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1.
Food Chem ; 334: 127567, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32707362

RESUMO

Fruit acidity is an important determinant of peach organoleptic quality, but its regulatory mechanism remains elusive. Measurement of organic acids in ripe fruits of seventy-five peach cultivars revealed the predominant components malate and citrate, accompanied by quinate. Organic acid accumulation increased at early stages of fruit growth, but exhibited a more dramatic reduction in low-acid cultivar during later stages of fruit development compared to high-acid cultivars. Low-acid cultivars showed citrate degradation and less transport of malate into the vacuole due to up- and down-regulation of a GABA pathway gene GAD and a malate transporter gene ALMT9, respectively. The NAD-MDH1 gene might control the rate-limiting step in malate synthesis, while three genes, PDK, PK, and ADH, could affect citrate synthesis through the pyruvate-to-acetyl-CoA-to-citrate pathway. Altogether, these results suggested that malate accumulation is controlled at the level of metabolism and vacuolar storage, while metabolism is crucial for citrate accumulation in peach.


Assuntos
Ácidos Carboxílicos/metabolismo , Frutas/metabolismo , Malatos/metabolismo , Proteínas de Plantas/genética , Prunus persica/metabolismo , Acetilcoenzima A/metabolismo , Ácido Cítrico/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Prunus persica/genética , Prunus persica/crescimento & desenvolvimento , Ácido Pirúvico/metabolismo , Vacúolos/metabolismo
2.
PLoS Genet ; 16(7): e1008884, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32639996

RESUMO

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.


Assuntos
Osso e Ossos/metabolismo , Calcinose/genética , Ácido Cítrico/metabolismo , Proteínas de Transporte de Fosfato/genética , Trifosfato de Adenosina/metabolismo , Animais , Desenvolvimento Ósseo/genética , Calcinose/metabolismo , Calcinose/patologia , Diferenciação Celular , Células Cultivadas , Difosfatos/metabolismo , Humanos , Fenômenos Mecânicos , Camundongos , Mutação/genética , Proteínas de Transporte de Fosfato/metabolismo
3.
Chemosphere ; 257: 127247, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32534296

RESUMO

Chelate-assisted phytoextraction by high biomass producing macrophyte plant Typha latifolia L. commonly known as cattail, is gaining much attention worldwide. The present study investigated the effects of Lead (Pb) and Mercury (Hg) on physiology and biochemistry of plant, Pb and Hg uptake in T. latifolia with and without citric acid (CA) amendment. The uniform seedlings of T. latifolia were treated with various concentrations in the hydroponics as: Pb and Hg (1, 2.5, 5 mM) each alone and/or with CA (5 mM). After four weeks of treatments, the results revealed that Pb and Hg significantly reduced the plant agronomic traits as compare to non-treated plants. The addition of CA improved the plant physiology and enhanced the antioxidant enzymes activities to overcome Pb and Hg induced oxidative damage and electrolyte leakage. Our results depicted that Pb and Hg uptake and accumulation by T. latifolia was dose depend whereas, the addition of CA further increased the concentration and accumulation of Pb and Hg by up to 22 & 35% Pb and 72 & 40% Hg in roots, 25 & 26% Pb and 85 & 60% Hg in stems and 22 & 15 Pb and 100 & 58% Hg in leaves respectively compared to Pb and Hg treated only plants. On other hand, the root-shoot translocation factor was ≥1 and bioconcentration factor was also ≥2 for both Pb & Hg. The results also revealed that T. latifolia showed greater tolerance towards Hg and accumulated higher Hg in all parts compared with Pb.


