Designed broad-specificity peroxygenases

Abstract

Non-natural unspecific peroxygenase proteins comprising at least four mutations from a wild-type sequence are provided. Methods of using the non-natural peroxygenase proteins for terpene oxyfunctionalization are also provided.

Description

DESIGNED BROAD-SPECIFICITY PEROXYGENASESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Israeli Patent Application No. 315983, filed September 26, 2024, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (YEDA-MAR-P-057-PCT.xml; Size: 78,815 bytes; and Date of Creation: September 25, 2025) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of computational protein design.BACKGROUND OF THE INVENTION

[0004] Selective oxyfunctionalization ranks among the most challenging and desirable reactions in synthetic chemistry. These reactions are often critical for selective C-H activation and alkene epoxidation of complex organic molecules.

[0005] Since their discovery in 2004, fungal unspecific peroxygenases (UPOs) have attracted great interest for their ability to perform versatile oxyfunctionalization reactions on a broad scope of substrates. These enzymes have several advantages relative to other versatile oxidases, such as P450s: they are typically stable, use pre-reduced hydrogen peroxide as co-substrate instead of molecular oxygen and expensive reductants such as NAD(P)H, exhibit a broad substrate scope and high turnover numbers (TON) up to 900,00010.

[0006] Oxyfunctionalization of small terpenes, which constitute the largest class of secondary plant metabolites, is of particular interest. Terpenes and their oxyfunctionalized derivatives, terpenoids, frequently showcase pharmacological activity and find applications in the flavor and fragrance industries. Chemo-, regio- and stereoselective oxygenationpresents a challenge for chemical catalysis, given that many terpenes feature multiple sites where C-H hydroxylation and C=C epoxidation which are chemically and energetically almost indistinguishable. Enzymes are naturally stereoselective and can position substrates to promote thermodynamically unfavorable reactions; thus, accurate control of the UPO active-site pocket may enable oxyfunctionalization reactions that overcome the dictates of chemical reactivity. Terpenes, for example rose ketones consisting of ionones and damascenes, can be oxy functionalized by several UPOs with different regioselectivities.

[0007] UPOs can be engineered for overexpression in rapidly proliferating host organisms, such as yeast, through the implementation of protein and signal-peptide engineering and promoter-shuffling techniques. Research has yielded enhanced variants characterized by increased activity, augmented thermo-, pH-, and solvent-stabilities, and large shifts in chemo-, regio-, and stereoselectivities. These studies frequently employed directed evolution, a prominent protein engineering approach that emulates the engineering prowess of natural evolution through iterative rounds of random or semi-rational mutation and selection of variants that exhibit desirable properties.

[0008] Despite yielding adequate results, directed evolution is labor intensive and time consuming. It is especially impractical when seeking enzymes that exhibit improvements across multiple substrates, as mutations that are favorable for one substrate rarely benefit others. Furthermore, measuring oxyfunctionalization activity is not amenable to chromogenic or fluorogenic measurement, limiting options for medium to high throughput screening which is often essential to successful in vitro evolution campaigns. These limitations are currently addressed through “smart” libraries, designed using insights from crystal structures, active-site composition, and mechanistic information. Critically, evolutionary processes rely on iterative selection of mutations that must at least be tolerated, whereas large gains in activity often demand epistatic combinations of mutations that may not be tolerated individually.

[0009] Recent developments in computational protein design and engineering can sometimes address these limitations by enabling one-shot, noniterative design. These methods leverage phylogenetic information and atomistic design calculations applied to crystallographic structures or Al-based model structures. For example, FuncLib is an automated method for designing diverse combinations of multipoint mutations within the active site of an enzyme (International Patent Publication W02020035865). This method uses phylogenetic analysis and Rosetta atomistic design calculations to generate variants that exhibit large diversity in active-site geometry and electrostatics without impairing thestability, foldability and primary activity of the enzyme. The resulting designs may exhibit strong epistatic dependencies among the mutations that are rarely observed in natural and lab-evolved variants; yet these designs can exhibit diverse activities suggesting that FuncLib may uncover sequences and activities that are difficult for evolutionary processes to reach. New peroxygenases variants with expanded specificity and improved function are greatly needed.SUMMARY OF THE INVENTION

[0010] The present invention provides non-natural peroxygenase proteins comprising at least four mutations from a wild-type sequence. Methods of using the non-natural peroxygenase proteins for terpene oxyfunctionalization are also provided.[Oi l] According to a first aspect, there is provided a non-natural unspecific peroxygenase protein comprising an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 51 and comprising at least 2 mutations selected from the group consisting of: mutation of phenylalanine 59 (F59) to tyrosine; mutation of leucine 60 (L60) to phenylalanine or isoleucine; mutation of phenylalanine 63 (F63) to leucine, isoleucine or glutamine; mutation of leucine 86 (L86) to isoleucine, valine or methionine; mutation of alanine 153 (Al 53) to leucine, isoleucine or glutamine; mutation of phenylalanine 154 (Fl 54) to leucine or isoleucine; mutation of tyrosine 156 (Y156) to leucine or isoleucine; mutation of glycine 157 (G157) to alanine or serine; mutation of serine 159 (SI 59) to alanine or tyrosine; and mutation of alanine 161 (A161) to leucine, phenylalanine or methionine.

[0012] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence consisting of SEQ ID NO: 51 and the at least 2 mutations.

[0013] According to some embodiments, the at least 2 mutations is at least 4 mutations.

[0014] According to some embodiments, the non-natural unspecific peroxygenase protein comprises at least one of: mutation of G157 to alanine or serine and mutation of A161 to leucine, phenylalanine or methionine.

[0015] According to some embodiments, the non-natural unspecific peroxygenase protein comprises at least two of: mutation of F63 to leucine, isoleucine or glutamine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0016] According to some embodiments, the non-natural unspecific peroxygenase protein comprises at least two of: mutation of L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0017] According to some embodiments, the non-natural unspecific peroxygenase protein comprises at least three of mutation of F63 to leucine, isoleucine or glutamine; mutation of L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0018] According to some embodiments, the non-natural unspecific peroxygenase protein comprises at least four of mutation of F63 to leucine, isoleucine or glutamine; mutation of L86 to isoleucine, valine or methionine; mutation of F154 to leucine or isoleucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0019] According to some embodiments, the non-natural unspecific peroxygenase protein consists of SEQ ID NO: 51 comprising four mutations selected from mutation of F63 to leucine, isoleucine or glutamine; mutation of L86 to isoleucine, valine or methionine; mutation of F 154 to leucine or isoleucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0020] According to some embodiments, the non-natural unspecific peroxygenase protein comprises mutation of F59 to tyrosine and not comprising mutation of L60, A153, F154, Y156 or S159.

[0021] According to some embodiments, the non-natural unspecific peroxygenase protein comprises mutation of F59 to tyrosine; mutation of F63 to leucine; mutation of G157 alanine and either mutation of L86 to isoleucine or mutation of A161 to phenylalanine.

[0022] According to some embodiments, the non-natural unspecific peroxygenase protein comprises Al 53 mutated to leucine, isoleucine or glutamine or Y156 mutated to leucine or isoleucine, but not both.

[0023] According to some embodiments, the non-natural unspecific peroxygenase protein comprises mutation of L60 to phenylalanine or isoleucine; mutation of G157 to alanine; mutation of Al 61 to leucine, phenylalanine or methionine and either mutation of L86 to isoleucine or mutation of Fl 54 to leucine or isoleucine.

[0024] According to some embodiments, the non-natural unspecific peroxygenase protein comprises mutation of Y156 to leucine or isoleucine and at least three of: mutation of F63 to leucine or isoleucine; mutation of L86 to valine or isoleucine; mutation of Fl 54 to leucine;mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0025] According to some embodiments, the Y156 is mutated to leucine.

[0026] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence selected from SEQ ID NO: 1-50.

[0027] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence consisting of an amino acid sequence selected from SEQ ID NO: 1-50.

[0028] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 4, comprising isoleucine at position 63, leucine at position 154, alanine at position 157 and phenylalanine at position 161 and comprising increased chemoselectivity for the formation of citral A from geraniol as compared to a natural peroxygenase comprising SEQ ID NO: 51.

[0029] According to some embodiments, the non-natural unspecific peroxygenase protein comprises F59, L60, L86, Al 53, Y156, and SI 59.

[0030] According to some embodiments, the non-natural unspecific peroxygenase protein comprises amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 4.

[0031] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence comprising or consisting of SEQ ID NO: 4.

[0032] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 11, comprising isoleucine at position 86, leucine at position 154, leucine at position 156 and phenylalanine at position 161 and comprising at least one of: increased conversion of P-ionone to 4- hydroxy-P-ionone and an increased S:R enantiomer ratio as compared to a natural peroxygenase comprising SEQ ID NO: 51.

[0033] According to some embodiments, the non-natural unspecific peroxygenase protein comprises F59, L60, F63, Al 53, G157, and SI 59.

[0034] According to some embodiments, the non-natural unspecific peroxygenase protein comprises amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 11.

[0035] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence comprising or consisting of SEQ ID NO: 11.

[0036] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 18, comprising phenylalanine at position 60, isoleucine at position 154, alanine at position 157 and leucine at position 161 and comprising at least one of: increased conversion of a-damascone to 3- hydroxy-a-damascone, increased conversion of P-damascone to 4-hydroxy-P-damascone, increased conversion of 6-damascone to 2-hydroxy-6-damascone; increased conversion of P-ionone to 4-hydroxy-P-ionone; and increased regioselectivity for the formation of 3- hydroxy-a-ionone from a-ionone as compared to a natural peroxygenase comprising SEQ ID NO: 51.

[0037] According to some embodiments, the non-natural unspecific peroxygenase protein comprises F59, F63, L86, A153, Y156, and S159.

[0038] According to some embodiments, the non-natural unspecific peroxygenase protein comprises amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 18.

[0039] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence comprising or consisting of SEQ ID NO: 18.

[0040] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 28, comprising leucine at position 63, leucine at position 154, leucine at position 156 and phenylalanine at position 161 and comprising increased chemoselectivity for the formation of 2,3-epoxy nerol from nerol as compared to a natural peroxygenase comprising SEQ ID NO: 51.

[0041] According to some embodiments, the non-natural unspecific peroxygenase protein comprises F59, L60, L86, Al 53, G157, and SI 59.

[0042] According to some embodiments, the non-natural unspecific peroxygenase protein comprises amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 28.

[0043] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence comprising or consisting of SEQ ID NO: 28.

[0044] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 29, comprising isoleucine at position 86, leucine at position 154, leucine at position 156 and alanine at position 157 and comprising increased chemoselectivity for the formation of citral B from nerol as compared to a natural peroxygenase comprising SEQ ID NO: 51.

[0045] According to some embodiments, the non-natural unspecific peroxygenase protein comprises F59, L60, F63, A153, S159 and A161.

[0046] According to some embodiments, the non-natural unspecific peroxygenase protein comprises amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 29.

[0047] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence comprising or consisting of SEQ ID NO: 29.

[0048] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 34, comprising leucine at position 63, isoleucine at position 86, leucine at position 154 and phenylalanine at position 161 and comprising at least one of: increased conversion of P-ionone to 4-hydroxy- P-ionone, an increased S:R enantiomer ratio and increased peroxidase activity as compared to a natural peroxygenase comprising SEQ ID NO: 51.

[0049] According to some embodiments, the non-natural unspecific peroxygenase protein comprises F59, L60, A153, Y156, G157, and S159.

[0050] According to some embodiments, the non-natural unspecific peroxygenase protein comprises amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 34.

[0051] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence comprising or consisting of SEQ ID NO: 34.

[0052] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 45, comprising isoleucine at position 63, leucine at position 156, serine at position 157 and leucine at position 161 and comprising at least one of: increased regioselectivity for the formation of 3-hydroxy-a-ionone from a-ionone, increased regioselectivity for the formation of 3- hydroxy-P-damascone from P-damascone, increased chemoselectivity for the formation of (+)-carveol from (R)-(+)limonene, and increased formation of isopiperitenol from (S)-(-)- limonene as compared to a natural peroxygenase comprising SEQ ID NO: 51.

[0053] According to some embodiments, the non-natural unspecific peroxygenase protein comprises F59, L60, L86, A153, F154 and S159.

[0054] According to some embodiments, the non-natural unspecific peroxygenase protein comprises amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 45.

[0055] According to some embodiments, the non-natural unspecific peroxygenase protein comprises an amino acid sequence comprising or consisting of SEQ ID NO: 45. /

[0056] According to another aspect, there is provided a method of terpene oxyfunctionalization, the method comprising contacting the terpene with a non-natural unspecific peroxygenase protein of the invention.

[0057] According to some embodiments, the terpene is selected from geraniol, nerol, S-(-)- limonene, R-(+)-limonene, a-damascone, P-damascone, 6-damascone, a-ionone and P- ionone.

