A heat-resistant, long half-life pantothenate synthetase mutant

Abstract

The application discloses a heat-resistant and long-half-life pantothenate synthetase mutant, relates to the field of biochemistry, and the nucleotide sequence of the pantothenate synthetase mutant N54R / K115R / Q148R is SEQ ID NO.1, and the amino acid sequence of the pantothenate synthetase mutant N54R / K115R / Q148R is SEQ ID NO.2.The relative enzyme activity of the pantothenate synthetase mutant is 43% higher than that of the original plasmid, and the catalytic efficiency is stronger.In addition, under the conditions of 35 DEG C, 40 DEG C and 45 DEG C, the enzyme activity residual rate and the half-life of the pantothenate synthetase mutant are significantly higher than those of the original plasmid, and the yield of D-pantothenic acid and the substrate conversion rate are significantly increased.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, specifically to a heat-resistant, long-half-life pantothenic acid synthase mutant, plasmid, strain, and its applications. Background Technology

[0002] Pantothenic acid, also known as vitamin B5, is an important food additive, feed additive, cosmetic additive, and also an important vitamin drug, widely used in many fields. Increasing pantothenic acid yield to reduce its usage cost is a pressing technical problem for researchers. Constructing pantothenic acid synthases is a crucial method for increasing pantothenic acid production; however, current pantothenic acid synthases are not heat-resistant and have short half-lives, resulting in cumbersome synthesis steps and low yields, failing to significantly reduce the synthesis cost of pantothenic acid. Therefore, developing a heat-resistant, long-half-life pantothenic acid synthase mutant is essential.

[0003] Chinese invention patent CN117946984B discloses a pantothenic acid synthase mutant and its preparation method, a construct and its construction method, a pantothenic acid-producing strain, its application, and a method for preparing pantothenic acid. The pantothenic acid synthase mutant is a primitive pantothenic acid synthase containing T29K mutations and / or L135P mutations. Compared with the primitive pantothenic acid synthase, its enzyme activity is significantly enhanced, which can improve the ability of the strain to metabolize and synthesize pantothenic acid, showing good application prospects. However, it lacks heat resistance, and the conditions for its conversion to produce pantothenic acid are relatively harsh, making industrial production difficult. Chinese invention patent CN113789307B discloses a pantothenic acid synthase mutant, its encoding gene, a vector, and its application. The 5th and / or 176th amino acids of its pantothenic acid synthase are obtained through site-directed mutagenesis, which can increase the pantothenic acid synthase activity by 1.25 times compared to the starting strain, making it more suitable for industrial applications, reducing production costs, and improving production efficiency. However, the half-life is not significantly extended. Summary of the Invention

[0004] To develop a heat-resistant, long-half-life pantothenic acid synthase mutant, the first aspect of the present invention provides a heat-resistant, long-half-life pantothenic acid synthase mutant N54R / K115R / Q148R, the nucleotide sequence of which is SEQ ID NO.1, and the amino acid sequence of which is SEQ ID NO.2.

[0005] The pantothenic acid synthase mutant was obtained by site-directed mutagenesis using an artificially synthesized fragment of the panC gene, which is optimized from wild-type Corynebacterium gluteni, as a template to obtain the pantothenic acid synthase mutant N54R / K115R / Q148R.

[0006] As one embodiment, the pantothenic acid synthase mutant N54R / K115R / Q148R includes N54R mutation, K115R mutation and Q148R mutation.

[0007] In one embodiment, the N54R mutation is performed by changing the 54th position of asparagine (N) to arginine (R); the K115R mutation is performed by changing the 115th position of lysine (K) to arginine (R); and the Q148R mutation is performed by changing the 148th position of glutamine (Q) to arginine (R).

[0008] This application introduces three key amino acid substitutions through site-directed mutagenesis. This mutation enhances the structural stability of the enzyme protein by changing the surface charge distribution and spatial conformation of the enzyme molecule, thereby improving thermal stability and half-life. At the same time, because the mutation site does not destroy the active site of the enzyme, the enzyme's catalytic activity is preserved and enhanced.

