Application of carbonyl reductase and mutant thereof in preparation of atomoxetine chiral intermediate

Abstract

The invention discloses carbonyl reductase and application of a mutant thereof in preparation of a atomoxetine chiral intermediate. The carbonyl reductase and the mutant thereof have a sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 33. According to the carbonyl reductase disclosed by the invention, the reaction conversion rate of the carbonyl reductase for generating (S)-3-chlorobenzene propanol from 3-chloropropiophenone reaches 99%, the optical purity is greater than 99%, and the carbonyl reductase shows excellent catalytic effect and conversion efficiency. The method based on the enzyme and the mutant is mild in reaction condition and environmentally friendly, has high regioselectivity and stereoselectivity, avoids chiral resolution and heavy metal residues in products, makes up for the defects of chemical methods, and has wide application prospects and high market value.

Description

Technical Field

[0001] The present invention relates to the field of biosynthesis technology, and particularly to the application of carbonyl reductase and its mutants in the preparation of tomoxetine chiral intermediates. Background Art

[0002] Tomoxetine is a selective norepinephrine reuptake inhibitor (SNRI), which can increase the level of norepinephrine in the brain, thereby improving the attention, impulse control, and behavioral regulation ability of patients with attention deficit hyperactivity disorder (ADHD). Different from traditional stimulant drugs (such as methylphenidate), tomoxetine is not addictive and is therefore more suitable for long-term use. In clinical applications, tomoxetine is mainly used for the treatment of ADHD in children, adolescents, and adults. Other studies have also shown that tomoxetine may have certain therapeutic effects on depression, anxiety, and neuropathic pain.

[0003] The chemical structure of tomoxetine contains a chiral center, and the main pharmacologically active ingredient is the (S)-enantiomer. Therefore, in the process of synthesizing tomoxetine, it is necessary to efficiently prepare its chiral intermediate, and (S)-3-chlorophenylpropanol is one of the key intermediates. The traditional chemical synthesis route of tomoxetine usually uses 3-chloropropiophenone or 3-hydroxy-3-phenylpropionitrile as the starting material, introduces a chiral center through chemical reduction, chiral resolution, or asymmetric synthesis, and finally obtains tomoxetine. Generally, 3-chloropropiophenone is first reduced to 3-chlorophenylpropanol by a chemical reducing agent (such as sodium borohydride), and then the racemic 3-chlorophenylpropanol is resolved using a chiral catalyst or resolving agent (such as tartaric acid) to obtain S-3-chlorophenylpropanol. However, the chiral control of this synthesis route is difficult, and the traditional chemical reduction and resolution methods are less efficient and also require expensive chiral catalysts or resolving agents. In addition, traditional methods often cause serious environmental pollution. The chemical reducing agents (such as sodium borohydride) and organic solvents used generate a large amount of waste, which does not conform to the principles of green chemistry, and the cost is difficult to control. The yield of the chiral resolution step is low, resulting in waste of raw materials and invisibly increasing the production cost.

[0004] The currently commonly used representative synthesis route of tomoxetine hydrochloride is as follows:

[0005] 1. Wang Ling et al. used 3-chloropropiophenone as the starting material, and after reduction with NaBH4, Mitsunobu reaction, methylation with methylamine, and resolution with mandelic acid, tomoxetine hydrochloride was obtained after salification with hydrochloric acid.

[0006]

[0007] 2. Kumar et al. used ethyl 3-oxo-3-phenylpropionate as the starting material. After chiral reduction of the carbonyl group by Baker's yeast, reaction with methylamine, reduction with LiAlH4, Boc protection, and reaction with o-cresol via the Mitsunobu reaction, the product tomoxetine hydrochloride was obtained by acidification with hydrochloric acid. However, the biocatalytic stereoselectivity of this method is not high, and the e.e. value of the final product is only 70%. Moreover, there are processes of Boc protection and deprotection in the reaction route, and the reaction steps are relatively long.

[0008]

[0009] 3. Vogl et al. used 3-chloropropiophenone as the starting material. After selective reduction of the carbonyl group by a carbonyl reductase (Candida tenuis xylosereductase, CtXR), the Mitsunobu reaction, and then amination reaction with methylamine, tomoxetine was obtained. In this method, the carbonyl reductase CtXR was used to catalytically reduce (S)-3-chlorophenylpropanol, but its substrate concentration was only 2 mM, the chiral purity was 98%, and the space-time yield of the product was low, which was not suitable for industrial production.

[0010]

[0011] The existing industrial preparation methods of tomoxetine mainly adopt traditional resolution methods, but the theoretical yield limit of this method is only 50%. In the existing biological preparation methods, due to problems such as low catalytic activity of carbonyl reductase, low substrate concentration, and low chiral purity of the product, it is difficult to meet the requirements of industrial production, especially unable to meet the quality control requirements of high optical purity APIs. Therefore, there is an urgent need for a chemical-enzyme combined method for the efficient industrial preparation of tomoxetine. Summary of the Invention

[0012] The present invention aims to at least solve one of the above technical problems in the prior art. For this reason, the object of the present invention is to provide the application of a carbonyl reductase and its mutants in the preparation of a tomoxetine chiral intermediate. In the present invention, a variety of carbonyl reductases and their mutants have been developed, which can effectively be used as catalytic enzymes for the synthesis of tomoxetine chiral intermediates. Based on the carbonyl reductase and its mutants, the reaction conditions for the synthesis of tomoxetine chiral intermediates are mild, environmentally friendly, have high regioselectivity and stereoselectivity, and avoid chiral resolution and heavy metal residues in the product, overcoming the deficiencies of chemical methods. Moreover, this method has a high conversion rate, the obtained intermediate has a high chiral purity, and the enzyme dosage is small, and the preparation cost is low, with extremely high practical value.

[0013] In the first aspect of the present invention, there is provided a carbonyl reductase or its mutant, and the carbonyl reductase includes an active fragment and an optional tag sequence.

