Improved biotechnological method for producing guanidinoacetic acid (GAA) by using NADH-dependent dehydrogenase

Abstract

The present invention relates to a microorganism transformed to be capable of producing guanidinoacetic acid (GAA) and containing at least one gene encoding a protein having the function of NADH-dependent dehydrogenase, and a method for the fermentative production of GAA using such a microorganism. The present invention also relates to a method for the fermentative production of creatine.

Description

Technical Field

[0001] Guanidinoacetic acid (GAA) is a colorless crystalline organic compound that is used as an animal feed additive (e.g., International Publication No. WO 2005 / 120246 pamphlet and U.S. Patent Application Publication No. US 2011 / 257075 specification). GAA is a natural precursor of creatine (e.g., 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 the fermentative production of GAA using such a microorganism. The present invention also relates to a method for the fermentative production of creatine. For the fermentation process, industrial feedstocks (e.g., ammonia, ammonium salts, glucose or sugar-containing substrates) are used as starting materials.

Background Art

[0003] In biological systems, GAA and ornithine are formed from arginine and glycine as starting materials by the catalysis 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. Muenchhoff et al. (FEBS Journal, 277, (2010), 3844-3860) also reported the first characterization of prokaryotic-derived AGAT by cloning and heterologously expressing the enzyme in E. coli. Sosio et al. (Cell Chemical Biology, 25, 540-549, May 17, 2018) elucidated the biosynthetic pathway for 25 pseudouridomycins in Streptomyces species. They described the formation of GAA and L-ornithine by the reaction of L-arginine and glycine, catalyzed by PumN, L-arginine:glycine-amidinotransferase (AGAT), as an intermediate reaction.

[0005] Fan Wenchao has disclosed a method for the production of creatine by fermentation of non-pathogenic microorganisms such as Corynebacterium glutamicum (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-amidino-transferase (EC: 2.1.4.1) and guanidinoacetic acid-N-methyltransferase (EC: 2.1.1.2). The microorganisms preferably overexpress glycine aminotransferase (L-arginine: glycine amidino-transferase) and guanidinoacetic acid-N-methyltransferase.

[0006] Chinese Patent Application Publication No. 113481139 describes the construction of recombinant Bacillus subtilis that produces GAA by introducing exogenous arginine: glycine amidino-transferase derived from Amycolatopsis kentuckyensis into the genome of wild-type Bacillus subtilis and knocking out the gcvP and / or argI genes in the genome.

[0007] Microorganisms capable of producing guanidinoacetic acid (GAA) have been published by Zhang et al. (ACS Synth. Biol., 2020, 9, 2066-275). They designed a reconstructed ornithine cycle in Escherichia coli by introducing heterologous AGAT from different species (e.g., Homo sapiens, Cylindrospermopsis raciborskii, Moorea producens), as well as introducing citrulline synthesis modules (e.g., overexpression of carAB, argF and argI) and arginine synthesis modules (e.g., overexpression of argG, argH; introduction of aspA) into E. coli.

[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 was provided by Park et al. (NATURE COMMUNICATIONS | DOI:10.1038 / ncomms5618). They proposed that L-arginine production increases gradually throughout the strain engineering process by random mutagenesis and screening of L-arginine producers of C. glutamicum strains that already produce L-arginine, such as ATCC 21831 (Nakayama and Yoshida, 1974, US Patent Application Publication No. 3849250), and by stepwise rational metabolic engineering based on the analysis of the entire metabolic system. 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 inactivates the argR gene, which encodes 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 the deletion of the argR repressor gene.

[0009] Kurahashi et al. (European Patent Application Publication No. 1057893) reported a method for enhancing the L-arginine-producing ability of microorganisms by using recombinant DNA technology to enhance L-arginine biosynthetic enzymes, for example, by introducing recombinant DNA containing vector DNA and a DNA fragment containing genes for acetylornithine deacetylase, N-acetylglutamate-γ-semialdehyde dehydrogenase, N-acetylglutamokinase, and argininosuccinase derived from a microorganism belonging to the genus Escherichia. To improve L-arginine production, the authors further proposed microorganisms having enhanced activity of intracellular glutamate dehydrogenase (GDH) and having the ability to produce L-arginine.

[0010] Finally, Schneider and Jankowitsch (International Publication No. 2021122400 pamphlet; European Patent Application Publication No. 3839051) proposed a method for the fermentative production of GAA by using microorganisms having improved L-arginine-producing ability by having heterologous AGAT from different species (e.g., Homo sapiens, Cylindrospemopsis raciborskii, Morea producens) and increasing the expression of enzymes in the L-arginine pathway, for example, enzymes having the function of carbamoyl phosphate synthetase and enzymes related to the arginine operon.

[0011] Regarding the second starting material for GAA biosynthesis, it may also be desirable to increase the supply of glycine to improve GAA biosynthesis in microorganisms that naturally provide a homologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT), or in microorganisms provided with a heterologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT).

[0012] Schneider and Jankowitsch (International Publication No. WO 2022 / 008280 pamphlet) disclose a method for producing GAA using a microorganism comprising at least one gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT) and at least one protein having the function of glyoxylate aminotransferase. It has been shown that the yield of GAA increases by using glyoxylate aminotransferase.

