Vitamin D3 hydroxylase mutants and their application in the preparation of 25-hydroxyvitamin D3

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to vitamin D3 hydroxylase mutants and their application in the preparation of 25-hydroxyvitamin D3. This invention obtains vitamin D3 hydroxylase mutants VM, MV, and VMV with higher activity by directed mutation of vitamin D3 hydroxylase Vdh-K1. Compared to the parental vitamin D3 hydroxylase Vdh-K1, the activities of mutants VM, MV, and VMV are increased by 2.4 times, 1.6 times, and 2.2 times, respectively. When used in the preparation of 25-hydroxyvitamin D3, the conversion rate and conversion efficiency of VD3 to 25-hydroxyvitamin D3 are significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to vitamin D3 hydroxylase mutants and their application in the preparation of 25-hydroxyvitamin D3. Background Technology

[0002] Vitamin D3 (VD3), also known as cholecalciferol, is a fat-soluble vitamin that plays a crucial role in maintaining calcium and phosphorus balance, bone health, and many other physiological processes. 25-hydroxyvitamin D3 (25(OH)VD3) is the active form of vitamin D3 in vivo, and compared to VD3, it can more directly and effectively treat diseases related to VD3 deficiency.

[0003] Currently, the preparation methods of 25(OH)VD3 are mainly divided into chemical synthesis and biotransformation. Biotransformation primarily relies on C25 hydroxylase to convert VD3 to 25(OH)VD3 in a one-step reaction. Compared with chemical synthesis, biotransformation has advantages such as less environmental pollution, higher selectivity, and milder reaction conditions. Vdh-K1 is derived from autotrophic pseudonocardia (…). Pseudonocardia autotrophica Vitamin D3 hydroxylase (Vdh-K1) has been found to possess C25 hydroxylation activity for VD3 with high selectivity. However, as a fat-soluble vitamin, VD3 requires hydrophobic interaction to bind to the hydrophobic pocket of Vdh-K1. The hydrophobicity and steric properties of this pocket directly affect the binding of VD3 to Vdh-K1, thus influencing the efficiency of the C25 hydroxylation reaction, i.e., the ability of VD3 to be catalytically converted to 25(OH)VD3. Improving the catalytic activity of Vdh-K1 is of great significance for enhancing its industrial application potential.

[0004] In view of this, this invention is hereby proposed. Summary of the Invention

[0005] Based on the above problems, the purpose of this invention is to provide a vitamin D3 hydroxylase mutant and its application in the preparation of 25(OH)VD3. This mutant has higher activity and its ability to catalyze the conversion of VD3 to 25(OH)VD3 is significantly improved.

[0006] The first aspect of the present invention provides a vitamin D3 hydroxylase mutant, which is based on the vitamin D3 hydroxylase with the amino acid sequence shown in SEQ ID NO.1, and the specific mutation sites include L171V+V172M, V172M+A177V, and L171V+V172M+A177V.

[0007] Furthermore, the amino acid sequence of the mutant with the mutation site L171V+V172M is shown in SEQ ID NO.3; the amino acid sequence of the mutant with the mutation site V172M+A177V is shown in SEQ ID NO.5; and the amino acid sequence of the mutant with the mutation site L171V+V172M+A177V is shown in SEQ ID NO.7.

[0008] A second aspect of the invention provides a gene encoding the above-mentioned vitamin D3 hydroxylase mutant.

[0009] A third aspect of the present invention provides a recombinant expression vector comprising the above-described gene.

[0010] Furthermore, the vector for the recombinant expression vector is plasmid pET28a.

[0011] A fourth aspect of the present invention provides a recombinant strain comprising the above-described recombinant expression vector.

[0012] Furthermore, the host cell of the recombinant strain is Escherichia coli BL21(DE3).

[0013] The fifth aspect of the present invention provides the use of the above-mentioned vitamin D3 hydroxylase mutant in the preparation of 25(OH)VD3.

[0014] Furthermore, a vitamin D3 hydroxylase mutant was used to convert VD3 into 25(OH)VD3.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. This invention obtains vitamin D3 hydroxylase mutants VM, MV, and VMV with higher activity by targeted mutation of vitamin D3 hydroxylase Vdh-K1. Compared with the parental vitamin D3 hydroxylase Vdh-k1, the activities of mutants VM, MV, and VMV are increased by 2.4 times, 1.6 times, and 2.2 times, respectively.

