Production of guanidinoacetic acid (GAA) from serine fermentation by using microorganisms having enhanced l-serine hydroxymethyltransferase activity

Abstract

The present invention provides: a microorganism which has been modified to express a gene encoding a protein having an arginine: glycine amidino transferase (AGAT) activity, and to increase glycine production from L-serine by means of an enhanced L-serine hydroxymethyltransferase activity; and a method for producing guanidinoacetic acid (GAA) by fermenting such a microorganism and a method for producing creatine.

Description

[0001] The present invention provides a microorganism that has been modified to express a gene encoding a protein having arginine:glycine amidinotransferase (AGAT) activity and to increase glycine production from L-serine through enhanced L-serine hydroxymethyltransferase activity; a method for producing guanidine acetic acid (GAA) by fermenting such a microorganism; and a method for producing creatine.

[0002] Guanidinoacetic acid (GAA) is the direct precursor of creatine, which is active in the energy homeostasis of vertebrates. GAA is used in feed formulations to improve feed conversion efficiency, animal health and meat quality. GAA is formed from L-arginine and glycine in vertebrates and occasionally in bacterial metabolism (secondary metabolism). This step is catalyzed by the enzyme arginine:glycine amidinotransferase (AGAT) [EC 2.1.4.1], whereby L-ornithine is produced as a by-product.

[0003]

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

[0005] Microorganisms capable of producing guanidine acetate (GAA) were published by Zhang et al. (ACS Synth. Biol. 2020, 9, 2066-275). They designed a reconstructed ornithine cycle in E. coli by introducing heterologous AGATs from different 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, argH; introduction of aspA) into E. coli.

[0006] Schneider and Jankowitsch (WO2021122400 A1) proposed a method for producing GAA using a microorganism with a gene encoding a protein having the function of L-arginine:glycine amidinotransferase and increased carbamyl phosphate synthase. Carbamyl phosphate is an important precursor for the biosynthesis of GAA but also for L-arginine and other compounds. Further, Wang et al. (Applied Microbiology and Biotechnology, 2021, vol. 105, pp. 3265-3276; https: / / doi.org / 10.1007 / s00253-021-11242-w) particularly emphasized that carbamyl phosphate is also essential for L-arginine production in Corynebacterium sp.

[0007] Jankowitsch et al. (WO2022243116 A1) also disclose, inter alia, a Corynebacterium glutamicum strain for the production of guanidinoacetic acid (GAA) expressing an L-arginine:glycine amidinotransferase (AGAT; EC:2.1.4.1) from productive Moore's algae, lacking lysE, lysG, and argR genes, and overexpressing the carAB operon (which encodes carbamoyl phosphate synthase). The microorganism may also have guanidinoacetic acid N-methyltransferase activity to further produce creatine.

[0008] In order to increase the production of GAA by using microorganisms, high intracellular amounts of the starting materials arginine and / or glycine are necessary.

[0009] Glycine can be produced in cells by organisms from various sources, including L-serine. Glycine can be produced directly from L-serine by an L-serine hydroxymethyltransferase (SHMT) [EC 2.1.2.1] encoded by the glyA gene in Escherichia coli and in Corynebacterium glutamicum. Figure 1 A schematic depiction of the production of glycine from serine is shown.

[0010] There are multiple publications focusing on producing L-serine in various microorganisms. For example, WO2016120326A1 discloses a method for producing L-serine by culturing bacteria, in which the expression of genes encoding polypeptides having serine deaminase activity and genes encoding polypeptides having serine hydroxymethyltransferase activity (i.e., sdaA, sdaB, tdcG, and glyA, respectively) has been weakened. For example, CN109797126 A proposes the use of bacteria with reduced serine O-acetyltransferase (CysE) expression for L-serine production. CN113621638A proposes a method for producing L-serine by fermentation of bacteria with knocked-out genes related to the L-serine degradation pathway (e.g., serine hydroxymethyltransferase glyA, homoserine dehydrogenase thrA, serine dehydratase sdaA, serine dehydratase isozyme sdaB, L-serine transporter sdaC, serine dehydratase isozyme tdcG and threonine dehydrogenase tdcB genes).

[0011] WO2011080301 A2 discloses Escherichia coli strains for producing L-methionine. One of these strains contains a missense mutation in the sdaA gene, resulting in an increase in the availability of serine for L-methionine production. Another of these strains contains an extra copy of the glyA gene. Comparison with strains containing a mutated glyA gene leads to the conclusion that the glyA gene product catalyzes both the conversion of L-serine to glycine and the degradation of L-threonine to glycine.

[0012] An object of the present invention is to provide a microorganism having the ability to produce GAA by using L-serine as a source of glycine, a method for producing GAA by culturing the microorganism, and a method for producing creatine.

[0013] The present invention therefore relates to microorganisms comprising at least one heterologous gene encoding a protein having the function of an L-arginine:glycine amidinotransferase and an overexpressed gene encoding a protein having the enzymatic activity of an L-serine hydroxymethyltransferase (SHMT) [EC 2.1.2.1].

[0014] L-Serine hydroxymethyltransferase (SHMT) [EC 2.1.2.1] is encoded by the glyA gene in Escherichia coli and in Corynebacterium glutamicum.

[0015] Overexpression of the gene encoding the protein having the enzymatic activity of L-serine hydroxymethyltransferase (e.g., glyA) can be achieved by increasing the copy number of the gene, and / or by functionally linking the gene to a strong promoter, and / or by enhancing the ribosome binding site, and / or by optimizing the codon usage of the start codon or the entire gene.

[0016] A heterologous gene means that the gene has been inserted into a host organism that does not naturally possess the gene. Insertion of a heterologous gene into a host is performed by recombinant DNA technology. Microorganisms that have undergone recombinant DNA technology are referred to as transgenic, genetically modified, or recombinant. A heterologous protein means a protein that does not naturally occur in the microorganism. A homologous or endogenous gene means that the gene itself (including its function) or the nucleotide sequence of the gene naturally occurs in the microorganism or is "native" in the microorganism. A homologous or native protein means a protein that naturally occurs in the microorganism.

[0017] Proteins that function as L-arginine:glycine amidinotransferases (AGATs) belong to the amidinotransferase family. The amidinotransferase family includes glycine (EC:2.1.4.1) and inositolamine (EC:2.1.4.2) amidinotransferases, which are enzymes involved in the biosynthesis of creatine and streptomycin, respectively. The family also includes arginine deiminases (EC:3.5.3.6). These enzymes catalyze the following reactions:

[0018]

[0019] 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 following PFAM family: amidinotransferase (PF02274) (Marchler-Bauer A et al., (2017), "CDD / SPARCLE: functional classification of proteins via subfamily domain architectures.", Nucleic Acids Res. 45(D1):D200-D203), also described in the following publications: Pissowotzki K et al., Mol Gen Genet 1991, 231:113-123 (PUBMED:1661369 EPMC:1661369); D'Hooghe I et al., J Bacteriol 1997, 179:7403-7409 (PUBMED:9393705 EPMC:9393705); Kanaoka M et al., Jpn J Cancer Res 1987,78:1409-1414(PUBMED:3123442EPMC:3123442).

