Serine hydroxymethyltransferase mutant and application thereof in production of L-serine

Abstract

The invention discloses a serine hydroxymethyltransferase mutant and an application thereof in production of L-serin.The serine hydroxymethyltransferase mutant with the specific enzyme activity remarkably improved is obtained by conducting saturated mutation on serine hydroxymethyltransferase from escherichia coli and conducting screening, and the serine hydroxymethyltransferase mutant is applied to synthesis of L-serine. The serine hydroxymethyltransferase disclosed by the invention can be used for effectively improving the efficiency of catalyzing glycine and formaldehyde to synthesize L-serine by using the serine hydroxymethyltransferase, reducing the enzyme dosage, remarkably reducing the production cost and reducing the separation difficulty of downstream products.

Description

(I) Technical Field

[0002] This invention belongs to the field of enzyme engineering technology and relates to a serine hydroxymethyltransferase mutant and its application in L-serine production. (II) Background Technology

[0004] L-Serine (L-Ser), a non-essential amino acid, is widely involved in several important metabolic processes, including amino acid synthesis, lipid synthesis, and purine synthesis. It is also a significant source of active one-carbon units in the body. In recent years, L-serine has shown great promise in the food, biopharmaceutical, and animal feed industries. In the food industry, L-serine is used as a food additive to supplement essential nutrients. In the pharmaceutical industry, L-serine and its derivatives are added to drugs to regulate cell proliferation and differentiation, redox balance, nervous system function, and immune function. In the animal feed industry, L-serine is also widely added to regulate intestinal health and improve growth performance in livestock.

[0005] Currently, the industrial production of L-serine mainly uses chemical methods, but these methods suffer from problems such as the generation of toxic byproducts, high costs, low conversion efficiency, and severe environmental pollution. Compared with traditional chemical synthesis methods, the biosynthetic method of producing L-serine from glycine via enzymatic catalysis has advantages such as mild reaction and being environmentally friendly, and has great development potential. However, the lack of highly active key enzymes and the low conversion efficiency severely limit the development of enzymatic synthesis of L-serine. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a serine hydroxymethyltransferase mutant and its application in L-serine production. This invention involves saturating and mutagenizing a serine hydroxymethyltransferase derived from Escherichia coli to screen for a serine hydroxymethyltransferase mutant with significantly increased enzyme activity. This mutant is then applied to the synthesis of L-serine, which can effectively improve the efficiency of serine hydroxymethyltransferase in catalyzing the synthesis of L-serine from glycine and formaldehyde, significantly reduce production costs, and reduce the difficulty of separating downstream products.

[0008] The technical solution adopted in this invention is:

[0009] In a first aspect, the present invention provides a serine hydroxymethyltransferase mutant, which is obtained by single or combined mutations at positions 5, 18, 116, 121, 127, 241, 245, 246, 257, 332, 335, 346, 355, or 356, 357, 358, or 396 of the amino acid sequence of the serine hydroxymethyltransferase derived from Escherichia coli shown in SEQ ID No. 2.

[0010] Furthermore, the mutation sites and mutation forms corresponding to the mutants are constituted as shown in Table 1, which includes single-point mutation forms and all possible combinations.

[0011] Table 1 Beneficial mutation sites and their mutation forms

[0012]

[0013] Note: a: indicates a synonymous mutation, where the codon for phenylalanine (F) at position 257 changes from TTC to TTT.

[0014] The amino acid sequence of the wild-type serine hydroxymethyltransferase described in this invention is shown in SEQ ID NO:2, and the corresponding encoding gene is shown in SEQ ID NO:1.

[0015] The mutants described in this invention include:

[0016] (1) Single-point mutants: E5R, A18X1, V116M, L121I, L127E, A241X2, S245X3, E246X4, F257X5, E332X6, A335X7, K346X8, S355X9, P356S, F357X10, V358X11, E396X12;

[0017] (2) Two-point mutants: E246A / E5R, E246A / A18X1, E246A / V116M, E246A / L121I, E246A / L127E, E246A / A241X2, E246A / S245X3, E246A / F257X5, E246A / E332X6, E246A / A335X7, E246A / K346X8, E246A / S355X9, E246A / P356S, E246A / F357X10, E246A / V358X11, E246A / E396X12;

[0018] (3) Three-point mutants: E246A / A241S / E5R, E246A / A241S / A18X1, E246A / A241S / V116M, E246A / A241S / L121I, E246A / A241S / L127E, E246A / A241S / S245X3, E246A / A241S / F257X5, E246A / A24 1S / E332X6, E246A / A241S / A335X7, E246A / A241S / K346X8, E246A / A241S / S355X9, E246A / A241S / P356S, E246A / A241S / F357X10, E246A / A241S / V358X11, E246A / A241S / E396X12;

