Efficient synthesis of guanidinoacetic acid by carbamyl phosphoric acid synthesis-enhanced multi-enzyme cascade system

Abstract

The invention discloses efficient synthesis of guanidinoacetic acid by a carbamyl phosphoric acid synthesis-enhanced multi-enzyme cascade system, and belongs to the field of biological catalysis engineering. The invention provides a dominant mutant E31K / G351N of L-arginine: glycine amidino transferase, the catalytic efficiency of guanidinoacetic acid is improved by 34.6% compared with that of wild type, an eight-enzyme synergistic arginine circulation system is subsequently constructed based on a mutant strain, 18.35 g / L GAA (56.75 mM) is realized by a 5L fermentation tank, the arginine conversion rate reaches 261.12%, and the yield of the glycine amidino transferase is greatly improved. The highest level of catalytic synthesis of guanidinoacetic acid by escherichia coli is publically reported at home and abroad at present. According to the research, an efficient technical route is provided for GAA industrial production, and a universal strategy framework is established for rational design of a multi-enzyme system and multi-gene co-expression optimization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological catalysis, and mainly relates to a high-efficiency synthesis of guanidinoacetic acid by a multi-enzyme cascade system with enhanced carbamoyl phosphate synthesis. BACKGROUND

[0002] Guanidinoacetic acid (GAA, CAS No. 352-97-6) is a natural amino acid derivative and the only precursor for creatine synthesis in vertebrates, which is synthesized by methylation reaction mediated by guanidinoacetate methyltransferase (GAMT) in vivo. As a feed additive, GAA has attracted much attention in the field of agriculture, which can effectively improve meat yield and feed conversion efficiency. In addition to agricultural applications, the potential of GAA in human nutrition and health is increasingly expanding. It not only can be used as a precursor for creatine, but also has the functions of regulating insulin sensitivity, relieving dietary arginine stress, regulating protein synthesis, affecting hormone secretion, promoting vasodilation, and possibly affecting neural regulation through intervention of the gamma-aminobutyric acid system. These multi-dimensional physiological effects indicate that GAA may provide a new strategy for brain health and metabolic syndrome intervention, and its application value has gone beyond the traditional category of creatine supplementation. In 2023, the global guanidinoacetic acid market size was about 1.04 billion US dollars, and it is expected to grow to 5.05 billion US dollars by 2033, with a compound annual growth rate of 4.3%.

[0003] Based on the important role and potential market value of guanidinoacetic acid, there have been reports on the development of its production method. In the early stage, it was achieved by condensation of glycine and cyanamide. Industrial production has developed routes such as thiourea-glycine method, glycine-monocyanamide method, and guanidine hydrochloride-chloroacetic acid method. Although process optimization has improved yield and reduced by-products, traditional chemical synthesis still faces problems such as harsh reaction conditions, use of toxic reagents, and environmental burden.

[0004] Compared with the traditional chemical synthesis, the biotechnological route has more green advantages. Microbial synthesis of GAA relies on L-arginine: glycine amidotransferase (AGAT) catalyzing the transamidination reaction of L-arginine and glycine to generate product GAA and by-product L-ornithine. Zhang Yiweng et al. reconstructed the ornithine cycle in Escherichia 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 the Bacillus subtilis strain to catalyze the synthesis of 4.26 g / L GAA, with a reaction time of 20 hours. The current microbial synthesis of GAA system is limited by the high cost of substrate L-arginine and insufficient product titer, which are the industrialization bottlenecks. Therefore, it is urgent to develop methods for directed evolution of AGAT enzyme to improve its industrial properties. In addition, due to the fact that L-arginine accounts for more than 60% of the production cost, an efficient arginine regeneration system is particularly important. SUMMARY

[0005] Based on the synthesis of GAA by exogenous introduction of AGAT enzyme, four enzymes from ornithine to arginine synthesis pathway are introduced based on arginine synthesis pathway: carbamoyl phosphate synthase II (CPSAB, EC 6.3.5.5); ornithine carbamoyltransferase (ArgI); arginine succinate synthase (ArgG); arginine succinate lyase (ArgH), so that the byproduct ornithine is recycled to the product arginine. In order to reduce the demand for exogenous substrate, glutamine synthetase (GlnA) and aspartate ammonia lyase (AspA) are further introduced to realize the endogenous synthesis of glutamine and aspartate. By quantitatively analyzing the specific activity of the key metabolic nodes, it is found that the catalytic activity of CPSAB enzyme responsible for the synthesis of carbamoyl phosphate and GlnA enzyme responsible for the synthesis of glutamine is significantly lower than that of the downstream enzyme. The core rate-limiting step of ornithine-arginine regeneration cycle, carbamoyl phosphate synthesis module, is optimized. Since the availability of glutamine directly affects the efficiency of carbamoyl phosphate synthesis, glutamine regeneration is first strengthened: three exogenous glutamine synthetases (GS) of Schistosoma japonicum, Corynebacterium glutamicum and Methylococcus capsulAtus, and endogenous GlnA of Escherichia coli are screened, and the glutamine synthetase M. capsulAtus (McGS) with the highest catalytic activity is selected. In view of the dependence of traditional CPSAB pathway on L-glutamine and ATP, carbamate kinase (CK) is introduced to form a complementary pathway. The enzyme directly synthesizes carbamoyl phosphate from ammonia and carbamoyl phosphate precursor with inorganic ammonia as nitrogen donor. By comparing the endogenous YahI of Escherichia coli with the exogenous CK of Giardia muris and Enterococcus, it is found that the activity of Giardia muris (GmCK) is the best.

[0006] In order to further improve the upper limit of GAA synthesis, six groups of AGAT enzymes of different sources are screened, and AtAGAT from Actinokineospora terrae is selected and subjected to enzyme modification. Based on the analysis of crystal structure, the amino acid residues in the active center within the range of The amino acid residues in the active center within the range of

[0007] In the present application, the glycine amidinotransferase is L-arginine: glycine amidinotransferase, which is two naming ways of the same enzyme.

[0008] The application provides an L-arginine:glycine amidinotransferase mutant, which is obtained by mutating glutamic acid at position 31 of L-arginine:glycine amidinotransferase with an amino acid sequence shown in SEQ ID NO. 1 into lysine or glutamine, and is named as AtE31K or AtE31Q.

[0009] Or glutamic acid at position 31 of L-arginine:glycine amidinotransferase with an amino acid sequence shown in SEQ ID NO. 1 into lysine or glutamine, and is named as AtE31K or AtE31Q.

[0010] Or glutamic acid at position 31 of L-arginine:glycine amidinotransferase with an amino acid sequence shown in SEQ ID NO. 1 into lysine or glutamine, and is named as AtE31K or AtE31Q.

[0011] The amino acid sequence of L-arginine:glycine amidinotransferase is shown in SEQ ID NO. 1:

[0012] MQQVSEVVNSWNEWDPLEEIVVGSADGANFEPTEPGNRPQIRNAPPGTPFPSGPKSAEAVDRANEELAGLVSLLESEGVRVRRPAPHDFSQSVRTPDFEAANQYCAVCPRDVMITIGNEIIEAPMSRRSRYFEFQPYRELVYEYWNADPRVVWTTAPKPSMADKMYRDGFWDWPLAERHERMHSFEFCVTQDEVVFDAADMSRFGRDIVVQESMTTNRAGISWLKRHLEPKGFRVRPVHFPLDFFPSHIDCTFVPLRPGLVLTNPDRPLREGEEKLFLANDWELVDAPEPTTGNDEMPEFCQSSKWLSMNVLSIGPNKVICEQQEKPLQELLYKLDFEVFPVPFRNVFEYGGSLHCATWDVRRVGAGEDYFPNSDYQPLA

[0013] The nucleotide sequence encoding the L-arginine:glycine amidinotransferase is shown in SEQ ID NO. 2:

[0014]

[0015] SEQ ID NO. 3 (AtE31Q / G351N):

[0016]

[0017] The present application also provides a gene encoding the above-mentioned glycine amidinotransferase mutant or a recombinant vector carrying the gene.

