Imine reductases for synthesis of high optical purity amines

Abstract

The present application relates to imine reductases useful in the synthesis of high optical purity amines, and specifically provides engineered polypeptides having improved imine reductase activity and / or stereoselectivity compared to wild-type sequences, and further, to the synthesis of high optical purity amines. The invention provides a fusion protein of the engineered polypeptide, a polynucleotide, a nucleic acid construct, an expression vector, a host cell, a composition and a method for catalytically synthesizing chiral amine by using the engineered polypeptide.

Description

Technical Field

[0001] The present application relates to the field of biocatalysis, and in particular to an imine reductase for synthesizing amines with high optical purity. Background Art

[0002] Many pharmaceutical intermediates and final drug substances are chiral amines, and there are strict requirements for the optical purity of chiral amines (usually the optical purity is expressed by the enantiomeric excess value "ee", which generally requires the optical purity of chiral amines to be greater than 99.0-99.5% ee, that is, in the two possible enantiomeric products R or S, the ratio of the desired enantiomer to the unwanted enantiomer is usually required to be greater than (200-400):1.

[0003] Cinacalcet (Structural Formula 1) is such a chiral amine molecule. It is a new type of calcium analog drug that can activate calcium receptors in the parathyroid glands, thereby reducing the secretion of parathyroid hormone (PTH) and is used to treat hyperparathyroidism.

[0004]

[0005] Current chemical catalysis of Cinacalcet involves the use of hazardous metal hydrides or high-pressure hydrogen in combination with toxic and expensive transition metal / ligand combinations, resulting in heavy waste streams that are environmentally unfriendly. Therefore, a mild and highly selective catalyst is needed to construct the chiral center of Cinacalcet. Enzymes are such a mild and selective catalyst.

[0006] The most effective enzyme-catalyzed reaction for Cinacalcet (Structural Formula 1) is to start from 1-acetonapthone (Structural Formula 2) and 3-trifluoromethylamphetamine (Structural Formula 3), to form an imine intermediate (Structural Formula 4, which may be a mixture of E isomers and Z isomers) in the reaction solvent or in the enzyme active site, and then reduced to the desired chiral amine (Structural Formula 1) under the action of "imine reductase" (IRED; EC1.5.1.x), as shown in the following formula:

[0007]

[0008] NAD(P)H coenzyme can be effectively recycled and regenerated by the well-known combinations of glucose dehydrogenase (GDH) / glucose, alcohol dehydrogenase (ADH) / isopropanol, formate dehydrogenase (FDH) / formate, lactate dehydrogenase / lactic acid, phosphite dehydrogenase (PDH) / phosphite, etc. As shown in the following formula, NAD(P)H coenzyme can be recycled by the combination of glucose dehydrogenase (GDH) and glucose.

[0009]

[0010] Although there are many reports on the use of enzymes in the pharmaceutical industry to produce chiral amines (especially imine reductase, EC2.6.1x), there are few reports on the successful synthesis of chiral amines using imine reductase. There is still a demand in the market for the use of biological enzymes to generate high optical purity cinacalcet from inexpensive and readily available achiral substrates (such as ketones shown in Structural Formula 2 and amines shown in Structural Formula 3). Summary of the invention

[0011] The present disclosure provides a method for developing an imine reductase and a variant thereof with high activity, high selectivity and stability, wherein the method obtains a polypeptide variant by engineering a wild-type imine reductase. The polypeptide variant can be used to convert 1-naphthone (Structural Formula 2) and 3-trifluoromethylamphetamine (Structural Formula 3) into (R)-cinacalcet (Structural Formula 1) having an optical purity of at least 10% ee, 20% ee, 50% ee, 90% ee, 95% ee, 98% ee, 99% ee, 99.5% ee, 99.6% ee, 99.7% ee, 99.8% ee or 99.9% ee.

[0012] Specifically, the present invention uses enzyme engineering technology to express the imine reductase gene in Escherichia coli, and constructs an imine reductase library including a site-saturated mutagenesis library and a combinatorial library to mutate and evolve the initial imine reductase (SEQ ID No: 12). At the same time, through the "Substrate Walking" strategy, a substrate analog with less steric hindrance is used as a substrate to screen the activity of two ketone substrates (Structure 2 and Structure X) and multiple amine substrates (Structure 3, Structure X), and finally obtain variants active for 1-naphthone (Structure 2) and 3-trifluoromethylamphetamine (Structure 3), which are converted into (R)-cinacalcet (Structure 1) through an intermediate (Structure 4). The mutant bacteria are cultured using a 96-well plate, and multiple rounds of high-throughput screening are completed, and finally an amine reductase and its variant method with high activity, high optical purity and stability are obtained, and this bioenzyme catalysis method is used to synthesize cinacalcet.

[0013] Although the original imine reductase (e.g., SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) did not show activity on the substrate pair of 1-acetonapthone (Structural Formula 2) and 3-trifluoromethylamphetamine (Structural Formula 3), an active mutant (e.g., SEQ ID No.: 93) was obtained by enzyme engineering. Through subsequent enzyme engineering evolution, mutants were obtained that could tolerate at least 1, 10, 20, 50 or 100 g / L of 1-acetonapthone (Structural Formula 2) substrate or 3-trifluoromethylamphetamine (Structural Formula 3) substrate or a combination of 1-acetonapthone (Structural Formula 2) and 3-trifluoromethylamphetamine (Structural Formula 3) substrates, in the presence of at least 10%, 20%, 30%, 40% or 50% (volume ratio v / v) of an organic solvent, at a pH of 5, 6, 7, 8, 9 or 10, in the presence of NAD or NADP, at a temperature of at least 25°C, at least 30°C, at least 40°C or at least 50°C, and a concentration ratio of 1-acetonapthone (Structural Formula 2) to 3-trifluoromethylamphetamine (Structural Formula 3) ranging from 1:20, 1:10, 1:5, 1:1, 5:1, 10:1 to 20:1. The NAD(P)H coenzyme is regenerated through cycles such as glucose dehydrogenase (GDH) / glucose, alcohol dehydrogenase (ADH) / isopropanol, formate dehydrogenase (FDH) / formate, lactate dehydrogenase / lactate, or phosphite dehydrogenase (PDH) / phosphite.

[0014] Engineered Peptides

[0015] In one aspect, the present disclosure provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference sequence SEQ ID NO:12, or a functional fragment thereof.

[0016] In another aspect, the disclosure provides an engineered polypeptide comprising an amino acid substitution at one or more positions selected from the group consisting of: 4, 5, 6, 8, 10, 14, 25, 51, 52, 58, 61, 69, 74, 75, 76, 77, 83, 85, 87, 90, 92, 99, 101, 102, 110, 113, 114, 115, 117, 123, 127, 128, 129, 130, 132, 137, 139, 141, 153, 155, 161 , 162, 172, 178, 180, 181, 182, 184, 185, 186, 188, 189, 191, 195, 201, 204, 215, 219, 220, 221, 224, 228, 231, 232, 236, 238, 244, 245, 246, 248, 249, 250, 251, 252, 253, 255, 269, 277 or 286, wherein the positions are numbered with reference to SEQ ID NO:12.

[0017] In some embodiments, the “one or more” includes 1 to 60, for example 1 to 30, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60.

[0018] In some embodiments, the amino acid substitution is selected from the group consisting of: 4Y, 5F, 5K, 5I, 6R, 8R, 8V, 8N, 8L, 10S, 10V, 14S, 25I, 51A, 52F, 58Q, 61Q, 69Y, 74L, 75S, 76P, 77S, 77A, 83I, 85Y, 85G, 87M, 87I, 90G, 92T, 92N, 92H, 92E, 92S, 99F, 101A, 102S, 110H, 113L, 113F, 113R , 113I, 114F, 114A, 114I, 115R, 117F, 123N, 123Q, 127G, 127A, 127I, 127L, 127Y, 127C, 128Y, 128E, 128L, 128M , 129A, 129W, 129D, 129P, 129Q, 129C, 129V, 129L, 130G, 130T, 130Q, 130V, 132C, 132F, 132G, 137H, 137T, 137S , 137N, 139Q, 141Y, 153V, 155V, 155W, 161G, 162K, 172M, 178L, 178Q, 178D, 180W, 181M, 181G, 182L, 184N, 184 V, 184S, 184G, 184A, 184C, 185M, 185L, 185V, 185C, 185I, 185S, 185A, 185G, 186W, 186G, 188A, 188G, 189H, 189 194R, 215R, 219S, 220P, 221C, 224W, 228Y, 231V, 232S, 236R, 238F, 244A, 245L, 245H, 245S, 246T, 248T, 249H, 249D, 250L, 250Y, 251A, 252T, 253M, 255H, 269L, 277I, 286A and 286M, wherein the positions are numbered with reference to SEQ ID NO:12.

[0019] In some embodiments, the engineered polypeptide contains an amino acid replacement at one or more positions selected from the group consisting of: 76, 77, 92, 99, 110, 114, 117, 127, 128, 129, 132, 139, 153, 155, 161, 180, 181, 185, 189, 220, 228, 236, 245, 250, 251, 253, 269, 286 compared to the reference sequence SEQ ID NO: 12, wherein the amino acid positions of the engineered polypeptide are numbered with reference to SEQ ID NO: 12.

