Squalene hopene cyclase (SHC) variants

Abstract

To provide a new and improved method for making (-)-Ambrox.SOLUTION: Provided is a process for preparing (-)-Ambrox or a mixture including (-)-Ambrox, the process including enzymatically converting a mixture of homofarnesol isomers into (-)-Ambrox or a mixture including (-)-Ambrox using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has at least about 70% identity to a specific sequence and has an amino acid modification at a specific position.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field The present invention generally relates to SHC / HAC enzymes and their variants. The present invention further relates to various uses of SHC / HAC enzymes and their variants, such as for the enzymatic conversion of (3E,7E)-homofarnesol (EEH) to (-)-ambrox or for the enzymatic conversion of E,E-bishomofarnesol (bisEEH) to ambra oxide. The present invention also relates to the products of the enzymatic reactions, such as (-)-ambrox or ambra oxide, produced using SHC / HAC enzymes and their variants, and various uses of the products. [Background technology]

[0002] background Squalene Hopene Cyclase (SHC) is a membrane-bound enzyme that acts as a biocatalyst for the cyclization of the linear triterpenoid squalene to hopene and hopanol.

[0003] Numerous wild-type and variant SHC enzymes from various bacteria have been demonstrated to be useful for converting (3E,7E)-homofarnesol to (-)-ambrox (e.g., WO 2016 / 170099; WO 2018 / 157021; Neumann & Simon 1986, Biol Chem Hoppe-Seyler 367, 723-729; JP2009060799; Seckler & Poralla 1986, Biochem Biophys Act 356-363; Ochs et al. 1990, J Bacteriol 174, 298-302; WO 2010 / 139719; US 8759043; WO 2012 / 066059; Seitz et al. 2012, J Molecular Catalysis B:Enzymatic 84, 72-77; and Seitz 2012 PhD thesis (http: / / elib.uni-stuttgart.de / handle / 1 1682 / 1400), the contents of which are incorporated herein by reference). It would be desirable to provide new and improved methods for making (-)-ambrox, for example, using new SHC enzymes or enzyme variants. It would also be desirable to provide new and improved methods for cyclizing other substrates to form compounds useful, for example, in or as fragrances. Summary of the Invention

[0004] overview According to a first aspect of the present invention, there is provided a process for preparing (-)-ambrox or a mixture containing (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme variant; wherein the SHC / HAC enzyme variant has at least about 70.0% identity to SEQ ID NO:1; wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid alterations, relative to SEQ ID NO:1, at positions corresponding to positions 132, 224, and 432 of SEQ ID NO:1, which are M132R, A224V, and I432T, respectively; and wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to SEQ ID NO:1, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to positions 81, 431, or 613 of SEQ ID NO:1.

[0005] According to a second aspect of the present invention there is provided (-)-ambrox obtained by or obtainable by the process of the first aspect of the present invention (including any embodiment thereof).

[0006] According to a third aspect of the present invention there is provided the use of (-)-ambrox according to the second aspect of the present invention (including any embodiment thereof) as part of a fragrance or cosmetic or consumer product.

[0007] According to a fourth aspect of the present invention there is provided a fragrance or cosmetic or consumer product comprising (-)-ambrox according to the second aspect of the present invention (including any embodiment thereof).

[0008] According to a fifth aspect of the present invention there is provided a SHC / HAC enzyme variant having an amino acid sequence having at least about 70.0% identity to SEQ ID NO: 1, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications, relative to SEQ ID NO:1, at positions corresponding to positions 132, 224, and 432 of SEQ ID NO:1, which are M132R, A224V, and I432T, respectively; and wherein the amino acid sequence of the SHC / HAC enzyme variant further has an amino acid modification, relative to SEQ ID NO:1, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to positions 81, 431, or 613 of SEQ ID NO:1.

[0009] According to a sixth aspect of the present invention there is provided a nucleic acid sequence encoding the SHC / HAC enzyme variant of the fifth aspect of the present invention (including any embodiment thereof).

[0010] According to a seventh aspect of the present invention there is provided a construct comprising a nucleic acid sequence of the sixth aspect of the present invention (including any embodiment thereof).

[0011] According to an eighth aspect of the present invention there is provided a vector comprising a construct of the seventh aspect of the present invention (including any embodiment thereof).

[0012] According to a ninth aspect of the present invention there is provided a recombinant host cell comprising a nucleic acid sequence of the sixth aspect of the invention, a construct of the seventh aspect of the invention or a vector of the eighth aspect of the invention (including any embodiment thereof).

[0013] According to a tenth aspect of the present invention, there is provided a process for preparing (-)-ambrox or a mixture comprising (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers comprising EEH to (-)-ambrox or a mixture comprising (-)-ambrox using an SHC / HAC enzyme variant; wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme; and wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications relative to the wild-type SHC / HAC enzyme at positions selected from those corresponding to positions 557, 81, 431, and 613 of SEQ ID NO:1.

[0014] According to an eleventh aspect of the present invention there is provided an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme; and wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications relative to the wild-type SHC / HAC enzyme at positions selected from those corresponding to positions 557, 81, 431, and 613 of SEQ ID NO:1.

[0015] According to a twelfth aspect of the present invention, there is provided a process for preparing (-)-ambrox or a mixture comprising (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers comprising EEH to (-)-ambrox or a mixture comprising (-)-ambrox using an SHC / HAC enzyme or an SHC / HAC enzyme variant; Here, the SHC / HAC enzyme or SHC / HAC enzyme variant has an amino acid sequence that has at least about 70.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0016] According to a thirteenth aspect of the present invention, there is provided an SHC / HAC enzyme or SHC / HAC enzyme variant, wherein the SHC / HAC enzyme or SHC / HAC enzyme variant has an amino acid sequence having at least about 70.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0017] According to a fourteenth aspect of the present invention, there is provided a process for preparing ambroxide, the process comprising enzymatically converting E,E-bishomofarnesol or a mixture of bishomofarnesol isomers containing E,E-bishomofarnesol to ambroxide or a mixture containing ambroxide using an SHC / HAC enzyme or SHC / HAC enzyme variant. The SHC / HAC enzyme or SHC / HAC enzyme variant may, for example, be according to any aspect of the present invention.

[0018] According to a fifteenth aspect of the present invention there is provided an amberoxide obtained by or obtainable by the process of the fourteenth aspect of the present invention (including any embodiment thereof).

[0019] According to a sixteenth aspect of the present invention there is provided the use of ambroxide according to the fifteenth aspect of the present invention (including any embodiment thereof) as part of a fragrance or cosmetic or consumer product.

[0020] According to a seventeenth aspect of the present invention there is provided a fragrance or cosmetic or consumer product comprising an ambroxide according to the fifteenth aspect of the present invention (including any embodiment thereof).

[0021] In certain embodiments of any aspect of the invention, the SHC / HAC enzyme variant has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and / or SEQ ID NO:30.

[0022] In some embodiments of any aspect of the invention, the SHC / HAC enzyme variant is encoded by a nucleic acid having a sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:22, and SEQ ID NO:23.

[0023] Certain embodiments of the present invention may provide one or more of the following advantages: • Improved EEH conversion rates, specifically in the first 12 hours or first 6 hours; • Cyclization of new substrates and / or identification of new products; • Improved productivity (g / l / hr or g / l / hr / g biocatalyst); • Industrial scale processes for preparing end products such as (-)-ambrox and ambroxide, and / or mixtures of their isomers; • Improved selectivity for EEH over other homofarnesol isomers.

[0024] The details, examples, and preferences provided with respect to any one or more specific aspects of the invention as further described herein will apply equally to all aspects of the invention. Any combination of the embodiments, examples, and preferences described herein in all possible variations thereof is covered by the present invention unless otherwise indicated herein or clearly contradicted by context. [Brief explanation of the drawings]

[0025] Brief description of the figure [Figure 1] Figure 1 shows the relative activity of SHC variants under the original and optimized reaction conditions. Reactions were performed with 4 g / L EEH and a biocatalyst loaded to an OD of 10.0, either at the original conditions (35°C, pH 5.4, 0.070% SDS) or with the T, pH, and [SDS] set to the conditions defined as optimal for each variant (optimized conditions).

[0026] [Figure 2]Figure 2 shows the relative activity of the SHC variants under optimized conditions compared to the parent 215G2 SHC enzyme. Reactions were performed with 4 g / L EEH and biocatalyst loaded to an OD of 10.0, with T, pH, and [SDS] set to the conditions defined as optimal for each variant.

[0027] [Figure 3] Figure 3 shows the conversion of EEH to (-)-ambrox by the SHC / HAC enzyme variants under optimal conditions compared to the parent SHC / HAC enzyme of 215G2. Reactions were performed with 125 g / L EEH and 250 g / L biocatalyst in the presence of 1.3% SDS at the T and pH conditions defined as optimal for each variant.

[0028] [Figure 4] Figure 4 shows the relative improvement in EEH conversion for the best SHC / HAC enzyme variants compared to the reference 215G2 SHC / HAC parent enzyme. Reactions were performed with 125 g / L EEH and 250 g / L biocatalyst in the presence of 1.3% SDS at T and pH conditions defined as optimal for each variant. At all time points, the EEH conversion obtained with 215G2 SHC was set to 100% (reference).

[0029] [Figure 5] Figure 5 shows the relative activity of the SHC / HAC variants compared to the parent (reference) 215G2 SHC / HAC enzyme under optimal conditions. Reactions were performed with 4 g / L bisEEH and biocatalyst loaded to an OD of 10.0. T, pH, and [SDS] were set to the conditions defined as optimal for each variant.

[0030] [Figure 6]Figure 6 shows the cyclization of bishomofarnesol with the parent (reference) 215G2 SHC / HAC enzyme and the new variant SHC / HAC enzymes. Reactions were performed with 125 g / L bisEEH and 250 g / L biocatalyst in the presence of 1.3% SDS at 35°C and pH 5.4 (all SHC enzymes).

[0031] [Figure 7] Figure 7 shows an amino acid sequence alignment between 215G2 SHC / HAC (SEQ ID NO: 10) and SHC / HAC enzyme variants #49 (SEQ ID NO: 2), #65 (SEQ ID NO: 3), #66 (SEQ ID NO: 4), #90C7 (SEQ ID NO: 17), #110B8 (SEQ ID NO: 5), and #115A7 (SEQ ID NO: 18).

[0032] [Figure 8] FIG. 8 shows an amino acid sequence alignment between 215G2 (SEQ ID NO: 10) and SHC / HAC enzyme variants derived from 215G2 with one or more of the substitutions V174I, F601Y, and L37Q.

[0033] [Figure 9A-1] Figure 9A shows an amino acid sequence alignment between wild-type AacSHC (SEQ ID NO: 1) and wild-type TelSHC (SEQ ID NO: 19), wild-type ApaSHC1 (SEQ ID NO: 20), wild-type ZmoSHC1 (SEQ ID NO: 11), wild-type ZmoSHC2 (SEQ ID NO: 12), wild-type BjpSHC (SEQ ID NO: 13), and wild-type GmoSHC (SEQ ID NO: 14) using CLUSTAL O (1.2.4). Amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted in white letters on a black background.

[0034] [Figure 9A-2]Figure 9A shows an amino acid sequence alignment between wild-type AacSHC (SEQ ID NO: 1) and wild-type TelSHC (SEQ ID NO: 19), wild-type ApaSHC1 (SEQ ID NO: 20), wild-type ZmoSHC1 (SEQ ID NO: 11), wild-type ZmoSHC2 (SEQ ID NO: 12), wild-type BjpSHC (SEQ ID NO: 13), and wild-type GmoSHC (SEQ ID NO: 14) using CLUSTAL O (1.2.4). Amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted in white letters on a black background.

[0035] [Figure 9B-1] Figure 9B shows an amino acid sequence alignment between wild-type AacSHC (SEQ ID NO: 1) and wild-type TelSHC (SEQ ID NO: 19), wild-type ApaSHC1 (SEQ ID NO: 20), wild-type ZmoSHC1 (SEQ ID NO: 11), wild-type ZmoSHC2 (SEQ ID NO: 12), wild-type BjpSHC (SEQ ID NO: 13), wild-type GmoSHC (SEQ ID NO: 14), wild-type BmeSHC (SEQ ID NO: 28), wild-type SalSHC (SEQ ID NO: 29), and wild-type ApaSHCA (SEQ ID NO: 30) using CLUSTAL O (1.2.4), with amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC highlighted in white letters on a black background.

[0036] [Figure 9B-2]Figure 9B shows an amino acid sequence alignment between wild-type AacSHC (SEQ ID NO: 1) and wild-type TelSHC (SEQ ID NO: 19), wild-type ApaSHC1 (SEQ ID NO: 20), wild-type ZmoSHC1 (SEQ ID NO: 11), wild-type ZmoSHC2 (SEQ ID NO: 12), wild-type BjpSHC (SEQ ID NO: 13), wild-type GmoSHC (SEQ ID NO: 14), wild-type BmeSHC (SEQ ID NO: 28), wild-type SalSHC (SEQ ID NO: 29), and wild-type ApaSHCA (SEQ ID NO: 30) using CLUSTAL O (1.2.4), with amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC highlighted in white letters on a black background.

[0037] [Figure 10] FIG. 10 is the reaction scheme used to produce the starting mixture of E,E / Z-bishomofarnesol. [Figure 11] Figure 11 is a reaction scheme showing the cyclization of bishomofarnesol with the SHC enzyme. The bishomofarnesol used consisted of a mixture of isomers.

[0038] [Figure 12] Figure 12 shows the conversion of EEH to (-)-ambrox by the SHC / HAC enzyme variants under optimal conditions compared to the 215G2 parent SHC / HAC enzyme. Reactions were performed with 125 g / L EEH and 250 g / L biocatalyst in the presence of 1.3% SDS at the T and pH conditions defined as optimal for each variant.

[0039] [Figure 13] Figure 13 shows the conversion of EEH to (-)-ambrox by the SHC / HAC enzyme variants under optimal conditions compared to the 215G2 parent SHC / HAC enzyme. Reactions were performed with 125 g / L EEH and 125 g / L biocatalyst in the presence of 0.65% SDS at the T and pH conditions defined as optimal for each variant.

[0040] [Figure 14] Figure 14 shows EEH conversion to (-)-ambrox by SHC / HAC enzyme variant SHC#65 under optimal conditions compared to the 215G2 parent SHC / HAC enzyme. Reactions were performed at 125 g / L to 300 g / L EEH and either 250 g / L biocatalyst or an [EEH]:[cell] ratio of 1. SDS was supplied at a constant [SDS]:[cell] ratio of 0.052. Reactions were performed at the T and pH conditions defined as optimal for SHC variant SHC#65.

[0041] [Figure 15] 15 shows EEH conversion to (-)-ambrox by wild-type AacSHC (SEQ ID NO:1), wild-type BjpSHC (SEQ ID NO:13), wild-type BmeSHC (SEQ ID NO:28), wild-type GmoSHC (SEQ ID NO:14), wild-type ApaSHCA (SEQ ID NO:30), wild-type ApaSHC1 (SEQ ID NO:20), wild-type SalSHC (SEQ ID NO:29), wild-type TelSHC (SEQ ID NO:19), wild-type ZmoSHC1 (SEQ ID NO:11), and wild-type ZmoSHC2 (SEQ ID NO:12). Reactions were performed at 4 g / L EEH for all listed wild-type SHC enzymes, and at 125 g / L EEH for all listed wild-type SHC enzymes except wild-type ApaSHCA (SEQ ID NO:30), wild-type SalSHC (SEQ ID NO:29), wild-type TelSHC (SEQ ID NO:19), and wild-type ZmoSHC2 (SEQ ID NO:12). Reactions were performed at T and pH conditions and SDS concentrations defined as optimal for each wild-type SHC enzyme listed. The SDS concentration was adjusted to maximize biocatalytic activity in 125 g / L EEH bioconversion.

[0042] [Figure 16]Figure 16 shows the conversion of EEH to (-)-ambrox by SHC / HAC. Bioconversion was performed with SHC#65 biocatalyst. Reactions were performed at 125 g / L EEH in the presence of 0.060-0.090% SDS, corresponding to [SDS]:[cell] ratios ranging from 0.040 to 0.060, at 45°C and pH 5.6 with 150 g / L cells. EEH conversion values ​​for 72 h of reaction are shown. DETAILED DESCRIPTION OF THE INVENTION

[0043] Array Overview SEQ ID NO: 1 is the wild-type Alicyclobacillus acidocaldarius (Aac) SHC amino acid sequence. SEQ ID NO:2 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, A557T, and H431L, and is sometimes referred to herein as SHC / HAC enzyme variant #49. SEQ ID NO:3 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, A557T, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #65. SEQ ID NO:4 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, Y81H, A557T, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #66. SEQ ID NO:5 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, Y81H, H431L, and A557T, and is sometimes referred to herein as SHC / HAC enzyme variant #110B8.

[0044] SEQ ID NO:6 is a nucleic acid sequence encoding the polypeptide of SEQ ID NO:2 (SHC / HAC enzyme variant #49). SEQ ID NO:7 is a nucleic acid sequence encoding the polypeptide of SEQ ID NO:3 (SHC / HAC enzyme variant #65). SEQ ID NO:8 is a nucleic acid sequence encoding the polypeptide of SEQ ID NO:4 (SHC / HAC enzyme variant #66). SEQ ID NO: 9 is a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 5 (SHC / HAC enzyme variant #110B8). SEQ ID NO: 10, sometimes referred to as 215G2, corresponds to the wild-type AacSHC amino acid sequence with the mutations M132R, A224V, and I432T.

[0045] SEQ ID NO: 11 is the wild-type amino acid sequence of ZmoSHC1. SEQ ID NO: 12 is the wild-type amino acid sequence of ZmoSHC2. SEQ ID NO: 13 is the wild-type amino acid sequence of BjpSHC / BjaSHC. SEQ ID NO: 14 is the wild-type amino acid sequence of GmoSHC. SEQ ID NO: 15 is the nucleotide sequence encoding wild-type AacSHC.

[0046] SEQ ID NO: 16 is the nucleotide sequence encoding 215G2 SHC. SEQ ID NO: 17 corresponds to SEQ ID NO: 1 with the substitutions M132R, A224V, I432T, T90A, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #90C7. SEQ ID NO: 18 corresponds to SEQ ID NO: 1 with the substitutions M132R, A224V, I432T, A172T, and M277K, and is sometimes referred to herein as SHC / HAC enzyme variant #115A7. SEQ ID NO: 19 is the wild-type amino acid sequence of TelSHC. SEQ ID NO: 20 is the wild-type amino acid sequence of ApaSHC1.

[0047] SEQ ID NO: 21 is a GmoSHC variant. SEQ ID NO: 22 is the nucleotide sequence encoding the polypeptide of SEQ ID NO: 17 (SHC / HAC enzyme variant #90C7). SEQ ID NO:23 is the nucleotide sequence encoding the polypeptide of SEQ ID NO:18 (SHC / HAC enzyme variant #115A7). SEQ ID NO: 24 is the amino acid sequence of the SHC / HAC variant 215G2 SHC with the additional natural mutation L37Q. SEQ ID NO: 25 is the amino acid sequence of the SHC / HAC variant 215G2 SHC with the additional mutation V174I.

