# US 8,771,998 B2 · Process for the production of gamma-aminobutyric acid > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,998 B2 - Document ID: US8771998B2 - Status: Granted - Kind: B2 - Application number: 12/918,241 - Filed: 2009-02-20 - Published: 2010-12-23 - Granted: 2014-07-08 - Earliest claimed: 2008-02-21 - Assignees: BASF SE - Applicants: Oskar Zelder; Weol Kyu Jeong; Corinna Klopprogge; Andrea Herold; Hartwig Schröder - Inventors: Oskar Zelder; Weol Kyu Jeong; Corinna Klopprogge; Andrea Herold; Hartwig Schröder - Web page: https://patentgrep.com/patent/US8771998B2 ## CPC - C12P 13/005: {Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids} - C12N 9/88: Lyases (4.) - C12P 13/02: Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} - 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)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to a novel method for the fermentative production of gamma-aminobutyric acid (GABA) by cultivating a recombinant microorganism expressing an enzyme having a glutamate decarboxylase activity. The present invention also relates to corresponding recombinant hosts, recombinant vectors, expression cassettes and nucleic acids suitable for preparing such hosts as well as to a method for preparing polyamides making use of GABA as obtained fermentative production. ## Claims ### Claim 1 A method for the fermentative production of gamma-aminobutyric acid (GABA), comprising cultivating a recombinant microorganism derived from a parent microorganism having the ability to produce glutamate and additionally having the ability to express a heterologous glutamate decarboxylase (E.C. 4.1.1.15) in a culture medium, wherein said microorganism is a Corynebacterium,wherein glutamate is converted to GABA in said recombinant microorganism, and wherein said heterologous glutamate decarboxylase is encoded by a nucleic acid composing: a) the nucleotide sequence from position 472 to 1200 of SEQ ID NO: 1 or from position 193 to 1605 of SEQ ID NO: 1; b) a nucleotide sequence encoding a glutamate decarboxylase comprising the amino acid sequence of SEQ ID NO: 2; or c) a nucleotide sequence encoding a glutamate decarboxylase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, wherein said glutamate decarboxylase comprises the amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or an amino acid sequence having at least 98% sequence identity to the amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2. ### Claim 2 (depends on 1) The method of claim 1, wherein the microorganism is Corynebacterium glutamicum. ### Claim 3 (depends on 1) The method of claim 1, wherein said heterologous glutamate decarboxylase is of eukaryotic origin. ### Claim 4 (depends on 1) The method of claim 1, wherein said heterologous glutamate decarboxylase is a plant glutamate decarboxylase or a chimeric glutamate decarboxylase comprising at least one amino acid sequence portion obtained from a plant glutamate decarboxylase. ### Claim 5 (depends on 4) The method of claim 4, wherein said plant glutamate decarboxylase is obtained from a plant of the genus Solanum, from Solanum tuberosum, or from Solanum lycopersicum. ### Claim 6 (depends on 1) The method of claim 1, wherein the heterologous glutamate decarboxylase comprises the amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or an amino acid sequence having at least 98% sequence identity to the amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2. ### Claim 7 (depends on 1) The method of claim 1, wherein, the heterologous glutamate decarboxylase is a chimeric glutamate decarboxylase comprising the amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or an amino acid sequence having at least 95% sequence identity thereto, wherein said chimeric glutamate decarboxylase further comprises at least one ammo acid sequence portion at the N-terminus, the C-terminus, or both the N-terminus and C-terminus, of said amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or an amino acid sequence having at least 95% sequence identity thereto, and wherein the at least one amino acid sequence portion is obtained from it corresponding amino acid sequence portion of a glutamate decarboxylase of Solanum tuberosum or a plant different from Solanum tuberosum. ### Claim 8 (depends on 7) The method of claim 7, wherein aid plant different from Solanum tuberosum is Solanum lycopersicum. ### Claim 9 (depends on 1) The method of claim 1, wherein said heterologous glutamate decarboxylase comprises the amino acid sequence of SEQ ID NO: 2 or is encoded by the nucleotide sequence from position 472 to 1200 of SEQ ID NO: 1 or from position 193 to 1605 of SEQ ID NO: 1. ### Claim 10 (depends on 1) The method of claim 1, wherein the heterologous glutamate decarboxylase is encoded by a nucleic acid sequence adapted to the codon usage of Corynebacterium. ### Claim 11 A chimeric glutamate decarboxylase comprising the amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or an amino acid sequence having at least 95% sequence identity thereto, wherein said chimeric glutamate decarboxylase further comprises at least one amino acid sequence portion at the N-terminus, the C-terminus, or both the N-terminus and C-terminus, of said amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or an amino acid sequence having at least 95% sequence identity thereto, wherein the at least one amino acid sequence portion is obtained from a corresponding amino acid sequence portion of a glutamate decarboxylase of a plant different from Solanum tuberosum, and wherein said chimeric glutamate decarboxylase encoded by a nucleic acid comprising: a) the nucleotide sequence from position 472 to 1200 of SEQ ID NO: 1 or from position 193 to 1605 of SEQ ID NO: 1; b) a nucleotide sequence encoding a glutamate decarboxylase comprising the amino acid sequence of SEQ ID NO: 2; or c) a nucleotide sequence encoding a glutamate decarboxylase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, wherein said glutamate decarboxylase comprises the amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or an amino acid sequence having at least 98% sequence identity to the amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2. ### Claim 12 (depends on 11) An isolated nucleic acid molecule encoding the chimeric glutamate decarboxylase of claim 11. ### Claim 13 (depends on 12) An expression cassette comprising the isolated nucleic acid molecule of claim 12 operatively linked to at least one regulatory nucleic acid sequence. ### Claim 14 (depends on 13) A recombinant vector comprising at least one expression cassette of claim 13. ### Claim 15 (depends on 14) A prokaryotic host transformed with the recombinant vector of claim 14. ### Claim 16 (depends on 15) The prokaryotic host of claim 15, wherein the prokaryotic host is a recombinant Corynebacterium. ### Claim 17 (depends on 16) The prokaryotic host of claim 16, wherein the prokaryotic host is recombinant Corynebacterium glutamicum. ### Claim 18 (depends on 1) The method of claim 1, further comprising recovering the GABA produced from the culture medium. ### Claim 19 (depends on 1) A method of preparing, a polyamide comprising: a) preparing GABA by the method of claim 1; b) isolating GABA; and c) polymerizing said GABA, optionally in the presence of at least one further suitable polyvalent co-monomer, selected from aminocarboxylic acids and hydroxycarboxylic acids. ### Claim 20 (depends on 11) The chimeric glutamate decarboxylase of claim 11, wherein said plant different from Solanum tuberosum is Solanum lycopersicum. ### Claim 21 A method for the fermentative production of gamma-aminobutyric acid (GABA), comprising cultivating a recombinant microorganism derived from a parent microorganism having the ability to produce glutamate and additionally having the ability to express a heterologous glutamate decarboxylase (E.C. 4.1.1.15) in a culture medium, wherein said microorganism is a Corynebacterium, wherein glutamate is converted to GABA in said recombinant microorganism, and wherein said heterologous glutamate decarboxylase is encoded by a nucleotide sequence which hybridizes to the complementary sequence of the nucleotide sequence from position 472 to 1200 of SEQ ID NO: 1 or from position 193 to 1605 of SEQ ID NO: 1 under stringent hybridization conditions comprising hybridization at 42°C in a solution consisting of 50% formamide, 5×SSC, 50mM sodium phosphate, 5×Denhardt Solution, 10% dextran sulfate and 20g/ml denatured, sheared salmon sperm DNA, followed by washing with 0.1×SSC at 65°C.