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Amélioration du champignon cellulolytique Trichoderma reesei par reproduction sexuée

Abstract : Genetic engineering of Trichoderma reesei is one of the solutions considered to reduce the cost of the enzymatic hydrolysis step in second-generation ethanol production processes. It allows the improvement of the secretory performances of this cellulase producer fungus and the properties of its enzymes. Like many industrial fungi, T. reesei has long been considered to possess exclusively an asexual cycle. The recent discovery of a sexual reproduction cycle in this filamentous fungus opens up new prospects of improvement for strains used in biotechnology. However, all industrial strains of T. reesei are derived from the wild isolate QM6a and therefore possess the MAT1-2 mating type and are female sterile.The aim of this work was to develop sexual reproduction as a genetic tool and to improve the performance of the industrial strains. In combination with the genetic engineering tools, it would enable combination of interesting traits or generation of new ones, improve strains stability by purging deleterious mutations, selection markers elimination and identification of genes and mutations responsible of interesting characteristics.The first part of this work was dedicated to the optimization of sexual reproduction and to the study of the steps and mechanisms of sexual reproduction between wild-type and hyperproducer strains. In a second step, we set up sexual reproduction between female sterile hyperproducer strains, using the original "assistant strain" strategy, which resulted in the partial restoration of their sexual reproduction. A study of the fertilization step was also initiated, as we suspect it to be the blocking point in our strategy. In a second part, we took advantage of sexual reproduction between wild-type and hyperproducer strains for (i) the generation of a MAT1-1 mating-type strain compatible with all industrial strains, female fertile and possessing improved β-glucosidase activity and (ii) the implementation of a bulk segregation analysis associated with high-throughput sequencing techniques to identify mutations involved in phenotypes of interest.
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Laetitia Chan Ho Tong. Amélioration du champignon cellulolytique Trichoderma reesei par reproduction sexuée. Biotechnologie. Institut agronomique, vétérinaire et forestier de France, 2017. Français. ⟨NNT : 2017IAVF0017⟩. ⟨tel-03013494⟩

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