An olefin reductase mutant and its application in the catalytic synthesis of sacubitril intermediates

Abstract

This invention belongs to the field of biotechnology and discloses an olefin reductase mutant and its application in the catalytic synthesis of sacubitril intermediates. This invention modifies an olefin reductase from *Clavisporalusitaniae* using gene mining technology, molecular docking, rational and semi-rational design, and site-directed mutagenesis to obtain a mutant with high yield of key sacubitril intermediates. The recombinant vector constructed from the mutant and recombinant genetically engineered bacteria are used to produce key sacubitril intermediates via whole-cell catalysis and pure enzyme catalysis, resulting in a significantly increased yield, 1.2-3 times that of the starting strain. Therefore, this invention provides a method for producing sacubitril intermediates with advantages such as mild reaction conditions, simple operation, short production cycle, environmental friendliness, and reduced production costs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an olefin reductase mutant and its application in the catalytic synthesis of sacubitril intermediates. Background Technology

[0002] Sacubitril / valsartan (LCZ696), the first orally administered angiotensin receptor neprilysin inhibitor, is an innovative drug for the treatment of heart failure. It is a salt complex containing sacubitril (AHU-377) and the ARB valsartan. Sacubitril is a prodrug that is metabolized in vivo into an active neprilysin inhibitor; valsartan blocks the AT1 receptor. LCZ696 is a valsartan and sacubitril sodium salt hydrate cocrystal, with the following chemical structure:

[0003] .

[0004] Sacubitril / valsartan sodium tablets are a salt complex crystal composed of the two components mentioned above in a 1:1 molar ratio. Sacubitril / valsartan can inhibit the activation of the RASS system, thereby dilating blood vessels and improving myocardial remodeling. On the other hand, it can also inhibit the degradation of BNP by endorphins, leading to an increase in endogenous BNP, which also dilates blood vessels, promotes diuresis, and prevents and reverses ventricular remodeling, thus slowing the progression of heart failure, improving symptoms, and improving cardiac function.

[0005] However, the current preparation process of sacubitril suffers from harsh reaction conditions and low conversion rates. Therefore, it is necessary to develop a method that is simple to operate, produces less pollution, is easy to separate and purify, and is easy to scale up, so as to realize the industrial production of sacubitril. Summary of the Invention

[0006] To address the needs of existing technologies, this invention provides an enzyme cascade method for synthesizing sacubitril precursors using enzyme catalysis as the core reaction. Simultaneously, the preparation method of the key substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pentane-2,4-dienoic acid) in the synthetic route is optimized. Through screening existing olefin reductases in the laboratory, an olefin reductase capable of efficiently catalyzing the construction of sacubitril precursors was obtained. This method is simple to operate, produces minimal pollution, is easy to separate and purify, and is suitable for scale-up and industrial production.

[0007] To this end, this invention modifies the olefin reductase ClER (GenBank No.: CH408080.1) from Clavisporalusitaniae using techniques such as gene mining, directed evolution, and rational design to obtain a mutant with a high yield of sacubitril intermediate, so as to utilize industrial microorganisms to produce sacubitril intermediate more efficiently.

[0008] Therefore, the first object of the present invention is to provide a mutant of the olefin reductase ClER (GenBank No.: CH408080.1) derived from *Clavispora lusitaniae*, wherein the mutation is based on the amino acid sequence of the olefin reductase ClER and contains the following mutations selected from one or more of the following amino acid residue sites: 33, 35, 66, 76, 108, 110, 184, 187, 189, 236, 243, 244, 288, 289, 343 and / or 370.

[0009] All of the above amino acid mutants possess the catalytic function of wild-type olefin reductases, as shown by ClER, in catalyzing sacubitril intermediates.

[0010] More specifically, the ClER olefin reductase mutant modifies ClER using any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or all of the following sixteen types of mutations:

[0011] X1. Mutate the threonine at position 33 of olefin reductase ClER to aspartic acid, glycine, or lysine.

[0012] X2. Mutate the methionine at position 35 of olefin reductase ClER to isoleucine, cysteine, or serine.

[0013] X3. Mutate the alanine at position 66 of olefin reductase ClER to threonine, isoleucine, or serine.

