A vitamin D3 C25 hydroxylase mutant and its genetically engineered bacteria and application

Abstract

The present invention discloses a C25 hydroxylase mutant of vitamin D3 and its genetically engineered bacteria and application, belonging to the field of genetic engineering and enzyme engineering technology. The vitamin D3 hydroxylase mutant of the present invention is to mutate the 293-position histidine of the CYP109E1-H enzyme gene in Bacillus megaterium H-1 into alanine, to obtain the vitamin D3 hydroxylase mutant CYP109E1-H1, and to use pMA5 as an expression plasmid to achieve heterologous expression of the vitamin D3 hydroxylase mutant in Bacillus subtilis. The present invention also provides the application of the mutant and the engineered bacteria in the preparation of calcidiol / or calcitriol, and the engineered bacteria provided by the present invention can improve the product concentration accumulation amount and shorten the reaction time when calcidiol / or calcitriol is synthesized, and meet the production demand of shortening the time cost in industrial production.

Description

Technical Field

[0001] The invention relates to a vitamin D3 C25 hydroxylase mutant and a genetically engineered bacterium and application thereof, belonging to the technical field of genetic engineering and enzyme engineering. Background Art

[0002] Vitamin D3 is an important fat-soluble vitamin in the body. It is mainly used as a precursor substance for calcium and phosphorus regulation. Under the irradiation of ultraviolet wavelength, 7-dehydrocholesterol in the body opens the C9 position of cyclopentane polyhydrogen phenanthrene to form the structure of VD3. VD3 has no biological activity in the body. However, VD3 can generate two active forms through hydroxylation reaction in the human body, namely 25(OH)VD3 and 1α,25(OH)2VD3. Among them, 25(OH)VD3 is its main active form. Calcifediol, also known as 25-hydroxyvitamin D3 [25(OH)VD3], is a biologically active derivative of vitamin D3. It is quickly absorbed in the human body and has the functions of regulating calcium and phosphorus balance, improving immunity, preventing osteoporosis, and resisting rickets.

[0003] As the demand for active VD3 increases, the traditional chemical synthesis method needs to consider the regioselectivity and stereoselectivity to specifically introduce hydroxyl groups at C1 and C25. The reaction is difficult and the process is complicated. It has the disadvantages of low yield, high cost, and serious environmental pollution. Chemical synthesis is not suitable for industrial production. In order to solve many problems in chemical synthesis, it is necessary to develop new production processes. Compared with chemical synthesis, microbial methods have the characteristics of fewer synthesis steps and environmental friendliness. In recent years, many scholars have widely invented P450 enzyme catalysis using vitamin D3 as a substrate.

[0004] Bacillus megaterium H-1 contains a C-25 hydroxylase CYP109E1-H that can selectively catalyze the substrate vitamin D3 to produce the product 25(OH)VD3. This process is mainly mediated by the cytochrome P450 enzyme system, specifically through the catalytic reaction of cytochrome P450 hydroxylase (CYP), and also requires redox partners (RPs) to assist in electron transfer, and the coenzyme NADPH provides electrons. However, the expression levels of P450 oxidoreductase and its reductase in Bacillus megaterium H-1 are very low, which greatly limits the production of 25(OH)VD3.

[0005] Therefore, it is of great industrial significance to combine genomics, bioinformatics analysis and metabolic engineering to explore and identify more new soluble enzyme genes and transform them, and screen out P450 enzymes that can efficiently convert substrates. Summary of the invention

[0006] The present invention provides an engineered strain of a VD3 hydroxylase mutant, provides a gene of the mutant, a recombinant plasmid containing the mutant gene, and a host cell containing the recombinant plasmid, realizes enhanced expression of VD3 hydroxylase, and the corresponding mutant is heterologously expressed and can effectively convert VD3 into 25(OH)VD3. By screening the electron transport chain system, it is found that the expression of CYP and a specific electron transport chain system in a specific host can effectively increase the yield of 25(OH)VD3, and the nucleotide sequence of CYP109E1-H of the parent Bacillus megaterium H-1 is shown in SEQ ID NO.1.

