A method for producing creatine by fermenting carbohydrate raw materials using microorganisms

Abstract

The present invention discloses a method for producing creatine by fermenting saccharide raw materials using microorganisms, and belongs to the field of genetic engineering technology. L-arginine-glycine amidinotransferase and guanidineacetic acid N-methyltransferase with high enzyme activity are overexpressed in Corynebacterium glutamicum var.CCTCC AB 2021051 using the pXMJ19 plasmid to construct a creatine synthesis pathway. The degradation of SAH is promoted by integrating the expression of the SAHase enzyme, thereby relieving the inhibitory effect of SAH on the key enzymes in creatine production and promoting the regeneration of SAH substrate SAM. The engineered bacteria are scaled up in a 5L tank, fermented for 60 hours, and the creatine yield reaches 14.2g / L. The production method of creatine by the present invention has the advantages of low production cost and high efficiency, which is conducive to scaled-up production.

Description

Technical Field

[0001] The invention relates to a method for producing creatine by fermenting saccharide raw materials using microorganisms, and belongs to the technical field of genetic engineering. Background Art

[0002] Creatine (CR), also known as N-methylguanidinoacetic acid, is an amino acid derivative that participates in ATP homeostasis in all cell populations and has the ability to buffer high-energy phosphates. Creatine is crucial for energy homeostasis in the brain, as creatine deficiency can lead to creatine deficiency syndrome (CDS), which can cause mental retardation and language delay. Evidence suggests that creatine, as a very safe medical and nutritional supplement, has beneficial effects on a variety of conditions, including muscular dystrophy, autism, cancer, diabetes, and metabolic disorders.

[0003] Creatine is obtained from the diet and endogenously synthesized. In vertebrates, creatine synthesis involves two stages: 1) guanidinoacetic acid is produced from arginine and glycine by the enzyme L-arginine:glycine amidinotransferase (AGAT, EC: 2.1.4.1); and 2) guanidinoacetic acid and S-adenosylmethionine are converted to creatine by the enzyme guanidinoacetic acid N-methyltransferase (GAMT, EC: 2.1.1.2). Creatine can also be supplemented through the consumption of fresh fish, meat, and dairy products, but in very small quantities. For those with creatine deficiency, additional supplementation through nutritional supplements is necessary.

[0004] Currently, the industrial production of creatine relies primarily on chemical methods. These traditional chemical preparation methods often involve the highly toxic cyanamide or have suboptimal creatine yields, resulting in significant safety risks and high production costs. Leakage of highly toxic substances can easily cause casualties in large-scale industrial production, and they are also inconsistent with environmental protection trends. To improve economic efficiency and safety, manufacturers are seeking biological alternatives to chemical synthesis for creatine production. Furthermore, as a widely used food additive, the search for a biological alternative to chemical production has become a research hotspot. Summary of the Invention

[0005] The present invention provides a recombinant strain capable of efficiently and cost-effectively synthesizing creatine and a method for its construction, as well as a method for high creatine production through one-step fermentation using glucose as a substrate. To address the above technical problems, the present invention provides a recombinant Corynebacterium glutamicum that simultaneously overexpresses L-arginine-glycine amidinotransferase and guanidineacetate N-methyltransferase by screening for key enzymes for creatine synthesis. The recombinant Corynebacterium glutamicum also incorporates S-adenosylhomocysteine ​​hydrolase, which relieves the feedback inhibition of the byproduct SAH on the creatine production enzyme and promotes SAH regeneration of the substrate SAM.

[0006] The present invention provides a recombinant Corynebacterium glutamicum, wherein the recombinant Corynebacterium glutamicum overexpresses L-arginine-glycine amidinotransferase AtAGAT derived from Actinokineospora terrae and guanidineacetate N-methyltransferase HsGAMT derived from Homosapiens; or the recombinant Corynebacterium glutamicum overexpresses L-arginine-glycine amidinotransferase AtAGAT derived from Actinokineospora terrae, guanidineacetate N-methyltransferase HsGAMT derived from Homosapiens and S-adenosylhomocysteine ​​hydrolase SAHase derived from Saccharomyces cerevisiae.

