Improved biotechnological methods for producing guanidinoacetic acid (GAA) by inactivation of amino acid exporters

Abstract

The present invention relates to microorganisms transformed with an inactivated amino acid exporter capable of producing guanidinoacetic acid (GAA), and to a method for the fermentative production of GAA using such microorganisms.The present invention also relates to a method for the fermentative production of creatine.

Description

[Technical field]

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

[0002] GAA is an organic compound used as an animal feed additive (WO2005120246 / US2011257075). GAA is a natural precursor of creatine. Therefore, supplementation with GAA allows optimal supply of creatine in the organism.

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

[0004] Guthmiller et al. (J Biol Chem. 1994 Jul 1;269(26):17556-60) characterized rat kidney AGAT by cloning and heterologous expression of the enzyme in E. coli. Muenchhoff et al. (FEBS Journal 277 (2010) 3844-3860) also reported the first characterization of AGAT from a prokaryote by cloning and heterologous expression of the enzyme in E. coli. To produce GAA from L-arginine and glycine by whole-cell catalysis, Zhang et al. engineered a reconstituted ornithine cycle in Escherichia coli by introducing heterologous AGATs from different species (e.g., Homo sapiens, Cylindrospermopsis raciborskii, Moorea producens) and introducing citrulline synthesis modules (e.g., overexpression of carAB, argF, and argl) and arginine synthesis modules (e.g., overexpression of argG, argH; introduction of aspA) into Escherichia coli (Yiwen Zhang, Hang Zhou, Yong Tao, and Baixue Lin, ACS Synth. Biol. 2020, 9, 2066-2075).

[0005] Several approaches to increase the production of one of the starting materials in GAA synthesis, i.e. L-arginine, in microorganisms, especially bacteria, are also known from the literature. An overview on the metabolic engineering of Corynebacterium glutamicum (C. glutamicum) for L-arginine production is provided by Park et al. (NATURE COMMUNICATIONS | DOI: 10.1038 / ncomms5618). They have already proposed a stepwise rational metabolic engineering based on a system-wide analysis of the metabolism resulting in a gradual increase in L-arginine production through random mutagenesis and screening and strain engineering steps for L-arginine production of L-arginine-producing C. glutamicum strains, such as ATCC21831 (Nakayama and Yoshida 1974, US Pat. No. 3,849,250). Yim et al. (J Ind MicrobiolBiotechnol (2011) 38:1911-1920) were able to show that inactivation of the argR gene, which encodes the central repressor protein ArgR that controls the L-arginine biosynthetic pathway, gave rise to improved arginine producing strains by disrupting the chromosomal argR gene in C. glutamicum. Ginesy et al. (Microbial Cell Factories (2015) 14:29) report successful engineering of E. coli to enhance arginine production. Among other things, they propose the deletion of the argR repressor gene.

[0006] Kurahashi et al. (European Patent Publication No. 1057893) report a method for increasing the L-arginine production capacity of a microorganism by improving L-arginine biosynthetic enzymes using recombinant DNA technology, for example by using a microorganism belonging to the genus Corynebacterium or Brevibacterium that harbors recombinant DNA having vector DNA and DNA fragments containing genes for acetylornithine deacetylase, N-acetylglutamate-γ-semialdehyde dehydrogenase, N-acetylglutaminase and argininosuccinase derived from a microorganism belonging to the genus Escherichia. For improved L-arginine production, the authors further propose a microorganism that has an increased activity of intracellular glutamate dehydrogenase (GDH) and L-arginine production capacity.

[0007] Suga et al. reported a method of using a genetically engineered strain in which a gene that inhibits the expression of the arginine biosynthesis operon argR was inactivated (US Pat. No. 7,160,705). In particular, a deletion in argR, which controls the arginine operon, was considered to be an important factor in arginine production. In Corynebacterium microorganisms, the argCJBDFR genes involved in arginine biosynthesis are organized in the form of an operon and are subject to feedback inhibition by intracellular arginine (Sakanyan et al., Microbiology, 142:9-108, 1996), which imposes limitations on the high yield of L-arginine production. The arginine operon is an operon consisting of genes encoding enzymes involved in the mechanism of L-arginine biosynthesis, and in particular, the arginine operon consists of genes encoding enzymes that constitute the cycle steps of L-arginine biosynthesis. In particular, the arginine operon consists of N-acetylglutamylphosphate reductase (ArgC), glutamate N-acetyltransferase (ArgJ), N-acetylglutamate kinase (ArgB), acetylornithine aminotransferase (ArgD), ornithine carbamoyltransferase (ArgF), and arginine repressor (ArgR). These enzymes are involved in the sequential enzymatic reactions of L-arginine biosynthesis.

[0008] According to the literature, the amino acid exporter LysE catalyzes the cellular excretion transport of not only L-lysine, but also L-arginine and L-citrulline. LysG activates the transcription of the gene lysE, which codes for the amino acid exporter LysE. LysG requires co-inducers, such as L-lysine, L-arginine, L-citrulline or L-histidine (Lubitz et al. (2016). "Roles of export genes cgmA and lysE for the production of L-arginine and L-citrulline by Corynebacterium glutamicum." Appl MicrobiolBiotechnol 100(19): 8465-8474). Lubitz et al. used the C. glutamicum strain ARG2 (Peters-Wendisch et al. (2014) Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production. J Biotechnol. doi:10.1016 / j.jbiotec.2014.01.023), which harbors a feedback-resistant allele of ArgB (ArgB fbr ) inactivation of the argR gene by deletion combined with plasmid-based expression of arginine. For this strain carrying both modifications, the authors describe the accumulation of L-arginine in the culture supernatant. Furthermore, Lubitz et al. describe that in such a strain, the inactivation of the genes lysE and cmg, which encodes a membrane protein, results in reduced arginine formation.

[0009] Ginesy et al. (M. Ginesy et al., Microbiol Cell Factories (2015) 14:29, DOI 10.1186 / s12934-015-0211-y) were able to show that arginine production using an E. coli production strain with, inter alia, a deleted argR repressor gene, can be increased by overexpression of the arginine exporter system.

[0010] However, to obtain a relatively high intracellular L-arginine concentration, it is necessary to prevent L-arginine efflux transport. The amino acid exporter LysE efficiently transports the substrate arginine out of the cell, thereby preventing intracellular arginine concentration and reducing substrate utilization. In addition, citrulline from arginine biosynthesis is also secreted into the medium by the active LysE exporter. LysE is regulated by the transcription 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).

[0011] Fan Wenchao has disclosed a method for producing creatine by fermentation of non-pathogenic microorganisms, such as Corynebacterium glutamicum (China Patent Publication No. 106065411). The microorganism has the following biotransformation functions: conversion of glucose to L-glutamic acid; conversion of L-glutamic acid to N-acetyl-L-glutamic acid; conversion of N-acetyl-L-glutamic acid to N-acetyl-L-glutamic semialdehyde; conversion of N-acetyl-L-glutamic semialdehyde to N-acetyl-L-ornithine; conversion of N-acetyl-L-ornithine to L-ornithine; conversion of L-ornithine to L-citrulline; conversion of L-citrulline to argininosuccinic acid; conversion of argininosuccinic acid to L-arginine; conversion of L-arginine to guanidinoacetate; and finally, conversion of guanidinoacetate to creatine. Fan Wenchao proposes that the microorganism overexpresses one or more enzymes selected from the group consisting of N-acetylglutamate synthase, N-acetylornithine-δ-aminotransferase, N-acetylornithinase, ornithine-carbamoyltransferase, argininosuccinate synthetase, glycine amidinotransferase (EC:2.1.4.1), and guanidinoacetate N-methyltransferase (EC:2.1.1.2). The microorganism preferably overexpresses glycine aminotransferase (L-arginine:glycine amidinotransferase) and guanidinoacetate N-methyltransferase.

