A glycosyltransferase mutant with improved thermostability and activity

Abstract

The present invention discloses a glycosyltransferase mutant with improved thermal stability and activity, belonging to the technical field of biocatalytic synthesis. The mutant is obtained by simultaneously mutating the 158th, 218th, and 369th positions on the basis of the glycosyltransferase YojK-I241T / G327N with the amino acid sequence shown in SEQ ID NO. 3. The present invention determines the mutation sites capable of improving the thermal stability and activity of YojK through rational design, and efficiently obtains the combined mutant YojK M1 . Compared with YojK M0 , the half-life of this variant at 50 °C is increased from 2.16 h to 58.64 h, and the enzyme activity is increased by 1.39 times; finally, through fed-batch reaction, it is shown that the conversion rate of this mutant is as high as 87.7% under the condition of 25 mmol / L Reb A, providing an efficient and green new route for the production of rebaudioside D.

Description

Technical Field

[0001] The invention relates to a glycosyltransferase mutant with improved thermal stability and activity, and belongs to the technical field of biocatalytic synthesis. Background Art

[0002] Excessive intake of high-calorie sugars leads to serious obesity, diabetes, hypertension, cardiovascular and cerebrovascular diseases worldwide. Therefore, steviol glycoside compounds from stevia have attracted widespread attention due to their high sweetness, low calories and high safety. Among them, stevioside and rebaudioside A, which are relatively abundant, have been widely used in beverages, food and other fields as sweeteners. They have a sweetness 250-300 times that of sucrose, but the bitter aftertaste in addition to sweetness seriously affects their taste as sweeteners. Rebaudioside D (Reb D), which has a lower content in steviol glycosides, has a higher sweetness than rebaudioside A (Reb A) and stevioside, and reduces the bitter aftertaste in addition to sweetness, so it has a better taste as a sweetener, and is considered to be a very potential next-generation sweetener. However, the content of Reb D in stevia dry leaves is only 0.4%-0.5%, which is only about one-tenth of the content of Reb A. This also makes the traditional method of extracting Reb A from leaves not suitable for the extraction of Reb D. The cumbersome and complicated extraction method makes it difficult to achieve large-scale production by extracting only from stevia leaves, and it is also difficult to meet market demand.

[0003] At present, the synthesis of Reb D by enzyme catalysis using Reb A, which is a high content in stevia, as a substrate is considered to be a feasible technical route to increase the production of Reb D. Through the continuous exploration and mining of relevant scientists, the glycosyltransferases (EUGT11, UGT91D2, and UGTSL2) related to the synthesis of Reb D using Reb A as a substrate have been discovered and identified. In addition, there are also successful cases of heterologous expression of these glycosyltransferases and heterologous biosynthesis of Reb D through microorganisms such as Escherichia coli, but the expression level of glycosyltransferases from plants is too low, which limits the production and market use of Reb D. In view of the above situation, our laboratory has previously mined and obtained the glycosyltransferase YojK from Bacillus subtilis BS168. The enzyme can be expressed in a soluble and efficient manner in Escherichia coli, and has the activity of catalyzing the synthesis of Reb D from Reb A in the presence of uridine diphosphate glucose (UDPG). Through directed evolution based on protein structure, the efficient mutant YojK-I241T / G327N was successfully obtained. However, it was found in the cascade reaction that the enzyme had poor thermal stability and it was difficult to meet the conditions for industrial production.

[0004] Therefore, modifying the mutant YojK-I241T / G327N to further improve its enzyme activity and thermal stability is of great significance for the industrial production of Reb D. Summary of the invention

[0005] The present invention successfully and efficiently obtains a combined mutant based on YojK-I241T / G327N. The present invention provides a glycosyltransferase mutant capable of efficiently catalyzing the biosynthesis of Reb D from Reb A, further improving its thermal stability and catalytic activity, and meeting the requirements of industrial production.

[0006] The present invention uses a mutant YojK-I241T / G327N (hereinafter referred to as YojK) of a glycosyltransferase derived from Bacillus subtilis BS168 constructed in the laboratory in the early stage. M0 ) was used as a template (its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4), and site-directed mutagenesis was performed to obtain the mutant YojK M1 .

