Patent Application
20250171790
The present application is accompanied by an ASCII text file as a computer readable form containing the sequence listing entitled, “2023 Jun. 23-003950USPCT_SL_ST25-02.txt”, created on Jun. 23, 2023, with a file size of 230,608 bytes, the content of which is hereby incorporated by reference in its entirety.
The present invention relates to a microorganism transformed to be capable of producing guanidinoacetic acid (GAA) and to a method for the fermentative production of GAA using such a microorganism. The present invention also relates to a method for the fermentative production of creatine.
GAA is an organic compound used as animal feed additive (WO2005120246 A1/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 method to produce GAA by a fermentative process using industrial feed stocks (e.g. ammonia, ammonium salts and glucose or sugar containing substrates) as starting material. 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), which is the first step in creatine biosynthesis (US20060200870 A1): L-arginine+glycineAGAT>L-ornithine+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 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. Sosio et al. (Cell Chemical Biology 25, 540-549, May 17, 2018) elucidated the biosynthetic pathway for pseudouridimycin in Streptomyces sp. They describe as an intermediate reaction the formation of GAA and L-ornithine by the reaction of L-arginine with glycine catalyzed by PumN, an L-arginine:glycine-amidinotransferase (AGAT). Humm et al. expressed a recombinant gene encoding human AGAT in Escherichia coli and identified cysteine-407 as an active-site residue of AGAT (Biochem. J. (1997) 322, 771-776).
Mijts et al. (WO 2018079687 A1) disclose in the context of the production of objective substances in microorganisms, e.g. vanillin and vanillic acid, that creatine can be produced from L-arginine and glycine. The authors further propose that this may be achieved by using L-arginine biosynthesis enzymes, glycine biosynthesis enzymes, and enzymes that catalyse the conversion of L-arginine and glycine into creatine. L-arginine and glycine can be combined to generate guanidinoacetate (GAA) and ornithine by the action of AGAT (EC 2.1.4.1) and that GAA can be methylated to generate creatine by the action of guanidinoacetate N-methyltransferase (GAMT, EC 2.1.1.2), using S-adenosyl methionine (SAM) as the methyl donor. The authors mention in the context of the production of polyamines that examples for L-arginine biosynthesis enzymes may also include the well-known L-ornithine biosynthesis enzymes, as well as the enzymes that are well-known from the so called L-ornithine cycle, i.e. carbamoyl phosphate synthase (carAB), ornithine carbamoyl transferase (argF, argI), argininosuccinate synthetase (argG), argininosuccinate lyase (argH) (cf. Marc et al., Eur. J. Biochem. 267, 5217-5226, 200).
However, several more specific approaches for increasing the production of one of the starting materials in GAA synthesis, i.e. L-arginine, in microorganisms, particularly bacteria, 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). They propose random mutagenesis and screening for L-arginine producers of already L-arginine producing C. glutamicum strains, e.g. of ATCC 21831 (Nakayama and Yoshida 1974, U.S. Pat. No. 3,849,250 A) and stepwise rational metabolic engineering based on system-wide analysis of metabolism results in a gradual increase in L-arginine production throughout the strain engineering steps. 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.
Kurahashi et al. (EP1057893 A1) report on methods for increasing the L-arginine producing ability of a microorganism by enhancing L-arginine biosynthesis enzymes utilizing recombinant DNA techniques, e.g. by utilizing a microorganism belonging to the genus Corynebacterium or Brevibacterium which is made to harbor a recombinant DNA comprising a vector DNA and a DNA fragment containing genes for acetylomithine deacetylase, N-acetylglutamic acid-γ-semialdehyde dehydrogenase, N-acetyl glutamokinase and argininosuccinase derived from a microorganism belonging to the genus Escherichia. For an improved L-arginine production the authors further propose a microorganism which is enhanced in an activity of intracellular glutamate dehydrogenase (GDH) and which has an L-arginine producing ability.
A method of using a genetic recombinant strain, wherein a gene which inhibits the expression of arginine-biosynthesizing operon, argR, was inactivated has been reported by Suga et al. (U.S. Pat. No. 7,160,705 B2). In particular, the deletion in argR, which controls the arginine operon, has been considered as an important factor in arginine production.
In a microorganism of Corynebacterium, the argCJBDFR gene, which is involved in arginine biosynthesis, is constituted in the form of an operon and is subjected to feedback-inhibition by intracellular arginine (Sakanyan et al., Microbiology, 142:9-108, 1996), thus imposing a limitation on its high yield L-arginine production.
However, Bae et al. (EP3153573 A1) in an attempt to increase the production yield of L-arginine in C. glutamicum, discovered that L-arginine can be produced in higher yield compared to the parental L-arginine-producing strain, by enhancing the activities of the arginine operon and ornithine carbamoyltransferase (ArgF, ArgF2), without any deletion in arginine repressor (argR).
The arginine operon is an operon consisting of genes encoding enzymes involved in the mechanism of L-arginine biosynthesis, and in particular, arginine operon consists of genes encoding enzymes constituting the cyclic steps of L-arginine biosynthesis. Specifically, the arginine operon consists of N-acetylglutamyl phosphate reductase (ArgC), glutamate N-acetyltransferase (ArgJ), N-acetylglutamate kinase (ArgB), acetylornithine aminotransferase (ArgD), ornithine carbamoyltransferase (ArgF), and the arginine repressor (ArgR). These enzymes are involved in the continuous enzyme reactions of L-arginine biosynthesis and are controlled by the arginine repressor encoded by argR (WO 2006/057450 A1).
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-citrulline; 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-δ-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.
Until now, microorganisms suitable for an increased production of GAA compared to their wildtype forms and a respective method for the production of GAA using such microorganisms have not been reported.
Therefore, the problem underlying the present invention is to provide 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 problem is solved by a microorganism comprising an increased activity of an enzyme having the function of a carbamoylphosphate synthase (EC 6.3.4.16) compared to the respective enzymic activity in the wildtype microorganism and comprising at least one heterologous gene coding for a protein having the function of an L-arginine:glycine amidinotransferase (AGAT).
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. Thus, the microorganism according to the present invention is recombinant.
The increased activity of the enzyme having the function of a carbamoylphosphate synthase may be achieved by a mutation and/or overexpression of a gene coding for the enzyme having the function of a carbamoylphosphate synthase.
The activity of the L-arginine:glycine amidinotransferase may also be increased by a mutation and/or overexpression of the gene coding for the L-arginine:glycine amidinotransferase.
Proteins having the function of an L-arginine:glycine amidinotransferase (AGAT) belong to the amidinotransferase family. The amidinotransferase family comprises glycine (EC: 2.1.4.1) and inosamine (EC: 2.1.4.2) amidinotransferases, enzymes involved in creatine and streptomycin biosynthesis respectively. This family also includes arginine deiminases, EC: 3.5.3.6. These enzymes catalyse the reaction: arginine+H2O<=>citrulline+NH3. Also found in this family is the Streptococcus anti tumour glycoprotein. Enzymes or proteins with an L-arginine:glycine-amidinotransferase (AGAT) activity are also described to possess a conserved domain that belongs to the PFAM Family: Amidinotransf (PF02274) (Marchler-Bauer A et al. (2017), “CDD/SPARCLE: functional classification of proteins via subfamily domain architectures.”, Nucleic Acids Res. 45 (D1): D200-D203.) as described also in the following publications: Pissowotzki K et al., Mol Gen Genet 1991; 231:113-123 (PUBMED: 1661369 EPMC: 1661369); D'Hooghe I et al., J Bacteriol 1997; 179:7403-7409 (PUBMED: 9393705 EPMC: 9393705); Kanaoka M et al., Jpn J Cancer Res 1987; 78:1409-1414 (PUBMED: 3123442 EPMC: 3123442). Particular examples of AGATs are those of Moorea producens, Homo sapiens, Rattus norvegicus, Galeopterus variegatus, and of Cylindrospermopsis raciborskii.