[0018] The present application also provides a recombinant cell expressing the above-mentioned mutant or carrying the above-mentioned gene or the recombinant vector, preferably, the recombinant cell is a bacterial or fungal host cell.

[0019] The present application also provides a recombinant enzyme catalyst containing the above-mentioned glycine amidinotransferase mutant, which is in any of the following forms:

[0020] (1) culturing a recombinant expression transformant containing the glycine amidinotransferase mutant, and isolating transformant cells containing the recombinant glycine amidinotransferase mutant enzyme;

[0021] (2) culturing a recombinant expression transformant containing the glycine amidinotransferase mutant, and isolating transformant cells containing the recombinant glycine amidinotransferase mutant enzyme, crushing the transformant cells containing the recombinant glycine amidinotransferase mutant enzyme to obtain a cell crushing solution;

[0022] (3) culturing a recombinant expression transformant containing the glycine amidinotransferase mutant, and isolating transformant cells containing the recombinant glycine amidinotransferase mutant enzyme, crushing the transformant cells containing the recombinant glycine amidinotransferase mutant enzyme to obtain a cell crushing solution, and freeze-drying the cell crushing solution of the recombinant glycine amidinotransferase mutant enzyme to obtain a freeze-dried enzyme powder.

[0023] The present application also provides a method for improving the catalytic synthesis of guanidinoacetic acid by glycine amidinotransferase, wherein the glycine amidinotransferase mutant is obtained by mutating glutamic acid at position 31 of the amino acid sequence shown in SEQ ID NO. 1 to glutamine or lysine; or the mutant is obtained by mutating glycine at position 351 of the amino acid sequence shown in SEQ ID NO. 1 to asparagine; or the mutant is obtained by mutating glutamic acid at position 31 of the amino acid sequence shown in SEQ ID NO. 1 to glutamine or lysine and mutating glycine at position 351 to asparagine.

[0024] The present application also provides a method for efficiently expressing the above-mentioned eight enzymes in Escherichia coli by two plasmids. The pRSFDuet-pETDuet double-plasmid system is used, and each plasmid expresses four genes, and the specific connection mode is as follows: Figure 8 .

[0025] The application in the preparation of guanidinoacetic acid is a whole-cell reaction.

[0026] The reaction system is:

[0027] The application provides a genetically engineered bacterium, which expresses L-arginine: glycine amidinotransferase AGAT from Actinokineosporaterrae or a mutant thereof; carbamate kinase CK from Giardia muris, the protein sequence of which is shown as SEQ ID NO. 4; glutamine synthetase GS from M. capsulatus, the nucleotide sequence of which is shown as SEQ ID NO. 5; and ornithine carbamoyltransferase ArgI from Escherichia coli, the nucleotide sequence of which is shown as SEQ ID NO. 6; carbamoyl phosphate synthetase IICPS AB, the nucleotide sequence of which is shown as SEQ ID NO. 7; arginine succinate synthetase ArgG, the nucleotide sequence of which is shown as SEQ ID NO. 8; aspartate ammonia-lyase AspA, the nucleotide sequence of which is shown as SEQ ID NO. 9; and arginine succinate lyase ArgH, the nucleotide sequence of which is shown as SEQ ID NO. 10.

[0028] SEQ ID NO. 4 (carbamate kinase)

[0029] MKTVVIALGGNAMLQPGQKGDFEVQLENVKAAIKEIHAIHRAGYRVVLTSGNGPQVGAIKLQNQSAASVSPEMPLYACGAMSQGLIGYMMVQELRNAFRSQKEATQCIGCLTQTLVDSGDPAFAAPSKPMGRFYTEEEARAMMAKDSTIMMKEDAGRGWRVVVPSPRPVEILEYPMIKKLVDDGVIVICTNGGGIPCKLEGERIVGVDAVIDKDMATSLLAQKLNSDYLLILTDVPYATINYRTSDQQEIKTVTVEEMMKLEQEGHFKDGSMKPKVRAAIEFTMKTGNPSIITSLTSAFDALEGNCGTRIVQ

[0030] SEQ ID NO. 5 (glutamine synthetase)

[0031]

[0032] SEQ ID NO. 6 (ornithine carbamoyltransferase)

[0033]

[0034] SEQ ID NO. 7 (carbamoyl phosphate synthase II)

[0035]

[0036] SEQ ID NO. 8 (Arginine succinyl- synthetase)

[0037]

[0038] SEQ ID NO. 9 (Aspartate ammonia-lyase)

[0039]

[0040] SEQ ID NO. 10 (Arginine succinylase)

[0041]

[0042] In an embodiment of the present application, the genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Saccharomyces.

[0043] In an embodiment of the present application, the genetically engineered bacteria are recombinant Escherichia coli.

[0044] In an embodiment of the present application, the recombinant Escherichia coli is an expression host of E. coli BL21(DE3).

[0045] In an embodiment of the present application, the recombinant vector is pET28a(+), pETDuet-1, pRSFDuet-1, pACYCDuet-1, or pRCDFDuet-1.

[0046] The present application also provides one or more recombinant cells expressing the above genes or carrying the above recombinant vector.

[0047] In an embodiment of the present application, the recombinant cells use Escherichia coli as an expression host.

[0048] In an embodiment of the present application, the recombinant bacteria are constructed by PCR amplifying L-arginine:glycine amidinotransferase (AGAT) or its mutant (AGATmut), carbamoyl phosphate synthase II (CPSAB), glutamine synthetase (GS), carbamate kinase (CK), ornithine carbamoyltransferase (ArgI), arginine succinyl synthetase (ArgG), aspartate ammonia-lyase (AspA), and arginine succinylase (ArgH), cloning into plasmid pETDuet-1 or pACYCDuet-1 or pRCDFDuet-1 and pRSFDuet-1, constructing recombinant plasmid pETDuet-1-ArgI-ArgG-AGATmut-ArgH or pACYCDuet-1-ArgI-ArgG-AGATmut-ArgH or pRCDFDuet-1-ArgI-ArgG-AGATmut-ArgH and pRSFDuet-1-CPSAB-GmCK-McGS-AspA, and co-transforming into E. coli BL21(DE3) to construct a recombinant Escherichia coli catalyst.

[0049] In an embodiment of the present application, the NCBI accession number of the L-arginine:glycine amidinotransferase is WP_092775660.1 or the mutant of the L-arginine:glycine amidinotransferase described above; the NCBI accession number of the glutamine synthetase is CAI8875598.1, the NCBI accession number of the carbamate kinase is TNJ29176.1, the NCBI accession number of the ornithine carbamoyltransferase is ACT45908, the NCBI accession number of the arginine succinyl synthetase is ACT44842, the NCBI accession numbers of the two subunits of carbamoyl phosphate synthase II are ACT41938 and ACT41939, the NCBI accession number of the aspartate ammonia-lyase is ACT45798, and the NCBI accession number of the arginine succinylase is ACT45638.

