Carbonyl reductase mutants and their use in chiral ketone resolution and chiral alcohol synthesis

Abstract

The application provides a carbonyl reductase mutant and application thereof in chiral ketone resolution and chiral alcohol synthesis. The mutant protein is a non-natural protein, and the mutant protein can recognize the chirality of a cyclic diketone, selectively reduce a certain configuration ketone in the cyclic diketone into a single configuration alcohol, and retain another configuration ketone. The carbonyl reductase mutant of the application is applied to a production process of a key starting material of melagatran to convert 100-500 g / L of a substrate for complete resolution, and retain (1R, 5S)-configuration ketone. The application lays a foundation for green biological manufacturing of (1R, 5S)-chiral ketone.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and enzyme engineering, and relates to carbonyl reductase mutants and their applications in chiral ketone resolution and chiral alcohol synthesis. Background Technology

[0002] Carbonyl reductases, also known as alcohol dehydrogenases, are a class of biocatalysts that reversibly reduce carbonyl groups to alcohols. To achieve catalytic activity, carbonyl reductases require a coenzyme to transfer hydride ions to the carbonyl substrate. In the catalytic process of carbonyl reductases, the amount of coenzyme used is the same as the molar amount of substrate. From an economic perspective, appropriate coenzyme regeneration systems are matched to carbonyl reductase catalytic reactions. With the development of directed evolution technology, the application of carbonyl reductases in the preparation of chiral alcohols is becoming increasingly widespread.

[0003] Mirogabalin (trade name: Tarlige®) is a third-generation gabapentin-type neuropathic pain medication developed by Daiichi Sankyo of Japan. It entered the Chinese market in June 2024, primarily for the treatment of diabetic peripheral neuropathy (DPNP). The structural formula of miregabalin is as follows:

[0004] .

[0005] In China, it has been approved for use in adults with diabetic peripheral neuropathy (DPNP); in Japan and Europe, it is also used for postherpetic neuralgia. Merogabalin's target: highly selective binding to the α2δ-1 subunit of voltage-gated calcium channels (significantly stronger affinity than pregabalin / gabapentin). Its pathway: inhibition of calcium... 2+ It reduces the release of pain mediators such as glutamate and substance P, blocking the transmission of nerve pain signals. Merogabalin has the following advantages: strong binding to α2δ-1 and slow dissociation; rapid dissociation to α2δ-2, resulting in fewer central side effects (dizziness / drowsiness).

[0006] According to the industrial synthetic route of melogabarin, (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one is the key starting material, which can currently only be obtained through resolution. The structural formula of (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one is as follows:

[0007] .

[0008] Daiichi Sankyo, the original developer of melogabalone, reported a method using chiral amines as catalysts to resolve racemic ketones in organic solvents, yielding (1R,5S)-chiral ketones and aromatic acid derivatives (optical resolution method for bicyclic compounds using asymmetric catalysts, CN201280061637.0). Since chemical catalysis inevitably involves the use of organic reagents, the development of green and efficient catalytic technologies is imperative. Subsequently, Daiichi Sankyo also reported a method using carbonyl reductases, but with a low substrate concentration of less than 70 g / L (optical resolution method for bicyclic compounds using enzymes, CN201380019311.6). While this method avoids the use of organic reagents in the reduction process, the low substrate concentration results in a lack of significant cost advantages in production. Summary of the Invention

[0009] This invention provides a carbonyl reductase mutant, its encoding gene, and its applications, improving enzyme activity and substrate structural diversity to meet industrial needs. This invention functionally determined and directed the evolution of an enzyme (OaCR, GenBank: YP_001370612.1) derived from Ochrobactrum anthropi ATCC 49188, obtaining a mutant with significantly enhanced enzyme activity. This mutant catalyzes the resolution of the racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one to generate (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one and (1S,5R,6S)-3-ethylbicyclo[3.2.0]hept-3-en-6-ol.

