P450 mutant and application thereof in synthesis of calcifediol

Abstract

The invention discloses a P450 mutant and application thereof in synthesis of calcifediol, and belongs to the technical field of bioengineering. According to the present invention, the P450 enzyme mutant enzyme is constructed, and the single plasmid three-enzyme co-expression system of the redox partner and the cofactor cycle enzyme is constructed, such that the sufficient electron transport chain is provided for the P450 enzyme, and the calcifediol can be efficiently synthesized under the mild condition by using the vitamin D3 as the substrate; the conversion rate of calcifediol can reach 63.1%, and the conversion rate of the reaction is greatly improved. By adopting the method disclosed by the invention, the catalytic efficiency of the catalyst is improved, and the reaction cost is reduced.

Description

Technical Field

[0001] The invention relates to a P450 mutant and application thereof in synthesizing calcifediol, belonging to the technical field of bioengineering. Background Art

[0002] Calcifediol (also known as 25-hydroxyvitamin D3), molecular formula is C 27 H 44 O is the main form of vitamin D3 in the human body. Calcifediol can not only treat vitamin D deficiency, but also maintain liver homeostasis and intestinal immunity. Calcifediol preparations can be used clinically to treat osteoporosis in the elderly, and also have a preventive effect on chronic kidney disease, hyperglycemia, and cholestatic liver disease.

[0003] At present, the industrial production methods of calcifediol mainly include total synthesis and semi-synthesis. The total synthesis method uses chain sterol as raw material and gradually passes through 25-hydroxycholesterol and 25-hydroxy-7-ketocholesterol to generate 25-hydroxy-7-dehydrocholesterol, and finally obtains the target product through photolysis and ring opening. The semi-synthesis method uses cholesterol as raw material to obtain 7-dehydrochain sterol through yeast multi-step fermentation, and then generates 25-hydroxy-7-dehydrocholesterol through chemical method, and obtains the target product through photolysis. The paths of these two types of reactions are long, and double bond protective agents and hydroxyl protective agents need to be used frequently, and finally they have to go through the step of photolysis and ring opening. This step of reaction has many by-products, low product yield, and strict control of light time and intensity, so the cost of industrial production is high. The biological synthesis has attracted the favor of many scholars due to its high selectivity, few by-products, mild reaction conditions and other advantages. With the development of biotechnology such as synthetic biology and enzyme engineering, the synthesis pathway of calcifediol has gradually begun to be constructed in engineered bacteria, and initial progress has been made. The one-step enzymatic production of calcifediol using relatively cheap raw material vitamin D3 as substrate is a potential biosynthetic method. This method only requires one step of enzymatic reaction, and does not require the addition of protective agents, nor does it require harsh conditions such as controlling strong light to achieve product synthesis. However, the catalytic efficiency of the currently reported biosynthetic methods for the substrate vitamin D3 is generally low and cannot meet the requirements of industrial production. Therefore, there is an urgent need for an efficient biological method for preparing calcifediol using low-cost vitamin D3 as substrate. Summary of the invention

[0004] The present invention provides a P450 enzyme mutant, which is obtained by mutating one or more of the amino acids at positions 73 and 81 on the basis of the starting sequence.

[0005] In one embodiment, the mutant is any one of the following (a) to (c):

[0006] (a) mutating the glutamine Q at position 73 of the P450 enzyme CYP109E1 as shown in SEQ ID NO.1 to methionine M, and the resulting mutant is named Q73M;

[0007] (b) mutating the glycine G at position 81 of the P450 enzyme CYP109E1 as shown in SEQ ID NO.1 to tryptophan W, and the resulting mutant was named G81W;

[0008] (c) The glutamine Q at position 73 of the P450 enzyme CYP109E1 as shown in the amino acid sequence of SEQ ID NO.1 was mutated to methionine M, and the glycine G at position 81 was mutated to tryptophan W. The resulting mutant was named Q73M / G81W.

[0009] In one embodiment, the amino acid sequence of the starting sequence CYP109E1 of the P450 enzyme is shown as SEQ ID NO.1, and the nucleotide sequence encoding the P450 enzyme is shown as SEQ ID NO.5.

