Methods for the fermentative production of guanidinoacetic acid using microorganisms containing heterologous L-threonine 3-dehydrogenase genes (tdh) and glycine C-acetyltransferase genes (kbl)

Abstract

The present invention relates to a microorganism containing at least one heterologous gene encoding L-arginine:glycine amidinotransferase (AGAT), at least one heterologous L-threonine 3-dehydrogenase gene (tdh), and at least one heterologous glycine C-acetyltransferase (2-amino-3-oxobutanoate coenzyme A ligase) gene (kbl), as well as a method for the fermentative production of guanidinoacetic acid (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 containing at least one heterologous gene encoding L-arginine:glycine amidinotransferase (AGAT), at least one heterologous L-threonine 3-dehydrogenase gene (tdh), and at least one heterologous glycine C-acetyltransferase (2-amino-3-oxobutanoate coenzyme A ligase) gene (kbl), as well as a method for the fermentative production of guanidinoacetic acid (GAA) using such a microorganism. The present invention also relates to a method for the fermentative production of creatine.

[0002] GAA is a colorless crystalline organic compound used as an animal feed additive (e.g., International Publication No. 2005120246 and US Patent No. 2011257075). GAA is the natural precursor of creatine (e.g., Humm et al., Biochem. J. (1997) 322, 771-776). Therefore, GAA supplementation can provide optimal creatine supply in organisms.

[0003] In biological systems, GAA and ornithine are formed from arginine and glycine as starting materials by the catalysis of L-arginine:glycine-amidinotransferase (AGAT; EC 2.1.4.1). The reaction is shown in Figure 1. This reaction is also the first step in creatine biosynthesis.

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

[0005] A microorganism capable of producing guanidinoacetic acid (GAA) was published by Zhang et al. (ACS Synth. Biol. 2020, 9, 2066-275). They engineered a reconstituted ornithine cycle in Escherichia coli by introducing heterologous AGATs from various species (e.g., Homo sapiens, Cylindrospermopsis raciborskii, Moorea producens) and by introducing citrulline synthesis modules (e.g., overexpression of carAB, argF, and argI) and arginine synthesis modules (e.g., overexpression of argG and argH; introduction of aspA) into E. coli.

[0006] Schneider and Jankowitsch (WO 2021122400) proposed a method for producing GAA using a microorganism with a gene encoding a protein with L-arginine:glycine amidinotransferase function and increased carbamoyl phosphate synthase activity. Carbamoyl phosphate is an important precursor not only for GAA biosynthesis but also for L-arginine and other compounds. Furthermore, Wang et al. (Applied Microbiology and Biotechnology, 2021, vol. 105, pp. 3265-3276; https: / / doi.org / 10.1007 / s00253-021-11242-w) specifically highlighted that carbamoyl phosphate is essential for L-arginine production in Corynebacterium species.

[0007] To increase GAA production using microorganisms, large amounts of the starting materials arginine and / or glycine are required intracellularly.

[0008] One approach to increasing the intracellular L-arginine concentration is to prevent L-arginine transport, as proposed by Jankowitsch et al. (WO 2022243116). To achieve this, these authors disclose a microorganism essentially comprising a heterologous AGAT (EC 2.1.4.1) gene, an overexpressed carAB operon, a deletion of the argR regulatory gene, and inactivation or deletion of genes encoding proteins with arginine exporter function, as well as inactivation or deletion of genes encoding the respective translational activators.

[0009] Glyoxylate can be used as a starting material to form glycine in the presence of an amino donor, such as an amino acid, and glyoxylate transaminase. Glyoxylate transaminase catalyzes the transfer of an amino group from an amino acid to glyoxylate. The product of this transfer is glycine and the corresponding α-keto acid. To increase the intracellular glycine concentration, Schneider and Jankowitsch (WO 2022008280) proposed providing a microorganism with a glyoxylate aminotransferase gene in addition to the AGAT gene, and demonstrated that the combination of increased glyoxylate aminotransferase activity and increased ability to produce L-arginine in the presence of enzymatic AGAT activity improved GAA production.

[0010] Glycine can also be produced from threonine via two different pathways (Figure 2). The first pathway is catalyzed by low-specificity L-threonine aldolase [EC 4.1.2.48] / L-threonine aldolase ([EC 4.1.2.5], Lta), an enzyme that directly cleaves L-threonine into glycine and acetaldehyde. The second pathway is a two-step process. In the first step, L-threonine is reacted with 2-amino-3-ketobutyrate (2-amino-3-ketobutanoate), catalyzed by L-threonine 3-dehydrogenase (EC 1.1.1.103, also known as threonine dehydrogenase (Tdh)). In the second step, 2-amino-3-ketobutyrate is cleaved into glycine and acetyl-CoA by the action of glycine C-acetyltransferase (EC 2.3.1.19) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase, or 2-amino-3-oxobutanoate coenzyme A ligase, Kbl).

[0011] Smirnov and Kotliarova (WO 2019130723) propose the use of a second pathway and disclose a method for producing glycine by fermenting bacteria modified to overexpress a gene encoding a protein having L-threonine 3-dehydrogenase activity (tdh) and a gene encoding a protein having 2-amino-3-oxobutanoate coenzyme A ligase activity (kbl).

[0012] It is an object of the present invention to provide microorganisms that have been transformed to be capable of producing GAA and that have additional methods for providing glycine as a starting material for GAA biosynthesis, and processes for producing GAA using such microorganisms.

[0013] The problem is solved by a microorganism having an increased ability to produce L-arginine from L-ornithine compared to the ability of a wild-type or unmodified microorganism, comprising at least one heterologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase, at least one heterologous gene encoding a protein having L-threonine 3-dehydrogenase activity (EC 1.1.1.103), and at least one heterologous gene encoding a protein having glycine C-acetyltransferase activity (EC 2.3.1.29).

[0014] A heterologous gene means that the gene is inserted into a host organism that does not naturally possess this gene. The insertion of a heterologous gene into a host is achieved by recombinant DNA technology. Microorganisms that have been subjected to recombinant DNA technology are called transgenic, genetically modified, or recombinant. A heterologous protein means a protein that does not naturally occur in the microorganism.

[0015] 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) amidinotransferase and inosamine (EC:2.1.4.2) amidinotransferase, 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 following reaction: [ka]

[0016] Streptococcal antitumor glycoproteins are also found in this family. Enzymes or proteins with L-arginine:glycine-amidinotransferase (AGAT) activity have also been described as having a conserved domain belonging to the PFAM family of amidinotransferases (PF02274) (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)), as described in the following publications (Marchler-Bauer A 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)). al. (2017), “CDD / SPARCLE: functional classification of proteins via subfamily domain architectures.”, Nucleic Acids Res.45(D1):D200-D203.).

