Fusion vitamin D hydroxylase and its application in the preparation of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a fusion vitamin D hydroxylase and its application in the preparation of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The fusion vitamin D hydroxylase comprises a Vdh-K1 heme domain (as shown in SEQ ID NO.1) and a CYP116B65 redox domain (as shown in SEQ ID NO.3), as well as a linker connecting the two. This fusion vitamin D hydroxylase is a self-sufficient enzyme that can stably catalyze the conversion of vitamin D2 and vitamin D3 without relying on an Fdx and Fdr-assisted electron transport system.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the fusion of vitamin D hydroxylase and its application in the preparation of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Background Technology

[0002] Vitamin D is a key fat-soluble nutrient for maintaining calcium and phosphorus balance and bone health in the human body. It mainly exists in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). 25-hydroxyvitamin D (including 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) is the main form of vitamin D in organisms and a key indicator for clinically assessing vitamin D nutritional status. Furthermore, it is a crucial precursor for vitamin D synthesis. Therefore, the efficient and environmentally friendly production of high-purity 25-hydroxyvitamin D has enormous application prospects and market demand in the pharmaceutical, health product, and diagnostic reagent fields.

[0003] Currently, the main methods for preparing 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 include chemical synthesis and biosynthesis. Chemical synthesis suffers from drawbacks such as cumbersome reaction steps, harsh reaction conditions, numerous byproducts, difficulty in product separation and purification, and severe environmental pollution. In contrast, biosynthesis, based on enzyme catalysis, offers advantages such as mild reaction conditions, high specificity, fewer byproducts, and environmental friendliness, making it a current research hotspot.

[0004] Vitamin D hydroxylase is a key rate-limiting enzyme in the biosynthesis of 25-hydroxyvitamin D, but natural vitamin D hydroxylases suffer from low catalytic activity, poor stability, and low expression levels in host cells, which restricts their industrial application. Although studies have modified them using genetic engineering techniques such as site-directed mutagenesis and codon optimization, the improvement in the overall catalytic performance of the enzyme remains limited. Furthermore, currently reported enzymes typically only specifically catalyze vitamin D2 or vitamin D3 and lack dual-substrate catalytic capabilities.

[0005] In view of this, this invention is hereby proposed. Summary of the Invention

[0006] Based on the above problems, the purpose of this invention is to provide a fused vitamin D hydroxylase and its application in the preparation of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0007] A first aspect of the present invention provides a fused vitamin D hydroxylase comprising a Vdh-K1 heme domain as shown in SEQ ID NO. 1 and a CYP116B65 redox domain as shown in SEQ ID NO. 3, and a linker connecting the two.

[0008] Furthermore, the amino acid sequence of the fused vitamin D hydroxylase is shown in SEQ ID NO.7 or SEQ ID NO.10.

[0009] A second aspect of the invention provides a gene encoding the aforementioned fused vitamin D hydroxylase.

[0010] A third aspect of the present invention provides a recombinant expression vector comprising the above-described genes.

[0011] Furthermore, the vector for the recombinant expression vector is plasmid pET28a.

[0012] A fourth aspect of the present invention provides a recombinant strain comprising the above-described recombinant expression vector.

[0013] Furthermore, the host cell of the recombinant strain is Escherichia coli BL21 DE3.

[0014] The fifth aspect of the present invention provides the use of the above-described fused vitamin D hydroxylase in the preparation of 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.

[0015] Furthermore, the application specifically involves using a fused vitamin D hydroxylase to convert vitamin D2 or vitamin D3 into 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention constructs a self-sufficient fused vitamin D hydroxylase by fusing the heme domain of type I P450 enzyme Vdh-K1 derived from autotrophic pseudonocardia with the redox domain of type VII P450 enzyme CYP116B65 derived from thermophilic amycobacterium through a linker. This enzyme can achieve stable catalytic conversion of vitamin D2 and vitamin D3 without relying on an electron transport system assisted by Fdx and Fdr. Attached Figure Description

[0018] Figure 1 A schematic diagram of the assembly of vitamin D hydroxylase;

[0019] Figure 2 The results of the determination of the catalytic activity of vitamin D hydroxylase on vitamin D2 and vitamin D3 were used to integrate the results. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] 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.

[0023] This invention provides a fused vitamin D hydroxylase comprising a Vdh-K1 heme domain as shown in SEQ ID NO.1 and a CYP116B65 redox domain as shown in SEQ ID NO.3, as well as a linker connecting the two.