[0019] (4) Four-point mutants: E246A / A241S / S245V / E5R, E246A / A241S / S245V / A18X1, E246A / A241S / S245V / V116M, E246A / A241S / S245V / L121I, E246A / A241S / S245V / L127E, E246A / A241S / S245V / F257X5, E246A / A241S / S245V / E332X6, E246A / A241S / S245V / A335X7, E246A / A241S / S245V / K346X8, E246A / A241S / S245V / S355X9, E246A / A241S / S 245V / P356S, E246A / A241S / S245V / F357X10, E246A / A241S / S245V / V358X11, E246A / A241S / S245V / E396X12;

[0020] (5) Five-point mutants: E246A / A241S / S245V / K346R / E5R, E246A / A241S / S245V / K346R / A18X1, E246A / A241S / S245V / K346R / V116M, E246A / A241S / S245V / K346R / L121I, E246A / A241S / S245V / K346R / L127E, E246A / A241S / S245V / K346R / F257X5, E246A / A241S / S24 5V / K346R / E332X6, E246A / A241S / S245V / K346R / A335X7, E246A / A241S / S245V / K346R / S355X9, E246A / A241S / S245V / K346R / P356S, E246A / A241S / S245V / K346R / F357X10, E246A / A241S / S245V / K346R / V358X11, E246A / A241S / S245V / K346R / E396X12;

[0021] (6) Six-point mutants: E246A / A241S / S245V / K346R / V116M / E5R, E246A / A241S / S245V / K346R / V116M / A18X1, E246A / A241S / S245V / K346R / V116M / L121I, E246A / A241S / S245V / K346R / V116M / L127E, E246A / A241S / S245V / K346R / V116M / F257X5, E246A / A241S / S245V / K346R / V116M / E332X6 , E246A / A241S / S245V / K346R / V116M / A335X7, E246A / A241S / S245V / K346R / V116M / S355X9, E246A / A241S / S245V / K346R / V116M / P356S, E 246A / A241S / S245V / K346R / V116M / F357X10, E246A / A241S / S245V / K346R / V116M / V358X11, E246A / A241S / S245V / K346R / V116M / E396X12;

[0022] (7) Seven - point mutants: E246A / A241S / S245V / K346R / V116M / V358I / E5R, E246A / A241S / S245V / K346R / V116M / V358I / A18X1, E246A / A241S / S245V / K346R / V116M / V358I / L121I, E246A / A241S / S245V / K346R / V116M / V358I / L127E, E246A / A241S / S245V / K346R / V116M / V358I / F257X5, E246A / A241S / S245V / K346R / V116M / V358I / E332X6, E246A / A241S / S245V / K346R / V116M / V358I / A335X7, E246A / A241S / S245V / K346R / V116M / V358I / S355X9, E246A / A241S / S245V / K346R / V116M / V358I / P356S, E246A / A241S / S245V / K346R / V116M / V358I / F357X10, E246A / A241S / S245V / K346R / V116M / V358I / E396X12;

[0023] (8) Eight - point mutants: E246A / A241S / S245V / K346R / V116M / V358I / E5R / A18X1, E246A / A241S / S245V / K346R / V116M / V358I / E5R / L121I, E246A / A241S / S245V / K346R / V116M / V358I / E5R / L127E, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257X5, E246A / A241S / S245V / K346R / V116M / V358I / E5R / E332X6, E246A / A241S / S245V / K346R / V116M / V358I / E5R / A335X7, E246A / A241S / S245V / K346R / V116M / V358I / E5R / S355X9, E246A / A241S / S245V / K346R / V116M / V358I / E5R / P356S, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F357X10, E246A / A241S / S245V / K346R / V116M / V358I / E5R / E396X12;

[0024] (9) Nine-point mutant: E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / A18X1,E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / L121I,E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / L127E,E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / E332X6,E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / A335X7,E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / S355X9,E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / P356S,E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / F357X10,E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) a / E396X12;

[0025] Where X1 represents the beneficial mutation forms G, L, M, E, F at site A18; X2 represents the beneficial mutation forms P, S, W, C, T at site A241; X3 represents the beneficial mutation forms D, K, C, W, F, I, G, L at site S245; X4 represents the beneficial mutation forms V, A, P, T at site E246; and X5 represents the beneficial mutation forms G, F257(TTT) at site F257. aThat is, the codon for phenylalanine mutates from TTC to TTT. X6 represents the beneficial mutation form C,I at the E332 site, X7 represents the beneficial mutation form I,F,Y,N,E,R,L at the A335 site, X8 represents the beneficial mutation form P,R,F,A,M,Q at the K346 site, X9 represents the beneficial mutation form G,E at the S355 site, X10 represents the beneficial mutation form K,I,G,S,M,P,A,H at the F357 site, X11 represents the beneficial mutation form Y,D,Q,I at the V358 site, and X12 represents the beneficial mutation form S,Q at the E396 site.

