Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis.

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid's precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis. KEY POINTS: • Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis. • Perillic acid was first synthesized de novo in engineered microorganisms. • The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

Transcriptome analysis of Reticulitermes flaviceps exposed to Mentha spicata essential oil and carvone indicates a potential neurotoxic mechanism of action characterized by intracellular calcium influx mediated by dopamine receptors.

Reticulitermes flaviceps is an economically important pest in agriculture, forestry, and construction. Recent studies have shown an increase in research focusing on the anti-termite properties of plant essential oils, however, there remains a lack of information regarding the specific molecular mechanism involved. In this study, RNA-seq analysis was conducted on termites exposed to Mentha spicata essential oil (EO) and carvone, leading to the discovery of various genes that were expressed differentially under different treatment conditions. Numerous genes that exhibited a response to M. spicata EO and carvone found to be associated with stress-related pathways, such as drug metabolism cytochrome P450, glutathione metabolism, fatty acid metabolism, citric acid cycle, neuroactive ligand-receptor interaction, cell apoptosis, the AMPK signalling pathway, the mTOR signalling pathway, the longevity regulation pathway, ubiquitin-mediated protein hydrolysis, and the calcium signalling pathway. The up-regulation of genes (SPHK) associated with calcium channels, such as SPHK, indicates a potential mechanism of neurotoxicity, while the up-regulation of apoptosis-associated genes, including ACTB_G1, PYG, SQSTM1, RNF31, suggests a potential mechanism of cytotoxicity. The metabolism of M. spicata EO induces oxidative stress, elevates free Ca2+ levels in mitochondria, and initiates the generation of reactive oxygen species (ROS), ultimately resulting in programmed cell necrosis and apoptosis, as well as facilitating cellular autophagy. The monoterpenes exhibited neurotoxic and cytotoxic effects on R. flaviceps and could be exploited to advance termiticide development and eco-friendly termite control.

Innovative Analogs of Unpasteurized Kombucha Beverages: Comparative Analysis of Mint/Nettle Kombuchas, Considering Their Health-Promoting Effect, Polyphenolic Compounds and Chemical Composition.

Increasing demand for functional beverages is attracting consumers' attention and driving research to expand our knowledge of fermentation using symbiotic culture of bacteria and yeast (SCOBY) and demonstrate the health effects of consuming kombucha. The objective of this study was to develop innovative recipes for unpasteurized mint/nettle kombucha analogs, and to compare the products obtained under varying conditions in terms of chemical composition, bioactive polyphenols and health-promoting activity. Four variants of kombucha beverages (K1-K4), differing in the addition of sucrose and fermentation temperature, were formulated. The fermentation process provided data indicating the increase of antidiabetic, anti-inflammatory and anticholinergic properties, while a decrease in antioxidant capacity was observed. The content of polyphenolics was the highest on the seventh day of fermentation. A higher fermentation temperature and a larger amount of sucrose accelerated the fermentation process, which may be crucial for shortening the production time of kombucha drinks.

Chemical Characterization of the Essential Oil Compositions of Mentha spicata and M. longifolia ssp. cyprica from the Mediterranean Basin and Multivariate Statistical Analyses.

This present study aims to characterize the essential oil compositions of the aerial parts of M. spicata L. and endemic M. longifolia ssp. cyprica (Heinr. Braun) Harley by using GC-FID and GC/MS analyses simultaneously. In addition, it aims to perform multivariate statistical analysis by comparing with the existing literature, emphasizing the literature published within the last two decades, conducted on both species growing within the Mediterranean Basin. The major essential oil components of M. spicata were determined as carvone (67.8%) and limonene (10.6%), while the major compounds of M. longifolia ssp. cyprica essential oil were pulegone (64.8%) and 1,8-cineole (10.0%). As a result of statistical analysis, three clades were determined for M. spicata: a carvone-rich chemotype, a carvone/trans-carveol chemotype, and a pulegone/menthone chemotype, with the present study result belonging to the carvone-rich chemotype. Carvone was a primary determinant of chemotype, along with menthone, pulegone, and trans-carveol. In M. longifolia, the primary determinants of chemotype were identified as pulegone and menthone, with three chemotype clades being pulegone-rich, combined menthone/pulegone, and combined menthone/pulegone with caryophyllene enrichment. The primary determinants of chemotype were menthone, pulegone, and caryophyllene. The present study result belongs to pulegone-rich chemotype.

Effects of Harvest Time and Hydrodistillation Time on Yield, Composition, and Antioxidant Activity of Mint Essential Oil.