[0020] In the microorganism of the present invention, the gene encoding the protein having the function of L-arginine:glycine amidinotransferase may be further overexpressed.

[0021] In some embodiments, the overexpression of a gene can be by increasing the copy number of the gene, and / or by enhancing the regulatory factor, for example, by functionally connecting the gene to a strong promoter, and / or by enhancing the ribosome bind site, and / or by optimizing the codon usage of the start codon or the whole gene, or by realizing by the combination of the selection comprising the aforementioned method. The enhancing of this type of regulatory factor that positively affects gene expression can for example be by being modified in the promoter sequence upstream of the structural gene to increase the efficiency of this promoter or by replacing the promoter fully with more effective or so-called strong promoter to realize.Promoter is located in the upstream of the gene.Promoter is such DNA sequence dna, and it is made up of approximately 40 to 50 base pairs, and has constituted the binding site and the transcription start point about the RNA polymerase holoenzyme, can influence the intensity of the expression of controlled polynucleotide or gene thus. In general, it is possible that by selecting a strong promoter, for example, by replacing the original promoter with a strong, natural (originally assigned to other genes) promoter, or by modifying certain regions of a given, natural promoter (for example, its so-called -10 and -35 districts) towards a consensus sequence to achieve overexpression or expression increase of a gene in bacteria, such as taught by M. Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for Corynebacterium glutamicum. An example of a "strong" promoter is superoxide dismutase (sod) promoter ("Psod"; Z. Wang et al., Eng. Life Sci. 2015, 15, 73-82). "Functionally connected" is understood to mean the sequential arrangement of a promoter and a gene, which results in transcription of the gene.

[0022] The genetic code is degenerate, which means that a certain amino acid can be encoded by many different triplets. The term "codon usage" refers to the observation that a certain organism typically will not use every possible codon for a certain amino acid with the same frequency. Instead, organisms typically will show some preference for specific codons, which means that these codons are found more frequently in the coding sequences of the organism's transcribed genes. If a gene that is foreign to its future host (i.e., 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).

[0023] In the microorganism of the present invention, the sdaA gene, which encodes a protein having the function of L-serine ammonia lyase [EC 4.3.1.17] (SDHL) (also referred to as L-serine deaminase or L-serine dehydratase activity), may also be inactivated or deleted.

[0024] exist Figure 2GAA production starting from serine is shown in .

[0025] Preferably, the microorganism of the present invention has an increased ability to produce L-arginine from L-ornithine compared to the ability of a wild-type microorganism.

[0026] Under the situation of the present invention, the microorganism with the generation L-arginic ability of increase means to exceed itself and need produce the L-arginic microorganism.About the example of such generation L-arginic microorganism be for example Corynebacterium glutamicum ATCC 21831 or by people such as Park (NATURE COMMUNICATIONS|DOI:10.1038 / ncomms5618) or by people such as Ginesy (Microbial Cell Factories (2015) 14:29) disclosed those.

[0027] In the microorganism of the present invention, increased ability to produce L-arginine can be achieved by inactivating or deleting the argR gene encoding the arginine-responsive repressor protein ArgR.

[0028] In the context of the present invention, inactivation of a gene means that the gene is expressed at a low level or is not expressed, or the activity is eliminated or reduced, compared to the parent strain or unmodified strain, although the gene is expressed. In the present invention, inactivation can be achieved by a mutation selected from an insertion mutation (wherein one or more base pairs are inserted into the gene) or a deletion mutation (wherein more than one base pair is deleted in the gene); or by introducing one or more mutations selected from the group consisting of base pair conversion or transversion mutations of nonsense codons into the gene; or by replacing the natural promoter of the gene with a weaker promoter of the gene.

[0029] In a particular embodiment of the present invention, the microorganism according to the invention further comprises at least one overexpressed gene (carAB) encoding an enzyme having the function of carbamoyl-phosphate synthase (EC 6.3.4.16, CarAB).

[0030] The microorganism according to the present invention may preferably further comprise at least one or more overexpressed genes selected from the group consisting of: a gene encoding a protein having the function of ornithine carbamoyltransferase (ArgF / ArgF2, EC 2.1.3.3) (e.g., argF / argF2 / argI), a gene encoding a protein having the function of argininosuccinate synthetase (ArgG, EC6.3.4.5) (e.g., argG), and a gene encoding a protein having the function of argininosuccinate lyase (ArgH, EC4.3.2.1) (e.g., argH).

[0031] Overexpression of a gene is typically achieved by increasing the copy number of the gene, and / or by functionally linking the gene to a strong promoter (e.g., as mentioned above, the superoxide dismutase (sod) promoter ("Psod"; Z. Wang et al., Eng. Life Sci. 2015, 15, 73-82), and / or by enhancing ribosome binding sites, and / or by optimizing the codon usage of the start codon or the entire gene, or by a combination of options including the aforementioned methods.

[0032] In order to obtain a relatively high L-arginine concentration in the cell, it is necessary to prevent L-arginine export. The amino acid export protein LysE hinders the intracellular arginine concentration and reduces substrate availability by effectively transporting the substrate arginine from the cell. In addition, citrulline from arginine biosynthesis is also secreted into the culture medium by the active LysE export protein. 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).

[0033] In a particular embodiment of the present invention, the lysEG gene encoding a protein having the functions of the arginine exporter LysE and its transcriptional activator LysG is inactivated or deleted in the microorganism according to the invention.

[0034] The microorganism of the present invention may belong to the genus Corynebacterium, preferably Corynebacterium glutamicum, or to the family Enterobacteriaceae, preferably Escherichia coli, or to the genus Pseudomonas, preferably Pseudomonas putida.

[0035] In Corynebacterium glutamicum, the gene encoding the protein with the function of arginine exporter is lysE and the gene encoding the transcriptional activator is lysG. In Escherichia coli, the gene encoding the protein with the function of arginine exporter is argO(ybjE). In Pseudomonas putida, the gene encoding the protein with the function of arginine exporter is lysE.

[0036] The protein having the function of L-arginine:glycine amidinotransferase (AGAT) in the microorganism of the present 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: 17. In a further embodiment of the present invention, the amino acid sequence of the L-arginine:glycine amidinotransferase is identical to the amino acid sequence according to SEQ ID NO: 17 of the productive Moorella sp., a filamentous cyanobacterium.

[0037] The above-mentioned problem is further solved by a method for the fermentative production of guanidinoacetic acid (GAA), comprising the steps of culturing the microorganism according to the present invention as defined above in a suitable culture medium, and accumulating GAA in the culture medium to form a fermentation broth containing GAA.