[0050] The present application also provides a method for catalytically synthesizing guanidinoacetic acid by using whole cells, which comprises catalytically converting guanidinoacetic acid by using the recombinant E. coli whole cells described above under aeration conditions with arginine and glycine as substrates. Preferably, the catalytic reaction conditions are as follows: whole cell reaction at 25-30°C and pH 7.0-8.0 for 24-60 hours; preferably, the addition amount of the genetically engineered bacteria 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 used: the initial glycine concentration is 40-80 mM, 120 mL of a glycine concentrate with a concentration of 75-100 g / L is fed at a rate of 5 mL / h, and the glycine addition amount is about 120-160 mM.

[0051] In an embodiment of the present application, 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), the aeration amount is 3 vvm, 70 g of wet bacteria is added, the volume is made up to 800 ml with 100 mM phosphate solution (pH 7.5), the feeding bottle is a 0.5 g / mL glucose solution (500 mL), ammonia water is used to control the pH to 8.0, and the reaction time is 60 h at a reaction temperature of 30°C.

[0052] In an embodiment of the present application, the whole cell catalyst is obtained by culturing the recombinant bacteria and inducing the expression of L-arginine:glycine amidinotransferase, carbamoyl phosphate synthase II, glutamine synthetase, carbamate kinase, ornithine carbamoyltransferase, arginine succinyl synthetase, aspartate ammonia-lyase, and arginine succinylase, and then collecting the recombinant bacteria.

[0053] In an embodiment of the present application, the full cell catalyst is obtained by inoculating 1% of recombinant E. coli co-expressing L-arginine:glycine amidinotransferase, carbamoyl phosphate synthetase II; glutamine synthetase; carbamate kinase; ornithine carbamoyltransferase; arginine succinyl synthetase; aspartate ammonia-lyase; arginine succinyl lyase into 100 mL LB medium, culturing at 37℃, adding 0.5 mM IPTG to induce protein expression when OD 600 reaches 0.4-0.6, and centrifuging at 8,000 x g for 10 min at low temperature after 16 h of induction at 20℃, and washing the bacterial cells twice with 0.9% sodium chloride solution to obtain the full cell catalyst.

[0054] In an embodiment of the present application, the reaction conditions of the full cell catalyst are as follows: the DO is controlled to be 30% by oxygen-dissolved linkage and agitation, the temperature is 30℃, the pH is 8.0, the aeration rate is 2 vvm, the glucose solution is fed at a rate of 20 mL / h for 0-20 h, the reaction mixture (120 mL of 100 mM phosphate buffer, pH 8.0, 9 g of glycine (120 mM), 6.96 g of arginine (40 mM), 15.8 g of ammonium bicarbonate (200 mM), and 0.19 g of magnesium chloride (2 mM)) is supplemented at a rate of 5 mL / h after 20 h, the glucose solution feeding rate is reduced to 10 mL / h, the glucose supply is stopped after 44 h, and the guanidinoacetic acid content starts to decrease after 60 h when the yield reaches the highest.

[0055] In an embodiment of the present application, the addition amounts of arginine and glycine are 60 mM (equivalent to 10.44 g / L) and 200 mM (equivalent to 15 g / L), respectively, and the addition amount of the catalyst is 46 g / L (wet weight of cells).

[0056] The present application provides the use of the above-mentioned recombinant E. coli or the above-mentioned method in the preparation of guanidinoacetic acid, and in any one of the following: (a) the preparation of feed additives; (b) the improvement of the meat yield and feed conversion rate of livestock and poultry; and (c) the synthesis of creatine precursors.

[0057] Advantages

[0058] (1)The application successfully obtains a high-activity mutant of L-arginine: glycine amidinotransferase, improves the catalytic efficiency of L-arginine: glycine amidinotransferase. By optimizing the carbamoyl phosphate synthesis pathway, the arginine regeneration system is improved. In E. coli, L-arginine: glycine amidinotransferase mutant, carbamoyl phosphate synthase II, glutamine synthetase, carbamate kinase, ornithine carbamoyltransferase, arginine succinate synthetase, aspartate ammonia lyase and arginine succinate lyase are co-expressed by double plasmids pRSFDuet-1 and pETDuet-1. Eight enzymes are efficiently expressed in a single cell, by-product ornithine is eliminated and converted into substrate arginine, the inhibition of ornithine on L-arginine: glycine amidinotransferase is avoided, and the economic problem of high cost of arginine is avoided. With a small amount of arginine and glycine as substrates, after multi-enzyme one-pot cascade catalysis, the final yield of guanidyl acetic acid can reach 18.35 g / L, and the conversion rate of arginine is 261.12%. The multi-enzyme coupling system constructed in the application solves the problems of high cost of arginine in the process of guanidyl acetic acid biosynthesis and the recycling of by-product ornithine, and improves the atomic utilization efficiency of the biological catalytic reaction process.

[0059] (2)The establishment of the 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 process of guanidyl acetic acid, has good atom economy and environmental friendliness, realizes efficient synthesis of guanidyl acetic acid from a small amount of arginine and glycine as substrates, and according to all the data, the yield of guanidyl acetic acid in the application is the highest level in the biological synthesis from E. coli. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 : Principle diagram of multi-enzyme catalytic efficient synthesis of guanidyl acetic acid GAA.

[0061] Figure 2 : Screening of key enzyme AGAT; wherein, A is the catalytic reaction of AGAT and the phylogenetic tree of AGAT systems of different species, and the gray part is the selected enzyme; B is the GAA produced by the selected AGAT under the condition of 25 DEG C after eight hours of whole cell catalysis; C is the soluble expression of AGAT of different species in BL21.

[0062] Figure 3: At AGAT structure and mutation analysis of key amino acid residues in the binding pocket; wherein, A is a multiple sequence alignment and sequence conservation analysis of At AGAT; B is the docking of At AGAT model with arginine and glycine and the visualization of its binding pocket; C is the energy change when the adjacent amino acids in the binding pocket are mutated to alanine; the red pentagon represents the potential mutation site; D is the energy change analysis of virtual saturation mutagenesis of part of the key amino acid residue sites; E is the virtual combinatorial mutagenesis heat map analysis of the potential key amino acid residue sites; F is the beneficial single-point and combined mutation enzyme activity and catalytic ability result graph.

[0063] Figure 4 : Comparison of crude enzyme catalytic ability of each step and optimization of CP synthesis module.

[0064] Figure 5 : Protein expression of engineering strain and screening of construction method.

[0065] Figure 6 : Optimization of reaction conditions of engineering strain.

[0066] Figure 7 : Engineering strain 5L fermenter catalysis results.

[0067] Figure 8 : Engineering strain plasmid construction sample diagram. DETAILED DESCRIPTION

[0068] The detection methods involved in the following examples are as follows:

[0069] SDS-PAGE gel electrophoresis:

[0070] Prepare BBI uncapped SDS-PAGE denaturing acrylamide color gel rapid preparation kit and prepare protein gel according to the instructions. Sample preparation: take the wet bacteria containing the target protein, prepare 20 g / L cell suspension with Tris-HCl buffer (pH 7.5, 100 mM), and place it in an ice bath for ultrasonic crushing (450 W, work for 2 s, pause for 4 s, for a total of 10 min). Centrifuge at 12,000 x g, 4°C for 1 min, separate the supernatant from the precipitate. Take 20 μL of supernatant, mix with 8 μL of loading buffer, heat in a boiling water bath for 10 min, centrifuge at 12,000 x g for 5 min, and reserve. Electrophoresis: protein gel loading amount: sample 10 μL, marker 3 μL; electrophoresis conditions: 160 V, 60 min; electrophoresis buffer (g / L): glycine 14.4, SDS 1, Tris 3. Staining: soak 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, and wash with water twice. Decolorization: soak in decolorizing solution (ethanol 10%, acetic acid 10%, water 80%) and gently shake, replace fresh decolorizing solution until the bands are clearly visible.