In this study, we assessed the effects of different harvest times (9 a.m., 1 p.m., and 5 p.m.) and hydrodistillation times (60, 90, and 120 min) on the yield, chemical composition, and antioxidant activity of the spearmint (Mentha spicata L.) essential oil (EO) sourced from the Amazon region. EO yield was ≥1.55% and was not significantly influenced (p ≥ 0.05) by the different harvest times and hydrodistillation times. Thirty-one different organic compounds were identified, of which menthol (91.56-95.68%), menthone (0.6-2.72%), and isomenthone (0.55-1.46%) were the major constituents. The highest menthol content in the EO was obtained from samples collected at 9 a.m., with a hydrodistillation time of 60-90 min, compared to other harvest and hydrodistillation times. This suggests that exposure to sun and light, which is greater at harvest times of 1 p.m. and 5 p.m., decreased the menthol content and altered the chemical composition of Mentha EO. Furthermore, the sample harvested at 9 a.m. and hydrodistilled for 60 min showed the highest antioxidant activity (61.67 equivalent mg of Trolox per g of EO), indicating that antioxidant activity is strongly affected by light exposure and the contact duration of the sample with boiling water during hydrodistillation.

Towards Bioprospection of Commercial Materials of Mentha spicata L. Using a Combined Strategy of Metabolomics and Biological Activity Analyses.

Spearmint (Mentha spicata L.) has been widely studied for its diversity of compounds for product generation. However, studies describing the chemical and biological characteristics of commercial spearmint materials from different origins are scarce. For this reason, this research aimed to bioprospecting spearmint from three origins: Colombia (Col), Mexico (Mex), and Egypt (Eg). We performed a biological activity analysis, such as FRAP, DPPH, and ABTS, inhibition potential of S. pyogenes, K. pneumoniae, E. coli, P. aeuroginosa, S. aureus, S aureus Methicillin-Resistant, and E. faecalis. Furthermore, we performed chemical assays, such as total polyphenol and rosmarinic acid, and untargeted metabolomics via HPLC-MS/MS. Finally, we developed a causality analysis to integrate biological activities with chemical analyses. We found significant differences between the samples for the total polyphenol and rosmarinic acid contents, FRAP, and inhibition analyses for Methicillin-Resistant S. aureus and E. faecalis. Also, clear metabolic differentiation was observed among the three commercial materials evaluated. These results allow us to propose data-driven uses for the three spearmint materials available in current markets.

Spearmint (Mentha spicata L.) Phytochemical Profile: Impact of Pre/Post-Harvest Processing and Extractive Recovery.

The purpose of this study was to chemically compare samples of Mentha spicata (marketing byproducts, production byproducts, and export material), cultivated in the open field and under greenhouse, using an integrated approach by HPLC/DAD and GC/MS analysis. The presence of phenolic compounds was higher in the marketing byproducts cultivated in the open field. Marketing byproducts also had the highest amount of carvone. For this reason, this byproduct was selected as a candidate for the development of natural ingredients. With the best selected material, the optimization of simultaneous high-intensity ultrasound-assisted extraction processes was proposed for the recovery of the compounds of interest. This extraction was defined by Peleg's equation and polynomial regression analysis. Modeling showed that the factors amplitude, time, and solvent were found to be significant in the recovery process (p < 0.005). The maximum amount of compounds was obtained using 90% amplitude for 5 min and ethanol/water mixture (80:20) for extraction to simultaneously obtain phenolic and terpenoid compounds. This system obtained the highest amount of monoterpenoid and sesquiterpenoid compounds from the essential oil of M. spicata (64.93% vs. 84.55%). Thus, with an efficient and eco-friendly method, it was possible to optimize the extraction of compounds in M. spicata as a starting point for the use of its byproducts.

Bioactive Profile of Distilled Solid By-Products of Rosemary, Greek Sage and Spearmint as Affected by Distillation Methods.

