A serine hydroxymethyltransferase mutant and a method for biosynthesizing L-glycine

Abstract

The present invention relates to a serine hydroxymethyltransferase mutant and a method for biosynthesizing L-glycine. In the present invention, serine hydroxymethyltransferase glyA is overexpressed in a host bacterium, and whole-cell catalysis is used to produce L-glycine; through site-directed mutagenesis, the catalytic direction of the enzyme is changed to make the catalysis favorable for the production of L-glycine; the mutant enzyme glyA A292V‑D392N The specific enzyme activity for catalyzing the production of L-glycine is increased to 1.52 U / mg, which is 204% higher than that of the original enzyme; the mutant enzyme glyA A292V‑D392N The conversion rate of whole-cell conversion for producing L-glycine can reach 61.8%, which is 2.8 times higher than that of the original enzyme glyA; after coupling serine hydroxymethyltransferase with the tetrahydrofolate cycle in the whole-cell catalysis system, only 5 mM to 10 mM of tetrahydrofolate is added, and the yield of L-glycine is 14.03 g / L at 12 h, and the substrate conversion rate is increased to 93.2%, effectively reducing the addition amount of the substrate tetrahydrofolate, reducing the catalytic cost, and greatly improving the production efficiency of L-glycine. The present invention is of great significance for the biosynthesis of L-glycine by biological methods.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and in particular to a serine hydroxymethyltransferase mutant and a method for biosynthesizing L-glycine by biological method. Background Art

[0002] L-Glycine, also known as aminoacetic acid, is a non-essential amino acid with the smallest relative molecular mass and the simplest molecular structure. As an important fine chemical intermediate, glycine is widely used in multiple industries, including but not limited to pesticides, pharmaceuticals, foods, animal feeds, and daily chemicals. Especially in the pesticide field, glycine is an important intermediate for the synthesis of the highly effective broad-spectrum herbicide glyphosate. According to statistics, more than 80% of the total domestic output of glycine is mainly used for the synthesis of glyphosate. With the large-scale cultivation of genetically modified crops such as soybeans and corn, the demand for glyphosate continues to grow, which has further promoted the improvement of the domestic industrial-grade glycine production capacity. Although there is an overall oversupply trend, the market is relatively stable.

[0003] At present, the methods for synthesizing L-glycine include chloroacetic acid ammonolysis method, Strecker method, hydantoin method, and biosynthesis method; the first three methods have problems such as high energy consumption, high product cost, and serious environmental pollution. In particular, the problems of the chloroacetic acid ammonolysis method are particularly prominent. Therefore, it is necessary to develop more environmentally friendly and efficient synthesis processes. Generally speaking, the technology for biosynthesizing L-glycine is still in the preliminary research stage, limited to laboratory development research, and there are still many technical problems to be overcome, such as low L-glycine yield, low enzyme activity, the need to find highly active enzymes with a wide adaptation range, more effective gene modification means, etc. Domestic scholars such as Zhang Zhenshu (Chinese Patent: CN111433366A, 2020) made ATP phosphoribosyltransferase mutate by genetic means, improving the ability of Corynebacterium microorganisms to produce glycine by nearly 2 times. In the long run, the biosynthesis method has advantages such as good selectivity, high product yield, mild reaction conditions, and environmental protection and pollution-free. These advantages indicate that the biosynthesis method has great development potential in the future production of L-glycine.

