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1.
Microb Cell Fact ; 18(1): 160, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547812

RESUMO

BACKGROUND: Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction. RESULTS: α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L. CONCLUSIONS: An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.


Assuntos
Cicloexenos/metabolismo , Engenharia Metabólica , Monoterpenos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Vitis/enzimologia , Vitis/genética
2.
Pestic Biochem Physiol ; 158: 185-200, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378356

RESUMO

The present work describes the antimicrobial action of 25 monoterpenes (six hydrocarbons, five ketones, two aldehydes, six alcohols and six acetate analogues) against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus and antifungal activity against Aspergillus flavus. The antibacterial activity was evaluated by broth microdilution technique as a minimum inhibitory concentration (MIC) and the antifungal activity was performed by mycelia radial growth technique as the effective concentration causing 50% inhibition of the mycelial growth (EC50). The results showed that thymol and α-terpineol were the most potent against E. coli (MIC = 45 and 55 mg/L, respectively) and S. aureus (MIC = 135 and 225 mg/L, respectively). The results also showed that thymol displayed the maximum antifungal action against A. flavus with EC50 20 mg/L. Furthermore, the antioxidant activity was determined using N,N-dimethyl-1,4-phenylenediamine (DMPD) and the results showed that geraniol were the most potent compound (IC50 = 19 mg/L). Molecular docking studies indicated that the compounds displayed different binding interactions with the amino acid residues at the catalytic sites of N5-carboxyaminoimidazole synthetase and oxysterol binding protein Osh4 enzymes. Non-covalent interactions including van der Waals, hydrogen bonding as well as hydrophobic were observed between the compounds and the enzymes. A significant relationship was found between the docking score and the biological activity of the tested monoterpenes compared to the ceftriaxone and carbendazim as standard bactericide and fungicide, respectively. In silico ADMET properties were also performed and displayed potential for the development of promising antimicrobial agents. For these reasons, these compounds may be considered as potential ecofriendly alternatives in food preservation to delay or prevent the microbial infection and prolong the shelf life of food products.


Assuntos
Anti-Infecciosos/farmacologia , Antioxidantes/farmacologia , Monoterpenos/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Cicloexenos/química , Cicloexenos/farmacologia , Escherichia coli/efeitos dos fármacos , Hidrocarbonetos/química , Hidrocarbonetos/farmacologia , Testes de Sensibilidade Microbiana , Monoterpenos/química , Monoterpenos/farmacologia , Staphylococcus aureus/efeitos dos fármacos
3.
J Agric Food Chem ; 67(33): 9265-9276, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31361479

RESUMO

Fungal infections significantly alter the emissions of volatile organic compounds (VOCs) by plants, but the mechanisms for VOCs affecting fungal infections of plants remain largely unknown. Here, we found that infection by Botrytis cinerea upregulated linalool production by strawberries and fumigation with linalool was able to inhibit the infection of fruits by the fungus. Linalool treatment downregulated the expression of rate-limiting enzymes in the ergosterol biosynthesis pathway, and this reduced the ergosterol content in the fungi cell membrane and impaired membrane integrity. Linalool treatment also caused damage to mitochondrial membranes by collapsing mitochondrial membrane potential and also downregulated genes involved in adenosine triphosphate (ATP) production, resulting in a significant decrease in the ATP content. Linalool treatment increased the levels of reactive oxygen species (ROS), in response to which the treated fungal cells produced more of the ROS scavenger pyruvate. RNA-Seq and proteomic analysis data showed that linalool treatment slowed the rates of transcription and translation.


