Bioactive properties of the aromatic molecules of spearmint (Mentha spicata L.) essential oil: a review.

Spearmint belongs to the genus Mentha in the family Labiatae (Lamiaceae), which is cultivated worldwide for its remarkable aroma and commercial value. The aromatic molecules of spearmint essential oil, including carvone, carveol, dihydrocarvone, dihydrocarveol and dihydrocarvyl acetate, have been widely used in the flavors and fragrances industry. Besides their traditional use, these aromatic molecules have attracted great interest in other application fields (e.g., medicine, agriculture, food, and beverages) especially due to their antimicrobial, antioxidant, insecticidal, antitumor, anti-inflammatory and antidiabetic activities. This review presents the sources, properties, synthesis and application of spearmint aromatic molecules. Furthermore, this review focuses on the biological properties so far described for these compounds, their therapeutic effect on some diseases, and future directions of research. This review will, therefore, contribute to the rational and economic exploration of spearmint aromatic molecules as natural and safe alternative therapeutics.

Alpha-terpineol prevents myocardial damage against isoproterenol-MI induced in Wistar-Kyoto rats: new possible to promote cardiovascular integrity.

Alpha-terpineol (TPN) is one of the major components of the resin obtained from Protium heptaphyllum. This plant has been utilized as medicine by Brazilian indigenous tribes to treat cardiovascular diseases. Scientific reports have shown that the TPN possesses vasorelaxant and antihypertensive effects. This study was conducted to assess the cardioprotective action of TPN against isoproterenol (ISO)-induced cardiotoxicity. Wistar rats were randomly divided into six groups. Rats were orally administered with TPN (25, 50, and 75 mg/kg, respectively) for 15 days, and ISO was administered (85 mg/kg, subcutaneously) on the 14th and 15th days. At the end of the experiment, the hemodynamic, baroreflex test, ECG, biochemical, histological, and morphometric changes were monitored from control and experimental groups, i.e., on the 15th day. ISO-induced myocardial infarcted rats showed an increase in mortality rates, cardiac marker enzymes, tachycardia, hypertrophy, myocardium necrosis, edema, hemorrhagic areas, infiltration of inflammatory cells like lymphocytes, and increased myocardial infarct size. However, pretreatment with TPN significantly inhibited these effects of ISO. The histopathological findings obtained for the myocardium further confirmed the biochemical results. Thus, the present study provides evidence for the efficacy of TPN against ISO-induced myocardial infarction in rats.

Diploid mint (M. longifolia) can produce spearmint type oil with a high yield potential.

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native (Mentha Spicata L) and Scotch (M. × gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (-)-carvone and (-)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F2 population (32/160) produced from these two accessions. Field-testing of ten of these F2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.

Increased carvone production in Escherichia coli by balancing limonene conversion enzyme expression via targeted quantification concatamer proteome analysis.

(-)-Carvone is a monoterpenoid with a spearmint flavor. A sustainable biotechnological production process for (-)-carvone is desirable. Although all enzymes in (-)-carvone biosynthesis have been functionally expressed in Escherichia coli independently, the yield was low in previous studies. When cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in a single strain, by-product formation (dihydrocarveol and dihydrocarvone) was detected. We hypothesized that P450 and CDH expression levels differ in E. coli. Thus, two strains independently expressing P450/CPR and CDH were mixed with different ratios, confirming increased carvone production and decreased by-product formation when CDH input was reduced. The optimum ratio of enzyme expression to maximize (-)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method. Thereafter, a single strain expressing both P450/CPR and CDH was constructed to imitate the optimum expression ratio. The upgraded strain showed a 15-fold improvement compared to the initial strain, showing a 44 ± 6.3 mg/L (-)-carvone production from 100 mg/L (-)-limonene. Our study showed the usefulness of the QconCAT proteome analysis method for strain development in the industrial biotechnology field.

α-Terpineol: An Aggregation Pheromone in Optatus palmaris (Coleoptera: Curculionidae) (Pascoe, 1889) Enhanced by Its Host-Plant Volatiles.

