Method for the targeted integration of multiple copies of a gene of interest in a yarrowia strain

摘要

This invention concerns a method for the targeted integration of at least three copies of a gene of interest in the genome of a Yarrowia strain including the steps of: (a) cultivating a Yarrowia strain, said strain including a deletion among at least three genes, the phenotype associated with each of these deletions corresponding to an auxotrophy or to a dominant character for this strain; (b) transforming said Yarrowia strain thus obtained with at least three recombinant vectors that include selection markers allowing, for this strain, the complementation of auxotrophy and, potentially, of the dominant character resulting from each of these deletions; and (c) selecting, on a minimum medium, the yeasts having integrated said at least three recombinant vectors. This invention also includes a method for producing a polypeptide of interest using this method as well as a method for obtaining a modified Yarrowia strain including a deletion among at least three genes, the phenotype associated with each of these deletions corresponding to an auxotrophy or to a dominant character for this strain.

主权项

1. A method for the targeted integration of at least three copies of a gene of interest in the genome of a Yarrowia strain including the following steps:
a) cultivation of a Yarrowia strain comprising a deletion in at least three genes wherein the deletion is a deletion in URA3, a deletion in LEU2 and the specific deletion GUT2-744 or wherein the deletion is a deletion in URA3, a deletion in LEU2 and the specific deletion Ade2-844 and wherein independent of the at least three genes, the phenotype associated with each of the deletions corresponds to an auxotrophy or to a dominant character for the strain; b) transformation of the auxotrophic and/or dominant character Yarrowia strain with at least three recombinant vectors that include selection markers allowing, for the Yarrowia strain, complementation of the auxotrophy and/or of the dominant phenotype resulting from each of the deletions, the recombinant vectors each including:
i) the sequence of the gene of interest,ii) a selection marker, andiii) two DNA sequences framing the sequence of the gene of interest and the selection marker, the two DNA sequences are homologous to the sequences corresponding to the ends of the targeted integration site in the genome of the Yarrowia strain in order to allow the targeted integration of the recombinant vector via homologous recombination; and c) selection on minimum medium of the yeasts having integrated the at least three recombinant vectors.