[0038] The method of the present invention may further comprise the step of separating GAA from the fermentation broth.

[0039] The method according to the present invention may further comprise the step of drying and / or granulating the fermentation broth containing GAA.

[0040] The present invention further relates to a microorganism as defined above, further comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase (EC: 2.1.1.2) activity. Preferably, the gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity is overexpressed.

[0041] The present invention also relates to a method for producing creatine by fermentation, comprising the steps of culturing a microorganism according to the present invention comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity in a suitable culture medium, and accumulating creatine in the culture medium to form a fermentation broth containing creatine.

[0042] Preferably, the method further comprises isolating creatine from the fermentation broth comprising creatine. Creatine can be extracted from the fermentation broth by an isoelectric point method and / or an ion exchange method. Alternatively, creatine can be further purified by recrystallization in water. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1: Schematic depiction of the production of glycine from serine via glycine by SHMT. Abbreviations: PGDH – phosphoglycerate dehydrogenase [EC 1.1.1.95], PSAT – phosphoserine aminotransferase [EC 2.6.1.52], PSP – phosphoserine phosphatase [EC 3.1.3.3], SDHL – L-serine ammonia lyase [EC 4.3.1.17], SHMT – serine hydroxymethyltransferase [EC 2.1.2.1], 3P HP – 3-phosphohydroxypyruvate, THF – (6S)-5,6,7,8-tetrahydrofolate, CH2-TFH – (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate.

[0045] Figure 2 : Schematic depiction of the production of GAA from serine via glycine by SHMT and AGAT. Abbreviations: PGDH – phosphoglycerate dehydrogenase [EC 1.1.1.95], PSAT – phosphoserine aminotransferase [EC 2.6.1.52], PSP – phosphoserine phosphatase [EC 3.1.3.3], SDHL – L-serine ammonia lyase [EC 4.3.1.17], SHMT – serine hydroxymethyltransferase [EC 2.1.2.1], AGAT – arginine:glycine amidinotransferase [EC 2.1.4.1], 3P HP – 3-phosphohydroxypyruvate, THF – (6S)-5,6,7,8-tetrahydrofolate, CH2-TFH – (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate.

[0046] Sequence Description

[0047] SEQ ID NO: 1 Synthetic oligonucleotide: DNA sequence of primer DargR_lf (Example 1).

[0048] SEQ ID NO: 2 Synthetic oligonucleotide: DNA sequence of primer DargR_lr (Example 1).

[0049] SEQ ID NO: 3 Synthetic oligonucleotide: DNA sequence of primer DargR_rf (Example 1).

[0050] SEQ ID NO: 4 Synthetic oligonucleotide: DNA sequence of primer DargR_rr (Example 1).

[0051] SEQ ID NO: 5 DNA sequence of plasmid pK18mobsacB_IBcg0054::Pg3-argFGH (Example 1).

[0052] SEQ ID NO: 6 Synthetic DNA fragment for the Pg3-argFGH operon (Example 1).

[0053] SEQ ID NO: 7 Synthetic oligonucleotide: DNA sequence of primer argFGH_f (Example 1).

[0054] SEQ ID NO: 8 Synthetic oligonucleotide: DNA sequence of primer argFGH_r (Example 1).

[0055] SEQ ID NO: 9 DNA sequence of plasmid pK19mobsacB-ΔlysEG (Example 1).

[0056] SEQ ID NO: 10 Synthetic oligonucleotide: DNA sequence of primer PsodcarAB-LA-F (Example 1).

[0057] SEQ ID NO: 11 Synthetic oligonucleotide: DNA sequence of primer PsodcarAB-LA-R (Example 1).

[0058] SEQ ID NO: 12 Synthetic oligonucleotide: DNA sequence of primer PsodcarAB-F (Example 1).

[0059] SEQ ID NO: 13 Synthetic oligonucleotide: DNA sequence of primer PsodcarAB-R (Example 1).

[0060] SEQ ID NO: 14 Synthetic oligonucleotide: DNA sequence of primer PsodcarAB-RA-F (Example 1).

[0061] SEQ ID NO: 15 Synthetic oligonucleotide: DNA sequence of primer PsodcarAB-RA-R (Example 1).

[0062] SEQ ID NO: 16 DNA sequence of a productive Moorella sp. gene with locus_tag BJP34_00300. It encodes L-arginine:glycine amidinotransferase (Example 1).

[0063] SEQ ID NO: 17 Amino acid sequence of L-arginine:glycine amidinotransferase from productive Moorella sp. (NCBI Accession No. WP_070390602) (Example 1).

[0064] SEQ ID NO: 18 DNA sequence of plasmid pLIB_P[AGAT-Mp] (Example 1).

[0065] SEQ ID NO: 19 DNA sequence of plasmid pLF338 (Example 1).

[0066] SEQ ID NO: 20 Synthetic oligonucleotide: DNA sequence of primer MW_21_80_fw (Example 1).

[0067] SEQ ID NO: 21 Synthetic oligonucleotide: DNA sequence of primer MW_21_81_rv (Example 1).

[0068] SEQ ID NO: 22 Synthetic oligonucleotide: DNA sequence of primer MW_21_82_fw (Example 1).

[0069] SEQ ID NO: 23 Synthetic oligonucleotide: DNA sequence of primer MW_21_83_rv (Example 1).

[0070] SEQ ID NO: 24 Synthetic oligonucleotide: DNA sequence of primer MW_21_84_fw (Example 1).

[0071] SEQ ID NO: 25 Synthetic oligonucleotide: DNA sequence of primer MW_21_85_rv (Example 1).

[0072] SEQ ID NO: 26 DNA sequence of plasmid pXMJ19[glyA_Cg] (Example 2).

[0073] SEQ ID NO: 27 Synthetic oligonucleotide: DNA sequence of primer oMC21 (Example 2).

[0074] SEQ ID NO: 28 Synthetic oligonucleotide: DNA sequence of primer oMC22 (Example 2).

[0075] SEQ ID NO: 29 DNA sequence of plasmid pK18mobsacB-sdaA (Example 4).

[0076] SEQ ID NO: 30 Synthetic oligonucleotide: DNA sequence of primer oMC58 (Example 4).

[0077] SEQ ID NO: 31 Synthetic oligonucleotide: DNA sequence of primer oMC59 (Example 4).

[0078] SEQ ID NO: 32 Synthetic oligonucleotide: DNA sequence of primer oMC60 (Example 4).

[0079] SEQ ID NO: 33 Synthetic oligonucleotide: DNA sequence of primer oMC61 (Example 4).

[0080] SEQ ID NO: 34 DNA sequence of plasmid pLIB_P (Example 5).

[0081] SEQ ID NO: 35 DNA sequence of plasmid pLIB_P[AGAT-Mp glyA_Cg] (Example 5).