[0014] In some embodiments of the present invention, the active fragment has an amino acid sequence with the following Genebank numbers:

[0015] AHC30845.1, ABP38340.1, ADM32128.1, 2FR0_A, XP_719854.1, 100034242, AJ544275.1, 6TQ8_A, RAW08233.1, and WP_003056551.1.

[0016] In some embodiments of the present invention, the tag sequence includes a Flag tag, a GST tag, a His tag, an HA tag, a C-Myc tag, and mCherry.

[0017] In some embodiments of the present invention, the tag sequence is a His6 tag.

[0018] In some embodiments of the present invention, the carbonyl reductase has the sequence shown in SEQ ID NO: 15.

[0019] In some embodiments of the present invention, the carbonyl reductase is the sequence shown in SEQ ID NO: 15.

[0020] In some embodiments of the present invention, the carbonyl reductase mutants include:

[0021] Based on the carbonyl reductase sequence described in the above aspect, mutants that retain catalytic activity after 1-5 amino acid substitutions, deletions, or additions.

[0022] In the present invention, the phrase "mutant that retains catalytic activity" refers to a mutant that retains the catalytic reduction activity of the carbonyl group.

[0023] In some embodiments of the present invention, the catalytic reduction reaction of the carbonyl group includes the reduction reaction of aldehyde groups, the reduction reaction of ketone groups, and the dehydrogenation reaction of alcohols.

[0024] In some embodiments of the present invention, "mutant that retains catalytic activity" refers to a mutant that retains the activity of catalytically converting 3-chloropropiophenone into the tomoxetine intermediate.

[0025] In some embodiments of the present invention, the tomoxetine intermediate is a chiral intermediate. In some embodiments of the present invention, the tomoxetine intermediate is (S)-3-chlorophenylpropanol.

[0026] In some embodiments of the present invention, the conversion rate of the mutant to 3-chloropropiophenone is greater than or equal to 40%.

[0027] In some embodiments of the present invention, the conversion rate of the mutant to 3-chloropropiophenone is greater than or equal to 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0028] In some embodiments of the present invention, the carbonyl reductase mutant is a mutant that retains catalytic activity after 1-3 amino acid substitutions, deletions or additions based on the carbonyl reductase sequence described in the above aspect.

[0029] In some embodiments of the present invention, the carbonyl reductase mutant is a mutant that retains catalytic activity after 1-3 amino acid substitutions, deletions or additions based on SEQ ID NO: 15.

[0030] In some embodiments of the present invention, the carbonyl reductase mutant is a mutant that retains catalytic activity after 1-3 amino acid substitutions based on the carbonyl reductase sequence described in the above aspect.

[0031] In some embodiments of the present invention, the carbonyl reductase mutant is a mutant that retains catalytic activity after 1-3 amino acid substitutions based on SEQ ID NO: 15.

[0032] In some embodiments of the present invention, the amino acid substitution of the carbonyl reductase mutant occurs at least at one position among valine (V) at position 48, phenylalanine (F) at position 76, asparagine (N) at position 129, tyrosine (Y) at position 146, tyrosine (Y) at position 179, and valine (V) at position 237 of SEQ ID NO: 15.

[0033] In some embodiments of the present invention, the amino acid substitution of the carbonyl reductase mutant occurs at 1-3 positions among valine (V) at position 48, phenylalanine (F) at position 76, asparagine (N) at position 129, tyrosine (Y) at position 146, tyrosine (Y) at position 179, and valine (V) at position 237 of SEQ ID NO: 15.

[0034] In some embodiments of the present invention, the amino acid substitution is substituting the amino acid at the above position with alanine (A).

[0035] In some embodiments of the present invention, when the carbonyl reductase mutant has an amino acid substitution at position 1 in SEQ ID NO: 15, it includes: valine (V) at position 48 is replaced with alanine (A), phenylalanine (F) at position 76 is replaced with alanine (A), asparagine (N) at position 129 is replaced with alanine (A), tyrosine (Y) at position 146 is replaced with alanine (A), tyrosine (Y) at position 179 is replaced with alanine (A), and valine (V) at position 237 is replaced with alanine (A).

[0036] In some embodiments of the present invention, when the carbonyl reductase mutant has amino acid substitutions at 2 positions in SEQ ID NO: 15, it includes: valine (V) at position 48 is replaced with alanine (A) and phenylalanine (F) at position 76 is replaced with alanine (A); valine (V) at position 48 is replaced with alanine (A) and asparagine (N) at position 129 is replaced with alanine (A); valine (V) at position 48 is replaced with alanine (A) and tyrosine (Y) at position 146 is replaced with alanine (A); valine (V) at position 48 is replaced with alanine (A) and tyrosine (Y) at position 179 is replaced with alanine (A); valine (V) at position 48 is replaced with alanine (A) and valine (V) at position 237 is replaced with alanine (A); phenylalanine (F) at position 76 is replaced with alanine (A) and asparagine (N) at position 129 is replaced with alanine (A); phenylalanine (F) at position 76 is replaced with alanine (A) and tyrosine (Y) at position 146 is replaced with alanine (A); phenylalanine (F) at position 76 is replaced with alanine (A) and tyrosine (Y) at position 179 is replaced with alanine (A); phenylalanine (F) at position 76 is replaced with alanine (A) and valine (V) at position 237 is replaced with alanine (A); asparagine (N) at position 129 is replaced with alanine (A) and tyrosine (Y) at position 146 is replaced with alanine (A); asparagine (N) at position 129 is replaced with alanine (A) and tyrosine (Y) at position 179 is replaced with alanine (A); asparagine (N) at position 129 is replaced with alanine (A) and valine (V) at position 237 is replaced with alanine (A); tyrosine (Y) at position 146 is replaced with alanine (A) and tyrosine (Y) at position 179 is replaced with alanine (A); tyrosine (Y) at position 146 is replaced with alanine (A) and valine (V) at position 237 is replaced with alanine (A); tyrosine (Y) at position 179 is replaced with alanine (A) and valine (V) at position 237 is replaced with alanine (A).

