Directed evolution improves the forward activity of 7β-hydroxysteroid dehydrogenase for efficient synthesis of ursodeoxycholic acid

Abstract

The present invention discloses a method for improving the forward enzymatic activity of 7β-hydroxysteroid dehydrogenase by directed evolution to efficiently synthesize ursodeoxycholic acid, belonging to the field of biocatalytic engineering. The present invention obtains a 7β-hydroxysteroid dehydrogenase mutant TEAE / F152L / W101N with improved forward reaction activity and substrate affinity. Compared with TEAE, the mutant enzyme has a 2.9-fold increase in forward enzymatic activity and a 3.6-fold decrease in reverse activity. The catalytic efficiency k of 7K-LCA is 1.3 times higher than that of TEAE. cat / K m The enzyme activity of the mutant TEAE / F152L / W101N increased by 4.3 times compared to the wild type. The catalytic efficiency for 7K-LCA increased by 58.56 times. This invention provides a universal technical platform for the directed evolution of hydroxysteroid dehydrogenases, provides excellent mutant enzymes for the efficient biosynthesis of ursodeoxycholic acid, and lays a solid research foundation for the efficient and low-cost preparation of ursodeoxycholic acid.

Description

Technical Field

[0001] This invention relates to directed evolution to enhance the forward enzyme activity of 7β-hydroxysteroid dehydrogenase for efficient synthesis of ursodeoxycholic acid, and belongs to the field of biocatalysis engineering. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is an important natural bile acid derivative with unique biological activities and a wide range of biomedical applications. It plays a unique pharmacological role in regulating bile acid metabolism, promoting bile excretion, and reducing cholesterol solubility. Clinically, it is widely used to treat gallstones, liver disease, gastrointestinal diseases, and biliary tract diseases. In particular, it plays a crucial role in improving liver function, reducing gallstone formation, and enhancing intestinal health. Furthermore, ursodeoxycholic acid can also be used as a hepatoprotective agent and immunosuppressant, especially after liver transplantation.

[0003] Ursodeoxycholic acid (UDCA) is primarily found in bear bile, but it can also be synthesized through chemical or biological methods. In vivo, UDCA biosynthesis mainly involves the conversion of cholesterol into other compounds. First, cholesterol is converted to 7α-hydroxycholesterol, which is further converted to chenodeoxycholic acid (CDCA). Finally, chenodeoxycholic acid is converted to UDCA by 7α / 7β-hydroxysteroid dehydrogenases (7α / 7β-HSDH) in the gut microbiota. The UDCA biosynthetic pathway mainly involves several key enzymes in the bile acid synthesis pathway, including cholesterol oxidase, 7α-hydroxylase, 7α-HSDH, and 7β-HSDH. 7α-HSDH and 7β-HSDH are the key enzymes determining the conversion of chenodeoxycholic acid to UDCA. Currently, there are three main synthetic pathways for ursodeoxycholic acid in vitro. The first pathway involves using primary hydrophilic bile acids as substrates, undergoing chemical oxidation followed by hydroxyl dehydrogenation to produce the first intermediate, 3α,7β,12-oxolithocholic acid. Then, under the action of 3α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, the second intermediate, 12-ketolithocholic acid, is produced. Finally, it is converted to ursodeoxycholic acid via the Wolff-Kishner reduction reaction. The second pathway involves the production of 7,12-oxolithocholic acid catalyzed by 7α-hydroxysteroid dehydrogenase and 12α-hydroxysteroid dehydrogenase. Subsequently, under the action of 7β-hydroxysteroid dehydrogenase, the second intermediate, 12-ketolithocholic acid, is generated, which is then converted to ursodeoxycholic acid via the Wolff-Kishner reduction reaction. The third pathway involves chenodeoxycholic acid, which is an isomer of the product. In the first step, the substrate is converted to the intermediate 7-keto-lithocholic acid (7K-LCA) by 7α-hydroxysteroid dehydrogenase (7α-HSDH), and UDCA is subsequently produced by 7β-hydroxysteroid dehydrogenase (7β-HSDH). Due to its relative simplicity, this pathway has become the preferred route for UDCA biosynthesis. Regardless of the chosen pathway, 7β-HSDH is the key enzyme in UDCA synthesis.

[0004] The catalytic efficiency and thermal stability of 7β-HSDH are generally much lower than those of 7α-HSDH. The catalytic activity of 7β-HSDH in reducing 7K-LCA to UDCA (forward activity) is usually much lower than that in the oxidation of UDCA to 7K-LCA (reverse activity). These factors make 7β-HSDH a ​​key limiting factor for the efficiency of UDCA synthesis under in vitro conditions. Summary of the Invention

[0005] This invention is based on Hyphomicrobium 7β-hydroxysteroid dehydrogenase sp. ( HsUsing 7β-HSDH as the template enzyme, structural analysis and virtual mutagenesis were used to screen for mutation hotspots. Through directed evolution, recombinant 7β-HSDH mutant strains with significantly enhanced positive reactivity and enzyme activity were obtained. This improved... Hs The activity of 7β-HSDH in catalyzing the conversion of 7K-LCA to UDCA was investigated, and the enzymatic properties of 7β-HSDH and its mutants were characterized in detail, enabling the efficient synthesis of UDCA from 7K-LCA. This invention lays a solid foundation for the industrial-scale, efficient preparation of ursodeoxycholic acid.

[0006] This invention provides a 7β-hydroxysteroid dehydrogenase mutant, wherein the mutant is a [missing information - likely a specific type of mutant]. Hs It is obtained by mutating at least one of positions 101 and 152 of 7β-HSDH; Hs 7β-HSDH has the amino acid sequence shown in SEQ ID NO. 1. Hs 7β-HSDH enzyme.

[0007] In one embodiment of the present invention, the 7β-hydroxysteroid dehydrogenase mutant includes one or more amino acid substitutions at positions 101 / 152. Specifically, amino acid at position 101 is substituted with asparagine N, and amino acid at position 152 is substituted with leucine L.

[0008] In one embodiment of the present invention, the Hs The catalytic efficiency of the 7β-HSDH mutant was measured by detecting the change in absorbance of NADH at 340 nm using a microplate reader, with 7-keto-lithocholic acid as the substrate and NADH as the cofactor. One unit of enzyme activity (U) is defined as the ability to catalyze the oxidation of 1 μmol of NADH to NAD per minute under the measured conditions. + The amount of enzyme.

[0009] This invention provides Hs The 7β-HSDH mutant, wherein the 7β-hydroxysteroid dehydrogenase mutant is composed of amino acid sequences as shown in SEQ ID NO.3. Hs It is obtained by simultaneously mutating positions 101 and / or 152 of 7β-HSDH.

[0010] This invention provides Hs The 7β-HSDH mutant, wherein the 7β-hydroxysteroid dehydrogenase mutant is composed of amino acid sequences as shown in SEQ ID NO.1. Hs The mutants obtained by mutating 7β-HSDH are: W101N, F152L, and W101N / F152L.

[0011] In one embodiment of the present invention, the mutant is obtained by mutating tryptophan at position 101 of 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to asparagine, and is named W101N.

[0012] Alternatively, the mutant is obtained by mutating phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to leucine, and is named F152L.

[0013] Alternatively, the mutant is obtained by mutating tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to asparagine, and simultaneously mutating phenylalanine at position 152 to leucine, and is named W101N / F152L.

