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1.
World J Microbiol Biotechnol ; 35(7): 109, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280382

RESUMO

Echinocandin B (ECB) is an important lipohexapeptide used for chemical manufacture of the antifungal agent anidulafungin. Sterigmatocystin (ST) is a polyketide mycotoxin produced by certain species of Aspergillus such as Aspergillus delacroxii SIPIW15, which could produce both ECB and ST. However, the presence of the potent carcinogen ST will greatly affect the quality and safety of ECB production. Therefore, it is essential to eliminate the ST biosynthesis and increase ECB titers in Asp. delacroxii SIPIW15. In this study, the polyketide synthase gene (stcA) required for biosynthesis of ST and its flanking region in Asp. delacroxii SIPIW15 were cloned, sequenced and analyzed firstly. Based on Agrobacterium-mediated transformation, the ΔstcA mutant AMT-1 was obtained and its yield of ECB was increased by 40% without ST detected at the same time as compared to the original strain. The results of the fed-batch experiments showed that the ECB yield of the ΔstcA strain AMT-1 was increased to 2163 ± 31 mg/l and no ST was detected in the 50 l bioreactor. This work suggested that the ΔstcA strain AMT-1 has the potential for application in ECB production improvement, and more importantly, to eliminate ST-related environmental pollution in ECB fermentation industry.


Assuntos
Aspergillus/genética , Aspergillus/metabolismo , Equinocandinas/biossíntese , Equinocandinas/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Policetídeo Sintases/genética , Esterigmatocistina/biossíntese , Agrobacterium/genética , Anidulafungina , Antifúngicos , Sequência de Bases , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , DNA Fúngico/isolamento & purificação , Fermentação , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Metabolismo Secundário/genética , Transformação Genética
2.
Nat Commun ; 10(1): 2760, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235787

RESUMO

Heart failure is a leading cause of mortality, yet our understanding of the genetic interactions underlying this disease remains incomplete. Here, we harvest 1352 healthy and failing human hearts directly from transplant center operating rooms, and obtain genome-wide genotyping and gene expression measurements for a subset of 313. We build failing and non-failing cardiac regulatory gene networks, revealing important regulators and cardiac expression quantitative trait loci (eQTLs). PPP1R3A emerges as a regulator whose network connectivity changes significantly between health and disease. RNA sequencing after PPP1R3A knockdown validates network-based predictions, and highlights metabolic pathway regulation associated with increased cardiomyocyte size and perturbed respiratory metabolism. Mice lacking PPP1R3A are protected against pressure-overload heart failure. We present a global gene interaction map of the human heart failure transition, identify previously unreported cardiac eQTLs, and demonstrate the discovery potential of disease-specific networks through the description of PPP1R3A as a central regulator in heart failure.


Assuntos
Redes Reguladoras de Genes/genética , Insuficiência Cardíaca/genética , Miócitos Cardíacos/patologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Benzenoacetamidas , Células Cultivadas , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fosfoproteínas Fosfatases/genética , Cultura Primária de Células , Piridinas , Locos de Características Quantitativas/genética , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA/métodos
3.
Nat Commun ; 10(1): 2701, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221965

RESUMO

One of the biggest hurdles for the development of metabolism-targeted therapies is to identify the responsive tumor subsets. However, the metabolic vulnerabilities for most human cancers remain unclear. Establishing the link between metabolic signatures and the oncogenic alterations of receptor tyrosine kinases (RTK), the most well-defined cancer genotypes, may precisely direct metabolic intervention to a broad patient population. By integrating metabolomics and transcriptomics, we herein show that oncogenic RTK activation causes distinct metabolic preference. Specifically, EGFR activation branches glycolysis to the serine synthesis for nucleotide biosynthesis and redox homeostasis, whereas FGFR activation recycles lactate to fuel oxidative phosphorylation for energy generation. Genetic alterations of EGFR and FGFR stratify the responsive tumors to pharmacological inhibitors that target serine synthesis and lactate fluxes, respectively. Together, this study provides the molecular link between cancer genotypes and metabolic dependency, providing basis for patient stratification in metabolism-targeted therapies.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Mutação com Ganho de Função , Perfilação da Expressão Gênica/métodos , Glicólise/efeitos dos fármacos , Glicólise/genética , Homeostase/efeitos dos fármacos , Homeostase/genética , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Seleção de Pacientes , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Serina/biossíntese , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biochim Biophys Acta Rev Cancer ; 1872(1): 24-36, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31152822