Assuntos
Botrytis/efeitos dos fármacos , Fragaria/metabolismo , Frutas/microbiologia , Monoterpenos/metabolismo , Doenças das Plantas/microbiologia , Compostos Orgânicos Voláteis/metabolismo , Trifosfato de Adenosina/metabolismo , Botrytis/crescimento & desenvolvimento , Fragaria/química , Fragaria/microbiologia , Frutas/química , Frutas/metabolismo , Interações Hospedeiro-Patógeno , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Monoterpenos/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Compostos Orgânicos Voláteis/farmacologia
4.
Bol. latinoam. Caribe plantas med. aromát ; 18(4): 435-443, jul. 2019. ilus, tab
Artigo em Inglês | LILACS | ID: biblio-1008258

RESUMO

Volatiles compounds are involved in defensive induction against insects, playing an important role in insect-plant interaction being induced by response to mechanical damage. However, they could decrease according to the domestication degree in cultivated plants. Currently, it has been established that secondary metabolites are reduced due to the domestication process in murtilla. Hence, the follow question emerges: Are volatile organic compounds induced by mechanical damage reduced in cultivated murtilla plants in relation to wild plants? Two cultivated ecotypes and their respective wild counterparts were sampled. Volatiles compounds were obtained using Porapak-Q columns and analyzed by gas chromatography. Results showed that compounds as 2-hexanone, α-pinene, 2-thujene, 3-thujene and 1,8- cineole were more abundant in wild plants exposed to a mechanical damage than cultivated plants. Hence, these compounds have been associated to induced defense, these results suggest that domestication reduced the induction of defensive volatiles in cultivated murtilla in response to mechanical damage.


Los compuestos volátiles están implicados en la defensa inducida contra insectos, desempeñando un papel importante en esta interacción. Sin embargo, estos compuestos podrían disminuir según el grado de domesticación. Actualmente, se ha reportado que algunos metabolitos secundarios son reducidos en plantas de murtilla domesticadas. Por lo tanto, surge la siguiente pregunta de investigación: ¿Los compuestos orgánicos volátiles inducidos por el daño mecánico son reducidos en plantas cultivadas de murtilla en comparación con plantas silvestres? Para dos ecotipos cultivados y sus respectivas contrapartes silvestres, los compuestos volátiles fueron capturados usando columnas de Porapak-Q y las muestras analizadas por cromatografía gaseosa. Los resultados mostraron que compuestos tales como 2- hexanona, α-pineno, 2-tujeno, 3-tujeno y 1,8-cineol fueron más abundantes en plantas silvestres expuestas a daño mecánico que en cultivadas. Debido a que estos compuestos se han asociado a defensa inducida, estos resultados sugieren que la domesticación reduce la inducción de volátiles en plantas cultivadas sometidas a daño mecánico.


Assuntos
Animais , Óleos Voláteis/metabolismo , Myrtaceae/metabolismo , Myrtaceae/microbiologia , Domesticação , Insetos/fisiologia , Sesquiterpenos/análise , Sesquiterpenos/metabolismo , Óleos Voláteis/química , Myrtaceae/química , Monoterpenos/análise , Monoterpenos/metabolismo , Meio Selvagem , Larva/fisiologia , Cromatografia Gasosa-Espectrometria de Massas
5.
BMC Plant Biol ; 19(1): 313, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307374

RESUMO

BACKGROUND: Essential oils (EOs) of Lavandula angustifolia, mainly consist of monoterpenoids and sesquiterpenoids, are of great commercial value. The multi-flower spiciform thyrse of lavender not only determines the output of EOs but also reflects an environmental adaption strategy. With the flower development and blossom in turn, the fluctuation of the volatile terpenoids displayed a regular change at each axis. However, the molecular mechanism underlying the regulation of volatile terpenoids during the process of flowering is poorly understood in lavender. Here, we combine metabolite and RNA-Seq analyses of flowers of five developmental stages at first- and second-axis (FFDSFSA) and initial flower bud (FB0) to discover the active terpenoid biosynthesis as well as flowering-related genes. RESULTS: A total of 56 mono- and sesquiterpenoids were identified in the EOs of L. angustifolia 'JX-2'. FB0' EO consists of 55 compounds and the two highest compounds, ß-trans-ocimene (20.57%) and (+)-R-limonene (17.00%), can get rid of 74.71 and 78.41% aphids in Y-tube olfactometer experiments, respectively. With sequential and successive blossoms, temporally regulated volatiles were linked to pollinator attraction in field and olfaction bioassays. In three characteristic compounds of FFDSFSA' EOs, linalyl acetate (72.73%) and lavandulyl acetate (72.09%) attracted more bees than linalool (45.35%). Many transcripts related to flowering time and volatile terpenoid metabolism expressed differently during the flower development. Similar metabolic and transcriptomic profiles were observed when florets from the two axes were maintained at the same maturity grade. Besides both compounds and differentially expressed genes were rich in FB0, most volatile compounds were significantly correlated with FB0-specific gene module. Most key regulators related to flowering and terpenoid metabolism were interconnected in the subnetwork of FB0-specific module, suggesting the cross-talk between the two biological processes to some degree. CONCLUSIONS: Characteristic compounds and gene expression profile of FB0 exhibit ecological value in pest control. The precise control of each-axis flowering and regular emissions at transcriptional and metabolic level are important to pollinators attraction for lavender. Our study sheds new light on lavender maximizes its fitness from "gene-volatile terpenoid-insect" three layers.