[0026] Furthermore, the mutant is obtained by performing one of the following mutations on the amino acid sequence shown in SEQ ID No. 2: (1) glutamic acid at position 246 is mutated to alanine, E246A; (2) glutamic acid at position 246 is mutated to alanine, and alanine at position 241 is mutated to serine, E246A / A241S; (3) glutamic acid at position 246 is mutated to alanine, alanine at position 241 is mutated to serine, and serine at position 245 is mutated to valine, E246A / A241S / S245V; (4) glutamic acid at position 246 is mutated to alanine, alanine at position 241 is mutated to serine, and serine at position 245 is mutated to valine. (5) The glutamic acid at position 246 is mutated to alanine, the alanine at position 241 is mutated to serine, the serine at position 245 is mutated to valine, the lysine at position 346 is mutated to arginine, and the threonine at position 116 is mutated to methionine, E246A / A241S / S245V / K346R / V116M; (6) The glutamic acid at position 246 is mutated to alanine, the alanine at position 241 is mutated to serine, the serine at position 245 is mutated to valine, and the lysine at position 346 is mutated to arginine. The acid at position 116 is mutated to arginine, the threonine at position 116 is mutated to methionine, and the valine at position 358 is mutated to isoleucine, E246A / A241S / S245V / K346R / V116M / V358I; (7) The glutamic acid at position 246 is mutated to alanine, the alanine at position 241 is mutated to serine, the serine at position 245 is mutated to valine, the lysine at position 346 is mutated to arginine, the threonine at position 116 is mutated to methionine, the valine at position 358 is mutated to isoleucine, and the methionine at position 5 is mutated to arginine, E246A / A241S / S245V / K3 46R / V116M / V358I / E5R; (8) Glutamic acid at position 246 is mutated to alanine, alanine at position 241 is mutated to serine, serine at position 245 is mutated to valine, lysine at position 346 is mutated to arginine, threonine at position 116 is mutated to methionine, valine at position 358 is mutated to isoleucine, methionine at position 5 is mutated to arginine, and the codon of phenylalanine (F) at position 257 is mutated from TTC to TTT, A241S / K346R / S245V / E246A / V116M / V358I / E5R / F257(TTT);(9) The glutamic acid at position 246 is mutated to alanine, the alanine at position 241 to serine, the serine at position 245 to valine, the lysine at position 346 to arginine, the threonine at position 116 to methionine, the valine at position 358 to isoleucine, the glutamic acid at position 5 to arginine, the codon for phenylalanine (F) at position 257 is changed from TTC to TTT, and the alanine at position 335 is changed to asparagine. The sequence is: E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / A335N.

[0027] Secondly, the present invention provides the coding gene of the serine hydroxymethyltransferase mutant, a recombinant expression vector comprising the coding gene, and a recombinant genetically engineered bacterium; the recombinant expression vector is based on pET-21b(+), and the recombinant genetically engineered bacterium is based on pET-21b(+). E. coli BL21(DE3) is the host bacterium.

[0028] Thirdly, the present invention provides an application of the aforementioned serine hydroxymethyltransferase mutant in catalyzing the production of L-serine from glycine.

[0029] Furthermore, the application method is as follows: using crude enzyme solution obtained by inducing expression of recombinant genetically engineered bacteria expressing the serine hydroxymethyltransferase mutant and then crushing and extracting the wet bacterial cells as a catalyst, pyridoxal phosphate is added, and the reaction is stirred at 30-40℃ and 200-400 rpm for 3 h. Then, the substrates glycine and tetrahydrofolate are added, the pH is adjusted to 8.2, and the reaction system is replenished with deionized water. A 37% formaldehyde aqueous solution is slowly added to prevent excessive formaldehyde from inhibiting enzyme activity. The reaction is stirred at 20-45℃ and 100-900 rpm until complete to obtain L-serine.

[0030] Furthermore, in the reaction system, the amount of crude enzyme solution added is 20-50 g / L (preferably 25 g / L) based on the weight of the wet bacterial cells before crushing; the final concentration of pyridoxal phosphate added is 0.05-0.2 g / L (preferably 0.06 g / L); the final concentration of glycine added is 10-300 g / L (preferably 200 g / L); and the final concentration of tetrahydrofolic acid added is 0.25-6 mM (preferably 6 mM). The amount of the 37% formaldehyde aqueous solution added is 20% based on the volume of the reaction system.

[0031] Furthermore, the crude enzyme solution is prepared according to the following method:

[0032] (1) The recombinant genetically engineered bacteria were inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 10 h. Then, the bacteria were transferred to LB liquid medium at an inoculation rate of 2% of the medium volume and cultured at 37℃ and 200 rpm for 2 h. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.06 g / L. The bacteria were induced to express serine hydroxymethyltransferase at 28℃ and 200 rpm for 16 h. The bacterial solution was collected, centrifuged, and then reconstituted with LB liquid medium to make the wet bacterial concentration 100 g / L, which was used as the whole bacterial solution.

[0033] (2) Add hexadecyltrimethylammonium bromide (CTAB) powder to the whole bacterial culture in step (1). After the CTAB is completely dissolved, place the mixture in a shaker at 37°C and 200 rpm for 1.5 h. Through the action of CTAB and the shaking treatment, the bacterial cells are broken, and the intracellular enzymes are released into the solution to obtain the crude enzyme solution. The final concentration of CTAB is 1 g / L.

