A mutant of serine hydroxymethyltransferase and its application

Abstract

The present invention discloses a mutant of serine hydroxymethyltransferase and its application. The amino acid sequence of the serine hydroxymethyltransferase is as shown in SEQ ID NO:1 in the sequence listing; the mutation occurs at one or more of the 19th, 310th, 138th, 168th, 363rd and 194th positions; wherein, the mutations corresponding to each position are M19K, S310K, K138R, K168R, F363R and G194A respectively. By site-directed mutagenesis of serine hydroxymethyltransferase derived from Pseudomonas aeruginosa strain 3Re2-7, the mutant improves the thermal stability of serine hydroxymethyltransferase and the catalytic activity of producing L-serine.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a mutant of serine hydroxymethyltransferase and its application. Background Art

[0002] L-Serine is a non-essential amino acid, scientifically named L-2-amino-3-hydroxypropionic acid, with the molecular formula C 3 H 7 NO 3 , and the structural formula is CH 2 OHCH(NH 2 )COOH, with a relative molecular weight of 105.09 and a melting point of 496 - 501K. It is easily soluble in water and almost insoluble in non-polar solvents. Its structural formula is as follows:

[0003]

[0004] L-Serine plays a role in the metabolism of fats and fatty acids and the growth of muscles, and also plays a role in the manufacture and processing of cell membranes, the synthesis of muscle tissues and the sheath surrounding nerve cells. In the pharmaceutical field, L-serine is commonly used in amino acid infusions and nutritional supplements. For example, the content of L-serine in the compound amino acid preparation for general nutritional therapy is 1.68 g / L, and the content of L-serine in the compound amino acid preparation for patients with liver injury is 5.00 g / L; L-serine can also be used as an intermediate for the synthesis of L-tryptophan, L-dopa, L-cysteine, phosphatidylserine, cycloserine, azaserine, etc. In the food industry, L-serine is a raw material for maple syrup flavor. In the feed field, L-serine as an additive can effectively promote the growth and development of poultry and livestock. In the cosmetics industry, L-serine is an important natural moisturizing factor, which can increase the vitality and moisture retention of epidermal cells, delay skin aging, and it also has antibacterial properties, improves skin elasticity, strengthens nutrition, and maintains the vitality of surface cells.

[0005] The main methods for preparing L-serine include protein hydrolysis extraction method, chemical method, fermentation method and enzymatic method. The protein hydrolysis method uses cocoon waste as raw material. After hydrolysis by boiling with hydrochloric acid at high temperature, L-serine is obtained through purification by ion exchange resin. Due to disadvantages such as low extraction efficiency, consumption of a large amount of acid, large amount of wastewater generated, and serious environmental pollution, this method has been gradually phased out by manufacturers; The chemical method can use different starting materials such as methyl acrylate, glycine or hydroxyacetaldehyde, acetyl compounds, etc., and obtain serine through different reaction routes. The disadvantages of this method are low product stereoselectivity, complex subsequent separation process, and high separation cost; The fermentation method uses glycine or sugars as raw materials, and through fermentation and culture of microorganisms such as Corynebacterium glycophilum, Nocardia butyrica and Sarcina alba, a fermentation broth containing L-serine is obtained. The fermentation broth is then subjected to subsequent extraction and separation to obtain L-serine. Since the acid production rate of this method is generally low, separation is difficult, and the extraction rate is low, the cost is relatively high, the price is relatively expensive, and it is not suitable for industrial production. The enzymatic method uses glycine and formaldehyde as raw materials, and uses serine hydroxymethyltransferase (serine hydroxymethyltransferase, SHMT, EC.2.1.2.1) to catalyze the reverse synthesis of L-serine with the assistance of pyridoxal phosphate (PLP) and tetrahydrofolic acid (THAF). Its mechanism of action is as follows:

[0006]

[0007]

[0008]

[0009] The enzymatic production of L-serine has the advantages of simple reaction process, few side reactions, short time, high production efficiency, single product and convenient extraction. However, wild serine hydroxymethyltransferase also has disadvantages such as low catalytic efficiency and poor thermal stability.

[0010] The current existing technologies related to L-serine are listed as follows:

[0011] Patent CN101787356B discloses a method for constructing and immobilizing a glyA gene engineering bacterium. This invention expresses the glyA gene derived from Escherichia coli E.coli k-12 in Escherichia coli, and the enzyme activity is 50-100 times that of the host bacterium. The glycine conversion rate for enzymatic production of L-serine can reach 70-80%.

[0012] Patent CN110872593A discloses a SHMT mutant derived from Escherichia coli. The catalytic enzyme activity of this mutant is 2 times higher than that of the wild-type enzyme. However, under the substrate condition of 200 g / L, its conversion rate is at most 85.6%, and there is still the disadvantage of low conversion rate.

[0013] Patent CN110205346A discloses a method for in vitro evolution of high - efficiency serine hydroxymethyltransferase. The steps include: performing PCR amplification on serine hydroxymethyltransferase genes from multiple sources; subjecting the serine hydroxymethyltransferase genes from multiple sources to low - energy ion implantation in vitro, constructing a vector and transforming a recipient bacterium; screening for high - activity serine hydroxymethyltransferase strains and sequencing. However, this patent does not disclose the beneficial effects of the serine hydroxymethyltransferase mutants obtained by using this method.

