Kluyveromyces as a host strain
AssigneeGist-Brocades N.V.
InventorsJohan A. Van den Berg, Albert J. J. Van Ooyen, Krijn Rietveld
Life of this patent
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Abstract
Kluyveromyces hosts and DNA expression cassettes for use in Kluyveromyces are provided for transcription of endogenous and/or exogenous DNA, and production of peptides, for enhancing production of an endogenous product, or producing an exogenous product. The Kluyveromyces hosts find particular use for secretion of a desired peptide product, where signal sequences may be native to the peptide or provided from endogenous or exogenous signal sequences, including synthetic sequences, functional in Kluyveromyces. A transformation procedure is provided for efficiently transforming Kluyveromyces.
Claims16
A method for transforming Kluyveromyces yeast cells which comprises:
- pretreating said yeast cells with a chaotropic ion at a concentration of about 2 mM to about 1 M then treating said yeast cells with about 25-50% polyethylene glycol, wherein said pretreating and said treating is for about 5 to 120 minutes;
- incubating said yeast cells with a DNA construct at about 20.degree. C. to 35.degree. C. for about 5 to 60 minutes;
- heating said yeast cells with polyethylene glycol at about 35.degree. C. to about 45.degree. C. for about 1 to 10 minutes;
- and
- growing said yeast cells.
A method for obtaining transformed Kluyveromyces yeast cells which comprises:
- pretreating said yeast cells with a chaotropic ion at a concentration of about 2 mM to about 1 M then treating said yeast cells with about 25-50% polyethylene glycol, wherein said pretreating and said treating is for about 5 to 120 minutes;
- incubating said yeast cells with a DNA construct at about 20.degree. C. to 35.degree. C. for about 5 to 60 minutes, wherein said DNA construct comprises a DNA sequence which confers resistance to an antibiotic;
- heating said yeast cells with polyethylene glycol at about 35.degree. C. to about 45.degree. C. for about 1 to 10 minutes;
- growing said yeast cells in the presence of a sufficient concentration of said antibiotic whereby transformants are selected for;
- and
- isolating said transformants.
A method for obtaining transformed Kluyveromyces yeast cells which comprises:
- contacting Kluyveromyces cells treated with a chaotropic ion and a DNA construct comprising a selection marker in the presence of a fusant under fusing conditions;
- and
- growing said cells under conditions whereby transformants comprising said DNA construct are selected for;
- and
- isolating said transformants.
Dependencies are inferred from the claim text.