Ketoreductases for synthesis of 1, 3-diol substituted indane compounds

Abstract

In particular, the present disclosure provides ketoreductases having modified enzymatic properties, including the ability to reduce hydroxyindanones, to provide diastereomerically pure 1, 3-indandiols that can be used in the synthesis of beretetifan. Also provided are polynucleotides encoding the ketoreductases, and host cells capable of expressing the ketoreductases. Also provided are purification methods for isolating the ketoreductases.

Description

Technical Field

[0001] The present invention relates to ketoreductases which are useful in biocatalytic and synthetic processes involving the reduction of ketones to chiral alcohols. Such enzymes are particularly useful for the preparation of 1,3-diol substituted indanes.

[0002] References to electronically submitted sequence listings

[0003] This application contains a sequence listing submitted electronically in XML format, and is hereby incorporated by reference in its entirety. The XML file was created on October 31, 2022, is named 25557-WO-PCT_SL.XML, and is 26,015 bytes in size. Background Art

[0004] Enzymes are polypeptides that are used to speed up chemical reactions in living cells (usually by several orders of magnitude). Without enzymes, most biochemical reactions would be too slow for even life processes to proceed. Enzymes display great specificity and are not permanently altered by the reactions they participate in. Since they do not change during the course of a reaction, enzymes are particularly cost-effective when used as catalysts for desired chemical transformations.

[0005] Ketoreductases, also known as alcohol dehydrogenases, are enzymatic reductants, which are a specific class of enzymes that catalyze the selective reduction of ketones to chiral alcohols. Enzymes belonging to the class of ketoreductases or carbonyl reductases can be used to synthesize optically active alcohols. Ketoreductases can selectively convert ketone or aldehyde substrates into the corresponding chiral alcohol products; in the reverse reaction, these enzymes can also convert alcohols into the corresponding ketones or aldehydes. The enzymatic reduction of ketones and aldehydes requires the participation of a cofactor that can act as an electron donor, while the enzymatic oxidation of alcohols requires the participation of a cofactor that can act as an electron acceptor.

[0006] Ketoreductases are well known in nature, and many genes encoding ketoreductases and ketoreductase sequences have been reported. See, for example, Candida magnolia (Genbank Accession No. JC7338; GI: 11360538), Candida parapsilosis (Genbank Accession No. 10BAA24528.1; GI: 2815409), Sporobolomyces salmonicolor (Genbank Accession No. AF160799; GI: 6539734), and Rhodococcus erythropolis (Genbank Accession No. AAN73270.1; GI: 34776951).

[0007] Ketoreductases are being used with increasing frequency to provide alternative synthetic pathways for key compounds. For example, Kosjek, B. et al. disclose the use of ketoreductases to asymmetric syntheses of chiral precursors 4,4-dimethoxy-2H-pyran-3-ol (Organic Process Research & Development, 2008, 12.584-588). When used, ketoreductases can be provided as purified enzymes or as complete cells expressing the desired ketoreductases. In view of their prospects for improved synthetic pathways, there is still a need to identify other ketoreductases that can be used to perform certain chemical transformations to prepare specific chiral alcohols. Summary of the invention

[0008] The present disclosure relates to ketoreductases capable of converting ketones to chiral alcohols, particularly on substituted indane backbones. In some embodiments, the subject ketoreductases described herein are capable of converting hydroxyindanones to diastereomerically pure 1,3-indandiols that can be used to synthesize belzutifan (3-[[(1S,2S,3R)-2,3-difluoro-2,3-dihydro-1-hydroxy-7-(methylsulfonyl)-1H-inden-4-yl]oxy]-5-fluorobenzonitrile). This agent is a hypoxia-inducible factor inhibitor and was recently approved by the U.S. Food and Drug Administration for the treatment of adult patients with von Hippel-Lindau disease who require therapy for associated renal cell carcinoma, central nervous system hemangioblastoma, or pancreatic neuroendocrine tumors without the need for immediate surgery.

[0009] Other embodiments describe methods for making the subject ketoreductases and methods of using the subject ketoreductases.

[0010] Other embodiments, aspects and features of the present invention will be further described in or will be apparent from the ensuing description, examples and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Depicted is an SDS-PAGE gel showing the removal of iPrOH-insoluble proteins from a lyophilized enzyme preparation. DETAILED DESCRIPTION OF THE INVENTION

[0013] definition

[0014] Certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure relates. Nevertheless, and unless otherwise indicated, the following definitions apply throughout this specification and claims. Chemical names, common names, and chemical structures may be used interchangeably to describe the same structure.

[0015] As used herein and throughout this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0016] The present disclosure also includes isotopically labeled compounds, which are identical to the compounds described herein, except for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F. 36 Cl and 123 I.

[0017] Certain isotopically labeled compounds (e.g. 3 H and 14 C-labeled compounds) are used in tissue distribution assays of compounds and / or substrates. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred because of their ease of preparation and detectability. Isotopic substitution at the position where epimerization occurs can slow down or reduce the epimerization process and thereby retain the more active or potent form of the compound for a longer period of time. Isotope-labeled compounds, particularly those containing isotopes with longer half-lives (T 1 / 2 >1 day) can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or in the Examples herein below, by substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent.

[0018] The compounds herein may contain one or more stereocenters and may occur as racemates, racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereomers. Additional asymmetric centers may be present depending on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomers of the compound as a pure or partially purified compound are included in the present disclosure. Any expression, structure, or name of a compound described herein that does not specify a specific stereochemistry is intended to encompass any and all isomers present and mixtures thereof in any ratio as described above. When specifying stereochemistry, the present disclosure is intended to encompass a specific isomer in pure form or as part of a mixture in any ratio with other isomers.

[0019] Based on their physicochemical differences, diastereomeric mixtures are separated into their individual diastereomers by methods well known to those skilled in the art (e.g., such as by chromatography and / or fractional crystallization). Enantiomer mixtures can be converted to diastereomeric mixtures by reacting the enantiomeric mixtures with an appropriate optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers, thereby achieving separation of the enantiomers. Enantiomers can also be separated using chiral HPLC columns.

[0020] All stereoisomers (e.g., geometric isomers, optical isomers, etc.) of the disclosed compounds (including salts and solvates of the compounds and salts, solvates and esters of prodrugs), such as stereoisomers that may exist due to asymmetric carbons on various substituents, including enantiomeric forms (even in the absence of asymmetric carbons), rotamer forms, atropisomers, and diastereomeric forms, are contemplated by the present disclosure. Individual stereoisomers of a compound may, for example, be substantially free of other isomers, or may be mixed, for example, as a racemate or mixed with all or other selected stereoisomers. Chiral centers may have the S or R configuration as defined by the IUPAC 1974 Recommendation.

[0021] The present disclosure further includes all compounds in isolated form and synthetic intermediates thereof.For example, the above-mentioned compounds are intended to encompass all forms of the compounds, such as any solvates, hydrates, stereoisomers and tautomers thereof.

[0022] "Ketoreductase" and "KRED" are used interchangeably herein and refer to polypeptides having the enzymatic ability to reduce a carbonyl group to its corresponding alcohol. More specifically, the disclosed ketoreductase polypeptides are capable of stereoselectively reducing fluorohydroxyindanone (6) (Figure 2) to fluorodiol (7) (Figure 2).

[0023]

[0024] Typically, the polypeptide utilizes the cofactor reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a reducing agent. Ketoreductases as used herein include naturally occurring (wild-type) ketoreductases, as well as non-naturally occurring engineered polypeptides generated by human manipulation.

[0025] "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 length or post-translational modifications (e.g., glycosylation or phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes D- and L-amino acids, as well as mixtures of D- and L-amino acids, and polymers comprising D- and L-amino acids, as well as mixtures of D- and L-amino acids. Proteins, polypeptides, and peptides may include tags, such as histidine tags, which should not be included when determining percent sequence identity.

[0026] "Amino acid" or "residue" used in the context of polypeptides disclosed herein refers to a specific monomer at a sequence position. Amino acids are represented herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may also be represented by their generally accepted single-letter codes.

[0027] Abbreviations for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (GIn or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0028] Abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise specified, abbreviated nucleosides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated as ribonucleosides or 2'-deoxyribonucleosides on an individual or collective basis. When a nucleic acid sequence is represented by a string of single-letter abbreviations, the sequence is represented in the 5' to 3' direction according to common convention, and phosphates are not indicated.

[0029] As used herein, "derived from" in the context of an enzyme refers to an original enzyme that is the basis of the enzyme and / or a gene encoding the enzyme. For example, the ketoreductase of SEQ ID NO: 2 is obtained by artificially evolving the gene encoding the ketoreductase of SEQ ID NO: 1 for multiple generations. Therefore, this evolved ketoreductase is "derived from" the ketoreductase of SEQ ID NO: 1.

[0030] "Reference sequence" refers to a designated sequence used as the 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. Typically, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of a nucleic acid or polypeptide. Since 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, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare sequence similarities in local regions.

[0031] In some embodiments, a "reference sequence" may be based on a primary amino acid sequence, wherein the reference sequence is a sequence that may have one or more changes in the primary sequence. For example, a reference sequence "based on SEQ ID NO: 1 having a proline at the residue corresponding to X190" refers to a reference sequence in which the corresponding residue at X190 in SEQ ID NO: 1 has been changed to a proline.

[0032] "Hydrophilic amino acids or residues" refer to amino acids or residues having side chains that exhibit a hydrophobicity of less than zero according to the standardized consensus hydrophobicity scale of Eisenberg et al. 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

[0033] "Acidic amino acids or residues" refer to hydrophilic amino acids or residues having side chains that, when the amino acid is contained in a peptide or polypeptide, exhibit a pK value of less than about 6. Acidic amino acids typically have negatively charged side chains at physiological pH due to the loss of hydrogen ions. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0034] "Basic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that, when the amino acid is contained in a peptide or polypeptide, exhibits a pKa value greater than about 6. Basic amino acids generally have positively charged side chains at physiological pH due to association with hydronium ions. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0035] "Polar amino acid or residue" refers to a hydrophilic amino acid or residue with a side chain that is uncharged at physiological pH but has at least one bond in which an electron pair shared by two atoms is more tightly held by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T).

[0036] "Hydrophobic amino acid or residue" refers to an amino acid or residue having a side chain that exhibits a hydrophobicity greater than zero according to the standardized consensus hydrophobicity scale of Eisenberg et al. 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

[0037] "Aromatic amino acid or residue" refers to a hydrophilic or hydrophobic amino acid or residue having a side chain containing at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), L-His (H) and L-Trp (W). L-His (H) histidine is also classified as a hydrophilic residue or constrained residue herein.

[0038] As used herein, "constrained amino acid or residue" refers to an amino acid or residue with constrained geometry. Herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring; proline has a constrained geometry because it also has a five-membered ring.

[0039] "Non-polar amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH and having a bond in which an electron pair shared by two atoms is typically held equally by each of the two atoms (i.e., the side chain is non-polar). Genetically encoded non-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

[0040] As used herein, "aliphatic amino acid or residue" refers to a hydrophobic amino acid or residue with an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile (I).

[0041] The ability of L-Cys(C) (and other amino acids with SH-containing side chains) to exist in a peptide as a reduced free SH or as an oxidized disulfide bridge affects whether L-Cys(C) contributes net hydrophobicity or hydrophilicity to the peptide. Although L-Cys(C) exhibits a hydrophobicity of 0.29 according to Eisenberg's standardized consensus scale (Eisenberg et al., 1984, supra), it should be understood that for the purposes of this disclosure, L-Cys(C) is classified as a unique group in its own right. It is noteworthy that cysteine ​​(or "L-Cys" or "[C]") is special in that it is able to form disulfide bridges with other L-Cys(C) amino acids or other sulfanyl- or sulfhydryl-containing amino acids. "Cysteine-like residues" include cysteine ​​and other amino acids containing sulfhydryl moieties, which can be used to form disulfide bridges.

