(S)-engineered alcohol nitrile hydrolase polypeptides and uses thereof

Abstract

The present disclosure relates to a method for producing a chiral beta-nitroalcohol compound. In particular, the present invention relates to an (S)-selective alcohol nitrile hydroformylase that can enantioselectively catalyze the Henry reaction, wherein an aldehyde or ketone compound is converted to the corresponding beta-nitroalcohol compound in the presence of a nitroalkane compound and an alcohol nitrile hydroformylase.

Description

[0001] Priority claim

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 350,047, filed on June 8, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been electronically filed in ST.26 format and is hereby incorporated by reference in its entirety. The ST.26 copy was created on June 7, 2023 and is named PAT059273-WO-PCT_SL.xml. Technical Field

[0005] The present disclosure relates to the field of biotechnology, in particular to engineered alcohol nitrile hydroformylase polypeptides and their use in industrial biocatalysis. In particular, the present disclosure relates to a method for producing a chiral β-nitro alcohol compound, wherein an aldehyde or ketone compound is converted to the corresponding β-nitro alcohol compound in the presence of a nitroalkane compound and an engineered alcohol nitrile hydroformylase. In particular, the present disclosure relates to an (S)-selective alcohol nitrile hydroformylase that enantioselectively catalyzes the Henry reaction. Background Art

[0006] Biocatalytic processes have become very important for the chemical industry. Of particular importance is the use of enzymes in cases where the properties of the biocatalyst are such that one of the two enantiomers reacts or forms preferentially in a chemical reaction with a chiral or prochiral compound.

[0007] The basic requirements for utilizing these favorable properties of enzymes are that they can be obtained in sufficient quantities (to meet the needs of industrial processes) at low cost and that they have sufficiently high reactivity, selectivity and high stability under the actual conditions of industrial processes.

[0008] β-Nitroalcohols are precursors of β-amino alcohols and are important chiral building blocks for the synthesis of biologically active compounds. The nitroaldol or Henry reaction is one of the classically named reactions in organic synthesis for C-C bond formation. Due to the potential to produce up to two new chiral centers, this reaction is crucial for synthetic applications that can enantioselectively and stereoselectively carry out nitroaldol addition. Although this reaction has been known for more than a century (Henry, 1895), stereospecific protocols using non-enzymatic organocatalysts or chiral metal catalysts have only recently been developed. The development of these methods still has many disadvantages, including long reaction times in the case of metal catalysts and sometimes extreme reaction conditions, or insufficient selectivity in the case of organic catalysts.

[0009] Hydroxynitrile lyases (HNLs), also commonly known as alcohol nitrile aldolases, belong to the class of enzymes called aldehyde lyases (EC 4.1.2.X). Essentially, HNLs catalyze the reversible, stereoselective cleavage of cyanocyanides to form hydrocyanic acid and aldehydes or ketones. Plants use this cyanogenic reaction to defend themselves against fungi or predators by releasing hydrogen cyanide in the cell. In the reverse of its natural reaction, HNLs also catalyze the stereoselective addition of hydrocyanic acid to aldehydes or ketones to produce enantiomeric cyanocyanides, which are commonly used as synthetic building blocks for various pharmaceuticals and agrochemicals (Dadashipour and Asano ACS Catalysis 2011 1(9), 1121-1149). For nitrile formation, HNL has shown a limited substrate range in terms of the nature of the electrophiles it accepts, including aliphatic and aromatic aldehydes or aliphatic ketone compounds, accepting only cyanide as a nucleophile (Liu et al. Front. Bioeng. Biotechnol. 2021 9:653682).

[0010] HNL from rubber tree (Hevea brasiliensis) has been the first described enzyme capable of catalyzing the enzymatic nitroaldol (Henry) reaction of aldehydes with nitromethane (Mandana Gruber-Khadjawi et al. Adv. Synth. Catal. [Advanced Synthesis and Catalysis] 2007, 349, 1445–1450). In recent years, examples of (R)-selective HNL catalyzed Henry reaction from Acidobacterium capsulatum, Granulicella tundricula (Bekerle-Bogner et al. ChemCatChem [Catalysis] 2016, 8, 2214) or Arabidobsis thaliana (Arabidobsis thaliana) (Fuhshuku et al. J. Biotechnol. [Biological Technology Magazine] 2011, 153, 153-159) have been described. Among them, the latter example from Arabidopsis thaliana (AtHNL) is the most extensively described (Fuhshuku et al. J. Biotechnol. 2011, 153, 153-159).

[0011] In addition to HNL, a TGase (protein-glutamine-glutamyltransferase; EC 2.3.2.13) from Streptorerticillium griseoverticillatum has also been described to catalyze the Henry reaction.

[0012] Therefore, it is extremely desirable to develop asymmetric syntheses of β-nitro alcohols. There is still a need for new alcohol nitrile hydroformylases that can enantioselectively catalyze the Henry reaction.

[0013] The present disclosure provides a series of engineered polypeptides with high stereoselectivity, which overcome the above-mentioned disadvantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A Comparative data for the conversion of trifluoroacetone (1) and nitromethane (2) to nitroalcohol compounds (IA) using wild-type (S)-cyanohydrin lyase from Baliospermum montanum (Uniprot ID: D1MX73) having SEQ ID NO:606 relative to the engineered polypeptide SEQ ID NO:2 are shown.

[0015] Figure 1B Comparative data showing the enantiomeric excess of wild-type (S)-cyanohydrin lyase from S. punctata having SEQ ID NO: 606 (Uniprot ID: D1MX73) relative to the engineered polypeptide SEQ ID NO: 2 in the reaction with trifluoroacetone (1) and nitromethane (2) to produce nitroalcohol compound (IA).

[0016] Figure 2A Comparative data on the conversion rate of the most active polypeptide in each round of evolution from the reaction of trifluoroacetone (1) and nitromethane (2) to produce nitroalcohol compound (IA) under the reaction conditions described in Example 13 are shown.

[0017] Figure 2B Comparative data of the enantiomeric excess of the most active polypeptide in each round of evolution from the reaction of trifluoroacetone (1) and nitromethane (2) to produce nitroalcohol compound (IA) under the reaction conditions described in Example 13 are shown.

[0018] Figure 2C Comparative data on the conversion rate of the most active polypeptide in each round of evolution from the reaction of trifluoroacetone (1) and nitromethane (2) to produce nitroalcohol compound (IA) under the reaction conditions described in Example 13 are shown.

[0019] Figure 2D Comparative data of the enantiomeric excess of the most active polypeptide in each round of evolution from the reaction of trifluoroacetone (1) and nitromethane (2) to produce nitroalcohol compound (IA) under the reaction conditions described in Example 13 are shown.

[0020] Figure 3A The activity of the free enzyme compared to the immobilized enzyme on amino or epoxy supports is shown.

[0021] Figure 3B Selectivity of the free enzyme compared to the immobilized enzyme on either amino or epoxy supports was shown.

[0022] Figure 4 The residue differences of the claimed alcohol nitrile hydroformylase polypeptide relative to SEQ ID No: 606 and SEQ ID No: 2 are shown. Summary of the invention

[0023] The present disclosure provides engineered polypeptides with high stereoselectivity, high catalytic activity and good stability, which can asymmetrically synthesize β-nitro alcohols, and in particular asymmetrically synthesize (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol. The present disclosure particularly relates to an (S)-selective alcohol nitrile hydroformylase that can enantioselectively catalyze the Henry reaction.

[0024] Surprisingly, the engineered polypeptides disclosed herein are particularly suitable for substrates containing electron-withdrawing groups. It was found that β-nitro alcohol products can be synthesized in high yield and high stereoselectivity by introducing electron-withdrawing substituents into aldehyde or ketone substrates.

[0025] The present disclosure also provides gene sequences of engineered polypeptides, recombinant expression vectors comprising these genes, engineered strains and efficient methods for producing the same, and reaction processes for asymmetric synthesis of β-nitro alcohols using the engineered polypeptides.

[0026] The engineered alcohol nitrile hydroformylase polypeptides disclosed herein have improved catalytic properties. Wild-type alcohol nitrile hydroformylase has low stereoselectivity for the product, and thus these engineered polypeptides are derived from the wild-type alcohol nitrile hydroformylase by substitution, insertion or deletion of multiple amino acid residues during directed evolution. The wild-type alcohol nitrile hydroformylase is from BmHNL, which consists of 263 amino acids and has the sequence shown in SEQ ID No: 606 (also available in UniProt under accession number D1MX73). The wild-type alcohol nitrile hydroformylase shows low stereoselectivity for the product. In the reaction of 1,1,1-trifluoropropan-2-one and nitromethane using SEQ ID No: 606 to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (i.e. (IA)), for IA, the enantiomeric excess (i.e. ee) is ≤2%.

[0027] In a first aspect, an engineered alcohol nitrile hydroformylase polypeptide is provided, wherein the engineered alcohol nitrile hydroformylase polypeptide is a polypeptide of the following (a) or (b):

[0028] (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 604 and 608 to 640; or

[0029] (b) A polypeptide having alcohol nitrile hydroformylase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity with one of the polypeptides listed in (a), and (ii) one or more amino acid residues substituted, deleted, added or inserted relative to the amino acid sequence listed in (a).

[0030] In a second aspect, an engineered alcohol nitrile hydroformylase polypeptide is provided, which is capable of coupling 1,1,1-trifluoropropan-2-one with nitromethane to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol with higher stereoselectivity and / or activity than SEQ ID NO:606 under suitable reaction conditions.

[0031] In a third aspect, an engineered alcohol nitrile hydroformylase polypeptide is provided, the engineered alcohol nitrile hydroformylase polypeptide comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 606, and the engineered alcohol nitrile hydroformylase polypeptide is capable of coupling 1,1,1-trifluoropropan-2-one with nitromethane under suitable reaction conditions to produce at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more enantiomeric excess of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol.

[0032] In yet another aspect, a polypeptide is provided, which is immobilized on a solid material by a chemical bond or a physical adsorption method, wherein the polypeptide is selected from the alcohol nitrile hydroformylase polypeptide as disclosed herein.

[0033] In yet another aspect, a polynucleotide is provided, which encodes the polypeptide of the present disclosure.

[0034] In yet another aspect, an expression vector is provided, comprising the polynucleotide as described in the present disclosure.

[0035] In yet another aspect, a host cell is provided, comprising the expression vector of the present disclosure, wherein the host cell is preferably Escherichia coli.

[0036] In yet another aspect, a method for preparing an alcohol nitrile hydroformylase polypeptide is provided, the method comprising the steps of culturing the host cell as disclosed herein and obtaining the alcohol nitrile hydroformylase polypeptide from the culture.

[0037] In yet another aspect, a catalyst for alcohol nitrile hydroformylase is provided, which can be obtained by culturing the host cell disclosed herein, wherein the catalyst for alcohol nitrile hydroformylase comprises cells or culture fluid containing the alcohol nitrile hydroformylase polypeptide, or an article processed therewith, wherein the article refers to an extract obtained from the culture of the transformant cell, an isolated product obtained by isolating or purifying the alcohol nitrile hydroformylase from the extract, or an immobilized product obtained by immobilizing the transformant cell, its extract, or an isolated product of the extract.

[0038] In yet another aspect, a method for asymmetric synthesis of β-nitro alcohols using an alcohol nitrile aldolase polypeptide is provided, the method comprising the step of contacting a nitroalkane and an aldehyde or ketone substrate with the alcohol nitrile aldolase polypeptide to obtain a β-nitro alcohol product.

[0039] In yet another aspect, a method for asymmetric synthesis of β-nitro alcohols using the engineered alcohol nitrile aldolase polypeptide disclosed herein is provided, the method comprising the step of contacting a nitroalkane and an aldehyde or ketone substrate with the alcohol nitrile aldolase polypeptide disclosed herein to obtain a β-nitro alcohol product.

[0040] In yet another aspect, a method for asymmetric synthesis of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA) is provided:

[0041]

[0042] The method comprises the step of contacting nitromethane and 1,1,1-trifluoropropan-2-one with an alcohol nitrile hydroformylase polypeptide as described in the present disclosure to obtain (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol having formula (IA).

[0043] In yet another aspect, a method for synthesizing (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol having formula (IB) is provided:

[0044]

[0045] The method comprises the step of contacting (IA) with hydrogen under suitable hydrogenation conditions to obtain (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol (IB), wherein (IA) is synthesized by the method described in the present disclosure.

[0046] In yet another aspect, a method for synthesizing (S)-3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide having formula (IC) is provided.

[0047]

[0048] The method comprises the step of contacting nitromethane and 1,1,1-trifluoropropan-2-one with an alcohol nitrile hydroformylase polypeptide as described in the present disclosure to obtain (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol having formula (IA). DETAILED DESCRIPTION

[0049] The present disclosure describes the directed evolution of HNLs to obtain nitro alcohols with excellent yields and enantiomeric excess even in the case of equimolar substrate ratios. This is the first description of HNLs accepting ketones as substrates for the Henry reaction, and the first description of an S-selective alcohol nitrile hydroformylase catalyzing the Henry reaction. HNLs derived from the organism B. maculatus (BmHNL) have also never been described as being able to catalyze the Henry reaction. A series of engineered polypeptides with high S-stereoselectivity are provided.

[0050] These engineered polypeptides were developed by directed evolution with selection against (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol, a compound of formula (IA) as defined herein.

[0051] The present disclosure describes an engineered polypeptide derived from BmHNL that catalyzes the Henry reaction of trifluoroacetone (1) and nitromethane (2) to produce a nitro alcohol compound (IA). All alcohol nitrile hydroformylases described to date require a large excess of nitro compound of 10-fold to even 45-fold to achieve a respectable conversion of >50%, which makes these processes have low atomic efficiency (<20%) and low economic feasibility, while the newly described polypeptide can achieve >80% conversion and >80% ee, or 100% conversion and >90% ee under equimolar conditions, wherein the substrate nitromethane (2) is in 1.2-fold excess. This is the first description of HNL accepting ketones as substrates for the Henry reaction, and also the first description of a highly S-selective alcohol nitrile hydroformylase catalyzing the Henry reaction (ee>80%).

[0052] The present disclosure also provides a method for asymmetric synthesis of β-nitro alcohols using the engineered alcohol nitrile aldolase polypeptide disclosed herein, the method comprising the step of contacting a nitroalkane and an aldehyde or ketone substrate with the alcohol nitrile aldolase polypeptide disclosed herein to obtain a β-nitro alcohol product.

[0053] In particular, the present disclosure provides a method for the asymmetric synthesis of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA) which stereoselectively produces the desired (S) enantiomer over the (R) enantiomer.

[0054] Solution 1

[0055]

[0056] Compound (IA) (i.e. (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol) is synthesized (ie, (S)-3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide (also referred to herein as (IC))), which is useful in treating cystic fibrosis.

[0057] Enzymatic synthesis of compound (IA) provides an attractive alternative to traditional chemical synthesis. The use of enzyme biocatalysts can also reduce chemical waste and shorten the total number of steps required in the synthesis.

[0058] definition

[0059] Unless otherwise expressly defined, the technical and scientific terms used in this disclosure have the meanings commonly understood by those skilled in the art to which the invention belongs. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology [Microbiology and Molecular Biology Dictionary] (2nd Edition 1994); The Cambridge Dictionary of Science and Technology [Cambridge Science and Technology Dictionary] (Walker edited, 1988); The Glossary of Genetics [Genetics Vocabulary], 5th Edition, R. Rieger et al. (eds.), Springer Verlag [Springer Publishing Company] (1991); and Hale & Marham, The Harper Collins Dictionary of Biology [Harper Collins Biology Dictionary] (1991). As used herein, unless otherwise indicated, the following terms have the meanings assigned to them below. The abbreviations for genetically encoded amino acids are conventional and are as follows:

[0060] Amino Acids Three letter abbreviation Single letter abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0061] For amino acid deletions, "-" is used and for stop codons, "*" is used. When three-letter abbreviations are used, unless specifically preceded by "L" or "D" or it is clear from the context in which the abbreviation is used, the amino acid may be about the α-carbon (C α) in the L- or D-configuration. For example, where "Ala" represents alanine, but the configuration about the α-carbon is not specified, "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively.

[0062] When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to common convention.

[0063] Abbreviations for genetically encoded nucleotides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise specified, abbreviated nucleotides may be ribonucleotides or 2'-deoxyribonucleotides. Nucleotides may be designated as ribonucleotides or 2'-deoxyribonucleotides on an individual basis or on a collective basis. When a nucleic acid sequence is presented as a string of single-letter abbreviations, the sequence is presented in the 5' to 3' direction according to common convention, and phosphates are not indicated.

[0064] "Amino acid difference" or "residue difference" refers to the difference in the amino acid residue at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence. The position of the amino acid difference is generally referred to as "Xn" in this article, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X2 as compared to SEQ ID NO:2" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 2 of SEQ ID NO:2. Therefore, if the reference polypeptide of SEQ ID NO:2 has leucine at position 2, then "the residue difference at position X2 as compared to SEQ ID NO:2" refers to the amino acid substitution of any residue other than leucine at the polypeptide position corresponding to position 2 of SEQ ID NO:2. In most examples herein, the specific amino acid residue difference at a position is expressed as "XnY", where "Xn" designates the corresponding position as described above, and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that is different from the reference polypeptide). In some examples (e.g., in Figure 4In some embodiments, the present disclosure also provides specific amino acid differences represented by the conventional symbol "AnB", wherein A is a single letter identifier of a residue in a reference sequence, "n" is the number of residue positions in a reference sequence, and B is a single letter identifier of a residue substitution in an engineered polypeptide sequence. In some examples, the engineered polypeptide of the present disclosure may include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of specific positions where residue differences exist relative to a reference sequence. In some embodiments, more than one amino acid residue may be used at a specific residue position of an engineered polypeptide, and multiple amino acid residues that can be used may be separated by " / " (e.g., X38F / X38F). "Deletion" refers to modifying a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion may include removing one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids of an enzyme, or up to 20% of the total number of amino acids constituting a reference enzyme, while retaining the enzymatic activity of the engineered alcohol nitrile hydroformylase and / or retaining the improved properties of the engineered alcohol nitrile hydroformylase. Deletions may involve internal and / or terminal portions of a polypeptide. In various embodiments, deletions may include continuous segments or may be discontinuous.

[0065] Unless stated otherwise, the term "and / or" means "and" or "or".

[0066] "Coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0067] "Comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein the sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the sequence portion in the comparison window can include 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 consecutive residues, and optionally includes 30, 40, 50, 100 or more residues.

[0068] Unless otherwise indicated, the term "compounds of the present disclosure, compounds of the disclosure or compound of the disclosure" refers to compounds having formula (1), (2), (I), (Ii), (II), (III), (IA), (IB), (IB).HCl, (IC), (ID), E1, E3, E4, E5, E6, exemplary compounds, salts thereof (particularly pharmaceutically acceptable salts thereof), hydrates, solvates, prodrugs, and all stereoisomers (including diastereomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitution), as well as inherently formed moieties.

[0069] "Conversion" refers to the enzymatic conversion of a substrate into a corresponding product. "Conversion percentage" or "conversion rate" refers to the percentage of substrate converted to product within a period of time under specified conditions. Therefore, the "enzyme activity" or "activity" of an alcohol nitrile hydroformylase polypeptide can be expressed as the "conversion percentage" of substrate to product.

[0070] In the context of a given amino acid or polynucleotide sequence numbering, "corresponding to", "referenced to" or "relative to" refers to the numbering of the residues of the reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given sequence are specified relative to the reference sequence, rather than being specified by the actual numerical positions of the residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence (e.g., an engineered alcohol nitrile hydroformylase) can be aligned with a reference sequence by introducing gaps to optimize the residue matching between the two sequences. In these cases, despite the presence of gaps, the residue numbering in a given amino acid or polynucleotide sequence is performed relative to the reference sequence to which it is aligned.

