(12) Patent Application Publication (10) Pub. No.: US 2007/0122887 A1 Klopprogge Et Al
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US 2007.0122887A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0122887 A1 Klopprogge et al. (43) Pub. Date: May 31, 2007 (54) GENE VARIANTS CODING FOR PROTEINS (30) Foreign Application Priority Data FROM THE METABOLIC PATHWAY OF FNE CHEMICALS Dec. 18, 2003 (DE)..................................... 103 59 661.5 (75) Inventors: Corinna Klopprogge, Mannheim (DE); Publication Classification Oskar Zelder, Speyer (DE); Burkhard Kroger, Limburgerhof (DE); Hartwig (51) Int. Cl. Schroder, Nublock (DE); Stefan CI2P I3/04 (2006.01) Haefner, Ludwigshafen (DE); Uwe CI2P I3/08 (2006.01) Ruffer, Meckenheim (DE); Claudia C7H 2L/04 (2006.01) Isabella Graef, Lampertheim (DE) CI2N 9/10 (2006.01) CI2N 15/74 (2006.01) Correspondence Address: CI2N I/2 (2006.01) LAHIVE & COCKFIELD, LLP ONE POST OFFICE SQUARE (52) U.S. Cl. ......................... 435/106; 435/115; 435/193; BOSTON, MA 02109-2127 (US) 435/252.3; 435/471; 536/23.2 (73) Assignee: BASF Aktiengesellschaft, Ludwigshafen (DE) (57) ABSTRACT (21) Appl. No.: 10/583,404 (22) PCT Filed: Dec. 16, 2004 The present invention relates to mutated nucleic acids and proteins of the metabolic pathway of fine chemicals, to (86). PCT No.: PCT/EPO4/14338 processes for preparing genetically modified producer organisms, to processes for preparing fine chemicals by S 371(c)(1), culturing said genetically modified organisms and to said (2), (4) Date: Jun. 16, 2006 genetically modified organisms themselves. US 2007/O 122887 A1 May 31, 2007 GENEVARLANTS CODNG FOR PROTEINS FROM and optimized C yield, there is a constant need for further THE METABOLIC PATHWAY OF FINE improving the productivity of said organisms. CHEMICALS 0010. It is an object of the present invention to provide 0001. The present invention relates to mutated nucleic further mutated genes and proteins which result in an acids and proteins of the metabolic pathway of fine chemi increase in productivity in producer organisms of fine cals, to processes for preparing genetically modified pro chemicals and thus in an improvement of biotechnological ducer organisms, to processes for preparing fine chemicals processes for preparing fine chemicals. by culturing said genetically modified organisms and to said genetically modified organisms themselves. 0011 We have found that this object is achieved by proteins having the function indicated in each case in table 0002 Many products and byproducts of naturally occur 1/column 7 and having an amino acid sequence which has in ring metabolic processes in cells are used in many branches at least one of the amino acid positions which, starting from of industry, including the food industry, the animal feed the amino acid sequence referred to in each case in table industry, the cosmetic industry and the pharmaceutical 1/column 2, correspond to the amino acid positions indi industry. These compounds, which are collectively referred cated for said amino acid sequence in table 1/column 4 a to as “fine chemicals, comprise, for example, organic acids, proteinogenic amino acid different from the particular amino both proteinogenic and nonproteinoge amino acids, nucle acid indicated in the same row in table 1/column 5, with the otides and nucleosides, lipids and fatty acids, diols, carbo proviso that the proteins according to table 2 are excepted. hydrates, aromatic compounds, vitamins and cofactors and also enzymes. 0012. The present invention provides novel nucleic acid molecules and proteins which, on the one hand, can be used 0003. They may be produced, for example, by large-scale for identifying or classifying Corynebacterium glutamicum fermentation of microorganisms which have been developed or related bacterial species and, on the other hand, result in to produce and secrete large amounts of one or more desired an increase in productivity in producer organisms of fine fine chemicals. chemicals and thus in an improvement of biotechnological 0004 An organism which is particularly suitable for this processes for preparing fine chemicals. purpose is Corynebacterium glutamicum, a Gram-positive, 0013 C. glutamicum is a Gram-positive, aerobic bacte nonpathogenic bacterium. Using strain selection, a number rium which is widely used in industry for large-scale pro of mutant strains have been developed which produce vari duction of a number of fine chemicals and also for the ous desirable compounds. The selection of strains which degradation of hydrocarbons (e.g. in the case of crude oil have been improved with respect to production of a particu spills) and for the oxidation of terpenoids. The nucleic acid lar compound is, however, a time-consuming and difficult molecules may therefore be used furthermore for identifying