[0004] The glyA gene is widely present in organisms, and the serine hydroxymethyltransferase (SHMT) encoded by it catalyzes the interconversion between L-serine and L-glycine. Currently, the glyA enzyme mainly tends to catalyze in the direction of L-serine. Researchers mainly use serine hydroxymethyltransferase to catalyze the production of L-serine from L-glycine (Chinese Patent: CN111961656A, 2020; Chinese Patent: CN110872593A, 2020). However, there is currently no research on using the reverse reaction of serine hydroxymethyltransferase to catalyze the production of L-glycine, and there is no study on the enzyme activity of glyA and its mutant enzymes in catalyzing the production of L-glycine. Therefore, this transferase and its mutants are of great significance for the biotransformation production of L-glycine. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a serine hydroxymethyltransferase mutant and a method for its biosynthesis of L-glycine. The present invention overexpresses the serine hydroxymethyltransferase glyA derived from Methanocaldococcus jannaschii in Escherichia coli BL21(DE3) for biocatalytic production of L-glycine by whole cells; through site-directed mutagenesis, the catalytic direction of the enzyme is changed to make the catalysis favorable for the production of L-glycine; at the same time, by coupling three enzymes, glycine decarboxylase (GcvP), glycine cleavage system H protein (GcvH), and glycine cleavage system aminomethyltransferase (GcvT) with the tetrahydrofolate cycle, the addition amount of the substrate tetrahydrofolate is reduced, the enzymatic catalytic system is optimized, the cost is reduced, and the catalytic efficiency of L-glycine is improved.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a serine hydroxymethyltransferase mutant, using the serine hydroxymethyltransferase with the amino acid sequence shown in SEQ ID NO.2 as the parent, including at least one mutation of mutating alanine at position 271 of the parent serine hydroxymethyltransferase to leucine, mutating alanine at position 292 to valine, and mutating aspartic acid at position 392 to asparagine.

[0008] In an embodiment of the present invention, alanine at position 292 of the parent serine hydroxymethyltransferase is mutated to valine and aspartic acid at position 392 is mutated to asparagine.

[0009] In an embodiment of the present invention, the serine hydroxymethyltransferase glyA is derived from Methanocaldococcus jannaschii; the nucleotide sequence of the serine hydroxymethyltransferase glyA is shown in SEQ ID NO.1.

[0010] The second object of the present invention is a gene encoding the serine hydroxymethyltransferase mutant.

[0011] The third object of the present invention is a recombinant plasmid carrying the said gene.

[0012] The fourth object of the present invention is a recombinant bacterium expressing the serine hydroxymethyltransferase mutant.

[0013] In one embodiment of the present invention, the host bacterium of the recombinant bacterium is Escherichia coli.

[0014] In one embodiment of the present invention, the recombinant Escherichia coli uses Escherichia coli E. coli BL21(DE3) as the host.

[0015] The fifth object of the present invention is a method for synthesizing L-glycine using the said recombinant bacterium, comprising the following steps:

[0016] Using the said recombinant bacterium as a catalyst, and using a buffer solution containing 50 mM - 200 mM L-serine, 5 mM - 10 mM tetrahydrofolic acid, 50 mM - 400 mM NaHCO3, 5 mM - 50 mM NADH, glycine decarboxylase, glycine cleavage system H protein and glycine cleavage system aminomethyltransferase as a reaction system to catalytically synthesize L-glycine.

[0017] In one embodiment of the present invention, the temperature of the catalytic synthesis is 30°C - 45°C, and the time is 12 h - 24 h;

[0018] And / or, the pH value of the reaction system is 7.0 - 9.0.

[0019] In one embodiment of the present invention, the nucleotide sequence of the glycine decarboxylase is as shown in SEQ ID NO.3;

[0020] And / or, the nucleotide sequence of the glycine cleavage system H protein is as shown in SEQ ID NO.4;

[0021] And / or, the nucleotide sequence of the glycine cleavage system aminomethyltransferase is as shown in SEQ ID NO.5.

[0022] The sixth object of the present invention is the application of the said serine hydroxymethyltransferase mutant or the said recombinant bacterium in the preparation of L-glycine or products containing L-glycine.

