A c1,25 position double-hydroxylase mutant of vitamin d3 and a genetically engineered bacterium thereof and application

Abstract

The application discloses a C1, 25-position double-hydroxylase mutant of vitamin D3 and a genetically engineered bacterium and application thereof, and belongs to the technical field of enzyme engineering. In the application, the glutamic acid at position 81 of vitamin D3 double-hydroxylase of Bacillus megaterium H-1 is mutated into alanine to obtain a vitamin D3 double-hydroxylase mutant; pMA5 is used as an expression plasmid to realize heterologous expression of the vitamin D3 double-hydroxylase mutant in Bacillus subtilis; and the application of the engineered bacterium of the application to the synthesis of calcitriol has the effects of improving the product concentration accumulation amount and shortening the reaction time consumption, and meets the production demand of shortening the time cost in industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a cytochrome P450 enzyme mutant for C-1 and C-25 double hydroxylation of vitamin D3 and an engineering bacterium and application, which can convert vitamin D3 into 1α, 25(OH)2VD3. BACKGROUND

[0002] Vitamin D3 (VD3) is a fat-soluble vitamin, has a cyclopentane polycyclic hydrophenanthrene structure, belongs to a steroid hormone, and is a precursor substance for calcium and phosphorus regulation. In the human body, 7-dehydrocholesterol in the skin is a precursor, is irradiated by ultraviolet wavelength, and is opened at a C9 position to form VD3. It is found that VD3 itself does not have physiological functions, can be converted into an active form after being metabolized in the human body, and the active form mainly has two kinds: 25(OH)VD3 and 1α, 25(OH)2VD3. Among them, 1α, 25(OH)2VD3 (calcitriol) is the main active form, and the 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 metabolite of vitamin D3 with biological activity, can be absorbed without liver and kidney conversion in the human body, can promote the absorption of calcium in the human body more quickly and effectively, helps to enhance the bone strength of the human body, and thus improves and prevents some diseases.

[0003] VD3 is related to calcium absorption and bone health, and the risk of suffering from chondropathy increases due to lack of vitamin D, so the demand for active VD3 of human beings is increasing. The method for industrial synthesis of hydroxylated derivatives of VD3 is usually a chemical synthesis method, which needs to introduce hydroxyl groups at C1 and C25 positions with region selectivity and stereoselectivity, has high reaction difficulty, a complex process, and many disadvantages such as many by-products, difficult separation and purification, and great environmental pollution, which greatly limits the industrial production of calcitriol. Due to the multiple negative limitations of the chemical synthesis method, developing a green and environmentally friendly synthesis method has become a new research trend in recent years. Compared with the chemical synthesis method, the microbial method has advantages such as high region and stereoselectivity, mild action, simple process, low cost, and few by-products, and has great industrial application potential. In recent years, the production of calcitriol by microbial transformation method taking VD3 as a substrate has been widely studied by many scholars. SUMMARY

[0004] The present application aims to provide an engineering strain of a VD3 double-hydroxylase mutant, provide a gene of the mutant, a recombinant plasmid containing the mutant gene, and a host cell containing the recombinant plasmid, realize the enhanced expression of the VD3 double-hydroxylase, and correspondingly realize the heterologous expression of the mutant, which can effectively convert VD3 into 1α, 25(OH)2VD3.

[0005] In one aspect, the present application provides a C1,25-hydroxylase mutant of vitamin D3, which has an amino acid sequence as shown in SEQ ID NO: 4. In one alternative embodiment, the mutant is obtained by replacing the glutamic acid at position 81 of CYP109A2-H of parent Bacillus megaterium H-1 having an amino acid sequence as shown in SEQ ID NO: 2 with alanine. In some embodiments, the gene encoding the C1,25-hydroxylase mutant of vitamin D3 has a nucleotide sequence as shown in SEQ ID NO: 3.

[0006] The inventors identified a C1,25-hydroxylase CYP109A2-H from Bacillus megaterium H-1, which can selectively catalyze the substrate vitamin D3 to generate the product 1α,25(OH)2VD3. The process is mainly mediated by cytochrome P450 enzyme system. However, the expression level of 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 mine and identify more new soluble enzyme genes and to screen P450 enzymes capable of efficiently converting substrates.

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

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

[0009] In one aspect, the present application provides an engineered strain for efficiently expressing vitamin D3 hydroxylase, which integrates the gene of the C1,25-hydroxylase mutant of vitamin D3 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 application provides the use of the engineered strain in the preparation of 1α,25(OH)2VD3. The structure of 1α,25(OH)2VD3 is as shown in the following formula:

[0011]

[0012] In some embodiments, the application comprises: using the engineered strain to catalyze the 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℃, and the time is 24-48 h.