[0014] X4. Mutate the tyrosine residue at position 76 of olefin reductase ClER to alanine, leucine, lysine, valine, or cysteine.

[0015] X5. Mutate the glutamic acid at position 108 of olefin reductase ClER to arginine, leucine, or glycine.

[0016] X6. Mutate the tryptophan at position 110 of olefin reductase ClER to serine, tyrosine, valine, threonine, lysine, phenylalanine, glycine, or aspartic acid.

[0017] X7. Mutate histidine at position 184 of olefin reductase ClER to asparagine and isoleucine.

[0018] X8. Mutate histidine at position 187 of olefin reductase ClER to aspartic acid, tyrosine, tryptophan, or valine.

[0019] X9. Mutate the tyrosine residue at position 189 of the olefin reductase ClER to glycine, aspartic acid, phenylalanine, or serine.

[0020] X10. Mutate the arginine at position 236 of olefin reductase ClER to proline, leucine, or phenylalanine.

[0021] X11. Mutate the phenylalanine at position 243 of olefin reductase ClER to aspartic acid and histidine.

[0022] X12. Mutate the glutamic acid at position 244 of the olefin reductase ClER to serine, methionine, alanine, glycine, or proline.

[0023] X13. Mutate glycine at position 288 of olefin reductase ClER to glutamine and histidine;

[0024] X14. Mutate valine at position 289 of olefin reductase ClER to serine, cysteine, or proline.

[0025] X15. Mutate the arginine at position 343 of olefin reductase ClER to alanine, leucine, and lysine.

[0026] X16. Mutate the tyrosine residue at position 370 of the olefin reductase ClER to asparagine, alanine, and histidine.

[0027] In one embodiment of the present invention, the ClER olefin reductase mutant is:

[0028] The protein obtained by mutating isoleucine at position 33 of olefin reductase ClER;

[0029] The protein obtained by mutating leucine at position 35 of olefin reductase ClER;

[0030] The protein obtained by mutating glycine at position 76 of the olefin reductase ClER.

[0031] The protein obtained by mutating valine at position 76 of olefin reductase ClER;

[0032] The protein obtained by mutating the 110th site of the olefin reductase ClER to threonine;

[0033] The protein obtained by mutating isoleucine at position 187 of olefin reductase ClER;

[0034] The protein obtained by mutating the olefin reductase ClER at position 243 to aspartic acid;

[0035] The protein obtained by mutating the 244th position of olefin reductase ClER to tyrosine;

[0036] The protein obtained by mutating valine at position 244 of olefin reductase ClER;

[0037] The protein obtained by mutating the 244th position of olefin reductase ClER to serine;

[0038] The protein obtained by mutating methionine at position 343 of the olefin reductase ClER.

[0039] In another embodiment of the present invention, the olefin reductase ClER mutant is:

[0040] The protein obtained by mutating positions 33 and 35 to isoleucine and leucine, respectively.

[0041] The protein obtained by mutating positions 35 and 76 to glycine and valine, respectively.

[0042] The protein obtained by mutating positions 35 and 110 to isoleucine and isoleucine, respectively.

[0043] The protein obtained by mutating positions 76 and 244 to valine and glycine, respectively.

[0044] Proteins obtained by mutating positions 35, 76, and 110 to isoleucine, isoleucine, and threonine, respectively.

[0045] Proteins obtained by mutating positions 35, 244, and 370 to cysteine, glycine, and valine, respectively.

[0046] Proteins obtained by mutating at positions 184, 187, and 243 to glycine, aspartic acid, and methionine, respectively.

[0047] Proteins obtained by mutating at positions 35, 76, 108, 243, and 289 to isoleucine, glycine, threonine, valine, and serine, respectively.

[0048] Proteins obtained by mutating positions 35, 76, 244, 288, and 370 to isoleucine, threonine, isoleucine, aspartic acid, and methionine, respectively.

[0049] The present invention also provides a recombinant strain, which is obtained by transferring the recombinant vector into a host cell. The host cell contains the recombinant vector or a gene containing the olefin reductase or a mutant integrated into its genome.

[0050] Specifically, its originating bacterium is Escherichia coli.

[0051] The present invention also provides the application of the olefin reductase mutant, the encoding gene, the recombinant vector, or the recombinant strain in the production of sacubitril intermediate.

