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1.
PLoS One ; 15(3): e0229738, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160222

RESUMO

Ultrasound has many uses, such as in medical imaging, monitoring of crystallization, characterization of emulsions and suspensions, and disruption of cell membranes in the food industry. It can also affect microbial cells by promoting or slowing their growth and increasing the production of some metabolites. However, the exact mechanism explaining the effect of ultrasound has not been identified yet. Most equipment employed to study the effect of ultrasound on microorganisms has been designed for other applications and then only slightly modified. This results in limited control over ultrasound frequency and input power, or pressure distribution in the reactor. The present study aimed to obtain a well-defined reactor by simulating the pressure distribution of a sonobioreactor. Specifically, we optimized a sonotrode to match the bottle frequency and compared it to measured results to verify the accuracy of the simulation. The measured pressure distribution spectrum presented the same overall trend as the simulated spectrum. However, the peaks were much less intense, likely due to non-linear events such as the collapse of cavitation bubbles. To test the application of the sonobioreactor in biological systems, two biotechnologically interesting microorganisms were assessed: an electroactive bacterium, Geobacter sulfurreducens, and a lignocellulose-degrading fungus, Fusarium oxysporum. Sonication resulted in increased malate production by G. sulfurreducens, but no major effect on growth. In comparison, morphology and growth of F. oxysporum were more sensitive to ultrasound intensity. Despite considerable morphological changes at 4 W input power, the growth rate was not adversely affected; however, at 12 W, growth was nearly halted. The above findings indicate that the novel sonobioreactor provides an effective tool for studying the impact of ultrasound on microorganisms.


Assuntos
Reatores Biológicos/microbiologia , Fusarium/crescimento & desenvolvimento , Geobacter/crescimento & desenvolvimento , Análise Numérica Assistida por Computador , Sonicação , Calorimetria , Simulação por Computador , Fusarium/ultraestrutura , Geobacter/metabolismo , Malatos/metabolismo , Metaboloma , Pressão , Vibração
2.
Exp Parasitol ; 208: 107792, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31707003

RESUMO

Nitazoxanide (NTZ) is a broad-spectrum drug used in intestinal infections, but still poorly explored in the treatment of parasitic tissular infections. This study aimed to evaluate the in vitro responses of the energetic metabolism of T. crassiceps cysticerci induced by NTZ. The organic acids of the tricarboxylic acid cycle, products derived from fatty acids oxidation and protein catabolism were analyzed. These acids were quantified after 24 h of in vitro exposure to different NTZ concentrations. A positive control group was performed with albendazole sulfoxide (ABZSO). The significant alterations in citrate, fumarate and malate concentrations showed the NTZ influence in the tricarboxylic acid (TCA) cycle. The non-detection of acetate confirmed that the main mode of action of NTZ is effective against T. crassiceps cysticerci. The statistical differences in fumarate, urea and beta-hydroxybutyrate concentrations showed the NTZ effect on protein catabolism and fatty acid oxidation. Therefore, the main energetic pathways such as the TCA cycle, protein catabolism and fatty acids oxidation were altered after in vitro NTZ exposure. In conclusion, NTZ induced a significant metabolic stress in the parasite indicating that it may be used as an alternative therapeutic choice for cysticercosis treatment. The use of metabolic approaches to establish comparisons between anti parasitic drugs mode of actions is proposed.


Assuntos
Antiparasitários/farmacologia , Taenia/efeitos dos fármacos , Tiazóis/farmacologia , Albendazol/análogos & derivados , Albendazol/farmacologia , Análise de Variância , Animais , Anti-Helmínticos/farmacologia , Citratos/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Meios de Cultura/química , Cysticercus/efeitos dos fármacos , Cysticercus/metabolismo , Metabolismo Energético/efeitos dos fármacos , Fumaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Neurocisticercose/tratamento farmacológico , Ácido Oxaloacético/metabolismo , Ácido Succínico/metabolismo , Taenia/metabolismo
3.
Cell Prolif ; 53(2): e12702, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31628715

