L-serine reaction system with methylobacterium extorquens intracellular THFA as cofactor and production method

Abstract

The invention provides an L-serine reaction system taking THFA in methylobacterium extorquens cells as a cofactor. The L-serine reaction system comprises a THFA solution from methylobacterium extorquens, EcSHMT, glycine, formaldehyde and PLP. The invention also provides a production method of L-serine. The method comprises the following steps: culturing methylobacterium extorquens to obtain a THFA solution; the EcSHMT is obtained; the method comprises the following steps: adding EcSHMT, glycine, formaldehyde and PLP into a THFA solution to construct a reaction system as shown in the specification, and carrying out enzymatic reaction to obtain the L-serine. The method has the advantages that a brand new L-serine production method with THFA in methylobacterium extorquens cells as a cofactor is provided, the extracted THFA has high activity, L-serine production without additional THFA can be realized by utilizing an enzymatic reaction system, the production cost of L-serine is greatly reduced, and the method is suitable for industrial production. And meanwhile, relatively high L-serine yield is ensured.

Description

Technical Field

[0001] This invention relates to the fields of biochemistry and biocatalysis, and particularly to a method using *Methylobacterium truncatum* (…). Methylobacterium extorquens L-serine reaction system and production method with intracellular THFA as cofactor. Background Technology

[0002] L-Serine, a non-essential amino acid, plays an indispensable role in the biosynthesis of various amino acids, phospholipids, choline, purines, and other important substances in organisms, and is an important carrier of active one-carbon units in the body. In recent years, it has been widely used in the food, biopharmaceutical, and cosmetic fields: in the food industry, it can be used as an additive to supplement nutrients; in the pharmaceutical industry, its derivatives, such as phosphatidylserine, can repair brain cells and prevent Alzheimer's disease, while diazoserine can be used as an anticancer agent; in the cosmetics industry, as a natural moisturizing factor, it can increase cell surface activity and delay aging.

[0003] Currently, L-serine production methods include protein hydrolysis, chemical synthesis, microbial fermentation, and enzymatic synthesis. Among these, protein hydrolysis, due to its low raw material cost, was one of the earliest methods applied to industrial amino acid production, but its downstream separation faces challenges. Chemical synthesis typically uses halogenated acrylic acid as a precursor, is complex, generates significant environmental pollution, and produces a racemic mixture of L- and D-serine, making separation difficult. Microbial fermentation, usually using *E. coli* or *Corynebacterium glutamicum* as hosts and glucose or sucrose as raw materials, can achieve high yields, but it relies on grain carbon sources, is susceptible to price fluctuations, and faces competition for food resources. Enzymatic synthesis uses glycine and formaldehyde as precursors and serine hydroxymethyltransferase (SHMT) as a biocatalyst to produce L-serine under mild reaction conditions. It has advantages such as high specificity, high selectivity, high product yield, and low environmental impact, but requires the addition of the expensive cofactor tetrahydrofolate (THFA), resulting in high production costs.

[0004] In the enzymatic synthesis of L-serine catalyzed by SHMT, THFA is an indispensable cofactor: it spontaneously combines with formaldehyde to form methylenetetrahydrofolate, which acts as a methyl carrier, binds to the enzyme, and transfers the methyl group to glycine. Subsequently, methylenetetrahydrofolate is regenerated into THFA and recycled. However, as mentioned earlier, THFA is expensive (market price is 2520 yuan / 0.5g, purity 65%), making it difficult to meet the needs of large-scale L-serine production.

[0005] Therefore, how to obtain THFA at low cost while ensuring a high yield of L-serine has become an urgent problem to be solved. Summary of the Invention

[0006] The purpose of this invention is to provide a method using *Methylobacterium truncatum* (… Methylobacterium extorquens This invention relates to an L-serine reaction system and production method using intracellular THFA as a cofactor, aiming to solve the problem of high L-serine production costs caused by the use of expensive THFA in existing enzymatic synthesis methods.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: An L-serine reaction system using intracellular THFA of *Bacillus thuringiensis* as a cofactor, comprising *Bacillus thuringiensis* (… Methylobacterium extorquens The solution contained THFA, serine hydroxymethyltransferase EcSHMT, glycine, formaldehyde, and pyridoxal phosphate PLP.