[0012] The problem underlying the present invention is to provide improved microorganisms transformed so as to be capable of producing guanidinoacetic acid (GAA) and to provide a method for the fermentative production of GAA using such microorganisms.

[0013] This problem is solved by a microorganism, which has an increased capacity to provide L-arginine compared to the capacity of a wild-type microorganism, which comprises at least one gene encoding a protein with the function of L-arginine:glycine amidinotransferase, and which has a reduced activity of a protein with the function of arginine exporter compared to the activity of the respective protein in a wild-type microorganism at the same state of the cell cycle.

[0014] This means that a protein having the function of an arginine exporter in a microorganism according to the invention exhibits, at any time and under any circumstance throughout the cell cycle, an artificially designed reduced activity compared to the activity of the respective protein in a wild-type microorganism at the same time and under any circumstance throughout the cell cycle.

[0015] The microorganism according to the invention is preferably a genetically modified microorganism (GMO) that does not occur in nature. In GMOs, the genetic material has been modified using genetic engineering techniques. Preferably, at least one gene encoding a protein with the function of L-arginine:glycine amidinotransferase has been introduced using genetic engineering techniques. Preferably, a reduction in the activity of a protein with the function of arginine exporter has also been achieved using genetic engineering techniques, compared to the activity of the respective protein in a wild-type microorganism.

[0016] Proteins with the function of L-arginine:glycine amidinotransferase (AGAT) belong to the amidinotransferase family, which includes glycine amidinotransferase (EC:2.1.4.1) and inosamine amidinotransferase (EC:2.1.4.2), enzymes involved in creatine and streptomycin biosynthesis, respectively. This family also includes arginine deiminase, EC:3.5.3.6. These enzymes catalyze the reaction arginine + H2O ⇔ citrulline + NH3. Streptococcus antitumor glycoproteins are also found in this family. Enzymes or proteins with L-arginine:glycine amidinotransferase (AGAT) activity have also been described that have a conserved domain belonging to the PFAM family: Amidinotransf (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., Jpn J It is also described in Cancer Res 1987;78:1409-1414 (PUBMED:3123442 EPMC:3123442).Specific examples of AGAT are those of Moorea producens, Homo sapiens, the brown rat (Rattus norvegicus), the Malaysian flying lemur (Galeopterus variegatus), and Cylindrospermopsis raciborskii.

[0017] In the context of the present invention, a microorganism with an improved ability to provide L-arginine refers to a microorganism that produces or recycles L-arginine beyond its own needs. This property may be achieved by selection of a microorganism that is a natural L-arginine producer or may acquire the ability to produce L-arginine by mutation. Examples of such L-arginine producing microorganisms are e.g. C. glutamicum ATCC21831 or those described in Park et al. (NATURE COMMUNICATIONS | DOI: 10.1038 / ncomms5618) or Ginesy et al. (Microbial Cell Factories (2015) 14:29).

[0018] In one embodiment of the present invention, the argR gene encoding the arginine-responsive repressor protein ArgR in the microorganism according to the present invention is weakened or deleted.

[0019] The activity of an enzyme having the function of carbamoyl phosphate synthase (EC 6.3.4.16, e.g. CarAB) in the microorganism according to the invention may be increased compared to the respective enzyme activity in a wild-type microorganism. This may be achieved by mutation and / or overexpression of the gene encoding the enzyme having the function of carbamoyl phosphate synthase.

[0020] Furthermore, in the microorganism according to the invention, at least one or more genes encoding enzymes of the biosynthetic pathway of L-ornithine and L-arginine may be overexpressed, including argF / argF2 encoding ornithine carbamoyltransferase, argG encoding argininosuccinate synthetase and argH encoding argininosuccinate lyase.

[0021] Additionally or alternatively, at least one or more genes encoding enzymes of the L-ornithine and L-arginine biosynthetic pathway, including gdh encoding glutamate dehydrogenase, argJ encoding ornithine acetyltransferase, argB encoding acetylglutamate kinase, argC encoding acetylglutamylphosphate reductase and argD encoding acetylornithine aminotransferase, may be overexpressed in the microorganism according to the invention.

[0022] 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 optimizing the codon usage of the start codon or the entire gene or by a combination with any selection of all of the above methods.

[0023] In the microorganism according to the invention, the gene encoding a protein with the function of L-arginine:glycine amidinotransferase may be heterologous.

[0024] The microorganism according to the invention is preferably recombinant and the gene encoding a protein with the function of L-arginine:glycine amidinotransferase (AGAT) is preferably heterologous.

[0025] A heterologous gene means that a gene has been inserted into a host organism that does not naturally possess this gene. The insertion of a heterologous gene into a host is accomplished by recombinant DNA technology. Microorganisms that have undergone recombinant DNA technology are said to be transgenic, genetically modified or recombinant.

[0026] In the microorganism of the present invention, the gene encoding a protein having the function of L-arginine:glycine amidinotransferase may further be overexpressed. Overexpression of the gene is generally 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 whole gene or by a combination with the selection of all the above methods.

[0027] The protein having the function of L-arginine:glycine amidinotransferase (AGAT) encoded by at least one respective gene in the microorganism of the invention may, for example, have an amino acid sequence according to SEQ ID NO: 13, i.e. an amino acid sequence which is at least 70% identical, preferably 80% or at least 90% identical to AGAT of Moorea producens ("AGAT_Mp"). In a further embodiment of the invention, the amino acid sequence of L-arginine:glycine amidinotransferase is identical to the amino acid sequence according to SEQ ID NO: 13 (see database UniPort, 15 February 2017, "Glycine amidinotransferase", XP055706853, EBI accession number UNIPROT: A0A1D8TKD3). The wild-type DNA sequence encoding Moorea producens AGAT is SEQ ID NO:12, and the corresponding DNA sequence with codons optimized for C. glutamicum is SEQ ID NO:14.

[0028] The protein having the function of L-arginine:glycine amidinotransferase encoded by at least one respective gene in the microorganism of the present invention may have, for example, an amino acid sequence that is at least 70% homologous, preferably at least 80% or at least 90% identical to the amino acid sequence of AGAT of Cylindrospermopsis raciborskii ATW205 (J. Muenchhoff et al., FEBS Journal 277 (2010) 3844-3860).

[0029] The protein having the function of L-arginine:glycine amidinotransferase encoded by at least one respective gene in the microorganism of the present invention may have an amino acid sequence which is at least 70% homologous, preferably at least 80% or at least 90% identical to the amino acid sequence of AGAT of the Malayan flying lemur (Galeopterus variegatus).

