13C-metabolic flux ratio and novel carbon path analyses confirmed that Trichoderma reesei uses primarily the respirative pathway also on the preferred carbon source glucose

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>Trichoderma reesei </it>is an important host organism for industrial enzyme production. It is adapted to nutrient poor environments where it is capable of producing large amounts of hydrolytic enzymes. In its natural environment <it>T. reesei </it>is expected to benefit from high energy yield from utilization of respirative metabolic pathway. However, <it>T. reesei </it>lacks metabolic pathway reconstructions and the utilization of the respirative pathway has not been investigated on the level of <it>in vivo </it>fluxes.</p> <p>Results</p> <p>The biosynthetic pathways of amino acids in <it>T. reesei </it>supported by genome-level evidence were reconstructed with computational carbon path analysis. The pathway reconstructions were a prerequisite for analysis of <it>in vivo </it>fluxes. The distribution of <it>in vivo </it>fluxes in both wild type strain and <it>cre1</it>, a key regulator of carbon catabolite repression, deletion strain were quantitatively studied by performing ¹³C-labeling on both repressive carbon source glucose and non-repressive carbon source sorbitol. In addition, the ¹³C-labeling on sorbitol was performed both in the presence and absence of sophorose that induces the expression of cellulase genes. Carbon path analyses and the ¹³C-labeling patterns of proteinogenic amino acids indicated high similarity between biosynthetic pathways of amino acids in <it>T. reesei </it>and yeast <it>Saccharomyces cerevisiae</it>. In contrast to <it>S. cerevisiae</it>, however, mitochondrial rather than cytosolic biosynthesis of Asp was observed under all studied conditions. The relative anaplerotic flux to the TCA cycle was low and thus characteristic to respiratory metabolism in both strains and independent of the carbon source. Only minor differences were observed in the flux distributions of the wild type and <it>cre1 </it>deletion strain. Furthermore, the induction of the hydrolytic gene expression did not show altered flux distributions and did not affect the relative amino acid requirements or relative anabolic and respirative activities of the TCA cycle.</p> <p>Conclusion</p> <p>High similarity between the biosynthetic pathways of amino acids in <it>T. reesei </it>and yeast <it>S. cerevisiae </it>was concluded. <it>In vivo </it>flux distributions confirmed that <it>T. reesei </it>uses primarily the respirative pathway also when growing on the repressive carbon source glucose in contrast to <it>Saccharomyces cerevisiae</it>, which substantially diminishes the respirative pathway flux under glucose repression.</p

Compartmentation of glycogen metabolism revealed from 13C isotopologue distributions

Background: Stable isotope tracers are used to assess metabolic flux profiles in living cells. The existing methods of measurement average out the isotopic isomer distribution in metabolites throughout the cell, whereas the knowledge of compartmental organization of analyzed pathways is crucial for the evaluation of true fluxes. That is why we accepted a challenge to create a software tool that allows deciphering the compartmentation of metabolites based on the analysis of average isotopic isomer distribution. Results: The software Isodyn, which simulates the dynamics of isotopic isomer distribution in central metabolic pathways, was supplemented by algorithms facilitating the transition between various analyzed metabolic schemes, and by the tools for model discrimination. It simulated 13C isotope distributions in glucose, lactate, glutamate and glycogen, measured by mass spectrometry after incubation of hepatocytes in the presence of only labeled glucose or glucose and lactate together (with label either in glucose or lactate). The simulations assumed either a single intracellular hexose phosphate pool, or also channeling of hexose phosphates resulting in a different isotopic composition of glycogen. Model discrimination test was applied to check the consistency of both models with experimental data. Metabolic flux profiles, evaluated with the accepted model that assumes channeling, revealed the range of changes in metabolic fluxes in liver cells. Conclusions: The analysis of compartmentation of metabolic networks based on the measured 13C distribution was included in Isodyn as a routine procedure. The advantage of this implementation is that, being a part of evaluation of metabolic fluxes, it does not require additional experiments to study metabolic compartmentation. The analysis of experimental data revealed that the distribution of measured 13C-labeled glucose metabolites is inconsistent with the idea of perfect mixing of hexose phosphates in cytosol. In contrast, the observed distribution indicates the presence of a separate pool of hexose phosphates that is channeled towards glycogen synthesis