[0071] Guanidinoacetic acid sample treatment and HPLC detection

[0072] It was found through research that citric acid can improve the solubility of guanidinoacetic acid (GAA). For the detection of fermentation solution for the scale-up synthesis of guanidinoacetic acid (GAA), the stirring was adjusted to 300 rpm before sampling to ensure uniform mixing of the solution. 50 mL of sample was taken, 1 mL of which was centrifuged for amino acid detection. The remaining sample was adjusted to pH 2.8 with citric acid, thoroughly mixed, and then left to stand for one hour. The sample was centrifuged, 50 μL of the supernatant was dissolved in 150 μL of pure water, 600 μL of pure acetonitrile was added, mixed, filtered with a 0.22 μm organic filter membrane, and analyzed by HPLC. For each strain, at least three independent experiments were performed.

[0073] Analysis method: The concentration of GAA was measured by HPLC (Agilent 1260 series, Hewlett-Packard) through a Lac-Amino column (4.5 mm x 300 mm). The analysis temperature was 35°C, the mobile phase (acetonitrile: water = 75:25) had a flow rate of 0.7 mL / min, and GAA was detected at an OD of 210 nm. The retention time of GAA was 19.5 minutes.

[0074] Amino acid detection

[0075] The concentration of amino acids, including arginine, glycine, ornithine, glutamic acid, glutamine, citrulline, aspartic acid, was measured by HPLC (Agilent 1260 series, Hewlett-Packard) through a C18 column (4.5 mm x 300 mm). The sample pretreatment, consisting of a derivatization reagent of 2,4-dinitrofluorobenzene / acetonitrile (1 :99) solution (50 μί) and 0.5 m NaHC03(100 μί), was added to 100 μί of the supernatant. The mixture was incubated at 60 °C for 60 min. Then, 750 μί of 0.01 m KH2P04was added to stop the reaction, mixed and left for 10 min. The reaction solution was analyzed at a flow rate of 1 mL / min using a linear gradient of two mobile phases (eluent A, acetonitrile; eluent B, 50 mM sodium acetate, linear gradient of eluent B 90% ~ 50%, for 25 min) at 30 °C, and detected at 360 nm.

[0076] Purification and enzyme activity assay of L-arginine:glycine amidinotransferase

[0077] All recombinant proteins have a 6xHistine tag at the N-terminus, and the proteins were purified using a nickel affinity chromatography column (Sangon Biotech, Shanghai, China). The purified proteins were used for kinetic assays. The HPLC was used to determine the activity of glycine amidinotransferase. The enzyme activity assay system (2 mL): 100 mM Tris-HCl (pH 8.0), 40 mM arginine, 40 mM glycine stock solution 0.95 mL and 0.05 mL of pure enzyme (1 mg / mL). The reaction was carried out in a 4 mL EP tube at 25 °C, 200 rpm for 1 h, then the reaction was stopped by boiling for two minutes, 200 μί of supernatant was added to 600 μί of acetonitrile, and the organic filter membrane was used for liquid phase detection. The boiled enzyme was used as a negative control. One enzyme activity unit was defined (U): the amount of enzyme required to catalyze the production of 1 μιτιοΙ of guanyl acetic acid (GAA) per minute under standard reaction conditions was one enzyme activity unit.

[0078] High-throughput screening of mutants

[0079] According to the virtual mutation results, several potential mutation sites in the substrate binding pocket are screened, degenerate primers (NNK-MNN) are used for random mutation and combined mutation of the mutation sites, and a mutant with improved catalytic ability is screened. All the single clone strains of random mutation are cultured in a 96-well plate and induced to express, and the collected strains are prepared into 1 mL reaction solution (pH 7.5), and the reaction mixture includes 30 mM arginine, 30 mM glycine, 220 rpm, 25°C reaction for 6 hours, heating to terminate the reaction, and the product GAA content is detected by high performance liquid chromatography, and compared with the wild type, the strain with high yield is selected for DNA sequencing to confirm the mutation results. In the following examples, the definition of 50 g / L of crude enzyme solution is as follows: adding the crude enzyme solution obtained by crushing 50 g of bacterial body to a 1L reaction system.

[0080] The culture and expression method of the recombinant Escherichia coli involved in the following examples is as follows:

[0081] (1) Preparation of seed solution

[0082] A single colony is inoculated into a test tube containing LB liquid medium containing (concentration: 50 mg / L) ampicillin or kanamycin, or containing both of the above two antibiotics, and cultured at 37°C, 200 rpm for 8-10 h to obtain a seed solution.

[0083] (2) Fermentation culture of recombinant Escherichia coli

[0084] The seed solution is inoculated into a LB liquid medium shake flask (100 mL liquid / 500 mL volume) containing (concentration: 50 mg / L) ampicillin or kanamycin, or containing both of the above two antibiotics, at an inoculation amount of 1% (v / v), and cultured at 37°C, 200 rpm until the OD 600 reaches 0.4-0.6, the final concentration of isopropyl-β-D-thiogalactoside (IPTG) is added to 0.5 mM, the temperature is adjusted to 20°C, and the culture is continued for 16 h to induce protein expression; and a fermentation broth is obtained.

[0085] Preparation method of crude enzyme solution involved in the following examples

[0086] The obtained fermentation broth is centrifuged at 8,000 x g, 10 min, 4°C, the supernatant is discarded after centrifugation, and the precipitate is washed twice with normal saline to obtain wet bacterial cells containing the target protein; the obtained wet bacterial cells are ultrasonically broken under the conditions of 400 W, 0°C, working for 2 s, intermittent for 4 s, and total time of 10 min to obtain a cell lysate which is the crude enzyme solution required to be added in the examples.

[0087] Example 1: Screening of AGAT enzyme

[0088] The specific steps are as follows:

[0089] (1) Chemically synthesize SsAGATase from Streptomyces sp. (NCBI No: WP_089510850.1), AtAGATase from Actinokineospora terrae (NCBI No: WP_092775660.1), CbAGATase from Cyanobacteria bacterium SBLK (NCBI No: MBP0019671.1), AdAGATase from Actinoplanes derwentensis (NCBI No: GID89974.1), PbAGATase from Pelagibacterales bacterium (NCBI No: GIR05490.1), respectively;

[0090] (2) Connect the genes encoding the enzymes obtained in step (1) to plasmid pET28a after codon optimization (can be synthesized by a company), respectively, to obtain recombinant plasmids; introduce the obtained recombinant plasmids into E. coli BL21 (DE3) respectively, to prepare recombinant E. coli respectively;

[0091] (3) After culturing the recombinant E. coli, perform catalytic reaction: 1 mL reaction solution (pH 7.5), reaction solution: 50 mM glycine, 50 mM arginine and 50 mM phosphate buffer (pH 7.5), 200 rpm, recombinant bacteria: 25 g / L, 25°C reaction for 8 hours. The results are shown in Figure 2 ;

[0092] The results show that AtAGATase from Actinokineospora terrae has the best effect, and 11.23 mM GAA is obtained after 8 hours of biotransformation using AtAGAT. Subsequent experiments further study AtAGATase from Actinokineospora terrae.

