Improved Methods and Enzymes

Abstract

Improved methods for producing amberketal and amberketal homologs and compositions containing them, improved squalene-hopene cyclase (SHC) enzymes for use in the methods, nucleic acid constructs and vectors encoding the enzymes, and host cells expressing the enzymes.

Description

[Technical field]

[0001] Field The present disclosure relates generally to improved methods for making amberketal and amberketal homologs. The disclosure further relates to improved SHC enzymes for use in the methods, nucleic acid constructs and vectors encoding the enzymes, and host cells expressing the enzymes. [Background technology]

[0002] background Amberketal provides a strong and persistent ambery and woody odor and is useful in fragrance compositions, either alone or in combination with other woody or ambery ingredients. Amberketal is traditionally prepared from manool through a number of chemical transformations. However, the supply of natural manool is limited. WO2021 / 209482 discloses a method for producing amberketal and amberketal homologs from polyunsaturated alcohols using the enzyme squalene-hopene cyclase (SHC). Summary of the Invention

[0003] overview One aspect of the present disclosure is a compound of formula (I): [ka] Formula (I) The present invention relates to a method for preparing a compound represented by formula (II): [ka] Formula (II) with a squalene-hopene cyclase (SHC) enzyme comprising an amino acid sequence having at least 70% identity or similarity to the sequence of SEQ ID NO:1, wherein the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:1, and wherein R is selected from the group consisting of H and C. 1 ~C 4 is selected from alkyl.

[0004] In some embodiments of the method for making a compound of formula (I), the method is such that the compound of formula (II) is such that the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomer).

[0005] A further aspect of the present disclosure is a compound of formula (I): [ka] Formula (I) The present invention relates to a method for preparing a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa), [ka] Formula (II) [ka] Formula (IIa) with a squalene-hopene cyclase (SHC) enzyme comprising an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:1 or the sequence of SEQ ID NOs:43-49, preferably having at least 70% identity or similarity to the sequence of SEQ ID NO:1 and comprising one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:1, and wherein R is selected from the group consisting of H and C. 1 ~C 4 is selected from alkyl.

[0006] In some embodiments of the method for making a mixture comprising a compound represented by formula (I), the method further comprises the step of: [ka] Formula (Ia) wherein R is selected from the group consisting of H and C 1 ~C 4 In some embodiments, the compound of formula (Ia) is selected from the group consisting of alkyl, [ka] Formula (V) wherein R is H and C 1 ~C 4 is selected from alkyl.

[0007] In some embodiments of the method for making a mixture comprising a compound represented by Formula (I), the method further comprises the step of: wherein the mixture comprising a compound represented by Formula (II) and a compound represented by Formula (IIa) comprises any one of the following: i) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomer). ii) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomer). iii) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomer). iv) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomer). v) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomers) and compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomers). vi) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomers) and compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomers). vii) Any combination of i) to vi).

[0008] In some embodiments of the method for making a mixture comprising a compound represented by Formula (I), the method further comprises the step of: - Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomers). - Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomers). - compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomers), as well as; - Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomers).

[0009] In some embodiments of the methods for making a compound represented by formula (I) and the methods for making a mixture comprising a compound represented by formula (I), a compound represented by formula (III) [ka] Formula (III) The compound represented by the formula: 1 ~C 4 is selected from alkyl.

[0010] In some embodiments of the methods for making a compound represented by formula (I) and the methods for making a mixture comprising a compound represented by formula (I), a compound represented by formula (IIIa): [ka] Formula (IIIa) Compounds having the relative configuration shown in are produced as by-products, where R is H and C 1 ~C 4 is selected from alkyl.

[0011] In some embodiments of the method for making a mixture comprising a compound represented by formula (I), a compound represented by formula (VI) [ka] Formula (VI) The compound represented by the formula: 1 ~C 4 is selected from alkyl.

[0012] In some embodiments of the method for making a mixture comprising a compound represented by formula (I), the mixture is represented by formula (VIa): [ka] Formula (VIa) Compounds having the relative configuration shown in are produced as by-products, where R is H and C 1 ~C 4 is selected from alkyl. In some embodiments of the methods for making a compound represented by formula (I) and the methods for making a mixture comprising a compound represented by formula (I), R is methyl.

[0013] In some embodiments of the methods for making a compound represented by Formula (I) and methods for making a mixture comprising a compound represented by Formula (I), the SHC enzyme comprises an amino acid sequence having at least 70% identity or similarity to the sequence of SEQ ID NO:1, and the SHC enzyme comprises 1-7, preferably 2-6, more preferably 3-5 amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:1.

[0014] In some embodiments of the methods for making a compound represented by Formula (I) and the methods for making a mixture comprising a compound represented by Formula (I), the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO: at one or more positions corresponding to positions 2, 5, 35, 166, 211, 212, 355, 483, and 539 of SEQ ID NO:1.

[0015] In some embodiments of the methods for making a compound of formula (I) and methods for making a mixture comprising a compound of formula (I), the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 166, 211, 212, 483, and 539 of SEQ ID NO:1, preferably corresponding to positions 2, 5, 35, 166, 211, 483, and 539.

[0016] In some embodiments of the methods for making a compound represented by formula (I) and the methods for making a mixture comprising a compound represented by formula (I), the SHC enzyme comprises an amino acid substitution relative to SEQ ID NO:1 selected from the following: (i) an asparagine (N) residue at a position corresponding to position 2 of SEQ ID NO:1; (ii) a proline (P) residue at a position corresponding to position 5 of SEQ ID NO:1; (iii) an alanine (A) residue at a position corresponding to position 35 of SEQ ID NO:1; (iv) a threonine (T) residue at a position corresponding to position 116 of SEQ ID NO:1; (v) an alanine (A) residue at a position corresponding to position 166 of SEQ ID NO:1; (vi) a valine (V) residue at a position corresponding to position 211 of SEQ ID NO:1; (vii) an arginine (R) residue at a position corresponding to position 212 of SEQ ID NO:1; (viii) a methionine (M) residue at a position corresponding to position 317 of SEQ ID NO:1; (ix) a threonine (T) residue at a position corresponding to position 355 of SEQ ID NO:1; (x) a threonine (T) residue at a position corresponding to position 382 of SEQ ID NO:1; (xi) a valine (V) residue at a position corresponding to position 399 of SEQ ID NO:1; (xii) a cysteine ​​(C) residue at a position corresponding to position 483 of SEQ ID NO:1; (xiii) a histidine (H) residue at a position corresponding to position 539 of SEQ ID NO:1; (xiv) an alanine (A) residue at a position corresponding to position 585 of SEQ ID NO:1; or (xv) any combination thereof.

[0017] In some embodiments of the methods for making a compound represented by formula (I) and the methods for making a mixture comprising a compound represented by formula (I), the SHC enzyme comprises an amino acid substitution relative to SEQ ID NO:1 selected from the following, which correspond to positions in SEQ ID NO:1: (i) I2N, T35A, A355T, and L539H; (ii) T166A; (iii) I2N and Y483C; (iv) I2N, Y483C, and L539H; (v) I2N, L5P, T35A, L539H; (vi) I2N, L5P, T35A, and Y483C; (vii) I2N, L5P, T35A, T166A, and L539H; (viii) I2N, L5P, T35A, T166A, E211V, and L539H; (ix) I2N, L5P, T35A, E211V, S212R, Y483C, and L539H; (x) I2N, T166A, and Y483C; (xi) I2N, T166A, Y483C, and L539H; (xii) I2N, T166A, E211V, and Y483C; or (xiii) I2N, T166A, E211V, Y483C, and L539H.

[0018] In some embodiments of the methods for making a compound represented by Formula (I) and the methods for making a mixture comprising a compound represented by Formula (I), the SHC enzyme comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N and T166A.

[0019] In some embodiments of the methods for making a compound represented by formula (I) and the methods for making a mixture comprising a compound represented by formula (I), the SHC enzyme further comprises one or more amino acid substitutions relative to SEQ ID NO:1 selected from L5P, T35A, E211V, Y483C, and L539H.

[0020] In some embodiments of the methods for making a compound represented by formula (I) and the methods for making a mixture comprising a compound represented by formula (I), the SHC enzyme further comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or 42, preferably SEQ ID NOs: 4, 6, 18, 20, 22, 24, 30, 32, 34, 36, 38, 40, or 42, more preferably SEQ ID NOs: 30, 32, 34, 36, 38, 40, or 42, and most preferably SEQ ID NOs: 30, 38, 40, 42.

[0021] A further aspect of the present disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding a squalenehopene cyclase (SHC) enzyme as described in any of the methods for making a compound of formula (I) and the methods for making a mixture comprising a compound of formula (I). A further aspect of the present disclosure relates to a vector comprising a nucleic acid molecule according to the present disclosure.

[0022] A further aspect of the present disclosure relates to a host cell comprising a nucleic acid molecule according to the present disclosure or a vector according to the present disclosure. Further aspects of the present disclosure relate to the squalene hopene cyclase (SHC) enzyme as described in any of the methods for making a compound of formula (I) and the methods for making a mixture comprising a compound of formula (I).

[0023] A further aspect of the present disclosure relates to a composition comprising a compound of formula (I) and a compound of formula (Ia), wherein the composition is obtained or obtainable by a process for making a mixture comprising a compound of formula (I) according to the present disclosure.

[0024] In some embodiments, the composition is such that the compound represented by formula (I) and the compound represented by formula (Ia) are in solid form, preferably in amorphous or crystalline form. In some embodiments, the composition is such that the compound represented by formula (Ia) has the configuration represented by formula (V).

[0025] A further aspect of the present disclosure relates to the use of a composition according to the present disclosure for the manufacture of a fragrance composition or a consumer product. A further aspect of the present disclosure relates to a fragrance composition or consumer product comprising a composition according to the present disclosure.

[0026] A further aspect of the present disclosure relates to a mixture comprising a product obtainable by a process as described in any of the methods for making a compound of the present disclosure, wherein the mixture comprises I, Ia, III, IIIa, IV, IVa, V, Va, VI, and / or VIa. A further aspect of the present disclosure relates to a composition according to the present disclosure, wherein the composition comprises a compound represented by formula (I) and / or a compound represented by formula (Ia), and further comprises III, IIIa, IV, IVa, V, Va, and VI and / or VIa. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] description There is still a need to provide new, more efficient, cost-effective and sustainable methods for producing amberketal and amberketal homologs.The financial viability and sustainability of the production method of amberketal and amberketal homologs can be enhanced by obtaining improved substrate conversion and production yield, reduced by-product yield, and improved overall reaction performance under industrially relevant conditions.As a result, there is still a need for improved production process of amberketal and amberketal homologs.As a result, there is still a need for improved SHC enzymes and host cells expressing the enzymes to produce amberketal and amberketal homologs.

[0028] The inventors have surprisingly found that the squalene-hopene cyclase (SHC) enzymes described herein are capable of converting a compound of formula (IIa) to a compound of formula (Ia) as described later herein. They are further capable of converting a compound of formula (II) and / or a compound of formula (IIa) (wherein the compound of formula (II) and the compound of formula (IIa) are included in a mixture) to a compound of formula (I) and a compound of formula (Ia), respectively. Furthermore, substitution of amino acid residues corresponding to one or more specific positions of the squalene-hopene cyclase (SHC) enzyme results in improved conversion of a compound of formula (II) to a compound of formula (I) and / or improved conversion of a compound of formula (IIa) to a compound of formula (Ia), as described later herein.

[0029] Specifically, as detailed elsewhere herein and in the Experimental Section, the methods, enzymes, and host cells described herein exert at least one, at least two, or all of the following advantageous effects: Improved conversion ratio of the compound represented by formula (II) and / or the compound represented by formula (IIa) Improved yield of the compound represented by formula (I) and / or the compound represented by formula (Ia) Improved reaction performance (e.g., conversion rate, productivity, yield at high substrate concentrations)

[0030] Consequently, aspects and embodiments of the present disclosure solve at least some of the problems and needs as described herein.

[0031] method The methods described herein may involve the enzymatic conversion of a compound represented by formula (II) to a compound represented by formula (I) by an SHC enzyme of the disclosure. The methods described herein may involve the enzymatic conversion of a compound represented by formula (IIa) to a compound represented by formula (Ia) by an SHC enzyme of the disclosure. The methods described herein may involve the enzymatic conversion of a compound represented by formula (II) and / or a compound represented by formula (IIa) (wherein the compound represented by formula (II) and / or the compound represented by formula (IIa) are included in a mixture) to a compound represented by formula (I) and / or a compound represented by formula (Ia), respectively, or to a mixture comprising a compound represented by formula (I) and / or a compound represented by formula (Ia).

[0032] Accordingly, in one aspect, the present disclosure provides a compound of formula (I): [ka] Formula (I) The present invention provides a method for making a compound represented by formula (II): [ka] Formula (II) The method includes contacting a compound represented by the formula:

[0033] In one aspect, the present disclosure provides a compound of formula (Ia): [ka] Formula (Ia) The present invention provides a method for making a compound represented by formula (IIa): [ka] Formula (IIa) The method includes contacting a compound represented by the formula:

[0034] In one aspect, the disclosure provides a method for making a mixture comprising a compound of formula (I) and / or a compound of formula (Ia), the method comprising contacting a compound of formula (II) and / or a compound of formula (IIa) with a squalene-hopene cyclase (SHC) enzyme as described herein. The compound of formula (II) and / or the compound of formula (IIa) may be present in the mixture.

[0035] In some embodiments, the squalene-hopene cyclase (SHC) enzyme comprises an amino acid sequence having at least 30%, 40%, 50%, 60%, or 70%, preferably at least 70%, identity or similarity to the sequence of SEQ ID NO:1 or SEQ ID NOs:43-49.

[0036] In preferred embodiments, the squalene-hopene cyclase (SHC) enzyme comprises an amino acid sequence having at least 30%, 40%, 50%, 60% or 70%, preferably at least 70%, identity or similarity to the sequence of SEQ ID NO: 19, wherein the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO: 1. Preferably, the one or more amino acid substitutions relative to SEQ ID NO: 1 are at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO: 1.

[0037] SHC enzymes according to the present disclosure are described in more detail later herein. R in all formulas described herein is H (hydrogen) and C 1 ~C 4 In some embodiments, R is selected from alkyl, in some embodiments, R is H (hydrogen), in some embodiments, R is ethyl, in some embodiments, R is n-propyl, in some embodiments, R is iso-propyl, in some embodiments, R is methyl.

[0038] Accordingly, in some embodiments, there is provided a method for making a compound of formula (I), the method comprising contacting a compound of formula (II) with a squalene-hopene cyclase (SHC) enzyme comprising an amino acid sequence having at least 70% identity or similarity to the sequence of SEQ ID NO:1, wherein the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:1, and wherein R is selected from the group consisting of H and C. 1 ~C 4 alkyl, preferably wherein R is methyl.

[0039] In some embodiments, a method for producing a mixture comprising a compound of formula (I) is provided, the method comprising contacting a mixture comprising a compound of formula (II) and a compound of formula (IIa) with a squalene-hopene cyclase (SHC) enzyme comprising an amino acid sequence having at least 70% identity or similarity to the sequence of SEQ ID NO:1 or SEQ ID NOs:43-49, preferably having at least 70% identity or similarity to the sequence of SEQ ID NO:1 and comprising one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:1, and wherein R is selected from the group consisting of H and C. 1 ~C 4 alkyl, preferably wherein R is methyl. In some embodiments, the mixture comprising a compound represented by formula (I) further comprises a compound represented by formula (Ia), preferably a compound represented by formula (Ia) having a configuration represented by a compound represented by formula (V), as described later herein.

[0040] As used herein, "contacting" may correspond to the physical interaction of a compound with the squalene-hopene cyclase (SHC) enzyme as described herein, thereby promoting a reaction catalyzed by the enzyme.

[0041] "Contacting with a compound of formula (II)" and "contacting with a compound of formula (IIa)" may correspond to contacting with a single isomer or with a mixture of isomers of these compounds. An "isomer" of a compound, as used herein, preferably refers to a stereoisomer of the compound.

[0042] The SHC enzyme may be produced in a host cell as described later herein. Such a host cell may be used in the methods described herein. In some embodiments, the SHC enzyme may be associated with a membrane (such as a cell membrane or a membrane in which the SHC enzyme is immobilized) to receive and / or interact with a substrate (e.g., a compound represented by formula (II) and / or a compound represented by formula (IIa)), which membrane (such as a cell membrane) may be part of a whole cell (e.g., a recombinant host cell such as those described later herein). The SHC enzyme may also be present in a crude cell extract or a cell-free extract. Accordingly, those skilled in the art will understand that "contacting" may also correspond to the physical interaction of a compound with a cell expressing the SHC enzyme as described later herein, with a membrane fraction of the cell, with a crude cell extract of the cell, or with a cell-free extract of the cell. The SHC enzyme may also be in an immobilized form (e.g., associated with an enzyme carrier) in which the SHC enzyme can interact with a substrate (e.g., a compound represented by formula (II) and / or a compound represented by formula (IIa)). The term "immobilized" is provided later in this specification. The SHC enzymes may also be used in soluble form.

[0043] Compounds represented by formula (II) and (IIa) Compounds of formula (II), compounds of formula (IIa), and mixtures containing them may alternatively be referred to herein as "substrates", "(bio)transformation substrates", or "reaction substrates", all terms being interchangeable. The numbering of the carbon atoms in compounds of formula (II) is as follows: [ka] Formula (II).

[0044] The numbering of the carbon atoms in the compound of formula (IIa) is as follows: [ka] Formula (IIa).

[0045] Compounds of formula (IIa) are "structural isomers" of compounds of formula (II). The SHC enzymes described herein are particularly suitable for converting compounds of formula (II) and / or compounds of formula (IIa) into useful products, as described later herein.

[0046] In embodiments involving contacting with a mixture of isomers of a compound of formula (II), at least one isomer is converted to a compound of formula (I). In embodiments involving contacting with a mixture of isomers of a compound of formula (IIa), at least one isomer is converted to a compound of formula (Ia). In embodiments involving contacting with a mixture comprising a compound of formula (II) and a compound of formula (IIa), the compound of formula (II) may be converted to a compound of formula (I) and / or the compound of formula (IIa) may be converted to a compound of formula (Ia).

[0047] Compounds of formula (II) and formula (IIa) may occur in four different isomeric forms, for example, as compounds of formula (II) or compounds of formula (IIa) having E,E-, Z,E-, Z,Z-, or E,Z-configurations (alternatively referred to herein as E,Z-, Z,E-, Z,Z-, or E,Z-isomers). In some embodiments, compounds of formula (II) have a double bond between C-8 and C-9 in the E-configuration and a double bond between C-4 and C-5 in the Z-configuration (E,Z-isomers). In some embodiments, compounds of formula (II) have a double bond between C-8 and C-9 in the E-configuration and a double bond between C-4 and C-5 in the E-configuration (E,E-isomers).

[0048] A compound of formula (II) having a double bond between C-8 and C-9 in the Z-configuration and a double bond between C-4 and C-5 in the E-configuration corresponds to the Z,E-isomer. A compound of formula (II) having a double bond between C-8 and C-9 in the Z-configuration and a double bond between C-4 and C-5 in the Z-configuration also corresponds to the Z,Z-isomer.

[0049] In some embodiments, the compound of formula (IIa) has a double bond between C-6 and C-7 in the E-configuration and a double bond between C-2 and C-3 in the Z-configuration (E,Z-isomer). In some embodiments, the compound of formula (IIa) has a double bond between C-6 and C-7 in the E-configuration and a double bond between C-2 and C-3 in the E-configuration (E,E-isomer).

[0050] A compound of formula (IIa) having a double bond between C-6 and C-7 in the Z-configuration and a double bond between C-2 and C-3 in the E-configuration corresponds to the Z,E-isomer. A compound of formula (IIa) having a double bond between C-6 and C-7 in the Z-configuration and a double bond between C-2 and C-3 in the Z-configuration also corresponds to the Z,Z-isomer.

[0051] In some embodiments, the compound represented by formula (II) is a mixture of two or more of its isomers. In some embodiments, the mixture includes the E,E-isomer and one or more other isomers of the compound represented by formula (II). In some embodiments, the mixture includes the E,Z-isomer and one or more other isomers of the compound represented by formula (II). As a result, in some embodiments, the mixture may include the E,E- and Z,E-isomers. In some embodiments, the mixture may include the E,E- and Z,Z-isomers. In some embodiments, the mixture may include the E,E- and E,Z-isomers. In some embodiments, the mixture may include the E,Z-isomer and the Z,E-isomer. In some embodiments, the mixture may include the E,Z- and Z,Z-isomers.

[0052] In some embodiments, the compound represented by formula (IIa) is a mixture of two or more of its isomers. In some embodiments, the mixture includes the E,E-isomer and one or more other isomers of the compound represented by formula (IIa). In some embodiments, the mixture includes the E,Z-isomer and one or more other isomers of the compound represented by formula (IIa). Consequently, in some embodiments, the mixture may include the E,E- and Z,E-isomers. In some embodiments, the mixture may include the E,E- and Z,Z-isomers. In some embodiments, the mixture may include the E,E- and E,Z-isomers. In some embodiments, the mixture may include the E,Z-isomer and the Z,E-isomer. In some embodiments, the mixture may include the E,Z- and Z,Z-isomers.

[0053] In some embodiments, the compound represented by formula (II) is a mixture of three or more of its isomers. In some embodiments, the mixture includes the E,E-isomer and two or more other isomers of the compound represented by formula (II). In some embodiments, the mixture includes the E,Z-isomer and two or more other isomers of the compound represented by formula (II). Consequently, in some embodiments, the mixture may include the E,E-, Z,E-, and Z,Z-isomers. In some embodiments, the mixture may include the E,E-, Z,E-, and Z,Z-isomers. In some embodiments, the mixture may include the E,E-, Z,E-, and E,Z-isomers. In some embodiments, the mixture may include the Z,E-, Z,Z-, and E,Z-isomers.

[0054] In some embodiments, the compound represented by formula (IIa) is a mixture of three or more of its isomers. In some embodiments, the mixture includes the E,E-isomer and two or more other isomers of the compound represented by formula (IIa). In some embodiments, the mixture includes the E,Z-isomer and two or more other isomers of the compound represented by formula (IIa). Consequently, in some embodiments, the mixture may include the E,E-, Z,E-, and Z,Z-isomers. In some embodiments, the mixture may include the E,E-, Z,E-, and Z,Z-isomers. In some embodiments, the mixture may include the E,E-, Z,E-, and E,Z-isomers. In some embodiments, the mixture may include the Z,E-, Z,Z-, and E,Z-isomers.

[0055] In some embodiments, the compound of formula (II) is a mixture containing the E,Z-, E,E-, Z,E-, and Z,Z-isomers. Preferred mixtures contain the E,Z- and / or E,E-isomers of the compound of formula (II), preferably the E,Z-isomer.

[0056] In some embodiments, the compound of formula (IIa) is a mixture containing the E,Z-, E,E-, Z,E-, and Z,Z-isomers. Preferred mixtures contain the E,Z- and / or E,E-isomers of the compound of formula (IIa), preferably the E,Z-isomer.

[0057] In some embodiments, the composition comprises the E,Z-isomer of the compound of formula (II) and / or the E,E-isomer of the compound of formula (II), preferably the E,Z-isomer of the compound of formula (II), and the E,Z-isomer of the compound of formula (IIa) and / or the E,E-isomer of the compound of formula (IIa), preferably the E,Z-isomer of the compound of formula (IIa). Optionally, the Z,E-isomer of the compound of formula (II), the Z,Z-isomer of the compound of formula (II), the Z,E-isomer of the compound of formula (IIa), and / or the Z,Z-isomer of the compound of formula (IIa) may be included in a mixture.

[0058] In some embodiments, the methods described herein comprise contacting the E,Z-isomer of a compound of formula (II) with a squalene-hopene cyclase (SHC) enzyme described herein. In some embodiments, the methods described herein comprise contacting the E,Z-isomer and / or the E,E-isomer of a compound of formula (IIa), preferably the E,Z-isomer of a compound of formula (IIa), with a squalene-hopene cyclase (SHC) enzyme described herein.

[0059] In some embodiments, the methods described herein comprise contacting a mixture comprising, consisting essentially of, or consisting of the E,E-isomer and the E,Z-isomer of a compound of formula (II) with a squalene-hopene cyclase (SHC) enzyme. In some embodiments, the mixture comprises at least one or both of the Z,E-isomer and the Z,Z-isomer of a compound of formula (II). In some embodiments, the mixture does not comprise either or both of the Z,E-isomer and the Z,Z-isomer of a compound of formula (II).

[0060] In some embodiments, the methods described herein comprise contacting a mixture comprising, consisting essentially of, or consisting of the E,E-isomer and the E,Z-isomer of a compound of formula (IIa) with a squalene-hopene cyclase (SHC) enzyme described herein. In some embodiments, the mixture comprises at least one or both of the Z,E-isomer and the Z,Z-isomer of a compound of formula (IIa). In some embodiments, the mixture does not comprise either or both of the Z,E-isomer and the Z,Z-isomer of a compound of formula (IIa).

[0061] In some embodiments, the methods described herein include contacting a mixture comprising, consisting essentially of, or consisting of the E,E-isomer of a compound of formula (II) and the E,Z-isomer of a compound of formula (II) and / or the E,E-isomer of a compound of formula (IIa) and / or the E,Z-isomer of a compound of formula (IIa) with a squalene-hopene cyclase (SHC) enzyme described herein. In some embodiments, the mixture comprises at least one or both of the Z,E-isomer and the Z,Z-isomer of a compound of formula (IIa). In some embodiments, the mixture comprises at least one or both of the Z,E-isomer and the Z,Z-isomer of a compound of formula (IIa). In some embodiments, the mixture does not comprise either or both of the Z,E-isomer and the Z,Z-isomer of a compound of formula (II). In some embodiments, the mixture does not comprise either or both of the Z,E-isomer and the Z,Z-isomer of a compound of formula (IIa).

[0062] In a mixture comprising the E,Z-isomer of a compound of formula (II) and one or more other E isomers of a compound of formula (II), the ratio of the E,Z-isomer to all other isomers may be equal to or greater than 10:90, or about 10:90. In some embodiments, the ratio is equal to or greater than 20:80, or about 20:80. In some embodiments, the ratio is equal to or greater than 30:70, or about 30:70. In some embodiments, the ratio is equal to or greater than 40:60, or about 40:60. In some embodiments, the ratio is equal to or greater than 50:50, or about 50:50. In some embodiments, the ratio is equal to or greater than 60:40, or about 60:40. In some embodiments, the ratio is equal to or greater than 70:30, or about 70:30. In some embodiments, the ratio is equal to or greater than 80:20, or about 80:20. In some embodiments, the ratio is equal to or greater than 85:15, or about 85:15. In some embodiments, the ratio is equal to or greater than 90:10, or about 90:10. In some embodiments, the ratio is equal to or greater than 95:5, or about 95:5. In some embodiments, the ratio is equal to or greater than 96:4, or about 96:4. In some embodiments, the ratio is equal to or greater than 97:3, or about 97:3. In some embodiments, the ratio is equal to or greater than 98:2, or about 98:2. In some embodiments, the ratio is equal to or greater than 99:1, or about 99:1.

[0063] In a mixture comprising the E,Z-isomer of a compound of formula (II) and one or more other E isomers of a compound of formula (II), the ratio of the E,Z-isomer to all other isomers may be equal to or less than 99:1, or about 99:1. In some embodiments, the ratio is equal to or less than 95:5, or about 95:5. In some embodiments, the ratio is equal to or less than 90:10, or about 90:10. In some embodiments, the ratio is equal to or less than 85:15, or about 85:15. In some embodiments, the ratio is equal to or less than 80:20, or about 80:20. In some embodiments, the ratio is equal to or less than 70:30, or about 70:30. In some embodiments, the ratio is equal to or less than 60:40, or about 60:40. In some embodiments, the ratio is equal to or less than, or about 50:50. In some embodiments, the ratio is equal to or less than, or about 40:60. In some embodiments, the ratio is equal to or less than, or about 30:70. In some embodiments, the ratio is equal to or less than, or about 20:80.