[0008] As one of the preferred embodiments of the present invention, the *Methylobacterium tumefaciens* is... Methylobacterium extorquens AM1 or based on the above Methylobacterium extorquens AM1 folC Overexpression strains.

[0009] As one of the preferred embodiments of the present invention, the folC The overexpression strain was constructed by assembling Gibson cells... folC The gene (encoding dihydrofolate synthase) and the vector pCM110 were ligated to obtain the recombinant plasmid pCM110- folC The recombinant plasmid pCM110- was converted using electroconversion. folC Conversion into Methylobacterium extorquens AM1; Transformants with correct sequencing results were cultured in MC medium (with 50 μg / ml kanamycin added) to obtain... folC Overexpression strain; the folC The gene sequence is shown in SEQ ID NO.1.

[0010] As one of the preferred embodiments of the present invention, the concentration of EcSHMT added to the reaction system is at least 1 mg / mL, more preferably 2 mg / mL.

[0011] As one of the preferred embodiments of the present invention, the concentrations of glycine, formaldehyde, and PLP added to the reaction system are 1~3M, with an initial maximum of 50mM, a replenishment rate of 25mM / h~100mM / h, and 0.4~1mM, respectively; more preferably, glycine is 1M, formaldehyde is initially 25mM with a replenishment rate of 25mM / h~75mM / h, and PLP is 0.4mM.

[0012] As one of the preferred embodiments of the present invention, the pH value of the reaction system is 7 to 8, more preferably 8.0.

[0013] A method for producing L-serine using intracellular THFA from *Thiago methylobacterium* as a cofactor includes the following steps: Culture *Trichoderma truncatum* to obtain THFA solution; Obtaining serine hydroxymethyltransferase EcSHMT; EcSHMT, glycine, formaldehyde, and pyridoxal phosphate (PLP) were added to the THFA solution to construct the reaction system shown above, and an enzymatic reaction was carried out to obtain L-serine.

[0014] As one of the preferred embodiments of the present invention, the step of culturing *Methylobacterium truncatum* to obtain a THFA solution includes: The target strain was cultured and grown in a simple inorganic salt medium with methanol as the carbon source, and the bacterial cells were collected. Add extraction solvent to the bacterial cells, resuspend, heat, centrifuge and collect the supernatant to obtain THFA solution.

[0015] As one of the preferred embodiments of the present invention, the target strain is cultured by shake flask culture or fermentation culture; when shake flask culture is used, the harvesting time of the strain is OD. 600 =4~6, more preferably OD 600 =6; When fermentation culture is used, the harvest time of the strain is OD. 600 =40~60, more preferably OD 600 =50.

[0016] As one of the preferred embodiments of the present invention, the simple inorganic salt culture medium is MC culture medium.

[0017] As one of the preferred embodiments of the present invention, the formulation of the extraction solvent is: 10~80mM KH2PO4, 0.5~2% sodium ascorbate, and 0.1~0.2% β-mercaptoethanol; more preferably: 50mM KH2PO4, 1% sodium ascorbate, and 0.1% β-mercaptoethanol.

[0018] As one of the preferred embodiments of the present invention, the heating parameters are: 98~100℃ for 3~8 minutes; more preferably, heating at 98℃ for 5 minutes.

[0019] As one of the preferred embodiments of the present invention, the step of obtaining serine hydroxymethyltransferase EcSHMT includes: Will EcSHMT The gene was ligated into the pET21a vector to construct the recombinant plasmid pET21a- EcSHMT ;The recombinant plasmid pET21a- EcSHMT Transformed into BL21(DE3), yielding the recombinant plasmid pET21a- EcSHMTEscherichia coli; the bacteria were inoculated into LB broth and cultured for 2-3 hours, and then IPTG was added for induction; after induction, the bacteria were harvested, lysed, and purified to obtain the target EcSHMT; EcSHMT The gene sequence is shown in SEQ ID NO.2.