RESUMO

OBJECTIVES: Carnosine (ß-alanyl-l-histidine) is a naturally occurring dipeptide that selectively inhibits cancer cell growth, possibly by influencing glucose metabolism. As its precise mode of action and its primary targets are unknown, we analysed carnosine's effect on metabolites and pathways in glioblastoma cells. MATERIALS AND METHODS: Glioblastoma cells, U87, T98G and LN229, were treated with carnosine, and metabolites were analysed by gas chromatography coupled with mass spectrometry. Furthermore, mitochondrial ATP production was determined by extracellular flux analysis and reaction products of carnosine were investigated using mass spectrometry. RESULTS: Carnosine decreased the intracellular abundance of several metabolites indicating a reduced activity of the pentose phosphate pathway, the malate-aspartate shuttle and the glycerol phosphate shuttle. Mitochondrial respiration was reduced in U87 and T98G but not in LN229 cells, independent of whether glucose or pyruvate was used as substrate. Finally, we demonstrate non-enzymatic reaction of carnosine with dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. However, glycolytic flux from glucose to l-lactate appeared not to be affected by the reaction of carnosine with the metabolites. CONCLUSIONS: Carnosine reacts non-enzymatically with glycolytic intermediates reducing the activity of the pentose phosphate pathway which is required for cell proliferation. Although the activity of the malate-aspartate and the glycerol phosphate shuttle appear to be affected, reduced mitochondrial ATP production under the influence of the dipeptide is cell-specific and appears to be independent of the effect on the shuttles.


Assuntos
Carnosina/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Via de Pentose Fosfato/fisiologia , Trifosfato de Adenosina/metabolismo , Ácido Aspártico/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Glucose/metabolismo , Gliceraldeído/metabolismo , Glicólise/fisiologia , Humanos , Malatos/metabolismo , Mitocôndrias/metabolismo , Fosfatos/metabolismo
4.
Physiol Plant ; 168(1): 133-147, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30740711

RESUMO

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part - inflorescence and non-reproductive part - receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids - citrate and malate - were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.


Assuntos
Ácido Cítrico/metabolismo , Ficus/metabolismo , Frutas/metabolismo , Malatos/metabolismo , Polinização , Frutas/genética , Regulação da Expressão Gênica de Plantas , Inflorescência/metabolismo
5.
Food Microbiol ; 86: 103265, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31703880

RESUMO

Malolactic fermentation (MLF) in Valtellina Superiore DOCG red wine was monitored in 4 cellars and the final products were analysed to determine the content of melatonin (MEL) and other tryptophan (TRP) derivatives, including tryptophan ethyl ester (TEE) and MEL isomers (MISs), and to isolate predominant O. oeni strains. MEL and TEE significantly increased in wines after MLF from two cellars out of four. Six strains were isolated during the MLF of red wines and under laboratory scale, in rich and synthetic wine cultural media, together with other four O. oeni strains able to trigger the MLF. Results showed that the presence of stressful growth factors, like ethanol and acid pH, has a pivotal role in triggering the release of TEE by oenococci. Indeed, all the strains became capable to produce also MEL and MISs, together with TEE. under harsh growth conditions, as in a synthetic wine medium. The production of these compounds was strain-dependent and a maximum amount of 0.0078 ±â€¯0.0023 ngT/mL (UMB472) and 619.85 ±â€¯196.16 ngT/mL (UMB436) of MEL and TEE was obtained, respectively. In particular, different MISs were detected under oenological and laboratory scale suggesting that other factors (i.e. technological and/or physico-chemical) could affect the synthesis of TRP derivatives.