[0064] In some embodiments, the ratio is equal to or less than 10:90, or is about 10:90. In mixtures comprising the E,Z-isomer of the compound of Formula (II) and one or more other isomers of the compound of Formula (II), the ratio of the E,Z-isomer to all other isomers may range from 10:90 to 99:1, from 10:90 to 90:1, from 20:80 to 80:20, from 50:50 to 80:20, or from 60:40 to 80:20.

[0065] In a mixture comprising the E,Z-isomer of the compound of formula (IIa) and one or more other E isomers of the compound of formula (IIa), the ratio of the E,Z-isomer to all other isomers may be equal to or greater than 10:90, or about 10:90. In some embodiments, the ratio is equal to or greater than 20:80, or about 20:80. In some embodiments, the ratio is equal to or greater than 30:70, or about 30:70. In some embodiments, the ratio is equal to or greater than 40:60, or about 40:60. In some embodiments, the ratio is equal to or greater than 50:50, or about 50:50. In some embodiments, the ratio is equal to or greater than 60:40, or about 60:40. In some embodiments, the ratio is equal to or greater than 70:30, or about 70:30. In some embodiments, the ratio is equal to or greater than or about 80:20. In some embodiments, the ratio is equal to or greater than or about 85:15. In some embodiments, the ratio is equal to or greater than or about 90:10. In some embodiments, the ratio is equal to or greater than or about 95:5. In some embodiments, the ratio is equal to or greater than or about 99:1.

[0066] In a mixture comprising the E,Z-isomer of the compound of formula (II)a and one or more other isomers of the compound of formula (IIa), the ratio of the E,Z-isomer to all other isomers may be equal to or less than 99:1, or about 99:1. In some embodiments, the ratio is equal to or less than 95:5, or about 95:5. In some embodiments, the ratio is equal to or less than 90:10, or about 90:10. In some embodiments, the ratio is equal to or less than 85:15, or about 85:15. In some embodiments, the ratio is equal to or less than 80:20, or about 80:20. In some embodiments, the ratio is equal to or less than 70:30, or about 70:30. In some embodiments, the ratio is equal to or less than 60:40, or about 60:40. In some embodiments, the ratio is equal to or less than, or about 50:50. In some embodiments, the ratio is equal to or less than, or about 40:60. In some embodiments, the ratio is equal to or less than, or about 30:70. In some embodiments, the ratio is equal to or less than, or about 20:80. In some embodiments, the ratio is equal to or less than, or about 10:90.

[0067] In mixtures comprising the E,Z-isomer of the compound of Formula (IIa) and one or more other isomers of the compound of Formula (IIa), the ratio of the E,Z-isomer to all other isomers may range from 10:90 to 99:1, from 10:90 to 90:1, from 20:80 to 80:20, from 50:50 to 80:20, or from 60:40 to 80:20.

[0068] In a mixture comprising the E,Z-isomer and the E,E-isomer of a compound of formula (II), the ratio of the E,Z-isomer to the E,E-isomer may be equal to or greater than 10:90, or about 10:90. In some embodiments, the ratio is equal to or greater than 20:80, or about 20:80. In some embodiments, the ratio is equal to or greater than 30:70, or about 30:70. In some embodiments, the ratio is equal to or greater than 40:60, or about 40:60. In some embodiments, the ratio is equal to or greater than 50:50, or about 50:50. In some embodiments, the ratio is equal to or greater than 60:40, or about 60:40. In some embodiments, the ratio is equal to or greater than 70:30, or about 70:30. In some embodiments, the ratio is equal to or greater than or about 80:20. In some embodiments, the ratio is equal to or greater than or about 85:15. In some embodiments, the ratio is equal to or greater than or about 90:10. In some embodiments, the ratio is equal to or greater than or about 95:5. In some embodiments, the ratio is equal to or greater than or about 99:1.

[0069] In a mixture comprising the E,Z-isomer and the E,E-isomer of a compound of formula (II), the ratio of the E,Z-isomer to the E,E-isomer may be equal to or less than 99:1, or about 99:1. In some embodiments, the ratio is equal to or less than 95:5, or about 95:5. In some embodiments, the ratio is equal to or less than 90:10, or about 90:10. In some embodiments, the ratio is equal to or less than 85:15, or about 85:15. In some embodiments, the ratio is equal to or less than 80:20, or about 80:20. In some embodiments, the ratio is equal to or less than 70:30, or about 70:30. In some embodiments, the ratio is equal to or less than 60:40, or about 60:40. In some embodiments, the ratio is equal to or less than, or about 50:50. In some embodiments, the ratio is equal to or less than, or about 40:60. In some embodiments, the ratio is equal to or less than, or about 30:70. In some embodiments, the ratio is equal to or less than, or about 20:80. In some embodiments, the ratio is equal to or less than, or about 10:90.

[0070] In a mixture comprising the E,Z- and E,E-isomers of a compound of formula (II), the ratio of the E,Z-isomer to the E,E-isomer can be from 10:90 to 99:1, or from about 10:90 to about 99:1, from 10:90 to 90:1, or from about 10:90 to about 90:1, from 20:80 to 80:20, or from about 20:80 to about 80:20, from 50:50 to 80:20, or from about 50:50 to about 80:20, or from 60:40 to 80:20, or from about 60:40 to about 80:20.

[0071] In a mixture comprising the E,Z-isomer and the E,E-isomer of the compound of formula (IIa), the ratio of the E,Z-isomer to the E,E-isomer may be equal to or greater than 10:90, or about 10:90. In some embodiments, the ratio is equal to or greater than 20:80, or about 20:80. In some embodiments, the ratio is equal to or greater than 30:70, or about 30:70. In some embodiments, the ratio is equal to or greater than 40:60, or about 40:60. In some embodiments, the ratio is equal to or greater than 50:50, or about 50:50. In some embodiments, the ratio is equal to or greater than 60:40, or about 60:40. In some embodiments, the ratio is equal to or greater than 70:30, or about 70:30. In some embodiments, the ratio is equal to or greater than or about 80:20. In some embodiments, the ratio is equal to or greater than or about 85:15. In some embodiments, the ratio is equal to or greater than or about 90:10. In some embodiments, the ratio is equal to or greater than or about 95:5. In some embodiments, the ratio is equal to or greater than or about 99:1.

[0072] In a mixture comprising the E,Z-isomer and the E,E-isomer of the compound of formula (IIa), the ratio of the E,Z-isomer to the E,E-isomer may be equal to or less than 99:1, or about 99:1. In some embodiments, the ratio is equal to or less than 95:5, or about 95:5. In some embodiments, the ratio is equal to or less than 90:10, or about 90:10. In some embodiments, the ratio is equal to or less than 85:15, or about 85:15. In some embodiments, the ratio is equal to or less than 80:20, or about 80:20. In some embodiments, the ratio is equal to or less than 70:30, or about 70:30. In some embodiments, the ratio is equal to or less than 60:40, or about 60:40. In some embodiments, the ratio is equal to or less than, or about 50:50. In some embodiments, the ratio is equal to or less than, or about 40:60. In some embodiments, the ratio is equal to or less than, or about 30:70. In some embodiments, the ratio is equal to or less than, or about 20:80. In some embodiments, the ratio is equal to or less than, or about 10:90.

[0073] In a mixture comprising the E,Z- and E,E-isomers of a compound of formula (IIa), the ratio of E,Z-isomer to E,E-isomer can be from 10:90 to 99:1, or from about 10:90 to about 99:1, from 10:90 to 90:1, or from about 10:90 to about 90:1, or from 20:80 to 80:20, or from about 20:80 to about 80:20, from 50:50 to 80:20, or from about 50:50 to about 80:20, or from 60:40 to 80:20, or from about 60:40 to about 80:20.

[0074] In a mixture comprising the E,Z-isomer of the compound of formula (II) and the E,Z-isomer of the compound of formula (IIa), the ratio of the E,Z-isomer of the compound of formula (II) to the E,Z-isomer of the compound of formula (IIa) may be equal to or greater than 10:90, or about 10:90. In some embodiments, the ratio is equal to or greater than 20:80, or about 20:80. In some embodiments, the ratio is equal to or greater than 30:70, or about 30:70. In some embodiments, the ratio is equal to or greater than 40:60, or about 40:60.

[0075] In some embodiments, the ratio is equal to or greater than 50:50, or about 50:50. In some embodiments, the ratio is equal to or greater than 60:40, or about 60:40. In some embodiments, the ratio is equal to or greater than 70:30, or about 70:30. In some embodiments, the ratio is equal to or greater than 80:20, or about 80:20. In some embodiments, the ratio is equal to or greater than 85:15, or about 85:15. In some embodiments, the ratio is equal to or greater than 90:10, or about 90:10. In some embodiments, the ratio is equal to or greater than 95:5, or about 95:5. In some embodiments, the ratio is equal to or greater than 99:1, or about 99:1.

[0076] In a mixture comprising the E,Z-isomer of the compound represented by formula (II) and the E,Z-isomer of the compound represented by formula (IIa), the ratio of the E,Z-isomer of the compound represented by formula (II) to the E,Z-isomer of the compound represented by formula (IIa) may be equal to or less than 99:1, or about 99:1. In some embodiments, the ratio is equal to or less than 95:5, or about 95:5. In some embodiments, the ratio is equal to or less than 90:10, or about 90:10. In some embodiments, the ratio is equal to or less than 85:15, or about 85:15. In some embodiments, the ratio is equal to or less than 80:20, or about 80:20. In some embodiments, the ratio is equal to or less than 70:30, or about 70:30. In some embodiments, the ratio is equal to or less than 60:40, or about 60:40. In some embodiments, the ratio is equal to or less than 50:50, or about 50:50. In some embodiments, the ratio is equal to or less than 40:60, or about 40:60. In some embodiments, the ratio is equal to or less than 30:70, or about 30:70. In some embodiments, the ratio is equal to or less than 20:80, or about 20:80. In some embodiments, the ratio is equal to or less than 10:90, or about 10:90.

[0077] In a mixture comprising the E,Z-isomer of the compound of formula (II) and the E,Z-isomer of the compound of formula (IIa), the ratio of the E,Z-isomer of the compound of formula (II) to the E,Z-isomer of the compound of formula (IIa) may be from 10:90 to 99:1, from 10:90 to 90:1, from 20:80 to 80:20, from 50:50 to 80:20, or from 60:40 to 80:20.

[0078] In a mixture comprising the E,Z-isomer of the compound of formula (II) and the E,E-isomer of the compound of formula (IIa), the ratio of the E,Z-isomer of the compound of formula (II) to the E,E-isomer of the compound of formula (IIa) may be equal to or greater than 10:90, or about 10:90. In some embodiments, the ratio is equal to or greater than 20:80, or about 20:80. In some embodiments, the ratio is equal to or greater than 30:70, or about 30:70. In some embodiments, the ratio is equal to or greater than 40:60, or about 40:60. In some embodiments, the ratio is equal to or greater than 50:50, or about 50:50. In some embodiments, the ratio is equal to or greater than 60:40, or about 60:40. In some embodiments, the ratio is equal to or greater than, or about 70:30. In some embodiments, the ratio is equal to or greater than, or about 80:20. In some embodiments, the ratio is equal to or greater than, or about 85:15. In some embodiments, the ratio is equal to or greater than, or about 90:10. In some embodiments, the ratio is equal to or greater than, or about 95:5. In some embodiments, the ratio is equal to or greater than, or about 99:1.

[0079] In a mixture comprising the E,Z-isomer of the compound of formula (II) and the E,E-isomer of the compound of formula (IIa), the ratio of the E,Z-isomer of the compound of formula (II) to the E,Z-isomer of the compound of formula (IIa) may be equal to or less than 99:1, or about 99:1. In some embodiments, the ratio is equal to or less than 95:5, or about 95:5. In some embodiments, the ratio is equal to or less than 90:10, or about 90:10. In some embodiments, the ratio is equal to or less than 85:15, or about 85:15. In some embodiments, the ratio is equal to or less than 80:20, or about 80:20. In some embodiments, the ratio is equal to or less than 70:30, or about 70:30. In some embodiments, the ratio is equal to or less than 60:40, or about 60:40. In some embodiments, the ratio is equal to or less than 50:50, or about 50:50. In some embodiments, the ratio is equal to or less than 40:60, or about 40:60. In some embodiments, the ratio is equal to or less than 30:70, or about 30:70. In some embodiments, the ratio is equal to or less than 20:80, or about 20:80. In some embodiments, the ratio is equal to or less than 10:90, or about 10:90.

[0080] In a mixture comprising the E,Z-isomer of the compound of formula (II) and the E,E-isomer of the compound of formula (IIa), the ratio of the E,Z-isomer of the compound of formula (II) to the E,Z-isomer of the compound of formula (IIa) may be from 10:90 to 99:1, from 10:90 to 90:1, from 20:80 to 80:20, from 50:50 to 80:20, or from 60:40 to 80:20. One of ordinary skill in the art will appreciate that the ratios discussed above may be determined, for example, by dividing the weights or concentrations of the stereoisomers.

[0081] The ratio of a given isomer to one or more other isomers in a mixture of isomers may be quantified using routine methods available to those skilled in the art, such as gas chromatography (optionally in combination with mass spectrometry) and nuclear magnetic resonance (NMR) spectroscopy. Examples of such methods are found in Encyclopedia of Analytical Science: 3 rd The methods can be found in standard handbooks in the art, such as the methods described in the Physics Letters, Vol. 13, No. 1, pp. 111-115, 1999, Edition, Eds. Paul Worsfold, Alan Townshend, Colin Poole, Manuel Miro, Elsevier (2019), which are incorporated herein by reference in their entirety. Those skilled in the art will appreciate that these methods may also be used to quantify the concentration of isomers in a mixture, such as an aqueous solution. The concentration of isomers in a mixture may be expressed using a number of quantitative units, such as molar concentration, molar concentration, mass percentage, parts per thousand (ppth), parts per million (ppm), and parts per billion (ppb). The interconversion of these units, as well as the calculation of the weight of an isomer in a given mixture based on the concentration values, are all well within the capabilities of those skilled in the art.

[0082] In some embodiments, R is selected from H (hydrogen), and C 1 ~C 4R is selected from alkyl, such as methyl, ethyl, n-propyl, or isopropyl. Preferably, R is methyl. The compound of formula (II) in which R is methyl may be referred to as hydroxyfarnesylacetone (HFA), which encompasses the respective compounds E,E-hydroxyfarnesylacetone (E,E-HFA), Z,E-hydroxyfarnesylacetone (Z,E-HFA), Z,Z-hydroxyfarnesylacetone (Z,Z-HFA), and E,Z-hydroxyfarnesylacetone (E,Z-HFA), as well as mixtures thereof. Among the isomers of hydroxyfarnesylacetone, E,Z-hydroxyfarnesylacetone is preferred.

[0083] Among the isomers of the compound represented by formula (IIa), the E,Z-isomer and the E,E-isomer are preferred, with the E,Z-isomer being more preferred.

[0084] Accordingly, in some embodiments, the mixture comprising a compound represented by Formula (II) and a compound represented by Formula (IIa) comprises any one of the following: i) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomer). ii) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomer). iii) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomer). iv) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomer). v) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomers) and compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomers). vi) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomers) and compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomers). vii) Any combination of i) to vi).

[0085] In some embodiments, the mixture comprising a compound represented by Formula (II) and a compound represented by Formula (IIa) comprises: - Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomers). - Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomers). - compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomers), as well as; - Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomers).

[0086] Such mixtures may optionally contain isomers of the compound of formula (II) and isomers of the compound of formula (IIa) in a specific ratio of E,Z-isomer of the compound of formula (II):E,E-isomer of the compound of formula (II):E,Z-isomer of the compound of formula (IIa):E,E-isomer of the compound of formula (IIa), such as, but not limited to, 37:9:29:16 or about 37:9:29:16, or 27:36:13:24 or about 27:36:13:24. Optionally, the mixtures contain the Z,E-isomer of the compound of formula (II), the Z,Z-isomer of the compound of formula (II), the Z,E-isomer of the compound of formula (IIa), and / or the Z,Z-isomer of the compound of formula (IIa).

[0087] Those skilled in the art will understand that in the context of this disclosure, not all of the compounds need to be converted to compounds of formula (I) upon "contacting with a compound of formula (II)". Similarly, not all of the compounds need to be converted to compounds of formula (Ia) upon "contacting with a compound of formula (IIa)". In some instances, reaction by-products (e.g., as described later herein) may be formed, or the compounds of formula (II) and / or formula (IIa) may not be fully converted. In other instances, in a mixture containing two or more isomers of a compound of formula (II), not all of the isomers are necessarily converted to compounds of formula (I). In other instances, in a mixture containing two or more isomers of a compound of formula (IIa), not all of the isomers are necessarily converted to compounds of formula (Ia). As another example, in a mixture containing a compound represented by formula (II) and a compound represented by formula (IIa), not all of the compound represented by formula (II) is necessarily converted to a compound represented by formula (I) and / or not all of the compound represented by formula (IIa) is necessarily converted to a compound represented by formula (Ia).

[0088] In some embodiments, not all of the compound represented by formula (II) is converted to the compound represented by formula (I) or to a reaction by-product, resulting in a product, such as a composition, that includes the compound represented by formula (II) and the compound represented by formula (I). In some embodiments, any unconverted compound represented by formula (II) in a product, such as a composition, may be isolated and / or purified from the product, such that the product is free of any compound represented by formula (II). In some embodiments, all of the compound represented by formula (II) is converted to the compound represented by formula (I) or to a reaction by-product.

[0089] In some embodiments, not all of the compound represented by formula (IIa) is converted to the compound represented by formula (Ia) or to a reaction by-product, resulting in a product, such as a composition, that includes the compound represented by formula (IIa) and the compound represented by formula (Ia). In some embodiments, any unconverted compound represented by formula (IIa) in a product, such as a composition, may be isolated and / or purified from the product, such that a product is obtained that does not include any compound represented by formula (IIa). In some embodiments, all of the compound represented by formula (IIa) is converted to the compound represented by formula (Ia) or to a reaction by-product.

[0090] In some embodiments, in a mixture containing a compound represented by formula (II) and a compound represented by formula (IIa), not all of the compound represented by formula (II) is converted to a compound represented by formula (I) or to a reaction by-product, and / or not all of the compound represented by formula (IIa) is converted to a compound represented by formula (Ia) or to a reaction by-product. In some embodiments, any unconverted compound represented by formula (II) and / or any unconverted compound represented by formula (IIa) in a product such as a composition may be isolated and / or purified from the product to obtain a product that does not contain any compound represented by formula (II) and / or any compound represented by formula (IIa). In some embodiments, all of the compound represented by formula (II) is converted to a compound represented by formula (I) or to a reaction by-product. In some embodiments, all of the compound represented by formula (IIa) is converted to a compound represented by formula (Ia) or to a reaction by-product.

[0091] Isolation and / or purification are discussed later in this specification. In embodiments where the compound of formula (II) and / or the compound of formula (IIa) corresponds to a mixture of isomers, the presence of various isomers may affect the conversion; for example, the reaction rate may be reduced.

[0092] Thus, the SHC enzymes described herein may be capable of converting the E,Z-isomer of a compound of formula (II) from a mixture of isomers of a compound of formula (II) to a compound of formula (I). The SHC enzymes described herein may be capable of converting the E,Z-isomer of a compound of formula (IIa) from a mixture of isomers of a compound of formula (IIa) to a compound of formula (Ia).

[0093] The SHC enzymes described herein may be capable of converting the E,Z-isomer of a compound of formula (II) from a mixture comprising an isomer of a compound of formula (II) and a compound of formula (IIa) to a compound of formula (I). The SHC enzymes described herein may be capable of converting the E,Z-isomer of a compound of formula (IIa) from a mixture comprising an isomer of a compound of formula (IIa) and a compound of formula (II) to a compound of formula (Ia).

[0094] The mixture may contain two isomers of the compound of formula (II), such as the E,Z-isomer and the E,E-isomer. The mixture may contain three isomers of the compound of formula (II), such as the E,Z-isomer, the E,E-isomer, and either the Z,E-isomer or the Z,Z-isomer. The mixture may contain four isomers of the compound of formula (II), namely the E,Z-isomer, the E,E-isomer, the Z,E-isomer, and the Z,Z-isomer. The presence of other isomers of the compound of formula (II) may also reduce the conversion ratio of the E,Z-isomer to the compound of formula (I). Without wishing to be bound by theory, a possible explanation may be that other isomers may compete with the E,Z-isomer of formula (II) for access to the SHC enzyme and thus act as competitive inhibitors and / or alternative substrates for the conversion of the E,Z-isomer of the compound of formula (II) to the compound of formula (I). Consequently, the reaction substrate may refer to an isomeric mixture of two to four isomers, preferably two isomers, of the compound of formula (IIa). In some embodiments, the reaction substrate comprises, consists essentially of, or consists of an isomeric mixture of the E,Z-isomer and the E,E-isomer of the compound of formula (II).

[0095] The mixture may contain two of the isomers of the compound represented by formula (IIa), for example the E,Z-isomer and the E,E-isomer. The mixture may contain three of the isomers of the compound represented by formula (IIa), for example the E,Z-isomer, the E,E-isomer, and either the Z,E-isomer or the Z,Z-isomer. The mixture may contain four isomers of the compound represented by formula (IIa), namely the E,Z-isomer, the E,E-isomer, the Z,E-isomer, and the Z,Z-isomer. Consequently, the reaction substrate may refer to an isomeric mixture of two to four isomers of the compound represented by formula (IIa), preferably two isomers. In some embodiments, the reaction substrate comprises, consists essentially of, or consists of an isomeric mixture of the E,Z-isomer and the E,E-isomer of the compound represented by formula (IIa).

[0096] The mixture may contain two isomers of the compound of formula (II), such as the E,Z-isomer and the E,E-isomer, and two isomers of the compound of formula (IIa), such as the E,Z-isomer and the E,E-isomer. The mixture may contain three isomers of the compound of formula (II), such as the E,Z-isomer, the E,E-isomer, and one of the Z,E-isomer or the Z,Z-isomer, and three isomers of the compound of formula (IIa), such as the E,Z-isomer, the E,E-isomer, and one of the Z,E-isomer or the Z,Z-isomer. The mixture may comprise the four isomers of the compound of formula (II), namely the E,Z-isomer, the E,E-isomer, the Z,E-isomer and the Z,Z-isomer, and the four isomers of the compound of formula (IIa), namely the E,Z-isomer, the E,E-isomer, the Z,E-isomer and the Z,Z-isomer.

[0097] Consequently, the reaction substrate may refer to an isomeric mixture of two to four isomers, preferably two isomers, of the compound represented by formula (II) and two to four isomers, preferably two isomers, of the compound represented by formula (IIa). In some embodiments, the reaction substrate comprises, consists essentially of, or consists of an isomeric mixture of the E,Z-isomer of the compound of formula (II), the E,E-isomer of the compound of formula (II), the E,Z-isomer of the compound of formula (IIa), and the E,E-isomer of the compound of formula (IIa).

[0098] Compounds of formula (II) and compounds of formula (IIa) may be synthesized following the general procedures outlined by Fujiwara et al. (Tetrahedron Letters, 1995 Vol 36(46), 8435-8438), which is incorporated herein by reference in its entirety. Additional general procedures are described in GB2108985.9, which is incorporated herein by reference in its entirety.

[0099] Alternatively, the compound of formula (II) may be obtained as briefly illustrated in FIG. 1, where R is optionally selected from H (hydrogen) and C alkyl groups such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4 is selected from alkyl.

[0100] Compounds represented by formula (I) and formula (Ia) As used herein, "making a compound of formula (I)" and "making a compound of formula (Ia)" may also be referred to as "producing" or "obtaining" the respective compound. It may also refer to "producing" or "obtaining" a mixture that comprises, consists essentially of, or consists of the respective compound.

[0101] The compounds of formula (I) and (Ia) contain a number of chiral carbon atoms. Thus, one or more isomers of the compounds of formula (I) and formula (Ia), such as, for example, enantiomers and diastereomers, may occur. In addition to the compounds of formula (I), the products produced by the methods described herein may contain one or more other isomers of the compounds of formula (I). In addition to the compounds of formula (Ia), the products produced by the methods described herein may contain one or more other isomers of the compounds of formula (Ia). In this regard, these other isomers may represent by-products of the enzymatic conversion. The isomers obtained by the methods described herein may depend on the isomers of the compounds of formula (II) and / or formula (IIa) that are contacted with the SHC enzyme as described herein.

[0102] As a non-limiting example, contacting a compound of formula (II) with an SHC enzyme as described herein produces a compound of formula (IV): [ka] Formula (IV) This may result in the preparation of a compound represented by the formula:

[0103] In some embodiments, R is selected from H (hydrogen), and C 1 ~C 4 It is selected from alkyl, such as methyl, ethyl, n-propyl, or isopropyl, and preferably, R is methyl.

[0104] The compound of formula (IV) where R is methyl is also known as (-)-epi-8-amberketal. The compound of formula (I) where R is methyl is also known as (+)-amberketal. As a result, in some embodiments, compounds of formula (I) and one or more other isomers of compounds of formula (I), including but not limited to compounds of formula (IV), are made, optionally wherein R is H, and C is an isomer such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4 Thus, products such as the compounds described later herein may include compounds of formula (I) and, optionally, one or more other isomers of compounds of formula (I), including, but not limited to, compounds of formula (IV), where R is H (hydrogen), and C alkyl, such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4 is selected from alkyl.

[0105] A preferred compound of formula (Ia) is represented by formula (V): [ka] Formula (V) It has a configuration represented by the following formula:

[0106] In some embodiments, R is selected from H (hydrogen), and C 1 ~C 4 R is selected from alkyl, such as methyl, ethyl, n-propyl, or isopropyl, and preferably R is methyl. Consequently, in some embodiments, the methods described herein result in the production of a compound represented by formula (V). Thus, products such as those described later herein may include a compound represented by formula (V) and, optionally, one or more other isomers of a compound represented by formula (Ia), optionally wherein R is H (hydrogen) and C is an isomer such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4is selected from alkyl.

[0107] In some embodiments, the methods described herein result in products, such as compounds subsequently described herein, which may include compounds represented by formula (I) and compounds represented by formula (V), optionally wherein R is H (hydrogen), and C, such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4 Optionally, the product may contain one or more other isomers of the compound of formula (I), such as, but not limited to, the compound of formula (IV), and / or one or more other isomers of the compound of formula (Ia).

[0108] In some embodiments, the ratio of the compound of formula (I) to all other combined isomers of the compound of formula (I) made by a method or contained in a product, such as a composition, as described herein is equal to or greater than 50:50, or about 50:50. In some embodiments, the ratio is equal to or greater than 55:45, or about 55:45. In some embodiments, the ratio is equal to or greater than 60:40, or about 60:40. In some embodiments, the ratio is equal to or greater than 65:35, or about 65:35. In some embodiments, the ratio is equal to or greater than 70:30, or about 70:30. In some embodiments, the ratio is equal to or greater than 75:25, or about 75:25. In some embodiments, the ratio is equal to or greater than 80:20, or about 80:20. In some embodiments, the ratio is equal to or greater than, or about 85:15. In some embodiments, the ratio is equal to or greater than, or about 90:10. In some embodiments, the ratio is equal to or greater than, or about 95:5. In some embodiments, the ratio is equal to or greater than, or about 99:1.

[0109] In some embodiments, the ratio of the compound of formula (V) to all other combined isomers of the compound of formula (Ia) made by a method or contained in a product, such as a composition, as described herein is equal to or greater than 50:50, or about 50:50. In some embodiments, the ratio is equal to or greater than 55:45, or about 55:45. In some embodiments, the ratio is equal to or greater than 60:40, or about 60:40. In some embodiments, the ratio is equal to or greater than 65:35, or about 65:35. In some embodiments, the ratio is equal to or greater than 70:30, or about 70:30. In some embodiments, the ratio is equal to or greater than 75:25, or about 75:25. In some embodiments, the ratio is equal to or greater than 80:20, or about 80:20. In some embodiments, the ratio is equal to or greater than, or about 85:15. In some embodiments, the ratio is equal to or greater than, or about 90:10. In some embodiments, the ratio is equal to or greater than, or about 95:5. In some embodiments, the ratio is equal to or greater than, or about 99:1.