[0010] The present invention also provides a gene encoding the P450 enzyme mutant.

[0011] In one embodiment, the nucleotide sequence of the P450 enzyme mutant is shown as SEQ ID NO.6.

[0012] The invention also provides a recombinant vector carrying the gene.

[0013] In one embodiment, the recombinant vector uses pET-28a as an expression vector.

[0014] The present invention also provides a microbial cell carrying the above gene or the above recombinant vector.

[0015] In one embodiment, the microbial cell uses bacteria or fungi as expression hosts.

[0016] In one embodiment, the microbial cell uses Escherichia coli BL21 (DE3) as an expression host.

[0017] The present invention also provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses the above-mentioned P450 enzyme CYP109E1 mutant.

[0018] In one embodiment, the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as an expression host and pET28a as an expression vector.

[0019] The present invention also provides a method for obtaining the above-mentioned P450 enzyme CYP109E1 mutant, the method comprising the following steps:

[0020] (1) Determine the mutation site based on the amino acid sequence of the P450 enzyme CYP109E1; design mutation primers for site-directed mutagenesis, and perform site-directed mutagenesis using a vector carrying the P450 enzyme CYP109E1 gene as a template; and construct a plasmid vector containing the mutant;

[0021] (2) transforming the plasmid vector containing the mutant into a host cell;

[0022] (3) Select positive clones for fermentation culture and purify the P450 enzyme CYP109E1.

[0023] In one embodiment, the host cell is Escherichia coli.

[0024] The present invention also provides a method for constructing a single-plasmid three-enzyme co-expression system, the method comprising the following steps:

[0025] (1) The gene sequences of ferredoxin (SelFdx) and ferredoxin reductase (SelFdR) from Synechococcus elongatus PCC 7942, and the gene sequence of glucose dehydrogenase (BsGDH) from Bacillus subtilis were amplified, respectively, and inserted into the expression plasmid pRSFDuet-1 to obtain plasmid pRSFDuet-SelFdx, plasmid pRSFDuet-SelFdR, and plasmid pRSFDuet-BsGDH, respectively;

[0026] (2) The plasmid pRSFDuet-SelFdx, the plasmid pRSFDuet-SelFdR, and the plasmid pRSFDuet-BsGDH were respectively digested with endonucleases, and then connected in sequence to transform the host Escherichia coli BL21 (DE3) to construct a single-plasmid three-enzyme co-expression system.

[0027] The present invention also provides a recombinant Escherichia coli containing a three-enzyme co-expression system, wherein the three-enzyme co-expression system uses the same plasmid to co-express genes encoding ferredoxin (SelFdx), ferredoxin reductase (SelFdR) and glucose dehydrogenase (BsGDH); the nucleotide sequence encoding the ferredoxin (SelFdx) is shown in SEQ ID NO.7; the nucleotide sequence encoding the ferredoxin reductase (SelFdR) is shown in SEQ ID NO.8; and the nucleotide sequence encoding the glucose dehydrogenase (BsGDH) is shown in SEQ ID NO.9.

[0028] In one embodiment, pRSFDuet is used as an expression vector to express the ferredoxin gene SelFdx, the ferredoxin reductase gene SelFdR and the glucose dehydrogenase gene BsGDH.

[0029] The present invention also provides a method for preparing a crude enzyme solution, which is characterized in that the bacterial suspension of the recombinant Escherichia coli containing the three-enzyme co-expression system is subjected to ultrasonic disruption and centrifugation to obtain a supernatant.

[0030] In one embodiment, the crude enzyme solution is prepared by culturing the recombinant Escherichia coli for a period of time, collecting bacterial cells and preparing a bacterial suspension with a concentration of 100 g / L, and then subjecting the suspension to ultrasonic disruption and centrifugation to obtain a supernatant.