[0007] In one embodiment of the present invention, the amino acid sequence of the L-arginine-glycine amidinotransferase AtAGAT is shown in SEQ ID NO.1, the amino acid sequence of the guanidineacetic acid N-methyltransferase HsGAMT is shown in SEQ ID NO.2, and the amino acid sequence of the S-adenosylhomocysteine ​​hydrolase SAHase is shown in SEQ ID NO.3.

[0008] In one embodiment of the present invention, the nucleotide sequence encoding the L-arginine-glycine amidinotransferase AtAGAT is shown as SEQ ID NO.4, the nucleotide sequence encoding the guanidineacetic acid N-methyltransferase HsGAMT is shown as SEQ ID NO.5, and the nucleotide sequence encoding the S-adenosylhomocysteine ​​hydrolase SAHase is shown as SEQ ID NO.6.

[0009] In one embodiment of the present invention, pXMJ19 plasmid is used as an expression vector.

[0010] In one embodiment of the present invention, Corynebacterium glutamicum var. CCTCC AB 2021051 (Cg AB2021051) or Corynebacterium glutamicum ATCC14067 (C.gATCC14067) is used as the expression host.

[0011] In one embodiment of the present invention, Cg AB2021051 is used as the creatine production strain, and pXMJ19 plasmid is used as the expression vector.

[0012] In one embodiment of the present invention, the S-adenosylhomocysteine ​​hydrolase SAHase is integrated into the host genome, and the integration site is a pseudogene site (C629_RS09275); the integrated expression of the S-adenosylhomocysteine ​​hydrolase SAHase promotes the hydrolysis of SAH, relieves its feedback inhibition on guanidineacetate N-methyltransferase, and promotes the regeneration of SAH to the expensive substrate SAM, thereby reducing the cost of exogenous addition.

[0013] The present invention also provides a method for preparing creatine by fermentation while promoting the regeneration of S-adenosylmethionine (SAM), wherein the method comprises fermenting any of the above-mentioned recombinant Corynebacterium glutamicum to prepare creatine.

[0014] In one embodiment of the present invention, the method uses glucose as a raw material and utilizes the above-mentioned recombinant Corynebacterium glutamicum to ferment and produce creatine.

[0015] In one embodiment of the present invention, the method is to add the above-mentioned recombinant Corynebacterium glutamicum to a seed culture medium, and culture it at 30° C. and 220 rpm for 24 hours to prepare a seed solution.

[0016] In one embodiment of the present invention, the fermentation conditions are as follows: the seed liquid is inoculated into the fermentation medium at an inoculum size of 8-12% (v / v), IPTG is added at a final concentration of 0.2-0.8 mM 10-15 hours after inoculation, and glycine is added at a final concentration of 2-12 g / L 0-10 hours after inoculation, and the dissolved oxygen level is maintained at 30%-40% during the fermentation process.

[0017] In one embodiment of the present invention, the fermentation conditions are as follows: the seed solution prepared above is inoculated into the fermentation medium at an inoculation rate of 10% (v / v), and the fermentation is carried out at 30° C. and 220 rpm for 60 h.

[0018] In one embodiment of the present invention, when the seed solution is transferred to the fermentation medium for fermentation culture, IPTG is added 12 hours after inoculation to a final concentration of 0.5 mM.

[0019] In one embodiment of the present invention, when the seed solution is transferred to a fermentation medium for fermentation culture, glycine is added 0 to 10 hours after inoculation, and the final concentration of the added glycine is 2 to 12 g / L.

[0020] In one embodiment of the present invention, during tank culture, the dissolved oxygen level during the fermentation process is maintained at 30%-40% by further adopting a method of speed coupling dissolved oxygen and manually increasing ventilation volume.

[0021] The present invention also provides the use of the above-mentioned S-adenosylhomocysteine ​​hydrolase SAHase derived from Saccharomyces cerevisiae in improving the fermentation production of creatine by recombinant Corynebacterium glutamicum while promoting the regeneration of the substrate S-adenosylmethionine SAM. The recombinant Corynebacterium glutamicum overexpresses L-arginine-glycine amidinotransferase AtAGAT derived from Actinokineospora terrae, guanidineacetic acid N-methyltransferase HsGAMT derived from Homo sapiens, and S-adenosylhomocysteine ​​hydrolase SAHase derived from Saccharomyces cerevisiae.

