Ketoreductase mutant and use thereof

Abstract

Provided are a ketoreductase mutant and a use thereof. The mutant is obtained by amino acid mutation on the basis of the amino acid sequence shown in SEQ ID NO. 2, and the mutation is (1) or (2): (1) any one of Y199A, Y199C, M196W, A146F, and A146L; (2) a combination of any one of Y199A, Y199C, M196W, A146F, and A146L and any one of N149L, N149I, I195L, and I195V. The obtained mutant having higher catalytic activity has a conversion rate increased by up to 2.4 times and a de value of more than 99.8%. The mutant has great potential in the biotransformation of steroidal compounds to prepare 24R-hydroxy-configured cholesterol.

Description

A ketoreductase mutant and its application Technical Field

[0001] The present invention relates to the technical field of genetic engineering and bioenzyme catalysis, and in particular to a ketoreductase and an application thereof. Background Art

[0002] Ketone reductases are ubiquitous in nature, found in animals, plants, and microorganisms. Microbial ketoreductases are the most widespread, with at least 200 species reported to participate in reduction reactions of various substances. Ketone reductases play a crucial role in the synthesis of chiral alcohols. Ketone reductases with excellent catalytic activity and stereoselectivity can effectively catalyze the asymmetric synthesis of chiral alcohols, significantly improving synthesis efficiency and simplifying product preparation and separation processes. For example, APIs and intermediates such as atorvastatin, duloxetine, and ticagrelor have been prepared using ketoreductases, reducing the need for metal chiral catalysts or resolving agents. Ketone reductases have become the most widely used enzymes in the pharmaceutical and chemical industries. Ketone reductases are a class of oxidoreductases that rely on NAD(H) or NADP(H) as coenzymes and catalyze the reversible reduction of aldehydes or ketones to the corresponding alcohols. Ketone reductases share a common molecular characteristic: they utilize NAD(H) or NADP(H) as coenzymes, the coenzyme binding region is located in the N-terminal Rossmann fold, and a conserved Gly (Gly)-rich sequence, GxxxGxG. The catalytic domain is located in the middle of the N-terminus, with a conserved catalytic triad consisting of Ser-Tyr-Lys. Enzymes in this family are generally composed of monomers, dimers, or tetramers, with a single subunit containing approximately 250 amino acids, and play a crucial role in the synthesis of chiral alcohols.

[0003] Squalamine, also known as (3β,5α,7α)-3-[[3-((4-aminobutyl)amino)propyl]amino]cholestane-7,24-diol-24-sulfonate, was originally extracted from the liver of spiny dogfish by Zasloff and his collaborators as a marine aminosterol compound. Squalamine has strong broad-spectrum antibacterial and anti-angiogenic effects.

[0004] Traditional squalamine synthesis processes primarily use 3β-hydroxy-5-cholenic acid, chenodeoxycholic acid, fucoxanthinol, dehydroepiandrosterone, or pregnenolone acetate as raw materials. For example, the chemical synthesis of squalamine using chenodeoxycholic acid methyl ester requires a nine-step reaction (Dong-Hui Zhang, Feng Cai, Xiang-Dong Zhou, et al. A concise and stereoselective synthesis of squalamine. Organic Letters, 2003, 5(18): 3257-3259). The key to this synthesis is the construction of a 24-chiral hydroxyl intermediate. This reaction route requires the C-7 hydroxyl group to be protected with MOMO, a highly toxic and carcinogenic reagent. The C-24 position undergoes nucleophilic addition to the compound under the control of a chiral reagent compound to obtain an R-configured hydroxyl group. Both chiral reagents and diisopropylzinc are expensive reagents, resulting in extremely high synthesis costs, requiring researchers to seek better alternative preparation methods.

[0005] Patent 202310759919.8 discloses a chemical-enzymatic method for preparing 3α,7α,24R-trihydroxy-5α-cholesterol. This method is highly advanced, but the ketoreductases used in this method, including one or more of KRED-101, KRED-171, and their mutants, only achieve a conversion efficiency of 64%.