[0010] This invention provides a mutant of carbonyl reductase whose protein sequence has at least 90% identity with SEQ ID NO: 1, and is obtained by substitution mutations at one or more of the sites I91, D94, C138, H142, K147, V150, P183, Y185, L191, I194, K198, L202, L205, H206, and R210 in the amino acid sequence shown in SEQ ID NO: 1.

[0011] Specifically, it includes one or more mutations at the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1:

[0012] The following mutations were observed: Isoleucine at position 91 was replaced by threonine (T), serine (S), and leucine (L); aspartic acid at position 94 was replaced by serine (S), asparagine (N), valine (V), and threonine (T); cysteine ​​at position 138 was replaced by glycine (G), alanine (A), and valine (V); histidine at position 142 was replaced by tryptophan (S), tyrosine (Y), and phenylalanine (F); lysine at position 147 was replaced by cysteine ​​(C), leucine (L), and glutamine (Q); valine at position 150 was replaced by threonine (T), serine (S), and methionine (M); proline at position 183 was replaced by leucine (L), valine (V), glycine (G), alanine (A), and threonine (T); and tyrosine at position 185 was replaced by leucine (L), valine (V), glycine (G), alanine (A), and threonine (T). The following mutations were made: Leucine at position 191 was mutated to glycine (G), methionine (M), leucine (L), and valine (V); leucine at position 191 was mutated to histidine (H) and threonine (T); isoleucine at position 194 was mutated to threonine (T), valine (V), serine (S), and leucine (L); lysine at position 198 was mutated to alanine (A), glycine (G), and threonine (T); leucine amide at position 202 was mutated to threonine (T), serine (S), valine (V), and alanine (A); leucine at position 205 was mutated to valine (V), glycine (G), and alanine (A); histidine at position 206 was mutated to phenylalanine (F), tryptophan (W), and tyrosine (Y); and arginine at position 210 was mutated to threonine (T) and serine (S).

[0013] Preferably, it includes any of the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1:

[0014] The 91st isoleucine is mutated to threonine (T), serine (S), and leucine amino acid (L).

[0015] The 94th aspartic acid is mutated to serine (S), asparagine (N), valine (V), and threonine (T).

[0016] The cysteine ​​at position 138 is mutated to glycine (G), alanine (A), or valine (V);

[0017] The histidine at position 142 is mutated to tryptophan (S), tyrosine (Y), and phenylalanine (F).

[0018] The lysine at position 147 is mutated to cysteine ​​(C), leucine (L), and glutamine (Q);

[0019] The valine at position 150 is mutated to threonine (T), serine (S), and methionine (M).

[0020] The proline at position 183 is mutated to leucine (L), valine (V), glycine (G), alanine (A), or threonine (T);

[0021] The tyrosine at position 185 is mutated to glycine (G), methionine (M), leucine (L), and valine (V); the leucine at position 191 is mutated to histidine (H) and threonine (T).

[0022] The isoleucine at position 194 is mutated to threonine (T), valine (V), serine (S), and leucine (L).

[0023] The 198th lysine residue is mutated to alanine (A), glycine (G), and threonine (T);

[0024] The 202nd position of leucine amide is mutated to threonine (T), serine (S), valine (V), and alanine (A).

[0025] The leucine at position 205 is mutated to valine (V), glycine (G), and alanine (A);

[0026] The histidine at position 206 is mutated to phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0027] Arginine at position 210 is mutated to threonine (T) and serine (S);

[0028] The proline (P) at position 183 is mutated to leucine (L), and the tyrosine (Y) at position 185 is mutated to valine (V).

[0029] The proline (P) at position 183 is mutated to leucine (L), and the leucine (L) at position 191 is mutated to histidine (H).

[0030] The proline (P) at position 183 is mutated to leucine (L), and the isoleucine (I) at position 194 is mutated to threonine (T).

[0031] The proline (P) at position 183 is mutated to leucine (L), and the isoleucine (I) at position 202 is mutated to alanine (A).

