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We introduce a novel computational approach, CoReCo, for comparative metabolic reconstruction and provide genome-scale metabolic network models for 49 important fungal species. Leveraging on the exponential growth in sequenced genome availability, our method reconstructs genome-scale gapless metabolic networks simultaneously for a large number of species by integrating sequence data in a probabilistic framework. High reconstruction accuracy is demonstrated by comparisons to the well-curated Saccharomyces cerevisiae consensus model and large-scale knock-out experiments. Our comparative approach is particularly useful in scenarios where the quality of available sequence data is lacking, and when reconstructing evolutionary distant species. Moreover, the reconstructed networks are fully carbon mapped, allowing their use in 13C flux analysis. We demonstrate the functionality and usability of the reconstructed fungal models with computational steady-state biomass production experiment, as these fungi include some of the most important production organisms in industrial biotechnology. In contrast to many existing reconstruction techniques, only minimal manual effort is required before the reconstructed models are usable in flux balance experiments. CoReCo is available at http://esaskar.github.io/CoReCo/., Author Summary Advances in next-generation sequencing technologies are revolutionizing molecular biology. Sequencing-enabled cost-effective characterization of microbial genomes is a particularly exciting development in metabolic engineering. There, considerable effort has been put to reconstructing genome-scale metabolic networks that describe the collection of hundreds to thousands of biochemical reactions available for a microbial cell. These network models are instrumental in understanding microbial metabolism and guiding metabolic engineering efforts to improve biochemical yields. We have developed a novel computational method, CoReCo, which bridges the growing gap between the availability of sequenced genomes and respective reconstructed metabolic networks. The method reconstructs genome-scale metabolic networks simultaneously for related microbial species. It utilizes the available sequencing data from these species to correct for incomplete and missing data. We used the method to reconstruct metabolic networks for a set of 49 fungal species providing the method protein sequence data and a phylogenetic tree describing the evolutionary relationships between the species. We demonstrate the applicability of the method by comparing a metabolic reconstruction of Saccharomyces cerevisiae to the manually curated, high-quality consensus network. We also provide an easy-to-use implementation of the method, usable both in single computer and distributed computing environments.

Background: The soft rot ascomycetal fungus Trichoderma reesei is utilized for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. T. reesei uses several different enzymes for the degradation of plant cell wall-derived material, including 9 characterized cellulases, 15 characterized hemicellulases and at least 42 genes predicted to encode cellulolytic or hemicellulolytic activities. Production of cellulases and hemicellulases is modulated by environmental and physiological conditions. Several regulators affecting the expression of cellulase and hemicellulase genes have been identified but more factors still unknown are believed to be present in the genome of T. reesei. Results: We have used transcriptional profiling data from T. reesei cultures in which cellulase/hemicellulase production was induced by the addition of different lignocellulose-derived materials to identify putative novel regulators for cellulase and hemicellulase genes. Based on this induction data, supplemented with other published genome-wide data on different protein production conditions, 28 candidate regulatory genes were selected for further studies and they were overexpressed in T. reesei. Overexpression of seven genes led to at least 1.5-fold increased production of cellulase and/or xylanase activity in the modified strains as compared to the parental strain. Deletion of gene 77513, here designated as ace3, was found to be detrimental for cellulase production and for the expression of several cellulase genes studied. This deletion also significantly reduced xylanase activity and expression of xylan-degrading enzyme genes. Furthermore, our data revealed the presence of co-regulated chromosomal regions containing carbohydrate-active enzyme genes and candidate regulatory genes. Conclusions: Transcriptional profiling results from glycoside hydrolase induction experiments combined with a previous study of specific protein production conditions was shown to be an effective method for finding novel candidate regulatory genes affecting the production of cellulases and hemicellulases. Recombinant strains with improved cellulase and/or xylanase production properties were constructed, and a gene essential for cellulase gene expression was found. In addition, more evidence was gained on the chromatin level regional regulation of carbohydrate-active enzyme gene expression.