Assuntos
Flores/genética , Redes Reguladoras de Genes , Lavandula/genética , Terpenos/metabolismo , Acetatos/metabolismo , Animais , Ecossistema , Flores/crescimento & desenvolvimento , Flores/metabolismo , Perfilação da Expressão Gênica , Insetos , Lavandula/crescimento & desenvolvimento , Lavandula/metabolismo , Monoterpenos/metabolismo , Odorantes , Óleos Voláteis/metabolismo , Óleos Vegetais/metabolismo , Polinização , RNA de Plantas , Análise de Sequência de RNA
6.
J Agric Food Chem ; 67(25): 7087-7097, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31199132

RESUMO

Limonene, a cyclic monoterpene, is widely used in food and cosmetics industries as well as in agriculture. In the work described herein, employing a systematic optimization strategy, we constructed an efficient platform for producing limonene via the heterologous mevalonate pathway in Escherichia coli. By site-directed mutation of EfMvaS and tuning the initial translation of EfMvaE and EfMvaSA110G through ribosome binding site engineering, the upstream module for overproducing mevalonate was obtained. Expression of MmMK with ScPMK, ScPMD, and ScIDI under FAB80 promoter resulted in an efficient midstream module to produce 181.73 mg/L of limonene. Subsequently, coexpression of SlNPPS and MsLS in the downstream module led to a great improvement of limonene production to 694.61 mg/L. Finally, metabolically engineered strain ELIM78 produced 1.29 g/L of limonene in 84 h by fed-batch fermentation in a shake-flask. This is the first report on limonene biosynthesis in E. coli using neryl pyrophosphate synthase, which has promising potential for producing other monoterpenes.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Limoneno/metabolismo , Técnicas de Cultura Celular por Lotes , Fermentação , Engenharia Metabólica , Ácido Mevalônico/metabolismo , Monoterpenos/metabolismo
7.
Food Chem Toxicol ; 131: 110582, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31220535

RESUMO

Alcoholism is a serious addiction that can lead to various health complications such as liver fibrosis, steatosis, and cirrhosis. Carvacrol is present in many plant-based essential oils and used as a preservative in the food industry. In this study, we have investigated the hepatoprotective role of carvacrol against ethanol-induced liver toxicity in mice. To determine the effect of carvacrol on liver injury parameters, 5 doses of 50% ethanol (10 mL/kg body weight) were orally administered every 12 h for inducing the hepatotoxicity in experimental mice. Interestingly, carvacrol pre-treatment (50 and 100 mg/kg) reversed the ethanol-induced effects on liver function, antioxidant markers, matrix metalloproteinases activities, and histological changes. Moreover, carvacrol binds to the active pocket of cytochrome P450 (Cyt P450) and inhibits its expression. Thus, our finding suggests carvacrol can be used as an adjuvant for the amelioration of alcohol-induced hepatotoxicity.


