WO2015092599A1 - Improved process for the production of gamma-aminobutyric acid (gaba) - Google Patents

Improved process for the production of gamma-aminobutyric acid (gaba) Download PDF

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Publication number
WO2015092599A1
WO2015092599A1 PCT/IB2014/066633 IB2014066633W WO2015092599A1 WO 2015092599 A1 WO2015092599 A1 WO 2015092599A1 IB 2014066633 W IB2014066633 W IB 2014066633W WO 2015092599 A1 WO2015092599 A1 WO 2015092599A1
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Prior art keywords
nucleic acid
glutamate
gaba
sequence
recombinant microorganism
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PCT/IB2014/066633
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French (fr)
Inventor
Oskar Zelder
Stefan Haefner
Hartwig Schröder
Robert THUMMER
Hyung-Hwan Hyun
Weol Kyu Jeong
Aline Raddatz
Karin SCHEIN-ALBRECHT
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Basf Se
Basf (China) Company Limited
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Publication of WO2015092599A1 publication Critical patent/WO2015092599A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01015—Glutamate decarboxylase (4.1.1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • GABA gamma-aminobutyric acid
  • the present invention relates to an improved method for the fermentative production of gamma-aminobutyric acid (GABA) by cultivating a recombinant microorganism expressing an ex- ogenous enzyme having a glutamate decarboxylase activity, wherein the recombinant microorganism has a reduced glutamate exporter expression and / or activity.
  • GABA gamma-aminobutyric acid
  • 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 pyrrolidone or polymers making use of GABA as obtained by fermentative production.
  • GABA (CAS number 56-12-2) is an important ubiquitous non-protein amino acid in both pro- karyotic and eukaryotic organisms. It shows different biological functions, for example as rep- resentative depressive neurotransmitter in the sympathetic nervous system and it is effective for lowering the blood pressure of experimental animals and humans.
  • the compound is synthesized by a glutamate decarboxylase (GAD) from glutamate.
  • GAD glutamate decarboxylase
  • GABA is used in different technical fields.
  • GABA-enriched food can be used as a dietary supplement and nutraceutical to help treat sleeplessness, depression and autonomic disorders, chronic alcohol-related symptoms, and to stimulate immune cells.
  • the compound can also be used as a raw material for the production of polyamides and of pyrrolidone.
  • WO2009103547A1 describes a method for fermentative production of GABA by expressing in a coryneform bacterium a glutamate decarboxylase (gad) gene.
  • the yield obtained by the method described in WO2009103547A1 is rather low.
  • the object of the present invention is, therefore, to provide an improved method for the fer- mentative production of GABA or corresponding salts thereof.
  • the objective problem has been solved in the present invention by amending the expression of certain enzymes influencing the GABA yield in the microorganism used for fermentation.
  • the objective problem has been solved by reducing the expression of a glutamate exporter. This solution has not been suggested by the prior art.
  • Vo et al. discloses the produc- tion of GABA in recombinant Escherichia coli, in which the expression of the gene coding for the glutamate/GABA antiporter is increased.
  • the above-mentioned problem was solved by the present invention teaching the fermentative production of GABA or a salt thereof by cultivating a recombinant glutamate producing microorganism expressing an exogenous GAD enzyme which enzyme converts glutamate that is formed in said microorganism to GABA, wherein the recombinant microorganism has a reduced glutamate exporter expression and / or 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 pyrrolidone or polymers making use of GABA as obtained by fermentative production.
  • Cre recombinase gene analysed by gel electrophoresis and subsequent coomassie brilliant blue staining S.M: protein size marker; Mbi- otech, Cat.No. 20030; Lane 1 : pCESCAT-icl.cre in C. glutamicum (no induction); Lane 2: pCESCAT-icl.cre in C. glutamicum (16hr induction). Fermentation time courses of C. glutamicum HH 105 which contains a GAD gene from potato-tomato and 4-aminobutyrate aminotransferase gene was disrupted (growth ( ⁇ ), glucose (°), GABA ( ⁇ ), mono-sodium glutamate (MSG) (°))-
  • glutamicum HH 105 which contains a GAD gene from potato-tomato and 4-aminobutyrate aminotransferase gene was disrupt- ed (growth ( ⁇ ), glucose (o), GABA ( ⁇ ), MSG ( ⁇ )).
  • FIG. 10 Fermentation time courses in fed-batch culture for the production of GABA by
  • C.glutamicum HH 105 (cell growth ( ⁇ ), GABA ( ⁇ ), mono-sodium glutamate (MSG) (X) and reducing sugar ( ⁇ * ⁇ )).
  • ⁇ cell growth
  • GABA ⁇
  • MSG mono-sodium glutamate
  • X reducing sugar
  • ⁇ * ⁇ reducing sugar
  • Figure 1 1 Fermentation time courses in fed-batch culture for the production of GABA by
  • C.glutamicum HH 106 (cell growth ( ⁇ ), GABA ( ⁇ ), mono-sodium glutamate (MSG) (X) and reducing sugar (* ⁇ )).
  • ⁇ cell growth
  • GABA ⁇
  • MSG mono-sodium glutamate
  • X reducing sugar
  • * ⁇ reducing sugar
  • FIG. 12 Fermentation time courses in fed-batch culture for the production of GABA by
  • Figure 13 Comparison of the fermentation time courses of C. glutamicum which comprises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for 4-aminobutyrate aminotransferase.
  • Figure 14 Comparison of the fermentation time courses of C. glutamicum which com- prises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for a glutamate exporter.
  • Figure 15 Comparison of the fermentation time courses of C. glutamicum which comprises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for 4-aminobutyrate aminotrans- ferase and a disruption of the gene coding for a glutamate exporter.
  • Figure 16 GABA production of various mutants of C. glutamicum (wt coryne HH09 ( ⁇ ),
  • Figure 19 Construction of additional plasmids containing the different promoters and
  • Homologues of a protein encompass peptides, oligopeptides, polypeptides, proteins and / or enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified protein in question and having similar functional activity as the unmodified protein from which they are derived.
  • “Homologues” of a nucleic acid encompass nucleotides and / or polynucleotides having nucleic acid substitutions, deletions and / or insertions relative to the unmodified nucleic acid in question, wherein the protein coded by such nucleic acids has similar functional activity as the unmodified protein coded by the unmodified nucleic acid from which they are de- rived.
  • homologues of a nucleic acid may encompass substitutions on the basis of the degenerative amino acid code.
  • identity refers in each case over the entire length of the respective nucleic acid sequence or amino acid sequence.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over a particular region, determining the number of positions at which the identical base or amino acid occurs in both sequences in order to yield the number of matched positions, dividing the number of such positions by the total number of positions in the region being compared and multiplying the result by 100.
  • GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage.
  • Global percentages of similarity/homology/identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics.
  • MatGAT an application that generates similarity/homology/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art.
  • the sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981 ) J. Mol. Biol 147(1 );195-7).
  • the sequence identity may also be calculated by means of the Vector NTI Suite 7.1 program of the company Informax (USA) employing the Clustal Method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr; 5(2): 151 -1 ) with the following settings: Multiple alignment parameter:
  • the identity may be determined according to Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids Res 31 (13):3497-500, the web page: http://www.ebi.ac.Uk/Tools/clustalw/index.html# and the following settings
  • a “deletion” refers to removal of one or more amino acids from a protein or to the removal of one or more nucleic acids from DNA or RNA.
  • An “insertion” refers to one or more amino acid residues or nucleic acid residues being in-duced into a predetermined site in a protein or the nucleic acid.
  • substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or beta-sheet structures).
  • a substitution refers to a replacement of one or more nucleotides with other nucleotides within a nucleic acid, wherein the protein coded by the modified nucleic acid has a similar function.
  • homologues of a nucleic acid encompass substitutions on the basis of the degenerative amino acid code.
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the protein and may range from 1 to 10 amino acids; insertions or deletion will usually be of the order of about 1 to 10 amino acid residues.
  • the amino acid substitutions are preferably conservative amino acid substitutions.
  • Con- servative substitution tables are well known in the art (see for example Taylor W.R. (1986) The classification of amino acid conservation J Theor Biol., 1 19:205-18 and Table 1 below).
  • Amino acid substitutions, deletions and / or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation.
  • encode or "coding for” is used for the capability of a nucleic acid to contain the information for the amino acid sequence of a protein via the genetic code, i.e., the succession of codons each being a sequence of three nucleotides, which specify which amino acid will be added next during protein synthesis.
  • the terms “encode” or “coding for” therefore includes all possible reading frames of a nucleic acid.
  • the terms “encode” or “coding for” also applies to a nucleic acid, which coding sequence is interrupted by non- coding nucleic acid sequences, which are removed prior translation, e.g., a nucleic acid sequence comprising introns.
  • domain refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other posi- tions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein.
  • hybridization includes "any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing" (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules.
  • Tm is used in reference to the "melting temperature.”
  • the melt- g ing temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • Hybridization conditions are known in the art and are for example described in Sambrook, J., Fritsch, E.F., Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31 -9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6.
  • the hybridization conditions for DNA:DNA hybrids are 0.1 x SSC and temperatures between about 20 °C to 45 °C, preferably between about 30 °C to 45 °C.
  • hybridization conditions are advantageously 0.1 x SSC and temperatures between about 30 °C to 55 °C, preferably between about 45 °C to 55 °C. These stated temperatures for hybridization are examples of calculated melting temperature values for a nucleic acid with a length of approx. 100 nucle- otides and a G + C content of 50 % in the absence of formamide.
  • Hybridization can in particular be carried out under stringent conditions.
  • stringent conditions refers to conditions, wherein 100 contigous nucleotides or more, 150 contigous nucleotides or more, 200 contigous nucleotides or more or 250 contigous nucleotides or more which are a fragment or identical to the complementary nucleic acid molecule (DNA, RNA, ssDNA or ssRNA) hybridizes under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 2 X SSC, 0.1 % SDS at 50°C or 65°C, preferably at 65°C, with a specific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA).
  • SDS sodium dodecyl sulfate
  • the hybridizing conditions are equivalent to hybridiza- tion in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C, more preferably the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M Na- P04, 1 mM EDTA at 50°C with washing in 0,1 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C.
  • the complementary nucleotides hybridize with a fragment or the whole of the nucleic acids.
  • preferred hybridization conditions encompass hybridisation at 65°C in 1x SSC or at 42°C in 1x SSC and 50% formamide, followed by washing at 65°C in 0.3x SSC or hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% forma- mide, followed by washing at 50°C in 2x SSC.
  • Further preferred hybridization conditions are 0.1 % SDS, 0.1 SSD and 65°C.
  • isolated nucleic acid or isolated protein refers to a nucleic acid or protein that is not located in its natural environment, in particular its natural cellular environment. Thus, an isolated nucleic acid or isolated protein is essentially separated from other components of its natural environment.
  • an isolated nucleic acid or an isolated protein can display a certain degree of impurity depending on the isolation procedure used.
  • the isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis.
  • a recombinant nucleic acid may also be in an isolated form.
  • Methods for purifying and synthesizing nucleic acids and proteins are well known in the art (as described for example in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid and / or protein refers to the nucleic acid and / or protein in question as found in a cell in its natural form (i.e., without there being any human intervention).
  • exogenous nucleic acid refers to a nucleic acid that has been introduced in an organism by means of genetechnology, i.e., the respective modification of the organism is not of natural origin.
  • An "exogenous" nucleic acid can either not occur in an organism in its natural form, be different from the nucleic acid in question as found in an organism in its natural form, or can be identical to a nucleic acid found in an organism in its natural form, but integrated not within its natural genetic environment.
  • exogenous is applied in the context of protein expression.
  • exogenous and heterologous are used interchangeably herein.
  • nucleic acid sequence means with regard to, for example, a nucleic acid sequence, a nucleic acid molecule, an expression cassette or a vector construct, all those constructions brought about by man by genetechnological methods in which either (a) the sequences of a nucleic acid or a part thereof, or
  • genetic control sequence(s) which is operably linked with a nucleic acid sequence, for example a promoter, or
  • nucleic acid sequence is not located in their natural genetic environment or have been modified by man by genetechnological methods, i.e., the nucleic acid sequence, the nucleic acid molecule, the expression cassette, the vector construct or the modification of the microorganism is not of natural origin.
  • the modification may take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.
  • the natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original organism or the presence in a genomic library or the combination with the natural promoter.
  • a naturally occurring expression cassette for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a protein useful in the methods of the present invention, as defined above - becomes a recombinant expression cassette when this expression cas- sette is modified by man by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350, WO 00/15815 or US200405323.
  • a naturally occurring expression cassette for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a protein useful in the methods of the present invention, as defined above - becomes a recombinant expression cassette when this expression cassette is not integrated in the natural genetic environment but in a different genetic environment.
  • microorganism means all generally unicellular organisms with dimensions be- neath the limits of vision which can be propagated and manipulated in a laboratory.
  • the microorganism can be, e.g., bacteria, yeast, fungus, or algae.
  • recombinant microorganism refers to a microorganism (e.g., bacteria, yeast, fungus, etc.) or microbial strain which has been genetically altered, modified or engineered (e.g., genetically engineered) such that it exhibits an altered, modified or different genotype as compared to the wild-type microorganism which it was derived from.
  • the "recombinant microorganism” comprises an exogenous, preferably, a recombinant, nucleic acid.
  • Recombinant microorganism and “genetically modified microorganism” are used herein interchangeably.
  • an “intermediary product” is understood as a product, which is transiently or continuously formed during a chemical or biochemical process, in a not necessarily analytically directly detectable concentration. Said “intermediary product” may be removed from said biochemical process by a second, chemical or biochemical reaction.
  • operatively linked is intended to mean that the nucleic acid to be expressed is linked to one or more regulatory sequences, including promoters, terminators, enhancers and / or other ex- pression control elements (e.g., polyadenylation signals), in a manner which allows for expression of the nucleic acid (e.g., in a host cell when the vector is introduced into the host cell).
  • regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of nucleic acid desired, and the like.
  • “Expression cassette” or “expression construct” means, according to the invention, one or more “regulatory elements”, which are operatively linked to one or more nucleic acids that are to be expressed.
  • “Expression cassette” or “expression construct” can be monocistronic or polycistronic.
  • regulatory element means, according to the invention, a nucleic acid suitable to drive, increase, decrease or terminate expression of a nucleic acid operatively linked thereto.
  • a “regulatory element” can be a promoter, enhancer, repressor, terminator or the like.
  • a “promoter”, a “nucleic acid with promoter activity” or a “promoter sequence” mean, according to the invention, a nucleic acid which, functionally associated with a nucleic acid that is to be transcribed, regulates the transcription of this nucleic acid.
  • terminal encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription.
  • the terminator can be derived from the endogenous gene or from an exogenous gene.
  • transgenic refers to an organism that exogenously contains the nucleic acid, recombinant construct, vector or expression cassette described herein or a part thereof or mutation which is preferably introduced by genetechnological methods.
  • wild-type means, according to the invention, the state prior to a certain modification and the state to which the state after the modification is compared to in order to evaluate the effect of the respective modification.
  • a wild-type microorganism refers to a micro- organism that has not been altered in the respective feature under investigation.
  • a wild- type microorganism can also be a recombinant microorganism itself with respect to a different modification.
  • a wild-type microorganism is preferably able to produce glutamate.
  • host cell describes a cell, preferably a cell of a microorganism, which shall be modified, e.g., by means of genetechnology.
  • Natural locus means the location on a specific chromosome, preferably the location between certain genes, more preferably the same sequence background as in the original organism which is transformed.
  • expression or “gene expression” means the transcription of a specific gene or specific genes or specific genetic vector construct.
  • expression or “gene expression” in particular means the transcription of a gene or genes or genetic vector construct into structural RNA (rRNA, tRNA), or imRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the result- ing RNA product.
  • expression can also include the translation of the imRNA and therewith the synthesis of the encoded protein, i.e., protein expression.
  • deregulation or “modulation” has to be understood in its broadest sense, and comprises an increase or decrease or of complete switch off of gene expression or protein activity by different means well known to those in the art. Suitable methods comprise for example an increase or decrease of the copy number of gene and / or protein molecules in an organism, or the modification of another feature of the gene or protein affecting the protein activity and / or expression, which then results in the desired effect on the metabolic pathway at issue, in particular the glutamate biosynthetic pathway or any pathway or enzymatic reaction coupled thereto.
  • Suitable genetic manipulation can also include, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by removing strong promoters, inducible promoters or multiple promoters), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, decreasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and / or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene routine in the art (including but not limited to use of antisense nucleic acid molecules, or other methods to knock-out or block expression of the target protein).
  • modifying proteins e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like
  • the term "increased expression” or “enhanced expression” or “overexpression” or “increase in content” as used herein means any form of expression of a gene or gene product that is additional to the original wild-type expression level.
  • the original wild-type expression level might also be zero (absence of expression).
  • An increase in expression can preferably range from 1 -100% or even more increase in expression, preferably, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 10% or more increase.
  • the term “decreased expression” or “reduced expression” or “decrease in content” or “disrupted expression” as used herein means any form of expression of a gene or gene product that is reduced to the original wild-type expression level.
  • a decrease in expression can preferably range from 1 -100% decrease in expression with 100% decrease in expression being no expression, preferably, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease.
  • the term "increased activity” or "enhanced activity” as used herein means a protein having increased enzymatic activity as compared to the wild-type activity.
  • An increase in enzymatic activity can be due to an amendment of the amino acid sequence, a conformational change or the effect of an activating substance on the protein.
  • An increase in activity can preferably range from 1 -100% or even more increase in activity, preferably, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1 10% or more increase.
  • reduced activity or “decreased activity” or “disrupted activity” as used herein means a protein having reduced enzymatic activity as compared to the wild-type activity.
  • An decrease in enzymatic activity can be due to an amendment of the amino acid sequence, a conformational change or the effect of an inhibitory substance on the protein.
  • a decrease in activity can preferably range from 1 -100% decrease in activity with 100% decrease in activity being no activity, preferably, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease.
  • splice variant encompasses variants of a nucleic acid sequence in which selected introns and / or exons or parts thereof have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened.
  • a splice variant can have one or more or even all introns removed or added or partially removed or partially added.
  • a cDNA is considered as a splice variant of the respective intron-containing genomic sequence and vice versa.
  • Such splice variants may be found in nature or may be manmade. Methods for predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex (2005) BMC Bioinfor- matics 6: 25).
  • introduction or “transformation” as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
  • the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome.
  • the host genome includes the chromosomal nucleic acid as well as extra-chromosomal nucleic acid.
  • GABA gamma-aminobutyric acid
  • gamma- aminobutyrate gamma- aminobutyrate
  • 4-aminobutanoat gamma-aminobutyric acid
  • GABA product as obtained according to the present invention may be in the form of the free acid, in the form of a partial or complete salt of said acid and base func- tional groups or in the form of mixtures of the non-charged acid and any of its salt or mixtures.
  • GABA salt comprises for example metal salts, as for example mono- or di-alkalimetal salts of GABA like mono-sodium di-sodium, mono-potassium and di-potassium salts as well as alkaline earth metal salts as for example the calcium or magnesium salts or the proto- nated form of GABA.
  • nucleic acid or protein refers to a nucleic acid or protein which differ from the respective nucleic acid or protein as defined herein, but still provides the same or similar functional activity.
  • similar functional activity means at least 50%, at least 60%, at least 70%, at least 80 %, at least 90 %, at least 95%, at least 98 %, or at least 99% of the respective nucleic acid or protein described herein.
  • the "similar functional activity” of a “functional equivalent” of a nucleic acid or protein can also be higher than the respective nucleic acid or protein described herein.
  • “functional equivalents” of an enzyme means enzymes, which, in a test used for enzymatic activity, display at least 50%, at least 60%, at least 70%, at least 80 %, at least 90 %, at least 95%, at least 98 %, or at least 99%of the activity of an enzyme, as defined herein.
  • “Functional equivalents” are in particu- lar mutants or fragments of the nucleotide or amino acid sequence, but can also be glycosylate variants or other side chain variants or fusion proteins or homologs. Methods for generation and identification of such "functional equivalents" are known in the art.
  • the invention is based at least in part on the finding that a reduced or increased expression and / or activity of one or more genes of interest involved in the metabolism of GABA in a microorganism leads to an improved GABA yield.
  • the genes of interest are one or more genes selected from the group consisting of glutamate exporter, GABA- aminotransferase, succinate semialdehyde dehydrogenase, and glutamate dehydrogenase.
  • the microorganism produces glutamate and preferably also expresses an exogenous glutamate decarboxylase. Suitable nucleic acids and proteins are described below.
  • GABA glutamate decarboxylase
  • the glutamate decarboxylase is according to EC 4.1.1.15.
  • the GAD of the present invention is of prokaryotic or eukaryotic origin, preferably, of bacterial, animal or plant origin.
  • the glutamate decarboxylase described herein can also be named as L-glutamic acid decarboxylase, L-glutamic decarboxylase, cysteic acid decarboxylase, L-glutamate a- decarboxylase, aspartate 1 -decarboxylase, aspartic a-decarboxylase, L-aspartate-a- decarboxylase, ⁇ -glutamate decarboxylase, or L-glutamate 1 -carboxy-lyase.
  • said exogenous glutamate decarboxylase is a plant glutamate decarboxylase, for example from the Solanum genus, preferably Solanum tuberosum, i.e., potato, or Solanum lycopersicum, i.e., tomato.
  • said exogenous glutamate decarboxylase is a bacterial glutamate decarboxylase, for example from a bacterium of the genus Escherichia, in particular from E. coli, preferably, GadA, GadB, GadC, or GadBC complex, or from the genus Lactobacillus, preferably, Lactobacillus plantarum, preferably ATCC8014, or from the genus Mycobacterium, preferably, Mycobacterium smegmatis, preferably ATCC607.
  • said glutamate decarboxylase is a chimeric glutamate decarboxylase.
  • the GAD comprises one or more parts of the amino acid sequence from one species and one or more parts of the amino acid sequence from another species. In total, there may be 1 to 10, in particular, 1 to 5, preferably 1 or 2 amino acid sequence portions derived from another species.
  • the GAD comprises an N-terminal and / or a C-terminal portion from one species and one central portion from another species.
  • Each of said portions may have a length of 10 to 500, 10 to 450, 10 to 400, 20 to 350, 40 to 300, 50 to 250, 60 to 200, 70 to 150 or 80 to 100, preferably 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive amino acid residues of the GAD sequence.
  • the GAD is a chimeric glutamate decarboxylase comprising at least one amino acid sequence portion derived from plant glutamate decarboxylase.
  • Said "at least one ami- no acid sequence portion derived from plant glutamate decarboxylase” comprises at least ten consecutive amino acid residues of said plant enzyme. In total, there may be 1 to 10, in particular, 1 to 5, preferably 1 or 2 amino acid sequence portions derived from said plant sequence.
  • the GAD comprises an N-terminal and / or a C-terminal portion from one plant, preferably from tomato (Solanum lycopersicum), and one central portion from another plant, preferably potato (Solanum tuberosum).
  • the codon usage of the GAD gene is adapted to the codon usage of the host cell, preferably, to the codon usage of a corynebacterial cell.
  • said exogenous glutamate decarboxylase is from Solanum tuberosum and comprises an amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or a sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity to an amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2.
  • the N-terminal part of the GAD is encoded by position 193 to 471 of SEQ ID NO:1 or a nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity thereto.
  • the C-terminal part of the GAD is encoded by position 1201 to 1605 of SEQ ID NO:1 or a nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity thereto.
  • the central part of the GAD is encoded by position 472 to 1200 of SEQ ID NO:1 or a nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity thereto.
  • the GAD is encoded by position 193 to 1605 of SEQ ID NO: 1 or a nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity thereto.
  • the GAD nucleic acid is an isolated nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72, or a functional equivalent, homologue, or a splice variant thereof;
  • a nucleic acid encoding a GAD protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73, or functional equivalent, homologue, or a splice variant thereof; preferably the G
  • nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GAD protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 2, 4, 6, 8, 1 1 , or 13; preferably the encoded protein converts glutamate to GABA; and
  • nucleic acid encoding the same GAD protein as the GAD nucleic acids of (i) to (iii) above, but differing from the GAD nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
  • the GAD nucleic acid is an isolated nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 , or a functional equivalent, hom- ologue, or a splice variant thereof;
  • a nucleic acid encoding a GAD protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, or functional equivalent, homologue, or a splice variant thereof; preferably the GAD protein has essentially the same biological activity as a GAD protein encoded by SEQ ID NO: ; preferably the GAD protein converts glutaa G
  • nucleic acid encoding the same GAD protein as the GAD nucleic acids of (i) to (iii) above, but differing from the GAD nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
  • the GAD protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
  • an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least
  • the GAD protein converts glutamate to GABA.
  • the GAD protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
  • an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least
  • GAD nucleic acids and amino acid sequences described herein are useful in the methods, cells and genetic constructs of the invention.
  • a microorganism for the fermentative production of GABA a microorganism is preferably used, which is reduced in its expression and / or activity of a GABA-aminotransferase, preferably an endogenous GABA-aminotransferase.
  • the GABA aminotransferase is according to EC 2.6.1 .19.
  • the GABA aminotransferase of the present invention is endogenous to the microorganism used in the methods of the present invention.
  • the GABA aminotransferase is a GABA aminotransferase from corynebacteria, preferably from Corynebacterium glutamicum. More preferably, GABA aminotransferase is a GABA aminotransferase according to Genbank accession number NP_599724 / Ncgl0462, or a functional equivalent thereof.
  • the GABA aminotransferase to be reduced in its expression and / or activity in the present invention is also named as ⁇ -alanine-oxoglutarate transaminase, aminobutyrate aminotransferase (ambiguous), ⁇ -alanine aminotransferase, p-alanine-oxoglutarate aminotransferase, ⁇ -aminobutyrate aminotransaminase (ambiguous), ⁇ -aminobutyrate transaminase (ambiguous), ⁇ -aminobutyrate-a-ketoglutarate aminotransferase, ⁇ -aminobutyrate-a- ketoglutarate transaminase, y-aminobutyrate:a-oxoglutarate aminotransferase, ⁇ - aminobutyric acid aminotransferase (ambiguous), ⁇ -aminobutyric acid transaminase (am- biguous), ⁇ -aminobuty
  • the GABA-aminotransferase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 14, 16, 34-41 , 74, 76, 78, 80, 82, 84, 86 or 88, or a functional equivalent, homologue, or a splice variant thereof;
  • a nucleic acid encoding a GABA-aminotransferase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 15, 17, 75, 77, 79, 81 , 83, 85, 87, or 89, or functional equivalent, homologue, or a splice variant thereof; preferably the G
  • nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GABA-aminotransferase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 15 or 17; preferably the encoded protein converts GABA to succinate semialdehyde; and
  • the GABA-aminotransferase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 16 or 14, preferably SEQ ID NO: 16, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid encoding a GABA-aminotransferase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least
  • the GABA- aminotransferase protein has essentially the same biological activity as a GABA- aminotransferase protein encoded by SEQ ID NO: 16 or 14, preferably SEQ ID NO:
  • GABA-aminotransferase protein converts GABA to succinate semi- aldehyde
  • nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GABA-aminotransferase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 17 or 15, preferably SEQ ID NO: 17; preferably the encoded protein converts GABA to succinate semialdehyde; and
  • nucleic acid encoding the same GABA-aminotransferase protein as the GABA- aminotransferase nucleic acids of (i) to (iii) above, but differing from the GABA- aminotransferase nucleic acids of (i) to (iii) above due to the degeneracy of the genet- ic code.
  • the GABA-aminotransferase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 14, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid encoding a GABA-aminotransferase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least
  • the GABA-aminotransferase protein has essentially the same biological activity as a GABA-aminotransferase protein encoded by SEQ ID NO: 14, preferably the GABA-aminotransferase protein converts GABA to succinate semialdehyde;
  • nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GABA-aminotransferase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 15, preferably the encoded protein converts GABA to succinate semialdehyde; and
  • nucleic acid encoding the same GABA-aminotransferase protein as the GABA- aminotransferase nucleic acids of (i) to (iii) above, but differing from the GABA- aminotransferase nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
  • the GABA-aminotransferase protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
  • GABA-aminotransferase nucleic acids and amino acid sequences described herein are useful in the methods, cells and genetic constructs of the invention.
  • a microorganism for the fermentative production of GABA a microorganism can preferably be used, which is reduced in its expression and / or activity of a glutamate exporter, preferably an endogenous glutamate exporter.
  • the glutamate exporter exports glutamate from the inside to the outside of the cell.
  • the glutamate exporter is a glutamate exporter from corynebacteria, prefer- ably from Corynebacterium glutamicum. More preferably, glutamate exporter is a glutamate exporter according to Genbank accession number NP_600492; NCgl 1221 , or a functional equivalent thereof.
  • the glutamate exporter nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof; (ii) a nucleic acid encoding a glutamate exporter protein
  • the glutamate exporter protein has essentially the same biological activity as a glutamate exporter protein encoded by SEQ ID NO: 18 or 20; preferably the glutamate exporter protein exports glutamate out of the cell;
  • nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a glutamate exporter protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 19 or 21 ; preferably the encoded protein exports glutamate out of the cell; and
  • glutamate exporter nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 20 or 18, preferably SEQ ID NO: 20, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid encoding a glutamate exporter protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least
  • the glutamate exporter protein has essentially the same biological activity as a glutamate exporter protein encoded by SEQ ID NO: 20 or 18, preferably SEQ ID NO: 20; preferably the glutamate exporter protein exports glutamate out of the cell;
  • nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a glutamate exporter protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the pol- ypeptide described in SEQ ID NO: 21 or 19, preferably SEQ ID NO: 21 ; preferably the encoded protein exports glutamate out of the cell; and
  • nucleic acid encoding the same glutamate exporter protein as the glutamate exporter nucleic acids of (i) to (iii) above, but differing from glutamate exporter nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
  • the glutamate exporter protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
  • nucleic acid having in increasing order of pref- erence at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least
  • glutamate exporter protein exports glutamate out of the cell.
  • the glutamate exporter nucleic acids and amino acid sequences described herein are useful in the methods, cells and genetic constructs of the invention. Succinate semialdehyde dehydrogenase nucleic acids and proteins
  • a microorganism for the fermentative production of GABA a microorganism can preferably be used, which is reduced in its expression and / or activity of a succinate semialdehyde dehydrogenase, preferably an endogenous succinate semialdehyde dehydrogenase.
  • the succinate semialdehyde dehydrogenase is a succinate semialdehyde dehydrogenase from corynebacteria, preferably from Corynebacterium glutamicum.
  • the succinate semialdehyde dehydrogenase is according to EC 1.2.1.24.
  • the succinate semialdehyde dehydrogenase is also named as succinate semialdehyde dehydrogenase (NAD + ), succinic semialdehyde dehydrogenase (NAD + ), succinyl semialdehyde dehydrogenase (NAD + ), succinate semialdehyde: NAD + oxidoreductase, or succinate- semialdehyde:NAD + oxidoreductase.
  • the succinate semialdehyde dehydrogenase nucleic acid is a nucleic acid mole- cule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 22, 24, 50-56, or 57, or a functional equivalent, homologue, or a splice variant thereof;
  • a nucleic acid encoding a succinate semialdehyde dehydrogenase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 23 or 25, or functional equivalent, homologue, or a splice variant thereof; preferably the succinate semialdehyde dehydrogenase protein has essentially the same biological
  • the succinate semialdehyde dehydrogenase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 22 or 24, preferably SEQ ID NO: 26, or a functional equivalent, homologue, or a splice variant thereof;
  • a nucleic acid encoding a succinate semialdehyde dehydrogenase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 25 or 23, preferably SEQ ID NO: 25, or functional equivalent, homologue, or a splice variant thereof; preferably the succinate semialdehyde dehydrogenas
  • nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a succinate semialdehyde dehydrogenase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 25 or 23, preferably SEQ ID NO: 25; preferably succinate semialdehyde dehydrogenase protein converts sue- cinate semialdehyde to succinate; and
  • the succinate semialdehyde dehydrogenase protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
  • an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least
  • succinate semialdehyde dehydrogenase nucleic acids and amino acid sequences described herein are useful in the methods, cells and genetic constructs of the invention. Glutamate dehydrogenase nucleic acids and proteins
  • a microorganism for the fermentative production of GABA a microorganism can preferably be used, which is increased in its expression and / or activity of a glutamate dehydrogenase, preferably by means of a recombinant exogenous glutamate dehydrogenase.
  • the glutamate dehydrogenase is a glutamate dehydrogenase from corynebacte- ria, preferably from Corynebacterium glutamicum.
  • the glutamate dehydrogenase is according to EC 1 .4.1 .3.
  • the glutamate dehydrogenase is also named as glutamic dehydrogenase, L- glutamate:NAD(P) + oxidoreductase (deaminating) or glutamate dehydrogenase [NAD(P)].
  • the glutamate dehydrogenase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
  • nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 1 1 , or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid encoding a glutamate dehydrogenase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least
  • the glutamate dehydrogenase protein has essentially the same biological activity as a glutamate dehydrogenase protein encoded by SEQ ID NO: 1 1 1 ; preferably the glutamate dehydrogenase protein converts L- glutamate to 2-oxoglutarate;
  • nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a glutamate dehydrogenase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 1 12; preferably the glutamate dehydro- genase protein converts L-glutamate to 2-oxoglutarate; and
  • nucleic acid encoding the same glutamate dehydrogenase protein as the glutamate dehydrogenase nucleic acids of (i) to (iii) above, but differing from glutamate dehydrogenase nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
  • the glutamate dehydrogenase protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of: (i) an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 12, or a functional equivalent, homologue, or a splice variant thereof; preferably the glutamate dehydrogenase protein has
  • an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
  • nucleic acid sequence represented by SEQ ID NO: 1 1 1 or a functional equivalent, homologue, or a splice variant thereof; preferably the glutamate dehydrogenase protein converts L-glutamate to 2-oxoglutarate.
  • the invention in particular relates to methods for the fermentative production of GABA.
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism and wherein the recombinant microorganism has a reduced or increased expression and / or activity of one or more genes of interest involved in the metabolism of GABA in the microorganism as compared to a microorganism that has not the reduced or increased expression and / or activity of the one or more genes of interest involved in the metabolism of GABA (i.e. wild- type microorganism).
  • a microorganism for the fermentative production of GABA a microorganism can preferably be used, which expresses an exogenous glutamate decarboxylase (GAD, preferably, EC 4.1.1.15).
  • GABA exogenous glutamate decarboxylase
  • the invention is preferably directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate produced by the recombinant microorganism is converted to GABA, and wherein the recombinant microorganism has a reduced or increased expression of one or more genes of interest involved in the metabolism of GABA in the microorganism and / or a reduced or increased activity of the protein encoded by said gene of interest (i.e., protein of interest).
  • the microorganism has no endogenous expression of an enzyme having a glutamate decarboxylase activity.
  • the microorganism is not an Escherichia coli cell.
  • the microorganism used in the method of the invention is recombinant due to a man made reduced or increased expression of one or more genes of interest involved in the metabolism of GABA in the microorganism and / or a reduced or increased activity of the protein encoded by said gene of interest (i.e., protein of interest), preferably by genetechnological methods (i.e., the reduction or the increase is genetically engineered), compared to the wild-type level of gene expression and/or protein activity, i.e., the modification of the microorganism is not of natural origin, and the microorganism can preferably be discriminated from the wild-type or natural variants of the wild-type by the presence of exogenous DNA sequences introduced into the nucleic acid (genomic or plasmid nucleic acid) of the host microorganis
  • the expression and/or activity is preferably reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even and preferably 100%, i.e., no detectable expression.
  • the gene of interest is preferably selected from the group consisting of a gene encoding for a gene encoding a glutamate exporter, a GABA-aminotransferase (preferably, EC 2.6.1.19), a gene encoding a succinate semialdehyde dehydrogenase (preferably, EC 1 .2.1.24), and a gene encoding a glutamate dehydrogenase (preferably, EC 1 .4.1 .3), as described above in more detail.
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins selected from the group consisting of a glutamate exporter, a GABA-aminotransferase (preferably, EC 2.6.1 .19), and a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24) and wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3).
  • a glutamate exporter preferably, a GABA-aminotransferase (preferably, EC 2.6.1 .19), and a
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- Jerusalem glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1 .19), a glutamate exporter, and a succinate semialdehyde dehydrogenase (preferably, EC
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- Jerusalem glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1 .19) and a glutamate exporter.
