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1.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668723

RESUMO

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glicerol/metabolismo , Malatos/metabolismo , Aerobiose , Bicarbonatos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Cinética , Malato Desidrogenase/metabolismo , Mutação/genética , Fosfoenolpiruvato Carboxilase/metabolismo
2.
PLoS Genet ; 17(1): e1009314, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33493203

RESUMO

The environmental conditions of microorganisms' habitats may fluctuate in unpredictable ways, such as changes in temperature, carbon source, pH, and salinity to name a few. Environmental heterogeneity presents a challenge to microorganisms, as they have to adapt not only to be fit under a specific condition, but they must also be robust across many conditions and be able to deal with the switch between conditions itself. While experimental evolution has been used to gain insight into the adaptive process, this has largely been in either unvarying or consistently varying conditions. In cases where changing environments have been investigated, relatively little is known about how such environments influence the dynamics of the adaptive process itself, as well as the genetic and phenotypic outcomes. We designed a systematic series of evolution experiments where we used two growth conditions that have differing timescales of adaptation and varied the rate of switching between them. We used lineage tracking to follow adaptation, and whole genome sequenced adaptive clones from each of the experiments. We find that both the switch rate and the order of the conditions influences adaptation. We also find different adaptive outcomes, at both the genetic and phenotypic levels, even when populations spent the same amount of total time in the two different conditions, but the order and/or switch rate differed. Thus, in a variable environment adaptation depends not only on the nature of the conditions and phenotypes under selection, but also on the complexity of the manner in which those conditions are combined to result in a given dynamic environment.


Assuntos
Adaptação Fisiológica/genética , Evolução Biológica , Código de Barras de DNA Taxonômico , Seleção Genética/genética , Aclimatação/genética , Análise por Conglomerados , Variação Genética/genética , Genoma Fúngico/genética , Glicerol/metabolismo , Glicerol/farmacologia , Fenótipo , Análise de Componente Principal , Saccharomyces cerevisiae/genética
3.
J Mater Chem B ; 9(2): 295-306, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33398322

RESUMO

The clinical transfusion of red blood cells (RBCs) has provided the greatest number of cryobiology applications in the case of rare blood groups and antibody problems, as well as civil and military disasters. The main technical difficulty with the current clinical technique is the removal of high concentration glycerol (20% or 40%) after thawing. Reducing the probability of intracellular ice formation (IIF) as well as preventing the solution effect are crucial to ensure RBCs avoid cryoinjury. Here, the non-permeating cryoprotectant trehalose was used to dehydrate RBCs before freezing. Furthermore, with the substitution of the low concentration glycerol (5% or 7.5%) for the intracellular remaining water, the bulk of RBCs were successfully cryopreserved to obtain a nearly 95% high survival rate with rapid cooling via EP tubes. Additionally, the washed RBCs after cryopreservation maintained their morphology, deformability, ATP, and 2-3 DPG levels, and all of them met the clinical standards for transfusion safety. Moreover, the whole addition and washing process was simple and easy to operate and could be completed within 30 min, which is crucial for emergency uses. This method will provide more potential for current clinical RBCs cryopreservation practices.


Assuntos
Criopreservação/métodos , Desidratação/genética , Eritrócitos/metabolismo , Glicerol/metabolismo , Trealose/química , Voluntários Saudáveis , Humanos
4.
Nat Commun ; 12(1): 678, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514753

RESUMO

Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. Ex-situ cloning-based methods are commonly used, but the target gene number and combinatorial library size are severely limited by cloning and transformation efficiencies. In-situ methods such as multiplex automated genome engineering (MAGE) depends on high-efficiency transformation and incorporation of heterologous DNA donors, which are limited to few microorganisms. Here, we describe a Base Editor-Targeted and Template-free Expression Regulation (BETTER) method for simultaneously diversifying multigene expression. BETTER repurposes CRISPR-guided base editors and in-situ generates large numbers of genetic combinations of diverse ribosome binding sites, 5' untranslated regions, or promoters, without library construction, transformation, and incorporation of DNA donors. We apply BETTER to simultaneously regulate expression of up to ten genes in industrial and model microorganisms Corynebacterium glutamicum and Bacillus subtilis. Variants with improved xylose catabolism, glycerol catabolism, or lycopene biosynthesis are respectively obtained. This technology will be useful for large-scale fine-tuning of multigene expression in both genetically tractable and intractable microorganisms.


