C1, 25-bit dihydroxylase mutant of vitamin D3 as well as genetically engineered bacterium and application of C1, 25-bit dihydroxylase mutant

Abstract

The invention discloses a C1, 25-bit dihydroxylase mutant of vitamin D3 as well as a genetically engineered bacterium and application thereof, and belongs to the technical field of enzyme engineering. The preparation method comprises the following steps: mutating 81-site glutamic acid of vitamin D3 dihydroxylase of bacillus megatherium H-1 into alanine to obtain a vitamin D3 dihydroxylase mutant; pMA5 is used as an expression plasmid to realize heterologous expression of the vitamin D3 dihydroxylase mutant in the bacillus subtilis; when the engineering bacteria disclosed by the invention are applied to calcitriol synthesis, the mutant has the effects of increasing the concentration accumulation amount of a product and shortening the reaction time consumption, and the production requirements of shortening the time cost in industrial production are met.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a cytochrome P450 enzyme mutant and an engineering bacterium for dihydroxylation of C-1 and C-25 positions of vitamin D3 and their application, which can convert vitamin D3 into 1α,25(OH)2VD3. Background Art

[0002] Vitamin D3 (VD3) is a fat-soluble vitamin with a cyclopentane polyhydrophenanthrene structure. It belongs to a steroid hormone and is a precursor substance regulated by calcium and phosphorus. In the human body, 7-dehydrocholesterol in the skin is used as a precursor. After irradiation with ultraviolet wavelengths, 7-dehydrocholesterol C9 position opens to form VD3. People have found that VD3 itself has no physiological function. After being metabolized in the human body, it can be converted into its active form. There are two main active forms: 25(OH)VD3 and 1α,25(OH)2VD3. Among them, 1α,25(OH)2VD3 (calcitriol) is its main active form, and its activity is more than 500 times higher than that of 25(OH)VD3. Calcitriol, also known as 1,25-hydroxyvitamin D3 (1α,25(OH)2VD3), is a biologically active metabolite of vitamin D3. It can be absorbed in the human body without conversion by the liver and kidneys. It can promote calcium absorption in the human body faster and more effectively, help the body strengthen bone strength, and thus improve and prevent some diseases.

[0003] VD3 is related to calcium absorption and bone health. Vitamin D deficiency increases the risk of osteomalacia, so the demand for active VD3 is increasing. The method for industrial synthesis of VD3 hydroxylated derivatives is usually chemical synthesis. This process requires the introduction of hydroxyl groups at C1 and C25 positions in a regioselective and stereoselective manner. The reaction is difficult, the process is complicated, and there are many by-products, difficult separation and purification, and large environmental pollution, which greatly limits the industrial production of calcitriol. Due to the various negative limitations of chemical synthesis, the development of green and environmentally friendly synthesis methods has become a new research trend in recent years. Compared with chemical synthesis, microbial methods have the advantages of high regioselectivity and stereoselectivity, mild action, simple process, low cost, and few by-products, and have great potential for industrial application. In recent years, the production of calcitriol by microbial transformation using VD3 as a substrate has been widely studied by many scholars. Summary of the invention

[0004] Purpose of the invention: The present invention provides an engineered strain of a VD3 dihydroxylase mutant, provides the gene of the mutant, a recombinant plasmid containing the mutant gene, and a host cell containing the recombinant plasmid, thereby achieving enhanced expression of VD3 dihydroxylase, and the corresponding mutant is heterologously expressed, which can effectively convert VD3 into 1α,25(OH)2VD3.

[0005] In one aspect, the present invention provides a vitamin D3 C1,25 dihydroxylase mutant, whose amino acid sequence is shown in SEQ ID NO: 4. In an optional embodiment, the mutant is obtained by replacing the 81st glutamic acid of CYP109A2-H of the parent Bacillus megaterium H-1 with alanine as shown in the amino acid sequence of SEQ ID NO: 2. In some embodiments, the encoding gene of the vitamin D3 C1,25 dihydroxylase mutant is shown in SEQ ID NO: 3.