[0022] In one embodiment of the present invention, the amino acid sequence of the L-arginine-glycine amidinotransferase AtAGAT is shown in SEQ ID NO.1, the amino acid sequence of the guanidineacetic acid N-methyltransferase HsGAMT is shown in SEQ ID NO.2, and the amino acid sequence of the S-adenosylhomocysteine ​​hydrolase SAHase is shown in SEQ ID NO.3.

[0023] In one embodiment of the present invention, the nucleotide sequence encoding the L-arginine-glycine amidinotransferase AtAGAT is shown as SEQ ID NO.4, the nucleotide sequence encoding the guanidineacetic acid N-methyltransferase HsGAMT is shown as SEQ ID NO.5, and the nucleotide sequence encoding the S-adenosylhomocysteine ​​hydrolase SAHase is shown as SEQ ID NO.6.

[0024] The present invention also provides the use of the recombinant Corynebacterium glutamicum in the simultaneous preparation of S-adenosylmethionine SAM and creatine, or in the preparation of a product containing S-adenosylmethionine SAM, or in the preparation of a product containing creatine.

[0025] In one embodiment of the present invention, the products include but are not limited to food, medicine, and health care products.

[0026] Beneficial effects

[0027] The invention constructs a recognized safe strain Cg AB-AG-2 which uses glucose as a substrate to synthesize creatine by a one-step fermentation method.

[0028] (1) Using the GRAS strain Corynebacterium glutamicum var.CCTCC AB2021051, which is a high-arginine-producing strain, as the starting strain, we screened for creatine synthase and optimal chassis strains with high enzyme activity, and constructed a recombinant Corynebacterium glutamicum Cg AB-AG-1 with L-arginine-glycine amidinotransferase and guanidineacetic acid N-methyltransferase activities. This method eliminates the need to add expensive L-arginine and guanidineacetic acid as raw materials, and can directly use cheap glucose as raw material for creatine production, thereby effectively reducing production costs.

[0029] (2) In order to relieve the inhibitory effect of SAH, a byproduct of creatine production, on guanidineacetic acid N-methyltransferase, the present invention integrated the expression of SAH hydrolase SAHase. While relieving feedback inhibition, the byproduct S-adenosylhomocysteine ​​SAH was used to regenerate the expensive substrate SAM, thereby obtaining the recombinant bacterium Cg AB-AG-2, which further enhanced creatine production.

[0030] (3) When the method provided by the present invention is used for fermentation tank culture, the recombinant bacteria Cg AB-AG-2 can accumulate 14.2 g / L of creatine in the fermentation broth of the fermentation tank, realizing the direct synthesis of creatine from glucose by a one-step fermentation method without the addition of arginine or guanidine acetate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Construction and verification results of the recombinant plasmid pXMJ19-AtAGAT.

[0032] Figure 2 : GAMT expression results from different sources in Cg AB2021051.

[0033] Figure 3 : Construction and verification results of the recombinant plasmid pXMJ19-AtAGAT-HsGAMT. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific examples so that those skilled in the art can better understand the present invention and can be implemented, but the examples are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0035] In the following examples, the host used to construct the recombinant plasmids was Escherichia coli BL21 (DE3), purchased from Bena Biotechnology, and the pXMJ19 plasmid and pK18mobSacB plasmid were purchased from the BioVector Plasmid Vector Bacteria Cell Gene Collection Center.

[0036] The host used for the expression vector in the following examples is Corynebacterium glutamicum var. CCTCC AB 2021051 (Cg AB2021051), which is described in the paper "Improvement of the intracellular environment for enhancing L-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply";

[0037] The Corynebacterium glutamicum ATCC14067 (Cg ATCC14067) was purchased from Xinyang Laiyao Biotechnology Co., Ltd.

[0038] The competent preparation of E. coli and the chemical transformation method involved in the following examples are as follows:

[0039] Prepare competent E. coli cells using the TakaRa Competent Cell Preparation Kit (refer to the manufacturer's instructions for detailed procedures). Heat shock the cells at 42°C and transform them into E. coli BL21. Positive transformants were screened on antibiotic-resistant plates. Plasmids were extracted and verified by PCR and sent to GeneWeichi Biotechnology for sequencing verification.

[0040] The extraction method of the relevant plasmids involved in the following examples:

[0041] When extracting plasmids from recombinant strains of Escherichia coli, centrifuge the culture solution of appropriate concentration and remove the supernatant. Use the Jereh mini plasmid extraction kit for extraction. Refer to the instructions for detailed procedures.