[0110] In some embodiments, the methods described herein produce only compounds of formula (I) and not other isomers of compounds of formula (I), such as compounds of formula (IV), optionally wherein R is H, and C is an isomer such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4 In some embodiments, the methods described herein produce only compounds of formula (V) and not other isomers of compounds of formula (Ia), optionally wherein R is selected from H (hydrogen) and C alkyl, such as methyl, ethyl, n-propyl, or isopropyl.1 ~C 4 is selected from alkyl.

[0111] In some embodiments, any isomer other than the compound represented by formula (I) and / or the compound represented by formula (V) may be separated from the product, such as the composition made by the method described herein, to obtain a product that does not contain any other isomers; for example, the compound represented by formula (IV), optionally where R is H (hydrogen), methyl, or ethyl, is separated from the product and is no longer present in the product. In other words, the composition as described herein may be, for example, a composition that includes 100 wt% of the compound represented by formula (I) and does not include other isomers of this compound (alternatively referred to as a 100:0 ratio herein). Similarly, the composition as described herein may be, for example, a composition that includes 100 wt% of the compound represented by formula (V) and does not include other isomers of the compound represented by formula (Ia). The composition as described herein may be, for example, a mixture that includes, consists essentially of, or consists of, preferably includes, the compound represented by formula (I) and the compound represented by formula (V). Separation methods are known to those skilled in the art and have been previously discussed herein.

[0112] In some embodiments, the ratio of the compound of formula (I) to all other combined isomers of the compound of formula (I) made by a method or contained in a product, such as a composition, as described herein is equal to or less than 99:1, or about 99:1. In some embodiments, the ratio is equal to or less than 98:2, or about 98:2. In some embodiments, the ratio is equal to or less than 97:3, or about 97:3. In some embodiments, the ratio is equal to or less than 96:4, or about 96:4. In some embodiments, the ratio is equal to or less than 95:5, or about 95:5.

[0113] In some embodiments, the ratio of the compound of formula (I) to all other isomers of the compound of formula (I) made by a method or included in a product, such as a composition, as described herein, may be from 50:50 to 100:0, or from about 50:50 to about 100:0, or from about 60:40 to 99:1, or from about 60:40 to about 99:1, or from about 70:30 to 98:2, or from about 70:30 to about 98:2, or from about 80:20 to about 97:3, or from about 90:10 to 97:3, or from about 90:10 to about 97:3.

[0114] In some embodiments, the ratio of the compound of formula (V) to all other combined isomers of the compound of formula (Ia) made by a method or contained in a product, such as a composition, as described herein is equal to or less than 99:1, or about 99:1. In some embodiments, the ratio is equal to or less than 98:2, or about 98:2. In some embodiments, the ratio is equal to or less than 97:3, or about 97:3. In some embodiments, the ratio is equal to or less than 96:4, or about 96:4. In some embodiments, the ratio is equal to or less than 95:5, or about 95:5.

[0115] In some embodiments, the ratio of the compound of Formula (V) to all other isomers of the compound of Formula (Ia) made by a method or included in a product, such as a composition, as described herein, may be from 50:50 to 100:0, or from about 50:50 to about 100:0, or from about 60:40 to 99:1, or from about 60:40 to about 99:1, or from about 70:30 to 98:2, or from about 70:30 to about 98:2, or from about 80:20 to about 97:3, or from about 90:10 to 97:3, or from about 90:10 to about 97:3.

[0116] In some embodiments, the ratio of the compound of formula (I) to the compound of formula (Ia) (such as the compound of formula (V)) made by a method or contained in a product, such as a composition, as described herein is equal to or less than 99:1 or about 99:1. In some embodiments, the ratio is equal to or less than 98:2 or about 98:2. In some embodiments, the ratio is equal to or less than 97:3 or about 97:3. In some embodiments, the ratio is equal to or less than 96:4 or about 96:4. In some embodiments, the ratio is equal to or less than 95:5 or about 95:5. In some embodiments, the ratio is equal to or less than 94:6 or about 94:6. In some embodiments, the ratio is equal to or less than 93:7 or about 93:7. In some embodiments, the ratio is equal to or less than 92:8, or about 92:8. In some embodiments, the ratio is equal to or less than 91:9, or about 91:9. In some embodiments, the ratio is equal to or less than 90:10, or about 90:10. In some embodiments, the ratio is equal to or less than 85:15, or about 85:15. In some embodiments, the ratio is equal to or less than 80:20, or about 80:20. In some embodiments, the ratio is equal to or less than 75:25, or about 75:25. In some embodiments, the ratio is equal to or less than 70:30, or about 70:30. In some embodiments, the ratio is equal to or less than 65:35, or about 65:35. In some embodiments, the ratio is equal to or less than, or about 60:40. In some embodiments, the ratio is equal to or less than, or about 55:45.In some embodiments, the ratio is equal to or less than 50:50, or about 50:50. In some embodiments, the ratio is equal to or less than 49:51, or about 49:51. In some embodiments, the ratio is equal to or less than 49:51, or about 49:51. In some embodiments, the ratio is equal to or less than 48:52, or about 48:52. In some embodiments, the ratio is equal to or less than 47:53, or about 47:53. In some embodiments, the ratio is equal to or less than 46:54, or about 46:54. In some embodiments, the ratio is equal to or less than 45:55, or about 45:55. In some embodiments, the ratio is equal to or less than 44:56, or about 44:56. In some embodiments, the ratio is equal to or less than, or about 43:57. In some embodiments, the ratio is equal to or less than, or about 42:58. In some embodiments, the ratio is equal to or less than, or about 41:59. In some embodiments, the ratio is equal to or less than, or about 40:60.

[0117] In some embodiments, the ratio of the compound of Formula (I) to the compound of Formula (Ia) (such as the compound of Formula (V)) made by a method or included in a product, such as a composition, as described herein, may be from 50:50 to 100:0, or from about 50:50 to about 100:0, or from about 60:40 to 99:1, or from about 60:40 to about 99:1, or from about 70:30 to 98:2, or from about 70:30 to about 98:2, or from about 80:20 to about 97:3, or from 90:10 to 97:3, or from about 90:10 to about 97:3, or from 93:7 to 97:3, or from about 97:3 to about 97:3.

[0118] The ratio of a given isomer of a compound of formula (I) and / or a compound of formula (Ia), such as a compound of formula (V), to one or more other isomers of the respective compound in a mixture of isomers, as well as the amount and concentration of the isomers, may be determined using routine methods available to those skilled in the art, such as gas chromatography (optionally on a chiral column) or NMR spectroscopy (optionally in the presence of a shift reagent), as previously discussed herein. The same methods may be used to determine the ratio of a given isomer of a compound of formula (I) to another isomer of a compound of formula (V) and / or a compound of formula (Ia).

[0119] The compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) produced by the method described herein may, for example, be included in a mixture. The compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) produced by the method described herein may, for example, be in solid form, preferably in amorphous or crystalline form. The compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) produced by the method described herein may, for example, be in a solid phase in a reaction mixture.

[0120] Such a form may be advantageous, since the presence of the compound in solid form / solid phase may simplify downstream processing after the preparation of the compound. As a non-limiting example, when a host cell expressing an SHC enzyme as described herein is used as a biocatalyst and the compound represented by formula (I) and / or the compound represented by formula (Ia) (such as the compound represented by formula (V)) is prepared in solid form (such as amorphous or crystalline form), the compound may be easily separated from the reaction mixture (which may also correspond to a cell culture as described later herein) via simple techniques such as filtration and / or centrifugation. Optionally, the obtained compound represented by formula (I) and / or the compound represented by formula (Ia) (such as the compound represented by formula (V)) may be further isolated and / or purified as described herein, but in any case, less material (e.g. solvent) and / or less energy input is required compared to the case where the compound represented by formula (I) and / or the compound represented by formula (Ia) (such as the compound represented by formula (V)) was not prepared in solid form (such as amorphous or crystalline form).

[0121] The compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) may be isolated and / or purified after it is made. As a result, in some embodiments, the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) is isolated. Optionally, the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) is purified. The term "isolation" as used herein refers to the separation (alternatively referred to herein as "extraction") of a compound such as the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) from its accompanying components. The degree of isolation or purity of a compound may be measured by any method commonly used in the art, such as gas chromatography (GC), chromatographic methods (such as HPLC), or NMR spectroscopy, all of which are known to those skilled in the art and are collected in standard handbooks such as the Encyclopedia of Analytical Science: 3rd Edition (see above).

[0122] Isolation may be accomplished by any method commonly used in the art. Examples of suitable methods include steam extraction, distillation, or organic solvent extraction using a water-immiscible solvent, which separates the reaction products and unreacted substrates from the biocatalyst that remains in the aqueous phase, followed by evaporation of the solvent to obtain the crude reaction product as determined by gas chromatographic analysis. These methods are known to those skilled in the art and are collected in standard handbooks such as the Encyclopedia of Analytical Science: 3rd Edition (see above).

[0123] As an example, the produced compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) may be extracted from the entire reaction mixture using an organic solvent such as a water-immiscible solvent (e.g., toluene). Alternatively, the produced compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) may be extracted from the solid-phase reaction mixture (e.g., obtained by centrifugation or filtration) using a water-miscible solvent (e.g., ethanol) or a water-immiscible solvent (e.g., toluene). As a further example, the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) may be present in the solid phase as a crystal or in an amorphous form, as previously discussed herein, and may be separated from the remaining solid phase (cell material or its debris) and from the liquid phase using filtration. As a further example, at temperatures above the melting point of the compound represented by formula (I) and / or the compound represented by formula (Ia) (such as the compound represented by formula (V)), the compound represented by formula (I) and / or the compound represented by formula (Ia) (such as the compound represented by formula (V)) may form an oil layer on top of the aqueous phase, which can be removed and collected. To ensure complete recovery of the compounds after the oil layer is removed, an organic solvent may be added to the aqueous phase containing the biomass to extract any remaining compounds represented by formula (I) (e.g., (+)-amberketal) and / or the compound represented by formula (Ia) (such as the compound represented by formula (V)). The organic layer may be combined with the oil layer, after which the whole is further processed to isolate and purify the compound represented by formula (I) and / or the compound represented by formula (Ia) (such as the compound represented by formula (V)). The compound of formula (I) and / or the compound of formula (Ia), such as the compound of formula (V), may further be selectively crystallized from the final product to remove by-products and any unreacted compound of formula (II) and / or the compound of formula (IIa).

[0124] Purification may be achieved by any method commonly used in the art, which methods are known to those skilled in the art and are summarized in standard handbooks such as the Encyclopedia of Analytical Science: 3rd Edition (see above). Further examples of isolation and purification are provided in the experimental section herein.

[0125] The term "selective crystallization" refers to a process step in which the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) is crystallized from the solvent to such an extent that the isolated crystalline material contains only the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) (or if it contains any by-products, then these are present only in olfactory acceptable amounts). The compound of formula (I) is free or substantially free of by-products, such as, for example, the compound of formula (III) or (IIIa) (described later herein). The compound of formula (Ia), preferably the compound of formula (V), is free or substantially free of by-products, such as, for example, the compound of formula (VI) or (VIa) (described later herein). The selective crystallization step may use a water-miscible solvent such as ethanol or the like. The selective crystallization of the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) may be influenced by the presence of the unreacted compound of formula (II) and / or the unreacted compound of formula (IIa) and also by the ratio of the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) to other detectable by-products. Even if only 10% conversion of the compound of formula (II) to the compound of formula (I) is obtained, selective crystallization of the compound produced may still be possible. Similarly, even if only 10% conversion of the compound of formula (IIa) to the compound of formula (Ia) (preferably to the compound of formula (V)) is obtained, selective crystallization of the compound produced may still be possible.

[0126] The purity of the resulting final compound of formula (I) and / or of the final compound of formula (Ia), such as the compound of formula (V), may be determined using routine gas chromatography (GC) techniques. Similar techniques may also be applied to mixtures comprising a compound of formula (I) and a compound of formula (Ia), such as the compound of formula (V).

[0127] The olfactive purity of a product comprising a compound of formula (I), a compound of formula (Ia) (such as a compound of formula (V)), or a mixture comprising a compound of formula (I) and a compound of formula (Ia) (such as a compound of formula (V)) may be determined by testing the crystalline material or a solution of the crystalline material in ethanol. A compound of formula (I), a compound of formula (Ia) (such as a compound of formula (V)), or a mixture comprising a compound of formula (I) and a compound of formula (Ia) (such as a compound of formula (V)) may be tested for its olfactory purity, quality, and its sensory profile by a trained olfactory expert or a panel of trained olfactory experts against a commercial reference for the compound of formula (I), a commercial reference for the compound of formula (Ia) (such as a compound of formula (V)), or a commercial reference mixture comprising the compound of formula (I) and the compound of formula (Ia) (such as a compound of formula (V)). The product may also be tested by trained olfactory experts in applied research to determine whether the material meets specifications regarding its olfactory profile, thus providing an olfactorily acceptable product.

[0128] The term "olfactorily pure", when used in reference to a product of the present disclosure, is intended to mean that a compound represented by formula (I), a compound represented by formula (Ia) (such as a compound represented by formula (V)), or a mixture comprising a compound represented by formula (I) and a compound represented by formula (Ia) (such as a compound represented by formula (V)) product is free of compounds (II), (IIa), (III), (IIIa), (IV), (IVa), (V), (Va), (VI), and / or (VIa), and / or any other materials found in the reaction mixture, or, if such compounds and / or materials are to be present, they are present in an amount that is olfactorily acceptable as that term is defined herein.

[0129] In an embodiment of the present disclosure, a mixture comprising a compound represented by formula (I), a compound represented by formula (Ia) (such as a compound represented by formula (V)), or a product of a compound represented by formula (I) and a compound represented by formula (Ia) (such as a compound represented by formula (V)) in olfactorily pure form contains less than 5% by weight of any of compounds (II), (IIa), (III), (IIIa), (IV), (IVa), (V), (Va), (VI), and / or (VIa), and / or any other materials found in the reaction mixture.

[0130] In more specific embodiments, a mixture comprising a compound of formula (I), a compound of formula (Ia) (such as a compound of formula (V)), or a product of a compound of formula (I) and a compound of formula (Ia) (such as a compound of formula (V)) in olfactorily pure form contains less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.05% by weight of each of compounds (II), (IIa), (III), (IIIa), (IV), (IVa), (V), (Va), (VI), and / or (VIa), and / or any other materials found in the reaction mixture.

[0131] In more specific embodiments, a mixture comprising a compound of formula (I), a compound of formula (Ia) (such as a compound of formula (V)), or a product of a compound of formula (I) and a compound of formula (Ia) (such as a compound of formula (V)) in olfactorily pure form contains less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.05% by weight of each of compounds (II), (IIa), (III), (IIIa), (IV), (IVa), (VI), and / or (VIa), and / or any other materials found in the reaction mixture.

[0132] Non-limiting examples of water-miscible and water-immiscible organic solvents suitable for use in the extraction and / or selective crystallization of the compound of Formula (I) and / or the compound of Formula (Ia) (such as the compound of Formula (V)) include aliphatic hydrocarbons, preferably having 5 to 8 carbon atoms such as pentane, cyclopentane, cyclohexane, heptane, octane, or cyclooctane, aromatic hydrocarbons such as toluene, xylene, chlorobenzene, or dichlorobenzene, aliphatic acyclic and cyclic ethers or alcohols, preferably having 4 to 8 carbon atoms such as ethanol, isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran, methyl tetrahydrofuran, or esters such as ethyl acetate or n-butyl acetate, or ketones such as methyl isobutyl ketone, or mixtures thereof. Preferred solvents are heptane, methyl tert-butyl ether (also known as MTBE, tert-butyl methyl ether, tertiary butyl methyl ether, and tBME), diisopropyl ether, tetrahydrofuran, methyltetrahydrofuran, ethyl acetate, and / or mixtures thereof. Preferably, a water-miscible solvent such as ethanol is used for the extraction of the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) from the solid phase of the reaction mixture. The use of ethanol may be advantageous because it is easy to handle, non-toxic, environmentally friendly, and can be produced using renewable raw materials.

[0133] The term "% purity" as used herein refers to the percentage of the compound in the material that is the desired compound in the material (e.g., expressed by the percentage ratio of the mass of the desired compound to the mass of the total material).In some embodiments, the compound represented by formula (I) (e.g., (+)-amberketal) is isolated from the obtained crude product and purified to a purity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0134] In some embodiments, the compound of formula (Ia), preferably the compound of formula (V), is isolated from the resulting crude product and purified to a purity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0135] In some embodiments, a product comprising a compound represented by formula (I) (e.g., (+)-amberketal) and a compound represented by formula (Ia) (e.g., a compound represented by formula (V)) is isolated from the resulting crude product and purified to a purity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0136] In some embodiments, the concentration of the compound of formula (Ia) and / or the compound of formula (Ia) (such as the compound of formula (V)) in the reaction mixture or culture broth obtained by the methods described herein is from 1 mg / L to 20000 mg / L (20 g / L), or from about 1 mg / L to about 20000 mg / L or more, for example, from 20 g / L to 200 g / L, or from about 20 g / L to about 200 g / L, or from 100 g / L to 500 g / L, or from about 100 g / L to about 500 g / L, or from 150 g / L to about 200 g / L. to 500 g / L, or from about 150 g / L to about 500 g / L, or from 250 g / L to 500 g / L, or from about 250 g / L to about 500 g / L, or from 300 g / L to 500 g / L, or from about 300 g / L to about 500 g / L, or from 350 g / L to 500 g / L, or from about 350 g / L to about 500 g / L, or from 400 g / L to 500 g / L, or from 450 g / L to 500 g / L, or from about 450 g / L to about 500 g / L. Exemplary concentration values ​​are 1 mg / L or more, 20 g / L or more, 50 g / L or more, 100 g / L or more, 150 g / L or more, 200 g / L or more, 250 g / L or more, 300 g / L or more, 350 g / L or more, 400 g / L or more, or 450 g / L or more.

[0137] Compounds represented by formulae (III) and (VI) In some embodiments, the compound of formula (III): [ka] Formula (III) In some embodiments, R is selected from H (hydrogen) and C 1 ~C 4R is selected from alkyl, such as methyl, ethyl, n-propyl, or isopropyl, and preferably R is methyl. For example, the compound represented by formula (III) can be represented by formula (IIIa): [ka] Formula (IIIa) wherein R is H (hydrogen) and C alkyl groups such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4 alkyl, preferably wherein R is methyl.

[0138] In some embodiments, the compound of formula (VI): [ka] Formula (VI) In some embodiments, R is selected from H (hydrogen) and C 1 ~C 4 R is selected from alkyl, such as methyl, ethyl, n-propyl, or isopropyl, and preferably R is methyl. For example, the compound represented by formula (VI) can be represented by formula (VIa): [ka] Formula (VIa) wherein R is H (hydrogen) and C alkyl groups such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4 alkyl, preferably wherein R is methyl.

[0139] One of skill in the art will appreciate that the production of a particular by-product, such as a compound of Formula (III), a compound of Formula (IIIa), a compound of Formula (VI), and / or a compound of Formula (VIa), may depend on the particular substrate used (e.g., a compound of Formula (II), a compound of Formula (IIa), or a mixture comprising a compound of Formula (II) and a compound of Formula (IIa)), as well as the biocatalyst (as described herein) and / or bioconversion reaction conditions used.

[0140] The methods described herein may, for example, produce one or more isomers of a compound represented by formula (III) and / or one or more isomers of a compound represented by formula (VI). Products, such as compositions described herein, may include one or more isomers of a compound represented by formula (III) and / or one or more isomers of a compound represented by formula (VI). Accordingly, in some embodiments, a compound represented by formula (III) having a configuration represented by formula (IIIa) and / or a compound represented by formula (VI) having a configuration represented by formula (VIa) (optionally, where R is H, and C-alkyl groups such as methyl, ethyl, n-propyl, or isopropyl) are prepared. 1 ~C 4 alkyl) is produced as a by-product. In some embodiments, a product, such as a composition, comprises a compound represented by formula (III) having a configuration represented by formula (IIIa). In some embodiments, a product, such as a composition, comprises a compound represented by formula (VI) having a configuration represented by formula (VIa). In some embodiments, the only compound represented by formula (III) made by or in the product of the methods described herein is a compound having a configuration represented by formula (IIIa). In some embodiments, the only compound represented by formula (VI) made by or in the product of the methods described herein is a compound having a configuration represented by formula (VIa).

[0141] In some embodiments, at least 50 wt% or about 50 wt% of the compound represented by formula (III) has the configuration shown in formula (IIIa). In some embodiments, at least 50 wt% or about 50 wt% of the compound represented by formula (VI) has the configuration shown in formula (VIa). For example, at least 60 wt% or about 60 wt%, at least 70 wt% or about 70 wt%, at least 80 wt% or about 80 wt%, or at least 90 wt% or about 90 wt% of the compound represented by formula (III) may have the configuration shown in formula (IIIa). For example, at least 60 wt% or about 60 wt%, at least 70 wt% or about 70 wt%, at least 80 wt% or about 80 wt%, or at least 90 wt% or about 90 wt% of the compound represented by formula (VI) may have the configuration shown in formula (VIa). In some embodiments, the compound having the configuration shown in formula (IIIa) is the only isomer of the compound represented by formula (III) made or contained in the product, i.e., 100 wt% of the compounds represented by formula (III) have the configuration shown in formula (IIIa). In some embodiments, the compound having the configuration shown in formula (IIIa) may be less than or equal to 99 wt% or about 99 wt%, less than or equal to 95 wt%, less than or equal to 95 wt%, less than or equal to 90 wt%, less than or equal to 85 wt%, less than or equal to 80 wt%, or less than or equal to 75 wt% or about 75 wt% of the compounds represented by formula (III). In some embodiments, the compound having the configuration shown in formula (VIa) is the only isomer of the compound represented by formula (VI) made or contained in a product, i.e., 100 wt % of the compound represented by formula (VI) has the configuration shown in formula (VIa).In some embodiments, the compound having the configuration shown in Formula (VIa) may be less than or equal to 99 wt% or about 99 wt%, less than or equal to 95 wt%, less than or equal to 95 wt%, less than or equal to 90 wt%, less than or equal to 85 wt%, less than or equal to 80 wt%, or less than or equal to 75 wt% or about 75 wt% of the compound represented by Formula (VI).

[0142] In some embodiments, 50wt% to 100wt%, or about 50wt% to about 100wt%, or 60wt% to 99wt%, or about 60wt% to about 99wt%, or 70wt% to 95wt%, or about 70wt% to about 95wt% of the compound represented by formula (III) has the configuration represented by formula (IIIa). In some embodiments, 50wt% to 100wt%, or about 50wt% to about 100wt%, or 60wt% to 99wt%, or about 60wt% to about 99wt%, or 70wt% to 95wt%, or about 70wt% to about 95wt% of the compound represented by formula (VI) has the configuration represented by formula (VIa).

[0143] Determining the ratios, amounts, and concentrations of the different isomers of the compound represented by formula (III) and / or the different isomers of the compound represented by formula (VI) in a mixture may be carried out by any of the methods previously discussed herein.

[0144] Suitable reaction conditions for the methods described herein are discussed later in this specification, and examples are further provided in the experimental section. Additional examples of suitable reaction conditions may be found in WO2021 / 209482, which is incorporated herein by reference in its entirety.

[0145] Products Obtained by the Methods Described Herein In certain aspects, products, such as compositions, made by the methods described herein are provided. As used herein, a "made product" may also be referred to as being "produced," "obtained by," or "obtainable by" the methods described herein.

[0146] In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (I) and a compound represented by formula (VI). In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (I) and a compound represented by formula (III). The composition may comprise one or more isomers of formula (III), such as a compound having a configuration represented by formula (IIIa). The composition may further comprise one or more isomers of formula (I), such as a compound represented by formula (IV). The composition may further comprise one or more isomers of a compound represented by formula (II), such as an unconverted or unreacted amount of an isomer of a compound represented by formula (II).

[0147] In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (I), a compound represented by formula (IV), and a compound represented by formula (III). In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (I), a compound represented by formula (IV), and a compound represented by formula (IIIa). In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (I) and a compound represented by formula (IIIa).

[0148] In some embodiments, the composition comprises, consists essentially of, or consists of a compound of formula (I) and one or more isomers of the compound of formula (I), such as a compound of formula (IV). The composition may further comprise, for example, a compound of formula (III), such as a compound of formula (IIIa). The composition may further comprise one or more isomers of a compound of formula (II), such as an unconverted or unreacted amount of an isomer of a compound of formula (II).

[0149] In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (Ia), preferably a compound represented by formula (V). In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (Ia), preferably a compound represented by formula (V), and a compound represented by formula (IV). The composition may comprise one or more isomers represented by formula (VI), such as a compound having a configuration represented by formula (VIa). The composition may further comprise one or more isomers represented by formula (Ia). The composition may further comprise one or more isomers of a compound represented by formula (IIa), such as an unconverted or unreacted amount of an isomer of a compound represented by formula (IIa).

[0150] In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (I) and a compound represented by formula (Ia). In some embodiments, the composition comprises, consists essentially of, or consists of a compound represented by formula (I) and a compound represented by formula (V). The composition may further comprise a compound represented by formula (IV). The composition may further comprise an isomer of a compound represented by formula (Ia). The composition may further comprise a compound represented by formula (III), e.g., a compound represented by formula (IIIa). The composition may further comprise a compound represented by formula (VI), e.g., a compound represented by formula (VIa). The composition may further comprise one or more isomers of a compound represented by formula (II), e.g., an unconverted or unreacted amount of an isomer of a compound represented by formula (IIa). The composition may further comprise one or more isomers of a compound represented by formula (IIa), e.g., an unconverted or unreacted amount of an isomer of a compound represented by formula (IIa). In some embodiments, the composition does not include a compound represented by formula (III). In some embodiments, the composition does not include a compound represented by formula (IIIa). In some embodiments, the composition does not include a compound represented by formula (VI). In some embodiments, the composition does not include a compound represented by formula (VIa).

[0151] In some embodiments, in the compounds of formula (I) and its isomers (e.g., compounds of formula (IV)), compounds of formula (Ia) and its isomers (e.g., compounds of formula (V)), compounds of formula (II) and its isomers, compounds of formula (IIa) and its isomers, compounds of formula (III) and its isomers (e.g., compounds of formula (IIIa)), and compounds of formula (VI) and its isomers (e.g., compounds of formula (VIa)) present in the compositions described herein, R is H (hydrogen) and C, such as methyl, ethyl, n-propyl, or isopropyl. 1 ~C 4 alkyl, preferably R is methyl.

[0152] In some embodiments, the ratio of the compound of formula (I) to the compound of formula (III) (e.g., the compound of formula (IIIa)) in the compositions described herein may be from 60:40 to 99:1, or from about 60:40 to about 99:1. In some embodiments, the ratio of the compound of formula (I) to the compound of formula (III) in the compositions described herein may be from 65:35 to 99:1, or from about 65:35 to about 99:1, from 70:30 to 99:1, or from about 70:30 to about 99:1, from 75:25 to 99:1, or from about 75:25 to about 99:1, from 80:20 to 99:1, or from about 80:20 to about 99:1, from 85:15 to 99:1, or from about 85:15 to about 99:1, or from 90:10 to 99:1. or from about 90:10 to about 99:1, or from about 95:5 to 99:1, or from about 95:5 to about 99:1, or from about 65:35 to 98:2, or from about 65:35 to about 98:2, or from about 70:30 to 97:3, or from about 70:30 to about 97:3, or from about 75:25 to 96:4, or from about 75:25 to about 96:4, or from about 80:20 to 95:5, or from about 80:20 to about 95:5, or from about 85:15 to 90:10, or from about 85:15 to about 90:10.

[0153] In some embodiments, the ratio of the compound of Formula (I) to the compound of Formula (II) in a composition, such as a crude product, described herein may be from 90:10 to 100:0, or from about 90:10 to about 100:0. In some embodiments, the ratio of the compound of formula (I) to the compound of formula (II) in a composition, such as a crude product, described herein may be from 92:8 to 100:0, or from about 92:8 to about 100:0, from 94:6 to 100:0, or from about 94:6 to about 100:0, from 95:5 to 100:0, or from about 95:5 to about 100:0, from 96:4 to 99.5:0.5, or from about 96:4 to about 99.5:0.5, from 97:3 to 99:1, or from about 97:3 to about 99:1, or from 98:2 to 99:1, or from about 98:2 to about 99:1.

