Methods for genetic engineering Kluyveromyces host cells

A method for modifying a target site in a Kluyveromyces host cell genome, the method comprising:

• (a) contacting a Kluyveromyces host cell, which has a reduced non-homologous end joining (NHEJ) activity, wherein the NHEJ activity is reduced by integrating a nucleic acid at YKU70 or YKU80, with:
• (i) a nuclease capable of cleaving the target site;
• and
• (ii) a donor DNA molecule comprising a nucleic acid sequence encoding a polypeptide, wherein the donor DNA molecule is capable of homologous recombination at the cleaved target site, whereby homologous recombination in the host cell results in integration of the donor linear nucleic acid at the target site;
• (b) contacting the host cell with a linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the host cell, whereby homologous recombination in the host cell converts the linear nucleic acid or the linear nucleic acid and the one or more additional linear nucleic acids into a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker;
• and
• (c) selecting a transformed host cell in which the donor DNA molecule is integrated into the target site wherein the host cell secretes the polypeptide encoded by the nucleic acid sequence.