The Annonaceae fruits weevil (Optatus palmaris) causes high losses to the soursop production in Mexico. Damage occurs when larvae and adults feed on the fruits; however, there is limited research about control strategies against this pest. However, pheromones provide a high potential management scheme for this curculio. Thus, this research characterized the behavior and volatile production of O. palmaris in response to their feeding habits. Olfactometry assays established preference by weevils to volatiles produced by feeding males and soursop. The behavior observed suggests the presence of an aggregation pheromone and a kairomone. Subsequently, insect volatiles sampled by solid-phase microextraction and dynamic headspace detected a unique compound on feeding males increased especially when feeding. Feeding-starvation experiments showed an averaged fifteen-fold increase in the concentration of a monoterpenoid on males feeding on soursop, and a decrease of the release of this compound males stop feeding. GC-MS analysis of volatiles identified this compound as α-terpineol. Further olfactometry assays using α-terpineol and soursop, demonstrated that this combination is double attractive to Annonaceae weevils than only soursop volatiles. The results showed a complementation effect between α-terpineol and soursop volatiles. Thus, α-terpineol is the aggregation pheromone of O. palmaris, and its concentration is enhanced by host-plant volatiles.

Production of complementary defense metabolites reflects a co-evolutionary arms race between a host plant and a mutualistic bark beetle-fungal complex.

Intra-specific variation in conifers has been extensively studied with respect to defense against herbivores and pathogens. While studies have shown the ability of individual or specific mixtures of compounds to influence insects and microbes, research testing biologically relevant mixtures of defense compounds reflecting intra-specific variation amongst tree populations to enemy complexes is needed. We characterized the variations in lodgepole pine monoterpenes from a progeny trial in western Canada and grouped trees in four clusters using their monoterpene profiles. We then selected 11 representative families across four clusters and amended their entire monoterpene profiles (with the exception of ß-phellandrene) in media to determine how representative families affect the performance of the mountain pine beetle or its fungal symbiont. We placed adult beetles or inoculated fungus on the amended media and measured beetle performance and fungal growth as a proxy to host suitability. We found that different clusters or families differentially influenced beetle or fungal responses. However, monoterpene profiles of trees suitable to the beetle or the fungus were dissimilar. These outcomes reflect a co-evolutionary arms-race between the host and the bark beetle-fungus complex, which has resulted in the production of complementary defense metabolites among different pine populations to enhance tree survival.

Screening a Strain of Klebsiella sp. O852 and the Optimization of Fermentation Conditions for Trans-Dihydrocarvone Production.

Flavors and fragrances have high commercial value in the food, cosmetic, chemical and pharmaceutical industries. It is interesting to investigate the isolation and characterization of new microorganisms with the ability to produce flavor compounds. In this study, a new strain of Klebsiella sp. O852 (accession number CCTCC M2020509) was isolated from decayed navel orange (Citrus sinensis (L.) Osbeck), which was proved to be capable of converting limonene to trans-dihydrocarvone. Besides, the optimization of various reaction parameters to enhance the trans-dihydrocarvone production in shake flask was performed for Klebsiella sp. O852. The results showed that the yield of trans-dihydrocarvone reached up to 1 058 mg/L when Klebsiella sp. O852 was incubated using LB-M medium for 4 h at 36 °C and 150 rpm, and the biotransformation process was monitored for 36 h after adding 1680 mg/L limonene/ethanol (final ethanol concentration of 0.8% (v/v)). The content of trans-dihydrocarvone increased 16 times after optimization. This study provided a basis and reference for producing trans-dihydrocarvone by biotransformation.

Bioproduction of α-terpineol and R-(+)-limonene derivatives by terpene-tolerant ascomycete fungus as a potential contribution to the citrus value chain.