[0058] According to some embodiments, the method is a method of producing a molecule selected from: 4-epoxy-a-damascone, 3-hydroxy-a-damascone, 4-CO-P-damascone, 4- hydroxy-P-damascone, 3-hydroxy-P-damascone, 2-hydroxy- 6-damascone, S-(-)-limonene, R-(+)-limonene, 3-hydroxy-ot-ionone, limonene oxide, (+)-carveol, isopiperitenol, 4- hydroxy-P-ionone, 2-hydroxy-P-ionone, citral A, citral B, 2,3-epoxy geraniol, 2,3-epoxy nerol, 7,1 l-epoxymega-stigma-5(6)-en-9-one, 6,7-epoxynerol and 6,7-epoxygeraniol.

[0059] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1: Terpene Substrates investigated in this study.

[0061] Figure 2: Total activity (sum of all products) in relation to the activity of wildtype (wt) / rUPO for all active substrates.

[0062] Figures 3A-3D: Relative abundance of selected products after conversion with different FuncLib designs. All data are analyzed by GC-MS. (3A) Conversion of a-, P- and 6-damascone. (3B) Conversion of (S)-limonene and (R)-limonene, stacked bar chart displays the relative abundance of products after conversion of (R)-limonene leading to carveol (22) (orange), limonene oxide (21) (red), mix lim-ol (23 / 24) (blue) and others (grey). Isopiperitenol elutes on the GC-MS simultaneously with a second limonene alcohol (presumambly limonene- lO-ol), elution peaks are not separable, but mass spectra indicate the presence of two different moieties, the sum of both products is referred to as mix lim-ol. (3C) Conversion of a-ionone. (3D) Conversion of P-ionone reveals differing relative productabundance, and increased enzyme activity compared to wildtype MthUPO. All measurements were performed in triplicates utilizing enzymes in supernatant from a microtiter plate.

[0063] Figures 4A-4G: Oxyfunctionalization of geraniol (1) and nerol (2) with FuncLib designs of MthUPO. (4A) Proportion of 2,3 -epoxide (32 / 34) (blue) and aldehyde (31 / 33) (orange) formation in the overall reaction. (4B) Regioselectivity of selected enzyme variants toward citral A (33) / citral B (31), neric acid and 2,3 -epoxy (32 / 34) and 6,7-epoxy formation. (4C) Turnover number of selected products, data are mean ± s.d. of measurements in triplicates. (4D-4G) Most representative structure of the binding mode of geraniol / nerol in FuncLib designs (4D) 2, (4E) 4, (4F) 26 and (4G) 28 as determined by clustering analysis.

[0064] Figures 5A-5B: Influence of FuncLib designs on stereoselectivity. (5A) Shifts in the diastereomeric ratio of trans-limonene oxide (orange) and cis-limonene oxide (blue). Measurements were performed as unique samples with enzyme from supernatant in microtiter plate scale. (5B) Shifts in enantiomeric ratio of 4-hydroxy-P-ionone (28) giving access to S-hydroxy-P-ionone with Var 11 and Var 34.DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention, in some embodiments, provides non-natural unspecific peroxygenase proteins. Methods of using the non-natural unspecific peroxygenase proteins for terpene oxyfunctionalization are also provided.

[0066] The invention is based, at least in part, on the generation of 50 MthUPO FuncLib designs. These variants were tested on several terpenes, including limonene, which displays poor starting regioselectivity, and geraniol and nerol with their products citral A and B, valuable compounds of the fragrance industry. All 50 FuncLib designs proved to be functionally secreted from yeast, allowing the comparison of their diverse influence on the various substrates and find large activity improvements and substantial shifts in chemo-, regio- and stereoselectivity through a limited experimental screening effort.

[0067] All FuncLib designs tested were functional and exhibited large and potentially useful changes in activity and selectivity profiles. Screening 50 designed variants in the current study led to substantial improvements in activity, chemo-, regio- and stereoselectivity such as >99 % chemo- and regioselectivity for the fragrant citral A (starting from 40 % for wildtype MthUPO), more than 1,900-fold activity increase for ABTS, variants with at leastdoubled activity for all substrates, 98 % regioselectivity and an inversion of enantioselectivity to an e.r. of 1 :99 (starting from 76:24) for (S)-4-hydroxy-P-ionone as well as novel products and significant regioselectivity shifts with limonene.

[0068] By a first aspect, there is provided a non-natural unspecific peroxygenase protein.

[0069] In some embodiments, the protein is a variant. In some embodiments, the protein is a mutant. In some embodiments, the protein is not naturally occurring. In some embodiments, the protein is derived from a naturally occurring unspecific peroxygenase. In some embodiments, the natural unspecific peroxygenase is a fungal unspecific peroxygenase (UPO). In some embodiments, the UPO is Myceliophthora thermophila UPO (AY / AUPO). In some embodiments, the amino acid sequence of wild-type MthUPO comprises SEQ ID NO: 51. In some embodiments, the amino acid sequence of wild-type MthUPO consists of SEQ ID NO: 51.

[0070] In some embodiments, the non-natural protein comprises at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity to the wild-type UPO. Each possibility represents a separate embodiment of the invention. In some embodiments, the non-natural protein comprises at least 85% sequence identity to the wild-type UPO. In some embodiments, the non-natural protein comprises at least 90% sequence identity to the wildtype UPO. In some embodiments, the non-natural protein comprises at least 95% sequence identity to the wild-type UPO. In some embodiments, the non-natural protein comprises at least 98% sequence identity to the wild-type UPO. In some embodiments, the non-natural protein comprises at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, or 98% sequence identity to SEQ ID NO: 51. Each possibility represents a separate embodiment of the invention. In some embodiments, the non-natural protein comprises at least 85% sequence identity to SEQ ID NO: 51. In some embodiments, the non-natural protein comprises at least 90% sequence identity to SEQ ID NO: 51. In some embodiments, the non-natural protein comprises at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, the non- natural protein comprises at least 98% sequence identity to SEQ ID NO: 51.

[0071] In some embodiments, the non-natural protein comprises at least one mutation in the wild-type sequence. In some embodiments, the non-natural protein comprises at least one mutation in SEQ ID NO: 51. In some embodiments, at least one is at least 2. In some embodiments, at least one is at least 3. In some embodiments, the at least one is at least 4. In some embodiments, at least 4 is 4. In some embodiments, the at least one mutation is within a substrate binding pocket of the protein. In some embodiments, the at least one mutation iswithin the catalytic site of the protein. In some embodiments, the substrate is a terpene. In some embodiments, the substrate binding pocket or catalytic site comprises amino acids 59, 60, 63, 86, 153, 154, 156, 159, 161, 206 and 210 of SEQ ID NO: 51. In some embodiments, the substrate binding pocket or catalytic site comprises amino acids 59, 60, 63, 86, 153, 154, 156, 159, and 161 of SEQ ID NO: 51. In some embodiments, the substrate is a terpene. In some embodiments, the substrate binding pocket or catalytic site comprises amino acids 55- 64, and 152-162. In some embodiments, the substrate is a terpene. In some embodiments, the substrate binding pocket or catalytic site comprises amino acids 55-64, 85-89, and 152- 162. In some embodiments, the substrate is a terpene. In some embodiments, the substrate binding pocket or catalytic site comprises amino acids 55-64, 85-89, 152-162 and 205-211. In some embodiments, the at least one mutation is within 8 angstroms of the substrate binding pocket. In some embodiments, the at least one mutation is within 5-8 angstroms of the substrate binding pocket. In some embodiments, the at least one mutation is within the substrate binding pocket or a first shell around the substrate binding pocket. In some embodiments, the first shell is a radius of 8 angstroms. In some embodiments, the first shell is a radius of 5-8 angstroms. In some embodiments, the mutation is not of a charged amino acid. In some embodiments, the mutation is not a mutation of any one of arginine, histidine lysine, glutamic acid and aspartic acid. In some embodiments, the mutation is not a mutation of any one of arginine, histidine lysine, glutamic acid, aspartic acid and threonine.

[0072] In some embodiments, the at least one mutation is mutation of phenylalanine 59 (F59) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of F59 of SEQ ID NO: 51. In some embodiments, F59 is mutated to tyrosine (F59Y). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 27 or 35. In some embodiments, F59 is not mutated to glutamine (F59Q).

[0073] In some embodiments, the at least one mutation is mutation of leucine 60 (L60) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of L60 of SEQ ID NO: 51. In some embodiments, L60 is mutated to phenylalanine (L60F). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 18, 32, or 33. In some embodiments, L60 is mutated to isoleucine (L60I). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, L60 is mutated to isoleucine or phenylalanine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 18, 19, 32 or 33. In some embodiments, L60 is not mutated to methionine (L60M).

[0074] In some embodiments, the at least one mutation is mutation of phenylalanine 63 (F63) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of F63 of SEQ ID NO: 51. In some embodiments, F63 is mutated to leucine (F63L). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2, 3, 6, 7, 12, 14, 15, 16, 17, 25, 27, 28, 31, 34, 35, 36, 38, 42, 43, 44, 47 or 49. In some embodiments, F63 is mutated to isoleucine (F63I). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 4, 20,24, 26 or 45. In some embodiments, F63 is mutated to glutamine (F63Q). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 13, 40 or 46. In some embodiments, F63 is mutated to leucine, isoleucine or glutamine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 6, 7, 12, 13, 14, 15, 16, 17, 20, 24, 25, 26, 27, 28, 31, 34, 35, 36, 38, 42, 43, 44, 45, 46, 47 or 49.

[0075] In some embodiments, the at least one mutation is mutation of leucine 86 (L86) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of L86 of SEQ ID NO: 51. In some embodiments, L86 is mutated to isoleucine (L86I). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 5, 7, 10, 11, 14, 16, 19, 22, 29, 30, 31, 33, 34, 35, 36, 37, 38, 40, 46 or 49. In some embodiments, L86 is mutated to valine (L86V). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 6, 9, 13, 15, 17,25, 39, or 41. In some embodiments, L86 is mutated to methionine (L86M). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, L86 is mutated to valine, isoleucine or methionine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 22, 25, 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 46 or 49. In some embodiments, L86 is not mutated to an aromatic amino acid. In some embodiments, L86 is not mutated to any of histidine, phenylalanine, tryptophan and tyrosine.

[0076] In some embodiments, the at least one mutation is mutation of alanine 153 (Al 53) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of Al 53 of SEQ ID NO: 51. In some embodiments, Al 53 is mutated to leucine (A153L). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 5, 15, 41, or 43. In some embodiments, A153 is mutated to isoleucine (A153I). In some embodiments, the non-natural protein comprises or consists of the amino acidsequence of SEQ ID NO: 2, 36, 38, 39 or 48. In some embodiments, A153 is mutated to glutamine (A153Q). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 42 or 50. In some embodiments, Al 53 is mutated to leucine, isoleucine or glutamine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 2, 5, 15, 36, 38, 39, 41, 42, 43, 48 or 50.

[0077] In some embodiments, the at least one mutation is mutation of phenylalanine 154 (Fl 54) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of F154 of SEQ ID NO: 51. In some embodiments, F154 is mutated to leucine (F154L). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 8, 9, 11, 23, 28, 29, 32, 37, 44, 48 or 49. In some embodiments, F154 is mutated to isoleucine (Fl 541). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 2, 10, 18, 20, 26, 34, 47 or 50. In some embodiments, Fl 54 is mutated to leucine or isoleucine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2, 4, 8, 9, 10, 11, 18, 20, 23, 26, 28, 29, 32, 34, 37, 44, 47, 48, 49 or 50. In some embodiments, Fl 54 is not mutated to alanine (F154A).

[0078] In some embodiments, the at least one mutation is mutation of tyrosine 156 (Y156) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of Y156 of SEQ ID NO: 51. In some embodiments, Y156 is mutated to leucine (Y156L). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 3, 6, 8, 11, 16, 21, 22, 24, 28, 29, 30, 44, 45. In some embodiments, Y156 is mutated to isoleucine (Y156I). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, Y156 is mutated to leucine or isoleucine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 3, 6, 8, 11, 16, 21, 22, 23, 24, 28, 29,30, 44, 45.

[0079] In some embodiments, the at least one mutation is mutation of glycine 157 (G157) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of G157 of SEQ ID NO: 51. In some embodiments, G157 is mutated to alanine (G157A). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 15, 17, 18, 19, 21, 23, 24, 25, 26, 27, 29, 30,31, 32, 33, 35, 41, 42, 43, 46, 48 or 50. In some embodiments, G157 is mutated to serine (G157S). In some embodiments, the non-natural protein comprises or consists of the aminoacid sequence of SEQ ID NO: 20, 22, 36, 37, 39, 40, 44, 45, 47 or 49. In some embodiments, G157 is mutated to alanine or serine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, G157 is not mutated to any amino acids other than alanine or serine.

