Browse Prior Art Database

Eubacterial RNA-polymerase mutants with altered product production

IP.com Disclosure Number: IPCOM000011583D
Publication Date: 2003-Mar-06

Publishing Venue

The IP.com Prior Art Database

Related People

Novozymes A/S: OWNER

Abstract

The present invention relates to an isolated mutant eubacterium comprising at least one mutation resulting in a substitution of at least one amino acid in the beta-subunit of the RNA-polymerase encoded for by the rpoB-gene providing an altered production of a product of interest when said production of a product of interest is compared to the production of the same product in an isogenic wild type strain grown at identical conditions, wherein the substitution of at least one amino acid occurs at any of the positions 469, 478, 482, 485, or 487 of SEQ ID NO:2, or at the equivalent positions in any eubacterial RNA-polymererase beta-subunit family member. Another aspect of the invention relates to a process for producing at least one product of interest in a mutant eubacterium and to a use of the mutant eubacterium according to the invention for producing at least one product of interest

This text was extracted from a Microsoft Word document.
This is the abbreviated version, containing approximately 3% of the total text.

TITLE: Eubacterial RNA-polymerase mutants with altered product production

FIELD OF INVENTION

                      The present invention relates to an isolated mutant eubacterium comprising at least one mutation resulting in a substitution of at least one amino acid in the beta-subunit of the RNA-polymerase encoded for by the rpoB-gene providing an altered production of a product of interest when said production of a product of interest is compared to the production of the same product in an isogenic parent strain grown at identical conditions. Another aspect of the invention relates to a process for producing at least one product of interest in a mutant eubacterium and to a use of the mutant eubacterium according to the invention for producing at least one product of interest.

BACKGROUND OF THE INVENTION

                      In the industrial production of polypeptides it is of interest to achieve a product yield as high as possible. One way to increase the yield is to increase the copy number of a gene encoding a polypeptide of interest. This can be done by placing the gene on a high copy number plasmid. However, plasmids are unstable and are often lost from the host cells if there is no selective pressure during the cultivation of the host cells. Another way to increase the copy number of the gene of interest is to integrate it into the host cell chromosome in multiple copies. It has previously been described how to integrate a gene into the chromosome by double homologous recombination without using antibiotic markers (Hone et al., Microbial Pathogenesis, 1988, 5: 407-418); integration of two genes has also been described (Novo Nordisk: WO 91/09129 and WO 94/14968). Integrating several copies of a gene into the chromosome of a host cell could lead to instability. Integration of two genes closely spaced in anti-parallel tandem to achieve better stability has been described (Novozymes: WO 99/41358)as well as the stable chromosomal multi-copy integration of genes (Novozymes: WO 02/00907).

                      Other ways of increasing the product yield would be to increase promoter activity of the specific promoter regulating the expression of a specific gene of interest. Also a more general increase in the activity of several promoters at the same time could lead to an improved product yield.

                      The most studied bacterial RNA-polymerase is the E. coli RNA-polymerase and it is representative of the enzymes isolated from a number of bacterial genera, including Salmonella, Serratia, Proteus, Aerobacter, and Bacillus (Fukuda et al., 1977, Mol. Gen. Genet. 154:135).

                      The RNA-polymerase core enzyme is composed of alpha, beta, and beta' subunits in the ratio 2:1:1, encoded for by the rpoA, rpoB, and rpoC genes respectively. Mutations that confer a cellular resistance to several antibiotics (e.g. rifampicin) are within the rpoB gene.

                      Rifampicin binds to the beta-...