[0024] It should be noted that cytochrome P450 enzymes are a class of heme-dependent monooxygenases known for their specific regions and stereoselective conversion, and they have great potential in the synthesis of 25-hydroxyvitamin D3. Vdh-K1 is a type I P450 enzyme derived from autotrophic pseudonocardia and is commonly used to catalyze the conversion of vitamin D3 to 25-hydroxyvitamin D3. However, its catalytic conversion function is highly dependent on the electron transport system assisted by redox proteins (Fdx) and redox protein reductases (Fdr), and the stability and efficiency of this system have a significant impact on catalytic activity. CYP116B65 is a type VII P450 enzyme (also known as a self-sufficient P450 enzyme) derived from *Amycium pyrenoidosa*. It naturally possesses redox domains, allowing it to function without the assistance of additional redox proteins. Furthermore, its electron transfer occurs within the enzyme itself, rather than between different enzymes, resulting in generally higher electron transfer efficiency compared to type I P450 enzymes. However, it exhibits extremely low catalytic activity for non-natural substrates such as vitamin D2 or vitamin D3, and even lacks effective substrate recognition and binding capabilities. In this embodiment, the heme domain of Vdh-K1 is fused with the redox domain of CYP116B65 via a linker to construct a self-sufficient fused vitamin D hydroxylase. This enzyme can achieve stable catalytic conversion of vitamin D2 and vitamin D3 without relying on an electron transfer system assisted by Fdx and Fdr.

[0025] In this embodiment, the linker is a wild-type linker with an amino acid sequence as shown in SEQ ID NO.4, or an optimized linker with an amino acid sequence as shown in SEQ ID NO.9. The wild-type linker is also derived from CYP116B65. The applicant discovered this through long-term research. By comparing the differences between the heme domain of Vdh-K1 and the heme domain of CYP116B65, the wild-type linker was modified and adjusted. The resulting optimized linker allows for a more stable relative position between the heme domain of Vdh-K1 and the redox domain of CYP116B65, ensuring stable and efficient electron transfer between them, thereby further improving the catalytic activity of the fused vitamin D hydroxylase.

[0026] In some specific embodiments, the fusion vitamin D hydroxylase containing a wild-type linker and the fusion vitamin D hydroxylase containing an optimized linker have amino acid sequences as shown in SEQ ID NO.7 or SEQ ID NO.10, respectively.

[0027] This embodiment further provides a gene encoding the vitamin D hydroxylase fused as described above, with nucleotide sequences shown in SEQ ID NO.8 or SEQ ID NO.11, respectively.

[0028] This embodiment further provides a recombinant expression vector containing the gene described above. The vector for the recombinant expression vector is plasmid pET28a. By constructing an operable linker structure containing the coding gene and the highly efficient expression regulatory element pET28a, this embodiment ensures stable transcription and induced expression of the fused vitamin D hydroxylase in the host cell, thereby improving the expression level and solubility of the recombinant protein.

[0029] This invention further provides a recombinant bacterial strain comprising the recombinant expression vector described above, to achieve high biomass expression and functional folding of vitamin D hydroxylase. In some preferred embodiments, the host cell of the recombinant bacterial strain is *Escherichia coli* BL21 DE3.

[0030] The present invention also provides the application of the fused vitamin D hydroxylase described above in the preparation of 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3. Specifically, without using Fdx and Fdr, the fused vitamin D hydroxylase is used directly to convert vitamin D2 or vitamin D3 into 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.

[0031] To make the technical solution of the present invention clearer, the following detailed description of the fused vitamin D hydroxylase is provided through several specific embodiments.

[0032] The experimental reagents and their components involved in the embodiments of this invention include:

[0033] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L;

[0034] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L;

[0035] Buffer A: 500 mM sodium chloride, 20 mM dipotassium hydrogen phosphate, 20 mM imidazole, 10% glycerol, pH 7.4;

[0036] Buffer B: 500 mM sodium chloride, 20 mM dipotassium hydrogen phosphate, 300 mM imidazole, 10% glycerol, pH 7.4;

[0037] Buffer C: 100 mM sodium chloride, 20 mM dipotassium hydrogen phosphate, pH 7.4.

[0038] 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.

[0039] Example: Construction and performance determination of vitamin D hydroxylase

[0040] 1. Obtain the Vdh-K1 gene (VK1) and the CYP116B65 gene (B65).

