Method for producing guanidinoacetic acid (GAA)

Abstract

The invention relates to a transforming microorganism capable of producing guanidinoacetic acid (GAA), which is improved by using carbamate kinase (2.7. 2.2) and L-arginine: glycine amidino transferase (2.1. 4.1), and a method for producing GAA by using the microorganism through fermentation. The microorganism can be further improved by deleting a central repressor protein argR, expressing glyoxylate aminotransferase (2.6. 1.-) or expressing NADH dependent amino acid dehydrogenase (1.4. 1.-). The invention also relates to a method for the fermentative production of creatine by further expression of guanidinoacetic acid N-methyltransferase (2.1. 1.2).

Description

[0001] Guanidinoacetic acid (GAA) is a colorless crystalline organic compound used as an animal feed additive (e.g., WO2005120246 A1 and US 2011257075 A1). GAA is a natural precursor to creatine (e.g., Humm et al., Biochem.J. (1997) 322, 771-776). Therefore, GAA supplementation can provide organisms with optimal creatine intake.

[0002] This invention relates to a microorganism capable of producing guanidinoacetic acid (GAA) after conversion, and a method for producing GAA using this microorganism through fermentation. This invention also relates to a method for the fermentation production of creatine.

[0003] In biological systems, arginine and glycine are used as starting materials to form GAA and ornithine under the catalysis of L-arginine:glycine-amidinyltransferase (AGAT; EC 2.1.4.1). This reaction is also the first step in the biosynthesis of creatine.

[0004]

[0005] Guthmiller et al. (J Biol Chem. 1994 Jul 1; 269(26): 17556-60) characterized the enzyme by cloning and heterologously expressing rat kidney AGAT in Escherichia coli (E. coli). Muenchhoff et al. (FEBS Journal 277(2010) 3844–3860) also reported the first characterization of prokaryotic AGAT by cloning and heterologously expressing AGAT in E. coli.

[0006] Fan Wenchao disclosed a method (CN 106065411 A) for producing creatine through fermentation by non-pathogenic microorganisms (such as Corynebacterium glutamicum). The microorganisms possess the following biotransformation functions: glucose is converted to L-glutamic acid; L-glutamic acid is converted to N-acetyl-L-glutamic acid; N-acetyl-L-glutamic acid is converted to N-acetyl-L-glutamic acid semialdehyde; N-acetyl-L-glutamic acid semialdehyde is converted to N-acetyl-L-ornithine; N-acetyl-L-ornithine is converted to L-ornithine; L-ornithine is converted to L-citrulline; L-citrulline is converted to argininosuccinic acid; argininosuccinic acid is converted to L-arginine; L-arginine is converted to guanidinoacetic acid; and finally, guanidinoacetic acid is converted to creatine. Fan Wenchao proposed that the microorganisms overexpress one or more enzymes, wherein the enzymes are selected from the group consisting of: N-acetylglutamate synthase, N-acetylornithine-δ-aminotransferase, N-acetylornithine, ornithine-carbamoyltransferase, argininosuccinate synthase, glycine amidotransferase (EC: 2.1.4.1), and guanidinoacetic acid N-methyltransferase (EC: 2.1.1.2). The microorganisms preferably overexpress glycine aminotransferase (L-arginine: glycine-midotransferase) and guanidinoacetic acid N-methyltransferase.

[0007] Zhang et al. published a study on a microorganism capable of producing guanidinoacetic acid (GAA) (ACS Synth. Biol. 2020, 9, 2066-275). They designed a reconstructed ornithine cycle in *E. coli* by introducing heterologous AGAT from different species (e.g., *Homo sapiens*, *Cylindrospermopsis raciborskii*, *Mooreaproducens*) and by introducing citrulline synthesis modules (e.g., overexpression of carAB, argF, and argl) and argine synthesis modules (e.g., overexpression of argG and argH; introduction of aspA) into *E. coli*.

[0008] Schneider and Jankowitsch (WO 2021122400 A1) proposed a method for producing GAA using microorganisms possessing genes encoding proteins with L-arginine:glycine-midinosyltransferase function and increased carbamoyl phosphate synthase. Carbamoyl phosphate is an important precursor in the biosynthesis of GAA and L-arginine and other compounds.

[0009] Several methods for increasing the yield of L-arginine, one of the starting materials for GAA synthesis in microorganisms, particularly bacteria, are also known in the literature. Park et al. (NATURE COMMUNICATIONS|DOI:10.1038 / ncomms5618) provided an overview of metabolic engineering for L-arginine production in Corynebacterium glutamicum. Yim et al. (JInd Microbiol Biotechnol (2011) 38:1911–1920) showed that by disrupting the chromosomal argR gene in Corynebacterium glutamicum, inactivating the argR gene encoding the central repressor protein ArgR that controls the L-arginine biosynthesis pathway, arginine-producing strains were improved. Ginesy et al. (Microbial Cell Factories (2015) 14:29) reported successful engineering for increasing arginine yield. They proposed the deletion of the argR repressor gene.

[0010] Wang et al (Applied Microbiology and Biotechnology, 2021, vol.105, pp.3265–3276; https: / / doi.org / 10.1007 / s00253-021-11242-w They emphasized that carbamoyl phosphate is also essential for L-arginine production in Corynebacterium. They showed that overexpression of the carAB gene encoding carbamoyl phosphate synthase and the introduction of a heterologous gene encoding carbamate kinase (CK) from *Enterococcus faecalis* can lead to increased L-arginine production, where CK catalyzes the synthesis of carbamoyl phosphate from inorganic ammonia, bicarbonate, and ATP. The advantage of using carbamate kinase is that this enzyme utilizes inorganic ammonium as a nitrogen source. Compared to carbamoyl phosphate synthases that use glutamine as a nitrogen source, carbamate kinase allows for a reduction in the total energy requirement for carbamoyl phosphate formation. Yan et al. (Fermentation2022, vol.8, no.3, 7 March 2022, p.116; doi:10.3390 / fermentation8030116) disclosed that by introducing a heterologous AGAT gene into Bacillus subtilis, optimizing the expression level of the AGAT gene, optimizing the natural ornithine cycle, and knocking out the first gene (glycine dehydrogenase gene gcvP) of the glycine degradation pathway, they were able to synthesize GAA using whole-cell catalysis in Bacillus subtilis.

[0011] Schneider and Jankowitsch (WO 2022008276 A1) proposed using recombinant microorganisms to produce GAA, wherein the recombinant microorganisms contain a gene encoding L-arginine:glycine amidoyltransferase (AGAT), and to increase the yield of one of the starting materials (glycine), also contain a reduced or missing malate synthase gene, as well as an overexpression gene encoding glyoxylate aminotransferase. They also disclosed that carbamate kinase (CK) may contribute to the production of arginine.

[0012] To increase GAA production using microorganisms, the cells of the microorganisms must contain a large amount of starting materials (arginine and / or glycine). Simultaneously, the byproduct of the AGAT reaction (ornithine) must be efficiently recovered into arginine to prevent carbon and energy loss.

[0013] The fundamental problem of the present invention is to provide a converted microorganism capable of producing guanidinoacetic acid (GAA), particularly said microorganism having an improved ability to provide L-arginine as a starting material for GAA biosynthesis by effectively recovering ornithine, and a method for fermenting GAA production using such microorganism.

