Method for efficiently synthesizing ursodesoxycholic acid by improving positive enzyme activity of 7 beta-hydroxysteroid dehydrogenase through directed evolution

Abstract

The invention discloses efficient synthesis of ursodesoxycholic acid by improving the positive enzyme activity of 7 beta-hydroxysteroid dehydrogenase through directed evolution, and belongs to the field of biological catalysis engineering. According to the invention, the 7beta-hydroxysteroid dehydrogenase mutant TEAE / F152L / W101N with improved positive reaction activity and improved affinity with a substrate is obtained. Compared with TEAE, the forward enzyme activity of the mutant enzyme is improved by 2.9 times, the reverse activity of the mutant enzyme is reduced by 3.6 times, the catalytic efficiency kcat / Km of the mutant enzyme to 7K-LCA is improved by 4.3 times, and compared with a wild type mutant TEAE / F152L / W101N, the enzyme activity of the mutant enzyme is improved by 206.23 times, and the catalytic efficiency of the mutant enzyme to 7K-LCA is improved by 58.56 times. The invention provides a universal technical platform for directed evolution of hydroxysteroid dehydrogenase, provides an excellent mutant enzyme for biological efficient synthesis of ursodesoxycholic acid, and lays a solid research foundation for efficient and low-cost preparation of ursodesoxycholic acid.

Description

Technical Field

[0001] The invention relates to directed evolution to improve the forward enzyme activity of 7β-hydroxysteroid dehydrogenase to efficiently synthesize ursodeoxycholic acid, and belongs to the field of biocatalytic engineering. Background Art

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

[0003] Ursodeoxycholic acid is mainly found in bear bile, but it can also be synthesized by chemical or biological methods. In vivo, the biosynthesis of ursodeoxycholic acid mainly involves the conversion of cholesterol into other compounds. First, cholesterol is converted into 7α-hydroxycholesterol, which is further converted into chenodeoxycholic acid (CDCA). Finally, chenodeoxycholic acid is converted into UDCA under the action of 7α / 7β-hydroxysteroid dehydrogenase (7α / 7β-HSDH) in the intestinal flora. The biosynthetic pathway of ursodeoxycholic acid mainly involves several key enzymes in the bile acid synthesis pathway, including cholesterol oxidase, 7α-hydroxylase, 7α-HSDH and 7β-HSDH, and 7α-HSDH and 7β-HSDH are the key enzymes that determine the conversion of chenodeoxycholic acid to ursodeoxycholic acid. There are currently three main in vitro synthetic pathways for ursodeoxycholic acid. The first pathway involves using primary hydrophilic bile acids as substrates, and then undergoing a hydroxyl dehydrogenation reaction through chemical oxidation to produce the first intermediate 3α, 7β, 12-oxolithocholic acid, and then producing the second intermediate 12-ketolithocholic acid under the action of 3α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, and finally converting it into ursodeoxycholic acid through a Wolff-Kishner reduction reaction; the second pathway produces 7,12-oxolithocholic acid catalyzed by 7α-hydroxysteroid dehydrogenase and 12α-hydroxysteroid dehydrogenase, and then, under the action of 7β-hydroxysteroid dehydrogenase, produces the second intermediate 12-ketolithocholic acid, and then converts it into ursodeoxycholic acid through a 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 option 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 usually much lower than those of 7α-HSDH. The activity of 7β-HSDH in catalyzing the reduction of 7K-LCA to UDCA (forward activity) is usually much lower than the activity of UDCA in oxidizing 7K-LCA to 7K-LCA (reverse activity). These factors make 7β-HSDH a ​​key limiting factor in the efficiency of UDCA synthesis under in vitro conditions. Summary of the invention

[0005] The present invention uses 7β-hydroxysteroid dehydrogenase (Hs7β-HSDH) from Hyphomicrobium sp. as a template enzyme, uses structural analysis and virtual mutation to screen mutation hotspots, and obtains a recombinant 7β-HSDH mutant strain with significantly improved forward reaction activity and enzyme activity through directed evolution. The activity of Hs7β-HSDH in catalyzing the conversion of 7K-LCA to UDCA is improved, and the enzymatic properties of 7β-HSDH and its mutants are characterized in detail, achieving efficient synthesis of UDCA from 7K-LCA. The present invention lays a solid foundation for the industrialized and efficient preparation of ursodeoxycholic acid.

[0006] The present invention provides a 7β-hydroxysteroid dehydrogenase mutant, wherein the mutant is obtained by mutating at least one of positions 101 and 152 of Hs7β-HSDH; the Hs7β-HSDH is an Hs7β-HSDH enzyme having an amino acid sequence as shown in SEQ ID NO.1.

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

[0008] In one embodiment of the present invention, the catalytic efficiency of the Hs7β-HSDH mutant is measured by using 7-keto-lithocholic acid as a substrate and NADH as a cofactor to react by detecting the change in the absorbance of NADH at 340 nm using an enzyme reader. One unit of enzyme activity (U) is defined as the amount of NADH that catalyzes the oxidation of 1 μmol of NADH to NAD per minute under the measurement conditions. + The amount of enzyme.

[0009] The present invention provides a Hs7β-HSDH mutant, wherein the 7β-hydroxysteroid dehydrogenase mutant is obtained by simultaneously mutating the 101st and / or 152nd positions of the Hs7β-HSDH with an amino acid sequence as shown in SEQ ID NO.3.

[0010] The present invention provides a Hs7β-HSDH mutant, wherein the 7β-hydroxysteroid dehydrogenase mutant is a mutant obtained by mutating the Hs7β-HSDH with an amino acid sequence as shown in SEQ ID NO.1: W101N, F152L, W101N / F152L.