[0041] Based on PDB:3A50, the crystal structure of Vdh-K1 from autotrophic pseudonocardia was obtained. The amino acid sequence of the heme domain of Vdh-K1 was extracted (as shown in SEQ ID NO: 1). After codon optimization, the optimized gene sequence was synthesized by a biotechnology company, yielding the gene VK1 with the nucleotide sequence shown in SEQ ID NO: 2. Similarly, based on NCBI:WP_378364562.1, the amino acid sequence of CYP116B65 from *Amycium pyridae* was obtained. The amino acid sequence of the redox domain (as shown in SEQ ID NO: 3) and the amino acid sequence of the wild-type linker (as shown in SEQ ID NO: 4) were extracted. After codon optimization, the redox domain gene (as shown in SEQ ID NO: 5) and the wild-type linker gene (as shown in SEQ ID NO: 6) were obtained. Since the redox domain and the wild-type linker are structurally continuous in the original protein, the two optimized gene sequences were merged (wild-type linker gene first) and then synthesized by a biotechnology company to obtain gene B65.

[0042] 2. Constructing a fusion vitamin D hydroxylase containing wild-type linkers

[0043] (1) Construction of plasmid pET28-KB65WT

[0044] Using plasmid pET28a as a template, PCR amplification was performed using primers PF / PR; using gene VK1 as a template, PCR amplification was performed using primers KF / KR; using gene B65 as a template, PCR amplification was performed using primers BF / BR. Primer information is shown in Table 1, PCR amplification reaction systems are shown in Table 2, and amplification procedures are shown in Table 3.

[0045] Table 1 Primers used to construct a fusion vitamin D hydroxylase containing a wild-type linker

[0046] .

[0047] Table 2 PCR reaction system

[0048] .

[0049] Table 3 PCR amplification program

[0050] .

[0051] After purifying the PCR products, recombination ligation was performed using a seamless cloning kit. The ligation products were then transformed into *E. coli* DH5α competent cells and screened on LB agar containing kanamycin. Transformants were sequenced and the plasmid pET28-KB65WT was extracted.

[0052] (2) Constructing a fusion vitamin D hydroxylase KB65WT containing a wild-type linker

[0053] The plasmid pET28-KB65WT was transformed into *E. coli* BL21 DE3 competent cells and screened on LB medium containing kanamycin. Transformants, after sequencing verification, were inoculated into 5 mL of LB liquid medium containing kanamycin and cultured overnight with shaking at 37°C and 200 rpm to obtain a fusion vitamin D hydroxylase seed culture containing wild-type linkers. This seed culture was then inoculated into 100 mL of LB liquid medium containing kanamycin and cultured with shaking at 37°C and 200 rpm until OD was obtained. 600 When the concentration of the sample is 0.6-0.8, add IPTG to a final concentration of 0.2 mM, ferrous sulfate to a final concentration of 0.1 mM, 5-aminolevulinic acid to a final concentration of 0.1 mM, cysteine ​​to a final concentration of 0.2 mM, and ferric chloride to a final concentration of 0.1 mM. Incubate overnight with shaking at 16°C and 120 r / min.

[0054] The bacterial cells were collected by centrifugation, resuspended in 10 mL of buffer A, and subjected to ultrasonic cell disruption (200 W power, 2 s on, 4 s off, 10 min total). 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 collection of the target protein. Finally, the target protein was desalted and concentrated in buffer C using an ultrafiltration tube to obtain a fusion vitamin D hydroxylase solution containing the wild-type linker. The obtained fusion vitamin D hydroxylase KB65WT containing the wild-type linker was sequenced; its amino acid sequence is shown in SEQ ID NO: 7, and its nucleotide sequence is shown in SEQ ID NO: 8.

[0055] 3. Constructing a fusion vitamin D hydroxylase KB65OT containing an optimized linker

[0056] Using plasmid pET28-KB65WT as a template, PCR amplification was performed on plasmid pET28-KB65WT using the primers OF / OR shown in Table 4. After PCR product recovery and purification, recombination ligation was performed using a seamless cloning kit. The ligation product was then transformed into *E. coli* DH5α competent cells and screened on LB medium containing kanamycin. After sequencing verification of the transformants, the plasmid pET28-KB65OT was extracted to obtain plasmid pET28-KB65OT. Subsequently, plasmid pET28-KB65OT was transformed into *E. coli* BL21 DE3 competent cells for expression, and the crude enzyme solution was purified to obtain a fusion vitamin D hydroxylase solution containing an optimized linker (amino acid sequence as shown in SEQ ID NO: 9). The obtained fusion vitamin D hydroxylase KB65OT containing the optimized linker was sequenced, and its amino acid sequence is shown in SEQ ID NO: 10, and its nucleotide sequence is shown in SEQ ID NO: 11.

[0057] Table 4. Sequence information of primers OF and OR

[0058] .

[0059] Figure 1 This is a schematic diagram of the assembly of a fusion vitamin D hydroxylase containing a linker, which is formed by the sequential connection of the heme domain of Vdh-K1, the linker, and the redox domain of CYP116B65.

