Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-30
2001-02-06
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06183953
ABSTRACT:
BACKGROUND OF THE INVENTION
Methylotrophic yeasts are those yeasts that are able to utilize methanol as a sole source of carbon and energy. Species of yeasts that have the biochemical pathways necessary for methanol utilization are classified in four genera, Hansenula, Pichia, Candida, and Torulopsis. These genera are somewhat artificial, having been based on cell morphology and growth characteristics, and do not reflect close genetic relationships (Billon-Grand,
Mycotaxon
35:201-204, 1989; Kurtzman,
Mycologia
84:72-76, 1992). Furthermore, not all species within these genera are capable of utilizing methanol as a source of carbon and energy. As a consequence of this classification, there are great differences in physiology and metabolism between individual species of a genus.
Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems. Some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Certain genes of methylotrophic yeasts are tightly regulated and highly expressed under induced or de-repressed conditions, suggesting that promoters of these genes might be useful for producing polypeptides of commercial value. See, for example, Faber et al.,
Yeast
11:1331, 1995; Romanos et al.,
Yeast
8:423, 1992; and Cregg et al.,
Bio/Technology
11:905, 1993.
Development of methylotrophic yeasts as hosts for use in recombinant protein production systems has been slow, due in part to a lack of suitable materials (e.g., promoters, selectable markers, and mutant host cells) and methods (e.g., transformation techniques). The most highly developed methylotrophic host systems utilize
Pichia pastoris
and
Hansenula polymorpha
(Faber et al.,
Curr. Genet.
25:305-310, 1994; Cregg et al., ibid.; Romanos et al., ibid.; U.S. Pat. No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,231; and U.S. Pat. No. 4,929,555).
More recently, materials and techniques useful for producing foreign proteins in
Pichia methanolica
have been developed (WIPO Publication WO 9717450). However, there remains a need in the art for additional techniques that can be used to manipulate the genome of
P. methanolica
so as to expand our understanding of this organism and produce strains that can be used in large-scale protein production systems.
One such needed tool is a technique for directed mutagenesis of
P. methanolica.
Directed mutagenesis allows the introduction of mutations into predetermined genomic loci, permitting the selective alteration of gene activity. Useful alterations include, for example, mutation of promoter sequences to increase gene expression, introduction of heterologous genes at particular sites, and generation of protease deficiencies and auxotrophies. Techniques developed for the budding yeast
Saccharomyces cerevisiae
are unsuitable for
P. methanolica.
For example, the “pop-in/pop-out” method developed by Scherer and Davis (
Proc. Natl. Acad. Sci. USA
76:1035, 1979) and summarized by Rothstein (
Methods Enzymol.
194:281, 1991) requires a selection against the presence of the URA3 marker, such as by addition of 5-FOA (5 fluoro orotic acid) to the culture medium. This method is unsuitable with
P. methanolica
because the cells are resistant to 5 fluoro-orotic acid (5-FOA), and no
P. methanolica
URA marker is available. The present invention provides methods for producing directed mutations in the genome of
P. methanolica,
cells having such mutations, and other, related advantages.
SUMMARY OF THE INVENTION
The present invention provides a method for altering a chromosomal locus of
Pichia methanolica
cells, comprising the steps of: (a) selecting a target chromosomal locus of the cells; (b) providing a population of
P. methanolica
cells each comprising a chromosomal copy of the selected target locus, wherein the cells are auxotrophic for adenine; (c) introducing into the cells a linear DNA construct comprising (i) a segment comprising a portion of the target chromosomal locus in which at least one nucleotide pair is altered, and (ii) a selectable marker that complements adenine auxotrophy; (d) culturing the cells from step (c) under conditions that are selective for the presence in the cells of the selectable marker; (e) identifying a subset of the cultured cells in which the segment of the DNA construct and the selectable marker have been chromosomally integrated by homologous recombination, resulting in tandem duplication of the target chromosomal locus; (f) culturing the identified subset of cells under conditions wherein cells prototrophic for adenine grow and exhibit a first phenotype, and cells auxotrophic for adenine grow and exhibit a second phenotype; (g) recovering cells that are auxotrophic for adenine; and (h) identifying a subset of the auxotrophic cells in which the segment of the DNA construct has been chromosomally integrated, whereby the target chromosomal locus is altered. Within one embodiment of the invention, a plurality of nucleotide pairs of the portion of the chromosomal locus are altered. Within a related embodiment, from 1 kbp to 2 kbp of the portion of the chromosomal locus is altered. Within another embodiment, the alteration is a deletion of at least one nucleotide pair. Within further embodiments, the target chromosomal locus encodes a protease, such as proteinase A or proteinase B, an alcohol oxidase, or a nutritional marker.
Within the method disclosed above, steps (a) through (h) can be repeated, whereby two or more chromosomal loci are altered. Within certain embodiments of the invention, a chromosomal locus encoding a protease and a second chromosomal locus encoding an alcohol oxidase are altered.
The invention also provides a
Pichia methanolica
cell produced by the method disclosed above.
These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawings.
REFERENCES:
patent: 5135868 (1992-08-01), Cregg
patent: 595 334 A2 (1994-05-01), None
patent: 92/17595 (1992-10-01), None
patent: 97/17450 (1997-05-01), None
Hiep et al.,Yeast9: 1189-1197, 1993.
Hiep et al.,Yeast9: 1251-1258, 1993.
Raymond et al.,Yeast14: 11-23, 1998.
Mezard et al.,Cell70: 659-670, 1992.
Rothstein,Methods in Enzymology194: 281-301, 1991.
Rothstein,Methods in Enzymology101: 202-211, 1983.
Arthur Lisa B.
Parker Gary E.
ZymoGenetics Inc.
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