Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-10-12
2003-03-18
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S254200, C435S320100, C435S471000, C435S477000
Reexamination Certificate
active
06534315
ABSTRACT:
The present invention relates to a yeast transformation tool or cassette family leaving in yeast no exogenous DNA fragment other than the fragments coding for proteins of interest.
The subject of the present invention is an integration-excision cassette which makes it possible to inactivate one or more alleles of a gene and/or to insert a new gene, leaving in the host strain only yeast DNA and possibly the new gene.
The present invention also relates to a high copy plasmid in yeast having no unnecessary or useless exogenous DNA fragment, i.e. no exogenous DNA that is not required for the yeast searched function.
The invention also relates to a method of transforming yeasts using the said tools or cassettes as well as the transformed strains obtained.
Improving the productivity and robustness of yeast strains is a constant concern to which recombinant DNA technologies may help to provide answers.
Gene replacement is a molecular biology technique which is frequently used in yeast. A DNA fragment is cloned into a vector and then it is introduced into the cell to be transformed. The DNA fragment integrates by homologous recombination into a targeted region of the recipient genome (Orr-Weaver T., Szostak J. and Rothstein R., 1981, Proc. Natl. Acad. Sci. USA, 78, pp. 6354-6358). However, the recombination event is rare in practice and only occurs in a few cells. Accordingly, selectable markers are inserted between the sequences bringing about the recombination in order to make it possible, after transformation, to isolate the cells where the integration of the DNA fragment occurred by identifying the markers corresponding, for example, to a resistance to an antibiotic. However, these selectable markers are difficult to eliminate, which has the disadvantage, on the one hand, of not being able to reuse the same marker for another transformation, and, on the other hand, of leaving in the host cell exogenous DNA fragments.
The problem is further complicated for the industrial strains of
Saccharomyces cerevisiae
which are distinguishable from the laboratory strains by the fact that they are both aneuploid and polyploid, that is to say that many genes are present in several copies: multiple copies in tandem, multiple copies dispersed in the genome, families of genes only slightly different in their sequence and for which no difference in activity has been detected such as the SUC gene for example (Olson M., 1991, Genome Structure and Organization, in
Saccharomyces cerevisiae
in the Molecular Biology of the Yeast Saccharomyces—Genome Dynamics, Protein Synthesis and Energetics; Ed. Broach, Jones, Pringle, CSHL Press, New York).
This particular ploidy makes any manipulation intended to inactivate a gene by disruption or deletion of its alleles difficult because it is necessary to inactivate all the copies of the gene, most often by repeating the inactivation operation.
The prototrophic character of industrial strains does not make it possible to use so-called “auxotrophic” markers and involves only the use of dominant markers in all the transformations, but in this case, it is necessary either to have several different markers, or to eliminate the marker after each transformation so as to be able to reuse it for a new transformation.
Another problem consists in the fact that to carry out any transformation at all the DNA construct used often contains DNA of non-yeast origin which is not always eliminated or not in its entirety. Foreign DNA therefore remains present in the genome of the yeast. However, for marketing in the food industry sector, it is important that the yeasts do not contain DNA not originating from strains belonging to the same genus, preferably to the same yeast species, with the sole possible exception of the part of a gene of interest encoding for a protein which is not naturally produced by the yeast strain, such as for example a xylanase, a malate permease, a malolactic enzyme, a lactate dehydrogenase.
In yeast, the integration of a gene of interest into a DNA fragment or target gene occurs according to the principle of homologous recombination. For that, an integration cassette contains a module comprising at least one yeast marker gene and the gene to be integrated, this module being flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site. These fragments will be termed hereinafter “recombinogenic” because they will bring about double homologous recombination allowing the insertion of the cassette. These recombinogenic fragments will be called hereinafter RS (=Recombinogenic Sequence). After transforming the yeast with the cassette by appropriate methods, a homologous recombination between the recombinogenic sequences of the construct and the corresponding regions of the target gene results in the inactivation of the target gene caused by the simultaneous integration of the construct, that is to say the replacement of the target gene by the integration cassette.
This technique also applies to the inactivation of a gene, which may be a disruption/interruption or a deletion; in the case of a total deletion, the entire target gene is exchanged; in the case of a disruption/interruption, the sequence of the target gene is interrupted, and in general, it is accompanied by a larger or smaller deletion; accordingly, the terminology “total or partial deletion” is sometimes used to designate both the deletion and the disruption of a gene, which has the result of blocking or modifying its expression. In these two particular cases, the cassette to be inserted into the target gene may contain only the selectable marker flanked by the recombinogenic homologous fragments.
The problem then posed is that of eliminating the unnecessary exogenous fragments, in particular the markers thus introduced, whose presence is generally considered to be inopportune. For that, it is possible to use the “pop-out” or spontaneous excision phenomenon in yeast. It is an intrachromosomal recombination event between identical or similar sequences which can occur naturally. A DNA loop forms between two similar direct sequences, and is then ejected, leaving in place one of the two recombined sequences. The frequency of this phenomenon increasing with the degree of sequence identity, it is possible to promote the elimination of a DNA fragment, for example of a selectable marker, by placing it between two identical direct sequences, termed direct repeat sequences (DRS). Thus, the marker is excised while a direct repeat sequence is conserved.
For example, an article describes a molecular construct (pNKY51) which makes it possible to disrupt or to delete a yeast gene (E. Alani, L. Cao and N. Kleckner, “A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains”, Genetics, 116, pp. 541-545, August 1987). The vector is an
Escherichia coli
plasmid containing the URA3 gene (marker) flanked by direct repeat sequences derived from the Salmonella histidine operon (hisG). This plasmid is digested in order to produce a module (hisG-URA3-hisG) which is then flanked on either side by recombinogenic homologous sequences of the target gene to be disrupted. The cassette thus produced is integrated into the target gene of a Ura
31
yeast, and the strains having integrated the cassette may be isolated by selecting the Ura
+
strains. After excising the cassette, the strains become Ura
−
again.
The construct is used to disrupt or delete the yeast TRP1, SPO13, HO, RAD50 and LEU2 genes. The multiple transformations are carried out either by crossing strains carrying one transformation each, or by a series of transformations with different disruption constructs and by repeated Ura
+
and Ura3
−
selection cycles in a single strain.
One of the two Salmonella direct repeat sequences hisG remains in the genome after each transformation.
This method makes it possible to reuse the marker (auxotrophic marker) for a new transformation.
This same type of method is described in the docu
Bauer Jürgen
Loiez Annie
Nacken Valerie
Foley & Lardner
Ketter James
La Societe Lesaffre et Cie
Loeb Bronwen M.
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