[0154] In some embodiments, the ratio of the compound represented by formula (Ia) (preferably a compound represented by formula (V)) to the compound represented by formula (VI) (e.g., a compound represented by formula (VIa)) in the compositions described herein may be from 60:40 to 99:1, or from about 60:40 to about 99:1. In some embodiments, the ratio of the compound represented by formula (Ia), preferably a compound represented by formula (V) to the compound represented by formula (VI) in the compositions described herein may be from 65:35 to 99:1, or from about 65:35 to about 99:1, from 70:30 to 99:1, or from about 70:30 to about 99:1, from 75:25 to 99:1, or from about 75:25 to about 99:1, from 80:20 to 99:1, or from about 80:20 to about 99:1, or from 85:15 to 99:1, or from about 90:10 to about 99: or from about 95:5 to about 99:1; 95:5 to 99:1 or from about 95:5 to about 99:1; 65:35 to 98:2 or from about 65:35 to about 98:2; 70:30 to 97:3 or from about 70:30 to about 97:3; 75:25 to 96:4 or from about 75:25 to about 96:4; 80:20 to 95:5 or from about 80:20 to about 95:5; 85:15 to 90:10 or from about 85:15 to about 90:10.

[0155] In some embodiments, the ratio of the compound represented by Formula (Ia) (preferably the compound represented by Formula (V)) to the compound represented by Formula (IIa) in a composition, such as a crude product, described herein may be from 90:10 to 100:0, or from about 90:10 to about 100:0. In some embodiments, the ratio of the compound of formula (I), preferably the compound of formula (V), to the compound of formula (II) in a composition, such as a crude product, described herein may be from 92:8 to 100:0, or from about 92:8 to about 100:0, from 94:6 to 100:0, or from about 94:6 to about 100:0, from 95:5 to 100:0, or from about 95:5 to about 100:0, from 96:4 to 99.5:0.5, or from about 96:4 to about 99.5:0.5, from 97:3 to 99:1, or from about 97:3 to about 99:1, or from 98:2 to 99:1, or from about 98:2 to about 99:1.

[0156] The determination of the ratio, amount and concentration of the compound of formula (I) and its isomers, such as the compound of formula (IV), the compound of formula (Ia) and its isomers, such as the compound of formula (V), the compound of formula (II) and its isomers, the compound of formula (IIa) and its isomers, such as the compound of formula (III) and its isomers, such as the compound of formula (IIIa), and the compound of formula (VI) and its isomers, such as the compound of formula (VI), in the composition may be carried out by any of the methods previously discussed herein.

[0157] In some embodiments, the compositions obtained or obtainable by the methods described herein comprise the compounds of Formula (I) and compounds of Formula (Ia) (such as the compounds of Formula (V)) in solid form, preferably in amorphous or crystalline form.

[0158] Fragrance Composition Products such as compositions made by the methods described herein may be included in a fragrance composition. Accordingly, there is further provided a use of a composition as described herein for the manufacture of a fragrance composition. In some embodiments, the fragrance composition comprises a compound represented by formula (I). Optionally, the fragrance composition comprises an isomer of a compound represented by formula (I), such as a compound represented by formula (IV). In some embodiments, the fragrance composition comprises a compound represented by formula (Ia), preferably a compound represented by formula (V). In some embodiments, the fragrance composition comprises a compound represented by formula (I) and a compound represented by formula (Ia). In some embodiments, the composition comprises a compound represented by formula (I) and a compound represented by formula (V). Optionally, the fragrance composition comprises an isomer of a compound represented by formula (Ia).

[0159] "Fragrance composition" as used herein includes any composition comprising a compound of formula (I), optionally one or more isomers of the compound of formula (I), such as the compound of formula (IV), and a substrate. It further includes any composition comprising a compound of formula (Ia) and a substrate. It further includes any composition comprising a compound of formula (V), optionally one or more other isomers of the compound of formula (Ia), and a substrate. It further includes any composition comprising a compound of formula (I), a compound of formula (Ia), and a substrate. It further includes any composition comprising a compound of formula (I), a compound of formula (V), and a substrate, optionally additionally comprising one or more isomers of the compound of formula (I) and / or one or more other isomers of the compound of formula (Ia).

[0160] As used herein, "substrate" may be understood to encompass all known fragrance ingredients selected from a wide range of currently available natural products and synthetic molecules, such as essential oils, alcohols, aldehydes and ketones, ethers and acetals, esters and lactones, macrocycles and heterocycles, and / or in admixture with one or more ingredients or excipients conventionally used in conjunction with substances in fragrance compositions (e.g., carrier materials, diluents, and other auxiliaries commonly used in the art; examples of which may be found in standard handbooks such as Perfume Engineering: Design, Performance and Classification (2012), Miguel Teixeira et al., Butterworth-Heinemann, UK, which is incorporated herein by reference in its entirety).

[0161] Suitable fragrance ingredients are further commercially available. Non-limiting examples of such ingredients include: essential oils and extracts, such as, for example, bead onion, costus root oil, oak moss absolute, geranium oil, tree moss absolute, basil oil, fruit oils such as bergamot oil and mandarin oil, myrtle oil, palmarosa oil, patchouli oil, petitgrain oil, jasmine oil, rose oil, sandalwood oil, absinthe oil, lavender oil and / or ylang-ylang oil; alcohols, such as cinnamic alcohol ((E)-3-phenylprop-2-en-1-ol); cis-3-hexenol ((Z)-hex-3-en-1-ol); citronellol (3,7-dimethyloct-6-en-1-ol); dihydromyrcenol (2,6-dimethyloct-7-en-2-ol); Ebanol (trademark) ((E)-3-methyl-5-(2,2,3-trimethylcyclopenta[1,2-diphenylphosphine]-2,4-diol); -3-en-1-yl)pent-4-en-2-ol;Eugenol (4-allyl-2-methoxyphenol);Ethylinalool ((E)-3,7-dimethylnona-1,6-dien-3-ol);Farnesol ((2E,6Z)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol);Geraniol ((E)-3,7-dimethylocta-2,6-dien-1-ol);Super Lily of the Valley (Super Muguet™ ((E)-6-ethyl-3-methyloct-6-en-1-ol); Linalool (3,7-dimethylocta-1,6-dien-3-ol); Menthol (2-isopropyl-5-methylcyclohexanol); Nerol (3,7-dimethyl-2,6-octadien-1-ol); Phenylethyl alcohol (2-phenylethanol); Rhodinol™ (3,7-dimethyloct-6-en-1-ol); Sandalore™ (3 -methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentan-2-ol; terpineol (2-(4-methylcyclohex-3-en-1-yl)propan-2-ol); or Timberol™ (1-(2,2,6-trimethylcyclohexyl)hexan-3-ol); 2,4,7-trimethylocta-2,6-dien-1-ol, and / or [1-methyl-2(5-methylhex-4-en-2-yl)cyclopropyl]-methanol; - aldehydes and ketones, such as anisaldehyde (4-methoxybenzaldehyde); alpha-amyl cinnamaldehyde (2-benzylideneheptanal); Georgywood™ (1-(1,2,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)ethanone); hydroxycitronellal (7-hydroxy-3,7-dimethyloctanal); Iso E Super (Iso E Super® (1-(2,3,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)ethanone); Isoraldeine® ((E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one); 3-(4-isobutyl-2-methylphenyl)propanal; maltol; methyl cedryl ketone; methyl ionone; verbenone; and / or vanillin; - ethers and acetals, such as, for example, Ambrox® (3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro-1H-benzo[e][1]benzofuran); geranyl methyl ether ((2E)-1-methoxy-3,7-dimethylocta-2,6-diene); rose oxide (4-methyl-2-(2-methylprop-1-en-1-yl)tetrahydro-2H-pyran); and / or Spirambrene® (2',2',3,7,7-pentamethylspiro[bicyclo[4.1.0]heptane-2,5'-[1,3]dioxane]); macrocycles, such as, for example, ambrettolide ((Z)-oxacycloheptadecan-10-en-2-one); ethylene brassylate (1,4-dioxacycloheptadecane-5,17-dione); and / or Exaltolide® (16-oxacyclohexadecane-1-one); and - Heterocycles, for example isobutylquinoline (2-isobutylquinoline).

[0162] As used herein, "carrier material" may be understood to be a material that is practically neutral from the viewpoint of odorant, i.e., a material that does not significantly modify the sensory properties of odorant. The term "diluent" may be understood to include any diluent that is conventionally used in conjunction with odorants, such as diethyl phthalate (DEP), dipropylene glycol (DPG), isopropyl myristate (IPM), triethyl citrate (TEC), and alcohol (e.g., ethanol). The term "auxiliary agent" may be understood to include any ingredient that may be employed in a fragrance composition because it is not particularly related to the olfactory performance of the composition. For example, an auxiliary agent, such as an antioxidant adjuvant, may be an ingredient that acts as an auxiliary to process the fragrance ingredient(s) or the composition containing the ingredient(s), or may improve the handling or storage of the fragrance ingredient or the composition containing the same. The antioxidant may be selected from, for example, Tinogard® TT (BASF), Tinogard® Q (BASF), tocopherol (including its isomers, CAS 59-02-9; 364-49-8; 18920-62-2; 121854-78-2), 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT, CAS 128-37-0), and the related phenol, hydroquinone (CAS 121-31-9). The auxiliary may also be an ingredient that provides additional benefits, such as imparting color or texture to the fragrance composition. The auxiliary may also be an ingredient that imparts increased light resistance or chemical stability to one or more ingredients contained in the fragrance composition. The fragrance ingredients, carrier materials, diluents and auxiliaries discussed herein should be understood as non-limiting examples; the skilled artisan will be aware of suitable substrates commonly used in the art, examples of which are available in standard handbooks such as Perfume Engineering: Design, Performance and Classification (see above).

[0163] The compounds of formula (I), compounds of formula (Ia) (such as compounds of formula (V)), and mixtures comprising compounds of formula (I) and compounds of formula (Ia) (such as compounds of formula (V)), as described herein, may further be included in a number of compositions, including but not limited to fine fragrance or consumer products, such as fabric care, toiletry, beauty and cleaning products, detergent products, and soap products, which encompass substantially all products in which currently available (+)-amberketal components are commercially used.

[0164] The present disclosure further provides a consumer product comprising a composition or fragrance composition as described herein (including any embodiment thereof). The consumer product may be, for example, a cosmetic product (e.g., an eau de parfum or eau de toilette), a cleaning product, a detergent product, or a soap product. Fragrances and consumer products containing mixtures comprising a compound of formula (I) and a compound of formula (Ia), such as a compound of formula (V), may advantageously exhibit unique olfactory properties.

[0165] Accordingly, in some embodiments, the fragrance composition or consumer product comprises a composition comprising a compound represented by formula (I) and a compound represented by formula (Ia) (such as a compound represented by formula (V)), wherein the composition is obtained or obtainable by the methods described herein. In some embodiments, the compound represented by formula (I) and the compound represented by formula (Ia) (such as a compound represented by formula (V)) are in solid form, preferably in amorphous or crystalline form.

[0166] Starting Materials and Intermediates In certain aspects, the disclosure provides starting materials and intermediates used in the methods described herein.

[0167] Also provided herein are mixtures that comprise, consist essentially of, or consist of a compound of formula (II). For example, the mixture may comprise, consist essentially of, or consist of a compound of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomer), and a compound of formula (IIa) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomer). In some embodiments, the mixture comprises three isomers of the compound of formula (II), such as the E,Z-isomer, the E,E-isomer, and either the Z,E-isomer or the Z,Z-isomer. In some embodiments, the mixture comprises all four isomers of the compound represented by Formula (II), namely, the E,Z-isomer, the E,E-isomer, the Z,E-isomer, and the Z,Z-isomer.

[0168] In some embodiments, R is selected from H (hydrogen), and C 1 ~C 4 R is selected from alkyl, such as methyl, ethyl, n-propyl, or isopropyl, and preferably R is methyl.

[0169] Also provided herein are mixtures that comprise, consist essentially of, or consist of a compound of formula (IIa). For example, the mixture may comprise, consist essentially of, or consist of a compound of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the E-configuration (E,E-isomer), and a compound of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomer). In some embodiments, the mixture comprises three isomers of the compound of formula (IIa), such as the E,Z-isomer, the E,E-isomer, and either the Z,E-isomer or the Z,Z-isomer. In some embodiments, the mixture comprises four isomers of the compound represented by Formula (IIa): the E,Z-isomer, the E,E-isomer, the Z,E-isomer, and the Z,Z-isomer.

[0170] In some embodiments, R is selected from H (hydrogen), and C 1 ~C 4 R is selected from alkyl, such as methyl, ethyl, n-propyl, or isopropyl, and preferably R is methyl.

[0171] Also provided herein are mixtures comprising, consisting essentially of, or consisting of a compound of formula (II) and a compound of formula (IIa). For example, the mixture may comprise, consist essentially of, or consist of a compound of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomer) and a compound of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomer). For example, the mixture may comprise, consist essentially of, or consist of compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomers) and compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomers). For example, the mixture may comprise, consist essentially of, or consist of compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomers) and compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomers). For example, the mixture may comprise, consist essentially of, or consist of a compound of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomer) and a compound of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomer).

[0172] For example, the mixture may comprise, consist essentially of, or consist of compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomers), compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomers), compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomers), and compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomers). Optionally, the mixture may further comprise one or more other isomers of the compound of formula (II) and / or the compound of formula (IIa).

[0173] In some embodiments, R is selected from H (hydrogen), and C 1 ~C 4 R is selected from alkyl, such as methyl, ethyl, n-propyl, or isopropyl, and preferably R is methyl.

[0174] In a mixture comprising the E,Z-isomer and the E,E-isomer of a compound of formula (II), the ratio of the E,Z-isomer to the E,E-isomer may be equal to or greater than, or about, 10:90. In some embodiments, the ratio is equal to or greater than 20:80, or about 20:80, equal to or greater than 30:70, or about 30:70, equal to or greater than 40:60, or about 40:60, equal to or greater than 50:50, or about 50:50, equal to or greater than 60:40, or about 60:40, equal to or greater than 70:30, or about 70:30, equal to or greater than 80:20, or about 85:15, equal to or greater than 90:10, or about 95:5, or equal to or greater than 99:1.

[0175] In a mixture comprising the E,Z- and E,E-isomers of a compound of formula (II), the ratio of the E,Z- to E,E-isomers may be less than or equal to 99:1, or about 99:1. In some embodiments, the ratio is less than or equal to 95:5, less than or equal to 90:10, less than or equal to 85:15, less than or equal to 80:20, less than or equal to 70:30, or less than or equal to 60:40. or less than about 60:40; equal to or less than about 50:50; equal to or less than about 40:60; equal to or less than about 30:70; equal to or less than about 20:80; or equal to or less than about 10:90.

[0176] In a mixture comprising the E,Z- and E,E-isomers of a compound of formula (II), the ratio of the E,Z-isomer to the E,E-isomer may be from 10:90 to 99:1, or from about 10:90 to about 99:1, from 10:90 to 90:10, or from about 10:90 to about 90:10, or from about 5:95 to about 95:5, or from about 4:96 to about 96:4, or from about 3:97 to about 97:3, or from about 2:98 to about 98:2, or from about 1:99 to about 99:1, or from about 20:80 to about 80:20, or from about 50:50 to 80:20, or from about 50:50 to about 80:20, or from 60:40 to 80:20, or from about 60:40 to about 80:20. Optionally, the mixture may further comprise one or more other isomers of the compound of formula (II) and / or the compound of formula (IIa).

[0177] In a mixture comprising the E,Z- and E,E-isomers of a compound of formula (IIa), the ratio of the E,Z-isomer to the E,E-isomer may be equal to or greater than 10:90, or may be about 10:90. In some embodiments, the ratio is equal to or greater than 20:80, or about 20:80, equal to or greater than 30:70, or about 30:70, equal to or greater than 40:60, or about 40:60, equal to or greater than 50:50, or about 50:50, equal to or greater than 60:40, or about 60:40, equal to or greater than 70:30, or about 70:30, equal to or greater than 80:20, or about 85:15, equal to or greater than 90:10, or about 95:5, or equal to or greater than 99:1.

[0178] In a mixture comprising the E,Z- and E,E-isomers of a compound of formula (IIa), the ratio of the E,Z- to E,E-isomers may be less than or equal to 99:1, or about 99:1. In some embodiments, the ratio is less than or equal to 95:5, less than or equal to 90:10, less than or equal to 85:15, less than or equal to 80:20, less than or equal to 70:30, or less than or equal to 60:40. or less than about 60:40; equal to or less than about 50:50; equal to or less than about 40:60; equal to or less than about 30:70; equal to or less than about 20:80; or equal to or less than about 10:90.

[0179] In a mixture comprising the E,Z-isomer and the E,E-isomer of the compound of formula (IIa), the ratio of the E,Z-isomer to the E,E-isomer may be from 10:90 to 99:1 or from about 10:90 to about 99:1, from 10:90 to 90:1 or from about 10:90 to about 90:1, from 20:80 to 80:20 or from about 20:80 to about 80:20, from 50:50 to 80:20 or from about 50:50 to about 80:20, or from 60:40 to 80:20 or from about 60:40 to about 80:20. Optionally, the mixture may further comprise one or more other isomers of the compound of formula (II) and / or of the compound of formula (IIa).

[0180] In a mixture comprising a compound of formula (II) and a compound of formula (IIa), the ratio of the compound of formula (II) to the compound of formula (IIa) may be equal to or greater than or about 50:50, equal to or greater than or about 60:40, equal to or greater than or about 70:30, equal to or greater than or about 80:20, equal to or greater than or about 85:15, equal to or greater than or about 90:10, equal to or greater than or about 95:5, or equal to or greater than or about 99:1.

[0181] In a mixture comprising a compound represented by Formula (II) and a compound represented by Formula (IIa), the ratio of the compound represented by Formula (II) to the compound represented by Formula (IIa) may be less than or equal to 99:1, or about 99:1. In some embodiments, the ratio may be less than or equal to 95:5, less than or equal to 90:10, less than or equal to 85:15, less than or equal to 80:20, less than or equal to 70:30, or less than or equal to 60:40. or less than about 60:40; equal to or less than about 50:50; equal to or less than about 40:60; equal to or less than about 30:70; equal to or less than about 20:80; or equal to or less than about 10:90.

[0182] In a mixture comprising a compound of formula (II) and a compound of formula (IIa), the ratio of the compound of formula (II) to the compound of formula (IIa) may be from 10:90 to 99:1 or from about 10:90 to about 99:1, from 10:90 to 90:1 or from about 10:90 to about 90:1, from 20:80 to 80:20 or from about 20:80 to about 80:20, from 50:50 to 80:20 or from about 50:50 to about 80:20, or from 60:40 to 80:20 or from about 60:40 to about 80:20.

[0183] Squalene-hopene cyclase (SHC) enzyme The methods described herein utilize the squalene-hopene cyclase (SHC) enzyme as described herein.

[0184] In some embodiments, the squalene-hopene cyclase enzyme described herein may comprise an amino acid sequence having at least 30%, 40%, 50%, 60%, or 70%, preferably at least 70%, identity or similarity to the sequence of SEQ ID NO: 1 or SEQ ID NOs: 43-49, preferably to the sequence of SEQ ID NO: 1. SEQ ID NO: 1 represents the SHC enzyme from Bacillus megaterium (BmeSHC). SEQ ID NO: 43 represents the SHC enzyme from Alicyclobacillus acidocaldarius (AacSHC). SEQ ID NOs: 44 and 45 represent the SHC enzyme from Zymomonas mobilis (ZmoSHC1 and ZmoSHC2, respectively). SEQ ID NO: 46 represents the SHC enzyme from Bradyrhizobium japonicum (BjaSHC). SEQ ID NO: 47 represents the SHC enzyme from Thermosynechococcus elongatus (TeISHC). SEQ ID NO: 48 represents the SHC enzyme from Acetobacter pasteurianus (ApaSHC). SEQ ID NO: 49 represents the SHC enzyme from Gluconobacter morbifer (GmoSHC). Further description of these enzymes may be found in WO2021 / 209482.

[0185] In some embodiments, the squalene-hopene cyclase (SHC) enzyme described herein has a similar affinity to the sequence of SEQ ID NO:1 or SEQ ID NOs:43-49, preferably to the sequence of SEQ ID NO:1 by at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61% , 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identity or similarity. In some embodiments, the identity or similarity is at least 30%. In some embodiments, the identity or similarity is at least 35%. In some embodiments, the identity or similarity is at least 40%. In some embodiments, the identity or similarity is at least 45%. In some embodiments, the identity or similarity is at least 50%. In some embodiments, the identity or similarity is at least 55%. In some embodiments, the identity or similarity is at least 60%. In some embodiments, the identity or similarity is at least 65%. In some embodiments, the identity or similarity is at least 70%. In some embodiments, the identity or similarity is at least 75%. In some embodiments, the identity or similarity is at least 80%. In some embodiments, the identity or similarity is at least 85%. In some embodiments, the identity or similarity is at least 90%. In some embodiments, the identity or similarity is at least 95%. In some embodiments, the identity or similarity is at least 95.5%. In some embodiments, the identity or similarity is at least 96%.In some embodiments, the identity or similarity is at least 96.5%. In some embodiments, the identity or similarity is at least 97%. In some embodiments, the identity or similarity is at least 97.5%. In some embodiments, the identity or similarity is at least 98%. In some embodiments, the identity or similarity is at least 98.5%. In some embodiments, the identity or similarity is at least 99%. In some embodiments, the identity or similarity is at least 99.5%. In some embodiments, the identity or similarity is less than 100%, i.e., the amino acid sequence is not identical to SEQ ID NO: 1 or SEQ ID NOs: 43-49, preferably SEQ ID NO: 1. The definitions of "identity" and "similarity" of sequences, as well as methods for their determination, are provided herein below in the section entitled "General Definitions".

[0186] The SHC enzymes described herein may be derived from the SHC enzyme represented by SEQ ID NO: 1 or SEQ ID NOs: 43-49, preferably from the SHC enzyme represented by SEQ ID NO: 1, by introducing modifications into the sequence. Such enzymes may also be referred to herein as "SHC variants", "SHC mutants", or "SHC derivatives". The SHC enzymes described herein may also be derived from other SHC variants by introducing additional modifications into the sequence of existing SHC variants. The SHC enzymes described herein may not be naturally occurring.

[0187] In other words, the term "variant", such as SHC variant, should be understood as a polypeptide (enzyme) described herein that contains one or more sequence modifications in comparison with the polypeptide from which it is derived. The polypeptide from which the variant is derived may also be referred to herein as a parent or reference polypeptide (i.e., parent SHC enzyme or reference SHC enzyme). The parent SHC enzyme may be a wild-type enzyme. The parent SHC enzyme may be a homolog, ortholog, or paralog of the wild-type polypeptide. The parent SHC enzyme may be another variant, i.e., an enzyme that originates from the introduction of additional modifications in its amino acid sequence compared to the previously obtained variant enzyme. Thus, the SHC enzymes described herein may originate from "old generations" of SHC variants and may exhibit improved properties compared to their parent SHC enzymes. Examples of sequence modifications that may be included in the variant enzymes are amino acid substitutions, deletions, insertions, N-terminal truncations, C-terminal truncations, or combinations thereof. Variant enzymes can be generated by, for example, random mutagenesis, site-specific mutagenesis, directed evolution, gene shuffling, CRISPR / Cas-mediated mutagenesis, etc. (examples of which are also described in In Vitro Mutagenesis: Methods and Protocols (Methods in Molecular Biology 1498), 1 st The SHC enzymes described herein may be synthetically produced or produced by cellular (or in vitro) production after modifying the nucleotide sequence encoding the enzyme using mutagenesis techniques known to those skilled in the art, such as those available in standard handbooks, such as The Journal of Clinical Chemistry, Vol. 13, No. 1, 1999, 1999 Edition, Reeves A. (Ed), Humana Press (2017), which is incorporated herein by reference in its entirety. In some embodiments, the SHC enzymes described herein are synthetically produced. In some embodiments, the SHC enzymes described herein are produced by recombinant host cells.

[0188] Sequence modifications of SHC described herein may be identified through direct comparison of their respective amino acid sequences or the nucleotide sequences of the nucleic acids encoding the enzymes when compared to its parent SHC enzyme, such as the SHC enzyme represented by SEQ ID NO: 1 or SEQ ID NOs: 43-49, preferably SEQ ID NO: 1, using standard bioinformatics algorithms available in the art and further discussed herein below in the section entitled "General Definitions." These algorithms typically utilize routine sequence alignment methods in which a particular nucleotide or amino acid residue corresponding to a particular position in a sequence is matched to the corresponding position in a reference sequence to which it is aligned.

[0189] Using SEQ ID NO:1 as an example, and using such methods, the skilled artisan can easily identify, by way of example, which amino acid positions of the SHC enzyme correspond to, for example, positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:1 (or other positions of SEQ ID NO:1) when SEQ ID NO:1 is used as a reference sequence and the SHC enzyme amino acid sequence in question is aligned thereto. Similarly, the positions of the corresponding nucleotides encoding specific amino acid residues may be identified when the nucleotide sequence 1 of the nucleic acid encoding SEQ ID NO:1 and the SHC enzyme in question are aligned instead. In this regard, the skilled artisan understands that the methionine (M) residue at the N-terminus of SEQ ID NO:1 corresponds to position 1, the serine (S) residue at the C-terminus of SEQ ID NO:1 corresponds to position 625, and the amino acids between the N-terminus and C-terminus of SEQ ID NO:1 correspond to positions 2 to 624, respectively.

[0190] Amino acid substitution refers to a sequence modification that replaces an amino acid residue (or a nucleotide in the nucleotide sequence of a nucleic acid encoding an amino acid sequence) in a parent (reference) amino acid sequence, resulting in a variant (derivative) sequence having the same number of amino acids. The amino acid substitution may correspond to a substitution by any other amino acid. The amino acid substitution may be conservative. A definition of a "conservative" substitution is provided later in the specification. The amino acid substitution may correspond to multiple specific amino acid positions of a parent SHC enzyme sequence, such as the sequence represented by SEQ ID NO: 1 or SEQ ID NOs: 43-49, preferably SEQ ID NO: 1. In embodiments where multiple amino acids have been substituted, they may correspond to consecutive positions, non-consecutive positions, or positions that are spatially separated in the polypeptide sequence.

[0191] In some embodiments, the SHC enzymes described herein comprise one or more amino acid substitutions relative to SEQ ID NO: 1. Preferred positions of substitutions may be selected from the group of positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO: 1. In some embodiments, the preferred SHC enzymes described herein comprise one or more amino acid substitutions relative to SEQ ID NO: 1 at one or more positions corresponding to positions 2, 5, 35, 166, 211, 212, 355, 483, and 539 of SEQ ID NO: 1. Preferably, the one or more amino acid substitutions relative to SEQ ID NO: 1 are at one or more positions corresponding to positions 2, 5, 35, 166, 211, 212, 483, and 539 of SEQ ID NO: 1. More preferably, the one or more amino acid substitutions relative to SEQ ID NO: 1 are at one or more positions corresponding to positions 2, 5, 35, 166, 211, 483, and 539 of SEQ ID NO: 1.

[0192] In some embodiments, the SHC enzymes described herein comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 amino acid substitutions relative to SEQ ID NO:1. In some embodiments, at least one amino acid is substituted relative to SEQ ID NO:1. In some embodiments, at least two amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least three amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least four amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least five amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least six amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least seven amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least eight amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least nine amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least ten amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least eleven amino acids are substituted relative to SEQ ID NO:1. In some embodiments, at least 12 amino acids are substituted relative to SEQ ID NO: 1. In some embodiments, at least 13 amino acids are substituted relative to SEQ ID NO: 1. In some embodiments, at least 14 amino acids are substituted relative to SEQ ID NO: 1. Preferred positions of substitution may be selected from the group of positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585, preferably 2, 5, 35, 166, 211, 212, 355, 483, and 539, more preferably 2, 5, 35, 166, 211, 212, 483, and 539, and most preferably 2, 5, 35, 166, 211, 483, and 539.

