RESUMO
Streptococcus thermophilus is a lactic acid bacterium adapted toward growth in milk and is a vital component of starter cultures for milk fermentation. Here, we combine genome-scale metabolic modeling and transcriptome profiling to obtain novel metabolic insights into this bacterium. Notably, a refined genome-scale metabolic model (GEM) accurately representing S. thermophilus CH8 metabolism was developed. Modeling the utilization of casein as a nitrogen source revealed an imbalance in amino acid supply and demand, resulting in growth limitation due to the scarcity of specific amino acids, in particular sulfur amino acids. Growth experiments in milk corroborated this finding. A subtle interdependency of the redox balance and the secretion levels of the key metabolites lactate, formate, acetoin, and acetaldehyde was furthermore identified with the modeling approach, providing a mechanistic understanding of the factors governing the secretion product profile. As a potential effect of high expression of arginine biosynthesis genes, a moderate secretion of ornithine was observed experimentally, augmenting the proposed hypothesis of ornithine/putrescine exchange as part of the protocooperative interaction between S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yogurt. This study provides a foundation for future community modeling of food fermentations and rational development of starter strains with improved functionality. IMPORTANCE Streptococcus thermophilus is one the main organisms involved in the fermentation of milk and, increasingly, also in the fermentation of plant-based foods. The construction of a functional high-quality genome-scale metabolic model, in conjunction with in-depth transcriptome profiling with a focus on metabolism, provides a valuable resource for the improved understanding of S. thermophilus physiology. An example is the model-based prediction of the most significant route of synthesis for the characteristic yogurt flavor compound acetaldehyde and identification of metabolic principles governing the synthesis of other flavor compounds. Moreover, the systematic assessment of amino acid supply and demand during growth in milk provides insights into the key challenges related to nitrogen metabolism that is imposed on S. thermophilus and any other organism associated with the milk niche.
Assuntos
Lactobacillus delbrueckii , Streptococcus thermophilus , Acetaldeído/metabolismo , Aminoácidos/metabolismo , Animais , Fermentação , Perfilação da Expressão Gênica , Ácido Láctico/metabolismo , Lactobacillus delbrueckii/genética , Leite/microbiologia , Nitrogênio/metabolismo , Ornitina , Streptococcus thermophilus/metabolismo , Iogurte/microbiologiaRESUMO
The blood-brain barrier (BBB) is a barrier that separates the blood from the brain tissue and possesses unique characteristics that make the delivery of drugs to the brain a great challenge. To achieve this purpose, it is necessary to design strategies to allow BBB passage, in order to reach the brain and target the desired anatomic region. The use of nanomedicine has great potential to overcome this problem, since one can modify nanoparticles with strategic molecules that can interact with the BBB and induce uptake through the brain endothelial cells and consequently reach the brain tissue. This review addresses the potential of nanomedicines to treat neurological diseases by using nanoparticles specially developed to cross the BBB.
Assuntos
Encéfalo/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Nanopartículas/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Humanos , Nanopartículas/química , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Preparações Farmacêuticas/administração & dosagem , FarmacocinéticaRESUMO
Robust and selective quantification methods are required to better analyze feed supplementation effectiveness with specific amino acids. In this work, a reversed-phase high-performance liquid chromatography method with fluorescence detection is proposed and validated for lysine quantification, one of the most limiting amino acids in ruminant nutrition and essential towards milk production. To assess and widen method applicability, different matrices were considered: namely Li2CO3 buffer (the chosen standard reaction buffer), phosphate buffer solution (to mimic media in cellular studies), and rumen inoculum. The method was validated for all three matrices and found to be selective, accurate (92% ± 2%), and precise at both the inter- and intra-day levels in concentrations up to 225 µM, with detection and quantification limits lower than 1.24 and 4.14 µM, respectively. Sample stability was evaluated when stored at room temperature, 4 °C, and -20 °C, showing consistency for up to 48 h regardless of the matrix. Finally, the developed method was applied in the quantification of lysine on real samples. The results presented indicate that the proposed method can be applied towards free lysine quantification in ruminant feeding studies and potentially be of great benefit to dairy cow nutrition supplementation and optimization.
Assuntos
Ração Animal/análise , Lisina/análise , Lisina/química , Aminoácidos/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Suplementos Nutricionais/análise , Reprodutibilidade dos Testes , Ruminantes/metabolismoRESUMO
A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show, in this study, that competitive exclusion, i.e., competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain dependent, affected by species coculturing and the growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in a loss of bioactivity, proving this gene to be important for manganese depletion. The presence of an mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from 10 major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others. In summary, competitive exclusion through ion depletion is herein reported as a novel mechanism in LAB to delay the growth of spoilage contaminants in dairy products.IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of "good bacteria" to protect food against spoilage organisms has a long, successful history, even though the molecular mechanisms are not fully understood. In this study, we show that the depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provide a link to the distinct, nonenzymatic, manganese-catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation.