Celiac disease (CD) is a common small intestinal injury caused by sensitivity to gliadin in genetically-predisposed individuals. The only susceptibility locus established is the HLA-DQ. We tested whether the chromosomal region of the CD28/CTLA4 genes on 2q33 is linked to CD. These genes encode receptors regulating the T-lymphocyte activation. Recently, this gene region was reported to be linked to the susceptibility to many autoimmune diseases, including insulin-dependent diabetes (IDDM12locus). It is thus an obvious candidate locus also for CD, since the intestinal injury is mediated by the immune system. Genetic linkage between seven marker loci in this gene region and CD was studied in 69 Finnish families. In the multipoint linkage analysis, the highest non-pararametric linkage score (NPL) was 1.75 (P=0.04) for D2S116, suggesting weak linkage for this candidate locus. To evaluate this finding, an additional 31 families were typed for all markers. In the combined set of 100 families the NPL score for marker D2S116 was 2.55 (P=0.006) and for other markers 1.90-2.47 (P=0.029-0.007), supporting genuine linkage at this region. Significantly, locus D2S116 also showed a clear allelic association in these 100 families (P=0.0001). The transmission/disequilibrium test (TDT) for locus D2S116 gave preliminary evidence for preferential maternal non-transmission of allele *136 to patients (TDTmax=8.3; P

We present a method for inferring the structure of ancestral metabolic networks directly from the networks of observed species and their phylogenetic tree. In particular, we aim to minimize the number of mutations on the phylogenetic tree, whilst keeping the ancestral networks structurally feasible, or gapless. In gapless metabolic networks all reactions are reachable from external substrates such as nutrients.

This paper aims to be a survey of recent experiments performed in the Neuromarketing field. Our purpose is to illustrate results obtained by employing the popular tools of investigation well known in the international neuroelectrical community such as the MEG, High Resolution EEG techniques and steady-state visually evoked potentials. By means of temporal and frequency patterns of cortical activations we intend to show how the neuroscientific community is nowadays sensible to the needs of companies and, at the same time, how the same tools are able to retrieve hidden information about the demands of consumers. These instruments could be of help both in pre- and post-design stage of a product, or a service, that a marketer is going to promote.

Background Trichoderma reesei is a soft rot Ascomycota fungus utilised for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. About 30 carbohydrate active enzymes (CAZymes) of T. reesei have been biochemically characterised. Genome sequencing has revealed a large number of novel candidates for CAZymes, thus increasing the potential for identification of enzymes with novel activities and properties. Plenty of data exists on the carbon source dependent regulation of the characterised hydrolytic genes. However, information on the expression of the novel CAZyme genes, especially on complex biomass material, is very limited. Results In this study, the CAZyme gene content of the T. reesei genome was updated and the annotations of the genes refined using both computational and manual approaches. Phylogenetic analysis was done to assist the annotation and to identify functionally diversified CAZymes. The analyses identified 201 glycoside hydrolase genes, 22 carbohydrate esterase genes and five polysaccharide lyase genes. Updated or novel functional predictions were assigned to 44 genes, and the phylogenetic analysis indicated further functional diversification within enzyme families or groups of enzymes. GH3 β-glucosidases, GH27 α-galactosidases and GH18 chitinases were especially functionally diverse. The expression of the lignocellulose degrading enzyme system of T. reesei was studied by cultivating the fungus in the presence of different inducing substrates and by subjecting the cultures to transcriptional profiling. The substrates included both defined and complex lignocellulose related materials, such as pretreated bagasse, wheat straw, spruce, xylan, Avicel cellulose and sophorose. The analysis revealed co-regulated groups of CAZyme genes, such as genes induced in all the conditions studied and also genes induced preferentially by a certain set of substrates. Conclusions In this study, the CAZyme content of the T. reesei genome was updated, the discrepancies between the different genome versions and published literature were removed and the annotation of many of the genes was refined. Expression analysis of the genes gave information on the enzyme activities potentially induced by the presence of the different substrates. Comparison of the expression profiles of the CAZyme genes under the different conditions identified co-regulated groups of genes, suggesting common regulatory mechanisms for the gene groups.

