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

Abstract

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 the production of glycine from L-serine, as well as methods for producing guanidinoacetic acid (GAA) by fermentation of such microorganisms, and methods for producing creatine.

Description

[Technical Field]

[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 increase the production of glycine from L-serine, as well as a method for producing guanidinoacetic acid (GAA) by fermentation of such a microorganism, and a method for producing creatine.

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

[0003] Guthmiller et al. (J Biol Chem. 1994 Jul 1;269(26):17556-60) characterized rat kidney AGAT by cloning and heterologously expressing the enzyme in Escherichia coli (E. coli). Muenchhoff et al. (FEBS Journal 277(2010)3844-3860) reported the first characterization of a prokaryotic AGAT, also by cloning and heterologously expressing the enzyme in E. coli.

[0004] A microorganism capable of producing guanidinoacetic acid (GAA) was published by Zhang et al. (ACS Synth. Biol. 2020, 9, 2066-275). They engineered a reconstituted ornithine cycle in Escherichia coli by introducing heterologous AGATs from various species (e.g., Homo sapiens, Cylindrospermopsis raciborskii, Moorea producens) and by introducing citrulline synthesis modules (e.g., overexpression of carAB, argF, and argI) and arginine synthesis modules (e.g., overexpression of argG and argH; introduction of aspA).

[0005] Schneider and Jankowitsch (WO 2021122400) proposed a method for producing GAA using a microorganism with a gene encoding a protein with L-arginine:glycine amidinotransferase function and increased carbamoyl phosphate synthase activity. Carbamoyl phosphate is an important precursor not only for GAA biosynthesis but also for L-arginine and other compounds. Furthermore, Wang et al. (Applied Microbiology and Biotechnology, 2021, vol. 105, pp. 3265-3276; https: / / doi.org / 10.1007 / s00253-021-11242-w) specifically emphasized that carbamoyl phosphate is essential for L-arginine production in Corynebacterium species.

[0006] Jankowitsch et al. (WO 2022243116) also disclose a Corynebacterium glutamicum strain derived from Moorea producens that expresses L-arginine:glycine amidinotransferase (AGAT; EC:2.1.4.1) for the production of guanidinoacetic acid (GAA), specifically, by deleting the lysE, lysG, and argR genes and overexpressing the carAB operon (encoding carbamoyl phosphate synthase). This microorganism may also possess guanidinoacetic acid N-methyltransferase activity for the additional production of creatine.

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

[0008] Glycine can be produced intracellularly by organisms from a variety of sources, including L-serine. Glycine can be produced directly from L-serine by L-serine hydroxymethyltransferase (SHMT) [EC 2.1.2.1], encoded by the glyA gene, in Escherichia coli (E. coli) and Corynebacterium glutamicum (C. glutamicum). Figure 1 shows a schematic diagram of glycine production from serine.

[0009] There are several publications focusing on the production of L-serine in various microorganisms. For example, International Publication No. 2016120326 discloses a method for producing L-serine by culturing bacteria in which the expression of a gene encoding a polypeptide having serine deaminase activity and the expression of genes encoding a polypeptide having serine hydroxymethyltransferase activity, i.e., sdaA, sdaB, tdcG, and glyA, are attenuated. For example, Chinese Patent No. 109797126 proposes the use of bacteria with reduced serine O-acetyltransferase (CysE) expression for the production of L-serine. Chinese Patent No. 113621638 proposes a method for producing L-serine by fermenting a bacterium in which genes related to the L-serine degradation pathway, such as the serine hydroxymethyltransferase glyA gene, the homoserine dehydrogenase thrA gene, the serine dehydratase sdaA gene, the serine dehydratase isozyme sdaB gene, the L-serine transporter sdaC gene, the serine dehydratase isozyme tdcG gene, and the threonine dehydrogenase tdcB gene, have been knocked out.

[0010] International Publication No. 2011080301 discloses Escherichia coli strains for the production of L-methionine. One of these strains contains a missense mutation in the sdaA gene, which results in increased serine utilization for the production of L-methionine. Another of these strains contains an additional copy of the glyA gene. Comparison with a strain containing the 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.

[0011] The object of the present invention is to provide a microorganism capable of producing GAA by using L-serine as a glycine source, a method for producing GAA by culturing the microorganism, and a method for producing creatine.