[0007] The present invention provides a glycosyltransferase mutant, wherein the mutant is obtained by simultaneously mutating positions 158, 218 and 369 based on the amino acid sequence of the glycosyltransferase YojK-I241T / G327N as shown in SEQ ID NO.3.

[0008] In one embodiment of the present invention, the mutant is obtained by mutating the serine at position 158 to glutamic acid, the alanine at position 218 to histidine, and the alanine at position 369 to lysine based on the amino acid sequence of the glycosyltransferase YojK-I241T / G327N shown in SEQ ID NO.3, and is named YojK M1 (or YojK M0 / S158E / A218H / A369K).

[0009] In one embodiment of the present invention, the glycosyltransferase mutant YojK M1 The amino acid sequence is shown in SEQ ID NO.1.

[0010] In one embodiment of the present invention, the glycosyltransferase mutant YojK M1 The nucleotide sequence is shown as SEQ ID NO.2.

[0011] The present invention also provides a gene encoding the glycosyltransferase mutant YojK M1 genes.

[0012] The invention also provides a recombinant vector carrying the gene.

[0013] In one embodiment of the present invention, the recombinant vector is pET-21b(+) as an expression vector.

[0014] The present invention also provides a recombinant cell expressing the glycosyltransferase mutant, or containing the gene, or containing the recombinant vector.

[0015] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0016] The present invention also provides a method for improving the thermal stability and activity of a glycosyltransferase, the method comprising: mutating the serine at position 158 of the glycosyltransferase YojK-I241T / G327N with an amino acid sequence as shown in SEQ ID NO.3 to glutamic acid, mutating the alanine at position 218 to histidine, and mutating the alanine at position 369 to lysine.

[0017] The present invention also provides a method for catalytically synthesizing rebaudioside D, the method comprising adding the mutant YojK to a reaction system containing rebaudioside A. M1 , or the recombinant cell, react to prepare rebaudioside D.

[0018] In one embodiment of the present invention, the reaction system contains 1-25 mmol / L rebaudioside A, 5-25% (v / v) DMSO, 25-50 mmol / L Tris-HCl buffer, and 150-300 mmol / L NaCl.

[0019] In one embodiment of the present invention, the reaction conditions are: 40-50° C. for 0-48 h, not 0.

[0020] The present invention also provides a method for preparing a glycosyltransferase mutant YojK M1 , or the use of the above-mentioned gene, or the above-mentioned recombinant cell in the preparation of a product containing rebaudioside D.

[0021] Beneficial Effects

[0022] (1) The present invention provides a glycosyltransferase YojK with improved catalytic activity and thermal stability M0 Mutant YojK M1 The catalytic efficiency of Reb A at 50 °C is YojK M0 The thermal stability is improved by 27 times.

[0023] (2) Using the glycosyltransferase mutant YojK of the present invention M1Catalytic synthesis of rebaudioside D, in the long-term fed-batch reaction, the conversion rate of mutant Reb A was relatively high compared with YojK M0 An increase of about 20%.

[0024] The experimental results confirm that the present invention can promote the industrial application of biosynthetic Reb D, save enzyme amount, increase utilization times, and reduce production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown are the SDS-PAGE results of YojK.

[0026] Figure 2 for YojK M0 And the optimal temperature test results of mutants.

[0027] Figure 3 for YojK M0 and mutant half-life test results.

[0028] Figure 4 for YojK M0 With YojK M1 Fed-batch test results. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0030] The glycosyltransferase involved in the following examples is M0 The mutant YojK-I241T / G327N (hereinafter referred to as YojK M0 ), whose amino acid sequence is shown in SEQ ID NO.3, and its construction method is described in the Chinese invention patent publication number CN 114164191 A. The construction methods of pET-21b(+)-YojK and pET-21b(+)-YojK-I241T / G327N involved in the following examples are described in the Chinese invention patent publication number CN 114164191 A.