[0193] In some embodiments, the SHC enzymes described herein comprise 1-7, preferably 2-6, more preferably 3-5 amino acid substitutions relative to SEQ ID NO: 1. In some embodiments, the SHC enzymes described herein comprise 1-7, preferably 2-6, more preferably 3-5 amino acid substitutions at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO: 1, preferably 2, 5, 35, 166, 211, 212, 355, 483, and 539, more preferably 2, 5, 35, 166, 211, 212, 483, and 539, and most preferably 2, 5, 35, 166, 211, 483, and 539 of SEQ ID NO: 1.

[0194] As used herein, "conservative" amino acid substitutions refer to the interchangeability of residues having similar side chains. Conservative amino acid substitutions may be made, by way of example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved.

[0195] Examples of similar classes of amino acid residues for conservative substitutions are provided in the table below. [Table A]

[0196] Alternative conservative amino acid residue substitution classes: [Table B]

[0197] Physical and functional classification of alternative amino acid residues: [Table C]

[0198] For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

[0199] Preferred substitutions that occur at preferred substitution positions that correspond to particular positions in SEQ ID NO:1 described herein are indicated below. In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the isoleucine (I) corresponding to position 2 of SEQ ID NO:1 is replaced by any amino acid, preferably by asparagine (N), serine (S), threonine (T), or glutamine (Q), more preferably by asparagine (N).

[0200] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the leucine (L) corresponding to position 5 of SEQ ID NO:1 is replaced by any amino acid, preferably by proline (P), methionine (M), or cysteine ​​(C), more preferably by proline (P). In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the threonine (T) corresponding to position 35 of SEQ ID NO:1 is replaced by any amino acid, preferably by alanine (A), isoleucine (I), valine (V), glycine (G), or leucine (L), more preferably by alanine (A).

[0201] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the isoleucine (I) corresponding to position 116 of SEQ ID NO:1 is replaced by any amino acid, preferably by threonine (T), asparagine (N), serine (S), or glutamine (Q), more preferably by threonine (T).

[0202] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the threonine (T) corresponding to position 166 of SEQ ID NO:1 is replaced by any amino acid, preferably alanine (A), isoleucine (I), valine (V), glycine (G), or leucine (L), more preferably alanine (A).

[0203] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which glutamic acid (E) corresponding to position 211 of SEQ ID NO:1 is replaced by any amino acid, preferably by valine (V), alanine (A), isoleucine (I), glycine (G), or leucine (L), more preferably by valine (V).

[0204] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the serine (S) corresponding to position 212 of SEQ ID NO:1 is replaced by any amino acid, preferably by arginine (R), lysine (K), or histidine (H), more preferably by arginine (R).

[0205] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the leucine (L) corresponding to position 317 of SEQ ID NO:1 is replaced by any amino acid, preferably by methionine (M), proline (P), or cysteine ​​(C), more preferably by methionine (M).

[0206] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which an alanine (A) corresponding to position 355 of SEQ ID NO:1 is replaced by any amino acid, preferably by threonine (T), asparagine (N), serine (S), or glutamine (Q), more preferably by threonine (T).

[0207] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the serine (S) corresponding to position 382 of SEQ ID NO:1 is replaced by any amino acid, preferably by threonine (T), asparagine (N), or glutamine (Q), more preferably by threonine (T).

[0208] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the isoleucine (I) corresponding to position 399 of SEQ ID NO:1 is replaced by any amino acid, preferably by valine (V), alanine (A), or glycine (G), leucine (L), and more preferably by valine (V).

[0209] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which a tyrosine (Y) corresponding to position 483 of SEQ ID NO:1 is replaced by any amino acid, preferably by cysteine ​​(C), methionine (M), or proline (P), more preferably by cysteine ​​(C).

[0210] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which the leucine (L) corresponding to position 539 of SEQ ID NO:1 is replaced by any amino acid, preferably by histidine (H), arginine (R), or lysine (K), more preferably by histidine (H).

[0211] In some embodiments, the SHC enzymes described herein comprise an amino acid sequence in which glutamic acid (E) corresponding to position 585 of SEQ ID NO:1 is replaced by any amino acid, preferably by alanine (A), valine (V), isoleucine (I), glycine (G), or leucine (L), more preferably by alanine (A).

[0212] In some embodiments, a preferred SHC enzyme as described herein comprises an amino acid sequence having at least 30%, 40%, 50%, 60%, or 70%, preferably at least 70%, identity or similarity to the sequence of SEQ ID NO:1, preferably wherein the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585, preferably 2, 5, 35, 166, 211, 212, 355, 483, and 539, more preferably 2, 5, 35, 166, 211, 212, 483, and 539, most preferably 2, 5, 35, 166, 211, 211, 483, and 539. In some embodiments, the identity or similarity to the sequence of SEQ ID NO:1 is at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 100%, 101 ... 5%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%.

[0213] In some embodiments, the SHC enzymes described herein comprise an amino acid substitution relative to SEQ ID NO:1 selected from the following: (i) an asparagine (N), serine (S), threonine (T), or glutamine (Q) residue at a position corresponding to position 2 of SEQ ID NO:1; (ii) a proline (P), methionine (M), or cysteine ​​(C) residue at a position corresponding to position 5 of SEQ ID NO:1; (iii) an alanine (A), isoleucine (I), valine (V), glycine (G), or leucine (L) residue at a position corresponding to position 35 of SEQ ID NO:1; (iv) a threonine (T), asparagine (N), serine (S), or glutamine (Q) residue at a position corresponding to position 116 of SEQ ID NO:1; (v) an alanine (A), isoleucine (I), valine (V), glycine (G), or leucine (L) residue at a position corresponding to position 166 of SEQ ID NO:1; (vi) a valine (V), alanine (A), isoleucine (I), glycine (G), or leucine (L) residue at a position corresponding to position 211 of SEQ ID NO:1; (vii) an arginine (R), lysine (K), or histidine (H) residue at a position corresponding to position 212 of SEQ ID NO:1; (viii) a methionine (M), proline (P), or cysteine ​​(C) residue at a position corresponding to position 317 of SEQ ID NO:1; (ix) a threonine (T), asparagine (N), serine (S), or glutamine (Q) residue at a position corresponding to position 355 of SEQ ID NO:1; (x) a threonine (T), asparagine (N), or glutamine (Q) residue at a position corresponding to position 382 of SEQ ID NO:1; (xi) a valine (V), alanine (A), glycine (G), or leucine (L) residue at a position corresponding to position 399 of SEQ ID NO:1; (xii) a cysteine ​​(C), methionine (M), or proline (P) residue at a position corresponding to position 483 of SEQ ID NO:1; (xiii) a histidine (H), arginine (R), or lysine (K) residue at a position corresponding to position 539 of SEQ ID NO:1; (xiv) an alanine (A), valine (V), isoleucine (I), glycine (G), or leucine (L) residue at a position corresponding to position 585 of SEQ ID NO:1; or (xv) any combination thereof.

[0214] In some embodiments, the SHC enzymes described herein comprise an amino acid substitution relative to SEQ ID NO:1 selected from the following: (i) an asparagine (N) residue at a position corresponding to position 2 of SEQ ID NO:1; (ii) a proline (P) residue at a position corresponding to position 5 of SEQ ID NO:1; (iii) an alanine (A) residue at a position corresponding to position 35 of SEQ ID NO:1; (iv) a threonine (T) residue at a position corresponding to position 116 of SEQ ID NO:1; (v) an alanine (A) residue at a position corresponding to position 166 of SEQ ID NO:1; (vi) a valine (V) residue at a position corresponding to position 211 of SEQ ID NO:1; (vii) an arginine (R) residue at a position corresponding to position 212 of SEQ ID NO:1; (viii) a methionine (M) residue at a position corresponding to position 317 of SEQ ID NO:1; (ix) a threonine (T) residue at a position corresponding to position 355 of SEQ ID NO:1; (x) a threonine (T) residue at a position corresponding to position 382 of SEQ ID NO:1; (xi) a valine (V) residue at a position corresponding to position 399 of SEQ ID NO:1; (xii) a cysteine ​​(C) residue at a position corresponding to position 483 of SEQ ID NO:1; (xiii) a histidine (H) residue at a position corresponding to position 539 of SEQ ID NO:1; (xiv) an alanine (A) residue at a position corresponding to position 585 of SEQ ID NO:1; or (xv) any combination thereof.

[0215] In some embodiments, the SHC enzymes described herein comprise an amino acid substitution relative to SEQ ID NO:1 selected from the following positions relative to SEQ ID NO:1: (i) 2, 35, 355, and 539; (ii) 166; (iii) 2 and 483; (iv) 2, 483, and 539; (v) 2, 5, 35, 539; (vi) 2, 5, 35, and 483; (vii) 2, 5, 35, 166, and 539; (viii) 2, 5, 35, 166, 211, and 539; (ix) 2, 5, 35, 211, 212, 483, and 539; (x) 2, 166, and 483; (xi) 2, 166, 483, and 539; (xii) 2, 166, 211, and 483; or (xiii) 2, 166, 211, 483, and 539.

[0216] In some embodiments, the SHC enzymes described herein comprise an amino acid substitution relative to SEQ ID NO:1 selected from the following: (i) I2N, T35A, A355T, and L539H; (ii) T166A; (iii) I2N and Y483C; (iv) I2N, Y483C, and L539H; (v) I2N, L5P, T35A, L539H; (vi) I2N, L5P, T35A, and Y483C; (vii) I2N, L5P, T35A, T166A, and L539H; (viii) I2N, L5P, T35A, T166A, E211V, and L539H; (ix) I2N, L5P, T35A, E211V, S212R, Y483C, and L539H; (x) I2N, T166A, and Y483C; (xi) I2N, T166A, Y483C, and L539H; (xii) I2N, T166A, E211V, and Y483C; or (xiii) I2N, T166A, E211V, Y483C, and L539H.

[0217] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, T35A, A355T, L539H. Optionally, it further comprises an E211V substitution relative to SEQ ID NO:1.

[0218] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitution relative to SEQ ID NO: 1: T166A. Optionally, it further comprises an E211V and / or a L539H substitution relative to SEQ ID NO: 1.

[0219] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, Y483C. Optionally, it further comprises an E211V and / or a L539H substitution relative to SEQ ID NO:1.

[0220] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, Y483C, L539H. Optionally, it further comprises an E211V substitution relative to SEQ ID NO:1.

[0221] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, L5P, T35A, L539H. Optionally, it further comprises an E211V substitution relative to SEQ ID NO:1.

[0222] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, L5P, T35A, Y483C. Optionally, it further comprises an E211V and / or an L539H substitution relative to SEQ ID NO:1.

[0223] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, L5P, T35A, T166A, L539H. Optionally, it further comprises an E211V substitution relative to SEQ ID NO:1.

[0224] In some embodiments, the SHC enzymes described herein comprise the following amino acid substitutions relative to SEQ ID NO:1: I2N, L5P, T35A, T166A, E211V, L539H. In some embodiments, the SHC enzymes described herein comprise the following amino acid substitutions relative to SEQ ID NO:1: I2N, L5P, T35A, E211V, S212R, Y483C, L539H.

[0225] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, T166A, Y483C. Optionally, it further comprises an E211V and / or an L539H substitution relative to SEQ ID NO:1.

[0226] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, T166A, Y483C, L539H. Optionally, it further comprises an E211V substitution relative to SEQ ID NO:1.

[0227] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N, T166A, E211V, Y483C. Optionally, it further comprises a L539H substitution relative to SEQ ID NO:1.

[0228] In some embodiments, the SHC enzymes described herein comprise the following amino acid substitutions relative to SEQ ID NO:1: I2N, T166A, E211V, Y483C, L539H.

[0229] In some embodiments, the SHC enzyme described herein comprises the following amino acid substitutions relative to SEQ ID NO: 1: I2N, T166A. Optionally, it further comprises an E211V and / or an L539H substitution relative to SEQ ID NO: 1. Optionally, it further comprises a Y483C substitution relative to SEQ ID NO: 1.

[0230] In some embodiments, any of the SHC enzymes described herein further comprise one or more substitutions relative to SEQ ID NO:1 selected from L5P, T35A, E211V, Y483C, and L539H. One of skill in the art will understand that the numbering of positions representing the amino acid substitutions described herein refers to the corresponding positions in SEQ ID NO:1, as discussed elsewhere herein.

[0231] In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or 42, preferably SEQ ID NOs: 4, 8, 18, 20, 22, 24, 30, 32, 34, 36, 38, 40, or 42, more preferably SEQ ID NOs: 30, 32, 34, 36, 38, 40, or 42, most preferably SEQ ID NOs: 30, 38, 40, or 42. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 30, 34, 36, 40, or 42. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 4. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:6. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:8. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:10. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:12. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:14. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:16. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:18. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:20.In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 22. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 24. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 26. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 28. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 30. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 32. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 34. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 36. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 38. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 40. In some embodiments, any of the SHC enzymes described herein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 42. The amino acid sequence may be at least 91% identical. The amino acid sequence may be at least 92% identical. The amino acid sequence may be at least 93% identical. The amino acid sequence may be at least 94% identical. The amino acid sequence may be at least 95% identical. The amino acid sequence may be at least 95.5% identical. The amino acid sequence may be at least 96% identical. The amino acid sequence may be at least 96.5% identical. The amino acid sequence may be at least 97% identical.The amino acid sequences may be at least 97.5% identical. The amino acid sequences may be at least 98% identical. The amino acid sequences may be at least 98.5% identical. The amino acid sequences may be at least 99% identical. The amino acid sequences may be at least 99.5% identical. The amino acid sequences may be identical.

[0232] In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 41, preferably SEQ ID NOs: 3, 7, 17, 19, 21, 23, 29, 31, 33, 35, 37, 39, or 41, more preferably SEQ ID NOs: 29, 31, 33, 35, 37, 39, or 41, most preferably SEQ ID NOs: 29, 37, 39, or 41. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 29, 33, 35, 39, or 41. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 3. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO:5. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO:7. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO:9. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO:11. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO:13. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO:15. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO:17.In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 19. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 21. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 23. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 25. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 27. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 29. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 31. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 33. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 35. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 37. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 39. In some embodiments, any of the SHC enzymes described herein are encoded by a nucleic acid comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 41.

[0233] The nucleotide sequences may be at least 91% identical. The nucleotide sequences may be at least 92% identical. The nucleotide sequences may be at least 93% identical. The nucleotide sequences may be at least 94% identical. The nucleotide sequences may be at least 95% identical. The nucleotide sequences may be at least 95.5% identical. The nucleotide sequences may be at least 96% identical. The nucleotide sequences may be at least 96.5% identical. The nucleotide sequences may be at least 97% identical. The nucleotide sequences may be at least 97.5% identical. The nucleotide sequences may be at least 98% identical. The nucleotide sequences may be at least 98.5% identical. The nucleotide sequences may be at least 99% identical. The nucleotide sequences may be at least 99.5% identical. The nucleotide sequences may be identical.

[0234] As used herein, the term "activity" or "enzyme activity" or "biological activity" refers to the ability of an enzyme to react with a substrate to provide a target product. "SHC activity" or "SHC enzyme activity" or "SHC biological activity" may refer, for example, to the ability of the SHC enzyme described herein to convert a compound represented by formula (II) to a compound represented by formula (I), such as their ability to convert hydroxyfarnesylacetone to (+)-amberketal. It may also refer, for example, to the ability of the SHC enzyme described herein to convert a compound represented by formula (IIa) to a compound represented by formula (Ia), preferably to a compound represented by formula (V). It may also refer, for example, to the ability of the SHC enzyme described herein to convert a compound represented by formula (II) to a compound represented by formula (I) and / or a compound represented by formula (IIa) to a compound represented by formula (Ia), such as a compound represented by formula (V), where the compound represented by formula (II) and the compound represented by formula (IIa) are included in a mixture as previously described herein.

[0235] An SHC enzyme that exhibits its enzymatic activity may also be referred to herein as a functional enzyme. Enzyme activity may be determined using what is known as an activity test, for example, via monitoring the increase in target product, the decrease in substrate (or starting material), or via a combination of these parameters as a function of time.

[0236] The SHC enzymes described herein may, for example, have increased enzymatic activity for the conversion of a compound represented by formula (II) (e.g., hydroxyfarnesylacetone) to a compound represented by formula (I) (e.g., (+)-amberketal) and / or may have increased enzymatic activity for the conversion of a compound represented by formula (IIa) to a compound represented by formula (Ia) (e.g., a compound represented by formula (V)) compared to its parent SHC enzyme. Increased enzyme activity may refer to any aspect of the enzymatic conversion of a compound of formula (II) to a compound of formula (I) and / or a compound of formula (IIa) to a compound of formula (Ia) (such as a compound of formula (V)), including, for example, increased total conversion (yield), increased conversion rate (for example, but not limited to, in the first 4 hours, or in the first 6 hours, or in the first 12 hours, or in the first 24 hours, or in the first 48 hours, or in the first 72 hours, or in the first 96 hours, or in the first 120 hours, or in the first 144 hours, or in the first 168 hours of the reaction), increased production of a compound of formula (I) and / or a compound of formula (Ia) (such as a compound of formula (V)), and / or decreased production of by-products. Increased enzyme activity may generally be defined in terms of increased productivity, which is the amount of compound of formula (I) and / or β-lactamase produced per hour of reaction time (typically measured from the start of the reaction), per gram of biocatalyst, and per liter of reaction. / or in terms of a compound of formula (Ia) (such as a compound of formula (V)).

[0237] In some embodiments, the use of an SHC enzyme according to the methods described herein provides at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (2-fold), 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, 64-fold, 65-fold, 66-fold, 67-fold, 68-fold, 69-fold, 70-fold, 71-fold, 72-fold, 73-fold, 74-fold, 75-fold, 76-fold, 77-fold x, 44x, 45x, 46x, 47x, 48x, 49x, 50x, 51x, 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x , 60x, 61x, 62x, 63x, 64x, 65x, 66x, 67x, 68x, 69x, 70x, 71x, 72x, 73x, 74x, 75x, resulting in 76x, 77x, 78x, 79x, 80x, 81x, 82x, 83x, 84x, 85x, 86x, 87x, 88x, 89x, 90x, 91x, 92x, 93x, 94x, 95x, 96x, 97x, 98x, 99x, 100x, 200x, 500x, or 1000x greater productivity.

[0238] Assays for determining and quantifying SHC enzyme activity are known in the art, and further examples are provided in the experimental section herein.As an example, the activity of the SHC enzyme described herein can be determined by incubating purified enzyme(s) or extracts from host cells that produced the enzyme(s) or from fully recombinant host cells with appropriate substrates under appropriate conditions, and performing analysis of the substrates and reaction products (e.g., by gas chromatography (GC) or HPLC analysis as discussed in standard handbooks in the art, such as Encyclopedia of Analytical Science: 3rd Edition (see above)).Further details of SHC enzyme activity assays and analysis of reaction products are provided in the examples.These assays may include producing the enzyme in recombinant host cells (e.g., E. coli).

[0239] The SHC enzymes described herein may provide, for example, increased total conversion of a compound represented by formula (II) compared to its parent SHC enzyme. Thus, a method using an SHC enzyme described herein may have increased total conversion of a compound represented by formula (II) compared to a method using its parent SHC enzyme. The SHC enzymes described herein may provide, for example, increased total conversion of a compound represented by formula (IIa) compared to its parent SHC enzyme. Thus, a method using an SHC enzyme described herein may have increased total conversion of a compound represented by formula (IIa) compared to a method using its parent SHC enzyme. The SHC enzymes described herein may provide, for example, increased total conversion of a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa) compared to its parent SHC enzyme. Thus, a method using an SHC enzyme described herein may result in increased total conversion of a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa) compared to a method using its parent SHC enzyme, where the compound represented by formula (II) and the compound represented by formula (IIa) are included in the mixture as previously described herein.

[0240] The SHC enzymes described herein may provide, for example, increased conversion of compounds of formula (II) and / or compounds of formula (IIa) compared to their parent SHC enzymes. Thus, methods using the SHC enzymes described herein may provide increased conversion of compounds of formula (II) and / or compounds of formula (IIa) compared to methods using their parent SHC enzymes. The SHC enzymes described herein may provide, for example, increased conversion of compounds of formula (II) and / or compounds of formula (IIa) over the first 2 hours, the first 4 hours, the first 6 hours, the first 8 hours, the first 12 hours, the first 24 hours, the first 36 hours, the first 48 hours, the first 72 hours, the first 96 hours, the first 120 hours, the first 144 hours, or the first 168 hours of the reaction compared to their parent SHC enzymes. Thus, a method using an SHC enzyme described herein may provide increased conversion of a compound of formula (II) and / or a compound of formula (IIa) over the first 2 hours, over the first 4 hours, over the first 6 hours, over the first 8 hours, over the first 12 hours, over the first 24 hours, over the first 36 hours, over the first 48 hours, over the first 72 hours, over the first 96 hours, over the first 120 hours, over the first 144 hours, or over the first 168 hours of the reaction, preferably over the first 24 hours, compared to a method using its parent SHC enzyme, for example.

[0241] In some embodiments, the total conversion and / or ratio of a compound represented by formula (II) and / or a compound represented by formula (IIa) exhibited by an SHC enzyme described herein is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (2-fold), 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold , 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, 50x, 51x, 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x, 60x, 61x, 62x, 63x, 64x, 65x, 66x, 67x, 68x, 69x, 70x, 71x times, 72x, 73x, 74x, 75x, 76x, 77x, 78x, 79x, 80x, 81x, 82x, 83x, 84x, 85x, 86x, 87x, 88x, 89x, 90x, 91x, 92x, 93x, 94x, 95x, 96x, 97x, 98x, 99x, 100x, 200x, 500x, or 1000x higher.

[0242] In some embodiments, improved total conversion and / or ratio of a compound represented by formula (II) and / or a compound represented by formula (IIa) exhibited by an SHC enzyme described herein compared to its parent SHC enzyme is obtained in a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa) described herein.

[0243] The SHC enzymes described herein may provide, for example, improved conversion of compounds represented by formula (II) to compounds represented by formula (I) (which may alternatively be defined as the yield of compounds represented by formula (I)) compared to the parent SHC enzyme. In other words, the SHC enzymes described herein may result in more grams / mole of compounds represented by formula (I) formed per gram / mole of compounds represented by formula (II) converted compared to the parent SHC enzyme. The SHC enzymes described herein may provide, for example, improved conversion of compounds represented by formula (IIa) to compounds represented by formula (Ia) (such as compounds represented by formula (V)) (which may alternatively be defined as the yield of compounds represented by formula (Ia)) compared to the parent SHC enzyme. In other words, the SHC enzymes described herein may result in more grams / mole of compounds represented by formula (Ia) (such as compounds represented by formula (V)) formed per gram / mole of compounds represented by formula (IIa) converted compared to the parent SHC enzyme.

[0244] In some embodiments, the conversion of a compound represented by formula (II) and / or a compound represented by formula (IIa) to a compound represented by formula (Ia) (e.g., a compound represented by formula (V)) achieved by the SHC enzymes described herein is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (2-fold), 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, , 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, 50x, 51x , 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x, 60x, 61x, 62x, 63x, 64x, 65x, 66x, 67x, 68x, 69x, 70x, 71x, 72x, 73x, 74x, 75x, 76x, 77x, 78x, 79x, 80x, 81x, 82x, 83x, 84x, 85x, 86x, 87x, 88x, 89x, 90x, 91x, 92x, 93x, 94x, 95x, 96x, 97x, 98x, 99x, 100x, 200x, 500x, or 1000x higher.

[0245] In some embodiments, the SHC enzymes described herein are given in mole percent and based on the moles of compound of formula (II) employed, the SHC enzymes are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 102, 104, 105, 106, 1 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, to effect conversion of a compound represented by formula (II) to a compound represented by formula (I), and / or conversion of a compound represented by formula (IIa) to a compound represented by formula (Ia) (such as a compound represented by formula (V)). Preferably the yield is from 5 to 100, 10 to 100, 20 to 100, 30 to 100, 35 to 100, more preferably from 35 to 100, 45 to 100, 50 to 100, 60 to 100, or 70 to 100 mole percent. Preferably the conversion is measured at or after 24 hours reaction time.

[0246] In some embodiments, the improved conversion of a compound represented by formula (II) to a compound represented by formula (I) and / or a compound represented by formula (IIa) to a compound represented by formula (Ia) (such as a compound represented by formula (V)) exhibited by the SHC enzymes described herein compared to their parent SHC enzymes described herein is obtained in a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa) described herein. Additional non-limiting parameters that may characterize the SHC enzymes described herein include, among others, specificity (e.g., substrate specificity, bond specificity, group specificity, optical specificity, cofactor specificity, geometric specificity), reaction rate, by-product formation, sensitivity to reaction conditions (e.g., pH, temperature, substrate concentration, concentration of solubilizing agent such as SDS), and resistance to product inhibition.

[0247] The SHC enzymes described herein may be compared to their parent enzymes under the same reaction conditions (e.g., the same pH, temperature, substrate concentration, concentration of solubilizing agent such as SDS) or under conditions individually defined as optimal for the activity of each enzyme, which may be the same or different from each other. The reaction capacity of an SHC enzyme, relative to its parent SHC enzyme, for any of the reaction conditions may be assessed using any of the parameters described above, such as productivity, total or increased conversion of a compound of formula (II) and / or a compound of formula (IIa), or yield of a compound of formula (I) and / or a compound of formula (Ia) (such as a compound of formula (V)), and may be, for example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (2-fold), 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47 x, 48x, 49x, 50x, 51x, 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x, 60x, 61x, 62x, 63x, 64x, 65x, 66x, 67x, The improvement may be up to 68-fold, 69-fold, 70-fold, 71-fold, 72-fold, 73-fold, 74-fold, 75-fold, 76-fold, 77-fold, 78-fold, 79-fold, 80-fold, 81-fold, 82-fold, 83-fold, 84-fold, 85-fold, 86-fold, 87-fold, 88-fold, 89-fold, 90-fold, 91-fold, 92-fold, 93-fold, 94-fold, 95-fold, 96-fold, 97-fold, 98-fold, 99-fold, 100-fold, 200-fold, 500-fold, or 1000-fold. Preferably, the reaction capacity is measured at or after a 24 hour reaction time.

[0248] The reaction capacity of the SHC enzyme as described herein may be assessed using any substrate concentration, e.g., a substrate concentration of at least 1 g / L or more. In embodiments in which a host cell expressing the SHC enzyme as described herein is utilized, the reaction capacity may be assessed using any substrate concentration as defined above and / or any cell concentration, e.g., a cell concentration of at least 1 g / L or more.

[0249] In particular, the SHC enzyme described herein may exhibit improved reaction capacity at high substrate concentrations compared to its parent SHC enzyme. A compound represented by formula (II) concentration of 50 g / L or more may be considered to be a high substrate concentration. In some embodiments, the SHC enzyme may exhibit improved reaction capacity at a compound represented by formula (II) concentration of 50 g / L or more, 60 g / L or more, 70 g / L or more, 80 g / L or more, 90 g / L or more, 100 g / L or more, 110 g / L or more, 120 g / L or more, 130 g / L or more, 135 g / L or more, 150 g / L or more, 175 g / L or more, or 200 g / L or more, or 250 g / L or more, preferably at a concentration of 135 g / L or more, compared to its parent SHC enzyme.

[0250] In some embodiments utilizing host cells expressing SHC enzymes as described herein, the SHC enzymes may exhibit improved reaction capacity at high cell concentrations compared to their parent SHC enzymes. A cell concentration of 50 g / L or more may be considered to be a high cell concentration. The SHC enzymes may exhibit improved reaction capacity at cell concentrations of 50 g / L or more, 60 g / L or more, 70 g / L or more, 80 g / L or more, 90 g / L or more, 100 g / L or more, 110 g / L or more, 120 g / L or more, 130 g / L or more, 150 g / L or more, 175 g / L or more, or 200 g / L or more, or 250 g / L or more, preferably at a concentration of 175 g / L or more, compared to their parent SHC enzymes.

[0251] In some embodiments, the improved reaction performance exhibited by the SHC enzymes described herein compared to their parent SHC enzymes is obtained in a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa) described herein. In some embodiments, the ratio of SHC enzyme to substrate, or the ratio of host cells expressing the SHC enzyme to substrate, may be adjusted to optimize the bioconversion reaction.