[0017] 2. When the vitamin D3 hydroxylase mutant of the present invention is used in the preparation of 25(OH)VD3, the conversion rate and conversion efficiency of VD3 to 25(OH)VD3 are significantly improved. Attached Figure Description

[0018] Figure 1 The results are the relative activity assay results of the vitamin D3 hydroxylase single-point mutant in Example 1;

[0019] Figure 2 The results are the relative activity assay results of the vitamin D3 hydroxylase combination mutant in Example 2;

[0020] Figure 3 The figure shows the trend of the concentration of product 25(OH)VD3 with reaction time in the reaction system of vitamin D3 hydroxylase mutant VM and vitamin D3 hydroxylase Vdh-k1 in Example 3. Detailed Implementation

[0021] The invention is further described below through specific embodiments. Unless otherwise specified, the technical means and materials involved in the following embodiments are all known to those skilled in the art, and suitable means and materials that can solve the corresponding technical problems can be selected. In addition, the embodiments should be understood as illustrative, not limiting the scope of the invention, and the essence and scope of the invention are defined only by the claims.

[0022] It should be understood that the scope of this invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to these embodiments that will be apparent to those skilled in the art or related fields without departing from the spirit and scope of this invention are covered within the scope of the appended claims.

[0023] It should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0024] The markers for the vitamin D3 hydroxylase mutant used in this invention are as follows:

[0025] The amino acid of the vitamin D3 hydroxylase mutation is represented by "the amino acid that was replaced at the original amino acid position". For example, V172M means that the amino acid at position 172 is replaced by M in the parental vitamin D3 hydroxylase, and the position number corresponds to the amino acid sequence number in SEQ ID NO.1.

[0026] This invention provides a vitamin D3 hydroxylase mutant, the amino acid sequence of which is shown in SEQ ID NO.3, SEQ ID NO.5, or SEQ ID NO.7.

[0027] This embodiment uses vitamin D3 hydroxylase Vdh-k1, whose amino acid sequence is shown in SEQ ID NO.1, as the parent. Structural analysis revealed that leucine at position 171, valine at position 172, and alanine at position 177 are potential mutation sites. Mutating these sites to one of valine, leucine, isoleucine, or methionine could potentially improve the affinity between the enzyme and the substrate. Based on this analysis, this embodiment first performs single-point mutations on Vdh-k1, screening for single-point mutants with significantly enhanced activity. Then, it further constructs combinatorial mutants, ultimately obtaining vitamin D3 hydroxylase mutants VM, MV, or VMV with significantly enhanced activity. The amino acid sequences of mutant VM are shown in SEQ ID NO.3, with mutation sites including L171V and V172M, specifically leucine and valine at positions 171 and 172 are mutated to valine and methionine, respectively. The amino acid sequences of mutant MV are shown in SEQ ID NO.5, with mutation sites including V172M and A177V, specifically valine and alanine at positions 172 and 177 are mutated to methionine and valine, respectively. The amino acid sequences of mutant VMV are shown in SEQ ID NO.7, with mutation sites including L171V, V172M, and A177V, specifically leucine, valine, and alanine at positions 171, 172, and 177 are mutated to valine, methionine, and valine, respectively. The activities of mutants VM, MV, and VMV were increased by 2.4-fold, 1.6-fold, and 2.2-fold compared to Vdh-K1, respectively.

[0028] The present invention further provides a gene encoding the vitamin D3 hydroxylase mutant as described above, the nucleotide sequence of which is shown in SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8.

[0029] This invention further provides a recombinant expression vector containing the gene described above. The vector for the recombinant vector is plasmid pET28a. This embodiment ensures stable transcription and induced expression of the vitamin D3 hydroxylase mutant gene in host cells by constructing an operable linker structure containing the coding gene and the highly efficient expression regulatory element pET28a, thereby improving the expression level and solubility of the recombinant expression vector.

[0030] The present invention further provides a recombinant strain comprising the recombinant expression vector described above, to achieve high biomass expression and functional folding of the vitamin D3 hydroxylase mutant. In some preferred embodiments, the host cell of the recombinant strain is Escherichia coli BL21(DE3).

