EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9 |
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Authors: | Mathew M Jessop‐Fabre Tadas Jako?iūnas Vratislav Stovicek Zongjie Dai Michael K Jensen Jay D Keasling Irina Borodina |
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Institution: | 1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, H?rsholm, Denmark;2. The Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden;3. Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden;4. Joint BioEnergy Institute, Emeryville, CA, USA;5. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;6. Department of Chemical and Biomolecular Engineering & Department of Bioengineering University of California, Berkeley, CA, USA |
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Abstract: | Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker‐free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone‐MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113‐7D) and a diploid industrial strain (Ethanol Red) for production of 3‐hydroxypropionic acid, where we tested three different acetyl‐CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone‐MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available. |
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Keywords: | CRISPR‐Cas9 3‐hydroxypropionic acid Metabolic engineering Saccharomyces cerevisiae |
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