[0023] The above technical solutions of the present invention have the following advantages compared with the prior art:

[0024] The present invention provides a serine hydroxymethyltransferase mutant and a method for biosynthesizing L - glycine. The serine hydroxymethyltransferase glyA derived from Methanocaldococcus jannaschii is overexpressed in a host bacterium, and L - glycine is produced by whole - cell biocatalysis; through site - directed mutagenesis, the specific enzyme activity of the mutant enzyme glyA A292V-D392N for catalyzing the production of L - glycine is increased to 1.52 U / mg, which is 204% higher than that of the original enzyme; the optimal temperature and optimal pH after mutation are 35 °C and 8.0 respectively; under these conditions, the mutant enzyme glyA A292V-D392N has a whole - cell bioconversion rate for producing L - glycine of 61.8%, which is 2.8 times higher than that of the original enzyme; after coupling the serine hydroxymethyltransferase with three enzymes gcvPHT of the tetrahydrofolate cycle in the whole - cell catalysis system, only 5 mM - 10 mM of tetrahydrofolate is added, and the L - glycine yield is 14.03 g / L, and the conversion rate of the substrate L - serine is increased to 93.5%. This strategy effectively reduces the addition amount of the substrate tetrahydrofolate, reduces the catalytic cost, greatly improves the production efficiency of L - glycine, and provides a basis for the industrial production of L - glycine by the microbial method. Brief Description of the Drawings

[0025] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings. Among them,

[0026] Figure 1 is the graph of the optimal temperature and optimal pH of glyA and its mutant enzyme in the embodiment of the present invention;

[0027] Figure 2 is the schematic diagram of the enzymatic catalysis for producing L - glycine coupled with the tetrahydrofolate cycle in the embodiment of the present invention;

[0028] Figure 3 is the liquid - phase chromatogram of the samples at 0 h (A) and 12 h (B) in the embodiment of the present invention;

[0029] Figure 4 is the yield of whole - cell catalysis for producing L - glycine in the embodiment of the present invention. Detailed Embodiments

[0030] The following further illustrates the present invention in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0031] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0032] The culture media involved in the following examples:

[0033] LB liquid medium: 1% NaCl, 1% tryptone, 0.5% yeast extract. 2% agar powder was added to the solid medium.

[0034] The detection methods involved in the following examples are as follows:

[0035] The content of L-glycine was determined by HPLC (Thermo Fisher), using a C18 chromatographic column. The mobile phase was an aqueous sodium acetate solution with a volume fraction of 92% and a methanol-acetonitrile solution with 8%. The flow rate was set at 1 mL / min, the column temperature was maintained at 40 °C, and quantitative detection was carried out at an ultraviolet wavelength of 338 nm.

[0036] Example 1: Construction of serine hydroxymethyltransferase recombinant Escherichia coli E.coli / pET28a-glyA

[0037] (1) Preparation of Escherichia coli competent cells

[0038] Streak Escherichia coli E.coli BL21 on an antibiotic-free LB plate, incubate in an incubator at 37 °C, pick colonies and inoculate them into a 10 mL LB vial for 12 h. Transfer with an inoculation amount of 1% to a 50 mL LB medium bottle. When the cell concentration reaches 0.4 - 0.6, prepare for the preparation of Escherichia coli competent cells. Pre-cool relevant reagents and instruments in advance. Prepare calcium chloride solution, calcium chloride and glycerol mixed solution, 1.5 mL EP tubes, and 50 mL centrifuge tubes and place them on ice. Control the temperature of the centrifuge to 4 °C. Aliquot 50 mL of the bacterial solution in a sterile workbench, centrifuge (8000 r·min -1 , 5 min), and discard the supernatant. Pipette 5 mL of calcium chloride solution and blow and suck to suspend, centrifuge (8000 r·min -1 , 5 min), and discard the supernatant. Pipette 5 mL of calcium chloride and glycerol mixed solution and blow and suck to suspend, and aliquot into pre-cooled and pre-equipped EP tubes, 100 μL per tube.

