A method for producing guanidinoacetic acid by fermenting a saccharide raw material using microorganisms

Abstract

The application discloses a method for producing guanidino acetic acid by fermenting saccharide raw materials by using microorganisms, and belongs to the technical field of genetic engineering. L-arginine-glycine amidinotransferase is overexpressed in different strains by using a shuttle plasmid pXMJ19 between Escherichia coli and Corynebacterium glutamicum, and Corynebacterium glutamicum var. CCTCC AB2021051 is screened as a guanidino acetic acid production strain through shake flask fermentation. The expression amount of the enzyme is improved by using RBS optimization, and the absorption and utilization of carbon and nitrogen sources by the strain are enhanced. After fermentation condition optimization, the guanidino acetic acid production reaches 27.2 g / L through 5L tank amplification production and 72h fermentation. The guanidino acetic acid produced by the method has the advantages of low production cost and high efficiency, and is beneficial to amplification production.

Description

Technical Field

[0001] This invention relates to a method for producing guanidinoacetic acid by microbial fermentation of saccharide raw materials, belonging to the field of industrial microbial technology. Background Technology

[0002] Guanidinoacetic acid (GAA) is a naturally occurring amino acid derivative that serves as a direct precursor to creatine and can act as a creatine source in tissues requiring high energy. Due to its cost-effectiveness and high stability in aqueous solutions, as well as its non-specific transport properties compared to creatine, GAA has been investigated as a nutritional and feed additive. Supplementation with GAA can restore cellular bioenergy in diseases characterized by low brain creatine and creatine synthesis enzyme mechanisms, including neurodegenerative diseases, brain tumors, or cerebrovascular diseases. Furthermore, GAA does not pose mutagenic or genotoxic issues and does not pose environmental risks. Given the wide range of applications of GAA, there is significant industrial demand for it.

[0003] GAA preparation methods include chemical synthesis, biotransformation, and microbial fermentation. Industrial-scale GAA production primarily involves chemical synthesis via the reaction of glycine or sodium glycine with guanylate acylating agents (such as O-alkylisourea or cyanamide). This process requires high temperature and pressure, making it environmentally unfriendly. Biotransformation of GAA mainly involves the production of guanidinoacetic acid and ornithine from arginine and glycine substrates under the catalysis of L-arginine-glycine amidoyltransferase (AGAT, EC: 2.1.4.1). Compared to chemical synthesis, this method offers advantages such as simpler steps, fewer byproducts, and environmental friendliness. However, biotransformation requires expensive arginine or glycine substrates, resulting in high costs. Furthermore, ornithine inhibits AGAT production, significantly hindering the industrialization of GAA production through biotransformation.

[0004] Compared to these methods, microbial fermentation has many advantages, including inexpensive raw materials, less environmental pollution, higher purity of natural products, and mild reaction conditions. Currently, the reported production strains used in microbial fermentation include *Escherichia coli*, *Corynebacterium glutamicum*, and *Pseudomonas putida*. Researchers have increased GAA production by overexpressing enzymes related to GAA synthesis in these strains and improving the supply of their precursor arginine. However, despite extensive metabolic engineering, low final GAA yield and low overall production rate remain drawbacks.

[0005] Corynebacterium glutamicum is widely used in the production of amino acids and their derivatives. Guanidioacetic acid (GAA), a derivative of arginine, is also commonly selected as a substrate strain from Corynebacterium glutamicum. Previously, there were reports of GAA production via fermentation using two strains, Corynebacterium glutamicum ATCC13032 and Corynebacterium um glutamicum ATCC21831, but the yield was not very high. Possible reasons include: low GAA synthase activity or poor heterologous expression, insufficient supply of the GAA precursor arginine, and low carbon and nitrogen source utilization efficiency of the producing strains.

[0006] Therefore, it is crucial to develop green synthetic routes for the efficient synthesis of GAA using inexpensive sugar raw materials as substrates. Furthermore, improving the utilization of substrates by the producing bacteria is a research hotspot for increasing GAA yield. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention employs overexpression of guanidinoacetic acid synthase in different bacterial strains, followed by shake-flask fermentation to screen a high-yielding GAA-producing strain as its chassis strain. RBS optimization further enhances GAA synthase expression, promoting GAA production. Simultaneously, to reduce production costs and improve the utilization of carbon and nitrogen sources by the producing strain, the fermentation process is optimized to achieve one-step GAA production.

