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 processes for oxidation of organic compound substrates using such enzymes. More specifically, provided herein are mutations in cytochrome P450 enzymes that enhance their peroxygenase activity and / or alter product selectivity compared to wild-type cytochrome P450 enzymes. Uses for such mutant cytochrome P450 enzymes are also provided, such as for biocatalytic oxidation of carbon-hydrogen bonds via a peroxygenase pathway, or for oxidizing a substrate which is an organic compound.

Description

MUTANT CYTOCHROME P450 ENZYMES WITH ENHANCED PEROXYGENASE ACTIVITY AND / OR ALTERED PRODUCT SELECTIVITYPRIORITY CLAIM

[0001] This application claims priority from Australian provisional patent application number 2023902798 filed on 31 August 2023, the contents of which are to be taken as incorporated herein by this reference.FIELD OF THE INVENTION

[0002] The present invention relates generally to mutant enzymes with enhanced properties and processes for oxidation of organic compound substrates using such enzymes.BACKGROUND OF THE INVENTION

[0003] Biocatalysts offer significant advantages over traditional homogeneous and heterogeneous inorganic catalytic systems, displaying biodegradability and low toxicity along with high specificity, turnover numbers and activity under moderate conditions. Accordingly, their application to industrial processes is being intensely pursued. However, a significant drawback of the majority of biocatalysts is their intrinsic fragility under typical commercial operating conditions e.g. high temperatures, organic solvents or extreme pH. Overcoming this challenge is an important step towards their widespread application and thus the advancement of ‘green’ and efficient industrial chemical processes.

[0004] Cytochromes P450 (P450s, CYPs) are a family of haem enzymes that carry out oxidative transformation reactions that are important in a range of settings, including xenobiotic detoxification, carcinogen activation, steroid biosynthesis, fatty acid metabolism, and reactions required for the survival of microorganisms on selected nutrients. Most commonly, cytochrome P450 enzymes catalyse the regio- and enantio-specific hydroxylation of carbon-hydrogen bonds and epoxidation reactions, but other more complex reactions are also supported. Cytochrome P450 enzymes have become a globally pursued target for biocatalysis as they catalyse a broad range of reactions not readily achievable by traditional synthetic approaches. They are potentially ideal biocatalysts for enabling new enzyme catalysed chemistry, offering advantages over synthetic methods for selective carbon-hydrogen bond hydroxylation under mild conditions.

[0005] However, while progress has been made on structural characterisation of certainthermostable P450 enzymes, such as the CYP119A1 (Sulfolobus acidocaldarius), CYP175A1 (Thermos thermophilus) and CYP231A2 (Picrophilus torridus) enzymes, the broader application of these as biocatalysts has stalled. Their low activity with available electron transfer partner proteins and surrogate oxygen donors, and the expense of required cofactors (NAD(P)H) limit their utility. While a few niche examples of the industrial application of P450 enzymes are known, their low stability and the requirement for electron transfer proteins and complex and expensive cofactors hampers the use of the majority of these enzymes as industrial biocatalysts.

[0006] There is therefore an ongoing need for improved cytochrome P450 enzymes, which can be used to catalyse selective oxidation reactions in complex organic molecules, to at least partially address one or more of the above-mentioned short-comings, or provide a useful alternative.

[0007] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.SUMMARY OF THE INVENTION

[0008] The present invention is predicated, in part, on the identification of mutations in cytochrome P450 enzymes that enhance peroxygenase activity and / or alter product selectivity compared to wild-type cytochrome P450 enzymes.

[0009] Accordingly, in a first aspect the present invention provides a mutant cytochrome P450 enzyme with enhanced peroxygenase activity and / or altered product selectivity, wherein the mutant cytochrome P450 enzyme comprises a substitution of at least two consecutive amino acid residues in the l-helix of the polypeptide chain of the enzyme compared to the l-helix of a wild-type cytochrome P450 enzyme, wherein the substitution is at a position corresponding to amino acid residues 23 and 24 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine and Glutamic acid (QE) amino acid residues at said position in the l-helix of the polypeptide chain of the enzyme.

[0010] In some embodiments, the mutant cytochrome P450 enzyme is derived from bacterial or archaeal species. In some embodiments, the mutant cytochrome P450 enzymeis 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 a wildtype cytochrome P450 enzyme comprising 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 a wildtype cytochrome P450 enzyme comprising an amino acid sequence which has at least about 40% amino acid sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67, and which comprises the amino acid consensus sequence X1X2X2X3X4X5X6 (SEQ ID NO: 32) in the l-helix of the polypeptide chain, wherein:Xi is Alanine (A) or Glycine (G);X2is Glycine (G) or Alanine (A);X3 is Histidine (H), Asparagine (N), Leucine (L), Alanine (A), Threonine (T), Isoleucine (I), or Phenylalanine (F);X4is Glutamic acid (E), Glycine (G), Leucine (L), or Aspartic acid (D);X5is Threonine (T), Alanine (A), or Asparagine (N);X6is Threonine (T), Isoleucine (I), Valine (V), Serine (S), or Alanine (A); andX7 is Threonine (T), Valine (V), Alanine (A), Tryptophan (W), Arginine (R), Serine (S), and Isoleucine (I).

[0013] In some embodiments, the mutant cytochrome P450 enzyme comprises a substitution of an amino acid residue immediately after the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. In some embodiments, the amino acid residue immediately after the Glutamine and Glutamic acid (QE) amino acid residues is substituted to a Proline (P) amino acid residue. In some embodiments, the mutant cytochrome P450 comprises a substitution of three consecutive amino acid residues at a position corresponding to amino acid residues 23 to 25 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine, Glutamic acid and Proline (QEP) amino acid residues at said position 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.

[0014] In some embodiments, the mutant cytochrome P450 enzyme comprises a substitution of two consecutive amino acid residues immediately after the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. In some embodiments, the two consecutive amino acid residues immediately after the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Proline and Glycine (PG) amino acid residues. In some embodiments, the mutant cytochrome P450 comprises a substitution of four consecutive amino acid residues at a position corresponding to amino acid residues 23 to 26 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues at said position in the l-helix of the polypeptide chain of the enzyme.

[0015] In some embodiments, the mutant cytochrome P450 enzyme comprises a substitution of three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. In some embodiments, the three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Glycine, Alanine and X (GAX) amino acid residues, wherein X represents any amino acid. In some embodiments, the three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Glycine, Alanine and Leucine (GAL) or Glycine, Alanine and Histidine (GAH). In some embodiments, the mutant cytochrome P450 comprises a substitution of five consecutive amino acid residues at a position 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 position in the l-helix of the polypeptide chain of the enzyme compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

[0016] In some embodiments, the mutant cytochrome P450 enzyme comprises a substitution of a seven consecutive amino acid section encompassing the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. In some embodiments, the seven consecutive amino acid section is substituted to Glycine, Alanine, X, Glutamine, Glutamic acid, Proline and Glycine (GAXQEPG) amino acid residues, wherein X represents any amino acid. In some embodiments, the mutant cytochrome P450 comprises a substitution of seven consecutiveamino acid residues at a position corresponding to amino acid residues 20 to 26 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises 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 said position in the l-helix of the polypeptide chain of the enzyme 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 a wildtype cytochrome P450 enzyme which is a member of a CYP family selected from the group consisting of the CYP119 family, the CYP231 family, the CYP175 family, the CYP199 family, the CYP154 family, the CYP102 family, the CYP107 family, the CYP109 family, the CYP116 family, and the CYP267 family.

[0018] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP119 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Threonine (ETTT) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Asparagine, Glutamic acid, and Threonine (AGNET) amino acid residues 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP119.

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

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

[0021] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP109 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues, or Glutamic acid and Alanine (EA) amino acid residues, 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, with 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, with 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) amino acid residues, or Alanine, Glycine, Asparagine, Glutamic acid, and Threonine (AGNET) amino acid residues, with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; 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), with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues,in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP109.

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

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

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

[0025] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP154 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Isoleucine (ETTI) amino acid residues with Glutamine, Glutamic acid, Proline and glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) 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) 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,in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP154.

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

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

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

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

[0030] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP267 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues, or Glutamic acid and Alanine (EA) amino acid residues, 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, 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, with 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, 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), 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, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP267.

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

[0032] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP102 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Serine (ETTS) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP102.

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

[0034] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP175 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Valine (ETV) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Valine and Alanine (ETVA) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP175.

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

[0036] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP199 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Aspartic acid and Threonine (DT) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Aspartic acid, Threonine, and Threonine (DTT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Aspartic acid, Threonine, Threonine and Valine (DTTV) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Leucine, Aspartic acid, and Threonine (AGLDT) 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP199.

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

[0038] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP231 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Threonine (ETTT) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Glycine, Glycine, Asparagine, Glutamic acid, and Threonine (GGNET) amino acid residues with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; or(v) Glycine, Glycine, Asparagine, Glutamic acid, Threonine, Threonine and Threonine (GGNETTT) amino acid residues (or equivalent amino acid residues) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP231 .

[0039] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP107 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Alanine (EA) amino acid residues, or Glutamic acid and Threonine (ET) amino acid residues, 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, 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, with 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, 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), with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues;in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP107.

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

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

[0042] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP116 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Valine (ETTV) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Alanine, Histidine, Glutamic acid, and Threonine (AAHET) amino acid residues with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; or(v) Alanine, Alanine, Histidine, Glutamic acid, Threonine, Threonine and Valine (AAHETTV) amino acid residues (or equivalent amino acid residues) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wildtype CYP1 16.

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

[0044] In some embodiments of the second aspect of the invention, the biocatalytic oxidation of carbon-hydrogen bonds takes place at 1 ,0°C to 99°C.

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

[0046] In some embodiments of the third aspect of the invention, the process further comprises adding a peroxide to the oxidation. In some embodiments, the peroxide is selected from the group consisting of hydrogen peroxide, t-butyl hydroperoxide and m- chloroperbenzoic acid. In some embodiments, the peroxide is hydrogen peroxide.

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

[0048] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure. Any example / embodiment of the present disclosure herein shall be taken to apply mutatis mutandis to any other example / embodiment of the disclosure unless specifically stated otherwise.BRIEF DESCRIPTION OF THE FIGURES

[0049] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description, taken in conjunction with the accompanying figures which illustrate certain embodiments of the present invention.

[0050] FIGURE 1 - 3-dimensional representations of cytochrome P450 enzyme structure. (A) ribbon presentation showing that the fold of cytochrome P450s is highly conserved. The I a-helix is labelled and highlighted in black. The oxygen binding groove located within this helix is also labelled. (B) illustration comparing the active site structures of CYP255A2 and CYP119A1 highlighting the oxygen binding groove of the l-helix.

[0051] FIGURE 2 - an amino acid sequence alignment of the l-helix region of selected wildtype cytochrome P450 enzyme sequences surrounding the two consecutive amino acid residues that are substituted in the mutant cytochrome P450 enzymes of the present invention. Previously studied P450s are given by their name e.g. CYP101A1 . UnclassifiedP450 sequences from other extremophiles are labeled P450a through to P450r. The two consecutive amino acid residues that are substituted are highlighted by the shaded region. Note CYP107A1 and CYP176A1 do not have a conserved acid-alcohol pair, EA and DN, respectively, which are to be substituted. P450c, P450h and P450i also do not contain an acid-alcohol pair, GT, EA and LT, respectively, which are to be substituted. The region of the l-helix of interest to this application is highlighted by the narrower solid box. The sequence of the entire l-helix is within the larger dashed box (based on the l-helix sequence of CYP101A1 , P450cam). The top sequence (SEQ ID NO: 1) is the consensus sequence for this alignment (the most common amino acid at each position).

[0052] FIGURE 3 - a synthetic scheme showing the metabolites generated from the oxidation of fatty acids by the GALQEPG mutant of CYP1 19A1 .

[0053] FIGURE 4 - a gas chromatogram (GC-MS) showing the analysis of the oxidation of a fatty acid (dodecanoic / lauric acid; 1 mM) by wild-type CYP119A1 (WT) and the CYP119A1 GALQEPG mutant (mutant), 3 pM at room temperature (RT) and 80 °C, 2 hours, using hydrogen peroxide (50 mM).

[0054] FIGURE 5 - a gas chromatogram (GC-MS) showing the analysis of the oxidation of dodecanoic (lauric) acid by the GALQEPG mutant of CYP119A1 (3 pM) at room temperature (RT) and 50 °C, 70 °C and 90 °C using hydrogen peroxide (50 mM).

[0055] FIGURE 6 - a synthetic scheme showing the oxidation of progesterone and androstenedione by a CYP154 GAHQEPG mutant using hydrogen peroxide.

[0056] FIGURE 7 - an HPLC chromatogram showing the analysis of the stereoselective oxidation of progesterone 1 mM by WT and the GAHQEPG mutant of CYP154C8 (1 pM) at room temperature (RT) and using 20 mM hydrogen peroxide.