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

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

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

[0014] The present invention provides a 7β-hydroxysteroid dehydrogenase mutant, wherein the mutant is obtained by mutating the valine at position 99 of the Hs7β-HSDH enzyme with an amino acid sequence as shown in SEQ ID NO.3 to threonine or aspartic acid;

[0015] or obtained by mutating the tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to isoleucine or glutamine;

[0016] or by mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to threonine or leucine;

[0017] or by mutating the tyrosine at position 201 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to phenylalanine;

[0018] or the 7β-hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID NO.3 is obtained by mutating the phenylalanine at position 152 to leucine, and simultaneously mutating the tryptophan at position 101 to asparagine, valine or proline;

[0019] or by mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to leucine, and mutating the tyrosine at position 201 to methionine;

[0020] or the amino acid sequence of Hs7β-HSDH 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;

[0021] In one embodiment of the present invention, the 7β-hydroxysteroid dehydrogenase having an amino acid sequence as shown in SEQ ID NO.3 is a combined mutant obtained by performing mutations on the basis of the Hs7β-HSDH mutant parent enzyme: A22T / Q23E / L45A / N46E;

[0022] SEQ ID NO.3:

[0023] MSYDAFADFRMIGHNVIITGGTENIGAGIARTLSGAGAKVMIADAEGDMAKETAAGIQKDTGNDCRGIKCDVTSLDDINAVVAETVKAFGGISTLVNNVGWGGRHDDPAAVTEEDFIASYKLNAISAYRMSM ACLPHLLKTKNATITNSGSFSASAPAYDILAYGTAKAALNQMMVSLAHMLAKKVRVNSVLIGTVMTAGYADAGIDPEMQERLSHPDNLTGRSGKPEDIANAMLWLCSPASGWVSGQIINVHGGGDVVRLFGE

[0024] The parent enzyme sequence of the Hs7β-HSDH mutant (SEQ ID NO.1) is:

[0025] MSYDAFADFFRMIGHNVIITGGAQNIGAGIARTLSGAGAKVMIADLNGDMAKETAAGIQKDTGNDCRGIKCDVTSLDDINAVVAETVKAFGGISTLVNNVGWGGRHDDPAAVTEEDFIASYKLNAISAYRMSM ACLPHLLKTKNATITNSGSFSASAPAYDILAYGTAKAALNQMMVSLAHMLAKKVRVNSVLIGTVMTAGYADAGIDPEMQERLSHPDNLTGRSGKPEDIANAMLWLCSPASGWVSGQIINVHGGGDVVRLFGE

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

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

[0028] ATGAGCTACGATGCCTTTGCAGATTTCCGTATGATTGGCCACAACGTTATCATCACTGGCGGTGCTCAGAACATTGGTGCGGGTATCGCGCGTACCCTGTCTGGTGCTGGCGCGAAAGTAATGATCGCCGACCTGAACGGCGATATGGCGAA AGAAACCGCGGCGGGCATTCAGAAAGACACGGGTAACGACTGCCGTGGCATCAAATGTGACGTGACGTCCCTGGATGACATCAACGCTGTTGTGGCGGAGACCGTTAAGGCATTCGGTGGCATTTCTACCCTGGTTAACAATGTTGGTTGGG GCGGCCGTCATGATGATCCGGCTGCTGTCACTGAGGAAGACTTCATCGCGTCTTACAAACTGAACGCAATCTCCGGTACCGCATGAGCATGGCCTGCCTGCCGCATCTGCTGAAAACCAAAAACGCCACCATCACCAACTCCGGTTCCTTT AGCGCTTCCGCACCTGCGTACGACATTCTGGCTTATGGCACCGCCAAAGCTGCTCTGAACCAAATGATGGTCTCTCTGGCTCATATGCTGGCGAAAAAAGTTCGCGTGAACTCCGTGCTGATCGGTACTGTAATGACGGCCGGCTATGCTGAT GCAGGCATCGACCCGGAAATGCAAGAACGTCTGAGCCATCCGGACAATCTGACCGGCCGTTCTGGTAAGCCAGAGGATATCGCGAACGCAATGCTGTGGCTGTGTTCCCCGGCGTCTGGCTGGGTTTCCGGGCCAGATTATCAACGTTCATGGTGGTGGTGACGTAGTTCGTCTGTTCGGTGAA

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

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

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

[0032] In one embodiment of the present invention, the recombinant vector uses pET28a plasmid, pRSF-Duet1 plasmid, pET21a plasmid or pGEX-6P-1 plasmid as an expression vector.

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

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

[0035] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as an expression host.

[0036] The present invention provides a recombinant vector carrying BsGDH (SEQ ID NO. 4) derived from Bacillus sp..

[0037] Bacillus sp.-derived BsGDH SEQ ID NO.4:

[0038] MYPDLKGKVVAITGASSGLGRAMAIRFGQEQAKVVINYYSNEKEAQTVKEEVQKAGGEAVIIQGDVTKEEDVKNIVQTAVKEFGTLDIMINNAGMENPVESHKMPLKDWNKVINTNLTGAFLGCREAIKY YVENDIQGNVINMSSVHEMIPWPLFVHYAASKGGIKLMTETLALEYAPKRIRVNNIGPGAINTPINAEKFADPVQKKDVESMIPMGYIGEPEEIAAVAVWLASKESSYVTGITLFADGGMTQYPSFQAGRG

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

[0040] ATGTATCCGGATTTAAAAGGAAAAGTCGTCGCCATTACAGGAGCATCATCAGGATTAGGAAGAGCGATGGCGATCCGCTTCGGGCAGGAGCAGGCGAAAGTCGTGATTAACTACTACAGTAATGAAAAAGAGGCTCAAACCGTAAAAGAAGAAGTTCAAAAAGCGGGCGGCGAAGCGGTCATTATTCAAGGTGAC GTTACAAAAGAAGAGGATGTCAAAAACATTGTGCAGACCGCGGTCAAGGAATTCGGCACATTAGATATCATGATCAACAACGCCGGCATGGAAAATCCGGTCGAGTCGCATAAAATGCCGCTAAAAGACTGGAACAAAGTCATCAACACCAACCTGACCGGCGCTTTTCTGGGATGCCGCGAAGCCATTAAATATT ACGTAGAGAATGATATTCAAGGAAACGTCATTAACATGTCGAGCGTACATGAAATGATTCCGTGGCCGCTGTTTGTCCACTATGCGGCAAGTAAAGGCGGCATTAAATTAATGACGGAAACATTGGCGCTTGAGTACGCGCCGAAGCGCATCCGTGTTAACAATATCGGGCCGGGCGCCATCAATACGCCGATCAA TGCGGAAAAGTTTGCGGATCCCGTTCAGAAAAAAGATGTGGAAAGCATGATTCCGATGGGGTATATCGGTGAGCCGGAAGAAATCGCGGCTGTCGCCGTCTGGCTTGCTTCAAAGGAATCAAGCTACGTGACCGGCATTACGCTGTTTGCTGACGGCGGAATGACACAATATCCGTCATTCCAGGCAGGCCGCGGT

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

[0042] The present invention also provides the enzymatic properties of Hs7β-HSDH of engineered Hyphomicrobium sp., including optimum temperature, pH, temperature stability, pH stability and kinetic parameters.

[0043] The present invention provides a method for improving the forward reaction activity of 7β-hydroxysteroid dehydrogenase and reducing the reverse activity. The method comprises the following steps: mutating the tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase with an amino acid sequence as shown in SEQ ID NO.3 into asparagine, and mutating the phenylalanine at position 152 into leucine.

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

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

[0046] (2) culturing the recombinant expression transformant, isolating transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid;

[0047] (3) culturing the recombinant expression transformant, isolating 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 lyophilized enzyme powder.

[0048] The present invention also provides a method for improving the forward reaction activity of 7β-hydroxysteroid dehydrogenase, the method comprising:

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

[0050] or mutating the tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID NO.3 to isoleucine or glutamine;

[0051] or mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID NO.3 to threonine or leucine;

[0052] or mutating the tyrosine at position 201 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to phenylalanine;

[0053] Or the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 is mutated to leucine, and the tryptophan at position 101 is mutated to asparagine, valine or proline;

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

[0055] Or the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with an amino acid sequence as 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.

[0056] The present invention also provides a method for preparing UDCA, wherein 7K-LCA is used as a substrate, 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 conversion of the substrate to prepare ursodeoxycholic acid.

[0057] Or the method is to use 7-keto-lithocholic acid as a substrate, NADH as a cofactor, glucose as a co-substrate, and use the above mutant or the above recombinant cell or the above recombinant enzyme preparation and a recombinant cell containing BsGDH to jointly catalyze the substrate conversion to prepare ursodeoxycholic acid.

[0058] In one embodiment of the present invention, the nucleotide sequence encoding the BsGDH is shown as 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 6.0-10.0 and temperature 20-45°C.

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

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

[0068] Beneficial Effects

[0069] (1) The present invention uses Hs7β-HSDH from Hyphomicrobium sp. as a template enzyme, analyzes its structure, and obtains a mutant TEAE / F152L with significantly improved activity in reducing 7K-LCA and a combined mutant TEAE / F152L / W101N with significantly improved activity in reducing 7K-LCA and significantly reduced activity in oxidizing UDCA. A 7β-hydroxysteroid dehydrogenase mutant with significantly improved forward catalytic activity and reduced reverse activity is obtained.

[0070] (2) The present invention studied the enzymatic properties of Hs7β-HSDH and its mutants TEAE / F152L and TEAE / F152L / W101N, determined the optimal temperature, pH and kinetic parameters of the enzyme, and verified that the enzyme activity and catalytic efficiency of the mutants were significantly improved. The enzyme activity of the mutants was increased by 3.2 and 2.9 times, respectively, compared with the starting point of the mutation, and the catalytic efficiency (k cat / K m ) were increased by 4.7 and 4.3 times, respectively; compared with the wild type, they were increased by 206.23 and 184.52 times, respectively, and the catalytic efficiency (k cat / K m ) were increased by 58.56 and 52.91 times respectively.

[0071] (3) Based on homology modeling, molecular docking analysis, affinity determination of ligand-enzyme complexes and dynamic simulation, the present invention has analyzed the molecular mechanism of the improved catalytic performance of Hs7β-HSDH mutants TEAE / F152L and TEAE / F152L / W101N, and provided a general technology for targeted improvement of catalytic performance such as substrate affinity of hydroxysteroid dehydrogenase.

[0072] (4) The present invention improves the catalytic activity of Hs7β-HSDH in catalyzing the reduction of 7K-LCA to UDCA, while reducing its activity in catalyzing the reduction of UDCA to 7K-LCA. In the reaction with 40 mM 7K-LCA and 40 mM NADH, the yields of mutants TEAE / F152L and TEAE / F152L / W101N reach 61.8% and 72.1%, respectively. Compared with the mutant starting enzyme TEAE, the yields are increased by 18.2% and 28.4%, respectively.

[0073] (5) The method of the present invention is used to construct a "coenzyme regeneration" reaction system, which is coupled with BsGDH and only 1 mM NADH is added to achieve efficient synthesis of UDCA. The yields of mutants TEAE / F152L and TEAE / F152L / W101N reach 82.4% and 92.8%, respectively. Compared with the mutant starting enzyme TEAE, the yields are increased by 23.9% and 34.3%, respectively. Compared with the reaction without adding cofactor regeneration, the yields are increased by 20.6% and 20.7%, respectively. After three batches of reactions, the average yield of mutant TEAE / F152L / W101N can still be maintained at 89.2%, and the theoretical space-time yield can reach 171 g / L / d. The whole-cell biocatalytic reaction is completed in one step, does not require cumbersome steps, does not generate unnecessary by-products, has mild reaction conditions, is environmentally friendly, and is a green and efficient method for biosynthesizing ursodeoxycholic acid. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0075] Figure 2 : Changes in affinity of mutant TEAE and wild-type enzyme for NADH.

[0076] Figure 3 :(a) Key amino acid residues of Hs7β-HSDH in the UDCA binding pocket; (b) Key amino acid residues of Hs7β-HSDH in the 7K-LCA binding pocket; (c) Key amino acid residues of Hs7β-HSDH in the NADH binding pocket; (d) Substrate and cofactor channels of Hs7β-HSDH.

[0077] Figure 4 : Energy changes when the amino acid near 7K-LCA in the substrate binding pocket of Hs7β-HSDH is mutated to alanine.

[0078] Figure 5 : A high-throughput method that measures the extent of reaction using the residual amount of cofactor NADH.

[0079] Figure 6 :Results of iterative saturation mutation; (a) the first round of saturation mutation; (b) the second round of saturation mutation; (c) the third round of saturation mutation; (d) comparison of enzyme activity and yield between the mutation starting point and the dominant mutant.

[0080] Figure 7 : SDS-PAGE analysis of wild-type and mutant enzyme expression; (a) SDS-PAGE analysis of wild-type and mutant supernatants; (b) SDS-PAGE analysis of wild-type and mutant pure enzymes; where, M, color pre-stained protein maker; 1, E.coli / pGEX-6p-Hs7β-HSDH; 2, E.coli / pGEX-6p-F152L; E.coli / pGEX-6p-F152L / W101N.

[0081] Figure 8 :Comparative enzyme activity determination of Hs7β-HSDH wild type and mutants; (a) optimal pH value; (b) optimal temperature; (c) thermal stability.

[0082] Fig. 9 : Dynamic simulation of Hs7β-HSDH and mutants; (a) Changes in the binding energy of Hs7β-HSDH and mutants with NADH and their binding entropy; (b) Changes in the root mean square fluctuation (RMSF) of the ligand; (c) Changes in the root mean square deviation (RMSD); (d) Changes in the root mean square fluctuation (RMSF) of Hs7β-HSDH; (e) Types of forces acting on amino acid residues in the binding pocket; (f) Spatial distance between Hs7β-HSDH wild type and 7K-LCA; (g) Spatial distance between Hs7β-HSDH mutants and 7K-LCA.

[0083] Fig.10 :(a) Time curve of UDCA synthesis catalyzed by Hs7β-HSDH and its mutants; (b) Yield determination of the system without coenzyme regeneration and the system with coenzyme regeneration; (c) UDCA yield of multiple batch reactions. DETAILED DESCRIPTION

[0084] Technical terms:

[0085] Ursodeoxycholic acid: 3α,7β-dihydroxy-5β-cholestane-24-acid, is a hydrophilic bile acid with multiple 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 hydrophobic and hydrophilic bile acids from damaging mitochondrial membranes and their cytotoxicity, inhibit hepatocyte apoptosis, significantly enhance liver function, and exhibit immunomodulatory and cytoprotective effects. It is one of the most commonly used prescription drugs in the clinical treatment of cholestatic diseases.

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

[0087] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a 7β-hydroxysteroid dehydrogenase mutant of the present invention and operably linked to control sequences that provide for its expression.

[0088] Fragment: The term "fragment" means a polypeptide that lacks one or more (e.g., several) amino acids at the amino and / or carboxyl terminus of the polypeptide; wherein the 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., not including the length of the zymogen region sequence) shown in SEQ ID NO.1.

[0089] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

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

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

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

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

[0094]

[0095] Reaction formula of the present invention is as follows:

[0096]

[0097] The culture medium involved in the following examples is as follows:

[0098] LB liquid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, natural pH.

[0099] The detection methods involved in the following embodiments are:

[0100] The detection method of 7β-hydroxysteroid dehydrogenase activity is:

[0101] The enzyme activity of 7β-HSDH in reducing 7K-LCA or oxidizing UDCA was measured at 30°C by measuring the absorbance change rate of NADH at 340nm. Enzyme activity assay system: 250μL reaction volume, 2mM NADH and 10mM 7K-LCA were added to 0.1M PBS buffer (pH7.5), incubated in a 30°C metal bath for 3min, 50μL of pure enzyme at an appropriate concentration was added, and the absorbance change at 340nm was scanned using the Bio-Tek Cytation 5 Cell Imaging Multifunctional Detection System.

[0102] Enzyme activity is defined as: under the above conditions, catalyzing 1 μmol NADH per minute or oxidizing NADH to NAD per minute + The amount of enzyme is defined as one unit U.

[0103] The calculation formula of enzyme activity is: enzyme activity (U) = EW × V × 10 3 / (6220×0.625).

[0104] The calculation formula of specific activity is: specific activity (U·mg -1 ) = enzyme activity (U) / protein amount (mg).

[0105] Where, EW: change in absorbance at 340 nm within 1 min; V: volume of reaction solution (mL); 6220: molar extinction coefficient (L·mol -1 cm -1 );0.5:optical path distance(cm).

[0106] Calculation of kinetic parameters:

[0107] According to the Michaelis equation v = V max ×[S] / K m +[S], calculate the K of the enzyme for different substrates m value.

[0108] Where, v: reaction rate (U·mg -1 );V max :Maximum reaction rate (U·mg -1 ); [S]: substrate concentration (mM); K m :The reaction speed v reaches half 1 / 2V max The substrate concentration at .

[0109] When the substrate concentration is saturated, k cat =V max / Et, calculate k cat value.

[0110] Among them, V max : maximum reaction rate; Et: enzyme concentration.

[0111] Determination of kinetic parameters:

[0112] The total reaction volume was 250 μL. 0.1 M PBS buffer (pH 7.5) and 10 mM 7K-LCA were added. The temperature was kept at 30 °C for 2 min. 50 μL of pure enzyme at an appropriate concentration was added. The specific enzyme activity under different concentrations (0.1-10 mM) of substrate 7K-LCA or product UDCA was measured. The enzyme kinetic curve was fitted by GraphPad software. The substrate concentration and specific enzyme activity were used as the horizontal and vertical coordinates, respectively. The Michaelis-Menten equation model was selected for nonlinear fitting to obtain V max Value and K m Value, obtained by data conversion cat Value and k cat / K m Values ​​(Table 4).

[0113] The high performance liquid chromatography (HPLC) detection method of ursodeoxycholic acid content is:

[0114] Agilent 1260 liquid chromatograph and Accucore-C18 LC column were used for detection, with 60% acetonitrile solution (containing 0.1% phosphoric acid) as the mobile phase and a flow rate of 1 mL min -1 , detection temperature 30°C, detection at UV 195nm. The substrate and product standards were dissolved in methanol or acetonitrile solution for detection to obtain the standard curves of UDCA and 7K-LCA.

[0115] Example 1: Mining of 7β-hydroxysteroid dehydrogenase and virtual screening of key residues for cofactor-dependent mutations

[0116] The specific steps are as follows:

[0117] (1) Discovery of Hs7β-HSDH

[0118] 7β-HSDH from different sources such as Hyphomicrobium sp., Mediterraneibacter sp., Eubacterium sp., Ruegeria sp., Stanieria sp. and Cereibacter sp. were screened, and the enzymatic activity of 7β-hydroxysteroid dehydrogenase and the initial conversion rate at 10 mM 7K-LCA were determined. The results are as follows: Figure 1 As shown, Hs7β-HSDH from Hyphomicrobium sp. showed the highest catalytic activity (8.4 U·mg -1 ) and initial conversion rate (48.9%), the enzyme was selected as the research object.

[0119] (2) Structural analysis of Hs7β-HSDH

[0120] 1) By docking Hs7β-HSDH with NADPH and NADH, several semi-conserved sites in the cofactor binding region, such as A22, Q23, N24, G26, D44, L45, and N46, were selected as potential key amino acid residues affecting cofactor binding, and these sites were selected for virtual mutation; by virtually mutating the above amino acid residues, a combined mutant A22D / Q23A / L45A / N46E, whose cofactor dependence was changed from NADPH to NADH, was obtained and named TEAE. The results are shown in the figure. Figure 2 As shown, the binding energy of mutant TEAE to NADH was significantly reduced, proving that its affinity for NADH was improved;

[0121] 2) By docking Hs7β-HSDH with UDCA, 7K-LCA and cofactor NADH, the results are as follows Figure 3 As shown, several semi-conserved sites, such as G20, N98, V99, G100, W101, G102, F152 and Y201, present in the substrate binding region, and the highly conserved catalytic triad S151-Y164-K168 were identified, and these semi-conserved sites were selected for virtual mutation.

[0122] (3) Screening of Hs7β-HSDH mutation hotspots

[0123] By virtually mutating the amino acid residues obtained by screening in step (2) to alanine, the results are as follows Figure 4 As shown, the binding energy of mutants V99, W101, G102, F152 and Y201 sites to the protein molecule showed significant improvement, proving that these sites are key residues for binding to the substrate 7K-LCA, and these sites were selected as mutation hotspots for directed evolution.

[0124] Example 2: Construction and protein expression of 7β-hydroxysteroid dehydrogenase and its mutants

[0125] The specific steps are as follows:

[0126] (1) Construction of pGEX-6p-Hs7β-HSDH

[0127] The 7β-hydroxysteroid dehydrogenase Hs7β-HSDH from Hyphomicrobium sp. (amino acid sequence as shown in SEQ ID NO.1) was codon-optimized (nucleotide sequence as shown in SEQ ID NO.2) and connected to the plasmid pGEX-6p-1 (which can be sent to the company for synthesis), and named pGEX-6p-Hs7β-HSDH.

[0128] First, pGEX-6p-Hs7β-HSDH was used as a template and primers L45A / N46E-F, L45A / N46E-R, A22D / Q23A-F, A22D / Q23A-R were used for amplification to obtain a recombinant vector containing a combined mutant A22T / Q23E / L45A / N46E (named TEAE) that was converted to NADH in a cofactor-dependent manner: pGEX-6p-TEAE;

[0129] Table 1: Primers

[0130]

[0131] (2) Introduction of random mutations

[0132] 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, and the mutation hotspots V99, W101, G102, F152 and Y201 were selected as the mutation starting points. Then, random mutations were introduced at the corresponding sites by whole plasmid PCR using degenerate primers (NNK-MNN);

[0133] Table 2: Primer design for 7β-hydroxysteroid dehydrogenase mutant enzyme gene

[0134]

[0135] The PCR reaction mixture system is as follows:

[0136] Upstream primer 1 μL, downstream primer 1 μL, template plasmid (pGEX-6p-TEAE) 1 μL, 2×Primstar 5 μL, ddH2O 2 μL. Pre-denaturation temperature 98°C, 30 s, denaturation temperature 98°C, 15 s, annealing temperature 55°C, 30 s, extension temperature 72°C, 90 s.

[0137] (3) Construction of recombinant strains

[0138] The wild-type recombinant expression vectors pGEX-6p-Hs7β-HSDH, pGEX-6p-TEAE and the recombinant expression vectors introduced with random mutations obtained in step (1) were transformed into E. coli BL21 (DE3) competent cells, respectively, to prepare recombinant expression strains: E. coli / pGEX-6p-Hs7β-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.

[0139] (4) Directed evolution of Hs7β-HSDH

[0140] like Figure 5 As shown in the figure, 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 introduced with random mutations in step (3) was cultured and induced for expression in a 96-well plate, and the wild type was used as a control. After centrifugation and removal of the supernatant, 1 mL of the reaction mixture (pH 7.5) was added in situ for resuspending. The reaction mixture included 10 mM NADH and 10 mM 7K-LCA (dissolved in 200 mM Tris-NaCl buffer solution) and reacted at 30°C for 12 hours with 200 oscillations per minute. After the reaction was completed, 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 that all substrates and products were completely dissolved. The absorbance was measured at a wavelength of 340 nm using a BioTek-Cytation-5 cell imaging multimode 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 rescreened by HPLC, and mutants with significantly increased ursodeoxycholic acid production were sequenced for further experimental analysis.

[0141] The results are as follows Figure 6As shown, through three rounds of iterative saturation mutagenesis, the recombinant strains of 7β-hydroxysteroid dehydrogenase mutants: 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, TEAE / F152L / W101N / Y201F were obtained.

[0142] (4) Expression and purification of 7β-hydroxysteroid dehydrogenase and its mutants

[0143] The recombinant strain obtained in step (3) was inoculated into a solution containing 100 μg mL -1 In LB liquid medium containing ampicillin, shake and culture at 37°C, 200 rpm for 8-10 hours to obtain seed solution;

[0144] The obtained seed solution was inoculated at a 1% (v / v) inoculum until 100 μg mL -1 The culture was incubated in ampicillin LB medium at 37°C with shaking at 200 rpm until OD 600 After reaching 0.6-0.8 (about 2 hours), 0.1 mM IPTG was added and culture was continued at 20°C for 16 hours. The cells were collected by centrifugation at 8,000×g and 4°C for 10 minutes.

[0145] The collected bacteria were resuspended in PBS buffer, and the cells were broken with an ultrasonic disruptor under ice bath conditions. The broken liquid was centrifuged at 12,000×g and 4°C for 30 minutes, and the supernatant was analyzed by SDS-PAGE. The supernatant was purified with a GSTrap HP affinity column. After purification, PreScission protease was added to cleave at 4°C for 10 hours to cut and remove the GST tag. The purified protein was analyzed by SDS-PAGE. The results are as follows: Figure 7 As shown, the pure enzyme was obtained for the next step of analysis.

[0146] (5) Detection of enzyme activity and conversion rate:

[0147] The enzyme activity and conversion rate of the pure enzyme solution of 7β-hydroxysteroid dehydrogenase and its mutant enzyme obtained in step (4) were detected respectively. The enzyme activities of different mutants were measured at pH 7.0 and 25°C with 7K-LCA (7β-HSDH reducing 7K-LCA to prepare UDCA) and UDCA as substrates (7β-HSDH oxidizing UDCA to prepare 7K-LCA);

[0148] At the same time, the yield of UDCA of each mutant was preliminarily detected under the conditions of pH 7.0, 25°C, 10mM 7K-LCA, and 10mM NADH. The results are shown in Table 3:

[0149] Table 3: Mutant enzyme activity and yield

[0150]

[0151]

[0152] Example 3: Enzymatic properties of 7β-hydroxysteroid dehydrogenase and its mutants

[0153] The specific steps are as follows:

[0154] (1) Determination of optimal pH and temperature

[0155] The optimum pH of Hs7β-HSDH and its mutants was determined by measuring the specific enzyme activity (with NADH as cofactor and 7K-LCA as substrate) at pH 6.0-10.0 (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0) and 25°C. The optimum temperature was determined by measuring the specific enzyme activity (with NADH as cofactor and 7K-LCA as substrate) at pH 7.5 and a temperature range of 20-45°C (20, 25, 30, 35, 40, 45). The results are shown in Table 1. Figure 8 As shown, Hs7β-HSDH and its mutants had the highest activity at pH 7.5 and 30°C.

[0156] Compared with the wild type, under the optimal conditions (pH 7.5, 30°C), the enzyme activities of the mutants were significantly higher than those of the wild type; the enzyme activities of TEAE / F152L, TEAE / F152L / W101N, TEAE, and the wild type were 26.81 U·mg -1 、23.99U·mg -1 、8.28U·mg -1 , 0.13U·mg -1The specific enzyme activities of mutants TEAE / F152L and TEAE / F152L / W101N were increased by 3.23 and 2.88 times, respectively, compared with the mutation starting point TEAE mutant, and by 206.23 and 184.52 times, respectively, compared with the wild type.

[0157] (2) Thermal stability determination

[0158] The thermal stability of the enzyme was evaluated at pH 7.5 and 30°C (with NADH as cofactor and 7K-LCA as substrate);

[0159] The results are as follows Figure 6 As shown in Figure c, compared with the wild type, the enzyme activities of the mutants were significantly improved, but their half-lives showed similar properties. The enzyme activities of all mutants and the wild type decreased by about 50% in about 3 hours, and the wild type and mutants basically lost their activity after incubation for 8 hours.

[0160] (3) Determination of kinetic parameters

[0161] Kinetic data were obtained using a BioTek-Cytation-5 cell imaging multimode reader at 30°C and pH 7.5. The assay was performed in 10 mM PBS (pH 7.5) containing 2.0 mM NADH, and 50 μM of enzyme and different concentrations of 7K-LCA or UDCA (0.1-10.0 mM) were used. All values ​​represent the average of three replicates.

[0162] The results are shown in Table 4:

[0163] Table 4: Kinetic parameters of 7β-hydroxysteroid dehydrogenase and its mutants

[0164]

[0165] Compared with the mutant starting point TEAE (with 7K-LCA as substrate), the enzyme activity of the mutant TEAE / F152L increased by 3.23 times, and the catalytic efficiency k cat / K m The enzyme activity of mutant TEAE / F152L / W101N increased by 4.71 times, and the catalytic efficiency k of 7K-LCA increased by 2.88 times. cat / K m Improved by 4.25 times;

[0166] Compared with the wild type (with 7K-LCA as substrate), the enzyme activity of mutant TEAE / F152L increased by 206.23 times, and the catalytic efficiency of 7K-LCA was cat / K mThe enzyme activity of mutant TEAE / F152L / W101N increased by 58.56 times, and the catalytic efficiency of 7K-LCA increased by 184.52 times. cat / K m Increased by 52.91 times.

[0167] Example 4: Binding energy determination of 7β-hydroxysteroid dehydrogenase and its mutants

[0168] Discovery Studio was used to analyze the binding energy and binding entropy of the protein model of the wild type and mutant with the substrate 7K-LCA complex. Fig. 9 The results show that the binding free energy (ΔG) of the wild type with the substrate 7K-LCA is -25.42 kcal·mol -1 The mutation starting point TEAE is -33.1 kcal·mol -1 Compared with the mutant TEAE, the binding free energy of the mutants TEAE / F152L and TEAE / F152L / W101N to the substrate 7K-LCA complex was significantly reduced, which was -49.4 kcal·mol -1 and -47.7 kcal·mol -1 .

[0169] In contrast, the changes in complex entropy for both mutants were small, approximately -20.19 kcal·mol -1 This indicates that the affinity of the mutant to 7K-LCA is significantly improved.

[0170] Example 5: Molecular dynamics simulation of 7β-hydroxysteroid dehydrogenase and its mutants

[0171] The specific steps are as follows:

[0172] (1)Root Mean Square Fluctuation analysis

[0173] The results are as follows Fig. 9 As shown in b and d, the RMSF values ​​of the TEAE / F152L and TEAE / F152L / W101N mutant enzymes deviate slightly from the RMSF values ​​of the wild-type enzyme, but the rigidity within the loop region is slightly enhanced, which leads to a slight improvement in the stability of the enzyme.

[0174] (2) Root-mean-square deviation analysis

[0175] The results are as follows Fig. 9As 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 reached equilibrium at about 20 ns. In contrast, the RMSD of the wild-type Hs7β-HSDH complex fluctuated more. It is worth noting that the RMSD values ​​of the two mutants were always lower than that of the wild-type complex, indicating that the mutants have better stability relative to the wild-type enzyme.

[0176] (3) Analysis of the interaction between residues and ligands

[0177] The results are as follows Fig. 9 As shown in Figure e, compared with TEAE, the mutation of W101 leads to a slight enhancement of the hydrogen bonding interaction between the adjacent residues N98 and R104 and the ligand. The mutation of the F152L site significantly strengthens the hydrogen bonding between Y164 / K168 in the catalytic triad and the substrate, and also slightly enhances the hydrophobic interaction and water bridge forces between the substrate and the substrate. These changes in intermolecular forces change the substrate binding pocket, making it more consistent with the conformation of the ligand, which has a positive effect on improving specific activity and catalytic efficiency. Fig. 9 As shown in fg, after the mutation of TEAE / F152, the spatial distance from 7K-LCA changes from Reduce to Similarly, after the mutation of W101, the spatial distance from 7K-LCA changed from Reduce to The reduction in distance leads to a decrease in the volume of the active site, which promotes a tighter binding between the enzyme and the substrate 7K-LCA, thereby reducing the binding energy.

[0178] Example 6: Synthesis of ursodeoxycholic acid catalyzed by 7β-hydroxysteroid dehydrogenase and its mutants

[0179] The specific steps are as follows:

[0180] (1) Preparation of substrate solution and culture of wet cells:

[0181] 7K-LCA solution: Dissolve 7K-LCA in 200 mM Tris-NaCl buffer and adjust the pH to 7.5;

[0182] Preparation of wet cells: The recombinant bacteria containing TEAE / F152L, TEAE / F152L / W101N, TEAE, and WT prepared according to the method of step (3) of Example 2 were inoculated into 100 μg mL -1 The cells were shaken and cultured in LB liquid medium containing ampicillin at 37°C and 200 rpm for 10 h to obtain seed solutions. The obtained seed solutions were inoculated at a 1% (v / v) inoculation volume until 100 μg mL-1 The culture was incubated in ampicillin LB medium 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 cultured at 20 °C for 16 h to induce the expression of the target protein, and fermentation broth was obtained respectively;

[0183] The obtained fermentation broth was 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, and E. coli / pGEX-6p-WT;

[0184] (2) Preparation of ursodeoxycholic acid:

[0185] The reaction mixture (10 mL) included: 200 mM Tris-NaCl buffer (pH 7.5), 40 mM NADH, 40 mM 7K-LCA and 20 g·L -1 The cultured wet cells obtained in step (1).

[0186] The reaction was carried out at 30°C with shaking at 200 rpm for 2 h, then heated in boiling water for 10 min to terminate the reaction, and 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 denatured cells and other insoluble impurities, and the supernatant was used to detect the product by HPLC.

[0187] The results are as follows Figure 7 As shown, after 3 h of reaction with 40 mM 7K-LCA and 40 mM NADH, the yields of mutants TEAE / F152L and TEAE / F152L / W101N reached 61.8% and 72.1%, respectively, which were increased by 18.2% and 28.4% compared with TEAE (43.6%), respectively, while the yield of the wild type was only 3.21%.

[0188] The production of TEAE / F152L, TEAE / F152L / W101N, TEAE, and WT were 24.72mM, 28.84mM, 17.44mM, and 1.28mM, respectively.

[0189] Example 7: Construction of a cofactor regeneration and efficient synthesis system for ursodeoxycholic acid

[0190] Coupling of the Hs7β-HSDH mutant to BsGDH for in situ regeneration of the coenzyme NAD +and NADH, achieving efficient synthesis of ursodeoxycholic acid at low cofactor concentrations.