[0052] The present invention further provides a method for producing sacubitril intermediates, comprising the steps of culturing the recombinant strain to produce sacubitril intermediates and isolating the sacubitril intermediates.

[0053] Furthermore, it also includes a step of purifying the sacubitril intermediate.

[0054] This invention discloses a novel modified olefin reductase mutant, which was obtained by mutating the amino acid sequence of an olefin reductase such as ClER at specific sites. All the resulting mutants possess the function of catalyzing the formation of sacubitril intermediates in conjunction with the wild-type ClER. Furthermore, the recombinant strains constructed from these mutants exhibit a significantly increased yield of sacubitril intermediates, 1.2-3 times that of the starting strain, demonstrating a substantial increase in sacubitril intermediate production. Moreover, the method for producing sacubitril intermediates provided by this invention offers advantages such as mild reaction conditions, simple operation, and environmental friendliness. Attached Figure Description

[0055] Figure 1 The plasmid map constructed for the olefin reductase recombinant vector.

[0056] Figure 2 The reaction route for the production of sacubitril intermediate from the substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pent-2,4-dienoic acid) catalyzed by olefin reductase.

[0057] Figure 3 HPLC chromatograms of sacubitril intermediate racemic standard and product prepared by olefin reductase.

[0058] Figure 4 The NMR spectrum of the intermediate product of sacubitril. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0061] Example 1: Discovery of olefin reductase

[0062] The olefin reductase ClER from Clavispora lusitaniae (GenBank ID: CH408080.1) was obtained by searching the NCBI database. The whole gene of ClER was synthesized and ligated into the pET24a expression vector that had been double-digested with NdeI and XhoI to obtain the recombinant expression vector pET24a-ClER. Figure 1 ).

[0063] olefin reductase amino acid sequence (SEQ ID) NO: 1): MVAVKPLKDTEIFKPTKVGNHELSNKIVYAPTTRMRAIADHTPSDLAYKYYDDRTKYPGSLVITEATLMSPKTGLYDRVPGIYTDEHVAGWKKITDK IHANGSKVSMQLWPLGRVADPVATKKAGYPLVAPSLIYPSEEAKKAAEEAGNPIHVLTTEEVEDLVNDFVHAAKKAVAAGVDYVEVHGAHGYLVDTFFQVSTN KRTDKYGGSIENRARFALEILDRLIEEIGAERVAIRISPWAKFQGILAEEGEVNPVAQFGYFLSELENRARAGKRIAYVSIVEPRVSGVIDVAGEDIQGDNSF VRSVWKGIVIKAGNYTYDAPEFKTLLQDVSDGKTLVAFARYFTSNPDLVQRLHDGADLTPYKRELFYAPSNWGYNTFTNAGETKTFSEEEESKRLPAPIDTKA.

[0064] The recombinant expression vector was transformed into a suitable microbial host. The host microorganism can be any conventional microorganism in the art, as long as it can stably replicate on its own and effectively express the olefin reductase gene. In this embodiment, the recombinant expression plasmid was introduced into E. coli BL21(DE3) competent cells via electroporation and cultured upside down on LB agar plates containing kanamycin resistance for 12-16 h. Positive transformants were selected for DNA sequencing verification; the correctly verified transformants were the olefin reductase gene-engineered strains.

[0065] Example 2: Obtaining olefin reductase or its mutant

[0066] 2.1 Construction of ClER single-point saturation mutant library

[0067] Based on gene mining, molecular docking, and rational analysis of wild-type olefin reductases, 16 sites were rationally designed according to the substrate binding pocket and substrate molecule interaction. Therefore, this invention selected 16 sites in the amino acid sequence of the olefin reductase ClER (GenBank ID: CH408080.1) from Clavispora lusitaniae for modification, namely positions 33, 35, 66, 76, 108, 110, 184, 187, 189, 236, 243, 244, 288, 289, 343, and 370. Single-site saturation mutant libraries were constructed for each site.

[0068] To obtain the ClER mutant of olefin reductase, the following experiments were conducted:

[0069] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template ClER 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL.

[0070] PCR amplification reaction conditions: 98 ℃: 2 min, (98 ℃: 10 s, 55 ℃: 15 s, 72 ℃: 35 s) 30 cycles, 72 ℃: 4 min.