[0008] The first objective of this invention is to provide a recombinant bacterium, which uses Corynebacterium glutamicum as a host and undergoes any of the modifications described in (a) to (c):

[0009] (a) Overexpression of L-arginine-glycine amidotransferase AGAT;

[0010] (b) Overexpression of L-arginine-glycine amidotransferase AGAT with RBS;

[0011] (c) Based on (a) or (b), integrate expression of the ammonium transporter AmtB.

[0012] In one embodiment, the AGAT is derived from Actinokineospora terrae, Homo sapiens, or Mooreaproducens.

[0013] In one embodiment, the ammonium transporter AmtB is expressed by a strong promoter.

[0014] In one implementation, after knocking out AmtR, the ammonium transporter AmtB is integrated at the AmtR site.

[0015] In one implementation, the strong promoter includes the tacM promoter.

[0016] In one embodiment, the AGAT is derived from the terrestrial actinomycete Actinokineospora terrae.

[0017] In one embodiment, the nucleotide sequence of the AGAT is shown in SEQ ID NO.1.

[0018] In one embodiment, the sequence of the RBS is as shown in any one of SEQ ID NO.4 to SEQ ID NO.9.

[0019] In one embodiment, the sequence of the RBS is as shown in SEQ ID NO.6 or SEQ ID NO.8.

[0020] In one embodiment, the nucleotide sequence of the ammonium transporter AmtB is shown in SEQ ID NO.10.

[0021] In one embodiment, the Corynebacterium glutamicum is selected from Corynebacterium glutamicum ATCC13032 (C.gATCC13032) or Corynebacterium glutamicum var.CCTCCAB 2021051 (C.gAB2021051).

[0022] In one implementation, C.gAB2021051 is used as the host.

[0023] In one embodiment, the pXMJ19 plasmid is used as the expression vector.

[0024] The second objective of this invention is a method for producing guanidinoacetic acid by fermentation, wherein the method utilizes the aforementioned recombinant bacteria to produce guanidinoacetic acid by fermentation.

[0025] In one implementation, the method includes the following steps:

[0026] (1) Activate and culture the recombinant bacteria, and obtain the seed culture of the recombinant bacteria.

[0027] (2) The seed culture was transferred to a culture medium for fermentation to prepare guanidinoacetic acid.

[0028] In one embodiment, in step (1), the recombinant bacteria are inoculated into a seed culture medium and cultured at 28–32°C and 200–220 r / min for 20–30 h to prepare a seed solution.

[0029] In one embodiment, in step (2), the seed liquid is inoculated into the fermentation medium at an inoculation rate of 8-12% (v / v), and IPTG with a final concentration of 0.2-0.8 mM is added after 10-15 hours of inoculation. Glycine with a final concentration of 4-12 g / L is added after 0-10 hours of inoculation.

[0030] In one embodiment, in step (2), the culture is carried out at 28–32°C and 200–220 r / min for 72–96 h.

[0031] In one implementation, the dissolved oxygen level is maintained at 30%-40%.

[0032] A third object of the present invention is to provide the use of the recombinant bacteria or the method in the preparation of guanidinoacetic acid or products containing guanidinoacetic acid.

[0033] Beneficial effects:

[0034] This invention utilizes the overexpression of L-arginine-glycine amidosyltransferase AGAT from different sources in various Corynebacterium glutamicum strains to produce GAA via fermentation. A high-GAA-producing strain, C.gAB2021051 / pXMJ19-AGAT, was screened, achieving a yield of 8.63 g / L, overcoming the low efficiency of guanidinoacetic acid (GAA) production via Corynebacterium glutamicum fermentation. Further optimization of the Recombinant Biosynthesis System (RBS) was employed to optimize AGAT enzyme expression, enhancing GAA production to over 9 g / L. Knocking out AmtR relieved its feedback inhibition on the ammonium transporter AmtB, and overexpression of AmtB with a strong promoter-initiated expression yielded a recombinant strain with a GAA yield of 12.5 g / L. The resulting recombinant strain was used for guanidinoacetic acid (GAA) fermentation. By enhancing the utilization of carbon and nitrogen sources, optimizing the timing and amount of glycine addition, and optimizing dissolved oxygen control, a yield of 27.2 g / L GAA was achieved in a 5L tank after 72 hours of fermentation. Attached Figure Description