[0037] In some embodiments of the present invention, when the carbonyl reductase mutant has amino acid substitutions at three positions in SEQ ID NO: 15, it includes: asparagine (N) at position 129 is replaced by alanine (A), tyrosine (Y) at position 146 is replaced by alanine (A), and tyrosine (Y) at position 179 is replaced by alanine (A).

[0038] In some embodiments of the present invention, when the carbonyl reductase mutant has amino acid substitutions at three positions in SEQ ID NO: 15, it includes: a carbonyl reductase mutant having the sequence shown in SEQ ID NO: 14.

[0039] In some embodiments of the present invention, the carbonyl reductase or its mutant is further modified, and the modifications include but are not limited to: glycosylation, phosphorylation, acetylation, methylation, ubiquitination or lipidation; or introduction of non-natural amino acids (such as α-aminoketone or β-aminoketone).

[0040] The second aspect of the present invention provides a biological product, which includes:

[0041] (1) A nucleic acid molecule encoding the carbonyl reductase or its mutant described in the above aspect;

[0042] (2) An expression vector containing the nucleic acid molecule described in (1);

[0043] (3) A transformant containing the nucleic acid molecule described in (1);

[0044] (4) A transformant containing the expression vector described in (2);

[0045] In some embodiments of the present invention, the expression vectors include viruses, plasmids and phages.

[0046] In the present invention, the term "expression vector" refers to a vector or expression system for integrating or inserting a foreign gene of interest.

[0047] In some embodiments of the present invention, the transformants include bacteria, fungi and animal and plant cells.

[0048] In the present invention, the term "transformant" refers to a recipient cell that has acquired a new genetic marker after incorporating or introducing a foreign gene.

[0049] In some embodiments of the present invention, the transformants do not involve animal and plant reproductive materials.

[0050] In some embodiments of the present invention, the construction of the expression vectors and transformants can be achieved based on any conventional technical means in the art.

[0051] The third aspect of the present invention provides the application of the carbonyl reductase or its mutant, or biological product described in the above aspect in an enzymatic reaction.

[0052] In some embodiments of the present invention, the enzymatic reaction includes the catalytic reduction reaction of a carbonyl group.

[0053] In some embodiments of the present invention, the catalytic reduction reaction of the carbonyl group includes the reduction reaction of an aldehyde group, the reduction reaction of a ketone group, and the dehydrogenation reaction of an alcohol.

[0054] The fourth aspect of the present invention provides the application of the carbonyl reductase or its mutant, or biological product described in the above aspect in the preparation of tomoxetine intermediate and / or tomoxetine.

[0055] In some embodiments of the present invention, the tomoxetine intermediate is a chiral intermediate.

[0056] In some embodiments of the present invention, the tomoxetine intermediate is (S)-3-chlorophenylpropanol.

[0057] The fourth aspect of the present invention provides a method for preparing a tomoxetine intermediate, comprising the following steps:

[0058] Using 3-chloropropiophenone as a substrate, adding the carbonyl reductase or its mutant, or biological product described above, then adding formate dehydrogenase or a biological material capable of expressing, secreting or producing formate dehydrogenase, ammonium formate and a coenzyme, and reacting at 25-35 °C for 2-24 h to obtain the tomoxetine intermediate.

[0059] In some embodiments of the present invention, the tomoxetine intermediate is (S)-3-chlorophenylpropanol.

[0060] In some embodiments of the present invention, the substrate further contains a solvent and / or a cosolvent.

[0061] In some embodiments of the present invention, the solvent and / or cosolvent includes at least one of ethyl acetate, butyl acetate, isobutyl acetate, dibutyl phthalate, ethyl laurate, dimethyl sulfoxide (DMSO), methyl tert-butyl ether, dimethyl phthalate and methanol.

[0062] In some embodiments of the present invention, the solvent and / or cosolvent is selected from at least one of dibutyl phthalate, ethyl laurate, methyl tert-butyl ether and dimethyl phthalate.

[0063] In some embodiments of the present invention, the solvent and / or cosolvent is dimethyl phthalate.

[0064] In some embodiments of the present invention, the coenzyme includes at least one of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide (NADH), and nicotinamide adenine dinucleotide (NAD+).

[0065] In some embodiments of the present invention, the coenzyme includes at least one of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), and nicotinamide adenine dinucleotide (NAD+).

[0066] In some embodiments of the present invention, a buffer solution is further added to the method.

[0067] In some embodiments of the present invention, the buffer solution includes at least one of PB buffer solution, Tris-HCl buffer solution, and TEA buffer solution.

[0068] In some embodiments of the present invention, the buffer solution is at least one of PB buffer solution or TEA buffer solution.

[0069] In some embodiments of the present invention, the pH of the buffer solution is 6-8.

[0070] In some embodiments of the present invention, the pH of the buffer solution is 6-7.5.

[0071] In some embodiments of the present invention, the concentration of the TEA buffer solution is 0.05-0.20 M, and the pH is 6-8; the concentration of the PB buffer solution is 0.05-0.10 M, and the pH is 6-7; the concentration of the Tris-HCl buffer solution is 0.05-0.10 M, and the pH value is 7-8.

[0072] In some embodiments of the present invention, the concentration of the carbonyl reductase or its mutant, or the biological product in the system is 10-200 g / L.

[0073] In some embodiments of the present invention, the concentration of the carbonyl reductase or its mutant, or the biological product in the system is 50-200 g / L.

[0074] In some embodiments of the present invention, the concentration of the formate dehydrogenase or the biological material capable of expressing, secreting, or producing formate dehydrogenase in the system is 10-200 g / L.

[0075] In some embodiments of the present invention, the concentration of the formate dehydrogenase or the biological material capable of expressing, secreting, or producing formate dehydrogenase in the system is 25-200 g / L.

[0076] In some embodiments of the present invention, the concentration of ammonium formate in the system is 0.2 - 1 M.

[0077] In some embodiments of the present invention, the concentration of ammonium formate in the system is 0.7 - 1 M.