The microorganism according to the present invention ideally has an improved ability to produce L-arginine compared with the ability of the wildtype microorganism. This property may be achieved by selection of microorganisms that are natural L-arginine producers or may have acquired the ability to produce L-arginine by mutation.
The microorganism according to the present invention and having an improved ability to produce L-arginine compared with the ability of the wildtype microorganism may have an increased activity of an enzyme having the function of an argininosuccinate lyase (E.C. 4.3.2.1) compared to the respective enzymic activity in the wildtype microorganism.
Furthermore, in the microorganism according to the present invention, the activity of an enzyme having the function of an ornithine carbamoyltransferase (EC 2.1.3.3) may be increased compared to the respective enzymic activity in the wildtype microorganism.
In the microorganism according to the present invention, the activity of an enzyme having the function of an argininosuccinate synthetase (E.C. 6.3.4.5) may be also increased compared to the respective enzymic activity in the wildtype microorganism.
Increased enzyme activities in microorganisms can be achieved, for example, by mutation of the corresponding endogenous gene. A further measure to increase enzymic activities may be to stabilize the mRNA coding for the enzymes.
The increased activities of the above-mentioned enzymes may also be achieved by overexpressing the genes coding for the respective enzymes. In other words, the problem is preferably solved by a microorganism having an improved ability to produce L-arginine compared with the ability of the wildtype organism and/or having at least one or more overexpressed genes (e.g. carA, carB) coding for a protein having the function of a carbamoylphosphate synthase (EC 6.3.4.16), and further comprising a gene coding for a protein having the function of an L-arginine:glycine amidinotransferase (AGAT, e.g. EC 2.1.4.1).
The microorganism according to the present invention preferably also comprises at least one or more overexpressed genes selected from the group consisting of a gene (e.g. argF/argF2/argI) coding for a protein having the function of an ornithine carbamoyltransferase (EC 2.1.3.3), a gene (e.g. argG) coding for a protein having the function of an argininosuccinate synthetase (E.C. 6.3.4.5), and a gene (e.g. argH) coding for a protein having the function of an argininosuccinate lyase (E.C. 4.3.2.1).
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 or a combination comprising a selection of all methods mentioned above.
In the context of the present invention, a microorganism having an improved ability to produce L-arginine means a microorganism producing L-arginine in excess of its own need. Examples for such L-arginine producing microorganisms are e.g. C. glutamicum ATCC 21831 or those disclosed by Park et al. (NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5618) or by Ginesy et al. (Microbial Cell Factories (2015) 14:29).
In one embodiment of the microorganism according to the present invention the arginine operon (argCJBDFR) may be overexpressed.
Alternatively, in the microorganism according to the present invention the argR gene coding for the arginine responsive repressor protein ArgR may be attenuated or deleted.
In a further embodiment of the present invention and, optionally in addition to the above-mentioned modifications, at least one or more of the genes coding for an enzyme of the biosynthetic pathway of L-arginine, comprising of gdh, argJ, argB, argC and/or argD coding for a glutamate dehydrogenase, an ornithine acetyltransferase, an acetylglutamate kinase, an acetylglutamylphosphate reductase and an acetylornithine aminotransferase, respectively, is overexpressed in the microorganism according to the present invention.
Table 1 shows the different names of enzymes involved in or contributing to arginine biosynthesis in different species, i.e. E. coli, C. glutamicum and Pseudomonas putida (P. putida).
In the microorganism of the present invention the gene coding for a protein having the function of an L-arginine:glycine amidinotransferase may further be overexpressed. 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 of the whole gene or a combination comprising a selection or all methods mentioned above.
E. coli
C. glutamicum
P. putida
putida)
The protein having the function of an L-arginine:glycine amidinotransferase (AGAT) in the microorganism of the present invention may comprise an amino acid sequence which is at least 70% homologous, preferably at least 80% or at least 90% homologous to the amino acid sequence according to SEQ ID NO: 2 or to SEQ ID NO: 4 (“AGAT_Mp” of Moorea producens). In a further embodiment of the present invention the amino acid sequence of the L-arginine:glycine amidinotransferase is identical to amino acid sequence according to SEQ ID NO: 2 or to SEQ ID NO: 4 (cf. Database UniProt, 15 Feb. 2017, “Glycine amidinotransferase”, XP055706853, EBI accession no. UNIPROT: A0A1D8TKD3). The sequence of wildtype DNA coding for the Moorea producens AGAT is SEQ ID NO: 1, the corresponding DNA sequence that has been codon optimized for C. glutamicum is SEQ ID NO: 3. The corresponding DNA sequence for the Moorea producens AGAT gene that has been codon optimized for P. putida is SEQ ID NO: 33.
The protein having the function of an L-arginine:glycine amidinotransferase in the microorganism of the present invention may comprise an amino acid sequence which is at least 70% homologous, preferably at least 80% or at least 90% homologous to the amino acid sequence according to SEQ ID NO:16 or to SEQ ID NO:26 (“AGAT_cyrA” of Cylindrospermopsis raciborskil ATW205). In a further embodiment of the present invention the amino acid sequence of the L-arginine:glycine amidinotransferase is identical to amino acid sequence according to SEQ ID NO: 16 or to SEQ ID NO: 26. The sequence of wildtype DNA coding for the Cylindrospermopsis raciborskil AGAT is SEQ ID NO: 15, the corresponding DNA sequence that has been codon optimized for C. glutamicum is SEQ ID NO: 25.
The protein having the function of an L-arginine:glycine amidinotransferase in the microorganism of the present invention may comprise an amino acid sequence which is at least 70% homologous, preferably at least 80% or at least 90% homologous to the amino acid sequence according to SEQ ID NO: 23 (“AGAT_Gv” of Galeopterus variegatus), preferably to SEQ ID NO: 24 or to SEQ ID NO: 32. In a further embodiment of the present invention the amino acid sequence of the L-arginine:glycine amidinotransferase is identical to amino acid sequence according to SEQ ID NO: 24 or to SEQ ID NO:32 The sequence of the corresponding Galeopterus variegatus AGAT DNA that has been codon optimized for C. glutamicum is SEQ ID NO: 31.
The protein having the function of an L-arginine:glycine amidinotransferase in the microorganism of the present invention may comprise an amino acid sequence which is at least 70% homologous, preferably at least 80% or at least 90% homologous to the amino acid sequence according to SEQ ID NO:18 (“AGAT_Hs” of Homo sapiens), preferably to SEQ ID NO: 20 or to SEQ ID NO:28, e.g. the amino acid sequence according to SEQ ID NO: 21 or to SEQ ID NO: 22 or to SEQ ID NO: 30 (each “AGAT Rn” of Rattus norvegicus: the corresponding DNA, codon optimized for C. glutamicum is SEQ ID NO: 29). In a further embodiment of the present invention the amino acid sequence of the L-arginine:glycine amidinotransferase is identical to amino acid sequence according to SEQ ID NO: 18. The sequence of wildtype DNA coding for the Homo sapiens AGAT is SEQ ID NO: 17, the corresponding DNA sequence that has been codon optimized for C. glutamicum is SEQ ID NO: 27.