[0093] Example 2: Preparation of L-arginine: glycine amidinotransferase mutants

[0094] The specific steps are as follows:

[0095] 1. Determine the mutation site

[0096] (1) Structure analysis of AtAGAT

[0097] By docking AtAGATase from Actinokineospora terrae with arginine and glycine, the results are as follows: Figure 3As shown, according to the scanning of the residues within the range of 5A around the ligand arginine, multiple semi-conserved sites such as E31, C108, D111, G351, R127, etc. existing in the substrate binding region were determined, and these semi-conserved sites were selected for virtual mutation.

[0098] (2) Screening of AtAGAT mutation hotspots

[0099] The amino acid residues obtained by screening in step (1) were subjected to virtual alanine mutation and further virtual saturation mutation, and the results are shown in Table 1. The binding energy of the mutant sites E31, G351, R127, etc. to the protein molecule showed a significant increase, proving that these sites are key residues involved in arginine catalysis, and these sites were selected as mutation hotspots for directed evolution.

[0100] Table 1: Mutation energy changes of virtual saturation mutation of AtAGAT key sites

[0101]

[0102] 2, Construction of AtAGAT and its mutants

[0103] The specific steps are as follows:

[0104] Construction of single mutants

[0105] (1) Construction of recombinant AtAGAT expression vector

[0106] The Actinokineospora terrae L-arginine:glycine amidinotransferase (AGAT) derived from (the amino acid sequence is shown as SEQ ID NO. 1) was codon-optimized (the nucleotide sequence is shown as SEQ ID NO. 2) and then connected to the plasmid pET28a (which can be synthesized by a company) to be named pET28a-AtAGAT.

[0107] (2) Introduction of random mutations

[0108] The mutation hotspots E31, G351, R127 in step 1 were selected as the starting point of mutation, and then random mutations were introduced at the corresponding sites by PCR using the degenerate primer (NNK-MNN) shown in Table 2. The PCR reaction mixture was as follows:

[0109] 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of template plasmid (pET28a-AtAGAT), 5 μL of 2×Primstar, and 2 μL of ddH2O. Pre-denaturation temperature 98℃, 30S, denaturation temperature 98℃, 15S, annealing temperature 55℃, 30S, extension temperature 72℃, 90S.

[0110] Table 2: Mutation sites and primers

[0111]

[0112] (3) Construction of recombinant strains

[0113] The wild-type recombinant expression vector pET28a-AtAGAT obtained in step (1) and the recombinant expression vector with introduced random mutations were transformed into E. coli BL21 (DE3) competent cells, respectively, to prepare recombinant expression strains.

[0114] (4) High-throughput screening of positive mutants

[0115] Establishment of high-throughput detection method:

[0116] Monoclonal inoculation into 96-well plates for heterologous expression. After expression was completed, the cells were collected and resuspended in 1 mL of reaction buffer containing 30 mM arginine and 30 mM glycine (pH 7.5), and the concentration of cell mass was 20 g / L. The enzyme reaction was incubated at 25°C, 220 rpm 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 GAA production increased by ≥20% compared to the wild type were selected for DNA sequencing verification, and the results are shown in Table 3.

[0117] Table 3: Mutation sequencing results and concentration of catalytic synthesis of guanidinoacetic acid

[0118]

[0119] Combined mutation of AtAGAT

[0120] The three positive mutations E31Q, E31K, and G351N described above were combined to further obtain high-activity mutants, and the specific steps are as follows:

[0121] The recombinant strain containing the three positive mutants E31Q, E31K, and G351N was inoculated into a test tube containing 5 mL of LB medium, 5 μL of kanamycin (concentration: 50 g / L) was added, and it was incubated in a constant temperature shaker at 37°C, 200 rpm for 8 h. Then, plasmid extraction was performed using Vazyme FastPure Plasmid Mini Kit-BOX 2, and the extraction steps were referred to the instructions provided in the kit. Two recombinant plasmids, pET28a(+)-AtAGAT E31Q and pET28a(+)-AtAGAT E31K were obtained. Subsequently, according to the mutation primers shown in Table 4, PCR reaction was performed using the above two plasmids as templates to introduce the second mutation site G351N.

[0122] Table 4: Site-directed mutation primers

[0123]

[0124] Wherein the PCR and template digestion and product purification process refer to the construction of single mutants. Obtain double mutant recombinant plasmid pET28a(+)-AtAGAT E31Q / G351N , pET28a(+)-AtAGAT E31K / G351N . Then according to the recombinant strain construction method in the construction of single mutants, the corresponding recombinant strains are obtained, and the double-point combination mutation is screened according to the single mutant screening method, and the results are shown in Table 5.

[0125] Table 5: Concentration of product guanidino acetic acid catalyzed by combination mutant

[0126]

[0127] The results show that after the combination mutation of E31Q and G351N with positive effect, the catalytic synthesis of guanidino acetic acid is weaker than that of single-point mutation E31Q and E31K, while E31K and G351N in combination mutation, the catalytic synthesis of guanidino acetic acid is further improved, which is increased by 36.9% compared with wild type.

[0128] Therefore, we construct guanidino acetic acid chassis cells based on AtE31K / G351Q.

[0129] Example 3: In vitro evaluation of synthetic pathway enzymes

[0130] Based on the pET28a plasmid system, five enzymes of the ornithine cycle core module are constructed and co-expressed in E. coli BL21(DE3): glycine amidinotransferase AGAT (EC: 2.1.4.1), carbamoyl phosphate synthase II (CPSAB, EC: 6.3.5.5), ornithine carbamoyltransferase (ArgI, EC: 2.1.3.3), argininosuccinate synthase (ArgG, EC: 6.3.4.5), argininosuccinate lyase (ArgH, EC: 4.3.2.1).

[0131] The specific steps are as follows:

[0132] (1) Preparation of crude enzyme solution

[0133] CPS AB enzyme, ArgI enzyme, ArgG enzyme, and ArgH enzyme are respectively connected to pET28a vector to prepare recombinant vectors, and the recombinant vectors are respectively introduced into E. coli to prepare recombinant E. coli;

[0134] The prepared recombinant E. coli was inoculated into LB medium containing kanamycin (concentration: 50 mg / L), and cultured at 37°C, 200 rpm for 8-10 h to prepare seed liquid. The seed liquid was inoculated into LB liquid medium containing kanamycin (concentration: 50 mg / L) at a inoculation amount of 1% (v / v), and cultured at 37°C, 200 rpm until the OD 600 The temperature was adjusted to 20°C, and the culture was continued for 16 h to induce protein expression. The culture liquid was collected, and the bacterial cells were collected by centrifugation. The bacterial cells were broken by ultrasonic treatment at 400 W, 0°C, 2 s of work, 4 s of interval, and 10 min of total time. The crude enzyme liquid was prepared.

[0135] The culture liquid was centrifuged to collect the bacterial cells, which were broken by ultrasonic treatment at 400 W, 0°C, 2 s of work, 4 s of interval, and 10 min of total time. The crude enzyme liquid was prepared.

[0136] (2) The feasibility of in vitro enzyme cascade regeneration of arginine was verified

[0137] To the crude enzyme liquid system containing ArgI (NCBI number: ACT45908) with a crude enzyme activity of 6.82 U, CPSAB (NCBI number: ACT41938) with a crude enzyme activity of 1.88 U, ArgG (NCBI number: ACT44842) with a crude enzyme activity of 7.09 U, and ArgH (NCBI number: ACT45638) with a crude enzyme activity of 8.34 U, 30 mM ornithine was added, and glutamine (10 mM), ATP (5 mM), ammonium bicarbonate (20 mM) and aspartic acid (10 mM) were used as auxiliary substrates. After 8 h of biological reaction (25°C, 200 rpm), 4.3 mM arginine was detected to accumulate;

[0138] The feasibility of in vitro enzyme cascade regeneration of arginine was preliminarily verified.

