Mutant cytochrome p450 enzymes with enhanced peroxygenase activity and / or altered product selectivity

Abstract

The present invention relates to mutant enzymes with enhanced properties and methods of using such enzymes to oxidize organic compound substrates. More specifically, provided herein are mutations in cytochrome P450 enzymes that enhance their peroxygenase activity and / or alter product selectivity as compared to wild-type cytochrome P450 enzymes. Also provided are uses of such mutant cytochrome P450 enzymes, e.g., for biocatalytic oxidation of carbon-hydrogen bonds by a peroxygenase pathway, or for oxidation of organic compound substrates.

Description

[0001] Priority Statement This application claims priority to Australian Provisional Patent Application No. 2023902798, filed on August 31, 2023, the contents of which are incorporated herein by reference.

[0002] Invention Field This invention generally relates to mutant enzymes with enhanced properties, and methods for oxidizing organic compound substrates using such enzymes. Background of the Invention Biocatalysts exhibit significant advantages over traditional homogeneous and heterogeneous inorganic catalytic systems, including biodegradability, low toxicity, and high specificity, high conversion number, and high activity under mild conditions. Therefore, there is intense effort being put into their application in industrial processes. However, a significant drawback of most biocatalysts is their inherent vulnerability under typical commercial operating conditions, such as high temperatures, organic solvents, or extreme pH levels. Overcoming this challenge is a crucial step towards their widespread application, thereby advancing “green” and efficient industrial chemical processes.

[0004] Cytochrome P450 (P450, CYP) is a family of heme enzymes that perform oxidative transformation reactions important in a range of environments, including xenobiotic detoxification, carcinogen activation, steroid biosynthesis, fatty acid metabolism, and reactions necessary for microbial survival by utilizing selected nutrients. Most commonly, cytochrome P450 enzymes catalyze regio- and enantio-specific hydroxylation of carbon-hydrogen bonds and epoxidation reactions, but they also support other more complex reactions. Cytochrome P450 enzymes have become a global target of biocatalysis efforts because they catalyze a wide range of reactions that are difficult to achieve using conventional synthetic methods. They are potentially ideal biocatalysts for achieving novel enzyme-catalyzed chemistry, offering advantages over synthetic methods in the selective hydroxylation of carbon-hydrogen bonds under mild conditions.

[0005] However, despite the presence of certain thermostable P450 enzymes (such as CYP119A1 (acidophilic thermosulfuric leaf mold)... Sulfolobus acidocaldarius )), CYP175A1 (Thermophilic bacteria ( Thermus thermophilu s)) and CYP231A2 (scorching acidophilic archaea ( Picrophilus torridus While progress has been made in the structural characterization of P450 enzymes, their wider application as biocatalysts has stalled. Their low activity with available electron transport chaperone proteins and alternative oxygen donors, as well as the cost of the required cofactor (NAD(P)H), limit their practicality. Although a few specific examples of industrial applications of P450 enzymes are known, their low stability and the need for electron transport proteins and complex, expensive cofactors hinder the application of most such enzymes as industrial biocatalysts.

[0006] Therefore, there remains a need for improved cytochrome P450 enzymes capable of catalyzing selective oxidation reactions of complex organic molecules, in order to at least partially address one or more of the aforementioned drawbacks, or to provide useful alternatives.

[0007] The discussions of literature, actions, materials, devices, articles, etc., in this specification are for the purpose of providing context for the invention only. This does not imply or represent that any or all of these matters constitute part of the prior art or common general knowledge in the relevant field of the invention prior to the priority date of the claims of this application. Invention Overview This invention is based in part on the identification of mutations in cytochrome P450 enzymes that enhance peroxyase activity and / or alter product selectivity compared to wild-type cytochrome P450 enzymes.

[0009] Therefore, in a first aspect, the present invention provides a mutant cytochrome P450 enzyme having enhanced peroxyase activity and / or altered product selectivity, wherein the mutant cytochrome P450 enzyme, compared to the wild-type cytochrome P450 enzyme, contains at least two consecutive amino acid residues substituted in the I-helix of the enzyme's polypeptide chain, wherein the substitutions are located at positions corresponding to amino acid residues 23 and 24 of SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glutamine and glutamate (QE) amino acid residues at said positions in the I-helix of the enzyme's polypeptide chain.

[0010] In some embodiments, the mutant cytochrome P450 enzyme is derived from bacterial or archaea species. In some embodiments, the mutant cytochrome P450 enzyme is an extremophilic cytochrome P450 enzyme. In some embodiments, the mutant cytochrome P450 enzyme is a thermophilic cytochrome P450 enzyme.

[0011] In some embodiments, the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67.

[0012] In some embodiments, the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme, comprising an amino acid sequence having at least about 40% amino acid sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67, and comprising an amino acid common sequence X1X2X2X3X4X5X6 (SEQ ID NO: 32) in the I-helix of the polypeptide chain, wherein: X1 is either alanine (A) or glycine (G); X2 is either glycine (G) or alanine (A); X3 is histidine (H), asparagine (N), leucine (L), alanine (A), threonine (T), isoleucine (I), or phenylalanine (F); X4 is glutamic acid (E), glycine (G), leucine (L), or aspartic acid (D); X5 is threonine (T), alanine (A), or asparagine (N); X6 is threonine (T), isoleucine (I), valine (V), serine (S), or alanine (A); and X7 consists of threonine (T), valine (V), alanine (A), tryptophan (W), arginine (R), serine (S), and isoleucine (I).

[0013] In some embodiments, the mutant cytochrome P450 enzyme contains a substitution of amino acid residues immediately following the glutamine and glutamate (QE) amino acid residues in the I-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. In some embodiments, the amino acid residues immediately following the glutamine and glutamate (QE) amino acid residues are substituted with proline (P) amino acid residues. In some embodiments, the mutant cytochrome P450 contains a substitution of three consecutive amino acid residues at positions 23 to 25 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glutamine, glutamate, and proline (QEP) amino acid residues at the positions in the I-helix of the enzyme's polypeptide chain compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

[0014] In some embodiments, the mutant cytochrome P450 enzyme contains a substitution of two consecutive amino acid residues immediately following the glutamine and glutamate (QE) amino acid residues in the I-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. In some embodiments, the two consecutive amino acid residues immediately following the glutamine and glutamate (QE) amino acid residues are substituted with proline and glycine (PG) amino acid residues. In some embodiments, the mutant cytochrome P450 contains a substitution of four consecutive amino acid residues at positions 23 to 26 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glutamine, glutamate, proline, and glycine (QEPG) amino acid residues at said positions in the I-helix of the enzyme's polypeptide chain.

[0015] In some embodiments, the mutant cytochrome P450 enzyme contains a substitution of three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues in the I-helix of the polypeptide chain. In some embodiments, the three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues are substituted with glycine, alanine, and X (GAX) amino acid residues, where X represents any amino acid. In some embodiments, the three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues are substituted with glycine, alanine, and leucine (GAL) or glycine, alanine, and histidine (GAH). In some embodiments, the mutant cytochrome P450 contains a substitution of five consecutive amino acid residues at positions 20 to 24 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) or glycine, alanine, histidine, glutamine, and glutamic acid (GAHQE) amino acid residues at said positions in the I-helix of the enzyme's polypeptide chain compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

[0016] In some embodiments, the mutant cytochrome P450 enzyme contains a substitution of a seven-amino acid segment containing glutamine and glutamate (QE) amino acid residues in the I-helix of its polypeptide chain. In some embodiments, the seven-amino acid segment is substituted with glycine, alanine, X, glutamine, glutamate, proline, and glycine (GAXQEPG) amino acid residues, where X represents any amino acid. In some embodiments, the mutant cytochrome P450 contains a substitution of seven-amino acid residues at positions corresponding to amino acid residues 20 to 26 of SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glycine, alanine, leucine, glutamine, glutamate, proline, and glycine (GALQEPG) or glycine, alanine, histidine, glutamine, glutamate, proline, and glycine (GAHQEPG) amino acid residues at the corresponding positions in the I-helix of the enzyme's polypeptide chain compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

[0017] In some embodiments, the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme, which is a member of the CYP family selected from the group consisting of the CYP119, CYP231, CYP175, CYP199, CYP154, CYP102, CYP107, CYP109, CYP116, and CYP267 families.

[0018] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP119 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP119: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and threonine (ETTT) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) The amino acid residues of alanine, glycine, asparagine, glutamic acid, and threonine (AGNET) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) Alanine, glycine, asparagine, glutamic acid, threonine, threonine and threonine (AGNETTT) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0019] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP119A1, the mutant cytochrome P450 enzyme, compared with the wild-type CYP119A1, contains a substitution of AGNETTT (corresponding to amino acid residues 209 to 215 of SEQ ID NO: 53) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain.

[0020] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP119A2, the mutant cytochrome P450 enzyme, compared with the wild-type CYP119A2, contains a substitution of AGNETTT (corresponding to amino acid residues 210 to 216 of SEQ ID NO: 54) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain.

[0021] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP109 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP109: (i) Glutamic acid and threonine (ET) amino acid residues, or glutamic acid and alanine (EA) amino acid residues, are replaced with glutamine and glutamic acid (QE) amino acid residues. (ii) Glutamic acid, threonine and alanine (ETA) amino acid residues, or glutamic acid, alanine and alanine (EAA) amino acid residues, or glutamic acid, threonine and threonine (ETT) amino acid residues, replaced by glutamine, glutamic acid and proline (QEP) amino acid residues. (iii) Glutamic acid, threonine, alanine and threonine (ETAT) amino acid residues, or glutamic acid, alanine, alanine and threonine (EAAT) amino acid residues, or glutamic acid, threonine, threonine and threonine (ETTT) amino acid residues, replaced by glutamine, glutamic acid, proline and glycine (QEPG) amino acid residues. (iv) Alanine, glycine, threonine, glutamic acid, and threonine (AGTET), or alanine, glycine, threonine, glutamic acid, and alanine (AGTEA), or alanine, glycine, asparagine, glutamic acid, and threonine (AGNET), substituted with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) Alanine, glycine, threonine, glutamic acid, threonine, alanine and threonine (AGTETAT) amino acid residues (or equivalent amino acid residues), or alanine, glycine, threonine, glutamic acid, alanine, alanine and threonine (AGTEAAT) amino acid residues (or equivalent amino acid residues), or alanine, glycine, asparagine, glutamic acid, threonine, threonine and threonine (AGNETTT) amino acid residues (or equivalent amino acid residues), substituted with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0022] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109B1, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP109B1, contains a substitution of ET (corresponding to amino acids 242 and 243 of SEQ ID NO: 63) with QE in the I-helix of the enzyme's polypeptide chain.

[0023] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109C1, the mutant cytochrome P450 enzyme contains, compared to the I-helix of wild-type CYP109C1, a substitution of AGTETAT (corresponding to amino acids 226 to 232 of SEQ ID NO: 58) with a substitution of GALQEPG in the I-helix of the enzyme's polypeptide chain.

[0024] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109E1, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP109E1, contains a substitution of ET (corresponding to amino acids 245 and 246 of SEQ ID NO: 64) with QE in the I-helix of the enzyme's polypeptide chain.

[0025] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP154 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP154: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and isoleucine (ETTI) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) Alanine, glycine, histidine, glutamic acid, and threonine (AGHET) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) The amino acid residues (or equivalent amino acid residues) of alanine, glycine, histidine, glutamic acid, threonine, threonine and isoleucine (AGHETTI) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) or amino acid residues of glycine, alanine, histidine, glutamine, glutamic acid, proline and glycine (GAHQEPG).

[0026] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme contains a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain, compared to the I-helix of wild-type CYP154C8.

[0027] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme, compared with the wild-type CYP154C8, contains a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GAHQEPG in the I-helix of the mutant enzyme's polypeptide chain.

[0028] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP154C8, contains a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 62) with QE in the I-helix of the mutant enzyme's polypeptide chain.

[0029] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type P450t, the mutant cytochrome P450 enzyme, compared to the wild-type P450t, contains a substitution of ET (corresponding to amino acids 244 and 245 of SEQ ID NO: 65) with QE in the I-helix of the mutant enzyme's polypeptide chain.

[0030] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP267 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP267: (i) Glutamic acid and threonine (ET) amino acid residues, or glutamic acid and alanine (EA) amino acid residues, are replaced with glutamine and glutamic acid (QE) amino acid residues. (ii) Glutamic acid, threonine and threonine (ETT) amino acid residues, or glutamic acid, alanine and threonine (EAT) amino acid residues, replaced with glutamine, glutamic acid and proline (QEP) amino acid residues. (iii) Glutamic acid, threonine, threonine and valine (ETTV) amino acid residues, or glutamic acid, alanine, threonine and valine (EATV) amino acid residues, replaced by glutamine, glutamic acid, proline and glycine (QEPG) amino acid residues. (iv) Alanine, glycine, histidine, glutamic acid, and threonine (AGHET) amino acid residues, or alanine, glycine, histidine, glutamic acid, and alanine (AGHEA) amino acid residues, substituted with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, histidine, glutamic acid, threonine, threonine and valine (AGHETTV) amino acid residues (or equivalent amino acid residues), or alanine, glycine, histidine, glutamic acid, alanine, threonine and valine (AGHEATV) amino acid residues (or equivalent amino acid residues), substituted with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues or glycine, alanine, histidine, glutamine, glutamic acid, proline and glycine (GAHQEPG) amino acid residues.

[0031] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP267B1, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP267B1, contains a substitution of ET (corresponding to amino acids 246 and 247 of SEQ ID NO: 59) with QE or a substitution of AGHETTV (corresponding to amino acids 243 to 249 of SEQ ID NO: 59) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

[0032] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP102 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP102: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and serine (ETTS) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) Alanine, glycine, histidine, glutamic acid, and threonine (AGHET) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, histidine, glutamic acid, threonine, threonine and serine (AGHETTS) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0033] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP102A1, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP102A1, contains a substitution of ET (corresponding to amino acids 268 and 269 of SEQ ID NO: 52) with QE, or a substitution of AGHETTS (corresponding to amino acids 265 to 271 of SEQ ID NO: 52) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

[0034] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP175 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP175: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) Glutamic acid, threonine and valine (ETV) amino acid residues are replaced with glutamine, glutamic acid and proline (QEP) amino acid residues; (iii) The amino acid residues of glutamic acid, threonine, valine and alanine (ETVA) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) Alanine, glycine, histidine, glutamic acid, and threonine (AGHET) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, histidine, glutamic acid, threonine, valine and alanine (AGHETVA) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0035] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP175A1, the mutant cytochrome P450 enzyme contains a substitution of AGHETVA (corresponding to amino acids 221 to 227 of SEQ ID NO: 55) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain, compared to the I-helix of wild-type CYP175A1.

[0036] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP199 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP199: (i) Aspartic acid and threonine (DT) amino acid residues are replaced with glutamine and glutamic acid (QE) amino acid residues; (ii) The amino acid residues of aspartic acid, threonine and threonine (DTT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of aspartic acid, threonine, threonine and valine (DTTV) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) Alanine, glycine, leucine, aspartic acid, and threonine (AGLDT) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, leucine, aspartic acid, threonine, threonine and valine (AGLDTTV) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0037] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP199A4, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP199A4, contains a substitution of DT (corresponding to amino acids 252 and 253 of SEQ ID NO: 60) with QE, a substitution of AGLDT (corresponding to amino acids 249 to 253 of SEQ ID NO: 60) with GALQE, or a substitution of AGLDTTV (corresponding to amino acids 249 to 255 of SEQ ID NO: 60) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

[0038] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP231 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP231: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and threonine (ETTT) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) The amino acid residues of glycine, glycine, asparagine, glutamic acid, and threonine (GGNET) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) The amino acid residues (or equivalent amino acid residues) of glycine, glycine, asparagine, glutamic acid, threonine, threonine and threonine (GGNETTT) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG).

[0039] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP107 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP107: (i) Glutamic acid and alanine (EA) amino acid residues, or glutamic acid and threonine (ET) amino acid residues, are replaced with glutamine and glutamic acid (QE) amino acid residues. (ii) Glutamic acid, alanine and serine (EAS) amino acid residues, or glutamic acid, alanine and threonine (EAT) amino acid residues, or glutamic acid, threonine and threonine (ETT) amino acid residues, replaced with glutamine, glutamic acid and proline (QEP) amino acid residues. (iii) Glutamic acid, alanine, serine and valine (EASV) amino acid residues, or glutamic acid, alanine, threonine and valine (EATV) amino acid residues, or glutamic acid, threonine, threonine and valine (ETTV) amino acid residues, replaced by glutamine, glutamic acid, proline and glycine (QEPG) amino acid residues. (iv) Alanine, glycine, phenylalanine, glutamic acid, and alanine (AGFEA) amino acid residues, or alanine, glycine, histidine, glutamic acid, and alanine (AGHEA) amino acid residues, or alanine, glycine, histidine, glutamic acid, and threonine (AGHET) amino acid residues, substituted with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, phenylalanine, glutamic acid, alanine, serine and valine (AGFEASV) amino acid residues (or equivalent amino acid residues), or alanine, glycine, histidine, glutamic acid, alanine, threonine and valine (AGHEATV) amino acid residues (or equivalent amino acid residues), or alanine, glycine, histidine, glutamic acid, threonine, threonine and valine (AGHETTV) amino acid residues (or equivalent amino acid residues), substituted with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0040] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP107PQ (P450h), the mutant cytochrome P450 enzyme, compared with the wild-type CYP107PQ (P450h), contains a substitution of EA (corresponding to amino acids 247 and 248 of SEQ ID NO: 40) with QE in the I-helix of the mutant enzyme's polypeptide chain.