[0017] In the context of the present invention, a microorganism with an increased ability to produce L-arginine refers to a microorganism that produces L-arginine beyond its own needs. Examples of such L-arginine-producing microorganisms include 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).

[0018] In the microorganism according to the present invention, the increased ability to produce L-arginine from L-ornithine compared to the ability of a wild-type or unmodified microorganism can be achieved by inactivating or deleting a gene encoding the arginine-responsive repressor protein ArgR, and / or by overexpressing a gene encoding an enzyme having the function of carbamoylphosphate synthase (EC 6.3.4.16, CarAB), and / or by overexpressing at least one or more genes selected from the group consisting of a gene encoding a protein having the function of ornithine carbamoyltransferase (EC 2.1.3.3) (e.g., argF / argF2 / argI), a gene encoding a protein having the function of argininosuccinate synthetase (EC 6.3.4.5) (e.g., argG), and a gene encoding a protein having the function of argininosuccinate lyase (EC 4.3.2.1) (e.g., argH).

[0019] To achieve a relatively high intracellular L-arginine concentration, it is necessary to prevent L-arginine transport. The amino acid exporter LysE efficiently transports the substrate arginine out of the cell, counteracting the intracellular arginine concentration and reducing substrate availability. In addition, citrulline from arginine biosynthesis is also secreted into the medium by the active LysE exporter. LysE is regulated by the transcriptional activator LysG (Bellmann, A., et al. (2001). "Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum." Microbiology (Reading) 147(Pt 7):1765-1774).

[0020] In a particular embodiment of the present invention, in the microorganism of the present invention, the gene encoding a protein having the function of an arginine exporter, and optionally the gene encoding its transcriptional activator, are inactivated or deleted.

[0021] In a further specific embodiment of the present invention, in the microorganism of the present invention, the lysEG gene / operon, which encodes a protein having the function of the arginine exporter LysE and which encodes its transcriptional activator LysG, is inactivated or deleted.

[0022] In the microorganism of the present invention, the gene encoding a protein having L-threonine 3-dehydrogenase (Tdh) activity may further be overexpressed.

[0023] In the microorganism of the present invention, a gene encoding a protein having glycine C-acetyltransferase (Kbl) activity may also be overexpressed.

[0024] Overexpression of a gene is generally achieved by increasing the copy number of the gene, and / or by operably linking the gene to a strong promoter, and / or by enhancing the ribosome binding site, and / or by codon usage optimization of the start codon or the entire gene, or by selection, or a combination including all of the above methods.

[0025] In particular, gene overexpression can be achieved by increasing the copy number of the gene and / or by enhancing regulatory factors, for example, by operably linking the gene to a strong promoter, enhancing the ribosome binding site, and / or optimizing the codon usage of the start codon or 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 the structural gene to increase promoter effectiveness, or by completely replacing the promoter with a more effective or so-called strong promoter. A promoter is located upstream of a gene. A promoter is a DNA sequence consisting of approximately 40 to 50 base pairs that constitutes a binding site for RNA polymerase holoenzyme and a transcription start site, thereby affecting the intensity of expression of the controlled polynucleotide or gene. Generally, as taught for example by M. Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for C. glutamicum, 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 promoter (originally assigned to another gene), or by modifying specific regions of a given native promoter (for example, its so-called -10 and -35 regions) toward consensus sequences. An example of a "strong" promoter is the superoxide dismutase (sod) promoter ("Psod"; Z. Wang et al., Eng. Life Sci. 2015, 15, 73-82). "Operative linkage" is understood to mean the contiguous arrangement of a promoter with a gene, resulting in transcription of the gene.

[0026] The genetic code is degenerate, which means that a particular amino acid can be coded by several different triplets. The term codon usage refers to the observation that a particular organism typically does not use all possible codons for a particular amino acid with the same frequency. Instead, organisms typically exhibit certain preferences for certain codons, which means that these codons are more frequently found in the coding sequences of the organism's transcribed genes. When a particular gene that is foreign to a future host, i.e., derived from a different species, is to be expressed in a future host organism, the coding sequence of the gene should be adjusted to the codon usage of the future host organism (i.e., codon usage optimization).

[0027] The microorganism of the present invention may belong to the genus Corynebacterium, preferably Corynebacterium glutamicum (C. glutamicum), or the genus Enterobacteriaceae, preferably Escherichia coli (E. coli), or the genus Pseudomonas, preferably Pseudomonas putida (P. putida).

[0028] In Corynebacterium glutamicum, the gene encoding the protein with arginine exporter function is lysE, and the gene encoding the transcriptional activator is lysG. In Escherichia coli, the gene encoding the protein with arginine exporter function is argO (ybjE). In Pseudomonas putida, the protein with arginine exporter function is lysE.

[0029] A protein having the function of L-arginine:glycine amidinotransferase (AGAT) in a microorganism of the invention may comprise an amino acid sequence that is at least 80% identical, preferably at least 90% identical to the amino acid sequence according to SEQ ID NO: 44. In a further embodiment of the invention, the amino acid sequence of the L-arginine:glycine amidinotransferase is identical to the amino acid sequence according to SEQ ID NO: 44.

[0030] A protein having L-threonine 3-dehydrogenase activity in a microorganism of the present invention may comprise an amino acid sequence that is at least 50-80% identical to the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In a further embodiment, a protein having L-threonine 3-dehydrogenase activity in a microorganism of the present invention may comprise an amino acid sequence that is identical to one of these sequences.

[0031] A protein having glycine C-acetyltransferase activity in a microorganism of the invention may comprise an amino acid sequence that is at least 50-80% identical to the amino acid sequence according to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. In a further embodiment, a protein having glycine C-acetyltransferase activity in a microorganism of the invention may comprise an amino acid sequence that is identical to one of these sequences.

[0032] The above-mentioned problem is further solved by a method for the fermentative production of guanidinoacetic acid (GAA), comprising the steps of culturing a microorganism according to the invention as defined above in a suitable medium and allowing GAA to accumulate in the medium to form a GAA-containing fermentation broth.

[0033] The method of the present invention may further comprise the step of isolating GAA from the fermentation broth.

[0034] The method according to the present invention may further comprise the step of drying and / or granulating the GAA-containing fermentation broth.

[0035] The present invention further relates to a microorganism as 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 an enzyme having the activity of guanidinoacetate N-methyltransferase is overexpressed.

[0036] The present invention also relates to a method for the fermentative production of creatine, comprising the steps of culturing in a suitable medium a microorganism according to the present invention, which comprises a gene encoding an enzyme having the activity of guanidinoacetate N-methyltransferase, and allowing creatine to accumulate in the medium to form a creatine-containing fermentation broth.