[0014] The problem is solved by microorganisms comprising at least one heterologous gene encoding a protein having the function of L-arginine:glycine amidotransferase (AGAT, e.g., EC2.1.4.1), and at least one gene encoding a protein having the function of carbamate kinase (CK, e.g., EC 2.7.2.2), and further comprising at least one gene encoding a protein having the function of NADH-dependent amino acid dehydrogenase.

[0015] Heterogeneous genes are genes inserted into host organisms that do not naturally possess the gene. This insertion is achieved through recombinant DNA technology. Microorganisms that have undergone recombinant DHA technology are called transgenic, genetically modified, or recombinant. Heterogeneous proteins are proteins that do not naturally exist in microorganisms. Homologous genes or endogenous genes are genes that include their own functions, or whose nucleotide sequences are naturally present in or "native" to microorganisms. Homologous proteins or native proteins are proteins that naturally exist in microorganisms.

[0016] In the microorganisms of the present invention, the protein having L-arginine:glycine-amidinyltransferase (AGAT) function comprises an amino acid sequence having at least 80% identity with the amino acid sequence according to SEQ ID NO: 9.

[0017] In the microorganisms of the present invention, at least one gene encoding a protein having a carbamate kinase (CK, e.g., EC 2.7.2.2) function is heterologous.

[0018] In the microorganisms of the present invention, the activity of at least one protein having carbamate kinase function is increased compared to the corresponding activity in wild-type microorganisms.

[0019] Generally, increased enzyme activity in microorganisms can be achieved through methods such as mutations in the corresponding endogenous genes. A further method to increase enzyme activity is to stabilize the mRNA encoding the enzyme. Increased enzyme activity in microorganisms can also be achieved through overexpression of the gene encoding the corresponding enzyme.

[0020] The microorganisms of the present invention may contain at least one heterologous gene encoding a carbamate kinase function. In the microorganisms of the present invention, at least one protein having carbamate kinase enzymatic activity may contain an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:6.

[0021] Gene overexpression is typically achieved by increasing the gene copy number and / or by functionally linking the gene to a strong promoter and / or by enhancing the ribosome binding site and / or by optimizing the start codon or codon selection of the entire gene, or a combination of all of the above methods.

[0022] A promoter is a DNA sequence of approximately 40-50 base pairs that forms the binding site of the RNA polymerase holoenzyme and the transcription start site, thereby influencing the expression intensity of controlled polynucleotides or genes. Generally, gene overexpression or increased expression in bacteria can be achieved by selecting strong promoters, for example, by replacing the original promoter with a strong, native (originally assigned to other genes) promoter or by modifying certain regions of a given native promoter (e.g., the so-called -10 and -35 regions of the native promoter) against a shared sequence, as taught by M. Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for Corynebacterium glutamicum. An example of a “strong” promoter is the superoxide dismutase (SOD) promoter (“Psod”; Z. Wang et al., Eng. Life Sci. 2015, 15, 73–82). “Functional linkage” is understood as the sequential arrangement of the promoter and gene that leads to gene transcription.

[0023] In one particular embodiment of the invention, a gene encoding a protein that functions as a carbamate kinase is functionally linked to a strong promoter. Preferably, the promoter is a superoxide dismutase (SOD) promoter (“Psod”).

[0024] In the microorganisms of this invention, the protein that functions as an NADH-dependent amino acid dehydrogenase can be a heterologous protein.

[0025] NADH-dependent amino acid dehydrogenases (AaDH) catalyze the amination reaction of keto acids with L-amino acids; NADH-dependent amino acid dehydrogenases play an important role in the assimilation or dissimilation of ammonium in cells.

[0026]

[0027] For example, R = CH3, H, CH2COOH

[0028] Because most of these amino acid dehydrogenases can use different α-keto acids as substrates, they are usually labeled with different EC numbers. However, all of these amino acid dehydrogenases have something in common: they all assimilate ammonium, or dissimilate ammonium in the reverse reaction.

[0029] Examples of different amino acid dehydrogenases are as follows:

[0030] Reaction EC 1.4.1.1: Alanine dehydrogenase:

[0031] Pyruvate + NH3 + NADH + H+ <-> L-alanine + H2O + NAD+

[0032] Reaction EC 1.4.1.10: Glycine dehydrogenase:

[0033] Glyoxylic acid + NH3 + NADH + H+ <-> glycine + H2O + NAD+

[0034] Reaction EC 1.4.1.21: Aspartate dehydrogenase:

[0035] Oxaloacetic acid + NH3 + NADH + H+ <-> L-aspartic acid + H2O + NAD+

[0036] Several NADH-dependent amino acid dehydrogenase (AaDH) proteins are known in the literature to accept a variety of substrates, and therefore can usually aminolate several different keto acids into the corresponding amino acids. (Fernandes et al.ProteinEngineering,Design&Selection,2015,vol.28no.2,pp.29–35; Giffin et al.,Journalof Bacteriology,2012,vol.194no.5,pp.1045-1054; Phogosee et al.,Archives ofMicrobiology,2018vol.200pp.719–727; Schuffenhauer et al.1999,vol.171,pp 417-423; Vancura et al.Eur J Biochem 1989,vol.179,pp.221–227; Yoshida and Freese, Biochim. Biophys. Acta, 1965, vol. 96, pp 248-262).

[0037] Therefore, there is no clear link between the reaction and the EC number. Table 1 shows some examples:

[0038]

[0039] Table 1: Examples of NADH-dependent amino acid dehydrogenase (AaDH) proteins

[0040] In the microorganisms of the present invention, at least one protein having the function of an NADH-dependent amino acid dehydrogenase may be selected from the group consisting of: alanine dehydrogenase (EC 1.4.1.1), glycine dehydrogenase (EC 1.4.1.10), and aspartate dehydrogenase (EC 1.4.1.21).

[0041] In the microorganisms of the present invention, the activity of at least one NADH-dependent amino acid dehydrogenase is increased compared to the individual activities in wild-type microorganisms.

[0042] At least one protein that functions as an NADH-dependent amino acid dehydrogenase is preferably heterologous.

[0043] The at least one NADH-dependent amino acid dehydrogenase contained in the microorganisms of the present invention may be selected from the group consisting of: alanine dehydrogenase (EC 1.4.1.1), glycine dehydrogenase (EC 1.4.1.10), and aspartate dehydrogenase (EC 1.4.1.21).

[0044] In the microorganisms of the present invention, proteins having the function of NADH-dependent amino acid dehydrogenases may contain amino acid sequences having at least 80% identity with the amino acid sequences according to SEQ ID NO: 13, according to SEQ ID NO: 21, according to SEQ ID NO: 22, according to SEQ ID NO: 23 or according to SEQ ID NO: 24.

[0045] The microorganisms of the present invention may further include at least one gene encoding a protein that functions as a glyoxylate aminotransferase.

[0046] Several glyoxylate aminotransferases are known, each with varying substrate specificity for its amino donor (see, for example, Kameyea et al. FEBS Journal 277 (2010) 1876–1885; Liepman and Olsen, Plant Physiol. Vol. 131, 2003, 215–227; Sakuraba et al., JOURNAL OF BACTERIOLOGY, Aug. 2004, p. 5513–5518; Takada and Noguchi, Biochem. J. (1985) 231, 157–163). Because most of these glyoxylate aminotransferases can use different amino acids as amino donors, they are usually labeled with different EC numbers. However, all these aminotransferases share a common characteristic: they all use glyoxylate as the acceptor molecule, or, in the reverse case, glycine as the donor molecule. Examples of proteins that function as glyoxylate aminotransferases are as follows:

[0047] Glycine aminotransferase (EC 2.6.1.4) catalyzed reaction:

[0048] L-glutamic acid + glyoxylic acid <=> α-ketoglutarate + glycine.

