Ketoreductase mutant and application thereof

Abstract

The invention discloses a ketoreductase mutant and application thereof.The mutant is obtained through amino acid mutation on the basis of an amino acid sequence shown in SEQ ID NO.1, and the amino acid mutation at least comprises (1) or a combination of (1) and (2): (1) any one or more than two of D42A, P89G, E12G, P158S and L125M; and (2) any one or more than two of A327V, A327P, K67R and D42V. The mutant can regioselectively and stereoselectively reduce 24-keto of a substrate to obtain a target product with a 24R-hydroxyl configuration, the conversion efficiency can reach 99% or above, the optical purity of the target product is not lower than 99.8%, and synthesis of a high-purity squalane key intermediate can be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and biological enzyme catalysis, and particularly relates to a ketoreductase mutant with high selectivity and application thereof in synthesis of chiral alcohol. BACKGROUND

[0002] Ketoreductase is a biological catalyst capable of catalyzing asymmetric reduction of ketone compounds to produce chiral alcohol, and is widely present in enzymes in organisms. According to the difference in structure and catalytic property, ketoreductase can be divided into short-chain dehydrogenase / reductase, medium-chain dehydrogenase / reductase and aldehyde-ketone reductase. Medium-chain ketoreductase is an excellent biological catalyst for reducing ketone compounds to generate optically pure chiral alcohol. A ketoreductase (RhKRED) from Rhodococcus sp. is a zinc-dependent medium-chain ketoreductase, each subunit containing 2 zinc ions, one of which coordinates the active center Cys, His, Cys and Asp, and participates in catalysis; the other zinc coordinates 4 Cys, maintains the integrity and stability of the enzyme structure. The asymmetric reduction step includes binding of NADH, catalysis of substrate (substrate binding, proton transfer, electron transfer) and product release, and has high efficiency and high stereoselectivity.

[0003] Squalamine, also known as 3β-[[3-((4-aminobutyl)amino)propyl]amino]-5α-cholestan-7α,24R-diol-24-sulfate, is initially proved to have strong bactericidal activity and is used as a broad-spectrum aminosterol antibiotic. It is subsequently found that squalamine can inhibit retinal neovascularization and is used for treating retinal diseases; inhibit mitogen-induced endothelial cell proliferation and migration and is used for treating cancer; inhibit the interaction of α-synuclein oligomers with lipid membranes and eliminate the toxicity of α-synuclein oligomers in human neuroblastoma cells, and is used for treating neurological diseases, and has high medicinal significance and value.

[0004] In recent years, there have been reports of synthesizing squalamine using porcine deoxycholic acid, soybean sterol, dichlorvos, methyl 3β-hydroxy-5-cholanenoate, 3β-hydroxy-5,24-cholestadiene, and chenodeoxycholic acid as raw materials. Studies of the reported synthetic routes have revealed that the main challenge in squalamine synthesis lies in the construction of the carbonyl group at the 3-position and the 24R-OH group in the key intermediate 1. For the construction of the 24R-hydroxyl group, chemical methods require the introduction of protecting groups and chiral reagents, and the reaction conditions are stringent. To address this issue, patent CN117126823B provides a one-step method for constructing 24R-OH using a ketone reductase mutant of Novosphingobium aromaticivorans, specifically catalyzing the conversion of 3α,7α-dihydroxy-5α-cholestane-24-one (A6) to 3α,7α,24R-trihydroxy-5α-cholestane (B6). In the process of oxidizing the key intermediate 7α,24R-dihydroxy-5α-cholestan-3-one (B1), the low selectivity of the oxidation process leads to the generation of various impurities (including A1) while obtaining B1, resulting in a low yield. The impurities are difficult to purify and remove, which in turn leads to a low purity of B1. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention provides a ketone reductase mutant capable of stereoselectively reducing 24-carbonyl steroidal compounds to chiral alcohols.