[0032] The proline (P) at position 183 is mutated to leucine (L), and the histidine (H) at position 142 is mutated to tyrosine (Y).

[0033] The proline (P) at position 183 is mutated to valine (V), and the tyrosine (Y) at position 185 is mutated to valine (V).

[0034] The proline (P) at position 183 is mutated to valine (V), and the leucine (L) at position 191 is mutated to histidine (H).

[0035] The proline (P) at position 183 is mutated to valine (V), and the leucine (L) at position 191 is mutated to histidine (T).

[0036] The proline (P) at position 183 is mutated to valine (V), and the isoleucine (I) at position 194 is mutated to threonine (T).

[0037] The proline (P) at position 183 is mutated to valine (V), and the isoleucine (I) at position 202 is mutated to alanine (A).

[0038] The proline (P) at position 183 is mutated to valine (V), and the histidine (H) at position 142 is mutated to tyrosine (Y).

[0039] The tyrosine (Y) at position 185 is mutated to valine (V), and the leucine (L) at position 191 is mutated to histidine (H);

[0040] The tyrosine (Y) at position 185 is mutated to valine (V), and the leucine (L) at position 191 is mutated to histidine (T).

[0041] The tyrosine (Y) at position 185 is mutated to valine (V), and the isoleucine (I) at position 194 is mutated to threonine (T).

[0042] The tyrosine (Y) at position 185 is mutated to valine (V), and the isoleucine (I) at position 202 is mutated to alanine (A).

[0043] The 185th tyrosine (Y) is mutated to valine (V), and the 142nd histidine (H) is mutated to tyrosine (Y).

[0044] The 185th tyrosine (Y) is mutated to leucine (L), and the 191st leucine (L) is mutated to histidine (H).

[0045] The 185th tyrosine (Y) is mutated to leucine (L), and the 191st leucine (L) is mutated to histidine (T).

[0046] The 185th tyrosine (Y) is mutated to leucine (L), and the 194th isoleucine (I) is mutated to threonine (T).

[0047] The 185th tyrosine (Y) is mutated to leucine (L), and the 202nd isoleucine (I) is mutated to alanine (A).

[0048] The 185th tyrosine (Y) is mutated to leucine (L), and the 142nd histidine (H) is mutated to tyrosine (Y).

[0049] The tyrosine (Y) at position 185 is mutated to glycine (G), and the leucine (L) at position 191 is mutated to histidine (H).

[0050] The tyrosine (Y) at position 185 is mutated to glycine (G), and the leucine (L) at position 191 is mutated to histidine (T).

[0051] The tyrosine (Y) at position 185 is mutated to glycine (G), and the isoleucine (I) at position 194 is mutated to threonine (T).

[0052] The tyrosine (Y) at position 185 is mutated to glycine (G), and the isoleucine (I) at position 202 is mutated to alanine (A).

[0053] The 185th tyrosine (Y) is mutated to glycine (G), and the 142nd histidine (H) is mutated to tyrosine (Y).

[0054] The 191st leucine (L) is mutated to histidine (H), and the 194th isoleucine (I) is mutated to threonine (T).

[0055] The 191st leucine (L) is mutated to histidine (H), and the 194th isoleucine (I) is mutated to valine (V).

[0056] The 191st leucine (L) is mutated to histidine (H), and the 202nd isoleucine (I) is mutated to alanine (A).

[0057] The 191st leucine (L) is mutated to histidine (H), and the 142nd histidine (H) is mutated to tyrosine (Y).

[0058] The 191st leucine (L) is mutated to threonine (T), and the 194th isoleucine (I) is mutated to threonine (T).

[0059] The 191st leucine (L) is mutated to threonine (T), and the 194th isoleucine (I) is mutated to valine (V).

[0060] The 191st leucine (L) is mutated to threonine (T), and the 202nd isoleucine (I) is mutated to alanine (A).

[0061] The 191st leucine (L) is mutated to threonine (T), and the 142nd histidine (H) is mutated to tyrosine (Y).

