 5 and 6. These four positions were mutagenized alone and in various combinations in both HPO and EAH and the mutant enzymes were characterized for changes in substrate selectivity, reaction product specificity, and kinetic properties. These mutations did not alter the regio- or stereo-specificity of either HPO or EAH, but specific combinations of the mutations did improve the catalytic efficiencies 10-15-fold. Molecular models and comparisons between HPO and EAH provide insights into the catalytic properties of these enzymes of specialized metabolism in plants.

Terpenes are structurally diverse compounds that are of interest because of their biological activities and industrial value. These compounds consist of chirally rich hydrocarbon backbones derived from terpene synthases, which are subsequently decorated with hydroxyl substituents catalyzed by terpene hydroxylases. Availability of these compounds is, however, limited by intractable synthetic means and because they are produced in low amounts and as complex mixtures by natural sources. We engineered yeast for sesquiterpene accumulation by introducing genetic modifications that enable the yeast to accumulate high levels of the key intermediate farnesyl diphosphate (FPP). Co-expression of terpene synthase genes diverted the enlarged FPP pool to greater than 80 mg/L of sesquiterpene. Efficient coupling of terpene production with hydroxylation was also demonstrated by coordinate expression of terpene hydroxylase activity, yielding 50 mg/L each of hydrocarbon and hydroxylated products. These yeast now provide a convenient format for investigating catalytic coupling between terpene synthases and hydroxylases, as well as a platform for the industrial production of high value, single-entity and stereochemically unique terpenes.