[0042] As used herein, "small amino acid or residue" refers to an amino acid or residue whose side chain consists of a total of three or fewer carbons and / or heteroatoms (excluding α-carbon and hydrogen). According to the above definition, small amino acids or residues can be further divided into aliphatic, non-polar, polar or acidic small amino acids or residues. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D).

[0043] "Hydroxyl-containing amino acid or residue" refers to an amino acid that contains a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

[0044] As used herein, "conservative amino acid substitutions" refer to the replacement of one residue with a different residue having a similar side chain, and thus generally involve replacing an amino acid in a polypeptide with an amino acid within the same or similarly defined amino acid class. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is replaced with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced with another amino acid with an acidic side chain (e.g., aspartic acid and glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0045] As used herein, "non-conservative substitutions" refer to substitutions of amino acids in a polypeptide with amino acids having significantly different side chain properties. Non-conservative substitutions may use amino acids between defined groups rather than within defined groups and affect: (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions may be substitutions of acidic amino acids for basic or aliphatic amino acids; substitutions of aromatic amino acids for small amino acids; and substitutions of hydrophilic amino acids for hydrophobic amino acids.

[0046] As used herein, "deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion may include the removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids that make up the reference enzyme, or up to 20% of the total number of amino acids that make up the reference enzyme, while retaining enzymatic activity and / or retaining improved properties of the evolved enzyme. Deletion may be directed to the internal and / or terminal portions of a polypeptide. In various embodiments, deletion may comprise a continuous segment or may be discontinuous. Deletion is typically represented by "-" in an amino acid sequence.

[0047] As used herein, "insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. The insertion may be located in an internal portion of the polypeptide or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions may be continuous stretches of amino acids or may be separated by one or more amino acids in a naturally occurring polypeptide.

[0048] The term "amino acid substitution group" or "substitution group" refers to a group of amino acid substitutions in a polypeptide sequence compared to a reference sequence. A substitution group can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions.

[0049] "Functional fragment" and "biologically active fragment" are used interchangeably herein and refer to a polypeptide having amino-terminal and / or carboxyl-terminal deletions and / or internal deletions, but wherein the remaining amino acid sequence is identical to the corresponding positions in the compared sequence and substantially retains all the activities of the full-length polypeptide.

[0050] As used herein, "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that are naturally associated with it. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or via in vitro synthesis). Recombinant polypeptides can be present in cells, in cell culture media, or prepared in various forms, such as lysates or isolated preparations. Therefore, in some embodiments, the recombinant polypeptide can be an isolated polypeptide.

[0051] As used herein, "substantially pure polypeptide" or "purified protein" refers to a composition in which the polypeptide species is the predominant species present (i.e., more abundant than any other single macromolecular species in the composition by mole or weight), and when the target species accounts for at least about 50% (in mole or weight percentage) of the macromolecular species present, it is generally a substantially purified composition. However, in some embodiments, the composition comprising the enzyme comprises an enzyme having a purity of less than 50% (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%). Typically, a substantially pure enzyme or polypeptide composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more (in mole or weight percentage) of all macromolecular species present in the composition. In some embodiments, the target species is purified to substantially homogeneity (i.e., contaminant species in the composition cannot be detected by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species.In some embodiments, an isolated recombinant polypeptide is a substantially pure polypeptide composition.

[0052] "Improved enzyme properties" refers to an enzyme that exhibits an improvement in any enzyme property compared to a reference enzyme. For the enzymes described herein, comparison is typically made to a wild-type enzyme, although in some embodiments the reference enzyme may be another improved enzyme. Enzyme properties that require improvement include, but are not limited to, enzyme activity (expressed as a percentage of available substrate conversion), thermal stability, pH activity spectrum, cofactor requirements, refractoriness to inhibitors (e.g., product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity).

[0053] "Increased enzyme activity" refers to an improved property of an enzyme, which can be represented by an increase in specific activity (e.g., product produced / time / protein weight) or an increase in the percentage of substrate converted to product (e.g., the percentage of a starting amount of substrate converted to product using a specified amount of enzyme in a specified time period) compared to a reference enzyme. Exemplary methods for determining enzyme activity are provided in the Examples. Any property related to enzyme activity can be affected, including classical enzyme properties. m 、V max or k cat , whose changes can result in increased enzyme activity. The improvement in enzyme activity can be from about 1.5 times to up to 2 times the enzyme activity of the corresponding wild-type enzyme. Compared with a naturally occurring enzyme or another enzyme from which the polypeptide is derived, the enzyme activity is 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times, 500 times, 1000 times, 3000 times, 5000 times, 7000 times or more. In a specific embodiment, the enzyme exhibits an improved enzyme activity higher than the parent enzyme, ranging from 150 to 3000 times, 3000 to 7000 times or 7000 times more. It is understood by those skilled in the art that the activity of any enzyme is diffusion-limited, and therefore the catalytic turnover rate cannot exceed the diffusion rate of the substrate (including any required cofactors). The theoretical maximum value or k of diffusion limitation is 1.5 times, 2.5 times, 3.5 times, 4.5 times, 5.5 times, 6.5 times, 7.5 times, 8.5 times, 9.5 times, 10.5 times, 11.5 times, 12.5 times, 13.5 times, 14.5 times, 15.5 times, 16.5 times, 17.5 times, 18.5 times, 19.5 times, 20.5 times, 21.5 times, 22.5 times, 23.5 times, 24.5 times, 25.5 times, 26.5 times, 27.5 times, 28.5 times, 29.5 times, 30.5 times, 31.5 times, 31.5 times, 32.5 times, 34.5 times, 35.5 times, 36.5 times, 37.5 times, 38.5 times, cat / K m Usually about 10 8 Up to 10 9 (M -1 s -1). Therefore, any improvement in enzyme activity will have an upper limit associated with the substrate diffusion rate of the enzyme action. Enzyme activity can be measured by any standard assay for measuring kinase activity, or measured via a coupled assay with a nucleoside phosphorylase, which can catalyze the reaction between a polypeptide product and a nucleoside base to provide nucleosides, or measured by any conventional method for measuring chemical reactions, including but not limited to HPLC, HPLC-MS, UPLC, UPLC-MS, TLC and NMR. Use a determined enzyme preparation, an assay specified under set conditions and one or more determined substrates to compare enzyme activity, as described in further detail herein. Typically, when comparing lysates, determine the amount of cell number and the protein measured, and use the same expression system and the same host cell to minimize the change in the amount of enzyme produced by the host cell and present in the lysate.

[0054] As used herein, "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably linked to an appropriate control sequence that can achieve expression of a polypeptide encoded in the DNA sequence in an appropriate host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgenic) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0055] As used herein, the term "expression" includes any step involved in the production of the polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0056] As used herein, the term "production" refers to the production of proteins and / or other compounds by a cell. The term is intended to encompass any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of a polypeptide from a cell.

[0057] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, a signal peptide, a terminator sequence, etc.) is "heterologous" if the two sequences are not related in nature to each other. For example, a "heterologous polynucleotide" is any polynucleotide that is introduced into a host cell by laboratory techniques, and the term includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0058] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector comprising a DNA as provided herein (e.g., a polynucleotide encoding a variant). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0059] The term "analog" refers to a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) with a reference polypeptide. In some embodiments, an "analog" refers to a polypeptide containing one or more non-naturally occurring amino acid residues, including but not limited to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids. In some embodiments, an analog further comprises one or more D-amino acid residues and non-peptide bonds between two or more amino acid residues.

[0060] As used herein, "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a system for numerically classifying enzymes based on the chemical reaction that the enzyme catalyzes.

[0061] As used herein, "ATCC" refers to the American Type Culture Collection, whose biobank collections include genes and strains.

[0062] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases it provides.

[0063] "Coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0064] "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 man, with the only exception that the wild type polypeptide or polynucleotide sequence identified herein may contain a tag, such as a histidine tag, which should not be included when determining the percentage of sequence identity. Herein, a "wild type" polypeptide or polynucleotide sequence may be represented as "WT".

[0065] "Recombinant" when used with respect to, for example, a cell, nucleic acid, or polypeptide, refers to a material or material corresponding to the natural or native form of that material that has been modified in a manner not originally found in nature, or is identical thereto but is produced or derived from synthetic materials and / or manipulated through the use of recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not present in the natural (non-recombinant) form of the cell or that express natural genes that are originally expressed at different levels.

[0066] "Percentage of sequence identity", "percentage of identity" and "percentage of identity" used herein refer to the comparison between polynucleotide sequences or polypeptide sequences, 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., intervals) compared to the reference sequence for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences, or the nucleic acid base or amino acid residue is aligned with the interval to produce 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 produce the percentage of sequence identity. Optimal alignment and determination of percentage of sequence identity are performed using BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410; and Altschul et al., 1977, Nucleic Acids Res. 3389-3402). Software for BLAST analysis can be publicly available through the website of the National Center for Biotechnology Information.

[0067] In brief, BLAST analysis involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial hit neighborhood words serve as seeds for initiating searches to find longer HSPs containing them. The hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the hits in each direction stops when: the cumulative alignment score drops by a number X from the maximum value it reaches; the cumulative score drops to zero or below 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 as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults 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 89:10915).

[0068] There are many other algorithms available which function similarly to BLAST in providing percent identity between two sequences. Sequences can be optimally aligned for comparison by, for example, 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 search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the GCG Wisconsin software package), or by visual inspection (see generally Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture of Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)). Additionally, sequence alignments and determination of 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.

[0069] "Substantially identical" means that a polynucleotide or polypeptide sequence has at least 80% sequence identity, preferably at least 85% sequence identity, more preferably at least 89% sequence identity, more preferably at least 95% sequence identity, and even more preferably at least 99% sequence identity compared to a reference sequence over a comparison window of at least 20 residue positions (usually a window of at least 30-50 residues), wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence comprising deletions or additions that total 20% or less of the reference sequence over the comparison window. In specific embodiments applied to polypeptides, the term "substantially identical" means that two polypeptide sequences share at least 80% sequence identity, preferably at least 89% sequence identity, more preferably at least 95% sequence identity or more (e.g., 99% sequence identity) when optimally aligned (e.g., by the programs GAP or BESTFIT using default interval weights). Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[0070] "Corresponding to," "reference," or "relative to," when used in the context of numbering a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a given reference sequence when comparing the given amino acid or polynucleotide sequence to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are assigned relative to a reference sequence, rather than according to the actual numerical positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence may be aligned to a reference sequence by introducing gaps to optimize the matching of residues between the two sequences. In these cases, the numbering of the residues in a given amino acid or polynucleotide sequence is made relative to the reference sequence to which it is aligned, despite the presence of gaps.

[0071] "Stereoselectivity" refers to the formation of one stereoisomer in preference to another stereoisomer in a chemical or enzymatic reaction. Stereoselectivity can be partial, where the formation of one stereoisomer is superior to that of the other, or can be thorough, where only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is called enantioselectivity, i.e., the fraction of one enantiomer in the sum of two (usually reported as a percentage). The art generally reports it (usually as a percentage) as the enantiomeric excess (EE) calculated thereby according to the formula [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity is called diastereoselectivity, i.e., the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), which is generally reported as diastereomeric excess (DE). Enantiomeric excess and diastereomeric excess are types of stereoisomer excess.

[0072] "Highly stereoselective" refers to a chemical or enzymatic reaction that is capable of converting a substrate to its corresponding product with a stereoisomeric excess of at least about 85%.

[0073] "Chemological selectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

[0074] "Conversion" refers to the enzymatic conversion of a substrate into a corresponding product. "Percent conversion" refers to the percentage of substrate that is converted to product under specific conditions and within a certain period of time. Thus, for example, the "enzyme activity" or "activity" of a polypeptide can be expressed as the "percent conversion" of substrate into product.