[0048] SEQ ID NO: 26 is the amino acid sequence of the SHC / HAC variant 215G2 SHC with the additional mutations V174I and F601Y. SEQ ID NO: 27 is the amino acid sequence of SHC / HAC variant 215G2 SHC with additional mutations L37Q, V174I, and F601Y. SEQ ID NO: 28 is the wild-type amino acid sequence of BmeSHC. SEQ ID NO: 29 is the wild-type amino acid sequence of SalSHC. SEQ ID NO: 30 is the wild-type amino acid sequence of ApaSHCA.

[0049] Detailed Description SHC / HAC enzymes and their variants As used herein, the term "SHC enzyme" refers to a wild-type (WT) squalene hopene cyclase enzyme that occurs naturally in thermophilic bacteria, such as, for example, Alicyclobacillus acidocaldarius. SHCs that act in the cyclization of homofarnesol to ambrox are also sometimes referred to as mofarnesol ambrox cyclase (HAC) enzymes. Thus, the term "SHC / HAC enzymes" is sometimes used herein.

[0050] As used herein, the term "variant" should be understood as a polypeptide that differs from a polypeptide from which it is derived by one or more changes in amino acid sequence. The polypeptide from which a variant is derived is also known as the parent or reference polypeptide. Typically, variants are artificially constructed, preferably by genetic engineering means. Typically, the polypeptide from which a variant is derived is a wild-type protein or a domain of a wild-type protein. However, variants usable in the present disclosure may also be derived from homologs, orthologs, or paralogs of the parent polypeptide or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent polypeptide. The change in amino acid sequence may be an amino acid exchange (substitution), insertion, deletion, N-terminal truncation, or C-terminal truncation, or any combination of these changes, and may occur at one or several sites.

[0051] As used herein, the term "SHC / HAC enzyme variant" refers to an enzyme derived from a wild-type SHC enzyme but having one or more amino acid modifications relative to the wild-type SHC enzyme, and thus not naturally occurring in prokaryotes. The one or more amino acid modifications may, for example, modify (e.g., increase) the enzyme's activity toward a substrate (e.g., EEH).

[0052] Assays for determining and quantifying the activity of SHC / HAC enzymes and / or SHC / HAC enzyme variants are described herein and are known in the art. For example, the activity of an SHC / HAC enzyme and / or SHC / HAC enzyme variant can be determined by incubating a purified SHC / HAC enzyme or enzyme variant, or an extract from a host cell or a complete recombinant host organism producing the SHC / HAC enzyme or enzyme variant, with an appropriate substrate under appropriate conditions and analyzing the reaction products (e.g., by gas chromatography (GC) or HPLC analysis). Further details of assaying the activity of an SHC / HAC enzyme and / or SHC / HAC enzyme variant and analyzing the reaction products are provided in the Examples. These assays involve producing an SHC / HAC enzyme variant in a recombinant host cell (e.g., E. coli).

[0053] As used herein, the term "activity" refers to the ability of an enzyme to react with a substrate to provide a desired product. Activity can be determined using what is known as an activity test to monitor the formation of the desired product. Derivatives of the SHC / HAC enzymes of the present disclosure can be characterized by their ability to cyclize homofarnesol (e.g., EEH) to (-)-ambrox and demonstrate biological activity, such as HAC activity. Derivatives of the SHC / HAC enzymes of the present disclosure can be characterized by their ability to cyclize bishomofarnesol (e.g., E,E-bishomofarnesol) to ambroxide.

[0054] "Biological activity," as used herein, refers to any activity that a polypeptide may exhibit, including, but not limited to, enzymatic activity; binding activity to another compound (e.g., binding to another polypeptide, especially binding to a receptor, or binding to a nucleic acid); inhibitory activity (e.g., enzyme inhibitory activity); activating activity (e.g., enzyme activating activity); or toxic effects. A variant is not required to exhibit such an activity to the same extent as the parent or wild-type polypeptide. A variant is considered a variant within the context of this application if it exhibits the relevant activity to at least 10% of the activity of the parent polypeptide. Similarly, a variant is considered a variant within the context of this application if it exhibits the relevant biological activity to at least 10% of the activity of the parent polypeptide (as the terms derivative and variant are used interchangeably throughout this disclosure). In other embodiments, the SHC / HAC enzyme variants of the present disclosure exhibit better yields than a reference SHC protein (e.g., a wild-type SHC / HAC enzyme or a known SHC / HAC enzyme variant). The term "yield" refers to grams of recoverable product per gram of feedstock (which may be calculated as a percent molar conversion rate). In additional embodiments, the SHC / HAC enzyme variants of the present disclosure exhibit modified (e.g., increased) productivity compared to a reference SHC protein (e.g., wild-type AacSHC or 215G2 AacSHC). The term "productivity" refers to the amount of recoverable product in grams per liter of reaction capacity per hour of bioconversion time (i.e., time after substrate is added). The term "productivity" also refers to the amount of recoverable product in grams per liter of reaction capacity per hour of bioconversion time (i.e., time after substrate is added) per gram of biocatalyst used in the reaction.

[0055] In further embodiments, the SHC / HAC enzyme variants of the disclosure exhibit modified yield compared to a reference SHC protein (e.g., wild-type AacSHC (SEQ ID NO: 1), or 215G2 AacSHC (SEQ ID NO: 10), or wild-type ZmoSHC1 (SEQ ID NO: 11), or wild-type ZmoSHC2 (SEQ ID NO: 12), or wild-type BjpSHC (SEQ ID NO: 13), or wild-type GmoSHC (SEQ ID NO: 14), or wild-type TelSHC (SEQ ID NO: 19), or wild-type ApaSHC1 (SEQ ID NO: 20), or wild-type BmeSHC (SEQ ID NO: 28), or wild-type SalSHC (SEQ ID NO: 29), or wild-type ApaSHCA (SEQ ID NO: 30)). The term "target yield factor" refers to the ratio between the obtained product concentration and the concentration of the SHC / HAC variant enzyme in the reaction medium (e.g., purified SHC / HAC enzyme variant or an extract from a recombinant host cell producing the SHC / HAC enzyme variant). In various embodiments, the SHC / HAC enzyme variants of the disclosure exhibit a modified (e.g., increased) fold increase in enzymatic activity (e.g., modified / increased homofarnesol ambrox cyclase (HAC) activity) compared to a reference SHC protein (e.g., SEQ ID NO:1, or SEQ ID NO:10, or SEQ ID NO:11, or SEQ ID NO:12, or SEQ ID NO:13, or SEQ ID NO:14, or SEQ ID NO:19, or SEQ ID NO:20, or SEQ ID NO:28, or SEQ ID NO:29, or SEQ ID NO:30). This increase in activity may be at least: 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and / or 100 fold.

[0056] As used herein, the term "amino acid modification" refers to the insertion of one or more amino acids between two amino acids, the deletion of one or more amino acids, or the substitution of one or more amino acids with one or more different amino acids (which may be conservative or non-conservative) compared to the amino acid sequence of a reference amino acid sequence. A substitution replaces the amino acids of the reference sequence with the same number of amino acids in the variant sequence. The reference amino acid sequence may be, for example, a wild-type (WT) amino acid sequence (e.g., SEQ ID NO: 1, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 19, or SEQ ID NO: 20, or SEQ ID NO: 28, or SEQ ID NO: 29, or SEQ ID NO: 30), or may be, for example, the sequence of an SHC / HAC enzyme variant (e.g., Aac 215G2 variant - SEQ ID NO: 10) itself.

[0057] Amino acid modifications can be readily identified by comparison of the amino acid sequence of the SHC / HAC enzyme variant with that of a reference amino acid sequence.

[0058] Conservative amino acid substitutions may be made, by way of illustration, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids, as outlined above, can be grouped into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0059] Consequently, as used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the replacement of Asp with Glu maintains one negative charge in the modified polypeptide. In addition, glycine and proline may be substituted for each other based on their ability to disrupt alpha-helices. Some preferred conservative substitutions within the above six groups are the replacements within the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (ii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. Given the known genetic code and recombinant and synthetic DNA techniques, skilled scientists can easily construct DNA encoding conservative amino acid variants.

[0060] As used herein, a "non-conservative substitution" or "non-conservative amino acid exchange" is defined as the replacement of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) through (6) as set forth above. Typically, the SHC / HAC enzyme variants described herein are prepared using non-conservative substitutions that alter the biological function (e.g., HAC activity) of the disclosed SHC / HAC enzyme variant. For ease of reference, the one-letter amino acid symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission are indicated as follows: A three-letter code is also provided for reference purposes.

[0061] [Table 1]

[0062] Amino acid modifications, such as amino acid substitutions, may be introduced using known protocols of recombinant genetic technology, including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in vitro transcription, which may be used to introduce such changes into a reference sequence to yield SHC / HAC enzyme variants. The enzyme variants may then be screened for SHC / HAC functional activity.

[0063] Suitable sources of SHC / HAC enzymes include, for example, Alicyclobacillus acidocaldarius (Aac), Zymomonas mobilis (Zmo), Bradyrhizobium japonicum (Bjp), Gluconobacter morbifer (Gmo), Burkholderia ambifaria, Bacillus anthracis, Methylococcus capsulatus, Frankia alni, Acetobacter pasteurianus(Apa), Thermosynechococcus elongatus(Tel), Streptomyces coelicolor(Sco), Rhodopseudomonas palustris(Rpa), Teredinibacter turnerae(Ttu), Pelobacter carbinolicus(Pca), Bacillus megaterium(Bme), Streptomyces albolongus(Sal), and Tetrahymena pyriformis (e.g., WO 2010 / 139719, US 2012 / 01345477, WO 2012 / 066059, the contents of which are incorporated herein by reference.

[0064] In particular, the SHC / HAC enzyme (e.g., an SHC / HAC enzyme from which an SHC / HAC enzyme variant may be derived) may be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, a Zymomonas mobilis SHC / HAC (ZmoSHC1) enzyme, a Bradyrhizobium japonicum (Bjp or Bja) SHC / HAC enzyme, a Gluconobacter morbifer (Gmo) SHC / HAC enzyme, an Acetobacter pasteurianus SHC / HAC (ApaSHC1) enzyme, or a Bacillus megaterium (Bme) SHC / HAC enzyme. In particular, the SHC / HAC enzyme (e.g., an SHC / HAC enzyme from which an SHC / HAC enzyme variant may be derived) may be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme.

[0065] For ease of reference, the designation "AacSHC" may be used to refer to the Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, "ZmoSHC" may be used to refer to the Zymomonas mobilis (Zmo) SHC / HAC enzyme, "BjpSHC" or "BjaSHC" may be used to refer to the Bradyrhizobium japonicum (Bjp) SHC / HAC enzyme, "ApaSHC" may be used to refer to the Acetobacter pasteurianus (Apa) SHC / HAC enzyme, "BmeSHC" may be used to refer to the Bacillus megaterium (Bme) SHC / HAC enzyme, "SalSHC" may be used to refer to the Streptomyces albolongus (Sal) SHC / HAC enzyme, and "GmoSHC" may be used to refer to the Gluconobacter morbifer (Gmo) SHC / HAC enzyme.

[0066] The enzyme sequences for AacSHC, ZmoSHC, and BjpSHC are disclosed in BASF WO 2010 / 139719, US 2012 / 01345477A1, Seitz et al. (cited above), and Seitz (2012 PhD thesis, cited above). Two different sequences have been disclosed for ZmoSHC, designated ZmoSHC1 and ZmoSHC2. The Gmo SHC / HAC enzyme sequence is disclosed in WO 2018 / 157021. The SalSHC enzyme is disclosed in Liu et al. (2020): A Novel Soluble Squalene-Hopene Cyclase and Its Application in Efficient Synthesis of Hopene, Frontiers in Bioengineering and Biotechnology, vol. 8, article 426, https: / / doi.org / 10.3389 / fbioe.2020.00426.

[0067] Table 1 discloses the sources and accession numbers of wild-type SHC enzymes. Table 1. Sources and accession numbers of wild-type (WT) SHC enzymes. [Table 2]

[0068] The sequences of wild-type AacSHC, wild-type ZmoSHC1, wild-type ZmoSHC2, wild-type BjpSHC, wild-type GmoSHC, wild-type TelSHC, and wild-type ApaSHC1, wild-type BmeSHC, wild-type SalSHC, and wild-type ApaSHCA are also disclosed herein (SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively).

[0069] Alignment of the WT SHC sequences prepared by Hoshino and Sato (2002, supra) indicated that multiple motifs were detected in all four sequences and consisted of the core sequence Gln-XXX-Gly-X-Trp, which was found six times in the SHC sequences of both Z. mobilis and A. acidocaldarius (see Figure 3 in Reipen et al. 1995, Microbiology 141, 155-161). Hoshino and Sato (2002, supra) reported that aromatic amino acids were unusually abundant in SHC and noted two characteristic motifs in SHC: one was a QW motif represented by a specific amino acid motif [(K / R)(G / A)X2-3(F / Y / W)(L / IV)3X3QX2-5GXW], and the other was a DXDDTA motif. Wendt et al. (1997, Science 277, 1811-1815 and 1999, J Mol Biol 286, 175-187) reported the X-ray structural analysis of the A. acidocaldarius SHC. The DXDDTA motif appears to correlate with the SHC active site.

[0070] As used herein, the reference AacSHC protein may refer to the wild-type AacSHC protein as disclosed in SEQ ID NO: 1. AacSHC has the activity of homofarnesol ambrox cyclase (HAC), which is useful for the production of ambrox derivatives through the biocatalytic reaction of SHC with a homofarnesol substrate. The primary reaction of AacSHC is the cyclization of linear or non-linear substrates, such as homofarnesol, to produce ambrox.

[0071] Functional homologs of the wild-type SHC / HAC enzymes or SHC / HAC enzyme variants described herein are also suitable for use in cyclization reactions, e.g., to produce (-)-ambrox, in a recombinant host. Thus, the recombinant host may include one or more heterologous nucleic acid(s) encoding a functional homolog of the above-described polypeptides and / or a heterologous nucleic acid encoding a SHC / HAC derivative enzyme as described herein.

[0072] A functional homolog is a polypeptide that has sequence similarity with a reference polypeptide and performs one or more of the biochemical or physiological functions of the reference polypeptide. Functional homologs and reference polypeptides may be naturally occurring polypeptides, and the sequence similarity may be due to convergent or divergent evolutionary events. Therefore, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of naturally occurring functional homologs, such as polypeptides encoded by mutants of a wild-type coding sequence, may themselves be functional homologs. Functional homologs can also be created for a given polypeptide through site-directed mutagenesis of the coding sequence, or for different naturally occurring polypeptides by combining domains from the coding sequences ("domain swapping"). Techniques for modifying genes encoding functional homologs described herein are known and include, among others, directed evolution, site-directed mutagenesis, and random mutagenesis techniques, and may be useful to increase a specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide:polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to nucleic acids encoding functionally homologous polypeptides.

[0073] Functional homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, performing a query on a nucleotide or polypeptide sequence database can identify homologs of nucleic acid sequences encoding SHC derivative polypeptides, etc.

[0074] Hybridization can also be used to identify functional homologs and / or to identify homology between two nucleic acid sequences as an indicator. Nucleic acid sequences encoding any of the proteins disclosed herein, or portions thereof, can be used as hybridization probes according to standard hybridization techniques. Hybridization of the probe to DNA or RNA from a test source (e.g., mammalian cells) is indicative of the presence of the relevant DNA or RNA in the test source. Hybridization conditions are known to those skilled in the art and can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY, 6.3.1-6.3.6, 1991. Moderate hybridization conditions are defined as equivalent to hybridization in 2x sodium chloride / sodium citrate (SSC) at 30°C, followed by a wash in 1x SSC, 0.1% SDS at 50°C. Highly stringent conditions are defined as equivalent to hybridization in 6x sodium chloride / sodium citrate (SSC) at 45°C, followed by a wash in 0.2x SSC, 0.1% SDS at 65°C. Sequence analysis to identify functional homologs can also involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using related amino acid sequences as reference sequences. Amino acid sequences are, in some cases, inferred from nucleotide sequences. Those polypeptides in the database with greater than 40% sequence identity are candidates for further evaluation for suitability for use in SHC / HAC biotransformation reactions. Amino acid sequence similarity allows for conservative amino acid substitutions, such as the substitution of one hydrophobic residue for another, or one polar residue for another. If desired, manual inspection of such candidates can be performed to narrow the number of candidates to be further evaluated. Manual inspection can be performed, for example, by selecting those candidates that appear to have conserved functional domains.

[0075] Typically, polypeptides exhibiting at least about 30% amino acid sequence identity are useful for identifying conserved regions. Conserved regions of related polypeptides exhibit at least 30%, 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%, or 69% amino acid sequence identity. In some embodiments, the conserved regions exhibit at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity. Sequence identity can be determined as described above and below.

[0076] The SHC / HAC enzymes or enzyme variants described herein and used in the methods described herein may be based on, for example, the amino acid sequence of SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or a variant, homologue, mutant, derivative, or fragment thereof. The SHC / HAC enzyme or enzyme variant may be, for example, SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, and may be at least 30%, 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%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, It may have an amino acid sequence that is 0%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the present invention.

[0077] Additionally, the reference SHC produced may be based on amino acid sequences produced from E. coli.

[0078] "Percent (%) identity" with respect to the nucleotide sequence of a gene is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to nucleotides in the DNA sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in a variety of ways that are within the skill in the art, illustratively using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide chain of amino acids, regardless of length or post-translational modification.

[0079] As used herein, the term "derivative" includes, but is not limited to, variants. The terms "derivative" and "variant" are used interchangeably herein.

[0080] In preferred embodiments, variant enzymes usable in the present disclosure exhibit a total of up to 200 changes (modifications) (i.e., replacements, insertions, deletions, N-terminal truncations, and / or C-terminal truncations) in the amino acid sequence (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200). The amino acid exchanges may be conservative and / or non-conservative. In a preferred embodiment, the variants usable in the present disclosure differ from the protein or domain from which they are derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid exchanges (preferably conservative amino acid changes). The variants may additionally or alternatively contain amino acid deletions, which may be N-terminal truncations, C-terminal truncations, or internal deletions, or any combination thereof. Such variants containing N-terminal truncations, C-terminal truncations, and / or internal deletions are referred to in the context of this application as "deletion variants" or "fragments." The terms "deletion variants" and "fragments" are used interchangeably herein. Deletion variants can be naturally occurring (for example, splice variants), or can be artificially constructed, preferably by genetic engineering means.Typically, the protein or protein domain from which deletion variants are derived is a wild-type protein.However, the deletion variants of the present disclosure can also be derived from homologs, orthologs, or paralogs of parent polypeptides or artificially constructed variants, provided that the deletion variants exhibit at least one biological activity of parent polypeptides.Preferably, a deletion variant (or fragment) has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids at its N-terminus, and / or at its C-terminus, and / or internally, compared to the parent polypeptide.