[0014] Zhao Qian et al. (Zhao Qian, Research on the properties of serine hydroxymethyltransferase encoding and its molecular dynamics simulation. Huazhong University of Science and Technology, 2015.) cloned and expressed SHMT from Escherichia coli SRZ018 in Escherichia coli. This SHMT can maintain more than 85% residual enzyme activity between pH 6.5 and 8.0; using the molecular dynamics simulation software Gromacs, glycine (G) at position 151 of SHMT was mutated to alanine (A), and it was predicted that the thermal stability of the protein would be improved. However, the thermal stability effect of the SHMT mutant in this study was only a prediction, and there was no data support for the actual effect.

[0015] Jiang Wei et al. (Jiang Wei, Analysis, directed evolution and application of serine hydroxymethyltransferase from Shewanella algae and Arthrobacter nicotianae. Huazhong Agricultural University, 2014) expressed the GlyA gene encoding SHMT from bacteria Shewanella algae and Arthrobacter nicotianae in Escherichia coli BL21(DE3), and site - directed mutagenized isoleucine (I) at position 249 of SHMT to leucine (L). The catalytic efficiency of the mutant was 2.78 times that of the wild - type. Using 10 g / L glycine and 26.6 mmol / L formaldehyde (fed - batch) as substrates, this SaSHMT could produce 77.76 mM L - serine through enzymatic conversion, and the molecular conversion rate of glycine to L - serine was 1.41 times that of Escherichia coli under the same conditions. However, the catalytic efficiency of the SHMT mutant in this study was still relatively low, and under the substrate condition of 10 g / L, its conversion rate was only 58.4%. Summary of the Invention

[0016] The technical problem to be solved by the present invention is to overcome the problems of low catalytic efficiency and poor stability of serine hydroxymethyltransferase in the prior art, and to provide a serine hydroxymethyltransferase (SHMT) mutant and a method for preparing L - serine using the same.

[0017] The first aspect of the present invention relates to a mutant of serine hydroxymethyltransferase, which is a site-directed mutation of the wild-type serine hydroxymethyltransferase with the amino acid sequence shown in SEQ ID No: 1. The mutation sites are one or more of the 19th, 310th, 138th, 168th, 363rd, and 194th positions;

[0018] Among them, the mutations corresponding to each site are as follows: the 19th position of methionine (M), the 310th position of serine (S) mutated to lysine (K), the 138th position of lysine (K), the 168th position of lysine (K), and the 363rd position of phenylalanine (F) mutated to arginine (R), and the 194th position of glycine (G) mutated to alanine (A). The wild-type serine hydroxymethyltransferase is derived from Pseudomonas aeruginosa strain 3Re2-7. The amino acid sequence of the wild-type serine hydroxymethyltransferase is shown in SEQ ID NO.1 in the sequence listing.

[0019] Preferably, the mutation in the present invention occurs at the 19th position of the wild-type serine hydroxymethyltransferase; the mutation preferably also occurs at the 138th, 168th, or 363rd position. In a preferred embodiment of the present invention, the mutation occurs at the 19th, 194th, and 138th or 168th positions.

[0020] Preferably, the mutation in the present invention occurs at the 310th position; the mutation preferably also occurs at the 138th, 168th, or 363rd position. In a preferred embodiment of the present invention, the mutation occurs at the 310th, 194th, and 138th or 168th positions.

[0021] In another preferred embodiment of the present invention, the mutation occurs at the 138th, 168th, 363rd, or 194th position.

[0022] The second aspect of the present invention relates to a nucleotide encoding the mutant as described in the first aspect of the present invention.

[0023] The third aspect of the present invention relates to an expression vector containing the nucleotide as described in the second aspect of the present invention.

[0024] The expression vectors described in the present invention may be plasmids of prokaryotic expression vector systems, including but not limited to pET series such as pET28a and pET3a; pBAD series such as pBAD-HisA, pBAD30, pGEX-2T, pXMJ19, and pexk99e, etc. They may also be plasmids of fungal expression systems, including but not limited to pPIC9K, pPIC9, PRS304, pUG6, pSH47, pUC110, pPZP-HYG2, and pFC330, etc. The preferred expression vector is pET28a or pPIC9K.

[0025] The fourth aspect of the present invention relates to a transformant (genetically engineered bacterium) containing the expression vector as described in the third aspect. The host cells of the transformant include but are not limited to Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, and Bacillus subtilis, etc.; preferably Escherichia coli or Pichia pastoris.

[0026] The experimental steps for obtaining the serine hydroxymethyltransferase mutant in the present invention are as follows:

[0027] Design site-directed mutagenesis primers, perform site-directed mutagenesis using the serine hydroxymethyltransferase of Pseudomonas aeruginosa strain 3Re2-7 as a template, and construct a mutant expression plasmid. Transform the mutant expression plasmid into competent host cells to obtain a recombinant mutant strain.

[0028] Ferment and culture the recombinant mutant strain, induce expression, and then obtain the serine hydroxymethyltransferase mutant.

[0029] The fifth aspect of the present invention relates to a method for preparing the mutant described in the first aspect. The method includes culturing the transformant as described in the fourth aspect to obtain a fermentation product, and obtaining the mutant from the fermentation product.