[0031] The present invention also provides a method for preparing calcifediol, which comprises: mixing the crude enzyme solution with any one of (a) to (c) containing a cofactor NADP + , glucose, vitamin D3 in a reaction system; wherein:

[0032] (a) the P450 enzyme mutant;

[0033] (b) the microbial cell;

[0034] (c) the recombinant Escherichia coli.

[0035] In one embodiment, when microbial cells or recombinant E. coli expressing a P450 enzyme mutant are used for catalysis, lysozyme is also added to the reaction system so that the P450 enzyme is released from the cells and fully contacts with the enzyme and substrate in the reaction system.

[0036] In one embodiment, the reaction system contains hydroxypropyl-β-cyclodextrin to reduce the wall adhesion phenomenon when vitamin D3 and calcifediol are mixed with water.

[0037] In one embodiment, the amount of crude enzyme solution added is 12.5% ​​of the total volume of the reaction solution.

[0038] In one embodiment, the recombinant Escherichia coli is added to the reaction system at a final concentration of 10 to 50 g / L.

[0039] In one embodiment, the mass volume percentage of the hydroxypropyl-β-cyclodextrin in the reaction system is 1.13-2.83%.

[0040] In one embodiment, the added amount of lysozyme is 1500 U / mg cell.

[0041] In one embodiment, the crude enzyme solution is added in an amount of 25% of the total volume.

[0042] In one embodiment, the vitamin D3 is vitamin D3 pre-dissolved in a co-solvent; the co-solvent includes but is not limited to one or more of DMSO, methanol, ethanol and DMF.

[0043] In one embodiment, the final concentration of vitamin D3 in the reaction system is 0.5-5 mM.

[0044] In one embodiment, the volume percentage of the co-solvent in the reaction system is 2-5%.

[0045] In one embodiment, NADP + The molar ratio of the vitamin D3 is 0.05 to 1.2:1.

[0046] In one embodiment, the molar ratio of glucose to vitamin D3 is 25 to 75:1.

[0047] In one embodiment, the reaction conditions are: pH 6.0-8.0, 37° C., and reaction time of 5-8 hours.

[0048] The present invention also provides the use of the above-mentioned P450 enzyme CYP109E1 mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant Escherichia coli in the preparation of calcifediol or a product containing calcifediol.

[0049] In one embodiment, the product is a chemical.

[0050] Beneficial Effects

[0051] (1) The present invention provides a P450 enzyme CYP109E1 mutant that can be used to catalyze the production of calcifediol, and the catalytic ability is significantly improved compared to the starting sequence.

[0052] (2) The present invention provides a method for producing calcifediol by using a single plasmid three-enzyme co-expression system and a P450 enzyme for co-catalysis. The single plasmid three-enzyme co-expression system is used to provide the electrons required for CYP109E1 catalysis.

[0053] (3) The P450 enzyme CYP109E1 mutant of the present invention increases the yield of vitamin D3 into calcifediol by 28.2% to 104.7% compared with the P450 enzyme CYP109E1. The P450 enzyme CYP109E1 mutant exhibits a higher hydroxylation activity. Under the conditions of 37°C and pH 7.0 for 8 hours, the maximum yield of calcifediol is 631.7 mg / L, and the yield reaches 63.1%. The method of the present invention improves the catalytic efficiency of the catalyst, reduces the cost of the reaction, and accelerates the industrialization process of producing calcifediol by enzyme conversion. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 : A synthetic method for synthesizing calcifediol catalyzed by vitamin D3.

[0055] Figure 2 : SDS-PAGE images of the inducible expression of glucose dehydrogenase BsGDH and redox chaperones SelFdR and SelFdx.

[0056] Figure 3 : Liquid phase detection spectrum of reaction substrate and product; i: Liquid phase detection of calcifediol standard, the retention time of calcifediol is 6.2min; ii: The reaction converts vitamin D3 to produce calcifediol for detection, the retention time of calcifediol is 6.2min; iii: Liquid phase detection of vitamin D3 standard, the retention time is 12.1min. DETAILED DESCRIPTION

[0057] The pET-28a(+) and pRSFDuet-1 plasmids involved in the following examples were purchased from Novagen (Madison, WI, USA), and restriction endonucleases, T4 DNA ligase, PrimeSTAR, etc. were purchased from TaKaRa (Dalian, China). Standard calcifediol was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., vitamin D3 was purchased from Tianjin Xiens Biochemical Technology Co., Ltd., and the remaining reagents were purchased from the market.

