Methods of producing ionones

Abstract

The present invention provides a method for the preparation of an ionone compound of formula (I). Included in this invention is a bioconversion or in vivo process for the preparation of a compound of formula (I) in recombinant cells. The present invention also provides recombinant cells which may be used in said process. The present invention further provides enal-cleaving enzymes, alcohol dehydrogenases, cytochrome P450 enzymes and ionylideneethane synthase enzymes for the preparation of an ionone compound of formula (I).

Description

[0001] METHODS OF PRODUCING IONONES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods of producing ionones, enzymes involved in ionone synthesis and cells for producing ionones.

[0004] BACKGROUND TO THE INVENTION

[0005] The aroma compounds known as ionones are an example of a plant volatile organic compound that are widely distributed in plant species. These are an economically important group of molecules because of their use in fragrance products, such as perfumes, and as flavouring agents. The compound alpha-ionone, for example, is a key component in the aroma of raspberry.

[0006] The biosynthetic pathway of ionones in plants starts from the group of compounds known as carotenoids, which are large terpenes found in plants, bacteria and fungi, and which are essential in absorbing light energy for use in photosynthesis and providing photoprotection. The ionones are a degradation product from carotenoids.

[0007] Terpenes are a class of compound found in most organisms (microorganisms, animals and plants). These compounds are generated from five-carbon units, called isoprene units derived from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). Terpenes are classified by the number of these isoprene units present in their structure. For example, monoterpenes, sesquiterpenes, and diterpenes contain 10, 15 and 20 carbon atoms, derived from 2, 3 and 4 isoprene units, respectively. Carotenoids are an example of a tetraterpene containing 40 carbon atoms derived from 8 isoprene units.

[0008] The biosynthetic production of terpenes generally requires the sequential condensation of dimethylallyl pyrophosphate (DMAPP) with isopentenyl pyrophosphate (IPP) to form acyclic prenyl pyrophosphates that are synthesized by prenyltransferases. These linear prenyl pyrophosphates may then be cyclised by a class of enzymes known as terpene synthases. For example, sesquiterpenes are derived from the condensation of DMAPP with two molecules of IPP catalysed by the enzyme famesyl pyrophosphate synthase (FPPS) to form famesylpyrophosphate. In turn, the acyclic famesyl pyrophosphate is then cyclised by sesquiterpene synthases to form the sesquiterpene product.

[0009] In the biosynthesis of carotenoids, DMAPP condenses with three molecules of IPP to generate the 20-carbon compound geranylgeranyl pyrophosphate by the action of geranylgeranyl pyrophosphate synthase. The first committed step in carotenoid biosynthesis is the condensation of two molecules of geranylgeranyl pyrophosphate to form the 40-carbon linear molecule phytoene by the action of phytoene synthase. Phytoene is then converted to all-trans lycopene. The route for the conversion of phytoene to lycopene is dependent on the organism. In bacteria, a single enzyme catalyses the conversion, whilst in plants and cyanobacteria, four separate enzymes are used. Lycopene is in turn cyclised to a carotenoid product such as alpha, beta, or gamma-carotene.

[0010] Ionones are synthesised via the degradation of carotenoids by the class of enzymes known as carotenoid dioxygenases.

[0011] (R)-alpha-ionone of formula I-Al described herein has been produced by fermentation in the yeast Saccharomyces cerevisiae (WO 2019 / 126777) and in the bacteria Escherichia coli (WO 2017 / 036495). However, these approaches required the metabolic engineering of (R)-alpha-ionone via the degradation of carotenoids. In order to produce (R)-alpha-ionone in Saccharomyces cerevisiae or Escherichia coli, it was therefore necessary to reconstitute the pathway for carotenoid biosynthesis, together with the enzymes for carotenoid degradation needed for ionone formation, which is energetically expensive and not carbon efficient.

[0012] There is therefore a need to develop a more sustainable process for the production of ionones that is more carbon efficient and does not utilise the carotenoid pathway. In addition, there is a need for methods for producing the diverse repertoire of ionone stereoisomers including (R)-gamma-ionone (formula I-A2 described herein), beta-ionone (formula I-B3 described herein), (S)-alpha-ionone (formula I-Cl described herein) and (S)-gamma-ionone (formula I-C2 described herein).

[0013] These problems are addressed by the present invention which provide methods for producing such compounds by a unique enzymatic route either in vivo and / or by bioconversion methods.

[0014] In particular, the inventors have surprisingly demonstrated that enal-cleaving enzymes are able to act on aldehyde precursors to generate specific ionone stereoisomers.In addition, the inventors have demonstrated that ionones of formula I-Al, formula I-A2, formula I-B3, formula I-D and formula I-E can be synthesized directly from famesyl pyrophosphate using an enzymatic pathway that utilises an ionylideneethane synthase, a cytochrome P450, an alcohol dehydrogenase and an enal-cleaving enzyme. The use of this pathway allows for the enantioselective production of ionones which is more carbon efficient than the biosynthetic pathway of ionones via the carotenoid pathway.

[0015] SUMMARY OF THE INVENTION

[0016] The inventors have demonstrated that the enal-cleaving enzymes are able to act on aldehyde precursors to generate specific ionones. Accordingly, the invention provides a method for preparing an ionone compound of formula I

[0017]

[0018] (formula I)

[0019] in the form of any one of its stereoisomers or a mixture thereof, wherein the method comprises:

[0020] contacting an ionylidene acetaldehyde compound of formula II

[0021]

[0022] (formula II)

[0023] in the form of any one of its stereoisomers or a mixture thereof, with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I;

[0024] wherein any bond having a dotted line ( - ) represents independently from each other either a single carbon-carbon bond or a carbon-carbon double bond, wherein one of the bonds having a dotted line ( - ) represents a carbon-carbon double bond, with the proviso that the compound does not comprise two cumulated carbon-carbon double bonds;

[0025] and wherein any wavy line represents a carbon-carbon bond linked to a carbon-carbon double bond which is either in the Z or in the E-configuration.The inventors have further established a complete biosynthetic pathway to ionone compounds from simple carbon precursors (such as glucose). Accordingly, the method of the invention may further comprise:

[0026] (A) contacting an ionylideneethanol compound of formula III

[0027]

[0028] (formula III)

[0029] in the form of any one of its stereoisomers or a mixture thereof, with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce the ionylidene acetaldehyde compound of formula II

[0030]

[0031] (formula II); and / or

[0032] (B) contacting an ionylideneethane compound of formula (IV)

[0033]

[0034] (formula IV)

[0035] in the form of any one of its stereoisomers or a mixture thereof, with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce the ionylideneethanol compound of formula III

[0036]

[0037] (formula III); and / or

[0038] (C) contacting a famesyl pyrophosphate compound of formula V

[0039]

[0040] (formula V)

[0041] in the form of any one of its stereoisomers or a mixture thereof, with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce the ionylideneethane compound of formula (IV)

[0042]

[0043] (formula IV).The biocatalytic steps may be coupled to preceding (upstream) or (downstream) enzymatic steps which allows for the provision of a biocatalytic multistep process for the enzymatic synthesis of ionones.

[0044] Accordingly, the invention provides a cell capable of producing an ionone compound of formula I, wherein the cell further comprises a heterologous enal-cleaving enzyme. The cell producing the ionone compound of formula I may further produce an ionylidene acetaldehyde compound of formula II and further comprises a heterologous alcohol dehydrogenase enzyme; and optionally the cell further produces an ionylideneethanol compound of formula III, and the cell further comprises a heterologous cytochrome P450 enzyme. The invention further provides a cell for producing an ionone of formula I, wherein the cell comprises a famesyl pyrophosphate of formula V, a heterologous ionylideneethane synthase enzyme, a heterologous cytochrome P450 enzyme, a heterologous alcohol dehydrogenase enzyme, and a heterologous enal-cleaving enzyme.

[0045] As will be described herein, the method may be a bioconversion or in vivo process.

[0046] BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1: GC-MS analysis of the product formed using E. coli cells modified to produce (R)-alpha-ionylideneethane and expressing the Bcaba3 enzyme (SEQ ID NO: 83). The total ion chromatogram is annotated with numbers corresponding to the identified sesquiterpene compounds: (2Z,4E)-(R)-alpha-ionylideneethane (IV-Al(i)); (2E,4E)-(R)-alpha-ionylideneethane (IV-Al(ii)); (E)-beta-famesene (VI-1), (3Z,6E)-alpha-famesene (VI-2); (3E,6E)-alpha-famesene (VI-3), (2Z,4E,6E)-allofamesene (VI-5); (2E,4E,6E)-allofamesene (VI-4), i.s., internal standard.

[0048] Figure 2: GC-MS analysis of the product formed using E. coli cells modified to produce (R)-alpha-ionylideneethane and expressing the Tnigaba3 enzyme (SEQ ID NO: 97). The total ion chromatogram is annotated with numbers corresponding to the identified sesquiterpene compounds: (2Z,4E)-(R)-alpha-ionylideneethane (IV-Al(i)); (2E,4E)-(R)-alpha-ionylideneethane (IV-Al(ii)); (E)-beta-famesene(VI-l), (3Z,6E)-alpha-famesene (VI-2); (3E,6E)-alpha-famesene (VI-3), (2Z,4E,6E)-allofamesene (VI-5); (2E,4E,6E)-allofamesene (VI-4), i.s., internal standard.Figure 3: GC-MS analysis of the product formed using E coli cells modified to produce (R)-gamma-ionylideneethane and expressing the Pcruaba3 enzyme (SEQ ID NO: 101). The total ion chromatogram is annotated with numbers corresponding to the identified sesquiterpene compounds: (2Z,4E)-(R)-gamma-ionylideneethane (IV-A2(i)); (2E,4E)-(R)-gamma-ionylideneethane (IV-A2(ii)); (3Z,6E)-alpha-famesene (VI-2); (3E,6E)-alpha-famesene (VI-3), (2Z,4E,6E)-allofamesene (VI-5); (2E,4E,6E)-allofamesene (VI-4), i.s., internal standard.

[0049] Figure 4: GC-MS analysis of the non-enzymatic conversion of (R)-alpha-ionylideneethane under aerobic conditions. The total ion chromatogram is annotated with numbers corresponding to the identified terpene compounds: (2Z,4E)-(R)-alpha-ionylideneethane (IV-Al(i)); (R)-alpha-ionone (I -Al).

[0050] Figure 5: GC-MS analysis of the product formed using E. coli cells modified to produce (R)-alpha-ionylideneethane in two-phase liquid culture with decane as a solvent overlay and cultures without solvent overlay. The main products in the total ion chromatograms are annotated with numbers corresponding to the identified sesquiterpene compounds or sesquiterpene derivatives: (2Z,4E)-(R)-alpha-ionylideneethane (IV-Al(i)); (R)-alpha-ionone (I -Al), i.s., internal standard.

[0051] Figure 6: (R)-alpha-ionylideneethane and (R)-alpha ionone (I-Al) titers obtained from engineered Saccharomyces cerevisiae strain YST361 cultures with different organic solvents and incubated at 30°C for either three (A) or seven (B) days.

[0052] Figure 7: GC-MS analysis of the product formed using E coli cells modified to produce (R)-alpha-ionylideneethanol and expressing the Bcaba3 (SEQ ID NO: 83) and Bcabal enzyme (SEQ ID NO: 46). The total ion chromatogram is annotated with numbers corresponding peaks of the identified sesquiterpene compounds: (2Z,4E)-(R)-alpha-ionylideneethane (IV-Al(i)); (2E,4E)-(R)-alpha-ionylideneethane (IV-Al(ii)); (E)-beta-famesene (VI-1), (3Z,6E)-alpha-famesene (VI-2); (3E,6E)-alpha-famesene (VI-3), (2Z,4E,6E)-allofamesene (VI-4); (2E,4E,6E)-allofamesene (VI-5); (2Z,4E)-(R)-gamma-ionylideneethane (IV-A2(i)); (2Z,4E)-(R)-alpha-ionylideneethanol (III-Al(i)); (2E,4E)-(R)-alpha-ionylideneethanol (III-Al(ii)); (2Z,4E)-(R)-alpha-ionylidene acetaldehyde (II-Al(i)); (2E,4E)-(R)-alpha-ionylidene acetaldehyde (II-Al(ii)). i.s., internal standard.

[0053] Figure 8: GC-MS analysis of the product formed using E coli cells modified to produce (R)-alpha-ionylideneethanol and expressing the Bcaba3 (SEQ ID NO: 83) together with fourdifferent cytochrome P450s: Bcabal (SEQ ID NO: 46), THV44259 (SEQ ID NO: 59), THY15918 (SEQ ID NO: 51) and XP 038762906 (SEQ ID NO: 55). The peaks in the total ion chromatograms are annotated with numbers corresponding to (2Z,4E)-(R)-alpha-ionylideneethane (IV-A(i)) and (2Z,4E)-(R)-alpha-ionylideneethanol (III-Al(i)).

[0054] Figure 9: GC-MS analysis of the product formed using E. coli cells modified to produce (R)-alpha-ionylideneethanol and expressing the Bcaba3 (SEQ ID NO: 83) together with four different cytochrome P450s. The peaks in the total ion chromatograms are annotated with numbers corresponding to (2Z,4E)-(R)-alpha-ionylideneethane (IV-Al(i)) and (2Z,4E)-(R)-alpha-ionylideneethanol (III-Al(i)). i.s, internal standard.

[0055] Figure 10: GC-MS analysis of the non-enzymatic conversion of (R)-alpha-ionylideneethanol under aerobic conditions. A representative chromatogram is presented for each incubation day and under aerobic condition (air, left part) or under anaerobic condition (argon, right part). The total ion chromatograms are annotated with numbers corresponding to the identified terpene compounds: (2Z,4E)-(R)-alpha-ionylideneethanol (III-Al(i)) and (2E,4E)-(R)-alpha-ionylideneethanol (III-Al(ii)) and (R)-alpha-ionone (I-Al).

[0056] Figure 11: GC-MS analysis of the product formed using E coli cells modified to produce (R)-alpha-ionone (I-Al) from a sesquiterpene precursor and expressing the Bcaba3 (SEQ ID NO: 83) and Bcabal (SEQ ID NO: 46) enzymes, an ADH and an enal-cleaving enzyme. A. cells expressing Bcaba3 (SEQ ID NO: 83) and Bcabal. B. cells expressing Bcaba3 (SEQ ID NO: 83), Bcabal (SEQ ID NO: 46) and an ADH. C, cells expressing Bcaba3 (SEQ ID NO: 83), Bcabal (SEQ ID NO: 46), an ADH and an enal-cleaving enzyme. The main peaks in the total ion chromatograms are annotated with numbers corresponding to the identified sesquiterpene compounds or sesquiterpene derivatives: (2Z,4E)-(R)-alpha-ionylideneethane (IV-Al(i)); (2Z,4E)-(R)-alpha-ionylideneethanol (III-Al(i)); (2Z,4E)-(R)-alpha-ionylidene acetaldehyde (II-Al(i)); (2E,4E)-(R)-alpha-ionylidene acetaldehyde (II-Al(ii)), (R)-alpha-ionone (I-Al). i.s., internal standard.

[0057] Figure 12: Chiral GC analysis of alpha-ionone produced by recombinant cells engineered to produced alpha-ionone from a sesquiterpene biosynthetic pathway. A. Chiral GC separation of (S)-alpha-ionone (I-Cl) and (R)-alpha-ionone (I-Al) of a synthetic alphaionone. B. GC chiral analysis of (R)-alpha-ionone (I-Al) produced by the engineered cells.

[0058] Figure 13: New biosynthetic pathway of (R)-alpha-ionone.Figure 14: GC-MS analysis of the product formed using E coli cells modified to produce (R)-alpha-ionone (I-Al) from a sesquiterpene precursor and expressing the Bcaba3 (SEQ ID NO: 83) and Bcabal (SEQ ID NO: 46) enzymes, SCH91-03945 (ADH) (SEQ ID NO: 36) and the enal-cleaving enzyme RinsDUF4334 (SEQ ID NO: 30). A. Cells expressing Bcaba3 (SEQ ID NO: 83), Bcabal (SEQ ID NO: 46) and SCH91 -03945 (SEQ ID NO: 36). B.

[0059] Cells expressing Bcaba3 (SEQ ID NO: 83), Bcabal (SEQ ID NO: 46), SCH91-03945 (SEQ ID NO: 36) and RinsDUF4334 (SEQ ID NO: 30). The main peaks in the total ion chromatograms are annotated with numbers corresponding to the identified sesquiterpene compounds or sesquiterpene derivatives: (2Z,4E)-(R)-alpha-ionylideneethane (IV-A(i)); (2Z,4E)-(R)-alpha-ionylidene acetaldehyde (II-Al(i)); (2E,4E)-(R)-alpha-ionylidene acetaldehyde (II-Al(ii)), (R)-alpha-ionone (I -Al), i.s., internal standard.

[0060] Figure 15: GC-MS analysis of compounds produced by engineered Saccharomyces cerevisae strain YST382 and the corresponding control strain. The internal standard (i.s.), (2Z,4E)-(R)-alpha-ionylideneethane (IV-A(i)), (2Z,4E)-(R)-alpha-ionylideneethanol (III-Al(i)), (2Z,4E)-(R)-alpha-ionylidene acetaldehyde (II-Al(i)) and (R)-alpha-ionone (I -Al) are labeled. MS profile for the peak corresponding to (R)-alpha-ionone (I-Al) is shown.

[0061] Figure 16: GC-MS analysis of the product formed using E coli cells modified to produce (R)-alpha-ionylideneethanol and expressing the Bcaba3-Del53 (SEQ ID NO: 89) and Skri_CYP2 enzymes (SEQ ID NO: 180). A. cells co-expressing Bcaba3-Del53 (SEQ ID NO: 89) and Skri_CYP2 enzymes (SEQ ID NO: 180). B. cells expressing Bcaba3-Del53 (SEQ ID NO: 89) only. The total ion chromatograms are annotated with numbers and names of the identified terpene compounds.

[0062] Figure 17: GC-MS analysis of the product formed using E coli cells modified to produce gamma-ionylideneethane and expressing the enzymes Sath_TPS-V5 (SEQ ID NO: 244), Alar_TPS-V2 (SEQ ID NO: 270), Myal_TPS-V2 (SEQ ID NO: 262), Myro_TPS-Vl (SEQ ID NO: 256). The total ion chromatogram is annotated with numbers corresponding to the identified sesquiterpene compounds: (2Z,4E)-gamma-ionylideneethane (IV-E(i));

[0063] (2Z,4E)-alpha-ionylideneethane (IV-D(i)); (E)-beta-famesene (VI-1); (2Z,4E,6E)-allofamesene (VI-5); (2E,4E,6E)-allofamesene (VI-4), i.s., internal standard.

[0064] Figure 18: GC-MS analysis of the product formed using E coli cells modified to produce (2Z,4E)-(R)-gamma-ionylideneethanol (III-A2(i)); and expressing Sath_Tps_V5 (SEQ ID NO: 244) and one of the following cytochrome P450 enzyme: CiCYP71AV8-P20(SEQ ID NO: 172), Bcabal (SEQ ID NO: 46), Pcrubal (SEQ ID NO: 176), Skri_CYP2 (SEQ ID NO: 180). The botom chromatogram was obtained from control cells expressing the synthase only. The peaks of (2Z,4E)-gamma-ionylideneethane (IV-Al(i)) and (2Z,4E)-gamma-ionylideneethanol (III-A2(i)) are annotated in the total ion chromatograms.

[0065] Figure 19A: GC-MS analysis of the product formed using E coli cells modified to produce gamma-ionone (I-A2) from a sesquiterpene precursor. All strain were engineered to express the Sath_TPS-V5 terpene synthase (SEQ ID NO: 244), the ThTerpADHl alcohol dehydrogenase (SEQ ID NO: 40), and the Rins-DUF4334 Enal-cleaving enzyme (SEQ ID NO: 30). The cells were transformed with a second plasmid to express different functional cytochrome P450 monooxyganses: Skri_CYP2-AlkJ-K (SEQ ID NO: 180) (A) Bcabal (SEQ ID NO: 46) (B), CiCYP71AV8-P20 (SEQ ID NO: 172) (C) The chromatogram (D) is obtained with control cells without heterologous cytochrome P450 enzyme. The peak marked with an * correspond to geranylactone derived from the degradation of farnesol present in this strain Figure 19B: GC chiral analysis of the gamma-ionone produced using engineered E. coli cells (and synthetic references of (R)-gamma-ionone and (S)- gamma-ionone.

[0066] Figure 20: GC-MS analysis of the product formed using E. coli cells modified to produce terpeneoids and expressing the Teni TPS (upper chromatogram) or the Skri TPS (lower chromatogram) enzymes. The peaks of (2Z,4E)-beta-ionylideneethane (1) and (2E,4E)-beta-ionylideneethane (2) are indicated.

[0067] Figure 21: GC-MS analysis of the product formed using E coli cells modified to produce beta-ionylideneethanol and expressing the Skri TPS (SEQ ID NO: 274) and a cytochrome P450 enzyme. A. Cells co-expressing Skri TPS (SEQ ID NO: 274) and Skri_CYP2 enzymes (SEQ ID NO: 180). B. Cells co-expressing Skri TPS (SEQ ID NO: 180) and CiCYP71AV8-P20 (SEQ ID NO: 172). C. Control cells expressing Skri TPS (SEQ ID NO: 274) only. The peaks of (2E,4E)-beta-ionylideneethane and (2E,4E)-beta-ionylideneethanol are annotated in the total ion chromatograms.

[0068] Figure 22: GC-MS analysis of the product formed using E coli cells modified to produce beta-ionone (I-B3) from a sesquiterpene precursor and expressing the Skri TPS, ThTerpADHl (SEQ ID NO: 40), Rins-DUF4334, and Skri_CYP2 (A) or CiCYP71AV8-P20 (SEQ ID NO: 172). C. Control cells with heterologous cytochrome P450.Figure 23: GCMS analysis demonstrating the production of (2Z,4E)-alpha-ionylideneethane (IV-D(i)) and (2E,4E)-alpha-ionylideneethane (IV-D(ii)) in E. coli cells expressing different alpha-ionylideneethane synthases.

[0069] Abbreviations used

[0070] ADH alcohol dehydrogenase

[0071] bp base pair

[0072] kb kilo base

[0073] DNA deoxyribonucleic acid

[0074] cDNA complementary DNA

[0075] CYP450 cytochrome P450

[0076] DMAPP dimethylallyl diphosphate

[0077] FMO flavin Monooxygenase

[0078] FPP famesyl diphosphate

[0079] GPP geranyl diphosphate

[0080] GGPP geranylgeranyl diphosphate

[0081] GGPS geranylgeranyl diphosphate synthase

[0082] GC gas chromatograph

[0083] IPP isopentenyl diphosphate

[0084] MS mass spectrometer / mass spectrometry

[0085] MV A mevalonic acid

[0086] PP diphosphate, pyrophosphate

[0087] PCR polymerase chain reaction

[0088] RNA ribonucleic acid

[0089] mRNA messenger ribonucleic acid

[0090] miRNA micro-RNA

[0091] siRNA small interfering RNA

[0092] rRNA ribosomal RNA

[0093] tRNA transfer RNA

[0094] TPP terpenyl diphosphateDefinitions

[0095] In order that the present disclosure can be more readily understood, certain terms and methodologies are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below.

[0096] Additional definitions are set forth throughout the application. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.

[0098] Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the detailed description and from the claims.

[0099] General terms:

[0100] As used in the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise. As an example, "an element" may mean one element or more than one element, i.e. “at least one element”.

[0101] For the descriptions herein and the appended claims, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are interchangeable and not intended to be limiting.

[0102] It is to be further understood that where descriptions of various embodiments use the term “comprising” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of”.The terms "purified", "substantially purified", and "isolated" as used herein refer to the state of being free of other, dissimilar compounds with which a compound of the invention is normally associated in its natural state, so that the "purified", "substantially purified", and "isolated" subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, or at least 50% or 75% of the mass, by weight, of a given sample. In one embodiment, these terms refer to the compound of the invention comprising at least 95, 96, 97, 98, 99 or 100%, of the mass, by weight, of a given sample. As used herein, the terms "purified," "substantially purified," and "isolated" when referring to a nucleic acid or protein, or nucleic acids or proteins, also refers to a state of purification or concentration different than that which occurs naturally, for example in a prokaryotic or eukaryotic environment, like, for example in a bacterial or fungal cell, or in the mammalian organism, especially human body. Any degree of purification or concentration greater than that which occurs naturally, including (1) the purification from other associated structures or compounds or (2) the association with structures or compounds to which it is not normally associated in said prokaryotic or eukaryotic environment, are within the meaning of "isolated”. The nucleic acid or protein or classes of nucleic acids or proteins, described herein, may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.

[0103] The term “about” indicates a potential variation of ± 25% of the stated value, in particular ± 15%, ± 10 %, more particularly ± 5%, ± 2% or ± 1%.

[0104] The term "substantially" describes a range of values of from about 80 to 100%, such as, for example, 85-99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9% and especially 99 to 99.9%.

[0105] “Predominantly” refers to a proportion in the range of above 50%, as for example in the range of 51 to 100%, particularly in the range of 75 to 99,9%, more particularly 85 to 98,5%, like 95 to 99%.