[0006] Another objective of this invention is to provide a mutant that stereoselectively reduces the 24-keto group region of A1 to the 24R-hydroxy configuration (target product B1), thereby achieving the synthesis of a key intermediate of squalamine.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] To achieve the objectives of this invention, a ketone reductase library was screened to obtain a ketone reductase (RhKRED) from Rhodococcus sp., which can realize the enzymatic conversion of A1 to B1, with the amino acid shown in SEQ ID NO.1, and has the activity of regio- and stereoselectively reducing the carbonyl group at position 24 to a 24R-hydroxyl group.

[0009] A ketone reductase mutant, said mutant being obtained by amino acid mutation based on the amino acid sequence shown in SEQ ID NO.1, said amino acid mutation including at least (1), or a combination of (1) and (2):

[0010] (1) Any one or more of D42A, P89G, E12G, P158S, and L125M;

[0011] (2) Any one or more of A327V, A327P, K67R, and D42V.

[0012] Preferably, the mutation is any combination of the following: D42A / P89G, E12G / D42V, P89G / P158S, E12G / D42V / K67R, D42A / P89G / P158S, D42A / P89G / P158S / A327P, D42A / P89G / P158S / A327V, D42A / P89G / L125M / P158S / A327P.

[0013] Preferably, the ketone reductase mutant is used for stereoselective reduction of 24-carbonyl steroidal compounds to prepare 24R-chiral alcohol compounds; the structure of the 24-carbonyl steroidal compound is such that the steroidal compound has a carbonyl group at position 24 of its side chain, and the 24R-chiral alcohol compound has an R-hydroxyl group at position 24 of its side chain.

[0014] Preferably, the 24-carbonyl steroid compound is any one of the following:

[0015]

[0016] Preferably, the 24R-chiral alcohol compound is any one of the following:

[0017]

[0018] Preferably, using a 24-carbonylsteroid compound as a substrate, an alcohol solvent and a buffer solution are added, and combined with an NADH regeneration reduction system, the ketone reductase mutant catalyzes the side-chain carbonyl asymmetric reduction reaction of the substrate to obtain a chiral alcohol compound.

[0019] The ketone reductase mutant provided by this invention can catalyze the conversion of A1 to B1 under NADH regeneration system. The enzymatic conversion reaction formula is as follows: Figure 1 As shown.

[0020] Preferably, the NADH regeneration and reduction system comprises glucose and glucose dehydrogenase; the alcohol solvent is one or more of methanol, ethanol, n-propanol, isopropanol, tert-butanol, and n-butanol; the pH of the buffer solution is 5-7, more preferably 5.8-6.8; and the reaction conditions are: temperature 30±15℃, rotation speed 200±100rpm, and time 6±3h.

[0021] A DNA molecule that encodes any of the above mutants.

[0022] A recombinant plasmid, wherein the recombinant plasmid is obtained by ligating the above-mentioned DNA molecule into an expression vector selected from any one of the following: 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-23b(+), pET-24b(+), pET-23 ... ET-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-4 3b(+), 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, p UC-18 and pUC-19.

[0023] A recombinant cell containing the aforementioned recombinant plasmid. The host cell of the recombinant cell is a prokaryotic cell or a eukaryotic cell, preferably a yeast cell.

[0024] Furthermore, the host cell is a competent cell, preferably Escherichia coli BL21(DE3).

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

[0026] (1) The present invention provides a ketone reductase mutant that is tolerant to high concentrations of substrates. This mutant can regionally and stereoselectively reduce the 24-keto group of the substrate to obtain the target product B1 with a 24R-hydroxy configuration, thereby achieving the synthesis of a key intermediate of high-purity squalamine.

[0027] (2) The mutant of this invention can achieve an A1 conversion yield of over 99% and the optical purity of the target product is not less than 99.8%, which is beneficial to simplifying the synthesis steps of squalamine and improving production efficiency. Attached Figure Description

[0028] Figure 1 The reaction formula is for the mutant to catalyze the reaction of A1 to obtain B1.

[0029] Figure 2 This is the HPLC chromatogram of A1 catalyzed by the parental RhKRED.