[0081] In some embodiments, the SHC / HAC enzyme variants described herein include only substitutions and do not include any deletions or insertions.

[0082] "Variant," as used herein, may alternatively or additionally be characterized by a degree of sequence identity with the parent polypeptide from which it is derived. Variants of the WT / reference SHC / HAC or SHC / HAC derivatives of the present disclosure share at least 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, It may have a sequence identity of 5%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0083] The expression "at least 30%, 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%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, The terms "3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity" are used throughout this specification in reference to sequence comparisons of polypeptides and polynucleotides. Polynucleotides belonging to any of the families of enzymes or proteins disclosed herein can be identified based on their similarity to related genes or proteins, respectively. For example, identification can be based on sequence identity.In a preferred embodiment, the disclosure provides a nucleic acid molecule comprising: (a) a nucleic acid molecule encoding a polypeptide of a wild-type SHC / HAC enzyme disclosed herein (e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30); (b) the nucleotide sequence of SEQ ID NO:15; and (c) a nucleic acid molecule comprising a segment of at least 30 (e.g., at least 30, 40, 50, 60, 80, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 850, 900, 950, 1000, or 1010) nucleotides of SEQ ID NO:15, and a nucleic acid molecule comprising at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 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%, 1010, 102 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%, at least 81%, at least 82%, at least 83% , at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the present invention.

[0084] Preferably, the polypeptide in question and the reference polypeptide exhibit the indicated sequence identity over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. Preferably, the polynucleotide in question and the reference polynucleotide exhibit the indicated sequence identity over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides. In cases where two sequences are compared and the reference sequence is not specified in comparison with the sequence for which the sequence identity percentage is to be calculated, unless otherwise specifically indicated, the sequence identity should be calculated with reference to the longer of the two sequences to be compared. Where a reference sequence is indicated, sequence identity is determined based on the full length of the reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30) unless otherwise specifically indicated.

[0085] For example, a peptide sequence consisting of 130 amino acids may exhibit a maximum percentage sequence identity of 20.6% (130 / 631 x 100) when compared to the full-length wild-type AacSHC having 631 amino acid residues, while a sequence having a length of 300 amino acids may exhibit a maximum percentage sequence identity of 47.5% (300 / 631 x 100).

[0086] Nucleotide and amino acid sequence similarity, i.e., percentage of sequence identity, can be determined through sequence alignment. Such alignments include, for example, https: / / www.ebi.ac.uk / Tools / msa / clustalo / The algorithm may be implemented using several algorithms known in the art, such as the GAP program (University of Iowa Mathematical Algorithm), or the mathematical algorithm of Myers and Miller (1989-Cabios 4:11-17), or the algorithm of Clone Manager 9, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). The preferred parameters used are the default parameters as set on https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0087] The grade of sequence identity (sequence matching) may be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score=100, word length=12, to obtain polynucleotide sequences homologous to those nucleic acids encoding related proteins.

[0088] BLAST protein searches are performed using the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to the SHC polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques, such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are mentioned in this application, these percentages are calculated with respect to the full length of the longer sequence, unless otherwise specifically indicated.

[0089] In a specific embodiment, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4). Particular SHC / HAC enzymes and enzyme variants that may be used in the methods described herein are described further below.

[0090] Aac 215G2 SHC / HAC variant with a novel mutation Surprisingly, it has been found that an SHC / HAC enzyme variant derived from the Aac SHC / HAC enzyme variant disclosed in WO 2016 / 170099 (215G2 SHC / HAC enzyme variant) provides improved enzymatic activity for the conversion of EEH to (-)-ambrox. Even more surprisingly, it has been found that an SHC / HAC enzyme variant derived from 215G2 provides improved enzymatic activity for the conversion of E,E-bishomofarnesol to ambroxoxide.

[0091] The new SHC / HAC enzyme variants have two or three amino acid alterations in addition to the amino acid substitutions already present in the 215G2 SHC / HAC enzyme variant.

[0092] Thus, provided herein are processes for producing (-)-ambrox by enzymatically converting EEH to (-)-ambrox. Also provided herein are processes for producing ambroxide by enzymatically converting E,E-bishomofarnesol to ambroxide. These processes may use any of the wild-type SHC / HAC enzymes or enzyme variants described herein, particularly the Aac 215G2 SHC / HAC variant described herein.

[0093] SHC / HAC enzyme variants of Aac 215G2 SHC / HAC have an amino acid sequence that is at least about 70.0% identical to SEQ ID NO: 1. For example, SHC / HAC enzyme variants may have at least about 75.0%, or at least about 80.0%, or at least about 85.0%, or at least about 90.0%, or at least about 95.0%, or at least about 95.5%, or at least about 96.0%, or at least about 96.5%, or at least about 97.0%, or at least about 97.5%, or at least about 98.0%, or at least about 98.5%, or at least about 99.0% identity to SEQ ID NO: 1.

[0094] An enzyme variant of Aac 215G2 SHC / HAC has less than 100% identity to SEQ ID NO: 1. For example, an SHC / HAC enzyme variant may have equal to or less than about 99.5% identity to SEQ ID NO: 1, or equal to or less than about 99.0% identity to SEQ ID NO: 1.

[0095] For example, an enzyme variant of Aac 215G2 SHC / HAC may have from about 70.0% to about 99.5%, or from about 80.0% to about 99.0%, or from about 85.0% to about 98.5%, or from about 90.0% to about 98.0% identity to SEQ ID NO:1.

[0096] "Percent (%) identity" with respect to a polypeptide or nucleotide sequence is defined as the percentage of amino acids or nucleotides in a candidate sequence that are identical to those in a reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, but without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways that are within the skill in the art, illustratively using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide chain of amino acids, regardless of length or post-translational modification.

[0097] Nucleotide and amino acid sequence similarity, i.e., percentage of sequence identity, can be determined through sequence alignment. Such alignments include, for example, https: / / www.ebi.ac.uk / Tools / msa / clustalo / The algorithm can be implemented using several algorithms known in the art, such as the GAP program (University of Iowa Mathematical Algorithm), or the Myers and Miller Mathematical Algorithm (1989-Cabios 4:11-17), preferably the Karlin and Altschul Mathematical Algorithm (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). The preferred parameters used are the default parameters as set on https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0098] Percentage sequence identity may be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score = 100, word length = 12, to obtain polynucleotide sequences homologous to those nucleic acids encoding related proteins. BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to polypeptides.

[0099] To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov Random Fields. When percentages of sequence identity are mentioned in this application, these percentages are calculated with respect to the full length of the longer sequence unless otherwise specifically indicated.

[0100] In a specific embodiment, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0101] In certain embodiments, the SHC / HAC enzyme variant may have less than or equal to about 30 amino acid modifications compared to SEQ ID NO: 1. For example, the SHC / HAC enzyme variant may have less than or equal to about 25, or less than or equal to about 20, or less than or equal to about 15, or less than or equal to about 10, or less than or equal to about 9, or less than or equal to about 8, or less than or equal to about 7, or less than or equal to about 6 amino acid modifications compared to SEQ ID NO: 1. For example, the SHC / HAC enzyme variant may have at least about 5 or at least about 6 amino acid modifications compared to SEQ ID NO: 1. The amino acid modifications may be, for example, insertions, deletions, and / or substitutions, as described above.

[0102] In some embodiments, the only amino acid modifications in the SHC / HAC enzyme variant compared to SEQ ID NO:1 are substitutions (ie, no insertions or deletions). Amino acid alterations are defined relative to a reference sequence. An amino acid alteration relative to a reference sequence means that the amino acid sequence of a variant sequence differs from the reference sequence.

[0103] Amino acids in a reference sequence and a variant sequence may be assigned numbers, where the numbering begins with the amino acid at the N-terminus of the polypeptide (i.e., the amino acid at the N-terminus of a polypeptide is numbered 1, the next amino acid is numbered 2, etc.). A "position" in a reference sequence refers to a particular amino acid residue present in the reference sequence, as identified by a particular numbered amino acid in the reference sequence. A "position" in a variant sequence refers to a particular amino acid residue present in the variant sequence, as identified by a particular numbered amino acid in the variant sequence.

[0104] Because a variant sequence may contain deletions or insertions compared to a reference sequence, amino acids in the variant sequence may be numbered differently than the same amino acids in the reference sequence. As an example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO: 1, the amino acid will retain the number 132 in the reference sequence, while the amino acid following the insertion will have the number 133 in the variant sequence. In this example, the position in the variant sequence that corresponds to position 132 in the reference sequence is position 133. Thus, an amino acid in a variant sequence that is retained from a reference sequence may be defined by referring to the "corresponding position" in the reference sequence. In other words, a "position" in a variant sequence may be defined by referring to the "corresponding position" in the reference sequence. Notably, a substitution in a variant sequence compared to a reference sequence may be defined by referring to the "corresponding position" in the reference sequence, despite any insertions and / or deletions in the reference sequence. If an amino acid in the reference sequence is deleted, there will be no "corresponding position" in the variant sequence. If there are no insertions or deletions (i.e., only substitutions) compared to the reference sequence, the "corresponding position" of the reference sequence will be the same as the position in the variant sequence.

[0105] The original amino acid modifications in the 215G2 SHC / HAC enzyme variant (compared to wild-type Aac SHC) are retained in the new Aac 215G2 SHC / HAC enzyme variant described herein. The original amino acid modifications in the 215G2 SHC / HAC enzyme variant compared to wild-type Aac SHC were substitutions M132R, A224V, and I432T (i.e., substitution of an M residue with an R residue at position 132, an A residue with a V residue at position 224, and an I residue with a T residue at position 432).

[0106] Thus, the new Aac 215G2 SHC / HAC enzyme variant has amino acid modifications compared to SEQ ID NO: 1 at positions corresponding to positions 132, 224, and 432 of SEQ ID NO: 1, which are M132R, A224V, and I432T, respectively. The numbers "132" in "M132R," "224" in "A224V," and "432" in "I432T" refer to the numbering of SEQ ID NO: 1 and do not necessarily correspond to the numbering of the variant sequence due to any additional insertions and / or deletions, as discussed above.

[0107] The amino acid sequence of the new Aac 215G2 SHC / HAC enzyme variant also has amino acid modifications, relative to SEQ ID NO: 1, at a position corresponding to position 557 of SEQ ID NO: 1 and at least one position corresponding to positions 81, 431, or 613 of SEQ ID NO: 1. These amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions).

[0108] For example, the amino acid sequence of the SHC / HAC enzyme variant may have amino acid modifications compared to SEQ ID NO: 1 at a position corresponding to position 557 of SEQ ID NO: 1 and at one position corresponding to positions 81, 431, or 613 of SEQ ID NO: 1. These amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions).

[0109] For example, the amino acid sequence of the SHC / HAC enzyme variant may have amino acid modifications relative to SEQ ID NO: 1 at a position corresponding to position 557 of SEQ ID NO: 1 and at two positions selected from positions corresponding to positions 81, 431, and 613 of SEQ ID NO: 1. These amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions).

[0110] For example, the amino acid sequence of the SHC / HAC enzyme variant may have amino acid modifications relative to SEQ ID NO: 1 at a position corresponding to position 557 of SEQ ID NO: 1, and at all positions corresponding to positions 81, 431, and 613 of SEQ ID NO: 1. These amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions).

[0111] For example, the SHC / HAC enzyme variant may have amino acid modifications relative to SEQ ID NO: 1 at positions corresponding to positions 431 and 557 of SEQ ID NO: 1. These amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). In some embodiments, the SHC / HAC enzyme variant has the sequence of SEQ ID NO: 2.

[0112] For example, the SHC / HAC enzyme variant may have amino acid modifications compared to SEQ ID NO: 1 at positions corresponding to positions 557 and 613 of SEQ ID NO: 1. These amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). In some embodiments, the SHC / HAC enzyme variant has the sequence of SEQ ID NO: 3.

[0113] For example, the SHC / HAC enzyme variant may have amino acid modifications relative to SEQ ID NO: 1 at positions corresponding to positions 81, 557, and 613 of SEQ ID NO: 1. These amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). In some embodiments, the SHC / HAC enzyme variant has the sequence of SEQ ID NO: 4.

[0114] For example, the SHC / HAC enzyme variant may have amino acid modifications relative to SEQ ID NO: 1 at positions corresponding to positions 81, 431, and 557 of SEQ ID NO: 1. These amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). In some embodiments, the SHC / HAC enzyme variant has the sequence of SEQ ID NO: 5.

[0115] An amino acid modification at a position corresponding to position 557 of SEQ ID NO: 1 may be, for example, A557X, which refers to the substitution of amino acid A at position 557 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 557.

[0116] The new amino acid (X) at the position corresponding to position 557 of SEQ ID NO: 1 may be, for example, Met, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 557 of SEQ ID NO: 1 may be a substitution of the amino acid of SEQ ID NO: 1 (i.e., A) with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the amino acid modification at the position corresponding to position 557 of SEQ ID NO: 1 may be a substitution of the amino acid of SEQ ID NO: 1 (i.e., A) with threonine (i.e., the amino acid modification at the position corresponding to position 557 of SEQ ID NO: 1 is A557T).

[0117] An amino acid modification at a position corresponding to position 81 of SEQ ID NO: 1 may be, for example, Y81X, which refers to the substitution of amino acid Y at position 81 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 81.

[0118] The new amino acid (X) at the position corresponding to position 81 of SEQ ID NO:1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Phe. For example, the amino acid modification at the position corresponding to position 81 of SEQ ID NO:1 can be a substitution of the amino acid of SEQ ID NO:1 (i.e., Y) with a basic amino acid (i.e., His, Lys, or Arg). For example, the amino acid modification at the position corresponding to position 81 of SEQ ID NO:1 can be a substitution of the amino acid of SEQ ID NO:1 (i.e., Y) with a histidine (i.e., the amino acid modification at the position corresponding to position 81 of SEQ ID NO:1 is Y81H).

[0119] An amino acid modification at a position corresponding to position 431 of SEQ ID NO: 1 may be, for example, H431X, which refers to the substitution of the amino acid H at position 431 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 431.

[0120] The new amino acid (X) at the position corresponding to position 431 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 431 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., H) with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the amino acid modification at the position corresponding to position 431 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., H) with leucine (i.e., the amino acid modification at the position corresponding to position 431 of SEQ ID NO:1 is H431L).

[0121] An amino acid modification at a position corresponding to position 613 of SEQ ID NO: 1 may be, for example, R613X, which refers to the substitution of the amino acid R at position 613 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 613.

[0122] The new amino acid (X) at the position corresponding to position 631 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 613 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., R) with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the amino acid modification at the position corresponding to position 613 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., R) with serine (i.e., the amino acid modification at the position corresponding to position 613 of SEQ ID NO:1 is R613S).

[0123] In some embodiments, the new Aac 215G2 SHC variant has the following amino acid substitution: (i) M132R, A224V, I432T, A557T, and H431L (SEQ ID NO: 2); or (ii) M132R, A224V, I432T, A557T, and R613S (SEQ ID NO: 3); or (iii) M132R, A224V, I432T, A557T, Y81H, and R613S (SEQ ID NO: 4); or (iv) M132R, A224V, I432T, A557T, Y81H, and H431L (SEQ ID NO: 5) Identical to SEQ ID NO: 1 except for:

[0124] The SHC / HAC enzyme variant may have one or more additional amino acid modifications, for example, at positions corresponding to positions 90, 172, and / or 277 of SEQ ID NO:1.

[0125] An amino acid modification at a position corresponding to position 90 of SEQ ID NO: 1 may be, for example, T90X, which refers to the substitution of the amino acid T at position 90 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 90.

[0126] The new amino acid (X) at the position corresponding to position 90 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 90 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., T) with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, Ile). For example, the amino acid modification at the position corresponding to position 90 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., T) with alanine (i.e., the amino acid modification at the position corresponding to position 90 of SEQ ID NO:1 is T90A).

[0127] An amino acid modification at a position corresponding to position 172 of SEQ ID NO: 1 may be, for example, A172X, which refers to the substitution of the amino acid T at position 172 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 172.

[0128] The new amino acid (X) at the position corresponding to position 172 of SEQ ID NO:1 may be, for example, Met, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 172 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., A) with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, Gln). For example, the amino acid modification at the position corresponding to position 172 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., A) with threonine (i.e., the amino acid modification at the position corresponding to position 172 of SEQ ID NO:1 is A172T).

[0129] An amino acid modification at a position corresponding to position 277 of SEQ ID NO: 1 may be, for example, M277X, which refers to the substitution of the amino acid M at position 277 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 277.

[0130] The new amino acid (X) at the position corresponding to position 277 of SEQ ID NO:1 may be, for example, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 277 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., M) with a basic amino acid (i.e., His, Lys, Arg). For example, the amino acid modification at the position corresponding to position 277 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., M) with a lysine (i.e., the amino acid modification at the position corresponding to position 277 of SEQ ID NO:1 is M277K).

[0131] The SHC / HAC enzyme variant may have one or more additional amino acid modifications, e.g., at positions corresponding to positions 37, 174, and / or 601 of SEQ ID NO: 1. For example, the SHC / HAC enzyme variant may have one or more amino acid substitutions (e.g., conservative or non-conservative substitutions) at positions corresponding to positions 37, 174, and / or 601 of SEQ ID NO: 1.

[0132] An amino acid modification at a position corresponding to position 37 of SEQ ID NO: 1 may be, for example, L37X, which refers to the substitution of the amino acid L at position 37 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 37.

[0133] The new amino acid (X) at the position corresponding to position 37 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 37 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., L) with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the amino acid modification at the position corresponding to position 37 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., L) with glutamine (i.e., the amino acid modification at the position corresponding to position 37 of SEQ ID NO:1 is L37Q).

[0134] An amino acid modification at a position corresponding to position 174 of SEQ ID NO: 1 may be, for example, V174X, which refers to the substitution of amino acid V at position 174 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 174.

[0135] The new amino acid (X) at the position corresponding to position 174 of SEQ ID NO:1 may be, for example, Met, Ala, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. The new amino acid (X) at the position corresponding to position 174 of SEQ ID NO:1 may be, for example, a hydrophobic amino acid (i.e., Met, Ala, Leu, or Ile). For example, the amino acid modification at the position corresponding to position 174 of SEQ ID NO:1 may be a substitution of the amino acid (i.e., V) of SEQ ID NO:1 with isoleucine (i.e., the amino acid modification at the position corresponding to position 174 of SEQ ID NO:1 is V174I).

[0136] An amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1 may be, for example, F601X, which refers to the substitution of amino acid F at position 601 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 601.