[0106] A “main product” in the context of the present invention designates a single compound or a group of at least 2 compounds, like 2, 3, 4, 5 or more, particularly 2 or 3 compounds, which single compound or group of compounds is “predominantly” prepared by a reaction as described herein, and is contained in said reaction in a predominant proportion based on the total amount of the constituents of the product formed by said reaction. Said proportion maybe a molar proportion, a weight proportion or, preferably based on chromatographic analytics, an area proportion calculated from the corresponding chromatogram of the reaction products.

[0107] A “side product” in the context of the present invention designates a single compound or a group of at least 2 compounds, like 2, 3, 4, 5 or more, particularly 2 or 3 compounds, which single compound or group of compounds is not “predominantly” prepared by a reaction as described herein.

[0108] Because of the reversibility of enzymatic reactions, the present invention relates, unless otherwise stated, to the enzymatic or biocatalytic reactions described herein in both directions of reaction.

[0109] "Functional mutants" of herein described polypeptides include the "functional equivalents" of such polypeptides as defined below.

[0110] The term "stereoisomers" includes conformational isomers and in particular configuration isomers.

[0111] Included in general are, according to the invention, all “stereoisomeric forms” of the compounds described herein, such as “constitutional isomers” and “stereoisomers”.

[0112] “Stereoisomeric forms” encompass in particular, “stereoisomers” and mixtures thereof, e.g. configuration isomers (optical isomers), such as enantiomers, or geometric isomers (diastereomers), such as E- and Z-isomers, and combinations thereof. If one or more asymmetric centers are present in one molecule, the invention encompasses all combinations of different conformations of these asymmetry centers, e.g. enantiomeric pairs.

[0113] “Stereoselectivity” describes the ability to produce a particular stereoisomer of a compound in a stereoisomerically pure form or to specifically convert a particular stereoisomer in an enzyme catalyzed method as described herein out of a plurality of stereoisomers. More specifically, this means that a product of the invention is enriched with respect to a specific stereoisomer, or an educt may be depleted with respect to a particular stereoisomer. This may be quantified via the purity %ee-parameter calculated according to the formula:

[0114] %ee = [XA-XB] / [ XA+XB]*I00,

[0115] wherein XAand XB represent the molar ratio (Molenbruch) of the stereoisomers A and B.

[0116] The terms “selectively converting” or “increasing the selectivity” in general means that a particular stereoisomeric form, as for example the E-form, of an unsaturated hydrocarbon, isconverted in a higher proportion or amount (compared on a molar basis) than the corresponding other stereoisomeric form, as for example Z-form, either during the entire course of said reaction (i.e. between initiation and termination of the reaction), at a certain point of time of said reaction, or during an “interval” of said reaction. In particular, said selectivity may be observed during an “interval” corresponding 1 to 99%, 2 to 95%, 3 to 90%, 5 to 85%, 10 to 80%, 15 to 75%, 20 to 70%, 25 to 65%, 30 to 60%, or 40 to 50% conversion of the initial amount of the substrate. Said higher proportion or amount may, for example, be expressed in terms of:

[0117] a higher maximum yield of an isomer observed during the entire course of the reaction or said interval thereof;

[0118] a higher relative amount of an isomer at a defined % degree of conversion value of the substrate; and / or

[0119] an identical relative amount of an isomer at a higher % degree of conversion value; each of which preferably being observed relative to a reference method, said reference method being performed under otherwise identical conditions with known chemical or biochemical means.

[0120] Generally also comprised in accordance with the invention are all “isomeric forms” of the compounds described herein, such as constitutional isomers and in particular stereoisomers and mixtures of these, such as, for example, optical isomers or geometric isomers, such as E and Z-isomers, and combinations of these. If several centers of asymmetry are present in a molecule, then the invention comprises all combinations of different conformations of these centers of asymmetry, such as, for example, pairs of enantiomers, or any mixtures of stereoisomeric forms.

[0121] “Yield" and / or the "conversion rate" of a reaction according to the invention is determined over a defined period of, for example, 4, 6, 8, 10, 12, 16, 20, 24, 36 or 48 hours, in which the reaction takes place. In particular, the reaction is carried out under precisely defined conditions, for example at “standard conditions” as herein defined.The different yield parameters ("Yield" or Yp / s; " Specific Productivity Yield"; or Space-Time- Yield (STY)) are well known in the art and are determined as described in the literature.

[0122] "Yield" and "Yp / s" (each expressed in mass of product produced / mass of material consumed) are herein used as synonyms.

[0123] The specific productivity-yield describes the amount of a product that is produced per h and L fermentation broth per g of biomass. The amount of wet cell weight stated as WCW describes the quantity of biologically active microorganism in a biochemical reaction. The value is given as g product per g WCW per h (i.e. g / gWCW'1h'1). Alternatively, the quantity of biomass can also be expressed as the amount of dry cell weight stated as DCW. Furthermore, the biomass concentration can be more easily determined by measuring the optical density at 600 nm (ODsoo) and by using an experimentally determined correlation factor for estimating the corresponding wet cell or dry cell weight, respectively.

[0124] If the present disclosure refers to features, parameters and ranges thereof of different degree of preference (including general, not explicitly preferred features, parameters and ranges thereof) then, unless otherwise stated, any combination of two or more of such features, parameters and ranges thereof, irrespective of their respective degree of preference, is encompassed by the disclosure of the present description.

[0125] Biochemical and biological terms:

[0126] The term "domain" refers to a set of amino acids or a partial sequence of amino acids residues conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between protein homologues, amino acids that are highly conserved at specific positions of such domain indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.

[0127] The term "motif" or consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain.The sequences of motifs can be described using the standard IUPAC one-letter codes for the amino acids. Ambiguities are indicated by listing the acceptable amino acids for a given position between brackets. For example, [LWI] stands for L (Leucine), W (Tryptophan) or I (Isoleucine). X represent positions where independently of each other any natural amino acid residue is present.

[0128] A “protein family” is defined as a group of proteins that share a common evolutionary origin reflected by their related functions, similarities in sequence, or similar primary, secondary or tertiary structure. Proteins within protein families are usually homologous and have similar structure of conserved functional domains and motifs.

[0129] Specialist databases exist for the identification of domains, for example, SMART (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi?GENOMIC=l) (Schultz etal. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Paysan-Lafosse et al (2022), Nucleic Acids Research; Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)).

[0130] Useful tools to search or predict protein domains or protein family signatures in protein sequence are for example the NCBI conserved domain search tool (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) or the InterProScan tool (http: / / www.ebi.ac.uk / interpro / search / sequence / ). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.

[0131] The term "Pfam" refers to a large collection of protein domains and protein families maintained by the Pfam Consortium and available at several sponsored world wide web sites, such as the InterPro consortium web site https: / / www.ebi.ac.uk / interpro / (European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL EBI). The latest release of Pfam is Pfam 35.0 (November 2021), based on the UniProt Reference Proteomes (El-Gebali S. et al, 2019, Nucleic Acids Res. 47, Database issue D427-D432). Pfam domains and families are identified using multiple sequence alignments and hidden Markov models (HMMs). Pfam-A family or domain assignments, are high quality assignments generated by acurated seed alignment using representative members of a protein family and profile hidden Markov models based on the seed alignment (Unless otherwise specified, matches of a queried protein to a Pfam domain or family are Pfam-A matches). All identified sequences belonging to the family are then used to automatically generate a full alignment for the family (Sonnhammer (1998) Nucleic Acids Research 26, 320-322; Bateman (2000) Nucleic Acids Research 26, 263-266; Bateman (2004) Nucleic Acids Research 32, Database Issue, D138-D141; Finn (2006) Nucleic Acids Research Database Issue 34, D247-251; Finn (2010) Nucleic Acids Research Database Issue 38, D211-222). By accessing the Pfam database, for example, using any of the above-reference websites, protein sequences can be queried against the HMMs using HMMER homology search software (e.g., HMMER2, HMMER3, or a higher version, hmmer.janelia.org / ). Significant matches that identify a queried protein as being in a pfam family (or as having a particular Pfam domain) are those in which the bit score is greater than or equal to the gathering threshold for the Pfam domain. Expectation values (e-values) can also be used as a criterion for inclusion of a queried protein in a Pfam or for determining whether a queried protein has a particular Pfam domain, where low e-values, much less than 1.0, for example less than 0.1, or less.

[0132] InterPro is another database of protein families providing a classification of protein sequences into families and identifies functionally important domains and conserved sites (Blum et al, Nucleic Acids Res. 2021 49(D1):D344-D354). The protein signatures are provided by multiple databases such as Pfam or SMART (Simple Modular Architecture Research Tool). InterProScan is a software that allows protein and nucleic acid sequences to be searched against InterPro's signatures.

[0133] The “E-value” (expectation value) is the number of hits that would be expected to have a score equal to or better than this value, by chance alone. This means that a good E-value which gives a confident prediction is much less than 1. E-values around 1 is what is expected by chance. Thus, the lower the E-value, the more specific the search for domains will be. Only positive numbers are allowed.

[0134] A “precursor” compound or molecule of a target compound or molecule as described herein is converted to said target compound, preferably through the enzymatic action of a suitable polypeptide performing at least one structural or functional change on said precursor molecule. For example, a “diphosphate precursor” (as for example a “terpenyl diphosphateprecursor”) is converted to said target compound (as for example a terpene alcohol) via enzymatic removal of the diphosphate moiety, for example by removal of mono- or diphosphate groups by a phosphatase enzyme. For example, a “non-cyclic precursor” (like a “non-cyclic terpenyl precursor”) may be converted to the cyclic target molecule (like a cyclic terpene compound) through the action of a cyclase or synthase enzyme, irrespective of the particular enzymatic mechanism of such enzyme, in one or more steps.

[0135] The enzyme nomenclature or enzyme classification (EC) established by the International Union of Biochemistry and Molecular Biology (IUBMB) is a system of naming and categorizing enzymes based on their catalytic activity and biochemical properties. The enzyme nomenclature is widely used in biochemistry to classify and categorize based on their function. The E.C. classification assigns each enzyme a number reflecting the reaction or the type of reaction catalyzed by this enzyme.

[0136] The enzyme classification can be explored using the ‘ExplorEnz’ database (https: / / www.enzyme-database.org / ) or International Union of Biochemistry and Molecular Biology (IUBMB) web site (https: / / iubmb.qmul.ac.uk). Information can be found about the classification and nomenclature of enzymes, their functions and properties. The database can be searched to find information for a specific enzyme family or enzyme.

[0137] Particular examples of suitable standard conditions for each of the above-described enzyme activities may be taken from the Examples below.

[0138] The “mevalonate pathway” also known as the “isoprenoid pathway” or “HMG-CoA reductase pathway” is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five-carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP). Key enzymes are acetoacetyl-CoA thiolase (atoB), HMG-CoA synthase (mvaS), HMG-CoA reductase (mvaA), mevalonate kinase (MvaKl), phosphomevalonate kinase (MvaK2), a mevalonate diphosphate decarboxylase (MvaD), and an isopentenyl diphosphate isomerase (idi). Combining the mevalonate pathway with enzyme activity to generate the terpene precursors GPP, FPP or GGPP, like in particular FPP synthase (ERG20), allows the recombinant cellular production of terpenes.As used herein, the term “host cell”, “recombinant cell” or “transformed cell” refers to a cell (or organism) altered to harbor at least one nucleic acid molecule, for instance, a recombinant gene encoding a desired protein or nucleic acid sequence which upon transcription yields at least one functional polypeptide of the present invention. The host cell is particularly a bacterial cell, a fungal cell or a plant cell or plants. The host cell may contain a recombinant gene or several genes, as for example organized as an operon, which has been integrated into the nuclear organelle genomes of the host cell. Alternatively, the host may contain the recombinant gene extra-chromosomally. Methods of introducing recombinant nucleic acid sequences into such host cells are well known in the art and constitute routine laboratory methodologies which do not need to be further described herein.

[0139] The term "heterologous" when used with respect to a polynucleotide (such as DNA or RNA), polypeptide or protein refers to a polynucleotide, polypeptide or protein that does not occur naturally as part of the recombinant cell, genome or DNA or RNA in which it is present, or that is found in a different number of copies, or under the control of a different control sequence, or in a cell or location or locations in the genome or DNA or RNA that differ from that in which it is found in nature. Heterologous polynucleotides, polypeptides or proteins are not endogenous to the cell into which they are introduced but have been obtained from another cell or synthetically or recombinantly produced.

[0140] The term "homologous" when used to indicate the relation between a given (recombinant) polynucleotide or polypeptide and a given host organism or host cell such as the recombinant cell as disclosed herein, is understood to mean that in nature the polynucleotide or polypeptide molecule is produced by a recombinant cell, host cell or organism of the same species, such as of the same variety or strain.

[0141] The term “organism” refers to any non-human multicellular or unicellular organism such as a plant, or a microorganism. Particularly, a micro-organism is a bacterium, a yeast, an algae or a fungus.

[0142] The term “plant” is used interchangeably to include plant cells including plant protoplasts, plant tissues, plant cell tissue cultures giving rise to regenerated plants, or parts of plants, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits and the like. Any plant can be used to carry out the methods of an embodiment herein.

[0143] The term "fermentative production" or "fermentation" refers to the ability of a microorganism (assisted by enzyme activity contained in or generated by said microorganism)to produce a chemical compound in cell culture utilizing at least one carbon source added to the incubation.

[0144] The term "fermentation broth" is understood to mean a liquid, particularly aqueous or aqueous / organic solution which is based on a fermentative process and has not been worked up or has been worked up, for example, as described herein.

[0145] An “enzymatically catalysed” or “biocatalytic” method means that said method is performed under the catalytic action of an enzyme, including enzyme mutants, as herein defined. Thus, the method can either be performed in the presence of said enzyme in isolated (purified, enriched) or crude form or in the presence of a cellular system, in particular, natural or recombinant microbial cells containing said enzyme in active form, and having the ability to catalyze the conversion reaction as disclosed herein.

[0146] The term “control sequence” as used herein refers to components involved in the regulation of the expression of a coding sequence in a specific organism or in vitro. Examples of control sequences are transcription initiation sequences, termination sequences, promoters, leaders, signal peptides, propeptides, prepropeptides, or enhancer sequences; Shine-Delgamo sequences, repressor or activator sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.

[0147] As used herein, the terms "culture broth," "culture medium," and "growth medium" can be used interchangeably to refer to a liquid or solid that supports growth of a cell. Typically, the culture medium may comprise a carbon source (e.g. one or more of glucose, fructose, sucrose, xylose, glycerol, plant biomass, celluloses, hemicelluloses, pectin, rhamnose, galactose, fucose, maltose, maltodextrin, ribose, ribulose, or starch, starch derivatives, lactose, fatty acids, triglycerides). Typically, the culture medium may also comprise a nitrogen source such as urea, or an ammonium salt such as ammonium sulphate, ammonium chloride, ammonium nitrate or ammonium phosphate. A culture broth may further comprise trace metals, vitamins, salts, amino acids, etc.. The trace metals may be divalent cations, including, but not limited to Mn2+, Mg2+, Fe2+, Cu2+etc. The culture may also include an organic overlay as a co-solvent, such as n-decane, n-dodecane, mineral oil or an adipate ester (such as esterex-A32).The term "derived from" also includes the terms "originates from," "obtained from," "obtainable from," "isolated from," and "created from," and typically indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material. As used herein, a substance (e.g., a nucleic acid molecule or polypeptide) "derived from" a cell preferably means that the substance is native to that microorganism.

[0148] As used herein, the term "fed-batch culture" or "semi-batch culture" are used interchangeably to refer to as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. In some embodiments, all the nutrients are fed into the bioreactor.

[0149] Chemical terms

[0150] The term “alpha, beta-unsaturated carbonyl” compound describes organic molecules containing an aldehyde or keto group of the general formula RaRbC=C(RC)-C=O, wherein the C=C bond may be of any stereoisomeric configuration and wherein residues Ra, Rband Rcmay be identical or different and may have the meanings as specified below for particular alpha, beta unsaturated carbonyl compounds.

[0151] An “ionone” compound in the context of the present invention will show the following basic structure of formula I. The term “ionone” encompasses any compounds of this basic structure, in any stereoisomeric form. The different stereoisomers of the ionone are disclosed in Table 1.

[0152]

[0153] (formula I)

[0154] In formula I the dotted line represents the potential positions of the double bond. The bond having a dotted line ( - ) represents independently from each other either a single carbon-carbon bond or a carbon-carbon double bond, wherein one of the bonds having a dotted line ( - ) represents a carbon-carbon double bond. The proviso is that the compound does not comprise two cumulated carbon-carbon double bonds.An “lonyhdene acetaldehyde” compound in the context of the present invention will show the following basic structure of formula II. The term “ionylidene acetaldehyde” encompasses any compounds of this basic structure, in any stereoisomeric form. The different stereoisomers of the ionylidene acetaldehyde are disclosed in Table 2.

[0155]

[0156] (formula II)

[0157] In formula II the dotted line represents the potential positions of the double bond. The bond having a dotted line ( - ) represents independently from each other either a single carbon-carbon bond or a carbon-carbon double bond, wherein one of the bonds having a dotted line ( - ) represents a carbon-carbon double bond. The proviso is that the compound does not comprise two cumulated carbon-carbon double bonds.

[0158] The wavy line represents a carbon-carbon bond linked to a carbon-carbon double bond which is either in the Z or in the E-configuration.

[0159] An “ionylideneethanol” compound in the context of the present invention will show the following basic structure of formula III. The term “ionylideneethanol” encompasses any compounds of this basic structure, in any stereoisomeric form. The different stereoisomers of the ionylideneethanol are disclosed in Table 3.

[0160]

[0161] (formula III)

[0162] In formula III the dotted line represents the potential positions of the double bond. The bond having a dotted line ( - ) represents independently from each other either a single carbon-carbon bond or a carbon-carbon double bond, wherein one of the bonds having a dotted line ( - ) represents a carbon-carbon double bond. The proviso is that the compound does not comprise two cumulated carbon-carbon double bonds.

[0163] The wavy line represents a carbon-carbon bond linked to a carbon-carbon double bond which is either in the Z or in the E-configuration.An “ionylideneethane” compound in the context of the present invention will show the following basic structure of formula IV. The term “ionylideneethane” encompasses any compounds of this basic structure, in any stereoisomeric form. The different stereoisomers of the ionylideneethane are disclosed in Table 4.

[0164]

[0165] (formula IV)

[0166] In formula IV the dotted line represents the potential positions of the double bond. The bond having a dotted line ( - ) represents independently from each other either a single carbon-carbon bond or a carbon-carbon double bond, wherein one of the bonds having a dotted line ( - ) represents a carbon-carbon double bond. The proviso is that the compound does not comprise two cumulated carbon-carbon double bonds.

[0167] The wavy line represents a carbon-carbon bond linked to a carbon-carbon double bond which is either in the Z or in the E-configuration.

[0168] A “famesyl pyrophosphate” compound in the context of the present invention will show the following basic structure of formula V. The term “famesyl pyrophosphate” encompasses any compounds of this basic structure, in any stereoisomeric form. The different stereoisomers of the famesyl pyrophosphate are disclosed in Table 5. “Diphosphate” and “pyrophosphate” as used herein are synonyms. The wavy line represents a carbon-carbon bond linked to a carbon-carbon double bond which is either in the Z or in the E-configuration.

[0169]

[0170] (formula V)

[0171] Overview of particular compound names disclosed herein and their structural formulae Ionones:

[0172] Table 1

[0173]

[0174]

[0175] lonylidene aldehydes:

[0176] Table 2

[0177]

[0178]

[0179]

[0180] It is known that the following pairs of ionylidene aldehydes interconvert in solution:

[0181] Pair 1: Formula II-Al(i) and Formula II-Al(ii);

[0182] Pair 2: Formula II-A2(i) and Formula II-A2(ii);

[0183] - Pair 3: Formula II-B3(i) and Formula II-B3(ii);

[0184] Pair 4: Formula Il-Cl(i) and Formula Il-Cl(ii);

[0185] Pair 5: Formula II-C2(i) and Formula II-C2(ii);

[0186] Pair 6: Formula II-D(i) and Formula II-D(ii);

[0187] Pair 7: Formula II-E(i) and Formula II-E(ii).

[0188] Ionylidene alcohols

[0189] Table 3

[0190]

[0191]

[0192] lonylideneethane

[0193] Table 4

[0194]

[0195]

[0196] Farnesyl pyrophosphate

[0197] Table 5

[0198]

[0199] Linear terpene products

[0200] Table 6

[0201]

[0202] DETAILED DESCRIPTION

[0203] The present inventors sought to identify improved methods for the preparation of an ionone compound of formula I:

[0204]

[0205] (formula I)and in particular for the ionone compounds of formula I-Al, formula I-A2, formula I-B3, formula I-Cl and / or formula I-C2.

[0206] The inventors developed a deep understanding of the biochemical route to the production of these ionone compounds and identified a biosynthetic pathway that does not require the degradation of carotenoids. The invention therefore represents a significant scientific and commercial advance in the preparation of ionone compounds of formula I.

[0207] In particular, the combination of enzymes in the elucidated biosynthetic pathways described herein, and the order of the enzymes used in the defined methods, have not been described before in the prior art.

[0208] In addition, and as will be discussed in more detail below, included in this invention is an in vivo process for the preparation of compounds of formula I in recombinant cells. This is the first time the production of a compound of formula I has been demonstrated using the biosynthetic pathway identified by the inventors in recombinant cells.

[0209] Methods of producing ionones

[0210] Use of enal-cleaving enzymes in the method of the invention

[0211] The invention provides a method for preparing an ionone compound of formula I

[0212]

[0213] (formula I)

[0214] in the form of any one of its stereoisomers or a mixture thereof, wherein the method comprises:

[0215] contacting an ionylidene acetaldehyde compound of formula II

[0216]

[0217] (formula II)

[0218] in the form of any one of its stereoisomers or a mixture thereof, with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I.The inventors provide the first demonstration that enal-cleaving enzymes can catalyse this reaction. Accordingly, the invention further provides the use of an enal-cleaving enzyme described herein for converting a compound of formula II to a compound of formula I.

[0219] In some embodiments, the compound of formula II is in the form of formula II-A and the enal-cleaving enzyme produces a compound of formula I-A.

[0220]

[0221] In some embodiments, the compound of formula II is in the form of formula II-D(i) or formula II-D(ii) and the enal-cleaving enzyme produces a compound of formula I-D.

[0222]

[0223] In some embodiments, the compound of formula II is in the form of formula II-E(i) or formula II-E(ii) and the enal-cleaving enzyme produces a compound of formula I-E.

[0224]

[0225] In some embodiments, the compound of formula II is in the form of formula II-Al(i) or formula II-Al(ii) and the enal-cleaving enzyme produces a compound of formula I -Al.

[0226]

[0227] In some embodiments, the compound of formula II is in the form of formula II-A2(i) or formula II- A2(ii) and the enal-cleaving enzyme produces a compound of formula I-A2.

[0228]

[0229] In some embodiments, the compound of formula II is in the form of formula II-B3 (i) or formula II-B3(ii) and the enal-cleaving enzyme produces a compound of formula I-B3.

[0230]

[0231] In some embodiments, the compound of formula II is in the form of formula Il-Cl(i) or formula Il-Cl(ii) and the enal-cleaving enzyme produces a compound of formula I-Cl.

[0232]

[0233] In some embodiments, the compound of formula II is in the form of formula II-C2(i) or formula II-C2(ii) and the enal-cleaving enzyme produces a compound of formula I-C2.

[0234]

[0235] An “enal cleaving enzyme”, “enal-cleaving protein” or “enal-cleaving polypeptide” is a polypeptide that catalyses the cleavage of a,P-unsaturated aldehyde carbon-carbon double bonds. Such a polypeptide may also be called an “a,P-unsaturated aldehyde C=C bond cleaving enzyme”, an “a,P-unsaturated aldehyde C=C cleaving enzyme” or an “enal C=C cleaving enzyme”. Examples of such enzymes based on protein domain organization, may also be described as a member of the DUF4334 protein family, and / or as a member of the “GXWXG protein family”.

[0236] “Enal-cleaving activity” is determined under “standard conditions” as described herein below. Enal-cleaving activity can be determined using recombinant enal-cleaving polypeptide expressing host cells, disrupted enal-cleaving polypeptide expressing cells, fractions of these or enriched or purified enal-cleaving polypeptide, in a culture medium or reaction medium, preferably buffered, having a pH in the range of 6 to 11, preferably 7 to 9, at a temperature in the range of about 20 to 45 °C, like about 25 to 40 °C, preferably 25 to 32 °C and in the presence of a reference substrate, as described herein, either added at an initial concentration in the range of 1 to 100 pM mg / ml, preferably 5 to 50 pM, in particular 30 to 40 pM, or endogenously produced by the host cell. The conversion reaction to form the respective cleavage product is conducted from 10 min to 5 h, preferably about 1 to 2 h. The cleavageproduct may then be determined in a conventional manner, for example after extraction with an organic solvent, like ethyl acetate.

[0237] The polypeptide having said enal-cleaving enzyme activity may be selected from the group of polypeptides containing:

[0238] a) at least one DUF4334 protein family domain having the Pfam ID number PF 14232 (in particular within the C-terminal region of their amino acid sequence);

[0239] b) at least one GXWXG (SEQ ID NO: 145) protein family domain having the Pfam ID number PF14231 (in particular within the N-terminal region of their amino acid sequence); and / or

[0240] c) a domain retaining at least 90% sequence identity to PF14232 or PF14231.

