In Vivo DNA Assembly in <i>Yarrowia lipolytica</i>

The oleaginous yeast <i>Yarrowia lipolytica</i> is an important platform organism for biotechnology applications. In this study, we established an <i>in vivo</i> DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of <i>Y. lipolytica</i>. Using the green fluorescent protein mNeonGreen as a model, we demonstrated 53% correct assembly of three DNA fragments with homology arms as short as 50 bp. The system was further validated by constructing 2-3 step biosynthetic pathways for pigments betaxanthin and betanin. To improve the homologous recombination efficiency of <i>Y. lipolytica</i>, we expressed <i>S. cerevisiae RAD52</i> (<i>ScRAD52</i>) or a Cas9-hBrex27 fusion protein. While <i>ScRAD52</i> expression impaired growth, the <i>cas9-hBrex27</i> fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The <i>in vivo</i> assembly method simplifies pathway construction and gene overexpression in <i>Y. lipolytica</i>.