[0034] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0035] This invention screens for serine hydroxymethyltransferases with enhanced enzyme activity through directed evolution. The specific enzyme activity (per unit wet weight of bacteria) of the serine hydroxymethyltransferase mutant is up to 5 times higher than that of the wild type.

[0036] The serine hydroxymethyltransferase mutant of the present invention is used to catalyze the production of L-serine from glycine. It reduces the amount of enzyme required by at least 50% while achieving a similar substrate conversion rate and a higher product yield, thereby effectively reducing production costs. (iv) Description of the attached drawings

[0038] Figure 1 This is an SDS-PAGE gel image of wild-type serine hydroxymethyltransferase.

[0039] Figure 2 This is a standard curve showing the concentration of L-serine standard samples versus the HPLC peak area.

[0040] Figure 3 This is a standard curve showing the relationship between the concentration of glycine standard samples and the HPLC peak area. (V) Detailed Implementation Methods

[0042] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0043] In this embodiment of the invention, the LB liquid culture medium consists of 10 g / L peptone, 10 g / L sodium chloride, and 5 g / L yeast extract, with deionized water as the solvent. The LB solid culture medium is the LB liquid culture medium with 20 g / L agar added.

[0044] Example 1: Construction and Induction of Expression Vector for Wild-Type Serine Hydroxymethyltransferase

[0045] 1. Construction of wild-type strains

[0046] With Escherichia coli ( Escherichia coli Using the genome of MG1655 as a template, the wild-type serine hydroxymethyltransferase SHMT gene fragment (NCBI accession number P0A825.1) was amplified by PCR and designated as serine hydroxymethyltransferase EcSHMT. The gene sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2. The EcSHMT gene was cloned into the NdeI and HindIII restriction sites of the expression plasmid pET21b to obtain the recombinant plasmid pET21b-EcSHMT. The recombinant plasmid pET21b-EcSHMT was transformed into the expression host *Escherichia coli*. E. coli BL21(DE3) was plated on LB agar plates containing 0.05 mg / mL ampicillin resistance and incubated overnight at 37°C inverted position. Randomly selected clones were then subjected to colony PCR identification and sequencing verification. The results showed that the recombinant expression plasmid pET21b-EcSHMT containing the serine hydroxymethyltransferase gene was successfully transformed into [the desired colony]. E. coli Recombinant genetically engineered bacteria were obtained from BL21(DE3). E. coli The recombinant plasmid pET21b-EcSHMT was extracted from the bacterial culture that had been successfully transformed (verified by sequencing) and stored at -80°C. Sequencing was performed by Beijing Qingke Biotechnology Co., Ltd.

[0047] 2. Induced expression of wild-type strains

[0048] Recombinant bacteria E. coliBL21(DE3) / pET21b-EcSHMT was inoculated into 10 mL of LB liquid medium and cultured at 37℃ and 200 rpm for 10 h. Then, 2 mL of the culture was transferred to 100 mL of LB liquid medium and cultured at 37℃ and 200 rpm for 2 h. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.06 g / L, and the culture was induced for serine hydroxymethyltransferase expression at 28℃ and 200 rpm for 16 h. The bacterial culture was collected, centrifuged, and then reconstituted with LB liquid medium to a wet cell concentration of 100 g / L, which was used as the whole bacterial culture. A portion of the whole bacterial culture was ultrasonically disrupted at 240 W for 2 s, paused for 3 s, and repeated for 20 min. 100 μL of the disrupted culture was centrifuged at 12000 rpm for 20 min, and the supernatant and precipitate were collected separately. The whole bacteria, supernatant, and precipitate were appropriately diluted and then subjected to SDS-PAGE for verification to detect the expression level of serine hydroxymethyltransferase. The results are as follows: Figure 1 As shown, serine hydroxymethyltransferase is expressed in almost completely soluble form.

[0049] SEQ ID No.1

[0050]

[0051] SEQ ID No.2

[0052] MLKREMNIADYDAELWQAMEQEKVRQEEHIELIASENYTSPRVMQAQGSQLTNKYAEGYPGKRYYGGCEYVDIVEQLAIDRAKELFGADYANVQPHSGSQANFAVYTALLEPGDTVLGMNLAHGGHLTHGSPVNFSGKLYNIVPYGIDATGHIDYADLEKQAKEHKPKMIIGGFSAAYSGVVDWAKMREIADSIGAYLFVDMAHVAGLV AAGVYPNPVPHAHVVTTTTHKTLAGPRGGLILAKGGSEELYKKLNSAVFPGGQGGPLMHVIAGKAVALKEAMEPEFKTYQQQVAKNAKAMVEVFLERGYKVVSG GTDNHLFLVDLVDKNLTGKEADAALGRANITVNKNSVPNDPKSPFVTSGIRVGTPAITRRGFKEAEAKELAGWMCDVLDSINDEAVIERIKGKVLDICARYPVYA

[0053] Example 2: Preparation of crude enzyme solution and enzyme-catalyzed reaction

[0054] 1. Preparation of crude enzyme solution

[0055] Hexadecyltrimethylammonium bromide (CTAB) powder was added to the whole bacterial culture of Example 1 to make the final concentration of CTAB 1 g / L. After the CTAB was completely dissolved, the mixture was placed in a shaker at 37°C and 200 rpm for 1.5 h. Through the action of CTAB and the shaking treatment, the bacterial cells were broken, and the intracellular enzymes were released into the solution, thereby obtaining the crude enzyme solution.