The microorganism of the present invention may belong to the genus Corynebacterium, preferably Corynebacterium glutamicum (C. glutamicum), or to the genus Enterobacteriaceae, preferably Escherichia coli (E. coli), or to the genus Pseudomonas, preferably Pseudomonas putida (P. putida).
Generally, increased enzyme activities in microorganisms can be achieved, for example, by mutation of the corresponding endogenous gene. Enzyme activities can also be enhanced by overexpression of the corresponding gene.
Generally, the overexpression of a gene, according to the present invention, is achieved by increasing the copy number of the gene and/or by an enhancement of regulatory factors, e.g. 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. The enhancement of such regulatory factors which positively influence gene expression can, for example, be achieved by modifying the promoter sequence upstream of the structural gene in order to increase the effectiveness of the promoter or by completely replacing said promoter with a more effective or a so-called strong promoter. Promoters are located upstream of the gene. 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. A “functional linkage” is understood to mean the sequential arrangement of a promoter with a gene, which leads to a transcription of the gene.
The genetic code is degenerated which means that a certain amino acid may be encoded by a number of different triplets. The term codon usage refers to the observation that a certain organism will typically not use every possible codon for a certain amino acid with the same frequency. Instead an organism will typically show certain preferences for specific codons meaning that these codons are found more frequently in the coding sequence of transcribed genes of an organism. If a certain gene foreign to its future host, i.e. from a different species, should be expressed in the future host organism the coding sequence of said gene should then be adjusted to the codon usage of said future host organism (i.e. codon usage optimization).
The above-mentioned problem is further solved by a method for the fermentative production of guanidino acetic acid (GAA), comprising the steps of a) cultivating the microorganism according to the present invention as defined above in a suitable medium under suitable conditions, and b) accumulating GAA in the medium to form an GAA containing fermentation broth.
The method according to the present invention may further comprise adding glycine and/or adding L-arginine and/or adding L-omithine to the medium. Preferably, the medium is supplemented with glycine in a concentration ranging from 0.1 to 300 g glycine/l medium, preferably 0.82 g glycine/l medium, and/or with L-arginine to obtain a concentration ranging from 0.1 to 200 g L-arginine/l medium, preferably 1.9 g L-arginine/l medium.
The method of the present invention may further comprise the step of isolating GAA from the fermentation broth.
The method according to the present invention may further comprise the step of drying and/or granulating the GAA containing fermentation broth.
The present invention further concerns a microorganism as defined above, further comprising a gene coding for an enzyme having the activity of a guanidinoacetate N-methyltransferase (EC: 2.1.1.2). Preferably, the gene coding for an enzyme having the activity of a guanidinoacetate N-methyltransferase is 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 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/or ion exchange method. Alternatively, creatine can be further purified by a method of recrystallization in water.
Kanamycin solution from Streptomyces kanamyceticus was purchased from Sigma Aldrich (St. Louis, USA, Cat. no. K0254). IPTG (Isopropyl β-D-1-thiogalactopyranoside) was purchased from Carl-Roth (Karlsruhe, Germany, Cat. no. 2316.4.). 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).
If not stated otherwise, cultivation/incubation procedures were performed as follows herewith:
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.
PCR with a proof reading (high fidelity) polymerase was used to amplify a desired segment of DNA for Sanger 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.
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.
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 μl 10 mM Tris*HCl (pH 8.5).
DNA concentration was measured using the NanoDrop Spectrophotometer ND-1000 from PEQLAB Biotechnologie GmbH, since 2015 VWR brand (Erlangen, Germany).
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 μl 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.
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), on Applied Biosystems® (Carlsbad, CA, USA) 3730xl DNA Analyzers. Clonemanager Professional 9 software from Scientific & Educational Software (Denver, USA) was used to visualise and evaluate the 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. Cultures of 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.
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 μl of a glycerol stock culture and the culture incubated for 24 h at 30° C. and 200 rpm. The composition of the seed medium (SM) is shown in table 5.
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 μl 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 μl of the resuspended cells from the precultures. The composition of the production medium (PM) is shown in table 6.
The main cultures were incubated for 72 h at 30° C. and 300 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 analysed 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 OD600. Another part of the culture was centrifuged and the concentration of GAA in the supernatant was analyzed as described below.
As yeast extract FM902 (Angel Yeast Co., LTD, Hubei, P.R.China) contains various peptides and amino acids, its content of L-arginine and glycine was measured as follows.
For measuring free amino acids, the samples were prepared by dissolving 1 g of yeast extract in 20 ml of water. The solution was filled up with water to a total volume of 25 ml, mixed thoroughly and filtered using a 0.2 μM nylon syringe filter.
For measuring total amino acids (free amino acids plus amino acids bound in peptides), the samples were prepared by dissolving 1 g yeast extract in 10 ml 6M HCl and incubating them for 24 h at 110° C. Then, water was added up to a total volume of 25 ml. The solution was mixed thoroughly and filtered using a 0.2 μM nylon syringe filter.
The concentrations of L-arginine and glycine in the samples were determined by ion exchange chromatography using a SYKAM S433 amino acid analyzer from SYKAM Vertriebs GmbH (Fürstenfeldbruck, Germany). As solid phase a column with spherical, polystyrene-based cation exchanger (Peek LCA N04/Na, dimension 150×4.6 mm) from SYKAM was used. Depending on the L-amino acid the separation takes place in an isocratic run using a mixture of buffers A and B for elution or by gradient elution using said buffers. As buffer A an aqueous solution containing in 20 | 263 g trisodium citrate, 120 g citric acid, 1100 ml methanol, 100 ml 37% HCl and 2 ml octanoic acid (final pH 3.5) was used. As buffer B an aqueous solution containing in 20 | 392 g trisodium citrate, 100 g boric acid and 2 ml octanoic acid (final pH 10.2) was used. The free amino acids were coloured with ninhydrin through post-column derivatization and detected photometrically at 570 nm.
Table 7 shows the content of free and total L-arginine and glycine determined in yeast extract FM902 (Angel Yeast Co., LTD, Hubei, P.R.China), as well as the resulting amounts in the production medium (PM).
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, 4.6×250 mm, 5 μm (Waters Corporation, Milford, USA) at 35° C. Mobile phase A was water with 10 mM ammonium formate and 0.2% formic acid. Mobile phase B was a mixture of 90% acetonitrile and 10% water, 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.
Moorea producens is a filamentous cyanobacterium. The genome of the Moorea producens strain PAL-Aug. 15, 2008-1 was published By Leao et al. (Leao T, Castelão G, Korobeynikov A, Monroe E A, Podell S, Glukhov E, Allen E E, Gerwick W H, Gerwick L, Proc Natl Acad Sci USA. 2017 March 21; 114 (12): 3198-3203. doi: 10.1073/pnas.1618556114; accession number CP017599.1). It contains an open reading frame putatively coding for a L-arginine:glycine amidinotransferase (AGAT. EC 2.1.4.1, accession number BJP34_00300, SEQ ID NO:1). SEQ ID NO:2 and SEQ ID NO: 4 show the derived amino acid sequence (accession number WP_070390602) which was designated as AGAT_Mp.
