P450 enzyme mutant and engineering bacteria thereof in synthesis of calcifediol

Abstract

The application belongs to the technical field of bio-chemical industry, and particularly relates to a P450 enzyme mutant and application of an engineering bacterium thereof in synthesis of calcifediol, the mutant is one or more point positions in an amino acid sequence based on a wild type P450 enzyme Vdh being mutated; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to GenBank accession number CP069288.1. Through systematic molecular dynamics simulation, conservation analysis and substrate binding pocket engineering on the Vdh enzyme, multiple key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I114R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, and the pure enzyme activity can reach 9.8 times of the wild type enzyme, greatly improving the conversion rate from vitamin D3 to calcifediol.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to the application of a P450 enzyme mutant and its engineered bacteria in the synthesis of calcidiol. Background Technology

[0002] Calcidiol (25-hydroxyvitamin D3) is one of the main active metabolites of vitamin D3 in the body. It plays an important physiological role in the regulation of calcium and phosphorus metabolism, immune regulation, and cell proliferation and differentiation, and is widely used in the prevention and treatment of diseases such as osteoporosis, renal osteodystrophy, and vitamin D deficiency. Furthermore, calcidiol is also a key precursor for the synthesis of calcitriol (1,25-dihydroxyvitamin D3) and other more active vitamin D derivatives, and has significant market demand in the pharmaceutical and health supplement industries.

[0003] Currently, the industrial production of calcidiol mainly relies on chemical synthesis and microbial fermentation. Chemical synthesis typically involves multiple reactions, using strong acids, strong bases, or toxic reagents. The reaction conditions are harsh, the steps are cumbersome, numerous byproducts are produced, and environmental pollution is significant. Microbial fermentation relies on the natural hydroxylation ability of certain strains (such as mycobacteria), but its conversion efficiency is low, the cycle is long, the byproducts are complex, and the costs of strain cultivation and product separation are high.

[0004] In recent years, enzymatic catalysis has become an important research direction for the synthesis of calcidiol due to its advantages such as mild reaction conditions, high selectivity, and environmental friendliness. Cytochrome P450 enzymes are a class of monooxygenases with regio and stereoselectivity that can catalyze the 25-hydroxylation of vitamin D3 to calcidiol. However, natural P450 enzymes generally suffer from bottlenecks in in vitro applications, such as low enzyme activity, poor stability, dependence on the expensive coenzyme NAD(P)H, and low electron transfer efficiency, which limit their potential for industrial application.

[0005] To improve the catalytic performance of P450 enzymes, researchers often employ protein engineering techniques to modify them. Methods such as rational design, directed evolution, and computational-aided design have been used to enhance the activity, stability, and adaptability of P450 enzymes. However, systematic studies on the modification of vitamin D325-hydroxylase (Vdh) derived from *Priestia megaterium* are still relatively few, especially those involving multi-strategy fusion (such as molecular dynamics simulations, conservation analysis, and substrate-binding pocket engineering) to achieve multi-site synergistic optimization, and further constructing co-expression engineered bacteria and coenzyme regeneration systems to comprehensively improve calcidiol synthesis efficiency.

[0006] Therefore, developing a highly active, stable P450 enzyme mutant suitable for industrial production and its efficient engineered strain is of great significance for promoting the green and economical production of calcidiol. Summary of the Invention

[0007] To address the problems mentioned in the background art, this invention proposes the application of a P450 enzyme mutant and its engineered bacteria in the synthesis of calcidiol. Through systematic molecular dynamics simulations, conservation analysis, and substrate binding pocket engineering of the Vdh enzyme, this invention successfully screened and obtained multiple key mutation sites. The constructed combined mutants (such as I114R / N173M / Q310R, Vdh-M3) exhibit extremely high hydroxylation activity, with their pure enzyme activity reaching 9.8 times that of the wild-type enzyme, thus improving the conversion rate of vitamin D3 to calcidiol.

[0008] The technical solution adopted by the present invention to solve its technical problem is as follows: a P450 enzyme mutant is provided, wherein the mutant is formed by mutating one or more of the amino acids at positions 81, 114, 167, 173, 187, 190, 268, 270, 290, 310, 318, 323, 335, 341, 366, 374, and 390 of the amino acid sequence of the wild-type P450 enzyme Vdh; the amino acid sequence of the wild-type P450 enzyme Vdh corresponds to GenBank accession number CP069288.1.