[0056] 2. Enzyme-catalyzed reaction

[0057] The enzyme-catalyzed reaction system consisted of 10 mL of the following components and procedures: 25 g / L glycine, 3 mM tetrahydrofolate, 16 mL / L β-mercaptoethanol, and 0.125 g / L pyridoxal phosphate (PLP) were added sequentially to 60 mM phosphate buffer (pH 8.0, formulation: accurately weigh 5.59 g of dipotassium hydrogen phosphate and 0.14 g of potassium dihydrogen phosphate, dissolved in 800 mL of water, and brought to a final volume of 1 L). After complete dissolution, 20 g / L (based on the weight of the wetted bacterial cells before lysis) of crude enzyme solution was added, followed by 200 μL of formaldehyde (final concentration 268 mM), and the mixture was thoroughly mixed. The system was incubated at 37℃ and 250 rpm with shaking for 15 min, followed by a boiling water bath for 15 min to terminate the reaction. The supernatant was collected by centrifugation and used for subsequent detection of the substrate glycine and the product L-serine, as well as for specific enzyme activity calculation.

[0058] 3. Determination of substrate and product concentrations

[0059] Dilute the supernatant from step 2 with ddH2O to an appropriate factor, mix thoroughly, and then transfer 100 μL of the diluted sample into a new 1.5 mL EP tube. Add 500 μL of borate buffer and 300 μL of derivatization reagent to the EP tube sequentially, and incubate at 60℃ and 600 rpm in the dark for 1 h for derivatization. Filter the derivatized sample through a 0.22 μm organic filter membrane to remove some impurities. Analyze the peak areas of glycine and L-serine in the sample using high-performance liquid chromatography (HPLC). Correct for glycine and L-serine standard curves, and calculate the actual glycine and L-serine content (denoted as c, g / L).

[0060] High-performance liquid chromatography (HPLC) was performed using a Thermo Fisher Vanquish Core HPLC system, C 18 Column (4.6) The parameters set during the detection process (250 mm, 5 μm) were as follows: injection volume 10 μL, flow rate 0.8 mL / min, column temperature 30℃, and UV detection wavelength 260 nm. Two mobile phases were used: mobile phase A (pure acetonitrile) and mobile phase B (826 mL ddH₂O, 170 mL acetonitrile, 2 mL triethylamine, and 2 mL acetic acid, pH 4.9). The HPLC elution program settings are shown in Table 2.

[0061] The borate buffer was a mixture of 0.2 mol / L boric acid and 0.05 mol / L borax prepared with deionized water, with a pH of 9.0; the derivatization reagent was 0.27 g CNBF (3,5-dinitro-4-chloro-trifluoromethylbenzene) added to 10 mL of acetonitrile.

[0062] L-Serine Standard Curve: L-Serine standard solutions of 0.1–2 g / L were prepared using deionized water and subjected to the same derivatization treatment as the samples. Peak areas at different concentrations were measured. A standard curve was plotted with peak area on the x-axis and L-Serine concentration on the y-axis. The results are shown below. Figure 2 As shown.

[0063] Glycine standard curve: Glycine standard solutions of 0.1–2 g / L were prepared using deionized water and subjected to the same derivatization treatment as the samples. Peak areas at different concentrations were measured. A standard curve was plotted with peak area on the x-axis and glycine concentration on the y-axis. The results are shown below. Figure 3 As shown.

[0064] Table 2 Liquid Chromatography Elution Procedure

[0065]

[0066] 4. Calculation of specific enzyme activity

[0067] Based on the definition of enzyme activity per unit cell (the amount of enzyme required to produce 1 μmol L-serine per minute per gram of wet cells is 1 U / g), the specific enzyme activity of the crude enzyme solution is calculated according to the formula.

[0068] Crude enzyme solution specific enzyme activity U / g =

[0069] c: Actual measured L-serine content in the conversion solution (g / L), 0.01: Total reaction volume 10 mL; 10 6 : Conversion factor from mol / L to μmol / L; 105: Molecular weight of L-serine (relative molar mass g / mol); 0.25: Mass of crude enzyme solution (g); 15: Represents reaction time of 15 min.