[0013] 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 and 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. It has also been shown that in a microorganism equipped with a gene encoding a protein having the function of AGAT, the yield of GAA increases when the activity of a protein having the function of malate synthase that uses glyoxylate as a substrate decreases (International Publication No. WO 2022 / 008276 pamphlet).

[0014] The drawback of glyoxylate aminotransferase is that the amino group of the amino donor usually originates from L-glutamate or L-glutamate is directly used. The biosynthesis of L-glutamate from 2-oxoglutarate generally requires NADPH. In C. glutamicum, NADPH is regenerated from NADP+ mainly via the oxidative pentose phosphate pathway (PPP) and partially by NADP-dependent isocitrate dehydrogenase and NADP-dependent malic enzyme (Marx, Achim, de Graaf, Albert A., Wiechert, Wolfgang, Lothar Eggeling and Sahm, Hermann, (1996), Biotechnology and Bioengineering, Vol. 49(2), 111-129, DOI: https: / / doi.org / 10.1002 / (SICI)1097-0290(19960120)49:2<111::AID-BIT1>3.0.CO;2-T). These pathways for NADPH regeneration result in carbon loss, thereby reducing the maximum theoretical yield. On the other hand, methods for enhancing the NADPH supply, such as overexpression of inorganic polyphosphate / ATP-NAD kinase ppnK (Yin, Lianghong; Zhao, Jianxun; Chen, Cheng; Hu, Xiaoqing; Wang, Xiaoyuan, (2014), Biotechnology and Bioprocess Engineering: BBE; Dordrecht Bd. 19, 1st ed: 132-142. DOI: 10.1007 / s12257-013-0416-z) or expression of a heterologous transhydrogenase gene (Yamauchi Y, Hirasawa T, Nishii M, Furusawa C, Shimizu H., (2014), J Gen Appl Microbiol., 2014;60(3):112-8.doi:10.2323 / jgam.60.112.PMID:25008167) are known.C. glutamicum naturally fixes NH3 with NADPH-dependent glutamate dehydrogenase (E. Kimura, (2005), "L-Glutamate Production" in Handbook of Corynebacterium glutamicum, ISBN 9780849318214, published by CRC Press on March 30, 2005). It is known that this is also possible with heterologous NADH-dependent glutamate dehydrogenase (Marx, Achim, Eikmanns, Bernhard J., Sahm, Hermann, de Graaf, Albert A and Lothar Eggeling, (1999), Metabolic Engineering, Volume 1, Issue 1, 1999, pp. 35 - 48, ISSN 1096 - 7176, DOI: https: / / doi.org / 10.1006 / mben.1998.0106). This reduces the NADPH requirement.

[0015] NADH-dependent amino acid dehydrogenases (AaDH) catalyze the amination reaction of keto acids to L-amino acids by utilizing NADH as a cosubstrate (instead of NADPH). Therefore, they provide alternative pathways for the assimilation or dissimilation of ammonium in cells.

Chemical formula

[0016] 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 dissimilate ammonium in the case of the reverse reaction.

[0017] 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+

[0018] 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).

[0019] Thus, most NADH - dependent amino acid dehydrogenases correspond to multiple reactions and thus multiple EC numbers. Table 1 shows some examples.

[0020]

Table 1

Prior art documents

Patent documents

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Summary of the Invention

Problems to be Solved by the Invention

[0023] Accordingly, the underlying problem of the present invention is to provide an improved microorganism transformed to be able to produce guanidinoacetic acid (GAA), in particular a microorganism with improved ability to provide glycine as a starting material for GAA biosynthesis, and a method for the fermentative production of GAA using such a microorganism.

Means for Solving the Problem

[0024] 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) and at least one heterologous gene encoding a protein having the function of NADH-dependent amino acid dehydrogenase.

Embodiments for Carrying out the Invention

[0025] 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.

[0026] A heterologous protein means a protein that does not naturally exist in a microorganism.

[0027] A homologous gene or an endogenous gene means that a gene or a nucleotide sequence of a gene containing such a function naturally exists in a microorganism or is "native" within a microorganism.

[0028] A homologous protein or a native protein means a protein that naturally exists in a microorganism.

[0029] Proteins having the function of L-arginine:glycine amidinotransferase (AGAT) belong to the amidinotransferase family. The amidinotransferase family includes glycine (EC:2.1.4.1) and inosamine (EC:2.1.4.2) amidinotransferases, which are enzymes involved in creatine and streptomycin biosynthesis, respectively. This family also includes arginine deiminase, EC:3.5.3.6. These enzymes catalyze the reaction: arginine + H2O <=> citrulline + NH3. A streptococcal antitumor glycoprotein is also found in this family. Enzymes or proteins having L-arginine:glycine-amidinotransferase (AGAT) activity have also been described as having a conserved domain belonging to the PFAM family Amidinotransf (PF02274) (Marchler-Bauer A et al., (2017), "CDD / SPARCLE: functional classification of proteins via subfamily domain architectures.", Nucleic Acids Res., 45(D1):D200~D203), and are also described in the following publications: Pissowotzki K et al., Mol Gen Genet, 1991;231:113~123 (PUBMED:1661369 EPMC:1661369); D’Hooghe I et al., J Bacteriol, 1997;179:7403~7409 (PUBMED:9393705 EPMC:9393705); Kanaoka M et al., Jpn J Cancer Res, 1987;78:1409~1414 (PUBMED:3123442 EPMC:3123442).