[0082] SEQ ID NO: 36 Synthetic oligonucleotide: DNA sequence of primer oMC23 (Example 5).

[0083] SEQ ID NO: 37 Synthetic oligonucleotide: DNA sequence of primer oMC24 (Example 5).

[0084] Experimental part

[0085] A) Materials and Methods

[0086] Chemicals

[0087] Unless otherwise stated, all chemicals were ordered as analytical grade from Merck, Sigma Aldrich, or Carl-Roth.

[0088] Molecular biology techniques

[0089] All oligonucleotide primers were synthesized by Eurofins Genomics Germany GmbH (Ebersberg, Germany). The genomic DNA of Corynebacterium glutamicum ATCC 13032 was isolated according to the manufacturer's instructions for DNeasy Blood & Tissue Kits (Qiagen, Catalog # 69504). The genomic DNA of Corynebacterium glutamicum ATCC 13032 was isolated according to the manufacturer's instructions for DNeasy Blood & Tissue Kits (Qiagen, Catalog # 69504). DNA digestion was routinely performed using restriction enzymes (NEB) or FastDigest restriction enzymes (FD; Thermo Fischer Scientific). The desired DNA region was amplified using polymerase chain reaction (PCR) using specific DNA oligos. The manufacturer's instructions were followed for use. High-Fidelity 2X Master Mix (NEB Catalog#M0492) and High-Fidelity DNA polymerase (NEB catalog #M0530) with proofreading activity. PCR and restriction fragments were cleaned up by using the QIAquick PCR Purification Kit (Qiagen, Catalog #28106) following the manufacturer's instructions. When necessary, DNA fragments were purified from agarose gels by using the QIAquick Gel Extraction Kit (Qiagen, Catalog #28706) following the manufacturer's instructions. Agarose gel electrophoresis was performed using 0.8-1.2% agarose (Roth, Catalog #3810.4) dissolved in 1X TAE buffer (Roth, Catalog #CL83.3). Gelstain (Roth, Catalog # 3865.1) was used for gel casting, and electrophoresis itself was performed at 150 V for 25-40 minutes as needed. Backbone and insert DNA assembly for plasmid cloning was performed by using HiFi DNA Assembly Master Mix (NEB, Catalog # E2621) was used. After DNA assembly, the reaction was transformed into E. coli competent cells to obtain individual clones (see DNA transformation). Correct assembly was verified by colony PCR or restriction analysis for the presence of inserts, and sequence integrity was confirmed by DNA sequencing. Taq DNA polymerase (Qiagen, catalog # 201203) or Fast PCR Master Mix (Takara, catalog #RR350) was used to confirm the presence of the desired DNA segment from the transformed cells. Plasmid DNA after cloning was routinely isolated using one of the following kits: QIAprep Spin Miniprep Kit (Qiagen, Catalog #27106), HiSpeed Plasmid Kit (Qiagen, Catalog #12643) following the manufacturer's instructions. DNA sequencing was performed by Eurofins Genomics Germany GmbH (Ebersberg, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

[0090] Strains and culture conditions

[0091] Corynebacterium glutamicum ATCC13032 (Kinoshita S, Udaka S, Shimono M., J. Gen. Appl. Microbiol. 1957, 3(3):193-205), a type strain / wild type of Corynebacterium glutamicum, is commercially available from the American Type Culture Collection (ATCC) or from the German Collection of Microorganisms and Cell Cultures GmbH under the accession number DSM 20300.

[0092] Corynebacterium glutamicum ATCC13032 and derivative thereof are at brain heart infusion (BHI; Merck Millipore, Catalog#1104930500) meat soup or in the CgXII minimal medium (people such as Keilhauer, 1993) that is supplemented with 10g / L glucose, 1g / L L-arginine and 4g / L L-serine, (as specifically indicated) grow routinely under 30 ℃.The accurate composition of the CgXII minimal medium without any carbon source is described in detail in Table 1.The genomic sequence of Corynebacterium glutamicum ATCC13032 can be at NCBI accession number: visit under NC_003450.By preparing the glycerine reserve of Corynebacterium glutamicum with aseptic 86% (w / v) glycerol solution phase mixture of 900 μ L overnight culture in BHI substratum (30 ℃, 200rpm, 10mL substratum) and 600 μ L, then it is stored in-80 ℃. Escherichia coli strains are grown routinely at 37°C in Lysogeny Broth (LB) substratum (Sigma, Catalog#L3022-1KG). Solid culture medium is supplemented with 1.5% (w / v) agar. When needed, antibiotic is used for selection purpose with following concentration: chloramphenicol-34mg / L, for Escherichia coli, and 7.5mg / L, for Corynebacterium glutamicum; Kanamycin-25-50mg / L, for Escherichia coli, and 15-25mg / L, for Corynebacterium glutamicum. By adding 0.5mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to substratum when cultivating beginning as final concentration, carry out the induction of the gene from pXMJ19.

[0093] Table 1: Composition of CgXII minimal medium without carbon source

[0094]

[0095]

[0096] DNA transformation

[0097] E. coli chemically or electrocompetent cells were used for cloning and amplification of plasmid DNA following the manufacturer's instructions (NEB, Catalog # C3019, C3020, C3040). After a recovery period, cells were plated on LB agar plates with appropriate antibiotics for selection.

[0098] A modified protocol was used to make the C. glutamicum strain electrocompetent: from a 1:1 flask grown at 33°C, 250 rpm to an OD of 0.3. 600 100 mL of BHI culture + 0.5 M sorbitol was inoculated with an overnight pre-culture of 100 mL and incubated at 33° C., 130 rpm until it reached an OD of 1.75. 600 . The culture was transferred to a 50mL falcon tube and collected by centrifugation (3400g, 10 minutes, 4°C). The cell pellets were combined and washed twice with 50mL of ice-cold Tris-glycerol buffer (1mM Tris-HCl pH 7.5, 10% (v / v) glycerol), followed by 2 washing steps with 50mL of ice-cold 10% (v / v) glycerol. The final cell pellet was resuspended in 800 μL of ice-cold 10% (v / v) glycerol. The electrocompetent cells were divided into 150 μL and stored at -80°C. For electroporation, the cell aliquots were thawed on ice and DNA was added to it. The cell-DNA mixture was transferred to a 2mm electroporation cup (Sigma-Aldrich, Catalog # Z706088-50EA), and using a Gene Pulser Xcell TM The cells were plated on a BHI agar plate with suitable antibiotics for selection, and incubated at 30°C for 2-3 days to obtain single cell colonies. The manufacturer's instructions were followed, except that the cells were resuspended in buffer P1 and incubated at 37°C for 2 hours before lysis, using QIAprep Spin Miniprep kit (Qiagen, Catalog #27106X4), which was used to separate and verify the correct maintenance of the replicative plasmid after conversion by plasmid DNA. The isolated plasmid DNA was then verified by restriction analysis.