Assuntos
Inibidores das Enzimas do Citocromo P-450/uso terapêutico , Fígado Gorduroso Alcoólico/prevenção & controle , Monoterpenos/uso terapêutico , Substâncias Protetoras/uso terapêutico , Animais , Autofagia/efeitos dos fármacos , Bebedeira , Domínio Catalítico , Inibidores das Enzimas do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado Gorduroso Alcoólico/patologia , Fígado/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Monoterpenos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/metabolismo , Ligação Proteica
8.
Food Chem ; 297: 124921, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253264

RESUMO

Plants conjugate monoterpenoids to sugars, rendering them non-volatile. Hydrolysis of these glycosidic precursors frees the volatile aroma compounds. Here, we profile intact monoterpenyl glycosides in six Vitis vinifera grape berry cultivars. Relative concentrations of twenty-six monoterpenyl glycosides, including nine new putatively identified compounds, were analyzed by UHPLC-QTOF MS/MS at three times during grape maturation (pre-véraison, véraison, and post-véraison). Total glycoside content reached a maximum in Muscat cultivars post-véraison but remained relatively constant in all other cultivars. Three types of monoterpenyl glycosides predominated in all samples: malonylated monoterpenol glucosides, monoterpenol hexose-pentoses, and monoterpendiol hexose-pentoses. The two Muscat cultivars were not differentiated at the earlier developmental stages but could be differentiated post-véraison. In contrast, similarities between Chardonnay and Pinot noir glycoside profiles developed post-véraison. Overall monoterpene glycoconjugation patterns may align with underlying genetic relationships among cultivars. By understanding monoterpene glycoconjugation in wine grapes, scientists and winemakers can better understand grape and wine aromas.


Assuntos
Monoterpenos/química , Vitis/química , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Frutas/química , Frutas/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Glicosilação , Monoterpenos/metabolismo , Análise de Componente Principal , Espectrometria de Massas em Tandem , Vitis/metabolismo
9.
J Sci Food Agric ; 99(12): 5606-5609, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31206173

RESUMO

BACKGROUND: There is a lack of information on the presence of volatile organic compounds including p-cymene in foods for dietary exposure assessment. p-Cymene is a monoterpene found in many plant species, which has been used as a flavouring agent in foods and also exhibits antioxidant and antimicrobial properties. While the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has no safety concerns at current levels of intake when used as a flavouring, the current study examines p-cymene levels in foods in general, not just from possible uses as a flavouring, as there could still be a potential health concern if there were high levels of exposure. RESULTS: 159 food composite samples from the 2014 Canadian Total Diet Study were analysed for p-cymene using a gas chromatographic-mass spectrometric method after headspace solid-phase microextraction. Concentrations of p-cymene in the composite samples of most food types were generally low, with a maximum level of 73.5 µg g-1 , detected in the composite sample of herbs and spices. Dietary exposures to p-cymene were estimated for different age groups of Canadian populations. CONCLUSIONS: Although p-cymene was detected in various foods, estimated dietary exposures to p-cymene for different age groups are well below the human intake threshold of toxicological concern established by JECFA of 1800 µg per person per day for structural class I flavours, which includes p-cymene. © 2019 Society of Chemical Industry.


Assuntos
Antioxidantes/análise , Exposição Dietética/análise , Monoterpenos/análise , Adolescente , Adulto , Idoso , Animais , Antioxidantes/metabolismo , Canadá , Criança , Pré-Escolar , Grão Comestível/química , Grão Comestível/metabolismo , Fast Foods/análise , Feminino , Aromatizantes/análise , Aromatizantes/metabolismo , Frutas/química , Frutas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lactente , Masculino , Carne/análise , Pessoa de Meia-Idade , Monoterpenos/metabolismo , Verduras/química , Verduras/metabolismo , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismo , Adulto Jovem
10.
Plant Sci ; 283: 256-265, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128696