RESUMO

Cancer cells constantly face a fluctuating nutrient supply and interference with adaptive responses might be an effective therapeutic approach. It has been discovered that in the absence of glucose, cancer cells can synthesize crucial metabolites by expressing phosphoenolpyruvate carboxykinase (PEPCK, PCK1 or PCK2) using abbreviated forms of gluconeogenesis. Gluconeogenesis, which in essence is the reverse pathway of glycolysis, uses lactate or amino acids to feed biosynthetic pathways branching from glycolysis. PCK1 and PCK2 have been shown to be critical for the growth of certain cancers. In contrast, fructose-1,6-bisphosphatase 1 (FBP1), a downstream gluconeogenesis enzyme, inhibits glycolysis and tumor growth, partly by non-enzymatic mechanisms. This review sheds light on the current knowledge of cancer cell gluconeogenesis and its role in metabolic reprogramming, cancer cell plasticity, and tumor growth.


Assuntos
Proliferação de Células/genética , Gluconeogênese/genética , Redes e Vias Metabólicas/genética , Neoplasias/genética , Aminoácidos/metabolismo , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Glucose/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo
5.
BMC Plant Biol ; 19(1): 252, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185903

RESUMO

BACKGROUND: Thermo-sensitive male-sterility based on Aegilops kotschyi cytoplasm (K-TCMS) plays an important role in hybrid wheat breeding. This has important possible applications in two-line hybrid wheat breeding but the genetic basis and molecular regulation mechanism related to fertility restoration are poorly understood. In this study, comparative transcriptome profiling based on RNA sequencing was conducted for two near-isogenic lines comprising KTM3315R and its sterile counterpart KTM3315A, a total of six samples (3 repetitions per group), in order to identify fertility restoration genes and their metabolic pathways. RESULTS: In total, 2642 significant differentially expressed genes (DEGs) were detected, among which 1238 were down-regulated and 1404 were up-regulated in fertile anthers. Functional annotation enrichment analysis identified important pathways related to fertility restoration, such as carbohydrate metabolism, phenylpropanoid metabolism and biosynthesis, as well as candidate genes encoding pectin methylesterase and flavanone 3-hydroxylase. Moreover, transcription factor analysis showed that a large number of DEGs were mainly involved with the WRKY, bHLH, and MYB transcription factor families. Determination of total soluble sugar and flavonoid contents demonstrated that important metabolic pathways and candidate genes are associated with fertility restoration. Twelve DEGs were selected and detected by quantitative reverse-transcribed PCR, and the results indicated that the transcriptome sequencing results were reliable. CONCLUSIONS: Our results indicate that identified DEGs were related to the fertility restoration and they proved to be crucial in Aegilops kotschyi cytoplasm. These findings also provide a basis for exploring the molecular regulation mechanism associated with wheat fertility restoration as well as screening and cloning related genes.


Assuntos
Aegilops/genética , Melhoramento Vegetal , Infertilidade das Plantas/genética , Proteínas de Plantas/genética , Transcriptoma , Triticum/genética , Citoplasma/fisiologia , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genética , Proteínas de Plantas/metabolismo , Triticum/fisiologia
6.
Genome Biol ; 20(1): 121, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196170

RESUMO

Genome-scale metabolic models (GEMs) computationally describe gene-protein-reaction associations for entire metabolic genes in an organism, and can be simulated to predict metabolic fluxes for various systems-level metabolic studies. Since the first GEM for Haemophilus influenzae was reported in 1999, advances have been made to develop and simulate GEMs for an increasing number of organisms across bacteria, archaea, and eukarya. Here, we review current reconstructed GEMs and discuss their applications, including strain development for chemicals and materials production, drug targeting in pathogens, prediction of enzyme functions, pan-reactome analysis, modeling interactions among multiple cells or organisms, and understanding human diseases.