[0080] In some embodiments, the at least one mutation is mutation of serine 159 (SI 59) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of SI 59 of SEQ ID NO: 51. In some embodiments, SI 59 is mutated to alanine (S159A). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 14, 21 or 25. In some embodiments, S159 is mutated to tyrosine (S159Y). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 31. In some embodiments, SI 59 is mutated to alanine or tyrosine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 14, 21, 25 or 31. In some embodiments, S159 is not mutated to glycine (S159G).

[0081] In some embodiments, the at least one mutation is mutation of alanine 161 (A161) of the wild-type UPO. In some embodiments, the at least one mutation is mutation of Al 61 of SEQ ID NO: 51. In some embodiments, Al 61 is mutated to leucine (Al 6 IL). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, 17, 18, 22, 26, 41, 43, 45 or 46. In some embodiments, A161 is mutated to phenylalanine (Al 6 IF). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 3, 4, 5, 7, 11, 16, 19, 21, 23, 27, 28, 34, 37, 38,39, 40, 42, 47 or 50. In some embodiments, A161 is mutated to methionine (A161M). In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 8, 9, 10, 12, 13, 14, 20, 24, 30, 32, 33 or 48. In some embodiments, A161 is mutated to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises or consists of the amino acid sequence of SEQ ID NO: 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 30, 32, 33, 35, 37, 38, 39,40, 41, 42, 43, 45, 46, 47, 48 or 50. In some embodiments, A161 is not mutated to valine (A161V).

[0082] In some embodiments, leucine 206 (L206) is not mutated. In some embodiments, L206 is not mutated to alanine (L206A). In some embodiments, L206 is also mutated. In some embodiments, methionine 210 (M210) is not mutated. In some embodiments, M210 isalso mutated. In some embodiments, glutamic acid 158 (El 58) is not mutated. In some embodiments, El 58 is not mutated to aspartic acid (E158D). In some embodiments, histidine 88 (H88) is not mutated. In some embodiments, H88 is not mutated to alanine (H88A). In some embodiments, arginine 47 (R47) is not mutated. In some embodiments, R47 is not mutated to leucine (R47L). In some embodiments, tyrosine 51 (Y51) is not mutated. In some embodiments, Y51 is not mutated to phenylalanine (Y51F). In some embodiments, phenylalanine 87 (F87) is not mutated. In some embodiments, F87 is not mutated to valine (F87V).

[0083] In some embodiments, the non-natural protein comprises at least four mutations selected from the group consisting of: mutation of phenylalanine 59 (F59) to tyrosine; mutation of leucine 60 (L60) to phenylalanine or isoleucine; mutation of phenylalanine 63 (F63) to leucine, isoleucine or glutamine; mutation of leucine 86 (L86) to isoleucine, valine or methionine; mutation of alanine 153 (Al 53) to leucine, isoleucine or glutamine; mutation of phenylalanine 154 (Fl 54) to leucine or isoleucine; mutation of tyrosine 156 (Y156) to leucine or isoleucine; mutation of glycine 157 (G157) to alanine or serine; mutation of serine 159 (S159) to alanine or tyrosine; and mutation of alanine 161 (A161) to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises at least four mutations selected from the group consisting of: mutation of leucine 60 (L60) to phenylalanine or isoleucine; mutation of phenylalanine 63 (F63) to leucine, isoleucine or glutamine; mutation of leucine 86 (L86) to isoleucine, valine or methionine; mutation of alanine 153 (Al 53) to leucine, isoleucine or glutamine; mutation of phenylalanine 154 (Fl 54) to leucine or isoleucine; mutation of tyrosine 156 (Y156) to leucine or isoleucine; mutation of glycine 157 (G157) to alanine or serine; mutation of serine 159 (SI 59) to alanine or tyrosine; and mutation of alanine 161 (A161) to leucine, phenylalanine or methionine.

[0084] In some embodiments, the non-natural protein comprises at least one mutation selected from: mutation of G157 to alanine or serine and mutation of A161 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises at least four mutations and at least one of the four is selected from: mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises four mutations and at least one of the four is selected from: mutation of G157 to alanine or serine and mutation of A161 to leucine, phenylalanine or methionine.

[0085] In some embodiments, the non-natural protein comprises at least two mutations selected from: mutation of F63 to leucine, isoleucine or glutamine; mutation of G157 toalanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises at least four mutations and at least two of the four are selected from: mutation of F63 to leucine, isoleucine or glutamine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises four mutations and at least two of the four are selected from: mutation of F63 to leucine, isoleucine or glutamine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0086] In some embodiments, the non-natural protein comprises at least two mutations selected from: mutation of L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises at least four mutations and at least two of the four are selected from: mutation of L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises four mutations and at least two of the four are selected from: mutation of L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0087] In some embodiments, the non-natural protein comprises at least three mutations selected from: mutation of F63 to leucine, isoleucine or glutamine; L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises at least four mutations and at least three of the four are selected from: mutation of F63 to leucine, isoleucine or glutamine; L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises four mutations and at least three of the four are selected from: mutation of F63 to leucine, isoleucine or glutamine; L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of A161 to leucine, phenylalanine or methionine.

[0088] In some embodiments, the non-natural protein comprises at least four mutations selected from: mutation of F63 to leucine, isoleucine or glutamine; L86 to isoleucine, valine or methionine; mutation of Fl 54 to leucine or isoleucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises at least four mutations and at least four of the four are selected from: mutation of F63 to leucine, isoleucine or glutamine; L86 to isoleucine, valine or methionine; mutation of Fl 54 to leucine or isoleucine; mutation of G157 to alanine orserine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises four mutations selected from: mutation of F63 to leucine, isoleucine or glutamine; L86 to isoleucine, valine or methionine; mutation of Fl 54 to leucine or isoleucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0089] In some embodiments, the non-natural protein comprises an amino acid sequence consisting of SEQ ID NO: 51 and at least one mutation. In some embodiments, the non- natural protein comprises an amino acid sequence consisting of SEQ ID NO: 51 and at least two mutations. In some embodiments, the non-natural protein comprises an amino acid sequence consisting of SEQ ID NO: 51 and at least three mutations. In some embodiments, the non-natural protein comprises an amino acid sequence consisting of SEQ ID NO: 51 and at least four mutations. In some embodiments, the non-natural protein comprises an amino acid sequence consisting of SEQ ID NO: 51 and four mutations. In some embodiments, the non-natural protein consists of SEQ ID NO: 51 comprising four mutations selected from mutation of F63 to leucine, isoleucine or glutamine; mutation of L86 to isoleucine, valine or methionine; mutation of Fl 54 to leucine or isoleucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

[0090] In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine and does not comprise mutation of L60. In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine and does not comprise mutation of A153. In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine and does not comprise mutation of Fl 54. In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine and does not comprise mutation of Y156. In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine and does not comprise mutation of S159. In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine (F59Y) and does not comprise mutation of L60, A153, F154, Y156 or S159. In some embodiments, the non-natural protein comprises F59Y and L60, A153, F154, Y156 and SI 59. In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine, mutation of F63 to leucine; mutation of G157 alanine and mutation of L86 to isoleucine. In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine, mutation of F63 to leucine; mutation of G157 alanine and mutation of Al 61 to phenylalanine. In some embodiments, the non-natural protein comprises mutation of F59 to tyrosine, mutation of F63 to leucine; mutation of G157 alanine and either mutation of L86 to isoleucine or mutation of Al 61 to phenylalanine.

[0091] In some embodiments, the non-natural protein comprises A153 mutated to leucine, isoleucine or glutamine or Y156 mutated to leucine or isoleucine, but not both. In some embodiments, the non-natural protein comprises a mutation of Al 53 and does not comprise a mutation of Y156. In some embodiments, the non-natural protein comprises a mutation of Y156 and does not comprise a mutation of Al 53. In some embodiments, the non-natural protein comprises Al 53 mutated to leucine, isoleucine or glutamine and comprises Y156. In some embodiments, the non-natural protein comprises Y156 mutated to leucine or isoleucine and comprises Al 53.

[0092] In some embodiments, the non-natural protein comprises mutation of L60 to phenylalanine or isoleucine; mutation of G157 to alanine; mutation of Al 61 to leucine, phenylalanine or methionine and mutation of L86 to isoleucine. In some embodiments, the non-natural protein comprises mutation of L60 to phenylalanine or isoleucine; mutation of G157 to alanine; mutation of Al 61 to leucine, phenylalanine or methionine and mutation of Fl 54 to leucine or isoleucine. In some embodiments, the non-natural protein comprises mutation of L60 to phenylalanine or isoleucine; mutation of G157 to alanine; mutation of A161 to leucine, phenylalanine or methionine and either mutation of L86 to isoleucine or mutation of Fl 54 to leucine or isoleucine. In some embodiments, the non-natural protein does not comprise mutation of L60 to phenylalanine (L60F) and mutation of F63. In some embodiments, the non-natural protein does not comprise L60F and F63I.

[0093] In some embodiments, the non-natural protein comprises mutation of Y156 to leucine or isoleucine and at least three of: mutation of L86 to valine or isoleucine; mutation of Fl 54 to leucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises mutation of Y156 to leucine or isoleucine and at least three of: mutation of F63 to leucine or isoleucine; mutation of Fl 54 to leucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises mutation of Y156 to leucine or isoleucine and at least three of: mutation of F63 to leucine or isoleucine; mutation of L86 to valine or isoleucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises mutation of Y156 to leucine or isoleucine and at least three of: mutation of F63 to leucine or isoleucine; mutation of L86 to valine or isoleucine; mutation of Fl 54 to leucine; and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, the non-natural protein comprises mutation of Y156 to leucine or isoleucine and at least three of: mutation of F63 to leucine or isoleucine; mutationof L86 to valine or isoleucine; mutation of Fl 54 to leucine; and mutation of G157 to alanine or serine. In some embodiments, the non-natural protein comprises mutation of Y156 to leucine or isoleucine and at least three of: mutation of F63 to leucine or isoleucine; mutation of L86 to valine or isoleucine; mutation of Fl 54 to leucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine. In some embodiments, Y156 mutated to leucine or isoleucine is Y156L.

[0094] In some embodiments, the non-natural protein comprises an amino acid sequence selected from SEQ ID NO: 1-50. In some embodiments, the non-natural protein consists of an amino acid sequence selected from SEQ ID NO: 1-50. In some embodiments, the non- natural protein comprises the amino acid sequence provided in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. Each possibility represents a separate embodiment of the invention. In some embodiments, the non-natural protein comprises amino acids 55-64, 85-89 and 152-162 or any one of SEQ ID NO: 1-50. In some embodiments, a non-natural protein comprising a cutoff level of identity with any one of SEQ ID NO: 1-50 comprises all of amino acids 55-64, 85-89 and 152-162 of the sequence.

[0095] In some embodiments, the non-natural protein comprises a threshold identity to SEQ ID NO: 51 and comprises the four mutations present in any one of SEQ ID NO: 1-50. In some embodiments, the non-natural protein comprises a threshold identity to any one of SEQ ID NO: 1-50 and comprises the four mutations that distinguish the sequence from SEQ ID NO: 51. It will be understood by a skilled artisan that additional mutations can be made throughout the body of the protein so long as the four mutations that identify each of SEQ ID NO: 1-50 are retained. In some embodiments, the threshold identity is 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97 or 98% identity. Each possibility represents a separate embodiment of the invention. In some embodiments, the threshold identity is 85%. In some embodiments, the threshold identity is 90%. In some embodiments, the threshold identity is 95%.

[0096] In some embodiments, the additional mutations are to L206, M210 or both. In some embodiments, the additional mutations are made outside of the substrate binding pocket. In some embodiments, the additional mutations are made outside of the substrate binding pocket or are to L206 or M210. In some embodiments, the additional mutations are made outside the substrate binding pocket and a shell around the pocket. In some embodiments, the additional mutations are made outside the substrate binding pocket and a shell around the pocket or are to L206 or M210. L206 and M210 were found to be substitutable positionsand thus mutation of them is possible. However, in the 50 Funclib designs no mutations at these positions occurred. In some embodiments, the shell is a radius of 8 angstroms. In some embodiments, the shell is 5-8 angstroms around the pocket. In some embodiments, the binding pocket comprises amino acids 59, 60, 63, 86, 153, 154, 156, 159, 161, 206 and 210 of SEQ ID NO: 51. In some embodiments, the binding pocket comprises amino acids 59, 60,63, 86, 153, 154, 156, 159, and 161 of SEQ ID NO: 51. In some embodiments, the binding pocket comprises amino acids 59, 60, 63, 86, 153, 154, 156, 159, 161, 206 and 210 of any one of SEQ ID NO: 1-50. In some embodiments, the binding pocket comprises amino acids 59, 60, 63, 86, 153, 154, 156, 159, and 161 of any one of SEQ ID NO: 1-50. In some embodiments, the substrate binding pocket comprises amino acids 55-64, 85-89, 152-162 and 205-211. In some embodiments, the substrate binding pocket comprises amino acids 55-64, 85-89, and 152-162. In some embodiments, the additional mutations are not to amino acids Cl 8, El 58 or H88. In some embodiments, the additional mutations are not to amino acids P17, C18, P19, E158 or H88. C18 (axial ligand) and E158 and H88 (catalytic cascade) were considered unsubstitutable positions and thus should not be modified in any future optimization of the sequence. The prolines sandwiching C18 are also functional and so may also be defined as unsubstitutable. As such, it will be understood that further mutations can be made throughout the body of the variant (such as by algorithms that improve stability and the like, e.g., the PROSS algorithm) so long as amino acids C18, E158 and H88 are not modified and such that the substrate binding pocket are not modified other than L206 and M2 10. In some embodiments, L206 and M210 also may not be modified.