[0062] The isoleucine (I) at position 194 is mutated to threonine (T), and the isoleucine (I) at position 202 is mutated to alanine (A).

[0063] The isoleucine (I) at position 194 is mutated to threonine (T), and the histidine (H) at position 142 is mutated to tyrosine (Y).

[0064] The isoleucine (I) at position 202 is mutated to alanine (A), and the histidine (H) at position 142 is mutated to tyrosine (Y).

[0065] The 185th tyrosine (Y) is mutated to valine (V), the 194th isoleucine (I) is mutated to threonine (T), and the 202nd isoleucine (I) is mutated to alanine (A).

[0066] The 185th tyrosine (Y) is mutated to valine (V), the 194th isoleucine (I) is mutated to threonine (T), the 202nd isoleucine (I) is mutated to alanine (A), and the 142nd histidine (H) is mutated to tyrosine (Y).

[0067] The 185th tyrosine (Y) is mutated to valine (V), the 191st leucine (L) is mutated to histidine (H), and the 202nd isoleucine (I) is mutated to alanine (A).

[0068] The 185th tyrosine (Y) is mutated to valine (V), the 191st leucine (L) is mutated to histidine (H), the 194th isoleucine (I) is mutated to threonine (T), and the 202nd isoleucine (I) is mutated to alanine (A).

[0069] The 185th tyrosine (Y) is mutated to glycine (G), the 191st leucine (L) is mutated to histidine (H), and the 202nd isoleucine (I) is mutated to alanine (A).

[0070] The 185th tyrosine (Y) is mutated to glycine (G), the 191st leucine (L) is mutated to histidine (H), the 194th isoleucine (I) is mutated to threonine (T), and the 202nd isoleucine (I) is mutated to alanine (A).

[0071] Histidine (H) at position 142 is mutated to tyrosine (Y), leucine (L) at position 191 is mutated to threonine (T), isoleucine (I) at position 194 is mutated to valine (V), and isoleucine (I) at position 202 is mutated to alanine (A).

[0072] The present invention also provides a nucleic acid encoding a mutant of the carbonyl reductase described above.

[0073] The present invention further provides a recombinant expression vector for the mutant of the carbonyl reductase.

[0074] The present invention also provides a recombinant strain of the aforementioned carbonyl reductase mutant.

[0075] A method for synthesizing (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one, comprising the steps of: contacting a carbonyl reductase mutant protein with a reaction substrate, adding a coenzyme cycle system to catalyze the reaction, thereby obtaining the (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one and (1S,5R,6S)-3-ethylbicyclo[3.2.0]hept-3-en-6-ol.

[0076] Specifically, the pH of the reaction system is 6.0-8.0, the reaction time is 5-24 hours, and the substrate concentration is 100-500 g / L.

[0077] More specifically, the reaction system needs to be controlled at a pH of around 6.5 using an inorganic base; the reaction time is 5-12 hours.

[0078] Optionally, it further includes the step of separating (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one and (1S,5R,6S)-3-ethylbicyclo[3.2.0]hept-3-en-6-ol.

[0079] This invention obtained a mutant capable of efficiently resolving racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one through directed evolution, with a substrate concentration of up to 500 g / L, laying the foundation for the green biomanufacturing of (1R,5S)-chiral ketones. Attached Figure Description

[0080] Figure 1 The gas phase spectrum of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one.

[0081] Figure 2 The reaction spectrum of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one. Detailed Implementation

[0082] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0083] Example 1: Construction of a carbonyl reductase mutant library and screening of mutants

[0084] I. Screening of carbonyl reductase library

[0085] Transformation reactions were performed to construct a laboratory-preserved enzyme library. The substrate was prepared using a phosphate buffered solution at pH 7.5: 100 mM (13.6 g / L), 75 mM (10 g / L) glucose, 5 U / mL glucose dehydrogenase (GDH), 10 g / L E. coli expressing the carbonyl reductase OaCR gene, and coenzyme NAD(P). + 0.3 g / L, reacted at 30 °C for 24 hours. Extracted with an equal volume of ethyl acetate, and detected by chiral GC. The gas chromatogram of the racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one (e.g.) was used for analysis. Figure 1 The comparison shows that carbonyl reductase OaCR has good stereoselectivity, such as... Figure 1 As shown. Figure 2 The reaction spectrum of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one is shown.