[0014] This invention provides a 7β-hydroxysteroid dehydrogenase mutant, wherein the mutant has the amino acid sequence shown in SEQ ID NO.3. Hs It is obtained by mutating valine at position 99 of the 7β-HSDH enzyme to threonine or aspartic acid;

[0015] Alternatively, it can be obtained by mutating tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to isoleucine or glutamine;

[0016] Alternatively, it can be obtained by mutating phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to threonine or leucine;

[0017] Alternatively, it can be obtained by mutating the tyrosine at position 201 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to phenylalanine;

[0018] Alternatively, it can be obtained by mutating phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to leucine, and simultaneously mutating tryptophan at position 101 to asparagine, valine, or proline.

[0019] Alternatively, it can be obtained by mutating phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to leucine, and simultaneously mutating tyrosine at position 201 to methionine.

[0020] Or the amino acid sequence is as shown in SEQ ID NO.3 HsThe 7β-HSDH is obtained by mutating phenylalanine at position 152 to leucine, mutating tryptophan at position 101 to asparagine, and mutating tyrosine at position 201 to cysteine, aspartic acid, arginine, or phenylalanine.

[0021] In one embodiment of the present invention, the 7β-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO.3 is... Hs The combined mutants obtained by mutating the parent enzyme 7β-HSDH mutant are: A22T / Q23E / L45A / N46E;

[0022] SEQ ID NO.3:

[0023] MSYDAFADFRMIGHNVIITGGTENIGAGIARTLSGAGAKVMIADAEGDMAKETAAGIQKDTGNDCRGIKCDVTSLDDINAVVAETVKAFGGISTLVNNVGWGGRHDDPAAVTEEDFIASYKLNAISAYRMSM ACLPHLLKTKNATITNSGSFSASAPAYDILAYGTAKAALNQMMVSLAHMLAKKVRVNSVLIGTVMTAGYADAGIDPEMQERLSHPDNLTGRSGKPEDIANAMLWLCSPASGWVSGQIINVHGGGDVVRLFGE

[0024] Hs The parental enzyme sequence of the 7β-HSDH mutant (SEQ ID NO.1) is as follows:

[0025] MSYDAFADFFRMIGHNVIITGGAQNIGAGIARTLSGAGAKVMIADLNGDMAKETAAGIQKDTGNDCRGIKCDVTSLDDINAVVAETVKAFGGISTLVNNVGWGGRHDDPAAVTEEDFIASYKLNAISAYRMSM ACLPHLLKTKNATITNSGSFSASAPAYDILAYGTAKAALNQMMVSLAHMLAKKVRVNSVLIGTVMTAGYADAGIDPEMQERLSHPDNLTGRSGKPEDIANAMLWLCSPASGWVSGQIINVHGGGDVVRLFGE

[0026] In one embodiment of the present invention, the nucleotide sequence encoding the parent enzyme of the 7β-hydroxysteroid dehydrogenase is shown in SEQ ID NO.2.

[0027] The nucleotide sequence of the parent enzyme of 7β-hydroxysteroid dehydrogenase is SEQ ID NO.2:

[0028] ATGAGCTACGATGCCTTTGCAGATTTCCGTATGATTGGCCACAACGTTATCATCACTGGCGGTGCTCAGAACATTGGTGCGGGTATCGCGCGTACCCTGTCTGGTGCTGGCGCGAAAGTAATGATCGCCGACCTGAACGGCGATATGGCGAAAGAAACCGCGGCGGGCATTCAGAAAGACACGGGTAACGACTGCCGT GGCATCAAATGTGACGTGACGTCCCTGGATGACATCAACGCTGTTGTGGCGGAGACCGTTAAGGCATTCGGTGGCATTTCTACCCTGGTTAACAATGTTGGTTGGGGCGGCCGTCATGATGATCCGGCTGCTGTCACTGAGGAAGACTTCATCGCGTCTTACAAACTGAACGCAATCTCCGCGTACCGCATGAGCATG GCCTGCCTGCCGCATCTGCTGAAAACCAAAAACGCCACCATCACCAACTCCGGTTCCTTTAGCGCTTCCGCACCTGCGTACGACATTCTGGCTTATGGCACCGCCAAAGCTGCTCTGAACCAAATGATGGTCTCTCTGGCTCATATGCTGGCGAAAAAAGTTCGCGTGAACTCCGTGCTGATCGGTACTGTAATGACG GCCGGCTATGCTGATGCAGGCATCGACCCGGAAATGCAAGAACGTCTGAGCCATCCGGACAATCTGACCGGCCGTTCTGGTAAGCCAGAGGATATCGCGAACGCAATGCTGTGGCTGTGTTCCCCGGCGTCTGGCTGGGTTTCCGGGCCAGATTATCAACGTTCATGGTGGTGGTGACGTAGTTCGTCTGTTCGGTGAA

[0029] The present invention also provides a gene encoding the above-mentioned mutant.

[0030] The present invention also provides a recombinant vector carrying the mutant or carrying the above-mentioned gene.

[0031] In one embodiment of the present invention, the recombinant vector is a pET series vector such as pET-28a plasmid, pET-21a plasmid, a pRSF series vector such as pRSF-Duet1 plasmid, or a pGEX series vector such as pGEX-6p-1 plasmid.

[0032] In one embodiment of the present invention, the recombinant vector is expressed using pET28a plasmid, pRSF-Duet1 plasmid, pET21a plasmid or pGEX-6P-1 plasmid.

[0033] The present invention also provides a recombinant cell expressing the above-mentioned mutant, carrying the above-mentioned gene, or carrying the above-mentioned recombinant vector.

[0034] In one embodiment of the present invention, the recombinant cells use bacteria or fungi as expression hosts.

[0035] In one embodiment of the present invention, the recombinant cells use Escherichia coli as the expression host.

[0036] This invention provides a portable Bacillus sp. source Bs Recombinant vector of GDH (SEQ ID NO.4).

[0037] carry Bacillus sp. source Bs GDH SEQ ID NO.4:

[0038] MYPDLKGKVVAITGASSGLGRAMAIRFGQEQAKVVINYYSNEKEAQTVKEEVQKAGGEAVIIQGDVTKEEDVKNIVQTAVKEFGTLDIMINNAGMENPVESHKMPLKDWNKVINTNLTGAFLGCREAIKY YVENDIQGNVINMSSVHEMIPWPLFVHYAASKGGIKLMTETLALEYAPKRIRVNNIGPGAINTPINAEKFADPVQKKDVESMIPMGYIGEPEEIAAVAVWLASKESSYVTGITLFADGGMTQYPSFQAGRG

[0039] The above encoding Bs The GDH nucleotide sequence is shown in SEQ ID NO.5:

[0040] ATGTATCCGGATTTAAAAGGAAAAGTCGTCGCCATTACAGGAGCATCATCAGGATTAGGAAGAGCGATGGCGATCCGCTTCGGGCAGGAGCAGGCGAAAGTCGTGATTAACTACTACAGTAATGAAAAAGAGGCTCAAACCGTAAAAGAAGAAGTTCAAAAAGCGGGCGGCGAAGCGGTCATTATTCAAGGTGAC GTTACAAAAGAAGAGGATGTCAAAAACATTGTGCAGACCGCGGTCAAGGAATTCGGCACATTAGATATCATGATCAACAACGCCGGCATGGAAAATCCGGTCGAGTCGCATAAAATGCCGCTAAAAGACTGGAACAAAGTCATCAACACCAACCTGACCGGCGCTTTTCTGGGATGCCGCGAAGCCATTAAATATT ACGTAGAGAATGATATTCAAGGAAACGTCATTAACATGTCGAGCGTACATGAAATGATTCCGTGGCCGCTGTTTGTCCACTATGCGGCAAGTAAAGGCGGCATTAAATTAATGACGGAAACATTGGCGCTTGAGTACGCGCCGAAGCGCATCCGTGTTAACAATATCGGGCCGGGCGCCATCAATACGCCGATCAA TGCGGAAAAGTTTGCGGATCCCGTTCAGAAAAAAGATGTGGAAAGCATGATTCCGATGGGGTATATCGGTGAGCCGGAAGAAATCGCGGCTGTCGCCGTCTGGCTTGCTTCAAAGGAATCAAGCTACGTGACCGGCATTACGCTGTTTGCTGACGGCGGAATGACACAATATCCGTCATTCCAGGCAGGCCGCGGT

[0041] In one embodiment of the present invention, the recombinant vector is pET28a, pRSF-Duet1, pET-Duet1, pACY-Duet1, pET21a or pGEX-6P-1 as the expression vector.

[0042] This invention also provides engineering methods. Hyphomicrobium sp. Hs Enzymatic properties of 7β-HSDH, including optimum temperature, pH, temperature stability, pH stability, and kinetic parameters.

[0043] This invention provides a method for improving the forward reaction activity of 7β-hydroxysteroid dehydrogenase while reducing its reverse reaction activity. The method involves mutating tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, to asparagine, and simultaneously mutating phenylalanine at position 152 to leucine.

[0044] The present invention also provides a recombinase catalyst containing the 7β-hydroxysteroid dehydrogenase mutant sequence, wherein the recombinase catalyst is any one of the following forms:

[0045] (1) Culturing the recombinant expression transformant and isolating transformant cells containing the recombinase;

[0046] (2) Cultivate the recombinant expression transformant, isolate the transformant cells containing the recombinase, and break the transformant cells containing the recombinase to obtain the cell lysate;

[0047] (3) Cultivate the recombinant expression transformant, isolate the transformant cells containing the recombinant enzyme, break the transformant cells containing the recombinant enzyme, obtain the cell lysate, and freeze-dry the cell lysate of the recombinant enzyme to obtain lyophilized enzyme powder.

[0048] This invention also provides a method for improving the forward reaction activity of 7β-hydroxysteroid dehydrogenase, the method being,

[0049] The valine at position 99 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, is mutated to threonine or aspartic acid.

[0050] Alternatively, tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, could be mutated to isoleucine or glutamine.

[0051] Alternatively, the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, may be mutated to threonine or leucine;

[0052] Alternatively, the tyrosine residue at position 201 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, may be mutated to phenylalanine;

[0053] Alternatively, the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, may be mutated to leucine, and the tryptophan at position 101 may be mutated to asparagine, valine, or proline.

[0054] Alternatively, the amino acid sequence of 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, may be mutated from phenylalanine at position 152 to leucine, and from tyrosine at position 201 to methionine.

[0055] Alternatively, the amino acid sequence of 7β-hydroxysteroid dehydrogenase, as shown in SEQ ID NO.3, may be mutated by replacing phenylalanine at position 152 with leucine, replacing tryptophan at position 101 with asparagine, and replacing tyrosine at position 201 with cysteine, aspartic acid, arginine, or phenylalanine.

[0056] The present invention also provides a method for preparing UDCA, wherein 7K-LCA is used as a substrate and NADH is used as a cofactor, and the above-mentioned recombinant cells or recombinant cells of the above-mentioned recombinant enzyme preparation are used to catalyze the substrate conversion to prepare ursodeoxycholic acid.

[0057] Alternatively, the method may involve using 7-keto-lithocholic acid as a substrate, NADH as a cofactor, and glucose as a co-substrate, employing the aforementioned mutant, recombinant cells, or recombinant enzyme preparation, and containing... Bs Ursodeoxycholic acid was prepared by co-catalyzing substrate conversion using recombinant GDH cells.

[0058] In one embodiment of the present invention, the encoding of the BsGDH The nucleotide sequence is shown in SEQ ID NO.4.

[0059] In one embodiment of the present invention, the reaction system contains 7-ketolithocholic acid and NADH.

[0060] In one embodiment of the present invention, the concentration of 7-ketolithocholic acid is 10~50 mM.

[0061] In one embodiment of the present invention, the concentration of NADH is 0.5~50 mM.

[0062] In one embodiment of the present invention, the reaction system contains 7-ketolithocholic acid, NADH, and glucose.

[0063] In one embodiment of the present invention, the concentration of glucose is 20-100 mM.

[0064] In one embodiment of the present invention, the concentration of NADH is 0.5~5 mM.

[0065] In one embodiment of the present invention, the reaction conditions are pH 6.0 to 10.0 and temperature 20 to 45°C.

[0066] The present invention also provides the use of the above-mentioned 7β-hydroxysteroid dehydrogenase mutant or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned recombinant Escherichia coli, or the above-mentioned method in the preparation of ursodeoxycholic acid or products containing ursodeoxycholic acid.

[0067] In one embodiment of the present invention, the product is a chemical product.

[0068] Beneficial effects

[0069] (1) This invention has mined from Hyphomicrobium sp. Hs Using 7β-HSDH as a template enzyme, structural analysis was performed, and directed evolution yielded the mutant TEAE / F152L, which exhibits significantly enhanced 7K-LCA reduction activity, and the combined mutant TEAE / F152L / W101N, which shows significantly enhanced 7K-LCA reduction activity while exhibiting significantly reduced UDCA oxidation activity. This resulted in a 7β-hydroxysteroid dehydrogenase mutant with significantly enhanced forward catalytic activity and reduced reverse catalytic activity.

[0070] (2) This invention studied Hs The enzymatic properties of 7β-HSDH and its mutants TEAE / F152L and TEAE / F152L / W101N were investigated, and the optimal temperature, pH, and kinetic parameters of the enzyme were determined. The enzyme activity and catalytic efficiency of the mutants were verified to be significantly improved; the enzyme activity of the mutants increased by 3.2 and 2.9 times respectively relative to the mutation initiation point, and the catalytic efficiency (…) k cat / K m The catalytic efficiency was increased by 4.7 and 4.3 times respectively compared to the wild type; and by 206.23 and 184.52 times respectively compared to the wild type. k cat / K m These figures increased by 58.56 and 52.91 times respectively.

[0071] (3) Based on homology modeling, molecular docking analysis, ligand-enzyme complex affinity determination, and dynamic simulation, this invention has resolved the following: Hs The molecular mechanism underlying the enhanced catalytic performance of the 7β-HSDH mutants TEAE / F152L and TEAE / F152L / W101N provides a general technique for directionally improving the substrate affinity and other catalytic properties of hydroxysteroid dehydrogenases.

[0072] (4) This invention improves HsThe catalytic activity of 7β-HSDH in reducing 7K-LCA to UDCA was reduced, while its activity in reducing UDCA to 7K-LCA was decreased. With 40 mM 7K-LCA and 40 mM NADH, the yields of the mutants TEAE / F152L and TEAE / F152L / W101N reached 61.8% and 72.1%, respectively, which were 18.2% and 28.4% higher than those of the mutant initiating enzyme TEAE.

[0073] (5) Using the method of the present invention, a "coenzyme regeneration" reaction system is constructed, and... BsGDH By coupling with only 1 mMNADH, highly efficient synthesis of UDCA can be achieved. The yields of the mutants TEAE / F152L and TEAE / F152L / W101N reached 82.4% and 92.8%, respectively. Compared with the mutant initiating enzyme TEAE, the yields increased by 23.9% and 34.3%, respectively, and compared with the reaction without cofactor regeneration, the yields increased by 20.6% and 20.7%, respectively. After three batches of reaction, the mutant TEAE / F152L / W101N maintained an average yield of 89.2%, with a theoretical space-time yield of 171 g / L / d. This one-step whole-cell biocatalysis reaction eliminates cumbersome steps, generates no unnecessary byproducts, and operates under mild conditions, making it a green and efficient method for the biosynthesis of ursodeoxycholic acid. Attached Figure Description

[0074] Figure 1 (a) Initial enzyme activity assay of 7β-HSDH from different sources; (b) Initial yield assay of 7K-LCA to UDCA catalyzed by 7β-HSDH from different sources.

[0075] Figure 2 Changes in the affinity of mutant TEAE for NADH compared to wild-type enzyme.

[0076] Figure 3 (a) UDCA combined with pocket Hs (a) Key amino acid residues of 7β-HSDH; (b) 7K-LCA binding pocket Hs Key amino acid residues of 7β-HSDH; (c) NADH binding pocket Hs Key amino acid residues of 7β-HSDH; (d) Hs 7β-HSDH substrate and cofactor channels.

[0077] Figure 4 : Hs Energy changes when an amino acid near 7K-LCA in the 7β-HSDH substrate binding pocket is mutated to alanine.

[0078] Figure 5 A high-throughput method for measuring the extent of a reaction by the residual amount of the cofactor NADH.

[0079] Figure 6 Results of iterative saturation mutations; (a) First round of saturation mutations; (b) Second round of saturation mutations; (c) Third round of saturation mutations; (d) Comparison of mutation initiation points with dominant mutant enzyme activity and yield.

[0080] Figure 7 SDS-PAGE analysis of enzyme expression in wild-type and mutant strains; (a) SDS-PAGE analysis of the supernatant of wild-type and mutant fragments; (b) SDS-PAGE analysis of purified enzymes in wild-type and mutant strains; where M is a color-prestained protein marker; 1, E. coli / pGEX-6p- Hs 7β-HSDH; 2, E. coli / pGEX-6p-F152L; E. coli / pGEX-6p-F152L / W101N.

[0081] Figure 8 : Hs Specific enzyme activity assays of 7β-HSDH wild-type and mutants; (a) optimal pH; (b) optimal temperature; (c) thermal stability.

[0082] Figure 9 : Hs Kinetic simulations of 7β-HSDH and mutants; (a) Hs (a) Changes in the binding energy and binding entropy of 7β-HSDH and its mutants with NADH; (b) Changes in the root mean square fluctuation (RMSF) of the ligands; (c) Changes in the root mean square deviation (RMSD); (d) Hs (e) Changes in the root mean square fluctuation (RMSF) of 7β-HSDH; (f) Types of interactions between amino acid residues in the binding bag; Hs Spatial distance between 7β-HSDH wild-type and 7K-LCA; (g) Hs Spatial distance between the 7β-HSDH mutant and the 7K-LCA.

[0083] Figure 10 (a) Hs (a) Time curves for the synthesis of UDCA catalyzed by 7β-HSDH and its mutants; (b) Yield determination of the regeneration system without coenzyme addition and the regeneration system with addition; (c) UDCA yield of multiple batches of reactions. Detailed Implementation

[0084] Technical terms:

[0085] Ursodeoxycholic acid (Usdeoxycholic acid): 3α,7β-dihydroxy-5β-cholestan-24-acid is a hydrophilic bile acid with various biological activities. It can promote the excretion of endogenous bile acids, increase the proportion of hydrophilic bile acids, protect hepatocytes and bile duct cells from the toxicity of hydrophilic bile acids, prevent the damage of mitochondrial membranes and their cytotoxic effects by hydrophobic hydrophilic bile acids, inhibit hepatocyte apoptosis, significantly enhance liver function, and exhibit immunomodulatory and cytoprotective effects. It is one of the most commonly prescribed drugs in the clinical treatment of cholestatic diseases.

[0086] Expression: The term “expression” includes any step involved in the generation of 7β-hydroxysteroid dehydrogenase mutants, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0087] Expression vector: The term “expression vector” refers to a straight or circular DNA molecule containing a polynucleotide encoding the 7β-hydroxysteroid dehydrogenase mutant of the present invention and operatively linked to a control sequence provided for its expression.

[0088] Fragment: The term "fragment" means a polypeptide that has one or more (e.g., several) amino acids missing from its amino and / or carboxyl terminus; wherein said fragment has 7β-hydroxysteroid dehydrogenase activity. In one aspect, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, of the number of amino acids 1 to 440 (i.e., excluding the zymogen region sequence length) shown in SEQ ID NO. 1.

[0089] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0090] The host cell can be any cell useful in the recombinant production of 7β-hydroxysteroid dehydrogenase mutants, such as prokaryotic or eukaryotic cells.

[0091] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus spp. Bacillus Clostridium ( Clostridium ), Enterococcus spp. Enterococcus ), Bacillus spp. Geobacillus Lactobacillus () Lactobacillus Lactococcus spp. Lactococcus), Bacillus spp. ( Oceanic Bacillus Staphylococcus spp. Staphylococcus Streptococcus spp. Streptococcus ) and Streptomyces ( Streptomyces Gram-negative bacteria include, but are not limited to, Campylobacter spp. Campylobacter ), Escherichia coli ( E. coli Flavobacterium ( Flavobacterium ), Fusobacterium genus ( Fusobacterium ), Helicobacter spp. Helicobacter ), Colistinia spp. ( Mycobacterium ), Neisseria spp. Neisseria ), Pseudomonas spp. Pseudomonas Salmonella ( Salmonella ), and Ureaplasma genus ( Ureaplasma ).

[0092] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0093] The UDCA (ursodeoxycholic acid), 7K-LCA (7-ketolithocholic acid), and glucose involved in the following examples were all purchased from Sinopharm Chemical Reagent Co., Ltd., with CAS numbers 128-13-2, 4651-67-6, and 141-53-7, respectively, and their structural formulas are as follows:

[0094]

[0095] UDCA

[0096]

[0097] 7K-LCA

[0098]

[0099] glucose

[0100] The reaction formula of this invention is as follows:

[0101]

[0102] The culture media involved in the following examples are as follows:

[0103] LB liquid medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, natural pH.

[0104] The detection methods involved in the following embodiments:

[0105] The method for detecting 7β-hydroxysteroid dehydrogenase activity is as follows:

[0106] The enzyme activity of 7β-HSDH in reducing 7K-LCA or oxidizing UDCA was measured at 30℃ by determining the rate of change in absorbance of NADH at 340 nm. The enzyme activity assay system consisted of a 250 μL reaction volume, with 2 mM NADH and 10 mM 7K-LCA added to 0.1 M PBS buffer (pH 7.5), incubated in a 30℃ metal bath for 3 min, followed by the addition of 50 μL of purified enzyme at an appropriate concentration. The absorbance at 340 nm was then scanned using a Bio-Tek Cytation 5 multi-functional cell imaging system.

[0107] Enzyme activity is defined as the ability of an enzyme to catalyze the oxidation of 1 μmol of NADH to NAD per minute under the conditions described above. + The amount of enzyme is defined as one unit, U.

[0108] The formula for calculating enzyme activity is: Enzyme activity (U) = EW × V × 10 3 / (6220 × 0.625).

[0109] Formula for calculating specific activity: Specific activity (U·mg) -1 = Enzyme activity (U) / Protein content (mg).

[0110] Where EW: change in absorbance at 340 nm within 1 min; V: volume of reaction solution (mL); 6220: molar extinction coefficient (L·mol⁻¹). -1 ·cm -1 ); 0.5: Optical path distance (cm).

[0111] Calculation of dynamic parameters:

[0112] According to the Michaelis equation v= V max ×[S] / K m +[S], calculate the enzyme's response to different substrates. K m value.

[0113] Where, v: reaction rate (U·mg) -1 ); V max Maximum reaction rate (U·mg) -1 [S]: Substrate concentration (mM); K m The reaction rate v reaches halfway. V max The substrate concentration at that time.

[0114] When the substrate concentration is saturated k cat =V max / Et, calculate k cat value.

[0115] in, V max : Maximum reaction rate; Et: Enzyme concentration.

[0116] Determination of kinetic parameters:

[0117] The total reaction volume was 250 µL. 0.1 M PBS buffer (pH 7.5) and 10 mM 7K-LCA were added, and the mixture was incubated at 30℃ for 2 min. Then, 50 μL of purified enzyme at an appropriate concentration was added. Specific enzyme activity was measured at different concentrations (0.1–10 mM) of substrate 7K-LCA or product UDCA. Enzyme kinetic curves were fitted using GraphPad software. Substrate concentration and specific enzyme activity were plotted on the x and y axes, respectively, and a Michaelis-Menten equation model was used for nonlinear fitting to obtain the desired results. V max Value and K m Values ​​are obtained through data conversion. k cat Value and k cat / K m Values ​​(Table 4).

[0118] The high-performance liquid chromatography (HPLC) method for detecting ursodeoxycholic acid content is as follows:

[0119] The analysis was performed using an Agilent 1260 high-performance liquid chromatograph and an Accucore-C18 LC column, with 60% acetonitrile solution (containing 0.1% phosphoric acid) as the mobile phase and a flow rate of 1 mL / min. -1 The detection temperature was 30 ℃, and the detection was performed at 195 nm ultraviolet light. The substrate and product standards were dissolved in methanol or acetonitrile solution for detection, yielding standard curves for UDCA and 7K-LCA.

[0120] Example 1: Discovery of 7β-hydroxysteroid dehydrogenase and virtual screening of key residues with cofactor-dependent mutations

[0121] The specific steps are as follows:

[0122] (1) Hs Mining of 7β-HSDH

[0123] Filtered Hyphomicrobium sp., Mediterraneibacter sp., Eubacterium sp., Ruegeria sp., Stanieria sp. and Cereibacter The activity of 7β-hydroxysteroid dehydrogenase and its initial conversion at 10 mM 7K-LCA were determined using 7β-HSDH from different sources, including sp. The results are as follows: Figure 1 As shown, from Hyphomicrobium sp. Hs 7β-HSDH exhibited the highest catalytic activity (8.4 U·mg) among the screened enzymes. -1 Based on the initial conversion rate (48.9%), this enzyme was selected as the research subject.

[0124] (2) Hs Structural analysis of 7β-HSDH

[0125] 1) By Hs 7β-HSDH was docked with NADPH and NADH. Several semi-conserved sites within the cofactor binding region, including A22, Q23, N24, G26, D44, L45, and N46, were selected as potential key amino acid residues affecting cofactor binding. These sites were then subjected to virtual mutations. By performing virtual mutations on these amino acid residues, a cofactor-dependent NADPH-to-NADH combination mutant, A22T / Q23E / L45A / N46E, was obtained and named TEAE. The results are as follows... Figure 2 As shown, the binding energy of the mutant TEAE to NADH was significantly reduced, demonstrating its increased affinity for NADH.

[0126] 2) By Hs 7β-HSDH was docked with UDCA, 7K-LCA and cofactor NADH, and the results were as follows: Figure 3 As shown, several semi-conserved sites in the substrate-binding region, including G20, N98, V99, G100, W101, G102, F152 and Y201, as well as the highly conserved catalytic triplet S151-Y164-K168, were identified, and virtual mutations were performed on these semi-conserved sites.

[0127] (3) Hs Screening for 7β-HSDH mutation hotspots

[0128] By virtually mutating the amino acid residues obtained from the screening in step (2) to alanine, the result is as follows: Figure 4 As shown, the binding energies of mutant sites V99, W101, G102, F152, and Y201 to protein molecules are significantly enhanced, proving that these sites are key residues for binding to the substrate 7K-LCA. These sites were selected as mutation hotspots for directed evolution.

[0129] Example 2: Construction and protein expression of 7β-hydroxysteroid dehydrogenase and its mutants

[0130] The specific steps are as follows:

[0131] (1) pGEX-6p- Hs7β- Construction of HSDH

[0132] Will come from Hyphomicrobium 7β-hydroxysteroid dehydrogenase sp. Hs 7β-HSDH (amino acid sequence as shown in SEQ ID NO.1), after codon optimization (nucleotide sequence as shown in SEQ ID NO.2), was ligated into plasmid pGEX-6p-1 (which can be sent to a company for synthesis), and named pGEX-6p- Hs 7β-HSDH.

[0133] First, let's take pGEX-6p- Hs 7 β- Using HSDH as a template, amplification was performed with primers such as L45A / N46E-F, L45A / N46E-R, A22T / Q23E-F, and A22T / Q23E-R to obtain a recombinant vector containing the combined mutant A22T / Q23E / L45A / N46E (named TEAE) that was converted to NADH in a cofactor-dependent manner: pGEX-6p-TEAE.

[0134] Table 1: Primers

[0135]

[0136] (2) Introduction of random mutations

[0137] The recombinant vector pGEX-6p-TEAE containing the combined mutant A22T / Q23E / L45A / N46E (named TEAE) obtained in step (1) was selected as the starting plasmid. Mutation hotspots V99, W101, G102, F152 and Y201 were selected as mutation origins. Then, random mutations were introduced at the corresponding sites by whole plasmid PCR using degenerate primers (NNK-MNN).

[0138] Table 2: Primer design for the mutant gene of 7β-hydroxysteroid dehydrogenase

[0139]

[0140] The PCR reaction mixture is as follows:

[0141] 1 μL upstream primer, 1 μL downstream primer, 1 μL template plasmid (pGEX-6p-TEAE), 5 μL 2×Primstar, 2 μL ddH2O. Pre-denaturation temperature: 98℃, 30 s; denaturation temperature: 98℃, 15 s; annealing temperature: 55℃, 30 s; extension temperature: 72℃, 90 s.

[0142] (3) Construction of recombinant strains

[0143] The wild-type recombinant expression vector pGEX-6p- obtained in step (1) Hs 7β-HSDH, pGEX-6p-TEAE, and recombinant expression vectors with introduced random mutations were transformed into [various gene expression vectors]. E. coli Recombinant expression strains were prepared from BL21 (DE3) competent cells: E . coli / pGEX-6p- Hs 7β-HSDH, E . coli / pGEX-6p-TEAE、 E . coli / pGEX-6p-TEAE-V99n、 E . coli / pGEX-6p-TEAE-W101n、 E . coli / pGEX-6p-TEAE-G102n、 E . coli / pGEX-6p-TEAE-F152n、 E . coli / pGEX-6p-TEAE-Y201n etc.

[0144] (4) Hs 7β-HSDH directed evolution

[0145] like Figure 5As shown, a high-throughput screening method was established to measure the reaction progress by the residual amount of NADH in the reaction mixture. First, the monoclonal recombinant strain with introduced random mutations in step (3) was cultured and induced to express in 96-well plates, with the wild type as a control. After centrifugation and removal of the supernatant, 1 mL of the reaction mixture (pH 7.5) was added in situ for resuspension. The reaction mixture contained 10 mM NADH and 10 mM 7K-LCA (dissolved in 200 mM Tris-NaCl buffer), and reacted at 30 °C for 12 hours with 200 oscillations per minute. After the reaction, the reaction mixture was heat-inactivated to terminate the enzyme activity. Subsequently, NaOH solution was added to the reaction mixture to adjust the pH to 11.0 to ensure complete dissolution of all substrates and products. The absorbance was measured at 340 nm using a BioTek-Cytation-5 cell imaging multimodal reader to determine the residual NADH concentration in the reaction mixture. Samples with significantly lower NADH content than the control group were screened out and re-screened by HPLC. Mutants with significantly increased ursodeoxycholic acid production were sequenced for further experimental analysis.

[0146] The results are as follows Figure 6 As shown, through three rounds of iterative saturation mutagenesis, recombinant strains of 7β-hydroxysteroid dehydrogenase mutants were obtained: TEAE, TEAE / V99T, TEAE / V99D, TEAE / W101I, TEAE / W101Q, TEAE / F152T, TEAE / F152L, TEAE / Y201F, TEAE / F152L / W101N, TEAE / F152L / W101V, TEAE / F152L / W101P, TEAE / F152L / Y201M, TEAE / F152L / W101N / Y201C, TEAE / F152L / W101N / Y201D, TEAE / F152L / W101N / Y201R, and TEAE / F152L / W101N / Y201F.

[0147] (4) Expression and purification of 7β-hydroxysteroid dehydrogenase and its mutants

[0148] The recombinant strain obtained in step (3) was inoculated into a solution containing 100 μg·mL⁻¹ -1 Seed culture was obtained by shaking ampicillin in LB liquid medium at 37°C and 200 rpm for 8-10 h.

[0149] The obtained seed cultures were inoculated at an inoculum size of 1% (v / v) to a concentration of 100 μg·mL⁻¹. -1 In ampicillin LB medium, cultured at 37°C with shaking at 200 rpm until OD. 600Once the culture volume reaches 0.6-0.8 (approximately 2 hours), add 0.1 mMIPTG and continue culturing at 20°C for 16 hours. Collect the cells by centrifugation at 8,000 × g, 4°C for 10 minutes.

[0150] The collected bacterial cells were resuspended in PBS buffer and sonicated on ice. The lysate was centrifuged at 12,000 × g at 4°C for 30 min. The supernatant was collected for SDS-PAGE analysis, and then purified using a GSTrap HP affinity column. After purification, PreScission protease was added and digested at 4°C for 10 h to remove the GST tag. The purified protein was analyzed by SDS-PAGE, and the results are shown below. Figure 7 As shown, the purified enzyme was obtained for the next step of analysis.

[0151] (5) Detection of enzyme activity and conversion rate:

[0152] The enzyme activity and conversion rate of the pure enzyme solutions of 7β-hydroxysteroid dehydrogenase and its mutant enzyme obtained in step (4) were detected respectively. The enzyme activity of different mutants was measured at pH 7.0 and 25℃ using 7K-LCA (7β-HSDH reduction of 7K-LCA to prepare UDCA) and UDCA as substrate (7β-HSDH oxidation of UDCA to prepare 7K-LCA).

[0153] Meanwhile, the yields of UDCA in each mutant were preliminarily determined under the conditions of pH 7.0, 25℃, 10 mM 7K-LCA, and 10 mM NADH. The results are shown in Table 3.

[0154] Table 3: Mutant enzyme activity and yield

[0155]

[0156] Example 3: Enzymatic properties of 7β-hydroxysteroid dehydrogenase and its mutants

[0157] The specific steps are as follows:

[0158] (1) Determination of optimal pH and temperature

[0159] Hs The optimal pH for 7β-HSDH and its mutants was determined by measuring specific enzyme activity (using NADH as a cofactor and 7K-LCA as a substrate) at a pH range of 6.0–10.0 (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0) and 25°C. The optimal temperature was determined by measuring specific enzyme activity (using NADH as a cofactor and 7K-LCA as a substrate) within a pH range of 7.5 and 20–45°C (20, 25, 30, 35, 40, 45°C). The results are as follows. Figure 8 As shown, Hs Both 7β-HSDH and its mutants exhibited the highest activity at pH 7.5 and 30°C.

[0160] Compared with the wild type, under optimal conditions (pH 7.5, 30℃), the enzyme activities of the mutants were significantly increased; the enzyme activities of TEAE / F152L, TEAE / F152L / W101N, TEAE, and wild type were 26.81 U·mg, respectively. -1 23.99 U·mg -1 8.28 U·mg -1 0.13 U·mg -1 The specific enzyme activities of the mutants TEAE / F152L and TEAE / F152L / W101N were increased by 3.23 times and 2.88 times, respectively, relative to the TEAE mutant, and by 206.23 times and 184.52 times, respectively, relative to the wild type.

[0161] (2) Thermal stability determination

[0162] The thermostability of the enzyme was evaluated at pH 7.5 and 30°C (using NADH as a cofactor and 7K-LCA as a substrate).

[0163] The results are as follows Figure 6 As shown in c, compared with the wild type, the enzyme activity of the mutants was significantly increased, but their half-life showed similar properties. The enzyme activity of all mutants and wild type decreased by about 50% after about 3 hours, and the wild type and mutants basically lost their activity after 8 hours of incubation.

[0164] (3) Measurement of dynamic parameters

[0165] Kinetic data were obtained at 30°C and pH 7.5 using a BioTek-Cytation-5 cell imaging multimodal reader. Measurements were performed in 10 mM PBS (pH 7.5) containing 2.0 mM NADH, using 50 μM of the enzyme and different concentrations of 7K-LCA or UDCA (0.1–10.0 mM). All values ​​represent the mean of three replicates.

[0166] The results are shown in Table 4:

[0167] Table 4: Kinetic parameters of 7β-hydroxysteroid dehydrogenase and its mutants

[0168]

[0169] Compared to the mutation origin TEAE (using 7K-LCA as a substrate), the mutant TEAE / F152L exhibits a 3.23-fold increase in enzyme activity and a higher catalytic efficiency for 7K-LCA. k cat / K m The enzyme activity of the mutant TEAE / F152L / W101N increased by 2.88 times, and its catalytic efficiency for 7K-LCA increased by 4.71 times. k cat / K m It increased by 4.25 times;

[0170] Compared to the wild type (using 7K-LCA as a substrate), the mutant TEAE / F152L exhibits a 206.23-fold increase in enzyme activity and a significantly higher catalytic efficiency for 7K-LCA. k cat / K m The enzyme activity of the mutant TEAE / F152L / W101N increased by 184.52 times, a 58.56-fold increase, and its catalytic efficiency for 7K-LCA increased. k cat / K m It increased by 52.91 times.

[0171] Example 4: Binding energy determination of 7β-hydroxysteroid dehydrogenase and its mutants

[0172] The binding energies and binding entropies of protein models of wild-type and mutant proteins with the substrate 7K-LCA were analyzed using Discovery Studio. The results are as follows: Figure 9 As shown in a, the results indicate that the binding free energy (ΔG) between the wild-type and the substrate 7K-LCA is -25.42 kcal·mol⁻¹. -1 The mutation origin TEAE is -33.1 kcal·mol⁻¹ -1 The binding free energy of the mutant TEAE / F152L and TEAE / F152L / W101N complexes with the substrate 7K-LCA was significantly reduced compared to that of the mutant TEAE, reaching -49.4 kcal·mol⁻¹. -1 and -47.7 kcal·mol -1 .

[0173] In contrast, the changes in complex entropy were small for both mutants, approximately -20.19 kcal·mol⁻¹. -1 This indicates that the mutant has a significantly enhanced affinity for 7K-LCA.

[0174] Example 5: Molecular dynamics simulation of 7β-hydroxysteroid dehydrogenase and its mutants

[0175] The specific steps are as follows:

[0176] (1) Root Mean Square Fluctuation analysis

[0177] The results are as follows Figure 9 As shown in b and d, the RMSF values ​​of the TEAE / F152L and TEAE / F152L / W101N mutant enzymes deviate little from those of the wild-type enzyme, but the rigidity of the loop region is slightly enhanced, which leads to a slight increase in enzyme stability.

[0178] (2) Root-mean-square deviation analysis

[0179] The results are as follows Figure 9 As shown in c, the root mean square deviation (RMSD) values ​​of the TEAE / F152L and TEAE / F152L / W101N mutant enzymes and the wild-type enzyme reach equilibrium at approximately 20 ns. In contrast, the wild-type... Hs The RMSD of the 7β-HSDH complex fluctuated more. Notably, the RMSD values ​​of both mutants were consistently lower than those of the wild-type complex, indicating that the mutants exhibited better stability compared to the wild-type enzyme.

[0180] (3) Analysis of the interaction forces between residues and ligands

[0181] The results are as follows Figure 9 As shown in Figure e, compared to TEAE, the mutation at W101 slightly enhances the hydrogen bonding interactions between adjacent residues N98 and R104 and the ligand. The mutation at the F152L site significantly strengthens the hydrogen bonding between Y164 / K168 in the catalytic ternary matrix and the substrate, while also slightly enhancing hydrophobic interactions and water bridging forces with the substrate. These changes in intermolecular forces alter the substrate binding pocket, making it more conformational to the ligand, thus positively impacting specific activity and catalytic efficiency. Figure 9 f- Figure 9 As shown in g, after the TEAE / F152 mutation, the spatial distance to 7K-LCA decreased from 4.0 Å to 3.5 Å; similarly, after the W101 mutation, the spatial distance to 7K-LCA decreased from 3.5 Å to 3.1 Å. This decrease in distance leads to a reduction in the volume of the active site, thereby promoting a tighter binding of the enzyme to the substrate 7K-LCA and consequently lowering the binding energy.

[0182] Example 6: Synthesis of ursodeoxycholic acid catalyzed by 7β-hydroxysteroid dehydrogenase and its mutant single enzyme.

[0183] The specific steps are as follows:

[0184] (1) Preparation of substrate solution and culture of wet cells:

[0185] 7K-LCA solution: Dissolve 7K-LCA in 200 mM Tris-NaCl buffer and adjust the pH to 7.5;

[0186] Preparation of wet cells: The recombinant bacteria containing TEAE / F152L, TEAE / F152L / W101N, TEAE, and WT, prepared according to step (3) of Example 2, were inoculated into cells containing 100 μg·mL⁻¹. -1 Seed culture of ampicillin was obtained by shaking in LB broth at 37°C and 200 rpm for 10 h. The seed culture was then inoculated at a rate of 1% (v / v) to a concentration of 100 μg / mL. -1 In ampicillin LB medium, cultured at 37°C with shaking at 200 rpm until OD. 600 After reaching 0.6~0.8 (about 2 h), 0.1 mM IPTG was added, and the mixture was cultured at 20°C for another 16 h to induce the expression of the target protein, and the fermentation broth was obtained.

[0187] The obtained fermentation broths were centrifuged at 12,000 × g and 4 °C, and then filtered to obtain wet cells. E . coli / pGEX-6p-TEAE / F152L、 E . coli / pGEX-6p-TEAE / F152L / W101N、 E . coli / pGEX-6p-TEAE、 E . coli / pGEX-6p-WT;

[0188] (2) Preparation of ursodeoxycholic acid:

[0189] The reaction mixture (10 mL) comprises: 200 mM Tris-NaCl buffer (pH 7.5), 40 mM NADH, 40 mM 7K-LCA, and 20 g·L⁻¹. -1 The cultured wet cells obtained in step (1).

[0190] The reaction was carried out at 30°C with shaking at 200 rpm for 2 h, and then the reaction was terminated by heating in boiling water for 10 min. Saturated NaOH solution was added to the reaction mixture to adjust the pH to 11 to ensure that the substrate and product were completely dissolved in the solution. The mixture was centrifuged at 12,000 × g for 10 min to remove degenerated cells and other insoluble impurities. The supernatant was collected and the product was analyzed by HPLC.

[0191] The results are as follows Figure 7 As shown, after reacting with 40 mM 7K-LCA and 40 mM NADH for 3 h, the yields of mutants TEAE / F152L and TEAE / F152L / W101N reached 61.8% and 72.1%, respectively. Compared with TEAE (43.6%), the yields increased by 18.2% and 28.4%, respectively, while the yield of wild type was only 3.21%.

[0192] The yields of TEAE / F152L, TEAE / F152L / W101N, TEAE, and WT are 24.72 mM, 28.84 mM, 17.44 mM, and 1.28 mM, respectively.

[0193] Example 7: Construction of a system for cofactor regeneration and efficient synthesis of ursodeoxycholic acid

[0194] Will Hs 7β-HSDH mutant and Bs GDH coupling for in-situ regeneration of coenzyme NAD + Together with NADH, it enables the efficient synthesis of ursodeoxycholic acid at low cofactor concentrations.

[0195] (1) Preparation of reaction substrate:

[0196] 1) 7K-LCA solution: Dissolve 7K-LCA in 200 mM Tris-NaCl buffer and adjust the pH to 7.5;

[0197] 2) Preparation of wet cells:

[0198] The recombinant bacteria containing TEAE / F152L, TEAE / F152L / W101N, TEAE, and WT, prepared according to step (3) of Example 2, were inoculated into a solution containing 100 μg·mL⁻¹. -1 Ampicillin was cultured in LB broth at 37°C with shaking at 200 rpm for 10 h to obtain seed culture. The seed culture was then inoculated at a rate of 1% (v / v) to a concentration of 100 μg / mL. -1 In ampicillin LB medium, cultured at 37°C with shaking at 200 rpm until OD. 600After reaching 0.6~0.8 (about 2 hours), 0.1 mM IPTG was added, and the mixture was cultured at 20°C for another 16 hours to obtain the fermentation broth.