[0030] A protein having the function of L-arginine:glycine amidinotransferase in the microorganism of the present invention may have an amino acid sequence which is at least 70% homologous, preferably at least 80% or at least 90% homologous to the amino acid sequence of the AGAT of homo sapiens, e.g. the AGAT of Homo sapiens itself (A. Humm, Biochem. J. (1997) 322, 771-776) or the AGAT of Rattus norvegicus.

[0031] Generally, according to the invention, overexpression of a gene is achieved by increasing the copy number of the gene and / or by enhancing a regulator, for example by functionally linking the gene with a strong promoter and / or by enhancing the ribosome binding site and / or by optimizing the codon usage of the start codon or the whole gene. The enhancement of such regulators, which have a positive effect on gene expression, can be achieved, for example, by modifying the promoter sequence upstream of the structural gene in order to increase the promoter's effectiveness or by completely replacing said promoter with a more effective or so-called strong promoter. The promoter is located upstream of the gene. A promoter is a DNA sequence consisting of about 40-50 base pairs, which constitutes a binding site for the RNA polymerase holoenzyme and a transcription initiation site, thereby making it possible to influence the strength of expression of the polynucleotide or gene to be controlled. In general, as taught, for example, by M. Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for C. glutamicum, overexpression or increased expression of genes can be achieved in bacteria by selection of a strong promoter, for example by replacement of the original promoter with a strong natural promoter (originally assigned to another gene) or by modification of certain regions of a given natural promoter (for example the so-called -10 and -35 regions) to a consensus sequence. An example of a "strong" promoter is the superoxide dismutase (sod) promoter ("Psod"; Z. Wang et al., Eng. Life Sci. 2015, 15, 73-82). "Operatively linked" is understood to mean a contiguous alignment of the promoter and the gene that causes transcription of the gene.

[0032] The genetic code is degenerate, meaning that a particular amino acid can be coded by many different triplets. The term codon usage generally refers to the observation that a given organism does not use all possible codons for a given amino acid with the same frequency. Instead, organisms generally show a certain preference for certain codons, meaning that these codons are more frequently found in the coding sequences of the organism's transcribed genes. When a given gene, foreign to a future host, i.e., heterologous, is to be expressed in a future host organism, the coding sequence of said gene should be adapted to the codon usage of said future host organism (i.e., codon usage optimization).

[0033] Table 1 shows the variously named enzymes involved or contributing to arginine biosynthesis in various species, namely, E. coli, C. glutamicum and Pseudomonas putida (P. putida). [Table 1-1] [Table 1-2]

[0034] In the microorganism of the present invention, the gene encoding the protein having the function of arginine exporter may be inactivated or deleted.Furthermore, in the microorganism of the present invention, the gene encoding the transcription activator of the gene encoding the protein having the function of arginine exporter may be deleted.

[0035] 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).

[0036] In Corynebacterium glutamicum, the gene encoding the protein with the function of arginine exporter is lysE, and the gene encoding the transcription activator is lysG. In Escherichia coli, the gene encoding the protein with the function of arginine exporter is argO(ybjE). In Pseudomonas putida, the protein with the function of arginine exporter is lysE.

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

[0038] The method according to the invention may further comprise adding glycine and / or L-arginine and / or L-ornithine to the medium. Preferably, the medium is supplemented with glycine in a concentration ranging from 0.1 to 300 g glycine per liter of medium, preferably 0.82 g glycine per liter of medium, and / or with L-arginine to obtain a concentration ranging from 0.1 to 200 g L-arginine per liter of medium, preferably 1.9 g L-arginine per liter of medium.

[0039] The method of the present invention may further comprise the step of isolating GAA from the fermentation broth.

[0040] The process according to the invention may further comprise the step of drying and / or granulating the GAA-containing fermentation broth.

[0041] 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.

[0042] The present invention also relates to a method for producing creatine by fermentation, comprising the steps of: a) culturing a microorganism according to the present invention having a gene encoding an enzyme having the activity of guanidinoacetate N-methyltransferase in a suitable medium under suitable conditions; and b) accumulating creatine in the medium to form a creatine-containing fermentation broth.

[0043] Preferably, the method further comprises isolating creatine from the creatine-containing fermentation broth. Creatine may be extracted from the fermentation broth by isoelectric focusing and / or ion exchange. Alternatively, creatine may be further purified by recrystallization in water.

[0044] Experimental Section A) Materials and Methods Chemicals Kanamycin solution from Streptomyces kanamyceticus was purchased from Sigma Aldrich (St. Louis, USA, Cat. no. K0254). Unless otherwise stated, all other chemicals were purchased at analytical purity from Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, USA) or Carl-Roth (Karlsruhe, Germany).

[0045] Culture for cell proliferation Unless otherwise stated, culture / incubation procedures were performed as follows: a. LB broth (MILLER) from Merck (Darmstadt, Germany; Cat. no. 110285) was used to cultivate E. coli strains in liquid medium. The liquid medium (10 ml of liquid medium per 100 ml Erlenmeyer flask with three baffles) was incubated at 30° C. and 200 rpm in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland).

[0046] b. LB agar (MILLER) from Merck (Darmstadt, Germany, Cat. no. 110283) was used for the cultivation of E. coli strains on agar plates. The agar plates were incubated at 30° C. in an INCU-Line® mini incubator from VWR (Radnor, USA).

[0047] Brain heart infusion broth (BHI) from c. Merck (Darmstadt, Germany, Cat. no. 110493) was used to cultivate C. glutamicum strains in liquid medium (10 ml of liquid medium per 100 ml Erlenmeyer flask with three baffles) and incubated at 30°C and 200 rpm in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland).

[0048] Brain Heart Agar (BHI-Agar) from d. Merck (Darmstadt, Germany, Cat. no. 113825) was used to cultivate C. glutamicum strains on agar plates. The agar plates were incubated at 30° C. in a Heraeus Instruments incubator equipped with a Kelvitron® temperature controller (Hanau, Germany).

[0049] e. For culturing C. glutamicum after electroporation, BHI agar (Merck, Darmstadt, Germany, Cat. no. 113825) was supplemented with sorbitol (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) 134 g / l, yeast extract (Oxoid / ThermoFisher Scientific, Waltham, USA, Cat. no. LP0021) 2.5 g / l and kanamycin 25 mg / l. Agar plates were incubated at 30° C. in a Heraeus Instruments incubator equipped with a Kelvitron® temperature controller (Hanau, Germany).

[0050] Determination of optical density of bacterial suspensions a. The optical density of the bacterial suspension in the shake flask cultures was determined at 600 nm (OD600) using a biophotometer from Eppendorf AG (Hamburg, Germany).

[0051] b. The optical density of bacterial suspensions produced in a Wouter Duetz (WDS) microfermentation system (24-well plates) was determined at 660 nm (OD660) using a GENios™ plate reader from Tecan Group AG (Maennedorf, Switzerland).

[0052] Centrifugation a. Bacterial suspensions with a maximum volume of 2 ml were centrifuged in 1.5 ml or 2 ml reaction tubes (e.g., Eppendorf Tubes® 3810X) using an Eppendorf 5417 R benchtop centrifuge (13,000 rpm for 5 min).

[0053] b. Bacterial suspensions with a maximum volume of 50 ml were centrifuged in 15 ml or 50 ml reaction tubes (e.g., Falcon™ 50 ml Conical Centrifuge Tubes) at 4,000 rpm for 10 min using an Eppendorf 5810 R benchtop centrifuge.