[0037] Preferably, the method further comprises 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. [Brief explanation of the drawings]

[0038] [Figure 1] Reaction of L-arginine and glycine catalyzed by L-arginine:glycine-amidinotransferase (AGAT; EC 2.1.4.1) to give L-ornithine and GAA. [Figure 2] Schematic diagram of two different pathways from L-threonine to glycine. Abbreviations: Tdh - L-threonine 3-dehydrogenase [EC 1.1.1.103], Kbl - glycine C-acetyltransferase [EC 2.3.1.19] (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase), Lta - low-specificity L-threonine aldolase [EC 4.1.2.48] or L-threonine aldolase [4.1.2.5].

[0039] A brief description of arrays SEQ ID NO: 1: Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from Aliivibrio fischeri (Tdh_Afi) (Example 1). SEQ ID NO: 2: Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from Bdellovibrio species (Tdh_Bds) (Example 1). SEQ ID NO: 3: Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from Bos taurus (Tdh(*S51M)_Bot) (Example 1). SEQ ID NO: 4. Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from Caulobacter sp. 17J65-9 (Tdh_Cs17) (Example 1). SEQ ID NO: 5. Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from Cupriavidus necator H16 (Tdh_Re:H16) (Example 1). SEQ ID NO: 6: Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from Corynebacterium xerosis (Tdh_Cx) (Example 1). SEQ ID NO: 7: Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from Chlamydia species 32-24 (Tdh_Ch32) (Example 1). SEQ ID NO: 8: Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from a Cyanobacteria bacterial isolate (MAG with accession number JAIEMW010000004.1) (Tdh_Cyb) (Example 1). SEQ ID NO: 9: Amino acid sequence of L-threonine 3-dehydrogenase (Tdh; EC 1.1.1.103) from Escherichia coli K12 (Tdh_Ec) (Example 1). SEQ ID NO: 10: Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) from Aliivibrio fischeri (Kbl_Afi) (Example 1). SEQ ID NO: 11 Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) from Bdellovibrio sp. (Kbl_Bds) (Example 1). SEQ ID NO: 12 Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) from Bos taurus (Kbl(*Q21M)_Bot) (Example 1). SEQ ID NO: 13. Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) from Caulobacter sp. 17J65-9 (Kbl_Cs17) (Example 1). SEQ ID NO: 14. Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) from Capricornis necator H16 (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) (Kbl_Re:H16) (Example 1). SEQ ID NO: 15: Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) from Corynebacterium xerosie (Kbl_Cx) (Example 1). SEQ ID NO: 16: Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) from Chlamydia species 32-24 (Kbl_Ch32) (Example 1). SEQ ID NO: 17: Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) from a cyanobacterial bacterial isolate (MAG with accession number JAIEMW010000004.1) (Kbl_Cyb) (Example 1). SEQ ID NO: 18. Amino acid sequence of glycine C-acetyltransferase (Kbl, EC 2.3.1.29) (alternative name: 2-amino-3-ketobutyrate coenzyme A ligase) from Escherichia coli K12 (Kbl_Ec) (Example 1). SEQ ID NO: 19 DNA sequence of plasmid pXMJ19[kbl_Afi tdh_Afi] (Example 1). SEQ ID NO: 20 DNA sequence of plasmid pXMJ19[tdh_Bds kbl_Bds] (Example 1). SEQ ID NO: 21 DNA sequence of plasmid pXMJ19 [kbl(*Q21M)_Bot tdh(*S51M)_Bot] (Example 1). SEQ ID NO: 22 DNA sequence of plasmid pXMJ19[kbl_Cs17 tdh_Cs17] (Example 1). SEQ ID NO: 23 DNA sequence of plasmid pXMJ19[kbl_Re:H16 tdh_Re:H16] (Example 1). SEQ ID NO: 24 DNA sequence of plasmid pXMJ19[tdh_Cx kbl_Cx] (Example 1). SEQ ID NO: 25 DNA sequence of plasmid pXMJ19[kbl_Ch32 tdh_Ch32] (Example 1). SEQ ID NO: 26 DNA sequence of plasmid pXMJ19[tdh_Cyb kbl_Cyb] (Example 1). SEQ ID NO: 27 DNA sequence of plasmid pXMJ19[kbl_Ec tdh_Ec] (Example 1). SEQ ID NO: 28 DNA sequence of synthetic oligonucleotide primer DargR_lf (Example 3). SEQ ID NO: 29 DNA sequence of synthetic oligonucleotide primer DargR_lr (Example 3). SEQ ID NO: 30 DNA sequence of synthetic oligonucleotide primer DargR_rf (Example 3). SEQ ID NO: 31 DNA sequence of synthetic oligonucleotide primer DargR_rr (Example 3). SEQ ID NO: 32 DNA sequence of plasmid pK18mobsacB_IBcg0054::Pg3-argFGH (Example 3). SEQ ID NO: 33 Synthetic DNA fragment for the Pg3-argFGH operon (Example 3). SEQ ID NO: 34: DNA sequence of synthetic oligonucleotide primer argFGH_f (Example 3). SEQ ID NO: 35: DNA sequence of synthetic oligonucleotide primer argFGH_r (Example 3). SEQ ID NO: 36 DNA sequence of plasmid pK19mobsacB-ΔlysEG (Example 3). SEQ ID NO: 37 DNA sequence of synthetic oligonucleotide primer PsodcarAB-LA-F (Example 3). SEQ ID NO: 38 DNA sequence of synthetic oligonucleotide primer PsodcarAB-LA-R (Example 3). SEQ ID NO: 39 DNA sequence of synthetic oligonucleotide primer PsodcarAB-F (Example 3). SEQ ID NO: 40 DNA sequence of synthetic oligonucleotide primer PsodcarAB-R (Example 3). SEQ ID NO: 41 DNA sequence of synthetic oligonucleotide primer PsodcarAB-RA-F (Example 3). SEQ ID NO: 42 DNA sequence of synthetic oligonucleotide primer PsodcarAB-RA-R (Example 3). SEQ ID NO: 43: DNA sequence of Moorea producens gene with locus_tag BJP34_00300, which encodes L-arginine:glycine amidinotransferase (Example 3). SEQ ID NO: 44. Amino acid sequence of Moorea producens L-arginine:glycine amidinotransferase (NCBI accession number WP_070390602) (Example 3). SEQ ID NO: 45 DNA sequence of plasmid pNP-6-36 (Example 3). SEQ ID NO: 46 DNA sequence of plasmid pLF338 (Example 3). SEQ ID NO: 47: DNA sequence of synthetic oligonucleotide primer MW_21_80_fw (Example 3). SEQ ID NO: 48: DNA sequence of synthetic oligonucleotide primer MW_21_81_rv (Example 3). SEQ ID NO: 49: DNA sequence of synthetic oligonucleotide primer MW_21_82_fw (Example 3). SEQ ID NO: 50 DNA sequence of synthetic oligonucleotide primer MW_21_83_rv (Example 3). SEQ ID NO: 51: DNA sequence of synthetic oligonucleotide primer MW_21_84_fw (Example 3). SEQ ID NO: 52 DNA sequence of synthetic oligonucleotide primer MW_21_85_rv (Example 3).