[0071] As used herein, the term "electron-withdrawing substituent" refers to a substituent with strong electron-withdrawing power. Exemplary electron-withdrawing groups include, but are not limited to, halogen atoms (such as fluorine atoms), haloalkyl groups (such as trifluoromethyl), carboxyl groups, alkoxycarbonyl groups (such as methoxycarbonyl), aryloxycarbonyl groups (such as phenoxycarbonyl), acyl groups (such as acetyl), acyloxy groups (such as acetoxy), cyano groups, aryl groups, alkenyl groups, nitro groups, sulfo groups, alkanesulfonyl groups, alkanesulfinyl groups, and alkoxysulfonyl groups, including any substituents disclosed herein, such as alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, heterocyclic groups, cycloalkyl groups, and arylalkyl groups containing these electron-withdrawing groups as substituents. Among them, preferred examples are fluorine-containing groups (such as fluorine atom and trifluoromethyl), acyloxy groups (such as acetoxy), cyano groups, nitro groups, sulfo groups, alkylsulfonyl groups (such as C1-C6 alkylsulfonyl groups), alkylsulfinyl groups (such as C1-C6 alkylsulfinyl groups) and alkoxysulfonyl groups (such as C1-C6 alkoxysulfonyl groups).

[0072] The terms "engineered alcohol nitrile aldolase," "engineered alcohol nitrile aldolase polypeptide," "alcohol nitrile aldolase polypeptide," "improved alcohol nitrile aldolase polypeptide," and "engineered polypeptide" are used interchangeably herein.

[0073] As used herein, "fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence. A fragment can be at least 10 amino acids long, at least 20 amino acids long, at least 50 amino acids long, or longer, and up to 70%, 80%, 90%, 95%, 98%, and 99% of a full-length alcohol nitrile hydroformylase polypeptide.

[0074] "Improved enzyme properties" refers to better or more desirable enzyme properties for a particular purpose compared to a reference alcohol nitrile aldolase (e.g., wild-type alcohol nitrile aldolase or another engineered alcohol nitrile aldolase). The engineered alcohol nitrile aldolase polypeptides of the present disclosure exhibit improved enzyme properties. Enzyme properties that are expected to be improved include, but are not limited to, enzyme activity (which can be expressed as a percentage of substrate conversion), thermal stability, solvent stability, pH activity profile, cofactor requirements, tolerance to inhibitors (e.g., substrate or product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity and diastereoselectivity).

[0075] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids from a reference polypeptide. In some embodiments, the improved engineered alcohol nitrile hydroformylase comprises the insertion of one or more amino acids in a naturally occurring alcohol nitrile hydroformylase polypeptide, and the insertion of one or more amino acids in other engineered alcohol nitrile hydroformylase polypeptides. Insertion can be made into an internal portion of a polypeptide or into a carboxyl or amino terminus. As used herein, insertion includes fusion proteins known in the art. Insertion can be a continuous segment of amino acids or separated by one or more amino acids in a naturally occurring or engineered polypeptide.

[0076] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other substances (e.g., proteins, lipids, and polynucleotides) associated with it in nature. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., in a host cell or in vitro synthesis). The engineered alcohol nitrile hydroformylase polypeptide can be present in a cell, in a cell culture medium, or prepared in various forms, such as a lysate or an isolated preparation. Therefore, in some embodiments, the engineered alcohol nitrile hydroformylase polypeptide can be an isolated polypeptide.

[0077] "Naturally occurring" or "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and has not been intentionally modified by human procedures.

[0078] As used herein, "alcohol nitrile aldolase" or "HNL" refers to a wild-type or engineered enzyme having alcohol nitrile aldolase activity.

[0079] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0080] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of its length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids. Preferably, the amino acids have the L configuration.

[0081] "Recombinant" or "engineered" or "non-naturally occurring" when used with reference to, for example, a cell, nucleic acid, or polypeptide, refers to a material or a material corresponding to the native or native form of that material that is modified in a manner not found in nature, or is identical thereto, but is produced or derived from synthetic materials and / or manipulation through the use of recombinant techniques.

[0082] "Reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a fragment of a full-length gene or polypeptide sequence. In general, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, or a full-length nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) contain a sequence that is similar between the two sequences (i.e., a portion of the complete sequence), and (2) can further contain a sequence that is different between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" is not intended to be limited to wild-type sequences, and can include engineered or altered sequences. For example, a "reference sequence based on SEQ ID NO:2 having proline at the residue corresponding to X35" refers to a reference sequence in which the corresponding residue at position X35 in SEQ ID NO:2 that is alanine has been changed to proline.

[0083] "Sequence identity" and "homology" are used interchangeably herein and refer to a comparison between polynucleotide sequences or polypeptide sequences ("sequence identity" and "homology" are usually expressed as percentages), and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences, or by aligning the nucleic acid base or amino acid residue with gaps to derive the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to derive the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used to align two sequences. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the similarity method search of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin package), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al. (eds.), Current Protocols, Joint Venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. [a joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc.], (1995 supplement) (Ausubel). Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990) J Mol Biol. 215:403-410; and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively.Software for performing BLAST analysis can be publicly obtained through the website of the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which, when compared with the words of the same length in the database sequence, match or satisfy some positive threshold score T. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits are used as seeds to initiate searches to find longer HSPs containing them. Then, the word hits are extended in both directions along each sequence, as far as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using parameters M (residue matching reward score; always>0) and N (penalty score of mismatched residues; always<0). For amino acid sequences, a scoring matrix is ​​used to calculate cumulative scores. Extension of word hits in each direction terminates when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score moves toward zero or lower due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains as defaults. For amino acid sequences, the BLASTP program uses by default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 89:10915). Exemplary determination of sequence alignment and percent sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0084] "Solvent stable" refers to an alcohol nitrile hydroformylase polypeptide that maintains similar activity (e.g., more than 50% to 80%) after exposure to different solvents (ethanol, isopropanol, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours).

[0085] "Stereoisomers" and similar expressions are used interchangeably herein to refer to all isomers that differ only in the orientation of their atoms in space. It includes enantiomers and compounds that have more than one stereocenter and are not mirror images of each other (ie, diastereomers).

[0086] "Stereoselectivity" refers to the preferential formation of one stereoisomer relative to another in a chemical reaction or enzymatic reaction. Stereoselectivity can be partial, in which one stereoisomer is formed in preference to another; or it can be complete in the case of forming only one stereoisomer. When the stereoisomers are enantiomers, the stereoselectivity is called enantioselectivity. It is usually reported as "enantiomeric excess" (abbreviated as ee). When the stereoisomers are diastereomers, the stereoselectivity is called diastereoselectivity. It is usually reported as "diastereomeric excess" (abbreviated as de). This fraction (typically a percentage) is usually reported in the art as the enantiomeric excess (i.e., ee) derived therefrom according to the following formula: [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer].

[0087] "Suitable reaction conditions" refer to those conditions in the reaction system (eg, catalyst loading, substrate loading, temperature, solvent, etc.) under which a substrate is converted to a desired product.

[0088] In the context of the biocatalytic methods of the present disclosure, "suitable reaction conditions" refer to those conditions (e.g., enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, cosolvent, etc.) under the biocatalytic reaction system under which the alcohol nitrile hydroformylase polypeptide of the present disclosure can convert the substrate into the desired product compound. Exemplary "suitable reaction conditions" are provided in the present disclosure and illustrated by examples.

[0089] "Thermostable" means an alcohol nitrile hydroformylase polypeptide that retains similar activity (eg, greater than 50%) after exposure to elevated temperature (eg, 30°C-85°C) for a period of time (eg, 0.5-24 h).

[0090] In addition, the use of terms representing monovalent groups and where divalent groups are appropriate should be understood to represent the corresponding divalent groups, and vice versa. Unless otherwise specified, the conventional definition of the term contrast and the valence of conventional stable atoms are assumed and embodied in all formulas and groups. The article "a / an" refers to one / kind or more than one / kind (e.g., at least one / kind) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0091] The term "substituted" means that the specified group or moiety carries one or more suitable substituents, wherein these substituents may be attached to the specified group or moiety at one or more positions. For example, an aryl substituted by a cycloalkyl group may indicate that the cycloalkyl group is attached to an atom of the aryl group either by a bond or by being fused to the aryl group and sharing two or more common atoms.

[0092] In the groups, radicals or moieties defined below, the number of carbon atoms is usually indicated before the group, e.g. C1-C8 alkyl means an alkyl group having 1 to 8 carbon atoms. In general, for groups containing two or more subgroups, the last mentioned group is the point of attachment of the group, e.g. "alkylaryl" means a monovalent group of the formula alkyl-aryl-, and "arylalkyl" means a monovalent group of the formula aryl-alkyl-.

[0093] The term "halogen" or "halo" means fluorine, chlorine, bromine, or iodine.

[0094] The term "nitro" shall mean the group -NO2.

[0095] As used herein, the term "alkyl" represents, for example, a saturated branched or straight chain hydrocarbon group having 1 to 50 carbon atoms, such as C1-C3 alkyl, C1-C6 alkyl, C2-C8 alkyl, C3-C8 alkyl, C1-C8 alkyl, C1-C 10 Alkyl, C1-C 20 Alkyl, C1-C 30 Alkyl, C1-C 40 Alkyl, C1-C 50 Alkyl, etc. Representative examples are methyl, ethyl, propyl (e.g., prop-1-yl, prop-2-yl (or isopropyl)), butyl (e.g., 2-methylprop-2-yl (or tert-butyl), but-1-yl, but-2-yl), pentyl (e.g., pent-1-yl, pent-2-yl, pent-3-yl), 2-methylbut-1-yl, 3-methylbut-1-yl, hexyl (e.g., hex-1-yl), heptyl (e.g., hept-1-yl), octyl (e.g., oct-1-yl), nonyl (e.g., non-1-yl), etc. The term "C1-C6 alkyl" should be interpreted accordingly.

[0096] As used herein, the term "alkenyl" represents a branched or straight chain hydrocarbon group having at least one double bond, for example, a C2-C3 alkenyl, a C2-C6 alkenyl, a C2-C7 alkenyl, a C2-C8 alkenyl, a C3-C5 alkenyl, a C1-C 10 Alkenyl, C1-C 20 Alkenyl, C1-C 30 Alkenyl, C1-C 40 Alkenyl, C1-C 50Representative examples are ethenyl or vinyl, propenyl (e.g., prop-1-enyl, prop-2-enyl), butadienyl (e.g., but-1,3-dienyl), butenyl (e.g., but-1-en-1-yl, but-2-en-1-yl), pentenyl (e.g., pent-1-en-1-yl, pent-2-en-2-yl), hexenyl (e.g., hex-1-en-2-yl, hex-2-en-1-yl), 1-ethylprop-2-enyl, 1,1-(dimethyl)prop-2-enyl, 1-ethylbut-3-enyl, 1,1-(dimethyl)but-2-enyl, and the like.

[0097] As used herein, the term "alkynyl" represents a branched or straight chain hydrocarbon group having at least one triple bond, for example, a C2-C3 alkynyl, C2-C6 alkynyl, C2-C7 alkynyl, C2-C8 alkynyl, C3-C5 alkynyl, C1-C 10 Alkynyl, C1-C 20 Alkynyl, C1-C 30 Alkynyl, C1-C 40 Alkynyl, C1-C 50 Representative examples are ethynyl, propynyl (e.g., prop-1-ynyl, prop-2-ynyl), butynyl (e.g., but-1-ynyl, but-2-ynyl), pentynyl (e.g., pent-1-ynyl, pent-2-ynyl), hexynyl (e.g., hex-1-ynyl, hex-2-ynyl), 1-ethylprop-2-ynyl, 1,1-(dimethyl)prop-2-ynyl, 1-ethylbut-3-ynyl, 1,1-(dimethyl)but-2-ynyl, and the like.

[0098] As used herein, the term "aryl" is intended to include monocyclic, bicyclic or polycyclic carbocyclic aromatic rings. Representative examples are phenyl, naphthyl (e.g., naphthalene-1-yl, naphthalene-2-yl), anthracenyl (e.g., anthracene-1-yl, anthracene-9-yl), phenanthrenyl (e.g., phenanthracene-1-yl, phenanthracene-9-yl), etc. Aryl is also intended to include monocyclic, bicyclic or polycyclic carbocyclic aromatic rings substituted by carbocyclic aromatic rings. Representative examples are biphenyl (e.g., biphenyl-2-yl, biphenyl-3-yl, biphenyl-4-yl), phenylnaphthyl (e.g., 1-phenylnaphthalene-2-yl, 2-phenylnaphthalene-1-yl), etc. Aryl is also intended to include partially saturated bicyclic or polycyclic carbocyclic rings with at least one unsaturated portion (e.g., benzo moiety). Representative examples are indanyl (e.g., indan-1-yl, indan-5-yl), indenyl (e.g., inden-1-yl, inden-5-yl), 1,2,3,4-tetrahydronaphthyl (e.g., 1,2,3,4-tetrahydronaphthyl-1-yl, 1,2,3,4-tetrahydronaphthyl-2-yl, 1,2,3,4-tetrahydronaphthyl-6-yl), 1,2-dihydronaphthyl (e.g., 1,2-dihydronaphthyl-1-yl, 1,2-dihydronaphthyl-4-yl, 1,2-dihydronaphthyl-6-yl), fluorenyl (e.g., fluoren-1-yl, fluoren-4-yl, fluoren-9-yl), etc. Aryl is also intended to include partially saturated bicyclic or polycyclic carbocyclic aromatic rings containing one or two bridges. Representative examples are benzonorbornyl (e.g., benzonorborn-3-yl, benzonorborn-6-yl), 1,4-ethano-1,2,3,4-tetrahydronaphthyl (e.g., 1,4-ethano-1,2,3,4-tetrahydronaphthyl-2-yl, 1,4-ethano-1,2,3,4-tetrahydronaphthyl-10-yl), etc. Aryl is also intended to include partially saturated bicyclic or polycyclic carbocyclic aromatic rings containing one or more spiro atoms. Representative examples are spiro[cyclopentane-1,1′-indene]-4-yl, spiro[cyclopentane-1,1′-indene]-4-yl, spiro[piperidin-4,1′-indene]-1-yl, spiro[piperidin-3,2′-indene]-1-yl, spiro[piperidin-4,2′-indene]-1-yl, spiro[piperidin-4,1′-indene]-3′-yl, spiro[pyrrolidine-3,2′-indene] -1-yl, spiro[pyrrolidine-3,1′-(3′,4′-dihydronaphthalene)]-1-yl, spiro[piperidine-3,1′-(3′,4′-dihydronaphthalene)]-1-yl, spiro[piperidine-4,1′-(3′,4′-dihydronaphthalene)]-1-yl, spiro[imidazolinidine-4,2′-indene]-1-yl, spiro[piperidine-4,1′-indene]-1-yl, etc.

[0099] Terminology C6-C 14 Aryl should be interpreted accordingly.

[0100] Preferably, aryl refers to a monocyclic or bicyclic carbocyclic aromatic ring.

[0101] The term "arylalkyl" (e.g., benzyl, phenethyl, 3-phenylpropyl, 1-naphthylmethyl, 2-(1-naphthyl)ethyl, etc.) represents an aryl group as defined above attached via an alkyl chain having the specified number of carbon atoms or a substituted alkyl group as defined above. 20 Arylalkyl should be interpreted accordingly.

[0102] Preferred examples of aryl include, but are not limited to, phenyl and naphthyl. In an embodiment, aryl is phenyl.

[0103] As used herein, the term "cycloalkyl" means a monocyclic or polycyclic saturated or partially unsaturated carbocyclic ring, for example containing 3-20 carbon atoms, for example containing 3-18 carbon atoms, wherein there are no shared delocalized π electrons (aromaticity) between the ring carbons. 20 Cycloalkyl" and "C3-C 10 The term polycyclic encompasses bridged (e.g., norbornane), fused (e.g., decalin), and spirocyclic cycloalkyl groups. Preferably, cycloalkyl groups such as "C3-C 20 Cycloalkyl" and "C3-C 18 "Cycloalkyl" is a monocyclic or spirocyclic hydrocarbon group of 3 to 20 and 3 to 18 carbon atoms, respectively.

[0104] Representative examples are spiro[2.5]octyl, spiro[4.5]decyl, cyclopropenyl, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, spiro[2.3]hexyl, spiro[3.3]heptyl, spiro[3.4]octyl, spiro[3.5]nonyl, spiro[4.5]decyl, spiro[5.5]undecyl, spiro[4.4]nonyl, bicyclo[2.2.2]octyl, bicyclo[2.2.2]octenyl, bicyclo[1.1.1]pentyl, decalinyl, bicyclo[3.3.0]octyl, adamantyl, norbornyl, norbornyl, nortricyclyl, bicyclo-[3.2.1]octyl, tricyclo[5.2.1.0] 2,6 ]decyl, bicyclo[2.2.1]heptyl, etc.

[0105] As used herein, the term "fluoroalkyl" refers to a straight or branched alkyl group as defined above, wherein some or all of the hydrogen atoms of these groups are replaced by fluorine atoms. The term C1-C6 fluoroalkyl group should be interpreted accordingly. Examples include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 3,3,3-trifluoropropyl-1-yl, 1,1,1-trifluoropropyl-2-yl, heptafluoroisopropyl, 1-fluorobutyl, 2-fluorobutyl, 3-fluorobutyl, 4-fluorobutyl, 4,4,4-trifluorobutyl, tert-butyl fluoride, etc.

[0106] As used herein, the term "haloalkyl" refers to an alkyl group as defined above substituted with one or more halo groups as defined herein. 20 Haloalkyl and C1-C6 haloalkyl should be interpreted accordingly. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2-fluoropropyl, 1,1,1-trifluoropropyl, 2,2-difluoropropyl, 3,3-difluoropropyl and 1-fluoromethyl-2-fluoroethyl, 1,3-dibromopropane-2-yl, 3-bromo-2-fluoropropyl, 1,1,2,2-tetrafluoropropyl and 1,4,4-trifluorobutane-2-yl.

[0107] As used herein, the term "heteroaryl" is intended to include monocyclic heterocyclic aromatic rings containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples are pyrrolyl, furanyl, thienyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazolyl, triazolyl (e.g., 1,2,4-triazolyl), oxadiazolyl (e.g., 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl), tetrazolyl, pyranyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, thiadiazinyl, aza Base, azecinyl, etc.

[0108] Heteroaryl is also intended to include bicyclic heterocyclic aromatic rings containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples are indolyl, isoindolyl, benzofuranyl, benzothiophenyl, indazolyl, benzopyranyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benzoxazinyl, benzotriazolyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, quinazolinyl, cinnolinyl, quinolinyl, isoquinolinyl, quinoxalinyl, oxazolopyridinyl, isoxazolopyridinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, imidazopyridinyl, imidazopyrimidinyl, pyrazolopyridinyl, pyrazolopyrimidinyl, pyrazolotriazinyl, triazolopyridinyl, triazolopyrimidinyl, imidazothiazolyl, triazolopyridinyl, triazolopyrimidinyl, and the like.

[0109] Heteroaryl is also intended to include polycyclic heterocyclic aromatic rings containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples are carbazolyl, phenoxazinyl, phenazinyl, acridinyl, phenothiazinyl, carbolinyl, phenanthrolinyl and the like.

[0110] Heteroaryl is also intended to include partially saturated monocyclic, bicyclic or polycyclic heterocyclic radicals containing one or more heteroatoms selected from oxygen, nitrogen and sulfur (O, N and S). Representative examples are imidazolinyl, indolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, dihydrobenzopyranyl, dihydropyridooxazinyl, dihydrobenzodioxinyl (e.g., 2,3-dihydrobenzo[b][1,4]dioxinyl), benzodioxolyl (e.g., benzo[d][1,3]dioxol), dihydrobenzoxazinyl (e.g., 3,4-dihydro-2H-benzo[b][1,4]oxazine), tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydroimidazo[4,5-c]pyridinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydroquinoxalinyl, etc.

[0111] The heteroaryl ring structure may be substituted with one or more substituents. These substituents themselves may be optionally substituted. The heteroaryl ring may be bonded via a carbon atom or a heteroatom.

[0112] The term "5-20 membered heteroaryl" should be interpreted accordingly.

[0113] As used herein, the term "monocyclic heteroaryl" is intended to include monocyclic heterocyclic aromatic rings as defined above.

[0114] As used herein, the term "bicyclic heteroaryl" is intended to include bicyclic heterocyclic aromatic rings as defined above.

