Method for the production of guanidinoacetic acid (GAA)

Abstract

The present invention relates to a method for the fermentative production of guanidinoacetic acid (GAA) using a microorganism transformed to be capable of producing GAA, which is improved by using carbamate kinase (2.7.2.2) and L-arginine:glycine amidinotransferase (2.1.4.1), and such a microorganism that can be further improved by deleting the central repressor protein argR and expressing glyoxylate aminotransferase (2.6.1.-) or NADH-dependent amino acid dehydrogenase (1.4.1-). The present invention also relates to a method for the fermentative production of creatine by further expressing guanidinoacetic acid N-methyltransferase (2.1.1.2).

Description

Technical Field

[0001] Guanidinoacetic acid (GAA) is a colorless crystalline organic compound that is used as an animal feed additive (for example, WO 2005 / 120246 pamphlet and US Patent Application Publication No. 2011 / 257075). GAA is a natural precursor of creatine (for example, Humm et al., Biochem. J., (1997), 322, 771-776). Thus, supplementation with GAA enables optimal supply of creatine in organisms.

[0002] The present invention relates to a microorganism transformed to be capable of producing guanidinoacetic acid (GAA), and a method for fermentative production of GAA using such a microorganism. The present invention also relates to a method for fermentative production of creatine.

Background Art

[0003] In biological systems, GAA and ornithine are formed from arginine and glycine as starting materials by the catalytic action of L-arginine:glycine amidinotransferase (AGAT; EC 2.1.4.1). This reaction is also the first step in creatine biosynthesis.

Chemical Formula

[0004] Guthmiller et al. (J Biol Chem., July 1, 1994; 269(26):17556-60) characterized rat kidney AGAT by cloning and heterologously expressing the enzyme in Escherichia coli (E. coli). Muenchhoff et al. (FEBS Journal, 277, (2010), 3844-3860) reported the first characterization of AGAT from prokaryotes, also by cloning and heterologously expressing the enzyme in E. coli.

[0005] Fan Wenchao has disclosed a method for the production of creatine by fermentation of non-pathogenic microorganisms such as Corynebacterium glutamicum (Specification of Chinese Patent Application Publication No. 106065411). The microorganisms have the following in vivo conversion functions: conversion of glucose to L-glutamic acid; conversion of L-glutamic acid to N-acetyl-L-glutamic acid; conversion of N-acetyl-L-glutamic acid to N-acetyl-L-glutamic acid semialdehyde; conversion of N-acetyl-L-glutamic acid semialdehyde to N-acetyl-L-ornithine; conversion of N-acetyl-L-ornithine to L-ornithine; conversion of L-ornithine to L-citrulline; conversion of L-citrulline to argininosuccinic acid; conversion of argininosuccinic acid to L-arginine; conversion of L-arginine to guanidinoacetic acid; and finally, conversion of guanidinoacetic acid to creatine. Fan Wenchao has proposed that the microorganisms overexpress one or more enzymes selected from the group consisting of N-acetylglutamate-synthetase, N-acetylornithine-δ-aminotransferase, N-acetylornithinase, ornithine-carbamoyltransferase, argininosuccinate synthetase, glycine-amidinotransferase (EC: 2.1.4.1) and guanidinoacetic acid-N-methyltransferase (EC: 2.1.1.2). The microorganisms preferably overexpress glycine aminotransferase (L-arginine: glycine amidinotransferase) and guanidinoacetic acid-N-methyltransferase.

[0006] Microorganisms capable of producing guanidinoacetic acid (GAA) have been published by Zhang et al. (ACS Synth. Biol., 2020, 9, 2066 - 275). They designed a reconstituted ornithine cycle in Escherichia coli by introducing heterologous AGATs from different species (e.g., Homo sapiens, Cylindrospermopsis raciborskii, Moorea producens), as well as introducing a citrulline synthesis module (e.g., overexpression of carAB, argF and argI) and an arginine synthesis module (e.g., overexpression of argG, argH; introduction of aspA) into E. coli.

[0007] Schneider and Jankowitsch (International Publication No. WO 2021 / 122400 pamphlet) proposed a method for producing GAA using a microorganism having a gene encoding a protein with the function of L - arginine:glycine amidinotransferase and increased carbamoyl phosphate synthetase. Carbamoyl phosphate is an important precursor not only for the biosynthesis of GAA, but also for L - arginine and other compounds.

[0008] Several approaches for increasing the production of one of the starting materials in GAA synthesis in microorganisms, particularly bacteria, namely L-arginine, are also known from the literature. An overview of the metabolic engineering of Corynebacterium glutamicum (C. glutamicum) for L-arginine production is provided by Park et al. (NATURE COMMUNICATIONS|DOI:10.1038 / ncomms5618). Yim et al. (J Ind Microbiol Biotechnol, (2011), 38:1911-1920) were able to show that disruption of the chromosomal argR gene in C. glutamicum inactivated argR, a gene encoding the central repressor protein ArgR that controls the L-arginine biosynthetic pathway, resulting in an improved arginine-producing strain. Ginesy et al. (Microbial Cell Factories, (2015), 14:29) reported the success of engineering Escherichia coli for enhanced arginine production. In particular, they proposed deletion of the argR repressor gene.

[0009] Wang et al. (Applied Microbiology and Biotechnology, 2021, 105, 3265-3276; https: / / doi.org / 10.1007 / s00253-021-11242-w) emphasized that carbamoyl phosphate is also essential for L-arginine production in Corynebacterium species. They showed, among other things, that overexpression of the carAB gene encoding carbamoyl phosphate synthetase and introduction of a heterologous gene (from Enterococcus faecalis) encoding carbamate kinase (CK), which catalyzes the synthesis of carbamoyl phosphate from inorganic ammonia, bicarbonate, and ATP, could lead to an increase in L-arginine production. The advantage of using carbamate kinase is due to the utilization of inorganic ammonium as a nitrogen source by this enzyme. Compared with carbamoyl phosphate synthetase using glutamine as a nitrogen source, carbamate kinase enables a reduction in the overall energy requirement for the formation of carbamoyl phosphate. Yan et al. (Fermentation, 2022, 8, 3, March 7, 2022, 116; doi:10.3390 / fermentation8030116) disclosed the biosynthesis of GAA by whole-cell catalysis by Bacillus subtilis 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 glycine dehydrogenase gene gcvP, the first gene of the glycine degradation pathway.

[0010] Schneider and Jankowitsch (International Publication No. WO 2022 / 008276) proposed producing GAA using a recombinant microorganism containing a gene encoding L-arginine:glycine amidinotransferase (AGAT), a reduced or deleted malate synthase gene to increase the production of glycine, one of the starting materials, and optionally an overexpressed gene encoding glyoxylate aminotransferase. They also disclosed that carbamate kinase (CK) can contribute to arginine production.