[0030] In the microorganism of the present invention, at least one protein having the function of NADH-dependent amino acid dehydrogenase can be NADH-dependent amino acid dehydrogenase. It can be selected from the group consisting of proteins having the functions of alanine dehydrogenase (EC 1.4.1.1), glycine dehydrogenase (EC 1.4.1.10), and aspartate dehydrogenase (EC 1.4.1.21).

[0031] In a further embodiment of the microorganism of the present invention, the activity of NADH-dependent amino acid dehydrogenase is increased as compared to the respective activity in the wild-type microorganism. Preferably, at least one gene encoding a protein having the enzyme activity of NADH-dependent amino acid dehydrogenase is overexpressed in the microorganism of the present invention as compared to the respective gene expression in the wild-type microorganism.

[0032] Preferably, the microorganism of the present invention has an increased ability to produce L-arginine from L-ornithine as compared to the ability of the wild-type microorganism.

[0033] In the context of the present invention, a microorganism having 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 Ginesy et al. (Microbial Cell Factories, (2015), 14:29).

[0034] In a specific embodiment of the present invention, the microorganism has an increased activity of an enzyme having the function of carbamoyl phosphate synthetase (EC 6.3.4.16) as compared to the respective enzyme activity in the wild-type microorganism.

[0035] The activity of the enzyme having the function of argininosuccinate lyase (E.C. 4.3.2.1) in the microorganism according to the present invention may be increased as compared with the respective enzyme activities in the wild-type microorganism.

[0036] Furthermore, in the microorganism according to the present invention, the activity of the enzyme having the function of ornithine carbamoyltransferase (EC 2.1.3.3) may be increased as compared with the respective enzyme activities in the wild-type microorganism.

[0037] In the microorganism according to the present invention, the activity of the enzyme having the function of argininosuccinate synthetase (E.C. 6.3.4.5) may also be increased as compared with the respective enzyme activities in the wild-type microorganism.

[0038] In a further embodiment of the microorganism according to the present invention, the expression of the gene encoding the protein having the function of malate synthase is attenuated as compared with the respective gene expressions in the wild-type microorganism, or the gene encoding the protein having the function of malate synthase is inactivated or deleted.

[0039] 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 as compared with the expression of the argR gene in the wild-type microorganism. Alternatively, the argR gene is inactivated or deleted.

[0040] In a further embodiment of the present invention, optionally in addition to the above modifications, at least one or more of the genes encoding the enzymes of the biosynthetic pathway of L-arginine, including gdh, argJ, argB, argC and / or argD, which respectively encode glutamate dehydrogenase, ornithine acetyltransferase, acetylglutamate kinase, acetylglutamyl phosphate reductase and acetylornithine aminotransferase, are overexpressed in the microorganism according to the present invention.

[0041] In the microorganism according to the present invention, the protein having the function of L-arginine:glycine amidinotransferase may be L-arginine:glycine amidinotransferase (AGAT, i.e., EC 2.1.4.1).

[0042] In the microorganism of the present invention, the gene encoding the protein having the function of L-arginine:glycine amidinotransferase may be further overexpressed.

[0043] The protein having the function of L-arginine:glycine amidinotransferase (AGAT) in the microorganism of the present invention may include an amino acid sequence that is at least 80% identical, preferably at least 90% identical to the amino acid sequence according to SEQ ID NO: 2. In a further embodiment of the present invention, the amino acid sequence of L-arginine:glycine amidinotransferase is identical to the amino acid sequence according to SEQ ID NO: 2.

[0044] In a specific embodiment according to the present invention, the protein having the function of NADH-dependent amino acid dehydrogenase includes an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 6, the amino acid sequence according to SEQ ID NO: 9, the amino acid sequence according to SEQ ID NO: 12, the amino acid sequence according to SEQ ID NO: 15, or the amino acid sequence according to SEQ ID NO: 18.

[0045] The microorganism of the present invention may belong to the genus Corynebacterium, preferably Corynebacterium glutamicum (C. glutamicum), or the family Enterobacteriaceae, preferably Escherichia coli (E. coli), or the genus Pseudomonas, preferably Pseudomonas putida (P. putida).

[0046] Generally, an increase in the enzymatic activity of a protein in a microorganism, particularly in the microorganism of the present invention, compared to the respective activities in the wild-type microorganism can be achieved, for example, by a mutation of the protein, particularly a mutation that confers feedback resistance to the product of an enzymatic reaction on the protein, or by overexpression of a gene encoding a protein having enzymatic activity, compared to the expression of the respective genes in the wild-type microorganism.

[0047] Overexpression of a gene is generally achieved by increasing the copy number of the gene and / or by functionally linking the gene to a strong promoter and / or by enhancing the ribosome binding site and / or by 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.