[0252] In some embodiments, the SHC enzyme or a host cell expressing the SHC enzyme has a substrate to substrate ratio of 0.1 to 4 to 1 or about 0.1 to 4 to 1 (0.1 to 4:1), 0.1 to 3 to 1 or about 0.1 to 3 to 1 (0.1 to 3:1), 0.1 to 2 to 1 or about 0.1 to 2 to 1 (0.1 to 2:1), 0.25 to 2 to 1 or about 0.25 to 2 to 1 (0.25 to 2:1), 0.5 to 2 to 1 or about 0.5 to 2 to 1 (0.5 to 2:1), 0. The weight ratio is 1 to 1 or about 0.1 to 1 (0.1:1), 0.5 to 1 or about 0.5 to 1 (0.5:1), 1 to 1 or about 1 to 1 (1:1), 1.5 to 1 or about 1.5 to 1 (1.5:1), or 2 to 1 or about 2 to 1 (2:1), preferably 0.1 to 1 or about 0.1 to 1 (0.1:1), 0.5 to 1 or about 0.5 to 1 (0.5:1), or 1 to 1 or about 1 to 1 (1:1).

[0253] As a result, the SHC enzymes described herein may exhibit at least one, at least two, at least three, or all of the following advantages compared to the parent SHC enzyme: Improved conversion ratio of the compound represented by formula (II) and / or the compound represented by formula (IIa) Improved yield of the compound represented by formula (I) and / or the compound represented by formula (Ia) • Improved reaction performance (e.g. conversion rate, productivity, yield at high substrate concentration).

[0254] As used herein, the "selectivity" of the SHC enzyme described herein may refer to the ability of the enzyme to react with a specific substrate compared to another substrate. As a non-limiting example, the SHC enzyme may be selective for the E,Z-isomer of the compound represented by formula (II) compared to the E,E-isomer or another isomer, meaning that the enzyme is more likely to convert the E,Z-isomer than the E,E-isomer or another isomer. As another non-limiting example, the SHC enzyme may be selective for the E,Z-isomer of the compound represented by formula (IIa) compared to the E,E-isomer or another isomer. As another non-limiting example, the SHC enzyme may be selective for a specific structural isomer of a compound, such as the compound represented by formula (II) or the compound represented by formula (IIa). As another non-limiting example, the SHC enzyme described herein and used in the methods described herein may illustratively have a selectivity for the compound represented by formula (II) that is equal to or greater than 75% or is about 75%. As further non-limiting examples, the SHC enzyme or its parent SHC enzyme may have a selectivity that is greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 95%. For example, the SHC enzyme or its parent SHC enzyme may have a selectivity of up to or about 100%, e.g., less than or equal to 100% (less than or equal to 99.5% or about 99.5%, less than or equal to 99% or about 99%, less than or equal to 98% or about 98%, or less than or equal to 97% or about 97%).

[0255] As another non-limiting example, the SHC enzyme described herein and used in the methods described herein may illustratively have a selectivity for compounds of formula (IIa) that is equal to or greater than or about 75%. As a further non-limiting example, the SHC enzyme or its parent SHC enzyme may have a selectivity that is equal to or greater than or about 80%, equal to or greater than or about 85%, equal to or greater than or about 90%, equal to or greater than or about 95%, or equal to or greater than or about 95%. For example, the SHC enzyme or its parent SHC enzyme may have a selectivity of up to or about 100%, e.g., less than or about 100% (such as less than or equal to 99.5% or about 99.5%, less than or equal to 99% or about 99%, less than or equal to 98% or about 98%, or less than or equal to 97% or about 97%, etc.).

[0256] The methods for making the compounds of formula (I) and / or compounds of formula (Ia) (such as compounds of formula (V)) disclosed herein may be carried out at the optimum temperature range or optimum temperature, and / or the optimum pH range or optimum pH, and / or the optimum concentration range or optimum solubilizing agent (such as SDS) concentration of the particular enzyme (such as a specific SHC variant) used as discussed later herein. Examples are further provided in the experimental section. Additional examples may be found in WO2021 / 209482.

[0257] Nucleic acids and vectors The SHC enzyme described herein may be encoded by a nucleotide sequence. The nucleic acid molecule comprising the nucleotide sequence may be, for example, an isolated nucleic acid molecule. Accordingly, the present disclosure further provides a nucleic acid molecule comprising a nucleotide sequence encoding the squalene hopene cyclase (SHC) enzyme as described herein.

[0258] The terms "nucleic acid" or "nucleic acid molecule" as used herein are used interchangeably to refer to a polynucleotide of the present disclosure, which may be DNA, cDNA, genomic DNA, synthetic DNA, or RNA, and may be double-stranded or single-stranded, the sense strand or the antisense strand.

[0259] The term specifically applies to polynucleotides encoding the SHC enzymes described herein, including full-length nucleotide sequences or fragments thereof that encode an SHC polypeptide or fragment thereof that exhibits its enzymatic activity. The term also encompasses separate molecules, such as cDNAs in which the corresponding genomic DNA of the cDNA has a different sequence due to introns, genomic fragments lacking at least one of the flanking genes, fragments of cDNA or genomic DNA produced by polymerase chain reaction (PCR) and lacking at least one of the flanking genes, restriction fragments lacking at least one of the flanking genes, and nucleic acids that are degenerate variants of cDNAs or naturally occurring nucleic acids.

[0260] The nucleic acid molecule may contain a codon-optimized sequence for expression in a specific host cell. "Codon optimization" as used herein refers to a process employed to modify an existing coding sequence or design a coding sequence, for example to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of the coding sequence. Codon optimization includes processes that include, but are not limited to, selecting codons for a coding sequence that match the codon preference of the expression host cell, for example, mammalian cells, insect cells, plant cells, or microbial cells, preferably microbial cells such as E. coli, and other codon preferences. Examples of microbial cells include eukaryotes such as yeast, filamentous fungi, and algae, and prokaryotes such as bacteria and archaea. Codon optimization also eliminates elements that potentially negatively affect RNA stability and / or translation, for example, termination sequences, TATA boxes, splice sites, ribosome entry sites, repeats and / or GC-rich sequences, and RNA secondary structure or instability motifs.

[0261] In this regard, nucleic acid molecules encoding SHC enzymes may contain the original nucleotide sequence as found in the source organism, or may contain sequences that have been codon-optimized for expression in a selected host cell, such as E. coli and other cells.

[0262] The present disclosure further provides a nucleic acid construct comprising a nucleotide sequence encoding an SHC enzyme as described herein, operably linked to a regulatory sequence, e.g., a transcription initiation sequence, such as a promoter sequence. As used herein, "nucleic acid construct" typically refers to an artificially created nucleic acid to be introduced into a target cell. Thus, the regulatory sequence operably linked to the nucleotide sequence encoding an SHC enzyme as described herein may not be associated with it in nature.

[0263] Optionally, other regulatory sequences, such as transcription terminators, enhancers, repressors, silencers, Kozak sequences, polyA sequences, and the like, may be operably linked to the nucleotide sequence encoding the SHC enzyme.

[0264] The above-mentioned regulatory sequences include, but are not limited to, inducible and non-inducible, constitutive, cell cycle-regulated, metabolically-regulated, enhancers, operators, silencers, repressors, and other elements known by those skilled in the art and which drive or otherwise regulate gene expression in cells. Such regulatory sequences include, but are not limited to, regulatory sequences that direct constitutive expression or regulatory sequences that allow inducible expression, such as, for example, the CUP-1 promoter, the Tet-repressor when employed in, for example, a Tet-on or Tet-off system, the Lac operon regulatory sequence, or the Trp operon regulatory sequence.

[0265] As a non-limiting example, isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effective inducer of gene expression, illustratively in the concentration range of 100 pM to 1.0 mM, when the Lac operon regulatory sequence is operably linked to a nucleotide sequence of interest. This compound is a molecular mimic of allolactose, a lactose metabolite that initiates transcription of the Lac operon, and therefore may be used to induce expression of a nucleotide sequence when the nucleotide sequence is under the control of the Lac operator.

[0266] The nucleic acid constructs described herein may further comprise nucleotide sequences encoding additional polypeptides, such as sequences that function as markers or reporters, and / or tags (e.g., His-tags) that allow for the isolation and / or purification of the encoded polypeptide (e.g., via affinity chromatography). In this regard, the nucleic acid constructs may comprise nucleotide sequences encoding "hybrid," "fusion," or "chimeric" proteins that represent fusions of SHC enzymes, such as markers, reporters, or tags. Fusion proteins may comprise one or more amino acids, such as, but not limited to, histidine (His), compared to the SHC enzymes from which they originate, usually at the N-terminus of the protein, but also fused at the C-terminus or within internal regions of the protein. Such fusion proteins or nucleic acid constructs encoding such proteins typically serve three purposes: (i) to increase the production of the recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the isolation and / or purification of the recombinant protein by providing a ligand for affinity purification. The SHC enzymes described herein may be said to be isolated when they are separated from the cellular or in vitro components used to produce them.

[0267] The marker may be a selectable marker. The term "selectable marker" refers to a polypeptide that can be used for the selection of host cells expressing it by conferring a selective advantage to the cells when exposed to selective conditions. A selectable marker may allow positive or negative selection. Suitable selectable markers are known in the art, and such markers and selection methods are discussed, for example, in standard publications such as Mortensen and Kingston (2009) Curr Protoc Mol Biol 86:9.5.1-9.5.13 (incorporated herein by reference in its entirety), and in standard handbooks such as Ausubel et al. (2003) and Sambrook and Green (2012) (see above). Those skilled in the art will understand that a particular selectable marker may allow positive or negative selection depending on the host cell and / or selection conditions applied. A positive selectable marker is a marker that allows the growth of a host cell when exposed to selective conditions, where growth would not otherwise occur. Negative selectable markers are markers that prevent the growth of a host cell when exposed to selective conditions. Non-limiting examples of suitable marker or reporter polypeptides that may be encoded by additional sequences included in the nucleotide construct include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), beta-galactosidase, and xanthine guanine phosphoribosyltransferase (XGPRT).

[0268] Examples of suitable tags include AviTag, calmodulin-tag, polyglutamate-tag, E-tag, FLAG-tag, HA-tag, His-tag, Myc-tag, S-tag, SBP-tag, Softag 1 and 3, Strep-tag, TC-tag, V5-tag, VSV-tag, X-press tag, isopeptag, SpyTag, BCCP, glutathione-S-transferase-tag, GFP-tag, Halo-tag, maltose binding protein-tag, Nus-tag, thioredoxin-tag, and Fc-tag.

[0269] Those skilled in the art are aware of suitable regulatory sequences and additional sequences that may be included in the nucleic acid construct of the present disclosure, as well as the molecular toolbox techniques that may be used to arrive at the nucleic acid construct described herein, examples of which may be found in standard handbooks such as Ausubel et al., Current Protocols in Molecular Biology, 3rd edition, John Wiley & Sons Inc (2003), and in Sambrook and Green, Molecular Cloning. A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press (2012); both of which are incorporated herein by reference in their entirety. Further examples may be found in WO2021 / 209482.

[0270] The present disclosure further provides a vector comprising a nucleic acid molecule or nucleic acid construct as described herein. As used herein, a "vector" is a nucleic acid molecule used as a vehicle to artificially introduce foreign genetic material into a cell in which it can be replicated and / or expressed. A vector may be linear or circular. A vector may be maintained in a host cell at low copy number (e.g., 1-2 copies per cell), medium copy number (e.g., 3-20 copies per cell), or high copy number (e.g., >20 copies per cell). Origins of replication for low, medium, and high copy vectors are known to those of skill in the art. A vector may be, for example, a plasmid, megaplasmid, cosmid, phagemid, phage, viral vector (e.g., adenoviral or retroviral vector), knockout or knockin construct, or artificial chromosome, such as a bacterial, yeast, plant, or mammalian artificial chromosome. A preferred vector is a plasmid. A skilled artisan will appreciate that the terms nucleic acid construct and vector may overlap, for example in the case of a plasmid.

[0271] The proteins encoded by the nucleic acid molecules, nucleic acid constructs or vectors described herein are preferably expressed upon their introduction into a host cell.

[0272] Host cells, methods of making the host cells, and methods of using the host cells to make compounds of formula (I) In one aspect, the present disclosure provides a host cell comprising a nucleic acid molecule, a nucleic acid construct, or a vector as described herein. The host cell preferably expresses (alternatively referred to herein as "producing") an SHC enzyme as described herein. The host cell of the present disclosure is alternatively referred to herein as a "cell," "recombinant cell," or "recombinant host cell." In this context, "recombinant" refers to a genetic modification that is introduced into the cell.

[0273] The host cell may be used in the methods described herein. For example, the method for producing the compound of formula (I) and / or the compound of formula (Ia) (such as the compound of formula (V)) as described herein may include culturing the host cell as described herein. The term "culturing" refers to the process of multiplying living cells so that they produce the SHC enzyme as described herein. Consequently, the benefits related to the SHC enzyme and the methods using the SHC enzyme described herein also apply to the host cell expressing the SHC enzyme and to the methods using the host cell.

[0274] The nucleic acid molecules, nucleic acid constructs or vectors described herein may be introduced into a host cell using standard molecular toolbox techniques available to the skilled artisan, which may vary depending on the host cell (e.g. prokaryotic or eukaryotic). Examples of such techniques are transfection or (viral) transduction. Further examples of such techniques may further be found in standard handbooks such as Ausubel et al. (2003), and Sambrook and Green (2012) (see above).

[0275] The introduced ("transforming") nucleic acid may or may not be integrated, i.e., covalently linked, into the chromosome of the cell. In prokaryotes and yeast, for example, the introduced nucleic acid may be maintained on an episomal element, such as a plasmid. For eukaryotic cells, a stably transfected cell is one in which the transfected nucleic acid is integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the eukaryotic cell's ability to establish cell lines or clones composed of a population of daughter cells containing the introduced nucleic acid. In prokaryotic and / or eukaryotic cells, the integration of the nucleic acid into the genome of the host cell may occur through cellular DNA repair mechanisms, such as, for example, homologous recombination, non-homologous end-joining, etc. Integration of the nucleic acid may be mediated by the introduction of a break in the host cell chromosome using a nuclease, such as, for example, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nuclease, or a recombinase (e.g., Cre recombinase). Nucleases and recombinases are known to those of skill in the art, and their use in transforming host cells is further described in Musunuru Kiran, Genome Editing: A Practical Guide to Research and Clinical Applications, 1 st Edition, Academic Press (2021), and Ghosh Dipanjan (Ed), Advances in CRISPR / Cas and Related Technologies, 1 st Edition, Academic Press (2021), both of which are incorporated herein by reference in their entireties.

[0276] Typically, the introduced nucleic acid is not native to the recipient host cell, but it is within the scope of the present disclosure to isolate a nucleic acid from a given host and then introduce one or more copies of that nucleic acid into the same host, e.g., to enhance production of the product of a gene or to alter the expression pattern of a gene, such as a gene expressing an SHC enzyme described herein. In some cases, the introduced nucleic acid will modify or even replace an endogenous nucleic acid sequence, e.g., by homologous recombination or site-directed mutagenesis.

[0277] Consequently, expression of an SHC enzyme by a host cell as described herein may refer to homologous expression (wherein the nucleotide sequence encoding the enzyme is naturally present in the cell) or heterologous expression (wherein the nucleotide sequence encoding the enzyme is not naturally present in the cell).

[0278] Suitable host cells may be selected from prokaryotic or eukaryotic cells, such as bacteria, archaea, yeast, filamentous fungi, algae, plant cells, animal cells, amphibian cells (including melanophore cells), insect cells, worm cells, and mammalian cells.

[0279] Algal host cells may be selected from a suitable group known in the art, such as Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria, Pleurochrysis carterae, and Sargassum. Yeast host cells may be selected from a suitable group known in the art, such as Saccharomyces (e.g., Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii), Candida (e.g., Candida utilis, Candida krusei), Schizosaccharomyces (e.g., Schizosaccharomyces pombe, Schizosaccharomyces japonicus), Pichia or Hansenula (e.g., Pichia pastoris or Pichia pastoris (Komagatella phaffi), or Hansenula polymorpha), Yarrowia, Kluyveromyces, and Brettanomyces (e.g., Brettanomyces claussenii).

[0280] Filamentous fungal host cells include Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Me The fungus may be selected from a suitable group known in the art, such as, for example, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria, and the like. Species include Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporiumpannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenaturn, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Penicillium chrysogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia Australeinsis、Thielavia fimeti、Thielavia microspora、Thielavia ovispora、Thielavia peruviana、Thielavia setosa、Thielavia spededonium、Thielavia subthermophila、Thielavia terrestris、Trichoderma harzianum、Trichoderma koningii、Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0281] Insect host cells and helminth cells may be selected from suitable groups known in the art, such as Sf9 cells, Sf21 cells, Spodoptora frugiperda cells, Caenorhabditis cells (such as Caenorhabditis elegans cells), and their derivatives. Mammalian host cells may be selected from suitable groups known in the art, such as human cells, Chinese Hamster Ovary (CHO) cells, COS cells (including Cos-1 and Cos-7), HEK293 cells, HEK293T cells, HEK293 T-RexTM cells, PerC6™ cells, HeLa cells, Jurcat cells, hybridomas, and their derivatives. Plant host cells may be selected from suitable groups known in the art, such as Arabidopsis.

[0282] Preferred host cells are bacterial host cells which may be selected from suitable groups known in the art. Bacterial host cells include Bacillus (e.g., Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus, Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis), Paenibacillus, Streptomyces, Micrococcus, Corynebacterium, Acetobacter, Cyanobacteria, Salmonella, Rhodococcus, Pseudomona s, Lactobacillus, Lactococcus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Thermus aquaticus, Pseudomonas stutzeri, Clostridium The host cells include both gram-negative and gram-positive bacteria, such as Escherichia (e.g., Escherichia coli) (including strains thereof), and Escherichia thermocellus and Escherichia (e.g., Escherichia coli) (including strains thereof). Among bacterial host cells, E. coli and its strains are preferred. Mutants, multiple libraries of plasmids, detailed computer models of metabolism, transformation methods, and other information for E. coli are available in the art, allowing rational design of various genetic modules to enhance the production yield of recombinant host cells expressing enzymes. Preferably, the E. coli host cell is an E. coli strain (including but not limited to K12 and BL21 strains) that are recognized as safe by industry and regulatory agencies. The use of E. coli as a host cell may be advantageous in producing a compound of formula (I) from a compound of formula (II), considering that a low-cost and industrially economical process can be relatively easily designed for this host cell.

[0283] Several host cells and strains from the groups discussed above are readily accessible to the public in a number of well-known collections, such as the American Type Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), the Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0284] In some embodiments, the host cell is a bacterial host cell selected from the group of Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus and strains thereof, preferably Escherichia coli and strains thereof. Further examples of suitable host cells and transformation methods may be found in WO2021 / 209482.

[0285] Cultivation of the host cells described herein may be carried out in a conventional manner. Suitable cell culture methods are known to those skilled in the art and are discussed, for example, in van't Riet, K. and Tramper, J., 1st edition, Basic Bioreactor Design, CRC Press, NY, 1991, which is incorporated herein by reference in its entirety. Such methods include, but are not limited to, submerged fermentation in liquid media, surface fermentation in liquid media, and solid-state fermentation. Culturing of cells may be carried out in a laboratory and / or industrial setting, for example, by cultivation in microtiter plates, shake flasks, small benchtop bioreactors, medium-sized bioreactors, and / or large-sized bioreactors. Suitable cell culture modes include, but are not limited to, continuous culture, batch culture, and / or fed-batch culture, and combinations thereof. Typically, cells are grown to a particular density (measurable, for example, as optical density (OD)) to produce sufficient biomass and / or SHC enzymes for the bioconversion reactions to occur as previously described herein.

[0286] In some embodiments, methods are provided for making a compound of formula (I) in a cell system, comprising producing an SHC enzyme described herein under conditions suitable for the cell system, feeding a compound of formula (II) to the cell system, converting the compound of formula (II) to a compound of formula (I) using the SHC enzyme produced using the cell system, harvesting the compound of formula (I) from the cell system, and optionally isolating and / or purifying the compound of formula (I).

[0287] In some embodiments, there is provided a method of making a compound of formula (Ia) (preferably a compound of formula (V)) in a cell system, comprising producing an SHC enzyme as described herein under conditions suitable for the cell system, delivering a compound of formula (IIa) to the cell system, converting the compound of formula (IIa) to a compound of formula (I) (preferably a compound of formula (V)) using the SHC enzyme produced using the cell system, harvesting the compound of formula (Ia) (preferably a compound of formula (V)) from the cell system, and optionally isolating and / or purifying the compound of formula (Ia) (preferably a compound of formula (V)).

[0288] In some embodiments, methods are provided for making a mixture comprising a compound of formula (I) and a compound of formula (Ia) in a cell system, comprising producing an SHC enzyme as described herein under conditions suitable for the cell system, feeding a mixture comprising a compound of formula (II) and a compound of formula (IIa) to the cell system, converting the compound of formula (II) to a compound of formula (I) and the compound of formula (IIa) to a compound of formula (Ia) using the SHC enzyme produced using the cell system, collecting the compound of formula (I) and the compound of formula (Ia) from the cell system, and optionally isolating and / or purifying the compound of formula (I) and / or the compound of formula (Ia).

[0289] In some embodiments, methods are provided for making a mixture comprising a compound of formula (I) and a compound of formula (V) in a cell system, comprising producing an SHC enzyme as described herein under conditions suitable for the cell system, feeding a mixture comprising a compound of formula (II) and a compound of formula (IIa) to the cell system, using the SHC enzyme produced using the cell system to convert the compound of formula (II) to a compound of formula (I) and the compound of formula (IIa) to a compound of formula (V), collecting the compound of formula (I) and the compound of formula (V) from the cell system, and optionally isolating and / or purifying the compound of formula (I) and / or the compound of formula (V).

[0290] Expression of other nucleic acids may serve to enhance the process, for example by enhancing the activity of the cell lines used in the bioconversion reactions described above.

[0291] In some embodiments, methods are provided for making a compound of formula (I) that include culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding an SHC enzyme as described herein, producing the SHC enzyme in the host cell, adding a compound of formula (II) to the cell culture, incubating the cell culture under conditions of pH, temperature, and optionally a solubilizing agent (such as SDS) suitable to promote conversion of the compound of formula (II) to a compound of formula (I), collecting the compound of formula (I), and optionally isolating and / or purifying the compound of formula (I).

[0292] In some embodiments, methods are provided for making a compound of formula (I) (preferably a compound of formula (V)) comprising culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding an SHC enzyme as described herein, producing the SHC enzyme in the host cell, adding a compound of formula (IIa) to the cell culture, incubating the cell culture under conditions of pH, temperature, and optionally a solubilizing agent (such as SDS) suitable to promote conversion of the compound of formula (IIa) to a compound of formula (Ia) (preferably a compound of formula (V)), collecting the compound of formula (Ia) (preferably a compound of formula (V)), and optionally isolating and / or purifying the compound of formula (I) (preferably a compound of formula (V)).

[0293] In some embodiments, methods are provided for making a mixture comprising a compound of formula (I) and a compound of formula (Ia), comprising culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding an SHC enzyme as described herein, producing the SHC enzyme in the host cell, adding a mixture comprising a compound of formula (II) and a compound of formula (IIa) to the cell culture, incubating the cell culture under conditions of pH, temperature, and optionally a solubilizing agent (such as SDS) suitable to promote conversion of the compound of formula (II) to a compound of formula (I) and conversion of the compound of formula (IIa) to a compound of formula (Ia), collecting the compound of formula (I) and the compound of formula (Ia), and optionally isolating and / or purifying the compound of formula (I) and / or the compound of formula (Ia).

[0294] In some embodiments, methods are provided for making a mixture comprising a compound of formula (I) and a compound of formula (V), comprising culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding an SHC enzyme as described herein, producing the SHC enzyme in the host cell, adding a mixture comprising a compound of formula (II) and a compound of formula (IIa) to the cell culture, incubating the cell culture under conditions of pH, temperature, and optionally a solubilizing agent (such as SDS) suitable to promote conversion of the compound of formula (II) to a compound of formula (I) and conversion of the compound of formula (IIa) to a compound of formula (V), collecting the compound of formula (I) and the compound of formula (V), and optionally isolating and / or purifying the compound of formula (I) and / or the compound of formula (V).

[0295] The bioconversion reaction may be enhanced by adding more biocatalyst, and optionally a solubilizing agent such as SDS, to the cell culture as described above.

[0296] Suitable cell culture conditions for growth and enzyme production by the host cells may vary depending on the host cell. Such conditions are known to those skilled in the art and are typically provided, for example, by the cell culture harvest from which the host cells may be obtained. The cell culture conditions and the bioconversion reaction conditions may be the same or different. Those skilled in the art further understand that the cells may be initially cultured under optimal conditions for cell growth and / or enzyme production, and subsequently the conditions may be adjusted to optimal conditions for the bioconversion reaction to occur, which may be the same or different.

[0297] The term "biocatalyst", as used herein, may refer to the SHC enzyme itself as described herein, but may also refer to a host cell expressing the enzyme, a membrane fraction of the host cell, a cell lysate, cell debris, or a cell-free extract, which share in common the presence of SHC enzyme activity.

[0298] In some embodiments, the biocatalyst is a recombinant host cell that produces the SHC enzyme, optionally in suspension or immobilized format.

[0299] In some embodiments, the biocatalyst is a membrane or liquid fraction prepared from a recombinant host cell producing an SHC enzyme, such as a crude or cell-free extract, using routine methods (e.g., as disclosed in Seitz (2012), Characterization of the substrate specificity of squalene-hopene cyclases (SHCs), PhD thesis, University of Stuttgart, available at http: / / dx.doi.org / 10.18419 / opus-1383, which is incorporated herein by reference in its entirety).

[0300] Biocatalysts include whole cells harvested from cell culture (e.g., from a bioreactor cell culture), as well as cells still in culture, which are then used later in the one-pot process described herein. Biocatalysts include intact recombinant host cells and / or their cellular debris.

[0301] Biocatalyst may be immobilized. Immobilization of host cell and / or SHC enzyme may be achieved by any means known to those skilled in the art, for example as discussed in Seitz et al. (see above) and in standard handbooks such as Guisan, JM, Bolivar, JM, Lopez-Gallego, F., Rocha-Martin, J. (Eds.), Immobilization of Enzymes and Cells: Methods and Protocols, Springer US, USA, 2020 (incorporated herein by reference in its entirety). An example of immobilization method involves polymerizing or solidifying the solution containing spores or cells. Examples of polymerizable or solidifyable solutions include alginate, lambda-carrageenan, chitosan, polyacrylamide, polyacrylamide-hydrazide, agarose, polypropylene, polyethylene glycol, dimethyl acrylate, polystyrene divinylbenzene, polyvinylbenzene, polyvinyl alcohol, epoxy carriers, cellulose, cellulose acetate, photocrosslinkable resins, prepolymers, urethane, and gelatin. Another example of an immobilization method involves cell adsorption onto a support. Examples of such supports include bone charcoal, cork, clay, resin, sand porous alumina beads, porous bricks, porous silica, celite, or wood chips. The host cells can colonize the support to form a biofilm. Another example of an immobilization method involves covalent coupling of the host cells to the support using chemical agents such as glutaraldehyde, o-dianisidine, polymeric isocyanates, silanes (as discussed, for example, in US 3,983,000; US 4,071,409; US 3,519,538, and US 3,652,761, all of which are incorporated by reference in their entireties), hydroxyethyl acrylate, transition metal activated supports, cyanuric chloride, sodium periodate, toluene, and the like.The cultured host cells can be immobilized at any phase of their growth, for example after reaching a desired cell density in culture.

[0302] In some embodiments, the host cells are cultured, harvested, washed, and optionally stored (eg, frozen or lyophilized) prior to their use in the bioconversion reaction.

[0303] In some embodiments, the host cells are cultured and then the culture conditions are adjusted prior to the bioconversion reaction to favor the reaction without harvesting and washing the cells. This one-step (or "one-pot") method may be advantageous as it may simplify the process. The culture medium used to grow the cells in these embodiments may also be used as the reaction mixture in the bioconversion reaction. The compound of formula (II), the compound of formula (IIa), and / or the mixture comprising the compound of formula (II) and the compound of formula (IIa) may be present in the culture from the beginning or may be added later to the culture phase of the method.

