Fusion P450 enzyme mutant, method for improving VD3 catalytic activity and method for generating 25 (OH) VD3 by whole cells

Abstract

The invention relates to the technical field of agricultural biology, in particular to a fusion P450 enzyme mutant, a method for improving VD3 catalytic activity and a method for generating 25 (OH) VD3 through whole cells. The invention constructs a fusion P450 enzyme VK1-CYP116B46-L21 and a self-sufficiency fusion P450 enzyme mutant with improved activity, wherein the self-sufficiency fusion P450 enzyme mutant is used for catalyzing vitamin D3 to synthesize calcifediol; vD3 is taken as a substrate, glucose, a cosolvent and a wet thallus induced by recombinant escherichia coli for simultaneously expressing fusion P450 enzyme and glucose dehydrogenase are added, and the highest yield of 25 (OH) VD3 within 24 hours after catalytic reaction can reach 3.26 g / L; whole cells are directly used as a catalyst, enzyme purification is not needed, glucose dehydrogenase is introduced, and glucose is used as a substrate to regenerate a cofactor NAD (P) H, so that addition of expensive cofactors is avoided, the production cost is reduced, and industrial production is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to a fusion P450 enzyme mutant, a method for improving the catalytic activity of VD3, and a method for producing 25(OH)VD3 by whole cell. BACKGROUND

[0002] Vitamin D3 (VD3), commonly known as cholecalciferol, plays a key role in many biological processes. Compared with VD3, 25-hydroxyvitamin D3 (25(OH)VD3) exhibits stronger water solubility and in vivo utilization efficiency.

[0003] Currently, the common methods for producing 25(OH)VD3 are chemical synthesis and biological transformation. Compared with the chemical synthesis route, the C25 hydroxylation enzyme catalysis using VD3 as the substrate only requires one reaction step, has high catalytic efficiency and excellent selectivity, and the reaction conditions are mild, which is less polluting to the environment. P450 is a member of the heme oxidase superfamily known for its special region and stereoselective transformation, and has great potential in the synthesis of 25(OH)VD3. However, the industrial application of P450 faces challenges such as the need for additional addition of redox partners, limited catalytic activity, low coupling efficiency, and insufficient stability, resulting in high production cost.

[0004] In the catalytic cycle of P450, the hydroxylation of the substrate depends on the efficiency of the transfer of electrons from the redox partner to the heme iron, and additional redox partner proteins are usually added to perform the function of electron transfer, resulting in an increase in the cost of P450 in industrial applications. In addition, the rate-limiting step in the P450 reaction is electron transfer, and when this process is not coordinated with the catalytic efficiency of the substrate, it can trigger uncoupling reactions, resulting in reduced P450 coupling efficiency, accumulation of reactive oxygen species, and decreased enzyme activity. Self-sufficient fusion P450 enzymes integrate heme catalysis and oxidoreductase domains on a single polypeptide chain, with the domains connected by a linker, which simplifies and facilitates electron transfer. Compared with multi-component P450, self-sufficient fusion P450 enzymes usually exhibit higher catalytic efficiency. Self-sufficient fusion P450 enzymes rely on their own conformational movements to achieve efficient inter-domain electron transfer, and the efficiency of electron transfer depends on the efficiency of the transfer of electrons from the reductase domain to the heme center, and the ability of the heme domain to utilize the transferred electrons for efficient catalysis. Utilizing a comprehensive strategy that simultaneously considers the electron transfer pathway and substrate specificity can effectively improve the efficiency of electron transfer. Therefore, developing a self-sufficient fusion P450 enzyme with C-25 hydroxylation activity of VD3 and improving its catalytic activity of VD3 by modifying the electron transfer pathway and substrate specificity has great industrial application potential. SUMMARY

[0005] The purpose of the present application is to provide a fusion P450 enzyme mutant.

[0006] Still another object of the present application is to provide a method for improving catalytic activity of a fusion P450 enzyme on VD3.