[0039] (2) Construction of recombinant Escherichia coli E.coli / pET28a-glyA

[0040] According to the gene sequence of serine hydroxymethyltransferase glyA from Methanocaldococcus jannaschii published on the NCBI website (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2), it was sent to GenScript (Suzhou) Co., Ltd. for synthesis. The PCR procedure was as follows: 95°C for 10 min; 95°C for 30 s; 58°C for 30 s; 72°C for 1 min; 72°C for 10 min, with 30 cycles. The obtained glyA gene fragment was purified and then ligated to the linearized plasmid pET-28a (P1 / P2) through the homologous recombinase In-Fusion Snap Assembly Master Mix (Takara), and transformed into competent E. coli BL21(DE3) cells to obtain transformants. The transformants were spread on LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. Positive colonies were picked, and colony PCR verification of single colonies was performed using P3 / P4 as primers through Taq DNA polymerase. After positive single colonies with the target band size were inoculated into a vial containing LB liquid medium and cultured for 12 h, plasmids were extracted. If the sequencing by GenScript (Suzhou) Co., Ltd. was correct, the recombinant Escherichia coli E. coli / pET28a-glyA was successfully constructed. The primer sequences involved are as follows:

[0041] P1: 5'-gggtcgcggatccgaattcgagctcatgttaaagcgtgaaatgaacat-3'

[0042] P2: 5'-tacccggtttacgcataacgtcgacaagcttgcggccgcact-3'

[0043] P3: 5'-agaggatcgagatctcgatcccgc-3'

[0044] P4: 5'-atccggatatagttcctcctttca-3'

[0045] Example 2: Construction of an engineered strain of serine hydroxymethyltransferase mutant

[0046] (1) Construction of single-point mutant strains E. coli / pET28a-glyA A271L 、E. coli / pET28a-glyA A292V 、E. coli / pET28a-glyA D392N

[0047] ​Since glyA is a bifunctional enzyme that can catalyze the production of L-serine from L-glycine or L-glycine from L-serine, in order to change the catalytic direction of this enzyme and make the catalysis favorable for the production of L-glycine, the present invention improves the enzyme activity of glyA for catalyzing the production of L-glycine by site-directed mutagenesis. Using pET28a-glyA as a template, PCR amplification of the mutation sites at 271, 292, and 392 was carried out with P5 / P6, P7 / P8, and P9 / P10 as primer pairs respectively, and then transformed into E. coli BL21(DE3) competent cells to obtain transformants. The transformants were spread on LB solid medium containing 50 μg / mL kanamycin and cultured at 37 °C for 12 h. Positive colonies were picked, and colony PCR verification of the single colonies was performed using P3 / P4 as primers and Taq DNA polymerase (ComWin Biotech, 2xEs Taq MasterMix). After the positive single colonies with the target band size were inoculated into a vial containing LB liquid medium and cultured for 12 h, the plasmids were extracted and sent to Genewiz for sequencing. If the mutation of the glyA mutation site was successfully verified, the single mutant strains E. coli / pET28a-glyA A271L 、E. coli / pET28a-glyA A292V 、E. coli / pET28a-glyA D392N were successfully constructed.

[0048] The primer sequences involved are as follows:

[0049] P5: 5’-ggcgtctacccgaacccgctgcctcatgctcacgttgttactacc-3’

[0050] P6: 5’-aacgtgagcatgaggcagcgggttcgggtagacgccagcagc-3’

[0051] P7: 5’-taccagcagcaggtcgtcaaaaacgctaaagcgatggtagaa-3’

[0052] P8: 5’-ctttagcgtttttgacgacctgctgctggtaagttttgaa-3’

[0053] P9: 5’-tgtgacgtgctggacaatatcaatgatgaagccgttatcgag-3’

[0054] P10: 5’-ggctggatgtgtgacgtgctggacaatatcaatgatgaagcc-3’

[0055] (2) Double mutant strain E. coli / pET28a-glyA A271L-A292V 、E. coli / pET28a-glyA A292V-D392N 、E. coli / pET28a-glyA D392N-A271L Construction

[0056] Using pET28a-glyA A271L 、pET28a-glyA A292V 、pET28a-glyA D392N as templates respectively, and using P7 / P8, P9 / P10, P5 / P6 as primers for PCR amplification to construct double mutant strains. The construction of the strains is the same as in step (1). If the sequencing is correct and it is verified that the mutation at the glyA mutation site is successful, then the double mutant strains E. coli / pET28a-glyA A271L-A292V 、E. coli / pET28a-glyA A292V-D392N 、E. coli / pET28a-glyA D392N-A271L are successfully constructed.

