Fusion vitamin d hydroxylase mutants and their use in the production of 25-hydroxyvitamin d

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a fusion vitamin D hydroxylase mutant and application thereof in preparation of 25-hydroxyvitamin D. The mutant is based on a fusion vitamin D hydroxylase KB65OT with an amino acid sequence shown in SEQ ID NO. 3, and phenylalanine at the 351th position is mutated into asparagine or glutamine, and alanine at the 355th position is mutated into arginine or lysine. The application improves the electron transfer efficiency in the enzyme by directional mutation of the fusion vitamin D hydroxylase KB65OT, and the obtained mutants M1, M2, M3 and M4 have high catalytic activity on vitamin D2 and vitamin D3.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a fusion vitamin D hydroxylase mutant and application thereof in preparation of 25-hydroxyvitamin D. BACKGROUND

[0002] Vitamin D is a key fat-soluble hormone precursor, which is essential for maintaining calcium and phosphorus homeostasis, bone health, immune regulation and cell growth and differentiation, and mainly includes vitamin D2 and vitamin D3. Vitamin D itself has no physiological activity and needs to be hydroxylated in vivo to convert into active form 25-hydroxyvitamin D (including 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3), which is not only a marker for measuring the level of vitamin D in the body, but also a key precursor for synthesizing vitamin D, and has wide application in the fields of medicine, health care products, and animal feed.

[0003] At present, the preparation methods of 25-hydroxyvitamin D mainly include chemical synthesis method and biological transformation method. The chemical synthesis method needs to go through multiple steps such as group protection, oxidation, and deprotection, and has defects such as harsh reaction conditions, large amount of organic solvent, serious environmental pollution, poor stereoselectivity of product, and high cost. The biological transformation method has become a research hotspot due to the advantages of mild reaction conditions, high specificity, and environmental friendliness.

[0004] The core of the biological transformation method for preparing 25-hydroxyvitamin D is to use vitamin D hydroxylase to catalyze the hydroxylation reaction of C-25 C-H bond of vitamin D. However, the existing natural vitamin D hydroxylase has problems such as low catalytic efficiency, poor stability, only catalytic activity to substrate vitamin D2 or vitamin D3, and insufficient affinity to substrate, which cannot meet the needs of industrial production. The fusion enzyme technology can significantly improve the expression amount, secretion efficiency, stability and catalytic activity of the target enzyme by fusing the target enzyme with a signal peptide, a tag protein or other functional domains, and is an effective strategy for enzyme molecular modification. However, the existing fusion vitamin D enzyme still cannot break through the bottleneck of single substrate specificity, and can only specifically catalyze vitamin D2 or vitamin D3, lacking the ability of high-efficiency catalysis of double substrates.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] In view of the above problems, the purpose of the present application is to provide a fusion vitamin D hydroxylase mutant and application thereof in preparation of 25-hydroxyvitamin D, which has high catalytic capacity for vitamin D2 or vitamin D3.

[0007] In a first aspect, the present application provides a fusion vitamin D hydroxylase mutant, which is based on a fusion vitamin D hydroxylase KB65OT with an amino acid sequence as shown in SEQ ID NO. 3, and has a phenylalanine at position 351 mutated into asparagine or glutamine, and an alanine at position 355 mutated into arginine or lysine.

[0008] Further, the amino acid sequence of the fusion vitamin D hydroxylase mutant is as shown in SEQ ID NO. 5 or SEQ ID NO. 7 or SEQ ID NO. 9 or SEQ ID NO. 11.

[0009] In a second aspect, the present application provides a gene encoding the above-mentioned fusion vitamin D hydroxylase mutant.

[0010] In a third aspect, the present application provides a recombinant expression vector comprising the above-mentioned gene.

[0011] Further, the carrier of the recombinant expression vector is plasmid pET28a.

[0012] In a fourth aspect, the present application provides a recombinant strain comprising the above-mentioned recombinant expression vector.