[0086] The amino acid sequence (SEQ ID No. 1) of carbonyl reductase OaCR is as follows: MRFDGKVAIVTGGASGIGEATVRAFVSEGANVVIADYSEHGQQLANELAGGTERAIFVKTDVTDTKAVQALIAKTVETYGRLDIMFANAGIAADGPIDELDETAWQKTIDINLTGVYLCDKYAIDQMRSQGGGVIVNCGSIHSHVGKSGVTAYAAAKGGVKLLTQTLAIDYGAQNIRVNAVCPGYIDTPLLKDIPDDKKQALVALHPIGRLGRAEEVASVVLFLASDESSFVTGASILVDGGYTAQ.

[0087] II. First Round of Revitalization Transformation

[0088] OaCR and coenzyme NADP were obtained through RoseTTAFold All-Atom simulation (Science 384, 291(2024)). + Docking results with racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one.

[0089] Amino acid sites within an 8 Å radius were analyzed, with S141 / Y153 / K157 forming a catalytic triplet. The following sites were selected from the analyzed structure to construct a mutant library: I91, A92, A93, D94, C138, G139, I141, H142, K147, S148, G149, V150, P183, G184, Y185, I186, L191, I194, P195, K198, L202, L205, H206, and R210.

[0090] Saturation mutations were performed separately, using degenerate codon NNK to design mutation primers, with pET21a-OaCR as the template. A two-step PCR method was used, employing high-fidelity polymerase Pfx.

[0091] 1. Construction of pET21a-LfCR plasmid

[0092] The wild-type amino acid sequence of carbonyl reductase is shown in SEQ ID NO:1. The corresponding nucleotide sequence was fully synthesized and cloned into the restriction endonuclease sites NdeI and XhoI of the pET-21a vector to obtain the recombinant plasmid pET21a-OaCR. This plasmid was further transformed into the expression host E. coli BL21(DE3), and positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET21a-OaCR.

[0093] 2. Construction of a carbonyl reductase mutant library

[0094] Using pET21a-OaCR as a template, PCR was performed using mutant primers designed for the mutation site. The PCR reaction system and reaction conditions are as follows:

[0095] 1) Step PCR reaction system and reaction conditions

[0096] Round 1: In a PCR reaction system with a total volume of 25 μL, add 15 ng of template, 12.5 μL of 2×Pfx Mix, 0.5 μL (10 μM) of each of the two mutant primers, and add sterile distilled water to a final volume of 25 μL.

[0097] The first step of the PCR reaction procedure is as follows: ① 98℃ pre-denaturation for 2 min, ② 98℃ denaturation for 30 sec, ③ Tm-5℃ annealing for 30 sec, ④ 72℃ extension for 30 sec, ⑤ 72℃ final extension for 5 min. Steps ② to ④ are performed for a total of 30 cycles.

[0098] 2) Second step PCR reaction system and reaction conditions

[0099] Round 2: In a PCR reaction system with a total volume of 50 μL, add 30 ng of template, 25 μL of 2×Pfx Mix, 1 μL of primer fragment (the product of the first round of PCR), and distilled water to a final volume of 50 μL.

[0100] The second step of the PCR reaction procedure is as follows: ① Pre-denaturation at 98℃ for 2 min, ② Denaturation at 98℃ for 30 sec, ③ Annealing at 55℃ for 30 sec, ④ Extension at 72℃ for 3 min, ⑤ Final extension at 72℃ for 10 min. Steps ② to ④ are performed for a total of 25 cycles.