[0054] DNA isolation Plasmid DNA was isolated from E. coli cells using the QIAprep Spin Miniprep kit from Qiagen (Hilden, Germany, Cat. No. 27106) according to the manufacturer's instructions.

[0055] Polymerase chain reaction (PCR) PCR with proofreading (high fidelity) polymerases was used to amplify desired fragments of DNA for Sanger sequencing or DNA assembly. Non-proofreading polymerase kits were used to determine the presence or absence of desired DNA fragments directly from E. coli or C. glutamicum colonies.

[0056] a. Phusion® High Fidelity DNA Polymerase Kit (Phusion Kit) from New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530) was used for template-directed amplification of selected DNA regions according to the manufacturer's instructions (see table). [Table 2]

[0057] b. The Taq PCR Core Kit (Taq Kit) from Qiagen (Hilden, Germany, Cat. No. 201203) was used to amplify the desired fragment of DNA to confirm its presence. The kit was used according to the manufacturer's instructions (see table). [Table 3]

[0058] c. SapphireAmp® Fast PCR Master Mix (Sapphire Mix) from Takara Bio Inc (Takara Bio Europe SAS, Saint-Germain-en-Laye, France, Cat. No. RR350A / B) was used as an alternative to confirm the presence of the desired fragment of DNA in cells taken from E. coli or C. glutamicum colonies according to the manufacturer's instructions (see table). [Table 4]

[0059] d. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany) using the phosphoramidite method described by McBride and Caruthers (1983).

[0060] e. As PCR template, appropriate dilutions of isolated plasmid DNA or total DNA isolated from liquid culture medium or total DNA contained in bacterial colonies were used (colony PCR). For said colony PCR, templates were prepared by picking cellular material from colonies on agar plates with a toothpick and placing the cellular material directly into a PCR reaction tube. The cellular material was heated for 10 seconds at 800W in a microwaveable Mikrowave & Grill from SEVERIN Elektrogeraete GmbH (Sundern, Germany), and then PCR reagents were added to the template in the PCR reaction tube.

[0061] f. All PCR reactions were carried out in a PCR cycle type Mastercycler or Mastercycler nexus gradient from Eppendorf AG (Hamburg, Germany).

[0062] Restriction enzyme digestion of DNA For restriction enzyme digestion, "FastDigest Restriction Endonuclease (FD)" (ThermoFisher Scientific, Waltham, USA) or New England BioLabs Inc. (Ipswich, USA) restriction endonucleases were used. The reactions were carried out according to the instructions in the manufacturer's manual.

[0063] Determining DNA fragment size a. The size of small DNA fragments (<1000 bps) was routinely determined by automated capillary electrophoresis using a QIAxcel from Qiagen (Hilden, Germany).

[0064] b. If DNA fragments needed to be isolated or were >1000 bps, DNA was separated by TAE agarose gel electrophoresis and stained with GelRed® Nucleic Acid Gel Stain (Biotium, Inc., Fremont, Canada). Stained DNA was visualized at 302 nm.

[0065] PCR amplification and purification of restriction fragments PCR amplification products and restriction fragments were cleaned up using the QIAquick PCR purification kit from Qiagen (Hilden, Germany; Cat. No. 28106) according to the manufacturer's instructions. DNA was eluted in 30 μl of 10 mM Tris*HCl (pH 8.5).

[0066] Determination of DNA concentration DNA concentrations were measured using a NanoDrop spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH, VWR brand since 2015 (Erlangen, Germany).

[0067] Assembly Cloning Plasmid vectors were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" from New England BioLabs Inc. (Ipswich, USA, Cat. No. E5520). The reaction mixture containing the linearized vector and at least one DNA insert was incubated at 50°C for 60 min. 0.5 μl of the assembly mixture was used for each transformation experiment.

[0068] Chemical transformation of E. coli For plasmid cloning, chemically competent "NEB® Stable Competent E. coli (high performance)" (New England BioLabs Inc., Ipswich, USA, Cat. No. C3040) was transformed according to the manufacturer's protocol. Successfully transformed cells were selected on LB agar supplemented with 25 mg / l kanamycin.

[0069] Transformation of C. glutamicum Transformation of C. glutamicum with plasmid DNA was performed by electroporation using a "Gene Pulser Xcell" (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) as described by Ruan et al. (2015). Electroporation was performed in 1 mm electroporation cuvettes (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) at 1.8 kV with a fixed time constant of 5 ms. Transformed cells were selected on BHI agar containing sorbitol 134 g / l, yeast extract 2.5 g / l and kanamycin 25 mg / l.

[0070] C. glutamicum strains Corynebacterium glutamicum ATCC13032 (DSM 20300, Kinoshita S, Udaka S, Shimono M., J. Gen. Appl. Microbiol. 1957; 3(3): 193-205), C. glutamicum wild-type strain is commercially available from the American Type Culture Collection (ATCC) or DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.

[0071] Corynebacterium glutamicum ATCC21831, an L-arginine producing C. glutamicum strain (U.S. Pat. No. 3,849,250), is commercially available from the American Type Culture Collection (ATCC).

[0072] Nucleotide sequence determination The nucleotide sequences of the DNA molecules were determined by Eurofins Genomics GmbH (Ebersberg, Germany) by cycle sequencing using the dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences USA 74, 5463-5467, 1977). The Clonemanager Professional 9 software from Scientific & Educational Software (Denver, USA) was used for visualization and evaluation of the sequences.

[0073] Glycerol stocks of E. coli and C. glutamicum strains For long-term storage of E. coli and C. glutamicum strains, glycerol stocks were prepared. Selected E. coli clones were cultured in 10 ml of LB medium supplemented with 2 g / l glucose. Selected C. glutamicum clones were cultured in 10 ml of double concentrated BHI medium supplemented with 2 g / l glucose. The medium for growing plasmid containing E. coli and C. glutamicum strains was supplemented with 25 mg / l kanamycin. The medium was placed in a 100 ml Erlenmeyer flask equipped with three baffles. This was inoculated with a loop of cells taken from a colony. The culture was then incubated at 30° C. and 200 rpm for 18 h. After said incubation period, 1.2 ml of 85% (v / v) sterile glycerol was added to the culture. The resulting glycerol-containing cell suspension was then divided into 2 ml aliquots and stored at -80°C.

[0074] GAA production in shake flask cultures Shake flask cultures in 250 mL baffled Erlenmeyer flasks were used for evaluation of GAA production of the strains.

[0075] Preculture of the strain was performed in 10 ml of seed medium (SM). This medium was placed in a 100 ml Erlenmeyer flask. It was inoculated with 100 μl of glucose stock culture and the culture was incubated at 30° C. and 200 rpm for 24 h. The composition of seed medium (SM) is shown in the table. Kanamycin was added to the culture as required to maintain the plasmid. [Table 5]

[0076] The optical density OD600 of the pre-culture was determined after said incubation period. A volume required to inoculate 2.5 ml of production medium (PM) to an OD600 of 0.5 was sampled from the pre-culture, centrifuged (1 min at 8000 g) and the supernatant was discarded. The cells were then suspended in 200 μl of production medium.