[0009] The amino acid sequence of the wild-type P450 enzyme is shown in SEQ ID NO. 1.

[0010] The amino acid sequence of mutant M3 is shown in SEQ ID NO. 2.

[0011] Furthermore, the mutant is any of the following types:

[0012] (1) A single-point mutant, wherein the amino acid mutation site is selected from any one of E81I, I114K, L167M, N173Q, N187V, V190S, R268K, D270R, A290R, Q310S, S318R, S323P, H335R, H341R, T366R, F374R, and V390I;

[0013] (2) Saturated mutants, whose amino acid mutation sites are selected from any one of positions 114, 173, 270, 310, and 335, and the corresponding amino acid after mutation is:

[0014] The amino acid residue at position I114 is mutated to R or H;

[0015] The amino acid residue at N173 is mutated to M or V;

[0016] The amino acid residue at position D270 is mutated to L;

[0017] The amino acid residue at position Q310 is mutated to R or K;

[0018] (3) A combination mutant containing at least two mutation sites as described in (1) or (2) above, and the combination mutant is selected from the double mutant I114R / N173M, I114R / Q310R; or the triple mutant I114R / N173M / Q310R.

[0019] The gene encoding the aforementioned P450 enzyme mutant.

[0020] Recombinant expression vectors carrying the above genes.

[0021] A genetically engineered bacterium comprising the above-mentioned recombinant expression vector, wherein the host bacterium of the engineered bacterium is Escherichia coli BL21(DE3).

[0022] A method for constructing a P450 enzyme mutant includes the following steps:

[0023] S1. Construction of single-point mutants: Using pET28(+)-Vdh plasmid as a template, the Quick-change site-directed mutagenesis method was used to perform PCR amplification with primers corresponding to the mutation site. After digesting the template with DpnI enzyme, the mutant plasmid was obtained.

[0024] S2. Construction of saturated mutants: Using pET28(+)-Vdh plasmid as a template, quick-change site-directed mutagenesis was performed at positions 114, 173, 270, 310, and 335 using primers containing degenerate codons. The saturated mutant plasmid was obtained after digestion of the template with DpnI enzyme.

[0025] S3. Construction of combined mutants: Using the single-point mutant plasmid obtained in S1 or S2 as a template, primers for other beneficial mutation sites are introduced iteratively to perform Quick-change site-directed mutagenesis to obtain double mutant or triple mutant plasmids.

[0026] S4. The mutant plasmid was transformed into E. coli BL21(DE3), and positive clones were obtained through resistance screening, thus obtaining engineered bacteria containing the target mutant.

[0027] Further, the primer sequences described in S1 are shown in SEQ ID NO. 3 to 36; the degenerate primer sequences described in S2 are shown in SEQ ID NO. 37 to 46; and the primer sequences used for the combined mutation in S3 are shown in SEQ ID NO. 47 to 60.

[0028] An application of a P450 enzyme mutant in the synthesis of calcidiol: using vitamin D3 as a substrate, in the presence of redox chaperone Fdr-Fdx and coenzyme, the mutant catalyzes the 25-hydroxylation reaction of vitamin D3 to generate calcidiol.

[0029] Furthermore, the redox chaperone Fdr-Fdx is derived from Bacillus megaterium, corresponding to the amino acid sequence of GenBank accession number CP009920.1, and is obtained by expressing the pETDuet1-Fdr-Fdx recombinant plasmid in Escherichia coli BL21(DE3).

[0030] This invention also provides the application of a P450 enzyme mutant, gene, recombinant expression vector, and genetically engineered bacteria in the preparation of calcidiol.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] (1) Through systematic molecular dynamics simulation, conservation analysis and substrate binding pocket engineering of Vdh enzyme, this invention successfully screened and obtained multiple key mutation sites. The constructed combined mutants (such as I114R / N173M / Q310R, Vdh-M3) exhibited extremely high hydroxylation activity, and their pure enzyme activity could reach 9.8 times that of the wild-type enzyme, which greatly improved the conversion rate of vitamin D3 to calcidiol.

[0033] (2) This invention is not a simple superposition of single-point mutations, but rather discovers synergistic effects between sites through iterative combination of beneficial mutations. For example, the activity of the double mutant I114R / Q310R (5.3 times) is significantly higher than that of its individual single-point mutations, while the activity of the triple mutant Vdh-M3 achieves an order-of-magnitude increase. This indicates that the modification strategy of this invention can effectively break through the performance bottleneck of single-point mutations.

