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1.
Phytochemistry ; 166: 112062, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31299395

RESUMO

Tripterygium wilfordii Hook. f. is a perennial woody vine member of the Celastraceae family. As a traditional Chinese medicine, it contains complex chemical components and exerts various pharmacological activities. In the present study, we identified a glucosyltransferase, TwUGT1, that can catalyze the synthesis of an abietane-type diterpene glucoside, namely, triptophenolide14-O-beta-D-glucopyranoside, and investigated the pharmacological activity of triptophenolide glucoside in diverse cancer cells. Triptophenolide glucoside exhibited significant inhibitory effects on U87-MG, U251, C6, MCF-7, HeLa, K562, and RBL-2H3 cells as determined by pharmacological analysis. The triptophenolide glucoside content of T. wilfordii was analyzed using Agilent Technologies 6490 Triple Quad LC/MS. The glucosyltransferase TwUGT1 belongs to subfamily 88 and group E in family 1. Molecular docking and site-directed mutagenesis of TwUGT1 revealed that the His30, Asp132, Phe134, Thr154, Ala370, Leu376, Gly382, His387, Glu395 and Gln412 residues play crucial roles in the catalytic activity of triptophenolide 14-O-glucosyltransferase. In addition, TwUGT1 was also capable of glucosylating phenolic hydroxyl groups, such as those in liquiritigenin, pinocembrin, 4-methylumbelliferone, phloretin, and rhapontigenin.


Assuntos
Biocatálise , Diterpenos/química , Diterpenos/metabolismo , Glucosídeos/química , Glucosiltransferases/metabolismo , Tripterygium/química , Glucosiltransferases/química , Simulação de Acoplamento Molecular , Conformação Proteica
2.
Phytochemistry ; 166: 112057, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31306913

RESUMO

Rice (Oryza sativa) leaves accumulate phytoalexins in response to pathogen attack. The major phytoalexins in rice are diterpenoids such as oryzalexins, momilactones, and phytocassanes. We measured the amount of oryzalexin A in leaves irradiated by UV light, treated with jasmonic acid, or inoculated with conidia of Bipolaris oryzae in the japonica cultivar Nipponbare and the indica cultivar Kasalath. Nipponbare leaves accumulated oryzalexin A at a high concentration, but Kasalath leaves did not. The locus responsible for this difference was mapped using backcrossed inbred lines and chromosome substitution lines. A region on Chr. 12 containing the KSL10 gene was responsible for the deficiency in oryzalexin A in the Kasalath cultivar. The amount of KSL10 transcript increased in Nipponbare leaves but not in Kasalath leaves in response to UV light irradiation, indicating that the suppressed expression of KSL10 caused the deficiency of oryzalexin A in Kasalath. We analyzed oryzalexin A accumulation in UV light-irradiated leaves of cultivars in the world rice core collection. There were cultivars that accumulated oryzalexin A and those that did not, and both of these chemotypes were found in japonica and indica subspecies. Furthermore, these chemotypes were found in the wild rice species Oryza rufipogon. The phylogenetic relationship of KSL10 sequences was not correlated to oryzalexin A chemotypes. These findings suggested that the biosynthesis of oryzalexin A was acquired by a common ancestor of O. rufipogon and was lost multiple times during the evolutionary process.


Assuntos
Diterpenos/química , Diterpenos/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Cruzamento
3.
Plant Sci ; 285: 184-192, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203883

RESUMO

Tripterygium wilfordii is known to contain various types of bioactive diterpenoids that exhibit many remarkable activities. Many studies have recently been targeted toward the elucidation of the diterpenoids biosynthetic pathways in attempts to obtain these compounds with a view to solving the dilemma of low yield in plants. However, the short-chain prenyltransferases (SC-PTSs) responsible for the formation of geranylgeranyl diphosphate (GGPP), a crucial precursor for synthesizing the skeleton structures of diterpenoids, have not been characterized in depth. Here, T. wilfordii transcriptome data were used to identify eight putative GGPPSs, including two small subunits of geranyl diphosphate synthase (GPPS.SSU). Of them, GGPPS1, GGPPS7, GGPPS8, GPPS.SSU II and GPPS.SSU were translocated mainly into chloroplasts, and GGPPS8 exhibited the optimal catalytic efficiency with respect to catalyzing the formation of GGPP. In addition, the expression pattern of GGPPS8 was similar to that of downstream terpene synthase genes that are directly correlated with triptolide production in roots, indicating that GGPPS8 was most likely to participate in triptolide biosynthesis in roots among the studied enzymes. GPPS.SSU was inactive alone but interacted with GGPPS1, GGPPS7 and GGPPS8 to change the product from GGPP to GPP. These findings implicate that these candidate genes can be regulated to shift the metabolic flux toward diterpenoid formation, increasing the yields of bioactive diterpenoids in plants.


