A recombinant Bacillus subtilis engineered strain and its application in biological preparation of ursodeoxycholic acid

Abstract

The present invention provides a recombinant Bacillus subtilis engineering strain and its application in the biological preparation of ursodeoxycholic acid, belonging to the field of bioengineering technology. The present invention provides a recombinant Bacillus subtilis engineering strain that simultaneously and efficiently and stably expresses 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH). During biosynthesis, chenodeoxycholic acid is used as a substrate and combined with oxidized coenzyme NADP under the action of 7α-HSDH. + , the 7α-hydroxyl group in chenodeoxycholic acid is oxidized to a carbonyl group, and the resulting intermediate 7-keto-lithocholic acid is reduced to ursodeoxycholic acid under the action of 7β-HSDH and NADPH. The recombinant Bacillus subtilis engineered strain can efficiently and stably biosynthesize ursodeoxycholic acid, and greatly reduces the purification work of the product in industrial applications, greatly reducing production costs.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant Bacillus subtilis engineering strain and application thereof in biological preparation of ursodeoxycholic acid. Background Art

[0002] Ursodeoxycholic acid, also known as 3α,7β-dihydroxy-5β-cholestane-24-acid, ursodeoxycholic acid, hyodeoxycholic acid, isodeoxycholic acid, dihydroxycholic acid, 3β,6α-dihydroxycholic acid and dihydroxycholic acid, can be used to increase bile acid secretion, change the composition of bile, reduce cholesterol and cholesterol lipid in bile, and facilitate the gradual dissolution of cholesterol in gallstones. It is used for cholesterol stones that are not suitable for surgical treatment, but cannot dissolve bile pigment stones, mixed stones and stones that are not X-ray transparent. It also has certain therapeutic effects on cholecystitis, cholangitis and indigestion.

[0003] There are three main traditional sources of ursodeoxycholic acid: natural sources, chemical synthesis and biosynthesis. Natural sources violate animal protection laws and are limited in source; although chemical synthesis is simple to operate and reagents are cheap, problems such as chemical toxic reagents and chiral difficulties in the process have become bottlenecks in industrial technology transformation; biosynthesis has the advantages of being green, simple and efficient, but due to low enzyme stability and complicated protein extraction and purification, it is limited to laboratory operations and difficult to industrialize.

[0004] The existing synthesis methods are shown in Table 1.

[0005] Table 1 Methods for synthesizing ursodeoxycholic acid in the prior art

[0006]

[0007]

[0008] Industrial synthesis mainly uses animal bile acid, which is easy to obtain, as raw materials, and adopts chemical synthesis technology to synthesize UCDA; the operation is simple, the reagents are cheap, and it is relatively mature in industry, but the problems of chemical toxic reagents and chiral difficulties in the process (as shown in the table) have become bottlenecks in industrial technology transformation. The biosynthesis method of generating UDCA with bile acid and chenodeoxycholic acid as raw materials under the action of biological enzyme catalysis has the advantages of being green, simple, and efficient, and has become a research hotspot in recent years. In addition, in terms of raw material cost, the bile acid used abroad comes from cow bile, the commercial value of bile is between 0.1-0.4 US dollars / L, and bile acid accounts for about 0.7% (w / w) of bile. In China, a large amount of chicken and goose bile sources make the raw materials not limited to cow bile, and compared with cow bile, chicken and goose bile has a higher content of chenodeoxycholic acid. Therefore, the synthesis of UDCA with chenodeoxycholic acid as raw material under enzyme catalysis has considerable prospects.

[0009] At present, biological methods are used to synthesize ursodeoxycholic acid. The method of selective oxidation of cheap and readily available chenodeoxycholic acid by 7-OH and subsequent reduction has received much attention. However, in the two-step reaction, the 7-OH in the first step needs to be oxidized to carbonylation and then the reaction system is treated to inactivate the 7a-HSDH enzyme. Then, the intermediate 7-keto-lithocholic acid is used as a substrate to synthesize ursodeoxycholic acid under the action of 7β-HSDH enzyme. At the same time, the current biological method mainly uses Escherichia coli as a host cell (CN109722442B, CN113061637A) to synthesize UDCA, but the steps involved in this method, such as cell wall disruption and protein renaturation, are cumbersome and costly. In addition, the influence of Escherichia coli endotoxins brings great difficulties to large-scale industrial production.

[0010] Compared with Escherichia coli, Bacillus subtilis not only has the advantages of non-pathogenicity, no endotoxicity, low codon preference, high solubility of expressed proteins, good biological activity, and high secretion, but its single-layer membrane structure makes protein secretion easier. In addition, the use of fusion expression of protein secretion signal peptides in Bacillus subtilis can guide the target protein to be secreted extracellularly more smoothly, thereby optimizing the extracellular secretion of heterologous proteins, greatly reducing the purification work of the required protein products in industrial applications, simplifying operations, and reducing production costs. Summary of the invention

[0011] In view of this, the object of the present invention is to provide a recombinant Bacillus subtilis engineered strain, which can efficiently produce ursodeoxycholic acid by a one-pot method by constructing a strain containing the coding genes 7α-HSDH and 7β-HSDH for the synthesis of key ursodeoxycholic acid catalytic enzymes.

[0012] The invention provides a recombinant Bacillus subtilis engineering strain, which takes Bacillus subtilis as a host bacterium and comprises a recombinant vector expressing a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a recombinant vector expressing a codon-optimized 7β-hydroxysteroid dehydrogenase gene, or a recombinant vector expressing a fusion gene formed by the codon-optimized 7α-hydroxysteroid dehydrogenase gene and the 7β-hydroxysteroid dehydrogenase gene.

[0013] Preferably, the nucleotide sequence of the codon-optimized 7α-hydroxysteroid dehydrogenase gene is as shown in SEQ ID NO: 1;

[0014] The nucleotide sequence of the codon-optimized 7β-hydroxysteroid dehydrogenase gene is shown in SEQ ID NO: 2;

[0015] The nucleotide sequence of the fusion gene formed by the codon-optimized 7α-hydroxysteroid dehydrogenase gene and the 7β-hydroxysteroid dehydrogenase gene is shown in SEQ ID NO:3.

[0016] Preferably, the strain of Bacillus subtilis includes WB800N.

[0017] The invention provides application of the recombinant Bacillus subtilis engineering strain in biological preparation of ursodeoxycholic acid.

[0018] The present invention provides a method for biologically preparing ursodeoxycholic acid based on the recombinant Bacillus subtilis engineered strain, comprising the following steps:

[0019] With chenodeoxycholic acid as a substrate, the culture supernatant of the recombinant Bacillus subtilis engineering strain is subjected to a catalytic reaction under the action of coenzyme NADP to obtain ursodeoxycholic acid.

[0020] Preferably, the temperature of the catalytic reaction is 25-37°C.

[0021] Preferably, the catalytic reaction time is 24 to 72 hours.

[0022] Preferably, the culture supernatant of the recombinant Bacillus subtilis engineered strain is cultured as follows: inoculating the seed solution into a liquid LB culture medium, and culturing at 37°C and 200rpm for 5 to 6 hours; when the bacteria grow to the logarithmic growth phase, adding isopropyl-β-D-thiogalactopyranoside at a final concentration of 1 mM, and continuing to culture at 25°C to 30°C and 200rpm for 18 hours, separating the culture solution, and obtaining a culture supernatant containing 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase.

[0023] Preferably, the catalytic reaction system is to add 0.25-0.75 g chenodeoxycholic acid and 2-5 mg NADP to every 100 ml of the culture supernatant of the recombinant Bacillus subtilis engineered strain;

[0024] Preferably, 0.5 g of chenodeoxycholic acid and 2 mg of NADP are added to 100 ml of the culture supernatant of the recombinant Bacillus subtilis engineered strain.

[0025] Preferably, the catalytic reaction further includes separating the product; the method for separating the product is to adjust the pH of the system after the catalytic reaction to 4, separate the crude ursodeoxycholic acid, mix the crude ursodeoxycholic acid and ethyl acetate and reflux them, and filter to obtain ursodeoxycholic acid with a purity greater than 85%.

[0026] The present invention forms a fusion gene by codon-optimized 7α-hydroxysteroid dehydrogenase gene and 7β-hydroxysteroid dehydrogenase gene and expresses them simultaneously in Bacillus subtilis to obtain a fusion enzyme conjugate, thereby forming a one-pot method for preparing ursodeoxycholic acid, which can fully and effectively utilize NADP +The conversion of 7α-HSDH to NADPH saves energy consumption in cells and improves yield. The one-step synthesis method of the fusion enzyme conjugate not only realizes the self-circulation of NADP+ / NADPH, but also exceeds 85% in conversion efficiency. The ursodeoxycholic acid enzyme catalysis process includes two steps. The step catalyzed by 7α-HSDH requires the consumption of NADP+, while the step catalyzed by 7β-HSDH requires the consumption of NADPH. Therefore, in order to solve the above problems, the present invention constructs a 7α / β-HSDH fusion enzyme conjugate to realize the self-circulation of NADP+ / NADPH and the one-step method simplifies the operation steps, laying a foundation for realizing the one-step synthesis of UDCA and its industrial production.

[0027] The present invention is achieved by utilizing the following technical solutions:

[0028] A nucleotide sequence of 7α-HSDH derived from Bacteroides fragiles, 7β-HSDH derived from Ruminococcusgnavus, and a fusion of the optimized 7α-hydroxysteroid dehydrogenase gene and the 7β-hydroxysteroid dehydrogenase gene is shown in SEQ ID NO:1.

[0029] The present invention constructs an engineered strain of Bacillus subtilis that stably and efficiently expresses 7α / β-HSDH, and the codon-optimized 7α / β-HSDH gene can be efficiently expressed in Bacillus subtilis WB800N. Bacillus subtilis has a better exogenous secretion ability, and can directly secrete the synthesized ursodeoxycholic acid into the culture medium outside the cell without the need to break the host cell, which effectively solves the interference of complex intracellular components on the target component during cell breakage, thereby greatly simplifying the extraction and purification steps. The ursodeoxycholic acid enzyme catalysis process includes a step catalyzed by 7α-HSDH that consumes NADP + , and the step catalyzed by 7β-HSDH consumes NADPH. Therefore, in order to solve the above problems, the present invention constructs a 7α / β-HSDH fusion enzyme conjugate to achieve NADP+ / NADPH self-circulation and a one-step simplified operation step, with a conversion efficiency of more than 85%, laying a foundation for the one-step synthesis and industrial production of ursodeoxycholic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the process of synthesizing ursodeoxycholic acid using chenodeoxycholic acid as substrate;

[0031] Figure 2 This is the spectrum of 7α-HSDH carrier;

[0032] Figure 3 This is the spectrum of 7β-HSDH carrier;

[0033] Figure 4 This is the 7αβ-HSDH vector spectrum;

[0034] Figure 5 This is the restriction enzyme digestion verification map of α-HSDH EcoRI vector, where the marker is 10000bp;

[0035] Figure 6 This is the 7β-HSDH EcoRI restriction enzyme digestion verification map, where the marker is 10000bp;

[0036] Figure 7 This is the 7αβ-HSDH EcoRI restriction enzyme digestion verification diagram, where the marker is 10000bp. DETAILED DESCRIPTION

[0037] The invention provides a recombinant Bacillus subtilis engineering strain, which takes Bacillus subtilis as a host bacterium and comprises a recombinant vector expressing a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a recombinant vector expressing a codon-optimized 7β-hydroxysteroid dehydrogenase gene, or a recombinant vector expressing a fusion gene formed by the codon-optimized 7α-hydroxysteroid dehydrogenase gene and the 7β-hydroxysteroid dehydrogenase gene.

[0038]

[0039] In the present invention, the method for constructing the recombinant Bacillus subtilis engineered strain preferably comprises the following steps:

[0040] Constructing a recombinant vector expressing a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a recombinant vector expressing a codon-optimized 7β-hydroxysteroid dehydrogenase gene, or a recombinant vector expressing a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a 7β-hydroxysteroid dehydrogenase gene;

[0041] A recombinant vector expressing a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a recombinant vector expressing a codon-optimized 7β-hydroxysteroid dehydrogenase gene, or a recombinant vector comprising the codon-optimized 7α-hydroxysteroid dehydrogenase gene and the 7β-hydroxysteroid dehydrogenase gene, is transformed into Bacillus subtilis, and a recombinant Bacillus subtilis engineering strain is obtained through screening.

[0042] In the present invention, the method for constructing a recombinant vector comprising a fusion gene formed by expressing a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a 7β-hydroxysteroid dehydrogenase gene is preferably to clone the fusion gene formed by expressing a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a 7β-hydroxysteroid dehydrogenase gene into a backbone vector to obtain a recombinant vector.

[0043] The present invention has no particular limitation on the cloning method, and any cloning method known in the art may be used, such as homologous recombination method.

[0044] The present invention has no particular limitation on the transformation method, and the transformation method known in the art can be used, for example, the Spizizen transformation method. For details, see the following documents (Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81: 741-746; Li Ruifang, Xue Wenwen, Huang Liang, et al. Preparation of Bacillus subtilis competent cells and study on plasmid transformation methods [J]. Biotechnology Bulletin, 2011 (05): 227-230. DOI: 10.13560 / j.cnki.biotech.bull.1985.2011.05.035; Li Chunyan, Feng Fengzhao, Feng Lu, Cheng Yi, Cheng Xiaosong. Optimization of Spizizen transformation of wild-type Bacillus subtilis N4 [J]. Journal of Northeast Agricultural University, 2015, 46 (02): 78-82+108.)

[0045] In the present invention, the screening method is preferably to pick a single colony into 4 ml of liquid LB culture medium (4 μl of 20 mg / ml chloramphenicol has been added), and culture it in a shaker at 37°C and 200 rpm overnight; the overnight cultured bacterial solution is taken out for microscopic examination, genome quick extraction, and PCR verification of the target gene.

[0046] The invention provides application of the recombinant Bacillus subtilis engineering strain in biological preparation of ursodeoxycholic acid.

[0047] The present invention provides a method for biologically preparing ursodeoxycholic acid based on the recombinant Bacillus subtilis engineered strain, comprising the following steps:

[0048] With chenodeoxycholic acid as a substrate, a catalytic reaction is carried out under the action of the culture supernatant of a recombinant Bacillus subtilis engineering strain and NADP to obtain ursodeoxycholic acid.

[0049] In the present invention, the culture supernatant of the recombinant Bacillus subtilis engineered strain is cultured as follows: seed liquid is inoculated into liquid LB culture medium, and cultured at 37°C and 200rpm for 5-6h; when the bacterial body grows to the logarithmic growth phase, isopropyl-β-D-thiogalactopyranoside is added with a final concentration of 1mM, and cultured at 25°C to 30°C and 200rpm for 18h, the obtained bacterial liquid is centrifuged, and the precipitate is discarded to obtain a culture supernatant containing 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase.

[0050] In the present invention, the method for preparing the seed solution is preferably to inoculate the recombinant Bacillus subtilis engineered strain into LB culture medium, and culture it at 25-30° C. to the logarithmic phase to obtain the seed solution.

[0051] In the present invention, the catalytic reaction system is preferably 0.25-0.75g chenodeoxycholic acid (CDCA) and 2-5mg NADP are added to the culture supernatant of the recombinant Bacillus subtilis engineering strain per 100ml, and more preferably 0.5g chenodeoxycholic acid (CDCA) and 2mg NADP are added to the culture supernatant of the recombinant Bacillus subtilis engineering strain per 100ml. After the catalytic reaction is completed, it is also preferably included to separate the product. The method for separating the product is preferably to add hydrochloric acid to the catalytic reaction system to adjust it to acidity (pH=4) to promote the crystallization of ursodeoxycholic acid (UDCA) product, centrifuge at 10000rmp for 20min to remove protein and solution, distill and remove the remaining liquid to obtain UDCA crude product, add the obtained UDCA crude product and ethyl acetate to the reaction container, heat and reflux for 1 hour, cool to room temperature, and filter to obtain UDCA with a purity greater than 85%. The temperature of the catalytic reaction is preferably 25-37°C, more preferably 30°C. The catalytic reaction time is preferably 24 to 72 hours, more preferably 36 to 48 hours, and most preferably 42 hours.

[0052] In the present invention, the recombinant Bacillus subtilis engineered strain can stably and efficiently express 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH). During biosynthesis, chenodeoxycholic acid (CDCA) is used as a starting substrate, and under the action of 7α-hydroxysteroid dehydrogenase (7α-HSDH), oxidized coenzyme NADP is combined. + , the 7α-hydroxyl group in chenodeoxycholic acid is oxidized to a carbonyl group, thereby generating an intermediate product 7-keto-lithocholic acid (7-K-LCA); 7-keto-lithocholic acid is a substrate of 7β-hydroxysteroid dehydrogenase (7β-HSDH), and 7-K-LCA is reduced to ursodeoxycholic acid under the action of 7β-HSDH combined with reduced coenzyme NADPH. It can be seen that the 7α-HSDH and 7β-HSDH prepared by the recombinant Bacillus subtilis engineering strain provided by the present invention have high biological activity, can efficiently and stably biosynthesize ursodeoxycholic acid, and Bacillus subtilis is a non-pathogenic microorganism, its own metabolites are low-toxic and harmless, and the purification work of the product during industrial application is greatly reduced, the product separation step is simplified, the complexity of the operation is reduced, and the production cost is greatly reduced.

[0053] The following examples further illustrate the present invention but should not be construed as limiting the present invention.

[0054] Example 1

[0055] Synthesis of 7α-hydroxysteroid dehydrogenase (7α-HSDH) and / or 7β-hydroxysteroid dehydrogenase (7β-HSDH) and construction of their recombinant vectors

[0056] According to the codon preference of Bacillus subtilis, the genes of 7α-HSDH (from Bacteroides fragiles, GenBank: OGX95366.1) and 7β-HSDH (from Ruminococcus gnavus, GenBank: R9UAM1) were codon optimized. The 7α-HSDH sequence and 7β-HSDH sequence were mined by gene mining method, and the codons were optimized according to the protein sequence to synthesize genes (SEQ ID NO:1 and SEQ ID NO:2), which were constructed into T50 expression vectors respectively, and the gene insertion site was XbaI. At the same time, the two genes were fused to synthesize a fusion gene (SEQ ID NO:3), which was inserted into the T50 expression vector, and the gene insertion site was XbaI. The spectrum of the recombinant expression vector is shown in Figure 2 to Figure 4 .

[0057] The artificially synthesized gene fragments were used as templates, and the primer pairs 7α-50for / 7α-50rev were used to amplify the 7α-HSDH gene fragment, 7β-50for / 7β-50rev were used to amplify the 7β-HSDH gene fragment, and 7αβ-50for / 7αβ-50rev were used to amplify the fusion fragment formed by 7α-HSDH and 7β-HSDH. The amplification reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0058] Table 1 Primer sequence information

[0059]

[0060] Table 2 Reaction system

[0061]

[0062] Table 3 Reaction procedure

[0063]

[0064] After the target fragment was amplified, it was connected to the expression vector T50 by Gibson method, and the obtained recombinant vector was transferred into the E. coli cloning strain, and a single colony was cultured to extract the plasmid, and the recombinant plasmid was verified by double restriction digestion with EcoRI. Figure 5-Figure 7 As shown, it proves that the recombinant plasmid was successfully constructed.

[0065] Example 2

[0066] Construction of an engineered strain expressing the fusion gene 7αβ-HSDH

[0067] The recombinant plasmid obtained in Example 1 was transformed into Bacillus subtilis competent cells WB800N by the Spizizen transformation method, and cultured on a solid LB medium containing ampicillin. The monoclonal colonies were picked and cultured in a liquid medium. The recombinant bacteria after screening were examined under a microscope, the genome was extracted quickly, the recombinant plasmid was verified by TaQ enzyme, and protein induction expression was performed preliminarily. The three enzymes 7α-HSDH (32kDa), 7β-HSDH (33.7kDa), and 7αβ-HSDH (64.3kDa) were analyzed and identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results all proved that the genes were correctly linked to the expression vector.