[0139] (3) To further reduce the substrate dependence, glutamine synthetase (GlnA, EC: 6.3.1.2) and aspartate ammonia lyase (AspA, EC: 4.3.1.1) were introduced. According to the method of step (1), glutamine synthetase crude enzyme liquid and aspartate ammonia lyase crude enzyme liquid were prepared, respectively.

[0140] To the reaction system of step (2), 4.15 U of GlnA (NCBI number: ACT45548) and 4.98 U of AspA (NCBI number: ACT45798) were additionally added. According to the method of step (2), the biological reaction was carried out for 8 h (25°C, 200 rpm).

[0141] The results show that the endogenous regeneration of glutamine and aspartate is achieved in vitro; the arginine yield of the optimized system (introducing GlnA enzyme and AspA enzyme) is increased to 7.4 mM (72% increase) under the same reaction conditions, but the conversion rate of ornithine is still limited to 24.6%.

[0142] To analyze the conversion bottleneck, the specific enzyme activity of the crude enzyme solution of the key nodes in the metabolic cycle was quantitatively analyzed in the present study. Figure 4 -A, B); it was found that the catalytic capacity of CPSAB and GlnA involved in the CP synthesis pathway was significantly lower than that of the key enzymes downstream of the cycle. Given that carbamoyl phosphate (CP) is the initial metabolite of the ornithine cycle, its synthesis efficiency directly determines the carbon and nitrogen flux input, and is particularly important for arginine regeneration. The insufficient catalytic capacity of CPSAB and GlnA will lead to limited CP capacity, and further cause substrate competition and imbalance of metabolic flow distribution. The above results show that strengthening the catalytic efficiency of the CP synthesis module is a key target for breaking the ornithine-arginine regeneration cycle bottleneck.

[0143] Example 4: Optimization of the carbamoyl phosphate CP synthesis system

[0144] The specific steps are as follows:

[0145] 1. Screening of key enzymes

[0146] (1) First, the regeneration efficiency of glutamine was optimized, by screening Schistosoma japonicum-derived glutamine synthetase GS (NCBI number Q86EI1), Corynebacterium glutamicum-derived glutamine synthetase GS (NCBI number AAD01244.2), Methylococcus capsulatus-derived glutamine synthetase GS (NCBI number CAI8875598.1), and comparing the three exogenous glutamine synthetases (GS) and the endogenous glutamine synthetase GlnA: according to the method of step (1) of Example 3, SjGS crude enzyme solution, CgGS crude enzyme solution, McGS crude enzyme solution, and GlnA crude enzyme solution were prepared, respectively; with E. coli BL21(DE3) expressing no enzyme as the control strain, after treatment according to the above method, as the WT group. The catalytic reaction system was: SjGS / CgGS / McGS / GlnA crude enzyme solution 50 g / L (based on the bacterial body before crushing), 50 mM glutamic acid, 50 mM ATP, 50 mM phosphate buffer (pH 7.2); after 8 h of reaction at 25°C, 200 rpm, the content of glutamic acid in the reaction solution was detected, and the performance of different enzymes was compared by the consumption of glutamic acid; the catalytic results showed that the effect of McGS from Methylococcus capsulatus (NCBI number CAI8875598.1) was the best, and the glutamic acid consumption was increased by about 60% compared with the endogenous enzyme GlnA of E. coli Figure 4 C).

[0147] (2) In order to strengthen the metabolic flux of CPS II, we introduced carbamate kinase CK to synthesize carbamoyl phosphate CP with inorganic ammonia as the ammonia donor, which cooperated with CSP II to catalyze and reduced the dependence on L-glutamine and ATP. By comparing the catalytic effects of endogenous YahI (EC: 2.7.2.2, NCBI number: ACT42177) of E. coli, and exogenous GmCK (NCBI number: TNJ29176.1) from Giardia muris and CK (NCBI number: WP_002367131.1) from Enterococcus, the EfCK was prepared according to the method of step (1) of Example 3. The E. coli BL21 (DE3) without expressing any enzyme was used as a control strain, and after the above treatment, it was used as the WT group. The catalytic reaction system was: YahI / GmCK / EfCK crude enzyme solution 50 g / L (based on the bacterial body before crushing), 50 mM ammonium bicarbonate, 50 mM ATP, 50 mM ornithine, ArgI crude enzyme solution (50 g / L). After 8 h of reaction at 25°C, 200 rpm, the content of citrulline in the reaction solution was detected; the results showed that the GmCK from Giardia muris had the best effect, and after joint catalysis with ArgI, 15.3 mM citrulline was obtained Figure 4 -D).

[0148] 2. Verification of the pathway of multi-enzyme combination catalytic synthesis of CP

[0149] The crude enzyme solution prepared by Example 3 and step 1 was 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.

[0150] CP-1 reaction system: 50 g / L ArgI crude enzyme solution was added to system A;

[0151] CP-2 reaction system: 50 g / L ArgI crude enzyme solution + 50 g / L CPSAB crude enzyme solution was added to system A;

[0152] CP-3 reaction system: 50 g / L ArgI crude enzyme solution + 50 g / L CPSAB + 50 g / L McGS crude enzyme solution was added to system A;

[0153] CP-4 reaction system: 50 g / L ArgI crude enzyme solution + 50 g / L CPSAB crude enzyme solution + 50 g / L McGS crude enzyme solution + 50 g / L GmCK crude enzyme solution was added to system A.

[0154] 3. Detection of citrulline content in different systems

[0155] The different reaction systems of step 2 were respectively placed under the condition of 25℃ and 200rpm for reaction, and the time was: 8h; after the reaction was completed, the production of citrulline in the reaction solution was respectively detected.

[0156] The experimental results: the production of citrulline in CP-1 reaction system was: 0.9mM; the production of citrulline in CP-2 reaction system was: 15.2mM; the production of citrulline in CP-3 reaction system was: 21.2mM; the production of citrulline in CP-4 reaction system was: 29.1mM;

[0157] The results show that: compared with the catalytic pathway of synthesizing CP only by CPSAB to synthesize citrulline by ArgI, in the system of adding McGS to realize the regeneration of substrate glutamine and GmCK to assist CPSAB to synthesize CP with inorganic ammonia as substrate, the content of citrulline is increased by 90%, reaching 29.1mM Figure 4 The related enzymes for synthesizing CP finally obtained in the application are:

[0158] GmCK with NCBI number: TNJ29176.1+McGS with NCBI number: CAI8875598.1+CPSAB with NCBI number: ACT41938 and ACT41939.

