Efficient synthesis of guanidino acetic acid by a multi-enzyme cascade system for the synthesis of carbamoyl phosphate enhanced

Abstract

The application discloses a high-efficiency synthesis of guanidino acetic acid by using a multi-enzyme cascade system with enhanced carbamoyl phosphate synthesis, and belongs to the field of biological catalysis engineering. The application provides an advantageous mutant E31K / G351N of L-arginine:glycine amidinotransferase, and the catalytic efficiency of guanidino acetic acid is increased by 34.6% compared with that of the wild type. An eight-enzyme synergistic arginine recycling system is constructed based on the mutant strain, and 18.35 g / L GAA (56.75 mM) is achieved in a 5L fermenter, and the arginine conversion rate reaches 261.12%, which is the highest level of guanidino acetic acid catalyzed by Escherichia coli reported at home and abroad. The research not only provides an efficient technical route for the industrial production of GAA, but also establishes a universal strategy framework for the rational design of multi-enzyme systems and the optimization of multi-gene co-expression.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology and mainly relates to the efficient synthesis of guanidinoacetic acid using a multi-enzyme cascade system enhanced by carbamoyl phosphate synthesis. Background Technology

[0002] Guanidinoacetic acid (GAA, CAS No.: 352-97-6) is a natural amino acid derivative and the only precursor for creatine synthesis in vertebrates. It is synthesized in vivo through a methylation reaction mediated by guanidinoacetic acid methyltransferase (GAMT). GAA has attracted considerable attention in agriculture as a feed additive, effectively improving meat yield and feed conversion efficiency. Beyond agricultural applications, GAA's potential in human nutrition and health is increasingly expanding. It not only serves as a creatine precursor but also regulates insulin sensitivity, alleviates dietary arginine stress, modulates protein synthesis, influences hormone secretion, and promotes vasodilation. Furthermore, it may affect neuroregulation by intervening in the γ-aminobutyric acid (GABA) system. These multidimensional physiological effects suggest that GAA may provide new strategies for intervention in brain health and metabolic syndrome, and its application value extends beyond the traditional scope of creatine supplementation.

[0003] Given the important role and potential market value of guanidinoacetic acid, its production methods have been developed and reported. Early methods involved the condensation of glycine and cyanamide. Industrial production has subsequently developed routes such as the thiourea-glycine method, the glycine-cyanamide method, and the guanidinium hydrochloride-chloroacetic acid method. Although process optimization has improved yields and reduced byproducts, traditional chemical synthesis still faces challenges such as harsh reaction conditions, the use of toxic reagents, and environmental burden.

[0004] In comparison, the biotechnology route has a greater advantage in terms of greenness. Microbial synthesis of GAA depends on L-arginine: glycine amidotransferase (AGAT) catalyzes the transamidination reaction of L-arginine and glycine, producing GAA as the product and L-ornithine as the byproduct. Zhang Yiwen et al. reconstructed the ornithine cycle in *E. coli* and coordinated the expression of seven enzymes, achieving a GAA yield of 8.6 g / L, with arginine and glycine conversion rates of 188.33% and 82.85%, respectively. Yan Kun et al. modified *Bacillus subtilis* strains to catalyze the synthesis of 4.26 g / L GAA in 20 hours. Currently, microbial GAA synthesis systems are limited by industrialization bottlenecks such as the high cost of the substrate L-arginine and insufficient product potency. Therefore, there is an urgent need to develop methods for directed evolution of AGAT enzymes to improve their industrial properties. Furthermore, since L-arginine accounts for more than 60% of the production cost, an efficient arginine regeneration system is particularly important. Summary of the Invention

[0005] This invention, based on the exogenous introduction of AGAT enzymes to synthesize GAA, introduces four enzymes from the ornithine to arginine synthesis pathway: carbamoyl phosphate synthase II (CPSAB, EC 6.3.5.5); ornithine carbamoyltransferase (ArgI); arginine succinate synthase (ArgG); and arginine succinate lyase (ArgH), enabling the byproduct ornithine to cycle to the product arginine. To reduce the demand for exogenous substrates, glutamine synthase (GlnA) and aspartate aminolyase (AspA) are further introduced to achieve the endogenous synthesis of glutamine and aspartate. Quantitative analysis of the crude enzyme specific activities of key metabolic nodes revealed that the catalytic activities of CPSAB enzymes responsible for carbamoyl phosphate synthesis and GlnA enzymes responsible for glutamine synthesis were significantly lower than those of downstream enzymes. This invention focuses on optimizing the core rate-limiting step of the ornithine-arginine regeneration cycle—the carbamoyl phosphate synthesis module. Given that glutamine availability directly affects the efficiency of carbamoyl phosphate synthesis, glutamine regeneration was first enhanced: screening... Schistosoma japonicum , Corynebacterium glutamicum and Methylococcus capsulAtus Three exogenous glutamine synthases (GS) and E. coli endogenous GlnA were selected, and the glutamine synthase with the highest catalytic activity was chosen. M. capsulAtus ( Mc GS). To address the dependence of the traditional CPSAB pathway on L-glutamine and ATP, a complementary pathway was introduced using carbamate kinase (CK). This enzyme uses inorganic ammonia as a nitrogen donor to directly synthesize carbamate phosphate from ammonia and carbamate phosphate precursors. By comparing endogenous YahI in *E. coli* with... Giardia muris , Enterococcus Exogenous CK was found Giardia muris ( Gm CK) has the best activity.

[0006] To further increase the upper limit of GAA synthesis, six AGAT enzymes from different sources were screened, and selected... Actinokineospora terrae Source At AGAT ， Enzyme modification was performed, and amino acid residues within 5 Å of the substrate in the active site were obtained based on crystal structure analysis. Alanine scanning, virtual saturation mutagenesis, and high-throughput screening were conducted to obtain three single-point mutants with enhanced activity, as well as a combined mutant with further enhanced activity obtained by combining the single-point mutants with combined mutagenesis.

[0007] In this invention, the glycine amidotransferase is L-arginine:glycine amidotransferase, which is two naming conventions for the same enzyme.

[0008] This invention provides an L-arginine:glycine amidoyltransferase mutant, wherein the L-arginine:glycine amidoyltransferase mutant is obtained by mutating the glutamic acid at position 31 of the L-arginine:glycine amidoyltransferase as shown in SEQ ID NO.1 to lysine or glutamine, and is named as follows: At E31K or At E31Q; Alternatively, it can be obtained by mutating the glycine at position 351 of L-arginine:glycine amidoyltransferase, as shown in SEQ ID NO.1, to asparagine, and named as... At G351N; Alternatively, the amino acid sequence shown in SEQ ID NO.1, where glutamate at position 31 of L-arginine:glycine amidoyltransferase is mutated to glutamine or lysine, and glycine at position 351 is mutated to asparagine, can be named as follows: At E31K / G351N or At E31Q / G351N; The amino acid sequence of L-arginine:glycine amidoyltransferase is shown in SEQ ID NO. 1: MQQVSEVVNSWNEWDPLEEIVVGSADGANFEPTEPGNRPQIRNAPPGTPFPSGPKSAEAVDRANEELAGLVSLLESEGVRVRRPAPHDFSQSVRTPDFEAANQYCAVCPRDVMITIGNEIIEAPMSRRSRYFEFQPYRELVYEYWNADPRVVWTTAPKPSMADKMYRDGFWDWPLAER HERMHSFEFCVTQDEVVFDAADMSRFGRDIVVQESMTTNRAGISWLKRHLEPKGFRVRPVHFPLDFFPSHIDCTFVPLRPGLVLTNPDRPLREGEEKLFLANDWELVDAPEPTTGNDEMPEFCQSSKWLSMNVLSIGPNKVICEQQEKPLQELLYKLDFEVFPVPFRNVFEYGGSLHC AT WDVRRVGAGEDYFPNSDYQPLA The nucleotide sequence encoding the L-arginine:glycine amidinosyltransferase is shown in SEQ ID NO. 2: SEQ ID NO. 3( At E31Q / G351N): The present invention also provides a gene encoding the above-mentioned glycine amidotransferase mutant or a recombinant vector carrying the gene.