[0304] Cultivation of the cells may occur using a culture medium (alternatively referred to herein as a growth medium) containing suitable nutrients such as carbon and nitrogen sources, and optionally additional compounds such as inorganic salts and vitamins. Suitable culture media may vary depending on the host cell and may be available from commercial suppliers or prepared using published compositions (e.g., in the catalog of the Centraalbureau Voor Schimmelcultures collection (CBS) publicly available for each host cell). Suitable carbon sources include any molecule that can be metabolized by a recombinant host cell to facilitate the growth and / or production of the SHC enzyme as described herein for the conversion of a compound represented by formula (II) to a compound represented by formula (I) and / or a compound represented by formula (IIa) to a compound represented by formula (Ia) (such as a compound represented by formula (V)). Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., pure or as found in mixtures such as molasses), fructose, xylose, glycerol, glucose, ethanol, cellulose, starch, cellobiose, or any other carbohydrate containing polymers, and mixtures thereof. Examples of suitable nitrogen sources include, but are not limited to, urea, ammonia, ammonium salts, nitrates, and mixtures thereof. Complex carbon and nitrogen sources, such as protein hydrolysates, tryptone, soybean meal, corn steep liquor, whey protein hydrolysates, egg protein hydrolysates, casein hydrolysates, yeast extracts, and the like, are also suitable.

[0305] In embodiments where the host cell is a yeast cell, preferred carbon sources may be selected from sucrose, fructose, xylose, ethanol, glycerol, glucose, and mixtures thereof. The host cells may be cultured in a rich medium (eg, LB medium, Bacto-tryptone yeast extract medium, etc.) or in a defined medium, such as defined minimal medium.

[0306] In some embodiments, a defined minimal medium such as M9A medium or another defined minimal medium is used for culturing the cells. M9A medium may contain: 14 g / L KH 2 PO 4 , 16g / LK 2 HPO 4 , 1g / L Na 3 Citrate.2H 2 O, 7.5 g / L (NH 4 ) 2 SO 4 , 0.25g / L MgSO 4 .7H 2 0, 0.015g / L CaCl 2 .2H 2 O, 5 g / L glucose, and 1.25 g / L yeast extract.

[0307] In some embodiments, a rich medium such as LB medium or another rich medium is used for culturing the cells. LB medium may contain: 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl. Further examples of mineral media and M9 mineral media may be found, for example, in US6524831B2 and US2003 / 0092143A1.

[0308] Additional examples of suitable minimal media may be prepared as follows: Per 350 ml of culture: 307 ml of H 2 35 ml of citric acid / phosphate stock solution (133 g / L KH 2 PO 4 , 40g / L (NH 4 ) 2 HPO 4 , 17g / L citric acid.H 22O and has a pH of 6.3, and the pH may be adjusted to 6.8 with 32% w / v NaOH. The solution may be autoclaved under routine conditions used in the art, and after autoclaving, 0.85 ml 50% w / v MgSO 4 .7H 2 O stock solution (see below), 0.035ml trace element stock solution (see below), 0.035ml thiamine stock solution (see below), and 7ml of 20% w / v glucose solution may be added.

[0309] The trace element stock solution may contain: 50 g / L Na in deionized water. 2 EDTA.2H 2 O, 20 g / L FeSO 4 .7H 2 O, 3g / LH 3 BO 3 , 0.9g / L MnSO 4 .2H 2 O, 1.1g / L CoCI 2 , 80g / L CuCI 2 , 240g / L NiSO 4 .7H 2 O, 100g / L Kl, 1.4g / L (NH 4 ) 6 Mo 7 O 24 .4H 2 O, 1g / L ZnSO 4 .7H 2 O. Thiamine stock solution may contain: 2.25 g / L thiamine. HCl in deionized water. MgSO 4 The stock solution may contain: 50% w / v MgSO in deionized water 4 .7H 2 O.

[0310] Typically, the optimum pH for growing cells in cell culture is from 4 to 8. The optimum pH for a bioconversion reaction may vary depending on the characteristics of the SHC enzyme used. The pH of the bioconversion reaction mixture may be from 4 to 8, preferably from 5 to 6.5, more preferably from 5.5 to 6.1. Adjustment and regulation of the pH in the cell culture or reaction mixture may be done by any suitable technique known by the skilled artisan, for example, by addition of stock solutions of acids and bases, or by addition of buffers. Non-limiting examples of buffers include citrate buffers and succinate buffers.

[0311] Typically, the optimum temperature for the cell culture and / or bioconversion reaction is from 15° C. to 60° C., preferably from 25° C. to 50° C., more preferably from 25° C. to 45° C. The optimum pH for the bioconversion reaction may vary depending on the characteristics of the SHC enzyme used. In some embodiments, the optimum temperature is 30° C. The temperature may be kept constant or may be varied throughout the cell culture and / or bioconversion reaction.

[0312] Specific optimal pH and temperature conditions for certain preferred enzymes described herein are given in Table 5.

[0313] Typically, the cultivation of cells is carried out under anaerobic, aerobic or oxygen-limited conditions. Oxygen requirements vary depending on the host cell and the cultivation mode and will be known to the skilled artisan. Aerobic conditions are conditions in which the oxygen consumption of the host cells is not limited by the availability of oxygen. Under oxygen-limited conditions, the oxygen consumption is limited by the availability of oxygen. Oxygen may be supplied to the culture by any known method, for example by shaking under an air atmosphere, by stirring, by sparging air and / or oxygen into the culture, etc.

[0314] Optionally, solubilizing agents, such as surfactants, detergents, solubility enhancers, water-miscible organic solvents, and the like, may be added to the cell culture or to the bioconversion reaction mixture. As used herein, the term "surfactant" refers to a component that reduces the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersing agents. Examples of surfactants include, but are not limited to, Triton X-100, Tween 80, taurodeoxycholate, sodium taurodeoxycholate, sodium dodecyl sulfate (SDS), and / or sodium lauryl sulfate (SLS).

[0315] Although Triton X-100 may be used to partially purify the SHC enzyme (in soluble or membrane fraction / suspended form), it may also be used in bioconversion reactions (see, e.g., the disclosure in Seitz (2012, supra), as well as the disclosures of Neumann and Simon (1986), Biol Chem 367:723-729, and JP2009060799, both of which are incorporated herein by reference in their entireties).

[0316] A preferred solubilizing agent is SDS. Without wishing to be bound by theory, the use of SDS with recombinant host cells may be advantageous because SDS may advantageously interact with the host cell membrane to make the SHC enzyme (which is a membrane-bound enzyme) more accessible to the compound of formula (II) and / or the compound of formula (IIa) substrate. In addition, the inclusion of suitable levels of SDS in the cell culture and / or bioconversion reaction mixture may improve the emulsion properties (e.g., of the compound of formula (II) and / or the compound of formula (IIa) in water) and / or may improve the access of the compound of formula (II) and / or the compound of formula (IIa) substrate to the SHC enzyme in the host.

[0317] Those skilled in the art understand that the optimal concentration of the solubilizing agent (e.g., SDS) used in the bioconversion reaction described herein may vary depending on the amount of cell biomass and the substrate concentration. The optimal concentration of the solubilizing agent (e.g., SDS) for the bioconversion reaction may also vary depending on the characteristics of the SHC enzyme used. The determination of the appropriate concentration can be made by routine experimentation. In the method of the present disclosure, the concentration ratio of SDS / cells may be preferably from 10:1 to 20:1, more preferably from 15:1 to 18:1, when the ratio of biocatalyst to compound represented by formula (II) and / or compound represented by formula (IIa) is 2:1 or about 2:1. In some embodiments, the concentration ratio of SDS / cells may be preferably at or about 10:1, 11:1 or about 11:1, 12:1 or about 12:1, 13:1 or about 13:1, 14:1 or about 14:1, 15:1 or about 15:1, 16:1 or about 16:1, 17:1 or about 17:1, 18:1 or about 18:1, 19:1 or about 19:1, or 20:1 or about 20:1 when the ratio of biocatalyst to compound of formula (II) and / or compound of formula (IIa) is at or about 2:1.

[0318] In the methods of the present disclosure, the SDS concentration may be, for example, from 0.001% to 0.03%, preferably from 0.01% to 0.025%, more preferably from 0.01% to 0.02% (w / v %). These ranges correspond to the ranges used in reactions containing cells at an OD of 10 or about 10 (measured at 650 nm). Those skilled in the art will understand that suitable SDS concentrations are not limited to these ranges and may be increased or decreased when the cell concentration increases or decreases, respectively, in order to maintain a constant SDS / cell concentration ratio.

[0319] Specific exemplary SDS concentrations for certain preferred enzymes described herein are provided in Table 5. Additional exemplary SDS concentrations for bioconversion reactions utilizing host cells as described herein are provided in Examples 8 and 9.

[0320] In embodiments in which a compound of Formula (II), a compound of Formula (IIa), or a mixture comprising a compound of Formula (II) and a compound of Formula (IIa) is added to a cell culture or reaction mixture, the addition ("feeding") may be accomplished using standard means available to one of skill in the art (e.g., through tubing, using a peristaltic pump, using an infusion syringe, etc.).

[0321] The compound of formula (II) and / or the compound of formula (IIa) may be soluble in oil and provided dissolved in oil. In the case where the biocatalyst as previously described herein is present in an aqueous phase, the addition of the compound of formula (II) and / or the compound of formula (IIa) will result in a three-phase system (comprising an aqueous phase, a solid phase, and an oil phase). This may be the case even when SDS is present in the cell culture and / or reaction mixture.

[0322] In some embodiments, the cell culture is a continuous culture. Such a culture may be advantageous in some cases, as it may result in improved production of the compound of formula (I) and / or the compound of formula (Ia), such as the compound of formula (V).

[0323] In some embodiments, the bioconversion of a compound represented by formula (II) to a compound represented by formula (I) in the presence of a host cell expressing an SHC enzyme as described herein is given in mole percent and based on the moles of compound represented by formula (II) employed, is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of a compound of formula (II) to a compound of formula (I). Preferably the yield is from 5 to 100, 10 to 100, 20 to 100, 30 to 100, 35 to 100, more preferably from 40 to 100, 45 to 100, 50 to 100, 60 to 100, or 70 to 100 mole percent.

[0324] In some embodiments, the bioconversion of a compound represented by formula (IIa) to a compound represented by formula (Ia) (preferably to a compound represented by formula (V)) in the presence of a host cell expressing an SHC enzyme as described herein is given in mole percent and based on the moles of compound represented by formula (IIa) employed, is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 10 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of a compound of formula (IIa) to a compound of formula (Ia) (preferably to a compound of formula (V)). Preferably the yield is from 5 to 100, 10 to 100, 20 to 100, 30 to 100, 35 to 100, more preferably from 40 to 100, 45 to 100, 50 to 100, 60 to 100, or 70 to 100 mole percent.

[0325] In some embodiments, the bioconversion of a compound represented by formula (II) to a compound represented by formula (I) and / or a compound represented by formula (IIa) to a compound represented by formula (Ia) in a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa) in the presence of a host cell expressing an SHC enzyme as described herein is given in mole percent and is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of a compound of formula (II) to a compound of formula (I) and / or a compound of formula (IIa) to a compound of formula (Ia). Preferably the yield of compound (I) is from 5 to 100, 10 to 100, 20 to 100, 30 to 100, 35 to 100, more preferably from 40 to 100, 45 to 100, 50 to 100, 60 to 100, or 70 to 100 mole percent. Preferably, the yield of compound (Ia) is from 5 to 100, 10 to 100, 20 to 100, 30 to 100, 35 to 100, more preferably from 40 to 100, 45 to 100, 50 to 100, 60 to 100, or 70 to 100 mole percent.

[0326] In some embodiments, the bioconversion of a compound represented by formula (II) to a compound represented by formula (I) and / or a compound represented by formula (IIa) to a compound represented by formula (V) in a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa) in the presence of a host cell expressing an SHC enzyme as described herein is given in mole percent and is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 1 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of a compound of formula (II) to a compound of formula (I) and / or a compound of formula (IIa) to a compound of formula (V). Preferably the yield of compound (I) is from 5 to 100, 10 to 100, 20 to 100, 30 to 100, 35 to 100, more preferably from 40 to 100, 45 to 100, 50 to 100, 60 to 100, or 70 to 100 mole percent. Preferably, the yield of compound (V) is from 5 to 100, 10 to 100, 20 to 100, 30 to 100, 35 to 100, more preferably from 40 to 100, 45 to 100, 50 to 100, 60 to 100, or 70 to 100 mole percent.

[0327] In some embodiments, the preferred rate of conversion of a compound represented by formula (II) and / or a compound represented by formula (IIa), and / or the resulting conversion of a compound represented by formula (II) to a compound represented by formula (I) and / or a compound represented by formula (IIa) to a compound represented by formula (Ia) (such as a compound represented by formula (V)) is determined over a defined period of time, e.g., 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 72, 96, 120, 142, 144, 150, or 168 hours, preferably 24 hours, during which a compound represented by formula (II) is converted to a compound represented by formula (I) and / or a compound represented by formula (IIa) is converted to a compound represented by formula (Ia) (such as a compound represented by formula (V)) by a recombinant host cell comprising a nucleotide sequence encoding an SHC enzyme as described herein (which is producing the SHC enzyme).

[0328] In some embodiments, the bioconversion reaction is carried out under temperature values ​​such as 25° C., 30° C., 35° C., 40° C., 50° C., or 60° C. In some embodiments, the resulting conversion of the compound represented by formula (II) to the compound represented by formula (I) and / or the compound represented by formula (IIa) to the compound represented by formula (Ia) (such as the compound represented by formula (V)), and / or the rate of conversion of the compound represented by formula (II) and / or the compound represented by formula (IIa) is determined by carrying out the reaction at a temperature range of 25° C. to 55° C., preferably 30° C. to 40° C., for a period of 24 to 72 hours. In some embodiments, the period is extended, for example, up to a total of 150 hours or longer.

[0329] In some embodiments, a recombinant host cell comprising a nucleotide sequence encoding an SHC enzyme described herein exhibits at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (or higher) activity compared to a recombinant host cell expressing a nucleotide sequence encoding a parent SHC enzyme under the same conditions (preferably under conditions individually defined as optimal for the activity of the SHC enzyme under consideration). 2x), 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 21x, 22x, 23x, 24x, 25x, 26x x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x x, 50x, 51x, 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x, 60x, 61x, 62x, 63x, 64x, 65x, 66x, 67x, 68x, 69x, 70x, 71x, 7 2x, 73x, 74x, 75x, 76x, 77x, 78x, 79x, 80x, 81x, 82x, 83x, 84x, 85x, 86x, 87x, 88x, 89x, 90x, 91x, 92x, 93x, 94x, 9 The conversion of a compound of formula (II) to a compound of formula (I), and / or a compound of formula (IIa) to a compound of formula (Ia) (such as a compound of formula (V)), and / or the conversion of a compound of formula (II) and / or a compound of formula (IIa) is shown to be 5 fold, 96 fold, 97 fold, 98 fold, 99 fold, 100 fold, 200 fold, 500 fold, or 1000 fold greater.

[0330] In some embodiments, the methods as described herein are performed at a host cell and / or compound of formula (II) and / or compound of formula (IIa) concentration (in liquid culture) of 5 g / L or more, 10 g / L or more, 20 g / L or more, 30 g / L or more, 40 g / L or more, 50 g / L or more, 60 g / L or more, 70 g / L or more, 80 g / L or more, 90 g / L or more, 100 g / L or more, 110 g / L or more, 120 g / L or more, 130 g / L or more, 135 g / L or more, 150 g / L or more, 175 g / L or more, or 200 g / L or more, or 250 g / L or more.

[0331] In some embodiments, the methods as described herein may be used with a 0.1 to 4:1 or about 0.1 to 4:1 (0.1 to 4:1), 0.1 to 3:1 or about 0.1 to 3:1 (0.1 to 3:1), 0.1 to 2:1 or about 0.1 to 2:1 (0.1 to 2:1), 0.25 to 2:1 or about 0.25 to 2:1 (0.25 to 2:1), 0.5 to 2:1 or about 0.5 to 2:1 (0.5 to 2:1), 0.1 to 1 or about 0.1 to 1 The method is carried out at a weight ratio of host cells to substrate of 0.1:1, 0.5:1 or about 0.5:1 (0.5:1), 1:1 or about 1:1 (1:1), 1.5:1 or about 1.5:1 (1.5:1), or 2:1 or about 2:1 (2:1), preferably 0.1:1 or about 0.1:1 (0.1:1), 0.5:1 or about 0.5:1 (0.5:1), or 1:1 or about 1:1 (1:1).

[0332] The SHC enzymes described herein may also exhibit improved reaction performance when compared to their parent enzymes at these concentrations, as previously described herein. The reaction capacity of the SHC enzymes described herein may be assessed using any of the parameters previously discussed herein, such as the productivity, total conversion or increased substrate conversion rate, or yield of a compound represented by formula (I) and / or a compound represented by formula (Ia) (e.g., a compound represented by formula (V)), which is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (2-fold), 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 32-fold, 34-fold, 36-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50 ... 0x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x , 50x, 51x, 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x, 60x, 61x, 62x, 63x, 64x, 65x, 66x, 67x, 68x, 69 fold, 70x, 71x, 72x, 73x, 74x, 75x, 76x, 77x, 78x, 79x, 80x, 81x, 82x, 83x, 84x, 85x, 86x, 87x, 88x, 89x, 90x, 91x, 92x, 93x, 94x, 95x, 96x, 97x, 98x, 99x, 100x, 200x, 500x, or up to 1000x improvement.

[0333] Table 1. Sequences [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

[0334] General information Unless otherwise stated, all technical and scientific terms used herein have the same meaning as customarily and commonly understood by one of ordinary skill in the art to which this disclosure belongs and are read in light of this disclosure.

[0335] Sequence identity In the context of this disclosure, a nucleic acid molecule, such as a nucleic acid molecule that encodes an SHC enzyme as described herein, is represented by a nucleic acid or nucleotide sequence that encodes an SHC enzyme as described herein.

[0336] It should be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct as identified herein by a given sequence identification number (SEQ ID NO:) is not limited to this particular sequence as disclosed. Each coding sequence as identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct, or is itself a protein fragment or polypeptide or construct or peptide or derived peptide.

[0337] Throughout this application it always refers to the SEQ ID NO of a particular nucleotide sequence (take SEQ ID NO:X as an example), which codes for a given protein fragment or polypeptide or peptide or derived peptide, which may be replaced by: i. a nucleotide sequence comprising a nucleotide sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:X; ii. a nucleotide sequence, the sequence of which differs from that of the nucleic acid molecule of (i) due to the degeneracy of the genetic code; or iii. A nucleotide sequence encoding an amino acid sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% amino acid identity or similarity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:X.

[0338] Another preferred level of sequence identity or similarity is 30%. Another preferred level of sequence identity or similarity is 40%. Another preferred level of sequence identity or similarity is 50%. Another preferred level of sequence identity or similarity is 60%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0339] Throughout this application, whenever a particular amino acid sequence of a SEQ ID NO is referred to (say SEQ ID NO: Y as an example), this may be replaced by a polypeptide represented by an amino acid sequence comprising a sequence having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity or similarity with the amino acid sequence of SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 30%. Another preferred level of sequence identity or similarity is 40%. Another preferred level of sequence identity or similarity is 50%. Another preferred level of sequence identity or similarity is 60%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

[0340] Each nucleotide sequence or amino acid sequence described herein, by virtue of its percentage of identity or similarity to a given nucleotide sequence or amino acid sequence, respectively, in further preferred embodiments, has at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62% , at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity or similarity to the

[0341] Each non-coding nucleotide sequence (i.e., of a promoter or of another regulatory region) may be replaced by a nucleotide sequence that contains a nucleotide sequence having at least 60% sequence identity or similarity with the sequence number of a particular nucleotide sequence (for example, sequence number A). Preferred nucleotide sequences include those that have at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111 %, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to the sequence. In a preferred embodiment, such a non-coding nucleotide sequence, such as a promoter, exhibits or exerts at least an activity of such a non-coding nucleotide sequence, such as the activity of a promoter as known to those skilled in the art.

[0342] The terms "homology", "sequence identity" and the like are used interchangeably herein. Sequence identity is described herein as the relationship between two or more amino acid (polypeptide or protein) sequences or between two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given SEQ ID NOs, or on a portion thereof. The portion preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NOs. In the art, "identity" also refers to the degree of sequence relatedness between such sequences, as determined by the match between a series of amino acid sequences or between nucleic acid sequences, as the case may be. The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes to the sequence of another polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004 (each of which is incorporated herein by reference).

[0343] "Sequence identity" and "sequence similarity" can be determined by alignment of two peptide sequences or two nucleotide sequences using a global or local alignment algorithm depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences over their entire length, whereas sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). When sequences share at least a certain minimum percentage of sequence identity (e.g., when optimally aligned by the programs EMBOSS needle or EMBOSS water using default parameters) (as described below), the sequences may then be referred to as "substantially identical" or "essentially similar".

[0344] Global alignment is preferably used to determine sequence identity when two sequences have similar lengths. When sequences have substantially different overall lengths, local alignment, such as using the Smith-Waterman algorithm, is preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the default parameters of EMBOSS needle and EMBOSS water are used, with gap open penalty = 10 (nucleotide sequence) / 10 (protein) and gap extension penalty = 0.5 (nucleotide sequence) / 0.5 (protein). For nucleotide sequences the default scoring matrix is ​​DNAfull, and for proteins the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference).

[0345] Alternatively, percentage similarity or identity may be determined by searching against public databases using algorithms such as FASTA, BLAST, and the like. Thus, the nucleic acid and protein sequences of some embodiments of the present disclosure may further be used as "query sequences" to perform searches against public databases, for example to identify other family or related sequences. Such searches may be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10 (incorporated herein by reference). BLAST nucleotide searches may be performed with the BLASTN program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present disclosure. BLAST protein searches may be performed with the BLASTx program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402, incorporated herein by reference. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information, accessible on the World Wide Web (www.ncbi.nlm.nih.gov / ).

[0346] Sequence identity analysis may be complemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 SuppI 1: 154-162) or Markov Random Fields. Optionally, in determining amino acid similarity, one skilled in the art may also take into account so-called conservative amino acid substitutions, as previously discussed herein.

[0347] Genes or coding sequences The term "gene" refers to a DNA fragment containing a region (transcribed region) that is transcribed in a cell into an RNA molecule (e.g., mRNA) and operably linked to a suitable regulatory region (e.g., a promoter). A gene will usually contain several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) (e.g., including a polyadenylation- and / or transcription termination site). A chimeric or recombinant gene is a gene that is not normally found in nature, such as a gene in which a promoter is not naturally associated with part or all of the transcribed DNA region. "Expression of a gene" refers to the process by which a DNA region operably linked to an appropriate regulatory region, specifically a promoter, is transcribed into biologically active RNA, i.e., RNA that can be translated into a biologically active protein or peptide.

[0348] Proteins and Amino Acids The terms "protein" or "polypeptide" or "amino acid sequence" are used interchangeably and refer to a molecule consisting of a chain of amino acids without reference to a particular mode of action, size, three-dimensional structure, or origin. In amino acid sequences as described herein, the amino acids or "residues" are designated by their three letter or one letter symbols. The three letter symbols as well as the corresponding one letter symbols are well known to those skilled in the art and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine. The residues may be any proteinogenic amino acid, but may also be non-proteinogenic amino acids, such as D-amino acids and modified amino acids formed by post-translational modifications, or any unnatural amino acid.

[0349] In this document and its claims, the verb "comprise" and its conjugations are used in their non-limiting sense to mean that the matter following the word is included, but not to exclude anything not specifically mentioned. In addition, the verb "consisting of" may be replaced by "consisting essentially of," which means that the composition as described herein may include additional component(s) other than the specifically identified components, which additional component(s) do not change the unique characteristics of the invention. In addition, the verb "consisting of" may be replaced by "consisting essentially of," which means that the method as described herein may include additional step(s) other than the specifically identified steps, which additional step(s) do not change the unique characteristics of the invention.

[0350] A reference to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one or only one of the element. Thus, the indefinite article "a" or "an" usually means "at least one".

[0351] As used herein, a particular value with "at least" means a value greater than or equal to the particular value. For example, "at least 2" should be understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... and so on.

[0352] Furthermore, the terms first, second, third, etc. in the description and in the claims are used to distinguish between similar elements and do not necessarily describe an order or chronology, and it is to be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments described herein are capable of operation in arrangements other than those described or illustrated herein.

[0353] The words "about" or "approximately" preferably when used in connection with a numerical value (for example, about 10) means that the value may be 1% more or 1% less than the given value (of 10).

[0354] In the context of the present disclosure, the term "and / or" is understood to mean that all members of the group connected by the term "and / or" are represented both cumulatively and alternatively with each other in any combination. For the representative expression "A, B, and / or C", the following disclosure should be understood under the conditions: i) (A or B or C), or ii) (A and B), or iii) (A and C), or iv) (B and C), or v) (A and B and C), or vi) (A and B or C), or vii) (A or B and C), or viii) (A and C or B).

[0355] Various embodiments are described herein. Each embodiment as identified herein may be combined unless otherwise indicated.

[0356] All patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except where the language of this disclosure controls, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that they contradict the express disclosures of this specification.

[0357] This disclosure is not limited by the methods, protocols, or materials described herein.Those skilled in the art will recognize many methods, protocols, and materials similar or equivalent to those described herein that can be used in the practice described herein.Indeed, this disclosure is in no way limited to the methods and materials described.It is also understood that this disclosure covers the generalization of the aspects of the following examples to the previous disclosure.

[0358] The present disclosure is further described by the following examples, which should not be construed as limiting its scope. [Brief description of the drawings]

[0359] Description of figures [Figure 1] Figure 1. Reaction scheme for the production of compounds represented by formula (II). For the compounds, R is optionally selected from H and C1-C4 alkyl. [Diagram 2] Figure 2. SHC enzyme activity of selected SHC variants. E,Z-HFA conversion is indicated relative to conversion in BmeSHC (2 g / l E,Z-HFA, OD650nm up to 10 cells, 0.005% SDS, 50 mM succinate / NaOH buffer (pH 5.2), 35°C, 250 rpm, 24 h) as tested during library screening and selection of improved variants. [Diagram 3]Figure 3. SHC enzyme activity of selected SHC variants. The reaction conditions were the same as those discussed in Figure 2. The biocatalysts used were produced in fermentation.

[0360] [Figure 4] Figure 4. SHC enzyme activity of selected SHC variants. E,Z-HFA conversion is indicated relative to conversion with wt BmeSHC (4 g / l E,Z-HFA, cells with OD650nm of 10, 0.004% SDS, 50 mM succinate / NaOH buffer (pH 5.2), 35°C, 250 rpm, 24 h) as tested during mutation studies and selection of improved variants. [Diagram 5] Figure 5. SHC enzyme activity of selected SHC variants. The reaction conditions were the same as those discussed in Figure 4. The biocatalysts used were produced in fermentation. [Figure 6] Figure 6. SHC enzyme activity of selected SHC variants. E,Z-HFA conversion is indicated relative to conversion with wt BmeSHC (4 g / l E,Z-HFA, cells with OD650nm ~10, 0.004% SDS, 50 mM succinate / NaOH buffer (pH 5.2), 35°C, 250 rpm, 24 h).

[0361] [Figure 7] Figure 7. Relative activity of wt and variant BmeSHC enzymes. Reactions were carried out in 135 g / l E,Z-HFA and 182 g / l cells at T, pH, and SDS (SDS:cell ratio) conditions defined as optimal for each of the variants. Conversion with wt BmeSHC is set as the reference (100). [Figure 8] Figure 8. Relative activity of BmeSHC#192 and BmeSHC#192 variants. Reactions were carried out in 135 g / l E,Z HFA and 182 g / l cells at T, pH, and SDS ([SDS]:[cell] ratio) conditions individually defined as optimal for each of the tested variants. Conversion in BmeSHC#192 is set to 100 as a reference. [Figure 9]Figure 9. Relative activity of BmeSHC#192 and BmeSHC#192 variants. Reactions were carried out in 100 g / l E,Z-HFA and 100 g / l cells at T, pH, and SDS ([SDS]:[cell] ratio) conditions individually defined as optimal for each of the tested variants. Conversion in BmeSHC#192 is set to 100 as a reference.