[0030] The sixth aspect of the present invention relates to the method for preparing L-serine, which includes: in the presence of pyridoxal phosphate (PLP) and tetrahydrofolic acid (THAF), using the mutant as described in the first aspect to catalyze the reaction of glycine and formaldehyde.

[0031] The seventh aspect of the present invention relates to the application of the mutant of the serine hydroxymethyltransferase, the nucleotide, the expression vector, or the genetically engineered bacterium in the production of L-serine.

[0032] Specifically, the following preparation method is preferably used to produce L-serine:

[0033] Using glycine and formaldehyde as raw materials, and using the mutant of the serine hydroxymethyltransferase as a catalyst, synthesize L-serine with the assistance of pyridoxal phosphate and tetrahydrofolic acid.

[0034] The following definitions are adopted in the present invention:

[0035] 1) Amino acid residues are in the form of three-letter abbreviations or single-letter symbols using the recognized IUPAC nomenclature. DNA nucleic acid sequences adopt the recognized IUPAC nomenclature.

[0036] 2) Identification of mutants: The mutated amino acids in SHMT mutants are represented by "the amino acid at the original amino acid position replaced". For example, M19K means that the amino acid at position 19 is replaced from methionine (Met) of wild-type serine hydroxymethyltransferase to lysine (Lys), and the position number corresponds to the amino acid sequence number of serine hydroxymethyltransferase in SEQ ID NO:1. For example, K138R / S310K means that the amino acids at positions 138 and 310 have both mutated.

[0037] Based on the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0038] The reagents and raw materials used in the present invention are all commercially available.

[0039] The positive and progressive effects of the present invention are as follows:

[0040] By site-directed mutagenesis of serine hydroxymethyltransferase derived from Pseudomonas aeruginosa strain 3Re2-7, the mutants have improved the thermal stability and catalytic activity of serine hydroxymethyltransferase for generating L-serine. Description of the Drawings

[0041] Figure 1 It is the optimal temperature of wild-type and mutant serine hydroxymethyltransferases in the examples.

[0042] Figure 2 It is the thermal stability of wild-type and mutant serine hydroxymethyltransferases in the examples. Detailed Embodiments

[0043] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0044] The plasmid pET28a was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; restriction endonucleases such as EcoRI and HindIII, Escherichia coli DH5α, BL21(DE3) competent cells, DNA marker, plasmid extraction kit, DNA gel recovery and purification kit, and T4 ligase were all purchased from Takara Bio Inc. (Dalian); DNA polymerase (Q5 High-Fidelity DNA Polymerase) was purchased from Gene Co., Ltd.; kanamycin sulfate and BCA protein content assay kit were both purchased from Biosharp; Ni-NTA protein purification kit was purchased from Sangon Biotech; chemical reagents were all of analytical grade from Sinopharm. The operating steps for plasmid extraction were referred to the instruction manual of the plasmid miniprep kit; the operating steps for DNA gel recovery were referred to the instruction manual of the DNA gel recovery kit; the operating steps for DNA fragment ligation were referred to the instruction manual of T4 ligase; the operating steps for protein purification were referred to the instruction manual of Ni-NTA protein purification kit; the operating steps for protein content were referred to the BCA protein content assay kit.

[0045] LB medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, sterilized at 121 °C for 20 min;

[0046] Fermentation medium (g / L): peptone 10, yeast extract 5, glycerol 10, disodium hydrogen phosphate dodecahydrate 20, potassium dihydrogen phosphate 7, ammonium chloride 5, anhydrous sodium sulfate 1, and magnesium sulfate heptahydrate 1, antifoaming agent 0.5 ml / L, sterilized at 121 °C for 30 min;

[0047] SHMT substrate reaction solution: 50 mmol / L phenylserine, 50 μmol / L pyridoxal phosphate, 0.03% (mass fraction) cetyltrimethylammonium bromide, pH 8.0.

[0048] Example 1 Construction of wild-type SHMT genetic engineering bacteria

[0049] According to the information published in the NCBI database, the sequence information of the serine hydroxymethyltransferase gene (GlyA) from Pseudomonas aeruginosa strain 3Re2-7 (SEQ ID No: 2) was retrieved. The gene sequence was codon-optimized according to the codon preference rule of Escherichia coli. After designing and adding EcoRI and HindIII restriction sites at both ends of the gene, it was sent to Wuhan Genecreate Biotechnology Co., Ltd. for artificial synthesis (SEQ ID No: 3).

[0050] The plasmid pET28a and the synthesized Gly gene fragment were double-digested with EcoRI / HindIII respectively. The reaction system was as follows: 1 μg of plasmid or Gly gene fragment, 5 μl of 10× buffer, 1 μl of EcoRI, 1 μl of HindIII, and the volume was made up to 50 μl with water. Digestion was carried out at 37 °C for 5 h. Detection was performed by 1% agarose gel electrophoresis, and nucleotide fragments of 5.4 kb or 1.3 kb were recovered using a DNA gel recovery and purification kit;

[0051] The GlyA gene DNA fragment was ligated to the pET28a vector using T4 DNA ligase. The ligation system was as follows: 0.5 μl of pET28a, 5 μl of Solution I, and 4.5 μl of Gly gene DNA fragment. Ligation was carried out overnight at 16 °C.