The gene cyrA from Cylindrospermopsis raciborskil AWT205 (accession number EU140798.1) codes for a L-arginine:glycine amidinotransferase (Mihali T K, Kellmann R. Muenchhoff J, Barrow K D, Neilan B A (2008) “Characterization of the gene cluster responsible for cylindrospermopsin biosynthesis.”, Appl Environ Microbiol., 74 (3): 716-22, doi: 10.1128/AEM.01988-07; SEQ ID NO:15). SEQ ID NO:16 and SEQ ID NO:26 show the derived amino acid sequence (accession number ABX60160) which was designated as AGAT_cyrA.
The cDNA sequence of the human L-arginine:glycine amidinotransferase was described by Humm et al., 1994 (Humm A, Huber R, Mann K (1994) “The amino acid sequences of human and pig I-arginine:glycine amidinotransferase.” FEBS Letters, Vol. 339 (1-2), 101-107. DOI: 10.1016/0014-5793 (94) 80394-3: accession number NM_001482.3, SEQ ID NO:17). The derived amino acid sequence (accession number NP_001473.1, SEQ ID NO:18) starts with a mitochondrial transit peptide (amino acids 1-37) which is absent in the mature enzyme. A truncated enzyme, starting at amino acid 56, was found to be active when expressed in E. coli (Humm A, Fritsche E, Mann K, Göhl M, Huber R (1997) “Recombinant expression and isolation of human L-arginine:glycine amidinotransferase and identification of its active-site cysteine residue.” Biochem. J. 322, 771-776, DOI: 10.1042/bj3220771). An N-terminal fusion of a 7 amino acid tag (SEQ ID NO:19) was shown to improve protein expression in E. coli (Hansted J G, Pietikäinen L, Hög F, Sperling-Petersen H U, Mortensen K K (2011) “Expressivity tag: A novel tool for increased expression in Escherichia coli.” Journal of Biotechnology 155 (2011) 275-283, DOI: 10.1016/j.jbiotec.2011.07.013). Accordingly a fusion protein consisting of the tag and the truncated AGAT was designed and it was designated as AGAT_Hs (SEQ ID NO:20 and SEQ ID NO:28).
The amino acid sequence of the L-arginine:glycine amidinotransferase from Rattus norvegicus (accession number NP_112293.1, SEQ ID NO:21) is highly similar to the human enzyme. As described for the human enzyme, the sequence was used to design a fusion protein consisting of the N-terminal expression tag and the truncated sequence of the enzyme. The resulting fusion protein was designated as AGAT_Rn (SEQ ID NO:22 and SEQ ID NO:30).
The sunda flying lemur Galeopterus variegatus has a predicted L-arginine:glycine amidinotransferase (accession number NP_112293.1, SEQ ID NO:23). As described for the human enzyme, its amino acid sequence was used to design a fusion protein consisting of the N-terminal expression tag and the truncated sequence of the enzyme. The resulting fusion protein was designated as AGAT_Gv (SEQ ID NO:24 and SEQ ID NO:32).
Using the software tool “GeneOptimizer” (Geneart/ThermoFisher Scientific, Waltham, USA) the amino acid sequences of AGAT_Mp, AGAT_cyrA, AGAT_Hs, AGAT_Rn and AGAT_Gv were translated back into DNA sequences and optimized for the codon usage of C. glutamicum. Their ends were expanded with sequences for assembly cloning and Shine-Dalgarno-Sequences were added upstream of the open reading frames. The resulting DNA sequences are SEQ ID NO:3 (coding for AGAT_Mp), SEQ ID NO:25 (coding for AGAT_cyrA), SEQ ID NO:27 (coding for AGAT_Hs), SEQ ID NO:29 (coding for AGAT_Rn) and SEQ ID NO:31 (coding for AGAT_Gv). They were ordered for gene synthesis from Invitrogen/Geneart (Thermo Fisher Scientific, Waltham, USA). The synthetic genes were delivered as parts of cloning plasmids which were designated as pMA-T_AGAT_Mp, pMA-T_AGAT_cyrA, pMA-T_AGAT_Hs, pMA-T_AGAT_Rn and pMA-T_AGAT_Gv.
The E. coli-C. glutamicum shuttle plasmid pEC-XK99E was digested using the restriction endonuclease SmaI. Terminal phosphates were removed using the “FastAP Thermosensitive Alkaline Phosphatase” (Thermo Fisher Scientific, Waltham, USA). The DNA was then purified with the “QIAquick PCR Purification Kit” (Qiagen GmbH, Hilden, Germany).
The cloning plasmid pMA-T_AGAT_Mp was digested with MluI+AatII and the resulting fragments were blunted using the “Fast DNA End Repair Kit” (Thermo Fisher Scientific, Waltham, USA). They were separated by agarose gel electrophoresis (0.8% agarose in TAE buffer) and the band corresponding to “AGAT_Mp” (1174 bp) was cut out. Its DNA was purified using the “QIAquick Gel Extraction Kit” (Qiagen GmbH, Hilden, Germany).
The AGAT_Mp fragment and the linearized pEC-XK99E were ligated using the “Ready-To-Go T4 DNA ligase” (GE Healthcare Europe GmbH, Freiburg, Germany). The ligation product was transformed into “NEB 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 clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmid was named pEC-XK99E_AGAT_Mp.
The E. coli-C. glutamicum shuttle plasmid pEKEx2 (Eikmanns, 1991) was digested using the restriction endonuclease PstI. The resulting fragments were blunted using the “Fast DNA End Repair Kit” (Thermo Fisher Scientific) and terminal phosphates were removed with “FastAP Thermosensitive Alkaline Phosphatase” (Thermo Fisher Scientific, Waltham, USA). The DNA was then purified with the “QIAquick PCR Purification Kit” (Qiagen GmbH, Hilden, Germany).
The cloning plasmids pMA-T_AGAT_Mp and pMA-T_AGAT_cyrA were digested with MluI+AatII and the resulting fragments were blunted using the “Fast DNA End Repair Kit” (Thermo Fisher Scientific, Waltham, USA). They were separated by agarose gel electrophoresis (0.8% agarose in TAE buffer) and the bands corresponding to AGAT_Mp (1174 bp) and AGAT_cyrA (1204 bp) were cut out. The DNA was purified using the “QIAquick Gel Extraction Kit” (Qiagen GmbH, Hilden, Germany).
Each AGAT fragment was ligated with the linearized pEKEx2 using the “Ready-To-Go T4 DNA ligase” (GE Healthcare Europe GmbH, Freiburg, Germany). The ligation products were transformed into “NEB 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 clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmids were named pEKEx2_AGAT_Mp and pEKEx2_AGAT_cyrA.
The cloning plasmids pMA-T_AGAT_Hs, pMA-T_AGAT_Rn and pMA-T_AGAT_Gv were digested with Eco31I and the products were purified using the “QIAquick Gel Extraction Kit” (Qiagen GmbH, Hilden, Germany).