[0241] In particular, a polypeptide of the invention having enal-cleaving enzyme activity is identified as a member of the DUF4334 protein family comprising said domain PF 14232 if it matches with said domain with an e-value of less than 1x1 O'5, or less than 1x1 O'10, or less than IxlO'15, or less than IxlO'20, or less than IxlO'25, or less than IxlO'30, or less than or equal to IxlO'35, in particular in a range of IxlO'20to IxlO'32and more particular in a range of IxlO'25to IxlO'31.

[0242] In particular, a polypeptide having enal-cleaving enzyme activity is identified as a member of GXWXG (SEQ ID NO: 145) protein family comprising said domain PF 14231 if it matches with an e-value of less than IxlO'5, or less than IxlO'10, or less than IxlO'15, or less than IxlO'20, or less than IxlO'25, or less than IxlO'30, or less than or equal to IxlO'35, in particular in a range of IxlO'20to IxlO'30.

[0243] As the query sequence the sequence of a polypeptide having enal-cleaving enzyme activity is applied.

[0244] For example, the following website may be applied for the search and calculating such e-value: http: / / www.ebi.ac.uk / Tools / hmmer / search / hmmscan or

[0245] http : / / www. ebi . ac.uk / Tools / pfa / pfams can / .

[0246] Furthermore, the polypeptide having said enal-cleaving enzyme activity may be selected from the group of polypeptides that comprise at least one or more sequence motifs / domains selected from:

[0247] G-[Y or “-”]-x-W-x-G-x-x-[F,L or I]-x-[T,S or R]-G-[H or A] (also expressed as GxxWxGxxxxxGx) set forth in SEQ ID NO: 146, or any partial motif thereof comprising up to 10 or up to 5 consecutive amino acid residues, as for example corresponding to residues inpositions 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, 1-8 or 9-13 of SEQ ID NO: 146. Here, X2 can be Y or can be deleted; X3 can be any naturally occurring amino acid; X5 can be any naturally occurring amino acid; X7 can be any naturally occurring amino acid; X8 can be any naturally occurring amino acid; X9 can be F, L, or I; XI 0 can be any naturally occurring amino acid; XI 1 can be R, S, or T; X13 can be H or A;

[0248] W-[F, Y, A or V]-G-[N or K]-x-[F or Y]-x-[S or D] (also expressed as WxGxxxxx) set forth in SEQ ID NO: 147, or any partial motif thereof comprising up to 4 consecutive amino acid residues, as for example corresponding to residues in positions 1-4, 2-5, 3-6, 4-7 or 5-8 of SEQ ID NO: 147. Here, X2 can be F, A, V, or Y; X4 can be N or K; X5 can be any naturally occurring amino acid; X6 can be F or Y; X7 can be any naturally occurring amino acid; X8 can be D or S;

[0249] [G or S]-x-[A or G]-x-[L, V or M]-x-x-x-x-[F, Y or L]-[R, Q or D]-[G or D]-x-V (also expressed as xxxxxxxxxxxxxV) set forth in SEQ ID NO: 148, or any partial motif thereof comprising up to 10 or up to 5 consecutive amino acid residues, as for example corresponding to residues in positions 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, 9-13, 10-14, 1-8 or 9-14 of SEQ ID NO: 148. Here XI can be G or S; X2 can be any naturally occurring amino acid; X3 can be A or G; X4 can be any naturally occurring amino acid; X5 can be L, M or V; X6 can be any naturally occurring amino acid; X7 can be any naturally occurring amino acid; X8 can be any naturally occurring amino acid; X9 can be any naturally occurring amino acid; XI 0 can be F, L, or Y; Xll can be R, Q or D; X12 can be G or D; X13 can be any naturally occurring amino acid; and

[0250] [M or L]-[V or I]-Y-D-x-x-[P or A] -[I, M or V]-x-[D or V]-x-[F, Y or L] (also expressed as xxYDxxxxxxxx) set forth in SEQ ID NO: 149, or any partial motif thereof comprising up to 10 or up to 5 consecutive amino acid residues, as for example corresponding to residues in positions 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, 1-6 or 7-12 of SEQ ID NO: 149. Here XI can be L or M; X2 can be I or V; X5 can be any naturally occurring amino acid; X6 can be any naturally occurring amino acid; X7 can be P or A; X8 can be I, M or V; X9 can be any naturally occurring amino acid; XI 0 can be D or V; XI 1 can be any naturally occurring amino acid; X12 can be F, Y or L;

[0251] wherein the numbering of X (e.g. X2) corresponds to its position in the relevant sequence. For example, X2 corresponds to X at position 2 in the relevant sequence; and in the above motifs, residues x represent independently of each other any natural amino acid residue,and wherein optionally in each of the above motifs, 1, 2, 3, 4 or 5 amino acid residues different from the x residues may be modified, for example by amino acid substitution, in particular by conservative substitutions, provided that the enzyme retains, at least to analytically detectable extent, enal-cleaving enzyme activity. The function of the square brackets has been described above.

[0252] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

[0253] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

[0254] In some embodiments, the enal-cleaving enzyme has an amino acid sequence of any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

[0255] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 1, 4, 6, 8, 10, 20, 22, 24, 26, 28, 30, or 32.

[0256] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 1, 4, 6, 8, 10, 20, 22, 24, 26, 28, 30, or 32.

[0257] In some embodiments, the enal-cleaving enzyme has an amino acid sequence of any one of SEQ ID NOs: 1, 4, 6, 8, 10, 20, 22, 24, 26, 28, 30, or 32.

[0258] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 1. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 1.

[0259] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 4. In some embodiments, the enal-cleaving enzyme comprises an amino acidsequence of SEQ ID NO: 4. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 4.

[0260] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 6. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 6.

[0261] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 8. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 8.

[0262] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 10. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 10.

[0263] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 12. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 12.

[0264] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 14. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 14.In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 16. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 16.

[0265] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 18. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 18.

[0266] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 20. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 20.

[0267] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 22. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 22. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 22.

[0268] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 24. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 24.

[0269] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity,at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 26. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 26.

[0270] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 28. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 28.

[0271] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 30. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 30.

[0272] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 32. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence of SEQ ID NO: 32. In some embodiments, the enal-cleaving enzyme has an amino acid sequence of SEQ ID NO: 32.

[0273] Non-limiting examples of enal-cleaving enzymes which can be used in the methods according to the present invention are provided in Table 7. Examples of enal-cleaving enzymes are presented in WO 2021 / 005097.

[0274] Table 7:

[0275]

[0276]

[0277]

[0278] * unless otherwise indicated the nucleic acid sequence has been codon optimized for expression in E. coli.

[0279] Non-enzymatic production of ionone

[0280] The inventors have demonstrated that under certain conditions the alcohol intermediate of the ionone can be oxidised non-enzymatically to generate the ionone. It is understood that the combination of the non-enzymatic production of the ionone from an alcohol intermediate, together with the enzymatic production of the alcohol intermediate according to the methods disclosed herein, may also provide a more carbon efficient pathway to ionone formation.

[0281] Accordingly, the invention also provides a method for preparing an ionone of formula I

[0282]

[0283] (formula I)

[0284] in the form of any one of its stereoisomers or a mixture thereof,

[0285] wherein the method comprises:

[0286] oxidising an ionylideneethanol compound of formula III

[0287]

[0288] (formula III)

[0289] in the form of any one of its stereoisomers or a mixture thereof,

[0290] non-enzymatically to produce the ionone compound of formula I.

[0291] In addition, it is envisaged that under certain conditions the aldehyde intermediate of the ionone can be oxidised non-enzymatically to generate the ionone. It is understood that the combination of the non-enzymatic production of the ionone from an aldehyde intermediate, together with the enzymatic production of the aldehyde intermediate according to the methods disclosed herein, may also provide a more carbon efficient pathway to ionone formation.Accordingly, the invention also provides a method for preparing an ionone of formula I

[0292]

[0293] (formula I)

[0294] in the form of any one of its stereoisomers or a mixture thereof,

[0295] wherein the method comprises:

[0296] e acetaldehyde compound of formula II

[0297]

[0298] (formula II)

[0299] in the form of any one of its stereoisomers or a mixture thereof, non-enzymatically to produce the ionone compound of formula I.

[0300] In some embodiments, the compound of formula II is in the form of formula II-A and the non-enzymatic oxidation produces a compound of formula I-A.

[0301]

[0302] In some embodiments, the compound of formula II is in the form of formula II-D(i) or formula II-D(ii) and the non-enzymatic oxidation produces a compound of formula I-D.

[0303]

[0304] In some embodiments, the compound of formula II is in the form of formula II-E(i) or formula II-E(ii) and the non-enzymatic oxidation produces a compound of formula I-E.

[0305]

[0306] In some embodiments, the compound of formula II is in the form of formula II-Al(i) or formula II-Al(ii) and the non-enzymatic oxidation produces a compound of formula I -Al.

[0307]

[0308] In some embodiments, the compound of formula II is in the form of formula II-A2(i) or formula II- A2(ii) and the non-enzymatic oxidation produces a compound of formula I-A2.

[0309]

[0310] In some embodiments, the method further comprises the enzymatic production of a compound of formula II-A, using the methods disclosed herein.

[0311] In some embodiments, the method further comprises the enzymatic production of a compound of formula II-D(i) or formula II-D(ii), using the methods disclosed herein.

[0312] In some embodiments, the method further comprises the enzymatic production of a compound of formula II-E(i) or formula II-E(ii), using the methods disclosed herein.

[0313] In some embodiments, the method further comprises the enzymatic production of a compound of formula II-Al(i) or formula II-Al(ii), using the methods disclosed herein.

[0314] In some embodiments, the method further comprises the enzymatic production of a compound of formula II-A2(i) or formula II-A2(ii), using the methods disclosed herein.

[0315] In some embodiments, the compound of formula II is in the form of formula II-B3 (i) or formula II-B3(ii) and the non-enzymatic oxidation produces a compound of formula I-B3.

[0316]

[0317] In some embodiments, the method further comprises the enzymatic production of a compound of formula II-B3 (i) or formula II-B3 (ii), using the methods disclosed herein.

[0318] In some embodiments, the compound of formula II is in the form of formula Il-Cl(i) or formula Il-Cl(ii) and the non-enzymatic oxidation produces a compound of formula I-Cl.

[0319]

[0320] In some embodiments, the compound of formula II is in the form of formula II-C2(i) or formula II-C2(ii) and the non-enzymatic oxidation produces a compound of formula I-C2.

[0321]

[0322] In some embodiments, the method further comprises the enzymatic production of a compound of formula Il-Cl(i) or formula Il-Cl(ii), using the methods disclosed herein.

[0323] In some embodiments, the method further comprises the enzymatic production of a compound of formula II-C2(i) or formula II-C2(ii), using the methods disclosed herein.

[0324] In some embodiments, the compound of formula III is in the form of formula III-A and the non-enzymatic oxidation produces a compound of formula I-A.

[0325]

[0326] In some embodiments, the compound of formula III is in the form of formula III-D(i) or formula III-D(ii) and the non-enzymatic oxidation produces a compound of formula I-D.

[0327]

[0328] In some embodiments, the compound of formula II is in the form of formula III-E(i) or formula III-E(ii) and the non-enzymatic oxidation produces a compound of formula I-E.

[0329]

[0330] In some embodiments, the method further comprises the enzymatic production of a compound of formula III-A, using the methods disclosed herein.

[0331] In some embodiments, the method further comprises the enzymatic production of a compound of formula III-D(i) or formula III-D(ii), using the methods disclosed herein.

[0332] In some embodiments, the method further comprises the enzymatic production of a compound of formula III-E(i) or formula III-E(ii), using the methods disclosed herein.

[0333] In some embodiments, the compound of formula III is in the form of formula III-Al(i) or formula III-Al(ii) and the non-enzymatic oxidation produces a compound of formula I-Al.

[0334]

[0335] In some embodiments, the compound of formula III is in the form of formula III-A2(i) or formula III-A2(ii) and the non-enzymatic oxidation produces a compound of formula I-A2.

[0336]

[0337] In some embodiments, the method further comprises the enzymatic production of a compound of formula III-Al(i) or formula III-Al(ii), using the methods disclosed herein.

[0338] In some embodiments, the method further comprises the enzymatic production of a compound of formula III-A2(i) or formula III-A2(ii), using the methods disclosed herein.

[0339] In some embodiments, the compound of formula III is in the form of formula III-B3(i) or formula III-B3(ii) and the non-enzymatic oxidation produces a compound of formula I-B3.

[0340]

[0341] In some embodiments, the method further comprises the enzymatic production of a compound of formula III-B3(i) or formula III-B3(ii) using the methods disclosed herein.

[0342] In some embodiments, the compound of formula III is in the form of formula III-Cl(i) or formula Ill-Cl(ii) and the non-enzymatic oxidation produces a compound of formula I-Cl.

[0343]

[0344] In some embodiments, the compound of formula III is in the form of formula III-C2(i) or formula III-C2(ii) and the non-enzymatic oxidation produces a compound of formula I-C2.

[0345]

[0346] In some embodiments, the method further comprises the enzymatic production of a compound of formula III-Cl(i) or formula Ill-Cl(ii), using the methods disclosed herein.

[0347] In some embodiments, the method further comprises the enzymatic production of a compound of formula III-C2(i) or formula III-C2(ii), using the methods disclosed herein.

[0348] In some embodiments, the non-enzymatic oxidation occurs upon exposure to air. In some embodiments, the non-enzymatic oxidation occurs upon exposure to Fenton’s reagent.

[0349] As will be discussed in more detail below, the methods described herein, may occur in recombinant cells. For the non-enzymatic oxidation of the ionone intermediates, the culture medium of the recombinant cells will impact the exposure of the ionone intermediates to air. For example, the use of an organic solvent overlay in the culture is often used to sequester the ionone product in the organic phase to reduce the amount of ionone lost through evaporation. However, the presence of the organic solvent overlay may decrease the exposure of the ionone intermediates to oxygen. The selection of the organic solvent overlay may therefore impact the non-enzymatic oxidation of the ionone intermediates. Accordingly, in some embodiments, when the methods disclosed herein occur in recombinant cells, the organic solvent overlay used in the culture medium may be mineral oil. In some embodiments, when the methods disclosed herein occur in recombinant cells, the organic solvent overlay used in the culture medium may be n-dodecane. In some embodiments, when the methods disclosed herein occur in recombinant cells, the organic solvent overlay used in the culture medium may be n-decane. In some embodiments, when the methods disclosed herein occur in recombinant cells, the organic solvent overlay used in the culture medium may be an adipate ester (for example esterex-A32).

[0350] Use of alcohol dehydrogenases in the method of the invention

[0351] In some embodiments, the method of the invention comprises contacting an ionylideneethanol compound of formula III

[0352]

[0353] (formula III) in the form of any one of its stereoisomers or a mixture thereof, with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce the ionylidene acetaldehyde compound of formula II

[0354]

[0355] (formula II).

[0356] The inventors provide the first demonstration that alcohol dehydrogenases can catalyse this reaction. Accordingly, the invention further provides the use of an alcohol dehydrogenase described herein for converting a compound of formula III to a compound of formula II.

[0357] In some embodiments, the compound of formula III is in the form of formula III-A and the alcohol dehydrogenase produces a compound of formula II-A.

[0358]

[0359] In some embodiments, the compound of formula III is in the form of formula III-D(i) or formula III-D(ii) and the alcohol dehydrogenase produces a compound of formula II-D(i) or formula II-D(ii).

[0360]

[0361] As discussed above, the aldehydes of formula II-D(i) and formula-D(ii) interconvert in solution.

[0362] In some embodiments, the compound of formula III is in the form of formula III-E(i) or formula III-E(ii) and the alcohol dehydrogenase produces a compound of formula II-E(i) or formula II-E(ii).

[0363]

[0364] As discussed above, the aldehydes of formula II-E(i) and formula-E(ii) interconvert in solution.

[0365] In some embodiments, the compound of formula III is in the form of formula III -Al (i) or formula III-Al(ii) and the alcohol dehydrogenase produces a compound of formula II-

[0366]

[0367] As discussed above, the aldehydes of formula II-Al(i) and formula-Al(ii) interconvert in solution.In some embodiments, the compound of formula III is in the form of formula III-A2(i) or formula III-A2(ii) and the alcohol dehydrogenase produces a compound of formula II-

[0368]

[0369] As discussed above, the aldehydes of formula II-A2(i) and formula-A2(ii) interconvert in solution.

[0370] In some embodiments, the compound of formula III is in the form of formula III-B3 (i) or formula III-B3 (ii) and the alcohol dehydrogenase produces a compound of formula II-B3 (i) or formula II-B3 (ii).

[0371]

[0372] As discussed above, the aldehydes of formula II-B3 (i) and formula II-B3 (ii) interconvert in solution.

[0373] In some embodiments, the compound of formula III is in the form of formula III-Cl(i) or formula Ill-Cl(ii) and the alcohol dehydrogenase produces a compound of formula Il-Cl(i) or formula Il-Cl(ii).

[0374]

[0375]

[0376] As discussed above, the aldehydes of formula Il-Cl(i) and formula-II-Cl(ii) interconvert in solution.

[0377] In some embodiments, the compound of formula III is in the form of formula III-C2(i) or formula III-C2(ii) and the alcohol dehydrogenase produces a compound of formula II-C2(i) or formula II-C2(ii).

[0378]

[0379] As discussed above, the aldehydes of formula II-C2(i) and formula-C2(ii) interconvert in solution.

[0380] An “alcohol dehydrogenase” (ADH) in the context of the present invention refers to a polypeptide having the ability to oxidize an alcohol to the corresponding aldehyde in the presence ofNAD+ or NADP+ as cofactor. Such enzymes are members of the E.C. families 1.1.1.1 (NAD+ dependent) or 1.1.1.2 (NADP+ dependent). ADHs, as used herein, may either be endogenously present in the respective biocatalytic process or may be exogenous.

[0381] “Alcohol dehydrogenase enzyme activity” is determined under “standard conditions” as described herein below. “Alcohol dehydrogenase enzyme activity” can be determined using recombinant alcohol dehydrogenase (ADH) polypeptide expressing host cells, disrupted ADH polypeptide expressing cells, fractions of these or enriched or purified ADH polypeptide, in a culture medium or reaction medium, preferably buffered, having a pH in the range of 6 to 11, preferably 7 to 9, at a temperature in the range of about 20 to 45 °C, like about 25 to 40 °C, preferably 25 to 32 °C and in the presence of a reference substrate, either added at an initialconcentration in the range of 1 to 100 pM, preferably 5 to 50 pM, in particular 30 to 40 pM. or endogenously produced by the host cell. For in-vitro assays a cofactor selected from NADH and NADPH has to be added in a suitable easily to be determined concentration. The conversion reaction to form the respective aldehyde compounds of formula II is conducted from 10 min to 5 h, preferably about 1 to 2 h. The oxidation product may then be determined in a conventional manner, for example after extraction with an organic solvent, like ethyl acetate.

[0382] A preferred embodiment of the invention is wherein the polypeptide having said ADH enzyme activity comprises at least one or more sequence motifs selected from:

[0383] . CHTD (SEQ ID NO: 150);

[0384] . GHEGxG (SEQ ID NO: 151);

[0385] . LxCGxxTGxGA (SEQ ID NO: 152);

[0386] . Gx[VI]GL (also expressed as GxxGL as set forth in SEQ ID NO: 153). Here X2 can be any naturally occurring amino acid and X3 can be V or I;

[0387] . LxxxG[LVI] [PAG] (also expressed as LxxxGxx as set forth in SEQ ID NO: 154). Here X6 can be L, V or I and X7 can be P, A or G;

[0388] . GxVxAI (SEQ ID NO: 155); and

[0389] . YxATKxA (SEQ ID NO: 156);

[0390] wherein

[0391] in the above motifs, residues x represent independently of each other any natural amino acid residue in a polypeptide having ADH activity unless otherwise stated. Ambiguities are indicated by listing the acceptable amino acids for a given position between brackets. For example, [VI] stands for V (valine), or I (isoleucine).

[0392] Preferably, the polypeptide having said ADH enzyme activity comprises: CHTD (SEQ ID NO: 150), GHEGxG (SEQ ID NO: 151), LxCGxxTGxGA (SEQ ID NO: 152) and Gx[VI]GL (SEQ ID NO: 153) motifs;

[0393] Preferably, the polypeptide having said ADH activity comprises: CHTD (SEQ ID NO 150), GHEGxG (SEQ ID NO: 151), LxCGxxTGxGA (SEQ ID NO: 152), Gx[VI]GL (SEQ ID NO: 153) and LxxxG[LVI] [PAG] (SEQ ID NO: 154) motifs.

[0394] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity,at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 34, 36, 38, 40, 42 or 44.

[0395] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence of any one of SEQ ID NOs: 34, 36, 38, 40, 42 or 44.

[0396] In some embodiments, the alcohol dehydrogenase has an amino acid sequence of any one of SEQ ID NOs: 34, 36, 38, 40, 42 or 44.

[0397] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 34. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence of any one of SEQ ID NO: 34. In some embodiments, the alcohol dehydrogenase has an amino acid sequence of SEQ ID NO: 34.

[0398] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 36. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence of any one of SEQ ID NO: 36. In some embodiments, the alcohol dehydrogenase has an amino acid sequence of SEQ ID NO: 36.

[0399] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 38. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the alcohol dehydrogenase has an amino acid sequence of SEQ ID NO: 38.

[0400] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 40. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the alcohol dehydrogenase has an amino acid sequence of SEQ ID NO: 40.

[0401] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity,at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 42. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence of any one of SEQ ID NO: 42. In some embodiments, the alcohol dehydrogenase has an amino acid sequence of SEQ ID NO: 42.

[0402] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 44. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the alcohol dehydrogenase has an amino acid sequence of SEQ ID NO: 44.

[0403] Non-limiting examples of alcohol dehydrogenases which can be used in the methods according to the invention are provided in Table 8.

[0404] Table 8:

[0405]

[0406] * unless otherwise indicated the nucleic acid sequence has been codon optimized for expression in E. coli.

[0407] Use of cytochrome P450s and cytochrome P450 reductases in the method of the invention

[0408] In some embodiments, the method of the invention comprises contacting an ionylideneethane compound of formula IV

[0409]

[0410] (formula IV)

[0411] in the form of any one of its stereoisomers or a mixture thereof, with a cytochrome P450 enzyme to produce the ionylideneethanol compound of formula III

[0412]

[0413] (formula III)

[0414] In some embodiments, the compound of formula IV is in the form of formula IV-A and the cytochrome P450 produces a compound of formula III-A

[0415]

[0416] In some embodiments, the compound of formula IV is in the form of formula IV-D(i) or formula IV-D(ii) and the cytochrome P450 produces a compound of formula III-D(i) or formula III-D(ii)

[0417]

[0418]

[0419] In some embodiments, the compound of formula IV is in the form of formula IV-E(i) or formula IV-E(ii) and the cytochrome P450 produces a compound of formula III-E(i) or formula III-E(ii)

[0420]

[0421] In some embodiments, the compound of formula IV is in the form of formula IV-Al(i) or formula IV-Al(ii) and the cytochrome P450 produces a compound of formula III-Al(i) or formula III- Al (i)

[0422]

[0423] In some embodiments, the compound of formula IV is in the form of formula IV-A2(i) or formula IV-A2(ii) and the cytochrome P450 produces a compound of formula III-A2(i) or formula III-A2(i). The inventors provide the first demonstration that the cytochrome P450s, as described herein, can catalyse this reaction. Accordingly, the invention further provides theuse of a cytochrome P450 described herein for converting a compound of formula IV-A2(i) or formula IV-A2(ii) to a compound of formula III-A2(i) or formula III-A2(ii).

[0424]

[0425] In some embodiments, the compound of formula IV is in the form of formula IV-B3(i) or formula IV-B3(ii) and the cytochrome P450 produces a compound of formula III-B3(i) or formula III-B3(ii). The inventors provide the first demonstration that the cytochrome P450s, as described herein, can catalyse this reaction. Accordingly, the invention further provides the use of a cytochrome P450 described herein for converting a compound of formula IV-B3(i) or formula IV-B3(ii) to a compound of formula III-B3 (i) or formula III-B3 (ii).

[0426]

[0427] In some embodiments, the compound of formula IV is in the form of formula IV-Cl(i) or formula IV-Cl(ii) and the cytochrome P450 produces a compound of formula III-Cl(i) or formula Ill-Cl(ii)

[0428]

[0429]

[0430] In some embodiments, the compound of formula IV is in the form of formula IV-C2(i) or formula IV-C2(ii) and the cytochrome P450 produces a compound of formula III-C2(i) or formula III-C2(i).

[0431]

[0432] Cytochrome P450s (CYPs or P450s) are key components of the cellular machinery involved in the biosynthesis and metabolism of a wide variety of endogenous and exogenous compounds. Cytochrome P450 enzymes are a large and diverse family of enzymes and are found in all domains of life including animals, plants, fungi and bacteria. A “cytochrome P450”, “CYP450”, “P450” or “CYP” in the context to the present invention refers to hemecontaining enzymes acting as monooxygenases having the ability to catalyse the insertion of one atom of oxygen into an organic substrate RH with the concomitant reduction of the other oxygen atom into water using the reducing power of NAD(P)H. This reaction typically follows the general form as shown below:

[0433] RH + O2+ H + NAD(P)H RH + H2O + NAD(P)+

[0434] In cytochrome P450 polypeptides, the mechanism involves two successive one-electron transfer steps where electrons originating from NAD(P)H are transferred to the CYP heme center via one or more redox partners. The different systems have been classified and described in literature, for example in Hanneman et al. in Biochimica et Biophysica Acta (2007) 1770: 330-344; Urlacher and Girhard in Trends in Biotechnology (2012) 30(1): 26-36;Sadeghi and Gilardi in Biotechnology and Applied Biochemistry (2013) 60(1): 102-110; Roberts et al. (2002) Journal of Bacteriology 184(14), 3898-3908.