[0020] In some embodiments, the engineered polypeptide contains an amino acid replacement at one or more positions selected from the group consisting of: 77, 92, 110, 114, 117, 127, 129, 132, 139, 153, 155, 161, 181, 185, 189, 220, 228, 236, 245, 250, 251, 253, or 286 compared to the reference sequence SEQ ID NO: 12, wherein the amino acid positions of the engineered polypeptide are numbered with reference to SEQ ID NO: 12.

[0021] In some embodiments, the engineered polypeptide contains an amino acid substitution at one or more positions selected from the group consisting of 77, 110, 127, 129, 153, 155, 220, or 236 compared to the reference sequence SEQ ID NO: 12, wherein the amino acid positions of the engineered polypeptide are numbered with reference to SEQ ID NO: 12.

[0022] In some embodiments, the engineered polypeptide contains an amino acid substitution at one or more positions selected from the group consisting of: 127, 128, 129, 130, 180, 249 compared to the reference sequence SEQ ID NO:93, wherein the positions are numbered with reference to SEQ ID NO:93.

[0023] In some embodiments, the engineered polypeptide contains an amino acid substitution selected from one or more of the following: 127Y, 128Y, 129W, 129Q, 129P, 129D, 129C, 130T, 180W, or 249D compared to the reference sequence SEQ ID NO:93, wherein the positions are numbered with reference to SEQ ID NO:93.

[0024] In some embodiments, the engineered polypeptide comprises an amino acid replacement at one or more (e.g., 2-15, 3-10, 3, 4, 5, 6, 7, 8, 9, or 10) positions selected from the group consisting of: 69, 76, 99, 101, 123, 127, 128, 129, 132, 180, 184, 189, 215, 250, 286 compared to the reference sequence SEQ ID NO: 121, wherein the positions are numbered with reference to SEQ ID NO: 121.

[0025] In some specific embodiments, the amino acid substitution positions include at least 180 and / or 286. In some further specific embodiments, the amino acid substitution positions also include one or more of 76, 99, 127, 128, 129, and 250.

[0026] In some specific embodiments, the amino acid replacement positions include at least 76 / 127 / 180, 99 / 286, 99 / 127 / 286, 99 / 127 / 129 / 286, 99 / 129 / 180 / 286, 99 / 127 / 128 / 129 / 286, 99 / 127 / 128 / 129 / 180 / 286, 127 / 286, 127 / 180 / 286, 127 / 129 / 180 / 286, 127 / 128 / 129 / 180 / 286, or 129 / 180 / 286.

[0027] In some specific embodiments, the amino acid replacement positions also include one or more of 69, 76, 99, 101, 123, 127, 128, 129, 132, 180, 184, 189, 215, 250 and 286.

[0028] In some embodiments, the amino acid substitution is selected from the group consisting of: 69Y, 76P, 99F, 101A, 123N, 127L, 127Y, 128Y, 128E, 129D, 129P, 132F, 180W, 184N, 189Y, 215R, 250Y, and 286M.

[0029] In some embodiments, the engineered polypeptide comprises at least one amino acid substitution selected from the following substitution groups compared to the reference sequence SEQ ID NO: 121: 76 / 99 / 127 / 128 / 129 / 180 / 286, 99 / 127 / 128 / 129 / 180 / 250 / 286, 99 / 101 / 128 / 129 / 180 / 286, 69 / 99 / 127 / 128 / 129 / 180 / 250 / 286, 76 / 99 / 127 / 128 / 129 / 180 / 184 / 286, 99 / 123 / 127 / 129 / 180 / 286, 76 / 99 / 127 / 128 / 129 / 180 / 184 / 286, 76 / 99 / 127 / 128 / 129 / 180 ... 6 / 132 / 180 / 250、99 / 101 / 123 / 127 / 128 / 129 / 180 / 184 / 286、69 / 101 / 127 / 129 / 180 / 250 / 286、180 / 189 / 286、76 / 127 / 129 / 180 / 286、76 / 99 / 127 / 129 / 215 / 250 / 286、76 / 123 / 127 / 128 / 129 / 180、76 / 127 / 132 / 180 / 189、69 / 76 / 99 / 123 / 129 / 180 / 250 / 286、127 / 132 / 180 / 189 / 250 / 286、101 / 127 / 180 / 189 / 286、99 / 123 / 127 / 128 / 129 / 184 / 250 / 286、99 / 101 / 132 / 180 / 189 / 215 / 286、69 / 99 / 101 / 127 / 128 / 129 / 184 / 215 / 250 / 286、76 / 99 / 180 / 215 / 250 / 286、76 / 99 / 1 9 / 127 / 180 / 250, wherein the positions are numbered with reference to SEQ ID NO: 121.

[0030] In some embodiments, the engineered polypeptide comprises at least one amino acid substitution selected from the following substitution groups compared to the reference sequence SEQ ID NO: 121: 76P / 99F / 127L / 128Y / 129D / 180W / 286M, 99F / 127L / 128Y / 129D / 180W / 250Y / 286M, 99F / 101A / 128Y / 129P / 180W / 286M, 69Y / 99F / 127L / 128Y / 129D / 180W / 250Y / 286M, 76P / 99F / 127L / 128Y / 129D / 180W / 184N / 286M, 99F / 123N / 127L / 129P / 180W / 286M. 32F / 180W / 250Y, 99F / 101A / 123N / 127L / 128Y / 129D / 180W / 184N / 286M, 69Y / 101A / 127L / 129P / 180W / 250Y / 286M, 180W / 189Y / 286M, 76P / 127L / 1 29P / 180W / 286M, 76P / 99F / 127L / 129P / 215R / 250Y / 286M, 76P / 123N / 12 7L / 128Y / 129D / 180W, 76P / 127Y / 132F / 180W / 189Y, 69Y / 76P / 99F / 123N / 129P / 180W / 250Y / 286M, 127Y / 132F / 180W / 189Y / 250Y / 286M, 101A / 127 Y / 180W / 189Y / 286M, 99F / 123N / 127L / 128Y / 129P / 184N / 250Y / 286M, 99 F / 101A / 132F / 180W / 189Y / 215R / 286M, 69Y / 99F / 101A / 127L / 128Y / 129 D / 184N / 215R / 250Y / 286M, 76P / 99F / 180W / 215R / 250Y / 286M, 76P / 99F / 1 23N / 127L / 128Y / 129D / 215R / 286M, 76P / 127L / 128Y / 129P / 250Y / 286M, 69Y / 99F / 101A / 132F / 180W / 189Y / 215R, 101A / 127Y / 128E / 189Y / 250Y / 286M, 69Y / 99F / 127Y / 189Y / 286M, 76P / 99F / 123N / 127L / 180W / 215R / 250Y / 286M, 76P / 99F / 123N / 180W, 76P / 99F / 127L / 180W / 250Y, wherein the positions are numbered with reference to SEQ ID NO:121.

[0031] In some embodiments, the engineered polypeptide contains an amino acid substitution at one or more positions selected from the group consisting of: 4, 5, 8, 10, 51, 83, 85, 87, 137, 172, 195, 219, 269, 277 compared to the reference sequence SEQ ID NO: 146, wherein the positions are numbered with reference to SEQ ID NO: 146.

[0032] In some embodiments, the engineered polypeptide contains one or more of the following amino acid substitutions compared to the reference sequence SEQ ID NO:146: 4Y, 5F, 5K, 5I, 8V, 8L, 8R, 10S, 51A, 83I, 85Y, 85G, 87M, 137H, 137T, 172M, 195S, 219S, 269L, 277I, wherein the positions are numbered with reference to SEQ ID NO:146.

[0033] In some embodiments, the engineered polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 or 244, or a functional fragment thereof.

[0034] In some embodiments, the engineered polypeptide has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the corresponding reference sequence.

[0035] The engineered polypeptides described herein have imine reductase activity. In some preferred embodiments, the engineered polypeptides have improved activity and / or stereoselectivity. In some further preferred embodiments, the engineered polypeptides convert substrates to chiral amines with greater than 50% ee, greater than 60% ee, greater than 70% ee, greater than 80% ee, greater than 90% ee, greater than 95% ee, greater than 98% ee, greater than 99% ee, greater than 99.5% ee, greater than 99.6% ee, greater than 99.7% ee, greater than 99.8% ee, or greater than 99.9% ee.

[0036] The engineered polypeptides described herein catalyze the and primary amines Generate chiral amine Among them, R 1 Selected from C 1-6 Alkyl; R 2 is selected from aryl; R 3 Selected from C 1-6 Alkyl, substituted or unsubstituted aryl-C 1-6 Alkylene; preferably, R 1 Selected from C 1-4 Alkyl; R 2 is phenyl or naphthyl; R 3 Selected from C 1-6 Alkyl, unsubstituted or halogen or halogenated C 1-4 Alkyl substituted phenyl-C 1-4 Alkylene; preferably, R 1 is methyl; R 2 is phenyl or naphthyl; R 3 Selected from n-butyl, isopentyl, benzyl, phenylpropyl or trifluoromethylphenylpropyl.