[0052] The ligation product was transformed into competent Escherichia coli DH5α cells by heat shock transformation. The transformed product was spread on an LB plate containing 100 μg / ml kanamycin sulfate and cultured overnight at 37 °C. Then, positive transformants were selected and sent to Wuhan Kingcare Biotechnology Co., Ltd. for sequencing. The transformants with correct sequencing were plasmid vectors GlyA-pET28a containing the wild-type serine hydroxymethyltransferase gene;

[0053] The transformants with correct sequencing were transferred to 5 ml of LB medium and cultured with shaking at 37 °C and 200 rpm for 16 h. 5 ml of the bacterial solution was taken, centrifuged at 10,000 rpm for 2 minutes, and the supernatant was discarded; the wet bacterial cells were used to extract plasmids using a plasmid miniprep kit. The plasmid was transformed into Escherichia coli BL21(DE3) by heat shock method and cultured overnight at 37 °C. Then, positive transformants were selected and sequenced again. The transformants with correct sequencing were the wild-type SHMT genetic engineering bacteria W1.

[0054] Example 2 Construction of SHMT mutant genetic engineering bacteria

[0055] Based on an online modeling website, mutation sites that might affect the activity and stability of serine hydroxymethyltransferase were screened out, and a list of single mutations and combined mutations was designed, as shown in Table 1:

[0056] Table 1

[0057] Mutant number Mutation site Mutant number Mutation site M1 M19K M10 S310K / K138R M2 S310K M11 S310K / K168R M3 K138R M12 S310K / F363R M4 K168R M13 M19K / K138R / G194A M5 F363R M14 M19K / K168R / G194A M6 G194A M15 S310K / K168R / G194A M7 M19K / K138R M16 S310K / F363R / G194A M8 M19K / K168R M17 M19K / F363R / G194A M9 M19K / F363R

[0058] Mutation primers for the mutation sites of M19K / S310K / K138R / K168R / F363R / G194A were designed, as shown in Table 2:

[0059] Table 2

[0060]

[0061] Single point mutation: Using GlyA FP / M19K RP, GlyA FP / S310K RP, GlyA FP / K138R RP, GlyA FP / K168R RP, GlyA FP / F363R RP, GlyA FP / G194A RP as primers respectively, and the GlyA-pET28a plasmid in Example 1 as the template, the left fragments of each mutation site were amplified by high-fidelity Q5 High-Fidelity DNA Polymerase PCR; Using M19K FP / GlyA RP, S310K FP / GlyA RP, K138R FP / GlyA RP, K168R FP / GlyA RP, F363R FP / GlyA RP, G194A FP / GlyA RP as primers respectively, and also using Gly-pET28a as the template to amplify the right fragments of the mutation sites; Then using GlyA FP / GlyA RP as the primer, and using the amplified left and right fragments corresponding to the mutations as templates respectively for overlap extension PCR to amplify the full-length GlyA single mutation fragments M1 - M6.

[0062] Combined mutation: Using the amplified single mutation DNA gene fragments as templates, referring to the method of the above primer combinations, first amplify the left fragments of the mutation sites, then amplify the right fragments of the mutation sites, and then using GlyA FP / GlyA RP as the primer, and using the amplified left and right fragments corresponding to the mutations as templates respectively for overlap extension PCR to amplify the full-length GlyA double mutation fragments M7 - M12; Similarly, using the amplified double mutation DNA gene fragments as templates to amplify the full-length GlyA triple mutation fragments M13 - M17.

[0063] The PCR reaction system (50 μl) is: 5×Q5 reaction buffer 10 μl, 10 mM dNTP 1 μl, each primer 2.5 μl, the template depends on the sample concentration, Q5 enzyme 0.5 μl, add water to 50 μl.

[0064] The final PCR products were detected by 1% agarose gel electrophoresis and purified and recovered using a DNA gel recovery kit.

[0065] The plasmid pET28a was double digested with EcoRI / HindIII, and the reaction system was: plasmid 1 μg, 10×buffer 5 μl, EcoRI 1 μl, HindIII 1 μl, add water to 50 μl. Digest at 37°C for 5 h. Detect by 1% agarose gel electrophoresis and recover the 5.4 kb linear fragment using a DNA gel recovery and purification kit.

[0066] The One-step cloning recombinant kit was used to recombine the above double-enzyme linearized fragments and the amplified and recovered mutant fragments respectively. The recombinant products were transformed into competent Escherichia coli DH5α cells, and the transformed products were spread on LB plates containing 100 μg / ml kanamycin sulfate. After overnight culture at 37 °C, positive transformants were selected and sent to Wuhan Genecreate Biotechnology Co., Ltd. for sequencing. The transformants with correct sequencing were the plasmid vectors of M1 - M17 mutant strains.

[0067] The transformants with correct sequencing were transferred to 5 ml of LB medium and cultured with shaking at 37 °C and 200 rpm for 16 h. 5 ml of the bacterial solution was taken, centrifuged at 10,000 rpm for 2 minutes, and the supernatant was discarded. The wet bacterial cells were used to extract plasmids with a plasmid miniprep kit. The plasmids were transformed into Escherichia coli BL21(DE3) by heat shock method. After overnight culture at 37 °C, positive transformants were selected and sequenced again. The transformants with correct sequencing were the M1 - M17 SHMT mutant genetically engineered bacteria.