[0435] Based on the number and organization of the redox partners, the cytochrome P450 systems can be organized in three types of topologies:

[0436] 3-component systems, i.e. systems wherein electrons originating fromNAD(P)H are transferred to the P450 protein via 2 separate redox partners, such as e.g. a FAD or FMN-containing flavodoxin reductase and a Ferredoxin protein bound to a Sulphur-iron cluster;

[0437] 2-component systems such as that wherein electrons originating from NAD(P)H are transferred to the P450 protein via a separate redox partner, i.e. a FMN / FAD-containing diflavin reductase, wherein the FMN / FAD-containing di-flavin reductase is also indicated as cytochrome P450 reductase (abbreviated CPR);

[0438] 1 -component systems wherein electrons originating fromNAD(P)H are transferred to the P450 domain via reductase domains which are fused to the P450 domain, such as e.g. FMN-containing flavin reductase domain and a Ferredoxin domain (abbreviated Fdx) bound to a Sulphur-iron cluster or, in alternative to the FMN-containing reductase and Fdx e.g., a reductase domain containing an FMN / FAD-containing di-flavin reductase, (such as in Figure 1 C of Sadeghi and Gilardi in Biotechnology and Applied Biochemistry (2013) 60(1): 102-110).

[0439] CYP polypeptides according to the disclosure may belong to 2-component cytochrome P450 systems (often indicated as Class II P450 systems e.g. in Hanneman et al. in Biochimica et Biophysica Acta (2007) 1770: 330-344 and Sadeghi and Gilardi in Biotechnology and Applied Biochemistry (2013) 60(1): 102-110), which require the cytochrome P450 protein on the one hand and may require a FAD / FMN- containing di-flavin P450 reductase (also indicated as cytochrome P450 reductase or CPR) to support the monooxygenase activity of the cytochrome P450 protein on the other hand.

[0440] Alternatively, CYP polypeptides according to the disclosure may belong to cytochrome P450 systems which may require electron transfer proteins flavodoxin reductase (FPR) and / or ferredoxin reductase (FDXR).

[0441] The substrates of CYPs include a wide range of molecules including terpene compounds. Generally, the oxidation of a particular substrate by a P450 enzyme occurs in a regioselective and stereoselective manner.Cytochrome P450s may contain a membrane anchor. The P450 membrane anchor is a hydrophobic region that embeds cytochrome P450 enzymes into the phospholipid bilayer of membranes. This anchoring participates in the positioning and function of P450 enzymes, facilitating their interaction with a membrane-bound CPR. The membrane anchor ensures the correct spatial orientation and stability of P450 enzymes, enabling efficient electron transfer and substrate access. The membrane anchor is a peptide located at the N-terminal end of the protein and is generally composed of 20-40 amino acids. The amino acid sequence of the P450 membrane anchor can be modified or replaced by a membrane anchor sequence from another P450 or by an artificial membrane anchor without affecting the substrate and enzyme selectivity. Such modifications can improve the expression of the protein in a heterologous host (Gillam et al (1997) Arch Biochem Biophys Vol. 346 Issue 1 Pages 81-90).

[0442] Alternatively, this membrane anchor can be deleted while retaining the enzymatic activity of the P450.

[0443] “Cytochrome P450 enzyme activity” may be determined under “standard conditions” as described herein below. “Cytochrome P450 enzyme activity” can be determined using recombinant cytochrome P450 polypeptide expressing host cells, disrupted cytochrome 450 polypeptide expressing cells, fractions of these or enriched or purified cytochrome P450 polypeptide, in a culture medium or reaction medium, preferably buffered, having a pH in the range of 6 to 11, preferably 7 to 9, at a temperature in the range of about 20 to 45 °C, like about 25 to 40 °C, preferably 25 to 32 °C and in the presence of a reference substrate, either added at an initial concentration in the range of 1 to 100 pM, preferably 5 to 50 pM. in particular 30 to 40 pM, or endogenously produced by the host cell. The conversion reaction to form the respective alcohol compounds of formula III is conducted from 10 min to 5 h, preferably about 1 to 2 h. The oxidation product may then be determined in a conventional manner, for example after extraction with an organic solvent, like ethyl acetate.

[0444] A preferred embodiment of the invention is wherein the polypeptide having said cytochrome P450 activity comprises at least one or more sequence motifs selected from:

[0445] F-G-x-G-x-x-x-C-[P or V]-G (also expressed as FGxGxxxCxG) set forth in SEQ ID NO: 157, or any partial motif thereof comprising up to 10 or up to 5 consecutive amino acid residues, as for example corresponding to residues in positions 1-5, 2-6, 3-7, 4-8, 5-9, or 6-10of SEQ ID NO: 157. Here X3, X5, X6, X7 can be any naturally occurring amino acid; X9 can be P or V;

[0446] P-E-R-[F or Y] (also expressed as PERx) set forth in SEQ ID NO: 158. Here X4 can be F orY;

[0447] L-x-x-L-x-x-V-[L, I or V]-x-E-x-x-R-[M or L]-[R or H]-x-[P or V] (also expressed as LxxLxxVxxExxRxxxx) set for in SEQ ID NO: 159, or any partial motif thereof comprising up to 10 or up to 5 consecutive amino acid residues, as for example corresponding to residues in positions 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, 9-13, 10-14, 11-15, 12-16 or 13-17. Here, X2, X3, X5, X6, X9, XI 1, X12 or X16 can be any naturally occurring amino acid; X8 can be L, I or V; X14 can be M or L; X15 can be R or H; X17 can be P or V.

[0448] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 49, 53, 57, 61, 65, 69, 74, or 79.

[0449] In some embodiments, the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 49, 53, 57, 61, 65, 69, 74, or 79.

[0450] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 174 or 178.

[0451] In some embodiments, the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 174 or 178.

[0452] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 49. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 49.

[0453] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ IDNO: 53. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 53.

[0454] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 57. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 57.

[0455] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 61. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 61.

[0456] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 65. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 65.

[0457] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 69. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 69.

[0458] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 74. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 74.

[0459] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 79. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 79.In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 174. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ IDNOs: 174.

[0460] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 178. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ IDNOs: 178.

[0461] In some embodiments, the cytochrome P450 further comprises a membrane anchor. In some embodiments, the membrane anchor is at the N-terminus of the cytochrome P450. In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82. In some embodiments, the membrane anchor comprises an amino acid sequence of any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82. In some embodiments, the membrane anchor has an amino acid sequence of any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82.

[0462] In some embodiments, the cytochrome P450 further comprises a membrane anchor. In some embodiments, the membrane anchor is at the N-terminus of the cytochrome P450. In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 175 or 179. In some embodiments, the membrane anchor comprises an amino acid sequence of any one of SEQ ID NOs: 175 or 179. In some embodiments, the membrane anchor has an amino acid sequence of any one of SEQ ID NOs: 175 or 179.

[0463] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 50. In some embodiments, the membrane anchor comprises an amino acid sequence ofSEQ ID NO: 50. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 50.

[0464] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 54. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 54. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 54.

[0465] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 58. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 58. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 58.

[0466] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 62. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 62. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 62.

[0467] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 66. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 66. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 66.

[0468] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 70. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 70. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 70.In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 75. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 75. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 75.

[0469] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 76. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 76. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 76.

[0470] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 81. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 81. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 81.

[0471] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 82. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 82. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 82.

[0472] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 175. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 175. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 175.

[0473] In some embodiments, the membrane anchor comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 179. In some embodiments, the membrane anchor comprises an amino acid sequence of SEQ ID NO: 179. In some embodiments, the membrane anchor has an amino acid sequence of SEQ ID NO: 179.

[0474] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80.

[0475] In some embodiments, the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80.

[0476] In some embodiments, the cytochrome P450 has an amino acid sequence of any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80.

[0477] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 172 or 176.

[0478] In some embodiments, the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 172 or 176.

[0479] In some embodiments, the cytochrome P450 has an amino acid sequence of any one of SEQ ID NOs: 172 or 176.

[0480] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 46. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 46.

[0481] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 51. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 51. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 51.In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 55. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 55.

[0482] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 59. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 59. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 59.

[0483] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 63. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 63. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 63.

[0484] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 67. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 67. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 67.

[0485] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 71. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 71.

[0486] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 73. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 73. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 73.

[0487] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 77. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 77. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 77.

[0488] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 80. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 80.

[0489] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 172. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 172. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 172.

[0490] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 176. In some embodiments, the cytochrome P450 comprises an amino acid sequence of SEQ ID NO: 176. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 176.

[0491] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 180. In some embodiments, the cytochrome P450 comprises an amino acid sequence ofSEQ ID NO: 180. In some embodiments, the cytochrome P450 has an amino acid sequence of SEQ ID NO: 180.

[0492] Non-limiting examples of CYP polypeptides which can be used in the methods according to the invention are provided in Table 9.

[0493] Table 9

[0494]

[0495]

[0496]

[0497] * unless otherwise indicated the nucleic acid sequence has been codon optimized for expression in E. coli.

[0498] Cytochrome P450s belonging to 2-component cytochrome P450 systems may require a FAD / FMN- containing di-flavin P450 reductase (also indicated as cytochrome P450 reductase or CPR) to support the monooxygenase activity of the cytochrome P450 protein.

[0499] Cytochrome P450 reductases are flavoproteins that serve as the primary electron donors to CYPs and aid in supporting the catalytic activity of CYP enzymes. The primary role of CPRs is to transfer electrons from NADPH (nicotinamide adenine dinucleotide phosphate) to CYPs. The electron transfer process involves the sequential reduction of FAD and FMN within the CPR, followed by the transfer of electrons from FMN to the heme iron of the CYP enzyme. This electron transfer activates the molecular oxygen for the subsequent oxidation of the substrate by the CYP. A CPR (cytochrome P450 reductase) is not selective for a particular P450 and a CPR can be combined with a P450 from another organism to form a functionally active P450 / CPR complex.In the methods according to the invention, the cytochrome P450 enzymes may therefore be used in conjunction with a cytochrome P450 reductase (CPR) to support monooxygenase activity.

[0500] Accordingly, in one embodiment, the method further comprises the use of a polypeptide capable of reducing a cytochrome P450. In one embodiment, the polypeptide capable of reducing a cytochrome P450 is a cytochrome P450 reductase.

[0501] In addition, as used herein a “cytochrome bs" or “CB5”, refers to a protein that comprises a lipid binding domain or cytochrome b5-like heme binding domain. In some embodiments, a lipid binding domain is a steroid binding domain. CB5 proteins are heme- or lipid- binding proteins. For example, a CB5 may be a steroid binding protein. CB5 proteins may serve as an electron transfer component of a redox reaction. For example, a CB5 may function as an obligate electron donor in an oxidative reaction. In some embodiments, a CB5 may serve as an electron-delivery partner for a cytochrome P450. A cytochrome P450 may receive electrons from cytochrome b and in turn, the cytochrome bs may receive electrons from either cytochrome bs reductase or cytochrome P450 reductase. In some embodiments, a CB5 may catalyze or promote electron transfer from NADPH to a cytochrome P450 enzyme. In some other embodiments, a CB5 may sterically interact with a cytochrome P450 enzyme to support an enzyme conformation that promotes higher activity, without a direct enzymatic role of the CB5 itself.

[0502] In one embodiment, the polypeptide capable of reducing a cytochrome P450 is a cytochrome bs.

[0503] In some embodiments, the polypeptide capable of reducing a cytochrome P450 comprises an amino acid having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 165, 168 or 170.

[0504] In some embodiments, the polypeptide capable of reducing a cytochrome P450 comprises an amino acid of any one of SEQ ID NOs: 165, 168 or 170.

[0505] In some embodiments, the polypeptide capable of reducing a cytochrome P450 cytochrome P450 has an amino acid sequence of any one of SEQ ID NOs: 165, 168 or 170.

[0506] In some embodiments, the polypeptide capable of reducing a cytochrome P450 comprises an amino acid having at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 165.

[0507] In some embodiments, the polypeptide capable of reducing a cytochrome P450 comprises an amino acid of SEQ ID NOs: 165.

[0508] In some embodiments, the polypeptide capable of reducing a cytochrome P450 cytochrome P450 has an amino acid sequence of SEQ ID NOs: 165.

[0509] In some embodiments, the polypeptide capable of reducing a cytochrome P450 comprises an amino acid having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 168.

[0510] In some embodiments, the polypeptide capable of reducing a cytochrome P450 comprises an amino acid of SEQ ID NOs: 168.

[0511] In some embodiments, the polypeptide capable of reducing a cytochrome P450 cytochrome P450 has an amino acid sequence of SEQ ID NOs: 168.

[0512] In some embodiments, the polypeptide capable of reducing a cytochrome P450 comprises an amino acid having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 170.

[0513] In some embodiments, the polypeptide capable of reducing a cytochrome P450 comprises an amino acid of SEQ ID NOs: 170.

[0514] In some embodiments, the polypeptide capable of reducing a cytochrome P450 cytochrome P450 has an amino acid sequence of SEQ ID NOs: 170.

[0515] Non-limiting example of polypeptides capable of reducing a cytochrome P450 are provided in Table 10.

[0516] Table 10

[0517]

[0518]

[0519] Use of ionylideneethane synthases in the method of the invention

[0520] In some embodiments, the method of the invention comprises contacting a famesyl pyrophosphate compound of formula V

[0521]

[0522] (formula V)

[0523] in the form of any one of its stereoisomers or a mixture thereof, with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce the ionylideneethane compound of formula IV

[0524]

[0525] (formula IV)

[0526] In some embodiments, the compound of formula V is in the form of formula V-l, or formula V -2 and the ionylideneethane synthase produces a compound of formula IV-A. In a preferred embodiment, the compound of formula V is in the form of formula V-l

[0527]

[0528] In some embodiments, the compound of formula V is in the form of formula V-l, or formula V -2 and the ionylideneethane synthase produces a compound of formula IV-D(i) or formula IV-D(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l. As highlighted in Example 22, the inventors have identified a wide variety of enzymes that are able to catalyse this reaction. The ionylideneethane synthases comprising anamino acid sequence of any one of SEQ IDNOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 222, 224, 226, 228, 230, 232, 234, 236, or 238 are able to produce a compound of formula IV-D(i) and the ionylideneethane synthase comprising an amino acid sequence of SEQ ID NO: 220 is able to produce a compound of formula IV-D(ii). Accordingly, the invention further provides the use of the ionylideneethane synthases described herein for converting a compound of formula V-l or formula V -2 to the compound of formula IV-D(i) or formula IV-D(ii).

[0529]

[0530] In some embodiments, the compound of formula V is in the form of formula V-l, or formula V-2 and the ionylideneethane synthase produces a compound of formula IV-Al(i) or formula IV-Al(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l.

[0531]

[0532] In some embodiments, the compound of formula V is in the form of formula V-l, or formula V-2 and the ionylideneethane synthase produces a compound of formula IV-E(i) or formula IV-E(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l. As highlighted in Example 2 and Example 15, the ionylideneethane synthases comprising an amino acid sequence of SEQ IDNOs: 101, 103, 105, 107, 109, 111, and 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272 are able to catalyse this reaction. Accordingly, the invention further provides the use of the ionylideneethane synthases described herein for converting a compound of formula V-l or formula V-2 to the compound of formula IV-E(i) or formula IV-E(ii).

[0533]

[0534] In some embodiments, the compound of formula V is in the form of formula V-l, or formula V-2 and the ionylideneethane synthase produces a compound of formula IV-A2(i) or formula IV-A2(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l.

[0535]

[0536] The inventors provide the first demonstration that an ionylideneethane synthase can catalyse this reaction. Accordingly, the invention further provides the use of the ionylideneethane synthase described herein for converting a compound of formula V-l or formula V-2 to the compound of formula IV-A2(i) or formula IV-A2(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l.

[0537] In some embodiments, the compound of formula V is in the form of formula V-l, or formula V-2 and the ionylideneethane synthase produces a compound of formula IV-B3(i) orformula IV-B3(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l.

[0538]

[0539] The inventors provide the first demonstration that an ionylideneethane synthase can catalyse this reaction. Accordingly, the invention further provides the use of the ionylideneethane synthase described herein for converting a compound of formula V-l or formula V-2 to the compound of formula IV-B3(i) or formula IV-B3(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l. As highlighted in Example 18, the ionylideneethane synthases comprising an amino acid sequence of SEQ ID NOs: 274, 276, and 278 are able to produce a compound of formula IV-B3(ii), and the ionylideneethane synthase comprising an amino acid sequence of SEQ ID NO: 280 is able to produce a compound of formula IV-B3(i).

[0540] In some embodiments, the compound of formula V is in the form of formula V-l, or formula V -2 and the ionylideneethane synthase produces a compound of formula IV-Cl(i) or formula IV-Cl(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l.

[0541]

[0542] In some embodiments, the compound of formula V is in the form of formula V-l, or formula V -2 and the ionylideneethane synthase produces a compound of formula IV-A2(i) or formula IV-A2(ii). In a preferred embodiment, the compound of formula V is in the form of formula V-l.

[0543]

[0544] As discussed above, isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are the universal precursors in the biosynthesis of terpenes. IPP and DMAPP are derived from the primary metabolism of the cells through for example the mevalonate (MV A) or the methylerythritol phosphate (MEP) pathways. These two building blocks are condensed by prenyltransferases to form the linear prenyl-diphosphates, such as for example geranyldiphosphate and famesyl-diphosphate composed of 10 and 15 carbons and precursors of the monoterpenes and sesquiterpenes, respectively. For the biosynthesis of famesyl-diphosphates, 3 isoprenes units from IPP and DMAPP are condensed by famesyl-diphosphate synthases.

[0545] The linear prenyl-diphosphates precursors are converted to linear or mostly cyclic terpene compounds by the action of a large group of enzymes known as terpene synthases or terpene cyclases. This enzymatic conversion involves as a first step the formation of a carbocation leading to a cyclization cascade controlled by the enzyme active site and leading to the formation of complex cyclic structures. According to the initiation mechanism of this cyclization, terpene synthases can be classified into Class I or class II terpene synthases (alternatively type I or type II terpene synthases). In Class I terpene synthase an allylic carbocation is generated by the ionization of the isoprenoid pyrophosphate of the substrate while in class II terpene synthases the terminal double bond of the substrate is protonated.

[0546] A “terpene synthase”, designates a polypeptide which converts a terpene precursor molecule to the respective terpene target molecule. Sesquiterpene synthases are terpene synthases converting famesyl-diphosphate to sesquiterpene compounds. “lonylideneethanesynthases” constitute a particular class of sesquiterpene synthases catalyzing a three-step reaction starting with the cleavage of the diphosphate moiety (type I). The resulting allylic cation undergoes isomerization and cyclization to yield an ionylideneethane structure. In particular some ionylideneethane synthases are known to catalyze the cyclization of famesyl-di phosphate to (2Z,4E)-(R)-alpha-ionylideneethane (formula IV-Al(i) herein) and (2E,4E)-(R)-alpha-ionylideneethane (formula IV-Al(ii) herein) (Takino et al (2018) Journal of the American Chemical Society 2018 Vol. 140 Issue 39 Pages 12392-12395 and

[0547] WO 2023 / 012111).

[0548] In particular, an “ionylideneethane synthase” in the context of the present invention refers to a terpene synthase that is able to catalyse the conversion of a compound of formula V to a compound of formula IV

[0549]

[0550] “Ionylideneethane enzymatic activity” may be determined under “standard conditions” as described herein below. “Ionylideneethane enzyme activity” can be determined using recombinant ionylideneethane synthase expressing host cells, disrupted ionylideneethane synthase polypeptide expressing cells, fractions of these or enriched or purified ionylideneethane synthase polypeptide, in a culture medium or reaction medium, having a pH in the range of 6 to 11, preferably 7 to 9, at a temperature in the range of about 20 to 45 °C, like about 25 to 40 °C, preferably 25 to 32 °C and in the presence of a reference substrate, either added at an initial concentration in the range of 1 to 100 pM, preferably 5 to 50 pM. in particular 30 to 40 pM, or endogenously produced by the host cell. The conversion reaction to form the respective terpene product of formula IV is conducted from 10 min to 5 h, preferably about 1 to 2 h. The product may then be determined in a conventional manner, for example after extraction with an organic solvent, like ethyl acetate.

[0551] A preferred embodiment of the invention is when the polypeptide having said ionylideneethane synthase activity comprises one or more sequence motifs selected from:A-x-A-[W or F]-E-Y-x-R (also expressed as AxAxEYxR) set forth in SEQ ID NO: 160, or any partial motif thereof comprising up to 4 consecutive amino acid residues, as for example corresponding to residues in positions 1-4, 2-5, 3-6, 4-7, or 5-8 of SEQ ID NO: 160. Here X2 or X7 can be any naturally occurring amino acid; X4 can be W or F;

[0552] W-x-R-Y-x-[A or G]-[F or W]-x-R (also expressed as WxRYxxxxR) as set forth in SEQ ID NO: 161, or any partial motif thereof comprising up to 4 consecutive amino acid residues, as for example corresponding to residues in positions 1-4, 2-5, 3-6, 4-7, 5-8 or 6-9 of SEQ ID NO: 161. Here X2, X5, X8 can be any naturally occurring amino acid; X6 can be A or G; X7 can be F or W;

[0553] WFRxRDxDxLxRF as set forth in SEQ ID NO: 162, or any partial motif thereof comprising up to 10 or up to 5 consecutive amino acid residues, as for example corresponding to residues in positions 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, or 9-13 of SEQ ID NO: 162. Here X4, X7, X9, or XI 1 can be any naturally occurring amino acid;

[0554] K-H-R-[S or A]-E-G-E-[T or I]-x-x-[T or S]-F-x-Y (also expressed as KHRxEGExxxxFxY) as set forth in SEQ ID NO: 163, or any partial motif thereof comprising up to 10 or up to 5 consecutive amino acid residues, as for example corresponding to residues in positions 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, 9-13 or 10-14 of SEQ ID NO: 163. Here X9, XI 0, XI 3 can be any naturally occurring amino acid; X4 can be S or A; X8 can be T or I; XI 1 can be T or S;

[0555] G-G-x-[I, L or V]-x-x-x-[M or T]-R-R-Y-R-[F or Y]-x-[E or D] (also expressed as GGxxxxxxRRYRxxx) as set forth in SEQ ID NO: 164, or any partial motif thereof comprising up to 10 or up to 5 consecutive amino acid residues, as for example corresponding to residues in positions 1-5, 2-6, 3-7, 4-8, 5-9, 6-10, 7-11, 8-12, 9-13, 10-14 or 11-15 of SEQ ID NO: 164. Here X3, X5, X6, X7, X14 can be any naturally occurring amino acid; X4 can be I, L or V; X8 can be M or T; XI 3 can be F or Y; X15 can be E or D.

[0556] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ IDNOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144.In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144.

[0557] In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of any one of SEQ ID NOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144.

[0558] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 83, 86, 89, 92, 95, or 97.

[0559] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 83, 86, 89, 92, 95, or 97.

[0560] In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of any one of SEQ ID NOs: 83, 86, 89, 92, 95, or 97.

[0561] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238.

[0562] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238.

[0563] In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238.

[0564] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 83. In some embodiments, the ionylideneethane synthase enzymecomprises an amino acid sequence of SEQ ID NO: 83. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 83.

[0565] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 86. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 86. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 86.

[0566] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 89. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 89. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 89.

[0567] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 92. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 92. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 92.

[0568] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 95. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 95. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 95.

[0569] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 97. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 97. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 97.In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 99. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 99. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 99.

[0570] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 83. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 83. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 83.

[0571] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 113. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 113. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 113.

[0572] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 114. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 114. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 114.

[0573] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 115. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 115. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 115.

[0574] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 116. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 116. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 116.

[0575] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 117. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 117. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 117.

[0576] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 118. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 118. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 118.

[0577] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 119. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 119. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 119.

[0578] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 120. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 120. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 120.

[0579] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 121. In some embodiments, the ionylideneethane synthase enzymecomprises an amino acid sequence of SEQ ID NO: 121. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 121.

[0580] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 122. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 122. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 122.

[0581] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 123. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 123. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 123.

[0582] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 124. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 124. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 124.

[0583] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 125. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 125. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 125.

[0584] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 126. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 126. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 126.In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 127. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 127. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 127.

[0585] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 128. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 128. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 128.

[0586] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 129. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 129. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 129.

[0587] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 130. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 130. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 130.

[0588] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 131. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 131. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 131.

[0589] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 132. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 132. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 132.

[0590] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 133. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 133. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 133.

[0591] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 134. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 134. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 134.

[0592] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 135. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 135. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 135.

[0593] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 136. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 136. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 136.

[0594] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 137. In some embodiments, the ionylideneethane synthase enzymecomprises an amino acid sequence of SEQ ID NO: 137. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 137.

[0595] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 138. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 138. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 138.

[0596] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 139. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 139. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 139.

[0597] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 140. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 140. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 140.

[0598] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 141. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 141. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 141.

[0599] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 142. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 142. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 142.In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 143. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 143. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 143.

[0600] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 144. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 144. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 144.

[0601] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 182. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 182. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 182.

[0602] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 184. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 184. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 184.

[0603] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 186. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 186. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 186

[0604] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 188. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 188. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 188.

