Method for fermentation production of guanidinoacetic acid

Abstract

The present invention relates to a microorganism transformed with L-arginine:glycine amidotransferase, glyoxylate aminotransferase, and reduced malate synthase to produce 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.

Description

Technical Field

[0001] The present invention relates to a microorganism transformed to be able to produce guanidinoacetic acid (GAA), and a method for fermentative production of GAA using such a microorganism. The present invention also relates to a method for fermentative production of creatine.

[0002] GAA is an organic compound used as an animal feed additive (U.S. Patent Application Publication No. 2011257075). GAA is a natural precursor of creatine (e.g., Humm et al., Biochem. J. (1997) 322, 771 - 776). Therefore, by adding GAA, optimal supply of creatine in vivo becomes possible.

[0003] The present invention relates to a method for producing GAA by a fermentation process using industrial raw materials (e.g., ammonia, ammonium salts, and glucose or sugar-containing substrates) as starting materials. In the biological system, using arginine and glycine as starting materials, GAA and L-ornithine are produced by the catalysis of L-arginine:glycine-amidinotransferase (AGAT; EC 2.1.4.1), which is the first step of creatine biosynthesis:

Chemical Formula

[0004] Guthmiller et al. (J Biol Chem. 1994 Jul 1;269(26):17556-60) elucidated the characteristics of rat kidney AGAT by cloning the enzyme in Escherichia coli (E. coli) and heterologously expressing it. Muenchhoff et al. (FEBS Journal 277 (2010) 3844-3860) also reported that they were the first to elucidate the characteristics of prokaryotic-derived AGAT by cloning the enzyme in Escherichia coli and heterologously expressing it. Sosio et al. (Cell Chemical Biology 25, 540-549, May 17, 2018) elucidated the biosynthetic pathway of pseudouridimycin in Streptomyces sp. They describe an intermediate reaction involving the reaction of L-arginine and glycine catalyzed by PumN, an L-arginine:glycine-amidinotransferase (AGAT), to produce GAA and L-ornithine.

[0005] Several approaches to increasing the production of L-arginine, one of the starting materials for GAA synthesis, in microorganisms, particularly bacteria, are known from the literature. An overview of metabolic engineering of Corynebacterium glutamicum (C. glutamicum) for L-arginine production is provided by Park et al. (NATURE COMMUNICATIONS | DOI: 10.1038 / ncomms5618). They propose a stepwise and rational metabolic engineering based on random mutagenesis and screening of L-arginine producers in already L-arginine-producing C. glutamicum strains, such as ATCC21831 (Nakayama and Yoshida 1974, U.S. Patent No. 3849250), as well as metabolic analysis of the entire system, gradually increasing L-arginine production through strain engineering steps. Yim et al. (J Ind Microbiol Biotechnol (2011) 38:1911-1920) demonstrated that disrupting the argR gene on the chromosome of C. glutamicum, thereby inactivating the gene encoding ArgR, a central repressor protein that regulates the L-arginine biosynthesis pathway, improved arginine production in the strain. Ginesy et al. (Microbial Cell Factories (2015) 14:29) reported successful engineering of E. coli to enhance arginine production. In particular, they proposed the deletion of the argR repressor gene.

[0006] A method using a recombinant strain in which the gene that suppresses the expression of the arginine biosynthesis operon argR has been inactivated has been reported by Suga et al. (U.S. Patent Application Publication No. 20070031946). In particular, the deletion of argR, which regulates the arginine operon, has been considered an important factor in arginine production.

[0007] Fan Wenchao discloses a method for producing creatine by fermentation of a non-pathogenic microorganism, such as C. glutamicum (Chinese Patent Application Publication No. 106065411). This microorganism has the following bioconversion 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 argininosuccinate; conversion of argininosuccinate to L-arginine; conversion of L-arginine to guanidinoacetic acid; and finally conversion of guanidinoacetic acid to creatine. Fan Wenchao proposes that this microorganism overexpresses one or more enzymes selected from the group consisting of N-acetylglutamate synthase, N-acetylornithine-δ-aminotransferase, N-acetylornithinase, ornithine-carbamoyltransferase, argininosuccinate synthetase, glycine amidinotransferase (EC:2.1.4.1), and guanidinoacetate-N-methyltransferase (EC:2.1.1.2). Preferably, this microorganism overexpresses glycine amidinotransferase (L-arginine:glycine amidinotransferase) and guanidinoacetate-N-methyltransferase.

[0008] As a second starting material for GAA biosynthesis, it would be desirable to increase the supply of glycine to improve GAA biosynthesis in microorganisms that naturally possess homologous genes encoding a protein with the function of L-arginine:glycine amidinotransferase (AGAT), or in microorganisms that possess heterologous genes encoding a protein with the function of L-arginine:glycine amidinotransferase (AGAT).

[0009] The so-called glyoxylate shunt pathway, naturally occurring in microorganisms such as Escherichia coli or C. glutamicum, is a side reaction of the tricarbonate (TCA) cycle (Krepus cycle) and includes the production of glyoxylate and succinate from isocitrate by isocitrate lyase, and the production of malate from glyoxylate and acetyl-CoA by malate synthase (Salusjarvi et al., Applied Microbiology and Biotechnology (2019) 103:2525-2535).

[0010] Glyoxylic acid may be used as a starting material for producing glycine in the presence of an amino acid donor and glyoxylic acid transaminase. Glyoxylic acid transaminase catalyzes the transfer of an amino group from an amino acid to glyoxylic acid. The products of this transfer are glycine and the corresponding α-keto acid.

[0011] In an attempt to improve glycolic acid production in C. glutamicum, Zahoor et al. (Journal of Biotechnology 192 (2014) 366-375) succeeded in increasing the supply of glyoxylic acid precursor in particular by deleting the malate synthase gene aceB.

[0012] Several glyoxylate aminotransferases are known, each exhibiting different substrate specificities for their respective amino acid donors (e.g., 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). Many of these glyoxylate aminotransferases can use different amino acids as amino donors and are therefore often annotated with different EC numbers. However, all of these aminotransferases share the commonality of using glyoxylate as the acceptor molecule, or glycine as the donor molecule in the reverse reaction.

[0013] Examples of proteins that have the function of glyoxylate aminotransferase are as follows: Glycine transaminase (EC2.6.1.4) catalyzes the following reaction: L-glutamic acid + glyoxylic acid <=> α-ketoglutaric acid + glycine.

[0014] Glycine:oxaloacetate transaminase (EC2.6.1.35) catalyzes the following reaction: L-aspartic acid + glyoxylic acid <=> oxaloacetate + glycine.

[0015] Alanine:glyoxylate transaminase (EC2.6.1.44) catalyzes the following reaction: L-alanine + glyoxylic acid <=> pyruvate + glycine.

[0016] Serine:glyoxylate transaminase (EC2.6.1.45) catalyzes the following reaction: L-serine + glyoxylic acid <=> 3-hydroxypyruvic acid + glycine.