Assuntos
Ácido Cítrico/metabolismo , Chumbo/metabolismo , Mercúrio/metabolismo , Poluentes do Solo/metabolismo , Typhaceae/fisiologia , Biodegradação Ambiental , Biomassa , Folhas de Planta/química , Raízes de Plantas/efeitos dos fármacos , Plântula/efeitos dos fármacos , Poluentes do Solo/análise , Typhaceae/efeitos dos fármacos
4.
Chemosphere ; 258: 127320, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32554008

RESUMO

Microorganisms play important roles in the degradation of volatile organic compounds. Aeromonas salmonicida strain (AEP-3) generated from biomass in the citric acid fermentation industry was screened and subjected to denaturing gradient gel electrophoresis (DGGE) fingerprinting and 16S rDNA gene sequencing. The growth conditions of AEP-3 in Luria-Bertani broth were optimized at 25 °C and approximately pH 7. AEP-3 was used to degrade ethyl formate, propionic aldehyde, or acetone alone and their mixture. The concentrations of ethyl formate, propionic aldehyde, and acetone were below 7500, 600, and 800 mg L-1, respectively, and their maximum degradation efficiencies were 100%, 92.41%, and 34.75%. AEP-3 first degraded acetone and propionic aldehyde in the mixture, followed by ethyl formate. The degradation pathways of these organic compounds in the mixture and their substrate interactions during degradation were explored. Propionic aldehyde was first converted into propionic acid in the metabolic process and was involved in the subsequent carboxylic acid cycle. By contrast, ethyl formate was first hydrolyzed into formic acid and ethanol. Then, formic acid participated in the cyclic metabolism of carboxylic acid, whereas, ethanol was hydrolyzed into acetaldehyde and acetic acid through alcohol and aldehyde dehydrogenase. Additionally, acetone directly interacted with nitrate in the medium under the action of hydrogen ions and produced carbon dioxide, water, and nitrogen. Overall, this study provides a new degrading bacterium biodegradability toward the exhaust gas of citric acid fermentation.


Assuntos
Acetona/metabolismo , Aeromonas salmonicida/metabolismo , Ésteres do Ácido Fórmico/metabolismo , Acetaldeído , Ácido Acético/metabolismo , Biodegradação Ambiental , Biomassa , Ácido Cítrico/metabolismo , Etanol/metabolismo , Fermentação , Formiatos , Propionatos/metabolismo
5.
Invest Ophthalmol Vis Sci ; 61(5): 3, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32392312

RESUMO

Purpose: The avian eye is an established model for exploring mechanisms that coordinate morphogenesis and metabolism during embryonic development. Less is known, however, about trafficking of bioenergetic and metabolic signaling molecules that are involved in retinal neurogenesis. Methods: Here we tested whether the known 3-day delayed neurogenesis occurring in the pigeon compared with the chick was associated with a deferred reshaping of eye metabolism in vivo. Developmental metabolic remodeling was explored using 1H-magnetic resonance spectroscopy of the whole eye and vitreous body, in ovo, in parallel with biochemical and molecular analyses of retinal, vitreous, and lens extracts from bird embryos. Results: Cross-species comparisons enabled us to show that a major glycolytic switch in the retina is related to neurogenesis rather than to eye growth. We further show that the temporal emergence of an interlocking regulatory cascade controlling retinal oxidative phosphorylation and glycolysis results in the exchange of lactate and citrate between the retina and vitreous. Conclusions: Our results point to the vitreous as a reservoir and buffer of energy metabolites that provides trophic support to oxidative neurons, such as retinal ganglion cells, in early development. Through its control of key glycolytic regulatory enzymes, citrate, exchanged between extracellular and intracellular compartments between the retina and vitreous, is a key metabolite in the initiation of a glycolytic switch.