[0009] A second aspect of the present invention provides a pantothenic acid synthase mutant plasmid comprising the above-mentioned pantothenic acid synthase mutant, wherein the vector of the pantothenic acid synthase mutant plasmid is pET28a.

[0010] A third aspect of the present invention provides a method for preparing a pantothenic acid synthase mutant plasmid, comprising the following steps:

[0011] Preparation of the original plasmid pET28a-panC;

[0012] Using the original plasmid pET28a-panC as a template, reverse PCR was performed on the template using primers SEQ ID NO.3 and SEQ ID NO.4. After template elimination, the PCR product was ligated to obtain the mutant plasmid pET28a-panC. N54R ;

[0013] Design primers SEQ ID NO.5 and SEQ ID NO.6 with pET28a-panC N54R Using a template, a reverse PCR reaction was performed. After removing the template, the PCR product was ligated to obtain the mutant plasmid pET28a-panC. N54R / K115R；

[0014] Design primers SEQ ID NO.7 and SEQ ID NO.8 with pET28a-panC N54R / K115R Using a template, a reverse PCR reaction was performed. After removing the template, the PCR product was ligated to obtain pET28a-panC. N54R / K115R / Q148R .

[0015] In one embodiment, the nucleotide sequence of SEQ ID NO.3 is: F-TCTATCTTTGTGCGCCCGCTGCAGTTTGAAGCAC; and the nucleotide sequence of SEQ ID NO.4 is: R-GGGCGCACAAAGATAGAGGCTACAACAGTATCG.

[0016] In one embodiment, the nucleotide sequence of SEQ ID NO.5 is: F-TCTATCGGTACTCGCCTGGAAGGTGCGTCTCGTCC; and the nucleotide sequence of SEQ ID NO.6 is: R-AGGCGAGTACCGATAGAGCCGGTGCGTGCCCA.

[0017] In one embodiment, the nucleotide sequence of SEQ ID NO.7 is: F-ACTTCGGTCGCAAAGACGCGCAGCAGGTAGCG; and the nucleotide sequence of SEQ ID NO.8 is: R-GTCTTTGCGACCGAAGTATGCGCGATCCGGAC.

[0018] In one embodiment, the reverse PCR reaction system includes sterile ddH2O, PCR buffer, deoxyribonucleoside triphosphate, forward and reverse primers, template plasmid, and DNA polymerase.

[0019] As one embodiment, the reverse PCR reaction system includes: 6 μL of sterile ddH2O, 5 μL of 10×PCR buffer, 5 μL of 2 mM deoxynucleoside triphosphates (dNTPs), 1 μL of 10 pmol / μL forward primer and 1 μL of 10 pmol / μL reverse primer, 1 μL of 50 ng / μL template plasmid, and 1 μL of KOD-Plus DNA polymerase.

[0020] As one implementation method, the conditions for the reverse PCR reaction include pre-denaturation: 94°C, 2 min, cycle number 1; denaturation-annealing-extension: 98°C, 10 s, 68°C, 1 min, cycle number 4; and holding at 4°C.

[0021] As one implementation method, the reaction conditions for ligating the PCR product are 16°C for 1 hour.

[0022] In one embodiment, the reaction system for ligating the PCR product consists of 2 μL of PCR product, 1 μL of high-efficiency DNA ligase, 1 μL of T4 polynucleotide kinase, and 6 μL of sterile ddH2O.

[0023] As one implementation method, the preparation method of the original plasmid pET28a-panC includes: using the panC gene as a template, performing a PCR reaction using primers F1 and R1 to add the same restriction sites as the plasmid vector to both ends of the panC gene, digesting the vector to obtain a linearized vector, and ligating it with the PCR product to obtain pET28a-panC.

[0024] As one embodiment, the nucleotide sequence of primer F1 is as follows: F1: 5'-CGCGGATCCGCGATGCAAGTCGCAACCACCAAAC-3'; (corresponding to SEQ ID NO.9 in the nucleotide sequence listing)

[0025] In one embodiment, the nucleotide sequence of primer R1 is as follows: R1: 5'-CCAAGCTTGGCTACAGTTCGATATTGTCGATC-3'. (Corresponding nucleotide sequence in SEQ ID NO.10)

[0026] As one implementation method, the PCR reaction conditions include: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 15 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 60 seconds, for 30 cycles; and final extension at 72°C for 5 minutes.