[0137] The new amino acid (X) at the position corresponding to position 601 of SEQ ID NO:1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Tyr. The new amino acid (X) at the position corresponding to position 601 of SEQ ID NO:1 can be, for example, an aromatic acid (i.e., Trp, Tyr, Phe). For example, the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 can be a substitution of the amino acid of SEQ ID NO:1 (i.e., F) with tyrosine (i.e., the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 is F601Y).

[0138] SHC / HAC enzyme variants may have one or more additional amino acid modifications, e.g., at positions corresponding to positions 77, 92, 129, 579, 601, and / or 605 of SEQ ID NO: 1. For example, SHC / HAC enzyme variants may have one or more amino acid substitutions (e.g., conservative or non-conservative substitutions) at positions corresponding to positions 77, 92, 129, 579, 601, and / or 605 of SEQ ID NO: 1.

[0139] An amino acid modification at a position corresponding to position 77 of SEQ ID NO: 1 may be, for example, T77X, which refers to the substitution of the amino acid T at position 77 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 77.

[0140] The new amino acid (X) at the position corresponding to position 77 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 77 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., T) with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the amino acid modification at the position corresponding to position 77 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., T) with alanine (i.e., the amino acid modification at the position corresponding to position 77 of SEQ ID NO:1 is T77A).

[0141] An amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1 may be, for example, I92X, which refers to the substitution of amino acid I at position 92 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 92.

[0142] The new amino acid (X) at the position corresponding to position 92 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Leu, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 92 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., I) with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the amino acid modification at the position corresponding to position 92 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., I) with valine (i.e., the amino acid modification at the position corresponding to position 92 of SEQ ID NO:1 is I92V).

[0143] An amino acid modification at a position corresponding to position 129 of SEQ ID NO: 1 may be, for example, F129X, which refers to the substitution of the amino acid F at position 129 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 129.

[0144] The new amino acid (X) at the position corresponding to position 129 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Tyr. For example, the amino acid modification at the position corresponding to position 129 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., F) with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the amino acid modification at the position corresponding to position 129 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., F) with leucine (i.e., the amino acid modification at the position corresponding to position 129 of SEQ ID NO:1 is F129L).

[0145] An amino acid modification at a position corresponding to position 579 of SEQ ID NO: 1 may be, for example, Q579X, which refers to the substitution of the amino acid Q at position 579 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 579.

[0146] The new amino acid (X) at the position corresponding to position 579 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the amino acid modification at the position corresponding to position 579 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., Q) with a basic amino acid (i.e., His, Lys, or Arg). For example, the amino acid modification at the position corresponding to position 579 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., Q) with a histidine (i.e., the amino acid modification at the position corresponding to position 579 of SEQ ID NO:1 is Q579H).

[0147] An amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1 may be, for example, F601X, which refers to the substitution of amino acid F at position 601 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 601.

[0148] The new amino acid (X) at the position corresponding to position 601 of SEQ ID NO:1 may be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Tyr. The new amino acid (X) at the position corresponding to position 601 of SEQ ID NO:1 may be, for example, an aromatic acid (i.e., Trp, Tyr, Phe). For example, the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 may be a substitution of the amino acid of SEQ ID NO:1 (i.e., F) with histidine (i.e., the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 is F601Y).

[0149] An amino acid modification at a position corresponding to position 605 of SEQ ID NO: 1 may be, for example, F605X, which refers to the substitution of amino acid F at position 605 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 605.

[0150] The new amino acid (X) at the position corresponding to position 605 of SEQ ID NO:1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Tyr. The new amino acid (X) at the position corresponding to position 605 of SEQ ID NO:1 can be, for example, an aromatic acid (i.e., Trp, Tyr, Phe). For example, the amino acid modification at the position corresponding to position 605 of SEQ ID NO:1 can be a substitution of the amino acid of SEQ ID NO:1 (i.e., F) with tryptophan (i.e., the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 is F605W).

[0151] For example, a SHC / HAC enzyme variant may have amino acid modifications (eg, substitutions) at positions corresponding to positions 132 and 432 of SEQ ID NO:1. For example, a SHC / HAC enzyme variant may have an amino acid modification (eg, a substitution) at a position corresponding to position 601 of SEQ ID NO:1. For example, a SHC / HAC enzyme variant may have amino acid modifications (eg, substitutions) at positions corresponding to positions 77, 92, and 129 of SEQ ID NO:1. For example, a SHC / HAC enzyme variant may have amino acid modifications (eg, substitutions) at positions corresponding to positions 579 and 601 of SEQ ID NO:1.

[0152] For example, a SHC / HAC enzyme variant may have amino acid modifications (eg, substitutions) at positions corresponding to positions 129, 132, and 432 of SEQ ID NO:1. For example, a SHC / HAC enzyme variant may have amino acid modifications (eg, substitutions) at positions corresponding to positions 132, 432, and 601 of SEQ ID NO:1. For example, a SHC / HAC enzyme variant may have amino acid modifications (eg, substitutions) at positions corresponding to positions 129, 132, 432, and 601 of SEQ ID NO:1.

[0153] The new SHC / HAC enzyme variants may have increased enzymatic activity for the conversion of EEH to (-)-ambrox or the conversion of bisEEH to ambroxide, for example, compared to the SHC / HAC enzyme of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10. Increased enzymatic activity may refer to any aspect of the enzymatic conversion of EEH to (-)-ambrox or the enzymatic conversion of bisEEH to ambroxide, including, for example, increased total conversion of EEH or bisEEH, increased conversion rate of EEH or bisEEH (e.g., in the first 6 hours or the first 12 hours of the reaction), increased production of (-)-ambrox or ambroxide, and decreased production of by-products. Increased enzymatic activity may be defined in terms of increased productivity, which may generally be defined in terms of (-)-ambrox or ambroxide produced per gram, per hour, and per liter of biocatalyst of the reaction.

[0154] The new SHC / HAC enzyme variants may provide increased conversion of EEH or E,E-bishomofarnesol (bisEEH) compared to, for example, the SHC / HAC wild-type enzyme of SEQ ID NO: 1 and / or the Aac 215G2 SHC / HAC enzyme variant of SEQ ID NO: 10. Accordingly, the processes described herein may have increased levels of EEH or bisEEH conversion compared to processes using the SHC / HAC wild-type enzyme of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10. The new SHC / HAC enzyme variants may provide increased EEH or bisEEH conversion rates compared to, for example, the SHC / HAC wild-type enzyme of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10. Accordingly, the processes described herein may have increased EEH or bisEEH conversion rates compared to the SHC / HAC wild-type enzyme of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10. The new SHC / HAC enzyme variants may provide increased EEH or bis-EEH conversion rates over the first 4 hours, or over the first 6 hours, or over the first 8 hours, or over the first 12 hours, or over the first 24 hours of the reaction, as compared to, for example, the SHC / HAC wild-type enzyme of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10. Thus, the processes described herein may have increased EEH or bis-EEH conversion rates over the first 4 hours, or over the first 6 hours, or over the first 8 hours, or over the first 12 hours, or over the first 24 hours of the reaction, as compared to the SHC / HAC wild-type enzyme of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10. This may be compared to using both enzymes (i.e., the new Aac 215G2 SHC / HAC enzyme variant and the wild-type enzyme of SEQ ID NO: 1 or the 215G2 enzyme of SEQ ID NO: 10) under the same reaction conditions (e.g., the same pH and temperature), or compared to using each enzyme under their respective optimized reaction conditions (e.g., optimized pH and temperature), which may be different from each other.

[0155] For example, the new SHC / HAC enzyme variants may provide, or the process may have, at least about 40% EEH or bis-EEH conversion in the first 12 hours of reaction. For example, the new SHC / HAC enzyme variants may provide, or the process may have, at least about 45%, or at least about 50%, or at least about 55%, or at least about 60% EEH or bis-EEH conversion in the first 12 hours of reaction. For example, the new SHC / HAC enzyme variants may provide, or the process may have, at least about 30% EEH or bis-EEH conversion in the first 6 hours of reaction. For example, the new SHC / HAC enzyme variants may provide, or the process may have, at least about 35%, or at least about 45%, or at least about 50%, or at least about 55% EEH or bis-EEH conversion in the first 12 hours of reaction. This may be compared to using both enzymes (i.e., the new SHC / HAC enzyme variant and the enzyme of SEQ ID NO: 1 or SEQ ID NO: 10) under the same reaction conditions (e.g., the same pH and temperature), or compared to using each enzyme under their respective optimized reaction conditions (e.g., optimized pH and temperature), which may be different from each other.

[0156] The conversion of EEH to (-)-ambrox, or bisEEH to ambroxide, may be determined, for example, using an activity assay as described above, and may be calculated as grams of recoverable product per gram of feedstock (which may be calculated as a percent molar conversion).

[0157] As used herein, any reference herein to a 99% / 100% conversion rate to (-)-ambrox for a homofarnesol substrate is a reference to a 99% / 100% conversion of the homofarnesol isomer (i.e., EEH) that can be converted to (-)-ambrox using an SHC / HAC enzyme or enzyme variant.

[0158] As used herein, any reference herein to 99% / 100% conversion of a bishomofarnesol substrate to amburoxide is a reference to 99% / 100% conversion of a bishomofarnesol isomer (i.e., bisEEH) that can be converted to amburoxide using an SHC / HAC enzyme or enzyme variant.

[0159] The optimum temperature for an SHC / HAC enzyme variant of Aac 215G2 SHC / HAC may be, for example, equal to or greater than about 35° C. For example, the optimum temperature for an SHC / HAC enzyme variant of Aac 215G2 SHC / HAC may range from about 40° C. to about 50° C., e.g., from about 42° C. to about 48° C., or from about 44° C. to about 46° C. For example, the optimum temperature for an SHC / HAC enzyme variant of Aac 215G2 SHC / HAC may be about 45° C. The processes for producing (-)-ambrox or ambroxide disclosed herein may be carried out at the optimum temperature for the SHC / HAC enzyme variant.

[0160] The optimum pH for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC may be, for example, equal to or greater than about 5.4. For example, the optimum pH for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC may range from about 5.2 to about 6.0, e.g., from about 5.4 to about 5.8, e.g., from about 5.6 to about 5.8. For example, the optimum pH for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC may be about 5.6 or about 5.8. The process for producing (-)-ambrox or ambroxide disclosed herein may be carried out at the optimum pH for the SHC / HAC enzyme variant.

[0161] The optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the process for making (-)-ambrox or ambroxide disclosed herein may be, for example, from about 0.010 w / w% to about 0.10 w / w%. For example, the optimal concentration of SDS may be from about 0.040 w / w% to about 0.080 w / w%, e.g., at 4 g / L of substrate (e.g., EEH or bisEEH), the OD 650nm When used with up to 10 cells, the optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the process for making (-)-ambrox or ambroxide disclosed herein may be, for example, about 1.0 w / w% to about 1.5 w / w% when a substrate (e.g., EEH or bisEEH) is used at 125 g / L with 250 g / L of cells. For example, the optimal concentration of SDS may be about 1.2 w / w% to about 1.4 w / w%, e.g., about 1.3 w / w% when a substrate (e.g., EEH or bisEEH) is used at 125 g / L with 250 g / L of cells.

[0162] The processes for making (-)-ambrox or ambrate oxide disclosed herein may be carried out at the optimum temperature range or optimum temperature, and / or optimum pH range or optimum pH, and / or optimum SDS concentration range or optimum SDS concentration for the particular enzyme used, as set forth in Tables 7, 9, or 11 in the Examples below.

[0163] Other variants with new mutations at positions corresponding to positions 81, 90, 172, 277, 431, 557, and / or 613 of SEQ ID NO: 1. As discussed above, it has surprisingly been found that SHC / HAC enzyme variants derived from the Aac SHC / HAC enzyme variant (215G2 SHC / HAC enzyme variant) disclosed in WO 2016 / 170099 provide improved enzymatic activity for the conversion of EEH to (-)-ambrox and for the conversion of bisEEH to ambroxide. The new SHC / HAC enzyme variants have two or three amino acid modifications in addition to the amino acid substitutions already present in the 215G2 SHC / HAC enzyme variant.

[0164] It is expected that other SHC / HAC enzyme variants having one or more of the new mutations identified at positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO: 1 will also provide enzymatic activity (e.g., improved enzymatic activity) for the conversion of EEH to (-)-ambrox or the conversion of bisEEH to ambroxide.

[0165] In particular, it is expected that SHC / HAC enzyme variants derived from other non-Aac species but with one or more of the new amino acid modifications identified at positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1 will also provide enzymatic activity for the conversion of EEH to (-)-ambrox or bisEEH to ambroxide. In particular, it is expected that enzyme variants derived from ZmoSHC1, ZmoSHC2, BjpSHC, GmoSHC, TelSHC, ApaSHC1, BmeSHC, SalSHC, or ApaSHCA with one or more of the new amino acid modifications identified at positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1 will also provide enzymatic activity for the conversion of EEH to (-)-ambrox or bisEEH to ambroxide.

[0166] Thus, provided herein are processes for producing (-)-ambrox by enzymatically converting EEH to (-)-ambrox. Also provided herein are processes for producing ambroxide by enzymatically converting E,E-bishomofarnesol to ambroxide. These processes may use any of the wild-type SHC / HAC enzymes or enzyme variants described herein.

[0167] Additionally, provided herein are SHC / HAC enzyme variants having at least about 70.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications, relative to the wild-type SHC / HAC enzyme, at positions selected from those corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1.

[0168] In particular, provided herein is a process for preparing (-)-ambrox or a mixture containing (-)-ambrox, the process comprising enzymatically converting EEH or a mixture containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC variant having at least about 70.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications, relative to the wild-type SHC / HAC enzyme, at positions selected from those corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1.

[0169] In particular, provided herein is a process for preparing ambroxide or a mixture containing ambroxide, the process comprising enzymatically converting bisEEH or a mixture containing bisEEH to ambroxide or a mixture containing ambroxide using an SHC / HAC variant having at least about 70.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications, relative to the wild-type SHC / HAC enzyme, at positions selected from positions corresponding to 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1.

[0170] A variant SHC / HAC enzyme may have, for example, an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme. For example, a variant SHC / HAC enzyme has an amino acid sequence that is at least about 75.0%, or at least about 80.0%, or at least about 85.0%, or at least about 90.0%, or at least about 95.0%, or at least about 95.5%, or at least about 96.5%, or at least about 97.0%, or at least about 97.5%, or at least about 98.0%, or at least about 98.5%, or at least about 99.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0171] For example, a SHC / HAC enzyme variant may have an amino acid sequence that is less than 100% identical to the amino acid sequence of a wild-type SHC / HAC enzyme, e.g., equal to or less than about 99.5% identity, or equal to or less than about 99.0% identity.

[0172] For example, a SHC / HAC enzyme variant may have from about 70.0% to about 99.5%, or from about 80.0% to about 99.0%, or from about 85.0% to about 98.5%, or from about 90.0% to about 98.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0173] The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, that of AacSHC (SEQ ID NO: 1), ZmoSHC1 (SEQ ID NO: 11), ZmoSHC2 (SEQ ID NO: 12), BjpSHC (SEQ ID NO: 13), GmoSHC (SEQ ID NO: 14), TelSHC (SEQ ID NO: 19), or ApaSHC1 (SEQ ID NO: 20), BmeSHC (SEQ ID NO: 28), SalSHC (SEQ ID NO: 29), or ApaSHCA (SEQ ID NO: 30). In particular, the amino acid sequence of the wild-type SHC / HAC enzyme may be that of AacSHC (SEQ ID NO: 1).

[0174] Thus, in some embodiments, an SHC / HAC enzyme variant may have an amino acid sequence having at least about 70.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20. For example, an SHC / HAC enzyme variant has an amino acid sequence having at least about 75.0%, or at least about 80.0%, or at least about 85.0%, or at least about 90.0%, or at least about 95.0%, or at least about 95.5%, or at least about 96.5%, or at least about 97.0%, or at least about 97.5%, or at least about 98.0%, or at least about 98.5%, or at least about 99.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0175] For example, an SHC / HAC enzyme variant may have an amino acid sequence that has less than 100% identity, e.g., equal to or less than about 99.5% identity, or equal to or less than about 99.0% identity, to, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0176] For example, the SHC / HAC enzyme variant may have from about 70.0% to about 99.5%, or from about 80.0% to about 99.0%, or from about 85.0% to about 98.5%, or from about 90.0% to about 98.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0177] "Percent (%) identity" with respect to a polypeptide or nucleotide sequence is defined as the percentage of amino acids or nucleotides in a candidate sequence that are identical to those in a reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, but without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways that are within the skill in the art, illustratively using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide chain of amino acids, regardless of length or post-translational modification.

[0178] Nucleotide and amino acid sequence similarity, i.e., percentage of sequence identity, can be determined through sequence alignment. Such alignments include, for example, https: / / www.ebi.ac.uk / Tools / msa / clustalo / The algorithm can be implemented using several algorithms known in the art, such as the GAP program (University of Iowa Mathematical Algorithm), or the Myers and Miller Mathematical Algorithm (1989-Cabios 4:11-17), preferably the Karlin and Altschul Mathematical Algorithm (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). The preferred parameters used are the default parameters as set on https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0179] Percentage sequence identity may be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score = 100, word length = 12, to obtain polynucleotide sequences homologous to those nucleic acids encoding related proteins. BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to polypeptides.

[0180] To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov Random Fields. When percentages of sequence identity are mentioned in this application, these percentages are calculated with respect to the full length of the longer sequence unless otherwise specifically indicated.

[0181] In a specific embodiment, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0182] In some embodiments, a SHC / HAC enzyme variant may have equal to or less than about 200 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO: 1, compared to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. For example, an SHC / HAC enzyme variant can have an RI of about 150 or less, or about 120 or less, or about 100 or less, or about 95 or less, or about 90 or less, or about 85 or less, or about 80 or less, or about 75 or less, or about 85 or less, or about 85 or less, or about 75 or less, compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. It may have less than or equal to about 70 amino acid modifications, or less than or equal to about 65, or less than or equal to about 60, or less than or equal to about 55, or less than or equal to about 50, or less than or equal to about 45, or less than or equal to about 40, or less than or equal to about 35, or less than or equal to about 30, or less than or equal to about 25, or less than or equal to about 20, or less than or equal to about 15, or less than or equal to about 10.

[0183] A variant SHC / HAC enzyme may have, for example, at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO: 1, compared to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.

[0184] For example, a variant SHC / HAC enzyme may have from about 1 to about 30 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20. For example, a variant SHC / HAC enzyme may have from about 2 to about 25 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, a variant SHC / HAC enzyme may have from about 3 to about 20 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, a variant SHC / HAC enzyme may have from about 4 to about 15 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, a variant SHC / HAC enzyme may have from about 5 to about 10 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0185] The amino acid modification may be, for example, an insertion, deletion, and / or substitution as described above. For example, the amino acid modification may be a substitution, for example, a non-conservative substitution.

[0186] In some embodiments, the only amino acid modifications are substitutions (i.e., no insertions or deletions) compared to the wild-type SHC / HAC enzyme (e.g., compared to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30).