[0006] Summary of the Invention

[0007] The present invention addresses the above-mentioned technical difficulties and provides a ketoreductase mutant, which utilizes ketoreductase (NaKRED) as a template to improve the biocatalytic activity of the enzyme. The mutant exhibits enhanced activity and selectivity, can combine with an NADH regeneration and reduction system to selectively reduce a steroid compound (1a) to produce 24R-hydroxylated cholesterol (1b), and can also improve conversion efficiency.

[0008] The technical solutions of the present invention are as follows:

[0009] The ketoreductase mutant provided by the present invention can catalyze 1a to produce 1b in one step under an NADH regeneration system. The enzymatic conversion reaction formula is shown in FIG1 .

[0010] To achieve the purpose of the invention, the present invention screened known ketoreductases and obtained a stereoselective ketoreductase (NaKRED) from Novosphingobium aromaticivorans as a template. By studying the binding pocket of the ketoreductase, a mutant was constructed, which can achieve the enzymatic conversion of 1a to 1b. The amino acid sequence shown in SEQ ID NO. 2 has been mutated at one or more amino acid sites and has the activity of selectively reducing the 24-position carbonyl group.

[0011] A ketoreductase mutant, wherein the mutant is obtained by amino acid mutation based on the amino acid sequence shown in SEQ ID NO. 2, wherein the mutation is (1) or (2):

[0012] (1) Any one of Y199A, Y199C, M196W, A146F, and A146L;

[0013] (2) A combination of any one of Y199A, Y199C, M196W, A146F, A146L and any one of N149L, N149I, I195L, and I195V.

[0014] Preferably, the mutation is any one of the following combinations: Y199A / N149L, Y199A / N149I, Y199A / I195L, Y199A / I195V.

[0015] A DNA molecule encoding the above-mentioned ketoreductase mutant.

[0016] A recombinant plasmid contains the DNA molecule.

[0017] According to the above recombinant plasmid, its vector is selected from any one of the following:

[0018] pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a (+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+) , pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80 , pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18 and pUC-19.

[0019] A recombinant bacterium contains the recombinant plasmid.

[0020] Preferably, the host cell of the recombinant bacteria is a yeast cell or Escherichia coli BL21 (DE3).

[0021] The application of the ketoreductase mutant is to combine the ketoreductase mutant with an NADH regeneration reduction system for catalytic reduction reaction to prepare chiral steroid compounds.

[0022] Preferably, the ketoreductase mutant is used in combination with an NADH regeneration system to catalyze an asymmetric carbonyl reduction reaction of the substrate to obtain cholesterol with a 24R-hydroxy configuration.

[0023] Preferably, the substrate is any one of the following structural formulas:

[0024] Preferably, the NADH regeneration system comprises glucose and glucose dehydrogenase; and the reaction conditions are: temperature 25±15° C., rotation speed 200±100 rpm, and time 12-24 h.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) The present invention changes the active pocket where the enzyme binds to the substrate through site-directed mutagenesis to obtain a mutant with higher catalytic activity.

[0027] (2) The ketoreductase mutant provided by the present invention uses inexpensive and readily available allochenodeoxycholic acid as a raw material to synthesize substrate 1a through a two-step reaction. The selective reduction conversion rate of the 24-carbonyl group is increased by 2.4 times compared to the template, and 1b with a de of over 99.8% can be obtained. This method not only has mild reaction conditions and few steps, but also can significantly reduce byproducts, making it environmentally friendly. Therefore, the ketoreductase mutant provided by the present invention has great potential in the biotransformation of steroid compounds to produce 24R-hydroxy-configured cholesterol. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows the reaction formula of ketoreductase NaKRED mutant catalyzing the conversion of 1a to 1b.

[0029] Figure 2 is the HPLC chart of template NaKRED catalyzing 1a.

[0030] Figure 3 shows the crystal structure of the R-configured product 1b obtained by catalysis of 1a by template NaKRED.

[0031] FIG4 is an HPLC chart of the Y199A / N149L mutant catalyzing 1a. DETAILED DESCRIPTION

[0032] The present invention is further described below with reference to the following examples, but is not limited to the examples in the present invention. Unless otherwise specified, the following experiments are all conventional methods, and the experimental materials used are all available from commercial companies unless otherwise specified.