[0058] The culture medium involved in the following examples is as follows:

[0059] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, sterilized at 121°C for 20 min.

[0060] LB solid medium: Add 2% agar to LB liquid medium.

[0061] TB liquid culture medium: KH2PO4 2.31 g / L, K2HPO4·3H2O 16.42 g / L, yeast powder 24 g / L, peptone 12 g / L, glycerol 4 g / L.

[0062] The detection methods involved in the following embodiments are as follows:

[0063] Detection of vitamin D3 and calcifediol content:

[0064] The system used 0.1M PBS buffer as the aqueous phase, vitamin D3 with a final concentration of 961.6 mg / L as the substrate, and a crude enzyme solution of a single plasmid three-enzyme co-expression system containing glucose dehydrogenase BsGDH and redox chaperones SelFdR and SelFdx (the crude enzyme solution accounted for 12.5% ​​of the total volume), and a cofactor NADP + 0.5mM, hydrogen donor glucose 50mM, cosolvent 4.375%, hydroxypropyl-β-cyclodextrin 2.25%, lysozyme 1500U / mg 表达P450酶细胞 , using whole cells to convert vitamin D3 to produce calcifediol. The whole cell concentration of the P450 enzyme CYP109E1 mutant was 20 g / L. After reacting in a constant temperature shaker at 37°C and 400 rpm for 8 hours, the reaction solution was sampled, a small amount of the sample was placed in an EP tube, and an equal volume of ethyl acetate was added to extract three times. After the organic phase was combined, the ethyl acetate was evaporated under reduced pressure to remove the ethyl acetate, and an equal volume of methanol was added to dissolve it again, and it was passed through a 0.22 μm organic membrane for HPLC analysis.

[0065] The above specific high performance liquid chromatography HPLC analysis method is:

[0066] C18 (5 μm, 250 × 4.6 mm) was used as the chromatographic column, filtered and ultrasonically degassed methanol was used as the organic phase, ultrapure water was used as the aqueous phase, and the gradient elution conditions were shown in Table 1. The injection volume was 20 μL, the column temperature was 40 °C, the wavelength of the UV detector was 264 nm, the flow rate was 0.8 mL / min, and the sample processing time was 20 min.

[0067] Table 1 Gradient elution conditions

[0068]

[0069] Under this detection condition, the retention times of calcifediol and vitamin D3 were 6.2 min and 12.1 min, respectively.

[0070]

[0071] Where: m 骨化二醇 Indicates the mass of calcifediol produced; m 维生素D3 represents the initial mass of vitamin D3; 384.63 and 400.64 represent the relative molecular masses of vitamin D3 and calcifediol, respectively.

[0072] Example 1: Construction of genetically engineered bacteria expressing CYP109E1

[0073] Using the nucleotide sequence of the target protein encoding gene in Bacillus megaterium (shown in SEQ ID NO.5) as a template, the plasmid pET-28a(+)-CYP109E1 was synthesized by entrusting Sangon Biotech Co., Ltd., wherein the target gene fragment was connected between BamHI and HindIII of the vector. The plasmid pET-28a(+)-CYP109E1 was transformed into Escherichia coli BL21 to obtain the engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1.

[0074] The engineered bacteria E.coli BL21 / pET-28a(+)-CYP109E1 was inoculated into LB liquid medium, and the seed solution was obtained after culturing for 12 hours. The seed solution was inoculated into fresh TB liquid medium at a 5% (v / v) inoculation amount. After culturing for 2 hours, IPTG with a final concentration of 0.4 mM was added, and the cells were cultured at 16°C for 18 hours to induce the expression of the recombinant target protein. 150 mL of the induced fermentation liquid was collected by centrifugation at 8000 r / min, and the cells were frozen and stored at -40°C.