[0037] In some embodiments, the substrate is selected from:

[0038]

[0039] In some embodiments, the engineered polypeptides described herein are used to catalyze the reaction of Compound 2 and Compound 3 to produce (R)-Cinacalcet.

[0040] In another aspect, the present invention also provides a fusion protein, which comprises the engineered polypeptide described in any of the above items.

[0041] In another aspect, the present invention also provides a polynucleotide encoding the engineered polypeptide described in any one of the above items.

[0042] In some embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241 or 243.

[0043] The polynucleotide can be prepared according to known standard solid phase synthesis methods. In some embodiments, base fragments can be synthesized separately and then connected (for example, by enzymatic ligation method or chemical ligation method or polymerase-mediated method) to form any desired continuous sequence. For example, polynucleotides or oligonucleotides of the present invention can be prepared by chemical synthesis (for example, using the classical phosphoramidite method described by Beaucage et al., Tet. Lett., 22: 1859-69

[1981] , or by Matthes et al., EMBO J., 3: 801-05

[1984] method described). According to the phosphoramidite method, oligonucleotides are synthesized (for example, in an automatic DNA synthesizer), purified, annealed, connected and cloned into appropriate vectors. In addition, basically any nucleic acid can be obtained from a variety of commercial sources (for example, The Midland Certified Reagent Company, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGen Inc. Chicago, IL, Operon Technologies Inc., Alameda, CA, and other commercial sources).

[0044] In another aspect, the present invention also provides a nucleic acid construct, which comprises the polynucleotide described above.

[0045] In some embodiments, the nucleic acid construct also comprises a regulatory sequence, including but not limited to a promoter operably connected to a protein coding sequence. Suitable promoters are known to those skilled in the art. In some embodiments, in order to obtain high expression levels in a specific host, it is advantageous to express the engineered polypeptide of the present invention under the control of a heterologous promoter. In some embodiments, the promoter sequence is operably connected to the 5' region of the engineered polypeptide coding sequence using any suitable method known in the art. Examples of promoters useful for expressing the engineered polypeptide of the present invention include but are not limited to promoters from fungi. In some embodiments, the nucleic acid construct of the present invention also comprises a suitable transcription terminator sequence, which is a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator having a function in a selected host cell can be used in the present invention.

[0046] In some embodiments, a suitable leader sequence is a portion of a cloned engineered polypeptide sequence, which is a sequence preceding the coding region in a structural gene, which can be transcribed but not translated. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the selected host cell can be used in the present invention.

[0047] In some embodiments, the sequences of the invention further include a polyadenylation sequence, which is a sequence operably linked to the 3' end of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal for adding polyadenylic acid residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the selected host cell can be used in the present invention.

[0048] In some embodiments, the regulatory sequence includes a signal peptide coding region encoding an amino acid sequence connected to the amino terminus of the polypeptide, and directs the encoded polypeptide to enter the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region, which is naturally connected to the segment of the coding region encoding the secreted polypeptide in the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. An exogenous signal peptide coding region may be required when the coding sequence does not naturally contain a signal peptide coding region.

[0049] Alternatively, the foreign signal peptide coding region can simply replace the natural signal peptide coding region to increase the secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide to enter the secretory pathway of the selected host cell can be used in the present invention.

[0050] In some embodiments, the regulatory sequence comprises a propeptide coding region that encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases a zymogen). Propolypeptides are generally inactive and can be converted to mature active transaminase polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide.

[0051] When both the signal peptide region and the propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located immediately adjacent to the amino terminus of the polypeptide and the signal peptide region is located immediately adjacent to the amino terminus of the propeptide region.

[0052] In some embodiments, regulatory sequences are also used to allow expression of polypeptides relative to the growth regulation of the host cell. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). In prokaryotic host cells, suitable regulatory sequences include but are not limited to lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include, for example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter and the Aspergillus oryzae glucoamylase promoter.

[0053] In another aspect, the present invention also provides an expression vector, which comprises the polynucleotide or nucleic acid construct described above.

[0054] The expression vector contains one or more expression regulatory regions such as a promoter and terminator, a replication origin, and the like, depending on the type of host into which the polynucleotide or nucleic acid construct is introduced.

[0055] In some embodiments, the various nucleic acids and control sequences described above are linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of a nucleic acid sequence encoding a polypeptide at such a site. Alternatively, in some embodiments, the nucleic acid sequence is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. When producing an expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked to an appropriate control sequence for expression.

[0056] Recombinant expression vectors include any suitable vectors (e.g., plasmids or viruses), which can be easily subjected to recombinant DNA procedures and can cause the expression of polynucleotide sequences. The selection of vectors generally depends on the compatibility of the vector with the host cell to be introduced into the vector. In some embodiments, the vector is a linear plasmid or a closed circular plasmid. In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector existing as an extrachromosomal entity, which replicates independently of chromosome replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome). In some embodiments, the vector includes any tool (means) for ensuring self-replication. Alternatively, in some other embodiments, after being introduced into the host cell, the vector is integrated into the genome and replicated together with the chromosome integrated therein. In addition, in other embodiments, a single vector or plasmid may be used, or two or more vectors or plasmids comprising the total DNA of the genome to be introduced into the host cell may be included together.

[0057] In another aspect, the present invention also provides a host cell, which comprises the polynucleotide, nucleic acid construct or expression vector described above.

[0058] In another aspect, the present invention also provides a composition comprising the engineered polypeptide, fusion protein, polynucleotide, nucleic acid construct, expression vector or host cell described in any one of the foregoing items.

[0059] In another aspect, the present invention also provides a method for preparing the engineered polypeptide described in any of the above items, comprising the steps of culturing the host cell under conditions allowing expression of the engineered polypeptide, and optionally recovering the engineered polypeptide from the cultured host cell culture.

[0060] Catalytic method

[0061] The present application creatively discovered an imine reductase and its variants that can directly synthesize Cinacalcet from 1-acetonapthone (Structural Formula 2) and 3-trifluoromethylamphetamine (Structural Formula 3) with high optical purity and high productivity, without the need for additional chemical conversion. At the same time, the NAD(P)H coenzyme required for the catalytic process can be recycled by glucose dehydrogenase (GDH) and glucose combination.

[0062] The present application also discovered an imine reductase variant having improved imine reductase activity compared to the original imine reductase. The imine reductase and its variant can be effectively used for the catalytic synthesis of cinacalcet, a chiral amine-containing substance, and have high optical purity.

[0063] Therefore, in another aspect, the present invention also provides a method for asymmetric catalytic synthesis of chiral amine (I), the method comprising reacting a ketone substrate (II) and an amine substrate (III) in the presence of any of the polypeptides, fusion proteins or compositions described above, and a cofactor;

[0064]

[0065] Among them, R 1 Selected from C 1-6 Alkyl; R 2 is selected from aryl; R 3 Selected from C 1-6 Alkyl, substituted or unsubstituted aryl-C 1-6 Alkylene.

[0066] In some embodiments, R 1 Selected from C 1-4 Alkyl; R 2 is phenyl or naphthyl; R 3 Selected from C 1-6 Alkyl, unsubstituted or halogen or halogenated C 1-4Alkyl substituted phenyl-C 1-4 Alkylene.

[0067] In some embodiments, R 1 is methyl; R 2 is phenyl or naphthyl; R 3 Selected from n-butyl, isopentyl, benzyl, phenylpropyl or trifluoromethylphenylpropyl.

[0068] In some embodiments, the reaction is carried out under one or more of the following conditions:

[0069] (1) The concentration of the ketone substrate (II) or the amine substrate (III) is 1 to 100 g / L; preferably, the concentration ratio of the ketone substrate (II) to the amine substrate (III) is 1:(0.05 to 20), for example, 1:(0.1-10), 1:(0.2-10), 1:(0.5-5), 1:(0.5-2), 1:(0.5-1.5), 1:(0.5-1.2) or 1:(0.5-0.8);

[0070] (2) The reaction is carried out in an organic solvent; preferably, the organic solvent is DMSO;

[0071] (3) The pH of the reaction system is 5 to 10, for example, 5, 6, 7, 8, 9 or 10;

[0072] (4) the cofactor NAD(P)H / NAD(P); preferably, the concentration of the cofactor is 0.1 to 2 g / L;

[0073] (5) The reaction temperature is 20-25°C, 20-30°C, 20-40°C or 20-50°C.

[0074] In some embodiments, the concentration of the ketone substrate (II) or the amine substrate (III) is 1-50 g / L, for example, 1-40 g / L, 1-30 g / L, 1-20 g / L, 1-10 g / L or 1-5 g / L.

[0075] In some embodiments, the engineered polypeptide has a concentration of 0.01 g / L-50 g / L, such as 0.05 g / L-40 g / L, 0.1 g / L-30 g / L, 0.1 g / L-20 g / L, 0.1 g / L-10 g / L, 0.1 g / L-5 g / L or 0.1 g / L-2 g / L, and another example is 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1 g / L, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L or 50 g / L.