[0031] This invention also provides the application of a vitamin D3 hydroxylase mutant in the preparation of 25(OH)VD3. Using the vitamin D3 hydroxylase mutant of this embodiment, VD3 is converted to 25(OH)VD3 with a significantly higher conversion rate than Vdh-K1.

[0032] To make the technical solution of the present invention clearer, the vitamin D3 hydroxylase mutant is described in detail below through several specific embodiments.

[0033] The experimental reagents and their components involved in the embodiments of this invention include:

[0034] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L;

[0035] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L;

[0036] Buffer A: 500 mM sodium chloride, 20 mM dipotassium hydrogen phosphate, 20 mM imidazole, 10% glycerol, pH 7.4;

[0037] Buffer B: 500 mM sodium chloride, 20 mM dipotassium hydrogen phosphate, 300 mM imidazole, 10% glycerol, pH 7.4;

[0038] Buffer C: 100 mM sodium chloride, 20 mM dipotassium hydrogen phosphate, pH 7.4.

[0039] The indicators and their measurement methods involved in the embodiments of this invention include:

[0040] The conversion rate (%) of vitamin D3 hydroxylase Vdh-K1 / mutant to 25(OH)VD3: The reaction system included 2 µM vitamin D3 hydroxylase or mutant, 4 µM redox protein reductase (Fdr), 12 µM redox protein (Fdx), 2 U glucose-6-phosphate dehydrogenase (G6PD), 10 mM glucose-6-phosphate, 10% (w / w) 2,6-dimethyl-β-cyclodextrin, 2 mM NADPH, 0.05 g / L VD3, 100 mM NaCl, 20 mM KH2PO4, and pH 7.4. The reaction system was subjected to hydroxylation at 30 °C. After the reaction, the product was analyzed by HPLC. The detection method was as follows: an Agilent 1260 Infinity II high-performance liquid chromatograph with a UV detector was used. The chromatographic column was a Poroshell 120 EC-C18 4 µm (4.6 × 150 mm). The column temperature was 40 °C, the flow rate was 1 ml / min, and the mobile phase was acetonitrile and water (acetonitrile 60%-100% for 0-12 min, 100% for 12-23 min, and 100%-60% for 23-30 min). The injection volume was 20 µL, and the detection wavelength was 265 nm. The concentration of 25(OH)VD3 in the reaction product was calculated based on the peak area, and then the conversion rate was calculated according to the following formula:

[0041] .

[0042] Relative activity is defined as the ratio of the conversion rate of the mutant to the conversion rate of Vdh-K1 after the reaction system has been reacted at 30 °C for 1 h.

[0043] Enzyme reaction rate: A higher enzyme reaction rate indicates a faster conversion of VD3 to 25(OH)VD3. The calculation formula is as follows:

[0044] .

[0045] Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0046] Example 1: Construction and screening of single-point mutants of vitamin D3 hydroxylase

[0047] (1) Obtain the vitamin D3 hydroxylase Vdh-k1 gene

[0048] The vitamin D3 hydroxylase crystal structure was obtained from PDB:3A50, and the amino acid sequence of vitamin D3 hydroxylase was extracted from it. After codon optimization of the sequence, the optimized gene sequence was synthesized by a biotechnology company, resulting in the vitamin D3 hydroxylase Vdh-k1 gene with the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2.

[0049] (2) Construction of plasmid pET28a-Vdhk1

[0050] The Vdh-k1 gene and the vector plasmid pET28a were amplified by PCR using primers VF / VR and 28-F / 28-R with homologous ends as shown in Table 1. The amplification reaction system is shown in Table 2, and the amplification program is shown in Table 3. After purification and recovery of the PCR amplification products, recombination ligation was performed using a seamless cloning kit. The ligation products were then transformed into E. coli DH5α competent cells and plated on LB solid medium containing kanamycin for screening. After sequencing verification of the transformants, the plasmid pET28a-Vdhk1 was extracted.

[0051] Table 1. Primers used to construct plasmid pET28a-Vdhk1

[0052] .

[0053] Table 2 PCR amplification reaction system

[0054] .

[0055] Table 3 PCR amplification program

[0056] .