[0078] In some embodiments of the present invention, the concentration of coenzyme in the system is 0.1 - 2.5 mM.

[0079] In some embodiments of the present invention, the concentration of coenzyme in the system is 1 - 2.5 mM.

[0080] In some embodiments of the present invention, the volume dosage of the solvent and / or cosolvent in the system is 5 - 50% (v / v).

[0081] In some embodiments of the present invention, the volume dosage of the solvent and / or cosolvent in the system is 10 - 50% (v / v).

[0082] In some embodiments of the present invention, the method further includes extracting and purifying the product.

[0083] In some embodiments of the present invention, the method specifically is: adding carbonyl reductase or its mutant, or biological product, formate dehydrogenase or biological material with the ability to express, secrete or produce formate dehydrogenase, buffer, substrate, cosolvent, ammonium formate and coenzyme into a reaction vessel, reacting at 25 - 35 °C for 2 - 24 h, and then obtaining the tomoxetine chiral intermediate after extraction, separation and rotary evaporation.

[0084] In some embodiments of the present invention, based on a 1 L reaction system, it contains 10 - 200 g of wet recombinant bacteria expressing carbonyl reductase or its mutant, 10 - 200 g of wet recombinant bacteria expressing formate dehydrogenase, 20 - 100 g of substrate, 50 - 500 mL of cosolvent, 0.2 - 1 moL of ammonium formate, 0.1 - 2.5 mmol of coenzyme, and the balance is buffer.

[0085] Among them, the wet recombinant bacteria expressing formate dehydrogenase, ammonium formate and coenzyme together construct a coenzyme recycling system.

[0086] In some embodiments of the present invention, based on a 1 L reaction system, it contains 10 - 200 g of wet recombinant bacteria expressing carbonyl reductase or its mutant, 25 - 75 g of wet recombinant bacteria expressing formate dehydrogenase, 20 - 100 g of substrate, 50 - 500 mL of cosolvent, 0.2 - 1 moL of ammonium formate, 0.1 - 2.5 mmol of coenzyme, and the balance is buffer.

[0087] In the present invention, the carbonyl reductase or its mutant can be in the form of a free enzyme (for example, used in the form of enzyme powder), or in the form of cells expressing the carbonyl reductase or its mutant (for example, wet bacterial cells). Or in other forms, such as the cell lysate supernatant expressing the carbonyl reductase or its mutant or whole-cell immobilization, or the immobilization of free enzyme powder.

[0088] In some embodiments of the present invention, the host microorganism for expressing the enzyme protein can be selected as Escherichia coli BL21(DE3), and the recombinant plasmid can be selected as the pET28 plasmid, such as pET28a+. All the molecular biology operations involved in the operation steps are conventional experimental operation methods well-known in the biological field, including gene acquisition (PCR), splicing of the plasmid and the target gene (i.e., vector construction), introduction of the plasmid containing the target gene fragment into the bacterial cells (i.e., transformation), cultivation of the bacterial cells in the medium and enzyme production (i.e., fermentation).

[0089] The fifth aspect of the present invention provides a method for preparing tomoxetine, comprising: preparing the tomoxetine intermediate using the method described in the above aspect, and then preparing tomoxetine.

[0090] The beneficial effects of the present invention are as follows:

[0091] 1. In the present invention, a variety of carbonyl reductases or their mutants have been developed, which can catalyze 3-chloropropiophenone to generate (S)-3-chlorophenylpropanol. Among them, for the reaction of 3-chloropropiophenone to generate (S)-3-chlorophenylpropanol, the conversion rate reaches 99%, and the optical purity is greater than 99%, showing excellent catalytic effects and conversion efficiency.

[0092] 2. The present invention uses a biocatalytic method to prepare the tomoxetine intermediate. It not only has mild reaction conditions, is environmentally friendly, has high regioselectivity and stereoselectivity, but also avoids chiral resolution and heavy metal residues in the product, making up for the deficiencies of chemical methods. Moreover, this method can obtain the target product with only one-step reaction, uses fewer reagents, has high catalytic activity, reduces production costs, and has broad application prospects and considerable market value. Description of the Drawings

[0093] Figure 1 is the plasmid map of the carbonyl reductase plasmid vector in the embodiment of the present invention.

[0094] Figure 2 is the technical roadmap of the present invention.

[0095] Figure 3 is the HPLC detection chart of 3-chloropropiophenone (substrate).

[0096] Figure 4It is the HPLC detection chart of 3-chlorophenylpropanol (product). Detailed implementation manners

[0097] The content of the present invention will be further described in detail through specific embodiments below. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are all conventional methods in the art.

[0098] In the following embodiments, the molecular biology operations involved are all conventional experimental operation methods well-known in the biological field, including gene acquisition (PCR), splicing of plasmid and target gene (i.e., vector construction), introduction of plasmid containing target gene fragment into bacterial cells (i.e., transformation), cultivation of bacteria in medium and enzyme production (i.e., fermentation).

[0099] In the following embodiments, the preparation method of the LB liquid medium used is as follows: calculated by the final concentration, 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, fully dissolved with water as a solvent and then sterilized at high temperature to obtain it.

[0100] The preparation method of the LB solid medium is: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L agar, fully dissolved with water as a solvent and then sterilized at high temperature to obtain it.

[0101] Example 1

[0102] In this example, a recombinant bacterial strain expressing carbonyl reductase was prepared. The specific preparation method is as follows:

[0103] (1) Preparation of carbonyl reductase plasmid vector:

[0104] Using the commercial plasmid pET28a+ (purchased from Tsingke), according to the conventional operations in the art, the corresponding target gene fragment (carbonyl reductase nucleotide sequence) was inserted into pET28a+ to obtain the carbonyl reductase plasmid vector (the plasmid map is as Figure 1 shown).

[0105] Among them, the target gene fragment was synthesized by Shanghai Sangon. The specific sequence information is as follows.