[0035] Figure 1 Validation results of bacteria P constructed from recombinant plasmid pXMJ19-AGAT;

[0036] Figure 2 Validation results of recombinant plasmid pXMJ19-AGAT by electroporation of C. gATCC13032 and C. gAB2021051 bacteria;

[0037] Figure 3 Results of the improvement of AGAT expression levels by different RBSs;

[0038] Figure 4 : Validation results of bacteria P constructed from recombinant plasmid pK18-AmtB. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] In the quantitative experiments described below, three replicates were performed, and the average value of the results was taken.

[0043] In the following examples, the host used to construct the recombinant plasmid was E. coli BL21(DE3), purchased from Benabio. The pXMJ19 plasmid and pK18mobSacB plasmid were purchased from BioVector Plasmid Vector Culture Collection Center.

[0044] In the following examples, the hosts used for the expression vector were Corynebacterium glutamicum ATCC13032 (C.gATCC13032) and Corynebacterium glutamicum var. CCTCAB 2021051 (C.gAB2021051).

[0045] The preparation and chemical transformation methods of competent E. coli involved in the following examples are as follows:

[0046] E. coli competent cells were prepared using the TakaRa Competent Cell Preparation Kit, with detailed instructions following the manufacturer's manual. Transformations were performed by heat shock at 42°C into E. coli BL21. Positive transformants were obtained after selection using antibiotic resistance plates, and plasmids were extracted for PCR verification and sequencing verification by Genewiz Biotechnology Co., Ltd.

[0047] The following examples illustrate the methods for extracting relevant plasmids:

[0048] When extracting plasmids from recombinant strains of Escherichia coli, centrifuge the culture solution of an appropriate concentration, remove the supernatant, and extract the plasmid using the Jereh mini plasmid extraction kit. For detailed operation, please refer to the instruction manual.

[0049] The PCR amplification system in the following examples was as follows: Primer F 1.0 μL, Primer R 1.0 μL, Template 1.0 μL, PhantaR Max (p515) DNA polymerases 25 μL, Nuclease-free water 22 μL.

[0050] The PCR amplification program in the following examples is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; annealing temperature is generally set to 58-60℃ for 30-60 s; 72℃ extension is set to amplify 1500 bp of gene per minute; denaturation to extension program for 30 cycles; 72℃ extension for another 5 min; storage at 4℃.

[0051] The preparation of competent Corynebacterium cells involved in the following examples: Bacterial culture was taken from frozen tubes using an inoculation loop, streaked onto BHI solid plates for activation, and incubated at 30°C for 16-24 hours. Single colonies from fresh plates were then inoculated into BHI liquid culture medium and incubated at 30°C and 180 rpm. -1 Incubate in a rotary shaker for 16-24 hours. Transfer to competent culture medium at an inoculum rate of 1-2% (v / v) and incubate at 30°C and 180 rpm. -1 Cultured in a rotary shaker, OD was tracked and measured. 600 , waiting for OD 600 When the bacterial culture reaches approximately 0.9, immediately place it in an ice bath. After 15 minutes, centrifuge it at 4°C and 6000 rpm using a refrigerated centrifuge. -1 Centrifuge for 10 min to collect the bacterial cells, then add 2 ml of pre-chilled 10% glycerol to wash the cells. Gently pipette the suspended cells and centrifuge again to collect the cells. Repeat this washing process 3 times. Finally, add an appropriate amount of 10% glycerol to suspend the cells. Aliquot 80 μL of cells into a 1.5 ml centrifuge tube. The cells can be used directly for electroporation or stored at -70°C.

[0052] The culture media involved in the following examples:

[0053] LB liquid medium (g / L): 5 yeast extract, 10 tryptone, 10 sodium chloride.

[0054] LB solid medium: Add 1.5-2.0% agar powder to the LB liquid medium.

[0055] BHI liquid culture medium (g / L): 39 brain and heart infusion broth.

[0056] BHI solid medium (g / L): Based on BHI liquid medium, add 1.5-2.0% agar powder.

[0057] Corynebacterium competent culture medium (g / L): 10 peptone, 5 yeast extract, 10 sodium chloride, 5 glucose, 1 ml Tween-80, 30 glycine.