Assuntos
Diterpenos/metabolismo , Farnesiltranstransferase/metabolismo , Proteínas de Plantas/metabolismo , Tripterygium/metabolismo , Clonagem Molecular , Filogenia , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Tripterygium/enzimologia , Tripterygium/genética , Técnicas do Sistema de Duplo-Híbrido
4.
J Agric Food Chem ; 67(23): 6523-6531, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31117507

RESUMO

Identification of diterpene synthase-encoding genes together with synthetic biology technology offers an opportunity for the biosynthesis of cis-abienol. The methylerythritol phosphate (MEP) and the mevalonate (MVA) pathways were both engineered for cis-abienol production in Escherichia coli, which improved the cis-abienol yield by approximately 7-fold and 31-fold, respectively, compared to the yield obtained by overexpression of the MEP pathway alone or the original MEP pathway. Furthermore, systematic optimization of cis-abienol biosynthesis was performed, such as diterpene synthase screening and two-phase cultivation. The combination of bifunctional class I/II cis-abienol synthase from Abies balsamea ( AbCAS) and class II abienol synthase from Salvia sclarea ( SsTPS2) was found to be the most effective. By using isopropyl myristate as a solvent in two-phase cultivation, cis-abienol production reached 634.7 mg/L in a fed-batch bioreactor. This work shows the possibility of E. coli utilizing glucose as a carbon source for cis-abienol biosynthesis through a modified pathway.


Assuntos
Diterpenos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Ácido Mevalônico/metabolismo , Naftóis/metabolismo , Abies/enzimologia , Vias Biossintéticas , Diterpenos/química , Fermentação , Glucose/metabolismo , Naftóis/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Salvia/enzimologia
5.
Med Sci Monit ; 25: 2935-2942, 2019 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-31005958

RESUMO

BACKGROUND Thyroid cancer causes considerable mortality and morbidity across the globe. Owing to the unavailability of biomarkers and the adverse effects of existing drugs, there is an urgent need to develop efficient chemotherapy for the treatment of thyroid cancers. Plants have served as exceptional source of drugs for the treatment of lethal diseases. The purpose of this study was to evaluate the anticancer effects of ferruginol against thyroid cancer cells. MATERIAL AND METHODS We monitored the cell proliferation rate using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected using 4',6-diamidino-2-phenylindole (DAPI), acridine orange/ethidium bromide (AO/EB), and annexin V/propidium iodide (PI) staining. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels were examined by fluorescence microscopy. Protein expressed was examined by western blotting. RESULTS We found that ferruginol exerted potent antiproliferative action against thyroid cancer cells, and an IC50 of 12 µM was observed for ferruginol against the MDA-T32 cell line. The toxic effects of ferruginol were less pronounced against normal cells. The anticancer effects of ferruginol were likely due to the induction of apoptosis which was also associated with upregulation of Bax and downregulation of Bcl-2. Ferruginol also caused ROS mediated alterations in the MMP of MDA-T32 cells. In MDA-T32 cells, ferruginol might also block the MAPK and PI3K/AKT signaling pathway, which is believed to be an important therapeutic target of anticancer drugs. CONCLUSIONS In conclusion, in view of the results of this study, it might be suggested that ferruginol might serve as an essential lead molecule for the treatment of thyroid cancer provided further in-depth studies especially studying ferruginol toxicological as well as in vivo studies are needed.