[0049] Glycine: Oxaloacetate aminotransferase (EC 2.6.1.35) catalyzed reaction:

[0050] L-Aspartic acid + glyoxylic acid <=> oxaloacetic acid + glycine.

[0051] Alanine: Glyoxylate aminotransferase (EC 2.6.1.44) catalyzed reaction:

[0052] L-alanine + glyoxylic acid <=> pyruvate + glycine.

[0053] Serine: Glyoxylate aminotransferase (EC 2.6.1.45) catalyzed reaction:

[0054] L-serine + glyoxylic acid <=> 3-hydroxypyruvate + glycine.

[0055] Methionine: Glyoxylate aminotransferase (EC 2.6.1.73) catalyzed reaction:

[0056] L-methionine + glyoxylic acid <=> 4-(meththio)-2-keto-butyric acid + glycine.

[0057] Aromatic amino acids: Glyoxylate aminotransferase (EC 2.6.1.60) catalyzed reaction:

[0058] Aromatic amino acids + glyoxylic acid <=> Aromatic keto acids + glycine.

[0059] Kynureanine: Glyoxylate aminotransferase (EC 2.6.1.63) catalyzed reaction:

[0060] Kynurenine + glyoxylic acid <=> 4-(2-aminophenyl)-2,4-diketo-butyric acid + glycine.

[0061] (S)-Ureido-glycine:glyoxylate aminotransferase (EC 2.6.1.112) catalyzed reaction:

[0062] (S)-Ureido-glycine + glyoxylic acid <=> N-carbamoyl-2-keto-glycine + glycine.

[0063] In another embodiment of the invention, the enzyme activity of at least one protein having the function of glyoxylate aminotransferase is increased compared to the respective enzyme activities in wild-type microorganisms.

[0064] At least one protein having the function of glyoxylate aminotransferase is preferably heterologous.

[0065] In a particular embodiment of the present invention, at least one protein having the function of glyoxylate aminotransferase is glycine:glyoxylate aminotransferase.

[0066] In the microorganisms of the present invention, proteins having glyoxylate aminotransferase activity may comprise an amino acid sequence having at least 80% identity with the amino acid sequence according to SEQ ID NO: 16, SEQ ID NO: 19 or SEQ ID NO: 20.

[0067] Compared to the ability of wild-type microorganisms, the ability of the microorganisms of the present invention to produce L-arginine from L-ornithine can be increased.

[0068] In the context of this invention, a microorganism with an increased capacity to produce L-arginine refers to a microorganism that produces L-arginine beyond its own needs. Examples of such L-arginine-producing microorganisms include, for example, Corynebacterium glutamicum ATCC 21831 or those disclosed by Park et al. (NATURE COMMUNICATIONS|DOI:10.1038 / ncomms5618) or Ginesy et al. (Microbial Cell Factories (2015) 14:29). Compared to the aforementioned microorganisms with an increased capacity to produce L-arginine, the secretion of L-arginine is not essential in GAA-producing strains because, within the framework of this invention, arginine is used intracellularly for GAA production.

[0069] In another embodiment of the invention, the expression of the argR gene, which encodes the argine repressor protein ArgR, is attenuated compared to the expression of the argR gene in wild-type microorganisms. Alternatively, the argR gene is absent.

[0070] The microorganisms of the present invention may belong to the genus Corynebacterium, the genus Bacillus (Yan, K., et al. (2022). "Biosynthesis of Guanidinoacetate by Bacillus subtilis Whole-Cell Catalysis." Fermentation 8(3):116), the family Enterobacteriaceae, or the genus Pseudomonas.

[0071] In a particular embodiment of the invention, the microorganism is Corynebacterium glutamicum or Escherichia coli.

[0072] The present invention also relates to a fermentation production method of guanidinoacetic acid (GAA), the method comprising the steps of: a) culturing the microorganism of the present invention in a culture medium, and b) accumulating GAA in the culture medium to form a fermentation broth containing GAA.

[0073] Preferably, the method further includes separating GAA from the fermentation broth containing GAA.

[0074] In one particular embodiment, the microorganism of the present invention further comprises a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity. The gene encoding the enzyme having guanidinoacetic acid N-methyltransferase activity can be overexpressed.

[0075] The present invention also relates to a method for the fermentation production of creatine, the method comprising the steps of: a) culturing the microorganism of the present invention in a suitable culture medium under suitable conditions, the microorganism further comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity, and b) accumulating creatine in the culture medium to form a creatine-containing fermentation broth.

[0076] Preferably, the method further includes separating creatine from the creatine-containing fermentation broth. Creatine can be extracted from the fermentation broth by isoelectric point method and / or ion exchange method. Alternatively, creatine can be further purified by recrystallization in water.

[0077] Experimental Section

[0078] A) Materials and Methods

[0079] Chemical reagents

[0080] Kanamycin solution from *Streptomyces kanamyceticus* was purchased from Sigma-Aldrich (St. Louis, USA, Cat. no. K0254). Unless otherwise specified, all other chemicals were analytical grade chemicals purchased from Merck (Darmstadt, Germany), Sigma-Aldrich (St. Louis, USA), or Carl-Roth (Karlsruhe, Germany).

[0081] Cell proliferation culture

[0082] Unless otherwise specified, the cultivation / incubation procedure shall be carried out as follows:

[0083] a. Escherichia coli strains were cultured in liquid medium using LB medium from Merck (Darmstadt, Germany; Cat. no. 110285). Liquid cultures (10 ml of liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were cultured in an Inforrs HT Multitron standard shaking incubator from Inforrs GmbH (Bottmingen, Switzerland) at 30°C and 200 rpm.

[0084] b. Use LB agar (MILLER) from Merck (Darmstadt, Germany, Cat. no. 110283) to culture *E. coli* strains on agar plates. In INCU- from VWR (Radnor, USA) Agar plates are cultured in a miniature incubator at 30°C.

[0085] c. Brain and Heart Infusion Broth (BHI) from Merck (Darmstadt, Germany, Cat. no. 110493) was used to culture Corynebacterium glutamicum in liquid medium. Liquid cultures (10 ml of liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were cultured in an Inforrs HT Multitron standard shaking incubator from Inforrs GmbH (Bottmingen, Switzerland) at 30°C and 200 rpm.

[0086] d. *Corynebacterium glutamicum* strains were cultured on agar plates using brain heart infusion agar (BHI-agar) from Merck (Darmstadt, Germany, Cat. no. 113825). The plates were equipped with… Agar plates were cultured at 30°C in an incubator with a temperature controller (Hanau, Germany) from Heraeus Instruments.

[0087] e. When culturing Corynebacterium glutamicum after electroporation, add 134 g / L sorbitol (Carl Roth GmbH+Co.KG, Karlsruhe, Germany), 2.5 g / L yeast extract (Oxoid / ThermoFisher Scientific, Waltham, USA, Cat.no.LP0021), and 25 mg / L kanamycin to BHI-agar (Darmstadt, Germany, Cat.no. LP0025). This should be done in a container equipped with… Agar plates were cultured at 30°C in an incubator from Heraeus Instruments with a temperature controller (Hanau, Germany).