Assuntos
Melatonina/biossíntese , Oenococcus/metabolismo , Triptofano/biossíntese , Etanol/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Malatos/metabolismo , Oenococcus/química , Triptofano/análogos & derivados , Triptofano/análise , Triptofano/química , Triptofano/metabolismo , Vinho/análise , Vinho/microbiologia
6.
Food Chem ; 309: 125726, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31704069

RESUMO

In our present study, we aimed to explore the effects of hot air and UV-C on anthocyanins and the interaction among anthocyanin, sucrose and organic acids in peaches during postharvest storage. Peaches were treated with hot air or UV-C and stored at 1 °C for 35 days. The results showed that both treatments significantly enhanced the accumulation of anthocyanins and suppressed the degradation of sucrose, citric and malic acids. An in vitro test verified that sucrose, citric and malic acid penetrated the tissue and then induced the biosynthesis of anthocyanins by up regulating anthocyanin-related enzymes. In addition, hot air and UV-C directly enhanced the activities and gene expression of related enzymes to promote the accumulation of anthocyanins. PAL, ANS and UFGT played crucial roles in the biosynthesis of anthocyanins in peach fruit after harvest, and these three enzymes can be stimulated by HA, UV-C, sucrose, citric and malic acid.


Assuntos
Antocianinas/metabolismo , Prunus persica/química , Raios Ultravioleta , Antocianinas/química , Armazenamento de Alimentos , Frutas/química , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Temperatura Alta , Malatos/química , Malatos/metabolismo , Prunus persica/metabolismo , Prunus persica/efeitos da radiação , Açúcares/química , Açúcares/metabolismo
7.
Food Chem ; 309: 125617, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31718833

RESUMO

In blueberry, sugars and organic acids determine fruit organoleptic quality and drastically change during fruit maturation. This study examined enzymes involved in the metabolism of sugars and organic acids during the three maturation phases (green, pink and blue). During maturation, an increase in sugar (mainly fructose and glucose) was associated with up-regulation of VcSPP (CUFF.32787.1), VcSPS (CUFF.14989.1), and VcINV (gene.g3367.t1.1, CUFF.8077.1 and CUFF.47310.2). A decrease in citrate was associated with VcACLY (CUFF.27347.1 and CUFF.28772.1) in the acetyl-CoA pathway and with VcGAD (CUFF.15663.1 and CUFF.13757.1) and VcGLT (CUFF.6416.1) in the GABA shunt. A decrease in malate was associated with VcMDH (CUFF.30072.1, CUFF.18332.1 and CUFF.24878.1) involved in malate biosynthesis, and with VcADH (gene.g1507.t1.1, CUFF.3210.1 and gene.g30667.t1.1) as well as VcPDC (CUFF.47532.1) involved in fermentation. Multi-isoforms of enzymes were divergent and differentially regulated, suggesting that they have specialized functions in these pathways. The information will contribute to the understanding of blueberry organoleptic quality.


Assuntos
Mirtilos Azuis (Planta)/fisiologia , Enzimas/metabolismo , Frutas/fisiologia , Proteínas de Plantas/metabolismo , Açúcares/metabolismo , Mirtilos Azuis (Planta)/metabolismo , Frutas/metabolismo , Malatos/metabolismo , Filogenia
8.
BMC Biotechnol ; 19(1): 72, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31684928

RESUMO

BACKGROUND: In filamentous fungi, transport of organic acids across the mitochondrial membrane is facilitated by active transport via shuttle proteins. These transporters may transfer different organic acids across the membrane while taking others the opposite direction. In Aspergillus niger, accumulation of malate in the cytosol can trigger production of citric acid via the exchange of malate and citrate across the mitochondrial membrane. Several mitochondrial organic acid transporters were recently studied in A. niger showing their effects on organic acid production. RESULTS: In this work, we studied another citric acid producing fungus, Aspergillus carbonarius, and identified by genome-mining a putative mitochondrial transporter MtpA, which was not previously studied, that might be involved in production of citric acid. This gene named mtpA encoding a putative oxaloacetate transport protein was expressed constitutively in A. carbonarius based on transcription analysis. To study its role in organic acid production, we disrupted the gene and analyzed its effects on production of citric acid and other organic acids, such as malic acid. In total, 6 transformants with gene mtpA disrupted were obtained and they showed secretion of malic acid at the expense of citric acid production. CONCLUSION: A putative oxaloacetate transporter gene which is potentially involved in organic acid production by A. carbonarius was identified and further investigated on its effects on production of citric acid and malic acid. The mtpA knockout strains obtained produced less citric acid and more malic acid than the wild type, in agreement with our original hypothesis. More extensive studies should be conducted in order to further reveal the mechanism of organic acid transport as mediated by the MtpA transporter.