By-products of essential oils (EOs) in the industry represent an exploitable material for natural and safe antioxidant production. One representative group of such by-products is distilled solid residues, whose composition is properly modulated by the distillation method applied for the recovery of EOs. Recently, in terms of Green Chemistry principles, conventional extraction and distillation processes are considered outdated and tend to be replaced by more environmentally friendly ones. In the present study, microwave-assisted hydro-distillation (MAHD) was employed as a novel and green method for the recovery of EOs from three aromatic plants (rosemary, Greek sage and spearmint). The method was compared to conventional ones, hydro-distillation (HD) and steam-distillation (SD), in terms of phytochemical composition of distilled solid residues, which was estimated by spectrophotometric and chromatographic methods. Total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity (ABTS, DPPH and FRAP) results highlighted the distilled solid residues as good sources of antioxidants. Moreover, higher antioxidant activity was achieved for MAHD extracts of solid residues in comparison to HD and SD extracts. A metabolomics approach was carried out on the methanolic extracts of solid residues obtained by different distillation methods using LC-MS analysis followed by multivariate data analysis. A total of 29 specialized metabolites were detected, and 26 of them were identified and quantified, presenting a similar phenolic profile among different treatments, whereas differences were observed among different species. Rosmarinic acid was the most abundant phenolic compound in all extracts, being higher in MAHD extracts. In rosemary and Greek sage extracts, carnosol and carnosic acid were quantified in significant amounts, while trimers and tetramers of caffeic acid (salvianolic acids isomers) were identified and quantified in spearmint extracts, being higher in MAHD extracts. The obtained results pointed out that MAHD extracts of distilled solid by-products could be a good source of bioactives with potential application in the food, pharmaceutical and cosmetic industries, contributing to the circular economy.

In vitro activity of essential oils against adult and immature stages of Ctenocephalides felis felis.

Essential oils (EOs) are considered a new class of ecological products aimed at the control of insects for industrial and domestic use; however, there still is a lack of studies involving the control of fleas. Ctenocephalides felis felis, the most observed parasite in dogs and cats, is associated with several diseases. The aim of this study was to evaluate the in vitro activity, the establishment of LC50 and toxicity of EOs from Alpinia zerumbet (Pers.) B. L. Burtt & R. M. Sm, Cinnamomum spp., Laurus nobilis L., Mentha spicata L., Ocimum gratissimum L. and Cymbopogon nardus (L.) Rendle against immature stages and adults of C. felis felis. Bioassay results suggest that the method of evaluation was able to perform a pre-screening of the activity of several EOs, including the discriminatory evaluation of flea stages by their LC50. Ocimum gratissimum EO was the most effective in the in vitro assays against all flea stages, presenting adulticide (LC50 = 5.85 µg cm-2), ovicidal (LC50 = 1.79 µg cm-2) and larvicidal (LC50 = 1.21 µg cm-2) mortality at low doses. It also presented an excellent profile in a toxicological eukaryotic model. These findings may support studies involving the development of non-toxic products for the control of fleas in dogs and cats.

Metabolic Engineering of the Native Monoterpene Pathway in Spearmint for Production of Heterologous Monoterpenes Reveals Complex Metabolism and Pathway Interactions.

Spearmint produces and stores large amounts of monoterpenes, mainly limonene and carvone, in glandular trichomes and is the major natural source of these compounds. Towards producing heterologous monoterpenes in spearmint, we first reduced the flux into the native limonene pathway by knocking down the expression of limonene synthase (MsLS) by RNAi method. The MsLS RNAi lines exhibited a huge reduction in the synthesis of limonene and carvone. Detailed GC-MS and LC-MS analysis revealed that MsLS RNAi plants also showed an increase in sesquiterpene, phytosterols, fatty acids, flavonoids, and phenolic metabolites, suggesting an interaction between the MEP, MVA shikimate and fatty acid pathways in spearmint. Three different heterologous monoterpene synthases namely, linalool synthase and myrcene synthase from Picea abies and geraniol synthase from Cananga odorata were cloned and introduced independently into the MsLS RNAi mutant background. The expression of these heterologous terpene synthases resulted mainly in production of monoterpene derivatives. Of all the introduced monoterpenes geraniol showed the maximum number of derivatives. Our results provide new insights into MEP pathway interactions and regulation and reveals the existence of mechanisms for complex metabolism of monoterpenes in spearmint.

Nicotine-induced oxidative stress, testis injury, AChE inhibition and brain damage alleviated by Mentha spicata.

Nicotine mediates some of the injurious effects caused by consuming tobacco products. The aim of this work is to investigate the protective effects of Mentha spicata extract (ME) supplementation on the testis and brain of nicotine-induced oxidative damage rats. ME extract showed interesting hydrogen peroxide-scavenging activity. HPLC-DAD analysis of ME revealed the presence of nine compounds among them gallic acid was the major one (165.44 µg/g ME). Thirty-two rats were randomly divided into four groups: control, a nicotine-treated group (1 mg/kg i.p.), a group receiving ME (100 mg/kg), and a group receiving both ME (100 mg/kg) and nicotine (1 mg/kg). After 2 months of treatment, the in vivo results showed that nicotine exhibited an increase in the body, brain, testis and accessory sex organ weights, sperm count and sperm motility. In addition, exposure to nicotine significantly (p < 0.01) increased acetylcholinesterase level (AChE) in brain, lipid peroxidation level in brain and testis as compared to control group. The antioxidant enzymes results showed that nicotine treatment induced a significant decrease (p < 0.01) in brain and testis antioxidant enzymes such as catalase, superoxide dismutase and glutathione peroxidase as compared to control group. Interestingly, pretreatment with ME significantly (p < 0.01) restored the majority of these biological parameters to normal levels, as well as a histological improvement. Obtained results suggest that ME contains promising substances that counteract the nicotine-intoxication and can be efficient in the prevention of brain and testis toxicity complications.