[0020] As one of the preferred embodiments of the present invention, in the step of carrying out the enzymatic reaction, the reaction temperature is 30~50℃, the stirring rate is 200~800rpm, and the reaction time is 10~24h; more preferably, the reaction temperature is 40℃, the stirring rate is 400rpm, and the reaction time is 10h.

[0021] The reaction principle is as follows: like Figure 1 As shown, the present invention Methylobacterium extorquens AM1 and folC The overexpressing strain is rich in THFA. By heating, the cells are broken up and the intracellular THFA is released from the protein, which can be used as a cofactor for enzymatic reactions. In the enzymatic reaction, THFA spontaneously combines with formaldehyde to form methylenetetrahydrofolate, which acts as a methyl carrier and binds to EcSHMT to transfer the methyl group to glycine to generate L-serine. Methylenetetrahydrofolate is then converted back into THFA and recycled.

[0022] The advantages of this invention compared to the prior art are: (1) The present invention utilizes Methylobacterium extorquens AM1 strain or based on this strain folC The overexpression strain produces THFA; the THFA from this source can be used for enzymatic reactions without purification, eliminating the need for additional expensive THFA, which greatly reduces the production cost of L-serine, while also providing a high L-serine yield. (2) Compared to Methylobacterium extorquens AM1 wild-type strain, through overexpression of the folC gene ( folC Overexpression strains can further increase Methylobacterium extorquens The intracellular THFA content of AM1 cells increased from 56 μg / L to 73 μg / L, a 1.3-fold increase, thus improving L-serine yield. (3) The present invention is used in the cultivation and collection of Methylobacterium extorquens AM1 / folC After overexpressing the bacterial cells, an extraction solvent is added to the cells, followed by resuspension and heating to release THFA. The antioxidants added to the extraction solution further prevent THFA from being oxidized and inactivated during the extraction process.

[0023] In summary, this invention provides a novel method for producing L-serine using intracellular THFA from *Thiago methylobacterium* as a cofactor. The extracted THFA has high activity, and the enzymatic reaction system enables the production of L-serine without the need for additional THFA, significantly reducing the production cost of L-serine while ensuring a high L-serine yield. Attached Figure Description

[0024] [[ID=2⑨]]Figure 1 This is a schematic diagram of the reaction principle of the present invention; Figure 2 The recombinant plasmid pCM110- in Example 1 of this invention folC Structural diagram; Figure 3 The recombinant plasmid pET21a- in Example 6 of this invention EcSHMT Structural diagram; Figure 4 This is an electrophoresis image of the purified EcSHMT protein from Example 6; Figure 5 It is in Example 7 [[ID=③6]]Methylobacterium extorquens AM1 wild-type strain and folC Comparison of intracellular THFA content in overexpression strains; Figure 6 The graph shows the serine production results of different groups in Example 8 (Figure A shows the serine production results of experimental group 1 and control group 1, and Figure B shows the serine production results of experimental group 2 and control group 2). Figure 7 This is a graph showing the serine yield and formaldehyde conversion rate under condition 1 in Example 9; Figure 8 This is a graph showing the results of serine yield and formaldehyde conversion under condition 2 in Example 9; ​ This is a graph showing the results of serine production and formaldehyde conversion in Example 10.

[0025] ​ This is a graph showing the results of serine production and formaldehyde conversion in Example 11. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0027] The culture medium formulations involved in the following examples are as follows: LB medium: 10 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract.

[0028] MC medium: Solution A: 75.68 mM (NH4)2SO4, 18.26 mM MgSO4·7H2O, 0.22 mM CaCl2·2H2O; Solution B: 45.53 mM Na3C6H3O7·2H2O, 1.2 mM ZnSO4·7H2O, 1.0 mM MnCl2·4H2O, 18 mM FeSO4·7H2O, 2 mM (NH4)6Mo7O 24 ·4H2O, 1mM CuSO4·5H2O, 2mM CoCl2·6H2O, 0.33mM Na2WO4·2H2O; Prepare solution C: 0.485mM H3BO3; Prepare solution D: 0.959M KH2PO4, 1.50M Na2HPO4·7H2O, adjust pH to 6.8 with NaOH; Take 100ml of solution A, 1ml of solution B, and 1ml of solution C, add 878ml of deionized water, sterilize, then add 10ml of sterilized solution D and 10ml of pure methanol.