RESUMO

Wounding is a key plant stress that results in a rapid, within seconds to a few minutes, release of ubiquitous stress volatiles and stored volatiles in species with storage structures. Understanding the timing and extent of wound-dependent volatile elicitation is needed to gain an insight into different emission controls, but real-time monitoring of plant emissions through wounding treatments has been hampered by the need to stop the measurements to perform the wounding, slow stabilization of gas flows upon chamber closure and smearing out the signal by large chambers and long sampling lines. We developed a novel leaf cutter that allows to rapidly perform highly precise leaf cuts within the leaf chamber. The cutter was fitted to the standard Walz GFS-3000 portable gas-exchange system leaf chamber and chamber exhaust air for analysis with a proton transfer reaction time-of-flight mass-spectrometer (PTR-TOF-MS) was taken right at the leaf chamber outlet. Wounding experiments in four species of contrasting leaf structure demonstrated significant species differences in timing, extent and blend of emitted volatiles, and showed unprecedently high emission rates of several stress volatiles and stored monoterpenes. In light of the rapid rise of release of de novo synthesized and stored volatiles, the results of this study suggest that past studies have underestimated the rate of elicitation and maximum emission rates of wound-dependent volatiles.


Assuntos
Folhas de Planta/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Botânica/instrumentação , Botânica/métodos , Monitorização Fisiológica/métodos , Monoterpenos/metabolismo , Phaseolus , Populus , Compostos Orgânicos Voláteis/análise , Zea mays
11.
Microb Cell Fact ; 18(1): 54, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30885220

RESUMO

BACKGROUND: Due to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose. RESULTS: Sixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines. CONCLUSION: Our results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.


Assuntos
Biocombustíveis/microbiologia , Lignina/metabolismo , Monoterpenos/metabolismo , Ustilaginales/metabolismo , Biomassa , Carotenoides/metabolismo , Fermentação
12.
J Chem Ecol ; 45(4): 339-347, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30854612

RESUMO

We report the identification of p-mentha-1,3-dien-8-ol, an unstable monoterpene alcohol, as a male-produced aggregation-sex pheromone component of the cerambycid beetle Paranoplium gracile (Leconte) (subfamily Cerambycinae, tribe Oemini), a species endemic to California. Headspace volatiles from live males contained a blend of nine cyclic terpenoids that were not detected in analogous samples from females. Volatiles produced by male Eudistenia costipennis Fall, also in the tribe Oemini, contained the same suite of nine compounds. Four compounds, dehydro-p-cymene, p-mentha-1,3-dien-8-ol, p,α,α-trimethylbenzyl alcohol, and an unidentified compound were found to elicit responses from antennae of P. gracile females in coupled gas chromatography-electroantennogram detection (GC-EAD) assays, whereas only p-mentha-1,3-dien-8-ol elicited responses from antennae of males. In field assays, p-mentha-1,3-dien-8-ol stabilized with the antioxidant butylated hydroxytoluene (BHT) attracted P. gracile of both sexes, indicating it functions as an aggregation-sex pheromone, as with other pheromones identified from its subfamily, the Cerambycinae. Adding four of the other compounds found in headspace samples to the dienol lure had no effect on attraction. Because of the instability of p-mentha-1,3-dien-8-ol, it seems likely that at least some of the compounds seen in the extracts of volatiles from both species are artefacts, rather than being components of the pheromone.


Assuntos
Álcoois/metabolismo , Besouros/metabolismo , Monoterpenos/metabolismo , Atrativos Sexuais/metabolismo , Álcoois/química , Animais , Antenas de Artrópodes/metabolismo , Cromatografia Gasosa/métodos , Besouros/classificação , Feminino , Masculino , Especificidade da Espécie
13.
Molecules ; 24(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889828

RESUMO

The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal-a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.