[0076] In some embodiments, the pH can be controlled by using a buffer. Suitable buffers for controlling the desired pH range are known in the art and include, but are not limited to, phosphate, acetate, borate, Tris, and the like.

[0077] In some embodiments, the reaction system further contains glucose dehydrogenase (GDH) / glucose, alcohol dehydrogenase (ADH) / isopropanol, formate dehydrogenase (FDH) / formic acid, lactate dehydrogenase / lactic acid or phosphite dehydrogenase (PDH) / phosphite, so as to achieve the recycling of cofactors. In some embodiments, the reaction system further contains GDH / glucose. In some embodiments, the GDH concentration is 0.1-2 g / L. In some embodiments, the glucose concentration is 1-20 g / L.

[0078] In some embodiments, the chiral amine is (R)-Cinacalcet;

[0079] Preferably, the optical purity of (R)-Cinacalcet is greater than 10% ee, greater than 20% ee, greater than 50% ee, greater than 60% ee, greater than 70% ee, greater than 80% ee, greater than 90% ee, greater than 95% ee, greater than 98% ee, greater than 99% ee, greater than 99.5% ee, greater than 99.6% ee, greater than 99.7% ee, greater than 99.8% ee or greater than 99.9% ee.

[0080] In another aspect, the present disclosure provides use of the engineered polypeptide, fusion protein, polynucleotide, nucleic acid construct, expression vector, host cell, composition, and catalytic method described in any of the foregoing items in the asymmetric catalytic synthesis of chiral amines, particularly (R)-Cinacalcet.

[0081] Definition of terms

[0082] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operating procedures used in the present disclosure are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0083] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to a molecule composed of two or more amino acids covalently linked by peptide bonds.

[0084] As used herein, the term "polynucleotide" or "nucleic acid" refers to a biological macromolecule obtained by polymerization of two or more nucleotides.

[0085] As used herein, the term "imine reductase" refers to an enzyme having imine reductase activity. Imine reductases include naturally occurring (wild-type) imine reductases and non-naturally occurring engineered polypeptides generated by human processing.

[0086] As used herein, the term "imine reductase activity" refers to an enzymatic activity that converts a carbonyl group of a ketone or aldehyde and an amino group of a primary or secondary amine (wherein the carbonyl and amino groups may be on different compounds or on the same compound) into a secondary or tertiary amine product compound in the presence of the cofactor NAD(P)H.

[0087] As used herein, the term "naturally occurring" or "wild type" refers to a form found in nature. For example, a naturally occurring or wild type polypeptide or polynucleotide sequence is a sequence that is present in an organism, can be isolated from a source in nature, and has not been manipulated by humans.

[0088] As used herein, the terms "recombinant" or "engineered" or "non-naturally occurring" when used to refer to, for example, a cell, a nucleic acid, or a polypeptide, refers to a material that is altered in a manner that does not otherwise exist in nature, or is identical to it but is produced or obtained from synthetic material and / or through the use of recombinant techniques, or a material that corresponds to the natural or native form of that material.

[0089] As used herein, the term "reference sequence" refers to the basic sequence for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a fragment of a full-length gene or polypeptide sequence. Generally speaking, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, or the total length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) include a sequence similar between the two sequences (that is, a part of a complete sequence), and (2) can further include a sequence different between the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is usually performed by comparing the sequences of the two polynucleotides or polypeptides in a "comparison window" to identify and compare the sequence similarity of a local region. In some embodiments, a "reference sequence" can be based on a basic amino acid sequence, wherein a reference sequence is a sequence that can have one or more variations in a basic sequence.

[0090] As used herein, the term "conservative amino acid substitution" refers to the replacement of one residue with a different residue having a similar side chain, and thus generally includes the replacement of an amino acid in a polypeptide with an amino acid in the same or similarly defined amino acid class. For example, but not limited to, an amino acid with an aliphatic side chain can be substituted with another aliphatic amino acid such as alanine, valine, leucine, and isoleucine; an amino acid with a hydroxyl side chain can be substituted with another amino acid with a hydroxyl side chain such as serine and threonine; an amino acid with an aromatic side chain can be substituted with another amino acid with an aromatic side chain such as phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain can be substituted with another amino acid with a basic side chain such as lysine and arginine; an amino acid with an acidic side chain can be substituted with another amino acid with an acidic side chain such as aspartic acid or glutamic acid; and a hydrophobic amino acid or a hydrophilic amino acid can be substituted with another hydrophobic amino acid or a hydrophilic amino acid, respectively.

[0091] As used herein, the term "stereoselectivity" refers to the preferential formation of a stereoisomer relative to another stereoisomer in a chemical or enzymatic reaction. Stereoselectivity can be partial, wherein the formation of one stereoisomer is superior to another isomer, or can be complete, wherein only one stereoisomer is formed. When stereoisomers are enantiomers, stereoselectivity is referred to as enantiomeric selectivity, the fraction (usually reported as percentage) of one enantiomer in the sum of two enantiomers. In the art, this fraction (typically percentage) is usually alternatively reported as enantiomeric excess (ee), which is calculated therefrom according to the following formula: [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. When the stereoisomers are diastereomers, the stereoselectivity is called diastereoselectivity, which is the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, often alternatively reported as diastereomeric excess (de). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

[0092] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyoma viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0093] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression regulatory sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by a host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0094] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0095] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0096] The compilation of the twenty conventional amino acids involved in the present disclosure follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present disclosure, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0097] As used herein, the term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight or branched chain alkane, for example, "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, and the like.

[0098] As used herein, the term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched chain alkane, for example, "C 1-6 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0099] As used herein, the term "aryl" refers to a group obtained by removing one hydrogen atom from the aromatic nucleus of an aromatic hydrocarbon, such as phenyl, naphthyl, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] The accompanying drawings herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0101] Figure 1 The reaction activity of the sixth generation mutant "Gen_6.0" at different enzyme loadings is shown. Among them, the y-axis is the signal intensity of (R)-Cinacalcet, and the x-axis is 5g / L, 10g / L and 20g / L enzyme loadings from left to right. Reaction conditions: 10% DMSO, 1g / L 1-naphthone (Structural Formula 2) and 1g / L 3-trifluoromethylamphetamine (Structural Formula 3), 10g / L glucose, 1g / L NADP, 1g / L NAD, 1g / L GDH, room temperature, reaction for 24 hours. Figure 1 It can be seen that as the enzyme loading increases, the activity of the imine reductase variant increases accordingly.

[0102] Figure 2The chromatographic mass spectrum (green) of the 4th generation mutant "Gen_4.0" with a (S)-Cinacalcet: (R)-Cinacalcet ratio of 1:0.8 and the chromatographic mass spectrum (red) of the reaction solution of the 5th generation mutant "Gen_5.0" with a (S)-Cinacalcet: (R)-Cinacalcet ratio of 1:10 are shown. Among them, the y-axis is the signal intensity of (S)-Cinacalcet and (R)-Cinacalcet, and the x-axis is the chromatographic elution time of (S)-Cinacalcet and (R)-Cinacalcet. Reaction conditions: 10% DMSO, 1g / L 1-naphthone (Structural Formula 2) and 1g / L 3-trifluoromethylamphetamine (Structural Formula 3), 10g / L glucose, 1g / L NADP, 1g / L NAD, 1g / L GDH, 30°C, reaction for 24 hours. By Figure 2 It can be seen that the optical purity of the fourth-generation mutant "Gen_4.0" is ~0% ee, and the reaction activity of the fifth-generation imine reductase mutant "Gen_5.0" obtained after enzyme engineering evolution is increased by 3 times, and the optical purity is increased to 80% ee. DETAILED DESCRIPTION

[0103] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0104] Example 1. Cultivation and production of cells overexpressing imine reductase

[0105] 1. Construction and expression of imine reductase library

[0106] The imine reductase gene was codon optimized to express in Escherichia coli. The optimized amino acid sequence is shown in SEQ ID NO:12. The gene was cloned into the expression vector pET-28a(+) driven by the T7 promoter. The pET-28a(+) expression vector contains the colE1 replication origin and the kanamycin resistance gene. The vector was transformed into Escherichia coli C43(DE3), and plasmid DNA was prepared from Escherichia coli C43(DE3) using standard methods.

[0107] According to MegaWHOP and SOE method, an imine reductase library including a site-saturation mutagenesis library and a combinatorial library was constructed, then cloned into a pET-28a (+) vector, transformed into Escherichia coli C43 (DE3) and expressed. For the construction of the site-saturation mutagenesis library, degenerate primers were designed using NNK to introduce saturation mutations at each desired position. Each degenerate primer was used to obtain a megaprimer by conventional PCR, and a complete plasmid with a single site saturation mutagenesis was generated by using a megaprimer PCR (MegaWHOP). All MegaWHOP products were then mixed and transformed to generate a pooled site-saturation mutagenesis library. Design sequence-defined primers to introduce programmed mutations in a combinatorial mutagenesis library. Each primer contained a mutation or multiple mutations or did not contain mutations in each region where a mutation was expected to be introduced. Multiple primers in each mutation region were mixed for initial PCR with equal molarity. Further PCR was performed to fuse all initial PCR products to generate a combinatorial library of all desired mutations. The SOE products were then assembled into the pET-28a(+) vector by the Gibson method and then transformed into E. coli C43(DE3) to obtain E. coli containing the plasmid of the gene of interest to generate a combined combinatorial library.

