Carbonyl reductase mutant and application thereof in synthesis of statin drug intermediates

Abstract

The invention provides a carbonyl reductase mutant. The carbonyl reductase mutant is obtained by performing single-point mutation or multi-point combined mutation on the 17th site, the 40th site and the 64th site of an amino acid sequence as shown in SEQ ID NO.1. The invention also provides a coding gene, a recombinant vector containing the coding gene, a co-expression engineering bacterium and an application of the co-expression engineering bacterium. Compared with a wild type enzyme, the carbonyl reductase mutant has high activity and high stereoselectivity, the enzyme activity can reach more than two times that of the wild type enzyme, the catalytic efficiency on a substrate precursor ketone is remarkably improved, the yield of a statin drug intermediate and chiral alcohol synthesized by an enzyme method of the carbonyl reductase mutant is remarkably improved, and the yield of a rosuvastatin intermediate (3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R) is remarkably improved. The highest yields of the (3R, 5R)-6-chloro-3, 5-dihydroxyhexanoic acid tert-butyl ester and the atorvastatin intermediate (3R, 5R)-6-cyano-3, 5-dihydroxyhexanoic acid tert-butyl ester reach 99% and 97.6% respectively, e.e. Is larger than 99%, and the method has high industrial application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a carbonyl reductase mutant and application thereof in the synthesis of third-generation statins. Background Art

[0002] Stereoselective carbonyl reductases (EC1.1.1.184) are enzymes that catalyze bidirectional, reversible redox reactions between alcohols and aldehydes / ketones. They require the coenzyme NAD(H) or NADP(H) as a hydrogen transfer agent, asymmetrically converting prochiral compounds to their corresponding chiral counterparts. They are among the most commonly used biocatalysts for the synthesis of chiral alcohols. Carbonyl reductases are widely available and are found in a variety of plants, bacteria, fungi, and animal tissues. They are primarily distributed in three superfamilies: short-chain dehydrogenases, medium-chain dehydrogenases, and aldehyde-keto reductases. While these three groups share similar catalytic functions, they differ significantly in their structures. The asymmetric reduction reaction catalyzed by carbonyl reductases follows a sequential Bi-Bi kinetic mechanism. The coenzyme NAD(P)H first binds to the enzyme to form the holoenzyme. The substrate then enters the enzyme's substrate binding pocket, receiving a [H] transferred by NAD(P)H and being reduced. Simultaneously, NAD(P)H loses [H] and is oxidized. The oxidized NADP is then oxidized. + Separated from the enzyme, the product is released from the active center, and finally the oxidized NADP is converted to + Reduction to NADPH for recycling. Stereoscopic carbonyl reductases have a broad substrate spectrum, catalyzing the conversion of various aldehyde- or carbonyl-containing substances into chiral alcohols. However, different chiral isomers typically exhibit different pharmacological activities. Therefore, the development of a highly active, stereoselective, and product-specific carbonyl reductase has high research and application value.

[0003] Statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors that lower low-density lipoprotein cholesterol (LDL-C) levels by inhibiting cholesterol synthesis in the liver. They are the first-line drug class for treating hypercholesterolemia. Rosuvastatin calcium is a third-generation, fully synthetic statin and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. It lowers low-density lipoprotein cholesterol (LDL-C) levels by inhibiting cholesterol synthesis in the liver. It is the first-line drug for treating hypercholesterolemia and is known as a "super statin." The chiral intermediate (tert-Butyl(3R,5S)-6-chloro-3,5-dihydroxyh-exanoate, (3R,5S)-CDHH) of rosuvastatin forms the drug's chiral side chain and pharmacophore, making it a key intermediate in the synthesis of rosuvastatin. It possesses two chiral centers and four isomers. The key to rosuvastatin synthesis lies in the construction of optically pure intermediates, which are currently prepared primarily through chemical and enzymatic methods. The key difficulty in chemically synthesizing the chiral side chains of statins lies in the construction of chiral centers, requiring expensive precious metal catalysts and the use of boron hydride as a strong reducing agent for asymmetric reduction of the carbonyl group. This makes it difficult to achieve high optical purity of the product at the chiral center. Enzymatic synthesis, however, has been adopted for the synthesis of (3R,5S)-CDHH due to its high optical purity, mild reaction conditions, high catalytic efficiency, and minimal pollution. Liu et al. used the carbonyl reductase RtSCR from the yeast Rhodosporidium toruloides to asymmetrically catalyze the synthesis of (3R,5S)-CDHH. Through molecular engineering and reaction system optimization, they achieved efficient substrate conversion, achieving a yield of 98.9% and an EE >99% at a substrate concentration of 1 M. Building on this, Zheng further coupled RtSCR9 with EsGDH to construct a dual-enzyme co-expression system, effectively regenerating NADPH. By adding substrate in a fed-batch manner, substrate conversion reached 98.5% with an EE >99%.