[0075] Example 2: Construction of a single plasmid three-enzyme co-expression system

[0076] The ferredoxin (SelFdx) gene (shown in SEQ ID NO.7) and the gene sequence of ferredoxin reductase (SelFdR) (shown in SEQ ID NO.8) from Synechococcus elongatus PCC 7942, and the gene sequence of glucose dehydrogenase (BsGDH) from Bacillus subtilis (shown in SEQ ID NO.9) were amplified respectively, and inserted between BamHI and HindIII of the expression plasmid pRSFDuet-1 to obtain plasmid pRSFDuet-SelFdx, plasmid pRSFDuet-SelFdR and plasmid pRSFDuet-BsGDH, respectively.

[0077] The plasmid pRSFDuet-SelFdx, the plasmid pRSFDuet-SelFdR, and the plasmid pRSFDuet-BsGDH were respectively digested with endonucleases, and connected in sequence to obtain the plasmid pRSFDuet-SelFdx-SelFdR-BsGDH, which was then transformed into the host Escherichia coli BL21 (DE3) to construct a single-plasmid three-enzyme co-expression strain E. coli BL21 / pRSFDuet-SelFdx-SelFdR-BsGDH.

[0078] Example 3: Expression of a single plasmid three-enzyme co-expression system and preparation of crude enzyme solution

[0079] The engineered bacteria E. coli BL21 / pRSFDuet-SelFdx-SelFdR-BsGDH constructed in Example 2 was inoculated into LB liquid medium, and the seed solution was obtained after culturing for 12 hours. The seed solution was inoculated into fresh TB liquid medium at an inoculum amount of 5% (v / v). After culturing for 2 hours, IPTG with a final concentration of 0.4 mM was added, and the cells were cultured at 16° C. for 18 hours to induce the expression of the recombinant target protein. 150 mL of the induced fermentation broth was collected by centrifugation at 8000 r / min, and the cells were frozen and stored at -40° C.

[0080] The collected bacteria were fully resuspended in 0.1M PBS buffer to a concentration of 100 g / L. The centrifuge tube was then placed in an ice bath and placed in an ultrasonic cell disruptor. The ultrasonic disruption conditions were: working time 2.5 s, interval time 1.5 s, total 20 min. The obtained disrupted liquid was subjected to low-temperature low-speed centrifugation at 4°C and 4200 rpm for 15 min, and the supernatant was collected to obtain the crude enzyme solution.

[0081] Example 4: Conversion of Vitamin D3 to Prepare Calcifediol

[0082] The crude enzyme solution of the three-enzyme co-expression system constructed by CYP109E1 protein and Example 3 was used to convert vitamin D3 to prepare calcifediol. A reaction system containing a final concentration of 2.5 mM vitamin D3 was prepared, wherein NADP + The molar ratio of glucose to vitamin D3 is 0.1:1, the molar ratio of glucose to vitamin D3 is 50:1, the wet bacterial concentration of CYP109E1 is 20g / L, and the volume ratio of the crude enzyme solution to the total system is 12.5%. The specific steps are:

[0083] In a 20 mL small brown bottle, 0.1 g of whole-cell E. coli BL21 / pET-28a(+)-CYP109E1 expressing CYP109E1 protein after induction culture according to the method of Example 1, 2.5 mL of 0.05 M PBS buffer, and 0.625 mL of lysozyme (enzyme activity ≥ 20000 U / mg, Sinopharm Chemical Reagent Co., Ltd., catalog number: 64006060) with a final concentration of 5 g / L were added, and the mixture was lysed at 37°C for 90 min, and 1.9 mg of NADP was added. + , 45.0mg hydrogen donor glucose, 112.5mg hydroxypropyl-β-cyclodextrin, add 0.1M PBS to 4781.3μL, and finally add 218.7μL vitamin D3 mother solution pre-dissolved in DMF, so that the final concentration of vitamin D3 in the 5mL reaction system is 961.6mg / L, start the reaction, adjust the system pH to 7.0, and react in a constant temperature shaker at 37℃ and 400rpm for 8h. After conversion, add an equal volume of ethyl acetate to the reaction liquid sample and shake vigorously in a vortex shaker, collect the upper organic phase by high-speed centrifugation, repeat the extraction twice, combine the organic phases, place in a vacuum centrifuge to volatilize and remove ethyl acetate, add an equal volume of methanol, shake to promote dissolution, filter through a 0.22μm organic filter membrane, and perform high-performance liquid chromatography HPLC analysis. The analysis results showed that the yield of calcifediol prepared by the recombinant bacteria E. coli BL21 / pET-28a(+)-CYP109E1 was 308.6 mg / L, and the conversion rate was 30.8%.