[0040] Experimental Section material and method chemicals Unless explicitly stated, all chemicals were ordered in analytical grade from Merck, Sigma Aldrich or Carl-Roth.

[0041] molecular biology technology All oligonucleotide primers were synthesized by Eurofins Genomics Germany GmbH (Ebersberg, Germany). Genomic DNA from C. glutamicum ATCC 13032 was isolated according to the manufacturer's instructions for the DNeasy Blood & Tissue Kits (Qiagen, catalog no. 69504). DNA digestion was routinely performed using either High-Fidelity (HF®) restriction enzymes (NEB) or FastDigest Restriction Enzymes (FD; Thermo Fischer Scientific) according to the manufacturer's instructions. Polymerase chain reaction (PCR) was used to amplify the desired DNA region using specific DNA oligos. Q5® High-Fidelity 2X Master Mix with proofreading activity (NEB catalog no. M0492) and Phusion® High-Fidelity DNA Polymerase (NEB catalog no. M0530) were used according to the manufacturer's instructions. PCR and restriction fragment cleanup was performed using a QIAquick PCR Purification Kit (Qiagen, Catalog No. 28106) according to the manufacturer's instructions. If necessary, DNA fragments were purified from agarose gels using a QIAquick Gel Extraction Kit (Qiagen, Catalog No. 28706) according to the manufacturer's instructions. Agarose gel electrophoresis was performed using 0.8-1.2% agarose (Roth, Catalog No. 3810.4) dissolved in 1X TAE buffer (Roth, Catalog No. CL83.3). Gels were cast using Roti® Gelstain (Roth, Catalog No. 3865.1), and electrophoresis itself was performed at 150V for 25-40 minutes, if necessary. DNA assembly of the backbone and insert for plasmid cloning was performed using NEBuilder® HiFi DNA Assembly Master Mix (NEB, Catalog No. E2621). After DNA assembly, the reaction was transformed into E. coli competent cells to obtain individual clones (see DNA transformation).Correct assembly was verified for the presence of the insert by either colony PCR or restriction analysis, and sequence integrity was confirmed by DNA sequencing. The presence of the desired DNA segment from transformed cells was confirmed using Taq DNA Polymerase (Qiagen, Cat. No. 201203) or SapphireAmp® Fast PCR Master Mix (Takara, Cat. No. RR350) according to the manufacturer's instructions. Plasmid DNA was routinely isolated after cloning using one of the following kits according to the manufacturer's instructions: QIAprep Spin Miniprep Kit (Qiagen, Cat. No. 27106), HiSpeed ​​Plasmid Kits (Qiagen, Cat. No. 12643). DNA sequencing was performed by either Eurofins Genomics Germany GmbH (Ebersberg, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

[0042] Strains and culture conditions The type strain / wild-type Corynebacterium glutamicum ATCC13032 (Kinoshita S, Udaka S, Shimono M., J. Gen. Appl. Microbiol. 1957;3(3):193-205) is commercially available at the American Type Culture Collection (ATCC) or at the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH under the deposit number DSM 20300.

[0043] E. coli W3110 (ATCC 27325, K12 W3110) is commercially available, for example, at the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH under the deposit number DSM 5911.

[0044] Corynebacterium glutamicum ATCC 13032 and its derivatives were routinely grown at 30 °C in Brain Heart Infusion (BHI; Merck Millipore, catalog no. 1104930500) broth or in CgXII minimal medium (Keilhauer et al., 1993) supplemented with 10 g / L glucose, 1 g / L L-arginine, and 0.5 g / L L-threonine, as specified. The exact composition of CgXII minimal medium without a carbon source is detailed in Table 1. The genome sequence of C. glutamicum ATCC 13032 is available under NCBI accession number NC_003450. Glycerol stocks of C. glutamicum were made by mixing 900 μL of an overnight culture in BHI medium (30°C, 200 rpm, 10 mL of medium) with 600 μL of sterile 86% (w / v) glycerol solution and then stored at -80°C. Cultures in 96-well deep-well plates (DWP) (Axygen, catalog number P-2ML-SQ-CS) for glycerol stocks were made by inoculating 1.8 mL of medium with a single colony. The plates were then covered with a gas-permeable membrane (M2P Labs, catalog number F-GP-10) and incubated overnight at 30°C with shaking at 1200 rpm in a Heidolph incubator 1000. Glycerol stocks were made by mixing 900 μL of overnight culture with 900 μL of 60% (v / v) glycerol, sealing the plates with aluminum sealing tape (Corning Costar, product number 6569), and storing them at -80°C. Unless otherwise indicated, E. coli strains were routinely grown at 37°C in Lysogeny Broth (LB) medium (Sigma, catalog number L3022-1KG) or SOB medium (Roth, catalog number AE27.1). Solid medium was supplemented with 1.5% (w / v) agar. Antibiotics were used for selection, as needed, at the following concentrations: chloramphenicol—34 mg / L for E. coli and 7.5 mg / L for C. glutamicum; kanamycin—25–50 mg / L for E. coli and 15–25 mg / L for C. glutamicum.Induction of genes from pXMJ19 was carried out by adding a final concentration of 0.1 or 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to the medium at the start of the culture, as described.

[0045] [Table 1]

[0046] DNA transformation Commercially available E. coli chemical or electrocompetent cells were used for cloning and amplification of plasmid DNA (NEB, catalog numbers C3019, C3020, C3040) according to the manufacturer's instructions. After a recovery period, cells were spread onto LB agar plates containing the appropriate antibiotic for selection.

[0047] E. coli W3110 cells were made chemically competent and transformed as follows: a single colony from the plate was used to inoculate 10 mL of SOB and grown overnight at 37 °C and 200 rpm as a preculture. A 1:100 dilution in fresh SOB medium was inoculated as the main culture, reaching an OD of 0.5–0.9. 600The culture was then cooled on ice for 15 min and aliquoted into Falcon tubes for centrifugation (15 min, 4 °C, 1100 g). The supernatant was discarded, and the cells were resuspended in 2 × 20 mL of ice-cold TfBI (30 mM potassium acetate, 50 mM MnCl2, 100 mM KCl, 10 mM CaCl2, 15% (v / v) glycerol) followed by centrifugation (15 min, 4 °C, 1100 g). The supernatant was then discarded, and the cell pellet was resuspended in a total of 5 mL of ice-cold TfBI (10 mM MOPS-KOH pH 7.0, 75 mM CaCl2, 10 mM KCl, 15% (v / v) glycerol). The cell suspension was incubated on ice for 20 min, after which 100 μL aliquots were made. Chemically competent cells were stored at -80 °C. For transformation of E. coli W3110 competent cells, an aliquot was thawed on ice for 5-15 minutes, then DNA was added and mixed gently. The mixture was further incubated on ice for 10 minutes. A heat shock was performed at 42°C for 45 seconds. Immediately after the heat shock, the cells were placed on ice. The cells were then recovered in 700 μL of SOB medium with incubation at 30°C for 1 hour. After the recovery period, the cells were spread onto appropriate selective agar plates and incubated at 30°C to obtain single colonies.

