Biomedical subjects

Eduardo Agosin

Publications and source records attributed to Eduardo Agosin.

11 recordsLinked to original sources

PubMed2006

Accurate and unambiguous tag-to-gene mapping in serial analysis of gene expression.

BACKGROUND: In this study, we present a robust and reliable computational method for tag-to-gene assignment in serial analysis of gene expression (SAGE). The method relies on current genome information and annotation, incorporation of several new features, and key improvements over alternative methods, all of which are important to determine gene expression levels more accurately. The method provides a complete annotation of potential virtual SAGE tags within a genome, along with an estimation of their confidence for experimental observation that ranks tags that present multiple matches in the genome. RESULTS: We applied this method to the Saccharomyces cerevisiae genome, producing the most thorough and accurate annotation of potential virtual SAGE tags that is available today for this organism. The usefulness of this method is exemplified by the significant reduction of ambiguous cases in existing experimental SAGE data. In addition, we report new insights from the analysis of existing SAGE data. First, we found that experimental SAGE tags mapping onto introns, intron-exon boundaries, and non-coding RNA elements are observed in all available SAGE data. Second, a significant fraction of experimental SAGE tags was found to map onto genomic regions currently annotated as intergenic. Third, a significant number of existing experimental SAGE tags for yeast has been derived from truncated cDNAs, which are synthesized through oligo-d(T) priming to internal poly-(A) regions during reverse transcription. CONCLUSION: We conclude that an accurate and unambiguous tag mapping process is essential to increase the quality and the amount of information that can be extracted from SAGE experiments. This is supported by the results obtained here and also by the large impact that the erroneous interpretation of these data could have on downstream applications.

Chromosome Mapping↗

PubMed2006

Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction: role of the TreYZ trehalose biosynthetic pathway.

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.

Bacterial Proteins↗

PubMed2005

Wine distillates: practical operating recipe formulation for stills.

Consumer perceptions of flavors are associated with the chemical composition of foods. However, consumer preferences change; therefore, it is necessary for food manufacturers to be able to adapt their products. Unlike in aged spirits, the chemical composition of young spirits is determined during distillation; therefore, this is where distillers must tailor their operating recipes to the new trends. Even for an experienced distiller, the complexity of the process makes adapting the operating recipe far from straightforward. In this study, we developed a methodology for generating practical recipes that makes use of computer simulations and optimization techniques. We used Pisco Brandy, a young Muscat wine distillate from Chile and Peru as our case study. Even so, because our methodology is independent of the chemical composition of the broth, it can be applied throughout the industry. Drawing on the experience and preferences of industry enologists, we designed a preferred distillate and used our methodology to obtain the appropriate recipe. This recipe was validated in lab scale experiments, and we obtained a much closer distillate to the desired prescription than commercial products.

Alcoholic Beverages↗

PubMed2005

Quantitative analysis of wine yeast gene expression profiles under winemaking conditions.

Wine fermentation is a dynamic and complex process in which the yeast cell is subjected to multiple stress conditions. A successful adaptation involves changes in gene expression profiles where a large number of genes are up- or downregulated. Functional genomic approaches are commonly used to obtain global gene expression profiles, thereby providing a comprehensive view of yeast physiology. We used SAGE to quantify gene expression profiles in an industrial strain of Saccharomyces cerevisiae under winemaking conditions. The transcriptome of wine yeast was analysed at three stages during the fermentation process, mid-exponential phase, and early- and late-stationary phases. Upon correlation with the yeast genome, we found three classes of transcripts: (a) sequences that corresponded to ORFs; (b) expressed sequences from intergenic regions; and (c) messengers that did not match the published reference yeast genome. In all fermentation phases studied, the most highly expressed genes related to energy production and stress response. For many pathways, including glycolysis, different transcript levels were observed during each phase. Different isoenzymes, including hexose transporters (HXT), were differentially induced, depending on the growth phase. About 10% of transcripts matched non-annotated ORF regions within the yeast genome and could correspond to small novel genes originally omitted in the first gene annotation effort. Up to 22% of transcripts, particularly at late-stationary phase, did not match any known location within the genome. As the available reference yeast genome was obtained from a laboratory strain, these expressed sequences could represent genes only expressed by an industrial yeast strain. Further studies are necessary to identify the role of these potential genes during wine fermentation.