[0108] 2. Obtaining Frozen Cell Pellets Containing Imine Reductase Gene

[0109] Use colony picker to select the recombinant Escherichia coli clone containing gene of interest into 96-well plates containing 200 μ L LB or TB, 1 g / L kanamycin (Kan) antibiotic.Cells are grown overnight in a humidity-controlled incubator at 85% humidity, 30 ℃ and 250 rpm.10 μ L aliquots are transferred to a new deep-well plate containing 380 μ L of TB containing 1 g / L Kan, placed in a humidity-controlled incubator at 85% humidity, 30 ℃ and 250 rpm for 2-3 hours, then induced with 10 μ L 40 mM IPTG and continued to incubate overnight.With 4000 rpm / 4 ℃ / 20 minutes centrifugal collection cell precipitation.Discard supernatant and the remaining cell precipitation in the plate is stored in -80 ℃ refrigerator until measuring.

[0110] Example 2. Determination of enzyme activity in 96-well plates

[0111] The frozen cell pellet obtained in Example 1 in a 96-well deep-well plate was thawed to room temperature and treated with 200 μL / well of a lysis solution consisting of 0.5 g / L polymixin B sulfate (PMBS), 1 g / L lysozyme, and 0.1% v / v DNAase I in 100 mM potassium phosphate buffer at pH 8 or pH 9 for 90 minutes at room temperature. The lysis mixture was centrifuged at 4000 rpm / 4°C for 20 min. A certain volume of lysate supernatant is added in another deep well plate, add a certain volume of 100mM pH 8 or pH 9 potassium phosphate buffers containing 10~50g / L glucose, 0.5~1g / L NADP, 0.5~1g / L NAD and 0.5~1g / L GDH, then add a certain volume of DMSO solution, this solution contains 5g / L~20g / L ketone substrate 1-naphthone (structural formula 2) and 5g / L~20g / L amine substrate 3-trifluoromethyl amphetamine (structural formula 3).The volume or concentration of lysate, glucose and coenzyme, substrate and DMSO are adjusted in each round of experiment as required.Plate is sealed and placed in rt, 30,40,45 or 50 ℃ shaking incubator and reacted 24 hours.React with 200 μ L acetonitrile (ACN) quenching and at room temperature centrifugal with 4000rpm / 10 minute. The centrifuged solution can be directly analyzed by LCMS / MS or further diluted for LCMS / MS analysis.

[0112] The optical purity analysis method used an Agilent Proshell 120Chiral-V column (4.6mm*100mm, 2.7μm), using 10mM ammonium formate as mobile phase A and methanol as mobile phase B. The column temperature was 30℃, the flow rate was 0.8mL / min, and the ratio of mobile phase A: mobile phase B was 10:90 for isocratic elution. The analysis time was 4.0 minutes. The mass spectrometer was Agilent 6460. The detailed mass spectrometry parameters were as follows: ESI positive ionization mode, MRM detection mode, parent ion m / z 358.0, daughter ion m / z 115.2, collision energy 50eV, gas temperature 300℃, dry gas nitrogen 5L / min, nebulizer pressure 45psi, sheath gas temperature 250℃, sheath gas flow nitrogen 11L / min, capillary voltage 3500V. Data analysis was completed by Agilent MassHunter workstation software. The retention times of (S)-Cinacalcet and (R)-Cinacalcet were 1.9 min and 2.7 min, respectively.

[0113] Example 3. Production of shake flask powder (SFP)

[0114] A single clone of E. coli containing a plasmid with the gene for the enzyme of interest was inoculated into a 15 mL culture tube containing 7 mL of TB medium, 10 g / L glucose and 1 g / L Kan. The tube was placed in a shaker at 250 rpm / 30°C. After 16-24 hours, 6 mL of this culture was inoculated into 250 mL of TB w / 1 g / L Kan in a 1000 mL baffled shake flask. The flask was placed in a shaker at 250 rpm / 30°C until OD ~ 0.6-0.8 was reached (usually after 2-4 hours), at which time 250 μL of 1 M IPTG was added. Continue shaking overnight (16-24 hours). Cells were collected by centrifugation at 4000 rpm / 4°C / 20 minutes. The supernatant was discarded and the remaining cell pellet was resuspended in 35 mL of 50 mM pH 8 or pH 9 triethanolamine*H 2 SO 4 Discard the supernatant and resuspend the remaining cell pellet in 4-5 mL of 50 mM pH 8 or pH 9 triethanolamine*H 2 SO 4 Buffer. Lyse cells at 35-40 kpsi using a Constant System "One-Shot" homogenizer. Centrifuge the lysed cell suspension at 7830 rpm / 4°C / 1 h. Pour the supernatant into a vial, freeze at -80°C for 1 hour, and then place in a freeze dryer overnight to obtain a dry powder.

[0115] Example 4. Determination of the activity of shake flask powder (SFP)

[0116] A certain volume of 100mM pH 8 or pH 9 potassium phosphate buffer was added to the wells of a 96-well deep well plate, followed by a certain volume of 100mM pH 8 or pH 9 potassium phosphate buffer containing 50g / L glucose, 0.5g / L NADP, 0.5g / L NAD and 0.5g / L GDH and a high concentration of substrate solution dissolved in an organic co-solvent DMSO. The volume or concentration of the enzyme powder solution, co-solvent DMSO, substrate, glucose and coenzyme was adjusted in each round of experiments as needed. The plate was sealed and placed in a shaking incubator at rt, 30, 40, 45 or 50°C for 24 hours. The reaction was quenched with 200μL acetonitrile (ACN) and centrifuged at 4000rpm / 10 minutes at room temperature. The solution after centrifugation can be directly subjected to LCMS / MS analysis, or it can be further diluted and analyzed by LCMS / MS testing. Optical purity analysis was performed according to the method in Example 2.

[0117] Example 5. Substrate Walking Strategy

[0118] The lysate or shake flask powder (concentration up to 40 g / L) of wild-type polypeptides (e.g., SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) had no activity against 1-acetonapthone (Structural Formula 2) and 3-trifluoromethylamphetamine (Structural Formula 3) substrates. This may be related to the fact that the active site pocket of the enzyme is too small to accommodate the bulky and hydrophobic imine intermediate substrate (Structural Formula 4). According to reports by Marx et al. (Adv. Synth. Catal. 2018, 360, 2157) and Saville et al. (Science 2010, 329, 305), the "Substrate Walking" strategy can be used to expand the active site. The wild-type polypeptides (e.g., Marx et al.) (SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) and their mutants were reacted in combination with different ketone substrates: 1-naphthyl acetonide (Structural Formula 2), acetophenone (Structural Formula 5), ​​and different amine substrates: 3-trifluoromethylamphetamine (Structural Formula 3), 3-phenyl-1-propylamine (Structural Formula 6), benzylamine (Structural Formula 7), isopentylamine (Structural Formula 8), butylamine (Structural Formula 9) to test whether the corresponding products can be generated.

[0119]

[0120] Subsequently, the active mutant group of less ketone / amine substrate pair (for example " structural formula 5 / structural formula 3 " combination or " structural formula 2 / structural formula 6 " combination) is tested on the substrate pair (1-acetonapthone (structural formula 2) and 3-trifluoromethyl amphetamine (structural formula 3)) that really needs.Surprisingly, using this strategy, in the mutant (for example SEQ ID No:54 and SEQ ID No:78) of sequence SEQ ID No:12, find that some mutants are active to 1-acetonapthone (structural formula 2) and 3-trifluoromethyl amphetamine (structural formula 3).More surprisingly, although the polypeptide of SEQ ID No:54 and SEQ ID No:78 only produces undesirable (S)-enantiomer, by subsequent enzyme engineering evolution, obtain the mutant (for example SEQ ID No:146,148,150,152,154 and 156) that can produce target (R)-enantiomer, its optical purity is all>99.5%ee.

[0121] Example 6. Initial activity determination

[0122] According to the procedures of Examples 1 and 2, 12 wild-type polypeptides (SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) were lysed, and the lysate was reacted with 1 g / L of 1-naphthone (Structural Formula 2) and 1 g / L of 3-trifluoromethylamphetamine (Structural Formula 3) in 10% DMSO at room temperature for 24 hours. The reaction mixture was quenched and LCMS / MS was determined as described in Example 5, and no signal of Cinacalcet (Structural Formula 1) was detected, and all were inactive.