[0071] The obtained PCR product was processed as follows: 1 µL of Dpn I enzyme was added to 50 μL of the PCR product to digest the plasmid template, and the mixture was treated at 37 ℃ for 2 h. 5 µL of the digested PCR product was electroporated into 100 µL of E. coli BL21(DE3) competent cells. The electroporated E. coli BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 µg / mL) LB agar plates and cultured at 37 ℃ for 14 h. Single colonies grew, which were the engineered strain of the olefin reductase ClER gene mutant. Recombinant plasmids containing the target nucleotides of this strain with undirected or directed mutations are the expression vectors for expressing the olefin reductase ClER gene mutant.

[0072] 2.2 Screening of ClER single-point saturation mutant libraries

[0073] After obtaining the above single colonies, using the pET-24a empty plasmid as a negative control and the ClER wild type as a positive control, negative, positive, and single colonies were picked with a sterile toothpick and placed in a 96-well deep-well plate containing 400 µL LB medium (LB medium contains 50 µg / mL kanamycin). After sealing, the plates were incubated at 37 ℃ with shaking at 800 rpm for 12 h.

[0074] Add 700 µL of TB medium (containing a final concentration of 50 µg / mL kanamycin) to a 96-well deep-well culture plate, and add IPTG to make a final concentration of 0.1 mM. Incubate at 20 °C with shaking at 800 rpm for 16 h for protein expression.

[0075] After bacterial collection, 400 µL of the prepared reaction solution was added to a 96-well deep-well plate. The final concentration of the substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pentane-2,4-dienoic acid) was 20 mM. The reaction was carried out at 30 °C with shaking at 800 rpm for 24 h. After that, the substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pentane-2,4-dienoic acid) was detected to be converted into sacubitril intermediate (enzymatic reaction route see [link]). Figure 2 ).

[0076] After 24 h of reaction, the 96-well deep-well culture plate was centrifuged at 4000 rpm for 30 min. 80 µL of the supernatant from the 96-well plate was transferred to a 96-well sample plate, diluted 5-fold with 320 µL of methanol, and detected using reverse-phase HPLC to screen for mutants with higher conversion rates than the wild type. Single colonies with conversion rates higher than the wild type, as determined by HPLC, were picked and sequenced for identification.

[0077] Preliminary screening yielded 16 mutants with relatively high activity: ClER-T33Y, ClER-M35V, ClER-Y76L, ClER-Y76I, ClER-Q108G, ClER-W110V, ClER-W110T, ClER-H184I, ClER-H187D, ClER-H187Y, ClER-H187V, ClER-Q244S, ClER-Q244G, ClER-G288C, ClER-G288T, and ClER-Y370M. These mutants all showed improved conversion rates of the substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pent-2,4-dienoic acid) compared to the wild type. The results are shown in Table 1.

[0078] Table 1. Yield ratio and ee value of recombinant engineered strain (R)-sacubazo.

[0079]

[0080] In summary, this invention is based on mutating a specific site of the wild-type olefin reductase ClER, resulting in mutants with significantly increased yield or chirality, which can significantly increase the yield of the (R)-sacubatrox key intermediate.

[0081] Example 3: Construction of the ClER combinatorial saturated mutant of olefin reductase or obtaining its mutants

[0082] 3.1 Construction of ClER combinatorial mutant library

[0083] Sixteen mutants selected from the 16 single-site saturated mutant libraries—T33Y, M35V, Y76L, Y76I, Q108G, W110V, W110T, H184I, H187D, H187Y, H187V, Q244S, Q244G, G288C, G288T, and Y370M—were subjected to combined mutations at a total of nine sites. The primer designs are shown in Table 2 below.

[0084] Table 2. Combinatorial saturated mutant libraries constructed based on olefin reductase ClER

[0085]

[0086] Note: Table 2 contains primers for multiple single-stranded DNAs. M represents A or C, R represents A or G, and W represents A or T.

[0087] To obtain the ClER mutant of olefin reductase, the following experiments were conducted:

[0088] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template ClER 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of the forward and reverse primers are shown in Table 2.

[0089] PCR amplification reaction conditions: 98 ℃: 2 min, (98 ℃: 10 s, 55 ℃: 15 s, 72 ℃: 30 s) 30 cycles, 72 ℃: 4 min.