[0057] (3) Constructing a single-point mutant of vitamin D3 hydroxylase

[0058] Using plasmid pET28a-Vdhk1 as a template, PCR amplification was performed on the plasmid using the site-directed mutagenesis primers listed in Table 4. A single-point mutation was performed on leucine at position 171, valine at position 172, or alanine at position 177 of vitamin D3 hydroxylase Vdh-k1, replacing one of valine, leucine, isoleucine, or methionine. For example, PCR amplification of plasmid pET28a-Vdhk1 was performed using primers L171V-F / L171V-R. The amplification reaction system and amplification program are shown in Tables 2 and 3. After purification and recovery of the PCR product, recombination ligation was performed using a seamless cloning kit. The ligation product was then transformed into *E. coli* DH5α competent cells and plated on LB solid medium containing kanamycin for screening. After sequencing verification of the transformants, the plasmid was extracted to obtain the single-point mutant plasmid pET28-L171V with the mutation site L171V. Following the above method, by changing the site-directed mutagenesis primers, single-point mutant plasmids of vitamin D3 hydroxylase, pET28-L171I, pET28-L171M, pET28-V172L, pET28-V172I, pET28-V172M, pET28-A177V, pET28-A177L, and pET28-A177I, were obtained.

[0059] Table 4 Primers for single-point mutation of vitamin D3 hydroxylase

[0060] .

[0061] (4) Expression and purification of vitamin D3 hydroxylase single-point mutant

[0062] The nine vitamin D3 hydroxylase single-point mutant plasmids were transformed into *E. coli* BL21(DE3) competent cells and plated on LB solid medium containing kanamycin for screening. After sequence verification, the transformants were inoculated into 5 ml of LB liquid medium containing kanamycin and cultured overnight with shaking at 37°C and 200 rpm to obtain the vitamin D3 hydroxylase single-point mutant seed culture. The seed culture was inoculated into 100 ml of LB liquid medium containing kanamycin and cultured with shaking at 37°C and 200 rpm. When OD600 = 0.6-0.8, IPTG (0.2 mM), ferrous sulfate (0.1 mM), and 5-aminolevulinic acid (0.1 mM) were added, and the culture was incubated overnight with shaking at 16°C and 120 rpm.

[0063] The bacterial cells were collected by centrifugation, resuspended in 10 ml of buffer A, and subjected to sonication (200 W, 2 seconds on, 4 seconds off, 10 min). The supernatant was then collected by centrifugation and purified using a Ni-NTA resin affinity column. After binding the crude enzyme solution to the resin, non-target proteins were eluted with 10 ml of buffer A, followed by elution with 10 ml of buffer B, and the target protein was collected. Finally, the target protein was desalted and concentrated in buffer C using an ultrafiltration tube to obtain a purified vitamin D3 hydroxylase single-point mutant enzyme solution.

[0064] The relative activity of the nine pure enzyme solutions obtained was determined, and the results are as follows: Figure 1 As shown in the figure, the relative activities of the single-point mutants V172M and A177V reached 1.35 and 1.25, respectively, which are significantly higher than those of Vdh-K1. This may be because the amino acid side chains of the mutants have more hydrophobic interactions with vitamin D3, which improves the affinity between the enzyme and the substrate.

[0065] Example 2 Construction and screening of vitamin D3 hydroxylase combinatorial mutants

[0066] Based on Example 1, the plasmid pET28a-V172M was further used as a template, and the multi-point mutation primers described in Table 5 were used to perform PCR amplification on the plasmid to construct a combined mutant.

[0067] The plasmid pET28a-V172M was amplified using primers VM-F / VM-R. The amplification reaction system and procedure are shown in Tables 2 and 3. After purification and recovery of the PCR product, recombination ligation was performed using a seamless cloning kit. The ligation product was then transformed into *E. coli* DH5α competent cells and plated on LB medium containing kanamycin for screening. After sequencing verification of the transformants, the plasmid was extracted to obtain the vitamin D3 hydroxylase combined mutant plasmid pET28a-VM, which contains mutation sites including L171V and V172M.

[0068] Following the method described above, plasmid pET28a-V172M was amplified using primers MV-F / MV-R to obtain a combined mutant plasmid pET28a-MV with mutation sites including V172M and A177V. Further amplification of plasmid pET28a-MV using primers VMV-F / VMV-R yielded a combined mutant plasmid pET28a-VMV with mutation sites including L171V, V172M, and A177V.

[0069] Table 5 Primers for multi-point mutation of vitamin D3 hydroxylase

[0070] .

