# US 11,028,384 B2 · Pyruvate carboxylase and pyruvate carboxylase-encoding DNA, plasmid containing said DNA and microorganism for the production thereof, and methods for the production of products the biosynthesis of which includes oxaloacetate as precursor, and chromosome > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,028,384 B2 - Document ID: US11028384B2 - Status: Granted - Kind: B2 - Application number: 16/606,230 - Filed: 2018-04-13 - Published: 2020-02-20 - Granted: 2021-06-08 - Earliest claimed: 2017-05-18 - Assignees: Forschungszentrum Juelich GmbH - Applicants: Forschungszentrum Juelich GmbH - Inventors: Maike Kortmann; Meike Baumgart; Michael Bott - Web page: https://patentgrep.com/patent/US11028384B2 ## CPC - C12N 9/93: {Ligases (6)} - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - C12N 1/205: Bacterial isolates - C12N 9/1217: {Phosphotransferases with a carboxyl group as acceptor (2.7.2)} - C12N 15/52: Genes encoding for enzymes or proenzymes - C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora - C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00) - C12Y 604/01001: Pyruvate carboxylase (6.4.1.1) - C12R 2001/15: Corynebacterium - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12Y: ENZYMES - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract A DNA sequence that includes at least 70% identity with respect to SEQ ID NO: 1 and further includes a triplet at position 1027-1029 that codes for alanine. ## Claims ### Claim 1 An isolated DNA sequence encoding a pyruvate carboxylase, comprising: at least 97% identity with respect to SEQ ID NO: 1, wherein a triplet at position 1027-1029 codes for alanine. ### Claim 2 (depends on 1) The DNA sequence according to claim 1, wherein the DNA has identity of at least 99% with respect to SEQ ID NO: 1. ### Claim 3 (depends on 1) The DNA sequence according to claim 1, wherein the DNA sequence is a DNA according to SEQ ID NO: 1. ### Claim 4 (depends on 1) The DNA sequence according to claim 1, further comprising, at position 3034-3036, a triplet which encodes for serine. ### Claim 5 (depends on 4) The DNA sequence according to claim 4, wherein the DNA has identity of at least 97% with respect to SEQ ID NO: 2. ### Claim 6 (depends on 4) The DNA sequence according to claim 4, wherein the DNA is according to SEQ ID NO: 2. ### Claim 7 A pyruvate carboxylase having at least 97% identity with respect to a pyruvate carboxylase according to SEQ ID NO: 3, wherein alanine is present at position 343. ### Claim 8 (depends on 7) The pyruvate carboxylase according to claim 7, wherein the pyruvate carboxylase has identity of at least 99% with respect to the pyruvate carboxylase according to SEQ ID NO: 3. ### Claim 9 (depends on 7) The pyruvate carboxylase according to claim 7, wherein the pyruvate carboxylase is a pyruvate carboxylase according to SEQ ID NO: 3. ### Claim 10 (depends on 7) The pyruvate carboxylase according to claim 7, further comprising serine at position 1012. ### Claim 11 (depends on 10) The pyruvate carboxylase according to claim 10, wherein the pyruvate carboxylase has identity of at least 97% with respect to the pyruvate carboxylase according to SEQ ID NO: 4. ### Claim 12 (depends on 10) The pyruvate carboxylase according to claim 10, wherein the pyruvate carboxylase is a pyruvate carboxylase according to SEQ ID NO: 4. ### Claim 13 (depends on 1) A vector, wherein the vector comprises a DNA sequence according to claim 1. ### Claim 14 (depends on 13) The vector according to claim 13, wherein the vector is a plasmid. ### Claim 15 (depends on 1) A microorganism, wherein the microorganism comprises a DNA sequence according to claim 1. ### Claim 16 (depends on 15) The microorganism according to claim 15, wherein the microorganism comprises a vector. ### Claim 17 (depends on 15) The microorganism according to claim 15, wherein the microorganism is a microorganism selected from the group consisting of the genera Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Vibrio, and Clostridium. ### Claim 18 (depends on 1) A method for the production of products whose biosynthesis includes oxaloacetate as a precursor, the method comprising: fermenting a microorganism containing a DNA sequence according to claim 1, which forms a pyruvate carboxylase, and performing an enrichment of the products in a medium or in cells. ### Claim 19 (depends on 18) The method according to claim 18, wherein a product is isolated. ### Claim 20 (depends on 18) The method according to claim 18, wherein a species from the group consisting of the genera Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Vibrio, and Clostridium is used as the microorganism. ### Claim 21 (depends on 18) The method according to claim 18, wherein an amino acid of the aspartate family is produced. ### Claim 22 (depends on 21) The method according to claim 21, wherein L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine, or L-methionine is produced. ### Claim 23 (depends on 18) The method according to claim 18, wherein substances from the glutamate family of amino acids are produced, such as L-glutamate, glutamine, L-arginine, L-proline, L-citrulline, or L-ornithine; intermediates of the citrate cycle, such as malate, fumarate, succinate, malate, fumarate citrate, isocitrate, or 2-oxoglutarate; diamines, such as diaminopentane or diaminobutane; as well as further metabolites prepared from oxaloacetate, such itaconate, ectoine, gamma aminobutyrate, butanol, 1-propanol, L-citrulline, L-ornithine, D-arginine, or 4-hydroxyproline. ### Claim 24 (depends on 18) The method according to claim 18, wherein the DNA sequence is more strongly expressed. ### Claim 25 (depends on 1) A chromosome including the DNA sequence according to claim 1.