The E. coli-C. glutamicum shuttle plasmid pEKEx2 (Eikmanns B J, Kleinertz E, Liebl W, Sahm H (1991) “A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing.”, Gene. 1991 Jun. 15: 102 (1): 93-8) was digested using the restriction endonucleases SbfI and BamHI. The DNA was purified with the “QIAquick PCR Purification Kit” (Qiagen GmbH, Hilden, Germany).
Each of the AGAT fragments was assembled with the digested pEKEx2 using the “NEBuilder HiFi DNA Assembly Cloning Kit” (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). The assembly products were transformed into “NEB 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 clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmids were designated as pEKEx2_AGAT_Hs, PEKEx2_AGAT_Rn and pEKEx2_AGAT_Gv respectively.
The argF gene was PCR amplified with the Phusion High-Fidelity DNA Polymerase Kit (New England BioLabs Inc., Ipswich, USA) using genomic DNA of C. glutamicum ATCC13032 and the oligonucleotide primer argF_1.p (SEQ ID NO:5) and argF_2.p (SEQ ID NO:6). The resulting PCR product was cloned into the plasmid pCR-Blunt II-TOPO (Thermo Fisher Scientific/Invitrogen, Waltham, USA) and a proper plasmid clone was identified by restriction enzyme digestion and DNA sequencing. This plasmid was named pCRII-argF.
The genes argG and argH were PCR amplified with the Phusion High-Fidelity DNA Polymerase Kit (New England BioLabs Inc., Ipswich, USA) using genomic DNA of C. glutamicum ATCC13032 and the oligonucleotide primer argG_1.p (SEQ ID NO:7) and argH_2.p (SEQ ID NO:8). The resulting PCR product was cloned into the plasmid pCR-Blunt II-TOPO (Thermo Fisher Scientific/Invitrogen, Waltham, USA) and a proper plasmid clone was identified by restriction enzyme digestion and DNA sequencing. The plasmid was named pCRII-argGH.
pCRII-argGH was cut using HpaI+AvrII and a restriction fragment of 2773 bps was isolated from an agarose gel. pCRII-argF was cut using SspI+AvrII and a restriction fragment of 4526 bps was isolated from an agarose gel. Both fragments were ligated and then transformed into E. coli. Proper plasmid clones were identified by restriction enzyme digestion and DNA sequencing. The resulting plasmid was named pCRII-argFGH.
pCRII-argFGH was cut using HpaI+AvrII and a restriction fragment of 2773 bps was isolated from an agarose gel. The plasmid pEC-XK99E was cut using Ecl136II+XbaI. A restriction fragment of 6999 bps was isolated from an agarose gel. Both fragments were ligated and then transformed into E. coli. Proper plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid pEC-XK99E_argFGH contains the genes argF, argG and argH from C. glutamicum.
pCRII-argFGH was cut using XbaI+SpeI and a restriction fragment of 3868 bps was isolated from an agarose gel. The plasmid pEC-XK99E_AGAT_Mp was cut using XbaI. A restriction fragment of 8188 bps was isolated from an agarose gel. Both fragments were ligated and then transformed into E. coli. Proper plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid pEC-XK99E_AGAT_Mp_argFGH contains the genes argF, argG and argH from C. glutamicum in combination with AGAT_Mp.
pCRII-argF was cut using KpnI+XbaI+AseI and the DNA was purified with the “QIAquick PCR Purification Kit” (Qiagen GmbH, Hilden, Germany). The plasmid pEC-XK99E_AGAT_Mp was cut using KpnI+XbaI, the DNA was purified with the “QIAquick PCR Purification Kit” (Qiagen GmbH. Hilden, Germany). Both eluates were mixed, the DNA fragments were ligated and the product was used to transform E. coli. Proper plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid pEC-XK99E_AGAT_Mp_argF contains the gene argF from C. glutamicum in combination with AGAT_Mp.
pCRII-argGH was cut using XbaI+SaII and a restriction fragment of 1798 bps was isolated from an agarose gel. The plasmid pEC-XK99E_AGAT_Mp was cut using XbaI+SaII, the DNA was purified with the “QIAquick PCR Purification Kit” (Qiagen GmbH, Hilden, Germany). The DNA fragments were ligated and the product was used to transform E. coli. Proper plasmid clones were identified by restriction digestion and DNA sequencing. The resulting plasmid pEC-XK99E_AGAT_Mp_argG contains the gene argG from C. glutamicum in combination with AGAT_Mp.
The enzymatic activity of the carbamoyl phosphate synthetase was increased by genomic insertion of the strong sod-promoter upstream of the carAB operon in ATCC13032. Therefore, the plasmid pK18mobsacB_Psod-carAB was constructed as follows. Plasmid pK18mobsacB was cut using EcoRI+HindIII and the linearized vector DNA (5670 bps) was cut out of an agarose gel. The DNA was extracted using the “QIAquick Gel Extraction Kit” (QIAGEN GmbH, Hilden, Germany).
For constructing the insert, three DNA fragments were created by PCR with the following pairs of primers (genomic DNA of ATCC13032 as template):
The product DNAs 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). Proper plasmid clones were identified by restriction digestion and DNA sequencing.
pK18mobsacB_Psod-carAB was used to integrate the strong sod-promoter upstream of the carAB genes into the genome of C. glutamicum ATCC13032. The plasmid was transformed into ATCC13032 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 μl) on BHI agar supplemented with 10% saccharose. These agar plates were incubated for 48 h at 33° C. The 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 for the appropriate integration of the sod promoter. The resulting strain was named ATCC13032_Psod-carAB.
Escherichia coli
E. coli reference strain (Blattner et al., 1997*))
Corynebacterium glutamicum
Corynebacterium glutamicum wild type strain
C. glutamicum variant producing L-arginine
producens); proprietary plasmid of Invitrogen/Geneart (Thermo
sapiens); proprietary plasmid of Invitrogen/Geneart (Thermo
norvegicus); proprietary plasmid of Invitrogen/Geneart (Thermo
variegatus); proprietary plasmid of Invitrogen/Geneart (Thermo
glutamicum)
glutamicum)
glutamicum)
glutamicum)
glutamicum)
The following strains of C. glutamicum were transformed with various plasmids:
The strains were transformed by electroporating with various plasmids (as shown in table 10). Plasmid containing cells were selected with 25 mg/l kanamycin.
Strains ATCC13032/pEC-XK99E_AGAT_Mp (carrying a gene for the AGAT enzyme from Moorea producens) and ATCC13032/pEC-XK99E (empty vector for control) were analyzed for their ability to produce GAA in batch cultivations using the system of Wouter Duetz (as described above). The production medium (PM) contained 40 g/l D-glucose as the main carbon source. Some batches were supplemented with L-arginine and/or glycine as indicated.
As shown in table 11, the control strain ATCC13032/pEC-XK99E could not produce GAA, even if the precursors L-arginine and glycine were provided. We conclude that it does not possess an intrinsic AGAT activity. Strain ATCC13032/pEC-XK99E_AGAT_Mp contains a polynucleotide coding for a putative AGAT from Moorea producens. It produced 25 mg/l GAA in unsupplemented PM. Supplementation of glycine resulted in a small increase to 31 mg/l GAA. Supplementation of glycine and L-arginine largely increased GAA production to 124 mg/l.
In an industrial GAA production process, supplementation of L-arginine would be rather costly when compared to primary substrates like ammonia, urea and glucose. It would therefore be desirable to generate GAA directly from such primary substrates.