Assuntos
Olho/embriologia , Glicólise , Ácido Láctico/metabolismo , Neurogênese , Animais , Aves , Ácido Cítrico/metabolismo , Espectroscopia de Ressonância Magnética , Mitocôndrias/metabolismo , Modelos Animais , Fosforilação Oxidativa , Oxigênio/metabolismo , Retina/embriologia , Retina/metabolismo , Corpo Vítreo/embriologia , Corpo Vítreo/metabolismo
6.
Sci Rep ; 10(1): 8536, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444674

RESUMO

Metformin is the first-line treatment for type 2 diabetes. Inhibition of hepatic gluconeogenesis is the primary contributor to its anti-diabetic effect. Metformin inhibits complex I and α-glycerophosphate shuttle, and the resultant increase in cytoplasmic NADH/NAD+ ratio diverts glucose precursors away from gluconeogenesis. These actions depend on metformin-mediated activation of AMP kinase (AMPK). Here we report on a hitherto unknown mechanism. Metformin inhibits the expression of the plasma membrane citrate transporter NaCT in HepG2 cells and decreases cellular levels of citrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, elicits a similar effect. The process involves a decrease in maximal velocity with no change in substrate affinity. The decrease in NaCT expression is associated with decreased mRNA levels. AMPK inhibits mTOR, and the mTOR inhibitor rapamycin also decreases NaCT expression. The transcription factor downstream of AMPK that is relevant to cAMP signaling is CREB; decreased levels of phospho-CREB seem to mediate the observed effects of metformin on NaCT. Citrate is known to suppress glycolysis by inhibiting phosphofructokinase-1 and activate gluconeogenesis by stimulating fructose-1,6-bisphophatase; therefore, the decrease in cellular levels of citrate would stimulate glycolysis and inhibit gluconeogenesis. These studies uncover a novel mechanism for the anti-diabetic actions of metformin.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Carcinoma Hepatocelular/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Metformina/farmacologia , Terapia de Alvo Molecular , Ribonucleotídeos/farmacologia , Simportadores/antagonistas & inibidores , Aminoimidazol Carboxamida/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ácido Cítrico/metabolismo , Glicólise , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transdução de Sinais , Simportadores/genética , Simportadores/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
7.
Biochem J ; 477(7): 1309-1321, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32227111

RESUMO

Metabolite production from carbon dioxide using sugar catabolism in cyanobacteria has been in the spotlight recently. Synechocystis sp. PCC 6803 (Synechocystis 6803) is the most studied cyanobacterium for metabolite production. Previous in vivo analyses revealed that the oxidative pentose phosphate (OPP) pathway is at the core of sugar catabolism in Synechocystis 6803. However, the biochemical regulation of the OPP pathway enzymes in Synechocystis 6803 remains unknown. Therefore, we characterized a key enzyme of the OPP pathway, glucose-6-phosphate dehydrogenase (G6PDH), and related enzymes from Synechocystis 6803. Synechocystis 6803 G6PDH was inhibited by citrate in the oxidative tricarboxylic acid (TCA) cycle. Citrate has not been reported as an inhibitor of G6PDH before. Similarly, 6-phosphogluconate dehydrogenase, the other enzyme from Synechocystis 6803 that catalyzes the NADPH-generating reaction in the OPP pathway, was inhibited by citrate. To understand the physiological significance of this inhibition, we characterized succinic semialdehyde dehydrogenase (SSADH) from Synechocystis 6803 (SySSADH), which catalyzes one of the NAD(P)H generating reactions in the oxidative TCA cycle. Similar to isocitrate dehydrogenase from Synechocystis 6803, SySSADH specifically catalyzed the NADPH-generating reaction and was not inhibited by citrate. The activity of SySSADH was lower than that of other bacterial SSADHs. Previous and this studies revealed that unlike the OPP pathway, the oxidative TCA cycle is a pathway with low efficiency in NADPH generation in Synechocystis 6803. It has, thus, been suggested that to avoid NADPH overproduction, the OPP pathway dehydrogenase activity is repressed when the flow of the oxidative TCA cycle increases in Synechocystis 6803.