[0071] Following the expression and purification methods described in Example 1, the three vitamin D3 hydroxylase combined mutant plasmids were expressed and purified. The relative activities of the three purified enzyme solutions were determined, and the results are as follows: Figure 2 As shown in the figure, the activity of the combined mutants is further improved compared with the single-point mutants. The activities of mutants VM, MV, and VMV are increased to 2.4 times, 1.6 times, and 2.2 times that of Vdh-K1, respectively. The activity of the vitamin D3 hydroxylase single-point mutant L171V is decreased compared with Vdh-K1, but the activity of the combined mutant VM, which includes mutation sites L171V and V172M, is significantly increased compared with the single-point mutant V172M. This may be because the leucine at position 171 is mutated into valine with a smaller side chain, which reduces the steric hindrance between valine and methionine at position 172, making the methionine side chain bind more firmly to vitamin D3.

[0072] The three vitamin D3 hydroxylase mutants were sequenced. The amino acid sequence of mutant VM is shown in SEQ ID NO.3 and the nucleotide sequence is shown in SEQ ID NO.4; the amino acid sequence of mutant MV is shown in SEQ ID NO.5 and the nucleotide sequence is shown in SEQ ID NO.6; and the amino acid sequence of mutant VMV is shown in SEQ ID NO.7 and the nucleotide sequence is shown in SEQ ID NO.8.

[0073] Example 3: Application of Vitamin D3 Hydroxylase Mutant in the Preparation of 25(OH)VD3

[0074] The vitamin D3 hydroxylase mutant VM and vitamin D3 hydroxylase Vdh-K1 obtained in Example 2 were used to prepare 25(OH)VD3. The preparation method was as follows:

[0075] Take 2 µM of mutant VM or Vdh-K1, add 4 µM of Fdr, 12 µM of Fdx, 2 U of G6PD, 20 mM of glucose-6-phosphate, 10% of 2,6-dimethyl-β-cyclodextrin, 2 mM of NADPH, 0.2 g / L of VD3, 100 mM of NaCl, and 20 mM of KH2PO4 to obtain a reaction system with pH 7.4. Place this reaction system at 30 °C for hydroxylation reaction.

[0076] During the above reaction process, samples were taken every 20 minutes, and HPLC analysis was performed on the samples according to the method in the conversion determination to determine the concentration of 25(OH)VD3 in the reaction product. The trend of the concentration of 25(OH)VD3 in the reaction system of mutant VM and Vdh-k1 with reaction time is shown below. Figure 3As shown in the figure, after 180 min of reaction, the concentration of 25(OH)VD3 in the reaction product of the mutant VM group was 65.75 mg / L, while the concentration of 25(OH)VD3 in the reaction product of the Vdh-K1 group was only 33.41 mg / L. Further calculation of the enzyme reaction rate during this time period showed that the enzyme reaction rate of the mutant VM group was 10.96 mg·L⁻¹. -1 ·μM -1 ·h -1 The enzyme reaction rate in the Vdh-K1 group was only 5.56 mg·L⁻¹. -1 ·μM -1 ·h -1 This indicates that, compared to Vdh-K1, the vitamin D3 hydroxylase mutant VM has a significantly improved ability to catalyze the conversion of vitamin D3 to 25(OH)VD3, making it more suitable for the preparation of 25(OH)VD3.

[0077] Finally, it should be noted that although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mutant of vitamin D3 hydroxylase characterized in that, The mutant is based on the vitamin D3 hydroxylase with amino acid sequence shown in SEQ ID NO. 1, and the specific mutation sites are L171V+V172M or V172M+A177V or L171V+V172M+A177V.

2. A gene encoding the vitamin D3 hydroxylase mutant according to claim 1.

3. A recombinant expression vector, characterized in that, The gene according to claim 2 is included.

4. The recombinant expression vector of claim 3, wherein, The carrier of the recombinant expression vector is plasmid pET28a.

5. A recombinant strain, characterized in that, The recombinant expression vector according to claim 3 or 4 is included.

6. The recombinant bacterial strain of claim 5, wherein The host cell of the recombinant strain is Escherichia coli BL21 (DE3).

7. Use of the vitamin D3 hydroxylase mutant according to claim 1 in the preparation of 25-hydroxyvitamin D3.

8. Use according to claim 7, wherein the compound is ###0002### The vitamin D3 is converted into 25-hydroxyvitamin D3 by using the vitamin D3 hydroxylase mutant.

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