[0048] Overexpression of genes in microorganisms, particularly the microorganisms of the present invention, can be achieved by increasing the copy number of the gene and / or enhancing regulatory factors as compared to the respective activities in wild-type microorganisms, for example, by 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. Enhancement of such regulatory factors that have a positive effect on gene expression can be achieved, for example, by modifying the promoter sequence upstream of the structural gene or completely replacing the promoter with a more effective or so-called strong promoter in order to enhance the effectiveness of the promoter. The promoter is located upstream of the gene. The promoter consists of about 40 to 50 base pairs and is a DNA sequence that constitutes the binding site for RNA polymerase holoenzyme and the transcription start point, and can thereby affect the intensity of 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), overexpression or increased expression of genes in bacteria can be achieved 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 a specific region of a given native promoter (e.g., its so-called -10 and -35 regions) towards the consensus sequence. 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 to mean the continuous arrangement of a promoter with a gene that results in the transcription of the gene.

[0049] The genetic code is degenerate, which means that a particular amino acid can be encoded by several different triplets. The term codon usage frequency refers to the finding that a particular organism typically does not use all of the codons possible for a particular amino acid with the same frequency. Instead, an organism will typically show a certain preference for particular codons, which means that these codons will be found more frequently in the coding sequences of the organism's transcribed genes. If a particular gene that is foreign to a future host, i.e., derived from a different species, is to be expressed in a future host organism, the coding sequence of said gene should be adjusted to the codon usage frequency of said future host organism (i.e., codon usage frequency optimization).

[0050] The above problem is further solved by a method for the fermentative production of GAA, comprising a) culturing a microorganism according to the invention as defined above in a suitable medium under suitable conditions, and b) accumulating guanidinoacetic acid (GAA) in the medium to form a GAA-containing fermentation broth.

[0051] The method according to the invention may further comprise isolating GAA from the fermentation broth.

[0052] The invention further relates to a microorganism as defined above, which further comprises a gene encoding an enzyme having the activity of guanidinoacetic acid N-methyltransferase (EC:2.1.1.2). Preferably, the gene encoding an enzyme having the activity of guanidinoacetic acid N-methyltransferase is overexpressed.

[0053] The invention also relates to a method for the fermentative production of creatine, comprising a) culturing a microorganism according to the invention, which 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.

[0054] Preferably, this method further comprises isolating creatine from the creatine-containing fermentation broth. Creatine can be extracted from the fermentation broth by the isoelectric point method and / or the ion exchange method. Alternatively, creatine can be further purified by a method of recrystallization in water.

[0055] Sequence: SEQ ID NO: 1 shows an open reading frame encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1; locus_tag BJP34_00300) from Moorena producens.

[0056] SEQ ID NO: 2 shows the amino acid sequence derived from SEQ ID NO: 1 (Genbank accession number WP_070390602).

[0057] SEQ ID NO: 3 shows a DNA sequence encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Moorena producens, optimized for the codon usage frequency of C. glutamicum. The 5' end of the optimized gene is 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 are added to the 3' end.

[0058] SEQ ID NO: 4 shows the Escherichia coli - C. glutamicum shuttle plasmid pLIB_P, consisting of an origin of replication from pBL1 (for C. glutamicum), an origin of replication from pSC101 (for Escherichia coli), and a kanamycin resistance gene. It has a strong promoter, two inverted BsaI sites, and a BioBricks Terminator BBa_B1006 following a unique NotI restriction site.

[0059] Open reading frame MRA_2804 of Mycobacterium tuberculosis H37Ra, encoding NADH-dependent amino acid dehydrogenase (Genbank accession CP000611 locus_tag = "MRA_2804"), is shown.

[0060] SEQ ID NO: 6 shows the amino acid sequence derived from SEQ ID NO: 5 (AaDH-Mt derived from Mycobacterium tuberculosis H37Ra).

[0061] SEQ ID NO: 7 shows the DNA sequence encoding NADH-dependent amino acid dehydrogenase of Mycobacterium tuberculosis H37Ra, optimized for the codon usage frequency of C. glutamicum. The DNA sequence is extended with a 5'-UTR consisting of a BsaI restriction site, a homologous region for assembly cloning, a strong Pg3N3 promoter, and a ribosome binding site. Further, the 3' end is extended with a random spacer sequence, a homologous region for assembly cloning, and a BsaI restriction site.

[0062] SEQ ID NO: 8 shows the open reading frame LJ00_13235 of Mycobacterium smegmatis MC2 155, probably encoding NADH-dependent amino acid dehydrogenase (Genbank accession CP009494 locus_tag = "LJ00_13235").

[0063] SEQ ID NO: 9 shows the amino acid sequence derived from SEQ ID NO: 8 (AaDH-Ms derived from Mycobacterium smegmatis MC2 155).

[0064] SEQ ID NO: 10 shows the DNA sequence encoding NADH-dependent amino acid dehydrogenase of Mycobacterium smegmatis MC2155, optimized for the codon usage frequency of C. glutamicum. The DNA sequence is extended with a 5'-UTR consisting of a BsaI restriction site, a homologous region for assembly cloning, a strong Pg3N3 promoter, and a ribosome binding site. Further, the 3' end is extended with a random spacer sequence, a homologous region for assembly cloning, and a BsaI restriction site.