[0004] Similarly, a chiral intermediate for the HMG-CoA inhibitor atorvastatin, tert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, can also be synthesized via biocatalysis. Shi et al. used the aldehyde-keto reductase KRED to reduce 450 g / L of the substrate (tert-butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate) to successfully obtain the stereospecific key intermediate, tert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, with a substrate conversion of 97.98% and an ee of 99.7% (CN114634944A). Wang et al. used a carbonyl reductase from Starmerella magnolia to catalyze the reduction of tert-butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate, achieving an ee ≥ 98% and a conversion ≥ 95% (CN116515780A).

[0005] Carbonyl reductases enable efficient asymmetric synthesis of statins and other drugs, and have high industrial application value. However, currently, the available sources of carbonyl reductases for industrial production are limited, and the specific catalytic mechanism has yet to be precisely elucidated. The discovery and modification of high-performance carbonyl reductases through protein engineering and the biosynthesis of statin pharmaceutical intermediates remain significant challenges in the green biomanufacturing of pharmaceutical chemicals. Summary of the Invention

[0006] To further improve the production efficiency and product purity of the chiral alcohols (3R,5S)-6-chloro-3,5-dihydroxyhexanoate or (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, both of which are statin intermediates, this paper investigates the stereospecific recombinant carbonyl reductase (SCR) derived from Rhodosporidium toruloides ZJB2014212. This study yields a highly active and stereoselective recombinant carbonyl reductase mutant, its encoding gene, and a recombinant vector and genetically engineered bacteria containing the mutant's encoding gene. This recombinant carbonyl reductase mutant can be used in the enzymatic synthesis of chiral alcohols, which are statin intermediates.

[0007] The technical solution adopted in the present invention is:

[0008] A carbonyl reductase mutant is obtained by performing single-point mutation or multi-point combined mutation at positions 17, 40 and 64 of the amino acid sequence shown in SEQ ID NO.1; the carbonyl reductase with the amino acid sequence shown in SEQ ID NO.1 is derived from Rhodosporidium toruloides.

[0009] Preferably, the mutation is one or more of the following combinations:

[0010] (1) Lysine 17 is mutated to any of valine, aspartic acid, or histidine;

[0011] (2) Arginine at position 40 is mutated to either aspartic acid or serine;

[0012] (3) Alanine at position 64 is mutated to any of valine, tyrosine, and serine.

[0013] Furthermore, it is preferred that the mutant is one of the following:

[0014] (1) mutating the lysine at position 17 of the amino acid sequence shown in SEQ ID NO. 1 to histidine (L17H);

[0015] (2) mutating the arginine at position 40 of the amino acid sequence shown in SEQ ID NO.1 to serine (R40S);

[0016] (3) The alanine at position 64 of the amino acid sequence shown in SEQ ID NO. 1 was mutated to valine (A64V);

[0017] (4) The lysine at position 17 of the amino acid sequence shown in SEQ ID NO. 1 was mutated to histidine, and the alanine at position 64 was mutated to valine (L17H / A64V);

[0018] (5) The lysine at position 17 of the amino acid sequence shown in SEQ ID NO. 1 was mutated to histidine, the arginine at position 40 was mutated to serine, and the alanine at position 64 was mutated to valine (L17H / R40S / A64V).

[0019] Due to the specificity of amino acid sequences, any fragment or variant of a peptide protein containing the amino acid sequence of the present invention, such as a conservative variant, biologically active fragment, or derivative thereof, falls within the scope of protection of the present invention, as long as the peptide protein fragment or peptide protein variant has at least 90% homology with the aforementioned amino acid sequence. Specifically, the alterations include deletions, insertions, or substitutions of amino acids in the amino acid sequence; for conservative alterations of the variant, the replaced amino acid has similar structural or chemical properties to the original amino acid, such as replacing isoleucine with leucine or replacing asparagine with glutamine. Enzyme variants may also have non-conservative alterations.

[0020] The present invention also provides a gene encoding the carbonyl reductase mutant.

[0021] Specifically, the nucleotide sequence of the coding gene corresponding to the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.2.

[0022] Due to the specificity of nucleotide sequences, any variant of the polynucleotide of the present invention, as long as it has more than 90% homology with the aforementioned polynucleotide, falls within the scope of protection of the present invention. The polynucleotide variant refers to a polynucleotide sequence with one or more nucleotide changes. This polynucleotide variant can be a natural or non-natural variant, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, but does not substantially change the function of the peptide protein it encodes.

[0023] The present invention also provides a recombinant vector containing the gene encoding the carbonyl reductase mutant. The recombinant vector comprises a polynucleotide operably linked to a control sequence suitable for directing expression in a host cell. Various conventional vectors in the art, such as various plasmids, phages, or viral vectors, linked to the nucleotide sequence of the novel carbonyl reductase mutant of the present invention, should all fall within the scope of protection of the present invention. The recombinant vector preferably uses the plasmid pET-28a(+) as an expression vector, and the gene encoding the novel carbonyl reductase mutant is linked to the plasmid pET-28a(+).