[0068] Example 3 Expression and purification of wild-type and mutant SHMT

[0069] The wild-type SHMT genetically engineered bacteria W1 and the mutant genetically engineered bacteria M1 - M17 were respectively inoculated into 5 ml of LB medium containing 100 μg / ml kanamycin sulfate and cultured at 37 °C / 200 rpm for 8 - 12 h. 2 ml of the culture was transferred to 50 ml of LB medium containing 100 μg / ml kanamycin sulfate and cultured at 37 °C / 200 rpm until the OD600 reached 0.4 - 1.0. Then, 0.1 - 0.5 mM / L IPTG was added to the medium and the culture was continued at 30 °C for another 4 - 16 hours to express the recombinant protein.

[0070] The fermentation broth was centrifuged at 4 °C and 8000 r / min for 15 min to collect the bacterial cell precipitate, which was washed and resuspended with buffer (20 mM Tris-HCl, Ph6.8). After ultrasonic disruption in an ice-water bath (the disruption conditions were 100 W, 3 s / 5 s, 30 min), it was centrifuged at 4 °C and 12,000 r / min for 20 min, and the supernatant was collected to obtain the crude enzyme solution.

[0071] The obtained crude enzyme solution was purified with a Ni-NTA protein purification kit, and the purification effect was examined by SDS-PAGE, and the protein concentration was determined by the BCA method.

[0072] Example 4 SHMT enzyme activity assay

[0073] The activity of SHMT is usually determined by the principle of its hydrolysis of DL-β-phenylserine to generate glycine and benzaldehyde: SHMT can catalyze the decomposition of D,L-phenylserine to produce glycine and benzaldehyde, and the latter has a characteristic strong absorption at 279 nm.

[0074] Definition of enzyme activity: Under the substrate conditions of 37 °C, 50 mmol / L phenylserine, 50 μmol / L pyridoxal phosphate, and 0.03% (mass fraction) cetyltrimethylammonium bromide, the conversion of 1 μmol of benzaldehyde within 1 minute is defined as 1 enzyme activity unit (IU).

[0075] Take 50 μl of the purified W1 and M1 - M17 pure proteases in Example 3, add them to a 1.5 ml EP tube, add 1 ml of SHMT substrate reaction solution, incubate at 37 °C in a water bath for 1 h, take the supernatant after heating to terminate the reaction, measure the A279 value, determine the concentration of benzaldehyde in the reaction solution according to the A279 value - benzaldehyde concentration regression equation, and calculate the SHMT enzyme activity. The results are shown in Table 3.

[0076] Table 3

[0077]

[0078]

[0079] The measurement results show that the enzyme activities of different mutants have all increased. Among them, the enzyme activity of M17 is the highest, being 441.25 U / mg, which is 2.83 times that of the wild-type W1 (155.62 U / mg), indicating that the enzyme has been modified by site-directed mutagenesis, improving its catalytic activity.

[0080] Example 5 Determination of the Optimal Temperature and Thermal Stability of SHMT

[0081] React the purified W1 and M1 - M17 pure proteases in Example 3 with the substrate reaction solution at different temperatures (25 - 60 °C), and then measure the enzyme activity according to the enzyme activity detection method in Example 4. Taking the maximum enzyme activity as 100%, calculate the relative enzyme activities at other temperatures. The results are as Figure 1 shown

[0082] Incubate at 25 °C - 60 °C for 30 min, and then measure the residual enzyme activity in the sample according to the enzyme activity detection method in Example 4. Taking the enzyme activity of the untreated sample as 100% to calculate the relative residual enzyme activity, and make a temperature - relative enzyme activity curve. When the residual enzyme activity reaches more than 85%, it is defined as having good thermal stability. The relative enzyme activity results are as Figure 1 shown. The optimal temperature of the SHMT mutant is the same as that of the wild-type, both being 40 °C. When the temperature is lower or higher than 40 °C, the SHMT mutant has higher catalytic activity, indicating that its operating temperature range is wider than that of the wild-type.

[0083] The enzyme was placed in a 50 mmol / L Tris-HCl buffer at pH 8.0 and incubated at 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, and 70 °C for 4 h, then cooled in an ice-water bath. Subsequently, the residual enzyme activity was measured at 37 °C according to the enzyme activity detection method in Example 4. The highest enzyme activity was defined as 100%, and the enzyme activities measured under different conditions were compared with it to calculate their relative enzyme activities. The thermal stabilities of various SHMTs were compared based on their relative enzyme activities. The results are as Figure 2 shown. The thermal stability of the SHMT mutant was significantly higher than that of the wild type. Especially when the temperature was higher than 55 °C, the relative enzyme activity of SHMT remained above 75%, while that of the wild type was already below 70%.