[0115] Examples of 5-20 membered heteroaryl groups include, but are not limited to, indolyl, imidazopyridinyl, isoquinolinyl, benzoxazolonyl, pyridinyl, pyrimidinyl, pyridonyl, benzotriazolyl, pyridazinyl, pyrazolotriazinyl, indazolyl, benzimidazolyl, quinolinyl, triazolyl (e.g., 1,2,4-triazolyl), pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, pyrrolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl), imidazolyl, pyrrolopyridinyl, tetrahydroindazolyl, quinoxalinyl, thiadiazole 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, pyrazinyl, oxazolopyridinyl, pyrazolopyrimidinyl, benzoxazolyl, indolinyl, isoxazolopyridinyl, dihydropyridooxazinyl, tetrazolyl, dihydrobenzodioxinyl (e.g., 2,3-dihydrobenzo[b][1,4]dioxinyl), benzodioxolyl (e.g., benzo[d][1,3]dioxole), and dihydrobenzoxazinyl (e.g., 3,4-dihydro-2H-benzo[b][1,4]oxazine).

[0116] As used herein, the term "heterocyclyl" represents a saturated or partially saturated monocyclic or polycyclic ring containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, S(═O) and S(═O)2, and wherein there are no shared delocalized π electrons (aromaticity) between the ring carbons or heteroatoms. The heterocyclyl ring structure may be substituted with one or more substituents. These substituents themselves may be optionally substituted. The heterocyclyl may be bonded via a carbon atom or a heteroatom. The term polycyclic encompasses bridged, fused and spirocyclic heterocyclyls.

[0117] Representative examples are aziridinyl (e.g., aziridin-1-yl), azetidinyl (e.g., azetidin-1-yl, azetidin-3-yl), pyrrolidinyl (e.g., pyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl), imidazolidinyl (e.g., imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl), oxazolidinyl (e.g., oxazolidin-2-yl, oxazolidin-3-yl, oxazolidin-4-yl), thiazolidinyl (e.g., thiazolidin-2-yl, thiazolidin-3-yl, thiazolidin-4-yl), isothiazolidinyl, piperidinyl (e.g., piperidin-1-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl), homopiperidinyl (e.g., homopiperidin-1-yl), , homopiperidin-2-yl, homopiperidin-3-yl, homopiperidin-4-yl), piperazinyl (e.g., piperazin-1-yl, piperazin-2-yl), morpholinyl (e.g., morpholin-2-yl, morpholin-3-yl, morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-2-yl, thiomorpholin-3-yl, thiomorpholin-4-yl), 1-oxothiomorpholinyl, 1,1-dioxo-thiomorpholinyl, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl, tetrahydrofuran-3-yl), tetrahydrothiophenyl, tetrahydro-1,1-dioxothiophenyl, tetrahydropyranyl (e.g., 2-tetrahydropyranyl), tetrahydrothiopyranyl (e.g., 2-tetrahydrothiopyranyl), 1,4-dioxanyl, 1,3-dioxanyl, and the like. Heterocyclyl is also intended to represent a saturated 6 to 8 membered bicyclic ring containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, S(═O) and S(═O)2. Representative examples are octahydroindolyl (e.g., octahydroindol-1-yl, octahydroindol-2-yl, octahydroindol-3-yl, octahydroindol-5-yl), decahydroquinolinyl (e.g., decahydroquinolin-1-yl, decahydroquinolin-2-yl, decahydroquinolin-3-yl, decahydroquinolin-4-yl, decahydroquinolin-6-yl), decahydroquinoxalinyl (e.g., decahydroquinoxalin-1-yl, decahydroquinoxalin-2-yl, decahydroquinoxalin-6-yl), etc. Heterocyclyl is also intended to represent a saturated 6 to 8 membered ring containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, S(═O) and S(═O)2, and having one or two bridges. Representative examples are 3-azabicyclo[3.2.2]nonyl, 2-azabicyclo[2.2.1]heptyl, 3-azabicyclo[3.1.0]hexyl, 2,5-diazabicyclo[2.2.1]heptyl, atropine, tropine, quinuclidinyl, 1,4-diazabicyclo[2.2.2]octyl, etc. Heterocyclyl is also intended to represent a 6- to 8-membered saturated ring containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, S(═O) and S(═O)2, and containing one or more spiro atoms.Representative examples are 1,4-dioxaspiro[4.5]decane (e.g., 1,4-dioxaspiro[4.5]decane-2-yl, 1,4-dioxaspiro[4.5]decane-7-yl), 1,4-dioxa-8-azaspiro[4.5]decane (e.g., 1,4-dioxa-8-azaspiro[4.5]decane-2-yl, 1,4-dioxa-8-azaspiro[4.5]decane-8-yl), 8-azaspiro[4.5]decane (e.g., 8-azaspiro[4.5]decane-1-yl, 8-azaspiro[4.5]decane-8-yl), 2-azaspiro[5.5]undecane (e.g., Such as 2-azaspiro[5.5]undecane-2-yl), 2,8-diazaspiro[4.5]decane (for example, 2,8-diazaspiro[4.5]decane-2-yl, 2,8-diazaspiro[4.5]decane-8-yl), 2,8-diazaspiro[5.5]undecane (2,8-diazaspiro[5.5]undecane-2-yl), 1,3,8-triazaspiro[4.5]decane (for example, 1,3,8-triazaspiro[4.5]decane-1-yl, 1,3,8-triazaspiro[4.5]decane-3-yl, 1,3,8-triazaspiro[4.5]decane-8-yl), etc.

[0118] The term "3- to 14-membered heterocyclyl" should be interpreted accordingly.

[0119] As used herein, the term "optional" or "optionally substituted" means that the event or situation described may or may not occur; for example, "optionally substituted aryl" refers to an aryl group that may or may not be substituted. The present description includes both substituted aryl and unsubstituted aryl.

[0120] The term "pharmaceutically acceptable" is defined as suitable for administration to humans without adverse events.

[0121] As used herein, the term "sponge nickel (sponge Nickel) and sponge cobalt (sponge Cobalt)" is intended to refer to nickel and cobalt catalysts having a skeleton structure formed by a nickel or cobalt alloy. Typically, these catalysts are formed by nickel or cobalt alloyed with aluminum and then removing the aluminum. The terms sponge nickel and sponge cobalt include registered trademarks and well-known Raney nickel (Raney nickel) and Raney cobalt (Raney cobalt) catalysts.

[0122] Certain defined terms may occur more than once in a formula, and in such cases, each term should be defined independently of the others.

[0123] Alcohol nitrile aldolase disclosed herein

[0124] In another aspect, the alcohol nitrile aldolase polypeptides disclosed herein can asymmetrically couple an aldehyde or ketone substrate and a nitroalkane substrate.

[0125] In some embodiments, the engineered alcohol nitrile hydroformylase polypeptide disclosed herein is capable of converting 1,1-trifluoropropan-2-one and nitromethane to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (i.e., (IA)) with a stereoselectivity at least equal to or greater than that of SEQ ID No: 606 and / or SEQ ID No: 2. The engineered alcohol nitrile hydroformylase polypeptide disclosed herein is capable of producing a β-nitro alcohol product, such as compound IA, with an enantiomeric excess of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0126] In some embodiments, the engineered alcohol nitrile hydroformylase polypeptide is capable of converting 1,1-trifluoropropan-2-one and nitromethane to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (i.e., (IA)) with greater stereoselectivity than the polypeptide of SEQ ID NO: 606 under suitable reaction conditions, such as those disclosed herein. These engineered alcohol nitrile hydroformylase polypeptides comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence of SEQ ID NO: NO:4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124、126、128、130、132、134、136、138、140、142、144、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、244、246、248、250、252、254、256、258、260、262、264、266、268、270、272、274、276、278、280、282、284、286、288、290、292、294、296、298、300、302、304、306、308、310、312、314、 316、318、320、322、324、326、328、330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、370、372、374、376、378、380、382、384、386、388、390、392、394、396、398、400、402、404、406、408、410、412、414、416、418、420、422、424、426、428、430、432、434、436、438、440、442、444、446、448、450、452、454、456、458、460、462、464、466、468、470、472、474、476、478、480、482、484、486、488、490、492、494、496、498、500、502、504、506、508、510、512、514、516、518、520、522、524、 602, 604, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638 and 640. The identity between two amino acid sequences or two nucleotide sequences can be obtained by algorithms commonly used in the art, and can be calculated by using NCBI Blastp and Blastn software, or by using the Clustal W algorithm (Nucleic Acid Research, 22 (22): 4673-4680, 1994). For example, using the Clustal W algorithm, the amino acid sequence identity between SEQ ID NO: 4 and SEQ ID NO: 606 is 92.78%.

[0127] The engineered alcohol nitrile aldolase polypeptides represented by SEQ ID NOs: 4 to 604 and 608 to 640 exhibit higher activity and / or stereoselectivity than SEQ ID NO: 606, as shown in the Examples.

[0128] In some embodiments, the engineered alcohol nitrile hydroformylase polypeptide comprises an amino acid sequence having an insertion of one or more amino acid residues in SEQ ID NO: 2 and having alcohol nitrile hydroformylase activity. For any embodiment of the engineered alcohol nitrile hydroformylase polypeptide disclosed herein, the insert may comprise 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, wherein the relevant functions and / or improved properties of the engineered alcohol nitrile hydroformylase described herein are maintained. The insert may be inserted at the amino terminus or carboxyl terminus, or at an internal portion of the alcohol nitrile hydroformylase polypeptide. In some embodiments, the insert may comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 amino acid residues. In some embodiments, the insertion may occur 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, 35, 40, 45, 50, 55, 60 times or more. In some embodiments, the insert may comprise 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 or more amino acid residues.

[0129] In some embodiments, the engineered alcohol nitrile hydroformylase polypeptides comprise an amino acid sequence that differs from the sequence of SEQ ID NO: 2 at one or more residue positions selected from the group consisting of: X2, X12, X28, X29, X32, X39, X50, X55, X64, X105, X111, X147, X152, X154, X160, X185, X196, X203, X208, X209, X232, X233, and X250. In some embodiments, the engineered alcohol nitrile hydroformylase polypeptides comprise an amino acid sequence comprising at least one of the following features (which features are amino acid residue substitutions compared to the reference sequence of SEQ ID NO:2): VX12I; SX28G; AX29W; NX32T; WX39F; WX39V; QX50E; QX50D; RX55G; LX64A; AX105G; DX111S; EX147K; TX152L; NX160M; TX185R; SX196G; YX203C; QX208R; QX208S; IX209V; SX232G; AX233G; or QX250G.

[0130] In specific embodiments, the engineered alcohol nitrile hydroformylase polypeptides comprise an amino acid sequence that differs from the sequence of SEQ ID NO: 2 at one or more residue positions selected from the group consisting of: X39, X105, and X154. In specific embodiments, the engineered alcohol nitrile hydroformylase polypeptides comprise an amino acid sequence comprising at least one of the following features (these features are amino acid residue substitutions compared to the reference sequence of SEQ ID NO: 2): WX39F; WX39V; or AX105G.

[0131] In some embodiments, the engineered alcohol nitrile hydroformylase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 606 at one or more residue positions selected from the group consisting of X2, X11, X12, X28, X29, X32, X33, X39, X43, X44, X46, X50, X55, X64, X80, X103, X105, X111, X118, X121, X147, X152, X154, X160, X172, X180, X185, X196, X203, X208, X209, X232, X233, X238, X241, X250, and X263. In some embodiments, the engineered alcohol nitrile hydroformylase polypeptide comprises an amino acid sequence that comprises at least one of the following features (these features are compared to SEQ ID NO: 606 amino acid residue substitutions of the reference sequence of NO:606): TX11S; IX12V; SX28G; AX29W; NX32T; AX33V; VX39F, IX43S; DX44N; RX46H; QX50E; QX50D; EX55R; EX55G; LX64A; SX80A; HX103V; AX105G; DX111S; YX118V; FX121Y; EX147K; TX152L; NX160M; LX172R; EX180L; TX185R; SX196G; YX203C; QX208R; QX208S; IX209V; SX232G; AX233G; QX238M; KX241R; QX250G; or AX263S.

[0132] In specific embodiments, the engineered alcohol nitrile hydroformylase polypeptides comprise an amino acid sequence that differs from the sequence of SEQ ID NO: 606 at one or more residue positions selected from the group consisting of: X2, X105, X111, X154, X160, X185, X209, X232, and X250. In specific embodiments, the engineered alcohol nitrile hydroformylase polypeptides comprise an amino acid sequence comprising at least one of the following features (these features are amino acid residue substitutions compared to the reference sequence of SEQ ID NO: 606): AX105G; DX111S; NX160M; TX185R; IX209V; SX232G; or QX250G.

[0133] In another aspect, the disclosure provides polynucleotides comprising sequences encoding engineered alcohol nitrile aldolase polypeptides, expression vectors, and host cells capable of expressing engineered alcohol nitrile aldolase polypeptides. In certain embodiments, the host cell can be a bacterial host cell, such as Escherichia coli. The host cell can be used to express and separate the engineered alcohol nitrile aldolase described herein, or alternatively directly used in the reaction of converting a substrate into a product.

[0134] In some embodiments, the engineered alcohol nitrile aldolase in the form of whole cells, crude extracts, isolated enzymes, or purified enzymes can be used alone or in an immobilized form (eg, immobilized on a resin).

[0135] Polynucleotides, control sequences, expression vectors and host cells useful for producing engineered alcohol nitrile aldolase polypeptides

[0136] In another aspect, the disclosure provides polynucleotides encoding engineered polypeptides having alcohol nitrile hydroformylase activity as described herein. The polynucleotides can be connected to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing the engineered polypeptide. An expression construct comprising a heterologous polynucleotide encoding an engineered alcohol nitrile hydroformylase can be introduced into a suitable host cell to express the corresponding engineered alcohol nitrile hydroformylase polypeptide.

[0137] As will be apparent to those skilled in the art, the availability of protein sequences and the knowledge of codons corresponding to a variety of amino acids provide an explanation of all possible polynucleotides encoding target protein sequences. The degeneracy of the genetic code (wherein the same amino acid is encoded by selectable or synonymous codons) allows the generation of a very large number of polynucleotides, all of which encode engineered alcohol nitrile hydroformylase polypeptides disclosed herein. Therefore, after determining a specific amino acid sequence, those skilled in the art can generate any number of different polynucleotides by modifying one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the disclosure specifically contemplates any change in polynucleotides, which can be prepared by selecting a combination based on possible codon selections for any polypeptide disclosed herein (comprising those amino acid sequences of the exemplary engineered polypeptides listed in Examples 6 to 12 and 19) and any fusion polypeptide disclosed by the even-numbered sequence identifiers of SEQ ID NO:4 to 604 and 608 to 640 in the sequence table cited.

[0138] In various embodiments, codons are preferably selected to accommodate the host cell in which the recombinant protein is produced. For example, codons preferred for bacteria are used to express genes in bacteria; codons preferred for yeast are used to express genes in yeast; and codons preferred for mammals are used to express genes in mammalian cells.

[0139] In some embodiments, the polynucleotides encode polypeptides comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the reference sequences of the even-numbered sequence identifiers of SEQ ID NOs: 4 to 604 and 608 to 640. Wherein these polypeptides have alcohol nitrile hydroformylase activity and one or more improved properties described herein, for example, the ability to convert 1,1,1-trifluoropropan-2-one to Compound (IA) with increased stereoselectivity compared to the polypeptide of SEQ ID NOs: 2 and / or 606.

[0140] In some embodiments, the polynucleotides encode engineered alcohol nitrile aldolase polypeptides comprising an amino acid sequence having the aforementioned percentages of identity and having one or more amino acid residue differences compared to SEQ ID NO:606.

[0141] In some embodiments, the disclosure provides an engineered polypeptide having alcohol nitrile hydroformylase activity, wherein the engineered polypeptide has at least 80% sequence identity to the reference sequence of SEQ ID NO: 2 and comprises a combination of residue differences selected from the following positions: X2, X12, X28, X29, X32, X39, X50, X55, X64, X105, X111, X147, X152, X154, X160, X185, X196, X203, X208, X209, X232, X233, X250.

[0142] In some embodiments, the polynucleotide encoding the engineered alcohol nitrile aldolase polypeptide comprises a sequence having odd-numbered sequence identifiers of SEQ ID NOs: 3-603 and 607-639.

[0143] In some embodiments, these polynucleotides encode polypeptides as described herein; however, at the nucleotide level, these polynucleotides have about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with reference polynucleotides encoding engineered alcohol nitrile hydroformylase polypeptides as described herein. In some embodiments, the reference polynucleotide is selected from sequences having odd sequence identifiers of SEQ ID NOs: 3 to 603 and 607 to 639.

[0144] An isolated polynucleotide encoding an engineered alcohol nitrile aldolase polypeptide can be manipulated in a variety of ways to achieve expression of the engineered polypeptide, including further modification of the sequence by codon optimization to improve expression, insertion into appropriate expression elements with or without additional control sequences, and transformation into a host cell suitable for expression and production of the engineered polypeptide.

[0145] Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide prior to inserting it into a vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided by Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F., ed., Greene Pub. Associates, 1998, updated in 2010.

[0146] In another aspect, the disclosure also relates to recombinant expression vectors, which, depending on the type of host into which they will be introduced, include polynucleotides encoding engineered alcohol nitrile hydroformylase polypeptides or variants thereof, and one or more expression regulatory regions, such as promoters and terminators, replication origins, etc. Alternatively, the nucleic acid sequence of the disclosure can be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate expression vector. When generating an expression vector, the coding sequence is located in the vector so that the coding sequence is connected to a suitable expression control sequence. The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be conveniently used in a recombinant DNA program and can result in the expression of a polynucleotide sequence. The selection of the vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid. The expression vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any tool for ensuring self-replication. Alternatively, the vector may be a vector that, when introduced into a host cell, is integrated into the genome and replicates together with the chromosome into which it is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell may be used.

[0147] Many expression vectors that can be used in embodiments of the present disclosure are commercially available. Exemplary expression vectors can be prepared by inserting a polynucleotide encoding an engineered alcohol nitrile hydroformylase polypeptide into a plasmid pACYC-Duet-1 (Novagen), a pBR322 vector (New England Biolabs), a pUC19 vector (New England Biolabs), or a pET T7 expression vector (Novagen).

[0148] In another aspect, the disclosure provides a host cell comprising a polynucleotide encoding an engineered alcohol nitrile aldolase polypeptide of the disclosure. The polynucleotide is connected to one or more control sequences for expressing the alcohol nitrile aldolase polypeptide in the host cell. Host cells for expressing polypeptides encoded by the expression vector of the disclosure are well known in the art, including but not limited to bacterial cells, such as Escherichia coli, Streptomyces cells and Salmonella typhimurium; fungi (such as Saccharomyces cerevisiae or Pichia pastoris); insect cells, such as Drosophila S2 and Spodoptera litura Sf9; animal cells, such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. An exemplary host cell is Escherichia coli BL21 (DE3). The above host cells can be wild-type or can be engineered cells by genome editing, such as knocking out the wild-type alcohol nitrile aldolase gene carried in the genome of the host cell. The culture medium and growth conditions suitable for the above host cells are well known in the art.

[0149] The polynucleotides for expressing the engineered alcohol nitrile hydroformylase can be introduced into the cells by various methods known in the art. Techniques include electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Different methods for introducing polynucleotides into cells are known to those skilled in the art.

[0150] Methods for producing engineered alcohol nitrile hydroformylase polypeptides

[0151] Engineered alcohol nitrile hydroformylase can be obtained by mutagenesis and / or directed evolution of polynucleotides encoding alcohol nitrile hydroformylase. Exemplary directed evolution techniques can be found in "Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing" (2009 John Wiley & Sons Asia (Pte) Ltd. ISBN: 978-0-470-82314-9).

[0152] When the sequence of the engineered polypeptide is known, the coding polynucleotide can be prepared by a standard solid phase method according to a known synthesis method. In certain embodiments, a fragment of up to about 100 bases can be synthesized separately and then connected (for example, by an enzyme or chemical connection method or a polymerase-mediated method) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using, for example, Beaucage et al., 1981, Tet Lett [Tetrahedron Express] 22: 1859-69 or Matthes et al., 1984, EMBO J. [European Molecular Biology Association Journal] 3: The classical phosphoramidite method described in 801-05 is prepared, as typically practiced in an automatic synthesis method. According to the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, connected and cloned into a suitable vector in, for example, a DNA automatic synthesizer. In addition, substantially any nucleic acid can be obtained from any of a variety of commercial sources.