[0009] The present invention also provides recombinant cells expressing the above-mentioned mutants or carrying the above-mentioned genes or the recombinant vectors, preferably, the recombinant cells using bacteria or fungi as host cells.

[0010] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned glycine amidotransferase mutant, which is any one of the following forms: (1) Culture recombinant expression transformants containing the glycine amidotransferase mutant and isolate transformant cells containing the recombinant glycine amidotransferase mutant enzyme; (2) Cultivate recombinant expression transformants containing the glycine amidotransferase mutant, isolate transformant cells containing the recombinant glycine amidotransferase mutant enzyme, and break the transformant cells containing the recombinant glycine amidotransferase mutant enzyme to obtain cell lysate; (3) Cultivate recombinant expression transformants containing the glycine amidotransferase mutant, isolate transformant cells containing the recombinant glycine amidotransferase mutant enzyme, break the transformant cells containing the recombinant glycine amidotransferase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant glycine amidotransferase mutant enzyme to obtain lyophilized enzyme powder.

[0011] The present invention also provides a method for improving the synthesis of guanidinoacetic acid catalyzed by glycine amidotransferase, wherein the method comprises: mutating the glutamic acid at position 31 of the glycine amidotransferase as shown in SEQ ID NO.1 to glutamine or lysine; or the mutant is obtained by mutating the glycine at position 351 of the glycine amidotransferase as shown in SEQ ID NO.1 to asparagine; or the mutant is obtained by mutating the glutamic acid at position 31 of the glycine amidotransferase as shown in SEQ ID NO.1 to glutamine or lysine, and simultaneously mutating the glycine at position 351 to asparagine.

[0012] This invention also provides a method for efficiently expressing and catalyzing the eight enzymes mentioned above in *E. coli* using two plasmids. The pRSFDuet-pETDuet dual plasmid system is employed, with each plasmid expressing four genes. The specific ligation method is as follows: Figure 8 .

[0013] In the application described above for the preparation of guanidinoacetic acid, the reaction system is a whole-cell reaction.

[0014] The reaction system is as follows: This invention provides a genetically engineered bacterium that expresses [a specific expression]. Actinokineosporaterrae L-arginine source: glycine amidotransferase AGAT or its mutant; Giardia muris The carbamate kinase CK derived from this source has the protein sequence shown in SEQ ID NO. 4; M. capsulAtus The glutamine synthase GS, derived from the source, has the nucleotide sequence shown in SEQ ID NO. 5; and the endogenous gene ornithine carbamoyltransferase ArgI, with the nucleotide sequence shown in SEQ ID NO. 6; carbamoyl phosphate synthase II CPS AB, with the nucleotide sequence shown in SEQ ID NO. 7; arginine succinate synthase ArgG, with the nucleotide sequence shown in SEQ ID NO. 8; aspartate aminotransferase AspA, with the nucleotide sequence shown in SEQ ID NO. 9; and arginine succinate lyase ArgH, with the nucleotide sequence shown in SEQ ID NO. 10.

[0015] SEQ ID NO. 4 (carbamate kinase) MKTVVIALGGNAMLQPGQKGDFEVQLENVKAAIKEIHAIHRAGYRVVLTSGNGPQVGAIKLQNQSAASVSPEMPLYACGAMSQGLIGYMMVQELRNAFRSQKEATQCIGCLTQTLVDSGDPAFAAPSKPMGRFYTEEEARAMMAKDSTIMMKEDAG RGWRVVVPSPRPVEILEYPMIKKLVDDGVIVICTNGGGIPCKLEGERIVGVDAVIDKDMATSLLAQKLNSDYLLILTDVPYATINYRTSDQQEIKTVTVEEMMKLEQEGHFKDGSMKPKVRAAIEFTMKTGNPSIITSLTSAFDALEGNCGTRIVQ SEQ ID NO. 5 (Glutamine synthase) SEQ ID NO.6 (ornithine carbamoyltransferase) SEQ ID NO. 7 (carbamoyl phosphate synthase II) SEQ ID NO. 8 (arginine succinate synthase) SEQ ID NO. 9 (Aspartate amino acid lyase) SEQ ID NO. 10 (arginine succinate lyase) In one embodiment of the present invention, the genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

[0016] In one embodiment of the present invention, the genetically engineered bacteria is recombinant Escherichia coli.

[0017] In one embodiment of the present invention, the recombinant Escherichia coli is... E. coli BL21 (DE3) expresses the host.

[0018] In one embodiment of the present invention, the recombinant vector is pET28a(+), pETDuet-1, pRSFDuet-1, pACYCDuet-1, and pRCDFDuet-1.

[0019] The present invention also provides one or more recombinant cells expressing the above-mentioned genes or carrying the above-mentioned recombinant vectors.

[0020] In one embodiment of the present invention, the recombinant cells use Escherichia coli as the expression host.

[0021] In one embodiment of the present invention, the recombinant bacteria are constructed by PCR amplification of L-arginine:glycine amidoyltransferase (A... G AT or its mutant (AGATmut), carbamoyl phosphate synthase II (CPSAB); glutamine synthase (GS); carbamate kinase (CK); ornithine carbamoyltransferase (ArgI); arginine succinate synthase (ArgG); aspartate aminotransferase (AspA); arginine succinate lyase (ArgH). These were cloned into plasmids pETDuet-1, pACYCDuet-1, pRCDFDuet-1, and pRSFFDuet-1 to construct the recombinant plasmid pETDuet-1-ArgI-ArgG-AG. AT mut-ArgH or pACYCDuet-1-ArgI-ArgG-AG AT mut-ArgH or pRCDFDuet-1-ArgI-ArgG-AG AT mut-ArgH and pRSFDuet-1-CPSAB- Gm CK- Mc GS-AspA co-converted to E. coli In BL21 (DE3), a recombinant Escherichia coli catalyst was constructed.

[0022] In one embodiment of the present invention, the NCBI accession number of the L-arginine:glycine amidoyltransferase is WP_092775660.1 or the above-mentioned L-arginine:glycine amidoyltransferase mutant; the NCBI accession number of the glutamine synthase is CAI8875598.1, the NCBI accession number of the carbamate kinase is TNJ29176.1, the NCBI accession number of the ornithine carbamate transferase is ACT45908, the NCBI accession number of the arginine succinate synthase is ACT44842, the NCBI accession numbers of the two subunits of carbamate phosphate synthase II are ACT41938 and ACT41939, the NCBI accession number of the aspartate aminotransferase is ACT45798, and the NCBI accession number of the arginine succinate lyase is ACT45638.

[0023] This invention also provides a method for the whole-cell catalytic synthesis of guanidinoacetic acid, wherein arginine and glycine are used as substrates, and the recombinant *E. coli* described above is used for whole-cell catalytic conversion to synthesize guanidinoacetic acid under aeration conditions. Preferably, the catalytic reaction conditions are: whole-cell reaction for 24-60 hours at pH 7.0-8.0 and 25-30°C; preferably, the amount of the genetically engineered bacteria added is 40-70 g / L. Preferably, the reaction system further comprises: 100-200 mM ammonium bicarbonate, 4-10 mM MgCl2, and 8%-12% (w / v) glucose; preferably, a dynamic feeding strategy is adopted: the initial glycine concentration is 40-80 mM, and 120 mL of concentrated glycine solution of 75-100 g / L is added at a rate of 5 mL / h, with the total amount of glycine added being approximately 120-160 mM.