[0029] The following embodiments involve ​ AM1 wild-type strain is a commercially available strain, ATCC: Methylorubrum extorquens (Urakami and Komagata) Green and Ardley -14718 | ATCC.

[0030] The primers used in the following examples are shown in Table 1.

[0031] Table 1 Primer sequences

[0032] Furthermore, unless otherwise specified, the culture media, carriers, plasmids, and reagents used in this invention are conventional culture media, carriers, plasmids, and reagents in this technical field; unless otherwise specified, the methods and equipment used in this invention are conventional methods and equipment in this technical field, and will not be described in detail again.

[0033] Example 1 ​ AM1 ​ Construction of overexpression strains: (1) Using folC-F (+Pmxa) and folC-R (+pCM110ter) as primers, PCR was performed to... ​ Gene fragment (SEQ ID NO.1) from ​ The amplified vector was then amplified from the AM1 genome. Simultaneously, the vector pCM110 was amplified using Pmxa-R and pCM110-ter-F primers. The relevant primers are shown in Table 1.

[0034] (2) The amplified material was assembled using Gibson assembly (50℃, 10 min). ​ The gene fragment and vector pCM110 were ligated and transformed into DH5α. The plasmid was extracted to obtain the recombinant plasmid pCM110- ​ (like ​ (As shown).

[0035] (3) The recombinant plasmid pCM110- was converted by electroconversion. ​ Conversion into ​ ​ AM1, select single clones and sequence them for verification; culture correctly sequenced transformants in MC medium (with 50 μg / ml kanamycin added) to obtain... ​ Overexpression strains.

[0036] Example 2 ​ Shake-flask culture of overexpression strains and preparation of THFA solution: (1) Take ​ AM1 ​ The overexpression strains were cultured in MC medium with methanol as the carbon source and grown to OD. 600 The bacterial culture volume was 200 mL, and the culture was centrifuged at 4℃ and 8000 rpm for 10 min to collect the bacterial cells.

[0037] (2) Add 10 ml of extraction solvent (50 mM KH2PO4, 1% sodium ascorbate, 0.1% β-mercaptoethanol) to the collected bacterial cells to resuspend the bacterial cells.

[0038] (3) Take the supernatant, heat it at 98°C for 5 minutes, and centrifuge it at 4°C and 10,000 rpm for 15 minutes to remove cell debris and obtain THFA solution.

[0039] Example 3 ​ Shake-flask culture of overexpression strains and preparation of THFA solution (THFA solution concentration factor doubled): (1) Take ​ AM1 ​ The overexpression strains were cultured in MC medium with methanol as the carbon source and grown to OD. 600 The bacterial culture volume was 400 mL, and the culture was centrifuged at 4℃ and 8000 rpm for 10 min to collect the bacterial cells.

[0040] (2) Add 10 ml of extraction solvent (50 mM KH2PO4, 1% sodium ascorbate, 0.1% β-mercaptoethanol) to the collected bacterial cells to resuspend the bacterial cells.

[0041] (3) Take the supernatant, heat it at 98°C for 5 minutes, and centrifuge it at 4°C and 10,000 rpm for 15 minutes to remove cell debris and obtain THFA solution.

[0042] Example 4 ​ Fermentation culture of overexpression strains and preparation of THFA solution: (1) Take ​ AM1 ​ The overexpression strain was cultured in a 1L fermenter with an initial methanol addition of 1% (MC medium), pH controlled at 6.8 (pH adjusted by adding ammonia), and dissolved oxygen set at 30%. Methanol was added in response to the increase in dissolved oxygen when the substrate was depleted. The bacterial culture was cultured to OD... 600 When the concentrations are 50 and 86, take 48 ml and 28 ml of bacterial culture, respectively. Centrifuge at 4℃ and 8000 rpm for 10 min to collect the bacterial cells.

[0043] (2) Add 10 ml of extraction solvent (50 mM KH2PO4, 1% sodium ascorbate, 0.1% β-mercaptoethanol) to the collected bacterial cells to resuspend the bacterial cells.

