Molecular Tools for Strain Improvement of Trichoderma spp.

Bischof, Robert H.; Schink, Bernhard

doi:10.1016/b978-0-444-59576-8.00012-6

Cited by 9 publications

(12 citation statements)

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“…As selectable marker the hygromycin phosphotransferase gene hph B from E. coli was used to generate single knockout strains (Mach et al ., ). For generation of double and triple knockout strains, the geneticin resistance gene ( npt2 , from E. coli (Bischof and Seiboth, )) and acetamidase gene ( amd S, from Aspergillus nidulans ; described for application as selection marker in Trichoderma in Penttilä et al ., ) were used as selectable markers (the primers for amplification of the marker genes are listed in http://onlinelibrary.wiley.com/doi/10.1111/mmi.13256/suppinfo). In the course of these experiments, also single knockout strains with npt2 ( hxk3 ) and amdS ( dam1 ) markers were generated (Table ) with which also the results from the hph B marker deletion strains could be verified.…”

Section: Methodsmentioning

confidence: 99%

“…T. reesei is a saprotroph and is widely used for biotechnological applications due to its remarkable cellulolytic potential. It is more amenable to molecular genetic manipulations than T. atroviride, and a range of different (Guangtao et al, 2009;Seiboth et al, 2011;Bischof and Seiboth, 2014). It has been established that GlcNAc is an efficiently consumable carbon source that promotes strong and fast growth of either species Seidl et al, 2006).…”

Section: Expression Of the Glcnac Cluster Genes Is Upregulated Duringmentioning

confidence: 99%

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The N‐acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80‐like transcription factor RON1

Kappel

Gaderer

Flipphi

et al. 2015

Molecular Microbiology

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SummaryChitin is an important structural constituent of fungal cell walls composed of N‐acetylglucosamine (GlcNAc) monosaccharides, but catabolism of GlcNAc has not been studied in filamentous fungi so far. In the yeast C andida albicans, the genes encoding the three enzymes responsible for stepwise conversion of GlcNAc to fructose‐6‐phosphate are clustered. In this work, we analysed GlcNAc catabolism in ascomycete filamentous fungi and found that the respective genes are also clustered in these fungi. In contrast to C . albicans, the cluster often contains a gene for an Ndt80‐like transcription factor, which we named RON1 (regulator of N‐acetylglucosamine catabolism 1). Further, a gene for a glycoside hydrolase 3 protein related to bacterial N‐acetylglucosaminidases can be found in the GlcNAc gene cluster in filamentous fungi. Functional analysis in T richoderma reesei showed that the transcription factor RON1 is a key activator of the GlcNAc gene cluster and essential for GlcNAc catabolism. Furthermore, we present an evolutionary analysis of Ndt80‐like proteins in Ascomycota. All GlcNAc cluster genes, as well as the GlcNAc transporter gene ngt1, and an additional transcriptional regulator gene, csp2, encoding the homolog of N eurospora crassa CSP2/GRHL, were functionally characterised by gene expression analysis and phenotypic characterisation of knockout strains in T . reesei.

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Section: Methodsmentioning

confidence: 99%

Section: Expression Of the Glcnac Cluster Genes Is Upregulated Duringmentioning

confidence: 99%

The N‐acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80‐like transcription factor RON1

Kappel

Gaderer

Flipphi

et al. 2015

Molecular Microbiology

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“…Sequencing and annotation of different Trichoderma genomes have enabled the functional analysis of the genes, such as selection of candidate genes and construction of DNA vectors and deletion cassettes [10,11].…”

Section: Introductionmentioning

confidence: 99%

Endo-β-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens

et al. 2019

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Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. β-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have β-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-β-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

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“…Trichoderma reesei is a widely employed microorganism for the production of technical enzymes, including cellulases and hemicellulases that are used by a variety of industries including the biorefinery industry [ 1 – 3 ]. As a result, a relatively well developed toolbox for the genetic engineering of T. reesei has become available over the past years [ 4 ]. Tuneable promoters are essential tools in this respect and represent a versatile means to alter gene expression at the level of gene transcription [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

“…In the context of industrial enzyme production, the use of an inexpensive repressing substance that is effective at low concentrations is mandatory. However, a repressible promoter meeting these specifications is currently not available for T. reesei , where tuneable expression is almost exclusively controlled by the promoters of the cellobiohydrolase cel7a or endoxylanase xyn1 [ 4 ]. In these two cases, the repression is mediated by d -glucose [ 6 , 7 ], which in turn leads not only to the repression of cellulases and xylanases but also to the repression of most of the other c arbohydrate a ctive en zymes (CAZYmes) [ 8 , 9 ] which is usually undesirable.…”

Section: Introductionmentioning

confidence: 99%

l-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei

et al. 2015

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BackgroundTrichoderma 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.ResultsWe 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0308-3) contains supplementary material, which is available to authorized users.

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Molecular Tools for Strain Improvement of Trichoderma spp.

Cited by 9 publications

References 100 publications

The N‐acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80‐like transcription factor RON1

The N‐acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80‐like transcription factor RON1

Endo-β-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens

l-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei

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