Improved biotechnological process for production of guanidinoacetic acid (GAA) by use of NADH-dependent dehydrogenase

Abstract

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

Description

[0001] Guanidine acetic acid (GAA) is a colorless crystalline organic compound that can be used as an animal feed additive (e.g., WO2005120246A1 and US2011257075 A1). GAA is a natural precursor of creatine (e.g., Humm et al., Biochem. J. (1997) 322, 771-776). Therefore, supplementing GAA can enable organisms to obtain ideal creatine supplementation.

[0002] The present invention relates to a microorganism capable of producing guanidinoacetic acid (GAA) after transformation, and a method for producing GAA by fermentation using the microorganism. The present invention also relates to a fermentation production method of creatine. For the fermentation method, industrial raw materials (such as ammonia, ammonium salts and glucose or sugar-containing substrates) are used as starting materials.

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

[0004]

[0005] Guthmiller et al. (J Biol Chem. 1994 Jul 1; 269 (26): 17556-60) characterized the enzyme by cloning and heterologously expressing rat kidney AGAT in E. coli. Muenchhoff et al. (FEBS Journal 277 (2010) 3844–3860) also reported the first characterization of prokaryotic AGAT by cloning and heterologously expressing AGAT in E. coli. Sosio et al. elucidated the biosynthetic pathway of 25-pseudouridimycin in Streptomyces. They described the reaction of L-arginine with glycine to form GAA and L-ornithine as an intermediate reaction, catalyzed by PumN (L-arginine:glycine amidinotransferase (AGAT)).

[0006] Fan Wenchao discloses a method for producing creatine by fermentation of a non-pathogenic microorganism (e.g., Corynebacterium glutamicum) (CN 106065411 A). The microorganism has the following bioconversion functions: glucose is converted into L-glutamic acid; L-glutamic acid is converted into N-acetyl-L-glutamic acid; N-acetyl-L-glutamic acid is converted into N-acetyl-L-glutamic acid semialdehyde; N-acetyl-L-glutamic acid semialdehyde is converted into N-acetyl-L-ornithine; N-acetyl-L-ornithine is converted into L-ornithine; L-ornithine is converted into L-citrulline; L-citrulline is converted into argininosuccinic acid; argininosuccinic acid is converted into L-arginine; L-arginine is converted into guanidinoacetic acid; and finally, guanidinoacetic acid is converted into creatine. Fan Wenchao proposed that the microorganism overexpresses one or more enzymes selected from the group consisting of: N-acetylglutamate synthase, N-acetylornithine-δ-aminotransferase, N-acetylornithine enzyme, ornithine-carbamoyltransferase, argininosuccinate synthetase, glycine amidinotransferase (EC:2.1.4.1) and guanidinoacetic acid N-methyltransferase (EC:2.1.1.2). The microorganism preferably overexpresses glycine aminotransferase (L-arginine:glycine-amidinotransferase) and guanidinoacetic acid N-methyltransferase.

[0007] CN 113481139 A describes the construction of recombinant Bacillus subtilis producing GAA by introducing exogenous arginine:glycine amidinotransferase from Amycolatopsis kentuckyensis into the genome of wild-type Bacillus subtilis and by knocking out the gcvP and / or argI genes in the genome.

[0008] Zhang et al. published a microorganism capable of producing guanidine acetate (GAA) (ACS Synth. Biol. 2020, 9, 2066-275). They designed an ornithine cycle reconstructed in Escherichia coli by introducing heterologous AGATs from different species (e.g., Homo sapiens, Cylindrospermopsisraciborskii, Moorea producens) and by introducing citrulline synthesis modules (e.g., overexpression of carAB, argF, and argl) and arginine synthesis modules (e.g., overexpression of argG and argH; introduction of aspA) in Escherichia coli.

[0009] Also known increase in the document at microorganism, particularly several methods of the output of one of the synthetic starting materials of GAA (i.e. L-arginine) of bacterium.People such as Park (NATURE COMMUNICATIONS | DOI:10.1038 / ncomms5618) have proposed the general introduction of the metabolic engineering of Corynebacterium glutamicum (C.glutamicum) for L-arginine production.They propose random mutagenesis, and screening has produced the Corynebacterium glutamicum bacterial strain (for example ATCC 21831) of L-arginine as L-arginine producer (Nakayama and Yoshida 1974, US3849250 A), the progressively rational metabolic engineering based on full system metabolic analysis causes L-arginine output to increase gradually in whole bacterial strain engineering step. Yim et al. (J Ind Microbiol Biotechnol (2011) 38: 1911–1920) showed that by destroying the chromosomal argR gene in Corynebacterium glutamicum, inactivating the gene argR encoding the central repressor protein ArgR that controls the L-arginine biosynthetic pathway led to an improvement in arginine production strains. Ginesy et al. (Microbial Cell Factories (2015) 14: 29) reported the successful engineering of Escherichia coli for improving arginine production. Among them, they proposed the deletion of the argR repressor gene.

[0010] Kurahashi et al. (EP 1057893 A1) report the method for improving the ability of microorganism to produce L-arginine by utilizing recombinant DNA technology to enhance L-arginine biosynthetic enzyme, for example, by utilizing the microorganism containing recombinant DNA belonging to Corynebacterium (Corynebacterium) or Brevibacterium (Brevibacterium), the recombinant DNA comprises vector DNA and DNA fragmentation, wherein contains the gene of acetyl-ornithine deacetylase, N-acetylglutamate-γ-semialdehyde dehydrogenase, N-acetylglutamate kinase and arginine succinate enzyme derived from the microorganism belonging to Escherichia (Escherichia).In order to increase the output of L-arginine, the author also proposes to use the microorganism having enhanced intracellular glutamate dehydrogenase (GDH) activity and having L-arginine production ability.

[0011] Finally, Schneider and Jankowitsch (2021122400A1; EP 3839051 A1) proposed a fermentative production method for GAA, wherein heterologous AGATs from different species (e.g., Homo sapiens, Pseudomonas, marine cyanobacteria) are used, and the ability to produce L-arginine is improved, for example, by increasing the expression of enzymes of the L-arginine pathway, such as enzymes having the function of carbamoyl phosphate synthase and enzymes associated with the arginine operon.

[0012] Regarding the second starting material for GAA biosynthesis, it is desirable to increase the supply of glycine to improve GAA biosynthesis in a microorganism that naturally has a homologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT) or a heterologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT).

[0013] Schneider and Jankowitsch (WO 2022008280 A1) disclose a method for producing GAA using a microorganism containing 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. By using glyoxylate aminotransferase, it is shown that the production of GAA is increased.

[0014] Several glyoxylate aminotransferases are known, which have different substrate specificities for amino donors (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, Aug. 2004, p. 5513-5518; Takada and Noguchi, Biochem. J. (1985) 231, 157-163). Since most of these glyoxylate aminotransferases can use different amino acids as amino donors, they are usually annotated with different EC numbers. However, all these aminotransferases have something in common: they all use glyoxylate as an acceptor molecule, or in the case of the reverse reaction, glycine as a donor molecule. It also shows that, in a microorganism having a gene encoding a protein having an AGAT function, a decrease in the activity of a protein having the function of malate synthase using glyoxylate as a substrate leads to an increase in GAA production (WO 2022008276 A1).