[0044] (3) Take the supernatant, heat it at 98℃ for 5 minutes, and centrifuge it at 4℃ and 10000 rpm for 15 minutes to remove cell debris and obtain THFA solution.

[0045] Example 5 ​ Fermentation culture of overexpression strains and preparation of THFA solution (THFA solution concentration factor doubled): (1) Take ​ AM1 ​ The overexpression strain was cultured in a 1L fermenter with an initial methanol addition of 1% (MC medium), pH controlled at 6.8 (adjusted by adding ammonia), and dissolved oxygen set at 30%. Methanol was added in response to the increase in dissolved oxygen when the substrate was depleted. The bacterial culture was cultured to OD... 600 When the concentration is 50, take a bacterial culture volume of 96 mL, centrifuge at 4℃ and 8000 rpm for 10 min, and collect the bacterial cells.

[0046] (2) Add 10 ml of extraction solvent (50 mM KH2PO4, 1% sodium ascorbate, 0.1% β-mercaptoethanol) to the collected bacterial cells to resuspend the bacterial cells.

[0047] (3) Take the supernatant, heat it at 98℃ for 5 minutes, and centrifuge it at 4℃ and 10000 rpm for 15 minutes to remove cell debris and obtain THFA solution.

[0048] Example 6: Obtaining the serine hydroxymethyltransferase EcSHMT: (1) ​The gene (SEQ ID NO.2) was ligated into the pET21a vector to construct the recombinant plasmid pET21a- ​ ( ​ ); recombinant plasmid pET21a- ​ Transformed into BL21(DE3), yielding the recombinant plasmid pET21a- ​ Escherichia coli.

[0049] (2) Inoculate the bacteria into LB broth and incubate for 2-3 hours until OD500 is reached. 600 Once the concentration reached 0.6, IPTG was added to a final concentration of 1 mM and induced overnight at 18°C.

[0050] (3) After induction, the bacteria were harvested, broken, and purified by nickel column to obtain the target EcSHMT protein (amino acid sequence as shown in SEQ ID NO.3).

[0051] ​ The purification results of EcSHMT protein (electrophoresis image).

[0052] Example 7, Wild-type strain and ​ Validation of THFA expression in overexpression strains: (1) Take ​ AM1 wild-type strain and ​ The overexpression strains were cultured in MC medium with methanol as the carbon source and grown to OD. 600 The bacterial culture volume was 10 mL, and the culture was centrifuged at 4℃ and 8000 rpm for 10 min to collect the bacterial cells.

[0053] (2) Add 500µL of extraction solvent (50mM KH2PO4, 1% sodium ascorbate, 0.1% β-mercaptoethanol) to the collected bacterial cells to resuspend the bacterial cells.

[0054] (3) Use a grinder to break the cells at a frequency of 50 Hz for 10 min, then centrifuge at 4 ℃ and 10,000 rpm for 15 min to remove cell debris.

[0055] (4) Take 400 μl of supernatant into an EP tube, add 20 μl of mouse serum, and incubate at 37°C for 3 h (this step is used to assist in the detection of THFA content); heat at 98°C for 5 minutes, and centrifuge at 4°C and 10,000 rpm for 15 min to remove impurities.

[0056] (6) Take the supernatant from the above steps, pass it through an SPE column to remove salt ions, and then pass it through a 0.22 μm filter membrane for sterilization. Use LC-MS / MS to quantify the intracellular THFA content.

[0057] The measurement results are as follows ​ As shown. By​ It can be seen that: This invention ​ AM1 wild-type strain and ​ Both overexpression strains effectively expressed THFA, with intracellular THFA levels of 56 μg / L and 73 μg / L, respectively; compared to the wild-type strain, ​ Overexpression strains can further increase ​ The intracellular THFA content of AM1 cells increased by 1.3 times.

[0058] Example 8: Preliminary validation and parameter optimization of L-serine production using intracellular THFA as a cofactor in *Thiazora mitochondrium*: (1) Following the method in Example 2, culture ​ Overexpressing strains yielded THFA solution.

[0059] (2) The serine hydroxymethyltransferase EcSHMT was obtained by referring to the method in Example 6.