[0041] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP107Mg, the mutant cytochrome P450 enzyme contains a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 66) with QE in the I-helix of the mutant enzyme's polypeptide chain compared to the I-helix of wild-type CYP107Mg.

[0042] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP116 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP116: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and valine (ETTV) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) The amino acid residues of alanine, alanine, histidine, glutamic acid, and threonine (AAHET) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) Alanine, alanine, histidine, glutamic acid, threonine, threonine and valine (AAHETTV) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0043] In a second aspect, the present invention provides the use of the mutant cytochrome P450 enzyme of the first aspect of the present invention in the biocatalytic oxidation of carbon-hydrogen bonds via the peroxygenase pathway.

[0044] In some embodiments of the second aspect of the invention, the biocatalytic oxidation of the carbon-hydrogen bond is carried out at 1.0°C to 99°C.

[0045] In a third aspect, the present invention provides a method for oxidizing an organic compound substrate, comprising oxidizing the organic compound substrate with a mutant cytochrome P450 enzyme of the first aspect of the present invention.

[0046] In some embodiments of the third aspect of the invention, the method further includes adding a peroxide to the oxidation state. In some embodiments, the peroxide is selected from the group consisting of hydrogen peroxide, tert-butylhydrogen peroxide, and m-chloroperoxybenzoic acid. In some embodiments, the peroxide is hydrogen peroxide.

[0047] Other aspects and embodiments of the invention are described herein.

[0048] The scope of this disclosure should not be limited to the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure. Unless otherwise specifically stated, any example / implementation of this disclosure herein should be considered applicable to any other example / implementation of this disclosure with necessary modifications.

[0049] Brief description of the attached diagram To further understand aspects and advantages of the invention, reference should be made to the following detailed description, taken in conjunction with the accompanying drawings, which illustrate certain embodiments of the invention.

[0050] Figure 1 – A three-dimensional schematic diagram of the cytochrome P450 enzyme structure. (A) The band diagram shows that the folding of cytochrome P450 is highly conserved. The I α-helix is ​​marked and highlighted in black. The oxygen-binding groove located within this helix is ​​also marked. (B) A diagram comparing the active site structures of CYP255A2 and CYP119A1, highlighting the oxygen-binding groove of the I-helix.

[0051] Figure 2 – An amino acid sequence alignment of the I-helix region of a selected wild-type cytochrome P450 enzyme sequence, surrounding the two consecutive amino acid residues substituted in the mutant cytochrome P450 enzyme of this invention. Previously studied P450s are given by their names, such as CYP101A1. Unclassified P450 sequences from other extremophiles are labeled P450a to P450r. The two consecutive substituted amino acid residues are highlighted by the shaded region. Note that CYP107A1 and CYP176A1 do not contain conserved acid-alcohol pairs to be substituted (EA and DN, respectively). P450c, P450h, and P450i also do not contain acid-alcohol pairs to be substituted (GT, EA, and LT, respectively). The I-helix region of interest in this application is highlighted by a narrower solid box. The entire I-helix sequence is enclosed in a larger dashed box (based on the I-helix sequences of CYP101A1 and P450cam). The sequence at the top (SEQ IDNO: 1) is the common sequence of this alignment (each position is the most common amino acid).

[0052] Figure 3– Shows the synthesis scheme of metabolites produced by the oxidation of fatty acids from the GALQEPG mutant of CYP119A1.

[0053] Figure 4 – Gas chromatograms (GC-MS) showing analysis of wild-type CYP119A1 (WT) and CYP119A1 GALQEPG mutant (mutant) (3 µM) at room temperature (RT) and 80 °C using hydrogen peroxide (50 mM) to oxidize fatty acids (dodecanoic acid / lauric acid; 1 mM) for 2 hours.

[0054] Figure 5 – Gas chromatograms (GC-MS) showing the analysis of CYP119A1 GALQEPG mutant (3 µM) oxidized with hydrogen peroxide (50 mM) at room temperature (RT) and 50 °C, 70 °C and 90 °C.

[0055] Figure 6 – This shows the synthetic protocol for the CYP154 GAHQEPG mutant using progesterone peroxide and androstenedione.

[0056] Figure 7 – HPLC chromatograms, showing the stereoselective oxidation of progesterone (1 mM) at room temperature (RT) using 20 mM hydrogen peroxide, WT and GAHQEPG mutants of CYP154C8 (1 μM).

[0057] Figure 8 – HPLC chromatograms, showing the stereoselective oxidation of progesterone (1 mM) with 2–40 mM hydrogen peroxide, GAHQEPG mutant of CYP154C8 (1 μM).

[0058] Figure 9 – HPLC chromatograms showing stereoselective oxidation of progesterone substrates (1 mM) by various CYP154C8 mutants (T258E, QE, and GAHQEPG) (1 μM) compared to wild-type (WT) CYP154C8 enzymes using 5 mM hydrogen peroxide.

[0059] Figure 10 – HPLC chromatograms showing the stereoselective oxidation of progesterone substrate (2 mM) by the CYP154C8 QE mutant (1 μM) using 5 mM hydrogen peroxide and different concentrations of DMSO as substrate solvents (15%, 20%, 25% or 30% v / v).

[0060] Figure 11– HPLC chromatograms showing the stereoselective oxidation of progesterone substrate (1 mM) by the CYP154C8 QE mutant (1 μM) using 5% DMSO as the substrate solvent and different concentrations of hydrogen peroxide (1 mM, 5 mM, 10 mM, 20 mM and 40 mM).

[0061] Figure 12 – HPLC chromatograms showing the results of oxidation of androstenedione (A) and testosterone (B) substrates (400 μM) using the P450t QE mutant (2 μM) with 10 mM hydrogen peroxide. P450t is a member of the CYP154C subfamily. In each HPLC chromatogram, the substrate is labeled “1”, the internal standard (9-hydroxyfluorene) is labeled “2”, and the oxidation product (16α-hydroxysteroid) is labeled “3”.

[0062] Figure 13 – Line graph showing the increase in 4-hydroxybenzoic acid product concentration over time in a time-progressive reaction of 3 µM enzyme with 4-methoxybenzoic acid driven by 5 mM H2O2, compared to: (A) wild-type (WT) P450 CYP199A4 enzyme; and (B) mutant (GALQE) P450 CYP199A4 enzyme.

[0063] Figure 14 – Line graph showing (A) the increase in 4-hydroxybenzoic acid concentration during the time-process reaction of 3 µM P450 CYP199A4 (QE and T252E mutants) with 4-methoxybenzoic acid (1 mM) driven by 10 mM H2O2. (B) HPLC analysis of the reaction.

[0064] Figure 15 – Line graphs showing (A) the increase in 4-hydroxybenzoic acid concentration during the time-processed reaction of 3 µM P450 CYP199A4 (QE mutant and WT) with 4-methoxybenzoic acid (1–5 mM) driven by 10 mM H2O2. (B) HPLC analysis of the reaction after 45 hours.

[0065] Figure 16 – GC-MS chromatogram showing the HazakQE mutant (2 μM) driven by 5 mM H2O2 (from *Thermospora kazakica*). Thermosporothrix hazakensis Analysis of the reaction of CYP102 enzyme with 250 μM myristic acid in Tris-HCl buffer (50 mM, pH 7.4) at 30 °C for 2 hours.

[0066] Figure 17 – Indicated from *Red Sun-dwelling Thermomyces* ( Meiothermus ruber The QE mutant of the thermophilic P450 enzyme ( Figure 2 The stability analysis and HPLC chromatograms of P450h (also known as CYP107PQ1) in the selective oxidation of β-ionone substrates to 4-hydroxy-β-ionone are shown below. (A) Line graph showing the catalytic activity of the mutant enzyme at different reaction temperatures. (B) HPLC chromatogram showing the catalytic activity of the mutant enzyme after heat treatment before oxidation. (C) HPLC chromatogram showing the catalytic activity of the mutant enzyme after storage at 30°C for a set time before oxidation.

[0067] Figure 18 – UV-Vis absorption spectra of the heme Soret band region of the P450h QE (CYP107PQ1QE) mutant enzyme in the presence or absence of (A) β-ionone substrate. Note: The spectral variation in the presence of the substrate is due to the oxidation of β-ionone, which exhibits low but significant absorption in the measured wavelength region.

[0068] Figure 19 – HPLC chromatograms demonstrating the oxidative superiority of the P450h QE (CYP107PQ1QE) mutant enzyme compared to the wild-type enzyme (A) and demonstrating the high enantioselectivity of the P450h QE (CYP107PQ1QE) mutant enzyme (B).

[0069] Figure 20 – HPLC chromatograms and line graphs demonstrating the stability of the P450h QE (CYP107PQ1QE) mutant enzyme in the oxidation of β-ionone to different types and concentrations of organic solvents. (A) HPLC chromatogram showing the yield of 4-hydroxy-β-ionone product using organic solvents acetonitrile, DMSO, ethanol, and isopropanol; (B) line graph showing the relative yield of 4-hydroxy-β-ionone product using different concentrations of DMSO or isopropanol; (C) HPLC chromatogram showing the yield of 4-hydroxy-β-ionone product using different concentrations of DMSO; and (D) HPLC chromatogram showing the yield of 4-hydroxy-β-ionone product using different concentrations of isopropanol.

[0070] Figure 21 – Line graphs demonstrating the stability of CYP109E1QE (A), CYP109B1QE (B), and P450h QE (CYP107PQ1QE) (C) mutant enzymes after heat treatment at different temperatures for 1 hour before oxidation of β-ionone substrate.

[0071] Figure 22 – HPLC chromatogram demonstrating the oxidative superiority of the CYP107MgQE mutant enzyme compared to the wild-type enzyme.

[0072] Detailed description of the invention This document refers to nucleotide sequences by their sequence identifiers (SEQ ID NO:). Table 1 provides a summary of the sequence identifiers. A sequence listing is also provided at the time of this application.

[0073] Table 1 Sequence Identifier Summary

[0074] As described above, this invention originates from the structural characterization and optimization of the catalytic activity of newly developed (mutant) thermostable mesophilic cytochrome P450 enzymes derived from protein engineering using equivalent wild-type P450 proteins. These mutant P450 enzymes have been modified to require only peroxides (e.g., hydrogen peroxide (H2O2)) as oxidants, thereby providing thermostable P450 peroxyses for biocatalytic hydroxylation reactions, with significantly enhanced activity compared to the wild-type form of the enzyme.

[0075] Therefore, certain disclosed embodiments provide mutant cytochrome P450 enzymes with one or more advantages, as well as uses and methods relating to these enzymes. For example, some advantages of some embodiments disclosed herein include one or more of the following: mutant cytochrome P450 enzymes with enhanced peroxyenzyme activity; mutant cytochrome P450 enzymes with enhanced stability that do not require additional protein chaperones or expensive nicotinamide cofactors; mutant cytochrome P450 enzymes with altered product selectivity; mutant cytochrome P450 enzymes containing amino acid substitutions in the I-helix that confer these advantageous properties; use of said mutant cytochrome P450 enzymes for improving the biocatalytic oxidation of carbon-hydrogen bonds; improved methods for oxidizing organic substrates; or providing commercial alternatives to existing cytochrome P450 enzymes. Other advantages of some embodiments of this disclosure are provided herein.

[0076] On one hand, the present invention provides a mutant cytochrome P450 enzyme having enhanced peroxyase activity and / or altered product selectivity, wherein the mutant cytochrome P450 enzyme, compared to the wild-type cytochrome P450 enzyme, contains at least two consecutive amino acid residues substituted in the I-helix of the enzyme's polypeptide chain, wherein the substitutions are located at positions corresponding to amino acid residues 23 and 24 of SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glutamine and glutamate (QE) amino acid residues at said positions in the I-helix of the enzyme's polypeptide chain. As will be understood by those skilled in the art, the term "peroxyase activity" as used herein refers to the ability of the mutant cytochrome P450 enzyme covered herein to catalyze the following reactions:

[0077] As used herein, references to cytochrome P450 enzymes also include references to cytochrome P450 protein, P450 protein, P450, CYP, CYP450, or any other synonym understood by those skilled in the art. Cytochrome P450 proteins, named for their absorption band at 450 nm in their carbon monoxide-bound ferrous form, are one of the largest superfamily of enzyme proteins, present in the genomes of almost all organisms. The cytochrome P450 superfamily is unusual among proteins and enzymes because they possess low-homology primary structures, despite highly conserved tertiary structures. All P450 protein structures determined to date exhibit a characteristic topology, with helical-rich domains stacked together with domains primarily containing β chains (Poulos TL). et al ., 1987, J. Mol. Biol (., 195: 687-700). The helix was named AL, the β chain was named β1-β5, and the overall topology is now referred to as the "P450 fold". In the secondary structural elements, the B and B' helices, BC loop, F and G helices, and FG loop located distal to the heme form a substrate-binding pocket. Sequence alignment can easily identify the residues within these helices and loops, but there is a high degree of variability within this general framework, both in terms of amino acid sequence and structural arrangement. This is what leads to the numerous specific, active, and selective modes of P450 catalysis.

[0078] P450 enzymes share a common overall folding and topological structure. For example... Figure 1 As shown in Figure A, the conserved P450 structural core is formed by a four-helix bundle consisting of three parallel helices (labeled D, L, and I) and one antiparallel helice E. The prosthetic heme group is confined between the distal I helix and the proximal L helix and binds to the adjacent Cys-heme-ligand ring. The long I helix forms the wall of the heme pocket and contains the characteristic amino acid sequence (A / G)GX(E / D)T (where “X” is any amino acid), centered on a kink (oxygen-binding groove) in the middle of the helix. A highly conserved threonine residue, following the acidic residue, is located at the active site and participates in catalysis.

[0079] At the molecular level, the amino acids that make up the I-helix of any particular or potential P450 enzyme can be referenced to a reference P450 enzyme (e.g., P450 enzyme). Figure 2The I-helix amino acid sequence of CYP101A1 (P450cam) shown is used to determine this. In this regard, once the amino acid sequence of a specific or potential P450 enzyme is obtained, it can be compared with a reference P450 enzyme sequence using sequence alignment tools. Optimal sequence alignment can be performed using computer-executed algorithms such as the BLAST family of programs, for example, Altschul. et al ., 1997, Nucl. Acids Res., As disclosed in 25: 3389-3402. Global alignment programs can also be used to align sequences of approximately equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss_needle / ), which is part of the EMBOSS software package (Rice Packet). et al ., 2000, Trends Genet ., 16: 276-277), and the GGSEARCH program (available at fasta.bioch.virginia.edu / fasta_www2 / fasta_www.cgi?rm=compare&pgm=gnw), which is part of the FASTA software package (Pearson W and Lipman D, 1988, ). Proc. Natl. Acad. Sci. USA These procedures are based on the Needleman-Wunsch algorithm, which is used to find the optimal alignment of two sequences along their entire length (including gaps). For a detailed discussion of sequence analysis, see Ausubel et al. ( , 85: 2444-2448). Current Protocols in Molecular Biology Unit 19.3 of John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998.

[0080] Figure 2 The image shows amino acid sequence alignments of many P450 enzymes in protein regions containing the I-helix. The I-helix sequence is based on the I-helix of CYP101A1 (P450cam) (Poulos TL). et al ., 1987, J. Mol. Biol ., 195(3): 687-700), in Figure 2 The numbers are indicated by dashed boxes. For CYP101A1, the I-helix is ​​represented by amino acid residues 234 (I) to 269 (P) of the CYP101A1 sequence shown in SEQ ID NO: 51. It should be noted that the amino acid numbering system for cytochrome P450 enzymes used in this paper is based on methionine residues containing the start codon.

[0081] Figure 2 Detailed information on the P450 enzyme, particularly regarding its nucleotide and amino acid sequences, can be obtained from the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov) or the UniProtKB / Swiss-Prot database (www.uniprot.org). Specific details are provided in Table 2 below. It should be noted that... Figure 2 The P450 enzymes included in Table 2 are only a representative list and are by no means exhaustive.