Assuntos
Aspergillus/metabolismo , Ácido Cítrico/metabolismo , Engenharia Metabólica/métodos , Proteínas Mitocondriais/metabolismo , Oxaloacetatos/metabolismo , Malatos/metabolismo
9.
PLoS One ; 14(10): e0223516, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613915

RESUMO

Citrus grandis seedlings were irrigated with nutrient solutions with four Al-P combinations [two Al levels (0 mM and 1.2 mM AlCl3·6H2O) × two P levels (0 µM and 200 µM KH2PO4)] for 18 weeks. Al dramatically inhibited the growth of C. grandis seedlings, as revealed by a decreased dry weight of roots and shoots. Elevating P level could ameliorate the Al-induced growth inhibition and organic acid (malate and citrate) secretion in C. grandis. Using a comparative proteomic approach revealed by the isobaric tags for relative and absolute quantification (iTRAQ) technique, 318 differentially abundant proteins (DAPs) were successfully identified and quantified in this study. The possible mechanisms underlying P-induced alleviation of Al toxicity in C. grandis were proposed. Furthermore, some DAPs, such as GLN phosphoribosyl pyrophosphate amidotransferase 2, ATP-dependent caseinolytic (Clp) protease/crotonase family protein, methionine-S-oxide reductase B2, ABC transporter I family member 17 and pyridoxal phosphate phosphatase, were reported for the first time to respond to Al stress in Citrus plants. Our study provides some proteomic details about the alleviative effects of P on Al toxicity in C. grandis, however, the exact function of the DAPs identified herein in response to Al tolerance in plants must be further investigated.


Assuntos
Alumínio/toxicidade , Citrus/metabolismo , Marcação por Isótopo/métodos , Fósforo/farmacologia , Raízes de Plantas/metabolismo , Biomassa , Ácido Cítrico/metabolismo , Citrus/efeitos dos fármacos , Citrus/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucose/metabolismo , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Malatos/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Raízes de Plantas/efeitos dos fármacos , Análise de Componente Principal , Amido/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
10.
Appl Microbiol Biotechnol ; 103(21-22): 9001-9011, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31641813

RESUMO

Optimization of export mechanisms for valuable extracellular products is important for the development of efficient microbial production processes. Identification of the relevant export mechanism is the prerequisite step for product export optimization. In this work, we identified transporters involved in malate export in an engineered L-malate-producing Escherichia coli strain using cheminformatics-guided genetics tests. Among all short-chain di- or tricarboxylates with known transporters in E. coli, citrate, tartrate, and succinate are most chemically similar to malate as estimated by their molecular signatures. Inactivation of three previously reported transporters for succinate, tartrate, and citrate, DcuA, TtdT, and CitT, respectively, dramatically decreased malate production and fermentative growth, suggesting that these transporters have substrate promiscuity for different short-chain organic acids and constitute the major malate export system in E. coli. Malate export deficiency led to an increase in cell sizes and accumulation of intracellular metabolites related to malate metabolism.


Assuntos
Transporte Biológico/genética , Proteínas de Transporte/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Malatos/metabolismo , Proteínas de Bactérias/genética , Ácido Cítrico/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Proteínas de Escherichia coli/genética , Fermentação/genética , Engenharia Genética , Transportadores de Ânions Orgânicos/genética , Ácido Succínico/metabolismo , Tartaratos/metabolismo
11.
Hum Genet ; 138(11-12): 1247-1257, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31538237

