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Development of synthetic biology tools and construction of Yarrowia lipolytica chassis strains for aromatic molecules production

Abstract : To produce biobased products by microbial cell factories in a time and cost-effective way, the development of robust, versatile and well-characterized chassis strains is essential. The availability of efficient, standardized and characterized synthetic biology tools is crucial to build these chassis in a fast and efficient manner. These rationally engineered strains, based on an optimized supply of precursors, provide the foundations for the synthesis of a wide range of products.During this project, synthetic biology tools dedicated to the yeast Yarrowia lipolytica were developed. A modular cloning technique based on Golden Gate (GG) was set-up for assembling up to 13 modular building blocks via a single-step, one-pot reaction. The toolkit, composed of 64 bricks, proved to be a powerful tool for rapidly engineer Y. lipolytica strains with complex pathways and is perfectly suited for combinatorial approaches. In addition, based on the GG assembly method, a versatile and modular CRISPR-Cas9 system was set up for genome editing. A set of six platform vectors was constructed to easily introduce any gRNA and to be used in a wide range of different Y. lipolytica genetic backgrounds including wild-type strains. Both toolkits are available to the community through Addgene. These newly developed synthetic biology tools were used for the construction of Y. lipolytica tailor-made chassis strains optimized for the synthesis of aromatic amino acids (AAA), which are the precursors of many aromatic compounds. This platform strain was further engineered as a cell factory to produce aromatic compounds with industrial interest, 2-Phenylethanol, naringenin and melanin, as proof of concept.Thus, through the expansion of Y. lipolytica toolbox and the construction of an AAA-chassis strain, this work greatly enlarges the scope of possible studies and applications for this industrially relevant microorganism.
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Submitted on : Monday, May 31, 2021 - 1:56:07 PM
Last modification on : Wednesday, June 2, 2021 - 5:36:02 PM

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  • HAL Id : tel-03243034, version 1

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Macarena Larroude. Development of synthetic biology tools and construction of Yarrowia lipolytica chassis strains for aromatic molecules production. Molecular biology. Université Paris-Saclay, 2020. English. ⟨NNT : 2020UPASA013⟩. ⟨tel-03243034⟩

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