[0077] The main culture was started by inoculating 100 μl of resuspended cells from the preculture into wells of a 24-well WDS plate containing 2.4 ml of production medium (PM), the composition of which is shown in the table. [Table 6]

[0078] The main culture was incubated at 30° C. and 200 rpm in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland) for 48 h until glucose was completely consumed. The glucose concentration in the suspension was analyzed using a blood glucose meter OneTouch Vita® from LifeScan (Johnson & Johnson Medical GmbH, Neuss, Germany).

[0079] After incubation, the culture suspension was transferred to a 50 ml centrifuge tube (e.g., Falcon™ 50 ml Conical Centrifuge Tubes). A portion of the culture suspension was appropriately diluted and the OD660 was measured. The other portion of the culture was centrifuged and the GAA concentration in the supernatant was analyzed as described below.

[0080] Quantification of GAA The samples were analyzed on an Agilent analytical system consisting of an HPLC "Infinity 1260" coupled with a mass spectrometer "Triple Quad 6420" (Agilent Technologies Inc., Santa Clara, USA). Chromatographic separation was performed on an Atlantis HILIC Silica column, 4.6 x 250 mm, 5 μm (Waters Corporation, Milford, USA) at 35 °C. Mobile phase A was water with 10 mM ammonium formate and 0.2% formic acid. Mobile phase B was a mixture of 90% acetonitrile and 10% water, to which 10 mM ammonium formate was added. The HPLC system started with 100% B and continued to 66% B with a linear gradient for 22 min and a constant flow rate of 0.6 mL / min. The mass spectrometer was operated in ESI positive ionization mode. For the detection of GAA, the m / z values ​​were monitored by using MRM fragmentation [M+H]+118-76. The limit of quantification (LOQ) for GAA was fixed at 7 ppm.

[0081] B) Experimental results Example 1: Cloning of plasmid pK19mobsacB-ΔlysEG for chromosomal deletion of genes lysE and lysG in C. glutamicum-based strains. The gene lysE encodes an exporter protein that catalyzes the efflux of L-lysine, L-arginine and L-citrulline in Corynebacterium glutamicum. Expression of lysE is positively regulated by the gene product of lysG. Both genes are located next to each other but are divergently transcribed.

[0082] To inactivate the transporter protein LysE and the positive regulator protein LysG, the plasmid pK19mobsacB-ΔlysEG (SEQ ID NO: 1) was constructed as described for pK18mobsacB-ΔlysEG in Vrljic et. al 1996 (Vrljic, M., et al. (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).

[0083] Example 2: Chromosomal deletion of the gene argR in ATCC13032 To improve intracellular L-arginine formation and L-arginine recycling from L-ornithine, the gene argR, which encodes the central repressor protein ArgR that controls the L-arginine biosynthetic pathway, was inactivated.

[0084] Therefore, the plasmid pK18mobsacB_DargR was constructed as follows: Plasmid pK18mobsacB (Schaefer, 1994) was cut with Xbal and the linearized vector DNA (5721 bps) was purified using the "QIAquick Gel Extraction Kit" (Qiagen GmbH, Hilden, Germany).

[0085] To assemble the insert, two DNA fragments were generated by PCR using the following pair of primers (genomic DNA of ATCC13032 as template): DargR_lf (SEQ ID NO: 2) + DargR_lr (SEQ ID NO: 3) = left homology arm (983 bps) DargR_rf (SEQ ID NO: 4) + DargR_rr (SEQ ID NO: 5) = left homology arm (984 bps)

[0086] The product DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0087] The linearized plasmid and PCR product were then assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). The resulting deletion vector was named pK18mobsacB_DargR. It was verified by restriction enzyme digestion and DNA sequencing.

[0088] For deletion of the argR gene, pK18mobsacB_DargR was transformed into ATCC13032 by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with sorbitol 134 g / l, yeast extract 2.5 g / l and kanamycin 25 mg / l. Agar plates were incubated at 33° C. for 48 h.

[0089] Individual colonies were transferred onto new agar plates (with kanamycin 25 mg / l) and incubated at 33 °C for 24 h. Liquid cultures of these clones were cultivated at 33 °C for 24 h in 10 ml of BHI medium placed in 100 ml Erlenmeyer flasks equipped with three baffles. 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% saccharose. These agar plates were incubated at 33 °C for 48 h. Colonies growing on the saccharose-containing agar plates were then tested for kanamycin sensitivity. For this, cellular material was removed from the colonies using a toothpick and transferred onto BHI agar containing kanamycin 25 mg / l and onto BHI agar containing 10% saccharose. These agar plates were incubated at 33 °C for 60 h. Clones that proved to be sensitive to kanamycin and resistant to saccharose were examined by PCR and DNA sequencing. The resulting strain was designated ATCC13032_DargR.

[0090] Example 3: Chromosomal insertion of the sod promoter upstream of the carAB operon in ATCC13032_DargR To improve L-arginine production, a strong sod-promoter was inserted upstream of the carAB operon into the genome of ATCC13032_DargR. Therefore, the plasmid pK18mobsacB_Psod-carAB was constructed as follows: pK18mobsacB was cut with EcoRI+HindIII and the linearized vector DNA (5670bps) was excised from an agarose gel. DNA was extracted using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0091] To assemble the insert, three DNA fragments were generated by PCR using the following primer pairs (genomic DNA of ATCC13032 as template): PsodcarAB-LA-F (SEQ ID NO: 6) + PsodcarAB-LA-R (SEQ ID NO: 7) = left homology arm (1025 bps) PsodcarAB-F (SEQ ID NO: 8) + PsodcarAB-R (SEQ ID NO: 9) = sod-promoter (250 bps) PsodcarAB-RA-F (SEQ ID NO: 10) + PsodcarAB-RA-R (SEQ ID NO: 11) = left homology arm (944 bps)

[0092] The product DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany). The linearized plasmid and PCR product were then assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). Appropriate plasmid clones were identified by restriction digestion and DNA sequencing.

[0093] The resulting plasmid, pK18mobsacB_Psod-carAB, was then transformed into ATCC13032_DargR by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with sorbitol 134 g / l, yeast extract 2.5 g / l and kanamycin 25 mg / l. Agar plates were incubated at 33° C. for 48 h.

[0094] Individual colonies were transferred onto new agar plates (with kanamycin 25 mg / l) and incubated at 33 °C for 24 h. Liquid cultures of these clones were cultivated at 33 °C for 24 h in 10 ml of BHI medium placed in 100 ml Erlenmeyer flasks equipped with three baffles. 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% saccharose. These agar plates were incubated at 33 °C for 48 h. Colonies growing on the saccharose-containing agar plates were then tested for kanamycin sensitivity. For this, cellular material was removed from the colonies using a toothpick and transferred onto BHI agar containing kanamycin 25 mg / l and onto BHI agar containing 10% saccharose. These agar plates were incubated at 33 °C for 60 h. Clones that proved sensitive to kanamycin and resistant to saccharose were checked for proper integration of the sod promoter by PCR and DNA sequencing. The resulting strain was named ATCC13032_DargR_Psod-carAB.