[0088] Measuring the optical density of bacterial suspensions

[0089] a. Using a Bio-Photometer from Eppendorf AG (Hamburg, Germany) at 600nm (OD) 600 The optical density of bacterial suspensions in shake flask cultures was measured.

[0090] b. Using materials from Tecan Group AG ( GENios (Switzerland) TM The board reader is at 600nm (OD) 600 The optical density of bacterial suspensions produced in a Wouter Duetz (WDS) micro-fermentation system (24-well plate) was measured.

[0091] Centrifugation

[0092] a. Using an Eppendorf 5417 R benchtop centrifuge, transfer a bacterial suspension with a maximum volume of 2 ml into 1.5 ml or 2 ml reaction tubes (e.g., Eppendorf). Centrifuge at 3810X (5 min, 13,000 rpm).

[0093] b. Using an Eppendorf 5810 R benchtop centrifuge, centrifuge a bacterial suspension with a maximum volume of 50 ml in 15 ml or 50 ml centrifuge tubes (e.g., Falcon™ 50 ml conical centrifuge tubes) at 4000 rpm for 10 min.

[0094] DNA isolation

[0095] Plasmid DNA from E. coli cells was isolated using the Qiagen QIAprep Spin Miniprep Kit (Hilden, Germany, Cat. No. 27106) according to the manufacturer's instructions.

[0096] Polymerase chain reaction (PCR)

[0097] PCR with a proofreading (high-fidelity) polymerase amplifies the desired DNA fragment for sequencing or DNA assembly cloning. Non-proofreading polymerase kits are used to determine the presence or absence of the desired DNA fragment directly from E. coli or Corynebacterium glutamicum colonies.

[0098] a. From New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530) The high-fidelity DNA polymerase kit (Phusion kit) is used for template-corrected amplification of selected DNA regions according to the manufacturer's instructions (see Table 2).

[0099]

[0100] Table 2: Use of materials from New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530) Thermal cycling conditions for PCR using a high-fidelity DNA polymerase kit

[0101] b. The Taq PCR Core Kit (Taq Kit) from Qiagen (Hilden, Germany, Cat. No. 201203) was used to amplify the desired DNA fragment to confirm its presence. The kit was used according to the manufacturer's instructions (see Table 3).

[0102]

[0103] Table 3: Thermal cycling conditions for PCR using the Taq PCR Core Kit (Taq Kit) from Qiagen

[0104] c. From Takara Bio Inc (Takara Bio Europe SAS, Saint-Germain-en-Laye, France, Cat. No. RR350A / B) Rapid PCR Master Mix (Sapphire Mix) is used as an alternative to confirm the presence of the desired DNA fragment extracted from cells from E. coli or Corynebacterium glutamicum colonies, according to the manufacturer’s instructions (see Table 4).

[0105]

[0106] Table 4: Use of products from Takara Bio Inc. Thermal cycling conditions for PCR using Rapid PCR Master Mix (SapphireMix)

[0107] d. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany).

[0108] e. Use an appropriately diluted solution of isolated plasmid DNA, or a total DNA solution isolated from liquid culture, or a total DNA solution contained in bacterial colonies (colony PCR) as a PCR template. For colony PCR, the template is prepared by removing cell material from colonies on an agar plate using a sterile toothpick and placing the cell material directly into a PCR reaction tube. (From SEVERIN) Cell material was heated at 800W for 10 seconds in a Mikrowave & Grill microwave oven from Sundern, Germany, and then PCR reagents were added to the template in the PCR reaction tube.

[0109] f. All PCR reactions were performed on a Mastercycler PCR instrument or a Mastercycler Nexus gradient PCR instrument from Eppendorf AG (Hamburg, Germany).

[0110] DNA restriction enzyme digestion

[0111] For restriction enzyme digestion, use FastDigest restriction endonuclease (FD) (ThermoFisher Scientific, Waltham, USA) or a restriction endonuclease from New England BioLabs Inc. (Ipswich, USA). Perform the reaction according to the manufacturer's instructions.

[0112] Determine the size of DNA fragments

[0113] a. The size of small DNA fragments (<1000bps) is usually determined by automated capillary electrophoresis using QIAxcel from Qiagen (Hilden, Germany).

[0114] b. If DNA fragments need to be separated, or if DNA fragments are >1000 bps, separate the DNA by TAE agarose gel electrophoresis and use... Nucleic acid gel staining was performed using a nucleic acid gel dye (Biotium, Inc., Fremont, Canada). The stained DNA was visible at 320 nm.

[0115] Purification of PCR amplification products and restriction fragments

[0116] Following the manufacturer's instructions, the PCR amplification products and restriction fragments were purified using the QIAquick PCR Purification Kit from Qiagen (Hilden, Germany; Cat. No. 28106). DNA was eluted with 30 μl of 10 mM Tris*HCl (pH 8.5).

[0117] DNA concentration measurement

[0118] DNA concentration was measured using a NanoDrop spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH (renamed VWR trademark in 2015) (Erlangen, Germany).

[0119] Assembly clone

[0120] Plasmid vectors were assembled using the NEBuilder HiFi DNA Assembly and Cloning Kit (Ipswich, USA, Cat. No. E5520), purchased from New England BioLabs Inc. The reaction mixture containing the linear vector and at least one DNA insert was incubated at 50°C for 60 min. 0.5 μl of the assembly mixture was used for each transformation experiment.

[0121] Chemical transformation of Escherichia coli

[0122] To perform plasmid cloning, chemically competent cells were transformed according to the manufacturer's protocol. Stable competent Escherichia coli (high-efficiency grade) (New England BioLabs Inc., Ipswich, USA, Cat. No. C3040). Successfully transformed cells were selected on LB agar supplemented with 25 mg / L kanamycin.

[0123] Transformation of Corynebacterium glutamicum

[0124] As described by Ruan et al. (2015), plasmid DNA was transformed into Corynebacterium glutamicum via electroporation using a Gene Pulser Xcell (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Electroporation was performed in 1 mm electroporation cuvettes (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) at a fixed time constant of 1.8 kV and 5 ms. Transformed cells were selected on BHI-agar containing 134 g / L sorbitol, 2.5 g / L yeast extract, and 25 mg / L kanamycin.

[0125] Determine nucleotide sequence

[0126] Cyclic sequencing was performed by Eurofins Genomics GmbH (Ebersberg, Germany), and the nucleotide sequence of the DNA molecules was determined using the dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences, USA 74, 5463–5467, 1977). Clonemanager Professional 9 software from Scientific & Educational Software (Denver, USA) was used for sequence visualization and evaluation, as well as computer assembly of the sequences.

[0127] Glycerin precursors of Escherichia coli and Corynebacterium glutamicum strains

[0128] For long-term storage of *E. coli* and *Corynebacterium glutamicum* strains, glycerol stock was prepared. Selected *E. coli* clones were cultured in LB medium supplemented with 2 g / L glucose. Selected *Corynebacterium glutamicum* clones were cultured in double-concentrated BHI medium supplemented with 2 g / L glucose. 25 mg / L kanamycin was added to the culture medium used for the growth of *E. coli* and *Corynebacterium glutamicum* strains containing plasmids. The culture medium was placed in 100 ml Erlenmeyer flasks with three baffles. One ring of cells extracted from the colonies was inoculated into each flask. The cultures were then incubated at 30 °C and 200 rpm for 18 h. After the incubation period, 1.2 ml of 85% (v / v) sterile glycerol was added to the cultures. The resulting glycerol containing the cell suspension was then divided into 2 ml aliquots and stored at -80 °C.