[0024] In one embodiment of the present invention, the whole-cell catalytic system comprises, but is not limited to, 60 mM arginine (equivalent to 10.44 g / L) and 200 mM glycine (equivalent to 15 g / L), with an aeration rate of 3 vvm, 70 g of wet bacteria added, and the volume adjusted to 800 ml with 100 mM phosphate solution (pH 7.5). The feed bottle contains 500 mL of 0.5 g / mL glucose solution, and the pH is controlled at 8.0 with ammonia. The reaction time is 60 h, and the reaction temperature is 30 °C.

[0025] In one embodiment of the present invention, the whole-cell catalyst is obtained by culturing recombinant bacteria and inducing the expression of L-arginine:glycine amidotransferase, carbamoyl phosphate synthase II; glutamine synthase; carbamate kinase; ornithine carbamoyltransferase; arginine succinate synthase; aspartate aminotransferase; and arginine succinate lyase, followed by collection of recombinant bacterial cells.

[0026] In one embodiment of the present invention, the whole-cell catalyst is obtained by inoculating recombinant *E. coli* co-expressing L-arginine:glycine amidotransferase, carbamoyl phosphate synthase II; glutamine synthase; carbamate kinase; ornithine carbamoyltransferase; arginine succinate synthase; aspartate aminotransferase; and arginine succinate lyase into 100 mL LB medium at a 1% inoculation rate and culturing at 37 °C. When OD... 600 When the protein concentration reaches 0.4–0.6, 0.5 mM IPTG is added to induce protein expression. After induction at 20 °C for 16 h, 8,000 × 10⁻⁶ ions are added. g Centrifuge at low temperature for 10 min, collect the bacterial cells, and wash the bacterial cells twice with 0.9% sodium chloride solution to obtain the final product.

[0027] In one embodiment of the present invention, the reaction conditions for whole-cell catalysis are as follows: dissolved oxygen-coupled stirring to control DO at 30%, temperature at 30 ℃, pH 8.0, aeration rate of 2 vvm, glucose solution fed at a rate of 20 mL / h from 0 to 20 h, after 20 h, the reaction mixture (120 mL 100 mM phosphate buffer, pH 8.0), 9 g glycine (120 mM), 6.96 g arginine (40 mM), 15.8 g ammonium bicarbonate (200 mM), and 0.19 g magnesium chloride (2 mM) are added at a rate of 5 mL / h, the glucose solution feeding rate is reduced to 10 mL / h, glucose supply is stopped after 44 h, the yield reaches its maximum at 60 h, and then the guanidinoacetic acid content begins to decrease.

[0028] In one embodiment of the present invention, the amounts of arginine and glycine added are 60 mM (equivalent to 10.44 g / L) and 200 mM (equivalent to 15 g / L), respectively, and the amount of catalyst added is 46 g / L (cell wet weight).

[0029] This invention provides the application of the above-described recombinant Escherichia coli or the above-described method in the preparation of guanidinoacetic acid, and its application in any of the following: (a) preparing feed additives; (b) improving livestock and poultry meat yield and feed conversion rate; (c) synthesizing creatine precursors.

[0030] Beneficial effects (1) This invention successfully obtained a high-activity mutant of L-arginine:glycine amidoyltransferase, which improved the catalytic efficiency of L-arginine:glycine amidoyltransferase. By optimizing the carbamoyl phosphate synthesis pathway, the arginine regeneration system was improved. In E. coli, the L-arginine:glycine amidoyltransferase mutant, carbamoyl phosphate synthase II, glutamine synthase, carbamate kinase, ornithine carbamoyltransferase, arginine succinate synthase, aspartate aminolysase, and arginine succinate lyase were co-expressed in a single cell using the dual plasmids pRSFDuet-1 and pETDuet-1. This successfully achieved efficient expression of eight enzymes in a single cell, eliminating the byproduct ornithine and converting it into the substrate arginine, thus avoiding the inhibition of L-arginine:glycine amidoyltransferase by ornithine and the economic problem of the high cost of arginine. Using small amounts of arginine and glycine as substrates, a one-pot cascade catalysis with multiple enzymes yielded a final guanidinoacetic acid (GAA) yield of 18.35 g / L and an arginine conversion rate of 261.12%. The multi-enzyme coupling system constructed in this invention solves the problem of high arginine costs and the recycling of the byproduct ornithine in the biosynthesis of GAA, thereby improving the atom utilization efficiency of the biocatalytic reaction process.

[0031] (2) The establishment of this whole-cell transformation system solves the problems of high temperature / strong acid environment, dependence on toxic reagents and environmental burden in the traditional chemical production of guanidinoacetic acid. It has good atom economy and environmental friendliness, and realizes efficient synthesis of guanidinoacetic acid with a small amount of arginine and glycine as substrates. According to all the data available, the yield of guanidinoacetic acid in this invention is the highest level in the biological synthesis from Escherichia coli. Attached Figure Description

[0032] Figure 1 Schematic diagram of the principle of efficient synthesis of guanidinoacetic acid (GAA) catalyzed by multiple enzymes.

[0033] Figure 2 Screening of the key enzyme AGAT; where A represents the catalytic reaction of AGAT and the phylogenetic tree of AGAT in different species, and the gray area represents the selected enzyme; B represents the GAA produced by the screened AGAT at 25 °C after eight hours of whole-cell catalysis; C represents the soluble expression of AGAT in BL21 in different species.

[0034] Figure 3 : At Structural analysis of AGAT and mutational analysis of key amino acid residues in its binding pocket; where A is... At Multiple sequence alignment and sequence conservation analysis of AGAT; B is to... AtVisualization of the AGAT model docking with arginine and glycine and its binding pocket; C shows the energy change when adjacent amino acids in the binding pocket mutate to alanine; red pentagons represent potential mutation sites; D shows the energy change analysis of virtual saturation mutagenesis at some sites of key amino acid residues; E shows the heatmap analysis of virtual combinatorial mutagenesis at potential key amino acid residue sites; F shows the results of beneficial single-site and combinatorial mutant enzyme activity and catalytic ability.

[0035] Figure 4 Comparison of crude enzyme catalytic capabilities at each step and optimization of the CP synthesis module.

[0036] Figure 5 Screening of protein expression and construction methods for engineered strains.

[0037] Figure 6 Optimization of reaction conditions for engineered strains.

[0038] Figure 7 AC: Catalytic results of engineered strains in a 5L fermenter.

[0039] Figure 8 : Sample diagram of plasmid construction for engineered strains. Detailed Implementation

[0040] The detection methods involved in the following embodiments are as follows: SDS-PAGE gel electrophoresis: Prepare the BBI Blocking-Free SDS-PAGE Denaturing Acrylamide Colored Gel Rapid Preparation Kit and prepare the protein gel according to the instructions. Sample preparation: Take wet bacterial cells containing the target protein, prepare a 20 g / L cell suspension with Tris-HCl buffer (pH 7.5, 100 mM), and sonicate on ice (450 W, 2 s on, 4 s off, 10 min total). 12,000 × g Centrifuge at 4 ℃ for 1 min to separate the supernatant and precipitate. Take 20 μL of the supernatant, mix with 8 μL of loading buffer, heat in a boiling water bath for 10 min, and centrifuge at 12,000 × 10⁻⁶. g Centrifuge for 5 min and set aside. Electrophoresis: Sample loading: 10 μL sample, 3 μL marker; Electrophoresis conditions: 160 V, 60 min; Electrophoresis buffer (g / L): glycine 14.4, SDS 1, Tris 3. Staining: Immerse the protein gel in staining solution (Coomassie Brilliant Blue R250 1 g, methanol 450 mL, acetic acid 100 mL, water 450 mL) for 15 min, then wash twice with water. Destaining: Immerse in destaining solution (ethanol 10%, acetic acid 10%, water 80%) and gently shake, replacing with fresh destaining solution until bands are clearly visible.