[0007] Still another object of the present application is to provide a method for producing 25-hydroxyvitamin D3 by a whole cell catalyst, thereby solving the problem of low yield of 25(OH)VD3 in the prior art due to low catalytic activity of a P450 enzyme on VD3, which makes it difficult to be used in industrial production.

[0008] The self-sufficient fusion P450 enzyme mutant with improved activity for catalyzing vitamin D3 to calcifediol according to the present application, the fusion P450 enzyme being P450 enzyme VK1-CYP116B46-L21, comprises a P450 enzyme Vdh-K1 heme catalytic domain derived from Pseudonocardia autotrophica and a redox domain of CYP116B46 of P450 enzyme derived from Tepidiphilus thermophiles, forming a wild-type fusion P450 enzyme VK1-CYP116B46-L21, the amino acid sequence of which is shown in SEQ ID NO: 1. Taking the wild-type P450 enzyme VK1-CYP116B46-L21 shown in SEQ ID NO: 1 as a template, a P450 enzyme mutant K180F is obtained by mutating the lysine K at position 180 to phenylalanine F, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0009] Further, the double-point mutant is A177M / K180F (SEQ ID NO: 3), P83A / K180F (SEQ ID NO: 4), K180F / F346K (SEQ ID NO: 5), and K180F / R354M (SEQ ID NO: 6).

[0010] Further, the triple-point mutant is P83A / A177M / K180F, the amino acid sequence of which is shown in SEQ ID NO: 7, P83A / K180F / F346K (SEQ ID NO: 8), P83A / K180F / R354M (SEQ ID NO: 9), A177M / K180F / F346K (SEQ ID NO: 10), A177M / K180F / R354M (SEQ ID NO: 11), and K180F / F346K / R354M (SEQ ID NO: 12).

[0011] Further, the four-point mutant is P83A / A177M / K180F / F346K (SEQ ID NO: 13), A177M / K180F / F346K / R354M (SEQ ID NO: 14), P83A / K180F / F346K / R354M (SEQ ID NO: 15), P83A / A177M / K180F / R354M (SEQ ID NO: 16).

[0012] Further, the five-point mutant is P83A / A177M / K180F / F346K / R354M, and the amino acid sequence is shown in SEQ ID NO: 17.

[0013] According to the specific embodiment of the present application, a co-expression plasmid comprising the fusion P450 enzyme mutant coding gene and the glucose dehydrogenase gene is provided.

[0014] The present application provides a recombinant cell comprising the co-expression plasmid of the fusion P450 enzyme or mutant and the glucose dehydrogenase coding gene.

[0015] In the embodiment of the present application, the recombinant cell is a recombinant Escherichia coli Escherichia coli BL21 (DE3).

[0016] The present application provides a whole cell catalyst containing the recombinant cell.

[0017] According to the method for catalytically preparing 25 (OH) VD3 of the present application, the method is to add the whole cell catalyst to a VD3 solution to catalytically prepare 25 (OH) VD3.

[0018] According to the method for catalytically preparing 25 (OH) VD3 of the present application, the steps of the method are as follows: The recombinant cell is cultured in LB medium at 37℃, and the OD 600 When the OD is 0.6-1.0, β-d-1-thiogalactopyranoside (IPTG) and 5-aminoacetylalanine salt (5-ALA) are added to a final concentration of 0.1-1.0 mM and 0.1-1.0 mM, respectively, and the induction is carried out at 16-37℃ for 5-20 h, and the wet bacteria are collected; the wet bacteria are inoculated into a VD3 solution, and the reaction is carried out at pH 6.5-8 and 20-40℃ for 2-24 h.

[0019] In some embodiments of the present application, the VD3 solution is prepared from VD3, purified water, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, a cosolvent, and a co-substrate.

[0020] In some embodiments of the present application, the cosolvent is hydroxypropyl-β-cyclodextrin and isopropyl alcohol / or ethanol.

[0021] In some embodiments of the present application, the initial concentration of the VD3 is 2-10 mM.

[0022] In some embodiments of the present application, the concentration of the hydroxypropyl-β-cyclodextrin is 20-100 g / L.

[0023] In some embodiments of the present application, the concentration of the isopropanol and / or ethanol is 5%-20% (v / v).

[0024] In some embodiments of the present application, the co-substrate is glucose.

[0025] In some embodiments of the present application, the concentration of the co-substrate is 25 g / L.

[0026] Advantages of the present application: The present application constructs a fusion P450 enzyme VK1-CYP116B46-L21, and constructs different mutants of the electron transfer path and the substrate binding pocket of VK1-CYP116B46-L21. After screening, a self-sufficient fusion P450 enzyme mutant with improved activity for catalyzing the synthesis of calcidiol from vitamin D3 is obtained. With VD3 as the substrate, glucose, a cosolvent, and the wet bacteria of recombinant E. coli expressing the fusion P450 enzyme and glucose dehydrogenase after induction are added, and the maximum yield of 25(OH)VD3 within 24 h after the catalytic reaction can reach 3.26 g / L. Directly using whole cells as catalysts without purifying enzymes, introducing glucose dehydrogenase, and using glucose as a substrate to regenerate co-factor NAD(P)H can avoid adding expensive co-factors and reduce production costs, which is conducive to industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The 25(OH)VD3 production capacity of wild-type VK1-CYP116B46-L21 and its mutants is shown. Figure 2 The 25(OH)VD3 yield in the whole-cell conversion reaction of substrate VD3 is shown. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0029] Test materials and reagents: 1. Strains and vectors: the expression host is E. coli Escherichia coliBL21(DE3), expression plasmid vector pETDuet1; 2. Enzymes: Endonuclease purchased from New England Biolabs, Inc., USA, Ligase; 3. Culture medium: LB medium (g / L): Tryptone 10 g, Yeast extract 5 g, Sodium chloride 10 g; TB medium (g / L): Tryptone 12 g, Yeast extract 24 g, Glycerol 5 g, Potassium phosphate monobasic 2.31 g, Potassium phosphate dibasic 16.37 g.

[0030] The primers used in the following examples are: VK1-F (SEQ ID NO: 18); VK1-CYP116B46-R (SEQ ID NO: 19); VK1-CYP116B46-F (SEQ ID NO: 20); CYP116B46-R (SEQ ID NO: 21); VK1-RBS-GDH-F (SEQ ID NO: 22); GDH-R (SEQ ID NO: 23); P83A-F (SEQ ID NO: 24); P83A-R (SEQ ID NO: 25); A177M-F (SEQ ID NO: 26); A177M-R (SEQ ID NO: 27); K180F-F (SEQ ID NO: 28); K180F-R (SEQ ID NO: 29); F346K-F (SEQ ID NO: 30); F346K-R (SEQ ID NO: 31); R354M-F (SEQ ID NO: 32); R354M-R (SEQ ID NO: 33); K180F / A177M-F (SEQ ID NO: 34); K180F / A177M-R (SEQ ID NO: 35).

[0031] The molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in the book "Molecular Cloning: A Laboratory Manual" (3rd edition) by J. Sambrook, or according to the instructions of the kit and product. Example 1: Preparation of recombinant strain BL21 (pETDuet1-VK1-CYP116B46-L21-GDH)

[0032] The plasmid pETDuet1 was double digested with restriction enzymes BamHI and Hind III; the primers VK1-F and VK1-CYP116B46-R with homologous ends were used to amplify the gene CYP116B46 from the autotrophic Pseudonocardia sp. (ATCC 31162) deposited in the American Type Culture Collection (ATCC) and the gene VK1 from the Escherichia coli BL21 (DE3) deposited in the China General Microbiological Culture Collection Center (CGMCC) (No. 2617). Pseudonocardia autotrophicaThe P450 enzyme Vdh-K1 gene sequence was obtained; primers VK1-CYP116B46-F and CYP116B46-R with homologous ends were used to amplify the gene from thermophilic bacteria ( ). Tepidiphilus thermophiles The gene sequence of the redox domain of the P450 enzyme CYP116B46 was obtained. The PCR product and enzyme digestion product were recovered and purified separately, and recombinant ligation was performed using a recombinase kit. The recombinant product was then transformed into *E. coli* Trans1-T1 competent cells and plated on LB agar (containing 100 μg / mL Ampicillin) for selection. After successful sequencing, the plasmid pETDuet1-VK1-CYP116B46-L21 was extracted.

[0033] The plasmid pETDuet1-VK1-CYP116B46-L21 was double-digested with restriction endonucleases Hind III and NotI. Primers with homologous ends VK1-RBS-GDH-F and GDH-R were used to amplify the plasmid derived from *Priscilla megaterium*. Priestia megaterium The glucose dehydrogenase GDH of (Gene ID: 64145183) was extracted. The PCR product and enzyme digestion product were separately recovered and purified, and recombinant ligation was performed using a recombinase kit. The recombinant product was then transformed into *E. coli* Trans1-T1 competent cells and plated on LB agar (containing 100 μg / mL Ampicillin) for selection. After successful sequencing, the plasmid pETDuet1-VK1-CYP116B46-L21-GDH was extracted and transformed into *E. coli* BL21(DE3) to obtain the recombinant strain BL21. Example 2: Construction and screening of mutants

[0034] Constructing mutants Three potential hotspots, P83, A177, and K180, located at the substrate-binding pocket entrance and potentially affecting substrate-enzyme binding, were identified, along with potential electron transport sites F346 and R354. Single-point saturation mutagenesis and iterative saturation mutagenesis were performed on these five amino acid residues to construct a mutant library. Based on VK1-CYP116B46-L21, mutation primers were designed according to the mutant, and the plasmid pETDuet1-VK1-CYP116B46-L21-GDH was used as a template for mutation. PCR products were digested with DpnI to remove the template. The digested PCR products were transformed into *E. coli* Tans1-T1 competent cells and sequenced for verification. Then, *E. coli* BL21(DE3) was transformed to obtain recombinant *E. coli* strain BL21. The names of each recombinant strain, the included plasmids, enzymes (wild-type WT or mutant), and amino acid sequences are shown below.

[0035] BL21 1, pETDuetl-VK1-CYP116B46-L21-GDH, WT, SEQ ID NO: 1; BL21 2, pETDuetl-VK1-CYP116B46-L21-P83A-GDH, P83A; BL21 3, pETDuetl-VK1-CYP116B46-L21-A177M-GDH, A177M; BL21 4, pETDuetl-VK1-CYP116B46-L21-K180F-GDH, K180F, SEQ ID NO: 2; BL21 5, pETDuetl-VK1-CYP116B46-L21-F346K-GDH, F346K; BL21 6, pETDuetl-VK1-CYP116B46-L21-R354M-GDH, R354M; BL21 7, pETDuetl-VK1-CYP116B46-L21-P83A-K180F-GDH, P83A / K180F, SEQ ID NO: 4; BL21 8, pETDuetl-VK1-CYP116B46-L21-A177M-K180F-GDH, A177M / K180F, SEQ ID NO: 3; BL21 9, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-GDH, K180F / F346K, SEQ ID NO: 5; BL21 10, pETDuetl-VK1-CYP116B46-L21-K180F-R354M-GDH, K180F / R354M, SEQ ID NO: 6; BL21 11, pETDuetl-VK1-CYP116B46-L21-P83A-A177M-GDH, P83A / A177M; BL21 12, pETDuetl-VK1-CYP116B46-L21-P83A-F346K-GDH, P83A / F346K; BL21 13, pETDuetl-VK1-CYP116B46-L21-P83A-R354M-GDH, P83A / R354M; BL2115, pETDuetl-VKl-CYP116B46-L21-A177M-R354M-GDH, A177M / R354M; BL2116, pETDuetl-VKl-CYP116B46-L21-F346K-R354M-GDH, F346K / R354M; BL2116, pETDuetl-VKl-CYP116B46-L21-F346K-R354M-GDH, F346K / R354M; BL2117, pETDuetl-VKl-CYP116B46-L21-P83A-A177M-K180F-GDH, P83A / A177M / K180F, SEQ ID NO: 7; BL2117, pETDuetl-VKl-CYP116B46-L21-P83A-A177M-K180F-GDH, P83A / A177M / K180F, SEQ ID NO: 7; BL2117, pETDuetl-VKl-CYP116B46-L21-P83A-A177M-K180F-GDH, P83A / A177M / K180F, SEQ ID NO: 7; BL2120, pETDuetl-VKl-CYP116B46-L21-P83A-K180F-F346K-GDH, P83A / K180F / F346K, SEQ ID NO: 8; BL2120, pETDuetl-VKl-CYP116B46-L21-P83A-K180F-F346K-GDH, P83A / K180F / F346K, SEQ ID NO: 8; BL2120, pETDuetl-VKl-CYP116B46-L21-P83A-K180F-F346K-GDH, P83A / K180F / F346K, SEQ ID NO: 8; BL2123, pETDuetl-VKl-CYP116B46-L21-A177M-K180F-F346K-GDH, A177M / K180F / F346K, SEQ ID NO: 10; BL2123, pETDuetl-VKl-CYP116B46-L21-A177M-K180F-F346K-GDH, A177M / K180F / F346K, SEQ ID NO: 10; BL2126, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH, K180F / F346K / R354M, SEQ ID NO: 12; BL2126, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH, K180F / F346K / R354M, SEQ ID NO: 12; BL2126, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH, K180F / F346K / R354M, SEQ ID NO: 12; BL2126, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH, K180F / F346K / R354M, SEQ ID NO: 12; BL2126, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH, K180F / F346K / R354M, SEQ ID NO: 12; BL2126, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH, K180F / F346K / R354M, SEQ ID NO: 12; BL2126, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH, K180F / F346K / R354M, SEQ ID NO: 12; BL2126, pETDuetl-VK1-CYP116B46-L21-K180F-F346K-R354M-GDH, K180F / F346K / R354M, SEQ ID NO: 12.

[0036] (2) Mutant screening: The 32 mutant strains obtained in step (1) were inoculated into LB medium, and cultured at 200 rpm and 37 °C for about 10 h. Then, the culture was inoculated into TB medium at a volume fraction of 1%, and cultured at 200 rpm and 37 °C until the OD 600=Approximately 0.8, add IPTG to a final concentration of 1.0 mM and 5-ALA of 1.0 mM, induce at 200 rpm and 25 ℃ for 18 h, centrifuge at 6000 rpm for 10 min to collect the bacterial cells, and then proceed with the substrate VD3. Whole-cell transformation reaction was used to screen for mutants with improved transformation efficiency.

[0037] Whole-cell transformation reaction system: VD3 substrate (final concentration 1 mM) and recombinant E. coli cells after induction (final cell concentration OD) were added to potassium phosphate buffer (50 mM, pH 7.4) supplemented with 50 g / L hydroxypropyl-β-cyclodextrin and 10% (v / v) ethanol. 600 = 10), and finally add glucose (final concentration 25 g / L). React at 25°C and 200 rpm for 1 h. After the reaction is complete, add an equal volume of ethyl acetate to terminate the reaction and extract. Vortex the mixture at high speed for 10 min, then centrifuge at high speed (12000 rpm, 10 min). Collect the upper organic phase, filter it through a 0.22 μm organic filter membrane, and perform HPLC analysis.