AIMS: The aims of this article were to select fungal species with high tolerance and high growth rate in mediums supplemented with limonene and citrus essential oils (CEOs), and to test the bioconversion capability of the chosen isolates for the bioproduction of aroma compounds. METHODS AND RESULTS: Based on the use of predictive mycology, 21 of 29 isolates were selected after assaying R-(+)-limonene and CEO tolerance (10 g l-1 ). With a dendrogram divisive coefficient of 0·937, the subcluster two with isolates Aspergillus niger LBM 055, Penicillium sp. LBM 150, Penicillium sp. LBM 151 and Penicillium sp. LBM 154 gathered the highest tolerance and mycelia growth speed. Ultrastructural analysis indicated that culture media containing limonene had no visible toxic activity that could promote morphological changes in the fungal cell wall. The biomass of A. niger LBM055 was distinctive in liquid media supplemented with R-(+)-limonene (0·57 ± 0·07 g) and it was selected to prove bioconversion capacity, under static and agitated conditions, and converted up to 98% of limonene, yielding a wide variety of products that were quantified by GC-FID. It was obtained at molecular weights less than limonene (64-100%), between limonene and α-terpineol (12-72%) and greater than α-terpineol (2-48%). CONCLUSIONS: Aspergillus niger LBM 055, Penicillium sp. LBM 150, Penicillium sp. LBM 151 and Penicillium sp. LBM 154 showed to the highest tolerance and growth rate in mediums supplemented with R-(+)-limonene and orange and lemon essential oils. Particularly, A. niger LBM055, showed limonene bioconversion capability and produced different molecular weights compounds such us α-terpineol. SIGNIFICANCE AND IMPACT OF THE STUDY: Different bioproducts can be obtained by changing operative condition with the same fungus, and this bioprocess aspect is a significant approach to be adopted on industrial scale leading to the creation of new natural flavours and fragrance compositions.

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation.

Monoterpenoids are industrially important natural products with applications in the flavours, fragrances, fuels and pharmaceutical industries. Most monoterpenoids are produced by plants, but recently two bacterial monoterpene synthases have been identified, including a cineole synthase (bCinS). Unlike plant cineole synthases, bCinS is capable of producing nearly pure cineole from geranyl diphosphate in a complex cyclisation cascade that is tightly controlled. Here we have used a multidisciplinary approach to show that Asn305 controls water attack on the α-terpinyl cation and subsequent cyclisation and deprotonation of the α-terpineol intermediate, key steps in the cyclisation cascade which direct product formation towards cineole. Mutation of Asn305 results in variants that no longer produce α-terpineol or cineole. Molecular dynamics simulations revealed that water coordination is disrupted in all variants tested. Quantum mechanics calculations indicate that Asn305 is most likely a (transient) proton acceptor for the final deprotonation step. Our synergistic approach gives unique insight into how a single residue, Asn305, tames the promiscuous chemistry of monoterpene synthase cyclisation cascades. It does this by tightly controlling the final steps in cineole formation catalysed by bCinS to form a single hydroxylated monoterpene product.

Exogenous bacterial composition changes dominate flavor deterioration of dried carrots during storage.

Flavor deterioration is a serious problem in dried carrots during storage and is frequently accompanied by water absorption and bacterial growth. To explore the underlying mechanism of flavor deterioration, relationship among water status, exogenous bacterial composition and flavor changes in dried carrots were analyzed at different water activities (aw, 0.43, 0.67, 0.76 and 0.84). Results suggested that the water molecules mobility significantly increased in the dried carrots at higher aw levels (0.67, 0.76 and 0.84), this was attributed to the raised content of bound water, rather than immobilized or free water. Consequently, this accelerated microbial growth and flavor deterioration. At aw = 0.84, the characteristic flavor compounds including 2,3-butanediol, pentanoic acid, hexanoic acid, heptanoic acid and nonanoic acid were lost. The disagreeable flavor compounds including terpenes were produced during the storage period. These were the main contributors of flavor deterioration in the dried carrots. Lactic acid bacteria, as the dominant bacteria in dried carrots during storage, were proved to be closely related to the production of o-cymene, ß-pinene and ß-myrcene. Moreover, the emergence of Pediococcus spp. was the major factor leading to the increase of γ-terpinene in dried carrots.

Profiling of volatile terpenes from almond (Prunus dulcis) young fruits and characterization of seven terpene synthase genes.

Almond (Prunus dulcis) is an agricultural and economically important fruit tree from the Rosaceae family used in the food industry. The monoterpenes and sesquiterpenes perform important ecological functions such as insecticidal and antifeedant activities against various insects. The young fruits of the different almond varieties were found to produce considerable amounts of terpene volatiles, including linalool and geraniol. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, existing genome databases of the Rosaceae were screened for almond genes with significant sequence similarity to other plants TPSs. Bioinformatics analysis led to the identification of seven putative TPSs genes with complete open reading frames. We characterized the enzymes encoded by these seven complementary DNAs: the monoterpene synthases PdTPS1, PdTPS3, PdTPS5, and PdTPS6 belong to the TPS-b clade, which catalyzes the formation of ß-phellandrene, geraniol, linalool, and farnesene, respectively. The sesquiterpene synthases PdTPS2 and PdTPS4, which belong to the TPS-a clade mainly catalyze the formation of bergamotene, while another sesquiterpene synthase, PdTPS7, from the TPS-g clade showed nerolidol synthase activity. The qRT-PCR analysis revealed that the various tissues of almond varieties showed differential transcription for all these PdTPSs genes.