[0007] The first object of the present invention is to provide a vitamin D3 C25 hydroxylase mutant, the amino acid sequence of the mutant is shown in SEQ ID NO.4; the present invention names the mutant CYP109E1-H1.

[0008] In one embodiment, the vitamin D3 C25 hydroxylase mutant is obtained by replacing the histidine at position 293 of CYP109E1-H of the parent Bacillus megaterium H-1 having an amino acid sequence as shown in SEQ ID NO.2 with alanine.

[0009] The second object of the present invention is to provide a nucleotide sequence encoding the above-mentioned C25 hydroxylase mutant. Optionally, the nucleotide sequence is shown in SEQ ID NO.3.

[0010] The third object of the present invention is to provide a recombinant expression plasmid for expressing the above-mentioned vitamin D3 C25 hydroxylase mutant, wherein the plasmid contains the nucleotide sequence of the vitamin D3 C25 hydroxylase mutant.

[0011] Optionally, the recombinant plasmid is constructed based on pMA5.

[0012] The fourth object of the present invention is to provide a genetically engineered bacterium, wherein the engineered bacterium is integrated with the nucleotide sequence of the C25 hydroxylase mutant or contains the above-mentioned recombinant expression plasmid.

[0013] In one embodiment, the genetically engineered bacteria uses pMA5 plasmid as an expression vector.

[0014] In one embodiment, the host microorganism comprises Bacillus subtilis.

[0015] Furthermore, the host microorganism is Bacillus subtilis WB600.

[0016] Furthermore, the engineered strain is constructed by using pMA5 as an expression vector, integrating the mutated CYP109E1-H1 gene into the expression vector pMA5, and transferring pMA5 into the host Bacillus subtilis WB600, and is named Bacillus subtilis WB600-pMA5-CYP109E1-H1.

[0017] In one embodiment, the genetically engineered bacteria contains a ferredoxin gene and a ferredoxin reductase gene, or a recombinant expression plasmid containing a ferredoxin gene and a ferredoxin reductase gene.

[0018] Optionally, the amino acid sequences of the ferredoxin protease gene and the ferredoxin reductase gene are shown as SEQ ID NO.6 and SEQ ID NO.8, respectively.

[0019] Optionally, the gene sequences of the ferredoxin protease gene and the ferredoxin reductase gene are shown as SEQ ID NO.5 and SEQ ID NO.7, respectively.

[0020] Furthermore, the engineered strain is obtained by using pMA5 as an expression vector, integrating the mutated CYP109E1-H1 gene, the ferredoxin protease gene (Fdx) and the ferredoxin reductase gene (FdR) into the expression vector pMA5, and transferring pMA5 into the host Bacillus subtilis WB600, and naming it Bacillus subtilis WB600-pMA5-CYP109E1-H1-Fdx-FdR.

[0021] The fifth object of the present invention is to provide a method for constructing the above-mentioned genetically engineered bacteria, which comprises: performing a point mutation on the hydroxylase CYP109E1-H gene at the C25 position, and transferring the mutated gene into Bacillus subtilis WB600 for expression. Optionally, the hydroxylase CYP109E1-H gene at the C-25 position is derived from Bacillus megaterium H-1.

[0022] In one embodiment, point mutagenesis is performed using inverse PCR.

[0023] In one embodiment, the restriction sites of the recombinant pMA5 plasmid are NdeⅠ and BamHI.

[0024] The sixth object of the present invention is to provide the use of the above-mentioned C25 hydroxylase mutant or the above-mentioned genetically engineered bacteria in the preparation of 25-hydroxyvitamin D3.