[0075] "Chiral alcohol" refers to a chiral alcohol of the formula R 1 -CH(OH)-R 2 Amines, where R 1 and R 2Different, and is used here in the broadest sense, including various different and mixed functional types of aliphatic and alicyclic compounds, characterized by the presence of a primary hydroxyl group bonded to a secondary carbon atom, which carries, in addition to a hydrogen atom, (i) a divalent group forming a chiral cyclic structure, or (ii) two substituents (other than hydrogen) that are different in structure or chirality from each other. The divalent group forming a chiral cyclic structure includes, for example, 2-methylbutane-1,4-diyl, pentane-1,4-diyl, hexane-1,4-diyl, hexane-1,5-diyl, 2-methylpentane-1,5-diyl. The two different substituents on the secondary carbon atom (the above R 1 and R 2 ) can also vary widely, including alkyl, aralkyl, aryl, halogen, hydroxy, lower alkyl, lower alkoxy, lower alkylthio, cycloalkyl, carboxyl, alkoxycarbonyl, carbamoyl, mono- and di-(lower alkyl) substituted carbamoyl, trifluoromethyl, phenyl, nitro, amino, mono- and di-(lower alkyl) substituted amino, alkylsulfonyl, arylsulfonyl, alkylformamide, arylformamide, etc., as well as alkyl, aralkyl or aryl substituted by the aforementioned.

[0076] Immobilized enzyme preparations have many recognized advantages. For example, they can provide the shelf life of enzyme preparations, they can improve reaction stability, they can impart stability in organic solvents, and they can help remove proteins from reaction streams. "Stable" refers to the ability of immobilized enzymes to maintain their structural conformation and / or their activity in solvent systems containing organic solvents. Stable immobilized enzymes have an activity loss of less than 10% per hour in solvent systems containing organic solvents. Stable immobilized enzymes have an activity loss of less than 9% per hour in solvent systems containing organic solvents. Preferably, stable immobilized enzymes have an activity loss of less than 8% per hour in solvent systems containing organic solvents. Preferably, stable immobilized enzymes have an activity loss of less than 7% per hour in solvent systems containing organic solvents. Preferably, stable immobilized enzymes have an activity loss of less than 6% per hour in solvent systems containing organic solvents. Preferably, stable immobilized enzymes have an activity loss of less than 5% per hour in solvent systems containing organic solvents. Preferably, stable immobilized enzymes have an activity loss of less than 4% per hour in solvent systems containing organic solvents. Preferably, stable immobilized enzymes have an activity loss of less than 3% per hour in solvent systems containing organic solvents. Preferably, the stable immobilized enzyme loses less than 2% of its activity per hour in a solvent system containing an organic solvent. Preferably, the stable immobilized enzyme loses less than 1% of its activity per hour in a solvent system containing an organic solvent.

[0077] "Thermostable" means that the polypeptide retains similar activity (eg, 60% to 80% or more) compared to the untreated enzyme after being exposed to high temperature (eg, 40°C to 80°C) for a period of time (eg, 0.5h to 24h).

[0078] "Solvent stability" means that the polypeptide maintains similar activity (e.g., 60% to 80% or more) compared to the untreated enzyme after being exposed to different concentrations (e.g., 5% to 99%) of solvents (isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5h to 24h).

[0079] "pH stable" means that the polypeptide retains similar activity (e.g., more than 60% to 80%) compared to the untreated enzyme after exposure to high or low pH (e.g., 4.5 to 6 or 8 to 12) for a period of time (e.g., 0.5 hour to 24 hours).

[0080] "Heat and solvent stable" means that the polypeptide is both heat stable and solvent stable.

[0081] As used herein, the terms "biocatalysis," "biocatalytic," "bioconversion," and "biosynthesis" refer to the use of enzymes to perform chemical reactions on organic compounds.

[0082] The term "effective amount" refers to an amount sufficient to produce the desired result. An effective amount can be determined by one of ordinary skill in the art using routine experiments.

[0083] The terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components that are separated from at least one other component with which they are naturally associated. The term "purified" does not require absolute purity, but is rather a relative definition.

[0084] "Control sequences" are defined herein as including all components necessary or advantageous for expressing a target polynucleotide and / or polypeptide. Each control sequence may be native or foreign to a nucleic acid sequence encoding a polypeptide. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. A control sequence includes at least a promoter and transcription and translation termination signals. A control sequence may be provided with a linker for the purpose of introducing specific restriction sites to facilitate connection of the control sequence to a target polynucleotide (e.g., the coding region of a nucleic acid sequence encoding a polypeptide).

[0085] "Operably linked" is defined herein as a configuration in which a control sequence is placed at an appropriate position (ie, in a functional relationship) relative to a polynucleotide sequence such that the control sequence directs the expression of the polynucleotide and / or a polypeptide encoded by the polynucleotide.

[0086] A "promoter sequence" is a nucleic acid sequence that is recognized by a host cell to express a polynucleotide. A control sequence may comprise an appropriate promoter sequence. A promoter sequence comprises a transcriptional control sequence that mediates the expression of a polynucleotide. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0087] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0088] abbreviation

[0089] ACN,MeCN Acetonitrile

[0090] DMSO Dimethyl sulfoxide

[0091] g

[0092] μg microgram

[0093] h hour

[0094] Hz Hertz

[0095] iPrOH Isopropyl alcohol

[0096] IPTG Isopropyl β-D-1-thiogalactopyranoside

[0097] J NMR coupling constants

[0098] L Liter

[0099] M mole, mol / L

[0100] mg milligram

[0101] min

[0102] mL,ml milliliters

[0103] mm Millimeters

[0104] mM millimole / liter

[0105] μm micrometer

[0106] nm nanometer

[0107] NaCl Sodium chloride

[0108] NADP Nicotinamide adenine dinucleotide phosphate

[0109] OD 600Optical density at 600nm wavelength

[0110] PTFE Polytetrafluoroethylene

[0111] RH Relative humidity

[0112] RPM, rpm revolutions per minute

[0113] RT Room temperature, about 25℃

[0114] TFA Trifluoroacetic acid

[0115] THF Tetrahydrofuran

[0116] UPLC Ultra-Performance Liquid Chromatography

[0117] vol volume

[0118] v / v Volume per volume

[0119] μg,ug microgram

[0120] μL,μl,uL,ul microliter

[0121] Ketoreductase

[0122] The present disclosure relates to ketoreductases capable of reducing ketones to chiral alcohols, particularly on substituted indane backbones. In some embodiments, the ketoreductases are capable of performing the following conversions:

[0123]

[0124] In specific embodiments, ketoreductases are used in conjunction with electrophilic fluorinating agents in the transformation:

[0125]

[0126] In some embodiments, the ketoreductases described herein have an amino acid sequence with one or more amino acid differences compared to a reference amino acid sequence of a commercially available non-wild-type ketoreductase that results in improved properties of the enzyme for defined ketone substrates.

[0127] The ketoreductases described herein are the product of directed evolution from a commercially available ketoreductase (SEQ ID NO: 1, Codexis, Inc.) identified by screening a collection of Codexis enzymes and having the amino acid sequence shown below:

[0128]

[0129] In some embodiments, the ketoreductases of the present disclosure may exhibit improvements relative to the ketoreductase of SEQ ID NO: 1, such as increased enzyme activity, stereoselectivity, stereospecificity, thermal stability, solvent stability, or reduced product inhibition.

[0130] In some embodiments, the ketoreductases of the present disclosure can exhibit improved enzymatic activity rates, i.e., conversion rates of substrates to products. In some embodiments, the ketoreductase polypeptide is capable of converting a substrate to a substrate at a rate that is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, 1000-fold, 3000-fold, 5000-fold, 7000-fold, or more than 7000-fold greater than the rate exhibited by the enzyme of SEQ ID NO: 1.

[0131] In some embodiments, such ketoreductase polypeptides are also capable of converting a substrate to a product in a stereometric excess of at least about 80%. In some embodiments, such ketoreductase polypeptides are also capable of converting a substrate to a product in a stereometric excess of at least about 90%. In some embodiments, such ketoreductase polypeptides are also capable of converting a substrate to a product in a stereometric excess of at least about 99%.

[0132] In some embodiments, the ketoreductase polypeptide is highly stereoselective, wherein the polypeptide is capable of reducing a substrate to a product in greater than about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% stereometric excess.

[0133] In some embodiments, the improved ketoreductase polypeptide of the present disclosure is a polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, which has an amino acid sequence as shown below:

[0134]

[0135] In some embodiments, the improved ketoreductase polypeptides of the present disclosure are based on the sequence formula of SEQ ID NO:2 and may comprise an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reference sequence of SEQ ID NO:2.

[0136] The differences between the variants and SEQ ID NO: 2 may be amino acid insertions, deletions, substitutions or any combination of such changes. In some embodiments, the amino acid sequence differences may comprise non-conservative, conservative amino acid substitutions and combinations of non-conservative and conservative amino acid substitutions.

[0137] In some embodiments, the ketoreductase polypeptide is a polypeptide selected from the group consisting of SEQ ID NOs: 2, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In specific embodiments, the ketoreductase polypeptide is a polypeptide selected from the group consisting of SEQ ID NOs: 2, 14, 15, and 16.

[0138] In a specific embodiment, the improved ketoreductase polypeptide of the present disclosure, wherein the amino acid sequence consists of SEQ ID NO: 2. In a specific embodiment, the improved ketoreductase polypeptide of the present disclosure consists of SEQ ID NO:2.

[0139] In one aspect, the improved ketoreductase polypeptides of the present disclosure are based on an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein at least one of the following conditions is met:

[0140] (a) amino acid (aa) residue 2 of SEQ ID NO:2 is not alanine; or

[0141] (b) aa residue 11 of SEQ ID NO:2 is not glutamic acid.

[0142] In certain embodiments, the improved ketoreductase polypeptides of the present disclosure satisfy both conditions (a) and (b). In specific embodiments, in the improved ketoreductase polypeptides of the present disclosure, aa residue 2 of SEQ ID NO: 2 is lysine. In specific embodiments, in the improved ketoreductase polypeptides of the present disclosure, aa residue 11 of SEQ ID NO: 2 is alanine.

[0143] In certain embodiments of this aspect, the improved ketoreductase polypeptides of the present disclosure are based on an amino acid sequence having at least 85%, 90%, 95% or 98% sequence identity to SEQ ID NO:2.

[0144] The applicant has found that a polypeptide having an aa residue other than an alanine residue at a position corresponding to position 2 of SEQ ID NO: 2 is better expressed by its parent polynucleotide. Similarly, the applicant has found that a polypeptide having an aa residue other than a glutamic acid residue at a position corresponding to position 11 of SEQ ID NO: 2 is also better expressed by its parent polynucleotide.

[0145] Other embodiments provide host cells comprising the polynucleotides and / or expression vectors described herein. The host cells can be Escherichia coli (E. coli), or they can be different organisms, such as Lactobacillus brevis (L. brevis). The host cells can be used to express and isolate the ketoreductases described herein, or alternatively, they can be used directly to convert substrates into stereoisomer products.

[0146] Whether the method is performed using whole cells, cell extracts or purified ketoreductases, a single ketoreductase may be used, or alternatively, a mixture of two or more ketoreductases may be used.

[0147] Some embodiments relate to ketoreductases capable of selectively preparing chiral alcohols in the synthesis of indandiols, particularly fluorine-substituted indandiols. In some embodiments, the ketoreductases are capable of performing the following transformations:

[0148]

[0149] In specific embodiments, ketoreductases in conjunction with electrophilic fluorinating agents are capable of performing the following transformations:

[0150]

[0151] The enzyme properties that are desired to be improved include, but are not limited to, enzymatic activity, thermal stability, pH activity spectrum, cofactor requirements, tolerance to inhibitors (such as product inhibition), stereospecificity, stereoselectivity, and solvent stability. The improvement may be directed to a single enzyme property, such as enzymatic activity, or a combination of different enzyme properties, such as enzymatic activity and stereoselectivity.