  • a GABA-aminotransferase preferably, EC 2.6.1 .19
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant micro- organism has a genetically engineered reduced expression and / or activity of a glutamate exporter compared to the wild-type.
  • a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA
  • an exogenous glutamate decarboxylase preferably, EC 4.1.1.15
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3).
  • a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3).
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins of interest se- lected from the group consisting of an endogenous glutamate exporter, an endogenous GABA-aminotransferase (preferably, EC 2.6.1 .19), and an endogenous succinate semialdehyde dehydrogenase (preferably, EC 1 .2.1 .24).
  • the recombinant microorganism has additionally or separately an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3).
  • Preferred is in addition to a reduction of the glutamate exporter expression a reduction of the GABA-aminotransferase expression and / or activity and / or an increase of the glutamate dehydrogenase (preferably, EC 1.4.1.3) expression and / or activity.
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins of interest selected from an endogenous GABA-aminotransferase (preferably, EC 2.6.1.19), an endoge- nous glutamate exporter, and an endogenous succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24).
  • an endogenous GABA-aminotransferase preferably, EC 2.6.1.19
  • an endoge- nous glutamate exporter preferably, EC 1.2.1.24
  • an endogenous succinate semialdehyde dehydrogenase preferably, EC 1.2.1.24
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- Jerusalem glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter.
  • a recombinant microorganism which pro- Jerusalem glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter.
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a GABA-aminotransferase (preferably, EC 2.6.1 .19).
  • a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a GABA-aminotransferase (preferably, EC 2.6.1 .19).
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter.
  • the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3), preferably by expression of an exogenous coding sequence under the control of an appropriate promoter (preferably, promoter CJ4 or Sod), preferably in a recombinant expression cassette.
  • a glutamate dehydrogenase preferably, EC 1.4.1.3
  • an appropriate promoter preferably, promoter CJ4 or Sod
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant micro- organism has a reduced expression and / or activity of a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24).
  • a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant micro- organism has a reduced expression and / or activity of a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24).
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter, a GABA- aminotransferase (preferably, EC 2.6.1.19), and a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24).
  • a glutamate exporter preferably, EC 2.6.1.19
  • succinate semialdehyde dehydrogenase preferably, EC 1.2.1.24
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter, a GABA- aminotransferase (preferably, EC 2.6.1.19), and a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24).
  • a glutamate exporter preferably, EC 2.6.1.19
  • succinate semialdehyde dehydrogenase preferably, EC 1.2.1.24
  • the recombinant microorganism has additionally or separately an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3), preferably by expression of an exogenous coding sequence under the control of an appropriate promoter (preferably, promoter CJ4 or Sod), preferably in a recombinant expression cassette.
  • a glutamate dehydrogenase preferably, EC 1.4.1.3
  • an exogenous coding sequence under the control of an appropriate promoter (preferably, promoter CJ4 or Sod), preferably in a recombinant expression cassette.
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- Jerusalem glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter and a GABA- aminotransferase (preferably, EC 2.6.1.19).
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a GABA-aminotransferase (prefera- bly, EC 2.6.1 .19) and a succinate semialdehyde dehydrogenase (preferably, EC 1 .2.1 .24).
  • a GABA-aminotransferase prefera- bly, EC 2.6.1 .19
  • succinate semialdehyde dehydrogenase preferably, EC 1 .2.1 .24.
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter and a succin- ate semialdehyde dehydrogenase (preferably, EC 1 .2.1.24).
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- Jerusalem glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more of an endogenous protein of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1.19), a glutamate exporter, and a succinate semialdehyde dehydrogenase (prefer- ably, EC 1.2.1.24).
  • a GABA-aminotransferase preferably, EC 2.6.1.19
  • glutamate exporter a glutamate exporter
  • succinate semialdehyde dehydrogenase prefer- ably, EC 1.2.1.24.
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more of an endogenous protein of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1.19), a glutamate exporter, and a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24) and wherein the recombinant microorganism has an increased expres- sion and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3).
  • a GABA-aminotransferase preferably, EC 2.6.1.19
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of an endogenous protein of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1.19), a glutamate exporter, and a succinate semialdehyde dehydrogenase (preferably, EC
  • a glutamate dehydrogenase preferably, EC 1.4.1.3
  • amino acid sequence and coding sequence of the GABA-aminotransferase, the glutamate exporter, the succinate semialdehyde dehydrogenase, and the glutamate dehydrogenase are preferably as described above in more detail.
  • the glutamate decarboxylase, the GABA-aminotransferase, the glutamate exporter, the succinate semialdehyde dehydrogenase, and the glutamate dehydrogenase protein comprises an amino acid sequence having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the sequence described herein in more detail, or a functional
  • nucleic acid having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the sequence described herein in more detail, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid encoding a protein having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the sequence described herein in more detail, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); or by
  • the glutamate decarboxylase has at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73
  • the GABA-aminotransferase has at least 70% sequence identity to SEQ I D NO: 15, 17, 75, 77, 79, 8 , 83, 85, 87, or 89
  • the glutamate exporter has at least 70% sequence identity to SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105
  • the succinate semialdehyde dehydrogenase has at least 70% sequence identity to SEQ ID NO: 23 or 25, and the glutamate dehydrogenase has at least 70% sequence identity to SEQ ID NO: 1 12.
  • the glutamate decarboxylase is encoded by a sequence with at least 70% sequence identity to SEQ I D NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72
  • the GABA-aminotransferase is encoded by a sequence with at least 70% sequence identity to SEQ ID NO: 14, 16, 34-41 , 74, 76, 78, 80, 82, 84, 86 or 88
  • the glutamate export- er is encoded by a sequence with at least 70% sequence identity to SEQ ID NO: 18, 20, 42- 49, 90, 92, 94, 96, 98, 100, 102, or 104
  • the succinate semialdehyde dehydrogenase is encoded by a sequence with at least 70% sequence identity to SEQ I D NO: 22, 24, 50-56, or 57
  • the glutamate decarboxylase is encoded by a sequence with at least 70% sequence identity to SEQ I
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, having at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more of an endogenous protein of interest selected from the group consisting of a GABA-aminotransferase (preferably, having at least 70% sequence identity to SEQ ID NO: 15, 17, 75, 77, 79,
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, having at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72), so that the glutamate is con- verted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more endogenous proteins of interest selected from the group consisting of a GABA-aminotransferase (preferably, having at least 70% sequence identity to SEQ ID NO: 15, 17, 75, 77, 79,
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, having at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72), so that the glutamate is con- verted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter (preferably, having at least 70% sequence identity to SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105; preferably encoded
  • recombinant microorganism of the method for the fermentative production of GABA further has a reduced expression and / or activity of an endogenous succinate semialdehyde dehydrogenase (preferably, having at least 70% sequence identity to SEQ ID NO: 23 or 25; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 22, 24, 50-56, or 57) and preferably wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, having at least 70% sequence identity to SEQ ID NO: 1 12; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 1 1 1 ).
  • an endogenous succinate semialdehyde dehydrogenase preferably, having at least 70% sequence identity to SEQ ID NO: 23 or 25; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 22, 24, 50-56, or 57
  • the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identi- ty to SEQ I D NO: 1 ), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more endogenous proteins of interest selected from the group consisting of a GABA-aminotransferase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 15 or 17; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 14
  • recombinant microorganism of the method for the fermentative production of GABA further has a reduced expression and / or activity of an endogenous succinate semialdehyde dehydrogenase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23 or 25; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 22 or 24) and preferably wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1 12; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ I D NO: 1 1 1 ).
  • an endogenous succinate semialdehyde dehydrogenase preferably, having at least 80%, at least 90%, or at least 9
  • isocitrate dehydrogenase icd, NCgl0634
  • the expression and / or activity of the isocitrate dehydrogenase is enhanced, the expression and / or activity of the phosphoenolpyruvate carboxylase (ppc, NCgl1523) is enhanced, the expression and / or activity of the 2-oxoglutarate dehydrogenase (odhA, NCgl1084) is enhanced or reduced, the expression and / or activity of the isocitrate lyase (aceA, NCgl2248) is enhanced or reduced, the expression and / or activity of the phosphoenolpyruvate carboxykinase (pck, NCgl2765) is enhanced or reduced and/or the expression and / or activity of the glutamine synthetase (glnA, NCgl2148) is enhanced or reduced. Enhancing or reducing the expression of a gene of interest or the activity of an encoded protein of interest in a host cell can be achieved by various methods well known in the art.
  • Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of an endogenous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the protein of interest.
  • endogenous promoters may be altered in vivo by mutation, deletion, and / or substitution (see, Kmiec, US 5,565,350; Zarling et al., W09322443) or isolated promoters may be introduced into a cell in the proper orientation and distance from a gene so as to control the expression of the gene.
  • Preferred promoters for gene expressing according to the present invention are the CJ4 promoter, preferably as in pCES208, or the pSod promoter, preferably as in pClick5aMCS.
  • Reducing the expression of a gene of interest or the activity of an encoded protein of interest in a host cell can be achieved by one or more methods selected from the group consisting of:
  • dsRNA double-strand ribonucleic acid molecule
  • a reduced expression of a gene of interest and/or the encoded protein can be achieved for instance by introducing into the gene of interest of the host cell one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the gene of interest.
  • mutants means the modification of the nucleic acid sequence of a nucleic acid sequence. Mutations can arise for example as the result of errors in the replication, or they can be caused by mutagens, or by means of genetechnolo- gy. While the spontaneous mutation rate in the cell genome of organisms is very low, the skilled worker is familiar with a multiplicity of biological, chemical or physical mutagens. Mutations comprise substitutions, additions, or deletions of one or more nucleic acid residues.
  • substitutions are understood as meaning the exchange of individual nucleic acid bases; one distinguishes between transitions (substitution of a purine base for a purine base, or of a pyrimidine base for a pyrimidine base) and transversions (substitution of a pyrimidine base for a purine base (or vice versa)).
  • Additions or insertions are understood as meaning the incorporation of additional nucleic acid residues into the DNA, it being possible to result in reading-frame shifts.
  • reading-frame shifts one distinguishes between "in-frame” insertions/additions and "out-of-frame” insertions.
  • the reading frame is retained, and a polypeptide which is enlarged by the number of the amino acids encoded by the inserted nucleic acids results.
  • out-of-frame inser- tions/additions, the original reading frame is lost, and the formation of a complete and functional polypeptide is no longer possible.
  • Deletions describe the loss of one or more base pairs, which likewise lead to "in-frame” or “out-of-frame” reading-frame shifts and the consequences which this entails regarding the formation of an intact protein.
  • mutations can be introduced into the endogenous gene of interest for generating a loss of function (e.g. generation of stop codons, reading-frame shifts, and mutations resulting in amino acid exchanges, which effect the protein activity). It is also possible to reduce, lessen or block the endogenous activity of the proteins of interest by methods known to the skilled worker, for example by mutating a the coding region for the active center, for binding sites, for localization signals, for domains, clusters and the like. The activity can also be reduced in accordance with the invention by mutations, which affect the secondary, tertiary or quaternary structure of the protein.
  • At least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, or more nucleotides can be deleted, added or mutated.
  • the start codon of a gene of interest can be deleted or changed, e.g., from ATG to GTG, CTG, or TTG, to achieve a reduction of the transcription of the gene of interest.
  • the order of the above mentioned alternative start codons indicates a subsequent re- duction of the transcription of the gene of interest, preferably in corynebacteria, more preferably in Corynebacterium glutamicum.
  • Chemical mutagens can be distinguished by their mechanism of action.
  • base analogs for example, 5-bromouracil, 2-aminopurine
  • mono- and bifunctional alkylating agents for example monofunctional agents such as ethylmethylsulfonate, dimethyl sulfate, or bifunctional agents such as dichloroethyl sulfite, mitomycin, nitrosoguanidine- dialkylnitrosamine, N-nitrosoguanidine derivatives
  • intercalating substances for example acridine, ethidium bromide.
  • Mutations are preferably inserted by an EMS mutagenesis (Birchler JA, Schwartz D. Biochem Genet.
  • Ionizing radiation is, for example, gamma-radiation (photo energy of approximately one megaelectron volt MeV), X-rays (photo energy of a plurality of or many kiloelectron volts keV) or else ultraviolet light (UV light, photon energy of above 3.1 eV). UV light causes the formation of dimers between bases; with thymidine dimers, which give rise to mutations, being the most frequent here.
  • Genetechnological methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art.
  • techniques for making substitution mutations at predetermined sites in DNA include but are not limited to M 13 mutagenesis, T7-Gene in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols, use of transposons (for example Tn5, Tn903, Tn916, Tn1000, Balcells et al., 1991 , May BP et al.
  • Point mutations may also be generated by means of DNA-RNA hybrids also known as "chimeraplasty"
  • sequence-specific recombination systems may be used, examples which may be mentioned being Cre/lox system of bacteriophage P1 , the FLP/FRT system from yeast, the Gin recombinase of phage Mu, the Pin recombinase from E. coli and the R/RS system of the pSR1 plasmid.
  • homologous recombination i.e., at least part of the target sequence is exchanged with a sequence comprising a deletion of at least one nucleotide via homologous recombination, as described herein.
  • homologous recombination can also be combined with one or more sequence-specific recombination systems, e.g., cre/lox system.
  • the mutation sites may be specifically targeted or randomly selected. If the mutations have been created randomly e.g. by transposon tagging or chemical mutagenesis, the skilled worked is able to specifically enrich selected mutation events in the inventive nucleic acids.
  • Suitable domains as targets for mutagenesis can be identified by suitable computer programs such as, for example, SMART or InterPRO, for example as described in Andersen P., The Journal of Biol. Chemistry, 279, 38, pp. 40053-40061 , 2004, and literature cited therein.
  • a reduced expression of a gene of interest can also be achieved for instance by introducing into the gene of interest one or more exogenous nucleic acid sequences and thereby disrupting the expression of the gene of interest. This results into a nonfunctional protein either due to frame-shift mutations or due to misfolding of the protein by the incorporation of one or more amino acids.
  • the transcription of the gene of interest can be inhibited by the introduction of a complete endogenous or exogenous gene or a fragment thereof resulting in the production of either nonfunctional proteins or the production of the gene product of the introduced sequence.
  • Insertion of one or more exogenous nucleic acid sequences into the gene of interest can be achieved by introducing a nucleic acid construct suitable for inducing a homologous recombination on the gene of interest (Ohtsuka M, Kimura M, Tanaka M, Inoko H. (2009) Curr Pharm Biotechnol. 10(2):244-51 ; Hohn B and Puchta (1999) H Proc Natl Acad Sci USA 96:8321 -8323; "Handbook of Corynebacterium", Lothar Eggeling & Michael Bott, CRC
  • the insertion of one or more exogenous nucleic acid sequences into the gene of interest is combined with a deletion of at least parts of the nucleic acid sequence of the gene of interest, i.e., at least parts of the nucleic acid sequence of the gene of interest are exchanged by the one or more exogenous nucleic acid sequences.
  • a nucleic acid construct is used which, for example, comprises at least part of an endogenous gene which is modified by a deletion, addition or substitution of at least one nucleotide in such a way that the functionality is reduced or completely eliminated.
  • the modified region is flanked at its 5' and 3' end by fur- ther nucleic acid sequences, which must be sufficiently long for allowing recombination.
  • Their length is, as a rule, in a range of from one hundred bases up to several kilobases (Thomas KR and Capecchi MR (1987) Cell 51 : 503; Strepp et al. (1998) Proc. Natl. Acad. Sci. USA 95(8): 4368-4373).
  • the host organism is transformed with the recombination construct using the methods described herein and clones, which have successfully undergone recombination are selected using for example a resistance to antibiotics.
  • the expression and / or activity of one or more genes coding for a glutamate exporter, a GABA-aminotransferase, and succinate semialdehyde dehydrogenase is reduced by introducing into the coding sequence an insertion and / or deletion, preferably, by means of homologous recombination as described herein (e.g., Fig. 3 and 4), and thereby disruption gene expression and / or altering the protein activity.
  • this approach is combined with one or more sequence-specific recombination systems, e.g., cre/lox system.
  • the promoter of the gene of interest can be changed to a weak promoter sequence under production conditions.
  • the promoter can be inactivated by exchanging one or more nucleotides crucial for promoter activity.
  • the endogenous promoter can be exchanged to an inducible promoter.
  • the promoter can be inducible by, e.g., temperature, pH, salinity, or chemical compounds like antibiotics (e.g., tetracyclin), sugars or hormones.
  • Changing the promoter sequence can be achieved for instance by mutagenesis, e.g., site-directed muta- genesis, or homologous recombination.
  • Suitable promoters for use in coryneform bacteria are also described in "Handbook of Corynebacterium” (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005), in Patek M, et al., 2013, Microbial Biotech- nology, vol. 6, p. 103-1 17, or in Patek M and Nesvera J, Promoters and Plasmid Vectors of Corynebacterium glutamicum, in Yukawa H and Inui M, Corynebacterium glutamicum, Biology and Biotechnology, Microbiology Monographs, 2013, VII, ISBN: 978-3-642-29856-1 , p. 51 -88. (c) Removing the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of a gene of interest
  • the complete coding sequence of the gene of interest can be removed.
  • This type of modification can also relate to the regulatory elements (for example the promoter sequence, the terminator sequence and / or one or more enhancer sequences) of the gene of interest, so that the coding sequence remains unaltered, but that expression (transcription and / or translation) does not take place and is reduced. Removal of the respective sequence can be achieved for instance by homologous recombination, as described herein.
  • a multiplicity of sequence-specific recombination systems may be used, examples which may be mentioned being Cre/lox system of bacteriophage P1 , the FLP/FRT system from yeast, the Gin recombinase of phage Mu, the Pin recombinase from E. coli and the R RS system of the pSR1 plasmid.
  • dsRNA double-strand ribonucleic acid molecule
  • the expression of the gene of interest and/or the encoded protein of the host cell can also be reduced by mean of a dsRNA molecule.
  • double-stranded RNA interference double-stranded RNA interference
  • dsRNAi double-stranded RNA interference
  • dsRNAi double-stranded RNA interference
  • RNAi is also doc- umented as an advantageous tool for the repression of genes in bacteria such as E. coli for example by Tchurikov et al. (J. Biol. Chem., 2000, 275 (34): 26523-26529). Fire et al.
  • RNAi for RNA interference.
  • dsRNAi methods are based on the phenomenon that the simultaneous introduction of complementary strand and counter- strand of a gene transcript brings about highly effective suppression of the expression of the gene in question.
  • the resulting phenotype is very similar to that of an analogous knock-out mutant (Waterhouse PM et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959-64).
  • RNAi method was able to show that the efficiency of the RNAi method is a function of the length of the duplex, the length of the 3'-end overhangs, and the sequence in these overhangs. Based on the work of Tuschl et al. and assuming that the underlining principles are conserved between different species specific guidelines to design RNAi molecules have been developed (e.g., Dykxhoorn et al., 2003, Nature, p. 457-467).
  • the double-stranded structure can be formed starting from a single, self-complementary strand or starting from two complementary strands.
  • "sense” and “antisense” sequence can be linked by a linking sequence ("linker") and form for example a hairpin structure (cf., e.g., WO 99/53050).
  • the linking sequence may take the form of an intron, which is spliced out following dsRNA synthesis.
  • the nucleic acid sequence encoding a dsRNA may contain further elements such as, for example, tran- scription termination signals or polyadenylation signals.
  • the two strands of the dsRNA are to be combined in a cell, this can be brought about in a variety of ways: a) transformation of the cell or of the organism with a vector encompassing the two expression cassettes; b) cotransformation of the cell or of the organism with two vectors, one of which encompasses the expression cassettes with the "sense” strand while the other encompasses the expression cassettes with the "antisense” strand; c) supertransformation of the cell or of the organism with a vector encompassing the expression cassettes with the "sense” strand, after the cell or the organism had already been transformed with a vector encompassing the expression cassettes with the "antisense” strand or vice versa; and / or d) introduction of a con- struct comprising two promoters that lead to transcription of the desired sequence from both directions.
  • the expression cassettes encoding the "antisense” or the "sense” strand of the dsRNA or the self-complementary strand of the dsRNA are preferably inserted into a vector and stably inserted into the genome of the host cell using the methods described herein in order to ensure permanent expression of the dsRNA. Transient expression with can also be useful.
  • the dsRNA is preferably expressed under the control of a promoter having all its cis-acting elements upstream of the sequence coding for the dsRNA (e.g., a DNA dependent RNA polymerase III type 3 promoter).
  • the length of the dsRNA can either be substantially similar to the length of the sequence of the gene of interest or can be a fragment thereof.
  • the double-strand ribonucleic acid molecule comprises a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
  • nucleic acid molecule having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the gene of interest;
  • nucleic acid molecule encoding a protein having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least
  • nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii);
  • the sense strand comprises a fragment of at least 17 nucleotides of the nucleic acid molecule of (i) or (ii), preferably, wherein sense and antisense strand are expressed as one self-complementary RNA molecule, wherein sense and antisense strand are separated by a linker sequence and thus, the self-complementary RNA molecule is capable of forming a hairpin structure.
  • the length of the fragment of the sense strand in the dsRNA molecule is at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, preferably at least 21 consecutive nucleotides, preferably 18-24 nucleotides, more preferably 21 -23 nucleotides, or more, for example approximately 25 nucleotides, or approximately 50 nucleotides, approximately 100 nucleotides, approximately 200 nucleotides or approximately 300 nucleotides.
  • the fragment of the sense strand in the dsRNA molecule has at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least
  • an antisense ribonucleic acid mole- cule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the gene of interest or a fragment thereof
  • the expression of the gene of interest and/or the encoded protein of the host cell can also be reduced by mean of an antisense RNA molecule.
  • antisense nucleic acid molecule hybridizes with, or binds to, the cellular mRNA and / or the genomic DNA encoding the target protein to be suppressed. This process sup- presses the transcription and/or translation of the target protein. Hybridization can be brought about in the conventional manner via the formation of a stable duplex or, in the case of genomic DNA, by the antisense nucleic acid molecule binding to the duplex of the genomic DNA by specific interaction in the large groove of the DNA helix.
  • an “antisense” nucleic acid molecule comprises a nucleotide sequence, which is at least in part complementary to a "sense" nucleic acid molecule encoding a protein, e.g., comple- mentary to the coding strand of a double-stranded DNA molecule or complementary to an encoding mRNA sequence. Accordingly, an antisense nucleic acid molecule can bind via hydrogen bonds to a sense nucleic acid molecule.
  • the antisense nucleic acid molecule can be complementary to an entire coding strand of a nucleic acid molecule conferring the ex- pression of the polypeptide of the invention or to only a portion thereof.
  • an antisense nucleic acid molecule can be antisense to a coding region of the coding strand of a nucleotide sequence of a nucleic acid molecule of the present invention or to a non-coding region.
  • the oligonucleotide can be complementary to the nucleic acid region, which encompasses the translation start for the protein.
  • the anti- sense nucleic acid molecule is antisense to a non-coding region of the mRNA flanking the coding region of a nucleotide sequence.
  • the non-coding region is preferably in the area of 50 bp, 100 bp, 200 bp or 300 bp, preferably 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp or 1000 bp up- and / or downstream from the coding region.
  • the non- coding region refers to 5' or 3' sequences which flank the coding region that are not trans- lated into a polypeptide, i.e., also referred to as 5' and 3' untranslated regions (5 ' -UTR or 3 ' -UTR).
  • the antisense ribonucleic acid molecule comprises a nucleic acid sequence complementary to the nucleic acid sequence of a nucleic acid molecule selected from (i) a nucleic acid molecule having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a gene of interest;
  • nucleic acid molecule encoding a protein having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least
  • nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii);
  • the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii).
  • sRNAs small RNAs
  • Storz G et al.
  • 201 1 Regulation by Small RNAs in Bacteria: Expanding Frontiers, Mol Cell, vol. 43(6): 880-891
  • inhibitory RNA molecules suitable for the present invention can be derived therefrom.
  • the sRNAs are designed to base pair at or near the ribosome binding site (RBS) of their targets and block translation by occluding ribosomes.
  • the sRNAs base may be designed to pair at more distant locations and thus interfere with ribosome binding by other mechanisms, or decrease mRNA stability.
  • the antisense nucleic acid sequence can be complementary to all of the transcribed mRNA of the protein; it may be limited to the coding region, or it may only consist of a fragment of the complementary sequence of the gene of interest, preferably of the coding region of the gene of interest or of the nucleotide sequence of the respective ribosome binding site (RBS).
  • RBS ribosome binding site
  • Antisense nucleic acid or sRNA sequences may thus have an advantageous length, for example, of at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, or at least 24 nucleotides, preferably at least 21 , preferably 18-24 nucleotides, more preferably 21 -23 nucleotides, or more, for example at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 nucleotides but they may also be longer and encompass at least 100, at least 200, at least 500, at least 1000, at least 2000 or at least 5000 nucleotides.
  • antisense nucleic acid molecules of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense molecule comprises or consists of a nucleotide sequence having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the reverse complementary sequence of the nucleotide sequence coding for a
  • the antisense molecule comprises or consists of a nucleotide sequence having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the reverse complementary sequence of a fragment of the nucleotide sequence coding for a glutamate exporter, a GABA-aminotransferase, or a succinate semialdehyde dehydrogenase of the nucleotide sequence of the respective ribosome binding
  • Antisense nucleic acid sequences can be expressed recombinantly or synthesized chemically or enzymatically using methods known to the skilled worker. Antisense nucleic acid sequences can be expressed transiently or stably integrated in the genome of the cell.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid molecule of interest, described further in the following subsection).
  • the expression of the gene of interest and / or the encoded protein of the host cell can be reduced by ribozyme which specifically cleaves ribonucleic acid molecules derived from the gene of interest.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity, which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. In this manner, ribozymes (for example "Hammerhead” ribozymes;
  • Haselhoff and Gerlach (1988) Nature 33410: 585-591 ) can be used to catalytically cleave the mRNA of a protein of interest to be suppressed and to prevent translation.
  • Catalytic RNA molecules or ribozymes can be adapted to any target RNA and cleave the diester backbone at specific positions, thus functionally deactivating the target RNA (Tanner NK (1999) FEMS Microbiol. Rev. 23(3): 257-275).
  • the ribozyme per se is not modified thereby, but is capable of cleaving further target RNA molecules in an analogous manner, thus ac- quiring the properties of an enzyme.
  • Methods for expressing ribozymes for reducing specific proteins are described in EP0291533, EP0321201 , and EP0360257.
  • Suitable target sequences and ribozymes can be identified for example as described by Steinecke P, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds, Academic Press, Inc. (1995), pp. 449-460 by calculating the secondary structures of ribozyme RNA and target RNA and by their interaction (Bayley CC et al. (1992) Plant Mol. Biol. 18(2): 353-361 ; Lloyd AM and Davis RW et al. (1994) Mol. Gen. Genet. 242(6): 653-657).
  • derivatives of the tetrahymena L-19 IVS RNA which have complementary regions to the mRNA of the protein to be suppressed can be constructed (see also US 4,987,071 and US 5, 1 16,742).
  • ribozymes can also be identified from a library of a variety of ribozymes via a selection process (Bartel D and Szostak JW (1993) Science 261 : 141 1 -1418).
  • the antisense nucleic acid molecule of the invention can also be a ribozyme.
  • the incorporation of ribozyme sequences into "antisense” RNAs imparts this en- zyme-like RNA-cleaving property to precisely these "antisense” RNAs and thus increases their efficiency when inactivating the target RNA.
  • the preparation and the use of suitable ribozyme "antisense” RNA molecules is described, for example, by Haseloff et al. (1988) Nature 33410: 585-591 .
  • the function or activity of a protein of a host cell can efficiently also be reduced by express- ing a dominant-negative variant of said protein.
  • the skilled worker is familiar with methods for reducing the function or activity of a protein by means of coexpression of its dominant- negative form (Lagna G and Hemmati-Brivanlou A (1998) Current Topics in Developmental Biology 36: 75-98; Perlmutter RM and Alberola-lla J (1996) Current Opinion in Immunology 8(2): 285-90; Sheppard D (1994) American Journal of Respiratory Cell & Molecular Biology 1 1 (1 ): 1 -6; Herskowitz I (1987) Nature 329 (6136): 219-22).
  • a dominant-negative protein variant can be accomplished for example by altering amino acid residues which are part of protein of interest and, as the result of their mutation the protein loses its function.
  • Amino acid residues which are preferably to be mutated are those which are conserved in the homologous proteins of different organisms. Such conserved regions can be determined for example by means of sequence alignments.
  • These mutations for obtaining a dominant-negative protein variant are preferably carried out at the level of the nucleic acid sequence coding for the protein of interest.
  • a suitable mutation can be realized for example by PCR-mediated in vitro mutagenesis using suitable oligonucleotide primers, by means of which the desired mutation is introduced. Methods which are known to the skilled worker are described above.
  • the reduced expression of a gene of interest and / or the encoded protein and / or the activity of the protein of interest of a host cell can also be achieved by introducing a factor which binds to the protein of interest or the DNA or RNA molecule encoding the protein of interest.
  • a factor which binds to the protein of interest or the DNA or RNA molecule encoding the protein of interest are specific DNA-binding factors, for example factors of the zinc finger transcription factor type.
  • These factors attach to the genomic sequence of the endogenous target gene, preferably in the regulatory regions, and bring about repression of the endogenous gene of interest.
  • the use of such a method makes possible the reduction in the expression of an endogenous gene without it being necessary to recombinantly manipu- late the sequence of the latter.
  • Such methods for the preparation of relevant factors are described in Dreier B et al.
  • This segment is preferably located in the promoter region. For the purposes of gene suppression, however, it may also be located in the region of the coding exons or introns.
  • the skilled worker can obtain the relevant segments for instance from Genbank by database search or starting from a cDNA whose gene is not present in Genbank by screening a genomic library for corresponding genomic clones.
  • factors which are introduced into a cell may also be those which themselves inhibit the target protein.
  • the protein-binding factors can, for example, be aptamers (Famu- lok M and Mayer G (1999) Curr. Top Microbiol. Immunol. 243: 123-36) or antibodies or anti- body fragments or single-chain antibodies (e.g., scFv). Obtaining these factors has been described, and the skilled worker is familiar therewith.
  • Such compounds can be added to the culture media or can be expressed by the host cell, e.g., by genetic modification of the host cell in order to produce the respective inhibitory factor.
  • Gene expression may also be suppressed by tailor-made low-molecular-weight synthetic compounds, for example of the polyamide type (Dervan PB and Biirli RW (1999) Current Opinion in Chemical Biology 3: 688-693; Gottesfeld JM et al. (2000) Gene Expr. 9(1 -2): 77- 91 ).
  • These oligomers consist of the units 3-(dimethyl-amino)propylamine, N-methyl-3- hydroxypyrrole, N-methyl-imidazole and N-methyl-pyrroles; they can be adapted to each portion of double-stranded DNA in such a way that they bind sequence-specifically to the large groove and block the expression of the gene sequences located in this position.
  • the method for the fermentative production of gamma-aminobutyric acid comprises reducing the expression and / or activity of the glutamate exporter in a recombinant microorganism comprising an exogenous nucleic acid encoding a glutamate decarboxylase by one or more measures selected from the group consisting of (a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous glu- tamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glu- tamate exporter in the recombinant microorganism;
  • dsRNA double-strand ribonucleic acid molecule
  • nucleic acid molecule having at least 70% identity with SEQ ID NO: 18, 20, 42- 49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid molecule encoding a protein having at least 70% identity with SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii);
  • nucleic acid molecule encoding the same glutamate exporter as any of the nu- cleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code
  • the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
  • an antisense ribonucleic acid mole- cule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
  • the method of for the fermentative production of gamma-aminobutyric acid comprises the steps of
  • the method of the present invention comprises the steps of
  • dsRNA double-strand ribonucleic acid molecule
  • nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii);
  • nucleic acid molecule encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code, or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
  • an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a frag- ment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
  • the method for the fermentative production of gamma-aminobutyric acid further comprises reducing the expression and / or activity of a GABA- aminotransferase in a recombinant microorganism comprising an exogenous nucleic acid encoding a glutamate decarboxylase and comprising a reduced expression and / or activity of the glutamate exporter by one or more measures selected from the group consisting of (a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous GABA-aminotransferase genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the GABA-aminotransferase in the recombinant microorganism;
  • dsRNA double-strand ribonucleic acid molecule
  • nucleic acid molecule having at least 70% identity with SEQ ID NO: 14, 16, 34- 41 , 74, 76, 78, 80, 82, 84, 86 or 88, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and (iv) a nucleic acid molecule encoding the same GABA-aminotransferase as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code,
  • the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
  • an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
  • the method of the present invention comprising the steps of
  • the method of the present invention comprising the steps of
  • the method further comprises the step of reducing or enhancing the expression and / or activity of one or more genes selected from the group consisting of a gene encoding for a a succinate semialdehyde dehydrogenase, a isocitrate dehydrogenase, a phos- phoenolpyruvate carboxylase, a 2-oxoglutarate dehydrogenase, a isocitrate lyase, a phos- phoenol pyruvate carboxy kinase, a glutamine synthetase, and glutamate dehydrogenase, preferably, reducing a succinate semialdehyde dehydrogenase expression and / or activity, and / or enhancing a glutamate dehydrogenase expression and / or activity, by means of one or more methods described herein.
  • Preferred is in addition to a reduction of the glutamate exporter expression and / or activity a reduction of the GABA-aminotransferase expression and / or activity and / or an increase of the glutamate dehydrogenase expression and / or activity.
  • the recombinant microorganisms as used according to the invention can be cultured continuously or discontinuously in the batch process or in the fed batch or repeated fed batch process.
  • a review of known methods of cultivation will be found in the textbook by Chmiel (Bioprocesstechnik 1. EinfCihrung in die Biovonstechnik (Gustav Fischer Verlag, Stuttgart, 1991 )) or in the textbook by Storhas (Bioreaktoren und periphere bamboo (Vie- weg Verlag, Braunschweig/Wiesbaden, 1994)).
  • the culture medium that is to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the handbook “Manual of Methods for General Bacteriology” of the American Soci- ety for Bacteriology (Washington D. C, USA, 1981 ).
  • These media that can be used according to the invention generally comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and / or trace elements.
  • Preferred sources of carbon are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add mixtures of various sources of carbon.
  • oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid or linoleic acid, alcohols such as glycerol, methanol or ethanol and organic acids such as acetic acid or lactic acid.
  • Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials con- taining these compounds.
  • sources of nitrogen include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex sources of nitrogen, such as corn-steep liquor, soybean flour, soybean protein, yeast extract, meat extract and others.
  • the sources of nitrogen can be used separately or as a mixture.
  • Inorganic salt compounds that may be present in the media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Inorganic sulfur-containing compounds for example sulfates, sulfites, dithionites, tetrathi- onates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as sources of sulfur.
  • Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as sources of phosphorus.
  • Chelating agents can be added to the medium, in order to keep the metal ions in solution.
  • suitable chelating agents comprise dihydroxyphenols, such as catechol or proto- catechuate, or organic acids, such as citric acid.
  • the fermentation media used according to the invention may also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
  • Growth factors and salts often come from complex components of the media, such as yeast extract, molasses, corn-steep liquor and the like.
  • suitable precursors can be added to the culture medium.
  • the precise composition of the compounds in the medium is strongly dependent on the particu- lar experiment and must be decided individually for each specific case. Information on media optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Publ. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3).
  • Growing media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.
  • biotin is contained in the culture medium.
  • the culture media comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and / or trace elements and biotin.
  • Biotin is contained in the culture medium preferably in a concentration of at least 2 g/L, at least 5 ⁇ g L, at least 10 g/L, at least 20 ⁇ g/L, at least 50 ⁇ g/L, at least 100 ⁇ g/L, at least 200 g/L, at least 300 g/L, at least 400 g/L, at least 500 ⁇ g/L, preferably at least 300 pg/L, preferably at 100-300 g/L, preferably 200-300 ⁇ g/L.
  • All components of the medium are sterilized, either by heating (20 min at 1.5 bar and 121 °C) or by sterile filtration.
  • the components can be sterilized either together, or if necessary separately.
  • All the components of the medium can be present at the start of growing, or optionally can be added continuously or by batch feed.
  • the temperature of the culture is normally between 15 °C and 45 °C, preferably 25 °C to 40 °C, preferably 30°C or 31.5°C, and can be kept constant or can be varied during the experiment.
