Selectivity of enzymes involved in the formation of opposite enantiomeric series of p-menthane monoterpenoids in peppermint and Japanese catnip.

Peppermint (Mentha x piperita L.) and Japanese catnip (Schizonepeta tenuifolia (Benth.) Briq.) accumulate p-menthane monoterpenoids with identical functionalization patterns but opposite stereochemistry. In the present study, we investigate the enantioselectivity of multiple enzymes involved in monoterpenoid biosynthesis in these species. Based on kinetic assays, mint limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, isopiperitenone reductase, and menthone reductase exhibited significant enantioselectivity toward intermediates of the pathway that proceeds through (-)-4S-limonene. Limonene synthase, isopiperitenol dehydrogenase and isopiperitenone reductase of Japanese catnip preferred intermediates of the pathway that involves (+)-4R-limonene, whereas limonene 3-hydroxylase was not enantioselective, and the activities of pulegone reductase and menthone reductase were too low to acquire meaningful kinetic data. Molecular modeling studies with docked ligands generally supported the experimental data obtained with peppermint enzymes, indicating that the preferred enantiomer was aligned well with the requisite cofactor and amino acid residues implicated in catalysis. A striking example for enantioselectivity was peppermint (-)-menthone reductase, which binds (-)-menthone with exquisite affinity but was predicted to bind (+)-menthone in a non-productive orientation that positions its carbonyl functional group at considerable distance to the NADPH cofactor. The work presented here lays the groundwork for structure-function studies aimed at unraveling how enantioselectivity evolved in closely related species of the Lamiaceae and beyond.

Colonization of Trichoderma viride Tv-1511 in peppermint (Mentha × piperita L.) roots promotes essential oil production by triggering ROS-mediated MAPK activation.

Peppermint (Mentha × piperita L.) is a flavoring additive used worldwide, and Trichoderma species are beneficial fungi that can stimulate growth and disease resistance of these plants. Here the growth conditions and metabolic processes of essential oil (EO) biosynthesis in response to inoculation with Trichoderma viride Tv-1511 were investigated. The results showed that T. viride Tv-1511 was able to colonize roots of peppermint to promote its growth and photosynthetic activity and induce higher levels of glandular trichomes and elevated EO yield and composition. GC-MS analysis showed that T. viride Tv-1511-inoculated peppermint produced higher concentrations of menthone, menthol, and pulegone and lower concentrations of menthofuran than un-inoculated seedlings, and qRT-PCR showed that T. viride Tv-1511 inoculation induced upregulation of Pr (pulegone reductase encoding gene) and Mr (menthone reductase encoding gene), whereas it led to the downregulation of Mfs (menthofuran synthase encoding gene). Furthermore, a mitogen-activated protein kinase (MAPK) in peppermint, which was determined to be an analog of Arabidopsis MPK6 protein, was found to be responsible for the modulation of EO metabolism at the transcriptional level and for enzymatic activation in the T. viride Tv-1511-inoculated peppermint. Notably, NADPH oxidase-dependent reactive oxygen species (ROS) production played vital roles in the root colonization of T. viride Tv-1511 and was also involved in the induction of MAPK activation. These data showed the beneficial effects of T. viride Tv-1511 on the seedling growth and EO yield of peppermint, and they elucidated that T. viride Tv-1511 improved the quantity and quality of EOs by regulating the genes that encode the enzymes involved in EO metabolism through a potential MAPK-mediated signaling pathways.

Co-expression of peppermint geranyl diphosphate synthase small subunit enhances monoterpene production in transgenic tobacco plants.