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

[0014] In a fifth aspect, the present application provides use of the above-mentioned fusion vitamin D hydroxylase mutant in the preparation of 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.

[0015] Further, the use specifically refers to conversion of vitamin D2 or vitamin D3 into 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 by the fusion vitamin D hydroxylase mutant.

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

[0017] The present application improves the electron transfer efficiency in the enzyme by simultaneously mutating the amino acids at positions 351 and 355 of the heme domain of the fusion vitamin D hydroxylase KB65OT, and the obtained mutants M1, M2, M3 and M4 have significantly improved catalytic activity on vitamin D2 and vitamin D3 compared with KB65OT. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Table 1 shows the catalytic activity of different fusion vitamin D hydroxylase mutants on vitamin D2 and vitamin D3. DETAILED DESCRIPTION

[0019] The present application will be further described by specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments can be known to those skilled in the art, and appropriate ones can be selected from the means and materials known to solve the corresponding technical problems. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the present application, the essence and scope of which are defined only by the claims.

[0020] It should be understood that the scope of the present application is not limited to the defined processes, properties or components, as these embodiments and others are merely illustrative of certain aspects of the present application. Indeed, various changes in the embodiments apparent to those skilled in the art or related fields can be made without departing from the essence and scope of the present application, which are encompassed by the scope of the appended claims.

[0021] It should be noted that, unless otherwise defined, the scientific and technical terms used in the context of the present application should have the meanings commonly understood by those of ordinary skill in the art.

[0022] The fusion vitamin D hydroxylase mutants employed in the present application are marked as follows:

[0023] The "amino acid replaced at the original amino acid position" is used to represent the amino acid of the fusion vitamin D hydroxylase mutant. For example, F351N indicates that the amino acid at position 351 is replaced by N from F of the parent fusion vitamin D hydroxylase KB65OT, and the position number corresponds to the amino acid sequence number in SEQ ID NO. 3.

[0024] The embodiments of the present application provide a fusion vitamin D hydroxylase mutant, which is based on the fusion vitamin D hydroxylase KB65OT with the amino acid sequence shown in SEQ ID NO. 3, and the phenylalanine at position 351 is mutated to asparagine or glutamine, and the alanine at position 355 is mutated to arginine or lysine.

[0025] In the present embodiment, the parent vitamin D hydroxylase KB65OT is obtained by connecting the heme domain derived from the type I P450 enzyme Vdh-K1 of Nocardiopsis sp. JS6 and the oxidoreductase domain derived from the type VII P450 enzyme CYP116B65 of Amycolatopsis sp. with a linker. The enzyme not only has the ability to catalyze the conversion of both vitamin D2 and vitamin D3, but is also a self-sufficient enzyme, and electron transfer occurs within the enzyme, without the need for additional oxidoreductase proteins to exhibit activity. However, the applicant found that the heme domain and the oxidoreductase domain are not naturally adapted, and the efficiency of electron transfer within the enzyme is low, thereby resulting in an unsatisfactory catalytic activity. Through analysis of the parent enzyme, the applicant found that the phenylalanine at position 351 and the alanine at position 355 in the heme domain are amino acids related to electron transfer, and mutating them to asparagine or glutamine, arginine or lysine, respectively, can improve the efficiency of electron transfer within the enzyme, thereby improving the catalytic activity. Based on this analysis, the present embodiment uses site-directed mutagenesis to simultaneously mutate the two amino acid sites selected from the same functional domain and adjacent in space by designing primers containing double-mutated bases, and successfully obtains vitamin D hydroxylase mutants M1, M2, M3 and M4 with significantly improved catalytic activity.