[0025] Beneficial Effects

[0026] The invention provides a Bacillus subtilis CYP109E1-H mutant and engineered bacteria and their applications, wherein the obtained mutant CYP109E1-H1 is heterologously expressed in Bacillus subtilis. In addition, when the engineered bacteria of CYP109E1-H1 constructed by the invention are applied to the synthesis of 25(OH)VD3, the reaction efficiency is improved and the reaction time is shortened, thus meeting the production demand of shortening the time cost in industrial production. Specifically:

[0027] (1) The yield of the mutated genetically engineered bacterium Bacillus subtilis WB600-pMA5-CYP109E1-H1 was much higher than that of the original strain WB600-pMA5-CYP109E1-H. The product concentration reached a maximum of 1.203 mg / L at 24 h. Compared with the original strain, the product concentration accumulation increased by 175.1% at 24 h of fermentation. During the fermentation process, the product concentration of the mutant strain WB600-pMA5-CYP109E1-H1 was also maintained at a high level.

[0028] (2) The wild-type strain and mutant strains WB600-pMA5-CYP109E1-H-Fdx-Fdr and WB600-pMA5-CYP109E1-H1-Fdx-Fdr co-expressing the redox chaperones Fdx and FdR all had higher yields than the Bacillus subtilis WB600-pMA5-CYP109E1-H that only heterologously expressed CYP109E1-H. At 36 hours, the product concentration of the co-expressed redox chaperone was 0.66 mg / L. After 36 hours, the product concentration was maintained at 0.60 mg / L. The addition of the redox chaperone has the advantage of improving the stability of product accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0030] Figure 1 The figure is a time course curve of the accumulation of 25(OH)VD3 products converted by the mutant recombinant bacteria obtained in Examples 5 and 6, wherein CYP109E1-H is VD3 hydroxylase, CYP109E1-H1 is a VD3 hydroxylase mutant, CYP109E1-H-Fdx-FdR is VD3 hydroxylase co-expressed with ferredoxin gene and ferredoxin reductase gene, and CYP109E1-H1-Fdx-FdR is VD3 hydroxylase mutant co-expressed with ferredoxin gene and ferredoxin reductase gene. DETAILED DESCRIPTION

[0031] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0032] The HPLC analysis method of the product is:

[0033] Chromatographic conditions: On an Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm), linear gradient elution was performed for 12 min using 50-100% acetonitrile-water as the mobile phase, followed by 100% acetonitrile elution for 13 min, followed by 50% acetonitrile-water column equilibration for 5 min. The single injection volume was 20 μL, the detection wavelength was 265 nm, the flow rate was 1.0 mL / min, and the column temperature was 40°C.

[0034] Example 1: Construction of VD3 Hydroxylase CYP109E1-H Strain and Acquisition of Genes of CYP Electron Transport Chain

[0035] A P450 enzyme gene sequence CYP109E1-H capable of hydroxylating vitamin D3 was amplified using the DNA sequence of Bacillus megaterium H-1 preserved in the laboratory as a template. The gene sequence of CYP109E1-H is shown in SEQ ID NO:1.

[0036] Table 1 Primer sequences

[0037]

[0038] The above primers were used to amplify the coding gene of CYP109E1-H using the genome of Bacillus megaterium H-1 as a template. The PCR reaction was carried out in a 25 μL system under the following conditions: 95°C pre-denaturation for 3 min before starting the cycle; 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, for a total of 34 cycles; 72°C final extension for 10 min. The PCR product was subjected to agarose gel electrophoresis and then cut and recovered, and ligated with T4 ligase overnight at 16°C. The ligation product was transformed into E. coli JM109 competent cells and cultured overnight on LB plates containing ampicillin resistance (50 mg / L). Positive transformants were screened, and plasmids were extracted after enrichment culture. The plasmid was named pMA5-CYP109E1-H. The successfully constructed recombinant expression plasmid was transformed into Bacillus subtilis WB600 by chemical transformation, spread on LB medium containing 50 mg / L kanamycin and cultured overnight, and positive transformants were picked to obtain heterologous expression CYP109E1-H recombinant Bacillus subtilis WB600-pMA5-CYP109E1-H.