Proteolytic processing is a key step in the production of polyphenol oxidases such as tyrosinases, converting the inactive proenzyme to an active form. In general, the fungal tyrosinase gene codes for a ~ 60 kDa protein that is, however, isolated as an active enzyme of ~ 40 kDa, lacking the C-terminal domain. Using the secreted tyrosinase 2 from Trichoderma reesei as a model protein, we performed a mutagenesis study of the residues in proximity of the experimentally determined cleavage site which are possibly involved in the proteolytic process. However, the mutant forms of tyrosinase 2 were not secreted in a full-length form retaining the C-terminal domain, but they were processed to give a ~ 45 kDa active form. Aiming at explaining this phenomenon, we analysed in silico the properties of the C-terminal domain of tyrosinase 2, of 23 previously retrieved homologous tyrosinase sequences from fungi (C. Gasparetti, G. Faccio, M. Arvas, J. Buchert, M. Saloheimo, K. Kruus, Appl. Microbiol. Biotechnol. 86 (2010) 213–226) and of nine well-characterised polyphenol oxidases. Based on the results of our study, we exclude the key role of specific amino acids at the cleavage site in the proteolytic process and report an overall higher sensitivity to proteolysis of the linker region and of the whole C-terminal domain of fungal tyrosinases.

Background Trichoderma reesei is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of T. reesei by high-resolution array comparative genomic hybridization (aCGH). Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general. Results We carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30) using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor) on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production. Conclusions aCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.

In the version of this article initially published, an author's name was misspelled as Barbote. The correct spelling is Barabote. The error has been corrected in the HTML and PDF versions of the article.

International audience; Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.

Trichoderma reesei Rut-C30 and its transformant producing Melanocarpusalbomyces laccase were cultivated in lactose-containing minimal medium inchemostats and subjected to proteome and transcriptome analysis to identifydifferentially expressed genes under these conditions. The cultures were carriedout using a dilution rate (0.03/h) previously shown to be optimal for productionof endogenous extracellular proteins under these conditions. The TRAC methodfor multiplexed transcriptional analysis was first applied to analyse theexpression levels of a selected set of genes and to monitor the steady state inthe chemostat cultures. The results indicated that UPR (unfolded proteinresponse) pathway was not activated as a response to laccase production. Onthe contrary, the expression levels of e.g. the foldase and chaperone genes pdi1and bip1 were expressed at a slightly higher level in the host. Also theexpression levels of the genes encoding the endogenous secreted cellulases,egl1 and cbh1, were slightly higher in the host strain. Both proteome andgenome wide transcriptional analysis using oligonucleotide microarrays revealedonly a modest number of cellular responses to laccase production. Altogether 32genes were differentially expressed between the strains, only five of the genesbeing upregulated in the laccase producing strain, and the rest having a higherexpression level in the host strain and including many genes encodingextracellular enzymes. Proteome analysis using 2D gel electrophoresis with DIGEshowed increased expression of a group of proteins including e.g. heat shockproteins as well ubiquitin associated proteins and proteins involved in ERassociated protein degradation. The result indicates that although laccaseexpression does not induce apparent UPR, other type stress responses areinduced e.g. activated protein degradation. The results were also compared tothose obtained in previous studies on the production human tissue plasminogenactivator (tPA) in T. reesei.

The recent sequencing efforts have revealed that fungal genomes contain many genes with little or no homology to known genes (20% - 40% of CDS with no hits in Interpro, www.ebi.ac.uk/interpro/index.html). Comparisons to other fungal genomes can add power to the genomic analysis by providing the evolutionary context of genes. Our goal is to compare the protein coding gene contents of fungal genomes and relate the differences to the physiological differences between species and taxonomic groups. However, no single repository currently provides consistent gene annotations of all sequenced fungi. We have produced consistent Interpro annotations and Tribe-MCL (www.ebi.ac.uk/research/cgg/tribe/) clustering of protein coding sequences of 16 sequenced fungal genomes. Our computational system is based on BioPerl (bio.perl.org) scripts, BioSQL schema for storing the sequences and annotations in a relational database and GBrowse (www.gmod.org) genome browser for visualising genomes, genes and gene clusters. By combining these data sources and technology we can carry out comparative genome analysis, offer a local comparative genome browser for experimental biologists and profit from the efforts of BioPerl bioinformatics community.