Assuntos
Genômica/tendências , Redes e Vias Metabólicas/genética , Modelos Biológicos , Animais , Humanos
7.
Microb Cell Fact ; 18(1): 104, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31170985

RESUMO

BACKGROUND: Methylocystis parvus is a type II methanotroph characterized by its high specific methane degradation rate (compared to other methanotrophs of the same family) and its ability to accumulate up to 50% of its biomass in the form of poly-3-hydroxybutyrate (PHB) under nitrogen limiting conditions. This makes it a very promising cell factory. RESULTS: This article reports the first Genome Scale Metabolic Model of M. parvus OBBP. The model is compared to Genome Scale Metabolic Models of the closely related methanotrophs Methylocystis hirsuta and Methylocystis sp. SC2. Using the reconstructed model, it was possible to predict the biomass yield of M. parvus on methane. The prediction was consistent with the observed experimental yield, under the assumption of the so called "redox arm mechanism" for methane oxidation. The co-consumption of stored PHB and methane was also modeled, leading to accurate predictions of biomass yields and oxygen consumption rates and revealing an anaplerotic role of PHB degradation. Finally, the model revealed that anoxic PHB consumption has to be coupled to denitrification, as no fermentation of PHB is allowed by the reconstructed metabolic model. CONCLUSIONS: The "redox arm" mechanism appears to be a general characteristic of type II methanotrophs, versus type I methanotrophs that use the "direct coupling" mechanism. The co-consumption of stored PHB and methane was predicted to play an anaplerotic role replenishing the serine cycle with glyoxylate and the TCA cycle with succinyl-CoA, which allows the withdrawal of metabolic precursors for biosynthesis. The stored PHB can be also used as an energy source under anoxic conditions when coupled to denitrification.


Assuntos
Hidroxibutiratos/metabolismo , Redes e Vias Metabólicas/genética , Metano/metabolismo , Methylocystaceae/metabolismo , Oxigênio/metabolismo , Poliésteres/metabolismo , Methylocystaceae/genética
8.
Nat Commun ; 10(1): 2581, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197173

RESUMO

Despite existing reports on differential DNA methylation in type 2 diabetes (T2D) and obesity, our understanding of its functional relevance remains limited. Here we show the effect of differential methylation in the early phases of T2D pathology by a blood-based epigenome-wide association study of 4808 non-diabetic Europeans in the discovery phase and 11,750 individuals in the replication. We identify CpGs in LETM1, RBM20, IRS2, MAN2A2 and the 1q25.3 region associated with fasting insulin, and in FCRL6, SLAMF1, APOBEC3H and the 15q26.1 region with fasting glucose. In silico cross-omics analyses highlight the role of differential methylation in the crosstalk between the adaptive immune system and glucose homeostasis. The differential methylation explains at least 16.9% of the association between obesity and insulin. Our study sheds light on the biological interactions between genetic variants driving differential methylation and gene expression in the early pathogenesis of T2D.


Assuntos
Metilação de DNA/fisiologia , Diabetes Mellitus Tipo 2/genética , Glucose/metabolismo , Insulina/metabolismo , Obesidade/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Simulação por Computador , Ilhas de CpG/genética , Diabetes Mellitus Tipo 2/metabolismo , Epigênese Genética/fisiologia , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Estudo de Associação Genômica Ampla/métodos , Homeostase/genética , Humanos , Masculino , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Obesidade/metabolismo , Polimorfismo de Nucleotídeo Único/fisiologia , Adulto Jovem
9.
BMC Bioinformatics ; 20(1): 227, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060515

RESUMO

BACKGROUND: Serratia marcescens is a chitinolytic bacterium that can potentially be used for consolidated bioprocessing to convert chitin to value-added chemicals. Currently, S. marcescens is poorly characterized and studies on intracellular metabolic and regulatory mechanisms would expedite development of bioprocessing applications. RESULTS: In this study, our goal was to characterize the metabolic profile of S. marcescens to provide insight for metabolic engineering applications and fundamental biological studies. Hereby, we constructed a constraint-based genome-scale metabolic model (iSR929) including 929 genes, 1185 reactions and 1164 metabolites based on genomic annotation of S. marcescens Db11. The model was tested by comparing model predictions with experimental data and analyzed to identify essential aspects of the metabolic network (e.g. 138 essential genes predicted). The model iSR929 was refined by integrating RNAseq data of S. marcescens growth on three different carbon sources (glucose, N-acetylglucosamine, and glycerol). Significant differences in TCA cycle utilization were found for growth on the different carbon substrates, For example, for growth on N-acetylglucosamine, S. marcescens exhibits high pentose phosphate pathway activity and nucleotide synthesis but low activity of the TCA cycle. CONCLUSIONS: Our results show that S. marcescens model iSR929 can provide reasonable predictions and can be constrained to fit with experimental values. Thus, our model may be used to guide strain designs for metabolic engineering to produce chemicals such as 2,3-butanediol, N-acetylneuraminic acid, and n-butanol using S. marcescens.