process. microorganism which can be used for production of fine 0005. It is possible to increase the productivity of pro chemicals, for example by fermentation processes. Although ducer organisms by genetic modifications. For example, the C. glutamicum itself is nonpathogenic, it is, however, related specific mutation of particular genes in a producer organism to other Corynebacterium species, such as Corynebacterium may result in an increase in productivity of a desired fine diphtheriae (the diphtheria pathogen), which are major chemical. pathogens in humans. The ability to identify the presence of Corynebacterium species may therefore also be of signifi 0006 EP 1 108 790 A2 describes, starting from the cant clinical importance, for example in diagnostic applica wild-type sequence encoding a Corynebacterium tions. Moreover, said nucleic acid molecules may serve as glutamicum homoserine dehydrogenase, a mutated nucleic reference points for mapping the C. glutamicum genome or acid sequence encoding a homoserine dehydrogenase which the genomes of related organisms. has, compared to the wild-type sequence, the mutation Val59Ala. Furthermore, a mutated nucleic acid sequence 0014. The proteins of the invention, also referred to as encoding a pyruvate carboxylase which has the mutation Metabolic-Pathway proteins or MP proteins hereinbelow, Pro458Ser in comparison with the Corynebacterium have the function indicated in each case in table 1/column 7. glutamicum wild-type amino acid sequence is described. Furthermore, they have in each case an amino add sequence Introduction of said mutations into Corynebacterium which has in at least one of the amino acid positions which, glutamicum results in an increase in the lysine yield. starting from the amino acid sequence referred to in each case in table 1/column 2, correspond to the amino acid 0007 WO 0063388 furthermore discloses a mutated ask positions indicated for said amino acid sequence in table gene which encodes an asparto-kinase having the mutation 1/column 4 a proteinogenic amino acid different from the T311. particular amino acid indicated in the same row in table 0008. Other mutations in genes and proteins of the 1/column 5. Corynebacterium glutamicum biosynthetic pathway of fine 0015 The “corresponding amino acid position prefer chemicals are described in WO 0340681, WO 0340357, WO ably means the amino acid position of the amino acid 0340181, WO 0340293, WO 0340292, WO 0340291, WO sequence of the MP proteins of the invention, which the 0340180, WO 0340290, WO 0346123, WO 0340289 and skilled worker can readily find WO O342389. a) by homology comparison of the amino acid sequence or 0009. Although the mutations known in the prior art already result in producer organisms having optimized pro b) by structural comparison of the secondary, tertiary and/or ductivity, i.e. optimized yield of the desired fine chemical quaternary structure of said amino acid sequence US 2007/O 122887 A1 May 31, 2007 with the amino acid sequence referred to in each case in biologically active section thereof. In a preferred embodi table 1/column 2 and having the amino acid position indi ment, the isolated MP protein or its section regulates tran cated for said amino acid sequence in each case in table Scriptionally, translationally or posttranslationally one or 1/column 4. more metabolic pathways in organisms, in particular in 0016 A preferred method of comparing homologies of corynebacteria and brevibacteria. the amino acid sequences is employed, for example, by the 0025 The MP polypeptide or a biologically active sec Lasergene Software from DNASTAR, inc. Madison, Wis. tion thereof may be functionally linked to a non-MP (USA), using the Clustal method (Higgins DG, Sharp PM. polypeptide to produce a fusion protein. In preferred Fast and sensitive multiple sequence alignments on a micro embodiments, the activity of this fusion protein is different computer. Comput Appl. Biosci. 1989 April; 5(2):151-1), from that of the MP protein alone and, in other preferred setting the following parameters: embodiments, said fusion protein regulates transcriptionally, translationally or posttranslationally one or more metabolic 0017 Multiple Alignment Parameter: pathways in organisms, in particular in corynebacteria and Gap penalty 10 brevibacteria, preferably in Corynebacterium glutamicum. In particularly preferred embodiments, integration of said Gap length penalty 10 fusion protein into a host cell modulates production of a 0018 Pairwise Alignment Parameter: compound of interest by the cell. K-tuple 1 0026. The invention furthermore relates to isolated nucleic acids encoding an above-described protein of the Gap penalty 3 invention. These nucleic acids are hereinbelow also referred Window 5 to as Metabolic-Pathway nucleic acids or MP nucleic acids or MP genes.