[0123] Example 7. Obtaining the initial activity of non-target products through the substrate "Substrate Walking" strategy

[0124] According to the procedures of Example 1 and Example 2, 12 wild-type polypeptides (SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) were cleaved, and the lysates were tested with all 10 possible ketone / amine substrate combinations in Example 6 (1 g / L ketone and 1 g / L amine, 1 g / L NADPH and 1 g / L NADH, 10% DMSO, reaction for 24 hours). The reaction mixture was quenched and LCMS / MS was measured as described in Example 5, and the results showed that the wild-type polypeptide SEQ ID No: 12 had the widest substrate acceptance range, including the substrate combinations "Structural Formula 5 / Structural Formula 3" and "Structural Formula 2 / Structural Formula 6" with relatively large molecular spaces.

[0125] Example 8. Improving the activity of non-target products through substrate walking strategy

[0126] According to the procedures of Example 1 and Example 2, a site-saturated mutagenesis library and a combinatorial library were constructed based on the wild-type sequence SEQ ID No: 12, and three different substrate combinations "Structure Formula 5 / Structure Formula 3", "Structure Formula 2 / Structure Formula 6" and the target substrate combination "1-naphthyl acetonide (Structure Formula 2) / 3-trifluoromethylamphetamine (Structure Formula 3)" were screened for activity, 1g / L ketone and 1g / L amine, 1g / L NADPH and 1g / L NADH, 10% DMSO, and reacted for 24 hours. The reaction mixture was quenched and LCMS / MS was determined as described in Example 4. The results show that the mutants have significantly improved activity for the substrate combinations "Structure Formula 5 / Structure Formula 3" and "Structure Formula 2 / Structure Formula 6". Table 1 and Table 2 are mutants with improved activity for the substrate combination "Structure Formula 5 / Structure Formula 3" and the substrate combination "Structure Formula 2 / Structure Formula 6", respectively. Relative activity is defined as the ratio of the LCMS / MS signal of the mutant to the LCMS / MS signal of the SEQ ID No: 12 parent.

[0127] According to the procedures of Example 3 and Example 4, the four mutants with higher activity improvement in Table 1 and Table 2 and the parent of SEQ ID No: 12 were respectively tested for activity, and the variant with the best performance (SEQ ID NO: 14) was selected as the parent for the next round of testing. In Tables 1 to 9, "nt" represents the coding sequence of the corresponding variant, and "aa" represents the amino acid sequence of the corresponding variant.

[0128] Table 1

[0129]

[0130]

[0131] +++: relative activity greater than 2.0 compared to the activity of SEQ ID NO: 12;

[0132] ++: compared with the activity of SEQ ID NO: 12, the relative activity is between 1.5 and 2.0; +: compared with the activity of SEQ ID NO: 12, the relative activity is between 1.0 and 1.5.

[0133] Table 2

[0134]

[0135]

[0136] ++++: relative activity greater than 20 compared to the activity of SEQ ID NO: 12;

[0137] +++: relative activity is between 15 and 20 compared with the activity of SEQ ID NO: 12;

[0138] ++: relative activity is between 10 and 15 compared with the activity of SEQ ID NO: 12;

[0139] +: Compared with the activity of SEQ ID NO: 12, the relative activity is between 5 and 10.

[0140] The imine reductase variant shown in SEQ ID NO: 14 was used as the parent for the next round of testing.

[0141] Example 9. Identification of Imine Reductase Generation 1 "Gen 1" (SEQ ID NO: 47)

[0142] According to the procedures of Example 1 and Example 2, a site combinatorial library was constructed based on the sequence SEQ ID No: 14, and three different substrate combinations "Structural Formula 5 / Structural Formula 3", "Structural Formula 2 / Structural Formula 6" and the target substrate combination "1-acetoacetyl naphthone (Structural Formula 2) / 3-trifluoromethylamphetamine (Structural Formula 3)" were screened for activity, 1g / L ketone and 1g / L amine, 1g / L NADPH and 1g / L NADH, 10% DMSO, and reacted for 24 hours. The reaction mixture was quenched as described in Example 5 and LCMS / MS was determined. The results show that an imine reductase mutant (SEQ ID No: 47, 48, 49, 50, 51, 52, 53) active against the target substrate combination 1-acetoacetyl naphthone (Structural Formula 2) / 3-trifluoromethylamphetamine (Structural Formula 3) was obtained, and the signal of Cinacalcet was detected. The results are shown in Table 3.

[0143] Table 3

[0144]

[0145]

[0146] +: The signal of Cinacalcet was detected on LCMS / MS.

[0147] The imine reductase variant shown in SEQ ID NO:47 was designated as imine reductase "Gen 1" and used as the parent for the next round of testing.

[0148] Example 10. Identification of Imine Reductase Generation 2 "Gen 2" (SEQ ID NO: 54)

[0149] According to the program of embodiment 1 and embodiment 2, mutant is cracked, lysate and the solution of 100mM pH 8 potassium phosphate buffers and DMSO containing 10g / L glucose, 1g / L NADP, 1g / L NAD, 1g / L GDH react, 10g / L ketone substrate 1-naphthone (structural formula 2) and 10g / L amine substrate 3-trifluoromethyl amphetamine (structural formula 3) are dissolved in DMSO, react 24 hours under room temperature.According to the quenching reaction mixture described in embodiment 5 and measure LCMS / MS.The results are shown in Table 4.

[0150] Table 4

[0151]

[0152]

[0153]

[0154] +++: relative activity greater than 2.0 compared with the imine reductase Gen 1 activity;

[0155] ++: relative activity is between 1.5 and 2.0 compared with the activity of imine reductase Gen 1;

[0156] +: Relative activity is between 1.2 and 1.5 compared to the activity of imine reductase Gen 1.

[0157] The imine reductase variant shown in SEQ ID NO:54 was designated as imine reductase "Gen 2" and used as the parent for the next round of testing.

[0158] Example 11. Identification of Imine Reductase Generation 3 "Gen 3" (SEQ ID NO: 78)

[0159] According to the program of embodiment 1 and embodiment 2, mutant is cracked, lysate and the solution of 100mM pH 8 potassium phosphate buffers and DMSO containing 10g / L glucose, 0.5g / L NADP, 0.5g / L NAD, 0.5g / L GDH react, 10g / L ketone substrate 1-naphthone (structural formula 2) and 10g / L amine substrate 3-trifluoromethyl amphetamine (structural formula 3) are dissolved in DMSO, react 24 hours under room temperature.According to the quenching reaction mixture described in embodiment 5 and measure LCMS / MS.The results are shown in Table 5.

[0160] Table 5

[0161]

[0162]

[0163] +++: relative activity greater than 3.0 compared with the imine reductase Gen 2 activity;

[0164] ++: relative activity is between 2.0 and 3.0 compared with the activity of imine reductase Gen 2;

[0165] +: Relative activity is between 1.2 and 2.0 compared to the activity of imine reductase Gen 2.

[0166] The imine reductase variant shown in SEQ ID NO:78 was designated as imine reductase "Gen 3" and used as the parent for the next round of testing.

[0167] Example 12. Identification of Imine Reductase Generation 4 "Gen 4" (SEQ ID NO: 93)

[0168] According to the program of Example 1 and Example 2, mutant is cracked, lysate and 100mM pH 8 potassium phosphate buffer containing 10g / L glucose, 1g / L NADP, 1g / L NAD, 1g / L GDH and the solution of DMSO are reacted, 10g / L ketone substrate 1-acetoacetyl ketone (structural formula 2) and 10g / L amine substrate 3-trifluoromethylamphetamine (structural formula 3) are dissolved in DMSO, and react 24 hours at room temperature.According to the quenching reaction mixture described in Example 5 and LCMS / MS are measured.The results are shown in Table 6.According to the program of Example 3 and Example 4, 7 mutants and SEQ ID No:78 parents with higher optical purity are selected to prepare shake flask powder (SFP) for activity test, and the best variant (SEQ ID NO:93) is selected as the parent (Parent) of the next round of test.

[0169] Table 6

[0170]

[0171]

[0172]

[0173] -: Compared with the activity of imine reductase Gen 3, the relative activity is between 0.8 and 1.0;

[0174] --: Compared with the activity of imine reductase Gen 3, the relative activity is between 0.5 and 0.8;

[0175] ---: Compared with the activity of imine reductase Gen 3, the relative activity is between 0.01 and 0.5.

[0176] The imine reductase variant shown in SEQ ID NO:93 was designated as imine reductase "Gen 4" and used as the parent for the next round of testing.

[0177] Example 13. Identification of Imine Reductase Generation 5 "Gen 5" (SEQ ID NO: 121)

[0178] According to the program of Example 1 and Example 2, mutant is cracked, lysate and 100mM pH 8 potassium phosphate buffer containing 10g / L glucose, 1g / L NADP, 1g / L NAD, 1g / L GDH and the solution of DMSO are reacted, 10g / L ketone substrate 1-acetoacetyl ketone (structural formula 2) and 10g / L amine substrate 3-trifluoromethylamphetamine (structural formula 3) are dissolved in DMSO, and react 24 hours at room temperature.According to the quenching reaction mixture described in Example 5 and measuring LCMS / MS.The results are shown in Table 7.According to the program of Example 3 and Example 4, 7 mutants with higher optical purity and SEQ ID No:93 parents are selected to prepare shake flask powder (SFP) for activity test, and the best performing variant (SEQ ID NO:121) is selected as the parent (Parent) of the next round of test.