[0060] 4. Determination of the catalytic activity of fused vitamin D hydroxylase

[0061] The catalytic activities of the above-mentioned fusion vitamin D hydroxylase KB65WT containing a wild-type linker and fusion vitamin D hydroxylase KB65OT containing an optimized linker were determined, with the linkerless fusion vitamin D hydroxylase KB65NO serving as a control. Among them:

[0062] The preparation method of the linkerless fused vitamin D hydroxylase KB65NO is as follows:

[0063] Using plasmid pET28-KB65WT as a template, PCR amplification was performed on plasmid pET28-KB65WT using the primers NF / NR shown in Table 5. After PCR product recovery and purification, 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 pET28-KB65NO was extracted to obtain plasmid pET28-KB65NO. Subsequently, plasmid pET28-KB65NO was transformed into *E. coli* BL21 DE3 competent cells for expression, and the crude enzyme solution was purified to obtain a linkerless fusion vitamin D hydroxylase solution. The obtained linkerless fusion vitamin D hydroxylase KB65NO was sequenced, and its amino acid sequence is shown in SEQ ID NO: 12, and its nucleotide sequence is shown in SEQ ID NO: 13.

[0064] Table 5. Sequence information of primers NF and NR

[0065] .

[0066] The method for determining the catalytic activity of vitamin D hydroxylase is as follows:

[0067] It is measured by the yield of substrate vitamin D2 or vitamin D3 converted into the corresponding product 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 per unit reaction. Specifically, under certain reaction conditions, a higher yield indicates better catalytic activity of the fused vitamin D hydroxylase.

[0068] The reaction system consisted of: 3 µM vitamin D hydroxylase, 1 U glucose-6-phosphate dehydrogenase, 10 mM glucose-6-phosphate, 10% (w / w) 2,6-dimethyl-β-cyclodextrin, 0.5 mM NADPH, 1 mM vitamin D2 (or vitamin D3), 100 mM NaCl, 20 mM KH2PO4, pH 7.4, with a total volume of 200 µL.

[0069] After reacting the above reaction system at 30 °C for 12 h, the product was extracted with twice the volume of ethyl acetate. The ethyl acetate was evaporated to dryness, and the product was redissolved in 400 µL of methanol. The product was then analyzed by high-performance liquid chromatography (HPLC) using the following method:

[0070] An Agilent 1260 Infinity II high-performance liquid chromatograph equipped 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-hydroxyvitamin D2 or 25-hydroxyvitamin D3 in the reaction product was calculated based on the detected peak area.

[0071] Results of catalytic activity assays for several enzymes are as follows: Figure 2 As shown in the figure, 25OH-VD2 represents 25-hydroxyvitamin D2, and 25OH-VD3 represents 25-hydroxyvitamin D3. The figure shows that the linkerless fusion vitamin D hydroxylase KB65NO has no conversion activity for either vitamin D2 or D3, indicating that the linker has a significant impact on the catalytic activity of the fusion enzyme. The fusion vitamin D hydroxylase KB65WT containing the wild-type linker has catalytic conversion activity for both vitamin D2 and D3; 3 µM of fusion vitamin D hydroxylase KB65WT can catalyze the conversion to 23 µM 25-hydroxyvitamin D2 and 17 µM 25-hydroxyvitamin D3. In contrast, 3 µM of fusion vitamin D hydroxylase KB65OT containing the optimized linker can catalyze the conversion to 197 µM 25-hydroxyvitamin D2 and 132 µM 25-hydroxyvitamin D3, which are 8.6 times and 7.8 times that of KB65WT, respectively. This indicates that the fusion vitamin D hydroxylase containing the optimized linker has higher catalytic activity for both vitamin D2 and D3.

[0072] 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 fusion of vitamin D hydroxylase, characterized in that, It comprises a Vdh-K1 heme domain with an amino acid sequence as shown in SEQ ID NO.1 and a CYP116B65 redox domain with an amino acid sequence as shown in SEQ ID NO.3, as well as a linker connecting the two; the amino acid sequence of the fused vitamin D hydroxylase is shown in SEQ ID NO.7 or SEQ ID NO.

10.

2. The gene encoding the vitamin D hydroxylase fusion as described in claim 1.

3. A recombinant expression vector comprising the gene as described in claim 2.

4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector is the plasmid pET28a.

5. A recombinant strain comprising the recombinant expression vector as described in claim 3 or 4.

6. The recombinant strain according to claim 5, characterized in that, The host cell for the recombinant strain is Escherichia coli BL21 DE3.

7. The use of the fusion vitamin D hydroxylase as described in claim 1 in the preparation of 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.

8. The application as described in claim 7, characterized in that, The fusion vitamin D hydroxylase is used to convert vitamin D2 or vitamin D3 into 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.

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