  • the pH value of the medium should be in the range from 5 to 8.5, preferably around 7.0, preferably pH 6.4, pH 6.5, pH 7.0 or pH 7.5.
  • the pH value for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid.
  • Antifoaming agents e.g. fatty acid polyglycol esters, can be used for controlling foaming.
  • suitable substances with selective action e.g. antibiotics
  • Oxygen or oxygen-containing gas mixtures e.g. the ambient air
  • Culture is continued until a maximum of the desired product has formed. This is normally achieved within 10 hours to 160 hours.
  • the methodology of the present invention can further include a step of recovering GABA from the culture media and / or the microbial cells.
  • the term "recovering” includes extracting, harvesting, isolating or purifying GABA.
  • the cells can be disrupted optionally by high-frequency ultrasound, by high pressure, e.g. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of several of the methods listed.
  • Recovering GABA from the culture media, the cells, or the disrupted cell fraction can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hex- ane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystalliza- tion, pH adjustment, lyophilization and the like.
  • a conventional resin e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.
  • a conventional adsorbent e.g., activated charcoal, silicic acid, silic
  • GABA can be recovered from culture media by first removing the microorganisms (e.g., by centrifugation). The remaining broth is then passed through or over a cation exchange resin to remove unwanted cations and then through or over an anion exchange resin to remove unwanted inorganic anions and organic acids.
  • a cation exchange resin to remove unwanted cations and then through or over an anion exchange resin to remove unwanted inorganic anions and organic acids.
  • the cells can be disrupted, the cellular debris removed (e.g., by centrifugation) and GABA can be isolated from the supernatant by any suitable method described above.
  • the cells are removed from the fermentation broth, preferably by centrifugation and / or filtration (preferably, using a 8kD-15kD membrane, preferably a 10 kD membrane), and GABA is recovered from the supernatant and / or permeate.
  • GABA can be extracted from the solution by ion exchange chromatography.
  • the cells are removed from the fermentation broth, preferably by centrifugation and / or filtration, and GABA is converted to 2-pyrrolidone in the supernatant and / or permeate by means of in-broth chemistry, preferably by heating the supernatant and / or permeate, preferably under increased pressure.
  • the GABA yield is preferably increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 90%, at least 100%, at least 1 10%, or even more.
  • the present invention is a recombinant prokaryotic or eukaryotic cell.
  • the recombinant prokaryotic or eukaryotic cell is a glutamate producing cell, preferably, without an endogenous expression of an enzyme having a glutamate decarboxylase activity.
  • the recombinant prokaryotic or eukaryotic cell is a cell of a microorganism.
  • the microorganism can include but is not limited to bacteria, yeast, fungus, or algae.
  • the recombinant prokaryotic cell is a recombinant bacteria, preferably, a recombinant coryneform bacteria.
  • the coryneform bacteria comprises aerobically growing, asporogenous, non-partially-acid-fast, irregularly shaped gram-positive rods.
  • a preferred coryneform bacteria is a Corynebacterium, preferably Corynebacterium glutami- cum.
  • glutamicum are Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum S91 14, Corynebacterium glutamicum ATCC 14067, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium acetoacidophi- lum ATCC 13870, Corynebacterium thermoaminogenes FERM BP-1539, Corynebacterium melassecola ATCC 17965, Corynebacterium glutamicum KFCC10065 and Corynebacte- rium glutamicum ATCC21608 (KFCC designates Korean Federation of Culture Collection, ATCC designates American type strain culture collection, FERM BP designates the collection of National institute of Bioscience and Human-Technology, Agency of Industrial Sci- ence and Technology, Japan).
  • Also preferred as a host cell are cells of the genus Brevibacterium, preferably Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC 14020.
  • Corynebacterium glutamicum strain ATCC 13032 or variants thereof, preferably Corynebacterium glutamicum HH09.
  • the variants of Corynebacterium glutamicum strain ATCC 13032 are preferably characterized by an increased glutamate production, preferably wherein the variants of Corynebacterium glutamicum strain ATCC 13032 are characterized by a ribosomal RNA sequence (rRNA) of the 16S, 23S and/or 5S rRNA, preferably the 16S rRNA, with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to the 16S, 23S and/or 5S rRNA, preferably the 16S rRNA, sequence of Corynebacterium glutamicum strain ATCC 13032.
  • rRNA ribosomal RNA sequence
  • the recombinant prokaryotic or eukaryotic cell preferably, a recombinant microorganism, is capable of producing GABA, preferably by converting glutamate to GABA.
  • a recombinant prokaryotic or eukaryotic cell capable of producing GABA is preferably obtained by modifying a suitable host cell.
  • a host cell a prokaryotic or eukaryotic cell is used that endogenously produces glutamate.
  • a host cell might be modified, preferably by genetechnological methods or by another method for genetic alteration (e.g.
  • the recombinant prokaryotic or eukaryotic cell has no endogenous expression of an enzyme having a glutamate decarboxylase activity.
  • the recombinant prokaryotic cell is not an Escherichia coli cell.
  • a prokaryotic or eukaryotic host cell capable of producing glutamate is transformed with one or more nucleic acid sequences cod- ing for a glutamate decarboxylase (preferably, EC 4.1.1.15) as described herein.
  • a glutamate decarboxylase preferably, EC 4.1.1.15
  • the recombinant prokaryotic or eukaryotic host cell is further modified by reducing the expression and / or activity of an endogenous glutamate exporter as described here- in and / or by reducing the expression and / or activity of an endogenous GABA- aminotransferase (preferably, EC 2.6.1.19) as described herein.
  • an endogenous GABA- aminotransferase preferably, EC 2.6.1.19
  • a preferred embodiment is a recombinant prokaryotic or eukaryotic cell capable of producing glutamate transformed with one or more nucleic acid sequences coding for a glutamate decarboxylase (preferably, EC 4.1.1.15) and further comprising one or more modifi- cations selected from
  • dsRNA double-strand ribonucleic acid molecule
  • nucleic acid molecule having at least 70% identity with SEQ ID NO: 18, 20, 42- 49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid molecule encoding a protein having at least 70% identity with SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii);
  • nucleic acid molecule encoding the same glutamate exporter as any of the nu- cleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code
  • the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
  • an antisense ribonucleic acid mole- cule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
  • the recombinant prokaryotic or eukaryotic cell additionally has one or more alterations selected from the group consisting of reduced expression and / or activity of a GABA-aminotransferase, reduced expression and / or activity of a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24) and increased expression of a glutamate dehy- drogenase (preferably, EC 1.4.1.3).
  • Preferred is in addition to a reduction of the glutamate exporter expression and / or activity a reduction of the GABA-aminotransferase expression and / or activity and / or an increase of the glutamate dehydrogenase expression and / or activity.
  • the present invention is a method for the production of a recom- binant microorganism, comprising the steps of
  • Preferred is a method for the production of a recombinant microorganism, comprising the steps of
  • dsRNA double-strand ribonucleic acid molecule
  • nucleic acid molecule having at least 70% identity with SEQ ID NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid molecule encoding a protein having at least 70% identity with SEQ I D NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, or a functional equivalent, homologue, or a splice variant thereof;
  • nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii);
  • nucleic acid molecule encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code, or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
  • an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
  • the method further comprises the step of reducing or enhancing in the microor- ganism the expression and / or activity of one or more genes selected from the group consisting of a gene coding for a GABA-aminotransferase, a succinate semialdehyde dehydrogenase, a isocitrate dehydrogenase, a phosphoenolpyruvate carboxylase, a 2-oxoglutarate dehydrogenase, a isocitrate lyase, a phosphoenolpyruvate carboxykinase, a glutamine synthetase, and glutamate dehydrogenase, preferably, reducing a glutamate exporter expres- sion and / or activity, and / or reducing a succinate semialdehyde dehydrogenase expression and / or activity, and / or enhancing a glutamate dehydrogenase expression and / or activity, by means
  • Preferred is in addition to a reduction of the glutamate exporter expression and / or activity a reduction of the GABA- aminotransferase expression and / or activity and / or an increase of the glutamate dehy- drogenase expression and / or activity.
  • the present invention provides an expression cassette, comprising at least one nucleic acid sequence as defined above, which sequence is operatively linked to at least one regulatory nucleic acid sequence; as well as a recombinant vector, comprising at least one such expression cassette.
  • nucleic acid sequences mentioned herein can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • Chemical synthesis of oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896- 897).
  • the accumulation of synthetic oligonucleotides and filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989).
  • Nucleic acid constructs according to the invention comprise in particular sequences selected from those specifically mentioned herein (e.g., overexpression constructs, antisense constructs, dsRNA constructs or homologous recombination constructs) and are preferably contained in a vector construct. Typically the nucleic acid constructs are functionally linked with one or more promoter sequence.
  • promoters are known in the art and may be introduced in an appropriate position (typically upstream) of nucleotide sequence so as to drive expression of a nucleic acid encoding the protein of interest. Promoters can be constitutive or non-constitutive. Non- constitutive promoters can be inducible. Preferred promoters for gene expressing according to the present invention are the CJ4 promoter, preferably as in pCES208, or the pSod promoter, preferably as in pClick5aMCS.
  • promoters for use in coryneform bacteria are also described in "Handbook of Corynebacterium” (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005), in Patek M, et al., 2013, Microbial Biotechnology, vol. 6, p. 103-1 17, or in Patek M and Nesvera J, Promoters and Plasmid Vectors of Corynebacterium glutamicum, in Yukawa H and Inui M, Corynebacterium glutamicum, Biology and Biotechnology, Microbiology Monographs, 2013, VI I, ISBN: 978-3- 642-29856-1 , p. 51 -88.
  • regulatory elements are targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, replication origins and the like. Suitable regulatory sequences are described for example in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the natural regulation of these sequences can still be present in front of the actual structural genes and optionally can have been altered genetically, so that natural regulation is switched off and the expression of the genes has been increased.
  • the nucleic acid construct can also be of a simpler design, i.e. without any additional regulatory signals being inserted in front of the coding sequence and without removing the natural promoter with its regulation.
  • a preferred nucleic acid construct advantageously also contains one or more of the aforementioned enhancer sequences, functionally associated with the promoter, which permit increased expression of the nucleic acid sequence. Additional advantageous sequences, such as other regulatory elements or terminators, can also be inserted at the 3' end of the DNA sequences.
  • One or more copies of the nucleic acids according to the invention can be contained in the construct.
  • the construct can also contain other markers, such as antibiotic resistances or auxotrophy-complementing genes, optionally for selection on the construct.
  • promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, ⁇ -lac-, lacl ⁇ T7-, T5-, T3-, gal-, trc-, ara-, rhaP (rhaP B AD)SP6-, lambda-PR- or in the lambda-PL promoter, which find application advantageously in Gram- negative bacteria.
  • promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, ⁇ -lac-, lacl ⁇ T7-, T5-, T3-, gal-, trc-, ara-, rhaP (rhaP B AD)SP6-, lambda-PR- or in the lambda-PL promoter, which find application advantageously in Gram- negative bacteria.
  • the nucleic acid construct is inserted in a host organism advantageously in a vector, for example a plasmid or a phage, which permits optimum expression of the genes in the host.
  • a vector for example a plasmid or a phage
  • vectors are also to be understood as meaning all other vectors known to a person skilled in the art, e.g. viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA.
  • viruses such as SV40, CMV, baculovirus and adenovirus
  • transposons e.g. viruses, such as SV40, CMV, baculovirus and adenovirus
  • transposons e.g. viruses, such as SV40, CMV, baculovirus and adenovirus
  • transposons e.g. viruses, such as SV40, CMV, baculovirus and
  • Suitable plasmids are, for example in corynebacteria, pCES208 or pClick5aMCS, in E. coli, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pi N-l 11 113 -B 1 , Agt1 1 or pBdCI ; in nocardioform actinomycetes pJAM2; in Streptomyces plJ 101 , pi J364, plJ702 or plJ361 ; in bacillus pUB1 10, pC194 or pBD214; in Corynebacterium pSA77 or pAJ667; in fungi pALS1 , plL2 or pBB1 16; in yeasts 2alpha
  • plasmids represent a small selection of the possible plasmids.
  • Other plasmids are well known to a person skilled in the art and will be found for example in the book “Cloning Vectors” (Eds. Pouwels P.H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).
  • the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can be inserted advantageously in the form of a linear DNA in the microorganisms and integrated into the genome of the host organism through heterologous or homologous recombination.
  • This linear DNA can comprise a linearized vector such as plasmid or just the nucleic acid construct or the nucleic acid according to the invention.
  • codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organism in question.
  • an expression cassette according to the invention is based on fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator signal or polyadenylation signal.
  • Common recombination and cloning techniques are used for this, as described for example in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) as well as in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al.,
  • Preferred vector constructs for overexpressing GAD in bacterial cells preferably coryneform bacteria are shown in Fig. 2 and Fig. 19.
  • the nucleotide sequence of the preferred vectors of Fig. 19 is shown in SEQ ID NO: 1 13-1 15.
  • the construct suitable for inducing homolo- gous recombination comprises a first nucleotide sequence comprising a fragment of the 3'- portion of the target sequence (preferably, at least 25, 50, 75, 100, 125, 150, 175, or at least 200 nucleotides in length) and a second nucleotide sequence comprising a fragment of the 5'-portion of the target sequence (preferably, at least 25, 50, 75, 100, 125, 150, 175, or at least 200 nucleotides in length) and a third nucleotide in between the first and the second sequence, wherein integration of the construct preferably via double crossing-over leads to a disruption of the expression of the target sequence, preferably because the third nucleotide sequence is integrated into the target sequence and differs from the target sequence by at least one nucleotide.
  • the third sequence comprises nucleotide sequence suitable to be excised via the cre-LoxP system.
  • the third sequence comprises a nucleotide sequence, preferably differing from the target sequence, flanked by two loxP sides.
  • the construct suitable for inducing homologous recombination is as shown in Fig. 3, wherein the target gene can be any gene of interest as described herein, preferably the GABA-aminotransferase and / or the glutamate exporter.
  • the vector construct comprise a 3'-portion of the GABA-aminotransferase coding sequence (preferably, at least 25, 50, 75, 100, 125, 150, 175, or at least 200 nucleotides in length) and a second nucleotide sequence comprising a fragment of the 5'-portion of the GABA-aminotransferase coding sequence (preferably, at least 25, 50, 75, 100, 125, 150, 175, or at least 200 nucleotides in length), wherein in between these two sequences is a third nucleotide sequence, preferably a kanamycin resistance gene.
  • the con- struct further comprises another antibiotics resistance gene, preferably an ampicillin resistance gene.
  • the vector construct comprises a nucleotide sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or even 100% sequence identity to SEQ I D NO: 1 16.
  • the fermentatively produced GABA may be applied for producing pyrrolidone by applying standard techniques of organic synthesis.
  • pyrrolidone can be synthesized by cyclization of GABA under removal of water.
  • the present invention relates to a method of preparing 2-pyrrolidone, which method comprises a) preparing GABA by a method as described herein; and
  • the method comprises
  • GABA is converted to 2-pyrrolidone by heating the fermentation broth after fer- mentation and removal of biomass.
  • Biomass can be removed preferably by means of cen- trifugation and preferably subsequent pasteurization and/or preferably subsequent ultrafil- tration.
  • the so processed fermentation broth can be concentrated prior the cyclization reaction.
  • prior cyclization GABA is purified from the fermentation broth, preferably by ion exchange chromatography, preferably by acidic ion exchange chromatography.
  • cyclization of said GABA to produce 2-pyrrolidone is achieved by heating the GABA solution, preferably the GABA containing processed fermentation broth. Heating is preferably at 170-300°C, preferably, at 200°C, at 210°C,at 220°C, preferably above 220°C, i.e., at 230°C, 240°C, 250°C, 260°C, 270°C, 280°C, 290°C, or 300°C, preferably between 220°C and 260°C, preferably between 200°C and 260°C, preferably between 200-240°C.
  • the heating time is between 5 min and 9 hours.
  • 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, or 60 min preferably 1.5 hours, 2 hours, 2.5 hours, or 3 hours, preferably 4 hours, 6 hours, 7 hours, 8 hours, or 9 hours.
  • the heating time is dependent on the temperature and the pressure applied to the cyclization reaction.
  • the cyclization reaction is performed at a pH between pH6 and pH 10, preferably between pH6 and pH9, more preferably between pH7 and pH9.
  • the cyclization reaction is performed at 200°C for 3 hours at pH7, pH8 or pH9 or at 240°C for 30 min at pH7, pH8 or pH9 or at 300°C for 10 min at pH 7, pH 8 or pH9.
  • the GABA concentration in the reaction solution is between 10-50%, preferably between 15-40%, preferably between 20-45%, preferably 30-40%, preferably 15% or pre- farably 40%.
  • the amount of sugars, preferably, glucose and/or molasses, in the GABA solution, preferably in the processed fermentation broth after fermentation reaction and removal of biomass is reduced, preferably is between 0-50% of their initial amount, preferably be- tween 10-50%, 10-20%, or 5-15%, preferably, 50%, 40%, 30%, 20%, 10%, 5%, or 0%.
  • salts and fermentation byproducts were removed or reduced in the processed fermentation broth after fermentation reaction and removal of biomass prior the cyclization reaction.
  • cyclization of said GABA to produce 2-pyrrolidone is achieved by removal of water, preferably by heating.
  • 2-pyrrolidone can be obtained from GABA solutions by heating and distillation under reduced pressure.
  • cyclization is carried out in a solvent that assists in the removal of water.
  • GABA solutions can be heated in a flask by using an oil bath (preferably, at 130°C-150°C) to remove the water.
  • the reaction is further heated, preferably 200°C-240°C, for 10-40min, preferably 20min, to proceed the cyclization reaction.
  • separation and purification of 2-pyrrolidone from byproducts and contaminants is achieved by distillation, preferably under reduced pressure (preferably, at 10-14 hPa at 1 10- 130°C, most preferably, at 12 hPa, 120°C).
  • 2-pyrrolidone can be obtained from GABA solutions, preferably from GABA in an aqueous solutions or in fermentation broth, preferably after removal of biomass, preferably by centrifugation, preferably with subsequent ultrafiltration, by heating with partial removal or without removal of water, preferably under increased pressure (preferably, 20-150 bar, preferably 50-150 bar, preferably 20-100 bar, more preferably 100 bar), preferably under temperature and pH conditions and for a time and with concentrations as described above.
  • water can be subsequently removed, preferably by distillation, or evaporation, preferably rotary evaporation.
  • 2-pyrrolidone can preferably be subsequently isolated by distillation. This can be done continuously or discontinuously.
  • prior cyclization GABA is purified from the fermentation broth, preferably by ion exchange chromatography, preferably by acidic ion exchange chromatography.
  • 2-pyrrolidone can be ob- tained by heating an aqueous GABA solution as described above, preferably at 180°C - 350°C, preferably at 200°C-250°C, preferably for 10 sec - 5h, preferably for 1 min - 40min, preferably for 10-40min, preferably 20min, preferably below 20min, preferably for 10sec- 5min (preferably, at 300°C for 30sec or 3 min or at 250°C for 5min or at 200°C for 30 min), preferably by heating the aqueous GABA containing fermentation broth after removal of the microbial cells, preferably under increased pressure.
  • cyclization of GABA to produce 2-pyrrolidone in the presence of water preferably a continuous laboratory reactor is used. Alternatively, cyclization can be done in a discontinuous reaction. Preferably, cyclization can be achieved by heating a GABA containing solution by means of a micro reactor, a microwave, or an autoclave.
  • the 2-pyrrolidone obtained from the GABA produced by a method of the present invention is further processed to venylpyrrolidone, preferably N- vinyl pyrrol idone (NVP).
  • NDP N- vinyl pyrrol idone
  • the 2-pyrrolidone obtained from the GABA produced by a method of the present invention is further processed to N-vinylpyrrolidone by reacting with acetylene, preferably by means of a Reppe synthesis.
  • vinylpyrrolidone can be polymerized to polyvinylpyrrolidone (PVP) or polyvinyl polypyrrol idone.
  • the present invention provides a process for the production of polymers, in particular polyamides, comprising a step as mentioned above for the production of GABA.
  • the GABA is preferably reacted in a known manner with itself or with at least one different co-monomer, selected from amino- and hydroxycarboxylic acids, by applying standard methods of polymer synthesis.
  • the present invention relates to a method of preparing a polymer, preferably a polyamide, which method comprises a) preparing GABA by a method as described herein; and b) polymerizing said GABA, optionally in the presence of at least one further suitable polyvalent copolymerizable co-monomer, selected, for example, from aminocarboxylic acids and hydroxycarboxylic acids.
  • the method comprises
  • polymerizing said GABA optionally in the presence of at least one further suitable polyvalent copolymerizable co-monomer, selected, for example, from aminocarboxylic acids and hydroxycarboxylic acids.
  • Suitable co-monomers are for example derived from C2-C31 , preferably C 4 -Cio-straight or branched chain monocarboxylic acids, carrying at least one reactive hydroxyl or amino group.
  • Such hydroxyl- or ami no-substituted, copolymerizable carboxylic acids are preferably derived from straight-chain or branched, saturated or mono- or poly-unsaturated C2-C30- monocarboxylic acids.
  • said hydroxyl- or amino-substituted, copolymerizable carboxylic acids carry a straight-chain mono- or polyunsaturated hydrocarbyl residue or a mixture of such residues with an average length of 1 -30, preferably 3-9 carbon atoms.
  • Particularly preferred residues of said hydroxyl- or amino-substituted, copolymerizable carbox- ylic acids are:
  • carboxylic acids as co-monomers for polymerization of GABA are RiCO(OH), wherein R1 is preferably selected from the group consisting of CH 2 (OH)-, CH 2 (NH 2 )-, C 2 H 4 (OH)-, C 2 H 4 (NH 2 )-, C 2 H 3 (OH) 2 -, C 2 H3(NH 2 )2-, C 3 H 4 (OH)-, C 3 H 4 (NH 2 )-, 0 3 ⁇ 3 ( ⁇ ) 2 -, or 0 3 ⁇ 3 ( ⁇ 2 )2.
  • the present invention further relates to a method of preparing vinylpyrrolidone, preferably N-vinylpyrrolidone, which method comprises
  • vinylpyrrolidone preferably by reacting said GABA with acetylene, preferably by means of a Reppe synthesis, to form vinylpyrrolidone, preferably N-vinylpyrrolidone.
  • vinylpyrrolidone is further processed by polymerization to form polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone.
  • Example 1 Culturing Corynebacterium glutamicum and fermentation
  • the pH was adjusted to 7.5 and then regulated at pH 6.4 with ammonia-water (25 %).
  • Glucose concentration was between 2 and 8 % during complete fermentation.
  • Sugar solution
  • Glucose and molasse mixture to a total concentration of 60 %.
  • the precultures for fermentation are grown in SF-Medium (see above) for 24 to 48 hours at 30 °C and at 200rpm (20 % volume of fermentation starting volume).
  • the fermentation is carried out in F-Medium.
  • the feed, 40 to 60% of the total volume consists of a sugar solution (see above) and is started at a total sugar concentration of ⁇ 2 %.
  • the fermentations were controlled by (i) a pH-value of 6.4, (ii) a total glucose concentration of 20 to 80 g/L and a stirrer speed of 300 to 900 rpm keeping a constant O2 partial pressure over the complete fermentation time. At the end of the fermentation the glucose was completely used up and the cells were harvested by centrifugation. The resulting fermentation broths were analyzed concerning production of GABA, glutamate and residual glucose concentration.
  • Alternative culture conditions are described as follows.
  • the pre-seed culture is prepared by inoculating a loopful of cells from slant culture into the flasks containing 25 ml_ of SF- medium and is cultivated at 31.5°C for 24h with shaking (250 rpm).
  • pre-seed culture was transferred into a 50ml of fresh SF-medium, and cultivated at 31.5°C with shaking (250 rpm).
  • the complete seed culture is transferred into a 5 L jar fermenter containing 1 ,600mL of production medium (F5H).
  • the fermentation medium (F5H) used for GABA production consisted of 40g of glucose, 40g of molasses (cane), 1.0g of (NhU ⁇ SC , 1.3g of KH2PO4, 0.8g of MgS0 4 -7H 2 0, 2ml of soybean protein hydrolysate, 600 g of thiamine monohydrochloride and 0.4g of neorin (an- tifoam agent) per liter and the pH was adjusted to 7.5 with ammonia water.
  • GAD genes e.g., tomato-potato-tomato chimera (SL- GAD), E. coli
  • SL- GAD tomato-potato-tomato chimera
  • E. coli E. coli
  • GAD expression vectors were prepared using PCR from different microorganisms such as Mycobacterium smegmatis (ATCC607) and Lactobacillus plantarum (ATCC8014).
  • the amplified GAD genes were placed under the control of the CJ4 or pSod promoter in a suitable expression vector.
  • Expression vectors were pClik5aMCS and pCES (pCES208), which are shown in Fig. 2 and also further described in WO2009103547A1 (cf. WO2009103547A1 , examples 1 -3).
  • the preculture for the production of electocompetent C. glutamicum cells is grown over night at 30 °C with 200 rpm in a rich medium (BHI, Bacto Brain Heart Infusion, Becton Dickinson REF 237500). 5 ml. of the preculture was used as inoculum for the main culture in 300 mL BHI medium. The main culture was grown to an OD of 1.6 (30 °C, 200 rpm) and the cells were harvested via centrifugation. The resulting pellet was washed twice with EP-washing-buffer (10 % glycerol, 8 mM Tris-HCI, pH 7.4) at 4 °C. In a third washing step the cells were resuspended in 10 % glycerol and centrifuged again. The resulting pellet was resuspended in 1 mL of 10 % glycerol at 4 °C.
  • BHI Bacto Brain Heart Infusion, Becton Dickinson REF 237500.
  • the electroporation was done in a BioRad Gene Pulser using cuvettes with a gap width of 2 mm.
  • the conditions for electroporation are 2.5 kV, 25 F and 400 ⁇ .
  • 150 ⁇ of cells were transformed with 10 to 15 ⁇ g of DNA.
  • the cells were transferred to 4 mL preheated (46 °C) BHIS medium (BHI with 91 g/L sorbitol) and waved for 6 min in a water bath at 46 °C. After this the cells were incubated for 1 to 2 h at 30 °C vigorously shaking at 200 rpm.
  • the cells were plated on agar plates with BHIS agar containing the respec- tive selection marker.
  • An alternative transformation protocol is described in the following.
  • An Erlenmeyer flask containing 10 mL of complex medium (BHI) was inoculated with a loopful of cells from a slant culture and incubated at 30°C for 24h with shaking (200 rpm).
  • 1 mL of culture broth grown up to an optical density of 0.3 at 610 nm were transferred into an Erlenmeyer flask containing 100 mL of EPO medium, and incubated at 18.0°C for 28h with shaking (120 rpm).
  • the cultivation was stopped when the turbidity of the culture broth optical density at 610 nm reached 0.8 to 1 .0, and the culture broth was chilled on ice for 10 min.
  • the cells were harvested by centrifugation at 4000xg for 10 min, washed four times with 50 ml 10 % (v/v) glycerol and resuspended in 0.5 ml 10% glycerol.
  • DNA (2 ⁇ ) was mixed with 50 ⁇ of the competent cells.
  • a single pulse of 2.5 kV was applied after transferring the mixture to a prechilled 0.2 cm electroporation cuvette. After the electroporation, cells were then incubated for 1 hour at 30.0°C to allow for recovery and expression of the antibiotic resistance marker. Afterwards cells were plated on selective Agar plates containing the respective antibiotic.
  • the EPO medium used for preparation of competent cells consisted of 20g of glucose, 0.2g of isonicotinic acid, 1.25g of glycine, 1 g of tween 80, 2.5g of NaCI, 5g of beef extract, 5g of yeast extract and 10g of peptone per liter.
  • Example 4 Evaluation of GABA production
  • Matrix Fermentation broths, samples are sterile filtrated before injection
  • GABA 10 g/L, 25 g/L, 50 g/L
  • the amount of GABA in the sample was determined by a standard calibration method. Standard samples containing GABA from 10 to 50 g/L were injected and the peak areas were used for calibration. Linear regression coefficient of the calibration curve was above 0.99. The peak area of the standards and the samples was used to calculate the amount of GABA by Waters LC Millenium software.
  • the pGem.T easy-gaba-T(F,B)-loxp.km vector (pGem.T easy - gabaT loxP; SEQ ID NO: 1 16) was constructed for knock-out of gaba-T gene by Cre-loxP system.
  • the strategy for deletion of 4-aminobutyrate aminotransferase gene was based on homologous recombina- tion comprising double crossing-over using a Cre-loxP system.
  • a fragment comprising loxP- Km R -loxP was inserted by a double cross-over into a 4-aminobutyrate aminotransferase gene in C. glutamicum wildtype HH09 after transformation (cf. Fig. 3 and Fig. 4) resulting in C.
  • the loxP-Km R -loxP fragment was subsequently removed by transforming a C. glutamicum HH 102, the recombinant strain with a loxP-Km R -loxP insert in a 4-aminobutyrate ami- notransferase gene, with a plasmid containing a Cre gene (pCES.CAT-ICL.CRE (pCICRE), cf. Fig. 6), and by expressing the Cre gene, resulting in C. glutamicum HH103.
  • the Cre recombinant gene in pCES.CAT-ICL.Cre vector was induced by 1 % ammonium acetate.
  • Cre recombinase was confirmed by gel electrophoresis and coomassie brilliant blue staining (cf. Fig. 7). Subsequently, the C. glutamicum HH 103 cells were pCICRE cured, resulting in C. glutamicum HH 104.
  • glutamicum HH 104 cells were transformed with a plasmid comprising a construct for expressing a potato-tomato chimeric GAD gene in C. glutamicum, resulting in C. glutamicum HH 105.
  • a plasmid comprising a construct for expressing a potato-tomato chimeric GAD gene in C. glutamicum, resulting in C. glutamicum HH 105.
  • An overview of the different transformants is shown in Table 4.
  • Fig. 8 shows a fermentation time courses of C. glutamicum HH 105 which contains a GAD gene from potato-tomato and in which the 4-aminobutyrate aminotransferase gene was eliminated by loxP-Km R -loxP disruption (growth ( ⁇ ), glucose (o), GABA ( ⁇ ), mono-sodium gluta- mate (MSG) (n)).
  • the production of GABA by the C. glutamicum HH 105 developed was performed by employing a fed-batch culture system, which produced 132g/L of GABA.
  • Flask-scale culture was performed and the production of GABA was analyzed by HPLC. Flask-scale culture was performed for 72 hours and collect- ed samples were used for the analysis of cell growth and GABA production. C. glutamicum wt-HH09, HH 104, HH 105, HH 106, HH 107 were used for the flask-scale culture and production of glutamic acid and GABA of each strain was analyzed by HPLC.
  • Glutamic acid was accumulated in C. glutamicum wt-HH09 and HH 104 which don't have the glutamate decarboxylase (gad) gene.
  • glutamic acid was not accumulated in C. glutamicum HH 105, HH106 and HH 107.
  • GABA was accumulated in C. glutamicum HH 105, HH 106 and HH 107 because glutamate decarboxylase (GAD) which converts glutamic acid to GABA was expressed in these strains.
  • GABA production of C. glutamicum HH 105, HH 106 and HH 107 was 20.47 g/L, 15.78 g/L and 26.95 g/L, respec- tively.
  • Table 5 Production of ⁇ -aminobutyric acid and glutamic acid by C. glutamicum wt-HH09 (wildtype), C. glutamicum HH 104 (gaba-t -), C. glutamicum HH105 (pClik5aMCS pSod SL_GAD to C. glutamicum HH 104), C. glutamicum HH 106 (Potato-tomato chimeric gad gene was integrated to C. glutamicum HH 104) and C. glutamicum HH 107 (pClik5aMCS pSod SL_GAD to C. glutamicum HH 106).
  • Strain was cultured in 500ml of Erlenmeyer flasks contain 50ml of medium at 30.0°C for 72 hours with shaking (250rpm)
  • C. glutamicum HH105 To optimize fermentation condition for the production of GABA, 5L-jar fermentation using C. glutamicum HH105, HH106 and HH 107 was performed.
  • C. glutamicum HH 105 cell growth was reached to 142.0 at OD610 after 45- hour cultivation, then the OD610 value was slowly decreased after 66-hour cultivation.
  • the growth of C. glutamicum HH 106 was reached to 173.0 at O D610 after 36-hour cultivation and C. glutamicum HH 107 was reached to 136.0 at OD610 after 33-hour cultivation then slowly decreased.
  • the GABA and glutamate production as well as the growth of the cells of C. glutamicum cells having the gene encoding the aminotransferase disrupted using the cre-loxP system as de- scribed above is also shown in Fig. 16-18.
  • the cells used for obtaining the data displayed in Fig. 16-18 have the chimeric GAD gene integrated in the chromosomal bioD gene and were cultured in flask-scale (wt coryne HH09 ( ⁇ ), 1xGAD; -bioD; -Aminotransferase (o), 1xGAD; - bioD; -Aminotransf. -Glut.Exp.
  • Fig. 16 shows an increased amount of GABA production for cells with a disrupted gene coding for the aminotransferase compared to cells expressing a glutamate decarboxylase gene (gad) together with a functional aminotransferase.
  • the knock out of the gene coding for a GABA-aminotransferase was achieved in C. glutamicum by inserting a GAD gene (potato-tomato chimera) by means of homologous recombination in the coding sequence of the GABA-aminotransferase of C. glutamicum cells expressing GAD (potato-tomato chimera) according to a protocol described in "Handbook of Corynebacterium” (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005). Thereby the expression of the GABA-aminotransferase gene was disrupted. Using this approach C.
  • FIG. 13 shows a comparison of the fermentation time courses of C. glutamicum cells which comprises a GAD gene and C. glutamicum cells which comprises a GAD gene in combination with a disruption of the gene coding for 4-aminobutyrate aminotransferase.
  • the disruption of the 4-aminobutyrate ami- notransferase gene leads to an increased GABA production of C. glutamicum.
  • Example 6 Integration of glutamate decarboxylase (gad) gene into the chromosomal DNA of GABA-aminotransferase (gaba-T) knock-outed C. glutamicum
  • the glutamate decarboxylase (gad) gene was integrated into the chromosomal DNA of GABA-aminotransferase(gaba-T) knock-outed C. glutamicum HH104.
  • the construction of integration plasmids was based on the sacB selection system.
  • pClik int sacB bioD plasmid was used to construct the plasmid for the integration.
  • the plasmids with glutamate decarboxylase (gad) gene were constructed using pClik int sacB bioD plasmid as a backbone.
  • pClik int sacB bioD plasmid has Hindlll restriction site between bioD L and bioD R.
  • Amplified fragments were ligated to pGEM-T Easy plasmid and then digested by Hind 111 restriction enzyme to ligate to pClik int sacB bioD plasmid.
  • glutamate decarboxylase (gad) gene was introduced to C. glutamicum HH 104 and integrated candidates were selected on LB plate containing 20 ug/ml kanamycin. Then, colony PCR was used to confirm selected colonies.
  • Glutamate decarboxylase (gad) gene integrated candidate strain selected on LB agar plate containing 20ug/ml kanamycin was cultivated in LB broth for 24hours. Then, the cultured strain was diluted by PBS and spread on LB agar plate containing 10% sucrose.
  • Glutamate decarboxylase (gad) gene integrated C. glutamicum HH104 was named C. glu- tamicum HH 106 and C. glutamicum HH 106 harboring pClik5aMCS-Psod.SL_GAD was named C. glutamicum HH 107.
  • Seed culture of C.glutamicum HH 105 was conducted by inoculating a loopful of cells from slant culture into the Erlenmeyer flasks containing 10 ml of SF-Medium and by cultivating at 31.5°C for 24 hours with shaking (250rpm).
  • One ml of seed culture broth was transferred into 25 ml of SF-Medium with various biotin concentrations, and cultivated at 31 .5°C for 72 hours with shaking (250rpm).
  • the samples can be taken at time intervals for analysis of growth and GABA production.
  • Table 8 Effect of biotin concentration on the production of GABA and glutamic acid by C.glutamicum HH 105.
  • Example 7.1 Fed-batch culture with biotin for the production of GABA
  • the pre-seed culture of C.glutamicum HH 105 was prepared by inoculating a loopful of cells from slant culture into the flasks containing 10ml_ of SF-Medium and cultivating at 31 .5°C for 24h with shaking (250rpm).
  • One ml of this pre-seed culture was transferred into a 50ml of SF-Medium, and cultivated at 31 .5°C with shaking (250rpm).