RESUMO

The reversible oxidation of L-malate to oxaloacetate is catalyzed by NAD(H)-dependent malate dehydrogenase (MDH). MDH plays essential roles in the malate-aspartate shuttle and the tricarboxylic acid cycle. These metabolic processes are important in mitochondrial NADH supply for oxidative phosphorylation. Recently, bi-allelic mutations in mitochondrial MDH2 were identified in patients with global developmental delay, epilepsy and lactic acidosis. We now report two patients from an extended consanguineous family with a deleterious variant in the cytosolic isoenzyme of MDH (MDH1). The homozygous missense variant in the NAD+-binding domain of MDH1 led to severely diminished MDH protein expression. The patients presented with global developmental delay, epilepsy and progressive microcephaly. Both patients had normal concentrations of plasma amino acids, acylcarnitines, lactate, and urine organic acids. To identify the metabolic consequences of MDH1 deficiency, untargeted metabolomics was performed on dried blood spots (DBS) from the patients and in MDH1 knockout HEK293 cells that were generated by Crispr/Cas9. Increased levels of glutamate and glycerol-3-phosphate were found in DBS of both patients. In MDH1 KO HEK293 cells, increased levels of glycerol-3-phosphate were also observed, as well as increased levels of aspartate and decreased levels of fumarate. The consistent finding of increased concentrations of glycerol-3-phosphate may represent a compensatory mechanism to enhance cytosolic oxidation of NADH by the glycerol-P-shuttle. In conclusion, MDH1 deficiency is a new metabolic defect in the malate-aspartate shuttle characterized by a severe neurodevelopmental phenotype with elevated concentrations of glycerol-3-phosphate as a potential biomarker.


Assuntos
Ácido Aspártico/metabolismo , Encefalopatias/metabolismo , Encefalopatias/patologia , Malato Desidrogenase/deficiência , Malatos/metabolismo , Doenças Metabólicas/etiologia , Idade de Início , Encefalopatias/complicações , Pré-Escolar , Feminino , Humanos , Masculino , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Metaboloma , Linhagem
12.
Microb Cell Fact ; 18(1): 154, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506101

RESUMO

BACKGROUND: Mitochondrial and cytoplasmic malate transporter proteins are responsible for transmembrane transport of malate, thereby linking malate metabolism in various subcellular regions of the cell. These transporters play an important role in fatty acid biosynthesis of oleaginous microorganisms. Our previous studies have found that lipid content of the recombinant Mucor circinelloides (M. circinelloides) strain with mitochondrial malate transporter (mt) gene overexpression was increased by 70%, while that of strain with mt gene knockout was decreased by 27%. However, the mechanism of malate transporter promoting the transport of mitochondrial malate and citrate related to lipid accumulation is not clear. Therefore, 13C-labeled glucose metabolic flux analysis was carried out to identify the metabolic network topology and estimate intracellular fluxes of genetically engineered M. circinelloides strains for the purpose of better understanding the roles of malate transporters in citrate transport systems and lipid accumulation. RESULTS: The metabolic flux distribution analysis suggested that tricarboxylic acid (TCA) cycle flux ratio of mt-overexpression strains was decreased compared to that of the control strain, but in contrast, glyoxylic acid (GOX) cycle flux ratio was increased. Accordingly, the mt-knockout strain showed an opposite phenomenon with a higher TCA cycle flux ratio and a lower GOX cycle flux ratio than the control strain. GOX cycle might be more effective than TCA cycle in producing malate and oxaloacetate replenishment. Moreover, a relatively higher flux ratio of the pentose phosphate (PP) pathway was obtained in mt-overexpression strains, but no significant difference in the malic enzyme flux between recombinant strains and the control strain. Our results confirmed that PP pathway might play an important role for supplying NADPH and malic enzyme is not a limiting factor for fatty acid synthesis in oleaginous fungus M. circinelloides strains. CONCLUSION: Intracellular metabolic flux information suggested that mt-overexpression strains had higher flux in PP pathway and GOX cycle, lower flux in TCA cycle, and no difference in malic enzyme cycle. Together, the role of malate transporter was assumed to further participate in transporting cycle of acetyl-CoA and drive PP pathway to supply NADPH required for lipid accumulation in recombinant M. circinelloides strains.