[0097] In some embodiments, the non-natural protein comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the non-natural protein consists of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 4. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 4 and comprises isoleucine at position 63, leucine at position 154, alanine at position 157 and phenylalanine at position 161. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 4 and comprises F59, L60, isoleucine at position 63, L86, A153, leucine at position 154, Y156, alanine at position 157, S159 and phenylalanine at position 161. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 4 comprises amino acids 59-86 and 153-161 of SEQ ID NO: 4. In some embodiments, the non-natural protein with a threshold identity to SEQ IDNO: 4 comprises increased peroxygenase activity. In some embodiments, peroxygenase activity is activity against a terpene. In some embodiments, increased activity is increased conversion of a terpene to an oxyfunctionalized molecule. In some embodiments, the nonnatural protein with a threshold identity to SEQ ID NO: 4 increases chemoselectivity for the formation of citral A from geraniol. In some embodiments, increases as compared to wildtype peroxygenase. In some embodiments, increases as compared to SEQ ID NO: 51.

[0098] In some embodiments, increases is substantially increases. In some embodiments, increases is detectably increases. In some embodiments, increases is significantly increases. In some embodiments, increases is by at least 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450 or 500%. Each possibility represents a separate embodiment of the invention. In some embodiments, increased is enhanced. In some embodiments, enhanced comprises increased chemoselectivity. In some embodiments, enhanced comprises increased regioselectivity. In some embodiments, enhanced comprises increased S:R enantiomer ratio in the produced molecule. In some embodiments, enhanced comprises production of a novel output molecule. In some embodiments, the novel output molecule is isopiperitenol.

[0099] In some embodiments, the non-natural protein comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the non-natural protein consists of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 11. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 11 and comprises isoleucine at position 86, leucine at position 154, leucine at position 156 and phenylalanine at position 161. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 11 and comprises F59, L60, F63, 186, A153, L154, LI 56, G157, SI 59 and F 161. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 11 comprises amino acids 59-86 and 153-161 of SEQ ID NO: 11. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 11 comprises increased peroxygenase activity. In some embodiments, peroxygenase activity is activity against a terpene. In some embodiments, increased activity is increased conversion of a terpene to an oxyfunctionalized molecule. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 11 increases conversion of P-ionone to 4-hydroxy- P-ionone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 11 increases increased S:R enantiomer ratio. In some embodiments, S:R enantiomerratio is in the produced molecule. In some embodiments, S:R ratio is for4-hydroxy-P-ionone. In some embodiments, the produced molecule is the produced oxyfunctionalized molecule. In some embodiments, increases as compared to wild-type peroxygenase. In some embodiments, increases as compared to SEQ ID NO: 51.

[0100] In some embodiments, the non-natural protein comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the non-natural protein consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 18. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 18 and comprises phenylalanine at position 60, isoleucine at position 154, alanine at position 157 and leucine at position 161. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 18 and comprises F59, F60, F63, L86, A153, 1154, Y156, Al 57, SI 59 and L161. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 comprises amino acids 59-86 and 153-161 of SEQ ID NO: 18. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 comprises increased peroxygenase activity. In some embodiments, peroxygenase activity is activity against a terpene. In some embodiments, increased activity is increased conversion of a terpene to an oxyfunctionalized molecule. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 increases oxyfunctionalization of 3-hydroxy-a- damascone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 increases oxyfunctionalization of 4-hydroxy-P-damascone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 increases oxyfunctionalization of 2-hydroxy-6-damascone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 increases conversion of a-damascone to 3-hydroxy-a-damascone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 increases conversion of P-damascone to 4-hydroxy-P-damascone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 increases conversion of 6-damascone to 2-hydroxy-6-damascone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 increases conversion of P- ionone to 4-hydroxy-P-ionone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 18 increases regioselectivity for the formation of 3- hydroxy-a-ionone from a-ionone. In some embodiments, increases as compared to wild-type peroxygenase. In some embodiments, increases as compared to SEQ ID NO: 51.

[0101] In some embodiments, the non-natural protein comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the non-natural protein consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 28. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 28 and comprises leucine at position 63, leucine at position 154, leucine at position 156 and phenylalanine at position 161. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 28 and comprises F59, L60, L63, L86, A153, L154, L156, G157, S159, F161. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 28 comprises amino acids 59-86 and 153-161 of SEQ ID NO: 28. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 28 comprises increased peroxygenase activity. In some embodiments, peroxygenase activity is activity against a terpene. In some embodiments, increased activity is increased conversion of a terpene to an oxyfunctionalized molecule. In some embodiments, the non- natural protein with a threshold identity to SEQ ID NO: 28 increases chemoselectivity for the formation of 2,3 -epoxy nerol from nerol. In some embodiments, increases as compared to wild-type peroxygenase. In some embodiments, increases as compared to SEQ ID NO: 51.

[0102] In some embodiments, the non-natural protein comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the non-natural protein consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 29. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 29 and comprises isoleucine at position 86, leucine at position 154, leucine at position 156 and alanine at position 157. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 29 and comprises F59, L60, F63, 186, Al 53, LI 54, LI 56, Al 57, SI 59 and A161. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 29 comprises amino acids 59-86 and 153-161 of SEQ ID NO: 29. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 29 comprises increased peroxygenase activity. In some embodiments, peroxygenase activity is activity against a terpene. In some embodiments, increased activity is increased conversion of a terpene to an oxyfunctionalized molecule. In some embodiments, the non-natural proteinwith a threshold identity to SEQ ID NO: 29 increases chemoselectivity for the formation of citral B from nerol. In some embodiments, increases as compared to wild-type peroxygenase. In some embodiments, increases as compared to SEQ ID NO: 51.

[0103] In some embodiments, the non-natural protein comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the non-natural protein consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 34. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 34 and comprises leucine at position 63, isoleucine at position 86, leucine at position 154 and phenylalanine at position 161. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 34 and comprises F59, L60, L63, 186, A153, L154, Y156, G157, SI 59 and F161. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 34 comprises amino acids 59-86 and 153-161 of SEQ ID NO: 34. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 34 comprises increased peroxygenase activity. In some embodiments, peroxygenase activity is activity against a terpene. In some embodiments, increased activity is increased conversion of a terpene to an oxyfunctionalized molecule. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 34 increases co conversion of P-ionone to 4- hydroxy-P-ionone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 34 increases increased S:R enantiomer ratio. In some embodiments, S:R enantiomer ratio is in the produced molecule. In some embodiments, S:R ratio is for 4- hydroxy-P-ionone. In some embodiments, the produced molecule is the produced oxyfunctionalized molecule. In some embodiments, increases as compared to wild-type peroxygenase. In some embodiments, increases as compared to SEQ ID NO: 51.

[0104] In some embodiments, the non-natural protein comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the non-natural protein consists of the amino acid sequence of SEQ ID NO: 45. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 45. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 45 and comprises isoleucine at position 63, leucine at position 156, serine at position 157 and leucine at position 161. In some embodiments, the non-natural protein comprises the amino acid sequence with at least a threshold level of identity to SEQ ID NO: 45 and comprises F59, L60, L63, L86, A153, F154, L156, S157,S159 and L161. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 45 comprises amino acids 59-86 and 153-161 of SEQ ID NO: 45. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 45 comprises increased peroxygenase activity. In some embodiments, peroxygenase activity is activity against a terpene. In some embodiments, increased activity is increased conversion of a terpene to an oxyfunctionalized molecule. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 45 increases regioselectivity for the formation of 3-hydroxy-a-ionone from a-ionone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 45 increases regioselectivity for the formation of 3- hydroxy-P-damascone from P-damascone. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 45 increases chemoselectivity for the formation of (+)-carveol from (R)-(+)limonene. In some embodiments, the non-natural protein with a threshold identity to SEQ ID NO: 45 increases formation of isopiperitenol from (S)-(-)- limonene. In some embodiments, increases as compared to wild-type peroxygenase. In some embodiments, increases as compared to SEQ ID NO: 51.

[0105] By another aspect, there is provided a method of terpene oxyfunctionalization, comprising contacting the terpene with a non-natural peroxygenase protein of the invention, thereby oxyfunctionalizing a terpene. By another aspect, there is provided a method of oxyfunctionalization a terpene, comprising contacting the terpene with a non-natural peroxygenase protein of the invention, thereby oxy functionalizing a terpene.

[0106] In some embodiments, the terpene is selected from geraniol, nerol, S-(-)-limonene, limonene, a-damascone, P-damascone, 6-damascone, a-ionone, and P-ionone. In some embodiments, limonene is selected from (R)-(+)-limonene and (S)-(-)-limonene. In some embodiments, the terpene is geraniol. In some embodiments, the terpene is nerol. In some embodiments, the terpene is limonene. In some embodiments, the terpene is S-(-)-limonene. In some embodiments, the terpene is R-(+)-limonene. In some embodiments, the terpene is a-damascone. In some embodiments, the terpene is P-damascone. In some embodiments, the terpene is 6-damascone. In some embodiments, the terpene is a-ionone. In some embodiments, the terpene is P-ionone.

[0107] In some embodiments, the method is a method of producing an output molecule selected from: 4-epoxy-a-damascone, 3-hydroxy-a-damascone, 4-CO-P-damascone (4-oxo- P-damascone), 4-hydroxy-P-damascone, 3-hydroxy-P-damascone, 2-hydroxy- 6- damascone, 3-hydroxy-a-ionone, limonene oxide, (+)-carveol, isopiperitenol, 4-hydroxy-P- ionone, 2-hydroxy-P-ionone, citral A, citral B, 2,3-epoxy geraniol, 2,3-epoxy nerol, 7,11-epoxymega-stigma-5(6)-en-9-one, 6,7-epoxynerol and 6,7-epoxygeraniol. In some embodiments, the method is a method of producing 4-epoxy-a-damascone. In some embodiments, the method is a method of producing 4-epoxy-a-damascone from a- damascone. In some embodiments, the method is a method of producing 3-hydroxy-a- damascone. In some embodiments, the method is a method of producing 3-hydroxy-a- damascone from a-damascone. In some embodiments, the method is a method of producing 4-CO-P-damascone. In some embodiments, the method is a method of producing 4-CO-P- damascone from P-damascone. In some embodiments, the method is a method of producing 4-hydroxy-P-damascone. In some embodiments, the method is a method of producing 4- hydroxy-P-damascone from P-damascone. In some embodiments, the method is a method of producing 3-hydroxy-P-damascone. In some embodiments, the method is a method of producing 3-hydroxy-P-damascone from P-damascone. In some embodiments, the method is a method of producing 2-hydroxy-6-damascone. In some embodiments, the method is a method of producing 2-hydroxy-6-damascone from 6-damascone. In some embodiments, the method is a method of producing 3-hydroxy-a-ionone. In some embodiments, the method is a method of producing 3-hydroxy-a-ionone from a-ionone. In some embodiments, the method is a method of producing limonene oxide. In some embodiments, limonene oxide is 1,2-epoxy limonene. In some embodiments, limonene oxide is trans-(R)-limonene oxide, cis-(R)-limonene oxide, trans-(S)-limonene oxide or cis-(S)-limonene oxide. In some embodiments, the ratio of trans to cis is shifted by the mutant protein. In some embodiments, the method is a method of producing (+)-carveol. In some embodiments, the method is a method of producing (+)-carveol.from (R)-(+)-limonene. In some embodiments, the method is a method of producing isopiperitenol. In some embodiments, the method is a method of producing isopiperitenol from (S)-(-)-limonene. In some embodiments, the method is a method of producing 4-hydroxy-P-ionone. In some embodiments, the method is a method of producing 4-hydroxy-P-ionone from P-ionone. In some embodiments, the method is a method of producing 2-hydroxy-P-ionone. In some embodiments, the method is a method of producing 2-hydroxy-P-ionone from P-ionone. In some embodiments, the method is a method of producing citral A. In some embodiments, the method is a method of producing citral A from geranial. In some embodiments, the method is a method of producing citral B. In some embodiments, the method is a method of producing citral B from neral. In some embodiments, the method is a method of producing 2,3 -epoxy geraniol. In some embodiments, the method is a method of producing 2,3-epoxy geraniol from geraniol. In some embodiments, the method is a method of producing 2,3-epoxy nerol. In some embodiments, the method is a method of producing 2,3-epoxy nerol from nerol.