[0060] (3) Set up two experimental groups and two control groups to compare the serine production of the enzyme-catalyzed reaction under different conditions.

[0061] Experimental Group 1: To ​ EcSHMT, glycine, formaldehyde, and pyridoxal phosphate (PLP) were added to the THFA solution from the overexpression strain to obtain an enzymatic reaction solution for enzymatic reaction. The enzymatic reaction solution contained: ​ The overexpression strain was derived from a THFA solution with a concentration of 55 μg / L, an EcSHMT concentration of 2 mg / mL, a glycine concentration of 200 mM, an initial formaldehyde concentration of 100 mM (no further addition), a PLP concentration of 0.4 mM, and a pH of 8.0. The enzymatic reaction conditions were: reaction temperature 40℃, stirring speed 400 rpm, and reaction time 8.5 h (until the enzymatic reaction reached equilibrium).

[0062] Control group 1: Basically the same as experimental group 1, the main difference being that a sufficient amount of commercially available THFA (4mM) was used instead of experimental group 1. ​ The overexpression strain was derived from THFA solution.

[0063] Experimental Group 2: Basically the same as Experimental Group 1, the main difference is that in the enzyme reaction solution: the concentration of formaldehyde added is 50mM (no additional addition), and the concentration of glycine is 250mM.

[0064] Control group 2: Basically the same as experimental group 2, the main difference being that a sufficient amount of commercially available THFA (4mM) was used instead of experimental group 2. ​ The overexpression strain was derived from THFA solution.

[0065] The serine production results of different groups are as follows: ​As shown: The L-serine yield measured in experimental group 1 was 46.7 mM, while that in control group 1 was 82.6 mM; the L-serine yield measured in experimental group 2 was 42.6 mM, while that in control group 2 was 47.6 mM.

[0066] The above results indicate that under the reaction conditions of 50 mM formaldehyde, ​ The THFA provided by the overexpression strain can achieve the same effect as commercial THFA; and under the reaction conditions of 100mM formaldehyde, ​ The THFA provided by the overexpressing strain was insufficient to consume formaldehyde in time, and the accumulation of high concentrations of formaldehyde led to a decrease in enzyme activity. Based on this, two key pieces of information can be obtained: a. the enzyme reaction rate (35 mM formaldehyde can be converted in 1.5 h, with a formaldehyde consumption rate of approximately 25 mM / h); b. the formaldehyde accumulation should ideally be below 50 mM, otherwise it will lead to a decrease in enzyme activity. Combining the above two key pieces of information, the subsequent formaldehyde feeding rate should be 25 mM / h.

[0067] Example 9: Further validation of L-serine production using intracellular THFA as a cofactor in *Thiazora thiazolinone* (obtained in shake flasks): Based on the results of Example 8, the formaldehyde replenishment rate can be determined. Next, different... ​ The serine yield and formaldehyde conversion rate were compared under conditions of THFA solution concentration and initial formaldehyde concentration from overexpression strains.

[0068] Condition 1: (1) Following the method in Example 2, culture ​ Overexpressing strains yielded THFA solution.

[0069] (2) The serine hydroxymethyltransferase EcSHMT was obtained by referring to the method in Example 6.

[0070] (3) To ​ EcSHMT, glycine, formaldehyde, and pyridoxal phosphate (PLP) were added to the THFA solution from the overexpression strain to obtain an enzymatic reaction solution for enzymatic reaction. The enzymatic reaction solution contained: ​ The overexpression strain was derived from a THFA solution with a concentration of 55 μg / L, an EcSHMT concentration of 2 mg / mL, a glycine concentration of 1 M, an initial formaldehyde concentration of 25 mM with hourly replenishment of 25 mM formaldehyde, a PLP concentration of 0.4 mM, and a pH of 8.0. The enzymatic reaction conditions were: reaction temperature 40℃, stirring rate 400 rpm, and reaction time 10 h.

[0071] The results of serine yield and formaldehyde conversion rate under condition 1 above are as follows: ​ As shown, the final measured L-serine yield was 131.5 mM, and the formaldehyde conversion rate was 54%.