[0034] (3) To address the inherent problems of low electron transfer efficiency and high coenzyme consumption in P450 enzyme reactions, this invention constructs an optimized Vdh mutant and its redox chaperone (Fdr-Fdx) for co-expression in the same engineered bacteria. This system significantly enhances the synergistic effect between the enzyme and the chaperone protein, improves electron transfer efficiency, and thus maintains a highly efficient catalytic reaction while reducing the amount of coenzyme NADH used.

[0035] (4) To further reduce production costs, this invention introduces glucose dehydrogenase (GDH) and inexpensive substrate glucose into the reaction system to construct a highly efficient NAD process. + / NADH recycling system. This design reduces the amount of expensive coenzyme NADH used from the millimolecular level to the catalyst amount (1 mM NAD). +At the same time, by utilizing the continuous regeneration and reducing power of glucose through oxidation, the cost of raw materials has been significantly reduced, laying an economic foundation for industrial application.

[0036] (5) The triple mutant Vdh-M3 and co-expressed engineered bacteria and coenzyme regeneration system of the present invention can achieve a calcidiol conversion rate of 71.30% under optimized reaction conditions. Compared with the unoptimized system (47.30%) and traditional chemical or microbial methods, it has achieved significant improvements in conversion efficiency, product selectivity and reaction economy. Attached Figure Description

[0037] Figure 1 The relative enzyme activity of the crude enzyme solution of the mutant in Example 6;

[0038] Figure 2 The relative enzyme activity of the pure enzyme solution of the mutant after saturation mutation in Example 7;

[0039] Figure 3 The relative enzyme activity is the pure enzyme solution of the mutant after combined mutation in Example 8. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1: Construction of wild-type Ecoli.BL21(DE3)-Vdh.

[0042] The P450 protein sequence (Vdh, GenBank: CP069288.1) from Priestia megaterium in GenBank was codon-optimized according to the E. coli preference principle, and a 6*His tag was added to the C-terminus of the protein. The whole gene was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the Vdh gene was inserted under the T7 promoter of pET-28a(+) using the principle of homologous recombination to obtain the expression plasmid pET28(+)-Vdh.

[0043] The constructed expression plasmid pET28(+)-Vdh was transformed into E. coli BL21(DE3), plated on LB agar plates containing 50 μg / mL kanamycin resistance, and incubated upside down at 37°C for 8-12 h. Positive clones were picked, which were wild-type Ecoli.BL21(DE3)-Vdh, and used to express recombinant Vdh.

[0044] Example 2: Expression and purification of Vdh.

[0045] Single colonies were picked from the plate and inoculated into LB liquid medium in a test tube containing a final concentration of 50 µg / mL kanamycin. The culture was carried out at 37°C and 180 rpm for 6-8 h. Then, 1% (v / v) of the culture was inoculated into fresh LB liquid medium in a shake flask containing 50 μg / mL kanamycin resistance. The culture was carried out at 37°C and 180 rpm until the bacterial OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 24°C for 12 h. After incubation, the culture was centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was discarded and the precipitate was collected to obtain wet bacterial cells expressing recombinant Vdh. The collected wet bacterial cells were resuspended in 50 mM sodium phosphate buffer (pH 7.0) at a concentration of 100 g / L to form a bacterial suspension. The suspension was then sonicated for 10 minutes (ice bath, 250 W power, 1 second operation followed by 2 seconds of rest). The cell lysate was collected and centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was collected as the crude enzyme solution. The amount of crude enzyme solution used subsequently was based on the amount of bacterial cells in the bacterial suspension before lysis.