[0179] Table 7

[0180]

[0181]

[0182]

[0183] +++: relative activity greater than 3.0 compared with the activity of imine reductase Gen 4;

[0184] ++: relative activity is between 2.0 and 3.0 compared with the activity of imine reductase Gen 4;

[0185] +: relative activity compared to imine reductase Gen 4 activity is between 1.0 and 2.0;

[0186] -: Compared with the activity of imine reductase Gen 4, the relative activity is between 0.8 and 1.0;

[0187] --: Compared with the activity of imine reductase Gen 4, the relative activity is between 0.5 and 0.8.

[0188] The imine reductase variant shown in SEQ ID NO: 121 was designated as imine reductase "Gen 5" and used as the parent for the next round of testing.

[0189] Example 14. Identification of Imine Reductase Generation 6 "Gen 6" (SEQ ID NO: 145 / 146)

[0190] According to the procedures of Example 1 and Example 2, the mutant is cracked, and the lysate is reacted with a solution of 100mM pH 9 potassium phosphate buffer and DMSO containing 20g / L glucose, 1g / L NADP, 1g / L NAD, 1g / L GDH, 20g / L ketone substrate 1-acetoacetyl naphthone (structural formula 2) and 20g / L amine substrate 3-trifluoromethylamphetamine (structural formula 3) dissolved in DMSO, and reacted for 24 hours at 40°C. According to the quenching reaction mixture described in Example 5 and LCMS / MS are measured. The results are shown in Table 8. According to the procedures of Example 3 and Example 4, 7 mutants with higher optical purity and SEQ ID No:121 parents are selected to prepare shake flask powder (SFP) for activity test, and the best performing variant (SEQ ID NO:146) is selected as the parent (Parent) of the next round of test.

[0191] Table 8

[0192]

[0193]

[0194]

[0195]

[0196] +++++: relative activity greater than 30 compared to imine reductase Gen 5 activity;

[0197] ++++: relative activity is between 20 and 30 compared with the activity of imine reductase Gen 5;

[0198] +++: relative activity is between 15 and 20 compared with the activity of imine reductase Gen 5;

[0199] ++: relative activity is between 10 and 15 compared with the activity of imine reductase Gen 5;

[0200] +: Relative activity is between 5 and 10 compared to the activity of imine reductase Gen 5.

[0201] The imine reductase variant represented by SEQ ID NO: 146 (whose encoding nucleic acid sequence is SEQ ID NO: 145) was designated as imine reductase "Gen 6" (sixth generation) and used as the parent for the next round of testing.

[0202] Example 15. Identification of Imine Reductase Generation 7 "Gen 7" (SEQ ID NO: 243 / 244)

[0203] According to the procedures of Example 1 and Example 2, the mutant is cracked, and the lysate is reacted with a solution of 100mM pH 8 potassium phosphate buffer and DMSO containing 10g / L glucose, 1g / L NADP, 1g / L NAD, 1g / L GDH, 10g / L ketone substrate 1-acetoacetyl naphthone (structural formula 2) and 10g / L amine substrate 3-trifluoromethylamphetamine (structural formula 3) in DMSO, and reacted for 24 hours at 40°C. According to the quenching reaction mixture described in Example 5 and LCMS / MS are measured. The results are shown in Table 9. According to the procedures of Example 3 and Example 4, 7 mutants with higher optical purity and SEQ ID No:146 parents are selected to prepare shake flask powder (SFP) for activity test, and the best performing variant (SEQ ID NO:244) is selected as the parent (Parent) of the next round of test.

[0204] Table 9

[0205]

[0206]

[0207] +: Relative activity compared to imine reductase Gen 6 activity is between 1.0 and 1.5.

[0208] The imine reductase variant represented by SEQ ID NO: 244 (whose encoding nucleic acid sequence is SEQ ID NO: 243) was designated as imine reductase "Gen 7".

[0209] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. An engineered polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference sequence SEQ ID NO: 12, or a functional fragment thereof.

2. An engineered polypeptide, comprising an amino acid substitution at one or more (e.g., 1 to 60) positions selected from the group consisting of: 4, 5, 6, 8, 10, 14, 25, 51, 52, 58, 61, 69, 74, 75, 76, 77, 83, 85, 87, 90, 92, 99, 101, 102, 110, 113, 114, 115, 117, 123, 127, 128, 129, 130, 132, 137, 139, 141, 153, 155 , 161, 162, 172, 178, 180, 181, 182, 184, 185, 186, 188, 189, 191, 195, 201, 204, 215, 219, 220, 221, 224, 228, 231, 232, 236, 238, 244, 245, 246, 248, 249, 250, 251, 252, 253, 255, 269, 277 or 286, wherein the positions are numbered with reference to SEQ ID NO:

12.

3. The engineered polypeptide of claim 2, wherein the amino acid substitution is selected from the group consisting of: 4Y, 5F, 5K, 5I, 6R, 8R, 8V, 8N, 8L, 10S, 10V, 14S, 25I, 51A, 52F, 58Q, 61Q, 69Y, 74L, 75S, 76P, 77S, 77A, 83I, 85Y, 85G, 87M, 87I, 90G, 92T, 92N, 92H, 92E, 92S, 99F, 101A, 102S, 110H, 113L, 114L, 3F, 113R, 113I, 114F, 114A, 114I, 115R, 117F, 123N, 123Q, 127G, 127A, 127I, 127L, 127Y, 127C, 128Y, 128E, 12 8L, 128M, 129A, 129W, 129D, 129P, 129Q, 129C, 129V, 129L, 130G, 130T, 130Q, 130V, 132C, 132F, 132G, 137H, 137 T, 137S, 137N, 139Q, 141Y, 153V, 155V, 155W, 161G, 162K, 172M, 178L, 178Q, 178D, 180W, 181M, 181G, 182L, 184 N, 184V, 184S, 184G, 184A, 184C, 185M, 185L, 185V, 185C, 185I, 185S, 185A, 185G, 186W, 186G, 188A, 188G, 189H , 189Y, 191V, 195S, 201I, 201T, 204R, 215R, 219S, 220P, 221C, 224W, 228Y, 231V, 232S, 236R, 238F, 244A, 245L, 245H, 245S, 246T, 248T, 249H, 249D, 250L, 250Y, 251A, 252T, 253M, 255H, 269L, 277I, 286A and 286M, wherein the positions are numbered with reference to SEQ ID NO:

12.

4. The engineered polypeptide according to any one of claims 1 to 3, wherein the engineered polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of: 76, 77, 92, 99, 110, 114, 117, 127, 128, 129, 132, 139, 153, 155, 161, 180, 181, 185, 189, 220, 228, 236, 245, 250, 251, 253, 269, 286 compared to the reference sequence SEQ ID NO: 12, wherein the amino acid positions of the engineered polypeptide are numbered with reference to SEQ ID NO: 12; Preferably, the engineered polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of 77, 92, 110, 114, 117, 127, 129, 132, 139, 153, 155, 161, 181, 185, 189, 220, 228, 236, 245, 250, 251, 253 or 286 compared to the reference sequence SEQ ID NO: 12, wherein the amino acid positions of the engineered polypeptide are numbered with reference to SEQ ID NO: 12; Preferably, the engineered polypeptide contains an amino acid substitution at one or more positions selected from the following positions: 77, 110, 127, 129, 153, 155, 220 or 236 compared to the reference sequence SEQ ID NO: 12, wherein the amino acid positions of the engineered polypeptide are numbered with reference to SEQ ID NO:

12.

5. The engineered polypeptide according to any one of claims 1 to 3, comprising an amino acid substitution at one or more positions selected from the group consisting of 127, 128, 129, 130, 180, 249 compared to the reference sequence SEQ ID NO: 93, wherein the positions are numbered with reference to SEQ ID NO: 93; Preferably, the engineered polypeptide contains an amino acid substitution selected from one or more of the following: 127Y, 128Y, 129W, 129Q, 129P, 129D, 129C, 130T, 180W or 249D compared to the reference sequence SEQ ID NO:93, wherein the positions are numbered with reference to SEQ ID NO:

93.