  • 50ml of this seed culture was transferred into a 5L jar fermenter containing 1 ,600mL of F-Medium.
  • the production of glutamic acid was decreased when biotin was added (100 g/I) by fed-batch culture.
  • the production of GABA was increased 23% (132 g/l to 162 g/l) and yield was increased 10% (0.30 to 0.33) when 100 of biotin was added compared with the biotin limitation by fed-batch culture.
  • GABA glutamate dehydrogenase
  • Glutamate dehydrogenase is an enzyme which converts a-ketoglutarate to L- glutamate in TCA cycle. To increase the production of GABA, GDH over expression plas- mid was constructed.
  • Sod and CJ4(S1 ) promoters were used to express GDH and sub-cloning to pGEM-T Easy vector was performed.
  • a plasmid vector which expresses both chimeric glutamate decarboxylase (GAD) and glutamate dehydrogenase (GDH) was constructed.
  • GDH-expressing plasmid was introduced to C. glutamicum HH 104 and plasmid introduction was confirmed by colony PCR.
  • the expression of glutamate dehydrogenase (GDH) was confirmed by SDS-PAGE and western-blotting.
  • GDH glutamate dehydrogenase
  • a potato-tomato chimeric GAD gene was inserted in the coding sequence of the glutamate exporter of C. glutamicum cells expressing GAD (potato-tomato chimera).
  • GAD potato-tomato chimera
  • the expression of the glutamate exporter gene was disrupted.
  • This modification resulted in C. glu- tamicum cells with two copies of the GAD gene (one copy in the BioD gene and one copy in the glutamate exporter gene).
  • the effect of this disruption of the glutamate exporter in C. glutamicum on the GABA production is shown in Fig. 14.
  • Fig. 14 shows a comparison of the fermentation time courses of C. glutamicum which comprises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for a glutamate exporter.
  • the disruption of the glutamate exporter gene leads to an increased GABA production of C. glutamicum.
  • the knock out of the gene coding for the glutamate exporter in C. glutamicum was achieved by means of homologous recombination without inserting a copy of the GAD gene in the glutamate exporter sequence according to a protocol described in "Handbook of Corynebacterium” (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005).
  • the GABA and glutamate production as well as the cell growth of C. glutamicum in which the gene coding for the glutamate exporter has been disrupted and which also lacks a functional aminotransferase due to a cre-loxP disruption as described in Example 5 is shown in Fig. 16-18.
  • Example 10 Combined knock out of the 4-aminobutyrate aminotransferase gene and the glutamate exporter gene in C. glutamicum
  • C. glutamicum For further improving the GABA-production in C. glutamicum the expression of the gene coding for the GABA-aminotransferase and the expression of the gene coding for the glutamate exporter was reduced by inserting a potato-tomato chimeric GAD gene in the respective coding sequences of the GABA-aminotransferase and the glutamate exporter in C. glutamicum cells expressing GAD (potato-tomato chimera). Using this approach C. glutamicum cells with three copies of the GAD gene were generated (one copy in the BioD gene, one copy in the GABA-aminotransferase gene and one copy in the glutamate exporter gene).
  • Fig. 15 shows a comparison of the fermentation time courses of C. glutamicum which comprises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for GABA-aminotransferase and a disruption of the gene coding for a glutamate exporter.
  • the combined knock out of the GABA-aminotransferase gene and the gene coding for the glutamate exporter leads to an improvement of the GABA production of C. glutamicum.
  • Fig. 15 The improvement in GABA production by reducing the expression of both the GABA-aminotransferase and the glutamate exporter (Fig. 15) was higher than the improvement of GABA production by reducing the expression of either the GABA- aminotransferase or of the glutamate exporter alone (Fig. 13 and Fig. 14, respectively).
  • Fig. 16-18 show the GABA and glutamate production as well as the growth of C.
  • glutamicum in flask-scale culture having a chimeric GAD gene integrated in the chromosomal bioD gene, a knock-out of the gene coding for the glutamate exporter and a knock-out of the gene coding for the GABA-aminotransferase (wt coryne HH09 ( ⁇ ), 1xGAD; -bioD; - Aminotransferase (o),1xGAD; -bioD; -Aminotransf. -Glut.Exp. (X), 1xGAD; -bioD; - Aminotransf.; -Glut.Exp. (*), 1xGAD; -bioD; intact Aminotransf.
  • Fig. 16 shows an increased amount of GABA production for cells with a disrupted gene coding for the aminotransferase and a disrupted gene coding for the glutamate exporter compared to cells expressing a glutamate decarboxylase gene (GAD) without disrupting the aminotransferase and the glutamate exporter gene.
  • GAD glutamate decarboxylase gene
  • the average yield of 2-pyrrolidone in the cyclization was 93-95% not regarding whether the cyclization was done at 200°C or 240°C.
  • the yield of the distillation steps was 92-96%, i.e. the overall yield of the downstreaming was 86-90% ( ⁇ 2% loss of 2-P to the distillation sump).
  • the core part of the continuous laboratory reactor was a heating section. This section was built up in a modular way to allow different heating length (up to 5 m) and residence times. Several temperature measurement points were available in the reactor and especially in the heating section to measure the reaction temperature in the broth along this part of the reactor.
  • the broth was heated with an electric heating system using different heating bands. These heating bands were not directly placed on the thin reactor pipe, but around some brass jackets/pipes which surrounded the thin reactor pipe in the heating section. One purpose of the brass pipes is to ensure a uniform temperature distribution along the heating section. Additionally this part of the reactor was well isolated to avoid heat losses.
  • the broth was cooled down using a thermostat on a defined temperature. Finally the broth flowed through one of the 100 bar liquid relief valves in the product recipient. For safety reasons two bow- out discs were integrated in the laboratory reactor as well as a safety vessel in case the pipe or liquid relief valve clogged for any reasons.
  • GABA was further purified prior to continuous cyclization, i.e. the fermentation broth after ultrafiltration was submitted to ionic exchange chromatography and the purified GABA redeluted in water.
  • ionic exchange chromatography the fermentation broth after ultrafiltration was filtered over Amberlite 252H. The Amberlite was washed with water and GABA was eluted with 5% aqueous NH3. The solution was concentrated (evaporation of NH3) and redeluted to approximately the same concentration as the fermentation broth (for better comparison with the unpurified broth in the cyclization). The yield of the purification was 97,5% (average yield of 4 runs).
  • the purity was increased from 88 wt% to >96 wt% (determination of GABA content in dry matter after drying of the broth before and after purification).
  • the amberlite was washed with water and reused for the next purification (3x).
  • the yield of the cyclization using purified GABA was 85-88%.
  • the overall yield after distilla- tion of 2-pyrrolidone was 79-83%.
  • Example 12 Polymerization of 2-pyrrolidone to PVP 2 kg of 2-pyrrolidone were prepared for further processing to PVP.
  • the raw material was distilled in a distillation column with high number of theoretical plates. Small fractions of 30 g were taken and analyzed by GC-MS. In the beginning of the distillation after complete removal of water the distillate so- lidified in the cooler, i.e. in the beginning the cooler has to be warmed.

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Abstract

The present invention relates to an improved method for the fermentative production of gamma-aminobutyric acid (GABA) by cultivating a recombinant microorganism expressing an enzyme having a glutamate decarboxylase activity, wherein the recombinant microorganism has an amended expression of certain enzymes influencing the GABA production and / yield. 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 pyrrolidone and polymers making use of GABA as obtained by fermentative production.

Description

Improved process for the production of gamma-aminobutyric acid (GABA)
This application claims priority to application number EP 13197718.3 filed December 17, 2013, which is incorporated herein by reference in their entirety.
Summary of the invention
The present invention relates to an improved method for the fermentative production of gamma-aminobutyric acid (GABA) by cultivating a recombinant microorganism expressing an ex- ogenous enzyme having a glutamate decarboxylase activity, wherein the recombinant microorganism has a reduced glutamate exporter expression and / or 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 pyrrolidone or polymers making use of GABA as obtained by fermentative production.
Background of the invention
GABA (CAS number 56-12-2) is an important ubiquitous non-protein amino acid in both pro- karyotic and eukaryotic organisms. It shows different biological functions, for example as rep- resentative depressive neurotransmitter in the sympathetic nervous system and it is effective for lowering the blood pressure of experimental animals and humans. The compound is synthesized by a glutamate decarboxylase (GAD) from glutamate.
GABA is used in different technical fields. For example, GABA-enriched food can be used as a dietary supplement and nutraceutical to help treat sleeplessness, depression and autonomic disorders, chronic alcohol-related symptoms, and to stimulate immune cells. The compound can also be used as a raw material for the production of polyamides and of pyrrolidone. WO2009103547A1 describes a method for fermentative production of GABA by expressing in a coryneform bacterium a glutamate decarboxylase (gad) gene. However, the yield obtained by the method described in WO2009103547A1 is rather low.
The object of the present invention is, therefore, to provide an improved method for the fer- mentative production of GABA or corresponding salts thereof.
The objective problem has been solved in the present invention by amending the expression of certain enzymes influencing the GABA yield in the microorganism used for fermentation. In particular, the objective problem has been solved by reducing the expression of a glutamate exporter. This solution has not been suggested by the prior art.
Vo et al. (Vo et al., 2012, Bioprocess Biosyst Eng, vol. 35, p. 645-650) discloses the produc- tion of GABA in recombinant Escherichia coli, in which the expression of the gene coding for the glutamate/GABA antiporter is increased.
None of these prior art documents either alone or in combination suggests to reduce the ex- pression and / or activity of a glutamate exporter in a microorganism expressing an exogenous enzyme having a glutamate decarboxylase activity.
Brief description of the invention The above-mentioned problem was solved by the present invention teaching the fermentative production of GABA or a salt thereof by cultivating a recombinant glutamate producing microorganism expressing an exogenous GAD enzyme which enzyme converts glutamate that is formed in said microorganism to GABA, wherein the recombinant microorganism has a reduced glutamate exporter expression and / or 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 pyrrolidone or polymers making use of GABA as obtained by fermentative production.
Brief description of the several views of the drawings
Simplified diagram for biosynthesis of GABA and GHB.
Construction of various plasmids containing the different promoters and
GAD genes.
Strategy and vector construct for deletion of 4-aminobutyrate aminotransferase gene by double crossing-over using a Cre-loxP system.
Insertion of loxP-KmR-loxP by double crossing-over into a 4-aminobutyrate aminotransferase gene in C. glutamicum wt-HH09.
Confirmation of the insertion of loxP-KmR-loxP into a 4-aminobutyrate aminotransferase gene by PCR and subsequent acrylamide electrophoresis. The plasmid pCESCAT-ICL-CRE containing a Cre recombinase gene for removal of loxP-KmR-loxP.
Expression of Cre recombinase gene analysed by gel electrophoresis and subsequent coomassie brilliant blue staining. S.M: protein size marker; Mbi- otech, Cat.No. 20030; Lane 1 : pCESCAT-icl.cre in C. glutamicum (no induction); Lane 2: pCESCAT-icl.cre in C. glutamicum (16hr induction). Fermentation time courses of C. glutamicum HH 105 which contains a GAD gene from potato-tomato and 4-aminobutyrate aminotransferase gene was disrupted (growth (·), glucose (°), GABA (■), mono-sodium glutamate (MSG) (°))-
Fermentation time courses in fed-bactch culture with biotin (100μ9/Ι) for the production of GABA by C. glutamicum HH 105 which contains a GAD gene from potato-tomato and 4-aminobutyrate aminotransferase gene was disrupt- ed (growth (·), glucose (o), GABA (■), MSG (□)).
Figure 10: Fermentation time courses in fed-batch culture for the production of GABA by
C.glutamicum HH 105 (cell growth (■), GABA (·), mono-sodium glutamate (MSG) (X) and reducing sugar (■*·)). Experiments were conducted in a fer- menter that contained 1600ml medium. Total sugar concentration in the fer- menter is controlled to be 1 %~8% by feeding the 60% glucose solution. The pH controlled at 7.5 with ammonia water.
Figure 1 1 : Fermentation time courses in fed-batch culture for the production of GABA by
C.glutamicum HH 106 (cell growth (■), GABA (·), mono-sodium glutamate (MSG) (X) and reducing sugar (*■)). Experiments were conducted in a fer- menter that contained 1600ml medium. Total sugar concentration in the fer- menter is controlled to be 1 %~8% by feeding the 60% glucose solution. The pH controlled at 7.5 with ammonia water.
Figure 12: Fermentation time courses in fed-batch culture for the production of GABA by
C.glutamicum HH 107 (cell growth (■), GABA (·), mono-sodium glutamate
(MSG) (X) and reducing sugar Experiments were conducted in a fer- menter that contained 1600ml medium. Total sugar concentration in the fer- menter is controlled to be 1 %~8% by feeding the 60% glucose solution. The pH controlled at 7.5 with ammonia water.
Figure 13: Comparison of the fermentation time courses of C. glutamicum which comprises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for 4-aminobutyrate aminotransferase.
Figure 14: Comparison of the fermentation time courses of C. glutamicum which com- prises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for a glutamate exporter.
Figure 15: Comparison of the fermentation time courses of C. glutamicum which comprises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for 4-aminobutyrate aminotrans- ferase and a disruption of the gene coding for a glutamate exporter.
Figure 16: GABA production of various mutants of C. glutamicum (wt coryne HH09 (■),
1xGAD; -bioD; -Aminotransferase (o), 1xGAD; -bioD; -Aminotransf. -Glut.Exp. (X), 1xGAD; -bioD; -Aminotransf.; -Glut.Exp. (±), 1xGAD; -bioD; intact Aminotransf. (= wtHH09 with 1xGAD in bioD) (♦), 1xGAD; -bioD; intact Ami- notransf. (= wtHH09 with 1xGAD in bioD) (+)).
Figure 17: OD measurement during GABA production of various mutants of C. glutamicum (wt coryne HH09 (■), 1xGAD; -bioD; -Aminotransferase (o), 1xGAD; - bioD; -Aminotransf. -Glut.Exp. (X), 1xGAD; -bioD; -Aminotransf.; -Glut.Exp. ( ), 1xGAD; -bioD; intact Aminotransf. (= wtHH09 with 1xGAD in bioD) (♦), 1xGAD; -bioD; intact Aminotransf. (= wtHH09 with 1xGAD in bioD) (+)).
Figure 18: Glutamate production of various mutants of C. glutamicum (wt coryne HH09 (■), 1xGAD; -bioD; -Aminotransferase (o), 1xGAD; -bioD; -Aminotransf. - Glut.Exp. (X), 1xGAD; -bioD; -Aminotransf.; -Glut.Exp. (±), 1xGAD; -bioD; intact Aminotransf. (= wtHH09 with 1xGAD in bioD) (♦), 1xGAD; -bioD; intact Aminotransf. (= wtHH09 with 1xGAD in bioD) (+)).
Figure 19: Construction of additional plasmids containing the different promoters and
GAD genes.
Figure 20: Brief description of the sequence listing. Detailed description of the invention
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the examples included herein. Definitions
Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided herein, definitions of common terms in molecular biology may also be found in Rieger et al., 1991 Glossary of genetics: classical and molecular, 5th Ed., Berlin: Spring- er-Verlag; and in Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998 Supplement). It is to be understood that as used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.
Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101 ; Grossman and Moldave (Eds.) 1980 Meth. Enzymol. 65; Miller (Ed.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose, 1981 Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, I RL Press, Oxford, UK; and Setlow and Hollaender 1979 Genetic
Engineering: Principles and Methods, Vols. 1 -4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.
"Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and / or enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified protein in question and having similar functional activity as the unmodified protein from which they are derived.
"Homologues" of a nucleic acid encompass nucleotides and / or polynucleotides having nucleic acid substitutions, deletions and / or insertions relative to the unmodified nucleic acid in question, wherein the protein coded by such nucleic acids has similar functional activity as the unmodified protein coded by the unmodified nucleic acid from which they are de- rived. In particular, homologues of a nucleic acid may encompass substitutions on the basis of the degenerative amino acid code.
The terms "identity", "homology" and "similarity" are used herein interchangeably. "Identity" or "homology" or "similarity" between two nucleic acids sequences or amino acid sequences refers in each case over the entire length of the respective nucleic acid sequence or amino acid sequence.
Preferably, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a particular region, determining the number of positions at which the identical base or amino acid occurs in both sequences in order to yield the number of matched positions, dividing the number of such positions by the total number of positions in the region being compared and multiplying the result by 100.
Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity/homology/identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity/homology/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981 ) J. Mol. Biol 147(1 );195-7). The sequence identity may also be calculated by means of the Vector NTI Suite 7.1 program of the company Informax (USA) employing the Clustal Method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr; 5(2): 151 -1 ) with the following settings: Multiple alignment parameter:
Gap opening penalty 10
Gap extension penalty 10
Gap separation penalty range 8
Gap separation penalty off
% identity for alignment delay 40
Residue specific gaps off
Hydrophilic residue gap off
Transition weighing 0 Pairwise alignment parameter:
FAST algorithm on
K-tuple size 1
Gap penalty 3
Window size 5
Number of best diagonals 5
Alternatively the identity may be determined according to Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids Res 31 (13):3497-500, the web page: http://www.ebi.ac.Uk/Tools/clustalw/index.html# and the following settings
DNA Gap Open Penalty 15.0
DNA Gap Extension Penalty 6.
DNA Matrix Identity
Protein Gap Open Penalty 10.0 Protein Gap Extension Penalty 0.2
Protein matrix Gonnet
Protein/DNA ENDGAP -1
Protein/DNA GAPDIST 4
A "deletion" refers to removal of one or more amino acids from a protein or to the removal of one or more nucleic acids from DNA or RNA.
An "insertion" refers to one or more amino acid residues or nucleic acid residues being in- traduced into a predetermined site in a protein or the nucleic acid.
A "substitution" refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or beta-sheet structures).
On the nucleic acid level a substitution refers to a replacement of one or more nucleotides with other nucleotides within a nucleic acid, wherein the protein coded by the modified nucleic acid has a similar function. In particular homologues of a nucleic acid encompass substitutions on the basis of the degenerative amino acid code.
Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the protein and may range from 1 to 10 amino acids; insertions or deletion will usually be of the order of about 1 to 10 amino acid residues. The amino acid substitutions are preferably conservative amino acid substitutions. Con- servative substitution tables are well known in the art (see for example Taylor W.R. (1986) The classification of amino acid conservation J Theor Biol., 1 19:205-18 and Table 1 below).
Table 1 : Examples of conserved amino acid substitutions
Figure imgf000008_0001
Amino acid substitutions, deletions and / or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation.
The terms "encode" or "coding for" is used for the capability of a nucleic acid to contain the information for the amino acid sequence of a protein via the genetic code, i.e., the succession of codons each being a sequence of three nucleotides, which specify which amino acid will be added next during protein synthesis. The terms "encode" or "coding for" therefore includes all possible reading frames of a nucleic acid. Furthermore, the terms "encode" or "coding for" also applies to a nucleic acid, which coding sequence is interrupted by non- coding nucleic acid sequences, which are removed prior translation, e.g., a nucleic acid sequence comprising introns.
The term "domain" refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other posi- tions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein.
Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31 , 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61 , AAAI Press, Menlo Park; Hu et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1 ): 276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31 :3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.
The term "hybridization" as used herein includes "any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing" (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules.
As used herein, the term "Tm" is used in reference to the "melting temperature." The melt- g ing temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acid molecules is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41 (% G+C), when a nucleic acid molecule is in aqueous solution at 1 M NaCI (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of Tm. Stringent conditions, are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
Hybridization conditions are known in the art and are for example described in Sambrook, J., Fritsch, E.F., Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31 -9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6. For example, depending on the particular nucleic acid, standard conditions mean temperatures between 42 and 58 °C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X SSC = 0.15 M NaCI, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50 % formamide, for example 42 °C in 5 x SSC, 50 % formamide. Advantageously, the hybridization conditions for DNA:DNA hybrids are 0.1 x SSC and temperatures between about 20 °C to 45 °C, preferably between about 30 °C to 45 °C. For DNA:RNA hybrids the hybridization conditions are advantageously 0.1 x SSC and temperatures between about 30 °C to 55 °C, preferably between about 45 °C to 55 °C. These stated temperatures for hybridization are examples of calculated melting temperature values for a nucleic acid with a length of approx. 100 nucle- otides and a G + C content of 50 % in the absence of formamide.
Hybridization can in particular be carried out under stringent conditions. The term "stringent conditions" refers to conditions, wherein 100 contigous nucleotides or more, 150 contigous nucleotides or more, 200 contigous nucleotides or more or 250 contigous nucleotides or more which are a fragment or identical to the complementary nucleic acid molecule (DNA, RNA, ssDNA or ssRNA) hybridizes under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 2 X SSC, 0.1 % SDS at 50°C or 65°C, preferably at 65°C, with a specific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA). Preferably, the hybridizing conditions are equivalent to hybridiza- tion in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C, more preferably the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M Na- P04, 1 mM EDTA at 50°C with washing in 0,1 X SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C. Preferably, the complementary nucleotides hybridize with a fragment or the whole of the nucleic acids. Alternatively, preferred hybridization conditions encompass hybridisation at 65°C in 1x SSC or at 42°C in 1x SSC and 50% formamide, followed by washing at 65°C in 0.3x SSC or hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% forma- mide, followed by washing at 50°C in 2x SSC. Further preferred hybridization conditions are 0.1 % SDS, 0.1 SSD and 65°C. The term "isolated nucleic acid" or "isolated protein" refers to a nucleic acid or protein that is not located in its natural environment, in particular its natural cellular environment. Thus, an isolated nucleic acid or isolated protein is essentially separated from other components of its natural environment. However, the skilled person in the art is aware that preparations of an isolated nucleic acid or an isolated protein can display a certain degree of impurity depending on the isolation procedure used. The isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis. In this regard, a recombinant nucleic acid may also be in an isolated form. Methods for purifying and synthesizing nucleic acids and proteins are well known in the art (as described for example in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Reference herein to an "endogenous" nucleic acid and / or protein refers to the nucleic acid and / or protein in question as found in a cell in its natural form (i.e., without there being any human intervention).
The term "exogenous" nucleic acid refers to a nucleic acid that has been introduced in an organism by means of genetechnology, i.e., the respective modification of the organism is not of natural origin. An "exogenous" nucleic acid can either not occur in an organism in its natural form, be different from the nucleic acid in question as found in an organism in its natural form, or can be identical to a nucleic acid found in an organism in its natural form, but integrated not within its natural genetic environment. The corresponding meaning of "exogenous" is applied in the context of protein expression. The terms "exogenous" and "heterologous" are used interchangeably herein.
For the purposes of the invention, "recombinant" means with regard to, for example, a nucleic acid sequence, a nucleic acid molecule, an expression cassette or a vector construct, all those constructions brought about by man by genetechnological methods in which either (a) the sequences of a nucleic acid or a part thereof, or
(b) genetic control sequence(s) which is operably linked with a nucleic acid sequence, for example a promoter, or
(c) a) and b)
are not located in their natural genetic environment or have been modified by man by genetechnological methods, i.e., the nucleic acid sequence, the nucleic acid molecule, the expression cassette, the vector construct or the modification of the microorganism is not of natural origin. The modification may take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original organism or the presence in a genomic library or the combination with the natural promoter.
For instance, a naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a protein useful in the methods of the present invention, as defined above - becomes a recombinant expression cassette when this expression cas- sette is modified by man by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350, WO 00/15815 or US200405323. Furthermore, a naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a protein useful in the methods of the present invention, as defined above - becomes a recombinant expression cassette when this expression cassette is not integrated in the natural genetic environment but in a different genetic environment.
The term "microorganism" means all generally unicellular organisms with dimensions be- neath the limits of vision which can be propagated and manipulated in a laboratory. In particular, the microorganism can be, e.g., bacteria, yeast, fungus, or algae.
The term "recombinant microorganism" refers to a microorganism (e.g., bacteria, yeast, fungus, etc.) or microbial strain which has been genetically altered, modified or engineered (e.g., genetically engineered) such that it exhibits an altered, modified or different genotype as compared to the wild-type microorganism which it was derived from. Preferably, the "recombinant microorganism" comprises an exogenous, preferably, a recombinant, nucleic acid. "Recombinant microorganism" and "genetically modified microorganism" are used herein interchangeably.
An "intermediary product" is understood as a product, which is transiently or continuously formed during a chemical or biochemical process, in a not necessarily analytically directly detectable concentration. Said "intermediary product" may be removed from said biochemical process by a second, chemical or biochemical reaction.
With respect to a vector construct and / or recombinant nucleic acid molecules, the term "operatively linked", "in operative linkage", "operably linked", "functionally linked", "in functional linkage" is intended to mean that the nucleic acid to be expressed is linked to one or more regulatory sequences, including promoters, terminators, enhancers and / or other ex- pression control elements (e.g., polyadenylation signals), in a manner which allows for expression of the nucleic acid (e.g., in a host cell when the vector is introduced into the host cell). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of nucleic acid desired, and the like.
"Expression cassette" or "expression construct" means, according to the invention, one or more "regulatory elements", which are operatively linked to one or more nucleic acids that are to be expressed. "Expression cassette" or "expression construct" can be monocistronic or polycistronic.
"Regulatory element" or "regulatory sequence" means, according to the invention, a nucleic acid suitable to drive, increase, decrease or terminate expression of a nucleic acid operatively linked thereto. A "regulatory element" can be a promoter, enhancer, repressor, terminator or the like.
A "promoter", a "nucleic acid with promoter activity" or a "promoter sequence" mean, according to the invention, a nucleic acid which, functionally associated with a nucleic acid that is to be transcribed, regulates the transcription of this nucleic acid.
The term "terminator" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the endogenous gene or from an exogenous gene.
As used herein, the term "transgenic" refers to an organism that exogenously contains the nucleic acid, recombinant construct, vector or expression cassette described herein or a part thereof or mutation which is preferably introduced by genetechnological methods.
The term "wild-type" means, according to the invention, the state prior to a certain modification and the state to which the state after the modification is compared to in order to evaluate the effect of the respective modification. A "wild-type microorganism" refers to a micro- organism that has not been altered in the respective feature under investigation. A "wild- type microorganism" can also be a recombinant microorganism itself with respect to a different modification. A wild-type microorganism is preferably able to produce glutamate.
The term "host cell" describes a cell, preferably a cell of a microorganism, which shall be modified, e.g., by means of genetechnology. Natural locus means the location on a specific chromosome, preferably the location between certain genes, more preferably the same sequence background as in the original organism which is transformed. The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific genetic vector construct. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic vector construct into structural RNA (rRNA, tRNA), or imRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the result- ing RNA product. The term "expression" or "gene expression" can also include the translation of the imRNA and therewith the synthesis of the encoded protein, i.e., protein expression. deregulation" or "modulation" has to be understood in its broadest sense, and comprises an increase or decrease or of complete switch off of gene expression or protein activity by different means well known to those in the art. Suitable methods comprise for example an increase or decrease of the copy number of gene and / or protein molecules in an organism, or the modification of another feature of the gene or protein affecting the protein activity and / or expression, which then results in the desired effect on the metabolic pathway at issue, in particular the glutamate biosynthetic pathway or any pathway or enzymatic reaction coupled thereto. Suitable genetic manipulation can also include, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by removing strong promoters, inducible promoters or multiple promoters), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, decreasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and / or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene routine in the art (including but not limited to use of antisense nucleic acid molecules, or other methods to knock-out or block expression of the target protein).
The term "increased expression" or "enhanced expression" or "overexpression" or "increase in content" as used herein means any form of expression of a gene or gene product that is additional to the original wild-type expression level. For the purposes of this invention, the original wild-type expression level might also be zero (absence of expression). An increase in expression can preferably range from 1 -100% or even more increase in expression, preferably, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 10% or more increase. The term "decreased expression" or "reduced expression" or "decrease in content" or "disrupted expression" as used herein means any form of expression of a gene or gene product that is reduced to the original wild-type expression level. A decrease in expression can preferably range from 1 -100% decrease in expression with 100% decrease in expression being no expression, preferably, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease.
The term "increased activity" or "enhanced activity" as used herein means a protein having increased enzymatic activity as compared to the wild-type activity. An increase in enzymatic activity can be due to an amendment of the amino acid sequence, a conformational change or the effect of an activating substance on the protein. An increase in activity can preferably range from 1 -100% or even more increase in activity, preferably, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1 10% or more increase.
The term "reduced activity" or "decreased activity" or "disrupted activity" as used herein means a protein having reduced enzymatic activity as compared to the wild-type activity. An decrease in enzymatic activity can be due to an amendment of the amino acid sequence, a conformational change or the effect of an inhibitory substance on the protein. A decrease in activity can preferably range from 1 -100% decrease in activity with 100% decrease in activity being no activity, preferably, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease.
The term "splice variant" as used herein encompasses variants of a nucleic acid sequence in which selected introns and / or exons or parts thereof have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Thus, a splice variant can have one or more or even all introns removed or added or partially removed or partially added. According to this definition, a cDNA is considered as a splice variant of the respective intron-containing genomic sequence and vice versa. Such splice variants may be found in nature or may be manmade. Methods for predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex (2005) BMC Bioinfor- matics 6: 25).
The term "introduction" or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The host genome includes the chromosomal nucleic acid as well as extra-chromosomal nucleic acid.
Unless otherwise stated the expressions "gamma-aminobutyric acid", "gamma- aminobutyrate", "4-aminobutanoat", "4-aminobutyric acid" and "GABA" are considered to be synonymous. The GABA product as obtained according to the present invention may be in the form of the free acid, in the form of a partial or complete salt of said acid and base func- tional groups or in the form of mixtures of the non-charged acid and any of its salt or mixtures.
A "GABA salt" comprises for example metal salts, as for example mono- or di-alkalimetal salts of GABA like mono-sodium di-sodium, mono-potassium and di-potassium salts as well as alkaline earth metal salts as for example the calcium or magnesium salts or the proto- nated form of GABA.
The terms "functional equivalent", "derivative" or "variant" of a nucleic acid or protein are used interchangeably herein and refer to a nucleic acid or protein which differ from the respective nucleic acid or protein as defined herein, but still provides the same or similar functional activity. Preferably "similar functional activity" means at least 50%, at least 60%, at least 70%, at least 80 %, at least 90 %, at least 95%, at least 98 %, or at least 99% of the respective nucleic acid or protein described herein. The "similar functional activity" of a "functional equivalent" of a nucleic acid or protein can also be higher than the respective nucleic acid or protein described herein. For example, "functional equivalents" of an enzyme means enzymes, which, in a test used for enzymatic activity, display at least 50%, at least 60%, at least 70%, at least 80 %, at least 90 %, at least 95%, at least 98 %, or at least 99%of the activity of an enzyme, as defined herein. "Functional equivalents", are in particu- lar mutants or fragments of the nucleotide or amino acid sequence, but can also be glycosylate variants or other side chain variants or fusion proteins or homologs. Methods for generation and identification of such "functional equivalents" are known in the art.
Detailed description
Nucleic acids and proteins
The invention is based at least in part on the finding that a reduced or increased expression and / or activity of one or more genes of interest involved in the metabolism of GABA in a microorganism leads to an improved GABA yield. Preferably, the genes of interest are one or more genes selected from the group consisting of glutamate exporter, GABA- aminotransferase, succinate semialdehyde dehydrogenase, and glutamate dehydrogenase. Preferably, the microorganism produces glutamate and preferably also expresses an exogenous glutamate decarboxylase. Suitable nucleic acids and proteins are described below.
Glutamate decarboxylase nucleic acids and proteins
For the fermentative production of GABA a microorganism is preferably used, which expresses glutamate decarboxylase (GAD).
The glutamate decarboxylase useful for the present invention is preferably an enzyme of any origin having the ability to convert glutamate into GABA, preferably the glutamate decarboxylase catalyzes the following reaction: L-glutamate = 4-aminobutanoate + C02. Preferably, the glutamate decarboxylase is according to EC 4.1.1.15. Preferably the GAD of the present invention is of prokaryotic or eukaryotic origin, preferably, of bacterial, animal or plant origin.
The glutamate decarboxylase described herein can also be named as L-glutamic acid decarboxylase, L-glutamic decarboxylase, cysteic acid decarboxylase, L-glutamate a- decarboxylase, aspartate 1 -decarboxylase, aspartic a-decarboxylase, L-aspartate-a- decarboxylase, γ-glutamate decarboxylase, or L-glutamate 1 -carboxy-lyase.
According to another embodiment of the invention, said exogenous glutamate decarboxylase is a plant glutamate decarboxylase, for example from the Solanum genus, preferably Solanum tuberosum, i.e., potato, or Solanum lycopersicum, i.e., tomato.
According to another embodiment of the invention, said exogenous glutamate decarboxylase is a bacterial glutamate decarboxylase, for example from a bacterium of the genus Escherichia, in particular from E. coli, preferably, GadA, GadB, GadC, or GadBC complex, or from the genus Lactobacillus, preferably, Lactobacillus plantarum, preferably ATCC8014, or from the genus Mycobacterium, preferably, Mycobacterium smegmatis, preferably ATCC607.
In one specific embodiment, said glutamate decarboxylase is a chimeric glutamate decarboxylase. Preferably, the GAD comprises one or more parts of the amino acid sequence from one species and one or more parts of the amino acid sequence from another species. In total, there may be 1 to 10, in particular, 1 to 5, preferably 1 or 2 amino acid sequence portions derived from another species. Preferably, the GAD comprises an N-terminal and / or a C-terminal portion from one species and one central portion from another species. Each of said portions may have a length of 10 to 500, 10 to 450, 10 to 400, 20 to 350, 40 to 300, 50 to 250, 60 to 200, 70 to 150 or 80 to 100, preferably 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive amino acid residues of the GAD sequence.
Preferably, the GAD is a chimeric glutamate decarboxylase comprising at least one amino acid sequence portion derived from plant glutamate decarboxylase. Said "at least one ami- no acid sequence portion derived from plant glutamate decarboxylase" comprises at least ten consecutive amino acid residues of said plant enzyme. In total, there may be 1 to 10, in particular, 1 to 5, preferably 1 or 2 amino acid sequence portions derived from said plant sequence. Preferably, the GAD comprises an N-terminal and / or a C-terminal portion from one plant, preferably from tomato (Solanum lycopersicum), and one central portion from another plant, preferably potato (Solanum tuberosum). Preferably, the codon usage of the GAD gene is adapted to the codon usage of the host cell, preferably, to the codon usage of a corynebacterial cell.
Preferably, said exogenous glutamate decarboxylase is from Solanum tuberosum and comprises an amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2 or a sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity to an amino acid sequence from Thr94 to Leu336 of SEQ ID NO: 2. Preferably the N-terminal part of the GAD is encoded by position 193 to 471 of SEQ ID NO:1 or a nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity thereto. Preferably the C-terminal part of the GAD is encoded by position 1201 to 1605 of SEQ ID NO:1 or a nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity thereto. Preferably the central part of the GAD is encoded by position 472 to 1200 of SEQ ID NO:1 or a nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity thereto. Preferably the GAD is encoded by position 193 to 1605 of SEQ ID NO: 1 or a nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98%, or at least 99 % sequence identity thereto. Preferably the GAD nucleic acid is an isolated nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a GAD protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73, or functional equivalent, homologue, or a splice variant thereof; preferably the GAD protein has essentially the same biological activity as a GAD protein encoded by SEQ ID NO: 1 , 3, 5, 7, 10, or 12; preferably the GAD protein converts glutamate to GABA;
(iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GAD protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 2, 4, 6, 8, 1 1 , or 13; preferably the encoded protein converts glutamate to GABA; and
(iv) a nucleic acid encoding the same GAD protein as the GAD nucleic acids of (i) to (iii) above, but differing from the GAD nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
Preferably the GAD nucleic acid is an isolated nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 , or a functional equivalent, hom- ologue, or a splice variant thereof;
(ii) a nucleic acid encoding a GAD protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, or functional equivalent, homologue, or a splice variant thereof; preferably the GAD protein has essentially the same biological activity as a GAD protein encoded by SEQ ID NO: ; preferably the GAD protein converts glutamate to GABA; a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GAD protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 2; preferably the encoded protein converts glutamate to GABA; and
a nucleic acid encoding the same GAD protein as the GAD nucleic acids of (i) to (iii) above, but differing from the GAD nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
Preferably, the GAD protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
(i) an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73, or a functional equivalent, homologue, or a splice variant thereof; preferably the GAD protein has essentially the same biological activity as a GAD protein encoded by SEQ ID NO: 2, 4, 6, 8, 1 1 , or 13; preferably the GAD protein converts glutamate to GABA; or
(ii) an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least
81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72, or a functional equivalent, homologue, or a splice variant thereof; preferably the GAD protein converts glutamate to GABA. Preferably, the GAD protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
(i) an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, or a functional equivalent, homologue, or a splice variant thereof; preferably the GAD protein has essentially the same biological activity as a GAD protein encoded by SEQ ID NO: 2; preferably the GAD protein converts glutamate to GABA; or
(ii) an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 , or a functional equivalent, homologue, or a splice variant thereof; preferably the GAD protein converts glutamate to GABA.