Assuntos
Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Serratia marcescens/genética
10.
Int J Mol Sci ; 20(9)2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-31060285

RESUMO

Internode length is an important agronomic trait affecting plant architecture and crop yield. However, few genes for internode elongation have been identified in tomato. In this study, we characterized an elongated internode inbred line P502, which is a natural mutant of the tomato cultivar 05T606. The mutant P502 exhibits longer internode and higher bioactive GA concentration compared with wild-type 05T606. Genetic analysis suggested that the elongated internode trait is controlled by quantitative trait loci (QTL). Then, we identified a major QTL on chromosome 2 based on molecular markers and bulked segregant analysis (BSA). The locus was designated as EI (Elongated Internode), which explained 73.6% genetic variance. The EI was further mapped to a 75.8-kb region containing 10 genes in the reference Heinz 1706 genome. One single nucleotide polymorphism (SNP) in the coding region of solyc02g080120.1 was identified, which encodes gibberellin 2-beta-dioxygenase 7 (SlGA2ox7). SlGA2ox7, orthologous to AtGA2ox7 and AtGA2ox8, is involved in the regulation of GA degradation. Overexpression of the wild EI gene in mutant P502 caused a dwarf phenotype with a shortened internode. The difference of EI expression levels was not significant in the P502 and wild-type, but the expression levels of GA biosynthetic genes including CPS, KO, KAO, GA20ox1, GA20ox2, GA20ox4, GA3ox1, GA2ox1, GA2ox2, GA2ox4, and GA2ox5, were upregulated in mutant P502. Our results may provide a better understanding of the genetics underlying the internode elongation and valuable information to improve plant architecture of the tomato.


Assuntos
Estudos de Associação Genética , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Locos de Características Quantitativas , Característica Quantitativa Herdável , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Endogamia , Lycopersicon esculentum/metabolismo , Redes e Vias Metabólicas/genética , Mutação , Filogenia
11.
Fungal Biol ; 123(5): 397-407, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31053329

RESUMO

Fungal secondary metabolites have important functions for the fungi that produce them, such as roles in virulence and competition. The hemibiotrophic pine needle pathogen Dothistroma septosporum has one of the lowest complements of secondary metabolite (SM) backbone genes of plant pathogenic fungi, indicating that this fungus produces a limited range of SMs. Amongst these SMs is dothistromin, a well-characterised polyketide toxin and virulence factor that is required for expansion of disease lesions in Dothistroma needle blight disease. Dothistromin genes are dispersed across six loci on one chromosome, rather than being clustered as for most SM genes. We explored other D. septosporum SM genes to determine if they are associated with gene clusters, and to predict what their likely products and functions might be. Of nine functional SM backbone genes in the D. septosporum genome, only four were expressed under a range of in planta and in culture conditions, one of which was the dothistromin PKS backbone gene. Of the other three expressed genes, gene knockout studies suggested that DsPks1 and DsPks2 are not required for virulence and attempts to determine a functional squalestatin-like SM product for DsPks2 were not successful. However preliminary evidence suggested that DsNps3, the only SM backbone gene to be most highly expressed in the early stage of disease, appears to be a virulence factor. Thus, despite the small number of SM backbone genes in D. septosporum, most of them appear to be poorly expressed or dispensable for virulence in planta. This work contributes to a growing body of evidence that many fungal secondary metabolite gene clusters might be non-functional and may be evolutionary relics.


Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Redes e Vias Metabólicas/genética , Metabolismo Secundário , Antraquinonas/metabolismo , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/isolamento & purificação , Perfilação da Expressão Gênica , Família Multigênica , Pinus/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia
12.
Mar Drugs ; 17(5)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035409

RESUMO

Short chain fatty acids (SCFAs) are valued as a functional material in cosmetics. Cyanobacteria can accumulate SCFAs under some conditions, the related mechanism is unclear. Two potential genes Synpcc7942_0537 (fabB/F) and Synpcc7942_1455 (fabH) in Synechococcus sp. PCC 7942 have homology with fabB/F and fabH encoding ß-ketoacyl ACP synthases (I/II/III) in plants. Therefore, effects of culture time and cerulenin on SCFAs accumulation, expression levels and functions of these two potential genes were studied. The results showed Synechococcus sp. PCC 7942 accumulated high SCFAs (C12 + C14) in early growth stage (day 4) and at 7.5g/L cerulenin concentration, reaching to 2.44% and 2.84% of the total fatty acids respectively, where fabB/F expression was down-regulated. Fatty acid composition analysis showed C14 increased by 65.19% and 130% respectively, when fabB/F and fabH were antisense expressed. C14 increased by 10.79% (fab(B/F)-) and 6.47% (fabH-) under mutation conditions, while C8 increased by six times in fab(B/F)- mutant strain. These results suggested fabB/F is involved in fatty acid elongation (C <18) and the elongation of cis-16:1 to cis-18:1 fatty acid in Synechococcus sp. PCC 7942, while fabH was involved in elongation of fatty acid synthesis, which were further confirmed in complementary experiments of E. coli. The research could provide the scientific basis for the breeding of SCFA-rich microalgae species.


Assuntos
Ácidos Graxos Voláteis/biossíntese , Microalgas/metabolismo , Synechococcus/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cosméticos/química , Redes e Vias Metabólicas/genética , Microalgas/genética , Homologia de Sequência de Aminoácidos , Synechococcus/genética
13.
Chin J Nat Med ; 17(4): 275-290, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31076131

RESUMO

Danggui Buxue Tang (DBT) is a famous Chinese medicinal decoction. Mechanism of DBT action is wide ranging and unclear. Exploring new ways of treatment with DBT is useful. Sprague-Dawley(SD) rats were randomly divided into 3 groups including control (NC, Saline), the DBT (at a dose of 8.10 g-1), and blood deficiency(BD) (Cyclophosphamide (APH)-andCyclophosphamide(CTX)-induced anaemia). A metabolomics approach using Liquid Chromatography-Quadrupole-Time-of-Flight/Mass Spectrometry (LC/Q-TOFMS) was developed to perform the plasma metabolic profiling analysis and differential metaboliteswerescreened according to the multivariate statistical analysiscomparing the NC and BD groups, andthe hub metabolites were outliers with high scores of the centrality indices. Anaemia disease-related protein target and compound of DBT databases were constructed. The TCMSP, ChemMapper and STITCH databases were used to predict the protein targets of DBT. Using the Cytoscape 3.2.1 to establish a phytochemical component-target protein interaction network and establish a component, protein and hub metabolite protein-protein interaction (PPI) network and merging the three PPI networks basing on BisoGenet. The gene enrichment analysis was used to analyse the relationship between proteins based on the relevant genetic similarity by ClueGO. The results shown DBT effectively treated anaemia in vivo. 11 metabolic pathways are involved in the therapeutic effect of DBT in vivo; S-adenosyl-l-methionine, glycine, l-cysteine, arachidonic acid (AA) and phosphatidylcholine(PC) were screened as hub metabolites in APH-and CTX-induced anaemia. A total of 288 targets were identified as major candidates for anaemia progression. The gene-set enrichment analysis revealed that the targets are involved in iron ion binding, haemopoiesis, reactive oxygen species production, inflammation and apoptosis. The results also showed that these targets were associated with iron ion binding, haemopoiesis, ROS production, apoptosis, inflammation and related signalling pathways. DBT can promote iron ion binding and haemopoiesis activities, restrain inflammation, production of reactive oxygen, block apoptosis, and contribute significantly to the DBT treat anaemia.