[0099] Allelic exchange in Corynebacterium glutamicum

[0100] Allele replacement (about gene deletion and gene integration at the regulation seat) is carried out by using pK18mobsacB (NCBI accession number: FJ437239) (people such as Schafer, 1994) derived plasmid, and described plasmid comprises upstream and downstream homology region (~300-1000bp) and desired insertion fragment (under the situation of integration).As mentioned previously, suitable plasmid is transformed in the Corynebacterium glutamicum bacterial strain by electroporation.After electroporation, on the BHI agar medium that comprises kanamycin, select the first recombination event, thereby produce intermediate bacterial strain.Screen intermediate bacterial strain via colony PCR.Next, on not having antibiotic BHI+10% (w / v) sucrose agar plate, intermediate bacterial strain is carried out counter-selection to promote the second recombination event.After counter-selection, screen sucrose resistance and kanamycin sensitive type bacterium colony to verify desired allele exchange and occur by colony PCR and dna sequencing (using suitable primer outside homology region).

[0101] Cultivation of the Corynebacterium glutamicum strain for GAA production in Example 3

[0102] Use suitable Corynebacterium glutamicum bacterial strain to inoculate the pre-culture from the glycerol stock.Pre-culture is carried out in the BHI substratum (when needing, having suitable antibiotic) of 10mL in the Erlenmeyer flask of 100mL band baffle, and incubated 24 hours under 30 ℃, 200rpm.Collect pre-culture by centrifugal (3100g, 10 minutes, under room temperature (RT)), and wash with the CgXII substratum (referring to Table 1) that does not have carbon source of 5mL.To be resuspended in the CgXII substratum that does not have carbon source of 2.5mL through the granular precipitation of washing, and use Ultraspec 2100pro (Amersham Biosciences) spectrophotometer to measure the OD of cell suspension. 600 An appropriate volume of washed cells was used to inoculate CgXII medium (with appropriate antibiotics, if needed) supplemented with 10 g / L glucose, 1 g / L L-arginine, and 4 g / L L-serine to a starting OD of 0.5. 600, as the main culture. Induction of genes from the pXMJ19-derived plasmid was performed by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to the production medium as a final concentration at the beginning of the culture. The main culture was transferred to a 48-well multitier plate (Beckman Coulter Life Sciences, Catalog # M2P-MTP-48-BOH1) using 800 μL / well and covered with sealing foil (Beckman Coulter Life Sciences / m2p Labs, Catalog # F-GPR48-10). The covered plate was placed on I (Beckman Coulter Life Sciences / m2p Labs) for incubation at 1400 rpm, 30°C for 27 hours. After 27 hours, the OD was measured in a spectrophotometer. 600 The cultures were spun down (2000 g, 10 min) and the supernatants were used to measure GAA production.

[0103] Cultivation of the Corynebacterium glutamicum strain for GAA production in Example 6

[0104] Use suitable Corynebacterium glutamicum bacterial strain to inoculate the pre-culture from the glycerol stock.Pre-culture is carried out in the BHI substratum (when needing, having suitable antibiotic) of 10mL in the Erlenmeyer flask of 100mL band baffle, and incubated 24 hours under 30 ℃, 200rpm.Collect pre-culture by centrifugal (3100g, 10 minutes, under room temperature (RT)), and wash with the CgXII substratum (referring to Table 1) that does not have carbon source of 5mL.To be resuspended in the CgXII substratum that does not have carbon source of 2.5mL through the granular precipitation of washing, and use Ultraspec 2100pro (Amersham Biosciences) spectrophotometer to measure the OD of cell suspension. 600 An appropriate volume of washed cells was used to inoculate CgXII medium (with appropriate antibiotics, if needed) supplemented with 10 g / L glucose, 1 g / L L-arginine, 1.275 g / L L-ornithine, and with or without 4 g / L L-serine (as specified in the text) to a starting OD of 0.5. 600, as the main culture. The main culture was transferred to a 48-well multitier plate (Beckman Coulter Life Sciences, Catalog # M2P-MTP-48-BOH1) using 800 μL / well and covered with sealing foil (Beckman Coulter Life Sciences / m2p Labs, Catalog # F-GPR48-10). The covered plate was placed on I (Beckman Coulter Life Sciences / m2p Labs) for incubation at 1400 rpm, 30° C. for 48 hours (when L-serine is present in the culture medium) and 72 hours (when L-serine is not present in the culture medium). OD was measured in a spectrophotometer after 48 or 72 hours, respectively. 600 The cultures were spun down (2000 g, 10 min) and the supernatants were used to measure GAA production.

[0105] Quantification of GAA

[0106] On Dionex Ultimate 3000 system (Thermo Scientific), quantitatively carry out GAA from culture supernatant by HPLC-UV.Sample is filtered with Sartorius Minisart NML Plus 0.2 μ m (Sartorius AG, Catalog#ST17823-K), then suitably dilute in mobile phase A (see below).Next, at 35 ℃, separate sample (10 μ L injection volumes) by using HyperCarb 100x4.6mm 7 μ m posts (Thermo Scientific, catalog number (Cat. No.) 35007-104630).Mobile phase A is made up of the 2.3g ammonium dihydrogen phosphate and the 2.6g diammonium hydrogen phosphate in the purified water being dissolved in 2L.Mobile phase B is made up of the 2.3g ammonium dihydrogen phosphate and the 2.6g diammonium hydrogen phosphate in the purified water being dissolved in 1.25L and the acetonitrile of 0.75L.In whole operation, keep the flow velocity of 1.0mL / minute. The column was pre-equilibrated with 100% mobile phase A, and a gradient between phases A and B was used: 0 to 8 minutes – linear gradient from 0% to 10% phase B; 8 to 10 minutes – linear gradient from 10% B to 40% B; 10 to 11 minutes – 40% B; 11.1 to 13 minutes – 0% B; and 13 to 14 minutes – constant at 0% B to re-equilibrate the column. The total run duration was 14 minutes. Under these conditions, GAA had a retention time of 5.8 minutes. GAA was detected using a UV detector (ThermoScientific Dionex Ultimate 3000DAD) at 200 nm (210 nm as a reference).

[0107] B) Experimental results

[0108] Example 1: Construction of LF-S-677a for chromosomal expression of AGAT and increased L-arginine availability in Corynebacterium glutamicum

[0109] 1.1. Chromosomal deletion of the argR gene in Corynebacterium glutamicum ATCC 13032 derivatives

[0110] To improve intracellular L-arginine formation and L-arginine recycling from L-ornithine, the argR gene encoding the central repressor protein ArgR that controls the L-arginine biosynthesis pathway was inactivated. Inactivation was achieved via allelic replacement using plasmid pK18mobsacB_DargR.