[0012] The present invention therefore relates to a microorganism comprising at least one heterologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase, in which the gene encoding a protein having the function of L-serine ammonia-lyase [EC 4.3.1.17] (SDHL), also called L-serine deaminase activity or L-serine dehydratase activity, has been inactivated or deleted.

[0013] A heterologous gene means that the gene is inserted into a host organism that does not naturally have this gene. The insertion of a heterologous gene into a host is achieved by recombinant DNA technology. Microorganisms that have been subjected to recombinant DNA technology are called transgenic, genetically modified, or recombinant. A heterologous protein means a protein that does not naturally occur in a microorganism. A homologous gene or endogenous gene means that the gene or the nucleotide sequence of the gene containing such a function naturally occurs in a microorganism or is "native" to a microorganism. A homologous protein or native protein means a protein that naturally occurs in a microorganism.

[0014] Proteins with the function of L-arginine:glycine amidinotransferase (AGAT) belong to the amidinotransferase family. The amidinotransferase family includes glycine (EC:2.1.4.1) amidinotransferase and inosamine (EC:2.1.4.2) amidinotransferase, enzymes involved in the biosynthesis of creatine and streptomycin, respectively. This family also includes arginine deiminase, EC:3.5.3.6. These enzymes catalyze the reaction: [ka]

[0015] Streptococcal antitumor glycoproteins are also found in this family. Enzymes or proteins with L-arginine:glycine-amidinotransferase (AGAT) activity have also been described as having a conserved domain belonging to the PFAM family of amidinotransferases (PF02274) (Marchler-Bauer A et al. (2017), "CDD / SPARCLE: functional classification of proteins via subfamily domain architectures.", Nucleic Acids Res. 45(D1):D200-D203.), and have been 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. al., Jpn J Cancer Res 1987;78:1409-1414(PUBMED:3123442 EPMC:3123442).

[0016] In the microorganism of the present invention, the gene encoding a protein having the function of L-arginine:glycine amidinotransferase may further be overexpressed.

[0017] Overexpression of a gene is generally achieved by increasing the copy number of the gene, and / or by operably linking the gene to a strong promoter, and / or by enhancing the ribosome binding site, and / or by codon usage optimization of the start codon or the entire gene, or by a combination including selection of all of the above methods.

[0018] In particular, gene overexpression can be achieved by increasing the copy number of the gene and / or by enhancing regulatory factors, for example, by operably linking the gene to a strong promoter, enhancing the ribosome binding site, and / or optimizing the codon usage of the start codon or the entire gene. Enhancement of such regulatory factors that positively affect gene expression can be achieved, for example, by modifying the promoter sequence upstream of the structural gene to increase promoter effectiveness, or by completely replacing the promoter with a more effective or so-called strong promoter. A promoter is located upstream of a gene. A promoter is a DNA sequence consisting of approximately 40 to 50 base pairs that constitutes a binding site for RNA polymerase holoenzyme and a transcription start site, thereby affecting the intensity of expression of the controlled polynucleotide or gene. In general, it is possible to achieve overexpression, or increased expression, of genes in bacteria by selecting a strong promoter, for example, by replacing the original promoter with a strong native promoter (originally assigned to another gene) or by modifying specific regions of a given native promoter (e.g., its so-called -10 and -35 regions) toward consensus sequences, as taught, for example, by M. Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for Corynebacterium glutamicum (C. glutamicum). An example of a "strong" promoter is the superoxide dismutase (sod) promoter ("Psod"; Z. Wang et al., Eng. Life Sci. 2015, 15, 73-82). "Operative linkage" is understood to mean the contiguous placement of a promoter with a gene, resulting in transcription of the gene.

[0019] Genetic code is degenerate, which means that a specific amino acid can be coded by several different triplets.The term codon usage refers to the observation that a specific organism typically does not use all possible codons for a specific amino acid at the same frequency.Instead, organisms typically show a certain preference for certain codons, which means that these codons are more frequently found in the coding sequence of the transcribed gene of organisms.When a specific gene that is foreign to a future host, i.e., derived from a different species, is to be expressed in a future host organism, the coding sequence of this gene should be adjusted to the codon usage of the future host organism (i.e., codon usage optimization).

[0020] 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], which is encoded by the glyA gene in Escherichia coli (E. coli) and Corynebacterium glutamicum (C. glutamicum).