[0068] 500 μl of Bacillus subtilis WB800N strain containing T50-7α / 7β-HSDH was added to 50 ml LB liquid medium, and 12.5 μl of 20 mg / ml chloramphenicol was added at the same time. The mixture was shaken and cultured overnight at 37°C 200 rpm as seed liquid. The overnight cultured seed liquid was taken out for microscopic examination, genome extraction, and PCR verification. The overnight cultured seed liquid was transferred to 500 ml LB fermentation medium containing 125 μl 20 mg / ml chloramphenicol at an inoculation volume of 1%, and cultured in a shaker at 37°C 200 rpm for 5-6 hours. When the bacterial cell volume grew to the logarithmic growth phase (OD 600 =0.6-1), add IPTG with a final concentration of 1 mM, induce for 18 h in a shaker at 30°C / 25°C at 200 rpm, centrifuge the induced bacterial solution at 4000 rpm for 30 min, discard the bacteria and keep the supernatant to obtain the supernatant containing 7α / 7β-HSDH.

[0069] Example 3

[0070] Method for converting ursodeoxycholic acid into recombinant bacterial fermentation broth

[0071] Take 100 ml of the supernatant containing 7α / 7β-HSDH prepared in Example 2, adjust the pH to 8.5 with NaOH, take 0.5 g of chenodeoxycholic acid and 2 mg of NADP + After being dissolved in 0.1 mol / L PBS (pH=8.0) solution, it was added dropwise to the supernatant, maintaining pH>7.5. The reaction progress was detected by thin layer chromatography. After 20 hours, the substrate reaction was complete. The reaction pH was adjusted to 6.0, and the precipitate was collected by filtration to obtain 0.45 g of ursodeoxycholic acid.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a recombinant Bacillus subtilis engineered strain in the biological preparation of ursodeoxycholic acid, wherein the recombinant Bacillus subtilis engineered strain uses Bacillus subtilis as a host bacterium and only contains a recombinant vector expressing a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a recombinant vector expressing a codon-optimized 7β-hydroxysteroid dehydrogenase gene, or only contains a recombinant vector expressing a fusion gene formed by a codon-optimized 7α-hydroxysteroid dehydrogenase gene and a 7β-hydroxysteroid dehydrogenase gene; the nucleotide sequence of the codon-optimized 7α-hydroxysteroid dehydrogenase gene is shown in SEQ ID NO: 1; The nucleotide sequence of the codon-optimized 7β-hydroxysteroid dehydrogenase gene is shown in SEQ ID NO: 2; The nucleotide sequence of the fusion gene formed by the codon-optimized 7α-hydroxysteroid dehydrogenase gene and the 7β-hydroxysteroid dehydrogenase gene is shown in SEQ ID NO: 3; The strain of Bacillus subtilis is WB800N.

2. A method for biologically preparing ursodeoxycholic acid based on the recombinant Bacillus subtilis engineered strain described in claim 1, characterized in that: The following steps are involved: With chenodeoxycholic acid as a substrate, the culture supernatant of the recombinant Bacillus subtilis engineering strain is subjected to a catalytic reaction under the action of the coenzyme NADP to obtain ursodeoxycholic acid.

3. The method according to claim 2, characterized in that: The temperature of the catalytic reaction is 25-37°C.

4. The method according to claim 2, characterized in that: The catalytic reaction time is 24 to 72 hours.

5. The method according to claim 2, characterized in that: The preparation method of the culture supernatant of the recombinant Bacillus subtilis engineering strain is as follows: seed liquid is inoculated into a liquid LB culture medium, and cultured at 37°C and 200rpm for 5-6h; when the bacteria grow to the logarithmic growth phase, isopropyl-β-D-thiogalactopyranoside with a final concentration of 1mM isopropyl-β-D-thiogalactopyranoside is added, and culture is continued for 18h at 25°C-30°C and 200rpm, and the culture liquid is separated to obtain a culture supernatant containing 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase.

6. The method according to claim 2, characterized in that: The catalytic reaction system is to add 0.25-0.75 g chenodeoxycholic acid and 2-5 mg NADP to every 100 ml of the culture supernatant of the recombinant Bacillus subtilis engineered strain; 0.5 g of chenodeoxycholic acid and 2 mg of NADP were added to 100 ml of the culture supernatant.

7. The method according to any one of claims 2 to 6, characterized in that: The method further comprises separating products after the catalytic reaction; the method for separating products comprises adjusting the pH value of the system after the catalytic reaction to 4, separating crude ursodeoxycholic acid, mixing the crude ursodeoxycholic acid and ethyl acetate for reflux, and filtering to obtain ursodeoxycholic acid with a purity greater than 85%.

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