[0082] Table 2 Representative P450 enzymes

[0083] As described above, the mutant cytochrome P450 enzyme of the present invention, compared to the wild-type cytochrome P450 enzyme, contains substitutions of at least two consecutive amino acid residues in the I-helix of its polypeptide chain. In this regard, SEQ ID NO: 1 is provided herein as a wild-type reference (common) sequence to highlight the positions of the amino acid substitutions present in the mutant cytochrome P450 enzyme. SEQ ID NO: 1 is Figure 2 The common sequence of the most common amino acids at each position in the I-helix region of the wild-type cytochrome P450 enzyme sequences (SEQ ID NO: 2 to 31) shown. For example, for wild-type CYP101A1, the at least two consecutive amino acid residues are represented by an acid-alcohol amino acid residue pair, namely amino acid residues 22 (D) and 23 (T) in the I-helix region of the sequence shown in SEQ ID NO: 20, or amino acid residues 252 (D) and 253 (T) in the full-length CYP101A1 sequence shown in SEQ ID NO: 51. Another example relates to wild-type cytochrome P450a, wherein the at least two consecutive amino acid residues are represented by amino acid residues 22 (E) and 23 (T) in the I-helix region of the sequence shown in SEQ ID NO: 2, or by amino acid residues 220 (E) and 221 (T) in the full-length P450a sequence shown in SEQ ID NO: 33.

[0084] for Figure 2The P450 enzyme sequence alignment shown highlights at least two consecutive amino acid residues of the wild-type enzyme substituted in the mutant cytochrome P450 enzyme by shaded areas. In some embodiments, the at least two consecutive amino acid residues of the wild-type enzyme substituted in the mutant enzyme may be acid-alcohol amino acid residue pairs represented by the following pairs: (i) glutamic acid and threonine (ET) amino acid residue pairs (e.g., in P450a, P450b, P450d, P450e, P450f, P450g, P450j, P450l, P450m, P450n, P450p, P450q, P450r, CYP102A1 ... (i) P119A1, CYP119A2, CYP175A1, CYP109C1, CYP267B1, CYP231A2 and CYP154C8; (ii) aspartic acid and threonine (DT) amino acid residue pairs (e.g. in P450k, P450o, CYP101A1 and CYP199A4); (iii) glutamic acid and serine (ES) amino acid residue pairs; or (iv) aspartic acid and serine (DS) amino acid residue pairs.

[0085] In some embodiments, at least two consecutive amino acid residues of the wild-type enzyme substituted in the mutant enzyme are not an acid-alcohol amino acid residue pair. In this regard, said at least two consecutive amino acid residues may be selected from the group consisting of: (i) glycine and threonine (GT) amino acid residue pairs (e.g., in P450c); (ii) glutamic acid and alanine (EA) amino acid residue pairs (e.g., in P450h and CYP107A1); (iii) leucine and threonine (LT) amino acid residue pairs (e.g., in P450i); and (iv) aspartic acid and asparagine (DN) amino acid residue pairs (e.g., in CYP176A1). (v) Histidine and threonine (HT) amino acid residue pairs; (vi) glutamic acid and asparagine (EN) amino acid residue pairs; (vii) glutamic acid and isoleucine (EI) amino acid residue pairs; (viii) valine and threonine (VT) amino acid residue pairs; (ix) alanine and threonine (AT) amino acid residue pairs; (x) phenylalanine and threonine (FT) amino acid residue pairs; (xi) isoleucine and threonine (IT) amino acid residue pairs; (xii) lysine and threonine (K) amino acid residue pairs. (xiii) Arginine and threonine (RT) amino acid residue pairs; (xiv) Alanine and asparagine (AN) amino acid residue pairs; (xv) Glycine and asparagine (GN) amino acid residue pairs; (xvi) Isoleucine and isoleucine (II) amino acid residue pairs; (xvii) Asparagine and proline (NP) amino acid residue pairs; (xviii) Isoleucine and serine (IS) amino acid residue pairs; (xix) Leucine and serine (LS) amino acid residue pairs; (xx) Valine and alanine (VA) amino acid residue pairs; (xxi) Isoleucine and alanine (IA) amino acid residue pairs; (xxii) Leucine and proline (LP) amino acid residue pairs; (xxiii) Glutamine and proline (QP) amino acid residue pairs; (xxiv) Serine and threonine (ST) amino acid residue pairs; (xxv) Proline and threonine (PT) amino acid residue pairs; and (xxvi) Methionine and alanine (MA) amino acid residue pairs.

[0086] This paper demonstrates that replacing at least two consecutive amino acid residues in the I-helix of the polypeptide chain of wild-type cytochrome P450 enzymes with a glutamine and glutamate (QE) amino acid residue pair can confer enhanced peroxyenzyme activity and / or altered product selectivity to the resulting mutant cytochrome P450 enzymes.

[0087] Those skilled in the art will recognize the mechanisms by which the mutant P450 enzymes covered by this invention are generated. For example, the nucleotide sequence encoding the relevant mutant P450 enzyme can be synthesized in a computer. Synthetic production using a high-throughput silicon platform (e.g., a platform provided by Twist Bioscience (South San Francisco, CA, USA)) allows for rapid, reliable, and faithful reproduction of the selected nucleotide sequence. The mutant P450 nucleotide sequence can also be synthesized as part of a prokaryotic, eukaryotic, or viral vector for future expression and mutagenesis (see, for example, Creative Biogene, NY, USA; GeneScript, NJ, USA; and DNAScript, Daly City, CA, USA). The advantage of synthetic DNA production is that it does not require the availability or pre-existing DNA material, nor does it depend on cloning the P450 nucleotide sequence into a suitable vector.

[0088] In other techniques, QE amino acid substitutions (and other amino acid substitutions covered by this invention) can be introduced by genetically modifying the corresponding wild-type P450 enzyme. In this regard, the mutant cytochrome P450 enzyme can be considered “derived” from the wild-type cytochrome P450 enzyme. In some embodiments, the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67.

[0089] Exemplary types of genetic modification considered herein include site-directed mutagenesis of endogenous wild-type P450 enzymes, PCR and gene shuffling methods, the use of multiple mutagenic oligonucleotides in a site-directed mutagenesis cycle, or the use of gene editing techniques. Thus, these genetic modification methods produce one or more polynucleotides encoding one or more different P450 mutant enzymes with desired amino acid substitutions in the I-helix.

[0090] Examples of gene editing technology may include the use of: Zinc finger nucleases (see Carroll D, 2011, Genetics 188: 773-782; Sander JD et al .,2011, Nat. Methods 8: 67-69; and Miller JC et al ., 2007, Nat. Biotechnol ., 25: 778-785). The transcription activator-like effector nuclease (TALEN) system (see Bogdanove AJ and Voytas DF, 2011). Science 333: 1843-1846; Streubel J et al ., 2012, Nat. Biotechnol ., 30: 593-595; Cermak T et al ., 2011, Nucl. Acids Res ., 39: e82; Chen K and Gao C, 2013, J. Genet. Genomics 40: 271-279; Voytas DF, 2013, Ann. Rev. Plant Biol ., 64: 327-350; and Wang Y et al ., 2014, Nat. Biotechnol ., 32: 947-951); and Gene editing methods or equivalent adaptation systems based on clustered regularly spaced short palindromic repeats ('CRISPR') (see Belhaj K) et al. , 2015, Current Opinion in Biotechnology , 32: 76-84; ShanQ et al ., 2014, Nature Protocols , 9: 2395-2410; and Wang Y et al., 2014, Nat. Biotechnol ., 32: 947-951).

[0091] The gene editing techniques described above can be used to efficiently introduce foreign DNA into the chromosome of a host cell and allow for the insertion of mutations at specific loci on the chromosome. For example, point mutations can be introduced into the P450 gene via knock-in, where the mutation encodes a desired (e.g., QE) amino acid substitution. In many cases, gene knock-in involves homologous recombination mechanisms. In this regard, gene editing techniques are capable of generating double-strand DNA breaks at desired gene loci. These controlled double-strand breaks can promote homologous recombination at that specific gene locus. This process relies on targeting specific nucleic acid molecule sequences with endonucleases, which recognize and bind to such sequences and induce double-strand breaks in the nucleic acid molecule.

[0092] Nuclease-mediated breaks in double-stranded DNA in the genome can be repaired through two main mechanisms: non-homologous end joining (NHEJ), which typically leads to the introduction of non-specific insertions and deletions (indels); or homologous directed repair (HDR), which incorporates a homologous strand as a repair template. When sequence-specific nucleases are delivered together with homologous donor DNA constructs containing the desired mutation, gene targeting efficiency is increased by 1000-fold compared to using donor constructs alone.

[0093] Alternative methods have been developed to accelerate genome modification processes by directly injecting the DNA or mRNA of site-specific nucleases into cells, generating DNA double-strand breaks (DSBs) at specific loci. These site-specific nuclease-induced DSBs can then be repaired via error-prone non-homologous end joining (NHEJ), thereby generating, for example, mutants carrying the desired substitution at the cleavage site. High-fidelity homologous recombination can generate P450 enzymes with targeted integration if a donor plasmid with flanking ends of the DSB is co-injected.

[0094] The CRISPR type II system has been used to edit the genomes of a wide range of species (see, for example, Friedland AE). et al ., 2013, Nat. Methods , 10(8): 741-743;Mali P et al ., 2013, Science , 339(6121):823-826; Hwang WY et al ., 2013, Nat. Biotechnol ., 31(3): 227-229;Jiang W et al ,2013, Nat. Biotechnol ., 31(3): 233-239;Jinek M et al ., 2013, eLife , 2: e00471;Cong L et al (., 2013, ibid.). CRISPR is particularly easy to customize because its active form consists of the invariant Cas9 protein and easily programmable single guide RNA (sgRNA). Among various CRISPR homologs, Streptococcus pyogenes (Streptococcus pyogenes) Streptococcus pyogenesCRISPR is the most well-characterized and widely used method. The Cas9-gRNA complex first probes the protospacer neighboring motif (PAM) sequence of the DNA (-NGG for Sp Cas9), followed by Watson-Crick base pairing between the sgRNA and the target DNA via a ratchet mechanism, forming an R-loop. After the formation of the ternary complex of Cas9, sgRNA, and target DNA, the Cas9 protein creates two nicks in the target DNA, resulting in blunt-ended double-strand breaks (DSBs), which are repaired via non-homologous end joining (NHEJ) or template-directed homologous recombination (HR). CRISPR methods are disclosed, for example, in US 8,697,359.

[0095] The techniques described above for generating the mutant P450 enzymes covered by this invention are merely representative and do not limit other possible mechanisms.

[0096] After generating the mutant P450 enzyme, checking or determining whether the desired amino acid substitution (e.g., QE) of the present invention has been successfully integrated can be achieved using allele-specific primers and probes. For example, for checking QE amino acid substitutions, PCR-based methods can use oligonucleotide primers that specifically bind to nucleotides encoding the QE amino acid (i.e., the CAA or CAG codon for Q; and the GAA or GAG codon for E). Such oligonucleotides that detect nucleotide variations in target sequences may be referred to by terms such as "allele-specific probe" or "allele-specific primer." The design and use of allele-specific probes for detecting known sequence variations are described, for example, in *Mutation Detection: A Practical Approach*, ed. Cotton. et al . Oxford University Press, 1998; Saiki et al ., 1986 ( Nature (324: 163-166); EP235726; and WO 89 / 11548. In one example, probes or primers can be designed to hybridize to a segment of target DNA containing a specific nucleotide encoding the QE amino acid, such that the actual encoding nucleotide is aligned with the 5' or 3' end of the probe or primer. In some assays, amplification may include labeled primers, thereby allowing detection of the amplification product of those primers.

[0097] In one type of PCR-based assay, allele-specific primers hybridize to a segment of target DNA that overlaps with a site encoding a nucleotide substitution of a desired amino acid (e.g., QE), and amplification of the allele form is initiated only when the primer and the allele form exhibit perfect complementarity (Gibbs, 1989). Nucleic Acid Res(17:2427-2448). Typically, the 3' terminal nucleotide of the primer is aligned and complementary to one of the nucleotides encoding the amino acid substitution being detected. This primer is used in conjunction with a second primer that hybridizes at the distal site. Amplification proceeds from both primers, producing a detectable product that indicates the presence of which allele form in the test sample. A second primer pair is typically used as a control, where one primer shows a single base mismatch with one of the nucleotides encoding the amino acid substitution, and the other primer is perfectly complementary to the distal site. A single base mismatch prevents amplification or significantly reduces amplification efficiency, resulting in either no detectable product formation or a small or slow amount of product formation. This method is generally most effective when the mismatch is located at the 3' terminal position of the oligonucleotide, as this position is most destructive to stability for extensions starting from the primer (see, for example, WO 93 / 22456).

[0098] In one example, the primer contains a sequence substantially complementary to a DNA segment containing an amino acid substitution encoding a nucleotide, except that the primer has a mismatched nucleotide at one of its three nucleotide positions at the 3' end, such that the mismatched nucleotide does not pair with a specific allele at the QE-encoding nucleotide site. The mismatched nucleotide in the primer can be the first, second, or third nucleotide starting from the last nucleotide at the 3' end of the primer. In some examples, the primer and / or probe are labeled with a detectable tag.

[0099] In alternative methods, tagged allele-specific primer pairs can be used to detect one of the amino acid substitutions encoding the nucleotide (Strom). et al ., 2005, Genet. Med (7:633-63). In one example, two tagged allele-specific primers overlap with the amino acid substitution encoding nucleotides in the target DNA; however, only the correctly hybridized primers will be extended to produce a tagged product. Non-complementary primers will not be extended or tagged due to 3' mismatched bases. The tagged extension product can be detected based on a detectable tag. Tagged extension primers can also be captured onto a solid support such as beads coupled to an anti-tag sequence. The immobilized extension primer product can be detected using commercially available methods such as the Luminex 100 LabMAP™ (Luminex Corporation, Austin TX).

[0100] The most common method for examining and / or detecting amino acid substitution encoding nucleotides covered by this invention is to amplify DNA segments containing these nucleotides using PCR, and then isolate and sequence (e.g., by Sanger sequencing) the amplified PCR fragments. For example, for Figure 2The P450 enzymes listed can be used to design primers for amplification procedures based on SEQ ID NO: 2 to 31 (or actually based on the corresponding full-length P450 enzyme sequences shown in SEQ ID NO: 33 to 62 or the P450 enzyme sequences shown in SEQ ID NO: 63 to 67). Procedures for primer design, PCR, isolation of amplified fragments, and sequencing of these fragments are known in the art and are found in standard textbooks such as Green MR and Sambrook J. Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory Press, 2012.

[0101] In addition to the QE amino acid substitutions described above, the mutant P450 enzyme of the present invention may also have other substitutions. In some embodiments, the mutant cytochrome P450 enzyme contains a substitution of an amino acid residue immediately following the glutamine and glutamate (QE) amino acid residues in the I-helix of the mutant cytochrome P450 enzyme polypeptide chain. Reference Figure 2 The representative wild-type cytochrome P450 enzyme sequence shown is equivalent to replacing the threonine (T), isoleucine (I), valine (V), serine (S), or alanine (A) amino acid residue immediately following the QE substitution.

[0102] In some embodiments, the amino acid residue immediately following the glutamine and glutamate (QE) amino acid residues is replaced with a proline (P) amino acid residue. Therefore, in some embodiments, the mutant cytochrome P450 contains a substitution of three consecutive amino acid residues at positions 23 to 25 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glutamine, glutamate, and proline (QEP) amino acid residues at the positions in the I-helix of the enzyme's polypeptide chain compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme. In this respect, the mutant cytochrome P450 enzyme will contain the QEP amino acid substitution in the I-helix compared to the wild-type cytochrome P450 enzyme. For wild-type cytochrome P450 enzymes, the amino acid residues that may be replaced by QEP include: (i) glutamic acid, threonine and threonine (ETT) amino acid residues; (ii) aspartic acid, threonine and threonine (DTT) amino acid residues; (iii) glutamic acid, threonine and alanine (ETA) amino acid residues; or (iv) glutamic acid, threonine and valine (ETV) amino acid residues.

[0103] In some embodiments, the mutant cytochrome P450 enzyme contains a substitution of two consecutive amino acid residues immediately following the glutamine and glutamate (QE) amino acid residues in the I-helix of the mutant cytochrome P450 enzyme polypeptide chain. (Reference) Figure 2 This would be equivalent to substituting the following amino acid residues immediately following the QE substitution: threonine (T) and threonine (T); threonine (T) and valine (V); isoleucine (I) and threonine (T); threonine (T) and alanine (A); threonine (T) and tryptophan (W); threonine (T) and arginine (R); valine (V) and valine (V); threonine (T) and serine (S); valine (V) and alanine (A); serine (S) and valine (V); alanine (A) and threonine (T); and threonine (T) and isoleucine (I).