Background Secretion stress is caused by compromised folding, modification or transport of proteins in the secretory pathway. In fungi, induction of genes in response to secretion stress is mediated mainly by the unfolded protein response (UPR) pathway. This study aims at uncovering transcriptional responses occurring in the filamentous fungi Trichoderma reesei exposed to secretion stress and comparing these to those found in the yeast Saccharomyces cerevisiae. Results Chemostat cultures of T. reesei expressing human tissue plasminogen activator (tPA) and batch bioreactor cultures treated with dithiothreitol (DTT) to prevent correct protein folding were analysed with cDNA subtraction and cDNA-amplified fragment length polymorphism (AFLP) experiments. ESTs corresponding to 457 unique genes putatively induced under secretion stress were isolated and the expression pattern of 60 genes was confirmed by Northern analysis. Expression of these genes was also studied in a strain over-expressing inositol-requiring enzyme 1 (IREI) protein, a sensor for the UPR pathway. To compare the data with that of S. cerevisiae, published transcriptome profiling data on various stress responses in S. cerevisiae was reanalysed. The genes up-regulated in response to secretion stress included a large number of secretion related genes in both organisms. In addition, analysis of T. reesei revealed up regulation of the cpc1 transcription factor gene and nucleosomal genes. The induction of the cpcA and histone gene H4 were shown to be induced also in cultures of Aspergillus nidulans treated with DTT. Conclusion Analysis of the genes induced under secretion stress has revealed novel features in the stress response in T. reesei and in filamentous fungi. We have demonstrated that in addition to the previously rather well characterised induction of genes for many ER proteins or secretion related proteins also other types of responses exist.

Motivation: cDNA amplified fragment length polymorphism (cDNA-AFLP) is one of the few genome-wide level expression profiling methods capable of finding genes that have not yet been cloned or even predicted from sequence but have interesting expression patterns under the studied conditions. In cDNA-AFLP, a complex cDNA mixture is divided into small subsets using restriction enzymes and selective PCR. A large cDNA-AFLP experiment can require a substantial amount of resources, such as hundreds of PCR amplifications and gel electrophoresis runs, followed by manual cutting of a large number of bands from the gels. Our aim was to test whether this workload can be reduced by rational design of the experiment. Results: We used the available genomic sequence information to optimize cDNA-AFLP experiments beforehand so that as many transcripts as possible could be profiled with a given amount of resources. Optimization of the selection of both restriction enzymes and selective primers for cDNA-AFLP experiments has not been performed previously. The in silico tests performed suggest that substantial amounts of resources can be saved by the optimization of cDNA-AFLP experiments. Availability: A Perl implementation of the optimization method is available upon request from the authors. Contact: Teemu.Kivioja@vtt.fi

Trichoderma reesei is an industrial protein production host famous for its exceptional protein secretion capability. Protein production can cause stress to cells by compromised protein folding or transport in the secretory pathway. T. reesei is known to respond to secretion stress by unfolded protein response (UPR) and repression through secretion stress (RESS). We have studied the effects of heterologous protein production in bioreactor cultivations on the transcriptome and proteome of T. reesei. As an example of secreted and UPR causing protein, a transformant producing human tissue plasminogen activator (tPA), was studied with semi-genomic transcription profiling methods, cDNA subtraction libraries and cDNA-AFLP and 2D proteomics. In addition effect of chemical dithiothreitol (DTT) and a transformant over-expressing IREI protein (UPR pathway sensor protein) were analysed. Data from various secretion stress transcription profiling experiments in S. cerevisiae were combined from literature and compared to results from T. reesei. The transcriptional responses of T. reesei and S. cerevisiae show clear overlap, especially with UPR related genes involved in protein translocation, folding and glycosylation in the ER, but also some interesting differences. As an example of a protein causing no UPR, a transformant producing Melanocarpus albomyces laccase was studied with oligonucleotide microarrays. Moderate downregulation of secreted proteins was detected.