Exploring chemical conversions in metabolic networks by tracing atoms transitions

Dissertação de mestrado em BioinformáticaUnderstanding the metabolic processes that occur in a cell has been fundamental to rationally design organisms for the production of a desired metabolite. The improvement in biosynthesis of certain metabolites can be supported by tracing the atoms transition in different chemical conversions, and this way tackle which metabolic pathways are more efficient or most promising for manipulation. In this work, a methodology to construct metabolic maps in which the carbon atom transitions are described was implemented. The carbon atom transition map for the central metabolism of Escherichia coli and Actinobacillus succinogenes were constructed. MetaCyc and KEGG databases were explored to obtain the metabolic information necessary to construct these maps and provided support for validation at some level.Compreender os processos metabólicos que ocorrem numa célula tem sido essencial para o desenho de estirpes capazes de sintetizar produtos de elevado interesse. A síntese de um determinado metabolito pode ser avaliada pela transição de átomos ao longo das diversas conversões químicas numa ou mais vias metabólicas, de modo a identificar quais as mais eficientes ou mais promissoras para manipulação. Neste trabalho foi implementada uma metodologia para a construção de mapas de transição dos átomos de carbono, que pode ser usado conjuntamente com modelos metabólicos. Neste caso, foram construídos os mapas de transição de átomos de carbono para o metabolismo central da Escherichia coli e da Actinobacillus succinogenes. As bases de dados MetaCyc e KEGG foram exploradas para obter as informações metabólicas necessárias à construção destes mapas

Computational discovery and analysis of metabolic pathways

Finding novel or non-standard metabolic pathways, possibly spanning multiple species, has important applications in fields such as metabolic engineering, metabolic network analysis, and metabolic network reconstruction. Traditionally, this has been a manual process, but the large volume of metabolic data now available has created a need for computational tools to automatically identify biologically relevant pathways. This thesis presents new algorithms for automatically finding biologically meaningful linear and branched metabolic pathways in multi-genome scale metabolic networks. These algorithms utilize atom mapping data, which provides the correspondence between atoms in the substrates to atoms in the products of a chemical reaction, to find pathways which conserve a given number of atoms between desired start and target compounds. The first algorithm presented identifies atom conserving linear pathways by explicitly tracking atoms during an exploration of a graph structure constructed from the atom mapping data. The explicit tracking of atoms enables finding branched pathways because it provides automatic identification of the reactions and compounds through which atoms are lost or gained. The thesis then describes two algorithmic approaches for identifying branched metabolic pathways based upon atom conserving linear pathways. One approach takes one linear pathway at a time and attempts to add branches that connect loss and gain compounds. The other approach takes a group of linear pathways and attempts to merge pathways that move mutually exclusive sets of atoms from the start to the target compounds. Comparisons to known metabolic pathways demonstrate that atom tracking causes the algorithms to avoid many unrealistic connections, often found in previous approaches, and return biologically meaningful pathways. While the theoretical complexity of finding even linear atom conserving pathways is high, by choosing the appropriate representations and heuristics, and perhaps due to the structure of the underlying data, the algorithms in this thesis have practical running times on real data. The results also demonstrate the potential of the algorithms to find novel or non-standard pathways that may span multiple organisms

Regulation of genes encoding enzymes involved in plant cell wall deconstruction in Trichoderma reesei