For this purpose, the L-arginine producer C. glutamicum ATCC21831 was transformed with
ATCC21831 was isolated as a canavanine resistant mutant and it was found to produce L-arginine. Its genome was sequenced by Park et al. (Nat Commun. 2014 Aug. 5; 5:4618. doi: 10.1038/ncomms5618; accession number CP007722) and the strain is publicly available from LGC Standards (LGC Standards GmbH, Wesel, Germany).
All transformed ATCC21831strains were analyzed for their ability to produce GAA in batch cultivations using the system of Wouter Duetz (as described above). The production medium (PM) contained 40 g/l D-glucose as the main carbon source. Some batches were supplemented with glycine and/or L-arginine.
As shown in table 12, ATCC21831/pEKEx2 did not produce GAA, even if the precursors L-arginine and glycine were present. We conclude that ATCC21831/pEKEx2 does not have an intrinsic AGAT activity. The transformed strains ATCC21831/pEKEx2_AGAT_Mp, ATCC21831/PEKEx2_AGAT_Hs, ATCC21831/PEKEx2_AGAT_Rn, ATCC21831/pEKEx2_AGAT_Gv, and ATCC21831/pEKEx2_AGAT_cyrA produced from about 1 up to 26 mg/l GAA in unsupplemented PM. We conclude that the precursors L-arginine and glycine were synthesized from the primary substrates D-glucose, ammonium and urea.
When glycine was added, GAA production of ATCC21831/pEKEx2_AGAT_Mp, ATCC21831/pEKEx2_AGAT_Hs, ATCC21831/pEKEx2_AGAT_Rn, ATCC21831/pEKEx2_AGAT_Gv, and ATCC21831/pEKEx2_AGAT_cyrA significantly increased compared to the non-supplemented experiments. When compared to strain ATCC13032/pEC-XK99E_AGAT_Mp (see table 10), the L-arginine producer ATCC21831 having an AGAT gene accumulates much more GAA when glycine is not limiting. We conclude that the capability of internally providing L-arginine improves GAA production.
In L-arginine production strains (e.g. ATCC21831) the intermediate L-ornithine is synthesized de novo and further converted into L-arginine. When such a strain is provided with an AGAT, the enzyme will produce an equal molar amount of GAA and L-ornithine. The formation of L-ornithine though consumes a significant amount of the primary C- and N-sources and it thus lowers the yield of GAA.
We found that enhancement of the biosynthetic pathway leading from L-ornithine to L-arginine improves GAA production, presumably by improving the recycling of L-ornithine to L-arginine.
Various strains, derived from ATCC13032, were cultivated using the Wouter Duetz system and then analyzed for their ability to produce GAA (tables 13, 14 and 15).
As shown in tables 13 to 15, strains lacking the AGAT gene did not produce a detectable amount of GAA.
The expression of AGAT_Mp in ATCC13032/pEC-XK89E_AGAT_Mp resulted in 124 mg/l GAA. Additional amplification of argG (strain ATCC13032/pEC-XK99E_AGAT_Mp_argG), argF (strain ATCC13032/pEC-XK98E_AGAT_Mp_argF) or argG+argH (strain ATCC13032/pEC-XK99E_AGAT_Mp_argGH) improved the production of GAA (cf. table 14).
In strain ATCC13032/pEC-XK99E_AGAT_Mp_argFGH the expression of the genes argF (coding for ornithine carbamoyltransferase). argG (coding for argininosuccinate synthetase) and argH (coding for argininosuccinate lyase) is enhanced. This further improved the production of GAA to 154 mg/l (cf. table 14).
The conversion of L-omithine to L-citrulline, catalyzed by the ornithine carbamoyltransferase, depends on the availability of the co-substrate carbamoyl phosphate. Carbamoyl phosphate is produced by the carbamoyl phosphate synthase, which is encoded by the genes carA and carB. In strain ATCC13032_Psod-carAB/pEC-XK99E_AGAT_Mp a genomic insertion of the strong sod-promoter enhances the expression of carA and carB. When compared to ATCC13032/pEC-XK99E_AGAT_Mp, this resulted in improved GAA production (156 mg/l vs. 124 mg/l).
In strain ATCC13032_Psod-carAB/pEC-XK99E_AGAT_Mp_argFGH the improved L-ornithine conversion (overexpression of argF, argG and argH) was combined with the improved carbamoyl phosphate biosynthesis (overexpression of carA and carB). This combination further improved GAA production to 171 mg/l.
For the heterologous expression of the AGAT from Moorea producens (EC 2.1.4.1, SEQ ID NO:2 and SEQ ID NO:4) in P. putida KT2440 the plasmid pACYCATh-5{PRha}[agat_Mp(coPp)] was constructed. A codon optimized AGAT_Mp gene was to be cloned under the control of the rhamnose inducible promoter Pra into the vector pACYATh-5. Downstream of the AGAT_Mp gene a terminator sequence is located. The AGAT_Mp gene was ordered for gene synthesis from Eurofins Genomics Germany GmbH (Ebersberg. Germany) and the DNA sequence of the gene fragment was codon-optimized for expression in P. putida KT2440 (SEQ ID NO:33). Upstream of the open reading frame a Shine-Dalgarno sequence was added. The PRha promoter cassette (SEQ ID NO: 34) and the terminator sequence (SEQ ID NO:35) were amplified from E. coli K12 genomic DNA. The vector is based on pACYC184 (New England BioLabs Inc., Ipswich, USA) and carries a p15A origin of replication for E. coli and a pVS1 origin of replication for the replication in P. putida KT2440. The pVS1 origin comes from the Pseudomonas plasmid pVS1 (Itoh Y, Watson J M, Haas D, Leisinger T, Plasmid 1984, 11 (3), 206-20). In the next step the AGAT_Mp gene fragment was amplified via PCR using the primer MW_20_01_fw (SEQ ID NO:36) and MW_20_02_Iv (SEQ ID NO: 37) and cloned into the vector pACYCATh-5 using the restriction sites ApaI/XhoI and NEBuilder® HiFi DNA Assembly Cloning Kit from (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). The assembled product was transformed into 10-beta electrocompetent E. coli cells (New England BioLabs Inc., Ipswich, USA, Cat. No. C3020K). Procedure of PCR purification, cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target gene was checked by restriction analysis and the authenticity of the introduced DNA fragments was verified by DNA sequencing. The resulting expression vector was named pACYCATh-5{PRha}[agat_Mp(coPp)] (SEQ ID NO:38, see table 17).
The P. putida strain KT2440 was transformed with the plasmid pACYCATh-5{PRha}[agat_Mp(coPp)] by means of electroporation and plated onto LB-agar plates supplemented with tetracycline (10 mg/l). Transformants were checked for the presence of the correct plasmid by plasmid preparation and analytic restriction analysis. The resulting strain was named P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp)] (see table 18).