Industrially exploited filamentous fungi are known for their extremely high capacity of protein secretion. High loads of protein, and especially foreign protein, in the secretory pathway form a challenge to the production organism and expose it to secretion stress. Unfolded protein response (UPR) denotes the induction mechanism of genes encoding ER-resident chaperones and foldases and numerous other genes involved in protein secretion. This induction is triggered when unfolded proteins accumulate into the ER. We have investigated several components of the UPR pathway, its regulatory range and its utilisation for improvement of protein production in filamentous fungi, predominantly Trichoderma reesei. We have shown that the UPR transcription factor gene hal1 is activated in filamentous fungi by a dual mechanism operational at the mRNA level. This mechanism includes a splicing event of an unconventional intron of only 20 nt in length and a truncation of the mRNA at the 5' flanking region. This truncation removes an upstream open reading frame from the mRNA, and we have shown that these uORFs are involved in translational control of the HAC1 protein formation. The regulatory range of secretion stress responses in T. reesei has been studied by subtraction library cloning, cDNA-AFLP and proteomics. These methods have revealed a high number of genes up-regulated by secretion stress, including ones involved in protein folding, glycosylation and trafficking in the secretory pathway. An interesting link between amino acid metabolism and secretion stress was also observed. The activated form of the hac1/hacA transcription factor gene was expressed from a constitutive promoter in T. reesei, Aspergillus niger var. awamori and S. cerevisiae, and in all cases the induction of the UPR pathway was observed. In A. niger the production of Trametes versicolor laccase was improved about 5-fold and that of bovine chymosin about 3-fold by the constitutive UPR induction. The production of Bacillus alpha-amylase and native invertase was enhanced in S. cerevisiae with this strategy. We have observed that concurrently with the induction of the UPR pathway, the genes encoding secreted proteins are rapidly down-regulated in T. reesei. This type of regulation can be caused by different secretion inhibitors and by foreign protein expression. The down-regulation is dependent on the promoter of the affected gene, suggesting that it is functional at the transcriptional level.

Trichoderma reesei is an industrial protein production host known for its exceptional protein secretion capability. This study aims at uncovering the transcriptional responses occurring in T. reesei cells exposed to secretion stress and comparing these responses to similar experiments carried out in S. cerevisiae. Secretion stress is caused by compromised protein folding or transport in the secretory pathway. It induces a number of genes involved in different aspects of secretion through the unfolded protein response (UPR) pathway. In T. reesei it has also been shown that secretion stress down-regulates genes encoding secreted proteins. We constructed cDNA subtraction libraries and made cDNA-AFLP (amplified fragment length polymorphism) experiments from cells under secretion stress. A transformant expressing human tissue plasminogen activator (tPA), treatment with the chemical DTT (dithiothreitol) that prevents correct protein folding and a transformant over-expressing IREI protein (sensor protein of the UPR pathway) were analysed. Around two hundred unique ESTs were retrieved by these methods and the expression pattern of about 50 was confirmed by Northern experiments. A rank sum test for the Northern data was used to define those genes that show upregulation in all the three conditions. Data from DTT and tunicamycin treatment, foreign protein production and IRE1 and HAC1 (UPR transcription factor) deletion experiments in S. cerevisiae were combined from litterature. The transcriptional responses of T. reesei and S. cerevisiae show clear overlap, especially with respect to genes involved in protein translocation, folding and glycosylation in the ER. However, there seems to be major differences in regulation of amino acid biosynthesis and nucleosome genes. The GCN4/CPC1 transcription factor and a limited set of its putative target genes are induced only in T. reesei. This response points to the upregulation of glutathione synthesis to relieve oxidative stress caused by compromised protein folding. Interestingly also a set of nucleosome genes is upregulated in T. reesei without a clear connection to cell cycle.