This study describes the regulation of genes encoding plant cell wall-degrading enzymes in the presence of different carbon sources from the biotechnologically important fungus Trichoderma reesei. It was shown that different carbon sources influence fungal growth rate, biomass production and subsequent enzyme secretion. Several genes were identified and suggested to play a role in the development of conidia and in maintaining polarised growth. RNA-sequencing studies showed an increase in transcript levels of genes encoding enzymes involved in plant cell wall degradation (CAZy) as well as of genes encoding lipases, expansins, hydrophobins, G-protein coupled receptors and transporters when mycelia were cultivated in the presence of a lignocellulosic substrate (wheat straw). The encoded non-CAZy proteins were proposed to have accessory roles in carbohydrate deconstruction. A model for solid substrate recognition in T. reesei was described, based on the comparison with the one proposed for Aspergillus niger. Post-transcriptional regulation mediated by regulatory RNAs was identified for nearly 2% of all T. reesei genes, including genes encoding cell wall-degrading enzymes. Transcriptional regulation studies confirmed that transcription patterns of genes encoding enzymes involved in polysaccharide degradation differed between different carbon sources and that they are fine-tuned and dependent on factors such as culture conditions, consumption rate, assimilation of glucose and the presence of several transcription factors. The analysis of the structure of chromatin in the promoter and coding regions of one of these genes, cbh1, revealed different nucleosome positioning patterns under repressing (glucose) and inducing (sophorose, cellulose) conditions. CRE1, the carbon catabolite repressor in T. reesei was shown to be involved in the repression of many CAZy and non-CAZy encoding genes. Furthermore, CRE1 was also shown to be important for nucleosome positioning within the cbh1 coding region under repressing conditions and proposed to do so by interaction with (a) yet unidentified protein(s)

Regulation of genes encoding enzymes involved in plant cell wall deconstruction in Trichoderma reesei

This study describes the regulation of genes encoding plant cell wall-degrading enzymes in the presence of different carbon sources from the biotechnologically important fungus Trichoderma reesei. It was shown that different carbon sources influence fungal growth rate, biomass production and subsequent enzyme secretion. Several genes were identified and suggested to play a role in the development of conidia and in maintaining polarised growth. RNA-sequencing studies showed an increase in transcript levels of genes encoding enzymes involved in plant cell wall degradation (CAZy) as well as of genes encoding lipases, expansins, hydrophobins, G-protein coupled receptors and transporters when mycelia were cultivated in the presence of a lignocellulosic substrate (wheat straw). The encoded non-CAZy proteins were proposed to have accessory roles in carbohydrate deconstruction. A model for solid substrate recognition in T. reesei was described, based on the comparison with the one proposed for Aspergillus niger. Post-transcriptional regulation mediated by regulatory RNAs was identified for nearly 2% of all T. reesei genes, including genes encoding cell wall-degrading enzymes. Transcriptional regulation studies confirmed that transcription patterns of genes encoding enzymes involved in polysaccharide degradation differed between different carbon sources and that they are fine-tuned and dependent on factors such as culture conditions, consumption rate, assimilation of glucose and the presence of several transcription factors. The analysis of the structure of chromatin in the promoter and coding regions of one of these genes, cbh1, revealed different nucleosome positioning patterns under repressing (glucose) and inducing (sophorose, cellulose) conditions. CRE1, the carbon catabolite repressor in T. reesei was shown to be involved in the repression of many CAZy and non-CAZy encoding genes. Furthermore, CRE1 was also shown to be important for nucleosome positioning within the cbh1 coding region under repressing conditions and proposed to do so by interaction with (a) yet unidentified protein(s)

Role of adipose tissue in the pathogenesis and treatment of metabolic syndrome

© Springer International Publishing Switzerland 2014. Adipocytes are highly specialized cells that play a major role in energy homeostasis in vertebrate organisms. Excess adipocyte size or number is a hallmark of obesity, which is currently a global epidemic. Obesity is not only the primary disease of fat cells, but also a major risk factor for the development of Type 2 diabetes, cardiovascular disease, hypertension, and metabolic syndrome (MetS). Today, adipocytes and adipose tissue are no longer considered passive participants in metabolic pathways. In addition to storing lipid, adipocytes are highly insulin sensitive cells that have important endocrine functions. Altering any one of these functions of fat cells can result in a metabolic disease state and dysregulation of adipose tissue can profoundly contribute to MetS. For example, adiponectin is a fat specific hormone that has cardio-protective and anti-diabetic properties. Inhibition of adiponectin expression and secretion are associated with several risk factors for MetS. For this purpose, and several other reasons documented in this chapter, we propose that adipose tissue should be considered as a viable target for a variety of treatment approaches to combat MetS