[0095] Example 4: Chromosomal deletion of genes lysE and lysG in ATCC13032_DargR_Psod-carAB The gene lysE encodes an exporter protein that catalyzes the efflux of L-lysine, L-arginine and L-citrulline in Corynebacterium glutamicum. Expression of lysE is positively regulated by the gene product of lysG. Both genes are located next to each other but are divergently transcribed. To delete the genes lysE and lysG, pK19mobsacB_DlysEG (see Example 1) was transformed into ATCC13032_DargR_Psod-carAB by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with sorbitol 134 g / l, yeast extract 2.5 g / l and kanamycin 25 mg / l. The agar plates were incubated at 33 °C for 48 h.

[0096] Individual colonies were transferred onto new agar plates (with kanamycin 25 mg / l) and incubated at 33 °C for 24 h. Liquid cultures of these clones were cultivated at 33 °C for 24 h in 10 ml of BHI medium placed in 100 ml Erlenmeyer flasks equipped with three baffles. 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% saccharose. These agar plates were incubated at 33 °C for 48 h. Colonies growing on the saccharose-containing agar plates were then tested for kanamycin sensitivity. For this, cellular material was removed from the colonies using a toothpick and transferred onto BHI agar containing kanamycin 25 mg / l and onto BHI agar containing 10% saccharose. These agar plates were incubated at 33 °C for 60 h. Clones that proved to be sensitive to kanamycin and resistant to saccharose were examined by PCR and DNA sequencing. The resulting strain was named ATCC13032_DargR_Psod-carAB_DlysEG.

[0097] Example 5: Cloning of the gene AGAT-Mp encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Moorea producens Moorea producens is a filamentous cyanobacterium. The genome of Moorea producens strain PAL-8-15-08-1 was published by Leao et al. (Leao T, Castelao G, Korobeynikov A, Monroe EA, Podell S, Glukhov E, Allen EE, Gerwick WH, Gerwick L, Proc Natl Acad Sci US A. 2017 Mar 21;114(12):3198-3203. doi: 10.1073 / pnas.1618556114; Genbank accession number CP017599.1). It contains an open reading frame encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1; locus_tag BJP34_00300 see SEQ ID NO: 12). SEQ ID NO: 13 shows the derived amino acid sequence (Genbank accession number WP_070390602).

[0098] This amino acid sequence was translated back into a DNA sequence (SEQ ID NO: 14) optimized for codon usage in C. glutamicum using the software tool "Optimizer" (http: / / genomes.urv.es / OPTIMIZER / ).

[0099] The fragment of the optimized gene consisting of base pairs 13 to 1142 was extended with a BsmBI restriction site at its 5' end. A second stop codon, the lysS terminator from C. glutamicum and a BsmBI restriction site were added at the 3' end. The resulting DNA sequence (SEQ ID NO: 15) was ordered for gene synthesis from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) and delivered as part of a cloning plasmid carrying an ampicillin resistance gene (designated pMA-RQ_AGAT_Mp_opt).

[0100] A second DNA fragment was designed, consisting of the sequence for assembly cloning, the promoter sequence, the ribosome binding site and the first 81 nucleotides of the optimized AGAT-Mp gene, which was ordered from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA; SEQ ID NO: 16) as a linear DNA string for gene synthesis.

[0101] Example 6: Cloning of expression plasmid pLIB_pBL1_AGAT-Mp The E. coli-C. glutamicum shuttle plasmid pLIB_pBL1 contains the origin of replication from pBL1, the pSC101 origin of replication, the kanamycin resistance gene and the BioBricks Terminator BBa_B1006 downstream of the NotI restriction site (SEQ ID NO: 17). It was digested with the restriction endonuclease NotI and the DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0102] A DNA fragment was amplified by PCR with pMA-RQ_AGAT_Mp_opt as template using primers AGAT_f (SEQ ID NO: 18) and AGAT_r (SEQ ID NO: 19). The PCR product was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0103] The linearized plasmid, promoter containing DNA string and PCR product were assembled using "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). The assembled product was transformed into "NEB Stable Competent E. coli (High Efficiency)" (New England Biolabs, Ipswich, USA) and cells were grown on LB agar containing 25 mg / l kanamycin. Appropriate plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pLIB_pBL1_AGAT-Mp.

[0104] Example 7: Chromosomal expression of arginine biosynthetic genes argF, argG, and argH under the control of the strong constitutive promoter Pg3 To increase the activity of ArgF, ArgG and ArgH, additional copies of the corresponding genes were inserted into the genome. A synthetic operon consisting of Pg3, argF, argG, argH and flanking regions for genome integration was designed (SEQ ID NO: 20).

[0105] The DNA sequence was ordered from Invitrogen / Geneart (Thermo Fisher Scientific, Waltham, USA) for gene synthesis and was delivered as part of a cloning plasmid carrying an ampicillin resistance gene (designated pMA-RQ_argFGH).

[0106] A DNA fragment was amplified by PCR with pMA-RQ_argFGH as template using primers argFGH_f (SEQ ID NO: 21) and argFGH_r (SEQ ID NO: 22). The PCR product was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0107] Plasmid pK18mobsacB (Schaefer, A. et al., Gene. 1994 Jul 22;145(1):69-73. doi: 10.1016 / 0378-1119(94)90324-7) was digested with EcoRI+HindIII and the linearized vector DNA (5670bps) was excised from an agarose gel. DNA was extracted using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0108] The linearized plasmid and PCR product were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). The assembly product was transformed into "NEB Stable Competent E. coli (High Efficiency)" (New England Biolabs, Ipswich, USA) and cells were grown on LB agar containing 25 mg / l kanamycin. Appropriate plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pK18_IBcg0054::Pg3-argFGH (SEQ ID NO: 23).

[0109] To insert the synthetic operon, pK18_IBcg0054::Pg3-argFGH was transformed into ATCC13032_DargR by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with sorbitol 134 g / l, yeast extract 2.5 g / l and kanamycin 25 mg / l. Agar plates were incubated at 33° C. for 48 h. Individual colonies were transferred onto new agar plates (with kanamycin 25 mg / l) and incubated at 33° C. for 24 h. Liquid cultures of these clones were grown in 10 ml of BHI medium in 100 ml Erlenmeyer flasks equipped with three baffles at 33° C. for 24 h. 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% saccharose. These agar plates were incubated at 33 °C for 48 h. Colonies growing on the saccharose-containing agar plates were then tested for kanamycin sensitivity. For this, cellular material was removed from the colonies using a toothpick and transferred onto BHI agar containing 25 mg / l kanamycin and onto BHI agar containing 10% saccharose. These agar plates were incubated at 33 °C for 60 h. Clones that were found to be sensitive to kanamycin and resistant to saccharose were examined by PCR and DNA sequencing. The resulting strain was designated ATCC13032_DargR_IBcg0054::Pg3-argFGH.

[0110] Example 8: Chromosomal deletion of genes lysE and lysG in ATCC13032_DargR_IBcg0054::Pg3-argFGH. The gene lysE encodes an exporter protein that catalyzes the efflux of L-lysine, L-arginine and L-citrulline in Corynebacterium glutamicum. The expression of lysE is positively regulated by the gene product of lysG. Both genes are located next to each other but are divergently transcribed. To delete the genes lysE and lysG, pK19mobsacB_DlysEG (see Example 1) was transformed into ATCC13032_DargR_IBcg0054::Pg3-argFGH by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with sorbitol 134 g / l, yeast extract 2.5 g / l and kanamycin 25 mg / l. The agar plates were incubated at 33 °C for 48 h.