[0152] Table 1 below provides a series of SEQ ID NOs and associated activities disclosed herein. Unless otherwise indicated, the following sequences are based on the ketoreductase sequence of SEQ ID NO: 1. In Table 1 below, one SEQ ID NO is listed per row. The column listing the number of mutations (i.e., residue changes) refers to the number of amino acid substitutions compared to the ketoreductase sequence of SEQ ID NO: 1. In the activity column of Table 1, + represents 30-50% (v / v) untreated lysate, ++ represents 15-30% (v / v) untreated lysate, +++ represents 15-30% iPrOH-treated lysate, ++++ represents 10-15% iPrOH-treated lysate, and +++++ represents 5-10% iPrOH-treated lysate. In the Selectivity column of the table, + indicates that the total diastereomeric ratio is <10:1, ++ indicates that the total dr is between 10:1 and 25:1, +++ indicates that the total dr is between 25:1 and 50:1, ++++ indicates that the total dr is between 50:1 and 99:1, and +++++ indicates that the total dr is >99:1. In the Solvent Tolerance column of the table, + indicates 0-12.5% ​​(v / v) total organic solvent, ++ indicates 12.5-18% (v / v) total organic solvent, +++ indicates 18-25% (v / v) total organic solvent, ++++ indicates 25-40% (v / v) total organic solvent, and +++++ indicates that 40-50% (v / v) total organic solvent is present. In the volumetric productivity column of the table, + represents 0-20 g / L of purified substrate, ++ represents 20-40 g / L of crude substrate, +++ represents 40-50 g / L of crude substrate, ++++ represents 50-60 g / L of crude substrate, and +++++ represents 60-80 g / L of crude substrate. In the iPrOH utilization column of the table, + represents 7.5-10% (v / v), ++ represents 11-13% (v / v), +++ represents 13.5% (v / v) iPrOH and 1.5% (v / v) acetone, ++++ represents 15% (v / v) iPrOH, and +++++ represents 16-18% (v / v) iPrOH.

[0153] The level of improvement in the properties reported in Table 1 was determined by setting up reactions with increasingly challenging conditions, including increasing the organic solvent (both composition and amount) and increasing the substrate concentration. The performance of each enzyme variant was measured by observing the amount of product formed on an Agilent UPLC instrument, typically after an overnight reaction as described in Example 2. When screening for improved diastereoselectivity, the amount of each product formed was measured by an Agilent SFC instrument, also as described in Example 2.

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] Polynucleotide encoding ketoreductase

[0160] In another aspect, the present disclosure provides polynucleotides encoding the ketoreductase polypeptides disclosed herein. The polynucleotides can be operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing a heterologous polynucleotide encoding a ketoreductase can be introduced into an appropriate host cell to express the corresponding ketoreductase polypeptide.

[0161] The availability of protein sequences, due to knowledge of the codons corresponding to a variety of amino acids, provides a description of all polynucleotides that can encode the subject matter. The degeneracy of the genetic code (where the same amino acid is encoded by an alternative or synonymous codon) allows a very large number of nucleic acids to be made, all of which encode the improved ketoreductases disclosed herein. Therefore, after determining a specific amino acid sequence, one skilled in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons without changing the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates every possible variation of a polynucleotide that can be generated by selecting a combination based on possible codon selections, and all such variations are considered to be specifically disclosed with respect to any polypeptide disclosed herein.

[0162] In various embodiments, codons are preferably selected to suit the host cell in which the protein is produced. For example, preferred codons used in bacteria are used to express genes in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. For example, the polynucleotide of SEQ ID NO: 3 encoding SEQ ID NO: 2 (see below) has been codon optimized for expression in E. coli.

[0163] On the other hand, the applicant has found that specific changes in the triplet codons in the polynucleotides encoding the ketoreductase polypeptides of the present disclosure confer increased polypeptide expression. These changes generally occur in the codons encoding the amino acid residues near the N-terminus of the polypeptide. For example, with reference to the polynucleotide of SEQ ID NO:17 encoding SEQ ID NO:1 (see below), the applicant noted that the codons (GCT) for the alanine residue at position 2, the codons (AAA) for the lysine residue at position 3, the codons (ATC) for the isoleucine residue at position 4, and the codons (GAA) for the glutamic acid residue at position 11 of the encoded polypeptide confer increased polypeptide expression. In some embodiments, the changes in the triplet codons at such positions do not result in changes in the amino acid residues in the encoded polypeptide. In other embodiments, the changes in the triplet codons at such positions do result in changes in the amino acid residues in the encoded polypeptide. Therefore, in one embodiment, the present disclosure provides polynucleotides encoding a polypeptide having at least 80% or 90% identity to SEQ ID NO:2, wherein at least one of the following conditions is met:

[0164] (a) the triplet codon encoding the amino acid residue at position 2 of the polypeptide is not GCT;

[0165] (b) the triplet codon encoding the amino acid residue at position 3 of the polypeptide is not AAA;

[0166] (c) the triplet codon encoding the amino acid residue at position 4 of the polypeptide is not ATC; or

[0167] (d) the triplet codon encoding the amino acid residue at position 11 of the polypeptide is not GAA.

[0168] In some embodiments, at least two of conditions (a)-(d) are met. In certain embodiments, at least three of conditions (a)-(d) are met. In specific embodiments, all four of conditions (a)-(d) are met.

[0169] In certain embodiments, it is not necessary to replace all codons to optimize the codon usage of the ketoreductase, because the native sequence will contain preferred codons, and because not all amino acid residues may require the use of preferred codons. Therefore, the codon-optimized polynucleotide encoding the ketoreductase may contain preferred codons at about 40%, 50%, 60%, 70%, 80% or more than 90% of the codon positions over the entire length of the coding region.

[0170] In various embodiments, the isolated polynucleotide encoding the improved ketoreductase polypeptide can be manipulated in a variety of ways to provide expression of the polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide prior to inserting it into a vector. The techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. The following provides guidance: Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3 rd Ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updated to 2006.

[0171] In some embodiments, the isolated polynucleotide encoding any ketoreductase polypeptide herein is manipulated in a variety of ways to promote the expression of the ketoreductase polypeptide. In some embodiments, the polynucleotide encoding the ketoreductase polypeptide comprises an expression vector in which one or more control sequences are present to regulate the expression of the ketoreductase polynucleotide and / or polypeptide. Depending on the expression vector utilized, it may be desirable or necessary to manipulate the isolated polynucleotide prior to inserting it into the vector. The techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, control sequences include, among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, a suitable promoter is selected based on the selection of the host cell. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters obtained from the Escherichia coli lactose operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (See, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75:3727-3731

[1978] ), and the tac promoter (See, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80:21-25

[1983] ). Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters obtained from genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from Aspergillus niger neutral α-amylase and Aspergillus oryzae triosephosphate isomerase), as well as mutants, truncations, and hybrids thereof. Exemplary yeast cell promoters can be derived from genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (See, e.g., Romanos et al., Yeast 8:423-488

[1992] ).

[0172] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence that is recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that functions in the selected host cell can be used in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin. Exemplary terminators for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells known in the art (see, e.g., Romanos et al., supra).

[0173] In some embodiments, the control sequence is also a suitable leader sequence (i.e., an mRNA non-translational region that is important for host cell translation). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the ketoreductase. Any suitable leader sequence that functions in the selected host cell can be used in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from genes related to: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtained from genes related to: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0174] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of the nucleic acid sequence and, when transcribed, recognized by the host cell as a signal to add polyadenylic acid residues to the transcribed mRNA). Any suitable polyadenylation sequence that functions in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, genes for: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin, and Aspergillus niger alpha-glucosidase. Polyadenylation sequences useful for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 15: 5983-5990

[1995] ).

[0175] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence connected to the amino terminus of the polypeptide and directs the encoded polypeptide to enter the cell secretory pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region, which is naturally connected to the coding region fragment encoding the secreted polypeptide in the translation reading frame. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region exogenous to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell can be used for the expression of the engineered polypeptide. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the following genes: Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Further signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0176] In some embodiments, regulatory sequences are also utilized. These sequences help regulate the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0177] On the other hand, the present invention relates to a recombinant expression vector comprising a polynucleotide encoding a ketoreductase polypeptide, and one or more expression regulatory regions, such as promoters and terminators, replication origins, etc., depending on the type of host into which they are to be introduced. In some embodiments, the various nucleic acids and control sequences described herein are linked together to produce a recombinant expression vector, the vector comprising one or more convenient restriction sites to allow insertion or substitution of the nucleic acid sequence encoding the enzyme polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of an expression vector, the coding sequence is located in a vector so that the coding sequence is operably linked to an appropriate control sequence for expression.

[0178] The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be conveniently used in recombinant DNA procedures and results in the expression of the enzyme polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is introduced. The vector can be a linear or closed circular plasmid.

[0179] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, and its replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any tool that ensures self-replication. In some alternative embodiments, the vector is a vector that, when introduced into a host cell, is integrated into the genome and replicates with the chromosome into which it is integrated. In addition, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids (which together contain the entire DNA to be introduced into the host cell genome) and / or a transposon are used.

[0180] In some embodiments, the expression vector contains one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or viral resistance, heavy metal resistance, prototrophy to auxotrophy, etc. Examples of bacterial selectable markers include, but are not limited to, dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from Aspergillus nidulans or Aspergillus oryzae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., from Streptomyces hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from Aspergillus nidulans or Aspergillus oryzae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0181] On the other hand, the present invention provides a host cell comprising at least one polynucleotide encoding at least one ketoreductase of the present disclosure, the polynucleotide being operably linked to one or more control sequences to express at least one ketoreductase in the host cell. Host cells suitable for expressing polypeptides encoded by the expression vector of the present invention are well known in the art and include, but are not limited to, bacterial cells, such as Escherichia coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); exemplary host cells also include various Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21). Examples of bacterial selective markers include, but are not limited to, dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol, and / or tetracycline resistance).

[0182] In some embodiments, the expression vector of the present invention contains elements that allow the vector to be integrated into the host cell genome or the vector to replicate autonomously in the cell independent of the genome. In some embodiments involving integration into the host cell genome, the vector relies on the nucleic acid sequence encoding the polypeptide or any other element of the vector to integrate the vector into the genome by homologous or nonhomologous recombination.

[0183] In some alternative embodiments, the expression vector contains additional nucleic acid sequences for guiding integration into the host cell genome by homologous recombination. The additional nucleic acid sequences enable the carrier to be integrated into the host cell genome at the precise position in the chromosome. In order to increase the possibility of integration at the precise position, the integration element preferably contains a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous to the corresponding target sequence, to improve the probability of homologous recombination. The integration element can be any sequence homologous to the target sequence in the host cell genome. In addition, the integration element can be a non-coding or encoding nucleic acid sequence. On the other hand, the carrier can be integrated into the host cell genome by non-homologous recombination.

[0184] For autonomous replication, the vector may further comprise an origin of replication so that the vector can autonomously replicate in the host cell in question. Examples of bacterial origins of replication are the origin of replication of P15A ori or plasmids pBR322, pUC19, pACYC177 (which contains P15A ori) or pACYC184 (which contains P15A ori), which allow replication in Escherichia coli, and the origin of replication of pUB110, pE194 or pTA1060, which allows replication in Bacillus. Examples of origins of replication for yeast host cells are 2 micron origins of replication ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6. The origin of replication may be a mutation that allows it to function as a temperature-sensitive construct in a host cell (see, e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433

[1978] ).

[0185] In some embodiments, more than one copy of the nucleic acid sequence of the present invention is inserted into the host cell to increase the production of the gene product. The copy number of the nucleic acid sequence can be increased by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence, wherein by culturing the cells in the presence of an appropriate selectable agent, cells containing amplified copies of the selectable marker gene and thus containing additional copies of the nucleic acid sequence can be selected.

[0186] Many expression vectors used in the present invention are commercially available. Suitable commercially available expression vectors include, but are not limited to pET E. coli T7 expression vector (Millipore Sigma) and p3xFLAGTM TMExpression vector (Sigma-Aldrich Chemicals). Other suitable expression vectors include, but are not limited to, pBluescriptII SK(-) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen), or pPoly (see, e.g., Lathe et al., Gene 57:193-201

[1987] ).