[0605] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 190. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 190. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 190.

[0606] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 192. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 192. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 192.

[0607] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 194. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 194. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 194.

[0608] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 196. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 196. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 196.

[0609] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 198. In some embodiments, the ionylideneethane synthase enzymecomprises an amino acid sequence of SEQ ID NO: 198. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 198.

[0610] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 200. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 200. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 200.

[0611] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 202. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 202. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 202.

[0612] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 204. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 204. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 204.

[0613] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 206. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 206. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 206.

[0614] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 208. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 208. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 208.In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 210. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 210. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 210.

[0615] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 212. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 212. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 212.

[0616] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 214. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 214. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 214.

[0617] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 216. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 216. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 216.

[0618] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 218. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 218. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 218.

[0619] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 220. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 220. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 220.

[0620] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 222. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 222. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 222.

[0621] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 224. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 224. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 224.

[0622] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 226. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 226. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 226.

[0623] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 228. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 228. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 228.

[0624] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 230. In some embodiments, the ionylideneethane synthase enzymecomprises an amino acid sequence of SEQ ID NO: 230. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 230.

[0625] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 232. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 232. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 232.

[0626] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 234. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 234. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 234.

[0627] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 236. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 236. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 236.

[0628] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 238. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 238. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 238.

[0629] The inventors have demonstrated that ionylideneethane synthases comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111, can convert a compound of formula V-l to a compound of formula IV-A2(i) or formula IV-A2(ii). Accordingly, insome embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111. Furthermore, the inventors have demonstrated that ionylideneethane synthases comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272, can convert a compound of formula V-l to a compound of formula IV-E(i) or formula IV-E(ii). Accordingly, in some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272.

[0630] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 101, 103, 105, 107, 109 or 111. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272.

[0631] In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272.

[0632] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 101. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 101. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 101.

[0633] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 103. In some embodiments, the ionylideneethane synthase enzymecomprises an amino acid sequence of SEQ ID NO: 103. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 103.

[0634] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 105. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 105. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 105.

[0635] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 107. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 107. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 107.

[0636] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 109. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 109. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 109.

[0637] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 111. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 111. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 111.

[0638] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 240. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 240. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 240.In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 242. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 242. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 242.

[0639] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 244. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 244. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 244.

[0640] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 246. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 246. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 246.

[0641] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 248. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 248. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 248.

[0642] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 250. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 250. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 250.

[0643] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 252. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 252. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 252.

[0644] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 254. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 254. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 254.

[0645] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 256. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 256. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 256.

[0646] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 258. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 258. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 258.

[0647] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 260. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 260. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 260.

[0648] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 262. In some embodiments, the ionylideneethane synthase enzymecomprises an amino acid sequence of SEQ ID NO: 262. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 262.

[0649] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 264. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 264. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 264.

[0650] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 266. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 266. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 266.

[0651] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 268. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 268. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 268.

[0652] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 270. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 270. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 270.

[0653] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 272. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 272. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 272.The inventors have demonstrated that ionylideneethane synthases comprising an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 274, 276, 278 or 280, can convert a compound of formula V to a compound of formula IV-B3(i) or formula IV-B3(ii). Accordingly, in some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 274, 276, 278 or 280.

[0654] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of any one of SEQ ID NOs: 274, 276, 278 or 280.

[0655] In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of any one of SEQ ID NOs: 274, 276, 278 or 280.

[0656] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 274. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 274. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 274.

[0657] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 276. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 276. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 276.

[0658] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 278. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 278. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 278.2025P00208WG

[0659] In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 280. In some embodiments, the ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 280. In some embodiments, the ionylideneethane synthase enzyme has an amino acid sequence of SEQ ID NO: 280.

[0660] Non-limiting examples of ionylideneethane synthases which can be used in the methods according to the invention are provided in Table 11A, Table 1 IB, Table 11C and Table 1 ID.

[0661] Table 11A

[0662]

[0663]

[0664] Table 11B

[0665]

[0666]

[0667]

[0668] * unless otherwise indicated the nucleic acid sequence has been codon optimized for expression in E. coli.

[0669] Table 11C

[0670]

[0671]

[0672]

[0673] * unless otherwise indicated the nucleic acid sequence has been codon optimized for expression in E. coli.

[0674] Table 11D

[0675]

[0676] * unless otherwise indicated the nucleic acid sequence has been codon optimized for expression in E. coli.

[0677] In addition, non-limiting examples of ionylideneethane synthases which can be used in the methods according to the invention are provided in Table 12.

[0678] Table 12

[0679]

[0680]

[0681] Engineered ionylideneethane synthases of the invention

[0682] As highlighted in Example 15, the inventors have surprisingly demonstrated that engineering the ionylideneethane synthases can result in variants with improved enzymatic activity. Accordingly, the invention further provides engineered ionylideneethane synthases. An engineered ionylideneethane synthase is an enzyme that has been modified to differ from the wild type form of the enzyme. The inventors have demonstrated that truncating the wild type ionylideneethane synthase enzyme at the N-terminus and / or replacing the N-terminus with a protein sequence of SEQ ID NO: 282, 283 or 284 may improve activity.

[0683] In some embodiments, the engineered ionylideneethane synthase has increased enzymatic activity in comparison to the corresponding wild type enzyme. In some embodiments, the enzymatic activity is at least 1.5X, at least 2X, at least 2.5X, at least 3X, at least 3.5X, at least 4X, at least 4.5X, at least 5X, at least 6X, at least 7X, at least 8X, at least9X, at least 10X, at least 15X, at least 20X, at least 25X, at least 3OX, at least 35X, at least 40X, at least 45X, at least 5 OX, at least 60X, at least 70X, at least 8OX, at least 90X, at least 1OOX or more in comparison to the corresponding wild type enzyme. The enzymatic activity may be measured by any suitable assay in the art. For example, the increase in enzymatic activity may be measured by the relative increase in the yield of the end product.

[0684] In some embodiments, the engineered ionylideneethane synthase enzyme is truncated at the N-terminus by at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 or at least 100 amino acids in comparison to the wild type enzyme.

[0685] In some embodiments, the engineered ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 282.

[0686] In some embodiments, the engineered ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 283.

[0687] In some embodiments, the engineered ionylideneethane synthase enzyme comprises an amino acid sequence of SEQ ID NO: 284.

[0688] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 242. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 242. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 242.

[0689] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 244. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 244. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 244.

[0690] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 246. In some embodiments, the engineered ionylideneethane synthasecomprises an amino acid sequence of SEQ ID NO: 246. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 246.

[0691] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 248. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 248. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 248.

[0692] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 252. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 252. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 252.

[0693] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 254. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 254. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 254.

[0694] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 256. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 256. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 256.

[0695] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 260. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 260. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 260.In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 262. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 262. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 262.

[0696] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 264. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 264. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 264.

[0697] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 268. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 268. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 268.

[0698] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 270. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 270. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 270.

[0699] In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 272. In some embodiments, the engineered ionylideneethane synthase comprises an amino acid sequence of SEQ ID NO: 272. In some embodiments, the engineered ionylideneethane synthase has an amino acid sequence of SEQ ID NO: 272.Further embodiments of the method of the invention

[0700]

[0701] of formula I-A

[0702] The inventors have demonstrated that a compound of formula I, and in particular of formula I-A (i.e. either formula I-Al or formula I-A2), can be prepared by the sequential biocatalytic conversion of a compound of formula V.

[0703] In some embodiments, the method of the invention is for preparing an ionone compound of formula I-A

[0704]

[0705] (formula I-A)

[0706] wherein the method comprises:

[0707] step (a) contacting a famesyl pyrophosphate compound of formula V

[0708]

[0709] in the form of any one of its stereoisomers or a mixture thereof, with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV-A:

[0710]

[0711] (formula IV -A);

[0712] step (b) contacting the ionylideneethane compound of formula IV-A:

[0713]

[0714] (formula IV-A)

[0715] with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-A:

[0716]

[0717] (formula III-A);step (c) contacting the ionylideneethanol compound of formula III-A:

[0718]

[0719] (formula III-A)

[0720] with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-A:

[0721] (formula II-A); and

[0722] e ionylidene acetaldehyde compound of formula II-A:

[0723]

[0724] (formula II-A)

[0725] with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-A:

[0726]

[0727] ,

[0728] (a) the famesyl pyrophosphate compound of formula V is in the form of formula V-l :

[0729]

[0730] (b) the ionylideneethane compound of formula IV is in the form of formula IV-Al(i) or formula IV-Al(ii):

[0731]

[0732] (formula IV-Al(ii);

[0733] (c) the ionylideneethanol compound of formula III is in the form of formula III -Al (i) or formula III- Al (ii):

[0734]

[0735] (formula III-Al(ii); and

[0736] (d) the ionylidene acetaldehyde compound of formula II is in the form of formula II-

[0737]

[0738] (formula II-Al(ii)).

[0739] In some embodiments, the method of the invention is for preparing an ionone compound of formula I-A2

[0740]

[0741] (formula I-A2), wherein:

[0742] (a) the famesyl pyrophosphate compound of formula V is in the form of formula V-l :

[0743]

[0744] (formula V-l);

[0745] (b) the ionylideneethane compound of formula IV is in the form of formula IV-A2(i) or formula IV-A2(ii):

[0746]

[0747] (formula IV -A2(ii);

[0748] (c) the ionylideneethanol compound of formula III is in the form of formula III-A2(i) or formula III- A2(ii):

[0749]

[0750] (formula III-A2(ii); and

[0751] (d) the ionylidene acetaldehyde compound of formula II is in the form of formula II-A2(i) or formula II-A2(ii):

[0752]

[0753] (formula II- A2(ii)).

[0754] The invention also provides a method for producing the compound of formula I-Al, wherein the method comprises step (a), step (b) and step (c), and wherein instead of step (d) the compound of formula II -Al (i) or formula II-Al(ii) is oxidised non-enzymatically to produce the ionone of compound I-Al.

[0755] The invention also provides a method for producing the compound of formula I-A2, wherein the method comprises step (a), step (b) and step (c), and wherein instead of step (d), the compound of formula II-A2(i) or formula II-A2(ii) is oxidised non-enzymatically to produce the ionone of compound I-A2.

[0756] The invention also provides a method for producing the compound of formula I-Al, wherein the method comprises step (a), and step (b) and wherein instead of step (c) and step (d) the compound of formula III-Al(i) or formula III-Al(ii) is oxidised non-enzymatically to produce the ionone of compound I-Al.

[0757] The invention also provides a method for producing the compound of formula I-A2, wherein the method comprises step (a), and step (b) and wherein instead of step (c) and step (d), the compound of formula III-A2(i) or formula III-A2(ii) is oxidised non-enzymatically to produce the ionone of compound I-A2.

[0758] The invention also provides a method for producing the compound of formula IV-A, for example formula IV-Al(i), formula IV-Al(ii), formula IV-A2(i) or formula IV-A2(ii), wherein the method comprises step (a).

[0759] The invention also provides a method for producing the compound of formula III-A, for example formula III-Al(i), formula III-Al(ii), formula III-A2(i) or formula III-A2(ii), wherein the method comprises steps (a) and (b).The invention also provides a method for producing the compound of formula II-A, for example formula II-Al(i), formula II-Al(ii), formula II-A2(i) or formula II-A2(ii), wherein the method comprises steps (a), (b) and (c).

[0760] Preparation of a compound of formula I-D

[0761] The inventors have demonstrated that a compound of formula I, and in particular of formula I-D, can be prepared by the sequential biocatalytic conversion of a compound of formula V.

[0762] In some embodiments, the method of the invention is for preparing an ionone compound of formula I-D

[0763]

[0764] (formula I-D)

[0765] wherein the method comprises:

[0766] step (a) contacting a famesyl pyrophosphate compound of formula V

[0767]

[0768] (formula V)

[0769] in the form of any one of its stereoisomers or a mixture thereof, with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV-D(i) or formula IV-D(ii):

[0770]

[0771] (formula IV -D(ii));

[0772] step (b) contacting the ionylideneethane compound of formula IV-D(i) or formula IV-

[0773]

[0774] (formula IV-D(ii));

[0775] with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-D(i) or formula D(ii):

[0776]

[0777] (formula III-D(ii));

[0778] step (c) contacting the ionylideneethanol compound of formula III-D(i) or formula III- D(n):

[0779]

[0780] (formula III-D(ii)) with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-D(i) or formula II-D(ii):

[0781]

[0782] (formula II-D(ii)); and step (d) contacting the ionylidene acetaldehyde compound of formula II-D(i) or formula

[0783]

[0784] (formula II-D(ii)) with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-D:

[0785]

[0786] (formula I-D).

[0787] Preparation of a compound of formula I-E

[0788] The inventors have demonstrated that a compound of formula I, and in particular of formula I-E, can be prepared by the sequential biocatalytic conversion of a compound of formula V.

[0789] In some embodiments, the method of the invention is for preparing an ionone compound of formula I-E

[0790]

[0791] (formula I-E)

[0792] wherein the method comprises:

[0793] step (a) contacting a famesyl pyrophosphate compound of formula V

[0794]

[0795] in the form of any one of its stereoisomers or a mixture thereof, with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV- E(i) or formula IV-E(ii):

[0796]

[0797] (formula IV -E(ii)); ng the ionylideneethane compound of formula IV-E(i) or formula IV-

[0798]

[0799] (formula I

[0800]

[0801] (formula IV -E(ii));

[0802] with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-E(i) or formula E(ii):

[0803]

[0804] (formula III-E(ii));

[0805] step (c) contacting the ionylideneethanol compound of formula III-E(i) or formula III-

[0806]

[0807] with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-E(i) or formula II-E(ii):

[0808]

[0809] (formula II-E(ii)); and step (d) contacting the ionylidene acetaldehyde compound of formula II-E(i) or formula

[0810]

[0811] (formula II-E(ii)) with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-E:

[0812]

[0813] (formula I-E).

[0814] Preparation of a compound of formula I-B3 from a compound of formula III-B3(i) or formula III-B3(ii),

[0815] A compound of formula I-B3, can be prepared by the sequential biocatalytic conversion of a compound of formula III-B3(i) or formula III-B3(ii).

[0816] In some embodiments, the method of the invention is for preparing an ionone compound of formula I-B3

[0817]

[0818] (formula I-B3)

[0819] wherein the method comprises:

[0820] step (a) contacting the ionylideneethanol compound of formula III-B3(i) or formula III-

[0821]

[0822] (formula III-B3(ii)) with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-B3 (i) or formula II-B3 (ii) :

[0823]

[0824] (formula II-B3(ii)); and step (b) contacting the ionylidene acetaldehyde compound of formula II-B3 (i) or formula II- B3(ii) with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-B3:

[0825]

[0826] (formula I-B3).

[0827] The invention also provides a method for producing the compound of formula II-B3 (i) or formula II-B3 (ii) wherein the method comprises step (a).

[0828] The compound of formula III-B3(i) or formula III-B3(ii) can be used as the starting substrate in the method of the invention, for example in the form of a purified compound preparation which is commercially available.

[0829] Preparation of a compound of formula I-B3 from a compound of formula V

[0830] The inventors have demonstrated that a compound of formula I, and in particular of formula I-B3, can be prepared by the sequential biocatalytic conversion of a compound of formula V.

[0831] In some embodiments, the method of the invention is for preparing an ionone compound of formula I-B3:

[0832]

[0833] (formula I-B3)

[0834] wherein the method comprises:

[0835] step (a) contacting a famesyl pyrophosphate compound of formula V

[0836]

[0837] (formula V)

[0838] in the form of any one of its stereoisomers or a mixture thereof,with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV- B3(i) or formula IV-B3(ii):

[0839]

[0840] (formula IV-B3(ii)) ing the ionylideneethane compound of formula IV-B3(i) or formula IV-

[0841]

[0842] (formula I

[0843]

[0844] (formula IV-B3(ii))

[0845] with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-B3(i) or formula III-B3(ii):

[0846]

[0847] (formula III-B3(ii))

[0848] step (c) contacting the ionylideneethanol compound of formula III-B3(i) or formula III- B3(ii):

[0849]

[0850] (formula III-B3(ii))

[0851] with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-B3(i) or formula II-B3 (ii) :

[0852]

[0853] (formula II-B3 (ii)); and

[0854] step (d) contacting the ionylidene acetaldehyde compound of formula II-B3 (i) or formula II-B3(ii):

[0855]

[0856] (formula II-B3 (ii)); with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-B:

[0857]

[0858] (formula I-B3).

[0859] The invention also provides a method for producing the compound of formula I-B3, wherein the method comprises step (a), step (b) and step (c), and wherein instead of step (d) the compound of formula II-B3 (i) or formula II-B3 (ii) is oxidised non-enzymatically to produce the ionone of compound I-B3.

[0860] The invention also provides a method for producing the compound of formula I-B3, wherein the method comprises step (a), and step (b) and wherein instead of step (c) and step (d) the compound of formula III-B3(i) or formula III-B3(ii) is oxidised non-enzymatically to produce the ionone of compound I-B3.

[0861] The invention also provides a method for producing the compound of formula IV-B3(i) or formula IV-B3(ii) wherein the method comprises step (a).

[0862] The invention also provides a method for producing the compound of formula III-B3(i) or formula III-B3(ii) wherein the method comprises steps (a) and (b).

[0863] The invention also provides a method for producing the compound of formula II-B3(i) or formula II-B3 (ii) wherein the method comprises steps (a), (b) and (c).

[0864] Preparation of a compound of formula III-B3(i) and formula III-B3(ii) from a compound of formula V

[0865] Intermediates in the pathway to ionone biosynthesis also have potential use. The inventors have demonstrated that a compound of formula III-B3 (i) and formula III-B3 (ii), can be prepared by the sequential biocatalytic conversion of a compound of formula V.

[0866] In some embodiments, the method of the invention is for preparing a compound of formula III-B3(i) or formula III-B3(ii):

[0867]

[0868] (formula III-B3(ii)) wherein the method comprises:

[0869] step (a) contacting a famesyl pyrophosphate compound of formula V

[0870]

[0871] (formula V)

[0872] in the form of any one of its stereoisomers or a mixture thereof,

[0873] with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV-B3(i) or formula IV-B3(ii):

[0874]

[0875] (formula IV-B3(ii)); and step (b) contacting the ionylideneethane compound of formula IV-B3(i) or formula IV-B3(ii):

[0876]

[0877] (formula IV -B3 (ii)) with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-B3(i) or formula III-B3(ii):

[0878]

[0879] (formula III-B3(ii)).

[0880]

[0881] of formula II-' i) or formula II-CKii)

[0882] A compound of formula I-Cl can be prepared by contacting a compound of formula II-C 1 (i) or formula Il-Cl(ii) with an enal-cleaving enzyme.

[0883] In some embodiments, the method of the invention is for preparing an ionone of formula I-Cl

[0884]

[0885] (formula I-Cl)

[0886] wherein the method comprises contacting an ionylidene acetaldehyde compound of

[0887]

[0888] yme to produce the ionone compound of formula I-Cl.

[0889] The compound of formula Il-Cl(i) or formula Il-Cl(ii) can be used as the starting substrate in the method of the invention, for example in the form of a purified compound preparation.

[0890]

[0891] A compound of formula I-C2 can be prepared by contacting a compound of formula II- C2(i) or formula II-C2(ii) with an enal-cleaving enzyme.

[0892] In some embodiments, the method of the invention is for preparing an ionone of formula I-C2

[0893]

[0894] (formula I-C2)

[0895] wherein the method comprises contacting an ionylidene acetaldehyde compound of ula II-C2(i

[0896]

[0897] (formula

[0898]

[0899] (formula II-C2(ii)) with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-C2.The compound of formula II-C2(i) or formula II-C2(ii) can be used as the starting substrate in the method of the invention, for example in the form of a purified compound preparation.

[0900]

[0901] of formula V

[0902] A compound of formula I, and in particular of formula I-Cl, can be prepared by the sequential biocatalytic conversion of a compound of formula V.

[0903] In some embodiments, the method of the invention is for preparing an ionone compound of formula I-Cl:

[0904]

[0905] (formula I-Cl)

[0906] wherein the method comprises:

[0907] step (a) contacting a famesyl pyrophosphate compound of formula V

[0908]

[0909] (formula V)

[0910] in the form of any one of its stereoisomers or a mixture thereof,

[0911] with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV-Cl(i) or formula IV-Cl(ii):

[0912]

[0913] (formula IV -C 1 (ii)) step (b) contacting the ionylideneethane compound of formula IV-Cl(i) or formula IV-Cl(n):

[0914]

[0915] (formula IV -C 1 (ii)) with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-Cl(i) or formula Ill-Cl(ii):

[0916]

[0917] (formula Ill-Cl(ii))step (c) contacting the ionylideneethanol compound of formula III-Cl(i) or formula III-

[0918]

[0919] (formula Ill-Cl(ii)) with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula Il-Cl(i) or formula Il-Cl(ii):

[0920]

[0921] (formula Il-Cl(ii)); and

[0922] step (d) contacting the ionylidene acetaldehyde compound of formula Il-Cl(i) or formula Il-Cl(ii):

[0923]

[0924] (formula Il-Cl(ii)); with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-Cl :

[0925]

[0926] (formula I-Cl).

[0927] The invention also provides a method for producing the compound of formula I-Cl, wherein the method comprises step (a), step (b) and step (c), and wherein instead of step (d) the compound of formula Il-Cl(i) or formula Il-Cl(ii) is oxidised non-enzymatically to produce the ionone of compound I-Cl.

[0928] The invention also provides a method for producing the compound of formula I-Cl, wherein the method comprises step (a), and step (b) and wherein instead of step (c) and step (d) the compound of formula III-Cl(i) or formula Ill-Cl(ii) is oxidised non-enzymatically to produce the ionone of compound I-Cl.

[0929] The invention also provides a method for producing the compound of formula IV-Cl(i) or formula IV-Cl(ii) wherein the method comprises step (a).The invention also provides a method for producing the compound of formula III-Cl(i) or formula Ill-Cl(ii) wherein the method comprises steps (a) and (b).

[0930] The invention also provides a method for producing the compound of formula Il-Cl(i) or formula Il-Cl(ii) wherein the method comprises steps (a), (b) and (c).

[0931] Preparation of a compound of formula I-C2 from a compound of formula V

[0932] A compound of formula I, and in particular of formula I-C2, can be prepared by the sequential biocatalytic conversion of a compound of formula V.

[0933] In some embodiments, the method of the invention is for preparing an ionone compound of formula I-C2:

[0934]

[0935] (formula I-C2)

[0936] wherein the method comprises:

[0937] step (a) contacting a famesyl pyrophosphate compound of formula V

[0938]

[0939] (formula V)

[0940] in the form of any one of its stereoisomers or a mixture thereof,

[0941] with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV-C2(i) or formula IV-C2(ii):

[0942]

[0943] (formula IV-C2(ii)) step (b) contacting the ionylideneethane compound of formula IV-C2(i) or formula IV-C2(ii):

[0944]

[0945] (formula IV-C2(ii)) with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-C2(i) or formula III-C2(ii):

[0946]

[0947] (formula III-C2(ii)) step (c) contacting the ionylideneethanol compound of formula III-C2(i) or formula III-

[0948]

[0949] (formula III-C2(ii)) with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-C2(i) or formula II-C2(ii):

[0950]

[0951] (formula II-C2(ii)); and

[0952] step (d) contacting the ionylidene acetaldehyde compound of formula II-C2(i) or formula II-C2(ii):

[0953]

[0954] (formula II-C2(ii)); with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-C2:

[0955]

[0956] (formula I-C2).

[0957] The invention also provides a method for producing the compound of formula I-C2, wherein the method comprises step (a), step (b) and step (c), and wherein instead of step (d) the compound of formula II-C2(i) or formula II-C2(ii) is oxidised non-enzymatically to produce the ionone of compound I-C2.

[0958] The invention also provides a method for producing the compound of formula I-C2, wherein the method comprises step (a), and step (b) and wherein instead of step (c) and step(d) the compound of formula III-C2(i) or formula III-C2(ii) is oxidised non-enzymatically to produce the ionone of compound I-C2.

[0959] The invention also provides a method for producing the compound of formula IV-C2(i) or formula IV-C2(ii) wherein the method comprises step (a).

[0960] The invention also provides a method for producing the compound of formula III-C2(i) or formula III-C2(ii) wherein the method comprises steps (a) and (b).

[0961] The invention also provides a method for producing the compound of formula II-C2(i) or formula II-C2(ii) wherein the method comprises steps (a), (b) and (c).

[0962] Preparation of a compound of formula V

[0963] In some embodiments, the invention provides a method of preparing a compound of formula I from the sequential biocatalytic conversion of a compound of formula V, for example wherein the compound of formula V is in the form of formula V-l, or formula V-2.

[0964] The compound of formula V can be used as the starting substrate in the method of the invention, for example in the form of a purified compound preparation.

[0965] However, a preferred embodiment of the invention is wherein the process of the invention further comprises providing a compound of formula V by the sequential biocatalytic conversion of precursor compounds to the compound of formula V.

[0966] As discussed above, terpenoids are a large family of structurally diverse natural compounds. All terpenoids derive biosynthetically from two five-carbon units, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP can be produced from different biosynthetic pathways such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, the mevalonate (MV A) pathway or alternative MVA pathways (Dellas, N., et al. (2013) eLife 2: e00672). Alternatively, IPP and DMAPP can also be formed by successive enzymatic phosphorylation or by enzymatic pyrophosphorylation of their corresponding alcohols, isoprenol and prenol (Ma, X, et al. (2022). J Agric Food Chem 70(11): 3512-3520).