[0072] Condition 2: Further increase the concentration of THFA solution (1) Following the method in Example 3, culture ​ Overexpressing strains yielded THFA solution.

[0073] (2) The serine hydroxymethyltransferase EcSHMT was obtained by referring to the method in Example 6.

[0074] (3) To ​ EcSHMT, glycine, formaldehyde, and pyridoxal phosphate (PLP) were added to the THFA solution from the overexpression strain to obtain an enzymatic reaction solution for enzymatic reaction. The enzymatic reaction solution contained: ​ The overexpression strain was derived from a THFA solution with a concentration of 120 μg / L, an EcSHMT concentration of 2 mg / mL, a glycine concentration of 1 M, an initial formaldehyde concentration of 50 mM with 25 mM formaldehyde added hourly, a PLP concentration of 0.4 mM, and a pH of 8.0. The enzymatic reaction conditions were: reaction temperature 40℃, stirring rate 400 rpm, and reaction time 30 h.

[0075] The results of serine yield and formaldehyde conversion rate under condition 2 above are as follows: ​ As shown: after 10 h of reaction, the yield of L-serine was 203.8 mM, and the formaldehyde conversion rate was 67%; after 21.5 h of reaction, the yield of L-serine was 316.6 mM.

[0076] Therefore, it can be seen that the method of the present invention is applicable to the shake flask production of L-serine, and the optimal parameters are glycine 1M, initial formaldehyde 25mM / 50mM, replenishment rate 25mM / h, and PLP 0.4mM.

[0077] Example 10: Further validation of L-serine production using intracellular THFA as a cofactor in *Trichoderma truncatum* (obtained through fermentation): (1) Following the method in Example 4, culture ​ Overexpressing strains yielded THFA solution.

[0078] (2) The serine hydroxymethyltransferase EcSHMT was obtained by referring to the method in Example 6.

[0079] (3) EcSHMT, glycine, formaldehyde, and pyridoxal phosphate (PLP) were added to the THFA solution to construct a reaction system for enzymatic reaction to obtain L-serine. In the reaction system: the concentration of THFA solution was 55 μg / L, the concentration of EcSHMT was 2 mg / mL, the concentration of glycine was 1 M, the initial concentration of formaldehyde was 50 mM with a replenishment rate of 25 mM / h, the concentration of PLP was 0.4 mM, and the pH of the reaction system was 8.0. The enzymatic reaction conditions were: reaction temperature 40℃, stirring speed 400 rpm, and reaction time 24 h.

[0080] The serine yield results under the above conditions are as follows: ​ As shown: OD 600 The AM1 fermentation lysate ratio was 50 to OD. 600 The fermentation lysate of 86 showed better catalytic performance in the SHMT reaction. OD 600 After providing THFA to 50% of AM1 fermentation lysate and reacting with SHMT for 20.75 h, the highest yield of L-serine was measured to be 413.8 mM.

[0081] Example 11: Further validation of L-serine production using intracellular THFA as a cofactor in *Trichoderma truncatum* (obtained through fermentation): (1) Following the method in Example 5, culture ​ Overexpressing strains yielded THFA solution.

[0082] (2) The serine hydroxymethyltransferase EcSHMT was obtained by referring to the method in Example 6.

[0083] (3) EcSHMT, glycine, formaldehyde, and pyridoxal phosphate (PLP) were added to the THFA solution to construct a reaction system for enzymatic reaction to obtain L-serine. In the reaction system: the concentration of THFA solution was 240 μg / L, the concentration of EcSHMT was 2 mg / mL, the concentration of glycine was 1 M, the initial concentration of formaldehyde was 0 mM, the reaction rate was 75 mM / h and 100 mM / h, the concentration of PLP was 0.4 mM, and the pH of the reaction system was 8.0. The enzymatic reaction conditions were: reaction temperature 40℃, stirring speed 400 rpm, and reaction time 12 h.

