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Gene coding for glucose-6-phosphate-dehydrogenase proteins

Gene coding for glucose-6-phosphate-dehydrogenase proteins description/claims

This application is a continuation of U.S. application Ser. No. 11/787,137, filed Apr. 13, 2007, which is a continuation of U.S. application Ser. No. 10/495,291, filed May 11, 2004, which is a 35 U.S.C. 371 National stage filing of International Application No. PCT/EP02/12556, filed Nov. 11, 2002, which claims priority to German Application No. 101 55 505.9, filed Nov. 13, 2001. The entire contents of each of these applications are hereby incorporated by reference herein.

This application incorporates herein by reference the sequence listing filed concurrently herewith, i.e., the file “seqlist” (24 KB) created on Mar. 20, 2008.

Particular products and byproducts of naturally occurring metabolic processes in cells are used in many branches of industry, including the food industry, the animal feed industry, the cosmetics industry and the pharmaceutical industry. These molecules which are collectively referred to as “fine chemicals” comprise organic acids, both proteinogenic and nonproteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors and enzymes. They are best produced by means of cultivating, on a large scale, bacteria which have been developed to produce and secrete large amounts of the molecule desired in each particular case. An organism which is particularly suitable for this purpose is Corynebacterium glutamicum, a Gram-positive nonpathogenic bacterium. Using strain selection, a number of mutant strains have been developed which produce various desirable compounds. The selection of strains which are improved with respect to the production of a particular molecule is, however, a time-consuming and difficult process.

The present invention provides novel nucleic acid molecules which can be used for identifying or classifying Corynebacterium glutamicum or related bacterial species. C. glutamicum is a Gram-positive, aerobic bacterium which is widely used in industry for the large-scale production of a number of fine chemicals and also for the degradation of hydrocarbons (for example in the case of crude oil spills) and for the oxidation of terpenoids. The nucleic acid molecules may therefore be used for identifying microorganisms which can be used for producing fine chemicals, for example by fermentation processes. Although C. glutamicum itself is nonpathogenic, it is, however, related to other Corynebacterium species such as Corynebacterium diphteriae (the diphtheria pathogen), which are major pathogens in humans. The ability to identify the presence of Corynebacterium species may therefore also be of significant clinical importance, for example in diagnostic applications. Moreover, said nucleic acid molecules may serve as reference points for mapping the C. glutamicum genome or genomes of related organisms.

These novel nucleic acid molecules encode proteins which are referred to as glucose-6-phosphate-dehydrogenase proteins.

Glucose-6-phosphate-dehydrogenase genes from Corynebacteria are described, for example, in EP 1108790A2. However, the genes described therein code for polypeptide sequences which are shorter than those described herein according to the invention. The N terminus of the glucose-6-phosphate-dehydrogenase described in EP 1108790A2 is truncated by 30 amino acids compared with the polypeptide sequence claimed herein.

Moritz et al. (Eur. J. Biochemistry 267, 3442-3452, 2000) describe the isolation of a glucose-6-phosphate-dehydrogenase from Corynebacterium glutamicum. The N-terminal protein sequencing described therein results in a polypeptide which starts with a serine and differs from the protein of the invention.

The invention relates to novel genes for glucose-6-phosphate-dehydrogenase, which start with the amino acid at position 1 or 2, i.e. Val or Ser and which encode at position 243 a proteinogenic amino acid which is not Ala (numbering based on SEQ ID NO: 2).

Particular preference is given to novel genes for glucose-6-phosphate-dehydrogenase, which start with the amino acid at position 1 and encode Thr at position 243 (numbering based on SEQ ID NO: 2).

The nucleic acid molecules of the invention can be used for genetic manipulation of an organism in order to make it a better and more efficient producer of one or more fine chemicals. The molecules of the invention can be modified so as to improve the yield, production and/or efficiency of production of one or more fine chemicals.

Furthermore, the molecules of the invention may be involved in one or more intracellular signal transduction pathways which influence the yields and/or the rate of production of one or more fine chemicals from C. glutamicum. Proteins which are required, for example, for importing one or more sugars from the extracellular medium (e.g. Hpr, enzyme I or a component of the enzyme II complex) are, if a sufficient amount of sugar is present in the cell, frequently posttranslationally modified so that they are no longer able to import said sugar. Although the amount of sugar at which the transport system is switched off is sufficient for maintaining normal cellular functions, it limits overproduction of the fine chemical of interest. It is therefore recommended to modify the proteins of the invention so that they no longer respond to such a negative regulation. As a result, it is possible to attain higher intracellular concentrations of one or more sugars and, by extension, a more efficient production or higher yields of one or more fine chemicals from organisms which contain said mutant proteins.

Appendix A defines hereinbelow the nucleic acid sequences of the sequence listing together with the sequence modifications at the relevant position, described in Table 1.

Appendix B defines hereinbelow the polypeptide sequences of the sequence listing together with the sequence modifications at the relevant position, described in Table 1.

In a further embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule which comprises a nucleotide sequence of Appendix A. The isolated nucleic acid molecule preferably corresponds to a naturally occurring nucleic acid molecule. The isolated nucleic acid more preferably encodes a naturally occurring C. glutamicum G6PD protein or a biologically active section thereof.

A further aspect of the invention relates to vectors, for example recombinant expression vectors, which contain the nucleic acid molecules of the invention and to host cells into which said vectors have been introduced. In one embodiment, a G6PD protein is prepared by using a host cell which is cultivated in a suitable medium. The G6PD protein may then be isolated from the medium or the host cell.

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