[0039] Example 2: Construction and heterologous expression of VD3 hydroxylase mutants

[0040] (1) Crystal structure simulation of VD3 hydroxylase CYP109E1-H from Bacillus megaterium H-1

[0041] Using the reported CYP109E1 (PDB code: 5I91.2) as a template (the amino acid similarity between the two is 96.78%), the molecular simulation docking software Autodock vina was used to simulate the docking of substrate VD3 and CYP109E1-H, and the 293rd histidine of the enzyme was selected as the active site of the present invention and mutated to alanine.

[0042] (2) Construction of VD3 hydroxylase mutant strain

[0043] Table 2 Primer sequences

[0044]

[0045] Note: The underlined sequence represents the mutated amino acid site

[0046] Primers H293A-F and H293A-R were designed using the principle of one-step reverse PCR. The plasmid PMA5-CYP109E1-H constructed in Example 1 was used as a template for PCR. The histidine at position 293 of VD3 hydroxylase was replaced with alanine. The plasmid obtained after mutation was named pMA5-CYP109E1-H1. The PCR reaction conditions were: 95°C for 3min, 34 cycles (95°C for 30S, 58°C for 30S, 72°C for 1.5min), and 72°C for 10min. PCR amplification system: 1μL template, 1μL upstream and downstream primers, 20μL Prime StarMax (Premix) DNA, and 17μL ddH2O. The PCR product was purified and recovered using a gel recovery kit, and the concentration of the recovered product was tested by electrophoresis. It was transformed into E.coil JM109 competent cells, coated with ampicillin LB plates, and positive colonies were picked. After overnight culture at 37°C in a shaking incubator, plasmid pMA5-CYP109E1-H1 was extracted and then transferred into Bacillus subtilis WB600. PCR verification was performed to obtain a mutant recombinant strain with heterologous expression, which was named WB600-pMA5-CYP109E1-H1.

[0047] Example 3: Construction of a recombinant expression plasmid with enhanced catalytic efficiency

[0048] In the CYP reaction process, not only NAD(P)H is needed to provide electrons, but also certain enzymes in the electron transport chain are needed to transfer electrons. The ferredoxin protease and ferredoxin reductase genes in the electron transport chain were synthesized by full gene synthesis, and the sequences are shown in SEQ ID NO. 5 and 7. Among them, Fdx (ferredoxin) is ferredoxin, and FdR (ferredoxinreductase) is ferredoxin reductase.

[0049] (1) Construction of recombinant non-mutated plasmid pMA5-CYP109E1-H-Fdx-Fdr with enhanced catalytic efficiency

[0050] The expression plasmid used in the present invention is the Bacillus subtilis expression plasmid pMA5, the restriction site is NdeⅠ&BamHⅠ, vitamin D3 hydroxylase CYP109E1-H (amino acid sequence as shown in SEQ ID NO.2) and the synthesized Fdx and FdR genes are connected to the vector pMA5 by a fusion PCR method and then transformed into a host E. coli JM109 competent cell to obtain an E. coli engineering bacterium containing the CYP gene and the redox chaperones Fdx and FdR recombinant plasmid pMA5-CYP109E1-H-Fdx-Fdr.

[0051] (2) Construction of the recombinant mutant plasmid pMA5-CYP109E1-H1-Fdx-Fdr with enhanced catalytic efficiency

[0052] The expression plasmid used in the present invention is the Bacillus subtilis expression plasmid pMA5, the restriction site is NdeⅠ&BamHⅠ, vitamin D3 hydroxylase CYP109E1-H1 (amino acid sequence as shown in SEQ ID NO.4) and the synthesized Fdx, FdR genes are connected to the vector pMA5 by a fusion PCR method and then transformed into a host E. coli JM109 competent cell to obtain the E. coli engineering bacteria containing the recombinant plasmid pMA5-CYP109E1-H1-Fdx-Fdr containing the CYP mutant gene and the redox partners Fdx, FdR.