[0057] Example 3: Induced expression and enzymatic property determination of serine hydroxymethyltransferase wild-type strain and mutant engineering strains

[0058] (1) Induced expression and specific enzyme activity determination of glyA enzyme

[0059] Inoculate single colonies of the serine hydroxymethyltransferase wild-type strain and mutant engineering strains successfully constructed in Example 1 and Example 2 into 10 mL of LB liquid medium, culture at 37 °C for 12 h, then inoculate with an inoculum size of 1% into 50 mL of LB liquid medium, and culture until the OD 600 is approximately 0.8. Add IPTG and culture at 20 °C for 16 h. Wash the cells three times with PBS, resuspend the collected cells with PBS again, and break the cells with an ultrasonic crusher, with 1 s of breaking and 3 s of pausing for Escherichia coli, for a total of 15 min. Centrifuge at 10000 rpm for 20 min, and take the supernatant to run a protein gel. Purify the crude enzyme solution through a protein purification nickel column, verify the pure enzyme by running a protein gel, and set aside for use.

[0060] Respectively, the wild-type pure enzyme glyA and the mutated pure enzyme glyA A271L 、glyA A292V 、glyA D392N 、glyA A271L-A292V 、glyA A292V-D392N 、glyA D392N-A271LAdd it to 100 mM Tris-HCl buffer, which contains 20 mM L-serine and 20 mM tetrahydrofolic acid. React at 37 °C for 20 min, add 10% trichloroacetic acid to terminate the reaction, centrifuge at 10000 rpm for 10 min, and detect the L-glycine content in the supernatant by HPLC to represent the glyA enzyme activity. The definition of one unit of glyA enzyme activity (1 U) is the amount of enzyme required to produce 1 μmol of L-glycine per minute.

[0061] The experimental results are shown in Table 1. The specific enzyme activity of the original glyA enzyme catalyzing the formation of L-glycine is 0.5 U / mg. The specific enzyme activities of the mutants A271L, A292V, D392N, A271L-A292V, A292V-D392N, and D392N-A271L catalyzing the formation of L-glycine are increased to 0.75 U / mg, 0.8 U / mg, 0.7 U / mg, 1.09 U / mg, 1.52 U / mg, and 1.22 U / mg, respectively, which are 50%, 60%, 40%, 118%, 204%, and 144% higher than the original enzyme. Among them, compared with the original enzyme, the best mutant site for catalyzing the formation of L-glycine after mutation is the double combination mutation of alanine at position 292 mutated to valine and aspartic acid at position 392 mutated to asparagine (the amino acid sequence after mutation is shown in SEQ ID NO.3).

[0062] Table 1 Specific enzyme activities of the original enzyme and mutant enzymes

[0063]

[0064] (2) Determination of the optimal temperature and optimal pH of the original enzyme and mutant enzymes

[0065] Determination of the optimal temperature: The reaction system contains Tris-HCl (pH 8.0) with a final concentration of 100 mM, 20 mM L-serine, 20 mM tetrahydrofolic acid, and 100 μL of glyA / glyA A292V-D392N pure enzyme. Place them at different temperatures from 25 - 60 °C (at intervals of 5 °C), sample and measure after 30 min, and determine the specific enzyme activity of glyA according to the above enzyme activity measurement method.

[0066] Determination of the optimal pH: 100 mM Tris-HCl (pH 8.0) contains 20 mM L-serine, 20 mM tetrahydrofolic acid, and 100 μL of glyA / glyA A292V-D392N pure enzyme is placed in different buffer systems with pH values ranging from 6.0 to 9.5, sampled and measured after 30 min, and the specific enzyme activity of glyA is determined according to the above enzyme activity measurement method.

[0067] The experimental results are as Figure 1As shown, the experimental results are expressed as relative enzyme activity, and the highest specific enzyme activity at different temperatures or pH values is defined as 100% relative enzyme activity. Overall, glyA and glyA A292V-D392N showed little change in relative enzyme activity at different temperatures and pH values. The relative enzyme activity first increased and then decreased with the increase of temperature. The optimal temperatures of glyA and glyA A292V-D392N were both 35 °C. The relative enzyme activities of glyA and glyA A292V-D392N first increased and then decreased, and the optimal pH values were both 8.0, indicating that the enzyme preferred an alkaline environment.