[0006] The inventors identified a C1,25-position dihydroxylase CYP109A2-H from Bacillus megaterium H-1, which can selectively catalyze the substrate vitamin D3 to generate the product 1α,25(OH)2VD3. This process is mainly mediated by the cytochrome P450 enzyme system. However, the expression level of the P450 enzyme system of Bacillus megaterium H-1 is very low, which greatly limits the production of 1α,25(OH)2VD3. Therefore, it is of great industrial significance to combine genomics, bioinformatics analysis and metabolic engineering methods to explore and identify more new soluble expressed enzyme genes and transform them, and screen out P450 enzymes that can efficiently convert substrates.

[0007] In one aspect, the present invention provides a gene encoding a C1,25-dihydroxylase mutant of vitamin D3. In some specific embodiments, the nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0008] In one aspect, the present invention provides a recombinant expression vector comprising the vitamin D3 C1,25-position dihydroxylase mutant gene. In some embodiments, the vitamin D3 C1,25-position dihydroxylase mutant gene is inserted into an original vector using a genetic engineering method to obtain a recombinant expression vector. In some specific embodiments, the original vector is pMA5.

[0009] In one aspect, the present invention provides an engineered strain that efficiently expresses vitamin D3 dihydroxylase, wherein the engineered strain is integrated with the gene of the vitamin D3 dihydroxylase mutant, or contains the recombinant expression vector. In some embodiments, the host bacteria is Bacillus subtilis. In some more specific embodiments, the host bacteria is Bacillus subtilis WB600.

[0010] In one aspect, the present invention provides the use of the engineered strain in the preparation of 1α,25(OH)2VD3. The structure of 1α,25(OH)2VD3 is shown in the following formula:

[0011]

[0012] In some embodiments, the application includes: using the engineered strain to catalyze a substrate vitamin D3. In some embodiments, the concentration of the substrate vitamin D3 is 0.2-1 g / L, the catalytic reaction temperature is 33-37° C., and the reaction time is 24-48 h.

[0013] Beneficial effects of the present invention: The Bacillus subtilis CYP109A2-H mutant and engineered bacteria and their applications provided by the present invention heterologously express the obtained mutant in Bacillus subtilis. In addition, when the engineered bacteria constructed by the present invention are applied to the synthesis of 1α,25(OH)2VD3, the reaction efficiency is improved and the reaction time is shortened, thus meeting the production demand of shortening the time cost in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a time course curve of the accumulation of 1α,25(OH)2VD3 products converted by the mutant recombinant bacteria obtained in Example 5, wherein CYP109A2-H is VD3 hydroxylase and CYP109A2-H1 is a VD3 hydroxylase mutant. DETAILED DESCRIPTION

[0015] The HPLC analysis method of the product is:

[0016] 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.

[0017] Example 1: Mining and amplification of VD3 dihydroxylase CYP109A2-H gene in Bacillus megaterium H-1

[0018] Transcriptome sequencing technology was used to screen the cytochrome P450 enzyme (CYPs) genes of Bacillus megaterium H-1 (this strain is recorded in the paper "Mining and Functional Characterization of Key Enzymes for C25 Hydroxylating Vitamin D3"; deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No.20362), and its family was analyzed through the NCBI database. A CYP was confirmed to be highly consistent with the known VD3 hydroxylase CYP109A2 gene sequence, and it was named CYP109A2-H. The gene sequence is shown in SEQ ID NO:1, and the complete target gene-derived CYP109A2-H amino acid sequence is SEQ ID NO:2.

[0019] Example 2: Construction and heterologous expression of VD3 dihydroxylase CYP109A2-H strain

[0020] The expression plasmid used in this study was the Bacillus subtilis expression plasmid pMA5, and the host bacteria was Bacillus subtilis WB600. The PCR product of CYP109A2-H was subjected to agarose gel electrophoresis, and the gel was cut and recovered, and then double-digested with NdeI and BamHI. The vector pMA5 was double-digested with NdeI and BamHI and then recovered. The digested fragments and the vector were connected by T4 ligase at 16°C overnight, the ligation product was transformed into E. coli JM109 competent cells, cultured overnight on an LB plate containing ampicillin resistance (50 mg / L), positive transformants were screened, and the plasmid was extracted after enrichment culture, named pMA5-CYP109A2-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 the positive transformants were picked to obtain heterologous expression recombinant Bacillus subtilis WB600-pMA5-CYP109A2-H.