Assuntos
Produtos Fermentados do Leite/microbiologia , Microbiologia de Alimentos , Fungos/fisiologia , Lactobacillus/fisiologia , Leveduras/fisiologiaRESUMO
Streptococcus pneumoniae is a frequent bacterial pathogen of the human respiratory tract causing pneumonia, meningitis and sepsis, a serious healthcare burden in all age groups. S. pneumoniae lacks complete respiratory chain and relies on carbohydrate fermentation for energy generation. One of the essential components for this includes the mannose phosphotransferase system (Man-PTS), which plays a central role in glucose transport and exhibits a broad specificity for a range of hexoses. Importantly, Man-PTS is involved in the global regulation of gene expression for virulence determinants. We herein report the three-dimensional structure of the EIIA domain of S. pneumoniae mannose phosphotransferase system (SpEIIA-Man). Our structure shows a dimeric arrangement of EIIA and reveals a detailed molecular description of the active site. Since PTS transporters are exclusively present in microbes and sugar transporters have already been suggested as valid targets for antistreptococcal antibiotics, our work sets foundation for the future development of antimicrobial strategies against Streptococcus pneumoniae.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Manose/metabolismo , Fosfotransferases/química , Fosfotransferases/metabolismo , Streptococcus pneumoniae/enzimologia , Cristalografia por Raios X , Especificidade por SubstratoRESUMO
Summary: Metabolite analogues (MAs) mimic the structure of native metabolites, can competitively inhibit their utilization in enzymatic reactions, and are commonly used as selection tools for isolating desirable mutants of industrial microorganisms. Genome-scale metabolic models representing all biochemical reactions in an organism can be used to predict effects of MAs on cellular phenotypes. Here, we present the metabolite analogues for rational strain improvement (MARSI) framework. MARSI provides a rational approach to strain improvement by searching for metabolites as targets instead of genes or reactions. The designs found by MARSI can be implemented by supplying MAs in the culture media, enabling metabolic rewiring without the use of recombinant DNA technologies that cannot always be used due to regulations. To facilitate experimental implementation, MARSI provides tools to identify candidate MAs to a target metabolite from a database of known drugs and analogues. Availability and implementation: The code is freely available at https://github.com/biosustain/marsi under the Apache License V2. MARSI is implemented in Python. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Metabolômica/métodos , Software , Bases de Dados Factuais , GenomaRESUMO
Receptors on the cell surfaces of bacterial hosts are essential during the infection cycle of bacteriophages. To date, the phage receptors of the industrial relevant dairy starter bacterium Streptococcus thermophilus remain elusive. Thus, we set out to identify cell surface structures that are involved in host recognition by dairy streptococcal phages. Five industrial S. thermophilus strains sensitive to different phages (pac type, cos type, and the new type 987), were selected to generate spontaneous bacteriophage-insensitive mutants (BIMs). Of these, approximately 50% were deselected as clustered regularly interspaced short palindromic repeat (CRISPR) mutants, while the other pool was further characterized to identify receptor mutants. On the basis of genome sequencing data, phage resistance in putative receptor mutants was attributed to nucleotide changes in genes encoding glycan biosynthetic pathways. Superresolution structured illumination microscopy was used to visualize the interactions between S. thermophilus and its phages. The phages were either regularly distributed along the cells or located at division sites of the cells. The cell wall structures mediating the latter type of phage adherence were further analyzed via phenotypic and biochemical assays. Altogether, our data suggested that phage adsorption to S. thermophilus is mediated by glycans associated with the bacterial cell surface. Specifically, the pac-type phage CHPC951 adsorbed to polysaccharides anchored to peptidoglycan, while the 987-type phage CHPC926 recognized exocellular polysaccharides associated with the cell surface.IMPORTANCEStreptococcus thermophilus is widely used in starter cultures for cheese and yoghurt production. During dairy fermentations, infections of bacteria with bacteriophages result in acidification failures and a lower quality of the final products. An understanding of the molecular factors involved in phage-host interactions, in particular, the phage receptors in dairy bacteria, is a crucial step for developing better strategies to prevent phage infections in dairy plants.
Assuntos
Parede Celular/metabolismo , Polissacarídeos/metabolismo , Fagos de Streptococcus/fisiologia , Streptococcus thermophilus/virologia , Parede Celular/virologia , Queijo/microbiologia , Fermentação , Genoma Viral , Fagos de Streptococcus/genética , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Iogurte/microbiologiaRESUMO
Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.