[0187] Amino acid alterations are defined relative to a reference sequence. An amino acid alteration relative to a reference sequence means that the amino acid sequence of a variant sequence differs from the reference sequence.

[0188] Amino acids in a reference sequence and a variant sequence may be assigned numbers, where the numbering begins with the amino acid at the N-terminus of the polypeptide (i.e., the amino acid at the N-terminus of a polypeptide is numbered 1, the next amino acid is numbered 2, etc.). A "position" in a reference sequence refers to a particular amino acid residue present in the reference sequence, as identified by a particular numbered amino acid in the reference sequence. A "position" in a variant sequence refers to a particular amino acid residue present in the variant sequence, as identified by a particular numbered amino acid in the variant sequence.

[0189] Because a variant sequence may contain deletions or insertions compared to a reference sequence, amino acids in the variant sequence may be numbered differently than the same amino acids in the reference sequence. As an example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO: 1, the amino acid will retain the number 132 in the reference sequence, while the amino acid following the insertion will have the number 133 in the variant sequence. In this example, the position in the variant sequence that corresponds to position 132 in the reference sequence is position 133. Thus, an amino acid in a variant sequence that is retained from a reference sequence may be defined by referring to the "corresponding position" in the reference sequence. In other words, a "position" in a variant sequence may be defined by referring to the "corresponding position" in the reference sequence. Notably, a substitution in a variant sequence compared to a reference sequence may be defined by referring to the "corresponding position" in the reference sequence, despite any insertions and / or deletions in the reference sequence. If an amino acid in the reference sequence is deleted, there will be no "corresponding position" in the variant sequence. If there are no insertions or deletions (i.e., only substitutions) compared to the reference sequence, the "corresponding position" of the reference sequence will be the same as the position in the variant sequence.

[0190] Wild-type SHC / HAC enzymes from different species have different polypeptide lengths. Wild-type sequences may be aligned using the algorithm described above to identify "corresponding positions" in two different wild-type SHC / HAC enzymes. Thus, an amino acid at a position in a variant sequence that corresponds to a position in a reference sequence may, for example, be a different amino acid residue and / or have a different number than that of the reference sequence. As an example, the amino acid M at position 132 of AacSHC (SEQ ID NO: 1) may correspond to the amino acid Y at position 185 of ZmoSHC1 (SEQ ID NO: 11).

[0191] Thus, an amino acid modification may be defined relative to two different reference sequences. For example, the amino acid modification may be a change relative to a first reference sequence (e.g., the sequence of a wild-type SHC / HAC enzyme from which the variant is derived), and the position of the amino acid modification in the variant sequence may be defined by reference to a second reference sequence (e.g., AacSHC (SEQ ID NO: 1)). Thus, an amino acid modification in a SHC / HAC enzyme variant may be compared to the first wild-type SHC / HAC enzyme at a position defined by reference to the second wild-type SHC / HAC enzyme.

[0192] The amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications, relative to the amino acid sequence of the wild-type SHC / HAC enzyme, at positions selected from those corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1. For example, the amino acid modifications may be at one or more positions selected from those corresponding to positions 81, 431, 557, and 613 of SEQ ID NO:1. The amino acid modification may be, for example, a substitution (e.g., a non-conservative substitution). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, the wild-type sequence may be SEQ ID NO:1.

[0193] In some embodiments, the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to the amino acid sequence of a wild-type SHC / HAC enzyme, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to positions 81, 431, or 613 of SEQ ID NO:1. The amino acid modification may be, for example, a substitution (e.g., a non-conservative substitution). The amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, the wild-type sequence may be SEQ ID NO:1.

[0194] For example, the amino acid sequence of the SHC / HAC enzyme variant may have an amino acid modification, relative to the amino acid sequence of the wild-type SHC / HAC enzyme, at a position corresponding to position 557 of SEQ ID NO:1 and at one position corresponding to positions 81, 431, or 613 of SEQ ID NO:1. The amino acid modification may be, for example, a substitution (e.g., a non-conservative substitution). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, the wild-type sequence may be SEQ ID NO:1.

[0195] For example, the amino acid sequence of the variant SHC / HAC enzyme may have amino acid modifications, relative to the amino acid sequence of the wild-type SHC / HAC enzyme, at a position corresponding to position 557 of SEQ ID NO:1 and at two positions selected from positions corresponding to positions 81, 431, and 613 of SEQ ID NO:1. The amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, the wild-type sequence may be SEQ ID NO:1.

[0196] For example, the amino acid sequence of the SHC / HAC enzyme variant may have an amino acid modification, relative to the amino acid sequence of a wild-type SHC / HAC enzyme, at a position corresponding to position 557 of SEQ ID NO:1, and all of the positions corresponding to positions 81, 431, and 613 of SEQ ID NO:1. The amino acid modification may be, for example, a substitution (e.g., a non-conservative substitution). The amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, the wild-type sequence may be SEQ ID NO:1.

[0197] For example, the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications, relative to the amino acid sequence of the wild-type SHC / HAC enzyme, at positions corresponding to positions 90 and 613 of SEQ ID NO: 1. The amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. For example, the wild-type sequence may be SEQ ID NO: 1.

[0198] For example, the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications, relative to the amino acid sequence of the wild-type SHC / HAC enzyme, at positions corresponding to positions 172 and 277 of SEQ ID NO: 1. The amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. For example, the wild-type sequence may be SEQ ID NO: 1.

[0199] For example, the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications, relative to the amino acid sequence of the wild-type SHC / HAC enzyme, at positions corresponding to positions 557 and 431 of SEQ ID NO: 1. The amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. For example, the wild-type sequence may be SEQ ID NO: 1.

[0200] For example, the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 613 of SEQ ID NO:1 compared to the amino acid sequence of the wild-type SHC / HAC enzyme. The amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, the wild-type sequence may be SEQ ID NO:1.

[0201] For example, the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 81, 557, and 613 of SEQ ID NO:1 compared to the amino acid sequence of the wild-type SHC / HAC enzyme. The amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, the wild-type sequence may be SEQ ID NO:1.

[0202] For example, the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 81, 431, and 557 of SEQ ID NO:1 compared to the amino acid sequence of the wild-type SHC / HAC enzyme. The amino acid modifications may be, for example, substitutions (e.g., non-conservative substitutions). The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, the wild-type sequence may be SEQ ID NO:1.

[0203] An amino acid modification at a position corresponding to position 557 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 557.

[0204] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 557 of SEQ ID NO: 1 can be, for example, Met, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the new amino acid in the SHC / HAC enzyme variant can be threonine.

[0205] An amino acid modification at a position corresponding to position 81 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 81.

[0206] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 81 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a basic amino acid (i.e., His, Lys, or Arg). For example, the new amino acid in the SHC / HAC enzyme variant can be histidine.

[0207] An amino acid modification at a position corresponding to position 90 of SEQ ID NO:1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO:1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 90.

[0208] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 90 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, Ile). For example, the new amino acid in the SHC / HAC enzyme variant can be alanine.

[0209] An amino acid modification at a position corresponding to position 172 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 172.

[0210] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 172 of SEQ ID NO: 1 can be, for example, Met, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, Gln). For example, the new amino acid in the SHC / HAC enzyme variant can be threonine.

[0211] An amino acid modification at a position corresponding to position 277 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 277.

[0212] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 277 of SEQ ID NO: 1 can be, for example, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a basic amino acid (i.e., His, Lys, Arg). For example, the new amino acid in the SHC / HAC enzyme variant can be lysine.

[0213] An amino acid modification at a position corresponding to position 431 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). As noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and the variant may additionally contain insertions and / or deletions, so the numbering of the new amino acid (X) in the variant sequence may not be 431.

[0214] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 431 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the new amino acid in the SHC / HAC enzyme variant can be leucine.

[0215] An amino acid modification at a position corresponding to position 613 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 613.

[0216] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 613 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the new amino acid in the SHC / HAC enzyme variant can be serine.

[0217] The amino acids and positions (e.g., amino acids at positions 81, 431, 557, and 613 of AacSHC) in the sequences of wild-type ZmoSHC1, ZmoSHC2, BjpSHC, GmoSHC, TelSHC, and ApaSHC1 (SEQ ID NOs: 11, 12, 13, 14, 19, and 20, respectively) corresponding to the amino acids of AacSHC (SEQ ID NO: 1) are shown in Figure 9A. The amino acids and positions (e.g., amino acids at positions 81, 431, 557, and 613 of AacSHC) in the sequences of wild-type ZmoSHC1, ZmoSHC2, BjpSHC, GmoSHC, TelSHC, ApaSHC1, BmeSHC, SalSHC, and ApaSHCA (SEQ ID NO: 11, 12, 13, 14, 19, 20, 28, 29, and 30, respectively) corresponding to the amino acids of AacSHC (SEQ ID NO: 1) are shown in Figure 9B.

[0218] Amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted in white text on a black background. Thus, the amino acids immediately above or below the highlighted amino acids are the amino acids and positions in ZmoSHC2, BjaSHC, GmoSHC, ApaSHC1, ApaSHC1, ZmoSHC1, and TelSHC that correspond to positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 of AacSHC (SEQ ID NO: 1). For example, the amino acid in BjaSHC, GmoSHC, ApaSHC1, and ZmoSHC1 that corresponds to amino acid Y at position 81 of AacSHC (SEQ ID NO: 1) is Y. The amino acid in ZmoSHC2 and TelSHC that corresponds to amino acid Y at position 81 of AacSHC (SEQ ID NO: 1) is F. Position 84 of TelSHC is the position of TelSHC that corresponds to position 81 of AacSHC (SEQ ID NO: 1).

[0219] In some embodiments, one or more of the original amino acid modifications in the 215G2 SHC / HAC enzyme variant (compared to wild-type Aac SHC (SEQ ID NO: 1)) may be retained in the new SHC / HAC enzyme variants described herein. The amino acid modifications in the 215G2 SHC / HAC enzyme variant compared to wild-type Aac SHC were substitutions M132R, A224V, and I432T (i.e., substitution of an M residue with an R residue at position 132, an A residue with a V residue at position 224, and an I residue with a T residue at position 432).

[0220] Thus, the amino acid sequence of the SHC / HAC enzyme variant may have one or more amino acid modifications compared to the amino acid sequence of the wild-type SHC / HAC enzyme at positions corresponding to positions 132, 224, and 432 of SEQ ID NO: 1. The amino acid modifications may be, for example, substitutions.

[0221] For example, the amino acid sequence of the SHC / HAC enzyme variant may have one, two, or three amino acid modifications compared to the amino acid sequence of the wild-type SHC / HAC enzyme at positions selected relative to positions corresponding to positions 132, 224, and 432 of SEQ ID NO:1.

[0222] For example, the amino acid sequence of the SHC / HAC enzyme variant may have amino acid modifications at positions corresponding to positions 132 and 432 of SEQ ID NO:1 compared to the amino acid sequence of the wild-type SHC / HAC enzyme.

[0223] For example, the amino acid sequence of the SHC / HAC enzyme variant may have amino acid modifications at positions corresponding to positions 132, 224, and 432 of SEQ ID NO:1 compared to the amino acid sequence of the wild-type SHC / HAC enzyme.

[0224] An amino acid modification at a position corresponding to position 132 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 132.

[0225] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 132 of SEQ ID NO: 1 can be, for example, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a basic amino acid (i.e., His, Lys, or Arg). For example, the new amino acid in the SHC / HAC enzyme variant can be arginine.

[0226] An amino acid modification at a position corresponding to position 224 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 224.

[0227] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 224 of SEQ ID NO: 1 can be, for example, Met, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a hydrophobic amino acid (i.e., Met, Val, Leu, or Ile). For example, the new amino acid in the SHC / HAC enzyme variant can be valine.

[0228] An amino acid modification at a position corresponding to position 432 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 432.

[0229] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 432 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the new amino acid in the SHC / HAC enzyme variant can be threonine.

[0230] The amino acids and positions (for example, at positions 132, 224, and 432 of AacSHC) in the sequences of wild-type ZmoSHC1, ZmoSHC2, BjpSHC, GmoSHC, TelSHC, and ApaSHC1 (SEQ ID NOs: 11, 12, 13, 14, 19, and 20, respectively) corresponding to the amino acids of AacSHC (SEQ ID NO: 1) are shown in Figure 9.

[0231] The SHC / HAC enzyme variants described herein may additionally have one or more other amino acid modifications (e.g., substitutions) at other positions of AacSHC identified in WO 2016 / 170099, for example.

[0232] Thus, the SHC / HAC enzyme variants described herein may have one or more additional amino acid modifications, relative to a wild-type SHC / HAC enzyme, at positions corresponding to positions 77, 92, 129, 579, 601, and / or 605 of SEQ ID NO: 1. For example, the SHC / HAC enzyme variants may have one or more amino acid substitutions (e.g., conservative or non-conservative substitutions), relative to a wild-type SHC / HAC enzyme, at positions corresponding to positions 77, 92, 129, 579, 601, and / or 605 of SEQ ID NO: 1.

[0233] An amino acid modification at a position corresponding to position 77 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 77.

[0234] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 77 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the new amino acid in the SHC / HAC enzyme variant can be alanine.

[0235] An amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 92.

[0236] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 92 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the new amino acid in the SHC / HAC enzyme variant can be valine.

[0237] An amino acid modification at a position corresponding to position 129 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 129.

[0238] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 129 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Tyr. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the new amino acid in the SHC / HAC enzyme variant can be leucine.

[0239] An amino acid modification at a position corresponding to position 579 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 579.

[0240] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 579 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a basic amino acid (i.e., His, Lys, Arg). For example, the new amino acid in the SHC / HAC enzyme variant can be histidine.

[0241] An amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 601.

[0242] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at a position corresponding to position 601 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Tyr. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be an aromatic amino acid (i.e., Trp, Tyr, Phe). For example, the new amino acid in the SHC / HAC enzyme variant can be tyrosine.

[0243] An amino acid modification at a position corresponding to position 605 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 605.

[0244] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at the position corresponding to position 605 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Tyr. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be an aromatic amino acid (i.e., Trp, Tyr, Phe). For example, the new amino acid in the SHC / HAC enzyme variant can be tryptophan.

[0245] The amino acid positions in the sequences of wild-type TelSHC (SEQ ID NO: 19), wild-type ApaSHC1 (SEQ ID NO: 20), wild-type ZmoSHC1 (SEQ ID NO: 11), wild-type ZmoSHC2 (SEQ ID NO: 12), wild-type BjpSHC (SEQ ID NO: 13), and wild-type GmoSHC (SEQ ID NO: 14) corresponding to amino positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted in white letters on a black background in wild-type AacSHC (SEQ ID NO: 1) in Figure 9A.

[0246] The amino acid positions in wild-type TelSHC (SEQ ID NO: 19), wild-type ApaSHC1 (SEQ ID NO: 20), wild-type ZmoSHC1 (SEQ ID NO: 11), wild-type ZmoSHC2 (SEQ ID NO: 12), wild-type BjpSHC (SEQ ID NO: 13), wild-type GmoSHC (SEQ ID NO: 14), wild-type BmeSHC (SEQ ID NO: 28), wild-type SalSHC (SEQ ID NO: 29), and wild-type ApaSHCA (SEQ ID NO: 30) corresponding to amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted in white letters on a black background in wild-type AacSHC (SEQ ID NO: 1) in Figure 9B.

[0247] A variant SHC / HAC enzyme may have, for example, one or more additional amino acid modifications relative to the amino acid sequence of a wild-type SHC / HAC enzyme at positions corresponding to positions 37, 174, and / or 601 of SEQ ID NO: 1. For example, a variant SHC / HAC enzyme may have one or more amino acid substitutions (e.g., conservative or non-conservative) relative to the amino acid sequence of a wild-type SHC / HAC enzyme at positions corresponding to positions 37, 174, and / or 601 of SEQ ID NO: 1.

[0248] An amino acid modification at a position corresponding to position 37 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 37.

[0249] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at a position corresponding to position 37 of SEQ ID NO: 1 can be, for example, Met, Ala, Val, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln), such as glutamine.

[0250] An amino acid modification at a position corresponding to position 174 of SEQ ID NO:1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO:1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 174.

[0251] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at a position corresponding to position 174 of SEQ ID NO: 1 can be, for example, Met, Ala, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile), such as isoleucine.

[0252] An amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1 relative to the amino acid sequence of a wild-type SHC / HAC enzyme may be, for example, a substitution of that amino acid in the wild-type SHC / HAC enzyme with a different amino acid (X). Because, as noted above, the wild-type sequence may have a different length relative to SEQ ID NO: 1, and because variants may additionally contain insertions and / or deletions, the numbering of the new amino acid (X) in a variant sequence may not be 601.

[0253] The new amino acid (X) in the amino acid sequence of the SHC / HAC enzyme variant at a position corresponding to position 601 of SEQ ID NO: 1 can be, for example, Met, Ala, Leu, Val, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Tyr. For example, the new amino acid (X) in the SHC / HAC enzyme variant can be an aromatic amino acid (i.e., Trp, Tyr, Phe), such as tyrosine.

[0254] The amino acid positions in the sequences of wild-type TelSHC (SEQ ID NO: 19), wild-type ApaSHC1 (SEQ ID NO: 20), wild-type ZmoSHC1 (SEQ ID NO: 11), wild-type ZmoSHC2 (SEQ ID NO: 12), wild-type BjpSHC (SEQ ID NO: 13), and wild-type GmoSHC (SEQ ID NO: 14) corresponding to amino positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted in white letters on a black background in wild-type AacSHC (SEQ ID NO: 1) in Figure 9A.

[0255] The amino acid positions in wild-type TelSHC (SEQ ID NO: 19), wild-type ApaSHC1 (SEQ ID NO: 20), wild-type ZmoSHC1 (SEQ ID NO: 11), wild-type ZmoSHC2 (SEQ ID NO: 12), wild-type BjpSHC (SEQ ID NO: 13), wild-type GmoSHC (SEQ ID NO: 14), wild-type BmeSHC (SEQ ID NO: 28), wild-type SalSHC (SEQ ID NO: 29), and wild-type ApaSHCA (SEQ ID NO: 30) corresponding to amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted in white letters on a black background in wild-type AacSHC (SEQ ID NO: 1) in Figure 9B.

[0256] Any combination of amino acid modifications described herein is contemplated, particularly combinations of amino acid modifications at positions corresponding to the combinations of amino acid modifications identified herein in AacSHC and in WO 2016 / 17009.