Prior Art Documents

Patent Documents

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Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0013] To increase the production of GAA using microorganisms, a large amount of the starting materials arginine and / or glycine is required inside the cells. At the same time, ornithine, which is a by-product of the AGAT reaction, must be efficiently recycled into arginine to prevent loss of carbon and energy.

[0014] The underlying problem of the present invention is to provide a microorganism transformed to be able to produce guanidinoacetic acid (GAA), in particular, a microorganism with improved ability to provide L-arginine as a starting material for GAA biosynthesis by efficient recycling of ornithine, and a method for fermentative production of GAA using such a microorganism.

Means for Solving the Problems

[0015] This problem is solved by a microorganism comprising at least one heterologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT, e.g., EC 2.1.4.1), comprising 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.

[0016] A heterologous gene means that the gene is inserted into a host organism that does not naturally have this gene. Insertion of a heterologous gene into a host is carried out by recombinant DNA technology. A microorganism subjected to recombinant DNA technology is called transgenic, genetically modified or recombinant. A heterologous protein means a protein that does not naturally exist in the microorganism. A homologous gene or endogenous gene means that a gene or nucleotide sequence of a gene containing such a function naturally exists within the microorganism or is "native" within the microorganism. A homologous protein or native protein means a protein that naturally exists in the microorganism.

[0017] In the microorganism according to the present invention, the protein having the function of L-arginine:glycine amidinotransferase (AGAT) comprises an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 9.

[0018] In the microorganism according to the present invention, at least one gene encoding a protein having the function of carbamate kinase (CK, e.g., EC 2.7.2.2) may be heterologous.

[0019] In the microorganism according to the present invention, the activity of at least one protein having the function of carbamate kinase is increased as compared to the respective activity in the wild-type microorganism.

[0020] Generally, an increase in enzyme activity in a microorganism can be achieved, for example, by mutation of the corresponding endogenous gene. A further means for increasing enzyme activity can be to stabilize the mRNA encoding the enzyme. An increase in enzyme activity in a microorganism can also be achieved by overexpression of the gene encoding each enzyme.

[0021] The microorganism according to the present invention may contain at least one heterologous gene encoding a protein having the function of carbamate kinase. In the microorganism according to the present invention, at least one protein having the enzyme activity of carbamate kinase may contain an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 6.

[0022] Overexpression of a gene is generally achieved by increasing the copy number of the gene and / or functionally linking the gene to a strong promoter and / or enhancing the ribosome binding site and / or optimizing the codon usage frequency of the start codon or the entire gene, or by a combination including the selection of all of the above methods.

[0023] The promoter consists of approximately 40 to 50 base pairs and is a DNA sequence that constitutes the binding site for the RNA polymerase holoenzyme and the transcription start point, and can affect the intensity of the expression of the polynucleotide or gene controlled thereby. Generally, for example, as taught for C. glutamicum by M. Patek et al. (Microbial Biotechnology, 6, (2013), 103 - 117), by selecting a strong promoter, for example, by replacing the original promoter with a strong native (originally assigned to another gene) promoter, or by modifying specific regions of a given native promoter (e.g., its so-called -10 and -35 regions) towards the consensus sequence, it is possible to achieve overexpression or increased expression of a gene in bacteria. An example of a "strong" promoter is the superoxide dismutase (sod) promoter ("Psod"; Z. Wang et al., Eng. Life Sci., 2015, 15, 73 - 82). "Functionally linked" is understood to mean the continuous arrangement of a promoter with a gene that results in the transcription of the gene.

[0024] In a specific embodiment of the present invention, the gene encoding the protein having the function of carbamate kinase is functionally linked to a strong promoter. Preferably, the promoter is the superoxide dismutase (sod) promoter ("Psod").

[0025] In the microorganism according to the present invention, the protein having the function of NADH-dependent amino acid dehydrogenase can be a heterologous protein.

[0026] NADH-dependent amino acid dehydrogenase (AaDH) catalyzes the amination reaction of keto acids to L-amino acids. NADH-dependent amino acid dehydrogenase is important for the assimilation or dissimilation of ammonium in cells.

Chemical formula

[0027] Most of these amino acid dehydrogenases can use different α-keto acids as substrates and are often annotated with different EC numbers. However, all of these amino acid dehydrogenases have in common that they assimilate ammonium or, in the case of the reverse reaction, dissimilate ammonium.

[0028] Examples of different amino acid dehydrogenases are as follows. Reaction EC 1.4.1.1: Alanine dehydrogenase: Pyruvate + NH3 + NADH + H+ <-> L-Alanine + H2O + NAD+ Reaction EC 1.4.1.10: Glycine dehydrogenase: Glyoxylate + NH3 + NADH + H+ <-> Glycine + H2O + NAD+ Reaction EC 1.4.1.21: Aspartate dehydrogenase: Oxaloacetate + NH3 + NADH + H+ <-> L-Aspartate + H2O + NAD+

[0029] Several NADH-dependent amino acid dehydrogenase (AaDH) proteins are known in the literature that accept a wide range of substrates and can thus, in many cases, aminate several different keto acids to the corresponding amino acids. (Fernandes et al., Protein Engineering, Design & Selection, 2015, 28(2), 29 - 35; Giffin et al., Journal of Bacteriology, 2012, 194(5), 1045 - 1054; Phogosee et al., Archives of Microbiology, 2018, 200, 719 - 727; Schuffenhauer et al., 1999, 171, 417 - 423; Vancura et al., Eur J Biochem, 1989, 179, 221 - 227, Yoshida and Freese, Biochim. Biophys. Acta, 1965, 96, 248 - 262)

[0030] Therefore, there is no clear link to the reaction for the EC number. Table 1 shows some examples.

[0031]

Table 1

[0032] In the microorganism according to the present invention, at least one protein having the function of NADH-dependent amino acid dehydrogenase can 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).

[0033] In the microorganism of the present invention, the activity of at least one NADH-dependent amino acid dehydrogenase may be increased as compared with the respective activities in the wild-type microorganism.

[0034] At least one protein having the function of NADH-dependent amino acid dehydrogenase is preferably heterologous.

[0035] At least one NADH-dependent amino acid dehydrogenase contained in the microorganism according to the present invention can 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).

[0036] In the microorganism according to the present invention, the protein having the function of NADH-dependent amino acid dehydrogenase may contain an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 13, the amino acid sequence according to SEQ ID NO: 21, the amino acid sequence according to SEQ ID NO: 22, the amino acid sequence according to SEQ ID NO: 23, or the amino acid sequence according to SEQ ID NO: 24.

[0037] The microorganism of the present invention may further contain at least one gene encoding a protein having the function of glyoxylate aminotransferase.

