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alpha SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei

Ouedraogo, Jean Paul ; Arentshorst, Mark ; Nikolaev, Igor ; Barends, Sharief ; Ram, Arthur FJ

Applied microbiology and biotechnology, 2015-12, Vol.99 (23), p.10083-10095 [Periódico revisado por pares]

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  • Título:
    alpha SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei
  • Autor: Ouedraogo, Jean Paul ; Arentshorst, Mark ; Nikolaev, Igor ; Barends, Sharief ; Ram, Arthur FJ
  • Assuntos: Hypocrea jecorina ; Saccharomyces cerevisiae
  • É parte de: Applied microbiology and biotechnology, 2015-12, Vol.99 (23), p.10083-10095
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    content type line 23
    ObjectType-Feature-2
  • Descrição: 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 alpha 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 alpha SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the alpha SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via alpha 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 Delta ku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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