Analysis of wheat straw grown Trichoderma reesei gene expression as influenced by L-methionine in the medium
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ABSTRACT: Wheat straw grown cultures of T. reesei QM9414 were supplemented with 100 µM L-methionine and the genome wide gene expression monitored in order to find novel L-Methionine repressible genes. Total RNA was isolated from independent duplicate shake flask cultures of T. reesei QM9414 pregrown on pretreated wheat straw. Global gene and analyzed using a 4 chip design where 2 chips each represented cultures with or without exogeneously added 100 µM L- Methionine.
Project description:Investigation of whole genome gene expression level changes in response to different light conditions of the T. reesei QM9414 parental strain and the deletion strains delta-phlp1, delta-gnb1 and delta gng1, cultivated on 1 % microcrystalline cellulose. The mutants analyzed in this study are further described in Tisch et al. 2011: Carbohydrate degradation is significantly regulated by light and the phosducin like protein PhLP1 in Trichoderma reesei (Hypocrea jecorina). We used two biological replicates of four T. reesei strains (QM9414, delta-phlp1, delta-gnb1 and delta-gng1), cultivated in constant light (LL, 1800 lux) or constant darkness (DD) on microcrystalline cellulose.
Project description:We used three biological replicas of T. reesei (QM9414), a M-NM-^Tlae1 mutant and a lae1-overexpressing strain in the chemostat on glucose at two different growth rates (0.075 and 0.020 h-1). Two to four subsequently achieved, independent steady-states were sampled and analysed for each dilution rate and fungal strain.
Project description:We have examined and compared the transcriptome of T. reesei growing on wheat straw and lactose as carbon sources under otherwise similar conditions. Gene expression on wheat straw exceeded that on lactose, and 1619 genes were found to be only induced on wheat straw but not on lactose. They comprised 30 % of the CAZome, but were also enriched in genes associated with phospholipid metabolism, DNA synthesis and repair and iron homeostatis. Two thirds of the CAZome was expressed both on wheat straw as well as on lactose, but 60 % of it at least >2-fold higher on the former. Major wheat straw specific genes comprised xylanases, chitinases and M-CM-^_-mannosidases. Interestingly, the latter two CAZyme families were significantly higher expressed in a strain in which xyr1 encoding the major regulator of cellulase and hemicellulase biosynthesis is non-functional, demonstrating that XYR1 is a repressor of these genes. We used two biological replicas of four T. reesei strains growing on glucose, lactose, and on wheat straw
Project description:The mitosporic fungus Trichoderma reesei is an industrial producer of enzymes for degradation of lignocellulosic polysaccharides to soluble monomers that can be fermented to biofuels. The genes encoding these enzymes in T. reesei have recently been shown to be clustered in the genome. Here we will show that the expression of these genes is epigenetically controlled at the heterochromatin level by the protein methyltransferase LAE1. Deletion of lae1 led to a loss of expression of the major cellulase and hemicellulase encoding genes, and resulted in an inability to grow on cellulose. The cellulase null phenotype was also seen with known soluble inducers of enzymes active on cellulose. In contrast, introduction of a second copy of lae1 or its enhanced expression under a strong constitutive promoter resulted in increased levels of cellulases. Thus, our data provides an experiment-based explanation for the advantage for clustering of cellulases in the genome of T. reesei, and imply that the heterochromatin structure is a major determinant of cellulase gene expression and hence an attractive target for strain improvement. We used two biological replicas of four T. reesei strains growing on lactose, the parent strain (QM9414), a delta-lae1, and two overexpressing strains (tef1:lae1 mutant 1 and mutant 2).
Project description:Lactose (1,4-0-M-CM-^_-d-galactopyranosyl-d-glucose), a by-product from cheese manufacture or whey processing industries, is known to induce the formation of plant biomass hydrolyzing enzymes needed for the biorefinery industry in the fungus Trichoderma reesei, but the reason for this induction and the underlying mechanism are not fully understood. Here, we used systems analysis of the Trichoderma reesei transcriptome during utilization of lactose. We found that the respective CAZome encoded glycosyl hydrolases specifically tailored for the attack of monocotyledon xyloglucan. In addition, genes for a high number of putative transporters of the major facilitator superfamily were also induced. Systematic knock out of them identified a gene whose knock-out completely impaired lactose utilization and cellulase induction in Trichoderma reesei. These data shed new light on the mechanism by which Trichoderma reesei metabolizes lactose and illuminate the key role of M-CM-^_-D-galactosides in habitat specificity of this fungus. We used two biological replicas of Trichoderma reesei growing on lactose, glucose and glycerol
Project description:Wheat straw grown cultures of T. reesei QM9414 were supplemented with 100 µM L-methionine and the genome wide gene expression monitored in order to find novel L-Methionine repressible genes.