[0021] GAA production starting from serine is shown in Figure 2.

[0022] 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.

[0023] In the context of the present invention, gene inactivation means that, although the gene is expressed, it is expressed at a low level or not expressed, or its activity is eliminated or reduced, compared with the parent strain or unmodified strain.In the present invention, inactivation can be achieved by a mutation selected from the following: an insertion mutation in which one or more base pairs are inserted into the gene, or a deletion mutation in which multiple base pairs are deleted within the gene; or by introducing one or more mutations from the group consisting of base pair transition or transversion mutation of nonsense codons into the gene; or by replacing the native promoter of the gene with a weaker promoter of the gene.

[0024] The microorganism according to the invention may further comprise at least one overexpressed gene encoding an enzyme with the function of carbamoyl phosphate synthase (EC 6.3.4.16, CarAB).

[0025] The microorganism of the present invention may further comprise at least one overexpressed gene 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 (EC 6.3.4.5, ArgG), and a gene encoding a protein having the function of argininosuccinate lyase (EC 4.3.2.1, ArgH).

[0026] To achieve a relatively high intracellular L-arginine concentration, it is necessary to prevent L-arginine transport. The amino acid exporter LysE efficiently transports the substrate arginine out of the cell, counteracting the intracellular arginine concentration and reducing substrate availability. In addition, citrulline from arginine biosynthesis is also secreted into the medium by the active LysE exporter. LysE is regulated by the transcriptional activator LysG (Bellmann, A., et al. (2001). "Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum." Microbiology (Reading) 147(Pt 7):1765-1774).

[0027] In a specific embodiment of the present invention, the lysEG gene encoding a protein having the functions of the arginine exporter LysE and its transcriptional activator LysG may be inactivated or deleted in the microorganism of the present invention.

[0028] The microorganism of the present invention may belong to the genus Corynebacterium, preferably Corynebacterium glutamicum (C. glutamicum), or the genus Enterobacteriaceae, preferably Escherichia coli (E. coli), or the genus Pseudomonas, preferably Pseudomonas putida (P. putida).

[0029] In Corynebacterium glutamicum, the gene encoding the protein with arginine exporter function is lysE, and the gene encoding the transcriptional activator is lysG. In Escherichia coli, the gene encoding the protein with arginine exporter function is argO (ybjE). In Pseudomonas putida, the protein with arginine exporter function is lysE.

[0030] A protein having the function of L-arginine:glycine amidinotransferase (AGAT) in a 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 filamentous cyanobacterium Moorea producens.

[0031] The above-mentioned problem is further solved by a method for the fermentative production of guanidinoacetic acid (GAA), comprising the steps of culturing a microorganism according to the invention as defined above in a suitable medium and allowing GAA to accumulate in the medium to form a GAA-containing fermentation broth.

[0032] The method of the present invention may further comprise the step of isolating GAA from the fermentation broth.

[0033] The method according to the present invention may further comprise the step of drying and / or granulating the GAA-containing fermentation broth.

[0034] The present invention further relates to a microorganism as defined above, further comprising a gene encoding an enzyme having the activity of guanidinoacetate N-methyltransferase (EC:2.1.1.2). Preferably, the gene encoding an enzyme having the activity of guanidinoacetate N-methyltransferase is overexpressed.

[0035] The present invention also relates to a method for the fermentative production of creatine, comprising the steps of culturing in a suitable medium a microorganism according to the present invention, which comprises a gene encoding an enzyme having the activity of guanidinoacetate N-methyltransferase, and allowing creatine to accumulate in the medium to form a creatine-containing fermentation broth.

[0036] Preferably, the method further comprises isolating creatine from the creatine-containing fermentation broth. Creatine can be extracted from the fermentation broth by isoelectric focusing and / or ion exchange. Alternatively, creatine can be further purified by recrystallization in water. [Brief explanation of the drawings]

[0037] [Figure 1] Schematic diagram of glycine production from serine via glycine by SHMT. Abbreviations: PGDH - phosphoglycerate dehydrogenase [EC 1.1.1.95], PSAT - phosphoserine transaminase [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. [Figure 2] Schematic diagram of GAA production from serine via glycine by SHMT and AGAT. Abbreviations: PGDH - phosphoglycerate dehydrogenase [EC 1.1.1.95], PSAT - phosphoserine transaminase [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, CH2TFH-(6R)-5,10-methylene-5,6,7,8-tetrahydrofolate.