[0153] In some embodiments, the present disclosure also provides a method for preparing or producing an engineered alcohol nitrile hydroformylase polypeptide, which is capable of converting 1,1,1-trifluoropropan-2-one to compound (IA) under suitable reaction conditions, wherein the method includes culturing a host cell capable of expressing a polynucleotide encoding the engineered polypeptide under culture conditions suitable for expressing the polypeptide. In some embodiments, the method for preparing the polypeptide further includes isolating the polypeptide. The engineered polypeptide can be expressed in a suitable cell and separated (or recovered) from the host cell and / or culture medium using any one or more well-known protein purification techniques, including lysozyme treatment, ultrasonic treatment, filtration, salting out, ultracentrifugation, chromatography, and the like.

[0154] Methods of using engineered alcohol nitrile aldolases and compounds made therefrom

[0155] The present disclosure also provides a method for preparing a wide range of compounds or structural analogs thereof using the engineered alcohol nitrile aldolase polypeptides disclosed herein.

[0156] Therefore, the present disclosure provides a method for asymmetric synthesis of β-nitro alcohols using the engineered alcohol nitrile aldolase polypeptide disclosed herein, the method comprising the step of contacting a nitroalkane and an aldehyde or ketone substrate with the alcohol nitrile aldolase polypeptide described herein to obtain a β-nitro alcohol product.

[0157] The selection of applicable electrophiles ranges from aromatic to heteroaromatic and aliphatic aldehydes and ketones. Depending on the substrate and the reaction system, yields up to at least 90% or >99% enantiomeric excess can be obtained by the methods of the present disclosure.

[0158] In embodiments, the aldehyde or ketone substrate comprises an electron withdrawing substituent.

[0159] In an embodiment, the present disclosure also provides a method for asymmetric synthesis of β-nitro alcohols using the engineered alcohol nitrile hydroformylase polypeptide disclosed herein, wherein the resulting β-nitro alcohol has a structure shown in formula (I):

[0160]

[0161] in:

[0162] R 1 and R 2 Each independently selected from H, alkyl such as C1-C 20 Alkyl, alkenyl such as C2-C 20 Alkenyl, alkynyl such as C2-C 20 Alkynyl, cycloalkyl such as C3-C 10 Cycloalkyl, aryl such as C6-C 14 Aryl, arylalkyl such as C7-C 20 Arylalkyl, heterocyclic group such as 3-14 membered heterocyclic group and heteroaryl such as 5-20 membered heteroaryl,

[0163] wherein alkyl, alkenyl and alkynyl are each optionally substituted with one or more R a , for example, one to six R a replace,

[0164] wherein cycloalkyl, aryl, arylalkyl, heterocyclyl and heteroaryl are each optionally substituted by one or more R b , for example, one to six R b replace;

[0165] R 3 and R 4 Each independently selected from H and alkyl such as C1-C 20 Alkyl, wherein the alkyl group is C1-C 20 The alkyl group is optionally substituted with one or more R b , for example, one to six R b replace;

[0166] Each R a Each occurrence is independently selected from cycloalkyl such as C3-C 10 Cycloalkyl, aryl such as C6-C 14 Aryl, arylalkyl such as C7-C 20 Arylalkyl, heterocyclic group such as 3-14 membered heterocyclic group and heteroaryl such as 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -OR c 、-NR c R c 、-(CH2) n COOR c、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c ;

[0167] Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c 、C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl;

[0168] Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 alkynyl, each of which is optionally substituted with one or more Rb, such as one to six Rb; and

[0169] n is 0, 1, 2, 3, 4, 5 or 6, such as 0, 1, 2 or 3;

[0170] The method comprises reacting a nitroalkane having the formula (II), namely R 3 R 4 CHNO2, and an aldehyde or ketone substrate having a formula (III) are contacted with an alcohol nitrile aldolase polypeptide as disclosed herein to obtain a β-nitro alcohol product having a formula (I),

[0171]

[0172] In an embodiment, the engineered alcohol nitrile aldolase polypeptide can be used in a method for preparing a β-nitroalcohol compound (eg, a compound having structural formula (I) as defined above).

[0173] The addition of a nitroalkane other than nitromethane to an aldehyde or ketone substrate generates two new stereocenters simultaneously. The addition of nitromethane to an aldehyde or ketone substrate generates a new stereocenter.

[0174] Thus, in embodiments, the β-nitro alcohol product (e.g., a compound of formula (I)) is present in diastereomeric excess relative to the other diastereomers. In embodiments, the β-nitro alcohol product is present in a diastereomeric excess of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0175] In embodiments, the β-nitro alcohol product (e.g., a compound of formula (I)) has an enantiomeric excess relative to the other enantiomer. In embodiments, the β-nitro alcohol product is present in an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0176] In embodiments, the alcohol nitrile hydroformylase polypeptides have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2 and are capable of coupling an aldehyde or ketone substrate, for example, of formula (III) and a nitroalkane substrate, for example, nitromethane, for example, of formula (II) to form a β-nitroalcohol product, for example, of formula (I), with higher conversion and / or higher stereoselectivity than SEQ ID NO: 606.

[0177] As disclosed herein, alcohol nitrile aldolase polypeptides useful in the methods of the present disclosure can be characterized by the ability to condense 1,1,1-trifluoropropan-2-one and nitromethane to form (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA).

[0178] Thus, in any embodiment of the methods disclosed herein, the method can be performed wherein the alcohol nitrile hydroformylase polypeptide is capable of coupling 1,1,1-trifluoropropan-2-one and nitromethane to form (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA) with a higher conversion rate and / or a higher stereoselectivity than SEQ ID NO:606, and has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2.

[0179] In an embodiment, the resulting β-nitro alcohol has the structure shown in formula (Ii):

[0180]

[0181] in:

[0182] R 1 and R 2 Each independently selected from H, alkyl such as C1-C 20 Alkyl, alkenyl such as C2-C 20 Alkenyl, alkynyl such as C2-C 20 Alkynyl, cycloalkyl such as C3-C 10 Cycloalkyl, aryl such as C6-C 14 Aryl, arylalkyl such as C7-C 20 Arylalkyl, heterocycloalkyl such as 3-14 membered heterocycloalkyl and heteroaryl such as 5-20 membered heteroaryl,

[0183] wherein alkyl, alkenyl and alkynyl are each optionally substituted with one or more R a , for example, one to six R a replace,

[0184] wherein cycloalkyl, aryl, arylalkyl, heterocycloalkyl and heteroaryl are each optionally substituted with one or more R b , for example, one to six R b replace;

[0185] Each R a Each occurrence is independently selected from cycloalkyl such as C3-C 10 Cycloalkyl, aryl such as C6-C 14 Aryl, arylalkyl such as C7-C 20 Arylalkyl, heterocycloalkyl such as 3-14 membered heterocycloalkyl and heteroaryl such as 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c ;

[0186] Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2)n -C(=O)R c 、-(CH2) n -C(=O)NR c R c 、C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl;

[0187] Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl, each of which is optionally substituted by one or more R b , for example, one to six R b substituted; and n is 0, 1, 2, 3, 4, 5 or 6, such as 0, 1, 2 or 3;

[0188] The method comprises the step of contacting nitromethane and an aldehyde or ketone substrate with an alcohol nitrile aldolase polypeptide according to the present disclosure to obtain a β-nitro alcohol product having formula (Ii), the aldehyde or ketone substrate having formula (III),

[0189]

[0190] In any of the embodiments of the methods disclosed herein, R 1 and R 2 Each independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocycloalkyl and 5-20 membered heteroaryl, wherein the C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Each alkynyl group is optionally substituted by one to six R a replace,

[0191] Among them, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocycloalkyl and 5-20 membered heteroaryl are each optionally substituted by one to six R b replace;

[0192] Each R a Each occurrence is independently selected from C3-C 10Cycloalkyl, C6-C 14 Aryl, 3-14 membered heterocycloalkyl, 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c ;

[0193] Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c 、C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl;

[0194] Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl; and

[0195] n is 0, 1, 2, 3, 4, 5 or 6, for example 0, 1, 2 or 3.

[0196] In any of the embodiments of the methods disclosed herein, R 1 and R 2 Each independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocycloalkyl and 5-20 membered heteroaryl,

[0197] Among them, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Each alkynyl group is optionally substituted by one to six R a replace,

[0198] Among them, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocycloalkyl and 5-20 membered heteroaryl are each optionally substituted by one to six R b replace;

[0199] Each R a Each occurrence is independently selected from C3-C 10 Cycloalkyl, C6-C 14 Aryl, 3-14 membered heterocyclic group, 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -OR c and-NR c R c ;

[0200] Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c 、-NR c R c 、C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl;

[0201] Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl.

[0202] In any of the embodiments of the methods disclosed herein, R 1 and R 2 Each independently selected from H, C1-C 20 Alkyl, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocyclic group and 5-20 membered heteroaryl group,

[0203] Among them, C1-C 20The alkyl group is optionally substituted by one to six R a replace,

[0204] Among them, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocyclyl and 5-20 membered heteroaryl are each optionally substituted by one to six R b replace,

[0205] Each R a Each occurrence is independently selected from halogen such as F, C1-C 20 Haloalkyl such as -CF3, -CN, -OR c and –NR c R c ;

[0206] Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c and-NR c R c ;

[0207] Each R c Each occurrence is independently selected from H and C1-C 20 alkyl.

[0208] In any of the embodiments of the methods disclosed herein, R 1 Selected from hydrogen and C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a Replacement, and R 2 It is C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace,

[0209] Each R a Each occurrence is independently selected from halogen such as F, C1-C 20 Halogenated alkyl such as C1-C 20 Fluoroalkyl, such as -CF3.

[0210] In any of the embodiments of the methods disclosed herein, R 1 and R 2 At least one of them is C1-C 20 Fluoroalkyl, for example C1-C6 fluoroalkyl.

[0211] In any of the embodiments of the methods disclosed herein, R1 is selected from hydrogen and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one to six F, and R 2 It is C1-C6 alkyl or phenyl.

[0212] In any of the embodiments of the methods disclosed herein, R 1 is selected from hydrogen and trifluoromethyl, and R2 is methyl or phenyl.

[0213] In any of the embodiments of the methods disclosed herein, R 3 and R 4 Each independently selected from H and C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace,

[0214] Each R a Each occurrence is independently selected from C3-C 10 Cycloalkyl, C6-C 14 Aryl, 3-14 membered heterocyclic group, 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -OR c and-NR c R c ;

[0215] Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl.

[0216] In any of the embodiments of the methods disclosed herein, R 3 and R 4 Each independently selected from H and C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace,

[0217] Each R a Each occurrence is independently selected from halogen such as F, C1-C 20 Haloalkyl such as -CF3, -OR c and –NR c R c ;

[0218] Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C20 Alkynyl.

[0219] In any of the embodiments of the methods disclosed herein, R 3 and R 4 Each independently selected from H and C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace,

[0220] Each R a Each occurrence is independently selected from halogen such as F, C1-C 20 Haloalkyl such as -CF3, -OR c and –NR c R c ;

[0221] Each R c Each occurrence is independently selected from H and C1-C 20 alkyl.

[0222] In any embodiments of the methods disclosed herein, the substrate is a ketone.

[0223] In any embodiments of the methods disclosed herein, the nitroalkane substrate is nitromethane or nitroethane.

[0224] In any embodiments of the methods disclosed herein, the ketone substrate is

[0225]

[0226] The present disclosure also provides a method for asymmetric synthesis of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA):

[0227]

[0228] The method comprises the step of contacting nitromethane and 1,1,1-trifluoropropan-2-one with an alcohol nitrile hydroformylase polypeptide as described in the present disclosure to obtain (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol having formula (IA).

[0229] As disclosed herein and illustrated in the examples, the present disclosure contemplates a range of suitable reaction conditions that can be used in the methods herein, including but not limited to pH, temperature, buffer, solvent system, substrate loading, mixtures of product stereoisomers (such as enantiomers), polypeptide loading, cofactor loading, pressure and reaction time. Additional suitable reaction conditions for carrying out the method of enzymatically converting a substrate compound into a product compound using the engineered alcohol nitrile aldolase polypeptides described herein can be readily optimized by routine experimentation, including but not limited to contacting the engineered alcohol nitrile aldolase polypeptides with the substrate compound under experimental reaction conditions of varying concentrations, pH, temperature, solvent conditions, and detecting the product compound, for example, using the methods described in the examples provided herein.

[0230] As described above, the engineered polypeptide having alcohol nitrile hydroformylase activity for use in the methods of the present disclosure generally comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a reference amino acid sequence selected from any one of the even-numbered sequences of SEQ ID NOs: 4 to 604 and 608 to 640.

[0231] In consideration of the amount of, for example, desired product compounds, the influence of substrate concentration on enzyme activity, the stability of enzyme under reaction conditions, and the conversion percentage of substrate to product, the substrate compound in the reaction mixture can vary. In any embodiment of the method disclosed herein, suitable reaction conditions include at least about 0.5 to about 200g / L, about 1 to about 200g / L, about 5 to about 150g / L, about 10 to about 150g / L, or about 50 to about 150g / L of a load, for example, an aldehyde or ketone substrate with formula (III). In an embodiment, suitable reaction conditions include at least about 0.5g / L, at least about 1g / L, at least about 5g / L, at least about 10g / L, at least about 15g / L, at least about 20g / L, at least about 30g / L, at least about 50g / L, at least about 75g / L, at least about 100g / L or even more loads, for example, an aldehyde or ketone substrate with formula (III). The substrate loading values ​​provided herein are based on the molecular weight of the aldehyde or ketone substrate, for example, having formula (III), however it is also contemplated that equimolar amounts of various hydrates and salts of the aldehyde or ketone substrate, for example, having formula (III) may also be used in the process.

[0232] In any embodiment of the method disclosed herein, the engineered alcohol nitrile hydroformylase polypeptide uses an aldehyde or ketone substrate and a nitroalkane compound to form a β-nitro alcohol product compound. In an embodiment, suitable reaction conditions include the presence of a nitroalkane in an amount of at least about 1 times the molar loading of an aldehyde or ketone substrate, for example, of formula (III). In an embodiment, the nitroalkane is present in an amount of 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the molar loading of an aldehyde or ketone substrate, for example, of formula (III).

[0233] In any embodiments of the methods disclosed herein, suitable reaction conditions include the nitroalkane present at a loading of 0.5 to about 200 g / L, about 1 to about 200 g / L, about 5 to about 150 g / L, about 10 to about 150 g / L, or about 50 to about 150 g / L of the nitroalkane substrate. In embodiments, suitable reaction conditions include at least about 0.5 g / L, at least about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, at least about 75 g / L, at least about 100 g / L, or even more loadings of the nitroalkane substrate.

[0234] In any embodiment of the method disclosed herein, reaction conditions may include suitable pH. As described above, the desired pH or desired pH range may be maintained by using an acid or base, a suitable buffer or a combination of a buffer and an added acid or base. The pH of the reaction mixture may be controlled before and / or during the reaction. In some embodiments, suitable reaction conditions include a solution pH of about 4 to about 8, a pH of about 5 to about 7, a pH of about 6 to about 7. In some embodiments, reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8.

[0235] In any embodiment of the method disclosed herein, considering, for example, the increase of reaction rate at a higher temperature, the activity of the enzyme in the sufficient reaction duration, suitable temperature can be used for reaction conditions. Therefore, in some embodiments, suitable reaction conditions include a temperature of about 10°C to about 60°C, about 25°C to about 50°C, about 25°C to about 40°C, about 25°C to about 30°C, or about 10°C to about 30°C. In some embodiments, suitable reaction temperature includes a temperature of about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a certain temperature during the entire reaction process. In some embodiments, the temperature during the enzymatic reaction can be regulated with a certain temperature spectrum during the reaction process.

[0236] The methods using the engineered alcohol nitrile hydroformylase are usually carried out in a solvent. However, these methods can also be carried out in the absence of a solvent.

[0237] Suitable solvents include water, aqueous buffer, organic solvents and / or co-solvent systems, which generally include aqueous solvents and organic solvents. The organic solvent can be any organic solvent. Preferably, it should not interfere with or inhibit the enzymatic reaction. In certain embodiments, the organic solvent is miscible with water or partially miscible with water. The organic solvent is especially an aprotic organic solvent.

[0238] The aqueous solution (water or aqueous co-solvent system) can be pH buffered or non-buffered. In some embodiments, the method using the engineered alcohol nitrile hydroformylase polypeptide is generally carried out in a solvent system comprising an organic solvent. In some embodiments, the method using the engineered alcohol nitrile hydroformylase polypeptide is generally carried out in an aqueous co-solvent system comprising an organic solvent.

[0239] In any embodiments of the methods disclosed herein, the organic solvent is selected from methanol, ethanol, propanol, isopropanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), isopropyl acetate, ethyl acetate, butyl acetate, 1-octanol, hexane, heptane, octane, methyl tert-butyl ether (MTBE), toluene, benzene, glycerol, polyethylene glycol, and ionic liquids such as 1-ethyl-4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, and 1-butyl-3-methylimidazolium hexafluorophosphate.

[0240] The organic solvent component of the aqueous co-solvent system can be miscible with the aqueous component to provide a single liquid phase, or can be miscible or immiscible with the aqueous component part to provide two liquid phases. Exemplary aqueous co-solvent systems comprise water and one or more organic solvents as defined herein. In general, the organic solvent component of the aqueous co-solvent system is selected so that it will not completely inactivate the alcohol nitrile hydroformylase. By using enzyme activity assays (such as those described herein) to measure the enzyme activity of specific engineered alcohol nitrile hydroformylase in a candidate solvent system with a defined target substrate, a suitable co-solvent system can be easily identified. In any embodiments of the methods disclosed herein, suitable reaction conditions include an aqueous co-solvent system comprising isopropyl acetate at a concentration of about 1% (v / v) to about 60% (v / v), about 1% (v / v) to about 50% (v / v), about 1% (v / v) to about 40% (v / v), about 2% (v / v) to about 40% (v / v), about 5% (v / v) to about 40% (v / v), about 10% (v / v) to about 40% (v / v), about 10% (v / v) to about 30% (v / v), or about 10% (v / v) to about 20% (v / v). In some embodiments of the method, suitable reaction conditions include an aqueous co-solvent system comprising isopropyl acetate at a concentration of at least about 1% (v / v), 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v), 25% (v / v), 30% (v / v), 35% (v / v), 40% (v / v), 45% (v / v), 50% (v / v), 55% (v / v), or 60% (v / v).

[0241] Suitable reaction conditions can include a combination of reaction parameters that provide for the biocatalytic conversion of a substrate compound into its corresponding product compound. Thus, in some embodiments of the method, the combination of reaction parameters comprises: (a) a substrate loading, such as a 1,1,1-trifluoropropan-2-one loading of about 5 g / L to about 150 g / L; (b) a nitroalkane loading, such as a nitromethane loading of about 2 times the molar amount of the substrate, such as 1,1,1-trifluoropropan-2-one; (c) an engineered polypeptide concentration of about at least 3 g / L; (d) an aqueous isopropyl acetate concentration of about 1% (v / v) to about 60% (v / v); (e) a pH of about 4.0 to 8.0; and (f) a temperature of about 10° C. to 30° C.

[0242] Exemplary reaction conditions include the assay conditions provided in the Examples section.

[0243] When performing the reactions described herein, the engineered alcohol nitrile aldolase polypeptide biocatalyst can be added to the reaction mixture in different formulations, such as frozen or lyophilized whole cells (FWC or LWC) transformed with genes encoding engineered alcohol nitrile aldolase polypeptides and / or cell lysates or lyophilized cell lysates of such cells, i.e., so-called shake flask powders (SFP), wherein cell debris is removed and / or further purified to fermentation powder (FP). Whole cells or cell extracts, lysates, and isolated enzymes transformed with genes encoding engineered alcohol nitrile aldolase can be used in a variety of different forms, including solids (e.g., freeze-dried solids, spray-dried solids, etc.) or semi-solids (e.g., coarse pastes). Cell extracts or cell lysates can be partially purified by precipitation (e.g., ammonium sulfate, polyethyleneimine, heat treatment, etc.) prior to lyophilization, followed by desalting procedures (e.g., ultrafiltration, dialysis, etc.). Any enzyme preparation can be stabilized by cross-linking or immobilization to a solid phase material (e.g., resin) using known cross-linking agents (e.g., glutaraldehyde).