[0033] Example 1 Synthesis and recombinant expression of template pET28a-NaKRED

[0034] According to the literature report that the ketoreductase NaKRED can reduce the potential chiral ketone according to the anti-prelog rule as a template (Wu K, Zheng K, Xiong L, et al. Efficient synthesis of an antiviral drug intermediate using an enhanced short-chain dehydrogenase in an aqueous-organic solvent system [J]. Applied microbiology and biotechnology, 2019, 103 (11): 4417-4427.), the optimized nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The optimized nucleotide sequence was synthesized into the pET28a vector and then transformed into Escherichia coli BL21 (DE3). After the culture was resumed according to the conventional slow-transfection method, an appropriate amount of bacterial solution was spread on an LB plate containing 50 μg / mL kanamycin and cultured inverted at 37 ° C overnight. Single colonies were picked from the plates for activation and inoculated into 25 mL of LB medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C, 220 rpm, until the OD reached 0.6-0.8. IPTG (0.2 mM) was added and the cells were shaken at 25°C, 180 rpm, and incubated for 20 h. The cells were harvested at 6500 rpm for 10 min at 4°C. The harvested cells were washed twice with reaction buffer, disrupted using an ultrasonic disruptor, and centrifuged at 12000 rpm for 15 min. The supernatant was used for activity assays and as a control for mutant activity screening. For a 1 mM substrate concentration, the enzyme dosage was 50-60 g / L of buffer, based on the weight of the wet cells.

[0035] Example 2 Construction of Ketoreductase NaKRED Mutants

[0036] Using the entire pET28a-KRED plasmid as a template, design mutagenesis primers, placing the base to be mutated in the middle of the primer. The PCR system is as follows: 1 ng of template, 1.5 μL of upstream and downstream primers, 25 μL of 2× PCR Buffer for KOD FX Neo, 1 μL of KOD FX Neo enzyme, and finally, add sterile distilled water to bring the volume to 50 μL.

[0037] PCR reaction procedure: (1) pre-denaturation at 94°C for 2 min; (2) denaturation at 98°C for 10 s; (3) annealing temperature set according to the mutant primer, annealing time 15 s; (4) extension at 68°C for 4 min 20 s, steps (2) to (4) were repeated 30 times, and the final extension was at 68°C for 10 min. The PCR product was stored at 12°C.

[0038] The PCR product was digested with Dpn I, and after template removal, the product was transformed into competent E. coli DH5α cells. The recovered culture was plated onto LB medium plates containing 50 μg / mL kanamycin and incubated inverted at 37°C overnight. Single clones were selected for single colony verification, and one to three colonies were selected for gene sequencing. Mutants with multiple mutation points were generated by performing additive mutations based on the single mutant using the same method described above.

[0039] The inducible expression of the mutants was consistent with the inducible expression of the above templates.

[0040] Example 3 Activity screening of mutants

[0041] The obtained wet cells were resuspended according to 1g (wet cells): 15mL (reaction buffer), and 900μL was used for ultrasonic disruption, with a break time of 1s and a rest time of 2s for a total of 10min. The disrupted liquid was centrifuged at 12000rpm for 15min. The supernatant was collected and added with 1mM substrate 1a (prepared to 100mM stock solution with methanol), 1mg / mL glucose dehydrogenase, 100mM glucose, 1mM NAD + The reaction system was stirred at 25°C and 220 rpm for 12-24 hours. After the reaction was complete, an equal volume of ethyl acetate was extracted. This extraction was repeated three times. The organic phases were combined, the solvent was dried by rotary evaporation, and then redissolved in 1 mL of methanol. The sample was filtered through a 0.22 μm organic filter membrane and the content was determined by HPLC.

[0042] Table 1 Activity test results of mutants

[0043] The above results were obtained by performing three parallel reactions under the same reaction conditions. Conversion is defined as: initial substrate concentration - final substrate concentration / initial substrate concentration (%); selectivity is defined as: diastereoselectivity (de) is defined as: [RS] / [R + S] %.

[0044] Based on the above activity test results, the Y199A / N149L mutant was purified.