[0187] Thus, in some embodiments, a vector comprising a sequence encoding at least one variant ketoreductase is transformed into a host cell to allow for vector proliferation and expression of one or more variant ketoreductases. In some embodiments, the transformed host cells are cultured in a suitable nutrient medium under conditions that allow for expression of one or more variant ketoreductases. Any suitable medium that can be used to culture the host cells can be used in the present invention, including but not limited to minimal or complex medium containing appropriate supplements. In some embodiments, host cells are grown in HTP medium. Suitable medium can be obtained from various commercial suppliers or can be prepared according to published recipes (e.g., in the catalog of the American Type Culture Collection).

[0188] Host cells for expressing ketoreductase

[0189] On the other hand, the present disclosure provides a host cell comprising a polynucleotide encoding an improved ketoreductase polypeptide of the present disclosure, the polynucleotide being operably linked to one or more control sequences to express ketoreductase in the host cell. Host cells for expressing ketoreductase polypeptides encoded by the expression vector of the present invention are well known in the art, and include but are not limited to bacterial cells, such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus amyloliquefaciens, Lactobacillus kefir (Lactobacilluskejir), Lactobacillus brevis (L.brevis), Lactobacillus minor (Lactobacillus minor), Streptomyces and Salmonella typhimurium (Salmonellatyphimurium) cells; Fungal cells, such as Saccharomyces cerevisiae or Pichia pastoris (ATCC Record No. 201178). Suitable culture media and growth conditions for the above-mentioned host cells are well known in the art.

[0190] The polynucleotide for expressing ketoreductase can be introduced into cells by various methods known in the art. The techniques include electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion, among others. Various methods for introducing polynucleotides into cells will be apparent to those skilled in the art.

[0191] In some embodiments of the invention, the filamentous fungal host cell is of any suitable genus and species, including, but not limited to, Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium , Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium and / or Volvariella, and / or their sexual or asexual forms, and their synonyms, basic synonyms or taxonomic equivalents.

[0192] In some embodiments of the invention, the host cell is a yeast cell, including but not limited to a cell of a Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerance, or Pichia spp. thermotolerans, Pichia alictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0193] In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include but are not limited to Gram-positive, Gram-negative and Gram-indeterminate bacterial cells. Any suitable bacterial organism can be used for the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium. rium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium um, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacteriu m), Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum,Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium anaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas. In some embodiments, the host cell is selected from the following species: Agrobacterium, Acinetobacter, Azotobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces, or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments, the bacterial host strain is an industrial strain. Many bacterial industrial strains are known and suitable for the present invention. In some embodiments of the present invention, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfates, and A. ureafaciens). In some embodiments of the invention,The bacterial host cell is a Bacillus species (e.g., B. thuringensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alcalophilus, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens). In some embodiments, the host cell is an industrial Bacillus strain, including but not limited to Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus clausii, Bacillus stearothermophilus or Bacillus amyloliquefaciens. In some embodiments, the Bacillus host cell is Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus and / or Bacillus amyloliquefaciens. In some embodiments, the bacterial host cell is a Clostridium species (e.g., C.acetobutylicum, C.tetani E88, C.lituseburense, C.saccharobutylicum, C.perfringens and C.beijerinckii). In some embodiments, the bacterial host cell is a species of the genus Corynebacterium (e.g., Corynebacterium glutamicum and Corynebacterium acetoacidophilum). In some embodiments, the bacterial host cell is a species of the genus Escherichia (e.g., E. coli). In some embodiments, the host cell is E. coli W3110. In some embodiments, the host is E. coli BL21 or BL21 (DE3). In some embodiments, the bacterial host cell is a species of the genus Erwinia (e.g., Erwinia uredovora, Erwinia carotovora, Erwinia ananas, Erwinia herbicola, Erwinia punctata, and Erwinia terreus). In some embodiments,The bacterial host cell is a Pantoea species (e.g., P. citrea and P. agglomerans). In some embodiments, the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and Pseudomonas species (P. sp.) D-0110). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, and S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Zymomonas mobilis and Zymomonas lipolytica).

[0194] Many prokaryotic and eukaryotic strains used in the present invention are readily available from a number of culture collections, such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms (DSM), the Netherlands Fungal Culture Collection (Centraalbureau Voor Schimmelcultures, CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, NRRL.

[0195] In some embodiments, host cells are genetically modified to have improved protein secretion, protein stability and / or the characteristics of other characteristics required for the expression and / or secretion of proteins.Genetic modification can be achieved by genetic engineering techniques and / or classical microbiological techniques (e.g., chemical or ultraviolet mutagenesis and subsequent selection).In fact, in some embodiments, host cells are produced using a combination of recombinant modification and classical selection techniques.Using recombinant techniques, nucleic acid molecules can be introduced, deleted, suppressed or modified in a manner that causes an increase in the output of ketoreductase variants in the host cell and / or in the culture medium.In a genetic engineering method, homologous recombination is used to induce targeted gene modification by specific targeting genes in vivo to suppress the expression of encoded proteins.In alternative methods, siRNA, antisense and / or ribozyme technology can be used to suppress gene expression.A variety of methods known in the art for reducing protein expression in cells include but are not limited to all or part of the deletion of protein encoding genes and site-specific mutagenesis to destroy the expression or activity of gene products. (See, e.g., Chaveroche et al., Nucl. Acids Res., 28:22e97

[2000] ; Cho et al., Molec. Plant Microbe Interact., 19:7-15

[2006] ; Maruyama and Kitamoto, Biotechnol. Lett., 30:1811-1817

[2008] ; Takahashi et al., Mol. Gen. Genom., 272:344-352

[2004] ; and You et al., Arch. Microbiol., 191:615-622

[2009] , the entire contents of which are incorporated herein by reference). Random mutagenesis followed by screening for desired mutations also works (See, e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8

[2003] ; and Firon et al., Eukary. Cell 2:247-55

[2003] , both incorporated by reference).

[0196] Introduction of a vector or DNA construct into a host cell can be accomplished using any suitable method known in the art, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, PEG-mediated transformation, electroporation, or other common techniques known in the art.

[0197] In some embodiments, the engineered host cells of the invention (i.e., "recombinant host cells") are cultured in conventional nutrient media modified to be suitable for activating promoters, selecting transformants, or amplifying ketoreductase polynucleotides. The culture conditions (e.g., temperature, pH, etc.) are those previously used by the host cell selected for expression and are well known to those skilled in the art. As previously mentioned, many standard references and texts are available for culturing and producing many cells, including cells of bacterial, plant, animal (especially mammalian) and archaeal origin.

[0198] In some embodiments, the cells expressing the ketoreductase of the present invention are grown under batch or continuous fermentation conditions. Classical "batch fermentation" is a closed system, wherein the composition of the culture medium is set at the beginning of the fermentation and is not subject to artificial changes during the fermentation process. A variant of the batch system is "fed batch fermentation", which can also be used in the present invention. In this variant, as the fermentation proceeds, the substrate is added in an incremental manner. When catabolite repression may inhibit the metabolism of the cell and the substrate amount in the culture medium is required to be limited, the fed batch system is useful. Batch fermentation and fed batch fermentation are very common and well-known in the art. "Continuous fermentation" is an open system, wherein the specified fermentation medium is continuously added to the bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation usually maintains the culture at a constant high density, wherein the cells are mainly in logarithmic phase growth. Continuous fermentation systems strive to maintain steady-state growth conditions. Methods for regulating the nutrients and growth factors of the continuous fermentation process and the technology for maximizing the product production rate are well-known in the field of industrial microbiology.

[0199] More than one copy of the nucleic acid sequence of the present invention can be inserted into the host cell to increase the production of the gene product. The copy number of the nucleic acid sequence can be increased by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence, wherein cells can be cultured in the presence of an appropriate selectable agent to select for amplified copies of the selectable marker gene, and thus cells containing additional copies of the nucleic acid sequence selected.

[0200] In some embodiments of the invention, cell-free transcription and translation systems can be used to produce ketoreductase. Several systems are commercially available, and these methods are well known to those skilled in the art.

[0201] Evolutionary approaches to ketoreductase

[0202] In some embodiments, in order to make the ketoreductase of the present disclosure, a ketoreductase that catalyzes the reduction reaction is obtained (or derived) from Escherichia coli. In some embodiments, the parent polynucleotide sequence is codon-optimized to enhance the expression of the ketoreductase in a specific host cell. The parent polynucleotide sequence encoding SEQ ID NO:2 (designated as SEQ ID NO:3) is codon-optimized for expression in Escherichia coli, and the codon-optimized polynucleotide is cloned into an expression vector so that the expression of the ketoreductase gene is under the control of the T7 promoter. The T7 polymerase required for expressing the target gene is under the control of the lac promoter, and both the target gene and the T7 polymerase are inhibited by lac1. The presence of IPTG activates the production of the T7 polymerase and eliminates inhibition, resulting in the expression of the ketoreductase protein. The clones expressing active ketoreductase in Escherichia coli were identified, and the genes were sequenced to confirm their identity.

[0203] The ketoreductase of the present disclosure can be obtained by mutagenesis and / or directed evolution methods for polynucleotides encoding parental sequences. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling, as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91: 10747-10751; WO 95 / 22625; WO 97 / 20078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Pat. No. 6,537,746. Other useful directed evolution procedures include the staggered extension process (StEP), in vitro recombination (Zhao et al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis (Black et al., 1996, Proc. Natl. Acad. Sci. USA 93:3525-3529), among others.

[0204] The clones obtained after the mutagenesis treatment are screened to obtain ketoreductases with the desired improved enzyme properties. The enzyme activity in the expression library can be measured using standard chemical analysis techniques (e.g., UPLC-MS) for measuring substrates and products, and standard biochemical techniques for monitoring the rate of decrease (via a decrease in absorbance or fluorescence) of the concentration of NADH or NADPH (as it is converted into NAD+ or NADP+). In this reaction, NADH or NADPH is consumed (oxidized) by the ketoreductase as the ketone substrate is reduced to the corresponding hydroxyl group by the ketoreductase. The rate of decrease of the concentration of NADH or NADPH per unit time, as measured by a decrease in absorbance or fluorescence, represents the relative (enzymatic) activity of the ketoreductase polypeptide in a fixed amount of lysate (or a lyophilized powder made therefrom). When the desired improved enzyme property is thermal stability, the enzyme activity can be measured after the enzyme preparation is placed at a specified temperature and the amount of enzyme activity remaining after the heat treatment is measured. A clone containing a polynucleotide encoding a ketoreductase is then isolated, sequenced to identify nucleotide sequence variations, if any, and used to express the enzyme in a host cell.

[0205] When the polypeptide sequence is known, the polynucleotide encoding the enzyme can be prepared by a standard solid phase method according to a known synthesis method. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then connected (e.g., by enzymatic or chemical connection methods, or polymerase-mediated methods) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides of the present invention can be prepared by chemical synthesis, for example, using the classical phosphoramidite method, such as Beaucage et al., 1981, Tet. Lett. 22: 1859-69, or such as Matthes et al., 1984, EMBO J. 3: 801-05, as it is usually practiced in an automatic synthesis method. According to the phosphoramidite method, oligonucleotides are synthesized in, for example, an automatic DNA synthesizer, purified, annealed, connected and cloned in an appropriate vector. In addition, essentially any nucleic acid can be obtained from a variety of commercial sources, such as The Midland Certified Reagent Company, Midland, Tex., The Great American Gene Company, Ramona, Calif., ExpressGen Inc. Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and many others.

[0206] Ketoreductases expressed in host cells can be recovered from cells and / or culture medium using one or more well-known protein purification techniques, including, but not limited to, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography. Suitable solutions for lysing and efficiently extracting proteins from bacteria (e.g., E. coli) are available from Sigma-Aldrich, St. Louis, Missouri, under the trade name Buy.

[0207] Chromatographic techniques for isolating ketoreductase polypeptides include, among others, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying a particular enzyme depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to those skilled in the art.

[0208] In some embodiments, affinity techniques can be used to separate improved ketoreductases. With respect to affinity chromatography purification, the protein sequence can be tagged with a recognition sequence to achieve purification. Common tags include cellulose binding domains, poly-His tags, dimeric His chelates, FLAG tags, and many other tags well known to those skilled in the art. Antibodies can also be used as affinity purification reagents. Any antibody that specifically binds to a ketoreductase polypeptide can be used.