[0108] In some embodiments, the method is a method of producing citral A from geraniol. In some embodiments, the method is a method of producing citral A from geraniol and the non-natural protein comprises or consists of SEQ ID NO: 4. In some embodiments, the method is a method of producing citral A from geraniol and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 4.

[0109] In some embodiments, the method is a method of producing 4-hydroxy-P-ionone from P-ionone. In some embodiments, the method is a method of producing 4-hydroxy-P- ionone from P-ionone and the non-natural protein comprises or consists of SEQ ID NO: 11. In some embodiments, the method is a method of producing 4-hydroxy-P-ionone from P- ionone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 11.

[0110] In some embodiments, the method is a method of producing 3-hydroxy-a-damascone from a-damascone. In some embodiments, the method is a method of producing 3-hydroxy- a-damascone from a-damascone and the non-natural protein comprises or consists of SEQ ID NO: 18. In some embodiments, the method is a method of producing 3-hydroxy-a- damascone from a-damascone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 18. In some embodiments, the method is a method of producing 4-hydroxy-P-damascone from P-damascone. In some embodiments, the method is a method of producing 4-hydroxy-P-damascone from P- damascone and the non-natural protein comprises or consists of SEQ ID NO: 18. In some embodiments, the method is a method of producing 4-hydroxy-P-damascone from P- damascone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 18. In some embodiments, the method is a method of producing 2-hydroxy-6-damascone from 6-damascone. In some embodiments, the method is a method of producing 2-hydroxy-6-damascone from 6-damascone and the non-natural protein comprises or consists of SEQ ID NO: 18. In some embodiments, the method is a method of producing 2-hydroxy-6-damascone from 6-damascone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 18. In some embodiments, the method is a method of producing 4-hydroxy-P-ionone from P-ionone. In some embodiments, the method is a method of producing 4-hydroxy-P-ionone from P-ionone and the non-natural protein comprises or consists of SEQ ID NO: 18. In some embodiments, the method is a method of producing 4-hydroxy-P-ionone from P-ionone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 18. In some embodiments, the method is a method of producing 3-hydroxy-a-ionone from a-ionone. In some embodiments, the method is a method of producing 3 -hydroxy-a-ionone from a-ionone and the non-natural protein comprises or consists of SEQ ID NO: 18. In some embodiments, the method is a method of producing 3- hydroxy-a-ionone from a-ionone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 18.

[0111] In some embodiments, the method is a method of producing 2,3-epoxy nerol from nerol. In some embodiments, the method is a method of producing 2,3-epoxy nerol from nerol and the non-natural protein comprises or consists of SEQ ID NO: 28. In some embodiments, the method is a method of producing 2,3-epoxy nerol from nerol and the non- natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 28.

[0112] In some embodiments, the method is a method of producing citral B from nerol. In some embodiments, the method is a method of producing citral B from nerol and the non- natural protein comprises or consists of SEQ ID NO: 29. In some embodiments, the method is a method of producing citral B from nerol and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 29.

[0113] In some embodiments, the method is a method of producing 4-hydroxy-P-ionone from P-ionone. In some embodiments, the method is a method of producing 4-hydroxy-P- ionone from P-ionone and the non-natural protein comprises or consists of SEQ ID NO: 34. In some embodiments, the method is a method of producing 4-hydroxy-P-ionone from P- ionone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 34.

[0114] In some embodiments, the method is a method of producing 3 -hydroxy-a-ionone from a-ionone. In some embodiments, the method is a method of producing 3-hydroxy-a- ionone from a-ionone and the non-natural protein comprises or consists of SEQ ID NO: 45. In some embodiments, the method is a method of producing 3 -hydroxy-a-ionone from a- ionone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 45. In some embodiments, the method is a method of producing 3-hydroxy-P-damascone from P-damascone. In some embodiments, the method is a method of producing 3-hydroxy-P-damascone from P-damascone and the non-natural protein comprises or consists of SEQ ID NO: 45. In some embodiments, the method is a method of producing 3-hydroxy-P-damascone from P-damascone and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ IDNO: 45. In some embodiments, the method is a method of producing (+)-carveol from (R)- (+)limonene. In some embodiments, the method is a method of producing (+)-carveol from (R)-(+)limonene and the non-natural protein comprises or consists of SEQ ID NO: 45. In some embodiments, the method is a method of producing (+)-carveol from (R)-(+)limonene and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 45. In some embodiments, the method is a method of producing isopiperitenol from (S)-(-)-limonene. In some embodiments, the method is a method of producing isopiperitenol from (S)-(-)-limonene and the non-natural protein comprises or consists of SEQ ID NO: 45. In some embodiments, the method is a method of producing isopiperitenol from (S)-(-)-limonene and the non-natural protein comprises or consists of an amino acid sequence with a threshold identity to SEQ ID NO: 45.

[0115] In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is a laboratory method.

[0116] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0117] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0118] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either ofthe terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0119] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0120] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0121] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0122] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0123] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0124] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0125] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and Methods

[0126] Chemicals: Solvents were used as provided without further purification from Carl Roth (Karlsruhe, DE) as GC ultragrade. The commercially available compounds were also used without further purification from the following suppliers:Sigma-Aldrich, St. Louis, US: ethanol (absolute), (S)-(-)-perillaldehyde, trans-(+)- limonene, (S)-(-)-limonene (>96 %), (-)-carveol (mixture of isomeres) (>97 %), 6- damascone (>90 %), (-)-limonene oxide (mixture of cis and trans) (99 %), [3- damascone (>90 %), (+)-valencene (>70 %), [3-ionone (96 %), hydrogen peroxide solution (30 % (w / w) in H2O, naphthalene (99 %), NBD (98 %), ABTS, DMPCarl Roth, Karlsruhe, DE: acetone (Rotisolv >99.9 % GC Ultra Grade), acetic acid ethyl ester (Rotisolv >99.9 % GC Ultra Grade),Fluka, Buchs, CH: (-)-Carvone (>99 %), (-)-perillyl alcoholTCI chemicals, Tokyo, Japan: 4-octanone (>98 %), citral (cis and trans mixture) (>96 %), (+)-limonene (>95 %), a-ionone (>90 %), nerol (>98 %), geraniol (>96 %).

[0127] For cultivation of S. cerevisiae cells D-Galactose, Peptone and Synthetic Complete Mixture (Kaiser) Drop-Out (-URA) were purchased from Formedium (Hunstanton, GB). Yeast nitrogen base (without amino acids) and Yeast extract were purchased from Carl Roth (Karlsruhe, DE). Bsal was purchased from New England Biolabs (Ipswich, US). BbsI and FastDigest Asci were purchased from ThermoFisherScientific (Waltham, US) and T4 DNA Ligase from Promega (Madison, US).

[0128] Bacterial and Yeast Strains: For all cloning purposes and plasmid propagation A. coll DH10B cells (ThermoFisherScientific, Waltham, US) were utilized. All work regarding S. cerevisiae was performed utilizing the INVScl strain (ThermoFisher Scientific, Waltham, US).

[0129] Oligonucleotides and gene parts: All oligonucleotides were purchased in the lowest purification grade “desalted” and minimal quantity at Eurofins Genomics (Ebersberg, DE). The genes of the MthUPO FuncLib library were purchased as gene parts from Twist Bioscience (San Francisco, US).

[0130] AlphaFol d2 model: The AlphaFold2 model of MthUPO was extracted from the AlphaFold Protein Structure Database (UniProt entry G2QID2). Source: alphafold.ebi.ac.uk / entry / G2QID2 (16.02.2023).

[0131] FuncLib design: A C-terminally truncated (-18 residues) AlphaFold2 model of MthUPO with its natural signal peptide was automatically refined by the FuncLib algorithm (using Rosetta) as protein structure and used. The loop at the original C terminus entered the substrate channel and active side during the simulations, impeding residue variation wherefore the truncated model was used for further calculations. Subsequently, positions C18 (axial ligand) as well as E158 and H88 (catalytic cascade) were defined as essential amino acids which must not be subject to any variation. The number of mutations per design was set to two to four with at least two variations between each design. An algorithm like PROSSI was not applied for introducing stabilizing mutations into the enzyme before running the FuncLib campaign, even though introducing several mutations in the active site may compromise enzyme stability. In previous works with MthUPO stability issues were not encountered after introducing mutations. Further prior work on AaeUPO variant PADA- I showed, that an aggressive FuncLib campaign on unspecific peroxygenase can be toleratedwithout introducing further stabilizing mutations beforehand (see Gomez de Santos, et al., 2023, “Repertoire of computationally designed peroxygenases for enantiodivergent C-H oxyfunctionalization reactions”. J. Am. Chem. Soc. 145, 3443-3453, the contents of which are hereby incorporated by reference in their entirety). Positions F59, L60, F63, L86, Al 53, Fl 54, Y156, G157, SI 59, A161, L206, and M210 were selected to be diversified. Charged amino acids (Arg, His, Lys, Glu, Asp) were excluded from the allowed sequence space to keep the hydrophobic character of the active site. Thr was also excluded as including all mutations to the initial sequence space file, that were found in previous projects (e.g. F59Q), exceeded the maximum permitted number of variants. The highly polar amino acid Thr was not considered to be of most interest for the project compared to other, more hydrophobic amino acids. Initial FuncLib runs showed sterically overlap of mutations containing aromatic identities at position L86, mutations larger than Ala and Ser at position G157 might obstruct access to the heme, therefore such mutations were prohibited. In the future the usage of AlphaFill or AlphaFold3 to include heme into the active site could prevent the introduction of mutations such as the forementioned. Finaly, the following sequence space was explored: Table 2.

[0132] Table 2: Explored sequence space during FuncLib creation.

[0133] Expression plasmids: Expression plasmid for MthUPO expression in S. cerevisiae were constructed as previously described - all parts are available at AddGene (Pullmann, etal., 2021, “A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases”, Commun. Biol. 4, 562, the contents of which are hereby incorporated by reference in their entirety). The gene parts of the FuncLib library were first cloned as individual level 0 standard modules into the universal Level 0 acceptor plasmid (pAGM9121) and afterward released upon Bsal restriction digest. Golden Gate reactions were performed to combine the MthUPO FuncLib genes with the Sce- a.galactosidase signal peptide and a TwinStrep-GFPl l-Tag for purification and secretion detection in a level 1 expression plasmid (pAGT572).

[0134] Microtiter plate cultivation of S. cerevisiae: The enzyme production in S. cerevisiae in microtiter plates was performed as in Pullmann, et al.

[0135] Shake flask cultivation of S. cerevisiae'. The enzyme production in S. cerevisiae in shake flasks was performed as described in Pullmann, et al.

[0136] Supernatant ultrafiltration and protein purification: The supernatant was concentrated approx. 20-fold by means of ultrafiltration as described in Pullmann, et al.

[0137] Heme CO complex measurements: Concentration determination of enzymes in concentrated supernatant were performed as described in Pullmann, et al.

[0138] Colorimetric screening assays. Four colorimetric assays were performed: ABTS, DMP, NBD and splitGFP-assay. All assays were performed as described in Dietz, et al., 2023, “Secretion and directed evolution of unspecific peroxygenases in S. cerevisiae”, Methods Enzymol. 693, 267-306, the contents of which are hereby incorporated by reference in their entirety. For NBD, ABTS and DMP, the absorbance difference between five min (tl) and 0 min (tO) was evaluated. The DMP assay was performed with enzyme secreted in medium without additional heme to avoid background reaction.

[0139] Bioconversion in microtiter plate: All reactions with non-colorimetric substrates were initially performed in microtiter plate. 100 pL supernanat derived from enzyme expression and secretion in S. cerevisiae in a microtiter plate were transferred to a 96-deep well plate (CR1496, EnzyScreen, Heemstede, NL). 400 pL of a reaction mastermix were added to achieve final concentrations of 1 mM substrate, 1 mM H2O2, 5 % acetone and 100 mM Kpi (pH 7.0). Reactions were performed for 1 h at 30 °C under continuous shaking at 300 rpm. The extraction was accomplished through the addition of 500 pL EtOAc (GC Ultra Grade) containing 0.25 mM of an internal standard and further shaking for 30 min at 25 °C and 300 rpm. Microtiter plates were centrifuged to separate the phases (3000 rpm, 10 min)and 300 pL of the organic phase were transferred to glass coated microtiter plate utilizing the Platemaster (Gilson, Middelton, US) for subsequent GC-MS analysis.