For the heterologous expression of AGAT_Mp from Moorea producens and argF (SEQ ID NO:39), argG (SEQ ID NO:41), argH (SEQ ID NO:43) from P. putida KT2440 the plasmid pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp] was constructed. The synthetic operon consisting of AGAT_Mp encoding an L-arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1, SEQ ID NO:2 and SEQ ID NO:4), argF encoding an omithine carbamoyltransferase (ArgF, EC 2.1.3.3, SEQ ID NO: 40), argG encoding an argininosuccinate synthase (ArgG, E.C. 6.3.4.5, SEQ ID NO:42) and argH encoding an argininosuccinate lyase (ArgH, E.C. 4.3.2.1, SEQ ID NO:44), respectively, was cloned under the control of the rhamnose inducible promoter Prha into the vector pACYCATh-5. Downstream of the synthetic operon a terminator sequence is located. The AGAT_Mp gene was ordered for gene synthesis from Eurofins Genomics Germany GmbH (Ebersberg, Germany) and the DNA sequence of the gene fragment was codon-optimized for expression in P. putida KT2440. The genes argFGH were also synthesized as gene fragment argFGH (SEQ ID NO: 45). The PRha promoter cassette (SEQ ID NO:34) and the terminator sequence (SEQ ID NO:35) were amplified from E. coli K12 genomic DNA. The vector is based on pACYC184 (New England BioLabs Inc., Ipswich, USA) and carries a p15A origin of replication for E. coli and a pVS1 origin of replication for the replication in P. putida KT2440. The pVS1 origin comes from the Pseudomonas plasmid pVS1 (Itoh Y, Watson J M, Haas D, Leisinger T, Plasmid 1984, 11 (3), 206-20). For cloning AGAT_Mp and argFGH were amplified via PCR. Primers used for cloning are listed in table 16. The PCR products were cloned into the vector pACYCATh-5 using the restriction sites ApaI/XhoI and NEBuilder® HIFI DNA Assembly Cloning Kit from (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520) to generate an optimized operon. For amplification the Phusion™ High-Fidelity Master Mix from New England Biolabs (Ipswich, USA) was used according to manufacturer's manual. The assembled product was transformed into 10-beta electrocompetent E. coli cells (New England BioLabs Inc., Ipswich, USA, Cat. No. C3020K). Procedure of PCR purification, cloning and transformation was carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced DNA fragments was verified by DNA sequencing. The resulting expression vector was named pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp] (SEQ ID NO:49, see table 17).
The P. putida strain KT2440 was transformed with the plasmid pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp] by means of electroporation and plated onto LB-agar plates supplemented with tetracycline (10 mg/l). Transformants were checked for the presence of the correct plasmid by plasmid preparation and analytic restriction analysis. The resulting strain was named P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp] (see table 18).
For the heterologous expression of AGAT_Mp from Moorea producens, argF (SEQ ID NO:39), argG (SEQ ID NO:41), argH (SEQ ID NO:43), carA (SEQ ID NO:50) and carB (SEQ ID NO:52) from P. putida KT2440 the plasmid pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp]{carAB_Pp}[carAB_Pp][ter] was constructed. The carA (SEQ ID NO:50) and carB gene (SEQ ID NO:52) encoding a carbamoyl-phosphate synthase (CarAB, EC 6.3.5.5, SEQ ID NO: 51 and SEQ ID NO:53) were amplified from genomic DNA of P. putida KT2440 including the native promoter of the carAB operon via PCR using the primer MW_20_35_fw (SEQ ID NO:54) and MW_20_36_rv (SEQ ID NO:55). For amplification the Phusion™ High-Fidelity Master Mix from New England Biolabs (Ipswich, USA) was used according to manufacturer's manual. The PCR product (SEQ ID NO:56) was cloned into the pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp] cut with Bsu361 using NEBuilder® HiFi DNA Assembly Cloning Kit from (New England BioLabs Inc., Ipswich, USA, Cat. No. E5520). The assembled product was transformed into 10-beta electrocompetent E. coli cells (New England BioLabs Inc., Ipswich, USA, Cat. No. C3020K). Procedure of PCR purification, cloning and transformation were carried out according to manufacturer's manual. The correct insertion of the target genes was checked by restriction analysis and the authenticity of the introduced DNA fragments was verified by DNA sequencing. The resulting expression vector was named pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp]{carAB_Pp}[carAB_Pp][ter] (SEQ ID NO:57, see table 17).
The P. putida strain KT2440 was transformed with the plasmid pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp]{carAB_Pp}[carAB_Pp][ter] by means of electroporation and plated onto LB-agar plates supplemented with tetracycline (10 mg/l). Transformants were checked for the presence of the correct plasmid by plasmid preparation and analytic restriction analysis. The resulting strain was named P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp]{carAB_Pp}[carAB_Pp][ter] (see table 18).
P. putida
P. putida KT2440/pACYCATh-5
P. putida
P. putida KT2440/pACYCATh-
P. putida
P. putida KT2440/pACYCATh-
P. putida
P. putida KT2440/pACYCATh-
Strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp)] carrying a gene for the AGAT enzyme from M. producens and P. putida KT2440/pACYCATh-5 (empty vector control) were analyzed for their ability to produce GAA in batch cultivation using shake flasks. On a LB agar plate containing 10 mg/l tetracycline an inoculation loop of glycerol cryoculture of the corresponding strains was streaked. The agar plates were incubated for 24 h at 30° C. A 100 ml flask with baffles containing 15 ml of seed medium (autoclaved: 4.4 g/L Na2HPO4×2 H2O, 1.5 g/L KH2PO4, 1 g/L NH4Cl, 10 g/L yeast extract, sterilized separately: 20 g/L glucose, 0.2 g/L MgSO4×7 H2O, 0.006 g/L FeCl3, 0.015 g/L CaCl2), 1 ml/L trace elements solution SL6 (sterile-filtered: 0.3 g/L H3BO3, 0.2 g/L CoCl2×6 H2O, 0.1 g/L ZnSO4×7 H2O, 0.03 g/L MnCl2×4H2O, 0.01 g/L CuCl2×2 H2O, 0.03 g/L Na2MoO4×2 H2O, 0.02 g/L NiCl2×6 H2O), pH 7) with 10 mg/l tetracycline was inoculated with a single colony of an agar plate and incubated in a shaking incubator for 18 h at 30° C. and 200 rpm to produce a preculture. The preculture was used to inoculate 40 ml of M12 medium (composition: (2.2 g/L (NH4)2SO4, 0.02 g/L NaCl, 0.4 g/L MgSO4×7H2O, 0.04 g/L CaCl2×2H2O, sterilized separately: 2 g/L KH2PO4, 8.51 g/L Na2HPO4, 20 g/L glucose, 10 ml/l trace elements solution M12 (sterile-filtered: 0.2 g/L ZnSO4×7 H2O, 0.1 g/L MnCl2×4H2O, 1.5 g/L Na3-citrate×2H2O, 0.1 g/L CuSO4×5 H2O, 0.002 g/L NiCl2×6 H2O, 0.003 g/L Na2MoO4×2 H2O, 0.03 g/L H3BO3, 1 g/L FeSO4×7 H2O), pH 7.4) with 10 mg/l tetracycline, 3.48 g/l arginine and 1.5 g/l glycine to a start OD600 of 0.1. The strains were cultivated for 48 h. At an OD600 of ˜0.5-0.8 gene expression was induced by adding 0.2% (w/V) rhamnose. 9 h and 24 h after induction 1.74 g/l arginine and 0.75 g/l glycine were spiked. At the end of cultivation samples were taken to determine the concentration of GAA produced.
The results are shown in table 19.