Motivation: Measuring transcriptional expression levels (transcriptional profiling) has become one of the most important methods in functional genomics. Still, new measuring methods are needed to obtain more reliable, quantitative data about transcription on a genomic scale. In this paper we concentrate on certain computational optimization problems arising in the design of one such novel method. From a computational point of view the key feature of the new method is that the hybridized probes are distinguished from each other based on their different size. Therefore the probes have to be assigned into pools such that the probes in the same pool have unique sizes different enough from each other. Identification of expressed RNA is given by probe pool and probe size while quantification is given by the label of the probe, e.g. fluorescence intensity. Results: We show how to computationally find the probes and assign them into pools for a whole genome such that (i) each gene has a specific probe suitable for amplification and hybridization, and (ii) the expression level measurement can be done in a minimal number of pools separable by electrophoresis in order to minimize the total experiment cost of the measurement. Our main result is a polynomial-time approximation algorithm for assigning the probes into pools. We demonstrate the feasibility of the procedure by selecting probes for the yeast genome and assigning them into less than 100 pools. The probe sequences and their assignment into pools are available for academic research on request from the authors. Contact: Teemu.Kivioja@cs.Helsinki.FI Keywords: transciptional profiling; hybridization probe selection; graph algorithms; degree-contrained subgraph.

Background Several dozen fungi encompassing traditional model organisms, industrial production organisms and human and plant pathogens have been sequenced recently and their particular genomic features analysed in detail. In addition comparative genomics has been used to analyse specific sub groups of fungi. Notably, analysis of the phylum Saccharomycotina has revealed major events of evolution such as the recent genome duplication and subsequent gene loss. However, little has been done to gain a comprehensive comparative view to the fungal kingdom. We have carried out a computational genome wide comparison of protein coding gene content of Saccharomycotina and Pezizomycotina, which include industrially important yeasts and filamentous fungi, respectively. Results Our analysis shows that based on genome redundancy, the traditional model organisms Saccharomyces cerevisiae and Neurospora crassa are exceptional among fungi. This can be explained by the recent genome duplication in S. cerevisiae and the repeat induced point mutation mechanism in N. crassa. Interestingly in Pezizomycotina a subset of protein families related to plant biomass degradation and secondary metabolism are the only ones showing signs of recent expansion. In addition, Pezizomycotina have a wealth of phylum specific poorly characterised genes with a wide variety of predicted functions. These genes are well conserved in Pezizomycotina, but show no signs of recent expansion. The genes found in all fungi except Saccharomycotina are slightly better characterised and predicted to encode mainly enzymes. The genes specific to Saccharomycotina are enriched in transcription and mitochondrion related functions. Especially mitochondrial ribosomal proteins seem to have diverged from those of Pezizomycotina. In addition, we highlight several individual gene families with interesting phylogenetic distributions. Conclusion Our analysis predicts that all Pezizomycotina unlike Saccharomycotina can potentially produce a wide variety of secondary metabolites and secreted enzymes and that the responsible gene families are likely to evolve fast. Both types of fungal products can be of commercial value, or on the other hand cause harm to humans. In addition, a great number of novel predicted and known enzymes are found from all fungi except Saccharomycotina. Therefore further studies and exploitation of fungal metabolism appears very promising.