[0159] Example 5: Construction of multi-enzyme co-expression engineering strain

[0160] AGAT mutant AtAGAT E31K / G351N and the rest of the proteins (sequences are shown in SEQ ID NO. 3-10) are integrated into the same strain of E. coli, and the specific operation steps are as follows:

[0161] (1) Screening of different vectors

[0162] In this study, the strain combination system (single strain / double strain system) containing K-4, C-4, A-4, S-4, K+C, K+S, K+A seven vectors was functionally screened; the corresponding recombinant vectors of the above combinations are shown in Table 6:

[0163] Table 6: Information of different vectors

[0164]

[0165] The specific construction steps are as follows:

[0166] In the early stage, we obtained the preferred AtAGAT mutant AtAGAT E31K / G351NThe strains were extracted by Vazyme FastPure Plasmid Mini Kit-BOX 2, and the extraction steps were referred to the instructions provided in the kit. The recombinant plasmid pET28a-AtAGAT E31K / G351N ; the other seven genes were respectively constructed into plasmid pET28a(+) according to Example 2 to obtain plasmids pET28a-McGS, pET28a-GmCK, pET28a-CPSAB, pET28a-ArgI, pET28a-ArgG, pET28a-AspA, pET28a-ArgH, which were used for metabolic pathway analysis;

[0167] The eight genes were constructed into plasmids pETDuet-1 and pRSFDuet-1 using the above plasmids as templates to obtain recombinant plasmids pACYCDuet-ArgI-ArgG-E31K / G351N-ArgH (C-4) and pRSFDuet-1-CPSAB-GmCK-McGS-AspA (K-4), as well as vectors A-4, S-4, K+C, K+S, K+A in Table 5;

[0168] (2) Construction and screening of recombinant strains containing different vectors

[0169] The recombinant plasmids K-4, C-4, A-4, S-4, K+A obtained in step (1) were introduced into E. coli BL21 (DE3) to prepare recombinant strains 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, respectively; and the obtained recombinant E. coli were cultured for expression;

[0170] The results showed that, Figure 5 In the A, C-4 failed to express the four required proteins, so for the four genes on it, pETDuet and pCDFDuet-1 were tried to express, and the effect was better than pACYCDuet.

[0171] (3) The recombinant plasmids K+C, K+S, K+A obtained in step (1) were introduced into E. coli BL21 (DE3) to prepare recombinant strains E. coli BL21 (DE3) / K+C, E. coli BL21 (DE3) / K+S, E. coli BL21 (DE3) / K+A, respectively. The obtained recombinant E. coli were cultured for expression;

[0172] The cultured recombinant strains E. coli BL21(DE3) / K+C, E. coli BL21(DE3) / K+S, and E. coli BL21(DE3) / K+A were added to a reaction system: 100 mM ammonium bicarbonate, 10 mM 6 water magnesium chloride, 5 mM arginine, 50 mM glycine, 1.2% glucose (the added amount of the recombinant bacteria was 50 g / L), and the reaction conditions were: 200 rpm, 12 h.

[0173] The culture conditions were the same as above, and the strains were adjusted to be E. coli BL21(DE3) / K-4 and E. coli BL21(DE3) / C-4 added to the above reaction system in a ratio of 1:1 (the added amount was 50 g / L) to obtain the K-4+C-4 group.

[0174] The culture conditions were the same as above, and the strains were adjusted to be E. coli BL21(DE3) / K-4 and E. coli BL21(DE3) / S-4 added to the above reaction system in a ratio of 1:1 (the added amount was 50 g / L) to obtain the K-4+S-4 group.

[0175] The culture conditions were the same as above, and the strains were adjusted to be E. coli BL21(DE3) / K-4 and E. coli BL21(DE3) / A-4 added to the above reaction system in a ratio of 1:1 (the added amount was 50 g / L) to obtain the K-4+A-4 group.

[0176] The results showed that, as shown in Table 1, the performance of the E. coli BL21(DE3) / K+A strain was the best. Figure 5

[0177] Example 6: Establishment of an efficient reaction system

[0178] The recombinant E. coli BL21(DE3) / K+A strain obtained in Example 5 was used as a preferred catalyst to establish a reaction system.

[0179] The prepared recombinant E. coli BL21(DE3) / K+A was inoculated into a kanamycin LB culture medium (the concentration was 50 mg / L), and was cultured at 37°C and 200 rpm for 8-10 h to prepare a seed liquid. The seed liquid was inoculated into a kanamycin LB liquid culture medium (100 mL liquid / 500 mL volume) at a 1% (v / v) inoculation amount, and was cultured at 37°C and 200 rpm until the OD 600 The temperature was adjusted to 20°C, and the culture was continued for 16 h to induce protein expression. The culture liquids were obtained, respectively.​

[0180] The obtained culture solution was centrifuged at 8,000 x g, 10 min, 4°C, and the supernatant was discarded after centrifugation. The precipitate was washed twice with normal saline to obtain a whole-cell catalyst. The whole cell was used as a catalyst to optimize the reaction conditions. The specific operation steps are as follows:

[0181] 1. Optimization of reaction conditions

[0182] (1) Optimization of the optimum induction temperature

[0183] Different induction temperatures, 16°C, 20°C, 25°C, and 30°C, were used for protein induction. The collected cells were reacted under the following conditions: 5 ml of phosphate buffer (pH 7.5), 30 g / L of cell wet weight, 100 mM ammonium bicarbonate, 10 mM magnesium chloride hexahydrate, 10 mM arginine, 40 mM glycine, and 1.2% glucose solution, 30°C, 220 rpm for 18 hours. The results are shown in Table 1, and the optimum induction temperature is 25°C. At this time, the content of guanidino acetic acid in the solution is 12.1 mM. Figure 6

[0184] (2) Optimization of the optimum reaction temperature

[0185] The collected cells were induced at 25°C, and the reaction conditions were the same as above (1) except for the temperature. The reaction was carried out at 15°C, 20°C, 25°C, 30°C, and 37°C, respectively. The results are shown in Table 2, and the optimum reaction temperature is 25°C. At this time, the content of guanidino acetic acid in the solution is 14.5 mM. Figure 6

[0186] (3) Optimization of the optimum pH

[0187] The collected cells were induced at 25°C, and the reaction conditions were the same as above (2) except for the pH. The reaction pH was controlled at 6.5, 7.0, 7.5, 8.0, and 8.5, respectively. The results are shown in Table 3, and the optimum reaction pH is 8.0. At this time, the content of guanidino acetic acid in the solution is 28.9 mM. Figure 6

[0188] (4) Optimization of the optimum reaction time

[0189] The collected cells were induced at 25°C, and the reaction conditions were the same as above (3). The samples were taken at different time periods for comparison with the strain not constructed with arginine cycle. The results are shown in Table 4, and the reaction reached a peak at 33 hours. At this time, the content of guanidino acetic acid in the solution is 33.6 mM, which is increased by 246% compared to the strain not constructed with arginine cycle. Figure 6

[0190] 2. Effect of the optimum reaction system

[0191] (1) Preparation of whole cells​​​​

[0192] The prepared recombinant E. coli BL21(DE3) / K+A was inoculated into LB medium containing kanamycin (concentration: 50 mg / L), and cultured at 37°C, 200 rpm for 8-10 h to prepare seed liquid. The seed liquid was inoculated into LB liquid medium containing kanamycin (concentration: 50 mg / L) at a seeding amount of 1% (v / v), and cultured at 37°C, 200 rpm until the OD 600 was reached. Then, 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 liquid was obtained. The obtained culture liquid was centrifuged at 8,000 x g, 10 min, 4°C. The supernatant was discarded after centrifugation, and the precipitate was washed twice with normal saline to obtain a whole-cell catalyst.

[0193] (2) Catalytic system:

[0194] The collected cells were reacted under the following conditions: 5 mL phosphate buffer (pH 8.0), cell wet weight 60 mg / mL, reaction system of 100 mM ammonium bicarbonate, 10 mM magnesium chloride hexahydrate, 10 mM arginine, 40 mM glycine and 1.2% glucose solution, 25°C, 220 rpm for 34 h.

[0195] The results showed that the GAA yield reached 33.6 mM, and the molar conversion rates of arginine and glycine were 336% and 84%, respectively, after 34 h of whole-cell catalytic reaction at an induction temperature of 25°C, a reaction temperature of 25°C, pH 8.0, and a cell concentration of 60 mg / mL (wet weight).