[0257] In some embodiments, the SHC / HAC enzyme variant has the following amino acid substitutions: (i) M132R, A224V, I432T, A557T, and H4331L (SEQ ID NO: 2); or (ii) M132R, A224V, I432T, A557T, and R613S (SEQ ID NO: 3); or (iii) M132R, A224V, I432T, A557T, Y81H, and R613S (SEQ ID NO: 4); or (iv) M132R, A224V, I432T, A557T, Y81H, and H431L (SEQ ID NO: 5); or (v) M132R, A224V, I432T, T90A, and R613S (SEQ ID NO: 17); or (vi) M132R, A224V, I432T, A172T, and M277K (SEQ ID NO: 18) Identical to SEQ ID NO: 1 except for:

[0258] The new SHC / HAC enzyme variants may have increased enzymatic activity for the conversion of EEH to (-)-ambrox or the conversion of bisEEH to ambroxide, for example, compared to the 215G2 SHC enzyme. Increased enzymatic activity may refer to any aspect of the enzymatic conversion of EEH to (-)-ambrox or the enzymatic conversion of bisEEH to ambroxide, including, for example, increased total conversion of EEH or bisEEH, increased conversion rate of EEH or bisEEH (e.g., in the first 6 hours or the first 12 hours of the reaction), increased production of (-)-ambrox or ambroxide, and decreased production of by-products. Increased enzyme activity may translate into increased productivity, which may generally be defined in terms of (-)-ambrox or amblyoxide produced per liter of reaction capacity and per hour of bioconversion time, or (-)-ambrox or amblyoxide produced per liter of reaction capacity, per hour of reaction time (i.e., time after substrate is added), and per gram of biocatalyst used in the reaction.

[0259] The new SHC / HAC enzyme variants may provide increased EEH or bis-EEH conversion, for example, compared to the 215G2 SHC enzyme. Thus, the processes described herein may have increased levels of EEH or bis-EEH conversion, compared to processes using the 215G2 SHC enzyme. The new SHC / HAC enzyme variants may provide increased EEH or bis-EEH conversion rates, for example, compared to the 215G2 SHC enzyme. Thus, the processes described herein may have increased EEH or bis-EEH conversion rates, compared to the 215G2 SHC enzyme.

[0260] The new SHC / HAC enzyme variant may provide, for example, an increased EEH or bis-EEH conversion rate over the first 4 hours, or over the first 6 hours, or over the first 8 hours, or over the first 12 hours, or over the first 24 hours of the reaction compared to the 215G2 SHC enzyme. Thus, the processes described herein may have an increased EEH or bis-EEH conversion rate over the first 4 hours, or over the first 6 hours, or over the first 8 hours, or over the first 12 hours, or over the first 24 hours of the reaction compared to the 215G2 SHC enzyme. This may be when compared to using both enzymes (i.e., the new SHC / HAC enzyme variant and the 215G2 SHC enzyme) under the same reaction conditions (e.g., the same pH and temperature), or when compared to using each enzyme under their respective optimized reaction conditions (e.g., optimized pH and temperature), which may be different from each other.

[0261] For example, the new SHC / HAC enzyme variants may provide, or the process may have, at least about 40% EEH or bis-EEH conversion in the first 12 hours of reaction. For example, the new SHC / HAC enzyme variants may provide, or the process may have, at least about 45%, or at least about 50%, or at least about 55%, or at least about 60% EEH or bis-EEH conversion in the first 12 hours of reaction. For example, the new SHC / HAC enzyme variants may provide, or the process may have, at least about 30% EEH or bis-EEH conversion in the first 6 hours of reaction. For example, the new SHC / HAC enzyme variants may provide, or the process may have, at least about 35%, or at least about 45%, or at least about 50%, or at least about 55% EEH or bis-EEH conversion in the first 12 hours of reaction. This may be compared to using both enzymes (i.e., the new SHC / HAC enzyme variant and the 215G2 SHC enzyme) under the same reaction conditions (e.g., the same pH and temperature), or compared to using each enzyme under their respective optimized reaction conditions (e.g., optimized pH and temperature), which may be different from each other.

[0262] The conversion of EEH to (-)-ambrox, or bisEEH to ambroxide, may be determined, for example, using an activity assay as described above, and may be calculated as grams of recoverable product per gram of feedstock (which may be calculated as a percent molar conversion).

[0263] As used herein, any reference herein to 99% / 100% conversion of a homofarnesol substrate to (-)-ambrox or of a bishomofarnesol substrate to amblyoxide is a reference to 99% / 100% conversion of the isomer that can be converted to (-)-ambrox or amblyoxide using the SHC / HAC enzyme or enzyme variant.

[0264] The temperature optimum for the SHC / HAC enzyme variant may be, for example, equal to or greater than about 35° C. For example, the temperature optimum for the SHC / HAC enzyme variant may range from about 40° C. to about 50° C., e.g., from about 42° C. to about 48° C., or from about 44° C. to about 46° C. For example, the temperature optimum for the SHC / HAC enzyme variant may be about 45° C. The processes for making (-)-ambrox or ambroxide disclosed herein may be carried out at the temperature optimum for the SHC / HAC enzyme variant.

[0265] The pH optimum for the SHC / HAC enzyme variant may be, for example, equal to or greater than about 5.4. For example, the pH optimum for the SHC / HAC enzyme variant may range from about 5.2 to about 6.0, e.g., from about 5.4 to about 5.8, e.g., from about 5.6 to about 5.8. For example, the pH optimum for the SHC / HAC enzyme variant may be about 5.6 or about 5.8. The processes for making (-)-ambrox or ambroxide disclosed herein may be carried out at the pH optimum for the SHC / HAC enzyme variant.

[0266] The optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the process for making (-)-ambrox or ambryoxide disclosed herein is, for example, 4 g / L of substrate (e.g., EEH or bisEEH) and OD 650nmWhen used with up to 10 cells, the optimal concentration of SDS may be from about 0.010 w / w% to about 0.10 w / w%. For example, the optimal concentration of SDS may be from about 0.040 w / w% to about 0.080 w / w%, e.g., at 4 g / L of substrate (e.g., EEH or bisEEH) and an OD 650nm The optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the process for producing (-)-ambrox or amblyoxide disclosed herein may be about 0.050 w / w% when used with up to 10 cells. For example, when a substrate (e.g., EEH or bisEEH) is used at 125 g / L with 250 g / L of cells, the optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the process for producing (-)-ambrox or amblyoxide disclosed herein may be about 1.0 w / w% to about 1.5 w / w%. For example, when a substrate (e.g., EEH or bisEEH) is used at 125 g / L with 125 g / L of cells, the optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the process for producing (-)-ambrox or amblyoxide disclosed herein may be about 0.45 w / w% to about 0.85 w / w%, e.g., about 0.65 w / w%, when a substrate (e.g., EEH or bisEEH) is used at 125 g / L with 125 g / L of cells. For example, the optimal concentration of SDS may be from about 1.2 w / w% to about 1.4 w / w%, e.g., about 1.3 w / w% when a substrate (e.g., EEH or bisEEH) is used at 125 g / L with 250 g / L of cells.

[0267] The processes for making (-)-ambrox or ambrate oxide disclosed herein may be carried out at the optimum temperature range or optimum temperature, and / or optimum pH range or optimum pH, and / or optimum SDS concentration range or optimum SDS concentration for the particular enzyme used, as set forth in Tables 7, 9, or 11 in the Examples below.

[0268] The following numbered paragraphs define further aspects of the present disclosure. 1. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme variant; wherein the SHC / HAC enzyme variant has at least about 70.0% identity to SEQ ID NO: 1; and wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications compared to the wild-type SHC / HAC enzyme at positions selected from positions corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1.

[0269] 2. A process for preparing amburoxide or a mixture containing amburoxide, the process comprising enzymatically converting (E,E)-bishomofarnesol (bisEEH) or a mixture of bishomofarnesol isomers containing bisEEH using an SHC / HAC enzyme variant to amburoxide or a mixture containing amburoxide; wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme; and wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications compared to the wild-type SHC / HAC enzyme at positions selected from positions corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1.

[0270] 3. The process of any preceding paragraph, wherein the wild-type SHC / HAC enzyme is SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20.

[0271] 4. The process of any preceding paragraph, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least about 90.0% or at least about 95.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0272] 5. The process of any of paragraphs 1-4, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 90 and 613 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0273] 6. The process of any of paragraphs 1-4, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications, relative to a wild-type SHC / HAC enzyme, at positions corresponding to positions 172 and 277 of SEQ ID NO:1.

[0274] 7. The process of any of paragraphs 1-4, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to position 81, 431, or 613 of SEQ ID NO:1.

[0275] 8. The process of paragraph 7, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 431 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0276] 9. The process of paragraph 7, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 613 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0277] 10. The process of paragraph 8 or 9, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at a position corresponding to position 81 of SEQ ID NO:1.

[0278] 11. The process of any preceding paragraph, wherein one or more, e.g., all, of the amino acid modifications at positions 81, 90, 172, 277, 431, 557, and 613 are substitutions, e.g., non-conservative substitutions.

[0279] 12. The amino acid modification at the position corresponding to position 81 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, e.g., histidine; and / or The amino acid modification at the position corresponding to position 90 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, e.g., alanine; and / or The amino acid modification at the position corresponding to position 172 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, e.g., threonine; and / or the amino acid modification at the position corresponding to position 277 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, e.g., lysine; and / or the amino acid modification at the position corresponding to position 431 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, e.g., leucine; and / or The amino acid modification at the position corresponding to position 557 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, e.g., threonine; and / or The process of any preceding paragraph, wherein the amino acid modification at the position corresponding to position 613 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC with a neutral hydrophilic amino acid, e.g., serine.

[0280] 13. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 132 of SEQ ID NO:1; an amino acid modification at a position corresponding to position 224 of SEQ ID NO:1; and Amino acid modification at a position corresponding to position 432 of SEQ ID NO: 1 The process of any preceding paragraph, having one or more additional amino acid modifications selected from:

[0281] 14. The amino acid modification at the position corresponding to position 132 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a basic amino acid, e.g., arginine; and / or the amino acid modification at the position corresponding to position 224 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., valine; and / or 14. The process of paragraph 13, wherein the amino acid modification at the position corresponding to position 432 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a neutral hydrophilic amino acid, e.g., threonine.

[0282] 15. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 77 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 129 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 579 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1; and / or Amino acid modification at a position corresponding to position 605 of SEQ ID NO: 1 The process of any preceding paragraph, having one or more additional amino acid modifications selected from:

[0283] 16. The amino acid modification at the position corresponding to position 77 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., alanine; and / or the amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., valine; and / or the amino acid modification at the position corresponding to position 129 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., leucine; and / or the amino acid modification at the position corresponding to position 579 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a basic amino acid, e.g., histidine; and / or the amino acid modification at the position corresponding to position 601 of SEQ ID NO: 1 is a substitution of an amino acid in the amino acid sequence of a wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tyrosine; and / or 16. The process of paragraph 15, wherein the amino acid modification at the position corresponding to position 605 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tryptophan.

[0284] 17. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 37 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 174 of SEQ ID NO: 1; and / or Amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1 The process of any preceding paragraph, having one or more additional amino acid modifications selected from:

[0285] 18. The amino acid modification at the position corresponding to position 37 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a neutral hydrophilic amino acid, e.g., glutamine; and / or the amino acid modification at the position corresponding to position 174 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., isoleucine; and / or 18. The process of paragraph 17, wherein the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tyrosine.

[0286] 19. The process of any preceding paragraph, wherein the SHC / HAC enzyme variant has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, or SEQ ID NO:18.

[0287] 20. The process of any preceding paragraph, wherein the process comprises culturing a recombinant host cell that produces the SHC / HAC enzyme variant.

[0288] 21. The process of paragraph 20, wherein the recombinant host cell comprises a nucleic acid sequence encoding an SHC / HAC enzyme selected from, e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:22, and SEQ ID NO:23.

[0289] 22. The process of any preceding paragraph in which (-)-ambrox is produced in admixture with at least one or more of by-products (II), (III), or (IV).

[0290] 23. A process according to any claim for preparing (-)-ambrox or a reaction mixture containing (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme variant; wherein the SHC / HAC enzyme variant has at least about 70.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20; wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications relative to a wild-type SHC / HAC enzyme at positions selected from positions corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1; and wherein the mixture of isomers containing EEH is selected from one or more of the group consisting of: [(3E,7E) and [(3Z,7E)], and / or [(3E,7E) and (3E,7Z)], and / or [(3Z,7E), (3E,7E), and (3E,7Z)] (also designated as [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively).

[0291] 24. A process according to any claim for preparing ambroxide or a mixture containing ambroxide, the process comprising enzymatically converting (E,E)-bishomofarnesol (EEH) or a mixture of bishomofarnesol isomers containing bisEEH using an SHC / HAC enzyme variant to ambroxide or a mixture containing ambroxide; wherein the SHC / HAC enzyme variant has at least about 70.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20; wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications relative to a wild-type SHC / HAC enzyme at positions selected from positions corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1; and wherein the mixture of isomers comprising bis-EEH is selected from one or more of the group consisting of: [(E,E) and [(Z,E)], and / or [(E,E) and (E,Z)], and / or [(Z,E), (E,E), and (E,Z)] (also designated as [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively).

[0292] 25. (-)-Ambrox, obtained by or obtainable by the process of any preceding paragraph, e.g., in amorphous or crystalline form.

[0293] 26. Ambroxide, e.g., in amorphous or crystalline form, obtained by or obtainable by the process of any preceding paragraph.

[0294] 27. Use of (-)-ambrox of paragraph 25 and / or ambroxide of paragraph 26 as part of a fragrance or cosmetic or consumer product.

[0295] 28. Fragrances or cosmetics or consumer products containing (-)-ambrox and / or ambroxide as specified in paragraph 25 and / or ambroxide as specified in paragraph 26.

[0296] 29. A SHC / HAC enzyme variant having an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid modifications, relative to the wild-type SHC / HAC enzyme, at positions selected from positions corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1.

[0297] 30. The SHC / HAC enzyme variant of paragraph 29, wherein the wild-type SHC / HAC enzyme is SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20.

[0298] 31. The SHC / HAC enzyme variant of paragraph 29 or 30, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least about 90.0% or at least about 95.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0299] 32. The SHC / HAC enzyme variant of any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 90 and 613 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0300] 33. The SHC / HAC enzyme variant of any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 172 and 277 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0301] 34. The SHC / HAC enzyme variant of any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to position 81, 431, or 613 of SEQ ID NO:1.

[0302] 35. The SHC / HAC enzyme variant of any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 431 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0303] 36. The SHC / HAC enzyme variant of any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 613 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0304] 37. The SHC / HAC enzyme variant of paragraph 35 or 36, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at a position corresponding to position 81 of SEQ ID NO:1.

[0305] 38. The SHC / HAC enzyme variant of any of paragraphs 29-37, wherein one or more, e.g., all, of the amino acid modifications at positions 81, 90, 172, 277, 431, 557, or 613 are substitutions, e.g., non-conservative substitutions.

[0306] 39. The amino acid modification at the position corresponding to position 81 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC with a basic amino acid, e.g., histidine; and / or The amino acid modification at the position corresponding to position 90 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, e.g., alanine; and / or The amino acid modification at the position corresponding to position 172 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, e.g., threonine; and / or the amino acid modification at the position corresponding to position 277 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, e.g., lysine; and / or the amino acid modification at the position corresponding to position 431 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, e.g., leucine; and / or The amino acid modification at the position corresponding to position 557 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, e.g., threonine; and / or The SHC / HAC enzyme variant of any of paragraphs 29 to 38, wherein the amino acid modification at the position corresponding to position 613 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of wild-type SHC / HAC with a neutral hydrophilic amino acid, e.g., serine.

[0307] 40. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 132 of SEQ ID NO:1; an amino acid modification at a position corresponding to position 224 of SEQ ID NO:1; and Amino acid modification at a position corresponding to position 432 of SEQ ID NO: 1 40. The SHC / HAC enzyme variant of any of paragraphs 29 to 39, having one or more additional amino acid modifications selected from:

[0308] 41. The amino acid modification at the position corresponding to position 132 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a basic amino acid, e.g., arginine; and / or the amino acid modification at the position corresponding to position 224 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., valine; and / or 41. The SHC / HAC enzyme variant of paragraph 40, wherein the amino acid modification at the position corresponding to position 432 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a neutral hydrophilic amino acid, e.g., threonine.

[0309] 42. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 77 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 129 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 579 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1; and / or Amino acid modification at a position corresponding to position 605 of SEQ ID NO: 1 42. The SHC / HAC enzyme variant of any of paragraphs 29 to 41, having one or more additional amino acid modifications selected from:

[0310] 43. The amino acid modification at the position corresponding to position 77 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., alanine; and / or the amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., valine; and / or the amino acid modification at the position corresponding to position 129 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., leucine; and / or the amino acid modification at the position corresponding to position 579 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a basic amino acid, e.g., histidine; and / or the amino acid modification at the position corresponding to position 601 of SEQ ID NO: 1 is a substitution of an amino acid in the amino acid sequence of a wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tyrosine; and / or 43. The SHC / HAC enzyme variant of paragraph 42, wherein the amino acid modification at the position corresponding to position 605 of SEQ ID NO:1 is a substitution of an amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tryptophan.

[0311] 44. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 37 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 174 of SEQ ID NO: 1; and / or Amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1 44. The SHC / HAC enzyme variant of any of paragraphs 29 to 43, having one or more additional amino acid modifications selected from:

[0312] 45. The amino acid modification at the position corresponding to position 37 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a neutral hydrophilic amino acid, e.g., glutamine; and / or the amino acid modification at the position corresponding to position 174 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., isoleucine; and / or 45. The SHC / HAC enzyme variant of paragraph 44, wherein the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tyrosine.

[0313] 46. ​​The SHC / HAC enzyme variant of any of paragraphs 29 to 45, wherein the SHC / HAC enzyme variant has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, and SEQ ID NO:18.

[0314] 47. A nucleic acid sequence encoding the SHC / HAC enzyme variant of any of paragraphs 29 to 46. 48. The nucleic acid sequence of paragraph 47, wherein the nucleic acid sequence is selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:22, and SEQ ID NO:23. 49. A construct comprising the nucleic acid sequence of paragraph 47 or 48. 50. A vector comprising the construct of paragraph 49.

[0315] 51. A recombinant host cell comprising a nucleic acid sequence of paragraph 47 or 48, a construct of paragraph 49, or a vector of paragraph 50. 52. The recombinant host cell of paragraph 51, wherein the construct is integrated into the genome of the host cell. 53. The recombinant host cell of paragraph 51 or 52, wherein the recombinant host cell is selected from a prokaryote, yeast, plant, and / or insect host cell.

[0316] 54. The recombinant host cell of any of paragraphs 51 to 53, wherein the recombinant host cell is a bacterium having a genus selected from Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus, for example, the recombinant host cell is E. coli.

[0317] 55. The process according to any one of paragraphs 1 to 24, wherein the mixture of homofarnesol isomers containing EEH comprises a mixture of EE:EZ isomers.

[0318] 56. The process according to paragraph 55, wherein the mixture of EE:EZ isomers is in a weight ratio of EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; or EE:EZ 66:34, or the mixture of EE:EZ isomers is selected from the group consisting of EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; and EE:EZ 66:34.

