Mikko Arvas

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).

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.

Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates

BackgroundTrichoderma 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.ResultsIn 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.ConclusionsIn 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.

Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production

BackgroundThe 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.ResultsWe 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.ConclusionsTranscriptional 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.

Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae.

BackgroundSecretion 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.ResultsChemostat 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.ConclusionAnalysis 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.

Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties.

BackgroundTrichoderma 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.ResultsWe 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.ConclusionsaCGH 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.

Comparative Genome-Scale Reconstruction of Gapless Metabolic Networks for Present and Ancestral Species

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/.

Correlation of gene expression and protein production rate - a system wide study

BackgroundGrowth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.ResultsWe have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response.ConclusionsBased on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR).

Comparison of protein coding gene contents of the fungal phyla Pezizomycotina and Saccharomycotina

BackgroundSeveral 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.ResultsOur 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.ConclusionOur 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.

Optimization of cDNA-AFLP experiments using genomic sequence data

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.

Characterisation of the gene cluster for l-rhamnose catabolism in the yeast Scheffersomyces (Pichia) stipitis

In Scheffersomyces (Pichia) stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription factor, TRC1, which is conserved among related species. Our transcriptome analysis shows that all the catabolic genes and all genes of the cluster are up-regulated on L-rhamnose. Among genes that were also up-regulated on L-rhamnose were two transcription factors including the TRC1. In addition, in 16 out of the 32 analysed fungal species only RHA1, LRA2 and LRA3 are physically clustered. The clustering of RHA1, LRA3 and TRC1 is also conserved in species not closely related to S. stipitis. Since the LRA4 is often not part of the cluster and it has several paralogues in L-rhamnose utilising yeasts we analysed the function of one of the paralogues, LRA41 by heterologous expression and biochemical characterization. Lra41p has similar catalytic properties as the Lra4p but the transcript was not up-regulated on L-rhamnose. The RHA1, LRA2, LRA4 and LADH genes were previously characterised in S. stipitis. We expressed the L-rhamnonate dehydratase, Lra3p, in Saccharomyces cerevisiae, estimated the kinetic constants of the protein and showed that it indeed has activity with L-rhamnonate.