Assuntos
Anemia/tratamento farmacológico , Anemia/metabolismo , Medicamentos de Ervas Chinesas/uso terapêutico , Metaboloma/efeitos dos fármacos , Metabolômica , Anemia/sangue , Anemia/induzido quimicamente , Animais , Cromatografia Líquida , Ciclofosfamida/toxicidade , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem
14.
BMC Plant Biol ; 19(1): 219, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31132986

RESUMO

BACKGROUND: Mature fruit cracking during the normal season in African Pride (AP) atemoya is a major problem in postharvest storage. Our current understanding of the molecular mechanism underlying fruit cracking is limited. The aim of this study was to unravel the role starch degradation and cell wall polysaccharide metabolism in fruit ripening and cracking after harvest through transcriptome analysis. RESULTS: Transcriptome analysis of AP atemoya pericarp from cracking fruits of ethylene treatments and controls was performed. KEGG pathway analysis revealed that the starch and sucrose metabolism pathway was significantly enriched, and approximately 39 DEGs could be functionally annotated, which included starch, cellulose, pectin, and other sugar metabolism-related genes. Starch, protopectin, and soluble pectin contents among the different cracking stages after ethylene treatment and the controls were monitored. The results revealed that ethylene accelerated starch degradation, inhibited protopectin synthesis, and enhanced the soluble pectin content, compared to the control, which coincides with the phenotype of ethylene-induced fruit cracking. Key genes implicated in the starch, pectin, and cellulose degradation were further investigated using RT-qPCR analysis. The results revealed that alpha-amylase 1 (AMY1), alpha-amylase 3 (AMY3), beta-amylase 1 (BAM1), beta-amylase 3 (BAM3), beta-amylase 9 (BAM9), pullulanase (PUL), and glycogen debranching enzyme (glgX), were the major genes involved in starch degradation. AMY1, BAM3, BAM9, PUL, and glgX all were upregulated and had higher expression levels with ethylene treatment compared to the controls, suggesting that ethylene treatment may be responsible for accelerating starch degradation. The expression profile of alpha-1,4-galacturonosyltransferase (GAUT) and granule-bound starch synthase (GBSS) coincided with protopectin content changes and could involve protopectin synthesis. Pectinesterase (PE), polygalacturonase (PG), and pectate lyase (PEL) all involved in pectin degradation; PE was significantly upregulated by ethylene and was the key enzyme implicated pectin degradation. CONCLUSION: Both KEGG pathway enrichment analysis of DEGs and material content analysis confirmed that starch decomposition into soluble sugars and cell wall polysaccharides metabolism are closely related to the ripening and cracking of AP atemoya. A link between gene up- or downregulation during different cracking stages of atemoya fruits and how their expression affects starch and pectin contents were established by RT-qPCR analysis.


Assuntos
Annona/genética , Etilenos/farmacologia , Frutas/crescimento & desenvolvimento , Reguladores de Crescimento de Planta/farmacologia , Polissacarídeos/metabolismo , Annona/metabolismo , Etilenos/administração & dosagem , Frutas/genética , Frutas/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas , Redes e Vias Metabólicas/genética
15.
BMC Plant Biol ; 19(1): 222, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138172

RESUMO

BACKGROUND: Forage and turf grasses are routinely cut and grazed upon throughout their lifecycle. When grasses are cut or damaged, they rapidly release a volatile chemical cocktail called green leaf volatiles (GLV). Previously we have shown that mechanical wounding or exposure to GLV released from cut grass, activated a Lt 46 kDa mitogen-activated protein kinase (MAPK) within 3 min and a 44 kDa MAPK within 15-20 min in the model grass species Lolium temulentum (Lt). Currently very little is known concerning the perception, signaling or molecular responses associated with wound stress in grasses. Since GLV are released during wounding, we wanted to investigate what genes and signaling pathways would be induced in undamaged plants exposed to GLV. RESULTS: RNA-Seq generated transcriptome of Lolium plants exposed to GLV identified 4308 up- and 2794 down-regulated distinct differentially-expressed sequences (DES). Gene Ontology analysis revealed a strong emphasis on signaling, response to stimulus and stress related categories. Transcription factors and kinases comprise over 13% of the total DES found in the up-regulated dataset. The analysis showed a strong initial burst within the first hour of GLV exposure with over 60% of the up-regulated DES being induced. Specifically sequences annotated for enzymes involved in the biosynthesis of jasmonic acid and other plant hormones, mitogen-activated protein kinases and WRKY transcription factors were identified. Interestingly, eleven DES for ferric reductase oxidase, an enzyme involved in iron uptake and transport, were exclusively found in the down-regulated dataset. Twelve DES of interest were selected for qRT-PCR analysis; all displayed a rapid induction one hour after GLV exposure and were also strongly induced by mechanical wounding. CONCLUSION: The information gained from the analysis of this transcriptome and previous studies suggests that GLV released from cut grasses transiently primes an undamaged plant's wound stress pathways for potential oncoming damage, and may have a dual role for inter- as well as intra-plant signaling.


Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Lolium/genética , Transcriptoma , Compostos Orgânicos Voláteis/farmacologia , Perfilação da Expressão Gênica , Genes de Plantas/genética , Lolium/metabolismo , Redes e Vias Metabólicas/genética , Folhas de Planta/química , Transdução de Sinais/genética , Compostos Orgânicos Voláteis/química
16.
Microb Cell Fact ; 18(1): 88, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122246

RESUMO

BACKGROUND: There have been many successful strategies to implement xylose metabolism in Saccharomyces cerevisiae, but no effort has so far enabled xylose utilization at rates comparable to that of glucose (the preferred sugar of this yeast). Many studies have pointed towards the engineered yeast not sensing that xylose is a fermentable carbon source despite growing and fermenting on it, which is paradoxical. We have previously used fluorescent biosensor strains to in vivo monitor the sugar signalome in yeast engineered with xylose reductase and xylitol dehydrogenase (XR/XDH) and have established that S. cerevisiae senses high concentrations of xylose with the same signal as low concentration of glucose, which may explain the poor utilization. RESULTS: In the present study, we evaluated the effects of three deletions (ira2∆, isu1∆ and hog1∆) that have recently been shown to display epistatic effects on a xylose isomerase (XI) strain. Through aerobic and anaerobic characterization, we showed that the proposed effects in XI strains were for the most part also applicable in the XR/XDH background. The ira2∆isu1∆ double deletion led to strains with the highest specific xylose consumption- and ethanol production rates but also the lowest biomass titre. The signalling response revealed that ira2∆isu1∆ changed the low glucose-signal in the background strain to a simultaneous signalling of high and low glucose, suggesting that engineering of the signalome can improve xylose utilization. CONCLUSIONS: The study was able to correlate the previously proposed beneficial effects of ira2∆, isu1∆ and hog1∆ on S. cerevisiae xylose uptake, with a change in the sugar signalome. This is in line with our previous hypothesis that the key to resolve the xylose paradox lies in the sugar sensing and signalling networks. These results indicate that the future engineering targets for improved xylose utilization should probably be sought not in the metabolic networks, but in the signalling ones.


Assuntos
Glucose , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae , Xilose , Transporte Biológico , Fermentação , Deleção de Genes , Glucose/genética , Glucose/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Plasmídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Xilose/genética , Xilose/metabolismo
17.
Microb Cell Fact ; 18(1): 90, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122258

RESUMO

BACKGROUND: Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (L-glutamate, L-aspartate, L-leucine and L-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear. RESULTS: In this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of L-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of L-leucine and L-valine, not only improved surfactin production but also increased the proportion of the C14 isoform. CONCLUSIONS: This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals.


Assuntos
Aminoácidos , Bacillus subtilis , Lipopeptídeos , Redes e Vias Metabólicas/genética , Tensoativos/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/genética , Lipopeptídeos/biossíntese , Lipopeptídeos/genética
18.
PLoS Comput Biol ; 15(4): e1006867, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30986217

RESUMO

Genome-scale metabolic models provide a valuable context for analyzing data from diverse high-throughput experimental techniques. Models can quantify the activities of diverse pathways and cellular functions. Since some metabolic reactions are only catalyzed in specific environments, several algorithms exist that build context-specific models. However, these methods make differing assumptions that influence the content and associated predictive capacity of resulting models, such that model content varies more due to methods used than cell types. Here we overcome this problem with a novel framework for inferring the metabolic functions of a cell before model construction. For this, we curated a list of metabolic tasks and developed a framework to infer the activity of these functionalities from transcriptomic data. We protected the data-inferred tasks during the implementation of diverse context-specific model extraction algorithms for 44 cancer cell lines. We show that the protection of data-inferred metabolic tasks decreases the variability of models across extraction methods. Furthermore, resulting models better capture the actual biological variability across cell lines. This study highlights the potential of using biological knowledge, inferred from omics data, to obtain a better consensus between existing extraction algorithms. It further provides guidelines for the development of the next-generation of data contextualization methods.