[0015] The shortcoming of glyoxylate aminotransferase is that the amino group of amino donor is usually derived from L-glutamic acid or directly uses L-glutamic acid.L-glutamic acid from 2-ketoglutaric acid biosynthesis usually requires NADPH.In Corynebacterium glutamicum, NADPH is mainly regenerated from NADP+ by oxidative pentose phosphate pathway (PPP), partly regenerated 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 NADPH regeneration pathways result in carbon losses, which reduce the maximum theoretical yield. On the other hand, methods for increasing the supply of NADPH are known, 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, Ed. 1: 132-142. DOI: 10.1007 / s12257-013-0416-z), or expression of heterologous transhydrogenase genes (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). Corynebacterium 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 also known that heterologous NADH-dependent glutamate dehydrogenase is possible (Marx, Achim, Eikmanns, Bernhard J., Sahm, Hermann, de Graaf, Albert A. and Lothar Eggeling (1999) Metabolic Engineering, Volume 1, Issue 1, 1999, Pages 35-48, ISSN 1096-7176, DOI: https: / / doi.org / 10.1006 / mben.1998.0106). This reduces the demand for NADPH.

[0016] NADH-dependent amino acid dehydrogenases (AaDH) catalyze the amination of keto acids to L-amino acids by utilizing NADH as a cosubstrate (rather than NADPH). They therefore provide an alternative pathway for the assimilation or dissimilation of ammonium within the cell.

[0017]

[0018] For example, R=CH3, H, CH2COOH

[0019] Since most of these amino acid dehydrogenases can use different α-keto acids as substrates, they are usually annotated with different EC numbers. However, all of these amino acid dehydrogenases have something in common: they assimilate ammonium, or, in the case of the reverse reaction, dissimilate ammonium.

[0020] Examples of different amino acid dehydrogenases are as follows:

[0021] Reaction EC 1.4.1.1: Alanine dehydrogenase:

[0022] Pyruvate + NH3 + NADH + H+ <-> L-alanine + H2O + NAD+

[0023] Reaction EC 1.4.1.10: Glycine dehydrogenase:

[0024] Glyoxylate + NH3 + NADH + H+ <-> Glycine + H2O + NAD+

[0025] Reaction EC 1.4.1.21: Aspartate dehydrogenase:

[0026] Oxaloacetate + NH3 + NADH + H+ <-> L-aspartate + H2O + NAD+

[0027] Several NADH-dependent amino acid dehydrogenase (AaDH) proteins are known in the literature that can accept a variety of substrates and can thus generally aminate several different keto acids to the corresponding amino acids. (Fernandes et al.ProteinEngineering,Design&Selection,2015,vol.28no.2,pp.29–35; Giffin et al.,Journalof Bacteriology,2012,vol.194no.5,pp.1045-1054; Phogosee et al.,Archives ofMicrobiology,2018vol.200pp.719–727; Schuffenhauer et al.1999,vol.171,pp 417-423; Vancura et al.Eur J Biochem 1989,vol.179,pp.221–227; Yoshida and Freese, Biochim. Biophys. Acta, 1965, vol. 96, pp 248-262).

[0028] Therefore, most NADH-dependent amino acid dehydrogenases correspond to multiple reactions and therefore have multiple EC numbers. Table 1 shows some examples:

[0029]

[0030] Table 1: Examples of NADH-dependent amino acid dehydrogenase (AaDH) proteins

[0031] The problem underlying the present invention is therefore to provide improved microorganisms which have been transformed to be able to produce guanidinoacetic acid (GAA), in particular microorganisms with an improved ability to provide glycine as a starting material for the biosynthesis of GAA, and methods for the fermentative production of GAA using such microorganisms.

[0032] The problem is solved by a microorganism comprising at least one heterologous gene encoding a protein having the function of an L-arginine:glycine amidinotransferase (AGAT, for example EC 2.1.4.1) and at least one heterologous gene encoding a protein having the function of an NADH-dependent amino acid dehydrogenase.

[0033] A heterologous gene is a gene inserted into a host organism that does not naturally possess the gene. The insertion of a heterologous gene into a host is done through recombinant DNA technology. Microorganisms that have undergone recombinant DHA technology are called transgenic, genetically modified, or recombinant.

[0034] A heterologous protein is a protein that does not naturally occur in the microorganism.

[0035] A homologous gene or endogenous gene refers to a gene including its own function, or the nucleotide sequence of the gene naturally exists in the microorganism, or is "native" to the microorganism.

[0036] Homologous proteins or native proteins are proteins that occur naturally in microorganisms.

[0037] Proteins with the function of L-arginine:glycine-guanyltransferase (AGAT) belong to the guanyltransferase family. The guanyltransferase family contains glycine guanyltransferase (EC:2.1.4.1) and inositolamine guanyltransferase (EC:2.1.4.2), which are involved in the biosynthesis of creatine and streptomycin, respectively. This family also contains arginine deiminases (EC:3.5.3.6). These enzymes catalyze the following reaction: arginine + H2O <=> citrulline + NH3. Streptomyces antitumor glycoproteins are also found in this family. Enzymes or proteins with L-arginine:glycine-amidinotransferase (AGAT) activity are also 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 subfamilydomain architectures.", Nucleic Acids Res. 45(D1):D200-D203.), which 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:3123442EPMC:3123442).

[0038] In the microorganism of the present invention, at least one protein having the function of NADH-dependent amino acid dehydrogenase can be a NADH-dependent amino acid dehydrogenase. It can be selected from the group consisting of: a protein having the function of alanine dehydrogenase (EC 1.4.1.1), a protein having the function of glycine dehydrogenase (EC 1.4.1.10), and a protein having the function of aspartate dehydrogenase (EC 1.4.1.21).

[0039] In another embodiment of the microorganism of the present invention, the activity of NADH-dependent amino acid dehydrogenase is increased compared to the respective activity in the wild-type microorganism. Preferably, at least one gene encoding a protein having the enzymatic activity of NADH-dependent amino acid dehydrogenase in the microorganism of the present invention is overexpressed compared to the expression of the respective gene in the wild-type microorganism.

[0040] Preferably, the ability of the microorganism of the present invention to produce L-arginine from L-ornithine is increased compared to the ability of a wild-type microorganism.

[0041] In the context of the present invention, the microorganism with the L-arginic ability of production increased refers to the microorganism producing the L-arginic acid that exceeds its own demand.The example of this L-arginic microorganism producing has, for example, Corynebacterium glutamicum ATCC 21831 or by those disclosed by people such as Park (NATURE COMMUNICATIONS|DOI:10.1038 / ncomms5618) or people such as Ginesy (Microbial Cell Factories (2015) 14:29).

[0042] In a particular embodiment of the invention, the activity of an enzyme having the function of carbamoyl-phosphate synthase (EC 6.3.4.16) in the microorganism is increased compared to the corresponding enzyme activity in the wild-type microorganism.

[0043] The activity of an enzyme having the function of argininosuccinate lyase (EC 4.3.2.1) in the microorganism of the present invention may be increased compared to the corresponding enzyme activity in a wild-type microorganism.

