Method for stable gene-amplification in a bacterial host cell

Patent metadata

Field	Value
Publication number	US-9458468-B2
Application number	US-201414180975-A
Country	US
Kind code	B2
Filing date	Feb 14, 2014
Priority date	Oct 31, 2003
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

A bacterial host cell is disclosed including at least two copies of an amplification unit in its genome, the amplification unit including: i) at least one copy of a gene of interest, and ii) an expressible conditionally essential gene, wherein the conditionally essential gene is either promoterless or transcribed from a heterologous promoter having an activity substantially lower than the endogenous promoter of the conditionally essential gene, and wherein the conditionally essential gene if not functional would render the cell auxotrophic for at least one specific substance or unable to utilize one or more specific sole carbon source; methods for producing a protein using the cell of the invention, and methods for constructing the cell of the invention.

First claim

The invention claimed is:

1. A bacterial host cell comprising at least two copies of an amplification unit, wherein the amplification unit is integrated in the genome of the bacterial host cell and the amplification unit comprises:
i) at least one copy of a gene of interest, and
ii) an expressible conditionally essential gene,
wherein the conditionally essential gene is either promoterless or transcribed from a heterologous promoter having an activity substantially lower than the endogenous promoter of said conditionally essential gene,
wherein the conditionally essential gene encodes a glutamyl-tRNA reductase, and
wherein the conditionally essential gene if not functional would render the cell auxotrophic for 5-amino levulinic acid.

2. The cell of claim 1 , wherein the bacterial host cell is a gram-positive cell.

3. The cell of claim 1 , wherein the bacterial host cell is a species of the genus Bacillus.

4. The cell of claim 1 , wherein the gene of interest encodes an enzyme with an activity selected from the group consisting of aminopeptidase, amylase, amyloglucosidase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, galactosidase, beta-galactosidase, glucoamylase, glucose oxidase, glucosidase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinase, peroxidase, phytase, phenoloxidase, polyphenoloxidase, protease, ribonuclease, transferase, transglutaminase, or xylanase.

5. The cell of claim 1 , wherein the gene of interest encodes an antimicrobial peptide.

6. The cell of claim 1 , wherein the gene of interest encodes a peptide with biological activity in the human body.

7. The cell of claim 1 , wherein the conditionally essential gene is at least 95% identical to a hemA sequence of Bacillus licheniformis.

8. The cell of claim 1 , wherein the conditionally essential gene is at least 97% identical to a hemA sequence of Bacillus licheniformis.

9. The cell of claim 1 , wherein the conditionally essential gene is a hemA sequence of Bacillus licheniformis.

10. The cell of claim 1 , wherein the amplification unit further comprises an antibiotic selection marker.

11. The cell of claim 1 , wherein the amplification unit further comprises a resolvase site or res-site.

12. The cell of claim 1 , wherein the conditionally essential gene is transcribed from a heterologous promoter having an activity level, when compared with the endogenous promoter of the conditionally essential gene, which is reduced by a factor or 2 to 100.

13. The cell of claim 1 , wherein the conditionally essential gene is promoterless.

14. The cell of claim 13 , wherein the gene of interest is located upstream of the conditionally essential gene in the amplification unit, and wherein the gene of interest and the conditionally essential gene are co-directionally transcribed.

15. The cell of claim 14 , wherein the conditionally essential gene is expressed by read-through transcription from the gene of interest.

16. A method for producing a protein encoded by a gene of interest, comprising
a) culturing the bacterial host cell of claim 1 ; and
b) recovering the protein.

17. The method of claim 16 , wherein the conditionally essential gene is at least 95% identical to a hemA sequence of Bacillus licheniformis.

18. The method of claim 16 , wherein the conditionally essential gene is at least 97% identical to a hemA sequence of Bacillus licheniformis.

19. The method of claim 16 , wherein the conditionally essential gene is a hemA sequence of Bacillus licheniformis.

20. The method of claim 16 , wherein the bacterial host cell is a species of the genus Bacillus.

Assignees

• Novozymes As

Inventors

• Olsen Carsten
• Rasmussen Michael Dolberg

Classifications

• C12Y302/01023

  Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase · CPC title

• C12Y201/01014

  5-Methyltetrahydropteroyltriglutamate--homocysteine S-methyltransferase (2.1.1.14) · CPC title

• C12N9/2471

  Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase · CPC title

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title