Analysis of Aspergillus nidulans metabolism at the genome-scale.

BACKGROUND: Aspergillus nidulans is a member of a diverse group of filamentous fungi, sharing many of the properties of its close relatives with significance in the fields of medicine, agriculture and industry. Furthermore, A. nidulans has been a classical model organism for studies of development biology and gene regulation, and thus it has become one of the best-characterized filamentous fungi. It was the first Aspergillus species to have its genome sequenced, and automated gene prediction tools predicted 9,451 open reading frames (ORFs) in the genome, of which less than 10% were assigned a function. RESULTS: In this work, we have manually assigned functions to 472 orphan genes in the metabolism of A. nidulans, by using a pathway-driven approach and by employing comparative genomics tools based on sequence similarity. The central metabolism of A. nidulans, as well as biosynthetic pathways of relevant secondary metabolites, was reconstructed based on detailed metabolic reconstructions available for A. niger and Saccharomyces cerevisiae, and information on the genetics, biochemistry and physiology of A. nidulans. Thereby, it was possible to identify metabolic functions without a gene associated, and to look for candidate ORFs in the genome of A. nidulans by comparing its sequence to sequences of well-characterized genes in other species encoding the function of interest. A classification system, based on defined criteria, was developed for evaluating and selecting the ORFs among the candidates, in an objective and systematic manner. The functional assignments served as a basis to develop a mathematical model, linking 666 genes (both previously and newly annotated) to metabolic roles. The model was used to simulate metabolic behavior and additionally to integrate, analyze and interpret large-scale gene expression data concerning a study on glucose repression, thereby providing a means of upgrading the information content of experimental data and getting further insight into this phenomenon in A. nidulans. CONCLUSION: We demonstrate how pathway modeling of A. nidulans can be used as an approach to improve the functional annotation of the genome of this organism. Furthermore we show how the metabolic model establishes functional links between genes, enabling the upgrade of the information content of transcriptome data.

Metabolic network driven analysis of genome-wide transcription data from Aspergillus nidulans.

BACKGROUND: Aspergillus nidulans (the asexual form of Emericella nidulans) is a model organism for aspergilli, which are an important group of filamentous fungi that encompasses human and plant pathogens as well as industrial cell factories. Aspergilli have a highly diversified metabolism and, because of their medical, agricultural and biotechnological importance, it would be valuable to have an understanding of how their metabolism is regulated. We therefore conducted a genome-wide transcription analysis of A. nidulans grown on three different carbon sources (glucose, glycerol, and ethanol) with the objective of identifying global regulatory structures. Furthermore, we reconstructed the complete metabolic network of this organism, which resulted in linking 666 genes to metabolic functions, as well as assigning metabolic roles to 472 genes that were previously uncharacterized. RESULTS: Through combination of the reconstructed metabolic network and the transcription data, we identified subnetwork structures that pointed to coordinated regulation of genes that are involved in many different parts of the metabolism. Thus, for a shift from glucose to ethanol, we identified coordinated regulation of the complete pathway for oxidation of ethanol, as well as upregulation of gluconeogenesis and downregulation of glycolysis and the pentose phosphate pathway. Furthermore, on change in carbon source from glucose to ethanol, the cells shift from using the pentose phosphate pathway as the major source of NADPH (nicotinamide adenine dinucleotide phosphatase, reduced form) for biosynthesis to use of the malic enzyme. CONCLUSION: Our analysis indicates that some of the genes are regulated by common transcription factors, making it possible to establish new putative links between known transcription factors and genes through clustering.

CreA influences the metabolic fluxes of Aspergillus nidulans during growth on glucose and xylose.

The physiological phenotype of Aspergillus nidulans was investigated for different genetic and environmental conditions of glucose repression through the quantification of in vivo fluxes in the central carbon metabolism using (13)C-metabolic-flux analysis. The particular focus was the role of the carbon repressor CreA, which is the major regulatory protein mediating carbon repression in many fungal species, in the primary metabolism of A. nidulans. Batch cultivations were performed with a reference strain and a deletion mutant strain (creADelta4) using [1-(13)C]glucose as carbon source. The mutant strain was also grown on a mixture of [1-(13)C]glucose and unlabelled xylose. Fractional enrichment data were measured by gas chromatography-mass spectrometry. A model describing the central metabolism of A. nidulans was used in combination with fractional enrichment data, and measurements of extracellular rates and biomass composition for the estimation of the in vivo metabolic fluxes. The creA-mutant strain showed a lower maximum specific growth rate than the reference strain when grown on glucose (0.11 and 0.25 h(-1), respectively), whereas the specific growth rate of the mutant strain grown on the glucose/xylose mixture was identical to that on glucose (0.11 h(-1)). Different patterns and increased levels of extracellular polyols were observed both upon deletion of the creA gene and upon addition of xylose to the growth medium of the mutant strain. Concerning metabolic fluxes, the major change observed in the flux distribution of A. nidulans upon deletion of the creA gene was a 20 % decrease in the flux through the oxidative part of the pentose-phosphate pathway. Addition of xylose to the growth medium of the mutant resulted in an increase of about 40 % in the activity of the oxidative part of the pentose-phosphate pathway, as well as decreases in the fluxes through the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle (in the range of 20-30 %). The derepression of key pathways leads to alterations in the demands for cofactors, thereby imposing changes in the central metabolism due to the coupling of the many different reactions via the redox and energy metabolism of the cells.

Reconstruction of the central carbon metabolism of Aspergillus niger.

The topology of central carbon metabolism of Aspergillus niger was identified and the metabolic network reconstructed, by integrating genomic, biochemical and physiological information available for this microorganism and other related fungi. The reconstructed network may serve as a valuable database for annotation of genes identified in future genome sequencing projects on aspergilli. Based on the metabolic reconstruction, a stoichiometric model was set up that includes 284 metabolites and 335 reactions, of which 268 represent biochemical conversions and 67 represent transport processes between the different intracellular compartments and between the cell and the extracellular medium. The stoichiometry of the metabolic reactions was used in combination with biosynthetic requirements for growth and pseudo-steady state mass balances over intracellular metabolites for the quantification of metabolic fluxes using metabolite balancing. This framework was employed to perform an in silico characterisation of the phenotypic behaviour of A. niger grown on different carbon sources. The effects on growth of single reaction deletions were assessed and essential biochemical reactions were identified for different carbon sources. Furthermore, application of the stoichiometric model for assessing the metabolic capabilities of A. niger to produce metabolites was evaluated by using succinate production as a case study.