Assuntos
Proteínas de Bactérias/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Via de Pentose Fosfato , Synechocystis/enzimologia , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Cinética , NADP/metabolismo , Fotossíntese/fisiologia
8.
Nat Rev Urol ; 17(4): 214-231, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32112053

RESUMO

Anabolic metabolism mediated by aberrant growth factor signalling fuels tumour growth and progression. The first biochemical descriptions of the altered metabolic nature of solid tumours were reported by Otto Warburg almost a century ago. Now, the study of tumour metabolism is being redefined by the development of new molecular tools, tumour modelling systems and precise instrumentation together with important advances in genetics, cell biology and spectroscopy. In contrast to Warburg's original hypothesis, accumulating evidence demonstrates a critical role for mitochondrial metabolism and substantial variation in the way in which different tumours metabolize nutrients to generate biomass. Furthermore, computational and experimental approaches suggest a dominant influence of the tissue-of-origin in shaping the metabolic reprogramming that enables tumour growth. For example, the unique metabolic properties of prostate adenocarcinoma are likely to stem from the distinct metabolism of the prostatic epithelium from which it emerges. Normal prostatic epithelium employs comparatively glycolytic metabolism to sustain physiological citrate secretion, whereas prostate adenocarcinoma consumes citrate to power oxidative phosphorylation and fuel lipogenesis, enabling tumour progression through metabolic reprogramming. Current data suggest that the distinct metabolic aberrations in prostate adenocarcinoma are driven by the androgen receptor, providing opportunities for functional metabolic imaging and novel therapeutic interventions that will be complementary to existing diagnostic and treatment options.


Assuntos
Adenocarcinoma/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proliferação de Células , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Glicólise , Humanos , Lipogênese , Masculino , Redes e Vias Metabólicas , Fosforilação Oxidativa , Microambiente Tumoral
9.
Nature ; 579(7800): 586-591, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214246

RESUMO

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods1, and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease2-4. Fructose intake triggers de novo lipogenesis in the liver4-6, in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates7. Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases8. However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota9, and this supplies lipogenic acetyl-CoA independently of ACLY10. Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA.


Assuntos
Acetatos/metabolismo , Açúcares da Dieta/metabolismo , Frutose/metabolismo , Microbioma Gastrointestinal/fisiologia , Lipogênese , Fígado/metabolismo , ATP Citrato (pro-S)-Liase/deficiência , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetato-CoA Ligase/deficiência , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Acetilcoenzima A/metabolismo , Animais , Ácido Cítrico/metabolismo , Açúcares da Dieta/administração & dosagem , Açúcares da Dieta/farmacologia , Ácidos Graxos/metabolismo , Frutose/administração & dosagem , Frutose/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Marcação por Isótopo , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Especificidade por Substrato
10.
J Biotechnol ; 312: 56-62, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32151642

RESUMO

Whole-cell biocatalysts have numerous advantages including ease of preparation and coenzyme recovery over purified industrially used enzymes. However, the cell membrane can occasionally hinder cytoplasmic diffusion of the substrate, resulting in reduced biotransformation efficiency. Psychrophiles can grow and reproduce at low temperatures; their cell membranes are highly flexible, and their permeability can be improved via heat treatment at a moderate temperature. The aim of this study was to generate a psychrophile-based simple biocatalyst (PSCats) using Shewanella livingstonensis Ac10. This biocatalyst contained two enzymes that were heterologously expressed and converted citric acid to itaconic acid, thereby serving as a potential platform replacing the petroleum-based counterparts. The efficiency of the biocatalyst was increased via heat treatment at 45 °C for 15 min, and itaconic acid productivity of the cells after heat treatment (1.41 g/L/h) was increased around 6-fold in comparison with those without heat treatment (0.22 g/L/h). A large part of the productivity remained (67.3 %) when the cells were reused for 5 times (10 h for each reaction). Therefore, the potential of this heat-permeabilized psychrophile host to increase the productivity of whole-cell biocatalyst was proved; however, further research is necessary to understand the underlying mechanism.


Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Enzimas/farmacologia , Shewanella/metabolismo , Succinatos/metabolismo , Aconitato Hidratase , Ácido Cítrico/metabolismo , Temperatura Baixa , Citoplasma/metabolismo , Escherichia coli/genética , Temperatura Alta , Metaboloma , Shewanella/genética , Shewanella/crescimento & desenvolvimento
11.
Microb Cell Fact ; 19(1): 76, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209089

RESUMO

BACKGROUND: Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited. RESULTS: In this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5'-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors. CONCLUSIONS: In this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.


Assuntos
Aspergillus niger/genética , Ácido Cítrico/metabolismo , Edição de Genes/métodos , Uridina/análogos & derivados , Uridina/metabolismo
12.
Nat Commun ; 11(1): 698, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019928

RESUMO

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization.


Assuntos
Aconitato Hidratase/metabolismo , Macrófagos/enzimologia , Óxido Nítrico/metabolismo , /metabolismo , Aconitato Hidratase/genética , Animais , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ácido Pirúvico/metabolismo
13.
Microb Cell Fact ; 19(1): 17, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000778

RESUMO

BACKGROUND: Citric acid, a commodity product of industrial biotechnology, is produced by fermentation of the filamentous fungus Aspergillus niger. A requirement for high-yield citric acid production is keeping the concentration of Mn2+ ions in the medium at or below 5 µg L-1. Understanding manganese metabolism in A. niger is therefore of critical importance to citric acid production. To this end, we investigated transport of Mn2+ ions in A. niger NRRL2270. RESULTS: we identified an A. niger gene (dmtA; NRRL3_07789), predicted to encode a transmembrane protein, with high sequence identity to the yeast manganese transporters Smf1p and Smf2p. Deletion of dmtA in A. niger eliminated the intake of Mn2+ at low (5 µg L-1) external Mn2+ concentration, and reduced the intake of Mn2+ at high (> 100 µg L-1) external Mn2+ concentration. Compared to the parent strain, overexpression of dmtA increased Mn2+ intake at both low and high external Mn2+ concentrations. Cultivation of the parent strain under Mn2+ ions limitation conditions (5 µg L-1) reduced germination and led to the formation of stubby, swollen hyphae that formed compact pellets. Deletion of dmtA caused defects in germination and hyphal morphology even in the presence of 100 µg L-1 Mn2+, while overexpression of dmtA led to enhanced germination and normal hyphal morphology at limiting Mn2+ concentration. Growth of both the parent and the deletion strains under citric acid producing conditions resulted in molar yields (Yp/s) of citric acid of > 0.8, although the deletion strain produced ~ 30% less biomass. This yield was reduced only by 20% in the presence of 100 µg L-1 Mn2+, whereas production by the parent strain was reduced by 60%. The Yp/s of the overexpressing strain was 17% of that of the parent strain, irrespective of the concentrations of external Mn2+. CONCLUSIONS: Our results demonstrate that dmtA is physiologically important in the transport of Mn2+ ions in A. niger, and manipulation of its expression modulates citric acid overflow.


Assuntos
Aspergillus niger/metabolismo , Ácido Cítrico/metabolismo , Proteínas Fúngicas/fisiologia , Manganês/metabolismo , Metiltransferases/fisiologia , Biotecnologia/métodos , Fermentação , Proteínas Fúngicas/genética , Mutação com Perda de Função , Metiltransferases/genética
14.
Crit Rev Biotechnol ; 40(2): 199-212, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31903797

RESUMO

Citric acid is considered one of the most valuable weak organic acids on the market and its production by biotechnological approaches is a very interesting topic. Despite the related scientific research, the literature still lacks a state of the art for the technological innovation change, necessary for a study of the inventions designed for real scale implementation. In this context, the present review looks to account for more than 100 worldwide patents (1929-2018), necessary for the identification of the innovative markets and the most promising fields for economic investments. This deepened study identified an increasing invention number, combined with the current worldwide citric acid export flows, with China as the leader (with an economic contribution of 75%, in 2017). In order to satisfy the requests of the market which has moved toward a circular economy, the possibility to use waste substrates represents one of the main options considered in the recent patents. The discussion highlights the sustainability improvement, achieved by the conversion from a submerged technology to a solid-state fermentation (koji process). The listed results are essential for both a scientific audience and the stakeholders involved in citric acid production, in order to have a complete and updated overview of this topic.