[0038] Several glyoxylate aminotransferases are known, and their substrate specificities for amino donors are different (see, for example, Kameya et al., FEBS Journal, 277, (2010), 1876-1885; Liepman and Olsen, Plant Physiol., Vol. 131, 2003, 215-227; Sakuraba et al., JOURNAL OF BACTERIOLOGY, August 2004, pages 5513-5518; Takada and Noguchi, Biochem. J., (1985), 231, 157-163). Most of these glyoxylate aminotransferases can use different amino acids as amino donors, so they are often annotated with different EC numbers. However, all of these aminotransferases have in common that they use glyoxylate as the acceptor molecule or, in the case of the reverse reaction, glycine as the donor molecule. Examples of proteins having the function of glyoxylate aminotransferase are as follows. Glycine transaminase (EC 2.6.1.4) catalyzes the following reaction. L-Glutamate + glyoxylate <=> α-Ketoglutarate + glycine. Glycine:oxaloacetate transaminase (EC 2.6.1.35) catalyzes the following reaction. L-Aspartate + glyoxylate <=> Oxaloacetate + glycine. Alanine:glyoxylate transaminase (EC 2.6.1.44) catalyzes the following reaction. L-Alanine + glyoxylate <=> Pyruvate + glycine. Serine:glyoxylate transaminase (EC 2.6.1.45) catalyzes the following reaction. L-Serine + glyoxylate <=> 3-Hydroxy-pyruvate + glycine. Methionine:glyoxylate transaminase (EC 2.6.1.73) catalyzes the following reaction. L-Methionine + glyoxylate <=> 4-(methylsulfonyl)-2-keto-butanoate + glycine. Aromatic amino acid:glyoxylate transaminase (EC 2.6.1.60) catalyzes the following reaction. Aromatic amino acid + glyoxylate <=> aromatic keto acid + glycine. Kynurenine:glyoxylate transaminase (EC 2.6.1.63) catalyzes the following reaction. Kynurenine + glyoxylate <=> 4-(2-aminophenyl)-2,4-diketo-butanoate + glycine. (S)-Ureido-glycine:glyoxylate transaminase (EC 2.6.1.112) catalyzes the following reaction. (S)-Ureido-glycine + glyoxylate <=> N-carbamoyl-2-keto-glycine + glycine.

[0039] In a further embodiment of the present invention, the enzymatic activity of at least one protein having the function of glyoxylate aminotransferase is increased as compared to the respective enzymatic activity in wild-type microorganisms.

[0040] At least one protein having the function of glyoxylate aminotransferase is preferably heterologous.

[0041] In a specific embodiment of the present invention, at least one protein having the function of glyoxylate aminotransferase is glycine:glyoxylate aminotransferase.

[0042] In the microorganism according to the present invention, the protein having the enzymatic activity of glyoxylate aminotransferase may comprise an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 16, the amino acid sequence according to SEQ ID NO: 19 or the amino acid sequence according to SEQ ID NO: 20.

[0043] The microorganism according to the present invention may have an increased ability to produce L-arginine compared to the ability of a wild-type microorganism.

[0044] In the context of the present invention, a microorganism with an increased ability to produce L-arginine means a microorganism that produces L-arginine in excess of its own requirements. Examples of such L-arginine-producing microorganisms are, for example, C. glutamicum ATCC 21831, or those disclosed by Park et al. (NATURE COMMUNICATIONS|DOI:10.1038 / ncomms5618), or by Ginesy et al. (Microbial Cell Factories, (2015), 14:29). In contrast to the previously described microorganisms with an increased ability to produce L-arginine, in the framework of the present invention for GAA production, since arginine is utilized intracellularly, L-arginine excretion is not necessary in the strains for GAA production.

[0045] In a further embodiment, in the microorganism according to the present invention, the expression of the argR gene encoding the arginine-responsive repressor protein ArgR is attenuated compared to the expression of the argR gene in a wild-type microorganism. Alternatively, the argR gene is deleted.

[0046] The microorganism 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.

[0047] In a specific embodiment of the present invention, the microorganism is Corynebacterium glutamicum (C. glutamicum) or Escherichia coli (E. coli).

[0048] The present invention further relates to a method for the fermentative production of guanidinoacetic acid (GAA), comprising: a) culturing a microorganism according to the present invention in a medium; and b) accumulating guanidinoacetic acid (GAA) in the medium to form a GAA-containing fermentation broth.

[0049] Preferably, the method further comprises isolating GAA from the GAA-containing fermentation broth.

[0050] In certain embodiments, the microorganism of the present invention further comprises a gene encoding an enzyme having the activity of guanidinoacetic acid N-methyltransferase. The gene encoding the enzyme having the activity of guanidinoacetic acid N-methyltransferase can be overexpressed.

[0051] The present invention also relates to a method for the fermentative production of creatine, comprising: a) culturing a microorganism according to the present invention, which further comprises a gene encoding an enzyme having the activity of guanidinoacetic acid N-methyltransferase, in a suitable medium under suitable conditions; and b) accumulating creatine in the medium to form a creatine-containing fermentation broth.

[0052] Preferably, the method further comprises isolating creatine from the creatine-containing fermentation broth. Creatine can be extracted from the fermentation broth by an isoelectric point method and / or an ion exchange method. Alternatively, creatine can be further purified by a method of recrystallization in water.

[0053] Experimental section A) Materials and methods Chemicals The kanamycin solution derived from Streptomyces kanamyceticus was purchased from Sigma Aldrich (St. Louis, USA, catalog number K0254). Unless otherwise specified, all other chemicals were purchased in analytically pure form from Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, USA) or Carl-Roth (Karlsruhe, Germany).

[0054] Culture for cell growth Unless otherwise specified, the culture / incubation procedures were performed as follows: a. An Escherichia coli strain was cultured in a liquid medium using LB broth (MILLER) manufactured by Merck (Darmstadt, Germany, catalog number 110285). The liquid culture (10 ml of liquid medium per 100 ml Erlenmeyer flask equipped with three baffles) was incubated at 30 °C and 200 rpm in an Infors HT Multitron standard incubator shaker manufactured by Infors GmbH (Bottmingen, Switzerland).

[0055] b. An Escherichia coli strain was cultured on an agar plate using LB agar (MILLER) manufactured by Merck (Darmstadt, Germany, catalog number 110283). The agar plate was incubated at 30 °C in an INCU-Line® mini incubator manufactured by VWR (Radnor, USA).

[0056] c. A C. glutamicum strain was cultured in a liquid medium using brain heart infusion broth (BHI) manufactured by Merck (Darmstadt, Germany, catalog number 110493). The liquid culture (10 ml of liquid medium per 100 ml Erlenmeyer flask equipped with three baffles) was incubated at 30 °C and 200 rpm in an Infors HT Multitron standard incubator shaker manufactured by Infors GmbH (Bottmingen, Switzerland).