[0101] After verifying the PCR products obtained in the above steps by agarose gel electrophoresis, the restriction endonuclease DpnI was added and digested at 37°C for 2 h. The digested products were then transformed into E. coli BL21(DE3) competent cells and plated on plates containing 10 μg / mL ampicillin antibiotic. The plates were then incubated at 37°C for approximately 12 h until single colonies grew, yielding a carbonyl reductase mutant library.

[0102] Meanwhile, the pET21a-LfCR mutant library plasmid was transformed into E. coli BL21(DE3) competent cells and plated on a plate containing 100 mg / mL ampicillin antibiotic. The plate was then incubated at 37°C for about 12 h, and a single colony was grown, thus obtaining a strain expressing the carbonyl reductase mutant.

[0103] 3. Induced expression of carbonyl reductase mutants

[0104] The monoclonal colonies obtained after culturing in step 2 above were picked and transferred to LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) containing 4 mL of ampicillin (100 mg / L) and cultured overnight at 37°C and 200 rpm to obtain the culture medium. The culture medium was then inoculated into fermentation medium (LB liquid medium) at a 1% (v / v) inoculation rate and cultured on a shaker at 37°C and 200 rpm until OD reached. 600 The concentration was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. The mixture was induced at 25°C and 200 rpm for 8-12 hours. The bacterial cells were collected by centrifugation at 6000 g, and then subjected to high-pressure disruption to obtain a crude carbonyl reductase solution for subsequent enzyme activity assays.

[0105] 4. Screening of carbonyl reductase mutants

[0106] The screening method involved detecting the decrease in NADPH at 340 nm. The specific reaction method was as follows: 10 mM substrate, 0.25 mg / mL NADPH, 10 μL crude enzyme solution, and potassium phosphate buffer to a final volume of 200 μL. NADPH exhibits characteristic absorbance at 340 nm. The decrease in absorbance at 340 nm was detected using a microplate reader. Higher enzyme activity indicated faster NADPH consumption and a steeper slope in the absorbance decrease curve. Beneficial mutation sites for enhancing enzyme activity were identified through screening: I91, D94, C138, H142, K147, V150, P183, Y185, L191, I194, K198, L202, L205, H206, and R210.

[0107] III. The Second Round of Revitalization Transformation

[0108] Based on the results of saturation mutation, single-site combination mutants were constructed by selecting sites with significantly improved activity. The resulting mutants were cultured in test tubes containing 4 mL of LB medium. The activity of the expressed protein was detected. The results of significantly improved activity are shown in Table 1. Mutants 89 and 92 had the highest activity, which was 175 and 201 times higher than that of the wild type, respectively.

[0109] Table 1. Relative activity of OaCR and its mutants against substrates

[0110] ;

[0111] ;

[0112] ;

[0113] Example 2: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hepten-3-en-6-one using mutant 53

[0114] Mutant 53 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 200 mL of potassium phosphate buffer (pH 7.0, 100 mM), along with 300 g / L racemic ketone 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 240 g / L glucose, and 0.3 g / L NADP. + The reaction was carried out at 30 U / mL LGDH and 10 g / L wet bacterial cells at 30 °C for 6 hours, with the pH controlled between 6.5 and 7.0 during the reaction. After the reaction, GC analysis showed a conversion rate of 49.8% and a substrate ee value of 99.6%.

[0115] Example 3: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one using mutant 60

[0116] Mutant 60 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 2, with a reaction time of 6 hours, a conversion rate of 49.9%, and a substrate ee value of 99.5%.

[0117] Example 4: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one using mutant 62

[0118] The mutant 62 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 2, with a reaction time of 5 hours, a conversion rate of 49.6%, and a substrate ee value of 99.4%.

[0119] Example 5: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hepten-3-en-6-one using mutant 81

[0120] Mutant 81 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 2, with a reaction time of 5 hours, a conversion rate of 49.4%, and a substrate ee value of 99.4%.