[0090] Overlap extension PCR procedure:

[0091] Round 1: R1-1, R1-2, R1-3, R1-4, R1-5, and R1-6 are mixed in a 1:1:1:1:1:1 ratio to form downstream primer R1. F1 and R1 amplify fragment 1 (approximately 274 bp). R2-1 and R2-2 are mixed in a 1:1 ratio to form downstream primer R2. F2 and R2 amplify fragment 2 (approximately 139 bp). R3-1 and R3-2 are mixed in a 2:1 ratio to form downstream primer R3. F3 and R3 amplify fragment 3 (approximately 162 bp).

[0092] Second round: Fragment 1, fragment 2, and fragment 3 were excised from the gel and recovered as templates. Using upstream primer F1 and mixed downstream primer R3 as upstream and downstream primers respectively, the overlapping extension PCR was used to amplify fragment 4.

[0093] Third round: Fragment 4 is used as a large primer to amplify the entire plasmid using the plasmid as a template.

[0094] The obtained PCR product was processed as follows: 1 µL of Dpn I enzyme was added to 20 μL of the PCR product to digest the plasmid template, and the mixture was treated at 37 ℃ for 2 h. 5 µL of the digested PCR product was electroporated into 100 µL of E. coli BL21(DE3) competent cells. The electroporated E. coli BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 µg / mL) LB agar plates and cultured at 37 ℃ for 14 h. Single colonies grew, which were the engineered strain of the olefin reductase ClER gene mutant. Recombinant plasmids containing the target nucleotides of this strain with undirected or directed mutations are the expression vectors for expressing the olefin reductase ClER gene mutant.

[0095] 3.2 Screening of ClER combinatorial mutant libraries

[0096] After obtaining the above single colonies, using the pET-24a empty plasmid as a negative control and the ClER wild type as a positive control, negative, positive, and single colonies were picked with a sterile toothpick and placed in a 96-well deep-well plate containing 400 µL LB medium (LB medium contains 50 µg / mL kanamycin). After sealing, the plates were incubated at 37 ℃ with shaking at 800 rpm for 12 h.

[0097] Add 700 µL of TB medium (containing a final concentration of 50 µg / mL kanamycin) to a 96-well deep-well culture plate, and add IPTG to make a final concentration of 0.1 mM. Incubate at 20 °C with shaking at 800 rpm for 16 h for protein expression.

[0098] After bacterial collection, 400 µL of the prepared reaction solution was added to a 96-well deep-well culture plate. The final concentration of the substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pent-2,4-dienoic acid) was 20 mM. The reaction was carried out at 30 ℃ and 800 rpm for 24 h. After that, the substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pent-2,4-dienoic acid) was detected to be converted into sacubitril intermediate.

[0099] After 24 h of reaction, the 96-well deep-well culture plate was centrifuged at 4000 rpm for 30 min. 80 µL of the supernatant from the 96-well plate was transferred to a 96-well sample plate, diluted 5-fold with 320 µL of methanol, and detected using reverse-phase HPLC to screen for mutants with higher conversion rates than the wild type. Single colonies with conversion rates higher than the wild type, as determined by HPLC, were picked and sequenced for identification.

[0100] The sequenced strains with higher concentrations than the wild type were activated by streak plating. Single colonies were picked and inoculated into 5 mL LB broth (containing a final concentration of 50 µg / mL kanamycin) and cultured overnight at 37 ℃ and 220 rpm. Then, a 5% inoculum was added to 50 mL TB broth (12 g / L peptone, 24 g / L yeast extract, 8 mL / L glycerol, 2.31 g KH₂PO₄, 16.43 g K₂HPO₄), and kanamycin was added to a final concentration of 50 µg / mL. The culture was maintained at 37 ℃ and 220 rpm until the OD₀ reached approximately 0.8, and then induced for 16 h with 0.1 mM IPTG.

[0101] After collecting the bacterial culture in a 50 mL centrifuge tube, centrifuge at 4000 rpm for 30 min, discard the supernatant, add 10 mL of buffer to resuspend and wash the bacteria, centrifuge again, and take 0.1 g / mL of whole cells to carry out 1 mL of reaction. The final concentration of the substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pent-2,4-dienoic acid) was 100 mM. After reacting at 30 ℃ and 800 rpm for 24 h, the substrate (4-hydroxy-2-methyl-5-(4-biphenyl)pent-2,4-dienoic acid) was detected to be converted into sacubitril intermediate.