Comparative laboratory efficacy of novel botanical extracts against Tribolium castaneum.

BACKGROUND: Citrus sinensis, Allium sativum, Ocimum basilicum, Mentha spicata, Curcuma longa, Carica papaya, Andrographis paniculata, Azadirachta indica, Cymbopogon citratus and Acorus calamus were compared in the form of extracts of chloroform, hexane and aqueous media for their effectiveness in repelling Tribolium castaneum. Filter paper of 9 cm in diameter was cut into halves. Using a micro-pipette, each extract was applied to one half of the filter paper. The volume applied was kept constant at 0.6 mL per 30 cm2 . The filter paper was dried and reattached leaving a 0.5 cm gap between, in a Petri-plate. Ten adults of T. castaneum were introduced and the repellency was observed after 30, 60 and 180 min. RESULTS: The aqueous extract of C. papaya exhibited the highest repellency (82-97%) followed by that of garlic bulbs (aqueous extract) with 86.67% repellency. Further, gunny bags were sprayed separately using bottle sprayers with the two most effective extracts at a constant volume of 42 mL per 575 cm2 . These treated gunny bags were compared with malathion-treated gunny bags in the insect management unit. The observations were taken after 2, 8 and 24 h. Papaya leaf extract-treated gunny bags showed relative repellent activity similar to that of malathion-treated gunny bags after 24 h of treatment. Thus, gas chromatography-mass spectrometry analysis of aqueous extract of papaya leaves was performed. CONCLUSIONS: It can be concluded that extract of papaya leaves containing 2-methoxy-4-vinylphenol (8.47% peak area) can be used as a repellent biopesticide. © 2019 Society of Chemical Industry.

A Polyphasic Approach for Assessing Eco-System Connectivity Demonstrates that Perturbation Remodels Network Architecture in Soil Microcosms.

Network analysis was used to show changes in network attributes by analyzing the relations among the main soil microbial groups in a potted tomato soil inoculated with arbuscular mycorrhizal fungus, treated with low doses of Mentha spicata essential oil, or both, and then exposed to tenfold higher oil addition (stress pulse). Pretreatments were chosen since they can induce changes in the composition of the microbial community. Cellular phospholipid fatty acids (PLFAs) and the activity of six soil enzymes, mainly involved in the N-cycle were measured. Networks were constructed based on correlated changes in PLFA abundances. The values of all parameters were significantly different from those of random networks indicating modular architecture. Networks ranked from the lowest to highest modularity: control, non-pretreated and stressed, inoculated and stressed, oil treated and stressed, inoculated and treated with oil and stressed. The high values of network density and 1st/2nd eigenvalue ratio are related to arylamidase activity while N-acetyl-glucosaminidase, acid phosphomoesterase, and asparaginase activities related to high values of the clustering coefficient index. We concluded that modularity may be an efficient indicator of changes in the network of interactions among the members of the soil microbial community and the modular structure of the network may be related to the activity of specific enzymes. Communities that were stressed without a pretreatment were relatively resistant but prone to sudden transition towards instability, while oil or inoculation pretreatments gave networks which could be considered adaptable and susceptible to gradual change.

Essential oils as tick repellents on clothing.

Essential oils show promise as natural alternatives to synthetic tick repellents, but few studies have investigated their repellent efficacy in vivo or under field conditions. Here, blanket-drags and standardised walks were employed to evaluate tick acquisition by 1 m2 cotton blankets or cotton trousers, respectively, in woodland edge habitats of known high tick abundance. Blankets and trousers had been treated with one of 5% oregano, rosemary, spearmint or thyme oils, 20% DEET (N,N-diethyl-3-methylbenzamide) (positive control) or ethanol excipient-only (negative control). The number of ticks present on the blankets or trousers differed significantly between treatments: spearmint oil treatments resulted in significantly fewer ticks than the negative controls for both blankets and trousers and significantly fewer ticks were present on the oregano oil treated blankets. For ticks that did attach to the trousers, the rate of drop off within 3 min was significantly higher for trousers treated with spearmint oil or thyme oil than ethanol, oregano oil and rosemary oil. No reduction in repellence was detected over a 24 h period between treatment and testing. The results suggest that 5% oregano and spearmint oils exhibit potential as natural clothing repellents, with an effective equivalence to 20% DEET.