[0046] The crude enzyme solution (100 g / L) was diluted to 50 g / L with sodium phosphate buffer (20 mM, pH 7.0). The protein was purified using a Ni affinity column (1.6 × 10 cm, Bio-Rad, USA). The specific procedures were as follows: ① Equilibrate the Ni column with 5 column volumes of binding buffer (containing 20 mM NaH2PO4·2H2O, 300 mM NaCl, pH 7.0) at a flow rate of 1 mL / min until the baseline stabilizes; ② Load the sample by injecting the crude enzyme solution into the Ni column at a flow rate of 1 mL / min, allowing the target protein Vdh to adsorb onto the Ni column; ③ Wash away contaminating proteins with 6 column volumes of washing buffer (containing 20 mM NaH2PO4·2H2O, 300 mM NaCl, 50 mM imidazole, pH 7.0) at a flow rate of 1 mL / min until the baseline stabilizes; ④ Elute the target protein with elution buffer (containing 20 mM NaH2PO4·2H2O, 300 mM NaCl, 50 mM imidazole, pH 7.0). Elute with NaH2PO4·2H2O, 300 mM NaCl, 500 mM imidazole (pH 7.0) at a flow rate of 1 mL / min, and collect the target protein Vdh. Wash the Ni column with 5 column volumes of binding buffer until the baseline stabilizes, and finally wash the Ni column with 5 column volumes of 20% ethanol. Store at 4°C. Dialyze the eluent containing the target protein Vdh to sodium phosphate buffer (20 mM, pH 7.0) at low temperature for 48 h, and collect the retentate as the purified enzyme Vdh.

[0047] Example 3: Construction and expression of the redox chaperone Ecoli.BL21(DE3)-Fdr-Fdx.

[0048] The redox chaperone protein sequences (Fdr, GenBank: CP009920.1; Fdx, GenBank: CP009920.1) from Bacillus megaterium in GenBank were codon-optimized according to the E. coli preference principle, and 6*His tags were added to the C-terminus of both proteins. The whole genome was synthesized by Beijing Qingke Biotechnology Co., Ltd. Using the principle of homologous recombination, the Fdr gene was inserted under the T7 promoter of MCS-1 of pETDuet-1, and the Fdx gene was inserted under the T7 promoter of MCS-2 of pETDuet-1, to obtain the expression plasmid pETDuet1-Fdr-Fdx. The constructed expression plasmid pETDuet1-Fdr-Fdx was transformed into *E. coli* BL21(DE3) and plated on LB agar plates containing 100 μg / mL ampicillin resistance. The plates were incubated upside down at 37°C for 8–12 h. Positive clones were selected, which were wild-type *E. coli* BL21(DE3)-Fdr-Fdx, for expression of recombinant Fdr-Fdx. The crude enzyme solution was prepared using the same steps as in Example 2, with the amount of Fdr-Fdx used based on the pre-lysis bacterial cell volume.

[0049] Example 4: Determination of enzyme activity of Vdh.

[0050] Enzyme activity detection standard conditions: 1 mM VD3 (dissolved in 2-hydroxypropyl-β-cyclodextrin), 5 mM NADH, appropriate amount of Vdh enzyme solution, crude enzyme solution Fdr-Fdx 20 g / L, added to 50 mM sodium phosphate buffer (pH 7.0), reacted at 30℃ for 10 minutes, and the sample was analyzed by HPLC after processing.

[0051] HPLC detection conditions: Inert Sustain™ C18 column (4.6×250 mm, 5µm, Shimadzu), mobile phase: methanol:water = 95:5, flow rate: 1.0 mL / min, detection wavelength: 265 nm, injection volume: 10 μL, column temperature: 25℃, sample processing time: 30 min.

[0052] Enzyme activity definition: Under the above reaction conditions, the amount of enzyme required to produce 1 μmol 25(OH)VD3 per minute is defined as one enzyme activity unit, U.

[0053] Example 5: Constructing a highly active Vdh through rational design.

[0054] 1. Use SWISS-MODEL to predict the three-dimensional structure of Vdh. Align the amino acid sequence of Vdh with the amino acid sequence of the template protein, and select the three-dimensional model with the highest GMQE score for modeling.

[0055] 2. Molecular dynamics (MD) simulations are performed using GROMACS. First, a protein complex file with ligands is prepared after docking with Autodock Vina. Then, preprocessing is performed by creating a topology file, defining the simulation box, adding solvents and ions, minimizing energy, and balancing NPT and NVT. The parameters to be simulated are modified in the md.mdp file, and the MD simulation is performed to generate a trajectory file. Finally, RMSD and RMSF are generated by inputting commands. RMSD analysis can assess the overall structural stability of the protein during the dynamic simulation, while RMSF analysis helps identify flexible regions within the protein.