[0191] (1) Preparation of reaction substrate:

[0192] 1) 7K-LCA solution: Dissolve 7K-LCA in 200 mM Tris-NaCl buffer and adjust the pH to 7.5;

[0193] 2) Preparation of wet cells:

[0194] The recombinant bacteria containing TEAE / F152L, TEAE / F152L / W101N, TEAE, and WT prepared according to step (3) of Example 2 were inoculated into 100 μg mL -1 The cells were shaken in LB liquid medium containing ampicillin at 37°C and 200 rpm for 10 h to obtain seed solutions. The obtained seed solutions were inoculated at a 1% (v / v) inoculation volume until 100 μg mL -1 The culture was incubated in ampicillin LB medium 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 culture was continued at 20 °C for 16 h to obtain fermentation broth;

[0195] The fermentation broth was centrifuged at 12,000 × g and 4°C and then filtered to obtain wet cells expressing Hs7β-HSDH mutant or wild-type enzyme: E. coli / pGEX-6p-TEAE / F152L, E. coli /

[0196] pGEX-6p-TEAE / F152L / W101N, E.coli / pGEX-6p-TEAE, E.coli / pGEX-6p-WT;

[0197] 3) Preparation of recombinant cells expressing BsGDH:

[0198] Bacillus sp.-derived BsGDH (SEQ ID NO. 5) was connected to the pET28a vector to construct the recombinant vector pET28a-BsGDH (which can be sent to the company for synthesis). The recombinant vector was transformed into E. coli BL21 (DE3) competent cells to obtain the recombinant cell E. coli / pET28a-BsGDH.

[0199] The recombinant bacteria verified by sequencing were inoculated into a solution containing 50 μg mL -1The kanamycin-containing LB liquid medium was cultured at 37°C, 200 rpm, and shaken for 10 h to obtain seed solution. The obtained seed solution was inoculated at a 1% (v / v) inoculation amount until 50 μg mL -1 The culture was incubated in LB medium containing kanamycin at 37°C with shaking at 200 rpm until the OD 600 After reaching 0.6-0.8 (about 2 hours), add 0.1 mM IPTG and continue to culture at 20°C for 16 hours to obtain fermentation broth;

[0200] The obtained fermentation broth was centrifuged at 12,000×g and 4°C and then filtered to obtain wet cells expressing BsGDH;

[0201] (2) Preparation of ursodeoxycholic acid:

[0202] The reaction mixture (10 mL) included 200 mM Tris-NaCl buffer (pH 7.5), 1 mM NADH, 40 mM 7K-LCA, 60 mM glucose, 20 g·L -1 Wet cells (E. coli /

[0203] 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 Wet cells expressing BsGDH after culture.

[0204] The reaction was carried out at 30°C and 200 rpm for 2 h, then heated in boiling water for 10 min to terminate the reaction, and 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 denatured cells, and the supernatant was used to detect the product by HPLC.

[0205] The results are as follows Fig.10 As shown, the yields of mutants TEAE / F152L and TEAE / F152L / W101N reached 82.4% and 92.8%, respectively, which were 23.9% and 34.3% higher than those of TEAE, and 20.6% and 20.7% higher than those of reactions without cofactor regeneration. After three batches of reactions, the average yield of mutant TEAE / F152L / W101N was still 89.2%, and the theoretical space-time yield could reach 171 g / L / d, while the yield of the wild-type enzyme was only 6.15%.

[0206] The production of TEAE / F152L, TEAE / F152L / W101N, TEAE, and WT were 37.12 mM, 32.96 mM, 23.60 mM, and 2.46 mM, respectively.

[0207] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A 7β-hydroxysteroid dehydrogenase mutant, characterized in that: The mutant is obtained by mutating the valine at position 99 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to threonine or aspartic acid; or obtained by mutating the tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to isoleucine or glutamine; or by mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to threonine or leucine; or by mutating the tyrosine at position 201 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to phenylalanine; or the 7β-hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID NO.3 is obtained by mutating the phenylalanine at position 152 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 as shown in SEQ ID NO.3 to leucine, and mutating the tyrosine at position 201 to methionine; Or 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 or 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 7β-hydroxysteroid dehydrogenase mutant sequence of claim 1, characterized in that: The recombinase catalyst is any one of the following forms: (1) culturing the recombinant expression transformant and isolating transformant cells containing the recombinase; (2) culturing the recombinant expression transformant, isolating transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid; (3) culturing the recombinant expression transformant, isolating 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 lyophilized enzyme powder.

7. A method for improving the forward reaction activity of 7β-hydroxysteroid dehydrogenase, characterized in that: The method is: The valine at position 99 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 is mutated to threonine or aspartic acid; or mutating the tryptophan at position 101 of the 7β-hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID NO.3 to isoleucine or glutamine; or mutating the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID NO.3 to threonine or leucine; or mutating the tyrosine at position 201 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 to phenylalanine; Or the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 is mutated to leucine, and the tryptophan at position 101 is mutated to asparagine, valine or proline; Or the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with the amino acid sequence as shown in SEQ ID NO.3 is mutated to leucine, and the tyrosine at position 201 is mutated to methionine; Or the phenylalanine at position 152 of the 7β-hydroxysteroid dehydrogenase with an amino acid sequence as 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, using the mutant according to claim 1 or the recombinant cell according to claim 4 or 5 or the recombinant enzyme preparation according to claim 6, and a recombinant cell containing glucose dehydrogenase to jointly catalyze the substrate to convert and prepare ursodeoxycholic acid; Or the method is to use 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 preparation 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; Preferably, the concentration of 7-keto-lithocholic acid is 10 mM to 50 mM; Preferably, the concentration of NADH is 0.5 mM to 50 mM; Preferably, the glucose concentration is 20mM to 100mM; Preferably, the reaction conditions are pH 6.0-8.0 and temperature 20°C-40°C; the reaction conditions are pH 6.5-10.0 and temperature 20-45°C.

10. 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 9 in the preparation of ursodeoxycholic acid or a product containing ursodeoxycholic acid.

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