Assuntos
Diterpenos de Abietano/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , China , Diterpenos/metabolismo , Diterpenos/farmacologia , Diterpenos de Abietano/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
6.
Int J Med Mushrooms ; 21(4): 401-411, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31002635

RESUMO

Recently, erinacine A-enriched Hericium erinaceus (EAHE) mycelia have demonstrated therapeutic efficacy in animal models of neurodegenerative disease, including Alzheimer and Parkinson disease. Despite promising results from animal models, there have been no reports on its toxicity after long-term consumption. Hence, the present study was designed to evaluate the safety of EAHE mycelia through a 13-week subchronic rodent feeding study. Following 13 weeks of EAHE mycelia feeding at dosages of 0, 875, 1750, and 2625 mg/kg body weight in both male and female Sprague-Dawley rats, findings revealed neither any mortalities nor noticeable toxicological effects in all the rats during the investigation period. Physiological parameters including body weight and feed consumption patterns were unaffected by EAHE mycelia administration. The hematological and biochemical parameters as well as histopathological studies revealed no significant differences between the treatment and control groups. Conclusively, the obtained results suggested that EAHE mycelia could be relatively unharmful when used over an extended period, supporting its safe use in food preparation.


Assuntos
Basidiomycota/química , Diterpenos/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Masculino , Micélio/química , Nível de Efeito Adverso não Observado , Ratos , Ratos Sprague-Dawley , Testes de Toxicidade
7.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(2): 174-179, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30975284

RESUMO

Objective To develop high-sensitivity and high-specificity monoclonal antibody against tiamulin (TML). Methods Using oximation and carbodiimide method, TML was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare the immunogen TML-BSA and coating antigen TML-OVA, respectively. BALB/c mice were immunized with TML-BSA. The anti-TML monoclonal antibody hybridoma cell lines were screened by indirect ELISA after cell fusion. The ascites were prepared by in vivo induction method. The monoclonal antibody was purified from ascites by caprylic acid-ammonium sulfate method and identified by SDS-PAGE. Results The hybridoma cell lines of 3B3 and 4A7 were successfully obtained. Their titers of cell supernatant were 1:1600 and 1:6400, respectively. After purification, the titer of monoclonal antibody 4A7 was 5.12×105. The half inhibitory concentration (IC50) of 4A7 was 0.049 ng/mL. The affinity constant Kaff of 4A7 was 1.47×109 L/mol. The cross-reaction rate of 4A7 was 2.7% with analog retapamulin, whereas no cross-reaction was detected with other antibiotics such as valnemulin and fleroxacin. Conclusion Anti-TML monoclonal antibody has been successfully developed.


Assuntos
Anticorpos Monoclonais , Animais , Antibacterianos/metabolismo , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Reações Cruzadas , Diterpenos/metabolismo , Ensaio de Imunoadsorção Enzimática , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C
8.
Chem Biodivers ; 16(6): e1900032, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30957403

RESUMO

The inhibition of carbohydrate-hydrolyzing enzymes in human digestive organs is crucial in controlling blood sugar levels, which is important in treating type 2 diabetes. In the current study, pahangensin A (1), a bis-labdanic diterpene characterized previously in the rhizomes of Alpinia pahangensis Ridl., was identified as an active dual inhibitor for α-amylase (IC50 =114.80 µm) and α-glucosidase (IC50 =153.87 µm). This is the first report on the dual α-amylase and α-glucosidase inhibitory activities of a bis-labdanic diterpene. The Lineweaver-Burk plots of compound 1 indicate that it is a mixed-type inhibitor with regard to both enzymes. Based on molecular docking studies, compound 1 docked in a non-active site of both enzymes. The dual inhibitory activity of compound 1 makes it a suitable natural alternative in the treatment of type 2 diabetes.


Assuntos
Alpinia/química , Diterpenos/química , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismo , Alpinia/metabolismo , Sítios de Ligação , Domínio Catalítico , Diterpenos/isolamento & purificação , Diterpenos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Cinética , Simulação de Acoplamento Molecular , Extratos Vegetais/química , alfa-Amilases/antagonistas & inibidores , alfa-Glucosidases/química
9.
Microb Cell Fact ; 18(1): 73, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31018856