Effect of exposure to light emitted diode (LED) lights on essential oil composition of sweet mint plants.

Sweet mint plants (Mentha spicata) were separately exposed to blue, red and green light emitted diodes (LED) lights and natural sunlight as a control. Leaves were analyzed periodically for their essential oil composition using head space GCMS analysis. Control experiments showed that the main essential oil components were carvone (80%), limonene (17%) and other minor terpenes (3%). Exposed plants to green and red lights showed that their headspace essential oils were similar to that of the control plants. However, blue LED lights caused complete disappearance of carvone and limonene, which were never recovered. Disappearance of carvone and limonene were associated with the appearance of new chemicals at concentrations of 65 and 21% that were identified as carvone oxide and eucalyptol, respectively.

Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase.

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (-)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.

Chemical composition, antioxidant, antimicrobial and Antiproliferative activities of essential oil of Mentha spicata L. (Lamiaceae) from Algerian Saharan atlas.

BACKGROUND: Mentha spicata (M. spicata) is a member of Lamiaceae that spreads mainly in the temperate and sub-temperate zones of the world. It is considered as a rich source of essential oils, which is widely used in pharmaceutical industries and food production. The objectives of the current study were to evaluate chemical composition, antioxidant, antimicrobial and antiproliferative activities associated with the essential oil of M. spicata cultivated in Algerian Saharan Atlas. METHODS: The aerial parts of M. spicata were subjected to hydrodistillation to produce the oil. Chemical identification of the oil composition was conducted by GC and GC-MS analyses. The antioxidant activity of the hydrodistilled oil was studied using DPPH, ABTS radical scavenging and ferric-reducing power assay. Antimicrobial potential was characterized against two microorganisms, signifying Gram positive, and Gram negative bacteria, and one Candida species. The microdilution method was employed to determine the minimum inhibitory concentration (MIC). The oil's antiproliferative effects against three human tumor cell lines were also investigated using the MTT assay, and the toxic doses that yielded 50% reduction of cell viability (LD50) were reported. RESULTS: Chemical analysis of the essential oil composition revealed 44 unique compounds with oxygenated monoterpenes (67.2%), followed by monoterpene hydrocarbons (20.8%), as the most abundant chemical components. Essential oil of M. spicata demonstrated moderate antioxidant activities as well as moderate to weak antimicrobial activities with best susceptibility observed for Gram positive bacteria towards the oil. In addition, anticancer activities that are associated with the oil against three human cancer cell lines were observed with LD50 values of 324 µg/mL, 279 µg/mL, 975 µg/mL against T47D, HCT-116 and MCF-7 cell lines, respectively. CONCLUSION: The results suggest that M. spicata essential oil may have potential value as a bioactive oil, for nutraceutical and medical applications, with its antioxidant, antimicrobial and antiproliferative activities.

Weed interference with peppermint (Mentha x piperita L.) and spearmint (Mentha spicata L.) crops under different herbicide treatments: effects on biomass and essential oil yield.

BACKGROUND: 'Minor crops' such as spearmint and peppermint are high added value crops, despite the fact that their production area is comparably small worldwide. The main limiting factor in mint commercial cultivation is weed competition. Thus, field experiments were carried out to evaluate the effects of weed interference on growth, biomass and essential oil yield in peppermint and spearmint under different herbicide treatments. RESULTS: The application of pendimethalin and oxyfluorfen provided better control of annual weeds resulting in higher crop yield. Additionally, when treated with herbicides both crops were more competitive against annual weeds in the second year than in the first year. All pre-emergence herbicides increased biomass yield, since pendimethalin, linuron and oxyfluorfen reduced the density of annual weeds by 71-92%, 63-74% and 86-95%, respectively. Weed interference and herbicide application had no effect on essential oil content; however, a relatively strong impact on essential oil production per cultivated area unit was observed, mainly due to the adverse effect of weed interference on plant growth. CONCLUSION: Considering that pendimethalin and oxyfluorfen were effective against annual weeds in both spearmint and peppermint crops, these herbicides should be included in integrated weed management systems for better weed management in mint crops. © 2017 Society of Chemical Industry.

Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis.

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C10) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.