[0038] The concentrations of VD3 and 25(OH)VD3 in the reaction solution were analyzed by HPLC: an Agilent C18 column (5 μm × 4.6 mm × 250 mm) was used, with methanol / acetonitrile (30:70, v / v) as the mobile phase; the detection wavelength was set to 264 nm; the flow rate was maintained at 1.0 mL / min; the injection volume was 10 μL; the column temperature was 30 °C; and the detection time was 15 min. The catalytic activity of the constructed mutants was evaluated by the conversion of substrate VD3. The mutants with higher 25(OH)VD3 production capacity than the wild type were obtained, such as... Figure 1 As shown in the figure, the activity of all mutants is improved to varying degrees. Among them, the five-point mutant VK1-CYP116B46 L21P83A / A177M / K180F / F346K / R354M has the highest 25(OH)VD3 production, which is 8.8 times that of the wild type. Example 3: Gram-scale preparation of 25(OH)VD3

[0039] The recombinant strain constructed in Example 2 E.coli BL21 32 / pETDuet1-VK1-CYP116B46-L21-P83A-A177M-K180F-F346K-R354M-GDH was inoculated into LB medium and cultured at 200 rpm and 37 ℃ for about 10 h. Then, it was inoculated into TB medium at 1% of the medium volume and cultured at 200 rpm and 37 ℃ until OD... 600= 0.8, 1.0 mM IPTG and 1.0 mM 5-ALA were added, and the bacteria were induced at 200 rpm and 25°C for 18 h. The bacteria were collected by centrifugation at 6000 rpm for 10 min, and then subjected to whole-cell conversion reaction of the substrate VD3.

[0040] In the whole-cell conversion reaction, the VD3 concentration of the whole reaction system was 10 mM, the reaction time was 24 h, and the rest of the operations were the same as in Example 2. After the reaction was completed, an equal volume of ethyl acetate was added to terminate the reaction and extract it. After the mixture was shaken at high speed for 10 min, it was centrifuged in a high-speed centrifuge (12000 rpm, 10 min). The upper organic phase was collected, filtered through a 0.22 μm organic filter membrane, and then loaded onto an HPLC for detection. The yield was as shown in Table 1. VK1-CYP116B46-L21 P83A / A177M / K180F / F346K / R354M achieved 82% substrate conversion in 24 h, and the yield was 8.16 mM (about 3.26 g / L). Figure 2

[0041] The above description is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above-described embodiments of the present application can be variously changed, that is, any simple, equivalent change and modification made according to the content of the claims and the specification of the present application are within the scope of the present application. The present application is not described in detail, and is all conventional technical content.​

Claims

1. A fusion P450 enzyme mutant with enhanced activity catalyzing the synthesis of 25(OH)VD3 from vitamin D3, characterized in that, The amino acid sequence of the fusion P450 enzyme mutant is shown in any one of SEQ ID NO:2 to SEQ ID NO:

17.

2. A fusion of the P450 enzyme gene, characterized in that, The fusion P450 enzyme gene encodes a protein with an amino acid sequence as shown in any one of SEQ ID NO:2 to SEQ ID NO:

17.

3. The recombinant expression vector comprising the P450 enzyme gene fused as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector includes the fusion P450 enzyme gene and glucose dehydrogenase gene as described in claim 2.

5. The recombinant strain comprising the fusion of the P450 enzyme gene and glucose dehydrogenase gene as described in claim 2.

6. The application of the recombinant strain according to claim 5 as a whole-cell catalyst for the synthesis of vitamin D3 into 25(OH)VD3.

7. A method for improving the ability of fused P450 enzymes to catalyze the synthesis of 25(OH)VD3 from vitamin D3, characterized in that, The method includes the following steps: Perform one or more of the following mutations on the fusion P450 enzyme with the amino acid sequence shown in SEQ ID NO:1: P83A, A177M, K180F, F346K or R354M.

8. A method for catalytically preparing 25(OH)VD3, characterized in that, The method includes the following steps: A recombinant strain comprising the fused P450 enzyme gene and glucose dehydrogenase gene as described in claim 2 was added to a solution containing VD3 and glucose to catalyze the preparation of 25(OH)VD3.

9. The method for catalytic preparation of 25(OH)VD3 according to claim 8, characterized in that, After adding the recombinant strain, the reaction was carried out at pH 6.5–8 and 20–40 °C for 2–24 h.

10. The method for catalytic preparation of 25(OH)VD3 according to claim 8, characterized in that, The initial concentration of VD3 is 2-10 mM, and the concentration of glucose is 25 g / L.

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