[0244] In any embodiment of the method disclosed herein, the reaction is carried out under suitable reaction conditions as described herein, wherein the engineered alcohol nitrile hydroformylase polypeptide is fixed to a solid support, such as a film, resin, solid carrier or other solid phase material. The solid support can be composed of an organic polymer, such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethylene oxide, polymethacrylate and polyacrylamide and copolymers and grafts thereof. The solid support can also be inorganic, such as glass, silica, controlled porous glass (CPG), reversed phase silica or metal, such as gold or platinum. The configuration of the solid support can be in the form of microbeads, balls, microparticles, particles, gels, films or surfaces. The surface can be planar, substantially planar or non-planar. The solid support can be porous or non-porous and can have swelling or non-swelling properties. The solid support can be configured in the form of a hole, depression or other container, vessel, feature or position. Solid supports that can be used to immobilize the engineered alcohol nitrile aldolase for reaction include, but are not limited to, microbeads or resins, such as polymethacrylates, such as polymethacrylates with epoxy functional groups, polymethacrylates with aminoepoxy functional groups, polymethacrylates, styrene / DVB copolymers, or polymethacrylates with octadecyl functional groups.

[0245] In particular embodiments, the solid support is microbeads or resins comprising polymethacrylate.

[0246] Exemplary solid supports include, but are not limited to, chitosan microbeads, Eupergit C, IB-150, IB-350, IB-C435, IB-A369, IB-A161, IB-A171, IBS500, IB-S861, SEPABEADS (Mitsubishi) (e.g., Sepabeads EC-EP, Sepabeads EC-HFA, Sepabeads EC-HG, Sepabeads EC-BU, Sepabeads EC-OD, Sepabeads EC-CM, Sepabeads EC-IDA, Sepabeads EC-EA, Sepabeads EC-HA, Sepabeads EC-QA, Sepabeads EXE, Sepabeads EXA), Dilbeads-TA, Amberzyme Oxirane, Amberlite XAD-7HP, Amberlite FPA98Cl, Amberlite IRA958Cl, AmberliteIRA67, Amberlite FPA90Cl, Amberlite FPA40Cl, Amberlite XAD18, Accurel EP100, ECR8206F / 5730, ECR8206 / 5803, ECR8206M / 5749, ReliZyme EP403, ReliZymeEP113, LewatitVP OC 1600, Diaion WA20, Diaion WA21J, Diaion WA30, Dowex66, Diaion HPA-25L, Lewatit VP OC 1064MD PH, Lewatit VP OC 1163, Lifetech ECR8304F, LifetechECR8309F, Lifetech ECR8315F, Lifetech ECR8204F, Lifetech ECR8285, LifetechECR1090M, Lifetech ECR1030M, Lifetech ECR8806M, Chromalite(MAM2 / F)D6591, Chromalite MIDA / M, Chromalite MIDA / M / Fe, Chromalite MIDA / M / Co, Chromalite MIDA / M / Ni, Chromalite MIDA / M / Cu and Chromalite MIDA / M / Zn.

[0247] In any of the embodiments of the methods disclosed herein, wherein the engineered polypeptide is expressed as a secreted polypeptide, culture medium containing the secreted polypeptide can be used in the methods herein.

[0248] In any embodiment of the methods disclosed herein, solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in a variety of different forms, including powders (e.g., freeze-dried powders, spray-dried powders, etc.), solutions, emulsions, suspensions, etc. The reactants can be easily freeze-dried or spray-dried using methods and instruments known to those skilled in the art. For example, the protein solution can be frozen at -80°C in small aliquots and then added to a pre-freeze drying chamber, and then a vacuum is applied.

[0249] In any embodiment of the method disclosed herein, the order of addition of the reactants is not critical. The reactants can be added to the solvent (e.g., single-phase solvent, two-phase aqueous co-solvent system, etc.) simultaneously, or alternatively, some reactants can be added separately, and some reactants can be added together at different time points. For example, the alcohol nitrile hydroformylase and substrate can be first added to the solvent. In order to improve the mixing efficiency when using the aqueous co-solvent system, the alcohol nitrile hydroformylase can be added first and mixed into the aqueous phase. Then the organic phase can be added, such as isopropyl acetate and mixed, followed by the addition of the substrate. Alternatively, these substrates can be pre-mixed in the organic phase before being added to the aqueous phase.

[0250] The method for carrying out the enzymatic reaction may include the additional step of separating the product of the enzymatic reaction. In particular, this step is carried out after the enzymatic reaction is completed. In particular, the product is separated from one or more (particularly substantially all) other components of the reaction mixture. For example, the product is separated from remaining substrate, by-product, enzyme and / or organic solvent. The separation of the product can be achieved by means and technology known in the art, including for example evaporation of the solvent, aggregation or crystallization and filtration, phase separation, chromatographic separation, etc.

[0251] The engineered alcohol nitrile hydroformylase polypeptide that can be used in any embodiment of the methods disclosed herein can comprise one or more sequences selected from the group consisting of the amino acid sequences corresponding to: SEQ ID NO:4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114 ,116,118,120,122,124,126,128,130,132,134,136,138,140,142,14 4, 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, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378 8、380、382、384、386、388、390、392、394、396、398、400、402、404、406、408、410、412、414、416、418、420、422、424、426、428、430、432、434、436、438、440、442、444、446、448、450、452、454、456、458、460、462、464、466、468、470、472、474、476、478、480、482、484、486、488、490、492、494、496、498、500、502、504、506、508、510、512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638 or 640.

[0252] In any embodiments of the methods disclosed herein, the β-nitroalcohol product, e.g., having formula (IA), is obtained in an enantiomeric excess (ee) of at least 55% ee, e.g., at least 65% ee, e.g., at least 75%, e.g., at least 80%, e.g., at least 90% ee, e.g., at least 95% ee, e.g., at least 99%.

[0253] In any embodiments of the methods disclosed herein, the β-nitroalcohol compound, for example having formula (IA), is obtained with a conversion rate of at least 10%, for example at least 20%, for example at least 30%, for example at least 40%, for example at least 50%, more preferably at least 75%, at least 85%, at least 90%, for example 95%.

[0254] The present disclosure also provides a method for synthesizing (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol having formula (IB):

[0255]

[0256] The method comprises the step of contacting (IA) with hydrogen under suitable hydrogenation conditions to obtain (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol (IB), wherein (IA) is synthesized by the method described in the present disclosure.

[0257] The hydrogen source can be selected from gaseous hydrogen (H2), hydrogen donors (transfer hydrogenation, such as formic acid or its salts), hydride reagents (LiAlH4), etc.

[0258] Suitable hydrogenation conditions include the presence of hydrogen, transition metal catalyst and suitable solvent. In an embodiment, the transition metal catalyst is selected from sponge metal, such as sponge nickel, sponge cobalt, Pd / C, Pt / C, PtO2, Rh / Al2O3 and Pd / BaSO4. In particular, the catalyst is sponge nickel. In an embodiment, the transition metal catalyst is present in a loading of at least 2wt.%, such as at least 5wt.%, at least 10wt.%. Sponge metal or Raney metal catalysts that can be used for reaction contain 0.1% to 10% by weight based on the weight of the catalyst, such as about 0.5% to 3% of platinum as a promoter. Sponge nickel promoted with platinum is a preferred catalyst, although sponge cobalt or Raney cobalt are very suitable. These catalysts can be used in a reaction medium in a loading of at least 2wt.%, such as at least 5wt.%, at least 10wt.%. In an embodiment, the transition metal catalyst is a metal-promoted sponge nickel, such as a platinum-promoted sponge nickel. In an embodiment, the transition metal catalyst is an unpromoted sponge nickel. In an embodiment, the solvent is selected from water, ethanol, methanol, n-propanol, isopropanol, ethyl acetate, isopropyl acetate, butyl acetate, tert-butyl methyl ether and tetrahydrofuran. In an embodiment, the reaction is carried out at a temperature of about 20°C-60°C, such as 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C. In an embodiment, the reaction is carried out at a pressure of about 0.1-40 bar. In an embodiment, the concentration of the compound of formula (IA) is at least 2wt.%, such as 2wt.%, 4wt.%, 8wt.%, 10wt.%. In an embodiment, the hydrogenation reaction is carried out in batches or in a flow.

[0259] In another embodiment, the above method for producing (IB) further comprises the step of converting the compound of formula (IB) into an acid salt. In another embodiment, the method comprises the additional step of crystallizing the acid salt. Preferably, the acid salt is an HCl salt. The crystallization step increases the enantiomeric purity of compound (IB). Advantageously, the ee of compound (IB) having about 92% before separation increases to about 98% after separation of the crystallized compound (IB) HCl salt.

[0260] The present disclosure also provides a method for synthesizing (S)-3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide having formula (IC)

[0261]

[0262] The method comprises the step of contacting nitromethane and 1,1,1-trifluoropropan-2-one with an alcohol nitrile hydroformylase polypeptide as described in the present disclosure to obtain (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol having formula (IA).

[0263] In a particular embodiment, the method further comprises the step of contacting the compound of formula (IA) with hydrogen under suitable hydrogenation conditions to obtain (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol (IB). In another embodiment, the method further comprises the step of converting the compound of formula (IB) into an acid salt. Preferably, the acid salt is an HCl salt (compound (IB). HCl).

[0264] In another embodiment, the method comprises the additional step of crystallizing the acid salt.

[0265] The additional steps required to synthesize compounds of formula (IC) from compounds of formula (IB) or (IB).HCl may be used according to the disclosure of EP 3555048 B1, for example according to Scheme 5 and

[0028] and the Examples herein.

[0266] Alternatively, the method may further comprise coupling the compound of formula (IB).HCl or crystallized (IB).HCl with a compound of formula (E6) in the presence of a coupling reagent under suitable reaction conditions,

[0267]

[0268] To produce a compound of formula (IC).

[0269] Suitable reaction conditions may be those commonly used for the amidation of carboxylic acids, such as reaction conditions known in the art. These may include suitable coupling agents, such as tetramethyl silicate (TMOS), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), for example in the presence of a suitable base (e.g., Et3N, DIEA, DMAP or pyridine), in a suitable solvent (e.g., THF, DCM or toluene), at a suitable temperature (e.g., between 0°C and 120°C). In a particular embodiment, the coupling agent is tetramethyl silicate. In a particular embodiment, suitable reaction conditions include Et3N, toluene and the reaction is heated to about 110°C.

[0270] The overall procedure for the synthesis of compound (IC) is described in Scheme 2, see below.

[0271] Solution 2

[0272]

[0273] abbreviation:

[0274] DMC = dimethyl carbonate [(CH3O)2CO]

[0275] TMPMgClLiCl = lithium chloride (2,2,6,6-tetramethylpiperidin-1-yl) magnesium salt (1-)

[0276] NH3aq = aqueous solution of ammonia

[0277] Cu cat.=Copper sulfate pentahydrate

[0278] TMOS = Methyl orthosilicate

[0279] NEt3 = triethylamine

[0280] In another embodiment, a method for preparing a compound of formula (IC) in free form or in a pharmaceutically acceptable salt form according to Scheme 2 is provided. In another embodiment of Scheme 2, compound (IB).HCl is synthesized according to the present disclosure.

[0281] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative rather than limiting.

[0282] Examples

[0283] The following examples, including experiments and results achieved, are provided for illustrative purposes only and should not be construed as limiting the present invention.

[0284] In the examples below, the following abbreviations apply: ppm (parts per million); M (mole); mM (millimole), uM and μM (micromole); nM (nanomole); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min (minute); h and hr (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); O D600 (optical density at 600 nm), CAM and cam (chloramphenicol); IPAc (isopropyl acetate), DMSO (dimethyl sulfoxide); FP (fermentation powder); FWC (frozen whole cells), LWC (lyophilized whole cells), PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (lysate broth); NM (nitromethane), MeOH (methanol), MTBE (methyl tert-butyl ether), TB (Terrific broth; 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate (pH 7.0), 1 mM MgSO4); PLP (pyridoxal 5'-phosphate), TEoA (triethanolamine buffer), HEPES (HEPES zwitterionic buffer; 4-(2-hydroxyethyl)-piperazineethanesulfonic acid); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); Escherichia coli W3110 (a commonly used laboratory strain of Escherichia coli, available from the Genetic Collection of Escherichia coli [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); GC (gas chromatography), MS (mass spectrometer), RF (Rapid Fire), FIOP (fold improvement over positive control); Microfluidics, Corp. (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Agilent Technologies, Inc. (Agilent Technologies, Inc., Santa Clara, CA); Corning, Inc. (Corning, Inc.), Palo Alto, CA; Dow Corning, Corp., Midland, MI; and Gene Oracle, Inc., Mountain View, CA.

[0285] Throughout the examples, "T100%conv" corresponds to the calculated conversion of each individual sample expressed as a percentage relative to the theoretical 100% conversion value. The amount of nitroaldol product produced in each individual sample was quantified by HPLC using an external standard with a known concentration of nitroaldol product. When 100% conversion was reached, the conversion of each individual sample was obtained by dividing the calculated amount of nitroaldol product produced by the theoretical amount of nitroaldol product, and then multiplying the obtained value by 100 to express the conversion result as a percentage.

[0286] Example 1

[0287] Production of engineered polypeptides in pCK110900

[0288] A polynucleotide (SEQ ID NO: 1) encoding a polypeptide having alcohol nitrile hydroformylase activity (SEQ ID NO: 2) was cloned into the pCK110900 vector system (see, e.g., FIG. 3 of U.S. Patent Application No. 2006 / 0195947A1, which is incorporated herein by reference in its entirety), and then expressed in E. coli W3110fhuA under the control of the lac promoter. The expression vector also contains a P15a origin of replication and a chloramphenicol (CAM) resistance gene. The polynucleotide and related polypeptides are the product of six rounds of directed evolution, which began with the (S)-hydroxynitrile lyase found in Sphaerocarpus punctatus (UniProt D1MX73).

[0289] Example 2

[0290] Preparation of cell pellet

[0291] Escherichia coli W3110fhuA cells were transformed with the pCK110900 plasmid containing the gene encoding the alcohol nitrile hydroformylase. The transformed cells were plated on lysing broth (LB) agar plates containing 1% glucose and 30 μg / mL CAM and grown overnight at 37°C. Subsequently, single colonies were picked in a 96-well format and grown at 30°C, 200rpm and 85% humidity in 190 μL LB medium containing 1% glucose and 30 μg / mL CAM. After overnight growth, 20 μL of the grown culture was transferred to a deep well plate containing 380 μL Terrific broth (TB) (containing 30 μg / mL CAM). The culture was grown for about 2.5 hours at 30°C, 250rpm and 85% humidity. When the optical density (OD 600 ) reached 0.4-0.8, the expression of the alcohol nitrile hydroformylase gene was induced by adding isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 1 mM. After induction, growth was continued for 18-20 hours at 30°C, 250 rpm and 85% humidity. The cells were harvested by centrifugation at 4,000 rpm for 10 minutes at 4°C; the supernatant was then discarded. The cell pellet was stored at -80°C until ready for use.

[0292] Example 3

[0293] Preparation of lysate and clarified lysate

[0294] Prior to the assay, the cell pellet was thawed and resuspended in 300 μL of lysis buffer (containing 1 g / L lysozyme, 0.5 g / L PMBS and 0.1 μL / mL or 0.2 U / ml commercial DNase (New England Biolabs, M0303L) in 0.1 M citrate buffer (pH 6.0). The plate was agitated by shaking at medium speed for 2.5 hours at room temperature on a microtiter plate shaker. The plate was then centrifuged at 4,000 rpm for 10 minutes at 4°C, and the clarified supernatant was used in the HTP assay reaction for activity assays, as described in the following examples.

[0295] Example 4

[0296] Preparation of frozen whole cells (FWC), lyophilized whole cells (LWC), shake flask powder (SFP), and fermentation powder (FP)

[0297] The shake flask procedure can be used to generate engineered alcohol nitrile hydroformylase polypeptide shake flask powder (SFP), which can be used for secondary screening assays and / or for the biocatalytic processes described herein. Compared to the cell lysate used in the HTP assay, the shake flask powder preparation of the enzyme can provide a more concentrated engineered enzyme preparation. To start the culture, a single colony from a plate or a glycerol stock of E. coli containing a plasmid encoding the engineered polypeptide of interest is inoculated into 25 mL of LB (supplemented with 30 μg / mL CAM and 1% glucose) in a 250 ml baffled shake flask. The culture is grown overnight (16-20 hours, OD 600 >3.8). Inoculate 5 mL of overnight culture into 1 L shake flask containing 250 mL TB medium (containing 30 μg / mL CAM). Incubate 250 mL culture at 30°C, 250 rpm for 3-3.5 hours until OD 600 The expression of the alcohol nitrile hydroformylase gene was induced by adding IPTG at a final concentration of 1 mM and continued to grow for another 18-20 hours. The culture was transferred to a centrifuge bottle, which was then centrifuged at 7,000 rpm for 5 minutes at 4°C to harvest the cells. The supernatant was discarded and the remaining cell pellet was lysed, or in some embodiments, stored as frozen whole cells (FWC) at -80°C for later use. For lysis, the cell pellet was resuspended in 30 mL of 50 mM citrate buffer (pH 6.0) and lysed using LM20 Processor system (microfluidics company) is cracked. Cell debris is removed by centrifugation at 14,000rpm for 30 minutes at 4°C. Clarified lysate is collected using standard methods known in the art, frozen at -80°C, and then lyophilized. Lyophilized frozen clarified lysate provides dry shake flask powder (SFP), and lyophilized FWC provides dry lyophilized whole cells (LWC), both of which contain crude engineered alcohol nitrile hydroformylase polypeptides. In order to obtain a large amount of enzymes, these enzymes are necessary for amplification reactions such as in Example 12, and standard methods known in the art are used to ferment cells in a bioreactor. Compared with SFP, due to lower salt content, the fermentation powder (FP) has a higher concentration of engineered alcohol nitrile hydroformylase polypeptides.

[0298] Example 5

[0299] Analytical methods for activity and selectivity evaluation

[0300] The results were analyzed by high pressure liquid chromatography (HPLC) using the method described in Table 5.1 or by Mass spectrometry (RF-MS) analysis of the improved activity of engineered alcohol nitrile hydroformylases. An HPLC method with UV-detection was developed to analyze the conversion of compounds (1) and (2) to compound (IA). Only the nitromethane substrate (2) and the nitroalcohol product (IA) could be detected, while there was no difference between the (R) enantiomers of (IA) vs. (IA). In Examples 9, 10 and 11, RF-MS was used for rapid quantification of product (IA). The corresponding method parameters are described in Table 5.2.

[0301] Table 5.1: HPLC method used for activity determination.

[0302]

[0303]

[0304] Table 5.2: RF-MS methods used for activity determination

[0305]

[0306]

[0307] Selected samples were analyzed for selectivity toward desired (S)-(IA) or undesired (R)-(IA) by gas chromatography (GC) using the method described in Table 5.3.

[0308] Table 5.3: GC method used for selectivity determination.

[0309]

[0310]

[0311] In Example 20, LC-MS was used to detect the products 1,1,1-trifluoro-2-methyl-3-nitrobutan-2-ol (4) and 1,1,1-trifluoro-3-nitro-2-phenylpropan-2-ol (8). The method parameters are described in Table 5.4.

[0312] Table 5.4: LC-MS method used for product detection.

[0313]

[0314]

[0315] In Example 20, GC-MS / FID was used to detect the product 1-nitro-2-phenylpropan-2-ol (6). The method parameters are described in Table 5.5.

[0316] Table 5.5: GC-MS / FID method used for product detection.

[0317]

[0318]

[0319] The methods provided herein can be used to analyze variants generated using the present invention. However, the present invention is not intended to be limited to the methods described herein, as other suitable methods known in the art are also applicable to analyzing variants provided herein and / or variants generated using the methods provided herein.