[0058] LBGS liquid sucrose medium (g / L) for screening Corynebacterium glutamicum: 10 peptone, 5 yeast extract, 10 NaCl, 10% sucrose.

[0059] Seed culture medium (g / L): 20g yeast extract, 20g ammonium sulfate, 1.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 1g calcium carbonate, 50g glucose.

[0060] Fermentation medium (g / L): 10 yeast extract, 40 ammonium sulfate, 1.5 potassium dihydrogen phosphate, 1 potassium chloride, 0.5 magnesium sulfate heptahydrate, 0.02 ferrous sulfate heptahydrate, 0.02 manganese sulfate monohydrate, 2 calcium carbonate, 100 glucose.

[0061] The detection methods involved in the following embodiments are as follows:

[0062] Guanidinoacetic acid was detected using Agilent high-performance liquid chromatography (HPLC) with a UV absorber. The column was a WatersXBridge BEH Amide 5μm column (4.6mm × 250mm). The mobile phase was 30% acetonitrile aqueous solution, the detection wavelength was 210nm, and the flow rate was 0.6mL / min.

[0063] Arginine was detected using an Agilent C18 column, 5 μm, 4.6 × 250 mm; the flow rate was 1.0 mL / min. -1 Column temperature 40℃; Detection wavelength 338nm; Mobile phase: Phase A: 8.0g sodium acetate (13.3g sodium acetate trihydrate) dissolved in 1000mL water, 225μL triethylamine added, pH adjusted to 7.20±0.05 with 5% acetic acid, and finally 5mL tetrahydrofuran added and mixed; Phase B: 6.0g sodium acetate dissolved in 200mL water, pH adjusted to 7.20±0.05 with 5% acetic acid, this solution added to 400mL HPLC-grade methanol and 400mL HPLC-grade acetonitrile, and mixed.

[0064] Example 1: Construction of recombinant bacteria Cg ATCC13032 / pXMJ19-AGAT and Cg AB2021051 / pXMJ19-AGAT

[0065] The specific steps are as follows:

[0066] 1. Construction of recombinant plasmid pXMJ19-AGAT

[0067] To obtain AGAT with higher enzyme activity, the Brenda database and NCBI BLAST were searched to obtain the gene sequences encoding AGAT from *Actinokineospora terrae*, *Homo sapiens*, and *Mooreaproducens* (as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, named AtAGAT, HsAGAT, and MpAGAT), which were then synthesized by Suzhou Genewiz Company. The three synthesized agat gene fragments were ligated to the linearized plasmid pXMJ19 obtained by primers P1 / P2 using the ClonExpress II One Step Cloning Kit (Novizan), and transformed into E. coli BL21(DE3) competent cells to obtain transformants. The transformants were plated on LB agar medium containing 10 mg / L chloramphenicol and incubated at 37°C for 12 h. Positive colonies were picked, and single colonies were verified by Taq DNA polymerase using primers P3 / P4 (see [link to Taq DNA polymerase]). Figure 1 A single positive colony with the target band size was inoculated into a vial containing LB liquid medium and cultured for 12 hours. Plasmids were extracted and sent to Genewiz for sequencing. The sequencing results were correct, and the recombinant plasmids pXMJ19-AtAGAT, pXMJ19-HsAGAT, and pXMJ19-MpAGAT were successfully constructed.

[0068] The primer sequences involved are as follows:

[0069] P1: 5'-CAGGAAACAGAATTAATTAAGCTT-3';

[0070] P2: 5'-AATTCAGCTTGGCTGTTTTGGC-3'.

[0071] P3: 5'-CTGGCAAATATTCTGAAATGAGCTG-3';

[0072] P4: 5'-GCAGTTCCCTACTCTCGCAT-3'.