[0041] Guanidinoacetic acid sample preparation and HPLC detection Studies have shown that citric acid can improve the solubility of guanidinoacetic acid (GAA). For the fermentation solution detection of scaled-up synthesis of GAA, the stirring was adjusted to 300 rpm before sampling to ensure uniform mixing. A 50 mL sample was taken, and 1 mL was centrifuged for amino acid detection. The remaining sample was adjusted to pH 2.8 with citric acid, thoroughly mixed, and allowed to stand for one hour. The sample was then centrifuged, and 50 µL of the supernatant was dissolved in 150 µL of purified water. 600 µL of pure acetonitrile was added, and the mixture was thoroughly mixed. The sample was filtered through a 0.22 μm organic filter membrane and analyzed by HPLC. At least three independent experiments were performed for each strain.

[0042] Analytical Method: The concentration of GAA was measured by HPLC (Agilent 1260 series, Hewlett-Packard) using a Lac-Amino column (4.5 mm × 300 mm). The analysis temperature was 35 °C, the mobile phase was acetonitrile:water = 75:25, the flow rate was 0.7 mL / min, and GAA was detected at 210 nm OD. The retention time of GAA was 19.5 min.

[0043] Amino acid detection The concentrations of amino acids, including arginine, glycine, ornithine, glutamic acid, glutamine, citrulline, and aspartic acid, were measured using HPLC (Agilent 1260 series, Hewlett-Packard) on a C18 column (4.5 mm × 300 mm). Sample pretreatment involved adding a derivatization reagent consisting of 50 μL of 2,4-dinitrofluorobenzene / acetonitrile (1:99) solution and 100 μL of 0.5 m NaHCO3 to 100 μL of the supernatant. The mixture was incubated at 60 °C for 60 min. Then, 750 μL of 0.01 m KH2PO4 was added to inhibit the reaction, and the mixture was mixed and allowed to stand for 10 min. The reaction solution was analyzed at 1 mL / min and detected at 360 nm using a linear gradient of two mobile phases (elution A: acetonitrile; eluent B: 50 mM sodium acetate, with a linear gradient of 90%–50% for 25 min).

[0044] Purification and Enzyme Activity Detection of L-Arginine: Glycine Interiminotransferase All recombinant proteins had a 6 × Histine tag at the N-terminus and were purified using a nickel affinity chromatography column (Sangon Biotech, Shanghai, China). The purified proteins were then subjected to kinetic assays. Glycine imidyltransferase activity was determined by HPLC. The enzyme activity assay system (2 mL) consisted of 0.95 mL of a stock solution of 100 mM Tris-HCl (pH 8.0), 40 mM arginine, and 40 mM glycine, and 0.05 mL of purified enzyme (1 mg / mL). The reaction was carried out in 4 mL EP tubes at 25°C and 200 rpm for 1 h, then terminated by boiling in water for 2 minutes. 200 µL of the supernatant was added to 600 µL of acetonitrile and filtered through an organic filter membrane for HPLC analysis. The boiled enzyme was used as a negative control. One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the production of 1 µmol of guanidinoacetic acid (GAA) per minute under standard reaction conditions.

[0045] High-throughput screening of mutants Several potential mutation sites in the substrate binding pocket were screened based on virtual mutation results. Random and combined mutations were performed on these sites using degenerate primers (NNK-MNN), and mutants exhibiting enhanced catalytic activity were selected. All randomly mutated monoclonal strains were cultured and induced to express the mutants in 96-well plates. The collected strains were prepared into 1 mL reaction solution (pH 7.5), with the reaction mixture containing 30 mM arginine and 30 mM glycine. The reaction was carried out at 220 rpm and 25°C for 6 hours, and the reaction was terminated by heating. The GAA content of the product was detected by high-performance liquid chromatography (HPLC). Strains with higher yields compared to the wild type were selected for DNA sequencing to confirm the mutation results. In the following examples, the crude enzyme solution of 50 g / L is defined as: adding 50 g of crude enzyme solution obtained after cell disruption to 1 L of the reaction system.

[0046] The recombinant Escherichia coli culture and expression methods involved in the following examples are as follows: (1) Preparation of seed liquid Pick a single colony from the plate and inoculate it into a test tube containing ampicillin or kanamycin (concentration: 50 mg / L), or LB liquid medium containing both of the above antibiotics, and incubate at 37 ℃ and 200 rpm for 8-10 h to obtain the seed culture.

[0047] (2) Fermentation culture of recombinant Escherichia coli Inoculate the seed culture at a 1% (v / v) inoculation rate into shake flasks containing ampicillin or kanamycin (concentration: 50 mg / L), or both of the above antibiotics (100 mL inoculation / 500 mL volume), and incubate at 37°C and 200 rpm until OD. 600Once the concentration reaches 0.4~0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, adjust the temperature to 20 ℃, and continue culturing for 16 h to induce protein expression; the fermentation broth is then obtained.

[0048] The following examples illustrate the preparation methods of crude enzyme solutions. The obtained fermentation broth was centrifuged at low temperature at 8,000 × 10⁻⁶. g Centrifuge at 4°C for 10 min, discard the supernatant, and wash the precipitate twice with physiological saline to obtain wet bacterial cells containing the target protein. The obtained wet bacterial cells are ultrasonically disrupted under the conditions of 400 W, 0°C, working for 2 s, intermittent for 4 s, for a total time of 10 min to obtain cell lysate, which is the crude enzyme solution to be added in the embodiments of the present invention.

[0049] Example 1: Screening of AGAT enzyme The specific steps are as follows: (1) Chemical synthesis separately Streptomyces sp. source Ss AGAT enzyme (NCBI code: WP_089510850.1), Actinokineospora terrae Source At AGAT enzyme (NCBI ID: WP_092775660.1), Cyanobacteria bacterium SBLK origin Cb AGAT enzyme (NCBI code: MBP0019671.1), Actinoplanes derwentensis Source Ad AGAT enzyme (NCBI ID: GID89974.1), Pelagibacterales bacterium Source Pb AGAT enzyme (NCBI code: GIR05490.1); (2) The genes encoding the enzymes obtained in step (1) are codon-optimized and ligated into plasmid pET28a (which can be sent to a company for synthesis) to obtain recombinant plasmids; the recombinant plasmids are then introduced into E. coli. E . Coli Recombinant Escherichia coli were prepared separately in BL21(DE3); (3) The recombinant E. coli were cultured and subjected to a catalytic reaction: 1 mL reaction solution (pH 7.5), containing 50 mM glycine, 50 mM arginine and 50 mM phosphate buffer (pH 7.5), 200 rpm, recombinant bacteria: 25 g / L, reaction at 25℃ for 8 hours. The results are as follows. Figure 2 As shown; The results show that: originating from Actinokineospora terrae of AtAGAT enzyme is most effective; after 8 hours of biotransformation, it is used... At AGAT was reacted to yield 11.23 mM GAA. Subsequent experiments used GAA derived from... Actinokineospora terrae of At Further research is needed on the AGAT enzyme.