[0320] Example 6

[0321] For improved compound (IA) production, round 7 evolution and screening of engineered polypeptides derived from SEQ ID NO: 2

[0322] The engineered polypeptides of Table 6-1 were generated using an engineered polynucleotide (SEQ ID NO: 1) encoding a polypeptide having the alcohol nitrile aldolase activity of SEQ ID NO: 2. These polypeptides exhibit improved alcohol nitrile aldolase activity under desired conditions compared to the starting polypeptide, for example, improved formation of a nitro alcohol compound (IA) that is produced in situ from the substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively. Some polypeptides that exhibit improved product formation of the nitro alcohol product (IA) compared to the starting polypeptide are shown in Table 6-1. Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 2 as described below and using the analytical methods described in Table 5-1.

[0323] Directed evolution begins with the polynucleotide set forth in SEQ ID NO: 1. Libraries of engineered polypeptides are generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce Compound (IA) as described below.

[0324] Enzyme assays were performed in 96-well deep-well plates (total volume 1.1 mL) with a total reaction volume of 100 μL per well. These reactions contained 91 v / v% of undiluted alcohol nitrile hydroformylase lysate (prepared as described in Example 3), 0.6 M trifluoroacetone (compound (1)), 0.6 M nitromethane (compound (2)), 0.1 M citrate buffer (pH 5.5). The reaction plates were heat-sealed and shaken at 150 rpm and 22° C.

[0325] After overnight incubation (about 22 hours), 300 μL / well MTBE is added to the reaction plate. The quenched reaction plate is sealed, mixed thoroughly, and centrifuged at 4,000rpm for 5min. Subsequently, an aliquot of 150 μL of the top organic phase is removed from each well and added to a shallow well 96-well plate. In order to determine selectivity, the plate is sealed and analyzed by chiral GC using the analytical method described in Table 5-3. In order to analyze activity by HPLC, the top organic phase of the reaction plate quenched by 20 μL / well is removed and added to a shallow well 96-well plate containing 180 μL of MeOH. The plate is sealed and mixed thoroughly. Using the analytical method described in Table 5-1, these samples are analyzed by HPLC to determine the activity of the enzyme variant. The selected alcohol nitrile hydroformylase variants showing faster formation of product (IA) relative to SEQ ID NO:2 are shown in Table 6.1.

[0326]

[0327]

[0328] Example 7

[0329] For improved compound (IA) production, round 8 of evolution and screening of engineered polypeptides derived from SEQ ID NO: 48

[0330] The engineered polypeptides of Table 7-1 were generated using polynucleotides from Example 6 SEQ ID NO: 47, which encodes the most active polypeptide having the alcohol nitrile aldolase activity of SEQ ID NO: 48. These polypeptides exhibit improved alcohol nitrile aldolase activity under the desired conditions compared to the starting polypeptide, for example, improved formation of nitro alcohol compound (IA), which is produced in situ from the substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively. Some polypeptides that exhibit improved product formation of nitro alcohol product (IA) compared to the starting polypeptide are shown in Table 7-1. Engineered polypeptides with amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 48, as described below and using the analytical methods described in Table 5-1.

[0331] Directed evolution begins with the polynucleotide set forth in SEQ ID NO: 47. Libraries of engineered polypeptides are generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce Compound (IA) as described below.

[0332] Enzyme assays were performed in 96-well deep-well plates (total volume 1.1 mL) with a total reaction volume of 100 μL per well. The reactions contained 20 v / v% undiluted alcohol nitrile hydroformylase lysate (prepared as described in Example 3), 0.62 M trifluoroacetone (compound (1)), 0.63 M nitromethane (compound (2)), and citrate buffer (pH 5.5). The reaction plates were heat-sealed and shaken at 150 rpm and 22° C.

[0333] After overnight incubation (about 22 hours), 600 μL / well MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000rpm for 15min. Subsequently, the 20 μL / well quenched reaction plate was removed and added to a shallow 96-well plate containing 180 μL of MeOH. The plate was sealed and mixed thoroughly. Using the analytical method described in Table 5-1, these samples were analyzed by HPLC to determine the activity of the enzyme variants. The selected alcohol nitrile hydroformylase variants showing faster formation of product (IA) relative to SEQ ID NO: 48 are shown in Table 7.1. Before continuing to evolve as described in Example 8, the selectivity of the (IA) product of the most active polypeptide SEQ ID NO: 74 was confirmed by chiral GC analysis using the analytical method described in Table 5-3. Under given reaction conditions, all variants showed> 75% (> 50% ee) of S-selectivity.

[0334]

[0335]

[0336] Example 8

[0337] For improved compound (IA) production, round 9 of evolution and screening of engineered polypeptides derived from SEQ ID NO: 74

[0338] The engineered polypeptides of Table 8-1 were generated using polynucleotides from Example 7 SEQ ID NO: 73, which encodes the most active polypeptide having the alcohol nitrile aldolase activity of SEQ ID NO: 74. These polypeptides exhibit improved alcohol nitrile aldolase activity under the desired conditions compared to the starting polypeptide, for example, improved formation of nitro alcohol compound (IA), which is generated in situ from the substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively. Some polypeptides that exhibit improved product formation of nitro alcohol product (IA) compared to the starting polypeptide are shown in Table 8-1. Engineered polypeptides with amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 74, as described below and using the analytical methods described in Table 8-1.

[0339] Directed evolution begins with the polynucleotide set forth in SEQ ID NO: 73. Libraries of engineered polypeptides are generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of a polypeptide to produce Compound (IA) as described below.

[0340] Enzyme assays were performed in 96-well deep-well plates (total volume 1.1 mL) with a total reaction volume of 100 μL per well. The reactions contained 15 v / v% undiluted alcohol nitrile hydroformylase lysate (prepared as described in Example 3), 0.62 M trifluoroacetone (compound (1)), 0.63 M nitromethane (compound (2)), citrate buffer (pH 6.0). The reaction plates were heat-sealed and shaken at 150 rpm and 22° C.

[0341] After overnight incubation (about 22 hours), 300 μL / well MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 15 min. Subsequently, the 20 μL / well quenched reaction plate was removed and added to a shallow 96-well plate containing 180 μL of MeOH. The plate was sealed and mixed thoroughly. Using the analytical method described in Table 5-1, these samples were analyzed by HPLC to determine the activity of the enzyme variants. The selected alcohol nitrile hydroformylase variants showing faster formation of product (IA) relative to SEQ ID NO: 74 are shown in Table 8.1. Please note that the alcohol nitrile hydroformylase polypeptide with SEQ ID NO: 142 was rationally constructed. It combines the beneficial mutations of polypeptides SEQ ID NO: 124 and SEQ ID NO: 140. Before continuing to evolve as described in Example 9, the high selectivity of the (IA) product of the most active polypeptides SEQ ID NO: 124, 140 and 142 was confirmed by chiral GC analysis using the analytical method described in Table 5-3. Under the given reaction conditions, all variants showed S-selectivity of >80% (>60% ee).

[0342]

[0343]

[0344] Example 9

[0345] For improved compound (IA) production, round 10 of evolution and screening of engineered polypeptides derived from SEQ ID NO: 142

[0346] The engineered polypeptides of Table 9-1 were generated using polynucleotides from Example 8 SEQ ID NO: 141, which encodes the most active polypeptide having the alcohol nitrile aldolase activity of SEQ ID NO: 142. These polypeptides exhibit improved alcohol nitrile aldolase activity under the desired conditions compared to the starting polypeptide, for example, improved formation of nitro alcohol compound (IA), which is generated in situ from the substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively. Some polypeptides that exhibit improved product formation of nitro alcohol product (IA) compared to the starting polypeptide are shown in Table 8-1. Engineered polypeptides with amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 74, as described below and using the analytical methods described in Table 8-1.

[0347] Directed evolution begins with the polynucleotide set forth in SEQ ID NO: 141. Libraries of engineered polypeptides are generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of a polypeptide to produce Compound (IA) as described below.

[0348] Enzyme assays were performed in 96-well deep-well plates (total volume 1.1 mL) with a total reaction volume of 100 μL per well. The reactions contained 10 v / v% undiluted alcohol nitrile hydroformylase lysate (prepared as described in Example 3), 0.6 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), citrate buffer (pH 6.0). The reaction plates were heat-sealed and shaken at 150 rpm and 22° C.

[0349] After overnight incubation (about 22 hours), 700 μL / well MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000rpm for 15min. Subsequently, the 20 μL / well quenched reaction plate was removed and added to a shallow 96-well plate containing 180 μL of MeOH. The plate was sealed and mixed thoroughly. These samples were diluted 1:400 with MeOH, and the samples were analyzed by RF-MS to determine the activity of the enzyme variants using the analytical method described in Table 5-2. The selected alcohol nitrile hydroformylase variants showing faster formation of product (IA) relative to SEQ ID NO: 142 are shown in Table 9.1. Before continuing to evolve as described in Example 10, the high selectivity of the (IA) product of the most active polypeptide SEQ ID NO: 156 was confirmed by chiral GC analysis using the analytical method described in Table 5-3. Under given reaction conditions, all variants showed> 90% (> 80% ee) of S-selectivity.

[0350]

[0351]

[0352]

[0353] Example 10

[0354] For improved stability and production of compound (IA) in isopropyl acetate, round 11 of evolution and screening of engineered polypeptide derived from SEQ ID NO: 156

[0355] The engineered polypeptides of Table 10-1 were generated using polynucleotides from Example 9 SEQ ID NO: 155 encoding the most active polypeptide having the alcohol nitrile aldolase activity of SEQ ID NO: 156. These polypeptides exhibit improved alcohol nitrile aldolase activity under the desired conditions compared to the starting polypeptide, for example, improved formation of nitro alcohol compound (IA), which is produced in situ from the substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively. Some polypeptides that exhibit improved product formation of nitro alcohol product (IA) compared to the starting polypeptide are shown in Table 10-1. Engineered polypeptides with amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 156 as described below and using the analytical methods described in Table 8-3.

[0356] Directed evolution begins with the polynucleotide set forth in SEQ ID NO: 155. Libraries of engineered polypeptides are generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce Compound (IA) as described below.

[0357] Enzyme assays were performed in 96-well deep well plates (total volume 1.1 mL) with a total reaction volume of 100 μL per well. The reactions contained 5 v / v% undiluted alcohol nitrile hydroformylase lysate (prepared as described in Example 3), 0.6 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), 30% isopropyl acetate in citrate buffer (pH 6.0). The reaction plates were heat sealed and shaken at 150 rpm and 22° C.

[0358] After overnight incubation (about 22 hours), 700 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 15 min.

[0359] Subsequently, the reaction plate of 20 μ L / well quenching is removed and added to a shallow well 96-well plate containing 180 μ L of MeOH. The plate is sealed and fully mixed. These samples are diluted with MeOH at 1:400, and the analytical method described in Table 5-2 is used to analyze the sample by RF-MS to determine the activity of the enzyme variant. The selected alcohol nitrile hydroformylase variants showing the faster formation of product (IA) relative to SEQ ID NO:156 are shown in Table 10.1. Before the continued evolution as described in Example 11, the high selectivity of the (IA) product of the most active polypeptide SEQ ID NO:234 ​​was confirmed by chiral GC analysis using the analytical method described in Table 5-3. Under given reaction conditions, all variants show>92% (>84%ee) of S-selectivity.

[0360]

[0361]

[0362]

[0363] Example 11

[0364] For improved compound (IA) production, round 12 of evolution and screening of engineered polypeptides derived from SEQ ID NO: 234

[0365] The engineered polypeptides of Table 11-1 were generated using the polynucleotide from Example 10 SEQ ID NO: 233 encoding the most active polypeptide having the alcohol nitrile hydroformylase activity of SEQ ID NO: 234.

[0366] Compared to the starting polypeptide, these polypeptides exhibit improved alcohol nitrile hydroformylase activity under the desired conditions, for example, improved formation of nitro alcohol compound (IA), which is generated in situ from the substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively. Some polypeptides that exhibit improved product formation of nitro alcohol product (IA) compared to the starting polypeptide are shown in Table 11-1. Engineered polypeptides with amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 234, as described below and using the analytical methods described in Table 8-3.

[0367] Directed evolution begins with the polynucleotide set forth in SEQ ID NO: 233. Libraries of engineered polypeptides are generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of a polypeptide to produce Compound (IA) as described below.

[0368] Enzyme assays were performed in 96-well deep well plates (total volume 1.1 mL) with a total reaction volume of 100 μL per well. The reactions contained 2.5 v / v% undiluted alcohol nitrile hydroformylase lysate (prepared as described in Example 3), 0.75 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), 30% isopropyl acetate in citrate buffer (pH 6.0). The reaction plates were heat sealed and shaken at 150 rpm and 22° C.

[0369] After overnight incubation (about 22 hours), 650 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 15 min.

[0370] Subsequently, the reaction plate of 15 μ L / well quenching was removed and added to a shallow well 96-well plate containing 185 μ L of MeOH. The plate was sealed and mixed thoroughly. These samples were diluted 1:400 with MeOH, and the activity of the enzyme variants was determined by RF-MS analysis using the analytical method described in Table 5-2. The selected alcohol nitrile hydroformylase variants that show a faster formation of product (IA) relative to SEQ ID NO:234 ​​are shown in Table 11.1. Before the continued evolution as described in Example 12, the high selectivity of the (IA) product of the most active polypeptide SEQ ID NO:410 was confirmed by chiral GC analysis using the analytical method described in Table 5-3. Under given reaction conditions, all variants showed>92% (>84%ee) of S-selectivity.

[0371]

[0372]

[0373]

[0374] Example 12

[0375] For improved compound (IA) production, round 13 of evolution and screening of engineered polypeptides derived from SEQ ID NO: 410

[0376] The engineered polypeptides of Table 12-1 were generated using polynucleotides from Example 11 SEQ ID NO: 409 encoding the most active polypeptide having the alcohol nitrile aldolase activity of SEQ ID NO: 410. These polypeptides exhibit improved alcohol nitrile aldolase activity under the desired conditions compared to the starting polypeptide, for example, improved formation of nitro alcohol compound (IA), which is generated in situ from the substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively. Some polypeptides that exhibit improved product formation of nitro alcohol product (IA) compared to the starting polypeptide are shown in Table 12-1. Engineered polypeptides with amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 410 as described below and using the analytical methods described in Table 8-3.

[0377] Directed evolution begins with the polynucleotide set forth in SEQ ID NO: 409. Libraries of engineered polypeptides are generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce Compound (IA) as described below.

[0378] Enzyme assays were performed in 96-well deep well plates (total volume 1.1 mL) with a total reaction volume of 100 μL per well. The reactions contained 5 v / v% undiluted alcohol nitrile hydroformylase lysate (prepared as described in Example 3), 0.75 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), 30% isopropyl acetate in citrate buffer (pH 6.0). The reaction plates were heat sealed and shaken at 150 rpm and 22° C.

[0379] After overnight incubation (about 22 hours), 650 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 15 min.

[0380] Subsequently, the reaction plate quenched by 15 μ L / well was removed and added to a shallow well 96-well plate containing 185 μ L of MeOH. The plate was sealed and mixed thoroughly. Using the analytical method described in Table 5-1, these samples were analyzed by HPLC to determine the activity of enzyme variants. The selected alcohol nitrile hydroformylase variants showing faster formation of product (IA) relative to SEQ ID NO:410 are shown in Table 12.1. Before continuing to evolve as described in Example 13, the analytical method described in Table 5-3 was used to confirm the S-high selectivity of >95% (>90%ee) of (IA) product of the most active polypeptide SEQ ID NO:414 by chiral GC analysis. Under given reaction conditions, all other variants also showed a high S-selectivity of >94% (>88%ee).

[0381]

[0382]

[0383]

[0384]

[0385] Example 13

[0386] Comparison of catalytic activity and selectivity of wild-type polypeptide SEQ ID NO: 606 and engineered polypeptide SEQ ID NO: 2

[0387] The polynucleotide SEQ ID NO:606 encoding the wild-type (S)-cyanohydrin lyase from Spermum maculatum (Uniprot ID: D1MX73) having SEQ ID NO:605 and the engineered polynucleotide SEQ ID NO:1 encoding the starting enzyme from Example 6 having SEQ ID NO:2 have been used for SFP production as described in Example 4.

[0388] The catalytic activity and selectivity of the substrate trifluoroacetone (1) and nitromethane (2) into the desired nitroalcohol compound (IA) were performed in a 96-well deep well plate (1.1 mL total volume) with a total reaction volume of 100 μL per well. The reactions contained 0 g / L, 0.049 g / L, 0.098 g / L, 0.195 g / L, 0.391 g / L, 0.781 g / L, 1.563 g / L, 3.125 g / L, 6.25 g / L, 12.5 g / L, 25 g / L, 50 g / L of SFP (prepared as described in Example 3), 0.623 M (70 g / L) trifluoroacetone (1), 0.631 M (39 g / L) nitromethane (2) in 100 mM citrate buffer (pH 5.5). Each reaction was performed in duplicate. Two reaction plates were set up in parallel, one for chiral GC analysis and the other for achiral LC analysis. Both reaction plates were heat sealed and shaken at 150 rpm and 22 ° C. After overnight incubation (about 22 hours), 450 μL / well of MTBE was added to the GC reaction plate, and 650 μL / well of MeOH was added to the LC reaction plate. The quenched reaction plates were sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 15 min.

[0389] Subsequently, the 15 μL / well quenched LC reaction plate was removed and added to a shallow 96-well plate containing 185 μL of MeOH. The plate was sealed and mixed thoroughly. These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 5-1.

[0390] To analyze selectivity and enantiomeric excess (ee), 150 μL / well of the top organic phase of the quenched GC reaction plate was removed and added to a shallow 96-well plate. The plate was then analyzed by chiral GC using the analytical method described in Table 5-3.

[0391] FIG1 clearly shows the higher selectivity and activity of SEQ ID 2 compared to the wild-type polypeptide SEQ ID 606.

[0392] Figure 1A and 1B The comparative data in show the conversion percentage and enantiomeric excess of trifluoroacetone (1) and nitromethane (2) into nitroalcohol compound (IA) by wild-type (S)-hydroxynitrile lyase from Sphaerocarpus punctatus having SEQ ID NO: 606 (Uniprot ID: D1MX73) relative to engineered polypeptide SEQ ID NO: 2, respectively. The experiment was performed in duplicate. The reaction mixture contained 0.623 M (70 g / L) trifluoroacetone (1), 0.631 M (39 g / L) nitromethane (2) and SFP catalyst concentration varied from 0-50 g / L in 100 mM citrate buffer (pH 5.5). The engineered polypeptide having SEQ ID NO: 2 showed higher selectivity and activity than the wild-type polypeptide SEQ ID NO: 606. Since the selective enzymatic reaction always competes in parallel with the non-selective chemical reaction, the selectivity (>90%) of the SEQ ID NO: 606 alcohol nitrile hydroformylase only produces a maximum of 25% of the actual ee. Therefore, the evolutionary activities described in this disclosure were proposed to further improve the activity of the enzyme while maintaining high selectivity.

[0393] In subsequent experiments, the best enzymes from each round of evolution have been used to convert the substrates trifluoroacetone (1) and nitromethane (2) into the desired nitroalcohol compounds (IA). Unless otherwise stated, the experimental procedures were the same as those of Figure 1A and 1B The same as in, showing the comparison with the wild type sequence. The enzyme SFP has been prepared as described in Example 3 and used at the following concentrations: 0 g / L, 0.049 g / L, 0.098 g / L, 0.195 g / L, 0.391 g / L, 0.781 g / L, 1.563 g / L, 3.125 g / L, 6.25 g / L, 12.5 g / L SFP. Two different conditions have been studied, (1st): the initial screening conditions of Examples 6 and 7, which are 0.623 M (70 g / L) trifluoroacetone (1), 0.631 M (39 g / L) nitromethane (2) in 100 mM citrate buffer (pH 5.5) (see Figure 2A , 2B) and (Second): Final screening conditions for Examples 11, 12 and 13 with 0.75 M (84 g / L) trifluoroacetone (1), 0.9 M (55 g / L) nitromethane (2), 30% isopropyl acetate in 100 mM citrate buffer (pH 6.0) (see Figure 2C , 2D ). Figure 2A and 2C shows the conversion percentage, while Figure 2B and 2D The enantiomeric excess of the most active polypeptide in each round for the conversion of trifluoroacetone (1) and nitromethane (2) to nitroalcohol compound (IA) is shown. As shown in FIG2 , the ranking of the different enzyme variants matches well with the progress from round to round under both conditions. The alcohol nitrile hydroformylase with SEQ ID NO: 414 showed the highest selectivity and conversion under both conditions. However, although SEQ ID NO: 414 with 12.5 g / L SFP loading reached 91% conversion and 87% ee under condition 1 (see Figure 2A , 2B ), but under the improved second reaction conditions, it achieved a loading of only 6.25 g / L, 100% conversion, and 93% ee (see Figure 2C , 2D ). The enzyme having SEQ ID NO: 414 is then used in an amplification reaction, see Example 14.

