ALANINE RACEMASE DOUBLE DELETION AND TRANSCOMPLEMENTATION

Abstract

The present invention relates to a bacterial host cell in which a first chromosomal gene encoding a first alanine racemase and a second chromosomal gene encoding a second alanine racemase have been inactivated. Said bacterial host cell comprises – either on a plasmid comprising at least one autonomous replication sequence or present as multiple copies in the chromosome – a gene expression cassette comprising a polynucleotide encoding at least one polypeptide of interest, operably linked to a promoter, and a polynucleotide encoding a third alanine racemase, operably linked to a promoter. The present invention further relates to a method for producing at least one polypeptide of interest based on cultivating the bacterial host cell of the present invention.

Description

Claims

1. A method for producing at least one polypeptide of interest, said method comprising the steps of a) providing a bacterial host cell belonging to the phylum of Firmicutes in which at least the following chromosomal genes have been inactivated: i. a first chromosomal gene encoding a first alanine racemase, andii. a second chromosomal gene encoding a second alanine racemase, and wherein the bacterial host cell comprises a plasmid comprising 1. at least one autonomous replication sequence,2. a polynucleotide encoding at least one polypeptide of interest, operably linked to a promoter, and3. a polynucleotide encoding a third alanine racemase, operably linked to a promoter, andb) cultivating the bacterial host cell under conditions conducive for maintaining said plasmid in the bacterial host cell and conducive for expressing said at least one polypeptide of interest, thereby producing said at least one polypeptide of interest.

2. The method of claim 1, wherein step a) comprises the following steps: a1) providing a bacterial host cell belonging to the phylum of Firmicutes, said host cell comprising i) a first chromosomal gene encoding a first alanine racemase, and ii) a second chromosomal gene encoding a second alanine racemase,a2) inactivating said first and said second chromosomal gene, anda3) introducing into said bacterial host cell a plasmid comprising 1. at least one autonomous replication sequence,2. a polynucleotide encoding at least one polypeptide of interest operably linked to a promoter, and3. a polynucleotide encoding a third alanine racemase operably linked to a promoter.

3. The method of claim 1, wherein the first chromosomal gene encoding the first alanine racemase and the second chromosomal gene encoding the second alanine racemase have been inactivated by mutation.

4. The method of claim 3, wherein the bacterial host cell belongs to the class of Bacilli.

5. The method of claim 1, wherein the host cell is a Bacillus licheniformis host cell, and wherein the first chromosomal gene encoding the first alanine racemase is the alr gene, and wherein the second chromosomal gene encoding the second alanine racemase is the yncD gene.

6. The method of claim 1, wherein the polynucleotide encoding the third alanine racemase is heterologous to the bacterial host cell and/or wherein the polynucleotide encoding at least one polypeptide of interest is heterologous to the bacterial host cell.

7. The method of claim 6, wherein the third alanine racemase comprises an amino acid sequence being at least 40% identical to SEQ ID NO: 4.

8. The method of claim 1, wherein the promoter which is operably linked to the polynucleotide encoding the third alanine racemase is a constitutive promoter.

9. The method of claim 1, wherein the promoter which is operably linked to the polynucleotide encoding the third alanine racemase is the promoter of the B. subtilis alrA gene.

10. The method of claim 1, wherein the polypeptide of interest is an enzyme.

11. The method of claim 1, further comprising step c) of purifying the polypeptide of interest.

12. A bacterial host cell belonging to the phylum of Firmicutes in which at least the following chromosomal genes have been inactivated: i. a first chromosomal gene encoding a first alanine racemase, andii. a second chromosomal gene encoding a second alanine racemase.

13. The bacterial host cell of claim 12, wherein said bacterial host cell comprises a plasmid comprising 1. at least one autonomous replication sequence,2. a polynucleotide encoding at least one polypeptide of interest, operably linked to a promoter, and3. a polynucleotide encoding a third alanine racemase operably linked to a promoter.

14. The bacterial host cell of claim 12, wherein said bacterial host cell comprises u) a non-replicative vector comprisingu1) optionally, a plus origin of replication (ori+),u2) a polynucleotide encoding at least one polypeptide of interest, operably linked to a promoter,u3) a polynucleotide encoding a third alanine racemase, operably linked to a promoter,u4) a polynucleotide which has homology to a chromosomal polynucleotide of the bacterial host cell to allow integration of the non-replicative vector into the chromosome of the bacterial host cell by recombination.