Assuntos
Aldeídos/metabolismo , Engenharia Metabólica/métodos , Monoterpenos/metabolismo , NADPH Desidrogenase/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Modelos Moleculares , Mutagênese/genética , Proteínas Mutantes/metabolismo , NADPH Desidrogenase/metabolismo , Oxirredução , Estereoisomerismo
14.
Int J Mol Sci ; 20(4)2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30791528

RESUMO

Bursaphelenchus xylophilus is fatal to the pine trees around the world. The production of the pine tree secondary metabolite gradually increases in response to a B. xylophilus infestation, via a stress reaction mechanism(s). α-pinene is needed to combat the early stages of B. xylophilus infection and colonization, and to counter its pathogenesis. Therefore, research is needed to characterize the underlying molecular response(s) of B. xylophilus to resist α-pinene. We examined the effects of different concentrations of α-pinene on the mortality and reproduction rate of B. xylophilus in vitro. The molecular response by which B. xylophilus resists α-pinene was examined via comparative transcriptomics of the nematode. Notably, B. xylophilus genes involved in detoxification, transport, and receptor activities were differentially expressed in response to two different concentrations of α-pinene compared with control. Our results contribute to our understanding of the molecular mechanisms by which B. xylophilus responds to monoterpenes in general, and the pathogenesis of B. xylophilus.


Assuntos
Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita , Monoterpenos/metabolismo , Nematoides , Pinus/genética , Pinus/metabolismo , Transcriptoma , Animais , Biologia Computacional/métodos , Resistência à Doença , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Anotação de Sequência Molecular , Monoterpenos/farmacologia , Nematoides/efeitos dos fármacos , Pinus/parasitologia , Reprodução/efeitos dos fármacos
15.
J Chem Ecol ; 45(2): 162-177, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30788656

RESUMO

The specificity of woody plant defense responses to different attacking herbivores is poorly known. We investigated the responses of black poplar (Populus nigra) to leaf feeding by three lepidopteran species (Lymantria dispar, Laothoe populi and Amata mogadorensis) and two leaf beetle species (Phratora vulgatissima and Chrysomela populi). Of the direct defenses monitored, increases in trypsin protease inhibitor activity and the salicinoid salicin were triggered by herbivore damage, but this was not herbivore-specific. Moreover, the majority of leaf salicinoid content was present constitutively and not induced by herbivory. On the other hand, volatile emission profiles did vary among herbivore species, especially between coleopterans and lepidopterans. Monoterpenes and sesquiterpenes were induced in damaged and adjacent undamaged leaves, while the emission of green leaf volatiles, aromatic and nitrogen-containing compounds (known to attract herbivore enemies) was restricted to damaged leaves. In conclusion, indirect defenses appear to show more specific responses to attacking herbivores than direct defenses in this woody plant.


Assuntos
Besouros/fisiologia , Lepidópteros/fisiologia , Populus/química , Compostos Orgânicos Voláteis/química , Animais , Álcoois Benzílicos/química , Álcoois Benzílicos/metabolismo , Besouros/crescimento & desenvolvimento , Glucosídeos/química , Glucosídeos/metabolismo , Herbivoria/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/fisiologia , Lepidópteros/crescimento & desenvolvimento , Monoterpenos/química , Monoterpenos/metabolismo , Reguladores de Crescimento de Planta/química , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/química , Folhas de Planta/metabolismo , Populus/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia , Compostos Orgânicos Voláteis/metabolismo , Compostos Orgânicos Voláteis/farmacologia
16.
J Chem Ecol ; 45(3): 227-240, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30796677

RESUMO

Two pine shoot beetles, Tomicus yunnanensis and Tomicus minor, are the most destructive pests of Pinus yunnanensis in southwestern China. We investigated behavioral responses within and between these two species during the shoot-feeding phase using walking bioassays. We also identified the pheromonal aspects of beetles by static solid phase microextraction (SPME) and hindgut extraction following interactive communication by gas chromatography-mass spectroscopy (GC-MS). Both species were significantly attracted by their own species and the same sex, and attraction was inhibited by exposure to additional beetles or to the hindgut extracts of beetles which had shown interaction. Female and male T. minor and T. yunnanensis hindguts contained 0.19, 0.09, 0.22, and 0.05 ng/individual of (-)-trans-verbenol, respectively; following interaction with additional beetles, this increased to 16.74-292.71 ng/individual in T. minor females. Mean concentration of verbenone detected in the hindguts of female/male individuals of T. minor and T. yunnanensis under natural conditions were 0.16, 0.06, 0.03, and 0.05 ng/individual, respectively, but these correspondingly increased to 5.90, 2.43, 0.06, and 0.19 ng/individual after exposure to additional insects. In T. yunnanensis, the amounts of detectable (-)-trans-verbenol and verbenone extracted from hindguts were lower than those from T. minor. The levels of cis-verbenol and (-)-trans-verbenol most attractive to walking T. yunnanensis and T. minor were 0.1 and 1.0 ng/µl, respectively. Verbenone was not attractive at any of the concentrations tested. The addition of verbenone to cis-verbenol or (-)-trans-verbenol reduced the attraction responses. We conclude that the (-)-trans-verbenol produced by these two pine shoot beetles is attractive at low concentrations and repellant at high concentrations, thereby fostering intraspecific competition. Verbenone is produced to prevent overcrowding via interspecific inhibition, and to expel beetles during shoot-feeding.