[0030] Figure 3 The crystal structure of the R-configuration product B1 obtained by parental RhKRED catalysis of A1 is shown.

[0031] Figure 4 The HPLC chromatogram of Al catalyzed by the mutant D42A / P89G / P158S / A327P in Example 5 is shown. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments, but is not limited to the embodiments described herein. Unless otherwise specified, the following experiments are all conventional methods, and the experimental materials used can be easily purchased from commercial companies unless otherwise specified.

[0033] Example 1: Screening of ketone reductases using A1 as a substrate

[0034] Ketoreductase screening system: 1 mg / mL A1, 2% (v / v) methanol for dissolution, 50 mg / mL crude enzyme solution, 100 mM phosphate buffer (pH 7.2), 1 mg / mL NADP + / NAD + 18 mg / mL glucose, 50 mg / mL glucose dehydrogenase, and a total reaction volume of 1 mL were used. The reaction tube was placed in a temperature-controlled shaker (25℃, 220 rpm) for 24 h. After the reaction, an equal volume of ethyl acetate was added to terminate the reaction and extract. After vortexing for 1–2 min, the mixture was centrifuged at 10,000 rpm and 25℃ for 5 min. The upper organic phase was collected, and the extraction was repeated three times (3 × 1 mL). The organic phases were combined. Ethyl acetate was removed from the organic phase by rotary evaporation. 1 mL of chromatographic grade methanol was added to dissolve the residual substrate. After filtration through a 0.22 μm organic filter membrane, HPLC analysis was performed. The substrate peak time was 5.8 min, and the target product peak time was 6.4 min. Preliminary screening showed that RhKRED possesses regio- and stereoselective activity for reducing the 24-carbonyl group of A1 to generate a 24R-hydroxyl group. The HPLC results are shown below. Figure 2 As shown.

[0035] Example 2: Synthesis and Recombinant Expression of Ketoreductase pET28a-RhKRED

[0036] Using pET28a as a template, the pET28a-RhKRED plasmid was constructed. The amino acid sequence of RhKRED is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. The pET28a-RhKRED plasmid was transformed into *E. coli* BL21(DE3) (with BamHI and HindIII restriction sites). After recovery culture using the standard slow transfection method, an appropriate amount of bacterial culture was plated onto LB agar plates containing 100 μg / mL kanamycin and incubated overnight at 37°C with the plates inverted. 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 and 220 rpm until the OD value reached 0.6–0.8. 0.4 mM IPTG was added as an inducer, and the culture was incubated at 25°C and 180 rpm with shaking for 20 h. The bacterial cells were collected at 6500 rpm and 4°C for 10 min, and the collected cells were washed twice with reaction buffer.

[0037] Single-crystal cultivation of the product in Example 3

[0038] An enzyme-catalyzed reaction was prepared on a certain scale at a substrate concentration of 1 mg / mL, with a total reaction volume of 60 mL: 1 mg / mL substrate, 2% (v / v) methanol for dissolution, and 1 mg / mL NAD. + NADH, 18 mg / mL glucose, 50 mg / mL glucose dehydrogenase, and 50 mg / mL crude RhKRED enzyme solution were mixed thoroughly and reacted at 25°C and 220 rpm for 24 h on a shaker. After the reaction, the mixture was extracted three times with equal volumes of ethyl acetate. The organic phases were combined and the ethyl acetate was removed by rotary evaporation. The product was purified by silica gel column chromatography (ethyl acetate / petroleum ether, 1 / 2). 15 mg of the purified product was dissolved in a mixture of acetonitrile and methanol (chromatographic grade, acetonitrile / methanol: 4 / 1), filtered to obtain a clear solution, and allowed to slowly evaporate at room temperature for 5–10 days to obtain colorless, transparent blocky crystals. The obtained crystals were collected by Rigaku single-crystal diffraction at 100 K (-173.15°C) using a monochromatic CuKα X-ray target. ) and Hybrid Pixel Array detector, single-crystal detection results are shown in Figure 3 The product was confirmed to be the key intermediate B1.