Assuntos
Antifúngicos/farmacologia , Produtos Fermentados do Leite/microbiologia , Debaryomyces/efeitos dos fármacos , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/metabolismo , Peptídeos/farmacologia , Antifúngicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Peptídeos/isolamento & purificaçãoRESUMO
Resveratrol is a polyphenol compound with great value in cancer therapy, cardiovascular protection, and neurodegenerative disorders. The mechanism by which resveratrol exerts such pleiotropic effects is not yet clear and there is a huge need to understand the influence of this compound on the regulation of lipid domains formation on membrane structure. The aim of the present study was to reveal potential molecular interactions between resveratrol and lipid rafts found in cell membranes by means of Förster resonance energy transfer, DPH fluorescence quenching, and triton X-100 detergent resistance assay. Liposomes composed of egg phosphatidylcholine, cholesterol, and sphingomyelin were used as model membranes. The results revealed that resveratrol induces phase separation and formation of liquid-ordered domains in bilayer structures. The formation of such tightly packed lipid rafts is important for different signal transduction pathways, through the regulation of membrane-associating proteins, that can justify several pharmacological activities of this compound.
Assuntos
Bicamadas Lipídicas/química , Lipossomos/química , Microdomínios da Membrana/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Galinhas , Colesterol/química , Óxidos N-Cíclicos/química , Difenilexatrieno/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Microdomínios da Membrana/química , Octoxinol/química , Fosfatidilcolinas/química , Resveratrol , Esfingomielinas/química , Estilbenos/química , TermodinâmicaRESUMO
Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.
Assuntos
Recombinação Genética , Fagos de Streptococcus/classificação , Fagos de Streptococcus/genética , Streptococcus thermophilus/virologia , Fagos Bacilares , Queijo/microbiologia , Queijo/virologia , Produtos Fermentados do Leite/microbiologia , Produtos Fermentados do Leite/virologia , Empacotamento do DNA , DNA Viral , Fermentação , Microbiologia de Alimentos , Genoma Viral , Lactococcus lactis/virologia , Microscopia Eletrônica de Transmissão , Filogenia , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Fagos de Streptococcus/isolamento & purificação , Fagos de Streptococcus/ultraestrutura , Proteínas Estruturais Virais/isolamento & purificação , Iogurte/microbiologia , Iogurte/virologiaRESUMO
Several strategies have been implemented to enhance brain drug delivery, and herein solid lipid nanoparticles functionalized with apolipoprotein E were tested in hCMEC/D3 cell monolayers. The mean diameter of 160 nm, negative charge of -12 mV, and their lipophilic characteristics make these nanosystems suitable for brain delivery. Confocal images and flow cytometry data showed a cellular uptake increase of 1.8-fold for SLN-Palmitate-ApoE and 1.9-fold for SLN-DSPE-ApoE when compared with the non-functionalized SLNs. Clathrin-mediated endocytosis was distinguished as the preferential internalization pathway involved in cellular uptake and nanoparticles could cross the blood-brain barrier predominantly by a transcellular pathway. The understanding of the mechanisms involved in the transport of these nanosystems through the blood-brain barrier may potentiate their application on brain drug delivery.
Assuntos
Apolipoproteínas E/metabolismo , Barreira Hematoencefálica/metabolismo , Portadores de Fármacos/metabolismo , Nanopartículas/metabolismo , Encéfalo/metabolismo , Linhagem Celular , Sistemas de Liberação de Medicamentos , Humanos , Metabolismo dos Lipídeos , Lipídeos/química , Palmitatos/metabolismo , Fosfatidiletanolaminas/metabolismoRESUMO
The aggregation of amyloid-ß peptide (Aß) has been linked to the formation of neuritic plaques, which are pathological hallmarks of Alzheimer's disease (AD). Various natural compounds have been suggested as therapeutics for AD. Among these compounds, resveratrol has aroused great interest due to its neuroprotective characteristics. Here, we provide evidence that grape skin and grape seed extracts increase the inhibition effect on Aß aggregation. However, after intravenous injection, resveratrol is rapidly metabolized into both glucuronic acid and sulfate conjugations of the phenolic groups in the liver and intestinal epithelial cells (within less than 2 h), which are then eliminated. In the present study, we show that solid lipid nanoparticles (SLNs) functionalized with an antibody, the anti-transferrin receptor monoclonal antibody (OX26 mAb), can work as a possible carrier to transport the extract to target the brain. Experiments on human brain-like endothelial cells show that the cellular uptake of the OX26 SLNs is substantially more efficient than that of normal SLNs and SLNs functionalized with an unspecific antibody. As a consequence, the transcytosis ability of these different SLNs is higher when functionalized with OX-26.