[0319] 57. The process according to paragraph 56, wherein the mixture of EE:EZ isomers is in a weight ratio of 80:20.

[0320] 58. The process according to any one of paragraphs 1 to 24 or any one of paragraphs 55 to 57, wherein the weight ratio of SHC / HAC biocatalyst to EEH or a mixture of homofarnesol isomers containing EEH (preferably a mixture of EE:EZ isomers in an 80:20 weight ratio) is in the range of about 0.5 to 2:1, or about 0.25 to 2:1, or about 0.1 to 2:1, or about 1:1, or about 0.5:1.

[0321] 59. The process according to paragraph 58, wherein the weight ratio of SHC / HAC biocatalyst to EEH or a mixture of homofarnesol isomers containing EEH (preferably a mixture of EE:EZ isomers in an 80:20 weight ratio) is in the range of about 1:1, or about 0.5:1, or about 0.1:1.

[0322] Additional SHC / HAC enzymes and enzyme variants Even more surprisingly, it was found that the SHC / HAC enzyme variants also act on other substrates, such as E,E-bishomofarnesol, to generate products such as ambroxide.

[0323] Additionally, it has surprisingly been found that certain wild-type SHC enzymes provide improved (i.e., higher) selectivity for EEH over other isomers of homofarnesol compared to WT AacSHC.

[0324] Thus, it is expected that additional wild-type SHC / HAC enzymes and additional variants of wild-type SHC / HAC enzymes will also provide enzymatic activity (e.g., improved enzymatic activity) for the conversion of EEH to (-)-ambrox and / or the conversion of bisEEH to ambroxide.

[0325] Thus, provided herein are processes for producing (-)-ambrox by enzymatically converting EEH to (-)-ambrox. Also provided herein are processes for producing ambroxide by enzymatically converting E,E-bishomofarnesol to ambroxide. These processes may use any of the wild-type SHC / HAC enzymes or enzyme variants described herein.

[0326] Additionally, provided herein are SHC / HAC enzymes or SHC / HAC enzyme variants having at least about 70.0% amino acid sequence identity to the wild-type SHC / HAC enzyme.

[0327] Among other things, provided herein is a process for preparing (-)-ambrox or a mixture containing (-)-ambrox, which process comprises enzymatically converting EEH or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme or an SHC / HAC enzyme variant having at least about 70.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0328] Among other things, provided herein is a process for preparing ambroxide or a mixture containing ambroxide, the process comprising enzymatically converting bis-EEH or a mixture of bishomofarnesol isomers containing bis-EEH to ambroxide or a mixture containing ambroxide using an SHC / HAC enzyme or an SHC / HAC enzyme variant having at least about 70.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0329] The "selectivity" of an enzyme or enzyme variant refers to the ability of the enzyme or enzyme variant to react with a particular substrate compared to another substrate. For example, a WT SHC enzyme or an enzyme variant of WT SHC that is selective for EEH over other isomers of homofarnesol, or selective for bisEEH over other isomers of bishomofarnesol, means that the WT SHC enzyme or enzyme variant of WT SHC is more likely to convert EEH than other isomers of homofarnesol, or more likely to convert bisEEH than other isomers of bishomofarnesol.

[0330] For example, the wt% of total products formed as a result of the reaction of the WT SHC enzyme or WT SHC enzyme variant with EEH may be at least about 1 percentage point greater than the wt% of total products formed as a result of the reaction of the WT AacSHC with EEH. For example, the wt% of total products formed as a result of the reaction of the WT SHC enzyme or WT SHC enzyme variant with EEH may be at least about 2, or at least about 3, or at least about 4 percentage points greater than the wt% of total products formed as a result of the reaction of the WT AacSHC with EEH. For example, the wt% of total products formed as a result of the reaction of the WT SHC enzyme or WT SHC enzyme variant with EEH may be up to about 40, or up to about 30, or up to about 20, or up to about 15, or up to about 10 percentage points greater than the wt% of total products formed as a result of the reaction of the WT AacSHC with EEH. For example, the wt % of total products formed as a result of the reaction of the WT SHC enzyme or WT SHC enzyme variant with EEH may be from about 1 to about 40 percentage points, or from about 2 to about 30 percentage points, or from about 3 to about 20 percentage points, or from about 4 to about 10 percentage points greater than the wt % of total products formed as a result of the reaction of the WT AacSHC with EEH. The total products formed as a result of the reaction of the WT SHC enzyme or WT SHC enzyme variant or the WT AacSHC or WT AacSHC variant may comprise, consist essentially of, or consist of the compound of formula (I) ((-)-ambrox) and the compound of formula (IV) described herein, for example, when EEH is used as a substrate. The total product formed as a result of the reaction of the WT SHC enzyme or WT SHC enzyme variant, or the WT AacSHC or WT AacSHC variant, when, for example, EEH is used as a substrate, can comprise, consist essentially of, or consist of compounds of formula (X) and / or formula (XII) described herein.

[0331] For example, the wt% of (-)-ambrox formed using a WT SHC enzyme or a WT SHC enzyme variant may be at least about 1 percentage point greater than the wt% of (-)-ambrox formed as a result of, for example, the reaction of WT AacSHC with EEH. For example, the wt% of (-)-ambrox formed as a result of the reaction of a WT SHC enzyme or an enzyme variant of WT SHC with EEH may be at least about 2, or at least about 3, or at least about 4 percentage points greater than the wt% of (-)-ambrox formed as a result of the reaction of WT AacSHC with EEH. For example, the wt% of (-)-ambrox formed as a result of the reaction of a WT SHC enzyme or an enzyme variant of WT SHC with EEH may be up to about 40, or up to about 30, or up to about 20, or up to about 15, or up to about 10 percentage points greater than the wt% of (-)-ambrox formed as a result of the reaction of WT AacSHC with EEH. For example, the wt% of (-)-ambrox formed as a result of the reaction of the WT SHC enzyme or an enzyme variant of WT SHC with EEH may be from about 1 to about 40 percentage points, or from about 2 to about 30 percentage points, or from about 3 to about 20 percentage points, or from about 4 to about 10 percentage points greater than the wt% of (-)-ambrox formed as a result of the reaction of the WT AacSHC with EEH.

[0332] The selectivity of a WT SHC enzyme or WT SHC enzyme variant may also be compared to that of a WT AacSHC or WT AacSHC variant by comparing the EEH:EZH conversion ratio (i.e., % conversion of EEH:% conversion of EZH) or the bisEEH:bisEZH conversion ratio (i.e., % conversion of bisEEH:% conversion of bisEZH) for reactions using each enzyme. This may be determined by measuring the amount of EEH and EZH or bisEEH and bisEZH remaining in the reaction mixture upon completion of the reaction. Alternatively, the selectivity of a WT SHC enzyme or WT SHC enzyme variant may also be compared to that of a WT AacSHC or WT AacSHC variant by comparing the ratio of products resulting from the conversion of EEH (compounds represented by Formulas I and IV) to EZH (compounds represented by Formulas II and III), respectively, or from the conversion of bisEEH (compounds represented by Formulas X and XII) to bisEZH (compounds represented by Formulas XI and XIII), respectively.

[0333] The SHC enzyme or WT SHC enzyme variant may, for example, provide an EEH:EZH conversion ratio of at least about 2.0 in a process for producing (-)-ambrox from a mixture containing EEH and EZH. For example, the WT SHC enzyme or WT SHC enzyme variant may provide an EEH:EZH conversion ratio of at least about 2.5, or at least about 3.0, or at least about 3.5 in a process for producing (-)-ambrox from a mixture containing EEH and EZH. For example, the WT SHC enzyme or WT SHC enzyme variant may provide an EEH:EZH conversion ratio of up to about 5.0, or up to about 4.5, or up to about 4.0 in a process for producing (-)-ambrox from a mixture containing EEH and EZH. For example, a WT SHC enzyme or WT SHC enzyme variant may provide an EEH:EZH conversion ratio ranging from about 2.0 to about 5.0, or from about 2.5 to about 4.5, or from about 3.0 to about 4.0 in a process for producing (-)-ambrox from a mixture comprising EEH and EZH. This may be in contrast to the conversion ratio provided by AacSHC, for example, in a process for producing (-)-ambrox from a mixture comprising EEH and EZH, which may be, for example, less than about 2.0.

[0334] Wild-type SHC / HAC enzymes (e.g., from which SHC / HAC enzyme variants may be derived) include, for example, Alicyclobacillus acidocaldarius (Aac), Zymomonas mobilis (Zmo), Bradyrhizobium japonicum (Bjp), Gluconobacter morbifer (Gmo), Burkholderia ambifaria, Bacillus anthracis, Methylococcus capsulatus, Frankia alni, Acetobacter pasteurianus (Apa), Thermosynechococcus elongatus (Tel), Streptomyces coelicolor (Sco), Rhodopseudomonas palustris (Rpa), Teredinibacter turnerae (Ttu), Pelobacter carbinolicus (Pca), Tetrahymena pyriformis (Tpy), Bacillus megaterium (Bme), or Streptomyces The SHC may be obtained from Saccharomyces cerevisiae (Sal) (see, for example, WO 2010 / 139719, US 2012 / 01345477, WO 2012 / 066059, the contents of which are incorporated herein by reference).

[0335] In particular, the wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) may be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, a Zymomonas mobilis (Zmo) SHC / HAC enzyme, a Bradyrhizobium japonicum (Bjp / Bja) SHC / HAC enzyme, an Acetobacter pasteurianus (Apa) SHC / HAC enzyme, a Bacillus megaterium (Bme) SHC / HAC enzyme, or a Gluconobacter morbifer (Gmo) SHC / HAC enzyme. In particular, the wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) may be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme.

[0336] If the WT SHC enzyme or WT SHC enzyme variant has a higher selectivity for EEH over other isomers of homofarnesol compared to AacSHC and / or variants of AacSHC, then the wild-type SHC / HAC enzyme (e.g., from which the SHC / HAC enzyme variant may be derived) is not an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme. In particular, if the WT SHC enzyme or WT SHC enzyme variant has a higher selectivity for EEH over other isomers of homofarnesol compared to WT AacSHC and / or variants of WT AacSHC, then the wild-type SHC / HAC enzyme (e.g., from which the SHC / HAC enzyme variant may be derived) may be selected from TelSHC1, ApaSHC1, ZmoSHC1, ZmoSHC2, BjaSHC, GmoSHC, BmeSHC, SalSHC, and ApaSHCA. For example, if the WT SHC enzyme or WT SHC enzyme variant has a higher selectivity for EEH over other isomers of homofarnesol compared to WT AacSHC and / or a variant of WT AacSHC, the wild-type SHC / HAC enzyme (e.g., from which the SHC / HAC enzyme variant may be derived) may be selected from ZmoSHC1, BjaSHC, GmoSHC, ApaSHC1, and BmeSHC.

[0337] For ease of reference, the designation "AacSHC" may be used to refer to the Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, "ZmoSHC" may be used to refer to the Zymomonas mobilis (Zmo) SHC / HAC enzyme, "BjpSHC" or "BjaSHC" may be used to refer to the Bradyrhizobium japonicum (Bjp) SHC / HAC enzyme, "GmoSHC" may be used to refer to the Gluconobacter morbifer (Gmo) SHC / HAC enzyme, and "BmeSHC" may be used to refer to the Bacillus megaterium SHC / HAC enzyme.

[0338] An SHC / HAC enzyme or SHC / HAC enzyme variant may, for example, have an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme. For example, an SHC / HAC enzyme or SHC / HAC enzyme variant may have an amino acid sequence that is at least about 75.0%, or at least about 80.0%, or at least about 85.0%, or at least about 90.0%, or at least about 95.0%, or at least about 95.5%, or at least about 96.5%, or at least about 97.0%, or at least about 97.5%, or at least about 98.0%, or at least about 98.5%, or at least about 99.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0339] The SHC / HAC enzyme has an amino acid sequence that is 100% identical to the wild-type SHC / HAC enzyme. A SHC / HAC enzyme variant has an amino acid sequence that is less than 100% identical to the amino acid sequence of a wild-type SHC / HAC enzyme, e.g., equal to or less than about 99.5% identity, or equal to or less than about 99.0% identity.

[0340] For example, a SHC / HAC enzyme variant may have from about 70.0% to about 99.5%, or from about 80.0% to about 99.0%, or from about 85.0% to about 98.5%, or from about 90.0% to about 98.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0341] The amino acid sequence of the wild-type SHC / HAC enzyme may be, for example, AacSHC (SEQ ID NO: 1), ZmoSHC1 (SEQ ID NO: 11), ZmoSHC2 (SEQ ID NO: 12), BjpSHC (SEQ ID NO: 13), GmoSHC (SEQ ID NO: 14), TelSHC (SEQ ID NO: 19), ApaSHC1 (SEQ ID NO: 20), BmeSHC (SEQ ID NO: 28), SalSHC (SEQ ID NO: 29), or ApaSHCA (SEQ ID NO: 30). For example, the wild-type SHC / HAC enzyme may be AacSHC (SEQ ID NO: 1).

[0342] Thus, in some embodiments, an SHC / HAC enzyme or SHC / HAC enzyme variant may have an amino acid sequence having at least about 70.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, an SHC / HAC enzyme or SHC / HAC enzyme variant has an amino acid sequence having at least about 75.0%, or at least about 80.0%, or at least about 85.0%, or at least about 90.0%, or at least about 95.0%, or at least about 95.5%, or at least about 96.5%, or at least about 97.0%, or at least about 97.5%, or at least about 98.0%, or at least about 98.5%, or at least about 99.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0343] For example, the SHC / HAC enzyme may have an amino acid sequence that is 100% identical to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0344] For example, an SHC / HAC enzyme variant may have an amino acid sequence that is less than 100% identical, e.g., equal to or less than about 99.5% identical, or equal to or less than about 99.0% identical, to, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0345] For example, the SHC / HAC enzyme variant may have from about 70.0% to about 99.5%, or from about 80.0% to about 99.0%, or from about 85.0% to about 98.5%, or from about 90.0% to about 98.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0346] "Percent (%) identity" with respect to a polypeptide or nucleotide sequence is defined as the percentage of amino acids or nucleotides in a candidate sequence that are identical to those in a reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, but without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways that are within the skill in the art, illustratively using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide chain of amino acids, regardless of length or post-translational modification.

[0347] Nucleotide and amino acid sequence similarity, i.e., percentage of sequence identity, can be determined through sequence alignment. Such alignments include, for example, https: / / www.ebi.ac.uk / Tools / msa / clustalo / The algorithm can be implemented using several algorithms known in the art, such as the GAP program (University of Iowa Mathematical Algorithm), or the Myers and Miller Mathematical Algorithm (1989-Cabios 4:11-17), preferably the Karlin and Altschul Mathematical Algorithm (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). The preferred parameters used are the default parameters as set on https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0348] Percentage sequence identity may be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score = 100, word length = 12, to obtain polynucleotide sequences homologous to those nucleic acids encoding related proteins. BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to polypeptides.

[0349] To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov Random Fields. When percentages of sequence identity are mentioned in this application, these percentages are calculated with respect to the full length of the longer sequence unless otherwise specifically indicated.

[0350] In a specific embodiment, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0351] In some embodiments, a SHC / HAC enzyme variant may have equal to or less than about 200 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO: 1, compared to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. For example, an SHC / HAC enzyme variant can have an RI of about 150 or less, or about 120 or less, or about 100 or less, or about 95 or less, or about 90 or less, or about 85 or less, or about 80 or less, or about 75 or less, or about 85 or less, or about 85 or less, or about 75 or less, compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. It may have less than or equal to about 70 amino acid modifications, or less than or equal to about 65, or less than or equal to about 60, or less than or equal to about 55, or less than or equal to about 50, or less than or equal to about 45, or less than or equal to about 40, or less than or equal to about 35, or less than or equal to about 30, or less than or equal to about 25, or less than or equal to about 20, or less than or equal to about 15, or less than or equal to about 10.

[0352] A variant SHC / HAC enzyme may have, for example, at least about one, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO: 1, compared to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.

[0353] For example, a variant SHC / HAC enzyme may have from about 1 to about 30 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:20. For example, a variant SHC / HAC enzyme may have from about 2 to about 25 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, a variant SHC / HAC enzyme may have from about 3 to about 20 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, a variant SHC / HAC enzyme may have from about 4 to about 15 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. For example, a variant SHC / HAC enzyme may have from about 5 to about 10 amino acid modifications compared to a wild-type SHC / HAC enzyme, e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0354] The amino acid modification may be, for example, an insertion, deletion, and / or substitution as described above. For example, the amino acid modification may be a substitution, for example, a non-conservative substitution.

[0355] In some embodiments, the only amino acid modifications are substitutions (i.e., no insertions or deletions) compared to the wild-type SHC / HAC enzyme (e.g., compared to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30).

[0356] Amino acid alterations are defined relative to a reference sequence. An amino acid alteration relative to a reference sequence means that the amino acid sequence of a variant sequence differs from the reference sequence.

[0357] Amino acids in a reference sequence and a variant sequence may be assigned numbers, where the numbering begins with the amino acid at the N-terminus of the polypeptide (i.e., the amino acid at the N-terminus of a polypeptide is numbered 1, the next amino acid is numbered 2, etc.). A "position" in a reference sequence refers to a particular amino acid residue present in the reference sequence, as identified by a particular numbered amino acid in the reference sequence. A "position" in a variant sequence refers to a particular amino acid residue present in the variant sequence, as identified by a particular numbered amino acid in the variant sequence.

[0358] Because a variant sequence may contain deletions or insertions compared to a reference sequence, amino acids in the variant sequence may be numbered differently than the same amino acids in the reference sequence. As an example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO: 1, the amino acid will retain the number 132 in the reference sequence, while the amino acid following the insertion will have the number 133 in the variant sequence. In this example, the position in the variant sequence that corresponds to position 132 in the reference sequence is position 133. Thus, an amino acid in a variant sequence that is retained from a reference sequence may be defined by referring to the "corresponding position" in the reference sequence. In other words, a "position" in a variant sequence may be defined by referring to the "corresponding position" in the reference sequence. Notably, a substitution in a variant sequence compared to a reference sequence may be defined by referring to the "corresponding position" in the reference sequence, despite any insertions and / or deletions in the reference sequence. If an amino acid in the reference sequence is deleted, there will be no "corresponding position" in the variant sequence. If there are no insertions or deletions (i.e., only substitutions) compared to the reference sequence, the "corresponding position" of the reference sequence will be the same as the position in the variant sequence.