[0065] SEQ ID NO: 11: Open reading frame HIR77_18035 of Bacillus subtilis subsp. subtilis str. 168, which probably encodes NADH-dependent amino acid dehydrogenase (Genbank accession CP053102 locus_tag = "HIR77_18035").

[0066] SEQ ID NO: 12: Amino acid sequence derived from SEQ ID NO: 11 (AaDH-Bs derived from Bacillus subtilis subsp. subtilis str. 168).

[0067] SEQ ID NO: 13: DNA sequence encoding NADH-dependent amino acid dehydrogenase of Bacillus subtilis subsp. subtilis str. 168, optimized for the codon usage frequency of C. glutamicum. The sequence is extended with a 5'-UTR consisting of a BsaI restriction site, a homologous region for assembly cloning, a strong Pg3N3 promoter and a ribosome binding site. Further, the 3'-end is extended with a random spacer sequence, a homologous region for assembly cloning and a BsaI restriction site.

[0068] SEQ ID NO: 14: Open reading frame CP974_05185 of Streptomyces fradiae ATCC10745, which probably encodes NADH-dependent amino acid dehydrogenase (Genbank accession CP023696 locus_tag = "CP974_05185").

[0069] SEQ ID NO: 15: Amino acid sequence derived from SEQ ID NO: 14 (AaDH-Sf derived from Streptomyces fradiae ATCC10745).

[0070] Disclosed is a DNA sequence encoding NADH-dependent amino acid dehydrogenase of Streptomyces fradiae ATCC 10745, which is optimized for the codon usage frequency of C. glutamicum. The sequence is extended with a 5'-UTR consisting of a BsaI restriction site, a homologous region for assembly cloning, a strong Pg3N3 promoter, and a ribosome binding site. Further, the 3' end is extended with a random spacer sequence, a homologous region for assembly cloning, and a BsaI restriction site.

[0071] SEQ ID NO: 17: Shows the open reading frame of Aphanothece halophytica CM1, which probably encodes NADH-dependent amino acid dehydrogenase (Genbank accession MG430510).

[0072] SEQ ID NO: 18: Shows the amino acid sequence derived from SEQ ID NO: 17 (AaDH-Ah from Aphanothece halophytica CM1).

[0073] SEQ ID NO: 19: Discloses a DNA sequence encoding NADH-dependent amino acid dehydrogenase of Aphanothece halophytica CM1, which is optimized for the codon usage frequency of C. glutamicum. The sequence is extended with a 5'-UTR consisting of a BsaI restriction site, a homologous region for assembly cloning, a strong Pg3N3 promoter, and a ribosome binding site. Further, the 3' end is extended with a random spacer sequence, a homologous region for assembly cloning, and a BsaI restriction site.

[0074] 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).

[0075] Bacterial strain Corynebacterium glutamicum type strain / wild type Corynebacterium glutamicum ATCC 13032 (Kinoshita S, Udaka S, Shimono M., J. Gen. Appl. Microbiol., 1957; 3(3): 193~205) is commercially available under the deposit number DSM 20300 at the American Type Culture Collection (ATCC) or DSMZ - German Collection of Microorganisms and Cell Cultures GmbH.

[0076] Culture for cell growth Unless otherwise specified, the culture / incubation procedures were carried out as follows: a. Escherichia coli strains were cultured in liquid medium using LB broth (MILLER) manufactured by Merck (Darmstadt, Germany, catalog number 110285). Liquid cultures (10 ml of liquid medium per 100 ml Erlenmeyer flask with three baffles) were incubated at 30 °C and 200 rpm in an Infors HT Multitron standard incubator shaker manufactured by Infors GmbH (Bottmingen, Switzerland).

[0077] b. Escherichia coli strains were cultured on agar plates using LB agar (MILLER) manufactured by Merck (Darmstadt, Germany, catalog number 110283). The agar plates were incubated at 30 °C in an INCU-Line® Mini Incubator manufactured by VWR (Radnor, USA).

[0078] c. C. glutamicum strains were cultured in liquid medium using Brain Heart Infusion Broth (BHI) manufactured by Merck (Darmstadt, Germany, catalog number 110493). Liquid cultures (10 ml of liquid medium per 100 ml Erlenmeyer flask with three baffles) were incubated at 30 °C and 200 rpm in an Infors HT Multitron standard incubator shaker manufactured by Infors GmbH (Bottmingen, Switzerland).

[0079] d. C. glutamicum strains were cultured on agar plates using Brain Heart Infusion 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).

[0080] e. To culture C. glutamicum after electroporation, BHI-agar (Merck, Darmstadt, Germany, catalog number 113825) was supplemented with 134 g / l of sorbitol (Carl Roth GmbH+Co.KG, Karlsruhe, Germany), 2.5 g / l of yeast extract (Oxoid / ThermoFisher Scientific, Waltham, USA, catalog number LP0021), and 25 mg / l of kanamycin. The agar plates were incubated at 30 °C in an incubator manufactured by Heraeus Instruments equipped with a Kelvitron® temperature controller (Hanau, Germany).