[0084] Example 5: Construction and screening of single mutants

[0085] Using the pET-28a(+)-CYP109E1 constructed in Example 1 as a template, primers for the Q73M and G81W mutation sites were designed as shown in Table 2, and mutants were constructed by whole plasmid PCR.

[0086] Table 2 Single mutant mutation primer sequences

[0087]

[0088] Construct the PCR amplification system: PrimeSTAR enzyme 25 μL, two primers for each mutation site 1 μL each, template (CYP109E1 WT)1μL, water 22μL; reaction conditions are: ①98℃5min; ②94℃30s; ③55℃30s; ④72℃3min20s; ⑤cycle ②~④30 times; ⑥72℃10min; ⑦12℃ insulation. The above reaction system was incubated at 37℃ for 1h to digest the plasmid template (digestion system: DpnI 1μL, PCR product of the above reaction 8μL, 10×T Buffer 1μL), and the digestion product obtained after digestion was introduced into E. coli BL21 competent cells by chemical transformation method. The specific steps of chemical transformation method are:

[0089] (1) Introduce 10 μL of digestion product into 100 μL of E. coli BL21 (DE3) competent cells;

[0090] (2) Ice bath for 10 min;

[0091] (3) Heat shock at 42°C in a water bath for 90 seconds, then quickly place in ice for 4 minutes;

[0092] (4) Add 800 μL of resistance-free LB medium, mix well, and culture at 37°C, 220 rpm for 1 h;

[0093] (5) Centrifuge at 5000 rpm for 2 min to collect the bacteria;

[0094] (6) Remove the supernatant and mix the remaining 100-200 μL by pipetting and apply it to an LB resistance plate containing 0.05 mg / mL kanamycin and culture at 37°C for about 12 h;

[0095] (7) Pick a single clone and place it in LB containing 0.05 mg / mL kanamycin resistance. After culturing at 200 rpm and 37°C for 12 h, send it to the company for sequencing. The one with correct sequencing is the positive transformant.

[0096] Genetically engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1 were prepared respectively. Q73M , E.coli BL21 / pET-28a(+)-CYP109E1 G81W .

[0097] Subsequently, the obtained genetically engineered bacteria were transformed to prepare calcifediol according to the method of Example 4, and better mutants were screened. The results are shown in Table 3. The engineered bacteria E. coli BL21 / pET-28a(+)-CYP109E1 Q73M , E.coliBL21 / pET-28a(+)-CYP109E1 G81WAll of them have good vitamin D3 hydroxylation effects, and the production is increased by 28.2% to 84.3% compared with the wild-type E. coli BL21 / pET-28a(+)-CYP109E1.

[0098] Table 3 Calcifediol production of wild type and single mutants

[0099]

[0100] Example 6: Construction and screening of double mutations

[0101] The double mutants of this example were constructed by whole plasmid PCR based on the corresponding single mutants using the primers in Table 4. For example, in mutant CYP109E1 G81W Based on the results, the double mutant CYP109E1 was expressed by whole plasmid PCR using the mutant primers Q73M-R and Q73M-F (Table 4). G81W / Q73M 's construction.

[0102] The preparation method of genetically engineered bacteria is shown in Example 5. The primers used are shown in Table 4. According to the method of Example 5, genetically engineered bacteria containing double mutants were prepared: E. coli BL21 / pET-28a(+)-CYP109E1 G81W / Q73M .