Assuntos
Comportamento Alimentar , Brotos de Planta , Gorgulhos/fisiologia , Animais , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Monoterpenos/metabolismo , Feromônios/metabolismo , Microextração em Fase Sólida , Especificidade da Espécie , Terpenos/metabolismo
17.
J Chem Ecol ; 45(4): 348-355, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30788655

RESUMO

Aggregation of the bean flower thrips, Megalurothrips sjostedti (Trybom) (Thysanoptera: Thripidae), has been observed on cowpea, Vigna unguiculata (L.) Walp. To understand the mechanism underpinning this behavior, we studied the responses of M. sjostedti to headspace volatiles from conspecifics in a four-arm olfactometer. Both male and female M. sjostedti were attracted to male, but not to female odor. Gas chromatography/mass spectrometry (GC/MS) analyses revealed the presence of two distinct compounds in male M. sjostedti headspace, namely (R)-lavandulyl 3-methylbutanoate (major compound) and (R)-lavandulol (minor compound); by contrast, both compounds were only present in trace amounts in female headspace collections. A behavioral assay using synthetic compounds showed that male M. sjostedti was attracted to both (R)-lavandulyl 3-methylbutanoate and (R)-lavandulol, while females responded only to (R)-lavandulyl 3-methylbutanoate. This is the first report of a male-produced aggregation pheromone in the genus Megalurothrips. The bean flower thrips is the primary pest of cowpea, which is widely grown in sub-Saharan Africa. The attraction of male and female M. sjostedti to these compounds offers an opportunity to develop ecologically sustainable management methods for M. sjostedti in Africa.


Assuntos
Atrativos Sexuais/metabolismo , Tisanópteros/metabolismo , Vigna/parasitologia , Animais , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Monoterpenos/metabolismo , Comportamento Sexual Animal , Tisanópteros/fisiologia
18.
J Tissue Viability ; 28(2): 94-99, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30792116

RESUMO

This study was undertaken to investigate the in vitro wound healing effects and the anti-inflammatory and antioxidant activities of terpinolene and α-phellandrene. The in vitro stimulatory effects on the proliferation and migration of fibroblasts were assessed using the scratch assay. The anti-inflammatory activity was evaluated using cell-based assays by investigating their influence on nitric oxide (NO), superoxide anion (O2•-), tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) production and using the TNF-α-induced nuclear factor kappa (NF-κB) assay. Antioxidant activity was determined by the ABTS cation radical scavenging capacity, ferric reducing/antioxidant potential (FRAP), and NO free radical scavenging assays. Terpinolene and α-phellandrene significantly increased the migration and proliferation of fibroblasts and suppressed the pro-inflammatory cytokines IL-6 and TNF-α in a dose-dependent manner. Terpinolene and α-phellandrene at a concentration of 100 µM significantly inhibited NO production (41.3 and 63.8%, respectively) in a macrophage cell-culture-based assay, and resulted in reductions in O2•- production of 82.1 ±â€¯3.5% and 70.6 ±â€¯4.3%, respectively. Moreover, these monoterpenes were verified to suppress NF-κB activity. In summary, terpinolene and α-phellandrene may contribute to broadening clinical options in the treatment of wounds by attenuating inflammation and oxidative stress in vitro.


