US4882279A
Expired / Lapsed
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Site selective genomic modification of yeast of the genus pichia
Assignee: Research Corp Technologies Inc
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Filing: 1985-10-25
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Granted: 1989-11-21
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Expires: 2005-10-25
Abstract
Abstract Method for the site specific genomic modification of yeasts of the genus Pichia and novel DNA sequences useful therefor are provided. Pichia is transformed with a serially arranged linear DNA fragment comprising first and second insertable DNA fragments, which flank a marker gene. The insertable DNA sequences are homologous to defined portions of the Pichia genome and integrate by recombination at such defined sites. The resulting transformed organism contains the marker gene and any additional DNA sequences which are positioned between the insertable DNA fragments. In addition, the resulting transformed organism has either a disrupted or deleted sequence at the site of the genomic modification. Enhanced production of heterologous gene products is observed when using as the host for expression strains in which the alcohol oxidase gene has been disrupted. Upon disruption of the primary alcohol oxidase gene of Pichia, the existence of a second alcohol oxidase gene in Pichia was also discovered.
Inventors
- 👤 James M. Cregg
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 22:14
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C12N15/815; C12N15/87; C12N15/90; C12N9/00; C12N9/0004; C12N9/0006; C12N2730/00; C12N2730/00011; C12N2730/10011; C12N2730/10111; C12N2730/10122
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
165 words
A process for the site selected genomic modification of Pichia pastoris at a predetermined genomic site which comprises transforming a host strain of Pichia pastoris with a serially arranged linear DNA fragment comprising a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment; wherein said first and second insertable DNA fragments are each at least about 200 nucleotides in length and have nucleotide sequences which are homologous with separate portions of the native Pichia pastoris genomic site at which genomic modification is to occur; wherein said first and second insertable DNA fragments are oriented with respect to one another in said linear DNA fragment as they are so oriented in the genomic with Pichia pastoris; wherein said marker gene is positioned between the first DNA insertable fragment and the second insertable DNA fragment and wherein said first and second insertable DNA fragments are selected from the group consisting of fragments of: the alcohol oxidase genes, and the histidinol dehydrogenase gene.
Claim 2
Dependent Claim
165 words
A process for the site selected genomic modification of Pichia pastoris at a predetermined genomic site which comprises transforming a host strain of Pichia pastoris with a serially arranged linear DNA fragment comprising a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment; wherein said first and second insertable DNA fragments are each at least about 200 nucleotides in length and have nucleotide sequences which are homologous with separate portions of the native Pichia pastoris genomic site at which genomic modification is to occur; wherein said first and second insertable DNA fragments are oriented with respect to one another in said linear DNA fragment as they are so oriented in the genomic with Pichia pastoris; wherein said marker gene is positioned between the first DNA insertable fragment and the second insertable DNA fragment and wherein said first and second insertable DNA fragments are selected from the group consisting of fragments of: the alcohol oxidase genes, and the histidinol dehydrogenase gene.
Claim 3
Dependent Claim
42 words
A process in accordance with claim 1 wherein said linear DNA fragment further comprises a heterologous gene, wherein said heterologous gene, as well as said selectable marker gene, is positioned between the first insertable DNA fragment and the second insertable DNA fragment.
Claim 4
Dependent Claim
61 words
A process in accordance with claim 1 wherein said linear DNA fragment further comprises a regulatory region, and a heterologous gene; wherein said heterologous gene is under the control of the regulatory region; and wherein the regulatory region-heterologous gene construct, in addition to said selectable marker gene, is positioned between the first insertable DNA fragment and the second insertable DNA fragment.
Claim 5
Dependent Claim
22 words
A process in accordance with claim 1 wherein said first and second insertable DNA fragments are fragments of the alcohol oxidase gene.
Claim 6
Dependent Claim
21 words
A process in accordance with claim 4 wherein said first and second insertable DNA fragments are fragments of the AOX1 gene.
Claim 7
Dependent Claim
21 words
A process in accordance with claim 4 wherein said first and second insertable DNA fragments are fragments of the AOX2 gene.
Claim 8
Dependent Claim
22 words
A process in accordance with claim 1 wherein said first and second insertable DNA fragments are fragments of the histidinol dehydrogenase gene.
Claim 9
Dependent Claim
131 words
An isolated serially arranged linear DNA fragment which comprises a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment; wherein said first and second insertable DNA fragments are each at least 200 nucleotides in length and have nucleotide sequences which are homologous with portions of the genomic DNA of Pichia pastoris; wherein said first and second insertable DNA fragments are oriented with respect to one another in said linear DNA fragment as they are so oriented in the genome of Pichia pastoris; wherein said marker gene is positioned between the first insertable DNA fragment, the second insertable DNA fragment, and wherein said first and second insertable DNA fragments are selected from the group consisting of fragments of: the alcohol oxidase genes, and the histidinol dehydrogenase gene.
Claim 10
Dependent Claim
131 words
An isolated serially arranged linear DNA fragment which comprises a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment; wherein said first and second insertable DNA fragments are each at least 200 nucleotides in length and have nucleotide sequences which are homologous with portions of the genomic DNA of Pichia pastoris; wherein said first and second insertable DNA fragments are oriented with respect to one another in said linear DNA fragment as they are so oriented in the genome of Pichia pastoris; wherein said marker gene is positioned between the first insertable DNA fragment, the second insertable DNA fragment, and wherein said first and second insertable DNA fragments are selected from the group consisting of fragments of: the alcohol oxidase genes, and the histidinol dehydrogenase gene.
Claim 11
Dependent Claim
44 words
A linear DNA fragment in accordance with claim 8 wherein said linear DNA fragment further comprises: a heterologous gene, wherein said heterologous gene, as well as said selectable marker gene is positioned between the first insertable DNA fragment and the second insertable DNA fragment.
Claim 12
Dependent Claim
62 words
A DNA fragment in accordance with claim 9 wherein said linear DNA fragment further comprises a resulatory region, and a heterologous gene; wherein said heterologous gene is under the control of the regulatory region; and wherein the regulatory region-heterologous gene construct, in addition to said selectable marker gene, is positioned between the first insertable DNA fragment and the second insertable DNA fragment.
Claim 13
Dependent Claim
23 words
A DNA fragment in accordance with claim 8 wherein said first and second insertable DNA fragments are fragments of the alcohol oxidase gene.
Claim 14
Dependent Claim
22 words
A DNA fragment in accordance with claim 11 wherein said first and second insertable DNA fragments are fragments of the AOX1 gene.
Claim 15
Dependent Claim
22 words
A DNA fragment in accordance with claim 11 wherein said first and second insertable DNA fragments are fragments of the AOX2 gene.
Claim 16
Dependent Claim
23 words
A DNA fragment in accordance with claim 8 wherein said first and second insertable DNA fragments are fragments of the histidinol dehydrogenase gene.
Claim 17
Dependent Claim
18 words
A linear DNA fragment in accordance with claim 8 wherein said DNA fragment further comprises bacterial plasmid DNA.
Claim 18
Dependent Claim
35 words
A closed circular plasmid comprising the linear DNA portion of claim 8, and bacterial plasmid DNA; wherein said bacterical plasmid DNA is positioned between the first insertable DNA fragment and the second insertable DNA fragment.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1992-11-20 | 1993-05-22 | 1993-11-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-11-20 | 1997-05-22 | 1997-11-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-11-20 | 2001-05-22 | 2001-11-21 | $7700.00 | Window Closed / Lapsed |