[0044] Furthermore, the activity of an enzyme having the function of ornithine carbamoyltransferase (EC 2.1.3.3) in the microorganism of the present invention may be increased compared to the corresponding enzyme activity in a wild-type microorganism.

[0045] The activity of an enzyme having the function of argininosuccinate synthetase (EC 6.3.4.5) in the microorganism of the present invention may also be increased compared to the corresponding enzyme activity in a wild-type microorganism.

[0046] In another embodiment of the present invention, the expression of the gene encoding the protein having the function of malate synthase is attenuated, or the gene encoding the protein having the function of malate synthase is inactivated or deleted compared to the expression of the corresponding gene in the wild-type microorganism.

[0047] In another embodiment of the present invention, the expression of the argR gene encoding the arginine response repressor protein ArgR is attenuated compared to the expression of the argR gene in the wild-type microorganism. Alternatively, the argR gene is inactivated or deleted.

[0048] In another embodiment of the present invention, and optionally in addition to the above-mentioned modifications, at least one or more genes encoding enzymes of the biosynthetic pathway of L-arginine are overexpressed in the microorganism of the present invention, said genes comprising gdh, argJ, argB, argC and / or argD encoding glutamate dehydrogenase, ornithine acetyltransferase, acetylglutamate kinase, acetylglutamyl phosphate reductase and acetyl-ornithine aminotransferase, respectively.

[0049] In the microorganism of the present invention, the protein having the function of L-arginine:glycine-guanyltransferase may be L-arginine:glycine-guanyltransferase (AGAT, i.e., EC 2.1.4.1).

[0050] In the microorganism of the present invention, a gene encoding a protein having the function of L-arginine:glycine-guanyltransferase may be further overexpressed.

[0051] In the microorganism of the present invention, the protein having the function of L-arginine:glycine-guanyl transferase (AGAT) can comprise an amino acid sequence having at least 80% identity, preferably at least 90% identity with the amino acid sequence according to SEQ ID NO: 2. In another embodiment of the present invention, the amino acid sequence of L-arginine:glycine-guanyl transferase is identical to the amino acid sequence according to SEQ ID NO: 2.

[0052] In a specific embodiment of the invention, the protein having the function of NADH-dependent amino acid dehydrogenase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 6, according to SEQ ID NO: 9, according to SEQ ID NO: 12, according to SEQ ID NO: 15 or according to SEQ ID NO: 18.

[0053] The microorganism of the present invention may belong to the genus Corynebacterium, preferably Corynebacterium glutamicum (C. glutamicum), or to the family Enterobacteriaceae, preferably Escherichia coli (E. coli), or to the genus Pseudomonas, preferably Pseudomonas putida (P. putida).

[0054] In general, an increase in the enzymatic activity of a protein in a microorganism, in particular in the microorganism of the present invention, compared to the corresponding activity in a wild-type microorganism can be achieved, for example, by mutation of the protein, in particular by mutations that confer feedback resistance to the protein, such as feedback resistance to the product of the enzyme-catalyzed reaction, or by overexpression of a gene encoding a protein with enzymatic activity compared to the expression of the corresponding gene in a wild-type microorganism.

[0055] Overexpression of a gene is usually 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 of the start codon or the entire gene or a combination of options including all of the above.

[0056] Compared to the corresponding activity in the wild-type microorganism, in the microorganism, the overexpression of the gene in the microorganism of the present invention is achievable, and this is by increasing the copy number of the gene and / or by enhancing regulatory factors, for example by gene and strong promoter functional connection and / or by enhancing ribosome binding site and / or by the codon selection optimization of start codon or whole gene to realize.It is achievable that the enhancing of this type of regulatory factor that has a positive impact on genetic expression is arranged, for example by modifying the promoter sequence upstream of the structural gene to improve the effectiveness of the promoter or by replacing the driver with more effective or so-called strong promoter fully.Promoter is located in the upstream of gene.Promoter is the DNA sequence dna that is composed of about 40-50 base pairs, and it constitutes the binding site and the transcription start point of RNA polymerase holoenzyme, thereby can affect the expression intensity of controlled polynucleotide or gene. In general, it is possible to realize overexpression or expression increase of gene in bacterium by selecting strong promoter, for example, by replacing original promoter with strong, natural (originally assigned to other genes) promoter or by modifying certain regions of given natural promoter (such as so-called -10 district and -35 district of natural promoter) for consensus sequence, such as M.Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for Corynebacterium glutamicum taught. An example of " strong " promoter is superoxide dismutase (sod) promoter (" Psod "; Z.Wang et al., Eng.Life Sci.2015,15,73–82). " Functional connection " is understood as the order of promoter and gene, which has caused the transcription of gene.

[0057] The genetic code is degenerate, which means that a certain amino acid may be encoded by several different triplets. The term codon usage refers to the observation that a certain organism does not usually use all possible codons for a certain amino acid with the same frequency. Instead, organisms usually show a certain preference for specific codons, which means that these codons are found more frequently in the coding sequences of the organism's transcribed genes. If a certain foreign gene of the future host (i.e., originating from a different species) should be expressed in the future host organism, the coding sequence of said gene should be adjusted to the codon usage of said future host organism (i.e., codon usage optimization).

[0058] The above problem is further solved by a method for the fermentative production of guanidine acetic acid (GAA), which comprises the following steps: a) culturing the microorganism of the present invention as defined above in a suitable culture medium under suitable conditions, and b) accumulating GAA in the culture medium to form a fermentation broth containing GAA.

[0059] The method of the present invention may further comprise the step of isolating GAA from the fermentation broth.

[0060] The present invention also relates to a microorganism as defined above, further comprising 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.

[0061] The present invention also relates to a method for producing creatine by fermentation, the method comprising the following steps: a) culturing the microorganism of the present invention in a suitable culture medium under suitable conditions, the microorganism comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity, and b) accumulating creatine in the culture medium to form a fermentation broth containing creatine.

[0062] Preferably, the method further comprises separating creatine from the fermentation broth containing creatine. 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.

[0063] sequence:

[0064] SEQ ID NO: 1: shows an open reading frame encoding L-arginine:glutamate amidinotransferase (AGAT, EC 2.1.4.1; locus_tag BJP34_00300) from a marine cyanobacterium.

[0065] SEQ ID NO: 2: shows the amino acid sequence derived from SEQ ID NO: 1 (Genbank Accession No. WP_070390602).

[0066] SEQ ID NO: 3: shows a DNA sequence encoding L-arginine:glutamate amidinotransferase (AGAT, EC 2.1.4.1) from marine cyanobacteria, which is optimized for codon usage in Corynebacterium glutamicum. The 5' end of the optimized gene is amplified with a BsaI restriction site, a 5'-UTR sequence for assembling clones, and a ribosome binding site, and a second stop codon, a sequence for assembling clones, and a BsaI site are added to the 3' end.

[0067] SEQ ID NO:4: have shown intestinal bacteria-Corynebacterium glutamicum shuttle plasmid pLIB_P, it is made up of replication origin (being used for Corynebacterium glutamicum), pSC101 replication origin (being used for intestinal bacteria) and the kanamycin resistance gene from pBL1.After unique NotI restriction site, it has a strong promoter, two reverse directional BsaI sites and BioBricks Terminator BBa_B1006.

