In Vivo DNA Assembly in Yarrowia lipolytica

Abstract

The oleaginous yeast Yarrowia lipolytica is an important platform organism for biotechnology applications. In this study, we established an in vivo DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of Y. lipolytica. 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 Y. lipolytica, we expressed S. cerevisiae RAD52 (ScRAD52) or a Cas9-hBrex27 fusion protein. While ScRAD52 expression impaired growth, the cas9-hBrex27 fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The in vivo assembly method simplifies pathway construction and gene overexpression in Y. lipolytica.

Keywords: Yarrowia lipolytica; homologous recombination; in vivo DNA assembly; pathway construction.

1

In vivo DNA assembly of green fluorescent protein mNG in Y. lipolytica. A. Schematic illustration of CRISPR-Cas9-mediated genomic integration of mNG via in vivo assembly of three DNA fragments: an upstream homology arm with promoter (PrTEF1), the mNG coding sequence, and a terminator (Tpex20) with downstream homology arm. Fragments were flanked by overlapping homology regions of varying lengths. B. Integration efficiency of mNG using different homology arm lengths (50 bp, 100 bp, 200 bp, and 400 bp). Values above the bars represent the average integration efficiency (%), calculated as the percentage of fluorescent colonies among total colonies. Error bars indicate the standard deviation from three biological replicates. C. Representative YPD plates showing fluorescent colonies 2 days post-transformation with DNA fragments containing different homology arm lengths.

2

In vivo assembly of multigene biosynthetic pathways in Y. lipolytica. A. Schematic representation of CRISPR-Cas9-mediated in vivo assembly of the betaxanthin (5 fragments) and betanin (7 fragments) biosynthetic pathways. B. Quantification of colony phenotypesyellow (betaxanthins), pink (betanin), and white (nonproducers)and integration efficiencies of betaxanthin (% yellow colonies/total colonies) and betanin (% pink colonies/total colonies) pathway in ST6512 (Δku70::cas9) and ST14915 (Δku70::cas9-hBrex27), respectively.