[0042] The amplification system of PCR in the following examples is:

[0043] Primer F 1.0μL, Primer R 1.0μL, Template 1.0μL, PhantaR Max(p515) DNApolymerases 25μL, Nuclease-free water 22μL.

[0044] The PCR amplification procedure in the following examples is:

[0045] Pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing temperature is generally set at 58-60°C for 30-60 seconds; extension at 72°C is set according to the time for amplifying 1500bp of gene per minute; denaturation to extension program for 30 cycles; extension at 72°C for another 5 minutes; storage at 4°C.

[0046] Preparation of Corynebacterium competent cells involved in the following examples:

[0047] Use an inoculating loop to dip the bacterial solution in the frozen tube, streak on a BHI solid plate for activation, place in a 30°C incubator for activation for 16-24 hours, pick a single colony from the fresh plate and inoculate it into BHI liquid medium, place in a 30°C, 220 rpm rotary shaker for 16-24 hours. Transfer 1-2% of the inoculum to the competent medium, place in a 30°C, 220 rpm rotary shaker, and track the OD 600 , wait for OD 600 When the p-value reaches approximately 0.9, immediately place the bacterial solution in an ice bath. After 15 minutes, centrifuge at 6000 rpm in a refrigerated centrifuge for 10 minutes at 4°C to collect the cells. Then, add 2 ml of pre-chilled 10% glycerol to wash the cells. Gently pipette to resuspend the cells and centrifuge again to collect the cells. Repeat this wash three times. Finally, add an appropriate amount of 10% glycerol to resuspend the cells. Aliquot 80 μl of cells into a 1.5 ml centrifuge tube. Use directly for electroporation transformation or store in a -70°C freezer.

[0048] Guanidineacetate N-methyltransferase activity assay method:

[0049] (1) Enzyme activity assay system: 20 mM Tris-HCl, 2 mM guanidine acetic acid, 0.5 mM S-adenosylmethionine, 1 mM dithiothreitol, and a certain amount of enzyme solution, react at 37°C for 30 min;

[0050] (2) Enzyme activity detection method: react at 37°C for 30 min, add 750 μL HClO4 to terminate the reaction, let it stand for 10 min, centrifuge to remove the precipitate, and take the supernatant for liquid phase detection.

[0051] Enzyme activity definition: Under standard reaction conditions, the amount of enzyme required to catalyze the production of 1 μmol of creatine per minute is 1 enzyme activity unit.

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

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

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

[0055] BHI liquid medium (g / L): 39 Brain Heart Infusion Broth.

[0056] BHI solid medium (g / L): Add 1.5-2.0% agar powder to BHI liquid medium.

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

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

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

[0060] Fermentation medium (g / L): 10% yeast extract powder, 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 examples are as follows:

[0062] Creatine and guanidine acetate were detected using a Thermo Fisher Scientific high-performance liquid chromatography (HPLC) equipped with a UV absorber. The column used was a Hypersil ODS C18 250 × 4 mm 5 μm column. The mobile phase consisted of 30 / 70 acetonitrile / 100 mM anhydrous sodium hydrogen phosphate. The detection wavelength was 210 nm, and the flow rate was 0.6 ml / min.

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

[0064] Example 1: Construction of recombinant plasmid pXMJ19-AtAGAT-HsGAMT

[0065] The specific steps are as follows:

[0066] 1. Construction of recombinant plasmid pXMJ19-AtAGAT containing L-arginine-glycine amidinotransferase

[0067] L-arginine-glycine amidinotransferase AtAGAT from Actinokineospora terrae was selected, and its nucleotide sequence is shown in SEQ ID NO.4. It was synthesized by Suzhou Jinweizhi Company to obtain the agat gene fragment.

[0068] The obtained agat gene fragment was ligated with the linearized plasmid pXMJ19 (P1 / P2 amplified) using the homologous recombinase ClonExpress II One Step Cloning Kit (Novozymes) to obtain a recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells to obtain transformants. The transformants were spread on LB solid medium containing 10 mg / L chloramphenicol and cultured at 37°C for 12 h. Positive colonies were picked and verified by colony PCR using Taq DNA polymerase using P3 / P4 as primers (e.g. Figure 1 A positive single colony with the target band size was inoculated into a vial containing LB liquid medium and cultured for 12 hours. The plasmid was extracted and sent to GeneWeiZ for sequencing. The sequence was correct, and pXMJ19-AtAGAT was successfully constructed.