[0068] SEQ ID NO: 5: shows the open reading frame MRA_2804 of Mycobacterium tuberculosis H37Ra, encoding NADH-dependent amino acid dehydrogenase (Genbank Accession No. CP000611 locus_tag="MRA_2804").

[0069] SEQ ID NO: 6: shows the amino acid sequence derived from SEQ ID NO: 5 (AaDH-Mt from Mycobacterium tuberculosis H37Ra).

[0070] SEQ ID NO: 7: shows the DNA sequence encoding the NADH-dependent amino acid dehydrogenase of Mycobacterium tuberculosis H37Ra, which has been optimized for the codon usage of Corynebacterium glutamicum. The 5'-UTR of the DNA sequence is amplified at the 5' end, which consists of a BsaI restriction site, a homology sequence for assembling clones, a strong promoter Pg3N3 and a ribosome binding site. In addition, the 3'-UTR is amplified at the 3' end: a random spacer sequence, a homology region for assembling clones and a BsaI restriction site.

[0071] SEQ ID NO: 8: shows the open reading frame LJ00_13235 of Mycobacterium smegmatis MC2 155, encoding a NADH-dependent amino acid dehydrogenase (Genbank Accession No. CP009494 locus_tag="LJ00_13235").

[0072] SEQ ID NO: 9: shows the amino acid sequence derived from SEQ ID NO: 8 (AaDH-Ms from Mycobacterium smegmatis MC2155).

[0073] SEQ ID NO: 10: Shown is the DNA sequence of the NADH-dependent amino acid dehydrogenase encoding Mycobacterium smegmatis MC2 155, which is optimized for the codon usage of Corynebacterium glutamicum. The 5'-UTR of the DNA sequence is amplified at the 5' end, which is composed of a BsaI restriction site, a homologous sequence for assembling clones, a strong promoter Pg3N3 and a ribosome binding site. In addition, the 3'-UTR is amplified at the 3' end: a random spacer sequence, a homologous sequence for assembling clones and a BsaI restriction site.

[0074] SEQ ID NO: 11: shows the open reading frame HIR77_18035 of Bacillus subtilis subsp. subtilis str. 168, which is predicted to encode an NADH-dependent amino acid dehydrogenase (Genbank Accession No. CP053102 locus_tag="HIR77_18035"). SEQ ID NO: 12: shows the amino acid sequence derived from SEQ ID NO: 11 (AaDH-Bs from Bacillus subtilis subsp. subtilis str. 168).

[0075] SEQ ID NO: 13: show the DNA sequence of the NADH-dependent amino acid dehydrogenase of coding Bacillus subtilis subtilis strain 168, it is optimized to be used for the codon usage of Corynebacterium glutamicum.The 5' end amplification of sequence has 5'-UTR, is made up of a BsaI restriction site, a homologous sequence for assembling clones, strong promoter Pg3N3 and a ribosome binding site.In addition, its 3' end amplification has: a random spacer sequence, a homologous sequence for assembling clones and a BsaI restriction site.

[0076] SEQ ID NO: 14: shows the open reading frame CP974_05185 of Streptomyces fradiae ATCC10745, which is speculated to encode an NADH-dependent amino acid dehydrogenase (Genbank Accession No. CP023696 locus_tag="CP974_05185").

[0077] SEQ ID NO: 15: shows the amino acid sequence derived from SEQ ID NO: 14 (AaDH-Sf from Streptomyces flexneri ATCC10745).

[0078] SEQ ID NO:16: show the DNA sequence of the NADH-dependent amino acid dehydrogenase of coding Streptomyces freundii ATCC10745, it has been optimized to be used for the codon usage of Corynebacterium glutamicum.5' end amplification of sequence has 5'-UTR, it is made up of a BsaI restriction site, a homologous sequence that is used to assemble clones, strong promoter Pg3N3 and a ribosome binding site.In addition, its 3' end amplification has: a random spacer sequence, a homologous sequence that is used to assemble clones and a BsaI restriction site.

[0079] SEQ ID NO: 17: shows the open reading frame of Aphanothece halophytica CM1 putatively encoding a NADH-dependent amino acid dehydrogenase (Genbank Accession No. MG430510).

[0080] SEQ ID NO: 18: shows the amino acid sequence derived from SEQ ID NO: 17 (AaDH-Ah from C. salinarum CM1).

[0081] SEQ ID NO: 19: shows the DNA sequence encoding the NADH-dependent amino acid dehydrogenase of C. salinarum CM1, which is optimized for codon usage of Corynebacterium glutamicum. The 5' end of the sequence is amplified with a 5'-UTR, which consists of a BsaI restriction site, a homologous sequence for assembling clones, a strong promoter Pg3N3 and a ribosome binding site. In addition, its 3' end is amplified with: a random spacer sequence, a homologous sequence for assembling clones and a BsaI restriction site.

[0082] A) Materials and Methods

[0083] Chemical reagents

[0084] Kanamycin solution from Streptomyces kanamyceticus was purchased from Sigma Aldrich (St. Louis, USA, Cat. no. K0254). Unless otherwise specified, all other chemical reagents were analytical grade chemicals purchased from Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, USA) or Carl-Roth (Karlsruhe, Germany).

[0085] Bacterial strains

[0086] Corynebacterium glutamicum ATCC13032 (Kinoshita S, Udaka S, Shimono M., J. Gen. Appl. Microbiol. 1957; 3(3): 193-205), Corynebacterium glutamicum strain / wild type, can be purchased from American Type Culture Collection (ATCC) or DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, and its deposit number is DSM 20300.

[0087] Cell proliferation culture

[0088] Unless otherwise specified, the incubation procedure was performed as follows:

[0089] a. LB medium from Merck (Darmstadt, Germany; Cat. no. 110285) was used to grow E. coli strains in liquid medium. Liquid cultures (10 ml of liquid medium in each 100 ml conical flask with 3 baffles) were grown in an Infors HT Multitron standard shaking incubator from Infors GmbH (Bottmingen, Switzerland) at 30°C and 200 rpm.

[0090] b. LB agar (MILLER) from Merck (Darmstadt, Germany, Cat. no. 110283) was used to culture E. coli strains on agar plates. The agar plates were incubated at 30°C in a microincubator.

[0091] C. use brain heart infusion broth (BHI) from Merck (Darmstadt, Germany, Cat.no.110493) to cultivate Corynebacterium glutamicum strain in liquid medium.In the Infors HT Multitron standard shaking incubator from Infors GmbH (Bottmingen, Switzerland), under the condition of 30 ℃ and 200rpm, cultivate liquid culture (10ml liquid medium in every 100ml conical flask with 3 baffles).

[0092] d. Brain heart infusion agar (BHI-agar) from Merck (Darmstadt, Germany, Cat. no. 113825) was used to culture the Corynebacterium glutamicum strains on agar plates. Agar plates were incubated at 30° C. in an incubator from Heraeus Instruments with a temperature controller (Hanau, Germany).