RESUMO

BACKGROUND: Diterpenoids are a large class of natural products with complex structures and broad commercial applications as food additives, important medicines, and fragrances. However, their low abundance in plants and high structural complexity limit their applications. Therefore, it is important to create an efficient diterpenoid-producing yeast cell factory of the production of various high-value diterpenoid compounds in a cost-effective manner RESULTS: In this study, 13R-manoyl oxide (13R-MO; 2.31 mg/L) was produced by expressing CfTPS2 and CfTPS3 from Coleus forskohlii in Saccharomyces cerevisiae. The 13R-MO titer was increased by 142-fold to 328.15 mg/L via the stepwise metabolic engineering of the original strain, including the overexpression of the rate-limiting genes (tHMG1 and ERG20) of the mevalonate pathway, transcription and protein level regulation of ERG9, Bts1p and Erg20F96Cp fusion, and the overexpression of tCfTPS2 and tCfTPS3 (excision of the N-terminal plastid transit peptide sequences of CfTPS2 and CfTPS3). The final titer of 13R-MO reached up to 3 g/L by fed-batch fermentation in a 5 L bioreactor. CONCLUSIONS: In this study, an efficient 13R-MO yeast cell factory was constructed, which achieved the de novo production of 3 g/L of 13R-MO from glucose. To the best of our knowledge, this is the highest 13R-MO titer reported to date. Furthermore, the metabolic engineering strategies presented here could be used to produce other valuable diterpenoid compounds in yeast.


Assuntos
Diterpenos/metabolismo , Engenharia Metabólica , Saccharomyces cerevisiae/metabolismo , Fermentação , Ácido Mevalônico/metabolismo , Saccharomyces cerevisiae/genética
10.
BMC Plant Biol ; 19(1): 114, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30909879

RESUMO

BACKGROUND: Horehound (Marrubium vulgare) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, MvCPS1 and MvELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds. RESULTS: Mining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential MvCPS1 and MvELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta, high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare. CONCLUSIONS: In a sequential reaction with the diterpene synthase pair MvCPS1 and MvELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/metabolismo , Marrubium/metabolismo , Proteínas de Plantas/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Flores/genética , Flores/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hidroxilação , Isomerismo , Marrubium/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Tabaco/genética
11.
Plant Mol Biol ; 100(1-2): 83-93, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847712

RESUMO

KEY MESSAGE: The novel AP2/ERF transcription factor SmERF128 positively regulates diterpenoid tanshinone biosynthesis by activating the expression of SmCPS1, SmKSL1, and SmCYP76AH1 in Salvia miltiorrhiza. Certain members of the APETALA2/ethylene-responsive factor (AP2/ERF) family regulate plant secondary metabolism. Although it is clearly documented that AP2/ERF transcription factors (TFs) are involved in sesquiterpenoid biosynthesis, the regulation of diterpenoid biosynthesis by AP2/ERF TFs remains elusive. Here, we report that the novel AP2/ERF TF SmERF128 positively regulates diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza. Overexpression of SmERF128 increased the expression levels of copalyl diphosphate synthase 1 (SmCPS1), kaurene synthase-like 1 (SmKSL1) and cytochrome P450 monooxygenase 76AH1 (SmCYP76AH1), whereas their expression levels were decreased when SmERF128 was silenced. Accordingly, the content of tanshinone was reduced in SmERF128 RNA interference (RNAi) hairy roots and dramatically increased in SmERF128 overexpression hairy roots, as demonstrated through Ultra Performance Liquid Chromatography (UPLC) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. Furthermore, SmERF128 activated the expression of SmCPS1, SmKSL1, and SmCYP76AH1 by binding to the GCC box, and to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs within their promoters during in vivo and in vitro assays. Our findings not only reveal the molecular basis of how the AP2/ERF transcription factor SmERF128 regulates diterpenoid biosynthesis, but also provide useful information for improving tanshinone production through genetic engineering.


