ArticleApplied microbiology and biotechnology2015
I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.
Article in Applied microbiology and biotechnology, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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16 citing papers in PubMed.
- Efficient clonal seeds sorting for apomictic hybrid rice using a pollen-specific gene switch system.Plant biotechnology journal · 2025Article
- The Construction of Heterothallic Strains ofBiomolecules · 2025Article
- Evolution and screening of Trichoderma reesei mutants for secreted protein production at elevated temperature.Journal of industrial microbiology & biotechnology · 2024Article
- Current genetic strategies to investigate gene functions in Trichoderma reesei.Microbial cell factories · 2023Review
- An overview on current molecular tools for heterologous gene expression in Trichoderma.Fungal biology and biotechnology · 2021Review
- Reduced viscosity mutants of Trichoderma reesei with improved industrial fermentation characteristics.Journal of industrial microbiology & biotechnology · 2021Article
- Precise Replacement ofG3 (Bethesda, Md.) · 2020Article
- Homing endonuclease I-SceI-mediated Corynebacterium glutamicum ATCC 13032 genome engineering.Applied microbiology and biotechnology · 2020Article
- Modification of transcriptional factor ACE3 enhances protein production inBiotechnology for biofuels · 2020Article
- Tracking localization and secretion of cellulase spatiotemporally and directly in livingBiotechnology for biofuels · 2019Article
- Genetic engineering of Trichoderma reesei cellulases and their production.Microbial biotechnology · 2017Review
- Expression of Talaromyces thermophilus lipase gene in Trichoderma reesei by homologous recombination at the cbh1 locus.Journal of industrial microbiology & biotechnology · 2017Article
- Combining manipulation of transcription factors and overexpression of the target genes to enhance lignocellulolytic enzyme production inBiotechnology for biofuels · 2017Article
- A set of isogenic auxotrophic strains for constructing multiple gene deletion mutants and parasexual crossings in Aspergillus niger.Archives of microbiology · 2016Article
- Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei.Microbial cell factories · 2016Review
- Modern Approaches to the Genome Editing of Antibiotic Biosynthetic Clusters in Actinomycetes.Acta naturaeArticle
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5 authors.
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Abstract
Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.
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