Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei.

BACKGROUND: Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp). RESULTS: PcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-ß-D-glucosaminidase in the supernatant. CONCLUSIONS: Heterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

N-acetylglucosamine, the building block of chitin, inhibits growth of Neurospora crassa.

N-acetylglucosamine (GlcNAc) is the monomer of the polysaccharide chitin, an essential structural component of the fungal cell wall and the arthropod exoskeleton. We recently showed that the genes encoding the enzymes for GlcNAc catabolism are clustered in several ascomycetes. In the present study we tested these fungi for growth on GlcNAc and chitin. All fungi, containing the GlcNAc gene cluster, could grow on GlcNAc with the exception of four independent Neurospora crassa wild-type isolates, which were however able to grow on chitin. GlcNAc even inhibited their growth in the presence of other carbon sources. Genes involved in GlcNAc catabolism were strongly upregulated in the presence of GlcNAc, but during growth on chitin their expression was not increased. Deletion of hxk-3 (encoding the first catabolic enzyme, GlcNAc-hexokinase) and ngt-1 (encoding the GlcNAc transporter) improved growth of N. crassa on GlcNAc in the presence of glycerol. A crucial step in GlcNAc catabolism is enzymatic conversion from glucosamine-6-phosphate to fructose-6-phosphate, catalyzed by the glucosamine-6-phosphate deaminase, DAM-1. To assess, if DAM-1 is compromised in N. crassa, the orthologue from Trichoderma reesei, Trdam1, was expressed in N. crassa. Trdam1 expression partially alleviated the negative effects of GlcNAc in the presence of a second carbon source, but did not fully restore growth on GlcNAc. Our results indicate that the GlcNAc-catabolism pathway is bypassed during growth of N. crassa on chitin by use of an alternative pathway, emphasizing the different strategies that have evolved in the fungal kingdom for chitin utilization.

Molecular and catalytic properties of fungal extracellular cellobiose dehydrogenase produced in prokaryotic and eukaryotic expression systems.

BACKGROUND: Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by lignocellulolytic fungi. cdh gene expression is high in cellulose containing media, but relatively low CDH concentrations are found in the supernatant of fungal cultures due to strong binding to cellulose. Therefore, heterologous expression of CDH in Pichia pastoris was employed in the last 15 years, but the obtained enzymes were over glycosylated and had a reduced specific activity. RESULTS: We compare the well-established CDH expression host P. pastoris with the less frequently used hosts Escherichia coli, Aspergillus niger, and Trichoderma reesei. The study evaluates the produced quantity and protein homogeneity of Corynascus thermophilus CDH in the culture supernatants, the purification, and finally compares the enzymes in regard to cofactor loading, glycosylation, catalytic constants and thermostability. CONCLUSIONS: Whereas E. coli could only express the catalytic dehydrogenase domain of CDH, all eukaryotic hosts could express full length CDH including the cytochrome domain. The CDH produced by T. reesei was most similar to the CDH originally isolated from the fungus C. thermophilus in regard to glycosylation, cofactor loading and catalytic constants. Under the tested experimental conditions the fungal expression hosts produce CDH of superior quality and uniformity compared to P. pastoris.

Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains.

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

The ß-importin KAP8 (Pse1/Kap121) is required for nuclear import of the cellulase transcriptional regulator XYR1, asexual sporulation and stress resistance in Trichoderma reesei.

The ascomycete Trichoderma reesei is an industrial producer of cellulolytic and hemicellulolytic enzymes, and serves as a prime model for their genetic regulation. Most of its (hemi-)cellulolytic enzymes are obligatorily dependent on the transcriptional activator XYR1. Here, we investigated the nucleo-cytoplasmic shuttling mechanism that transports XYR1 across the nuclear pore complex. We identified 14 karyopherins in T. reesei, of which eight were predicted to be involved in nuclear import, and produced single gene-deletion mutants of all. We found KAP8, an ortholog of Aspergillus nidulans KapI, and Saccharomyces cerevisiae Kap121/Pse1, to be essential for nuclear recruitment of GFP-XYR1 and cellulase gene expression. Transformation with the native gene rescued this effect. Transcriptomic analyses of Δkap8 revealed that under cellulase-inducing conditions 42 CAZymes, including all cellulases and hemicellulases known to be under XYR1 control, were significantly down-regulated. Δkap8 strains were capable of forming fertile fruiting bodies but exhibited strongly reduced conidiation both in light and darkness, and showed enhanced sensitivity towards abiotic stress, including high osmotic pressure, low pH and high temperature. Together, these data underscore the significance of nuclear import of XYR1 in cellulase and hemicellulase gene regulation in T. reesei, and identify KAP8 as the major karyopherin required for this process.

Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei.

More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei.

Genome sequencing of the Trichoderma reesei QM9136 mutant identifies a truncation of the transcriptional regulator XYR1 as the cause for its cellulase-negative phenotype.

BACKGROUND: Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a. RESULTS: In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn2Cys6 transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be expressed over the basal constitutive level. CONCLUSION: The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype.

Nucleo-cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei.

Trichoderma reesei is a model for investigating the regulation of (hemi-)cellulase gene expression. Cellulases are formed adaptively, and the transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are main regulators of their expression. We quantified the nucleo-cytoplasmic shuttling dynamics of GFP-fusion proteins of both transcription factors under cellulase and xylanase inducing conditions, and correlated their nuclear presence/absence with transcriptional changes. We also compared their subcellular localization in conidial germlings and mature hyphae. We show that cellulase gene expression requires de novo biosynthesis of XYR1 and its simultaneous nuclear import, whereas carbon catabolite repression is regulated through preformed CRE1 imported from the cytoplasmic pool. Termination of induction immediately stopped cellulase gene transcription and was accompanied by rapid nuclear degradation of XYR1. In contrast, nuclear CRE1 rapidly decreased upon glucose depletion, and became recycled into the cytoplasm. In mature hyphae, nuclei containing activated XYR1 were concentrated in the colony center, indicating that this is the main region of XYR1 synthesis and cellulase transcription. CRE1 was found to be evenly distributed throughout the entire mycelium. Taken together, our data revealed novel aspects of the dynamic shuttling and spatial bias of the major regulator of (hemi-)cellulase gene expression, XYR1, in T. reesei.

L-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is the main producer of lignocellulolytic enzymes that are required for plant biomass hydrolysis in the biorefinery industry. Although the molecular toolbox for T. reesei is already well developed, repressible promoters for strain engineering and functional genomics studies are still lacking. One such promoter that is widely employed for yeasts is that of the L-methionine repressible MET3 gene, encoding ATP sulphurylase. RESULTS: We show that the MET3 system can only be applied for T. reesei when the cellulase inducing carbon source lactose is used but not when wheat straw, a relevant lignocellulosic substrate for enzyme production, is employed. We therefore performed a transcriptomic screen for genes that are L-methionine repressible in a wheat straw culture. This analysis retrieved 50 differentially regulated genes of which 33 were downregulated. Among these, genes encoding transport proteins as well as iron containing DszA like monooxygenases and TauD like dioxygenases were strongly overrepresented. We show that the promoter region of one of these dioxygenases can be used for the strongly repressible expression of the Aspergillus niger sucA encoded extracellular invertase in T. reesei wheat straw cultures. This system is also portable to other carbon sources including D-glucose and glycerol as demonstrated by the repressible expression of the Escherichia coli lacZ encoded ß-galactosidase in T. reesei. CONCLUSION: We describe a novel, versatile set of promoters for T. reesei that can be used to drive recombinant gene expression in wheat straw cultures at different expression strengths and in an L-methionine repressible manner. The dioxygenase promoter that we studied in detail is furthermore compatible with different carbon sources and therefore applicable for manipulating protein production as well as functional genomics with T. reesei.

Xylanase gene transcription in Trichoderma reesei is triggered by different inducers representing different hemicellulosic pentose polymers.

The ascomycete Trichoderma reesei is a paradigm for the regulation and production of plant cell wall-degrading enzymes, including xylanases. Four xylanases, including XYN1 and XYN2 of glycosyl hydrolase family 11 (GH11), the GH10 XYN3, and the GH30 XYN4, were already described. By genome mining, we identified a fifth xylanase, XYN5, belonging to GH11. Transcriptional analysis reveals that the expression of all xylanases but xyn3 is induced by D-xylose, dependent on the cellulase and xylanase regulator XYR1 and negatively regulated by the carbon catabolite repressor CRE1. Impairment of D-xylose catabolism at the D-xylose reductase and xylitol dehydrogenase step strongly enhanced induction by D-xylose. Knockout of the L-xylulose reductase-encoding gene lxr3, which connects the D-xylose and L-arabinose catabolic pathways, had no effect on xylanase induction. Besides the induction by D-xylose, the T. reesei xylanases were also induced by L-arabinose, and this induction was also enhanced in knockout mutants in L-arabinose reductase (xyl1), L-arabitol dehydrogenase (lad1), and L-xylulose reductase (lxr3). Induction by L-arabinose was also XYR1 dependent. Analysis of intracellular polyols revealed accumulation of xylitol in all strains only during incubation with D-xylose and accumulation of L-arabitol only during incubation with L-arabinose. Induction by L-arabinose could be further stimulated by addition of D-xylose. We conclude that the expression of the T. reesei xylanases can be induced by both D-xylose and L-arabinose, but independently of each other and by using different inducing metabolites.