[0027] As one implementation method, the PCR reaction (20 μL) system includes: 1 μL template (panC gene), 1 μL F1, 1 μL R1, 1 μL dNTP, 10 μL DNA polymerase, and 6 μL ddH2O.

[0028] As one implementation method, the enzyme digestion reaction conditions are 37°C for 20 min.

[0029] As one embodiment, the enzyme digestion reaction system includes: 25 μL of pET28a vector plasmid, 5 μL of 10× buffer, 2 μL of BamHI (restriction endonuclease), 2 μL of HindIII (restriction endonuclease), and 16 μL of ddH2O.

[0030] As one implementation method, the reaction conditions for ligating the PCR product are 37°C for 30 min.

[0031] In one embodiment, the reaction system for ligating the PCR product includes: 4.5 μL of pET28a vector, 1.5 μL of panC gene, 4 μL of 5×CEⅡ Buffer, 2 μL of ExnaseⅡ, and 8 μL of ddH2O.

[0032] A fourth aspect of the present invention provides a genetically engineered strain comprising the above-described pantothenic acid synthase mutant plasmid, wherein the genetically engineered strain uses Escherichia coli BL21 as the host cell.

[0033] As one implementation method, the construction and induced expression of the genetically engineered strain are as follows:

[0034] 1 μL of the original plasmid and mutant plasmid that were correctly sequenced were added to E. coli BL21 competent cells, placed on ice for 30 min, then heat-shocked in a water bath at 42℃ for 60 s, and incubated on ice for 5 min.

[0035] Add 500 μL of LB liquid culture and allow it to recover at 37℃ with shaking for 1 h. Then take 100 μL of the bacterial culture and spread it on an LB culture plate.

[0036] Pick a single colony and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37°C and 120 rpm for 12-16 h to obtain seed culture.

[0037] Inoculate the seed culture into LB medium at a volume ratio of 2% and incubate at 37°C until OD. 600 The value was 0.6-0.8, then the temperature was lowered to 18℃, and IPTG with a final concentration of 0.1mM was added to induce expression for 16-18h to obtain the fermentation broth, thus achieving induced expression.

[0038] In one embodiment, the fermentation broth is post-processed to achieve protein separation and purification. The protein separation and purification method includes the following steps:

[0039] The fermentation broth was centrifuged at 8000 rpm for 15 min at 4 °C. The bacterial cells in the fermentation broth were collected, resuspended in 50 mmol / L sodium carbonate-sodium bicarbonate buffer (pH 9.0), and then the bacterial cells were lysed using a high-pressure homogenizer (750 bar, 10 min). The supernatant after centrifugation at 12000 rpm for 1 h at 4 °C to remove cell debris was the crude enzyme solution.

[0040] In one embodiment, the nucleotide sequence of the panC gene is SEQ ID NO.11, and the amino acid sequence of the panC gene is SEQ ID NO.12.

[0041] A fifth aspect of the present invention provides a pantothenic acid synthase obtained by expression in the above-described genetically engineered strain.

[0042] A sixth aspect of the present invention provides an application of a pantothenic acid synthase, said pantothenic acid synthase being used to catalyze the reaction of D-pantothenic acid with β-alanine to generate D-pantothenic acid.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) The pantothenic acid synthase mutant of the present invention is obtained by designing site-directed mutagenesis primers to obtain pantothenic acid synthase mutants N54R / K115R / Q148R containing N54R mutation, K115R mutation and Q148R mutation. Its relative enzyme activity is increased by 43% compared with the original plasmid and its catalytic efficiency is stronger.

[0045] (2) The pantothenic acid synthase mutant of the present invention has a significantly higher enzyme activity residual rate than the original plasmid at 35℃, 40℃ and 45℃, which can adapt to the medium temperature reaction conditions in industrial production, relax the reaction conditions, and facilitate industrial scale-up production.