[0057] FIGURE 8 - an HPLC chromatogram showing the analysis of the stereoselective oxidation of progesterone (1 mM) by the GAHQEPG mutant of CYP154C8 (1 pM) at room temperature (RT) and using 2 - 40 mM hydrogen peroxide.

[0058] FIGURE 9 - an HPLC chromatogram showing the stereoselective oxidation of progesterone substrate (1 mM) by various CYP154C8 mutants (T258E, QE andGAHQEPG) (1 pM) compared to wildtype (WT) CYP154C8 enzyme, and using 5 mM hydrogen peroxide.

[0059] FIGURE 10 - an HPLC chromatogram showing the stereoselective oxidation of progesterone substrate (2 mM) by the CYP154C8 QE mutant (1 pM) using 5 mM hydrogen peroxide and varying concentrations of DMSO as substrate solvent (15%, 20%, 25% or 30% v / v).

[0060] FIGURE 11 - an HPLC chromatogram showing the stereoselective oxidation of progesterone substrate (1 mM) by the CYP154C8 QE mutant (1 pM) using 5% DMSO as substrate solvent and hydrogen peroxide at varying concentrations (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 pM) by a P450t QE mutant (2 pM) using 10 mM hydrogen peroxide. P450t is a member of the CYP154C subfamily. In each HPLC chromatogram, the substrate is labelled “1 ”, the internal standard (9- hydroxyfluorene) is labelled “2”, and the product of oxidation (16a-hydroxy steroid) is labelled “3”.

[0062] FIGURE 13 - line graphs showing the increase in concentration of 4-hydroxybenzoic acid product over time comparing: (A) the P450 CYP199A4 wild-type (WT) enzyme; and (B) the P450 CYP199A4 mutant (GALQE) enzyme during time course reactions for 3 pM enzyme with 4-methoxybenzoic acid, driven by 5 mM H2O2.

[0063] FIGURE 14 - line graphs showing (A) the increase in concentration of 4- hydroxybenzoic acid over time course reactions for 3 pM P450 CYP199A4 (QE and T252E mutants) with 4-methoxybenzoic acid (1 mM), driven by 10 mM H2O2. (B) HPLC analysis of the reactions.

[0064] FIGURE 15 - line graphs showing (A) the increase in concentration of 4- hydroxybenzoic acid over time course reactions for 3 pM P450 CYP199A4 (QE mutant and WT) with 4-methoxybenzoic acid (1-5 mM), driven by 10 mM H2O2. (B) HPLC analysis of the 45-hour reactions.

[0065] FIGURE 16 - a GC-MS chromatogram showing the analysis of the 2-hour reaction of the HazakQE mutant (2 pM) (the CYP102 enzyme from Thermosporothrix hazakensis) with 250 pM myristic acid driven by 5 mM H2O2at 30 °C in Tris-HCI buffer (50 mM, pH 7.4).

[0066] FIGURE 17 - a graph and HPLC chromatograms showing analysis of the stability of a QE mutant of the thermophilic P450 enzyme from Meiothermus ruber (P450h in Figure 2) (also known as CYP107PQ1) with respect to selective oxidation of p-ionone substrate to 4- hydroxy-p-ionone. (A) A line graph showing catalytic activity of the mutant enzyme at various reaction temperatures. (B) A HPLC chromatogram showing catalytic activity of the mutant enzyme after heat treatment of the mutant enzyme prior to the oxidation reaction. (C) A HPLC chromatogram showing catalytic activity of the mutant enzyme after storage of the mutant enzyme at 30 °C for set periods of time prior to the oxidation reaction.

[0067] FIGURE 18 - UV-vis absorbance spectroscopy of the Soret band region of the heme of a P450h QE (CYP107PQ1 QE) mutant enzyme in the presence (A) or absence (B) of p- ionone substrate. Note: the changes in the spectrum in the presence of substrate are due to oxidation of p-ionone which has a low but significant absorption in the measured wavelength region.

[0068] FIGURE 19 - HPLC chromatograms demonstrating oxidation superiority of a P450h QE (CYP107PQ1 QE) mutant enzyme compared to wild type enzyme (A) and demonstrating high enantioselectivity of the P450h QE (CYP107PQ1 QE) mutant enzyme (B).

[0069] FIGURE 20 - HPLC chromatograms and a line graph demonstrating the stability of a P450h QE (CYP107PQ1 QE) mutant enzyme to different types and concentrations of organic solvents in the oxidation of p-ionone. (A) HPLC chromatogram showing 4-hydroxy- p-ionone product yield using the organic solvents acetonitrile, DMSO, ethanol and isopropanol; (B) line graph showing relative 4-hydroxy-p-ionone product yield using various concentrations of DMSO or isopropanol; (C) HPLC chromatogram showing 4-hydroxy-p- ionone product yield using varying concentrations of DMSO; and (D) HPLC chromatogram showing 4-hydroxy-p-ionone product yield using varying concentrations of isopropanol.

[0070] FIGURE 21 - line graphs demonstrating the stability of a CYP109E1 QE (A), a CYP109B1 QE (B), and a P450h QE (CYP107PQ1QE) (C) mutant enzyme to heat treatment for 1 hour at varying temperatures prior to oxidation of p-ionone substrate.

[0071] FIGURE 22 - a HPLC chromatogram demonstrating oxidation superiority of a CYP107MgQE) mutant enzyme compared to wild type enzyme.DETAILED DESCRIPTION OF THE INVENTION

[0072] Nucleotide sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of the sequence identifiers is provided in Table 1. A sequence listing has also been provided at the time of filing this application.TABLE 1Summary of Sequence Identifiers

[0073] As set out above, the present invention arises from the structural characterisation and optimisation of the catalytic activity of newly developed (mutant) thermostable and mesophilic cytochrome P450 enzymes using protein engineering of equivalent wild-type P450 proteins. These mutant P450 enzymes have been modified to only require peroxides, such as hydrogen peroxide (H2O2), as an oxidant thereby providing thermostable P450 peroxygenase enzymes for biocatalytic hydroxylation reactions with significantly enhanced activity compared to the wild-type form of the enzyme.

[0074] Accordingly, certain disclosed embodiments provide mutant cytochrome P450 enzymes, and uses and processes with respect to these enzymes, that have one or more advantages. For example, some of the advantages of some embodiments disclosed herein include one or more of the following: mutant cytochrome P450 enzymes with enhanced peroxygenase activity; mutant cytochrome P450 enzymes with enhanced stability that do not require additional protein partners or expensive nicotinamide cofactors; mutant cytochrome P450 enzymes with altered product selectivity; mutant cytochrome P450 enzymes comprising amino acid substitutions in the l-helix which impart these advantageous properties; use of said mutant cytochrome P450 enzymes for improved biocatalytic oxidation of carbon-hydrogen bonds; improved processes for oxidising organic substrates; or the provision of a commercial alternative to existing cytochrome P450 enzymes. Other advantages of some embodiments of the present disclosure are provided herein.

[0075] In one aspect, the present invention provides a mutant cytochrome P450 enzyme with enhanced peroxygenase activity and / or altered product selectivity, wherein the mutantcytochrome P450 enzyme comprises a substitution of at least two consecutive amino acid residues in the l-helix of the polypeptide chain of the enzyme compared to the l-helix of a wild-type cytochrome P450 enzyme, wherein the substitution is at a position corresponding to amino acid residues 23 and 24 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine and Glutamic acid (QE) amino acid residues at said position in the l-helix of the polypeptide chain of the enzyme. As would be understood by a person skilled in the art, the term “peroxygenase activity” as used herein refers to the ability of the mutant cytochrome P450 enzymes encompassed herein to catalyse the following reaction:RH + H2O2-► ROH + H2O

[0076] As used herein, reference to a cytochrome P450 enzyme also encompasses reference to a cytochrome P450 protein, a P450 protein, P450, CYP, CYP450, or any other synonym as would be understood by those skilled in the art. Cytochrome P450 proteins, named forthe absorption band at 450 nm of their carbon-monoxide-bound ferrous form, are one of the largest superfamilies of enzyme proteins and can be found in the genomes of virtually all organisms. The cytochrome P450 superfamily are unusual among proteins and enzymes in that they have primary structures with low homology, despite having a highly conserved tertiary structure. The structure of all P450 proteins determined to date show a characteristic topography incorporating a helix-rich domain packed against a mainly p strand domain (Poulos TL et al., 1987, J. Mol. Biol., 195: 687-700). The helices are termed A-L, and the p strands p1-p5, with the overall topography now being known as the “P450 fold”. Of the secondary structural elements, the B and B' helices, the BC loop, the F and G helices and the FG loop on the distal side of the haem form the substrate binding pocket. Sequence alignments readily identify 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, and it is this that gives rise to the myriad specificity, activity and selectivity patterns of P450 catalysis.

[0077] P450 enzymes share a common overall fold and topology. As shown in Figure 1A, the conserved P450 structural core is formed by a four-helix bundle composed of three parallel helices labeled D, L, and I and one antiparallel helix E. The prosthetic heme group is confined between the distal I helix and proximal L helix and bound to the adjacent Cys-heme-ligand loop. The long I helix forms a wall of the heme pocket and contains the signature amino acid sequence (A / G)GX(E / D)T (“X” being any amino acid) which iscentered at a kink in the middle of the helix (the oxygen binding groove). The highly conserved threonine preceded by an acidic residue is positioned in the active site and is involved in catalysis.

[0078] At the molecular level, the amino acids constituting the l-helix of any particular or potential P450 enzyme can be determined with reference to the l-helix amino acid sequence of a reference P450 enzyme, for example CYP101 A1 (P450cam) as shown in Figure 2. In this regard, once the amino acid sequence of a particular or potential P450 enzyme has been obtained, sequence alignment tools can be used to compare the sequence to a reference P450 enzyme sequence. Optimal alignment of sequences may be conducted by computerized implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al., 1997, Nucl. Acids Res., 25: 3389-3402. Global alignment programs may also be used to align sequences of roughly 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 package (Rice P 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 package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). These programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can be found in Unit 19. 3 of Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15,1998).

[0079] Figure 2 shows an amino acid sequence alignment of a number of P450 enzymes in the region of these proteins which includes the l-helix. The sequence of the l-helix is represented by the dashed box in Figure 2 based on the l-helix of CYP101A1 (P450cam) (Poulos TL et al., 1987, J. Mol. Biol., 195(3): 687-700). With respect to CYP101A1 , the I- helix is represented by amino acid residues 234 (I) to 269 (P) of the CYP101A1 sequence set forth in SEQ ID NO: 51 . It is to be noted that the amino acid numbering system for the cytochrome P450 enzymes adopted herein is based on inclusion of the methionine residue of the start codon.

[0080] Details of the P450 enzymes in Figure 2, particularly with respect to nucleotide and amino acid sequence information, may be accessed from the GenBank database at the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov), or from theUniProtKB / Swiss-Prot database (www.uniprot.org). Particular details are provided below in Table 2. It is to be noted that the P450 enzymes included in Figure 2 and Table 2 are merely a representative list and are by no means exhaustive.TABLE 2Representative P450 Enzymes

[0081] As indicated above, the mutant cytochrome P450 enzymes of the present invention comprise a substitution of at least two consecutive amino acid residues in the l-helix of the polypeptide chain of the enzyme compared to the l-helix of a wild-type cytochrome P450 enzyme. In this regard, SEQ ID NO: 1 has been provided herein as a wild-type reference (consensus) sequence to highlight the position of the amino acid substitutions present in the mutant cytochrome P450 enzyme. SEQ ID NO: 1 is a consensus sequence of the most common amino acid at each position in the l-helix region of the wild-type cytochrome P450 enzyme sequences shown in Figure 2 (SEQ ID NOs: 2 to 31). For example, with respect to wild-type CYP101 A1 , the at least two consecutive amino acid residues are represented by an acid-alcohol pair of amino acid residues, namely amino acid residues 22 (D) and 23 (T) of the l-helix region of the sequence set forth in SEQ ID NO: 20, or amino acid residues 252 (D) and 253 (T) of the full-length CYP101A1 sequence set forth in SEQ ID NO: 51. Another example is with respect 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) of the l-helix region of the sequence set forth in SEQ ID NO: 2, or amino acid residues 220 (E) and 221 (T) of the full-length P450a sequence set forth in SEQ ID NO: 33.

[0082] For the P450 enzyme sequence alignments shown in Figure 2, the at least two consecutive amino acid residues of the wild-type enzyme that are substituted in the mutant cytochrome P450 enzyme are highlighted by the shaded region. In some embodiments, the at least two consecutive amino acid residues of the wild-type enzyme that are substituted in the mutant enzyme may be an acid-alcohol pair of amino acid residues represented by a pair of (i) Glutamic acid and Threonine (ET) amino acid residues (for example in P450a, P450b, P450d, P450e, P450f, P450g, P450j, P450I, P450m, P450n, P450p, P450q, P450r,CYP102A1 , CYP119A1 , CYP119A2, CYP175A1 , CYP109C1 , CYP267B1 , CYP231 A2 and CYP154C8); (ii) a pair of Aspartic acid and Threonine (DT) amino acid residues (for example in P450k, P450o, CYP101A1 , and CYP199A4); (iii) a pair of Glutamic acid and Serine (ES) amino acid residues; or (iv) a pair of Aspartic acid and Serine (DS) amino acid residues.