[0093] e. When culturing Corynebacterium glutamicum after electroporation, 134 g / l sorbitol (Carl Roth GmbH+Co.KG, Karlsruhe, Germany), 2.5 g / l yeast extract (Oxoid / ThermoFisher Scientific, Waltham, USA, Cat.no.LP0021) and 25 mg / l kanamycin were added to BHI-agar (Darmstadt, Germany, Cat.no.113825). Agar plates were incubated at 30° C. in an incubator from Heraeus Instruments with a temperature controller (Hanau, Germany).

[0094] Determine the optical density of bacterial suspension

[0095] a. Using a Bio-Photometer from Eppendorf AG (Hamburg, Germany) at 600 nm (OD 600 ) was used to measure the optical density of the bacterial suspension in shake flask culture.

[0096] b. Using the Tecan Group AG ( GENios in Switzerland TM The plate reader was set at 600 nm (OD 600 ) was used to measure the optical density of the bacterial suspension produced in the Wouter Duetz (WDS) microfermentation system (24-well plate).

[0097] Centrifugation

[0098] a. Use an Eppendorf 5417R benchtop centrifuge to centrifuge a maximum volume of 2 ml of bacterial suspension in a 1.5 ml or 2 ml reaction tube (e.g. Eppendorf 3810X) and centrifuged (5 min, 13.000 rpm).

[0099] b. Centrifuge a bacterial suspension with a maximum volume of 50 ml in a 15 ml or 50 ml centrifuge tube (eg, Falcon™ 50 ml conical centrifuge tube) at 4000 rpm for 10 min using an Eppendorf 5810R tabletop centrifuge.

[0100] DNA Isolation

[0101] Plasmid DNA from E. coli cells was isolated using the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany, Cat. No. 27106) according to the manufacturer's instructions.

[0102] Polymerase chain reaction (PCR)

[0103] Use PCR with proofreading (high fidelity) polymerase to amplify the desired DNA fragment for sequencing or DNA assembly.Non-proofreading polymerase kits are used to determine the presence or absence of the desired DNA fragment derived directly from Escherichia coli or Corynebacterium glutamicum colonies.

[0104] a. From New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530) A high-fidelity DNA polymerase kit (Phusion kit) was used for template-corrected amplification of the selected DNA region according to the manufacturer's instructions (see Table 2).

[0105] Table 2: Use of the products from New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530) Thermal cycling conditions for PCR using the High-Fidelity DNA Polymerase Kit

[0106]

[0107] b. Taq PCR Core Kit (Taq Kit) from Qiagen (Hilden, Germany, Cat. No. 201203) was used to amplify the desired DNA fragment to confirm its presence. The kit was used according to the manufacturer's instructions (see Table 3).

[0108] Table 3: Thermal cycling conditions for PCR using the Taq PCR Core Kit from Qiagen (Taq Kit)

[0109]

[0110]

[0111] c. From Takara Bio Inc (Takara Bio Europe SAS, Saint-Germain-en-Laye, France, Cat. No. RR350A / B) Rapid PCR Master Mix (Sapphire Mix) was used according to the manufacturer's instructions as an alternative to confirm the presence of the desired DNA fragment in cells extracted from colonies of E. coli or C. glutamicum (see Table 4).

[0112] Table 4: Use of Thermal cycling conditions for PCR with Fast PCR Master Mix (SapphireMix)

[0113]

[0114] d. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany).

[0115] e. Use an appropriately diluted isolated plasmid DNA solution or a total DNA solution isolated from a liquid culture or a total DNA solution contained in a bacterial colony (colony PCR) as a PCR template. For the colony PCR, the template is prepared by removing the cell material from the colony on the agar plate with a sterile toothpick and placing the cell material directly into a PCR reaction tube. The cell material was heated in a microwave oven of the Mikrowave & Grill type from Sigma-Aldrich GmbH (Sundern, Germany) at 800 W for 10 sec., and the PCR reagents were subsequently added to the template in the PCR reaction tube.

[0116] f. All PCR reactions were performed in a Mastercycler PCR instrument or a Mastercycler nexus gradient PCR instrument from Eppendorf AG (Hamburg, Germany).

[0117] Restriction enzyme digestion of DNA

[0118] For restriction enzyme digestion, "FastDigest restriction endonuclease (FD)" (ThermoFisher Scientific, Waltham, USA) or restriction endonucleases from New England BioLabs Inc. (Ipswich, USA) were used. The reaction was performed according to the instructions of the manufacturer's manual.

[0119] Determine the size of DNA fragments

[0120] a. The size of small DNA fragments (<1000 bps) is typically determined by automated capillary electrophoresis using QIAxcel from Qiagen (Hilden, Germany).

[0121] b. If DNA fragments need to be separated, or if the DNA fragments are >1000 bps, separate the DNA by TAE agarose gel electrophoresis and use Nucleic acid gel stain (Biotium, Inc., Fremont, Canada) was used for staining. The stained DNA was visible at 320 nm.

[0122] Purification of PCR amplification products and restriction fragments

[0123] PCR amplification products and restriction fragments were purified using the QIAquick PCR purification kit from Qiagen (Hilden, Germany; Cat. No. 28106) according to the manufacturer's instructions. DNA was eluted with 30 μl of 10 mM Tris*HCl (pH 8.5).

[0124] Determination of DNA concentration

[0125] DNA concentration was measured using a NanoDrop spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH (renamed VWR brand in 2015) (Erlangen, Germany).

[0126] Assembling clones

[0127] Plasmid vectors were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" purchased from New England BioLabs Inc. (Ipswich, USA, Cat. No. E5520). The reaction mixture containing the linear vector and at least one DNA insert was incubated at 50°C for 60 min. 0.5 μl of the assembly mixture was used for each transformation experiment.

[0128] Chemical transformation of E. coli

[0129] For plasmid cloning, chemically competent “ Stable competent Escherichia coli (high efficiency grade)" (New England BioLabs Inc., Ipswich, USA, Cat. No. C3040). Successfully transformed cells were selected on LB agar supplemented with 25 mg / l kanamycin.

[0130] Transformation of Corynebacterium glutamicum

[0131] As described by Ruan et al. (2015), plasmid DNA was transformed into Corynebacterium glutamicum using "Gene Pulser Xcell" (Bio-Rad LaboratoriesGmbH, Feldkirchen, Germany) by electroporation. Electroporation was performed with a fixed time constant of 1.8kV and 5ms in a 1mm electroporation cuvette (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). The cells transformed were selected on the BHI-agar containing 134g / l sorbitol, 2.5g / l yeast extract and 25mg / l kanamycin.

[0132] Determine the nucleotide sequence

[0133] Cycle sequencing was performed by Eurofins Genomics GmbH (Ebersberg, Germany) using the dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences USA 74, 5463-5467, 1977) to determine the nucleotide sequence of the DNA molecule. Clonemanager Professional 9 software from Scientific & Educational Software (Denver, USA) was used to visualize and evaluate the sequences and computer assembly of the sequences.