[0070] Example 3: Construction of a single mutant library of serine hydroxymethyltransferase and screening of dominant mutants

[0071] 1. Screening for mutation sites:

[0072] To improve the activity of serine hydroxymethyltransferase and its affinity for coenzymes, thereby reducing the amount of enzyme and coenzyme required, 3D models such as PDB accession numbers 1DFO, 1EQB, and 3G8M were used as references. Through structural comparison, the approximate interaction mode of serine hydroxymethyltransferase-PLP-Gly-5,10-methylenetetrahydrofolate (MTHF) was determined. The results showed that the active site of serine hydroxymethyltransferase is assembled from three subunits. PLP, Gly, and formaldehyde each interact with the amino acid residues of the active site of only one subunit, while the methylenetetrahydropteroyl glutamate of the coenzyme MTHF is exposed at the interaction interface of the three subunits. Based on the above analysis, the amino acid residues in the active pocket of serine hydroxymethyltransferase that directly interact with PLP, substrates (including formaldehyde and Gly), and coenzyme MTHF were preliminarily identified. Combined with the interaction information reported in the literature, the amino acid residues within 5 Å around MTHF that may interact with it (derived from 3 different subunits) were identified as the hot spots for modification, with particular attention paid to the amino acid residues around the p-aminobenzoylglutamate moiety. At the same time, the sites involved in the literature were included in the candidate sites, and a total of 25 candidate saturation mutation sites were identified (Table 3).

[0073] Table 3. Selection of saturation mutation sites for serine hydroxymethyltransferase

[0074]

[0075] 2. Unit point mutation

[0076] Using the recombinant plasmid pET21b-EcSHMT from Example 1 as a template, site-directed saturation mutagenesis was performed at 25 sites using primers listed in Table 4. The mutagenesis was then transformed into *E. coli* BL21(DE3) using the same method as in Example 1. Colonies were picked from the plates for colony PCR and sequencing verification, resulting in the construction of 475 single-saturation mutants. Induction expression and SDS-PAGE verification were performed in batches using the same method as in Example 1.

[0077] The specific enzyme activity of crude enzyme solutions of 475 single-saturation mutants was determined using the same method as in Example 2 and compared with that of crude wild-type serine hydroxymethyltransferase. The results are shown in Table 5. Among the 475 mutants, 60 single-saturation mutants showed a significant increase in specific enzyme activity compared to the wild type. Among them, A241T, E246A, F257G, A335N, K346P, K346R, S355E, and V358Q were the preferred single-point mutants, with specific enzyme activity increased by more than 3 times compared to the wild-type enzyme. Next, V116M, E332C, K346F, K346A, and V358Y were the second-best single-point mutants, with specific enzyme activity increased by more than 2 times. The remaining 45 single-point mutants showed a specific enzyme activity increased by 1-2 times and can be used for further combined mutations.

[0078] Table 4. Primers used for single mutations

[0079]

[0080] Note: The underlined NNK degenerate codon can encode 32 possible codons, covering 20 natural amino acids and 1 stop codon. The remaining 19 amino acid mutants were obtained by bacterial selection and sequencing.

[0081] Table 5. Summary of screening results for single-point saturation mutation libraries

[0082]

[0083] Note: + indicates an increase of 0-100% in enzyme activity, ++ indicates an increase of 100-200% in enzyme activity, +++ indicates an increase of 201-300% in enzyme activity, and ++++ indicates an increase of 300%-400% in enzyme activity.

[0084] Example 4: Construction, expression, and screening of serine hydroxymethyltransferase combinatorial mutants

[0085] Based on the selection of E246A (denoted as M1) through single-site saturation mutation screening, further research on combined saturation mutations was conducted. All advantageous mutations were combined and superimposed to construct a library of two to nine-site combined mutants (a total of 315 strains), specifically including: 56 two-site mutants, 47 three-site mutants, 38 four-site mutants, 33 five-site mutants, 39 six-site mutants, 35 seven-site mutants, 34 eight-site mutants, and 33 nine-site mutants. The specific enzyme activity of the crude enzyme solutions of the 315 combined mutants was measured using the same method as in Example 2 and compared with that of wild-type serine hydroxymethyltransferase. Eight representative mutants with significantly enhanced enzyme activity were screened from each combined mutant, as shown in Table 6. Among them, the seven-site combined mutant E246A / A241S / S245V / K346R / T116M / V358I / E5R showed the most significant increase in cell enzyme activity, demonstrating a synergistic effect of the mutation sites.

[0086] Table 6. Summary of representative combination mutation screening results

[0087]

[0088] Note: + indicates that the specific enzyme activity of the combined mutant is increased by 0-10% compared with the parent, ++ indicates an increase of 10-20%, and +++ indicates an increase of 20-30%. a: indicates a synonymous mutation in which the codon of phenylalanine (F) at position 257 is changed from TTC to TTT.

[0089] Example 5: Application of dominant mutant in the catalytic synthesis of L-serine

[0090] The dominant mutants A241T, E246A, F257G, A335N, K346P, K346R, S355E, V358Q, M2, M3, M4, M5, M6, M7, M8, and M9 obtained in Examples 3 and 4 were induced to express according to the method in Example 1, and crude enzyme solution was prepared as a catalyst according to the method in Example 2. The amount of catalyst used was based on the weight of the wet bacterial cells before lysis.