[0083] In some embodiments, the at least two consecutive amino acid residues of the wildtype enzyme that are substituted in the mutant enzyme are not an acid-alcohol pair of amino acid residues. In this regard, the at least two consecutive amino acid residues may be selected from the group consisting of: (i) a pair of Glycine and Threonine (GT) amino acid residues (for example in P450c); (ii) a pair of Glutamic acid and Alanine (EA) amino acid residues (for example in P450h and CYP107A1); (iii) a pair of Leucine and Threonine (LT) amino acid residues (for example in P450i); (iv) a pair of Aspartic acid and Asparagine (DN) amino acid residues (for example in CYP176A1); (v) a pair of Histidine and Threonine (HT) amino acid residues; (vi) a pair of Glutamic acid and Asparagine (EN) amino acid residues; (vii) a pair of Glutamic acid and Isoleucine (El) amino acid residues; (viii) a pair of Valine and Threonine (VT) amino acid residues; (ix) a pair of Alanine and Threonine (AT) amino acid residues; (x) a pair of Phenylalanine and Threonine (FT) amino acid residues; (xi) a pair of Isoleucine and Threonine (IT) amino acid residues; (xii) a pair of Lysine and Threonine (KT) amino acid residues; (xxiii) a pair of Arginine and Threonine (RT) amino acid residues; (xiv) a pair of Alanine and Asparagine (AN) amino acid residues; (xv) a pair of Glycine and Asparagine (GN) amino acid residues; (xvi) a pair of Isoleucine and Isoleucine (II) amino acid residues; (xvii) a pair of Asparagine and Proline (NP) amino acid residues; (xviii) a pair of Isoleucine and Serine (IS) amino acid residues; (xix) a pair of Leucine and Serine (LS) amino acid residues; (xx) a pair of Valine and Alanine (VA) amino acid residues; (xxi) a pair of Isoleucine and Alanine (IA) amino acid residues; (xxii) a pair of Leucine and Proline (LP) amino acid residues; (xxiii) a pair of Glutamine and Proline (QP) amino acid residues; (xxiv) a pair of Serine and Threonine (ST) amino acid residues; (xxv) a pair of Proline and Threonine (PT) amino acid residues; and (xxvi) a pair of Methionine and Alanine (MA) amino acid residues.

[0084] It has been shown herein that substitution of at least two consecutive amino acid residues in the l-helix of the polypeptide chain of a wild-type cytochrome P450 enzyme with a pair of Glutamine and Glutamic acid (QE) amino acid residues imparts enhanced peroxygenase activity and / or an altered product selectivity on the resultant mutant cytochrome P450 enzyme.

[0085] Mechanisms to produce the mutant P450 enzymes encompassed by the present invention would be known by a skilled addressee. For example, a nucleotide sequence encoding a relevant mutant P450 enzyme can be synthesized in silico. Synthetic production using high-throughput silicon platforms, such as those offered by Twist Bioscience (South San Francisco, CA, USA), allows rapid, reliable, and faithful reproduction of the nucleotide sequence of choice. Mutant P450 nucleotide sequences can also be synthesised 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 there is no requirement to obtain or have pre-existing DNA material, or rely on cloning P450 nucleotide sequences into suitable vectors.

[0086] In other techniques, the QE amino acid substitution (and other amino acid substitutions encompassed by the present invention) may be introduced by genetic modification of the equivalent wild-type P450 enzyme. In this regard, the mutant cytochrome P450 enzyme could be considered to be “derived” from a wild-type cytochrome P450 enzyme. In some embodiments, the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme comprising 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.

[0087] Exemplary types of genetic modification contemplated herein include site-directed mutagenesis of an endogenous wild-type P450 enzyme, PCR and gene shuffling methods, the use of multiple mutagenic oligonucleotides in cycles of site-directed mutagenesis, or the use of gene editing techniques. The genetic modification methods therefore produce one or more polynucleotides encoding one or more different P450 mutant enzymes having the desired amino acid substitutions in the l-helix,

[0088] Examples of gene editing techniques 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); transcription activator-like effector nuclease (TALEN) systems (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., 201 1 , 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); andclustered regularly interspaced short palindromic repeats (‘CRISPR’) based gene editing methods or equivalently adapted systems (see Belhaj K ef al., 2015, Current Opinion in Biotechnology, 32: 76-84; Shan Q et al., 2014, Nature Protocols, 9: 2395-2410; and Wang Y et al., 2014, Nat. Biotechnol., 32: 947-951).

[0089] Use of the gene editing technologies referred to above allow for effective introduction of exogenous DNA into the chromosome of a host cell and allows insertion of a mutation at a particular locus on a chromosome. For example, point mutations can be introduced into a P450 gene by knock-in, wherein said mutations encode the desired (e.g. QE) amino acid substitutions. In many circumstances, gene knock-in involves homologous recombination mechanisms. In this regard, gene editing techniques enable the creation of double-strand DNA breaks at desired locus sites. These controlled double-strand breaks can promote homologous recombination at such specific locus sites. This process relies on targeting specific sequences of nucleic acid molecules, with endonucleases that recognize and bind to such sequences and induce a double-strand break in the nucleic acid molecule.

[0090] A nuclease-mediated double-stranded DNA break in the genome can be repaired by two main mechanisms: Non-Homologous End Joining (NHEJ), which frequently results in the introduction of non-specific insertions and deletions (indels), or homology directed repair (HDR), which incorporates a homologous strand as a repair template. When a sequence-specific nuclease is delivered along with a homologous donor DNA construct containing a desired mutation, gene targeting efficiencies are increased by 1000-fold compared to just the donor construct alone.

[0091] Alternative methods have been developed to accelerate the process of genome modification by directly injecting DNA or mRNA of site-specific nucleases into a cell to generate a DNA double strand break (DSB) at a specified locus. DSBs induced by these site-specific nucleases can then be repaired by either error-prone non-homologous end joining (NHEJ) resulting, for example, in mutants carrying the desired substitution at the cut site. If a donor plasmid with homology to the ends flanking the DSB is co-injected, high- fidelity homologous recombination can produce P450 enzymes with targeted integrations.

[0092] The CRISPR type II system has been used to edit the genomes of a broad spectrum 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 \NY et al., 2013, Nat. Biotechnol., 31 (3): 227-229; Jiang Wet al., 2013, Nat. Biotechnol., 31 (3): 233-239; Jinek M et al., 2013, eLife,2: e00471 ; Cong L et al., 2013, supra). CRISPR is particularly customizable because the active form consists of an invariant Cas9 protein and an easily programmable single guide RNA (sgRNA). Of the various CRISPR orthologs, the Streptococcus pyogenes (Sp) CRISPR is the most well-characterized and widely used. The Cas9-gRNA complex first probes DNA for the protospacer-adjacent motif (PAM) sequence (-NGG for Sp Cas9), after which Watson-Crick base-pairing between the sgRNA and target DNA proceeds in a ratchet mechanism to form an R-loop. Following formation of a ternary complex of Cas9, sgRNA, and target DNA, the Cas9 protein generates two nicks in the target DNA, creating a blunt double-strand break (DSB) that is repaired by the non-homologous end joining (NHEJ) pathway or template-directed homologous recombination (HR). CRISPR methods are disclosed, for example, in US 8,697,359.

[0093] The aforementioned technologies to produce the mutant P450 enzymes encompassed by the present invention are merely representative and are not limiting to other mechanisms that may be employed.

[0094] Checking for, or determining successful integration of, the desired amino acid (e.g. QE) substitutions of the present invention following production of a mutant P450 enzyme can be achieved using allele-specific primers and probes. For example, with respect to checking for the QE amino acids substitution, PCR-based approaches can use oligonucleotide primers which specifically bind to the nucleotides encoding the QE amino acids (that is CAA or CAG codons for Q; and GAA or GAG codons for E). Such oligonucleotides which detect nucleotide variations in target sequences may be referred to by such terms as "allele-specific probes", or "allele-specific primers". The design and use of allele-specific probes for detecting known sequence variations is described in, for example, 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, a probe or primer may be designed to hybridize to a segment of target DNA that includes the specific nucleotides encoding the QE amino acids such that the actual coding nucleotides align with eitherthe 5' most end orthe 3' most end of the probe or primer. In some assays, the amplification may include a labeled primer, thereby allowing detection of the amplification product of that primer.

[0095] In one type of PCR-based assay, an allele-specific primer hybridizes to a segment of target DNA that overlaps with the site of the desired amino acid (e.g. QE) substitution encoding nucleotides and only primes amplification of an allelic form to which the primerexhibits perfect complementarity (Gibbs, 1989, Nucleic Acid Res. 17:2427-2448). Typically, the primer's 3'-most nucleotide is aligned with and complementary to one of the amino acid substitution encoding nucleotides being tested for. This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two primers, producing a detectable product that indicates which allelic form is present in the test sample. A control is usually performed with a second pair of primers, one of which shows a single base mismatch with one of the amino acid substitution encoding nucleotides and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification or substantially reduces amplification efficiency, so that either no detectable product is formed or it is formed in lower amounts or at a slower pace. The method generally works most effectively when the mismatch is at the 3'-most position of the oligonucleotide because this position is most destabilizing to elongation from the primer (see for example WO 93 / 22456).

[0096] In one example, a primer contains a sequence substantially complementary to a segment of DNA containing the amino acid substitution encoding nucleotides except that the primer has a mismatched nucleotide in one of the three nucleotide positions at the 3'- most end of the primer, such that the mismatched nucleotide does not base pair with a particular allele at the QE encoding nucleotide site. The mismatched nucleotide in the primer can be the first, second or the third nucleotide from the last nucleotide at the 3'-most position of the primer. In some examples, primers and / or probes are labeled with detectable labels.

[0097] In an alternative approach, tagged allele specific primer pairs can be used to detect one of the amino acid substitution encoding nucleotides (Strom et al., 2005, Genet. Med. 7:633-63). In one example, two tagged allele-specific primers overlap the amino acid substitution encoding nucleotides in the target DNA; however, only the correctly hybridized primer(s) will be extended to generate a labeled product(s). A non-complementary primer will not be extended or labeled due to the 3' mismatched base. The labeled extended product can be detected based on the detectable label. The tagged extended primers can also be captured on a solid support such as beads that are coupled to anti-tag sequences. The immobilized extended primer product can be detected by commercially available means such as Luminex 100 LabMAP™ (Luminex Corporation, Austin TX).

[0098] The most common approach to check for and / or detect the amino acid substitution encoding nucleotides encompassed by the present invention is amplification of a segmentof DNA comprising the nucleotides using PCR, and then isolating and sequencing (for example by Sanger sequencing) the PCR fragment that has been amplified. For example, with respect to the P450 enzymes listed in Figure 2, primers for the amplification procedure can be designed based on SEQ ID NOs: 2 to 31 (or indeed based on the equivalent full- length P450 enzyme sequences set forth in SEQ ID NOs: 33 to 62 or the P450 enzyme sequences set forth in SEQ ID NOs: 63 to 67). Procedures for designing primers, conducting PCR, isolating amplified fragments, and sequencing said fragments are known in the art, and are provided in standard texts such as Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.

[0099] The mutant P450 enzymes of the present invention may have substitutions in addition to the QE amino acids substitution referred to above. In some embodiments, the mutant cytochrome P450 enzyme comprises a substitution of an amino acid residue immediately after the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. With reference to the representative wild-type cytochrome P450 enzyme sequences shown in Figure 2, this would equate to a substitution of the Threonine (T), Isoleucine (I), Valine (V), Serine (S) or Alanine (A) amino acid residue, immediately following the QE substitution.

[0100] In some embodiments, the amino acid residue immediately after the Glutamine and Glutamic acid (QE) amino acid residues is substituted to a Proline (P) amino acid residue. Accordingly, in some embodiments the mutant cytochrome P450 comprises a substitution of three consecutive amino acid residues at a position corresponding to amino acid residues 23 to 25 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine, Glutamic acid and Proline (QEP) amino acid residues at said position 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. In this regard, the mutant cytochrome P450 enzyme would comprise a QEP amino acid substitution in the l-helix compared to a wild-type cytochrome P450 enzyme. With respect to a wild-type cytochrome P450 enzyme, the amino acid residues that may be substituted to 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.

[0101] In some embodiments, the mutant cytochrome P450 enzyme comprises a substitution of two consecutive amino acid residues immediately after the Glutamine andGlutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. With reference to Figure 2, this would equate to a substitution of the: 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), amino acid residues immediately following the QE substitution.

[0102] In some embodiments, the two consecutive amino acid residues immediately after the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Proline and Glycine (PG) amino acid residues. Accordingly, in some embodiments the mutant cytochrome P450 comprises a substitution of four consecutive amino acid residues at a position corresponding to amino acid residues 23 to 26 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues at said position in the l-helix of the polypeptide chain of the enzyme. In this regard, the mutant cytochrome P450 enzyme would comprise a QEPG amino acid substitution in the l-helix compared to a wild-type cytochrome P450 enzyme. With respect to a wild-type cytochrome P450 enzyme, the amino acid residues that may be substituted to 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.