The GAD nucleic acids and amino acid sequences described herein are useful in the methods, cells and genetic constructs of the invention.
GABA-aminotransferase nucleic acids and proteins
For the fermentative production of GABA a microorganism is preferably used, which is reduced in its expression and / or activity of a GABA-aminotransferase, preferably an endogenous GABA-aminotransferase. The GABA aminotransferase to be reduced in its expression and / or activity in the present invention is preferably an enzyme having the ability to catalyze the following reaction: 4- aminobutanoate + 2-oxoglutarate = succinate semialdehyde + L-glutamate. Preferably the GABA aminotransferase is according to EC 2.6.1 .19. Preferably the GABA aminotransferase of the present invention is endogenous to the microorganism used in the methods of the present invention. Preferably, the GABA aminotransferase is a GABA aminotransferase from corynebacteria, preferably from Corynebacterium glutamicum. More preferably, GABA aminotransferase is a GABA aminotransferase according to Genbank accession number NP_599724 / Ncgl0462, or a functional equivalent thereof. The GABA aminotransferase to be reduced in its expression and / or activity in the present invention is also named as β-alanine-oxoglutarate transaminase, aminobutyrate aminotransferase (ambiguous), β-alanine aminotransferase, p-alanine-oxoglutarate aminotransferase, γ-aminobutyrate aminotransaminase (ambiguous), γ-aminobutyrate transaminase (ambiguous), γ-aminobutyrate-a-ketoglutarate aminotransferase, γ-aminobutyrate-a- ketoglutarate transaminase, y-aminobutyrate:a-oxoglutarate aminotransferase, γ- aminobutyric acid aminotransferase (ambiguous), γ-aminobutyric acid transaminase (am- biguous), γ-aminobutyric acid-a-ketoglutarate transaminase, γ-aminobutyric acid-a- ketoglutaric acid aminotransferase, γ-aminobutyric acid-2-oxoglutarate transaminase, γ- aminobutyric transaminase (ambiguous), 4-aminobutyrate aminotransferase (ambiguous), 4-aminobutyrate-2-ketoglutarate aminotransferase, 4-aminobutyrate-2-oxoglutarate ami- notransferase, 4-aminobutyrate-2-oxoglutarate transaminase, 4-aminobutyric acid 2- ketoglutaric acid aminotransferase, 4-aminobutyric acid aminotransferase (ambiguous), aminobutyrate transaminase (ambiguous), GABA aminotransferase (ambiguous), GABA transaminase (ambiguous), GABA transferase, GABA-a-ketoglutarate aminotransferase, GABA-a-ketoglutarate transaminase, GABA-a-ketoglutaric acid transaminase, GABA-a- oxoglutarate aminotransferase, GABA-2-oxoglutarate aminotransferase, GABA-2- oxoglutarate transaminase, GABA-oxoglutarate aminotransferase, GABA-oxoglutarate transaminase, glutamate-succinic semialdehyde transaminase, 4-aminobutanoate:2- oxoglutarate aminotransferase, or 4-aminobutyrate-2-oxoglutarate transaminase. Preferably, the GABA-aminotransferase is a GABA-aminotransferase from corynebacteria, preferably from Corynebacterium glutamicum.
Preferably the GABA-aminotransferase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 14, 16, 34-41 , 74, 76, 78, 80, 82, 84, 86 or 88, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a GABA-aminotransferase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 15, 17, 75, 77, 79, 81 , 83, 85, 87, or 89, or functional equivalent, homologue, or a splice variant thereof; preferably the GABA- aminotransferase protein has essentially the same biological activity as a GABA- aminotransferase protein encoded by SEQ ID NO: 14 or 16; preferably the GABA- aminotransferase protein converts GABA to succinate semialdehyde;
(iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GABA-aminotransferase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 15 or 17; preferably the encoded protein converts GABA to succinate semialdehyde; and
(iv) a nucleic acid encoding the same GABA-aminotransferase protein as the GABA- aminotransferase nucleic acids of (i) to (iii) above, but differing from the GABA- aminotransferase nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code. Preferably the GABA-aminotransferase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 16 or 14, preferably SEQ ID NO: 16, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a GABA-aminotransferase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 17 or 15, preferably SEQ ID NO: 17, or functional equivalent, homologue, or a splice variant thereof; preferably the GABA- aminotransferase protein has essentially the same biological activity as a GABA- aminotransferase protein encoded by SEQ ID NO: 16 or 14, preferably SEQ ID NO:
16; preferably the GABA-aminotransferase protein converts GABA to succinate semi- aldehyde;
(iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GABA-aminotransferase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 17 or 15, preferably SEQ ID NO: 17; preferably the encoded protein converts GABA to succinate semialdehyde; and
(iv) a nucleic acid encoding the same GABA-aminotransferase protein as the GABA- aminotransferase nucleic acids of (i) to (iii) above, but differing from the GABA- aminotransferase nucleic acids of (i) to (iii) above due to the degeneracy of the genet- ic code.
Preferably the GABA-aminotransferase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 14, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a GABA-aminotransferase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least
78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 15, or functional equivalent, homologue, or a splice variant thereof; preferably the GABA-aminotransferase protein has essentially the same biological activity as a GABA-aminotransferase protein encoded by SEQ ID NO: 14, preferably the GABA-aminotransferase protein converts GABA to succinate semialdehyde;
(iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a GABA-aminotransferase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 15, preferably the encoded protein converts GABA to succinate semialdehyde; and
(iv) a nucleic acid encoding the same GABA-aminotransferase protein as the GABA- aminotransferase nucleic acids of (i) to (iii) above, but differing from the GABA- aminotransferase nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
Preferably, the GABA-aminotransferase protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
(i) an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 15, 17, 75, 77, 79, 81 , 83, 85, 87, or 89, or a functional equivalent, homologue, or a splice variant thereof; preferably the GABA-aminotransferase protein has essentially the same biological activity as a GABA-aminotransferase protein encoded by SEQ I D NO: 14 or 16; preferably the GABA-aminotransferase protein converts GABA to succinate semialdehyde; or (ii) an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least
81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 14, 16, 34-41 , 74, 76, 78, 80, 82, 84, 86 or 88, or a functional equivalent, homologue, or a splice variant thereof; preferably the GABA-aminotransferase protein converts GABA to succinate semialdehyde.
The GABA-aminotransferase nucleic acids and amino acid sequences described herein are useful in the methods, cells and genetic constructs of the invention.
Glutamate exporter nucleic acids and proteins
For the fermentative production of GABA a microorganism can preferably be used, which is reduced in its expression and / or activity of a glutamate exporter, preferably an endogenous glutamate exporter.
Preferably, the glutamate exporter exports glutamate from the inside to the outside of the cell. Preferably, the glutamate exporter is a glutamate exporter from corynebacteria, prefer- ably from Corynebacterium glutamicum. More preferably, glutamate exporter is a glutamate exporter according to Genbank accession number NP_600492; NCgl 1221 , or a functional equivalent thereof.
Preferably the glutamate exporter nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof; (ii) a nucleic acid encoding a glutamate exporter protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least
78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or
105, or functional equivalent, homologue, or a splice variant thereof; preferably the glutamate exporter protein has essentially the same biological activity as a glutamate exporter protein encoded by SEQ ID NO: 18 or 20; preferably the glutamate exporter protein exports glutamate out of the cell;
(iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a glutamate exporter protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 19 or 21 ; preferably the encoded protein exports glutamate out of the cell; and
(iv) a nucleic acid encoding the same glutamate exporter protein as the glutamate exporter nucleic acids of (i) to (iii) above, but differing from glutamate exporter nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code. Preferably the glutamate exporter nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 20 or 18, preferably SEQ ID NO: 20, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a glutamate exporter protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 21 or 19, preferably SEQ ID NO: 21 , or functional equivalent, homologue, or a splice variant thereof; preferably the glutamate exporter protein has essentially the same biological activity as a glutamate exporter protein encoded by SEQ ID NO: 20 or 18, preferably SEQ ID NO: 20; preferably the glutamate exporter protein exports glutamate out of the cell;
(iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a glutamate exporter protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the pol- ypeptide described in SEQ ID NO: 21 or 19, preferably SEQ ID NO: 21 ; preferably the encoded protein exports glutamate out of the cell; and
(iv) a nucleic acid encoding the same glutamate exporter protein as the glutamate exporter nucleic acids of (i) to (iii) above, but differing from glutamate exporter nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
Preferably, the glutamate exporter protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
(i) an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, or a functional equivalent, homologue, or a splice variant thereof; preferably the glutamate exporter protein has essentially the same biological activity as a glutamate exporter protein encoded by SEQ ID NO: 18 or 20; preferably the glutamate exporter protein exports glutamate out of the cell; or
(ii) an amino acid sequence encoded by a nucleic acid having in increasing order of pref- erence at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least
75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID
NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof; preferably the glutamate exporter protein exports glutamate out of the cell. The glutamate exporter nucleic acids and amino acid sequences described herein are useful in the methods, cells and genetic constructs of the invention. Succinate semialdehyde dehydrogenase nucleic acids and proteins
For the fermentative production of GABA a microorganism can preferably be used, which is reduced in its expression and / or activity of a succinate semialdehyde dehydrogenase, preferably an endogenous succinate semialdehyde dehydrogenase.
Preferably, the succinate semialdehyde dehydrogenase is a succinate semialdehyde dehydrogenase from corynebacteria, preferably from Corynebacterium glutamicum. Preferably the succinate semialdehyde dehydrogenase catalyzes the following reaction: succinate semialdehyde + NAD+ + Η20 = succinate + NADH + 2 H+. Preferably, the succinate semialdehyde dehydrogenase is according to EC 1.2.1.24.
The succinate semialdehyde dehydrogenase is also named as succinate semialdehyde dehydrogenase (NAD+), succinic semialdehyde dehydrogenase (NAD+), succinyl semialdehyde dehydrogenase (NAD+), succinate semialdehyde: NAD+ oxidoreductase, or succinate- semialdehyde:NAD+ oxidoreductase.
Preferably the succinate semialdehyde dehydrogenase nucleic acid is a nucleic acid mole- cule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 22, 24, 50-56, or 57, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a succinate semialdehyde dehydrogenase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 23 or 25, or functional equivalent, homologue, or a splice variant thereof; preferably the succinate semialdehyde dehydrogenase protein has essentially the same biological activity as a succinate semialdehyde dehydrogenase protein encoded by SEQ ID NO: 22 or 24; pref- erably succinate semialdehyde dehydrogenase protein converts succinate semialdehyde to succinate; (iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a succinate semialdehyde dehydrogenase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially iden- tical properties to the polypeptide described in SEQ ID NO: 23 or 25; preferably succinate semialdehyde dehydrogenase protein converts succinate semialdehyde to succinate; and
(iv) a nucleic acid encoding the same succinate semialdehyde dehydrogenase protein as the succinate semialdehyde dehydrogenase nucleic acids of (i) to (iii) above, but dif- fering from succinate semialdehyde dehydrogenase nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
Preferably the succinate semialdehyde dehydrogenase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 22 or 24, preferably SEQ ID NO: 26, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a succinate semialdehyde dehydrogenase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 25 or 23, preferably SEQ ID NO: 25, or functional equivalent, homologue, or a splice variant thereof; preferably the succinate semialdehyde dehydrogenase protein has essentially the same biological activity as a succinate semialdehyde dehydrogenase protein encoded by SEQ ID NO: 24 or 22, preferably SEQ ID NO: 24; preferably succinate semialdehyde dehydrogenase protein converts succinate semialdehyde to succinate;
(iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a succinate semialdehyde dehydrogenase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 25 or 23, preferably SEQ ID NO: 25; preferably succinate semialdehyde dehydrogenase protein converts sue- cinate semialdehyde to succinate; and
(iv) a nucleic acid encoding the same succinate semialdehyde dehydrogenase protein as the succinate semialdehyde dehydrogenase nucleic acids of (i) to (iii) above, but differing from succinate semialdehyde dehydrogenase nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
Preferably, the succinate semialdehyde dehydrogenase protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of:
(i) an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 25 or 23, or a functional equivalent, homologue, or a splice variant thereof; preferably the succinate semialdehyde dehydrogenase protein has essentially the same biological activity as a succinate semialdehyde dehydrogenase protein encoded by SEQ ID NO: 24 or 22; preferably succinate semialdehyde dehydrogenase protein converts succinate semial- dehyde to succinate; or
(ii) an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least
81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 22, 24, 50-56, or 57, or a functional equivalent, homologue, or a splice variant thereof; preferably succinate semialdehyde dehydrogenase protein converts succinate semialdehyde to succinate.
The succinate semialdehyde dehydrogenase nucleic acids and amino acid sequences described herein are useful in the methods, cells and genetic constructs of the invention. Glutamate dehydrogenase nucleic acids and proteins
For the fermentative production of GABA a microorganism can preferably be used, which is increased in its expression and / or activity of a glutamate dehydrogenase, preferably by means of a recombinant exogenous glutamate dehydrogenase.
Preferably, the glutamate dehydrogenase is a glutamate dehydrogenase from corynebacte- ria, preferably from Corynebacterium glutamicum. Preferably the glutamate dehydrogenase catalyzes the following reaction: L-glutamate + H2O + NAD(P)+ = 2-oxoglutarate + N H3 + NAD(P)H + H+. Preferably, the glutamate dehydrogenase is according to EC 1 .4.1 .3. The glutamate dehydrogenase is also named as glutamic dehydrogenase, L- glutamate:NAD(P)+ oxidoreductase (deaminating) or glutamate dehydrogenase [NAD(P)].
Preferably the glutamate dehydrogenase nucleic acid is a nucleic acid molecule consisting of or comprising a nucleic acid selected from the group consisting of:
(i) a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 1 1 , or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a glutamate dehydrogenase protein comprising an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least
78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 12, or functional equivalent, homologue, or a splice variant thereof; preferably the glutamate dehydrogenase protein has essentially the same biological activity as a glutamate dehydrogenase protein encoded by SEQ ID NO: 1 1 1 ; preferably the glutamate dehydrogenase protein converts L- glutamate to 2-oxoglutarate;
(iii) a nucleic acid molecule which hybridizes with a complementary sequence of any of the nucleic acid molecules of (i) or (ii) under high stringency hybridization conditions; preferably encoding a glutamate dehydrogenase protein; preferably wherein the nucleic acid molecule codes for a polypeptide which has essentially identical properties to the polypeptide described in SEQ ID NO: 1 12; preferably the glutamate dehydro- genase protein converts L-glutamate to 2-oxoglutarate; and
(iv) a nucleic acid encoding the same glutamate dehydrogenase protein as the glutamate dehydrogenase nucleic acids of (i) to (iii) above, but differing from glutamate dehydrogenase nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code. Preferably, the glutamate dehydrogenase protein is a protein consisting of or comprising an amino acid sequence selected from the group consisting of: (i) an amino acid sequence having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 12, or a functional equivalent, homologue, or a splice variant thereof; preferably the glutamate dehydrogenase protein has essentially the same biological activity as a glutamate dehydro- genase protein encoded by SEQ ID NO: 1 1 1 ; preferably glutamate dehydrogenase protein converts L-glutamate to 2-oxoglutarate; or
(ii) an amino acid sequence encoded by a nucleic acid having in increasing order of preference at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence represented by SEQ ID NO: 1 1 1 , or a functional equivalent, homologue, or a splice variant thereof; preferably the glutamate dehydrogenase protein converts L-glutamate to 2-oxoglutarate.
Methods for the production of GABA
The invention in particular relates to methods for the fermentative production of GABA. In particular, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism and wherein the recombinant microorganism has a reduced or increased expression and / or activity of one or more genes of interest involved in the metabolism of GABA in the microorganism as compared to a microorganism that has not the reduced or increased expression and / or activity of the one or more genes of interest involved in the metabolism of GABA (i.e. wild- type microorganism).
For the fermentative production of GABA a microorganism can preferably be used, which expresses an exogenous glutamate decarboxylase (GAD, preferably, EC 4.1.1.15). Thus, the invention is preferably directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate produced by the recombinant microorganism is converted to GABA, and wherein the recombinant microorganism has a reduced or increased expression of one or more genes of interest involved in the metabolism of GABA in the microorganism and / or a reduced or increased activity of the protein encoded by said gene of interest (i.e., protein of interest). Preferably, the microorganism has no endogenous expression of an enzyme having a glutamate decarboxylase activity. Preferably, the microorganism is not an Escherichia coli cell. The microorganism used in the method of the invention is recombinant due to a man made reduced or increased expression of one or more genes of interest involved in the metabolism of GABA in the microorganism and / or a reduced or increased activity of the protein encoded by said gene of interest (i.e., protein of interest), preferably by genetechnological methods (i.e., the reduction or the increase is genetically engineered), compared to the wild-type level of gene expression and/or protein activity, i.e., the modification of the microorganism is not of natural origin, and the microorganism can preferably be discriminated from the wild-type or natural variants of the wild-type by the presence of exogenous DNA sequences introduced into the nucleic acid (genomic or plasmid nucleic acid) of the host microorganism in the course of reducing or increasing expression of one or more genes of interest and / or in the course of reducing or increasing activity of the protein encoded by said gene of interest. Hence, this does not include microbial variants generated by natural mutations not induced by man.
In case of an reduced expression and/or activity, the expression and/or activity is preferably reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even and preferably 100%, i.e., no detectable expression.
The gene of interest is preferably selected from the group consisting of a gene encoding for a gene encoding a glutamate exporter, a GABA-aminotransferase (preferably, EC 2.6.1.19), a gene encoding a succinate semialdehyde dehydrogenase (preferably, EC 1 .2.1.24), and a gene encoding a glutamate dehydrogenase (preferably, EC 1 .4.1 .3), as described above in more detail. Preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins selected from the group consisting of a glutamate exporter, a GABA-aminotransferase (preferably, EC 2.6.1 .19), and a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24) and wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3). More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- duces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1 .19), a glutamate exporter, and a succinate semialdehyde dehydrogenase (preferably, EC
1.2.1.24).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- duces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1 .19) and a glutamate exporter.
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant micro- organism has a genetically engineered reduced expression and / or activity of a glutamate exporter compared to the wild-type.
Preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3). More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins of interest se- lected from the group consisting of an endogenous glutamate exporter, an endogenous GABA-aminotransferase (preferably, EC 2.6.1 .19), and an endogenous succinate semialdehyde dehydrogenase (preferably, EC 1 .2.1 .24). Preferably, the recombinant microorganism has additionally or separately an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3).
Preferred is in addition to a reduction of the glutamate exporter expression a reduction of the GABA-aminotransferase expression and / or activity and / or an increase of the glutamate dehydrogenase (preferably, EC 1.4.1.3) expression and / or activity.
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more proteins of interest selected from an endogenous GABA-aminotransferase (preferably, EC 2.6.1.19), an endoge- nous glutamate exporter, and an endogenous succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- duces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter.
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a GABA-aminotransferase (preferably, EC 2.6.1 .19).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter.
Preferably, the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3), preferably by expression of an exogenous coding sequence under the control of an appropriate promoter (preferably, promoter CJ4 or Sod), preferably in a recombinant expression cassette.
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant micro- organism has a reduced expression and / or activity of a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24). More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter, a GABA- aminotransferase (preferably, EC 2.6.1.19), and a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24). More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter, a GABA- aminotransferase (preferably, EC 2.6.1.19), and a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24).
Preferably, the recombinant microorganism has additionally or separately an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3), preferably by expression of an exogenous coding sequence under the control of an appropriate promoter (preferably, promoter CJ4 or Sod), preferably in a recombinant expression cassette.
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- duces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter and a GABA- aminotransferase (preferably, EC 2.6.1.19). More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a GABA-aminotransferase (prefera- bly, EC 2.6.1 .19) and a succinate semialdehyde dehydrogenase (preferably, EC 1 .2.1 .24).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter and a succin- ate semialdehyde dehydrogenase (preferably, EC 1 .2.1.24).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which pro- duces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more of an endogenous protein of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1.19), a glutamate exporter, and a succinate semialdehyde dehydrogenase (prefer- ably, EC 1.2.1.24).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more of an endogenous protein of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1.19), a glutamate exporter, and a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24) and wherein the recombinant microorganism has an increased expres- sion and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, EC 4.1.1.15), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of an endogenous protein of interest selected from the group consisting of a GABA-aminotransferase (preferably, EC 2.6.1.19), a glutamate exporter, and a succinate semialdehyde dehydrogenase (preferably, EC
1.2.1.24) and wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, EC 1.4.1.3).
The amino acid sequence and coding sequence of the GABA-aminotransferase, the glutamate exporter, the succinate semialdehyde dehydrogenase, and the glutamate dehydrogenase are preferably as described above in more detail.
According to the methods for the fermentative production of GABA, as described herein in more detail, the glutamate decarboxylase, the GABA-aminotransferase, the glutamate exporter, the succinate semialdehyde dehydrogenase, and the glutamate dehydrogenase protein comprises an amino acid sequence having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the sequence described herein in more detail, or a functional equivalent, homologue thereof, or the glutamate decarboxylase, the GABA- aminotransferase, the glutamate exporter, the succinate semialdehyde dehydrogenase, and the glutamate dehydrogenase protein comprises an amino acid sequence encoded by
(i) a nucleic acid having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the sequence described herein in more detail, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid encoding a protein having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the sequence described herein in more detail, or a functional equivalent, homologue, or a splice variant thereof;
(iii) a nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); or by
(iv) a nucleic acid encoding the same protein as any of the nucleic acids of (i) to (iii)
above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
Preferably, according to the methods for the fermentative production of GABA, as described herein in more detail, the glutamate decarboxylase has at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73, the GABA-aminotransferase has at least 70% sequence identity to SEQ I D NO: 15, 17, 75, 77, 79, 8 , 83, 85, 87, or 89, the glutamate exporter has at least 70% sequence identity to SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, the succinate semialdehyde dehydrogenase has at least 70% sequence identity to SEQ ID NO: 23 or 25, and the glutamate dehydrogenase has at least 70% sequence identity to SEQ ID NO: 1 12.
Preferably, according to the methods for the fermentative production of GABA, as described herein in more detail, the glutamate decarboxylase is encoded by a sequence with at least 70% sequence identity to SEQ I D NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72, the GABA-aminotransferase is encoded by a sequence with at least 70% sequence identity to SEQ ID NO: 14, 16, 34-41 , 74, 76, 78, 80, 82, 84, 86 or 88, the glutamate export- er is encoded by a sequence with at least 70% sequence identity to SEQ ID NO: 18, 20, 42- 49, 90, 92, 94, 96, 98, 100, 102, or 104, the succinate semialdehyde dehydrogenase is encoded by a sequence with at least 70% sequence identity to SEQ I D NO: 22, 24, 50-56, or 57, and the glutamate dehydrogenase is encoded by a sequence with at least 70% se- quence identity to SEQ I D NO: 1 1 1.
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, having at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more of an endogenous protein of interest selected from the group consisting of a GABA-aminotransferase (preferably, having at least 70% sequence identity to SEQ ID NO: 15, 17, 75, 77, 79, 81 , 83, 85, 87, or 89; preferably encoded by a sequence having at least 70% sequence identity to SEQ I D NO: 14, 16, 34-41 , 74, 76, 78, 80, 82, 84, 86 or 88), a glutamate exporter (preferably, having at least 70% sequence identity to SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105; preferably encoded by a sequence hav- ing at least 70% sequence identity to SEQ ID NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104), and a succinate semialdehyde dehydrogenase (preferably, having at least 70% sequence identity to SEQ ID NO: 23 or 25; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 22, 24, 50-56, or 57) and wherein the recombinant microorganism has preferably an increased expression and / or activity of a glu- tamate dehydrogenase (preferably, having at least 70% sequence identity to SEQ ID NO: 1 12; preferably encoded by a sequence having at least 70% sequence identity to SEQ I D NO: 1 1 1 ).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, having at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72), so that the glutamate is con- verted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more endogenous proteins of interest selected from the group consisting of a GABA-aminotransferase (preferably, having at least 70% sequence identity to SEQ ID NO: 15, 17, 75, 77, 79, 81 , 83, 85, 87, or 89; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 14, 16, 34-41 , 74, 76, 78, 80, 82, 84, 86 or 88) and a glutamate exporter (preferably, having at least 70% sequence identity to
SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105; preferably encoded by a sequence having at least 70% sequence identity to SEQ I D NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104).
More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, having at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72), so that the glutamate is con- verted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of a glutamate exporter (preferably, having at least 70% sequence identity to SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105; preferably encoded by a se- quence having at least 70% sequence identity to SEQ ID NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104).
Preferably, wherein recombinant microorganism of the method for the fermentative production of GABA further has a reduced expression and / or activity of an endogenous succinate semialdehyde dehydrogenase (preferably, having at least 70% sequence identity to SEQ ID NO: 23 or 25; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 22, 24, 50-56, or 57) and preferably wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, having at least 70% sequence identity to SEQ ID NO: 1 12; preferably encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 1 1 1 ). More preferably, the invention is directed to a method for the fermentative production of GABA, which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identi- ty to SEQ I D NO: 1 ), so that the glutamate is converted to GABA, and wherein the recombinant microorganism has a reduced expression and / or activity of one or more endogenous proteins of interest selected from the group consisting of a GABA-aminotransferase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 15 or 17; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 14 or 16) and a glutamate exporter (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ I D NO: 19 or 21 ; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 18 or 20) and / or wherein the recombinant microorganism has preferably an increased expression of a glutamate dehydrogenase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1 12; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identi- ty to SEQ I D NO: 1 1 1 ).
Preferably, wherein recombinant microorganism of the method for the fermentative production of GABA further has a reduced expression and / or activity of an endogenous succinate semialdehyde dehydrogenase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 23 or 25; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 22 or 24) and preferably wherein the recombinant microorganism has an increased expression and / or activity of a glutamate dehydrogenase (preferably, having at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1 12; preferably encoded by a sequence having at least 80%, at least 90%, or at least 95% sequence identity to SEQ I D NO: 1 1 1 ).
In another embodiment, the expression of one or more genes or a sequence with the respective enzymatic activity having at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 97% identity to the gene sequences selected from the group consisting of isocitrate dehydrogenase (icd, NCgl0634), phosphoenolpyruvate carboxylase (ppc, NCgl 1523), 2-oxoglutarate dehydrogenase (odhA, NCgl1084), isocitrate lyase (aceA, NCgl2248), phosphoenolpyruvate carboxykinase (pck, NCgl2765) and glutami- ne synthetase (glnA, NCgl2148) or the activity of the respective encoded proteins is modi- fied or is additionally modified to improve fermentative GABA production. Preferably, the expression and / or activity of the isocitrate dehydrogenase (icd, NCgl0634) is enhanced, the expression and / or activity of the phosphoenolpyruvate carboxylase (ppc, NCgl1523) is enhanced, the expression and / or activity of the 2-oxoglutarate dehydrogenase (odhA, NCgl1084) is enhanced or reduced, the expression and / or activity of the isocitrate lyase (aceA, NCgl2248) is enhanced or reduced, the expression and / or activity of the phosphoenolpyruvate carboxykinase (pck, NCgl2765) is enhanced or reduced and/or the expression and / or activity of the glutamine synthetase (glnA, NCgl2148) is enhanced or reduced. Enhancing or reducing the expression of a gene of interest or the activity of an encoded protein of interest in a host cell can be achieved by various methods well known in the art.
Methods for increasing expression and activity of genes or gene products are well documented in the art and include, for example, insertion of an exogenous gene, replace an ex- isting gene by another gene, increase the number of copies of the gene or genes, overex- pression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of an endogenous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the protein of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and / or substitution (see, Kmiec, US 5,565,350; Zarling et al., W09322443) or isolated promoters may be introduced into a cell in the proper orientation and distance from a gene so as to control the expression of the gene. Preferred promoters for gene expressing according to the present invention, for instance, the expression of the GAD gene, are the CJ4 promoter, preferably as in pCES208, or the pSod promoter, preferably as in pClick5aMCS.
Reducing the expression of a gene of interest or the activity of an encoded protein of interest in a host cell can be achieved by one or more methods selected from the group consisting of:
(a) introducing into the coding sequence, the promoter sequence, the terminator se- quence, and / or one or more enhancer sequences of a gene of interest of the host cell one or more mutations or exogenous nucleic acid sequences;
(b) exchanging the promoter sequence of a gene of interest of the host cell to a weak, inactive or inducible promoter sequence;
(c) removing the complete coding sequence, the promoter sequence, the terminator se- quence and / or one or more enhancer sequences of a gene of interest of the host cell;
(d) introducing into the host cell a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand of the gene of interest or a fragment thereof;
(e) introducing into the host cell an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the gene of interest or a fragment thereof;
(f) introducing into the host cell a ribozyme which specifically cleaves ribonucleic acid molecules derived from the gene of interest;
(g) introducing into the host cell an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the gene of interest; and (h) introducing into the host cell a factor which binds to the protein of interest or the DNA or RNA molecule encoding the protein of interest. These methods of reducing the expression of a gene of interest or the activity of an encoded protein of interest in a host cell are described in more detail below. However, the skilled worker recognizes that a series of further methods is available for influencing the expression of a gene of interest and the encoded protein in the desired manner. (a) Introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of a gene of interest one or more mutations or exogenous nucleic acid sequences
A reduced expression of a gene of interest and/or the encoded protein can be achieved for instance by introducing into the gene of interest of the host cell one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the gene of interest.
For the purposes of the present invention, "mutations" means the modification of the nucleic acid sequence of a nucleic acid sequence. Mutations can arise for example as the result of errors in the replication, or they can be caused by mutagens, or by means of genetechnolo- gy. While the spontaneous mutation rate in the cell genome of organisms is very low, the skilled worker is familiar with a multiplicity of biological, chemical or physical mutagens. Mutations comprise substitutions, additions, or deletions of one or more nucleic acid residues. Substitutions are understood as meaning the exchange of individual nucleic acid bases; one distinguishes between transitions (substitution of a purine base for a purine base, or of a pyrimidine base for a pyrimidine base) and transversions (substitution of a pyrimidine base for a purine base (or vice versa)).
Additions or insertions are understood as meaning the incorporation of additional nucleic acid residues into the DNA, it being possible to result in reading-frame shifts. In the case of such reading-frame shifts, one distinguishes between "in-frame" insertions/additions and "out-of-frame" insertions. In the case of the "in-frame" insertions/additions, the reading frame is retained, and a polypeptide which is enlarged by the number of the amino acids encoded by the inserted nucleic acids results. In the case of "out-of-frame" inser- tions/additions, the original reading frame is lost, and the formation of a complete and functional polypeptide is no longer possible.
Deletions describe the loss of one or more base pairs, which likewise lead to "in-frame" or "out-of-frame" reading-frame shifts and the consequences which this entails regarding the formation of an intact protein.
For instance, mutations can be introduced into the endogenous gene of interest for generating a loss of function (e.g. generation of stop codons, reading-frame shifts, and mutations resulting in amino acid exchanges, which effect the protein activity). It is also possible to reduce, lessen or block the endogenous activity of the proteins of interest by methods known to the skilled worker, for example by mutating a the coding region for the active center, for binding sites, for localization signals, for domains, clusters and the like. The activity can also be reduced in accordance with the invention by mutations, which affect the secondary, tertiary or quaternary structure of the protein.
For instance, at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, or more nucleotides can be deleted, added or mutated.
Alternatively, the start codon of a gene of interest can be deleted or changed, e.g., from ATG to GTG, CTG, or TTG, to achieve a reduction of the transcription of the gene of interest. The order of the above mentioned alternative start codons indicates a subsequent re- duction of the transcription of the gene of interest, preferably in corynebacteria, more preferably in Corynebacterium glutamicum.
A multiplicity of chemical, physical and biological mutagens are known in the art.
Chemical mutagens can be distinguished by their mechanism of action. Thus, there are base analogs (for example, 5-bromouracil, 2-aminopurine), mono- and bifunctional alkylating agents (for example monofunctional agents such as ethylmethylsulfonate, dimethyl sulfate, or bifunctional agents such as dichloroethyl sulfite, mitomycin, nitrosoguanidine- dialkylnitrosamine, N-nitrosoguanidine derivatives) or intercalating substances (for example acridine, ethidium bromide). Mutations are preferably inserted by an EMS mutagenesis (Birchler JA, Schwartz D. Biochem Genet. 1979 Dec; 17(1 1 -12): 1 173-80; Hoffmann GR. Mutat Res. 1980 Jan;75(1 ):63- 29). Physical mutagens are, for example, ionizing radiation. Ionizing radiation is, for example, gamma-radiation (photo energy of approximately one megaelectron volt MeV), X-rays (photo energy of a plurality of or many kiloelectron volts keV) or else ultraviolet light (UV light, photon energy of above 3.1 eV). UV light causes the formation of dimers between bases; with thymidine dimers, which give rise to mutations, being the most frequent here.
Genetechnological methods (as an example of a biological mutagen) for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include but are not limited to M 13 mutagenesis, T7-Gene in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols, use of transposons (for example Tn5, Tn903, Tn916, Tn1000, Balcells et al., 1991 , May BP et al. (2003) Proc Natl Acad Sci U S A. Sep 30;100(20):1 1541 -6.) or mutagenesis by means of T-DNA insertion. Point mutations may also be generated by means of DNA-RNA hybrids also known as "chimeraplasty"
(Cole-Strauss et al. (1999) Nucl. Acids Res. 27(5): 1323-1330; Kmiec (1999) Gene Therapy American Scientist 87(3): 240-247). Also sequence-specific recombination systems may be used, examples which may be mentioned being Cre/lox system of bacteriophage P1 , the FLP/FRT system from yeast, the Gin recombinase of phage Mu, the Pin recombinase from E. coli and the R/RS system of the pSR1 plasmid.
Another means for introducing deletions is homologous recombination, i.e., at least part of the target sequence is exchanged with a sequence comprising a deletion of at least one nucleotide via homologous recombination, as described herein. Homologous recombination can also be combined with one or more sequence-specific recombination systems, e.g., cre/lox system. The mutation sites may be specifically targeted or randomly selected. If the mutations have been created randomly e.g. by transposon tagging or chemical mutagenesis, the skilled worked is able to specifically enrich selected mutation events in the inventive nucleic acids. Suitable domains as targets for mutagenesis can be identified by suitable computer programs such as, for example, SMART or InterPRO, for example as described in Andersen P., The Journal of Biol. Chemistry, 279, 38, pp. 40053-40061 , 2004, and literature cited therein. A reduced expression of a gene of interest can also be achieved for instance by introducing into the gene of interest one or more exogenous nucleic acid sequences and thereby disrupting the expression of the gene of interest. This results into a nonfunctional protein either due to frame-shift mutations or due to misfolding of the protein by the incorporation of one or more amino acids.
For instance, the transcription of the gene of interest can be inhibited by the introduction of a complete endogenous or exogenous gene or a fragment thereof resulting in the production of either nonfunctional proteins or the production of the gene product of the introduced sequence.