[0104] In some embodiments, the two consecutive amino acid residues immediately following the glutamine and glutamate (QE) amino acid residues are replaced with proline and glycine (PG) amino acid residues. Therefore, in some embodiments, the mutant cytochrome P450 contains a substitution of four consecutive amino acid residues at positions 23 to 26 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glutamine, glutamate, proline, and glycine (QEPG) amino acid residues at these positions in the I-helix of the enzyme's polypeptide chain. In this respect, the mutant cytochrome P450 enzyme, compared to the wild-type cytochrome P450 enzyme, will contain the QEPG amino acid substitution in the I-helix. For wild-type cytochrome P450 enzymes, the amino acid residues that may be replaced by QEPG include: (i) glutamic acid, threonine, threonine and threonine (ETTT) amino acid residues; (ii) aspartic acid, threonine, threonine and valine (DTTV) amino acid residues; (iii) glutamic acid, threonine, alanine and threonine (ETAT) amino acid residues; (iv) glutamic acid, threonine, threonine and isoleucine (ETTI) amino acid residues; (v) glutamic acid, threonine, threonine and valine (ETTV) amino acid residues; (vi) glutamic acid, threonine, threonine and serine (ETTS) amino acid residues; or (vii) glutamic acid, threonine, valine and alanine (ETVA) amino acid residues.

[0105] In some embodiments, the mutant cytochrome P450 enzyme contains a substitution of three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues in the I-helix of the mutant cytochrome P450 enzyme polypeptide chain. (Reference) Figure 2This would be equivalent to replacing the following amino acid residues immediately preceding the QE substitution: alanine (A), glycine (G), and asparagine (N); alanine (A), glycine (G), and histidine (H); alanine (A), glycine (G), and leucine (L); alanine (A), glycine (G), and alanine (A); alanine (A), glycine (G), and threonine (T); glycine (G), glycine (G), and leucine (L); glycine (G), glycine (G), and isoleucine (I); alanine (A), glycine (G), and phenylalanine (F); and glycine (G), glycine (G), and asparagine (N).

[0106] In some embodiments, the three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues are replaced with glycine, alanine, and X (GAX) amino acid residues, where X represents any amino acid. In this respect, the mutant cytochrome P450 enzyme, compared to the wild-type cytochrome P450 enzyme, will have the GAXQE amino acid substituted in the I-helix. In some embodiments, the three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues are replaced with glycine, alanine, and leucine (GAL) or glycine, alanine, and histidine (GAH). In this respect, the mutant cytochrome P450 enzyme, compared to the wild-type cytochrome P450 enzyme, will have the GALQE or GAHQE amino acid substituted in the I-helix. Therefore, in some embodiments, the mutant cytochrome P450 contains a substitution of five consecutive amino acid residues at positions 20 to 24 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) or glycine, alanine, histidine, glutamine, and glutamic acid (GAHQE) amino acid residues at the positions in the I-helix of the enzyme's polypeptide chain compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme. For wild-type cytochrome P450 enzymes, the amino acid residues that may be replaced by GALQE or GAHQE include: (i) alanine, glycine, asparagine, glutamic acid, and threonine (AGNET) amino acid residues; (ii) alanine, glycine, leucine, aspartic acid, and threonine (AGLDT) amino acid residues; (iii) alanine, glycine, threonine, glutamic acid, and threonine (AGTET) amino acid residues; or (iv) alanine, glycine, histidine, glutamic acid, and threonine (AGHET) amino acid residues.

[0107] In some embodiments, the mutant cytochrome P450 enzyme includes a substitution of a seven-amino acid segment containing glutamine and glutamate (QE) amino acid residues in the I-helix of the mutant cytochrome P450 enzyme polypeptide chain. In some embodiments, this includes substitution of the QE amino acid residue, substitution of the three amino acid residues immediately preceding the pair, and substitution of the two amino acid residues immediately following the pair. Reference Figure 2 This is equivalent to replacing the seven consecutive amino acids present within the solid box in the diagram.

[0108] In some embodiments, the seven consecutive amino acid segments are replaced with glycine, alanine, X, glutamine, glutamic acid, proline, and glycine (GAXQEPG) amino acid residues, where X represents any amino acid. In some embodiments, the seven amino acid segments are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline, and glycine (GALQEPG) amino acid residues or glycine, alanine, histidine, glutamine, glutamic acid, proline, and glycine (GAHQEPG) amino acid residues. Therefore, in some embodiments, the mutant cytochrome P450 contains a substitution of seven consecutive amino acid residues at positions 20 to 26 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glycine, alanine, leucine, glutamine, glutamic acid, proline, and glycine (GALQEPG) or glycine, alanine, histidine, glutamine, glutamic acid, proline, and glycine (GAHQEPG) amino acid residues at the positions in the I-helix of the enzyme's polypeptide chain compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

[0109] In addition to the one or more mutations described above, the mutant cytochrome P450 enzyme of the present invention may have 1, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more other mutations, such as substitutions, insertions, or deletions. These additional mutations may or may not enhance the peroxidase activity of the mutant P450 enzyme. Other mutations may be within or outside the active site. For example, mutations may be in the second sphere, i.e., residues that affect or contact the position or orientation of one or more amino acids in the active site. Insertions are typically at the N-terminus and / or C-terminus. Thus, the enzyme may comprise a short peptide or a full-length protein of up to 20 amino acids, fused to either end or both ends, for example to aid protein purification by affinity chromatography or immobilization on a solid matrix. Deletions typically include the deletion of amino acids that do not participate in catalysis, such as those outside the active site (thus the enzyme is a mutant fragment of a naturally occurring enzyme). In some embodiments, the additional mutation may be a protein fusion between a portion of the P450 enzyme and another enzyme (e.g., an oxidase) that generates peroxides in situ using other substrates. Therefore, these additional mutations are covered by the present invention, provided that the mutant enzyme retains at least the substitutions containing the QE substitutions described above.

[0110] Other mutations within the active site typically alter the position and / or conformation of the substrate when it binds to the active site. Mutations may make the oxidizable site on the substrate more accessible to the heme group. Therefore, mutations can be substitutions with amino acids having smaller or larger, or more or less polar side chains.

[0111] Additional mutations can include amino acid residue changes that increase enzyme stability. These mutations typically prevent protein oligomerization; for example, dimerization in P450cam (CYP101A1) is eliminated by preferably replacing cysteine ​​(C) at amino acid residue 335 with alanine (A). Other mutations can also inhibit oligomerization caused by contact between hydrophobic plaques on the protein surface. Further mutations include insertions / deletions that aid in enzyme purification and / or fixation, as well as mutations that allow the protein to be prepared in a soluble form, such as by introducing deletions or multiple histidine tags, or by mutating the N-terminal membrane anchoring sequence.

[0112] The mutant P450 enzymes covered by this invention also include natural and artificial homologs, including those homologs with amino acid identity as low as 40% with each other or their wild-type counterparts. In fact, amino acid identity among different families of P450 enzymes is known to be as low as 20%. In the systematic classification of the P450 superfamily (Nelson DR, 2006, Cytochrome P450 Nomenclature, 2004. In: Phillips, IR, Shephard, EA (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ; and Nebert DW...), et al (See *The P450 Gene Superfamily: Recommended Nomenclature*, 2009, published online: 25 Mar 2009 https: / / doi.org / 10.1089 / dna.1987.6.1). Enzymes with only 40% amino acid identity are grouped into the same family, while closely related members within the family (>55% identity) are grouped into subfamilies. In fact, what is conserved within a family is detailed molecular structure, substrate specificity, and product selectivity, rather than the typically low sequence identity.

[0113] Therefore, in some embodiments, the mutant cytochrome P450 enzyme of the present invention may be derived from the wild-type cytochrome P450 enzyme, comprising an amino acid sequence having at least about 40% amino acid sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67, and containing the amino acid common sequence X1X2X3X4X5X6X7 (SEQ ID NO: 32) in the I-helix of the polypeptide chain, wherein: X1 is either alanine (A) or glycine (G); X2 is either glycine (G) or alanine (A); X3 is histidine (H), asparagine (N), leucine (L), alanine (A), threonine (T), isoleucine (I), or phenylalanine (F); X4 is glutamic acid (E), glycine (G), leucine (L), or aspartic acid (D); X5 is threonine (T), alanine (A), asparagine (N), or serine (S); X6 is threonine (T), isoleucine (I), valine (V), serine (S), or alanine (A); and X7 consists of threonine (T), valine (V), alanine (A), tryptophan (W), arginine (R), serine (S), and isoleucine (I).

[0114] In some embodiments, the mutant cytochrome P450 enzyme may be derived from the wild-type cytochrome P450 enzyme, comprising an amino acid sequence having at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67, and comprising the aforementioned common sequence.

[0115] When comparing an amino acid sequence with any of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67 to calculate the percentage of identity, the amino acid sequences should be compared over the following comparison windows: at least 10 amino acids, at least 50 amino acids, at least 100 amino acid residues, at least 200 amino acid residues, at least 400 amino acid residues, and / or the full length of the sequence. The comparison window may contain approximately 20% or less of additions or deletions (i.e., vacancies) compared to a reference sequence (excluding additions or deletions) to achieve optimal alignment of the two sequences. Optimal alignment of the sequences used for the comparison window can be performed using computer-executed algorithms (such as those described above).

[0116] To generate a specific wild-type P450 enzyme protein for subsequent mutagenesis, the nucleotide sequence encoding the enzyme can be amplified by PCR from genomic DNA isolated from a source expressing the enzyme. Typical sources include humans, animals, plants, fungi, protozoa, bacteria, and archaea, as further described below. DNA amplification methods are well known in the art and generally rely on knowledge of the P450 nucleotide sequence to be amplified.

[0117] Furthermore, the nucleotide sequence encoding a specific P450 enzyme can be readily determined by back-translating the amino acid sequence into one or more nucleotide sequences using a codon frequency table. Moreover, the nucleotide sequence encoding a specific P450 enzyme can be codon-optimized for use in a specific host and can be generated by back-translating the amino acid sequence using a codon frequency table specific to that host organism. Codon optimization is a process used to improve gene expression and increase the translation efficiency of the P450 gene of interest, achieved by adapting to the codon preferences of the host organism. A range of exemplary codon frequency tables include those presented in the GenScript codon usage frequency table tool (such as those disclosed at http: / / www.genscript.com / cgi-bin / tools / codon_freq_table).

[0118] The nucleotide sequences encoding the wild-type and mutant cytochrome P450 enzymes of this invention can be cloned into suitable vectors for expression in specific host cell types. Typical host cells will include *Escherichia coli* (E. coli). Escherichia coli ), Pseudomonas spp. Pseudomonas sp.), Flavobacteria, fungal cells, Rhodococcus (sp.), Rhodococcus sp.) and Bacillus spp. Bacillus (sp.). Other host cells were also considered. The P450 protein can then be generated and isolated from the host cell for downstream applications. Methods for converting and expressing the introduced nucleotide sequence in various host cell types are well known in the art, and the present invention contemplates the use of any suitable method.

[0119] To enable the expression of an introduced nucleic acid in a cell, the introduced nucleic acid may be operatively linked to one or more transcriptional control sequences, where appropriate. The term "transcriptional control sequence" should be understood as a nucleic acid sequence that includes any transcriptional process that affects the operatively linked nucleic acid. Transcriptional control sequences may include, for example, leader sequences, polyadenylated sequences, promoters, enhancers, or upstream activating sequences, as well as transcription terminators. Typically, a transcriptional control sequence includes at least a promoter. As used herein, the term "promoter" describes any nucleic acid that confers, activates, or enhances the expression of a nucleic acid molecule in a cell.

[0120] In some embodiments, at least one transcription control sequence is operatively linked to a nucleic acid encoding an associated P450 gene. For the purposes of this specification, a transcription control sequence is considered "operatively linked" to a given gene or other nucleotide sequence when it is capable of promoting, inhibiting, or otherwise regulating transcription of the gene or other nucleotide sequence.

[0121] Promoters can constitutively or differentially regulate the expression of operablely linked nucleotide sequences relative to the cell, tissue, organ, or developmental stage in which expression occurs. Therefore, promoters used according to the present invention may include, for example, constitutive promoters, inducible promoters, tissue-specific promoters, or activatable promoters. Such promoters are known in the art.

[0122] As mentioned above, typical sources of P450 enzymes include humans, animals, insects, plants, fungi, protozoa, bacteria, and archaea. Humans encode up to 60 P450 enzymes, belonging to 18 families and 43 subfamilies. An overview can be found at Human Cytochrome P450s – Cytochrome P450 Homepage (uthsc.edu). P450 enzymes are widely expressed in almost all animal species and have been extensively studied in mice, rats, dogs, and zebrafish to facilitate the application of these model organisms in drug discovery and toxicology. P450 enzymes are also present in sponges, sea urchins, and the cephalochordate Florida amphioxus (…). Branchiostoma floridae Cytochrome P450 enzymes are involved in various processes of plant growth, development, and defense. It is estimated that P450 genes account for approximately 1% of the plant genome.

[0123] In some embodiments, the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme from bacterial or archaeal species. The distribution of P450 in bacteria is variable; many bacteria have not been identified with P450 (e.g., *Escherichia coli*), while some bacteria, primarily actinomycetes, possess numerous P450s. P450s identified in bacteria to date are generally involved in the biotransformation of xenobiotic compounds (e.g., from *Streptomyces griseus*). Streptomyces griseolus CYP105A1 in Streptomyces albopictus metabolizes sulfonylurea herbicides into less toxic derivatives) or is part of a specific metabolite biosynthetic pathway (e.g., CYP170B1 in Streptomyces albopictus). Streptomyces albus (It catalyzes the production of sesquiterpenoid albaflavenone). The CYP105 family is highly conserved and is represented in every Streptomyces genome sequenced to date. Due to the soluble nature of bacterial and archaea P450 enzymes, they are generally considered easier to manipulate than predominantly membrane-bound eukaryotic P450s.

[0124] Examples of bacterial species include, but are not limited to, thermoautotrophic carbonaceous actinomycetes (…). Carbonactinospora thermoautotrophica (Kitasatosporales, Actinomycetes), Bacillus pyriformis ( Thermogemmatispora onikobensis(Thermogemmatisporales bacteria – Ktedonobacteria), Red erythrophyte (Thermales bacteria – Deinococci), Rhodopseudomonas xylanophilus ( Rubrobacter xylanophilus (Bacteria of the order Rubrobacterales – class Rubrobacteria), halophilic thermophilic Bifidobacterium ( Dichotomicrobium thermohalophilum (Hyphomicrobiales, class Alphaproteobacteria, genus Thermomicrobiales) Thermomicrobium sp.) (Bacteria of the order Thermomicrobiales – class Thermomicrobia), *Pseudomonas putida* (sp.) (Bacteria of the order Thermomicrobiales – class Thermomicrobia) Pseudomonas putida (Bacteria of the order Pseudomonadales – class Gammaproteobacteria), *Priscilla megaterium* ( Priestia megaterium ) and Bacillus subtilis ( Bacillus subtilis (Bacteria of the order Bacillales – class Bacilli), red sugar polysporum ( Saccharopolyspora erythraea (Pseudonocardiales, Bacteria – Actinomycetes), Cellulose-rich bacteria ( Sorangium cellulosum (Polyangiales bacteria – Polyangia), thermophilic bacteria (Thermophilales bacteria – Anomococci), Rhodopseudomonas palustris ( Rhodopseudomonas palustris (Bacteria of the order Micromycetes – class Alpha-Proteobacteria), genus Streptomyces ( Streptomyces sp.) (bacteria of the order Kitasporales – class Actinobacteria), Rhodococcus spp. and Nocardia guinea pig otitis media (sp.) Nocardia otitidiscaviarum (Mycobacteriales, Actinomycetes), Griffithwald Magnetella ( Magnetospirillum gryphiswaldense (Rhodospirillales, α-Proteobacteria) and Kazakh Thermosporophytes (Ktedonobacterales, α-Proteobacteria).

[0125] Examples of archaea species include, but are not limited to, heat-loving acidophilic archaea (Archaeology: Thermoplasmatales – Class: Thermoplasmata), acidophilic archaea (… Cuniculiplasma divulgatum (Pyrogenic order Archaea–Pyrogenic class), *Nyctalmonella acidophilus* ( Thermogymnomonas acidicola (Pyrogenic order Archaea–Pyrogenic class), Diplococcus acidophilus ( Sulfodiicoccus acidiphilus (Sulfolobales, Archaea – Thermoprotei), Bryllium ( Acidianus brierleyi (Sulphophyllaceae, Archaea–Thermoproteobacteria), Halophilus warwickii ( Haloferax volcanii (Haloferacales, Archaea–Halobacteria) and acidophilic thermosulfuric leaf fungi (Sulfuric leaf fungi, Archaea–Thermoproteobacteria).

[0126] In some embodiments, the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme, which is an extremophile cytochrome P450 enzyme. This enzyme originates from extremophiles and can maintain activity and stability under a variety of extreme conditions considered destructive to mesophilic enzymes. For example, extremophile cytochrome P450 enzymes can tolerate extreme temperatures, high salt, high alkalinity, or high acidity. Some extremophile cytochrome P450 enzymes can tolerate other extreme conditions, including high pressure, high levels of denaturants, and solvents encountered in industrial processes.

[0127] In some embodiments, the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme, which is a thermophilic cytochrome P450 enzyme. Thermophiles are extremophiles that thrive at relatively high temperatures (e.g., 41 to 122°C). Many thermophiles are archaea, but some are bacteria and fungi. Therefore, thermophilic P450 enzymes are enzymes that function at high temperatures. Such enzymes have advantages due to their use in commercial applications.