[0111] Individual colonies were transferred onto new agar plates (with kanamycin 25 mg / l) and incubated at 33 °C for 24 h. Liquid cultures of these clones were cultivated at 33 °C for 24 h in 10 ml of BHI medium placed in 100 ml Erlenmeyer flasks equipped with three baffles. 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% saccharose. These agar plates were incubated at 33 °C for 48 h. Colonies growing on the saccharose-containing agar plates were then tested for kanamycin sensitivity. For this, cellular material was removed from the colonies using a toothpick and transferred onto BHI agar containing kanamycin 25 mg / l and onto BHI agar containing 10% saccharose. These agar plates were incubated at 33 °C for 60 h. Clones that proved to be sensitive to kanamycin and resistant to saccharose were examined by PCR and DNA sequencing. The resulting strain was designated ATCC13032_DargR_IBcg0054::Pg3-argFGH_DlysEG.

[0112] Example 9: Chromosomal deletion of genes lysE and lysG in ATCC21831_DlysEG. The gene lysE encodes an exporter protein that catalyzes the efflux of L-lysine, L-arginine and L-citrulline in Corynebacterium glutamicum. Expression of lysE is positively regulated by the gene product of lysG. Both genes are located next to each other but are divergently transcribed. To delete the genes lysE and lysG, pK19mobsacB_DlysEG (see Example 1) was transformed into ATCC21831_DlysEG by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on BHI agar supplemented with sorbitol 134 g / l, yeast extract 2.5 g / l and kanamycin 25 mg / l. The agar plates were incubated at 33 °C for 48 h.

[0113] Individual colonies were transferred onto new agar plates (with kanamycin 25 mg / l) and incubated at 33 °C for 24 h. Liquid cultures of these clones were cultivated at 33 °C for 24 h in 10 ml of BHI medium placed in 100 ml Erlenmeyer flasks equipped with three baffles. 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% saccharose. These agar plates were incubated at 33 °C for 48 h. Colonies growing on the saccharose-containing agar plates were then tested for kanamycin sensitivity. For this, cellular material was removed from the colonies using a toothpick and transferred onto BHI agar containing kanamycin 25 mg / l and onto BHI agar containing 10% saccharose. These agar plates were incubated at 33 °C for 60 h. Clones that proved to be sensitive to kanamycin and resistant to saccharose were examined by PCR, and the resulting strain was designated ATCC21831_DlysEG.

[0114] Example 10: Transformation of C. glutamicum strains with pLIB_pBL1_AGAT-MP The following strains of C. glutamicum were transformed with pLIB_pBL1_AGAT-Mp by electroporation and plasmid-containing cells were selected with 25 mg / l kanamycin. The resulting plasmid-containing strains are shown in the table. [Table 7]

[0115] C. glutamicum ATCC13032: A commonly used wild-type strain (Kinoshita et al., J. Gen. Appl. Microbiol. 1957; 3(3): 193-205) · ATCC13032_DargR_Psod-carAB: increased L-arginine production capacity due to reduced activity of the ArgR regulator protein and integration of the strong sod promoter upstream of carAB in ATCC13032. ATCC13032_DargR_Psod-carAB_DlysEG: Increased L-arginine production capacity by reducing the activity of the ArgR regulator protein and integrating a strong sod promoter upstream of carAB in ATCC13032. Reduced L-arginine efflux transport activity. · ATCC13032_DargR_IBcg0054::Pg3-argFGH: reduced activity of ArgR regulator protein and increased L-arginine production capacity by genomic integration of an argFGH expression cassette under the control of the strong promoter Pg3 in ATCC13032. ATCC13032_DargR_IBcg0054::Pg3-argFGH_DlysEG: Increased L-arginine production capacity by reducing the activity of the ArgR regulator protein and genomic integration of an argFGH expression cassette under the control of the strong promoter Pg3 in ATCC13032. Reduced L-arginine efflux transport activity. · C. glutamicum strain ATCC21831 (Park et al., Nat Commun. 2014 Aug 5; 5:4618) synthesizes L-arginine from primary substrates such as ammonia and glucose. C. glutamicum strain ATCC21831_DlysEG (Park et al., Nat Commun. 2014 Aug 5; 5:4618) synthesizes L-arginine from primary substrates such as ammonia and glucose. Reduced efflux transport activity of L-arginine.

[0116] Example 11: Effect of increasing L-arginine production capacity and decreasing arginine efflux transport on GAA production To evaluate the combined effect of increased L-arginine production capacity and reduced L-arginine efflux transport on GAA production, strains ATCC13032, ATCC13032 / pLIB_pBL1, ATCC13032 / pLIB_pBL1_AGAT-Mp, ATCC13032_DargR_Psod-carAB / pLIB_pBL1_AGAT-Mp, ATCC13032_DargR_Psod-carAB_DlysEG / pLIB_pBL1_AGAT-Mp, ATCC13032_DargR_IBcg0054::Pg3-argFGH / pLIB_pBL1_AGAT-Mp and ATCC13032_DargR_IBcg0054::Pg3-argFGH_DlysEG / pLIB_pBL1_AGAT-Mp were cultured in Wouter culture medium. Cultures were grown in the Duetz system and the resulting GAA titers were determined. [Table 8]

[0117] As shown in the table, ATCC13032_DargR_Psod-carAB / pLIB_pBL1_AGAT-Mp and ATCC13032_DargR_IBcg0054::Pg3-argFGH / pLIB_pBL1_AGAT-Mp, which have a polynucleotide encoding AGAT from Moorea producens, a deleted argR gene, and increased expression of the carAB or argFGH genes, produce 2.2 g / l or 1.7 g / l of GAA, respectively.

[0118] Strains ATCC13032_DargR_Psod-carAB_DlysEG / pLIB_pBL1_AGAT-Mp and ATCC13032_DargR_IBcg0054::Pg3-argFGH_DlysEG / pLIB_pBL1_AGAT-Mp also have a polynucleotide encoding AGAT from Moorea producens, have a deleted argR gene, and increase the expression of the carAB or argFGH genes. In addition, the gene lysEG is inactivated, which reduces L-arginine efflux transport activity. These strains produce 2.3 g / l and 1.9 g / l of GAA, respectively, which is improved compared to strains without reduced L-arginine efflux transport. The inventors concluded that the combination of increased enzyme AGAT activity, L-arginine production activity, and reduced L-arginine efflux transport improves GAA production.

[0119] Example 12: The effect of increasing L-arginine production capacity and decreasing arginine efflux transport on GAA production using the L-arginine producing strain ATCC21831. To evaluate the combined effect of increased L-arginine production capacity and reduced L-arginine efflux transport on GAA production, strains ATCC21831, ATCC21831 / pLIB_pBL1 and ATCC21831 / pLIB_pBL1_AGAT-Mp were cultivated in the Wouter Duetz system in production medium and the resulting GAA titers were determined. [Table 9]

[0120] As shown in Table 9, ATCC21831, ATCC21831 / pLIB_pBL1 and ATCC21831_DlysEG / pLIB_pBL1 are unable to produce GAA.

[0121] The strain ATCC21831 / pLIB_pBL1_AGAT-Mp also has a polynucleotide encoding AGAT from Moorea producens and produces 5.0 g / L of GAA. Furthermore, as shown in Table 8, the strain ATCC13032 / pLIB_pBL1_AGAT-Mp, which has a polynucleotide encoding AGAT from Moorea producens but cannot supply additional L-arginine, produces only 1.1 g / L of GAA.