[0038] A brief description of arrays SEQ ID NO: 1 DNA sequence of plasmid pK18mobsacB-sdaA SEQ ID NO: 2 DNA sequence of synthetic oligonucleotide primer oMC58 SEQ ID NO: 3 DNA sequence of synthetic oligonucleotide primer oMC59 SEQ ID NO: 4 DNA sequence of synthetic oligonucleotide primer oMC60 SEQ ID NO: 5 DNA sequence of synthetic oligonucleotide primer oMC61 SEQ ID NO: 6 DNA sequence of Moorea producens gene with locus_tag BJP34_00300, which encodes L-arginine:glycine amidinotransferase SEQ ID NO: 7. Amino acid sequence of Moorea producens L-arginine:glycine amidinotransferase (NCBI accession number WP_070390602) SEQ ID NO: 8 DNA sequence of plasmid pLIB_P SEQ ID NO: 9 DNA sequence of plasmid pLIB_P[AGAT-Mp] (Example 1)

[0039] Experimental Section A) Materials and Methods chemicals Unless explicitly stated, all chemicals were ordered in analytical grade from Merck, Sigma Aldrich or Carl-Roth.

[0040] molecular biology technology All oligonucleotide primers were synthesized by Eurofins Genomics Germany GmbH (Ebersberg, Germany). Genomic DNA from C. glutamicum ATCC 13032 was isolated according to the manufacturer's instructions for the DNeasy Blood & Tissue Kits (Qiagen, catalog no. 69504). DNA digestion was routinely performed using either High-Fidelity (HF®) restriction enzymes (NEB) or FastDigest Restriction Enzymes (FD; Thermo Fischer Scientific) according to the manufacturer's instructions. Polymerase chain reaction (PCR) was used to amplify the desired DNA region using specific DNA oligos. Q5® High-Fidelity 2X Master Mix with proofreading activity (NEB catalog no. M0492) and Phusion® High-Fidelity DNA Polymerase (NEB catalog no. M0530) were used according to the manufacturer's instructions. PCR and restriction fragment cleanup was performed using a QIAquick PCR Purification Kit (Qiagen, Catalog No. 28106) according to the manufacturer's instructions. If necessary, DNA fragments were purified from agarose gels using a QIAquick Gel Extraction Kit (Qiagen, Catalog No. 28706) according to the manufacturer's instructions. Agarose gel electrophoresis was performed using 0.8-1.2% agarose (Roth, Catalog No. 3810.4) dissolved in 1X TAE buffer (Roth, Catalog No. CL83.3). Gels were cast using Roti® Gelstain (Roth, Catalog No. 3865.1), and electrophoresis itself was performed at 150V for 25-40 minutes, if necessary. DNA assembly of the backbone and insert for plasmid cloning was performed using NEBuilder® HiFi DNA Assembly Master Mix (NEB, Catalog No. E2621). After DNA assembly, the reaction was transformed into E. coli competent cells to obtain individual clones (see DNA transformation).Correct assembly was verified for the presence of the insert by either colony PCR or restriction analysis, and sequence integrity was confirmed by DNA sequencing. The presence of the desired DNA segment from transformed cells was confirmed using Taq DNA Polymerase (Qiagen, Cat. No. 201203) or SapphireAmp® Fast PCR Master Mix (Takara, Cat. No. RR350) according to the manufacturer's instructions. Plasmid DNA was routinely isolated after cloning using one of the following kits according to the manufacturer's instructions: QIAprep Spin Miniprep Kit (Qiagen, Cat. No. 27106), HiSpeed ​​Plasmid Kits (Qiagen, Cat. No. 12643). DNA sequencing was performed by either Eurofins Genomics Germany GmbH (Ebersberg, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

[0041] Strains and culture conditions The type strain / wild-type Corynebacterium glutamicum ATCC13032 (Kinoshita S, Udaka S, Shimono M., J. Gen. Appl. Microbiol. 1957;3(3):193-205) is commercially available from the American Type Culture Collection (ATCC) or DSMZ - German Collection of Microorganisms and Cell Cultures GmbH under the deposit number DSM 20300.

