caa a22 4500 605504733 CHVBK 20210128100618.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6829-1 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6829-1 I- Sce I-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei [Elektronische Daten] [Jean Ouedraogo, Mark Arentshorst, Igor Nikolaev, Sharief Barends, Arthur Ram] 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. The Author(s), 2015 Meganuclease nationallicence DNA repair nationallicence Counter selection nationallicence Targeted integration nationallicence Hypocrea jecorina nationallicence Ouedraogo Jean Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Kluyver Centre for Genomics of Industrial Fermentation, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands aut Arentshorst Mark Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Kluyver Centre for Genomics of Industrial Fermentation, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands aut Nikolaev Igor Dupont Industrial Biosciences, Archimedesweg 30, 2333 CN, Leiden, The Netherlands aut Barends Sharief Dupont Industrial Biosciences, Archimedesweg 30, 2333 CN, Leiden, The Netherlands aut Ram Arthur Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Kluyver Centre for Genomics of Industrial Fermentation, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10083-10095 0175-7598 99:23<10083 2015 99 253 https://doi.org/10.1007/s00253-015-6829-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6829-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ouedraogo Jean Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Kluyver Centre for Genomics of Industrial Fermentation, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands aut NATIONALLICENCE 700 1- Arentshorst Mark Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Kluyver Centre for Genomics of Industrial Fermentation, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands aut NATIONALLICENCE 700 1- Nikolaev Igor Dupont Industrial Biosciences, Archimedesweg 30, 2333 CN, Leiden, The Netherlands aut NATIONALLICENCE 700 1- Barends Sharief Dupont Industrial Biosciences, Archimedesweg 30, 2333 CN, Leiden, The Netherlands aut NATIONALLICENCE 700 1- Ram Arthur Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Kluyver Centre for Genomics of Industrial Fermentation, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10083-10095 0175-7598 99:23<10083 2015 99 253