[0017] Methionine:glyoxylate transaminase (EC2.6.1.73) catalyzes the following reaction: L-methionine + glyoxylic acid <=> 4-(methylsulfanyl)-2-keto-butanoic acid + glycine.

[0018] Aromatic amino acid: Glyoxylate transaminase (EC2.6.1.60) catalyzes the following reaction: Aromatic amino acids + glyoxylic acid <=> aromatic keto acids + glycine.

[0019] Kynurenine:glyoxylate transaminase (EC2.6.1.63) catalyzes the following reaction: Kynurenine + Glyoxylic acid <=> 4-(2-aminophenyl)-2,4-diketo-butanoic acid + Glycine.

[0020] (S)-Ureidoglycine:glyoxylate transaminase (EC2.6.1.112) catalyzes the following reaction: (S)-Ureido-glycine + Glyoxylic acid <=> N-Carbamoyl-2-keto-glycine + Glycine.

[0021] However, endogenous glyoxylate aminotransferases from Escherichia coli and C. glutamicum have not been reported to date. Furthermore, microorganisms suitable for producing large amounts of GAA compared to wild-type GAA, and methods for producing GAA using such microorganisms, have not been reported.

[0022] Therefore, the fundamental problem of the present invention is to provide microorganisms transformed to produce guanidinoacetic acid (GAA), in particular microorganisms with improved ability to supply glycine as a starting material for GAA biosynthesis, and a method for fermentation production of GAA using such microorganisms.

[0023] This problem is solved by a microorganism comprising at least one gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT, e.g., EC 2.1.4.1) and comprising at least one protein having the function of glyoxylate aminotransferase.

[0024] Preferably, in the microorganism according to the invention, the enzymatic activity of at least one protein having the function of glyoxylate aminotransferase is increased as compared to the respective enzymatic activities in wild-type microorganisms.

[0025] The microorganism according to the invention may comprise at least one gene encoding a protein having the enzymatic activity of glyoxylate aminotransferase. The at least one protein having the enzymatic activity of glyoxylate aminotransferase may be homologous or heterologous. Preferably, at least one gene encoding a protein having the enzymatic activity of glyoxylate aminotransferase is overexpressed in the microorganism of the invention.

[0026] Preferably, the microorganism of the invention has an improved L-arginine-producing ability as compared to the ability of wild-type microorganisms.

[0027] In the context of the present invention, a microorganism having an improved L-arginine-producing ability means a microorganism that produces L-arginine in excess of its own required amount. 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).

[0028] In certain embodiments of the present invention, the microorganism exhibits increased activity of the enzyme having the function of carbamoyl phosphate synthase (EC 6.3.4.16) compared to the respective enzyme activity in the wild-type microorganism.

[0029] In the microorganism according to the present invention, the activity of the enzyme having the function of argininosuccinate lyase (EC 4.3.2.1) may be increased compared to the activity of the respective enzymes in wild-type microorganisms.

[0030] Furthermore, in the microorganisms according to the present invention, the activity of the enzyme having the function of ornithine carbamoyltransferase (EC2.1.3.3) is increased compared to the activity of the respective enzymes in wild-type microorganisms.

[0031] In the microorganism according to the present invention, the activity of the enzyme having the function of argininosuccinate synthetase (EC 6.3.4.5) may also be increased compared to the respective enzyme activities in wild-type microorganisms.

[0032] Increasing enzyme activity in microorganisms can be achieved, for example, by mutating the corresponding endogenous gene. Further strategies for increasing enzyme activity may involve stabilizing the mRNA encoding the enzyme. Additionally, increasing the activity of the enzymes can also be achieved by overexpressing the gene encoding each enzyme.

[0033] In a further embodiment of the microorganism according to the present invention, the activity of the malate synthase-functioning protein is reduced compared to the respective activities in wild-type microorganisms.

[0034] The activity of a protein having malate synthase function can be reduced by mutating the protein to one with lower enzymatic activity than the wild-type protein, by reducing the expression of the gene encoding the enzyme having malate synthase function compared to the expression of the respective gene in wild-type microorganisms, by reducing the efficiency of translation, for example by changing the ATG start codon to GTG, by introducing a secondary structure into the 5' untranslated region of mRNA, by reducing codon use, or by deleting the gene encoding the enzyme having malate synthase function.

[0035] Furthermore, the microorganism according to the present invention preferably includes at least one overexpressed gene selected from the group consisting of a gene encoding a protein having the function of ornithine carbamoyltransferase (EC2.1.3.3) (e.g., argF / argF2 / argI), a gene encoding a protein having the function of argininosuccinate synthetase (EC6.3.4.5) (e.g., argG), and a gene encoding a protein having the function of argininosuccinate lyase (EC4.3.2.1) (e.g., argH).

[0036] Furthermore, the microorganism according to the present invention may be overexpressed in the arginine operon (argCJBDFR).

[0037] Alternatively, in the microorganism according to the present invention, the argR gene encoding the arginine-responsive repressor protein ArgR may be attenuated or deleted.

[0038] In further embodiments of the present invention, and as necessary, in addition to the above modifications, at least one or more 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, are overexpressed in the microorganism according to the present invention.

[0039] Table 1 shows the different names of enzymes involved in or contributing to arginine biosynthesis in different species, namely Escherichia coli, C. glutamicum, and Pseudomonas putida.

[0040] [Table 1-1] [Table 1-2]

[0041] Gene overexpression is generally achieved by increasing the copy number of a gene, and / or functionally linking the gene to a strong promoter, and / or strengthening ribosome binding sites, and / or optimizing the use of the start codon or the codon of the entire gene, or a combination of the above methods or a selection of them.

[0042] In a further embodiment of the present invention, the gene encoding the protein having the function of L-arginine:glycine amidinotransferase in the microorganism according to the present invention is heterologous.

[0043] A heterologous gene is a gene that has been inserted into a host organism that does not originally possess that gene. The insertion of heterologous genes into a host organism is carried out using recombinant DNA technology. Microorganisms that have undergone recombinant DNA technology are called transgenic, genetically modified, or recombinant organisms.

[0044] A heterologous protein refers to a protein that does not naturally exist in microorganisms.

[0045] Homologous genes or endogenous genes mean that the gene or the nucleotide sequence of a gene that contains its function is naturally present in a microorganism or is "native" to that microorganism.

[0046] Homologous proteins, or native proteins, refer to proteins that are naturally present in microorganisms.

[0047] Proteins with 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 the biosynthesis of creatine and streptomycin, respectively. This family also includes arginine deiminase, EC:3.5.3.6. These enzymes catalyze the reaction: arginine + H2O <=> citrulline + NH3. This family also includes Streptococcus antitumor glycoprotein. Furthermore, enzymes or proteins possessing L-arginine:glycine-amidinotransferase (AGAT) activity are also described as having a conserved domain belonging to the PFAM family: Amidinotransf(PF02274), as stated 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) (Marchler-Bauer A et al.). (2017), “CDD / SPARCLE: functional classification of proteins via subfamily domain architectures.”, Nucleic Acids Res. 45(D1):D 200-D203.).