[0128] In some embodiments, the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme, which is a member of the CYP family selected from the following groups: CYP119 family (archaic – e.g., species of *Sulfolobus* and *Sulfurisphaera*), CYP231 family (archaic – e.g., species of *Picrophilus*), CYP175 family (bacterial – e.g., species of *Thermus*), CYP199 family (bacterial – widely distributed in Alpha-Proteobacteria and Actinomycetes, e.g., species of *Rhodopseudmonas*, *Amycolatopsis*, *Rhodococcus*, and *Streptomyces*), and CYP154 family (bacterial – widely distributed in Actinomycetes, e.g., *Bifidobacterium thermophilum*). (Thermobifida, Nocardia, and Streptomyces species), the CYP102 family (bacteria – widely distributed in Bacillus, Alpha-Proteobacteria, and Actinobacteria classes, such as Prestia, Bacillus, Rhodopseudomonas, and Streptomyces species), and the CYP107 and CYP109 families (bacteria – widely distributed in many bacteria, including Myxococcia). Bacillus, Alpha-Proteobacteria, and Actinomycetes, such as species of *Priestella*, *Bacillus*, *Sorangium*, *Gordonia*, and *Streptomyces*; the CYP267 family (bacteria of the Myxococci and Planctomycetia classes, including *Sorangium* and *Pirellula* species); and the CYP116 family (bacteria of the Actinomycetes class, such as species of *Amycium* and *Rhodococcus*).

[0129] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP119 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP119, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) glutamate and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) glutamate, threonine, and threonine (ETT) amino acid residues are replaced with glutamine, glutamate, and proline (QEP) amino acid residues; (iii) glutamate, threonine, threonine, and threonine... (ETTT) amino acid residues are replaced with glutamine, glutamic acid, proline, and glycine (QEPG) amino acid residues; (iv) alanine, glycine, asparagine, glutamic acid, and threonine (AGNET) amino acid residues are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) alanine, glycine, asparagine, glutamic acid, threonine, threonine, and threonine (AGNETTT) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline, and glycine (GALQEPG) amino acid residues.

[0130] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP119A1, the mutant cytochrome P450 enzyme, compared with the wild-type CYP119A1, contains a substitution of AGNETTT (corresponding to amino acid residues 209 to 215 of SEQ ID NO: 53) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain.

[0131] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP119A2, the mutant cytochrome P450 enzyme, compared with the wild-type CYP119A2, contains a substitution of AGNETTT (corresponding to amino acid residues 210 to 216 of SEQ ID NO: 54) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain.

[0132] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP109 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP109, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) glutamate and threonine (ET) amino acid residues, or glutamate and alanine (EA) amino acid residues, substituted with glutamine and glutamate (QE) amino acid residues; (ii) glutamate, threonine, and alanine (ETA) amino acid residues. (iii) Amino acid residues, or glutamic acid, alanine and alanine (EAA) amino acid residues, or glutamic acid, threonine and threonine (ETT) amino acid residues, substituted with glutamine, glutamic acid and proline (QEP) amino acid residues; (iv) amino acid residues of alanine, glycine, threonine, glutamic acid, and threonine (AGTET), or amino acid residues of alanine, glycine, threonine, glutamic acid, and alanine (AGTEA), or amino acid residues of alanine, glycine, asparagine, glutamic acid, and threonine (AGNET), substituted with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) amino acid residues of alanine, glycine, threonine, glutamic acid, threonine, and alanine. The amino acid residues of threonine (AGTETAT) (or equivalent amino acid residues), or amino acid residues of alanine, glycine, threonine, glutamic acid, alanine, alanine and threonine (AGTEAAT) (or equivalent amino acid residues), or amino acid residues of alanine, glycine, asparagine, glutamic acid, threonine, threonine and threonine (AGNETTT) (or equivalent amino acid residues), are replaced with amino acid residues of glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG).

[0133] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109B1, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP109B1, contains a substitution of ET (corresponding to amino acids 242 and 243 of SEQ ID NO: 63) with QE in the I-helix of the enzyme's polypeptide chain.

[0134] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109C1, the mutant cytochrome P450 enzyme contains a substitution of AGTETAT (corresponding to amino acids 226 to 232 of SEQ ID NO: 58) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain compared to the I-helix of wild-type CYP109C1.

[0135] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109E1, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP109E1, contains a substitution of ET (corresponding to amino acids 245 and 246 of SEQ ID NO: 64) with QE in the I-helix of the enzyme's polypeptide chain.

[0136] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP154 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP154, contains the following substitutions in the I-helix of the mutant enzyme: (i) glutamate and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) glutamate, threonine, and threonine (ETT) amino acid residues are replaced with glutamine, glutamate, and proline (QEP) amino acid residues; (iii) glutamate, threonine, threonine, and isoleucine (ETTI) amino acid residues are replaced with glutamine, glutamate, and proline (QEP) amino acid residues. (iv) Alanine, proline, and glycine (QEPG) amino acid residues; or (v) alanine, glycine, histidine, glutamic acid, and threonine (AGHET) amino acid residues replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) alanine, glycine, histidine, glutamic acid, threonine, threonine, and isoleucine (AGHETTI) amino acid residues (or equivalent amino acid residues) replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline, and glycine (GALQEPG) amino acid residues or glycine, alanine, histidine, glutamine, glutamic acid, proline, and glycine (GAHQEPG) amino acid residues.

[0137] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme contains a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain, compared to the I-helix of wild-type CYP154C8.

[0138] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme, compared with the wild-type CYP154C8, contains a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GAHQEPG in the I-helix of the mutant enzyme's polypeptide chain.

[0139] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP154C8, contains a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 62) with QE in the I-helix of the mutant enzyme's polypeptide chain.

[0140] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type P450t, the mutant cytochrome P450 enzyme, compared to the wild-type P450t, contains a substitution of ET (corresponding to amino acids 244 and 245 of SEQ ID NO: 65) with QE in the I-helix of the mutant enzyme's polypeptide chain. The P450t enzyme is a member of the CYP154 family of cytochrome P450 enzymes, more specifically a member of the CYP154C subfamily.

[0141] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP267 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP267, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) glutamate and threonine (ET) amino acid residues, or glutamate and alanine (EA) amino acid residues, substituted with glutamine and glutamate (QE) amino acid residues; (ii) glutamate, threonine, and threonine (ETT) amino acid residues, or glutamate, alanine, and threonine (EAT) amino acid residues, substituted with glutamine, glutamate, and proline (QEP) amino acid residues; (iii) glutamate, threonine, threonine, and valine (ETTV) amino acid residues, or glutamate, alanine, threonine, and valine (EATV) amino acid residues, substituted with glutamine, glutamate, and proline. (iv) amino acid residues of alanine, glycine, histidine, glutamic acid and threonine (AGHET), or amino acid residues of alanine, glycine, histidine, glutamic acid and alanine (AGHEA), substituted with amino acid residues of glycine, alanine, leucine, glutamine and glutamic acid (GALQE); or (v) amino acid residues of alanine, glycine, histidine, glutamic acid, threonine, threonine and valine (AGHETTV) (or equivalent amino acid residues), or amino acid residues of alanine, glycine, histidine, glutamic acid, alanine, threonine and valine (AGHEATV) (or equivalent amino acid residues), substituted with amino acid residues of glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) or amino acid residues of glycine, alanine, histidine, glutamine, glutamic acid, proline and glycine (GAHQEPG).

[0142] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP267B1, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP267B1, contains a substitution of ET (corresponding to amino acids 246 and 247 of SEQ ID NO: 59) with QE, or a substitution of AGHETTV (corresponding to amino acids 243 to 249 of SEQ ID NO: 59) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain.

[0143] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP102 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP102, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) glutamate and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) glutamate, threonine, and threonine (ETT) amino acid residues are replaced with glutamine, glutamate, and proline (QEP) amino acid residues; (iii) glutamate, threonine, threonine, and... The serine (ETTS) amino acid residues are replaced with glutamine, glutamic acid, proline, and glycine (QEPG) amino acid residues; (iv) alanine, glycine, histidine, glutamic acid, and threonine (AGHET) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) alanine, glycine, histidine, glutamic acid, threonine, and serine (AGHETTS) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline, and glycine (GALQEPG) amino acid residues.

[0144] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP102A1, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP102A1, contains a substitution of ET (corresponding to amino acids 268 and 269 of SEQ ID NO: 52) with QE, or a substitution of AGHETTS (corresponding to amino acids 265 to 271 of SEQ ID NO: 52) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

[0145] In some embodiments, when the wild-type cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP175 family, the mutant cytochrome P450 enzyme, compared to wild-type CYP175, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) glutamate and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) glutamate, threonine, and valine (ETV) amino acid residues are replaced with glutamine, glutamate, and proline (QEP) amino acid residues; (iii) glutamate, threonine, valine, and alanine amino acid residues. (iv) Alanine, glycine, histidine, glutamic acid, and glycine (QEPG) amino acid residues are replaced with glycine, alanine, leucine, glutamine, and threonine (AGHET); or (v) alanine, glycine, histidine, glutamic acid, threonine, valine, and alanine (AGHETVA) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline, and glycine (GALQEPG) amino acid residues.

[0146] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP175A1, the mutant cytochrome P450 enzyme contains a substitution of AGHETVA (corresponding to amino acids 221 to 227 of SEQ ID NO: 55) with GALQEPG in the I-helix of the mutant enzyme's polypeptide chain, compared to the I-helix of wild-type CYP175A1.

[0147] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP199 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP199, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) aspartic acid and threonine (DT) amino acid residues are replaced by glutamine and glutamate (QE) amino acid residues; (ii) aspartic acid, threonine, and threonine (DTT) amino acid residues are replaced by glutamine, glutamate, and proline (QEP) amino acid residues; (iii) aspartic acid, threonine, and threonine... (iv) Replace valine (DTTV) amino acid residues with glutamine, glutamic acid, proline, and glycine (QEPG) amino acid residues; (iv) Replace alanine, glycine, leucine, aspartic acid, and threonine (AGLDT) amino acid residues with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Replace alanine, glycine, leucine, aspartic acid, threonine, threonine, and valine (AGLDTTV) amino acid residues (or equivalent amino acid residues) with glycine, alanine, leucine, glutamine, glutamic acid, proline, and glycine (GALQEPG) amino acid residues.

[0148] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP199A4, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP199A4, contains a substitution of DT (corresponding to amino acids 252 and 253 of SEQ ID NO: 60) with QE, a substitution of AGLDT (corresponding to amino acids 249 to 253 of SEQ ID NO: 60) with GALQE, or a substitution of AGLDTTV (corresponding to amino acids 249 to 255 of SEQ ID NO: 60) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

[0149] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP231 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP231: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and threonine (ETTT) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) The amino acid residues of glycine, glycine, asparagine, glutamic acid, and threonine (GGNET) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) The amino acid residues (or equivalent amino acid residues) of glycine, glycine, asparagine, glutamic acid, threonine, threonine and threonine (GGNETTT) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG).

[0150] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP107 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP107: (i) Glutamic acid and alanine (EA) amino acid residues, or glutamic acid and threonine (ET) amino acid residues, are replaced with glutamine and glutamic acid (QE) amino acid residues. (ii) Glutamic acid, alanine and serine (EAS) amino acid residues, or glutamic acid, alanine and threonine (EAT) amino acid residues, or glutamic acid, threonine and threonine (ETT) amino acid residues, replaced with glutamine, glutamic acid and proline (QEP) amino acid residues. (iii) Glutamic acid, alanine, serine and valine (EASV) amino acid residues, or glutamic acid, alanine, threonine and valine (EATV) amino acid residues, or glutamic acid, threonine, threonine and valine (ETTV) amino acid residues, replaced by glutamine, glutamic acid, proline and glycine (QEPG) amino acid residues. (iv) Alanine, glycine, phenylalanine, glutamic acid, and alanine (AGFEA) amino acid residues, or alanine, glycine, histidine, glutamic acid, and alanine (AGHEA) amino acid residues, or alanine, glycine, histidine, glutamic acid, and threonine (AGHET) amino acid residues, substituted with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, phenylalanine, glutamic acid, alanine, serine and valine (AGFEASV) amino acid residues (or equivalent amino acid residues), or alanine, glycine, histidine, glutamic acid, alanine, threonine and valine (AGHEATV) amino acid residues (or equivalent amino acid residues), or alanine, glycine, histidine, glutamic acid, threonine, threonine and valine (AGHETTV) amino acid residues (or equivalent amino acid residues), substituted with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0151] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP107PQ (P450h), the mutant cytochrome P450 enzyme, compared with the wild-type CYP107PQ (P450h), contains a substitution of EA (corresponding to amino acids 247 and 248 of SEQ ID NO: 40) with QE in the I-helix of the mutant enzyme's polypeptide chain.

[0152] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP107Mg, the mutant cytochrome P450 enzyme contains a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 66) with QE in the I-helix of the mutant enzyme's polypeptide chain compared to the I-helix of wild-type CYP107Mg.

[0153] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP116 family, the mutant cytochrome P450 enzyme contains the following substitutions in the I-helix of its polypeptide chain compared to the wild-type CYP116: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and valine (ETTV) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) The amino acid residues of alanine, alanine, histidine, glutamic acid, and threonine (AAHET) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) Alanine, alanine, histidine, glutamic acid, threonine, threonine and valine (AAHETTV) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

[0154] As described above, the mutant cytochrome P450 enzymes of the present invention exhibit enhanced peroxyase activity and / or altered product selectivity relative to wild-type cytochrome P450 enzymes. Enhanced peroxyase activity can be characterized by increased coupling efficiency / activity or increased product formation rate for one or more oxidized substrates. Coupling efficiency / activity refers to the proportion of consumed nicotinamide cofactor converted into oxidized organic products. The increased coupling efficiency / activity or product formation rate may or may not be common to all substrates utilized by the mutant cytochrome P450 enzyme. Mutant cytochrome P450 enzymes typically exhibit coupling efficiency / activity that is at least about 10%, about 20%, about 50%, about 100%, about 500%, about 1000%, or about 1500% higher than that of wild-type enzymes. The product formation rate of mutant cytochrome P450 enzymes may also be at least about 50%, about 100%, about 150%, about 500%, about 1000%, about 2000%, about 5000%, or about 10000% higher than that of wild-type enzymes.

[0155] It should be understood that the mutant cytochrome P450 enzymes of the present invention may also exhibit other altered properties relative to wild-type cytochrome P450 enzymes and mutants disclosed in the literature, such that the effects may include, but are not limited to, enhanced peroxyenzyme activity. For example, the mutant enzyme may exhibit altered substrate specificity, allowing preferential utilization of specific substrates, or may exhibit peroxyenzyme activity when wild-type enzymes or known mutants cannot oxidize substrate organic compounds.

[0156] The mutant cytochrome P450 enzyme of the present invention may also exhibit altered product selectivity, wherein a minor proportion of the product produced by the wild type becomes the major product of the mutant, or a minor proportion of a new product produced by the wild type (or a new product not produced by the wild type at all) becomes the majority or major product. Other altered properties of the mutant enzyme and other altered properties of the oxidation method performed by the mutant enzyme are described below.

[0157] In a further embodiment, the invention also relates to the use of mutant cytochrome P450 enzymes as described herein in the biocatalytic oxidation of carbon-hydrogen bonds via the peroxygenase pathway.

[0158] In a further embodiment, the present invention relates to a method for oxidizing an organic compound substrate, comprising oxidizing the organic compound substrate with a mutant cytochrome P450 enzyme as described herein.

[0159] The substrate used in the oxidation method can be any organic compound, more typically any organic compound that can be oxidized by peroxyenase. The suitability of any organic compound for peroxyenase oxidation can be routinely determined by the methods described herein.

[0160] Oxidation causes the formation of CO bonds in compounds, typically through the oxidation of carbon-hydrogen bonds to form alcohols, but can also involve the oxidation of C=C bonds to form epoxides. Therefore, oxidation can introduce alcohols, aldehydes, ketones, carboxylic acids, or epoxy groups. Alternatively, oxidation may lead to further oxidation of the oxygen-containing group, such as converting an alcohol group to an aldehyde, ketone, or carboxylic acid. In the same substrate molecule, one, two, or more carbon atoms may be attacked. Oxidation can also lead to N-dealkylation and O-dealkylation, or S-oxidation of the substrate molecule. In some cases, oxidation may lead to the breaking of C / C bonds or the formation of C / C or CX bonds (where X is a heteroatom, such as N, rather than oxygen).

[0161] Oxidation typically produces one, two, or more oxidation products. These different products may originate from different carbon atoms being attacked and / or from varying degrees of oxidation occurring on a given carbon atom.

