Improved biotechnological process for production of guanidinoacetic acid (GAA) by targeted introduction or by increasing activity of transmembrane outward transporters

Abstract

The present invention relates to a microorganism comprising at least one gene encoding a protein having the function of an L-arginine: glycine amidino transferase and having increased transmembrane outward transporter activity; and to methods for the fermentative production of guanidinoacetic acid (GAA) and to methods for the fermentative production of creatine using such microorganisms.

Description

Technical Field

[0001] Guanidinoacetic acid (GAA) is a colorless crystalline organic compound used as an animal feed additive (e.g., WO2005120246A1 and US2011257075 A1). GAA is a natural precursor of creatine (e.g., Humm et al., Biochem. J. (1997) 322, 771-776). Thus, supplementation with GAA allows for optimal supply of creatine in organisms.

[0002] The present invention relates to microorganisms capable of being transformed to produce guanidinoacetic acid (GAA) and methods for fermenting such microorganisms to produce GAA. The present invention also relates to methods for fermenting to produce creatine. Background Art

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

[0004]

[0005] Guthmiller et al. (J Biol Chem. 1994 Jul 1; 269(26):17556-60) have characterized rat kidney AGAT by cloning and heterologous expression of the enzyme in Escherichia coli (E. coli). Muenchhoff et al. (FEBS Journal 277(2010) 3844-3860) have also reported the first characterization of AGAT from prokaryotes by cloning and heterologous expression of the enzyme in E. coli.

[0006] Fan Wenchao disclosed a method for producing creatine by fermenting non-pathogenic microorganisms such as Corynebacterium glutamicum (CN106065411A). The microorganisms have the following biotransformation functions: converting glucose into L-glutamic acid; converting L-glutamic acid into N-acetyl-L-glutamic acid; converting N-acetyl-L-glutamic acid into N-acetyl-L-glutamic semialdehyde; converting N-acetyl-L-glutamic semialdehyde into N-acetyl-L-ornithine; converting N-acetyl-L-ornithine into L-ornithine; converting L-ornithine into L-citrulline; converting L-citrulline into argininosuccinic acid; converting argininosuccinic acid into L-arginine; converting L-arginine into guanidinoacetic acid; and finally converting guanidinoacetic acid into creatine. Fan Wenchao proposed that the microorganisms overexpress one or more enzymes selected from N-acetylglutamate synthase, N-acetylornithine-δ-aminotransferase, N-acetylornithinase, ornithine carbamoyltransferase, argininosuccinate synthase, glycine amidinotransferase (L-arginine: glycine amidinotransferase, EC: 2.1.4.1), and guanidinoacetic acid N-methyltransferase (EC: 2.1.1.2). The microorganisms preferably overexpress glycine aminotransferase (L-arginine: glycine amidinotransferase) and guanidinoacetic acid N-methyltransferase.

[0007] In WO2009129558 A1, the production of guanidinoacetic acid (GAA) as the first step in the biosynthesis of cylindrospermopsin by cyanobacterial cells expressing CyrA encoding L-arginine: glycine amidinotransferase (AGAT) was disclosed, and CyrK was involved as a putative cylindrospermopsin exporter. In WO2020191079A1, the production of GAA from L-arginine and the export of GAA regulated by SAT1 in isolated Th17 cells were disclosed.

[0008] Zhang et al. published a microorganism capable of producing guanidinoacetic acid (GAA) (ACS Synth. Biol. 2020, 9, 2066 - 275). They designed a recombinant ornithine cycle in Escherichia coli by introducing heterologous AGAT from different species (such as Homo sapiens, Cylindrospermopsis raciborskii, Moorena producens) and by introducing citrulline synthesis components (such as overexpression of argF and argI) and arginine synthesis components (such as overexpression of argG, argH; introduction of aspA) into Escherichia coli. CN111748506 A also discloses the production of GAA by Escherichia coli (E. coli) expressing AGAT, while CN113487139 A discloses the production of GAA by Bacillus subtilis expressing GAA.

[0009] Schneider and Jankowitsch (WO 2021122400 A1) proposed a method for producing GAA using a microorganism having a gene encoding a protein with the function of L - arginine:glycine amidinotransferase (AGAT) and an increased carbamoyl phosphate synthase. Carbamoyl phosphate is an important precursor for GAA biosynthesis, but also an important precursor for the biosynthesis of L - arginine and other compounds.

[0010] To increase GAA production using microorganisms, a large amount of the starting materials arginine and / or glycine are required intracellularly. At the same time, the by - product L - ornithine of the AGAT reaction must be effectively recycled to L - arginine to prevent loss of carbon and energy. Several pathways for increasing the production of one of the starting materials (i.e., L - arginine) in the synthesis of GAA in microorganisms (especially bacteria) are also known from the literature. Park et al. (NATURE COMMUNICATIONS|DOI: 10.1038 / ncomms5618) provided a review of the metabolic engineering of Corynebacterium glutamicum for L - arginine production. Yim et al. (J Ind Microbiol Biotechnol (2011) 38:1911 - 1920) could show that inactivation of the argR gene encoding the central repressor protein ArgR controlling the L - arginine biosynthetic pathway in Corynebacterium glutamicum led to an improved arginine - producing strain. Ginesy et al. (Microbial Cell Factories (2015) 14:29) reported the successful engineering of Escherichia coli to enhance arginine production. Among them, they proposed the deletion of the argR repressor gene. The deletion or inactivation of the argR repressor gene is also disclosed in combination with the production of GAA in WO2022008276A1 and WO2022008280A1.

[0011] On the other hand, an increased intracellular glycine concentration is also required to increase the production of GAA. As disclosed in WO2022008276 A1 and WO2022008280 A1, this is achieved by the de novo synthesis of glycine. Alternatively, glycine can be added to the culture medium, and membrane transporters facilitate the uptake.

[0012] If GAA production depends on the de novo synthesis of arginine and glycine, an efficient interconnection of precursor supply (arginine and glycine), product recycling (ornithine), and export (GAA) is required. Although ornithine recycling occurs through the reactions of the urea cycle, the transport of GAA across the bacterial cell membrane is not understood at all.

[0013] Generally, chemicals can cross biological membranes either by passive diffusion or by active transport. Transporters are responsible for the transport of compounds that cannot diffuse through the membrane. These proteins allow active transport or facilitate the diffusion of substances and are essential elements of living cells. Transporters can be specialized for the import of compounds, from the extracellular environment into the cytoplasm, or for the export of compounds, from the cytoplasm into the extracellular environment. Members of the first group are called importers or uptake systems, and members of the second group are called exporters.

[0014] Transporters are classified in databases such as the "Transporter Classification Database" (Nucleic Acids Res (2006) 34: 181-6). The classification is based on sequence similarity, and information on the known substrates of specific transporters is provided in the database. Some transporters have a very limited and specific substrate spectrum, while other transporters have a very broad substrate spectrum. In addition, different classes of transporters can transport the same compound. Despite the classification, it is not yet possible to predict the substrate transported by a specific transporter based on its protein sequence.

[0015] It is important to note that the sequence similarity of membrane proteins (including transporters) is less obvious compared to globular soluble proteins. This depends on the fact that the main functional structural element of membrane proteins, the transmembrane helix, can be composed of different amino acids without changing its nature as a transmembrane helix. Therefore, the same protein functionality can be observed in proteins sharing <20% sequence identity (Relation between sequence and structure in membraneproteins; M. Olivella, A. Gonzalez, L. Pardo and X. Deupi; Bioinformatics 2013 Vol. 29 Issue 13 Pages 1589-92; Accession Number: 23677941 DOI: 10.1093 / bioinformatics / btt249).

[0016] To optimize the production of chemical compounds by bacteria for industrial applications, efficient export of the product from the cell is crucial. For example, the lysine exporter LysE (encoded by lysE, i.e., NCgl1214) plays an important role in product secretion in Corynebacterium glutamicum strains (J Bacteriol (1995) 177: 4021-7). On the one hand, the accumulation of intracellular products inhibits their own production by affecting the chemical equilibrium of the final reaction or by regulating key metabolic reactions (Biosci Biotechnol Biochem (2001) 65: 1149-54). On the other hand, the accumulation of high concentrations of compounds inside the cell can have a harmful effect on growth and survival (Microbiology (2001) 147: 1765-74).

[0017] The serE gene from Corynebacterium glutamicum (C.glutamicum) (= NCgl0580 = cg0701 = Cgl0605 = CGL_RS03040; SEQ ID NO: 5) encodes a secondary transporter (TC 2.A.7; Saier MH Jr, Tran CV, Barabote RD. "TCDB: the Transporter Classification Database for membrane transport protein analyses and information." Nucleic Acids Res. 2006 Jan 1; 34 (Database issue): D181-6. doi: 10.1093 / nar / gkj001. PMID: 16381841; PMCID: PMC1334385; RefSeq WP_011013759; SEQ ID NO: 6) of the "Drug / Metabolite Transporter (DMT) Superfamily".

[0018] It was found that the SerE transporter exports L-serine and L-threonine outward and its overexpression improves the production of L-serine (Zhang X, Gao Y, Chen Z, Xu G, Zhang X, Li H, Shi J, Koffas MAG, Xu Z. “High-yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum.” Microb Cell Fact. 2020 May 29;19(1):115. doi:10.1186 / s12934-020-01374-5. PMID: 32471433; PMCID: PMC7260847). Overexpression of SerE also improves the production of L-cysteine (Kishino M, Kondoh M, Hirasawa T. “Enhanced L-cysteine production by overexpressing potential L-cysteine exporter genes in an L-cysteine-producing recombinant strain of Corynebacterium glutamicum.” Biosci Biotechnol Biochem. 2019 Dec;83(12):2390-2393. doi:10.1080 / 09168451.2019.1659715. Epub 2019 Sep 6. PMID: 31671040).

[0019] The thrE gene from Corynebacterium glutamicum (=NCgl2533=cg2905=Cgl2622=CGL_RS13090;SEQ ID NO:9) may encode a secondary transporter of the "threonine / serine exporter (ThrE)" family (TC 2.A.79, Saier MH Jr, Tran CV, Barabote RD.,, TCDB: the Transporter Classification Database for membrane transport protein analyses and information.” Nucleic Acids Res. 2006 Jan 1; 34(Database issue): D181-6.doi: 10.1093 / nar / gkj001.PMID: 16381841; PMCID: PMC1334385; RefSeq WP_003853939; Seq ID NO: 10). In the genome of Corynebacterium glutamicum strain ATCC13032, it is annotated as a threonine / serine exporter family protein (RefSeq accession number NC_003450, locus_tag=“CGL_RS13090”; also see Kalinowski J et al., 2003, J Biotechnol 104:5-25.10.1016 / S0168-1656(03)00154-8). Homologous proteins are present in selected bacteria, archaea, and fungal eukaryotes (Yen MR, Tseng YH, Simic P, Sahm H, Eggeling L, Saier MH Jr., Res Microbiol. 2002 Jan-Feb; 153(1):19-25.doi: 10.1016 / s0923-2508(01)01281-5.PMID: 11881894).

[0020] ThrE shows 10 putative transmembrane α-helical spanners, and it catalyzes the proton motive force (pmf)-dependent efflux of L-threonine and L-serine (Simic P, Sahm H, Eggeling L., J Bacteriol. 2001 Sep;183(18):5317-24.doi:10.1128 / JB.183.18.5317-5324.2001.PMID:11514515;PMCID:PMC95414;Simic et al., Appl Environ Microbiol. 2002;68:3321–3327.doi:10.1128 / AEM.68.7.3321-3327.2002) as well as L-proline (Liu et al., Nat Commun. 2022 Feb 16;13(1):891.doi:10.1038 / s41467-022-28501-7.PMID:35173152;PMCID:PMC8850433). Expression of thrE from Corynebacterium glutamicum in Escherichia coli threonine producers significantly improves L-threonine production (Kruse et al., Appl Microbiol Biotechnol. 2002 Jul;59(2-3):205-10.doi:10.1007 / s00253-002-0987-7.Epub 2002 Apr 4.PMID:12111147). Although having a similar function to the L-threonine efflux proteins of Escherichia coli (RhtA, RhtB, and RhtC), BLAST comparisons show no significant identity with those.

[0021] The gene NCgl2566 from Corynebacterium glutamicum (=cg2941 = Cgl2656 = CGL_RS13250; SeqID NO: 13) may encode a secondary transporter of the "LysE superfamily" (RefSeq WP_011015285; Seq ID NO: 14).

[0022] In the genome of Corynebacterium glutamicum strain ATCC13032, it is annotated as a "LysE family translocator" (RefSeq accession number NC_003450, locus_tag = "CGL_RS13250"). Through a BLASTp search against the "Transporter Classification Database" (https: / / tcdb.org / progs / blast.php) and through the information on the website, this protein can be correspondingly designated as a member of the LysE superfamily (Tsu, Brian V.; Saier, Milton H. (2015) The LysE Superfamily of Transport Proteins Involved in Cell Physiology and Pathogenesis. PloS One 10(10). doi:10.1371 / journal.pone.0137184. ISSN 1932-6203. PMC 4608589. PMID 26474485.). More specifically, NCgl2566 can be designated as a member of the RhtB family (TC#2.A.76) (see https: / / www.tcdb.org / search / result.php?tc = 2.A.76).

[0023] Zakataeva (Zakataeva NP et al., Microb Cell Fact. 2020 May 29;19(1):115. doi:10.1186 / s12934-020-01374-5. PMID: 32471433; PMCID: PMC7260847) showed that the deletion of rhtB in Escherichia coli led to increased resistance to several amino acids, including L-threonine and L-serine. Overexpression of NCgl2566 in Corynebacterium glutamicum improved L-cysteine production (Kinisho et al., Biosci Biotechnol Biochem. 2019 Dec;83(12):2390-2393. doi:10.1080 / 09168451.2019.1659715. Epub 2019 Sep 6. PMID: 31671040).

[0024] CN108486133A discloses an L-serine transporter and its application. It has been found that the NCgl0580 sequence in Corynebacterium glutamicum has the function of an L-serine transporter, which is called serE. Its overexpression improved the production of L-serine in Corynebacterium glutamicum.

[0025] WO 2021049866 A1 discloses L-threonine exporter variants and methods for preparing L-threonine using the same, in particular the expression of a variant of the Escherichia coli threonine exporter RhtC in Corynebacterium glutamicum, whereby the native thrE gene of Corynebacterium glutamicum is deleted.

[0026] WO 2012134253 A2 relates to Corynebacterium sp. transformed with a fructokinase gene derived from Escherichia sp. and a process for preparing L-amino acids using the same. To improve the export of L-threonine in Corynebacterium glutamicum, the expression of thrE was increased. Summary of the Invention

[0027] A potential problem of the present invention is to provide a microorganism transformed to be capable of producing guanidinoacetic acid (GAA) in higher amounts and a method for producing GAA using such a microorganism.

[0028] This problem is solved by a microorganism comprising at least one heterologous or synthetic gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT, e.g., EC 2.1.4.1) and having an overexpressed gene encoding a transmembrane exporter, wherein the transmembrane exporter is preferably selected from proteins of the drug / metabolite exporter (DMT) superfamily (TC.2.A.7), proteins of the 10TMS drug / metabolite exporter (DME) family (TC.2.A.7.3), proteins of the threonine / serine exporter (ThrE) family (TC.2.A.79), or proteins of the homoserine / threonine (RhtB) family (TC.2.A.76).

[0029] A microorganism in the context of the present invention is an organism of microscopic size, which can exist in single-cell form or as a cell colony. Microorganisms include most single-celled organisms from all three domains of life. The domains Archaea and Bacteria only include microorganisms. Among the third-domain eukaryotic single-celled organisms such as yeasts, protists and protozoa belong to the group of microorganisms. In the context of the present invention, a multicellular organism or a part thereof, such as a cell culture, in particular a culture of animal cells (e.g., human cells), is not considered a microorganism. In the context of the present invention, animal cells (e.g., human cells) are explicitly excluded from the definition of microorganisms.

[0030] The transmembrane exporter can be a transmembrane transporter annotated as a serine or threonine exporter.

[0031] Preferably, the transmembrane exporter has at least 20%, 30%, 40%, 50%, 60%, 70%, at least 80% or at least 90% identity, preferably to a protein comprising the amino acid sequence according to SEQ ID NO: 6 (SerE) or a protein comprising the amino acid sequence according to SEQ ID NO: 10 (ThrE) or a protein comprising the amino acid sequence according to SEQ ID NO: 14.

[0032] In this context, the degree of relatedness of protein sequences is expressed as identity, which more specifically means how much of the sequences are identical according to the results of a blast search given as a percentage (definition see https: / / www.ncbi.nlm.nih.gov / books / NBK62051 / ).

[0033] In a further embodiment of the invention, the transmembrane exporter is encoded by the open reading frame of the Corynebacterium glutamicum NCgl0580 gene according to SEQ ID NO: 5 (i.e., the serE gene from Corynebacterium glutamicum = cg0701 = Cgl0605 = CGL_RS03040).

[0034] In another embodiment of the invention, the transmembrane exporter is encoded by the open reading frame of the Corynebacterium glutamicum NCgl2533 (thrE) gene according to SEQ ID NO: 9.

[0035] In another embodiment of the invention, the transmembrane exporter is encoded by the open reading frame of the Corynebacterium glutamicum NCgl2566 gene according to SEQ ID NO: 13.

[0036] The microorganism according to the invention is preferably a genetically modified organism that does not occur naturally. In such a microorganism, the genetic material has been altered using genetic engineering techniques. According to the invention, the open reading frame of the gene encoding a protein having the function of L-arginine:glycine amidinotransferase (AGAT, e.g., EC 2.1.4.1) is heterologous or synthetic, which means that at least one gene encoding a protein having the function of L-arginine:glycine amidinotransferase has been introduced using genetic engineering techniques.

[0037] A heterologous or synthetic gene means that the gene has been inserted into a host organism that does not naturally have this gene. Insertion of a heterologous or synthetic gene into a host is carried out by recombinant DNA technology. A microorganism that has undergone recombinant DNA technology is called transgenic, genetically modified, or recombinant. A heterologous protein means a protein that does not naturally occur in the microorganism. A homologous or endogenous gene means that the gene (including its own function) or the nucleotide sequence of the gene naturally exists in the microorganism or is "natural" in the microorganism. A homologous or natural protein means a protein that naturally occurs in the microorganism.

[0038] In the microorganism according to the invention, the activity of the protein having L-arginine:glycine amidinotransferase (AGAT) function is preferably increased by overexpression of the gene encoding the protein having L-arginine:glycine amidinotransferase function, as compared to the corresponding activity in the wild-type organism.

[0039] In the microorganism according to the invention, the protein having L-arginine:glycine amidinotransferase (AGAT) function comprises an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 2.

[0040] Increased enzyme activity in a microorganism is generally achieved by overexpressing the gene encoding the corresponding enzyme.

[0041] Overexpression of a 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 start codon or codon usage of the entire gene and / or by modifying the activity of the protein that regulates gene expression or a selected combination including all of the above methods.

[0042] In the microorganism of the invention, overexpression of the gene encoding the transmembrane efflux protein can be achieved by increasing the copy number of the gene and / or by functionally linking the gene to a strong promoter and / or by enhancing the ribosome binding site and / or by optimizing the start codon or codon usage of the entire gene and / or by modifying the activity of the protein that regulates gene expression.

[0043] A promoter is a DNA sequence consisting of approximately 40 - 50 base pairs that constitutes the binding site for the RNA polymerase holoenzyme and the transcription start point, thereby influencing the expression intensity of the controlled polynucleotide or gene. Generally, overexpression or an increase in gene expression in bacteria is possible by using strong promoters, for example, by replacing the original promoter with a strong promoter or by placing an additional strong promoter upstream of the gene of interest. Such strong promoter sequences can be natural (e.g., other sequences originally designated for the same species), they can be heterologous (e.g., DNA from other species), or they can be partially or completely of synthetic origin. Overexpression can also be achieved by modifying certain regions of a given natural promoter (e.g., its so-called -10 and -35 regions), as taught, for example, by M. Patek et al. (Microbial Biotechnology 6 (2013), 103 - 117) regarding Corynebacterium glutamicum. Compared to the capabilities of wild-type microorganisms, the microorganisms according to the present invention can have an increased ability to produce L-arginine from L-ornithine.

[0044] In the context of the present invention, a microorganism having an improved ability to produce L-arginine means a microorganism that produces L-arginine in excess of its own needs. Examples of such L-arginine-producing microorganisms are, for example, Corynebacterium glutamicum ATCC 21831 or those disclosed by Park et al. (NATURE COMMUNICATIONS|DOI: 10.1038 / ncomms5618) or Ginesy et al. (Microbial Cell Factories (2015) 14:29). Compared to previously described microorganisms having an increased ability to produce L-arginine, L-arginine secretion is not desired in the strains used for GAA production because arginine is utilized intracellularly for GAA production within the framework of the present invention.

[0045] In a further embodiment of the microorganism according to the present invention, the expression of the argR gene encoding the arginine-responsive repressor protein ArgR is attenuated compared to the expression of the argR gene in the wild-type microorganism. Alternatively, the argR gene is inactivated or deleted.

[0046] The microorganisms of the present invention can belong to the genus Corynebacterium, the genus Bacillus (Yan, K. et al. (2022) “Biosynthesis of Guanidinoacetate by Bacillus subtilis Whole-Cell Catalysis.” Fermentation 8(3):116), the Enterobacteriaceae family, or the genus Pseudomonas.

[0047] In a specific embodiment of the present invention, the microorganism is Corynebacterium glutamicum (C. glutamicum) or Escherichia coli (E. coli).

[0048] The present invention further relates to a method for fermentative production of guanidinoacetic acid (GAA), which comprises the steps of culturing a microorganism according to the present invention in a culture medium and accumulating GAA in the culture medium to form a fermentation broth containing GAA.

[0049] Preferably, the method further comprises separating GAA from the fermentation broth containing GAA.

[0050] In a specific embodiment, the microorganism of the present invention further comprises a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity. The gene encoding the enzyme having guanidinoacetic acid N-methyltransferase activity may be overexpressed.

[0051] The present invention also relates to a method for fermentative production of creatine, comprising the steps of: a) culturing a microorganism according to the present invention in a suitable culture medium under suitable conditions, the microorganism further comprising a gene encoding an enzyme having guanidinoacetic acid N-methyltransferase activity, and b) accumulating creatine in the culture medium to form a fermentation broth containing creatine.

[0052] Preferably, the method further comprises separating creatine from the fermentation broth containing creatine. Creatine can be extracted from the fermentation broth by isoelectric point method and / or ion exchange method. Alternatively, creatine can be further purified by recrystallization in water.

[0053] Brief Description of the Sequences

[0054] SEQ ID NO: 1: DNA sequence of locus_tag BJP34_00300 encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) of Moorena producens strain PAL-8-15-08-1

[0055] SEQ ID NO: 2: Derived amino acid sequence from locus_tag BJP34_00300 (RefSeq accession number WP_070390602)

[0056] SEQ ID NO: 3: Synthetic DNA fragment of a plasmid for cloning the expression of L-arginine:glycine amidinotransferase for Moorena producens strain PAL-8-15-08-1

[0057] SEQ ID NO: 4: DNA sequence of the shuttle plasmid pLIB_P for gene expression in Corynebacterium glutamicum

[0058] SEQ ID NO: 5: DNA sequence of the open reading frame serE (NCgl0580 = CGL_RS03040) of Corynebacterium glutamicum strain ATCC13032

[0059] SEQ ID NO: 6: Derived amino acid sequence from the open reading frame serE of Corynebacterium glutamicum strain ATCC13032 (RefSeq accession number WP_011013759)

[0060] SEQ ID NO: 7: PCR primer serE_f_NotI for amplifying ORF serE

[0061] SEQ ID NO: 8: PCR primer serE_r_NotI for amplifying ORF serE

[0062] SEQ ID NO: 9: DNA sequence of the open reading frame thrE (NCgl2533 = CGL_RS13090) of Corynebacterium glutamicum strain ATCC13032

[0063] SEQ ID NO: 10: Derived amino acid sequence from the open reading frame thrE of Corynebacterium glutamicum strain ATCC13032 (RefSeq accession number WP_003853939)

[0064] SEQ ID NO: 11: PCR primer thrE_f_NotI for amplifying ORF thrE

[0065] SEQ ID NO: 12: PCR primer thrE_r_NotI for amplifying ORF thrE

[0066] SEQ ID NO: 13: DNA sequence of the open reading frame NCgl2566 (CGL_RS13250) of Corynebacterium glutamicum ATCC13032

[0067] SEQ ID NO: 14: Derived amino acid sequence from the open reading frame NCgl2566 of Corynebacterium glutamicum strain ATCC13032 (RefSeq accession number RefSeq WP_011015285)

[0068] SEQ ID NO: 15: PCR primer NCgl2566_f_NotI for amplifying ORF NCgl2566

[0069] SEQ ID NO: 16: PCR primer NCgl2566_r_NotI for amplifying ORF NCgl2566

[0070] SEQ ID NO: 17: PCR primer DargR_lf for amplifying the left homologous arm of the argR deletion plasmid pK18mobsacB_DargR

[0071] SEQ ID NO: 18: PCR primer DargR_lr for amplifying the left homologous arm of the argR deletion plasmid pK18mobsacB_DargR

[0072] SEQ ID NO: 19: PCR primer DargR_rf for amplifying the right homologous arm of the argR deletion plasmid pK18mobsacB_DargR

[0073] SEQ ID NO: 20: PCR primer DargR_rr for amplifying the right homologous arm of the argR deletion plasmid pK18mobsacB_DargR Example

[0074] A) Materials and methods

[0075] Chemicals

[0076] Kanamycin solution from Streptomyces kanamyceticus was purchased from Sigma Aldrich (St. Louis, USA, Cat. no. K0254). All other chemicals were purchased in analytical grade from Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, USA) or Carl-Roth (Karlsruhe, Germany) if not stated otherwise.

[0077] Cultivation for cell proliferation

[0078] If not stated otherwise, the cultivation / incubation procedures were carried out as follows:

[0079] a. LB broth (MILLER) from Merck (Darmstadt, Germany; Cat. no. 110285) was used for cultivating Escherichia coli strains in liquid medium. The liquid culture (10 ml liquid medium / 100 ml Erlenmeyer with 3 baffles) was incubated at 30 °C and 200 rpm in an Infors HT Multitron standard shaking incubator from Infors GmbH (Bottmingen, Switzerland).

[0080] b. LB agar (MILLER) from Merck (Darmstadt, Germany, Cat. no. 110283) was used to culture Escherichia coli strains on agar plates. The agar plates were incubated at 30 °C in a microincubator from VWR (Radnor, USA).

[0081] c. Brain heart infusion (BHI) from Merck (Darmstadt, Germany, Cat. no. 110493) was used to culture Corynebacterium glutamicum strains in liquid medium. The liquid cultures (10 ml liquid medium / 100 ml Erlenmeyer with 3 baffles) were incubated at 30 °C and 200 rpm in an Infors HT Multitron standard shaking incubator from Infors GmbH (Bottmingen, Switzerland).

[0082] d. Brain heart agar (BHI-agar) from Merck (Darmstadt, Germany, Cat. no. 113825) was used to culture Corynebacterium glutamicum strains on agar plates. The agar plates were incubated at 30 °C in an incubator from Heraeus Instruments with a temperature controller (Hanau, Germany).

[0083] e. To culture Corynebacterium glutamicum after electroporation, BHI-agar (Merck, Darmstadt, Germany, Cat. no. 113825) was supplemented with 134 g / l sorbitol (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), 2.5 g / l yeast extract (Oxoid / ThermoFisher Scientific, Waltham, USA, Cat. no. LP0021) and 25 mg / l kanamycin. The agar plates were incubated at 30 °C in an incubator from Heraeus Instruments with a temperature controller (Hanau, Germany).

[0084] Determination of the optical density of the bacterial suspension

[0085] a. The optical density of the bacterial suspension in shake flask cultures was determined at 600 nm (OD600) using a Bio-Photometer from Eppendorf AG (Hamburg, Germany).

[0086] b. The optical density of the bacterial suspension produced in the Wouter Duetz (WDS) microfermentation system (24-well plate) was measured at 660 nm (OD660) using a GENios plate reader from Tecan Group AG ( TM Switzerland).

[0087] Centrifugation

[0088] a. The bacterial suspension with a maximum volume of 2 ml was centrifuged (5 minutes, 13,000 rpm) in 1.5 ml or 2 ml reaction tubes (e.g., Eppendorf 3810X) using an Eppendorf 5417R benchtop centrifuge.

[0089] b. The bacterial suspension with a maximum volume of 50 ml was centrifuged at 4,000 rpm for 10 minutes in 15 ml or 50 ml centrifuge tubes (e.g., FalconTM 50 ml Conical Centrifuge Tubes) using an Eppendorf 5810R benchtop centrifuge.

[0090] DNA isolation

[0091] Plasmid DNA was isolated from Escherichia coli cells using the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany, Cat. No. 27106) according to the manufacturer's instructions.

[0092] Polymerase chain reaction (PCR)

[0093] The desired segment of DNA was amplified by PCR using a proofreading (high-fidelity) polymerase for sequencing or DNA assembly. A non-proofreading polymerase kit was used to determine the presence or absence of the desired DNA fragment directly from Escherichia coli or Corynebacterium glutamicum colonies.

[0094] a. According to the manufacturer's instructions (see Table 1), the selected DNA region was amplified with template-correction using the High-Fidelity DNA Polymerase Kit (PhusionKit) from New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530).

[0095]

[0096] Table 1: Using Thermal cycling conditions for PCR using the High-Fidelity DNA Polymerase Kit.

[0097] b. Amplify the desired segment of DNA to confirm its presence using the Taq PCR Core Kit (Taq Kit) from Qiagen (Hilden, Germany, Cat. No. 201203). Use the kit according to the manufacturer's instructions (see Table 2).

[0098]

[0099]

[0100] Table 2: Thermal cycling conditions for PCR using the Taq PCR Core Kit (Taq Kit) from Qiagen.

[0101] c. Use the Fast PCR MasterMix (Sapphire Mix) from Takara Bio Inc (Takara Bio Europe S.A.S., Saint-Germain-en-Layye, France, Cat. No. RR350A / B) as an alternative to confirm the presence of the desired DNA segment in cells taken from Escherichia coli or Corynebacterium glutamicum colonies (see Table 3). Fast PCR MasterMix (Sapphire Mix) for confirmation of the presence of the desired DNA segment in cells taken from Escherichia coli or Corynebacterium glutamicum colonies (see Table 3).

[0102]

[0103] Table 3: Thermal cycling conditions for PCR using the Fast PCR Master Mix (Sapphire Mix) from Takara Bio Inc.

[0104] d. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany).

[0105] e. As the PCR template, use isolated plasmid DNA or an appropriately diluted solution of total DNA isolated from a liquid culture or total DNA contained in a bacterial colony (colony PCR). For the colony PCR, prepare the template by picking cell material from a colony on an agar plate with a sterile toothpick and placing the cell material directly into the PCR reaction tube. In SEVERIN In the microwave oven type Mikrowave&Grill of GmbH (Sundern, Germany), the cell material was heated at 800 W for 10 seconds, and then the PCR reagent was added to the template in the PCR reaction tube.

[0106] f. All PCR reactions were carried out in a PCR cycler type Mastercycler or Mastercycler nexus gradient from Eppendorf AG (Hamburg, Germany).

[0107] Restriction enzyme digestion of DNA

[0108] For restriction enzyme digestion, "FastDigest restriction endonuclease (FD)" (ThermoFisher Scientific, Waltham, USA) or restriction endonucleases from New England BioLabs Inc. (Ipswich, USA) were used. The reaction was carried out according to the instructions in the manufacturer's manual.

[0109] Determination of the size of DNA fragments

[0110] a. The size of small DNA fragments (<1000 bp) was usually determined by using automated capillary electrophoresis with QIAxcel from Qiagen (Hilden, Germany).

[0111] b. If DNA fragments needed to be separated, or if the DNA fragments were >1000 bp, the DNA was separated by TAE agarose gel electrophoresis and stained with Nucleic Acid Gel Stain (Biotium, Inc., Fremont, Canada). The stained DNA was visualized at 302 nm.

[0112] PCR amplification and purification of restriction fragments

[0113] According to the manufacturer's instructions, the PCR amplification fragments and restriction fragments were cleaned up using the QIAquick PCR Purification Kit from Qiagen (Hilden, Germany; Cat. No. 28106). The DNA was eluted with 30 μl of 10 mM Tris*HCl (pH 8.5).

[0114] Determination of DNA concentration

[0115] The DNA concentration was measured using a NanoDrop spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH, 2015 VWR brand (Erlangen, Germany).

[0116] Assembly cloning

[0117] Plasmid vectors were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" purchased from New England BioLabs Inc. (Ipswich, USA, Cat. No. E5520). The reaction mixture containing the linear vector and at least one DNA insert was incubated at 50 °C for 60 minutes. 0.5 μl of the assembly mixture was used for each transformation experiment.

[0118] Chemical transformation of Escherichia coli

[0119] For plasmid cloning, chemically competent " Stable Competent E. coli (High Efficiency)" (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.

[0120] Transformation of Corynebacterium glutamicum

[0121] Transformation of Corynebacterium 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 carried out in a 1 mm electroporation cuvette (Bio-Rad Laboratories GmbH, Feldkirchen, Germany) at 1.8 kV and a fixed time constant set to 5 ms. Transformed cells were selected on BHI-agar containing 134 g / l sorbitol, 2.5 g / l yeast extract, and 25 mg / l kanamycin.

[0122] Corynebacterium glutamicum strains

[0123] The wild-type strain of Corynebacterium glutamicum, Corynebacterium glutamicum ATCC 13032 (DSM 20300, 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 from the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH.

[0124] Determination of the nucleotide sequence

[0125] The nucleotide sequence of the DNA molecule was determined by cycle sequencing by Eurofins Genomics GmbH (Ebersberg, Germany) using the dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences USA 74, 5463-5467, 1977). The sequences and the computer assembly of the sequences were visualized and evaluated using the CloneManager Professional 9 software from Scientific & Educational Software (Denver, USA).

[0126] Glycerol stocks of Escherichia coli and Corynebacterium glutamicum strains

[0127] For long-term storage, glycerol stocks of Escherichia coli and Corynebacterium glutamicum were prepared. The selected Escherichia coli clones were cultured in 10 ml of LB medium supplemented with 2 g / l glucose. The selected Corynebacterium glutamicum clones were cultured in 10 ml of double-strength BHI medium supplemented with 2 g / l glucose. The media containing Escherichia coli and Corynebacterium glutamicum strains for growing plasmids were supplemented with 25 mg / l kanamycin. The media were contained in 100 ml Erlenmeyer flasks with 3 baffles. The media were inoculated with a cell loop taken from the colonies. The cultures were then incubated at 30 °C and 200 rpm for 18 h. After the incubation period, 1.2 ml of 85% (v / v) sterile glycerol was added to the cultures. The resulting cell suspensions containing glycerol were then aliquoted in 2 ml portions and stored at -80 °C.

[0128] GAA production in small-scale cultures

[0129] The GAA-production of the strains was evaluated using a milliliter-scale cultivation system according to Duetz (2007). For this purpose, a 24-deep well microplate (24-well WDS plate) from EnzyScreen BV (Heemstede, Netherlands, Cat. no. CR1424) filled with 2.5 ml of medium per well was used.

[0130] The pre-cultivation of the strains was carried out in 10 ml of seed medium (SM). The medium was contained in a 100 ml Erlenmeyer with 3 baffles. The medium was inoculated with 100 μl of the glycerol stock culture, and the culture was incubated at 30 °C and 200 rpm for 24 hours. The composition of the seed medium (SM) is shown in Table 4.

[0131] Ingredient Concentration (g / l) Yeast extract FM902 (Angel Yeast Co., LTD, Hubei, P.R. China) 10 Urea 1.5 <![CDATA[KH 2 PO 4 > 0.5 <![CDATA[K 2 HPO 4 > 0.5 <![CDATA[MgSO 4 *7H 2 O]]> 1 Biotin 0.0001 Thiamine hydrochloride 0.0001 <![CDATA[FeSO 4 *7H 2 O]]> 0.01 <![CDATA[MnSO 4 *H 2 O]]> 0.01 Glucose 20 Kanamycin 0.025 pH = 7.0

[0132] Table 4: Seed medium (SM)

[0133] After the incubation period, the optical density OD600 of the pre-culture was determined. A volume of the pre-culture was sampled to inoculate 2.5 ml of production medium (PM) to an OD600 of 0.1, centrifuged (1 minute at 8000 g), and the supernatant was discarded. The cells were then resuspended in 100 μl of production medium.

[0134] The main culture was initiated by inoculating each well of a 24-well WDS-plate containing 2.4 ml of production medium (PM) with 100 μl of the resuspended cells from the pre-culture. The composition of the production medium (PM) is shown in Table 5.

[0135]

[0136]

[0137] Table 5: Production medium (PM)

[0138] The main culture was incubated in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland) at 30 °C and 225 rpm for 72 hours until the glucose was completely consumed. The glucose concentration in the suspension was analyzed using a OneTouch blood glucose meter from LifeScan (Johnson & Johnson Medical GmbH, Neuss, Germany). Analyze the glucose concentration in the suspension.

[0139] After cultivation, the culture suspension was transferred to a deep well microplate. A portion of the culture suspension was appropriately diluted to measure the OD600. Another portion of the culture was centrifuged and the concentration of GAA in the supernatant was analyzed as described below.

[0140] Quantification of GAA

[0141] The samples were analyzed using an analytical system from Agilent consisting of HPLC “Infinity 1260” connected to a mass analyzer “Triple Quad 6420” (Agilent Technologies Inc., Santa Clara, USA). Chromatographic separation was carried out at 35 °C on an Atlantis HILIC Silica column, 4.6×250 mm, 5 μm (Waters Corporation, Milford, USA). Mobile phase A was water containing 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, followed by a linear gradient for 22 minutes and a constant flow rate of 0.6 mL / min to 66% B. The mass analyzer was operated in the ESI positive ionization mode. To detect GAA, the m / z value was monitored by using MRM fragmentation [M+H]+118-76. The limit of quantification (LOQ) of GAA was fixed at 7 ppm.

[0142] B) Experimental results

[0143] Example 1: Cloning of the gene AGAT-Mp encoding L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Moorena producens

[0144] Moorena producens is a filamentous cyanobacterium. The genome of the Moorena producens strain PAL-8-15-08-1 was published by Leo et al. (Leo T, Castao G, Korobeynikov A, Monroe EA, Podell S, Glukhov E, Allen EE, Gerwick WH, Gerwick L, Proc Natl Acad Sci USA. 2017 Mar 21;114(12):3198-3203. doi: 10.1073 / pnas;1618556114; RefSeq 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 shown in SEQ ID NO: 1). SEQ ID NO: 2 shows the derived amino acid sequence (RefSeq accession number WP_070390602).

[0145] The amino acid sequence was translated back into a DNA sequence optimized for codon usage in Corynebacterium glutamicum using the software tool "Optimizer" (http: / / genomes.urv.es / OPTIMIZER / ). The 5' end of the optimized gene was amplified with BsaI restriction sites, a 5'-UTR sequence for assembly cloning, and a ribosome binding site. A second stop codon, a sequence for assembly cloning, and a BsaI site were added at the 3' end. The resulting DNA sequence was named AGAT-Mp-insert (SEQ ID NO: 3), which was customized for gene synthesis from Eurofins Genomics GmbH (Ebersberg, Germany) and delivered as part of a cloning plasmid with an ampicillin resistance gene (designated pEX-A258_AGAT-Mp).

[0146] The Escherichia coli - Corynebacterium glutamicum shuttle plasmid pLIB_P consists of a pBL1 origin of replication (for Corynebacterium glutamicum), a pSC101 origin of replication (for Escherichia coli), and a kanamycin resistance gene. It has a strong promoter, two BsaI sites in opposite orientations, and the BioBricks terminator BBa_B1006 (SEQ ID NO: 4) after a unique NotI restriction site.

[0147] pLIB_P was digested with the restriction endonuclease BsaI, and the DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0148] Digest the cloning plasmid pEX-A258_AGAT-Mp with the restriction endonuclease BsaI, and purify the DNA using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0149] Combine the DNA solutions of BsaI-digested pLIB_P and pEX-A258_AGAT-Mp, and use the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, catalog number E5520) to assemble the matching sequence ends. Transform the product into NEB Stable competent Escherichia coli (high efficiency) "(New England Biolabs, Ipswich, USA), and grow the cells on LB agar containing 25 mg / l kanamycin. Identify the appropriate plasmid clones by restriction digestion and DNA sequencing. The resulting plasmid was named pLIB_P_AGAT-Mp.

[0150] Example 2: Cloning of the expression plasmid pLIB_serE_AGAT-Mp encoding the SerE protein (NCgl0580) from Corynebacterium glutamicum and L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Moorenaproducens:

[0151] The expression plasmid pLIB_serE_AGAT-Mp contains the serE gene (NCgl0580, SEQ ID NO:5) from Corynebacterium glutamicum and its promoter located upstream of the AGAT gene.

[0152] Perform PCR using the primers serE_f_NotI (Seq ID NO:7) and serE_r_NotI (Seq ID NO:8) and the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. The resulting PCR product contains the serE gene (NCgl0580) plus a 41 bp sequence upstream of the atg start codon. It is purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0153] Digest plasmid pLIB_P_AGAT-Mp with the restriction endonuclease NotI and purify the DNA using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0154] Combine the DNA of NotI-digested pLIB_P_AGAT-Mp with the PCR product containing serE and use the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520) to assemble the matching sequence ends.

[0155] Transform the product into NEB Stable competent Escherichia coli (high efficiency) "(New England Biolabs, Ipswich, USA), and grow the cells on LB agar containing 25 mg / l kanamycin. Identify the appropriate plasmid clones by restriction digestion and DNA sequencing. The resulting plasmid was named pLIB_serE_AGAT-Mp.

[0156] Example 3: Cloning of the expression plasmid pLIB_thrE_AGAT-Mp encoding the ThrE protein (NCgl2533) from Corynebacterium glutamicum and the L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Moorenaproducens

[0157] The expression plasmid pLIB_thrE_AGAT-Mp contains the thrE gene (NCgl2533, SEQ ID NO:9) from Corynebacterium glutamicum located upstream of the AGAT gene and its promoter.

[0158] Perform PCR using the primers thrE_f_NotI (Seq ID NO:11) and thrE_r_NotI (Seq ID NO:12) and using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. The resulting PCR product contains the thrE gene (NCgl2533) plus a 114 bp sequence upstream of the atg start codon. It is purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0159] The plasmid pLIB_P_AGAT-Mp was digested with the restriction endonuclease NotI, and the DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0160] The DNA of NotI-digested pLIB_P_AGAT-Mp was combined with the PCR product containing thrE, and the matching sequence ends were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520).

[0161] The product was transformed into " Stable Competent E.coli (High Efficiency)" (New England Biolabs, Ipswich, USA), and the 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_thrE_AGAT-Mp.

[0162] Example 4: Cloning of the expression plasmid pLIB_NCgl2566_AGAT-Mp encoding the NCgl2566 protein from Corynebacterium glutamicum and the L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Moorena producens

[0163] The expression plasmid pLIB_NCgl2566_AGAT-Mp contains the gene NCgl2566 (SEQ ID NO:13) from Corynebacterium glutamicum upstream of the AGAT gene and its promoter.

[0164] PCR was performed using the primers NCgl2566_f_NotI (SEQ ID NO:15) and NCgl2566_r_NotI (SEQ ID NO:16), and using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. The resulting PCR product contained the gene NCgl2566 plus a 141 bp sequence upstream of the gtg start codon. It was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0165] The plasmid pLIB_P_AGAT-Mp was digested with the restriction endonuclease NotI, and the DNA was purified using the "QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0166] The DNA of NotI-digested pLIB_P_AGAT-Mp was combined with the PCR product containing NCgl2566, and the matching sequence ends were assembled using the "NEBuilder HiFi DNA Assembly Cloning Kit" (New England BioLabs Inc., Ipswich, USA, catalog number E5520).

[0167] The product was transformed into NEB Stable competent Escherichia coli (high efficiency) "(New England Biolabs, Ipswich, USA), and the 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_NCgl2566_AGAT-Mp.

[0168] Example 5: Chromosomal deletion of the gene argR (NCgl1345) in ATCC13032

[0169] To improve intracellular L-arginine formation and L-arginine recovery from L-ornithine, the gene argR (NCgl1345) encoding the central repressor protein ArgR that controls the L-arginine biosynthetic pathway was inactivated.

[0170] Thus, the plasmid pK18mobsacB_DargR was constructed as follows. The plasmid pK18mobsacB ( 1994) was cut with XbaI and the linearized vector DNA (5721 bp) was purified using the "QIAquick Gel Extraction Kit" (Qiagen GmbH, Hilden, Germany).

[0171] To construct the insert, two DNA fragments were generated by PCR using the following primer pairs (genomic DNA of ATCC13032 as the template):

[0172] DargR_lf (SEQ ID NO: 17), +DargR_lr (SEQ ID NO: 18)

[0173] = left homologous arm (983 bp)

[0174] 10 DargR_rf (SEQ ID NO: 19), +DargR_rr (SEQ ID NO: 20)

[0175] = left homologous arm (984 bp)

[0176] The PCR products were purified using the "QIA quick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

[0177] Then, the linearized plasmid and the PCR products were 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.

[0178] To delete the argR gene, pK18mobsacB_DargR was transformed into Corynebacterium glutamicum ATCC 13032 (Kinoshita et al., J. Gen. Appl. Microbiol. 1957; 3(3): 193 - 205) by electroporation. Chromosomal integration (resulting from the first recombination event) was selected by plating on 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 h.

[0179] Single colonies were transferred to fresh agar plates (with 25 mg / l kanamycin) and incubated at 33 °C for 24 h. The liquid cultures of these clones were grown in 10 ml of BHI medium contained in a 100 ml Erlenmeyer with 3 baffles at 33 °C for 24 h. To isolate clones that had undergone the second recombination event, aliquots were taken from each liquid culture, appropriately diluted, and plated (usually 100 μl to 200 μl) on BHI agar supplemented with 10% sucrose. These agar plates were incubated at 33 °C for 48 h. Then, the kanamycin sensitivity of the colonies growing on the agar plates containing sucrose was examined. For this purpose, cell material was removed from the colonies using toothpicks and transferred to BHI agar containing 25 mg / l kanamycin and BHI agar containing 10% sucrose. The agar plates were incubated at 33 °C for 60 h.

[0180] Clones that were shown to be sensitive to kanamycin and resistant to sucrose were examined by PCR and DNA sequencing.

[0181] The obtained strain was named ATCC13032_DargR.

[0182] Example 6: Transformation of Corynebacterium glutamicum ATCC13032_DargR with expression plasmids

[0183] The strain ATCC13032_DargR was transformed by electroporation with the expression plasmids pLIB_P_AGAT-Mp, pLIB_serE_AGAT-Mp, pLIB_thrE_AGAT-Mp and pLIB_NCgl2566__AGAT-Mp. Plasmid-containing cells were selected with 25 mg / l kanamycin. The obtained plasmid-containing strains are shown in Table 6.

[0184]

[0185] Table 6: Plasmids containing derivatives of Corynebacterium glutamicum ATCC13032

[0186] Example 7: Effect of overexpression of cycA on transporter genes

[0187] To evaluate the effect of overexpression of transporter genes on GAA production, the strains ATCC13032_DargR / pLIB_P_AGAT-Mp, ATCC13032_DargR / pLIB_serE_AGAT-Mp, ATCC13032_DargR / pLIB_thrE_AGAT-Mp and ATCC13032_DargR / pLIB_NCgl2566_AGAT-Mp were cultured in a production medium in a Wouter Duetz system, and the resulting GAA titers were determined as described above.

[0188] Strain GAA ATCC13032_DargR / pLIB_P_AGAT-Mp 286 mg / l ATCC13032_DargR / pLIB_serE_AGAT-Mp 1009 mg / l ATCC13032_DargR / pLIB_thrE_AGAT-Mp 398 mg / l ATCC13032_DargR / pLIB_NCgl2566_AGAT-Mp 319 mg / l

[0189] Table 7: Effect of overexpression of transporter genes on GAA production

[0190] Culturing the strains ATCC13032_DargR / pLIB_serE_AGAT-Mp, ATCC13032_DargR / pLIB_thrE_AGAT-Mp and ATCC13032_DargR / pLIB_NCgl2566_AGAT-Mp led to higher GAA titers compared to ATCC13032 / pLIB_P_AGAT-Mp (see Table 7). We conclude that overexpression of the transporter improved GAA production.

Claims

1. A microorganism comprising at least one heterologous or synthetic gene encoding a protein having the function of L-arginine:glycine amidinotransferase, and having an overexpressed gene encoding a transmembrane efflux transporter, wherein the transmembrane efflux transporter is selected from proteins of the drug / metabolite transporter (DMT) superfamily (TC.2.A.7), proteins of the 10TMS drug / metabolite efflux transporter (DME) family (TC.2.A.7.3), proteins of the threonine / serine efflux transporter (ThrE) family (TC.2.A.79), or proteins of the homoserine / threonine (RhtB) family (TC.2.A.76).

2. The microorganism of claim 1, wherein the transmembrane efflux transporter has at least 20% identity with a protein comprising the amino acid sequence according to SEQ ID NO:6 or a protein comprising the amino acid sequence according to SEQ ID NO:10 or a protein comprising the amino acid sequence according to SEQ ID NO:

14.

3. The microorganism of claim 1 or 2, wherein the transmembrane efflux transporter has at least 70% identity with a protein comprising the amino acid sequence according to SEQ ID NO:6 or a protein comprising the amino acid sequence according to SEQ ID NO:10 or a protein comprising the amino acid sequence according to SEQ ID NO:

14.

4. The microorganism of claim 2 or 3, wherein the transmembrane efflux transporter is encoded by the open reading frame of the Corynebacterium glutamicum gene NCgl0580 according to SEQ ID NO:

5.

5. The microorganism of claim 2 or 3, wherein the transmembrane efflux transporter is encoded by the open reading frame of the Corynebacterium glutamicum gene NCgl2533 according to SEQ ID NO:

9.

6. The microorganism of claim 2 or 3, wherein the transmembrane efflux transporter is encoded by the open reading frame of the Corynebacterium glutamicum gene NCgl2566 according to SEQ ID NO:

13.

7. The microorganism of any one of the preceding claims, wherein overexpression of the gene encoding the transmembrane efflux transporter is achieved by increasing the copy number of the gene and / or by functionally linking the gene to a strong promoter and / or by enhancing the ribosome binding site and / or by optimizing the start codon or codon usage of the entire gene.

8. The microorganism of any one of the preceding claims, wherein the microorganism has an increased ability to produce L-arginine from L-ornithine compared to the ability of a wild-type microorganism.

9. The microorganism of claim 8, wherein the expression of the argR gene encoding the arginine-responsive repressor ArgR is attenuated compared to the expression of the argR gene in the wild-type microorganism, or wherein the argR gene is inactivated or deleted.

10. A method for fermentative production of guanidinoacetic acid (GAA), which comprises the following steps: Cultivate the microorganism as defined in any one of the preceding claims in a culture medium and accumulate GAA in the culture medium to form a fermentation broth containing GAA.

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

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

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

14. A method for fermentative production of creatine, the method comprising the steps of: cultivating the microorganism as defined in any one of claims 12 or 13 and accumulating creatine in the culture medium to form a fermentation broth containing creatine.

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

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