# US 8,465,946 B2 · Expression system for the antibiotic-free production of polypeptides

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- Number: US 8,465,946 B2
- Document ID: US8465946B2
- Status: Granted
- Kind: B2
- Application number: 12/598,712
- Filed: 2008-02-12
- Published: 2010-09-30
- Granted: 2013-06-18
- Earliest claimed: 2007-05-04
- Assignees: AB Enzymes Oy
- Applicants: Céline Cadot; Tina Ploss; Ruth Schwerdtfeger; Bruno Winter
- Inventors: Céline Cadot; Tina Ploss; Ruth Schwerdtfeger; Bruno Winter
- Web page: https://patentgrep.com/patent/US8465946B2

## CPC

- C12N 15/75: for Bacillus
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- 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 a microbial expression system for the production of polypeptides based on the use of extrachromosomal DNA, whereby no antibiotic marker genes for the selection of the host cell but DNA sequences that encode glycerine-3-phosphate dehydrogenase are used, and, thus, the production of the desired polypeptide, e.g., xylanase, does not need the addition of antibiotics. The expression system is free from antibiotic-resistance genes. The invention further relates to a DNA sequence that encodes a polypeptide with glycerine-3-phosphate dehydrogenase activity as well as a polypeptide with glycerine-3-phosphate dehydrogenase activity.

## Claims

### Claim 1

An expression system for the production of one or more target polypeptide/target polypeptides, comprising a host cell in whose genome the DNA sequence that encodes glycerine-3-phosphate dehydrogenase is inactivated or partially or completely deleted and which is transformed by an extrachromosomal element that comprises a DNA sequence that encodes the target polypeptide(s) and glycerine-3-phosphate dehydrogenase, whereby not only the host cell genome but also the extrachromosomal element do not carry an antibiotic-resistance gene.

### Claim 2 (depends on 1)

The expression system according to claim 1 wherein the extrachromosomal element is a plasmid, a phage, a phagemid or a transposon.

### Claim 3 (depends on 1)

The expression system according to claim 1 wherein the extrachromosomal element comprises a DNA sequence that corresponds to the DNA sequence that encodes the endogenous glycerine-3-phosphate dehydrogenase of the host cell, a DNA sequence that encodes a related glycerine-3-phosphate dehydrogenase or a foreign glycerine-3-phosphate dehydrogenase.

### Claim 4 (depends on 1)

The expression system according to claim 1 wherein the DNA sequence that encodes glycerine-3-phosphate dehydrogenase is under the control of a weak promoter.

### Claim 5 (depends on 4)

The expression system according to claim 4 wherein the weak promoter is located upstream of the gpsA gene in FIG. 7 and is derived from plasmid pUB 110.

### Claim 6 (depends on 4)

The expression system according to claim 4 wherein the weak promoter is the ptsH promoter from B. subtilis.

### Claim 7 (depends on 1)

The expression system according to claim 1 wherein the host cell is derived from a Gram-positive bacterial cell.

### Claim 8 (depends on 7)

The expression system according to claim 7 wherein the host cell is derived from a cell of the genus Staphylococcus, Corynebacterium or Bacillus.

### Claim 9 (depends on 8)

The expression system according to claim 8 wherein the host cell is derived from Bacillus amyloliquefaciens.

### Claim 10 (depends on 9)

The expression system according to claim 9 wherein the host cell is Bacillus amyloliquefaciens RH 1626 deposited under accession number DSM 18878.

### Claim 11 (depends on 1)

The expression system according to claim 1 wherein the host cell is derived from a Gram-negative bacterial cell.

### Claim 12 (depends on 1)

The expression system according to claim 1 wherein the target peptide is selected from the group consisting of hydrolytic enzymes, pectin-degrading enzymes, proteolytic enzymes and lipolytic enzymes.

### Claim 13 (depends on 1)

The expression system according to claim 1 wherein the DNA sequence that encodes glycerine-3-phosphate dehydrogenase is selected from the group consisting of
a) a nucleotide sequence according to SEQ ID NO: 1,
b) a DNA sequence that encodes a protein sequence according to SEQ ID NO: 2,
c) a DNA sequence that hybridizes under stringent conditions with a sequence according to a) or b),
d) a DNA sequence that is related to the sequences according to a), b) or c) due to the degeneracy of the genetic code, and
e) complementary strands to the sequences a) to d).