[0048] In the microorganism of the present invention, a gene encoding a protein having the function of L-arginine:glycine amidinotransferase may be further overexpressed. Gene overexpression is generally achieved by increasing the copy number of the gene, and / or functionally linking the gene to a strong promoter, and / or strengthening the ribosome binding site, and / or optimizing the use of the start codon or the codon of the entire gene, or a combination of the above methods or a selection of all of them.

[0049] The protein having the function of L-arginine:glycineamidinotransferase in the microorganism of the present invention may contain an amino acid sequence that is at least 80% homologous, preferably at least 90% homologous, to the amino acid sequence determined by SEQ ID NO:11. In a further embodiment of the present invention, the amino acid sequence of L-arginine:glycineamidinotransferase is the same as the amino acid sequence determined by SEQ ID NO:11.

[0050] In certain embodiments of the present invention, the protein having glyoxylate aminotransferase enzymatic activity in the microorganism according to the present invention comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence specified by SEQ ID NO:2, the amino acid sequence specified by SEQ ID NO:5, or the amino acid sequence specified by SEQ ID NO:8.

[0051] The microorganisms of the present invention may belong to the genus Corynebacterium, preferably Corynebacterium glutamicum (C. glutamicum), or to the genus Enterobacteriaceae, preferably Escherichia coli (E. coli), or to the genus Pseudomonas, preferably Pseudomonas putida (P. putida).

[0052] In microorganisms, particularly those of the present invention, increased enzymatic activity of proteins compared to their respective activities in wild-type microorganisms can be achieved, for example, by mutations in the proteins, especially mutations that confer feedback resistance to the products of enzyme-catalyzed reactions to the proteins, or by increased expression of genes encoding enzyme-active proteins compared to the expression of their respective genes in wild-type microorganisms.

[0053] Increased or overexpression of genes in microorganisms, particularly those of the present invention, compared to their respective activities in wild-type microorganisms, can be achieved by increasing the copy number of the gene and / or enhancing regulatory factors, such as functionally linking the gene to a strong promoter and / or strengthening the ribosome binding site and / or optimizing the use of the start codon or the codon of the entire gene. Enhancement of such regulatory factors that positively affect gene expression can be achieved, for example, by modifying the promoter sequence upstream of a structural gene to increase the effectiveness of the promoter, or by completely replacing the aforementioned promoter with a more effective or so-called strong promoter. The promoter is located upstream of the gene. A promoter is a DNA sequence consisting of approximately 40-50 base pairs that constitutes the binding site and transcription start site of the RNA polymerase holoenzyme, thereby influencing the strength of expression of the polynucleotide or gene being regulated. In general, it is possible to achieve overexpression or increased expression of a gene in bacteria by selecting a strong promoter, for example, by replacing the original promoter with a strong, native (originally assigned to another gene) promoter, or by modifying specific regions of a given native promoter (e.g., its so-called -10 and -35 regions) toward the consensus sequence, as taught by M. Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for C. glutamicum, for example. An example of a “strong” promoter is the superoxide dimstase (SOD) promoter (“PsOD”; Z. Wang et al., Eng. Life Sci. 2015, 15, 73-82). “Functionally linked” is understood to mean that the promoter is sequentially positioned with the gene, thereby resulting in the transcription of the gene.

[0054] The degenerate nature of the genetic code means that a given amino acid may be coded by many different triplets. The term codon use refers to the observation that an organism does not typically use all possible codons corresponding to a given amino acid with equal frequency. Instead, an organism typically exhibits a certain preference for certain codons, and these codons are found more frequently in the coding sequences of the organism's transcription genes. If a gene that is exotic to a future host, i.e., from a different species, is to be expressed in the future host organism, then the coding sequence of that gene should be adapted to the codon use of that future host organism (i.e., codon use optimization).

[0055] The aforementioned problems can be further solved by a method for fermentation production of guanidinoacetic acid (GAA), comprising the steps of: a) culturing the microorganism according to the present invention as defined above in a suitable medium under suitable conditions; and b) accumulating GAA in the medium to produce a GAA-containing fermentation broth.

[0056] The method according to the present invention may further include the step of isolating GAA from the fermentation broth.

[0057] The method according to the present invention may further include the step of drying and / or granulating the fermentation broth containing GAA.

[0058] The present invention further relates to the microorganism defined above, further comprising a gene encoding an enzyme having the activity of guanidinoacetate-N-methyltransferase (EC:2.1.1.2). Preferably, the gene encoding the enzyme having the activity of guanidinoacetate-N-methyltransferase is overexpressed.

[0059] The present invention also relates to a method for fermentation production of creatine, comprising the steps of: a) culturing a microorganism according to the present invention, which contains a gene encoding an enzyme having guanidinoacetate-N-methyltransferase activity, in a suitable medium under suitable conditions; and b) accumulating creatine in the medium to produce a creatine-containing fermentation broth.

[0060] Preferably, the method further includes the step of isolating creatine from the creatine-containing fermentation broth. Creatine can be extracted from the fermentation broth by isoelectric focusing and / or ion exchange. Alternatively, creatine can be further purified by recrystallization in water.

[0061] Examples A) Materials and methods chemicals Kanamycin solution derived from Streptomyces kanamyceticus was purchased from Sigma Aldrich (St. Louis, USA, Cat. no. K0254). IPTG (isopropyl-β-thiogalactopyranoside) was purchased from Carl-Roth (Karlsruhe, Germany, Cat. no. 2316.4). Unless otherwise noted, all other chemicals were purchased in analytically pure form from Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, USA), or Carl-Roth (Karlsruhe, Germany).

[0062] Cell culture for cell proliferation Unless otherwise specified, the culture / incubation procedure is as follows: a. E. coli strains were cultured in liquid medium using LB broth (MILLER) from Merck (Darmstadt, Germany; Cat. no. 110285). The liquid culture (10 ml of liquid medium per 100 ml Erlenmeyer flask with three baffles) was incubated at 30°C and 200 rpm in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland).

[0063] E. coli strains were cultured on agar plates using LB agar (MILLER) from b. Merck (Darmstadt, Germany, Cat. no. 110283). The agar plates were incubated at 30°C in an INCU-Line® mini incubator from VWR (Radnor, USA).

[0064] C. glutamicum strains were cultured in liquid medium using Brain Heart Infusion Medium (BHI) from c. Merck (Darmstadt, Germany, Cat. no. 110493). The liquid culture medium (10 ml of liquid medium per 100 ml Erlenmeyer flask with three baffles) was incubated at 30°C and 200 rpm in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland).

[0065] C. glutamicum strains were cultured on agar plates using Brainheart Agar (BHI Agar) from d. Merck (Darmstadt, Germany, Cat. no. 113825). The agar plates were incubated at 30°C in a Kelvitron® temperature-controlled incubator from Heraeus Instruments (Hanau, Germany).