[0024] The present invention also provides a genetically engineered bacterium containing a gene encoding the carbonyl reductase mutant, which is obtained by constructing a recombinant vector containing the gene encoding the carbonyl reductase mutant. The recombinant genetically engineered bacterium is generally constructed by ligating the gene encoding the carbonyl reductase mutant with the expression vector pET-28a(+) to construct a recombinant vector containing the gene encoding the carbonyl reductase mutant, and then transforming the recombinant vector into a host cell to obtain a genetically engineered bacterium containing the recombinant plasmid. The host cell can be a bacterium, a fungus, a plant cell, or an animal cell, with Escherichia coli BL21 (DE3) being preferred as the expression host.

[0025] The coding gene of the carbonyl reductase mutant is introduced into a host cell through genetic engineering technology to construct a genetically engineered bacterium and express the bacterium to obtain the carbonyl reductase mutant of the present invention.

[0026] The carbonyl reductase mutant can be obtained by the following method:

[0027] A recombinant vector containing the encoding gene of the carbonyl reductase mutant is constructed, the recombinant vector is transformed into a host bacterium, the obtained genetically engineered bacteria are induced and cultured, the culture fluid is separated to obtain wet bacterial cells containing the carbonyl reductase mutant, the wet bacterial cells are crushed to obtain a crude enzyme liquid of the carbonyl reductase mutant M6, and the crude enzyme liquid is purified to obtain a pure enzyme of the carbonyl reductase mutant.

[0028] In the present invention, the carbonyl reductase mutant can be used in various forms. For example, resting cells or wet cells expressing the carbonyl reductase mutant of the present invention can be used, or various forms such as crude enzyme solution, pure enzyme, or crude enzyme powder can be used, or an immobilized enzyme can be used.

[0029] Preferably, in order to obtain higher transformation efficiency and reduce costs, wet cells are preferably used.

[0030] The present invention also provides the use of the carbonyl reductase mutant in the synthesis of statin intermediates, wherein the statin intermediate is rosuvastatin intermediate (3R, 5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester or atorvastatin intermediate (3R, 5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester.

[0031] Preferably, the application method is: using wet cells obtained by fermentation culture of genetically engineered bacteria containing a gene encoding a carbonyl reductase mutant, crude enzyme liquid after crushing the wet cells, or extracted pure enzyme as a catalyst, (S)-6-chloro-5-hydroxy-3-oxohexanoic acid tert-butyl ester ((S)-CHOH) or (R)-6-cyano-5-hydroxy-3-oxohexanoic acid tert-butyl ester as a substrate, isopropanol as a cosubstrate, and NADPH as a coenzyme, respectively, in a buffer solution with a pH value of 6.0 to 8.0 to form a reaction system to prepare rosuvastatin intermediate (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester or atorvastatin intermediate (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester;

[0032] The reaction temperature is preferably 25-40°C, the stirring speed is preferably 200-800 rpm, and the reaction time is preferably 1-10 hours.

[0033] The buffer solution is preferably a 0.1 mM potassium phosphate buffer solution with a pH of 7.0.

[0034] The volume amount of isopropyl alcohol is preferably 20%-40% of the volume of the reaction system.

[0035] When the catalyst is in the form of wet cells, the amount of carbonyl reductase mutant wet cells used is 10-40 g / L buffer based on the weight of the wet cells. Furthermore, since the wet cells contain coenzymes, there is no need to add additional NADPH.

[0036] The concentration of the substrate is 100-400 g / L.

[0037] The wet cells can be prepared as follows: a genetically engineered bacterium containing a gene encoding a carbonyl reductase mutant is inoculated into an LB liquid culture medium containing a final concentration of 50 mg / L kanamycin resistance, cultured at 37° C. and 200 rpm for 8 hours, then inoculated into a fresh LB liquid culture medium containing a final concentration of 50 mg / L kanamycin resistance at a volume concentration of 2%, cultured at 37° C. and 200 rpm for 2 hours, IPTG with a final concentration of 0.1 to 0.3 mM is added, and induction culture is carried out at 28° C. and 200 rpm for 12 hours, followed by centrifugation at 4° C. and 8000 rpm for 15 minutes, discarding the supernatant, and collecting the wet cells.

[0038] In the present invention, the coenzyme NADPH is added to form an enzyme-enzyme coupled coenzyme regeneration system, and isopropanol is added as a cosubstrate. A carbonyl reductase mutant first combines with the coenzyme NADPH to form the holoenzyme. The substrate enters the substrate binding pocket of the enzyme, receiving a [H] transferred by NADPH and being reduced. Simultaneously, NADPH loses [H] and is oxidized. Oxidized NADP then separates from the enzyme, releasing the product from the active center. Finally, a chiral substance or enzyme is used to reduce the oxidized NADP to NADPH for recycling.