Insertion of one or more exogenous nucleic acid sequences into the gene of interest can be achieved by introducing a nucleic acid construct suitable for inducing a homologous recombination on the gene of interest (Ohtsuka M, Kimura M, Tanaka M, Inoko H. (2009) Curr Pharm Biotechnol. 10(2):244-51 ; Hohn B and Puchta (1999) H Proc Natl Acad Sci USA 96:8321 -8323; "Handbook of Corynebacterium", Lothar Eggeling & Michael Bott, CRC
Press Taylor & Francis Group, (2005)). Preferably, the insertion of one or more exogenous nucleic acid sequences into the gene of interest is combined with a deletion of at least parts of the nucleic acid sequence of the gene of interest, i.e., at least parts of the nucleic acid sequence of the gene of interest are exchanged by the one or more exogenous nucleic acid sequences. To generate a homologously-recombinant organism with reduced activity, a nucleic acid construct is used which, for example, comprises at least part of an endogenous gene which is modified by a deletion, addition or substitution of at least one nucleotide in such a way that the functionality is reduced or completely eliminated. In the case of conventional homologous recombination, the modified region is flanked at its 5' and 3' end by fur- ther nucleic acid sequences, which must be sufficiently long for allowing recombination. Their length is, as a rule, in a range of from one hundred bases up to several kilobases (Thomas KR and Capecchi MR (1987) Cell 51 : 503; Strepp et al. (1998) Proc. Natl. Acad. Sci. USA 95(8): 4368-4373). In the case of homologous recombination, the host organism is transformed with the recombination construct using the methods described herein and clones, which have successfully undergone recombination are selected using for example a resistance to antibiotics. Preferably, the expression and / or activity of one or more genes coding for a glutamate exporter, a GABA-aminotransferase, and succinate semialdehyde dehydrogenase is reduced by introducing into the coding sequence an insertion and / or deletion, preferably, by means of homologous recombination as described herein (e.g., Fig. 3 and 4), and thereby disruption gene expression and / or altering the protein activity. Preferably, this approach is combined with one or more sequence-specific recombination systems, e.g., cre/lox system.
(b) Exchanging the promoter sequence of a gene of interest to a weak, inactive or inducible promoter sequence
For reducing the expression of a gene of interest of a host cell and/or the encoded protein the promoter of the gene of interest can be changed to a weak promoter sequence under production conditions. Alternatively, the promoter can be inactivated by exchanging one or more nucleotides crucial for promoter activity. Furthermore, the endogenous promoter can be exchanged to an inducible promoter. The promoter can be inducible by, e.g., temperature, pH, salinity, or chemical compounds like antibiotics (e.g., tetracyclin), sugars or hormones. Changing the promoter sequence can be achieved for instance by mutagenesis, e.g., site-directed muta- genesis, or homologous recombination.
Suitable promoters for use in coryneform bacteria, preferably in Corynebacterium glutami- cum, are also described in "Handbook of Corynebacterium" (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005), in Patek M, et al., 2013, Microbial Biotech- nology, vol. 6, p. 103-1 17, or in Patek M and Nesvera J, Promoters and Plasmid Vectors of Corynebacterium glutamicum, in Yukawa H and Inui M, Corynebacterium glutamicum, Biology and Biotechnology, Microbiology Monographs, 2013, VII, ISBN: 978-3-642-29856-1 , p. 51 -88. (c) Removing the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of a gene of interest
In order to reduce the expression of a gene of interest and/or the encoded protein of a host cell also the complete coding sequence of the gene of interest can be removed. This type of modification can also relate to the regulatory elements (for example the promoter sequence, the terminator sequence and / or one or more enhancer sequences) of the gene of interest, so that the coding sequence remains unaltered, but that expression (transcription and / or translation) does not take place and is reduced. Removal of the respective sequence can be achieved for instance by homologous recombination, as described herein. A multiplicity of sequence-specific recombination systems may be used, examples which may be mentioned being Cre/lox system of bacteriophage P1 , the FLP/FRT system from yeast, the Gin recombinase of phage Mu, the Pin recombinase from E. coli and the R RS system of the pSR1 plasmid.
(d) Introducing a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand of the gene of interest or a fragment thereof
The expression of the gene of interest and/or the encoded protein of the host cell can also be reduced by mean of a dsRNA molecule.
The method of regulating genes by means of double-stranded RNA ("double-stranded RNA interference"; dsRNAi) has been described extensively for animal, yeast, fungi and plant organisms such as Neurospora, Zebrafish, Drosophila, mice, planaria, humans, Trypanosoma, petunia or Arabidopsis (for example Matzke MA et al. (2000) Plant Mol. Biol. 43: 401 - 415; Fire A. et al. (1998) Nature 391 : 806-81 1 ; WO 99/32619; WO 99/53050; WO00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). In addition RNAi is also doc- umented as an advantageous tool for the repression of genes in bacteria such as E. coli for example by Tchurikov et al. (J. Biol. Chem., 2000, 275 (34): 26523-26529). Fire et al.
named the phenomenon RNAi for RNA interference. The techniques and methods described in the above references are expressly referred to. dsRNAi methods are based on the phenomenon that the simultaneous introduction of complementary strand and counter- strand of a gene transcript brings about highly effective suppression of the expression of the gene in question. The resulting phenotype is very similar to that of an analogous knock-out mutant (Waterhouse PM et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959-64).
Tuschl et al. (Gens Dev., 1999, 13 (24): 3191-3197) was able to show that the efficiency of the RNAi method is a function of the length of the duplex, the length of the 3'-end overhangs, and the sequence in these overhangs. Based on the work of Tuschl et al. and assuming that the underlining principles are conserved between different species specific guidelines to design RNAi molecules have been developed (e.g., Dykxhoorn et al., 2003, Nature, p. 457-467).
The double-stranded structure can be formed starting from a single, self-complementary strand or starting from two complementary strands. In a single, self-complementary strand, "sense" and "antisense" sequence can be linked by a linking sequence ("linker") and form for example a hairpin structure (cf., e.g., WO 99/53050). Preferably, the linking sequence may take the form of an intron, which is spliced out following dsRNA synthesis. The nucleic acid sequence encoding a dsRNA may contain further elements such as, for example, tran- scription termination signals or polyadenylation signals. If the two strands of the dsRNA are to be combined in a cell, this can be brought about in a variety of ways: a) transformation of the cell or of the organism with a vector encompassing the two expression cassettes; b) cotransformation of the cell or of the organism with two vectors, one of which encompasses the expression cassettes with the "sense" strand while the other encompasses the expression cassettes with the "antisense" strand; c) supertransformation of the cell or of the organism with a vector encompassing the expression cassettes with the "sense" strand, after the cell or the organism had already been transformed with a vector encompassing the expression cassettes with the "antisense" strand or vice versa; and / or d) introduction of a con- struct comprising two promoters that lead to transcription of the desired sequence from both directions.
The expression cassettes encoding the "antisense" or the "sense" strand of the dsRNA or the self-complementary strand of the dsRNA are preferably inserted into a vector and stably inserted into the genome of the host cell using the methods described herein in order to ensure permanent expression of the dsRNA. Transient expression with can also be useful. The dsRNA is preferably expressed under the control of a promoter having all its cis-acting elements upstream of the sequence coding for the dsRNA (e.g., a DNA dependent RNA polymerase III type 3 promoter). The length of the dsRNA can either be substantially similar to the length of the sequence of the gene of interest or can be a fragment thereof.
In order to bring about an efficient reduction in the expression of the gene of interest expression 100% sequence identity between dsRNA and the gene of interest is not necessarily required. Accordingly, there is the advantage that the method tolerates sequence devia- tions as they can exist as the result of genetic mutations, polymorphisms or evolutionary divergences.
Hence, preferably, the double-strand ribonucleic acid molecule (dsRNA) comprises a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the gene of interest;
(ii) a nucleic acid molecule encoding a protein having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with protein of interest; and
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii);
or wherein the sense strand comprises a fragment of at least 17 nucleotides of the nucleic acid molecule of (i) or (ii), preferably, wherein sense and antisense strand are expressed as one self-complementary RNA molecule, wherein sense and antisense strand are separated by a linker sequence and thus, the self-complementary RNA molecule is capable of forming a hairpin structure.
The length of the fragment of the sense strand in the dsRNA molecule is at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, preferably at least 21 consecutive nucleotides, preferably 18-24 nucleotides, more preferably 21 -23 nucleotides, or more, for example approximately 25 nucleotides, or approximately 50 nucleotides, approximately 100 nucleotides, approximately 200 nucleotides or approximately 300 nucleotides. Preferably, the fragment of the sense strand in the dsRNA molecule has at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the corresponding fragment of the sense strand of the gene of interest.
(e) Introducing into the recombinant microorganism an antisense ribonucleic acid mole- cule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the gene of interest or a fragment thereof
The expression of the gene of interest and/or the encoded protein of the host cell can also be reduced by mean of an antisense RNA molecule.
Methods for suppressing a specific protein by preventing the accumulation of its mRNA by means of "antisense" technology can be used widely and has been described extensively in the art. The antisense nucleic acid molecule hybridizes with, or binds to, the cellular mRNA and / or the genomic DNA encoding the target protein to be suppressed. This process sup- presses the transcription and/or translation of the target protein. Hybridization can be brought about in the conventional manner via the formation of a stable duplex or, in the case of genomic DNA, by the antisense nucleic acid molecule binding to the duplex of the genomic DNA by specific interaction in the large groove of the DNA helix. An "antisense" nucleic acid molecule comprises a nucleotide sequence, which is at least in part complementary to a "sense" nucleic acid molecule encoding a protein, e.g., comple- mentary to the coding strand of a double-stranded DNA molecule or complementary to an encoding mRNA sequence. Accordingly, an antisense nucleic acid molecule can bind via hydrogen bonds to a sense nucleic acid molecule. The antisense nucleic acid molecule can be complementary to an entire coding strand of a nucleic acid molecule conferring the ex- pression of the polypeptide of the invention or to only a portion thereof. Accordingly, an antisense nucleic acid molecule can be antisense to a coding region of the coding strand of a nucleotide sequence of a nucleic acid molecule of the present invention or to a non-coding region. Thus, for example, the oligonucleotide can be complementary to the nucleic acid region, which encompasses the translation start for the protein. Advantageously, the anti- sense nucleic acid molecule is antisense to a non-coding region of the mRNA flanking the coding region of a nucleotide sequence. Thus, the non-coding region is preferably in the area of 50 bp, 100 bp, 200 bp or 300 bp, preferably 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp or 1000 bp up- and / or downstream from the coding region. Preferably, the non- coding region refers to 5' or 3' sequences which flank the coding region that are not trans- lated into a polypeptide, i.e., also referred to as 5' and 3' untranslated regions (5'-UTR or 3'-UTR).
Preferably, the antisense ribonucleic acid molecule comprises a nucleic acid sequence complementary to the nucleic acid sequence of a nucleic acid molecule selected from (i) a nucleic acid molecule having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a gene of interest;
(ii) a nucleic acid molecule encoding a protein having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or 100% identity with a protein of interest; and
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii);
or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii).
Gene regulation by small RNAs (sRNAs) in bacteria is described in the art (e.g., Storz G, et al., 201 1 , Regulation by Small RNAs in Bacteria: Expanding Frontiers, Mol Cell, vol. 43(6): 880-891 ) and inhibitory RNA molecules suitable for the present invention can be derived therefrom. Preferably the sRNAs are designed to base pair at or near the ribosome binding site (RBS) of their targets and block translation by occluding ribosomes. Alternatively, the sRNAs base may be designed to pair at more distant locations and thus interfere with ribosome binding by other mechanisms, or decrease mRNA stability. Hence, the antisense nucleic acid sequence can be complementary to all of the transcribed mRNA of the protein; it may be limited to the coding region, or it may only consist of a fragment of the complementary sequence of the gene of interest, preferably of the coding region of the gene of interest or of the nucleotide sequence of the respective ribosome binding site (RBS). Antisense nucleic acid or sRNA sequences may thus have an advantageous length, for example, of at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, or at least 24 nucleotides, preferably at least 21 , preferably 18-24 nucleotides, more preferably 21 -23 nucleotides, or more, for example at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 nucleotides but they may also be longer and encompass at least 100, at least 200, at least 500, at least 1000, at least 2000 or at least 5000 nucleotides.
Given the coding strand sequences encoding the polypeptide of the present invention, e.g., having above mentioned activity, antisense nucleic acid molecules of the invention can be designed according to the rules of Watson and Crick base pairing. Preferably, the antisense molecule comprises or consists of a nucleotide sequence having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the reverse complementary sequence of the nucleotide sequence coding for a glutamate exporter, a GABA-aminotransferase, or a succinate semialdehyde dehydrogenase or of the nucleotide sequence of the respective ribosome binding site. More preferably, the antisense molecule comprises or consists of a nucleotide sequence having at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the reverse complementary sequence of a fragment of the nucleotide sequence coding for a glutamate exporter, a GABA-aminotransferase, or a succinate semialdehyde dehydrogenase of the nucleotide sequence of the respective ribosome binding site, wherein the fragment has at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, or at least 24 nucleotides, preferably at least 21 , preferably 18-24 nucleotides, more preferably 21 -23 nucleotides, or more, for example at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 00, at least 200, at least 500, or at least 000. Antisense nucleic acid sequences can be expressed recombinantly or synthesized chemically or enzymatically using methods known to the skilled worker. Antisense nucleic acid sequences can be expressed transiently or stably integrated in the genome of the cell. The antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid molecule of interest, described further in the following subsection).
(f) Introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules derived from the gene of interest
The expression of the gene of interest and / or the encoded protein of the host cell can be reduced by ribozyme which specifically cleaves ribonucleic acid molecules derived from the gene of interest.
Ribozymes are catalytic RNA molecules with ribonuclease activity, which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. In this manner, ribozymes (for example "Hammerhead" ribozymes;
Haselhoff and Gerlach (1988) Nature 33410: 585-591 ) can be used to catalytically cleave the mRNA of a protein of interest to be suppressed and to prevent translation. Catalytic RNA molecules or ribozymes can be adapted to any target RNA and cleave the diester backbone at specific positions, thus functionally deactivating the target RNA (Tanner NK (1999) FEMS Microbiol. Rev. 23(3): 257-275). The ribozyme per se is not modified thereby, but is capable of cleaving further target RNA molecules in an analogous manner, thus ac- quiring the properties of an enzyme. Methods for expressing ribozymes for reducing specific proteins are described in EP0291533, EP0321201 , and EP0360257.
Suitable target sequences and ribozymes can be identified for example as described by Steinecke P, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds, Academic Press, Inc. (1995), pp. 449-460 by calculating the secondary structures of ribozyme RNA and target RNA and by their interaction (Bayley CC et al. (1992) Plant Mol. Biol. 18(2): 353-361 ; Lloyd AM and Davis RW et al. (1994) Mol. Gen. Genet. 242(6): 653-657). For example, derivatives of the tetrahymena L-19 IVS RNA, which have complementary regions to the mRNA of the protein to be suppressed can be constructed (see also US 4,987,071 and US 5, 1 16,742). As an alternative, such ribozymes can also be identified from a library of a variety of ribozymes via a selection process (Bartel D and Szostak JW (1993) Science 261 : 141 1 -1418).
It is advantageous to combine the above-described antisense strategy with a ribozyme method. Further the antisense nucleic acid molecule of the invention can also be a ribozyme. The incorporation of ribozyme sequences into "antisense" RNAs imparts this en- zyme-like RNA-cleaving property to precisely these "antisense" RNAs and thus increases their efficiency when inactivating the target RNA. The preparation and the use of suitable ribozyme "antisense" RNA molecules is described, for example, by Haseloff et al. (1988) Nature 33410: 585-591 .
(g) Introducing an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the gene of interest
The function or activity of a protein of a host cell can efficiently also be reduced by express- ing a dominant-negative variant of said protein. The skilled worker is familiar with methods for reducing the function or activity of a protein by means of coexpression of its dominant- negative form (Lagna G and Hemmati-Brivanlou A (1998) Current Topics in Developmental Biology 36: 75-98; Perlmutter RM and Alberola-lla J (1996) Current Opinion in Immunology 8(2): 285-90; Sheppard D (1994) American Journal of Respiratory Cell & Molecular Biology 1 1 (1 ): 1 -6; Herskowitz I (1987) Nature 329 (6136): 219-22).
A dominant-negative protein variant can be accomplished for example by altering amino acid residues which are part of protein of interest and, as the result of their mutation the protein loses its function. Amino acid residues which are preferably to be mutated are those which are conserved in the homologous proteins of different organisms. Such conserved regions can be determined for example by means of sequence alignments. These mutations for obtaining a dominant-negative protein variant are preferably carried out at the level of the nucleic acid sequence coding for the protein of interest. A suitable mutation can be realized for example by PCR-mediated in vitro mutagenesis using suitable oligonucleotide primers, by means of which the desired mutation is introduced. Methods which are known to the skilled worker are described above.
(h) Introducing a factor which binds to the protein of interest or the DNA or RNA molecule encoding the protein of interest.
The reduced expression of a gene of interest and / or the encoded protein and / or the activity of the protein of interest of a host cell can also be achieved by introducing a factor which binds to the protein of interest or the DNA or RNA molecule encoding the protein of interest. Preferably, such factors are specific DNA-binding factors, for example factors of the zinc finger transcription factor type. These factors attach to the genomic sequence of the endogenous target gene, preferably in the regulatory regions, and bring about repression of the endogenous gene of interest. The use of such a method makes possible the reduction in the expression of an endogenous gene without it being necessary to recombinantly manipu- late the sequence of the latter. Such methods for the preparation of relevant factors are described in Dreier B et al. ((2001) J. Biol. Chem. 276(31 ): 29466-78 and (2000) J. Mol. Biol. 303(4): 489-502), Beerli RR et al. ((1998) Proc. Natl. Acad. Sci. USA 95(25): 14628-14633; (2000) Proc. Natl. Acad. Sci. USA 97(4): 1495-1500 and (2000) J. Biol. Chem. 275(42): 32617-32627)), Segal DJ and Barbas CF (3rd (2000) Curr. Opin. Chem. Biol. 4(1 ): 3410- 39), Kang JS and Kim JS ((2000) J. Biol. Chem. 275(12): 8742-8748), Kim JS et al. ((1997) Proc. Natl. Acad. Sci. USA 94(8): 3616-3620), Klug A ((1999) J. Mol. Biol. 293(2): 215-218), Tsai SY et al. ((1998) Adv. Drug Deliv. Rev. 30(1 -3): 23-31 ), Mapp AK et al. ((2000) Proc. Natl. Acad. Sci. USA 97(8): 3930-3935), Sharrocks AD et al. ((1997) Int. J. Biochem. Cell Biol. 29(12): 1371 -1387) and Zhang L et al. ((2000) J. Biol. Chem. 275(43): 33850-33860). These factors can be selected using any portion of a gene. This segment is preferably located in the promoter region. For the purposes of gene suppression, however, it may also be located in the region of the coding exons or introns. The skilled worker can obtain the relevant segments for instance from Genbank by database search or starting from a cDNA whose gene is not present in Genbank by screening a genomic library for corresponding genomic clones.
Furthermore, factors which are introduced into a cell may also be those which themselves inhibit the target protein. The protein-binding factors can, for example, be aptamers (Famu- lok M and Mayer G (1999) Curr. Top Microbiol. Immunol. 243: 123-36) or antibodies or anti- body fragments or single-chain antibodies (e.g., scFv). Obtaining these factors has been described, and the skilled worker is familiar therewith. Such compounds can be added to the culture media or can be expressed by the host cell, e.g., by genetic modification of the host cell in order to produce the respective inhibitory factor. Gene expression may also be suppressed by tailor-made low-molecular-weight synthetic compounds, for example of the polyamide type (Dervan PB and Biirli RW (1999) Current Opinion in Chemical Biology 3: 688-693; Gottesfeld JM et al. (2000) Gene Expr. 9(1 -2): 77- 91 ). These oligomers consist of the units 3-(dimethyl-amino)propylamine, N-methyl-3- hydroxypyrrole, N-methyl-imidazole and N-methyl-pyrroles; they can be adapted to each portion of double-stranded DNA in such a way that they bind sequence-specifically to the large groove and block the expression of the gene sequences located in this position. Suitable methods have been described in Bremer RE et al. ((2001 ) Bioorg. Med. Chem. 9(8): 2093-103], Ansari AZ et al. [(2001 ) Chem. Biol. 8(6): 583-92), Gottesfeld JM et al. ((2001 ) J. Mol. Biol. 309(3): 615-29), Wurtz NR et al. ((2001 ) Org. Lett 3(8): 1201 -3), Wang CC et al. ((2001 ) Bioorg. Med. Chem. 9(3): 653-7), Urbach AR and Dervan PB ((2001) Proc. Natl. Acad. Sci. USA).
Thus, preferably, the method for the fermentative production of gamma-aminobutyric acid (GABA) comprises reducing the expression and / or activity of the glutamate exporter in a recombinant microorganism comprising an exogenous nucleic acid encoding a glutamate decarboxylase by one or more measures selected from the group consisting of (a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous glu- tamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glu- tamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous gluta- mate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the pro- moter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter;
(d) introducing into the recombinant microorganism a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO: 18, 20, 42- 49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid molecule encoding a protein having at least 70% identity with SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, or a functional equivalent, homologue, or a splice variant thereof;
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and
(iv) a nucleic acid molecule encoding the same glutamate exporter as any of the nu- cleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code,
or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid mole- cule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a glutamate exporter in the recombinant microor- ganism or an expression cassette expressing said ribozyme;
(g) introducing into the recombinant microorganism an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the glutamate exporter or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the glutamate exporter or the DNA or RNA molecule encoding the glutamate exporter, or an expres- sion cassette expressing said factor, and thereby inhibiting expression and / or activity of the glutamate exporter.
Preferably, the method of for the fermentative production of gamma-aminobutyric acid (GABA) comprises the steps of
(A) transforming into a microorganism an exogenous nucleic acid comprising a nucleic acid sequence encoding a glutamate decarboxylase (preferably, EC 4.1 .1 .15) in functional linkage with a promoter;
(B) reducing or enhancing the expression and / or activity of one or more genes selected from the group consisting of a gene encoding for a glutamate exporter, a GABA- aminotransferase, a succinate semialdehyde dehydrogenase, a isocitrate dehydrogenase, a phosphoenolpyruvate carboxylase, a 2-oxoglutarate dehydrogenase, a isocitrate lyase, a phosphoenolpyruvate carboxykinase, a glutamine synthetase and glutamate dehydrogenase, which are described above in more detail, by means of one or more methods described herein;
(C) culturing the recombinant microorganism; and preferably
(D) isolating GABA produced by the recombinant microorganism.
Preferably, the method of the present invention comprises the steps of
(A) transforming into a microorganism an exogenous nucleic acid comprising a nucleic acid sequence encoding a glutamate decarboxylase (preferably, EC 4.1 .1 .15) in functional linkage with a promoter;
(B) applying one or more of the methods steps selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter;
(d) introducing into the recombinant microorganism a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof; (ii) a nucleic acid molecule encoding a protein having at least 70% identity with SEQ I D NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, or a functional equivalent, homologue, or a splice variant thereof;
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and
(iv) a nucleic acid molecule encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code, or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a frag- ment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a glutamate exporter in the recombinant microorganism or an expression cassette expressing said ribozyme; (g) introducing into the recombinant microorganism an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the glutamate exporter or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the glu- tamate exporter or the DNA or RNA molecule encoding the glutamate exporter, or an expression cassette expressing said factor, and thereby inhibiting expression and / or activity of the glutamate exporter;
and thereby reducing the expression and / or activity of the glutamate exporter in the recombinant microorganism;
(C) culturing the recombinant microorganism; and
(D) isolating GABA produced by the recombinant microorganism.
Thus, preferably, the method for the fermentative production of gamma-aminobutyric acid (GABA) further comprises reducing the expression and / or activity of a GABA- aminotransferase in a recombinant microorganism comprising an exogenous nucleic acid encoding a glutamate decarboxylase and comprising a reduced expression and / or activity of the glutamate exporter by one or more measures selected from the group consisting of (a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous GABA-aminotransferase genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the GABA-aminotransferase in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous GABA- aminotransferase coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a GABA-aminotransferase;
(d) introducing into the recombinant microorganism a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complemen- tary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO: 14, 16, 34- 41 , 74, 76, 78, 80, 82, 84, 86 or 88, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid molecule encoding a protein having at least 70% identity with
SEQ ID NO: 15, 17, 75, 77, 79, 81 , 83, 85, 87, or 89, or a functional equivalent, homologue, or a splice variant thereof;
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and (iv) a nucleic acid molecule encoding the same GABA-aminotransferase as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code,
or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a GABA-aminotransferase in the recombinant microorganism or an expression cassette expressing said ribozyme;
(g) introducing into the recombinant microorganism an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the GABA-aminotransferase or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the GABA- aminotransferase or the DNA or RNA molecule encoding the GABA-aminotransferase, or an expression cassette expressing said factor, and thereby inhibiting expression and / or activity of the GABA-aminotransferase;
and thereby reducing the expression and / or activity of the GABA-aminotransferase in the recombinant microorganism. Preferably, the method of the present invention comprising the steps of
(A) transforming into a microorganism an exogenous nucleic acid comprising a nucleic acid sequence encoding a glutamate decarboxylase (preferably, EC 4.1 .1 .15) in func- tional linkage with a promoter;
(B) applying one or more of the methods steps selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter; and thereby reducing the expression and / or activity of the glutamate exporter in the recombinant microorganism;
(C) applying one or more of the methods steps selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous GABA-aminotransferase genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the GABA-aminotransferase in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous GABA-aminotransferase coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence; and
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a GABA- aminotransferase;
and thereby reducing the expression and / or activity of the GABA-aminotransferase in the recombinant microorganism;
(D) culturing the recombinant microorganism; and
(E) isolating GABA produced by the recombinant microorganism.
Preferably, the method of the present invention comprising the steps of
(A) transforming into a microorganism an exogenous nucleic acid comprising a nucleic acid sequence encoding a glutamate decarboxylase (preferably, EC 4.1 .1 .15) in functional linkage with a promoter; (B) applying one or more of the methods steps selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter; and thereby reducing the expression and / or activity of the glutamate exporter in the recombinant microorganism;
(C) increasing the expression and / or activity of a glutamate dehydrogenase in the recombinant microorganism;
(D) culturing the recombinant microorganism; and
(E) isolating GABA produced by the recombinant microorganism. Preferably, the method further comprises the step of reducing or enhancing the expression and / or activity of one or more genes selected from the group consisting of a gene encoding for a a succinate semialdehyde dehydrogenase, a isocitrate dehydrogenase, a phos- phoenolpyruvate carboxylase, a 2-oxoglutarate dehydrogenase, a isocitrate lyase, a phos- phoenol pyruvate carboxy kinase, a glutamine synthetase, and glutamate dehydrogenase, preferably, reducing a succinate semialdehyde dehydrogenase expression and / or activity, and / or enhancing a glutamate dehydrogenase expression and / or activity, by means of one or more methods described herein. Preferred is in addition to a reduction of the glutamate exporter expression and / or activity a reduction of the GABA-aminotransferase expression and / or activity and / or an increase of the glutamate dehydrogenase expression and / or activity.
The recombinant microorganisms as used according to the invention can be cultured continuously or discontinuously in the batch process or in the fed batch or repeated fed batch process. A review of known methods of cultivation will be found in the textbook by Chmiel (Bioprocesstechnik 1. EinfCihrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991 )) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vie- weg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium that is to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the handbook "Manual of Methods for General Bacteriology" of the American Soci- ety for Bacteriology (Washington D. C, USA, 1981 ).
These media that can be used according to the invention generally comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and / or trace elements. Preferred sources of carbon are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add mixtures of various sources of carbon. Other possible sources of carbon are oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid or linoleic acid, alcohols such as glycerol, methanol or ethanol and organic acids such as acetic acid or lactic acid.
Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials con- taining these compounds. Examples of sources of nitrogen include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex sources of nitrogen, such as corn-steep liquor, soybean flour, soybean protein, yeast extract, meat extract and others. The sources of nitrogen can be used separately or as a mixture.
Inorganic salt compounds that may be present in the media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Inorganic sulfur-containing compounds, for example sulfates, sulfites, dithionites, tetrathi- onates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as sources of sulfur.
Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as sources of phosphorus.
Chelating agents can be added to the medium, in order to keep the metal ions in solution. Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or proto- catechuate, or organic acids, such as citric acid.
The fermentation media used according to the invention may also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from complex components of the media, such as yeast extract, molasses, corn-steep liquor and the like. In addition, suitable precursors can be added to the culture medium. The precise composition of the compounds in the medium is strongly dependent on the particu- lar experiment and must be decided individually for each specific case. Information on media optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Publ. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3). Growing media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.
Preferably, biotin is contained in the culture medium. Preferably, the culture media comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and / or trace elements and biotin. Biotin is contained in the culture medium preferably in a concentration of at least 2 g/L, at least 5 μg L, at least 10 g/L, at least 20 μg/L, at least 50 μg/L, at least 100 μg/L, at least 200 g/L, at least 300 g/L, at least 400 g/L, at least 500 μg/L, preferably at least 300 pg/L, preferably at 100-300 g/L, preferably 200-300 μg/L.
All components of the medium are sterilized, either by heating (20 min at 1.5 bar and 121 °C) or by sterile filtration. The components can be sterilized either together, or if necessary separately. All the components of the medium can be present at the start of growing, or optionally can be added continuously or by batch feed.
The temperature of the culture is normally between 15 °C and 45 °C, preferably 25 °C to 40 °C, preferably 30°C or 31.5°C, and can be kept constant or can be varied during the experiment. The pH value of the medium should be in the range from 5 to 8.5, preferably around 7.0, preferably pH 6.4, pH 6.5, pH 7.0 or pH 7.5. The pH value for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, e.g. fatty acid polyglycol esters, can be used for controlling foaming. To maintain the stability of plasmids, suitable substances with selective action, e.g. antibiotics, can be added to the medium. Oxygen or oxygen-containing gas mixtures, e.g. the ambient air, are fed into the culture in order to maintain aerobic conditions. Culture is continued until a maximum of the desired product has formed. This is normally achieved within 10 hours to 160 hours.
The methodology of the present invention can further include a step of recovering GABA from the culture media and / or the microbial cells. The term "recovering" includes extracting, harvesting, isolating or purifying GABA. For isolation of GABA after cultivation of the recombinant microorganisms the cells can be disrupted optionally by high-frequency ultrasound, by high pressure, e.g. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of several of the methods listed. Recovering GABA from the culture media, the cells, or the disrupted cell fraction can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hex- ane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystalliza- tion, pH adjustment, lyophilization and the like. For example, GABA can be recovered from culture media by first removing the microorganisms (e.g., by centrifugation). The remaining broth is then passed through or over a cation exchange resin to remove unwanted cations and then through or over an anion exchange resin to remove unwanted inorganic anions and organic acids. For extracting GABA from the microbial cells, the cells can be disrupted, the cellular debris removed (e.g., by centrifugation) and GABA can be isolated from the supernatant by any suitable method described above. Preferably, the cells are removed from the fermentation broth, preferably by centrifugation and / or filtration (preferably, using a 8kD-15kD membrane, preferably a 10 kD membrane), and GABA is recovered from the supernatant and / or permeate. Preferably, GABA can be extracted from the solution by ion exchange chromatography. Alternatively, the cells are removed from the fermentation broth, preferably by centrifugation and / or filtration, and GABA is converted to 2-pyrrolidone in the supernatant and / or permeate by means of in-broth chemistry, preferably by heating the supernatant and / or permeate, preferably under increased pressure.
By applying the method described above the GABA yield is preferably increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 90%, at least 100%, at least 1 10%, or even more.
Recombinant cells
Another embodiment of the present invention is a recombinant prokaryotic or eukaryotic cell. Preferably, the recombinant prokaryotic or eukaryotic cell is a glutamate producing cell, preferably, without an endogenous expression of an enzyme having a glutamate decarboxylase activity. Preferably, the recombinant prokaryotic or eukaryotic cell is a cell of a microorganism. The microorganism can include but is not limited to bacteria, yeast, fungus, or algae. Preferably, the recombinant prokaryotic cell is a recombinant bacteria, preferably, a recombinant coryneform bacteria. Preferably the coryneform bacteria comprises aerobically growing, asporogenous, non-partially-acid-fast, irregularly shaped gram-positive rods. A preferred coryneform bacteria is a Corynebacterium, preferably Corynebacterium glutami- cum. Non-limiting examples of suitable strains of the genus Corynebacterium, and the species Corynebacterium glutamicum (C. glutamicum), are Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum S91 14, Corynebacterium glutamicum ATCC 14067, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium acetoacidophi- lum ATCC 13870, Corynebacterium thermoaminogenes FERM BP-1539, Corynebacterium melassecola ATCC 17965, Corynebacterium glutamicum KFCC10065 and Corynebacte- rium glutamicum ATCC21608 (KFCC designates Korean Federation of Culture Collection, ATCC designates American type strain culture collection, FERM BP designates the collection of National institute of Bioscience and Human-Technology, Agency of Industrial Sci- ence and Technology, Japan).
Also preferred as a host cell are cells of the genus Brevibacterium, preferably Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC 14020.
Mostly preferred is Corynebacterium glutamicum strain ATCC 13032 or variants thereof, preferably Corynebacterium glutamicum HH09. The variants of Corynebacterium glutamicum strain ATCC 13032 are preferably characterized by an increased glutamate production, preferably wherein the variants of Corynebacterium glutamicum strain ATCC 13032 are characterized by a ribosomal RNA sequence (rRNA) of the 16S, 23S and/or 5S rRNA, preferably the 16S rRNA, with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to the 16S, 23S and/or 5S rRNA, preferably the 16S rRNA, sequence of Corynebacterium glutamicum strain ATCC 13032.
Preferably, the recombinant prokaryotic or eukaryotic cell, preferably, a recombinant microorganism, is capable of producing GABA, preferably by converting glutamate to GABA. A recombinant prokaryotic or eukaryotic cell capable of producing GABA is preferably obtained by modifying a suitable host cell. Preferably, as a host cell a prokaryotic or eukaryotic cell is used that endogenously produces glutamate. Alternatively, a host cell might be modified, preferably by genetechnological methods or by another method for genetic alteration (e.g. random mutagenesis and selection), in order to obtain a host cell capable of glutamate production or in order to obtain a host cell capable of increased glutamate production. Preferably, the recombinant prokaryotic or eukaryotic cell has no endogenous expression of an enzyme having a glutamate decarboxylase activity. Preferably, the recombinant prokaryotic cell is not an Escherichia coli cell. Preferably, in order to obtain a recombinant prokaryotic or eukaryotic cell capable of producing GABA a prokaryotic or eukaryotic host cell capable of producing glutamate is transformed with one or more nucleic acid sequences cod- ing for a glutamate decarboxylase (preferably, EC 4.1.1.15) as described herein. Thereby, the recombinant prokaryotic or eukaryotic cell can convert glutamate to GABA.
Preferably, the recombinant prokaryotic or eukaryotic host cell is further modified by reducing the expression and / or activity of an endogenous glutamate exporter as described here- in and / or by reducing the expression and / or activity of an endogenous GABA- aminotransferase (preferably, EC 2.6.1.19) as described herein. Thus, a preferred embodiment is a recombinant prokaryotic or eukaryotic cell capable of producing glutamate transformed with one or more nucleic acid sequences coding for a glutamate decarboxylase (preferably, EC 4.1.1.15) and further comprising one or more modifi- cations selected from
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glu- tamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the pro- moter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter;
(d) introducing into the recombinant microorganism a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO: 18, 20, 42- 49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid molecule encoding a protein having at least 70% identity with SEQ ID NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, or a functional equivalent, homologue, or a splice variant thereof;
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and
(iv) a nucleic acid molecule encoding the same glutamate exporter as any of the nu- cleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code,
or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid mole- cule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a glutamate exporter in the recombinant microor- ganism or an expression cassette expressing said ribozyme;
(g) introducing into the recombinant microorganism an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the glu- tamate exporter or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the glutamate exporter or the DNA or RNA molecule encoding the glutamate exporter, or an expression cassette expressing said factor, and thereby inhibiting expression and / or activity of the glutamate exporter;
and thereby reducing the expression and / or activity of the glutamate exporter in the recombinant microorganism.
Preferably, the recombinant prokaryotic or eukaryotic cell additionally has one or more alterations selected from the group consisting of reduced expression and / or activity of a GABA-aminotransferase, reduced expression and / or activity of a succinate semialdehyde dehydrogenase (preferably, EC 1.2.1.24) and increased expression of a glutamate dehy- drogenase (preferably, EC 1.4.1.3). Preferred is in addition to a reduction of the glutamate exporter expression and / or activity a reduction of the GABA-aminotransferase expression and / or activity and / or an increase of the glutamate dehydrogenase expression and / or activity. For producing the recombinant prokaryotic or eukaryotic cell of the present invention, common cloning and transfection methods that are familiar to a person skilled in the art are used, for example co-precipitation, protoplast transformation, protoplast fusion, electro- poration, retroviral transfection, particle bombardment, agrobacterium mediated transformation and the like, in order to secure expression of the stated nucleic acids in the respec- tive expression system. Suitable systems are described for example in Current Protocols in Molecular Biology, F. Ausubel et al., Publ. Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, or Leach, J., Lang, B. R. a. and Yoder, O. C. (1982) Methods for selection of mutants and in vitro culture of Cochliobolus heterostrophus. J Gen Microbiol, 128: 1719-1729, or E. Risseeuw et al.: "Integration of an insertion -type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by Gap repair." Molecular and Cellular Biology, vol. 16, No. 10, Oct. 1996, pp. 5924-5932, or M. Ward et al.: "Transformation of Aspergillus awamori and A niger by electroporation" Experimental Mycology, vol. 13, 1989, pp. 289- 293, and "Handbook of Corynebacterium" (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005). Suitable transformation vectors and expression systems are described herein.