A novel L-xylulose reductase essential for L-arabinose catabolism in Trichoderma reesei.

L-Xylulose reductases belong to the superfamily of short chain dehydrogenases and reductases (SDRs) and catalyze the NAD(P)H-dependent reduction of L-xylulose to xylitol in L-arabinose and glucuronic acid catabolism. Here we report the identification of a novel L-xylulose reductase LXR3 in the fungus Trichoderma reesei by a bioinformatic approach in combination with a functional analysis. LXR3, a 31 kDa protein, catalyzes the reduction of L-xylulose to xylitol via NADPH and is also able to convert D-xylulose, D-ribulose, L-sorbose, and D-fructose to their corresponding polyols. Transcription of lxr3 is specifically induced by L-arabinose and L-arabitol. Deletion of lxr3 affects growth on L-arabinose and L-arabitol and reduces total NADPH-dependent LXR activity in cell free extracts. A phylogenetic analysis of known L-xylulose reductases shows that LXR3 is phylogenetically different from the Aspergillus niger L-xylulose reductase LxrA and, moreover, that all identified true L-xylulose reductases belong to different clades within the superfamily of SDRs. This indicates that the enzymes responsible for the reduction of L-xylulose in L-arabinose and glucuronic acid catabolic pathways have evolved independently and that even the fungal LXRs of the L-arabinose catabolic pathway have evolved in different clades of the superfamily of SDRs.

L-xylo-3-hexulose reductase is the missing link in the oxidoreductive pathway for D-galactose catabolism in filamentous fungi.

In addition to the well established Leloir pathway for the catabolism of d-galactose in fungi, the oxidoreductive pathway has been recently identified. In this oxidoreductive pathway, D-galactose is converted via a series of NADPH-dependent reductions and NAD(+)-dependent oxidations into D-fructose. The pathway intermediates include galactitol, L-xylo-3-hexulose, and d-sorbitol. This study identified the missing link in the pathway, the L-xylo-3-hexulose reductase that catalyzes the conversion of L-xylo-3-hexulose to D-sorbitol. In Trichoderma reesei (Hypocrea jecorina) and Aspergillus niger, we identified the genes lxr4 and xhrA, respectively, that encode the l-xylo-3-hexulose reductases. The deletion of these genes resulted in no growth on galactitol and in reduced growth on D-galactose. The LXR4 was heterologously expressed, and the purified protein showed high specificity for L-xylo-3-hexulose with a K(m) = 2.0 ± 0.5 mm and a V(max) = 5.5 ± 1.0 units/mg. We also confirmed that the product of the LXR4 reaction is D-sorbitol.

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei.

Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, ß-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing ('ChIP-seq') showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified.

The intracellular galactoglycome in Trichoderma reesei during growth on lactose.

Lactose (1,4-0-ß-D-galactopyranosyl-D-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for-among other purposes-use in biofuel and biorefinery industries. The mechanism how lactose induces cellulase formation in T. reesei is enigmatic, however. Previous results from our laboratory raised the hypothesis that intermediates from the two galactose catabolic pathway may give rise to the accumulation of intracellular oligogalactosides that could act as inducer. Here we have therefore used high-performance anion-exchange chromatography-mass spectrometry to study the intracellular galactoglycome of T. reesei during growth on lactose, in T. reesei mutants impaired in galactose catabolism, and in strains with different cellulase productivities. Lactose, allo-lactose, and lactulose were detected in the highest amounts in all strains, and two trisaccharides (Gal-ß-1,6-Gal-ß-1,4-Glc/Fru and Gal-ß-1,4-Gal-ß-1,4-Glc/Fru) also accumulated to significant levels. Glucose and galactose, as well as four further oligosaccharides (Gal-ß-1,3/1,4/1,6-Gal; Gal-ß-1,2-Glc) were only detected in minor amounts. In addition, one unknown disaccharide (Hex-ß-1,1-Hex) and four trisaccharides were also detected. The accumulation of the unknown hexose disaccharide was shown to correlate with cellulase formation in the improved mutant strains as well as the galactose pathway mutants, and Gal-ß-1,4-Gal-ß-1,4-Glc/Fru and two other unknown hexose trisaccharides correlated with cellulase production only in the pathway mutants, suggesting that these compounds could be involved in cellulase induction by lactose. The nature of these oligosaccharides, however, suggests their formation by transglycosylation rather than by glycosyltransferases. Based on our results, the obligate nature of both galactose catabolic pathways for this induction must have another biochemical basis than providing substrates for inducer formation.