[0134] Glycerol stocks of Escherichia coli and Corynebacterium glutamicum strains

[0135] For long-term storage of Escherichia coli and Corynebacterium glutamicum bacterial strain, prepared glycerol stock.Selected Escherichia coli clone was cultivated in the LB substratum that had added 2g / l glucose.Selected Corynebacterium glutamicum clone was cultivated in the double concentrated BHI substratum that had added 2g / l glucose.25mg / l kanamycin was added in the substratum of the growth of Escherichia coli and Corynebacterium glutamicum bacterial strain that was used to contain plasmid.Substratum was loaded in the 100ml conical flask with 3 baffles.Wherein inoculated a circle of cells extracted from bacterium colony.Then culture was cultivated 18h ​​under 30 ℃ and 200rpm.After the described cultivation period, 85% (v / v) sterile glycerol of 1.2ml was added in culture.Then the glycerol that contains the cell suspension that obtains is divided into 2ml parts, and is stored under-80 ℃.

[0136] GAA production in small-scale cultures

[0137] The milliliter scale culture system according to Duetz (2007) was used to assess the GAA production of the strains. For this purpose, 24-well deep well microplates (24-well WDS plates) from EnzyScreen BV (Heemstede, Netherlands, Cat. no. CR1424) were used, with 2.5 ml of culture medium per well.

[0138] The pre-culture of the strain was carried out in 10 ml of seed medium (SM). The medium was placed in a 100 ml conical flask with 3 baffles. 100 μl of glycerol stock culture was inoculated therein and the culture was cultured at 30°C and 200 rpm for 24 h. Table 5 shows the composition of the seed medium (SM).

[0139] Table 5: Seed medium (SM)

[0140] Components Concentration (g / l) Yeast extract FM902 (Angel Yeast Co., LTD, Hubei, PRChina) 10 Urea 1.5 <![CDATA[KH2PO4]]> 0.5 <![CDATA[K2HPO4]]> 0.5 <![CDATA[MgSO4*7H2O]]> 1 Biotin 0.0001 Thiamine Hydrochloride 0.0001 <![CDATA[FeSO4*7H2O]]> 0.01 <![CDATA[MnSO4*H2O]]> 0.01 glucose 20 Kanamycin 0.025 pH=7.0

[0141] After the incubation period, the optical density OD of the preculture was determined. 600 . Inoculate 2.5 ml of production medium (PM) to make OD 600 The required volume of 0.1 was sampled from the preculture, centrifuged (1 min, 8000 g), and the supernatant discarded. The cells were then resuspended in 100 μl of production medium.

[0142] The main culture was started by inoculating 100 μl per well of the resuspended cells from the preculture into the wells of a 24-well WDS plate containing 2.4 ml of production medium (PM). Table 6 shows the composition of the production medium (PM).

[0143] Table 6: Production medium (PM)

[0144] Components Concentration (g / l) 3-(N-Morpholino)propanesulfonic acid (MOPS) 40 Yeast extract FM902 (Angel Yeast Co., LTD, Hubei, PRChina) 1.5 <![CDATA[(NH4)2SO4]]> 10 <![CDATA[NH4Cl]]> 15 <![CDATA[Sodium citrate * 2H2O]]> 10 Urea 1 <![CDATA[KH2PO4]]> 0.5 <![CDATA[K2HPO4]]> 0.5 Ammonium acetate 7.7 <![CDATA[MgSO4*7H2O]]> 1 Biotin 0.0001 Thiamine Hydrochloride 0.0001 <![CDATA[FeSO4*7H2O]]> 0.01 <![CDATA[MnSO4*H2O]]> 0.01 <![CDATA[ZnSO4*7H2O]]> 0.00002 <![CDATA[CuSO4*5H2O]]> 0.0004 Defoamer XFO-1501 (Ivanhoe Industries Inc., Zion, USA) 0.5 glucose 40 L-Arginine 1.9 Kanamycin 0.025 pH=7.2

[0145] The main culture was incubated for 72 h in an Infors HT Multitron standard shaking incubator from Infors GmbH (Bottmingen, Switzerland) at 30°C and 225 rpm until glucose was completely consumed. A blood glucose meter OneTouch from LifeScan (Johnson & Johnson Medical GmbH, Neuss, Germany) was used. The glucose concentration in the suspension was analyzed.

[0146] After incubation, transfer the culture suspension to a deep-well microplate. Dilute a portion of the culture suspension appropriately to measure OD 600Another portion of the culture was centrifuged and the supernatant was analyzed for GAA concentration as described below.

[0147] Quantification of GAA

[0148] The samples were analyzed using an analytical system from Agilent, which consisted of an HPLC "Infinity 1260" coupled to a mass analyzer "Triple Quad 6420" (Agilent Technologies Inc., Santa Clara, USA). Chromatographic separation was performed on an Atlantis HILIC Silica column (4.6X250mm, 5μm) (Waters Corporation, Milford, USA) at 35°C. Mobile phase A was water containing 10mM ammonium formate and 0.2% formic acid. Mobile phase B was a mixture of 90% acetonitrile and 10% water, to which 10mM ammonium formate was added. The HPLC system was started at 100% B, followed by a linear gradient for 22min, at a constant flow rate of 0.6mL / min to 66% B. The mass analyzer was operated in ESI positive ionization mode. MRM fragments [M+H]+118–76 were used to monitor m / z values ​​to detect GAA. The limit of quantification (LOQ) of GAA was fixed at 7ppm.

[0149] B) Experimental results

[0150] Example 1: Cloning of the gene AGAT-Mp encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from marine cyanobacteria

[0151] Marine cyanobacteria are filamentous cyanobacteria. Leao et al. published the genome of the marine cyanobacteria strain PAL-8-15-08-1 (Leao T, G, Korobeynikov A, Monroe EA, Podell S, Glukhov E, Allen EE, Gerwick WH, Gerwick L, Proc Natl Acad Sci US A. 2017 Mar 21; 114(12): 3198-3203. doi: 10.1073 / pnas.1618556114; Genbank accession number CP017599.1). It contains an open reading frame encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1; locus_tag BJP34_00300 shown in SEQ ID NO: 1). SEQ ID NO: 2 shows the derived amino acid sequence (Genbank accession number WP_070390602).

[0152] Use software tool " Optimizer " (http: / / genomes.urv.es / OPTIMIZER / ) that amino acid sequence is reversely translated into the DNA sequence that is optimized to be used for the codon DNA sequence of Corynebacterium glutamicum.The 5 ' end amplification of optimized gene has: a BsaI restriction site, a 5 '-UTR sequence and a ribosome bind site that are used to assemble clones.Its 3 ' end amplification has: a second termination codon, a sequence that is used to assemble clones and a BsaI restriction site.The DNA sequence dna (SEQ ID NO:3) obtained is purchased from Eurofins Genomics GmbH (Ebersberg, Germany) to be used for gene synthesis, and delivers (designated as pEX-A258_AGAT-Mp) as a part of the cloning plasmid with ampicillin resistance gene.

[0153] Intestinal bacteria-Corynebacterium glutamicum shuttle plasmid pLIB_P is made up of replication origin (being used for Corynebacterium glutamicum), pSC101 replication origin (being used for intestinal bacteria) and the kanamycin resistance gene from pBL1.After unique NotI restriction enzyme site, it has a strong promoter, two reverse directional BsaI sites and BioBricks Terminator BBa_B1006 (SEQ ID NO:5).

[0154] pLIB_P was digested with restriction endonuclease BsaI, and the DNA was purified using the “QIAquick PCR Purification Kit” (Qiagen GmbH, Hilden, Germany).