[0106] The amino acid sequence of AKR26 refers to Genebank accession number AHC30845.1. On the basis of this sequence, a His tag (His6, HHHHHH, SEQ ID NO: 1) was added at the C-terminus.

[0107] The amino acid sequence of AKR27 refers to Genebank accession number ABP38340.1. On the basis of this sequence, a His tag was added at the C-terminus.

[0108] The amino acid sequence of AKR28 is shown in Genebank accession number ADM32128.1, and a His tag is added to the C-terminus based on this sequence.

[0109] The amino acid sequence of AKR29 is shown in Genebank accession number 2FR0_A, and a His tag is added to the C-terminus based on this sequence.

[0110] The amino acid sequence of AKR30 is shown in Genebank accession number XP_719854.1, and a His tag is added to the C-terminus based on this sequence.

[0111] The amino acid sequence of ADH14 is shown in Genebank accession number 100034242, and a His tag is added to the C-terminus based on this sequence.

[0112] The amino acid sequence of ADH16 is shown in Genebank accession number AJ544275.1, and a His tag is added to the C-terminus based on this sequence.

[0113] The amino acid sequence of ADH19 is shown in Genebank accession number 6TQ8_A, and a His tag is added to the C-terminus based on this sequence.

[0114] The amino acid sequence of ADH21 is shown in Genebank accession number RAW08233.1, and a His tag is added to the C-terminus based on this sequence. Specifically: MGFTSLPETVPTTMSAMLLTGHGDVDKLVYRQDVATPRPAAGEVLV KVTATAKNNTDRKAREGLYPTKDKGEVASFAMGGSPTLTFPRIQGADVVGQVVAVGDGVDASRLGERGLLDFNLYADERRDINLTPDYYGHGADGGYAEYIAVPSDQFHHVPNPELSDAELAAMGMCSYQTAYHMMTSAGIKAGERVLVTGASGGVGTALIQLCRIVGAVPYALSQRDKAQALLNLGAEAVLDRSDMSDFTEQVKAVTDGAPIDAVMDLVGGEMTDRFIDTMIFDMNARASYPRLSIAGASGGNVSEIMWTRIYLYQVQIFGVSHGTREEAEQLVAWIRDGQLKPVLHAAFKLSELHEAERYFVNRGSNYLGKIVIVPDSEWAQHGAPHALEGHQHHHHHH (SEQ ID NO: 15).

[0115] The amino acid sequence of ADH25 is shown in Genebank accession number WP_003056551.1, and a His tag is added to the C-terminus based on this sequence.

[0116] Of course, according to the need, no additional tag sequence can be introduced.

[0117] (2) Obtaining recombinant bacteria expressing carbonyl reductase:

[0118] Escherichia coli BL21(DE3) is used as a transformant for the expression of the enzyme protein. The specific steps are as follows: The plasmid vectors constructed in the above steps are respectively transformed into Escherichia coli cells, and then spread on an LB plate medium supplemented with 50 mg / L kanamycin and cultured at 37 °C for 12 hours. Then, single colonies are picked and transferred into an LB liquid medium supplemented with kanamycin to a final concentration of 50 mg / L and cultured at 37 °C for 12 hours. Then, plasmids are extracted from the screened cells and transfected into competent cells of Escherichia coli BL21(DE3), and spread on an LB plate medium containing 20 mg / L chloramphenicol and cultured at 37 °C for 12 hours to obtain recombinant bacteria expressing carbonyl reductase (AKR26, AKR27, AKR28, AKR29, AKR30, ADH14, ADH16, ADH19, ADH21, ADH25).

[0119] Example 2

[0120] (1) The recombinant bacteria expressing carbonyl reductase obtained in Example 1 are inoculated into 10 mL of LB liquid medium (inoculation amount 1%), and cultured at 37 °C and 180 rpm for 8 - 10 h. Then, the cultured bacterial solution is inoculated into 100 mL of LB culture medium at an inoculation amount of 1% (v / v) and cultured at 37 °C and 180 rpm for 2 - 2.5 h until the OD600 value is 0.6 - 0.8. Then, IPTG with a final concentration of 0.1 mM is added, and induced culture is carried out at 24 °C and 180 rpm for 12 h. The cultured bacterial solution is placed in a 500 mL centrifuge cup, centrifuged at 8000 rpm and 4 °C at a high speed for 10 min, the supernatant is discarded, and the cells are collected and weighed.

[0121] (2) Take 0.15 g of the wet cells of the recombinant bacteria expressing carbonyl reductase collected respectively, add them into 2 mL EP tubes, and set a blank control group. Add 0.15 g of the wet cells of formate dehydrogenase to each EP tube, and then add 700 μL of PB buffer (0.1 M, pH 7), 100 μL of 7 M ammonium formate solution, 100 μL of 25 mM NADP+ solution, and 100 μL of the methyl tert-butyl ether solution of 3-chloropropiophenone (the final concentration of 3-chloropropiophenone in the system is 50 g / L). Place the EP tubes in a thermostatic mixer and shake and react at 30 °C and 1200 rpm for 4 h. The technical route diagram is as Figure 2 shown.

[0122] Among them, the preparation method of the wet cells of formate dehydrogenase is the same as that of the wet cells of the recombinant bacteria expressing carbonyl reductase. Among them, the plasmid vector of formate dehydrogenase is also constructed based on the commercial plasmid pET28a+. Among them, the inserted target gene fragment is the formate dehydrogenase gene sequence with GenBank number AXT18256.1. The synthesis of the plasmid was completed by Sangon Biotech (Shanghai) Co., Ltd. The transformant is also Escherichia coli BL21(DE3), and the screening method is the same as that of the recombinant bacteria of carbonyl reductase.

[0123] (3) After the reaction is completed, take 200 μL of the reaction solution from each of the EP tubes in different groups, add 200 μL of ethyl acetate, shake and mix well for 2 min, centrifuge at 12000 rpm for 1 min, take the supernatant, and perform HPLC detection to confirm the conversion rate.