[0084] Example 6 Fermentation of Wild-Type SHMT and Mutants in a Fermenter

[0085] 1 mL of glycerol stock cultures of wild-type SHMT W1 and mutants M2, M6, M7, and M17 were respectively activated on an LB slant medium and cultured at 37 °C for 16 h. One inoculation loop of bacterial growth was picked from the slant medium and transferred to 100 mL of LB seed medium, and cultured with shaking at 37 °C and 160 rpm for 10 - 16 h. The seeds were inoculated into a 5-L fermenter containing 3 L of fermentation medium at a volume ratio of 5%. The temperature was controlled at 37 °C ± 1 °C. During fermentation, ammonia water was added to control the pH at 7.0 ± 0.1, the aeration rate was controlled at 0.5 vvm - 3.0 vvm, and the rotation speed was controlled at 200 rpm - 800 rpm. The dissolved oxygen was maintained at 30% by adjusting the rotation speed and aeration rate. During fermentation, 80% glycerol was added dropwise to control the glycerol residue in the fermentation broth at 1 - 3%. After 7 - 8 h of fermentation, the temperature was decreased and stabilized at 25 - 30 °C, and IPTG with a final concentration of 0.1 mM was added to induce for about 16 h. When the OD of the fermentation no longer increased, the fermentation ended.

[0086] The fermentation broth was centrifuged at 6000 rpm for 20 min to discard the supernatant, and the cells were obtained. The cells were washed and resuspended with a buffer (20 mM Tris-HCl, pH 6.8), and then ultrasonically disrupted in an ice-water bath (the disruption conditions were 100 W, 3 s / 5 s, 30 min). After that, it was centrifuged at 4 °C and 12000 r / min for 20 min, and the supernatant was collected to obtain the crude enzyme solution.

[0087] Example 7 Production of L-Serine by SHMT

[0088] The conversion substrate was prepared in a closed container: 25% glycine, 0.05 moL / L phosphate, 0.6% tetrahydrofolic acid, and 0.01% PLP were added to a 50 mmol / L Tris-HCl buffer at pH 8.0.

[0089] Under the protection of nitrogen gas flow, crude enzyme solution of fermentation was added to the conversion substrate at 37 °C. The weight ratio of the crude enzyme solution to glycine was 1:5. Meanwhile, formaldehyde solution was added dropwise. The volume ratio of the formaldehyde solution to the conversion substrate solution was 1:200. After 30 h of conversion, the conversion solution was centrifuged for the determination of the content of L-serine.

[0090] The content of L-serine was determined by paper chromatography. The detection method referred to the paper chromatography detection method recorded in Ma Lizhou et al. (Research on the Bioconversion Preparation of L-serine. Jiangnan University, 2008.). The detection conditions were as follows:

[0091] Developing agent: n-butanol - acetone - water - ammonia water (100:40:38:2),

[0092] Eluent: V(0.1% copper sulfate):V(75% ethanol) = 2:38

[0093] Developer: 0.5 g of ninhydrin was dissolved in 100 ml of acetone

[0094] L-serine and glycine standard solutions (0.2 μl - 1.2 μl) and the samples to be tested of the conversion solutions of W1, M2, M6, M7, and M17 were spotted on filter paper respectively. After being placed in a chromatography chamber for saturation for one hour, they were immersed in the developing agent for development. After development was completed, they were taken out, dried, and evenly sprayed with the developer. They were developed at 105 °C for 5 min. The L-serine color spots were cut off and eluted with 5.0 ml of eluent for a certain time. The A512 value was measured. According to the A512 value - L-serine concentration regression equation, the L-serine concentration in the reaction solution was determined, and the glycine conversion rate was calculated. The results are shown in Table 4:

[0095] Table 4

[0096]

[0097] The concentration of L-serine in the conversion solution of wild-type SHMT was 286.3 g / L, while the product concentration of L-serine in the conversion solutions of mutants was all above 300 g / L, indicating that site-directed mutagenesis of SHMT improved the catalytic activity of the enzyme. Among them, the catalytic efficiency of M2 increased by 7.2%, the catalytic efficiency of M6 increased by 13%, the catalytic efficiency of M7 increased by 15.8%, and the catalytic efficiency of M17 increased by 17.2%.