[0069] Prepare Cg AB2021051 competent cells and electroporate the constructed pXMJ19-AtAGAT recombinant plasmid. Electroporate at 1.8 kV for 5 ms. After electroporation, add 800 μl of BHI medium for recovery, heat shock at 46°C for 5 minutes, and incubate at 30°C at 200 rpm for 2 hours. Transformants were plated on BHI solid medium containing 10 mg / L chloramphenicol and incubated at 30°C. Positive colonies were isolated and verified by colony PCR using Taq DNA polymerase using primers P3 / P4 to obtain the recombinant strain Cg AB2021051 / pXMJ19-AtAGAT.

[0070] The primer sequences involved are as follows:

[0071] P1: 5'-CAGGAAACAGAATTAATTAAGCTT-3';

[0072] P2: 5'-AATTCAGCTTGGCTGTTTTGGC-3';

[0073] P3: 5'-CTGGCAAATATTCTGAAATGAGCTG-3';

[0074] P4: 5'-GCAGTTCCCTACTCTCGCAT-3'.

[0075] 2. Screening of Guanidineacetate N-methyltransferase

[0076] In order to obtain GAMT with higher enzyme activity, the Brenda database and NCBI BLAST were searched, and guanidine acetate N-methyltransferases from Isokutzneria albicans, Pseudomonas hornifera, Dokshima pacifica, Frankia, and Homo sapiens were obtained, named AaGAMT (NCBI No.: SDM24932.1), AkGAMT (NCBI No.: SDU01164.1), DpGAMT (NCBI No.: SNR77205.1), FsGAMT (NCBI No.: WP_009399062.1), and HsGAMT (SEQ ID NO. 4), which were synthesized by Suzhou Jinweizhi Co., Ltd.

[0077] (1) The obtained gamt gene fragments were ligated with the linearized plasmid pXMJ19 obtained after double digestion with HindⅢ and EcoRI, and then transformed into E. coli BL21 according to the above step 1. The positive transformants were picked to extract the plasmids and send them for sequencing. The results were correct, and pXMJ19-AaGAMT, pXMJ19-AkGAMT, pXMJ19-DpGAMT, pXMJ19-FsGAMT, and pXMJ19-HsGAMT were successfully constructed.

[0078] (2) The recombinant plasmids constructed above were electroporated into Cg AB2021051 competent cells according to the above method to obtain recombinant bacteria Cg AB2021051 / pXMJ19-AaGAMT, Cg AB2021051 / pXMJ19-AkGAMT, C.gAB2021051 / pXMJ19-DpGAMT, Cg AB2021051 / pXMJ19-FsGAMT, and C.gAB2021051 / pXMJ19-HsGAMT, which were named: Cg AB-01, Cg AB-02, Cg AB-03, Cg AB-04, and Cg AB-05, respectively.

[0079] (3) The recombinant bacteria constructed above and the starting strain Cg AB2021051 were streaked and activated on BHI solid medium, and single colonies were picked and inoculated into 10 ml of BHI liquid medium. After culturing for 18-20 h, the inoculum was transferred to a 250 ml conical flask containing 50 ml of BHI liquid medium at a 1% (v / v) inoculation volume and cultured for 4-5 h. 25 μl of 1 M IPTG was added for induction. The entire culture process was carried out in a reciprocating shaker at 30°C and 180 rpm.

[0080] After induction culture for 10-12 hours, the supernatant was collected by centrifugation. Before the crude enzyme solution was ultrasonically disrupted, a certain amount of lysozyme was added and ice-bathed for 2-3 hours to promote the cell wall destruction of Corynebacterium glutamicum. The working frequency of the ultrasonic disruptor was 2 seconds on and 5 seconds off, with a total working time of 30 minutes. Centrifuge at 10000 rpm for 20 minutes, and take the supernatant to run a protein gel to verify the expression results (such as Figure 2 As shown), it was found that AaGAMT, AkGAMT, and DpGAMT could not be expressed normally, while FsGAMT and HsGAMT could be expressed in a soluble manner; crude enzyme solutions were prepared respectively.

