EP0134048A1
Expired / Lapsed
EP
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Molecular cloning and expression in industrial microorganism species
Assignee: DSM BV
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Filing: 1984-07-06
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Granted: —
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Expires: 2004-07-06
Abstract
Abstract Novel methods and novel industrial unicellular microorganism strains, particularly industrial Bacillus strains, are provided for enhanced production of endogenous and exogenous polypeptides. Cloning vehicles containing one or more copies of the gene expressing the polypeptide of interest are introduced into a compatible host, the transformed hosts treated to form protoplasts and then fused with protoplast acceptors prepared from industrial strains. Efficient transfer of the vehicle containing the genes of interest is achieved, with the resulting modified industrial strains being effective producers of the desired polypeptide product.
Inventors
- 👤 Johan Pieter Marinus Sanders
- 👤 Johannes Abel Van Den Berg
- 👤 Peter Michael Andreoli
- 👤 Yvonne Johanna Vos
- 👤 Jan Hendrik Van Ee
- 👤 Leo J.S.M. Mulleners
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 17:24
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2405; C12N9/2408; C12N9/2411; C12N9/2414; C12N9/2417; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/75; C12N9/48; C12N9/50; C12N9/52; C12N9/54; C12N9/64; C12N9/6421; C12N9/6478; C12N9/6481
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
95 words
A method for efficiently introducing into an unicellular microorganism strain host DNA capable of stable maintenance and expression of a polypeptide of interest in said unicellular microorganism strain host, said method comprising: combining in the presence of a fusogen, first readily transformable host cells containing said DNA as an extrachromosomal element capable of maintenance in said first host and said unicellular microorganism strain host cells under conditions providing for selection of said strain host cells, whereby said DNA is transferred to said strain host cells; and selecting unicellular microorganism strain host cells containing said DNA.
Claim 2
Independent Claim
95 words
A method for efficiently introducing into an unicellular microorganism strain host DNA capable of stable maintenance and expression of a polypeptide of interest in said unicellular microorganism strain host, said method comprising: combining in the presence of a fusogen, first readily transformable host cells containing said DNA as an extrachromosomal element capable of maintenance in said first host and said unicellular microorganism strain host cells under conditions providing for selection of said strain host cells, whereby said DNA is transferred to said strain host cells; and selecting unicellular microorganism strain host cells containing said DNA.
Claim 3
Dependent Claim
17 words
A method according to Claim 1, wherein the unicellular microorganism strain host is an industrial strain host.
Claim 4
Dependent Claim
19 words
A method according to Claim 2, wherein the industrial strain host is resistent to genetic modification by a vector.
Claim 5
Dependent Claim
17 words
A method according to Claim 3, wherein the vector is a plasmid, a phage or a cosmid.
Claim 6
Dependent Claim
26 words
A method according to Claim 2, wherein the industrial strain host has a production level of at least 0.5% w/v secreted protein under industrial fermentation conditions.
Claim 7
Dependent Claim
120 words
A method according to claim 1 for producing a microorganism having increased ability to produce a desired protein or polypeptide, said method comprising: (a) fragmenting DNA containing a gene coding for the desired protein or polypeptide (b) ligating a vector to said fragmented DNA (c) transforming auxiliary cells with said ligated vector (d) selecting the transformed cells for the presence and optionally the expression of the desired gene and optionally inactivating said transformed auxiliary cells (e) fusing protoplasts of cells from a selected host microorganism and said optionally inactivated transformed auxiliary cells, and (f) selecting the fused regenerated cells containing the gene coding for the desired protein or polypeptide and having increased ability to produce the desired protein or polypeptide.
Claim 8
Dependent Claim
26 words
The method of claim 6 wherein the DNA containing a gene coding for the desired protein or polypeptide is fragmented by one or more restriction enzymes.
Claim 9
Dependent Claim
22 words
The method of claim 1 wherein the unicellular microorganism strain host is selected from the group consisting of bacteria, yeasts and fungi.
Claim 10
Dependent Claim
104 words
A method according to claim 1 for efficiently introducing into an industrial Bacillus strain host DNA capable of stable maintenance and expression of a polypeptide of interest in said industrial Bacillus strain host, said method comprising: combining in the presence of a fusogen, first readily transformable prokaryotic host cells containing said DNA as an extrachromosomal element capable of maintenance in said first host and said industrial Bacillus strain host cells under conditions providing for selection of said industrial Bacillus strain host cells, whereby said DNA is transferred to said industrial Bacillus strain host cells; and selecting industrial Bacillus strain host cells containing said DNA.
Claim 11
Dependent Claim
15 words
A method according to claim 9, wherein said industrial Bacillus strain is the strain licheniformis.
Claim 12
Dependent Claim
17 words
A method according to claim 9, wherein said polypeptide is an endogenous enzyme of said Bacillus strain.
Claim 13
Dependent Claim
20 words
A method according to claim 11, wherein said endogenous enzyme is an alpha-amylase, preferably a thermostable alpha-amylase, or a protease.
Claim 14
Dependent Claim
24 words
A method according to claim 1, wherein said conditions providing for selection of said unicellular microorganism strain is employing first killed prokaryotic host cells.