[0098] Among them, the average value of L-serine produced by M17 was 335.5 (g / L), which was equivalent to 3.19 M, and was 41 (3.19 / 0.07776) times the product concentration obtained by the known mutant I249L in the prior art. The conversion rate was equivalent to 1.64 times of it (95.9% / 58.4%). SEQUENCE LISTING <110> Wuhan Yuanda Hongyuan Co., Ltd. <120> A mutant of serine hydroxymethyltransferase and its application <130> P20016599C <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 417 <212> PRT <213> Pseudomonas aeruginosa <400> 1 Met Phe Ser Arg Asp Leu Thr Ile Ala Lys Tyr Asp Ala Asp Leu Phe 1 5 10 15 Ala Ala Met Glu Gln Glu Ala Gln Arg Gln Glu Glu His Ile Glu Leu 20 25 30 Ile Ala Ser Glu Asn Tyr Thr Ser Pro Ala Val Met Glu Ala Gln Gly 35 40 45 Ser Val Leu Thr Asn Lys Tyr Ala Glu Gly Tyr Pro Gly Lys Arg Tyr 50 55 60 Tyr Gly Gly Cys Glu Tyr Val Asp Val Val Glu Gln Leu Ala Ile Asp 65 70 75 80 Arg Ala Lys Gln Leu Phe Gly Ala Asp Tyr Ala Asn Val Gln Pro His 85 90 95 Ala Gly Ser Gln Ala Asn Ser Ala Val Tyr Leu Ala Leu Leu Ser Ala 100 105 110 Gly Asp Thr Ile Leu Gly Met Ser Leu Ala His Gly Gly His Leu Thr 115 120 125 His Gly Ala Ser Val Ser Ser Ser Gly Lys Leu Tyr Asn Ala Ile Gln 130 135 140 Tyr Gly Ile Asp Gly Asn Gly Leu Ile Asp Tyr Asp Glu Val Glu Arg 145 150 155 160 Leu Ala Leu Glu His Lys Pro Lys Met Ile Val Ala Gly Phe Ser Ala 165 170 175 Tyr Ser Gln Val Leu Asp Phe Pro Arg Phe Arg Glu Ile Ala Asp Lys 180 185 190 Val Gly Ala Tyr Leu Phe Val Asp Met Ala His Val Ala Gly Leu Val 195 200 205 Ala Ala Gly Val Tyr Pro Asn Pro Val Pro Phe Ala Asp Val Val Thr 210 215 220 Thr Thr Thr His Lys Thr Leu Arg Gly Pro Arg Gly Gly Leu Ile Leu 225 230 235 240 Ala Arg Ala Asn Ala Asp Ile Glu Lys Lys Leu Asn Ser Ala Val Phe 245 250 255 Pro Gly Ala Gln Gly Gly Pro Leu Glu His Val Ile Ala Ala Lys Ala 260 265 270 Ile Cys Phe Lys Glu Ala Leu Gln Pro Glu Phe Lys Thr Tyr Gln Gln 275 280 285 Gln Val Val Lys Asn Ala Lys Ala Met Ala Gly Val Phe Ile Glu Arg 290 295 300 Gly Phe Asp Val Val Ser Gly Gly Thr Glu Asn His Leu Phe Leu Leu 305 310 315 320 Ser Leu Ile Lys Gln Glu Ile Ser Gly Lys Asp Ala Asp Ala Ala Leu 325 330 335 Gly Lys Ala Phe Ile Thr Val Asn Lys Asn Ser Val Pro Asn Asp Pro 340 345 350 Arg Ser Pro Phe Val Thr Ser Gly Leu Arg Phe Gly Thr Pro Ala Val 355 360 365 Thr Thr Arg Gly Phe Lys Glu Ala Glu Cys Lys Glu Leu Ala Gly Trp 370 375 380 Ile Cys Asp Ile Leu Ala Asp Leu Asn Asn Glu Ala Val Ile Asp Ala 385 390 395 400 Val Arg Glu Lys Val Lys Ala Ile Cys Lys Lys Leu Pro Val Tyr Gly 405 410 415 Ala <210> 2 <211> 1254 <212> DNA <213> Pseudomonas aeruginosa <400> 2 atgttcagcc gtgatttgac tattgccaag tacgacgccg atctctttgc cgccatggag 60 caagaagctc agcgccagga agagcacatt gagctgatcg cttcggaaaa ctacaccagc 120 cccgcggtga tggaagctca aggctcggta ctgaccaaca agtacgccga aggctacccg 180 ggcaagcgtt actacggtgg ttgcgagtac gtcgacgtgg tcgagcaact ggccatcgat 240 cgcgccaagc aactgttcgg tgccgactat gccaacgtcc agccgcacgc tggctcccaa 300 gccaactccg ccgtgtacct ggcgctgctg tcggccggcg acaccatcct gggcatgagc 360 ctggcccacg gcggtcacct gacccacggc gccagcgttt cctcctccgg caagctgtac 420 aacgccatcc agtacggcat cgacggcaac ggcctgatcg actacgacga agtcgagcgc 480 ctggccctcg agcacaagcc gaaaatgatc gtggccggtt tctccgccta ctcccaggtc 540 ctcgacttcc cgcgtttccg cgagatcgcc gacaaggtcg gtgcctacct gttcgtcgac 600 atggcccacg ttgccggcct ggtcgccgct ggcgtctacc cgaacccggt gccattcgcc 660 gacgtggtca ccaccaccac ccacaagacc ctgcgtggtc cacgtggcgg cctgatcctg 720 gcgcgcgcca atgccgacat cgagaagaag ctgaactccg ccgtcttccc gggcgcccag 780 ggtggcccgc tggagcacgt gatcgcagcc aaggcgatct gcttcaagga agccctgcaa 840 cctgagttca agacttacca gcaacaagta gtgaagaatg ccaaggccat ggccggcgtg 900 ttcatcgagc gcggcttcga cgtggtgtcc ggcggtactg aaaaccacct gttcctgctg 960 tcgctgatca agcaagagat ctccggcaaa gacgccgacg cggcgctggg caaggcgttc 1020 atcaccgtca acaagaactc ggtgccaaac gacccacgct ctccgttcgt cacctcgggc 1080 ctgcgtttcg gcacccctgc cgtgaccact cgtggtttca aggaagccga gtgcaaggaa 1140 ctggctggct ggatctgcga catcctggcg gacctgaaca acgaagcggt catcgacgcc 1200 gtacgtgaga aggtcaaggc tatctgcaag aagctgccag tctacggcgc ttaa 1254 <210> 3 <211> 1253 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized serine hydroxymethyltransferase gene <400> 3 atgttttctc gtgatctgac tatcgcaaaa tacgatgctg acctgttcgc tgctatggaa 60 caagaagctc aacgtcagga agagcatatc gaactgatcg catccgagaa ctacacctct 120 ccggctgtta tggaagccca gggctccgtg ctgactaaca aatacgcgga gggttacccg 180 ggcaaacgtt actacggcgg ttgtgaatat gtggatgttg ttgaacagct ggcaattgac 240 cgtgccaagc agctgtttgg tgcggactac gcaaacgttc agccacacgc tggctctcag 300 gcgaactccg ctgtgtacct ggcgctgctg tctgctggtg ataccatcct gggcatgtct 360 ctggctcatg gtggtcacct gactcacggc gcttccgttt ctagctctgg taaactgtat 420 aacgccatcc agtatggtat cgacggtaac ggcctgatcg actacgacga agtagaacgc 480 ctggcgctgg aacacaagcc gaagatgatt gtcgcaggct tctccgcata cagccaggtg 540 ctggatttcc cgcgtttccg tgaaatcgcg gacaaggtag gtgcctacct gtttgttgac 600 atggcgcatg ttgctggtct ggttgcggcc ggtgtttatc cgaacccggt accgttcgca 660 gatgtggtga ccaccacgac ccataaaacc ctgcgtggtc cacgtggtgg tctgatcctg 720 gctcgtgcta acgctgacat cgagaaaaaa ctgaatagcg ctgtgttccc gggtgcacaa 780 ggtggtccgc tggaacatgt tatcgctgct aaagccattt gcttcaaaga agcgctgcag 840 ccagagttca aaacttatca gcagcaggtt gtgaaaaacg cgaaggcaat ggctggtgtc 900 tttatcgaac gtggcttcga cgttgttagc ggtggtaccg agaaccacct gttcctgctg 960 tctctgatca agcaagagat ttctggcaaa gatgcggatg ctgcgctggg caaagcgttt 1020 atcactgtta acaaaaacag cgtaccgaat gatccgcgtt ctccgtttgt taccagcggt 1080 ctgcgtttcg gtaccccggc agttactacc cgcggtttca aagaggctga atgcaaagag 1140 ctggctggct ggatctgcga tatcctggca gatctgaaca acgaggcggt aatcgatgca 1200 gttcgcgaga aggtgaaagc aatttgcaaa aaactgccgg tttacggcgc taa 1253 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> GlyA FP <400> 4 ggtcgcggat ccgaattcat gttttctcgt gatctg 36 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> GlyA RP <400> 5 gtgcggccgc aagcttttag cgccgtaaac cggca 35 <210> 6 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> M19K FP <400> 6 gctgacctgt tcgctgctaa agaacaagaa gctcaacg 38 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> M19K RP <400> 7 cgttgagctt cttgttcttt agcagcgaac aggtca 36 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> S310K FP <400> 8 tggcttcgac gttgttaagg gtggtaccga gaacca 36 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> S310K RP <400> 9 tggttctcgg taccaccctt aacaacgtcg aagcca 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> K138R FP <400> 10 ccgtttctag ctctggtcgt ctgtataacg ccatcc 36 <210> 11 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> K138R RP <400> 11 tggatggcgt tatacagacg accagagcta gaaacgg 37 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> K168R FP <400> 12 cgctggaaca caagccgcgc atgattgtcg caggct 36 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> K168R RP <400> 13 agcctgcgac aatcatgcgc ggcttgtgtt ccagcg 36 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F363R FP <400> 14 ttaccagcgg tctgcgtcgt ggtaccccgg cagtta 36 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F363R RP <400> 15 taactgccgg ggtaccacga cgcagaccgc tggtaa 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> G194A FP <400> 16 atcgcggaca aggtagctgc ctacctgttt gttgac 36 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> G194A RP <400> 17 gtcaacaaac aggtaggcag ctaccttgtc cgcgat 36