Assuntos
Aspergillus niger/metabolismo , Biotecnologia , Ácido Cítrico/metabolismo , Fermentação , China , Invenções
15.
J Biotechnol ; 309: 100-106, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31926980

RESUMO

S-adenosyl-l-methionine (SAM) has been attracting increasing attention because of its significance in the pharmaceutical industry; however, the high cost of this compound limits its application. Tofu yellow serofluid exhibits high nutritional value and is not costly; therefore, it can be utilized as a substrate in the fermentation industry. In the current study, Saccharomyces cerevisiae was cultured in the tofu yellow serofluid fermentation medium for the SAM biosynthesis. The optimum tofu yellow serofluid fermentation medium contained 70 g/L of glucose, 30 % of yellow serofluid, 20 g/L of l-methionine, and 2.5 g/L of ammonium citrate. Under these conditions, the optimum feeding strategy was established. The results revealed that the dry cell weight (DCW) reached 123.1 g/L, the maximum production of SAM was 16.14 g/L, the highest SAM productivity reached 1.048 g/L/h, and SAM content was determined at 131.1 mg/g DCW. Furthermore, addition of tofu yellow serofluid reduced the average cost of SAM by 31.9 % to compare with the culture process without addition of tofu yellow serofluid. Thus, the tofu yellow serofluid fermentation medium improved the production of SAM and significantly reduced the production costs.


Assuntos
Fermentação , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Alimentos de Soja , Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Ácido Cítrico/metabolismo , Meios de Cultura/química , Etanol/metabolismo , Metionina/metabolismo , Compostos de Amônio Quaternário/metabolismo
16.
Nat Commun ; 11(1): 458, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974386

RESUMO

Assembly of the peptidoglycan is crucial in maintaining viability of bacteria and in defining bacterial cell shapes, both of which are important for existence in the ecological niche that the organism occupies. Here, eight crystal structures for a member of the cell-shape-determining class of Campylobacter jejuni, the peptidoglycan peptidase 3 (Pgp3), are reported. Characterization of the turnover chemistry of Pgp3 reveals cell wall D,D-endopeptidase and D,D-carboxypeptidase activities. Catalysis is accompanied by large conformational changes upon peptidoglycan binding, whereby a loop regulates access to the active site. Furthermore, prior hydrolysis of the crosslinked peptide stem from the saccharide backbone of the peptidoglycan on one side is a pre-requisite for its recognition and turnover by Pgp3. These analyses reveal the noncanonical nature of the transformations at the core of the events that define the morphological shape for C. jejuni as an intestinal pathogen.


Assuntos
Campylobacter jejuni/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Ácido Cítrico/química , Ácido Cítrico/metabolismo , Cristalografia por Raios X , Endopeptidases/genética , Hidrólise , Metaloproteases/química , Modelos Moleculares , Mutação , Peptidoglicano/química , Conformação Proteica , Fatores de Virulência/química
17.
Chemosphere ; 247: 125936, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31978664