15. The bacterial host cell of claim 14, wherein the non-replicative vector lacks a polynucleotide encoding a replication polypeptide being capable of maintaining said vector in the bacterial host cell.

16. The bacterial host cell of claim 14, wherein said bacterial host cell further comprises v) a replicative vector comprising v1) a plus origin of replication (ori+),v2) a polynucleotide encoding a replication polypeptide, operably linked to a promoter, andv3) optionally, a polynucleotide encoding for a counterselection polypeptide, operably linked to a promoter, wherein the replication polypeptide encoded by the polynucleotide v2) is capable of maintaining the non-replicative vector and the replicative vector in the bacterial host cell.

17. The bacterial host cell of claim 16, wherein the non-replicative vector and the replicative vector are derived from a single vector which, when present in the bacterial host cell, forms the non-replicative and the replicative vector, wherein said single vector comprises i) a first portion comprising elements u1), u2), u3) and u4) of the non-replicative vector, but lacking a polynucleotide encoding a replication polypeptide, andii) a second portion comprising elements v1), v2) and v3) of the replicative vector,wherein the plus origin of replication u1) and the plus origin of replication v1) are present in the single vector in the same orientation, andwherein, upon introduction of said single vector into the bacterial host cell, the first portion of the single vector forms the non-replicative vector and the second portion forms the replicative vector.

18. A method for producing a bacterial host cell comprising, at at least one genomic locus, multiple copies of a non-replicative vector, comprising (a) providing a bacterial host cell belonging to the phylum of Firmicutes in which at least the following chromosomal genes have been inactivated: i. a first chromosomal gene encoding a first alanine racemase, andii. a second chromosomal gene encoding a second alanine racemase(b) introducing, into said bacterial host cell: (b1) a non-replicative vector comprising u1) optionally, a plus origin of replication (ori+),u2) a polynucleotide encoding at least one polypeptide of interest, operably linked to a promoter,u3) a polynucleotide encoding a third alanine racemase, operably linked to a promoter,u4) a polynucleotide which has homology to a chromosomal polynucleotide of the bacterial host cell to allow integration of the non-replicative vector into the chromosome of the bacterial host cell by recombination,(b2) a non-replicative vector and a replicative vector,the non-replicative vector comprising u1) optionally, a plus origin of replication (ori+),u2) a polynucleotide encoding at least one polypeptide of interest, operably linked to a promoter,u3) a polynucleotide encoding a third alanine racemase, operably linked to a promoter,u4) a polynucleotide which has homology to a chromosomal polynucleotide of the bacterial host cell to allow integration of the non-replicative vector into the chromosome of the bacterial host cell by recombination, and the replicative vector comprisingv1) a plus origin of replication (ori+),v2) a polynucleotide encoding a replication polypeptide, operably linked to a promoter, andv3) optionally, a polynucleotide encoding for a counterselection polypeptide, operably linked to a promoter, wherein the replication polypeptide encoded by the polynucleotide v2) is capable of maintaining the non-replicative vector and the replicative vector in the bacterial host cell, or (b3) a single vector comprisingi) a first portion comprising elements u1), u2), u3) and u4) of the non-replicative vector, but lacking a polynucleotide encoding a replication polypeptide, andii) a second portion comprising elements v1), v2) and v3) of the replicative vector,wherein the plus origin of replication u1) and the plus origin of replication v1) are present in the single vector in the same orientation, andwherein, upon introduction of said single vector into the bacterial host cell, the first portion of the single vector forms the non-replicative vector and the second portion forms the replicative vector, and(c) cultivating the host cell under conditions allowing the integration of multiple copies of the non-replicative vector introduced in step (b1) or (b2), or derived from the single vector introduced in step (b3) into at least one genomic locus of the bacterial host cell, and optionally(d) selecting a host cell comprising, at at least one genomic locus, multiple copies of the non-replicative vector.

19. The method of claim 18, wherein the host cell is cultivated in the presence of an effective amount of an alanine racemase inhibitor and/or wherein the host cell is cultivated under conditions to effectively express the counterselection polypeptide, optionally in the presence of an effective amount of a counterselection agent for the counterselection polypeptide.