[0394] Example 14

[0395] Production of nitroalcohol compound (IA) from substrates trifluoroacetone (1) and nitromethane (2) using engineered polypeptide SEQ ID NO: 414 as catalyst

[0396]

[0397] The most active polypeptide having alcohol nitrile hydroformylase activity from Example 12, namely SEQ ID NO: 414, was produced as fermentation powder and used to convert the substrates trifluoroacetone (1) and nitromethane (2) into nitro alcohol compound (IA). 0.504 g of polypeptide SEQ ID NO: 414 was dissolved in sodium citrate buffer (4.0 mL, 0.1 M, pH 6.0 ± 0.5) and loaded into a reactor precooled to 10° C. A 10° C. solution of trifluoroacetone (12.13 g, 108.3 mmol) in sodium citrate buffer (27.8 mL, 0.1 M, pH 6.0) and additional sodium citrate buffer (78.6 mL, 0.1 M, pH 6.0) were charged into the reactor. The internal temperature and stirring were set to 22°C and 350rpm before adding nitromethane (13.2g, 11.7mL, 216.5mmol) followed by isopropyl acetate (10.2g, 11.7mL). Additional isopropyl acetate (21.1g, 24.3mL) was added and stirred at 22°C for 22h. CellFlock 40 (6.0g) and isopropyl acetate (5.0g, 5.7mL) were added to the emulsion and stirred at 22°C for 2h. The resulting suspension was filtered to provide a clear biphasic mixture. The aqueous layer was separated and extracted with isopropyl acetate. The combined organic layers were concentrated at 50°C jacket temperature and 180-200mbar. Some isopropyl acetate was added to the distillation residue obtained, which was concentrated again. This sequence was repeated once more to obtain the nitro alcohol at the desired concentration in isopropyl acetate (91.7 g solution containing 16.84 g nitro alcohol, 90.8% yield, er = 96:4). 1 H NMR(DMSO-d6):6.96(s,OH),4.92(d,1H),4.82(d,1H),1.48(s,3H)

[0398] Example 15

[0399] Production of nitroalcohol compound (IA) from substrates trifluoroacetone (1) and nitromethane (2) using whole cells containing engineered polypeptide SEQ ID NO: 254

[0400] The polynucleotide having SEQ ID NO: 233 in Example 10, encoding the most active polypeptide having alcohol nitrile hydroformylase activity having SEQ ID NO: 234 in Example 10, was produced in three different enzyme formulations (e.g., SFP, FWC or LWC) as described in Example 4. All three formulations successfully formed nitroalcohol compound (IA), which was generated in situ from the substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively.

[0401] Enzyme assays were performed in 96-well deep well plates (total volume 1.1 mL) with a total reaction volume of 100 μL per well. The reactions contained one of the three alcohol nitrile hydroformylase formulations (SFP, LWC, or FWC) and 0.62 M trifluoroacetone (compound (1)), 0.98 M nitromethane (compound (2)), 30% isopropyl acetate in citrate buffer (pH 6.0). The reaction plates were heat sealed and shaken at 150 rpm and 22°C.

[0402] After overnight incubation (about 22 hours), 300 μL / well MTBE is added to the reaction plate. The quenched reaction plate is sealed, mixed thoroughly, and centrifuged at 4,000rpm for 5min. Subsequently, an aliquot of 150 μL of the top organic phase is removed from each well and added to a shallow well 96-well plate. In order to determine the selectivity, the plate is sealed and analyzed by chiral GC using the analytical method described in Table 5-3. In order to analyze the activity by HPLC, the top organic phase of the reaction plate quenched by 20 μL / well is removed and added to a shallow well 96-well plate containing 180 μL of MeOH. The plate is sealed and mixed thoroughly. Using the analytical method described in Table 5-1, these samples are analyzed by HPLC to determine the activity of the enzyme variant.

[0403] Three formulations derived from the same amount of cells normalized to 20 g / l SFP loading were used: catalyst in SFP, FWC or LWC, and after 20 hours of reaction time, 50% conversion to (IA) was observed with the SFP formulation, 55% conversion was observed with the LWC formulation, and 65% conversion was observed with the FWC. All samples showed 93% S-selectivity.

[0404] Example 16

[0405] Immobilization of engineered polypeptide SEQ ID NO:410

[0406] The fermentation powder of polypeptide SEQ ID NO:410 with alcohol nitrile hydroformylase activity is fixed on solid methoxy methacrylate carrier. This can filter and recycle polypeptide faster, thereby making it possible to provide more flexible method selection for nitroaldol reaction from substrate (1) and (2) to product (IA). As proof of concept, polypeptide SEQ ID NO:410 is cross-linked on epoxy resin (ECR8204F) or amino-functionalized methacrylate resin (ECR8304F) from Purolite Inc. For this reason, 6g of each resin is used and all washing steps are carried out in a ratio of 1:4 (resin: washing solution). Resin is initially washed with fixed buffer, 1M (for ECR8204F) and 50mM (for ECR8304F) sodium citrate buffer (pH 6.0). For crosslinking on amino-functionalized methacrylate resin (ECR8304F), hatch 2 hours with 1% glutaraldehyde solution, and wash activated resin three times with fixed buffer.Afterwards, two kinds of resins and the freeze-dried polypeptide SEQID:410 solution under 15mg / mL are incubated overnight in fixed buffer.Two types of resins (ECR8304F and ECR8204F) are washed once with 20mM sodium citrate buffer (pH 6.0) and washed twice with 0.5M saline.Afterwards, two kinds of resins are washed three times with 50mM sodium citrate buffer (pH 6.0).

[0407] These resins were then used under reaction conditions similar to those in Example 12, with 170 mg of immobilized enzyme incubated at 22° C. for 22 h in 1 ml of 0.1 M citrate buffer (pH 6) containing 0.85 M (1), 1.02 M (2) and isopropyl acetate (IPAc) at concentrations between 20% and 88%. After overnight incubation, 650 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 15 min. Subsequently, the 15 μL / well quenched reaction plate was removed and added to a shallow 96-well plate containing 185 μL of MeOH. The plate was sealed and mixed thoroughly. These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 5-1. Figure 3 shows the activity of the free enzyme compared to the immobilized enzyme on the amino or epoxy supports (see Figure 3A ) and selectivity (see Figure 3B As shown in Figure 3, the conversion rate (see Figure 3A ) and fixed yields, amino resins outperformed epoxy resins, while selectivity was similar in all cases (see Figure 3B). High isopropyl acetate (IPAc) concentrations (ranging between 20% and 88%) were used in the screening. Note that the volume of the substrate solution itself was 12%, and for the sample containing 88 vol% IPAc, the IPAc was saturated with water before the reaction.

[0408] The condition of amino resin has been further optimized, and optimal conditions have been used for the fixed reaction of amplification. For this reason, 30g of ECR8304F from Purolite Co., Ltd. is used, and all washing steps are carried out with a ratio of 1:4 (resin: washing solution). Initially, 50mM sodium citrate (pH 6.0) fixed buffer solution was used to wash the resin. For cross-linking, 1% glutaraldehyde solution was incubated for 2 hours, and the activated resin was washed three times with fixed buffer solution. Afterwards, two kinds of resins were incubated overnight in fixed buffer solution with the FP solution of polypeptide SEQ ID:410 under 20mg / mL. Two kinds of resins were washed once with 20mM sodium citrate buffer solution (pH 6.0), washed twice with 0.5M saline and washed twice with buffer solution. The program produces a fixed yield of 70%-80% and about 30% of the total recovery enzyme activity. 30g of the immobilized polypeptide SEQ ID NO:410 thus generated was incubated at 22°C for 22h in 180ml 0.1M citrate buffer (pH 6) containing 0.61M (1), 1.22M (2) and 30% IPAc, resulting in 80% yield and 92% ee (96% S-selectivity). This immobilized enzyme was recycled twice by filtering the reaction mixture, and no significant loss of reaction yield and selectivity was observed. The second recycling was carried out at 4°C after a total storage time of 25 days.

[0409] Example 17

[0410] Hydrogenation of compounds of formula (IA)

[0411]

[0412] Batch Procedure

[0413] (IA) in isopropyl acetate (70.0 g, 13.7 wt.-%, 9.6 g pure (IA)) was placed in a pressure vessel. A water-wet nickel sponge catalyst (Raney Ni) (0.96 g, 10 wt.-% to pure (IA)) was added, followed by methanol (20 g, 15.8 mL). The vessel was made inert with nitrogen and the atmosphere was then changed to hydrogen. The resulting suspension was stirred at 40 ° C under a positive pressure of hydrogen (20 bar) for 6 h. The reaction mixture was then cooled to room temperature and filtered in a manner to keep the filter residue wet to avoid ignition on contact with air. The conversion to (IB) was measured using GC (99% conversion).

[0414] HCl (1.25M in MeOH, 2.0 eq.) was added and stirred at room temperature for 6 h. The resulting solution was filtered and then concentrated at 40 °C jacket temperature and 60 mbar. Some isopropyl acetate was added to the distillation residue obtained and it was concentrated again. This sequence was repeated twice more to obtain the HCl salt of the amino alcohol without methanol. Acetonitrile was added and the obtained solution was heated to 35 °C jacket temperature, then n-heptane was slowly added, followed by a suspension of the product seed material in n-heptane (one part (IB).HCl and 33 parts n-heptane). Additional n-heptane was added and the resulting mixture was slowly cooled to 5 °C jacket temperature. Stirring was continued for 18 h. The suspension was filtered and the filter cake was washed with some n-heptane. The filter cake was dried under vacuum, then dissolved in acetonitrile and heated to 35 °C jacket temperature. n-heptane was added and then a suspension of the product seed material was added, which consisted of one part (IB).HCl and 33 parts n-heptane. Additional n-heptane was added and the resulting suspension was cooled to -1°C jacket temperature. Stirring was continued for 18 h. The resulting suspension was then filtered and the filter cake was washed with n-heptane. The filter cake was dried under vacuum at room temperature to provide the desired product, i.e., crystalline (IB).HCl (6.1 g, 61% yield, er 99:1). 1 H NMR (DMSO-d6): 8.32 (br s, NH3+), 6.84 (br s, OH), 2.99 (s, 2H), 1.39 (s, 3H). 13 CNMR(DMSO-d6):125.8(CF3),70.4(C-CF3),42.2(CH2),18.6(CH3).

[0415] Mobile Program

[0416] The stainless steel column with an internal diameter of 9.5mm and a length of 90mm is filled with a mixture of water-wet sponge-nickel (14.5g) and inert silicon carbide (9.58g) fully mixed in advance. The column has liquid and gas inlet, and contains a 5-μm sieve plate to prevent solid material from leaving the column at the outlet. At startup, the column of the loaded catalyst is placed in a water-ice bath, and rinsed with a 1:4v / v mixture of methanol and isopropyl acetate for no less than 12 column volumes. The liquid stream is then converted to a solution of 0.25M (IA) in a 1:4v / v mixture of methanol and isopropyl acetate. The feed is also precooled to 0 to 5 degrees Celsius in an ice bath. The feed is supplied to the column at a flow rate of 3mL / min by a gear pump. Hydrogen is produced by the electrolysis of water, and supplied with a flow rate of 99.0mL / min by a mass flow controller, and the whole device is pressurized to 10 bars using a back pressure regulator. Taking into account the presence of hydrogen, an apparent residence time of 31 seconds was calculated. Each pass through the cartridge provided between 50A% and 60A% product conversion, after which the solution containing the product was added back to the feed bottle and passed through the cartridge again until a total of 3 passes had been performed.

[0417] For the experiment of 180 minutes duration, a total of 17.92 g of starting material was used in a volume of 500 mL solvent (4.0 wt%). The conversion of the product after 3 passes was 98 A%, and after workup of the liquid fraction to the hydrochloride salt of the product (according to the standard crystallization method using HCl / methanol as described above), an isolated yield of 75% was observed with a quality of 99 A% HPLC.

[0418] Example 18

[0419] Coupling methods for synthesizing compounds (IC)

[0420]

[0421] E6 (41.4 g, 152 mMol) and crystallized (IB).HCl (30 g, 167 mMol) were loaded into a reactor, followed by 410 ml of toluene. Under stirring, triethylamine (38 g, 380 mMol) was added, followed by tetramethyl silicate (TMOS) (46 g, 304 mMol). The reaction mixture was heated to 110 ° C and stirred for 3 to 16 h, while slowly distilling out the methanol formed by the reaction. The completion of the reaction was monitored by HPLC. An aqueous NaOH solution (330 ml, 2 moles) was added and the mixture was stirred at 50 ° C for 2 h. The aqueous phase was separated and discarded. At 50 ° C, an aqueous HCl solution (330 ml, 10%) was added and the aqueous phase was discarded. After washing with water, the organic solution was dried and concentrated by distillation. (IC) was crystallized by adding 240 ml of n-heptane and cooling to 0 ° C. After drying under vacuum at 40 ° C, a compound (IC) with a yield of 80% (44 g) was obtained. 1 H-NMR(DMSO-d6): δppm 8.29(m,1H),7.67(s,1H),6.68(br,2H),6.28(s,1H),3.92(s,3H),3.69-3.43(m,2H),1.25(s,3H). 13 C-NMR (DMSO-d6): δppm 18.95, 42.2, 53.5, 72.6-71.9 (m), 116.5-115.6 (m), 124.9-121.0 (2C, m), 126.3, 128.3 (m), 140.9, 148.5, 166.3.

[0422] Example 19

[0423] For improved compound (IA) production, round 14 of evolution and screening of engineered polypeptides derived from SEQ ID NO: 606

[0424] The engineered polypeptides of Table 19-1 were generated using polynucleotides from Example 12 SEQ ID NO: 413, which encodes the most active polypeptide having the alcohol nitrile aldolase activity of SEQ ID NO: 414. These polypeptides exhibit improved alcohol nitrile aldolase activity under desired conditions, e.g., higher thermal stability and improved formation of nitro alcohol compound (IA), which is generated in situ from substrates trifluoroacetone and nitromethane, i.e., compounds (1) and (2), respectively, compared to the starting polypeptide. Some polypeptides that exhibit improved product formation of nitro alcohol product (IA) compared to the starting polypeptide are shown in Table 19-1. Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 414, as described below and using the analytical methods described herein.

[0425] Directed evolution begins with the polynucleotide set forth in SEQ ID NO: 413. Libraries of engineered polypeptides are generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of a polypeptide to produce Compound (IA) as described below.

[0426] Enzyme assays were performed in 96-well deep-well plates (total volume 1.1 mL), with a total reaction volume of 100 μL per well. These reactions contained 5 v / v% undiluted alcohol nitrile hydroformylase lysate (prepared as described in Example 3), then pre-incubated for 1 h at 4 ° C ("no heat incubation" was noted in Table 19-1) or 52 ° C ("heat incubation" was noted in Table 19-1). All other conditions were different from the previous round of evolution: 30% isopropyl acetate in 0.75 M trifluoroacetone (compound (1)), 0.9 M nitromethane (compound (2)), citrate buffer (pH 6.0). The reaction plates were heat-sealed and shaken at 150 rpm and 22 ° C.

[0427] After overnight incubation (about 22 hours), 650 μL / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 15 min.

[0428] The reaction plate of 15 μ L / well quenching was then removed and added to a shallow 96-well plate containing 185 μ L of MeOH. The plate was sealed and mixed thoroughly. The activity of these samples was determined by HPLC using the analytical method described in Table 5-1. The selected alcohol nitrile hydroformylase variants that form product (IA) faster than SEQ ID NO:414 are shown in Table 19-1. The S-high selectivity of >95% (>90%ee) of the (IA) product of the most active polypeptide SEQ ID NO:610 and 620 was confirmed by chiral GC analysis using the analytical method described in Table 5-3.

[0429]

[0430]

[0431] Example 20

[0432] Substrate scope of evolved alcohol nitrile aldolase

[0433]

[0434] The most active polypeptides with alcohol nitrile aldolase activity, namely SEQ ID NO: 2, 156, 414 and 606, from different rounds of evolution were produced as shake flask powders (SFP) and tested for their alcohol nitrile aldolase activity on different substrate mixtures. Reaction 1 was a reference reaction to form nitro alcohol compound (IA) using trifluoroacetone (1) and nitromethane (2) as substrates. In reaction 2, the alcohol nitrile aldolase was incubated with trifluoroacetone (1) and nitroethane (3) to study the formation of 1,1,1-trifluoro-2-methyl-3-nitrobutan-2-ol (4). In reaction 3, acetophenone (5) and nitromethane (2) were tested as substrates to investigate the formation of the product 1-nitro-2-phenylpropan-2-ol (6), and in reaction 4, 2,2,2-trifluoro-1-phenylethan-1-one (7) and nitromethane (2) were tested as substrates to investigate the formation of 1,1,1-trifluoro-3-nitro-2-phenylpropan-2-ol (8).

[0435] In the first experimental setup, the activity of the alcohol nitrile hydroformylase variants in reactions 1, 2 and 3 was investigated. The enzyme assay was performed in a 96-well deep well plate (total volume 2 mL) with a total reaction volume of 400 μL per well. The reaction mixture contained 0.1 M sodium citrate buffer (pH 6.0), 10 vol% iPrOAc, and in reaction 1: 1 g / L (9 mM) trifluoroacetone (1) and 11 g / L (180 mM) nitromethane (2); in reaction 2: 1 g / L (9 mM) trifluoroacetone (1) and 13.5 g / L (180 mM) nitroethane (3); and in reaction 3: 1.1 g / L (9 mM) acetophenone (5) and 11 g / L (180 mM) nitromethane (2). The reaction plates were heat sealed and shaken at 200 rpm and 22°C.

[0436] After overnight incubation (about 22 hours), 1.2 ml / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 10 min. Subsequently, the quenched reaction of 200 μL / well was transferred to a shallow well 96-well plate for HPLC-MS analysis to determine the activity of the enzyme variant using the analytical method described in Table 5.4. The relative product peak areas observed for reaction devices 1, 2, and 3 are shown in Table 20-1. The quality of products (IA) and (4) can be confirmed via LC-MS. In reaction 3, product formation of (6) was not observed in LC-MS using the analytical method described in Table 5.4 and in GC-MS / FID using the analytical method described in Table 5.5, indicating that there was no enzyme activity when acetophenone was used as a substrate. The results of reaction 2 show that the substrate acceptance of the evolved alcohol nitrile hydroformylase to nitroethane (3) increased, while the activity of the wild-type enzyme SEQ ID606 was not detected. After initial activity was detected for SEQ ID 2, >25-fold increases in enzyme productivity were achieved in subsequent rounds of evolution, as can be seen when comparing 100 g / L of the less evolved SEQ ID 2 which produced lower product formation than 4 g / L of SEQ ID 414.

[0437] The enzyme assay to obtain the results shown in Table 20-1-Reaction 4 was performed in a 96-well deep well plate (total volume 1.1 mL) with a total reaction volume of 200 μL per well. The reaction mixture contained 0.1 M sodium citrate buffer (pH 6.0), 20 vol% iPrOAc and 5.2 g / L (30 mM) 2,2,2-trifluoro-1-phenylethan-1-one (7) and 36.6 g / L (600 mM) nitromethane (2). The reaction plate was heat sealed and shaken at 200 rpm and 22°C.