[0050] Example 2: Preparation of L-arginine:glycine amidinosyltransferase mutant The specific steps are as follows: 1. Identify the mutation site (1) At Structural Analysis of AGAT By coming from Actinokineospora terrae of At The AGAT enzyme was docked with arginine and glycine, and the results were as follows: Figure 3 As shown, based on the scanning of residues within 5A near the ligand arginine, several semi-conserved sites in the substrate-binding region, such as E31, C108, D111, G351, and R127, were identified, and virtual mutations were performed on these semi-conserved sites.

[0051] (2) At Screening of AGAT mutation hotspots The amino acid residues obtained in step (1) were subjected to virtual alanine mutation and further virtual saturation mutation. The results are shown in Table 1. The binding energy of mutant sites E31, G351, R127 and other sites to protein molecules was significantly increased, which proved that these sites are key residues involved in arginine catalysis. These sites were selected as mutation hotspots for directed evolution.

[0052] Table 1: At Mutagenicity of virtual saturation mutations at key AGAT sites

[0053] 2. At Construction of AGAT and its mutants The specific steps are as follows: Construction of single mutants (1) Reorganization At Construction of AGAT expression vector Source Actinokineospora terrae L-arginine:glycine amidotransferase (AGAT) (amino acid sequence as shown in SEQ ID NO.1), after codon optimization (nucleotide sequence as shown in SEQ ID NO.2), was ligated into plasmid pET28a (which can be sent to a company for synthesis), and named pET28a- At AGAT.

[0054] (2) Introduction of random mutations The mutation hotspots E31, G351, and R127 from step 1 were selected as mutation origins. Then, random mutations were introduced at the corresponding sites by PCR using the degenerate primers (NNK-MNN) shown in Table 2. The PCR reaction mixture is as follows: 1 μL of upstream primer, 1 μL of downstream primer, template plasmid (pET28a- At AGAT) 1 μL, 2 × Primstar 5 μL, ddH2O 2 μL. Pre-denaturation temperature 98℃, 30 s, denaturation temperature 98℃, 15 s, annealing temperature 55℃, 30 s, extension temperature 72℃, 90 s.

[0055] Table 2: Mutation sites and primers

[0056] (3) Construction of recombinant strains The wild-type recombinant expression vector pET28a- obtained in step (1) At AGAT and recombinant expression vectors with introduced random mutations were transformed into [various vectors]. E. coli Recombinant expression strains were prepared from BL21 (DE3) competent cells.

[0057] (4) High-throughput screening of positive mutants Establishment of high-throughput detection methods: Single clones were inoculated into 96-well plates for heterologous expression. After expression, cells were collected and resuspended in 1 mL of reaction buffer (pH 7.5) containing 30 mM arginine and 30 mM glycine, with a cell concentration of 20 g / L. The enzyme reaction was incubated at 25°C and 220 rpm with shaking for 6 hours, and the reaction was terminated by heat inactivation. The concentration of guanidinoacetic acid (GAA) was determined by high-performance liquid chromatography (HPLC). Mutants with ≥20% higher GAA yield than wild-type were screened and their DNA was sequenced for verification. The results are shown in Table 3.

[0058] Table 3: Sequencing results of mutants and concentration of guanidinoacetic acid catalyzed for synthesis

[0059] At Combination mutations of AGAT Combination mutations were performed on the three positive mutations E31Q, E31K, and G351N to obtain highly active mutants. The specific steps are as follows: Recombinant bacterial strains containing three positive mutants, E31Q, E31K, and G351N, were inoculated into test tubes containing 5 mL of LB medium. 5 μL of kanamycin (50 g / L) was added, and the tubes were incubated at 37 ℃ and 200 rpm for 8 h. Plasmid extraction was then performed using the Vazyme FastPure Plasmid Mini Kit-BOX 2, following the instructions provided in the kit. Two recombinant plasmids, pET28a(+)-, were obtained. At AGAT E31Q and pET28a(+)- At AGAT E31K Subsequently, using the mutation primers shown in Table 4 and the two plasmids mentioned above as templates, a PCR reaction was performed to introduce the second mutation site G351N.

[0060] Table 4: Site-directed mutagenesis primers

[0061] The PCR, template digestion, and product purification procedures followed the same procedures as for the construction of a single mutant. The double-mutant recombinant plasmid pET28a(+)- was obtained. At AGAT E31Q / G351N pET28a(+)- At AGAT E31K / G351N Subsequently, the recombinant strains were obtained according to the method described in the construction of single mutants, and the double-point combination mutations were screened according to the single mutant screening method. The results are shown in Table 5.

[0062] Table 5: Concentration of guanidinoacetic acid, the product synthesized by the combined mutants

[0063] The results showed that the combined mutations of E31Q and G351N, which had a positive effect, were weaker than the single-point mutations of E31Q and E31K in catalyzing the synthesis of guanidinoacetic acid. However, the combined mutations of E31K and G351N further improved the ability of guanidinoacetic acid synthesis, increasing it by 36.9% compared with the wild type.

[0064] Therefore, we take At Guanidoacetic acid chassis cells were constructed based on E31K / G351N.

[0065] Example 3: In vitro evaluation of enzymes in the synthetic pathway This invention is based on the pET28a plasmid system, in E. coliFive enzymes that form the core module of the ornithine cycle were constructed and co-expressed in BL21 (DE3): glycine amidotransferase AGAT (EC: 2.1.4.1), carbamoyl phosphate synthase II (CPSAB, EC: 6.3.5.5), ornithine carbamoyltransferase (ArgI, EC: 2.1.3.3), arginine succinate synthase (ArgG, EC: 6.3.4.5), and arginine succinate lyase (ArgH, EC: 4.3.2.1).

[0066] The specific steps are as follows: (1) Preparation of crude enzyme solution CPS AB enzyme, ArgI enzyme, ArgG enzyme, and ArgH enzyme were respectively ligated into the pET28a vector to prepare recombinant vectors. The recombinant vectors were then introduced into Escherichia coli to prepare recombinant Escherichia coli. The prepared recombinant *E. coli* were inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 8–10 h to prepare seed culture. The seed culture was then inoculated at a rate of 1% (v / v) into shake flasks containing 50 mg / L kanamycin in LB liquid medium (100 mL / 500 mL volume) and cultured at 37°C and 200 rpm until OD (dose eluent) was reached. 600 When the concentration reached 0.4-0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM. The temperature was adjusted to 20 °C, and the culture was continued for 16 h to induce protein expression. The culture media were then obtained. After centrifuging the culture medium, the bacterial cells were collected and subjected to ultrasonic treatment at 400 W, 0℃, for 2 seconds followed by 4 seconds of rest, for a total time of 10 minutes to break the cell walls, thereby preparing crude enzyme solutions.

[0067] (2) The feasibility of in vitro enzyme cascade regeneration of arginine was verified. Adding 30 mM ornithine to a crude enzyme solution containing ArgI (NCBI ID: ACT45908) with a crude enzyme activity of 6.82 U, CPSAB (NCBI ID: ACT41938) with a crude enzyme activity of 1.88 U, ArgG (NCBI ID: ACT44842) with a crude enzyme activity of 7.09 U, and ArgH (NCBI ID: ACT45638) with a crude enzyme activity of 8.34 U, and using glutamine (10 mM), ATP (5 mM), ammonium bicarbonate (20 mM), and aspartic acid (10 mM) as auxiliary substrates, after 8 h of biological reaction (25℃, 200 rpm), an accumulation of 4.3 mM arginine was detected. The feasibility of in vitro enzyme cascade regeneration of arginine has been preliminarily verified.

