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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.

SUBMITTER: Herold S

PROVIDER: S-EPMC3629775 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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

Herold Silvia S Bischof Robert R Metz Benjamin B Seiboth Bernhard B Kubicek Christian P CP

Eukaryotic cell 20130104 3

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 reg ...[more]

PMID: 23291620

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Project description:Trichoderma reesei is known for its ability to produce large amounts of extracellular proteins and is one of the most important industrially used filamentous fungus. Xylanase regulator 1 (XYR1) as the master regulator is responsible for the activation of cellulase and hemicellulase gene expression, normally under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the carbon catabolite repression, allowing cellulase and hemicellulase production even in the presence of glucose. These mutations also change the profile of produced proteins, shifting it more towards xylanase production, and increase the overall protein production in inducing conditions. However, how these mutations alter the metabolism and other cellular processes to cause these changes remains unclear. In the present study, our aim was to explore changes caused by a point mutation in xyr1 on transcriptomic and metabolic level to better understand the reasons behind the increased protein production in both repressing glucose and inducing lactose conditions. We observed that the xyr1 mutant strain built more biomass and produced more extracellular proteins during growth on lactose compared to the wild type xyr1 strain. Genes involved in oxidoreductive D-galactose catabolism pathway were upregulated in the xyr1 mutant strain, potentially contributing to the more efficient utilization of lactose. The shift from utilizing lactose to using glucose as carbon source seemed faster in the xyr1 mutant strain. Clustering and enrichment analysis showed over-representation of mitochondria-related Gene Ontology terms in clusters where gene expression was higher in the xyr1 mutant, indicating that mitochondria play a role in the altered metabolic state associated with the xyr1 mutation. Metabolomics revealed that free tyrosine was more abundant in the xyr1 mutant strain in all measured timepoints, whereas multiple fatty acids were less abundant in the mutant strain on glucose. The results contribute to more in-depth knowledge on T. reesei physiology and aid in finding new targets for improved protein production.

Project description:BackgroundFilamentous 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.ResultsIn 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.ConclusionsAn 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.

Dataset Information

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

Publications

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

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