[0057] d. C. glutamicum strains were cultured on agar plates using brain heart agar (BHI-agar) manufactured by Merck (Darmstadt, Germany, catalog number 113825). The agar plates were incubated at 30 °C in an incubator manufactured by Heraeus Instruments equipped with a Kelvitron® temperature controller (Hanau, Germany).

[0058] e. To culture C. glutamicum after electroporation, BHI-agar (Merck, Darmstadt, Germany, catalog number 113825) was supplemented with 134 g / l sorbitol (Carl Roth GmbH+Co.KG, Karlsruhe, Germany), 2.5 g / l yeast extract (Oxoid / ThermoFisher Scientific, Waltham, USA, catalog number LP0021) and 25 mg / l kanamycin. The agar plates were incubated at 30 °C in an incubator manufactured by Heraeus Instruments equipped with a Kelvitron® temperature controller (Hanau, Germany).

[0059] Determination of the optical density of bacterial suspensions a. The optical density of the bacterial suspension in the shake flask culture was determined at 600 nm (OD600) using a Bio-Photometer manufactured by Eppendorf AG (Hamburg, Germany).

[0060] b. The optical density of the bacterial suspension produced in a Wouter Duetz (WDS) microfermentation system (24-well plate) was measured at 660 nm (OD660) using a GENios® plate reader manufactured by Tecan Group AG (Mannedorf, Switzerland).

[0061] Centrifugation a. A bacterial suspension with a maximum volume of 2 ml was centrifuged in a 1.5 ml or 2 ml reaction tube (e.g., Eppendorf Tubes® 3810X) using an Eppendorf 5417 R benchtop centrifuge (at 13,000 rpm for 5 minutes).

[0062] b. A bacterial suspension with a maximum volume of 50 ml was centrifuged in a 15 ml or 50 ml centrifuge tube (e.g., Falcon® 50 ml Conical Centrifuge Tube) using an Eppendorf 5810 R benchtop centrifuge at 4,000 rpm for 10 minutes.

[0063] DNA Isolation Plasmid DNA was isolated from E. coli cells using the QIAprep Spin Miniprep Kit (Hilden, Germany, catalog number 27106) from Qiagen according to the manufacturer's instructions.

[0064] Polymerase Chain Reaction (PCR) PCR using a proofreading (high-fidelity) polymerase was used to amplify the desired segment of DNA for sequencing or DNA assembly cloning. A non-proofreading polymerase kit was used to directly determine the presence or absence of the desired DNA fragment from E. coli or C. glutamicum colonies.

[0065] a. The Phusion® High-Fidelity DNA Polymerase Kit (Phusion Kit) from New England BioLabs Inc. (Ipswich, USA, catalog number M0530) was used according to the manufacturer's instructions for template-corrected amplification of the selected DNA region (see Table 2).

[0066]

Table 2

[0067] b. The desired segment of DNA was amplified to confirm its presence using the Taq PCR Core Kit (Taq Kit) manufactured by Qiagen (Hilden, Germany, catalog number 201203). The kit was used according to the manufacturer's instructions (see Table 3).

[0068]

Table 3

[0069] c. To confirm the presence of the desired DNA segment in cells collected from E. coli or C. glutamicum colonies, the SapphireAmp® Fast PCR Master Mix (Sapphire Mix) manufactured by Takara Bio Europe S.A.S. (Saint-Germain-en-Laye, France, catalog number RR350A / B) was used as an alternative according to the manufacturer's instructions (see Table 4).

[0070]

Table 4

[0071] d. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany).

[0072] e. As the PCR template, an appropriately diluted solution of isolated plasmid DNA or total DNA isolated from a liquid culture, or total DNA contained in bacterial colonies (colony PCR) was used. For the colony PCR, the template was prepared by collecting cell material with a sterile toothpick from colonies on an agar plate and directly placing the cell material into a PCR reaction tube. The cell material was heated at 800 W for 10 seconds using a microwave type Mikrowave&Grill manufactured by SEVERIN Elektrogerate GmbH (Sundern, Germany), and then the PCR reagent was added to the template in the PCR reaction tube.

[0073] f. All PCR reactions were carried out using a PCR cycler type Mastercycler or Mastercycler nexus gradient manufactured by Eppendorf AG (Hamburg, Germany).

[0074] Restriction enzyme digestion of DNA For restriction enzyme digestion, either "FastDigest restriction endonuclease (FD)" (ThermoFisher Scientific, Waltham, USA) or a restriction endonuclease manufactured by New England BioLabs Inc. (Ipswich, USA) was used. The reactions were carried out according to the instructions of the manufacturer's manual.

[0075] Determination of the size of DNA fragments a. The size of small DNA fragments (<1000 bps) was usually determined by capillary electrophoresis using QIAxcel manufactured by Qiagen (Hilden, Germany).

[0076] b. If it was necessary to isolate the DNA fragment or if the DNA fragment was >1000 bps, the DNA was separated by TAE agarose gel electrophoresis and stained with GelRed® Nucleic Acid Gel Stain (Biotium, Inc., Fremont, Canada). The stained DNA was visualized at 302 nm.

[0077] Purification of PCR amplification products and restriction fragments The PCR amplification products and restriction fragments were cleaned up using the QIAquick PCR Purification Kit manufactured by Qiagen (Hilden, Germany, catalog number 28106) according to the manufacturer's instructions. The DNA was eluted with 30 μl of 10 mM Tris*HCl (pH 8.5).

[0078] Determination of DNA concentration The DNA concentration was measured using a NanoDrop Spectrophotometer ND-1000 manufactured by PEQLAB Biotechnologie GmbH (Erlangen, Germany), which has been the VWR brand since 2015.

[0079] Assembly cloning The plasmid vector was assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" purchased from New England BioLabs Inc. (Ipswich, USA, catalog number E5520). A reaction mix containing a linear vector and at least one DNA insert was incubated at 50 °C for 60 minutes. 0.5 μl of the assembly mixture was used for each transformation experiment.

[0080] Chemical transformation of E. coli For plasmid cloning, chemically competent "NEB® Stable Competent E. coli (High Efficiency)" (New England BioLabs Inc., Ipswich, USA, catalog number C3040) was transformed according to the manufacturer's protocol. Cells that were successfully transformed were selected on LB agar supplemented with 25 mg / l kanamycin.

[0081] Transformation of C. glutamicum The transformation of C. glutamicum with plasmid DNA was carried out by electroporation using a "Gene Pulser Xcell" (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) as described by Ruan et al. (2015). The electroporation was performed in a 1 mm electroporation cuvette (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) at a fixed time set at 1.8 kV and 5 ms. The transformed cells were selected on BHI agar containing 134 g / l sorbitol, 2.5 g / l yeast extract and 25 mg / l kanamycin.