[0111] pK18mobsacB_DargR was cloned as follows:

[0112] Backbone: pK18mobsacB linearized with XbaI. Insert: PCR of ATCC 13032 genomic DNA using oligos DargR_lf (SEQ ID NO: 1) and DargR_lr (SEQ ID NO: 2), PCR of ATCC 13032 genomic DNA using oligos DargR_rf (SEQ ID NO: 3) and DargR_rr (SEQ ID NO: 4). Assembly was performed using NEBuilder HiFi DNA Assembly.

[0113] 1.2. Chromosomal Expression of Arginine Biosynthesis Genes argF, argG, and argH in Corynebacterium glutamicum ATCC 13032 Derivatives under the Control of the Strong Constitutive Promoter Pg3

[0114] To enhance the activity of ArgF, ArgG and ArgH, additional copies of the corresponding genes were inserted into the genome. This was achieved via allelic replacement using the plasmid pK18mobsacB_IBcg0054::Pg3-argFGH (SEQ ID NO: 5).

[0115] pK18mobsacB_IBcg0054::Pg3-argFGH (SEQ ID NO: 5) was cloned as follows:

[0116] First, a synthetic operon was designed consisting of Pg3, argF, argG, argH, and flanking regions for genomic integration (SEQ ID NO: 6). The DNA sequence was ordered from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) for gene synthesis and delivered as part of a cloning plasmid with an ampicillin resistance gene (designated pMA-RQ_argFGH).

[0117] Backbone: pK18mobsacB linearized with EcoRI and HindIII. Insert: PCR of pMA-RQ_argFGH using oligos argFGH_f (SEQ ID NO: 7) and argFGH_r (SEQ ID NO: 8). Assembly was performed using NEBuilder HiFi DNA Assembly.

[0118] 1.3. Chromosomal deletion of the lysE and lysG genes in derivative strains of Corynebacterium glutamicum ATCC 13032

[0119] Gene lysE encodes the export protein of the outflow of catalysis L-lysine, L-arginine and L-citrulline in Corynebacterium glutamicum.The expression of lysE is positively regulated by the gene product of lysG.These two genes are adjacent to each other, but the transcription mode is different.In order to make translocator LysE and positive regulatory protein LysG inactivation, as described for pK18mobsacB-Δ lysEG in (Vrljic et al., 1996), construct plasmid pK19mobsacB-Δ lysEG (SEQ ID NO:9).The disappearance of lysEG uses the pK19mobsacB-Δ lysEG of reconstruction to realize via allele replacement.

[0120] 1.4. Chromosomal insertion of the sod promoter upstream of the carAB operon in a Corynebacterium glutamicum ATCC 13032 derivative strain

[0121] In order to improve the production of L-arginine, a strong sod-promoter was inserted upstream of the carAB operon in the genome. This was achieved via allele replacement using plasmid pK18mobsacB_PsodcarAB.

[0122] pK18mobsacB_PsodcarAB was cloned as follows:

[0123] Backbone: pK18mobsacB linearized with EcoRI and HindIII. Insert: PCR of ATCC 13032 genomic DNA using oligomers PsodcarAB-LA-F (SEQ ID NO: 10) and PsodcarAB-LA-R (SEQ ID NO: 11), PCR of ATCC 13032 genomic DNA using oligomers PsodcarAB-F (SEQ ID NO: 12) and PsodcarAB-R (SEQ ID NO: 13), and PCR of ATCC 13032 genomic DNA using oligomers PsodcarAB-RA-F (SEQ ID NO: 14) and PsodcarAB-RA-R (SEQ ID NO: 15). Assembly was performed using NEBuilder HiFi DNA Assembly.

[0124] 1.5. Chromosomal insertion of the gene AGAT-Mp encoding L-arginine:glycine amidinotransferase (AGAT; EC 2.1.4.1) from productive Murraya algae

[0125] 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. Productive Moorella is a filamentous cyanobacterium. The genome of productive Moorella strain PAL-8-15-08-1 can be accessed 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:16). SEQ ID NO:17 shows the corresponding amino acid sequence (NCBI accession number WP_070390602). As an intermediate step, the AGAT-Mp expression cassette was assembled into a plasmid to facilitate future cloning, thereby generating pLIB_P[AGAT-Mp] (SEQ ID NO:18). The pLIB_P[AGAT-Mp] comprises a codon optimized version of a promoter sequence, a ribosome bind site and the AGAT-Mp gene. The corresponding gene product has the identical amino acid sequence that provides among the SEQ ID NO:17. Use pLF338 (SEQ ID NO:19), via allelic replacement, the AGAT-Mp expression cassette is integrated in the genome of Corynebacterium glutamicum at the intergenic region of NCgl0013_NCgl0014. pLF338 is a pK18mobsacB derivative, which comprises the homology region needed for the integration at the intergenic region of NCgl0013_NCgl0014 and the expression cassette about the AGAT-Mp gene.

[0126] The cloning of pLF388 was performed in multiple parts. First, pLF337 was constructed as a pK18mobsacB derivative containing homology regions for integration into the genome.

[0127] pLF337 was cloned as follows:

[0128] Backbone: pK18mobsacB linearized with EcoRI. Insert: PCR of ATCC 13032 genomic DNA using oligos MW_21_80_fw (SEQ ID NO: 20) and MW_21_81_rv (SEQ ID NO: 21), PCR of ATCC 13032 genomic DNA using oligos MW_21_82_fw (SEQ ID NO: 22) and MW_21_83_rv (SEQ ID NO: 23). Assembly was performed using NEBuilder HiFi DNA Assembly.

[0129] Finally, the AGAT-Mp expression cassette from pLIB_P[AGAT-Mp] was cloned into pLF337, generating pLF338 as a plasmid for chromosomal integration of AGAT in C. glutamicum.

[0130] pLF338 was cloned as follows:

[0131] Backbone: pLF337 linearized with AscI. Insert: PCR of pLIB_P[AGAT-Mp] with oligos MW_21_84_fw (SEQ ID NO: 24) and MW_21_85_rv (SEQ ID NO: 25). Assembly was performed using NEBuilder HiFi DNA Assembly.

[0132] Example 2: Overexpression of the glyA gene in Corynebacterium glutamicum

[0133] In Corynebacterium glutamicum, the glyA gene encoding has serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) active protein (people such as Simic, 2002).This protein catalysis L-serine is converted into glycine, and it can further be used (referring to embodiment 1.5) to produce GAA by AGAT (L-arginine: glycine amidinotransferase).Therefore, to be cloned into pXJM19 (a kind of well-known intestinal bacteria-Corynebacterium glutamicum shuttle vector) (people such as Jakoby, 1999) from the glyA gene of Corynebacterium glutamicum, thereby produce pXMJ19[glyA_Cg] (SEQ ID NO:26). This plasmid was transformed into LF-S-677a (genotype: ATCC 13032ΔargR IBcg0054::Pg3-argFGHΔlysEG Psod-carABintNCgl0013-NCgl0014::(P2947MU)[agat_Mp]).