[0042] Corynebacterium glutamicum ATCC 13032 and its derivatives were routinely grown at 30 °C in Brain Heart Infusion (BHI; Merck Millipore, catalog no. 1104930500) broth or in CgXII minimal medium (Keilhauer et al., 1993) supplemented with 10 g / L glucose, 1 g / L L-arginine, and 4 g / L L-serine, as specified. The exact composition of CgXII minimal medium without a carbon source is detailed in Table 1. Glycerol stocks of C. glutamicum were made by mixing 900 μL of an overnight culture in BHI medium (30 °C, 200 rpm, 10 mL of medium) with 600 μL of sterile 86% (w / v) glycerol solution and then stored at -80 °C. E. coli strains were routinely grown at 37°C in Lysogeny Broth (LB) medium (Sigma, catalog no. L3022-1KG). Solid medium was supplemented with 1.5% (w / v) agar. When necessary, antibiotics were used for selection at the following concentrations: chloramphenicol—34 mg / L for E. coli and 7.5 mg / L for C. glutamicum; kanamycin—25–50 mg / L for E. coli and 15–25 mg / L for C. glutamicum.

[0043] [Table 1]

[0044] DNA transformation E. coli chemical or electrocompetent cells were used for cloning and amplification of plasmid DNA (NEB, catalog numbers C3019, C3020, C3040) according to the manufacturer's instructions. After a recovery period, cells were spread onto LB agar plates containing the appropriate antibiotic for selection.

[0045] A modified protocol was used to make C. glutamicum strains electrocompetent: 100 mL cultures of BHI + 0.5 M sorbitol were grown at 33 °C and 250 rpm from an overnight preculture to an OD of 0.3. 600Inoculate to an OD of 1.75 600 The cells were incubated at 33°C and 130 rpm until a 50% saturation was reached. The culture was transferred to a 50 mL Falcon tube and collected by centrifugation (3400 g, 10 min, 4°C). The cell pellets were combined and washed twice with 50 mL of ice-cold Tris-glycerol buffer (1 mM Tris-HCl pH 7.5, 10% (v / v) glycerol), followed by two washing steps with 50 mL of ice-cold 10% (v / v) glycerol. The final cell pellet was resuspended in 800 μL of ice-cold 10% (v / v) glycerol. Electrocompetent cells were aliquoted in 150 μL aliquots and stored at -80°C. For electroporation, an aliquot of cells was thawed on ice and DNA was added. The cell-DNA mixture was transferred to a 2 mm electroporation cuvette (Sigma-Aldrich, catalog no. Z706088-50EA), and a 2.5 kV, 200 Ω, 25 μF pulse with a 5 ms time constant was triggered using a Gene Pulser Xcell™ (Bio-Rad). The cuvette was briefly placed on ice, and the contents were transferred to 4 mL of BHI (preheated to 46°C), followed by a 6-minute heat shock at 46°C. Cells were allowed to recover in BHI medium at 30°C and 200 rpm for 50 minutes. After recovery, cells were spread onto BHI agar plates containing the appropriate antibiotic for selection and incubated at 30°C for 2–3 days to obtain single-cell colonies. Correct maintenance of replicative plasmids after transformation was verified by plasmid DNA isolation using a QIAprep Spin Miniprep Kit (Qiagen, catalog no. 27106X4) according to the manufacturer's instructions, except that cells were resuspended in buffer P1 and incubated at 37°C for 2 hours before lysis. The isolated plasmid DNA was then verified by restriction analysis.

[0046] Allelic exchange in C. glutamicum Allelic replacement for both gene deletion and gene integration at defined loci was performed using pK18mobsacB (NCBI accession number: FJ437239) (Schafer et al., 1994) derivative plasmids containing upstream and downstream homologous regions (approximately 300–1000 bp) and, in the case of integration, the desired insert. The appropriate plasmids were transformed into C. glutamicum strains by electroporation, as previously described. After electroporation, the first recombination event resulting in an intermediate strain was selected on BHI agar plates containing kanamycin. The intermediate strains were then screened by colony PCR. To promote the second recombination event, the intermediate strains were then counterselected on BHI + 10% (w / v) sucrose without antibiotic agar plates. After counterselection, sucrose-resistant and kanamycin-sensitive colonies were screened by colony PCR and DNA sequencing using appropriate primers outside the homologous regions to verify that the desired allelic exchange had occurred.