[0209] Methods of using ketoreductase

[0210] The ketoreductases described herein can catalyze the reaction of substituted indanone compounds, such as fluorohydroxyindanone (6)

[0211]

[0212] Reduction to fluorodiol (7)

[0213]

[0214] In specific embodiments, the ketoreductases described herein can be used immediately following electrophilic fluorination of hydroxyindanone (5) to provide fluorodiol (7).

[0215]

[0216] In some embodiments, the method for preparing fluorodiol (7) comprises: contacting hydroxyindanone (5) with a fluorinating agent under acidic conditions to obtain fluorohydroxyindanone (6); contacting fluorohydroxyindanone (6) with a ketoreductase disclosed herein under reaction conditions suitable for reducing or converting fluorohydroxyindanone (6) to fluorodiol (7). Fluorodiol (7) is used as an intermediate in the synthesis of benzyltransferase (WELIREG). Therefore, in the method for preparing benzyltransferase, the method may include a step in which hydroxyindanone (5) is converted to fluorodiol (7) using a ketoreductase disclosed herein. In some embodiments, the diastereomeric excess of the product is greater than about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 6099.7%, 99.8% or 99.9% compared to the corresponding (1R) alcohol product.

[0217] As described herein, in some embodiments, the ketoreductase can comprise an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence comprising the sequence of SEQ ID NO: 2. In some embodiments, these ketoreductase polypeptides can have one or more modifications to the amino acid sequence of SEQ ID NO: 2. The modifications can include substitutions, deletions, or insertions. The substitutions can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions.

[0218] In some embodiments of the method for reducing a substrate to a product, the substrate is reduced to the product in greater than about 99% diastereomeric excess, wherein the ketoreductase polypeptide comprises a sequence corresponding to SEQ ID NO:2.

[0219] In another embodiment of the method for reducing a substrate to a product, at least about 95% of the substrate is converted to the product in less than about 24 hours when performed using more than about 60 g / L of substrate and less than about 5 g / L of polypeptide, wherein the polypeptide comprises an amino acid sequence corresponding to SEQ ID NO: 2.

[0220] As known to those skilled in the art, ketoreductases that catalyze reduction reactions generally require cofactors. Reduction reactions catalyzed by ketoreductases described herein also generally require cofactors, although many embodiments of engineered ketoreductases require far fewer cofactors than reactions catalyzed by wild-type ketoreductases. As used herein, the term "cofactor" refers to a non-protein compound that works in combination with a ketoreductase. Cofactors suitable for use with engineered ketoreductases described herein include, but are not limited to, NADP+ (nicotinamide adenine dinucleotide phosphate), NADPH (reduced form of NADP+), NAD+ (nicotinamide adenine dinucleotide), and NADH (reduced form of NAD+). Typically, a reduced form of the cofactor is added to the reaction mixture. Optionally, a cofactor regeneration system can be used to regenerate a reduced form of NAD(P)H from an oxidized form of NAD(P)+.

[0221] The term "cofactor regeneration system" refers to a group of reactants that participate in a reaction in which an oxidized form of a cofactor is reduced (e.g., NADP+ to NADPH). By means of the cofactor regeneration system, the oxidized cofactor is regenerated into a reduced form, and the cofactor is oxidized by the reduction of a ketone substrate catalyzed by a ketoreductase. The cofactor regeneration system comprises a stoichiometric reductant, which is a source of reducing hydrogen equivalents and is capable of reducing the oxidized form of the cofactor. The cofactor regeneration system may further comprise a catalyst, such as an enzyme catalyst that catalyzes the reduction of the oxidized form of the cofactor by the reductant. In some embodiments, the ketoreductase of the present disclosure itself can act as an enzyme catalyst that catalyzes the reduction of the oxidized form of the cofactor by the reductant. Therefore, in such embodiments, the ketoreductase exerts a dual catalytic effect. Cofactor regeneration systems that regenerate NADH or NADPH from NAD+ or NADP+, respectively, are known in the art and can be used in the methods described herein.

[0222] Suitable exemplary cofactor regeneration systems that can be used include, but are not limited to, glucose and glucose dehydrogenase, formate and formate dehydrogenase, glucose-6-phosphate and glucose-6-phosphate dehydrogenase, secondary alcohols (e.g., isopropanol) and secondary alcohol dehydrogenases, phosphorous acid and phosphorous acid dehydrogenases, molecular hydrogen and hydrogenases, etc. These systems can be used in combination with NADP+ / NADPH or NAD+ / NADH as cofactors. Electrochemical regeneration using hydrogenases can also be used as cofactor regeneration systems. See, e.g., U.S. Patent Nos. 5,538,867 and 6,495,023, both of which are incorporated herein by reference. Chemical cofactor regeneration systems comprising a metal catalyst and a reducing agent (e.g., molecular hydrogen or formic acid) are also suitable. See, e.g., PCT Publication WO 2000 / 053731, which is incorporated herein by reference.

[0223] The terms "glucose dehydrogenase" and "GDH" are used interchangeably herein and refer to an NAD+ or NADP+ dependent enzyme that catalyzes the conversion of D-glucose and NAD+ or NADP+ into gluconic acid and NADH or NADPH, respectively.

[0224] Glucose dehydrogenases suitable for implementing the methods described herein include naturally occurring glucose dehydrogenases, and non-naturally occurring glucose dehydrogenases. The coding genes of naturally occurring glucose dehydrogenases have been reported in the literature. For example, the subtilis 61297 GDH gene is expressed in Escherichia coli, and the physicochemical properties displayed are reported to be the same as those of the enzyme produced in its natural host (Vasantha et al., 1983, Proc. Natl. Acad. Sci. USA 80: 785). The gene sequence of the subtilis 61297 GDH gene corresponds to Genbank accession number M12276, reported by Lampel et al. (Lampel et al., 1986, J. Bacterial. 166: 238-243), and the corrected form is reported as Genbank accession number D50453 by Yamane et al. (Yamane et al., 1996, Microbiology 142: 3047-3056). Naturally occurring GDH genes also include those encoding GDH from Bacillus cereus ATCC 14579 (Nature, 2003, 423: 87-91; Genbank Accession No. AE017013) and Bacillus megaterium (Eur. J. Biochem., 1988, 174: 485-490, Genbank Accession No. X12370; J. Ferment. Bioeng., 1990, 70: 363-369, Genbank Accession No. GI216270). PCT Publication WO 2005 / 018579 provides glucose dehydrogenases from Bacillus species, the disclosure of which is incorporated herein by reference.

[0225] Non-naturally occurring glucose dehydrogenases can be prepared using known methods, such as, for example, mutagenesis, directed evolution, etc. One of ordinary skill in the art can readily identify glucose dehydrogenases (GDHs), whether naturally occurring or non-naturally occurring, having suitable activity, including using the assay methods described in Example 4 of PCT Publication No. WO 2005 / 018579, the disclosure of which is incorporated herein by reference.

[0226] The reduction reaction catalyzed by ketoreductase as described herein is usually carried out in a solvent. Suitable solvents include water, organic solvents (such as ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.) and ionic liquids (such as 1-ethyl-4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate hexafluorophosphate, etc.). In some embodiments, aqueous solvents are used, including water and aqueous co-solvent systems.

[0227] Exemplary aqueous co-solvent systems have water and one or more organic solvents. Typically, the organic solvent component of the aqueous co-solvent system is selected so that it does not completely inactivate the ketoreductase. Suitable co-solvent systems can be readily determined by measuring the enzymatic activity of a given engineered ketoreductase using a defined target substrate in a candidate solvent system using an enzyme activity assay such as those described herein.

[0228] The organic solvent component of the aqueous co-solvent system can be miscible with the aqueous component to provide a single liquid phase; or it can be partially miscible or immiscible with the aqueous component to provide two liquid phases. Typically, when an aqueous co-solvent system is adopted, it is selected as two-phase, wherein water is dispersed in an organic solvent, or vice versa. Typically, when utilizing an aqueous co-solvent system, it is desirable to select an organic solvent that is easy to separate from the aqueous phase. Typically, the ratio of water to organic solvent in the co-solvent system is typically in the range of about 90:10 to about 10:90 (v / v) of organic solvent: water, and between 80:20 to 20:80 (v / v) of organic solvent: water. The co-solvent system can be preformed before being added to the reaction mixture, or can be formed in situ in the reaction vessel.

[0229] Aqueous solvents (water or aqueous co-solvent systems) can be pH buffered or unbuffered. Typically, reduction can be performed at a pH of about 10 or lower, typically in the range of about 5 to about 10. In some embodiments, reduction is performed at a pH of about 9 or lower, typically in the range of about 5 to about 9. In some embodiments, reduction is performed at a pH of about 8 or lower, typically in the range of about 5 to about 8, and typically in the range of about 6 to about 8. It is also possible to reduce at a pH of about 7.8 or lower, or at a pH of 7.5 or lower. Alternatively, reduction can be performed at a neutral pH (i.e., pH about 7).

[0230] In the reduction reaction process, the pH of the reaction mixture can change. The pH of the reaction mixture can be maintained within the desired pH or desired pH range by adding acid or alkali during the reaction. Alternatively, the pH can be controlled by using an aqueous solvent comprising a buffer. Suitable buffers for maintaining the desired pH range are known in the art, and include, for example, phosphate buffers, triethanolamine buffers, etc. A combination of buffering and acid or alkali addition can also be used.

[0231] When a glucose / glucose dehydrogenase cofactor regeneration system is used, as shown in equation (1), if the generated aqueous gluconic acid is not otherwise neutralized, the co-generation of gluconic acid (pKa=3.6) will cause the pH of the reaction mixture to drop. The pH of the reaction mixture can be maintained at a desired level by standard buffering techniques, wherein the buffer neutralizes the gluconic acid to a provided buffer capacity, or by simultaneously adding a base during the conversion process to maintain the pH of the reaction mixture at a desired level. A combination of buffering and base addition can also be used. Suitable buffers for maintaining a desired pH range are described above. Suitable bases for neutralizing gluconic acid include organic bases, such as amines, alcohol salts, etc., and inorganic bases, such as hydroxide salts (such as NaOH), carbonates (such as NaHCO3), bicarbonates (such as K2CO3), alkaline phosphates (such as K2HPO4, Na3PO4), etc. Simultaneous addition of a base during the conversion process can be performed manually while monitoring the pH of the reaction mixture, or more conveniently, by using an automatic titrator as a pH constant. A combination of partial buffer capacity and base addition can also be used for process control.

[0232] When base addition is used to neutralize the gluconic acid released during the ketoreductase catalyzed reduction reaction, the progress of the conversion can be monitored by the amount of base added to maintain the pH. Typically, the base added to the unbuffered or partially buffered reaction mixture during the reduction is added in aqueous solution.

[0233] In some embodiments, the cofactor regeneration system may include a formate dehydrogenase. The terms "formate dehydrogenase" and "FDH" are used interchangeably herein and refer to NAD + or NADP + dependent enzyme that catalyzes the conversion of formate and NAD + or NADP +to carbon dioxide and NADH or NADPH, respectively. Formate dehydrogenases suitable for use as cofactor regeneration systems in the ketoreductase-catalyzed reduction reactions described herein include both naturally occurring formate dehydrogenases and non-naturally occurring formate dehydrogenases. Formate dehydrogenases include those corresponding to SEQ ID NO:70 (Pseudomonas species) and SEQ ID NO:72 (Candida boidinii) of PCT Publication WO 2005 / 018579, which are encoded by polynucleotide sequences corresponding to SEQ ID NO:69 and 71 of PCT Publication WO 2005 / 018579, respectively, the disclosures of which are incorporated herein by reference. The formate dehydrogenases employed in the methods described herein, whether naturally occurring or non-naturally occurring, may exhibit a rate of at least about 1 μmol / min / mg, sometimes at least about 10 μmol / min / mg, or at least about 10 2 μmol / min / mg, up to about 10 3 The invention discloses a method for preparing a novel nanostructured ...