[0967] These terpene building blocks are condensed successively to form linear terpenoid precursors with various length and multiple of five carbon such as geranyl-diphosphate (GPP), famesyl-diphosphate (FPP) or geranylgeranyl-diphosphate (GGPP) containing 10, 15 and 20 carbons, respectively. The condensation of the IPP and DMAPP is performed by aclass of enzyme named prenyltransferases. Prenyltransferase enzymes catalyze the initial condensation reaction between IPP and DMAPP to give GPP, and the subsequent addition of IPP molecules to give FPP and then GGPP (Ogura, K., and Koyama, T. (1998). Chem. Rev.

[0968] 98, 1263-1276). The successive condensation of DMAPP and IPP to FPP can be performed by:

[0969] i. The successive action of 2 prenyltransferases, a GPP synthase and an FPP synthase catalysing the synthesis of GPP and the addition of one IPP to GPP respectively; and / or

[0970] ii. the action of 1 prenyltransferase, for example a FPP synthase catalysing the condensation of one DMAPP and two IPP.

[0971] The pathways leading to IPP and DMAPP and to geranyl-diphosphate (GPP), famesyl-diphosphate (FPP) or geranylgeranyl-diphosphate (GGPP) is involved in the synthesis of terpenoids, a diverse class of molecules that play essential roles in primary metabolism and various cellular processes. Terpenoids are involved in numerous biological functions, including the synthesis of sterols, such as cholesterol in animals and phytosterols in plants, as well as the production of hormones, vitamins (such as vitamin E and K), and signaling molecules (such as ubiquinone and dolichol). Additionally, terpenoids are crucial for the formation of membrane lipids and post-translational modifications of proteins.

[0972] Therefore, the pathway leading to GPP, FPP and GGPP as described above is an important component of primary metabolism in all organisms as it provides the necessary precursors for the synthesis of essential terpenoid compounds involved in various physiological processes essential for the growth of the cells.

[0973] Hence, in a preferred embodiment of the invention, the method of the invention further comprises preparing the famesyl pyrophosphate compound of formula V from IPP and DMAPP using one or more prenyltransferases, preferably a famesyl pyrophosphate synthase.

[0974] The term ‘prenyltransferases’ represents a group of enzymes having the ability to condense successively five-carbon units such as isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form linear terpenyl-diphosphate compounds such as geranyl-diphosphate (GPP), famesyl-diphosphate (FPP) or geranylgeranyl-diphosphate(GGPP) containing 10, 15 and 20 carbons, respectively. Some prenyl transferases can add 5-carbon units to linear terpenyl-diphosphate compounds thereby extending the carbon chain length. An example of prenyl transferase are famesyl pyrophosphate synthases (FPP synthases) having the ability of producing FPP from IPP and DMAPP or by adding 5 carbons to GPP or condensing IPP and two molecules of DMAPP.

[0975] Examples of prenyltransferase enzymes that can be used in the method of the invention are well known in the art. For example, as discussed in the general methods of the Examples, a famesyl pyrophosphate synthase from Saccharomyces cerevisiae (ERG20) has been used.

[0976] Preferably, the prenyltransferase is an FPP synthase. In some embodiments, the FPP synthase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 166.

[0977] Accordingly, this embodiment of the invention provides a compound of formula V by the sequential biocatalytic conversion of precursor compounds to the compound of formula V.

[0978] In some embodiments, the method further comprises the preparation of IPP and DMAPP.

[0979] One means for the preparation of IPP and DMAPP is via the “mevalonate pathway”. The “mevalonate pathway” also known as the “isoprenoid pathway” or “HMG-CoA reductase pathway” is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five-carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP). Combining the mevalonate pathway with enzyme activity to generate the terpene precursors GPP, FPP or GGPP allows the recombinant cellular production of terpenes. The pathway is well known in the art. The list of enzymes required for the conversion of acetyl- CoA to IPP and DMAPP is provided below:

[0980] . Acetyl-CoA acetyltransferase (ACAT)

[0981] . 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)

[0982] . 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase)

[0983] . Mevalonate kinase

[0984] . Phosphomevalonate kinase. Mevalonate diphosphate decarboxylase

[0985] . Isopentenyl diphosphate isomerase.

[0986] An alternative means for the preparation of IPP, and DMAPP is via the methylerythritol phosphate (MEP). The pathway is well known in the art. The list of enzymes required for the conversion of glyceraldehyde 3-phosphate (GAP) and pyruvate to IPP and DMAPP is provided below:

[0987] . 1 -Deoxy -D-xylulose 5-phosphate synthase (DXS)

[0988] . 1 -Deoxy -D-xylulose 5-phosphate reductoisomerase (DXR)

[0989] . 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT, IspD)

[0990] . 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK, IspE)

[0991] . 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS, IspF)

[0992] . 4-hydroxy-3-methylbut-2-en-l-yl diphosphate synthase (HDS, IDS)

[0993] . 4-hydroxy-3-methylbut-2-en-l-yl diphosphate reductase (HDR)

[0994] Further alternative pathways to the preparation of IPP and DMAPP are known, see for example: Rinaldi, M. A., et al. (2022). Natural Product Reports 39(1): 90-118. https: / / doi.org / 10.1039 / DlNP00025J (see part 3 of this article).

[0995] The inventors have therefore provided a complete biocatalytic route for the preparation of a compound of formula I, and in particular a compound of formula I -Al, a compound of formula I-A2, a compound of formula I-B3, a compound of formula I-D, and a compound of formula I-E from a simple carbon source (for example glucose). This multistep biocatalytic process has for the first time been described herein and constitutes a significant advance in the preparation of such compounds.

[0996] Reaction conditions for the method of the invention

[0997] The method of the present invention may be an in vivo process or a bioconversion process.

[0998] The term in vivo process (or whole-cell production, or in-vivo production, or in-vivo biosynthesis, or fermentation or cultivation) refers to a process of using a metabolically activecell where the primary metabolism is active to produce the precursors for the methods of the invention (preferably a microbial cell) to convert a carbon source to a new compound, such as the conversion of a carbon source to a terpene or terpene-derived compound.

[0999] Preferred sources of carbon are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add mixtures of various sources of carbon. Other possible sources of carbon are oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid or linoleic acid, alcohols such as glycerol, methanol or ethanol and organic acids such as acetic acid or lactic acid.

[1000] The cell capable of producing the desired compound, such as an ionone of formula I, expresses all enzymes of one or more biosynthetic pathways needed for the production of the desired compound. At least some of the enzymes involved in the process are part of the cell’s primary metabolism. For example, the cell may express the enzymes of a pathway to convert a carbon source (e.g., glucose, glycerol, isoprenol, prenol, CO2) to terpenoid precursors (e.g., IPP, DMAPP, FPP, GGPP) and a pathway converting the terpene precursor to a terpene or terpene derived molecule such as a compound of formula I. The enzymes may be present naturally (endogenously) in the cell or the cell can be modified to introduce nucleic acid sequences encoding the enzymes of interest (such enzymes are also termed heterologous enzymes).

[1001] It is important to point out that, until the present invention, the in vivo production of compounds of formula I-Al had been limited to using biosynthetic routes that involved degradation of carotenoids (WO 2019 / 126777 and WO 2017 / 036495), or that used the non-enzymatic oxidation of the compound of formula IV-Al(i) (WO 2023 / 012111). The present invention provides an important advance of these methods by using a different biosynthetic pathway.

[1002] Alternatively, the methods of the present invention may be performed under bioconversion, also known as biotransformation conditions. Bioconversion processes refer to processes of conversion of compounds to different products using a biological process or agent such as enzymes or whole cells (preferably a microbial cell). Bioconversion does notinclude the use of a cell’s primary metabolism (as defined above) to produce the precursors for the processes of the invention. A bioconversion process can comprise multistep reactions each performed by a different enzyme, for example each of the enzymatic reaction steps disclosed in the methods herein. The compounds used in bioconversion process can be extracted from a natural source or produced using a separate chemical or a biochemical process.

[1003] The at least one polypeptide / enzyme which is present during the bioconversion method of the invention or an individual step of the multistep method as defined herein above, can be present in living cells naturally or recombinantly producing the enzyme or enzymes, in harvested cells, dead cells, in permeabilized cells, in crude cell extracts, in purified extracts, or in essentially pure or completely pure form, i.e. under bioconversion conditions. Such extracts may comprise membrane fraction or a liquid fraction prepared from the recombinant host cell that expresses at least one polypeptide / enzyme. The cells may be immobilized on a suitable substrate as is known in the art. At least one polypeptide / enzyme may be present in solution or as an enzyme immobilized on a carrier. One or several enzymes may simultaneously be present in soluble and / or immobilized forms.

[1004] It can be understood by the skilled person that there may be advantages for the use of an in vivo process.

[1005] In particular, a bioconversion process involves multiple steps, typically:

[1006] Preparation or isolation of the starting compound to be transformed. The compound can be prepared using a chemical or biochemical process or by extraction from a natural source.

[1007] Production of the enzymes or (living) cells used for the bioconversion. Biotransformation reaction by contacting the compound with the enzymes or (living) cells.

[1008] Product Recovery and Refinement.

[1009] In comparison, an in vivo process requires a limited number of steps, generally limited to:

[1010] The cultivation of the microorganism under conditions suitable for the production of the desired compound.Harvesting the cells or growing medium and purification of the desired compound.

[1011] In a bioconversion process, the addition of a detergent is often required to facilitate the solubilization of the compound or to maximize the contact with the biocatalyst. In an in vivo process, the reactants and enzymes are produced in the cells and the addition of a detergent is not needed.

[1012] Therefore, the in vivo process is usually more efficient and cost-effective than a bioconversion process.

[1013] Laboratory methods that can be used in in vivo and bioconversion processes of the invention are well known in the art. There follows a discussion on some of the methods that can be used.

[1014] The bioconversion processes according to the invention can be performed in common reactors, which are known to those skilled in the art, and in different ranges of scale, e.g. from a laboratory scale (few milliliters to dozens of liters of reaction volume) to an industrial scale (several liters to thousands of cubic meters of reaction volume). If the polypeptide is used in a form encapsulated by non-living, optionally permeabilized cells, in the form of a more or less purified cell extract or in purified form, a chemical reactor can be used. The chemical reactor usually allows controlling the amount of at least one enzyme, the amount of at least one substrate, the pH, the temperature and the circulation of the reaction medium.

[1015] Where the process of the invention is in vivo then it is preferred that the reaction is performed in a fermenter, where parameters necessary for suitable living conditions for the living cells (e.g. culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, and the like) can be controlled.

[1016] The term "fermentative production" or "fermentation" refers to the ability of a microorganism (assisted by enzyme activity contained in or generated by said microorganism) to produce a chemical compound in cell culture utilizing at least one carbon source added to the incubation.

[1017] The term "fermentation broth" or "fermentation medium" is understood to mean a liquid, particularly aqueous or aqueous / organic solution which is based on a fermentative process and has not been worked up or has been worked up, for example, as described herein.

[1018] Those skilled in the art are familiar with chemical reactors or bioreactors, e.g. with procedures for up-scaling chemical or biotechnological methods from laboratory scale toindustrial scale, or for optimizing process parameters, which are also extensively described in the literature (for biotechnological methods see e.g. Crueger und Crueger, Biotechnologie -Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Munchen, Wien, 1984).

[1019] The culture medium that is to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D. C., USA, 1981).

[1020] These media that can be used according to the invention may comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and / or trace elements.

[1021] Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials containing these compounds. Examples of sources of nitrogen include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex sources of nitrogen, such as com-steep liquor, soybean flour, soy-bean protein, yeast extract, meat extract and others. The sources of nitrogen can be used separately or as a mixture.

[1022] Inorganic salt compounds that may be present in the media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[1023] Inorganic sulfur-containing compounds, for example sulfates, sulfites, di-thionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as sources of sulfur.

[1024] Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used as sources of phosphorus.

[1025] Chelating agents can be added to the medium, in order to keep the metal ions in solution. Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[1026] The fermentation media used according to the invention may also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from complex components of the media, such as yeast extract, molasses, com-steep liquor and the like. In addition, suitable precursors can be added to the culture medium.The precise composition of the compounds in the medium is strongly dependent on the particular experiment and must be decided individually for each specific case. Information on media optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (1997) Growing media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.

[1027] All components of the medium are sterilized, either by heating (20 min at 1.5 bar and 121 °C) or by sterile filtration. The components can be sterilized either together or if necessary, separately. All the components of the medium can be present at the start of growing, or optionally can be added continuously or by batch feed.

[1028] The temperature of the culture is normally between 15 °C and 45 °C, preferably 25 °C to 40 °C and can be kept constant or can be varied during the experiment. The pH value of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH value for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, e.g. fatty acid polyglycol esters, can be used for controlling foaming. To maintain the stability of plasmids, suitable substances with selective action, e.g. antibiotics, can be added to the medium. Oxygen or oxygen-containing gas mixtures, e.g. the ambient air, are fed into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20 °C to 45 °C. Culture is continued until a maximum of the desired product has formed. This is normally achieved within 1 hour to 160 hours.

[1029] Where the method of the invention is an in vivo process or bioconversion method, the culture medium may further contain an organic solvent overlay. The use of an organic solvent overlay in the culture is often used to sequester an ionone product in the organic phase to reduce the amount of ionone lost through evaporation. However, the presence of the organic solvent overlay may decrease the exposure of the ionone intermediates to oxygen. The degree of in situ recovery of metabolic engineering products may depend on the availability of dissolved oxygen in microbial cultures, especially when the conversion to the ionone occurs via a non-enzymatic oxidation. In some embodiments, the organic solvent overlay may be mineral oil. In some embodiments, the organic solvent overlay may be n-dodecane. In some embodiments, the organic solvent overlay may be n-decane. In some embodiments, the organic solvent overlay may be an adipate ester (for example esterex-A32). As disclosedherein, the enzymatic production of the ionones of formula I is not reliant on the exposure of an ionone intermediate with air. Accordingly, an organic solvent overlay that lowers the exposure of an ionone intermediate to oxygen may be used.

[1030] Where the process of the invention is a bioconversion, cells containing the at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or radiofrequency pulses, French presses, or chemical means, such as hypotonic media, lytic enzymes and detergents present in the medium, or combination of such methods. Examples for detergents are SDS, digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween ® 20, deoxycholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-l-propansulfonate), Nonidet ® P40 (Ethylphenolpoly(ethyleneglycolether), and the like. As stated above, where the process of the invention is an in vivo process, then a detergent is not required for the reasons stated herein.

[1031] The conversion reaction can be carried out batch wise, semi-batch wise or continuously. Reactants (and optionally nutrients) can be supplied at the start of reaction or can be supplied subsequently, either semi-continuously or continuously.

[1032] The bioconversion reaction of the invention, depending on the particular reaction type, may be performed in an aqueous, aqueous-organic or non-aqueous reaction medium.

[1033] An aqueous or aqueous-organic medium may contain a suitable buffer in order to adjust the pH to a value in the range of 5 to 11, like 6 to 10.

[1034] In an aqueous-organic medium an organic solvent miscible, partly miscible or immiscible with water may be applied. Non-limiting examples of suitable organic solvents are listed below. Further examples are mono- or polyhydric, aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols like glycerol.

[1035] The non-aqueous medium may contain is substantially free of water, i.e. will contain less than about 1 wt.-% or 0.5 wt.-% of water.

[1036] Bioconversion methods may also be performed in an organic non-aqueous medium. As suitable organic solvents there may be mentioned aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; aromatic carbohydrates, like benzene, toluene, xylenes, chlorobenzene or di chlorobenzene, aliphatic acyclic and ethers, like di ethylether, methyl-tert. -butylether, ethyltert. -butylether, dipropylether, diisopropylether, dibutylether; or mixtures thereof.The concentration of the reactants / substrates may be adapted to the optimum bioconversion reaction conditions, which may depend on the specific enzyme applied. For example, the initial substrate concentration may be in the 0.1 to 0.5 M, as for example 10 to 100 mM.

[1037] The bioconversion reaction temperature may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the reaction may be performed at a temperature in a range of from 0 to 70 °C, as for example 20 to 50 or 25 to 40 °C. Examples for reaction temperatures are about 30 °C, about 35 °C, about 37 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C and about 60 °C.

[1038] The bioconversion may proceed until equilibrium between the substrate and then product(s) is achieved, but may be stopped earlier. Usual process times are in the range from 1 minute to 25 hours, in particular 10 min to 6 hours, as for example in the range from 1 hour to 4 hours, in particular 1.5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.

[1039] Advantageously, microorganisms such as bacteria, fungi or yeasts are used as host organisms. Advantageously, gram-positive or gram-negative bacteria are used, preferably bacteria of the families Enter obacter iaceae. Pseudomonadaceae, Rhizobiaceae.

[1040] Streptomycetaceae, Streptococcaceae or Nocar diaceae. especially preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Lactococcus, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus. The genus and species Escherichia coli is quite especially preferred. Furthermore, other advantageous bacteria are to be found in the group of alpha-Proteobacteria, beta-Proteobacteria or gamma-Proteobacteria.

[1041] Advantageously also yeasts of families like Saccharomyces or Pichia are suitable hosts.

[1042] Preferably, the cell is a bacterium or a fungal cell, in particular a yeast cell. Preferably, the cell is a unicellular organism, a cultured cell derived from a multi-cellular organism, a cell present in a cultured tissue derived from a multicellular organism, or a cell present in a living multicellular organism. Preferably, the cell is a bacterial cell of the genus Escherichia, preferably E. coli, or a yeast cell of the genus Saccharomyces, preferably S. cerevisiae, of the genus Yarrowia, preferably Y. lipolytica, or of the genus Pichia, preferably P. pastoris.

[1043] Alternatively, entire plants or plant cells may serve as natural or recombinant host. As non-limiting examples, the following plants or cells derived therefrom may be mentioned: thegenera Nicotiana. in particular Nicotiana benthamiana and Nicotiana tabacum (tobacco); as well as Arabidopsis , in particular Arabidopsis thaliana.

[1044] Recombinant cells

[1045] The inventors have demonstrated the in vivo production of compounds of formula I using a unique biocatalytic pathway in recombinant cells expressing the enzymes disclosed herein. In addition to cells expressing the entirety of the biocatalytic pathway, it will also be understood that cells comprising part of the biocatalytic pathway will also have utility in the methods of the invention, in particular in the in vivo and / or bioconversion methods disclosed herein.

[1046] Accordingly, the invention provides a cell comprising, capable of producing or producing an ionone compound of formula I, wherein the cell comprises a nucleic acid encoding a heterologous enal-cleaving enzyme, as described herein. In some embodiments, the cell further comprises a nucleic acid encoding a heterologous alcohol dehydrogenase enzyme, as described herein. In some embodiments, the cell further comprises a nucleic acid encoding a heterologous cytochrome P450 enzyme, as described herein. In some embodiments, the cell further comprises a nucleic acid encoding a heterologous ionylideneethane synthase, as described herein. In some embodiments, the cell further comprises famesyl pyrophosphate of formula V.

[1047] The invention further provides a cell comprising, capable of producing or producing an ionone compound of formula I or an ionylidene acetaldehyde compound of formula II, wherein the cell further comprises a nucleic acid encoding a heterologous alcohol dehydrogenase enzyme, as described herein. In some embodiments, the cell further comprises a nucleic acid encoding a heterologous cytochrome P450 enzyme, as described herein. In some embodiments, the cell further comprises a nucleic acid encoding a heterologous ionylideneethane synthase, as described herein. In some embodiments, the cell further comprises famesyl pyrophosphate of formula V.

[1048] The invention further provides a cell comprising, capable of producing or producing an ionone compound of formula I or an ionylideneethanol compound of formula III, wherein the further cell comprises a nucleic acid encoding a heterologous cytochrome P450 enzyme, as described herein. In some embodiments, the cell further comprises a nucleic acid encoding aheterologous ionylideneethane synthase, as described herein. In some embodiments, the cell further comprises famesyl pyrophosphate of formula V.

[1049] The invention further provides a cell comprising, capable of producing or producing an ionone compound of formula I or an ionylideneethane compound of formula IV, wherein the cell further comprises a nucleic acid encoding a heterologous ionylideneethane synthase, as described herein. In some embodiments, the cell further comprises famesyl pyrophosphate of formula V.

[1050] It is understood that when a cell compises a nucleic acid sequence encoding an enzyme the enzyme is expressed under suitable fermentative conditions.

[1051] The cell capable of producing an ionone compound of formula I, preferably expresses all the enzymes indicated in the pathway of Figure 13.

[1052] From the above it is understood that a cell capable of producing a desired compound, such as an ionone compound of formula I, can produce the desired compound when subjected to fermentation or cultivation under conditions suitable for production of the desired compound.

[1053] The invention further provides a cell for producing an ionone of formula I, wherein the cell comprises a famesyl pyrophosphate of formula V, a nucleic acid encoding a heterologous ionylideneethane synthase enzyme as described herein, a nucleic acid encoding a heterologous cytochrome P450 enzyme as described herein, a nucleic acid encoding a heterologous alcohol dehydrogenase enzyme as described herein, and a nucleic acid encoding a heterologous enal-cleaving enzyme as described herein.

[1054] In some embodiments, the cell as described herein produces at least 50 wt% of the compound of formula I relative to the total amount of the compounds of formula I, formula II, formula III, formula IV and V, such as at least 60wt%, such as at least 70wt%, such as at least 80wt% relative to the total amount of the compounds of formula I, formula II, formula III, formula IV and V.

[1055] In some embodiments, the ionone compound of formula I is in the form of:

[1056]

[1057] formula I-A.

[1058] In some embodiments, the ionone compound of formula I is in the form of:

[1059]

[1060] formula I-D.

[1061] In some embodiments, the ionone compound of formula I is in the form of:

[1062]

[1063] In some embodiments, the ionone compound of formula I is in the form of:

[1064]

[1065]

[1066] In some embodiments, the ionylidene acetaldehyde compound of formula II is in the form of:

[1067]

[1068] formula II-D(ii).

[1069] nd of formula II is in the

[1070]

[1071] formula II-E(ii).

[1072] In some embodiments, the ionylidene acetaldehyde compound of formula II is in the form of:

[1073]

[1074] formula II-Al(ii) In some embodiments, the ionylidene acetaldehyde compound of formula II is in the form of:

[1075]

[1076] formula II- A2(ii)

[1077] In some embodiments, the ionylideneethanol compound of formula III is in the form of:

[1078]

[1079] In some embodiments, the ionylideneethanol compound of formula III is in the form of:

[1080]

[1081] formula III-D(ii). In some embodiments, the ionylideneethanol compound of formula III is in the form of:

[1082]

[1083] formula III-E(ii).

[1084] In some embodiments, the ionylideneethanol compound of formula III is in the form of:

[1085]

[1086] formula III-Al(ii) In some embodiments, the ionylideneethanol compound of formula III is in the form of:

[1087]

[1088] formula III-A2(ii).

[1089] In some embodiments, the ionylideneethane compound of formula IV is in the form of:

[1090]

[1091] formula IV-A.In some embodiments, the ionylideneethane compound of formula IV is in the form of:

[1092]

[1093] formula IV -D(ii).

[1094] In some embodiments, the ionylideneethane compound of formula IV is in the form of:

[1095]

[1096] formula IV -E(ii).

[1097] In some embodiments, the ionylideneethane compound of formula IV is in the form of:

[1098]

[1099] formula IV -Al (ii).

[1100] In some embodiments, the ionylideneethane compound of formula IV is in the form of:

[1101]

[1102] formula IV -A2(ii).

[1103] In some embodiments, the ionone compound of formula I is in the form of:

[1104]

[1105] formula I-B3.

[1106] In some embodiments the ionylidene acetaldehyde compound of formula II is in the form of:

[1107]

[1108] formula II-B3(ii).

[1109] In some embodiments the ionylideneethanol compound of formula III is in the form of:

[1110]

[1111] formula III-B3(ii). In some embodiments the ionylideneethane compound of formula IV is in the form of

[1112]

[1113] formula IV-B3(ii).

[1114] In some embodiments, the ionone compound of formula I is in the form of:

[1115]

[1116] formula I-C2.

[1117] In some embodiments the ionylidene acetaldehyde compound of formula II is in the form of:

[1118]

[1119] formula Il-Cl(ii).

[1120] In some embodiments the ionylidene acetaldehyde compound of formula II is in the form of:

[1121]

[1122] formula II-C2(ii).

[1123] In some embodiments the ionylideneethanol compound of formula III is in the form of:

[1124]

[1125] formula Ill-Cl(ii). In some embodiments the ionylideneethanol compound of formula III is in the form of:

[1126]

[1127] formula III-C2(ii). In some embodiments the ionylideneethane compound of formula IV is in the form of

[1128]

[1129] formula IV-Cl(ii).

[1130] In some embodiments the ionylideneethane compound of formula IV is in the form of

[1131]

[1132] formula IV-C2(ii).

[1133] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-Al, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one ofSEQ IDNOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one ofSEQ ID NOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144. In some embodiments, wherein the ionone compound of formula I is in the form of formula I-Al, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 83, 86, 89, 92, 95, or 97, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 83, 86, 89, 92, 95, or 97.

[1134] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-D or formula I-Al, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238.

[1135] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-A2, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity,at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111.

[1136] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-E or formula I-A2, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272.

[1137] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-B3, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 274, 276, 278 or 280, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 274, 276, 278 or 280.

