EP0238023B1
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus
Assignee: Novozymes AS
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Filing: 1987-03-16
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Granted: 1993-12-22
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Expires: 2007-03-16
Abstract
Abstract Proteins (I) are expressed in Aspergillus oryzae by first transforming the host with a recombinant DNA cloning vector which can integrate into the genome (in one or more copies) and which comprises (1) sequences facilitating gene expression; (2) a selection marker, and (3) the sequence coding for (I). The host does not contain a gene for the selection marker. The transformants are then grown in a suitable culture medium to produce (I). Also claimed is a promoter for protein expression in Aspergillus; it comprises the TAKA-amylase promoter (or funtional parts), opt. preceded by upstream activating sequences. Two pref. base sequences for this promoter are reproduced in the specification (about 1200 bases).
Inventors
- 👤 Esper Boel
- 👤 Tove Christensen
- 👤 Helle Fabricius Wöldike
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-09 05:07
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/80; C12N15/11; C12N15/52; C12N15/67; C12N9/00; C12N9/14; C12N9/16; C12N9/18; C12N9/20; C12N9/24; C12N9/2402; C12N9/2405; C12N9/2408; C12N9/2411; C12N9/2414; C12N9/2417; C12N9/242; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6478; C12N9/6481
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
103 words
A process for expression of a protein product in Aspergillus oryzae comprising the steps of: (a) providing a recombinant DNA cloning vector system capable of integration into the genome of an Aspergillus oryzae host in one or more copies and comprising: DNA-sequences encoding functions facilitating gene expression; a suitable marker for selection of transformants; and a DNA-sequence encoding the desired protein product; (b) transforming the Aspergillus oryzae host which does not harbour a functional gene for the chosen selection marker with the recombinant DNA cloning vector system from step a; and (c) culturing the transformed Aspergillus oryzae host in a suitable culture medium.
Claim 2
Dependent Claim
103 words
A process for expression of a protein product in Aspergillus oryzae comprising the steps of: (a) providing a recombinant DNA cloning vector system capable of integration into the genome of an Aspergillus oryzae host in one or more copies and comprising: DNA-sequences encoding functions facilitating gene expression; a suitable marker for selection of transformants; and a DNA-sequence encoding the desired protein product; (b) transforming the Aspergillus oryzae host which does not harbour a functional gene for the chosen selection marker with the recombinant DNA cloning vector system from step a; and (c) culturing the transformed Aspergillus oryzae host in a suitable culture medium.
Claim 3
Dependent Claim
26 words
A process according to claim 1, wherein the DNA-sequence encoding functions facilitating gene expression comprises a promoter, transcription initiation sites, and transcription terminator and polyadenylation functions.
Claim 4
Dependent Claim
15 words
A process according to claim 2, wherein the promoter is preceded by upstream activating sequences.
Claim 5
Dependent Claim
33 words
A process according to claim 1, wherein the selection marker is derived from the gene for A. nidulans or A. niger argB, A. nidulans trpC, A. nidulans amdS, Neurospora crassae Pyr4 or DHFR.
Claim 6
Dependent Claim
29 words
A process according to claim 4, wherein the selection marker is the ArgB gene derived from A. nidulans or A. niger or the amdS gene derived from A. nidulans.
Claim 7
Dependent Claim
40 words
A process according to claim 3, wherein the promoter and upstream activating sequences are derived from a gene encoding an extracellular or intracellular protein, such as an amylase, a glucoamylase, a protease, a lipase, a cellulase or a glycolytic enzyme.
Claim 8
Dependent Claim
43 words
A process according to claim 6, wherein the promoter and upstream activating sequences are derived from the gene for A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral a-amylase, A. niger acid stable a-amylase, A. niger glucoamylase or Rhizomucor miehei lipase.
Claim 9
Dependent Claim
20 words
A process according to claim 7, wherein the promoter is the A. oryzae TAKA amylase promoter or functional parts thereof.
Claim 10
Dependent Claim
22 words
A process according to claim 8, wherein the promoter and upstream activating sequences have the following sequence: or a functional part thereof.
Claim 11
Dependent Claim
22 words
A process according to claim 8, wherein the promoter and upstream activating sequences have the following sequence: or a functional part thereof.
Claim 12
Dependent Claim
30 words
A process according to claim 10, wherein the sequence in claim 10 is preceded by the 1.05 kb unsequenced upstream region from position 0 to 1.05 in plasmid pTAKA 17.
Claim 13
Dependent Claim
25 words
A process according to claim 1, wherein the vector system further comprises a preregion providing for secretion of the expressed product into the culture medium.
Claim 14
Dependent Claim
50 words
A process according to claim 12, wherein the preregion is derived from a glucoamylase or an amylase gene from an Aspergillus species, an amylase gene from a Bacillus species, a lipase or proteinase gene from Rhizomucor miehei, the gene for the a-factor from S. cerevisiae or the calf prochymosin gene.
Claim 15
Dependent Claim
44 words
A process according to claim 13, wherein the preregion is derived from the gene for A. oryzae TAKA amylase, A. niger neutral a-amylase, A. niger acid-stable a-amylase, B. licheniformis a-amylase, the maltogenic amylase from Bacillus NCIB 11837, B. stearothermophilus a-amylase or B. licheniformis subtilisin.
Claim 16
Dependent Claim
17 words
A process according to claim 14, wherein the preregion is the TAKA-amylase preregion with the following sequence
Claim 17
Dependent Claim
37 words
A process according to claim 1, wherein the vector system comprises two vectors, where on contains the selection marker and the other contains DNA-sequences encoding functions facilitating gene expression and a DNA-sequence encoding the desired protein product.
Claim 18
Dependent Claim
46 words
A process for production of a protein product in Aspergillus oryzae, wherein an Aspergillus oryzae strain being transformed with a recombinant DNA cloning vector system as described in claim 1 is cultured in a suitable culture medium and the product is recovered from the culture medium.
References Cited (Backward Citations)
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