[0021] Example 3: Screening of mutation sites of VD3 dihydroxylase CYP109A2-H

[0022] CYP109A2-H was modeled using the reported CYP109A2 (PDB code: 8ABS.1) as a template (the amino acid similarity between the two is 97.02%). The molecular simulation docking software AutodockVina was used to simulate the docking of the substrate VD3 and CYP109A2-H, and the amino acid sites located in the active pocket were screened. The 81st glutamate of the enzyme was confirmed as the active site of this study through alanine scanning, and it was mutated to alanine.

[0023] Example 4: Construction and heterologous expression of VD3 hydroxylase mutants

[0024] Primers E81A-F and E81A-R were designed, and the constructed pMA5-CYP109A2-H was used as a template to replace the 81st glutamic acid of VD3 dihydroxylase with alanine by one-step inverse PCR. The PCR reaction conditions were: 95℃3min, 34 cycles (95℃30s, 58℃30s, 72℃3min), 72℃10min. PCR amplification system: 2μL template, 2μL upstream and downstream primers, 20μL Prime Star Max (Premix) DNA, 14μL 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, the plasmid was extracted and then transferred into Bacillus subtilis WB600 to obtain the mutant recombinant strain WB600-pMA5-CYP109A2-H1.

[0025] Table 1 Primer sequences

[0026]

[0027] Note: The underlined sequence represents the mutated amino acid site

[0028] Example 5: Fermentation verification of recombinant mutant engineered strains

[0029] The recombinant Bacillus subtilis WB600-pMA5-CYP109A2-H and WB600-pMA5-CYP109A2-H1 were cultured in LB medium for 12 hours, then transferred to fermentation medium TB, added with 0.25 g / L VD3 and transformed at 37°C. Samples were taken every 12 hours for HPLC analysis. The results are shown in Figure 1 As shown, the product concentration of CYP109A2-H reached a peak of 0.71 mg / L at 32 hours; the yield of CYP109A2-H1 after mutation was much higher than that of the starting strain CYP109A2-H, and the product concentration reached a maximum of 1.72 mg / L at 24 hours. Compared with the starting strain, the product concentration accumulation increased by 142.3%.

[0030] The dual hydroxylase mutant of the present invention can catalyze VD3 to directly generate high-value substance calcitriol, which is of great significance for the microbial production of calcitriol. The output of calcitriol generated by the mutant catalyzed by the mutant is 1.72 mg / L, which is 142.3% higher than that of the wild type.

[0031] SEQ ID NO:1CYP109A2-H

[0032]

[0033]

[0034] SEQ ID NO:2 CYP109A2-H amino acid sequence

[0035] SEQ ID NO:3 CYP109A2-H1 mutant

[0036]

[0037] SEQ ID NO:4 CYP109A2-H1 mutant amino acid sequence

[0038]

Claims

1. A vitamin D3 C1,25 dihydroxylase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

4.

2. The vitamin D3 C1,25 dihydroxylase mutant according to claim 1, characterized in that: The encoding gene is shown in SEQ ID NO:

3.

3. A gene encoding the C1,25 dihydroxylase mutant of vitamin D3 according to claim 1.

4. The vitamin D3 C1,25 dihydroxylase mutant gene according to claim 3, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO:

3.

5. A recombinant expression vector, characterized in that: It comprises the vitamin D3 C1,25 dihydroxylase mutant gene as described in claim 3 or 4.

6. The recombinant expression vector according to claim 5, characterized in that The original vector was pMA5.

7. An engineered strain that efficiently expresses vitamin D3 dihydroxylase, characterized in that: The engineered strain is integrated with the gene of the vitamin D3 dihydroxylase mutant described in claim 3 or 4, or contains the recombinant expression vector described in claim 5 or 6.

8. Use of the engineered strain according to claim 7 in the preparation of 1α,25(OH)2VD3.

9. The use according to claim 8, characterized in that: The application includes: using the engineered strain to catalyze substrate vitamin D3.

10. The use according to claim 9, characterized in that: The concentration of substrate vitamin D3 is 0.2-1 g / L, the catalytic reaction temperature is 33-37° C., and the reaction time is 24-48 h.

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