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In Vivo DNA Assembly in Yarrowia lipolytica.

Wei Jiang1, Ioannis Georgiadis1, Tommaso Fumagalli1,2

  • 1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.

ACS Synthetic Biology
|September 30, 2025
PubMed
Summary

This study introduces a new CRISPR-Cas9 DNA assembly system for Yarrowia lipolytica, enabling efficient integration of multiple gene fragments. This method simplifies the construction of complex biosynthetic pathways in this key biotechnology platform organism.

Keywords:
Yarrowia lipolyticahomologous recombinationin vivo DNA assemblypathway construction

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Area of Science:

  • Biotechnology
  • Synthetic Biology
  • Molecular Biology

Background:

  • Yarrowia lipolytica is a valuable yeast platform for biotechnological applications.
  • Efficient genomic integration of multiple DNA fragments is crucial for metabolic engineering in Y. lipolytica.

Purpose of the Study:

  • To establish an in vivo DNA assembly system for Y. lipolytica using CRISPR-Cas9.
  • To enhance homologous recombination efficiency for improved genetic engineering.

Main Methods:

  • Developed an in vivo CRISPR-Cas9 based DNA assembly system.
  • Demonstrated assembly of three DNA fragments using short homology arms (50 bp).
  • Tested expression of S. cerevisiae RAD52 (ScRAD52) and a Cas9-hBrex27 fusion protein to improve integration.

Main Results:

  • Achieved 53% correct assembly of three DNA fragments using the new system.
  • Successfully constructed 2-3 step biosynthetic pathways for betaxanthin and betanin.
  • The Cas9-hBrex27 fusion protein significantly enhanced integration efficiency, especially for multifragment assemblies, while ScRAD52 impaired growth.

Conclusions:

  • The developed in vivo DNA assembly system streamlines genetic engineering in Y. lipolytica.
  • The Cas9-hBrex27 fusion protein is a promising tool for improving homologous recombination efficiency.
  • This method simplifies pathway construction and gene overexpression for biotechnology applications.