[0031] The recombinant plasmid pET-21b(+)-YojK involved in the following examples M0 The construction method is recorded in the Chinese invention patent publication number CN114164191A.

[0032] The culture medium involved in the following examples is as follows:

[0033] LB solid plate: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 20 g / L agar powder.

[0034] LB liquid medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl.

[0035] 2xYT liquid medium: 16 g / L peptone, 10 g / L yeast powder, 5 / L NaCl.

[0036] The detection methods involved in the following embodiments are as follows:

[0037] Detection of glycosyltransferase activity: Detect enzyme activity by liquid phase analysis of glycosylation reaction results.

[0038] The glycosylation reaction was carried out in a 200 μL reaction system, and the reaction system was as follows: 50 mmol / L Tris-HCl pH 8.0, 5 mmol / L UDPG, 10 mmol / L MnCl 2 , 1mmol / L Reb A, glycosyltransferase concentration of 10μM. React at 35℃ for 20min. After the reaction, dilute with an equal volume of methanol, centrifuge at 20000×g for 5min, filter with 0.22μM filter membrane, and then perform ultra-high performance liquid chromatography (UPLC) to detect and analyze the yield of Reb D. UPLC uses Waters BEH C181.7μM reverse column, injection volume 4μL, column temperature 40℃, mobile phase A pipeline: acetonitrile, B pipeline: 1.38g / L NaH 2 PO 4 Buffer (pH 2.6), flow rate 0.3 mL / min, the specific procedure is shown in Table 1:

[0039] Table 1: UPLC analysis procedure

[0040]

[0041] Enzyme activity is defined as the amount of enzyme required to react 1 micromole of Reb A in one minute.

[0042] Example 1: Construction of recombinant Escherichia coli containing glycosyltransferase mutants

[0043] 1. YojK M0 Construction of the / S158E mutant

[0044] The recombinant plasmid pET-21b(+)-YojK M0As a template, the whole plasmid PCR was performed using the designed corresponding primers S158E_F and S158E_R (primers are shown in Table 2) to construct the recombinant plasmid pET-21b(+)-YojK carrying the mutant M0 / S158E.

[0045] Table 2: Primer names and primer sequences

[0046] Primer Name Primer Sequence D87I_F aaagcctgtaagATCatcgcgactcatatttatgaggaagtca D87I_R gcgatGATcttacaggctttcaaaaaagcgca V115I_F tcgcgggtaaaATTattgccaacatgctgaagctg V115I_R gcaatAATtttacccgcgagaagatggtgat S158E_F attatgaaGAGtaccagcagcttgcagaaacg S158E_R gctggtaCTCttcataatgaggcgaatcttcaagtgatc D169K_F acgttaaatgaaAAGtttcaagcagagatcaagaagccatt D169K_R aaaCTTttcatttaacgtttctgcaagctgct L199N_F cagccaAACgctgagcaatttggcgagcg L199N_R ttgctcagcGTTtggctgaaatccccttgatgtaaag A218H_F agaaagaCATggaaacaatgatttcccatttgatcagatt A218H_R attgtttccATGtctttctgtaatggaaggaccgacaaat L253F_F aatcaatgcTTTgaagtgtgtaaggactttgacggtaaag L253F_R cacttcAAAgcattgattaaaaaactgcttttgattatt T273I_F atattaaaATTagtgagttaaacgacattccggagaat T273I_R actcactAATtttaatatgcttgccgatggaaagca A369K_F cgttcgtatAAGgaaaaggcaaaagaaattggacaatcact A369K_R cttttcCTTatacgaacgattattcattacttcttgtat

[0047] The PCR amplification system (50 μL) was as follows: template DNA 0.1 ng-1 ng, 2× Prime star max Buffer 25 μL, 1 μL each of the upstream and downstream mutation primers, and the rest was washed with ddH 2 O was added to the total volume. PCR reaction parameters: (1) 95℃ pre-denaturation for 30s; (2) 95℃ denaturation for 15s; (3) 63℃ annealing for 15s; (4) 72℃ extension for 35s, steps (2)-(4) cycled 30 times; (5) 72℃ full extension for 10min, stored at 4℃. After the PCR product was positive after 0.9% agarose gel electrophoresis analysis, the DNA product was purified using the Kangwei Gel Extraction Kit.