Assuntos
Doença de Alzheimer/metabolismo , Lipídeos/química , Nanopartículas/química , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Estilbenos/administração & dosagem , Vitis/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Extrato de Sementes de Uva/administração & dosagem , Extrato de Sementes de Uva/química , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Permeabilidade/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , ResveratrolRESUMO
Resveratrol belongs to the large group of biologically active polyphenol compounds, with several beneficial health effects including antioxidant activity, anti-inflammatory action, cardiovascular protection, neuroprotection, and cancer chemoprevention. In the present study, the possibility that the effects of resveratrol described above are caused by resveratrol membrane interactions and structural modifications of lipid bilayers is evaluated. In this context, it is possible that resveratrol interacts selectively with lipid domains present in biological membranes, thereby modulating the localization of the anchored proteins and controlling their intracellular cascades. This study was conducted in a synchrotron particle accelerator, where the influence of resveratrol in the structural organization of lipid domains in bilayers was investigated using small- and wide-angle X-ray scattering (SAXS and WAXS) techniques. Membrane mimetic systems composed of egg l-α-phosphatidylcholine (EPC), cholesterol (CHOL), and sphingomyelin (SM), with different molar ratios, were used to access the effects of resveratrol on the order and structure of the membrane. The results revealed that resveratrol induces phase separation, promoting the formation of lipid domains in EPC, EPC:CHOL [4:1], and EPC:CHOL:SM [1:1:1] bilayers, which brings some structural organization to membranes. Therefore, resveratrol controls lipid packing of bilayers by inducing the organization of lipid rafts. Moreover, the formation of lipid domains is important for modulating the activity of many receptors, transmembrane proteins, and enzymes whose activity depends on the structural organization of the membrane and on the presence or absence of these organized domains. This evidence can thereby explain the therapeutic effects of resveratrol.
Assuntos
Bicamadas Lipídicas/química , Estilbenos/química , Animais , Bovinos , Colesterol/química , Gema de Ovo , Fosfatidilcolinas/química , Resveratrol , Espalhamento a Baixo Ângulo , Ovinos , Esfingomielinas/química , Síncrotrons , Difração de Raios XRESUMO
PURPOSE: Nanostructured Lipid Carriers (NLCs) loading oxaprozin were developed to address an effective drug packaging and targeted delivery, improving the drug pharmacokinetics and pharmacodynamics properties and avoiding the local gastric side-effects. Macrophages actively phagocyte particles with sizes larger than 200 nm and, when activated, over-express folate beta receptors - features that in the case of this work constitute the basis for passive and active targeting strategies. METHODS: Two formulations containing oxaprozin were developed: NLCs with and without folate functionalization. In order to target the macrophages folate receptors, a DSPE-PEG2000-FA conjugate was synthesized and added to the NLCs. RESULTS: These formulations presented a relatively low polydispersity index (approximately 0.2) with mean diameters greater than 200 nm and zeta potential inferior to -40 mV. The encapsulation efficiency of the particles was superior to 95% and the loading capacity was of 9%, approximately. The formulations retained the oxaprozin release in simulated gastric fluid (only around 10%) promoting its release on simulated intestinal fluid. MTT and LDH assays revealed that the formulations only presented cytotoxicity in Caco-2 cells for oxaprozin concentrations superior to 100 µM. Permeability studies in Caco-2 cells shown that oxaprozin encapsulation did not interfered with oxaprozin permeability (around 0.8 × 10(-5) cm/s in simulated intestinal fluid and about 1.45 × 10(-5) cm/s in PBS). Moreover, in RAW 264.7 cells NLCs functionalization promoted an increased uptake over time mainly mediated by a caveolae uptake mechanism. CONCLUSIONS: The developed nanoparticles enclose a great potential for oxaprozin oral administration with significant less gastric side-effects.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacocinética , Portadores de Fármacos/química , Nanopartículas/química , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Propionatos/administração & dosagem , Propionatos/farmacocinética , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Células CACO-2 , Linhagem Celular , Ácido Fólico/química , Humanos , Camundongos , Oxaprozina , Permeabilidade , Propionatos/efeitos adversosRESUMO
The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding α-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876-1878, 2001). To resolve this discrepancy, the enzyme encoded by butA Cg was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by 1H-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (â¼20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. V max values for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 µmol min-1 mg protein-1, and K m values were 0.23 ± 0.02 and 1.49 ± 0.07 mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,butA Cg ). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butA Cg (from 0.30 ± 0.03 to 0.004 ± 0.001 µmol min-1 mg protein-1), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.