[0124] Among them, the calculation formula of the conversion rate is as follows:

[0125]

[0126] Among them, the substrate refers to 3-chloropropiophenone, and the product is 3-chloropropanol.

[0127] The HPLC detection results are as Figures 3 - 4 and Table 1 show.

[0128] Table 1 Conversion rates and e.e. values of carbonyl reductase in each group

[0129] Carbonyl reductase Conversion rate e.e. value Carbonyl reductase Conversion rate e.e. value AKR26 47.65％ S form, 55.64% ADH14 80.21％ R form, 68.98% AKR27 2.49％ R form, 68.42% ADH16 15.83％ S form, 79.88% AKR28 52.34％ R form, 76.88% ADH19 30.43％ R form, 88.48% AKR29 46.98％ S form, 46.24% ADH21 90.45％ S form, 80.36% AKR30 90.86％ S form, 10.48% ADH25 65.33％ S form, 70.44%

[0130] The results show that the conversion rates of carbonyl reductases AKR30 and ADH21 are the highest. Among them, the chirality of carbonyl reductase ADH21 is better, which is of S type and the e.e. value is 80.36%.

[0131] Example 3

[0132] Based on the results of the above examples, taking ADH21 as the original template, prepare carbonyl reductase mutants, and the specific steps are as follows.

[0133] (1) Construct a single mutant library:

[0134] Take the recombinant strain of carbonyl reductase ADH21 prepared in the above example. After activation, extract the plasmid (pET28a(+)-ADH21) from it. Using pET28a(+)-ADH21 as a template, design alanine scanning primers (as shown in Table 2), and perform polymerase chain reaction (PCR). Among them, the PCR amplification system (25 μL) is shown in Table 3 to obtain the amplification product. Then, after digestion with DpnⅠ, transfer the recombinant plasmid into Escherichia coli BL21(DE3) competent cells, and inoculate the clones into 10 mL of LB plate medium and culture at 37 °C for 12 - 16 h.

[0135] Table 2 Alanine scanning primers

[0136]

[0137]

[0138] Table 3 PCR amplification system (25 μL)

[0139] Component Content Forward primer (100 μM) 1 μL Reverse primer (100 μM) 1 μL 2×Phanta buffer (Novoprotein) 12.5 μL dNTP mixture (each 10 mM) 0.5 μL Plasmid template 1 μL DNA polymerase Phanta (Novoprotein) 0.5 μL Ultra-pure water 8.5 μL Total volume 25 μL

[0140] The PCR amplification procedure refers to the instruction manual of Phanta Super-Fidelity DNA polymerase. Pre-denature at 95 °C for 5 min; denature at 95 °C for 15 s, anneal at 55 °C for 15 s, extend at 72 °C for 4 min, and perform 30 cycles; then perform final extension at 72 °C for 10 min and keep at 16 °C for insulation.

[0141] Then randomly select positive clones and the original strain on the plate, inoculate them into 10 mL of LB liquid medium (added with 50 mg / L kanamycin), and culture at 37 °C and 200 rpm in a temperature-controlled shaker for 8 - 10 h. Inoculate the culture solution into 100 mL of LB liquid medium (added with 50 mg / L kanamycin) at an inoculation amount of 1%, and culture at 37 °C and 180 rpm in a temperature-controlled shaker for 2 - 2.5 h (OD600 value is 0.6 - 0.8). Then add the inducer IPTG with a final concentration of 0.1 mM, and continue to culture at 24 °C and 180 rpm in a temperature-controlled shaker for 12 h. Then place the cultured bacterial solution in an 800 mL centrifuge cup, centrifuge at 8000 rpm and 4 °C for 10 min in a low-temperature high-speed centrifuge, discard the supernatant, collect the bacterial cells and weigh them.

[0142] Use the method in the above example to test the conversion effect of each mutant on 3-chloropropiophenone, and screen mutant strains with conversion rate and chirality as indicators.

[0143] The screened mutant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored in a -80°C refrigerator.

[0144] (2) Iterative mutation (construction of multi-mutation mutants):

[0145] Plasmids were extracted from the recombinant strains of ADH21-V48A, ADH21-F76A, ADH21-N129A, ADH21-Y146A, ADH21-Y179A, and ADH21-V237A screened in the above steps. Using the extracted plasmids as templates, PCR amplification was performed with the alanine scanning primers shown in Table 2 (the system is the same as Table 3), and recombinant strains were constructed and screened according to the method in the above steps.

[0146] Finally, double-mutation mutants ADH21-V48A / F76A, ADH21-V48A / N129A, ADH21-V48A / Y146A, ADH21-V48A / Y179A, ADH21-V48A / V237A, ADH21-F76A / N129A, ADH21-F76A / Y146A, ADH21-F76A / Y179A, ADH21-F76A / V237A, ADH21-N129A / Y146A, ADH21-N129A / Y179A, ADH21-N129A / V237A, ADH21-Y146A / Y179A, ADH21-Y146A / V237A, and ADH21-Y179A / V237A were obtained.

[0147] Using the same method, triple-mutation mutants were constructed, and the triple-mutation mutant ADH21-N129A / Y146A / Y179A was obtained.

[0148] Among them, the amino acid sequence of the triple-mutation mutant ADH21-N129A / Y146A / Y179A is shown as follows:

[0149] MGFTSLPETVPTTMSAMLLTGHGDVDKLVYRQDVATPRPAAGEVLVKVTATAKNNTDRKAREGLYPTKDKGEVASFAMGGSPTLTFPRIQGADVVGQVVAVGDGVDASRLGERGLLDFNLYADERRDIALTPDYYGHGADGGYAEAIAVPSDQFHHVPNPELSDAELAAMGMCSYQTAAHMMTSAGIKAGERVLVTGASGGVGTALIQLCRIVGAVPYALSQRDKAQALLNLGAEAVLDRSDMSDFTEQVKAVTDGAPIDAVMDLVGGEMTDRFIDTMIFDMNARASYPRLSIAGASGGNVSEIMWTRIYLYQVQIFGVSHGTREEAEQLVAWIRDGQLKPVLHAAFKLSELHEAERYFVNRGSNYLGKIVIVPDSEWAQHGAPHALEGHQHHHHHH (SEQ ID NO: 14).