[0047] Cultivation of C. glutamicum strains for GAA production in Example 3 The appropriate C. glutamicum strain was used to inoculate a preculture from a glycerol stock. The preculture was grown in 10 mL of BHI medium containing the appropriate antibiotics, as needed, in a 100 mL baffled Erlenmeyer flask and incubated at 30°C and 200 rpm for 24 h. The preculture was harvested by centrifugation (3100 g, 10 min, room temperature (RT)) and washed with 5 mL of CgXII medium without a carbon source (see Table 1). The washed pellet was resuspended in 2.5 mL of CgXII medium without a carbon source, and the OD of the cell suspension was measured using an Ultraspec 2100 pro (Amersham Biosciences) spectrophotometer. 600 An appropriate volume of washed cells was used to grow the main culture in CgXII medium supplemented with 10 g / L glucose, 1 g / L L-arginine, 1.275 g / L L-ornithine, and 3 g / L glycine, with appropriate antibiotics as needed, to a starting OD of 0.5. 600The main culture was transferred to a 48-well multi-layer plate (Beckman Coulter Life Sciences, Catalog No. M2P-MTP-48-BOH1) using 800 μL / well, and the plate was covered with sealing foil (Beckman Coulter Life Sciences / m2p Labs, Catalog No. F-GPR48-10).

[0048] The covered plate was placed in a BioLector® I (Beckman Coulter Life Sciences / m2p Labs) and incubated at 1400 rpm and 30°C for 27 hours. After 27 hours, the OD 600 was measured spectrophotometrically, the cultures were centrifuged (2000 g, 10 min), and the supernatants were used to measure GAA production.

[0049] Quantification of GAA GAA from culture supernatants was quantified by HPLC-UV using a Dionex Ultimate 3000 System (Thermo Scientific). Samples were filtered using a Sartorius Minisart NML Plus 0.2 μm filter (Sartorius AG, catalog number ST17823-K) and then appropriately diluted with mobile phase A (see below). Samples (10 μL injection volume) were then separated at 35°C using a HyperCarb 100x4.6 mm 7 μm column (Thermo Scientific catalog number 35007-104630). Mobile phase A consisted of 2.3 g of ammonium dihydrogen phosphate and 2.6 g of diammonium hydrogen phosphate dissolved in 2 L of purified water. Mobile phase B consisted of 2.3 g of ammonium dihydrogen phosphate and 2.6 g of diammonium hydrogen phosphate dissolved in 1.25 L of purified water and 0.75 L of acetonitrile. A 1.0 mL min. was used during the run. -1The flow rate was maintained constant. The column was pre-equilibrated with 100% mobile phase A, and a gradient between phases A and B was used: 0–8 min: linear gradient of phase B increasing from 0 to 10%; 8–10 min: linear gradient increasing from 10 to 40% B; 10–11 min: 40% B; 11.1–13 min: 0% B, and 13–14 min: constant at 0% B to re-equilibrate the column. The total run time was 14 min. Under these conditions, the retention time of GAA was 5.8 min. GAA was detected at 200 nm (210 nm as reference) using a UV detector (ThermoScientific Dionex Ultimate 3000 DAD).

[0050] B) Experimental results Example 1: Construction of an sdaA inactivated strain in C. glutamicum To improve L-serine utilization by preventing its conversion to pyruvate, the sdaA gene of C. glutamicum was inactivated. The sdaA gene of C. glutamicum encodes a protein with L-serine ammonia-lyase (EC 4.3.1.17) activity (abbreviated as SDHL). Alternative names for SDHL are serine deaminase, L-serine dehydratase, and especially L-serine deaminase. Inactivation was achieved via allelic replacement with the plasmid pK18mobsacB-sdaA (SEQ ID NO: 1) in C. glutamicum ATCC 13032, resulting in the C. glutamicum ATCC 13032 ΔsdaA strain.

[0051] pK18mobsacB-sdaA was cloned as follows: Backbone: BamHI, SalI linearized pK18mobsacB. Insert: PCR of ATCC 13032 gDNA with oligos oMC58 (SEQ ID NO: 2) and oMC59 (SEQ ID NO: 3), PCR of ATCC 13032 gDNA with oligos oMC60 (SEQ ID NO: 4) and oMC61 (SEQ ID NO: 5). Assembled via HiFi Assembly mix.

