Engineering enzyme for preparing hydroxylated indanone intermediate capable of being used for synthesizing beretevane

Abstract

The present disclosure provides enzymes derived from the fungus Fusarium oxysporum c8D ("FoPip4H enzyme") having improved properties compared to naturally occurring wild type enzymes, including the ability to hydroxylate certain substituted indanones to provide optically pure 3-hydroxyindanones. The invention also provides a polynucleotide encoding the FoPip4H enzyme, a host cell capable of expressing the FoPip4H enzyme, and a method for synthesizing (R)-4-fluoro-3-hydroxy-7-(methylsulfonyl)-2, 3-dihydro-1H-inden-1-one, which is a useful intermediate in the synthesis of betefan, by using the FoPip4H enzyme.

Description

Technical Field

[0001] The present invention relates to providing a hydroxylase derived from the fungus Fusarium oxysporum c8D, which can be used in biocatalytic and synthetic processes involving oxidation of indanone to provide chiral alcohols. Such enzymes are particularly suitable for providing optically pure 3-hydroxy indanone, which is a useful intermediate in the preparation of benzyl alcohol.

[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 25554-WO-PCT_SL.XML, and is 24,536 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] M. Hibi et al. disclose a protein derived from the fungus Fusarium oxysporum c8D, which is capable of hydroxylating pipecolic acid. Appl Environ Microbiol. 2016 Apr 1; ​​82(7): 2070-2077. The reference discloses that the enzyme is part of the Fe(II) / α-ketoglutarate-dependent dioxygenase superfamily. It further discloses that 4-hydroxyamino acids (e.g., (4S)-hydroxy-L and D-piperidinic acid) can be efficiently produced with high optical purity.

[0006] Recently, the new HIF-2α inhibitor 3-[(1S,2S,3R)-2,3-difluoro-1-hydroxy-7-methylsulfonyl-indan-4-yl]oxy-5-fluoro-benzonitrile (hereinafter referred to as bezutifan) was approved by the US Food and Drug Administration for the treatment of adult patients with von Hippel-Lindau (VHL) disease who do not require immediate surgery and who need treatment for related renal cell carcinoma (RCC), central nervous system (CNS) hemangioblastoma, or pancreatic neuroendocrine tumor (pNET).

[0007]

[0008] In a recent clinical study, 49% of patients with VHL disease-related RCC treated with benzylpenicillin had a confirmed objective response; most patients had a reduction in renal tumor size. In addition, 30% of patients with CNS hemangioblastoma responded to benzylpenicillin treatment, and 91% of patients with pancreatic neuroendocrine tumors responded after treatment. Benzylpenicillin can be used as an alternative therapy or supplemental surgical treatment for patients with VHL disease. The researchers speculate that benzylpenicillin may delay or eliminate the need for serial surgery, which would bring a large complication burden to such patients. (Jonasch, E. et al., NEngl JJ Med 2021; 385: 2036-2046).

[0009] In the study of beztifan for the treatment of RCC, the drug showed excellent in vitro efficacy and pharmacokinetic characteristics as well as in vivo efficacy in mouse models, and showed encouraging results in patients with advanced RCC (Xu, Rui et al., J. Med. Chem. 62: 6876-6893 (2019)). In a recent clinical study of patients who had previously received treatment for advanced clear cell RCC, the confirmed objective response rate was 25% and the median progression-free survival was 14.5 months. (Choueri, TK et al., Nature Medicine vol. 27, 802-805 (2021)).

[0010] Due to its therapeutic effect in treating patients with VHL disease and its potential in treating RCC patients, efficient processes are needed to prepare large-scale quantities of benzyltransferase to support commercial supply and ongoing clinical research. U.S. Patent Application Publication No. US2022 / 0881407A1 discloses methods for preparing certain substituted indanes (including benzyltransferase). U.S. Provisional Application No. 63 / 191,356, filed on May 21, 2021, discloses crystalline forms of certain synthetic intermediates and certain methods for isolating such forms, which are conducive to the preparation of benzyltransferase.

[0011] While the above publications and applications describe useful and scalable methods for preparing benzyltransferase, further methods for preparing the compounds are also valuable, particularly those that minimize the use of hazardous reagents and transition metal catalysts. In particular, it is desirable to explore new transformation methods to provide optically pure intermediates that minimize the burden on the environment by using biocatalysts (e.g., enzymes). The present disclosure provides such enzymes and processes for preparing useful intermediates for preparing benzyltransferase. Summary of the invention

[0012] The present disclosure relates to an enzyme derived from the fungus Fusarium oxysporum c8D (hereinafter referred to as "FoPip4H enzyme"), which is capable of hydroxylating substituted indanones to provide optically pure alcohols. In some embodiments, the target FoPip4H enzyme described herein is capable of converting 4-fluoro-7-(methylsulfonyl)-2,3-dihydro-1H-inden-1-one (II) into stereoisomerically pure (R)-4-fluoro-3-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1H-inden-1-one (I), which can be used to synthesize benzylthiocarbamate, i.e., 3-[[(1S,2S,3R)-2,3-difluoro-2,3-dihydro-1-hydroxy-7-(methylsulfonyl)-1H-inden-4-yl]oxy]-5-fluorobenzonitrile.

[0013] Additional embodiments describe methods for preparing the target FoPip4H enzyme and methods of using the target FoPip4H enzyme.

[0014] In one aspect, the disclosure provides a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence has at least 95% sequence identity to SEQ ID NO: 2. In certain embodiments, the amino acid sequence has at least 98% sequence identity to SEQ ID NO: 2. In specific embodiments, the amino acid sequence consists of SEQ ID NO: 2. In specific embodiments, the polypeptide consists of SEQ ID NO: 2.

[0015] In another aspect, the present disclosure provides a polynucleotide encoding any one of the above polypeptides. In a specific embodiment, the polynucleotide comprises SEQ ID NO:3.

[0016] In another aspect, the disclosure provides an expression vector comprising any of the above polynucleotides, the polynucleotide being operably linked to one or more control sequences, the control sequences being suitable for directing the expression of the encoded polypeptide in a host cell. In some embodiments, the control sequences comprise a promoter. In certain embodiments, the promoter comprises an E. coli promoter.

[0017] In another aspect, the disclosure provides a host cell comprising any of the above expression vectors. In certain embodiments, the host cell is Escherichia coli.

[0018] In another aspect, the present disclosure provides a method for preparing a compound according to formula (I) with at least 60% enantiomeric excess (ee),

[0019]

[0020] The method comprises contacting an indanone according to formula (II) with alpha-ketoglutarate in the presence of the above-mentioned polypeptide to provide a compound of formula (I).