6. The engineered polypeptide according to any one of claims 1 to 3, wherein the engineered polypeptide comprises an amino acid substitution at one or more (e.g., 2-15, 3-10, 3, 4, 5, 6, 7, 8, 9 or 10) positions selected from the group consisting of 69, 76, 99, 101, 123, 127, 128, 129, 132, 180, 184, 189, 215, 250, 286, compared to the reference sequence SEQ ID NO: 121, wherein the positions are numbered with reference to SEQ ID NO: 121; preferably, the amino acid substitution positions include at least 180 and / or 286; further preferably, the amino acid substitution positions also include one or more of 76, 99, 127, 128, 129, 250; Preferably, the engineered polypeptide comprises at least one amino acid substitution selected from the following substitution groups compared to the reference sequence SEQ ID NO: 121: 76 / 99 / 127 / 128 / 129 / 180 / 286, 99 / 127 / 128 / 129 / 180 / 250 / 286, 99 / 101 / 128 / 129 / 180 / 286, 69 / 99 / 127 / 128 / 129 / 180 / 250 / 286, 76 / 99 / 127 / 128 / 129 / 180 / 184 / 286, 99 / 123 / 127 / 129 / 180 / 286, 76 / 99 / 127 / 128 / 129 / 180 / 184 / 286, 76 / 99 / 127 / 128 / 129 / 180 ... 6 / 132 / 180 / 250、99 / 101 / 123 / 127 / 128 / 129 / 180 / 184 / 286、69 / 101 / 127 / 129 / 180 / 250 / 286、180 / 189 / 286、76 / 127 / 129 / 180 / 286、76 / 99 / 127 / 129 / 215 / 250 / 286、76 / 123 / 127 / 128 / 129 / 180、76 / 127 / 132 / 180 / 189、69 / 76 / 99 / 123 / 129 / 180 / 250 / 286、127 / 132 / 180 / 189 / 250 / 286、101 / 127 / 180 / 189 / 286、99 / 123 / 127 / 128 / 129 / 184 / 250 / 286、99 / 101 / 132 / 180 / 189 / 215 / 286、69 / 99 / 101 / 127 / 128 / 129 / 184 / 215 / 250 / 286、76 / 99 / 180 / 215 / 250 / 286、76 / 99 / 1 23 / 127 / 128 / 129 / 215 / 286, 76 / 127 / 128 / 129 / 250 / 286, 69 / 99 / 101 / 132 / 180 / 189 / 215, 101 / 127 / 128 / 189 / 250 / 286, 69 / 99 / 127 / 189 / 286, 76 / 99 / 123 / 127 / 180 / 215 / 250 / 286, 76 / 99 / 123 / 180, 76 / 99 / 127 / 180 / 250, wherein the positions are numbered with reference to SEQ ID NO: 121; Preferably, the engineered polypeptide comprises at least one amino acid substitution selected from the following substitution groups compared to the reference sequence SEQ ID NO: 121: 76P / 99F / 127L / 128Y / 129D / 180W / 286M, 99F / 127L / 128Y / 129D / 180W / 250Y / 286M, 99F / 101A / 128Y / 129P / 180W / 286M, 69Y / 99F / 127L / 128Y / 129D / 180W / 250Y / 286M, 76P / 99F / 127L / 128Y / 129D / 180W / 184N / 286M, 99F / 123N / 127L / 129P / 180W / 286M, 32F / 180W / 250Y, 99F / 101A / 123N / 127L / 128Y / 129D / 180W / 184N / 286M, 69Y / 101A / 127L / 129P / 180W / 250Y / 286M, 180W / 189Y / 286M, 76P / 127L / 1 29P / 180W / 286M, 76P / 99F / 127L / 129P / 215R / 250Y / 286M, 76P / 123N / 12 7L / 128Y / 129D / 180W, 76P / 127Y / 132F / 180W / 189Y, 69Y / 76P / 99F / 123N / 129P / 180W / 250Y / 286M, 127Y / 132F / 180W / 189Y / 250Y / 286M, 101A / 127 Y / 180W / 189Y / 286M, 99F / 123N / 127L / 128Y / 129P / 184N / 250Y / 286M, 99 F / 101A / 132F / 180W / 189Y / 215R / 286M, 69Y / 99F / 101A / 127L / 128Y / 129 D / 184N / 215R / 250Y / 286M, 76P / 99F / 180W / 215R / 250Y / 286M, 76P / 99F / 1 23N / 127L / 128Y / 129D / 215R / 286M, 76P / 127L / 128Y / 129P / 250Y / 286M, 69Y / 99F / 101A / 132F / 180W / 189Y / 215R, 101A / 127Y / 128E / 189Y / 250Y / 286M, 69Y / 99F / 127Y / 189Y / 286M, 76P / 99F / 123N / 127L / 180W / 215R / 250Y / 286M, 76P / 99F / 123N / 180W, 76P / 99F / 127L / 180W / 250Y, wherein the positions are numbered with reference to SEQ ID NO:

121.

7. The engineered polypeptide according to any one of claims 1 to 3, comprising an amino acid substitution at one or more positions selected from the group consisting of: 4, 5, 8, 10, 51, 83, 85, 87, 137, 172, 195, 219, 269, 277 compared to the reference sequence SEQ ID NO: 146, wherein the positions are numbered with reference to SEQ ID NO: 146; Preferably, the engineered polypeptide contains one or more of the following amino acid substitutions compared to the reference sequence SEQ ID NO: 146: 4Y, 5F, 5K, 5I, 8V, 8L, 8R, 10S, 51A, 83I, 85Y, 85G, 87M, 137H, 137T, 172M, 195S, 219S, 269L, 277I, wherein the positions are numbered with reference to SEQ ID NO:

146.

8. The engineered polypeptide of any one of claims 2-7, wherein the engineered polypeptide has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence.

9. The engineered polypeptide according to any one of claims 1 to 8, comprising an amino acid sequence as shown in SEQ ID NO: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242 or 244, or a functional fragment thereof.

10. The engineered polypeptide according to any one of claims 1 to 9, wherein the engineered polypeptide has imine reductase activity; Preferably, the engineered polypeptide has improved activity and / or stereoselectivity; Preferably, the engineered polypeptide converts the substrate to a chiral amine with greater than 50% ee, greater than 60% ee, greater than 70% ee, greater than 80% ee, greater than 90% ee, greater than 95% ee, greater than 98% ee, greater than 99% ee, greater than 99.5% ee, greater than 99.6% ee, greater than 99.7% ee, greater than 99.8% ee or greater than 99.9% ee.

11. A fusion protein, comprising the engineered polypeptide according to any one of claims 1 to 10.

12. A polynucleotide encoding the engineered polypeptide according to any one of claims 1 to 10; Preferably, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241 or 243.

13. A nucleic acid construct comprising the polynucleotide of claim 12. 14 . An expression vector comprising the polynucleotide according to claim 12 or the nucleic acid construct according to claim 12 . 15 . A host cell comprising the polynucleotide of claim 12 , the nucleic acid construct of claim 13 , or the expression vector of claim 14 .

16. A composition comprising the engineered polypeptide of any one of claims 1-10, the fusion protein of claim 11, the polynucleotide of claim 12, the nucleic acid construct of claim 13, the expression vector of claim 14 or the host cell of claim 15.

17. A method for preparing the engineered polypeptide according to any one of claims 1 to 10, comprising culturing the host cell according to claim 15 under conditions that allow expression of the engineered polypeptide, and optionally recovering the engineered polypeptide from the culture of the cultivated host cell.

18. A method for asymmetric catalytic synthesis of chiral amines (I), the method comprising reacting a ketone substrate (II) and an amine substrate (III) in the presence of the polypeptide according to any one of claims 1 to 10, the fusion protein according to claim 11 or the composition according to claim 12, and a cofactor; in, R1 is selected from C 1-6 Alkyl; R2 is selected from aryl; R3 is selected from C 1-6 Alkyl, substituted or unsubstituted aryl-C 1-6 Preferably, R1 is selected from C 1-4 Alkyl; R2 is phenyl or naphthyl; R3 is selected from C 1-6 Alkyl, unsubstituted or halogen or halogenated C 1-4 Alkyl substituted phenyl-C 1-4 Alkylene; preferably, R1 is methyl; R2 is phenyl or naphthyl; R3 is selected from n-butyl, isopentyl, benzyl, phenylpropyl or trifluoromethylphenylpropyl.

19. The method of claim 18, wherein the reaction is carried out under one or more of the following conditions: (1) The concentration of the ketone substrate (II) or the amine substrate (III) is 1 to 100 g / L; preferably, the concentration ratio of the ketone substrate (II) to the amine substrate (III) is 1:(0.05 to 20); (2) The reaction is carried out in an organic solvent; preferably, the organic solvent is DMSO; (3) The pH of the reaction system is 5 to 10; (4) The cofactor is NAD(P)H / NAD(P); preferably, the concentration of the cofactor is 0.1-2 g / L; (5) The reaction temperature is 20-25°C, 20-30°C, 20-40°C or 20-50°C.

20. The method of claim 18 or 19, wherein the chiral amine is (R)-Cinacalcet; Preferably, the optical purity of (R)-Cinacalcet is greater than 10% ee, greater than 20% ee, greater than 50% ee, greater than 60% ee, greater than 70% ee, greater than 80% ee, greater than 90% ee, greater than 95% ee, greater than 98% ee, greater than 99% ee, greater than 99.5% ee, greater than 99.6% ee, greater than 99.7% ee, greater than 99.8% ee or greater than 99.9% ee.

21. Use of the engineered polypeptide according to any one of claims 1 to 10, the fusion protein according to claim 11, the polynucleotide according to claim 12, the nucleic acid construct according to claim 13, the expression vector according to claim 14, the host cell according to claim 15, the composition according to claim 16, or the method according to claim 18 or 19 in the asymmetric catalytic synthesis of chiral amines, particularly (R)-cinacalcet.

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