Assuntos
Diterpenos/metabolismo , Proteínas de Plantas/metabolismo , Salvia miltiorrhiza/metabolismo , Fatores de Transcrição/metabolismo , Diterpenos/isolamento & purificação , Diterpenos de Abietano/biossíntese , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Motivos de Nucleotídeos/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , Interferência de RNA , Salvia miltiorrhiza/genética , Fatores de Transcrição/isolamento & purificação
12.
Phytochemistry ; 161: 149-162, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30733060

RESUMO

In plant terpene biosynthesis, oxidation of the hydrocarbon backbone produced by terpene synthases is typically carried out by cytochrome P450 oxygenases (CYPs). The modifications introduced by CYPs include hydroxylations, sequential oxidations at one position and ring rearrangements and closures. These reactions significantly expand the structural diversity of terpenoids, but also provide anchoring points for further decorations by various transferases. In recent years, there has been a significant increase in reports of CYPs involved in plant terpene pathways. Plant diterpenes represent an important class of metabolites that includes hormones and a number of industrially relevant compounds such as pharmaceutical, aroma or food ingredients. In this review, we provide a comprehensive survey on CYPs reported to be involved in plant diterpene biosynthesis to date. A phylogenetic analysis showed that only few CYP clans are represented in diterpene biosynthesis, namely CYP71, CYP85 and CYP72. Remarkably few CYP families and subfamilies within those clans are involved, indicating specific expansion of these clades in plant diterpene biosynthesis. Nonetheless, the evolutionary trajectory of CYPs of specialized diterpene biosynthesis is diverse. Some are recently derived from gibberellin biosynthesis, while others have a more ancient history with recent expansions in specific plant families. Among diterpenoids, labdane-related diterpenoids represent a dominant class. The availability of CYPs from diverse plant species able to catalyze oxidations in specific regions of the labdane-related backbones provides opportunities for combinatorial biosynthesis to produce novel diterpene compounds that can be screened for biological activities of interest.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/metabolismo , Plantas/metabolismo , Diterpenos/química , Filogenia , Plantas/química
13.
Molecules ; 24(4)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769848

RESUMO

Extracts of bled resin from Azorella compacta, of the Azorelloideae family from the Andes (>4000 m), were analyzed by gas chromatography-mass spectrometry. The mass spectra of the dominant compounds of the resin and its hydrogenation products were documented. The most abundant compounds were oxygenated diterpenoids, namely mulinadien-20-oic (Δ11,13 and Δ11,14) acids, azorell-13-en-20-oic acid, 13α,14ß-dihydroxymulin-11-en-20-oic acid, and azorellanol, with a group of azorellenes and mulinadienes. The mass spectra of the novel diterpenoid hydrocarbons with the azorellane and mulinane skeletons were also presented. This study documents the molecular diversity of these diterpenoid classes, and could be of great utility for future organic geochemical, environmental, archeological, pharmaceutical, and forensic chemistry studies.


Assuntos
Apiaceae/química , Diterpenos/química , Altitude , Apiaceae/crescimento & desenvolvimento , Apiaceae/metabolismo , Biomarcadores/química , Diterpenos/isolamento & purificação , Diterpenos/metabolismo
14.
BMC Plant Biol ; 19(1): 82, 2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782123

RESUMO

BACKGROUND: There exist differences in morphological traits and phytochemical compositions between field- and mountain-cultivated Panax ginseng (FCG and MCG), which might be attributed to variations of terpenoids metabolism adapting to different growth conditions. The present work aims to uncover these variations. RESULTS: Among 26,648 differentially expressed genes, 496 genes distributed in seven dominant terpenoids pathways were identified. Diterpenoids and triterpenoids biosynthesis genes were significantly higher-expressed in FCG root. Conversely, biosynthesis of carotenoids was significantly more active in MCG root. Additionally, terpenoids backbones, monoterpenoids, sesquiterpenoids, and terpenoid-quinones biosyntheses were neither obviously inclined. Our determination also revealed that there were more gibberellins and steroids accumulated in FCG root which might be responsible for its quick vegetative growth, and enriched abscisic acid and germacrenes as well as protopanaxatriol-type ginsenosides might be major causes of enhanced stress-resistance in MCG root. CONCLUSIONS: The study firstly provided an overview of terpenoids metabolism in roots of FCG and MCG in elucidating the underlying mechanisms for their different morphological appearances and phytochemical compositions.