[0068] (3) To further reduce substrate dependence, we introduced glutamine synthase (GlnA, EC: 6.3.1.2) and aspartate ammonia-lyase (AspA, EC: 4.3.1.1) and prepared crude glutamine synthase and crude aspartate ammonia-lyase respectively according to the method in step (1). Add GlnA (NCBI number: ACT45548) with a crude enzyme activity of 4.15 U and AspA (NCBI number: ACT45798) with a crude enzyme activity of 4.98 U to the reaction system in step (2), and carry out a biological reaction for 8 hours (25℃, 200 rpm) according to the method in step (2). The results showed that endogenous regeneration of glutamine and aspartic acid was achieved in vitro. The optimized system (with the introduction of GlnA and AspA enzymes) increased the arginine yield to 7.4 mM (an increase of 72%) under the same reaction conditions, but the ornithine conversion rate was still limited to 24.6%.

[0069] To clarify the transformation bottleneck, this study further quantitatively analyzed the crude enzyme solution specific enzyme activity at key nodes in the metabolic cycle. Figure 4 (A, B) It was found that CPSAB and GlnA, involved in the CP synthesis pathway, exhibited significantly lower catalytic activity in the cycle compared to key downstream enzymes. Given that carbamoyl phosphate (CP) is an initial metabolite in the ornithine cycle, its synthesis efficiency directly determines the carbon and nitrogen flux input, which is particularly important for arginine regeneration. Insufficient catalytic activity of CPSAB and GlnA leads to limited CP library capacity, thereby causing substrate competition and an imbalance in metabolic flux distribution. These results indicate that enhancing the catalytic efficiency of the CP synthesis module is a key target for overcoming the bottleneck in the ornithine-arginine regeneration cycle.

[0070] Example 4: Optimization of the CP synthesis system for carbamoyl phosphate The specific steps are as follows: 1. Screening of key enzymes (1) First, the regeneration efficiency of glutamine was optimized by screening... Schistosoma japonicum The source of glutamine synthase GS (NCBI code Q86EI1) Corynebacterium glutamicum The source of glutamine synthase GS (NCBI ID AAD01244.2) Methylococcus capsulatus The three exogenous glutamine synthases (GS) and endogenous glutamine synthase GlnA were compared using the method described in step (1) of Example 3. SjGS crude enzyme solution, Cg GS crude enzyme solution, Mc GS crude enzyme solution 、 Crude GlnA enzyme solution; without expressing any enzyme. E. coli BL21 (DE3) was used as the control strain and, after treatment according to the above method, was assigned to the WT group. The catalytic reaction system was as follows: Sj GS / Cg GS / Mc GS / 50 g / L crude GlnA enzyme solution (based on cell count before lysis), 50 mM glutamate, 50 mM ATP, and 50 mM phosphate buffer (pH 7.2) were reacted at 25℃ and 200 rpm for 8 h. The glutamate content in the reaction solution was then measured, and the performance of different enzymes was compared based on glutamate consumption. Catalytic results showed that: Methylococcus capsulatus (Source: NCBI ID CAI8875598.1) Mc GS showed the best effect, with its glutamate consumption increasing by about 60% compared to the endogenous E. coli enzyme GlnA. Figure 4 -C).

[0071] (2) To enhance the metabolic flux of the CPSII step, we introduced carbamate kinase CK, which uses inorganic ammonia as an ammonia donor to synthesize carbamate phosphate (CP), to synergistically catalyze CPSII and reduce dependence on L-glutamine and ATP. By comparing endogenous YahI (EC: 2.7.2.2, NCBI ID: ACT42177) and exogenous YahI from *E. coli*... Giardia muris Source Gm CK （ NCBI number: TNJ29176.1) and Enterococcus The source is CK (NCBI ID: WP_002367131.1). Ef Catalytic effect of CK: Following the method of step (1) in Example 3, crude YahI enzyme solution was prepared respectively. Gm CK crude enzyme solution, Ef CK crude enzyme solution; without expressing any enzyme. E. coli BL21 (DE3) was used as the control strain and, after treatment according to the above method, was assigned to the WT group. The catalytic reaction system was: YahI / Gm CK / Ef CK crude enzyme solution (50 g / L, based on cell volume before disruption), 50 mM ammonium bicarbonate, 50 mM ATP, 50 mM ornithine, and ArgI crude enzyme solution (50 g / L) were added. After reacting at 25℃ and 200 rpm for 8 h, the citrulline content in the reaction solution was measured. The results showed that... Giardia muris Source GmCK showed the best effect, and when co-catalyzed with ArgI, it yielded 15.3 mM citrulline. Figure 4 -D).

[0072] 2. Validation of the multi-enzyme combination catalytic synthesis pathway for CP The crude enzyme solutions prepared in Example 3 and Step 1 were used to determine the reaction system; System A: 40 mM ornithine, 20 mM glutamine, 50 mM ATP, 50 mM ammonium bicarbonate, 5 mM magnesium chloride.

[0073] CP-1 reaction system: Add 50 g / L crude ArgI enzyme solution to system A; CP-2 reaction system: Add 50 g / L ArgI crude enzyme solution + 50 g / L CPSAB crude enzyme solution to system A; CP-3 reaction system: Add 50 g / L crude ArgI enzyme solution + 50 g / L CPSAB + 50 g / L [unclear] to system A Mc GS crude enzyme solution; CP-4 reaction system: Add 50 g / L ArgI crude enzyme solution + 50 g / L CPSAB crude enzyme solution + 50 g / L Mc GS crude enzyme solution + 50 g / L Gm CK crude enzyme solution.

[0074] 3. Detection of citrulline content in different systems The different reaction systems in step 2 were placed at 25°C and 200 rpm for 8 hours. After the reaction was completed, the yield of citrulline in the reaction solution was measured.

[0075] Experimental results: The yield of citrulline in the CP-1 reaction system was 0.9 mM; the yield of citrulline in the CP-2 reaction system was 15.2 mM; the yield of citrulline in the CP-3 reaction system was 21.2 mM; and the yield of citrulline in the CP-4 reaction system was 29.1 mM. The results showed that, compared to the catalytic pathway of CP synthesis via ArgI to citrulline using only CPSAB, the addition of [a specific ingredient]... Mc GS achieves substrate glutamine regeneration and Gm In the system where CK uses inorganic ammonia as a substrate to assist in the synthesis of CP from CPSAB, the citrulline content increased by 90%, reaching 29.1 mM ( Figure 4 -E). The enzymes related to the synthesis of CP in this invention are ultimately obtained as follows: The NCBI number is TNJ29176.1 Gm CK+ NCBI number: CAI8875598.1Mc CPSABs with GS+NCBI numbers ACT41938 and ACT41939.

[0076] Example 5: Construction of multi-enzyme co-expression engineered strains AGAT mutant At AGAT E31K / G351N The proteins, along with the remaining proteins (sequences shown in SEQ ID NO. 3~10), were integrated into the same strain of *E. coli*. The specific steps are as follows: (1) Screening of different vectors This study screened the functions of bacterial strain combinations (single / double bacterial systems) containing seven vectors: K-4, C-4, A-4, S-4, K+C, K+S, and K+A. The recombinant vectors corresponding to the above combinations are shown in Table 6 below. Table 6: Information on Different Carriers