[0081] Determination of the optical density of the bacterial suspension a. The optical density of the bacterial suspension in the shaking flask culture was determined at 600 nm (OD600) using a Bio-Photometer manufactured by Eppendorf AG (Hamburg, Germany).

[0082] 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 branded plate reader manufactured by Tecan Group AG (Mannedorf, Switzerland).

[0083] Centrifugation a. A bacterial suspension with a maximum volume of 2 ml was centrifuged (for 5 minutes at 13,000 rpm) using an Eppendorf 5417 R benchtop centrifuge in 1.5 ml or 2 ml reaction tubes (e.g., Eppendorf Tubes® 3810X).

[0084] b. A bacterial suspension with a maximum volume of 50 ml was centrifuged for 10 minutes at 4,000 rpm using an Eppendorf 5810 R benchtop centrifuge in 15 ml or 50 ml centrifuge tubes (e.g., Falcon® 50 ml Conical Centrifuge Tube).

[0085] DNA Isolation Plasmid DNA was isolated from E. coli cells using a QIAprep Spin Miniprep Kit (Catalog No. 27106) manufactured by Qiagen according to the manufacturer's instructions.

[0086] 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. 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.

[0087] a. The Phusion (registered trademark) High-Fidelity DNA Polymerase Kit (Phusion Kit) manufactured by 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).

[0088] [Table 2]

[0089] b. The Taq PCR Core Kit (Taq Kit) manufactured by Qiagen (Hilden, Germany, catalog number 201203) was used to amplify the desired segment of DNA to confirm its presence. The kit was used according to the manufacturer's instructions (see Table 3).

[0090] [Table 3]

[0091] c. To confirm the presence of the desired DNA segment in cells collected from E. coli or C. glutamicum colonies, the SapphireAmp (registered trademark) 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).

[0092] [Table 4]

[0093] d. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany).

[0094] As the e.PCR template, an appropriately diluted solution of isolated plasmid DNA or total DNA isolated from a liquid culture, or total DNA contained in a bacterial colony (colony PCR) was used. For the colony PCR, the template was prepared by collecting cell material from colonies on an agar plate with a sterile toothpick 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.

[0095] f. All PCR reactions were performed using a PCR cycler type Mastercycler or Mastercycler nexus gradient manufactured by Eppendorf AG (Hamburg, Germany).

[0096] 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 reaction was carried out according to the instructions in the manufacturer's manual.

[0097] Determination of the Size of DNA Fragments a. The size of small DNA fragments (<1000 bps) was usually determined by automated capillary electrophoresis using QIAxcel manufactured by Qiagen (Hilden, Germany).

[0098] b. When it was necessary to isolate the DNA fragment or when 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.

[0099] Purification of PCR Amplification Products and Restriction Fragments The PCR amplification products and restriction fragments were cleaned up using the QIAquick PCR Purification Kit (Hilden, Germany, catalog number 28106) from Qiagen according to the manufacturer's instructions. The DNA was eluted with 30 μl of 10 mM Tris*HCl (pH 8.5).

[0100] Determination of DNA Concentration The DNA concentration was measured using a NanoDrop Spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH (Erlangen, Germany), which has been a VWR brand since 2015.

[0101] 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 the 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.

[0102] Chemical Transformation of Escherichia 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 had successfully undergone transformation were selected on LB agar supplemented with 25 mg / l kanamycin.

[0103] Transformation of C. glutamicum Transformation of C. glutamicum with plasmid DNA was performed by electroporation using a “Gene Pulser Xcell” (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) as described by Ruan et al. (2015). Electroporation was carried out in a 1 mm electroporation cuvette (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) at a fixed time set at 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.

[0104] 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 sequences were visualized and evaluated using Clonemanager Professional 9 software (Scientific & Educational Software, Denver, USA) for sequence and in silico assembly of the sequences.

[0105] 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 2-fold 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. Then, the cultures were 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. Then, 2 ml aliquots of the resulting cell-containing glycerol suspension were dispensed and stored at -80 °C.

[0106] GAA production in small-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.

[0107] 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.

[0108]

Table 5

[0109] After the incubation period, the optical density OD600 of the preculture was determined. The volume required 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.

[0110] The main culture was started by inoculating 100 μl of the resuspended cells from the preculture into each well 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.

[0111]

Table 6

[0112] The main culture was incubated for 72 hours at 30 °C and 225 rpm in an Infors HT Multitron standard incubator shaker manufactured by 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).

[0113] 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.

[0114] Quantification of GAA The sample was analyzed using an Agilent analytical system consisting of HPLC "Infinity 1260" combined with a mass spectrometer "Triple Quad 6420" (Agilent Technologies Inc., Santa Clara, USA). Chromatographic separation was performed at 35 °C on an Atlantis HILIC silica column, 4.6×250 mm, 5 μm (Waters Corporation, Milford, USA). Mobile phase A was water containing 10 mM ammonium formate and 0.2% formic acid. Mobile phase B was a mixture of 90% acetonitrile and 10% water, to which 10 mM ammonium formate was added. The HPLC system was started with 100% B and continued to 66% B at a constant flow rate of 0.6 mL / min with a linear gradient for 22 minutes. The mass spectrometer was operated in the ESI positive ionization mode. The m / z values were monitored by using MRM fragmentation [M+H]+118~76 for the detection of GAA. The limit of quantification (LOQ) of GAA was fixed at 7 ppm.

Example

[0115] B) Experimental results Example 1: Cloning of the gene AGAT-Mp encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Morea production Morea productionis is a filamentous cyanobacterium. The genome of Morea productionis 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 U S A., March 21, 2017; 114(12):3198-3203, doi:10.1073 / pnas.1618556114; Genbank accession number CP017599.1). This contains an open reading frame encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1; locus_tag BJP34_00300 shown in SEQ ID NO:1). SEQ ID NO:2 shows the amino acid sequence derived therefrom (Genbank accession number WP_070390602).

[0116] 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 (SEQ ID NO:3) was ordered from Eurofins Genomics GmbH (Ebersberg, Germany) for gene synthesis and delivered as part of a cloning plasmid having an ampicillin resistance gene (designated pEX-A258_AGAT-Mp).

[0117] The Escherichia coli-Corynebacterium glutamicum shuttle plasmid pLIB_P consists of an origin of replication derived from pBL1 (for C. glutamicum), the pSC101 origin of replication (for E. coli), and a kanamycin resistance gene. Following a unique NotI restriction site, it has a strong promoter, two inverted BsaI sites, and the BioBricks Terminator BBa_B1006 (SEQ ID NO: 5).

[0118] pLIB_P was digested using the restriction endonuclease BsaI, and the DNA was purified with the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0119] The cloning plasmid pEX-A258_AGAT-Mp was digested using the restriction endonuclease BsaI, and the DNA was purified with the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0120] The DNA solutions of pLIB_P and pEX-A258_AGAT-Mp digested with BsaI were combined, and the matching sequence ends were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, catalog number E5520). 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_P_AGAT-Mp.

[0121] Example 2: Synthesis of the gene 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: 7). SEQ ID NO: 8 shows the amino acid sequence derived therefrom.

[0122] Using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA), the open reading frame was 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, a strong Pg3N3 promoter and a ribosome binding site. Furthermore, the 3' end was extended with a random spacer sequence, a homologous region for assembly cloning and a BsaI restriction site. The resulting DNA sequence (SEQ ID NO: 9) 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.

[0123] Example 3: Synthesis of a gene encoding NADH-dependent AaDH derived from Mycobacterium smegmatis MC2 155 The open reading frame LJ00_13235 of Mycobacterium smegmatis MC2 155 probably encodes a NADH-dependent amino acid dehydrogenase (Genbank accession CP009494 locus_tag = "LJ00_13235", SEQ ID NO: 11). SEQ ID NO: 12 shows the amino acid sequence derived therefrom.

[0124] The open reading frame was optimized for codon usage 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 (SEQ ID NO: 13) 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.

[0125] Example 4: Synthesis of the gene encoding NADH-dependent AaDH from Bacillus subtilis 168 The open reading frame HIR77_18035 of Bacillus subtilis 168 probably encodes a NADH-dependent amino acid dehydrogenase (Genbank accession CP053102 locus_tag = "HIR77_18035", SEQ ID NO: 15). SEQ ID NO: 16 shows the amino acid sequence derived therefrom.

[0126] The open reading frame was optimized for codon usage 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. Further, the 3'-end was extended with a random spacer sequence, homologous regions for assembly cloning, and a BsaI restriction site. The resulting DNA sequence (SEQ ID NO: 17) was ordered for gene synthesis from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) and delivered as part of a cloning plasmid carrying the ampicillin resistance gene.

[0127] Example 5: Synthesis of the gene encoding NADH-dependent AaDH from Streptomyces fradiae ATCC 10745 The open reading frame CP974_05185 of Streptomyces fradiae ATCC 10745 probably encodes a NADH-dependent amino acid dehydrogenase (Genbank accession CP023696 locus_tag = "CP974_05185", SEQ ID NO: 19). SEQ ID NO: 20 shows the amino acid sequence derived therefrom.

[0128] Using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA), the open reading frame was optimized for the codon usage frequency of C. glutamicum. 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 (SEQ ID NO: 21) was ordered for gene synthesis from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) and delivered as part of a cloning plasmid carrying the ampicillin resistance gene.

[0129] Example 6: Synthesis of the gene encoding NADH-dependent AaDH from Afanotess halophytica CM1 Afanotess halophytica CM1 probably has an open reading frame encoding NADH-dependent amino acid dehydrogenase (Genbank accession MG430510, SEQ ID NO: 23). SEQ ID NO: 25 shows the amino acid sequence derived therefrom.

[0130] The open reading frame was optimized for codon usage 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. Further, the 3'-end was extended with a random spacer sequence, homologous regions for assembly cloning, and a BsaI restriction site. The resulting DNA sequence (SEQ ID NO: 24) was ordered for gene synthesis from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) and delivered as part of a cloning plasmid carrying the ampicillin resistance gene.

[0131] Example 7: Cloning of plasmids for co-expression of amino acid dehydrogenase and AGAT-Mp To enable combinatorial expression of AGAT-Mp and each amino acid dehydrogenase, the dehydrogenase genes were cloned into plasmid pLIB_P_AGAT-Mp.