[0140] Bioconversion in single vials: All bioconversions for regioselectivity determination were performed in triplicate in single vials with direct addition of H2O2. 250 nM enzyme supernatant, derived from enzyme expression and secretion in S. cerevisiae in a shake flask after ultrafiltration and concentration determination were transferred to a glass vial. A reaction mastermix was added to achieve final concentrations of 1 mM substrate, 5 % acetone, 1 mM H2O2 and 100 mM Kpi (pH 7.0) to give a total volume of 500 pL. Reactions were performed for 1 h at 30 °C under continuous shaking. The extraction was accomplished through the addition of 500 pL EtOAc (GC Ultra Grade) containing 0.25 mM of an internal standard, the organic phase was transferred to a new glass vial for subsequent GC-MS analysis.

[0141] Bioconversion in single vials with syringe pump. All bioconversion for TON determination were performed in triplicates in single vials utilizing a syringe pump system. 250 nM enzyme supernatant, derived from enzyme expression and secretion in S. cerevisiae in a shake flask after ultrafiltration and concentration determination were transferred to a glass vial. A reaction mastermix was added to achieve final concentrations of 1 mM substrate, 5 % acetone and 100 mM Kpi (pH 7.0) to give a total volume of 400 pL. 100 pL H2O2 (stock solution 5 mM, final concentration 1 mM) were added over the period of the reaction via a syringe pump. Reactions were performed for 1 h at 30 °C under continuous shaking. The extraction was accomplished through the addition of 500 pL EtOAc (GC Ultra Grade) containing 0.25 mM of an internal standard, the organic phase was transferred to a new glass vial for subsequent GC-MS analysis.

[0142] GC-MS analysis: All GC-MS measurements were performed on a Shimadzu GCMS- QP2010 Ultra (Shimadzu, Kyoto, JP) with helium as carrier gas. The detector voltage of the secondary electron multiplier was adjusted in relation to the tuning results with perfluorotributylamine. The GC-MS parameters were controlled with GCMS Real Time Analysis, and for data evaluation, GCMS Postrun Analysis (GCMSsolution Version 4.45, Shimadzu, Kyoto, JP) was used.

[0143] Non-chiral gas chromatography-mass spectrometry (GC-MS): Measurements were performed on a SH-Rxi-5Sil MS column (30 m x 0.25 mm, 0.25 pm film, Shimadzu, Kyoto, JP). 1 pl of each sample was injected with a split ratio of 1 :20 (inlet temperature 200°C). Ionization was obtained by electron impact with a voltage of 70 V, and the temperature ofthe ion source was 280 °C. Calibration and quantification were implemented in scan mode. All initial screening measurements were performed in unicast.

[0144] Chiral gas chromatography-mass spectrometry (GC-MS): Measurements were performed on a Lipodex E column (25 m x 0.25 mm, Macherey -Nagel, Duren, DE). 1 pl of each sample was injected with a split ratio of 1 : 10 (inlet temperature 200°C). Ionization was obtained by electron impact with a voltage of 70 V, and the temperature of the ion source was 200 °C. Calibration and quantification were implemented in scan mode.

[0145] Quantum Mechanics (QM) calculations: A truncated computational model has been used to model the C-H activation and epoxidation of geraniol (1) and nerol (2) substrates. The truncated model [Fe=O(Por)(SCH3)(substrate)] includes: the active Fe-oxo species (Fe=O), the porphyrin pyrrole core (Por), a methyl thiolate group (-SCH3) to mimic Cysteine axial ligand, and 1 or 2 as substrate. Density Functional Theory (DFT) calculations were carried out using Gaussian 16 software package. Geometry optimizations and frequency calculations were performed using the unrestricted hybrid (U)B3LYP functional with an ultrafine integration grid, and including the CPCM polarizable conductor model (di chloromethane, a = 8.9) to have an estimation of the dielectric permittivity in the enzyme active site. 6-3 IG(d) basis set was used for all atoms but Fe, where SDD basis set and related SDD pseudopotential were employed. The optimized geometries were verified as minima by a vibrational frequency analysis and transition states geometries have a single imaginary frequency consistent with the reaction coordinate. Enthalpies and entropies were calculated for 1 atm and 298.15 K. A correction to the harmonic oscillator approximation, as discussed by Truhlar and co-workers, was also applied to the enthalpy calculations by raising all frequencies below 100 cm-1 to 100 cm-1 using Goodvibes v.1.0.1 python script. Single point energy calculations were performed using the functional (U)B3LYP with the Def2TZVP basis set on all atoms, and within the CPCM polarizable conductor model (di chloromethane, s = 8.9) and an ultrafine integration grid. Empirical Grimme D3 dispersion corrections with Becke-Johnson (GD3BJ) damping are also included in single point calculations. All structures have a total neutral charge and calculations were performed with doublet (d) or quartet (q) multiplicities consistent with the expected electronic states of the Fe. Figure of DFT structures were rendered using CYLview and MolUP VMD extensionlwas used for output visualization.

[0146] Homology model and Molecular Dynamics (MD) simulations: Homology model for MthUPO structure (283 residues) obtained from our previous work has been used as starting point. Mutations were introduced using the Mutagenesis tool in PyMOL. MolecularDynamics (MD) simulations in explicit water were performed using the AMBER18 package. Parameters for the geraniol (1) and nerol (2) substrates were generated within the antechamber module in AMBER18 package using the general AMBER force field (gaff2), with partial charges set to fit the electrostatic potential generated at the B3LYP / 6-31G(d) level by the RESP model. The charges were calculated according to the Merz-Singh- Kollman scheme using the Gaussian 16 package. Parameters for the heme compound I (Cpd I) and the axial Cys were taken from reference. The protein was solvated in a pre-equilibrated cubic box with a 12-A buffer of TIP3P water molecules using the AMBER18 leap module, resulting in the addition of ~12500 solvent molecules. The systems were neutralized by addition of explicit counterions (Na+ and C1-). All subsequent calculations were done using the AMBER force field 14 Stony Brook (ffl4SB). A two-stage geometry optimization approach was performed. The first stage minimizes the positions of solvent molecules and ions imposing positional restraints on solute by a harmonic potential with a force constant of 500 kcal mol-1 A-2, and the second stage is an unrestrained minimization of all the atoms in the simulation cell. The systems were gently heated using six 50 ps steps, incrementing the temperature by 50 K for each step (0-300 K) under constant-volume and periodic- boundary conditions. Water molecules were treated with the SHAKE algorithm such that the angle between the hydrogen atoms was kept fixed. Long-range electrostatic effects were modelled using the particle-mesh-Ewald method. An 8 A cutoff was applied to Lennard- Jones and electrostatic interactions. Harmonic restraints of 30 kcal mol-1 were applied to the solute, and the Langevin equilibration scheme was used to control and equalize the temperature. The time step was kept at 1 fs during the heating stages, allowing potential inhomogeneities to self-adjust. Each system was then equilibrated for 2 ns with a 2 fs time step at a constant pressure of 1 atm and temperature of 300 K without restraints. Once the systems were equilibrated in the NPT ensemble, production trajectories were then run under the NVT ensemble and periodic-boundary conditions. In particular, a total of 1500 ns from 3 independent replicas (500 ns each) were accumulated for the each of the following systems: Design 2 (F63L / A153I / F154EG157A), Design 4 (F63EF154L / G157A / A161F), Design 26 (F63EF154EG157A / A161L), and Design 28 (F63L / F154L / Y156L / A161F). Trajectories were processed and analyzed using the CPPtraj module from Ambertools utilities. VMD visualization software was used to visualize MD simulations. Protein structures were rendered using PyMOL.

[0147] Docking and protocol used for substrate-bound MD simulations: Docking calculations were performed using AutoDock Vina. The most populated clusters (based onbackbone clustering analysis) obtained from MD simulations carried out in the absence of substrate were used, and docking predictions were then utilized as starting points for substrate-bound MD simulations. The following systems were prepared: Design 4 + geraniol, Design 2 + geraniol, Design 26 + nerol, and Design 28 + nerol. Same protocol for MD simulations described above has been employed. 3 replicas of 500 ns were carried out on each system without any external restraints on the substrate, thus accumulating a total of 1500 ns for each system. Trajectories were processed and analyzed using the CPPtraj module from Ambertools utilities. VMD visualization software was used to visualize MD simulations.33 Protein structures were rendered using PyMOL.

[0148] Further FuncLib designs for comparison with Munch, et al. (Munch et al., 2023, “Computational-Aided Engineering of a Selective Unspecific Peroxygenase toward Enantiodivergent P-Ionone Hydroxylation”, ACS Catal. 13, 8963-8972, the contents of which are hereby incorporated by reference in their entirety): The starting structure for FuncLib design is derived from the most populated cluster of the parent F63I holoenzyme, determined by MD simulations. Key substrate-interacting positions (L56, L60, F63, L86, Fl 54, A161, F206, and M210) were selected based on previous work for mutation to reduced alphabet A, V, I, L, and F (M allowed at position 210). The Cl 8 axial ligand remained fixed, with one to two mutations per design. The heme cofactor and the structural Mg ion are kept on the FuncLib model. In total, 509 variants were generated in the first round with 267 variants with lower Rosetta scores than the parent enzyme.

[0149] The second round of the FuncLib evolution was setup again using the most populated cluster structure of the previous hit 12 (L60F / F63EA161 V) as a parent holoenzyme and the same parameters as the ones used before. In total 197 variants were generated in the second round with 65 variants with lower Rosetta scores than the parent enzyme.

[0150] Preparative work:

[0151] 2,3-Epoxy nerol: Nerol (590 mg, 3.8 mmol) was dissolved in acetone (7.5 mL) and poured into a solution of potassium phosphate buffer (100 mM, 111 mL, pH 7.0) and 250 nM MthUPO Var 28 (stock solution 20.5 pM, 1.8 mL). 30 mM hydrogen peroxide (stock solution 150 mM, 30 mL) was added via a syringe pump over the period of the reaction time. The solution (total: 150 mL) was stirred at 30 °C over night. Afterwards the mixture was extracted three times with ethyl acetate. The organic phase was washed with brine, dried with sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel using n-hexane / ethyl acetate (9 / 1 —2 / 1) obtaining 156 mg (70 %) 2,3-epoxy nerol as a pale-yellow oil. GC-MS analysis showed impurities of silicone grease leading to a total purity of 85% which corresponds to a yield of 132 mg (55 %) of 2,3-epoxy nerol.

[0152] 1H-NMR (400 MHz, CDC13): 8 5.07 (t, J 8 Hz, 1H), 8 3.37 (m, 1H), 8 3.65 (m, 1H), 8 2.94 (q, J 4 Hz, 1H), 8 2.17-2.01 (m, 2H), 8 1.68-1.63 (m, 4H), 8 1.60 (s, 3H), 8 1.33 (s, 3H).

[0153] Isopiperitenol: (S)-(-)-limonene (360 mg, 2.4 mmol) was dissolved in acetone (7.5 mL) and poured into a solution of potassium phosphate buffer (100 mM, 93 mL, pH 7.0) and 200 nM MthUPO Var 28 (stock solution 1.5 pM, 20 mL). 20 mM hydrogen peroxide (stock solution 100 mM, 30 mL) was added via a syringe pump over the period of the reaction time. The solution (total: 150 mL) was stirred at 30 °C overnight. Afterwards the mixture was extracted three times with ethyl acetate. The organic phase was washed with brine, dried with sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel using n-hexane / ethyl acetate (19 / 1 — 5 / 1) obtaining 3 mg (1 %) isopiperitenol as a pale-yellow oil. GC-MS analysis showed impurities of silicone grease leading to an even lower yield of isolated product.

[0154] 1H-NMR (400 MHz, CDC13): 8 5.42 (s, 1H), 8 4.86 (d, J 20 Hz, 2H), 8 4.15-4.05 (m, 2H), 8 2.11 (s, 1H), 8 2.05 (m, 1H), 8 1.71 (s, 3H), 8 1.68 (s, 3H), 1.41 (d, J 8 Hz, 2H).

[0155] Citral A and B: The product was available as mix of both isomers citral A and B, in a first step the ratio of both products and thus the purity of each isomer was determined. Afterwards, five stock solutions (15 mM, 10 mM, 5 mM, 2 mM, and 1 mM) in acetone were prepared. In each case 0.025 mL were added to 0.475 mL of buffered solution (0.1 M Kpi, pH 7) with final concentrations of 0.75 mM, 0.5 mM, 0.2 mM, 0.1 mM, and 0.05 mM. 0.5 mM p ionone was used as internal standard.

