(254e) Efficient Site-Specific Integration of Pathways in Yarrowia Lipolytica Enabled By Serine Integrases (Faculty Candidate)

The oleaginous yeast Yarrowia lipolytica is a promising platform to produce lipids, oleochemicals and acetyl-CoA derived compounds due to its high flux towards acetyl-CoA and ability to grow on a wide range of substrates. Integrated expression of pathway genes is superior to plasmid-based expression due to concern of plasmid instability. Strain engineering of Y. lipolytica often requires iterative integration of gene expression cassettes into genome. Due to the limited selection markers in this yeast, integration and curing of selection marker is necessary. Existing genome integration methods such as homologous recombination and CRISPR-Cas9 mediated homology directed repair are generally time-consuming and require long homologous arms. Bacteriophage serine integrases are highly specific enzymes which mediates recombination between the short attachment sites of the phage genome and its bacterial host. In this work, we first screened a pool of serine integrases to identify the best candidate in Y. lipolytica in terms of integration efficiency. It was found that the marker-less integration efficiency of a green fluorescent protein (GFP) cassette reached 100%. We hypothesized that serine integrases would efficiently integrate large DNA fragments including pathways of genes. Therefore, we investigated the integration of a metabolic pathway for β-carotene biosynthesis. Last, we demonstrate rapid assembly and integration of a library of metabolic pathways for β-carotene biosynthesis. Our results indicate that serine integrases are a promising tool to accelerate the genome engineering of Y. lipolytica.