[0056] 3. Using the protein blast tool in the NCBI database, with the amino acid sequence of Vdh as a probe, we searched for enzyme proteins highly similar to the Vdh sequence. We selected all amino acid sequences with greater than 40% identity to the Vdh sequence for multiple sequence alignment. Using the ESPript 3.0 tool, we performed multiple sequence alignment on these homologous proteins. By aligning a series of homologous protein sequences, we can identify highly conserved amino acid residues during evolution. These conserved residues are often closely related to protein activity, binding sites, or structural stability. In protein engineering, by purposefully modifying these key residues, we can change the activity, selectivity, or stability of proteins. Combining molecular dynamics simulations and protein multiple sequence alignment methods, we selected important amino acid residue sites to construct a mutant library. The screening principle is as follows:

[0057] (1) RMSF analysis helps to identify flexible regions in protein structure, including the active site of Vdh and more active flexible regions. Excluding the relevant active sites and mutating these unstable amino acid residues with large fluctuations into other amino acid residues may help to improve the hydroxylation activity of Vdh.

[0058] (2) Protein multiple sequence alignment can reveal the evolutionary homology of related proteins. It provides key information for understanding the functional regions, active sites, structural domains of proteins and their conservation in different species. It is essential for designing and modifying proteins to improve their activity, stability or change their specific functions. Selecting relatively conserved amino acid residue sites and mutating them with other amino acid residues that occur more frequently in homologous proteins is expected to improve the hydroxylation activity of Vdh.

[0059] (3) The hydroxylation activity of P450 enzymes mainly depends on amino acid residues near the substrate binding pocket, and these residues also affect the regioselectivity of P450 enzymes. Arginine plays an important role in the substrate binding pocket, with some affecting the 25-hydroxylation activity of P450 enzymes and others inhibiting the hydroxylation activity in other regions. Therefore, non-conserved amino acid sites near the substrate binding pocket are preferentially mutated to arginine.

[0060] Example 6: Construction, expression and screening of Vdh mutants.

[0061] 1. Construct the Vdh mutant.

[0062] Based on the above principles, the following 17 single-point mutants were constructed: E81I, I114K, L167M, N173Q, N187V, V190S, R268K, D270R, A290R, Q310S, S318R, S323P, H335R, H341R, T366R, F374R, and V390I. Using the pET28a-Vdh plasmid from Example 1 as a template, and employing the principle of Quick-change site-directed mutagenesis, single-point mutagenesis was first performed using the primers listed in Table 1, mutating the amino acids at each site.

[0063] Table 1. Primer sequence listing for site-directed mutagenesis

[0064]

[0065]

[0066] PCR amplification was performed using the high-fidelity enzyme PrimeSTAR Max DNA Polymerase Ver.2 (Takara). After PCR, the original template was digested with DpnI enzyme (Takara) at 37 °C for 2 h to obtain the expression plasmid pET28(+)-Vdh mutant.

[0067] 2. Expression and screening of mutant engineered bacteria.

[0068] The mutant plasmid constructed in step 1 was transformed into the host bacterium *E. coli* BL21(DE3). A crude enzyme solution was prepared using the method described in Example 2, and its relative enzyme activity was determined according to the method described in Example 4. The results are as follows: Figure 1 As shown: with the activity of the crude enzyme solution of wild-type Vdh as 100%, the enzyme activities of the I114K, D270R, N173Q, Q310S, and H335R mutants were 2.2 times, 2.6 times, 1.7 times, 2.3 times, and 2.9 times that of wild-type Vdh, respectively, demonstrating that these sites have a significant impact on the hydroxylation activity of Vdh. In Example 7, saturation mutation studies were conducted on these sites.

[0069] Example 7: Enhancing Vdh activity through saturation mutation

[0070] 1. Site-directed saturation mutation.

[0071] The Quick-change mutation method was used, with the pET28-Vdh plasmid constructed by the method in Example 1 as a template, and the primers in Table 2 were used to select sites for saturation mutation.

[0072] Table 2. Primer sequence listing for saturation mutagenesis

[0073]

[0074] In the above, N = A, T, G, C; K = G, T; M = A, C.

[0075] 2. Expression and screening of mutant engineered bacteria.

[0076] The mutated plasmid from step 1 was transformed into the host bacterium *E. coli* BL21(DE3), and a pure enzyme solution was prepared using the method described in Example 2. Enzyme activity was then determined according to the method described in Example 4. The results are as follows: Figure 2 As shown: After saturation mutations at sites 114, 173, 270, and 310, the activities of the mutants I114R, I114H, D270L, N173M, N173V, Q310R, and Q310K were 3.1 times, 2.8 times, 2.5 times, 3.4 times, 2.7 times, 3.8 times, and 3.4 times that of the wild-type Vdh pure enzyme, respectively. This represents a further improvement in enzyme activity compared to the beneficial mutants at the same sites in Example 6. There was no significant improvement at site 335.