[0066] e. To culture C. glutamicum after electroporation, BHI agar (Merck, Darmstadt, Germany, Cat. no. 113825) was mixed with 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. The agar plates were incubated at 30°C in a Heraeus Instruments Kelvitron® temperature-controlled incubator (Hanau, Germany).

[0067] Measurement of the optical density of bacterial suspensions a. The optical density of bacterial suspensions in shaking flask cultures was measured at 600 nm (OD600) using a BioPhotometer from Eppendorf AG (Hamburg, Germany).

[0068] The optical density of bacterial suspensions produced in a Wouter Duetz (WDS) microfermentation system (24-well plate) was measured at 660 nm (OD660) using a GENios® plate reader from b.Tecan Group AG (Maennedorf, Switzerland).

[0069] Centrifugal separation a. A bacterial suspension with a maximum capacity of 2 ml was placed in a 1.5 ml or 2 ml reaction tube (e.g., Eppendorf Tubes® 3810X) and centrifuged using an Eppendorf 5417R benchtop centrifuge (5 minutes, 13,000 rpm).

[0070] b. A bacterial suspension with a maximum capacity of 50 ml was placed in a 15 ml or 50 ml centrifuge tube (e.g., a Falcon® 50 ml conical centrifuge tube) and centrifuged at 4,000 rpm for 10 minutes using an Eppendorf 5810R benchtop centrifuge.

[0071] DNA isolation Plasmid DNA was isolated from E. coli cells using the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany, Cat. No. 27106) according to the manufacturer's instructions.

[0072] Polymerase chain reaction (PCR) Using proofreading (high-fidelity) polymerase PCR, desired segments of DNA were amplified for Sanger sequencing or DNA assembly. The presence or absence of desired DNA fragments was determined directly from E. coli or C. glutamicum colonies using a non-proofreading polymerase kit.

[0073] a. The selected DNA region was template-corrected and amplified using the Phusion® High-Fidelity DNA Polymerase Kit (Phusion kit) from New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530) according to the manufacturer's instructions (see Table 2).

[0074] [Table 2]

[0075] The desired DNA segment was amplified and its presence confirmed using the Taq PCR Core Kit (Taq kit) from b. Qiagen (Hilden, Germany, Cat. No. 201203). The kit was used according to the manufacturer's instructions (see Table 3).

[0076] [Table 3]

[0077] c. Using SapphireAmp® Fast PCR Master Mix (Sapphire Mix) from Takara Bio Inc. (Takara Bio Europe SAS, Saint-Germain-en-Laye, France, Cat. No. RR350A / B), the presence of the desired DNA segment in cells isolated from E. coli or C. glutamicum colonies was confirmed according to the manufacturer's instructions (see Table 4).

[0078] [Table 4]

[0079] d. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany) using the phosphoramidite method described by McBride and Caruthers (1983).

[0080] For the ePCR template, an appropriate diluted solution of either isolated plasmid DNA, total DNA isolated from liquid culture, or total DNA contained in bacterial colonies was used (colony PCR). In the aforementioned colony PCR, the template was prepared by collecting cellular material from colonies on agar plates using a toothpick and directly placing this cellular material into a PCR reaction tube. This cellular material was heated for 10 seconds at 800W in a Mikrowave & Grill type microwave oven from SEVERIN Elektrogeraete GmbH (Sundern, Germany), and the PCR reagent was added to the template in the PCR reaction tube.

[0081] f. All PCR reactions were performed using a Mastercycler or Mastercycler nexus gradient type PCR cycler at Eppendorf AG (Hamburg, Germany).

[0082] Restriction enzyme digestion of DNA For restriction enzyme digestion, we used either FastDigest restriction endonuclease (FD) (ThermoFisher Scientific, Waltham, USA) or restriction endonuclease from New England BioLabs Inc. (Ipswich, USA). The reaction was carried out according to the manufacturer's instructions.

[0083] Measurement of DNA fragment size a. The size of small DNA fragments (<1000 bps) was typically measured by automated capillary electrophoresis using QIAxcel from Qiagen (Hilden, Germany).

[0084] b. When DNA fragments needed to be isolated, or when the DNA fragments were larger than 1000 bps, the DNA was separated by TAE agarose gel electrophoresis and stained using GelRed® nucleic acid gel staining (Biotium, Inc., Fremont, Canada). The stained DNA was visualized at 302 nm.

[0085] Purification of PCR amplifications and restriction fragments PCR amplifications and restriction fragments were cleaned up 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).

[0086] Measurement of DNA concentration DNA concentration was measured using a NanoDrop spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH (VWR brand since 2015 (Erlangen, Germany)).

[0087] Assembly cloning 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 insertion was incubated at 50°C for 60 minutes. 0.5 μl of the assembly mixture was used for each transformation experiment.

[0088] Chemical transformation of E. coli For plasmid cloning, chemically competent "NEB® Stable Competent E. coli (High Efficiency)" (New England BioLabs Inc., Ipswich, USA, Cat. No. C3040) was transformed according to the manufacturer's protocol. Successfully transformed cells were selected on LB agar medium supplemented with 25 mg / l kanamycin.

[0089] Transformation of C. glutamicum Plasmid-DNA transformation of C. glutamicum was performed by electroporation using a "Gene Pulser Xcell" (Bio-Rad Laboratories GmbH, Feldkirchen, Germany), as described by Ruan et al. (2015). Electroporation was performed in a 1 mm electroporation cuvette (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) at a fixed time constant of 1.8 kV and 5 ms. Transformed cells were selected on BHI agar containing 134 g / l sorbitol, 2.5 g / l yeast extract, and 25 mg / l kanamycin.

[0090] Determination of nucleotide sequences The nucleotide sequence of the DNA molecule was determined by cycle sequencing using the dideoxy chain termination method developed by Sanger et al. (Proceedings of the National Academy of Sciences USA 74, 5463 - 5467, 1977) at Eurofins Genomics GmbH (Ebersberg, Germany). Clonemanager Professional 9 software from Scientific & Educational Software (Denver, USA) was used for sequence visualization and evaluation.

[0091] Glycerol stocks of Escherichia coli and C. glutamicum strains Glycerol stocks were prepared for the long-term storage of Escherichia coli and C. glutamicum. Selected Escherichia coli were cultured in 10 ml of LB medium supplemented with 2 g / l glucose. Selected C. glutamicum were cultured in 10 ml of 2x concentrated BHI medium supplemented with 2 g / l glucose. 25 mg / l kanamycin was added to the culture medium for plasmid-containing Escherichia coli and C. glutamicum strains. The medium was placed in a 100 ml Erlenmeyer flask with three baffles. Loops of cells collected from colonies were inoculated into this medium. The culture was then incubated at 30°C and 200 rpm for 18 hours. After the incubation period, 1.2 ml of 85% (v / v) sterile glycerol was added to the culture medium. The resulting glycerol-containing cell suspension was then dispensed into 2 ml portions and stored at -80°C.