[0129] GAA yield from milliliter-scale culture

[0130] The milliliter-scale culture system of Duetz (2007) was used to evaluate the GAA production of the strain. For this purpose, 24-well deep-well microculture plates (24-well WDS plates) from EnzyScreen BV (Heemstede, Netherlands, Cat. no. CR1424) were used, with 2.5 ml of culture medium in each well.

[0131] Pre-culture of the strain was carried out in 10 ml of seed culture medium (SM). The medium was placed in a 100 ml Erlenmeyer flask equipped with three baffles. 100 μl of the original glycerol culture was inoculated into the flask, and the culture was incubated at 30 °C and 200 rpm for 24 h. Table 5 shows the composition of the seed culture medium (SM).

[0132]

[0133] Table 5: Seed Culture Medium (SM)

[0134] After the culture period, the optical density (OD) of the preculture was measured. 600 Inoculation with 2.5 ml of production medium (PM) resulted in OD... 600 Take a sample from the pre-culture at the required volume of 0.1, centrifuge (1 min, 8000 g), and discard the supernatant. Then resuspend the cells in 100 μl of production medium.

[0135] The primary culture was initiated by seeding 100 μl of resuspended cells from the preculture into each well of a 24-well WDS plate containing 2.4 ml of production medium (PM). Table 6 shows the composition of the production medium (PM).

[0136]

[0137] Table 6: Production Culture Medium (PM)

[0138] The primary culture was cultured for 72 hours at 30°C and 225 rpm in an Inforrs HT Multitron standard shaking incubator from Inforrs GmbH (Bottmingen, Switzerland) until glucose was completely consumed. A OneTouch blood glucose meter from LifeScan (Johnson & Johnson Medical GmbH, Neuss, Germany) was used. The glucose concentration in the suspension was analyzed.

[0139] After incubation, the culture suspension was transferred to a deep-well microplate. A portion of the culture suspension was appropriately diluted to measure OD. 600 Another portion of the culture was centrifuged, and the concentration of GAA in the supernatant was analyzed as described below.

[0140] B) Example

[0141] Example 1: Cloning of plasmid pK18mobsacB_DargR for genomic deletion of the argR gene in Corynebacterium glutamicum.

[0142] To enhance intracellular L-arginine formation and recovery of L-arginine from L-ornithine, it is necessary to delete the argR gene, which encodes the central repressor protein ArgR that controls the L-arginine biosynthesis pathway. Therefore, plasmid pK18obsacB_DargR was constructed as follows. The plasmid pK18mobsacB (…) was modified using the restriction endonuclease XbaI. The DNA was digested using the Genbank accession FJ437239 in 1994, and the linearized vector DNA (5721 bps) was purified using the QIAquick gel extraction kit.

[0143] To construct the insert, two DNA fragments were created via high-fidelity PCR using the following primer pairs (using DNA from ATCC13032 as a template):

[0144] DargR_lf(SEQ ID NO:1),+DargR_lr(SEQ ID NO:2)

[0145] =Left homologous arm (983bps)

[0146] DargR_rf(SEQ ID NO:3),+DargR_rr(SEQ ID NO:4)

[0147] =Left homologous arm (984bps)

[0148] The PCR products were purified using the QIAquick PCR Purification Kit.

[0149] Linearized plasmids and PCR products were assembled using the NEBuilder HiFi DNA Assembly and Cloning Kit. The assembled products were transformed into NEB Stable competent *E. coli* (high-efficiency grade) cells and grown on LB agar containing 25 mg / L kanamycin. Suitable plasmid clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmid was named pK18mobsacB_DargR.

[0150] Example 2: Chromosomal deletion of the argR gene in ATCC13032

[0151] Corynebacterium glutamicum ATCC13032 (Kinoshita S, Udaka S, Shimono M., J. Gen. Appl. Microbiol. 1957; 3(3): 193-205) (Corynebacterium glutamicum strain / wild type) can be purchased from American Type Culture Collection (ATCC) or DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, with accession number DSM 20300.

[0152] To induce argR gene deletion, plasmid pK18mobsacB_DargR was transformed into Corynebacterium glutamicum ATCC13032 via electroporation. The transformed plasmids were then inoculated onto BHI-agar supplemented with 134 g / L sorbitol, 2.5 g / L yeast extract, and 25 mg / L kanamycin to select for chromosome integration (obtained from the first recombination event). The agar plates were incubated at 33°C for 48 h.

[0153] Single colonies were transferred to fresh agar plates (containing 25 mg / L kanamycin) and incubated at 33°C for 24 h. Liquid cultures of these clones were then incubated at 33°C for 24 h in 10 ml BHI medium contained in 100 ml Erlenmeyer flasks with three baffles. To isolate clones that encountered a second recombination event, aliquots were taken from each liquid culture, appropriately diluted, and inoculated onto BHI agar plates containing 10% sucrose. The agar plates were incubated at 33°C for 48 h. The colonies grown on the sucrose-containing agar plates were then subjected to kanamycin susceptibility testing. For this purpose, cell material was removed from the colonies with a toothpick, and the colonies were transferred to BHI agar plates containing 25 mg / L kanamycin and BHI agar plates containing 10% sucrose. The agar plates were incubated at 33°C for 60 h. Clones sensitive to kanamycin and tolerant to sucrose were confirmed by PCR and DNA sequencing. The strain with the argR gene deletion obtained was named ATCC13032_DargR.

[0154] Example 3: Cloning plasmid pK18obsacB_CK for integrating the genome of the carbamate kinase (CK, EC 2.7.2.2) gene from Enterococcus faecalis ATCC 29212 into Corynebacterium glutamicum.

[0155] The coding sequence arcC of Enterococcus faecalis ATCC 29212 encodes carbamate kinase (Marina et al., Eur J Biochem. 1998 Apr 1; ​​253(1):280-91. doi:10.1046 / j.1432-1327.1998.2530280.x; Genbank accession AJ223332, SEQ ID NO: 5). SEQ ID NO: 6 shows the derived amino acid sequence (Genbank accession number CAA11271).

[0156] The coding sequence was optimized for use in Corynebacterium glutamicum using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA). The resulting optimized coding sequence was named CK.

[0157] Using an optimized coding sequence, a DNA fragment was designed for genomic integration between the NCgl0291 and NCgl0292 genes in Corynebacterium glutamicum. This fragment consists of the following elements: a BsaI restriction enzyme site, and a homologous sequence for assembly cloning into pK18mobsacB. 1994; Genbank accession FJ437239), a left homologous arm downstream of NCgl0291 for integration, a strong sod-promoter from Corynebacterium glutamicum, an optimized CK gene, BioBricks Terminator BBa_B1006, a right homologous arm for genome integration, a second homologous sequence for cloning, and a BsaI site. The obtained DNA sequence was named CK-insert (SEQ ID NO: 7). This DNA sequence was purchased from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) for gene synthesis and delivered as part of a cloning plasmid containing an ampicillin resistance gene.

[0158] The cloning plasmid containing the CK-insert was digested using the restriction endonuclease BsaI, and the DNA was purified using the QIAquick PCR Purification Kit.

[0159] The plasmid pK18mobsacB was digested using the restriction endonuclease SmaI, and the DNA was purified using the QIAquick PCR Purification Kit.