Project description:Investigation of whole genome gene expression level changes in response to different light conditions of the T. reesei QM9414 deletion strains delta-blr1, delta-blr2 and delta env1 cultivated on 1% microcrystalline cellulose. Perception and proper interpretation of environmental signals is crucial for survival in any natural habitat. Although the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina) is predominantly known for its capability of efficient plant cell wall degradation, recent studies show that it has not lost its evolutionary heritage. Transmission of nutrient signals via the heterotrimeric G protein pathway has been shown to be influenced by light. We show that this interconnection is mainly established by the light regulatory protein ENV1 and the phosducin-like protein PhLP1 via mutual transcriptional regulation and influence on GNB1 (G protein beta subunit) function. ENV1 thereby exerts a more severe effect on gene transcription than BLR1 or BLR2. Lack of either one of the photoreceptors or PhLP1, GNB1 or GNG1 leads to a partial shutdown of processes upregulated in light, indicating that heterotrimeric G protein signalling exerts its major function in light and is a target of the light response machinery. Consequently, signals transmitted via the G protein pathway are of different relevance in light and darkness. Investigation of regulation of glycoside hydrolases as one of the major output pathways of this mechanism revealed that 79% of all genes belonging to this group, representing all GH-families available in T. reesei, are potentially responsive to light. We conclude that ENV1 is a key factor in connecting nutrient signalling with light response and establishes a signalling output pathway independent of BLR1 and BLR2. We used two biological replicates of three T. reesei strains (delta-blr1, delta-blr2 and delta-env1), cultivated in constant light (LL, 1800 lux) or constant darkness (DD) on microcrystalline cellulose. The strains used in this study were cultivated, hybridized and analyzed together with strains and samples from GSE27581; the corresponding wild-type strain QM9414 samples have accession numbers GSM683732, GSM683733, GSM683734 and GSM683735.
Project description:The asexual spore or conidium plays a critical role in the life cycle of many filamentous fungi, being the primary means for dispersion in the environment. To investigate the transcriptional changes taking place during the sporulation phase in T. reesei, which culminates with the production of the conidiospores, microarray experiments were performed. Among the 1,994 distinct genes displaying >90 % confidence , a total of 900 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. The main functional categories (FunCat) overrepresented among upregulated genes were those involving solute transport, metabolism, transcriptional regulation, secondary metabolite synthesis, lipases, proteases and particularly cellulases and hemicellulases. Categories overrepresented among downregulated genes were especially those associated with ribosomal and mitochondrial functions. The upregulation of cellulase and hemicellulase genes was depending on the function of the positive transcriptional regulator XYR1, but the latter exerted no influence on sporulation itself. At least 20 % of the significantly regulated genes occured non-randomly distributed within the T. reesei genome suggesting an epigenetic component in the regulation of conidiation. The significant upregulation of cellulases and hemicellulases during conidiation, and thus cellulase and hemicelulase content in the spores of T. reesei lend to hypothesize that the ability to hydrolyse plant biomass is a major trait of this fungus to break dormancy and reinitiate vegetative growth after a period of facing unfavorable conditions One control and three sample points, checked by a marker enzyme for sporulation and by microscopy, were done in two biologicvao replicas
Project description:Comparison of T. reesei grown on lactose fed chemostat cultivations in different growth rates and cell densities The analysis is further described in paper Correlation of gene expression and protein production rate - a system wide study. Mikko Arvas, Tiina Pakula, Bart Smit, Jari Rautio, Heini Koivistoinen, Paula Jouhten, Erno Lindfors, Marilyn Wiebe, Merja Penttilä and Markku Saloheimo, Submitted. Abstract: Background: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 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. Results: We 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. Conclusions: 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). A nine chip study using total RNA recovered from three separate cultures of T. reesei RutC-30 grown with growth rate 0.03, three separate cultures grown with growth rate 0.06 and three separate cultures grown with growth rate 0.03 in high cell density.
Project description:Investigation on expression levels of normal tissue from prostate cancer patients on locus 8q24. The region chr8:127640000-129120000 is tiled with isothermal probes (hg17) 7 chip study, using 7 independent samples.