Production of guanidinoacetic acid (GAA) from serine fermentation by attenuating L-serine ammonia lyase activity in microorganisms

Abstract

The present invention provides: a microorganism which has been modified to express a gene encoding a protein having arginine: glycine amidino transferase (AGAT) activity, and which has been modified to increase glycine production from L-serine; 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 microorganisms that have been modified to express a gene encoding a protein having arginine:glycine amidinotransferase (AGAT) activity and to increase glycine production from L-serine; and methods for producing guanidine acetic acid (GAA) by fermenting such microorganisms and methods 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, and Moore's algae) 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] Therefore, the present invention relates to microorganisms comprising at least one heterologous gene encoding a protein having the function of an L-arginine:glycine amidinotransferase and in which the gene encoding a protein having the function of an L-serine ammonia lyase [EC 4.3.1.17] (SDHL) (also referred to as an L-serine deaminase or L-serine dehydratase activity) is inactivated or deleted.

[0014] 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.

[0015] 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:

[0016]

[0017] 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).

[0018] In the microorganism of the present invention, the gene encoding the protein having the function of L-arginine:glycine amidinotransferase may be further overexpressed.

[0019] Typically, overexpression of a gene is achieved by increasing the copy number of the gene, and / or by functionally linking the gene to a strong promoter, 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 comprising all of the above-mentioned methods.

[0020] 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 use of the codons of the start codon or the whole gene. The enhancing of this type of regulatory factor that positively affects gene expression can for example be by modifying the promoter sequence upstream of the structural gene to increase the efficiency of the promoter or by replacing the promoter fully with more effective or so-called strong promoter to achieve. Promoter is located in the upstream of the gene. Promoter is such DNA sequence dna, which is made up of approximately 40 to 50 base pairs, and constitutes the binding site and the transcription start point about the RNA polymerase holoenzyme, which can affect 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 promoters and genes, which results in transcription of the gene.

[0021] 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).

[0022] The microorganism of the present invention may further comprise an overexpressed gene encoding an enzyme having the function of L-serine hydroxymethyltransferase (SHMT) [EC 2.1.2.1]. L-serine hydroxymethyltransferase (SHMT) [EC 2.1.2.1] is encoded by the glyA gene in Escherichia coli and in Corynebacterium glutamicum.

[0023] exist Figure 2 GAA production starting from serine is shown in .

[0024] In the microorganism of the present invention, the expression of the argR gene encoding the arginine-responsive repressor protein ArgR may be inactivated or deleted.

[0025] 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.

[0026] The microorganism according to the present invention may further comprise at least one overexpressed gene encoding an enzyme having the function of carbamoyl-phosphate synthase (EC 6.3.4.16, CarAB).

[0027] The microorganism of the present invention may 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 (EC 2.1.3.3, ArgF, ArgF2), a gene encoding a protein having the function of argininosuccinate synthetase (EC6.3.4.5, ArgG), and a gene encoding a protein having the function of argininosuccinate lyase (EC4.3.2.1, ArgH).

[0028] 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).

[0029] 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 can be inactivated or deleted in the microorganism of the present invention.

[0030] 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.

[0031] 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.

[0032] 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: 7. 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: 7 of the productive Moorella sp., a filamentous cyanobacterium.

[0033] 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.

[0034] The method of the present invention may further comprise the step of separating GAA from the fermentation broth.

[0035] The method according to the present invention may further comprise the step of drying and / or granulating the fermentation broth containing GAA.

[0036] 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.

[0037] 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.

[0038] 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

[0040] 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.

[0041] 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], 3PHP – 3-phosphohydroxypyruvate, THF – (6S)-5,6,7,8-tetrahydrofolate, CH2-TFH – (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate.

[0042] Sequence Description

[0043] SEQ ID NO: 1 DNA sequence of plasmid pK18mobsacB-sdaA.

[0044] SEQ ID NO: 2 Synthetic oligonucleotide: DNA sequence of primer oMC58.

[0045] SEQ ID NO: 3 Synthetic oligonucleotide: DNA sequence of primer oMC59.

[0046] SEQ ID NO: 4 Synthetic oligonucleotide: DNA sequence of primer oMC60.

[0047] SEQ ID NO: 5 Synthetic oligonucleotide: DNA sequence of primer oMC61.

[0048] SEQ ID NO: 6 DNA sequence of a productive Moorella sp. gene with locus_tag BJP34_00300. It encodes L-arginine:glycine amidinotransferase.

[0049] SEQ ID NO: 7 Amino acid sequence of L-arginine:glycine amidinotransferase from productive Moorella sp. (NCBI Accession No. WP_070390602).

[0050] SEQ ID NO:8 DNA sequence of plasmid pLIB_P.

[0051] SEQ ID NO: 9 DNA sequence of plasmid pLIB_P[AGAT-Mp] (Example 1).

[0052] Experimental part

[0053] A) Materials and Methods

[0054] Chemicals

[0055] Unless otherwise stated, all chemicals were ordered as analytical grade from Merck, Sigma Aldrich, or Carl-Roth.