Monoterpenes are important for plant survival and useful to humans. In addition to their function in plant defense, monoterpenes are also used as flavors, fragrances and medicines. Several metabolic engineering strategies have been explored to produce monoterpene in tobacco but only trace amounts of monoterpenes have been detected. We investigated the effects of Solanum lycopersicum 1-deoxy-d-xylulose-5-phosphate synthase (SlDXS), Arabidopsis thaliana geranyl diphosphate synthase 1 (AtGPS) and Mentha × piperita geranyl diphosphate synthase small subunit (MpGPS.SSU) on production of monoterpene and geranylgeranyl diphosphate (GGPP) diversities, and plant morphology by transient expression in Nicotiana benthamiana and overexpression in transgenic Nicotiana tabacum. We showed that MpGPS.SSU could enhance the production of various monoterpenes such as (-)-limonene, (-)-linalool, (-)-α-pinene/ß-pinene or myrcene, in transgenic tobacco by elevating geranyl diphosphate synthase (GPS) activity. In addition, overexpression of MpGPS.SSU in tobacco caused early flowering phenotype and increased shoot branching by elevating contents of GA3 and cytokinins due to upregulated transcript levels of several plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway genes, geranylgeranyl diphosphate synthases 3 (GGPPS3) and GGPPS4. Our method would allow the identification of new monoterpene synthase genes using transient expression in N. benthamiana and the improvement of monoterpene production in transgenic tobacco plants.

PEG-induced osmotic stress in Mentha x piperita L.: Structural features and metabolic responses.

The present study investigated whether osmotic stress induced by the exposure of peppermint (Mentha x piperita L.) to moderate and severe stress for short periods of time changes the plant's physiological parameters, leaf anatomy and ultrastructure and essential oil. Plants were exposed to two levels of polyethyleneglycol (50 g L(-1) and 100 g L(-1) of PEG) in a hydroponic experiment. The plants exposed to 50 g L(-1) maintained metabolic functions similar to those of the control group (0 g L(-1)) without changes in gas exchange or structural characteristics. The increase in antioxidant enzyme activity reduced the presence of free radicals and protected membranes, including chloroplasts and mitochondria. In contrast, the osmotic stress caused by 100 g L(-1) of PEG inhibited leaf gas exchange, reduced the essential oil content and changed the oil composition, including a decrease in menthone and an increase in menthofuran. These plants also showed an increase in peroxidase activity, but this increase was not sufficient to decrease the lipid peroxidation level responsible for damaging the membranes of organelles. Morphological changes were correlated with the evaluated physiological features: plants exposed to 100 g L(-1) of PEG showed areas with collapsed cells, increases in mesophyll thickness and the area of the intercellular space, cuticle shrinkage, morphological changes in plastids, and lysis of mitochondria. In summary, our results revealed that PEG-induced osmotic stress in M. x piperita depends on the intensity level of the osmotic stress applied; severe osmotic stress changed the structural characteristics, caused damage at the cellular level, and reduced the essential oil content and quality.

Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.

Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6.

MAPK-mediated regulation of growth and essential oil composition in a salt-tolerant peppermint (Mentha piperita L.) under NaCl stress.

Peppermint (Mentha × piperita L.) is an important and commonly used flavoring agent worldwide, and salinity is a major stress that limits plant growth and reduces crop productivity. This work demonstrated the metabolic responses of essential oil production including the yield and component composition, gene expression, enzyme activity, and protein activation in a salt-tolerant peppermint Keyuan-1 with respect to NaCl stress. Our results showed that Keyuan-1 maintained normal growth and kept higher yield and content of essential oils under NaCl stress than wild-type (WT) peppermint.Gas chromatography-mass spectrometry (GC-MS) and qPCR results showed that compared to WT seedlings, a 150-mM NaCl stress exerted no obvious changes in essential oil composition, transcriptional level of enzymes related to essential oil metabolism, and activity of pulegone reductase (Pr) in Keyuan-1 peppermint which preserved the higher amount of menthol and menthone as well as the lower content of menthofuran upon the 150-mM NaCl stress. Furthermore, it was noticed that a mitogen-activated protein kinase (MAPK) protein exhibited a time-dependent activation in the Keyuan-1 peppermint and primarily involved in the modulation of the essential oil metabolism in the transcript and enzyme levels during the 12-day treatment of 150 mM NaCl. In all, our data elucidated the effect of NaCl on metabolic responses of essential oil production, and demonstrated the MAPK-dependent regulation mechanism of essential oil biosynthesis in the salt-tolerant peppermint, providing scientific basis for the economic and ecological utilization of peppermint in saline land.