[0081] (4) The crude enzyme solution was added to the same enzyme activity assay system, and the activity of guanidine acetate N-methyltransferase from different sources was measured according to the above enzyme activity assay method. The results are shown in Table 1.

[0082] Table 1: Comparison of GAMT enzyme activity from different sources

[0083]

[0084] Note: NT means no enzyme activity was detected.

[0085] The results showed that HsGAMT from Homo sapiens had better catalytic ability, with a crude enzyme activity of 0.23 U. This enzyme was selected for subsequent experiments. Its gene sequence is shown in SEQ ID NO.5.

[0086] 3. Construction of recombinant plasmid pXMJ19-AtAGAT-HsGAMT

[0087] The linearized plasmid pXMJ19-AtAGAT and HsGAMT gene fragments were obtained using primer pairs P5 / P6 and P7 / P8, respectively, with pXMJ19-AtAGAT constructed in step 1 and pXMJ19-HsGAMT constructed in step 2 as templates. The fragments were ligated using the homologous recombination enzyme ClonExpress II One Step Cloning Kit (Norwegian) and transformed into E. coli BL21 (DE3) competent cells according to the above method. Positive colonies were picked and single colonies were verified by colony PCR using Taq DNA polymerase using primers P3 / P4 (as shown in Figure 2). Figure 3 A positive single colony with the target band size was inoculated into a vial containing LB liquid medium and cultured for 12 h. The plasmid was extracted and sent to GeneWeichi for sequencing. The sequence was correct, and pXMJ19-AtAGAT-HsGAMT was successfully constructed.

[0088] The primer sequences involved are as follows:

[0089] P5: 5'-TGGCACATCATCATCATCATCACTAAATGAGCGCCCCCAGC-3';

[0090] P6: 5'-GTGACCAAAGGCTGAGAATTCAGCTTGGCTGTTTTGG-3';

[0091] P7: 5'-ATGAGCGCCCCCAGCGCGAC-3';

[0092] P8: 5'-GCCCCTGGTGACCAAAGGCTGA-3';

[0093] Example 2: Screening of Creatine-producing Strains by Fermentation

[0094] The specific steps are as follows:

[0095] (1) The pXMJ19-AtAGAT-HsGAMT recombinant plasmid constructed in Example 1 was electroporated into C.gAB2021051 and Cg ATCC14067 competent cells, respectively. After colony PCR verification, recombinant bacteria C.gAB2021051 / pXMJ19-AtAGAT-HsGAMT and Cg ATCC14067 / pXMJ19-AtAGAT-HsGAMT were prepared, respectively; they were named Cg AB-AG-1 and C.gAT-AG-1, respectively.

[0096] (2) The recombinant bacteria Cg AB-AG-1, Cg AT-AG-1 and the starting strains Cg AB2021051 and C.gATCC14067 were streaked and activated on BHI solid culture medium, and single colonies were picked and inoculated into seed culture medium. The cultures were cultured at 30°C and 220 rpm for 24 h to prepare seed solutions.

[0097] The prepared seed liquid was transferred to a 250 ml shake flask containing 30 ml of fermentation medium at a 10% (v / v) inoculation rate and cultured in a reciprocating shaker at 30°C and 220 rpm. After fermentation for 12 h, a final concentration of 0.5 mM IPTG and 2 g / L glycine were added; and fermentation was continued at 30°C and 220 rpm for 60 h.

[0098] After fermentation, it was found that after overexpressing AGAT and GAMT enzymes in Cg AB2021051 and Cg ATCC14067, the recombinant bacteria had the ability to produce guanidine acetate and creatine.

[0099] Compared with the trace amounts of guanidine acetate and creatine in Cg AT-AG-1 (1.2 and 0.54, respectively), Cg AB-AG-1 can produce 3.56 g / L creatine and accumulate 5.34 g / L guanidine acetate. This is mainly because Cg ATCC14067 cannot provide enough arginine for the production of guanidine acetate and creatine, while Cg AB2021051 can achieve the direct synthesis of creatine from glucose in a one-step fermentation method without the addition of exogenous arginine. Therefore, Cg AB-AG-1 was selected for subsequent research.

