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Growth, Enzymatic, and Transcriptomic Analysis of xyr1 Deletion Reveals a Major Regulator of Plant Biomass-Degrading Enzymes in Trichoderma harzianum.

ABSTRACT: The regulation of plant biomass degradation by fungi is critical to the carbon cycle, and applications in bioproducts and biocontrol. Trichoderma harzianum is an important plant biomass degrader, enzyme producer, and biocontrol agent, but few putative major transcriptional regulators have been deleted in this species. The T. harzianum ortholog of the transcriptional activator XYR1/XlnR/XLR-1 was deleted, and the mutant strains were analyzed through growth profiling, enzymatic activities, and transcriptomics on cellulose. From plate cultures, the Δxyr1 mutant had reduced growth on D-xylose, xylan, and cellulose, and from shake-flask cultures with cellulose, the Δxyr1 mutant had ~90% lower β-glucosidase activity, and no detectable β-xylosidase or cellulase activity. The comparison of the transcriptomes from 18 h shake-flask cultures on D-fructose, without a carbon source, and cellulose, showed major effects of XYR1 deletion whereby the Δxyr1 mutant on cellulose was transcriptionally most similar to the cultures without a carbon source. The cellulose induced 43 plant biomass-degrading CAZymes including xylanases as well as cellulases, and most of these had massively lower expression in the Δxyr1 mutant. The expression of a subset of carbon catabolic enzymes, other transcription factors, and sugar transporters was also lower in the Δxyr1 mutant on cellulose. In summary, T. harzianum XYR1 is the master regulator of cellulases and xylanases, as well as regulating carbon catabolic enzymes.

SUBMITTER: Wang L

PROVIDER: S-EPMC10887015 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Growth, Enzymatic, and Transcriptomic Analysis of xyr1 Deletion Reveals a Major Regulator of Plant Biomass-Degrading Enzymes in Trichoderma harzianum.

Wang Lunji L Zhao Yishen Y Chen Siqiao S Wen Xian X Anjago Wilfred Mabeche WM Tian Tianchi T Chen Yajuan Y Zhang Jinfeng J Deng Sheng S Jiu Min M Fu Pengxiao P Zhou Dongmei D Druzhinina Irina S IS Wei Lihui L Daly Paul P

Biomolecules 20240124 2

The regulation of plant biomass degradation by fungi is critical to the carbon cycle, and applications in bioproducts and biocontrol. Trichoderma harzianum is an important plant biomass degrader, enzyme producer, and biocontrol agent, but few putative major transcriptional regulators have been deleted in this species. The T. harzianum ortholog of the transcriptional activator XYR1/XlnR/XLR-1 was deleted, and the mutant strains were analyzed through growth profiling, enzymati ...[more]

PMID: 38397385

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Project description:BackgroundTrichoderma 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) is the master regulator responsible for the activation of cellulase and hemicellulase gene expression under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the need for inducing carbon source, allowing high (hemi)cellulase 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.ResultsIn this study, we aimed 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. As expected, the expression of many carbohydrate-active enzyme (CAZy) genes was increased in the xyr1 mutant in both conditions. However, their induction was higher under inducing conditions. 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. In addition to CAZy genes, 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.ConclusionsThe results contribute to more in-depth knowledge on T. reesei physiology growing under inducing and repressing carbon sources and gives new insights on the function of the master regulator XYR1. The vast data generated serve as a source for new targets for improved protein production.

Project description:Trichoderma harzianum, whose gene expression is tightly controlled by the transcription factors (TFs) XYR1 and CRE1, is a potential candidate for hydrolytic enzyme production. Here, we performed a network analysis of T. harzianum IOC-3844 and T. harzianum CBMAI-0179 to explore how the regulation of these TFs varies between these strains. In addition, we explored the evolutionary relationships of XYR1 and CRE1 protein sequences among Trichoderma spp. The results of the T. harzianum strains were compared with those of Trichoderma atroviride CBMAI-0020, a mycoparasitic species. Although transcripts encoding carbohydrate-active enzymes (CAZymes), TFs, transporters, and proteins with unknown functions were coexpressed with cre1 or xyr1, other proteins indirectly related to cellulose degradation were identified. The enriched GO terms describing the transcripts of these groups differed across all strains, and several metabolic pathways with high similarity between both regulators but strain-specific differences were identified. In addition, the CRE1 and XYR1 subnetworks presented different topology profiles in each strain, likely indicating differences in the influences of these regulators according to the fungi. The hubs of the cre1 and xyr1 groups included transcripts not yet characterized or described as being related to cellulose degradation. The first-neighbor analyses confirmed the results of the profile of the coexpressed transcripts in cre1 and xyr1. The analyses of the shortest paths revealed that CAZymes upregulated under cellulose degradation conditions are most closely related to both regulators, and new targets between such signaling pathways were discovered. Although the evaluated T. harzianum strains are phylogenetically close and their amino acid sequences related to XYR1 and CRE1 are very similar, the set of transcripts related to xyr1 and cre1 differed, suggesting that each T. harzianum strain used a specific regulation strategy for cellulose degradation. More interestingly, our findings may suggest that XYR1 and CRE1 indirectly regulate genes encoding proteins related to cellulose degradation in the evaluated T. harzianum strains. An improved understanding of the basic biology of fungi during the cellulose degradation process can contribute to the use of their enzymes in several biotechnological applications and pave the way for further studies on the differences across strains of the same species.

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.

Dataset Information

Growth, Enzymatic, and Transcriptomic Analysis of xyr1 Deletion Reveals a Major Regulator of Plant Biomass-Degrading Enzymes in Trichoderma harzianum.

Publications

Growth, Enzymatic, and Transcriptomic Analysis of <i>xyr1</i> Deletion Reveals a Major Regulator of Plant Biomass-Degrading Enzymes in <i>Trichoderma harzianum</i>.

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