[0048] A modified protocol was used to make C. glutamicum strains electrocompetent: 100 mL cultures of BHI + 0.5 M sorbitol were grown at 33 °C and 250 rpm from overnight precultures to an OD of 0.3. 600 Inoculate to an OD of 1.75 600The cells were incubated at 33°C and 130 rpm until a 50% saturation was reached. The culture was transferred to a 50 mL Falcon tube and collected by centrifugation (3400 g, 10 min, 4°C). The cell pellets were combined and washed twice with 50 mL of ice-cold Tris-glycerol buffer (1 mM Tris-HCl pH 7.5, 10% (v / v) glycerol), followed by two washing steps with 50 mL of ice-cold 10% (v / v) glycerol. The final cell pellet was resuspended in 800 μL of ice-cold 10% (v / v) glycerol. Electrocompetent cells were aliquoted in 150 μL aliquots and stored at -80°C. For electroporation, an aliquot of cells was thawed on ice and DNA was added. The cell-DNA mixture was transferred to a 2 mm electroporation cuvette (Sigma-Aldrich, catalog no. Z706088-50EA), and a 2.5 kV, 200 Ω, 25 μF pulse with a 5 ms time constant was triggered using a Gene Pulser Xcell™ (Bio-Rad). The cuvette was briefly placed on ice, and the contents were transferred to 4 mL of BHI (preheated to 46°C), followed by a 6-minute heat shock at 46°C. Cells were allowed to recover in BHI medium at 30°C and 200 rpm for 50 minutes. After recovery, cells were spread onto BHI agar plates containing the appropriate antibiotic for selection and incubated at 30°C for 2–3 days to obtain single-cell colonies. Correct maintenance of replicative plasmids after transformation was verified by plasmid DNA isolation using a QIAprep Spin Miniprep Kit (Qiagen, catalog no. 27106X4) according to the manufacturer's instructions, except that cells were resuspended in buffer P1 and incubated at 37°C for 2 hours before lysis. The isolated plasmid DNA was then verified by restriction analysis.

[0049] Allelic exchange in C. glutamicum Allelic replacement for both gene deletion and gene integration at defined loci was performed using pK18mobsacB (NCBI accession number: FJ437239) (Schafer et al., 1994) derivative plasmids containing upstream and downstream homologous regions and, in the case of integration, the desired insert. The appropriate plasmids were transformed into C. glutamicum strains by electroporation as previously described. After electroporation, the first recombination event resulting in an intermediate strain was selected on BHI agar plates containing kanamycin. The intermediate strains were screened for single-crossover events by colony PCR. Positive intermediate strains were then counterselected on antibiotic-free BHI + 10% (w / v) sucrose agar plates to promote a second recombination event. After counterselection, sucrose-resistant and kanamycin-sensitive colonies were screened by colony PCR and DNA sequencing using appropriate primers outside the homologous regions to confirm that the desired allelic exchange had occurred.

[0050] Cultivation of E. coli and C. glutamicum strains for glycine production as described in Example 2 Precultures were performed in 96-well DWPs filled with 1.7 mL of the corresponding medium (SOB + 20 mM glucose + chloramphenicol (34 mg / L) for E. coli W3110 derivatives, and BHI + chloramphenicol (7.5 mg / L) for C. glutamicum derivatives). Each well was inoculated from a glycerol stock of the appropriate strain. The plates were then covered with a gas-permeable membrane (M2P Labs, catalog no. F-GP-10) and incubated overnight at 30 °C with shaking at 1200 rpm in a Heidolph incubator 1000. Fresh DWPs were pre-filled with 1.45 mL of production medium for E. coli W3110 derivative strains (see Table 2) and 1.5 mL of CgXII medium + 2% glucose + 2 g / L L-threonine + 34 mg / L chloramphenicol. The basic components of CgIII medium are detailed in Table 1, except that a concentration of 30 mg / L of protocatechuic acid was used instead of 2.5 g / L. Pre-filled DWPs were inoculated with 250 μL of preculture for E. coli and 200 μL of preculture for C. glutamicum strains to a starting OD of approximately 0.3. 600 After inoculation, the DWP was covered with a gas-permeable membrane and incubated in a Heidolph incubator 1000 at 30°C and 1200 rpm. For both E. coli and C. glutamicum strains, gene expression was induced with 0.5 mM IPTG after 5.5 hours of incubation. After 50 hours, OD 600 was measured spectrophotometrically, the cultures were centrifuged (2000 g, 15 min, 20°C), and the supernatants were used to measure GAA production.

[0051] [Table 2]

[0052] Cultivation of C. glutamicum strains for GAA production as described in Example 4 The appropriate C. glutamicum strain was used to inoculate a preculture from a glycerol stock. The preculture was grown in 10 mL of BHI medium containing the appropriate antibiotics, as needed, in a 100 mL baffled Erlenmeyer flask and incubated at 30°C and 200 rpm for 24 h. The preculture was harvested by centrifugation (3100 g, 10 min, room temperature (RT)) and washed with 5 mL of CgXII medium without a carbon source (see Table 1). The washed pellet was resuspended in 2.5 mL of CgXII medium without a carbon source, and the OD of the cell suspension was measured using an Ultraspec 2100 pro (Amersham Biosciences) spectrophotometer. 600 An appropriate volume of washed cells was used to culture in CgXII medium supplemented with 10 g / L glucose, 1 g / L L-arginine, and 0.5 g / L L-threonine, with appropriate antibiotics as needed, to a starting OD of 0.5 as the main culture. 600 The culture was inoculated to 0.1 μL / well. The genes from the pXMJ19-derived plasmid were induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to the production medium at the start of the culture. The main culture was transferred to a 48-well multi-layer plate (Beckman Coulter Life Sciences, Cat. No. M2P-MTP-48-BOH1) using 800 μL / well, and the plate was covered with sealing foil (Beckman Coulter Life Sciences / m2p Labs, Cat. No. F-GPR48-10). The covered plate was placed in a BioLector® I (Beckman Coulter Life Sciences / m2p Labs) and incubated at 1400 rpm and 30°C for 48 hours. After 48 hours, the OD 600 was measured spectrophotometrically, the cultures were centrifuged (2000 g, 10 min), and the supernatants were used to measure GAA production.