[0077] Example 8: Beneficial site combination mutations enhance Vdh activity

[0078] 1. Double mutation.

[0079] The pET28-Vdh plasmid with single-point mutations of I114R, I114H, D270L, N173M, N173V, Q310R, Q310K, and H335R was subjected to iterative combination mutations to introduce beneficial mutations at other sites. Using the Quick-change mutation method, the pET28-Vdh plasmid with the single-point mutation constructed in Example 7 was used as a template, and amplification was performed using primers listed in Tables 1 and 3 to obtain the pET28-Vdh double mutant.

[0080] Table 3. List of double-mutant primer sequences

[0081]

[0082] After transforming the above-mentioned mutant plasmid into the host bacterium E. coli BL21(DE3), pure enzyme solution was prepared using the methods in Examples 1 and 2, and enzyme activity was determined according to the method in Example 4. The results are as follows: Figure 3 As shown, the I114R / N173M and I114R / Q310R double mutants showed the greatest increase in enzyme activity, which was 4.6 times and 5.3 times that of the wild-type Vdh, respectively.

[0083] 2. Three mutations.

[0084] The pET-Vdh plasmids with double mutations of I114R / N173M and I114R / Q310R were further mutated to introduce beneficial mutations at other mutation sites, including sites 270, 173, 310, and 335. The Quick-change mutation method was used, with the pET-Vdh plasmid containing double mutations of I114R / N173M or I114R / Q310R as a template, and the amplification primers in Table 3 were used to induce mutations.

[0085] The amino acid sequence of mutant M3 is shown in SEQ ID NO. 2. After transforming the above mutant plasmid into the host bacterium *E. coli* BL21(DE3), pure enzyme solutions were prepared using the methods in Examples 1 and 2, and enzyme activity was determined according to the method in Example 4. The results are as follows: Figure 3 As shown, the I114R / N173M / Q310R (Vdh-M3) triple mutant exhibits significantly increased activity, reaching 9.8 times that of the wild-type pure enzyme. In contrast, triple mutants with beneficial amino acid mutations at other sites show no significant change in activity compared to single-site or double-site mutations. Further introduction of beneficial amino acid mutations at other sites into this triple mutant Vdh-M3 resulted in no significant change in activity or even a decrease.

[0086] Example 9: Application of mutant Vdh-M3 in enzymatic synthesis of calcidiol.

[0087] The highly active mutant Vdh-M3 was used in the preparation of calcidiol. Vitamin D3 and NADH were used as substrates. In a reaction system containing 20 mL sodium phosphate buffer (50 mM, pH 7.0), 10 mM vitamin D3 (dissolved in 2-hydroxypropyl-β-cyclodextrin), 15 mM NADH, 50 g / L crude Vdh-M3 enzyme solution, and 40 g / L crude Fdr-Fdx enzyme solution were added to a final concentration of 10 mM vitamin D3 (dissolved in 2-hydroxypropyl-β-cyclodextrin), 15 mM NADH, 50 g / L crude Vdh-M3 enzyme solution, and 40 g / L crude Fdr-Fdx enzyme solution. The reaction was carried out at 30 °C for 19 h, and the HPLC method described in Example 4 was used for detection. After 19 h of reaction, calcidiol reached 4.73 mM, with a conversion rate of 47.30%.

[0088] Example 10: Application of co-expressed engineered bacteria in the enzymatic synthesis of calcidiol.

[0089] In Example 9, VdhM3 was used in the preparation of calcidiol. However, due to the poor electron transfer efficiency from Fdr to Fdx and finally to the Vdh heme cofactor during the hydroxylation reaction, a large amount of the redox chaperone Fdr-Fdx was used, resulting in low utilization and significant loss of the coenzyme NADH. Therefore, Vdh-M3 and Fdr-Fdx were co-expressed to improve their synergistic effect, further enhance the electron transfer efficiency during the Vdh hydroxylation reaction, reduce electron loss, and synthesize calcidiol more efficiently. The pET28-VdhM3 strain constructed in Example 8 and the pETDuet1-Fdr-Fdx strain constructed in Example 3 were simultaneously transformed into *E. coli* BL21(DE3). The transformed strains were plated on LB agar plates containing 50 μg / mL kanamycin resistance and 100 μg / mL ampicillin resistance, and incubated upside down at 37°C for 8–12 h. Positive clones were then selected, which were the co-expression strains *Ecoli.BL21(DE3)-VdhM3-Fdr-Fdx*. Crude enzyme solution was prepared using the same steps as in Example 2, with the amount of VdhM3-Fdr-Fdx used based on the corresponding pre-lysis bacterial cell volume.