[0068] Example 4: Whole-cell catalyzed production of L-glycine by wild-type serine hydroxymethyltransferase and mutant engineering bacteria

[0069] 1. Strains: E. coli / pET28a-glyA and E. coli / pET28a-glyA A292V-D392N were induced to express, and the cells were washed three times with PBS, and the cells were collected.

[0070] 2. Whole-cell catalyzed production of L-glycine: In 100 mM Tris-HCl (pH 8.0) buffer containing 100 mM L-serine and 100 mM tetrahydrofolic acid, the volume of wild-type / mutant bacteria added was 5 mL. The reaction was carried out at 35 °C for 24 h, and the reaction was terminated by adding 10% trichloroacetic acid. The supernatant was centrifuged at 10000 rpm for 10 min, and the yield and conversion rate of L-glycine were detected by HPLC.

[0071] The experimental results are as shown in Figure 4 and Table 2. For the whole-cell catalyzed production of L-glycine by the wild-type enzyme glyA, the yield was 1.66 g / L and the conversion rate was 22.12% in 12 h. For the whole-cell catalyzed production of L-glycine by the mutant bacteria glyA A292V-D392N the yield was 4.64 g / L and the conversion rate was 61.8% in 12 h, which was 2.8 times higher than that of the wild-type enzyme glyA, greatly improving the ability of serine hydroxymethyltransferase to catalyze the production of L-glycine from L-serine.

[0072] Table 2 Conversion rates of wild-type and mutant enzyme glyA A292V-D392N for whole-cell catalyzed production of L-glycine

[0073]

[0074] Example 5: Coupled tetrahydrofolic acid cycle whole-cell catalyzed production of L-glycine

[0075] 1. Construction of recombinant Escherichia coli E. coli / pET28a-gcvP, E. coli / pET28a-gcvH, and E. coli / pET28a-gcvT

[0076] In Example 4, the addition amount of the substrate tetrahydrofolic acid is 100 mM. Since tetrahydrofolic acid is expensive, in order to reduce the addition amount of tetrahydrofolic acid in catalytic synthesis, the present invention introduces a tetrahydrofolic acid cycle pathway, which involves three enzymes, gcvP, gcvH, and gcvT, that jointly catalyze ammonium ions, CO2, and methylene-tetrahydrofolic acid to generate tetrahydrofolic acid, as Figure 2 shown. According to the gene sequences of gcvP, gcvH, and gcvT of the tetrahydrofolic acid cycle pathway from Escherichia coli published on the NCBI website (the nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6), using the Escherichia coli BL21 genome as a template and P11 / P12, P13 / P14, and P15 / P16 as primers, they were obtained by PCR amplification. The PCR program was: 95°C, 10 min; 95°C, 30 s; 58°C, 30 s; 72°C, 1 min; 72°C, 10 min, for 30 cycles. The obtained gcvP, gcvH, and gcvT gene fragments were purified and then ligated to the linearized plasmid pET-28a (P17 / P18) through the homologous recombinase In-Fusion Snap Assembly Master Mix (Takara), and transformed into E. coli BL21(DE3) competent cells to obtain transformants. The transformants were spread on LB solid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 h. Positive colonies were picked and verified by colony PCR of single colonies using P3 / P4 as primers through Taq DNA polymerase; after positive single colonies with the target band size were inoculated into a vial containing LB liquid medium and cultured for 12 h, the plasmid was extracted and sent to Genewiz for correct sequencing, then the recombinant Escherichia coli E. coli / pET28a-gcvP, E. coli / pET28a-gcvH, and E. coli / pET28a-gcvT were successfully constructed. The primer sequences involved are as follows:

[0077] P11: 5’-tgggtcgcggatccgaattcatgacacagacgttaagcca-3’