[0026] The amino acid sequence of mutant M1 is shown in SEQ ID NO. 5, and the mutation site includes F351N+A355K, specifically, the phenylalanine and alanine at positions 351 and 355 are mutated to asparagine and lysine, respectively; the amino acid sequence of mutant M2 is shown in SEQ ID NO. 7, and the mutation site includes F351N+A355R, specifically, the phenylalanine and alanine at positions 351 and 355 are mutated to asparagine and arginine, respectively; the amino acid sequence of mutant M3 is shown in SEQ ID NO. 9, and the mutation site includes F351Q+A355K, specifically, the phenylalanine and alanine at positions 351 and 355 are mutated to glutamine and lysine, respectively; and the amino acid sequence of mutant M4 is shown in SEQ ID NO. 11, and the mutation site includes F351Q+A355R, specifically, the phenylalanine and alanine at positions 351 and 355 are mutated to glutamine and arginine, respectively.

[0027] The present embodiment further provides a vitamin D hydroxylase mutant gene encoding the vitamin D hydroxylase mutant as described above, and the nucleotide sequence is shown in SEQ ID NO. 6 or SEQ ID NO. 8 or SEQ ID NO. 10 or SEQ ID NO. 12.

[0028] The embodiment of the present application further provides a recombinant expression vector comprising the gene as described above. The carrier of the recombinant expression vector is plasmid pET28a. The present embodiment ensures stable transcription and induced expression of the fusion vitamin D hydroxylase mutant gene in the host cell by constructing an operable connection structure comprising the gene encoding and the high-efficiency expression regulatory element pET28a, thereby improving the expression amount and solubility of the recombinant expression vector.

[0029] The embodiment of the present application further provides a recombinant strain comprising the recombinant expression vector as described above, so as to realize high-biomass expression and functional folding of the fusion vitamin D hydroxylase mutant. In some preferred embodiments, the host cell of the recombinant strain is Escherichia coli BL21 DE3.

[0030] The embodiment of the present application further provides an application of the fusion vitamin D hydroxylase mutant in preparing 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3. The application specifically refers to converting vitamin D2 or vitamin D3 into 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 by using the fusion vitamin D hydroxylase mutant of the present embodiment.

[0031] In order to make the technical solutions of the present application clearer, the fusion vitamin D hydroxylase mutant is described in detail below through a plurality of specific embodiments.

[0032] The experimental reagents and components involved in the embodiments of the present application include:

[0033] LB liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, and 10 g / L of sodium chloride;

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

[0035] Buffer A: 500 mM of sodium chloride, 20 mM of potassium phosphate dibasic, 20 mM of imidazole, 10% of glycerol, and pH 7.4;

[0036] Buffer B: 500 mM of sodium chloride, 20 mM of potassium phosphate dibasic, 300 mM of imidazole, 10% of glycerol, and pH 7.4;

[0037] Buffer C: 100 mM of sodium chloride, 20 mM of potassium phosphate dibasic, and pH 7.4.

[0038] The indexes and determination methods involved in the embodiments of the present application include:

[0039] Fusion vitamin D hydroxylase catalytic activity: measured by the yield of substrate vitamin D2or vitamin D3converted to the corresponding product 25-hydroxy vitamin D2or 25-hydroxy vitamin D3per unit reaction. Specifically, the higher the yield under certain reaction conditions, the more excellent the catalytic activity of the fusion vitamin D hydroxylase.

[0040] The reaction system comprises: fusion vitamin D hydroxylase / mutant 3 µM, glucose-6-phosphate dehydrogenase 1 U, glucose-6-phosphate 10 mM, 2,6-dimethyl-β-cyclodextrin 10 % (w / w), NADPH 0.5 mM, vitamin D2(or vitamin D3) 1 mM, NaCl 100 mM, KH2PO420 mM, pH 7.4, total volume 200 µL.