[0082] Determination of nucleotide sequence The nucleotide sequence of the DNA molecule was determined by cycle sequencing using the dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences USA, 74, 5463-5467, 1977) by Eurofins Genomics GmbH (Ebersberg, Germany). The sequence was visualized and evaluated using Clonemanager Professional 9 software (Scientific & Educational Software, Denver, USA) for the in silico assembly of the sequence.

[0083] Glycerol stocks of E. coli strains and C. glutamicum strains For the long-term preservation of Escherichia coli strains and C. glutamicum strains, glycerol stocks were prepared. The selected E. coli clones were cultured in 10 ml of LB medium supplemented with 2 g / l glucose. The selected C. glutamicum was cultured in 10 ml of 2x concentrated BHI medium supplemented with 2 g / l glucose. The media for growing the E. coli strains and C. glutamicum strains containing the plasmid were supplemented with 25 mg / l kanamycin. The media were placed in 100 ml Erlenmeyer flasks equipped with three baffles. A loop of cells taken from the colonies was inoculated. The cultures were then incubated at 30 °C and 200 rpm for 18 hours. After the incubation period, 1.2 ml of 85% (v / v) sterile glycerol was added to the cultures. The resulting cell-containing glycerol suspension was then aliquoted into 2 ml portions and stored at -80 °C.

[0084] GAA production in milliliter-scale cultures The GAA production of the strains was evaluated using the milliliter-scale culture system by Duetz, (2007). For this purpose, a 24-deep well microplate (24-well WDS plate) from EnzyScreen BV (Heemstede, the Netherlands, catalog number CR1424) filled with 2.5 ml of medium per well was used.

[0085] The preculture of the strains was carried out in 10 ml of seed medium (SM). The medium was placed in a 100 ml Erlenmeyer flask equipped with three baffles. This was inoculated with 100 μl of the glycerol stock culture, and the culture was incubated at 30 °C and 200 rpm for 24 hours. The composition of the seed medium (SM) is shown in Table 5.

[0086]

Table 5

[0087] After the incubation period, the optical density OD600 of the preculture was determined. An amount necessary to inoculate 2.5 ml of production medium (PM) to an OD600 of 0.1 was sampled from the preculture, centrifuged (1 minute at 8000 g), and the supernatant was discarded. The cells were then resuspended in 100 μl of production medium.

[0088] Main cultures were initiated by inoculating 100 μl each of the resuspended cells from the preculture into the wells of a 24 Well WDS-Plate containing 2.4 ml of production medium (PM). The composition of the production medium (PM) is shown in Table 6.

[0089]

Table 6

[0090] The main cultures were incubated for 72 hours at 30 °C and 225 rpm in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland) until the glucose was completely consumed. The glucose concentration in the suspension was analyzed with a OneTouch Vita® glucometer from LifeScan (Johnson & Johnson Medical GmbH, Neuss, Germany).

[0091] After culturing, the culture suspension was transferred to a deep well microplate. A portion of the culture suspension was appropriately diluted and the OD600 was measured. Another portion of the culture was centrifuged and the concentration of GAA in the supernatant was analyzed as described below.

[0092] B) Examples Example 1: Cloning of plasmid pK18mobsacB_DargR for genomic deletion of the gene argR in C. glutamicum To enhance intracellular L-arginine formation from L-ornithine and L-arginine recycling, we decided to delete the gene argR encoding the central repressor protein ArgR that controls the L-arginine biosynthetic pathway. Therefore, the plasmid pK18mobsacB_DargR was constructed as follows.

[0093] The plasmid pK18mobsacB (Schafer, 1994; Genbank accession FJ437239) was digested using the restriction endonuclease XbaI, and the linearized vector DNA (5721 bp) was purified using the "QIAquick Gel Extraction Kit".

[0094] To construct the insert, two DNA fragments were generated by high-fidelity PCR using the following primer pairs (using the DNA of ATCC13032 as the template): DargR_lf (SEQ ID NO: 1), +DargR_lr (SEQ ID NO: 2) = left homology arm (983 bps) DargR_rf (SEQ ID NO: 3), +DargR_rr (SEQ ID NO: 4) = left homology arm (984 bps)

[0095] The PCR products were purified using the "QIAquick PCR Purification Kit".

[0096] The linearized plasmid and the PCR products were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit". The assembly products were transformed into "NEB Stable Competent E.coli (High Efficiency)", and the cells were grown on LB agar containing 25 mg / l kanamycin. Appropriate plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pK18mobsacB_DargR.

[0097] Example 2: Chromosomal deletion of the argR gene in ATCC 13032 The Corynebacterium glutamicum type strain / wild type C. glutamicum ATCC 13032 (Kinoshita S, Udaka S, Shimono M., J. Gen. Appl. Microbiol., 1957; 3(3): 193 - 205) is commercially available with the deposit number DSM 20300 at the American Type Culture Collection (ATCC) or DSMZ - German Collection of Microorganisms and Cell Cultures GmbH.

[0098] To delete the argR gene, plasmid pK18mobsacB_DargR was transformed into C. glutamicum ATCC 13032 by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with 134 g / l sorbitol, 2.5 g / l yeast extract and 25 mg / l kanamycin. The agar plates were incubated at 33 °C for 48 hours.

[0099] Individual colonies were transferred onto fresh agar plates (containing 25 mg / l kanamycin) and incubated at 33 °C for 24 hours. Liquid cultures of these clones were cultured at 33 °C for 24 hours in 10 ml of BHI medium contained in 100 ml Erlenmeyer flasks equipped with three baffles. To isolate the clones that encountered the second recombination event, aliquots were taken from each liquid culture, appropriately diluted, and 100 μl was seeded onto BHI agar supplemented with 10% sucrose. The agar plates were incubated at 33 °C for 48 hours. Subsequently, the colonies grown on the sucrose-containing agar plates were examined for kanamycin sensitivity. To do so, a toothpick was used to pick up cell material from the colonies and transfer it onto BHI agar containing 25 mg / l kanamycin and onto BHI agar containing 10% sucrose. The agar plates were incubated at 33 °C for 60 hours. The clones found to be sensitive to kanamycin and resistant to sucrose were examined by PCR and DNA sequencing. The resulting strain lacking the argR gene was named ATCC13032_DargR.

[0100] Example 3: Cloning of plasmid pK18mobsacB_CK for genomic integration of the carbamate kinase (CK, EC 2.7.2.2) gene derived from Enterococcus faecalis ATCC 29212 into C. glutamicum. The coding sequence arcC of Enterococcus faecalis ATCC 29212 encodes carbamate kinase (Marina et al., Eur J Biochem., April 1, 1998; 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 amino acid sequence (Genbank accession CAA11271) derived therefrom.

[0101] The coding sequence was optimized for the codon usage frequency of C. glutamicum using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA). The obtained optimized coding sequence was named CK.