[0056] Molecular biology techniques

[0057] 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 MasterMix (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).

[0058] Strains and culture conditions

[0059] 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.

[0060] Corynebacterium glutamicum ATCC13032 and derivative thereof are in brain heart infusion (BHI; Merck Millipore, Catalog#1104930500) broth 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 precise composition of the CgXII minimal medium without any carbon source is described in detail in Table 1.The glycerol stock of Corynebacterium glutamicum is prepared by mixing the overnight culture in BHI substratum (30 ℃, 200rpm, 10mL substratum) of 900 μ L with aseptic 86% (w / v) glycerol solution phase mixture of 600 μ L, then it is stored in-80 ℃. Escherichia coli strain is grown routinely at 37 ℃ in LysogenyBroth (LB) substratum (Sigma, Catalog#L3022-1KG).Solid culture medium is supplemented with 1.5% (w / v) agar.When needed, use antibiotic to be used for selection purpose with following concentration: chloramphenicol-34mg / L, is used for intestinal bacteria, and 7.5mg / L, is used for Corynebacterium glutamicum; Kantlex-25-50mg / L, is used for intestinal bacteria, and 15-25mg / L, is used for Corynebacterium glutamicum.

[0061] Table 1: Composition of CgXII minimal medium without carbon source

[0062]

[0063]

[0064] DNA transformation

[0065] 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.

[0066] 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.

[0067] Allelic exchange in Corynebacterium glutamicum

[0068] 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).

[0069] Cultivation of the Corynebacterium glutamicum strain for GAA production in Example 3

[0070] 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 3 g / L glycine 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).

[0071] Place the covered plate on I (Beckman Coulter Life Sciences / m2pLabs) 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.

[0072] Quantification of GAA

[0073] 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).

[0074] B) Experimental results

[0075] Example 1: Construction of sdaA-inactivated strain in Corynebacterium glutamicum

[0076] In order to improve L-Serine availability by stopping it to be 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 especially serine deaminase, L-Serine dehydratase and L-Serine deaminase.In Corynebacterium glutamicum ATCC 13032, realize inactivation via allelic replacement with plasmid pK18mobsacB-sdaA (SEQ ID NO:1), thereby produce bacterial strain Corynebacterium glutamicum ATCC 13032 Δ sdaA.

[0077] pK18mobsacB-sdaA was cloned as follows:

[0078] Backbone: pK18mobsacB linearized with BamHI and SalI.

[0079] Inserts: PCR of ATCC 13032 gDNA using oligomers oMC58 (SEQ ID NO: 2) and oMC59 (SEQ ID NO: 3), PCR of ATCC 13032 gDNA using oligomers oMC60 (SEQ ID NO: 4) and oMC61 (SEQ ID NO: 5).

[0080] Assembled using HiFiAssembly Mix.

[0081] For making sdaA inactivation, pK18mobsacB-sdaA is transformed among the Corynebacterium glutamicum ATCC13032 by electroporation.Select chromosomal integration (producing from the first recombination event) by carrying out plating on the BHI agar that is supplemented with 134g / l sorbitol, 2.5g / l yeast extract and 25mg / l kanamycin.With agar plate 33 ℃ of following incubations 48 hours.Individual bacterium colony is transferred to fresh agar plate (having 25mg / l kanamycin) and 33 ℃ of following incubations 24 hours.The liquid culture of these clones is cultivated 24 hours at 33 ℃ in the 10ml BHI substratum that is included in the 100ml conical flask with 3 baffles.In order to separate the clone that has run into the second recombination event, from each liquid culture, get aliquot, suitably dilute, and on the BHI agar that is supplemented with 10% sucrose, carry out plating (typically, 100 to 200 μ l). With these agar plates 33 ℃ of following incubations 48 hours.Then, check the bacterium colony that grows on the described agar plate that comprises sucrose with regard to kanamycin sensitivity.For this reason, use toothpick to remove cell material from bacterium colony and transfer it on the BHI agar that comprises 25mg / l kanamycin and transfer on the BHI agar that comprises 10% sucrose.With agar plates 33 ℃ of following incubations 60 hours.Check through PCR and DNA sequencing through being proved for kanamycin sensitivity and for the clone that sucrose is had to resistance.With the bacterial strain named after Corynebacterium glutamicum ATCC 13032 Δ sdaA of gained.

[0082] Example 2: Plasmid-based expression of AGAT

[0083] 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:6). SEQ ID NO:7 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:9). pLIB_P[AGAT-Mp] contains a codon-optimized version of the promoter sequence, ribosome binding site, and AGAT-Mp gene. The corresponding gene product has the same amino acid sequence as given in SEQ ID NO:7.

[0084] Plasmid pLIB_P (SEQ ID NO: 8) contains the replication origin from pBL1 for Corynebacterium glutamicum, the replication origin from pSC101 for Escherichia coli, a kanamycin resistance gene, a strong promoter (P from (Rytter et al., 2014) 294MU ) and BioBricks terminator BBa_B1006.

[0085] A codon-optimized version of the AGAT-Mp gene comprising an RBS and flanking Eco31I (BsaI) sites was ordered from Eurofins Genomics. This was delivered as a synthetic gene cloned in pEX-A258. The codon-optimized AGAT-Mp was cloned into pLIB_P, thereby generating pLIB_P[AGAT-Mp].

[0086] pLIB_P[AGAT-Mp] was cloned as follows:

[0087] Backbone: pLIB_P linearized with Eco31I.

[0088] Insert: 1.3 kb fragment of pEX-A258-AGAT-Mp digested with Eco31I.

[0089] Assembled using HiFi Assembly Mix.

[0090] pLIB_P[AGAT-Mp] plasmid is transformed into bacterial strain Corynebacterium glutamicum ATCC 13032ΔsdaA (it creates in embodiment 1) and bacterial strain Corynebacterium glutamicum ATCC 13032 (reference strain) and produces with test from the GAA of these bacterial strains.Choose the cell that comprises plasmid with 25mg / l kanamycin.

[0091] The confirmed strains were named Corynebacterium glutamicum ATCC 13032pLIB_P[AGAT-Mp] and Corynebacterium glutamicum ATCC 13032ΔsdaA pLIB_P[AGAT-Mp], respectively.

[0092] Example 3: Effects of inactivation of L-serine ammonia lyase (SDHL) and induction of L-arginine:glycine amidinotransferase (AGAT) on GAA production

[0093] In order to evaluate the effect of inactivation of L-serine ammonia lyase (SDHL) and the implementation of L-arginine:glycine amidinotransferase (AGAT) on GAA production, the strains Corynebacterium glutamicum ATCC 13032 pLIB_P[AGAT-Mp] and Corynebacterium glutamicum ATCC 13032 ΔsdaA pLIB_P[AGAT-Mp] were cultivated in CGXII medium in a BioLector system and the resulting GAA titers were determined.

[0094] Table 2: GAA production by Corynebacterium glutamicum ATCC 13032 ΔsdaA with L-arginine:glycine amidinotransferase (AGAT)

[0095]

[0096] Table 2 shows that C. glutamicum ATCC 13032 ΔsdaA pLIB_P[AGAT-Mp] produced 220 mg / L of GAA, which is an improved GAA production compared to 193 mg / L of GAA using the reference strain C. glutamicum ATCC 13032 pLIB_P[AGAT-Mp].

[0097] Therefore, we concluded that inactivation of L-serine ammonia lyase improved GAA production in strains fulfilling L-arginine:glycine amidinotransferase (AGAT).

[0098] Citations

[0099] Jakoby,M.,Ngouoto-Nkili,C.-E.,&Burkovski,A.(1999).Construction andapplication of new Corynebacterium glutamicum vectors.BiotechnologyTechniques,13(6),437-441.https: / / doi.org / 10.1023 / A:1008968419217

[0100] Keilhauer,C.,Eggeling,L.,&Sahm,H.(1993).Isoleucine synthesis inCorynebacterium glutamicum:molecular analysis of the ilvB-ilvN-ilvC operon.JBacteriol,175(17),5595-5603.

[0101] https: / / doi.org / 10.1128 / jb.175.17.5595-5603.1993

[0102] 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.

[0103] https: / / doi.org / 10.1073 / pnas.1618556114

[0104] 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.

[0105] https: / / doi.org / 10.1007 / s00253-013-5481-x

[0106] 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.

[0107] https: / / doi.org / 10.1016 / 0378-1119(94)90324-7

[0108] 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.

[0109] https: / / doi.org / 10.1128 / AEM.68.7.3321-3327.2002

[0110] 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.

[0111] 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 L-arginine:glycine amidinotransferase, wherein a gene encoding a protein having the function of L-serine ammonia lyase is inactivated or deleted. 2 . The microorganism according to claim 1 , further comprising an overexpressed gene encoding an enzyme having the function of L-serine hydroxymethyltransferase.

3. The microorganism according to claim 1 or claim 2, wherein the argR gene encoding the arginine-responsive repressor protein ArgR is inactivated or deleted.

4. 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.

5. 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.

6. 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.

7. A method for the fermentative production of guanidinoacetic acid (GAA), comprising the steps of culturing a 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. The method of claim 7 , further comprising isolating GAA from the fermentation broth comprising GAA. 9 . The microorganism according to claim 1 , further comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity. 10 . The microorganism according to claim 9 , wherein the gene encoding the enzyme having guanidinoacetic acid N-methyltransferase activity is overexpressed.

11. A method for producing creatine by fermentation, comprising the steps of culturing the microorganism defined in any one of claims 9 or 10, and accumulating creatine in the culture medium to form a fermentation broth containing creatine.

12. The method of claim 11, further comprising isolating creatine from the fermentation broth containing creatine.

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