[0041] After the above reaction system is reacted at 30 ℃ for 12 h, the product is extracted using two volumes of ethyl acetate, the ethyl acetate is evaporated, the product is redissolved with 400 µL of methanol, and high performance liquid chromatography detection is performed, and the detection method is as follows:

[0042] An Agilent 1260 Infinity II high performance liquid chromatograph with a UV detector is used, the chromatographic column is Poroshell 120 EC-C18 4 µm 4.6×150 mm, the column temperature is 40 ℃, the flow rate is 1 ml / min, the mobile phase is acetonitrile and water (0-12 min acetonitrile 60%-100%, 12-23 min acetonitrile 100%, 23-30 min acetonitrile 100%-60%), the injection amount is 20 µL, and the detection wavelength is 265 nm. The concentration of 25-hydroxy vitamin D2or 25-hydroxy vitamin D3in the reaction product is calculated according to the peak area detected.

[0043] In the following examples, the molecular biology experimental methods not specifically described are performed according to the specific methods listed in the book "Molecular Cloning Experiment Guide" (third edition) by J. Sambrook, or according to the kit and product instructions.

[0044] Example Construction of fusion vitamin D hydroxylase mutants and performance determination

[0045] (1) Obtain fusion vitamin D hydroxylase KB65OT gene

[0046] The heme domain amino acid sequence of Vdh-K1 was extracted from the Vdh-K1 crystal structure derived from N. autotrophica according to PDB:3A50, and the codon-optimized gene sequence was synthesized by a biological company after optimization, to obtain the gene VK1 with the nucleotide sequence as shown in SEQ ID NO:1. The CYP116B65 amino acid sequence derived from A. thermoluteus was obtained according to NCBI:WP_378364562.1, and the oxidoreductase domain and linker amino acid sequence were extracted therefrom, and the codon-optimized gene sequence was synthesized by a biological company after optimization, to obtain the gene B65 with the nucleotide sequence as shown in SEQ ID NO:2.

[0047] The plasmid pET28a was used as a template for PCR amplification using primers P-F / P-R; the gene VK1 was used as a template for PCR amplification using primers K-F / K-R; and the B65 gene was used as a template for PCR amplification using primers B-F / B-R. After purification of the PCR products, recombination was performed by a seamless cloning kit, and then the ligation product was 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 plasmid pET28-KB65OT containing the fusion vitamin D hydroxylase KB65OT gene. The amino acid sequence of the fusion vitamin D hydroxylase KB65OT is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4.

[0048] The primer information is shown in Table 1, the PCR amplification reaction system is shown in Table 2, and the amplification program is shown in Table 3.

[0049] Table 1 Primers used for constructing the plasmid pET28-KB65OT

[0050] .

[0051] Table 2 PCR reaction system

[0052] .

[0053] Table 3 PCR amplification program

[0054] .

[0055] (2) Construction of a fusion vitamin D hydroxylase mutant

[0056] The plasmid pET28-KB65OT was used as a template, and the site-directed mutation primers M1-F / M1-R, M2-F / M2-R, M3-F / M3-R and M4-F / M4-R shown in Table 4 were used for PCR amplification, so as to realize site-directed mutation of phenylalanine at position 351 and alanine at position 355 of the fusion vitamin D hydroxylase KB65OT. Specifically, the plasmid pET28-KB65OT was subjected to PCR amplification by using the primers M1-F / M1-R, and the amplification reaction system and amplification procedure were shown in Tables 2 and 3. After the PCR product was purified and recovered, recombination was performed by using a seamless cloning kit, and then the ligation product was transformed into E. coli DH5α competent cells, and was coated on LB solid medium containing kanamycin for screening. After the transformants were verified by sequencing, the plasmid was extracted, and the mutant plasmid pET28-M1 with mutation point positions including F351N and A355K was obtained.

[0057] Referring to the above method, the mutant plasmid pET28-M2 with mutation point positions including F351N and A355R was obtained by using the primers M2-F / M2-R; the mutant plasmid pET28-M3 with mutation point positions including F351Q and A355K was obtained by using the primers M3-F / M3-R; and the mutant plasmid pET28-M4 with mutation point positions including F351Q and A355R was obtained by using the primers M4-F / M4-R.