Claims

1. A mutant of serine hydroxymethyltransferase, characterized in that the amino acid sequence of the serine hydroxymethyltransferase is as shown in SEQ ID NO:1 in the sequence listing; the mutant is selected from the following mutations: (1) G194A at position 194; or (2) G194A at position 194, M19K at position 19, and K138R at position 138; or (3) G194A at position 194, M19K at position 19, and K168R at position 168; or (4) G194A at position 194, S310K at position 310, and K168R at position 168; or (5) G194A at position 194, S310K at position 310, and F363R at position 363; or (6) G194A at position 194, M19K at position 19, and F363R at position 363.

2. A nucleotide encoding the mutant according to claim 1.

3. An expression vector containing the nucleotide according to claim 2.

4. The expression vector according to claim 3, characterized in that the expression vector is pET28a or pPIC9K.

5. A transformant containing the expression vector according to claim 3 or 4.

6. The transformant according to claim 5, characterized in that the host cell used when obtaining the transformant is Escherichia coli or Pichia pastoris.

7. A method for preparing the mutant according to claim 1, which comprises culturing the transformant according to claim 5 or 6 to obtain a fermentation product, and obtaining the mutant from the fermentation product.

8. A method for preparing L-serine, characterized in that in the presence of pyridoxal phosphate and tetrahydrofolic acid, the mutant according to claim 1 is used to catalyze the reaction of glycine and formaldehyde.

9. Use of the mutant according to claim 1, the nucleotide according to claim 2, the expression vector according to claim 3 or 4, or the transformant according to claim 5 or 6 in the preparation of L-serine.

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