[0100] Example 3: Removal of the feedback inhibition of the byproduct S-adenosylhomocysteine ​​on the GAMT enzyme

[0101] Through the above fermentation, it was found that the intermediate product guanidine acetic acid still accumulated to a large extent and was not completely used to produce creatine.

[0102] It was found that while GAMT enzyme produces creatine, its byproduct S-adenosylhomocysteine ​​SAH has a large feedback inhibition effect on GAMT enzyme. In order to promote the production of creatine, it is necessary to relieve its feedback inhibition. Therefore, S-adenosylhomocysteine ​​hydrolase was integrated and expressed in the above-mentioned engineered bacteria to promote the degradation of SAH. At the same time, in order to increase the availability of the substrate SAM, we used SAH for the regeneration of SAM.

[0103] The specific steps are as follows:

[0104] (1) Using the Saccharomyces cerevisiae genome as a template and P9 / P10 as primers, PCR amplification was performed to obtain a 1350 bp S-adenosylhomocysteine ​​hydrolase (SAHase) gene fragment (shown in SEQ ID NO. 6).

[0105] Using the Cg AB2021051 genome as a template and P11 / P12 and P13 / P14 as primers, PCR was performed to amplify the upstream and downstream homology arms (C629_RS09275 site). Fusion extension PCR was performed to obtain a 2350 bp gene fragment, which was ligated into the pK18mobsacB vector to obtain pK18-SAHase. Sequencing results showed that the gene sequence was correct.

[0106] (2) The recombinant plasmid pK18-SAHase was electroporated into Cg AB-AG-1 (Cg AB2021051 / pXMJ19-AtAGAT-HsGAMT). After the colonies grew, they were identified and expanded and then transferred to LBGS liquid sucrose medium for screening. The screened bacterial liquid was first streaked on a BHI plate without antibiotics. Then, the colonies grown on the antibiotic-free plate were streaked on a plate containing Kan+BHI. The colonies that grew on the Kan+BHI plate but not on the antibiotic-free BHI plate were selected for culture. The genome sequencing was successful, and the strain Cg AB2021051-9275::SAHase / pXMJ19-AtAGAT-HsGAMT was obtained and named: Cg AB-AG-2.

[0107] The primer sequences involved are as follows:

[0108] P9: 5'-ATGTCTGCTCCAGCTCAAAACTACA-3';

[0109] P10: 5'-TCAATATCTGTAGTGGTCGGCCTTG-3';

[0110] P11: 5'-AGACATCTACAGCCTTACCGATTTCGAATCT-3';

[0111] P12: 5'-GGGTAAAAAATCCTTTCGTAGGTTTCCGC-3';

[0112] P13: 5'-CACCATTCCGTGTGAACAAGCTG-3';

[0113] P14: 5'-CATCTGCCCATACAACGTTGCACC-3'.

[0114] (3) Verification of the ability of Cg AB-AG-2 to produce creatine by fermentation:

[0115] After the recombinant bacteria Cg AB-AG-2 was streaked and activated on BHI solid medium, single colonies were picked and inoculated into seed medium and cultured at 30°C and 220 rpm for 24 h to prepare seed solutions.

[0116] The prepared seed liquid was transferred to a 250 ml shake flask containing 30 ml of fermentation medium at an inoculum rate of 10% (V / V) and cultured in a reciprocating shaker at 30°C and 220 rpm. After 12 h of fermentation, a final concentration of 0.5 mM IPTG and 2 g / L glycine were added, and fermentation was continued at 30°C and 220 rpm for 60 h.

[0117] At the end of fermentation, the creatine production was 5.37 g / L and the guanidine acetic acid accumulation was 3.46 g / L, which were 50.8% higher than those of Cg AB-AG-1.

[0118] Example 4: Creatine production by 5L tank fermentation of recombinant strain Cg AB-AG-2

[0119] The specific steps are as follows:

[0120] (1) Seed culture:

[0121] A single colony of the recombinant strain Cg AB-AG-2 prepared in Example 3 was picked from the activated plate and inoculated into 10 ml of BHI medium and cultured at 30°C, 200 rpm for 24 h to prepare a culture solution. The culture solution was then transferred at a 2% (v / v) inoculum volume to a 250 ml shake flask containing 30 ml of seed medium and cultured at 30°C, 200 rpm for 24 h to prepare a first-level seed solution. The first-level seed solution was transferred at a 10% (v / v) inoculum volume to a 1000 ml shake flask containing 200 ml of seed medium and cultured in a shaker at 30°C, 200 rpm for 20 h to prepare a second-level seed solution.