[0162] Oxidation can occur at the ring carbon atom or the substituent carbon atom, or both. At least the initial oxidation will involve attacking the CH bond (which may be activated or deactivated) or attacking the carbon-carbon double bond (usually forming an epoxide). Typically, an activated CH bond refers to a carbon atom in the benzyl or allyl position. Aromatic rings and alkene double bonds activate the CH bond through radical intermediates generated in the stabilizing reaction pathway or any charge accumulation. The carbon atom in the CH bond can be a primary, secondary, or tertiary carbon. Oxidation may lead to dehydrogenation, forming a C=C double bond, rather than inserting an oxygen atom. This is most likely to occur when the alkyl substituent is branched, or when dehydrogenation results in a C=C bond conjugated to the aromatic ring system, or when dehydrogenation leads to the formation of the aromatic ring system itself.

[0163] The substrate can be a natural substrate of the wild-type cytochrome P450 enzyme, or a substrate that is not normally a substrate of the wild-type enzyme but can be used as a substrate by the mutant enzyme. Examples of natural substrates of cytochrome P450 enzymes are branched and straight-chain fatty acids, as well as saturated and unsaturated fatty acids, which are hydroxylated by the wild-type cytochrome P450 enzyme at the α and β, ω, ω-1, ω-2, and ω-3 carbons. Preferred examples are substrates with a length in the range of 8 to 18 carbons, such as hexadecanoic acid, heptadecanoic acid, tetradecanoic acid, dodecanoic acid, undecanoic acid, decanoic acid, nonanoic acid, and octanoic acid.

[0164] In some embodiments, the substrate may include alkanes, alkylbenzenes, steroids, terpenes, norisoprenoids, and fatty acids. In some embodiments, the substrate is a short-chain alkanes, medium-chain alkanes, long-chain alkanes, or cycloalkanes. The term alkane refers to an hydrocarbon having the general formula C1. n H 2n+2 Acyclic or unbranched hydrocarbons.

[0165] Short-chain alkanes typically have 1 to about 9 carbon atoms, more preferably 1 to 8, 1 to 6, or 1 to 4 carbon atoms. The C1-C8 alkyl group or part can be straight-chain or branched. When it is a C1-C4 alkyl part, it can be, for example, methyl, ethyl, n-propyl, propyl, sec-butyl, and tert-butyl.

[0166] Alkylbenzene has one or more alkyl groups or portions substituted at positions on the benzyl aromatic ring. The number of carbon atoms in the alkyl groups or portions is typically from 1 to about 8 carbon atoms, more preferably from 1 to 8, 1 to 6, or 1 to 4 carbon atoms.

[0167] In some embodiments, one, two, three, or more substituents may be present on the backbone of a short-chain or medium-chain alkane, or directly substituted on a benzyl ring, or on an alkyl substituent of an alkylbenzene. Any combination of the following substituents may be present. Substituents are typically halogen atoms or alkyl or alkenyl groups, generally having one to six carbons, and are optionally substituted by one or more halogens. Substituents may also contain one, two, or more oxygen, halogen, or nitrogen atoms, and may be, for example, alcohols, aldehydes, ketones, ethers, amines, or epoxy groups.

[0168] Examples of preferred short-chain alkane substrates include, but are not limited to, pentane, 3-methylpentane, 2-methylbutane, butane, propane, ethane, methane, octane, and nonane. Examples of preferred alkylbenzene and alkyl aromatic substrates include, but are not limited to, propylbenzene, ethylbenzene, butanene, cumene, tert-butanene, o-xylene, m-xylene, p-cymene, and ethyl anisole. Other preferred aromatic compounds are those having naphthalene, biphenyl, indole, indance, and fluorene skeletons.

[0169] It is important to note that organic compounds such as butane, naphthalene, and especially propane, tert-butylbenzene, and o-xylene are widely classified as “non-natural” substrates of wild-type cytochrome P450 enzymes, but can be oxidized by the mutant cytochrome P450 enzyme of this invention. Non-natural substrates can be defined as molecules that, when incubated with wild-type cytochrome P450, show no detectable coupling rate and / or product formation. Non-natural substrates may also include molecules oxidized by wild-type cytochrome P450 enzymes at a rate approximately 10% lower than that of natural substrates, such that they may not be considered true substrates.

[0170] The oxidation method using the mutant cytochrome P450 enzyme of the present invention can be distinguished from oxidation methods using another wild-type or mutant cytochrome P450 enzyme in terms of improved coupling efficiency / activity or product formation rate as defined above. The method of the present invention can also be characterized by the formation of specific products from the oxidized substrate, which are typically not formed by the wild-type cytochrome P450 enzyme or another mutant cytochrome P450 enzyme, or are formed in negligible amounts, i.e., less than about 10%, about 8%, about 5%, about 2%, about 1%, or less of the total product amount. For example, the oxidation of propylbenzene may produce 2-propylphenol or 1-phenyl-2-propanol with high selectivity, or the oxidation of ethylbenzene may produce 2-phenylethanol and styrene.

[0171] The method using the mutant cytochrome P450 enzyme of the present invention may also exhibit altered proportions or amounts of oxidation products compared to oxidation methods performed with wild-type cytochrome P450 enzymes or other mutant cytochrome P450 enzymes. When altered product proportions exist, the product formation rate of a particular oxidation product is generally increased relative to the corresponding method performed with wild-type cytochrome P450 enzymes or other mutant cytochrome P450 enzymes. The increase in yield of a particular oxidation product may be at least about 10%, about 20%, about 50%, more preferably about 100%, about 200%, about 300%, about 500%, or more than the amount of said oxidation product in the product mixture formed by wild-type cytochrome P450 enzymes or other mutant cytochrome P450 enzymes.

[0172] This method is typically carried out in the presence of cytochrome P450 enzymes, substrates, and natural cofactors of the enzymes, said natural cofactors may include one or more of electron transport chaperones, oxygen, nicotinamide cofactors, hydrogen peroxide, and water. In some embodiments, the method is carried out in situ by generating hydrogen peroxide cofactors using electrochemical, biocatalytic, or photochemical methods known in the art (see, for example, Hobisch M...). et al ., 2021, Biotechnology Advances ,51: 107615;Yun CH et al ., 2022, Chem. Sci ., 13(42): 12260-12279; and Bormann S et al ., 2020, Biotechnol. Bioeng ., 118(1): 7-16).

[0173] While hydrogen peroxide is a commonly used cofactor, any other peroxide source, including organic alternatives, may be used. The suitability of any peroxide source for the oxidation method can be conventionally determined by the methods described herein. In some embodiments, the peroxide is selected from the group consisting of hydrogen peroxide, cumene hydrogen peroxide, iodobenzoylbenzene, tert-butyl hydrogen peroxide, and m-chloroperoxybenzoic acid. In some preferred embodiments, the peroxide is selected from the group consisting of hydrogen peroxide, tert-butyl hydrogen peroxide, and m-chloroperoxybenzoic acid. Preferably, the peroxide is hydrogen peroxide.

[0174] In this method, the enzyme concentration is typically about 10. -8 To about 10 -2 M, preferably about 10 -7 To about 10 -4 M. Typically, the method is carried out at the temperature and / or pH at which the enzyme functions, for example when the enzyme has at least about 20%, 50%, 80% or higher of its peak activity. Typically, the pH is about 2 to about 12, for example about 5 to about 9 or about 6 to about 8, preferably about 7 to about 7.8 or about 7.4. Typically, the temperature is about 10°C to about 90°C, for example about 25°C to about 75°C or about 30°C to about 60°C. In some embodiments, the method is carried out at about 10°C to about 90°C.

[0175] The present invention is further illustrated in the following embodiments. These embodiments are for the purpose of describing particular implementations only and are not intended to limit the above description.

[0176] Example 1 Production of mutant cytochrome P450 enzymes Genes encoding wild-type and mutant P450 enzymes were obtained in plasmids pET28b (between Nco I and Xho I restriction sites) or pET29b (between Nde I and Xho I restriction sites) provided by Twist Bioscience. The NcoI site (CCATGG) or NdeI site (CATATG) contains the start codon. The codon-optimized sequence was cloned into the pET28 vector between the Nco I and Xho I restriction sites when a 6x histidine purification tag was used. At the N-terminus, a 6x histidine tag and a tobacco etched virus (TEV) cleavage sequence (N-terminal adapted amino acid sequence: GGSSHHHHHHSSGENLYFQGHM) (SEQ ID NO: 68) were introduced before the Nde I restriction site, followed by the sequence of the gene of interest. Following the gene sequence were two stop codons and Kpn I and Hind III restriction sites, followed by the Xho I restriction site of the vector.

[0177] These plasmids were transformed into *E. coli* BL21 (DE3) competent cells and grown on LB agar plates in the presence of kanamycin (30 µg / mL). Single colonies were added to 500 mL of LB medium containing trace element solutions (1.5 mL solution containing CaCl2, ZnSO4·7H2O, MnSO4·H2O, Na2-EDTA, FeCl3·6H2O, CuSO4·5H2O, and CoCl2·6H2O) in the presence of antibiotics and incubated at 37 °C and 85 rpm. After 10 hours of incubation, the incubation temperature was lowered to 20 °C (or maintained at 37 °C for thermophilic enzymes), and 0.02% v / v benzyl alcohol and 2% v / v ethanol were added. After another 30 minutes, protein production was induced using 100 μM isopropyl-β-D-1-thiogalactoside (IPTG). Cells were regenerated for 24-48 hours and harvested by centrifugation (5000 g, 10 minutes, 4°C).

[0178] Example 2 Purification of mutant cytochrome P450 enzyme protein The *E. coli* cell pellet (from 2–3 L of growth medium) was stored at -20°C before purification. For His-tagged proteins, the cell pellet was resuspended in 200 mL of Buffer T (50 mM Tris buffer, pH 7.4–8.0, 1 mM dithiothreitol-DTT – hereinafter referred to as Buffer T). The mixture was lysed by sonication (35 20-second pulses, 40-second intervals; 70% amplitude). During sonication, the cell mixture was cooled on ice and frequently stirred. To remove cell debris, the lysed cell mixture was centrifuged at 35,000 g (17,010 rpm) for 30 min at 4°C. The crude protein mixture was purified by ammonium sulfate precipitation. After adding 30% ammonium sulfate, the mixture was centrifuged at 20,442 g (13,000 rpm) for 10 min at 4°C. The supernatant containing the P450 enzyme was retained, and the pellet was discarded. Increase the ammonium sulfate concentration to 60%, and centrifuge the mixture at 20,442 g (13,000 rpm) for 15 minutes at 4°C. Discard the supernatant and redissolve the precipitate containing the P450 enzyme in buffer T. Desalt the protein using a Sephadex G-25 coarse-particle column, using buffer T as the elution buffer. Collect the red fraction and purify it by ion exchange chromatography using a DEAE Sepharose column. Elute the protein using a 100–400 mM KCl gradient in buffer T at a flow rate of 6 mL / min. -1 The red fractions were combined and concentrated by ultrafiltration.

[0179] For proteins tagged with 6 x histidine, cells were resuspended in 50 mM Tris buffer (pH 7.4–8.0, containing 0.1% v / v 2-mercaptoethanol, 2 mL benzyl sulfonyl fluoride PMSF (10 mM stock solution), and 10% glycerol). Cells were lysed by sonication on ice for 30 cycles (20 sec on, 40 sec off), and cell debris was removed by centrifugation (37,000 g, 20 min, 4 °C). The supernatant was loaded onto a His-trap column (GE Healthcare) equilibrated with sample loading buffer (20 mM sodium phosphate, 20 mM imidazole, 0.5 M NaCl, pH 7.4–8.0). The column was washed with 5 column volumes of sample loading buffer, and His-tagged proteins were eluted with sample elution buffer (20 mM sodium phosphate buffer, pH 7.4–8.0, 0.5 M NaCl, and 500 mM imidazole). The fraction containing P450 was concentrated by ultrafiltration (10 kDa cutoff membrane), and then the protein was loaded onto a Sephadex G-25 medium particle column (250 mm x 40 mm; GE Healthcare) and desalted using 50 mM Tris buffer (pH 7.4–8.0).

[0180] All proteins were further purified by size exclusion chromatography using buffer T as the elution buffer. A HiPrep 16 / 6-Sephacryl S-200 HR column (GE Healthcare) was used, with a flow rate of 1 mL / min. -1 The highest purity fractions were combined, concentrated, filtered sterile, and stored in ~50% glycerol at -20°C. Before use, glycerol was removed using a PD-10 column (Cytiva) with Tris buffer (50 mM, pH 7.4 - 9.0) as the eluent.

[0181] Example 3 Mutant cytochrome P450 enzyme peroxidase reaction Unless otherwise specified, the peroxyase oxidation assays described in Examples 4 through 10 below were performed in a total volume of 600–1000 μL, consisting of Tris buffer (50 mM, pH 7.4–9.0), 1–3 μM enzyme, and 0.250–2 mM substrate (from a 50–100 mM stock solution in EtOH or DMSO solvent). The reaction was initiated by adding H₂O₂ (2–60 mM) and incubated for 1–4 hours. Samples were analyzed by HPLC or gas chromatography-mass spectrometry (GC-MS).

[0182] For HPLC analysis, take 132 μL of the sample aliquot and use 10 μL of 10 mg / mL solution. -1 The catalase was quenched with bovine liver catalase solution and 66 μL of acetonitrile (MeCN). Finally, 2 μL of internal standard (10 mM 9-hydroxyfluorene in EtOH solution) was added, and each mixture was centrifuged and analyzed by HPLC (20-95% acetonitrile aqueous gradient, acidified with 0.1% trifluoroacetic acid).

[0183] HPLC analysis was performed using a Shimadzu Prominence LC-20AD liquid chromatograph equipped with a DGU-20A. 5R Degassing unit, SIL-20A autosampler, SPD-20A UV-Vis detector, CTO-20A column oven and Kinetex XB-C18 reversed-phase LC column (100 Å pore size, 250 × 4.6 mm, 5 μm; Phenomenex).

[0184] For GC-MS analysis, 595 μL of the reaction mixture was mixed with 5 μL of internal standard solution (octanoic acid, 20 mM stock solution, EtOH). These mixtures were extracted with 3 x 400 μL of ethyl acetate and dried over anhydrous MgSO4. The mixture could then be used directly for analysis, or derivatized as follows: the organic solvent was removed under a nitrogen stream, and the residue was dissolved in anhydrous acetonitrile. 15 μL of bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) was added to the acetonitrile to derivatize the substrate and metabolites. These samples were incubated at 37°C for 2 hours before being used directly for GC-MS analysis.

[0185] GC-MS was performed on a Shimadzu GC-2010 equipped with a GC-MS-QP2010S detector. Interface and injection port temperatures were maintained at 280 °C and 250–300 °C, respectively. The column (DB5ms; 30 m x 0.25 mm x 0.25 µm) was held at 70 °C or 120 °C for 3 min, then at 7.5 °C for 3 min. -1 The rate at which the temperature is raised to 240°C is maintained at 240°C for 6 minutes.

[0186] Figure 1 B shows a comparison of the active site structures of CYP255A2 and CYP119A1, highlighting the oxygen-binding groove of the I-helix. The acid-alcohol amino acid residue pairs responsible for most of the monooxygenase activity of P450 (E212 and T213 in CYP119A1) are replaced by amide and acid pairs (Q248 and E249) in the peroxygenase CYP255A2.

[0187] Thermostable peroxyseic enzyme variants of CYP119A1 are based on the substitution of seven amino acids near the oxygen-binding groove of the enzyme. Figure 1 B). Activity was optimized by generating other mutations in this region. These seven residues of the I-helix in CYP119A1 were replaced with residues from CYP255A2 ( Figure 1 B). In general, the amino acids of CYP119A1 209 AGNETTT 215 It was replaced with the sequence GALQEPG.

[0188] As described below, the substitution of these seven amino acids has also been demonstrated to function in CYP119A1 (thermally stable P450) and CYP154C8 (GAHQEPG) (the P450 of oxidative steroids). It is also shown below that fewer substitutions can also be effective, including QE and GALQE substitutions in CYP199A4 and other enzymes. Figure 6-22 All of these showed increased peroxygenase activity compared to WT and single mutants with threonine changed to E (e.g., T252E in CYP199A4 with T changed to E).

[0189] Example 4 Mutant cytochrome P450 enzyme (CYP119) oxidizes fatty acids Metabolites generated from the oxidation of fatty acids by a CYP119A1 peroxyase mutant, such as Figure 3 As shown. Figure 4 Gas chromatographic analysis of 3 μM WT CYP119A1 and the peroxygenase mutant (mutant) treated with hydrogen peroxide (50 mM) for 2 h at room temperature (RT) and 80 °C with dodecanoic acid / lauric acid (1 mM). Figure 5 Gas chromatographic analysis of the oxidation of dodecanoic acid (lauric acid) by the GALQEPG variant of CYP119 (3 ​​μM) with hydrogen peroxide (50 mM) at room temperature (RT) and at 50 °C, 70 °C, and 90 °C is shown. This result highlights the ability of the CYP119 variant to function efficiently at high temperatures. It also highlights the complete conversion of all substrates to the product in the mutant.