[0438] After overnight incubation (about 22 hours), 0.6 ml / well of MeOH was added to the reaction plate. The quenched reaction plate was sealed, mixed thoroughly, and centrifuged at 4,000 rpm for 10 min. Subsequently, 100 μL / well of the quenched reaction was transferred to a shallow 96-well plate containing 100 μL MeOH / well. The samples were then analyzed by LC-MS analysis to determine the activity of the enzyme variants using the analytical method described in Table 5.4. The results of reaction 4 show that the conversion of the evolved alcohol nitrile hydroformylase to product (8) increases. The most evolved alcohol nitrile hydroformylase with SEQ ID 414 shows the highest product formation in all tested substrate combinations.

[0439] Table 20-1: Peak areas of products (IA), (4) and (6) depending on the enzyme SEQ ID 2, 156, 414 or 606 used.

[0440]

[0441]

Claims

1. An engineered alcohol nitrile hydroformylase polypeptide, wherein the engineered alcohol nitrile hydroformylase polypeptide is a polypeptide of the following (a) or (b): (a) a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 8 4, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 1 18, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 14 8、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、244、246、248、250、252、254、256、258、260、262、264、266、268、 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 39 0, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510,512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638 and 640; or (b) A polypeptide having alcohol nitrile hydroformylase activity, the polypeptide comprising an amino acid sequence having (i) at least 80% sequence identity with one of the polypeptides listed in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to the amino acid sequence listed in (a).

2. An engineered alcohol nitrile hydroformylase polypeptide capable of coupling 1,1,1-trifluoropropan-2-one with nitromethane to produce (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol with higher stereoselectivity and / or activity than SEQ ID NO: 606 under suitable reaction conditions.

3. An engineered alcohol nitrile hydroformylase polypeptide, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 606, wherein the engineered alcohol nitrile hydroformylase polypeptide is capable of coupling 1,1,1-trifluoropropan-2-one with nitromethane under suitable reaction conditions to produce at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more enantiomeric excess of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol.

4. The alcohol nitrile hydroformylase polypeptide of claim 3, wherein the suitable reaction conditions comprise about 5 g / L to about 150 g / L 1,1,1-trifluoropropan-2-one, a nitromethane loading of about 2 times the molar amount of 1,1,1-trifluoropropan-2-one, at least 3 g / L of the alcohol nitrile hydroformylase polypeptide, an isopropyl acetate concentration of about 20% (v / v) to about 60% (v / v), a pH of about 4.0 to about 8.0, and a temperature of about 10°C to 30°C.

5. The alcohol nitrile hydroformylase polypeptide of claim 3 or 4, wherein the amino acid sequence of the alcohol nitrile hydroformylase comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 2 at one or more amino acid residues selected from the group consisting of: 2, 12, 28, 29, 32, 39, 50, 55, 64, 105, 111, 147, 152, 154, 160, 185, 196, 203, 208, 209, 232, 233 and 250, wherein the numbering refers to SEQ ID NO: 2, and wherein the polypeptide has alcohol nitrile hydroformylase activity.

6. The alcohol nitrile hydroformylase polypeptide according to claim 5, wherein the amino acid sequence of the alcohol nitrile hydroformylase comprises one or more of the following amino acid residues: or X2 is absent; X12 is I; X28 is G; X29 is W; X32 is T; X39 is F or V; X50 is E or D; X55 is G; X64 is A; X105 is G; X111 is S; X147 is K; X152 is L; X154 is absent; X160 is M; X185 is R; X196 is G; X203 is C; X208 is R or S; X209 is V; X232 is G; X233 is G; or X250 is G; wherein numbering refers to SEQ ID NO:

2.

7. The alcohol nitrile hydroformylase polypeptide of claims 3 to 6, wherein the amino acid sequence of the alcohol nitrile hydroformylase comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 2 at one or more amino acid residues selected from the group consisting of: 39, 105 and 154, wherein the numbering refers to SEQ ID NO: 2, and wherein the polypeptide has alcohol nitrile hydroformylase activity.

8. The alcohol nitrile hydroformylase polypeptide according to claim 7, wherein the amino acid sequence of the alcohol nitrile hydroformylase comprises one or more of the following amino acid residues: X39 is F or V; X105 is G; or X154 is absent; wherein numbering refers to SEQ ID NO:

2.

9. The alcohol nitrile hydroformylase polypeptide of any one of claims 3 and 4, wherein the amino acid sequence of the alcohol nitrile hydroformylase comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 606 at one or more amino acid residues selected from the group consisting of: 2, 11, 12, 28, 29, 32, 33, 39, 43, 44, 46, 50, 55, 64, 80, 103, 105, 111, 118, 121, 147, 152, 154, 160, 172, 180, 185, 196, 203, 208, 209, 232, 233, 238, 241, 250, and 263, wherein the numbering refers to SEQ ID NO: 606, and wherein the polypeptide has alcohol nitrile hydroformylase activity.

10. The alcohol nitrile hydroformylase polypeptide according to claim 9, wherein the amino acid sequence of the alcohol nitrile hydroformylase comprises one or more of the following amino acid residues: X2 does not exist; X11 is S; X12 is V; X28 is G; X29 is W; X32 is T; X33 is V; X39 is F, X43 is S; X44 is N; X46 is H; X50 is E or D; X55 is R or G; X64 is A; X80 is A; X103 is V; X105 is G; X111 is S; X118 is V; X121 is Y; X1 47 is K; X152 is L; X154 is absent; X160 is M; X172 is R; X180 is L; X185 is R; X196 is G; X203 is C; X208 is R or S; X209 is V; X232 is G; X233 is G; X238 is M; X241 is R; X250 is G; or X263 is S; wherein numbering refers to SEQ ID NO:

606.

11. The alcohol nitrile hydroformylase polypeptide of any one of claims 3, 4 and 9, wherein the amino acid sequence of the alcohol nitrile hydroformylase comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 606 at one or more amino acid residues selected from the group consisting of: 2, 105, 111, 154, 160, 185, 209, 232 and 250, wherein the numbering refers to SEQ ID NO: 606, and wherein the polypeptide has alcohol nitrile hydroformylase activity.

12. The alcohol nitrile hydroformylase polypeptide according to claim 11, wherein the amino acid sequence of the alcohol nitrile hydroformylase comprises one or more of the following amino acid residues: X2 does not exist; X105 is G; X111 is S; X154 does not exist; X160 is M; X185 is R; X209 is V; X232 is G; or X250 is G; wherein numbering refers to SEQ ID NO:

606.

13. A polypeptide, which is immobilized on a solid material by chemical bonding or physical adsorption, wherein the polypeptide is selected from the alcohol nitrile hydroformylase polypeptide according to any one of claims 1 to 12.

14. A polynucleotide encoding the polypeptide of any one of claims 1 to 13.

15. The polynucleotide of claim 14, wherein the polynucleotide sequence is SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 4 3, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 8 3, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 11 7, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 14 7, 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, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267 , 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387 ,389,391,393,395,397,399,401,403,405,407,409,411,413,415,417,419,421,423,425,427,429,431,433,435,437,439,441,443,445,447,449,451,453,455,457,459,461,463,465,467,469,471,473,475,477,479,481,483,485,487,489,491,493,495,497,499,501,503,505,507,509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 57 3. 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637 or 639.

16. An expression vector comprising the polynucleotide according to any one of claims 14 and 15.

17. The expression vector of claim 16, comprising a plasmid, a cosmid, a phage or a viral vector.

18. A host cell comprising the expression vector according to any one of claims 16 and 17, wherein the host cell is preferably Escherichia coli.

19. A method for preparing an alcohol nitrile hydroformylase polypeptide, the method comprising the steps of culturing the host cell according to claim 18 and obtaining the alcohol nitrile hydroformylase polypeptide from the culture.

20. An alcohol nitrile hydroformylase catalyst, which can be obtained by culturing the host cell according to claim 18 or according to the method according to claim 19, wherein the alcohol nitrile hydroformylase catalyst comprises cells or culture fluid containing the alcohol nitrile hydroformylase polypeptide, or an article processed therewith, wherein the article refers to an extract obtained from the culture of the transformant cell, an isolated product obtained by isolating or purifying the alcohol nitrile hydroformylase from the extract, or an immobilized product obtained by immobilizing the transformant cell, its extract or the isolated product of the extract.

21. A method for asymmetric synthesis of β-nitro alcohols, comprising the step of contacting a nitroalkane and an aldehyde or ketone substrate with the alcohol nitrile aldolase polypeptide according to any one of claims 1 to 13 to obtain a β-nitro alcohol product.

22. The method of claim 21, wherein the aldehyde or ketone substrate comprises an electron withdrawing substituent.

23. The method of any one of claims 21 and 22, wherein the resulting β-nitro alcohol has the structure shown in formula (I): in: R 1 and R 2 Each independently selected from H, alkyl such as C1-C 20 Alkyl, alkenyl such as C2-C 20 Alkenyl, alkynyl such as C2-C 20 Alkynyl, cycloalkyl such as C3-C 10 Cycloalkyl, aryl such as C6-C 14 Aryl, arylalkyl such as C7-C 20 Arylalkyl, heterocyclic group such as 3-14 membered heterocyclic group and heteroaryl such as 5-20 membered heteroaryl, wherein the alkyl, alkenyl and alkynyl groups are each optionally substituted with one or more R a , for example, one to six R a replace, wherein the cycloalkyl, aryl, arylalkyl, heterocyclyl and heteroaryl groups are each optionally substituted with one or more R b , for example, one to six R b replace; R 3 and R 4 Each independently selected from H and alkyl such as C1-C 20 Alkyl, wherein the alkyl group is C1-C 20 The alkyl group is optionally substituted with one or more R b , for example, one to six R b replace; Each R a Each occurrence is independently selected from cycloalkyl such as C3-C 10 Cycloalkyl, aryl such as C6-C 14 Aryl, arylalkyl such as C7-C 20 Arylalkyl, heterocyclic group such as 3-14 membered heterocyclic group and heteroaryl such as 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c ; Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c 、C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl; Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl, each of which is optionally substituted by one or more R b , for example, one to six R b Replacement; and n is 0, 1, 2, 3, 4, 5 or 6, such as 0, 1, 2 or 3; The method comprises reacting a nitroalkane having the formula (II), namely R 3 R 4 CHNO2, and an aldehyde or ketone substrate having a formula (III) are contacted with the alcohol nitrile aldolase polypeptide according to any one of claims 1 to 13 to obtain a β-nitro alcohol product having a formula (I), 24. The method of claim 23, wherein R 3 and R 4 Each independently selected from H and C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace, Each R a Each occurrence is independently selected from C3-C 10 Cycloalkyl, C6-C 14 Aryl, 3-14 membered heterocyclic group, 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -OR c and-NR c R c ; Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl.

25. The method of any one of claims 23 and 24, wherein R 3 and R 4 Each independently selected from H and C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace, Each R a Each occurrence is independently selected from halogen such as F, C1-C 20 Haloalkyl such as -CF3, -OR c and –NR c R c ; Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl.

26. The method of any one of claims 23 to 25, wherein R 3 and R 4 Each independently selected from H and C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace, Each R a Each occurrence is independently selected from halogen such as F, C1-C 20 Haloalkyl such as -CF3, -OR c and –NR c R c ; Each R c Each occurrence is independently selected from H and C1-C 20 alkyl.

27. The process of any one of claims 21 to 26, wherein the resulting β-nitro alcohol has the structure shown in formula (Ii): in: R 1 and R 2 Each independently selected from H, alkyl such as C1-C 20 Alkyl, alkenyl such as C2-C 20 Alkenyl, alkynyl such as C2-C 20 Alkynyl, cycloalkyl such as C3-C 10 Cycloalkyl, aryl such as C6-C 14 Aryl, arylalkyl such as C7-C 20 Arylalkyl, heterocyclic group such as 3-14 membered heterocyclic group and heteroaryl such as 5-20 membered heteroaryl, wherein the alkyl, alkenyl and alkynyl groups are each optionally substituted with one or more R a , for example, one to six R a replace, wherein the cycloalkyl, aryl, arylalkyl, heterocyclyl and heteroaryl groups are each optionally substituted with one or more R b , for example, one to six R b replace; Each R a Each occurrence is independently selected from cycloalkyl such as C3-C 10 Cycloalkyl, aryl such as C6-C 14 Aryl, arylalkyl such as C7-C 20 Arylalkyl, heterocyclic group such as 3-14 membered heterocyclic group and heteroaryl such as 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c ; Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c 、C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl; Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl, each of which is optionally substituted by one or more R b , for example, one to six R b Replacement; and n is 0, 1, 2, 3, 4, 5 or 6, such as 0, 1, 2 or 3; The method comprises reacting nitromethane and an aldehyde or ketone substrate with The step of contacting the alcohol nitrile hydroformylase polypeptide according to any one of claims 1 to 13 to obtain a β-nitro alcohol product having formula (Ii), wherein the aldehyde or ketone substrate has formula (III), 28. The method of any one of claims 23 to 27, wherein R 1 and R 2 Each independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocyclyl and 5-20 membered heteroaryl, wherein the C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Each alkynyl group is optionally substituted by one to six R a replace, Wherein the C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocyclyl and 5-20 membered heteroaryl are each optionally substituted by one to six R b replace; Each R a Each occurrence is independently selected from C3-C 10 Cycloalkyl, C6-C 14 Aryl, 3-14 membered heterocyclic group, 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c ; Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c 、-NR c R c 、-(CH2) n COOR c 、-(CH2) n -C(=O)R c 、-(CH2) n -C(=O)NR c R c 、C1-C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Alkynyl; Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl; and n is 0, 1, 2, 3, 4, 5 or 6, for example 0, 1, 2 or 3.

29. The method of any one of claims 23 to 28, wherein R 1 and R 2 Each independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocyclyl and 5-20 membered heteroaryl, wherein the C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Each alkynyl group is optionally substituted by one to six R a replace, Wherein the C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocyclyl and 5-20 membered heteroaryl are each optionally substituted by one to six R b replace; Each R a Each occurrence is independently selected from C3-C 10 Cycloalkyl, C6-C 14 Aryl, 3-14 membered heterocyclic group, 5-20 membered heteroaryl, halogen such as F, halogenated alkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -OR c and-NR c R c ; Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c 、-NR c R c 、C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl; Each R c Each occurrence is independently selected from H, C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl.

30. The method of any one of claims 23 to 29, wherein R 1 and R 2 Each independently selected from H, C1-C 20 Alkyl, C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocyclic group and 5-20 membered heteroaryl group, Wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace, Wherein the C3-C 10 Cycloalkyl, C6-C 14 Aryl, C7-C 20 Arylalkyl, 3-14 membered heterocyclyl and 5-20 membered heteroaryl are each optionally substituted by one to six R b replace, Each R a Each occurrence is independently selected from halogen such as F, C1-C 20 Haloalkyl such as -CF3, -CN, -OR c and –NR c R c ; Each R b Each occurrence is independently selected from halogen such as F, haloalkyl such as C1-C 20 Haloalkyl such as -CF3, -CN, -NO2, -OR c and-NR c R c ;as well as Each R c Each occurrence is independently selected from H and C1-C 20 alkyl.

31. The method of any one of claims 23 to 30, wherein R 1 Selected from hydrogen and C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace, and R 2 It is C1-C 20 Alkyl, wherein the C1-C 20 The alkyl group is optionally substituted by one to six R a replace, Each R a Each occurrence is independently selected from halogen such as F, C1-C 20 Halogenated alkyl such as C1-C 20 Fluoroalkyl, such as -CF3.

32. The method of any one of claims 23 to 31, wherein R 1 and R 2 At least one of them is C1-C 20 Fluoroalkyl, for example C1-C6 fluoroalkyl.

33. The method of any one of claims 23 to 32, wherein R 1 is selected from hydrogen and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one to six F, and R 2 It is C1-C6 alkyl or phenyl.

34. The method of any one of claims 23 to 33, wherein R 1 is selected from hydrogen and trifluoromethyl, and R 2 It is methyl or phenyl.

35. The method of any one of claims 21 to 34, wherein the substrate is a ketone.

36. The method of any one of claims 21 to 35, wherein the ketone substrate is 37. The method of any one of claims 21 to 36, wherein the nitroalkane substrate is nitromethane or nitroethane.

38. A method for asymmetric synthesis of (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (IA): The method comprises the step of contacting nitromethane and 1,1,1-trifluoropropan-2-one with the alcohol nitrile hydroformylase polypeptide according to any one of claims 1 to 13 to obtain (S)-1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol having formula (IA).

39. The method of any one of claims 21 to 38, wherein the β-nitro alcohol is present in an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

40. The method of any one of claims 21 to 39, wherein the reaction is carried out in a solvent.

41. The method of claim 40, wherein the solvent is selected from the group consisting of polar solvents, non-polar solvents, and ionic liquids.

42. The method of any one of claims 21 to 41, wherein the reaction is carried out in a solvent, wherein the solvent is selected from the group consisting of water, methanol, ethanol, n-propanol, isopropanol, isopropyl acetate, dimethyl sulfoxide, dimethylformamide, ethyl acetate, butyl acetate, 1-octanol, hexane, heptane, octane, methyl tert-butyl ether, toluene, 1-ethyl-4-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole hexafluorophosphate, glycerol, and polyethylene glycol.

43. The process of any one of claims 40 to 42, wherein the reaction is carried out in the presence of a co-solvent.

44. The method of claim 43, wherein the co-solvent is selected from dimethyl sulfoxide (DMSO) and alcohols, such as methanol, ethanol, n-propanol, isopropanol.

45. The process of any one of claims 21 to 44, wherein the reaction is carried out at a temperature of 10°C to 30°C.

46. ​​The method of any one of claims 21 to 45, wherein the reaction is carried out at a pH of 4.0 to 8.

0.

47. The method of any one of claims 21 to 46, wherein the aldehyde or ketone substrate is present at a loading of 5 g / L to 150 g / L.

48. The method of any one of claims 21 to 47, wherein the nitroalkane is present in a loading of 5 g / L to 150 g / L.

49. The method of any one of claims 21 to 48, wherein the nitroalkane is present in an excess of 2 stoichiometric equivalents relative to the aldehyde or ketone substrate.

50. The method of any one of claims 21 to 49, wherein the alcohol nitrile hydroformylase polypeptide is present at a concentration of at least 3 g / L enzyme.

51. The method of any one of claims 21 to 50, wherein the solvent is present in a concentration of 0 to 500 g / L, such as 100 to 200 g / L, such as 196 g / L.

52. A method for synthesizing (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol having formula (IB): The method comprises the step of contacting (IA) with hydrogen under suitable hydrogenation conditions to obtain (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol (IB), wherein (IA) is synthesized by the method of any one of claims 21 to 51.

53. The process of claim 52, wherein the reaction is carried out in the presence of a catalyst, optionally wherein the catalyst is selected from the group consisting of Raney nickel, Raney cobalt, Pd / C and Pt / C.

54. The method of claim 53, wherein the catalyst is present at a loading of at least 2 wt.%, such as at least 5 wt.%, at least 10 wt.%.

55. The method of any one of claims 52 to 54, wherein the reaction is carried out in the presence of activated carbon.

56. The method of any one of claims 52 to 55, wherein the reaction is carried out in a solvent, wherein the solvent is selected from water, methanol, ethanol, propanol, isopropanol, ethyl acetate, isopropyl acetate, methyl tert-butyl ether.

57. The method of any one of claims 52 to 56, wherein the reaction is carried out at a temperature of 20°C to 60°C, such as 20°C, 30°C, 40°C, 50°C.

58. The method of any one of claims 52 to 57, wherein the concentration of the compound of formula (IA) is at least 2 wt.%, such as 2 wt.%, 4 wt.%, 8 wt.%, 10 wt.%.

59. The method of any one of claims 52 to 58, wherein the method is performed in batch or flow.

60. The process of any one of claims 52 to 59, further comprising the step of converting the compound of formula (IB) into an acid salt.

61. The method of claim 60, further comprising the step of recrystallizing the acid salt.

62. The method of any one of claims 60 and 61, wherein the acid salt is an HCl salt.

63. A method for synthesizing (S)-3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide having formula (IC) The method comprises the steps according to claim 38.

64. The method of claim 63, further comprising the steps of any one of claims 39 to 51.

65. The method of any one of claims 63 and 64, further comprising the steps of claim 52.

66. The method of claim 65, further comprising the steps of any one of claims 53 to 62.

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