Assuntos
Oxirredutases do Álcool/metabolismo , Butileno Glicóis/metabolismo , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/metabolismo , Engenharia Metabólica , Acetoína/metabolismo , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Carboxiliases/genética , Carboxiliases/metabolismo , Corynebacterium glutamicum/genética , Escherichia coli/genética , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Espectroscopia de Ressonância Magnética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: The present study takes advantage of the beneficial effects of resveratrol as a neuroprotective compound. Resveratrol-loaded solid lipid nanoparticles were functionalized with apolipoprotein E which can be recognized by the LDL receptors overexpressed on the blood-brain barrier. RESULTS: Transmission electron microscopy images revealed spherical nanoparticles, dynamic light scattering gave a Z-average lower than 200 nm, and a zeta potential of around -13 mV and very high resveratrol entrapment efficiency (ca. 90 %). In vitro cytotoxic effects were assessed by MTT and LDH assays in hCMEC/D3 cell line and revealed no toxicity up to 50 µM over 4 h of incubation. The permeability through hCMEC/D3 monolayers showed a significant increase (1.8-fold higher) for resveratrol-loaded solid lipid nanoparticles functionalized with apolipoprotein E when compared to non-functionalized ones. CONCLUSIONS: In conclusion, these nanosystems might be a promising strategy for resveratrol delivery into the brain, while protecting it from degradation in the blood stream. Graphical abstract .
Assuntos
Apolipoproteínas/administração & dosagem , Encéfalo/efeitos dos fármacos , Lipídeos/administração & dosagem , Nanopartículas/administração & dosagem , Estilbenos/administração & dosagem , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Humanos , Fármacos Neuroprotetores/administração & dosagem , Tamanho da Partícula , ResveratrolRESUMO
Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer's disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin pathway in Escherichia coli. We propose a novel biosynthetic pathway from the amino acid, tyrosine, utilizing nine heterologous enzymes. The pathway proceeds via the synthesis of two flavanones never produced in microorganisms before--garbanzol and resokaempferol. We show for the first time a functional biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols.
Assuntos
Escherichia coli/metabolismo , Flavonoides/biossíntese , Flavanonas/biossíntese , Flavonoides/química , Flavonóis , Tirosina/metabolismoRESUMO
BACKGROUND: 2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions. RESULTS: A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1). CONCLUSIONS: We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.
Assuntos
Butileno Glicóis/metabolismo , Corynebacterium glutamicum/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Butileno Glicóis/química , Corynebacterium glutamicum/metabolismo , Glucose/metabolismo , L-Lactato Desidrogenase/deficiência , L-Lactato Desidrogenase/genética , Lactococcus lactis/genética , Engenharia Metabólica , Família Multigênica , Oxigênio/metabolismo , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismoRESUMO
Nanotechnology can be an important tool to improve the permeability of some drugs for the blood-brain barrier. In this work we created a new system to enter the brain by functionalizing solid lipid nanoparticles with apolipoprotein E, aiming to enhance their binding to low-density lipoprotein receptors on the blood-brain barrier endothelial cells. Solid lipid nanoparticles were successfully functionalized with apolipoprotein E using two distinct strategies that took advantage of the strong interaction between biotin and avidin. Transmission electron microscopy images revealed spherical nanoparticles, and dynamic light scattering gave a Z-average under 200 nm, a polydispersity index below 0.2, and a zeta potential between -10 mV and -15 mV. The functionalization of solid lipid nanoparticles with apolipoprotein E was demonstrated by infrared spectroscopy and fluorimetric assays. In vitro cytotoxic effects were evaluated by MTT and LDH assays in the human cerebral microvascular endothelial cells (hCMEC/D3) cell line, a human blood-brain barrier model, and revealed no toxicity up to 1.5 mg ml(-1) over 4 h of incubation. The brain permeability was evaluated in transwell devices with hCMEC/D3 monolayers, and a 1.5-fold increment in barrier transit was verified for functionalized nanoparticles when compared with non-functionalized ones. The results suggested that these novel apolipoprotein E-functionalized nanoparticles resulted in dynamic stable systems capable of being used for an improved and specialized brain delivery of drugs through the blood-brain barrier.
Assuntos
Apolipoproteínas E/química , Barreira Hematoencefálica/metabolismo , Portadores de Fármacos/química , Nanopartículas/química , Nanotecnologia/métodos , Linhagem Celular , Sobrevivência Celular , HumanosRESUMO
Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression.