7beta-hydroxysteroid dehydrogenase mutants and methods for making same

Abstract

The present application relates to the technical field of microorganism and enzyme engineering, and particularly relates to 7beta-hydroxysteroid dehydrogenase mutant and a preparation method thereof, and particularly relates to semi-rational design of 7beta-hydroxysteroid dehydrogenase mutant. Ruminococcus torques The present application takes 7beta-hydroxysteroid dehydrogenase mutant T189V / V207M / S70P and T189V / V207M / Q133R obtained by the semi-rational design method of ancestor enzyme reconfiguration assisted enzyme directed evolution and taking 7beta-hydroxysteroid dehydrogenase mutant T189V / V207M of Ruminococcus as a template, the former significantly improves high-temperature stability, and the latter significantly improves specific enzyme activity.

Description

Technical Field

[0001] This invention relates to the fields of microbial and enzyme engineering technology, and particularly to 7β-hydroxysteroid dehydrogenase mutants and their preparation methods, specifically to the semi-rational design of 7β-hydroxysteroid dehydrogenase mutants. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is the active ingredient in the precious traditional Chinese medicine bear bile. Its chemical name is 3α,7β-dihydroxy-5β-cholestan-24-acid, also known as ursodeoxycholic acid. It is a drug approved by the US FDA for the treatment of primary biliary cirrhosis. It is also used to treat cholestatic liver diseases such as primary sclerosing cholangitis, alcoholic and fatty liver disease, viral hepatitis, and drug-induced hepatitis. It is also the first-line drug for dissolving cholesterol gallstones and is used to treat various gallstone diseases.

[0003] Currently, there are three main methods for synthesizing UDCA using biocatalysis: Route 1: Extracting CDCA from chickens, ducks, and geese, and then synthesizing UDCA using a one-pot, four-enzyme process; Route 2: Using phytosterols as starting materials, converting them into dioxol (BA) via mycobacteria, then synthesizing 7-oxo-LCA through a chemical reaction, and finally reacting it with 7β-HSDH and GDH to generate UDCA; Route 3: Using phytosterols as starting materials, converting them into dioxol (BA) via mycobacteria, then synthesizing lithocholic acid (LCA) through a chemical reaction, and finally reacting it with P450 enzyme and GDH to generate UDCA. However, in Route 3, the P450 enzyme for 7β-hydroxylation of LCA has poor substrate compatibility (usually only reacting with 1 mM LCA), and generates a large amount of the 6β-hydroxylated isomer MDCA. Therefore, the first two routes remain the mainstream methods for industrial production of UDCA, and both routes require the participation of 7β-HSDH, indicating that 7β-HSDH plays a crucial role in the synthesis of UDCA.

[0004] In existing technologies, 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutants T189V & V207M are used to efficiently convert CDCA to UDCA through a two-step enzymatic cascade reaction. At a substrate concentration of 100 mM, the final conversion rate is higher than 99% (e.g., patent CN107099516A). However, in actual production, the stability of the 7β-HSDH mutants T189V & V207M still cannot meet the requirements for maintaining high activity over a long period of time. Therefore, it is crucial to further improve the thermostability and enzyme activity of 7β-HSDH. Summary of the Invention

[0005] In view of this, the present invention proposes a 7β-hydroxysteroid dehydrogenase mutant and its preparation method. Based on the 7β-hydroxysteroid dehydrogenase mutants T189V & V207M, through a semi-rational design method of ancestral enzyme reconstruction and directed evolution of helper enzymes, mutants T189V / V207M / S70P and T189V / V207M / Q133R were obtained, further improving the thermostability and enzyme activity of 7β-HSDH.

[0006] This invention utilizes ancestral enzyme reconstruction and directed evolution technology to further improve the thermal stability and enzyme activity of 7β-HSDH. The theoretical basis for this technology is that early Earth environments were typically harsher, characterized by higher temperatures, more extreme pH levels, and higher salt concentrations. To survive and function in such environments, early enzymes had to possess inherent stability and robustness. As life evolved, organisms occupied more diverse ecological niches (e.g., the mammalian gut is homeothermic, while the deep sea is high-pressure and low-temperature). To achieve higher catalytic efficiency or more precise regulation in these specific, relatively mild environments, enzymes sometimes sacrifice some stability for optimized catalytic activity. This is like a "universal wrench" being meticulously crafted into a "specialized screwdriver"—extremely efficient for a specific task, but potentially more delicate and fragile in structure. Therefore, compared to modern enzymes, ancestral enzymes typically have tighter packaging and more salt bridges, hydrogen bonds, and hydrophobic interactions. These stronger intramolecular forces endow enzymes with greater intrinsic rigidity, much like adding more load-bearing walls and steel reinforcement to a building. This rigidity means that enzymes require more energy to be destroyed, thus resulting in higher thermal stability.

[0007] In 7β-HSDH homology sequence alignment, amino acid residues with high frequency at a certain site are beneficial to the thermal stability of the protein. If the protein to be modified does not have this amino acid residue at that site, mutating that site to obtain it can improve its thermal stability. The protein sequence composed of the amino acids with the highest frequency at each site is called the consensus sequence of that protein family, and is generally considered to be the closest to the ancestral sequence, thus possessing better thermal stability. When screening mutation sites and mutation methods, the following factors should be considered: ① whether the amino acid residue is located on the protein surface; ② whether the amino acid residue is far from the active site, substrate, or prosthetic binding site; ③ whether the mutation disrupts existing helices, hydrogen bonds, or salt bridges; ④ whether the mutation can introduce new forms of interaction, etc.

[0008] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a 7β-hydroxysteroid dehydrogenase mutant, which is obtained by mutating any of the following sites in the amino acid sequence shown in SEQ ID NO.1: position 31, position 34, position 39, position 67, position 70, position 88, position 124, position 133, position 134, position 161, or position 162.

[0009] This invention involves mutation of 7β-HSDH-T189V&V207M, and its amino acid sequence is shown in SEQ ID NO.1: MNLREKYGEWGIILGATEGVGKAFAEKIASEGMSVVLVGRREEKLQELGKSISETYGVDHMVIRADFAQSDCTDKIFEATKDLDMGFMSYVACFHTFGKLQDTPWEKHEQMINVNVMTFLKCFYHYMGIFAK QDRGAVINVSSLTAISSSPYNAQYGAGKSYIKKLTEAVAAECESTNVDVEVITLGTVITPSLLSNLPGGPAGEAMMKTAMTPEACVEEAFDNLGKSLSVIAGEHNKANVHNWQANKTDDEYIRYMGSFYSNN.

[0010] Based on the above technical solutions, preferably, the SEQ ID NO.1 sequence is optimized using E. coli codons, and the resulting nucleotide sequence is shown in SEQ ID NO.2: .

[0011] Based on the above technical solutions, preferably, the 7β-hydroxysteroid dehydrogenase mutant includes replacing glutamine at position 133 with arginine (Q133R).

[0012] Based on the above technical solutions, the amino acid sequence of the 7β-hydroxysteroid dehydrogenase mutant is further preferred as shown in SEQ ID NO.3.

[0013] The amino acid sequence of the 7β-hydroxysteroid dehydrogenase mutant T189V / V207M / Q133R is shown in SEQ ID NO.3: MNLREKYGEWGIILGATEGVGKAFAEKIASEGMSVVLVGRREEKLQELGKSISETYGVDHMVIRADFAQSDCTDKIFEATKDLDMGFMSYVACFHTFGKLQDTPWEKHEQMINVNVMTFLKCFYHYMGIFAK RDRGAVINVSSLTAISSSPYNAQYGAGKSYIKKLTEAVAAECESTNVDVEVITLGTVITPSLLSNLPGGPAGEAMMKTAMTPEACVEEAFDNLGKSLSVIAGEHNKANVHNWQANKTDDEYIRYMGSFYSNN.

[0014] Preferably, those having one or more amino acids substituted in the amino acid sequence shown in SEQ ID NO.3, achieving more than 95% homology, are all protected by this invention.

[0015] In a second aspect, a gene encoding a 7β-hydroxysteroid dehydrogenase mutant as described above is provided, the gene sequence of which is shown in SEQ ID NO.4.

[0016] The gene sequence of the 7β-hydroxysteroid dehydrogenase mutant T189V / V207M / Q133R is shown in SEQ ID NO.4: .

[0017] Based on the above technical solutions, preferably, the 7β-hydroxysteroid dehydrogenase mutant includes replacing the serine at position 70 with proline (S70P).

[0018] Based on the above technical solutions, the amino acid sequence of the 7β-hydroxysteroid dehydrogenase mutant is further preferred as shown in SEQ ID NO.5.

[0019] The amino acid sequence of the 7β-hydroxysteroid dehydrogenase mutant T189V / V207M / S70P is shown in SEQ ID NO. 5: MNLREKYGEWGIILGATEGVGKAFAEKIASEGMSVVLVGRREEKLQELGKSISETYGVDHMVIRADFAQPDCTDKIFEATKDLDMGFMSYVACFHTFGKLQDTPWEKHEQMINVNVMTFLKCFYHYMGIFAK QDRGAVINVSSLTAISSSPYNAQYGAGKSYIKKLTEAVAAECESTNVDVEVITLGTVITPSLLSNLPGGPAGEAMMKTAMTPEACVEEAFDNLGKSLSVIAGEHNKANVHNWQANKTDDEYIRYMGSFYSNN.

[0020] Preferably, those having one or more amino acids substituted in the amino acid sequence shown in SEQ ID NO.5, achieving more than 95% homology, are all protected by this invention.

[0021] Thirdly, a gene encoding a 7β-hydroxysteroid dehydrogenase mutant as described above is provided, the gene sequence of which is shown in SEQ ID NO.6.

[0022] The gene sequence of the 7β-hydroxysteroid dehydrogenase mutant T189V / V207M / S70P is shown in SEQ ID NO. 6: .

[0023] Fourthly, a recombinant expression vector is provided, which includes the gene as described above.

[0024] Fifthly, a recombinant strain is provided, comprising the gene as described above or the recombinant expression vector as described above.

[0025] Sixthly, a method for preparing the 7β-hydroxysteroid dehydrogenase mutant as described above is provided, comprising the following steps: S1, sequence alignment analysis of key amino acid sites in ancestral sequences to screen out amino acid residues to be mutated; S2, amplification of the 7β-hydroxysteroid dehydrogenase mutant gene; S3, construct a recombinant expression vector by inserting the 7β-hydroxysteroid dehydrogenase mutant gene into the empty vector to obtain the recombinant expression vector; S4. Construct recombinant strains by transforming the recombinant expression vector into host bacteria to obtain recombinant strains; The expression of the S5, 7β-hydroxysteroid dehydrogenase mutant was achieved by activating and expanding the recombinant strain to induce the expression of the 7β-hydroxysteroid dehydrogenase mutant, followed by cell disruption to obtain crude enzyme solution.

[0026] Based on the above technical solutions, preferably, all sequences with a sequence similarity higher than 30% to SEQ ID NO.1 are searched in the NCBI database using the Blast (Basic Local Alignment Search Tool), and duplicate sequences are deleted. Amino acid analysis software is used to perform sequence alignment analysis on amino acid sites with a conservation rate of 30% or higher, and sites where amino acid residues at the same positions as those in SEQ ID NO.1 differ are also marked. The three-dimensional structure of 7β-hydroxysteroid dehydrogenase (PDB: 5FYD) is analyzed using protein crystal structure analysis software. The catalytic residues are 143S-156Y-160K. Hotspot amino acids near the catalytic residues are removed, and hotspot amino acid residues on the protein surface are retained; these are the candidate mutant amino acid residues.

[0027] Based on the above technical solutions, the preferred method is to design site-directed mutagenesis primers targeting the amino acid residues to be mutated, use pET28a-7βHSDH-T189V&V207M plasmid as a template to perform whole plasmid PCR, and directly transform the PCR product into BL21(DE3) competent cells and then plate it on kanamycin-resistant LB plates to construct the mutant strain.

[0028] The 7β-hydroxysteroid dehydrogenase mutant and its preparation method of the present invention have the following advantages over the prior art: 1. The 7β-hydroxysteroid dehydrogenase mutants T189V / V207M / S70P and T189V / V207M / Q133R obtained by screening in this invention have significantly improved high-temperature stability and significantly improved specific enzyme activity.

[0029] 2. This invention is based on semi-rational design and analysis of 7β-HSDH-T189V & V207M and their ancestral sequences, and screens out a series of potential mutation sites, which increases the probability of obtaining positive mutants. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a diagram illustrating the conservation of the 7β-HSDH amino acid sequence of the present invention; Figure 2 The pET28a-7βHSDH-T189V&V207M plasmid spectrum of the present invention; Figure 3 The image shows the SDS-PAGE electrophoresis diagram of 7β-HSDH-T189V&V207M and its mutants of the present invention, where 1 to 11 represent E31R, S34N, G39A, F67L, S70P, M88L, Y124H, Q133R, D134G, S161A, and Y162F mutants, respectively, and M represents 7β-HSDH-T189V&V207M; Figure 3 Figure (a) in the diagram represents the M and 1-6 mutants. Figure 3 Figure (b) in the diagram represents the 7-11 mutant. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1 Ancestor sequence alignment analysis of 7β-HSDH was used to screen for key amino acid sites.

[0034] The amino acid sequence of 7β-HSDH-T189V&V207M is shown in SEQ ID NO.1. All sequences with a similarity greater than 30% to SEQ ID NO.1 were searched in the NCBI database using the Blast (Basic Local Alignment Search Tool), and duplicate sequences were removed. Sequence alignment analysis was performed using amino acid analysis software to identify amino acid sites with a conservation rate of 30% or higher. Sites with different amino acid residues at the same positions as those in SEQ ID NO.1 were also labeled. Figure 1 As shown, Figure 1The red pentagrams indicate some hotspot amino acids, and the query represents SEQ ID NO.1. The three-dimensional structure of 7β-hydroxysteroid dehydrogenase (PDB: 5FYD) was analyzed using protein crystal structure analysis software. The catalytic residues are 143S-156Y-160K. After removing hotspot amino acids near the catalytic residues and retaining those on the protein surface, the following amino acid residues remain as potential mutants: E31R, S34N, G39A, F67L, S70P, M88L, Y124H, Q133R, D134G, S161A, and Y162F.

[0035] Example 2 Construction of 7β-HSDH-T189V & V207M strains.

[0036] The sequence SEQ ID NO.1 from Example 1 was reverse transcribed to form a DNA sequence. After codon optimization for *E. coli*, the resulting nucleotide sequence is shown in SEQ ID NO.2. The SEQ ID NO.2 nucleotide sequence was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for gene synthesis and inserted into the Nco I and Xho I restriction sites of the pET28a plasmid to form the pET28a-7βHSDH-T189V&V207M expression plasmid. The plasmid map is shown below. Figure 2 As shown, after transforming this plasmid into BL21(DE3) competent cells, it was plated on kanamycin-resistant LB plates and cultured overnight at 37°C. The resulting single colony was the pET28a-7βHSDH-T189V&V207M expression strain.

[0037] Example 3 Construction of mutant strains of 7β-HSDH-T189V & V207M.

[0038] Based on 7β-HSDH-T189V&V207M, mutants E31R, S34N, G39A, F67L, S70P, M88L, Y124H, Q133R, D134G, S161A, and Y162F were constructed. Site-directed mutagenesis primers were designed (as shown in Table 1). For each mutant, using the pET28a-7βHSDH-T189V&V207M plasmid from Example 2 as a template, full plasmid PCR was performed using a pair of primers corresponding to the mutation sites in Table 1. The PCR products were directly transformed into BL21(DE3) competent cells and plated on kanamycin-resistant LB plates. Single colonies were grown overnight at 37°C. Three single colonies were selected for sequencing, and the correctly sequenced strains were preserved and named, such as pET28a-7βHSDH-E31R for E31R, and so on for the others.

[0039] Table 1

[0040] Example 4: Expression of 7β-HSDH-T189V & V207M and their mutants and preparation of crude enzyme solution.

[0041] 7β-HSDH-T189V & V207M, and the 11 mutant strains constructed in Example 3 were inoculated into LB broth containing 50 μg / ml kanamycin for seed culture, and then transferred to LB broth containing 50 μg / ml kanamycin for expansion culture. OD 600 When the concentration of 7β-HSDH-T189V & V207M reached approximately 0.8, IPTG was added to a final concentration of 0.8 mM and the mixture was induced at 25°C for 18 h at 220 rpm. After induction, the bacterial cells were collected by centrifugation. 1 g of each of the 7β-HSDH-T189V & V207M and its mutant cells were weighed and resuspended in 10 ml of 50 mM phosphate buffer (pH 8.0). After ultrasonic disruption, the cells were centrifuged at 12000 rpm for 30 min. The supernatant was collected as the crude enzyme supernatant sample. The centrifuged precipitate was resuspended in 10 ml of 50 mM phosphate buffer (pH 8.0) as the precipitate sample. The crude enzyme solution and precipitate sample of 7β-HSDH-T189V & V207M and its mutants were simultaneously subjected to SDS-PAGE electrophoresis. The results are shown below. Figure 3 As shown, the expression and solubility of the 12 proteins were good.

[0042] Example 5 Enzyme activity and stability analysis of 7β-HSDH-T189V & V207M and their mutants.

[0043] Enzyme activity definition: Unit enzyme activity is defined as the amount of enzyme required to consume 1 micromole of NADPH per minute under the following reaction conditions (enzyme activity (U / mL) = ΔOD / min * Vt * df / (6.22 * 1.0 * Vs).

[0044] Reagent I: 100 mM pH 8.0 potassium phosphate buffer, filtered through a 0.45 μm filter (0.09348 M K₂HPO₄ and 0.006523 M KH₂PO₄ were added to purified water to prepare 1 L); Reagent II: 7-ketolithocholic acid (7-KLCA) (0.276 mg / mL): Weigh 276 mg of 7-KLCA and dissolve it in 1 L of Reagent I until no particulate matter is found. If necessary, sonicate to dissolve. After preparation, aliquot and store at -20℃ for later use; Reagent III: 8.0 mM NADPH, dissolved in 100 mM pH 8.0 potassium phosphate. After preparation, aliquot and store at -20℃; Sample to be tested: Dilute with Reagent I to a suitable range (control the slope change of the sample per second within 0.001~0.003; if it does not meet this range, change the sample dilution factor).

[0045] Procedure: ① Add 2.9 mL of Reagent II and 50 μL of Reagent III to a 3 mL cuvette, mix well, and preheat at 37℃ for 5 min; ② Add 50 μL of the enzyme solution to be tested, mix well, and start the reaction; ③ Measure the rate of change of absorbance per second ΔA at 37℃ within 40 s at 340 nm. Activity calculation (U / mL) = (ΔA × Vt × df × 60) / (6.22 × Vs × 1.0). Where, Vt: total volume of reaction solution (3.00 mL); Vs: enzyme solution volume (0.05 mL); 1.0: optical path length (cm); df: dilution factor; 6.22: extinction coefficient of NADPH at 340 nm wavelength; 60: 1 min = 60 s. The results are shown in Table 2.

[0046] Table 2

[0047] According to the data in Table 2, compared with the control M1 (7β-HSDH-T189V & V207M), mutants 1, 2, 5, 8, 9, and 11 (E31R, S34N, S70P, Q133R, D134G, and Y162F mutants) showed higher enzyme activities, with initial relative activities all exceeding 90% (94%–118%). Among them, mutant 8, Q133R, had the highest relative enzyme activity, with an increase of 1.18 times.

[0048] The six mutants and control M1 were incubated in a 45℃ water bath for 0, 20 min, 40 min, and 60 min. After incubation, their thermal stability was tested, and the results are shown in Table 3.

[0049] Table 3

[0050] Results analysis: Mutant No. 5 (S70P) has the highest thermal stability. After treatment at 45℃ for one hour, the enzyme activity was still 98% retained, with almost no loss of enzyme activity.

[0051] The mutant 5 (S70P) and the control M1 were incubated at 55°C to test their thermal stability. The results are shown in Table 4.

[0052] Table 4

[0053] Results analysis: The S70P mutation increased the residual activity of 7β-HSDH-T189V&V207M after incubation at 55℃ for 60 min from 8% to 31%. This structure is consistent with the proline theory of protein evolution, that is, proline (Pro) residues play a special role in protein structure and its thermal stability. It is believed that in protein engineering to improve the thermal stability of enzymes, as long as the main chain conformation does not change abruptly, Pro can be introduced into appropriate β turns or random coils. Through its rigid pyrrolidine ring, it reduces the skeletal entropy of the protein during unfolding, thereby making its surrounding conformation more reasonable.

[0054] In summary, we obtained mutants T189V / V207M / S70P and T189V / V207M / Q133R through a semi-rational design method of ancestral enzyme reconstructed helper enzyme directed evolution. The former significantly improved high-temperature stability, while the latter significantly improved specific enzyme activity.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A 7β-hydroxysteroid dehydrogenase mutant, characterized in that: The amino acid sequence of the 7β-hydroxysteroid dehydrogenase mutant is shown in SEQ ID NO.3 or SEQ ID NO.

5.

2. A gene encoding the 7β-hydroxysteroid dehydrogenase mutant as described in claim 1, characterized in that: The gene sequence of the 7β-hydroxysteroid dehydrogenase mutant is shown in SEQ ID NO.

4.

3. A gene encoding the 7β-hydroxysteroid dehydrogenase mutant as described in claim 1, characterized in that: The gene sequence of the 7β-hydroxysteroid dehydrogenase mutant is shown in SEQ ID NO.

6.

4. A recombinant expression vector, characterized in that: Includes the gene described in claim 2 or 3.

5. A recombinant bacterial strain, characterized in that: Includes the gene as described in claim 2 or 3 or the recombinant expression vector as described in claim 4.

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