[0102] Using the optimized coding sequence, a DNA fragment for genomic integration between genes NCgl0291 and NCgl0292 in C. glutamicum was designed. It consists of the following elements: BsaI restriction site, homologous sequences for assembly cloning into pK18mobsacB (Schafer, 1994; Genbank accession FJ437239), left homologous arm for integration downstream of NCgl0291, strong sod-promoter from C. glutamicum, optimized CK gene, BioBricks Terminator BBa_B1006, right homologous arm for genomic integration, second homologous sequence for assembly cloning, and BsaI site. The obtained DNA sequence was named CK-insert (SEQ ID NO: 7). This was ordered for gene synthesis from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) and delivered as part of a cloning plasmid with an ampicillin resistance gene.

[0103] The cloning plasmid containing the CK-insert was digested using the restriction endonuclease BsaI, and the DNA was purified with the "QIAquick PCR Purification Kit".

[0104] The plasmid pK18mobsacB was digested using the restriction endonuclease SmaI, and the DNA was purified with the "QIAquick PCR Purification Kit".

[0105] The DNA of both digestion plasmids was ligated, and the matching 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)", and the cells were grown on LB agar containing 25 mg / l kanamycin. Appropriate plasmid clones were identified by restriction digestion and DNA sequencing. The obtained plasmid was named pK18mobsacB_CK.

[0106] Example 4: Integration of the carbamate kinase gene CK into the genome of C. glutamicum For the genomic integration of the carbamate kinase gene CK into C. glutamicum ATCC13032 and ATCC13032_DargR, the strains were transformed with plasmid pK18mobsacB_CK using electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with 134 g / l sorbitol, 2.5 g / l yeast extract, and 25 mg / l kanamycin. The agar plates were incubated at 33 °C for 48 h.

[0107] Individual 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 grown at 33 °C for 24 h in 10 ml of BHI medium contained in 100 ml Erlenmeyer flasks equipped with three baffles. To isolate clones that had encountered a second recombination event, aliquots were taken from each liquid culture, appropriately diluted, and 100 μl was seeded onto BHI agar supplemented with 10% sucrose. The agar plates were incubated at 33 °C for 48 h. Colonies that grew on the sucrose-containing agar plates were then examined for kanamycin sensitivity. To do so, cell material was removed from the colonies using toothpicks and transferred onto BHI agar containing 25 mg / l kanamycin and onto BHI agar containing 10% sucrose. The agar plates were incubated at 33 °C for 60 h. Clones that were found to be sensitive to kanamycin and resistant to sucrose were examined by PCR and DNA sequencing for proper integration of the CK gene. The resulting strains were named ATCC13032_CK and ATCC13032_DargR_CK, respectively.

[0108]

Table 7

[0109] Example 5: Cloning of the gene AGAT-Mp encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Moorena producens Molina Production is a filamentous cyanobacterium. The genome of Molina Production strain PAL-8-15-08-1 was published by Leao et al. (Leao T, Castelao G, Korobeynikov A, Monroe EA, Podell S, Glukhov E, Allen EE, Gerwick WH, Gerwick L, Proc Natl Acad Sci USA., March 21, 2017; 114(12): 3198-3203, doi: 10.1073 / pnas.1618556114; Genbank accession number CP017599.1). It contains an open reading frame encoding L-arginine: glycine amidinotransferase (AGAT, EC 2.1.4.1 shown in SEQ ID NO: 8; locus_tag BJP34_00300). SEQ ID NO: 9 shows the amino acid sequence derived therefrom (Genbank accession number WP_070390602).

[0110] Using the software tool "Optimizer" (http: / / genomes.urv.es / OPTIMIZER / ), the amino acid sequence was reverse-translated into a DNA sequence optimized for the codon usage frequency of C. glutamicum. The 5' end of the optimized gene was extended with a BsaI restriction site, a 5'-UTR sequence for assembly cloning, and a ribosome binding site. A second stop codon, a sequence for assembly cloning, and a BsaI site were added to the 3' end. The resulting DNA sequence was named AGAT-Mp-insert (SEQ ID NO: 10). This was ordered for gene synthesis from Eurofins Genomics GmbH (Ebersberg, Germany) and delivered as part of a cloning plasmid with an ampicillin resistance gene.

[0111] The cloning plasmid containing the AGAT-Mp-insert was digested using the restriction endonuclease BsaI, and the DNA was purified with the "QIAquick PCR Purification Kit".

[0112] The Escherichia coli - C. glutamicum shuttle plasmid pLIB_P consists of an origin of replication derived from pBL1 (for C. glutamicum), the pSC101 origin of replication (for Escherichia coli), and a kanamycin resistance gene. Following a unique NotI restriction site, it has a strong promoter, two reverse BsaI sites, and the BioBricks Terminator BBa_B1006 (SEQ ID NO: 11).

[0113] pLIB_P was digested using the restriction endonuclease BsaI, and the DNA was purified with the "QIAquick PCR Purification Kit".

[0114] The DNA solutions of both BsaI-digested plasmids were combined, and the matching sequence ends were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit". The product was transformed into "NEB Stable Competent E. coli (High Efficiency)", and the cells were grown on LB agar containing 25 mg / l kanamycin. Appropriate plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pLIB_P_AGAT-Mp.

[0115] Example 6: Synthesis of the gene AaDH-Mt encoding NADH-dependent AaDH derived from Mycobacterium tuberculosis H37Ra The open reading frame MRA_2804 of Mycobacterium tuberculosis H37Ra probably encodes a NADH-dependent amino acid dehydrogenase (Genbank accession CP000611 locus_tag = "MRA_2804", SEQ ID NO: 12). SEQ ID NO: 13 shows the amino acid sequence derived therefrom.

[0116] The open reading frame was optimized for codon usage frequency in C. glutamicum using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA). The resulting sequence was extended with a 5'-UTR consisting of a BsaI restriction site, homologous regions for assembly cloning, a strong Pg3N3 promoter, and a ribosome binding site. Furthermore, the 3'-end was extended with a random spacer sequence, homologous regions for assembly cloning, and a BsaI restriction site. The resulting DNA sequence was named AaDH-Mt-insert (SEQ ID NO: 14). This was ordered for gene synthesis from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) and delivered as part of a cloning plasmid with an ampicillin resistance gene. The optimized gene was named AaDH-Mt.

[0117] Example 7: Synthesis of the gene AtGGT1 encoding glyoxylate aminotransferase derived from Arabidopsis thaliana The gene GGT1 of Arabidopsis thaliana (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 reactions of glyoxylate + L-alanine = glycine + pyruvate (EC 2.6.1.44), 2-oxoglutarate + L-alanine = L-glutamate + pyruvate (EC 2.6.1.2), and 2-oxoglutarate + glycine = glyoxylate + L-glutamate (EC 2.6.1.4; Liepman AH, Olsen LJ., Plant Physiol., January 2003; 131(1):215-27.doi:10.1104 / pp.011460).