[0021]

[0022] In particular embodiments of the process, the compound according to formula (I) is prepared with at least 80%, 90%, 95%, 98% or 99% ee.

[0023] In some embodiments, the method further comprises a reducing agent selected from the group consisting of L-cysteine, ascorbic acid, dithiothreitol, D-cysteine, L-homocysteine, and D-cysteine ​​ethyl ester.

[0024] In certain embodiments, the method is performed using a buffer selected from the group consisting of phosphate buffer, 2-morpholineethanesulfonic acid, Bis Tris, PIPES, citrate, bicine, and TEOA.

[0025] In some embodiments, the method further comprises an iron salt selected from the group consisting of: Mohr's salt ((NH 4 ) 2 Fe(SO 4 ) 2 6H 2 O) and ferric chloride.

[0026] 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. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition

[0029] 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.

[0030] As used herein and throughout this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0031] 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 Oh, 17 Oh, 31 P, 32 P, 35 S, 18 F, 36 Cl and 123 I.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] "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.

[0038] "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.

[0039] 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).

[0040] 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.

[0041] As used herein, "derived from" in the context of an enzyme refers to an origin enzyme and / or a gene encoding the enzyme as the basis of the enzyme. For example, the FoPip4H enzyme of SEQ ID NO: 2 is obtained by artificial evolution of the gene encoding the wild-type (wt) FoPip4H enzyme of SEQ ID NO: 1 for multiple generations. Therefore, this evolved FoPip4H enzyme is "derived from" the FoPip4H of SEQ ID NO: 1.

[0042] "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.

[0043] 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 leucine at the residue corresponding to X96" refers to a reference sequence in which the corresponding residue at X96 in SEQ ID NO: 1 has been changed to a leucine.

[0044] "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).

[0045] "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).

[0046] "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).

[0047] "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).

[0048] "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).

[0049] "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.

[0050] 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.

[0051] "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).

[0052] 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).

[0053] 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.

[0054] 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).

[0055] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides covalently linked together. A polynucleotide may be composed entirely of ribonucleotides (i.e., RNA), entirely of 2'deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2'deoxyribonucleotides. Although nucleosides are usually linked together via standard phosphodiester bonds, polynucleotides may contain one or more non-standard bonds. A polynucleotide may be single-stranded or double-stranded, or a polynucleotide may contain both single-stranded and double-stranded regions. In addition, although a polynucleotide is usually composed of naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may contain one or more modified and / or synthetic nucleobases, such as inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

[0056] "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).

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] "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.

[0063] 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.

[0064] 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.

[0065] "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).

[0066] "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 kcat , 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] As used herein, "ATCC" refers to the American Type Culture Collection, whose biobank collections include genes and strains.

[0075] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases it provides.

[0076] "Coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

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

[0078] "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.

[0079] As used herein, "percentage of sequence identity", "percentage of identity" and "percentage of identity" refer to a 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., gaps) compared to the reference sequence for 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 where the nucleic acid base or amino acid residue is aligned with a gap 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 the 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 performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information.

[0080] 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).

[0081] 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.

[0082] "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.

[0083] "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.

[0084] "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.

[0085] "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%.

[0086] "Chemological selectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

[0087] "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.

[0088] "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, the secondary carbon atom carrying, in addition to a hydrogen atom, (i) a divalent group forming a chiral cyclic structure, or (ii) two substituents (except 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.

[0089] 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.

[0090] "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).

[0091] "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).

[0092] "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).

[0093] "Heat and solvent stable" means that the polypeptide is both heat stable and solvent stable.

[0094] As used herein, the terms "biocatalysis," "biocatalytic," "bioconversion," and "biosynthesis" refer to the use of enzymes to perform chemical reactions on organic compounds.

[0095] 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.

[0096] 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.

[0097] "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).

[0098] "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.

[0099] 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.

[0100] "Co-substrates" of the FoPip4H enzyme refer to α-ketoglutarate and co-substrate analogs that can replace α-ketoglutarate in the hydroxylation of indanone substrate analogs. Co-substrate analogs include, for example, but are not limited to, 2-oxoadipate (see, for example, Majamaa et al., 1985, Biochem. J. 229: 127-133).

[0101] "FoPip4H enzyme" or "FoPip4H polypeptide" refers to a polypeptide having the enzymatic ability to oxidize a cyclic substrate to provide an alcohol-substituted cyclic structure. More specifically, the disclosed FoPip4H polypeptide is capable of stereoselectively hydroxylating a substituted indanone of formula (II) to a substituted 3-hydroxyindanone of formula (I) (as shown above). The FoPip4H enzyme used herein includes naturally occurring (wild-type) hydroxylases as well as non-naturally occurring engineered polypeptides produced by artificial manipulation.

[0102] 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.

[0103] abbreviation

[0104] ACN, MeCN Acetonitrile

[0105] Bicine [Bis(2-hydroxyethyl)amino]acetic acid

[0106] Bis Tris 2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol

[0107] DMSO Dimethyl sulfoxide

[0108] g

[0109] h hour

[0110] Hz Hertz

[0111] IPA Isopropyl Alcohol

[0112] IPTG Isopropyl β-D-1-thiogalactopyranoside

[0113] J NMR coupling constants

[0114] L Liter

[0115] M mole, mol / L

[0116] mg milligram

[0117] min

[0118] mL, ml milliliter

[0119] mm Millimeters

[0120] mM millimole / liter

[0121] nm nanometer

[0122] NaCl Sodium chloride

[0123] NADP Nicotinamide adenine dinucleotide phosphate

[0124] OD 600 Optical density at 600nm wavelength

[0125] PIPES Piperazine-N,N-bis(2-ethanesulfonic acid)

[0126] PTFE Polytetrafluoroethylene

[0127] RH Relative humidity

[0128] RPM, rpm revolutions per minute

[0129] RT Room temperature, about 25℃

[0130] SFC Supercritical Fluid Chromatography

[0131] T50 is the temperature at which 50% enzyme activity is lost.

[0132] TEOA Triethanolamine

[0133] TFA Trifluoroacetic acid

[0134] THF Hydrofuran

[0135] UPLC Ultra-Performance Liquid Chromatography

[0136] vol volume

[0137] v / v Volume per volume

[0138] μg, ug microgram

[0139] μL, μl, uL, ul microliter

[0140] FoPip4H enzyme

[0141] The present disclosure relates to FoPip4H enzymes capable of hydroxylating substituted indanones to provide optically pure alcohols. In some embodiments, the FoPip4H enzyme is capable of the following transformations:

[0142]

[0143] In certain embodiments, the FoPip4H enzymes described herein have an amino acid sequence having one or more amino acid differences compared to a reference amino acid sequence of wild-type FoPip4H, which results in the enzyme having improved properties for a given indanone substrate.