[0039] The reaction formula is as follows:

[0040]

[0041] The present invention also provides a method for preparing a rosuvastatin intermediate or an atorvastatin intermediate, which comprises:

[0042] The invention relates to a method for preparing a rosuvastatin intermediate (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester or an atorvastatin intermediate (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester, using wet cells obtained by fermentation culture of a genetically engineered bacterium containing a gene encoding a carbonyl reductase mutant, a crude enzyme solution after crushing the wet cells, or an extracted pure enzyme as a catalyst, with (S)-6-chloro-5-hydroxy-3-oxohexanoic acid tert-butyl ester ((S)-CHOH) or (R)-6-cyano-5-hydroxy-3-oxohexanoic acid tert-butyl ester as a substrate, isopropanol as a cosubstrate, and NADPH as a coenzyme, respectively, in a buffer solution with a pH value of 6.0-8.0 to prepare a rosuvastatin intermediate (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester or an atorvastatin intermediate (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester.

[0043] The beneficial effects of the present invention are as follows: the carbonyl reductase mutant provided by the present invention has high activity and high stereoselectivity compared to the wild-type enzyme, and the enzyme activity can reach more than 2 times that of the wild-type, significantly improving the catalytic efficiency of the substrate precursor ketone, and significantly improving the yield of chiral alcohols. The carbonyl reductase mutant is applied to the bioenzymatic synthesis of chiral alcohols, and the maximum yields of rosuvastatin intermediate (3R, 5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester and atorvastatin intermediate (3R, 5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester reach 99.9% and 98.1%, respectively, with ee>99.9%. In addition, in the reaction system for catalytic preparation of chiral alcohols, the present invention constructs a single enzyme double substrate coupling system and selects isopropanol as the auxiliary substrate of the coenzyme regeneration system, which can effectively save production costs and is of great significance for the industrial application of bioenzymatic catalytic preparation of chiral alcohols. Compared with chemical methods for preparing chiral alcohols, the present invention utilizes asymmetric catalytic reduction of precursor ketones by recombinant carbonyl reductase mutants to synthesize chiral alcohols, thereby improving the optical purity of the product, achieving milder reaction conditions, lowering equipment requirements, reducing reaction costs, and being environmentally friendly, thereby having great industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Figure 3 is an SDS-PAGE image of carbonyl reductase and its mutants, wherein lane M is a protein molecular weight marker, lane 1 is pure enzyme of the original carbonyl reductase strain SCR, lane 2 is pure enzyme of SCR-L17H; lane 3 is pure enzyme of SCR-R40S, lane 4 is pure enzyme of SCR-A64V, lane 5 is pure enzyme of SCR-L17H / A64V, and lane 6 is pure enzyme of SCR-L17H / R40S / A64V. DETAILED DESCRIPTION

[0045] The following describes the embodiments of the present invention by specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the case of no conflict, the features in the following examples and embodiments can be combined with each other. In the embodiments of the present invention, unless otherwise specified, the methods used are all conventional methods, and the reagents used can be obtained from commercial sources.

[0046] Example 1: Construction of novel carbonyl reductase mutants

[0047]

Construction and induced expression of carbonyl reductase engineered bacteria

[0048] The carbonyl reductase encoding gene SEQ ID NO. 2 was ligated with the expression vector pET28b to construct a heterologous expression recombinant plasmid containing the carbonyl reductase encoding gene. The expression recombinant plasmid was transformed into host bacteria E. coli BL21 (DE3) competent cells to obtain recombinant genetically engineered bacteria E. coli BL21 (DE3) / pET28b (+) -SCR containing the recombinant plasmid.

[0049] After the carbonyl reductase engineered bacteria E. coli BL21 (DE3) / pET28b (+) -SCR was thawed on ice, the bacterial solution was streaked onto a kanamycin-resistant LB plate and incubated at 37 ° C for 12 hours. A single colony was picked and inoculated into a 10 mL LB test tube with a final concentration of 50 mg / L kanamycin resistance, and cultured at 37 ° C, 200 rpm for 8 hours for later use. Take 2 mL of the bacterial solution from the test tube and inoculate it into a 100 mL LB shake flask with a final concentration of 50 mg / L kanamycin resistance, cultured at 37 ° C, 200 rpm for 2 hours, add 100 μL of 0.1 M IPTG, cultured at 28 ° C, 200 rpm for 12 hours, centrifuged at 4 ° C, 8000 rpm for 15 minutes to collect the bacteria, and refrigerated at -20 ° C for later use.

[0050] Obtaining single-point mutants of carbonyl reductase

[0051] The prepared vector pET28b(+)-SCR was used as a template and the site-directed mutagenesis primers described below (Table 1) were used to introduce mutations by PCR. The mutation sites are underlined.

[0052] Table 1. Primer list

[0053]

[0054] The PCR reaction procedure was as follows: 98°C for 5 min, 98°C for 30 s, 55°C for 30 s, and 72°C for 3 min 30 s, repeated for 30 cycles; followed by extension at 72°C for 5 min. The PCR product was inactivated by treatment with Dpn I at 37°C for 3 h, then transformed into E. coli BL21(DE3) recipient strains and plated onto LB plates containing a final concentration of 50 mg / L kanamycin. After incubation at 37°C for 12 h, single colonies were randomly selected for sequencing analysis, yielding the carbonyl reductase mutants SCR-L17V, SCR-L17H, SCR-L17D, SCR-R40D, SCR-R40S, SCR-A64V, SCR-A64S, and SCR-A64Y.

[0055]

Obtaining multi-point combination mutants of carbonyl reductase

[0056] Using the single mutant plasmid as a template, new mutation sites were added, and double mutants were obtained through PCR, transformation, sequencing and other steps.

[0057] Specific examples are as follows:

[0058] The carbonyl reductase mutant SCR-L17H was named R1. The mutation was introduced by PCR using plasmid DNA containing the SCR-L17H gene as a template. The multi-point combination mutagenesis primers are as follows, and the mutation sites are underlined:

[0059] L17HA64V-F: 5'-ACAGCACGAT GTA AGCGATGAAG-3';

[0060] L17HA64V-R: 5'-CTTCATCGCT TAC ATCGGTGCTGT-3'.

[0061] The PCR reaction procedure was as follows: 98°C for 5 minutes, 98°C for 30 seconds, 55°C for 30 seconds, and 72°C for 3 minutes and 30 seconds, repeated for 30 cycles; followed by extension at 72°C for 5 minutes. The PCR product was inactivated by treatment with Dpn I at 37°C for 3 hours, then transformed into E. coli BL21(DE3) recipient strains, plated on LB plates containing a final concentration of 50 mg / L kanamycin, and incubated at 37°C for 12 hours. Randomly selected single colonies were sequenced and analyzed, resulting in a recombinant carbonyl reductase multi-point mutant, SCR-L17H / A64V, designated R2.

[0062] Using plasmid DNA containing the SCR-L17H / A64V gene as a template, mutations were introduced by PCR. The multi-point combination mutagenesis primers are as follows, and the mutation sites are underlined:

[0063] L17H R40S A64V-F: 5'-CACCGGTCGT AGC GCCGACGTTG-3′;

[0064] L17H R40S A64V-R: 5'-CAACGTCGGC GCT ACGACCGGTG-3'.

[0065] The PCR reaction procedure was as follows: 98°C for 5 minutes, 98°C for 30 seconds, 55°C for 30 seconds, and 72°C for 3 minutes and 30 seconds, repeated for 30 cycles; followed by extension at 72°C for 5 minutes. The PCR product was inactivated by treatment with Dpn I at 37°C for 3 hours, then transformed into E. coli BL21(DE3) recipient strains, plated on LB plates containing a final concentration of 50 mg / L kanamycin, and incubated at 37°C for 12 hours. Randomly selected single colonies were sequenced and analyzed, resulting in a recombinant carbonyl reductase multi-point mutant, SCR-L17H / R40S / A64V, designated R3.

[0066] Example 2: Screening of optimal novel carbonyl reductase mutants

[0067] The novel carbonyl reductase mutant obtained in Example 1 was subjected to enzyme activity assay analysis to determine the optimal mutant.

[0068] Preparation of wet cells: The genetically engineered bacteria containing the gene encoding the novel carbonyl reductase mutant were inoculated into LB liquid medium containing a final concentration of 50 mg / L kanamycin resistance, cultured at 37°C, 200 rpm for 8 h, then inoculated into fresh LB liquid medium containing a final concentration of 50 mg / L kanamycin resistance at a volume concentration of 2%, cultured at 37°C, 200 rpm for 2 h, IPTG was added at a final concentration of 0.1 mM, and the culture was induced at 28°C, 200 rpm for 12 h. The culture was centrifuged at 4°C, 8000 rpm for 15 min, the supernatant was discarded, and the wet cells were collected.

[0069] The carbonyl reductase wet cells are further ultrasonically disrupted to obtain a carbonyl reductase crude enzyme solution. The specific steps are:

[0070] The wet cells were washed three times with physiological saline, and the collected wet cells were resuspended in 100 mM potassium phosphate buffer, pH 7.0, to a 50 g / L cell solution. Ultrasonic disruption was performed on ice (65% power, 1 s duration, 2 s rest, 20 min continuous disruption) to obtain a cell disruption solution. The cell disruption solution was centrifuged, and the resulting supernatant was the desired crude enzyme solution.

[0071] The crude enzyme solution can be further purified using anion exchange columns and dialyzed for desalination to obtain pure enzyme. The specific purification steps are:

[0072] DEAE Sefinose TMA 6FF anion exchange column was equilibrated with 100 mM potassium phosphate buffer, pH 7.0, at a flow rate of 2 mL / min. After the baseline leveled out, the crude enzyme solution was loaded onto the column at a flow rate of 1.0 mL / min. Elution was first performed with 20 mM Tris-HCl buffer, pH 7.0, at a flow rate of 2 mL / min. After the baseline leveled out, a gradient elution was performed with 1 M NaCl in 20 mM Tris-HCl buffer, pH 7.0, to collect the active fractions, yielding pure carbonyl reductase. The collected enzyme was then dialyzed overnight against ultrapure water to remove residual high NaCl concentrations. The entire purification and dialysis process was performed at 4°C. The pure enzyme of the original carbonyl reductase strain SCR, pure enzyme of SCR-L17H, pure enzyme of SCR-R40S, pure enzyme of SCR-A64V, pure enzyme of SCR-L17H / A64V and pure enzyme of SCR-L17H / R40S / A64V were obtained respectively.

