Modification of 7beta hydroxysteroid dehydrogenase and application of 7beta hydroxysteroid dehydrogenase in synthesis of ursodesoxycholic acid

Abstract

The invention discloses a modified 7beta-hydroxysteroid dehydrogenase (7beta-HSDH), which has higher activity and stability in the synthesis process of ursodesoxycholic acid (UDCA), and takes NAD (P) H as a coenzyme. According to the 7beta-hydroxysteroid dehydrogenase mutant, the V104 site in the 7beta-hydroxysteroid dehydrogenase is subjected to saturated mutation modification, and the mutant with enhanced activity and changed coenzyme specificity is screened out. The mutant enzyme can keep high catalytic efficiency under the condition of taking NADH as a coenzyme, so that the synthesis yield of UDCA is remarkably increased, and the production cost is reduced. The enzyme mutant disclosed by the invention has a wide application prospect in industrial production, is particularly suitable for the production process of large-scale biosynthesis of UDCA, can effectively reduce the production cost, improve the product yield and reduce the environmental pollution and the production cost, and provides a technical support for wide application of UDCA.

Description

Technical Field

[0001] The present invention relates to the modification and application of ursodeoxycholic acid (UDCA) synthase, and belongs to the field of biotechnology. Background Art

[0002] Ursodeoxycholic acid (UDCA), an endogenous bile acid with important pharmacological activities, demonstrates unique application value in both traditional Chinese medicine and modern clinical treatment. As the primary component of bile, bile acids and their conjugates play an essential physiological role in human fat metabolism, making them an important target for the treatment of various metabolic diseases. Numerous clinical studies have demonstrated significant therapeutic effects of UDCA in dissolving gallstones, improving adverse reactions after liver transplantation, treating reflux gastritis, alleviating biliary pancreatitis, intervening in primary biliary cirrhosis, and preventing and treating drug-induced hepatitis. These clinical applications fully demonstrate the clinical potential of UDCA as a multifunctional therapeutic agent.

[0003] The current state of UDCA production relies primarily on chemical synthesis and biotransformation. Although chemical synthesis has matured over the years, its inherent complexity, operational hazards, and inevitable byproduct formation severely limit production efficiency and product quality. These bottlenecks not only result in low raw material utilization but also in significant resource waste. In contrast, enzyme-catalyzed biotransformation offers unique advantages due to its mild reaction conditions, high selectivity, and environmental friendliness. Among these, the biotransformation route using chenodeoxycholic acid (CDCA) as a substrate has attracted considerable research attention due to its broad availability and high conversion efficiency. CDCA, a diastereomer of UDCA, is abundant in poultry slaughter byproducts such as chickens, ducks, and geese. CDCA can be efficiently converted to the target product, UDCA, by specifically altering the spatial conformation of the C-7 hydroxyl group. This conversion process relies primarily on the synergistic catalysis of 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH). In the field of UDCA biosynthesis research, 7α-HSDH is relatively well-studied and has relatively mature industrial applications, while systematic research on 7β-HSDH lags behind, limiting the optimization and upgrading of UDCA biosynthesis processes. Precisely regulating the catalytic activity and stability of 7β-HSDH and modifying its coenzyme specificity through semi-rational design can not only improve UDCA production efficiency, but also effectively reduce pharmaceutical costs and improve drug quality. This is of great significance for the industrial feasibility of UDCA-based drugs. Therefore, modifying 7β-HSDH to improve its catalytic activity and stability and modify its coenzyme dependency could provide a feasible solution for large-scale, low-cost production of UDCA, with broad application prospects. Summary of the Invention

[0004] To address the low yield of UCDA biosynthesis, the present invention established an efficient high-throughput screening method for 7β-HSDH. Using 7β-HSDH from Roseococcus sp. SDR as a motherboard, 7β-HSDH with higher catalytic efficiency and stability was obtained through enzyme engineering technology, thereby improving the synthesis efficiency and production yield of UDCA.

[0005] The technical solutions adopted in the present invention are as follows:

[0006] The present invention uses 7β-HSDH from Roseococcus sp. SDR as a motherboard, evaluates the influence of non-conserved amino acid residues near the active site of the 7β-HSDH enzyme on the catalytic activity of the 7β-HSDH enzyme through alanine scanning technology, determines the key sites that affect the activity and stability of the 7β-HSDH enzyme, and performs saturation mutagenesis on the key sites to screen out mutants with better activity and stability. The mutants can maintain high catalytic efficiency under conditions where NADH is used as a coenzyme, thereby significantly improving the synthesis yield of 7β-HSDH and reducing production costs.

[0007] The amino acid sequence of the 7β-HSDH enzyme is shown in SEQ ID No. 1.

[0008] The 7β-HSDH mutant of the present invention is composed of any of the following amino acid sequences:

[0009] (1) The valine at position 104 of the amino acid sequence shown in SEQ ID NO.1 is mutated to proline or tyrosine, and the sequence thereof is shown in SEQ ID NO.2 and SEQ ID NO.3;

[0010] (2) Leucine at position 45 of the amino acid sequence shown in SEQ ID NO.1 is mutated to isoleucine, the sequence of which is shown in SEQ ID NO.4;

[0011] (3) Aspartic acid at position 46 of the amino acid sequence shown in SEQ ID NO.1 is mutated to lysine, and its sequence is shown in SEQ ID NO.5;

[0012] (4) The amino acid sequence shown in SEQ ID NO.1 has leucine at position 45 mutated to isoleucine, and aspartic acid at position 46 mutated to lysine. The sequence is shown in SEQ ID NO.6;

[0013] (5) The amino acid sequence shown in SEQ ID NO.1 has asparagine at position 98 mutated to tyrosine, and valine at position 258 mutated to phenylalanine. The sequence is shown in SEQ ID NO.7;

[0014] The present invention provides the coding gene of the 7β-HSDH mutant, and the coding gene can be obtained by cloning, artificial full-sequence synthesis or genetic engineering to transform the parent.

[0015] The present invention also provides a recombinant plasmid vector carrying the mutant. The recombinant expression vector can be constructed by conventional means by ligating a commercially available empty vector to ensure normal replication and expression of the corresponding mutant in the host. Preferably, the recombinant expression vector is pET28a(+).

[0016] The present invention also provides a recombinant microorganism comprising the above-mentioned recombinant plasmid vector. The recombinant microorganism is a conventional host cell in the art, capable of stably replicating and propagating the recombinant plasmid vector, and capable of effectively expressing the encoded mutant. Preferably, the recombinant microorganism is Escherichia coli BL21 (DE3).

[0017] The present invention provides a method for real-time detection of 7β-HSDH mutant activity, enabling rapid and efficient high-throughput screening on a microplate reader. The activity detection method can be performed in a 96-well microplate format. Real-time detection can be performed by mixing crude or pure 7β-HSDH enzyme solution (powder) after cell lysis and centrifugation with a substrate mixture.

[0018] The crude / pure enzyme solution (powder) can be obtained by conventional methods and conditions in the art. The preferred culture medium is LB medium, including 5g / L yeast extract, 10g / L tryptone, and 10g / L sodium chloride. The preferred culture conditions are as follows: the recombinant microorganism is inoculated into LB medium containing kanamycin and incubated at 37°C and 220rpm for 6-10h; then, the inoculation amount is transferred to a 500 / 1000mL conical flask (20% volume of LB medium, containing the corresponding antibiotic) at 1-2% (v / v), and incubated at 37°C and 220rpm for 2-5h; after the OD600 value of the bacterial solution reaches 0.6-0.8, IPTG is added at a final concentration of 0.2mM and cultured overnight on a shaker at 16°C and 200rpm to induce the Escherichia coli engineered bacteria to fully express the product. The induced bacterial solution is collected using a low-temperature centrifuge, washed twice with PBS buffer, and finally resuspended with PBS buffer with 1 / 10 volume of the culture solution. Resuspended bacteria can be used to break the cell wall to release 7β-HSDH crude enzyme solution, or to obtain 7β-HSDH crude enzyme powder by vacuum freeze-drying, or to obtain the corresponding pure enzyme solution / powder by protein tag purification.

[0019] The substrate mixture preferably comprises: 0.01-1 mM NADH, 0.01-2 mM 7-OXO-LCA (7-oxo-lithocholic acid), and 10-100 mM PBS buffer (pH 7.4). The preferred reaction temperature is 30°C, and the reaction time is 1-60 min. 7β-HSDH7-OXO-LCA is reduced to form UDCA, while NADH is oxidized to NAD+. NADH has a maximum absorption peak at 340 nm, so the reaction rate can be reflected by measuring the change in absorbance at 340 nm.

[0020] The present invention provides a method for detecting the stability of 7β-HSDH mutants. Pure enzyme solutions with the same protein concentration but heat-treated for different times are mixed with the above-mentioned substrate mixture and then tested. The preferred reaction conditions are the same as above.

[0021] Beneficial effects of the present invention:

[0022] This invention utilizes enzyme engineering to modify 7β-hydroxysteroid dehydrogenase, significantly enhancing its catalytic activity and stability. The V104P variant demonstrates a 1.16-fold increase in UDCA synthesis compared to the wild-type, while also significantly improving its stability. These improvements increase production efficiency, shorten production cycles, and reduce costs, providing an economical and feasible solution for the industrialized production of UCDA, promising broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :SDS-PAGE images of 7β-HSDH wild type and several dominant mutants

[0024] Figure 2 : NADH standard curve

[0025] Figure 3 :Activity determination of 7β-HSDH wild type and several dominant mutants

[0026] Figure 4 :Stability determination of 7β-HSDH wild type and several dominant mutants

[0027] Figure 5 :Structural analysis of mutants DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, and the scope of protection of the present invention is not limited to these embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] In the following examples, unless otherwise specified, all methods are conventional.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] Example 1: Semi-rational design of 7β-HSDH

[0032] The 7β-HSDH described in the present invention is derived from Roseococcus sp. SDR (WP_213611802.1). The amino acid sequence of 7β-HSDH and its related homologous sequence data were obtained from the NCBI database. The ConSurf online tool (ConSurf|Evolutionary conservation profiles of proteins) was used for multiple sequence alignment, and 7β-HSDH homologous protein sequences from multiple different species were selected. The alignment results were analyzed to identify highly conserved regions and potential beneficial mutation sites. At the same time, homology modeling was performed and after docking with the substrate, the 7β-HSDH enzyme active site was evaluated using pymol. The role of non-conserved amino acid residues in the enzyme catalysis process.

[0033] Discovery Studio virtual mutagenesis was used to calculate the binding free energies for the ligands 7-OXO-LCA and NADH, and alanine scanning was performed for virtual screening. All residues were mutated to alanine. Calculations were performed using the CHARMm force field at 25°C and a pH 7.4 aqueous environment. Virtual alanine scanning was used to identify sites with the highest binding free energies for the substrates. Binding interaction analysis revealed that sites Y201 and V104 exhibited strong binding energies for both the substrates 7-OXO-LCA and NADH, so point saturation mutagenesis was performed on these two sites for subsequent screening and validation.

[0034] At the same time, by comparing with non-homologous 7β-HSDH sequences reported in the literature, it was found that L45I / D46K and N98 / V258F mutations are potential dominant mutation sites of 7β-HSDH derived from Roseococcus sp. SDR.

[0035] Example 2: Construction, expression and purification of recombinant bacteria

[0036] The mutants and parental amino acid sequences were submitted to GenScript for codon optimization and synthesis. The plasmid vector pET-28a(+) was used, with restriction enzyme sites NdeI and HindIII. The synthesized recombinant plasmid was transfected into Escherichia coli BL21(DE3) purchased from Shanghai Weidi Biotechnology Co., Ltd. according to the manufacturer's instructions. Single colonies were plated and cultured overnight to obtain distinct colonies. Conspicuous single colonies were selected and added to 4 mL of LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C, 220 rpm, and shaken for 10 hours. A 1-2% transfer of the colony to 100 mL of LB liquid medium containing 50 μg / mL kanamycin was performed and incubated at 37°C, 220 rpm, until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.2 mM, and protein expression was induced overnight by shaking at 30°C, 220 rpm. Collect the cells by centrifugation at 4°C and resuspend in 10 ml of 50 mM PBS (pH 7.4). Repeat the centrifugation and resuspension process twice to wash the cells. Disrupt the cells using an ultrasonic cell disruptor and remove the precipitate after centrifugation at 4°C to obtain a crude enzyme solution. The 96-well plate screening culture procedure is similar to the above steps, except that the culture volume is reduced to 0.8 ml and a high-speed shaker is used to prevent sedimentation during the culture process.

[0037] The crude enzyme solution after centrifugation was filtered through a 0.45 μm filter to remove impurities and then purified using a Ni-NTA affinity column. Ni-NTA filler was purchased from Nanjing GenScript Biotech Co., Ltd. The target protein was purified according to the manufacturer's instructions. The purified enzyme solution was collected and desalted using a 10 kDa ultrafiltration tube before use in the reactions described in the subsequent examples. SDS-PAGE electrophoresis images of the crude and pure enzymes are shown in the accompanying figures.