[0078] P12: 5’-tcgagtgcggccgcaagcttttactggtattcgctaatcg-3’

[0079] P13: 5’-tgggtcgcggatccgaattcatgagcaacgtaccagcaga-3’

[0080] p14: 5'-tcgagtgcggccgcaagcttttactcgtcttctaacaatg-3'

[0081] p15: 5'-tgggtcgcggatccgaattcatggcacaacagactccttt-3'

[0082] p16: 5'-tcgagtgcggccgcaagctttcacgcgacggctttgccgt-3'

[0083] p17: 5'-atgggtcgcggatccgaattc-3'

[0084] p18: 5'-aagcttgcggccgcactcgag-3'

[0085] 2. Coupled tetrahydrofolate cycle whole-cell catalysis for the production of L-glycine

[0086] After induction and expression according to Example 4, cells were collected to obtain E. coli / pET28a-glyA A292V-D392N thalli. According to Example 3, after induction and expression of E. coli / pET28a-gcvP, E. coli / pET28a-gcvH, and E. coli / pET28a-gcvT, the cells were lysed, and the supernatant was reserved for use.

[0087] Whole-cell catalysis system: 200 mM L-serine, 5 mM - 10 mM tetrahydrofolate, 50 mM - 400 mM NaHCO3, 5 mM - 50 mM NADH, cell mass of 4 mL - 8 mL, 2 mL - 4 mL of cell supernatant of gcvP, gcvH, and gcvT, pH 7.0 - 9.0, temperature 30°C - 45°C, react for 24 h, sample and determine the yield and conversion rate of L-glycine by HPLC. The liquid phase peak diagram of the sample is as Figure 3 shown.

[0088] The experimental results are shown in Figure 4 as follows: After coupling serine hydroxymethyltransferase with the tetrahydrofolate cycle in the catalysis system, only 5 mM - 10 mM of tetrahydrofolate was added. At 12 h, the yield of L-glycine was 14.03 g / L, and the conversion rate increased to 93.5%. This strategy effectively reduced the addition amount of the substrate tetrahydrofolate, reduced the catalytic cost, and greatly improved the production efficiency of L-glycine.

[0089] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A serine hydroxymethyltransferase mutant, characterized in that The serine hydroxymethyltransferase with an amino acid sequence as shown in SEQ ID NO.2 is used as a parent, and the alanine at position 292 of the parent serine hydroxymethyltransferase is mutated to valine and the aspartic acid at position 392 is mutated to asparagine.

2. A gene encoding the serine hydroxymethyltransferase mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. A recombinant bacterium expressing the serine hydroxymethyltransferase mutant according to claim 1.

5. The recombinant bacterium according to claim 4, characterized in that The recombinant bacteria is Escherichia coli.

6. A method for synthesizing L-glycine using the recombinant bacteria according to claim 4 or 5, characterized in that: The following steps are involved: The recombinant bacteria according to claim 4 or 5 is used as a catalyst, and a buffer containing 50 mM to 200 mM L-serine, 5 mM to 10 mM tetrahydrofolate, 50 mM to 400 mM NaHCO3, 5 mM to 50 mM NADH, glycine decarboxylase, glycine cleavage system H protein and glycine cleavage system aminomethyltransferase is used as a reaction system to catalyze the synthesis of L-glycine.

7. The method according to claim 6, characterized in that The catalytic synthesis temperature is 30°C to 45°C, and the time is 12 h to 24 h; And / or, the pH value of the reaction system is 7.0-9.

0.

8. The method according to claim 6, characterized in that The nucleotide sequence of the glycine decarboxylase is shown in SEQ ID NO.3; And / or, the nucleotide sequence of the glycine cleavage system H protein is shown in SEQ ID NO.4; And / or, the nucleotide sequence of the glycine cleavage system aminomethyltransferase is shown as SEQ ID NO.

5.

9. Use of the serine hydroxymethyltransferase mutant according to claim 1 or the recombinant bacterium according to claim 4 or 5 in the preparation of L-glycine or a product containing L-glycine.

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