[0122] (2) Fermentation culture in 5L fermenter:

[0123] The cultured secondary seed liquid was transferred to a 5-L fermentation tank containing 2-L fermentation medium and fermented for 60 hours at 30°C using a method of speed-coupled dissolved oxygen and manual ventilation to maintain the dissolved oxygen level at 30%-40%. IPTG was added at a final concentration of 0.5 mM for induction after 15 hours of culture. After 24 hours of fermentation, glycine was added as a mother liquor at a flow rate of 10 ml / h to complete the reaction.

[0124] After the fermentation, the fermentation broth was collected and the contents of arginine, guanidinoacetic acid and creatine were detected by HPLC.

[0125] The results showed that the recombinant strain Cg AB-AG-2 could accumulate 14.2 g / L of creatine under the above fermentation process and conditions, achieving a one-step fermentation method for creatine production from carbohydrate raw materials without the addition of arginine or guanidine acetate.

[0126] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Corynebacterium glutamicum, characterized in that by Corynebacterium glutamicum var. CCTCCAB 2021051 was used as the expression host, overexpressing L-arginine-glycine amidinotransferase At AGAT, guanidineacetate N-methyltransferase Hs GAMT and S-adenosylhomocysteine ​​hydrolase SAHase; the L-arginine-glycine amidinotransferase At The amino acid sequence of AGAT is shown in SEQ ID NO.

1. The guanidine acetate N-methyltransferase Hs The amino acid sequence of GAMT is shown in SEQ ID NO.2, and the amino acid sequence of the S-adenosylhomocysteine ​​hydrolase SAHase is shown in SEQ ID NO.

3.

2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that Encoding the L-arginine-glycine amidinotransferase At The nucleotide sequence of AGAT is shown in SEQ ID NO.4, encoding the guanidine acetate N-methyltransferase Hs The nucleotide sequence of GAMT is shown in SEQ ID NO.5, and the nucleotide sequence encoding the S-adenosylhomocysteine ​​hydrolase SAHase is shown in SEQ ID NO.

6.

3. The recombinant Corynebacterium glutamicum according to claim 2, characterized in that The pXMJ19 plasmid was used as the expression vector.

4. A method for preparing creatine by fermentation while promoting the regeneration of the substrate S-adenosylmethionine (SAM), characterized in that: The method is to prepare the recombinant Corynebacterium glutamicum by fermentation according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The fermentation conditions are as follows: inoculating the recombinant Corynebacterium glutamicum into a seed culture medium to prepare a seed solution; inoculating the seed solution into a fermentation medium at an inoculum rate of 8-12% (v / v), adding IPTG with a final concentration of 0.2-0.8 mM 10-15 hours after inoculation, and adding glycine with a final concentration of 2-12 g / L 0-10 hours after inoculation; and maintaining the dissolved oxygen level at 30%-40% during the fermentation process.

6. Use of S-adenosylhomocysteine ​​hydrolase SAHase derived from Saccharomyces cerevisiae in improving the fermentation production of creatine by recombinant Corynebacterium glutamicum while promoting the regeneration of substrate S-adenosylmethionine SAM, characterized in that: The recombinant Corynebacterium glutamicum Corynebacterium glutamicum var. CCTCC AB 2021051 was used as the expression host, overexpressing Actinokineospora terrae L-arginine-glycine amidinotransferase At AGAT, derived from Homo sapiens Guanidine acetate N-methyltransferase Hs GAMT and S-adenosylhomocysteine ​​hydrolase SAHase from Saccharomyces cerevisiae.

7. The use according to claim 6, characterized in that The L-arginine-glycine amidinotransferase At The amino acid sequence of AGAT is shown in SEQ ID NO.

1. The guanidine acetate N-methyltransferase Hs The amino acid sequence of GAMT is shown in SEQ ID NO.2, and the amino acid sequence of the S-adenosylhomocysteine ​​hydrolase SAHase is shown in SEQ ID NO.

3.

8. Use of the recombinant Corynebacterium glutamicum according to any one of claims 1 to 3 in the simultaneous preparation of S-adenosylmethionine (SAM) and creatine.

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