[0160] The DNA from two enzyme-digested plasmids was ligated, and the matched sequence ends were assembled using the NEBuilder HiFi DNA Assembly Cloning Kit. The assembly product was transformed into NEB Stable competent E. coli (high-efficiency grade), and cells were grown on LB agar containing 25 mg / L kanamycin. Suitable plasmid clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmid was named pK18mobsacB_CK.

[0161] Example 4: Integration of the carbamate kinase gene CK into the genome of Corynebacterium glutamicum

[0162] To integrate the carbamate kinase gene CK into Corynebacterium glutamicum ATCC13032 and ATCC13032_DargR, these strains were transformed with plasmid pK18mobsacB_CK via electroporation. The transformed strains were plated on BHI-agar supplemented with 134 g / L sorbitol, 2.5 g / L yeast extract, and 25 mg / L kanamycin to select for chromosome integration (obtained from the first recombination event). The agar plates were incubated at 33°C for 48 h.

[0163] Single colonies were transferred to fresh agar plates (containing 25 mg / L kanamycin) and incubated at 33°C for 24 h. Liquid cultures of these clones were incubated at 33°C for 24 h in 10 ml BHI medium contained in 100 ml Erlenmeyer flasks with three baffles. To isolate clones that encountered a second recombination event, aliquots were taken from each liquid culture, appropriately diluted, and inoculated onto BHI agar plates supplemented with 10% sucrose. The agar plates were incubated at 33°C for 48 h. The colonies grown on sucrose-containing agar plates were then subjected to kanamycin susceptibility testing. For this purpose, cell material was removed from the colonies with a toothpick and transferred to BHI agar plates containing 25 mg / L kanamycin and 10% sucrose. The agar plates were incubated at 33°C for 60 h. Clones sensitive to kanamycin and tolerant to sucrose were confirmed by PCR and DNA sequencing to determine appropriate integration of the CK gene. The obtained strains were named ATCC13032_CK and ATCC13032_DargR_CK, respectively.

[0164]

[0165] Table 7: List of strains

[0166] Example 5: Cloning of the AGAT-Mp gene encoding L-arginine:glycine amidoside transferase (AGAT, EC 2.1.4.1) derived from marine cyanobacteria (Moorena producens).

[0167] Marine cyanobacteria are filamentous cyanobacteria. Leao et al. published the genome of the marine cyanobacteria strain PAL-8-15-08-1 (Leao T, G, Korobeynikov A, Monroe EA, Podell S, Glukhov E, Allen EE, Gerwick WH, Gerwick L, Proc Natl Acad Sci US A. 2017 Mar 21; 114(12):3198-3203. doi:10.1073 / pnas.1618556114; Genbank accession number CP017599.1). It contains an open reading frame encoding L-arginine:glycine amidoyltransferase (AGAT, EC 2.1.4.1; locus_tag BJP34_00300 shown in SEQ ID NO: 8). SEQ ID NO: 9 shows the derived amino acid sequence (Genbank accession number WP_070390602).

[0168] Using the software tool "Optimizer" (http: / / genomes.urv.es / OPTIMIZER / ), the amino acid sequence was reverse-translated into a DNA sequence optimized for codon usage in Corynebacterium glutamicum. The optimized gene's 5' end was amplified with: a BsaI restriction enzyme site, a 5'-UTR sequence for cloning, and a ribosome binding site. A second stop codon, a sequence for cloning, and a BsaI restriction enzyme site were added to its 3' end. The resulting DNA sequence was named AGAT-Mp-insert (SEQ ID NO: 10). This DNA sequence was purchased from Eurofins Genomics GmbH (Ebersberg, Germany) for gene synthesis and delivered as part of a cloning plasmid containing an ampicillin resistance gene.

[0169] The cloning plasmid containing AGAT-Mp-insert was digested using the restriction endonuclease BsaI, and the DNA was purified using the QIAquick PCR Purification Kit.

[0170] The Escherichia coli-Corynebacterium glutamate shuttle plasmid pLIB_P consists of the origin of replication of pBL1 (for Corynebacterium glutamate), the origin of replication of pSC101 (for Escherichia coli), and a kanamycin resistance gene. Following a unique NotI restriction site, it has a strong promoter, two inverted BsaI sites, and BioBricks Terminator BBa_B1006 (SEQ ID NO: 11).

[0171] pLIB_P was digested using the restriction endonuclease BsaI, and the DNA was purified using the QIAquick PCR Purification Kit.

[0172] Two DNA solutions of plasmids digested with BsaI were ligated, and the matched sequence ends were assembled using the NEBuilder HiFi DNA Assembly Cloning Kit. The products were transformed into NEB Stable competent E. coli (high-efficiency grade) and grown on LB agar containing 25 mg / L kanamycin. Suitable plasmid clones were identified by restriction enzyme digestion and DNA sequencing. The obtained plasmid clone was named pLIB_P_AGAT-Mp.

[0173] Example 6: Synthesis of the AaDH-Mt gene encoding NADH-dependent AaDH derived from Mycobacterium tuberculosis H37Ra

[0174] It is speculated that the open reading frame MRA_2804 of Mycobacterium tuberculosis H37Ra encodes an NADH-dependent amino acid dehydrogenase (Genbank accession number CP000611locus_tag="MRA_2804", SEQ ID NO: 12). SEQ ID NO: 13 shows the derived amino acid sequence (Genbank accession number WP_070390602).

[0175] The open reading frame (ORF) was optimized for codon usage in *Corynebacterium glutamicum* using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA). The obtained sequence had a 5'-UTR amplified at the 5' end, consisting of a BsaI restriction enzyme site, a homologous region for cloning, a strong promoter Pg3N3, and a ribosome binding site. Additionally, the 3' end was amplified with a random spacer sequence, a homologous sequence for cloning, and a BsaI restriction enzyme site. The obtained DNA sequence was named AaDH-Mt-insert (SEQ ID NO: 14). This DNA sequence was purchased from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) for gene synthesis and delivery as part of a cloning plasmid containing an ampicillin resistance gene. The optimized gene was named AaDH-Mt.

[0176] Example 7: Synthesis of the AtGGT1 gene encoding glyoxylate aminotransferase derived from Arabidopsis thaliana

[0177] The Arabidopsis thaliana GGT1 gene (Genbank accession number NM_102180, SEQ ID NO: 15) encodes glutamate:glyoxylate aminotransferase (Genbank accession number NP_564192, SEQ ID NO: 16). This protein has been shown to catalyze the following reactions: glyoxylate + L-alanine = glycine + pyruvate (EC 2.6.1.44), 2-ketoglutarate + L-alanine = L-glutamate + pyruvate (EC 2.6.1.2), and 2-ketoglutarate + glycine = glyoxylate + L-glutamate (EC 2.6.1.4; Liepman AH, Olsen LJ., Plant Physiol. 2003 Jan; 131(1):215-27. doi:10.1104 / pp.011460).

[0178] The amino acid sequence of the GGT1 protein was reverse-translated into an optimized DNA sequence for use with codons in Corynebacterium glutamicum using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA). The obtained sequence had a 5'-UTR amplified at the 5' end, consisting of a BsaI restriction enzyme site, a homologous sequence for cloning, and a ribosome binding site. Additionally, a homologous sequence for cloning and a BsaI restriction enzyme site were amplified at the 3' end. The obtained DNA sequence was named AtGGT1-insert (SEQ ID NO: 17). This DNA sequence was purchased from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) for gene synthesis and delivery as part of a cloning plasmid containing an ampicillin resistance gene. The optimized gene was named AtGGT1.