Assuntos
Metabolismo dos Lipídeos , Malatos/metabolismo , Mucor/metabolismo , Transportadores de Ânions Orgânicos/fisiologia , Transporte Biológico , Glioxilatos/metabolismo , Análise do Fluxo Metabólico/métodos , Via de Pentose Fosfato
13.
Am J Hum Genet ; 105(3): 534-548, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31422819

RESUMO

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.


Assuntos
Alelos , Ácido Aspártico/metabolismo , Encefalopatias/genética , Proteínas de Ligação a Ácido Graxo/genética , Malatos/metabolismo , Mutação , Animais , Criança , Pré-Escolar , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Sequenciamento Completo do Exoma
14.
BMC Biotechnol ; 19(1): 58, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31382948

RESUMO

BACKGROUND: Efficient xylose fermentation still demands knowledge regarding xylose catabolism. In this study, metabolic flux analysis (MFA) and metabolomics were used to improve our understanding of xylose metabolism. Thus, a stoichiometric model was constructed to simulate the intracellular carbon flux and used to validate the metabolome data collected within xylose catabolic pathways of non-Saccharomyces xylose utilizing yeasts. RESULTS: A metabolic flux model was constructed using xylose fermentation data from yeasts Scheffersomyces stipitis, Spathaspora arborariae, and Spathaspora passalidarum. In total, 39 intracellular metabolic reactions rates were utilized validating the measurements of 11 intracellular metabolites, acquired by mass spectrometry. Among them, 80% of total metabolites were confirmed with a correlation above 90% when compared to the stoichiometric model. Among the intracellular metabolites, fructose-6-phosphate, glucose-6-phosphate, ribulose-5-phosphate, and malate are validated in the three studied yeasts. However, the metabolites phosphoenolpyruvate and pyruvate could not be confirmed in any yeast. Finally, the three yeasts had the metabolic fluxes from xylose to ethanol compared. Xylose catabolism occurs at twice-higher flux rates in S. stipitis than S. passalidarum and S. arborariae. Besides, S. passalidarum present 1.5 times high flux rate in the xylose reductase reaction NADH-dependent than other two yeasts. CONCLUSIONS: This study demonstrated a novel strategy for metabolome data validation and brought insights about naturally xylose-fermenting yeasts. S. stipitis and S. passalidarum showed respectively three and twice higher flux rates of XR with NADH cofactor, reducing the xylitol production when compared to S. arborariae. Besides then, the higher flux rates directed to pentose phosphate pathway (PPP) and glycolysis pathways resulted in better ethanol production in S. stipitis and S. passalidarum when compared to S. arborariae.


Assuntos
Fermentação , Análise do Fluxo Metabólico/métodos , Metaboloma , Metabolômica/métodos , Saccharomycetales/metabolismo , Frutosefosfatos/metabolismo , Glucose-6-Fosfato/metabolismo , Glicólise , Malatos/metabolismo , Espectrometria de Massas/métodos , Modelos Biológicos , Via de Pentose Fosfato , Ribulosefosfatos/metabolismo , Saccharomycetales/classificação , Leveduras/classificação , Leveduras/metabolismo
15.
Appl Microbiol Biotechnol ; 103(18): 7687-7702, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31388732

RESUMO

In recent years, there is an increasing interest from the winemaking industry for the use of mixed fermentations with Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae, due to their ability to modulate metabolites of oenological interest. The current study was carried out to elucidate the effect of this fermentation protocol on the growth and malolactic activity of lactic acid bacteria (LAB) used for malolactic fermentation (MLF) and on the chemical and volatile profile of Nebbiolo wines and their chromatic characteristics. To this end, two LAB species, namely Lactobacillus plantarum and Oenococcus oeni, were inoculated at the beginning and at the end of the alcoholic fermentation (AF) performed by pure and mixed yeast using the abovementioned yeasts. The different yeast inoculation protocols and the combination of species tested influenced greatly the interactions and behavior of the inoculated yeasts and LAB during AF and MLF. For both LAB species, inoculation timing was critical to how rapidly MLF started and finished. Fermentation inoculated with L. plantarum, at the beginning of the AF, completed MLF faster than those inoculated with O. oeni. The presence of Starm. bacillaris in mixed fermentation promoted LAB growth and activity, in particular, O. oeni. Furthermore, LAB species choice had a greater impact on the volatile and chromatic profile of the wines than inoculation time. These findings reveal new knowledge about the importance of LAB species choice and inoculation time to ensure fast MLF completion and to improve wine characteristics in mixed fermentation with Starm. bacillaris and S. cerevisiae.