[0092] GAA production in milliliter-scale cultures GAA production of bacterial strains was evaluated using the milliliter-scale culture system developed by Duetz (2007). For this purpose, 24 deep-well microplates (24-well WDS plates) of EnzyScreen BV (Heemstede, Netherlands, Cat. no. CR1424) were used, with 2.5 ml of culture medium per well.

[0093] The bacterial strains were pre-cultured in 10 ml of seed medium (SM). The medium was placed in a 100 ml Erlenmeyer flask with three baffles. 100 μl of glycerol stock culture solution was inoculated into this, and the mixture was incubated at 30°C and 200 rpm for 24 hours. The composition of the seed medium (SM) is shown in Table 5.

[0094] [Table 5]

[0095] After the aforementioned incubation period, the optical density (OD600) of the pre-culture medium was measured. A sample was taken from the pre-culture medium to obtain the amount required to inoculate 2.5 ml of production medium (PM) so that the OD600 would be 0.1. The sample was then centrifuged (at 8000 g for 1 minute) and the supernatant was discarded. The cells were then resuspended in 100 μl of production medium.

[0096] Main culture was started by inoculating 100 μl of cells, resuspended from pre-culture, 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.

[0097] [Table 6]

[0098] The main culture medium was incubated in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland) at 30°C and 300 rpm for 72 hours until the glucose was completely consumed. The glucose concentration in the suspension was analyzed using a LifeScan (Johnson & Johnson Medical GmbH, Neuss, Germany) OneTouch Vita® blood glucose meter.

[0099] After culturing, the culture suspension was transferred to a deep-well microplate. A portion of the culture suspension was appropriately diluted to measure the OD660. Another portion of the culture was centrifuged, and the GAA concentration in the supernatant was analyzed as described below.

[0100] Measurement of L-arginine and glycine content in yeast peptone FM902 Since yeast extract FM902 (Angel Yeast Co., LTD, Hubei, PR China) contains various peptides and amino acids, its L-arginine and glycine content was measured as follows.

[0101] To measure free amino acids, a sample was prepared by dissolving 1 g of yeast extract in 20 ml of water. Water was added to this solution to make a total volume of 25 ml, and after thorough mixing, the solution was filtered using a 0.2 μM nylon syringe filter.

[0102] To measure total amino acids (free amino acids + amino acids bound to peptides), 1 g of yeast extract was dissolved in 10 ml of 6 M HCl and incubated at 110°C for 24 hours to prepare the sample. Water was then added to make a total volume of 25 ml. This solution was thoroughly mixed and filtered using a 0.2 μM nylon syringe filter.

[0103] The concentrations of L-arginine and glycine in the samples were measured by ion-exchange chromatography using a SYKAM S433 amino acid analyzer from SYKAM Vertriebs GmbH (Fuerstenfeldbruck, Germany). A column equipped with a SYKAM spherical polystyrene-based cation exchanger (Peek LCA N04 / Na, dimensions 150 × 4.6 mm) was used as the solid phase. Depending on the type of L-amino acid, separation was performed by isocratic elution using a mixture of buffers A and B, or by gradient elution using the aforementioned buffers. Buffer A was an aqueous solution containing 263 g of trisodium citrate, 120 g of citric acid, 1100 ml of methanol, 100 ml of 37% HCl, and 2 ml of octanoic acid in 20 L (final pH 3.5). Buffer B was an aqueous solution containing 392 g of trisodium citrate, 100 g of boric acid, and 2 ml of octanoic acid in 20 L (final pH 10.2). Free amino acids were stained with ninhydrin after derivatization following column chromatography and detected by photometer at 570 nm.

[0104] Table 7 shows the free and total L-arginine and glycine content measured in yeast extract FM902 ((Angel Yeast Co., LTD, Hubei, PR China)) and the resulting amounts in the production medium (PM).

[0105] [Table 7]

[0106] Quantitative determination of GAA The samples were analyzed using an Agilent analytical system consisting of an HPLC "Infinity 1260" and a mass spectrometer "Triple Quad 6420" (Agilent Technologies Inc., Santa Clara, USA). Chromatographic separation was performed at 35°C using 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 at 100% B and then linearly gradientd to 66% B at a constant flow rate of 0.6 mL / min for 22 minutes. The mass spectrometer was operated in ESI positive ionization mode. To detect GAA, the m / z value was monitored using the MRM fragment [M+H]+118-76. The limit of quantification (LOQ) for GAA was fixed at 7 ppm.

[0107] B) Experimental results Example 1: Cloning of the GGT1 gene encoding glyoxylate aminotransferase from Arabidopsis thaliana. The Arabidopsis thaliana gene GGT1 (Genbank accession number NM_102180, SEQ ID NO:1) encodes glutamate:glyoxylate aminotransferase (Genbank accession number NP_564192, SEQ ID NO:2). This protein has been found to catalyze the reactions glyoxylate + L-alanine = glycine + pyruvate (EC2.6.1.44), 2-oxoglutarate + L-alanine = L-glutamate + pyruvate (EC2.6.1.2), and 2-oxoglutarate + glycine = glyoxylate + L-glutamate (EC2.6.1.4; Liepman AH, Olsen LJ., Plant Physiol. 2003 Jan;131(1):215-27. doi: 10.1104 / pp.011460).

[0108] The amino acid sequence of the GGT1 protein was retranslated into a DNA sequence optimized for C. glutamicum codon use using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA). The Shine-Dalgano sequence was added directly upstream of the open reading frame (AGGAAAGGAGAGGATTG; Shi F, Luan M, Li Y, AMB Express. 2018 Apr 18;8(1):61. doi: 10.1186 / s13568-018-0595-2), and the ends of the resulting sequence were extended with motifs for subsequent subcloning. The obtained DNA sequence AtGGT1_opt_RBS (SEQ ID NO:3) was ordered from Eurofins Genomics GmbH (Ebersberg, Germany) for gene synthesis and provided as part of a cloning plasmid conferring resistance to ampicillin (denoted as pEX-A258-AtGGT1_opt_RBS).

[0109] Example 2: Cloning of the GGT2 gene encoding glyoxylate aminotransferase from Arabidopsis thaliana. The Arabidopsis thaliana gene AOAT2 (synonym: GGT2) (Genbank accession number NM_001036185, SEQ ID NO: 4) encodes alanine-2-oxoglutarate aminotransferase 2 (Genbank accession number NP_001031262, SEQ ID NO: 5). This protein was found to catalyze the reactions glyoxylic acid + L-alanine = glycine + pyruvate (EC2.6.1.44), 2-oxoglutaric acid + L-alanine = L-glutamic acid + pyruvate (EC2.6.1.2), and 2-oxoglutaric acid + glycine = glyoxylic acid + L-glutamic acid (EC2.6.1.4; Liepman AH, Olsen LJ. (2003), Plant Physiol. 2003 Jan;131(1):215-27. doi: 10.1104 / pp.011460).