[0179] Example 8: Cloning of plasmids co-expressing AGAT-Mp and AaDH-Mt

[0180] To achieve co-expression of AGAT-Mp and AaDH-Mt, the synthetic gene AaDH-Mt was cloned into plasmid pLIB_P_AGAT-Mp.

[0181] The plasmid pLIB_P_AGAT-Mp was digested with the restriction endonuclease NotI, and the DNA was purified using the QIAquick PCR Purification Kit. The plasmid containing the synthetic sequence AaDH-Mt-insert (SEQ ID NO: 14) was digested with the restriction endonuclease BsaI, and the obtained DNA was purified using the QIAquick PCR Purification Kit.

[0182] A pseudoDNA with compatible ends was designed for cloning. It was named dummy-insert (SEQ ID NO: 18) and was purchased as a linear double-stranded DNA fragment from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) for gene synthesis.

[0183] DNA from pLIB_P_AGAT-Mp, digested with NotI, was ligated with a plasmid containing an AaDH-Mt-insert, digested with BsaI, using the NEBuilder HiFi DNA Assembly Cloning Kit. The product was transformed into NEB Stable competent E. coli (high-efficiency grade) (New England Biolabs, Ipswich, USA), and cells were grown on LB agar containing 25 mg / L kanamycin. Suitable plasmid clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmid clone was named pLIB_AaDH-Mt_AGAT-Mp.

[0184] Example 9: Cloning of plasmids co-expressing AGAT-Mp, AaDH-Mt, and AtGGT1

[0185] To achieve the co-expression of AGAT-Mp, AaDH-Mt, and AtGGT1, the synthetic genes AaDH-Mt and AtGGT1 were cloned into the plasmid pLIB_P_AGAT-Mp.

[0186] The plasmid pLIB_P_AGAT-Mp was digested with restriction endonuclease NotI, and the DNA was purified using the QIAquick PCR Purification Kit. The plasmid containing the synthetic sequence AaDH-Mt-insert (SEQ ID NO: 14) was digested with restriction endonuclease BsaI, and the obtained DNA was purified using the QIAquick PCR Purification Kit. The plasmid containing the synthetic sequence AtGGT1-insert (SEQ ID NO: 17) was digested with restriction endonuclease BsaI, and the obtained DNA was purified using the QIAquick PCR Purification Kit.

[0187] The DNA of pLIB_P_AGAT-Mp, digested with NotI, was ligated with plasmids containing AaDH-Mt-inserts, digested with BsaI, and plasmids containing AtGGT1-inserts, digested with BsaI, using the NEBuilder HiFi DNA Assembly Cloning Kit. The products were transformed into NEB Stable competent E. coli (high-efficiency grade) cells and grown on LB agar containing 25 mg / L kanamycin. Suitable plasmid clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmid clone was named pLIB_AaDH-Mt_AtGGT1_AGAT-Mp.

[0188] Example 10: Cloning of plasmids co-expressing AGAT-Mp and AtGGT1

[0189] To achieve co-expression of AGAT-Mp and AtGGT1, the gene AaDH-Mt was deleted from plasmid pLIB_AaDH-Mt_AtGGT1_AGAT-Mp.

[0190] The plasmid pLIB_AaDH-Mt_AtGGT1_AGAT-Mp was digested with restriction endonucleases SacI and SalI, and the DNA was purified using the QIAquick PCR Purification Kit. The ends of the linear DNA were passivated using a Rapid DNA End Repair Kit (Thermo Fisher Scientific, Waltham, USA), and the DNA was purified. The DNA was self-ligated using a Rapid Ligation Kit (Thermo Fisher Scientific, Waltham, USA). The ligation product was transformed into NEB Stable competent E. coli (high-efficiency grade), and cells were grown on LB agar containing 25 mg / L kanamycin. Suitable plasmid clones were identified by restriction enzyme digestion, and the resulting plasmid was named pLIB_AtGGT1_AGAT-Mp.

[0191] Example 11: Transformation of Corynebacterium glutamicum strain with multiple plasmids

[0192] The following Corynebacterium glutamicum strains were transformed by electroporation with various plasmids (Table 8). Cells containing plasmids were selected using 25 mg / L kanamycin.

[0193] Corynebacterium glutamicum ATCC13032: Commonly used wild-type strain (Kinoshita et al., J. Gen. Appl. Microbiol. 1957; 3(3): 193-205)

[0194] • Corynebacterium glutamicum ATCC13032_DargR: Chromosomal deletion of argR in Corynebacterium glutamicum ATCC13032

[0195] • Corynebacterium glutamicum ATCC13032_CK: Chromosomal insertion of the CK gene between NCgl0291 and NCgl0292 in Corynebacterium glutamicum ATCC13032.

[0196] • Corynebacterium glutamicum ATCC13032_DargR_CK: Chromosomal deletion of argR in Corynebacterium glutamicum ATCC13032 and chromosomal insertion of CK gene between NCgl0291 and NCgl0292.

[0197]

[0198] Table 8: List of Corynebacterium glutamicum strains containing plasmids

[0199] Example 12: Effect of L-arginine:glycine amidosyltransferase gene on GAA production

[0200] To evaluate the effect of the L-arginine:glycine amidosyltransferase gene (AGAT-Mp) on GAA production, strains ATCC13032 / pLIB_P and ATCC13032 / pLIB_P_AGAT-Mp were cultured in production medium of the Wouter-Duetz system, and the resulting GAA titers were determined as described above.

[0201] strains GAA ATCC13032 / pLIB_P Undetectable ATCC13032 / pLIB_P_AGAT-Mp 120mg / l

[0202] Table 9: Effect of L-arginine:glycine amidotransferase gene expression on GAA production

[0203] Compared to strains lacking the L-arginine:glycine amidotransferase gene, culturing strains possessing the L-arginine:glycine amidotransferase gene resulted in the production of GAA (see Table 9). We conclude that the presence of a heterologous L-arginine:glycine amidotransferase gene enables the production of GAA.

[0204] Example 13: Effect of carbamate kinase gene on GAA production

[0205] To assess the effect of the carbamate kinase gene on GAA production, strains ATCC13032 / pLIB_P_AGAT-Mp and ATCC13032_CK / pLIB_P_AGAT-Mp were cultured in production medium in the Wouter-Duetz system, and the resulting GAA titers were determined as described above.

[0206] strains GAA ATCC13032 / pLIB_P_AGAT-Mp 120mg / l ATCC13032_CK / pLIB_P_AGAT-Mp 223mg / l

[0207] Table 10: Effect of carbamate kinase gene expression on GAA production

[0208] Compared to strains lacking the carbamate kinase gene, culturing strains with the carbamate kinase gene resulted in higher GAA titers (see Table 10). We conclude that the presence of the carbamate kinase gene increases GAA production.

[0209] Example 14: The effect of the combination of carbamate kinase gene and glyoxylate aminotransferase gene on GAA production

[0210] To assess the effect of the combined presence of the carbamate kinase gene (CK) and glyoxylate aminotransferase gene (AtGGT1) on GAA production, strains ATCC13032_CK / pLIB_P_AGAT-Mp and ATCC13032_CK / pLIB_AtGGT1_AGAT-Mp were cultured in production medium of the Wouter-Duetz system, and the resulting GAA titers were determined as described above.

