Expression method

Patent metadata

Field	Value
Publication number	US-9725704-B2
Application number	US-201314374556-A
Country	US
Kind code	B2
Filing date	Jan 29, 2013
Priority date	Jan 31, 2012
Publication date	Aug 8, 2017
Grant date	Aug 8, 2017

Abstract

In a microbial fermentation, the aim is to increase the product yield of protein. This is achieved by a method in which an expression construct is introduced into a microorganism of the species Bacillus pumilus which comprises a promoter and a nucleic acid coding for the protein, and the protein is expressed in said expression construct.

First claim

The invention claimed is:

1. A method for producing a protein by means of a microorganism comprising
(a) introducing an expression construct into a microorganism which comprises a promoter and a nucleic acid coding for the protein;
(b) expressing the protein in the microorganism,
wherein the microorganism belongs to the species Bacillus pumilus ; and
wherein the microorganism is sporulation-inhibited as a result of deleting the gene spoIV (yqfD) or parts thereof.

2. The method according to claim 1 , wherein the promoter comprises a nucleic acid sequence which is selected from
(a) nucleic acid sequence which is at least 95% identical to the nucleic acid sequence given in SEQ ID No. 1;
(b) nucleic acid sequence which is at least 95% identical to the nucleic acid sequence given in SEQ ID No. 2;
(c) nucleic acid sequence which is at least 95% identical to the nucleic acid sequence given in SEQ ID No. 3; or
(d) nucleic acid sequence which is at least 95% identical to the nucleic acid sequence given in SEQ ID No. 4.

3. The method according to claim 1 , wherein the protein is not naturally present in the microorganism.

4. The method according to claim 1 , wherein the protein is an enzyme.

5. The method according to claim 1 , wherein the microorganism is Bacillus pumilus DSM 14395.

6. The method according to claim 1 , wherein the microorganism is genetically modified.

7. A microorganism obtainable by a method comprising
(a) introducing an expression construct into a microorganism which comprises a promoter and a nucleic acid coding for the protein;
(b) expressing the protein in the microorganism,
wherein the microorganism belongs to the species Bacillus pumilus ; and
wherein the microorganism is sporulation-inhibited as a result of deleting the gene spoIV (yqfD) or parts thereof.

8. The microorganism according to claim 7 , wherein
(a) the promoter comprises a nucleic acid sequence which is selected from
(i) nucleic acid sequence which is at least 95% identical to the nucleic acid sequence given in SEQ ID No. 1;
(ii) nucleic acid sequence which is at least 95% identical to the nucleic acid sequence given in SEQ ID No. 2,
(iii) nucleic acid sequence which is at least 95% identical to the nucleic acid sequence given in SEQ ID No. 3; or
(iv) nucleic acid sequence which is at least 95% identical to the nucleic acid sequence given in SEQ ID No. 4;
or
(b) the protein is not naturally present in the microorganism, or
(c) the protein is an enzyme
or
(d) the microorganism is Bacillus pumilus DSM 14395, or
(e) the microorganism is genetically modified.

9. The method according to claim 4 , wherein the enzyme is an acidic cellulase, alpha-amylase, alpha-acetodecarboxylase, aminopetidase, amylase, arabanase, beta-glucanase, beta-glucosidase, beta-mannosidase, carageenase, carbohydrase, catalase, cellobiose-oxidase, cellulase, chymosin, endo-1,3-beta-glucanase, endo-1,3(4)-beta-glucanase, endo-1,4-beta-xylanase, endopeptidase, esterase, exopeptidase, G4-amylase, glucoamylase, glucose oxidase, glucosidase, glycolipase, hemicellulase, laccase, lipase, lysophospholipase, maltogenic amylase, mannanase, neutral protease, nuclease, oxidase, oxidoreductase, pectate lyase, pectinase, pectin esterase, pentosanase, perhydrolase, phospholipase, phytase, polygalacturonase, protease, proteinase, pullulanase, rennet enzyme, rhamnogalacturonase, subtilisin, tannase, transferase, transglutaminase, xanthanase, xylanase, xyloglucanase or mixtures thereof.

10. The method according to claim 9 , wherein the enzyme is an alpha-amylase, a protease, or a mixture thereof.

11. The method of claim 8 , wherein the enzyme is an acidic cellulase, alpha-amylase, alpha-acetodecarboxylase, aminopetidase, amylase, arabanase, beta-glucanase, beta-glucosidase, beta-mannosidase, carageenase, carbohydrase, catalase, cellobiose-oxidase, cellulase, chymosin, endo-1,3-beta-glucanase, endo-1,3(4)-beta-glucanase, endo-1,4-beta-xylanase, endopeptidase, esterase, exopeptidase, G4-amylase, glucoamylase, glucose oxidase, glucosidase, glycolipase, hemicellulase, laccase, lipase, lysophospholipase, maltogenic amylase, mannanase, neutral protease, nuclease, oxidase, oxidoreductase, pectate lyase, pectinase, pectin esterase, pentosanase, perhydrolase, phospholipase, phytase, polygalacturonase, protease, proteinase, pullulanase, rennet enzyme, rhamnogalacturonase, subtilisin, tannase, transferase, transglutaminase, xanthanase, xylanase, xyloglucanase or mixtures thereof.

12. The method of claim 11 , wherein the enzyme is an alpha-amylase, a protease, or a mixture thereof.

Assignees

• Basf Se

Inventors

• Kueppers Tobias
• Steffen Victoria
• Eichstadt Renee Charlott
• Evers Stefan
• Maurer Karl-Heinz
• Bongaerts Johannes
• Hellmuth Hendrik
• Weber Thomas
• O'Connell Timothy

Classifications

• C12N9/2411 Primary

  Amylases · CPC title

• C12N9/54

  bacteria being Bacillus · CPC title

• C12N9/2414

  Alpha-amylase (3.2.1.1.) · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N2510/02

  Cells for production · CPC title