Thus, another embodiment the present invention is a method for the production of a recom- binant microorganism, comprising the steps of
(A) transforming into a microorganism an exogenous nucleic acid comprising a nucleic acid encoding a glutamate decarboxylase (preferably, EC 4.1 .1 .15) in functional linkage with a promoter; and
(B) reducing or enhancing the expression and / or activity of one or more genes selected from the group consisting of a gene encoding for a glutamate exporter, a GABA- aminotransferase, a succinate semialdehyde dehydrogenase, a isocitrate dehydrogenase, a phosphoenol pyruvate carboxylase, a 2-oxoglutarate dehydrogenase, a isocitrate lyase, a phosphoenol pyruvate carboxykinase, a glutamine synthetase and glutamate dehydrogenase, which are described herein in more detail, by means of one or more methods described herein.
Preferred is a method for the production of a recombinant microorganism, comprising the steps of
(A) transforming into a microorganism an exogenous nucleic acid comprising a nucleic acid encoding a glutamate decarboxylase (preferably, EC 4.1 .1 .15) in functional linkage with a promoter; and
(B) applying one or more of the methods steps selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and / or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter;
(d) introducing into the recombinant microorganism a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO: 18, 20, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104, or a functional equivalent, homologue, or a splice variant thereof;
(ii) a nucleic acid molecule encoding a protein having at least 70% identity with SEQ I D NO: 19, 21 , 91 , 93, 95, 97, 99, 101 , 103, or 105, or a functional equivalent, homologue, or a splice variant thereof;
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and
(iv) a nucleic acid molecule encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code, or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a glutamate exporter in the recombinant microorganism or an expression cassette expressing said ribozyme;
(g) introducing into the recombinant microorganism an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the glutamate exporter or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the glutamate exporter or the DNA or RNA molecule encoding the glutamate exporter, or an expression cassette expressing said factor, and thereby inhibiting expres- sion and / or activity of the glutamate exporter;
and thereby reducing the expression and / or activity of the glutamate exporter in the recombinant microorganism.
Preferably, the method further comprises the step of reducing or enhancing in the microor- ganism the expression and / or activity of one or more genes selected from the group consisting of a gene coding for a GABA-aminotransferase, a succinate semialdehyde dehydrogenase, a isocitrate dehydrogenase, a phosphoenolpyruvate carboxylase, a 2-oxoglutarate dehydrogenase, a isocitrate lyase, a phosphoenolpyruvate carboxykinase, a glutamine synthetase, and glutamate dehydrogenase, preferably, reducing a glutamate exporter expres- sion and / or activity, and / or reducing a succinate semialdehyde dehydrogenase expression and / or activity, and / or enhancing a glutamate dehydrogenase expression and / or activity, by means of one or more methods described herein. Preferred is in addition to a reduction of the glutamate exporter expression and / or activity a reduction of the GABA- aminotransferase expression and / or activity and / or an increase of the glutamate dehy- drogenase expression and / or activity.
The recombinant cells described herein are useful for the methods described herein, in particular, for the methods for the production of GABA. Expression constructs and vector constructs In another embodiment, the present invention provides an expression cassette, comprising at least one nucleic acid sequence as defined above, which sequence is operatively linked to at least one regulatory nucleic acid sequence; as well as a recombinant vector, comprising at least one such expression cassette.
All the nucleic acid sequences mentioned herein (single-stranded and double-stranded DNA and RNA sequences, for example cDNA and mRNA) can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. Chemical synthesis of oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896- 897). The accumulation of synthetic oligonucleotides and filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989).
Nucleic acid constructs according to the invention comprise in particular sequences selected from those specifically mentioned herein (e.g., overexpression constructs, antisense constructs, dsRNA constructs or homologous recombination constructs) and are preferably contained in a vector construct. Typically the nucleic acid constructs are functionally linked with one or more promoter sequence.
Various promoters are known in the art and may be introduced in an appropriate position (typically upstream) of nucleotide sequence so as to drive expression of a nucleic acid encoding the protein of interest. Promoters can be constitutive or non-constitutive. Non- constitutive promoters can be inducible. Preferred promoters for gene expressing according to the present invention are the CJ4 promoter, preferably as in pCES208, or the pSod promoter, preferably as in pClick5aMCS.
Further suitable promoters for use in coryneform bacteria, preferably in Corynebacterium glutamicum, are also described in "Handbook of Corynebacterium" (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005), in Patek M, et al., 2013, Microbial Biotechnology, vol. 6, p. 103-1 17, or in Patek M and Nesvera J, Promoters and Plasmid Vectors of Corynebacterium glutamicum, in Yukawa H and Inui M, Corynebacterium glutamicum, Biology and Biotechnology, Microbiology Monographs, 2013, VI I, ISBN: 978-3- 642-29856-1 , p. 51 -88.
Apart from promoters and terminators, examples of other regulatory elements that may be mentioned are targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, replication origins and the like. Suitable regulatory sequences are described for example in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). In addition to these regulatory sequences, the natural regulation of these sequences can still be present in front of the actual structural genes and optionally can have been altered genetically, so that natural regulation is switched off and the expression of the genes has been increased. The nucleic acid construct can also be of a simpler design, i.e. without any additional regulatory signals being inserted in front of the coding sequence and without removing the natural promoter with its regulation. Instead, the natural regulatory sequence is silenced so that regulation no longer takes place and gene expression is increased. A preferred nucleic acid construct advantageously also contains one or more of the aforementioned enhancer sequences, functionally associated with the promoter, which permit increased expression of the nucleic acid sequence. Additional advantageous sequences, such as other regulatory elements or terminators, can also be inserted at the 3' end of the DNA sequences. One or more copies of the nucleic acids according to the invention can be contained in the construct. The construct can also contain other markers, such as antibiotic resistances or auxotrophy-complementing genes, optionally for selection on the construct.
Examples of suitable regulatory sequences are contained in promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ιρρ-lac-, lacl^ T7-, T5-, T3-, gal-, trc-, ara-, rhaP (rhaPBAD)SP6-, lambda-PR- or in the lambda-PL promoter, which find application advantageously in Gram- negative bacteria. Other advantageous regulatory sequences are contained for example in the Gram-positive promoters ace, amy and SP02, in the yeast or fungal promoters ADC1 , MFalpha, AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH. Artificial promoters can also be used for regulation.
For expression, the nucleic acid construct is inserted in a host organism advantageously in a vector, for example a plasmid or a phage, which permits optimum expression of the genes in the host. In addition to plasmids and phages, vectors are also to be understood as meaning all other vectors known to a person skilled in the art, e.g. viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally. These vectors represent a further embodiment of the invention. Suitable plasmids are, for example in corynebacteria, pCES208 or pClick5aMCS, in E. coli, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pi N-l 11113-B 1 , Agt1 1 or pBdCI ; in nocardioform actinomycetes pJAM2; in Streptomyces plJ 101 , pi J364, plJ702 or plJ361 ; in bacillus pUB1 10, pC194 or pBD214; in Corynebacterium pSA77 or pAJ667; in fungi pALS1 , plL2 or pBB1 16; in yeasts 2alphaM, pAG-1 , YEp6, YEp13 or pE BLYe23 or in plants pLGV23, pGHIac+, pBIN 19, pAK2004 or pDH51. The aforementioned plasmids represent a small selection of the possible plasmids. Other plasmids are well known to a person skilled in the art and will be found for example in the book "Cloning Vectors" (Eds. Pouwels P.H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Further suitable expression vectors for use in coryneform bacteria, preferably in Corynebac- terium glutamicum, are described in "Handbook of Corynebacterium" (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005) or in Patek M and Nesvera J, Promoters and Plasmid Vectors of Corynebacterium glutamicum, in Yukawa H and Inui M, Corynebacterium glutamicum, Biology and Biotechnology, Microbiology Monographs, 2013, VII, ISBN: 978-3-642-29856-1 , p. 51 -88.
In a further embodiment of the vector, the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can be inserted advantageously in the form of a linear DNA in the microorganisms and integrated into the genome of the host organism through heterologous or homologous recombination. This linear DNA can comprise a linearized vector such as plasmid or just the nucleic acid construct or the nucleic acid according to the invention.
For optimum expression of exogenous genes in organisms, it is advantageous to alter the nucleic acid sequences in accordance with the specific codon usage employed in the organism. The codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organism in question.
The production of an expression cassette according to the invention is based on fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator signal or polyadenylation signal. Common recombination and cloning techniques are used for this, as described for example in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) as well as in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al.,
Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
Preferred vector constructs for overexpressing GAD in bacterial cells, preferably coryneform bacteria are shown in Fig. 2 and Fig. 19. The nucleotide sequence of the preferred vectors of Fig. 19 is shown in SEQ ID NO: 1 13-1 15.
Preferred constructs are suitable for inducing homologous recombination when transformed in the prokaryotic or eukaryotic cell. Preferably, the construct suitable for inducing homolo- gous recombination comprises a first nucleotide sequence comprising a fragment of the 3'- portion of the target sequence (preferably, at least 25, 50, 75, 100, 125, 150, 175, or at least 200 nucleotides in length) and a second nucleotide sequence comprising a fragment of the 5'-portion of the target sequence (preferably, at least 25, 50, 75, 100, 125, 150, 175, or at least 200 nucleotides in length) and a third nucleotide in between the first and the second sequence, wherein integration of the construct preferably via double crossing-over leads to a disruption of the expression of the target sequence, preferably because the third nucleotide sequence is integrated into the target sequence and differs from the target sequence by at least one nucleotide. Preferably, the third sequence comprises nucleotide sequence suitable to be excised via the cre-LoxP system. Preferably, the third sequence comprises a nucleotide sequence, preferably differing from the target sequence, flanked by two loxP sides. Preferably, the construct suitable for inducing homologous recombination is as shown in Fig. 3, wherein the target gene can be any gene of interest as described herein, preferably the GABA-aminotransferase and / or the glutamate exporter.
Preferably, the vector construct comprise a 3'-portion of the GABA-aminotransferase coding sequence (preferably, at least 25, 50, 75, 100, 125, 150, 175, or at least 200 nucleotides in length) and a second nucleotide sequence comprising a fragment of the 5'-portion of the GABA-aminotransferase coding sequence (preferably, at least 25, 50, 75, 100, 125, 150, 175, or at least 200 nucleotides in length), wherein in between these two sequences is a third nucleotide sequence, preferably a kanamycin resistance gene. Preferably, the con- struct further comprises another antibiotics resistance gene, preferably an ampicillin resistance gene. Preferably, the vector construct comprises a nucleotide sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or even 100% sequence identity to SEQ I D NO: 1 16. Methods for the production of pyrrolidone
In another aspect of the invention, the fermentatively produced GABA may be applied for producing pyrrolidone by applying standard techniques of organic synthesis. In general, pyrrolidone can be synthesized by cyclization of GABA under removal of water. Thus, the present invention relates to a method of preparing 2-pyrrolidone, which method comprises a) preparing GABA by a method as described herein; and
b) cyclization of said GABA to produce 2-pyrrolidone.
Preferably, the method comprises
a) preparing GABA by a method as described herein;
b) isolating GABA; and
c) cyclization of said GABA to produce 2-pyrrolidone.
Preferably, GABA is converted to 2-pyrrolidone by heating the fermentation broth after fer- mentation and removal of biomass. Biomass can be removed preferably by means of cen- trifugation and preferably subsequent pasteurization and/or preferably subsequent ultrafil- tration. Preferably, the so processed fermentation broth can be concentrated prior the cyclization reaction.
Preferably, prior cyclization GABA is purified from the fermentation broth, preferably by ion exchange chromatography, preferably by acidic ion exchange chromatography.
Preferably, cyclization of said GABA to produce 2-pyrrolidone is achieved by heating the GABA solution, preferably the GABA containing processed fermentation broth. Heating is preferably at 170-300°C, preferably, at 200°C, at 210°C,at 220°C, preferably above 220°C, i.e., at 230°C, 240°C, 250°C, 260°C, 270°C, 280°C, 290°C, or 300°C, preferably between 220°C and 260°C, preferably between 200°C and 260°C, preferably between 200-240°C.
Preferably, the heating time is between 5 min and 9 hours. Preferably, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, or 60 min, preferably 1.5 hours, 2 hours, 2.5 hours, or 3 hours, preferably 4 hours, 6 hours, 7 hours, 8 hours, or 9 hours. Preferably, the heating time is dependent on the temperature and the pressure applied to the cyclization reaction.
Preferably, the cyclization reaction is performed at a pH between pH6 and pH 10, preferably between pH6 and pH9, more preferably between pH7 and pH9.
Preferably, the cyclization reaction is performed at 200°C for 3 hours at pH7, pH8 or pH9 or at 240°C for 30 min at pH7, pH8 or pH9 or at 300°C for 10 min at pH 7, pH 8 or pH9.
Preferably, the GABA concentration in the reaction solution is between 10-50%, preferably between 15-40%, preferably between 20-45%, preferably 30-40%, preferably 15% or pre- farably 40%.
Preferably, the amount of sugars, preferably, glucose and/or molasses, in the GABA solution, preferably in the processed fermentation broth after fermentation reaction and removal of biomass, is reduced, preferably is between 0-50% of their initial amount, preferably be- tween 10-50%, 10-20%, or 5-15%, preferably, 50%, 40%, 30%, 20%, 10%, 5%, or 0%. Preferably, salts and fermentation byproducts were removed or reduced in the processed fermentation broth after fermentation reaction and removal of biomass prior the cyclization reaction. Preferably, cyclization of said GABA to produce 2-pyrrolidone is achieved by removal of water, preferably by heating. Preferably, 2-pyrrolidone can be obtained from GABA solutions by heating and distillation under reduced pressure. Preferably, cyclization is carried out in a solvent that assists in the removal of water. Preferably, GABA solutions can be heated in a flask by using an oil bath (preferably, at 130°C-150°C) to remove the water. Af- ter removing the water from the solution, preferably, the reaction is further heated, preferably 200°C-240°C, for 10-40min, preferably 20min, to proceed the cyclization reaction. Preferably, separation and purification of 2-pyrrolidone from byproducts and contaminants is achieved by distillation, preferably under reduced pressure (preferably, at 10-14 hPa at 1 10- 130°C, most preferably, at 12 hPa, 120°C).
Alternatively, 2-pyrrolidone can be obtained from GABA solutions, preferably from GABA in an aqueous solutions or in fermentation broth, preferably after removal of biomass, preferably by centrifugation, preferably with subsequent ultrafiltration, by heating with partial removal or without removal of water, preferably under increased pressure (preferably, 20-150 bar, preferably 50-150 bar, preferably 20-100 bar, more preferably 100 bar), preferably under temperature and pH conditions and for a time and with concentrations as described above. Preferably, water can be subsequently removed, preferably by distillation, or evaporation, preferably rotary evaporation. 2-pyrrolidone can preferably be subsequently isolated by distillation. This can be done continuously or discontinuously. Preferably, prior cyclization GABA is purified from the fermentation broth, preferably by ion exchange chromatography, preferably by acidic ion exchange chromatography.Preferably, 2-pyrrolidone can be ob- tained by heating an aqueous GABA solution as described above, preferably at 180°C - 350°C, preferably at 200°C-250°C, preferably for 10 sec - 5h, preferably for 1 min - 40min, preferably for 10-40min, preferably 20min, preferably below 20min, preferably for 10sec- 5min (preferably, at 300°C for 30sec or 3 min or at 250°C for 5min or at 200°C for 30 min), preferably by heating the aqueous GABA containing fermentation broth after removal of the microbial cells, preferably under increased pressure.
For cyclization of GABA to produce 2-pyrrolidone in the presence of water preferably a continuous laboratory reactor is used. Alternatively, cyclization can be done in a discontinuous reaction. Preferably, cyclization can be achieved by heating a GABA containing solution by means of a micro reactor, a microwave, or an autoclave.
In a preferred embodiment, the 2-pyrrolidone obtained from the GABA produced by a method of the present invention is further processed to venylpyrrolidone, preferably N- vinyl pyrrol idone (NVP). Preferably, the 2-pyrrolidone obtained from the GABA produced by a method of the present invention is further processed to N-vinylpyrrolidone by reacting with acetylene, preferably by means of a Reppe synthesis. Preferably, vinylpyrrolidone can be polymerized to polyvinylpyrrolidone (PVP) or polyvinyl polypyrrol idone.
Method for the production of polymers In a further aspect of the invention, the present invention provides a process for the production of polymers, in particular polyamides, comprising a step as mentioned above for the production of GABA. The GABA is preferably reacted in a known manner with itself or with at least one different co-monomer, selected from amino- and hydroxycarboxylic acids, by applying standard methods of polymer synthesis. Thus, the present invention relates to a method of preparing a polymer, preferably a polyamide, which method comprises a) preparing GABA by a method as described herein; and b) polymerizing said GABA, optionally in the presence of at least one further suitable polyvalent copolymerizable co-monomer, selected, for example, from aminocarboxylic acids and hydroxycarboxylic acids. Preferably, the method comprises
a) preparing GABA by a method as described herein;
b) isolating GABA; and
c) polymerizing said GABA, optionally in the presence of at least one further suitable polyvalent copolymerizable co-monomer, selected, for example, from aminocarboxylic acids and hydroxycarboxylic acids.
Suitable co-monomers are for example derived from C2-C31 , preferably C4 -Cio-straight or branched chain monocarboxylic acids, carrying at least one reactive hydroxyl or amino group. Such hydroxyl- or ami no-substituted, copolymerizable carboxylic acids are preferably derived from straight-chain or branched, saturated or mono- or poly-unsaturated C2-C30- monocarboxylic acids. In particular, said hydroxyl- or amino-substituted, copolymerizable carboxylic acids carry a straight-chain mono- or polyunsaturated hydrocarbyl residue or a mixture of such residues with an average length of 1 -30, preferably 3-9 carbon atoms. Particularly preferred residues of said hydroxyl- or amino-substituted, copolymerizable carbox- ylic acids are:
- saturated, straight-chain residues like CH3-, C2H5-; C3H7-; C4H9-; C5H 11-; C6H13-; C7H15-; C8H17-; C9H19-; C10H21-; C11 H23-; C12H25-; C13H27-; C14H29-; C15H31-; C16H33-; C17H35-; C18H37-; C19H39-; C20H41-; -C21 H43-; C23H47-; C24H49-; C25H51-; C29H59-; C30H61-;
- saturated, branched residues like 1SO-C3H7-; 1SO-C4H9-; 1S0-C18H37-;
- mono-unsaturated, straight-chain residues like C2H3-; C3H5-; C15H29-; C17H33-; C21 H41-;
- two-fold unsaturated, straight-chain like C5H7-; C17H31-,
wherein the residues are modified so that they carry at least one functional substituent, selected from hydroxyl and amino groups, required for copolymerization. Preferred carboxylic acids as co-monomers for polymerization of GABA are RiCO(OH), wherein R1 is preferably selected from the group consisting of CH2(OH)-, CH2(NH2)-, C2H4(OH)-, C2H4(NH2)-, C2H3(OH)2-, C2H3(NH2)2-, C3H4(OH)-, C3H4(NH2)-, 03Η3(ΟΗ)2-, or 03Η3(ΝΗ2)2.
The present invention further relates to a method of preparing vinylpyrrolidone, preferably N-vinylpyrrolidone, which method comprises
a) preparing GABA by a method as described herein; and
b) processing said GABA to vinylpyrrolidone, preferably by reacting said GABA with acetylene, preferably by means of a Reppe synthesis, to form vinylpyrrolidone, preferably N-vinylpyrrolidone. Preferably, said vinylpyrrolidone is further processed by polymerization to form polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone. Examples
The following examples only serve to illustrate the invention. The numerous possible varia- tions that are obvious to a person skilled in the art also fall within the scope of the invention.
Unless otherwise stated the following experiments have been performed by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering, fermentative production of chemical compounds by cultivation of microorganisms, in particular of Corynebacteria, and in the analysis and isolation of products. See also Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and Chmiel et al. (Biopro- cesstechnik 1. Einfuhrung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991 ) and "Handbook of Corynebacterium" (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005).
Example 1 : Culturing Corynebacterium glutamicum and fermentation
Cells of Corynebacterium glutamicum were cultured under the following conditions. Shaking flask medium (1 L) (SF-Medium):
Glucose 80 g
Molasse (cane) 40 g
FeS04x7H20 10 mg
Figure imgf000076_0001
Thiamine hydrochloride 1 mg
(NH4)2S04 30 g
KH2P04 1.3 g
Figure imgf000076_0002
Soytone (Bacto) 5-10 g
Biotin 100 Mm (0 - 400 μΜ)
CaC03 (Riedel de Haen) 50 g
Feed after 24 h (if longer incubation necessary):
Glucose 1.25 g
(NH4)2S04 0.3 g
Fermentation medium (1 L) (F-Medium):
Glucose
Molasse
(NH4)2S04 1 - 40 g
KH2P04 1.3 g MgSO4 x 7H20 0.8 g
Soytone 5 g
Thiamine monochloride 1 mg
Biotin 200 μ
The pH was adjusted to 7.5 and then regulated at pH 6.4 with ammonia-water (25 %).
Glucose concentration was between 2 and 8 % during complete fermentation. Sugar solution:
Glucose and molasse mixture to a total concentration of 60 %.
Shaking flask experiments were carried out in SF-Medium (see above) at 30 °C and at 200 rpm. The growth of C. glutamicum strains is monitored via the optical density at 600 nm over time after the residual CaCC>3 is dissolved by the addition of 1 HCI.
The precultures for fermentation are grown in SF-Medium (see above) for 24 to 48 hours at 30 °C and at 200rpm (20 % volume of fermentation starting volume). The fermentation is carried out in F-Medium. The feed, 40 to 60% of the total volume, consists of a sugar solution (see above) and is started at a total sugar concentration of ~ 2 %.
The fermentations were controlled by (i) a pH-value of 6.4, (ii) a total glucose concentration of 20 to 80 g/L and a stirrer speed of 300 to 900 rpm keeping a constant O2 partial pressure over the complete fermentation time. At the end of the fermentation the glucose was completely used up and the cells were harvested by centrifugation. The resulting fermentation broths were analyzed concerning production of GABA, glutamate and residual glucose concentration. Alternative culture conditions are described as follows. The pre-seed culture is prepared by inoculating a loopful of cells from slant culture into the flasks containing 25 ml_ of SF- medium and is cultivated at 31.5°C for 24h with shaking (250 rpm). One ml of pre-seed culture was transferred into a 50ml of fresh SF-medium, and cultivated at 31.5°C with shaking (250 rpm). The complete seed culture is transferred into a 5 L jar fermenter containing 1 ,600mL of production medium (F5H).
Culture conditions of 5 L jar fermenter: agitation speed 850 rpm, air flow rate 2.0 vvm, temperature 31 .5°C, pH 7.5 (with ammonia water). The total sugar concentration in the fermenter was controlled to be 2%-8% by feeding of sugar solutions. The studies on a fermen- tation time course was conducted using a 5 L-jar fermenter (Kobiotech Co., Inchon, Korea). The fermentation medium (F5H) used for GABA production consisted of 40g of glucose, 40g of molasses (cane), 1.0g of (NhU^SC , 1.3g of KH2PO4, 0.8g of MgS04-7H20, 2ml of soybean protein hydrolysate, 600 g of thiamine monohydrochloride and 0.4g of neorin (an- tifoam agent) per liter and the pH was adjusted to 7.5 with ammonia water.
Example 2: Cloning of a glutamate decarboxylase (gad) gene
Identification, cloning and expression of GAD genes (e.g., tomato-potato-tomato chimera (SL- GAD), E. coli) was done as described in WO2009103547A1. Furthermore, GAD expression vectors were prepared using PCR from different microorganisms such as Mycobacterium smegmatis (ATCC607) and Lactobacillus plantarum (ATCC8014). For expression in C. glu- tamicum the amplified GAD genes were placed under the control of the CJ4 or pSod promoter in a suitable expression vector. Expression vectors were pClik5aMCS and pCES (pCES208), which are shown in Fig. 2 and also further described in WO2009103547A1 (cf. WO2009103547A1 , examples 1 -3).
Alternative GAD expression vectors based on pClik under the control of the pSod promoter are shown in Figure 19 (pClik MCS-SL-GAD, SEQ ID NO: 1 13; pClik MCS-E.coli-GAD, SEQ ID NO: 1 14; pClik MCS-M .smegmatis, SEQ ID NO: 1 15). The transformants were isolated, cultured in the shake flasks, and their productivities of GABA were evaluated (cf. Table 2 and 3).
Example 3: Transformation of Corynebacterium glutamicum
Cells of Corynebacterium glutamicum were transformed by means of electroporation.
Therefore, the preculture for the production of electocompetent C. glutamicum cells is grown over night at 30 °C with 200 rpm in a rich medium (BHI, Bacto Brain Heart Infusion, Becton Dickinson REF 237500). 5 ml. of the preculture was used as inoculum for the main culture in 300 mL BHI medium. The main culture was grown to an OD of 1.6 (30 °C, 200 rpm) and the cells were harvested via centrifugation. The resulting pellet was washed twice with EP-washing-buffer (10 % glycerol, 8 mM Tris-HCI, pH 7.4) at 4 °C. In a third washing step the cells were resuspended in 10 % glycerol and centrifuged again. The resulting pellet was resuspended in 1 mL of 10 % glycerol at 4 °C.
The electroporation was done in a BioRad Gene Pulser using cuvettes with a gap width of 2 mm. The conditions for electroporation are 2.5 kV, 25 F and 400 Ω. 150 μί of cells were transformed with 10 to 15 μg of DNA. After the electroporation the cells were transferred to 4 mL preheated (46 °C) BHIS medium (BHI with 91 g/L sorbitol) and waved for 6 min in a water bath at 46 °C. After this the cells were incubated for 1 to 2 h at 30 °C vigorously shaking at 200 rpm. The cells were plated on agar plates with BHIS agar containing the respec- tive selection marker. An alternative transformation protocol is described in the following. An Erlenmeyer flask containing 10 mL of complex medium (BHI) was inoculated with a loopful of cells from a slant culture and incubated at 30°C for 24h with shaking (200 rpm). 1 mL of culture broth grown up to an optical density of 0.3 at 610 nm were transferred into an Erlenmeyer flask containing 100 mL of EPO medium, and incubated at 18.0°C for 28h with shaking (120 rpm). The cultivation was stopped when the turbidity of the culture broth optical density at 610 nm reached 0.8 to 1 .0, and the culture broth was chilled on ice for 10 min. Then the cells were harvested by centrifugation at 4000xg for 10 min, washed four times with 50 ml 10 % (v/v) glycerol and resuspended in 0.5 ml 10% glycerol. DNA (2 μΙ) was mixed with 50 μΙ of the competent cells. A single pulse of 2.5 kV was applied after transferring the mixture to a prechilled 0.2 cm electroporation cuvette. After the electroporation, cells were then incubated for 1 hour at 30.0°C to allow for recovery and expression of the antibiotic resistance marker. Afterwards cells were plated on selective Agar plates containing the respective antibiotic.
The EPO medium used for preparation of competent cells consisted of 20g of glucose, 0.2g of isonicotinic acid, 1.25g of glycine, 1 g of tween 80, 2.5g of NaCI, 5g of beef extract, 5g of yeast extract and 10g of peptone per liter. Example 4: Evaluation of GABA production
Quantitative analysis of glucose, glutamic acid / mono sodium glutamate (MSG) and GABA in fermentation broths was done by HPLC with the following parameters (if not stated otherwise):
Column: Aminex HPX-87 C, 300*7.8 mm (Biorad)
Precolumn: Carbo-C
Temperature: 30 °C
Flow rate: 1.00 mL/min
Injection volume: 5.0 il
Detection: Rl- Detector
Maximum pressu 200 bar
Matrix: Fermentation broths, samples are sterile filtrated before injection Calibration: GABA: 10 g/L, 25 g/L, 50 g/L
2-Pyrrolidon: 25 g/L, 50 g/L
Retention time: GABA: ~ 18 min
2-Pyrrolidon: -22 min
Under above method, major estimated components in the cell culture sample matrix could be well separated from GABA, without interfering GABA's quantitation.
The amount of GABA in the sample was determined by a standard calibration method. Standard samples containing GABA from 10 to 50 g/L were injected and the peak areas were used for calibration. Linear regression coefficient of the calibration curve was above 0.99. The peak area of the standards and the samples was used to calculate the amount of GABA by Waters LC Millenium software.
The accumulation of GABA in recombinant strains containing a GAD gene was measured for instance as indicated in Table 2 (cf. WO2009103547A1 , here method as described in exam- pie 4 of WO2009103547A1 ).
Table 2: GABA production in shaking flask culture
Strains GABA (mmol/g cell)
ATCC 13032 0.0
+ pClik5aMCS 0.0
+ pClik5aMCS gadA 0.2
+ pClik5aMCS gadBC 0.4
+ pClik5aMCS Psod SL_gad 1.2
The GABA production of wildtype C. glutamicum ATCC13032 and wildtype C. glutamicum HH09 (a derivative of C. glutamicum ATCC 13032 with high glutamate production) transformed with pClick5aMCS vector containing chimeric GAD gene from potato-tomato, pCES.E.GAD vector containing GAD gene from E.coli and pCES.M.GAD vector containing GAD gene from M.smegmatis, respectively, was also determined as shown in Table 3. Table 3: Growth and GABA production by recombinant strains.
Concentration
GAD gene Growth Concentration
Transformant Promoters of glutamic from (610nm) of GABA (g/L)
acid (g/L) pClick5aMCS to Potato-
C. glutamicum tomato pSod 14.02 1 .90 0.29
ATCC13032 chimeric
pCES.E.GAD to
C. glutamicum E.coli CJ4 9.82 0.73 0.33
ATCC13032
pCES.M.GAD to
C. glutamicum M.smegmatis CJ4 13.24 0.73 0.30
ATCC13032
pClick5aMCS to Potato-
C. glutamicum wt- tomato pSod 13.75 12.12 17.91
HH09 chimeric Example 5: Knock out of the 4-aminobutyrate aminotransferase gene in C. glutamicum
To further increase the GABA production by reducing the amount of GABA processed to succinate semialdehyde the gene coding for a GABA-aminotransferase (gaba-T) was knocked out in C. glutamicum cells (cf. Fig. 1 ).
The pGem.T easy-gaba-T(F,B)-loxp.km vector (pGem.T easy - gabaT loxP; SEQ ID NO: 1 16) was constructed for knock-out of gaba-T gene by Cre-loxP system. The strategy for deletion of 4-aminobutyrate aminotransferase gene was based on homologous recombina- tion comprising double crossing-over using a Cre-loxP system. A fragment comprising loxP- KmR-loxP was inserted by a double cross-over into a 4-aminobutyrate aminotransferase gene in C. glutamicum wildtype HH09 after transformation (cf. Fig. 3 and Fig. 4) resulting in C. glutamicum HH 102. The insertion of loxP-KmR-loxP into a 4-aminobutyrate aminotransferase gene was confirmed by PCR (cf. Fig. 5) based on the expected fragment size indicated in Fig. 4.
The loxP-KmR-loxP fragment was subsequently removed by transforming a C. glutamicum HH 102, the recombinant strain with a loxP-KmR-loxP insert in a 4-aminobutyrate ami- notransferase gene, with a plasmid containing a Cre gene (pCES.CAT-ICL.CRE (pCICRE), cf. Fig. 6), and by expressing the Cre gene, resulting in C. glutamicum HH103. The Cre recombinant gene in pCES.CAT-ICL.Cre vector was induced by 1 % ammonium acetate. Expression of Cre recombinase was confirmed by gel electrophoresis and coomassie brilliant blue staining (cf. Fig. 7). Subsequently, the C. glutamicum HH 103 cells were pCICRE cured, resulting in C. glutamicum HH 104.
In order to enable GABA production C. glutamicum HH 104 cells were transformed with a plasmid comprising a construct for expressing a potato-tomato chimeric GAD gene in C. glutamicum, resulting in C. glutamicum HH 105. An overview of the different transformants is shown in Table 4.
Table 4: overview of the different transformants of C. glutamicum
Transformant Characteristics
C. glutamicum wt-HH09 -
C. glutamicum HH102 Agaba-T ('loxP-knY-loxP' ingegrated)
C. glutamicum HH103 Agaba-T (krr\r eliminated), harboring pCICRE
C. glutamicum HH104 Agaba-T, pCICRE cured
C. glutamicum HH105 Agaba-T, harboring pClik5aMSC-SL_GAD
C. glutamicum HH106 Agaba-T, SL_GAD integrated Transformant Characteristics
Agaba-T, SL_GAD integrated
C. glutamicum HH107
harboring pClik5aMSC-SL_GAD
The GABA production of these modified C. glutamicum cells is shown in Fig. 8. Fig. 8 shows a fermentation time courses of C. glutamicum HH 105 which contains a GAD gene from potato-tomato and in which the 4-aminobutyrate aminotransferase gene was eliminated by loxP-KmR-loxP disruption (growth (·), glucose (o), GABA (■), mono-sodium gluta- mate (MSG) (n)).The production of GABA by the C. glutamicum HH 105 developed was performed by employing a fed-batch culture system, which produced 132g/L of GABA.
To confirm the production of GABA, flask-scale culture was performed and the production of GABA was analyzed by HPLC. Flask-scale culture was performed for 72 hours and collect- ed samples were used for the analysis of cell growth and GABA production. C. glutamicum wt-HH09, HH 104, HH 105, HH 106, HH 107 were used for the flask-scale culture and production of glutamic acid and GABA of each strain was analyzed by HPLC.
Glutamic acid was accumulated in C. glutamicum wt-HH09 and HH 104 which don't have the glutamate decarboxylase (gad) gene. On the other hand, glutamic acid was not accumulated in C. glutamicum HH 105, HH106 and HH 107. In addition, GABA was accumulated in C. glutamicum HH 105, HH 106 and HH 107 because glutamate decarboxylase (GAD) which converts glutamic acid to GABA was expressed in these strains. GABA production of C. glutamicum HH 105, HH 106 and HH 107 was 20.47 g/L, 15.78 g/L and 26.95 g/L, respec- tively.
Table 5: Production of γ-aminobutyric acid and glutamic acid by C. glutamicum wt-HH09 (wildtype), C. glutamicum HH 104 (gaba-t -), C. glutamicum HH105 (pClik5aMCS pSod SL_GAD to C. glutamicum HH 104), C. glutamicum HH 106 (Potato-tomato chimeric gad gene was integrated to C. glutamicum HH 104) and C. glutamicum HH 107 (pClik5aMCS pSod SL_GAD to C. glutamicum HH 106). Strain was cultured in 500ml of Erlenmeyer flasks contain 50ml of medium at 30.0°C for 72 hours with shaking (250rpm)
Cone, of
Cell growth Cone, of Glutamic acid
Strain GABA
(OD 610nm) (g/L)
(g/L)
C. glutamicum
9.46 0 23.35
wt-HH09
C. glutamicum
10.4 25.84
HH104
C. glutamicum
15.5 20.47 0.07
HH105 Cone, of
Cell growth Cone, of Glutamic acid
Strain GABA
(OD 610nm) (g/L)
(g/L)
C. glutamicum
9.21 15.78 0.06
HH106
C. glutamicum
11.3 26.95 0.12
HH107
To optimize fermentation condition for the production of GABA, 5L-jar fermentation using C. glutamicum HH105, HH106 and HH 107 was performed. In the case of C. glutamicum HH 105, cell growth was reached to 142.0 at OD610 after 45- hour cultivation, then the OD610 value was slowly decreased after 66-hour cultivation. The growth of C. glutamicum HH 106 was reached to 173.0 at O D610 after 36-hour cultivation and C. glutamicum HH 107 was reached to 136.0 at OD610 after 33-hour cultivation then slowly decreased.