[0052] To inactivate sdaA, pK18mobsacB-sdaA was transformed into C. glutamicum ATCC 13032 by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating onto BHI agar supplemented with 134 g / L sorbitol, 2.5 g / L yeast extract, and 25 mg / L kanamycin. The agar plates were incubated at 33°C for 48 hours. Individual colonies were transferred to fresh agar plates (containing 25 mg / L kanamycin) and incubated at 33°C for 24 hours. Liquid cultures of these clones were grown in 10 ml of BHI medium contained in 100 ml Erlenmeyer flasks with three baffles at 33°C for 24 hours. To isolate clones that had undergone a second recombination event, aliquots were taken from each liquid culture, appropriately diluted, and plated (typically 100–200 μl) on BHI agar supplemented with 10% sucrose. These agar plates were incubated at 33°C for 48 hours. Colonies grown on sucrose-containing agar plates were then examined for kanamycin sensitivity. To do so, cellular material was removed from the colonies using a toothpick and transferred onto BHI agar containing 25 mg / L kanamycin 20 and BHI agar containing 10% sucrose. The agar plates were incubated at 33°C for 60 hours. Clones found to be sensitive to kanamycin and resistant to sucrose were examined by PCR and DNA sequencing. The resulting strain was designated C. glutamicum ATCC 13032 ΔsdaA.

[0053] Example 2: Plasmid-based AGAT expression A heterologous gene encoding L-arginine:glycine amidinotransferase (AGAT), EC 2.1.4.1, is required for the formation of guanidinoacetic acid (GAA) from L-arginine and glycine. Moorea producens is a filamentous cyanobacterium. The genome of M. producens strain PAL-8-15-08-1 is available under GenBank accession number CP017599.1 (Leao et al., 2017). It contains an open reading frame encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1; locus_tag BJP34_00300) shown in SEQ ID NO: 6. SEQ ID NO: 7 shows the corresponding amino acid sequence (NCBI accession number WP_070390602). As an intermediate step to facilitate subsequent cloning, the AGAT-Mp expression cassette was assembled into a plasmid, pLIB_P[AGAT-Mp] (SEQ ID NO: 9). pLIB_P[AGAT-Mp] contains a promoter sequence, a ribosome binding site, and a codon-optimized version of the AGAT-Mp gene. The corresponding gene product has the same amino acid sequence as shown in SEQ ID NO: 7.

[0054] Plasmid pLIB_P (SEQ ID NO: 8) contains the pBL1-derived replication origin for C. glutamicum, the pSC101 replication origin for E. coli, a kanamycin resistance gene, and a strong promoter (P from (Rytter et al., 2014)). 294MU ) and BioBricks Terminator BBa_B1006.

[0055] A codon-optimized version of the AGAT-Mp gene containing the RBS and flanked by Eco31I (BsaI) sites was ordered from Eurofins Genomics. It was delivered as a synthetic gene cloned into pEX-A258. The codon-optimized AGAT-Mp was cloned into pLIB_P to yield pLIB_P[AGAT-Mp].

[0056] pLIB_P[AGAT-Mp] was cloned as follows: Backbone: Eco31I linearized pLIB_P. Insert: 1.3 kb fragment from Eco31I digest of pEX-A258-AGAT-Mp. Assembled via HiFi Assembly mix.

[0057] The pLIB_P[AGAT-Mp] plasmid was transformed into the C. glutamicum ATCC 13032 ΔsdaA strain (prepared in Example 1) and the C. glutamicum ATCC 13032 strain (reference strain) to test GAA production from these strains. Plasmid-containing cells were selected using 25 mg / L kanamycin.

[0058] The confirmed strains were designated C. glutamicum ATCC 13032 pLIB_P[AGAT-Mp] and C. glutamicum ATCC 13032 ΔsdaA pLIB_P[AGAT-Mp], respectively.

[0059] Example 3: Effect of inactivation of L-serine ammonia-lyase (SDHL) and implementation of L-arginine:glycine amidinotransferase (AGAT) on GAA production To evaluate the effect of inactivating L-serine ammonia lyase (SDHL) and implementing L-arginine:glycine amidinotransferase (AGAT) on GAA production, the C. glutamicum ATCC 13032 pLIB_P[AGAT-Mp] strain and the C. glutamicum ATCC 13032 ΔsdaA pLIB_P[AGAT-Mp] strain were cultivated in the BioLector system in CGXII medium, and the resulting GAA titers were determined.