[0073] SDS-PAGE diagram of the wild-type carbonyl reductase and its mutants, where lane M is the protein molecular weight marker, lane 1 is the pure enzyme of the original carbonyl reductase strain SCR, lane 2 is the pure enzyme of SCR-L17H; lane 3 is the pure enzyme of SCR-R40S, lane 4 is the pure enzyme of SCR-A64V, lane 5 is the pure enzyme of SCR-L17H / A64V, and lane 6 is the pure enzyme of SCR-L17H / R40S / A64V.

[0074] This example also provides the use of a carbonyl reductase mutant in the preparation of rosuvastatin intermediate 6-chloro-(3R,5S)-dihydroxyhexanoic acid tert-butyl ester, the use comprising:

[0075] In a 5 mL reaction system, the substrate (S)-CHOH was dissolved in 2 mL of isopropanol (final concentration of 100 g / L), 2 mL of 0.1 mM, pH 7.0 KH2PO4-K2HPO4 buffer solution was added, and 1 mL of recombinant carbonyl reductase mutant wet bacteria (using 15 g / L buffer) was added. The mixture was incubated in a 30°C water bath with a magnetic stirrer at 600 rpm for 20 min. After the reaction was completed, 5 mL of 30% acetonitrile aqueous solution was added to terminate the reaction. A 200 μL sample was taken, separated and purified, and diluted 50-fold with 30% acetonitrile. The enzyme activity was detected by HPLC analysis. The results are shown in Table 2.

[0076] Through site-directed mutagenesis technology, the catalytic activity of carbonyl reductase towards substrates was further improved. After combining mutations at the optimal different mutation sites, the optimal mutant obtained was R3, namely SCR-L17H R40SA64V.

[0077] Table 2. Comparison of relative enzyme activities

[0078]

[0079] Example 3: Application of mutant L17H in the preparation of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

[0080] Furthermore, the single-point mutant SCR L17H with better activity in Example 2 was selected and applied to prepare a rosuvastatin intermediate. The preparation method is as follows:

[0081] The composition of the catalytic system and catalytic conditions are as follows:

[0082] In a 10mL reaction system, the substrate (S)-CHOH was dissolved in 4mL of isopropanol (final concentration 400g / L), 6mL of 0.1mM KH2PO4-K2HPO4 buffer solution, pH 7.0, and 1mL of wet cells of the recombinant carbonyl reductase mutant (using 15g / L buffer) were added. The reaction was incubated in a 30°C water bath with a magnetic stirrer at 600rpm. Samples were taken periodically, each in a 200μL volume. After separation and purification, the sample was diluted 200-fold with 30% acetonitrile, and the conversion was determined by HPLC. Results showed that after 7.5h of catalysis, the yield of (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester reached over 99.9%, with an ee >99.9%.

[0083] Example 4: Application of mutant R40S in the preparation of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

[0084] Furthermore, the single point mutant SCR R40S with better activity in Example 2 was selected for use in the preparation. The preparation method of the rosuvastatin intermediate is as follows:

[0085] The composition of the catalytic system and catalytic conditions are as follows:

[0086] In a 10mL reaction system, the substrate (S)-CHOH was dissolved in 4mL of isopropanol (final concentration 400g / L), 6mL of 0.1mM KH2PO4-K2HPO4 buffer solution, pH 7.0, and 1mL of wet cells of the recombinant carbonyl reductase mutant (using 15g / L buffer) were added. The reaction was incubated in a 30°C water bath with a magnetic stirrer at 600rpm. Samples were taken periodically, each in a 200μL volume. After separation and purification, the sample was diluted 200-fold with 30% acetonitrile, and the conversion was determined by HPLC. Results showed that after 7h of catalysis, the yield of (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester reached over 99.9%, with an ee >99.9%.

[0087] Example 5: Use of mutant A64V in the preparation of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

[0088] Furthermore, the single point mutant SCRA64V with better activity in Example 2 was selected for use in the preparation. The preparation method of the rosuvastatin intermediate is as follows:

[0089] The composition of the catalytic system and catalytic conditions are as follows:

[0090] In a 10mL reaction system, the substrate (S)-CHOH was dissolved in 4mL of isopropanol (final concentration 400g / L), 6mL of 0.1mM KH2PO4-K2HPO4 buffer solution, pH 7.0, and 1mL of wet cells of the recombinant carbonyl reductase mutant (using 15g / L buffer) were added. The reaction was incubated in a 30°C water bath with a magnetic stirrer at 600rpm. Samples of 200μL were taken periodically during the reaction, separated and purified, and diluted 200-fold with 30% acetonitrile. The conversion was determined by HPLC. Results showed that after 6.5h of catalysis, the yield of (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester reached over 99.9%, with an ee >99.9%.

