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There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of Saccharomyces cerevisiae will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid. Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR–Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously. Our study provides a foundation for efficient engineering of industrial yeast cell factories.
Bibliographical noteThis is an open access article under the CCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
- Genome editing
- Industrial yeast
- Chemical production
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- 1 Finished
BioREFINE-2G: Development of 2nd Generation Biorefineries Production of Dicarboxylic Acids and Bio-based Polymers Derived Thereof
01/10/2013 → 30/09/2017