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

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

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production.ResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.ConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism

Whole-genome metabolic model of Trichoderma reesei built by comparative reconstruction

Background: Trichoderma reesei is one of the main sources of biomass-hydrolyzing enzymes for the biotechnology industry. There is a need for improving its enzyme production efficiency. The use of metabolic modeling for the simulation and prediction of this organism's metabolism is potentially a valuable tool for improving its capabilities. An accurate metabolic model is needed to perform metabolic modeling analysis. Results: A whole-genome metabolic model of T. reesei has been reconstructed together with metabolic models of 55 related species using the metabolic model reconstruction algorithm CoReCo. The previously published CoReCo method has been improved to obtain better quality models. The main improvements are the creation of a unified database of reactions and compounds and the use of reaction directions as constraints in the gap-filling step of the algorithm. In addition, the biomass composition of T. reesei has been measured experimentally to build and include a specific biomass equation in the model. Conclusions: The improvements presented in this work on the CoReCo pipeline for metabolic model reconstruction resulted in higher-quality metabolic models compared with previous versions. A metabolic model of T. reesei has been created and is publicly available in the BIOMODELS database. The model contains a biomass equation, reaction boundaries and uptake/export reactions which make it ready for simulation. To validate the model, we dem1on-strate that the model is able to predict biomass production accurately and no stoichiometrically infeasible yields are detected. The new T. reesei model is ready to be used for simulations of protein production processes.Peer reviewe

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

<p>Abstract</p> <p>Background</p> <p>Growth 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 <it>Saccharomyces cerevisiae </it>chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus <it>Trichoderma reesei </it>(<it>Hypocrea jecorina</it>) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.</p> <p>Results</p> <p>We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of <it>T. reesei</it>. 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.</p> <p>Conclusions</p> <p>Based 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).</p

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

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

Genome Sequencing and Analysis of the Biomass-Degrading Fungus \u3ci\u3eTrichoderma reesei\u3c/i\u3e (syn. \u3ci\u3eHypocrea jecorina\u3c/i\u3e)

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

Genome wide analysis of protein production load in Trichoderma reesei

Background: The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is a widely used industrial host organism for protein production. In industrial cultivations, it can produce over 100 g/l of extracellular protein, mostly constituting of cellulases and hemicellulases. In order to improve protein production of T. reesei the transcriptional regulation of cellulases and secretory pathway factors have been extensively studied. However, the metabolism of T. reesei under protein production conditions has not received much attention. Results: To understand the physiology and metabolism of T. reesei under protein production conditions we carried out a well-controlled bioreactor experiment with extensive analysis. We used minimal media to make the data amenable for modelling and three strain pairs to cover different protein production levels. With RNA-sequencing transcriptomics we detected the concentration of the carbon source as the most important determinant of the transcriptome. As the major transcriptional response concomitant to protein production we detected the induction of selected genes that were putatively regulated by xyr1 and were related to protein transport, amino acid metabolism and transcriptional regulation. We found novel metabolic responses such as production of glycerol and a cellotriose-like compound. We then used this cultivation data for flux balance analysis of T. reesei metabolism and demonstrate for the first time the use of genome wide stoichiometric metabolic modelling for T. reesei. We show that our model can predict protein production rate and provides novel insight into the metabolism of protein production. We also provide this unprecedented cultivation and transcriptomics data set for future modelling efforts. Conclusions: The use of stoichiometric modelling can open a novel path for the improvement of protein production in T. reesei. Based on this we propose sulphur assimilation as a major limiting factor of protein production. As an organism with exceptional protein production capabilities modelling of T. reesei can provide novel insight also to other less productive organisms.Peer reviewe

Succession of physiological stages hallmarks the transcriptomic response of the&nbsp;fungus Aspergillus niger to lignocellulose.

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production.ResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.ConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism