Improved biotechnological process to produce guanidinoacetic acid (GAA) by targeted introduction or by increasing the activity of a transmembrane transport protein belonging to the Amino Acid-Polyamine-Organocation Superfamily

Guanidino acetic acid (GAA) is a colorless crystalline organic compound used as animal feed additive (e.g. W02005120246 A1 and US2011257075 A1). GAA is a natural precursor of creatine (e.g. Humm et al., Biochem. J. (1997) 322, 771-776). Therefore, the supplementation of GAA allows for an optimal supply of creatine in the organism.

The present invention pertains to a microorganism transformed to be capable of producing guanidinoacetic acid (GAA) and to a method for the fermentative production of GAA using such microorganism. The present invention also relates to a method for the fermentative production of creatine.

In biological systems GAA and ornithine are formed from arginine and glycine as starting materials by the catalytic action of an L-arginine:glycine-amidinotransferase (AGAT; EC 2.1 .4.1). This reaction is also the first step in creatine biosynthesis.

Arginine Glycine Ornithine Guanidinoacetic acid (GAA)

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

Fan Wenchao discloses a method for the production of creatine by fermentation of non-pathogenic microorganisms, such as Corynebacterium glutamicum (CN106065411 A). The microorganism has the following biotransformation functions: glucose conversion 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 acid semialdehyde; conversion of N-acetyl-L-glutamic acid semialdehyde to N- acetyl-L- ornithine; conversion of N-acetyl-L-ornithine to L-ornithine; conversion of L-ornithine to L-citru Hine; conversion of L-citrulline to arginino-succinic acid; conversion of arginino-succinic acid to L- arginine; conversion of L-arginine to guanidinoacetic acid; and, finally, conversion of guanidinoacetic acid to creatine. Fan Wenchao proposes, that the microorganism overexpresses one or more enzymes selected from the group consisting of N-acetylglutamate-synthase, N- acetylornithine-6-aminotransferase, N-acetylornithinase, ornithine-carbamoyl transferase, argininosuccinate synthetase, glycine amidino-transferase (EC: 2.1. 4.1), and guanidinoacetate N- methyltransferase (EC: 2.1 .1 .2). The microorganism overexpresses preferably glycine aminotransferase (L-arginine:glycine amidinotransferase) and guanidinoacetate N- methyltransferase.

A microorganism capable of producing guanidinoacetic acid (GAA) was published by Zhang et al. (ACS Synth. Biol. 2020, 9, 2066-275). They designed a reconstituted the ornithine cycle in E. coli by introducing a heterologous AGAT from different species (e.g., Homo sapiens, Cylindrospermopsis raciborskii, Moorea producens) and by introducing a citrulline synthesis module (e.g. ovexpression of argF and argl) and an arginine synthesis module (e.g. overexpression of argG, argH introduction of aspA) into E. coli.

Schneider and Jankowitsch (WO2021122400 A1) propose a method to produce GAA using a microorganism having a gene coding for a protein having the function of an L-arginine:glycine amidinotransferase (AGAT) and an increased carbamoyl phosphate synthase. The carbamoyl phosphate is an important precursor for the biosynthesis of GAA but also for L-arginine and other compounds.

To increase the production of GAA using a microorganism an intracellular high amount of the starting materials arginine and/or glycine are necessary. At the same time the byproduct of the AGAT reaction, L-ornithine, has to be recycled to L-arginine efficiently in order to prevent loss of carbon and energy.

Several approaches for increasing the production of one of the starting materials in GAA synthesis, i.e. L-arginine, in microorganisms are also known from literature. An overview for the metabolic engineering of Corynebacterium glutamicum (C. glutamicum) for L-arginine production is provided by Park et al. (NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5618). Yim et al. (J Ind Microbiol Biotechnol (2011) 38:1911-1920) could show that inactivation of the argR, gene coding for the central repressor protein ArgR controlling the L-arginine biosynthetic pathway, by disrupting the chromosomal argR gene in C. glutamicum leads to an improved arginine-producing strain.

Ginesy et al. (Microbial Cell Factories (2015) 14:29) report the successful engineering of E. coli for enhanced arginine production. Among other, they proposed the deletion of the argR repressor gene.

On the other hand, there is also a need of an increased intracellular glycine concentration in order to increase the production of GAA. This is achieved by the internal synthesis of glycine.

Alternatively, glycine can be added to the medium and membrane transport proteins facilitate the uptake. CycA from E. coli has been characterized as a secondary, H ⁺ dependent uptake transporter for D- alanine, D-serine, glycine, L-alanine, D-cycloserine (Robbins73, Cosloy73) and beta-alanine (Schneider et al., Appl Microbiol Biotechnol. 2004 Oct;65(5):576-82. doi: 10.1007/s00253-004- 1636-0. Epub 2004 Jun 24. PMID: 15221223). Also, Pulvermacher et al. (Microbiology (2009), 155, 106-114; DOI 10.1099/mic.0.023598-0) report that in Escherichia coli, the cycA gene encodes a permease for the transport of D-alanine, D-serine and glycine into the cell. Hook et al. (Microorganisms. 2022 Mar 17;10(3):647. doi: 10.3390/microorganismsl 0030647. PMID: 35336222) found that a-aminobutyrate, norvaline and L-valine are also substrates for CycA and overexpression of cycA improved L-isoleucine production by reducing the formation of unwanted by-products.

A BLAST search of the CycA protein from C. glutamicum ATCC13032 (SEQ ID NO:4) against Escherichia coli (E. coli) MG1655 proteins revealed, that CycA of C. glutamicum is 66% identical to CycA (= b4208) of E. coli.

The cycA gene (=NCglO453 = cg0555 = Cgl0470 = CGL_RS02385 = dagA SEQ ID NO:3) from C. glutamicum likely codes for a transmembrane transport protein belonging to the “Amino Acid- Polyamine-Organocation (APC) Superfamily (TC 2.A.3)” (Saier et al., Nucleic Acids Res. 2021 Jan 8;49(D1):D461-D467. doi: 10.1093/nar/gkaa1004) of secondary transporters. In the genome sequence of C. glutamicum strain ATCC13032 the cycA product is annotated as a D-serine/D- alanine/glycine transporter (Genbank accession NC_003450, locus_tag=" CGL_RS02385”; see also Kalinowski et al. 2003, J Biotechnol 104:5-25. 10.1016/S0168-1656(03)00154-8). However, the CycA protein has not been biochemically characterized yet and accordingly, its function in C. glutamicum is unknown.

The problem underlying the present inventions is to provide a microorganism transformed to be capable for producing guanidinoacetic acid (GAA) in higher amounts and a method for producing GAA using such microorganism.

The problem is solved by a microorganism comprising at least one heterologous gene coding for a protein having the function of a L-arginine:glycine amidinotransferase (AGAT, e.g. EC2.1.4.1) and having an overexpressed gene coding for a transmembrane transport protein with an amino acid sequence having at least 50 % identity to the sequence according to SEQ ID NO: 4. The amino acid sequence of the transmembrane transport protein may be at least 50 %, 66 %, 70 %, 80 % or 90 % identical to the sequence according to SEQ ID NO: 4. Preferably, the amino acid sequence of the transmembrane transport protein is at least 66 % identical, more preferably identical to the sequence according to SEQ ID NO: 4.

SEQ ID NO:3 shows the nucleotide sequence of the respective cycA gene. SEQ ID NO:4 is the amino acid sequence of the CycA protein of C. glutamicum. The CycA protein of C. glutamicum is a transmembrane transport protein belonging to the Amino Acid-Polyamine-Organocation Superfamily (TC 2. A.3) of transporters (Saier MH, Reddy VS, Moreno-Hagelsieb G, Hendargo KJ, Zhang Y, Iddamsetty V, Lam KJK, Tian N, Russum S, Wang J, Medrano-Soto A. “The Transporter Classification Database (TCDB): 2021 update.” Nucleic Acids Res. 2021 Jan 8;49(D1):D461-D467. doi: 10.1093/nar/gkaa1004).

A microorganism in the context of the present invention is an organism of microscopic size, which may exist in its single-celled form or as a colony of cells. Microorganisms include most unicellular organisms from all three domains of life. The domains Archaea and Bacteria contain microorganisms only. Among the third domain Eukaryota unicellular organism like yeasts, protists and protozoans belong to the group of microorganisms. Multicellular organisms or parts of them, like cell cultures, in particular cultures of animal cells, e.g. of human cells, are not considered as microorganism in the context of the present invention. Animal cells, e.g. human cells, are explicitly excluded from the definition of microorganisms in the context of the present invention.

The microorganism according to the present invention is capable to produce GAA in higher amounts without the addition of glycine to the production medium

A heterologous gene means that the gene has been inserted into a host organism which does not naturally have this gene. Insertion of the heterologous gene in the host is performed by recombinant DNA technology. Microorganisms that have undergone recombinant DNA technology are called transgenic, genetically modified or recombinant. A heterologous protein means a protein that is not naturally occurring in the microorganism. A homologous or endogenous gene means that the gene including its function as such or the nucleotide sequence of the gene is naturally occurring in the microorganism or is “native” in the microorganism. A homologous or a native protein means a protein that is naturally occurring in the microorganism.

In the microorganism according to the present invention the activity of the protein having the function of an L-arginine:glycine amidinotransferase (AGAT) is preferably increased compared to the respective activity in the wildtype organism. Preferably, the AGAT activity is increased by overexpression of the gene coding for the protein having the function of an L-arginine:glycine amidinotransferase.

In the microorganism according to the present invention the protein having the function of an L- arginine:glycine amidinotransferase (AGAT) comprises an amino acid sequence which is at least 80 % homologous to the amino acid sequence according to SEQ ID NO:2.

Overexpression of a gene is generally achieved by increasing the copy number of the gene and/or by functionally linking the gene with a strong promoter and/or by enhancing the ribosomal binding site and/or by codon usage optimization of the start codon or of the whole gene and/or by modifying activities of proteins modulating gene expression or a combination comprising a selection of all methods mentioned above.

A promoter is a DNA sequence consisting of about 40 to 50 base pairs and which constitutes the binding site for an RNA polymerase holoenzyme and the transcriptional start point, whereby the strength of expression of the controlled polynucleotide or gene can be influenced. Generally, it is possible to achieve an overexpression or an increase in the expression of genes in bacteria by selecting strong promoters, for example by replacing the original promoter with strong, native (originally assigned to other genes) promoters or by modifying certain regions of a given, native promoter (for example its so-called -10 and -35 regions) towards a consensus sequence, e.g. as taught by M. Patek et al. (Microbial Biotechnology 6 (2013), 103-117) for C. glutamicum. An example for a “strong” promoter is the superoxide dismutase (sod) promoter (“Psod”; Z. Wang et al., Eng. Life Sci. 2015, 15, 73-82). A “functional linkage” is understood to mean the sequential arrangement of a promoter with a gene, which leads to a transcription of the gene.

In the present context, the extent to which protein sequences are related is expressed as identity meaning more specifically, how much of the sequence is identical according to the results of a blast search given as percentage (see https://www.ncbi.nlm.nih.gov/books/NBK62051/ for definition).

The microorganism according to the present invention may have an increased ability to produce L- arginine from L-ornithine compared with the ability of the wildtype microorganism.

In the context of the present invention, a microorganism having an increased ability to produce L- arginine means a microorganism producing L-arginine in excess of its own need. Examples for such L-arginine producing bacteria are e.g. C. glutamicum ATCC 21831 orthose disclosed by Park et al. (NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5618) or by Ginesy et al. (Microbial Cell Factories (2015) 14:29). In contrast to formerly described bacteria having an increased ability to produce L-arginine, L-arginine excretion is not necessary in strains for GAA production since arginine is utilized inside the cell in the framework of the present invention for GAA production.

In a further embodiment the microorganism according to the present invention the expression of an argR gene coding for the arginine responsive repressor protein ArgR is attenuated compared to the expression of the argR gene in the wildtype microorganism. Alternatively, the argR gene is deleted.

The microorganism of the present invention may belong to the genus Corynebacterium, to the genus Bacillus (Yan, K., et al. (2022). "Biosynthesis of Guanidinoacetate by Bacillus subtilis Whole- Cell Catalysis." Fermentation 8(3):116), to the genus Enterobacteriaceae or to the genus Pseudomonas. In a particular embodiment of the present invention the microorganism is Corynebacterium glutamicum (C. glutamicum) or Escherichia coli (E. coli).

The present invention further concerns a method for the fermentative production of guanidino acetic acid (GAA), comprising the steps of cultivating the microorganism according to the present invention in a medium, and accumulating GAA in the medium to form a GAA containing fermentation broth.

The method according to the present invention may be further characterized in that no glycine is added to the medium.

Preferably, the method further comprises isolating GAA from the GAA containing fermentation broth.

In a particular embodiment the microorganism of the present invention further comprises a gene coding for an enzyme having the activity of a guanidinoacetate N-methyltransferase. The gene coding for an enzyme having the activity of a guanidinoacetate N-methyltransferase may be overexpressed.

The present invention also concerns a method for the fermentative production of creatine, comprising the steps of a) cultivating the microorganism according to the present invention further comprising a gene coding for an enzyme having the activity of a guanidinoacetate N- methyltransferase in a suitable medium under suitable conditions, and b) accumulating creatine in the medium to form a creatine containing fermentation broth.

Preferably, the method further comprises isolating creatine from the creatine containing fermentation broth. Creatine may be extracted from fermentation broth by isoelectric point method and I or ion exchange method. Alternatively, creatine can be further purified by a method of recrystallization in water.

Brief description of the sequences

SEQ ID NO:1 : Open reading frame coding for a L-arginine:glycine amidinotransferase (AGAT_Mp, EC 2.1 .4.1 ; locus_tag BJP34_00300) of Moorena producens.

SEQ ID NO:2: The derived AGAT_Mp amino acid sequence (Genbank accession Number WP_070390602)

SEQ ID NO:3: Open reading frame NCglO453 (=cycA) of Corynebacterium glutamicum ATCC13032 (NCBI Reference Sequence: NC_003450.3)

SEQ ID NO:4: The derived amino acid sequence of NCglO453 (=cycA) (NCBI Reference Sequence: WP_231838291 .1) SEQ ID NO:5: AGAT-Mp insert ordered for gene synthesis

SEQ ID NO:6: E. coli-C. glutamicum shuttle plasmid pLIB_P

SEQ ID NO:7: PCR primer cycA_f_Notl

SEQ ID NO:8: PCR primer cycA_r_Notl

SEQ ID NO:9: PCR primer DargRJf

SEQ ID NQ:10: PCR primer DargRJr

SEQ ID NO:11 : PCR primer DargR_rf

SEQ ID NO:12: PCR primer DargR_rr

A) MATERIALS and METHODS

Chemicals

Kanamycin solution from Streptomyces kanamyceticus was purchased from Sigma Aldrich (St. Louis, USA, Cat. no. K0254). If not stated otherwise, all other chemicals were purchased analytically pure from Merck (Darmstadt, Germany), Sigma Aldrich (St. Louis, USA) or Carl-Roth (Karlsruhe, Germany).

Cultivation for cell proliferation

If not stated otherwise, cultivation I incubation procedures were performed as follows herewith: a. LB broth (MILLER) from Merck (Darmstadt, Germany; Cat. no. 110285) was used to cultivate E. coli strains in liquid medium. The liquid cultures (10 ml liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were incubated in the Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland) at 30°C and 200 rpm. b. LB agar (MILLER) from Merck (Darmstadt, Germany, Cat. no. 110283) was used for 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). c. Brain heart infusion broth (BHI) from Merck (Darmstadt, Germany, Cat. no. 110493) was used to cultivate C. glutamicum strains in liquid medium. The liquid cultures (10 ml liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were incubated in the Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland) at 30°C and 200 rpm. d. Brain heart agar (BHI-agar) from Merck (Darmstadt, Germany, Cat. no. 113825) was used for cultivation of C. glutamicum strains on agar plates. The agar plates were incubated at 30°C in an incubator from Heraeus Instruments with Kelvitron® temperature controller (Hanau, Germany). e. For cultivating C. 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 Kelvitron® temperature controller (Hanau, Germany).

Determining optical density of bacterial suspensions a. The optical density of bacterial suspensions in shake flask cultures was determined at 600 nm (OD600) using the Bio-Photometer from Eppendorf AG (Hamburg, Germany). b. The optical density of bacterial suspensions produced in the Wouter Duetz (WDS) micro fermentation system (24-Well Plates) was determined at 660 nm (OD660) with the GENios™ plate reader from Tecan Group AG (Mannedorf, Switzerland).

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® 381 OX) using an Eppendorf 5417 R benchtop centrifuge (5 min. at 13.000 rpm). b. Bacterial suspensions with a maximum volume of 50 ml were centrifuged in 15 ml or 50 ml centrifuge tubes (e.g. FalconTM 50 ml Conical Centrifuge Tubes) using an Eppendorf 5810 R benchtop centrifuge for 10 min. at 4.000 rpm.

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 instructions of the manufacturer.

Polymerase chain reaction (PCR)

PCR with a proof reading (high fidelity) polymerase was used to amplify a desired segment of DNA for sequencing or DNA assembly. Non-proof-reading polymerase Kits were used for determining the presence or absence of a desired DNA fragment directly from E. coli or C. glutamicum colonies. a. The Phusion® High-Fidelity DNA Polymerase Kit (Phusion Kit) from New England BioLabs Inc. (Ipswich, USA, Cat. No. M0530) was used for template-correct amplification of selected DNA regions according to the instructions of the manufacturer (see Table 1).

Table 1 : Thermocycling conditions for PCR with Phusion® High-Fidelity DNA Polymerase Kit from New England BioLabs Inc. b. Taq PCR Core Kit (Taq Kit) from Qiagen (Hilden, Germany, Cat. No.201203) was used to amplify a desired segment of DNA to confirm its presence. The kit was used according to the instructions of the manufacturer (see Table 2).

Table 2: Thermocycling conditions for PCR with Taq PCR Core Kit (Taq Kit) from Qiagen. c. SapphireAmp® Fast PCR Master Mix (Sapphire Mix) from Takara Bio Inc (Takara Bio Europe S.A.S., Saint-Germain-en-Laye, France, Cat. No. RR350A/B) was used as an alternative to confirm the presence of a desired segment of DNA in cells taken from E. coli or C. glutamicum colonies according to the instructions of the manufacturer (see Table 3).

Table 3: Thermocycling conditions for PCR with SapphireAmp® Fast PCR Master Mix (Sapphire Mix) from Takara Bio Inc. d. All oligonucleotide primers were synthesized by Eurofins Genomics GmbH (Ebersberg, Germany). e. As PCR template either a suitably diluted solution of isolated plasmid DNA or of total DNA isolated from a liquid culture or the total DNA contained in a bacterial colony (colony PCR) was used. For said colony PCR the template was prepared by taking cell material with a sterile toothpick from a colony on an agar plate and placing the cell material directly into the PCR reaction tube. The cell material was heated for 10 sec. with 800 W in a microwave oven type Mikrowave & Grill from SEVERIN Elektrogerate GmbH (Sundern, Germany) and then the PCR reagents were added to the template in the PCR reaction tube. f. All PCR reactions were carried out in PCR cyclers type Mastercycler or Mastercycler nexus gradient from Eppendorf AG (Hamburg, Germany).

Restriction enzyme digestion of DNA

For restriction enzyme digestions either „FastDigest restriction endonucleases (FD)“ (ThermoFisher Scientific, Waltham, USA) or restriction endonucleases from New England BioLabs Inc. (Ipswich, USA) were used. The reactions were carried out according to the instructions of the manufacturer’s manual.

Determining the sizes of DNA fragments a. The sizes of small DNA fragments (<1000 bps) were usually determined by automatic capillary electrophoresis using the QIAxcel from Qiagen (Hilden, Germany). b. If DNA fragments needed to be isolated or if the DNA fragments 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.

Purification of PCR amplificates and restriction fragments

PCR amplificates 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 with 30 pl 10 mM TrisTICI (pH 8.5).

Determining DNA concentration

DNA concentration was measured using the NanoDrop Spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH, since 2015 VWR brand (Erlangen, Germany).

Assembly cloning

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 mix, containing the linear vector and at least one DNA insert, was incubated at 50°C for 60 min. 0.5 pl of Assembly mixture was used for each transformation experiment.

Chemical transformation of E. coli

For plasmid cloning, chemically competent “NEB® Stable Competent E. coli (High Efficiency)" (New England BioLabs Inc., Ipswich, USA, Cat. No. C3040) were transformed according to the manufacturer's protocol. Successfully transformed cells were selected on LB agar supplemented with 25 mg/l kanamycin.

Transformation of C. glutamicum

Transformation of C. glutamicum with plasmid-DNA was conducted via 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 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.

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

Determining nucleotide sequences

Nucleotide sequences of 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).

Clonemanager Professional 9 software from Scientific & Educational Software (Denver, USA) was used to visualize and evaluate the sequences as well as for in silico assembly of sequences.

Glycerol stocks of E. coli and C. glutamicum strains

For long time storage of E. coli- and C. glutamicum strains glycerol stocks were prepared. Selected E. coli clones were cultivated in 10 ml LB medium supplemented with 2 g/l glucose. Selected C. glutamicum clones were cultivated in 10 ml twofold concentrated BHI medium supplemented with 2 g/l glucose. Media for growing plasmid containing E. coli- and C. glutamicum strains were supplemented with 25 mg/l kanamycin. The medium was contained in 100 ml Erlenmeyer flasks with 3 baffles. It was inoculated with a loop of cells taken from a colony. The culture was then incubated for 18 h at 30°C and 200 rpm. After said incubation period 1.2 ml 85 % (v/v) sterile glycerol were added to the culture. The obtained glycerol containing cell suspension was then aliquoted in 2 ml portions and stored at -80°C.

GAA production in small-scale cultivations

The millilitre-scale cultivation system according to Duetz (2007) was used to assess the GAA- production of the strains. For this purpose, 24-deepwell microplates (24 well WDS plates) from EnzyScreen BV (Heemstede, Netherlands, Cat. no. CR1424) filled with 2.5 ml medium per well were used.

Precultures of the strains were done in 10 ml seed medium (SM). The medium was contained in a 100 ml Erlenmeyer flask with 3 baffles. It was inoculated with 100 pl of a glycerol stock culture and the culture was incubated for 24 h at 30°C and 200 rpm. The composition of the seed medium (SM) is shown in Table 4.

Table 4: Seed medium (SM)

After said incubation period the optical densities OD600 of the precultures were determined. The volume, needed to inoculate 2.5 ml of production medium (PM) to an OD600 of 0.1 , was sampled from the preculture, centrifuged (1 min at 8000 g) and the supernatant was discarded. Cells were then resuspended in 100 pl of production medium.

The main cultures were started by inoculating the 2.4 ml production medium (PM) containing wells of the 24 Well WDS-Plate with each 100 pl of the resuspended cells from the precultures. The composition of the production medium (PM) is shown in Table 5.

Table 5: Production medium (PM)

The main cultures were incubated for 72 h at 30 °C and 225 rpm in an Infors HT Multitron standard incubator shaker from Infors GmbH (Bottmingen, Switzerland) until complete consumption of glucose. The glucose concentration in the suspension was analyzed with the blood glucose-meter OneTouch Vita® from LifeScan (Johnson & Johnson Medical GmbH, Neuss, Germany).

After cultivation the culture suspensions were transferred to a deep well microplate. A part of the culture suspension was suitably diluted to measure the GD600. Another part of the culture was centrifuged and the concentration of GAA in the supernatant was analyzed as described below.

Quantification of GAA

Samples were analyzed with an analyzing system from Agilent, consisting of a HPLC “Infinity 1260” coupled with a mass analyzer “Triple Quad 6420” (Agilent Technologies Inc., Santa Clara, USA). Chromatographic separation was done on the Atlantis HILIC Silica column, 4,6X250mm, 5pm (Waters Corporation, Milford, USA) at 35°C. Mobile phase A was water with 10mM ammonium formate and 0.2% formic acid. Mobile phase B was a mixture of 90% acetonitrile and 10 % water, 10 mM ammonium formate were added to the mixture. The HPLC system was started with 100 % B, followed by a linear gradient for 22 min and a constant flow rate of 0,6 mL/min to 66 % B. The mass analyzer was operated in the ESI positive ionization mode. For detection of GAA the m/z values were monitored by using an MRM fragmentation [M+H] + 118 - 76. The limit of quantification (LOQ) for GAA was fixed to 7 ppm.

B) EXPERIMENTAL RESULTS

Example 1 : Cloning of the gene AGAT-Mp coding for an L-arginine:glycine amidinotransferase (AGAT, EC 2.1 .4.1) from Moorena producens

Moorena producens is a filamentous cyanobacterium. The genome of the Moorena 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 U S A. 2017 Mar 21 ;114(12):3198-3203. doi: 10.1073/pnas.16185561 14; Genbank accession Number CP017599.1). It contains an open reading frame coding for a 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 (Genbank accession Number WP_070390602).

Using the software tool ..Optimizer" (http://genomes.urv.es/OPTIMIZER/) the amino acid sequence was translated back into a DNA sequence optimized for the codon usage of C. glutamicum. The 5’- end of the optimized gene was expanded with a Bsal restriction site, a 5’-UTR sequence for assembly cloning and a ribosomal binding site. At the 3’-end a second stop-codon, a sequence for assembly cloning and a Bsal-site was added. The resulting DNA sequence was named AGAT-Mp- insert (SEQ ID NO:5) was ordered for gene synthesis from Eurofins Genomics GmbH (Ebersberg, Germany) and it was delivered as part of a cloning plasmid with an ampicillin resistance gene (designated as pEX-A258_AGAT-Mp).

The E. coli-C. glutamicum shuttle plasmid pLIB_P consists of the replication origin from pBL1 (for C. glutamicum), the pSC101 replication origin (for E. coli) and a kanamycin resistance gene. Following a unique Not restriction site it has a strong promoter, two inversely orientated Bsal-sites and the BioBricks Terminator BBa_B1006 (SEQ ID NO:6). pLIB_P was digested using the restriction endonuclease Bsal and the DNA was purified with the ..QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

The cloning plasmid pEX-A258_AGAT-Mp was digested using the restriction endonuclease Bsal and the DNA was purified with the ..QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

The DNA solutions of Bsal digested pLIB_P and pEX-A258_AGAT-Mp were joined, and matching sequence ends were assembled using the “NEBuilder HiFi DNA Assembly Cloning Kit” (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). The 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. Proper plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pLIB_P_AGAT-Mp.

Example 2: Cloning of the expression plasmid pLIB_cycA_AGAT-Mp coding for CycA (NCglO453) from Corynebacterium glutamicum and the L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) from Moorena producens

The expression plasmid pLIB_cycA_AGAT-Mp contains the cycA gene (NCglO453) from Corynebacterium glutamicum postitioned upstream of the AGAT-Mp gene and its promoter. A PCR was conducted using the primers cycA_f_Notl (Seq ID NO:7) and cycA_r_Notl (Seq ID NO:8) and genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. The resulting PCR product contained the cycA gene (NCglO453) plus 72 bp sequence upstream of the ata start codon. It was purified with the „QIAquick PCR Purification Kit“ (Qiagen GmbH, Hilden, Germany).

The Plasmid pLIB_P_AGAT-Mp was digested using the restriction endonuclease Notl and the DNA was purified with the „QIAquick PCR Purification Kit“ (Qiagen GmbH, Hilden, Germany).

The DNA of Notl-digested pLIB_P_AGAT-Mp was joined with the cycA containing PCR product and the matching sequence ends were assembled using the “NEBuilder HiFi DNA Assembly Cloning Kit” (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520).

The 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. Proper plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid was named pLIB_cycA_AGAT-Mp.

Example 3: Chromosomal deletion of the gene argR in ATCC13032

To improve intracellular L-Arginine formation and L-Arginine recycling from L-Ornithine, the gene argR coding for the central repressor protein ArgR controlling the L-arginine biosynthetic pathway was inactivated.

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

For constructing the insert, two DNA fragments were created by PCR with the following pairs of primers (genomic DNA of ATCC13032 as template):

DargRJf (SEQ ID NO:9), + DargRJr (SEQ ID NQ:10)

= left homology arm (983 bps)

DargR_rf (SEQ ID NO:11), + DargR_rr (SEQ ID NO:12)

= left homology arm (984 bps)

The PCR products were purified using the „QIAquick PCR Purification Kit" (Qiagen GmbH, Hilden, Germany).

The linearized plasmid and the PCR products 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.

For deleting the argR gene, pK18mobsacB_DargR was transformed into Corynebacterium glutamicum ATCC13032 (Kinoshita et al., J. Gen. Appl. Microbiol. 1957; 3(3): 193-205) by electroporation. Chromosomal integration (resulting from a 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 for 48 h at 33°C.

Individual colonies were transferred onto fresh agar plates (with 25 mg/l kanamycin) and incubated for 24 h at 33°C. Liquid cultures of these clones were cultivated for 24 h at 33°C in 10 ml BHI medium contained in 100 ml Erlenmeyer flasks with 3 baffles. To isolate clones that have encountered a second recombination event, an aliquot was taken from each liquid culture, suitably diluted and plated (typically 100 to 200 pl) on BHI agar supplemented with 10 % saccharose.

These agar plates were incubated for 48 h at 33°C. Colonies growing on the saccharose containing agar plates were then examined for kanamycin sensitivity. To do so a toothpick was used to remove cell material from the colony and to transfer it onto BHI agar containing 25 mg/l kanamycin and onto BHI agar containing 10 % saccharose. The agar plates were incubated for 60 h at 33°C. 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.

Example 4: Transformation of C. glutamicum ATCC13032_DargR with the expression plasmids

Strain ATCC13032_DargR was transformed by electroporation with the expression plasmids pLIB_P_AGAT-Mp and pLIB_cycA_AGAT-Mp. Plasmid containing cells were selected with 25 mg/l kanamycin. The resulting plasmid containing strains are shown in Table 6.

Table 6: Plasmid containing derivatives of C. glutamicum ATCC13032 Example 5: Impact of overexpression of cycA on GAA production

To assess the impact of the overexpression of cycA on GAA production, strains ATCC13032_DargR/pLIB_P_AGAT-Mp and ATCC13032_DargR/pLIB_cycA_AGAT-Mp were cultivated in the Wouter Duetz system in production medium without glycine addition and the resulting GAA titers were determined as described above.

Table 7: Impact of the overexpression of cycA on GAA production

The cultivation of strain with the cycA gene containing plasmid resulted in a higher GAA titer, compared to ATCC13032/pLIB_P_AGAT-Mp (see Table 7). We conclude that the overexpression of the cycA gene improves the production of GAA.