[0144] The FoPip4H enzyme described herein is the product of directed evolution of wild-type FoPip4H c8D (SEQ ID NO: 1), which was identified by screening a group of enzymes referenced in the literature and has an amino acid sequence as shown below (including an added histidine tag):

[0145]

[0146] In some embodiments, the FoPip4H enzymes of the present disclosure may exhibit improvements relative to the FoPip4H enzyme of SEQ ID NO: 1, such as increased enzyme activity, stereoselectivity, stereospecificity, thermal stability, solvent stability, reduced product inhibition, or reduced overoxidation.

[0147] In some embodiments, the FoPip4H enzymes of the present disclosure can exhibit improved enzyme activity rates, i.e., rates of conversion of indanone substrates to products. In some embodiments, the FoPip4H polypeptides are capable of converting substrates to products at a rate that is at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 25 times, 50 times, 100 times, 150 times, 200 times, 400 times, 1000 times, 3000 times, 5000 times, 7000 times, or more than 7000 times greater than the rate exhibited by the enzyme of SEQ ID NO: 1.

[0148] In some embodiments, such FoPip4H polypeptides are also capable of converting an indanone substrate into a product with an enantiomeric excess of at least 60%. In some embodiments, such FoPip4H polypeptides are also capable of converting a substrate into a product with an enantiomeric excess of at least 90%. In some embodiments, such FoPip4H polypeptides are also capable of converting a substrate into a product with an enantiomeric excess of at least about 99%.

[0149] In some embodiments, the FoPip4H polypeptide is highly enantioselective, wherein the polypeptide can reduce a substrate to a product with an enantiomeric excess of greater than about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0150] In some embodiments, the improved FoPip4H polypeptide of the present disclosure is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein SEQ ID NO: 2 has an amino acid sequence as described below:

[0151]

[0152] In some embodiments, the improved FoPip4H polypeptides disclosed herein 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 SEQ ID NO:2.

[0153] 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 a combination of non-conservative and conservative amino acid substitutions.

[0154] In a specific embodiment, the improved FoPip4H polypeptide of the present disclosure comprises an amino acid sequence consisting of SEQ ID NO: 2. In a specific embodiment, the improved FoPip4H polypeptide of the present disclosure comprises SEQ ID NO: 2.

[0155] Additional embodiments provide host cells comprising polynucleotides and / or expression vectors described herein. The host cell may be Escherichia coli (E. coli), or it may be a different organism, such as Lactobacillus brevis. The host cell may be used for expression and isolation of the FoPip4H enzyme described herein, or, alternatively, it may be used directly to convert a substrate into a stereoisomeric product.

[0156] Whether the method is performed using whole cells, cell extracts or purified FoPip4H enzymes, a single FoPip4H enzyme may be used, or, alternatively, a mixture of two or more FoPip4H enzymes may be used.

[0157] Table 1 below provides a list of FoPip4H polypeptides (identified by the SEQ ID NOs disclosed herein) and their associated properties. Unless otherwise indicated, the following sequences are based on the FoPip4H sequence of SEQ ID NO: 1. In the table below, each row lists one SEQ ID NO. The column listing the number of mutations (i.e., residue changes) refers to the number of amino acid substitutions compared to the FoPip4H sequence of SEQ ID NO: 1.

[0158] In the thermal stability column of the table: + indicates T 50 <25℃; ++ means T 50 >25℃<45℃;+++ indicates T 50 >45°C. In the conversion column in the table: + means <10% conversion of substrate to product; ++ means 10%-60% conversion; +++ means >60% conversion. The conversion analysis was performed based on the assumption of 25 wt.% enzyme and 10 g / L substrate. In the selectivity column in the table: + means <60% enantiomeric excess (ee); ++ means 60%-90% ee; +++ means >90% ee. In the peroxidation column in the table: +++ means >15% peroxidation; ++ means 5%-15% peroxidation; + means <5% peroxidation.

[0159]

[0160]

[0161]

[0162] Polynucleotide encoding FoPip4H enzyme

[0163] In another aspect, the present disclosure provides polynucleotides encoding the FoPip4H 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 FoPip4H can be introduced into an appropriate host cell to express the corresponding FoPip4H polypeptide.

[0164] Due to the knowledge of the codons corresponding to various amino acids, the availability of protein sequences 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 alternative or synonymous codons) allows a very large number of nucleic acids to be produced, all of which encode the improved FoPip4H enzymes disclosed herein. Therefore, after determining a specific amino acid sequence, one skilled in the art can simply modify the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein to produce any number of different nucleic acids. In this regard, the present disclosure specifically contemplates every possible polynucleotide variation 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.

[0165] 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 has been codon-optimized for expression in E. coli.

[0166] In certain embodiments, it is not necessary to replace all codons to optimize the codon usage of the FoPip4H enzyme, 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 FoPip4H enzyme 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.

[0167] In various embodiments, the isolated polynucleotide encoding the improved FoPip4H 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. Guidance is provided by Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd edition. d Ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updated to 2006.

[0168] In some embodiments, the isolated polynucleotide encoding any FoPip4H polypeptide herein is manipulated in a variety of ways to promote the expression of the FoPip4H polypeptide. In some embodiments, the polynucleotide encoding the FoPip4H polypeptide includes an expression vector in which one or more control sequences are present to regulate the expression of the FoPip4H polynucleotide and / or polypeptide. Depending on the expression vector utilized, it may be desirable or necessary to manipulate the isolated polynucleotide before 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] ).

[0169] 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 genes related to: 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 genes related to: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., above).

[0170] In some embodiments, the control sequence is also a suitable leader sequence (i.e., an untranslated region of an mRNA 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 FoPip4H. 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 for: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from genes for: 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).

[0171] 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, is 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] ).

[0172] 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.

[0173] 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.

[0174] On the other hand, the present invention relates to a recombinant expression vector comprising a polynucleotide encoding a FoPip4H 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 a nucleic acid sequence encoding an enzyme polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting a 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] In another aspect, the present invention provides a host cell comprising at least one polynucleotide encoding at least one FoPip4H of the present disclosure, the polynucleotide being operably linked to one or more control sequences to express at least one FoPip4H 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 strains of Escherichia coli (e.g., W3110 (ΔfhuA) and BL21). 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, and / or tetracycline resistance).