[0121] Example 6: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hepten-3-en-6-one using mutant 63

[0122] Mutant 63 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 2, with a reaction time of 6 hours, a conversion rate of 49.8%, and a substrate ee value of 99.7%.

[0123] Example 7: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hepten-3-en-6-one using mutant 70

[0124] The mutant 70 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 2, with a reaction time of 6 hours, a conversion rate of 49.7%, and a substrate ee value of 99.8%.

[0125] Example 8: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one using mutant 84

[0126] The mutant 84 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 2, with a reaction time of 6 hours, a conversion rate of 49.8%, and a substrate ee value of 99.3%.

[0127] Example 9: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hepten-3-en-6-one using mutant 86

[0128] Following the method described in Example 1, mutant 86 was induced and expressed. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 500 mL of potassium phosphate buffer (pH 7.0, 100 mM), along with 400 g / L racemic ketone 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 320 g / L glucose, and 0.2 g / L NADP. + The reaction was carried out at 30 U / mL LGDH and 10 mg / mL whole cells at 30°C for 6 hours, with the pH controlled between 6.5 and 7.0 during the reaction. After the reaction, GC analysis showed a conversion rate of 49.5% and a substrate ee value of 99.5%.

[0129] Example 10: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one using mutant 87

[0130] The mutant 87 was obtained by inducing expression according to the method of Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 9, the reaction time was 6 hours, the conversion rate was 49.9%, and the substrate ee value was 99.8%.

[0131] Example 11: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hepten-3-en-6-one using mutant 88

[0132] The mutant 88 was obtained by inducing expression according to the method of Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 9, the reaction time was 6 hours, the conversion rate was 50.1%, and the substrate ee value was 99.3%.

[0133] Example 12: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hepten-3-en-6-one using mutant 89

[0134] The mutant 89 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 9, with a reaction time of 6 hours, a conversion rate of 49.7%, and a substrate ee value of 99.7%.

[0135] Example 13: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one using mutant 90

[0136] The mutant 90 was obtained by inducing expression according to the method of Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 9, with a reaction time of 6 hours, a conversion rate of 49.5%, and a substrate ee value of 99.4%.

[0137] Example 14: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one using mutant 91

[0138] Mutant 91 was obtained by inducing expression according to the method of Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 9, the reaction time was 6 hours, the conversion rate was 49.6%, and the substrate ee value was 99.6%.

[0139] Example 15: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one using mutant 92

[0140] The mutant 92 was obtained by inducing expression according to the method of Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 9, the reaction time was 6 hours, the conversion rate was 49.9%, and the substrate ee value was 99.9%.

[0141] Example 16: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hepten-3-en-6-one using mutant 92

[0142] Following the method described in Example 1, mutant 92 was induced and expressed. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 500 mL of potassium phosphate buffer (pH 7.0, 100 mM), along with 500 g / L racemic ketone 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 400 g / L glucose, 0.2 g / L NADP+, 30 U / mL LDH, and 10 mg / mL whole cells. The reaction was carried out at 30°C for 6 hours, with the pH maintained between 6.5 and 7.0 during the reaction. After the reaction, GC analysis showed a conversion rate of 49.8% and a substrate ee value of 99.8%.

[0143] Example 17: Whole-cell catalytic resolution of racemic ketone 3-ethylbicyclo[3.2.0]hept-3-en-6-one using mutant 92

[0144] Following the method described in Example 1, mutant 92 was induced and expressed. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 1000 mL of potassium phosphate buffer (pH 7.0, 100 mM), along with 500 g / L racemic ketone 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 400 g / L glucose, 0.2 g / L NADP+, 30 U / mL LDH, and 10 mg / mL whole cells. The reaction was carried out at 30°C for 6 hours, with the pH maintained between 6.5 and 7.0 during the reaction. After the reaction, GC analysis showed a conversion rate of 49.7% and a substrate ee value of 99.7%.