[0122] The strain ATCC21831_DlysEG / pLIB_pBL1_AGAT-Mp also contains a polynucleotide encoding AGAT from Moorea producens, but additionally the gene lysEG is inactivated in the strain, resulting in reduced L-arginine efflux transport activity. This strain produces 5.6 g / l GAA, which is an improvement compared to the strain without reduced L-arginine efflux transport.

[0123] The inventors concluded that a combination of increasing the enzyme AGAT activity and ability to provide L-arginine, and reducing L-arginine efflux transport, improves GAA production.

[0124] Summary of arrays: SEQ ID NO:1 shows the DNA sequence encoding the plasmid pK19mobsacB-DlysEG, assembled as described in Vrljic et al. 1996 (Vrljic, M., et al. (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). SEQ ID NO: 2 shows the DNA sequence encoding primer DargR_lf. SEQ ID NO: 3: Shows the DNA sequence encoding primer DargR_lr. SEQ ID NO: 4 shows the DNA sequence encoding plasmid DargR_rf. SEQ ID NO: 5 shows the DNA sequence encoding primer DargR_rr. SEQ ID NO: 6 shows the DNA sequence encoding primer PsodcarAB-LA-F. SEQ ID NO: 7: Shows the DNA sequence encoding primer PsodcarAB-LA-R. SEQ ID NO: 8: Shows the DNA sequence encoding primer PsodcarAB-F. SEQ ID NO: 9 shows the DNA sequence encoding primer PsodcarAB-R. SEQ ID NO: 10 shows the DNA sequence encoding primer PsodcarAB-RA-F. SEQ ID NO: 11 shows the DNA sequence encoding primer PsodcarAB-RA-R. SEQ ID NO: 12: Shows the open reading frame encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1; locus_tag BJP34_00300). SEQ ID NO: 13: Shows the amino acid sequence derived from SEQ ID NO: 12 (Genbank accession number WP_070390602). SEQ ID NO: 14: DNA sequence encoding L-arginine:glycine amidinotransferase from Moorea producens (AGAT, EC 2.1.4.1) optimized for codon usage in C. glutamicum. SEQ ID NO: 15: DNA sequence encoding the codon usage optimized L-arginine:glycine amidinotransferase from Moorea producens (AGAT, EC 2.1.4.1) from C. glutamicum derived from SEQ ID NO: 14 with a fragment consisting of base pairs 13 to 1142 extended with a BsmBI restriction site at the 5' end, in addition to which a second stop codon, the lysS terminator from C. glutamicum and a BsmBI restriction site were added at the 3' end. SEQ ID NO: 16: A DNA fragment consisting of a sequence for assembly cloning, a promoter sequence, a ribosome binding site and the first 81 nucleotides of the optimized AGAT-Mp gene. SEQ ID NO: 17: shows the DNA sequence of shuttle plasmid pLIB_pBL1, which has the origin of replication from pBL1, the pSC101 origin of replication, the kanamycin resistance gene and the BioBricks Terminator BBa_B1006 downstream of the NotI restriction site. SEQ ID NO: 18 shows the DNA sequence of primer AGAT_f. SEQ ID NO: 19 shows the DNA sequence of primer AGAT_r. Sequence number 20: Shows the DNA sequence of promoter Pg3-argFGH. Sequence number 21: shows the DNA sequence of primer argFGH_f. Sequence number 22: shows the DNA sequence of primer argFGH_r. Sequence number 23: Shows the DNA sequence of plasmid pK18_IBcg0054::Pg3-argFGH.

Claims

1. A microorganism comprising: has an increased ability to provide L-arginine compared to the ability of a wild-type microorganism; having at least one gene encoding a protein having the function of L-arginine:glycine amidinotransferase; the activity of a protein having arginine exporter function is reduced compared to the activity of the respective protein in said wild-type microorganism at the same stage of the cell cycle; Microorganisms.

2. The microorganism according to claim 1, wherein the activity of the arginine-responsive repressor protein ArgR is weakened or deleted.

3. 3. The microorganism according to claim 1 or 2, in which the enzymatic activity having the function of carbamoyl phosphate synthase is increased compared to the respective enzymatic activity in the wild-type microorganism.

4. 4. The microorganism according to claim 3, wherein the increase in the activity of the enzyme having the function of carbamoyl phosphate synthase is achieved by mutation and / or overexpression of a gene encoding an enzyme having the function of carbamoyl phosphate synthase.

5. argF / argF2, which encodes ornithine carbamoyltransferase; argG, which encodes argininosuccinate synthetase; and argH, encoding argininosuccinate lyase 3. The microorganism according to claim 1, wherein at least one or more genes encoding enzymes in the biosynthetic pathway of L-ornithine and L-arginine are overexpressed.

6. 3. The microorganism according to claim 1 or 2, wherein the gene gdh encoding glutamate dehydrogenase is overexpressed.

7. 3. The microorganism of claim 1, wherein the gene encoding the protein having the function of L-arginine:glycine amidinotransferase is heterologous.

8. 3. The microorganism according to claim 1 or 2, wherein the protein having the function of L-arginine:glycine amidinotransferase has an amino acid sequence which is at least 70% identical to the amino acid sequence according to SEQ ID NO:

13.

9. 3. The microorganism according to claim 1 or 2, wherein the gene encoding the protein having the function of an arginine exporter is inactivated or deleted.

10. The microorganism according to claim 1 or 2, wherein a gene encoding a transcription activator of the gene encoding the protein having the function of an arginine exporter is deleted.

11. The microorganism according to claim 1 or 2, wherein the microorganism belongs to the genus Corynebacterium, Enterobacteriaceae or Pseudomonas.

12. The microorganism according to claim 11, wherein the microorganism is Corynebacterium glutamicum, the gene encoding the protein having the function of arginine exporter is lysE, and the gene encoding the transcription activator is lysG.

13. The microorganism according to claim 11 , wherein the microorganism is Escherichia coli and the gene encoding the protein having the function of an arginine exporter is argO(ybjE).

14. The microorganism according to claim 11, wherein the microorganism is Pseudomonas putida and the protein having the function of an arginine exporter is lysE.

15. 1. A method for the fermentative production of guanidinoacetic acid (GAA), comprising: a) culturing a microorganism according to claim 1 or 2 in a suitable medium under suitable conditions; b) accumulating GAA in the medium to form a GAA-containing fermentation broth; A method comprising:

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

17. 16. The method of claim 15, further comprising drying and / or granulating the GAA-containing fermentation broth.

18. The microorganism according to claim 1 or 2, further comprising a gene encoding an enzyme having guanidinoacetate N-methyltransferase activity.

19. 19. The microorganism of claim 18, wherein the gene encoding an enzyme having the activity of guanidinoacetate N-methyltransferase is overexpressed.

20. 1. A method for the fermentative production of creatine, comprising: a) culturing the microorganism of claim 18 in a suitable medium under suitable conditions; b) accumulating creatine in the medium to form a creatine-containing fermentation broth; A method comprising:

21. 21. The method of claim 20, further comprising isolating creatine from the creatine-containing fermentation broth.