[0110] The amino acid sequence of the GGT2 protein was retranslated into a DNA sequence optimized for C. glutamicum codon use using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA). The Shine-Dalgano sequence was added directly upstream of the open reading frame (AGGAAAGGAGAGGATTG; Shi, 2018), and the ends of the resulting sequence were extended with motifs for subsequent subcloning. The resulting DNA sequence AtGGT2_opt_RBS (SEQ ID NO: 6) was ordered from Eurofins Genomics GmbH (Ebersberg, Germany) for gene synthesis and provided as part of a cloning plasmid conferring resistance to ampicillin (denoted as pEX-A258-AtGGT2_opt_RBS).

[0111] Example 3: Cloning of the gene agt, which encodes glyoxylate aminotransferase from the hyperthermophilic archaeon (Thermococcus litoralis). The gene agt (Genbank accession number AB033996, SEQ ID NO:7) of a hyperthermophilic archaeon encodes alanine:glyoxylate aminotransferase (Genbank accession number BAB40321, SEQ ID NO:8). This protein has been found to catalyze the reactions glyoxylate + L-alanine = glycine + pyruvate (EC2.6.1.44) and glyoxylate + L-serine = glycine + 3-hydroxypyruvic acid (EC2.6.1.45;Sakuraba, H. et al., J Bacteriol. 2004 Aug; 186(16): 5513-5518. doi: 10.1128 / JB.186.16.5513-5518.2004).

[0112] The amino acid sequence of the Agt protein was retranslated into a DNA sequence optimized for C. glutamicum codon use using the software tool "Codon Optimization Tool" (Integrated DNA Technologies Inc., Coralville, Iowa, USA). The Shine-Dalgano sequence was added directly upstream of the open reading frame (AGGAAAGGAGAGGATTG; Shi F, Luan M, Li Y, AMB Express. 2018 Apr 18;8(1):61. doi: 10.1186 / s13568-018-0595-2), and the ends of the resulting sequence were extended with motifs for subsequent subcloning. The resulting DNA sequence (SEQ ID NO: 9) was ordered from Eurofins Genomics GmbH (Ebersberg, Germany) for gene synthesis and provided as part of a cloning plasmid conferring resistance to ampicillin (denoted as pEX-A258-AGT_Tl_opt_RBS).

[0113] Example 4: Cloning of the gene AGAT_Mp encoding L-arginine:glycine amidinotransferase (AGAT, EC2.1.4.1) from Moorea producens. Moorea producens is a filamentous cyanobacterium. The genome of Moorea producens 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 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 that putatively encodes L-arginine:glycine amidinotransferase (AGAT, EC2.1.4.1; locus_tag BJP34_00300, shown in SEQ ID NO:10). SEQ ID NO:11 indicates the originating amino acid sequence (Genbank accession number WP_070390602).

[0114] Using the software tool "Codon Optimization Tool" (Geneart / Thermo Fisher Scientific, Waltham, USA), this amino acid sequence was retranslated into a DNA sequence optimized for C. glutamicum codon usage. Its ends were extended with a sequence for assembly cloning, and a Shine-Dalgano sequence (AGGA) was added 5 base pairs upstream of the open reading frame. The resulting DNA sequence (SEQ ID NO: 12) was ordered from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) for gene synthesis and provided as part of a cloning plasmid (denoted as pMA-T_AGAT_Mp).

[0115] Example 5: Cloning of AGAT_Mp into the expression plasmid pEC-XK99E The Escherichia coli-C. glutamicum shuttle plasmid pEC-XK99E (Genbank accession number AY219682) was digested using the restriction endonuclease SmaI. Terminal phosphates were removed using FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, USA). Subsequently, the DNA was purified using the QIA quick PCR Purification Kit (Qiagen GmbH, Hilden, Germany).

[0116] The cloning plasmid pMA-T_AGAT_Mp was digested with MluI + AatII, and the resulting fragments were blunted using the Fast DNA End Repair Kit (Thermo Fisher Scientific, Waltham, USA). These fragments were separated by agarose gel electrophoresis (0.8% agarose in TAE buffer), and the band corresponding to "AGAT_Mp" (1174 bp) was cleaved. The DNA was purified using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany).

[0117] The AGAT_Mp fragment and linearized pEC-XK99E were ligated using "Ready-To-Go T4 DNA ligase" (GE Healthcare Europe GmbH, Freiburg, Germany). The ligation product was transformed into "NEB Stable Competent E. coli (High Efficiency)" (New England Biolabs, Ipswich, USA), and the cells were grown on LB agar medium containing 25 mg / l kanamycin. Appropriate clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmid was named pEC-XK99E_AGAT_Mp.

[0118] Example 6: Cloning of the glyoxylate aminotransferase gene into the expression plasmid pEC-XK99E_AGAT_Mp Plasmid pEC-XK99E_AGAT_Mp was digested using the restriction endonuclease BamHI, and terminal phosphate groups were removed using FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, USA). The digested DNA was then purified using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany).

[0119] The cloning plasmids pEX-A258-AtGGT1_opt_RBS, pEX-A258-AtGGT2_opt_RBS, and pEX-A258-AGT_Tl_opt_RBS were digested with BamHI and BsaI, respectively. The cleaved plasmids were purified using the "QIAquick Gel Extraction Kit" (Qiagen GmbH, Hilden, Germany).

[0120] The digested pEC-XK99E_AGAT_Mp was ligated with each digested cloning plasmid using "Ready-To-Go T4 DNA ligase" (GE Healthcare Europe GmbH, Freiburg, Germany). The ligation products were transformed into "NEB Stable Competent E. coli (High Efficiency)" (New England Biolabs, Ipswich, USA), and the cells were grown on LB agar medium containing 25 mg / l kanamycin. Appropriate clones were identified by restriction enzyme digestion and DNA sequencing. The obtained plasmids are shown in Table 8. These provide the AGAT_Mp gene and their respective glyoxylate aminotransferases in operon-like structures under the control of a strong IPTG-inducible trc promoter.

[0121] [Table 8]

[0122] Example 7: Chromosomal insertion of the sod promoter upstream of the carAB operon in ATCC13032 To enhance L-arginine production, a strong sod promoter was inserted into the ATCC13032 genome upstream of the carAB operon. Therefore, the plasmid pK18mobsacB_Psod-carAB was constructed as follows: pK18mobsacB (Schaefer, A. et al., Gene. 1994 Jul 22;145(1):69-73. doi: 10.1016 / 0378-1119(94)90324-7) was digested with EcoRI+HindIII, and the linearized vector DNA (5670 bps) from the agarose gel was digested. DNA was extracted using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0123] To construct the insert, three DNA fragments were generated by PCR using the following primer pairs (using the genomic DNA of ATCC13032 as a template): PsodcarAB-LA-F(SEQ ID NO:13)+PsodcarAB-LA-R(SEQ ID NO:14) =Left homology arm (1025bps) PsodcarAB-F(SEQ ID NO:15)+PsodcarAB-R(SEQ ID NO:16) = sod-promoter(250bps) PsodcarAB-RA-F(SEQ ID NO:17)+PsodcarAB-RA-R(SEQ ID NO:18) =Right homology arm (944bps) Product DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany). The linearized plasmid and PCR product were then assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). Appropriate plasmid clones were identified by restriction digestion and DNA sequencing.