[0179] 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.

[0180] 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.

[0181] For autonomous replication, the vector may further comprise an origin of replication, such that the vector is capable of autonomous replication in the host cell in question. Examples of bacterial origins of replication are the P15A ori or the origin of replication of plasmids pBR322, pUC19, pACYC177 (which contains the P15A ori) or pACYC184 (which contains the P15A ori), which allow replication in E. 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 the 2 micron origins of replication ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one with a mutation that enables it to function as a temperature-sensitive function in the host cell (see, e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75: 1433

[1978] ).

[0182] 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.

[0183] 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] ).

[0184] Therefore, in some embodiments, a vector comprising a sequence encoding at least one variant FoPip4H is transformed into a host cell to allow vector proliferation and expression of one or more variant FoPip4H. In some embodiments, the transformed host cells are cultured in a suitable nutrient medium under conditions that allow expression of one or more variant FoPip4H. 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 culture media 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).

[0185] Host cells for expressing FoPip4H enzyme

[0186] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding an improved FoPip4H polypeptide of the present disclosure, the polynucleotide being operably linked to one or more control sequences to express the FoPip4H enzyme in the host cell. Host cells for expressing the FoPip4H polypeptide 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 kejir, Lactobacillus brevis, Lactobacillus minor, Streptomyces, and Salmonella typhimurium; fungal cells such as Saccharomyces cerevisiae or Pichia pastoris (ATCC Record No. 201178). Appropriate culture media and growth conditions for the above host cells are well known in the art.

[0187] The polynucleotide for expressing FoPip4H polypeptide 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, etc. Various methods for introducing polynucleotides into cells are obvious to those skilled in the art.

[0188] 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, Cotiolus, 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.

[0189] 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 thermotolerans, Pichia willowii, or Pichia pastoris. salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0190] 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. protophonniaae, 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. licheniformnis, 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. grlseus, and S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Zymomonas mobilis and Zymomonas lipolytica).

[0191] 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.

[0192] In some embodiments, the host cell is genetically modified to have features that improve protein secretion, protein stability and / or other characteristics required for the expression and / or secretion of the protein. 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, a combination of recombinant modification and classical selection techniques is used to produce host cells. Using recombinant techniques, nucleic acid molecules can be introduced, deleted, suppressed or modified in a manner that results in an increase in the yield of FoPip4H variants in the host cell and / or culture medium. In a genetic engineering method, homologous recombination is used to induce targeted gene modification by specifically targeting genes in vivo to inhibit the expression of encoded proteins. In alternative methods, siRNA, antisense and / or ribozyme technology can be used to inhibit gene expression. A variety of methods for reducing protein expression in cells are known in the art, including but not limited to the deletion of all or part of a protein-encoding gene and site-specific mutagenesis to destroy the expression or activity of a gene product. (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).

[0193] 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.

[0194] 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 FoPip4H polynucleotides. The culture conditions (e.g., temperature, pH, etc.) are those previously used by the host cells 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.

[0195] In some embodiments, cells expressing the FoPip4H enzyme of the present disclosure are grown under batch or continuous fermentation conditions. Classical "batch fermentation" is a closed system in which 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, the substrate is added in increments as the fermentation proceeds. The fed batch system is useful when catabolite repression may inhibit the metabolism of the cell and a limited amount of substrate in the culture medium is required. Batch fermentation and fed batch fermentation are very common and well-known in the art. "Continuous fermentation" is an open system in which a specified fermentation medium is continuously added to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation typically maintains the culture at a constant high density, where the cells are mainly in logarithmic growth. Continuous fermentation systems strive to maintain steady-state growth conditions. Methods for regulating nutrients and growth factors in continuous fermentation processes and techniques for maximizing product production rates are well known in the field of industrial microbiology.

[0196] 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.

[0197] In some embodiments of the invention, a cell-free transcription and translation system can be used to produce FoPip4H polypeptides. Several systems are commercially available and these methods are well known to those skilled in the art.

[0198] Evolutionary approach for FoPip4H enzyme

[0199] In some embodiments, to make the FoPip4H polypeptides of the present disclosure, a FoPip4H enzyme that catalyzes the reduction reaction is obtained (or derived) from E. coli. In some embodiments, the parent polynucleotide sequence is codon-optimized to enhance the expression of the FoPip4H polypeptide in a specific host cell. The parent polynucleotide sequence (designated as SEQ ID NO: 3) is codon-optimized for expression in E. coli, and the codon-optimized polynucleotide is cloned into an expression vector such that the expression of the FoPip4H gene is under the control of the T7 promoter. The T7 polymerase required to express the gene of interest is under the control of the lac promoter, and both the gene of interest and the T7 polymerase are inhibited by lac1. The presence of IPTG activates T7 polymerase expression and eliminates inhibition, resulting in the production of the FoPip4H gene. A clone expressing active FoPip4H in E. coli was identified, and the gene was sequenced to confirm its identity.

[0200] The FoPip4H polypeptides disclosed herein can be obtained by mutagenesis and / or directed evolution methods on 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 available directed evolution procedures include the staggered extension process (StEP), in vitro recombination (Zhao et al., 1998, Nat. Biotechno1. 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.

[0201] The clones obtained after the mutagenesis treatment are screened to obtain FoPip4H polypeptides with the desired improved enzyme properties. Enzyme activity in the expression library can be measured using standard chemical analysis techniques for measuring substrates and products (e.g., UPLC-MS). 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. The clone containing the polynucleotide encoding the FoPip4H polypeptide is then isolated, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in a host cell.

[0202] When the polypeptide sequence is known, polynucleotides encoding the enzyme can be prepared by standard solid phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation 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 described by Beaucage et al., 1981, Tet. Lett. 22: 1859-69, or, for example, the method described by Matthes et al., 1984, EMBO J. 3: 801-05, as it is commonly practiced in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized, for example, in an automated DNA synthesizer, purified, annealed, ligated 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.

[0203] The FoPip4H enzyme expressed in the host cells can be recovered from the 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 CelLytic Buy.

[0204] Chromatographic techniques for separating FoPip4H 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.

[0205] In some embodiments, affinity techniques can be used to isolate the improved FoPip4H enzyme. Regarding 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 known to those skilled in the art. Antibodies can also be used as affinity purification reagents. Any antibody that specifically binds to the FoPip4H polypeptide can be used.

[0206] Methods of using FoPip4H enzyme

[0207] The FoPip4H enzyme described herein can catalyze the hydroxylation of a substituted indanone substrate (II).