[0073] 2. Overexpression of AGAT enzyme in Cg ATCC13032 and Cg AB2021051

[0074] Cg ATCC13032 and Cg AB2021051 competent cells were prepared according to the above method. The recombinant plasmid obtained in step 1 was electroporated into the competent cells. Electroporation was performed at 1.8 kV for 5 ms. After electroporation, 800 μl of BHI medium was added for recovery, followed by heat shock at 46°C for 5 min, and then incubation at 30°C and 200 rpm for 2 h. Transformants were plated on solid medium containing 10 mg / L chloramphenicol-resistant BHI and incubated at 30°C. Positive colonies were picked, and single colonies were verified by Taq DNA polymerase using primers P3 / P4 from step 1 (see [link to step 1]). Figure 2 The recombinant bacteria Cg ATCC13032 / pXMJ19-AtAGAT, Cg ATCC13032 / pXMJ19-HsAGAT, Cg ATCC13032 / pXMJ19-MpAGAT, Cg AB2021051 / pXMJ19-AtAGAT, Cg AB2021051 / pXMJ19-HsAGAT, and Cg AB2021051 / pXMJ19-MpAGAT were obtained respectively.

[0075] Example 2: Production of guanidinoacetic acid by recombinant bacteria in shake flasks

[0076] Method for producing GAA by shake-flask fermentation: The recombinant bacteria constructed in Example 1 and the starting strains C.gATCC13032 and CgAB2021051 were activated by streaking on BHI solid medium, and single colonies were picked and inoculated into seed medium. The culture was carried out at 30°C for 24 h to prepare seed liquid.

[0077] The prepared seed culture was transferred to a 250 ml shake flask containing 30 ml of fermentation medium at an inoculation rate of 10% (V / V). The culture was carried out in a reciprocating shaker at 30°C and 220 rpm. After 12 h of fermentation, 0.5 mM IPTG was added and the culture was continued for another 84 h.

[0078] After fermentation was completed, the fermentation broth was collected, and the contents of arginine and guanidinoacetic acid were detected by HPLC. The results are shown in Table 1.

[0079] Table 1. Content of guanidinoacetic acid produced by shake-flask fermentation with different recombinant bacteria.

[0080]

[0081] Fermentation revealed that Cg ATCC13032 produced only 0.41 g / L arginine, while Cg AB202105 produced 19.2 g / L arginine. Arginine, as a substrate for GAA synthesis, is crucial for its accumulation. Both strains exhibited GAA production capacity after AGATase overexpression. In Cg AB202105, AGATase overexpression resulted in higher GAA yields than the AGATase-overexpressing recombinant strain Cg ATCC13032. Furthermore, fermentation results showed that the AGATase derived from *Actinokineospora terrae* was the most effective, with Cg AB202105 / pXMJ19-AtAGAT accumulating 8.63 g / L GAA, 24 times the yield of Cg ATCC13032 / pXMJ19-AtAGAT. This further demonstrates that arginine accumulation promotes GAA synthesis. In summary, C.gAB202105 was selected as the production strain of guanidinoacetic acid, and the recombinant strain CgAB202105 / pXMJ19-AtAGAT was used for subsequent research, which was named CgAB-A1 here.

[0082] Example 3: RBS optimization of GAA synthase AGAT expression

[0083] The above shake-flask fermentation for GAA production revealed that arginine, an important substrate for GAA production from Cg AB-A1 fermentation, was not fully utilized by the AGAT enzyme. AGAT expression in Cg AB-A1 was found to be poor. Therefore, RBS optimization was used to improve the expression level of the GAA synthase AGAT.

[0084] The specific steps are as follows:

[0085] 1. Design of RBS expression regulation

[0086] Upload the tac promoter initiation sequence from the pXMJ19 plasmid used for gene expression and the 5' initiation sequence of the AtAGAT gene to the Salis Lab website (http: / / salislab.net / software / ). By inputting the translation initiation efficiency (RBS intensity) and confirming the host bacteria used, you can obtain RBS sequences with different expression intensities. Select the six RBS sequences with different expression intensities shown in Table 2.

[0087] Table 2 RBS sequences

[0088]

[0089]

[0090] 2. Construct recombinant bacteria carrying different RBS.

[0091] Using primer pairs P5 and P6, P7 and P8, P9 and P10, P11 and P12, P13 and P14, and P15 and P16 respectively, and using pXMJ19-AtAGAT obtained in Example 1 as a template, reverse PCR amplification was performed using the above PCR amplification system and PCR amplification reaction conditions to obtain linearized pXMJ19-AtAGAT carrying different RBS. After digestion with DpnI, the cells were transformed into competent E. coli BL21(DE3) bacterial cells. Clones with AGAT activity were screened on LB solid plates (containing 10 mg / L chloramphenicol). Plasmids were extracted from the cloned colonies, and DNA sequencing confirmed that pXMJ19-AtAGAT carrying different RBS was successfully constructed.