MAPK-mediated enhanced expression of vacuolar H(+)-ATPase confers the improved adaption to NaCl stress in a halotolerate peppermint (Mentha piperita L.).

Vacuolar H(+)-ATPase (V-H(+)-ATPase) has been proved to be of importance in maintenance of ion homeostasis inside plant cells under NaCl stress. In this study, the expression levels and salt-tolerate function of V-H(+)-ATPase genes were investigated in the roots and leaves of a halotolerate peppermint (Mentha × piperita L.) Keyuan-1 treated with different concentrations of NaCl. Results showed that the expressions of V-H(+)-ATPase in the transcriptional, protein and activity levels were significantly enhanced in the halotolerate peppermint Keyuan-1 compared to the wild-type (WT) peppermint under 50, 100, and 150 mM NaCl treatment. Moreover, inhibition experiments exhibited that V-H(+)-ATPase activity played vital roles in the salt tolerance of peppermint Keyuan-1 to 150 mM NaCl stress through increasing the vacuolar H(+) pumping activity and Na(+) compartmentalization capacity. Furthermore, results of Western blots showed that the activity of a mitogen-activated protein kinase (MAPK) was significantly increased under different concentrations of NaCl in the halotolerate peppermint Keyuan-1, which was much higher than that of WT peppermint. Further experiments with inhibitors suggested that this MAPK protein was responsible for the enhanced expression of V-H(+)-ATPase in the halotolerate peppermint Keyuan-1. In response to NaCl stress, increase of cytoplasmic calcium concentration ([Ca(2+)]cyt) occurred upstream of MAPK activation in the halotolerate peppermint Keyuan-1. In all, these findings demonstrated that increased V-H(+)-ATPase activity was positively correlated with the enhanced salt tolerance in the halotolerate peppermint Keyuan-1, providing the theoretic basis for the further development and utilization of peppermint in saline areas.

Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint.

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined.

Structure of a heterotetrameric geranyl pyrophosphate synthase from mint (Mentha piperita) reveals intersubunit regulation.

Terpenes (isoprenoids), derived from isoprenyl pyrophosphates, are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. Divergent evolution of homomeric prenyltransferases (PTSs) has allowed PTSs to optimize their active-site pockets to achieve catalytic fidelity and diversity. Little is known about heteromeric PTSs, particularly the mechanisms regulating formation of specific products. Here, we report the crystal structure of the (LSU . SSU)(2)-type (LSU/SSU = large/small subunit) heterotetrameric geranyl pyrophosphate synthase (GPPS) from mint (Mentha piperita). The LSU and SSU of mint GPPS are responsible for catalysis and regulation, respectively, and this SSU lacks the essential catalytic amino acid residues found in LSU and other PTSs. Whereas no activity was detected for individually expressed LSU or SSU, the intact (LSU . SSU)(2) tetramer produced not only C(10)-GPP at the beginning of the reaction but also C(20)-GGPP (geranylgeranyl pyrophosphate) at longer reaction times. The activity for synthesizing C(10)-GPP and C(20)-GGPP, but not C(15)-farnesyl pyrophosphate, reflects a conserved active-site structure of the LSU and the closely related mustard (Sinapis alba) homodimeric GGPPS. Furthermore, using a genetic complementation system, we showed that no C(20)-GGPP is produced by the mint GPPS in vivo. Presumably through protein-protein interactions, the SSU remodels the active-site cavity of LSU for synthesizing C(10)-GPP, the precursor of volatile C(10)-monoterpenes.

Cloning and functional characterisation of a cis-muuroladiene synthase from black peppermint (Menthaxpiperita) and direct evidence for a chemotype unable to synthesise farnesene.