[1138] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-B3, and the ionylideneethanol compound is in the form of formula III-B3(ii), and the ionylidene aldehyde compound is in the form of formula II-B3 (ii), the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 274, 276 or 278, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 274, 276 or 278.

[1139] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-B3, and the ionylideneethanol compound is in the form of formula III-B3(i), and the ionylidene aldehyde compound is in the form of formula II-B3 (i), the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95%identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 280, optionally wherein the ionylideneethane synthase enzyme comprises or consists of SEQ ID NO: 280.

[1140] In some embodiments, the cytochrome P450 enzyme comprises an amino acid sequence having 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 49, 53, 57, 61, 65, 69, 74, or 79, optionally wherein the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 49, 53, 57, 61, 65, 69, 74, or 79.

[1141] In some embodiments, the cytochrome P450 enzyme comprises an amino acid sequence having 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 174 or 178, optionally wherein the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 174 or 178.

[1142] In some embodiments, the cytochrome P450 enzyme comprises an amino acid sequence further comprising an N-terminal membrane anchor, wherein the N-terminal membrane anchor has an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82, further optionally wherein the N-terminal membrane anchor comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82.

[1143] In some embodiments, the cytochrome P450 enzyme comprises an amino acid sequence further comprising an N-terminal membrane anchor, wherein the N-terminal membrane anchor has an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 175 or 179, further optionally wherein the N-terminal membrane anchor comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 175 or 179.

[1144] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80, optionally wherein the cytochromeP450 comprises an amino acid sequence of any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80.

[1145] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 172 or 176, optionally wherein the cytochrome P450 comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 172 or 176.

[1146] In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 180, optionally wherein the cytochrome P450 comprises or consists of an amino acid sequence of SEQ ID NO: 180.

[1147] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity any one of SEQ ID NOs: 34, 36, 38, 40, 42 or 44, optionally wherein the alcohol dehydrogenase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 34, 36, 38, 40, 42 or 44.

[1148] In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, optionally wherein the enal-cleaving enzyme comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. In some embodiments, the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 1, 4, 6, 8, 10, 20, 22, 24, 26, 28, 30, or 32, optionally wherein the enal-cleaving enzyme comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1, 4, 6, 8, 10, 20, 22, 24, 26, 28, 30, or 32.

[1149] In some embodiments, wherein the ionone compound of formula I is in the form of formula I -Al, the ionylideneethane synthase enzyme comprises an amino acid sequencehaving at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 89, optionally wherein the ionylideneethane synthase enzyme comprises an amino acid sequence comprising SEQ ID NO: 89, and the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71 or 77, optionally wherein the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71 or 77.

[1150] In some embodiments, wherein the ionone compound of formula I is in the form of formula I -Al, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 83 or 89, optionally wherein the ionylideneethane synthase enzyme comprises an amino acid sequence comprising SEQ ID NO: 83 or 89, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 46, optionally wherein the cytochrome P450 enzyme comprises an amino acid sequence comprising SEQ ID NO: 46, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 34 or 36, optionally wherein the alcohol dehydrogenase enzyme comprises an amino acid sequence comprising SEQ ID NO: 34 or 36, and the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 1, 4, 6, 8, 10, 20, 22, 24, 26, 28, 30, or 32, optionally wherein the enal-cleaving enzyme comprises an amino acid sequence comprising SEQ ID NO: 1, 4, 6, 8, 10, 20, 22, 24, 26, 28, 30, or 32.

[1151] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-E or formula I-A2, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99%identity to SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272, optionally wherein the ionylideneethane synthase enzyme comprises an amino acid sequence comprising SEQ ID NO: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 180, 67, 46, 172, or 176, optionally wherein the cytochrome P450 enzyme comprises an amino acid sequence comprising SEQ ID NO: 180, 67, 46, 172 or 176, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 40, optionally wherein the alcohol dehydrogenase enzyme comprises an amino acid sequence comprising SEQ ID NO: 40, and the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 30, optionally wherein the enal-cleaving enzyme comprises an amino acid sequence comprising SEQ ID NO: 30.

[1152] In some embodiments, wherein the ionone compound of formula I is in the form of formula I-B3, the ionylideneethane synthase enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 274, 276, 278 or 280, optionally wherein the ionylideneethane synthase enzyme comprises an amino acid sequence comprising any one of SEQ ID NOs: 274, 276, 278 or 280, the cytochrome P450 comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 180 or 67, optionally wherein the cytochrome P450 enzyme comprises an amino acid sequence comprising any one of SEQ ID NOs: 180 or SEQ ID NO: 67, the alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 40, optionally wherein the alcohol dehydrogenase enzyme comprises an amino acid sequence comprising SEQ IDNO: 40, and the enal-cleaving enzyme comprises an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 30, optionally wherein the enal-cleaving enzyme comprises an amino acid sequence comprising SEQ ID NO: 30.

[1153] A “cell” as defined herein is an organism suitable for genetic manipulation and which may be cultured at cell densities useful for industrial production of a target product. A suitable organism may be a microorganism, for example one which may be maintained in a fermentation device. With regard to the present disclosure, it is understood that cells, such as e.g., microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). A cell may be a cell found in nature or a cell derived from a parent cell after genetic manipulation or classical mutagenesis.

[1154] A particular cell is meant to be “capable of producing famesyl pyrophosphate (FPP)” when it produces FPP naturally or when it does not produce FPP naturally but is transformed to produce FPP as described above herein. Cells transformed to produce a higher amount of FPP than the naturally occurring organism or cell are also encompassed by the “cells capable of producing FPP”.

[1155] The “mevalonate pathway” also known as the “isoprenoid pathway” or “HMG-CoA reductase pathway” is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five-carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP). Key enzymes are acetoacetyl-CoA thiolase (atoB), HMG-CoA synthase (mvaS), HMG-CoA reductase (mvaA), mevalonate kinase (MvaKl), phosphomevalonate kinase (MvaK2), a mevalonate diphosphate decarboxylase (MvaD), and an isopentenyl diphosphate isomerase (idi). Combining the mevalonate pathway with enzyme activity to generate the terpene precursors GPP, FPP or GGPP, like in particular FPP synthase (ERG20), allows the recombinant cellular production of terpenes.

[1156] A cell may be a prokaryotic, archaebacterial or eukaryotic cell.

[1157] A prokaryotic cell may, but is not limited to, a bacterial cell. A bacterial cell may be Gram-negative or Gram-positive bacteria. Examples of bacteria include, but are not limited to,bacteria belonging to the genus Bacillus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus), Acinetobacter. Nocardia, Xanthobacter. Escherichia (e.g., E. coll), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P. aeruginosa, P. fluor escens), Salmonella (e.g., S. typhimurium, S. typhi), Anabaena, Caulobactert, Gluconobacter , Rhodobacter, Paracoccus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter , Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus. Bacteria also include, but are not limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria green sulfur bacteria purple sulfur bacteria and purple non-sulfur bacteria.

[1158] A eukaryotic cell may be a fungus, such as a filamentous fungus or yeast. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Aspergillus (e.g. A. niger,A oryzae,A. nidulans),Agaricus,Aureobasidium, Coprinus, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium (e.g. P. chrysogenum, P. camember ti), Piromyces, Phanerochaete, Pleurotus, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia (e.g. Rasamsonia emersonii), Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma.

[1159] Yeast cells may be selected from the genera: Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), Kluyveromyces, Candida (e.g., C. rugosa, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis), Pichia (e.g., P. pastoris), Schizosaccharomyces, Issatchenkia, Zygosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia (e.g., Y. lipolytica, formerly classified as Candida lipolytica .

[1160] The cell may be an algae, a microalgae or a marine eukaryote. The cell may be a Labyrinthulomycetes cell, preferably of the order Thraustochytriales , more preferably of the family Thraustochytriaceae, more preferably a member of a genus selected from the group consisting of Aurantiochytrium, Oblongichytrium, Schizochytrium, Thraustochytrium, and Ulkenia, even more preferably Schizochytrium sp. ATCC# 20888.

[1161] The recombinant cell as disclosed herein may belong to one of the genera Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces , Pachysolen, Yarrowia, Yamadazyma or Escherichia, for example a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, an Issatchenkia orientalis cell or an Escherichia coli cell.Therefore, in one embodiment a recombinant cell capable of producing the ionones described herein, which have been modified to express the enzymes according to the disclosure may be a prokaryote, eukaryote or archaeal cell, particularly a plant cell or a cell selected from a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, an Issatchenkia orientalis cell, Pichia pastoris or an Escherichia coli cell.

[1162] Product isolation

[1163] The methodology of the present invention can further include a step of recovering an end product or an intermediate product, optionally in stereoisomerically or enantiomerically substantially pure form. The term “recovering” includes extracting, harvesting, isolating or purifying the compound from culture or reaction media. Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like.

[1164] The compounds produced in any of the processes described herein can be converted to derivatives such as, but not limited to hydrocarbons, esters, amides, glycosides, ethers, epoxides, aldehydes, ketones, alcohols, diols, acetals or ketals. The terpene compound derivatives can be obtained by a chemical method such as, but not limited to oxidation, reduction, alkylation, acylation and / or rearrangement. Alternatively, the terpene compound derivatives can be obtained using a biochemical method by contacting the terpene compound with an enzyme such as, but not limited to an oxidoreductase, a monooxygenase, a dioxygenase, a transferase or a terpene cyclase. The biochemical conversion can be performed in vitro using isolated enzymes, enzymes from lysed cells or bioconversion using whole cells.Polypeptides and nucleic acids of the invention or used in the process of the invention

[1165] The generic terms “polypeptide” or “peptide”, which may be used interchangeably, refer to a natural or synthetic linear chain or sequence of consecutive, peptidically linked amino acid residues, comprising about 10 to up to more than 1.000 residues. Short chain polypeptides with up to 30 residues are also designated as “oligopeptides”.

[1166] The term “protein” refers to a macromolecular structure consisting of one or more polypeptides. The amino acid sequence of its polypeptide(s) represents the “primary structure” of the protein. The amino acid sequence also predetermines the “secondary structure” of the protein by the formation of special structural elements, such as alpha-helical and beta-sheet structures formed within a polypeptide chain. The arrangement of a plurality of such secondary structural elements defines the “tertiary structure” or spatial arrangement of the protein. If a protein comprises more than one polypeptide chains said chains are spatially arranged forming the “quaternary structure” of the protein. A correct spatial arrangement or “folding” of the protein is prerequisite of protein function. Denaturation or unfolding destroys protein function. If such destruction is reversible, protein function may be restored by refolding.

[1167] A typical protein function referred to herein is an “enzyme function”, i.e. the protein acts as biocatalyst on a substrate, for example a chemical compound, and catalyzes the conversion of said substrate to a product. An enzyme may show a high or low degree of substrate and / or product specificity.

[1168] A “polypeptide” referred to herein as having a particular “activity” thus implicitly refers to a correctly folded protein showing the indicated activity, as for example a specific enzyme activity.

[1169] Thus, unless otherwise indicated the term “polypeptide” also encompasses the terms “protein” and “enzyme”.

[1170] Similarly, the term “polypeptide fragment” encompasses the terms “protein fragment ‘ and “enzyme fragment”.

[1171] The term “isolated polypeptide” refers to an amino acid sequence that is removed from its natural environment by any method or combination of methods known in the art and includes recombinant, biochemical and synthetic methods.“Target peptide” refers to an amino acid sequence which targets a protein, or polypeptide to intracellular organelles, i.e., mitochondria, or plastids, or to the extracellular space (secretion signal peptide). A nucleic acid sequence encoding a target peptide may be fused to the nucleic acid sequence encoding the amino terminal end, e.g., N-terminal end, of the protein or polypeptide, or may be used to replace a native targeting polypeptide.

[1172] The present invention also relates to "functional equivalents" (also designated as “analogs” or “functional mutations”) of the polypeptides specifically described herein.

[1173] For example, "functional equivalents" refer to polypeptides which, in a test used for determining enzymatic activity display at least a 1 to 10 %, or at least 20 %, or at least 50 %, or at least 75 %, or at least 90 % higher or lower activity, as that of the polypeptides specifically described herein.

[1174] "Functional equivalents”, according to the invention, also cover particular mutants, which, in at least one sequence position of an amino acid sequences stated herein, have an amino acid that is different from that concretely stated one, but nevertheless possess one of the aforementioned biological activities, as for example enzyme activity. "Functional equivalents" thus comprise mutants obtainable by one or more, like 1 to 20, in particular 1 to 15 or 5 to 10 amino acid additions, substitutions, in particular conservative substitutions, deletions and / or inversions, where the stated changes can occur in any sequence position, provided they lead to a mutant with the profile of properties according to the invention. Functional equivalence is in particular also provided if the activity patterns coincide qualitatively between the mutant and the unchanged polypeptide, i.e. if, for example, interaction with the same agonist or antagonist or substrate, however at a different rate, (i.e. expressed by a EC50 or IC50 value or any other parameter suitable in the present technical field) is observed. Examples of suitable (conservative) amino acid substitutions are shown in the following table:

[1175] Original residue Examples of substitution

[1176] Ala Ser

[1177] Arg Lys

[1178] Asn Gin; His

[1179] Asp Glu

[1180] Cys Ser

[1181] Gin AsnGlu Asp

[1182] Gly Pro

[1183] His Asn ; Gin

[1184] lie Leu; Vai

[1185] Leu lie; Vai

[1186] Lys Arg ; Gin ; Glu

[1187] Met Leu ; lie

[1188] Phe Met ; Leu ; Tyr

[1189] Ser Thr

[1190] Thr Ser

[1191] Trp Tyr

[1192] Tyr Trp ; Phe

[1193] Vai lie; Leu

[1194] "Functional equivalents" in the above sense are also "precursors" of the polypeptides described herein, as well as "functional derivatives" and "salts" of the polypeptides.

[1195] "Precursors" are in that case natural or synthetic precursors of the polypeptides with or without the desired biological activity.

[1196] The expression "salts" means salts of carboxyl groups as well as salts of acid addition of amino groups of the protein molecules. Salts of carboxyl groups can be produced in a known way and comprise inorganic salts, for example sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases, for example amines, such as triethanolamine, arginine, lysine, piperidine and the like. Salts of acid addition, for example salts with inorganic acids, such as hydrochloric acid or sulfuric acid and salts with organic acids, such as acetic acid and oxalic acid, are also covered by the invention.

[1197] "Functional derivatives" of polypeptides according to the invention can also be produced on functional amino acid side groups or at their N-terminal or C-terminal end using known techniques. Such derivatives comprise for example aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, produced by reaction with acyl groups; or O-acyl derivatives of free hydroxyl groups, produced by reaction with acyl groups.

[1198] ’’Functional equivalents” naturally also comprise polypeptides that can be obtained from other organisms, as well as naturally occurring variants. For example, areas of homologoussequence regions can be established by sequence comparison, and equivalent polypeptides can be determined on the basis of the concrete parameters of the invention.

[1199] "Functional equivalents" also comprise “fragments”, like individual domains or sequence motifs, of the polypeptides according to the invention, or N- and or C-terminally truncated forms, which may or may not display the desired biological function. Preferably such “fragments” retain the desired biological function at least qualitatively.

[1200] "Functional equivalents" are, moreover, fusion proteins, which have one of the polypeptide sequences stated herein or functional equivalents derived there from and at least one further, functionally different, heterologous sequence in functional N-terminal or C-terminal association (i.e. without substantial mutual functional impairment of the fusion protein parts). Non-limiting examples of these heterologous sequences are e.g. signal peptides, histidine anchors or enzymes.

[1201] “Functional equivalents” which are also comprised in accordance with the invention are homologs to the specifically disclosed polypeptides. These have at least 60%, preferably at least 75%, in particular at least 80 or 85%, such as, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, homology (or identity) to one of the specifically disclosed amino acid sequences, calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448. A homology or identity, expressed as a percentage, of a homologous polypeptide according to the invention means in particular an identity, expressed as a percentage, of the amino acid residues based on the total length of one of the amino acid sequences described specifically herein.

[1202] The identity data, expressed as a percentage, may also be determined with the aid of BLAST alignments, algorithm blastp (protein-protein BLAST), or by applying the Clustal settings specified herein below.

[1203] In the case of a possible protein glycosylation, "functional equivalents" according to the invention comprise polypeptides as described herein in deglycosylated or glycosylated form as well as modified forms that can be obtained by altering the glycosylation pattern.

[1204] Functional equivalents or homologues of the polypeptides according to the invention can be produced by mutagenesis, e.g. by point mutation, lengthening or shortening of the protein or as described in more detail below.Functional equivalents or homologs of the polypeptides according to the invention can be identified by screening combinatorial databases of mutants, for example shortening mutants. For example, a variegated database of protein variants can be produced by combinatorial mutagenesis at the nucleic acid level, e.g. by enzymatic ligation of a mixture of synthetic oligonucleotides. There are a great many methods that can be used for the production of databases of potential homologues from a degenerated oligonucleotide sequence. Chemical synthesis of a degenerated gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene can then be ligated in a suitable expression vector. The use of a degenerated genome makes it possible to supply all sequences in a mixture, which code for the desired set of potential protein sequences. Methods of synthesis of degenerated oligonucleotides are known to a person skilled in the art.

[1205] In the prior art, several techniques are known for the screening of gene products of combinatorial databases, which were produced by point mutations or shortening, and for the screening of cDNA libraries for gene products with a selected property. These techniques can be adapted for the rapid screening of the gene banks that were produced by combinatorial mutagenesis of homologues according to the invention. The techniques most frequently used for the screening of large gene banks, which are based on a high-throughput analysis, comprise cloning of the gene bank in expression vectors that can be replicated, transformation of the suitable cells with the resultant vector database and expression of the combinatorial genes in conditions in which detection of the desired activity facilitates isolation of the vector that codes for the gene whose product was detected. Recursive Ensemble Mutagenesis (REM), a technique that increases the frequency of functional mutants in the databases, can be used in combination with the screening tests, in order to identify homologues.

[1206] An embodiment provided herein provides orthologs and paralogs of polypeptides disclosed herein as well as methods for identifying and isolating such orthologs and paralogs. A definition of the terms “ortholog” and “paralog” is given below and applies to amino acid and nucleic acid sequences.

[1207] The polypeptides of the invention include all active forms, including active subsequences, e.g., catalytic domains or active sites, of an enzyme of the invention. In one aspect, the invention provides catalytic domains or active sites as set forth below. In one aspect, the invention provides a peptide or polypeptide comprising or consisting of an active site domain as predicted through use of a database such as Pfam(http: / / pfam.wustl.edu / hmmsearch.shtml) (which is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, The Pfam protein families database, A. Bateman, E. Bimey, L. Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. L. Sonnhammer, Nucleic Acids Research, 30(l):276-280, 2002) or equivalent, as for example InterPro and SMART databases (http: / / www.ebi.ac.uk / interpro / scan.html, http: / / smart.embl-heidelberg.de / ).

[1208] The invention also encompasses “polypeptide variant” having the desired activity, wherein the variant polypeptide is selected from an amino acid sequence having at least 40%, 45%, 50%. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a specific, in particular natural, amino acid sequence as referred to by a specific SEQ ID NO and contains at least one substitution modification relative to said SEQ ID NO.

[1209] Coding nucleic acid sequences applicable according to the invention

[1210] The terms “nucleic acid sequence,” “nucleic acid,” “nucleic acid molecule” and “polynucleotide” are used interchangeably meaning a sequence of nucleotides. A nucleic acid sequence may be a single-stranded or double-stranded deoxyribonucleotide, or ribonucleotide of any length, and include coding and non-coding sequences of a gene, exons, introns, sense and anti-sense complimentary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers and nucleic acid probes. The skilled artisan is aware that the nucleic acid sequences of RNA are identical to the DNA sequences with the difference of thymine (T) being replaced by uracil (U). The term “nucleotide sequence” should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid.

[1211] An “isolated nucleic acid” or “isolated nucleic acid sequence” relates to a nucleic acid or nucleic acid sequence that is in an environment different from that in which the nucleic acid or nucleic acid sequence naturally occurs and can include those that are substantially free from contaminating endogenous material.The term “naturally-occurring” as used herein as applied to a nucleic acid refers to a nucleic acid that is found in a cell of an organism in nature and which has not been intentionally modified by a human in the laboratory.

[1212] A “fragment” of a polynucleotide or nucleic acid sequence refers to contiguous nucleotides that is particularly at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp and / or at least 60 bp in length of the polynucleotide of an embodiment herein. Particularly the fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 contiguous nucleotides of the polynucleotide of an embodiment herein. Without being limited, the fragment of the polynucleotides herein may be used as a PCR primer, and / or as a probe, or for anti-sense gene silencing or RNAi.

[1213] “Recombinant nucleic acid sequences” are nucleic acid sequences that result from the use of laboratory methods (for example, molecular cloning) to bring together genetic material from more than on source, creating or modifying a nucleic acid sequence that does not occur naturally and would not be otherwise found in biological organisms.

[1214] “Recombinant DNA technology” refers to molecular biology procedures to prepare a recombinant nucleic acid sequence as described, for instance, in Laboratory Manuals edited by Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.

[1215] The term “gene” means a DNA sequence comprising a region, which is transcribed into a RNA molecule, e.g., an mRNA in a cell, operably linked to suitable regulatory regions, e.g., a promoter. A gene may thus comprise several operably linked sequences, such as a promoter, a 5’ leader sequence comprising, e.g., sequences involved in translation initiation, acoding region of cDNA or genomic DNA, introns, exons, and / or a 3’non-translated sequence comprising, e.g., transcription termination sites.

[1216] “Polycistronic” refers to nucleic acid molecules, in particular mRNAs, that can encode more than one polypeptide separately within the same nucleic acid molecule.

[1217] A “chimeric gene” refers to any gene which is not normally found in nature in a species, in particular, a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature. For example, the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region. The term “chimeric gene” is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or to an antisense, i.e., reverse complement of the sense strand, or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription). The term "chimeric gene" also includes genes obtained through the combination of portions of one or more coding sequences to produce a new gene.

[1218] A “3’ UTR” or “3’ non-translated sequence” (also referred to as “3’ untranslated region,” or “3 ’end”) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal such as AAUAAA or variants thereof. After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the site of translation, e.g., cytoplasm.

[1219] The term “primer” refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and is used for polymerization of a nucleic acid sequence complementary to the template.

[1220] The term “selectable marker” refers to any gene which upon expression may be used to select a cell or cells that include the selectable marker. Examples of selectable markers aredescribed below. The skilled artisan will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.

[1221] The invention also relates to nucleic acid sequences that code for polypeptides as defined herein.

[1222] In particular, the invention also relates to nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, e.g. cDNA, genomic DNA and mRNA), coding for one of the above polypeptides and their functional equivalents, which can be obtained for example using artificial nucleotide analogs.

[1223] The invention relates both to isolated nucleic acid molecules, which code for polypeptides according to the invention or biologically active segments thereof, and to nucleic acid fragments, which can be used for example as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the invention.

[1224] The present invention also relates to nucleic acids with a certain degree of “identity” to the sequences specifically disclosed herein. "Identity" between two nucleic acids means identity of the nucleotides, in each case over the entire length of the nucleic acid.

[1225] The “identity” between two nucleotide sequences (the same applies to peptide or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues) or that are identical in...

Claims

1. CLAIMS1. A method for preparing an ionone compound of formula I(formula I)in the form of any one of its stereoisomers or a mixture thereof, wherein the method comprises:contacting an ionylidene acetaldehyde compound of formula II(formula II)in the form of any one of its stereoisomers or a mixture thereof,with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I;wherein any bond having a dotted line ( - ) represents independently from each other either a single carbon-carbon bond or a carbon-carbon double bond, wherein one of the bonds having a dotted line ( - ) represents a carbon-carbon double bond, with the proviso that the compound does not comprise two cumulated carbon-carbon double bonds; and wherein any wavy line represents a carbon-carbon bond linked to a carbon-carbon double bond which is either in the Z or in the E-configuration.

2. The method of claim 1, wherein the method further comprises:(A) contacting an ionylideneethanol compound of formula III(formula III)in the form of any one of its stereoisomers or a mixture thereof,with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce the ionylidene acetaldehyde compound of formula II(formula II); and / or249(B) contacting an ionylideneethane compound of formula (IV)(formula IV)in the form of any one of its stereoisomers or a mixture thereof, with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce the ionylideneethanol compound of formula III(formula III); and / or(C) contacting a famesyl pyrophosphate compound of formula V(formula V)in the form of any one of its stereoisomers or a mixture thereof, with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce the ionylideneethane compound of formula (IV)(formula IV).

3. The method of claim 1 or claim 2, for preparing an ionone compound of formula I, wherein the compound of formula I is in the form of formula I -A:(formula I-A)wherein the method comprises:(a) contacting a famesyl pyrophosphate compound of formula V(formula V) in the form of any one of its stereoisomers or a mixture thereof, with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV-A:(formula IV -A);(b) contacting the ionylideneethane compound of formula IV -A:(formula IV-A)with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-A:(formula III-A);(c) contacting the ionylideneethanol compound of formula III-A:(formula III-A) with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-A:(formula II-A); and(d) contacting the ionylidene acetaldehyde compound of formula II-A:(formula II-A)with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-A:(formula I-A).

