7beta-HSDH enzyme mutant derived from active ruminococcus and application of 7beta-HSDH enzyme mutant
By mutating and optimizing the 7β-HSDH enzyme derived from active rumenococci, the problems of insufficient thermal stability and catalytic activity of natural enzymes in industrial production have been solved, achieving more efficient and economical ursodeoxycholic acid production suitable for industrial applications.
Patent Information
- Application Number
- CN202511587539.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-03
- Publication Date
- 2025-12-12
AI Technical Summary
In existing technologies, natural 7β-HSDH enzymes exhibit poor thermal stability and low catalytic activity under industrial production conditions, resulting in low enzyme utilization efficiency and high production costs, making it difficult to meet the needs of large-scale industrial production.
By performing single-point or combined mutations on 7β-HSDH enzymes derived from active rumenococci, their protein sequences were optimized to obtain 7β-HSDH enzyme mutants with improved thermostability and catalytic activity. These mutants included mutation sites such as V91N, C93G, T103I, and I147Y. The optimized enzyme mutants exhibited higher stability and activity under high-temperature conditions.
The thermostability of 7β-HSDH enzyme was improved by 1.3-11.8℃, and the enzyme activity was increased to 90-230%. In particular, the Tm value of the (E182L, Q155L, T103I) mutant was close to 60℃, and the activity was 1.7 times higher than that of the wild type, making it suitable for large-scale production of ursodeoxycholic acid and reducing production costs.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a 7β-HSDH enzyme mutant derived from Ruminococcus gnavus and application thereof. BACKGROUND
[0002] 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC 1.1.1.201) is a key oxidoreductase in the bile acid metabolic pathway, which can specifically catalyze the dehydrogenation reaction of the 7β-hydroxyl group of bile acids such as ursodeoxycholic acid (UDCA). Ursodeoxycholic acid is an important drug for the treatment of liver diseases, and is widely used in the treatment of gallstones, cholesteric cholecystitis and primary biliary cirrhosis, etc., and has a huge market demand.
[0003] At present, the preparation methods of UDCA mainly include chemical synthesis, animal organ extraction and microbial transformation. Among them, the biological enzyme catalysis method with natural 7β-HSDH as the core shows great application potential due to its mild reaction conditions, strong specificity, environmental friendliness and other advantages. However, the industrial application of this technology still faces severe challenges, and the bottleneck is mainly that the 7β-HSDH enzyme from natural sources usually has poor thermal stability, low catalytic activity, and is easily inactivated under industrial production conditions (such as high temperature and organic solvents). This leads to low efficiency of enzyme use and high production cost, which is difficult to meet the needs of large-scale industrial production.
[0004] Protein rational design and non-rational directed evolution are effective strategies to solve the defects of natural enzymes. By modifying the encoding gene, enzyme mutants with superior performance (such as enhanced stability, activity and substrate tolerance) can be obtained. At present, although there are research reports on 7β-HSDH, there is still a lack of 7β-HSDH mutants with high thermal stability and high catalytic activity in the prior art, which greatly limits the efficiency and economy of the downstream biological catalysis process.
[0005] The sources of wild-type 7βHSDH enzymes are very wide. At present, domestic and foreign scientific researchers have screened a large number of 7βHSDH enzyme-producing microorganisms and cloned their encoding genes. The wild-type 7β-HSDH enzyme from Ruminococcus gnavus was reported in the previous study to have the second highest activity among the reported species (Huang, Bin et al. “Sequence and structure-guided discovery of a novel NADH-dependent 7β-hydroxysteroid dehydrogenase for efficient biosynthesis of ursodeoxycholic acid.” Bioorganic chemistry vol. 131 (2023): 106340). Therefore, further improving the thermal stability and activity of the 7β-HSDH based on this source as the research object has greater value for developing 7β-HSDH suitable for industrial production processes. SUMMARY
[0006] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a Ruminococcus gnavus-derived 7β-HSDH enzyme mutant and its application.
[0007] The present application achieves the above-mentioned purpose through the following technical solutions: As a first aspect of the present application, a Ruminococcus gnavus-derived 7β-HSDH enzyme mutant is provided, which is obtained by single mutation or combined mutation of the protein sequence of the wild-type 7β-HSDH enzyme derived from Ruminococcus gnavus as shown in SEQ ID NO. 1; The single mutation point position at least includes one of V91N, C93G, T103I, I147Y, S148T, W152Y, Q155L, L164A, E182L, P202N, P202K, Q203R, K238R, E250W, D251L, D251R or D252E; The combined mutation point position at least includes one group of the following (1)-(4): (1) E182L, T103I; (2) E182L, Q155L; (3) E182L, Q155L, T103I; or (4) E182L, Q155L, C93G.
[0008] As a further optimization scheme of the present application, the 7β-HSDH enzyme mutant is based on a single mutation point of V91N on the protein sequence of the wild-type 7β-HSDH enzyme, and the amino acid sequence of the 7β-HSDH enzyme mutant is shown as SEQ ID NO. 2.
[0009] As a further optimization scheme of the present application, the 7β-HSDH enzyme mutant is based on a combination of mutation points of E182L, Q155L and T103I on the protein sequence of the wild-type 7β-HSDH enzyme, and the amino acid sequence of the 7β-HSDH enzyme mutant is shown as SEQ ID NO. 3.
[0010] As a second aspect of the present application, a polynucleotide encoding the 7β-HSDH enzyme mutant as described in any of the above is also provided.
[0011] As a further optimization scheme of the present application, the polynucleotide sequence is shown as SEQ ID NO. 4 or SEQ ID NO. 5.
[0012] As a third aspect of the present application, a recombinant plasmid containing the gene as described in any of the above, which is an expression vector capable of translating and expressing the 7β-HSDH enzyme mutant as described in any of the above, is also provided.
[0013] As a further optimization scheme of the present application, the expression vector is a pET-28a vector.
[0014] As a fourth aspect of the present application, a host cell expressing the 7β-HSDH enzyme mutant as described in any of the above is also provided.
[0015] As a fifth aspect of the present application, the use of the 7β-HSDH enzyme mutant as described in any of the above, the polynucleotide as described in any of the above, the recombinant plasmid as described in any of the above or the host cell as described above in the production of ursodeoxycholic acid is also provided.
[0016] As a further optimization scheme of the present application, 3α-hydroxy-7-oxo-5β-cholanic acid is used as the substrate.
[0017] The present application has the following beneficial effects: The present application takes 7beta-HSDH enzyme from Ruminococcus gnavus as the research object, and provides a series of single-point mutations and combination mutations of 7beta-HSDH enzyme. The thermal stability of these 7beta-HSDH enzyme mutants is increased by 1.3-11.8 DEG C compared with that of the wild-type 7beta-HSDH enzyme, and the activity is maintained at 90-230%. Among them, the thermal stability and enzyme activity of (E182L, Q155L, T103I) mutant and V91N mutant are improved compared with those of the wild-type 7beta-HSDH enzyme. The enzyme activity of the V91N mutant is 2.3 times that of the wild-type 7beta-HSDH enzyme, the enzyme activity is significantly improved, the use efficiency is improved, the cost is saved, the Tm value of (E182L, Q155L, T103I) is close to 60 DEG C, and the activity is 1.7 times that of the wild type, so that the enzyme tolerance to high temperature is improved. Therefore, the 7beta-HSDH enzyme mutant provided by the present application has a wider application condition than the wild-type 7beta-HSDH enzyme, and is more suitable for efficient production of ursodeoxycholic acid from 3alpha-hydroxy-7-oxo-5beta-cholanic acid (7-ketolithocholic acid) by biological transformation method, which is beneficial to large-scale production and industrial application. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 Purification results of 17 single mutant proteins of 7beta-HSDH provided by the present application; Figure 2 Purification results of 4 combination mutant proteins of 7beta-HSDH provided by the present application; Figure 3 Activity determination results of 17 single mutant proteins and 4 combination mutant proteins of 7beta-HSDH provided by the present application; Figure 4 Affinity purification results of wild-type 7beta-HSDH enzyme and high-quality 7beta-HSDH enzyme mutant protein provided by the present application; Figure 5 QC detection results of wild-type 7beta-HSDH and high-quality 7beta-HSDH enzyme mutant protein provided by the present application. DETAILED DESCRIPTION
[0019] The following detailed description of the application will be further described in conjunction with the drawings, and it is necessary to point out here that the following detailed description is only used to further illustrate the application, and cannot be understood as limiting the protection scope of the application. Those skilled in the art can make some non-essential improvements and adjustments to the application according to the above application content.
[0020] 1、Materials The methods used in the present application are conventional methods known to those skilled in the art, and if no specific conditions are indicated, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If no manufacturer is indicated for the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.
[0021] 2. Method 2.1. Construction of wild-type 7β-HSDH enzyme and mutant protein plasmids The gene sequences of the wild-type 7β-HSDH enzyme and mutant proteins derived from Ruminococcus gnavus were obtained by gene synthesis method. The protein sequence of the wild-type 7β-HSDH enzyme is SEQ ID NO. 1 (Uniprot: A7B4V1), and all mutant proteins are designed with corresponding mutant primers based on the sequence of the wild-type 7β-HSDH enzyme, and are constructed according to the method of molecular cloning: The single-point mutation sites include V91N; C93G; T103I; I147Y; S148T; W152Y; Q155L; L164A; E182L; P202N; P202K; Q203R; K238R; E250W; D251L; D251R; D252E, a total of 17; The combined mutation sites include (E182L, T103I); (E182L, Q155L); (E182L, Q155L, T103I); (E182L, Q155L, C93G), a total of 4.
[0022] The wild-type 7β-HSDH enzyme and mutant proteins are constructed on the modified pET-28a vector (Jinsirui), and the StrepII-8His-TEV-GG tag sequence is fused behind the T7 promoter of the vector. The StrepII-8His-TEV-GG tag sequence is SEQ ID NO. 6 (wherein Strep II and 8His are tag sequences for affinity purification, "TEV" is a TEV protease cleavage site for subsequent removal of the tag in the purification process, and "GG" is a tag sequence). The gene sequences of the recombinant proteins after construction are verified by a sequencing company to be correct.
[0023] 2.2. Small-scale expression and purification of 7β-HSDH enzyme mutants 2.2.1. Small-scale expression of 7β-HSDH enzyme mutants Small-scale expression of 7β-HSDH enzyme mutants was carried out in a cell-free manner, using cell-free reagents purchased from Biortus, and the specific expression protocol referred to in the article [Levine, M. Z., et al. (2019). Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology]. It mainly includes the following two steps: (1) Preparation of E. coli crude extract: inoculate BL21 (DE3) bacterial solution into 2xYT medium, and culture at 37°C until OD 600 0.6-0.8, add isopropyl β-D-1-thiogalactopyranoside (IPTG). When OD 600 ≈3, centrifuge the bacteria. After washing each gram of wet bacteria with S30 buffer (10 mM Tris-acetate, pH 8.2, 14 mM magnesium acetate, 50 mM potassium acetate, 2 mM DTT) at 4°C for 3 times, add 1 mL S30 Buffer to 1 g of bacteria to resuspend the bacteria, and then use an ultrasonic disrupter to perform ultrasonic treatment on the cells. After centrifugation at 13000xg for 10 minutes, transfer the supernatant to a nuclease-free tube, freeze it in liquid nitrogen, and store it in a -80°C refrigerator.
[0024] (2) Protein expression in a cell-free system: add plasmid, cell extract, reaction buffer (phosphoenolpyruvate, PEG mixture, potassium glutamate, magnesium glutamate, glutamic acid, 20 kinds of amino acids, reaction energy substances (nicotinamide adenine dinucleotide, adenosine 5'-triphosphate disodium salt, cytidine acid disodium, guanosine 5'-monophosphate disodium hydrate and uridine acid disodium salt, calcium folate salt and tRNA), glucose, spermidine and 1, 4-diaminobutane, etc. to a 15 mL nuclease-free centrifuge tube according to the proportion in the literature. The reaction system is 200 μL, and the reaction conditions are 37°C, 200 rpm, and 4 hours. After centrifugation at 12000 rpm for 10 minutes, collect the supernatant and precipitate, and take samples at each step for loading fixation.
[0025] 2.2.2, Small-scale purification of 7β-HSDH enzyme mutants The supernatant after expression in the cell-free system was added to 50 μL Strep-Tactin® XT filler that had been treated with buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP) at 4°C for 30 minutes. The incubated sample was centrifuged at 12000 rpm at 4°C for 10 minutes, 1 mL of buffer was added, and the sample was washed 3 times. Then, 100 μL of elution buffer (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP, 75 mM biotin) was added, and the sample was washed 2 times. The sample was centrifuged at 12000 rpm at 4°C for 5 minutes, and the eluted sample was collected. A small amount of sample was taken from each step of processing, fixed with loading buffer, and subjected to SDS-PAGE detection.
[0026] The experimental results are shown in Figure 1 All the single mutant proteins were expressed obviously.
[0027] 2.3, Thermal stability detection of wild-type 7β-HSDH enzyme and its mutant proteins The detection technology for thermal stability test of wild-type 7β-HSDH enzyme and its mutant proteins is protein thermal melting technology (Protein Thermal Shift, ThermoFluor). The protein structure has the feature that the hydrophobic region structure is hidden inside. When the temperature rises, the protein structure will be opened, exposing the hydrophobic region, and the fluorescent dye SYPRO Orange can bind to this region and be excited to fluorescence. According to the change of fluorescence signal intensity, a melting curve is formed, and the temperature corresponding to the maximum value of the derivative of the melting curve is the melting point temperature (Tm). The more stable the protein, the higher the measured Tm value.
[0028] The specific operation is as follows: 5 μg of wild-type 7β-HSDH enzyme or its mutant protein was taken and added to a 96-well PCR plate, 10×SYPRO Orange fluorescent dye was added to the corresponding well, the 96-well PCR plate was placed in a qPCR instrument, the instrument parameters were set, and the protein melting curve was calculated by setting a gradient of 1°C per minute from 25°C to 99°C.
[0029] The Tm value results of wild-type 7β-HSDH enzyme and all single mutant proteins are shown in Table 1.
[0030] Table 1 Tm value determination results of wild-type 7β-HSDH enzyme and its single mutant proteins ; As shown in Table 1, among the 17 single mutant proteins, the Tm values of all the single mutant proteins were increased, with an increase of 1.3-5.7℃, wherein the Tm value of E182L was increased by 5.7℃ compared with the wild type.
[0031] Further, the single mutant sites with significantly increased Tm values were combined to obtain four groups of combined mutant sites. The expression and purification of the combined mutant proteins corresponding to the four groups of combined mutant sites were the same as those of the single mutant proteins, and the results are shown in Table 3. Figure 2
[0032] After obtaining the proteins, the combined mutant proteins were also subjected to Tm value detection, and the detection results are shown in Table 2.
[0033] Table 2 Tm value determination results of wild type 7β-HSDH enzyme and its combined mutant proteins ; As can be seen from Table 2, the Tm values of the four combined mutant proteins were significantly increased compared with the wild type, with an increase of about 8.3-11.8℃. Among them, the Tm value of the (E182L, Q155L, C93G) mutant protein was increased by more than 60℃, and the Tm value of the (E182L, Q155L, T103I) mutant protein was also close to 60℃.
[0034] 2.4, Activity determination of wild type 7β-HSDH enzyme and its mutant proteins The activity determination of 7β-HSDH enzyme mutant is to take 3α-hydroxy-7-oxo-5β-cholanic acid (7-Ketolithocholic acid, 7-KLCA) as the substrate, take NADPH as the coenzyme, and calculate the activity of 7β-HSDH by detecting the number of NADPH converted into NADP+. The enzyme activity parameter is expressed by the intensity of absorbance signal value (340 nm) per nanomole of 7β-HSDH enzyme mutant per second. The specific experimental operation is as follows: Preparation of buffer: 50 mM Tris-HCl pH 7.0, substrate 1 mM 7-KLCA, coenzyme 0.5 mM NADPH, reaction temperature 25 ℃, 30 nM different concentrations of 7β-HSDH mutant. 20 μL of dissolved 7-KLCA was transferred to a 384-well plate and two duplicate wells were set up, 20 μL of NADPH was added to each well, and 20 μL of different 7β-HSDH enzyme mutants was added to the corresponding wells, and then centrifuged and shaken to mix immediately. The fluorescence signal value generated by the reaction was collected using a TECAN F200 enzyme reader. Data analysis was performed using GraphPad Prism9 analysis software, and the enzyme activity parameters of the 7β-HSDH enzyme mutants to be tested were finally obtained.
[0035] The activity of the wild-type 7β-HSDH enzyme was determined in the same way.
[0036] The results are shown in Figure 3 All single mutants had a certain degree of improvement in activity compared with the wild type, with an increase of about 1.1-2.3 times. The activity of the (E182L, Q155L, T103I) combined mutant protein increased by 1.7 times, and the activities of the other three mutant proteins were not significantly reduced compared with the wild-type 7β-HSDH enzyme, and all maintained 90% activity.
[0037] 2.5, Expression and purification of high-quality mutant protein of 7β-HSDH enzyme In order to further study the function of the high-quality mutant protein of 7β-HSDH enzyme, the (E182L, Q155L, T103I) combined mutant protein with a Tm value increase of 11.1℃ and an activity increase of 1.7 times, and the V91N single mutant protein with the most obvious activity increase were selected, and their amino acid sequences are SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and their nucleotide sequences are SEQ ID NO. 4 and SEQ ID NO. 5, respectively. Heterologous expression was carried out in E. coli, and the expressed protein was purified.
[0038] 2.5.1, Expression of 7β-HSDH high-quality mutant protein The above 7β-HSDH (V91N) mutant protein, 7β-HSDH (E182L, Q155L, T103I) mutant protein and wild-type 7β-HSDH recombinant plasmid were transformed into BL21 (DE3) strain, and the strain was inoculated into 50 ml LB liquid medium and cultured at 37℃ overnight. The bacteria cultured overnight were inoculated into 1L LB liquid medium at a ratio of 1:100, and cultured at 37℃ until the OD 600When the value of OD600 / OD700 is 0.6-0.8, 0.5 mM IPTG is added, and the bacteria are cultured at 15°C overnight, and the bacteria are collected by centrifugation at 5000 rpm for purification.
[0039] 2.5.2. Purification of high-quality mutant proteins of 7β-HSDH Affinity chromatography The collected bacterial blocks are weighed, and a corresponding volume of lysis buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol) is added at a ratio of 1:10. The bacterial cells are broken using a high-pressure homogenizer, and the supernatant is collected by high-speed centrifugation at 16000 rpm. All recombinant 7β-HSDH mutants have a Strep II tag, and the Strep-Tactin XT affinity chromatography column is used to enrich and purify the proteins.
[0040] The specific process is as follows: First, the Strep-Tactin XT affinity chromatography column is washed with lysis buffer for 10 column volumes, and then the lysis supernatant is loaded onto the Strep-Tactin XT FF affinity chromatography column. Elution is performed using lysis buffer containing 75 mM biotin. The eluted proteins are detected by SDS-PAGE, and the protein concentration is determined by Nanodrop to calculate the protein yield.
[0041] The results of protein purification are shown in Figure 4 The purification results of wild-type 7β-HSDH enzyme and 7β-HSDH (V91N) mutant protein and 7β-HSDH (E182L, Q155L, T103I) mutant protein show that the purity of both is high.
[0042] (2) Enzymatic digestion and reverse affinity chromatography To obtain higher-purity proteins, the above-mentioned affinity chromatography samples are added to a certain amount of TEV enzyme, and the enzyme is digested at 4°C overnight. The cut supernatant is further purified using a HisFF chromatography column. Because the wild-type 7β-HSDH enzyme and its mutant proteins do not have affinity tags after enzyme digestion, they will not bind to the affinity column and will flow out of the column (referred to as the breakthrough liquid), so the breakthrough liquid is collected.
[0043] (3) Gel filtration chromatography and QC detection The permeate was concentrated to approximately 2 mL and then subjected to gel filtration chromatography. The gel chromatography column was a Superdex 200 Increase 10 / 300GL, and the buffer consisted of 20 mM Tris–HCl pH 7.5, 150 mM NaCl, and 1 mM DTT. The gel filtration samples were collected and subjected to protein content analysis, specifically SDS-PAGE purity determination, mass spectrometry analysis, and analytical molecular sieve detection.
[0044] Test results as follows Figure 5 As shown in the SDS-PAGE results, the purity of both the wild-type 7β-HSDH enzyme and the 7β-HSDH mutant protein is greater than 99%. Mass spectrometry results also show that the molecular weight of the tested samples is basically consistent with the target protein, indicating that the purified protein is the target protein. Furthermore, analytical molecular sieve results show that all proteins are in a near-dimer state in solution.
[0045] 3. Conclusion The above description indicates that this invention uses wild-type 7β-HSDH enzyme derived from active rumenococci as the research object. Single or combined mutations were performed on the protein sequence of wild-type 7β-HSDH enzyme to obtain several mutant proteins. These 7β-HSDH enzyme mutants exhibit improved thermostability by 1.3-11.8℃ and maintained activity at 90-230% compared to wild-type 7β-HSDH enzyme. Furthermore, two superior 7β-HSDH enzyme mutant proteins, the V91N mutant protein and the (E182L, Q155L, T103I) mutant proteins, were selected, which respectively demonstrate better enzyme activity and thermostability compared to wild-type 7β-HSDH enzyme. Therefore, compared to wild-type 7β-HSDH enzyme, these mutants have broader application conditions and stronger practical application value, making them more suitable for large-scale production and industrial use.
[0046] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
Claims
1. A 7β-HSDH enzyme mutant of rumenococcus equii origin, characterized in that, The 7β-HSDH enzyme mutant is obtained from the protein sequence of wild-type 7β-HSDH enzyme derived from Lactobacillus agilis shown as SEQ ID NO. 1 by single mutation or combined mutation; The single mutation point position at least includes one of V91N, C93G, T103I, I147Y, S148T, W152Y, Q155L, L164A, E182L, P202N, P202K, Q203R, K238R, E250W, D251L, D251R or D252E; The combined mutation point position at least includes one group of (1)-(4): (1) E182L, T103I; (2) E182L, Q155L; (3) E182L, Q155L, T103I; or (4) E182L, Q155L, C93G.
2. A V. ruminis derived 7P-HSDH enzyme mutant according to claim 1, characterized in that, The 7β-HSDH enzyme mutant based on the single mutation point position V91N on the protein sequence of wild-type 7β-HSDH enzyme has an amino acid sequence shown as SEQ ID NO.
2.
3. A V. ruminis derived 7P-HSDH enzyme mutant according to claim 1, characterized in that, The 7β-HSDH enzyme mutant based on the combined mutation point position E182L, Q155L and T103I on the protein sequence of wild-type 7β-HSDH enzyme has an amino acid sequence shown as SEQ ID NO.
3.
4. A polynucleotide encoding the 7β-HSDH enzyme mutant according to any one of claims 1-3.
5. The polynucleotide of claim 4, wherein, The polynucleotide sequence is shown as SEQ ID NO. 4 or SEQ ID NO.
5.
6. A recombinant plasmid comprising the polynucleotide of any one of claims 4-5, characterized in that, The recombinant plasmid is an expression vector capable of translating and expressing the 7β-HSDH enzyme mutant according to any one of claims 1-3.
7. The recombinant plasmid according to claim 5, wherein The expression vector is pET-28a vector.
8. A host cell expressing the 7β-HSDH enzyme mutant according to any one of claims 1-3.
9. Use of the 7β-HSDH enzyme mutant according to any one of claims 1-3, the gene according to any one of claims 4-5, the recombinant plasmid according to any one of claims 6-7 or the host cell according to claim 8 in the preparation of ursodeoxycholic acid.
10. Use according to claim 9, characterized in that, 3α-hydroxy-7-oxo-5β-cholanic acid as a substrate.