[0196] 3. Top tank fermentation

[0197] The fermentation was carried out in a 5L fermenter containing 3L fermentation medium (5g / L yeast extract, 10g / L peptone, 4g / L K2HPO4, 3g / L NaCl, 2.1g / L citric acid monohydrate, 0.5g / L ferric ammonium citrate, 10g / L glycerol, 0.5g / L MgSO4.7H2O. Feed medium: 250g / L glycerol, 50g / L peptone, 25g / L yeast extract, 2.5g / L MgSO4, 3.5g / L (NH4)2SO4, 3g / L K2HPO4, 3g / L KH2PO4, 4g / L NaCl). The pH was controlled by automatic addition of ammonia. The gas flow rate was 3L / min. The agitation speed was 400rpm. To prepare the seed, a single colony was inoculated into 5ml LB medium and incubated at 37°C for 12h, then inoculated into 200ml LB medium at 1% inoculum, and incubated for 12h before transferring into the 5L fermenter. When the initial glycerol was almost consumed, the dissolved oxygen started to rebound, and when the dissolved oxygen reached 30%, the feed was started, and the feed reached 8ml (OD 600 When the initial glycerol was almost consumed, the dissolved oxygen started to rebound, and when the dissolved oxygen reached 30%, the feed was started, and the feed reached 8ml (OD

[0198] Scale-up of GAA biotransformation. After 24h cultivation, the bacteria in the 5L fermenter were centrifuged at 6,000xg, 4°C for 10min, and 75g of the collected bacteria were resuspended in 800ml of biotransformation solution. The biotransformation solution was phosphate buffer (16.282g K2HPO4, 887.8mg KH2PO4) containing 20mM arginine, 80mM glycine, 100mM NH4HCO3, and 2mM MgCl2. Glucose solution (500g / L) was fed at a rate of 20ml / h before the first feed. The agitation speed was linked to the dissolved oxygen control, DO was set at 30%, the temperature was set at 25°C, and the pH was maintained at 8.0 by automatic addition of ammonia. After 20h biotransformation, the reaction mixture was supplemented with 5ml / h of 120ml phosphate buffer (100mM, pH 8.0), 9g glycine (120mM), 6.96g arginine (40mM), 15.8g NH4HCO3 (200mM), 0.19g MgCl2 (2mM), and glucose solution (500g / L) was fed at a rate of 10ml / h. The glucose supply was stopped after 44h, and the total reaction time was 60h. The results showed that 18.35g / L GAA (156.75mM) was achieved in the 5L fermenter, and the arginine conversion rate reached 261.12%.

[0199] Although the present application has been disclosed with reference to preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the scope of protection of the present application should be defined by the claims.

Claims

1. A mutant glycine amidinotransferase, characterized in that the mutant is obtained by mutating glutamic acid at position 31 of glycine amidinotransferase with amino acid sequence as shown in SEQ ID NO. 1 into glutamine or lysine; or the mutant is obtained by mutating glycine at position 351 of glycine amidinotransferase with amino acid sequence as shown in SEQ ID NO. 1 into asparagine; or the mutant is obtained by mutating glutamic acid at position 31 of glycine amidinotransferase with amino acid sequence as shown in SEQ ID NO. 1 into glutamine or lysine and mutating glycine at position 351 into asparagine.

2. A gene encoding the mutant of glycine amidinotransferase according to claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant according to claim 1 or carrying the gene according to claim 2 or the recombinant vector, preferably, the recombinant cell is a bacterial or fungal host cell.

4. A recombinant enzyme catalyst comprising the mutant glycine amidinotransferase of claim 1, wherein, is any one of the following forms: (1) culturing the recombinant expression transformant containing the mutant of glycine amidinotransferase, and isolating the transformant cell containing the recombinant mutant of glycine amidinotransferase enzyme; (2) culturing the recombinant expression transformant containing the mutant of glycine amidinotransferase, and isolating the transformant cell containing the recombinant mutant of glycine amidinotransferase enzyme, crushing the transformant cell containing the recombinant mutant of glycine amidinotransferase enzyme to obtain a cell crushing solution; (3) culturing the recombinant expression transformant containing the mutant of glycine amidinotransferase, and isolating the transformant cell containing the recombinant mutant of glycine amidinotransferase enzyme, crushing the transformant cell containing the recombinant mutant of glycine amidinotransferase enzyme to obtain a cell crushing solution, and freeze-drying the recombinant mutant of glycine amidinotransferase enzyme to obtain a freeze-dried enzyme powder.

5. A method for improving the catalysis of glycine amidinotransferase for the synthesis of guanidinoacetic acid, characterized in that, the mutant is obtained by mutating glutamic acid at position 31 of glycine amidinotransferase with amino acid sequence as shown in SEQ ID NO. 1 into glutamine or lysine; or the mutant is obtained by mutating glycine at position 351 of glycine amidinotransferase with amino acid sequence as shown in SEQ ID NO. 1 into asparagine; or the mutant is obtained by mutating glutamic acid at position 31 of glycine amidinotransferase with amino acid sequence as shown in SEQ ID NO. 1 into glutamine or lysine and mutating glycine at position 351 into asparagine.

6. A genetically engineered bacterium, characterized by, the genetically engineered bacteria express the mutant of glycine amidinotransferase according to claim 1, and carbamate kinase CK, glutamine synthetase GS, ornithine carbamoyltransferase ArgI, carbamoyl phosphate synthase II, arginine succinyl synthetase ArgG, aspartate ammonia-lyase AspA, and arginine succinyl-lyase ArgH; Preferably, the carbamate kinase CK is derived from Giardia muris, the glutamine synthetase 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 succinyl synthetase ArgG is derived from Escherichia coli, the aspartate ammonia-lyase AspA is derived from Escherichia coli; the arginine succinylase ArgH is derived from Escherichia coli.

7. The genetically engineered bacteria according to claim 6, characterized in that, The genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast; Preferably, the amino acid sequence of the carbamate kinase CK is shown in SEQ ID NO. 4; the nucleotide sequence encoding the glutamine synthetase 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 succinyl synthetase ArgG is shown in SEQ ID NO. 8; the nucleotide sequence encoding the aspartate ammonia-lyase AspA is shown in SEQ ID NO. 9; and the nucleotide sequence encoding the arginine succinylase ArgH is shown in SEQ ID NO.

10.

8. A method for the preparation of guanidino acetic acid (GAA) characterized in that, The method is to add the genetically engineered bacteria or the lysate or the fermentation broth thereof according to claim 6 or 7 to a reaction system containing arginine and glycine, to catalyze and synthesize guanidino acetic acid; Preferably, the catalytic reaction conditions are: whole-cell reaction at pH 7.0-8.0, 25-30°C for 24-60 hours; Preferably, the genetically engineered bacteria are added in an amount of 40-70 g / L.

9. The production method according to claim 8, characterized by, The reaction system further comprises: 100-200 mM ammonium bicarbonate, 4-10 mM MgCl2, 8%-12% (w / v) glucose; A dynamic feeding strategy is adopted: the initial glycine concentration is 40-80 mM, and 120 mL of a concentrated solution of 75-100 g / L glycine is added at a rate of 5 mL / h, and the glycine addition amount is about 120-160 mM.

10. The mutant of claim 1, the gene or recombinant vector of claim 2, the recombinant cell of claim 3, the recombinant enzyme catalyst of claim 4, the method of claim 5, the genetically engineered bacteria of claim 6 or 7, or the method of claim 8 or 9, for use in any one of the following: (a) preparation of a feed additive; (b) increasing the yield of livestock meat and feed conversion rate; (c) synthesis of a creatine precursor.

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