[0134] pXMJ19[glyA_Cg] was cloned as follows:

[0135] Backbone: pXMJ19 linearized with HindIII and SalI. Insert: PCR of ATCC 13032 genomic DNA using oligos oMC21 (SEQ ID NO: 27) and oMC22 (SEQ ID NO: 28). Assembly was performed using HiFi Assembly Mix.

[0136] Example 3: Effect of increased serine hydroxymethyltransferase (SHMT) activity on GAA production by glyA_Cg

[0137] Corynebacterium glutamicum bacterial strain LF-S-677a (genotype: ATCC 13032 Δ argR IBcg0054:: Pg3-argFGH Δ lysEG Psod-carAB intNCgl0013-NCgl0014:: (P2947MU) [agat_Mp]) (its construction is described among the embodiment 1) serves as our basic bacterial strain.This bacterial strain has the L-arginine ability that increases (by the disappearance or weakening of ArgR and LysEG, the enhancing of ArgF, ArgG and ArgH, and the enhancing of CarAB) and has been equipped with L-arginine: glycine amidinotransferase (AGAT-Mp) heterologously.Being equipped with pXMJ19 [glyA_Cg] so that after AGAT-Mp supply glycine to produce GAA to this bacterial strain.Surprisingly, this is enough to generate the GAA production of the amount that increases compared to starting bacterial strain, as shown in Table 2.

[0138] Table 2: GAA production by Corynebacterium glutamicum LF-S-677a strain with enhanced serine hydroxymethyltransferase (SHMT)

[0139]

[0140] au – arbitrary unit.

[0141] Table 2 shows that C. glutamicum LF-S-677a+pXMJ19[glyA_Cg] with enhanced SHMT activity can produce 90 mg / L of GAA, compared to only 14 mg / L of GAA in the reference strain LF-S-677a. 600 The yield was higher in strains with enhanced SHMT activity compared to the parent (4.02 vs 0.61, respectively). Therefore, we conclude that enhancement of serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) activity improves GAA production in strains that have been modified to have a combination of AGAT activity, increased ability to provide L-arginine, and reduced L-arginine export.

[0142] Example 4: Construction of sdaA deletion strain in Corynebacterium glutamicum

[0143] In order to improve L-Serine availability by stopping it from being converted into pyruvic acid, the sdaA gene inactivation of Corynebacterium glutamicum is made.The sdaA gene encoding of Corynebacterium glutamicum has the active protein (being abbreviated as SDHL) of L-Serine ammonia lyase (EC 4.3.1.17).The alternative name of SDHL is serine deaminase, L-Serine dehydratase and L-Serine deaminase in particular.In Corynebacterium glutamicum ATCC 13032, realize inactivation via allelic replacement with plasmid pK18mobsacB-sdaA (SEQ ID NO:29), thereby produce bacterial strain Corynebacterium glutamicum ATCC 13032 Δ sdaA.

[0144] pK18mobsacB-sdaA (SEQ ID NO: 29) was cloned as follows:

[0145] Backbone: pK18mobsacB linearized with BamHI and SalI. Insert: PCR of ATCC 13032 gDNA using oligos oMC58 and oMC59, and PCR of ATCC 13032 gDNA using oligos oMC60 and oMC61. Assembly was performed using HiFi Assembly Mix.

[0146] Example 5: Plasmid-based expression of AGAT alone and together with SHMT

[0147] As mentioned in embodiment 1.5, for forming guanidine acetic acid (GAA) from L-arginine and glycine, need encoding L-arginine: the heterologous gene of glycine amidinotransferase (AGAT) (EC 2.1.4.1).In the present embodiment, use the identical L-arginine described in embodiment 1.5: glycine amidinotransferase (AGAT, EC 2.1.4.1; There is following aminoacid sequence SEQ ID NO:17), to be used for the expression based on plasmid in Corynebacterium glutamicum.Use the plasmid that is called pLIB_P (SEQ ID NO:34) as the basic carrier for expression, described plasmid has the replication origin from pBL1 for Corynebacterium glutamicum, the pSC101 replication origin for escherichia coli, kanamycin resistance gene, strong promoter (from (Rytter et al., 2014) P 294MU) and BioBricks terminator BBa_B1006. A codon-optimized version of the AGAT-Mp gene, including an RBS and flanked by Eco31I (BsaI) sites, was ordered from Eurofins Genomics. It was delivered as a synthetic gene cloned in pEX-A258. The codon-optimized AGAT-Mp was cloned into pLIB_P (SEQ ID NO: 34), generating pLIB_P[AGAT-Mp] (SEQ ID NO: 18).

[0148] pLIB_P[AGAT-Mp] (SEQ ID NO: 18) was cloned as follows:

[0149] Backbone: pLIB_P linearized with Eco31I. Insert: 1.3 kb fragment of pEX-A258-AGAT-Mp digested with Eco31I. Assembly was performed using HiFi Assembly Mix.

[0150] As mentioned in embodiment 2, in Corynebacterium glutamicum, the glyA genes encoding has serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) active protein.This protein catalysis L-serine is converted into glycine, and it can further be used to produce GAA by AGAT (L-arginine: glycine amidinotransferase).Therefore, to be cloned in pLIB_P[AGAT-Mp] to produce the active plasmid of coding AGAT and SHMT from the glyA gene (being called glyA_Cg) of Corynebacterium glutamicum.The plasmid of gained is called as pLIB_P[AGAT-Mp glyA_Cg](SEQ ID NO:35).

[0151] pLIB_P[AGAT-Mp glyA_Cg] (SEQ ID NO: 35) was cloned as follows:

[0152] Backbone: pLIB_P[AGAT-Mp] linearized with BamHI Insert: PCR of ATCC 13032 genomic DNA using oligos oMC23 (SEQ ID NO: 36) and oMC24 (SEQ ID NO: 37). Assembly was performed using HiFi Assembly Mix.

[0153] The pLIB_P, pLIB_P[AGAT-Mp] and pLIB_P[AGAT-Mp glyA_Cg] plasmids were transformed into the strain Corynebacterium glutamicum ATCC 13032 ΔsdaA (which was created in Example 4) to test GAA production from this strain.

[0154] Example 6: Effects of L-serine ammonia lyase (SDHL) deletion, enhanced serine hydroxymethyltransferase (SHMT) and L-arginine:glycine amidinotransferase (AGAT) on GAA production

[0155] Corynebacterium glutamicum ATCC 13032 Δ sdaA (its structure is described among the embodiment 4) serves as our basic bacterial strain.In this bacterial strain, the sdaA gene of coding has the active protein (being abbreviated as SDHL) of L-serine ammonia lyase (EC 4.3.1.17) is inactivated to increase L-serine availability.L-serine can be used to form glycine by serine hydroxymethyltransferase (SHMT), and it is by L-arginine: glycine amidinotransferase (AGAT) is used to form GAA.Surprisingly, this is enough to generate the GAA production of the amount that increases compared to starting bacterial strain, and in substratum, outside L-serine (referring to Table 3) and not providing in substratum under the situation of outside L-serine (referring to Table 4).