Assuntos
Panax/metabolismo , Raízes de Plantas/metabolismo , Terpenos/metabolismo , Produção Agrícola , Diterpenos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas
15.
Appl Microbiol Biotechnol ; 103(4): 1599-1616, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30613899

RESUMO

Indole-diterpenes (IDTs) such as the aflatrems, janthitrems, lolitrems, paspalitrems, penitrems, shearinines, sulpinines, and terpendoles are biogenetically related but structurally varied tremorgenic and neurotoxic mycotoxins produced by fungi. All these metabolites derive from the biosynthetic intermediate paspaline, a frequently occurring IDT on its own right. In this comprehensive review, we highlight the similarities and differences of the IDT biosynthetic pathways that lead to the generation of the main paspaline-derived IDT subgroups. We survey the taxonomic distribution and the regulation of IDT production in various fungi and compare the organization of the known IDT biosynthetic gene clusters. A detailed assessment of the highly diverse biological activities of these mycotoxins leads us to emphasize the significant losses that paspaline-derived IDTs cause in agriculture, and compels us to warn about the various hazards they represent towards human and livestock health. Conversely, we also describe the potential utility of these versatile molecules as lead compounds for pharmaceutical drug discovery, and examine the prospects for their industrial scale manufacture in genetically manipulated IDT producers or domesticated host microorganisms in synthetic biological production systems.


Assuntos
Diterpenos/toxicidade , Fungos/metabolismo , Indóis/toxicidade , Micotoxinas/toxicidade , Neurotoxinas/toxicidade , Tremor/induzido quimicamente , Vias Biossintéticas/genética , Diterpenos/metabolismo , Fungos/genética , Humanos , Indóis/metabolismo , Micotoxinas/metabolismo , Neurotoxinas/metabolismo
16.
Braz J Microbiol ; 50(1): 79-84, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30645731

RESUMO

Sclareol is an important intermediate for ambroxide synthesis industries. Hyphozyma roseonigra ATCC 20624 was the only reported strain capable of degrading sclareol to the main product of sclareol glycol, which is the precursor of ambroxide. To date, knowledge is lacking about the effects of sclareol on cells and the proteins involved in sclareol metabolism. Comparative proteomic analyses were conducted on the strain H. roseonigra ATCC 20624 by using sclareol or glucose as the sole carbon source. A total of 79 upregulated protein spots with a > 2.0-fold difference in abundance on 2-D gels under sclareol stress conditions were collected for further identification. Seventy spots were successfully identified and finally integrated into 30 proteins. The upregulated proteins under sclareol stress are involved in carbon metabolism and nitrogen metabolism, and replication, transcription, and translation processes. Eighteen upregulated spots were identified as aldehyde dehydrogenases, which indicating that aldehyde dehydrogenases might play an important role in sclareol metabolism. Overall, this study may lay the fundamentals for further cell engineering to improve sclareol glycol production.


Assuntos
Ascomicetos/metabolismo , Diterpenos/metabolismo , Proteínas Fúngicas/genética , Ascomicetos/química , Ascomicetos/genética , Carbono/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Proteômica
17.
Plant Physiol ; 179(3): 1001-1012, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30643014

RESUMO

Transcription activator-like effectors (TALEs) are bacterial Type-III effector proteins from phytopathogenic Xanthomonas species that act as transcription factors in plants. The modular DNA-binding domain of TALEs can be reprogrammed to target nearly any DNA sequence. Here, we designed and optimized a two-component AND-gate system for synthetic circuits in plants based on TALEs. In this system, named split-TALE (sTALE), the TALE DNA binding domain and the transcription activation domain are separated and each fused to protein interacting domains. Physical interaction of interacting domains leads to TALE-reconstitution and can be monitored by reporter gene induction. This setup was used for optimization of the sTALE scaffolds, which result in an AND-gate system with an improved signal-to-noise ratio. We also provide a toolkit of ready-to-use vectors and single modules compatible with Golden Gate cloning and MoClo syntax. In addition to its implementation in synthetic regulatory circuits, the sTALE system allows the analysis of protein-protein interactions in planta.