[0190] Example 5 Mutant cytochrome P450 enzyme (CYP154) oxidizes progesterone and androstenedione. The CYP154 mutant uses hydrogen peroxide to oxidize progesterone and androstenedione as the final product, such as Figure 6 As shown.

[0191] Figure 7The HPLC analysis of stereoselective oxidation of progesterone (1 mM) by Streptomyces CYP154C8 WT and GAHQEPG mutants (1 μM) at room temperature (RT) using 20 mM hydrogen peroxide is shown.

[0192] Figure 8 HPLC analysis of the stereoselective oxidation of progesterone (1 mM) by the GAHQEPG mutant of CYP154C8 at room temperature (RT) using 2–40 mM hydrogen peroxide is presented. These results highlight the ability of this variant to function even at low concentrations of hydrogen peroxide.

[0193] Figure 9 HPLC analysis of the peroxidase activities of CYP154C8 mutants (T258E, QE, and GAHQEPG) compared to wild-type (WT) enzymes was presented. Stereoselective oxidation of progesterone substrate (1 mM) by each mutant of CYP154C8 and WT was performed at room temperature (RT) for 4 h using 5.0 mM hydrogen peroxide and 5% v / v ethanol as substrate solvent. The results showed that each mutant exhibited higher conversion rates for progesterone oxidation compared to WT. Under these conditions, the QE mutant showed the highest oxidation efficiency, followed by the GAHQEPG mutant, and then the T258E mutant.

[0194] Figure 10 The effect of different DMSO concentrations (as substrate solvent) on the oxidation assay of the CYP154C8 QE mutant is shown. Stereoselective oxidation of progesterone substrate (2 mM) by the CYP154C8 QE mutant (1 μM) was performed at room temperature (RT) for 4 h using 5.0 mM hydrogen peroxide. HPLC results show the stability of the oxidative activity of the CYP154C8 QE mutant in the presence of organic solvents (up to 30% v / v DMSO in this case).

[0195] Figure 11 The effect of different hydrogen peroxide concentrations (1 mM, 5 mM, 10 mM, 20 mM, and 40 mM) on the oxidation assay of the CYP154C8 QE mutant was shown. Stereoselective oxidation of progesterone (1 mM) by the CYP154C8 QE mutant (1 μM) was performed at room temperature (RT) for 4 hours using different concentrations of hydrogen peroxide as substrate and 5% v / v DMSO as substrate solvent. HPLC results showed the stability of the oxidative activity of the CYP154C8 QE mutant at hydrogen peroxide concentrations exceeding 10 mM (and up to 40 mM).

[0196] Figure 12The P450t QE mutant was shown to use androstenedione peroxide (androstenedione) Figure 12 A) and testosterone ( Figure 12 B) Substrate Results. P450t is a member of the CYP154C subfamily, derived from *Nocardia otitis media* in guinea pigs. The reaction mixture contained 400 μM of each substrate, 10 mM hydrogen peroxide, and 2 μM of mutant enzyme in Tris buffer at pH 7.4. This indicates that other cytochrome P450 enzymes can be mutated, and other substrates can be oxidized, leading to increased oxidation efficiency.

[0197] Example 6 Mutant cytochrome P450 enzyme (CYP199) oxidizes 4-methoxybenzoic acid like Figure 13 As shown, in a time-progression reaction of 3 µM P450 CYP199A4 (WT and GALQE mutants) with 4-methoxybenzoic acid driven by 5 mM H2O2, the WT enzyme produced 61.6 ± 0.2 μM of product, while the GALQE mutant produced approximately 3 times more product (176 ± 3 μM product) under low peroxide concentrations.

[0198] Figure 14 Results of a time-process reaction of 3 µM P450 CYP199A4 (QE and T252E mutants) with 4-methoxybenzoic acid (1 mM) driven by 10 mM H2O2 are shown. These results highlight the improved performance of the QE mutant relative to the T252E single mutant.

[0199] Figure 15 Results of time-process reactions of 3 μM P450 CYP199A4 (QE mutant and WT) with 4-methoxybenzoic acid (1–5 mM) driven by 10 mM H2O2 are presented. These results highlight the increased product formation and catalyst lifetime of the QE variants when the substrate concentration is maintained at higher levels. Figure 11 B shows the HPLC analysis of the reaction after 45 hours.

[0200] Example 7 Mutant cytochrome P450 enzyme (CYP102) oxidizes myristic acid Figure 16The GC-MS analysis shows the reaction of 2 μM HazakQE with 250 μM myristic acid driven by 5 mM H2O2 in Tris-HCl buffer (50 mM, pH 7.4) at 30 °C for 2 h. This enzyme is a thermophilic CYP102 enzyme from *Thermospora kazakhstan*. The enzyme was heated at 50 °C for 30 min before the reaction. This enzyme... Figure 1 The CYP102A1 enzyme amino acid sequence provided in the diagram shows greater than 40% amino acid sequence identity, highlighting how enzymes with similar sequence identity can be identified. The P450 reaction is indicated by a dark line, while the control reaction, which omits the P450 enzyme but includes all other reaction components (including H2O2), is shown by a light line. The major metabolites are ω-3, ω-2, and ω-1 hydroxylated products.

[0201] Example 8 Mutant cytochrome P450 enzyme (P450h / CYP107) oxidizes β-ionone Figure 17 The thermophilic P450 enzyme from *Thermophilus rubrum* was shown. Figure 2 Stability analysis of the QE mutant of P450h (CYP107PQ1QE) in the selective oxidation of β-ionone substrates to 4-hydroxy-β-ionone. P450h is a member of the CYP107 family, also referred to herein as CYP107PQ1. The reaction conditions were 1 μM P450h QE mutant (CYP107PQ1QE) enzyme, 1 mM β-ionone (from a 100 mM stock solution in DMSO solvent), and 10 mM H2O2. Reaction temperature and time were varied as described below.

[0202] Figure 17 Figure A shows the effect of reaction temperature on the catalytic activity of the P450h QE mutant (CYP107PQ1QE) and the rate of 4-hydroxy-β-ionone product formation at different time points up to 4 hours (at 45°C, substrate evaporation occurred in longer reaction times). It is clear that the P450h QE mutant (CYP107PQ1QE) enzyme maintains its activity at different reaction temperatures, including 30°C and 45°C.

[0203] Figure 17 B presents an HPLC analysis of the β-ionone oxidation reaction of the P450h QE mutant (CYP107PQ1QE). The stability of the mutant enzyme was measured after heat treatment at different temperatures for 1 h before addition to the β-ionone substrate. The oxidation reaction was carried out at 30 °C for 2 h. These results indicate that the mutant enzyme is stable even after preheating at temperatures up to 60 °C to 65 °C.

[0204] Figure 17C demonstrates HPLC analysis of the β-ionone oxidation reaction of the P450h QE mutant (CYP107PQ1QE) after pre-storage at 30°C for a set period. The oxidation reaction was carried out at 30°C for 2 hours. These results demonstrate the ability to store mutant cytochrome P450 enzymes at ambient temperature (rather than in a refrigerator or freezer) for extended periods (including up to 1 year) without loss of activity.

[0205] Figure 18 Stability analysis of the heme of the P450h QE mutant (CYP107PQ1QE) enzyme in the selective oxidation of β-ionone substrate to 4-hydroxy-β-ionone was presented. Reaction conditions were 2 μM P450h QE mutant (CYP107PQ1QE) and 10 mM H2O2, substrate-free (A); and 2 μM P450h QE mutant (CYP107PQ1QE) and 20 mM H2O2, substrate-containing (B). These results demonstrate the stability of the heme of the mutant cytochrome P450 enzyme against 20 mM hydrogen peroxide in the presence of 1 mM substrate.

[0206] Figure 19 The catalytic activity of the P450h QE mutant enzyme (CYP107PQ1QE) relative to the wild-type enzyme (CYP107PQ1) in the selective oxidation of β-ionone substrate (1 mM) to 4-hydroxy-β-ionone is shown in (A), as well as the enantioselectivity of the mutant (B). The reaction conditions were 1 μM P450h QE mutant (CYP107PQ1QE) or wild-type enzyme, and 10 mM H2O2, reacted at 30 °C for 2 h. The HPLC chromatogram in (A) shows that for the mutant enzyme, 4-hydroxy-β-ionone is the major product on HPLC at a retention time of 16 min, while the wild-type enzyme produces little or no product. Figure 19 B shows the enantioselectivity-HPLC analysis of the mutagenesis-catalyzed 4-hydroxy-β-ionone compared to the chemically synthesized racemic product. This demonstrates the high enantioselectivity of the reaction.

[0207] Figure 20 The effects of different organic solvents on the catalytic activity of the P450h QE mutant (CYP107PQ1QE) enzyme in the presence of 1 mM β-ionone substrate (A and B) or 2 mM β-ionone substrate (C and D), 10 mM hydrogen peroxide, and 1 uM enzyme were shown. All reactions were carried out at 30 °C for 2 h. Figure 20 The HPLC chromatogram of A shows that the β-ionone substrate was efficiently converted to 4-hydroxy-β-ionone in the presence of a series of different organic solvents (each at 10% v / v). Figure 20Figure B is a graph comparing the effects of different concentrations of the organic solvents DMSO and isopropanol on the relative yield (ratio of product peak area to internal standard peak area) of 4-hydroxy-β-ionone. Figure 20 C is an HPLC chromatogram comparing the effect of different concentrations of DMSO solvent on the product yield. Figure 20 D is an HPLC chromatogram comparing the effect of different concentrations of isopropanol solvent on the product yield. These results demonstrate the stability of the mutant cytochrome P450 enzyme to a range of organic solvents at different concentrations.

[0208] Example 9 Mutant cytochrome P450 enzymes (CYP109 and P450h / CYP107) oxidize β-ionone Figure 21 The stability of cytochrome P450 enzymes CYP109B1 and CYP109E1, as well as the QE mutant of the P450h (CYP107PQ1) enzyme mentioned in the above examples, after heat treatment at different temperatures for 1 hour before adding the β-ionone substrate to the reaction is shown. The reaction conditions were 1 μM enzyme, 1 mM β-ionone (from a 100 mM stock solution in DMSO solvent), and 10 mM H2O2. (A) CYP109E1QE mutant; (B) CYP109B1QE mutant; (C) CYP107PQ1QE mutant. In each figure, the y-axis represents the relative yield of the 4-hydroxy-β-ionone product (the ratio of product to internal standard), and the x-axis represents the temperature at which the enzyme was pretreated before the reaction. These figures demonstrate that the mutant cytochrome P450 enzymes are temperature stable.

[0209] Example 10 Mutant cytochrome P450 enzyme (CYP107) oxidizes progesterone Figure 22 The ability of the CYP107MgQE mutant enzyme to catalyze the conversion of progesterone using hydrogen peroxide as the sole oxidant was demonstrated. The oxidation reaction was carried out at 30°C for 1 hour using 5 μM enzyme and 500 μM progesterone in the presence of 10 mM hydrogen peroxide. In the wild-type CYP107Mg enzyme, no oxidation products were observed, indicating that the wild-type enzyme cannot act as a peroxyenase in its native state. However, the CYP107MgQE mutant enzyme clearly possesses the ability to oxidize progesterone.

[0210] The foregoing embodiments demonstrate that the mutant cytochrome P450 enzyme of the present invention exhibits increased peroxyenzyme activity compared to the corresponding wild-type enzyme. The mutant cytochrome P450 enzyme of the present invention has been shown to be stable to hydrogen peroxide in the presence of the substrate and capable of hydroxylating fatty acid substrates with high substrate conversion rates (>99%) and good conversion numbers. The thermostability of the mutant P450 enzyme derived from thermophilic bacteria has been maintained, and catalytic activity at high temperatures (50°C to 90°C) has been demonstrated. The generation of an efficient thermostable heme peroxyenzyme establishes a system for achieving H2O2-dependent specific CH bond hydroxylation. This overcomes many major drawbacks of using cytochrome P450 enzymes for this purpose, namely the need for expensive nicotinamide cofactors and additional electron transport chaperones, and the low activity and low stability of most of these heme-thiolate enzymes. This work not only expands the toolbox of enzymatic CH activation but also provides a simple, inexpensive, and clean method for achieving this challenging reaction.

[0211] Throughout this specification, unless the context otherwise requires, the words “including / contains / comprises” or variations thereof shall be understood to imply inclusion of the stated element or integer or group of elements or integers, but not to exclude any other element or integer or group of elements or integers.

[0212] It is important to note that when describing a range of values, it should be clearly understood that the range encompasses the upper and lower limits of that range, as well as all values ​​or subranges between these limits, as if each value and subrange were explicitly stated. Unless otherwise stated, the expression "about X% to Y%" has the same meaning as "about X% to about Y%".

[0213] The term “about” as used in this specification means approximately or close to, and in the context of the numerical values ​​or ranges described herein, it means covering a variation of + / -10% or less, + / -5% or less, + / -1% or less, or + / -0.1% or less relative to the numerical values ​​or ranges described or claimed.

[0214] It should also be noted that, as used in this article, unless the context otherwise indicates, the singular forms of “a,” “an,” and “the” include the plural aspect.

[0215] The headings used herein are included for the reader's convenience only and should not be used to limit the scope of the entire disclosure or the subject matter found in the claims. The headings should not be used to interpret the claims or limit their scope.

[0216] The description provided herein relates to several embodiments that may share common characteristics and features. It should be understood that one or more features of one embodiment may be combined with one or more features of other embodiments. Furthermore, individual features or combinations of features of an embodiment may constitute additional embodiments.

[0217] Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., "for example") provided herein is intended only to better illustrate exemplary embodiments and, unless otherwise required, does not constitute a limitation on the scope of the claimed invention. No language in the specification should be construed as indicating that any unclaimed element is essential.

[0218] It will be apparent to those skilled in the art that, although the invention has been described in detail to a certain extent for the purposes of clarity and understanding, various modifications and changes may be made to the embodiments and methods described herein without departing from the scope of the inventive concept disclosed herein.

[0219] Those skilled in the art will understand that the invention described herein is readily adaptable to variations and modifications beyond those specifically described. It should be understood that the invention encompasses all such variations and modifications. The invention also includes all steps, features, compositions, and compounds mentioned or indicated in this specification, whether individually or collectively, and any and all combinations of any two or more of the stated steps or features.

[0220] Finally, references are made to standard molecular biology textbooks that incorporate methods for implementing the basic techniques covered by this invention. See, for example, Green MR and Sambrook J. Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.

[0221] sequence list

Claims

1. A mutant cytochrome P450 enzyme having enhanced peroxyase activity and / or altered product selectivity, wherein the mutant cytochrome P450 enzyme, compared to the wild-type cytochrome P450 enzyme, contains at least two consecutive amino acid residues substituted in the I-helix of the enzyme's polypeptide chain, wherein the substitutions are located at positions corresponding to amino acid residues 23 and 24 of SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme contains glutamine and glutamate (QE) amino acid residues at said positions in the I-helix of the enzyme's polypeptide chain.

2. The mutant cytochrome P450 enzyme according to claim 1, wherein the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme, and the wild-type cytochrome P450 enzyme is derived from a bacterial or archaea species.

3. The mutant cytochrome P450 enzyme according to claim 1 or claim 2, wherein the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme, and the wild-type cytochrome P450 enzyme is an extremophile cytochrome P450 enzyme.

4. The mutant cytochrome P450 enzyme according to any one of claims 1 to 3, wherein the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme, and the wild-type cytochrome P450 enzyme is a thermophilic cytochrome P450 enzyme.

5. The mutant cytochrome P450 enzyme according to any one of claims 1 to 4, wherein the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to 67.

6. The mutant cytochrome P450 enzyme according to any one of claims 1 to 5, wherein the mutant cytochrome P450 enzyme is derived from wild-type cytochrome P450 enzyme, comprising an amino acid sequence having at least about 40% amino acid sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67, and comprising an amino acid common sequence X1X2X2X3X4X5X6 (SEQ ID NO: 32) in the I-helix of the polypeptide chain, wherein: X1 is either alanine (A) or glycine (G); X2 is either glycine (G) or alanine (A); X3 is histidine (H), asparagine (N), leucine (L), alanine (A), threonine (T), isoleucine (I), or phenylalanine (F); X4 is glutamic acid (E), glycine (G), leucine (L), or aspartic acid (D); X5 is threonine (T), alanine (A), or asparagine (N); X6 is threonine (T), isoleucine (I), valine (V), serine (S), or alanine (A); and X7 consists of threonine (T), valine (V), alanine (A), tryptophan (W), arginine (R), serine (S), and isoleucine (I).

7. The mutant cytochrome P450 enzyme according to any one of claims 1 to 6, wherein the mutant cytochrome P450 enzyme contains a substitution of an amino acid residue immediately following the glutamine and glutamate (QE) amino acid residues in the I-helix of the polypeptide chain of the mutant cytochrome P450 enzyme.

8. The mutant cytochrome P450 enzyme according to claim 7, wherein the amino acid residue immediately following the glutamine and glutamate (QE) amino acid residues is replaced with proline (P) amino acid residues.