Asymmetric Whole-Cell Bio-Reductions of (R)-Carvone Using Optimized Ene Reductases.

(2R,5R)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of (R)-carvone. Escherichia coli (E. coli) cells overexpressing an ene reductase from Nostoc sp. PCC7120 (NostocER1) in combination with a cosubstrate regeneration system proved to be very effective biocatalysts for this reaction. However, the industrial applicability of biocatalysts is strongly linked to the catalysts' activity. Since the cell-internal NADH concentrations are around 20-fold higher than the NADPH concentrations, we produced E. coli cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These E. coli whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM (R)-carvone. 287 mM (2R,5R)-dihydrocarvone were formed within 5 h with a diasteromeric excess of 95.4% and a yield of 95.6%. Thus, the whole-cell biocatalysts were strongly improved by using NADH-accepting enzymes, resulting in an up to 2.1-fold increased initial product formation rate leading to a 1.8-fold increased space-time yield when compared to literature.

Functions of mountain pine beetle cytochromes P450 CYP6DJ1, CYP6BW1 and CYP6BW3 in the oxidation of pine monoterpenes and diterpene resin acids.

The mountain pine beetle (MPB; Dendroctonus ponderosae) is a forest insect pest that attacks several different pine (Pinus) species in its native range of distribution in western North America. MPB are exposed for most of their life cycle to the chemical defenses of their hosts. These defenses are dominated by oleoresin secretions containing mostly various monoterpenes and diterpene resin acids (DRAs). Cytochrome P450 enzymes (P450s) of the MPB are thought to be involved in the metabolism of at least some of these defense compounds. Here we describe the cloning and characterization of three MPB P450s, CYP6DJ1, CYP6BW1 and CYP6BW3, and their functions in the oxidation of various monoterpenes and diterpene resin acids. CYP6DJ1 oxidizes the monoterpenes (+)-(4R)-limonene, (-)-(4S)-limonene and terpinolene and produces (4R,8R)-limonene-8,9-epoxide, (4R,8S)-limonene-8,9-epoxide, (4S,8S)-limonene-8,9-epoxide, (4S,8R)-limonene-8,9-epoxide, perilla alcohol and several unidentified oxidized compounds. These products of CYP6DJ1 were also identified in extracts of MPB treated with the same monoterpenes. CYP6BW1 and CYP6BW3 both oxidize the DRAs abietic acid, dehydroabietic acid, neoabietic acid, levopimaric acid, palustric acid, and isopimaric acid, producing hydroxylated and epoxidized DRAs. CYP6DJ1, CYP6BW1 and CYP6BW3 appear to contribute to the metabolism of oleoresin terpenes as part of the MPB's ability to cope with host defenses.

Photosynthetic generation of heterologous terpenoids in cyanobacteria.

The work aims to convert the secondary slow metabolism of the terpenoid biosynthetic pathway into a primary activity in cyanobacteria and to generate heterologous products using these photosynthetic microorganisms as cell factories. Case study is the production of the 10-carbon monoterpene ß-phellandrene (PHL) in Synechocystis sp. PCC 6803 (Synechocystis). Barriers to this objective include the slow catalytic activity of the terpenoid metabolism enzymes that limit rates and yield of product synthesis and accumulation. "Fusion constructs as protein overexpression vectors" were applied in the overexpression of the geranyl diphosphate synthase (GPPS) and ß-phellandrene synthase (PHLS) genes, causing accumulation of GPPS up to 4% and PHLS up to 10% of the total cellular protein. Such GPPS and PHLS protein overexpression compensated for their slow catalytic activity and enabled transformant Synechocystis to constitutively generate 24 mg of PHL per g biomass (2.4% PHL:biomass, w-w), a substantial improvement over earlier yields. The work showed that a systematic overexpression, at the protein level, of the terpenoid biosynthetic pathway genes is a promising approach to achieving high yields of prenyl product biosynthesis, on the way to exploiting the cellular terpenoid metabolism for commodity product generation.

Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants.

This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8C9 to the corresponding alkene was also detected.