[0234] As used herein, the term "formic acid" refers to the formate anion (HCO2 - ), formic acid (HCO2H), and mixtures thereof. Formic acid can be provided in the form of a salt, usually an alkali metal salt or an ammonium salt (e.g. HCO2Na, KHCO2NH4, etc.), in the form of formic acid, usually an aqueous solution of formic acid, or a mixture thereof. Formic acid is a medium-strong acid. In aqueous solution within a few pH units of its pKa (pKa in water = 3.7), formic acid is present as HCO2 - and HCO2H exist in equilibrium concentrations. At pH values ​​above about pH 4, formic acid is primarily present as HCO2 -Existence. When formic acid is provided in the form of formic acid, the reaction mixture is generally buffered or reduced in acidity by adding alkali to provide the desired pH, generally about pH 5 or higher. Suitable alkalis for neutralizing formic acid include, but are not limited to, organic bases, such as amines, alcohol salts, etc., and inorganic bases. When a glucose / glucose dehydrogenase cofactor regeneration system is adopted, if the aqueous gluconic acid generated is not otherwise neutralized, the co-generation of gluconic acid (pKa=3.6) can cause the pH of the reaction mixture to decrease. The pH of the reaction mixture can be maintained at a desired level by standard buffering techniques, wherein the buffer neutralizes the gluconic acid to the buffer capacity provided, or by simultaneously adding alkali during the conversion process, the pH of the reaction mixture is maintained at a desired level. A combination of buffering and alkali addition can also be used. Suitable buffers for maintaining a desired pH range are described above. Suitable alkalis for neutralization are, for example, hydroxide salts (such as NaOH), carbonates (such as NaHCO3), bicarbonates (such as K2CO3), alkaline phosphates (such as K2HPO4, Na3PO4), etc.

[0235] When formic acid and formate dehydrogenase are used as the cofactor regeneration system, the pH of the reaction mixture can be maintained at the desired level by standard buffering techniques, wherein the buffer releases protons until the buffer capacity is provided, or acid is added simultaneously during the conversion process. Suitable acids added to maintain pH during the reaction include organic acids, such as carboxylic acids, sulfonic acids, phosphonic acids, etc., inorganic acids, such as hydrohalic acids (such as hydrochloric acid), sulfuric acid, phosphoric acid, etc., acidic salts, such as dihydrogen phosphates (such as KH2PO4), hydrogen sulfates (such as NaHSO4), etc. Some embodiments utilize formic acid, whereby both the formic acid concentration and pH in the solution can be maintained.

[0236] When acid addition is used to maintain pH during the reduction reaction using a formate / formate dehydrogenase cofactor regeneration system, the progress of the conversion can be monitored by the amount of acid added to maintain the pH. Typically, the acid added to the unbuffered or partially buffered reaction mixture during the reduction process is added in an aqueous solution.

[0237] In carrying out the embodiments of the ketoreductase-catalyzed reduction reaction described herein using a cofactor regeneration system, the cofactor can be initially provided in an oxidized form or a reduced form. As described above, the cofactor regeneration system converts the oxidized cofactor to its reduced form, which is then used for the reduction of the ketoreductase substrate.

[0238] In some embodiments, a cofactor regeneration system is not used. For reduction reactions performed without a cofactor regeneration system, the cofactor is added to the reaction mixture in reduced form.

[0239] In some embodiments, when the method is performed using intact cells of a host organism, the intact cells may provide the cofactors naturally.Alternatively or in combination, the cells may provide glucose dehydrogenase naturally or recombinantly.

[0240] When performing stereoselective reduction reactions as described herein, ketoreductases and any enzymes comprising optional cofactor regeneration systems can be added to the reaction mixture in the form of purified enzymes, in the form of intact cells transformed with genes encoding the enzymes, and / or in the form of cell extracts and / or lysates of such cells. The genes encoding ketoreductases and optional cofactor regeneration enzymes can be transformed into host cells separately, or transformed into the same host cell together. For example, in some embodiments, one group of host cells can be transformed with genes encoding ketoreductases, and another group can be transformed with genes encoding cofactor regeneration enzymes. Two groups of transformed cells can be used together in the reaction mixture in the form of intact cells, or in the form of lysates or extracts derived therefrom. In other embodiments, host cells can be transformed with genes encoding both ketoreductases and cofactor regeneration enzymes.

[0241] Whole cells transformed with genes encoding ketoreductase and / or optional cofactor regenerating enzymes, or cell extracts and / or lysates thereof, can be used in a variety of different forms, including solid (such as lyophilized, spray-dried, etc.) or semi-solid (such as a crude paste).

[0242] Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment, etc.) followed by a desalting procedure (such as ultrafiltration, dialysis, etc.) prior to lyophilization. Any cell preparation can be stabilized by cross-linking using known cross-linking agents.

[0243] In some embodiments, ketoreductases with improved purity may be desired. In such embodiments, clarified cell lysates used to obtain ketoreductases may be pre-treated with isopropanol to a volume % of isopropanol of 25%-30%. The isopropanol treated lysate may be incubated at 30°C for 1 hour to overnight. The isopropanol treated lysate may then be centrifuged and the precipitate may be removed. The supernatant may then be transferred to a petri dish and frozen at -80°C for a minimum of 2 hours. Lyophilization may then be performed using standard automated protocols. Figure 1 As shown in , gel electrophoresis using sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel (also known as SDS-PAGE) showed that proteins insoluble in isopropanol (iPrOH) were removed to obtain purified ketoreductase. Figure 1In the figure, lane 1 (labeled as standard) is the marker; lane 2 (labeled as P012024-B07) is the crude enzyme preparation; lane 3 (labeled as B07, 30% IPA / 30% IPA / 30C / 1h) is the crude enzyme preparation treated with 30% iPrOH solution for 1 hour and centrifuged; and lane 4 (labeled as 4 precipitate-B07 / 30% IPA) is the solid from the centrifugation. Figure 1 As shown in , lane 3 shows that many bands in the enzyme preparation were removed after treatment with iPrOH. Lane 4 shows the protein, which was precipitated and removed by centrifugation.

[0244] Suitable conditions for carrying out the ketoreductase-catalyzed reduction reactions described herein include a wide variety of conditions that can be readily optimized by routine experimentation, including, but not limited to, contacting the engineered ketoreductase and substrate at the experimental pH and temperature, and detecting the product, e.g., using the methods described in the Examples provided herein.

[0245] Ketoreductase-catalyzed reductions are typically carried out at a temperature in the range of about 15° C. to about 75° C. For some embodiments, the reaction is carried out at a temperature in the range of about 20° C. to about 55° C. In still other embodiments, the reaction is carried out at a temperature in the range of about 20° C. to about 45° C. The reaction may also be carried out under ambient conditions.

[0246] The reduction reaction is usually allowed to proceed until a substantially complete or nearly complete reduction of the substrate is achieved. The reduction of the substrate to the product can be monitored using known methods by detecting the substrate and / or the product. Suitable methods include gas chromatography, HPLC, etc. The conversion yield of the alcohol reduction product generated in the reaction mixture is usually greater than about 50%, also greater than about 60%, also greater than about 70%, also greater than about 80%, also greater than 90%, and usually greater than about 97%. Example

[0247] Example 1: Enzyme preparation

[0248] The E. coli cultures each carrying a plasmid encoding a ketoreductase (as described above, represented by the following amino acid sequences shown in SEQ ID NOs. 1, 2, 4-16) were serially diluted to 10 using Luria-Bertani broth (cell culture medium) as a diluent. -4 , 10 -5 and 10 -6 Dilution. Spread 100 μL of each dilution on a Petri dish containing LB agar supplemented with 50 μg / mL kanamycin. Place the plate in a 30°C incubator overnight.

[0249] The Luria-Bertani culture fluid (cell culture medium) (500mL LB+50μg / mL kanamycin) of 200μL per hole is divided equally into the 96-well shallow well plate of labeling. The shallow well plate is loaded into the plate stacker of the bacterium colony picker. The agar plate containing enough dilutions to separate the bacterium colonies of most bacterium colonies from each other (referred to as single bacterium colonies by those skilled in the art) is selected into the unique hole of the shallow well plate. Make the bacterium colony grow overnight at 200rpm, 30℃ and 85%RH.

[0250] 390 μL of Terrific Broth (TB) growth medium (available from ThermoFisher Scientific, catalog # A1374301) (TB + 50 μg / mL kanamycin) was aliquoted into labeled 96-well deep well subculture plates. 13 μL of overnight growth culture was transferred from each well of the master shallow well plate to the corresponding labeled deep well subculture plate. The plate was sealed with a breathable membrane and shaken at 250 rpm for 2-2.5 hours at 30°C and 85% RH. After shaking, the optical density (OD) of at least one plate was measured for growth. 600 , optical density at 600nm wavelength). When the OD 600 When pH was in the range of 0.4-0.8, the deep well plates were induced with 4 μL of 1 M IPTG solution per well. The plates were resealed and incubated at 30 °C and 85% RH with shaking at 250 rpm for 18-20 hours.

[0251] After incubation, all deep well plates were centrifuged at 4000 rpm for 15 minutes at 4°C. After centrifugation, the supernatant was discarded. The plates were heat sealed and stored at -80°C.

[0252] Remove the cell culture plates from -80°C storage and thaw at RT. Prepare 100 mM potassium phosphate (pH 8.0), 1 mg / mL lysozyme, 0.50 mg / mL polymyxin B sulfate (PMBS), 3 units / mL DNase I, 4 mM MgSO4, and 1 mg / mL NADP. + Lysis buffer. 200 μL lysis buffer was aliquoted into each well. The lysis mixture was vibrated at 1000 rpm on a plate shaker at RT for 1-1.5 hours. The lysis mixture was then centrifuged at 4000 rpm for 15 minutes at 4°C to prepare an enzyme-containing lysate solution (in the supernatant). In some cases, the lysate solution was further incubated with isopropanol for 1-5 hours in a solution of 25-30% isopropanol. After incubation, the lysate was centrifuged as described above.

[0253] Example 2: Ketoreductase reaction in a well plate

[0254]

[0255] The reaction buffer was prepared by resuspending the solid flow substrate (an impure product of a previous chemical fluorination reaction (6), see Example 5 below) to a concentration of 75-100 g / L in a 16:16:10:58 (v / v) solution of acetonitrile:methanol:isopropanol:potassium phosphate buffer (pH 8.0). The pH was then further adjusted to 8.0 using aqueous sodium hydroxide.

[0256] 80 μL of reaction buffer was added to a 0.3 mL round bottom well plate, followed by 20 μL of the enzyme-containing lysate solution of Example 1. The plate was heat sealed and shaken at 35° C. and 1000 rpm overnight.

[0257] After shaking overnight, 30 μL from the reaction was added to a new round-bottom well plate containing 240 μL acetonitrile. Allow the mixture to age for 1 hour, at which point 30 μL was added to the top of the filter stack containing an additional 240 μL of 20% (v / v) acetonitrile / water (filter plate above the round-bottom plate, with 0.20 μM hydrophilic PTFE, commercially available from Millipore MSRLN2250). The well plate was then centrifuged at 4000 rpm for 3 minutes at 4°C. The filter plate was removed, and the clear solution in the receiving plate was heat-sealed.

[0258] The filtered solution was analyzed by ultra performance liquid chromatography (UPLC), using a high throughput screening method to monitor the substrate and the degradation of the substrate, as well as the peak area of ​​the product. UPLC was performed on an Agilent instrument with a Waters HSS T3 1.8 μm, 2.1 x 75 mm column, using an isocratic method of 14% CH3CN + 0.1% TFA / H2O + 0.1% TFA over 1.1 minutes; flow rate 1 mL / min. The two diastereomers of the starting material eluted at 0.53 minutes and 0.66 minutes, respectively, the diastereomer of the desired product (7) eluted at 0.46 minutes, and the diastereomers of the undesired product (7-1, 7-2, 7-3) eluted at 0.48 minutes, 0.88 minutes and 0.47 minutes, respectively. Enantioselectivity was determined by supercritical fluid chromatography on a Daicel CHIRALPAK IG-3 3.0 μm, 50 x 4.6 mm column using an Agilent instrument. Mobile phase A was supercritical CO2 and mobile phase B was isopropanol. The flow rate was 2.5 mL / min; linear gradient from 18-37% B over 1.5 minutes, 37% B over 0.2 minutes, linear gradient to 18% B over 0.05 minutes, and 0.15 minutes equilibrium time at 18% B (total time 1.9 minutes). The two diastereomers of the starting material eluted at 0.79 minutes and 1.6 minutes, respectively, the diastereomer of the desired product (7) eluted at 1.1 minutes, and the diastereomers of the undesired product (7-1, 7-2, 7-3) eluted at 1.3 minutes, 0.9 minutes, and 0.6 minutes, respectively.