Assuntos
Plantas/genética , Biologia Sintética/métodos , Efetores Semelhantes a Ativadores de Transcrição/fisiologia , Xanthomonas/genética , Diterpenos/metabolismo , Engenharia Genética/métodos , Naftóis/metabolismo , Mapeamento de Interação de Proteínas , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismo
18.
Phytochemistry ; 156: 151-158, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30296708

RESUMO

Seven undescribed hydroxylated cassane-type furanoditerpenoids were isolated from pupal cases formed from the secretion/excretion of the larvae of the wild bruchid seed beetle Sulcobruchus sauteri in infested Caesalpinia decapetala seeds, and their structures were elucidated by interpreting their spectra. The hydroxylated furanoditerpenoids found in the pupal cases were not present in the seeds of the host plant. Caesalacetal and caesaljapin obtained from the intact seeds exhibited larvicidal activity against the larvae of Aedes albopictus, while the hydroxylated furanoditerpenoids isolated from the pupal cases were inactive. The larvae of S. sauteri are proposed to detoxify larvicidal diterpenoids that occur in the seeds of the host plant by regiospecific hydroxylation.


Assuntos
Caesalpinia/metabolismo , Diterpenos/metabolismo , Furanos/metabolismo , Pupa/metabolismo , Sementes/metabolismo , Animais , Caesalpinia/química , Besouros , Diterpenos/química , Diterpenos/isolamento & purificação , Furanos/química , Furanos/isolamento & purificação , Hidroxilação , Conformação Molecular , Pupa/química , Sementes/química
19.
Gene ; 679: 195-201, 2018 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-30194986

RESUMO

The aim of this study was to verify the effects of TwIDI (GenBank: KT279355.1) on triptolide and celastrol accumulation in the biosynthesis of terpenoids in Tripterygium wilfordii and the regulation of the expression of related genes in the triptolide and celastrol biosynthesis pathway. After bioinformatics analysis of TwIDI, we cloned the full-length CDS and a specific 398 bp fragment to construct overexpression and RNAi vectors, respectively. The specific amplification of hygromycin and kanamycin resistance gene fragments confirmed that the expression vectors had been successfully delivered into Tripterygium wilfordii suspension cells. qRT-PCR was used to detect the expression of TwIDI and related genes in the triptolide and celastrol biosynthesis pathway. The expression of TwIDI was increased to 157% of the control group (empty vector) in the overexpression group, and was reduced to 71% of the control group in the RNAi group. Notably, the expression of other genes in the triptolide and celastrol biosynthesis pathway also showed differences. For example, TwMCS was reduced to 62% of the control when TwIDI was overexpressed and increased to 188% in the RNAi group. The expression of TwDXS did not change significantly both during TwIDI overexpression and RNAi group. The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC, revealing that the contents of triptolide and celastrol were increased 1.36- and 1.20-fold over the control group in the overexpression group, and decreased to 0.16 and 0.36 of the control group in the RNAi group. Based on these findings, the effect on the accumulation of active terpenoids in Tripterygium wilfordii and the feedback regulation of genes in the triptolide and celastrol biosynthesis pathway was verified through TwIDI overexpression and RNAi experiments.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/genética , Diterpenos/metabolismo , Fenantrenos/metabolismo , Tripterygium/genética , Triterpenos/metabolismo , Vias Biossintéticas , Regulação para Baixo , Compostos de Epóxi/metabolismo , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/genética , Interferência de RNA , Tripterygium/metabolismo , Regulação para Cima
20.
Mar Drugs ; 16(8)2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096866

RESUMO

Five new cembranoid-related diterpenoids, namely, flexibilisins D and E (1 and 2), secoflexibilisolides A and B (3 and 4), and flexibilisolide H (5), along with nine known compounds (6⁻14), were isolated from the soft coral Sinularia flexibilis. Their structures were established by extensive spectral analysis. Compound 3 possesses an unusual skeleton that could be biogenetically derived from cembranoids. The cytotoxicity and anti-inflammatory activities of the isolates were investigated, and the results showed that dehydrosinulariolide (7) and 11-epi-sinulariolide acetate (8) exhibited cytotoxicity toward a limited panel of cancer cell lines and 14-deoxycrassin (9) displayed anti-inflammatory activity by inhibition of superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophils.


Assuntos
Antozoários/metabolismo , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Diterpenos/farmacologia , Neutrófilos/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/metabolismo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Citocalasina B/farmacologia , Diterpenos/química , Diterpenos/isolamento & purificação , Diterpenos/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Superóxidos/metabolismo
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