[0053] Cultivation of C. glutamicum strains for GAA production as described in Example 5 The appropriate C. glutamicum strain was used to inoculate a preculture from a glycerol stock. The preculture was grown in 10 mL of BHI medium containing the appropriate antibiotics, as needed, in a 100 mL baffled Erlenmeyer flask and incubated at 30°C and 200 rpm for 24 h. The preculture was harvested by centrifugation (3100 g, 10 min, room temperature (RT)) and washed with 5 mL of CgXII medium without a carbon source (see Table 1). The washed pellet was resuspended in 2.5 mL of CgXII medium without a carbon source, and the OD of the cell suspension was measured using an Ultraspec 2100 pro (Amersham Biosciences) spectrophotometer. 600 An appropriate volume of washed cells was used to culture in CgXII medium supplemented with 8 g / L L-threonine and 2 g / L L-arginine, with appropriate antibiotics as needed, to a starting OD of 0.5 as the main culture. 600 The medium was inoculated to 0.5% CO₂. L-threonine was added instead of glucose as the main carbon source in the medium. At the start of the culture, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the production medium as a final concentration to induce genes from the pXMJ19-derived plasmid. The main culture was transferred to a 48-well multi-layer plate (Beckman Coulter Life Sciences, Catalog No. M2P-MTP-48-BOH1) using 800 μL / well, and the plate was covered with sealing foil (Beckman Coulter Life Sciences / m2p Labs, Catalog No. F-GPR48-10). The covered plate was placed in a BioLector® I (Beckman Coulter Life Sciences / m2p Labs) and incubated at 1400 rpm and 30°C for 48 hours. After 48 hours, the OD 600 was measured spectrophotometrically, the cultures were centrifuged (2000 g, 15 min, 14°C), and the supernatants were used to measure GAA production.

[0054] Quantification of GAA GAA from culture supernatants was quantified by HPLC-UV using a Dionex Ultimate 3000 System (Thermo Scientific). Samples were filtered using a Sartorius Minisart NML Plus 0.2 μm filter (Sartorius AG, catalog number ST17823-K) and then appropriately diluted with mobile phase A (see below). Samples (10 μL injection volume) were separated at 35°C using a HyperCarb 100x4.6 mm 7 μm column (Thermo Scientific catalog number 35007-104630). Mobile phase A consisted of 2.3 g of ammonium dihydrogen phosphate and 2.6 g of diammonium hydrogen phosphate dissolved in 2 L of purified water. Mobile phase B consisted of 2.3 g of ammonium dihydrogen phosphate and 2.6 g of diammonium hydrogen phosphate dissolved in 1.25 L of purified water and 0.75 L of acetonitrile. A 1.0 mL min. was used during the run. -1 The flow rate was maintained constant. The column was pre-equilibrated with 100% mobile phase A, and a gradient between phases A and B was used: 0–8 min—linear gradient of phase B increasing from 0 to 10%; 8–10 min—linear gradient increasing from 10 to 40% B; 10–11 min—40% B; 11.1–13 min—0% B; and 13–14 min—constant at 0% B to re-equilibrate the column. The total run time was 14 min. Under these conditions, the retention time of GAA was 5.8 min. GAA was detected at 200 nm (210 nm as reference) using a UV detector (ThermoScientific Dionex Ultimate 3000 DAD).

[0055] Experimental results Example 1: Cloning of the tdh-kbl operon for expression in C. glutamicum and E. coli Glycine is biosynthetically produced by cells in a two-step process using L-threonine 3-dehydrogenase (EC 1.1.1.103), also known as threonine dehydrogenase (Tdh), followed by glycine C-acetyltransferase (EC 2.3.1.29), which converts threonine to glycine and acetyl-CoA, alternatively known as 2-amino-3-ketobutyrate coenzyme A ligase (Kbl).

[0056] The threonine dehydrogenase gene product is encoded by the tdh gene, and the glycine C-acetyltransferase gene product is encoded by the kbl gene. These genes are naturally encoded as an operon in organisms. Although the gene order of the operon may vary depending on the biological source, for simplicity, we refer to them collectively as the tdh-kbl operon. We selected a total of nine Tdh and Kbl proteins from Aliivibrio fischeri, Bdellovibrio species, Bos taurus, Caulobacter species 17J65-9, Capriavidus necator H16, Corynebacterium xerosus, a cyanobacterial bacterial isolate (MAG with accession number JAIEMW010000004.1), and Escherichia coli K12 (see Tables 3 and 4 for details). The B. taurus proteins were organized into a synthetic operon by adding a ribosome-binding sequence (RBS) upstream of each gene. For the B. taurus Tdh and Kbl proteins, truncated versions lacking the (putative) mitochondrial signaling peptide, designated Tdh(*S51M)_Bot (SEQ ID NO: 3) and Kbl(*Q21M)_Bot (SEQ ID NO: 13), were used.

[0057] [Table 3]

[0058] [Table 4]

[0059] [Table 5]

[0060] Example 2: Effect of Tdh-Kbl activity on glycine production from L-threonine A wild-type strain of E. coli was used as a platform for glycine production from L-threonine by overexpression of the tdh-kbl operon, which results in enhanced activity of L-threonine 3-dehydrogenase (EC 1.1.1.103) and glycine C-acetyltransferase (EC 2.3.1.29). E. coli W3110 (available as DSM 5911 and ATCC 27325) was transformed with the plasmids listed in Table 5 and the empty vector pXJM19 as a reference. These strains were then tested for glycine production in minimal medium supplemented with L-threonine, as described in the Materials and Methods section. As shown in Table 6, introduction of the Tdh and Kbl proteins was sufficient to increase glycine production compared to the starting strain.

[0061] [Table 6]

[0062] Table 6 shows that E. coli W3110 strains containing various Tdh and Kbl proteins produced 10 to 110 mg / L of glycine from L-threonine, whereas no glycine was detected in the reference strain W3110+pXJM19. Therefore, we conclude that introducing or enhancing the activity of Tdh and Kbl proteins can improve glycine production in microorganisms such as E. coli.

[0063] Example 3: Chromosomal expression of AGAT in C. glutamicum and construction of LF-S-677a for increased L-arginine availability 3.1 Chromosomal deletion of the argR gene in C. glutamicum ATCC 13032 derivatives To improve intracellular L-arginine formation from L-ornithine and L-arginine recycling, we inactivated the argR gene, encoding the central repressor protein ArgR, which controls the L-arginine biosynthetic pathway. Inactivation was achieved via allelic replacement with the plasmid pK18mobsacB_DargR.

[0064] pK18mobsacB_DargR was cloned as follows: Backbone: XbaI-linearized pK18mobsacB. Insert: PCR of ATCC 13032 genomic DNA with oligos DargR_lf (SEQ ID NO: 28) and DargR_lr (SEQ ID NO: 29), PCR of ATCC 13032 genomic DNA with oligos DargR_rf (SEQ ID NO: 30) and DargR_rr (SEQ ID NO: 31). Assembled via NEBuilder HiFi DNA Assembly.