[0090] To reduce the amount of NADH used and lower costs, a NADH recycling system and glucose dehydrogenase (GDH) were introduced. GDH (GenBank: KC426949.1) derived from Bacillus subtilis in GenBank was codon-optimized according to the principle of E. coli preference, and a 6*His tag was added to the C-terminus of the protein. The whole gene was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the GDH gene was inserted under the T7 promoter of pET-28a(+) using the principle of homologous recombination to obtain the expression plasmid pET28(+)-GDH. The constructed expression plasmid pET28(+)-GDH was transformed into *E. coli* BL21(DE3) and plated on LB agar plates containing 50 μg / mL kanamycin resistance. The plates were incubated upside down at 37°C for 8–12 h. Positive clones were selected, which were wild-type *E. coli* BL21(DE3)-GDH, for expressing recombinant GDH. Crude enzyme solution was prepared using the same steps as in Example 2, with the amount of GDH used based on the pre-lysis bacterial cell volume.

[0091] Add 10 mM vitamin D3 (dissolved in 2-hydroxypropyl-β-cyclodextrin) and 1 mM NAD+ to a reaction system containing 20 mL sodium phosphate buffer (50 mM, pH 7.0). +The crude enzyme solution of VdhM3-Fdr-Fdx (40 g / L), crude enzyme solution of GDH (5 g / L), and 20 mM glucose were reacted at 30 °C for 19 h. The HPLC method described in Example 4 was used for detection. After 19 h of reaction, calcidiol reached 7.13 mM, with a conversion rate of 71.30%.

[0092] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A P450 enzyme mutant, characterized in that, The mutant is obtained by mutating positions 114, 173, and 310 of the amino acid sequence of the wild-type P450 enzyme Vdh, as shown in SEQ ID NO.2; the amino acid sequence of the wild-type P450 enzyme Vdh corresponds to GenBank accession number CP069288.1, as shown in SEQ ID NO.

1.

2. The gene encoding the P450 enzyme mutant according to any one of claims 1.

3. A recombinant expression vector carrying the gene of claim 2.

4. A genetically engineered bacterium, characterized in that, The recombinant expression vector of claim 3 is included, wherein the host bacterium of the engineered bacteria is Escherichia coli BL21(DE3).

5. A method for constructing the P450 enzyme mutant as described in claim 1, characterized in that, Includes the following steps: S1. Construction of saturated mutants: Using pET28(+)-Vdh plasmid as a template, Quick-change site-directed mutagenesis was performed at positions 114, 173, and 310 using primer sequences containing degenerate codons. The saturated mutant plasmid was obtained after digestion of the template with DpnI enzyme. S2. The mutant plasmid was transformed into E. coli BL21(DE3), and positive clones were obtained through resistance screening, thus obtaining engineered bacteria containing the target mutant.

6. The method for constructing a P450 enzyme mutant according to claim 5, characterized in that, Primer sequences containing degenerate codons in S1 are shown in SEQ ID NO. 37 to 46.

7. The application of the P450 enzyme mutant as described in claim 1 in the synthesis of calcidiol, characterized in that, Using vitamin D3 as a substrate, in the presence of the redox chaperone Fdr-Fdx and coenzyme, the mutant catalyzes the 25-hydroxylation reaction of vitamin D3 to generate calcidiol.

8. The application of the P450 enzyme mutant according to claim 7 in the synthesis of calcidiol, characterized in that, The redox chaperone Fdr-Fdx is derived from Bacillus megaterium, corresponding to the amino acid sequence of GenBank accession number CP009920.1, and was obtained by expressing the pETDuet1-Fdr-Fdx recombinant plasmid in Escherichia coli BL21(DE3).

9. The application of the P450 enzyme mutant of claim 1, the gene of claim 2, the recombinant expression vector of claim 3, and the genetically engineered bacteria of claim 4 in the preparation of calcidiol.

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