EasyCloneYALI: CRISPR/Cas9-based synthetic toolbox for engineering of the yeast Yarrowia lipolytica

Carina Holkenbrink, Marie I. Dam, Kanchana R. Kildegaard, Johannes Beder, Jonathan Dahlin, David Doménech Belda, Irina Borodina*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

The oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid-derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well-characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, we present the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology. The toolbox allows marker-free integration of gene expression vectors into characterized genome sites as well as marker-free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80% were achieved with transformation protocols using non-replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.
Original languageEnglish
Article number1700543
JournalBiotechnology Journal
Volume13
Issue number9
Number of pages23
ISSN1860-6768
DOIs
Publication statusPublished - 2018

Bibliographical note

This is an open access article under the terms of the Creative Commons Attribution License

Keywords

  • CRISPR/Cas9
  • Integrative vectors
  • Metabolic engineering
  • Yarrowia lipolytica
  • Yeast

Cite this

Holkenbrink, Carina ; Dam, Marie I. ; Kildegaard, Kanchana R. ; Beder, Johannes ; Dahlin, Jonathan ; Belda, David Doménech ; Borodina, Irina. / EasyCloneYALI: CRISPR/Cas9-based synthetic toolbox for engineering of the yeast Yarrowia lipolytica. In: Biotechnology Journal. 2018 ; Vol. 13, No. 9.
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abstract = "The oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid-derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well-characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, we present the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology. The toolbox allows marker-free integration of gene expression vectors into characterized genome sites as well as marker-free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80{\%} were achieved with transformation protocols using non-replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.",
keywords = "CRISPR/Cas9, Integrative vectors, Metabolic engineering, Yarrowia lipolytica, Yeast",
author = "Carina Holkenbrink and Dam, {Marie I.} and Kildegaard, {Kanchana R.} and Johannes Beder and Jonathan Dahlin and Belda, {David Dom{\'e}nech} and Irina Borodina",
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language = "English",
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EasyCloneYALI: CRISPR/Cas9-based synthetic toolbox for engineering of the yeast Yarrowia lipolytica. / Holkenbrink, Carina; Dam, Marie I.; Kildegaard, Kanchana R.; Beder, Johannes; Dahlin, Jonathan; Belda, David Doménech; Borodina, Irina.

In: Biotechnology Journal, Vol. 13, No. 9, 1700543, 2018.

Research output: Contribution to journalJournal articleResearchpeer-review

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AU - Holkenbrink, Carina

AU - Dam, Marie I.

AU - Kildegaard, Kanchana R.

AU - Beder, Johannes

AU - Dahlin, Jonathan

AU - Belda, David Doménech

AU - Borodina, Irina

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AB - The oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid-derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well-characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, we present the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology. The toolbox allows marker-free integration of gene expression vectors into characterized genome sites as well as marker-free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80% were achieved with transformation protocols using non-replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.

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