[0208]

[0209] To obtain substituted hydroxyindanone (I)

[0210]

[0211] Hydroxyindanone (I) is an intermediate in the synthesis of WELIREG. Therefore, in the method for preparing WELIREG, the method may include the step of converting substituted indanone (II) into substituted hydroxyindanone (I) using the FoPip4H enzyme disclosed herein.

[0212] In the embodiments herein and as illustrated in the Examples, a range of various suitable reaction conditions that can be used in the methods include, but are not limited to, substrate loading, co-substrate loading, reducing agent, divalent transition metal, pH, temperature, buffer, solvent system, polypeptide loading, and reaction time. Further suitable reaction conditions for implementing the methods of biocatalytically converting substrate compounds to product compounds using the engineered FoPip4H polypeptides described herein can be readily optimized by routine experimentation, including, but not limited to, contacting the engineered FoPip4H polypeptide with the substrate compound under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound, given the guidance provided herein.

[0213] Suitable reaction conditions for using engineered FoPip4H polypeptides typically include a co-substrate, which is used in a stoichiometric amount in the hydroxylation reaction. Typically, the co-substrates of the FoPip4H enzyme are α-ketoglutarate, also known as α-ketoglutaric acid and 2-oxoglutaric acid. Other α-ketoglutaric acid analogs that can serve as co-substrates for the FoPip4H enzyme can be used. An exemplary analog that can serve as a co-substrate is 2-oxoadipic acid. Since the co-substrate is used in a stoichiometric amount, the co-substrate is present in an amount equal to or greater than the substrate compound, i.e., the molar concentration of the co-substrate is equal to or greater than the molar concentration of the substrate compound. In some embodiments, suitable reaction conditions may include a co-substrate molar concentration that is at least 1 times, 1.5 times, 2 times, 3 times, 4 times, or 5 times higher than the molar concentration of the substrate compound or more.

[0214] The substrate compound in the reaction mixture can vary, taking into account, for example, the desired amount of the product compound, the effect of the substrate concentration on the enzyme activity, the stability of the enzyme under the reaction conditions, and the percent conversion of the substrate to the product. In some embodiments, suitable reaction conditions include a substrate compound loading of 0.5 to 200 g / L, 1 to 200 g / L, 5 to 100 g / L, or 10 to 50 g / L. The substrate loading values ​​provided herein are based on the molecular weight of the substituted indanone (II), however, it is also contemplated that equimolar amounts of various hydrates and salts of the substituted indanone (II) may also be used in the method.

[0215] In carrying out the FoPip4H enzyme-mediated method described herein, the engineered polypeptide can be added to the reaction mixture in the form of a purified enzyme, a partially purified enzyme, a whole cell transformed with a gene encoding an enzyme, a cell extract and / or lysate of such a cell, and / or an enzyme immobilized on a solid support. Whole cells or cell extracts transformed with a gene encoding an engineered FoPip4H enzyme, their lysates, and isolated enzymes can be used in a variety of different forms, including solids (e.g., freeze-dried, spray-dried, etc.) or semi-solids (e.g., coarse pastes). Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment, etc.), followed by a desalting procedure (e.g., ultrafiltration, dialysis, etc.) before freeze-drying. Any enzyme preparation (including whole cell preparations) can be stabilized by cross-linking or immobilization to a solid phase (e.g., Eupergit C, etc.) using a known cross-linking agent (e.g., such as glutaraldehyde).

[0216] The improved activity and / or stereoselectivity of the engineered FoPip4H polypeptides disclosed herein provide methods in which a higher conversion percentage can be achieved with a lower concentration of the engineered polypeptide. In some embodiments of the method, suitable reaction conditions include an amount of the engineered polypeptide of 1% (w / w) to 100% (w / w), 1% (w / w) to 30% (w / w), 2.5% (w / w) to 20% (w / w / ), or from 2.5% (w / w) to 10% (w / w). In certain embodiments of the method, suitable reaction conditions include an amount of the engineered polypeptide of 1% (w / w), 2% (w / w), 5% (w / w), 7.5% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 75% (w / w), 100% (w / w) or more of the substrate compound loading.

[0217] In some embodiments, the engineered polypeptide is present at 0.01 g / L to 50 g / L; 0.1 g / L to 40 g / L; 1 g / L to 40 g / L; or 2 g / L to 10 g / L.

[0218] In some embodiments, the reaction conditions also include a divalent transition metal that can act as a cofactor in the oxidation reaction. Typically, the divalent transition metal cofactor is ferrous ion (Fe +2 Ferrous ions can be provided in various forms, such as ferrous sulfate (FeSO 4 ), ferrous chloride (FeCl 2 ), ferrous carbonate (FeCO 3 ) and organic acid salts (e.g., citrate, lactate, and fumarate). An exemplary source of ferrous sulfate is Mohr's salt, which is ammonium ferrous sulfate (NH 4 ) 2 Fe(SO 4 ) 2 , and can be obtained in anhydrous or hydrated form (e.g., hexahydrate). Although ferrous ion is a common transition metal cofactor in naturally occurring FoPip4H enzymes and functions effectively in engineered enzymes, it should be understood that other divalent transition metals that can act as cofactors can also be used in the method. In some embodiments, the divalent transition metal cofactor can include Mn +2 and Cr +2 In some embodiments, the reaction conditions may include a divalent transition metal cofactor, particularly Fe +2 , and its concentration is 0.1mM to 100mM, 0.5mM to 80mM, 10mM to 60mM or 40-60mM.

[0219] In some embodiments, the reaction conditions may further include the step of reacting the ferric iron (Fe +3 ) is reduced to ferrous ions (Fe +2 ). In some embodiments, the reducing agent can be L-cysteine, ascorbic acid, dithiothreitol, D-cysteine, L-homocysteine, or D-cysteine ​​ethyl ester. In some embodiments, the reducing agent includes cysteine, typically L-cysteine. Although cysteine ​​is not required for the hydroxylation reaction, its presence enhances the activity of the enzyme. Without being bound by theory, it is believed that cysteine ​​maintains or regenerates the enzyme-Fe +2 In some embodiments, the reducing agent, particularly L-cysteine, is present in an amount of 1 mM to 100 mM, 5 mM to 80 mM, or 50 to 70 mM.

[0220] In some embodiments, the reaction conditions include molecular oxygen, i.e., O 2Without being bound by theory, one oxygen atom of the molecular oxygen is incorporated into the substrate compound to form the hydroxylated product compound. 2 It may be naturally present in the reaction solution, or may be introduced or supplemented into the reaction by artificial means. In some embodiments, the reaction conditions may include the use of air, O 2 Gas or other O 2 Gas forced aeration (e.g., bubbling). In some embodiments, the aeration can be performed by using O 2 Or containing O 2 The gas increases the reaction pressure to increase the O 2 This can be done by 2 This is achieved by carrying out the reaction in a gas pressurized container.