[0103] In some embodiments, the mutant cytochrome P450 enzyme comprises a substitution of three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. With reference to Figure 2, this would equate to a substitution of the: 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), amino acid residues immediately before the QE substitution.

[0104] In some embodiments, the three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Glycine, Alanine and X (GAX) amino acid residues, wherein X represents any amino acid. In this regard, the mutant cytochrome P450 enzyme would comprise a GAXQE amino acid substitution in the l-helix compared to a wild-type cytochrome P450 enzyme. In some embodiments, the three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Glycine, Alanine and Leucine (GAL) or Glycine, Alanine and Histidine (GAH). In this regard, the mutant cytochrome P450 enzyme would comprise a GALQE or GAHQE amino acid substitution in the l-helix compared to a wild-type cytochrome P450 enzyme. Accordingly, in some embodiments the mutant cytochrome P450 comprises a substitution of five consecutive amino acid residues at a position 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 position in the l-helix of the polypeptide chain of the enzyme compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme. With respect to a wild-type cytochrome P450 enzyme, the amino acid residues that may be substituted to 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 acids 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.

[0105] In some embodiments, the mutant cytochrome P450 enzyme comprises a substitution of a seven consecutive amino section encompassing the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme. In some embodiments, this comprises the QE amino acid residue substitution, substitution of the three amino acid residues immediately before this pair, and substitution of the two amino acid residues immediately after this pair. With reference to Figure 2, this would equate to a substitution of the seven consecutive amino acids present in the solid box of the Figure.

[0106] In some embodiments, the seven consecutive amino acid section is substituted to Glycine, Alanine, X, Glutamine, Glutamic acid, Proline and Glycine (GAXQEPG) amino acid residues, wherein X represents any amino acid. In some embodiments, the seven amino acid section is substituted with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Prolineand Glycine (GALQEPG) amino acid residues or Glycine, Alanine, Histidine, Glutamine, Glutamic acid, Proline and Glycine (GAHQEPG) amino acid residues. Accordingly, in some embodiments the mutant cytochrome P450 comprises a substitution of seven consecutive amino acid residues at a position corresponding to amino acid residues 20 to 26 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises 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 said position in the l-helix of the polypeptide chain of the enzyme compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

[0107] The mutant cytochrome P450 enzymes of the present invention may have 1 , 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more other mutations in addition to the one or more mutations specified above, such as substitutions, insertions or deletions. These additional mutations may or may not enhance peroxygenase activity of the mutant P450 enzyme. The other mutations may be in the active site or outside the active site. For example, the mutations may be in the second sphere, i.e. residues which affect or contact the position or orientation of one or more of the amino acids in the active site. An insertion will typically be N and / or C terminal. Therefore, the enzyme may contain a short peptide of up to 20 amino acids or a full-length protein fused to either or both of the termini, e.g. to aid protein purification by affinity chromatography or immobilisation on a solid matrix. A deletion typically comprises the deletion of amino acids which are not involved in catalysis, such as those outside the active site (thus the enzyme is a mutated fragment of a naturally occurring enzyme). In some embodiments, an additional mutation may be in the form of a protein fusion between a portion of the P450 enzyme and another enzyme that generates peroxide in situ using other substrates (such as oxidase enzymes). These additional mutations are therefore encompassed by the present invention provided that the mutant enzyme maintains a substitution comprising at least the QE substitution referred to above.

[0108] Other mutations in the active site typically alter the position and / or conformation of the substrate when it is bound in the active site. The mutations may make the site on the substrate which is to be oxidized more accessible to the haem group. Therefore, the mutations may be substitutions to an amino acid which has a smaller or larger, or more or less polar, side chain.

[0109] Additional mutations can include amino acid residue changes that can increase the stability of the enzyme. These mutations typically prevent oligomerisation of the protein,e.g. dimerisation of P450cam (CYP101A1) has been removed by substitution of Cysteine (C), preferably to Alanine (A), at amino acid residue 335. Other mutations can also inhibit oligomerisation arising from contacts between hydrophobic patches on protein surfaces. Still further mutations include insertions / deletions that aid enzyme purification and / or immobilisation, and mutations that allow the protein to be prepared in soluble form, for example by the introduction of deletions or a poly-histidine tag, or by mutation of the N- terminal membrane anchoring sequence.

[0110] The mutant P450 enzymes encompassed by the present invention also include natural and artificial homologues, including those with as little as 40% amino acid identity to each other or to their wild-type counterparts. Indeed, it is known that P450 enzymes in different families have amino acid identities as low as 20%. In the systematic classification of the P450 superfamily (Nelson DR, 2006, Cytochrome P450 Nomenclature, 2004. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ; and Nebert DW ef a / ., 2009, The P450 Gene Superfamily: Recommended Nomenclature, Published Online: 25 Mar 2009 https: / / doi.Org / 10.1089 / dna.1987.6.1), enzymes with just 40% amino acid identity are therefore placed within the same family, and closely related members of a family (>55% identity) are grouped into sub-families. It is in fact the detailed molecular structure, substrate specificity and product selectivity that are conserved within a family rather than sequence identity, which is often low.

[0111] Accordingly, in some embodiments a mutant cytochrome P450 enzyme of the present invention may be derived from a wild-type cytochrome P450 enzyme which comprises an amino acid sequence which has at least about 40% amino acid sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67, and which comprises the amino acid consensus sequence X1X2X3X4X5X6X7 (SEQ ID NO: 32) in the l-helix of the polypeptide chain, wherein: Xi is Alanine (A) or Glycine (G); X2is Glycine (G) or Alanine (A);X3 is Histidine (H), Asparagine (N), Leucine (L), Alanine (A), Threonine (T), Isoleucine (I), or Phenylalanine (F);X4is Glutamic acid (E), Glycine (G), Leucine (L), or Aspartic acid (D);X5is Threonine (T), Alanine (A), Asparagine (N), or Serine (S);X6is Threonine (T), Isoleucine (I), Valine (V), Serine (S), or Alanine (A); andX7 is Threonine (T), Valine (V), Alanine (A), Tryptophan (W), Arginine (R), Serine (S), and Isoleucine (I).

[0112] In some embodiments, the mutant cytochrome P450 enzyme may be derived from a wild-type cytochrome P450 enzyme which comprises an amino acid sequence which has 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 to an amino acid sequence set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67, and which comprises the aforementioned consensus sequence.

[0113] When comparing an amino acid sequence to an amino acid sequence set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67 to calculate a percentage identity, the amino acid sequences should be compared over a comparison window of: 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 over the full length of the sequences. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms such as those mentioned above.

[0114] For the production of a particular wild-type P450 enzyme protein for subsequent mutagenesis, a nucleotide sequence encoding the enzyme may be PCR amplified from genomic DNA isolated from a source expressing the enzyme. Typical sources include humans, animals, plants, fungi, protists, bacteria and archaea, as described further below. Methods for DNA amplification are well known in the art and typically rely on knowledge of the P450 nucleotide sequence which is to be amplified.

[0115] Furthermore, nucleotide sequences encoding a particular P450 enzyme can be readily ascertained through the use of codon frequency tables in order to reverse translate an amino acid sequence into one or more nucleotide sequences. Moreover, nucleotide sequences encoding a particular P450 enzyme can be codon optimised for use in particular hosts and may be generated by reverse-translating an amino acid sequence via a codonfrequency table specific for a particular host organism. Codon optimization is a process used to improve gene expression and increase the translational efficiency of a P450 gene of interest by accommodating codon bias of the host organism. A range of example codon frequency tables include those presented in the GenScript Codon Usage Frequency Table Tool as published at http: / / www.genscript.com / cgi-bin / tools / codon_freq_table.

[0116] Nucleotide sequences encoding wild-type and mutant cytochrome P450 enzymes of the present invention can be cloned into a suitable vector for expression in particular host cell types. Typical host cells would include Escherichia coli, Pseudomonas sp. Flavobacteria, fungi cells, Rhodococcus sp. and Bacillus sp. Other host cells are contemplated. P450 protein can then be produced and isolated from the host cell for downstream applications. Methods for transformation and expression of an 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.

[0117] In order to effect expression of an introduced nucleic acid in a cell, where appropriate, the introduced nucleic acid may be operably connected to one or more transcriptional control sequences. The term “transcriptional control sequence” should be understood to include any nucleic acid sequence which effects the transcription of an operably connected nucleic acid. A transcriptional control sequence may include, for example, a leader, polyadenylation sequence, promoter, enhancer or upstream activating sequence, and transcription terminator. Typically, a transcriptional control sequence at least includes a promoter. The term “promoter” as used herein, describes any nucleic acid which confers, activates or enhances expression of a nucleic acid molecule in a cell.

[0118] In some embodiments, at least one transcriptional control sequence is operably connected to a nucleic acid encoding the relevant P450 gene. For the purposes of the present specification, a transcriptional control sequence is regarded as “operably connected” to a given gene or other nucleotide sequence when the transcriptional control sequence is able to promote, inhibit or otherwise modulate the transcription of the gene or other nucleotide sequence.

[0119] A promoter may regulate the expression of an operably connected nucleotide sequence constitutively, or differentially, with respect to the cell, tissue, organ or developmental stage at which expression occurs. As such, the promoter used in accordance with the present invention may include, for example, a constitutive promoter,an inducible promoter, a tissue-specific promoter, or an activatable promoter. Such promoters would be known in the art.

[0120] As indicated above, typical sources of P450 enzymes include humans, animals, insects, plants, fungi, protists, bacteria and archaea. Humans encode up to 60 P450 enzymes divided among 18 families and 43 subfamilies. A summary can be found at Human Cytochrome P450s - Cytochrome P450 Homepage (uthsc.edu). P450 enzymes are widely expressed across virtually all animal species, and have been extensively studied in mice, rats, dogs, and zebrafish to facilitate use of these model organisms in drug discovery and toxicology. P450 enzymes are also found in sponges, sea urchins, and in the cephalochordate Branchiostoma floridae. Cytochrome P450 enzymes are involved in a variety of processes of plant growth, development, and defense. It is estimated that P450 genes make up approximately 1 % of the plant genome.

[0121] In some embodiments, the mutant cytochrome P450 enzyme is derived from a wildtype cytochrome P450 enzyme which is from a bacterial or archaeal species. In bacteria, the distribution of P450s is variable with many bacteria having no identified P450s (e.g. E.coli), whereas some bacteria, predominantly actinomycetes, have numerous P450s. Those so far identified in bacteria are generally involved in either biotransformation of xenobiotic compounds (e.g. CYP105A1 from Streptomyces griseolus metabolizes sulfonylurea herbicides to less toxic derivatives) or are part of specialised metabolite biosynthetic pathways (e.g. CYP170B1 catalyses production of the sesquiterpenoid albaflavenone in Streptomyces albus). The CYP105 family is highly conserved with a representative in every streptomycete genome sequenced so far. Due to the solubility of bacterial and archaeal P450 enzymes, they are generally regarded as easier to work with than the predominantly membrane bound eukaryotic P450s.

[0122] Examples of bacterial species include, but are not limited to, Carbonactinospora thermoautotrophica (Kitasatosporales bacteria - Actinomycetes), Thermogemmatispora onikobensis (Thermogemmatisporales bacteria - Ktedonobacteria), Meiothermus ruber (Thermales bacteria - Deinococci), Rubrobacter xylanophilus (Rubrobacterales bacteria - Rubrobacteria), Dichotomicrobium thermohalophilum (Hyphomicrobiales bacteria - Alphaproteobacteria), Thermomicrobium sp. (Thermomicrobiales bacteriaThermomicrobia), Pseudomonas putida (Pseudomonadales bacteriaGammaproteobacteria), Priestia megaterium and Bacillus subtilis (Bacillales bacteria - Bacilli), Saccharopolyspora erythraea (Pseudonocardiales bacteria - Actinomycetes),Sorangium cellulosum (Polyangiales bacteria - Polyangia), Thermus thermophilus (Thermales bacteria - Deinococci), Rhodopseudomonas palustris (Hyphomicrobiales bacteria - Alphaproteobacteria), Streptomyces sp. (Kitasatosporales bacteria - Actinomycetes), Rhodococcus sp. and Nocardia otitidiscaviarum (Mycobacteriales bacteria - Actinomycetes), Magnetospirillum gryphiswaldense (Rhodospirillales bacteria - Alphaproteobacteria) and Thermosporothrix hazakensis (Ktedonobacterales bacteria - Ktedonobacteria).