[0118] Using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA), the amino acid sequence of the GGT1 protein was reverse translated into a DNA sequence optimized for the codon usage frequency of C. glutamicum. The resulting sequence was extended with a 5'-UTR consisting of a BsaI restriction site, a homologous region for assembly cloning, and a ribosome binding site. Further, the 3' end was extended with a homologous region for assembly cloning and a BsaI restriction site. The resulting DNA sequence was named AtGGT1-insert (SEQ ID NO: 17). This was ordered for gene synthesis from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) and delivered as part of a cloning plasmid with an ampicillin resistance gene. The optimized gene was named AtGGT1.

[0119] Example 8: Cloning of Plasmids for Co-Expression of AGAT-Mp and AaDH-Mt For the combined expression of AGAT-Mp and AaDH-Mt, the synthetic gene AaDH-Mt was cloned into the plasmid pLIB_P_AGAT-Mp.

[0120] The plasmid pLIB_P_AGAT-Mp was digested using the restriction endonuclease NotI, and the DNA was purified with the "QIAquick PCR Purification Kit". A plasmid containing the synthetic sequence AaDH-Mt-insert (SEQ ID NO: 14) was digested using the restriction endonuclease BsaI, and the resulting DNA was purified with the "QIAquick PCR Purification Kit".

[0121] A dummy DNA with compatible ends for assembly cloning was designed. This was named dummy insert (SEQ ID NO: 18) and ordered for gene synthesis from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) as a linear double-stranded DNA fragment.

[0122] By using the "NEBuilder HiFi DNA Assembly Cloning Kit", the DNA of NotI-digested pLIB_P_AGAT-Mp was ligated to the BsaI-digested plasmid containing the AaDH-Mt-insert and a dummy insert. The product was transformed into "NEB Stable Competent E.coli (High Efficiency)" (New England Biolabs, Ipswich, USA), and the cells were grown on LB agar containing 25 mg / l kanamycin. Appropriate plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pLIB_AaDH-Mt_AGAT-Mp.

[0123] Example 9: Cloning of Plasmids for Co-Expression of AGAT-Mp, AaDH-Mt and AtGGT1 For the combined expression of AGAT-Mp, AaDH-Mt and AtGGT1, the synthetic genes AaDH-Mt and AtGGT1 were cloned into the plasmid pLIB_P_AGAT-Mp.

[0124] The plasmid pLIB_P_AGAT-Mp was digested using the restriction endonuclease NotI, and the DNA was purified with the "QIAquick PCR Purification Kit". The plasmid containing the synthetic sequence AaDH-Mt-insert (SEQ ID NO: 14) was digested using the restriction endonuclease BsaI, and the resulting DNA was purified with the "QIAquick PCR Purification Kit". The plasmid containing the synthetic sequence AtGGT1-insert (SEQ ID NO: 17) was digested using the restriction endonuclease BsaI, and the resulting DNA was purified with the "QIAquick PCR Purification Kit".

[0125] Using the 「NEBuilder HiFi DNA Assembly Cloning Kit」, the NotI-digested DNA of pLIB_P_AGAT-Mp was ligated with the BsaI-digested plasmid containing the AaDH-Mt insert and the BsaI-digested plasmid containing the AtGGT1 insert. The product was transformed into 「NEB Stable Competent E.coli(High Efficiency)」, and the cells were grown on LB agar containing 25 mg / l kanamycin. Appropriate plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pLIB_AaDH-Mt_AtGGT1_AGAT-Mp.

[0126] Example 10: Cloning of Plasmids for Co-expression of AGAT-Mp and AtGGT1 For the combined expression of AGAT-Mp and AtGGT1, the gene AaDH-Mt was deleted from the plasmid pLIB_AaDH-Mt_AtGGT1_AGAT-Mp.

[0127] The plasmid pLIB_AaDH-Mt_AtGGT1_AGAT-Mp was digested using the restriction endonucleases SacI and SalI, and the DNA was purified with the 「QIAquick PCR Purification Kit」. The ends of the linear DNA were blunted using the Fast DNA End Repair Kit (Thermo Fisher Scientific, Waltham, USA), and the DNA was purified. The DNA was subjected to self-ligation using the Rapid Ligation Kit (Thermo Fisher Scientific, Waltham, USA). The ligation product was transformed into 「NEB Stable Competent E.coli(High Efficiency)」, and the cells were grown on LB agar containing 25 mg / l kanamycin. Appropriate plasmid clones were identified by restriction digestion, and the resulting plasmid was named pLIB_AtGGT1_AGAT-Mp.

[0128] Example 11: Transformation of C. glutamicum strains with various plasmids The following strains of C. glutamicum were transformed with various plasmids by electroporation (Table 8). Plasmid-containing cells were selected with 25 mg / l kanamycin. · C. glutamicum ATCC13032: A commonly used wild-type strain (Kinoshita et al., J. Gen. Appl. Microbiol., 1957; 3(3): 193-205) · C. glutamicum ATCC13032_DargR: Chromosomal deletion of argR in C. glutamicum ATCC13032 · C. glutamicum ATCC13032_CK: Chromosomal insertion of the CK gene between NCgl0291 and NCgl0292 in C. glutamicum ATCC13032 · C. glutamicum ATCC13032_DargR_CK: Chromosomal deletion of argR and chromosomal insertion of the CK gene between NCgl0291 and NCgl0292 in C. glutamicum ATCC13032

[0129] [Table 8]

[0130] Example 12: Effect of the L-arginine:glycine amidinotransferase gene on GAA production To evaluate the effect of the L-arginine:glycine amidinotransferase gene (AGAT-Mp) on GAA production, the ATCC13032 / pLIB_P strain and the ATCC13032 / pLIB_P_AGAT-Mp strain were cultured in a Wouter Duetz system in a production medium, and the resulting GAA titers were determined as described above.

[0131] [Table 9]

[0132] Cultivation of the strain having the L-arginine:glycine amidinotransferase gene resulted in GAA production as compared with the strain lacking the L-arginine:glycine amidinotransferase gene (see Table 9). The inventors conclude that the presence of the heterologous L-arginine:glycine amidinotransferase gene enables the production of GAA.

[0133] Example 13: Effect of the carbamate kinase gene on GAA production To evaluate the effect of the carbamate kinase gene on GAA production, the ATCC13032 / pLIB_P_AGAT-Mp strain and the ATCC13032_CK / pLIB_P_AGAT-Mp strain were cultured in a production medium using the Wouter Duetz system, and the resulting GAA titer was determined as described above.

[0134] [Table 10]

[0135] Cultivation of the strain having the carbamate kinase gene resulted in a higher GAA titer as compared with the strain lacking the carbamate kinase gene (see Table 10). The inventors conclude that the presence of the carbamate kinase gene improves the production of GAA.