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies. Monissa elintarvike-, kangas-, puunjalostus- ja energiateollisuuden prosesseissa tarvitaan entsyymejä. Näillä muunnellaan biomassan ominaisuuksia, esimerkiksi juoksutetaan juustoa, valkaistaan farkkuja tai paperia. Entsymaattinen käsittely on usein ympäristöystävällisempää kuin vastaava käsittely kemikaaleilla. Homesieniä käytetään entsyymien ja muiden proteiinien tuottamiseen. Proteiinit voivat olla peräisin myös muista eliöistä, esimerkiksi kasveista tai nisäkkäistä. Trichoderma reesei on trooppinen home, joka on tunnettu proteiinin tuottokyvystään ja sitä käytetään laajasti Suomessa ja muualla maailmassa. Tässä työssä on vertailtu home- ja hiivasienten perimiä laskennallisin metodein. Hiivoilla on puolet pienemmät perimät kuin homeilla ja ne tuottavat vähemmän proteiineja. Tehdyn vertailun perusteella ymmärrämme paremmin sieniperimien suhteita sienten havaittaviin ominaisuuksiin eli ilmiasuun. Onnistunut sienten perimän muuntelu niiden tuotto ominaisuuksien parantamiseksi edellyttää perimän ja ilmiasun suhteiden yksityiskohtaista ymmärtämistä. Perimässä oleva tieto muuttuu eliöiden ominaisuuksiksi monen välivaiheen kautta. Siksi pelkän perimän laskennallisen analyysin pohjalta on vaikea päätellä miten eliön perimää tulisi muunnella sen ominaisuuksien parantamiseksi. Perimä ilmentyy eri elinolosuhteissa eri tavoin ja ilmentymisen havainnoiminen on yksi tärkeimpiä tapoja tutkia eliön perimän ja ilmiasun suhdetta. Tässä työssä on kehitetty niin laskennallisia kuin laboratoriometodeja ilmentymisen tutkimiseen ja testattu niiden soveltuvuutta T. reesei:lle. Lisäksi T. reesei:n perimän ilmentymistä tutkittiin proteiinin tuoton kannalta kiinnostavissa olosuhteissa ja uusia perimän ilmentymisen muutoksia havaittiin. Ne saattavat osittain selittää homeiden erinomaista proteiinin tuottokykyä.

Low il-2 concentration favors generation of early memory t cells over terminal eefectors during car t-cell expansion

Trichoderma reesei has a long history in industrial enzyme production because of its high protein secretion capability and easy processability. Despite its many positive features, further strain engineering is still being pursued to remove any remaining protein production and secretion bottlenecks. Systems biology methods were applied to study protein production in selected proprietary T. reesei strains. Transcriptional analysis using microarrays was performed to identify differentially expressed genes in various cultivation conditions and high-density Comparative Genomic Hybridization (CGH) was used to characterize genomic alterations in the strains. Combined data from CGH and transcriptional analyses was utilized to identify target genes for further improvement of protein production/secretion. Deletions and/or overexpressions of 16 genes involved in protein transport, post-translational modification, basic energy metabolism or regulatory functions were studied in detail.

The role of the fungal genome annotation team at LANL is to provide high quality manually curated annotation of fungal genomic sequence. The JGI-LANL and fungal research community are teaming up to publish a full analysis and annotation of Trichoderma reesei and Aspergillus niger chromosomes. Work on the T. reesei genome was completed earlier this year and will appear in publication in the coming months. We begin our analysis by running each genome through an automated pipeline that combines evidence-based and abintio methods. All information is displayed before manual review in the JGI’s Genome Portal, available at http://genome.jgi.doe.gov/. Utilizing a combination of the Genome Portal, local tools and the Apollo editing system each predicted gene is then manually reviewed by comparing the predicted gene to EST and mRNA sequences from the T. reesei and A. niger genomes as well as closely related species, such as Neurospora crassa, Fusarium graminarium, Magnaporthe grisea and other Apergillus species. This suite of tools allows a distributed group of annotators from the fungal research community to correct errors as necessary and enrich the automated information with comments and human annotations. All manual annotations are immediately viewable and searchable in the Genome Portal.