RESUMO

Silver nanoparticles (AgNPs) can gradually accumulate in algae to exert their toxicity; however, there is little knowledge about their bioaccumulation dynamics. For the first time, this study reports the effect of surface charge of AgNPs on their bioaccumulation dynamics in freshwater algae (Chlorella vulgaris) using biodynamic modeling. Polyethylene-coated AgNPs (PEI-AgNPs) and citrate-coated AgNPs (Cit-AgNPs) were selected as positively and negatively charged AgNPs, i.e., P-AgNPs and N-AgNPs, respectively. Their uptake and elimination dynamics were investigated at a concentration of 50% inhibition of growth rate values (EC50) and 10% inhibition of growth rate values (EC10). The one-component model can generally well simulate the algal uptake and elimination kinetics of N-AgNPs but not of P-AgNPs. At both concentrations, the uptake rate constants (ku) for P-AgNPs were ∼20 times higher than that for N-AgNPs. The parameters of biphasic elimination kinetics revealed that P-AgNPs were eliminated faster than N-AgNPs during depuration compared to in subsequent processes. Compared with N-AgNPs, extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and dark-field imaging revealed that P-AgNPs can be rapidly absorbed on the algal cell surface membrane owing to their remarkably lower energy barrier between algal cells, resulting in a faster adsorption/uptake process and aggregation of algal cells. Our results clearly demonstrate that the AgNPs exhibited surface charge-dependent bioaccumulation dynamics in algal cells. Thus, AgNP surface charge primarily influences the AgNP accumulation dynamics in algal cells.


Assuntos
Chlorella vulgaris/metabolismo , Nanopartículas Metálicas/análise , Prata/metabolismo , Adsorção , Bioacumulação , Chlorella vulgaris/efeitos dos fármacos , Ácido Cítrico/metabolismo , Água Doce
18.
Biosci Biotechnol Biochem ; 84(1): 198-207, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31566090

RESUMO

High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, ß-glucosidase and ß-xylosidase activity in the mutants was ~3, ~2, ~4 and ~2.5 times higher than the parental strain RIB2604 on koji-making conditions, respectively. Citric acid production and mycelia growth of the mutants, however, approximately halved to that of the parent. Compared to the parent, the alcohol yield from rice and sweet potato shochu mash of the mutant increased ~5.7% and 3.0%, respectively. The mutant strains showed significantly low glucose assimilability despite the fructose one was almost normal, and they had a single missense or nonsense mutation in the glucokinase gene glkA. The recombinant strain that was introduced at one of the mutations, glkA Q300K, demonstrated similar but not identical phenotypes to the mutant strain. This result indicates that glkA Q300K is one of the major mutations in 2-DG resistant strains.


Assuntos
Aspergillus/genética , Aspergillus/isolamento & purificação , Separação Celular/métodos , Códon sem Sentido/genética , Genes Fúngicos/genética , alfa-Glucosidases/metabolismo , Aspergillus/classificação , Aspergillus/metabolismo , Catepsina A/metabolismo , Ácido Cítrico/metabolismo , Desoxiglucose/farmacologia , Farmacorresistência Fúngica , Etanol/metabolismo , Fermentação , Alimentos e Bebidas Fermentados/microbiologia , Frutose/metabolismo , Glucoquinase/genética , Glucose/metabolismo , Ipomoea batatas/química , Oryza/química , Fenótipo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Xilosidases/metabolismo , alfa-Amilases/metabolismo , beta-Glucosidase/metabolismo
19.
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
20.
Chem Commun (Camb) ; 56(1): 74-77, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31790117

RESUMO

We developed a new method for the de novo formation of fluorophores based on citrate (DNFC) in biological samples. Use of an amide coupling reagent and microwave irradiation greatly facilitates the fluorophore formation on peptides and proteins with N-terminal cysteine or serine. Since N-terminal cysteine and serine can form thiazolopyridone- or oxazolopyridone-based fluorophores emitting blue and green fluorescence, respectively, by the DNFC staining, each organelle, cell and tissue exhibited a characteristic fluorescence distribution. The DNFC staining is able to provide a new potential protocol for future cell imaging, histology and diagnosis.


Assuntos
Corantes Fluorescentes/metabolismo , Sondas Moleculares/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Linhagem Celular Tumoral , Ácido Cítrico/metabolismo , Cisteína/química , Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Humanos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/química , Células NIH 3T3 , Peptídeos/química , Estudo de Prova de Conceito , Proteínas/química , Piridonas/química , Piridonas/metabolismo , Serina/química , Tiazóis/química , Tiazóis/metabolismo
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