[0091] Example 6: Application of combined mutants in the preparation of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

[0092] Furthermore, the multi-point combination mutant SCR L17H / R40S / A64V with the best activity in Example 2 was selected for use in the preparation. The preparation method of the rosuvastatin intermediate is as follows:

[0093] The composition of the catalytic system and catalytic conditions are as follows:

[0094] In a 10mL reaction system, the substrate (S)-CHOH was dissolved in 4mL of isopropanol (final concentration 400g / L), 6mL of 0.1mM KH2PO4-K2HPO4 buffer solution, pH 7.0, and 1mL of wet cells of the recombinant carbonyl reductase mutant (using 15g / L buffer) were added. The reaction was incubated in a 30°C water bath with a magnetic stirrer at 600rpm. Samples were taken periodically, each in a 200μL volume. After separation and purification, the sample was diluted 200-fold with 30% acetonitrile, and the conversion was determined by HPLC. Results showed that after 6h of catalysis, the yield of (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester reached over 99.9%, with an ee >99.9%.

[0095] In addition, this example also used the optimal mutant R3 SCR L17H / R40S / A64V to catalyze the preparation of rosuvastatin intermediates in a 100 mL reaction system. In the 100 mL reaction system, the final concentration of the substrate (S)-CHOH was 400 g / L, and whole-cell E. coli BL21 (DE3) / pET28b-SCR was used. L17H / R40S / A64VThe reaction was carried out using a bacterial suspension as a catalyst (final concentration of 15 g / L), 40 ml of isopropanol as a co-substrate, and a 30°C water bath with agitation at 600 rpm. Samples of 200 μL were taken periodically during the reaction, purified, and diluted 200-fold with pure acetonitrile. The conversion was determined by HPLC. Results showed that after 6 hours of catalysis, the yield of tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate reached over 99.9%, with an ee exceeding 99%.

[0096] Example 7: Use of mutant L17H in the preparation of tert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate

[0097] The mutant SCR L17H with better activity in Example 2 was selected for the preparation of tert-butyl (3R,5R)-6-cyano-dihydroxyhexanoate using tert-butyl 6-cyano-(5R)-3-oxohexanoate as a substrate through a bioconversion reaction.

[0098] The composition of the catalytic system and catalytic conditions are as follows:

[0099] To a 10mL reaction system, 6mL of potassium phosphate buffer (pH 7.0), 30g / L of wet cells of the recombinant carbonyl reductase mutant SCR-L17H, and 4mL of isopropanol were added. The reaction mixture was incubated at 30°C in a water bath with a magnetic stirrer at 600rpm. Samples of 200μL were taken periodically and diluted 50-fold with 30% acetonitrile aqueous solution. The conversion was determined by HPLC. Results showed that at a substrate concentration of 200g / L, the yield of the product, tert-butyl (3R,5R)-6-cyano-dihydroxyhexanoate, reached 85.6% after 3h of catalysis, with an ee >99.9%.

[0100] Example 8: Application of mutant R40S in the preparation of tert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate

[0101] The mutant SCR R40S with better activity in Example 2 was selected for the preparation of tert-butyl (3R,5R)-6-cyano-dihydroxyhexanoate using tert-butyl 6-cyano-(5R)-3-oxohexanoate as a substrate through a bioconversion reaction.

[0102] The composition of the catalytic system and catalytic conditions are as follows:

[0103] To a 10mL reaction system, 6mL of potassium phosphate buffer (pH 7.0), wet cells of the recombinant carbonyl reductase mutant SCR-R40S (30g / L buffer), and 4mL of isopropanol were added. The reaction mixture was incubated at 30°C in a water bath with a magnetic stirrer at 600rpm. Samples of 200μL were taken periodically and diluted 50-fold with 30% acetonitrile aqueous solution. The conversion was determined by HPLC. Results showed that at a substrate concentration of 200g / L, the yield of the product, tert-butyl (3R,5R)-6-cyano-dihydroxyhexanoate, reached 89.2% after 3h of catalysis, with an ee >99.9%.

[0104] Example 9: Use of mutant A64V in the preparation of tert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate

[0105] The mutant SCRA64V with the better activity in Example 2 was selected for the preparation of tert-butyl (3R,5R)-6-cyano-dihydroxyhexanoate using tert-butyl 6-cyano-(5R)-3-oxohexanoate as a substrate through a biotransformation reaction.

[0106] The composition of the catalytic system and catalytic conditions are as follows:

[0107] To a 10mL reaction system, 6mL of potassium phosphate buffer (pH 7.0), wet cells of the recombinant carbonyl reductase mutant SCR-A64V (30g / L buffer), and 4mL of isopropanol were added. The reaction mixture was incubated at 30°C in a water bath with a magnetic stirrer at 600rpm. Samples of 200μL were taken periodically and diluted 50-fold with 30% acetonitrile aqueous solution. The conversion was determined by HPLC. Results showed that at a substrate concentration of 200g / L, the yield of the product, tert-butyl (3R,5R)-6-cyano-dihydroxyhexanoate, reached 90.6% after 3h of catalysis, with an ee >99.9%.

[0108] Example 10: Application of combined mutants in the preparation of tert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate

[0109] The mutant SCR L17H / R40S / A64V with the best activity in Example 2 was selected for the preparation of tert-butyl (3R,5R)-6-cyano-dihydroxyhexanoate using tert-butyl 6-cyano-(5R)-3-oxohexanoate as a substrate through a bioconversion reaction.

[0110] The composition of the catalytic system and catalytic conditions are as follows:

[0111] To a 10mL reaction system, 6mL of potassium phosphate buffer (pH 7.0), 30g / L of wet cells of the recombinant carbonyl reductase mutant SCR-L17H / R40S / A64V, and 4mL of isopropanol were added. The reaction mixture was incubated at 30°C in a water bath with a magnetic stirrer at 600rpm. Samples of 200μL were taken periodically and diluted 50-fold with 30% acetonitrile aqueous solution. The conversion was determined by HPLC. Results showed that at a substrate concentration of 200g / L, the yield of the product, tert-butyl (3R,5R)-6-cyano-dihydroxyhexanoate, reached 98.1% after 3h of catalysis, with an ee >99.9%.

[0112] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that The carbonyl reductase mutant is obtained by performing single-point mutation or multi-point combined mutation at positions 17, 40 and 64 of the amino acid sequence shown in SEQ ID NO.

1.

2. The carbonyl reductase mutant according to claim 1, characterized in that The mutation is one or more of the following combinations: (1) Lysine 17 is mutated to any of valine, aspartic acid, or histidine; (2) Arginine at position 40 is mutated to either aspartic acid or serine; (3) Alanine at position 64 is mutated to any of valine, tyrosine, and serine.

3. The carbonyl reductase mutant according to claim 1, characterized in that The carbonyl reductase mutant is one of the following: (1) mutating the lysine at position 17 of the amino acid sequence shown in SEQ ID NO.1 to histidine; (2) mutating the arginine at position 40 of the amino acid sequence shown in SEQ ID NO.1 to serine; (3) mutating the alanine at position 64 of the amino acid sequence shown in SEQ ID NO.1 to valine; (4) mutating the lysine at position 17 of the amino acid sequence shown in SEQ ID NO.1 to histidine, and mutating the alanine at position 64 to valine; (5) The lysine at position 17 of the amino acid sequence shown in SEQ ID NO. 1 was mutated to histidine, the arginine at position 40 was mutated to serine, and the alanine at position 64 was mutated to valine.

4. A gene encoding the carbonyl reductase mutant according to any one of claims 1 to 3.

5. A recombinant vector comprising a gene encoding the carbonyl reductase mutant according to claim 4.

6. A recombinant genetically engineered bacterium constructed with the recombinant vector according to claim 5 and containing the gene encoding the carbonyl reductase mutant.

7. Use of the carbonyl reductase mutant according to any one of claims 1 to 3 in the synthesis of a statin intermediate, wherein the statin intermediate is rosuvastatin intermediate (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester or atorvastatin intermediate (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester.

8. The use according to claim 7, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation and culture of genetically engineered bacteria containing a gene encoding a carbonyl reductase mutant, crude enzyme liquid after crushing the wet bacteria, or extracted pure enzyme as a catalyst; using (S)-6-chloro-5-hydroxy-3-oxohexanoic acid tert-butyl ester ((S)-CHOH) or (R)-6-cyano-5-hydroxy-3-oxohexanoic acid tert-butyl ester as a substrate, isopropanol as a cosubstrate, and NADPH as a coenzyme; forming a reaction system in a buffer solution with a pH value of 6.0 to 8.0, and accordingly preparing rosuvastatin intermediate (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester or atorvastatin intermediate (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester.

9. The use according to claim 8, characterized in that The buffer solution is a 0.1 mM potassium phosphate buffer solution with a pH of 7.0; the volume of isopropanol is preferably 20%-40% of the volume of the reaction system; when the catalyst is in the form of wet bacteria, the amount of the carbonyl reductase mutant wet bacteria is 10-40 g / L of buffer solution based on the weight of the wet bacteria; and the concentration of the substrate is 100-400 g / L.

10. A method for preparing a rosuvastatin intermediate or an atorvastatin intermediate, characterized in that The method is: The method comprises the following steps: using wet cells obtained by fermentation culture of genetically engineered bacteria containing a gene encoding a carbonyl reductase mutant, crude enzyme liquid after crushing the wet cells, or extracted pure enzyme as catalysts, using (S)-6-chloro-5-hydroxy-3-oxohexanoic acid tert-butyl ester ((S)-CHOH) or (R)-6-cyano-5-hydroxy-3-oxohexanoic acid tert-butyl ester as substrates, isopropanol as a cosubstrate, and NADPH as a coenzyme, respectively, to form a reaction system in a buffer solution with a pH value of 6.0-8.0, thereby preparing rosuvastatin intermediate (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester or atorvastatin intermediate (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester.

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