[0060] [Table 2]

[0061] 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].

[0062] Therefore, we conclude that inactivation of L-serine ammonia-lyase improves GAA production in strains implementing L-arginine:glycine amidinotransferase (AGAT).

[0063] References Jakoby, M., Ngouoto-Nkili, C.-E., & Burkovski, A. (1999). Construction and application of new Corynebacterium glutamicum vectors. Biotechnology Techniques, 13(6), 437-441. https: / / doi.org / 10.1023 / A:1008968419217 Keilhauer, C., Eggeling, L., &Sahm, H. (1993). Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon.J Bacteriol,175(17),5595-5603.https: / / doi.org / 10.1128 / jb.175.17.5595-5603.1993 Leao,T.,Castelao,G.,Korobeynikov,A.,Monroe,E.A.,Podell,S.,Glukhov,E.,Allen,E.E.,Gerwick,W.H.,&Gerwick,L.(2017).Comparative genomics uncovers the prolific and distinctive metabolic potential of the cyanobacterial genus Moorea.Proc Natl Acad Sci U S A,114(12),3198-3203.https: / / doi.org / 10.1073 / pnas.1618556114 Rytter,J.V.,Helmark,S.,Chen,J.,Lezyk,M.J.,Solem,C.,&Jensen,P.R.(2014).Synthetic promoter libraries for Corynebacterium glutamicum.Appl Microbiol Biotechnol,98(6),2617-2623.https: / / doi.org / 10.1007 / s00253-013-5481-x Schafer,A.,Tauch,A.,Jager,W.,Kalinowski,J.,Thierbach,G.,&Puhler,A.(1994).Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene,145(1),69-73.https: / / doi.org / 10.1016 / 0378-1119(94)90324-7 Simic,P.,Willuhn,J.,Sahm,H.,&Eggeling,L.(2002).Identification of glyA(encoding serine hydroxymethyltransferase)and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum.Appl Environ Microbiol,68(7),3321-3327.https: / / doi.org / 10.1128 / AEM.68.7.3321-3327.2002 Vrljic,M.,Sahm,H.,&Eggeling,L.(1996).A new type of transporter with a new type of cellular function:L-lysine export from Corynebacterium glutamicum.Mol Microbiol,22(5),815-826.https: / / doi.org / 10.1046 / j.1365-2958.1996.01527.x

Claims

1. A microorganism comprising at least one heterologous gene encoding a protein having the function of L-arginine:glycine amidinotransferase, in which the gene encoding a protein having the function of L-serine ammonia-lyase has been inactivated or deleted.

2. 2. The microorganism according to claim 1, further comprising an overexpressed gene encoding an enzyme having the function of L-serine hydroxymethyltransferase.

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

4. 4. A microorganism according to any one of claims 1 to 3, further comprising at least one overexpressed gene encoding an enzyme with the function of carbamoyl phosphate synthase.

5. 5. The microorganism according to claim 1, further comprising at least one overexpressed gene selected from the group consisting of a gene encoding a protein having the function of ornithine carbamoyltransferase, a gene encoding a protein having the function of argininosuccinate synthetase, and a gene encoding a protein having the function of argininosuccinate lyase.

6. 6. The microorganism according to claim 1, wherein the lysEG gene encoding a protein having the functions of arginine exporter LysE and its transcription activator LysG is inactivated or deleted.

7. 10. A method for the fermentative production of guanidinoacetic acid (GAA), comprising culturing a microorganism defined in any one of claims 1 to 6 in a medium and allowing GAA to accumulate in the medium to form a GAA-containing fermentation broth.

8. 8. The method of claim 7, further comprising isolating GAA from the GAA-containing fermentation broth.

9. The microorganism according to any one of claims 1 to 6, further comprising a gene encoding an enzyme having guanidinoacetate N-methyltransferase activity.

10. 10. The microorganism of claim 9, wherein the gene encoding an enzyme having guanidinoacetate N-methyltransferase activity is overexpressed.

11. 11. A method for the fermentative production of creatine, comprising culturing a microorganism as defined in claim 9 or 10, and allowing creatine to accumulate in the medium to form a creatine-containing fermentation broth.

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

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