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A Toolbox for Accelerating Strain Engineering of Oleaginous Yeast Yarrowia Lipolytica

The non-conventional oleaginous yeast Y. lipolytica is valued for its capability to accumulate high amounts of storage lipids and to degrade hydrophobic substrates. It is an attractive host for the production of lipids and fatty acid-derived compounds and for bioremediation, however the strain development is hampered by the lack of genetic tools. We seek to develop a genome editing toolbox, which will allow rapid iterative combinatorial strain engineering, i.e., repeatedly introducing combinations of multiple edits, such as gene knock-ins/knock-outs. First, we developed a set of standardized integration vectors for gene overexpression from high expression genome spots. Second, we adopted CRISPR-Cas9 technology for Y. lipolytica to enable genome editing with high efficiency without the need of a selection marker. We demonstrate the utility of the toolbox by engineering a strain for production of a potential platform chemical, a-ketoglutarate, from renewable feedstocks.