[0013] The Bacillus subtilis CYP109A2-H mutant and the engineered bacteria provided by the application can be heterologously expressed in Bacillus subtilis. In addition, when the engineered bacteria constructed by the application are applied to the synthesis of 1α, 25(OH)2VD3, the reaction efficiency is improved and the reaction time is shortened, which meets the production demand of shortening the time cost in industrial production. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0016] Chromatographic conditions: linear gradient elution for 12 min on an Agilent TC-C18 chromatographic column (250 mm x 4.6 mm, 5 μm) with 50-100% acetonitrile-water as the mobile phase, followed by 100% acetonitrile elution for 13 min, and then column equilibration with 50% acetonitrile-water for 5 min. The single injection amount is 20 μL, the detection wavelength is 265 nm, the flow rate is 1.0 mL / min, and the column temperature is 40℃.

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

[0018] The cytochrome P450 enzyme (CYPs) gene of Bacillus megaterium H-1 (this strain is recorded in the paper "C25 hydroxylated vitamin D3 key enzyme mining and functional characterization"; preserved in China General Microbiological Culture Collection Center, the preservation number is: CGMCC No. 20362) was screened by transcriptome sequencing technology, and its family was analyzed by NCBI database. It is confirmed that a CYP is highly consistent with the known VD3 hydroxylase CYP109A2 gene sequence, which is named CYP109A2-H, and the gene sequence is shown as SEQ ID NO: 1, and the complete CYP109A2-H amino acid sequence deduced from the target gene is SEQ ID NO: 2.

[0019] Example 2: VD3 double-hydroxylase CYP109A2-H strain construction and heterologous expression

[0020] The expression plasmid used in this study is Bacillus subtilis expression plasmid pMA5, and the host strain is Bacillus subtilis WB600. The PCR product of CYP109A2-H was subjected to agarose gel electrophoresis, and the gel was recovered and subjected to NdeI and BamHI double digestion. The vector pMA5 was subjected to NdeI and BamHI double digestion and recovered. The digested fragments and the vector were ligated by T4 ligase at 16°C overnight, and the ligation product was transformed into E. coli JM109 competent cells, which were cultured overnight on LB plates containing ampicillin (50 mg / L). Positive transformants were selected and enriched for plasmid extraction, named pMA5-CYP109A2-H. The successfully constructed recombinant expression plasmid was transformed into Bacillus subtilis WB600 by chemical transformation method, and was plated on LB medium containing 50 mg / L kanamycin and cultured overnight. Positive transformants were selected to obtain heterologous expression recombinant Bacillus subtilis WB600-pMA5-CYP109A2-H.

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

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

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

[0024] The primer E81A-F, E81A-R is designed to replace the glutamic acid at the 81st position of the VD3 double-hydroxylase with alanine by one-step reverse PCR technology using the pMA5-CYP109A2-H already constructed as a template. The PCR reaction conditions are as follows: 95°C for 3 min, 34 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 3 min, and 72°C for 10 min. The PCR amplification system is as follows: 2 μL of template, 2 μL of each of the upper and lower primers, 20 μL of Prime Star Max (Preamix) DNA, and 14 μL of ddH2O. The PCR product is purified and recovered by using a gel recovery kit, and the concentration of the recovered product is tested by electrophoresis. The product is transformed into E. coli JM109 competent cells, spread on ampicillin LB plates, and positive colonies are picked. After overnight culture at 37°C on a shaker, the plasmid is extracted, and then transferred into B. 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 mutant amino acid site

[0028] Example 5: Fermentation verification of the recombinant mutant engineering strain

[0029] The recombinant B. subtilis WB600-pMA5-CYP109A2-H and WB600-pMA5-CYP109A2-H1 are respectively cultured in LB medium for 12 h, then transferred into fermentation medium TB, 0.25 g / L of VD3 is added, and the transformation is carried out at 37°C. Sampling is performed every 12 h for HPLC analysis. The results are shown in Table 2. Figure 1 As shown in Table 2, the product concentration of CYP109A2-H reaches a peak of 0.71 mg / L at the 32nd hour; the yield of the mutant CYP109A2-H1 is much higher than that of the starting strain CYP109A2-H, and the product concentration reaches a maximum of 1.72 mg / L at the 24th hour, which is increased by 142.3% compared with the starting strain.

[0030] The double-hydroxylase mutant of the application can catalyze VD3 to directly generate the high-value substance calcitriol, which is of great significance for the microbial production of calcitriol. The yield of the mutant catalyzing the generation of calcitriol is 1.72 mg / L, which is increased by 142.3% compared with the wild type.

[0031] SEQ ID NO: 1 CYP109A2-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. A gene encoding the vitamin D3 C1,25 dihydroxylase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: It comprises the gene according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The original vector was pMA5.

5. An engineered strain that efficiently expresses vitamin D3 dihydroxylase, characterized in that: The engineered strain is integrated with the gene according to claim 2, or contains the recombinant expression vector according to claim 3 or 4.

6. Use of the engineered strain according to claim 5 in the preparation of 1α,25(OH)2VD3.

7. The use according to claim 6, characterized in that The application includes: using the engineered strain to catalyze substrate vitamin D3.

8. The use according to claim 7, characterized in that The concentration of substrate vitamin D3 is 0.2~1 g / L, the catalytic reaction temperature is 33~37℃, and the reaction time is 24~48h.

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