[0065] 3.2 Chromosomal expression of arginine biosynthetic genes argF, argG, and argH under the control of the strong constitutive promoter Pg3 in C. glutamicum ATCC 13032 derivative strains To enhance the activity of ArgF, ArgG, and ArgH, additional copies of the corresponding genes were inserted into the genome via allelic replacement with the plasmid pK18mobsacB_IBcg0054::Pg3-argFGH (SEQ ID NO: 32).

[0066] pK18mobsacB_IBcg0054::Pg3-argFGH (SEQ ID NO: 32) was cloned as follows: First, we designed a synthetic operon consisting of pg3, argF, argG, argH, and flanking regions for genome integration (SEQ ID NO: 33). The DNA sequence was ordered from Invitrogen / GeneArt (Thermo Fisher Scientific, Waltham, USA) for gene synthesis and delivered as part of a cloning plasmid (designated pMA-RQ_argFGH) carrying an ampicillin resistance gene. Backbone: EcoRI- and HindIII-linearized pK18mobsacB. Insert: PCR of pMA-RQ_argFGH with oligos argFGH_f (SEQ ID NO: 34) and argFGH_r (SEQ ID NO: 35). Assembled using NEBuilder HiFi DNA Assembly.

[0067] 3.3 Chromosomal deletion of the genes lysE and lysG in C. glutamicum ATCC 13032 derivative strains The gene lysE encodes an exporter protein that catalyzes the efflux of L-lysine, L-arginine, and L-citrulline in Corynebacterium glutamicum. Expression of lysE is positively regulated by the gene product of lysG. Both genes are located adjacent to each other but are divergently transcribed. To inactivate the transporter protein LysE and the positive regulatory protein LysG, the plasmid pK19mobsacB-ΔlysEG (SEQ ID NO: 36) was constructed as described for pK18mobsacB-ΔlysEG (Vrljic et al., 1996). Deletion of lysEG was achieved via allelic replacement using the reconstructed pK19mobsacB-ΔlysEG.

[0068] 3.4 Chromosomal insertion of the sod promoter upstream of the carAB operon in C. glutamicum ATCC 13032 derivatives To improve L-arginine production, a strong sod promoter was inserted upstream of the carAB operon in the genome. This was achieved via allelic replacement with the plasmid pK18mobsacB_PsodcarAB.

[0069] pK18mobsacB_PsodcarAB was cloned as follows: Backbone: EcoRI, HindIII-linearized pK18mobsacB. Insert: PCR of ATCC 13032 genomic DNA with oligos PsodcarAB-LA-F (SEQ ID NO: 37) and PsodcarAB-LA-R (SEQ ID NO: 38), PCR of ATCC 13032 genomic DNA with oligos PsodcarAB-F (SEQ ID NO: 39) and PsodcarAB-R (SEQ ID NO: 40), PCR of ATCC 13032 genomic DNA with oligos PsodcarAB-RA-F (SEQ ID NO: 41) and PsodcarAB-RA-R (SEQ ID NO: 42). Assembled via NEBuilder HiFi DNA Assembly.

[0070] 3.5 Chromosomal insertion of the gene AGAT-Mp encoding L-arginine:glycine amidinotransferase (AGAT; EC 2.1.4.1) from Moorea producens A heterologous gene encoding L-arginine:glycine amidinotransferase (AGAT), EC 2.1.4.1, is required for the formation of guanidinoacetic acid (GAA) from L-arginine and glycine. Moorea producens is a filamentous cyanobacterium. The genome of M. producens strain PAL-8-15-08-1 is available under GenBank accession number CP017599.1 (Leao et al., 2017). 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: 43). SEQ ID NO: 44 shows the corresponding amino acid sequence (NCBI accession number WP_070390602). The AGAT-Mp expression cassette was assembled into a plasmid as an intermediate step to facilitate subsequent cloning, resulting in pNP-6-36 (SEQ ID NO: 45). pNP-6-36 contains a promoter sequence, a ribosome binding site, and a codon-optimized version of the AGAT-Mp gene. The corresponding gene product has the same amino acid sequence as shown in SEQ ID NO: 44. The AGAT-Mp expression cassette was integrated into the genome of C. glutamicum at the intergenic region of NCgl0013_NCgl0014 via allelic replacement using pLF338 (SEQ ID NO: 46). pLF338 is a pK18mobsacB derivative that contains the homology region required for integration at the intergenic region of NCgl0013_NCgl0014 and the expression cassette of the AGAT-Mp gene.

[0071] The cloning of pLF388 was carried out in multiple parts: first, pLF337 was constructed as a pK18mobsacB derivative containing homologous regions for integration into the genome.

[0072] pLF337 was cloned as follows: Backbone: EcoRI-linearized pK18mobsacB. Insert: PCR of ATCC 13032 genomic DNA with oligos MW_21_80_fw (SEQ ID NO: 47) and MW_21_81_rv (SEQ ID NO: 48), PCR of ATCC 13032 genomic DNA with oligos MW_21_82_fw (SEQ ID NO: 49) and MW_21_83_rv (SEQ ID NO: 50). Assembled via NEBuilder HiFi DNA Assembly.

[0073] Finally, the AGAT-Mp expression cassette from pNP-6-36 was cloned into pLF337 to obtain pLF338 as a plasmid for chromosomal integration of AGAT in C. glutamicum.

[0074] pLF338 was cloned as follows: Backbone: AscI-linearized pLF337. Insert: PCR of pNP-6-36 with oligos MW_21_84_fw (SEQ ID NO: 51) and MW_21_85_rv (SEQ ID NO: 52). Assembled via NEBuilder HiFi DNA Assembly.

[0075] Example 4: Effect of Tdh-Kbl activity on GAA production from glucose, L-threonine, and L-arginine C. glutamicum strain LF-S-677a (genotype: ATCC 13032 ΔargR IBcg0054::Pg3-argFGH ΔlysEG Psod-carAB intNCgl0013-NCgl0014::{P2947MU}[agat_Mpr]), the construction of which is described in Example 3, served as our base strain. This strain has an increased ability to donate L-arginine (by deletion or attenuation of ArgR and LysEG, and enhancement of ArgF, ArgG, and ArgH and CarAB) and is equipped with a heterologous L-arginine:glycine amidinotransferase (AGAT-Mp). This base strain was further modified by overexpression of the tdh-kbl_Afi operon using pXMJ19[kbl_Afi tdh_Afi] (SEQ ID NO: 19), resulting in enhanced activity of L-threonine 3-dehydrogenase (EC 1.1.1.103) and glycine C-acetyltransferase (EC 2.3.1.29). These strains were tested for GAA production in production minimal medium supplemented with 10 g / L glucose, 1 g / L L-arginine, and 0.5 g / L L-threonine (see the Materials and Methods section for a full description). Surprisingly, as shown in Table 7, introduction of the Tdh and Kbl proteins was sufficient to increase GAA production compared to the starting strain.