[0124] Next, the obtained plasmid pK18mobsacB_Psod-carAB was transformed into ATCC13032 by electroporation. Chromosome integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with 134 g / l sorbitol, 2.5 g / l yeast extract, and 25 mg / l kanamycin. The agar plates were incubated at 33°C for 48 hours.

[0125] Individual colonies were transferred to fresh agar plates (containing 25 mg / l kanamycin) and incubated at 33°C for 24 hours. The liquid cultures of these clones were cultured at 33°C for 24 hours in 10 ml of BHI medium in a 100 ml Erlenmeyer flask with three baffles. To isolate clones that had experienced a second recombination event, aliquots were taken from each liquid culture, appropriately diluted, and plated onto BHI agar supplemented with 10% sucrose (typically 100-200 μl). These agar plates were incubated at 33°C for 48 hours. Colonies growing on sucrose-containing agar plates were then examined for kanamycin sensitivity. To do this, cytoplasm was removed from the colonies using a toothpick and transferred to BHI agar containing 25 mg / l kanamycin and BHI agar containing 10% sucrose. The agar plates were incubated at 33°C for 60 hours. Clones that were sensitive to kanamycin and resistant to saccharose were examined by PCR and DNA sequencing to determine whether the sod promoter was properly incorporated. The resulting strain was named ATCC13032_Psod-carAB.

[0126] Example 8: Chromosomal deletion of gene aceB (NCgl2247) in C. glutamicum ATCC13032_Psod-carAB To reduce the metabolic flux from glyoxylic acid to L-malate, the gene aceB (NCgl2247), which encodes malate synthase (EC2.3.3.9), was deleted in the ATCC13032_Psod-carAB strain.

[0127] Therefore, plasmid pK18mobsacB_DaceB was constructed as follows: Plasmid pK18mobsacB (Schaefer, 1994) was cleaved with XbaI, and the linearized vector DNA (5721 bps) was purified using the "QIAquick Gel Extraction Kit" (Qiagen GmbH, Hilden, Germany).

[0128] To construct the insert, two DNA fragments were generated by PCR using the following primer pair (using the genomic DNA of ATCC13032 as a template): 1f-aceB-D2_vec(SEQ ID NO:19)+1r-aceB-D2_aceB(SEQ ID NO:20) =Left homology arm (1065bps) 2f-aceB-D2_aceB(SEQ ID NO:21)+2r-aceB_D2_Vec(SEQ ID NO:22) =Left homology arm (1080bps) Product DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany). The linearized plasmid and PCR product were then assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). The resulting deletion vector was named pK18mobsacB_DaceB. It was confirmed by restriction enzyme digestion and DNA sequencing.

[0129] To delete the aceB gene, pK18mobsacB_DaceB was transformed into ATCC13032_Psod-carAB by electroporation. Chromosome integration (resulting from the first recombination event) was selected by plating on BHI agar medium supplemented with 134 g / l sorbitol, 2.5 g / l yeast extract, and 25 mg / l kanamycin. The agar plates were incubated at 33°C for 48 hours.

[0130] Individual colonies were transferred to fresh agar plates (containing 25 mg / l kanamycin) and incubated at 33°C for 24 hours. The liquid cultures of these clones were cultured at 33°C for 24 hours in 10 ml of BHI medium in a 100 ml Erlenmeyer flask with three baffles. To isolate clones that had experienced a second recombination event, aliquots were taken from each liquid culture, appropriately diluted, and plated onto BHI agar supplemented with 10% sucrose (typically 100-200 μl). These agar plates were incubated at 33°C for 48 hours. Colonies grown on sucrose-containing agar plates were then examined for kanamycin sensitivity. To do this, cytoplasm was removed from the colonies using a toothpick and transferred to BHI agar containing 25 mg / l kanamycin and BHI agar containing 10% sucrose. The agar plates were incubated at 33°C for 60 hours. Clones that were sensitive to kanamycin and resistant to saccharose were examined by PCR and DNA sequencing to determine whether the sod promoter was properly incorporated. The resulting strain was named ATCC13032_DaceB.

[0131] [Table 9]

[0132] Example 9: Transformation of C. glutamicum strain using various expression plasmids The following C. glutamicum strains were transformed with plasmids by electroporation (Table 10). Cells containing the plasmids were selected with 25 mg / l kanamycin.

[0133] • C. glutamicum ATCC13032: Commonly used wild-type strain (Kinoshita et al., J. Gen. Appl. Microbiol. 1957; 3(3): 193-205) • C. glutamicum ATCC13032_Psod-carAB: Enhanced L-arginine production capacity in ATCC13032 through chromosomal integration of a strong sod promoter upstream of carAB. • C. glutamicum ATCC13032_Psod-carAB_DaceB: In ATCC13032, chromosomal deletion of the aceB gene leads to decreased malate synthase activity and improved L-arginine production capacity due to chromosomal integration of a strong sod promoter upstream of carAB.

[0134] [Table 10]

[0135] Example 10: Effect of increased glyoxylate aminotransferase activity on GAA production To evaluate the effect of increased glyoxylate aminotransferase enzymatic activity on GAA production, strains ATCC13032 / pEC-XK99E, ATCC13032 / pEC-XK99E_AGAT_Mp, ATCC13032 / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032 / pEC-XK99E_AGAT_Mp_AtGGT2, and ATCC13032 / pEC-XK99E_AGAT_Mp_AGT_Tl were cultured in a Wouter Duetz system, and the resulting GAA titers were measured. The production medium (PM) contained 40 g / l D-glucose and 1.90 g / L L-arginine, but glycine was not added.

[0136] [Table 11]

[0137] As shown in Table 11, the ATCC13032 / pEC-XK99E strain did not produce any detectable amount of GAA.

[0138] The ATCC13032 / pEC-XK99E_AGAT_Mp strain possesses a polynucleotide encoding AGAT derived from Moorea producens, which provides AGAT enzyme activity. This strain produced 122 mg / L of GAA.

[0139] The ATCC13032 / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032 / pEC-XK99E_AGAT_Mp_AtGGT2, and ATCC13032 / pEC-XK99E_AGAT_Mp_AGT_Tl strains exhibit increased AGAT enzyme activity and glyoxylate aminotransferase enzyme activity. These strains produced 246 mg / l, 255 mg / l, and 199 mg / l of GAA, respectively.

[0140] We concluded that increasing the enzymatic activity of glyoxylate aminotransferase in the presence of AGAT enzyme activity improves GAA production.

