Xylanase gene transcription in Trichoderma reesei is triggered by different inducers representing different hemicellulosic pentose polymers.
ABSTRACT: 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.
Project description:Xyr1 (xylanase regulator 1) of the ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) was recently demonstrated to play an essential role in the transcriptional regulation of the xyn1 (xylanase 1-encoding) gene expression. Consequently, this study reports on the deletion of the xyr1 gene from the H. jecorina genome. Comparative studies of the growth behavior of the different mutant strains (deleted and retransformed xyr1) grown on various carbon sources pointed to the strongly reduced ability of the xyr1 deletion strain to utilize D-xylose and xylan. Transcriptional analysis of the xyl1 (D-xylose reductase 1-encoding) gene as well as measurements of corresponding enzymatic activities gave evidence that Xyr1 takes part in the control of the fungal D-xylose pathway, in particular in the regulation of D-xylose reductase. It could be demonstrated that the uptake of D-xylose into the fungal cell is uninfluenced in the Deltaxyr1 strain. Furthermore, transcriptional regulation of the major hydrolytic enzyme-encoding genes xyn1 and xyn2 (xylanases 1 and 2), cbh1 and cbh2 (cellobiohydrolases 1 and 2), and egl1 (endoglucanase 1) is strictly dependent on Xyr1. Regulation of the respective genes via Xyr1 is not affected by the substances mediating induction (xylose, xylobiose, and sophorose) and is indispensable for all modes of gene expression (basal, derepressed, and induced). Moreover, Xyr1, it was revealed, activated transcriptional regulation of inducer-providing enzymes such as beta-xylosidase BXLI and beta-glucosidase BGLI but was not shown to be involved in the regulation of BGLII.
Project description:<h4>Background</h4>Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.<h4>Results</h4>Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.<h4>Conclusions</h4>The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.
Project description:Background:The filamentous ascomycete T. reesei is industrially used to produce cellulases and xylanases. Cost-effective production of cellulases is a bottleneck for biofuel production. Previously, different strain and process optimizations were deployed to enhance enzyme production rates. One approach is the overexpression of the main activator Xyr1 and a second is the construction of synthetic transcription factors. Notably, these genetic manipulations were introduced into strains bearing the wild-type xyr1 gene and locus. Results:Here, we constructed a Xyr1-deficient strain expressing a non-functional truncated version of Xyr1. This strain was successfully used as platform strain for overexpression of Xyr1, which enhanced the cellulase and xylanase production rates under inducing conditions, with the exception of lactose-there the cellulase production was severely reduced. Further, we introduced fusion transcription factors consisting of the DNA-binding domain of Xyr1 and the transactivation domain of either Ypr1 or Ypr2 (regulators of the sorbicillinoid biosynthesis gene cluster). The fusion of Xyr1 and Ypr2 yielded a moderately transactivating transcription factor, whereas the fusion of Xyr1 and Ypr1 yielded a highly transactivating transcription factor that induced xylanases and cellulases nearly carbon source independently. Especially, high production levels of xylanases were achieved on glycerol. Conclusion:During this study, we constructed a Xyr1-deficient strain that can be fully reconstituted, which makes it an ideal platform strain for Xyr1-related studies. The mere overexpression of Xyr1 turned out not to be a successful strategy for overall enhancement of the enzyme production rates. We gained new insights into the regulatory properties of transcription factors by constructing respective fusion proteins. The Xyr1-Ypr1-fusion transcription factor could induce xylanase production rates on glycerol to outstanding extents, and hence could be deployed in the future to utilize crude glycerol, the main co-product of the biodiesel production process.
Project description:BACKGROUND:Trichoderma reesei is an organism involved in degradation of (hemi)cellulosic biomass. Consequently, the corresponding enzymes are commonly used in different types of industries, and recently gained considerable importance for production of second-generation biofuel. Many industrial T. reesei strains currently in use are derived from strain Rut-C30, in which cellulase and hemicellulase expression is released from carbon catabolite repression. Nevertheless, inducing substances are still necessary for a satisfactory amount of protein formation. RESULTS:Here, we report on a T. reesei strain, which exhibits a very high level of xylanase expression regardless if inducing substances (e.g. D-xylose, xylobiose) are used. We found that a single point mutation in the gene encoding the Xylanase regulator 1 (Xyr1) is responsible for this strong deregulation of endo-xylanase expression and, moreover, a highly elevated basal level of cellulase expression. This point mutation is localized in a domain that is common in binuclear zinc cluster transcription factors. Only the use of sophorose as inducer still leads to a slight induction of cellulase expression. Under all tested conditions, the formation of cbh1 and cbh2 transcript level strictly follows the transcript levels of xyr1. The correlation of xyr1 transcript levels and cbh1/cbh2 transcript levels and also their inducibility via sophorose is not restricted to this strain, but occurs in all ancestor strains up to the wild-type QM6a. CONCLUSIONS:Engineering a key transcription factor of a target regulon seems to be a promising strategy in order to increase enzymes yields independent of the used substrate or inducer. The regulatory domain where the described mutation is located is certainly an interesting research target for all organisms that also depend so far on certain inducing conditions.