[0091] The final concentration of the catalytic reaction system (500 mL) consisted of: 25 g / L crude enzyme solution and 0.06 g / L PLP, stirred at 37℃ and 250 rpm for 3 h to ensure sufficient contact. Then, 200 g / L glycine and 6 mM tetrahydrofolate were added sequentially, and after complete dissolution, the pH of the reaction solution was adjusted to 8.2 with 20% NaOH. The volume of the reaction solution was then brought to 500 mL with ddH2O. A 37% formaldehyde aqueous solution was slowly added (100 mL of formaldehyde aqueous solution was added over 16-21 h) to prevent excessive formaldehyde from inhibiting enzyme activity. The reaction was continued at 37℃ and 250 rpm for 16-21 h.

[0092] After the reaction was completed, the reaction solution was thoroughly mixed and diluted with deionized water to an appropriate factor so that the concentrations of substrate and product were within the range of the standard curve. The yields of the substrate glycine and the product L-serine in the reaction solution were determined according to the method in Example 2. Under the same conditions, the catalyst was replaced with crude enzyme solution of wild-type serine hydroxymethyltransferase, and the addition amount was changed to 50 g / L. The results are shown in Table 7. The catalyst dosage of each mutant was reduced, and higher substrate conversion and product yield were achieved.

[0093] Table 7. Catalytic activity of mutants

[0094]

Claims

1. A serine hydroxymethyltransferase mutant, characterized in that, The mutant is a single mutation or combined mutation of the 5th, 18th, 116th, 121st, 127th, 241st, 245th, 246th, 257th, 332nd, 335th, 346th, 355th, or 356th, 357th, 358th or 396th amino acid in the serine hydroxymethyltransferase amino acid sequence derived from Escherichia coli shown in SEQ ID No.

2.

2. The mutant of claim 1, wherein The mutant includes: (1) single point mutant: E5R, A18X1, V116M, L121I, L127E, A241X2, S245X3, E246X4, F257X5, E332X6, A335X7, K346X8, S355X9, P356S, F357X10, V358X11, E396X12; wherein X1 represents G, L, M, E, F; X2 represents P, S, W, C, T; X3 represents D, K, C, W, F, I, G, L; X4 represents V, A, P, T; X5 represents G, F257(TTT), wherein F257(TTT) represents the codon of phenylalanine at position 257 being mutated from TTC to TTT; X6 represents C, I; X7 represents I, F, Y, N, E, R, L; X8 represents P, R, F, A, M, Q; X9 represents G, E; X10 represents K, I, G, S, M, P, A, H; X11 represents Y, D, Q, I; X12 represents S, Q; (2) double point mutant: E246A / E5R, E246A / A18X1, E246A / V116M, E246A / L121I, E246A / L127E, E246A / A241X2, E246A / S245X3, E246A / F257X5, E246A / E332X6, E246A / A335X7, E246A / K346X8, E246A / S355X9, E246A / P356S, E246A / F357X10, E246A / V358X11, E246A / E396X12; (3) three point mutant: E246A / A241S / E5R, E246A / A241S / A18X1, E246A / A241S / V116M, E246A / A241S / L121I, E246A / A241S / L127E, E246A / A241S / S245X3, E246A / A241S / F257X5, E246A / A241S / E332X6, E246A / A241S / A335X7, E246A / A241S / K346X8, E246A / A241S / S355X9, E246A / A241S / P356S, E246A / A241S / F357X10, E246A / A241S / V358X11, E246A / A241S / E396X12; (4) Four-point mutants: E246A / A241S / S245V / E5R, E246A / A241S / S245V / A18X1, E246A / A241S / S245V / V116M, E246A / A241S / S245V / L121I, E246A / A241S / S245V / L127E, E246A / A241S / S245V / F257X5, E246A / A241S / S245V / E332X6, E246A / A241S / S245V / A335X7, E246A / A241S / S245V / K346X8, E246A / A241S / S245V / S355X9, E246A / A241S / S245V / P356S, E246A / A241S / S245V / F357X10, E246A / A241S / S245V / V358X11, E246A / A241S / S245V / E396X12; (5) Five-point mutants: E246A / A241S / S245V / K346R / E5R, E246A / A241S / S245V / K346R / A18X1, E246A / A241S / S245V / K346R / V116M, E246A / A241S / S245V / K346R / L121I, E246A / A241S / S245V / K346R / L127E, E246A / A241S / S245V / K346R / F257X5, E246A / A241S / S245V / K346R / E332X6, E246A / A241S / S245V / K346R / A335X7, E246A / A241S / S245V / K346R / S355X9, E246A / A241S / S245V / K346R / P356S, E246A / A241S / S245V / K346R / F357X10, E246A / A241S / S245V / K346R / V358X11, E246A / A241S / S245V / K346R / E396X12; (6) Six-point mutants: E246A / A241S / S245V / K346R / V116M / E5R, E246A / A241S / S245V / K346R / V116M / A18X1, E246A / A241S / S245V / K346R / V116M / L121I, E246A / A241S / S245V / K346R / V116M / L127E, E246A / A241S / S245V / K346R / V116M / F257X5, E246A / A241S / S245V / K346R / V116M / E332X6, E246A / A241S / S245V / K346R / V116M / A335X7, E246A / A241S / S245V / K346R / V116M / S355X9, E246A / A241S / S245V / K346R / V116M / P356S, E246A / A241S / S245V / K346R / V116M / F357X10, E246A / A241S / S245V / K346R / V116M / V358X11, E246A / A241S / S245V / K346R / V116M / E396X12; (7) Seven-point mutants: E246A / A241S / S245V / K346R / V116M / V358I / E5R, E246A / A241S / S245V / K346R / V116M / V358I / A18X1, E246A / A241S / S245V / K346R / V116M / V358I / L121I, E246A / A241S / S245V / K346R / V116M / V358I / L127E, E246A / A241S / S245V / K346R / V116M / V358I / F257X5, E246A / A241S / S245V / K346R / V116M / V358I / E332X6, E246A / A241S / S245V / K346R / V116M / V358I / A335X7, E246A / A241S / S245V / K346R / V116M / V358I / S355X9, E246A / A241S / S245V / K346R / V116M / V358I / P356S, E246A / A241S / S245V / K346R / V116M / V358I / F357X10, E246A / A241S / S245V / K346R / V116M / V358I / E396X12; (8) Eight-point mutants: E246A / A241S / S245V / K346R / V116M / V358I / E5R / A18X1, E246A / A241S / S245V / K346R / V116M / V358I / E5R / L121I, E246A / A241S / S245V / K346R / V116M / V358I / E5R / L127E, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257X5, E246A / A241S / S245V / K346R / V116M / V358I / E5R / E332X6, E246A / A241S / S245V / K346R / V116M / V358I / E5R / A335X7, E246A / A241S / S245V / K346R / V116M / V358I / E5R / S355X9, E246A / A241S / S245V / K346R / V116M / V358I / E5R / P356S, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F357X10, E246A / A241S / S245V / K346R / V116M / V358I / E5R / E396X12; (9) Nine-point mutants: E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / A18X1, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / L121I, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / L127E, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / E332X6, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / A335X7, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / S355X9, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / P356S, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / F357X10, E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257(TTT) / E396X12.