P. putida KT2440/pACYCATh-5
P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp)]
As can be seen in table 19 the strain P. putida KT2440/pACYCATh-5 {PRha}[agat_Mp(coPp)] equipped with the AGAT_Mp gene from M. producens was able to produce about 81.5 mg/l GAA. The control strain P. putida KT2440/pACYCATh-5 could not produce any GAA at all.
Strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp)] carrying a gene for the AGAT enzyme from M. producens and strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp] carrying additionally the arginine biosynthesis genes argFGH were analyzed for their ability to produce GAA in batch cultivation using shake flasks. On an LB agar plate containing mg/l tetracycline an inoculation loop of glycerol cryoculture of the corresponding strains was streaked. The agar plate was incubated for 24 h at 30° C. A 100 ml flask with baffles containing 15 ml of seed medium (autoclaved: 4.4 g/L Na2HPO4×2 H2O, 1.5 g/L KH2PO4, 1 g/L NH4Cl, 10 g/L yeast extract, sterilized separately: 20 g/L glucose, 0.2 g/L MgSO4×7 H2O, 0.006 g/L FeCl3, 0.015 g/L CaCl2), 1 ml/L trace elements solution SL6 (sterile-filtered: 0.3 g/L H3BO3, 0.2 g/L CoCl2×6 H2O, 0.1 g/L ZnSO4×7 H2O, 0.03 g/L MnCl2×4H2O, 0.01 g/L CuCl2×2 H2O, 0.03 g/L Na2MoO4×2 H2O, 0.02 g/L NiCl2×6 H2O). pH 7) with 10 mg/l tetracycline was inoculated with a single colony of an agar plate and incubated in a shaking incubator for 18 h at 30° C. and 200 rpm to produce a preculture. The preculture was used to inoculate 40 ml of M12 medium (composition: (2.2 g/L (NH4)2SO4, 0.02 g/L NaCl. 0.4 g/L MgSO4×7H2O, 0.04 g/L CaCl2×2H2O, sterilized separately: 2 g/L KH2PO4, 8.51 g/L Na2HPO4, 20 g/L glucose, 10 ml/l trace elements solution M12 (sterile-filtered: 0.2 g/L ZnSO4×7 H2O, 0.1 g/L MnCl2×4H2O, 1.5 g/L Na3-citrate×2H2O, 0.1 g/L CuSO4×5 H2O, 0.002 g/L NiCl2×6 H2O, 0.003 g/L Na2MoO4×2 H2O, 0.03 g/L H3BO3, 1 g/L FeSO4×7 H2O), pH 7.4) with 10 mg/l tetracycline, 3.48 g/l arginine and 1.5 g/l glycine to a start OD600 of 0.1. The strains were cultivated for 48 h. At an OD600 of ˜0.5-0.8 gene expression was induced by adding 0.2% (w/v) rhamnose. 9 h and 24 h after induction 1.74 g/l arginine and 0.75 g/l glycine were spiked. At the end of cultivation samples were taken to determine the concentration of GAA produced.
The results are shown in table 20.
P. putida KT2440/pACYCATh-5
P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp)]
P. putida KT2440/pACYCATh-5(PRha}[agat_Mp(coPp) argFGH_Pp]
As can be seen in table 20 the strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp)] equipped with the AGAT_Mp gene from M. producens was able to produce about 81.5 mg/l GAA. Additionally implementation of argF, argG and argH in strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp] improved GAA production to 169.5 mg/l.
Strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp)] carrying a gene for the AGAT enzyme from M. producens, strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp argFGH_Pp] carrying additionally the arginine biosynthesis genes argFGH and strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp]{carAB_Pp}[carAB_Pp][ter] carrying additionally the carbamoyl-phosphate synthase genes carAB were analyzed for their ability to produce GAA in batch cultivation using shake flasks. The conversion of L-ornithine to L-citrulline, catalyzed by the ornithine carbamoyltransferase, depends on the availability of the co-substrate carbamoyl phosphate. Carbamoyl phosphate is produced by the carbamoyl phosphate synthase, which is encoded by the genes carA and carB. On an LB agar plate containing 10 mg/l tetracycline an inoculation loop of glycerol cryoculture of the corresponding strains was streaked. The agar plate was incubated for 24 h at 30° C. A 100 ml flask with baffles containing 15 ml of seed medium (autoclaved: 4.4 g/L Na2HPO4×2 H2O, 1.5 g/L KH2PO4, 1 g/L NH4Cl, 10 g/L yeast extract, sterilized separately: 20 g/L glucose, 0.2 g/L MgSO4×7 H2O, 0.006 g/L FeCl3, 0.015 g/L CaCl2), 1 ml/L trace elements solution SL6 (sterile-filtered: 0.3 g/L H3BO3, 0.2 g/L CoCl2×6 H2O, 0.1 g/L ZnSO4×7 H2O, 0.03 g/L MnCl2×4H2O, 0.01 g/L CuCl2×2 H2O, 0.03 g/L Na2MoO4×2 H2O. 0.02 g/L NiCl2×6 H2O), pH 7) with 10 mg/l tetracycline was inoculated with a single colony of an agar plate and incubated in a shaking incubator for 18 h at 30° C. and 200 rpm to produce a preculture. The preculture was used to inoculate 40 ml of M12 medium (composition: (2.2 g/L (NH4)2SO4, 0.02 g/L NaCl, 0.4 g/L MgSO4×7H2O, 0.04 g/L CaCl2×2H2O, sterilized separately: 2 g/L KH2PO4, 8.51 g/L Na2HPO4, 20 g/L glucose, 10 ml/l trace elements solution M12 (sterile-filtered: 0.2 g/L ZnSO4×7 H2O, 0.1 g/L MnCl2×4H2O, 1.5 g/L Na3-citrate×2H2O, 0.1 g/L CuSO4×5 H2O, 0.002 g/L NiCl2×6 H2O, 0.003 g/L Na2MoO4×2 H2O, 0.03 g/L H3BO3. 1 g/L FeSO4×7 H2O), pH 7.4) with 10 mg/l tetracycline, 3.48 g/l arginine and 1.5 g/l glycine to a start OD600 of 0.1. The strains were cultivated for 48 h. At an OD600 of ˜0.5-0.8 gene expression was induced by adding 0.2% (w/v) rhamnose. 4 h/18 h/23 h after induction 6.97 g/l Arg/1.5 g/l Gly, 2.34 g/L Arg/0.75 g/L Gly and 6.97 g/l Arg/1.5 g/l Gly were spiked. At the end of cultivation samples were taken to determine the concentration of GAA produced.
The results are shown in table 21.
P. putida KT2440/pACYCATh-5
P. putida KT2440/pACYCATh-5(PRha}[agat_Mp argFGH_Pp]
P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp)
As can be seen in table 21 the strain equipped with the AGAT_Mp gene from M. producens and the argFGH genes from P. putida was able to produce 589 mg/l GAA. Additionally implementation of carAB in strain P. putida KT2440/pACYCATh-5{PRha}[agat_Mp(coPp) argFGH_Pp]{carAB_Pp}[carAB_Pp][ter] improved GAA production to 693 mg/l.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19218204.6 | Dec 2019 | EP | regional |
This application is the National Stage entry under § 371 of International Application No. PCT/EP2020/085882, filed on Dec. 14, 2020, and which claims the benefit of priority to European Application No. 19218204.6, filed on Dec. 19, 2019. The content of each of these applications is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/085882 | 12/14/2020 | WO |