[0092] P5: 5'-GAATTAATTAAGCTTAAAGGAGGAAAATCTACTAGATGCGCACCGAT-3';

[0093] P6: 5'-ATCGGTGCGCATCTAGTAGATTTTCCCTCCTTTAAGCTTAATTAATTC-3'.

[0094] P7: 5'-GAATTAATTAAGCTTAAAGGACAAAAACTACTAGATGCGCACCGAT-3';

[0095] P8: 5'-ATCGGTGCGCATCTAGTAGTTTTTGTCCTTTAAGCTTAATTAATTC-3'.

[0096] P9: 5'-GAATTAATTAAGCTTAAAGGAGGATTAGTACTAGATGCGCACCGAT-3';

[0097] P10: 5'-ATCGGTGCGCATCTAGTACTAATCCTCCTTTAAGCTTAATTAATTC-3'.

[0098] P11: 5'-GAATTAATTAAGCTTGAAAAGGACATGAACGTACTAGATGCGCACCGAT-3';

[0099] P12: 5'-ATCGGTGCGCATCTAGTACGTTCATGTCCTTTCAAGCTTAATTAATTC-3'.

[0100] P13: 5'-GAATTAATTAAGCTTAAAGGAGGACAACTATACTAGATGCGCACCGAT-3';

[0101] P14: 5'-ATCGGTGCGCATCTAGTATAGTTGTCCTCCTTTAAGCTTAATTAATTC-3'.

[0102] P15: 5'-GAATTAATTAAGCTTGAAGGAGATATACCTACTAGATGCGCACCGAT-3';

[0103] P16: 5'-ATCGGTGCGCATCTAGTAGGTATATCTCCTTCAAGCTTAATTAATTC-3'.

[0104] pXMJ19-AtAGAT carrying different RBS was electroporated into Cg AB2021051 competent cells. Single colonies were verified by Taq DNA polymerase using primers P3 / P4 from Example 1, resulting in recombinant bacteria carrying different RBS: Cg AB-A1-R1, Cg AB-A1-R2, Cg AB-A1-R3, Cg AB-A1-R4, Cg AB-A1-R1, and Cg AB-A1-R6.

[0105] 3. Verify the ability of recombinant bacteria with different RBS to produce GAA through fermentation.

[0106] Recombinant bacteria carrying different RBS were fermented to produce GAA according to the shake-flask fermentation method described in Example 2. After fermentation, the fermentation broth was collected, and the contents of arginine and guanidinoacetic acid were detected by HPLC. The results are shown in Table 3. Figure 3 As shown.

[0107] Table 3. GAA production capacity of different RBS recombinant bacteria during fermentation.

[0108]

[0109]

[0110] Fermentation revealed that recombinant bacteria carrying different RBS strains exhibited varying GAA production capabilities, with RBS3 showing the best performance. C.gAB-A1-R3 produced 10.21 g / L of GAA, representing an 18.3% increase compared to CgAB-A1. CgAB-A1-R3 was then used in subsequent GAA production and named CgAB-A2.

[0111] Example 4: Optimizing fermentation conditions to promote GAA production

[0112] The specific steps are as follows:

[0113] 1. Integrated expression of ammonium transporter AmtB promotes nitrogen source absorption and utilization.