Assuntos
Redes e Vias Metabólicas , Modelos Biológicos , Algoritmos , Animais , Linhagem Celular Tumoral , Biologia Computacional , Interpretação Estatística de Dados , Perfilação da Expressão Gênica , Genômica/estatística & dados numéricos , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Humanos , Redes e Vias Metabólicas/genética , Neoplasias/genética , Neoplasias/metabolismo , Análise de Componente Principal
19.
Methods Mol Biol ; 1945: 141-160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945245

RESUMO

ML-Rules is a rule-based language for multi-level modeling and simulation. ML-Rules supports dynamic nesting of entities and applying arbitrary functions on entity attributes and content, as well as for defining kinetics of reactions. This allows describing and simulating complex cellular dynamics operating at different organizational levels, e.g., to combine intra-, inter-, and cellular dynamics, like the proliferation of cells, or to include compartmental dynamics like merging and splitting of mitochondria or endocytosis. The expressiveness of the language is bought with additional efforts in executing ML-Rules models. Therefore, various simulators have been developed from which the user and automatic procedures can select. The experiment specification language SESSL facilitates design, execution, and reuse of simulation experiments. The chapter illuminates the specific features of ML-Rules as a rule-based modeling language, the implications for an efficient execution, and shows ML-Rules at work.


Assuntos
Biologia Computacional/métodos , Modelos Biológicos , Software , Algoritmos , Proliferação de Células/genética , Simulação por Computador , Endocitose/genética , Humanos , Cinética , Redes e Vias Metabólicas/genética , Mitocôndrias/genética
20.
MBio ; 10(2)2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967465

RESUMO

Methylomicrobium buryatense 5GB1 is an obligate methylotroph which grows on methane or methanol with similar growth rates. It has long been assumed that the core metabolic pathways must be similar on the two substrates, but recent studies of methane metabolism in this bacterium suggest that growth on methanol might have significant differences from growth on methane. In this study, both a targeted metabolomics approach and a 13C tracer approach were taken to understand core carbon metabolism in M. buryatense 5GB1 during growth on methanol and to determine whether such differences occur. Our results suggest a systematic shift of active core metabolism in which increased flux occurred through both the Entner-Doudoroff (ED) pathway and the partial serine cycle, while the tricarboxylic acid (TCA) cycle was incomplete, in contrast to growth on methane. Using the experimental results as constraints, we applied flux balance analysis to determine the metabolic flux phenotype of M. buryatense 5GB1 growing on methanol, and the results are consistent with predictions based on ATP and NADH changes. Transcriptomics analysis suggested that the changes in fluxes and metabolite levels represented results of posttranscriptional regulation. The combination of flux balance analysis of the genome-scale model and the flux ratio from 13C data changed the solution space for a better prediction of cell behavior and demonstrated the significant differences in physiology between growth on methane and growth on methanol.IMPORTANCE One-carbon compounds such as methane and methanol are of increasing interest as sustainable substrates for biological production of fuels and industrial chemicals. The bacteria that carry out these conversions have been studied for many decades, but gaps exist in our knowledge of their metabolic pathways. One such gap is the difference between growth on methane and growth on methanol. Understanding such metabolism is important, since each has advantages and disadvantages as a feedstock for production of chemicals and fuels. The significance of our research is in the demonstration that the metabolic network is substantially altered in each case and in the delineation of these changes. The resulting new insights into the core metabolism of this bacterium now provide an improved basis for future strain design.


Assuntos
Regulação Bacteriana da Expressão Gênica , Metano/metabolismo , Metanol/metabolismo , Methylococcaceae/genética , Methylococcaceae/metabolismo , Isótopos de Carbono/análise , Perfilação da Expressão Gênica , Marcação por Isótopo , Análise do Fluxo Metabólico , Redes e Vias Metabólicas/genética , Metabolômica , Methylococcaceae/crescimento & desenvolvimento
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