[0221] During the reaction process, the pH value of the reaction mixture can change. The pH value of the reaction mixture can be maintained at a desired pH value or a desired pH value range. This can be achieved by adding acid or alkali before and / or during the reaction. Alternatively, the pH value can be controlled by using a buffer. Therefore, in some embodiments, the reaction conditions include a buffer. Suitable buffers for maintaining a desired pH range are known in the art, and include, for example, but not limited to, borate, phosphate, 2-(N-morpholinyl) ethanesulfonic acid (MES), 3-(N-morpholinyl) propanesulfonic acid (MOPS), acetate, triethanolamine and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) etc. In some embodiments, the buffer is a phosphate. In some embodiments of the method, suitable reaction conditions include a concentration of from about 0.001 to about 0.2M, 0.003 to about 0.1M or 0.005 to about 0.05M buffer (e.g., phosphate). In some embodiments, the reaction conditions comprise a buffer (e.g., phosphate) at a concentration of 0.001, 0.002, 0.003, 0.004, 0.005, or 0.008 M. In some embodiments, the reaction conditions comprise water as a suitable solvent in the absence of a buffer.

[0222] In the embodiment of the method, reaction conditions can include suitable pH. The desired pH or desired pH range can be maintained by using an acid or base, a suitable buffer or a combination of buffering and adding an acid or base. The pH of the reaction mixture can be controlled before and / or during the reaction process. In some embodiments, suitable reaction conditions include solution pH from about 4 to about 10, pH from about 5 to about 10, pH from about 5 to about 9, pH from about 6 to about 9, pH from about 6 to about 8. In some embodiments, reaction conditions include solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10.

[0223] In the embodiments of the present method, suitable temperature can be used as reaction condition, for example, taking into account the increase of reaction rate at higher temperature and the activity of enzyme during reaction time period. Therefore, in some embodiments, suitable reaction condition comprises that temperature is 10 ℃ to 30 ℃, for example 25 ℃ to 30 ℃. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout the entire reaction process. In some embodiments, the temperature during the enzymatic reaction can be regulated by the temperature curve during the reaction process.

[0224] The method of the present invention is usually carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymer solvents and / or co-solvent systems, and the co-solvent system usually comprises an aqueous solvent, an organic solvent and / or a polymer solvent. The aqueous solvent (water or aqueous co-solvent system) can be pH buffered or non-buffered. In some embodiments, the method using the engineered FoPip4H polypeptide can be carried out in an aqueous co-solvent system, and the co-solvent system comprises an organic solvent (e.g., ethanol, isopropanol (i-PrOH), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.), and an ionic or polar solvent (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, etc.). In some embodiments, the cosolvent can be a polar solvent, such as a polyol, dimethyl sulfoxide (DMSO) or a lower alcohol. The non-aqueous cosolvent component of the aqueous cosolvent system can be miscible with the aqueous component to provide a single liquid phase, or can be partially miscible or immiscible with the aqueous component to provide two liquid phases. An exemplary aqueous cosolvent system can include water and one or more cosolvents selected from organic solvents, polar solvents and polyol solvents. In general, the cosolvent component of the aqueous cosolvent system is selected so that it does not adversely inactivate the FoPip4H enzyme under reaction conditions. By utilizing an enzyme activity assay (such as those described herein), the enzyme activity of a specific engineered FoPip4H enzyme on a specified target substrate can be easily confirmed by measuring the enzyme activity of the specific engineered FoPip4H enzyme in the selected solvent system.

[0225] In one embodiment, the method using the FoPip4H polypeptide can be carried out in a co-solvent system of water and 1-octanol. The ratio of water to octanol can be 5:1 to 50:1, such as 10:1 to 40:1 or 20:1 to 40:1.

[0226] In some embodiments, the reaction conditions may include a defoamer, which helps reduce or prevent the formation of foam in the reaction solution, such as when mixing or spraying the reaction solution. Defoamers include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkylamines, alkylamides, alkyl sulfates, etc.), and hydrophobic substances (e.g., treated silica, polypropylene, etc.), some of which also act as surfactants. Exemplary defoamers include Glanapon 2000 Konz, (Dow Corning), polyethylene glycol copolymers, oxylated / ethoxylated alcohols, and polydimethylsiloxane. In some embodiments, the defoaming agent may be present at 0.001% (v / v) to about 1% (v / v), 0.003 to 0.02% (v / v), or 0.005 to 0.01% (v / v).

[0227] In some embodiments, reaction conditions can include a surfactant for stabilizing or enhancing the reaction. Surfactant can include nonionic, cationic, anionic and / or amphipathic surfactants. Exemplary surfactants include, for example, but not limited to, nonylphenol polyoxyethylene ether (NP40), Triton X-100, polyoxyethylene stearylamine, hexadecyl trimethyl ammonium bromide, sodium oleamidosulfate, polyoxyethylene sorbitan monostearate, hexadecyl dimethylamine, etc. Any surfactant that can stabilize or enhance the reaction can be used. The concentration of the surfactant used in the reaction can generally be from 0.1 to 50 mg / ml, particularly 1 to 20 mg / ml.

[0228] The amounts of reactants used in the hydroxylase reaction will generally depend on the amount of product desired and will vary with the amount of FoPip4H substrate used. One of ordinary skill in the art will readily appreciate how to vary these amounts to adapt them to the desired productivity and scale of production. Example

[0229] Example 1: Enzyme preparation

[0230] Each of the E. coli cultures containing a plasmid encoding the FoPip4 enzyme (represented by the amino acid sequence shown in SEQ ID NOS. 1, 2, and 4-19 below) was serially diluted to 10 using Luria-Bretani Broth (cell culture medium) as a diluent. -4 , 10 -5 and 10 -6 Dilutions. Spread 100 μL of each dilution on a Petri dish containing LB agar supplemented with 30 μg / mL kanamycin and 1% (w / v) glucose. Place the plate in a 37°C incubator overnight.

[0231] The Luria-Bertani Broth (cell culture medium) (500mL LB+30μg / mL kanamycin+1% (w / v) glucose) of 200μL per well is divided 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. The bacterium colony is grown overnight at 200rpm, 30℃ and 85%RH.