Assuntos
Fermentação , Ácido Láctico/metabolismo , Lactobacillus plantarum/metabolismo , Malatos/metabolismo , Interações Microbianas , Oenococcus/metabolismo , Lactobacillales/crescimento & desenvolvimento , Lactobacillales/metabolismo , Vinho/análise , Vinho/microbiologia
16.
Proc Natl Acad Sci U S A ; 116(32): 15907-15913, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31320588

RESUMO

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional ß-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.


Assuntos
Leucina/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Succinatos/metabolismo , Aerossóis , Animais , Biocatálise , Ligantes , Liases/metabolismo , Malatos/metabolismo , Camundongos Endogâmicos C57BL , Filogenia , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Tuberculose/microbiologia , Tuberculose/patologia
17.
Photosynth Res ; 142(2): 153-167, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31325077

RESUMO

The engineering process of C4 photosynthesis into C3 plants requires an increased activity of phosphoenolpyruvate carboxylase (PEPC) in the cytosol of leaf mesophyll cells. The literature varies on the physiological effect of transgenic maize (Zea mays) PEPC (ZmPEPC) leaf expression in Oryza sativa (rice). Therefore, to address this issue, leaf-atmosphere CO2 and 13CO2 exchanges were measured, both in the light (at atmospheric O2 partial pressure of 1.84 kPa and at different CO2 levels) and in the dark, in transgenic rice expressing ZmPEPC and wild-type (WT) plants. The in vitro PEPC activity was 25 times higher in the PEPC overexpressing (PEPC-OE) plants (~20% of maize) compared to the negligible activity in WT. In the PEPC-OE plants, the estimated fraction of carboxylation by PEPC (ß) was ~6% and leaf net biochemical discrimination against 13CO2[Formula: see text] was ~ 2‰ lower than in WT. However, there were no differences in leaf net CO2 assimilation rates (A) between genotypes, while the leaf dark respiration rates (Rd) over three hours after light-dark transition were enhanced (~ 30%) and with a higher 13C composition [Formula: see text] in the PEPC-OE plants compared to WT. These data indicate that ZmPEPC in the PEPC-OE rice plants contributes to leaf carbon metabolism in both the light and in the dark. However, there are some factors, potentially posttranslational regulation and PEP availability, which reduce ZmPEPC activity in vivo.


Assuntos
Atmosfera/química , Dióxido de Carbono/metabolismo , Isótopos de Carbono/química , Oryza/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/metabolismo , Zea mays/enzimologia , Zea mays/genética , Respiração Celular , Malatos/metabolismo , Células do Mesofilo/metabolismo , Fotossíntese , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas
18.
Plant Cell Physiol ; 60(10): 2319-2330, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268146

RESUMO

Fruit set is an important yield-related parameter, which varies drastically due to genetic and environmental factors. Here, two commercial cultivars of Capsicum chinense (Biquinho and Habanero) were evaluated in response to light intensity (unshaded and shaded) and N supply (deficiency and sufficiency) to understand the role of source strength on fruit set at the metabolic level. We assessed the metabolic balance of primary metabolites in source leaves during the flowering period. Furthermore, we investigated the metabolic balance of the same metabolites in flowers to gain more insights into their influence on fruit set. Genotype and N supply had a strong effect on fruit set and the levels of primary metabolites, whereas light intensity had a moderate effect. Higher fruit set was mainly related to the export of both sucrose and amino acids from source leaves to flowers. Additionally, starch turnover in source leaves, but not in flowers, had a central role on the sucrose supply to sink organs at night. In flowers, our results not only confirmed the role of the daily supply of carbohydrates on fruit set but also indicated a potential role of the balance of amino acids and malate.