[0039] Example 4 Construction of the ketoreductase pET28a-RhKRED mutant

[0040] Table 1 Primer sequences of the mutants

[0041]

[0042]

[0043] The PCR system consisted of: 1 ng template, 1 μL each of forward and reverse primers, 0.3 μL DMSO, 12.5 μL 2×Phanta Flash MasterMix, and sterile water to a final volume of 25 μL.

[0044] PCR reaction procedure: (1) 95℃ pre-denaturation for 3 min; (2) 98℃ denaturation for 30 s; (3) set the annealing temperature according to the Tm value of the mutation primer, and annealing time for 15 s; (4) 72℃ extension for 35 s, perform 32 cycles of steps (2) to (4), and finally extend at 72℃ for 5 min, and store the PCR product at 12℃.

[0045] The PCR products were digested with Dpn I enzyme to remove the template, and then transformed into *E. coli* DH5α competent cells. The recovered bacterial culture was plated on LB agar plates containing 100 μg / mL kanamycin and incubated overnight at 37°C. One to three single colonies were selected for gene sequencing. Multipoint mutants were obtained by continuously stacking mutations using the same method described above.

[0046] The induced expression of the mutant is consistent with that of the template described above.

[0047] Example 5: Activity screening of mutants catalyzing the conversion of A1 to B1

[0048] Mutant activity screening system: 6 mg / mL A1, 10% (v / v) methanol for dissolution, 50 mg / mL crude enzyme solution, 100 mM phosphate buffer (pH 6.0), 2 mg / mL NAD + The reaction mixture consisted of NADH, 18 mg / mL glucose, and 50 mg / mL glucose dehydrogenase, with a final volume of 1 mL. The entire reaction system was incubated at 40 °C and 220 rpm for 6 h. After the reaction was complete, an equal volume of ethyl acetate was used for extraction. This extraction was repeated three times. The organic phases were combined, the solvent was evaporated to dryness by rotary evaporation, and the mixture was redissolved in methanol. The solution was filtered through a 0.22 μm organic filter membrane, and the content was determined by HPLC.

[0049] Table 2 Results of activity detection of mutants

[0050]

[0051]

[0052] The conversion rate is defined as: (initial substrate concentration - final substrate concentration) / initial substrate concentration * 100%; the stereoselectivity is defined as: the diastereoselectivity (de) is defined as: [RS] / [R+S] * 100%.

[0053] Example 6 Catalyzing the formation of B6 from A6

[0054] Catalytic reaction system: 6 mg / mL A6, 10% (v / v) methanol for dissolution, 50 mg / mL D42A / P89G / P158S / A327P crude enzyme solution, 100 mM phosphate buffer (pH 6.0), 2 mg / mL NAD + The reaction mixture consisted of NADH, 18 mg / mL glucose, and 50 mg / mL glucose dehydrogenase, with a final volume of 1 mL. The entire reaction system was incubated at 40 °C and 220 rpm for 6 h, achieving a conversion rate of 95.1% and a de concentration (de) > 99.8%.

[0055] Example 7 Catalyzing the production of B2 from A2

[0056] Catalytic reaction system: 2 mg / mL A2, 10% (v / v) methanol for dissolution, 50 mg / mL D42A / P89G / P158S / A327P crude enzyme solution, 100 mM phosphate buffer (pH 6.0), 2 mg / mL NAD + The reaction mixture consisted of NADH, 18 mg / mL glucose, and 50 mg / mL glucose dehydrogenase, with a final volume of 1 mL. The entire reaction system was incubated at 40°C and 220 rpm for 6 hours, achieving a conversion rate of 97.1% and a decomposition rate (de>99.8%).

[0057] Example 8: Isopropanol-mediated wild-type RhKRED-catalyzed reaction

[0058] Catalytic reaction system: 2 mg / mL Al, 5% (v / v) isopropanol, 50 mg / mL wild-type crude enzyme solution, 100 mM phosphate buffer (pH 6.0), 2 mg / mL NAD + / NADH, final volume 1 mL. The entire reaction system was reacted at 40℃ and 220 rpm for 6 h to produce B1, with a conversion rate of 10.1% and de>99.8%.

