Transformation systems for the yeast candida utilis and the expression of heterologous genes therewith
AssigneeKirin Beer Kabushiki Kaisha
InventorsKeiji Kondo, Susumu Kajiwara, Norihiko Misawa
Life of this patent
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Abstract
An reproducible transformation system of a yeast of Candida utilis, a process for expressing a heterologous gene in the transformation system, a vector which can be used in the transformation system and the expression method, and a novel DNA group are disclosed. In particular, the process for expressing a heterologous gene in Candida utilis comprises transforming Candida utilis with a vector comprising a drug-resistance marker, a sequence homologous to the chromosomal DNA of the Candida utilis yeast, and the heterologous gene, culturing the transformant, and isolating the expression product of the heterologous gene.
Claims101
A DNA sequence comprising a gene sequence encoding a ribosomal protein L41 of Candida utilis.
A DNA sequence comprising a gene which encodes a cycloheximide-resistant L41 protein having an amino acid sequence shown in FIG. 14 (SEQ ID NO:
- 6) wherein glutamine is substituted for proline at position 56.
A DNA fragment of about 13.5 kb which is represented by the restriction enzyme map in FIG. 6(b) and comprises an rRNA gene group of Candida utilis, or a partial DNA sequence thereof.
A DNA sequence comprising a gene sequence encoding orotidine-5'-phosphate decarboxylase (URA3) protein which was isolated from Candida utilis and which complements the ura3 mutation of Saccharomyces cerevisiae.
A phosphoglycerate kinase (PGK) gene promoter sequence of Candida utilis.
A PGK gene terminator sequence of Candida utilis.
A gene expression unit comprising a PGK gene promoter sequence of Candida utilis and a PGK gene terminator sequence of Candida utilis.
A DNA sequence comprising a PGK gene promoter sequence of Candida utilis functionally linked to a heterologous gene sequence.
A glyceraldehyde-3-phosphate dehydrogenase (GAP) gene promoter sequence of Candida utilis.
A GAP gene terminator sequence of Candida utilis.
A gene expression unit comprising a GAP gene promoter sequence of Candida utilis, and a GAP gene terminator sequence of Candida utilis.
A DNA sequence comprising a GAP gene promoter sequence of Candida utilis functionally linked to a heterologous gene sequence.
A plasma membrane proton ATPase (PMA) gene promoter sequence of Candida utilis.
A PMA gene terminator sequence of Candida utilis.
A gene expression unit comprising a PMA gene promoter sequence of Candida utilis and a PMA gene terminator sequence of Candida utilis.
A DNA sequence comprising a PMA gene promoter sequence of Candida utilis functionally linked to a heterologous gene sequence.
A circular integration vector comprising a sequence homologous to a portion of the chromosomal DNA of Candida utilis ("homologous DNA sequence"), and a marker gene for selecting a transformant, and optionally a heterologous gene, wherein the heterologous gene is incorporated into the chromosomal DNA of Candida utilis by homologous recombination when the vector is cut within the homologous DNA sequence with a restriction enzyme into a linear form and introduced into Candida utilis.
A DNA fragment at least 1.8 kb long having an autonomous replicability which can maintain a vector containing said DNA fragment as an extrachromosomal element in Candida utilis, said vector further characterized by its ability to transform a host at high frequency.
A DNA fragment, wherein said autonomously replicable DNA fragment comprises a DNA sequence shown in FIGS. 41 and 42 (SEQ ID NO:
- 11) or a partial sequence thereof which retains an autonomous replicability.
A DNA fragment, wherein said autonomously replicable DNA fragment comprises a DNA sequence shown in FIGS. 43 and 44 (SEQ ID NO:
- 12) or a partial sequence thereof which retains an autonomous replicability.
A vector for transformation of Candida utilis, comprising:
- a DNA fragment which imparts upon said vector autonomous replicability and which enhances the transformation frequency of the host selected from the group comprising:
- the DNA fragment shown in FIGS. 41 and 42 (SEQ ID NO:
- 11) or a functional fragment thereof;
- and
- the DNA fragment shown in FIGS. 43 and 44 (SEQ ID NO:
- 12) or a functional fragment further thereof.
A vector comprising a DNA sequence containing a heterologous gene and a sequence homologous to the chromosomal DNA of Candida utilis ("homologous DNA sequence") and a marker gene, wherein said heterologous gene is incorporated into the chromosomal DNA of Candida utilis by homologous recombination when the vector is cut within the homologous DNA sequence with a restriction enzyme into a linear form.
A transformant of Candida utilis which is transformed with a DNA sequence containing a heterologous gene.
A DNA fragment comprising the base sequence shown in FIG. 48 (SEQ ID NO:
- 13) or a partial sequence thereof which has a transcriptional promoter activity in Candida utilis.
Dependencies are inferred from the claim text.