[0232] 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 the pH was in the range of 0.4-0.8, the deep well plates were induced with 40 μL of induction medium (2.2 mM IPTG, final concentration 0.2 mM) per well. The plates were resealed and incubated at 30°C and 85% RH with shaking at 250 rpm for 18-20 hours.

[0233] Alternatively, Studier induction ZYM-5052 medium can also be used to replace the TB growth medium with induction medium for expression. 390 μL ZYM-5052 growth medium (available from Teknova as catalog #3S2000) is aliquoted into a labeled 96-well deep well subculture plate. 13 μL of overnight growth culture is transferred from each well of the main shallow well plate to the corresponding labeled deep well subculture plate. The culture plate is sealed with a breathable membrane and shaken at 250 rpm for 20-22 hours at 30°C and 85% RH.

[0234] After incubation, all deep-well plates were centrifuged at 4000 rpm for 15 min at 4°C. After centrifugation, the supernatant was discarded. The plates containing the cell pellets were heat-sealed and stored at -80°C.

[0235] Remove the culture plate containing the cell pellet from -80 °C storage and thaw at RT. Prepare 10 mM potassium phosphate (pH 7.0), 0.5 mg / mL lysozyme, 0.25 mg / mL polymyxin B sulfate (PMBS), 1 unit / mL DNase I, and 4 mM MgSO4 400 μL of lysis buffer was aliquoted into each well. The lysis mixture was shaken at 1000 rpm on a plate shaker at RT for 1.5 to 2 hours. The lysis mixture was then centrifuged at 4000 x g for 10 minutes at 4°C to prepare an enzyme-containing lysate solution (in the supernatant).

[0236] Example 2: Hydroxylase reaction in a well plate

[0237] 5 mg of ground substrate was dispensed into each well of a 1 mL round bottom well plate using a Chemspeed automated solid dispenser. Reaction buffer was prepared by mixing 448 mM α-ketoglutarate, 63 mM L-cysteine, 49 mM Mohr's salt in 10 mM potassium phosphate and adjusted to a final pH of 6.5.

[0238] 115 μL of reaction buffer was added to a 1 mL round bottom well plate. Then 10 μL of the enzyme lysate solution of Example 1 was added. The plate was sealed with a gas permeable sealing film and shaken overnight at 30° C., 250 rpm and 85% RH.

[0239] For the thermal stability test, the same reaction setup as above was used, but first 50 μL of the enzyme-containing lysate solution of Example 1 was transferred to a Biorad hard-shell PCR plate and heat treated at the enzyme's T50 for 10 minutes, and then 10 μL of the heat-treated lysate was transferred to the reaction plate to start the reaction. The residual activity of the heat-treated supernatant was determined compared to the untreated supernatant.

[0240] After shaking overnight, 250 μ L DMSO was added to each hole of the reaction plate. The plate was heat sealed and vibrated at 1000 rpm for 10 minutes at RT. 180 μ L 50% ACN / water mixture was divided into filter stackers (filter plates on top of round bottom plates, with 0.20 μ M hydrophilic PTFE, can be purchased from Millipore MSRLN2250). The reaction plate was not sealed. The 10 μ L reaction mixtures from the reaction plate were transferred to the corresponding filter plates, and a receiving plate was placed below. The plate was then centrifuged at 4 ℃ for 3 minutes at 4000 rpm. The filter plates were removed, and the clear solution in the heat-sealed receiving plate was removed.

[0241] The filtered solution was analyzed by ultra-performance liquid chromatography (UPLC) using a high-throughput screening approach to monitor substrate, product, and product peroxidation peak areas. The structure of the peroxidation product is shown below.

[0242]

[0243] UPLC was performed on an Acquity UPLC@HSS T3 1.8 μm 2.1x50 mm column using the following gradient method where mobile phase component A was water with 0.1% TFA and component B was ACN with 0.1% TFA: 12% B from 0-0.7 min, gradient from 12% to 95% B from 0.7-0.8 min, hold at 95% B from 0.8-1.2 min, gradient from 95% to 12% B from 1.2-1.25 min, and hold at 12% B from 1.25-1.5 min. The column temperature was set at 40°C, the flow rate was 0.75 mL / min, and the absorbance at 254 and 290 nm was measured. The starting material eluted at 1.1 min, the desired product eluted at 0.61 min, and the undesired peroxidation product eluted at 0.48 min.

[0244] In addition to UPLC analysis, plate reader analysis was also used as a method to measure peroxidation. After the overnight reaction, the plate was centrifuged at 1000x g for 10 minutes. 190uL of water was aliquoted into each well of a Greiner 96-well black plate. 10μL of the overnight reaction was transferred to the Greiner plate containing water. The absorbance at 300 and 400nm was measured and the ratio of 300 and 400nm was used as a representative of the relative amount of desired product to unwanted peroxidation.

[0245] Example 3: Enzyme preparation in shake flasks

[0246] 10 μL of E. coli cells were inoculated into 5 mL of Luria-Bertani Broth (cell culture medium) (250 mL of LB + 30 μg / mL of kanamycin + 1% glucose), each of which contained a plasmid encoding the FoPip4H enzyme, which can be represented by the amino acid sequence shown in SEQ ID NO. 1, 2, and 4-19 below, and the cells had been stored at -80°C in 20% glycerol and aliquoted into labeled 15 mL cell culture tubes. The cell culture tubes were sealed and incubated at 30°C with shaking at 250 rpm for 20-24 hours.

[0247] 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 (available from ThermoFisher Scientific, catalog # A1374301) (TB + 30 μg / mL kanamycin) to a final volume of 250 mL. The flask was shaken at 250 rpm for 3-4 hours at 30°C. After shaking, the OD was measured for growth 600 , until OD 600At this point, 0.2 mM IPTG (50 μL 1 MIPTG) was added to the culture to induce expression, and the culture was grown at 30°C at 250 rpm for 20-24 hours.

[0248] 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 mL of 10 mM potassium phosphate buffer (pH = 7) per gram of well-cell pellet.

[0249] 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. The clear 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.

[0250] Example 4: Hydroxylase reaction in vials, conversion and selectivity determination

[0251] Hydroxylase reaction of indanone substrate of formula (II) was performed in a vial with selected variants of FoPip4H enzyme to further evaluate the conversion rate and selectivity (i.e., enantioselectivity) of the variant. α-Ketoglutarate (2.8 g, 19.2 mmol), Mohr's salt (0.86 g, 2.2 mmol) and cysteine ​​(0.34 g, 2.8 mmol) were added to a 50 mL vial. The mixture was dissolved in 25 mL of water, the pH was adjusted to 6.0 with 5N NaOH, and the final volume was adjusted to 44.5 mL with water to provide a reaction solution. In a new 50 mL vial, indanone substrate of formula (II) (0.2 g, 0.88 mmol) and FoPip4H enzyme (15 mg, 7.5 wt %, from Example 3) were added to provide a solid vial. 4.45 mL of the reaction solution was transferred to a solid vial and 0.3 mL of 1-octanol was added. The solution was stirred for 24 hours and then sampled.