[0114] To promote nitrogen uptake by the producing bacteria, a single-copy ammonium transporter AmtB was integrated and expressed under the tacM promoter. At the same time, the feedback inhibition of the N transcription factor AmtR was relieved by knocking out AmtR. Therefore, AmtB was integrated into the AmtR site to achieve the integration of AmtB and the knockout of AmtR. First, the AmtB gene fragment was obtained by PCR using primer pairs P17 and P18 with the *Corynebacterium glutamicum* genome as a template. The fragment was then ligated to the linearized pXMJ19 vector using EcoRI and Hind III restriction sites to obtain pXMJ19-tacM-AmtB. Next, the tacM-AmtB fragment was obtained by PCR using primer pairs P19 and P20 with pXMJ19-tacM-AmtB as a template. Then, the tacM-AmtB fragment was obtained by PCR using primer pairs P21 and P22, and P23 and P24 with the *Corynebacterium glutamicum* genome as a template. A fragment with 500bp homologous arms upstream and downstream of the integration site was obtained by PCR using primer pairs P21 and P24. Finally, the tacM-AmtB fragment with 500bp homologous arms upstream and downstream of the integration site was obtained by three-fragment fusion PCR using primers P21 and P24. The obtained fragment was then simultaneously ligated to the pK18mobsacB vector using EcoRI and Hind III restriction sites. After enzyme digestion (III), the nucleic acid was purified by gel electrophoresis and ligated with homologous recombinase at 37°C. The ligation product was then transferred into E. coli BL21(DE3). Transformants were picked and cultured for 9 hours, followed by plasmid extraction and PCR verification (e.g., ...). Figure 4 The extracted plasmid was sent to Suzhou Genewiz for sequencing. DNAMAN software analysis confirmed the sequence was correct, indicating successful construction of pK18-AmtB.

[0115] P17: 5'-CCCAAGCTTAAAGGAGGGAAATCATGAGCGCAGATCAAATCGC-3';

[0116] P18: 5'-CCGGAATTCTTAGTGGTGGTGGTGGTGGTGACGAATTTCCGGCCCG-3'.

[0117] P19: 5'-TTGACAATTAATCATCGGCTCGTATAATGTG-3';

[0118] P20: 5'-TTAACGAATTTCCGGCCCTGTGGTG-3'.

[0119] P21: 5'-CCGGAATTCGCGGCGATCGCTGAAGTAGA-3';

[0120] P22: 5'-AGGCCAAAACTTTAAGGAAGTAGAATTACGCTTTGACAATTAATCAT-3'.

[0121] P23: 5'-CCGGAAATTCGTTAACCTCGCTGTATTATCACTTCCCG-3';

[0122] P24: 5'-CCCAAGCTTTCAAGCGTTTTTTCGACCCGG-3'.

[0123] The pK18-amtB plasmid was electroporated into Cg AB-A2. After colony growth, identification, and amplification, the culture was transferred to LBGS liquid sucrose medium for screening. The selected bacterial culture was first streaked onto antibiotic-free BHI plates. Then, the colonies that grew on the antibiotic-free plates were streaked onto plates containing Kan+BHI. Colonies that grew on Kan+BHI plates but not on antibiotic-free BHI plates were selected for further culture. The genome was successfully sequenced, and strain Cg AB-A3 was obtained.

[0124] The ability of Cg AB-A3 to produce GAA was verified by the shake-flask fermentation method in Example 2. After 72 hours of fermentation, the bacteria grew normally. The residual glucose concentration at the end of fermentation was lower than that of Cg AB-A2, and the GAA yield was 12.5 g / L, which was 22.4% higher than that of Cg AB-A2.

[0125] 2. Effect of substrate glycine addition time on GAA yield

[0126] To investigate whether the timing of glycine addition affects GAA yield, 8 g / L glycine was added to the transfer shake-flask fermentation medium at 0 h, 2 h, 4 h, 6 h, and 8 h. Fermentation-related parameters were measured every 12 h, and the OD value of the bacterial culture was determined using a UV spectrophotometer. 562 The absorbance at nm was used to determine the amount of residual glucose using an SBA biosensor, and the yield of GAA in the fermentation broth was determined using HPLC.

[0127] The yield of guanidinoacetic acid was determined by adding 8 g / L of glycine over 6 hours, resulting in a maximum yield of 14.51 g / L. The residual sugar content and OD were also measured. 562 Its growth was found to be good.

[0128] 3. Effect of substrate glycine addition on GAA yield

[0129] Because excessively high glycine concentrations can be toxic to cell growth, while excessively low concentrations result in insufficient substrate and reduced GAA accumulation, determining the optimal amount of glycine added is crucial for GAA accumulation.

[0130] To investigate whether the timing of glycine addition affected GAA yield, glycine concentrations of 4 g / L, 6 g / L, 8 g / L, 10 g / L, and 12 g / L were added during 6 hours of transfer shake-flask fermentation. Fermentation-related parameters were measured every 12 hours, and the OD value of the bacterial culture was determined using a UV spectrophotometer. 562 The absorbance at nm was used to determine the amount of residual glucose using an SBA biosensor, and the yield of GAA in the fermentation broth was determined using HPLC.