Metabolic engineering of inducer formation for cellulase and hemicellulase gene expression in Trichoderma reesei.

The filamentous fungus T. reeseiis today a paradigm for the commercial scale production of different plant cell wall degrading enzymes mainly cellulases and hemicellulases. Its enzymes have a long history of safe use in industry and well established applications are found within the pulp, paper, food, feed or textile processing industries. However, when these enzymes are to be used for the saccharification of cellulosic plant biomass to simple sugars which can be further converted to biofuels or other biorefinery products, and thus compete with chemicals produced from fossil sources, additional efforts are needed to reduce costs and maximize yield and efficiency of the produced enzyme mixtures. One approach to this end is the use of genetic engineering to manipulate the biochemical and regulatory pathways that operate during enzyme production and control enzyme yield. This review aims at a description of the state of art in this area.

Identification of a permease gene involved in lactose utilisation in Aspergillus nidulans.

Lactose is intracellularly hydrolysed by Aspergillus nidulans. Classical mutation mapping data and the physical characteristics of the previously purified glycosyl hydrolase facilitated identification of the clustered, divergently transcribed intracellular ß-galactosidase (bgaD) and lactose permease (lacpA) genes. At the transcript level, bgaD and lacpA were coordinately expressed in response to d-galactose, lactose or l-arabinose, while no transcription was detectable in the additional presence of glucose. In contrast, creA loss-of-function mutants derepressed for both genes to a considerable extent (even) under non-inducing or repressing growth conditions. Lactose- and d-galactose induction nevertheless occurred only in the absence of glucose, indicating a regulatory role for CreA-independent repression. Remarkably, bgaD deletion mutants grew normal on lactose. In contrast, lacpA deletants grew at a much slower rate in lactose liquid medium than wild-type while strains that carried more than one copy of lacpA grew faster, showing that transport is the limiting step in lactose catabolism. The effect of lacpA gene deletion on lactose uptake was exacerbated at lower substrate concentrations, evidence for the existence of a second transport system with a lower affinity for this disaccharide in A. nidulans.

A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background.

Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on D: -glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background.

Expression of biomass-degrading enzymes is a major event during conidium development in Trichoderma reesei.

The conidium plays a critical role in the life cycle of many filamentous fungi, being the primary means for survival under unfavorable conditions. To investigate the transcriptional changes taking place during the transition from growing hyphae to conidia in Trichoderma reesei, microarray experiments were performed. A total of 900 distinct genes were classified as differentially expressed, relative to their expression at time zero of conidiation, at least at one of the time points analyzed. The main functional categories (FunCat) overrepresented among the upregulated genes were those involving solute transport, metabolism, transcriptional regulation, secondary metabolite synthesis, lipases, proteases, and, particularly, cellulases and hemicellulases. Categories overrepresented among the downregulated genes were especially those associated with ribosomal and mitochondrial functions. The upregulation of cellulase and hemicellulase genes was dependent on the function of the positive transcriptional regulator XYR1, but XYR1 exerted no influence on conidiation itself. At least 20% of the significantly regulated genes were nonrandomly distributed within the T. reesei genome, suggesting an epigenetic component in the regulation of conidiation. The significant upregulation of cellulases and hemicellulases during this process, and thus cellulase and hemicellulase content in the spores of T. reesei, contributes to the hypothesis that the ability to hydrolyze plant biomass is a major trait of this fungus enabling it to break dormancy and reinitiate vegetative growth after a period of facing unfavorable conditions.

Differential regulation of the cellulase transcription factors XYR1, ACE2, and ACE1 in Trichoderma reesei strains producing high and low levels of cellulase.

Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly being investigated for second-generation biofuel production from lignocellulosic biomass. The induction mechanisms of T. reesei cellulases have been described recently, but the regulation of the genes involved in their transcription has not been studied thoroughly. Here we report the regulation of expression of the two activator genes xyr1 and ace2, and the corepressor gene ace1, during the induction of cellulase biosynthesis by the inducer lactose in T. reesei QM 9414, a strain producing low levels of cellulase (low producer). We show that all three genes are induced by lactose. xyr1 was also induced by d-galactose, but this induction was independent of d-galactose metabolism. Moreover, ace1 was carbon catabolite repressed, whereas full induction of xyr1 and ace2 in fact required CRE1. Significant differences in these regulatory patterns were observed in the high-producer strain RUT C30 and the hyperproducer strain T. reesei CL847. These observations suggest that a strongly elevated basal transcription level of xyr1 and reduced upregulation of ace1 by lactose may have been important for generating the hyperproducer strain and that thus, these genes are major control elements of cellulase production.

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing.

Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.