[0053] Example 4: Construction of recombinant engineering bacteria with enhanced catalytic efficiency

[0054] The Bacillus subtilis expression plasmids pMA5-CYP109E1-H-Fdx-Fdr and pMA5-CYP109E1-H1-Fdx-Fdr obtained in Example 3 were transformed into Bacillus subtilis WB600 competent cells, cultured overnight on LB plates containing Kana resistance (50 mg / L), screened for positive transformants WB600-pMA5-CYP109E1-H-Fdx-Fdr and WB600-pMA5-CYP109E1-H1-Fdx-Fdr, and picked the transformants to 10 mL LB liquid culture medium and cultured at 37° C. for 36 h.

[0055] Example 5: Fermentation verification of recombinant mutant engineered strains

[0056] The recombinant Bacillus subtilis WB600-pMA5-CYP109E1-H and WB600-pMA5-CYP109E1-H1 constructed in Example 1 and Example 2 were cultured in LB medium for 12 h, then transferred to fermentation medium TB, 0.25 g / L of VD3 was added for transformation, and samples were taken every 12 h for HPLC analysis.

[0057] The results are as follows Figure 1 As shown in the figure, the product concentration of WB600-pMA5-CYP109E1-H reached a peak of 0.687 mg / L at the 12th hour, and the product concentration reached 0.66 mg / L at the 24th hour; the yield of the mutated WB600-pMA5-CYP109E1-H1 was much higher than that of the starting strain WB600-pMA5-CYP109E1-H, and the product concentration reached 0.805 mg / L at the 12th hour, and its product concentration reached a maximum of 1.203 mg / L at 24 hours. Compared with the starting strain, the accumulated product concentration of the mutant strain WB600-pMA5-CYP109E1-H1 increased by 175.1% after 24 hours of fermentation, and during the fermentation process, the product concentration of the mutant strain WB600-pMA5-CYP109E1-H1 was also maintained at a high level.

[0058] Example 6: Fermentation verification of recombinant mutant engineered strains with enhanced catalytic efficiency

[0059] The recombinant Bacillus subtilis WB600-pMA5-CYP109E1-H-Fdx-Fdr and WB600-pMA5-CYP109E1-H1-Fdx-Fdr of Example 4 were cultured in LB medium for 12 h, then transferred to fermentation medium TB, 0.25 g / L of VD3 was added for transformation, and samples were taken every 12 h for HPLC analysis.

[0060] The results are as follows Figure 1As shown, the wild-type WB600-pMA5-CYP109E1-H-Fdx-Fdr and mutant strain WB600-pMA5-CYP109E1-H1-Fdx-Fdr co-expressing redox chaperones Fdx and FdR both have higher yields than the Bacillus subtilis WB600-pMA5-CYP109E1-H that only performs heterologous expression. At 36h, the product concentrations of the co-expressed wild-type and co-expressed mutants were both 0.66mg / L. After 36h, the product concentrations of both were also maintained at 0.60mg / L, indicating that the addition of the redox chaperone has the advantage of improving the stability of product accumulation.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A vitamin D3 C25 hydroxylase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

4.

2. A nucleotide encoding the C25 hydroxylase mutant according to claim 1.

3. A recombinant expression plasmid for expressing the C25 hydroxylase mutant of vitamin D3 according to claim 1.

4. The recombinant expression plasmid according to claim 3, characterized in that: The plasmid contains the nucleotide according to claim 2.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria is integrated with the nucleotide of the C25 hydroxylase mutant according to claim 2 or contains the recombinant expression plasmid according to claim 3.

6. The genetically engineered bacterium according to claim 5, characterized in that: The engineering bacteria uses pMA5 plasmid as an expression vector and Bacillus subtilis WB600 as an expression host.

7. The genetically engineered bacterium according to claim 5, characterized in that: The genetically engineered bacteria are integrated with a ferredoxin gene and a ferredoxin reductase gene, or a recombinant expression plasmid containing a ferredoxin gene and a ferredoxin reductase.

8. Use of the C25 hydroxylase mutant according to claim 1 in the preparation of 25-hydroxyvitamin D3.

9. Use of the genetically engineered bacteria according to any one of claims 5 to 7 in the preparation of 25-hydroxyvitamin D3.

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