[0046] (3) The pantothenic acid synthase mutant of the present invention has a half-life of 76.5%, 79.2% and 77.5% longer than the original plasmid at 35℃, 40℃ and 45℃, respectively, which prolongs the effective catalytic time of the enzyme, reduces the amount of enzyme preparation added and lowers the production cost.

[0047] (4) The pantothenic acid synthase mutant of the present invention increases the yield of D-pantothenic acid by 16.8% and the substrate conversion rate by 3.16% compared with the original plasmid, significantly improving the economic benefits of industrial production.

[0048] (5) The pantothenic acid synthase mutant of the present invention is constructed by modifying the genome to create an engineered strain that can catalyze the production of D-pantothenic acid from glucose, thereby increasing the enzyme activity of pantothenic acid synthase, increasing the yield of pantothenic acid, and reducing the cost of producing pantothenic acid. Attached Figure Description

[0049] Figure 1 The relative enzyme activity curves of pantothenic acid synthases constructed from the original plasmid and mutant plasmid at 35°C.

[0050] Figure 2 The relative enzyme activity curves of pantothenic acid synthases constructed from the original plasmid and mutant plasmid at 40℃.

[0051] Figure 3 The relative enzyme activity curves of pantothenic acid synthases constructed from the original plasmid and mutant plasmid at 45℃.

[0052] In the figure, the original plasmid corresponds to the wild type, and the mutant plasmid corresponds to Cg. Detailed Implementation

[0053] Example

[0054] A heat-resistant, long-half-life pantothenic acid synthase mutant, wherein the nucleotide sequence of the pantothenic acid synthase mutant N54R / K115R / Q148R is SEQ ID NO.1 and the amino acid sequence of the pantothenic acid synthase mutant N54R / K115R / Q148R is SEQ ID NO.2.

[0055] A method for preparing a pantothenic acid synthase mutant plasmid includes the following steps:

[0056] Preparation of the original plasmid pET28a-panC;

[0057] Using the original plasmid pET28a-panC as a template, a reverse PCR reaction was performed on the template using primer SEQ ID NO.3. After template elimination, the PCR product was ligated to obtain the mutant plasmid pET28a-panC. N54R ;

[0058] Design primer SEQ ID NO.4 with pET28a-panC N54R Using a template, a reverse PCR reaction was performed. After removing the template, the PCR product was ligated to obtain the mutant plasmid pET28a-panC. N54R / K115R；

[0059] Design primer SEQ ID NO.5 with pET28a-panC N54R / K115R Using a template, a reverse PCR reaction was performed. After removing the template, the PCR product was ligated to obtain pET28a-panC. N54R / K115R / Q148R .

[0060] The reverse PCR reaction system includes: 6 μL of sterile ddH2O, 5 μL of 10×PCR buffer, 5 μL of 2 mM deoxynucleoside triphosphates (dNTPs), 1 μL of 10 pmol / μL forward primer and 1 μL of 10 pmol / μL reverse primer, 1 μL of 50 ng / μL template plasmid, and 1 μL of KOD-Plus DNA polymerase.

[0061] The conditions for the reverse PCR reaction include: pre-denaturation: 94℃, 2 min, cycle number 1; denaturation-annealing-extension: 98℃, 10 s, 68℃, 1 min, cycle number 4; and incubation: 4℃.

[0062] The reaction conditions for ligating the PCR product were 16°C for 1 hour.

[0063] The reaction system for ligating the PCR product consisted of 2 μL of PCR product, 1 μL of high-efficiency DNA ligase, 1 μL of T4 polynucleotide kinase, and 6 μL of sterile ddH2O.

[0064] The preparation method of the original plasmid pET28a-panC includes: using the panC gene as a template, performing a PCR reaction with primers F1 and R1 to add the same restriction sites as the plasmid vector to both ends of the panC gene, digesting the vector to obtain a linearized vector, and ligating it with the PCR product to obtain pET28a-panC.

[0065] The PCR reaction conditions included: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 15 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 60 seconds, for 30 cycles; and final extension at 72°C for 5 minutes.