Project description:<h4>Background</h4>Filamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions. Xylanase has been reported as the crucial accessory enzyme in the degradation of lignocellulose for higher accessibility of cellulase. In addition, the efficient hydrolysis of xylan needs the co-work of multiple xylanolytic enzymes, which rise an increasing demand for the high yield of xylanase for efficient biomass degradation.<h4>Results</h4>In this study, a xylanase hyper-producing system in T. reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII. The expressed xylanase cocktail contained 5256 U/mL xylanase activity and 9.25 U/mL β-xylosidase (pNPXase) activity. Meanwhile, the transcription level of the xylanolytic genes in the strain with XYR1 overexpressed was upregulated, which was well correlated with the amount of XYR1-binding sites. In addition, the higher expression of associated xylanolytic enzymes would result in more efficient xylan hydrolysis. Besides, 2310-3085 U/mL of xylanase activities were achieved using soluble carbon source, which was more efficient and economical than the traditional strategy of xylan induction. Unexpectedly, deletion of ace1 in C30OExyr1 did not give any improvement, which might be the result of the disturbed function of the complex formed between ACE1 and XYR1. The enzymatic hydrolysis of alkali pretreated corn stover using the crude xylanase cocktails as accessory enzymes resulted in a 36.64% increase in saccharification efficiency with the ratio of xylanase activity vs FPase activity at 500, compared to that using cellulase alone.<h4>Conclusions</h4>An efficient and economical xylanase hyper-producing platform was developed in T. reesei RUT-C30. The novel platform with outstanding ability for crude xylanase cocktail production would greatly fit in biomass degradation and give a new perspective of further engineering in T. reesei for industrial purposes.
Project description: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.
Project description:Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei.
Project description:Two major xylanases (XYN I and XYN II) of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) are simultaneously expressed during growth on xylan but respond differently to low-molecular-weight inducers. In vivo footprinting analysis of the xylanase1 (xyn1) promoter revealed three different nucleotide sequences (5'-GGCTAAATGCGACATCTTAGCC-3' [an inverted repeat of GGCTAA spaced by 10 bp], 5'-CCAAT-3', and 5'-GGGGTCTAGACCCC-3' [equivalent to a double Cre1 site]) used to bind proteins. Binding to the Cre1 site is only observed under repressed conditions, whereas binding to the two other motifs is constitutive. Applying heterologously expressed components of the H. jecorina cellulase regulators Ace1 and Ace2 and the xylanase regulator Xyr1 suggests that Ace1 and Xyr1 but not Ace2 contact both GGCTAA motifs. H. jecorina transformants containing mutated versions of the xyn1 promoter, leading to elimination of protein binding to the left or the right GGCTAA box revealed either strongly reduced or completely eliminated induction of transcription. Elimination of Cre1 binding to its target released the basal transcriptional level from glucose repression but did not influence the inducibility of xyn1 expression. Mutation of the CCAAT box prevents binding of the Hap2/3/5 complex in vitro and is partially compensating for the loss of transcription caused by the mutation of the right GGCTAA box. Finally, evidence for a competition of Ace1 and Xyr1 for the right GGCTAA box is given. These data prompted us to hypothesize that xyn1 regulation is based on the interplay of Cre1 and Ace1 as a general and specific repressor with Xyr1 as transactivator.
Project description: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.
Project description:Plant pathogenic fungi must be able to degrade host cell walls in order to penetrate and invade plant tissues. Among the plant cell wall degrading enzymes (PCWDEs) produced, xylanases are of special interest since its degradation target, xylan, is one of the main structural polysaccharides in plant cell walls. In the biotrophic fungus <i>Ustilago maydis</i>, attempts to characterize PCWDEs required for virulence have been unsuccessful, most likely due to functional redundancy. In previous high-throughput screening, we found one xylanase to be important for <i>U. maydis</i> infection. Here, we characterize the entire <i>U. maydis</i> endo-xylanase family, comprising two enzymes from the glycoside hydrolase (GH) 10 family, Xyn1 and Xyn2, one from GH11, Xyn11A, and one from GH43, Xyn3. We show that all endo-xylanases except Xyn3 are secreted and involved in infection in a non-redundant manner, suggesting different roles for each xylanase in this process. Taking a closer look inside the plant during the pathogenic process, we observed that all secreted xylanases were necessary for fungal proliferation. Finally, we found that at least Xyn11A accumulated in the apoplast of the infected plant after three days, highlighting the role of these enzymes as important secreted proteins during fungal proliferation inside plant tissues.