[0252] Conversion was determined by comparing the peak areas of the product and starting material on an Agilent UPLC equipped with a Waters Aquity HSS T3 column. The mobile phase was 0.1% phosphoric acid in water and acetonitrile. The product and starting material were identified by comparison with pure standards.

[0253] The enantioselectivity (ee) was determined by comparing the peak areas of the enantiomers separated on a Waters SFC column. The SFC column was equipped with a Chiralpak AD-3 column and used methanol and CO. 2As mobile phase. Enantiomers are determined by comparison with pure standards.

[0254] Example 5: T 50 Determination of

[0255] 15 mg of each selected FoPip4H enzyme was dissolved in 10 mM potassium phosphate buffer at pH 6.0. The individual solutions were kept at a temperature ranging from 40-65°C for one hour and then centrifuged to clarify the mixture. The solutions were then used to set up the assay as described in Example 2.

[0256] Example 6: Preparation of (R)-4-fluoro-3-hydroxy-7-(methylsulfonyl)-2,3-dihydro-1H-inden-1-one (I)

[0257]

[0258] Water (34.5 L, 23 vol.) was added to the 100 L reactor and adjusted to 20-30° C. After adding α-ketoglutaric acid (2.11 kg, 14.46 mol, 2.20 eq.), N 2 The solution was bubbled until the dissolved oxygen (dO) level was <2%. L-cysteine ​​(0.26 kg, 2.10 mol, 0.32 eq) was added and the pH was adjusted to 6.8 with 10 N NaOH. Mohr's salt (0.64 kg, 1.64 mol, 0.25 eq) was added and the pH was then adjusted to 6.0 with 5 N NaOH. 1-octanol (1.13 L, 0.75 vol) was added followed by freeze-dried baking powder containing FoPip4H (0.11 kg, 7.5 wt%) and then sulfone (II) (1.5 kg, 6.57 mol, 1.00 eq). The dO level of the reaction was adjusted to 100% and the suspension was stirred for 24 to 48 hours before sulfuric acid was added to adjust the pH to 4.7. To the mixture was added (NH 4 ) 2 (SO 4) (9.0 kg, 68.1 mol) and 50% MeCN in toluene solution (42 L, 28 volumes) were added, followed by the addition of CELITE (3.0 kg), and the mixture was heated to 45° C. for 2 hours and then cooled to 25° C. The mixture was filtered and the organic layer was separated. The filter cake was washed twice with 25% MeCN / toluene (15 L, 10 volumes). The aqueous layer was back-extracted twice with the filter cake washing solvent, and the organic layers were combined and washed with water (1.1 L, 0.75 volumes). The organic layer was concentrated to 10 volumes at 50° C. under vacuum, then stirred at 55° C. for 2 hours, cooled to 25° C. over 4 hours and stirred for an additional 10 hours. The slurry was filtered and washed three times with 5% MeCN / toluene (1.5 L, 1 volume). The filter cake was dried under vacuum to obtain 1.39 kg of hydroxysulfone (I) (99 wt %, 5.63 mol, 86% yield). 1 H NMR (599.90 MHz, DMSO-d 6 )δ8.11 (dd, J=8.4and 4.4Hz, 1H, CH), 7.78 (t, J=8.5Hz, 1H, CH), 5.97 (d, J=7.3Hz, 1H, OH), 5.46 (td, J=7.1 and 2.2Hz, 1H, CH), 3.42 (s, 3H, CH3), 3.20 (dd, J=18.8and 6.8Hz, 1H, CH'H"), 2.59 (dd, J=18.8and 2.2Hz, 1H, CH'H")ppm. 13 C{ 1 H) NMR (150.85 MHz, DMSO-d 6 )δ200.17(s, C=O), 162.85(d, J CF =259.9Hz, CF), 145.05 (d, J CF =19.2Hz, C), 136.02(d, J CF =5.1Hz, C), 133.24(d, J CF =4.0Hz, C), 132.32(d, J CF =8.5Hz, CH), 121.39(d, J CF =21.0Hz, CH), 63.85 (s, CH), 47.22 (s, CH2), 42.65 (s, CH3)ppm. 19 F NMR (564.47 MHz, DMSO-d 6 )δ-110.86(dd,J HF =8.6and 4.5Hz,1F)ppm.

[0259] sequence:

[0260]

[0261]

[0262]

[0263]

[0264]

[0265] 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 according to claim 1, wherein the amino acid sequence has at least 95% sequence identity with SEQ ID NO:

2.

3. The polypeptide according to 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.

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

2.

6. A polynucleotide encoding a polypeptide according to any one of claims 1 to 5.

7. The polynucleotide according to claim 6, wherein the polynucleotide comprises SEQ ID NO:

3.

8. An expression vector comprising a polynucleotide according to any one of claims 6 and 7, said polynucleotide being operably linked to one or more control sequences suitable for directing the expression of the encoded polypeptide in a host cell.

9. The expression vector according to claim 8, wherein the control sequence comprises a promoter.

10. The expression vector according to claim 9, wherein the promoter comprises an E. coli promoter.

11. A host cell comprising the expression vector according to claim 8.

12. The host cell according to claim 11, wherein the host cell is Escherichia coli.

13. A process for preparing a compound according to formula (I) with at least 60% ee, The method comprises contacting an indanone according to formula (II) with alpha-ketoglutarate in the presence of a polypeptide according to claim 1 to provide a compound of formula (I), 14. The method according to claim 13, further comprising a reducing agent selected from the group consisting of L-cysteine, ascorbic acid, dithiothreitol, D-cysteine, L-homocysteine ​​and D-cysteine ​​ethyl ester.

15. The method according to claim 14, further comprising a buffer selected from the group consisting of phosphate buffer, 2-morpholineethanesulfonic acid, BisTris, PIPES, citrate, bicine and TEOA.

16. The method according to claim 14, further comprising an iron salt selected from the group consisting of Mohr's salt ((NH4)2Fe(SO4)26H2O) and ferric chloride.

17. The method according to claim 13, wherein the FoPip4 hydroxylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

2.

18. The process according to claim 13, wherein the compound of formula (I) is prepared with at least 95% ee.

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