[0123] Examples of archaeal species include, but are not limited to, Picrophiius torridus (Thermoplasmatales archaea - Thermoplasmata), Cuniculiplasma divulgatum (Thermoplasmatales archaea - Thermoplasmata), Thermogymnomonas acidicola (Thermoplasmatales archaea - Thermoplasmata), Sulfodiicoccus acidiphilus (Sulfolobales archaea - Thermoprotei), Acidianus brierleyi (Sulfolobales archaea - Thermoprotei), Haloferax volcanii (Haloferacales archaea - Halobacteria), and Sulfolobus acidocaldarius (Sulfolobales archaea - Thermoprotei).

[0124] In some embodiments, the mutant cytochrome P450 enzyme is derived from a wildtype cytochrome P450 enzyme which is an extremophilic cytochrome P450 enzyme. Such an enzyme is derived from extremophilic microorganisms and the enzyme is able to stay active and stable under various extreme conditions, which are considered destructive for mesophilic enzymes. For example, an extremophilic cytochrome P450 enzyme is able to withstand conditions such as extreme temperatures, high salt, high alkalinity or acidity. Some extremophilic cytochrome P450 enzymes are tolerant to other extreme conditions including high pressure, and high levels of denaturants and solvents as encountered in industrial processes.

[0125] In some embodiments, the mutant cytochrome P450 enzyme is derived from a wildtype cytochrome P450 enzyme which is a thermophilic cytochrome P450 enzyme. A thermophile is a type of extremophile that thrives at relatively high temperatures (e.g. between 41 to 122 °C). Many thermophiles are archaea, though some of them are bacteria and fungi. Thermophilic P450 enzymes are therefore enzymes that function at high temperatures. Such enzymes have advantages for their use in commercial applications.

[0126] In some embodiments, the mutant cytochrome P450 enzyme is derived from a wildtype cytochrome P450 enzyme which is a member of a CYP family selected from the group consisting of the CYP119 family (Archaeal - e.g. Sulfolobus and Sulfurisphaera species),the CYP231 family (Archaeal - e.g. Picrophilus species), the CYP175 family (Bacterial - e.g. Thermus species), the CYP199 family (Bacterial - widespread in Alphaproteobacteria and Actinomycetia e.g. Rhodopseudmonas, Amycolatopsis, Rhodococcus and Streptomyces species), the CYP154 family (Bacterial - widespread in Actinomycetia e.g. Thermobifida, Nocardia and Streptomyces species), the CYP102 family (Bacterial - widespread in Bacillia, Alphaproteobacteria and Actinomycetia e.g. Prestia, Bacillus, Rhodopseudmonas, and Streptomyces species), the CYP107 and CYP109 families (Bacterial - widespread in many bacteria including Myxococcia, Bacillia, Alphaproteobacteria and Actinomycetia e.g. Prestia, Bacillus, Sorangium, Gordonia, and Streptomyces species), the CYP267 family (Bacterial - Myxococcia, and Planctomycetia including Sorangium and Pirellula species), and the CYP116 family (Bacterial - Actinomycetia e.g. Amycolaptosis and Rhodococcus species).

[0127] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP119 family, the mutant cytochrome P450 enzyme comprises a substitution of: (i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues; (ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues; (iii) Glutamic acid, Threonine, Threonine and Threonine (ETTT) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues; (iv) Alanine, Glycine, Asparagine, Glutamic acid, and Threonine (AGNET) amino acid residues 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP119.

[0128] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP119A1 , the mutant cytochrome P450 enzyme comprises a substitution of AGNETTT (corresponding to amino acid residues 209 to 215 of SEQ ID NO: 53) with GALQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the I- helix of wild-type CYP119A1 .

[0129] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP119A2, the mutant cytochrome P450 enzyme comprises a substitution ofAGNETTT (corresponding to amino acid residues 210 to 216 of SEQ ID NO: 54) with GALQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the I- helix of wild-type CYP119A2.

[0130] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP109 family, the mutant cytochrome P450 enzyme comprises a substitution of: (i) Glutamic acid and Threonine (ET) amino acid residues, or Glutamic acid and Alanine (EA) amino acid residues, 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, with 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, with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues; (iv) Alanine, Glycine, Threonine, Glutamic acid, and Threonine (AGTET) amino acid residues, or Alanine, Glycine, Threonine, Glutamic acid, and Alanine (AGTEA) amino acid residues, or Alanine, Glycine, Asparagine, Glutamic acid, and Threonine (AGNET) amino acid residues, with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; 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), with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP109.

[0131] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109B1 , the mutant cytochrome P450 enzyme comprises a substitution of ET (corresponding to amino acids 242 and 243 of SEQ ID NO: 63) with QE in the l-helix of the polypeptide chain of the enzyme compared to the l-helix of wild-type CYP109B1 .

[0132] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109C1 , the mutant cytochrome P450 enzyme comprises a substitution ofAGTETAT (corresponding to amino acids 226 to 232 of SEQ ID NO: 58) with GALQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wildtype CYP109C1.

[0133] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP109E1 , the mutant cytochrome P450 enzyme comprises a substitution of ET (corresponding to amino acids 245 and 246 of SEQ ID NO: 64) with QE in the l-helix of the polypeptide chain of the enzyme compared to the l-helix of wild-type CYP109E1 .

[0134] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP154 family, the mutant cytochrome P450 enzyme comprises a substitution of: (i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues; (ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues; (iii) Glutamic acid, Threonine, Threonine and Isoleucine (ETTI) amino acid residues with Glutamine, Glutamic acid, Proline and glycine (QEPG) amino acid residues; (iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) 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) or 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, in the l-helix of the mutant enzyme compared to the l-helix of wild-type CYP154.

[0135] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme comprises a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GALQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wildtype CYP154C8.

[0136] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme comprises a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GAHQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wildtype CYP154C8.

[0137] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP154C8, the mutant cytochrome P450 enzyme comprises a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 62) with QE in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP154C8.

[0138] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type P450t, the mutant cytochrome P450 enzyme comprises a substitution of ET (corresponding to amino acids 244 and 245 of SEQ ID NO: 65) with QE in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type P450t. The P450t enzyme is a member of the CYP154 family, and more specifically a member of the CYP154C subfamily, of cytochrome P450 enzymes.

[0139] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP267 family, the mutant cytochrome P450 enzyme comprises a substitution of: (i) Glutamic acid and Threonine (ET) amino acid residues, or Glutamic acid and Alanine (EA) amino acid residues, 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, 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, with 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, 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), with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) or Glycine, Alanine, Histidine, Glutamine, Glutamic acid, Proline and Glycine (GAHQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP267.

[0140] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP267B1 , the mutant cytochrome P450 enzyme comprises a substitution of ET (corresponding to amino acids 246 and 247 of SEQ ID NO: 59) with QE, or AGHETTV(corresponding to amino acids 243 to 249 of SEQ ID NO: 59) with GALQEPG, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP267B1 .

[0141] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP102 family, the mutant cytochrome P450 enzyme comprises a substitution of: (i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues; (ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues; (iii) Glutamic acid, Threonine, Threonine and Serine (ETTS) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues; (iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP102.

[0142] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP102A1 , the mutant cytochrome P450 enzyme comprises a substitution of ET (corresponding to amino acids 268 and 269 of SEQ ID NO: 52) with QE, or AGHETTS (corresponding to amino acids 265 to 271 of SEQ ID NO: 52) with GALQEPG, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP102A1.

[0143] In some embodiments, when the wild-type cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP175 family, the mutant cytochrome P450 enzyme comprises a substitution of: (i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues; (ii) Glutamic acid, Threonine and Valine (ETV) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues; (iii) Glutamic acid, Threonine, Valine and Alanine (ETVA) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues; (iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; or (v) Alanine, Glycine, Histidine, Glutamic acid, Threonine, Valine andAlanine (AGHETVA) amino acid residues (or equivalent amino acid residues) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to wild-type CYP175.

[0144] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP175A1 , the mutant cytochrome P450 enzyme comprises a substitution of AGHETVA (corresponding to amino acids 221 to 227 of SEQ ID NO: 55) with GALQEPG, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wildtype CYP175A1.

[0145] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP199 family, the mutant cytochrome P450 enzyme comprises a substitution of: (i) Aspartic acid and Threonine (DT) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues; (ii) Aspartic acid, Threonine, and Threonine (DTT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues; (iii) Aspartic acid, Threonine, Threonine and Valine (DTTV) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues; (iv) Alanine, Glycine, Leucine, Aspartic acid, and Threonine (AGLDT) 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP199.

[0146] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP199A4, the mutant cytochrome P450 enzyme comprises a substitution of DT (corresponding to amino acids 252 and 253 of SEQ ID NO: 60) with QE, AGLDT (corresponding to amino acids 249 to 253 of SEQ ID NO: 60) with GALQE, or AGLDTTV (corresponding to amino acids 249 to 255 of SEQ ID NO: 60) with GALQEPG, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP199A4.

[0147] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP231 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Threonine (ETTT) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Glycine, Glycine, Asparagine, Glutamic acid, and Threonine (GGNET) amino acid residues with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; or(v) Glycine, Glycine, Asparagine, Glutamic acid, Threonine, Threonine and Threonine (GGNETTT) amino acid residues (or equivalent amino acid residues) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP231 .

[0148] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP107 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Alanine (EA) amino acid residues, or Glutamic acid and Threonine (ET) amino acid residues, 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, 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, with 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) aminoacid residues, 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), with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP107.

[0149] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP107PQ (P450h), the mutant cytochrome P450 enzyme comprises a substitution of EA (corresponding to amino acids 247 and 248 of SEQ ID NO: 40) with QE in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wildtype CYP107PQ (P450h).

[0150] In some embodiments, when the mutant cytochrome P450 enzyme is derived from wild-type CYP107Mg, the mutant cytochrome P450 enzyme comprises a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 66) with QE in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP107Mg.

[0151] In some embodiments, when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP116 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Valine (ETTV) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Alanine, Histidine, Glutamic acid, and Threonine (AAHET) amino acid residues with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; or(v) Alanine, Alanine, Histidine, Glutamic acid, Threonine, Threonine and Valine (AAHETTV) amino acid residues (or equivalent amino acid residues) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wildtype CYP116.

[0152] As indicated above, the mutant cytochrome P450 enzymes of the present invention display an enhanced peroxygenase activity and / or an altered product selectivity with respect to a wild-type cytochrome P450 enzyme. Enhanced peroxygenase activity may be characterised in terms of an increased coupling efficiency / activity or an increased product formation rate with one or more substrates for oxidation. Coupling efficiency / activity refers to how much nicotinamide cofactor consumed is converted into oxidised organic product. The increased coupling efficiency / activity or product formation rate may or may not be shared across all substrates utilised by the mutant cytochrome P450 enzyme. The mutant cytochrome P450 enzymes typically display a coupling efficiency / activity which is at least about 10%, about 20%, about 50%, about 100%, about 500%, about 1000%, or about 1500% greater, than that of the wild-type enzyme. The mutant cytochrome P450 enzymes may also have a product formation rate which is at least about 50%, about 100%, about 150%, about 500%, about 1000%, about 2000%, about 5000%, or about 10000% greater, than that of a wild-type enzyme.

[0153] It is to be understood that the mutant cytochrome P450 enzymes of the present invention may also display other altered characteristics with respect to a wild-type cytochrome P450 enzyme and mutants disclosed in the literature, such that the effects may include, but may also not be limited to, enhanced peroxygenase activity. For example, the mutant enzyme may display an altered substrate specificity, allowing preferential utilization of specific substrates, or may display peroxygenase activity where the wild-type enzyme or known mutants are not able to oxidize the substrate organic compound.

[0154] The mutant cytochrome P450 enzymes of the present invention may also display altered product selectivity where a product formed in minor proportions by the wild-type becomes the dominant product for the mutant, or new products formed in minor proportions, or not at all, by the wild-type become the majority or dominant product. Further altered characteristics of the mutant enzymes and of the oxidation processes carried out by the mutant enzymes are described below.

[0155] In a further embodiment, the present invention also relates to a use of the mutant cytochrome P450 enzyme as described herein in the biocatalytic oxidation of carbonhydrogen bonds via a peroxygenase pathway.

[0156] In a further embodiment, the present invention relates to a process for oxidizing a substrate which is an organic compound, comprising oxidizing said organic compound substrate with a mutant cytochrome P450 enzyme as described herein.

[0157] The substrate for the oxidation process may be any organic compound, more typically any organic compound capable of being oxidized by a peroxygenase enzyme. The suitability of any organic compound for oxidation by a peroxygenase enzyme may be routinely determined by the methods described herein.

[0158] The oxidation process causes the formation of a C-O bond in the compound, generally as the alcohol from the oxidation of a carbon-hydrogen bond, but an epoxide may be formed from the oxidation of a C=C bond. The oxidation may thus introduce an alcohol, aldehyde, ketone, carboxylic acid, or epoxide group. Alternatively, the oxidation may cause the further oxidation of an oxygen containing group, such as converting an alcohol group into an aldehyde, ketone, or carboxylic acid. 1 , 2 or more carbon atoms may be attacked in the same substrate molecule. Oxidation can also result in N- and O-dealkylation, or S- oxidation, of the substrate molecule. In certain instances, the oxidation may result in C-C bond cleavage or C-C or C-X (where X is a heteroatom, other than oxygen e.g. N) bond formation can occur.