[0059] Example 9: Isopropanol-mediated D42A / P89G / P158S / A327P catalytic reaction

[0060] 6 mg / mL A1, 23% (v / v) isopropanol, 50 mg / mL D42A / P89G / P158S / A327P crude enzyme solution, 100 mM phosphate buffer (pH 6.0), 2 mg / mL NAD + The final volume of NADH was 1 mL. The entire reaction system was reacted at 40 °C and 220 rpm for 6 h to produce B1, with a conversion rate of 99.0% and de > 99.8%.

[0061] Example 10: Isopropanol-mediated reaction catalyzed by D42A / P89G / L125M / P158S / A327P

[0062] 6 mg / mL A1, 23% (v / v) isopropanol, 50 mg / mL D42A / P89G / L125M / P158S / A327P crude enzyme solution, 100 mM phosphate buffer (pH 6.0), 2 mg / mL NAD + / NADH, final volume 1 mL. The entire reaction system was reacted at 40℃ and 220 rpm for 6 h to produce B1, with a conversion rate of 99.5% and de>99.8%.

[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A ketone reductase mutant, characterized in that, The mutant is obtained by amino acid mutation based on the amino acid sequence shown in SEQ ID NO.1, and the amino acid mutation includes at least (1), or a combination of (1) and (2): (1) Any one or more of D42A, P89G, E12G, P158S, and L125M; (2) Any one or more of A327V, A327P, K67R, and D42V.

2. The ketone reductase mutant according to claim 1, characterized in that, The mutation is any combination of the following: D42A / P89G, E12G / D42V, P89G / P158S, E12G / D42V / K67R, D42A / P89G / P158S, D42A / P89G / P158S / A327P, D42A / P89G / P158S / A327V, D42A / P89G / L125M / P158S / A327P.

3. The application of the ketone reductase mutant according to claim 1 or 2, characterized in that, The ketone reductase mutant is used for stereoselective reduction of 24-carbonyl steroidal compounds to prepare 24R-chiral alcohol compounds; the structure of the 24-carbonyl steroidal compound is that the steroidal compound has a carbonyl group at position 24 of its side chain, and the 24R-chiral alcohol compound has an R-hydroxyl group at position 24 of its side chain.

4. The application according to claim 3, characterized in that, The 24-carbonyl steroid compound is:

5. The application according to claim 4, characterized in that, The 24R-chiral alcohol compound is:

6. The application according to claim 3, 4, or 5, characterized in that, Using a 24-carbonylsteroid compound as a substrate, an alcohol solvent and a buffer solution were added, and a NADH regeneration reduction system was used to catalyze the side-chain carbonyl asymmetric reduction reaction of the substrate by the ketone reductase mutant, to obtain a chiral alcohol compound.

7. The application according to claim 6, characterized in that, The NADH regeneration and reduction system consists of glucose and glucose dehydrogenase; the alcohol solvent is one or more of methanol, ethanol, n-propanol, isopropanol, tert-butanol, and n-butanol; the pH of the buffer solution is 5-7; and the reaction conditions are: temperature 30±15℃, rotation speed 200±100rpm, and time 6±3h.

8. A DNA molecule, characterized in that, Encode the mutant as described in claim 1 or 2.

9. A recombinant plasmid, characterized in that, The recombinant plasmid is obtained by ligating the DNA molecule of claim 8 into the following expression vector, wherein the expression vector is selected from any one of the following: 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-23b(+), pET-24b(+), pET-25b(+), pET-23b(+), pET-24b(+), pET-25b(+), pET-23b(+), pET-24b(+), pET-25b(+), pET-23b(+), pET-23b(+), pET-24b(+), pET-23 ... ET-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-4 3b(+), 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, p UC-18 and pUC-19.

10. A recombinant cell, characterized in that, It contains the recombinant plasmid as described in claim 9.

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