Production of GABA and glutamic acid in C. glutamicum HH105 was 161.18 g/L and 0.27 g/L after 120-hour cultivation, respectively. In C. glutamicum HH 106, 140.76 g/L of GABA and 140.76 g/L of glutamic acid were produced after 105-hour cultivation. Production of GABA and glutamic acid in C. glutamicum HH 107 was 199.29 g/L and 0.80 g/L after 108- hour cultivation, respectively (cf. Table 6). As a result, the production of GABA in C. glutamicum HH105, HH 106 and HH 107 was non-growth associated.
Table 6: GABA yield of production in a fermenter scale
Figure imgf000083_0001
Respective fermentation time courses in fed-batch culture for the production of GABA by C. glutamicum HH 105, HH106, and HH 107 are shown in Fig. 10, 1 1 , and 12, respectively.
The GABA and glutamate production as well as the growth of the cells of C. glutamicum cells having the gene encoding the aminotransferase disrupted using the cre-loxP system as de- scribed above is also shown in Fig. 16-18. The cells used for obtaining the data displayed in Fig. 16-18 have the chimeric GAD gene integrated in the chromosomal bioD gene and were cultured in flask-scale (wt coryne HH09 (■), 1xGAD; -bioD; -Aminotransferase (o), 1xGAD; - bioD; -Aminotransf. -Glut.Exp. (X), 1xGAD; -bioD; -Aminotransf.; -Glut.Exp. ( ), 1xGAD; - bioD; intact Aminotransf. (= wtHH09 with 1xGAD in bioD) (♦), 1xGAD; -bioD; intact Ami- notransf. (= wtHH09 with 1xGAD in bioD) (+)). The data of cells characterized as 1xGAD; - bioD; -Aminotransf.; -Glut. Exp. were obtained from two different cellular clones ((X) and (A), respectively). The same applies to cells characterized as 1xGAD; -bioD; intact aminotransf. (= wtHH09 with 1xGAD in bioD) ((♦) and (+), respectively).
Fig. 16 shows an increased amount of GABA production for cells with a disrupted gene coding for the aminotransferase compared to cells expressing a glutamate decarboxylase gene (gad) together with a functional aminotransferase.
Alternatively, the knock out of the gene coding for a GABA-aminotransferase was achieved in C. glutamicum by inserting a GAD gene (potato-tomato chimera) by means of homologous recombination in the coding sequence of the GABA-aminotransferase of C. glutamicum cells expressing GAD (potato-tomato chimera) according to a protocol described in "Handbook of Corynebacterium" (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005). Thereby the expression of the GABA-aminotransferase gene was disrupted. Using this approach C. glutamicum cells with two copies of the GAD gene were generated (one copy in the BioD gene and one copy in the GABA-aminotransferase). The effect of this knock out of the 4-aminobutyrate aminotransferase gene on the GABA production of C. glutamicum is shown in Fig. 13. Figure 13 shows a comparison of the fermentation time courses of C. glutamicum cells which comprises a GAD gene and C. glutamicum cells which comprises a GAD gene in combination with a disruption of the gene coding for 4-aminobutyrate aminotransferase. The disruption of the 4-aminobutyrate ami- notransferase gene leads to an increased GABA production of C. glutamicum.
Example 6: Integration of glutamate decarboxylase (gad) gene into the chromosomal DNA of GABA-aminotransferase (gaba-T) knock-outed C. glutamicum
The glutamate decarboxylase (gad) gene was integrated into the chromosomal DNA of GABA-aminotransferase(gaba-T) knock-outed C. glutamicum HH104. The construction of integration plasmids was based on the sacB selection system. pClik int sacB bioD plasmid was used to construct the plasmid for the integration. The plasmids with glutamate decarboxylase (gad) gene were constructed using pClik int sacB bioD plasmid as a backbone. pClik int sacB bioD plasmid has Hindlll restriction site between bioD L and bioD R. To ligate the gad gene, primers with Hindl ll site were synthesized as shown in Table 7 and PCR was performed using pClik5aMCS-Psod.C.gad plasmid as a template. Recombinant PCR was performed to amplify the fragment, Psod chimeric gad 6XHis groEL using Psod chimeric gad 6XHis and 6XHis groEL as templates. Table 7: Primers used in PCR amplification
Figure imgf000085_0001
Amplified fragments were ligated to pGEM-T Easy plasmid and then digested by Hind 111 restriction enzyme to ligate to pClik int sacB bioD plasmid.
For the integration of glutamate decarboxylase (gad) gene to bioD gene of C. glutamicum HH 104, constructed plasmid was introduced to C. glutamicum HH 104 and integrated candidates were selected on LB plate containing 20 ug/ml kanamycin. Then, colony PCR was used to confirm selected colonies.
PCR result showed that the plasmid for the integration was successfully integrated to bioD gene of C. glutamicum HH 104 by single crossover recombination.
After the integration of glutamate decarboxylase (gad) gene into the chromosomal DNA of C. glutamicum HH 104, sacB system was used to remove selection marker (Kmr). Glutamate decarboxylase (gad) gene integrated candidate strain selected on LB agar plate containing 20ug/ml kanamycin was cultivated in LB broth for 24hours. Then, the cultured strain was diluted by PBS and spread on LB agar plate containing 10% sucrose. After 24-hour incubation, grown colonies were tooth-picked on LB agar plate containing 10% sucrose and LB agar plate containing 10% sucrose and 20ug/ml kanamycin, then the colonies grown only on LB agar plate containing 10% sucrose were selected and removal of selection marker (Kmr) was confirmed by PCR.
Glutamate decarboxylase (gad) gene integrated C. glutamicum HH104 was named C. glu- tamicum HH 106 and C. glutamicum HH 106 harboring pClik5aMCS-Psod.SL_GAD was named C. glutamicum HH 107.
Example 7: Effect of biotin concentration on the production of GABA
Seed culture of C.glutamicum HH 105 was conducted by inoculating a loopful of cells from slant culture into the Erlenmeyer flasks containing 10 ml of SF-Medium and by cultivating at 31.5°C for 24 hours with shaking (250rpm). One ml of seed culture broth was transferred into 25 ml of SF-Medium with various biotin concentrations, and cultivated at 31 .5°C for 72 hours with shaking (250rpm). The samples can be taken at time intervals for analysis of growth and GABA production.
Table 8: Effect of biotin concentration on the production of GABA and glutamic acid by C.glutamicum HH 105.
ConcentraConcentration
Trans- Growth Concentration
tion of biotin of glutamic acformant (610nm) of GABA (g/L)
(MQ/L) id (g/L)
0 8.34 13.92 14.1 1
20 1 1 .69 16.73 0.13
C. glutamic 50 12.52 17.31 0.20
urn HH105 100 12.12 18.03 0.00
200 13.26 28.06 0.12
300 12.62 31 .80 0.00
As shown in Table 8, the production of glutamic acid was decreased when biotin was added (above 20 pg/l). The production of GABA was increased 2.0 fold (13.92 g/l to 28.06 g/l) when 200 g of biotin was added compared with the biotin limitation by flask culture. Example 7.1 : Fed-batch culture with biotin for the production of GABA
The pre-seed culture of C.glutamicum HH 105 was prepared by inoculating a loopful of cells from slant culture into the flasks containing 10ml_ of SF-Medium and cultivating at 31 .5°C for 24h with shaking (250rpm). One ml of this pre-seed culture was transferred into a 50ml of SF-Medium, and cultivated at 31 .5°C with shaking (250rpm). 50ml of this seed culture was transferred into a 5L jar fermenter containing 1 ,600mL of F-Medium.
Culture conditions of 5L jar fermenter: agitation speed 850rpm, air flow rate 2.0 vvm, temperature 31.5°C, pH 7.5(with ammonia water). The total sugar concentration in the ferment- er was controlled to be 2%-8% by feeding of sugar solutions. The studies on a fermentation time course was conducted using a 5L-jar fermenter (Kobiotech Co., Inchon, Korea).
As shown in Fig. 9, the production of glutamic acid was decreased when biotin was added (100 g/I) by fed-batch culture. The production of GABA was increased 23% (132 g/l to 162 g/l) and yield was increased 10% (0.30 to 0.33) when 100 of biotin was added compared with the biotin limitation by fed-batch culture.
The production of GABA by the C.glutamicum HH 105 developed was performed by employing a fed-batch culture with biotin, which produced 162g/L of GABA. Example 8: Expression of glutamate dehydrogenase (GDH) for the enhanced production of GABA.
Glutamate dehydrogenase (GDH) is an enzyme which converts a-ketoglutarate to L- glutamate in TCA cycle. To increase the production of GABA, GDH over expression plas- mid was constructed.
Sod and CJ4(S1 ) promoters were used to express GDH and sub-cloning to pGEM-T Easy vector was performed. As a result, a plasmid vector which expresses both chimeric glutamate decarboxylase (GAD) and glutamate dehydrogenase (GDH) was constructed.
The constructed GDH-expressing plasmid was introduced to C. glutamicum HH 104 and plasmid introduction was confirmed by colony PCR. The expression of glutamate dehydrogenase (GDH) was confirmed by SDS-PAGE and western-blotting. As a result, glutamate dehydrogenase (GDH), approximately 49kDa, was successfully expressed in C. glutamicum HH 104.
To confirm the effect of GDH overexpression for the production of GABA, flask-scale culture was carried out. The result is shown in Table 9. Table 9: Amount of GABA with GDH overexpression
Cell arowth Cone, of
Strain Cone, of Glutamic acid
(OD 610nm) GABA
(g/i)
(g/i)
C. alutamicum
^HH104 14.5 0 23.5
C. alutamicum
'HH105 17.9 10.0 4.3
C. alutamicum
HH104 harborina 18.0 19.1 16.0
plasmid exDressing
GDH and GAD
As shown in Table 9, the production of GABA and glutamic acid in C. glutamicum HH 104 harboring the plasmid expressing GDH and GAD was higher than C. glutamicum HH 105. Example 9: Knock out of the glutamate exporter gene in C. glutamicum
For reducing the expression of the gene coding for the glutamate exporter in C. glutamicum a potato-tomato chimeric GAD gene was inserted in the coding sequence of the glutamate exporter of C. glutamicum cells expressing GAD (potato-tomato chimera). Thereby the expression of the glutamate exporter gene was disrupted. This modification resulted in C. glu- tamicum cells with two copies of the GAD gene (one copy in the BioD gene and one copy in the glutamate exporter gene). The effect of this disruption of the glutamate exporter in C. glutamicum on the GABA production is shown in Fig. 14. Fig. 14 shows a comparison of the fermentation time courses of C. glutamicum which comprises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for a glutamate exporter. The disruption of the glutamate exporter gene leads to an increased GABA production of C. glutamicum.
Alternatively, the knock out of the gene coding for the glutamate exporter in C. glutamicum was achieved by means of homologous recombination without inserting a copy of the GAD gene in the glutamate exporter sequence according to a protocol described in "Handbook of Corynebacterium" (Lothar Eggeling & Michael Bott, CRC Press Taylor & Francis Group, 2005). The GABA and glutamate production as well as the cell growth of C. glutamicum in which the gene coding for the glutamate exporter has been disrupted and which also lacks a functional aminotransferase due to a cre-loxP disruption as described in Example 5 is shown in Fig. 16-18.
Example 10: Combined knock out of the 4-aminobutyrate aminotransferase gene and the glutamate exporter gene in C. glutamicum
For further improving the GABA-production in C. glutamicum the expression of the gene coding for the GABA-aminotransferase and the expression of the gene coding for the glutamate exporter was reduced by inserting a potato-tomato chimeric GAD gene in the respective coding sequences of the GABA-aminotransferase and the glutamate exporter in C. glutamicum cells expressing GAD (potato-tomato chimera). Using this approach C. glutamicum cells with three copies of the GAD gene were generated (one copy in the BioD gene, one copy in the GABA-aminotransferase gene and one copy in the glutamate exporter gene).
The effect of combined disruption of the GABA-aminotransferase gene and the glutamate exporter gene on the GABA production of C. glutamicum is shown in Fig. 15. Fig. 15 shows a comparison of the fermentation time courses of C. glutamicum which comprises a GAD gene and C. glutamicum which comprises a GAD gene in combination with a disruption of the gene coding for GABA-aminotransferase and a disruption of the gene coding for a glutamate exporter. The combined knock out of the GABA-aminotransferase gene and the gene coding for the glutamate exporter leads to an improvement of the GABA production of C. glutamicum. The improvement in GABA production by reducing the expression of both the GABA-aminotransferase and the glutamate exporter (Fig. 15) was higher than the improvement of GABA production by reducing the expression of either the GABA- aminotransferase or of the glutamate exporter alone (Fig. 13 and Fig. 14, respectively). Fig. 16-18 show the GABA and glutamate production as well as the growth of C. glutamicum in flask-scale culture having a chimeric GAD gene integrated in the chromosomal bioD gene, a knock-out of the gene coding for the glutamate exporter and a knock-out of the gene coding for the GABA-aminotransferase (wt coryne HH09 (■), 1xGAD; -bioD; - Aminotransferase (o),1xGAD; -bioD; -Aminotransf. -Glut.Exp. (X), 1xGAD; -bioD; - Aminotransf.; -Glut.Exp. (*), 1xGAD; -bioD; intact Aminotransf. (= wtHH09 with 1xGAD in bioD) (♦), 1xGAD; -bioD; intact Aminotransf. (= wtHH09 with 1xGAD in bioD) (+)). The data of cells characterized as 1xGAD; -bioD; -Aminotransf.; -Glut.Exp. were obtained from two different cellular clones ((X) and respectively). The same applies to cells characterized as 1xGAD; -bioD; intact aminotransf. (= wtHH09 with 1xGAD in bioD) ((♦) and (+), respectively).
Fig. 16 shows an increased amount of GABA production for cells with a disrupted gene coding for the aminotransferase and a disrupted gene coding for the glutamate exporter compared to cells expressing a glutamate decarboxylase gene (GAD) without disrupting the aminotransferase and the glutamate exporter gene. In Fig. 16, the lack of further increase in GABA production by additionally disrupting the gene coding for the glutamate exporter is most likely due to the production scale (flask scale). Due to the activity of the glutamate decarboxylase the relatively low amount of glutamate in flask scale is rapidly converted to GABA. In a fermentation scale, there would be higher concentrations of glutamic acid (cf. glutamic acid concentration in Table 5 and results underlying Table 6) and thus less rapid conversion of most of the glutamate to GABA by GAD. Under these conditions the glutamate exporter would play a more prominent role by exporting excessive glutamate that has not been converted to GABA by GAD out of the cell. Consequently, the effect of the knockout of the glutamate exporter should become more visible. Thus, in fermentation scale the increase in GABA production by additionally disrupting the gene coding for the glutamate exporter would be more prominent.
Example 11 : Conversion of GABA to 2-pyrrolidone a) Discontinuous conversion of GABA to 2-pyrrolidone
GABA containing fermentation broth was obtained by culturing C. glutamicum as described above. After fermentation, biomass was removed by centrifugation and the supernatant was subjected to ultrafiltration. The processed fermentation broth was subsequently heated in a microwave, i.e. at T < 200°C and in an autoclave at T = 220°C - 260°C.
First the cyclization of GABA within the fermentation broth to 2-pyrrolidone was done in the microwave at pH7, pH8, or pH9 and 190-200°C. Since the pressure in the microwave is limited to 20 bar, the maximum temperature possible was 200°C. The reaction time was set to 5 h. The 2-pyrrolidone yield in the reaction media after cyclization obtained in this study was 90-95%.
In another experiment cyclization of GABA to 2-pyrrolidone was done in the microwave with an aqueous solution of commercial gamma-aminobutyric acid (15% aq) at pH 3-9 and 200°C at 20 bar for 5 h. The yield of 2-pyrrolidone was higher at higher temperature and higher pH. At pH 3-4 the yield (in solution) was only 30-60% after 5 h, at pH 6-9 the yield is
90-95%, provided that the temperature is high enough (> 190°C - 200°C). At pH 10 the yield drops to 70-80%. Since the temperature in the microwave is limited to 200°C, additional experiments were done in an autoclave. In the autoclave (1.2 I) the cyclization of fermentative GABA was done at 200°C and 240°C for 6 h and 9 h, respectively. Next the water was removed in a rotary evaporator, pluriol was added to keep the distillation sump stirrable and 2-pyrrolidone was distilled in a simple laboratory distillation (distillation of 2-pyrrolidone at 1 10-1 15°C at 1 10 mbar).
The average yield of 2-pyrrolidone in the cyclization was 93-95% not regarding whether the cyclization was done at 200°C or 240°C. The yield of the distillation steps was 92-96%, i.e. the overall yield of the downstreaming was 86-90% (<2% loss of 2-P to the distillation sump).
When a solution of commercial GABA was used, the yield of 2-pyrrolidone was comparable with the yield of 2-pyrrolidone from fermentative GABA, i.e. impurities from the fermentation broth do not seem to influence the yield. The fermentation conditions and the treatment of the broth before cyclization (removal of the biomass by centrifugation and pasteurization vs. centrifugation and ultrafiltration) do not have any significant influence on the yield of the cyclization either. b) Continuous conversion of GABA to 2-pyrrolidone
To produce 2-pyrrolidone from GABA cyclization trials were run in a continuous laboratory reactor. The continuous laboratory reactor was designed to run at temperatures up to 300°C and a pressure of 100 bar. A feed recipient was filled with GABA broths with different GABA concentrations. A HPLC pump was used for conveying of the broth through a thin pipe (used inside pipe diameter: 4.55 mm; outside diameter: 6.35 mm). Different volumetric flow rates were run.
To process the cyclization in the presence of water, it was necessary to keep the reactor under pressure. For this purpose, two parallel connected 100 bar liquid relief valves were installed. The reaction pressure of 100 bars was chosen to make possible trials at high temperatures in a liquid phase and avoid water evaporation. The core part of the continuous laboratory reactor was a heating section. This section was built up in a modular way to allow different heating length (up to 5 m) and residence times. Several temperature measurement points were available in the reactor and especially in the heating section to measure the reaction temperature in the broth along this part of the reactor.
The broth was heated with an electric heating system using different heating bands. These heating bands were not directly placed on the thin reactor pipe, but around some brass jackets/pipes which surrounded the thin reactor pipe in the heating section. One purpose of the brass pipes is to ensure a uniform temperature distribution along the heating section. Additionally this part of the reactor was well isolated to avoid heat losses. The broth was cooled down using a thermostat on a defined temperature. Finally the broth flowed through one of the 100 bar liquid relief valves in the product recipient. For safety reasons two bow- out discs were integrated in the laboratory reactor as well as a safety vessel in case the pipe or liquid relief valve clogged for any reasons.
Using this continuous cyclization procedure for GABA containing fermentation broth, the yield of the cyclization was 88-94%. When the products after continuous cyclization were further distilled, 2-pyrrolidone was obtained in an overall yield of 85-86%. The continuous cyclization of GABA to 2-pyrrolidone was also studied with an aqueous solution of commercial GABA. Previous results on the dependence of the yield on the pH were confirmed, i.e. at pH between pH = 7 and pH = 9, the yield is in the range of -90-95%.
When the concentration of GABA in water was raised from 15% according to the fermentation broth to 40%, the yield increased by -2-5% and the cyclization was slightly faster.
In another experiment, GABA was further purified prior to continuous cyclization, i.e. the fermentation broth after ultrafiltration was submitted to ionic exchange chromatography and the purified GABA redeluted in water. For ionic exchange chromatography the fermentation broth after ultrafiltration was filtered over Amberlite 252H. The Amberlite was washed with water and GABA was eluted with 5% aqueous NH3. The solution was concentrated (evaporation of NH3) and redeluted to approximately the same concentration as the fermentation broth (for better comparison with the unpurified broth in the cyclization). The yield of the purification was 97,5% (average yield of 4 runs). The purity was increased from 88 wt% to >96 wt% (determination of GABA content in dry matter after drying of the broth before and after purification). The amberlite was washed with water and reused for the next purification (3x). After the first use of the ionic exchange resin the pH of the broth was adjusted to pH = 2 prior to purification (first purification at pH = 6.8 = pH of broth after ultrafiltration).
The yield of the cyclization using purified GABA was 85-88%. The overall yield after distilla- tion of 2-pyrrolidone was 79-83%.
Example 12: Polymerization of 2-pyrrolidone to PVP 2 kg of 2-pyrrolidone were prepared for further processing to PVP. The cyclization of GABA to 2-pyrrolidone was done in a 20 I - autoclave (3 runs). The average yield of the cyclization was 94%. After removal of the water, the raw material was distilled in a distillation column with high number of theoretical plates. Small fractions of 30 g were taken and analyzed by GC-MS. In the beginning of the distillation after complete removal of water the distillate so- lidified in the cooler, i.e. in the beginning the cooler has to be warmed.
After the distillation the material was distilled once more for further removal of color and subsequently subjected to vinylation and then polymerization. The results were throughout positive, i.e. no difference could be detected between PVP based on fermentative GABA and PVP based on todays standard.

Claims

A method for the fermentative production of gamma-aminobutyric acid (GABA), which method comprises the cultivation of a recombinant microorganism which produces glutamate and expresses an exogenous glutamate decarboxylase, so that the glutamate is converted to GABA, and wherein compared to the wild-type the recombinant microorganism has a reduced expression and/or activity of an endogenous glutamate exporter by means of genetechnological methods, wherein the recombinant microorganism has no endogenous expression of an enzyme having a glutamate decarboxylase activity.
The method of claim 1 , wherein the microorganism is a coryneform bacterium, preferably a Corynebacterium, most preferably Corynebacterium glutamicum.
The method of claim 1 or 2, wherein the glutamate exporter comprises an amino acid sequence having at least 70% identity with SEQ ID NO: 21 , 19, 91 , 93, 95, 97, 99, 101 , 103, or 105, or
wherein the glutamate exporter comprises an amino acid sequence encoded by
(i) a nucleic acid having at least 70% identity with SEQ ID NO: 20, 18, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104;
(ii) a nucleic acid encoding a protein having at least 70% identity with SEQ ID NO:
21 , 19, 91 , 93, 95, 97, 99, 101 , 103, or 105;
(iii) a nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); or by
(iv) a nucleic acid encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code.
The method of anyone of claims 1 -3, wherein the glutamate decarboxylase comprises an amino acid sequence having at least 70% identity with SEQ ID NO: 2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73, or
wherein the glutamate decarboxylase comprises an amino acid sequence encoded by
(i) a nucleic acid having at least 70% identity with SEQ ID NO: 1 , 3, 5, 7, 10, 12, 26-33, 58, 60, 62, 64, 66, 68, 70, or 72;
(ii) a nucleic acid encoding a protein having at least 70% identity with SEQ ID NO:
2, 4, 6, 8, 1 1 , 13, 59, 61 , 63, 65, 67, 69, 71 , or 73;
(iii) a nucleic acid capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); or by
(iv) a nucleic acid encoding the same glutamate decarboxylase as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code. The method of anyone of claims 1 -4, wherein the expression and / or activity of the glutamate exporter in the recombinant microorganism comprising an exogenous nucleic acid encoding a glutamate decarboxylase is reduced by one or more measures selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and/or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter;
(d) introducing into the recombinant microorganism a double-stranded ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO: 20, 18, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104;
(ii) a nucleic acid molecule encoding a protein having at least 70% identity with SEQ I D NO: 21 , 19, 91 , 93, 95, 97, 99, 101 , 103, or 105;
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and
(iv) a nucleic acid molecule encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code,
or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a glutamate exporter in the recombinant microorganism or an expression cassette expressing said ribozyme;
(g) introducing into the recombinant microorganism an exogenous nucleic acid mol- ecule encoding a dominant-negative polypeptide capable of suppressing activity of the glutamate exporter or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the glutamate exporter or the DNA or RNA molecule encoding the glutamate exporter, or an expression cassette expressing said factor, and thereby inhibiting expression and/or activity of the glutamate exporter.
The method of any one of claims 1 -5 comprising the steps of
(A) transforming into a microorganism an exogenous nucleic acid comprising a nucleic acid sequence encoding a glutamate decarboxylase in functional linkage with a promoter;
(B) applying one or more of the methods steps selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and/or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter;
(d) introducing into the recombinant microorganism a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO:
20, 18, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104;
(ii) a nucleic acid molecule encoding a protein having at least 70% identity with SEQ I D NO: 21 , 19, 91 , 93, 95, 97, 99, 101 , 103, or 105;
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and
(iv) a nucleic acid molecule encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code, or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a glutamate exporter in the recombinant microorganism or an expression cassette expressing said ribozyme;
(g) introducing into the recombinant microorganism an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the glutamate exporter or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the glutamate exporter or the DNA or RNA molecule encoding the glutamate exporter, or an expression cassette expressing said factor, and thereby inhibiting expression and/or activity of the glutamate exporter; and thereby reducing the expression and / or activity of the glutamate exporter in the recombinant microorganism;
(C) culturing the recombinant microorganism; and
(D) isolating GABA produced by the recombinant microorganism.
The method of anyone of claims 1 -6, wherein the expression of the glutamate exporter in the recombinant microorganism comprising an exogenous nucleic acid encoding a glutamate decarboxylase is reduced by introducing into the recombinant microorganism an exogenous nucleic acid construct capable of inducing a homologous recombination on at least one endogenous gene encoding the glutamate exporter and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism.
The method of anyone of claims 1 -7, wherein the glutamate decarboxylase is integrated in the BioD gene of the microorganism.
The method of anyone of claims 1 -6, wherein the expression of the glutamate exporter in the recombinant microorganism comprising an exogenous nucleic acid encoding a glutamate decarboxylase is reduced by exchanging the start codon of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism from ATG to GTG, CTG, or TTG. 10. The method of anyone of claims 1 -9, wherein the recombinant microorganism additionally has one or more alterations selected from the group consisting of reduced ex- pression and/or activity of a GABA-aminotransferase, reduced expression and/or activity of a succinate semialdehyde dehydrogenase, and increased expression of a glutamate dehydrogenase.
The method of claim 10, wherein the glutamate decarboxylase is a glutamate decarboxylase according to EC 4.1.1.15, wherein the GABA-aminotransferase is a GABA- aminotransferase according to EC 2.6.1.19, wherein the succinate semialdehyde dehydrogenase is a succinate semialdehyde dehydrogenase according to EC 1.2.1.24 and/or wherein the glutamate dehydrogenase is a glutamate dehydrogenase according to EC 1 .4.1.3.
The method of claim 10 or 1 1 , wherein the amino acid sequence of the GABA- aminotransferase has at least 70% sequence identity to SEQ ID NO: 15, 17, 75, 77, 79, 81 , 83, 85, 87, or 89, the amino acid sequence of the succinate semialdehyde dehydrogenase has at least 70% sequence identity to SEQ ID NO: 25 or 23, and/or the amino acid sequence of the glutamate dehydrogenase has at least 70% sequence identity to SEQ ID NO: 1 12.
A recombinant prokaryotic or eukaryotic cell transformed with one or more exogenous nucleic acid sequences coding for a glutamate decarboxylase, wherein the prokaryotic or eukaryotic cell is further modified by means of genetechnological methods by one or more modifications selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and/or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter;
(d) introducing into the recombinant microorganism a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO: 20, 18, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104;
(ii) a nucleic acid molecule encoding a protein having at least 70% identity with SEQ I D NO: 21 , 19, 91 , 93, 95, 97, 99, 101 , 103, or 105; (iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and
(iv) a nucleic acid molecule encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code,
or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the complement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a glutamate exporter in the recombinant microorganism or an expression cassette expressing said ribozyme;
(g) introducing into the recombinant microorganism an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the glutamate exporter or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the glutamate exporter or the DNA or RNA molecule encoding the glutamate exporter, or an expression cassette expressing said factor, and thereby inhibiting expression and/or activity of the glutamate exporter;
and thereby reducing the endogenous expression and / or activity of the glutamate exporter in the recombinant microorganism compared to the wild-type, wherein the recombinant microorganism has no endogenous expression of an enzyme having a glutamate decarboxylase activity.
The recombinant prokaryotic or eukaryotic cell of claim 13, wherein the recombinant prokaryotic or eukaryotic cell additionally has one or more alterations selected from the group consisting of reduced expression and/or activity of a GABA- aminotransferase, reduced expression and/or activity of a succinate semialdehyde dehydrogenase, and increased expression of a glutamate dehydrogenase.
The prokaryotic cell of claim 13 or 14, wherein the prokaryotic cell is a coryneform bacterium, preferably a Corynebacterium, most preferably Corynebacterium glutami- cum.
Use of a prokaryotic or eukaryotic cell of anyone of claims 13-15 for the fermentative production of gamma-aminobutyric acid (GABA). A method for the production of a recombinant microorganism comprising the steps of
(A) transforming into a microorganism an exogenous nucleic acid comprising a nucleic acid encoding a glutamate decarboxylase in functional linkage with a promoter; and
(B) applying one or more of the methods steps selected from the group consisting of
(a) introducing into the coding sequence, the promoter sequence, the terminator sequence, and/or one or more enhancer sequences of one or more endogenous glutamate exporter genes of the recombinant microorganism one or more mutations or exogenous nucleic acid sequences and thereby disrupting the expression of the glutamate exporter in the recombinant microorganism;
(b) exchanging the endogenous promoter sequence of one or more endogenous glutamate exporter coding sequences of the recombinant microorganism to a weak, inactive or inducible promoter sequence;
(c) removing in the recombinant microorganism the complete coding sequence, the promoter sequence, the terminator sequence and / or one or more enhancer sequences of one or more endogenous genes coding for a glutamate exporter;
(d) introducing into the recombinant microorganism a double-strand ribonucleic acid molecule (dsRNA) comprising a sense strand and an antisense strand that is complementary to the sense strand, wherein the sense strand comprises a nucleic acid molecule selected from the group consisting of:
(i) a nucleic acid molecule having at least 70% identity with SEQ ID NO:
20, 18, 42-49, 90, 92, 94, 96, 98, 100, 102, or 104;
(ii) a nucleic acid molecule encoding a protein having at least 70% identity with SEQ I D NO: 21 , 19, 91 , 93, 95, 97, 99, 101 , 103, or 105;
(iii) a nucleic acid molecule capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to (i) or (ii); and
(iv) a nucleic acid molecule encoding the same glutamate exporter as any of the nucleic acids of (i) to (iii) above, but differing from the nucleic acids of (i) to (iii) above due to the degeneracy of the genetic code,
or wherein the sense strand comprises a fragment of at least 17 consecutive nucleotides of the nucleic acid molecule of (i) or (ii);
(e) introducing into the recombinant microorganism an antisense ribonucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence of the nucleic acid molecule of any of (i) to (iv) or comprising a fragment of at least 17 consecutive nucleotides of the com- plement of the nucleic acid molecule (i) or (ii);
(f) introducing into the recombinant microorganism a ribozyme which specifically cleaves ribonucleic acid molecules encoding a glutamate exporter in the recombinant microorganism or an expression cassette expressing said ribozyme;
(g) introducing into the recombinant microorganism an exogenous nucleic acid molecule encoding a dominant-negative polypeptide capable of suppressing activity of the glutamate exporter or an expression cassette expressing said dominant negative polypeptide; and
(h) introducing into the recombinant microorganism a factor which binds to the glutamate exporter or the DNA or RNA molecule encoding the glutamate exporter, or an expression cassette expressing said factor, and thereby inhibiting expression and/or activity of the glutamate exporter;
and thereby reducing the endogenous expression and / or activity of the glutamate exporter in the recombinant microorganism compared to the wild-type, wherein the recombinant microorganism has no endogenous expression of an enzyme having a glutamate decarboxylase activity.
A method of producing 2-pyrrolidone, which method comprises the steps of a) preparing GABA by a method of anyone of claims 1 to 12; and
b) cyclization of said GABA to produce 2-pyrrolidone.
A method of preparing a polyamide, which method comprises
a) preparing GABA by a method of anyone of claims 1 to 12; and
b) polymerizing said GABA, optionally in the presence of at least one further suitable polyvalent co-monomer, selected from aminocarboxylic acids and hy- droxycarboxylic acids.
A method of producing vinylpyrrolidone, which method comprises
a) preparing GABA by a method of anyone of claims 1 to 12; and
b) processing said GABA to vinylpyrrolidone.
PCT/IB2014/066633 2013-12-17 2014-12-05 Improved process for the production of gamma-aminobutyric acid (gaba) WO2015092599A1 (en)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101785150B1 (en) * 2015-07-02 2017-10-17 울산대학교 산학협력단 Method for producing gamma aminobutyric acid by using modular scaffolds
CN107674855A (en) * 2017-06-14 2018-02-09 漳州卫生职业学院 A kind of Corynebacterium glutamicum for producing gamma aminobutyric acid and its construction method and application
EP3401390A4 (en) * 2016-04-27 2019-01-02 Qilu University Of Technology Method for constructing engineered corynebacterium glutamicum bacteria having high production of l-lysine
CN110283763A (en) * 2019-04-19 2019-09-27 中国科学院天津工业生物技术研究所 Utilize the recombinant bacterium and its construction method of glycerol production γ-aminobutyric acid
US10597429B2 (en) 2015-12-02 2020-03-24 Basf Se Method of producing proteins in filamentous fungi with decreased CLR1 activity
CN110951664A (en) * 2019-12-27 2020-04-03 江南大学 Recombinant corynebacterium glutamicum and application thereof in production of 2-pyrrolidone
CN112251428A (en) * 2020-12-21 2021-01-22 中国科学院天津工业生物技术研究所 Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid
US11299522B2 (en) 2015-12-02 2022-04-12 Basf Se Method of producing proteins in filamentous fungi with decreased CLR2 activity
CN114752544A (en) * 2022-06-16 2022-07-15 森瑞斯生物科技(深圳)有限公司 Method for producing gamma-aminobutyric acid by one-step method and strain construction thereof
CN117165504A (en) * 2023-08-03 2023-12-05 天津世纪伟康生物科技有限公司 Engineering bacterium for efficiently producing gamma-aminobutyric acid by fermentation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009103547A1 (en) * 2008-02-21 2009-08-27 Basf Se Process for the production of gamma-aminobutyric acid
US7794989B2 (en) * 2004-12-28 2010-09-14 Ajinomoto Co., Inc. L-glutamic acid-producing microorganism and a method for producing L-glutamic acid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7794989B2 (en) * 2004-12-28 2010-09-14 Ajinomoto Co., Inc. L-glutamic acid-producing microorganism and a method for producing L-glutamic acid
WO2009103547A1 (en) * 2008-02-21 2009-08-27 Basf Se Process for the production of gamma-aminobutyric acid

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101785150B1 (en) * 2015-07-02 2017-10-17 울산대학교 산학협력단 Method for producing gamma aminobutyric acid by using modular scaffolds
US11299522B2 (en) 2015-12-02 2022-04-12 Basf Se Method of producing proteins in filamentous fungi with decreased CLR2 activity
US10597429B2 (en) 2015-12-02 2020-03-24 Basf Se Method of producing proteins in filamentous fungi with decreased CLR1 activity
EP3401390A4 (en) * 2016-04-27 2019-01-02 Qilu University Of Technology Method for constructing engineered corynebacterium glutamicum bacteria having high production of l-lysine
CN107674855A (en) * 2017-06-14 2018-02-09 漳州卫生职业学院 A kind of Corynebacterium glutamicum for producing gamma aminobutyric acid and its construction method and application
CN110283763A (en) * 2019-04-19 2019-09-27 中国科学院天津工业生物技术研究所 Utilize the recombinant bacterium and its construction method of glycerol production γ-aminobutyric acid
CN110951664B (en) * 2019-12-27 2021-09-24 江南大学 Recombinant corynebacterium glutamicum and application thereof in production of 2-pyrrolidone
CN110951664A (en) * 2019-12-27 2020-04-03 江南大学 Recombinant corynebacterium glutamicum and application thereof in production of 2-pyrrolidone
CN112251428A (en) * 2020-12-21 2021-01-22 中国科学院天津工业生物技术研究所 Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid
CN112251428B (en) * 2020-12-21 2021-03-02 中国科学院天津工业生物技术研究所 Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid
CN114752544A (en) * 2022-06-16 2022-07-15 森瑞斯生物科技(深圳)有限公司 Method for producing gamma-aminobutyric acid by one-step method and strain construction thereof
CN114752544B (en) * 2022-06-16 2022-09-06 森瑞斯生物科技(深圳)有限公司 Method for producing gamma-aminobutyric acid by one-step method and strain construction thereof
CN117165504A (en) * 2023-08-03 2023-12-05 天津世纪伟康生物科技有限公司 Engineering bacterium for efficiently producing gamma-aminobutyric acid by fermentation method and application thereof

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