[0038] Example 3: 7β-HSDH enzyme activity assay

[0039] Protein concentration was determined by the Coomassie Brilliant Blue method and compared with a bovine serum albumin standard to determine the concentration of purified 7β-HSDH protein. Based on the measured protein concentration, the pure enzyme solution was diluted to the same concentration using PBS buffer. The assay was performed in a 96-well plate. 180 μL of substrate premix (20 μL of 5 mM NADH, 20 μL of 10 mM 7-OXO-LCA, and 140 μL of 1× PBS buffer) was added to each well, with triplicate additions. The microplate reader was set to measure absorbance at 340 nm, with 21 readings, a 60-second kinetic interval, and oscillation between readings (5 seconds on, 55 seconds off). 20 μL of the corresponding pure enzyme solution was added to the wells containing 180 μL of substrate premix. The absorbance change at 340 nm was measured and compared with NADH standards of varying concentrations to calculate the reaction activity.

[0040] Example 4: 7β-HSDH stability determination

[0041] The protein concentration was determined using the Coomassie Brilliant Blue method and compared with a bovine serum albumin standard to determine the concentration of the purified 7β-HSDH protein. Based on the measured protein concentration, the pure enzyme solution was diluted with PBS buffer to the same concentration. The pure enzyme solution was then heat-treated at 30°C for 0, 5, 10, 20, 40, and 60 minutes, followed by centrifugation at 4°C. The enzyme activity of 7β-HSDH after heat treatment for different times was determined using the aforementioned enzyme activity assay, and the corresponding stability changes were plotted.

[0042] Example 5: Analysis of 7β-HSDH dominant mutants

[0043] After screening, further analysis was conducted on sites such as V104Y and V104P. Molecular docking and kinetic simulations were performed using yasara with substrates. It was found that the V104Y and V104P mutants significantly altered the enzyme conformation, further affecting its activity. Furthermore, mutation of I45, the site that binds to the NADH six-membered ring, to phenylalanine increased its Pi-Pi stacking capacity.

[0044] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It should be noted that changes and improvements made by those skilled in the art without departing from the technical solution of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for modifying ursodeoxycholic acid (UDCA) synthase, characterized in that: The alanine scanning technique was used to evaluate the effects of non-conserved amino acid residues near the active site of 7β-HSDH on the catalytic activity of 7β-HSDH enzyme, identify the key sites affecting the activity and stability of 7β-HSDH synthase, and perform saturation mutagenesis on the key sites.

2. A 7β-HSDH mutant, characterized in that: It is composed of any of the following amino acid sequences: (1) The valine at position 104 of the amino acid sequence shown in SEQ ID NO.1 is mutated to proline or tyrosine, and the sequence thereof is shown in SEQ ID NO.2 and SEQ ID NO.3; (2) Leucine at position 45 of the amino acid sequence shown in SEQ ID NO.1 is mutated to isoleucine, the sequence of which is shown in SEQ ID NO.4; (3) Aspartic acid at position 46 of the amino acid sequence shown in SEQ ID NO.1 is mutated to lysine, and its sequence is shown in SEQ ID NO.5; (4) The amino acid sequence shown in SEQ ID NO.1 has leucine at position 45 mutated to isoleucine, and aspartic acid at position 46 mutated to lysine. The sequence is shown in SEQ ID NO.6; (5) The amino acid sequence shown in SEQ ID NO.1 has asparagine at position 98 mutated to tyrosine, and valine at position 258 mutated to phenylalanine. The sequence is shown in SEQ ID NO.

7.

3. An isolated DNA molecule, characterized in that Encodes the amino acid sequence of claim 2.

4. A recombinant plasmid vector, characterized in that: The vector comprises the nucleic acid according to claim 3.

5. A host cell, characterized in that The invention comprises the recombinant plasmid vector according to claim 4 and is capable of expressing the modified enzyme during the production process of UDCA.

6. Use of the 7β-HSDH mutant according to claim 2 in the synthesis of UDCA and its derivatives.

7. The use according to claim 6, characterized in that: UDCA synthesis can be achieved by 7β-HSDH mutant-catalyzed reduction of the substrate 7-OXO-LCA, a process that requires the participation of the coenzyme NAD(P)H.

8. The use according to claim 7, characterized in that: The concentration of the coenzyme NADH is 0.01-1 mM.

9. The use according to claim 7, characterized in that: The concentration of the substrate 7-OXO-LCA was 0.01-2 mM.

10. The use according to claim 7, characterized in that: The reaction temperature is 25-40°C, and the reaction time is 0-6h.

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