[0048] The resulting plasmid pET-21b(+)-YojK M0 The mutants were sequenced and identified by sequencing and transformed into competent E. coli BL21 (DE3) cells. The mutants were screened on LB solid plates containing 100 μg / mL ampicillin to obtain the recombinant strains: E. coli BL21 (DE3) / pET-21b (+) -YojK M0 / S158E.

[0049] 2. Contains YojK M0 / T45E、YojK M0 / D87I、YojK M0 / V115I、YojK M0 / S158E, YojK M0 / D169K、YojK M0 / G204D、YojK M0 / A218H、YojK M0 / Q247D、YojK M0 / L253F、YojK M0 / T273I and YojK M0 Construction of recombinant bacteria with single-point mutant of / A369K

[0050] The specific construction method is the same as step 1, and the primers are shown in Table 2.

[0051] Recombinant bacteria were prepared: E. coli BL21(DE3) / pET-21b(+)-YojK M0 / D87I、E.coli BL2l(DE3) / pET-21b(+)-YojK M0 / V115I、E.coli BL2l(DE3) / pET-21b(+)-YojK M0 / S158E, E.coliBL21(DE3) / pET-21b(+)-YojK M0 / D169K、E.coli BL2l(DE3) / pET-21b(+)-YojK M0 / L199N、E.coli BL2l(DE3) / pET-21b(+)-YojK M0 / A218H、E.coli BL2l(DE3) / pET-21b(+)-YojK M0 / L253F、E.coli BL2l(DE3) / pET-21b(+)-YojK M0 / T273I and E.coli BL21(DE3) / pET-21b(+)-YojK M0 / A369K.

[0052] 3. Construction of recombinant Escherichia coli containing combined mutants

[0053] YojK M0 As a template, the first step of plasmid construction was carried out with the designed A218H corresponding primers to obtain the combined mutant YojK M0 / S158E / A218H;

[0054] Then YojK M0 / S158E / A218H was used as a template, and the designed A369K corresponding primers were used to construct the second step of plasmid to obtain the combined mutant YojK M1 (YojK M0 / S158E / A218H / A369K);

[0055] The primers are shown in Table 1, and the specific implementation method is the same as step 1.

[0056] The recombinant bacteria containing the combined mutants prepared according to the above method: E. coli BL21 (DE3) / pET-21b (+) -YojK M1 .

[0057] As controls, recombinant bacteria were prepared according to the above method: E. coli BL21 (DE3) / pET-21b (+) -YojK, E. coli BL21 (DE3) / pET-21b (+) -YojK-I241T / G327N (E. coli BL21 (DE3) / pET-21b (+) -YojK M0 )

[0058] Example 2: YojK M0 Preparation of pure enzymes of its mutants

[0059] (1) Enzyme production by shake flask fermentation

[0060] The genetically engineered bacteria obtained in Example 1 were streaked on LB plates containing ampicillin (100 μg / mL) and cultured at 37°C for 12 h. Single colonies were picked and inoculated into LB liquid culture medium containing ampicillin (100 μg / mL) for shake flask fermentation. The culture was carried out at 37°C and 200 rpm for 12 h to obtain seed solution.

[0061] 5 mL of seed solution was transferred to 500 mL of ampicillin (100 μg / mL) 2xYT liquid medium for shake flask fermentation and cultured at 37°C until OD 600 When it reaches 0.6-0.8, add IPTG with a final concentration of 0.1mM, lower the temperature to 18°C, induce enzyme production, and collect the bacteria by centrifugation after culturing for 12 hours.

