Yeast cells comprising at least two copies of a desired gene integrated into the chromosomal genome at more than one non-ribosomal RNA encoding domain, particularly with Kluyveromyces
First Claim
1. A yeast cell comprising at least two copies of a desired gene integrated into its chromosomes, wherein said chromosome(s) comprise at least two DNA domains each suitable for integration of one or more copies of said desired gene, which domains comprise diploid loci or multiple copies of a single locus and are non-ribosomal RNA encoding DNA domains, and wherein at least two of said non-ribosomal RNA encoding DNA domains have at least one copy of the said desired gene integrated.
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Abstract
The present invention provides for a yeast cell comprising at least two copies of a desired gene integrated into its chromosomal genome, wherein said genome comprises at least two DNA domains suitable for integration of one or more copies of said desired gene, which domains share substantial sequence homology and are non-ribosomal RNA encoding DNA domains, and wherein at least two of said substantially homologous non-ribosomal RNA encoding DNA domains have at least one copy of the said desired gene integrated. The invention also provides methods for making yeast cells according to the invention, as well as the use thereof for making a protein, a peptide or a metabolite.
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Citations
45 Claims
- 1. A yeast cell comprising at least two copies of a desired gene integrated into its chromosomes, wherein said chromosome(s) comprise at least two DNA domains each suitable for integration of one or more copies of said desired gene, which domains comprise diploid loci or multiple copies of a single locus and are non-ribosomal RNA encoding DNA domains, and wherein at least two of said non-ribosomal RNA encoding DNA domains have at least one copy of the said desired gene integrated.
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18. A method of obtaining a yeast cell that produces a desired protein or peptide comprising the steps of:
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(a) transforming a yeast cell having a genome which comprises at least two DNA domains suitable for integration of one or more copies of a desired gene, comprise diploid loci or multiple copies of a single locus and are non-ribosomal RNA encoding DNA domains, with a DNA molecule comprising a desired gene which codes for the said protein or peptide, or a precursor thereof, and a all or a portion of said domain;
(b) selecting or screening for cells having obtained at least one copy of the said desired gene integrated into at least one of said non-ribosomal RNA encoding DNA domains suitable for integration of one or more copies of a desired gene;
(c) propagating the cells obtained in (b) and screening or selecting for cells having obtained at least one copy of the said desired gene integrated into at least two of said non-ribosomal RNA encoding DNA domains. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26)
(d) propagating the cells obtained in (c) and screening or selecting for cells having obtained at least one copy of the said desired gene integrated into an additional copy of said non-ribosomal RNA encoding DNA domains.
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20. The method of claim 19 comprising in addition to step (a), (b) (c) and (d) the step of:
(e) repeating step (d) until each copy of said non-ribosomal RNA encoding DNA domains has obtained at least one integrated copy of the said desired gene.
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21. The method of claim 18 wherein, a bidirectional selectable marker is used in the transformation of the yeast cells in step (a), and wherein removal of the selectable marker is effected prior to step (c).
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22. A method of claim 18 wherein the yeast cell is a Kluyveromyces cell.
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23. The method of claim 20 wherein the yeast cell is a Kluyveromyces cell.
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24. The method of claim 21, wherein the bi-directional selectable marker is a dominant marker.
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25. The method of claim 21, wherein subsequent to the removal of the bi-directional selectable marker, step (a) is repeated at least once.
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26. The method of claim 24, wherein the selectable marker gene is an acetamidase gene.
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27. A method of obtaining a yeast cell that produces a desired protein or peptide comprising the steps of:
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a) transforming a yeast cell having a genome which comprises at least two DNA domains suitable for integration of one or more copies of a desired gene, which domains comprise diploid loci or multiple copies of a single locus and are non-ribosomal RNA encoding DNA domains, with a DNA molecule comprising a desired gene which codes for the said protein or peptide, or a precursor thereof, and all or a portion of said domain;
b) selecting or screening for cells having obtained at least one copy of the said desired gene integrated into at least one of said non-ribosomal RNA encoding DNA domains suitable for integration of, one or more copies of a desired gene;
c) transforming at cell obtained in (b) with a second DNA molecule comprising a selectable marker gene, and all or a portion of said domain;
d) selecting or screening for cells having obtained at least one copy of the said selectable marker gene integrated into one of said nonribosomal RNA encoding DNA domains without one or more copies of the desired gene;
e) propagating the cells obtained in (d) and screening or selecting for cells having lost the selectable marker gene and having obtained at least one copy of the said desired gene integrated into at least two of said non-ribosomal RNA encoding DNA domains. - View Dependent Claims (28, 29, 30, 31)
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32. A method of obtaining a yeast cell that produces a desired protein or peptide, wherein said method comprises the steps of:
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a) obtaining a transformed yeast cell comprising at least two DNA domains each suitable for integration of one or more copies of said desired gene, which domains comprise diploid loci or multiple copies of a single locus, and at least one copy of a desired gene integrated in a first DNA domain, wherein the desired gene is flanked by direct repeats;
b) isolating single colonies from the progeny of the cell obtained in (a);
c) screening for single colony isolates having cells which have obtained at least one additional tandemly integrated copy of the desired gene in at least one DNA domain other than the first DNA domain and, optionally;
d) repeating steps (a) to (c) on the cells obtained in (c) until a desired achievable number equal to or greater than two of tandemly repeated copies of the desired gene is obtained. - View Dependent Claims (33, 34, 35)
e) propagating the cells obtained in (c) or (d) and screening or selecting for cells having obtained copies of said desired gene integrated into at least two of said non-ribosomal RNA encoding DNA domains, and, optionally;
f) repeating the propagation and screening or selecting of (e) on the cells obtained in (e) until a desired achievable number of said non-ribosomal RNA encoding DNA domains equal to or greater than two have obtained integrated copies of said desired gene.
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34. The method of claim 32 wherein the yeast cell is a Kluyveromyces cell.
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35. The method of claim 33 wherein the yeast cell is a Kluyveromyces cell.
- 36. A yeast cell of the genus Kluyveromyces which is non-haploid for at least one locus, and which yeast cell has in each allele of the said non-haploid locus one or more copies of a desired gene integrated.
- 42. A Kluyveromyces yeast cell having at least 3 copies of a gene encoding lactase, or a mutant thereof, incorporated into at least two substantially homologous non-ribosomal RNA encoding DNA domains in its chromosomal genome.
- 44. Kluyveromyces yeast cell having at least 3 copies of a gene encoding chymosin, or a precursor or a mutant thereof, incorporated into at least two substantially homologous non-ribosomal RNA encoding DNA domains in its chromosomal genome.
Specification