[0156] Table 3: GAA production by Corynebacterium glutamicum ATCC 13032 ΔsdaA with L-arginine:glycine amidinotransferase (AGAT) and enhanced serine hydroxymethyltransferase (SHMT), with L-serine added to the culture medium

[0157]

[0158] Table 3 shows that when 4 g / L of L-serine was added to the culture medium, the C. glutamicum ATCC 13032ΔsdaA with AGAT and enhanced SHMT activity could produce 165 mg / L of GAA, compared to only 153 mg / L of GAA in the reference strain ATCC 13032ΔsdaApLIB_P[AGAT-Mp]. Similarly, GAA / OD 600 The yield was higher in the strain with enhanced SHMT activity compared to the parent (14.73 vs 9.27, respectively).

[0159] Table 4: GAA production by Corynebacterium glutamicum ATCC 13032 ΔsdaA with L-arginine:glycine amidinotransferase (AGAT) and enhanced serine hydroxymethyltransferase (SHMT) without addition of L-serine to the culture medium

[0160]

[0161] Similarly, the results in Table 4 show that when L-serine is not added to the culture medium, C. glutamicum ATCC 13032ΔsdaA with AGAT and enhanced SHMT activity can produce 66 mg / L of GAA, compared to only 52 mg / L of GAA in the reference strain ATCC 13032ΔsdaA pLIB_P[AGAT-Mp]. Similarly, GAA / OD 600 The yield of 1.547kJ / mL is higher in the bacterial strain with enhanced SHMT activity, compared to parent (respectively, 3.51vs 2.73).Table 4 shows that Corynebacterium glutamicum ATCC 13032 ΔsdaA can provide enough L-serine to improve GAA production via AGAT and the enhanced GAA with SHMT protein.Therefore, we draw a conclusion that the existence of AGAT, serine hydroxymethyltransferase (SHMT; EC2.1.2.1) active enhancing improve GAA production in the bacterial strain having deleted L-serine ammonia lyase therein.

[0162] Citations

[0163] Jakoby,M.,Ngouoto-Nkili,C.-E.,&Burkovski,A.(1999).Construction and application of new Corynebacterium glutamicum vectors.BiotechnologyTechniques,13(6),437-441.https: / / doi.org / 10.1023 / A:1008968419217

[0164] Keilhauer,C.,Eggeling,L.,&Sahm,H.(1993).Isoleucine synthesis inCorynebacterium glutamicum:molecular analysis of the ilvB-ilvN-ilvC operon.JBacteriol,175(17),5595-5603.

[0165] https: / / doi.org / 10.1128 / jb.175.17.5595-5603.1993

[0166] 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 theprolific and distinctive metabolic potential of the cyanobacterial genusMoorea.Proc Natl Acad Sci U S A,114(12),3198-3203.

[0167] https: / / doi.org / 10.1073 / pnas.1618556114

[0168] Rytter,J.V.,Helmark,S.,Chen,J.,Lezyk,M.J.,Solem,C.,&Jensen,P.R.(2014).Synthetic promoter libraries for Corynebacterium glutamicum.ApplMicrobiol Biotechnol,98(6),2617-2623.

[0169] https: / / doi.org / 10.1007 / s00253-013-5481-x

[0170] Schafer,A.,Tauch,A.,Jager,W.,Kalinowski,J.,Thierbach,G.,&Puhler,A.(1994).Small mobilizable multi-purpose cloning vectors derived from theEscherichia coli plasmids pK18 and pK19:selection of defined deletions in thechromosome of Corynebacterium glutamicum.Gene,145(1),69-73.

[0171] https: / / doi.org / 10.1016 / 0378-1119(94)90324-7

[0172] Simic,P.,Willuhn,J.,Sahm,H.,&Eggeling,L.(2002).Identification of glyA(encoding serine hydroxymethyltransferase)and its use together with theexporter ThrE to increase L-threonine accumulation by Corynebacteriumglutamicum.Appl Environ Microbiol,68(7),3321-3327.

[0173] https: / / doi.org / 10.1128 / AEM.68.7.3321-3327.2002

[0174] Vrljic,M.,Sahm,H.,&Eggeling,L.(1996).A new type of transporter with anew type of cellular function:L-lysine export from Corynebacteriumglutamicum.Mol Microbiol,22(5),815-826.

[0175] https: / / doi.org / 10.1046 / j.1365-2958.1996.01527.x

Claims

1. A microorganism comprising at least one heterologous gene encoding a protein having the function of an L-arginine:glycine amidinotransferase and an overexpressed gene encoding a protein having the enzymatic activity of an L-serine hydroxymethyltransferase.

2. microorganism according to claim 1, the overexpression of the gene of the protein with the enzymatic activity of wherein said coding has L-serine hydroxymethyl transferase is by increasing the copy number of said gene, and / or by functionally connecting said gene to a strong promoter, and / or by enhancing the ribosome bind site, and / or by optimizing the codon usage of the start codon or the whole gene.

3. The microorganism according to claim 1 or claim 2, wherein a gene encoding a protein having the function of L-serine ammonia lyase is inactivated or deleted.

4. The microorganism according to any one of the preceding claims, wherein the expression of the argR gene encoding the arginine-responsive repressor protein ArgR is inactivated or deleted.

5. The microorganism according to any one of the preceding claims, further comprising at least one overexpressed gene encoding an enzyme having the function of carbamoyl-phosphate synthase.

6. The microorganism according to any one of the preceding claims, further comprising at least one or more overexpressed genes selected from the group consisting of a gene encoding a protein having the function of ornithine transcarbamylase, a gene encoding a protein having the function of argininosuccinate synthetase, and a gene encoding a protein having the function of argininosuccinate lyase.

7. The microorganism according to any one of the preceding claims, wherein the lysEG gene encoding a protein having the function of the arginine exporter LysE and its transcriptional activator LysG is inactivated or deleted.

8. A method for the fermentative production of guanidinoacetic acid (GAA), comprising the steps of culturing the microorganism as defined in any one of the preceding claims in a culture medium, and accumulating GAA in the culture medium to form a fermentation broth comprising GAA.

9. The method of claim 8, further comprising isolating GAA from the fermentation broth comprising GAA. 10 . The microorganism according to claim 1 , further comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity. The microorganism according to claim 10 , wherein the gene encoding the enzyme having guanidinoacetic acid N-methyltransferase activity is overexpressed.

12. A method for producing creatine by fermentation, comprising the steps of culturing the microorganism defined in any one of claims 10 or 11, and accumulating creatine in the culture medium to form a fermentation broth containing creatine.

13. The method of claim 12, further comprising isolating creatine from the fermentation broth containing creatine.

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