[0077] The specific construction steps are as follows: Previously, we obtained the preferred embodiment from Example 2. At AGAT mutant At AGAT E31K / G351N Plasmid extraction was performed on the strain using the Vazyme FastPure Plasmid Mini Kit-BOX 2, following the instructions provided in the kit. The recombinant plasmid pET28a- was obtained. At AGAT E31K / G351N The remaining seven genes were constructed into plasmid pET28a(+) according to Example 2, resulting in plasmid pET28a- Mc GS, pET28a- Gm CK, pET28a-CPSAB, pET28a-ArgI, pET28a-ArgG, pET28a-AspA, and pET28a-ArgH are used for the analysis of metabolic pathways. Using the aforementioned plasmids as templates, eight genes were constructed into plasmids pETDuet-1 and pRSFDuet-1, resulting in recombinant plasmids pACYCDuet-ArgI-ArgG-E31K / G351N-ArgH(C-4) and pRSFDuet-1-CPSAB- Gm CK- Mc GS-AspA (K-4), and the carriers A-4, S-4, K+C, K+S, and K+A in Table 5; (2) Construction and screening of recombinant strains containing different vectors The recombinant plasmids K-4, C-4, A-4, S-4, and K+A obtained in step (1) were introduced into Escherichia coli, respectively.E. coli Recombinant strains were prepared from BL21(DE3). E. coli BL21 (DE3) / K-4, E. coli BL21 (DE3) / C-4, E. coli BL21 (DE3) / A-4、 E. coli BL21 (DE3) / S-4, E. coli BL21 (DE3) / K+A; the resulting recombinant E. coli were cultured and expressed. The results showed that Figure 5 In the study, it was found that C-4 failed to express the four required proteins. Therefore, two plasmids, pETDuet and pCDFDuet-1, were used to express the four genes on it, and the results were better than those of pACYCDuet.

[0078] (3) The recombinant plasmids K+C, K+S, and K+A obtained in step (1) were introduced into Escherichia coli respectively. E. coli Recombinant strains were prepared from BL21(DE3). E. coli BL21 (DE3) / K+C, E. coli BL21 (DE3) / K+S, E. coli BL21 (DE3) / K+A. The resulting recombinant E. coli were cultured and expressed. The cultured recombinant strains were respectively E. coli BL21 (DE3) / K+C, E. coli BL21 (DE3) / K+S, E. coli BL21 (DE3) / K+A was added to the reaction system: 100 mM ammonium bicarbonate, 10 mM magnesium chloride hexahydrate, 5 mM arginine, 50 mM glycine, and 1.2% glucose (the amount of recombinant bacteria added was 50 g / L). The reaction conditions were: 200 rpm and 12 h.

[0079] The culture conditions are the same as above, but the strain is adjusted to... E. coli BL21 (DE3) / K-4 and E. coli BL21 (DE3) / C-4 was added to the above reaction system at a ratio of 1:1 (addition amount: 50 g / L) to obtain the K-4+ C-4 group; The cultivation conditions are the same as above. E. coli BL21 (DE3) / K-4 and E. coliBL21 (DE3) / S-4 was added to the above reaction system at a ratio of 1:1 (addition amount: 50 g / L) to obtain the K-4+ S-4 group; The cultivation conditions are the same as above. E. coli BL21 (DE3) / K-4 and E. coli BL21 (DE3) / A-4 was added to the above reaction system at a ratio of 1:1 (addition amount: 50 g / L) to obtain the K-4+ A-4 group; The results showed that, Figure 5 As shown in B, E. coli The BL21 (DE3) / K+A strain exhibited the best performance.

[0080] Example 6: Establishing an efficient reaction system The recombinant engineered strain obtained in Example 5 E. coli BL21 (DE3) / K+A was selected as the preferred catalyst for establishing the reaction system.

[0081] The prepared recombinant Escherichia coli E. coli BL21 (DE3) / K+A was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 8–10 h to prepare a seed culture. The seed culture was then inoculated at a rate of 1% (v / v) into shake flasks containing 50 mg / L kanamycin in LB liquid medium (100 mL / 500 mL volume) and cultured at 37°C and 200 rpm until OD (dose retardation). 600 When the concentration reached 0.4-0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM. The temperature was adjusted to 20 °C, and the culture was continued for 16 h to induce protein expression. The culture media were then obtained. The obtained culture medium was centrifuged at low temperature at 8,000 °C. × g Centrifuge at 4 °C for 10 min, discard the supernatant, wash the precipitate twice with physiological saline to obtain whole-cell catalyst. The specific steps for optimizing reaction conditions using whole cells as catalyst are as follows: 1. Optimization of reaction conditions (1) Optimization of the optimal induction temperature Protein induction was performed at different induction temperatures of 16℃, 20℃, 25℃, and 30℃. The collected cells were reacted under the following conditions: 5 ml phosphate buffer (pH 7.5), cell wet weight 30 g / L, a reaction system consisting of 100 mM ammonium bicarbonate, 10 mM magnesium chloride hexahydrate, 10 mM arginine, 40 mM glycine, and 1.2% glucose solution, at 30℃ and 220 rpm for 18 hours. The results are as follows:Figure 6 As shown in A, the optimal induction temperature is 25℃, at which point the guanidinoacetic acid content in the solution is 12.1 mM.

[0082] (2) Optimization of the optimal reaction temperature Cells were induced and collected at 25℃, and the reaction conditions were the same as above (1), except that the temperature was the same. The cells were placed in shakers at 15℃, 20℃, 25℃, 30℃, and 37℃ respectively. The results are as follows: Figure 6 As shown in A, the optimal reaction temperature is 25℃, at which point the guanidinoacetic acid content in the solution is 14.5 mM.

[0083] (3) Optimization of optimal pH Five systems were selected, with cell collection induced at 25℃ and reaction conditions identical to (2) above, except for pH. The pH values ​​were controlled at 6.5, 7.0, 7.5, 8.0, and 8.5. The results are as follows: Figure 6 As shown in A, the optimal reaction pH is 8.0, at which point the guanidinoacetic acid content in the solution is 28.9 mM.

[0084] (4) Optimization of optimal reaction time Cells were induced and collected at 25℃, with reaction conditions as described above (3). Samples were taken and tested at different time points compared to strains without arginine cycling. Results are as follows: Figure 6 As shown in B, the reaction peaked at 33 hours. At this point, the concentration of guanidinoacetic acid in the solution was 33.6 mM, representing a 246% increase compared to the reaction without a cycle.

[0085] 2. Effects under optimal reaction system (1) Preparation of whole cells The prepared recombinant Escherichia coli E. coli BL21 (DE3) / K+A was inoculated into LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 8–10 h to prepare a seed culture. The seed culture was then inoculated at a rate of 1% (v / v) into shake flasks containing 50 mg / L kanamycin in LB liquid medium (100 mL / 500 mL volume) and cultured at 37°C and 200 rpm until OD (dose retardation). 600 Once the concentration reached 0.4–0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM. The temperature was adjusted to 25 °C, and the culture was continued for 16 h to induce protein expression. The culture media were then collected. The culture media were centrifuged at low temperature at 8,000 rpm. × g Centrifuge at 4 °C for 10 min, discard the supernatant, wash the precipitate twice with physiological saline to obtain whole-cell catalyst; (2) Catalytic system: The collected cells were reacted under the following conditions: 5 mL phosphate buffer (pH 8.0), 60 mg / mL wet cell weight, 100 mM ammonium bicarbonate, 10 mM magnesium chloride hexahydrate, 10 mM arginine, 40 mM glycine and 1.2% glucose solution, at 25°C and 220 rpm for 34 hours.

[0086] The results showed that with an induction temperature of 25 ℃, a reaction temperature of 25 ℃, a pH of 8.0, and a cell concentration of 60 mg / mL (wet weight), after 34 h of whole-cell catalytic reaction, the GAA yield reached 33.6 mM, and the molar conversion rates of arginine and glycine were 336% and 84%, respectively.