Cluster Analysis↗

PubMed2005

Heterologous expression of Escherichia coli ppsA (phosphoenolpyruvate synthetase) and galU (UDP-glucose pyrophosphorylase) genes in Corynebacterium glutamicum, and its impact on trehalose synthesis.

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on a Corynebacterium glutamicum strain expressing the Escherichia coli enzyme UDP-glucose pyrophosphorylase (GalU). Biochemical network analysis suggest a further bottleneck for trehalose synthesis resulting from the coupling of phosphotransferase (PTS) mediated glucose uptake, and glucose catabolism in C. glutamicum. To overcome this coupling, we propose the expression of E. coli phosphoenolpyruvate synthetase (PpsA), in addition to GalU expression, in C. glutamicum. Although GalU expression improved trehalose synthesis in C. glutamicum, the simultaneous expression of GalU and PpsA did not result in a further increase in trehalose yield, but resulted in an increased catabolic rate of glucose, which could be ascribed to the operation of a futile cycle between phosphoenolpyruvate and pyruvate. The impact of GalU and PpsA expression on polysaccharide content, side product excretion and metabolic fluxes is discussed, as well as alternative ways to decouple glucose uptake and catabolism, in order to increase trehalose yield.

Corynebacterium glutamicum↗

PubMed2004

Overproduction of trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum.

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.

Corynebacterium↗

PubMed2004

Biomass content governs fermentation rate in nitrogen-deficient wine musts.

Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen diminishes a yeast's metabolic activity, as well as the biomass yield, although it has not been clear which of these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter the chemistry of the must.

Biomass↗

PubMed2004

Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum.

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.

Corynebacterium↗

PubMed2004

Osmotic stress response: quantification of cell maintenance and metabolic fluxes in a lysine-overproducing strain of Corynebacterium glutamicum.

Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysine-overproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30- and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.

Biomass↗

PubMed2003

Modeling of yeast metabolism and process dynamics in batch fermentation.

Much is known about yeast metabolism and the kinetics of industrial batch fermentation processes. In this study, however, we provide the first tool to evaluate the dynamic interaction that exists between them. A stoichiometric model, using wine fermentation as a case study, was constructed to simulate batch cultures of Saccharomyces cerevisiae. Five differential equations describe the evolution of the main metabolites and biomass in the fermentation tank, while a set of underdetermined linear algebraic equations models the pseudo-steady-state microbial metabolism. Specific links between process variables and the reaction rates of metabolic pathways represent microorganism adaptation to environmental changes in the culture. Adaptation requirements to changes in the environment, optimal growth, and homeostasis were set as the physiological objectives. A linear programming routine was used to define optimal metabolic mass flux distribution at each instant throughout the process. The kinetics of the process arise from the dynamic interaction between the environment and metabolic flux distribution. The model assessed the effect of nitrogen starvation and ethanol toxicity in wine fermentation and it was able to simulate fermentation profiles qualitatively, while experimental fermentation yields were reproduced successfully as well.

Adaptation, Physiological↗

PubMed2003

Beta-glucosidase from the grape native yeast Debaryomyces vanrijiae: purification, characterization, and its effect on monoterpene content of a Muscat grape juice.

Six hundred ten yeast colonies isolated from various vineyards in Chile were screened for the presence of a beta-glucosidase activity as well as the resistance to glucose and ethanol inhibition. Among them, Debaryomyces vanrijiae was found to produce high levels of an extracelular beta-glucosidase which was tolerant to glucose (K(i) = 439 mM) and ethanol inhibitions. The enzyme (designated DV-BG) was purified to apparent homogeneity, respectively, by gel filtration, ion-exchange, and chromatofocusing techniques. Its molecular weight was 100 000, and its pI 3.0, optimum pH, and temperature activities were 5.0 and 40 degrees C, respectively, and had a V(max) of 47.6 micromol min(-)(1) mg(-)(1) and a K(m) of 1.07 mM. The enzyme was active against different beta-d-glucosides including glucosidic flavor precursors. The disaccharidic flavor precursors were not substrates for the enzyme. When added to a Muscat grape juice, the concentration of several monoterpenes increased as the consequence of its hydrolytic activity.

Ascomycota↗