Assuntos
Edição de Genes/métodos , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Família Multigênica/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos/genética , Glicerol/metabolismo , Licopeno/metabolismo , Redes e Vias Metabólicas/genética , Transformação Bacteriana , Xilose/metabolismo
5.
Nucleic Acids Res ; 49(2): 745-759, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367825

RESUMO

Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/genética , Sistemas CRISPR-Cas , Respiração Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/química , Ativação Enzimática , Etanol/metabolismo , Glicerol/metabolismo , Glicólise , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , RNA-Seq , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Fatores de Transcrição/deficiência
6.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348713

RESUMO

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h-1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h-1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.


Assuntos
Aldeído Liases/genética , Vias Biossintéticas/genética , Di-Hidroxiacetona/biossíntese , Escherichia coli K12/enzimologia , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Glicerol/metabolismo , Alelos , Frutosefosfatos/metabolismo , Deleção de Genes , Glucose/metabolismo , Glucosefosfato Desidrogenase/genética , Glicerol Quinase/genética , Isoenzimas/genética , Via de Pentose Fosfato/genética , Fosfofrutoquinases/química , Fosfofrutoquinases/genética , Desidrogenase do Álcool de Açúcar/genética
7.
Nat Commun ; 11(1): 5666, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168815

RESUMO

Aquaporin 3 (AQP3) is a transporter of water, glycerol and hydrogen peroxide (H2O2) that is expressed in various epithelial cells and in macrophages. Here, we developed an anti-AQP3 monoclonal antibody (mAb) that inhibited AQP3-facilitated H2O2 and glycerol transport, and prevented liver injury in experimental animal models. Using AQP3 knockout mice in a model of liver injury and fibrosis produced by CCl4, we obtained evidence for involvement of AQP3 expression in nuclear factor-κB (NF-κB) cell signaling, hepatic oxidative stress and inflammation in macrophages during liver injury. The activated macrophages caused stellate cell activation, leading to liver injury, by a mechanism involving AQP3-mediated H2O2 transport. Administration of an anti-AQP3 mAb, which targeted an extracellular epitope on AQP3, prevented liver injury by inhibition of AQP3-mediated H2O2 transport and macrophage activation. These findings implicate the involvement of macrophage AQP3 in liver injury, and provide evidence for mAb inhibition of AQP3-mediated H2O2 transport as therapy for macrophage-dependent liver injury.


Assuntos
Anticorpos Monoclonais/farmacologia , Aquaporina 3/antagonistas & inibidores , Aquaporina 3/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Aquaporina 3/genética , Células CHO , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Quimiocina CCL4/efeitos adversos , Cricetulus , Modelos Animais de Doenças , Descoberta de Drogas , Glicerol/metabolismo , Peróxido de Hidrogênio/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medicina Molecular , NF-kappa B/metabolismo , Estresse Oxidativo , Transdução de Sinais , Transcriptoma
8.
PLoS Genet ; 16(8): e1008996, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841242

RESUMO

The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Glucose/metabolismo , Quinases da Glicogênio Sintase/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Aspergillus nidulans/enzimologia , Repressão Catabólica/genética , Fungos/genética , Fungos/metabolismo , Glicerol/metabolismo , Concentração Osmolar , Fosforilação/genética , Mapas de Interação de Proteínas/genética , Proteínas Repressoras/genética , Xilose/metabolismo
9.
Nat Commun ; 11(1): 4107, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796836

RESUMO

Foamy macrophages, which have prominent lipid droplets (LDs), are found in a variety of disease states. Toll-like receptor agonists drive triacylglycerol (TG)-rich LD development in macrophages. Here we explore the basis and significance of this process. Our findings indicate that LD development is the result of metabolic commitment to TG synthesis on a background of decreased fatty acid oxidation. TG synthesis is essential for optimal inflammatory macrophage activation as its inhibition, which prevents LD development, has marked effects on the production of inflammatory mediators, including IL-1ß, IL-6 and PGE2, and on phagocytic capacity. The failure of inflammatory macrophages to make PGE2 when TG-synthesis is inhibited is critical for this phenotype, as addition of exogenous PGE2 is able to reverse the anti-inflammatory effects of TG synthesis inhibition. These findings place LDs in a position of central importance in inflammatory macrophage activation.


Assuntos
Inflamação/metabolismo , Lipidômica/métodos , Triglicerídeos/metabolismo , Animais , Células Cultivadas , Citometria de Fluxo , Glucose/metabolismo , Glicerol/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos/fisiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica , Palmitatos/metabolismo , Análise de Sequência de RNA
10.
Arch Biochem Biophys ; 691: 108481, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32735865

RESUMO

Aquaporins (AQPs) facilitate water and glycerol movement across membranes. AQP7 is the main aquaglyceroporin in pancreatic ß-cells and was proposed to play a role in insulin exocytosis. Although AQP7-null mice display adult-onset obesity, impaired insulin secretion and insulin resistance, AQP7 loss-of-function homozygous mutations in humans do not correlate with obesity nor type-2 diabetes. In addition, AQP12 is upregulated in pancreatitis. However, the implication of this isoform in endocrine pancreas inflammation is still unclear. Here, we investigated AQP7 and AQP12 involvement in cellular and inflammatory processes using RIN-m5F beta cells, a model widely used for their high insulin secretion. AQP7 and AQP12 expression were directly associated with cell proliferation, adhesion and migration. While tumor necrosis factor-alpha (TNFα)-induced inflammation impaired AQP7 expression and drastically reduced insulin secretion, lipopolysaccharides (LPS) prompted AQP7 upregulation, and both TNFα and LPS upregulated AQP12. Importantly, cells overexpressing AQP12 are more resistant to inflammation, revealing lower levels of proinflammatory markers. Altogether, these data document AQP7 involvement in insulin secretion and AQP12 implication in inflammation, highlighting their fundamental role in pancreatic ß-cell function.


Assuntos
Aquaporinas/metabolismo , Inflamação/metabolismo , Células Secretoras de Insulina/metabolismo , Fenótipo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Regulação para Baixo/efeitos dos fármacos , Glicerol/metabolismo , Inflamação/induzido quimicamente , Lipopolissacarídeos , Ratos , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Água/metabolismo
11.
Int J Food Microbiol ; 329: 108651, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32512285

RESUMO

In Latin, 'pulcherrima' is a superlative form of an adjective that translates as beautiful. Apart from being 'the most beautiful' yeast, Metschnikowia pulcherrima has a remarkable potential in production of wines with lower ethanol content. The oenological performance of six M. pulcherrima strains was hereby tested in sequential cultures with Saccharomyces cerevisiae. The best-performing strain MP2 was further characterised in fermentations with different S. cerevisiae inoculation delays in both white grape juice and Chemically Defined Grape Juice Medium (CDGJM). The analysis of main metabolites, undertaken prior to sequential inoculations and upon fermentation completion, highlighted metabolic interactions and carbon sinks other than ethanol in MP2 treatments. Depending on the inoculation delay, MP2 white wines contained between 0.6% and 1.2% (v/v) less ethanol than the S. cerevisiae monoculture, with even larger decreases detected in the CDGJM. The MP2 treatments also contained higher concentrations of TCA cycle by-products (i.e. fumarate and succinate) and glycerol, and lower concentrations of acetic acid. The analysis of volatile compounds showed increased production of acetate esters and higher alcohols in all MP2 wines, alongside other compositional alterations arising from the S. cerevisiae inoculation delay.


Assuntos
Fermentação , Microbiologia de Alimentos/métodos , Metschnikowia/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho/microbiologia , Ácido Acético/metabolismo , Etanol/metabolismo , Glicerol/metabolismo , Fatores de Tempo , Vitis/metabolismo , Vitis/microbiologia , Vinho/análise
12.
Obesity (Silver Spring) ; 28(7): 1254-1262, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32568464

RESUMO

OBJECTIVE: The aim of this study was to determine the effects of empagliflozin on glycerol-derived hepatic gluconeogenesis in adults with obesity without type 2 diabetes mellitus (T2DM) using oral carbon 13 (13 C)-labeled glycerol. METHODS: A randomized, double-blind, placebo-controlled trial was performed in participants with magnetic resonance imaging assessment of body fat and measurement of glycerol-derived 13 C enrichment in plasma glucose by nuclear magnetic resonance spectroscopy following ingestion of [U-13 C3 ]glycerol. Participants were randomized to oral empagliflozin 10 mg once daily or placebo for 3 months. Glycerol-derived 13 C enrichment studies were repeated, and treatment differences in the mean percentage of 13 C glycerol enrichment in glucose were compared using mixed linear models. RESULTS: Thirty-five participants completed the study. Empagliflozin increased glycerol-derived 13 C enrichment between baseline and follow-up by 6.5% (P = 0.005), consistent with less glycerol from visceral adipose tissue (VAT). No difference was found with placebo. Glycerol-derived 13 C enrichment was lower in participants with high VAT compared with low VAT by 12.6% (P = 0.04), but there was no heterogeneity of the treatment effect by baseline VAT. Glycerol-derived 13 C enrichment was inversely correlated with VAT but was not correlated with weight loss. CONCLUSIONS: VAT is associated with endogenous glycerol-derived hepatic gluconeogenesis, and empagliflozin reduces endogenous glycerol gluconeogenesis in adults with obesity without T2DM. These findings suggest a mechanism by which sodium-glucose cotransporter 2 inhibitors may prevent T2DM in obesity.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Gluconeogênese/efeitos dos fármacos , Glucosídeos/uso terapêutico , Glicerol/metabolismo , Fígado/efeitos dos fármacos , Obesidade/tratamento farmacológico , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Adulto , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Método Duplo-Cego , Feminino , Humanos , Gordura Intra-Abdominal , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/metabolismo , Placebos , Perda de Peso/efeitos dos fármacos
13.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1101-1112, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597060

RESUMO

As an important platform compound, 3-hydroxypropionic acid (3-HP) can be used as a substrate to synthesize a variety of biological products with commercial potential. The titer of 3-HP by wild-type bacteria is low, which severely limits the large-scale application and production of 3-HP. By modifying the genes related to the metabolic pathway, engineered bacteria using cheap substrates as carbon sources are constructed, the aim of reducing production cost and increasing output is realized. In this paper, the recent progress in the synthesis of 3-HP by metabolic engineering at home and abroad is reviewed. The advantages and disadvantages of glycerol pathway, malonyl-CoA pathway and beta-alanine pathway for synthesis of 3-HP are also summarized and analyzed, and the future development of 3-HP is prospected.


Assuntos
Microbiologia Industrial , Ácido Láctico/análogos & derivados , Engenharia Metabólica , Glicerol/metabolismo , Microbiologia Industrial/tendências , Ácido Láctico/biossíntese , Redes e Vias Metabólicas/genética
14.
PLoS Comput Biol ; 16(6): e1007533, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32542021

RESUMO

Metabolism underpins the pathogenic strategy of the causative agent of TB, Mycobacterium tuberculosis (Mtb), and therefore metabolic pathways have recently re-emerged as attractive drug targets. A powerful approach to study Mtb metabolism as a whole, rather than just individual enzymatic components, is to use a systems biology framework, such as a Genome-Scale Metabolic Network (GSMN) that allows the dynamic interactions of all the components of metabolism to be interrogated together. Several GSMNs networks have been constructed for Mtb and used to study the complex relationship between the Mtb genotype and its phenotype. However, the utility of this approach is hampered by the existence of multiple models, each with varying properties and performances. Here we systematically evaluate eight recently published metabolic models of Mtb-H37Rv to facilitate model choice. The best performing models, sMtb2018 and iEK1011, were refined and improved for use in future studies by the TB research community.


Assuntos
Genoma Bacteriano , Redes e Vias Metabólicas , Mycobacterium tuberculosis/genética , Teorema de Bayes , Biomassa , Carbono/metabolismo , Colesterol/metabolismo , Meios de Cultura , Reações Falso-Positivas , Genótipo , Glicerol/metabolismo , Modelos Biológicos , Mycobacterium tuberculosis/metabolismo , Fenótipo , Valor Preditivo dos Testes , Software , Biologia de Sistemas , Termodinâmica
15.
Arch Anim Nutr ; 74(4): 325-342, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32362138

RESUMO

The objective of this study was to evaluate the effects of in ovo injection with glycerol (GLY) and insulin-like growth factor (IGF-I) on hatchability, biochemical parameters, intestinal morphometry, performance, and carcass characteristics of broiler chickens. A total of 400 fertilised eggs were distributed into five experimental groups. The treatments were arranged as non-injected (control), saline solution injected (0.9% NaCl solution), GLY solution injected (10 nmol/ml), IGF-I solution injected (100 ng/ml), and GLY + IGF-I solution injected. At 17.5 d of incubation, 0.5 ml of each solution was injected into the amniotic fluid of each egg of the injected groups. The injection of different solutions did not influence the hatchability and incubation time of the eggs. Compared to intact eggs, IGF-I and IGF-I+ GLY increased (p < 0.01) the blood IGF-I at hatching. Higher hepatic glycogen was observed (p < 0.05) with GLY or IGF-I. The tested substances decreased (p = 0.02) the fructose 1,6-biphosfate phosphatase activity but did not affect glycaemia. No difference in performance was observed in the first week. Higher feed intake and weight gain with lower feed conversion ratio was obtained ( p  < 0.05) with IGF-I at 14 d. At 21 d, higher weight gain was obtained (p = 0.05) with IGF-I, GLY, IGF-I, and GLY + IGF-I, resulting (p < 0.01) in birds with greater weight gain at 35 and 42 d of age. GLY provided higher villus height in the ileum at hatching and at 7 d of age. The tested solutions increased the relative weight of the liver at hatching. At 42 d of age, no carcass characteristics were influenced. It is concluded that GLY and IGF-I, together or separately, can be used in the in ovo feeding to improve the post-hatch performance of broilers, without affecting hatchability and carcass composition.


Assuntos
Galinhas/fisiologia , Glicerol/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Intestinos/efeitos dos fármacos , Ração Animal/análise , Animais , Peso Corporal/efeitos dos fármacos , Galinhas/anatomia & histologia , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Glicerol/administração & dosagem , Injeções/veterinária , Fator de Crescimento Insulin-Like I/administração & dosagem , Intestinos/anatomia & histologia , Óvulo/efeitos dos fármacos
16.
Appl Environ Microbiol ; 86(15)2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32444469

RESUMO

Rhizobia are nitrogen-fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria in the soil and rhizosphere. Here, we show that free-living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) or the related glycoside SQ-glycerol using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway, resulting in production of sulfolactate (SL) as the major metabolic end product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter, sulfo-ED enzymes, and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates. Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose isomerase and a nonspecific aldehyde reductase, respectively. The discovery of a rhizobium isolate with the ability to degrade SQ builds our knowledge of how these important symbiotic bacteria persist within soil.IMPORTANCE Sulfonate sulfur is a major form of organic sulfur in soils but requires biomineralization before it can be utilized by plants. Very little is known about the biochemical processes used to mobilize sulfonate sulfur. We show that a rhizobial isolate from soil, Rhizobium leguminosarum SRDI565, possesses the ability to degrade the abundant phototroph-derived carbohydrate sulfonate SQ through a sulfoglycolytic Entner-Doudoroff pathway. Proteomics and metabolomics demonstrated the utilization of this pathway during growth on SQ and provided evidence for gluconeogenesis. Unexpectedly, off-cycle sulfoglycolytic species were also detected, pointing to the complexity of metabolic processes within cells under conditions of sulfoglycolysis. Thus, rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.


Assuntos
Proteínas de Bactérias/metabolismo , Glicerol/metabolismo , Metilglucosídeos/metabolismo , Proteoma/metabolismo , Rhizobium leguminosarum/metabolismo , Proteômica
17.
Am J Physiol Endocrinol Metab ; 319(1): E117-E132, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32369418

RESUMO

One of the primary metabolic functions of a mature adipocyte is to supply energy via lipolysis, or the catabolism of stored lipids. Adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL) are critical lipolytic enzymes, and their phosphorylation generates phospho-binding sites for 14-3-3 proteins, a ubiquitously expressed family of molecular scaffolds. Although we previously identified essential roles of the 14-3-3ζ isoform in murine adipogenesis, the presence of 14-3-3 protein binding sites on ATGL and HSL suggests that 14-3-3ζ could also influence mature adipocyte processes like lipolysis. Here we demonstrate that 14-3-3ζ is necessary for lipolysis in male mice and fully differentiated 3T3-L1 adipocytes, as depletion of 14-3-3ζ significantly impaired glycerol and free fatty acid (FFA) release. Unexpectedly, reducing 14-3-3ζ expression was found to significantly impact adipocyte maturity, as observed by reduced abundance of peroxisome proliferator-activated receptor (PPAR)γ2 protein and expression of mature adipocyte genes and those associated with de novo triglyceride synthesis and lipolysis. The impact of 14-3-3ζ depletion on adipocyte maturity was further examined with untargeted lipidomics, which revealed that reductions in 14-3-3ζ abundance promoted the acquisition of a lipidomic signature that resembled undifferentiated preadipocytes. Collectively, these findings reveal a novel aspect of 14-3-3ζ in adipocytes, as reducing 14-3-3ζ was found to have a negative effect on adipocyte maturity and adipocyte-specific processes like lipolysis.


Assuntos
Proteínas 14-3-3/genética , Adipócitos/metabolismo , Adipogenia/genética , Lipólise/genética , Proteínas 14-3-3/metabolismo , Células 3T3-L1 , Animais , Diferenciação Celular , Ácidos Graxos não Esterificados/metabolismo , Glicerol/metabolismo , Lipase/genética , Lipase/metabolismo , Lipidômica , Masculino , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/metabolismo , Esterol Esterase/genética , Esterol Esterase/metabolismo
18.
Int J Food Microbiol ; 324: 108615, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32371236

RESUMO

In recent years, CRISPR/Cas9-based genetic editing has become a mainstay in many laboratories including manipulations done with yeast. We utilized this technique to generate a self-cloned wine yeast strain that overexpresses two genes of oenological relevance i.e. the glycerol-3-phosphate dehydrogenase 1 (GPD1) and the alcohol acetyltransferase 1 (ATF1) directly implicated in glycerol and acetate ester production respectively. Riesling wine made from the resulting strain showed increased glycerol and acetate ester levels compared to the parental strain. In addition, significantly less acetic acid levels were measured in wine made with yeast containing both genetic alterations compared to wine made with the strain that only overexpresses GPD1. Thus, this strain provides an alternative strategy for alleviating the accumulation of acetic acid once glycerol production is favoured during alcoholic fermentation with the addition of dramatically increasing acetate esters production.


Assuntos
Sistemas CRISPR-Cas , Saccharomyces cerevisiae/genética , Vinho/microbiologia , Ácido Acético/análise , Ácido Acético/metabolismo , Fermentação , Edição de Genes , Glicerol/análise , Glicerol/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Fenótipo , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vinho/análise
19.
World J Microbiol Biotechnol ; 36(3): 49, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157439

RESUMO

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.


Assuntos
Glicerol/metabolismo , Engenharia Metabólica/métodos , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Aquaporinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capsicum/microbiologia , China , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Glicerolfosfato Desidrogenase/genética , Redes e Vias Metabólicas/genética , Fenazinas/metabolismo , Proteínas Repressoras/genética , Rizosfera
20.
Science ; 367(6482): 1147-1151, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139546

RESUMO

Mycobacterium tuberculosis has an unusual outer membrane that lacks canonical porin proteins for the transport of small solutes to the periplasm. We discovered that 3,3-bis-di(methylsulfonyl)propionamide (3bMP1) inhibits the growth of M. tuberculosis, and resistance to this compound is conferred by mutation within a member of the proline-proline-glutamate (PPE) family, PPE51. Deletion of PPE51 rendered M. tuberculosis cells unable to replicate on propionamide, glucose, or glycerol. Growth was restored upon loss of the mycobacterial cell wall component phthiocerol dimycocerosate. Mutants in other proline-glutamate (PE)/PPE clusters, responsive to magnesium and phosphate, also showed a phthiocerol dimycocerosate-dependent growth compromise upon limitation of the corresponding substrate. Phthiocerol dimycocerosate determined the low permeability of the mycobacterial outer membrane, and the PE/PPE proteins apparently act as solute-specific channels.


Assuntos
Amidas/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Permeabilidade da Membrana Celular , Farmacorresistência Bacteriana/genética , Deleção de Genes , Lipídeos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia
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