Using oligonucleotide primers designed to the known gene sequence of an (E)-beta-farnesene (EbetaF) synthase, two cDNA sequences (MxpSS1 and MxpSS2) were cloned from a black peppermint (Menthaxpiperita) plant. MxpSS1 encoded a protein with 96% overall amino acid sequence identity with the EbetaF synthase. Recombinant MxpSS1 produced in Escherichia coli, after removal of an N-terminal thioredoxin fusion, had a K(m) for FPP of 1.91+/-0.1 microM and k(cat) of 0.18 s(-1), and converted farnesyl diphosphate (FPP) into four products, the major two being cis-muurola-3,5-diene (45%) and cis-muurola-4(14),5-diene (43%). This is the first cis-muuroladiene synthase, to be characterised. MxpSS2 encoded a protein with only two amino acids differing from EbetaF synthase. Recombinant MxpSS2 protein showed no activity towards FPP. One of the two mutations, at position 531 (leucine in MxpSS2 and serine in EbetaF synthase) was shown, by structural modelling to occur in the J-K loop, an element of the structure of sesquiterpene synthases known to be important in the reaction mechanism. Reintroduction of the serine at position 531 into MxpSS2 by site-directed mutagenesis restored EbetaF synthase activity (K(m) for FPP 0.98+/-0.12 microM, k(cat) 0.1 s(-1)), demonstrating the crucial role of this residue in the enzyme activity. Analysis, by GC-MS, of the sesquiterpene profile of the plant used for the cloning, revealed that EbetaF was not present, confirming that this particular mint chemotype had lost EbetaF synthase activity due to the observed mutations.

Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint.

The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene reductases of mint involved in (-)-menthol biosynthesis. The isolation of the genes specifying redox enzymes of monoterpene biosynthesis in mint indicates that these genes arose from different ancestors and not by simple duplication and differentiation of a common progenitor, as might have been anticipated based on the common reaction chemistry and structural similarity of the substrate monoterpenes.

Monoterpene metabolism. Cloning, expression, and characterization of menthone reductases from peppermint.

(-)-Menthone is the predominant monoterpene produced in the essential oil of maturing peppermint (Mentha x piperita) leaves during the filling of epidermal oil glands. This early biosynthetic process is followed by a second, later oil maturation program (approximately coincident with flower initiation) in which the C3-carbonyl of menthone is reduced to yield (-)-(3R)-menthol and (+)-(3S)-neomenthol by two distinct NADPH-dependent ketoreductases. An activity-based in situ screen, by expression in Escherichia coli of 23 putative redox enzymes from an immature peppermint oil gland expressed sequence tag library, was used to isolate a cDNA encoding the latter menthone:(+)-(3S)-neomenthol reductase. Reverse transcription-PCR amplification and RACE were used to acquire the former menthone:(-)-(3R)-menthol reductase directly from mRNA isolated from the oil gland secretory cells of mature leaves. The deduced amino acid sequences of these two reductases share 73% identity, provide no apparent subcellular targeting information, and predict inclusion in the short-chain dehydrogenase/reductase family of enzymes. The menthone:(+)-(3S)-neomenthol reductase cDNA encodes a 35,722-D protein, and the recombinant enzyme yields 94% (+)-(3S)-neomenthol and 6% (-)-(3R)-menthol from (-)-menthone as substrate, and 86% (+)-(3S)-isomenthol and 14% (+)-(3R)-neoisomenthol from (+)-isomenthone as substrate, has a pH optimum of 9.3, and K(m) values of 674 mum, > 1 mm, and 10 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.06 s(-1). The recombinant menthone:(-)-(3R)-menthol reductase has a deduced size of 34,070 D and converts (-)-menthone to 95% (-)-(3R)-menthol and 5% (+)-(3S)-neomenthol, and (+)-isomenthone to 87% (+)-(3R)-neoisomenthol and 13% (+)-(3S)-isomenthol, displays optimum activity at neutral pH, and has K(m) values of 3.0 mum, 41 mum, and 0.12 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.6 s(-1). The respective activities of these menthone reductases account for all of the menthol isomers found in the essential oil of peppermint. Biotechnological exploitation of these genes could lead to improved production yields of (-)-menthol, the principal and characteristic flavor component of peppermint.

Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites.

A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa. Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions. Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro. Additionally, using mixed cultures of E. coli expressing different classes of modifying genes (OMTs and GTs), the production of polymethylated flavonoid glucosides was observed. This report demonstrates the potential to increase the structural diversity of plant secondary metabolites using a multi-enzyme, bio-fermentation approach.

Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint.

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1). Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.