[0150] (3) Catalytic activity detection:

[0151] Using 3-chloropropiophenone as the substrate, the single-mutation mutants, double-mutation mutants, and triple-mutation mutant strains prepared in the above steps were subjected to catalytic activity detection to compare the catalytic activities of each mutant. The specific steps were as follows:

[0152] The detection method was the same as that in the above example. Specifically, the reaction system was set to 1 mL, with 100 g / L of wet cell weight as the catalyst dosage, and the final substrate concentration was 50 g / L. The PB buffer solution with pH 7.0 and 100 mM was used as the reaction medium. The concentration of ammonium formate was 0.7 M, the concentration of formate dehydrogenase was 100 g / L, and the concentration of NADPH was 2.5 mM. Additionally, 0.1 mL of methyl tert-butyl ether was added as a co-solvent. The system was vortexed and reacted at 30 °C and 1200 rpm for 2 h. Then, 200 μL of the reaction solution was taken, 50 μL of 6 M hydrochloric acid was added to terminate the reaction, and then 200 μL of ethyl acetate was added for extraction twice. The ethyl acetate phases were combined, and the concentrations and chirality of 3-chloropropiophenone (substrate) and 3-chloropropanol (product) were detected by HPLC, and the conversion rate was calculated. ADH21 was used as a control.

[0153] The results are shown in Table 4.

[0154] Table 4 Conversion rates and e.e. values of mutants

[0155]

[0156] As can be seen from the results, among the single mutant mutants, ADH21-V48A, ADH21-F76A, ADH21-N129A, ADH21-Y146A, ADH21-Y179A, and ADH21-V237A have better conversion rates and chirality. Considering the effects of all mutants, ADH21-N129A / Y146A / Y179A has the best catalytic effect.

[0157] Example 4

[0158] In this example, the inventors used ADH21-N129A / Y146A / Y179A as the catalytic enzyme and used ethyl acetate, butyl acetate, isobutyl acetate, dibutyl phthalate, ethyl laurate, DMSO (dimethyl sulfoxide), methyl tert-butyl ether, dimethyl phthalate, methanol, etc. as co-solvents to screen the best co-solvent suitable for this catalytic enzyme.

[0159] The specific experimental steps are as follows:

[0160] Construct a reaction system according to the method in the above example. Specifically: 1 g each of wet cells of ADH21-N129A / Y146A / Y179A and wet cells of formate dehydrogenase, 6 mL of PB buffer (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP+ solution, and 2 mL of a co-solvent solution of 500 g / L 3-chloropropiophenone. Shake the reaction at 30 °C and 800 rpm.

[0161] Take 1 mL of the reaction solution at 2 h of the reaction and detect the conversion rate and e.e. value according to the HPLC detection method in the above example. Specifically, add an equal volume of ethyl acetate for extraction, centrifuge, and take the upper organic layer for HPLC detection.

[0162] The results are shown in Table 5.

[0163] Table 5 Conversion rates and e.e. values under different co-solvent conditions

[0164] Cosolvent Conversion rate e.e. value Ethyl acetate 45.87％ S form, 80.44% Butyl acetate 72.35％ S form, 92.38% Isobutyl acetate 76.98％ S form, 94.32% Dibutyl phthalate >99％ S form, 97.22% Ethyl laurate 96.88％ S form, 98.24% DMSO (Dimethyl sulfoxide) 89.99％ S form, 68.18% Methyl tert-butyl ether >99％ S form, 94.88% Dimethyl phthalate >99％ S form, 99.34% Methanol 78.35％ S form, 96.34%

[0165] The results show that when the co-solvent is dimethyl phthalate, the highest conversion rate is >99%, and its chirality is S-type with an e.e. value of 99.34%.

[0166] Example 5

[0167] In this example, the inventors used ADH21-N129A / Y146A / Y179A as the catalytic enzyme and tested the effects of different buffers and different pH values on the conversion rate and e.e. value to screen the best buffer and pH suitable for this catalytic enzyme.

[0168] The specific experimental steps are as follows:

[0169] Construct a reaction system according to the method in the above embodiment. Specifically, 1 g of each of ADH21-N129A / Y146A / Y179A wet cells and formate dehydrogenase wet cells, 6 mL of buffer solution (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP+ solution, and 2 mL of methyl tert-butyl ether solution of 500 g / L 3-chloropropiophenone. Shake and react at 30 °C and 800 rpm.

[0170] Take 1 mL of the reaction solution at 1 h of reaction, and detect the conversion rate and e.e. value according to the HPLC detection method in the above embodiment. Specifically, add an equal volume of ethyl acetate for extraction, centrifuge, and take the upper organic phase for HPLC detection.

[0171] The results are shown in Table 6.

[0172] Table 6 Conversion rate and e.e. value under different buffer conditions

[0173] Buffer Conversion rate e.e. value PB buffer at pH 7.0 51.24％ R form, 99.24% Tris-HCl buffer at pH 7.0 46.44％ R form, 99.20% TEA buffer at pH 7.0 57.37％ R form, 99.22%

[0174] The results show that the conversion rate is the highest when the buffer is TEA buffer with pH 7.0.

[0175] Using the same method and system, adjust the pH of the TEA buffer, and test the conversion rate and e.e. value at different pH values.

[0176] The results are shown in Table 7.

[0177] Table 7 Conversion rate and e.e. value under different pH conditions

[0178] Buffer Conversion rate TEA buffer at pH 6.0 87.34％ TEA buffer at pH 6.5 >99％ TEA buffer at pH 7.0 93.87％ TEA buffer at pH 7.5 81.43％ TEA buffer at pH 8.0 63.72％

[0179] The results show that when the buffer is TEA buffer with pH 6.5, the conversion rate is the highest, which is >99%.