[0362] example Example 1: Evolution of SHC enzymes: library screening, BmeSHC variants, and novel mutations An enzyme evolution program was performed using the gene encoding the Bacillus megaterium SHC enzyme as a template. A library of approximately 11'300 SHC variants was generated and screened for variants that showed increased ability to cyclize E,Z-hydroxyfarnesylacetone (E,Z-HFA) to (+)-amberketal. Gene expression for SHC production was performed in E. coli MC1061(DE3): 0.5 ml cultures were grown in auto-inducing medium and incubated at 37°C for 2 h, followed by incubation at 20°C for 22 h (250 rpm). Cells were harvested by centrifugation and washed with 50 mM succinate / NaOH buffer (pH 5.2).

[0363] SHC activity screening was performed in 96 deep-well plates. 0.5 ml reactions were run in 50 mM succinate / NaOH buffer (pH 5.2). They contained 2 g / l E,Z-HFA and 0.004% sodium dodecyl sulfate (SDS), and the ODs that produced SHC variants were 650nm The reaction was run at 35° C. under constant agitation (orbital shaking, 250 rpm) for 3 h and solvent extracted for GC-FID analysis to determine the conversion of E,Z-HFA to (+)-amber ketal as described in Example 7.

[0364] Of the approximately 11'300 variants produced, 316 were selected for validation. The conditions described above were applied to the library screening.

[0365] Of the above 316 variants, 82 were selected for larger scale validation. 20 ml cultures were run in autoinduction medium following the culture scheme and cell harvest described above. SHC activity was assayed in the settings described above. Reactions consisted of 2 g / l or 4 g / l E,Z-HFA, OD 650nm The mixture contained up to 10 or 20 cells, 0.01% or 0.005% SDS depending on the cell concentration (constant ratio of SDS / cells). Reactions were incubated at 35° C. for 2 h, 4 h, or 6 h (250 rpm) prior to solvent extraction for GC-FID analysis to determine the conversion of E,Z-HFA to (+)-amber ketal as described in Example 7.

[0366] 23 of the above 82 variants were selected for the final validation step. 20 ml cultures were run in autoinduction medium (2 h at 37° C., followed by 22 h incubation at 20° C. (180 rpm)). Cells were harvested by centrifugation, washed, and assayed for OD in 50 mM succinate / NaOH buffer (pH 5.2). 650nm The reaction was concentrated to an OD of 200. Activity was assayed in 96 deep-well plates. Reactions in 50 mM succinate / NaOH buffer (pH 5.2) contained 2 g / l, 4 g / l, or 8 g / l E,Z-HFA, OD 650nm The cultures contained 0.0025% or 0.005% SDS (constant ratio of SDS / cells) depending on the cell concentration, with up to 5 or 10 cells. Reactions were sampled over time, solvent extracted, and analyzed by gas chromatography to determine the conversion of E,Z-HFA to (+)-amber ketal as described in Example 7.

[0367] Among the seven variants that improved the E,Z-HFA cyclization activity depending on the conditions (substrate concentration, reaction time) applied to the activity test, the mutations listed in Table 2 were found. These variants were selected for thorough characterization. 650nmTheir activities (E,Z-HFA conversion relative to that with wt BmeSHC) in reactions containing up to 10 cells are shown in Figure 2. The activities of these variants when produced by fermentation are shown in Figure 3. The results indicated that the activity of the biocatalysts was strongly dependent on how they were produced (flask culture vs. fermentation, auto-induction medium vs. minimal medium).

[0368] Table 2: Mutations in selected BmeSHC variants [Table 2]

[0369] Example 2: Mutation Study 1 Mutational studies were performed to determine the effect of variants 3G6 and 50D3 on the cyclization of E,Z-HFAs to (+)-amberketal. All viable combinations of the 3G6 and 50D3 mutations were studied alone and in combination with the Y483C, L5P, and Y483C+L5P mutations. 176 additional variants were constructed and tested for their activity in cyclizing E,Z-HFAs to (+)-amberketal.

[0370] Culture and gene expression were performed in microtiter plates as described for library screening (Example 1). SHC activity was determined by incubation with 2 g / l and 4 g / l E,Z-HFA in 0.5 ml reactions; OD 650nm The assay was performed with 10 cells, 0.004% SDS in 50 mM succinate / NaOH buffer (pH 5.2) at 250 rpm. The reactions were incubated for 3 or 6 h prior to solvent extraction and GC analysis as described in Example 7. The mutations of the selected variants are shown in Table 3, and the activity of the variants (E,Z-HFA conversion relative to wt BmeSHC after 24 h of reaction) is shown in Figure 4. The activity of these biocatalysts produced by fermentation is shown in Figure 5. The results indicated that the activity of the biocatalysts was strongly dependent on how the cells were produced.

[0371] A combination mutation study allowed the identification of five beneficial mutations: I2N, Y483C, L539H, L5P, and T35A. Table 3: Mutations in selected BmeSHC variants [Table 3]

[0372] Example 3: Mutation Study 2 Mutations identified as beneficial during mutation study 1 (Example 2) were combined with mutations E211V and T166A, also identified as beneficial. E211V and / or T166A were added to SHC variants #15, #21, #42, #47, #56, and #96: 21 additional variants were constructed.

[0373] Cultivation and gene expression were performed in microtiter plates as described for library screening (Example 1). SHC activity was determined by incubation with 4 g / l E,Z-HFA; OD 650nm were assayed in 0.5 ml reactions containing 10 cells, 0.004% SDS in 50 mM succinate / NaOH buffer (pH 5.2) at 250 rpm. Reactions were incubated at 35°C and 250 rpm for 3, 6, or 24 h prior to solvent extraction and GC analysis. The mutations of selected additional variants are shown in Table 4, and the activity of the variants (E,Z-HFA conversion relative to wt BmeSHC after 3 h, 6 h, and 24 h) is shown in Figure 6.

[0374] SHC variants #179, #182, #188, #192, and #193 all showed between 4.5- and 6.5-fold improvement (E,Z-HFA conversion after 24 h of reaction) over wild-type BmeSHC.

[0375] Table 4: Mutations in selected BmeSHC variants [Table 4]

[0376] Example 4: Biocatalyst production (fermentation) For SHC enzyme production in Escherichia coli, the gene encoding the desired wild-type or variant squalene hopene cyclase enzyme was inserted into plasmid pET-28a(+), where the gene is under the control of an IPTG-inducible T7 promoter. The plasmid was transformed into E. coli strain BL21(DE3) using standard heat shock transformation procedures.

[0377] Culture medium The minimal medium used as the initial setting for biocatalyst production contained: 10% 10x citric acid / phosphate buffer (133g / l KH in deionized water) 2 PO 4 , 40g / l (NH 4 ) 2 HPO 4 , 17g / l citric acid.H 2 0, pH adjusted to 6.8 using 32% NaOH), 2.43% MgSO 4 Solution (50% w / v MgSO in deionized water 4 .7H 2 O), ● 0.01% trace element solution (50g / l Na in deionized water) 2 EDTA.2H 2 O, 20g / l FeSO 4 .7H 2 O, 3g / l H 3 BO 3 , 0.9g / l MnSO 4 .2H 2 O, 1.1g / l CoCl 2 , 80g / l CuCl 2 , 240g / l NiSO 4 .7H 2 O, 100g / l KI, 1.4g / l (NH 4 ) 6 Mo 7 O 24 .4H 2O, 1g / l ZnSO 4 .7H 2 O), ● 0.01% thiamine solution (2.25 g / l thiamine.HCl in deionized water), • 2% glucose solution (20% w / v glucose in deionized water).

[0378] The citrate / phosphate buffer was first sterilized by autoclaving, and other components added thereafter were added from sterile solutions sterilized by autoclaving or filter sterilization (0.2 μm).

[0379] fermentation The fermentation was carried out in a 750 ml InforsHT reactor. 168 ml deionized water was added to the fermenter. The reactor was fitted with all the required probes (pO 2 The reactor was then autoclaved after being equipped with a C+N feed bottle and a sodium hydroxide bottle (pH, sampling, antifoam). After autoclaving, the following was added to the reactor: ●20ml 10x phosphate / citrate buffer ●14ml 50% glucose 0.53ml MgSO 4 solution ●2ml (NH 4 ) 2 SO 4 Solution (50% (w / v) (NH) in deionized water 4 ) 2 SO 4 ) ●0.020ml trace element solution ● 0.400ml thiamine solution ●0.200ml Kanamycin solution (50mg / ml)

[0380] The operating parameters were: pH = 6.95, pO 2 = 40%, T = 30°C, 300 rpm. Cascade: rpm fixed at 300, min 300, max 1000, flow (l / min) fixed at 0.1, min 0, max 0.6. Antifoam control: 1:9.

[0381] The seed culture was grown in LB medium (+kanamycin) at 37° C. and 220 rpm for 8 h. The fermenter was incubated at OD 650nm The seed culture was inoculated until the pH reached 0.4-0.5. The fermentation was initially run in batch mode for 11.5 h, after which the following was obtained after sterilization: 17.5 ml (NH 4 ) 2 SO 4 Solution, 1.8ml MgSO 4 solution, 0.018 ml trace element solution, 0.360 ml thiamine solution, and 0.180 ml kanamycin solution were added to the feed solution (sterile glucose solution (143 ml H 2 C+N feed was started at 0.05g / ml (O+35g glucose). The feed was run at a constant flow rate of approximately 4.2 ml / h. Glucose and NH were added to assess the availability of C- and N-sources during the culture. 4 + Glucose levels usually remain very low.

[0382] Cultures were grown for a total of approximately 25 hours and typically reached OD 650nm The pH reached 40-45. SHC production was then induced by adding IPTG to the fermenter to a concentration of 1 mM and incubated at 30°C and 2 = 20% for approximately 16 h. At the end of the induction, cells were harvested by centrifugation, washed with citrate / sodium phosphate buffer (pH 5.6) and stored as pellets at 4°C or -20°C until further use.

[0383] Example 5: Optimized reaction conditions for BmeSHC variants The reaction conditions for the selected SHC variants were individually optimized with respect to temperature, pH, and SDS concentration. The biocatalysts were prepared by fermentation as described in Example 4.

[0384] 2-5 ml volume of 4 g / l E,Z-HFA and OD 650nmReactions with cells (expressing variant SHC enzymes) loaded at 10.0 were run in 0.1 M citrate / sodium phosphate buffer (pH 5.0-6.8) at temperatures ranging from 27°C to 50°C in the presence of 0.010-0.020% SDS and under constant agitation (Heidolph synthesis 1 Liquid device, 800 rpm). Reaction conditions defined as optimized were confirmed / adjusted (pH) in 0.1 M succinate / NaOH buffer. The introduced mutations had some influence on the optimum SDS concentration and pH more than the variants. A large variability was observed for the optimum temperature.

[0385] Table 5: Optimized reaction conditions for wild-type and variant BmeSHC enzymes. 1. [Table 5] 1 The optimum value for the wild-type Bme SHC enzyme is provided for comparison purposes. 2 O.D. 650nm in reactions containing up to 10 cells.

[0386] Example 6: Performance of SHC variants in 135g / l E,Z-hydroxyfarnesylacetone bioconversion Biocatalysts produced by fermentation of E. coli strains transformed with plasmids carrying genes encoding the selected BmeSHC wt or variant SHC enzymes were used for 135 g / l E,Z-HFA bioconversion. 4 ml reactions were run in a Radleys Carousel Plus / Monoblock 16. They contained 135 g / l E,Z-HFA, 182 g / l cells and were run under conditions defined as optimal with respect to temperature, pH, and SDS concentration.

[0387] Figure 7 shows the relative activity of wt and variant BmeSHC enzyme in terms of conversion of E,Z-HFA to (+)-amberketal as a function of time. Complete conversion was achieved by the best variants #179, #189, #192, and #193 in 24-48 h, whereas wt BmeSHC required 72 h to reach complete conversion.

[0388] Example 7: GC-FID analysis Samples were extracted (by vigorous shaking) with an appropriate volume of MBTE for quantification of the substrate and their content in the reaction products. The solvent fraction was separated from the aqueous phase by centrifugation (tabletop centrifuge) prior to GC-FID analysis. 1 μl of the solvent phase was injected onto a 30 m x 0.32 mm x 0.25 μm DB-Wax column (split ratio 10). The column was run at a constant flow (4 ml / min H 2 ) with a temperature gradient of 200°C, 25°C / min to 240°C, 120°C / min to 240°C, and 4 min at 240°C. Split flow: 10 ml / min, split ratio: 5. Inlet temperature: 250°C, detector temperature: 150°C. This resulted in separation of E,Z-HFA and (+)-amber ketal. The E,Z-HFA conversion was calculated from the areas of the (+)-amber ketal peak and the E,Z-HFA peak using the following formula: EZHFA Conversion (%) = 100 x (Area アンベルケタールピーク / (area アンベルケタールピーク + area EZHFAピーク ))

[0389] Example 8: Cyclization of E,Z-hydroxyfarnesylacetone E,Z-Hydroxyfarnesylacetone was cyclized using BmeSHC variant #192. The reaction contained 9.9 g E,Z-hydroxyfarnesylacetone, 364 g / l cells producing BmeSHC variant #192, 1.15 g SDS (10% SDS) and was run at 30° C. under constant stirring in 0.1 M succinate / NaOH buffer (pH 5.6) (115 ml total volume in a 250 ml flask (Radleys Monoblock)). E,Z-hydroxyfarnesylacetone was completely converted in approximately 142 hours.

[0390] The reaction mixture was extracted five times with 100 ml MTBE, the solvent phase was collected by centrifugation (30 min, 3579 g, room temperature), and the solvent phase was pooled and extracted with MgSO 4 Drying on HCl and evaporation of the solvent by rotary evaporation gave 20.9 g crude product.

[0391] The crude product was dissolved in ethanol and crystallized by addition of water. 8 g of crystalline (+)-amberketal was recovered with a purity of >99% according to GC analysis.

[0392] Example 9: Cyclization of E,Z-hydroxyfarnesylacetone from a mixture of hydroxyfarnesylacetone isomers and structural isomers of hydroxyfarnesylacetone A mixture of the following four compounds was cyclized using BmeSHC variant #192: a) The E,Z-isomer of the compound of formula (II) in which R is methyl (E,Z-hydroxyfarnesylacetone) b) The E,E-isomer of the compound of formula (II) in which R is methyl (E,E-hydroxyfarnesylacetone) c) E,Z-isomers of compounds of formula (IIa) in which R is methyl d) E,E-isomer of the compound of formula (IIa) in which R is methyl The ratio of a:b:c:d in this example was 37:9:29:16.

[0393] The reaction contained 135 g / L of the four-compound mixture, and 364 g / L cells that produced BmeSHC variant #192, 2.05 g SDS (10.25% SDS), and was run in 0.1 M succinate / NaOH buffer (pH 5.6) under constant agitation at 30° C. (200 ml total volume in a 250 ml DASBox fermenter). The reaction was run for a total of 150 hours, where the E,Z-hydroxyfarnesylacetone conversion was approximately 80%.

[0394] The reaction was extracted seven times with 100 ml MTBE, and the solvent phases were collected by centrifugation (30 min, 3579 g, room temperature), pooled, and diluted with MgSO 4 Drying on HCl and evaporation of the solvent by rotary evaporation gave 27.6 g crude product.

[0395] The reaction product was purified by flash chromatography using n-heptane / MTBE as the solvent system. The product-containing fractions were pooled and the solvent was evaporated to give 7.1 g crude product. The crude product was dissolved in ethanol and crystallized by addition of water to give two product fractions containing a compound of formula (I) and a compound of formula (V), where R is methyl.

[0396] The main product fraction (crystalline, 5.4 g) contained compounds of formula (I) and formula (V) in a ratio of 93:7 (>99% purity according to GC analysis). The second crop fraction (oily-crystalline, 708 mg) contained the compound of formula (I) and the compound of formula (V) in a ratio of 42:58 (96.8% purity).

[0397] Example 10: Mutations in structural elements related to enzyme stability A model of the BmeSHC enzyme was created by homology modeling using the crystal structure of Alicyclobacillus acidocaldarius SHC (PDB ID: 2 SQC). Structural elements that influence the stability of the enzyme include, but are not limited to, for example, glycine residues that may destabilize an α-helix or amino acid residues responsible for the formation of salt bridges.

[0398] The squalene-hopene cyclase enzyme family is characterized by QW repeats (glutamine (Q)-tryptophan (W) motifs) that constrict the protein structure through an intricate network of interactions (Wendt et al., The structure of the membrane protein squalene-hopene cyclase at 2.0 A resolution, J. Mol. Biol 286, 175-187 (1999)). By comparison of the QW repeats in BmeSHC and the QW repeats in homologs of BmeSHC, the BmeSHC#192 variants listed in Table 6 were designed with mutations directed to the QW repeats.

[0399] Table 6. Mutations in structural elements responsible for enzyme stability. [Table 6]

[0400] Example 11: E,Z-Hydroxyfarnesylacetone conversion in BmeSHC#192 variants The variant biocatalysts listed in Table 6 were produced by fermentation as described in Example 4. For each of the variants, the reaction conditions were individually optimized with respect to the reaction parameters temperature, pH, and SDS concentration along with the produced biocatalyst as described in Example 5. The optimized reaction conditions for selected BmeSHC#192 variants are listed in Table 7.

[0401] Table 7: Optimized reaction conditions for BmeSHC#192 variants. [Table 7] 1 O.D. 650nmin reactions containing up to 10 cells (approximately 9 g / l cells).

[0402] The biocatalyst was used with 182 g / l cells in the 135 g / l E,Z-HFA bioconversion: 4 ml reactions were run in a Radleys Carousel Plus under conditions individually defined as optimal for each variant with respect to temperature, pH, and SDS concentration.

[0403] Figure 8 shows the relative activity of the parent and variant BmeSHC#192 enzymes in terms of conversion of E,Z-HFA to (+)-amberketal as a function of time. Enhancing the stability of the enzyme by addressing structural elements such as the QW repeats allowed to increase the enzyme activity. The initial reaction rate measured in terms of conversion after 3 hours of reaction was increased for all variants tested. E,Z-hydroxyfarnesylacetone conversion after 42.5 h and 70 h of reaction was higher for variants other than the two variants BmeSHC#192_v70 and BmeSHC#192_v72 compared to the parent BmeSHC#192.

[0404] Example 12: E,Z-Hydroxyfarnesylacetone conversion in BmeSHC#192 variant at a cell:substrate ratio of 1 Biocatalysts of variants BmeSHC#192_v70, BmeSHC#192_v71, and BmeSHC#192_v75 (Table 6) were produced by fermentation as described in Example 4. Biocatalysts were used in the bioconversion at a cell:substrate ratio of 1 (100 g / l E,Z-HFA, 100 g / l cells):4 ml reactions were run in a Radleys Carousel Plus under conditions individually defined as optimal for each variant in terms of temperature, pH, and SDS concentration (Table 7).

[0405] Figure 9 shows the relative activity of the parent and variant BmeSHC#192 enzymes measured in terms of conversion of E,Z-HFA to (+)-amberketal as a function of time. Biocatalysts producing variants BmeSHC#192_v70, BmeSHC#192_v71, and BmeSHC#192_v75 performed better than the biocatalyst producing the parent enzyme BmeSHC#192: an increase of about 1.25-1.35-fold was observed for those variants over the parent enzyme variants in E,Z-HFA conversion.

Claims

1. Formula (I) 【Chemical 1】 Formula (I) wherein the method comprises preparing a compound of formula (II) 【Chemistry 2】 Formula (II) with a squalene-hopene cyclase (SHC) enzyme comprising an amino acid sequence having at least 70% identity or similarity to the sequence of SEQ ID NO:1, wherein the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:1, and wherein R is selected from the group consisting of H and C. 1 ~C 4 The method according to claim 1, wherein the alkyl is selected from the group consisting of alkyl, methyl ...

2. 2. The method of claim 1, wherein the compound of formula (II) has a double bond between C-8 and C-9 in the E-configuration and a double bond between C-4 and C-5 in the Z-configuration (E,Z-isomer).

3. Formula (I) 【Chemistry 3】 Formula (I) wherein the method comprises preparing a mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa), 【Chemistry 4】 Formula (II) 【Chemistry 5】 Formula (IIa) with a squalene-hopene cyclase (SHC) enzyme comprising an amino acid sequence having at least 70% identity or similarity to the sequence of SEQ ID NO:1 or the sequence of SEQ ID NOs:43-49, preferably having at least 70% identity or similarity to the sequence of SEQ ID NO:1 and comprising one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:1, and wherein R is selected from the group consisting of H and C. 1 ~C 4 The method according to claim 1, wherein the alkyl is selected from the group consisting of alkyl, methyl ...

4. The mixture containing the compound represented by formula (I) is a compound represented by formula (Ia) 【Chemistry 6】 Formula (Ia) In the formula, R is H and C 1 ~C 4 4. The method of claim 3, further comprising a compound represented by the formula:

5. The compound of formula (Ia) is a compound of formula (V) 【Chemistry 7】 Formula (V) In the formula, R is H and C 1 ~C 4 5. The method of claim 4, wherein the alkyl group has a configuration represented by:

6. The mixture comprising the compound represented by formula (II) and the compound represented by formula (IIa) is any one of the following: i) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomer). ii) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomer). iii) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomer). iv) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomer). v) Compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomers), and compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomers). vi) Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomers), and compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomers). vii) Any combination of i) to vi) 4. The method of claim 3, comprising:

7. A mixture comprising a compound represented by formula (II) and a compound represented by formula (IIa) is prepared by the following method: - compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (E,Z-isomers), - compounds of formula (II) in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is also in the E-configuration (E,E-isomers), - compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is in the Z-configuration (E,Z-isomers), and - Compounds of formula (IIa) in which the double bond between C-6 and C-7 is in the E-configuration and the double bond between C-2 and C-3 is also in the E-configuration (E,E-isomers).

4. The method of claim 3, comprising:

8. Formula (III) 【Chemistry 8】 Formula (III) In the formula, R is H and C 1 ~C 4 4. The method of claim 1 or 3, wherein a compound represented by the formula: is produced as a by-product, wherein the compound is selected from the group consisting of alkyl,

9. Formula (IIIa): 【Chemistry 9】 Formula (IIIa) In the formula, R is H and C 1 ~C 4 4. The method of claim 1 or 3, wherein a compound having the relative configuration shown in is produced as a by-product, wherein the compound is selected from the group consisting of alkyl,

10. Formula (VI) 【Chemistry 10】 Formula (VI) In the formula, R is H and C 1 ~C 4 4. The method of claim 3, wherein a compound represented by the formula: is produced as a by-product, wherein the compound is selected from the group consisting of alkyl,

11. Formula (VIa): 【Chemistry 11】 Formula (VIa) In the formula, R is H and C 1 ~C 4 4. The method of claim 3, wherein a compound having the relative configuration shown in is produced as a by-product, wherein the compound is selected from the group consisting of alkyl, aryl ...

12. 4. The method of claim 1 or 3, wherein R is methyl.

13. 4. The method of claim 1 or 3, wherein the SHC enzyme comprises an amino acid sequence having at least 70% identity or similarity to the sequence of SEQ ID NO:1, and wherein the SHC enzyme comprises 1 to 7, preferably 2 to 6, more preferably 3 to 5 amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 116, 166, 211, 212, 317, 355, 382, ​​399, 483, 539, and 585 of SEQ ID NO:

1.

14. 4. The method of claim 1 or 3, wherein the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO:1 at one or more positions corresponding to positions 2, 5, 35, 166, 211, 212, 355, 483, and 539 of SEQ ID NO:

1.

15. 4. The method of claim 1 or 3, wherein the SHC enzyme comprises one or more amino acid substitutions relative to SEQ ID NO: 1 at one or more positions corresponding to positions 2, 5, 35, 166, 211, 212, 483, and 539 of SEQ ID NO: 1, preferably at positions 2, 5, 35, 166, 211, 483, and 539.

16. The SHC enzyme is: (xvi) an asparagine (N) residue at a position corresponding to position 2 of SEQ ID NO:1; (xvii) a proline (P) residue at a position corresponding to position 5 of SEQ ID NO:1; (xviii) an alanine (A) residue at a position corresponding to position 35 of SEQ ID NO: 1; (xix) a threonine (T) residue at a position corresponding to position 116 of SEQ ID NO: 1; (xx) an alanine (A) residue at a position corresponding to position 166 of SEQ ID NO: 1; (xxi) a valine (V) residue at a position corresponding to position 211 of SEQ ID NO: 1; (xxii) an arginine (R) residue at a position corresponding to position 212 of SEQ ID NO: 1; (xxiii) a methionine (M) residue at a position corresponding to position 317 of SEQ ID NO: 1; (xxiv) a threonine (T) residue at a position corresponding to position 355 of SEQ ID NO: 1; (xxv) a threonine (T) residue at a position corresponding to position 382 of SEQ ID NO: 1; (xxvi) a valine (V) residue at a position corresponding to position 399 of SEQ ID NO: 1; (xxvii) a cysteine ​​(C) residue at a position corresponding to position 483 of SEQ ID NO: 1; (xxviii) a histidine (H) residue at a position corresponding to position 539 of SEQ ID NO: 1; (xxix) an alanine (A) residue at a position corresponding to position 585 of SEQ ID NO: 1; or (xxx) any combination thereof 4. The method of claim 1 or 3, comprising an amino acid substitution relative to SEQ ID NO: 1 selected from:

17. The SHC enzyme has the following structure in SEQ ID NO:1: (xiv) I2N, T35A, A355T, and L539H; (xv) T166A; (xvi) I2N and Y483C; (xvii) I2N, Y483C, and L539H; (xviii) I2N, L5P, T35A, L539H; (xix) I2N, L5P, T35A, and Y483C; (xx) I2N, L5P, T35A, T166A, and L539H; (xxi) I2N, L5P, T35A, T166A, E211V, and L539H; (xxii) I2N, L5P, T35A, E211V, S212R, Y483C, and L539H; (xxiii) I2N, T166A, and Y483C; (xxiv) I2N, T166A, Y483C, and L539H; (xxv) I2N, T166A, E211V, and Y483C; or (xxvi) I2N, T166A, E211V, Y483C, and L539H 4. The method of claim 1 or 3, comprising an amino acid substitution relative to SEQ ID NO: 1 selected from the corresponding positions of:

18. 4. The method of claim 1 or 3, wherein the SHC enzyme comprises the following amino acid substitutions relative to SEQ ID NO:1: I2N and T166A.

19. 4. The method of claim 1 or 3, wherein the SHC enzyme further comprises an amino acid substitution relative to SEQ ID NO: 1 selected from L5P, T35A, E211V, Y483C, and L539H.

20. 4. The method of claim 1 or 3, wherein the SHC enzyme further comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or 42, preferably SEQ ID NOs: 4, 6, 18, 20, 22, 24, 30, 32, 34, 36, 38, 40, or 42, more preferably SEQ ID NOs: 30, 32, 34, 36, 38, 40, or 42, most preferably SEQ ID NOs: 30, 38, 40, 42.

21. 10. A nucleic acid molecule comprising a nucleotide sequence encoding a squalene hopene cyclase (SHC) enzyme as defined in claim 1.

22. A vector comprising the nucleic acid molecule of claim 21.

23. 23. A host cell comprising the nucleic acid molecule of claim 21 or the vector of claim 22.

24. 2. The squalene hopene cyclase (SHC) enzyme as defined in claim 1.

25. 5. A composition comprising a compound of formula (I) and / or a compound of formula (Ia), wherein the composition is obtained or obtainable by the method of claim 4.

26. 26. The composition according to claim 25, wherein the compound of formula (I) and / or the compound of formula (Ia) is in solid form, preferably in amorphous or crystalline form.

27. 26. The composition of claim 25, wherein the compound of formula (Ia) has the configuration of formula (V).

28. 26. Use of a composition according to claim 25 for the manufacture of a fragrance composition or a consumer product.

29. 26. A fragrance composition or consumer product comprising a composition as claimed in claim 25.

30. 4. A mixture comprising a product obtainable by the process of claim 3, wherein the mixture comprises from I, Ia, III, IIIa, IV, IVa, V, Va, VI, and / or VIa.

31. 26. The composition of claim 25, wherein the composition further comprises III, IIIa, IV, IVa, V, Va, VI, and / or VIa.

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