[0058] Table 4 Mutant primers of the fusion vitamin D hydroxylase KB65OT

[0059] .

[0060] (3) Expression and purification of the fusion vitamin D hydroxylase mutants

[0061] The above four fusion vitamin D hydroxylase mutant plasmids were respectively transformed into E. coli BL21 DE3 competent cells, and were coated on LB medium containing kanamycin for screening. After the transformants were verified by sequencing, they were inoculated into 5 mL LB liquid medium containing kanamycin, and were cultured at 37°C and 200 r / min, overnight, to obtain seed liquid of the fusion vitamin D hydroxylase mutants. The seed liquid was inoculated into 100 mL LB liquid medium containing kanamycin, and was cultured at 37°C and 200 r / min. When the OD 600 =0.6-0.8, 0.2 mM IPTG, 0.1 mM ferrous sulfate, 0.1 mM 5-aminolevulinic acid, 0.2 mM cysteine and 0.1 mM ferric chloride were added, and the culture was carried out at 16°C and 120 r / min, overnight.

[0062] The bacteria were collected by centrifugation, the bacteria were suspended with 10 mL of buffer A, and the bacteria were broken by ultrasonic wave (power of 200 W, 2 s on and 4 s off, total time of 10 min), and then the supernatant was collected by centrifugation, and the crude enzyme solution was purified by using a Ni-NTA resin affinity column. After the crude enzyme solution was combined with the resin, 10 mL of buffer A was used to elute the non-target proteins, and then 10 mL of buffer B was used to elute and collect the target proteins, and finally the target proteins were desalted and concentrated in buffer C by using an ultrafiltration tube, and four fusion vitamin D hydroxylase mutant pure enzyme solutions were obtained.

[0063] The obtained fusion vitamin D hydroxylase mutants were sequenced, wherein the amino acid sequence of the mutant M1 is shown as SEQ ID NO: 5, and the nucleotide sequence is shown as SEQ ID NO: 6; the amino acid sequence of the mutant M2 is shown as SEQ ID NO: 7, and the nucleotide sequence is shown as SEQ ID NO: 8; the amino acid sequence of the mutant M3 is shown as SEQ ID NO: 9, and the nucleotide sequence is shown as SEQ ID NO: 10; the amino acid sequence of the mutant M4 is shown as SEQ ID NO: 11, and the nucleotide sequence is shown as SEQ ID NO: 12.

[0064] The catalytic activity of the fusion vitamin D hydroxylase KB65OT and the mutants was determined, and the determination results are shown in the following table. Figure 1 As can be seen from the figure, the catalytic activity of the mutants is significantly higher than that of KB65OT, whether for vitamin D2 or vitamin D3. When catalyzing the conversion of vitamin D2, the 25-hydroxyvitamin D2 yield of the mutants M1, M2, M3 and M4 is 2.65, 2.92, 2.24 and 2.32 times that of KB65OT, respectively; when catalyzing the conversion of vitamin D3, the 25-hydroxyvitamin D3 yield of the mutants M1, M2, M3 and M4 is 3.85, 3.78, 2.93 and 3.49 times that of KB65OT, respectively.

[0065] Finally, it should be pointed out that although the present application 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 replacements to part of the technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A fusion vitamin D hydroxylase mutant characterized in that, The mutant is based on the fusion vitamin D hydroxylase KB65OT with the amino acid sequence shown in SEQ ID NO. 3, in which the phenylalanine at position 351 is mutated to asparagine or glutamine, and the alanine at position 355 is mutated to arginine or lysine.

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

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

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

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

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 fusion vitamin D hydroxylase mutant according to claim 1 in the preparation of 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.

8. Use according to claim 7, wherein the compound is ###0002### Conversion of vitamin D2 or vitamin D3 to 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 using the fusion vitamin D hydroxylase mutant. Conversion of vitamin D2 or vitamin D3 to 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 using the fusion vitamin D hydroxylase mutant.

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