3. The mutant of claim 1, wherein The mutant is obtained by mutating the amino acid sequence shown in SEQ ID No. 2 as follows: (1) E246A; (2) E246A / A241S; (3) E246A / A241S / S245V; (4) E246A / A241S / S245V / K346R; (5) E246A / A241S / S245V / K346R / V116M; (6) E246A / A241S / S245V / K346R / V116M / V358I; (7) E246A / A241S / S245V / K346R / V116M / V358I / E5R; (8) A241S / K346R / S245V / E246A / V116M / V358I / E5R / F257TTT, the codon of phenylalanine at position 257 is mutated from TTC to TTT; (9) E246A / A241S / S245V / K346R / V116M / V358I / E5R / F257TTT / A335N.

4. A recombinant genetically engineered bacterium expressing the mutant serine hydroxymethyltransferase of claim 1.

5. Use of the mutant serine hydroxymethyltransferase of claim 1 in catalyzing the generation of L-serine from glycine.

6. The use according to claim 5, wherein the compound is ###0002### The method of the use is as follows: crude enzyme solution obtained by inducing expression of the recombinant genetically engineered bacterium expressing the mutant serine hydroxymethyltransferase, breaking and extracting the wet bacterium, is used as a catalyst, pyridoxal phosphate is added, after stirring at 200-400 rpm for 3 h at 30-40℃, the substrate glycine and tetrahydrofolic acid are added, the pH is adjusted to 8.2, deionized water is added to the reaction system, 37% formaldehyde aqueous solution is slowly added to prevent inhibition of enzyme activity caused by excessive formaldehyde, and the reaction is stirred at 100-900 rpm at 20-45℃ until completion, thereby obtaining L-serine.

7. Use according to claim 6, wherein In the reaction system, the amount of crude enzyme solution added is 20-50 g / L based on the weight of the wet bacterium before breaking, the final concentration of pyridoxal phosphate added is 0.05-0.2 g / L; the final concentration of glycine added is 10-300 g / L; and the final concentration of tetrahydrofolic acid added is 0.25-6 mM.

8. The use according to claim 6, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The crude enzyme solution is prepared as follows: (1) The recombinant genetically engineered bacterium is inoculated into LB liquid medium, cultured at 37℃ and 200 rpm for 10 h, then transferred to LB liquid medium at an inoculation amount of 2% of the volume of the medium, and cultured at 37℃ and 200 rpm for 2 h, isopropyl-β-D-thiogalactopyranoside is added to a final concentration of 0.06 g / L, and expression of serine hydroxymethyltransferase is induced at 28℃ and 200 rpm for 16 h, the bacterial solution is collected, centrifuged, and then re-dissolved in LB liquid medium to obtain a bacterial solution with a wet bacterium concentration of 100 g / L, which is used as a whole bacterium solution; (2) Cetyltrimethylammonium bromide powder is added to the whole bacterium solution of step (1), and after complete dissolution, the mixture is placed in a 37℃, 200 rpm shaking incubator for 1.5 h to obtain a crude enzyme solution.

Images