[0062] (2) Preparation of crude enzyme solution

[0063] The collected bacteria were added with an appropriate amount (10 times the volume) of lysis buffer (50mmol / L Tris-HCl, 300mmol / L NaCl, 20mmol imidazole, pH=8) to resuspend the bacteria, and after being broken by a high-pressure homogenizer, the supernatant was collected by centrifugation at 4°C, 20000xg for 30min; crude enzyme solutions were prepared respectively; protein gel images are shown in FIG. Figure 1 As shown, the results showed that the glycosyltransferase was expressed.

[0064] (3) Enzyme purification

[0065] The crude enzyme solution was treated with Ni + The column was purified by affinity chromatography. After loading, 10 times the volume of lysis buffer was used to wash the impurities, and the target protein was eluted with elution buffer. The eluted target protein was collected and desalted by a desalting column (HistrpTM 5mLDesalting), and the desalting buffer (25mmol / L Tris-HCl, 150mmol / L NaCl, 10% glycerol) was used. After desalting, it was concentrated to 10mg / mL and then subjected to subsequent reactions to prepare pure enzyme solutions.

[0066] (4)YojK M0 Enzyme activity test of its mutants in catalyzing the reaction of Reb A to synthesize Reb D

[0067] The purified wild-type enzyme YojK, YojK-I241T / G327N (YojK M0 ) and the mutants prepared as above were subjected to glycosylation reaction.

[0068] The relative enzyme activity of the mutants (expressed as YojK) can be obtained by liquid phase analysis. M0 The results are shown in Table 3.

[0069] Table 3: Relative enzyme activities of mutants

[0070]

[0071] The results showed that mutants A218H and YojK M1 The enzyme activity is the highest.

[0072] Example 3: YojK M0 Testing of denaturation temperature of its mutants

[0073] According to the steps in Example 2, YojK M0 Pure enzymes and mutants were used to test the denaturation temperature of the enzyme.

[0074] The specific steps are as follows:

[0075] The test sample enzyme concentration was 1 mg / mL, the buffer was 25 mmol / L Tris-HCl pH 8.0, 150 mmol / L NaCl. The test was performed using Nano-DSC, the baseline test range was 30-90°C, the sample test temperature range was 30-80°C, and the temperature rise rate was 1 min / °C. The measured data are shown in Table 4. Compared with YojK M0 , the denaturation temperature of the mutant was increased.

[0076] Table 4: Changes in relative enzyme activity and denaturation temperature

[0077]

[0078] ΔT m : With YojK M0 The difference in denaturation temperature.

[0079] Example 4: YojK M0 Testing of the optimal temperature of its mutants

[0080] According to the steps in Example 3, YojK M0Pure enzymes of the mutants were used to test the optimal temperature of the enzymes.

[0081] The specific steps are as follows:

[0082] The 200 μL reaction system contained 25 mmol / L Tris-HCl, pH 8.0, 150 mmol / L NaCl, 10 mmol / L UDPG, 4 mmol / L Reb A, and 10 μM pure enzyme; the reaction temperature test range was 30-50°C; the reaction was carried out in a reactor, the reaction time was 20 min, and the reaction oscillation speed was 800 rpm;

[0083] The reaction system was heated at 95°C for 10 min to terminate the reaction, and the product was determined according to the detection method mentioned above.

[0084] The results are as follows Figure 2 As shown, the results show that YojK M0 The optimum temperature of the single point mutation YojK is 45℃. M0 / A218H and YojK M0 / The optimum temperature of A369K increased by 5℃ to 50℃;

[0085] And YojK M0 / S158E and YojK M1 The optimum temperature of YojK M0 The temperature increased by 10℃ and was 55℃. The results showed that the mutant obtained significantly improved the thermal stability of the enzyme.

[0086] Example 5: YojK M0 Testing of half-life of its mutants

[0087] According to the steps in Example 3, YojK M0 Pure enzymes and mutants were used to test the half-life of the enzyme.