4. The method of any one of claims 1 to 3, wherein:(A) the ionone compound of formula I or formula I-A is in the form of (R)-alpha-ionone of formula I-Al:(formula I-Al); optionally wherein:(I)(i) the ionylidene acetaldehyde compound of formula II or formula II-A is in the form of formula II-Al(i):(formula II-Al(i));and / or(ii) the ionylideneethanol compound of formula III or formula III-A is in the form of formula III-Al(i):(formula III-Al(i)); and / or(iii) the ionylideneethane compound of formula IV or formula IV-A is in the form of formula IV-Al(i):(formula IV -Al (i)); or(II)(i) the ionylidene acetaldehyde compound of formula II or formula II-A is in the form of formula II-Al(ii):(formula II-Al(ii)); and / or(ii) the ionylideneethanol compound of formula III or formula III-A is in the form of formula III-Al(ii):(formula III-Al(ii)); and / or252(m) the ionylideneethane compound of formula IV or formula IV-A is in the form of formula IV-Al(ii):(formula IV -Al (ii)); or(B) the ionone compound of formula I or formula I-A is in the form of (R)-gamma-ionone of formula I-A2:(formula I-A2); optionally wherein(I)(i) the ionylidene acetaldehyde compound of formula II or formula II-A is in the form of(formula II-A2(i)); and / or(ii) the ionylideneethanol compound of formula III or formula III-A is in the form of formula III-A2(i):(formula III-A2(i)); and / or(iii) the ionylideneethane compound of formula IV or formula IV-A is in the form of formula IV-A2(i):(formula IV -A2(i)); or(II)(i) the ionylidene acetaldehyde compound of formula II or formula II-A is in the form of(formula II-A2(ii)); and / or(ii) the ionylideneethanol compound of formula III or formula III-A is in the form of formula III-A2(ii):(formula III-A2(ii)); and / or(iii) the ionylideneethane compound of formula IV or formula IV-A is in the form of(formula IV-A2(ii)).

5. The method of claim 1 or claim 2, for preparing an ionone of formula I, wherein the compound of formula I is in the form of formula I-B3 :(formula I-B3)wherein the method comprises:(A)(a) contacting a famesyl pyrophosphate compound of formula V(formula V)in the form of any one of its stereoisomers or a mixture thereof, with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV-B3(i):(formula IV-B3(i));(b) contacting the ionylideneethane compound of formula IV-B3(i):(formula IV-B3(i))with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-B3(i):(formula III-B3(i));(c) contacting the ionylideneethanol compound of formula III-B3(i):(formula III-B3(i))with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-B3(i):(formula II-B3(i)); and(d) contacting the ionylidene acetaldehyde compound of formula II-B3 (i) :(formula II-B3(i))with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-B3:(formula I-B3); and / or(B)(a) contacting a famesyl pyrophosphate compound of formula V(formula V) in the form of any one of its stereoisomers or a mixture thereof,with an ionylideneethane synthase enzyme under conditions suitable for the ionylideneethane synthase enzyme to produce an ionylideneethane compound of formula IV-B3(ii):(formula IV-B3(ii));(b) contacting the ionylideneethane compound of formula IV-B3(ii):255(formula IV-B3(ii)) with a cytochrome P450 enzyme under conditions suitable for the cytochrome P450 enzyme to produce an ionylideneethanol compound of formula III-B3(ii):(formula III-B3(ii));(c) contacting the ionylideneethanol compound of formula III-B3(ii):(formula III-B3(ii)) with an alcohol dehydrogenase enzyme under conditions suitable for the alcohol dehydrogenase enzyme to produce an ionylidene acetaldehyde compound of formula II-B3(ii):(formula II-B3(ii)); and (d) contacting the ionylidene acetaldehyde compound of formula II-B3 (ii) :(formula II-B3 (ii))with an enal-cleaving enzyme under conditions suitable for the enal-cleaving enzyme to produce the ionone compound of formula I-B3:(formula I-B3).

6. The method of claim 1, wherein the ionone compound of formula I is in the form of formula I-Cl orI-C2:(formula I-C2), wherein:256(a) the famesyl pyrophosphate compound of formula V is in the form of formula V- 1:(formula V)(b) the ionylideneethane compound of formula IV is in the form of formula IV-Cl(i):(formula IV-Cl(i));(c) the ionylideneethanol compound of formula III is in the form of formula III- Cl(i):(formula III-Cl(i)), and(d) the ionylidene acetaldehyde compound of formula II is in the form of formula II- Cl(i):(formula Il-Cl(i)); or(B)(a) the famesyl pyrophosphate compound of formula V is in the form of formula V-(formula V)(b) the ionylideneethane compound of formula IV is in the form of formula IV- Cl(ii):(formula IV-Cl(ii));(c) the ionylideneethanol compound of formula III is in the form of formula III- Cl(n):257(formula Ill-Cl(ii)); and(d) the ionylidene acetaldehyde compound of formula II is in the form of formula II- Cl(ii):(C)(a) the famesyl pyrophosphate compound of formula V is in the form of formula V- 1:(formula V)(b) the ionylideneethane compound of formula IV is in the form of formula IV-C2(i):(formula IV-C2(i)),(c) the ionylideneethanol compound of formula III is in the form of formula III-C2(i)(formula III-C2(i)), and(d) the ionylidene acetaldehyde compound of formula II is in the form of formula II-C2(i):(formula II-C2(i)); or(D)(a) the famesyl pyrophosphate compound of formula V is in the form of formula V- 1:(formula V)(b) the ionylideneethane compound of formula IV is in the form of formula IV- C2(ii):258(formula IV-C2(ii));(c) the ionylideneethanol compound of formula III is in the form of formula III- C2(ii):(formula III-C2(ii)), and(d) the ionylidene acetaldehyde compound of formula II is in the form of formula II- C2(ii):(formula II-C2(ii)).

7. The method of any one of claims 2 to 6, wherein the famesyl pyrophosphate compound of formula V is:(a) in the form of 2E,6E-famesyl diphosphate (formula V-l):(b) is in the form of 2Z,6E-famesyl diphosphate (formula V-2):(formula V-2).

8. The method of any one of claims 3, 4 or 7, wherein:(A)(I)(a) the famesyl pyrophosphate compound of formula V is in the form of formula V-1:(b) the ionylideneethane compound of formula IV is in the form of formula IV-Al(i):259(formula IV-Al(i));(c) the ionylideneethanol compound of formula III is in the form of formula III- AI(i):(formula III-Al(i)), and(d) the ionylidene acetaldehyde compound of formula II is in the form of formula II- AI(i):(formula II-Al(i)); and / or(II)(a) the famesyl pyrophosphate compound of formula V is in the form of formula V-(b) the ionylideneethane compound of formula IV is in the form of formula IV- Al(ii):(formula IV -Al (ii));(c) the ionylideneethanol compound of formula III is in the form of formula III- Al(n):(formula III-Al(ii)); and(d) the ionylidene acetaldehyde compound of formula II is in the form of formula II-260(a) the famesyl pyrophosphate compound of formula V is in the form of formula V-1:(b) the ionylideneethane compound of formula IV is in the form of formula IV-A2(i):(formula IV-A2(i)),(c) the ionylideneethanol compound of formula III is in the form of formula III-A2(i)(formula III-A2(i)), and(d) the ionylidene acetaldehyde compound of formula II is in the form of formula II-A2(i):& o (formula II-A2(i)); and / or(II)(a) the famesyl pyrophosphate compound of formula V is in the form of formula V-1:(formula V-l);(b) the ionylideneethane compound of formula IV is in the form of formula IV-A2(ii):(formula IV-A2(ii));261(c) the ionylideneethanol compound of formula III is in the form of formula III- A2(ii):(formula III- A2(ii)), and (d) the ionylidene acetaldehyde compound of formula II is in the form of formula II- A2(ii):(formula II-A2(ii)).

9. The method of any one of claims 1 to 8, wherein the enal-cleaving enzyme comprises:(A) at least one or more motifs selected from:(i) GxxWxGxxxxxGx (SEQ ID NO: 146), wherein X2 can be Y or can be deleted, X3, X5, X7, X8 and X10 can be any naturally occurring amino acid, X9 can be F, L, or I, XI 1 can be R, S, or T, and X13 can be H or A;(ii) WxGxxxxx (SEQ ID NO: 147), wherein X2 can be F, A, V, or Y; X4 can be N or K; X5 and X7 can be any naturally occurring amino acid, X6 can be F or Y, X8 can be D or S;(iii) xxxxxxxxxxxxxV (SEQ ID NO: 148), wherein XI can be G or S, X2, X4, X6, X7, X8, X9 or XI 3 can be any naturally occurring amino acid, X3 can be A or G, X5 can be L, M or V, X10 can be F, L, or Y, XI 1 can be R, Q or D, and X12 can be G or D;(iv) xxYDxxxxxxxx (SEQ ID NO: 149), wherein XI can be L or M; X2 can be I or V; X5, X6, X9, or XI 1 can be any naturally occurring amino acid, X7 can be P or A, X8 can be I, M or V, XI 0 can be D or V, and X12 can be F, Y or L; and / or(B) an amino acid sequence at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, optionally wherein the enal-cleaving enzyme comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.26210. The method of any one of claims 1 to 9, wherein the alcohol dehydrogenase enzyme comprises:(A) at least one or more motifs selected from:(i) CHTD (SEQ ID NO: 150);(ii) GHEGxG (SEQ ID NO: 151), wherein X5 can be any naturally occurring amino acid;(iii) LxCGxxTGxGA (SEQ ID NO: 152), wherein X2, X5, X6, and X9 can be any naturally occurring amino acid;(iv) GxxGL (SEQ ID NO: 153), wherein X2 can be any naturally occurring amino acid and X3 can be V or I;(v) LxxxGxx (SEQ ID NO: 154), wherein X2, X3 and X4 can be any naturally occurring amino acid, wherein X6 can be L, V or I and wherein X7 can be P, A or G;(vi) GxVxAI (SEQ ID NO: 155), wherein X2 and X4 can be any naturally occurring amino acid;(vii) YxATKxA (SEQ ID NO: 156), wherein X2 and X6 can be any naturally occurring amino acid; and / or(B) an amino acid sequence having at least at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity any one of SEQ ID NOs: 34, 36, 38, 40, 42 or 44, optionally wherein the alcohol dehydrogenase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 34, 36, 38, 40, 42 or 44;11. The method of any one of claims 1 to 10, wherein:(I) the ionylideneethane synthase enzyme comprises:(A) at least one or more motifs selected from:(i) AxAxEYxR (SEQ ID NO: 160), wherein X2 or X7 can be any naturally occurring amino acid; X4 can be W or F;(ii) WxRYxxxxR (SEQ ID NO: 161), wherein X2, X5, X8 can be any naturally occurring amino acid; X6 can be A or G; X7 can be F or W;(iii) WFRxRDxDxLxRF (SEQ ID NO: 162), wherein X4, X7, X9, or XI 1 can be any naturally occurring amino acid;263(iv) KHRxEGExxxxFxY (SEQ ID NO: 163), wherein X9, X10, X13 can be any naturally occurring amino acid; X4 can be S or A; X8 can be T or I; XI 1 can be T or S;(v) GGxxxxxxRRYRxxx (SEQ ID NO: 164), wherein X3, X5, X6, X7, X14 can be any naturally occurring amino acid; X4 can be I, L or V; X8 can be M or T; XI 3 can be F or Y; X15 can be E or D; and / or(B) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144; and / or (C) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238; and / or(II) the ionylideneethane synthase enzyme comprises:(A) at least one or more motifs selected from:(i) AxAxEYxR (SEQ ID NO: 160), wherein X2 or X7 can be any naturally occurring amino acid; X4 can be W or F;(ii) WxRYxxxxR (SEQ ID NO: 161), wherein X2, X5, X8 can be any naturally occurring amino acid; X6 can be A or G; X7 can be F or W;(iii) WFRxRDxDxLxRF (SEQ ID NO: 162), wherein X4, X7, X9, or XI 1 can be any naturally occurring amino acid;(iv) KHRxEGExxxxFxY (SEQ ID NO: 163), wherein X9, X10, X13 can be any naturally occurring amino acid; X4 can be S or A; X8 can be T or I; XI 1 can be T or S;264(v) GGxxxxxxRRYRxxx (SEQ ID NO: 164), wherein X3, X5, X6, X7, X14 can be any naturally occurring amino acid; X4 can be I, L or V; X8 can be M or T; XI 3 can be F or Y; X15 can be E or D; and / or(B) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111, optionally wherein the ionylideneethane synthase enzyme comprises any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111; and / or(C) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272; and / or (III) the ionylideneethane synthase enzyme comprises:(A) at least one or more motifs selected from:(i) AxAxEYxR (SEQ ID NO: 160), wherein X2 or X7 can be any naturally occurring amino acid; X4 can be W or F;(ii) WxRYxxxxR (SEQ ID NO: 161), wherein X2, X5, X8 can be any naturally occurring amino acid; X6 can be A or G; X7 can be F or W;(iii) WFRxRDxDxLxRF (SEQ ID NO: 162), wherein X4, X7, X9, or XI 1 can be any naturally occurring amino acid;(iv) KHRxEGExxxxFxY (SEQ ID NO: 163), wherein X9, X10, X13 can be any naturally occurring amino acid; X4 can be S or A; X8 can be T or I; XI 1 can be T or S;(v) GGxxxxxxRRYRxxx (SEQ ID NO: 164), wherein X3, X5, X6, X7, X14 can be any naturally occurring amino acid; X4 can be I, L or V; X8 can be M or T; XI 3 can be F or Y; X15 can be E or D; and / or(B) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 274, 276, 278 or 280, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 274, 276, 278 or 280; and / or265(IV) the cytochrome P450 enzyme comprises:(A) at least one or more motifs selected from:(i) FGxGxxxCxG (SEQ ID NO: 157), wherein X3, X5, X6, X7 can be any naturally occurring amino acid and X9 can be P or V;(ii) PERx (SEQ ID NO: 158), wherein X4 can be F or Y;(iii) LxxLxxVxxExxRxxxx (SEQ ID NO: 159), wherein X2, X3, X5, X6, X9, XI 1, X12 or X16 can be any naturally occurring amino acid; X8 can be L, I or V; X14 can be M or L; X15 can be R or H; X17 can be P or V; and / or(B) an amino acid sequence having 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 49, 53, 57, 61, 65, 69, 74, or 79, optionally wherein the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 49, 53, 57, 61, 65, 69, 74, or 79;(C) an amino acid sequence further comprising an N-terminal membrane anchor; optionally wherein the N-terminal membrane anchor has an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82, further optionally wherein the N-terminal membrane anchor comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82; and / or(D) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80, optionally wherein the cytochrome P450 comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80; and / or (E) an amino acid sequence having 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 174 or 178, optionally wherein the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 174 or 178;(F) an amino acid sequence further comprising an N-terminal membrane anchor; optionally wherein the N-terminal membrane anchor has an amino acid sequence having at266least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 175 or 179, further optionally wherein the N-terminal membrane anchor comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 175 or 179; and / or(G) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 172 or 176, optionally wherein the cytochrome P450 comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 172 or 176; and / or(H) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 180, optionally wherein the cytochrome P450 comprises or consists of an amino acid sequence of SEQ ID NO: 180.

12. The method of any one of claims 2 to 11, wherein the method further comprises preparing the famesyl pyrophosphate compound from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) using one or more prenyltransferase enzymes; preferably, a famesyl pyrophosphate synthase.

13. The method of any one of claims 1 to 12, wherein:(a) the method is an in vivo or a bioconversion process; and / or(b) the method is performed in a cell capable of functionally expressing a heterologous ionylideneethane synthase enzyme, a heterologous cytochrome P450 enzyme, a heterologous alcohol dehydrogenase enzyme, and / or a heterologous enal-cleaving enzyme.

14. A cell capable of producing an ionone compound of formula I under suitable fermentative conditions, wherein the cell expresses a heterologous enal-cleaving enzyme.

15. The cell of claim 14, wherein the cell produces an ionone compound of formula I:267(A) and further produces an ionylidene acetaldehyde compound of formula II, wherein the cell further expresses a heterologous alcohol dehydrogenase enzyme;(B) and optionally further produces an ionylideneethanol compound of formula III, wherein the cell expresses a heterologous cytochrome P450 enzyme.

16. The cell of claim 14 or 15, wherein the cell further:(a) expresses an ionylideneethane synthase enzyme; and(b) comprises famesyl pyrophosphate of formula V.

17. The cell of claims 14 to 16, wherein the cell comprises a famesyl pyrophosphate of formula V, a heterologous ionylideneethane synthase enzyme, a heterologous cytochrome P450 enzyme, a heterologous alcohol dehydrogenase enzyme, and a heterologous enal- cleaving enzyme.

18. The cell of claims 14 to 16, wherein the cell produces at least 50 wt% of the compound of formula I.

19. The cell of any one of claims 14 to 18, wherein the ionone compound of formula I is in the form of:(a)formula (I-Al);(b)formula (I-A2);(c)(formula I-B3);268(d)(formula I-Cl); or (e)(formula I-C2).

20. The cell of any one of claims 15 to 19, wherein:(I) the ionylidene acetaldehyde compound of formula II is in the form of:(a)formula II-A1 (i); (b)formula II-Al(ii) (c)formula II-A2(i); (d)formula II-A2(ii); (e)(formula II-B3(i)); (f)269; d / or (II) the ionylideneethanol compound of formula III is in the form of:(a)formula III-Al(i); (b)formula III-Al(ii) (c)formula III-A2(i); (d)270formula III- A2(ii); (e)(formula III-B3(i)); (f)(g)(h)(i)(formula III-C2(i)); or (j)(formula III-C2(ii)); and / or (III) the ionylideneethane compound of formula IV is in the form of:(a)formula IV-Al(i);(b)27172(formula IV-C2(ii).

21. The cell of any one of claims 14 to 20, wherein:(I) the enal-cleaving enzyme comprises:(A) at least one or more motifs selected from:(i) GxxWxGxxxxxGx (SEQ ID NO: 146), wherein X2 can be Y or can be deleted, X3, X5, X7, X8 and X10 can be any naturally occurring amino acid, X9 can be F, L, or I, XI 1 can be R, S, or T, and X13 can be H or A;(ii) WxGxxxxx (SEQ ID NO: 147), wherein X2 can be F, A, V, or Y; X4 can be N or K; X5 and X7 can be any naturally occurring amino acid, X6 can be F or Y, X8 can be D or S;(iii) xxxxxxxxxxxxxV (SEQ ID NO: 148), wherein XI can be G or S, X2, X4, X6, X7, X8, X9 or XI 3 can be any naturally occurring amino acid, X3 can be A or G, X5 can be L, M or V, X10 can be F, L, or Y, XI 1 can be R, Q or D, and X12 can be G or D;(iv) xxYDxxxxxxxx (SEQ ID NO: 149), wherein XI can be L or M; X2 can be I or V; X5, X6, X9, or XI 1 can be any naturally occurring amino acid, X7 can be P or A, X8 can be I, M or V, X10 can be D or V, and X12 can be F, Y or L; and / or(B) an amino acid sequence at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, optionally wherein the enal-cleaving enzyme comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32; and / or(II) the alcohol dehydrogenase enzyme comprises:(A) at least one or more motifs selected from:(i) CHTD (SEQ ID NO: 150);(ii) GHEGxG (SEQ ID NO: 151), wherein X5 can be any naturally occurring amino acid;273(iii) LxCGxxTGxGA (SEQ ID NO: 152), wherein X2, X5, X6, and X9 can be any naturally occurring amino acid;(iv) GxxGL (SEQ ID NO: 153), wherein X2 can be any naturally occurring amino acid and X3 can be V or I;(v) LxxxGxx (SEQ ID NO: 154), wherein X2, X3 and X4 can be any naturally occurring amino acid, wherein X6 can be L, V or I and wherein X7 can be P, A or G;(vi) GxVxAI (SEQ ID NO: 155), wherein X2 and X4 can be any naturally occurring amino acid;(vii) YxATKxA (SEQ ID NO: 156), wherein X2 and X6 can be any naturally occurring amino acid; and / or(B) an amino acid sequence having at least at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity any one of SEQ ID NOs: 34, 36, 38, 40, 42 or 44, optionally wherein the alcohol dehydrogenase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 34, 36, 38, 40, or 44; and / or(III) the cytochrome P450 enzyme comprises:(A) at least one or more motifs selected from:(i) FGxGxxxCxG (SEQ ID NO: 157), wherein X3, X5, X6, X7 can be any naturally occurring amino acid and X9 can be P or V;(ii) PERx (SEQ ID NO: 158), wherein X4 can be F or Y;(iii) LxxLxxVxxExxRxxxx (SEQ ID NO: 159), wherein X2, X3, X5, X6, X9, XI 1, XI 2 or XI 6 can be any naturally occurring amino acid; X8 can be L, I or V; X14 can be M or L; X15 can be R or H; X17 can be P or V; and / or(B) an amino acid sequence having 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 49, 53, 57, 61, 65, 69, 74, or 79, optionally wherein the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 49, 53, 57, 61, 65, 69, 74, or 79;(C) an amino acid sequence further comprising an N-terminal membrane anchor; optionally wherein the N-terminal membrane anchor has an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least27490% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82, further optionally wherein the N-terminal membrane anchor comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 50, 54, 58, 62, 66, 70, 75, 76, 81 or 82; and / or(D) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80, optionally wherein the cytochrome P450 comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 46, 51, 55, 59, 63, 67, 71, 73, 77, or 80;(E) an amino acid sequence having 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 174 or 178, optionally wherein the cytochrome P450 comprises an amino acid sequence of any one of SEQ ID NOs: 174 or 178;(F) an amino acid sequence further comprising an N-terminal membrane anchor; optionally wherein the N-terminal membrane anchor has an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 175 or 179, further optionally wherein the N-terminal membrane anchor comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 175 or 179; and / or(G) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 172 or 176, optionally wherein the cytochrome P450 comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 172 or 176; and / or(H) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 180, optionally wherein the cytochrome P450 comprises or consists of an amino acid sequence of SEQ ID NO: 180; and / or(IV) the ionylideneethane synthase enzyme comprises:(A)(a) at least one or more motifs selected from:(i) AxAxEYxR (SEQ ID NO: 160), wherein X2 or X7 can be any naturally occurring amino acid; X4 can be W or F;(ii) WxRYxxxxR (SEQ ID NO: 161), wherein X2, X5, X8 can be any naturally occurring amino acid; X6 can be A or G; X7 can be F or W;(iii) WFRxRDxDxLxRF (SEQ ID NO: 162), wherein X4, X7, X9, or XI 1 can be any naturally occurring amino acid;(iv) KHRxEGExxxxFxY (SEQ ID NO: 163), wherein X9, X10, X13 can be any naturally occurring amino acid; X4 can be S or A; X8 can be T or I; XI 1 can be T or S;(v) GGxxxxxxRRYRxxx (SEQ ID NO: 164), wherein X3, X5, X6, X7, X14 can be any naturally occurring amino acid; X4 can be I, L or V; X8 can be M or T; XI 3 can be F or Y; X15 can be E or D; and / or(b) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 83, 86, 89, 92, 95, 97, 99, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144; and / or (c) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, or 238; or(B)(a) at least one or more motifs selected from:(i) AxAxEYxR (SEQ ID NO: 160), wherein X2 or X7 can be any naturally occurring amino acid; X4 can be W or F;(ii) WxRYxxxxR (SEQ ID NO: 161), wherein X2, X5, X8 can be any naturally occurring amino acid; X6 can be A or G; X7 can be F or W;(iii) WFRxRDxDxLxRF (SEQ ID NO: 162), wherein X4, X7, X9, or XI 1 can be any naturally occurring amino acid;(iv) KHRxEGExxxxFxY (SEQ ID NO: 163), wherein X9, X10, X13 can be any naturally occurring amino acid; X4 can be S or A; X8 can be T or I; XI 1 can be T or S;(v) GGxxxxxxRRYRxxx (SEQ ID NO: 164), wherein X3, X5, X6, X7, X14 can be any naturally occurring amino acid; X4 can be I, L or V; X8 can be M or T; XI 3 can be F or Y; X15 can be E or D; and / or(b) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111, optionally wherein the ionylideneethane synthase enzyme comprises any one of SEQ ID NOs: 101, 103, 105, 107, 109 or 111; and / or(c) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, and 272; or(C) the ionylideneethane synthase enzyme comprises:(a) at least one or more motifs selected from:(i) AxAxEYxR (SEQ ID NO: 160), wherein X2 or X7 can be any naturally occurring amino acid; X4 can be W or F;(ii) WxRYxxxxR (SEQ ID NO: 161), wherein X2, X5, X8 can be any naturally occurring amino acid; X6 can be A or G; X7 can be F or W;(iii) WFRxRDxDxLxRF (SEQ ID NO: 162), wherein X4, X7, X9, or XI 1 can be any naturally occurring amino acid;(iv) KHRxEGExxxxFxY (SEQ ID NO: 163), wherein X9, X10, X13 can be any naturally occurring amino acid; X4 can be S or A; X8 can be T or I; XI 1 can be T or S;277(v) GGxxxxxxRRYRxxx (SEQ ID NO: 164), wherein X3, X5, X6, X7, X14 can be any naturally occurring amino acid; X4 can be I, L or V; X8 can be M or T; XI 3 can be F or Y; X15 can be E or D; and / or(b) an amino acid sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 274, 276, 278 or 280, optionally wherein the ionylideneethane synthase enzyme comprises or consists of any one of SEQ ID NOs: 274, 276, 278 or 280.

22. The method of claim 12, or the cell of any one of claims 14 to 21, wherein the cell is a bacterial cell, a plant cell, a fungal cell such as a yeast cell; preferably, the recombinant cell is of the genus Escherichia, Saccharomyces , Yarrowia or Pichia.278

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