Assuntos
Inflamação/fisiopatologia , Monoterpenos/metabolismo , Terpenos/metabolismo , Cicatrização/fisiologia , Análise de Variância , Humanos , Inflamação/metabolismo , Monoterpenos/análise , Estresse Oxidativo/fisiologia , Terpenos/análise
19.
Phytochemistry ; 159: 119-126, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30611871

RESUMO

The medicinal value of the monoterpenoid indole alkaloids (MIAs) such as 3',4'-anhydrovinblastine, as well as their chemical complexity have stimulated extensive efforts to understand the biochemical and molecular pathways involved in their biosynthesis in plants such as Catharanthus roseus, Rawvolfia serpentina and others. Ethyl methane sulphonate (EMS) mutagenesis has been used successfully together with simple MIA thin layer chromatography screening to identify C. roseus mutants with altered MIA profiles. This study describes the isolation of very low iridoid and MIA containing C. roseus mutant (M2-1582) that accumulates MIAs when the plant is provided with secologanin by feeding mutant roots or by grafting the mutant scion onto wild type roots. The observed low iridoid and MIA content was correlated with lowered expression of BIS1/BIS2 transcription factors and several genes involved in secologanin biosynthesis that are expressed in internal phloem parenchyma cells of leaves. When exogenous secologanin was applied to the roots of the mutant plant, secologanin levels rose more than 13-fold, while two major MIAs catharanthine and vindoline rose more than 8- and 4- fold, respectively. Grafting the mutant on WT stocks led to 27-, 11- and 27-fold increases in secologanin, catharanthine and vindoline, respectively in leaves of the scion one week after graft initiation. Other minor MIAs (serpentine, anhydrovinblastine, vindolidine, deacetylvindoline, tabersonine and 16-methoxytabersonine) that were not detected in the mutant, became detectable in leaves of the scion. These results provide strong evidence for a secologanin transport mechanism that mobilizes this iridoid between different plant organs in C. roseus and that secologanin transport to the mutant across the graft union permits the formation of MIAs in leaves of the mutant.


Assuntos
Catharanthus/metabolismo , Alcaloides Indólicos/metabolismo , Glucosídeos Iridoides/metabolismo , Monoterpenos/metabolismo , Mutação , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Fatores de Transcrição/metabolismo , Transporte Biológico , Catharanthus/genética , Cromatografia em Camada Delgada , Regulação para Baixo
20.
Z Naturforsch C ; 74(3-4): 63-70, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30645192

RESUMO

Biocatalysis has developed enormously in the last decade and now offers solutions for the sustainable production of chiral and highly functionalised asset molecules. Products generated by enzymatic transformations are already being used in the food, feed, chemical, pharmaceutical and cosmetic industry, and the accessible compound panoply is expected to expand even further. In particular, the combination of stereo-selective enzymes in linear cascade reactions is an elegant strategy toward enantiomeric pure compounds, as it reduces the number of isolation and purification steps and avoids accumulation of potentially unstable intermediates. Here, we present the set-up of an enzyme cascade to selectively convert citral to (-)-iso-isopulegol by combining an ene reductase and a squalene hopene cyclase. In the initial reaction step, the ene reductase YqjM from Bacillus subtilis selectively transforms citral to (S)-citronellal, which is subsequently cyclised exclusively to (-)-iso-isopulegol by a mutant of the squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). With this approach, we can convert citral to an enantiopure precursor for isomenthol derivatives.


Assuntos
Alicyclobacillus/enzimologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , FMN Redutase/genética , Transferases Intramoleculares/genética , Terpenos/síntese química , Aldeídos/química , Aldeídos/metabolismo , Alicyclobacillus/genética , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Clonagem Molecular , Ciclização , Escherichia coli/enzimologia , Escherichia coli/genética , FMN Redutase/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Microbiologia Industrial/métodos , Transferases Intramoleculares/metabolismo , Cinética , Monoterpenos/síntese química , Monoterpenos/química , Monoterpenos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Terpenos/metabolismo
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