[0156] 2,3-Epoxy geraniol and nerol: Four stock solutions (15 mM, 10 mM, 5 mM, and 2 mM) in acetone were prepared. In each case 0.025 mL were added to 0.475 mL of buffered solution (0.1 M Kpi, pH 7) with final concentrations of 0.75 mM, 0.5 mM, 0.2 mM, and 0.1 mM. 0.5 mM P ionone was used as internal standard.

[0157] Carveol: Four stock solutions (10 mM, 5 mM, 2 mM, and 1 mM) in acetone were prepared. In each case 0.025 mL were added to 0.475 mL of buffered solution (0.1 M Kpi, pH 7) with final concentrations of 0.5 mM, 0.2 mM, 0.1 mM, and 0.05 mM. 0.25 mM 4- octanone was used as internal standard. The calibration curve of carveol was used for TONdetermination of both carveol and mix-10-ol (consisting of isopiperitenol and another product, maybe limonene- lO-ol, that elute at the same time).

[0158] Calibration curves: In every case, each sample was prepared in triplicate. The samples were extracted with 0.5 mL EtOAc containing an internal standard by vortexing for 30 seconds and centrifuged at 3000 rpm for 1 min to separate the aqueous and the organic phase. The organic phase was transferred to a GC vial with glass insert and analyzed via GC- MS.

[0159] Identification of products: Identification of the different products was performed through mass spectrum comparison with the literature, elution time comparison with product standards and NMR for isopiperitenol. To ensure correct epoxide identification the injection temperature was lowered to 200 °C for side product identification runs.

[0160] Tagging: The signal peptide from a Galactosidase (S. ce evisiae. SEQ ID NO: 53) was used to ensure expression and secretion of the enzyme in S. cerevisiae. The TwinStrep- GFP11 tag (SEQ ID NO: 54) in pAGT572_Nemo 2.0 was used for enzyme isolation from S. cerevisiae.Example 1: Modelling and design calculations

[0161] Because the molecular structure of Myceliophthora thermophila UPO (AYz / rUPO) has not been determined experimentally, a truncated AlphaFol d2 model (G2QID2) of the core enzymatic domain (amino acid positions 1-227) was used. Because AlphaFol d2 does not model cofactors, the model was compared with the structure of an artificial unspecific peroxygenase (artUPO) (PDB entry 7ZNV; rcsb.org / structure / 7ZNV; SEQ ID NO: 52). Visual inspection verified that the side chain conformations of amino acids that are in direct contact with heme in other UPOs were aligned with those observed in experimentally determined UPO. The proximal axial ligand Cysl8 and the catalytic dyad Glul58 / His88 were kept fixed in their modelled conformations in all design calculations to maintain the core catalytic activity, and active-site positions were chosen for design to alter the activesite cavity. The active site of the enzyme covers amino acids 55-64 (55, L56, A57, S58, F59, L60, F61, D62, F63, A64), 85-89 (185, L86, E87, H88, D89), 152-162 (D152, A153, F154, T155, Y156, G157, E158, S159, A160, A161, Y162) and 205-211 (D205, L206, N207, S208, S209, M10, E21 l).The designs were selected to encode 2-4 mutations from wild type and at least two mutations different from other variants, and the 50 lowest-energy FuncLib designs were selected for experimental screening (see Materials and Methods for further details on FuncLib setup for A / AUPO diversification). Among these 50 designs, somepositions exhibited no mutation (Leu206 and Met210) or only low sequence diversity, reflecting the high sequence conservation and energetic sensitivity of active-site positions, whereas others showed radical mutations with position Glyl57 mutated in 88 % of the designs. The designs are summarized in Table 3.

[0162] Table 3: 50 variants of SEQ ID NO: 51Example 2: Designs generate chemically challenging oxyfunctionalized terpenes

[0163] Ten small cyclic and non-cyclic terpenes and terpenoids (Fig. 1) were selected to assess the catalytic capabilities of the designed MthUPO variants. Additionally, three standard colorimetric substrates that serve as indicators for peroxygenase or peroxidase activity (ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), DMP (2,6- dimethoxyphenol), and NBD) were chosen and a splitGFP assay was performed to determine the protein secretion levels independently of activity.

[0164] Amongst the substrates, the oxyfunctionalized products of geraniol and nerol are of outstanding interest, as partial oxidation of their terminal alcohol to aldehyde produces citral A (geranial) and citral B (neral), which are commonly used aroma compounds in perfumery due to their strong lemon (citrus) scents. Moreover, citral is a key building block for the synthesis of vitamin A and shows anti-inflammatory, anti-tumor, and anti-bacterial activity. Limonene and its oxyfunctionalized products are widely used in the fragrance and polymer industry, and also have important pharmacological effects. Wildtype MthUPO, however, yields a diverse array of products from limonene, including multiple oxyfunctionalizations, similar to the results observed with PaDa-I and artUPO (see Melling, et al., “Complementary specificity of unspecific peroxygenases enables access to diverse products from terpene oxygenation”, Chem Catalysis 2024, Vol 4:2, 100889, the contents of which are hereby incorporated by reference in their entirety). Additionally, the volatility of limonene renders upscaling reactions for product isolation and identification challenging.

[0165] MthUPO is readily secreted in functional form from yeast, and remarkably, all 50 designs were successfully expressed and secreted. Secretion levels were between 0.6-fold and 1.9-fold of wildtype levels (Fig. 2), indicating that active-site design using FuncLib preserves the expressibility of the parental enzyme. Furthermore, among the designed enzymes, improvements in activity relative to the wild type were measured for nine of the ten terpenes, with only valencene (10), the only substrate tested comprising two conjugated rings, not showing detectable activity in the wild type or any of the designs (Fig. 2). The greatest improvement was seen in designs 3 and 47 measured by ABTS (1,880-fold and 1,950-fold increase in absorbance related to product formation, respfectively). Design 34 demonstrated a 200-fold activity improvement by DMP. Both design 3 and 47 performed excellently as well for DMP, but underperformed with all other substrates, indicating that those designs improved the peroxidase rather than peroxygenase activity. By contrast, designs 22 and 25 showed twofold improvement on the peroxygenase model substrate NBD.

[0166] As can be seen in Figure 2, All FuncLib designs showed at least baseline activity toward geraniol (1) and nerol (2), confirming that all designs were functionally secreted. The largest activity improvement toward geraniol was seen with design 4 exhibiting 3.8-fold improvement compared to the wild type. The highest activity for nerol (2) is seen by design 24 with 6.2-fold improvement. The highest overall activity toward all damascenes and a- ionone is seen in design 18 with 3.1 to 6.7-fold increase. The highest activity on P-ionone (9) was revealed by design 11 with a sevenfold increase relative to the wildtype. For limonene, the activity gain reached 1.7 and 2.2-fold with design 2 and 29, respectively, for(R)-(+)-limonene and (S)-(-)-limonene. Thus, a variety of different designs exhibited excellent levels of oxyfunctionalization of different substrates.Example 3: Novel products and outstanding improvements in regioselectivity

[0167] Aside from the increase in activity, dramatic shifts in regioselectivity among the designs were also observed (Fig. 3A-D). Design 45 displays a notable shift in the formation of 3-hydroxy-P-damascone (15), which increased from 3% for wild type MthUPO to 46% (Fig. 3A). This regioselectivity shift is remarkable, as the aliphatic C3 -position is substantially less activated compared to the allylic C4-position of the main product 4- hydroxy-P-damascone (14). Thus, the designs can significantly increase the production of thermodynamically unfavorable products.

[0168] Shifts in regioselectivity for limonene were also observed. For instance, wildtype MthUPO mainly generates the epoxide limonene oxide (21), whereas design 45 mainly produces (+)-carveol (22) (Fig. 3B), alongside a 5.6-fold increase in turnover number (TON). This change in chemoselectivity is noteworthy because hydroxylation is energetically less favored than epoxidation, in addition to accessing a valuable fragrance and pharmacologically active compound. The main product of (S)-(-)-limonene with design 45 was identified as isopiperitenol (19), which could only be found in trace amounts after conversion by wild type MthUPO. This activity is boosted 25-fold, reaching up to 310 TONs.

[0169] a- and P-ionone are mainly converted by MthUPO to 3-hydroxy-a-ionone (26) and 4-hydroxy-P-ionone (28), respectively. With the FuncLib designs the main products remained 26 and 28, respectively, but the regioselectivity increased strongly. For the formation of 26 it increased from 91 % (wildtype MthUPO) to 98 % (design 18) and 99 % (design 45), respectively (Fig. 3C). For 28 the improvement was from 92 % (wildtype MthUPO) to 98 % (design 34) (Fig. 3D).

[0170] Overall, these experiments revealed large shifts in the main substrates, the formation of novel products, and an increase from satisfactory to outstanding regioselectivities, thus underscoring the efficacy of FuncLib in generating useful functional diversity. In addition, some FuncLib designs significantly enhanced reactivities and regioisomers that are energetically disfavored, demonstrating how alternative poses of the substrate within the active-site pocket can dramatically impact the outcome of the reactions (see below).Example 4: Large chemoselectivity shifts among FuncLib designs

[0171] FuncLib modification also leads to large shifts in chemoselectivity as demonstrated by geraniol (1) or nerol (2). Design 4 displays excellent selectivity (>99 %) for citral A (geranial, 19, Fig. 4A-C) and a 4.5-fold improvement in TON compared to wildtype Af / AUPO, which only showed 40 % chemoselectivity for converting the terminal alcohol group. The best chemoselectivity shift toward citral B (neral, 17) was achieved with design 26 with 89 % relative abundance reaching 11,170 TONs, which corresponds to a 4.5-fold activity gain, but only a minor chemoselectivity shift compared to the 72 % of wild type MthUPO. Further products are 2,3-epoxides, with a relative abundance of up to 58 % for 2,3-epoxy geraniol (20) with design 2 and 69 % for 2,3-epoxy nerol (18) with design 24. Some designs further exhibited overoxidation from nerol to neric acid, a reaction that was not observed with geraniol. Being able to selectively oxidize the primary alcohols of nerol and geraniol is of great importance to industrial applications. Upscaling reactions with design 28 were successfully performed and yielded more than 150 mg of 2,3-epoxy nerol, which was subsequently used to determine TONs of both nerol and geraniol 2,3 oxides. Design 2 displayed a 1.8-fold increase in TON for 20 when using geraniol. Design 28 displayed a 14.3-fold boost for 18 compared to the wild type MthUPO enzyme, resulting in 9,470 TONs for product 18 (Fig. 4C).Example 5: Molecular basis of chemoselectivity changes

[0172] To shed light on the molecular basis for the chemoselectivity changes observed on the non-cyclic terpene substrates, computational modeling was conducted based on DFT calculations using truncated models and molecular dynamics (MD) simulations. For both geraniol and nerol, DFT calculations in the absence of enzyme show that allylic hydroxylation on the terminal carbon atom (Cl) and epoxidation at the 2,3 double bond are the energetically most favored oxyfunctionalizations, with the terminal allylic C-H activation (via Hydrogen Atom Transfer, HAT) slightly preferred (Fig. 4D-4G). This is in line with the major products detected experimentally for these substrates with all variants.

[0173] Molecular Dynamics (MD) simulations (see Materials and Methods) were conducted to analyze the large selectivity shifts seen in design 4 versus design 2 for geraniol oxidation and design 28 versus 26 for nerol. According to these simulations, geraniol establishes a persistent polar interaction with the Glut 58 sidechain of design 4, thus positioning both Cl- H and C2-C3 bonds near the catalytically active oxo-ferryl cation radical complex species (Compound I, see Fig. 4E). The presence of the bulky Phel61 (mutated from the wild type Ala) side chain prevents the substrate from approaching the catalytic species in alternativenear-attack conformations. Considering that terminal allylic hydroxylation is energetically slightly preferred, the citral A product is preferentially formed from this binding mode. On the other hand, design 2 led mainly to 2,3 -epoxy geraniol with high activity and moderate chemoselectivity (1.8-fold increase and 58% selectivity). The models show that design 2 has a less dense active site and the C2-C3 double bond of geraniol may approach the catalytic iron-oxo group. At the same time, Cl-H bonds are not geometrically well oriented for effective HAT (Fig. 4D), leading to effective epoxidation by subtle substrate repositioning in the active site as compared to design 4.

[0174] A similar scenario is found for nerol and designs 26 and 28. In design 26, nerol is preferentially bound by placing both the allylic terminal position and the C2-C3 double bond geometrically preorganized for oxyfunctionalization (Fig. 4F). Nevertheless, mutations included in design 28, particularly Tyrl56Leu, induce structural changes in the active-site cavity that favor the positioning of a water molecule that persistently interacts with the Glul58 and Tyrl62 backbones. These structural changes, stemming from cooperation among several mutations, reposition nerol in the active site placing the C2-C3 double bond in a near-attack conformation for epoxidation by Compound I while geometrically disfavoring terminal C-H activation (Fig. 4G).Example 6: FuncLib designs impact stereoselectivity of limonene oxide and 4- hydroxy-p-ionone.