[0259] Example 3: Enzyme preparation in shake flasks

[0260] 10 μL of E. coli cells were inoculated into 5 mL of Luria-Bertani broth (cell culture medium) (250 mL of LB + 50 μg / ml of kanamycin + 1% glucose), each carrying a plasmid encoding a ketoreductase, which can be represented by the amino acid sequence shown in SEQ ID NO. 1, 2, and 4-19 as follows, which has been stored at -80°C in 20% glycerol and aliquoted into labeled 15 mL cell culture tubes as described below. The cell culture tubes were sealed and incubated at 30°C with shaking at 250 rpm for 20-24 hours.

[0261] After overnight growth, overnight growth culture (2-5 mL cell culture (with a starting OD of 0.2) was plated. 600)) was added to 250 mL of Terrific Broth (TB) growth medium (commercially available from ThermoFisher Scientific, catalog #A1374301) (TB + 50 μg / ml kanamycin) to a final volume of 250 mL. The flask was shaken at 250 rpm at 30°C for 3-4 hours. After shaking, the OD was measured for growth 600 , until OD 600 At this point, 1 mM IPTG (250 μL of 1 M IPTG) was added to the culture to induce expression, and the culture was grown at 30°C at 250 rpm for 20-24 hours.

[0262] After the additional growth period, the culture was transferred to a centrifuge bottle of known weight and centrifuged at 4000 rpm for 20 minutes at 4°C. After centrifugation, the supernatant was discarded and the cell pellet remaining in the bottle was weighed. The weight of the cell pellet was calculated by subtracting the known weight of the bottle, and the cell pellet was resuspended in 5 volumes of 50 mM sodium phosphate buffer (pH = 7) at 5 times the volume of the cell pellet weight.

[0263] The cells in the resuspended cell pellet were lysed using a microfluidizer, and the cell lysate was collected and centrifuged at 10,000 rpm for 60 minutes at 4°C. In some cases, the clarified lysate was further treated with isopropanol for 1-5 hours in a 25-30% isopropanol solution. After incubation, the lysate was centrifuged as described above. The clarified supernatant was transferred to a petri dish and frozen at -80°C for about 2 hours. The samples were lyophilized using a standard automated protocol.

[0264] Example 4: Ketoreductase reaction

[0265] Dissolve 0.90 g NADP in 60 mL of 200 mM K2HPO4 aqueous solution +, followed by 2.4 g of the enzyme powder prepared in Example 3. 135 mL of isopropanol was added to the solution, and the pH was adjusted to 8.0 with a 5 N NaOH aqueous solution. The above enzyme solution was added to a quenched chemical fluorination reaction mixture containing 60 g of fluoroketone substrate (6). The temperature was set to 33 ° C, and the reaction mixture was stirred for 18 hours. The enzymatic reaction mixture was quenched with 900 mL of ethyl acetate, cooled to 20 ° C and filtered. The two-phase mixture produced by the filtrate was separated, and the organic layer was washed with a 40% (w / v) (NH4)2SO4 aqueous solution (2x240 mL), and then washed with a 25% (w / v) K2HPO4 aqueous solution (2x120 mL). The organic phase volume was reduced to 900 mL by distillation and mixed with 500 mL of ethyl acetate. The volume was reduced to 900 mL again, mixed with 200 mL of ethyl acetate, and concentrated to 900 mL by distillation for the third time. CUNO#5 (7.8g) is added to saturated solution, and the mixture is stirred at room temperature for 45 minutes, then filtered by CELITE.Filtrate is distilled to 600mL at 60 ℃, then cooled to 30 ℃ through 45 minutes.Supersaturated solution is stirred with 350rpm and inoculated with product crystal (600mg).Toluene (518mL) is added through 4 hours, and at 29-33 ℃, is distilled to 360mL through 1.5 hours, then cooled to 20 ℃ through 2 hours.After aging for 14 hours at 20 ℃, filter slurries, and by solid 120mL 1:4 (v / v) ethyl acetate: toluene washing, then with 120mL toluene washing.Wet cake is placed in 45 ℃ of vacuum dryings 1 day, thereby providing fluorodiol (7).

[0266] Example 5: Preparation of (1S,2R,3S)-2,4-difluoro-7-(methylsulfonyl)-2,3-dihydro-1H-indene-1,3-diol (7)

[0267]

[0268] A five-gallon Hastelloy C-276 reactor was charged with acetonitrile (2.5 L), methanol (2.5 L), (5) (1.0 kg, 1.0 equiv), methanesulfonic acid (118 g, 0.3 equiv) and SELECTFLUOR (1.596 kg, 1.1 equiv). The vessel was pressure purged five times with N2, and the resulting mixture was then stirred and heated to 60°C for 16 hours. Methyl acetoacetate (95 g, 0.2 equiv) and water (369 g, 5 equiv) were charged, and the batch was further aged at 60°C for 2 hours. Subsequently, the batch was cooled to 20°C, and 0.2M K2HPO4 (7.75 L) was added. 50 wt% NaOH (344 g, 1.05 equiv) was added over 15 minutes to neutralize the acidic solution (final pH = 6.3). The mixture was stirred and then transferred to a high-density polyethylene tank. The reaction mixture was then vacuum transferred to a 30 L glass reactor. Isopropanol (2.25 L) was added to the reaction followed by a solution containing KRED and NADP (1.5 L 0.2 M K2HPO4, containing 40 g KRED and 15 g NADP). 5N NaOH (288 g, 0.3 eq) was added over 25 minutes to adjust the pH of the solution (final pH = 8.1). The solution was stirred and heated to 34 ° C for 17 hours. The reaction volume was reduced to 10 L by batch concentration. When the batch volume reached 10 L, DIH2O (1 L) was added and the batch was concentrated to a total volume of 10 L. CELITE (44 g, 0.044 w / w) and (NH4)2SO4 (5.8 kg, 5.8 w / w) treated with K2CO3 were charged to the reactor and the batch was stirred and heated to 50 ° C for 2 hours. At 50 ℃, charge EtOAc (15L) and mix for 30 minutes. Subsequently, the reaction is cooled to 20 ℃ and aged for 30 minutes. The slurry is filtered by 2' filter tanks with inner lining polypropylene and filter paper, and the waste cake (2x5L) is washed with EtOAc. The filtrate is merged into a 50L glass reactor, discharged and the aqueous phase is discarded. The organic layer is washed with 40% (w / w) (NH4)2SO4 (2x4L), 25% (w / w) K2HPO4 (2L) and 50% (w / w) K2HPO4 (2L) successively. The gained organic phase is concentrated to 15L, and constant volume distillation is carried out by adding fresh EtOAc, until water content <0.3wt%. The batch is transferred in a 50L round-bottomed flask, CUNO#5 (130g, 0.13w / w) is added, and the mixture is stirred for 30 minutes under ambient conditions. The slurry was filtered through a 10" filter pot lined with polypropylene and filter paper, and the filter cake was washed with EtOAc (2 x 2 L). The filtrate was transferred to a 20 L glass container and concentrated to 10 L at 60 °C, then cooled to 30 °C and seeded with (7) (10 g, 0.01 w / w).The obtained slurries were aged for 1 hour at 30 ° C, then toluene (8.5L) was added over 4 hours, and further aged for 9 hours. The mixture was concentrated to 6L and then constant volume distilled by adding fresh toluene (2L). The slurries were cooled to 20 ° C over 30 minutes, aged for 1 hour and filtered. The product cake was washed with 1: 4EtOAc / toluene (2L) and toluene (2L), and then dried in 40 ° C vacuum ovens to obtain (7) (950g, 85% yield, 96.7% area %, 96.3wt% by LC) as an off-white solid. 1 H NMR (599.90 MHz, DMSO-d6) δ 7.92 (ddd, J = 8.6, 4.7 and 0.7 Hz, 1H, CH), 7.46 (t, J = 8.9 Hz, 1H, CH), 6.14 (d, J = 7.1 Hz, 1H, OH), 5.96 (d, J = 6.9 Hz, 1H, OH), 5.56 (dd, J = 6.8, 5.2 and 3.2 Hz, 1H, CH), 5.40 (ddd, J = 14.1, 7.0 and 5.2 Hz, 1H, CH), 4.89 (dt, J = 51.1 and 5.2 Hz, 1H, CH), 3.31 (s, 3H, CH3) ppm. 13 C{ 1 H}NMR(150.85MHz,DMSO-d6)δ162.31(d,J CF =258.7Hz,CF),142.85(dd,J CF =6.0 and 2.9 Hz, C), 133.89 (d, J CF =3.4Hz,C),132.20(d,J CF =8.9Hz,C),130.53(dd,J CF =16.4 and 10.1 Hz, CH), 117.25 (d, J CF =21.4Hz,CH),97.82(d,J CF =194.0Hz,CH),73.17(d,J CF =25.2Hz,CH),68.95(d,J CF =17.9Hz,CH),44.93(s,CH3)ppm. 19 F NMR(564.47MHz,DMSO-d6)δ-111.51(dd,J HF =9.9 and 4.6 Hz, 1F), -203.88 (dd, J HF =51.0 and 13.9 Hz, 1F) ppm. Sequence:

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276] It should be appreciated that the above-discussed and other various features and functions and their alternatives may be combined into many other different systems or applications as desired. In addition, those skilled in the art may subsequently make various currently unforeseen or unanticipated alternatives, modifications, changes or improvements therein, which are also intended to be encompassed in the following claims.

Claims

1. A polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

2.

2. The polypeptide of claim 1, wherein the amino acid sequence has at least 95% sequence identity with SEQ ID NO:

2.

3. The polypeptide of claim 1, wherein the amino acid sequence has at least 98% sequence identity with SEQ ID NO:

2.

4. The polypeptide according to claim 1, wherein the amino acid sequence consists of SEQ ID NO:

2. The polypeptide according to claim 1 , which consists of SEQ ID NO:

2.

6. The polypeptide according to claim 1, wherein at least one of the following conditions is met: (a) amino acid (aa) residue 2 of SEQ ID NO:2 is not alanine; or (b) aa residue 11 of SEQ ID NO:2 is not glutamic acid.

7. A polynucleotide encoding the polypeptide according to any one of claims 1 to 6.

8. The polynucleotide of claim 7, wherein the polynucleotide comprises SEQ ID NO:

3.

9. The polynucleotide according to claim 7, wherein at least one of the following conditions is met: (a) the triplet codon encoding the amino acid residue at position 2 of the polypeptide is not GCT; (b) the triplet codon encoding the amino acid residue at position 3 of the polypeptide is not AAA; (c) the triplet codon encoding the amino acid residue at position 4 of the polypeptide is not ATC; or (d) the triplet codon encoding the amino acid residue at position 11 of the polypeptide is not GAA.

10. An expression vector comprising a polynucleotide according to any one of claims 7 to 9, said polynucleotide being operably linked to one or more control sequences suitable for directing the expression of the encoded polypeptide in a host cell.

11. The expression vector of claim 10, wherein the control sequence comprises a promoter.

12. The expression vector of claim 11, wherein the promoter comprises an E. coli promoter. A host cell comprising the expression vector according to claim 11 . The host cell according to claim 12 , wherein the host cell is Escherichia coli.

15. A method for precipitating protein from a cell lysate containing a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2, the method comprising treating a cell lysate containing the polypeptide to produce a cell lysate composition comprising >20% isopropanol.

16. The method of claim 15, wherein the volume percentage of isopropanol in the cell lysate composition is about 25%.

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