[0103] Table 4 Double mutant mutation primer sequences

[0104]

[0105] The mutant strain with correct sequencing was inoculated into LB seed medium and cultured at 220 rpm and 37 °C for 10 h. The seed solution was inoculated into fresh TB liquid medium at a 5% (v / v) inoculation rate and cultured at 220 rpm and 37 °C until OD 600 = about 0.8, add IPTG with a final concentration of 0.4mM for induction, and the induction conditions are 220rpm, 16℃ for 18h.

[0106] Then, the obtained genetically engineered bacteria were transformed according to the method of Example 4 to prepare calcifediol.

[0107] After the reaction, the yield of calcifediol was determined by HPLC. The double mutant genetic engineering bacteria: E. coli BL21 / pET-28a(+)-CYP109E1 G81W / Q73M It has a good hydroxylation effect on calcifediol, and the conversion rate of the prepared calcifediol is 63.1%, and the corresponding output is 631.7 mg / L.

[0108] Comparative Example:

[0109] The specific implementation method is the same as that in Example 5, except that mutants G81L, Q73E, Q77F, Q73D, P71L, N74C, A291C, G81W / A291C, G81W / Q77F, G81W / Q73E, G81W / Q73D, G81W / N74C, G81W / P71L, L292T, F391W, N7L, N7I, N7F, Q73D / N74M, Q73M / N74Y, etc. were also constructed, and calcifediol was prepared according to the method of Example 4. The results are shown in Table 5.

[0110] Table 5 Calcifediol production of wild type and single mutants

[0111]

[0112] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A P450 enzyme mutant, characterized in that: It is obtained by mutating one or more of the amino acids at positions 73 and 81 based on the starting sequence shown in SEQ ID NO.

1.

2. The mutant according to claim 1, characterized in that The mutant is any one of (a) to (c): (a) mutating the glutamine at position 73 of the P450 enzyme CYP109E1 as shown in SEQ ID NO.1 to methionine; (b) mutating the glycine at position 81 of the P450 enzyme CYP109E1 as shown in SEQ ID NO.1 to tryptophan; (c) The glutamine at position 73 of the P450 enzyme CYP109E1 whose amino acid sequence is shown in SEQ ID NO. 1 is mutated to methionine, and the glycine at position 81 is mutated to tryptophan.

3. A gene encoding the P450 enzyme mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. A microbial cell carrying the gene according to claim 3 or the recombinant vector according to claim 4.

6. A recombinant Escherichia coli, characterized in that The P450 enzyme mutant according to claim 1 or 2 is expressed.

7. A recombinant Escherichia coli containing a three-enzyme co-expression system, characterized in that: The three-enzyme co-expression system uses pRSFDuet as an expression vector to co-express the coding genes of ferredoxin, ferredoxin reductase and glucose dehydrogenase; and expresses the ferredoxin gene SelFdx, the ferredoxin reductase gene SelFdR and the glucose dehydrogenase gene BsGDH.

8. A crude enzyme solution containing ferredoxin, ferredoxin reductase and glucose dehydrogenase, characterized in that: The recombinant Escherichia coli according to claim 7 is cultured for a period of time, and a crude enzyme solution containing ferredoxin, ferredoxin reductase and glucose dehydrogenase is collected.

9. A method for preparing calcifediol, characterized in that: The crude enzyme solution of claim 8 is mixed with any one of (a) to (c) in the presence of a cofactor NADP + , glucose, hydroxypropyl-β-cyclodextrin, vitamin D3 reaction system; wherein: (a) the P450 enzyme mutant according to claim 1 or 2; (b) the microbial cell according to claim 4; (c) The recombinant Escherichia coli according to claim 6.

10. Use of the P450 enzyme mutant according to claim 1 or 2, or the gene according to claim 3, or the recombinant vector according to claim 4, or the recombinant microbial cell according to claim 5, or the recombinant Escherichia coli according to any one of claims 6 to 7 in the preparation of calcifediol or a product containing calcifediol.

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