[0132] Plasmid pLIB_P_AGAT-Mp was digested using the restriction endonuclease NotI, and the DNA was purified with the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0133] Each of the five plasmids containing the synthetic amino acid dehydrogenase genes was digested using the restriction endonuclease BsaI, and the resulting DNA was purified with the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0134] The DNA of NotI-digested pLIB_P_AGAT-Mp was ligated to each BsaI-digested amino acid dehydrogenase gene, and the matching sequence ends were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, catalog number E5520).

[0135] 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 obtained plasmids are shown in Table 7.

[0136]

Table 7

[0137] Example 8: Transformation of C. glutamicum ATCC13032 with the expression plasmid Corynebacterium glutamicum ATCC13032 (Kinoshita et al., J. Gen. Appl. Microbiol., 1957; 3(3): 193 - 205) was transformed with the expression plasmid by electroporation, and plasmid-containing cells were selected with 25 mg / l kanamycin. The obtained plasmid-containing strains are shown in Table 8.

[0138]

Table 8

[0139] Example 9: Effect of AaDH activity on GAA production To evaluate the effect of the expression of the AaDH gene on GAA production, the ATCC13032 / pLIB_P_AGAT-Mp strain, ATCC13032 / pLIB_AaDH-Mt_AGAT-Mp strain, ATCC13032 / pLIB_AaDH-Ms_AGAT-Mp strain, ATCC13032 / pLIB_AaDH-Bs_AGAT-Mp strain, ATCC13032 / pLIB_AaDH-Sf_AGAT-Mp strain, and ATCC13032 / pLIB_AaDH-Ah_AGAT-Mp strain were cultured in a production medium using the Wouter Duetz system, and the resulting GAA titer was determined as described above.

[0140]

Table 9

[0141] Cultivation of the strain containing the AaDH gene resulted in a higher GAA titer compared to ATCC13032 / pLIB_P_AGAT-Mp (see Table 9). The inventors conclude that the expression of the gene encoding NADH-dependent amino acid dehydrogenase improves the production of GAA.

Claims

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

2. 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.

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

4. 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.

5. The microorganism according to claim 4, wherein the activity of the enzyme having the function of carbamoyl phosphate synthase is increased compared to the respective enzyme activities in the wild-type microorganism.

6. The microorganism according to claim 4, further comprising an enzyme having the function of argininosuccinate lyase, having increased activity compared to the respective enzyme activities in the wild-type microorganism.

7. The microorganism according to claim 4, further comprising an enzyme having the function of ornithine carbamoyltransferase, having increased activity compared to the respective enzyme activities in the wild-type microorganism.

8. The microorganism according to claim 4, further comprising an enzyme having the function of argininosuccinate synthetase, having increased activity compared to the respective enzyme activities in the wild-type microorganism.

9. The microorganism according to claim 8, wherein the increase in the activity of the enzyme is achieved by overexpressing the gene encoding each of the enzymes.

10. The microorganism according to claim 1, wherein the expression of a gene encoding a protein having the function of malate synthase is attenuated compared to the expression of each of the genes in the wild-type microorganism, or the gene encoding a protein having the function of malate synthase is inactivated or deleted.

11. The microorganism according to claim 4, wherein the expression of the argR gene encoding the arginine-responsive repressor protein ArgR is attenuated compared to the expression of the argR gene in the wild-type microorganism, or the argR gene is inactivated or deleted.

12. The microorganism according to claim 4, wherein at least one of the genes encoding enzymes in the L-arginine biosynthesis pathway, including gdh, argJ, argB, argC and / or argD, which encode glutamate dehydrogenase, ornithine acetyltransferase, acetylglutamate kinase, acetylglutamyl phosphate reductase and acetylornithine aminotransferase, respectively, is overexpressed.

13. The microorganism according to claim 1, wherein the protein having the function of L-arginine:glycine amidinotransferase contains an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO:

2.

14. The microorganism according to claim 1, wherein the protein having the function of an NADH-dependent amino acid dehydrogenase includes an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, or SEQ ID NO:

18.

15. The microorganism according to claim 1, belonging to the genus Corynebacterium, the genus Enterobacteria seaae, or the genus Pseudomonas.

16. a) A method for fermentation production of guanidinoacetic acid (GAA), comprising the steps of: a) culturing a microorganism according to any one of claims 1 to 15 in a suitable medium under suitable conditions; and b) accumulating guanidinoacetic acid (GAA) in the medium to form a GAA-containing fermentation broth.

17. The method according to claim 16, further comprising isolating GAA from the GAA-containing fermented broth.

18. The microorganism according to claim 1, further comprising a gene encoding an enzyme having guanidinoacetate N-methyltransferase activity.

19. The microorganism according to claim 18, wherein the gene encoding an enzyme having guanidinoacetate N-methyltransferase activity is overexpressed.

20. a) A method for fermentation production of creatine, comprising the steps of: a) culturing the microorganism described in claim 18 or 19 in a suitable medium under suitable conditions; and b) accumulating creatine in the medium to form a creatine-containing fermentation broth.

21. The method according to claim 20, further comprising isolating creatine from the creatine-containing fermented broth.