[0159] The oxidation typically gives rise to 1 , 2 or more oxidation products. These different products may result from different carbon atoms being attacked and / or from different degrees of oxidation occurring at a given carbon atom.

[0160] The oxidation may occur on either a ring carbon atom or a substituent carbon atom or both. At least the initial oxidation will involve attack of a C-H bond which may be activated or non-activated or attack at a carbon-carbon double bond (typically giving an epoxide). Generally, an activated C-H bond is where the carbon atom is in a benzylic or allylic position. Aromatic rings and olefinic double bonds activate C-H bonds to attack by stabilizing the radical intermediate or any build-up of charge generated during the reaction pathway. The carbon of the C-H bond may be primary, secondary or tertiary. The oxidation may occur to result in dehydrogenation leading to a C=C double bond formation rather than insertion ofan oxygen atom. This is most likely to occur when the alkyl substituent is branched, or dehydrogenation leads to a C=C bond that is conjugated to an aromatic system, or dehydrogenation leads to the formation of an aromatic system.

[0161] The substrate can either be a natural substrate of a wild-type cytochrome P450 enzyme or a substrate which is not normally a substrate for the wild-type enzyme, but which is capable of being utilized as such in the mutant enzyme. Examples of natural substrates for the cytochrome P450 enzymes are branched and straight chain fatty acids, and saturated and unsaturated fatty acids, which are hydroxylated by wild type cytochrome P450 enzymes at the a and p, co, co-1 , co-2 and co-3 carbons. Preferred examples are those which range in length from 8 to 18 carbons e.g. hexadecanoic acid, heptadecanoic acid, tetradecanoic acid, dodecanoic acid, undecanoic acid, decanoic acid, nonanoic acid and octanoic acid.

[0162] In some embodiments, the substrate may include alkanes, alkylbenzenes, steroids, terpenoids, norisoprenoids and fatty acids. In some embodiments, the substrate is a shortchain alkane, a medium-chain alkane, a long-chain alkane, or a cyclic alkane. The term alkane refers to acyclic branched or unbranched hydrocarbons having the general formula CnH2n+2

[0163] A short-chain alkane has typically from 1 to about 9 carbon atoms, more preferably 1 to 8, 1 to 6, or 1 to 4 carbon atoms. A Ci-C8alkyl group or moiety can be linear or branched. Where it is a C1-C4 alkyl moiety, it can be, for example, methyl, ethyl, n-propyl, propyl, sec-butyl and t-butyl.

[0164] An alkylbenzene has one or more alkyl groups or moieties substituted at positions on the benzyl aromatic ring. The numbers of carbon atoms in the alkyl groups or moieties can be typically from 1 to about 8 carbon atoms, more preferably 1 to 8, 1 to 6, or 1 to 4 carbon atoms.

[0165] In some embodiments there may be 1 , 2, 3 or more substituents present on the backbone of the short-chain or medium-chain alkane or directly substituted on the benzyl ring, or on the alkyl substituent of the alkylbenzene. Any combination of the following substituents may be present. The substituent is typically a halogen atom or an alkyl or alkenyl group, which generally has 1 to 6 carbons, the substituent optionally being substituted with one or more halogens. The substituent may also comprise 1 , 2 or moreoxygen, halogen or nitrogen atoms and for example may be an alcohol, aldehyde, ketone, ether, amine or epoxide group.

[0166] Examples of preferred short-chain alkane substrates include, but are not limited to pentane, 3-methylpentane, 2-methylbutane, butane, propane, ethane and methane, octane and nonane. Examples of preferred alkylbenzene and alkylaromatic substrates include, but are not limited to propylbenzene, ethylbenzene, butylbenzene, cumene, t-butylbenzene, o- xylene, m-xylene, p-cymene and ethylanisole. Other preferred aromatic compounds are those with a naphthalene, biphenyl, indole, indance and fluorene skeleton.

[0167] It is to be noted that organic compounds such as butane, naphthalene, and in particular, propane, t-butylbenzene and o-xylene are broadly classified as “non-natural” substrates for the wild-type cytochrome P450 enzyme, but are capable of being oxidized by the mutant cytochrome P450 enzymes of the invention. A non-natural substrate can be defined as a molecule which has no detectable coupling rate and / or product formation when incubated with the wild-type cytochrome P450. Non-natural substrates may also include molecules which are oxidized at less than about 10% of the rate for a natural substrate by the wild-type cytochrome P450 enzyme such that they may not be regarded as a bona fide substrate.

[0168] The oxidation process carried out with a mutant cytochrome P450 enzyme of the present invention may be differentiated from that carried out by another wild-type or mutant cytochrome P450 enzyme in terms of an improved coupling efficiency / activity or rate of product formation, as defined above. The processes of the invention may also be characterized by formation of a specific product from the oxidized substrate, typically one which is not formed by the wild-type cytochrome P450 enzyme or another mutant cytochrome P450 enzyme, or one which is formed in negligible quantities, i.e. less than about 10%, about 8%, about 5%, about 2%, about 1%, or less, of the total amount of product. For example, oxidation of propylbenzene may produce 2-propylphenol, or 1- phenyl-2-propanol with high selectivity, or oxidation of ethylbenzene may produce 2- phenylethanol and styrene.

[0169] Processes carried out with the mutant cytochrome P450 enzyme of the invention may also display an altered ratio or number of oxidation products, as compared to the oxidation process carried out by a wild-type cytochrome P450 enzyme or other mutant cytochrome P450 enzyme. Where an altered ratio of products is present, the productformation rate for a specific oxidation product is typically increased with reference to the corresponding process carried out by a wild-type cytochrome P450 enzyme or other mutant cytochrome P450 enzymes. The increase in the prevalence of a specific oxidation product may be at least about 10%, about 20%, about 50%, more preferably about 100%, about 200%, about 300%, about 500%, or more, over the amount of said oxidation product in the product mixture as formed by the wild-type cytochrome P450 enzyme or other mutant cytochrome P450 enzymes.

[0170] The process is typically carried out in the presence of the cytochrome P450 enzyme, the substrate and natural co-factors of the enzyme which may include one or more of electron transfer partners, oxygen, nicotinamide co-factors, hydrogen peroxide, and water. In some embodiments, the process is carried out by generating the hydrogen peroxide cofactor in situ using electrochemical, biocatalytic, or photochemical methods known to those in the art (for example see 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).

[0171] While the co-factor hydrogen peroxide is typically used, any other peroxide sources can also be used including organic surrogates. The suitability of any peroxide source for use in the oxidation process may be routinely determined by the methods described herein. In some embodiments, the peroxide is selected from the group consisting of hydrogen peroxide, cumene hydroperoxide, iodosylbenzene, t-butyl hydroperoxide and m- chloroperbenzoic acid. In certain preferred embodiments, the peroxide is selected from the group consisting of hydrogen peroxide, t-butyl hydroperoxide and m-chloroperbenzoic acid. Preferably, the peroxide is hydrogen peroxide.

[0172] In the process, the concentration of the enzyme is typically from about 10’8to about 10’2M, preferably from about 10’7to about 10’4M. Generally, the process is carried out at a temperature and / or pH at which the enzyme is functional, such as when the enzyme has at least about 20%, 50%, 80% or more of peak activity. Typically, the pH is from about 2 to about 12, such as 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, such as about 25°C to about 75°C or about 30°C to about 60°C. In some embodiments, the process is carried out at about 10°C to about 90°C.

[0173] The invention is further illustrated in the following examples. The examples are forthe purpose of describing particular embodiments only and are not intended to be limiting with respect to the above description.EXAMPLE 1Production of Mutant Cytochrome P450 Enzymes

[0174] Codon optimized versions of genes encoding wild-type and mutant P450 enzymes were obtained in the plasmid, pET28b (between the Neo I -Xho I restriction sites) or pET29b (between the Nde I - Xho I restriction sites) by Twist Bioscience. The Ncol site (CCATGG) or Ndel site (CATATG) contain the start codon. Where a 6 x histidine purification tag was used the codon optimized sequence was cloned into the pET28 vector between the Neo I and Xho I restriction sites. A 6 x histidine tag and Tobacco Etch Virus (TEV) cleavage sequence was incorporated at the N-terminus before the Nde I restriction site (the amino acid sequence of N-terminal adaption was GGSSHHHHHHSSGENLYFQGHM) (SEQ ID NO: 68) followed by the sequence of the gene of interest. The gene sequence was followed by two stop codons and Kpn I and Hind III restriction sites before the Xho I restriction site of the vector.

[0175] These plasmids were transformed into E. coli BL21 (DE3) competent cells and grown on an LB agar plate in the presence of kanamycin (30 pg / mL). A single colony was added to 500 mL of LB media containing trace elements solution (1.5 mis of solution containing CaCI2, ZnSO4.7H2O, MnSO4.H2O, Na2-EDTA, FeCI3.6H2O, CuSO4.5H2O, and CoCI2.6H20) in the presence of antibiotic and incubated at 37 °C at 85 rpm. After 10 h incubation the temperature of incubation was either reduced 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 an additional 30 min, 100 pM isopropyl B-D-1 -thiogalactopyranoside (IPTG) was used to induce protein production. Cells were grown for a further 24-48 hrs and harvested by centrifugation (5000 g, 10 min, 4 °C).EXAMPLE 2Mutant Cytochrome P450 Enzyme Protein Purification

[0176] The Escherichia coli cell pellets (from 2- 3 L of growth media) were stored at -20 °C prior to purification. For non-His tagged proteins, the cell pellet was resuspended in 200 mL buffer T (50 mM Tris buffer, pH 7.4 - 8.0, 1 mM dithiothreitol - DTT - henceforth known as Buffer T). The mixture was lysed by sonication (35 x 20 s pulses with 40 s intervals; 70% amplitude). During sonication, the cell mixture was cooled on ice and the mixture stirred frequently. To eliminate the cell debris, the lysed cell mixture was centrifuged for 30min at 4 °C at 35,000 g (17,010 rpm). The crude protein mixture was purified via ammonium sulfate precipitation. After addition of 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. The ammonium sulfate concentration was increased to 60% and the mixture was centrifuged at 20,442 g (13,000 rpm) for 15 min at 4 °C. The supernatant was discarded, and the pellet containing the P450 enzyme was redissolved in buffer T. The protein was desalted using a Sephadex G-25 coarse grain column, using buffer T as the eluent. The red-coloured fractions were collected and purified by ion-exchange chromatography, using a DEAE Sepharose column. The protein was eluted using a gradient of 100 - 400 mM KCI in buffer T; the flow rate was 6 mL min-1. The red-coloured fractions were pooled and concentrated by ultrafiltration.

[0177] For 6 x histidine tagged proteins the cells were resuspended in 50 mM Tris buffer (pH 7.4 to 8.0 containing 0.1% v / v 2-mercaptoethanol, 2 mL phenylmethylsulfonyl fluoride PMSF (10 mM stock) and 10% glycerol). The cells were lysed by sonication on ice for 30 cycles (20 s on, 40 s 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 NaCI, pH 7.4-8.0). The column was washed with 5 column volumes of the sample loading buffer before elution of the His-tagged protein with sample elution buffer (20 mM sodium phosphate buffer, pH 7.4-8.0, 0.5 M NaCI and 500 mM imidazole). The P450 containing fractions were concentrated by ultrafiltration (10 kDa exclusion membrane) and afterwards the protein was loaded to a Sephadex G-25 medium grain column (250 mm x 40 mm; GE Healthcare) to desalt using 50 mM Tris buffer, pH 7.4-8.0.

[0178] All proteins were further purified by size-exclusion chromatography, using buffer T as the eluent. The column used was a HiPrep 16 / 6- Sephacryl S-200 HR column (GE Healthcare) and the flow rate was 1 mL min-1. The highest- purity fractions were combined, concentrated, filter-sterilised and stored in ~50% glycerol at -20 °C. Glycerol was removed before use using a PD-10 column (Cytiva), using Tris buffer (50 mM, pH 7.4 - 9.0) as the eluent.EXAMPLE 3Mutant Cytochrome P450 Enzyme Peroxide Turnover Reactions

[0179] Unless specifically stated otherwise, the peroxygenase oxidation assays for the experiments described in Examples 4 to 10 below were run in a total volume of 600 - 1000pL consisting of Tris buffer (50 mM, pH 7.4 - 9.0), 1 - 3 pM enzyme and 0.250 - 2 mM substrate (from a 50 - 100 mM stock solution in EtOH or DMSO). The reactions were started by addition of H2O2(2 - 60 mM) and incubated for 1-4 hrs. Samples were analysed by HPLC or Gas Chromatography-Mass Spectrometry (GC-MS).

[0180] For HPLC analysis, 132 pL aliquots were removed and quenched with 10 pL of 10 mg mL-1bovine liver catalase solution and 66 pL of acetonitrile (MeCN). Finally, 2 pL of internal standard (10 mM 9-hydroxyfluorene in EtOH) was added and each mixture was centrifuged and analysed via HPLC (20 - 95% gradient of acetonitrole in H2O, acidified with 0.1 % trifluoroacetic acid).