[0076] [Table 7]

[0077] Table 7 shows that C. glutamicum LF-S-677a+pXMJ19[kbl_Afi tdh_Afi] with enhanced Tdh and Kbl activities can produce 128 mg / L of GAA from glucose, L-threonine, and L-arginine, compared to only 89 mg / L of GAA in the reference strain LF-S-677a. GAA yield / OD 600was similarly higher in strains with Tdh and Kbl compared to the parent (6.7 vs. 4.6, respectively). We therefore conclude that introduction of Tdh and Kbl activity improves GAA production in strains already modified to have a combination of AGAT activity, increased ability to provide L-arginine, and reduced L-arginine transport.

[0078] Example 5: Effect of Tdh-Kbl activity on GAA production from L-threonine and L-arginine C. glutamicum strain LF-S-677a (genotype: ATCC 13032 ΔargR IBcg0054::Pg3-argFGH ΔlysEG Psod-carAB intNCgl0013-NCgl0014::{P2947MU}[agat_Mpr]), the construction of which is described in Example 3, served as our base strain. This strain has an increased ability to donate L-arginine (by deletion or attenuation of ArgR and LysEG, and enhancement of ArgF, ArgG, and ArgH and CarAB) and is equipped with a heterologous L-arginine:glycine amidinotransferase (AGAT-Mp). This base strain was further modified by overexpression of the tdh-kbl_Afi operon using pXMJ19[kbl_Afi tdh_Afi] (SEQ ID NO: 19), resulting in enhanced activity of L-threonine 3-dehydrogenase (EC 1.1.1.103) and glycine C-acetyltransferase (EC 2.3.1.29). These strains were tested for GAA production in production minimal medium supplemented with 8 g / L L-threonine and 2 g / L L-arginine (see the Materials and Methods section for a complete description). As shown in Table 8, introduction of the Tdh and Kbl proteins was sufficient to increase GAA production compared to the starting strain.

[0079] [Table 8]

[0080] Table 8 shows that C. glutamicum LF-S-677a+pXMJ19[kbl_Afi tdh_Afi] with enhanced Tdh and Kbl activities can produce 68 mg / L of GAA from L-threonine and L-arginine, compared to only 19 mg / L of GAA in the reference strain LF-S-677a. GAA yield / OD 600 was similarly higher in strains with Tdh and Kbl compared to the parent (21.3 vs. 6.3, respectively). We therefore conclude that the introduction of Tdh and Kbl activity improves GAA production in strains already modified to have AGAT activity.

[0081] quotation Jakoby, M., Ngouoto-Nkili, C.-E., & Burkovski, A. (1999). Construction and application of new Corynebacterium glutamicum vectors. Biotechnology Techniques, 13(6), 437-441. https: / / doi.org / 10.1023 / A:1008968419217 Keilhauer, C., Eggeling, L., &Sahm, H. (1993). Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon.J Bacteriol,175(17),5595-5603.https: / / doi.org / 10.1128 / jb.175.17.5595-5603.1993 Leao,T.,Castelao,G.,Korobeynikov,A.,Monroe,E.A.,Podell,S.,Glukhov,E.,Allen,E.E.,Gerwick,W.H.,&Gerwick,L.(2017).Comparative genomics uncovers the prolific and distinctive metabolic potential of the cyanobacterial genus Moorea.Proc Natl Acad Sci U S A,114(12),3198-3203.https: / / doi.org / 10.1073 / pnas.1618556114 Liu,J.Q.,Nagata,S.,Dairi,T.,Misono,H.,Shimizu,S.,&Yamada,H.(1997).The GLY1 gene of Saccharomyces cerevisiae encodes a low-specific L-threonine aldolase that catalyzes cleavage of L-allo-threonine and L-threonine to glycine--expression of the gene in Escherichia coli and purification and characterization of the enzyme.Eur J Biochem,245(2),289-293.https: / / doi.org / 10.1111 / j.1432-1033.1997.00289.x Schafer,A.,Tauch,A.,Jager,W.,Kalinowski,J.,Thierbach,G.,&Puhler,A.(1994).Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene,145(1),69-73.https: / / doi.org / 10.1016 / 0378-1119(94)90324-7 Vrljic,M.,Sahm,H.,&Eggeling,L.(1996).A new type of transporter with a new type of cellular function:L-lysine export from Corynebacterium glutamicum.Mol Microbiol,22(5),815-826.https: / / doi.org / 10.1046 / j.1365-2958.1996.01527.x

Claims

1. A microorganism having an increased ability to produce L-arginine from L-ornithine compared to the ability of a wild-type or unmodified microorganism, comprising at least one heterologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase, at least one heterologous gene encoding a protein having L-threonine 3-dehydrogenase activity, and at least one heterologous gene encoding a protein having glycine C-acetyltransferase activity.

2. 2. The microorganism according to claim 1, wherein the increased ability to produce L-arginine from L-ornithine compared to the ability of the wild-type or unmodified microorganism is achieved by inactivating or deleting a gene encoding the arginine-responsive repressor protein ArgR.

3. 3. The microorganism according to claim 1, wherein the increased ability to produce L-arginine from L-ornithine compared to the ability of the wild-type or unmodified microorganism is achieved by overexpressing a gene encoding an enzyme having the function of carbamoyl phosphate synthase.

4. 4. The microorganism according to claim 1, wherein the increased ability to produce L-arginine from L-ornithine compared to the ability of the wild-type or unmodified microorganism is achieved by overexpressing at least one or more genes selected from the group consisting of a gene encoding a protein having the function of ornithine carbamoyltransferase, a gene encoding a protein having the function of argininosuccinate synthetase, and a gene encoding a protein having the function of argininosuccinate lyase.

5. 5. The microorganism according to claim 1, wherein a gene encoding a protein having arginine exporter function is inactivated or deleted.

6. 6. The microorganism according to claim 1, wherein the gene encoding a protein having L-threonine 3-dehydrogenase activity is overexpressed.

7. 7. The microorganism according to claim 1, wherein the gene encoding a protein having glycine C-acetyltransferase activity is overexpressed.

8. 8. The microorganism according to any one of claims 1 to 7, which belongs to the genus Corynebacterium, Enterobacteriaceae or Pseudomonas.

9. The microorganism according to claim 8, which is Corynebacterium glutamicum.

10. 10. A method for the fermentative production of guanidinoacetic acid (GAA), comprising culturing a microorganism defined in any one of claims 1 to 9 in a medium and allowing GAA to accumulate in the medium to form a GAA-containing fermentation broth.

11. 11. The method of claim 10, further comprising isolating GAA from the GAA-containing fermentation broth.

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

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

14. 14. A method for the fermentative production of creatine, comprising culturing a microorganism as defined in claim 12 or 13, and allowing creatine to accumulate in the culture medium to form a creatine-containing fermentation broth.

15. 15. The method of claim 14, further comprising isolating creatine from the creatine-containing fermentation broth.

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