# US 8,569,029 B2 · DNase expression in recombinant host cells

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- Number: US 8,569,029 B2
- Document ID: US8569029B2
- Status: Granted
- Kind: B2
- Application number: 13/532,268
- Filed: 2012-06-25
- Published: 2012-10-18
- Granted: 2013-10-29
- Earliest claimed: 2006-11-30
- Assignees: Novozymes A/S
- Applicants: Michael Dolberg Rasmussen; Jon Martin Persson
- Inventors: Michael Dolberg Rasmussen; Jon Martin Persson
- Web page: https://patentgrep.com/patent/US8569029B2

## CPC

- C12N 1/08: Reducing the nucleic acid content
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/75: for Bacillus
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to cells producing at least one polypeptide of interest and expressing one or more recombinant nuclease encoding gene(s) thereby producing the nuclease(s), and methods for producing a polypeptide of interest essentially free from contaminating DNA, said method comprising the steps of: (a) cultivating a cell that produces at least one polypeptide of interest and expresses one or more recombinant nuclease encoding gene(s) thereby producing the nuclease(s); and (b) isolating the polypeptide of interest.

## Claims

### Claim 1

A Bacillus cell transformed with a first expression vector comprising a nucleic acid encoding at least one enzyme of interest and a second expression vector comprising a nucleic acid encoding at least one nuclease, wherein the Bacillus cell is able to secrete the enzyme(s) of interest and nuclease(s) into a fermentation broth resulting in reduced DNA in the fermentation broth compared to an otherwise isogenic Bacillus cell without the vector that encodes at least one nuclease.

### Claim 2 (depends on 1)

The Bacillus cell of claim 1, wherein the cell is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis cell.

### Claim 3 (depends on 1)

The Bacillus cell of claim 1, wherein the at least one enzyme of interest is a lyase, a ligase, a hydrolase, an oxidoreductase, a transferase, or an isomerase.

### Claim 4 (depends on 1)

The Bacillus cell of claim 1, wherein the cell is a Bacillus licheniformis cell.

### Claim 5 (depends on 1)

The Bacillus cell of claim 1, wherein the at least one enzyme of interest is a hydrolase.

### Claim 6 (depends on 1)

The Bacillus cell of claim 1, wherein the at least one nuclease is transcribed from a constitutive promoter.

### Claim 7 (depends on 1)

The Bacillus cell of claim 1, wherein the at least one nuclease is transcribed from a regulated promoter.

### Claim 8 (depends on 7)

The Bacillus cell of claim 7, wherein the regulated promoter is upregulated in response to phosphate-limitation in the growth medium.

### Claim 9 (depends on 1)

A method for producing an enzyme of interest significantly free from contaminating DNA, said method comprising the steps of: cultivating in a growth medium the Bacillus cell of claim 1 and separating the cells from the growth medium.

### Claim 10 (depends on 9)

The method of claim 9, comprising the step of isolating the enzyme of interest from the growth medium.

### Claim 11 (depends on 9)

The method of claim 9, wherein the cell is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis cell.

### Claim 12 (depends on 9)

The method of claim of claim 9, wherein the at least one enzyme of interest is a lyase, a ligase, a hydrolase, an oxidoreductase, a transferase, or an isomerase.

### Claim 13 (depends on 9)

The method of claim 9, wherein the one or more recombinant nuclease encoding gene(s) is integrated into the genome of said cell.

### Claim 14 (depends on 9)

The method of claim 9, wherein the cell is a B. licheniformis cell.

### Claim 15 (depends on 9)

The method in accordance with claim 9, wherein the enzyme is a lyase.

### Claim 16 (depends on 9)

The method in accordance with claim 9, wherein the enzyme is a ligase.

### Claim 17 (depends on 9)

The method in accordance with claim 9, wherein the enzyme is a hydrolase.

### Claim 18

The method in accordance with 9, wherein the enzyme is an oxidoreductase.

### Claim 19 (depends on 9)

The method in accordance with claim 9, wherein the enzyme is a transferase.

### Claim 20 (depends on 9)

The method in accordance with of claim 9, wherein the enzyme is or an isomerase.