[0211] strains GAA ATCC13032_CK / pLIB_P_AGAT-Mp 223mg / l ATCC13032_CK / pLIB_AtGGT1_AGAT-Mp 337mg / l

[0212] Table 11: Effects of the combination of carbamate kinase gene and glyoxylate aminotransferase gene on GAA production

[0213] Compared to strains lacking the glyoxylate aminotransferase gene, culturing strains possessing both the carbamate kinase gene and the glyoxylate aminotransferase gene resulted in higher GAA titers (see Table 11). We conclude that the combined presence of the carbamate kinase gene and the glyoxylate aminotransferase gene increases GAA production.

[0214] Example 15: The effect of the combination of carbamate kinase gene and NADH-dependent amino acid dehydrogenase gene on GAA production.

[0215] To assess the effect of the combined presence of the carbamate kinase gene (CK) and the NADH-dependent amino acid dehydrogenase gene (AaDH-Mt) on GAA production, strains ATCC13032_CK / pLIB_P_AGAT-Mp and ATCC13032_CK / pLIB_AaDH-Mt_AGAT-Mp were cultured in production medium on a Wouter-Duetz system, and the resulting GAA titers were determined as described above.

[0216] strains GAA ATCC13032_CK / pLIB_P_AGAT-Mp 223mg / l ATCC13032_CK / pLIB_AaDH-Mt_AGAT-Mp 545mg / l

[0217] Table 12: The effect of the combination of carbamate kinase gene and NADH-dependent amino acid dehydrogenase gene on GAA production.

[0218] Compared to strains lacking the NADH-dependent amino acid dehydrogenase gene, strains possessing both the carbamate kinase gene and the NADH-dependent amino acid dehydrogenase gene resulted in higher GAA titers (see Table 12). We conclude that the combined presence of the carbamate kinase gene and the NADH-dependent amino acid dehydrogenase gene increases GAA production.

[0219] Example 16: The effect of the combination of carbamate kinase gene, NADH-dependent amino acid dehydrogenase gene, and glyoxylate aminotransferase gene on GAA production.

[0220] To evaluate the effect of the combined presence of the carbamate kinase gene (CK), glyoxylate aminotransferase gene (AtGGT1), and NADH-dependent amino acid dehydrogenase gene (AaDH-Mt) on GAA production, strains ATCC13032_CK / pLIB_AtGGT1_AGAT-Mp, ATCC13032_CK / pLIB_AaDH-Mt_AGAT-Mp, and ATCC13032_CK / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp were cultured in production medium on a Wouter-Duetz system, and the resulting GAA titers were determined as described above.

[0221] strains GAA ATCC13032_CK / pLIB_AtGGT1_AGAT-Mp 337mg / l ATCC13032_CK / pLIB_AaDH-Mt_AGAT-Mp 545mg / l ATCC13032_CK / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp 657mg / l

[0222] Table 13: Effects of the combination of carbamate kinase gene, NADH-dependent amino acid dehydrogenase gene, and glyoxylate aminotransferase gene on GAA production.

[0223] Compared to strains lacking NADH-dependent amino acid dehydrogenase genes or glyoxylate aminotransferase genes, strains possessing carbamate kinase genes, NADH-dependent amino acid dehydrogenase genes, and glyoxylate aminotransferase genes resulted in higher GAA titers (see Table 13). We conclude that the combined presence of carbamate kinase genes, NADH-dependent amino acid dehydrogenase genes, and glyoxylate aminotransferase genes increases GAA production.

[0224] Example 17: The effect of the combination of argR gene deletion and the presence of carbamate kinase gene, NADH-dependent amino acid dehydrogenase gene and glyoxylate aminotransferase gene on GAA production.

[0225] To evaluate the effect of the combination of argR gene (DargR) deletion and the presence of carbamate kinase gene (CK), glyoxylate aminotransferase gene (AtGGT1), and NADH-dependent amino acid dehydrogenase gene (AaDH-Mt) on GAA production, strains ATCC13032 / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp, ATCC13032_DargR / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp, and ATCC13032_DargR_CK / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp were cultured in production medium of the Wouter-Duetz system, and the resulting GAA titers were determined as described above.

[0226] strains GAA ATCC13032 / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp 377mg / l ATCC13032_DargR / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp 589mg / l ATCC13032_DargR_CK / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp 845mg / l

[0227] Table 14: Effects of the combination of argR gene deletion and the presence of carbamate kinase gene, NADH-dependent amino acid dehydrogenase gene, and glyoxylate aminotransferase gene on GAA production.

[0228] Compared to strains possessing the wild-type argR gene, strains with a combination of argR gene deletion and the presence of glyoxylate aminotransferase and NADH-dependent amino acid dehydrogenase genes resulted in higher GAA titers when cultured (see Table 14). Compared to the two strains lacking the carbamate kinase gene, strains with a combination of argR gene deletion and the presence of carbamate kinase, glyoxylate aminotransferase, and NADH-dependent amino acid dehydrogenase genes resulted in higher GAA titers when cultured (see Table 14).

[0229] We conclude that the combination of argR gene deletion and the presence of carbamate kinase gene, glyoxylate aminotransferase gene, and NADH-dependent amino acid dehydrogenase gene increases GAA production.

Claims

1. A microorganism comprising at least one heterologous gene encoding a protein having an L-arginine:glycine amidinotransferase function and comprising at least one gene encoding a protein having a carbamate kinase function, and further comprising at least one gene encoding a protein having a NADH-dependent amino acid dehydrogenase function. 2 . The microorganism according to claim 1 , wherein the activity of the at least one protein having a carbamate kinase function is increased compared to the respective activity in the wild-type microorganism.

3. The microorganism according to claim 1 or 2, comprising at least one heterologous gene encoding a protein having a carbamate kinase function. The microorganism according to claim 1 , wherein the activity of the protein having the NADH-dependent amino acid dehydrogenase function is increased compared to the respective activities in a wild-type microorganism.

5. The microorganism according to any one of the preceding claims, wherein the protein having the function of NADH-dependent amino acid dehydrogenase is a heterologous protein.

6. The microorganism according to any one of the preceding claims, further comprising at least one gene encoding a protein having the function of a glyoxylate aminotransferase.

7. A microorganism according to any one of the preceding claims, wherein the ability of the microorganism to produce L-arginine from L-ornithine is increased compared to the ability of a wild-type microorganism.

8. The microorganism according to claim 7, wherein the expression of the argR gene encoding the arginine response repressor protein ArgR is attenuated compared to the expression of the argR gene in the wild-type microorganism, or wherein the argR gene is deleted.

9. The microorganism according to any one of the preceding claims, wherein the microorganism belongs to the genus Corynebacterium, to the genus Bacillus, to the family Enterobacteriaceae or to the genus Pseudomonas.

10. A fermentation production method of guanidinoacetic acid (GAA), the method comprising the following steps: a) culturing a microorganism as defined in any one of the preceding claims in a culture medium, and b) accumulating GAA in the culture medium to form a fermentation broth containing GAA.

11. The method according to claim 10, further comprising separating GAA from the GAA-containing fermentation broth.

12. The microorganism according to any one of claims 1 to 9, further comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity. 13 . The microorganism according to claim 12 , wherein the gene encoding the enzyme having the activity of guanidinoacetic acid N-methyltransferase is overexpressed.

14. A fermentation production method of creatine, the method comprising the following steps: a) culturing the microorganism as defined in any one of claims 12 or 13, and b) accumulating creatine in the culture medium to form a fermentation broth containing creatine.

15. The method according to claim 14, further comprising separating creatine from the fermentation broth containing creatine.

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