[0066] The PCR reaction (20 μL) system includes: 1 μL template (panC gene), 1 μL F1, 1 μL R1, 1 μL dNTP, 10 μL DNA polymerase, and 6 μL ddH2O.

[0067] The enzyme digestion reaction conditions were 37℃ for 20 min.

[0068] The enzyme digestion reaction system includes: 25 μL of pET28a vector plasmid, 5 μL of 10× buffer, 2 μL of BamHI (restriction endonuclease), 2 μL of HindIII (restriction endonuclease), and 16 μL of ddH2O.

[0069] The reaction conditions for ligating the PCR product were 37°C for 30 min.

[0070] The reaction system for ligating the PCR products includes: 4.5 μL of pET28a vector, 1.5 μL of panC gene, 4 μL of 5×CEⅡ Buffer, 2 μL of ExnaseⅡ, and 8 μL of ddH2O.

[0071] A genetically engineered strain, its construction and induced expression method are as follows:

[0072] 1 μL of the original plasmid and mutant plasmid with correct sequencing were added to E. coli BL21 competent cells, placed on ice for 30 min, then heat-shocked in a water bath at 42℃ for 60 s, and incubated on ice for 5 min.

[0073] Add 500 μL of LB liquid culture and allow it to recover at 37℃ with shaking for 1 h. Then take 100 μL of the bacterial culture and spread it on an LB culture plate.

[0074] Pick a single colony and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37°C and 120 rpm for 12-16 h to obtain seed culture.

[0075] Inoculate the seed culture into LB medium at a volume ratio of 2% and incubate at 37°C until OD.600 The value was 0.6-0.8, then the temperature was lowered to 18℃, and IPTG with a final concentration of 0.1mM was added to induce expression for 16-18h to obtain the fermentation broth, thus achieving induced expression.

[0076] The post-treatment of the fermentation broth achieves protein separation and purification. The method for protein separation and purification includes the following steps:

[0077] The fermentation broth was centrifuged at 8000 rpm for 15 min at 4 °C. The bacterial cells in the fermentation broth were collected, resuspended in 50 mmol / L sodium carbonate-sodium bicarbonate buffer (pH 9.0), and then the bacterial cells were lysed using a high-pressure homogenizer (750 bar, 10 min). The supernatant after centrifugation at 12000 rpm for 1 h at 4 °C to remove cell debris was the crude enzyme solution.

[0078] Performance testing

[0079] 1. Relative enzyme activity assay: The test system (10 mL) included: 50 mM sodium phosphate buffer (pH 9.0), 25 mM pantothenic acid, 25 mM β-alanine, 4.5 mM ATP, 10 mM magnesium chloride, 15 mM potassium chloride, and 3 mg / mL crude enzyme solution. The reaction was carried out at 37℃ for 45 min. One unit of activity was defined as the generation of 1 μM pantothenic acid per minute, and the enzyme activity of the original plasmid was taken as 100%. The relative enzyme activity of the mutant plasmid was calculated. The test results are shown in Table 1.

[0080] 2. Thermal stability test: The test system (10 mL) included: 50 mM sodium phosphate buffer (pH 9.0), 25 mM pantothenic acid, 25 mM β-alanine, 4.5 mM ATP, 10 mM magnesium chloride, 15 mM potassium chloride, and 3 mg / mL crude enzyme solution. The test system was incubated at 35℃, 40℃, and 45℃, with enzyme activity at 0 h considered 100%. Samples were taken at different time points (2, 4, 6, 8, 10, 12, and 14 h) to detect enzyme activity, and the half-life at these three temperatures was obtained. The test results are shown below. Figure 1-3 See Table 1.

[0081] 3. D-Pantothenic Acid Production Test: The D-pantothenic acid production reaction system (10 mL) included: 12 g / L D-pantothenic acid, 6 g / L β-alanine, 25 mM magnesium chloride, 30 mM potassium chloride, and 3 mg / mL crude enzyme solution. The remaining D-pantothenic acid and the yield of generated D-pantothenic acid were measured, and the conversion rate was calculated. The conversion rate was [(initial moles of D-pantothenic acid - remaining moles of D-pantothenic acid) / initial moles of D-pantothenic acid] × 100%. The test results are shown in Table 1.

