Method for non-homologous transformation of Yarrowia lipolytica

Details Method for non-homologous transformation of Yarrowia lipolytica Method for non-homologous transformation of Yarrowia lipolytica

2001-04-27

2003-06-24

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S243000, C435S252300, C435S254100, C435S440000, C435S471000, C435S473000, C435S483000, C435S484000

Reexamination Certificate

active

06582951

ABSTRACT:

The invention relates to tools for expressing heterologous genes in
Yarrowia lipolytica.
The yeast
Y. lipolytica
is being increasingly used as a host for expressing genes of interest; in this context, “integrating” vectors, which allow the insertion of a segment of DNA bearing the gene of interest into the chromosomal DNA, are in particular used. For example, Application EP 138 508 discloses the transformation of
Yarrowia lipolytica
using vectors capable of integrating into the chromosomal DNA by recombination of
Y. lipolytica
sequences borne by said vectors, with the homologous sequences present on the chromosomal DNA of the host cell.
Among the
Yarrowia lipolytica
sequences used for constructing integrating vectors, mention will be made in particular of the sequences termed: “zeta sequences”, which correspond to the LTRs (long terminal repeats) of the Ylt1 retrotransposon of
Yarrowia lipolytica;
these sequences have been described by SCHMID-BERGER et al. [J. Bacteriol., 2477-2482 (1994)], who indicate that they are present at a high copy number (approximately 35 copies of the complete retrotransposon and approximately 30 copies of the isolated zeta sequence) in the chromosomal DNA of certain strains of
Yarrowia lipolytica.
When a vector containing an insert flanked by zeta sequences is used to transform one of these strains of Yarrowia, the insert DNA integrates by homologous recombination with zeta sequences of the chromosomal DNA. In this way, transformed Yarrowia cells containing several copies of a heterologous sequence, integrated in tandem at chromosomal zeta sites, are obtained.
The inventors have now noted that, surprisingly, when vectors bearing inserts flanked by zeta sequences are used to transform Yarrowia cells lacking these sequences, the insert DNA integrates, however, into the chromosomal DNA, in the form of several copies dispersed in the genome.
A subject of the present invention is a method for integrating a gene of interest into the genome of a strain of Yarrowia, using a recombinant vector bearing an insert flanked by zeta sequences and comprising said gene of interest, which method is characterized in that said recombinant vector is used to transform a strain of Yarrowia, the genome of which lacks zeta sequences.


REFERENCES:
patent: 0 138 508 (1985-04-01), None
patent: 0 220 864 (1987-05-01), None
Barth and Gallardin, The dimorphic fungus Yarrowia lipolytica, In Non-conventional yeasts in biotechnology (Wolf K Ed.), Springer-Verlag, Berlin, pp. 313-388, 1996.*
C. Neuvéglise, et al., Gene, vol. 213, pp. 37-46, “A Shuttle Mutagenesis System for Tagging Genes in the Yeast Yarrowia Lipolytica”, 1998.
M-T. Le Dall, et al., Current Genetics, vol. 26, pp. 38-44, “Multiple-Copy Integration in the Yeast Yarrowia Lipolytica”, 1994.
N. Schmid-Berger, et al., Journal of Bacteriology, vol. 176, No. 9, pp. 2477-2482, “Ylt1, A Highly Repetitive Retrotransposon in the Genome of the Dimorphic Fungus Yarrowia Lipolytica”, May 1994.

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