[0045] Example 4 Activation of Y199A / N149L mutant and large-scale induced expression

[0046] Add 10 μL of Y199A / N149L glycerol stock stored at -80°C to 4 mL of LB medium containing 50 μg / mL of K-1β-lactamase and incubate overnight at 37°C, 220 rpm. Add 2.5 mL of the culture to 250 mL of LB medium containing 50 μg / mL of kanamycin and incubate at 37°C, 220 rpm, until the OD reaches 0.6-0.8. Induce with 0.2 mM IPTG at 25°C, 180 rpm, and induction for 20 hours. Harvest the culture at 6500 rpm, 4°C, for 10 minutes, wash twice with purification equilibration buffer, and store the resulting wet cells at -80°C until further use.

[0047] The collected wet cells were resuspended in a ratio of 1 g (wet cells): 15 mL (reaction buffer) and ultrasonically disrupted in an ice bath, with the cell suspension on for 1 second and off for 2 seconds, for a total of 45 minutes. The cell lysate was centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was filtered through a 0.22 μm filter for subsequent purification. The purification method was nickel affinity chromatography: the purification column was prepacked with a 5 mL column. The entire purification system was first equilibrated with equilibration solution, and the sample was loaded at a flow rate of 1 mL / min. Unbound proteins were further removed with equilibration solution, and then gradient elution was performed with elution buffer (500 mM NaCl, 500 mM imidazole). The target protein was eluted at an imidazole concentration of approximately 150 mM. The target protein was collected, concentrated, and desalted using ultrafiltration tubes (50 kDa cutoff) to a final volume of 1 mL. The protein solution was then aliquoted and stored at -80°C until further use.

[0048] Example 5 Enzyme activity detection of Y199A / N149L mutant

[0049] Ketoreductase is an NADH-dependent oxidoreductase, NADH has a maximum UV absorption at 340 nm, and NAD + Since there is no maximum absorption at this location, the activity of the mutant is evaluated by monitoring the change in absorbance at 340 nm. The specific method is as follows: 0.5 μM pure enzyme, 0.5 mM 1a, and 1 mM NADH are added to a 250 μL reaction system (100 mM PBS buffer, pH 7.2) and the absorbance change is monitored over a 5-minute reaction. Immediately after the reaction, an equal volume of ethyl acetate is used for extraction. This extraction is repeated three times. The organic phases are combined and the solvent is evaporated using a nitrogen blowdown device. Finally, the product is dissolved in an equal volume of methanol and filtered through a 0.22 μm filter membrane for sample preparation. Product formation is detected by HPLC.

[0050] Table 2 Enzyme activity results of Y199A / N149L mutant

[0051] The above is only a relatively better specific implementation method of the present invention, but is not limited to this method. All easily conceivable improvements within the scope of the patent application of the present invention are within the scope of protection of the present invention.

Claims

1. A ketoreductase mutant, characterized in that The mutant is obtained by amino acid mutation based on the amino acid sequence shown in SEQ ID NO.2, wherein the mutation is (1) or (2): (1) Any one of Y199A, Y199C, M196W, A146F, A146L; (2) A combination of any one of Y199A, Y199C, M196W, A146F, A146L and any one of N149L, N149I, I195L and I195V.

2. The ketoreductase mutant according to claim 1, characterized in that The mutation is any one of the following combinations: Y199A / N149L, Y199A / N149I, Y199A / I195L, Y199A / I195V.

3. A DNA molecule, characterized in that Encoding the ketoreductase mutant according to claim 1 or 2.

4. A recombinant plasmid, characterized in that: Containing the DNA molecule according to claim 3.

5. A recombinant bacterium, characterized in that: Contains the recombinant plasmid according to claim 4.

6. The recombinant bacterium according to claim 5, characterized in that The host cells are yeast cells or Escherichia coli BL21 (DE3).

7. Use of the ketoreductase mutant according to claim 1 or 2, characterized in that: The ketoreductase mutants were combined with the NADH regeneration reduction system to catalyze the reduction reaction to prepare chiral steroid compounds.

8. The use according to claim 7, characterized in that: In combination with the NADH regeneration system, the ketoreductase mutant is used to catalyze the asymmetric carbonyl reduction reaction of the substrate to obtain cholesterol with a 24R-hydroxy configuration.

9. The use according to claim 8, characterized in that: The substrate is any one of the following structural formulas:

10. The use according to claim 9, characterized in that: The NADH regeneration system comprises glucose and glucose dehydrogenase; the reaction conditions are as follows: temperature 25±15° C., rotation speed 200±100 rpm, and time 12-24 h.

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