[0102] High-performance liquid chromatography (HPLC) detection: 100 μL of the reaction solution was quenched with 100 μL of acetonitrile containing 50% formic acid, then diluted with 800 μL of acetonitrile. The mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane. The content of (R)-sacubazoline intermediate was detected using a Shimadzu LC-2030 chromatograph.

[0103] Liquid chromatography detection conditions: column: Daicel AD-RH (4.6 mm × 250 mm × 5 µM); detection wavelength: 254 nm;

[0104] Column temperature: 28 ℃; flow rate: 1 mL / min; sample loading volume: 10 µL; detection time: 9 min; mobile phase A: 0.2% acetic acid aqueous solution (2 mL acetic acid dissolved in 1 L ultrapure water); mobile phase B: pure acetonitrile; mobile phase A: mobile phase B = 40:60.

[0105] A detection method was established for the intermediate racemic standard of sacubitril in vitro, and its absorption peak was measured at 254 nm to establish a reference peak in the liquid chromatography spectrum.

[0106] High-performance liquid chromatography (HPLC) analysis showed that the elution times of (±)-sacubatrox intermediates were all between 7 and 8 minutes (Figure 3). The results indicated that the modified recombinant strains exhibited improved production capacity of (R)-sacubatrox intermediates compared to the wild type (NMR data of the enzymatically separated products are shown in Figure 4). Compared to the wild type, the mutant strains produced higher yields than wild-type single clones, and single colonies were selected for sequencing identification.

[0107] Through preliminary screening, nine mutants with relatively high activity were obtained: ClER-T33I / M35L, ClER-M35G / Y76V, ClER-M35I / W110I, ClER-Y76V / Q244G, ClER-M35I / Y76I / W110T, ClER-M35C / Q244G / Y370V, ClER-H184G / H187D / F243M, ClER-M35I / Y76G / Q108T / F243V / V289S, and ClER-M35I / Y76T / Q244I / G288D / Y370M. These mutants showed improved conversion rates of (R)-sacubatrox intermediates compared to ClER. The results are shown in Table 3.

[0108] Table 3. Yield ratio and ee value of recombinant engineered strain (R)-sacubazoline

[0109]

[0110] In summary, this invention, based on the combined saturation mutation of wild-type olefin reductase ClER, yielded nine multi-site combined mutants. The optimal mutant ClER-M35C / Q244G / Y370V showed a significant increase in yield, with a conversion rate 1.24 times that of the starting strain and a chiral selectivity (ee value) 2.91 times that of the starting strain, which can significantly increase the yield of (R)-sacubatrox intermediate.

[0111] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An olefin reductase mutant, characterized in that, Based on the amino acid sequence of the olefin reductase shown in SEQ ID NO: 1, there exists only one of the following substitution mutations: M35V; T33I / M35L; M35G / Y76V; M35I / W110I; M35I / Y76I / W110T; M35C / Q244G / Y370V; M35I / Y76G / Q108T / F243V / V289S; M35I / Y76T / Q244I / G288D / Y370M.

2. A gene encoding an olefin reductase mutant as described in claim 1.

3. A recombinant vector containing the encoding gene as described in claim 2.

4. A recombinant strain containing the recombinant vector as described in claim 3.

5. The recombinant strain according to claim 4, characterized in that, Its originating bacteria is Escherichia coli.

6. The application of the olefin reductase mutant of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the recombinant strain of claim 4 or 5 in the production of sacubitril intermediate, wherein, The substrate used was 4-hydroxy-2-methyl-5-(4-biphenyl)pent-2,4-dienoic acid.

7. A method for producing a sakubaqu intermediate, characterized in that, The method includes the steps of culturing the recombinant strain of claim 4 or 5 to produce sacubitril intermediate; and isolating the sacubitril intermediate, wherein the substrate used is 4-hydroxy-2-methyl-5-(4-biphenyl)pent-2,4-dienoic acid.

8. The method as described in claim 7, characterized in that, It also includes the step of purifying the sacubitril intermediate.

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