Background. Several dozens of fungi encompassing traditional model organisms, industrial production organisms and human and plant pathogens have been sequenced recently and their particular genomic features analysed in detail. In addition comparative genomics has been applied to make analyses within sub groups of fungi. Notably, analysis of the phylum Saccharomycotina has revealed major events of evolution such as the recent genome duplication and subsequent gene loss. However, little has been done to gain a comprehensive comparative view to the fungal kingdom. We have carried out a computational genome wide comparison of protein coding gene content of the phyla Saccharomycotina and Pezizomycotina, which include industrially important yeasts and filamentous fungi, respectively. Results. Our analysis shows that based on genome redundancy the traditional model organisms Saccharomyces cerevisiae and Neurospora crassa are exceptional among fungi. This can be explained by the recent genome duplication in S. cerevisiae and the repeat induced point mutation mechanism (RIP) in N. crassa. Interestingly, in Pezizomycotina a subset of protein families related to plant biomass degradation and secondary metabolism are the only ones showing signs of recent expansion. In addition, Pezizomycotina have a wealth of phylum-specific poorly characterised genes with a wide variety of predicted functions. These genes are well conserved in Pezizomycotina, but show no signs of recent expansion. The genes found in all fungi except Saccharomycotina are slightly better characterised and predicted to encode mainly enzymes. The gene classes specific to Saccharomycotina are enriched in transcription and mitochondrion related functions. Especially mitochondrial ribosomal proteins seem to have diverged from those of Pezizomycotina. Conclusions. Our analysis predicts that all Pezizomycotina unlike Saccharomycotina can potentially produce a wide variety of secondary metabolites and secreted enzymes and that the respective genetic systems are likely to evolve fast. Both types of fungal products can be of commercial value, but on the other hand can cause harm to humans. In addition, a great number of novel predicted and known enzymes are found from all fungi except Saccharomycotina. Therefore further studies and exploitation of fungal metabolism appears very promising.

Trichoderma reesei is known for its extremely high capacity of protein secretion. High loads of protein, and especially foreign protein, in the secretory pathway form a challenge to the production organism and expose it to secretion stress. Unfolded protein response (UPR) denotes the induction mechanism of genes encoding ER-resident chaperones and foldases and numerous other genes involved in protein secretion. This induction is triggered when unfolded proteins accumulate into the ER. We have cloned the transcription factor involved in UPR induction, HACI, from T. reesei. Our results indicate that the hac1 gene is activated by a dual mechanism operational at the mRNA level. This mechanism includes a splicing event of an unconventional intron of only 20 nt in length and a truncation of the mRNA at the 5' flanking region. This truncation removes an upstream open reading frame from the mRNA, and we have shown that these uORFs are involved in translational control of the HAC1 protein formation. We have observed that concurrently with the induction of the UPR pathway, the genes encoding secreted proteins are rapidly down-regulated in Trichoderma reesei. This type of regulation can be caused by different secretion inhibitors and by foreign protein expression. The down-regulation is dependent on the promoter of the affected gene, suggesting that it is functional at the transcriptional level. The down-regulation of genes encoding secreted proteins during secretion stress has not been reported before from any other experimental system and thus it could be unique for filamentous fungi.

Despite the extensive research the exact function many yeast genes remains unknown. Comparisons to other fungal genomes can add power to the genomic analysis by providing the evolutionary context of genes. Our goal is to compare the protein coding gene contents of fungal genomes and relate the differences to the physiological differences between species and taxonomic groups. We have produced consistent Interpro annotations and clustering of protein coding sequences of 16 sequenced fungal genomes of which 8 are yeasts. Our computational system is based on BioPerl scripts and BioSQL schema for storing the sequences and annotations in a relational database. The clustering of protein sequences is done with Tribe-MCL graph clustering software using distances based on Blast E-values. We have discovered that the number of genes belonging to protein clusters having members from all fungal species studied is negatively correlated with genome size, i.e. larger fungal genomes are likely to have more specialized functions not present in species with smaller genomes. In contrast, based on protein clustering Pezizomycotina and Saccharomycotina seem to differ in their level of paralogy, i.e. in number of duplicated genes. In Saccharomycotina average level of paralogy is positively correlated to the size of the genome. In Pezizomycotina, possibly due to Repeat Induced Point mutations (RIP), no clear correlation exists. Using Generic Genome Browser we have created a web-based system that allows the scientists at VTT to easily utilize comparative information in their work. In particular, we link the Interpro entries and clusters so that a user can for a particular protein family browse the neighborhood of the family members detected by Interpro to assess the true extend of the family. In addition, a user can easily find the clusters and Interpro entries that have interesting species distributions. We also link the S. cerevisiae metabolic model iND750 to the comparative data.