Assuntos
Aminoácidos/metabolismo , Capsicum/fisiologia , Nitrogênio/metabolismo , Amido/metabolismo , Sacarose/metabolismo , Biomassa , Capsicum/genética , Capsicum/crescimento & desenvolvimento , Capsicum/efeitos da radiação , Flores/genética , Flores/crescimento & desenvolvimento , Flores/fisiologia , Flores/efeitos da radiação , Frutas/genética , Frutas/crescimento & desenvolvimento , Frutas/fisiologia , Frutas/efeitos da radiação , Genótipo , Luz , Malatos/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação
19.
Metab Eng ; 55: 152-160, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31306776

RESUMO

Carbon dioxide (CO2) is an appealing carbon feedstock for the sustainable production of biocommodities. Here we designed three in vitro artificial enzymatic pathways featuring the ATP-excess, ATP-deficit, and ATP-balanced pathways for the biotransformation of starch and CO2 to malate. This ATP-balanced pathway without exogenous ATP donors can auto-regulate its carbon fluxes from glyceraldehyde 3-phosphate to 3-phosphoglycerate via either the ATP-generating pathway (a part of glycolysis) or no-ATP-generating pathway from a hyperthermophilic archaeon Thermococcus kodakarensis. The ATP-balanced pathway enabled to produce up to 52.4 mM malate with 95.3% of the theoretical yield, that is, 2 mol of malate synthesized from 1 mol of glucose of starch and 2 mol of CO2. This pathway also enabled to produce high-yield malate regardless of ATP/ADP ratios. Anaerobic reaction conditions and/or the addition of a reducing agent dithiothreitol were of importance for creating an anoxic environment for biocatalysis of enzyme cocktails and for mitigating the deactivation of enzymes and degradation of intermediates. This new pathway could provide a green route for direct conversion of CO2 to many building blocks, a promising alternative of petrochemical-based production of biocommodities.


Assuntos
Proteínas Arqueais/química , Dióxido de Carbono/química , Malatos/síntese química , Amido/química , Thermococcus/enzimologia , Proteínas Arqueais/genética , Dióxido de Carbono/metabolismo , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Malatos/química , Malatos/metabolismo , Engenharia Metabólica , Amido/metabolismo , Thermococcus/genética
20.
J Agric Food Chem ; 67(32): 8773-8782, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31283205

RESUMO

Conquering rapid postripeness and deterioration of Agaricus bisporus is quite challenging. We previously observed that methyl jasmonate (MeJA) pretreatment postponed the deterioration of A. bisporus, but the mechanism is unknown. Here, a nontargeted metabolomics analysis by ultrahigh-pressure liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) revealed that MeJA increased the synthesis of malate by inhibiting the decomposition of fumarate and cis-aconitate. MeJA maintained energy supply by enhancing ATP content and energy charge level and improving hexokinase and glucose-6-phosphate dehydrogenase activities as well. These results promoted ATP supply by maintaining glycolysis, the TCA cycle, and the pentose phosphate pathway. In addition, we revealed that the delayed deterioration was attributed to MeJA treatment which stimulated the energy status of A. bisporus by reducing the respiration rate and nutrient decomposition, thus maintaining energy production. Our results provide a new insight into the role of MeJA treatment in delaying deterioration of A. bisporus through ATP production and supply.


Assuntos
Acetatos/farmacologia , Agaricus/efeitos dos fármacos , Agaricus/metabolismo , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Ácido Aconítico/metabolismo , Trifosfato de Adenosina/metabolismo , Agaricus/química , Agaricus/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão/métodos , Metabolismo Energético/efeitos dos fármacos , Fumaratos/metabolismo , Malatos/metabolismo , Metabolômica , Espectrometria de Massas em Tandem/métodos
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