Claims

1. A mutant of carbonyl reductase, characterized in that, The mutant protein sequence corresponds to one or more mutations at the following sites in the amino acid sequence shown in SEQ ID NO: 1: The following mutations occurred: Isoleucine at position 91 was replaced by threonine, serine, and leucine; aspartic acid at position 94 was replaced by serine, asparagine, valine, and threonine; cysteine ​​at position 138 was replaced by glycine, alanine, and valine; histidine at position 142 was replaced by tryptophan, tyrosine, and phenylalanine; lysine at position 147 was replaced by cysteine, leucine, and glutamine; valine at position 150 was replaced by threonine, serine, and methionine; proline at position 183 was replaced by leucine, valine, glycine, alanine, and threonine; and so on. The tyrosine at position 191 is mutated to glycine, methionine, leucine, and valine; the leucine at position 194 is mutated to histidine and threonine; the isoleucine at position 195 is mutated to threonine, valine, serine, and leucine; the lysine at position 198 is mutated to alanine, glycine, and threonine; the leucine amide at position 202 is mutated to threonine, serine, valine, and alanine; the leucine at position 205 is mutated to valine, glycine, and alanine; the histidine at position 206 is mutated to phenylalanine, tryptophan, and tyrosine; and the arginine at position 210 is mutated to threonine and serine.

2. The mutant of the carbonyl reductase as claimed in claim 1, characterized in that, It includes the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: The isoleucine at position 91 is mutated to threonine, serine, and leucine amino acid; The 94th aspartic acid is mutated to serine, asparagine, valine, or threonine. The cysteine ​​at position 138 is mutated to glycine, alanine, or valine. The histidine at position 142 is mutated to tryptophan, tyrosine, and phenylalanine. The lysine at position 147 was mutated to cysteine, leucine, or glutamine. The valine at position 150 is mutated into threonine, serine, or methionine. The proline at position 183 is mutated to leucine, valine, glycine, alanine, or threonine; The tyrosine residue at position 185 is mutated to glycine, methionine, leucine, or valine. The leucine at position 191 is mutated to histidine and threonine; The isoleucine at position 194 is mutated into threonine, valine, serine, and leucine; The lysine at position 198 is mutated to alanine, glycine, or threonine. The leucine aminoamide at position 202 is mutated to threonine, serine, valine, or alanine. The leucine at position 205 is mutated into valine, glycine, or alanine. The histidine at position 206 is mutated to phenylalanine, tryptophan, or tyrosine; or Arginine at position 210 is mutated into threonine and serine.

3. The nucleic acid encoding a mutant of the carbonyl reductase as described in claim 1 or 2.

4. A recombinant expression vector containing the nucleic acid encoding as described in claim 3.

5. Recombinant bacteria containing the encoded nucleic acid as described in claim 3, or the recombinant expression vector as described in claim 4.

6. A synthesis (1 R 5 S The method for 3-ethylbicyclo[3.2.0]hept-3-en-6-one, characterized in that, The steps include: contacting the mutant carbonyl reductase of claim 1 or 2 with the reaction substrate, adding a coenzyme cycle system to catalyze the reaction, thereby obtaining the (1) R 5 S )-3-ethylbicyclo[3.2.0]hepta-3-en-6-one and (1 S 5 R 6 S )-3-ethylbicyclo[3.2.0]hept-3-en-6-ol.

7. The method as described in claim 6, characterized in that, The pH of the reaction system is 6.0-8.0, the reaction time is 5-24 hours, and the substrate concentration is 100-500 g / L.

8. The method as described in claim 6, characterized in that, The reaction system was controlled at pH 6.5 using an inorganic base; the reaction time was 5-12 hours.

9. The method according to any one of claims 6 to 8, characterized in that, It also includes the step of separating (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one and (1S,5R,6S)-3-ethylbicyclo[3.2.0]hept-3-en-6-ol.

10. The use of the mutant carbonyl reductase as described in claim 1 or 2 in the chiral ketone resolution and chiral alcohol synthesis.

Images