[0175] The major influence of the FuncLib library on stereoselectivity is shown by shifts in the diastereomeric ratio of 1,2-epoxy limonene (Fig. 5A) ranging from 86: 14 (design 28) for trans-(R)-limonene oxide to 23:77 (design 43) for cis-(R)-limonene oxide and 94:6 (design 28 and 34) for trans-(S)-limonene oxide to 10:90 (design 6) for cis-(S)-limonene oxide. Further, outstanding changes are seen in the enantioselectivity of 4-hydroxy-P-ionone (28) from an enantiomeric ratio of 76:24 for wildtype MthUPO to 1 :99 for both design 11 and 34 producing (S)-4-hydroxy-P-ionone (Fig. 5B). Lack of enantioselectivity of the FuncLib designs towards the (R)-4-hydroxyl-P-ionone product is not surprising as the key position for R-selectivity control in P-ionone hydroxylation (Leu206) was not diversified in the FuncLib library (Table 1). These shifts confirm that FuncLib not only influences regio- and chemoselectivity but can further exert major influences on stereoselectivity in accordance with previous work on small aromatic substrates.

[0176] Table 1 : List of variants. Header row provides the amino acid position and the amino acid present in the WT sequence. Mutations are bolded. The final line provides all acceptable amino acids at the position. The WT amino acid is given in bold and italics.

[0177] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A non-natural unspecific peroxygenase protein comprising an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 51 and comprising at least 2 mutations selected from the group consisting of: mutation of phenylalanine 59 (F59) to tyrosine; mutation of leucine 60 (L60) to phenylalanine or isoleucine; mutation of phenylalanine 63 (F63) to leucine, isoleucine or glutamine; mutation of leucine 86 (L86) to isoleucine, valine or methionine; mutation of alanine 153 (Al 53) to leucine, isoleucine or glutamine; mutation of phenylalanine 154 (Fl 54) to leucine or isoleucine; mutation of tyrosine 156 (Y156) to leucine or isoleucine; mutation of glycine 157 (G157) to alanine or serine; mutation of serine 159 (SI 59) to alanine or tyrosine; and mutation of alanine 161 (A161) to leucine, phenylalanine or methionine.

2. The non-natural unspecific peroxygenase protein of claim 1, comprising an amino acid sequence consisting of SEQ ID NO: 51 and said at least 2 mutations.

3. The non-natural unspecific peroxygenase protein of claim 1 or 2, wherein said at least 2 mutations is at least 4 mutations.

4. The non-natural unspecific peroxygenase protein of any one of claims 1 to 3, comprising at least one of: mutation of G157 to alanine or serine and mutation of A161 to leucine, phenylalanine or methionine.

5. The non-natural unspecific peroxygenase protein of any one of claims 1 to 4, comprising at least two of: mutation of F63 to leucine, isoleucine or glutamine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

6. The non-natural unspecific peroxygenase protein of any one of claims 1 to 5, comprising at least two of: mutation of L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

7. The non-natural unspecific peroxygenase protein of any one of claims 1 to 6, comprising at least three of: mutation of F63 to leucine, isoleucine or glutamine; mutation of L86 to isoleucine, valine or methionine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

8. The non-natural unspecific peroxygenase protein of any one of claims 1 to 7, comprising at least four of mutation of F63 to leucine, isoleucine or glutamine; mutation of L86 to isoleucine, valine or methionine; mutation of Fl 54 to leucine or isoleucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

9. The non-natural unspecific peroxygenase protein of claim 8, consisting of SEQ ID NO: 51 comprising four mutations selected from mutation of F63 to leucine, isoleucine or glutamine; mutation of L86 to isoleucine, valine or methionine; mutation of Fl 54 to leucine or isoleucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

10. The non-natural unspecific peroxygenase protein of any one of claims 1 to 8, comprising mutation of F59 to tyrosine and not comprising mutation of L60, A153, F154, Y156 or S159.

11. The non-natural unspecific peroxygenase protein of claim 10, comprising mutation of F59 to tyrosine; mutation of F63 to leucine; mutation of G157 alanine and either mutation of L86 to isoleucine or mutation of A161 to phenylalanine.

12. The non-natural unspecific peroxygenase protein of any one of claims 1 to 8, comprising Al 53 mutated to leucine, isoleucine or glutamine or Y156 mutated to leucine or isoleucine, but not both.

13. The non-natural unspecific peroxygenase protein of any one of claims 1 to 8, comprising mutation of L60 to phenylalanine or isoleucine; mutation of G157 to alanine; mutation of Al 61 to leucine, phenylalanine or methionine and either mutation of L86 to isoleucine or mutation of F154 to leucine or isoleucine.

14. The non-natural unspecific peroxygenase protein of any one of claims 1 to 8, comprising mutation of Y156 to leucine or isoleucine and at least three of mutation of F63 to leucine or isoleucine; mutation of L86 to valine or isoleucine; mutation of Fl 54 to leucine; mutation of G157 to alanine or serine and mutation of Al 61 to leucine, phenylalanine or methionine.

15. The non-natural unspecific peroxygenase protein of claim 14, wherein said Y156 is mutated to leucine.

16. The non-natural unspecific peroxygenase protein of any one of claims 1 to 8, comprising an amino acid sequence selected from SEQ ID NO: 1-50.

17. The non-natural unspecific peroxygenase protein of claim 16, comprising an amino acid sequence consisting of an amino acid sequence selected from SEQ ID NO: 1-50.

18. The non-natural unspecific peroxygenase protein of any one of claims 1 to 9 and 16 to 17, comprising an amino acid sequence with at least 85% identity to SEQ ID NO: 4, comprising isoleucine at position 63, leucine at position 154, alanine at position 157 and phenylalanine at position 161 and comprising increased chemoselectivity for the formation of citral A from geraniol as compared to a natural peroxygenase comprising SEQ ID NO: 51.

19. The non-natural unspecific peroxygenase protein of claim 18, comprising F59, L60, L86, A153, Y156, and S159.

20. The non-natural unspecific peroxygenase protein of claim 18 or 19, comprising amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 4.

21. The non-natural unspecific peroxygenase protein of any one of claims 18 to 20, comprising an amino acid sequence comprising or consisting of SEQ ID NO: 4.

22. The non-natural unspecific peroxygenase protein of any one of claims 1 to 6 and 14 to 17, comprising an amino acid sequence with at least 85% identity to SEQ ID NO: 11, comprising isoleucine at position 86, leucine at position 154, leucine at position 156 and phenylalanine at position 161 and comprising at least one of: increased conversion of P-ionone to 4-hydroxy-P-ionone and an increased S:R enantiomer ratio as compared to a natural peroxygenase comprising SEQ ID NO: 51.

23. The non-natural unspecific peroxygenase protein of claim 22, comprising F59, L60, F63, A153, G157, and S159.

24. The non-natural unspecific peroxygenase protein of claim 22 or 23, comprising amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 11.

25. The non-natural unspecific peroxygenase protein of any one of claims 22 to 24, comprising an amino acid sequence comprising or consisting of SEQ ID NO: 11.

26. The non-natural unspecific peroxygenase protein of any one of claims 1 to 6, 13 and 16 to 17, comprising an amino acid sequence with at least 85% identity to SEQ ID NO: 18, comprising phenylalanine at position 60, isoleucine at position 154, alanine at position 157 and leucine at position 161 and comprising at least one of: increased conversion of a-damascone to 3-hydroxy-a-damascone, increased conversion of P- damascone to 4-hydroxy-P-damascone, increased conversion of 6-damascone to 2- hydroxy-6-damascone; increased conversion of P-ionone to 4-hydroxy-P-ionone; and increased regioselectivity for the formation of 3-hydroxy-a-ionone from a-ionone as compared to a natural peroxygenase comprising SEQ ID NO: 51.

27. The non-natural unspecific peroxygenase protein of claim 26, comprising F59, F63, L86, A153, Y156, and S159.

28. The non-natural unspecific peroxygenase protein of claim 26 or 27, comprising amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 18.

29. The non-natural peroxygenase protein of any one of claims 26 to 28, comprising an amino acid sequence comprising or consisting of SEQ ID NO: 18.

30. The non-natural unspecific peroxygenase protein of any one of claims 1 to 6 and 14 to 17, comprising an amino acid sequence with at least 85% identity to SEQ ID NO:28, comprising leucine at position 63, leucine at position 154, leucine at position 156 and phenylalanine at position 161 and comprising increased chemoselectivity for the formation of 2,3-epoxy nerol from nerol as compared to a natural peroxygenase comprising SEQ ID NO: 51.

31. The non-natural unspecific peroxygenase protein of claim 30, comprising F59, L60, L86, A153, G157, and S159.

32. The non-natural unspecific peroxygenase protein of claim 30 or 31, comprising amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 28.

33. The non-natural unspecific peroxygenase protein of any one of claims 30 to 32, comprising an amino acid sequence comprising or consisting of SEQ ID NO: 28.

34. The non-natural unspecific peroxygenase protein of any one of claims 1 to 6 and 14 to 17, comprising an amino acid sequence with at least 85% identity to SEQ ID NO:29, comprising isoleucine at position 86, leucine at position 154, leucine at position 156 and alanine at position 157 and comprising increased chemoselectivity for the formation of citral B from nerol as compared to a natural peroxygenase comprising SEQ ID NO: 51.

35. The non-natural unspecific peroxygenase protein of claim 34, comprising F59, L60, F63, A153, S159 and A161.

36. The non-natural peroxygenase protein of claim 34 or 35, comprising amino acids 55- 64, 85-89, and 152-162 of SEQ ID NO: 29.

37. The non-natural unspecific peroxygenase protein of any one of claims 34 to 36, comprising an amino acid sequence comprising or consisting of SEQ ID NO: 29.

38. The non-natural unspecific peroxygenase protein of any one of claims 1 to 9 and 14 to 17, comprising an amino acid sequence with at least 85% identity to SEQ ID NO: 34, comprising leucine at position 63, isoleucine at position 86, leucine at position 154 and phenylalanine at position 161 and comprising at least one of: increased conversion of P-ionone to 4-hydroxy-P-ionone, an increased S:R enantiomer ratio and increased peroxidase activity as compared to a natural peroxygenase comprising SEQ ID NO: 51.

39. The non-natural unspecific peroxygenase protein of claim 38, comprising F59, L60, A153, Y156, G157, and S159.

40. The non-natural unspecific peroxygenase protein of claim 38 or 39, comprising amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 34.

41. The non-natural unspecific peroxygenase protein of any one of claims 38 to 40, comprising an amino acid sequence comprising or consisting of SEQ ID NO: 34.

42. The non-natural unspecific peroxygenase protein of any one of claims 1 to 6 and 14 to 17, comprising an amino acid sequence with at least 85% identity to SEQ ID NO: 45, comprising isoleucine at position 63, leucine at position 156, serine at position 157 and leucine at position 161 and comprising at least one of: increased regioselectivity for the formation of 3-hydroxy-a-ionone from a-ionone, increased regioselectivity for the formation of 3-hydroxy-P-damascone from P-damascone, increased chemoselectivity for the formation of (+)-carveol from (R)-(+)limonene, and increased formation of isopiperitenol from (S)-(-)-limonene as compared to a natural peroxygenase comprising SEQ ID NO: 51.

43. The non-natural unspecific peroxygenase protein of claim 42, comprising F59, L60, L86, A153, F154 and S159.

44. The non-natural unspecific peroxygenase protein of claim 42 or 43, comprising amino acids 55-64, 85-89, and 152-162 of SEQ ID NO: 45.

45. The non-natural unspecific peroxygenase protein of any one of claims 42 to 44, comprising an amino acid sequence comprising or consisting of SEQ ID NO: 45.

46. A method of terpene oxy functionalization, the method comprising contacting said terpene with a non-natural unspecific peroxygenase protein of any one of claims 1 to 45, thereby oxyfunctionalizing a terpene.

47. The method of claim 45, wherein said terpene is selected from geraniol, nerol, S-(-)- limonene, R-(+)-limonene, a-damascone, P-damascone, 6-damascone, a-ionone and P-ionone.

48. The method of claim 46 or 47, wherein said method is a method of producing a molecule selected from: 4-epoxy-a-damascone, 3-hydroxy-a-damascone, 4-CO-P- damascone, 4-hydroxy-P-damascone, 3-hydroxy-P-damascone, 2-hydroxy- 6- damascone, S-(-)-limonene, R-(+)-limonene, 3-hydroxy-a-ionone, limonene oxide, (+)-carveol, isopiperitenol, 4-hydroxy-P-ionone, 2-hydroxy-P-ionone, citral A, citral B, 2,3-epoxy geraniol, 2,3-epoxy nerol, 7,1 l-epoxymega-stigma-5(6)-en-9-one, 6,7- epoxynerol and 6,7-epoxygeraniol.

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