[0141] Example 11: The effect of combining increased glyoxylate aminotransferase activity and improved L-arginine production capacity on GAA production. To evaluate the effect of a combination of increased glyoxylate aminotransferase activity and improved L-arginine production capacity on GAA production, ATCC13032 / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032 / pEC-XK99E_AGAT_Mp_AtGGT2, ATCC13032 / pEC-XK99E_AGAT_Mp_AGT_Tl, ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AtGGT2, and ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AGT_Tl were cultured in a Wouter Duetz system, and the resulting GAA titers were measured. By inserting a strong sod promoter upstream of the chromosomal genes carA and carB, the latter three strains exhibit improved L-arginine production capacity. The production medium (PM) contains 40 g / l D-glucose and 1.90 g / L L-arginine, but without the addition of glycine.

[0142] [Table 12]

[0143] The ATCC13032 / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032 / pEC-XK99E_AGAT_Mp_AtGGT2, and ATCC13032 / pEC-XK99E_AGAT_Mp_AGT_Tl strains exhibit increased enzyme activity of AGAT enzyme activity and glyoxylate aminotransferase. As shown in Table 12, they produced 246 mg / l, 255 mg / l, and 199 mg / l of GAA, respectively.

[0144] Furthermore, the ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AtGGT2, and ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AGT_Tl strains also possess increased AGAT enzyme activity and glyoxylate aminotransferase enzyme activity. In addition, they exhibit improved L-arginine production capacity. These strains produced 325 mg / l, 322 mg / l, and 316 mg / l of GAA, respectively.

[0145] We concluded that the presence of AGAT enzyme activity leads to increased glyoxylate aminotransferase activity and improved L-arginine production capacity, thereby enhancing GAA production.

[0146] Example 12: Combined effects of increased glyoxylate aminotransferase activity, decreased malate synthase activity, and improved L-arginine production capacity on GAA production. To evaluate the combined effects of increased glyoxylate aminotransferase activity, decreased malate synthase activity, and improved L-arginine production capacity on GAA production, we used strains ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AtGGT2, and ATCC13032_Psod-carA The B / pEC-XK99E_AGAT_Mp_AGT_Tl strain, ATCC13032_Psod-carAB_DaceB / pEC-XK99E_AGAT_Mp_AtGGT1 strain, ATCC13032_Psod-carAB_DaceB / pEC-XK99E_AGAT_Mp_AtGGT2 strain, and ATCC13032_Psod-carAB_DaceB / pEC-XK99E_AGAT_Mp_AGT_Tl strain were cultured in a Wouter Duetz system, and the resulting GAA titers were measured. Due to the deletion of the aceB gene, the latter three strains have reduced malate synthase activity. The production medium (PM) contained 40 g / l D-glucose and 1.90 g / L L-arginine, but no glycine was added.

[0147] [Table 13]

[0148] The strains ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AtGGT2, and ATCC13032_Psod-carAB / pEC-XK99E_AGAT_Mp_AGT_Tl exhibit increased AGAT enzyme activity, enhanced glyoxylate aminotransferase enzyme activity, and improved L-arginine production capacity. As shown in Table 13, these strains produced 325 mg / l, 322 mg / l, and 316 mg / l of GAA, respectively.

[0149] The ATCC13032_Psod-carAB_DaceB / pEC-XK99E_AGAT_Mp_AtGGT1, ATCC13032_Psod-carAB_DaceB / pEC-XK99E_AGAT_Mp_AtGGT2, and ATCC13032_Psod-carAB_DaceB / pEC-XK99E_AGAT_Mp_AGT_Tl strains also exhibit increased AGAT enzyme activity, increased glyoxylate aminotransferase enzyme activity, and improved L-arginine production capacity. Furthermore, they possess reduced malate synthase enzyme activity. These strains produced 362 mg / l, 354 mg / l, and 331 mg / l of GAA, respectively.

[0150] We concluded that GAA production is enhanced by a combination of increased enzymatic AGAT activity, increased glyoxylate aminotransferase activity, decreased malate synthase activity, and improved L-arginine production capacity.

Claims

1. A microorganism comprising at least one gene encoding a protein having the function of L-arginine:glycine amidinotransferase and at least one gene encoding a protein having the function of glyoxylate aminotransferase, wherein the microorganism is Corynebacterium glutamicum, the enzymatic activity of the at least one protein having the function of glyoxylate aminotransferase is increased compared to the respective enzymatic activity in the wild-type microorganism, the microorganism has an improved L-arginine production capacity compared to the capacity of the wild-type microorganism, and the protein having the function of L-arginine:glycine amidinotransferase contains an amino acid sequence that is at least 90% identical to the amino acid sequence according to SEQ ID NO:

11.

2. The microorganism according to claim 1, wherein the microorganism has the activity of an enzyme having the function of carbamoyl phosphate synthase, and the activity is increased compared to the activity of each enzyme in the wild-type microorganism.

3. The microorganism according to claim 1 or 2, wherein the microorganism further comprises an enzyme having the function of ornithine carbamoyltransferase, and its activity is increased compared to the activity of each enzyme in the wild-type microorganism.

4. The microorganism according to any one of claims 1 to 3, wherein the microorganism further comprises an enzyme having the function of argininosuccinate synthetase, the activity of which is increased compared to the activity of each enzyme in the wild-type microorganism.

5. The microorganism according to any one of claims 1 to 4, wherein the increase in the activity of the enzyme is achieved by overexpressing the gene encoding each enzyme.

6. A microorganism according to any one of claims 1 to 5, wherein the arginine operon (argCJBDFR) is overexpressed.

7. The microorganism according to any one of claims 1 to 6, 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 wild-type microorganisms, or the argR gene is deleted.

8. The microorganism according to any one of claims 1 to 5 or 7, 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.

9. The microorganism according to any one of claims 1 to 8, wherein the gene encoding the protein having the function of L-arginine:glycine amidinotransferase is heterogeneous.

10. The microorganism according to any one of claims 1 to 9, wherein the gene encoding a protein having the function of L-arginine:glycine amidinotransferase is overexpressed.

11. The microorganism according to any one of claims 1 to 10, wherein the protein having the enzymatic activity of glyoxylate aminotransferase comprises the amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO:

8.

12. A method for fermenting and producing guanidinoacetic acid (GAA), comprising: a) culturing a microorganism according to any one of claims 1 to 11 in a suitable medium under suitable conditions; and b) accumulating GAA in the medium to produce a GAA-containing fermentation broth.

13. The method according to claim 12, further comprising the step of isolating GAA from the GAA-containing fermentation broth.

14. The method according to claim 12 or 13, further comprising the step of drying and / or granulating the GAA-containing fermentation broth.

15. The microorganism according to any one of claims 1 to 11, further comprising a gene encoding an enzyme having guanidinoacetate-N-methyltransferase activity.

16. The microorganism according to claim 15, wherein the gene encoding the enzyme having the activity of guanidinoacetate-N-methyltransferase is overexpressed.

17. A method for fermenting and producing creatine, comprising: a) culturing a microorganism according to claim 15 or 16 in a suitable medium under suitable conditions; and b) accumulating creatine in the medium to produce a creatine-containing fermentation broth.

18. The method according to claim 17, further comprising the step of isolating creatine from the creatine-containing fermented broth.

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