[0180] Example 6

[0181] In this example, the inventors used ADH21-N129A / Y146A / Y179A as the catalytic enzyme, and respectively tested the effects of different concentrations of ADH21-N129A / Y146A / Y179A (wet cell concentration), formate dehydrogenase wet cell concentration, ammonium formate concentration, NADP+ or NAD+ concentration, and the proportion of co-solvent in the system (%, volume) on the conversion rate and e.e. value, in order to screen the relevant system parameters suitable for this catalytic enzyme.

[0182] The specific experimental steps are as follows:

[0183] Construct the reaction system according to the method in the above embodiment, specifically: 1 g of each of the wet cells of ADH21-N129A / Y146A / Y179A and the wet cells of formate dehydrogenase, 6 mL of PB buffer (0.1 M, pH 7), 1 mL of ammonium formate solution, 1 mL of NADP+ or NAD+ solution, and 2 mL of a methyl tert-butyl ether solution of 500 g / L 3-chloropropiophenone. Perform the oscillating reaction at 30 °C and 800 rpm.

[0184] Take 1 mL of the reaction solution at 6 h of the reaction, and detect the conversion rate and e.e. value according to the HPLC detection method in the above embodiment. Specifically, add an equal volume of ethyl acetate for extraction, centrifuge, and take the upper organic phase for HPLC detection.

[0185] The results are shown in Table 7.

[0186] Table 7 Conversion rates and e.e. values under different system parameter conditions

[0187]

[0188]

[0189] It can be found from the above results that high conversion rates and high e.e. values can be achieved under the conditions of relevant parameters (concentration or usage amount).

[0190] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A carbonyl reductase or its mutant, characterized in that, The carbonyl reductase includes an active fragment and an optional tag sequence; The amino acid sequence of the active fragment has the following Genebank numbers: AHC30845.1, ABP38340.1, ADM32128.1, 2FR0_A, XP_719854.1, 100034242, AJ544275.1, 6TQ8_A, RAW08233.1, and WP_003056551.1; The tag sequence includes a Flag tag, a GST tag, a His tag, an HA tag, a C-Myc tag, and mCherry; the carbonyl reductase mutant includes a mutant that retains catalytic activity after 1-5 amino acid substitutions, deletions, or additions based on the carbonyl reductase sequence; Preferably, the conversion rate of the mutant to 3-chloropropiophenone is greater than or equal to 40%, preferably greater than or equal to 50%.

2. The carbonyl reductase or its mutant according to claim 1, wherein The carbonyl reductase mutant is a mutant that retains catalytic activity after 1-3 amino acid substitutions, deletions, or additions based on the carbonyl reductase sequence described in claim 1; a mutant that retains catalytic activity; Preferably, the carbonyl reductase mutant is a mutant that retains catalytic activity after 1-3 amino acid substitutions, deletions, or additions based on SEQ ID NO: 15; Preferably, the carbonyl reductase mutant has an amino acid substitution at least at one position of valine (V) at position 48, phenylalanine (F) at position 76, asparagine (N) at position 129, tyrosine (Y) at position 146, tyrosine (Y) at position 179, and valine (V) at position 237 in SEQ ID NO:

15.

3. A biological product, characterized in that, The biological product includes: (1) a nucleic acid molecule encoding the carbonyl reductase or its mutant according to any one of claims 1-2; (2) an expression vector containing the nucleic acid molecule described in (1); (3) a transformant containing the nucleic acid molecule described in (1); (4) a transformant containing the expression vector described in (2); wherein the transformant does not involve animal and plant reproductive materials; Preferably, the expression vector includes a virus, a plasmid, and a phage; Preferably, the transformant includes bacteria, fungi, and animal and plant cells.

4. Use of the carbonyl reductase or its mutant according to any one of claims 1-2, or the biological product according to claim 3 in an enzymatic reaction; Preferably, the enzymatic reaction includes a catalytic reduction reaction of a carbonyl group; Preferably, the catalytic reduction reaction of the carbonyl group includes a reduction reaction of an aldehyde group, a reduction reaction of a ketone group, and a dehydrogenation reaction of an alcohol.

5. Use of the carbonyl reductase or its mutant according to any one of claims 1-2, or the biological product according to claim 3 in the preparation of atomoxetine intermediate and / or atomoxetine; Preferably, the atomoxetine intermediate is a chiral intermediate; Preferably, the atomoxetine intermediate is (S)-3-chlorophenylpropanol.

6. A method for preparing an atomoxetine intermediate, comprising the following steps: Using 3-chloropropiophenone as a substrate, adding the carbonyl reductase or its mutant as described in any one of claims 1-2, or the biological product as described in claim 3, then adding formate dehydrogenase or a biological material having the ability to express, secrete or produce formate dehydrogenase, ammonium formate and coenzyme, and reacting at 25-35 °C for 2-24 h to obtain the tomoxetine intermediate; Preferably, the tomoxetine intermediate is (S)-3-chlorophenylpropanol.

7. The method according to claim 6, characterized in that, The substrate further contains a solvent and / or a cosolvent; Preferably, the solvent and / or cosolvent includes at least one of ethyl acetate, butyl acetate, isobutyl acetate, dibutyl phthalate, ethyl laurate, dimethyl sulfoxide (DMSO), methyl tert-butyl ether, dimethyl phthalate and methanol; Preferably, the solvent and / or cosolvent is selected from at least one of dibutyl phthalate, ethyl laurate, methyl tert-butyl ether and dimethyl phthalate.

8. The method according to claim 6, characterized in that, The coenzyme includes at least one of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide (NAD+).

9. The method according to claim 6, characterized in that, In the method, a buffer solution is further added; Preferably, the buffer solution includes at least one of PB buffer solution, Tris-HCl buffer solution and TEA buffer solution; Preferably, the pH of the buffer solution is 6-8.

10. A method for preparing atomoxetine, comprising: Using the method described in any one of claims 6-8 to prepare the tomoxetine intermediate, and then prepare tomoxetine.

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