[0131] The results showed that the highest GAA yield (17.32 g / L) was achieved when 10 g / L glycine was added. The residual sugar content and OD were also measured. 562 Its growth was found to be good.

[0132] Example 5: Production of guanidinoacetic acid using recombinant strain Cg AB-A35L fermenter

[0133] The specific steps are as follows:

[0134] 1. Dissolved oxygen control optimization

[0135] Dissolved oxygen (DO) levels in the fermenter affect cell growth and GAA synthesis, with rotation speed and aeration rate being two major factors influencing DO levels. It has been reported that the optimal DO level for *Corynebacterium glutamicum* fermentation of amino acids is 30%-40%. Therefore, this invention utilizes a method of coupling rotation speed with DO and manually increasing aeration rate to maintain the DO level at 30%-40% during fermentation, exploring the impact of DO control on *Corynebacterium glutamicum* cell growth and GAA synthesis.

[0136] 2. Fermentation of engineered bacteria in a 5L tank to produce guanidinoacetic acid

[0137] Seed culture: A single colony of the recombinant bacteria Cg AB-A3 prepared in Example 4 was picked from the activation plate and inoculated into 10 ml of BHI medium. The culture was incubated at 30°C and 200 rpm for 24 h. Then, at an inoculation rate of 2% (v / v), it was transferred to a 250 ml shake flask containing 30 ml of seed medium and incubated at 30°C and 200 rpm for 24 h to prepare the primary seed culture. The primary seed culture was then transferred at a transfer rate of 10% (v / v) to a 1000 ml shake flask containing 200 ml of seed medium and incubated at 30°C and 200 rpm for 20 h to prepare the secondary seed culture.

[0138] Fermentation culture in a 5L fermenter: The cultured secondary seed culture was transferred to a 5L fermenter containing 2L of fermentation medium. Fermentation was carried out at 30°C, and the dissolved oxygen level was maintained at 30%-40% during the fermentation process by using a combination of rotation speed and dissolved oxygen and manual increase of aeration rate. Fermentation was carried out for 96 hours. IPTG at a final concentration of 0.5mM was added at 15 hours of culture for induction. After 20 hours of fermentation, glycine at a flow rate of 10ml / min was added to the mother liquor at a concentration of 200g / L.

[0139] After fermentation, the fermentation broth was collected, and the contents of arginine and guanidinoacetic acid were determined by HPLC.

[0140] The results showed that the recombinant strain Cg AB-A3 could accumulate 27.2 g / L of guanidinoacetic acid under the above fermentation process and conditions, achieving one-step fermentation of sugar raw materials to produce guanidinoacetic acid without the addition of arginine.

[0141] 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 recombinant bacterium, characterized in that, The recombinant bacteria is Corynebacterium glutamicum. Corynebacterium glutamicum Using var. CCTCC AB 2021051 as the host, the following modifications are made: Overexpression of L-arginine-glycine amidosyltransferase AGAT with RBS, the sequence of which is shown in SEQ ID NO.6; after knocking out AmtR, integration and expression of the ammonium transporter AmtB at the AmtR site. The AGAT is derived from *Zoospora geysersis*. Actinokineospora terrae, The nucleotide sequence of the gene encoding AGAT is shown in SEQ ID NO.

1.

2. The recombinant bacteria according to claim 1, characterized in that, The ammonium transporter AmtB is expressed by a strong promoter.

3. A method for producing guanidinoacetic acid by fermentation, characterized in that, The method involves fermenting guanidinoacetic acid using the recombinant bacteria described in claim 1 or 2, and the method includes the following steps: (1) Activate and culture the recombinant bacteria, and obtain the seed culture of the recombinant bacteria. (2) The seed culture was transferred to a culture medium for fermentation to prepare guanidinoacetic acid.

4. The method according to claim 3, characterized in that, In step (2), the seed liquid is inoculated into the culture medium. After 10-15 h of inoculation, IPTG with a final concentration of 0.2-0.8 mM is added. After 0-10 h of inoculation, glycine with a final concentration of 4-12 g / L is added.

5. The method according to claim 4, characterized in that, Maintain dissolved oxygen levels at 30%-40%.

6. The use of the recombinant bacteria according to claim 1 or 2 in the preparation of products containing guanidinoacetic acid.

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