[0082] Table 1

[0083]

[0084] Depend on Figure 1-3 As shown in Table 1:

[0085] The mutant plasmid pET28a-panC of this application N54R / K115R / Q148R The enzyme activity was increased by 43% compared to the original plasmid; the D-pantothenic acid yield was increased; the conversion rate was increased by 3.16%; the thermal stability at 35, 40 and 45℃ was improved; the half-life at 35℃ was increased from 6.8h to 12.0h compared to the wild-type enzyme, the half-life at 40℃ was increased from 5.3h to 9.5h, and the half-life at 45℃ was increased from 4.0h to 7.1h.

Claims

1. A heat-resistant, long-half-life pantothenic acid synthase mutant, characterized in that, The nucleotide sequence of the pantothenic acid synthase mutant N54R / K115R / Q148R is SEQ ID NO.1, and the amino acid sequence of the pantothenic acid synthase mutant N54R / K115R / Q148R is SEQ ID NO.

2.

2. The heat-resistant, long-half-life pantothenic acid synthase mutant according to claim 1, characterized in that, The pantothenic acid synthase mutant N54R / K115R / Q148R includes the N54R mutation, the K115R mutation, and the Q148R mutation.

3. A pantothenic acid synthase mutant plasmid, characterized in that, Includes the pantothenic acid synthase mutant according to any one of claims 1-2, wherein the vector of the pantothenic acid synthase mutant plasmid is pET28a.

4. A method for preparing a pantothenic acid synthase mutant plasmid according to claim 3, characterized in that, Includes the following steps: Preparation of the original plasmid pET28a-panC; Using the original plasmid pET28a-panC as a template, reverse PCR was performed on the template using primers SEQ ID NO.3 and SEQ ID NO.

4. After template elimination, the PCR product was ligated to obtain the mutant plasmid pET28a-panC. N54R ; Design primers SEQ ID NO.5 and SEQ ID NO.6 with pET28a-panC N54R Using a template, a reverse PCR reaction was performed. After removing the template, the PCR product was ligated to obtain the mutant plasmid pET28a-panC. N54R / K115R ; Design primers SEQ ID NO.7 and SEQ ID NO.8 with pET28a-panC N54R / K115R Using a template, a reverse PCR reaction was performed. After removing the template, the PCR product was ligated to obtain pET28a-panC. N54R / K115R / Q148R .

5. The method for preparing the pantothenic acid synthase mutant plasmid according to claim 4, characterized in that, The reverse PCR reaction system includes sterile ddH2O, PCR buffer, deoxyribonucleoside triphosphate, forward and reverse primers, template plasmid, and DNA polymerase.

6. The method for preparing the pantothenic acid synthase mutant plasmid according to claim 4, characterized in that, The conditions for the reverse PCR reaction include pre-denaturation: 94℃, 2 min, cycle number 1; denaturation-annealing-extension: 98℃, 10 s, 68℃, 1 min, cycle number 4; Insulation: 4℃.

7. The method for preparing the pantothenic acid synthase mutant plasmid according to claim 4, characterized in that, The preparation method of the original plasmid pET28a-panC includes: using the panC gene as a template, performing a PCR reaction with primers F1 and R1 to add the same restriction sites as the plasmid vector to both ends of the panC gene, digesting the vector to obtain a linearized vector, and ligating it with the PCR product to obtain pET28a-panC.

8. A genetically engineered strain, characterized in that, The invention includes the pantothenic acid synthase mutant plasmid as described in claim 3, wherein the genetically engineered strain uses Escherichia coli BL21 as the host cell.

9. A pantothenic acid synthase, characterized in that, It is obtained by expression of the genetically engineered strain described in claim 8.

10. An application of a pantothenic acid synthase, characterized in that, The pantothenic acid synthase is used to catalyze the reaction of D-pantothenic acid with β-alanine to generate D-pantothenic acid.

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