[0155] The cloning plasmid pEX-A258_AGAT-Mp was digested with restriction endonuclease BsaI, and the DNA was purified using the “QIAquick PCR Purification Kit” (Qiagen GmbH, Hilden, Germany).

[0156] Connect the DNA solution of pLIB_P and pEX-A258_AGAT-Mp digested by BsaI, and use "NEBuilder HiFi DNA assembly cloning kit" (New England BioLabs Inc., Ipswich, USA, Cat.No.E5520) to assemble the sequence ends of the match. The product is transformed into "NEB Stable competent Escherichia coli (high efficiency level)" (New England Biolabs, Ipswich, USA), and grow cells on LB agar containing 25mg / l kanamycin. Appropriate plasmid clones are identified by restriction digestion and DNA sequencing. The plasmid obtained is named pLIB_P_AGAT-Mp.

[0157] Example 2: Synthesis of a gene encoding NADH-dependent AaDH from Mycobacterium tuberculosis H37Ra

[0158] The open reading frame MRA_2804 of Mycobacterium tuberculosis H37Ra is speculated to encode a NADH-dependent amino acid dehydrogenase (Genbank accession number CP000611 locus_tag="MRA_2804", SEQ ID NO: 7). SEQ ID NO: 8 shows the derived amino acid sequence.

[0159] Use software tool " codon optimization tool " (Integrated DNA Technologies Inc., Coralville, Iowa, USA) open reading frame is optimized for the codon usage of Corynebacterium glutamicum.The 5 ' end amplification of the sequence obtained has 5 '-UTR, which is composed of a BsaI restriction site, a homologous sequence for assembling clones, strong promoter Pg3N3 and a ribosome bind site.In addition, its 3 ' end amplification has: a random spacer sequence, a homologous sequence for assembling clones and a BsaI restriction site.The dna sequence dna obtained (SEQ ID NO:9) is purchased from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) to be used for gene synthesis, and is sent as a part of the cloning plasmid with ampicillin resistance gene.

[0160] Example 3: Synthesis of a gene encoding NADH-dependent AaDH from Mycobacterium smegmatis MC2155

[0161] The open reading frame LJ00_13235 of Mycobacterium smegmatis MC2 155 is speculated to encode a NADH-dependent amino acid dehydrogenase (Genbank accession number CP009494 locus_tag="LJ00_13235", SEQ ID NO: 11). SEQ ID NO: 12 shows the derived amino acid sequence.

[0162] Use software tool " codon optimization tool " (Integrated DNA Technologies Inc., Coralville, Iowa, USA) open reading frame is optimized for the codon usage of Corynebacterium glutamicum.The sequence amplification obtained has 5 '-UTR, and it is made up of a BsaI restriction site, a homologous sequence that is used to assemble clones, strong promoter Pg3N3 and a ribosome bind site.In addition, its 3 ' end amplification has: a random spacer sequence, a homologous sequence that is used to assemble clones and a BsaI restriction site.The dna sequence dna (SEQ ID NO:13) obtained is purchased from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) to be used for gene synthesis, and is sent as a part of the cloning plasmid with ampicillin resistance gene.

[0163] Example 4: Synthesis of a gene encoding NADH-dependent AaDH from Bacillus subtilis 168

[0164] The open reading frame HIR77_18035 of Bacillus subtilis 168 was speculated to encode a NADH-dependent amino acid dehydrogenase (Genbank accession number CP053102 locus_tag="HIR77_18035", SEQ ID NO: 15). SEQ ID NO: 16 shows the derived amino acid sequence.

[0165] Use software tool " codon optimization tool " (Integrated DNA Technologies Inc., Coralville, Iowa, USA) open reading frame is optimized for the codon usage of Corynebacterium glutamicum.The sequence amplification obtained has 5 '-UTR, is made up of a BsaI restriction site, a homologous sequence that is used to assemble clones, strong promoter Pg3N3 and a ribosome bind site.In addition, its 3 ' end amplification has: a random spacer sequence, a homologous sequence that is used to assemble clones and a BsaI restriction site.The dna sequence dna obtained (SEQ ID NO:17) is purchased from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) to be used for gene synthesis, and is sent as a part of the cloning plasmid with ampicillin resistance gene.

[0166] Example 5: Synthesis of a gene encoding NADH-dependent AaDH from Streptomyces freundii ATCC10745

[0167] The open reading frame CP974_05185 of Streptomyces freundii ATCC10745 was predicted to encode a NADH-dependent amino acid dehydrogenase (Genbank accession number CP023696 locus_tag="CP974_05185", SEQ ID NO: 19). SEQ ID NO: 20 shows the derived amino acid sequence.

[0168] Use software tool " codon optimization tool " (Integrated DNA Technologies Inc., Coralville, Iowa, USA) open reading frame is optimized to be used for the codon usage of Corynebacterium glutamicum.The sequence amplification obtained has 5 '-UTR, and it is made up of a BsaI restriction site, a homologous sequence that is used to assemble clones, strong promoter Pg3N3 and a ribosome bind site.In addition, its 3 ' end amplification has: a random spacer sequence, a homologous sequence that is used to assemble clones and a BsaI restriction site.The dna sequence dna (SEQ ID NO:21) obtained is purchased from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) to be used for gene synthesis, and sends as the part of the cloning plasmid with ampicillin resistance gene.

[0169] Example 6: Synthesis of a gene encoding NADH-dependent AaDH from C. salinarum CM1

[0170] C. salina CM1 has an open reading frame that is putatively encoding a NADH-dependent amino acid dehydrogenase (Genbank Accession No. MG430510, SEQ ID NO: 23). SEQ ID NO: 25 shows the derived amino acid sequence.

[0171] Use software tool " codon optimization tool " (Integrated DNA Technologies Inc., Coralville, Iowa, USA) open reading frame is optimized to be used for the codon usage of Corynebacterium glutamicum.The sequence amplification obtained has 5 '-UTR, and it is made up of a BsaI restriction site, a homologous sequence that is used to assemble clones, strong promoter Pg3N3 and a ribosome bind site.In addition, its 3 ' end amplification has: a random spacer sequence, a homologous sequence that is used to assemble clones and a BsaI restriction site.The dna sequence dna (SEQ ID NO:24) obtained is purchased from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) to be used for gene synthesis, and is sent as a part of the cloning plasmid with ampicillin resistance gene.

[0172] Example 7: Cloning of a plasmid for co-expression of amino acid dehydrogenase and AGAT-Mp

[0173] For the combined expression of AGAT-Mp and each amino acid dehydrogenase, the dehydrogenase genes were cloned into the plasmid pLIB_P_AGAT-Mp.

[0174] Plasmid pLIB_P_AGAT-Mp was digested with restriction endonuclease NotI and DNA was purified using the “QIAquick PCR Purification Kit” (Qiagen GmbH, Hilden, Germany).

[0175] Each of the five plasmids containing the synthetic amino acid dehydrogenase gene was digested with the restriction endonuclease BsaI, and the resulting DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0176] The DNA of pLIB_P_AGAT-Mp digested with NotI was ligated with each of the amino acid dehydrogenase genes digested with BsaI using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520), and the matching sequence ends were assembled.