[0136] Example 14: Effect of the combination of the presence of the carbamate kinase gene and the glyoxylate aminotransferase gene on GAA production To evaluate the effect of the combination of the presence of the carbamate kinase gene (CK) and the glyoxylate aminotransferase gene (AtGGT1) on GAA production, the ATCC13032_CK / pLIB_P_AGAT-Mp strain and the ATCC13032_CK / pLIB_AtGGT1_AGAT-Mp strain were cultured in a production medium using the Wouter Duetz system, and the resulting GAA titer was determined as described above.

[0137] [Table 11]

[0138] When culturing a strain having a carbamate kinase gene and a glyoxylate aminotransferase gene, a higher GAA titer was obtained compared to a strain lacking the glyoxylate aminotransferase gene (see Table 11). The inventors conclude that the combination of the presence of the carbamate kinase gene and the glyoxylate aminotransferase gene improves the production of GAA.

[0139] Example 15: Influence of the combination of the presence of the carbamate kinase gene and the NADH-dependent amino acid dehydrogenase gene on GAA production To evaluate the influence of the combination of the presence of the carbamate kinase gene (CK) and the NADH-dependent amino acid dehydrogenase gene (AaDH-Mt) on GAA production, the ATCC13032_CK / pLIB_P_AGAT-Mp strain and the ATCC13032_CK / pLIB_AaDH-Mt_AGAT-Mp strain were cultured in a production medium using the Wouter Duetz system, and the obtained GAA titer was determined as described above.

[0140] [Table 12]

[0141] When culturing a strain having a carbamate kinase gene and a NADH-dependent amino acid dehydrogenase gene, a higher GAA titer was obtained compared to a strain lacking the NADH-dependent amino acid dehydrogenase gene (see Table 12). The inventors conclude that the combination of the presence of the carbamate kinase gene and the NADH-dependent amino acid dehydrogenase gene improves the production of GAA.

[0142] Example 16: Influence of the combination of the presence of the carbamate kinase gene, the NADH-dependent amino acid dehydrogenase gene and the glyoxylate aminotransferase gene on GAA production To evaluate the effect of the combination of the presence of the carbamate kinase gene (CK), glyoxylate aminotransferase gene (AtGGT1), and NADH-dependent amino acid dehydrogenase gene (AaDH-Mt) on GAA production, the ATCC13032_CK / pLIB_AtGGT1_AGAT-Mp strain, ATCC13032_CK / pLIB_AaDH-Mt_AGAT-Mp strain, and ATCC13032_CK / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp strain were cultured in a production medium using the Wouter Duetz system, and the resulting GAA titer was determined as described above.

[0143]

Table 13

[0144] When culturing strains having a combination of the carbamate kinase gene, NADH-dependent amino acid dehydrogenase gene, and glyoxylate aminotransferase gene, higher GAA titers were obtained compared to strains lacking either the NADH-dependent amino acid dehydrogenase gene or the glyoxylate aminotransferase gene (see Table 13). The inventors conclude that the combination of the presence of the carbamate kinase gene, NADH-dependent amino acid dehydrogenase gene, and glyoxylate aminotransferase gene improves the production of GAA.

[0145] Example 17: Effect of deletion of the argR gene in combination with the presence of the carbamate kinase gene, NADH-dependent amino acid dehydrogenase gene, and glyoxylate aminotransferase gene on GAA production To evaluate the effect of the deletion of the argR gene (DargR) in combination with the presence of the carbamate kinase gene (CK), the glyoxylate aminotransferase gene (AtGGT1), and the NADH-dependent amino acid dehydrogenase gene (AaDH-Mt) on GAA production, the ATCC13032 / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp strain, the ATCC13032_DargR / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp strain, and the ATCC13032_DargR_CK / pLIB_AaDH-Mt_AtGGT1_AGAT-Mp strain were cultured in a Wouter Duetz system in a production medium, and the resulting GAA titers were determined as described above.

[0146]

Table 14

[0147] When culturing a strain lacking the argR gene in combination with the presence of the glyoxylate aminotransferase gene and the NADH-dependent amino acid dehydrogenase gene, the GAA titer was higher than that of the strain having the wild-type argR gene (see Table 14).

[0148] When culturing a strain lacking the argR gene in combination with the presence of the carbamate kinase gene, the glyoxylate aminotransferase gene, and the NADH-dependent amino acid dehydrogenase gene, a higher GAA titer was obtained compared to both strains lacking the carbamate kinase gene (see Table 14).

[0149] The inventors conclude that the deletion of the argR gene in combination with the presence of the carbamate kinase gene, the glyoxylate aminotransferase gene, and the NADH-dependent amino acid dehydrogenase gene improves the production of GAA.

Claims

1. A microorganism comprising at least one heterologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase, at least one gene encoding a protein having the function of carbamate kinase, and further comprising at least one gene encoding a protein having the function of NADH-dependent amino acid dehydrogenase.

2. The microorganism according to claim 1, wherein the activity of at least one protein having the function of a carbamate kinase is increased compared to the activity of each of the proteins in the wild-type microorganism.

3. The microorganism according to claim 1, comprising at least one heterologous gene encoding a protein having the function of a carbamate kinase.

4. The microorganism according to claim 1, wherein the activity of the protein having the function of NADH-dependent amino acid dehydrogenase is increased compared to the respective activities in the wild-type microorganism.

5. The microorganism according to claim 1, wherein the protein having the function of an NADH-dependent amino acid dehydrogenase is a heterologous protein.

6. The microorganism according to claim 1, further comprising at least one gene encoding a protein having the function of glyoxylate aminotransferase.

7. The microorganism according to claim 1, wherein the ability to produce L-arginine from L-ornithine is increased compared to the ability of the wild-type microorganism.

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

9. The microorganism according to claim 1, belonging to the genera Corynebacterium, Bacillus, Enterobacteria seaae, or Pseudomonas.

10. a) A step of culturing a microorganism according to any one of claims 1 to 9 in a culture medium; b) A step of accumulating guanidinoacetic acid (GAA) in the culture medium to form a GAA-containing fermentation broth, comprising these steps: a) a step of culturing a microorganism according to any one of claims 1 to 9 in a culture medium; b) a step of accumulating guanidinoacetic acid (GAA) in the culture medium to form a GAA-containing fermentation broth.

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

12. The microorganism according to any one of claims 1 to 9, further comprising a gene encoding an enzyme having guanidinoacetate N-methyltransferase activity.

13. The microorganism according to claim 12, wherein the gene encoding an enzyme having guanidinoacetate N-methyltransferase activity is overexpressed.

14. a) A step of culturing the microorganism described in claim 12 in a culture medium; b) A step of accumulating creatine in a culture medium to form a creatine-containing fermentation broth.

15. The method according to claim 14, further comprising isolating creatine from the creatine-containing fermented broth.