[0084] The serine yield under the above conditions is as follows: ​ As shown: With a formaldehyde addition rate of 100 mM / h, the L-serine yield was 390 mM after 5.6 h of reaction. With a formaldehyde addition rate of 75 mM / h, the L-serine yield was 579.1 mM after 11 h of reaction. Too slow a formaldehyde addition rate leads to an excessively long reaction time, making THFA and PLP in the system more susceptible to oxidation and reducing enzyme activity, thus decreasing the final serine yield. Too rapid an addition rate results in the accumulation of unconsumed formaldehyde, leading to enzyme inactivation. The final OD... 600 When providing THFA to the fermentation crushing supernatant of 50% for the SHMT reaction, the optimal formaldehyde addition rate is 25~75mM / h.

[0085] As can be seen from the above experiments, the method of the present invention is also applicable to the fermentation production of L-serine.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An L-serine reaction system using intracellular THFA of *Trichoderma truncatula* as a cofactor, characterized in that, This includes THFA solution derived from *Thiazaria truncatula*, serine hydroxymethyltransferase (EcSHMT), glycine, formaldehyde, and pyridoxal phosphate (PLP).

2. The reaction system according to claim 1, characterized in that, The methylphenidate is Methylobacterium extorquens AM1 or based on the above Methylobacterium extorquens AM1 folC Overexpression strains.

3. The reaction system according to claim 2, characterized in that, The folC The overexpression strain was constructed by assembling Gibson cells... folC The gene and vector pCM110 were ligated to obtain the recombinant plasmid pCM110- folC The recombinant plasmid pCM110- was converted using electroconversion. folC Conversion into Methylobacterium extorquens AM1; Transformants with correct sequencing were cultured in MC medium to obtain... folC Overexpression strain; the folC The gene sequence is shown in SEQ ID NO.

1.

4. The reaction system according to claim 1, characterized in that, The concentration of EcSHMT added to the reaction system is at least 1 mg / mL; the concentrations of glycine, formaldehyde, and PLP added to the reaction system are 1~3 M, with an initial maximum of 50 mM, a replenishment rate of 25 mM / h~100 mM / h, and 0.4~1 mM, respectively; the pH value of the reaction system is 7~8.

5. A method for producing L-serine using intracellular THFA from *Thiazonia truncatula* as a cofactor, characterized in that, Includes the following steps: Culture *Trichoderma truncatum* to obtain THFA solution; Obtaining serine hydroxymethyltransferase EcSHMT; EcSHMT, glycine, formaldehyde, and pyridoxal phosphate PLP were added to the THFA solution to construct the reaction system as described in any one of claims 1 to 4, and an enzymatic reaction was carried out to obtain L-serine.

6. The production method according to claim 5, characterized in that, The step of culturing *Methylobacterium truncatum* to obtain a THFA solution includes: The target strain was cultured in a simple inorganic salt medium with methanol as the carbon source, and the bacterial cells were collected. Add extraction solvent to the bacterial cells, resuspend, heat, centrifuge and collect the supernatant to obtain THFA solution.

7. The production method according to claim 6, characterized in that, The target strain is cultured in shake flasks or fermented; when shake flask culture is used, the harvest time of the strain is OD. 600 =4~6; When using fermentation culture, the harvest time of the strain is OD 600 =40~60.

8. The production method according to claim 6, characterized in that, The simple inorganic salt culture medium is MC medium; the extraction solvent is: 10~80mM KH2PO4, 0.5~2% sodium ascorbate, and 0.1~0.2% β-mercaptoethanol; The heating parameters are: 98~100℃, 3~8 minutes.

9. The production method according to claim 5, characterized in that, The steps for obtaining the serine hydroxymethyltransferase EcSHMT include: Will EcSHMT The gene was ligated into the pET21a vector to construct the recombinant plasmid pET21a- EcSHMT ;The recombinant plasmid pET21a- EcSHMT Transformed into BL21(DE3), yielding the recombinant plasmid pET21a- EcSHMT Escherichia coli; the bacteria were inoculated into LB broth and cultured for 2-3 hours, and then IPTG was added for induction; after induction, the bacteria were harvested, lysed, and purified to obtain the target EcSHMT; EcSHMT The gene sequence is shown in SEQ ID NO.

2.

10. The production method according to claim 5, characterized in that, In the step of carrying out the enzymatic reaction, the reaction temperature is 30~50℃, the stirring rate is 200~800rpm, and the reaction time is 10~24h.

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