[0181] HPLC analysis was performed using a Shimadzu Prominence LC-20AD Liquid Chromatograph equipped with a DGU-20A5R degassing unit, SIL-20A auto sampler, SPD- 20A UV-Vis detector, a CTO-20A column oven, and a Kinetex XB-C18 reversed-phase LC column (100 A pore size, 250 x 4.6 mm, 5 pm; Phenomenex).

[0182] For GC-MS analysis, 595 pL of the reaction mixture was mixed with 5 pL of an internal standard solution (octanoic acid, 20 mM stock solution in EtOH). These mixtures were extracted with 3 x 400 pL of ethyl acetate and dried with anhydrous MgSO4. These could then be used directly for analysis or derivatized as follows; the organic solvent was then removed under a stream of dinitrogen gas and the residue was dissolved in anhydrous acetonitrile. Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS), 15 pL, was added to the acetonitrile to derivatize the substrate and metabolites. After leaving these samples for 2 hours at 37 °C they were used directly for GC-MS analysis.

[0183] GC-MS was carried out on a Shimadzu GC-2010 with GC-MS-QP2010S detector. The interface and injection port temperatures were held at 280 °C and 250 - 300 °C, respectively. The column (DB5ms; 30 m x 0.25 mm x 0.25 pm) was held at 70 °C or 120 °C for 3 min, and the temperature was then increased to 240 °C at a rate of 7.5 °C min-1and held at 240 °C for 6 min.

[0184] A comparison of the active site structures of CYP255A2 and CYP119A1 highlighting the oxygen binding groove of the l-helix is shown in Figure 1 B. The acid-alcohol pair of amino acid residues (E212 and T213 in CYP119A1) responsible for the monooxygenase activity of most P450s is replaced with an amide and acid pair (Q248 and E249) in the peroxygenase CYP255A2.

[0185] The peroxygenase variant of the thermostable CYP119A1 , was based on the replacement of seven amino acids in the vicinity ofthe oxygen binding groove of the enzyme (Figure 1 B). The activity was optimised by generating other mutations in this region. These seven residues of the l-helix in CYP1 19A1 were replaced with those of CYP255A2 (Figure 1 B). Overall, the amino acids209AGNETTT215of CYP1 19A1 were replaced with the sequence GALQEPG.

[0186] As described below, it has also been demonstrated that this switch of the seven amino acids works in CYP119A1 - thermostable P450, and CYP154C8 (GAHQEPG) a steroid oxidizing P450. It has also been demonstrated below that fewer substitutions can also work including the QE and GALQE substitution in CYP199A4 and other enzymes (Figures 6-22). These all show improvements in peroxygenase activity compared to the WT and the single threonine to E mutant (e.g. T to E - T252E in CYP199A4).EXAMPLE 4Mutant Cytochrome P450 Enzyme (CYP119) Oxidation of Fatty Acids

[0187] The metabolites generated from the oxidation of fatty acids by the peroxygenase mutant variant of CYP119A1 is shown in Figure 3. Figure 4 shows gas chromatography analysis of dodecanoic / lauric acid (1 mM) by WT CYP119A1 and the peroxygenase mutant (mutant), 3 |j.M at room temperature (RT) and 80 °C, 2 hours, using hydrogen peroxide (50 mM) is shown. Figure 5 shows the gas chromatography analysis of the oxidation of dodecanoic (lauric) acid by the GALQEPG variant of CYP119 (3 |j.M) at room temperature (RT) and 50 °C, 70 °C and 90 °C using hydrogen peroxide (50 mM). This result highlights the ability ofthe CYP119 variant to function efficiently at high temperatures. It also highlights the complete conversion of all of the substrate to product in the mutant.EXAMPLE 5Mutant Cytochrome P450 Enzyme (CYP154) Oxidation of Progesterone and Androstenedione

[0188] The end products of the oxidation of progesterone and androstenedione by a CYP154 mutant using hydrogen peroxide is shown in Figure 6.

[0189] Figure 7 shows the HPLC analysis of the stereoselective oxidation of progesterone (1 mM) by WT and the GAHQEPG mutant of Streptomyces sp. CYP154C8 (1 |j.M) at room temperature (RT) and using 20 mM hydrogen peroxide.

[0190] Figure 8 shows the HPLC analysis of the stereoselective oxidation of progesterone (1 mM) by the GAHQEPG mutant of CYP154C8 (1 pM) at room temperature (RT) and using 2 - 40 mM hydrogen peroxide. This result highlights the ability of this variant to function at low concentrations of hydrogen peroxide.

[0191] Figure 9 shows HPLC analysis of peroxygenase activity of CYP154C8 mutants (T258E, QE and GAHQEPG) compared to wildtype (WT) enzyme. The stereoselective oxidation of progesterone substrate (1 mM) by each mutant of CYP154C8 and WT CYP154C8 (1 pM) was conducted at room temperature (RT) over 4 hours using 5.0 mM hydrogen peroxide and 5% v / v ethanol as substrate solvent. The results show that there is a higher turnover of progesterone oxidation for each mutant compared to WT. The QE mutant demonstrated the highest oxidation efficiency, followed by the GAHQEPG mutant, then the T258E mutant under these conditions.

[0192] Figure 10 shows the effect of different DMSO (as substrate solvent) concentrations in oxidation assays of the CYP154C8 QE mutant enzyme. The stereoselective oxidation of progesterone substrate (2 mM) by the CYP154C8 QE mutant (1 pM) was conducted at room temperature (RT) over 4 hours using 5.0 mM hydrogen peroxide. The HPLC results show stability of the CYP154C8 QE mutant oxidation activity to the presence of organic solvent (in this case DMSO up to 30% v / v).

[0193] Figure 11 shows the effect of different hydrogen peroxide concentrations (1 mM, 5 mM, 10 mM, 20 mM and 40 mM) in oxidation assays of the CYP154C8 QE mutant enzyme. The stereoselective oxidation of progesterone (1 mM) by the CYP154C8 QE mutant (1 pM) was conducted at room temperature (RT) over 4 hours using various concentrations of hydrogen peroxide as substrate and 5% v / v DMSO as substrate solvent. The HPLC results show stability of the CYP154C8 QE mutant oxidation activity to hydrogen peroxide concentrations in excess of 10 mM (and up to 40 mM).

[0194] Figure 12 shows the results of oxidation of androstenedione (Figure 12A) and testosterone (Figure 12B) substrates by a P450t QE mutant using hydrogen peroxide. P450t is a member of the CYP154C subfamily and is derived from Nocardia otitidiscaviarum. The reaction mixture contained 400 pM of each substrate in Tris buffer at pH 7.4 with 10 mM hydrogen peroxide and 2 pM of the mutant enzyme. This shows that other cytochrome P450 enzymes can be mutated, and other substrates can be oxidised, leading to improved oxidation efficiency.EXAMPLE 6Mutant Cytochrome P450 Enzyme (CYP199) Oxidation of 4-methoxybenzoic acid

[0195] As shown in Figure 13, during time course reactions for 3 pM P450 CYP199A4 (WT and GALQE mutant) with 4-methoxybenzoic acid, driven by 5 mM H2O2, the WT enzyme generated 61 .6 ± 0.2 pM product, whereas the GALQE mutant generated about 3-fold more product (176 ± 3 pM product) at low peroxide concentrations.

[0196] Figure 14 shows the results of time course reactions for 3 pM P450 CYP199A4 (QE and T252E mutant) with 4-methoxybenzoic acid (1 mM), driven by 10 mM H2O2. These results highlight the improved performance of the QE mutant relative to the T252E single mutant.

[0197] Figure 15 shows the results of time course reactions for 3 uM P450 CYP199A4 (QE mutant and WT) with 4-methoxybenzoic acid (1-5 mM), driven by 10 mM H2O2. These results highlight the increase in product formation with the QE variant and the lifetime of the catalyst when substrate concentration is maintained at higher levels. Figure 1 1 B shows the HPLC analysis of the 45-hour reactions.EXAMPLE 7Mutant Cytochrome P450 Enzyme (CYP102) Oxidation of Myristic Acid

[0198] Figure 16 shows the GC-MS analysis of the 2-hour reaction of HazakQE (2 pM) with 250 pM myristic acid driven by 5 mM H2O2at 30 °C in Tris-HCI buffer (50 mM, pH 7.4). This enzyme is a thermophilic CYP102 enzyme from Thermosporothrix hazakensis. The enzyme was heated at 50 °C for 30 minutes prior to the reaction. This enzyme shares greater than 40% amino acid sequence identity to the CYP102A1 enzyme amino acid sequence provided in Figure 1 highlighting how enzymes of similar sequence identity can be identified. The P450 reaction is represented by the dark line, and the control reaction omitting the P450 enzyme but containing all other reaction components (including the H2O2) is shown by the lighter line. The major metabolites were the co-3, OJ-2 and co-1 hydroxylation products.EXAMPLE 8Mutant Cytochrome P450 Enzyme (P450h / CYP107) Oxidation of -ionone

[0199] Figure 17 shows analysis of the stability of a QE mutant of the thermophilic P450 enzyme from Meiothermus ruber (P450h in Figure 2) with respect to selective oxidation of p-ionone substrate to 4-hydroxy-p-ionone. P450h is a member of the CYP107 family andis also referred to herein as CYP107PQ1 . Reaction conditions were 1 JJ.M P450h QE mutant (CYP107PQ1 QE) enzyme, 1 mM p-ionone (from a 100 mM stock in DMSO) and 10 mM H2O2. Reaction temperatures and times were varied as indicated below.

[0200] Figure 17A is a graph showing the effect of reaction temperature on the catalytic activity of the P450h QE mutant (CYP107PQ1 QE) and 4-hydroxy-p-ionone product formation rate at different time points up to 4 hours (substrate evaporation occurred for reactions at 45 °C over longer reaction times). It is clear that the P450h QE mutant (CYP107PQ1 QE) enzyme maintains activity at various reaction temperatures, including 30 °C and 45 °C.

[0201] Figure 17B represents HPLC analysis of p-ionone oxidation reactions of the P450h QE mutant (CYP107PQ1 QE). The stability of the mutant enzyme to heat treatment for 1 hour at different temperatures prior to adding the enzyme to the p-ionone substrate was measured. Oxidation reactions were performed at 30 °C for 2 hours. These results demonstrate that the mutant enzyme is stable to preheating at temperatures up to 60 °C to 65 °C.

[0202] Figure 17C represents HPLC analysis of p-ionone oxidation reactions of the P450h QE mutant (CYP107PQ1 QE) after prior storage of the enzyme for set periods of time at 30 °C. Oxidation reactions were performed at 30 °C for 2 hours. These results demonstrate the ability to store the mutant cytochrome P450 enzymes at ambient temperatures (rather than in fridges or freezers) for extended periods of time, including up to at least 1 year, without loss of activity.

[0203] Figure 18 shows stability analysis of the heme of the P450h QE mutant (CYP107PQ1 QE) enzyme with respect to selective oxidation of p-ionone substrate to 4- hydroxy-p-ionone. Reaction conditions were 2 |j.M P450h QE mutant (CYP107PQ1 QE) and 10 mM H2O2 in the absence of substrate (A) and 2 |j.M P450h QE mutant (CYP107PQ1 QE) and 20 mM H2O2 in the presence of substrate (B). These results demonstrate the stability of the heme of mutant cytochrome P450 enzymes to 20 mM hydrogen peroxide in the presence of 1 mM substrate.

[0204] Figure 19 shows the catalytic activity of the P450h QE mutant (CYP107PQ1 QE) enzyme with respect to selective oxidation of p-ionone substrate (1 mM) to 4-hydroxy-p- ionone compared to wild type (CYP107PQ1) enzyme (A), and with respect toenantioselectivity of the mutant (B). Reaction conditions were 1 JJ.M P450h QE mutant (CYP107PQ1 QE) or wild type enzyme, and 10 mM H2O2for 2 hours at 30 °C. The HPLC chromatogram in (A) shows 4-hydroxy-p-ionone as a major product on HPLC at the retention time of 16 minutes for the mutant enzyme compared to little or no product for the wild type enzyme. Figure 19B shows enantioselective-HPLC analysis of enantio-selectivity of the mutant enzyme catalysed 4-hydroxy-p-ionone in comparison to the racemic product synthesized chemically. This demonstrates the high enantioselectivity of the reaction.

[0205] Figure 20 shows the effect of the use of different organic solvents on the catalytic activity of the P450h QE mutant (CYP107PQ1QE) enzyme in the presence of 1 mM p- ionone substrate (A and B) or 2 mM p-ionone substrate (C and D), 10 mM hydrogen peroxide, and 1 uM enzyme. All reaction were performed at 30 °C for 2 hours. The HPLC chromatogram of Figure 20A shows the efficient conversion of p-ionone substrate to 4- hydroxy-p-ionone in the presence of a range of different organic solvents each at 10% v / v. Figure 20B is a graph comparing the effect of organic solvents DMSO and isopropanol at varying concentrations on relative 4-hydroxy-p-ionone product yield (ratio of product peak and internal standard peak area). Figure 20C is a HPLC chromatogram comparing the effects of varying the concentration of DMSO solvent on product yield, and Figure 20D is a HPLC chromatogram comparing the effects of varying the concentration of isopropanol solvent on product yield. These results demonstrate the stability of the mutant cytochrome P450 enzymes to different concentrations of a range of organic solvents.EXAMPLE 9Mutant Cytochrome P450 Enzyme (CYP109 and P450h / CYP107) Oxidation of - ionone

[0206] Figure 21 shows analysis of the stability of a QE mutant of the cytochrome P450 enzymes CYP109B1 and CYP109E1 , and the P450h (CYP107PQ1) enzyme referred to in the Examples above when the enzyme was heat treated for 1 hour at different temperatures before the p-ionone substrate was added to the reaction. Reaction conditions were 1 |j.M enzyme, 1 mM p-ionone (from a 100 mM stock in DMSO) and 10 mM H2O2. (A) CYP109E1 QE mutant; (B) CYP109B1 QE mutant; (C) CYP107PQ1 QE mutant. The y-axis in each graph represents relative 4-hydroxy-p-ionone product yield (ratio of product and internal standard) and the x-axis represents the temperature at which the enzyme was treated prior to the reaction. These graphs establish that the mutant cytochrome P450 enzymes are temperature stable.EXAMPLE 10Mutant Cytochrome P450 Enzyme (CYP107) Oxidation of Progesterone

[0207] Figure 22 shows the ability of a CYP107MgQE mutant enzyme to catalyse the conversion of progesterone using hydrogen peroxide as the sole oxidant. The oxidation reactions were performed using 5 uM of the enzyme and 500 pM of progesterone in the presence of 10 mM hydrogen peroxide for 1 hour at 30 °C. In the wild type CYP107Mg enzyme, no oxidation product was observed indicating that the wild type enzyme cannot act as a peroxygenase in its native state. However, the CYP107MgQE mutant enzyme clearly had an ability to oxidise progesterone.

[0208] The aforementioned examples show that the mutant cytochrome P450 enzymes of the present invention have increased peroxygenase activity compared to counterpart wildtype enzymes. The mutant cytochrome P450 enzymes of the present invention were shown to be stable to hydrogen peroxide in the presence of substrate and capable of hydroxylating fatty acid substrates with high levels of substrate conversion (>99%) and good turnover numbers). The thermostability of the mutant P450 enzymes derived from thermophilic bacteria was maintained and catalytic activity at high temperatures, 50 °C to 90 °C, was demonstrated. The generation of an efficient thermostable heme peroxygenase establishes a system to achieve H2O2-dependent specific C-H bond hydroxylation. This overcomes many of the major drawbacks associated with using cytochrome P450 enzyme for this purpose, namely, the requirement for expensive nicotinamide cofactors and additional electron transfer partners, and the low activity and stability of the majority of these hemethiolate enzymes. This work not only expands the toolkit for enzymatic C-H activation, but also enables for a simple, cheap, and clean method by which to undertake this challenging reaction.

[0209] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0210] It is to be noted that where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all numerical values or sub-ranges in between these limits as if each numerical value and subrange is explicitly recited. The statement "about X% to Y%" has the same meaning as "about X% to about Y%," unless indicated otherwise.

[0211] The term “about” as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of + / - 10% or less, + / - 5% or less, + / - 1 % or less, or + / - 0.1 % or less of and from the numerical value or range recited or claimed.

[0212] It is also to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

[0213] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure orthe claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0214] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0215] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[0216] It will be apparent to the person skilled in the art that while the invention has been described in some detail forthe purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0217] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations ofany two or more of the steps or features.

[0218] Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention. See, for example, Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.SEQUENCES

Claims

CLAIMS1 . A mutant cytochrome P450 enzyme with enhanced peroxygenase activity and / or altered product selectivity, wherein the mutant cytochrome P450 enzyme comprises a substitution of at least two consecutive amino acid residues in the l-helix of the polypeptide chain of the enzyme compared to the l-helix of a wild-type cytochrome P450 enzyme, wherein the substitution is at a position corresponding to amino acid residues 23 and 24 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine and Glutamic acid (QE) amino acid residues at said position in the l-helix of the polypeptide chain of the enzyme.

2. The mutant cytochrome P450 enzyme of claim 1 , wherein the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is from a bacterial or archaeal species.

3. The mutant cytochrome P450 enzyme of claim 1 or claim 2, wherein the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is an extremophilic cytochrome P450 enzyme.

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

5. The mutant cytochrome P450 enzyme of any one of claims 1 to 4, wherein the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme comprising 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 of any one of claims 1 to 5, wherein the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme comprising an amino acid sequence which has at least about 40% amino acid sequence identity to an amino acid sequence set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 31 or SEQ ID NO: 33 to SEQ ID NO: 67, and which comprises the amino acid consensus sequence X1X2X2X3X4X5X6 (SEQ ID NO: 32) in the l-helix of the polypeptide chain, wherein:Xi is Alanine (A) or Glycine (G);X2is Glycine (G) or Alanine (A);X3 is Histidine (H), Asparagine (N), Leucine (L), Alanine (A), Threonine (T), Isoleucine (I), or Phenylalanine (F);X4is Glutamic acid (E), Glycine (G), Leucine (L), or Aspartic acid (D);X5is Threonine (T), Alanine (A), or Asparagine (N);X6is Threonine (T), Isoleucine (I), Valine (V), Serine (S), or Alanine (A); andX7 is Threonine (T), Valine (V), Alanine (A), Tryptophan (W), Arginine (R), Serine (S), and Isoleucine (I).

7. The mutant cytochrome P450 enzyme of any one of claims 1 to 6, wherein the mutant cytochrome P450 enzyme comprises a substitution of an amino acid residue immediately after the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme.

8. The mutant cytochrome P450 enzyme of claim 7, wherein the amino acid residue immediately after the Glutamine and Glutamic acid (QE) amino acid residues is substituted to a Proline (P) amino acid residue.

9. The mutant cytochrome P450 enzyme of claim 7 or claim 8, wherein the mutant cytochrome P450 comprises a substitution of three consecutive amino acid residues at a position corresponding to amino acid residues 23 to 25 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine, Glutamic acid and Proline (QEP) amino acid residues at said position in the l-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 of any one of claims 1 to 6, wherein the mutant cytochrome P450 enzyme comprises a substitution of two consecutive amino acid residues immediately after the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme.

11. The mutant cytochrome P450 enzyme of claim 10, wherein the two consecutive amino acid residues immediately after the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Proline and Glycine (PG) amino acid residues.

12. The mutant cytochrome P450 enzyme of claim 10 or claim 11 , wherein the mutant cytochrome P450 comprises a substitution of four consecutive amino acid residues at aposition corresponding to amino acid residues 23 to 26 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues at said position in the l-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 of any one of claims 1 to 12, wherein the mutant cytochrome P450 enzyme comprises a substitution of three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme.

14. The mutant cytochrome P450 enzyme of claim 13, wherein the three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Glycine, Alanine and X (GAX) amino acid residues, wherein X represents any amino acid.

15. The mutant cytochrome P450 enzyme of claim 13 or claim 14, wherein the three consecutive amino acid residues immediately before the Glutamine and Glutamic acid (QE) amino acid residues are substituted to Glycine, Alanine and Leucine (GAL) or Glycine, Alanine and Histidine (GAH).

16. The mutant cytochrome P450 enzyme of any one of claims 13 to 15, wherein the mutant cytochrome P450 comprises a substitution of five consecutive amino acid residues at a position 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 position in the l-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 of any one of claims 1 to 16, wherein the mutant cytochrome P450 enzyme comprises a substitution of a seven consecutive amino acid section encompassing the Glutamine and Glutamic acid (QE) amino acid residues in the l-helix of the polypeptide chain of the mutant cytochrome P450 enzyme.

18. The mutant cytochrome P450 enzyme of claim 17, wherein the seven consecutive amino acid section is substituted to 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 of claim 17 or claim 18, wherein the mutant cytochrome P450 comprises a substitution of seven consecutive amino acid residues at a position corresponding to amino acid residues 20 to 26 of SEQ ID NO: 1 , and wherein the mutant cytochrome P450 enzyme comprises 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 said position in the l-helix of the polypeptide chain of the enzyme compared to the corresponding amino acid residues in the wild-type cytochrome P450 enzyme.

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

21. The mutant cytochrome P450 enzyme of clam 20, wherein when the mutant cytochrome P450 enzyme is derived from a wild-type cytochrome P450 enzyme which is a member of the CYP1 19 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Threonine (ETTT) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Asparagine, Glutamic acid, and Threonine (AGNET) amino acid residues 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues;in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP119.

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 comprises a substitution of AGNETTT (corresponding to amino acid residues209 to 215 of SEQ ID NO: 53) with GALQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP119A1 .

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 comprises a substitution of AGNETTT (corresponding to amino acid residues210 to 216 of SEQ ID NO: 54) with GALQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP119A2.

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 which is a member of the CYP109 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues, or Glutamic acid and Alanine (EA) amino acid residues, 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, with 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, with 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) amino acid residues, or Alanine, Glycine, Asparagine, Glutamic acid, and Threonine (AGNET) amino acid residues, with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; 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), with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP109.

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

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

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

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 which is a member of the CYP154 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Isoleucine (ETTI) amino acid residues with Glutamine, Glutamic acid, Proline and glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) 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) 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, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP154.

29. 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 comprises a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GALQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP154C8.

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 comprises a substitution of AGHETTI (corresponding to amino acids 254 to 260 of SEQ ID NO: 62) with GAHQEPG in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP154C8.

31. 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 comprises a substitution of ET (corresponding to amino acids 257 and 258 of SEQ ID NO: 62) with QE in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP154C8.

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

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 which is a member of the CYP267 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues, or Glutamic acid and Alanine (EA) amino acid residues, 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, 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, with 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, 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), 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, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP267.

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

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 which is amember of the CYP102 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Serine (ETTS) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP102.

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

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 which is a member of the CYP175 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Valine (ETV) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Valine and Alanine (ETVA) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Histidine, Glutamic acid, and Threonine (AGHET) 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP175.

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

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 which is a member of the CYP199 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Aspartic acid and Threonine (DT) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Aspartic acid, Threonine, and Threonine (DTT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Aspartic acid, Threonine, Threonine and Valine (DTTV) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Glycine, Leucine, Aspartic acid, and Threonine (AGLDT) 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) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues, in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP199.

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

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 which is a member of the CYP231 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Threonine (ETTT) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Glycine, Glycine, Asparagine, Glutamic acid, and Threonine (GGNET) amino acid residues with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; or(v) Glycine, Glycine, Asparagine, Glutamic acid, Threonine, Threonine and Threonine (GGNETTT) amino acid residues (or equivalent amino acid residues) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP231 .

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 which is a member of the CYP107 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Alanine (EA) amino acid residues, or Glutamic acid and Threonine (ET) amino acid residues, 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, 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 Glutamicacid, Threonine, Threonine and Valine (ETTV) amino acid residues, with 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, 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), with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP107.

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

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

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 which is a member of the CYP116 family, the mutant cytochrome P450 enzyme comprises a substitution of:(i) Glutamic acid and Threonine (ET) amino acid residues with Glutamine and Glutamic acid (QE) amino acid residues;(ii) Glutamic acid, Threonine and Threonine (ETT) amino acid residues with Glutamine, Glutamic acid and Proline (QEP) amino acid residues;(iii) Glutamic acid, Threonine, Threonine and Valine (ETTV) amino acid residues with Glutamine, Glutamic acid, Proline and Glycine (QEPG) amino acid residues;(iv) Alanine, Alanine, Histidine, Glutamic acid, and Threonine (AAHET) amino acid residues with Glycine, Alanine, Leucine, Glutamine, and Glutamic acid (GALQE) amino acid residues; or(v) Alanine, Alanine, Histidine, Glutamic acid, Threonine, Threonine and Valine (AAHETTV) amino acid residues (or equivalent amino acid residues) with Glycine, Alanine, Leucine, Glutamine, Glutamic acid, Proline and Glycine (GALQEPG) amino acid residues; in the l-helix of the polypeptide chain of the mutant enzyme compared to the l-helix of wild-type CYP116.

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

47. The use of claim 46, wherein the biocatalytic oxidation of carbon-hydrogen bonds takes place at 1 ,0°C to 99°C.

48. A process for oxidizing a substrate which is an organic compound, comprising oxidizing said organic compound substrate with a mutant cytochrome P450 enzyme of any one of claims 1 to 45.

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

50. The process of claim 49, wherein the peroxide is selected from the group consisting of hydrogen peroxide, t-butyl hydroperoxide and m-chloroperbenzoic acid.51 . The process of claim 50, wherein the peroxide is hydrogen peroxide.

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