[0199] The obtained fermentation broth was centrifuged at 12,000 × g and 4 °C, and then filtered to obtain the expression... Hs Wet cells containing 7β-HSDH mutant or wild-type enzyme: E . coli / pGEX-6p-TEAE / F152L、 E . coli pGEX-6p-TEAE / F152L / W101N E . coli / pGEX-6p-TEAE、 E . coli / pGEX-6p-WT;

[0200] 3) Expression Bs Preparation of recombinant GDH cells:

[0201] Will Bacillus sp. source Bs GDH (SEQ ID NO.5) was ligated into the pET28a vector to construct the recombinant vector pET28a- Bs GDH (can be sent to the company for synthesis). The recombinant vector is converted to... E. coli Recombinant cells were obtained from BL21 (DE3) competent cells. E. coli / pET28a- Bs GDH.

[0202] The recombinant bacteria, validated by sequencing, were inoculated into a solution containing 50 μg / mL. -1 Kanamycin was cultured in LB liquid medium at 37°C and 200 rpm with shaking for 10 h to obtain a seed culture. The seed culture was then inoculated at a rate of 1% (v / v) to a concentration of 50 μg / mL. -1 In LB medium containing kanamycin, cultured at 37°C and 200 rpm with shaking until OD. 600 After reaching 0.6~0.8 (about 2 hours), add 0.1 mM IPTG and continue culturing at 20°C for 16 hours to obtain the fermentation broth;

[0203] The obtained fermentation broth was centrifuged at 12,000 × g and 4 °C, then filtered to obtain the expression... Bs GDH wet cells;

[0204] (2) Preparation of ursodeoxycholic acid:

[0205] The reaction mixture (10 mL) comprises 200 mM Tris-NaCl buffer (pH 7.5), 1 mM NADH, 40 mM 7K-LCA, 60 mM glucose, and 20 g·L⁻¹. -1 Express Hs Wet cells containing 7β-HSDH mutant or wild-type enzyme ( E . coli / pGEX-6p-TEAE / F152L、 E . coli / pGEX-6p-TEAE / F152L / W101N、 E . coli / pGEX-6p-TEAE、 E . coli / pGEX-6p-WT) and 20 g·L -1 Post-culture expression Bs GDH wet cells.

[0206] The reaction was carried out at 30°C with shaking at 200 rpm for 2 h, and then the reaction was terminated by heating in boiling water for 10 min. Saturated NaOH solution was added to the reaction mixture to adjust the pH to 11 to ensure that the substrate and product were completely dissolved in the solution. The mixture was centrifuged at 12,000 × g for 10 min to remove degenerated cells, and the supernatant was used for product detection by HPLC.

[0207] The results are as follows Figure 10 As shown, the yields of the mutants TEAE / F152L and TEAE / F152L / W101N reached 82.4% and 92.8%, respectively, representing increases of 23.9% and 34.3% compared to TEAE, and increases of 20.6% and 20.7% compared to the reaction without cofactor regeneration. After three batches of reaction, the mutant TEAE / F152L / W101N maintained an average yield of 89.2%, with a theoretical space-time yield of 171 g / L / d, while the yield of the wild-type enzyme was only 6.15%.

[0208] The yields of TEAE / F152L, TEAE / F152L / W101N, TEAE, and WT are 37.12 mM, 32.96 mM, 23.60 mM, and 2.46 mM, respectively.

[0209] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A 7β-hydroxysteroid dehydrogenase mutant, characterized in that The mutant is, The phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID NO. 3 is mutated to leucine; or by mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3 to leucine, and simultaneously mutating the tryptophan at position 101 to asparagine, valine, or proline; or by mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3 to leucine, and simultaneously mutating the tyrosine at position 201 to methionine; Alternatively, the amino acid sequence of 7β-hydroxysteroid dehydrogenase as shown in SEQ ID NO. 3 is obtained by mutating the phenylalanine at position 152 to leucine, mutating the tryptophan at position 101 to asparagine, and mutating the tyrosine at position 201 to cysteine, aspartic acid, arginine or phenylalanine.

2. A gene encoding the mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. A recombinant cell expressing the mutant according to claim 1, carrying the gene according to claim 2, or carrying the recombinant vector according to claim 3.

5. The recombinant cell according to claim 4, characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

6. A recombinant enzyme catalyst comprising the sequence of the 7β-hydroxysteroid dehydrogenase mutant according to claim 1, characterized in that: The recombinase catalyst is any one of the following forms: (1) culturing the recombinant expression transformant of the 7β-hydroxysteroid dehydrogenase mutant and isolating the transformant cells containing the recombinant enzyme; (2) culturing the recombinant expression transformant of the 7β-hydroxysteroid dehydrogenase mutant, isolating the transformant cells containing the recombinant enzyme, and disrupting the transformant cells containing the recombinant enzyme to obtain a cell disrupted liquid; (3) culturing the recombinant expression transformant of the 7β-hydroxysteroid dehydrogenase mutant, isolating the transformant cells containing the recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant enzyme to obtain a freeze-dried enzyme powder.

7. A method for increasing the forward reaction activity of 7β-hydroxysteroid dehydrogenase, characterized in that: The method is: The phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID NO. 3 is mutated to leucine; or mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3 to leucine, and simultaneously mutating the tryptophan at position 101 to asparagine, valine, or proline; or mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3 to leucine, and mutating the tyrosine at position 201 to methionine; Alternatively, the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3 is mutated to leucine, the tryptophan at position 101 is mutated to asparagine, and the tyrosine at position 201 is mutated to cysteine, aspartic acid, arginine or phenylalanine.

8. A method for preparing ursodeoxycholic acid, characterized in that: The method comprises using 7-keto-lithocholic acid as a substrate and NADH as a cofactor, employing the mutant according to claim 1 or the recombinant cell according to claim 4 or 5 or the recombinant enzyme catalyst according to claim 6, and a recombinant cell containing glucose dehydrogenase to jointly catalyze the substrate and convert it into ursodeoxycholic acid; Or the method is, using 7-keto-lithocholic acid as a substrate, NADH as a cofactor, and the mutant according to claim 1 or the recombinant cell according to claim 4 or 5 or the recombinant enzyme catalyst according to claim 6 to catalyze the conversion of the substrate to prepare ursodeoxycholic acid.

9. The method according to claim 8, characterized in that The reaction system contains 7-keto-lithocholic acid, NADH and glucose.

10. The method according to claim 9, characterized in that The concentration of the 7-keto-lithocholic acid is 10 mM to 50 mM.

11. The method according to claim 9, characterized in that The concentration of NADH is 0.5 mM~50 mM.

12. The method according to claim 9, characterized in that The glucose concentration is 20 mM~100 mM.

13. The method according to claim 9, characterized in that The reaction conditions are pH 6.0~8.0 and temperature 20℃~40℃.

14. Use of the 7β-hydroxysteroid dehydrogenase mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, the recombinant cell according to claim 4 or 5, the recombinant enzyme catalyst according to claim 6, or the method according to any one of claims 7 to 13 in the preparation of ursodeoxycholic acid or a product containing ursodeoxycholic acid.

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