[0088] The specific steps are as follows:

[0089] Yj Y M0 The mutants were in 25mmol / L Tris-HCl, pH8.0, 150mmol / L NaCl buffer, the enzyme concentration was 1mg / mL, the sample was incubated at 50℃ for a corresponding time, and then 20μL of the incubated enzyme solution was taken out and added to a 180μL system containing 25mmol / L Tris-HCl, pH 8.0, 150mmol / L NaCl, 10mmol / L UDPG, 4mmol / L Reb A for reaction at 35℃ for 20min. The reaction termination and product determination were all subject to Example 4. The reaction results are shown in Figure 3 shown.

[0090] The results showed that the half-life of the mutant was significantly prolonged. M0 The half-life is 2.16h, YojK M1 The half-life of YojK is 58.64h. M1 The half-life of YojK M0 Compared with the half-life of

[0091] Example 6: YojK M0 With YojK M1 Fed-batch preparation of Reb D

[0092] According to the steps in Example 3, YojK M0 The pure enzyme of the mutant was used to test the actual effect of the enzyme in the fed-batch reaction. The specific steps are as follows:

[0093] (1) Add YojK to a buffer solution containing 25 mmol / L Tris-HCl pH 8.0 and 150 mmol / L NaCl. M0 Enzyme, pure enzyme concentration was 500 mg / L, reaction system was obtained, reaction temperature was 45°C, 5 mmol / L Reb A and 10 mmol / L UDPG were added at time points of 0 h, 1 h, 2 h, 4 h and 6 h, respectively.

[0094] Reaction termination: The reaction system was heated at 95°C for 10 min to determine the reaction termination.

[0095] The substrate and product contents were determined at 1, 2, 3, 4, 6, 8, 10 and 12 h and their yields were calculated. Figure 4 shown.

[0096] (2) The mutant enzyme and the pure enzyme concentration of 500 mg / L were added to 25 mmol / L Tris-HCl pH 8.0 and 150 mmol / L NaCl buffers to obtain a reaction system. The reaction temperature was 50°C, and 5 mmol / L Reb A and 10 mmol / L UDPG were added at 0 h, 1 h, 2 h, 4 h and 6 h, respectively.

[0097] Reaction termination: The reaction system was heated at 95°C for 10 min to determine the reaction termination.

[0098] The substrate and product contents were determined at 1, 2, 3, 4, 6, 8, 10 and 12 h and their yields were calculated. Figure 4 shown.

[0099] The results showed that after a total of 25 mM Reb A was added in batches, YojK M1With YojK M0 In comparison, the conversion rate increased by 23.7% to 87.7%, and the yield was 23.73 mg / mL.

[0100] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A glycosyltransferase mutant, It is characterized in that The mutant is obtained by mutating the serine at position 158 to glutamic acid, the alanine at position 218 to histidine, and the alanine at position 369 to lysine based on the amino acid sequence of the glycosyltransferase YojK-I241T / G327N shown in SEQ ID NO.3; the glycosyltransferase mutant YojK M1 The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the glycosyltransferase mutant according to claim 1.

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

4. A recombinant cell expressing the glycosyltransferase mutant according to claim 1, or containing the gene according to claim 2, or containing the recombinant vector according to claim 3.

5. The recombinant cell according to claim 4, It is characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

6. A method for improving the thermal stability and activity of glycosyltransferase, It is characterized in that The method comprises the following steps: mutating the serine at position 158 of the glycosyltransferase YojK-I241T / G327N of the amino acid sequence shown in SEQ ID NO.3 to glutamic acid, mutating the alanine at position 218 to histidine, and mutating the alanine at position 369 to lysine.

7. A method for catalytic synthesis of rebaudioside D, It is characterized in that The method comprises adding the mutant described in claim 1 or the recombinant cell described in claim 4 or 5 to a reaction system containing rebaudioside A, and preparing rebaudioside D through reaction.

8. The method according to claim 7, It is characterized in that The reaction system contains 1-25 mmol / L rebaudioside A, 5-25% (v / v) DMSO, 25-50 mmol / L Tris-HCl buffer, and 150-300 mmol / L NaCl.

9. Use of the glycosyltransferase mutant according to claim 1, or the gene according to claim 2, or the recombinant cell according to claim 4 or 5 in the preparation of a product containing rebaudioside D.

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