[0087] 3. Fermentation in the container Fermentation was conducted in 5 L petri dishes containing 3 L of fermentation medium (5 g / L yeast extract, 10 g / L peptone, 4 g / L K₂HPO₄, 3 g / L sodium chloride, 2.1 g / L citric acid monohydrate, 0.5 g / L ferric ammonium citrate, 10 g / L glycerol, 0.5 g / L anhydrous magnesium sulfate; fed medium: 250 g / L glycerol, 50 g / L peptone, 25 g / L yeast extract, 2.5 g / L magnesium sulfate, 3.5 g / L ammonium sulfate, 3 g / L dipotassium hydrogen phosphate and potassium dihydrogen phosphate, 4 g / L sodium chloride). pH was controlled by automatic ammonia addition. The gas flow rate was 3 L / min. The stirring speed was 400 rpm. To prepare the seed culture, streaks were applied to LB agar plates resistant to kanamycin and ampicillin and incubated overnight at 37°C. Single colonies were then picked and incubated in 5 mL of LB medium for 12 h. A 1% inoculum was then added to 200 mL of LB medium, and after 12 h, the culture was transferred to a 5 L fermenter. When the initial glycerol was almost depleted, dissolved oxygen began to rebound. Feeding was initiated when dissolved oxygen reached 30%, with a feed volume of 8 mL (OD200) being added. 600 Start cooling (25℃) and reducing the rotation speed (500 rpm) at 15), and add 1 / 1000 of IPTG to induce protein production.

[0088] Scale up the biotransformation of GAA. After culturing for 24 hours, the bacteria in a 5 L fermenter were passed through a 6,000 mL flow hood. × gCentrifuge at 4℃ for 10 minutes, weigh 75 g of the collected strain, and dilute to 800 ml with biotransformation solution. The biotransformation solution was phosphate buffer (16.282 g dipotassium hydrogen phosphate, 887.8 mg potassium dihydrogen phosphate) containing 20 mM arginine, 80 mM glycine, 100 mM ammonium bicarbonate, and 2 mM magnesium chloride. Glucose solution (500 g / L) was fed at a rate of 20 mL / h before the first feeding. The stirring speed was linked to dissolved oxygen control (DO) at 30%, the temperature was set at 25℃, and the pH was maintained at 8.0 by automatic ammonia addition. After 20 hours of biotransformation, the reaction mixture (120 mL phosphate buffer (100 mM, pH 8.0), 9 g glycine (120 mM), 6.96 g arginine (40 mM), 15.8 g ammonium bicarbonate (200 mM), 0.19 g magnesium chloride (2 mM)) was added at a rate of 5 mL / h, and glucose solution (500 g / L) was fed at a rate of 10 mL / h. Glucose supply was stopped after 44 hours, and the total reaction time was 60 hours.

[0089] The results showed that the 5 L fermenter achieved 18.35 g / L GAA (156.75 mM) and the arginine conversion rate reached 261.12%.

[0090] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A glycine amidotransferase mutant, characterized in that, The mutant is obtained by mutating the glutamic acid at position 31 of the glycine amidotransferase shown in SEQ ID NO.1 to glutamine or lysine; Alternatively, the mutant can be obtained by mutating glutamic acid at position 31 of the glycine amidotransferase as shown in SEQ ID NO.1 to glutamine or lysine, and simultaneously mutating glycine at position 351 to asparagine.

2. The gene encoding the glycine amidotransferase mutant of claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector.

4. The recombinant cell according to claim 3, characterized in that, The recombinant cells are bacteria or fungi as host cells.

5. A recombinant enzyme catalyst containing the glycine amidotransferase mutant of claim 1, characterized in that, It is any of the following forms: (1) Culture recombinant expression transformants containing the glycine amidotransferase mutant and isolate transformant cells containing the recombinant glycine amidotransferase mutant enzyme; (2) Cultivate recombinant expression transformants containing the glycine amidotransferase mutant, isolate transformant cells containing the recombinant glycine amidotransferase mutant enzyme, and break the transformant cells containing the recombinant glycine amidotransferase mutant enzyme to obtain cell lysate; (3) Cultivate recombinant expression transformants containing the glycine amidotransferase mutant, isolate transformant cells containing the recombinant glycine amidotransferase mutant enzyme, break the transformant cells containing the recombinant glycine amidotransferase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant glycine amidotransferase mutant enzyme to obtain lyophilized enzyme powder.

6. A method for improving the synthesis of guanidinoacetic acid catalyzed by glycine amidotransferase, characterized in that, The method is as follows: mutating the glutamic acid at position 31 of the glycine amidotransferase shown in SEQ ID NO.1 to glutamine or lysine; Alternatively, the glutamic acid at position 31 of the glycine amidotransferase, as shown in SEQ ID NO.1, may be mutated to glutamine or lysine, while the glycine at position 351 may be mutated to asparagine.

7. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria expressed the glycine amidotransferase mutant of claim 1, as well as carbamate kinase CK, glutamine synthase GS, ornithine carbamate transferase ArgI, carbamate phosphate synthase II, arginine succinate synthase ArgG, aspartate aminotransferase AspA, and arginine succinate lyase ArgH.

8. The genetically engineered bacteria according to claim 7, characterized in that, The carbamate kinase CK is derived from Giardia muris The glutamine synthase GS is derived from M. capsulAtus The ornithine carbamoyltransferase ArgI is derived from Escherichia coli, the carbamoyl phosphate synthase II is derived from Escherichia coli, the arginine succinate synthase ArgG is derived from Escherichia coli, the aspartate aminotransferase AspA is derived from Escherichia coli, and the arginine succinate lyase ArgH is derived from Escherichia coli.

9. The genetically engineered bacterium according to claim 8, characterized in that, The genetically engineered bacteria are Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or yeast.

10. The genetically engineered bacterium according to claim 9, characterized in that, The amino acid sequence of the carbamate kinase CK is shown in SEQ ID NO. 4; the nucleotide sequence encoding the glutamine synthase GS is shown in SEQ ID NO. 5; the nucleotide sequence encoding the ornithine carbamoyltransferase ArgI is shown in SEQ ID NO. 6; the nucleotide sequence encoding the carbamoyl phosphate synthase II is shown in SEQ ID NO. 7; the nucleotide sequence encoding the arginine succinate synthase ArgG is shown in SEQ ID NO. 8; the nucleotide sequence encoding the aspartate aminotransferase AspA is shown in SEQ ID NO. 9; and the nucleotide sequence encoding the arginine succinate lyase ArgH is shown in SEQ ID NO.

10.

11. A method for preparing guanidinoacetic acid (GAA), characterized in that, The method involves adding the genetically engineered bacteria or its lysate or fermentation broth as described in any one of claims 7 to 10 to a reaction system containing arginine and glycine, and catalyzing the conversion to synthesize guanidinoacetic acid.

12. The preparation method according to claim 11, characterized in that, The catalytic reaction conditions are: whole-cell reaction for 24-60 hours at pH 7.0-8.0 and 25-30℃.

13. The preparation method according to claim 12, characterized in that, The amount of the genetically engineered bacteria added is 40~70g / L.

14. The preparation method according to claim 13, characterized in that, The reaction system also includes: 100-200 mM ammonium bicarbonate, 4-10 mM MgCl2, and 8%-12% (w / v) glucose; A dynamic feeding strategy was adopted: the initial glycine concentration was 40-80 mM, and 120 mL of concentrated glycine solution of 75-100 g / L was added at a rate of 5 mL / h, with the glycine addition amount being 120-160 mM.

15. The use of the mutant of claim 1, or the gene or recombinant vector of claim 2, or the recombinant cell of claim 3 or 4, or the recombinase catalyst of claim 5, or the method of claim 6, or the genetically engineered bacteria of any one of claims 7 to 10, in any of the following: (a) Preparation of feed additives; (b) Increase livestock and poultry meat production and feed conversion rate; (c) Synthesize creatine precursor.

Images