[0177] The product was transformed into "NEB Stable competent E. coli (high efficiency grade)" (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 plasmids are listed in Table 7.

[0178] Table 7: Plasmids for co-expression of amino acid dehydrogenase and AGAT-Mp

[0179] Plasmids AaDH gene pLIB_AaDH-Mt_AGAT-Mp AaDH from Mycobacterium tuberculosis H37Ra pLIB_AaDH-Ms_AGAT-Mp AaDH from Mycobacterium smegmatis MC2 155 pLIB_AaDH-Bs_AGAT-Mp AaDH from Bacillus subtilis 168 pLIB_AaDH-Sf_AGAT-Mp AaDH from Streptomyces freundii ATCC10745 pLIB_AaDH-Ah_AGAT-Mp AaDH from C. salinarum CM1

[0180] Example 8: Transformation of Corynebacterium glutamicum ATCC13032 with expression plasmid

[0181] Transform Corynebacterium glutamicum ATCC13032 (Kinoshita et al., J. Gen. Appl. Microbiol. 1957; 3 (3): 193-205) with expression plasmid by electroporation, and select the cell containing plasmid with 25mg / l kanamycin. The bacterial strain containing plasmid obtained is listed in Table 8.

[0182] Table 8: Plasmids containing derivatives of Corynebacterium glutamicum ATCC13032

[0183] Recipient strain Plasmids Resulting strain ATCC13032 pLIB_P_AGAT-Mp ATCC13032 / pLIB_P_AGAT-Mp ATCC13032 pLIB_AaDH-Mt_AGAT-Mp ATCC13032 / pLIB_AaDH-Mt_AGAT-Mp ATCC13032 pLIB_AaDH-Ms_AGAT-Mp ATCC13032 / pLIB_AaDH-Ms_AGAT-Mp ATCC13032 pLIB_AaDH-Bs_AGAT-Mp ATCC13032 / pLIB_AaDH-Bs_AGAT-Mp ATCC13032 pLIB_AaDH-Sf_AGAT-Mp ATCC13032 / pLIB_AaDH-Sf_AGAT-Mp ATCC13032 pLIB_AaDH-Ah_AGAT-Mp ATCC13032 / pLIB_AaDH-Ah_AGAT-Mp

[0184] Example 9: Effect of AaDH activity on GAA production

[0185] To evaluate the effect of AaDH gene expression on GAA production, strains ATCC13032 / pLIB_P_AGAT-Mp, ATCC13032 / pLIB_AaDH-Mt_AGAT-Mp, ATCC13032 / pLIB_AaDH-Ms_AGAT-Mp, ATCC13032 / pLIB_AaDH-Bs_AGAT-Mp, ATCC13032 / pLIB_AaDH-Sf_AGAT-Mp, and ATCC13032 / pLIB_AaDH-Ah_AGAT-Mp were cultured in production medium in the Wouter Duetz system, and the resulting GAA titers were determined as described above.

[0186] Table 9: Effect of expression of NADH-dependent amino acid dehydrogenases on GAA production

[0187] strains GAA ATCC13032 / pLIB_P_AGAT-Mp 122mg / l ATCC13032 / pLIB_AaDH-Mt_AGAT-Mp 414mg / l ATCC13032 / pLIB_AaDH-Ms_AGAT-Mp 275mg / l ATCC13032 / pLIB_AaDH-Bs_AGAT-Mp 391mg / l ATCC13032 / pLIB_AaDH-Sf_AGAT-Mp 202mg / l ATCC13032 / pLIB_AaDH-Ah_AGAT-Mp 361mg / l

[0188] Cultivation of the strain containing the AaDH gene resulted in higher GAA titers compared to ATCC13032 / pLIB_P_AGAT-Mp (see Table 9). We concluded that expression of a gene encoding a NADH-dependent amino acid dehydrogenase can increase 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 and at least one heterologous gene 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 NADH-dependent amino acid dehydrogenase function is increased compared to the corresponding activity in the wild-type microorganism.

3. The microorganism according to claim 1 or 2, wherein the protein having the function of 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 any one of claims 1 to 3, wherein the ability of the microorganism to produce L-arginine from L-ornithine is increased compared to the ability of a wild-type microorganism.

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

6. The microorganism according to claim 4 or 5, wherein the microorganism further comprises an enzyme having the function of argininosuccinate lyase, the activity of which is increased compared to the corresponding enzyme activity in the wild-type microorganism.

7. The microorganism according to any one of claims 4 to 6, wherein the microorganism further comprises an enzyme having the function of ornithine carbamoyltransferase, wherein the activity of the enzyme is increased compared to the corresponding enzyme activity in the wild-type microorganism.

8. The microorganism according to any one of claims 4 to 7, wherein the microorganism further comprises an enzyme having the function of argininosuccinate synthetase, the activity of which is increased compared to the corresponding enzyme activity in the wild-type microorganism.

9. A microorganism according to any one of claims 4 to 8, wherein the increase in enzyme activity is achieved by overexpression of a gene encoding the respective enzyme.

10. The microorganism according to any one of the preceding claims, wherein the expression of the gene encoding the protein having the function of malate synthase is attenuated compared to the expression of the corresponding gene in the wild-type microorganism, or the gene encoding the protein having the function of malate synthase is inactivated or deleted.

11. The microorganism according to any one of claims 4 to 10, wherein the expression of the argR gene encoding the arginine response repressor protein ArgR is attenuated compared to the expression of the argR gene in the wild-type microorganism or wherein the argR gene is inactivated or deleted.

12. according to the microorganism described in claim 4-11 any one, wherein at least one or more genes of the enzyme of the biosynthetic pathway of coding L-arginine are overexpressed, and said gene comprises gdh, argJ, argB, argC and / or argD encoding glutamate dehydrogenase, ornithine acetyltransferase, acetylglutamate kinase, acetylglutamyl phosphate reductase and acetyl-ornithine aminotransferase respectively.

13. The microorganism according to any one of the preceding claims, wherein the protein having the function of L-arginine:glycine-amidinotransferase (AGAT) comprises an amino acid sequence having at least 80% identity with the amino acid sequence according to SEQ ID NO:

2.

14. A microorganism according to any of the preceding claims, wherein the protein having the function of a NADH-dependent amino acid dehydrogenase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 6, according to SEQ ID NO: 9, according to SEQ ID NO: 12, according to SEQ ID NO: 15 or according to SEQ ID NO:

18.

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

16. A method for producing guanidine acetic acid (GAA) by fermentation, the method comprising the following steps: a) culturing the microorganism according to any one of the preceding claims in a suitable culture medium under suitable conditions, and b) accumulating GAA in the culture medium to form a fermentation broth containing GAA.

17. The method of claim 16, further comprising separating GAA from the GAA-containing fermentation broth.

18. The microorganism according to any one of claims 1 to 15, further comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity.

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

20. A method for producing creatine by fermentation, the method comprising the following steps: a) culturing the microorganism as defined in any one of claims 18 or 19 in a suitable culture medium under suitable conditions, and b) accumulating creatine in the culture medium to form a fermentation broth containing creatine.

21. The method of claim 20, further comprising isolating creatine from the fermentation broth containing creatine.

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