9. The mutant cytochrome P450 enzyme according to claim 7 or claim 8, wherein the mutant cytochrome P450 comprises a substitution of three consecutive amino acid residues at positions 23 to 25 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme comprises glutamine, glutamic acid, and proline (QEP) amino acid residues at the positions in the I-helix of the polypeptide chain of the mutant enzyme, compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

10. The mutant cytochrome P450 enzyme according to any one of claims 1 to 6, wherein the mutant cytochrome P450 enzyme comprises a substitution of two consecutive amino acid residues immediately following the glutamine and glutamate (QE) amino acid residues in the I-helix of the polypeptide chain of the mutant cytochrome P450 enzyme.

11. The mutant cytochrome P450 enzyme according to claim 10, wherein the two consecutive amino acid residues immediately following the glutamine and glutamate (QE) amino acid residues are replaced with proline and glycine (PG) amino acid residues.

12. The mutant cytochrome P450 enzyme according to claim 10 or claim 11, wherein the mutant cytochrome P450 comprises a substitution of four consecutive amino acid residues at positions 23 to 26 corresponding to amino acid residues in SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme comprises glutamine, glutamic acid, proline, and glycine (QEPG) amino acid residues at the positions in the I-helix of the polypeptide chain of the enzyme, compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

13. The mutant cytochrome P450 enzyme according to any one of claims 1 to 12, wherein the mutant cytochrome P450 enzyme comprises a substitution of three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues in the I-helix of the polypeptide chain of the mutant cytochrome P450 enzyme.

14. The mutant cytochrome P450 enzyme according to claim 13, wherein the three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues are replaced with glycine, alanine and X (GAX) amino acid residues, wherein X represents any amino acid.

15. The mutant cytochrome P450 enzyme according to claim 13 or claim 14, wherein the three consecutive amino acid residues immediately preceding the glutamine and glutamate (QE) amino acid residues are replaced with glycine, alanine and leucine (GAL) or glycine, alanine and histidine (GAH).

16. The mutant cytochrome P450 enzyme according to any one of claims 13 to 15, wherein the mutant cytochrome P450 comprises a substitution of five consecutive amino acid residues at positions corresponding to amino acid residues 20 to 24 of SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme comprises glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) or glycine, alanine, histidine, glutamine, and glutamic acid (GAHQE) amino acid residues at said positions in the I-helix of the polypeptide chain of the mutant enzyme, compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

17. The mutant cytochrome P450 enzyme according to any one of claims 1 to 16, wherein the mutant cytochrome P450 enzyme comprises a segment of seven consecutive amino acid residues containing glutamine and glutamic acid (QE) in the I-helix of the polypeptide chain of the mutant cytochrome P450 enzyme.

18. The mutant cytochrome P450 enzyme according to claim 17, wherein the seven consecutive amino acid segments are replaced with glycine, alanine, X, glutamine, glutamic acid, proline, and glycine (GAXQEPG) amino acid residues, wherein X represents any amino acid.

19. The mutant cytochrome P450 enzyme according to claim 17 or claim 18, wherein the mutant cytochrome P450 comprises a substitution of seven consecutive amino acid residues at positions corresponding to amino acid residues 20 to 26 of SEQ ID NO: 1, and wherein the mutant cytochrome P450 enzyme comprises, at said positions in the I-helix of the polypeptide chain of the enzyme, glycine, alanine, leucine, glutamine, glutamic acid, proline, and glycine (GALQEPG) or glycine, alanine, histidine, glutamine, glutamic acid, proline, and glycine (GAHQEPG) amino acid residues compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

20. The mutant cytochrome P450 enzyme according to any one of claims 1 to 19, wherein the mutant cytochrome P450 enzyme is derived from the wild-type cytochrome P450 enzyme, and the wild-type cytochrome P450 enzyme is a member of the CYP family selected from the group consisting of the CYP119 family, CYP231 family, CYP175 family, CYP199 family, CYP154 family, CYP102 family, CYP107 family, CYP109 family, CYP116 family and CYP267 family.

21. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP119 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP119, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and threonine (ETTT) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) The amino acid residues of alanine, glycine, asparagine, glutamic acid, and threonine (AGNET) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) Alanine, glycine, asparagine, glutamic acid, threonine, threonine and threonine (AGNETTT) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

22. The mutant cytochrome P450 enzyme of claim 21, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP119A1, the mutant cytochrome P450 enzyme, compared with the wild-type CYP119A1, contains a substitution of AGNETTT (corresponding to amino acid residues 209 to 215 of SEQ ID NO: 53) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

23. The mutant cytochrome P450 enzyme of claim 21, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP119A2, the mutant cytochrome P450 enzyme, compared with the wild-type CYP119A2, contains a substitution of AGNETTT (corresponding to amino acid residues 210 to 216 of SEQ ID NO: 54) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

24. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP109 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP109, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and threonine (ET) amino acid residues, or glutamic acid and alanine (EA) amino acid residues, are replaced with glutamine and glutamic acid (QE) amino acid residues. (ii) Glutamic acid, threonine and alanine (ETA) amino acid residues, or glutamic acid, alanine and alanine (EAA) amino acid residues, or glutamic acid, threonine and threonine (ETT) amino acid residues, replaced by glutamine, glutamic acid and proline (QEP) amino acid residues. (iii) Glutamic acid, threonine, alanine and threonine (ETAT) amino acid residues, or glutamic acid, alanine, alanine and threonine (EAAT) amino acid residues, or glutamic acid, threonine, threonine and threonine (ETTT) amino acid residues, replaced by glutamine, glutamic acid, proline and glycine (QEPG) amino acid residues. (iv) Alanine, glycine, threonine, glutamic acid, and threonine (AGTET), or alanine, glycine, threonine, glutamic acid, and alanine (AGTEA), or alanine, glycine, asparagine, glutamic acid, and threonine (AGNET), substituted with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) Alanine, glycine, threonine, glutamic acid, threonine, alanine and threonine (AGTETAT) amino acid residues (or equivalent amino acid residues), or alanine, glycine, threonine, glutamic acid, alanine, alanine and threonine (AGTEAAT) amino acid residues (or equivalent amino acid residues), or alanine, glycine, asparagine, glutamic acid, threonine, threonine and threonine (AGNETTT) amino acid residues (or equivalent amino acid residues), substituted with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

25. The mutant cytochrome P450 enzyme according to claim 24, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP109B1, the mutant cytochrome P450 enzyme, compared with the I-helix of wild-type CYP109B1, contains a substitution of ET (corresponding to amino acids 242 and 243 of SEQ ID NO: 63) with QE in the I-helix of the polypeptide chain of the enzyme.

26. The mutant cytochrome P450 enzyme according to claim 24, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP109C1, the mutant cytochrome P450 enzyme, compared with the I-helix of wild-type CYP109C1, contains a substitution of AGTETAT (corresponding to amino acids 226 to 232 of SEQ ID NO: 58) with GALQEPG in the I-helix of the polypeptide chain of the enzyme.

27. The mutant cytochrome P450 enzyme according to claim 24, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP109E1, the mutant cytochrome P450 enzyme, compared with the I-helix of wild-type CYP109E1, contains a substitution of ET (corresponding to amino acids 245 and 246 of SEQ ID NO: 64) with QE in the I-helix of the polypeptide chain of the enzyme.

28. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP154 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP154, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and isoleucine (ETTI) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) Alanine, glycine, histidine, glutamic acid, and threonine (AGHET) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) The amino acid residues (or equivalent amino acid residues) of alanine, glycine, histidine, glutamic acid, threonine, threonine and isoleucine (AGHETTI) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) or amino acid residues of glycine, alanine, histidine, glutamine, glutamic acid, proline and glycine (GAHQEPG).

29. The mutant cytochrome P450 enzyme according to claim 28, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme, compared with the wild-type CYP154C8, contains a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

30. The mutant cytochrome P450 enzyme of claim 28, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme, compared with the wild-type CYP154C8, contains a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GAHQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

31. The mutant cytochrome P450 enzyme according to claim 28, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme, compared with the wild-type CYP154C8, contains a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 62) with QE in the I-helix of the polypeptide chain of the mutant enzyme.

32. The mutant cytochrome P450 enzyme according to claim 28, wherein when the mutant cytochrome P450 enzyme is derived from wild-type P450t, the mutant cytochrome P450 enzyme, compared with the wild-type P450t, contains a substitution of ET (corresponding to amino acids 244 and 245 of SEQ ID NO: 65) with QE in the I-helix of the polypeptide chain of the mutant enzyme.

33. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP267 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP267, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and threonine (ET) amino acid residues, or glutamic acid and alanine (EA) amino acid residues, are replaced with glutamine and glutamic acid (QE) amino acid residues. (ii) Glutamic acid, threonine and threonine (ETT) amino acid residues, or glutamic acid, alanine and threonine (EAT) amino acid residues, replaced with glutamine, glutamic acid and proline (QEP) amino acid residues. (iii) Glutamic acid, threonine, threonine and valine (ETTV) amino acid residues, or glutamic acid, alanine, threonine and valine (EATV) amino acid residues, replaced by glutamine, glutamic acid, proline and glycine (QEPG) amino acid residues. (iv) Alanine, glycine, histidine, glutamic acid, and threonine (AGHET) amino acid residues, or alanine, glycine, histidine, glutamic acid, and alanine (AGHEA) amino acid residues, substituted with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, histidine, glutamic acid, threonine, threonine and valine (AGHETTV) amino acid residues (or equivalent amino acid residues), or alanine, glycine, histidine, glutamic acid, alanine, threonine and valine (AGHEATV) amino acid residues (or equivalent amino acid residues), substituted with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues or glycine, alanine, histidine, glutamine, glutamic acid, proline and glycine (GAHQEPG) amino acid residues.

34. The mutant cytochrome P450 enzyme according to claim 33, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP267B1, the mutant cytochrome P450 enzyme, compared with the I-helix of wild-type CYP267B1, contains a substitution of ET (corresponding to amino acids 246 and 247 of SEQ ID NO: 59) with QE, or a substitution of AGHETTV (corresponding to amino acids 243 to 249 of SEQ ID NO: 59) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

35. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP102 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP102, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and serine (ETTS) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) Alanine, glycine, histidine, glutamic acid, and threonine (AGHET) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, histidine, glutamic acid, threonine, threonine and serine (AGHETTS) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

36. The mutant cytochrome P450 enzyme according to claim 35, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP102A1, the mutant cytochrome P450 enzyme, compared with the I-helix of wild-type CYP102A1, contains a substitution of ET (corresponding to amino acids 268 and 269 of SEQ ID NO: 52) with QE, or a substitution of AGHETTS (corresponding to amino acids 265 to 271 of SEQ ID NO: 52) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

37. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP175 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP175, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) Glutamic acid, threonine and valine (ETV) amino acid residues are replaced with glutamine, glutamic acid and proline (QEP) amino acid residues; (iii) The amino acid residues of glutamic acid, threonine, valine and alanine (ETVA) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) Alanine, glycine, histidine, glutamic acid, and threonine (AGHET) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, histidine, glutamic acid, threonine, valine and alanine (AGHETVA) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

38. The mutant cytochrome P450 enzyme according to claim 37, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP175A1, the mutant cytochrome P450 enzyme, compared with the wild-type CYP175A1, contains a substitution of AGHETVA (corresponding to amino acids 221 to 227 of SEQ ID NO: 55) with GALQEPG in the I-helix of the polypeptide chain of the mutant enzyme.

39. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP199 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP199, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Aspartic acid and threonine (DT) amino acid residues are replaced with glutamine and glutamic acid (QE) amino acid residues; (ii) The amino acid residues of aspartic acid, threonine and threonine (DTT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of aspartic acid, threonine, threonine and valine (DTTV) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) Alanine, glycine, leucine, aspartic acid, and threonine (AGLDT) are replaced with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, leucine, aspartic acid, threonine, threonine and valine (AGLDTTV) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

40. The mutant cytochrome P450 enzyme according to claim 39, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP199A4, the mutant cytochrome P450 enzyme, compared with the I-helix of wild-type CYP199A4, contains a substitution of DT (corresponding to amino acids 252 and 253 of SEQ ID NO: 60) with QE, a substitution of AGLDT (corresponding to amino acids 249 to 253 of SEQ ID NO: 60) with GALQE, or a substitution of AGLDTTV (corresponding to amino acids 249 to 255 of SEQ ID NO: 60) with GALQEPG.

41. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP231 family, the mutant cytochrome P450 enzyme, compared with the I-helix of wild-type CYP231, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and threonine (ETTT) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) The amino acid residues of glycine, glycine, asparagine, glutamic acid, and threonine (GGNET) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) The amino acid residues (or equivalent amino acid residues) of glycine, glycine, asparagine, glutamic acid, threonine, threonine and threonine (GGNETTT) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG).

42. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP107 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP107, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and alanine (EA) amino acid residues, or glutamic acid and threonine (ET) amino acid residues, are replaced with glutamine and glutamic acid (QE) amino acid residues. (ii) Glutamic acid, alanine and serine (EAS) amino acid residues, or glutamic acid, alanine and threonine (EAT) amino acid residues, or glutamic acid, threonine and threonine (ETT) amino acid residues, replaced with glutamine, glutamic acid and proline (QEP) amino acid residues. (iii) Glutamic acid, alanine, serine and valine (EASV) amino acid residues, or glutamic acid, alanine, threonine and valine (EATV) amino acid residues, or glutamic acid, threonine, threonine and valine (ETTV) amino acid residues, replaced by glutamine, glutamic acid, proline and glycine (QEPG) amino acid residues. (iv) Alanine, glycine, phenylalanine, glutamic acid, and alanine (AGFEA) amino acid residues, or alanine, glycine, histidine, glutamic acid, and alanine (AGHEA) amino acid residues, or alanine, glycine, histidine, glutamic acid, and threonine (AGHET) amino acid residues, substituted with glycine, alanine, leucine, glutamine, and glutamic acid (GALQE) amino acid residues; or (v) Alanine, glycine, phenylalanine, glutamic acid, alanine, serine and valine (AGFEASV) amino acid residues (or equivalent amino acid residues), or alanine, glycine, histidine, glutamic acid, alanine, threonine and valine (AGHEATV) amino acid residues (or equivalent amino acid residues), or alanine, glycine, histidine, glutamic acid, threonine, threonine and valine (AGHETTV) amino acid residues (or equivalent amino acid residues), substituted with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

43. The mutant cytochrome P450 enzyme according to claim 42, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP107PQ (P450h), the mutant cytochrome P450 enzyme, compared with the wild-type CYP107PQ (P450h), contains a substitution of EA (corresponding to amino acids 247 and 248 of SEQ ID NO: 40) with QE in the I-helix of the polypeptide chain of the mutant enzyme.

44. The mutant cytochrome P450 enzyme according to claim 42, wherein when the mutant cytochrome P450 enzyme is derived from wild-type CYP107Mg, the mutant cytochrome P450 enzyme, compared with the wild-type CYP107Mg, contains a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 66) with QE in the I-helix of the polypeptide chain of the mutant enzyme.

45. The mutant cytochrome P450 enzyme of claim 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme that is a member of the CYP116 family, the mutant cytochrome P450 enzyme, compared to the I-helix of wild-type CYP116, contains the following substitutions in the I-helix of the mutant enzyme's polypeptide chain: (i) Glutamic acid and threonine (ET) amino acid residues are replaced with glutamine and glutamate (QE) amino acid residues; (ii) The amino acid residues of glutamic acid, threonine and threonine (ETT) are replaced with amino acid residues of glutamine, glutamic acid and proline (QEP); (iii) The amino acid residues of glutamic acid, threonine, threonine and valine (ETTV) are replaced with amino acid residues of glutamine, glutamic acid, proline and glycine (QEPG); (iv) The amino acid residues of alanine, alanine, histidine, glutamic acid, and threonine (AAHET) are replaced with amino acid residues of glycine, alanine, leucine, glutamine, and glutamic acid (GALQE); or (v) Alanine, alanine, histidine, glutamic acid, threonine, threonine and valine (AAHETTV) amino acid residues (or equivalent amino acid residues) are replaced with glycine, alanine, leucine, glutamine, glutamic acid, proline and glycine (GALQEPG) amino acid residues.

46. ​​Use of the mutant cytochrome P450 enzyme according to any one of claims 1 to 45 in the biocatalytic oxidation of carbon-hydrogen bonds via the peroxygenase pathway.

47. The use according to claim 46, wherein the biocatalytic oxidation of the carbon-hydrogen bond is carried out at 1.0°C to 99°C.

48. A method for oxidizing an organic compound substrate, comprising oxidizing the organic compound substrate with a mutant cytochrome P450 enzyme according to any one of claims 1 to 45.

49. The method of claim 48, further comprising adding a peroxide to the oxidation.

50. The method of claim 49, wherein the peroxide is selected from the group consisting of hydrogen peroxide, tert-butyl hydroperoxide, and m-chloroperoxybenzoic acid.

51. The method of claim 50, wherein the peroxide is hydrogen peroxide.

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