Claim 15
Independent Claim
102 words
A method for efficiently introducing into an industrial Bacillus strain host DNA capable of stable maintenance and expression of a polypeptide of interest in said industrial Bacillus strain host, said method comprising: combining in the presence of a fusogen first killed Bacillus host cells readily capable of transformation and containing said DNA in a plasmid construct having a replication system capable of functioning in Bacillus and said industrial Bacillus strain host cells, where said cells are present as protoplasts, and whereby said DNA is transferred to said industrial Bacillus strain host cells; and selecting industrial Bacillus strain host cells containing said DNA.
Claim 16
Independent Claim
102 words
A method for efficiently introducing into an industrial Bacillus strain host DNA capable of stable maintenance and expression of a polypeptide of interest in said industrial Bacillus strain host, said method comprising: combining in the presence of a fusogen first killed Bacillus host cells readily capable of transformation and containing said DNA in a plasmid construct having a replication system capable of functioning in Bacillus and said industrial Bacillus strain host cells, where said cells are present as protoplasts, and whereby said DNA is transferred to said industrial Bacillus strain host cells; and selecting industrial Bacillus strain host cells containing said DNA.
Claim 17
Dependent Claim
16 words
A method according to claim 14, wherein said commercial Bacillus strain host is the strain licheniformis.
Claim 18
Dependent Claim
16 words
A method according to claim 14, wherein said polypeptide of interest is a Bacillus endogenous enzyme.
Claim 19
Dependent Claim
20 words
A method according to claim 16, wherein said DNA becomes integrated into the chromosome of said industrial Bacillus strain host.
Claim 20
Dependent Claim
19 words
A method according to claim 16, wherein said enzyme is an alpha-amylase, preferably a thermostable alpha-amylase or a protease.
Claim 21
Dependent Claim
34 words
A method for producing a polypeptide of interest in an industrial Bacillus strain which comprises: growing industrial Bacillus strain host cells prepared according to Claim 9; and isolating the polypeptide expressed by said DNA.
Claim 22
Dependent Claim
29 words
A method according to claim 19, wherein said polypeptide is secreted by said industrial Bacillus host strain cells; and said isolating is separating said polypeptide from said nutrient medium.
Claim 23
Dependent Claim
20 words
A method according to claim 20, wherein said secreted polypeptide is an alpha-amylase, preferably a thermostable alpha-amylase or a protease.
Claim 24
Dependent Claim
61 words
An industrial Bacillus host strain cell produced in accordance with the method of claim 12 and capable of producing at least 150% of the amount of alpha-amylase based on the amount of alpha-amylase produced by the parent cell or at least 150% of the amount of a protease based on the amount of said protease produced by the parent cell, respectively.
Claim 25
Dependent Claim
14 words
An industrial Bacillus strain cell according to claim 22, wherein said strain is licheniformis.
Claim 26
Dependent Claim
27 words
The method of claim 6 wherein the ligated product of step (b) is a plasmid selected from the group consisting of pGB33, pGB34, pLC28, pLC83 and pLC87.
Claim 27
Dependent Claim
45 words
A plasmid selected from the group consisting of the plasmid pGB33 deposited with CBS under No. 466.83, the plasmid pGB34 deposited with CBS under No. 467.83, the plasmid pLC28 deposited with CBS under No. 469.83 and the plasmid pLC83 deposited withe CBS under No. 468.83.
Claim 28
Dependent Claim
45 words
A plasmid selected from the group consisting of the plasmid pGB33 deposited with CBS under No. 466.83, the plasmid pGB34 deposited with CBS under No. 467.83, the plasmid pLC28 deposited with CBS under No. 469.83 and the plasmid pLC83 deposited withe CBS under No. 468.83.
Claim 29
Dependent Claim
24 words
A genetically engineered microorganism having increased ability to produce a desired protein or polypeptide, obtained by the method of any one of claims 1-24.
Claim 30
Dependent Claim
23 words
The genetically engineered microorganism of claim 18 which comprises a plasmid selected from the group consisting of pGB33, pGB34, pLC28, pLC83 and pLC87.
Claim 31
Dependent Claim
60 words
A genetically engineered microorganism of the genus Bacillus containing inserted DNA which microorganism is formed by protoplast fusion of cells of a Bacillus host microorganism which is resistant to genetic modification by a vector and cells of an optionally inactivated auxiliary strain which is capable of uptaking genetic information and in which the desired genetic information has been previously inserted.
Claim 32
Dependent Claim
60 words
A genetically engineered microorganism of the genus Bacillus containing inserted DNA which microorganism is formed by protoplast fusion of cells of a Bacillus host microorganism which is resistant to genetic modification by a vector and cells of an optionally inactivated auxiliary strain which is capable of uptaking genetic information and in which the desired genetic information has been previously inserted.
Claim 33
Dependent Claim
21 words
The genetically engineered microorganism of claim 28 which produces alpha-amylase and which contains at least two stable genes coding for alpha-amylase.
Claim 34
Dependent Claim
22 words
Use of a genetically engineered microorganism as defined in any one of claims 26-29, for the production of a protein or polypeptide.
Claim 35
Dependent Claim
16 words
A method according to claim 14, wherein said polypeptide of interest is a Bacillus exogenous enzyme.
Claim 36
Dependent Claim
20 words
A method according to claim 31, wherein said DNA becomes integrated into the chromosome of said industrial Bacillus strain host.
Claim 37
Dependent Claim
12 words
A method according to claim 31, wherein said enzyme is a chymosin.
References Cited (Backward Citations)
EP0032238A2
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EP0034470A2
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EP0036259A2
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