[0359] Because a variant sequence may contain deletions or insertions compared to a reference sequence, amino acids in the variant sequence may be numbered differently than the same amino acids in the reference sequence. As an example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO: 1, the amino acid will retain the number 132 in the reference sequence, while the amino acid following the insertion will have the number 133 in the variant sequence. In this example, the position in the variant sequence that corresponds to position 132 in the reference sequence is position 133. Thus, an amino acid in a variant sequence that is retained from a reference sequence may be defined by referring to the "corresponding position" in the reference sequence. In other words, a "position" in a variant sequence may be defined by referring to the "corresponding position" in the reference sequence. Notably, a substitution in a variant sequence compared to a reference sequence may be defined by referring to the "corresponding position" in the reference sequence, despite any insertions and / or deletions in the reference sequence. If an amino acid in the reference sequence is deleted, there will be no "corresponding position" in the variant sequence. If there are no insertions or deletions (i.e., only substitutions) compared to the reference sequence, the "corresponding position" of the reference sequence will be the same as the position in the variant sequence.

[0360] Thus, an amino acid modification may be defined relative to two different reference sequences. For example, the amino acid modification may be a change relative to a first reference sequence (e.g., the sequence of a wild-type SHC / HAC enzyme from which the variant is derived), and the position of the amino acid modification in the variant sequence may be defined by reference to a second reference sequence (e.g., AacSHC (SEQ ID NO: 1)). Thus, an amino acid modification in a SHC / HAC enzyme variant may be compared to the first wild-type SHC / HAC enzyme at a position defined by reference to the second wild-type SHC / HAC enzyme.

[0361] SHC / HAC enzyme variants may, for example, have one or more of the particular substitutions or combinations of substitutions defined above with reference to SEQ ID NO:1.

[0362] In particular, the SHC / HAC enzyme variant may have one or more of the specific substitutions or combinations of substitutions at one or more positions corresponding to positions 77, 81, 90, 92, 129, 132, 172, 224, 277, 431, 432, 557, 579, 601, 605, and 613 of SEQ ID NO:1.

[0363] Each of these substitutions may be independently as defined above, for example, in the subsections entitled "Variant of Aac 215G2" and "Other variants with new mutations at positions corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO: 1."

[0364] In particular, the SHC / HAC enzyme variant may have one or more of the following substitution combinations: (i) substitutions at positions corresponding to positions 132, 224, and 432 of SEQ ID NO:1; (ii) substitutions at positions corresponding to positions 132, 224, 432, 557, and 431 of SEQ ID NO:1; (iii) substitutions at positions corresponding to positions 132, 224, 432, 557, and 613 of SEQ ID NO:1; (iv) substitutions at positions corresponding to positions 132, 224, 432, 557, 81, and 613 of SEQ ID NO:1; (v) substitutions at positions corresponding to positions 132, 224, 432, 557, 81, and 431 of SEQ ID NO:1; (vi) substitutions at positions corresponding to positions 132, 224, 432, 90, and 613 of SEQ ID NO:1; (vii) substitutions at positions corresponding to positions 132, 224, 432, 172, and 277 of SEQ ID NO:1; (viii) substitutions at positions corresponding to positions 132, 224, 432, and 37 of SEQ ID NO:1; (ix) substitutions at positions corresponding to positions 132, 224, 432, and 174 of SEQ ID NO:1; (x) substitutions at positions corresponding to positions 132, 224, 432, 174, and 601 of SEQ ID NO:1; (xi) Substitutions at positions corresponding to positions 132, 224, 432, 37, 174, and 601 of SEQ ID NO:1.

[0365] In some embodiments, the SHC / HAC enzyme variant is identical to SEQ ID NO: 1 except for the following amino acid substitutions: (i) M132R, A224V, I432T, A557T, and H431L (SEQ ID NO: 2); or (ii) M132R, A224V, I432T, A557T, and R613S (SEQ ID NO: 3); or (iii) M132R, A224V, I432T, A557T, Y81H, and R613S (SEQ ID NO: 4); or (iv) M132R, A224V, I432T, A557T, Y81H, and H431L (SEQ ID NO: 5); or (v) M132R, A224V, I432T, T90A, and R613S (SEQ ID NO: 17); or (vi) M132R, A224V, I432T, A172T, and M277K (SEQ ID NO: 18); or (vii) M132R, A224V, I432T, and L37Q (SEQ ID NO: 24); or (viii) M132R, A224V, I432T, V174I (SEQ ID NO: 25); or (ix) M132R, A224V, I432T, V174I, and F601Y (SEQ ID NO: 26); or (x) M132R, A224V, I432T, L37Q, V174I, and F601Y (sequence number 27).

[0366] The following numbered paragraphs define additional aspects of the present disclosure: 1. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme or an SHC / HAC enzyme variant; wherein the SHC / HAC enzyme or SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme; Here, the WT SHC / HAC enzyme has a higher selectivity for EEH over other isomers of homofarnesol.

[0367] 2. A process for preparing ambroxide or a mixture containing ambroxide, the process comprising enzymatically converting E,E-bishomofarnesol (bisEEH) or a mixture of bishomofarnesol isomers containing bisEEH using an SHC / HAC enzyme or an SHC / HAC enzyme variant to ambroxide or a mixture containing ambroxide; Here, the SHC / HAC enzyme or SHC / HAC enzyme variant has an amino acid sequence that has at least about 70.0% identity to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0368] 3. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, the process comprising enzymatically converting 3E,7E-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme or an SHC / HAC enzyme variant; wherein the SHC / HAC enzyme or SHC / HAC enzyme variant has at least about 70.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30; and wherein the mixture of isomers containing EEH is selected from one or more of the group consisting of: [(3E,7E) and [(3Z,7E)], and / or [(3E,7E) and (3E,7Z)], and / or [(3Z,7E), (3E,7E), and (3E,7Z)] (also designated as [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively).

[0369] 4. A process for preparing amburoxide or a mixture containing amburoxide, the process comprising enzymatically converting E,E-bishomofarnesol (bisEEH) or a mixture of bishomofarnesol isomers containing bisEEH using an SHC / HAC enzyme or an SHC / HAC enzyme variant to amburoxide or a mixture containing amburoxide; wherein the SHC / HAC enzyme or SHC / HAC enzyme variant has at least about 70.0% identity to SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30; and wherein the mixture of isomers containing EEH is selected from one or more of the group consisting of: [(E,E) and [(Z,E)], and / or [(E,E) and (E,Z)], and / or [(Z,E), (E,E), and (E,Z)] (also designated as [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively).

[0370] 5. The process of paragraphs 1 or 3, wherein the SHC / HAC enzyme has a higher selectivity for EEH over other isomers of homofarnesol compared to that of WT AacSHC.

[0371] 6. The process of any of paragraphs 1, 3, or 5, wherein the wt% of total products formed as a result of the reaction of the SHC / HAC enzyme or enzyme variant with EEH is at least about 1 percentage point, e.g., at least about 2 percentage points, or at least about 3 percentage points higher than the wt% of total products formed as a result of the reaction of the AacSHC with EEH.

[0372] 7. The process of any of paragraphs 1, 3, 5 or 6, wherein the EEH:EZH conversion ratio is at least about 2.0, e.g., at least about 2.5, or at least about 3.0.

[0373] 8. The process of any of paragraphs 1 through 7, wherein the wild-type SHC / HAC enzyme is obtained from Alicyclobacillus acidocaldarius (Aac), Zymomonas mobilis (Zmo), Bradyrhizobium japonicum (Bjp), Gluconobacter morbifer (Gmo), Burkholderia ambifaria, Bacillus anthracis, Methylococcus capsulatus, Frankia alni, Acetobacter pasteurianus (Apa), Thermosynechococcus elongatus (Tel), Streptomyces coelicolor (Sco), Rhodopseudomonas palustris (Rpa), Teredinibacter turnerae (Ttu), Pelobacter carbinolicus (Pca), Tetrahymena pyriformis, Bacillus megaterium, or Streptomyces albolongus.

[0374] 9. The process of any of paragraphs 1-8, wherein the wild-type SHC / HAC enzyme is SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, e.g., the wild-type SHC / HAC enzyme is SEQ ID NO:1.

[0375] 10. The process of paragraph 1, wherein the mixture of isomers of homofarnesol is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E)], [(3Z,7E), (3E / 7E), and (3E,7Z)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)], and / or [(3E,7E) and (3E,7Z)].

[0376] 11. The process of any preceding paragraph, wherein the process uses a solubilizing agent selected from Triton X-100, Tween 80, taurodeoxycholate, sodium taurodeoxycholate, sodium dodecyl sulfate (SDS), and / or sodium lauryl sulfate (SLS).

[0377] 12. The process of any of paragraphs 1-11, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least about 90.0%, or at least about 95.0%, identical to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0378] 13. The process of any preceding paragraph, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at one or more positions selected from positions corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1.

[0379] 14. The process of any of paragraphs 1 to 13, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 90 and 613 of SEQ ID NO:1.

[0380] 15. The process of any of paragraphs 1 to 13, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 172 and 277 of SEQ ID NO:1.

[0381] 16. The process of any of paragraphs 1-13, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to position 81, 431, or 613 of SEQ ID NO:1.

[0382] 17. The process of paragraph 16, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 431 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0383] 18. The process of paragraph 16, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 613 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0384] 19. The process of paragraph 17 or 18, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at a position corresponding to position 81 of SEQ ID NO:1.

[0385] 20. The process of any preceding paragraph, wherein one or more, e.g., all, of the amino acid modifications at positions 81, 90, 172, 277, 431, 557, or 613 are substitutions, e.g., non-conservative substitutions.

[0386] 21. The amino acid modification at the position corresponding to position 81 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC with a basic amino acid, e.g., histidine; and / or The amino acid modification at the position corresponding to position 90 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, e.g., alanine; and / or The amino acid modification at the position corresponding to position 172 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, e.g., threonine; and / or the amino acid modification at the position corresponding to position 277 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, e.g., lysine; and / or the amino acid modification at the position corresponding to position 431 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, e.g., leucine; and / or The amino acid modification at the position corresponding to position 557 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, e.g., threonine; and / or The process of any preceding paragraph, wherein the amino acid modification at the position corresponding to position 613 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC with a neutral hydrophilic amino acid, e.g., serine.

[0387] 22. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 132 of SEQ ID NO:1; an amino acid modification at a position corresponding to position 224 of SEQ ID NO:1; and Amino acid modification at a position corresponding to position 432 of SEQ ID NO: 1 The process of any preceding paragraph, having one or more amino acid modifications selected from:

[0388] 23. The amino acid modification at the position corresponding to position 132 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a basic amino acid, e.g., arginine; and / or the amino acid modification at the position corresponding to position 224 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., valine; and / or 23. The process of paragraph 22, wherein the amino acid modification at the position corresponding to position 432 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a neutral hydrophilic amino acid, e.g., threonine.

[0389] 24. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 77 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 129 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 579 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1; and / or Amino acid modification at a position corresponding to position 605 of SEQ ID NO: 1 The process of any preceding paragraph, having one or more amino acid modifications selected from:

[0390] 25. The amino acid modification at the position corresponding to position 77 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., alanine; and / or the amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., valine; and / or the amino acid modification at the position corresponding to position 129 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., leucine; and / or the amino acid modification at the position corresponding to position 579 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a basic amino acid, e.g., histidine; and / or the amino acid modification at the position corresponding to position 601 of SEQ ID NO: 1 is a substitution of an amino acid in the amino acid sequence of a wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tyrosine; and / or 25. The process of paragraph 24, wherein the amino acid modification at the position corresponding to position 605 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tryptophan.

[0391] 26. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 37 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 174 of SEQ ID NO: 1; and / or Amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1 The process of any preceding paragraph, having one or more additional amino acid modifications selected from:

[0392] 27. The amino acid modification at the position corresponding to position 37 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a neutral hydrophilic amino acid, e.g., glutamine; and / or the amino acid modification at the position corresponding to position 174 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., isoleucine; and / or 27. The process of paragraph 26, wherein the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tyrosine.

[0393] 28. The process of any preceding paragraph, wherein the SHC / HAC enzyme variant has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0394] 29. The process of any preceding paragraph, wherein the process comprises culturing a recombinant host cell that produces the SHC / HAC enzyme variant.

[0395] 30. The process of paragraph 29, wherein the recombinant host cell comprises a nucleic acid sequence encoding an SHC / HAC enzyme (e.g., selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:22, or SEQ ID NO:23).

[0396] 31. The process of any preceding paragraph in which (-)-ambrox is produced in admixture with at least one or more of by-products (II), (III), or (IV).

[0397] 32. (-)-Ambrox, obtained by or obtainable by the process of any preceding paragraph, e.g., in amorphous or crystalline form. 33. Ambroxide, e.g., in amorphous or crystalline form, obtained by or obtainable by the process of any preceding paragraph.

[0398] 34. Use of (-)-ambrox of paragraph 32 and / or ambroxide of paragraph 33 as part of a fragrance or cosmetic or consumer product. 35. Fragrances or cosmetics or consumer products containing (-)-ambrox of paragraph 32 and / or ambroxide of paragraph 33.

[0399] 36. An SHC / HAC enzyme or SHC / HAC enzyme variant having an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0400] 37. The SHC / HAC enzyme or enzyme variant of paragraph 36, wherein the wild-type SHC / HAC enzyme is SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, e.g., SEQ ID NO:1.

[0401] 38. The SHC / HAC enzyme or enzyme variant of paragraph 36 or 37, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least about 90.0% or at least about 95.0% identical to the amino acid sequence of a wild-type SHC / HAC enzyme.

[0402] 39. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36-38, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at one or more positions selected from positions corresponding to positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO:1.

[0403] 40. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 90 and 613 of SEQ ID NO:1.

[0404] 41. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 172 and 277 of SEQ ID NO:1.

[0405] 42. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36-39, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to position 81, 431, or 613 of SEQ ID NO:1.

[0406] 43. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 431 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0407] 44. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications at positions corresponding to positions 557 and 613 of SEQ ID NO:1 compared to a wild-type SHC / HAC enzyme.

[0408] 45. The SHC / HAC enzyme or enzyme variant of paragraph 43 or 44, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, relative to a wild-type SHC / HAC enzyme, at a position corresponding to position 81 of SEQ ID NO:1.

[0409] 46. ​​The SHC / HAC enzyme or enzyme variant of any of paragraphs 36-45, wherein one or more, e.g., all, of the amino acid modifications at positions 81, 90, 172, 277, 431, 557, or 613 are substitutions, e.g., non-conservative substitutions.

[0410] 47. The amino acid modification at the position corresponding to position 81 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC with a basic amino acid, e.g., histidine; and / or The amino acid modification at the position corresponding to position 90 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, e.g., alanine; and / or The amino acid modification at the position corresponding to position 172 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, e.g., threonine; and / or the amino acid modification at the position corresponding to position 277 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, e.g., lysine; and / or the amino acid modification at the position corresponding to position 431 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, e.g., leucine; and / or The amino acid modification at the position corresponding to position 557 of SEQ ID NO: 1 is a substitution of the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, e.g., threonine; and / or 47. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 46, wherein the amino acid modification at the position corresponding to position 613 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of wild-type SHC / HAC with a neutral hydrophilic amino acid, e.g., serine.

[0411] 48. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 132 of SEQ ID NO:1; an amino acid modification at a position corresponding to position 224 of SEQ ID NO:1; and Amino acid modification at a position corresponding to position 432 of SEQ ID NO: 1 48. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 47, having one or more amino acid modifications selected from:

[0412] 49. The amino acid modification at the position corresponding to position 132 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a basic amino acid, e.g., arginine; and / or the amino acid modification at the position corresponding to position 224 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., valine; and / or 49. The SHC / HAC enzyme or enzyme variant of paragraph 48, wherein the amino acid modification at the position corresponding to position 432 of SEQ ID NO:1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a neutral hydrophilic amino acid, e.g., threonine.

[0413] 50. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 77 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 129 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 579 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1; and / or Amino acid modification at a position corresponding to position 605 of SEQ ID NO: 1 50. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 49, having one or more amino acid modifications selected from:

[0414] 51. The amino acid modification at the position corresponding to position 77 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, for example, alanine; and / or the amino acid modification at a position corresponding to position 92 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., valine; and / or the amino acid modification at the position corresponding to position 129 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., leucine; and / or the amino acid modification at the position corresponding to position 579 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a basic amino acid, e.g., histidine; and / or the amino acid modification at the position corresponding to position 601 of SEQ ID NO: 1 is a substitution of an amino acid in the amino acid sequence of a wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tyrosine; and / or 51. The SHC / HAC enzyme or enzyme variant of paragraph 50, wherein the amino acid modification at the position corresponding to position 605 of SEQ ID NO:1 is a substitution of an amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tryptophan.

[0415] 52. The amino acid sequence of the SHC / HAC enzyme variant, compared to the amino acid sequence of wild-type SHC / HAC, is as follows: an amino acid modification at a position corresponding to position 37 of SEQ ID NO: 1; and / or an amino acid modification at a position corresponding to position 174 of SEQ ID NO: 1; and / or Amino acid modification at a position corresponding to position 601 of SEQ ID NO: 1 52. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 51, having one or more amino acid modifications selected from:

[0416] 53. The amino acid modification at the position corresponding to position 37 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a neutral hydrophilic amino acid, e.g., glutamine; and / or the amino acid modification at the position corresponding to position 174 of SEQ ID NO: 1 is a substitution of the amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with a hydrophobic amino acid, e.g., isoleucine; and / or 53. The SHC / HAC enzyme or enzyme variant of paragraph 52, wherein the amino acid modification at the position corresponding to position 601 of SEQ ID NO:1 is a substitution of an amino acid in the amino acid sequence of the wild-type SHC / HAC enzyme with an aromatic amino acid, e.g., tyrosine.

[0417] 54. The SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 53, wherein the SHC / HAC enzyme variant has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0418] 55. A nucleic acid sequence encoding an SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 54. 56. The nucleic acid sequence of paragraph 55, wherein the nucleic acid sequence is selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:22, or SEQ ID NO:23.

[0419] 57. A construct comprising the nucleic acid sequence of paragraph 55 or 56. 58. A vector comprising the construct of paragraph 57. 59. A recombinant host cell comprising a nucleic acid sequence of paragraph 52 or 53, a construct of paragraph 57, or a vector of paragraph 58.

[0420] 60. The recombinant host cell of paragraph 59, wherein the construct is integrated into the genome of the host cell. 61. The recombinant host cell of paragraph 59 or 60, wherein the recombinant host cell is selected from a prokaryote, yeast, plant, and / or insect host cell.

[0421] 62. The recombinant host cell of any of paragraphs 59 to 61, wherein the recombinant host cell is a bacterium having a genus selected from Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus, for example, the recombinant host cell is E. coli.

[0422] 63. The process according to any one of paragraphs 1 to 31, wherein the mixture of homofarnesol isomers containing EEH comprises a mixture of EE:EZ isomers.

[0423] 64. The process according to paragraph 63, wherein the mixture of EE:EZ isomers is selected from the group consisting of EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; and EE:EZ 66:34, or the mixture of EE:EZ isomers is EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; or EE:EZ 66:34.

[0424] 65. The process according to paragraph 64, wherein the mixture of EE:EZ isomers is in a weight ratio of 80:20.

[0425] 66. The process according to any one of paragraphs 1-31 or 63-65, wherein the weight ratio of SHC / HAC enzymes to EEH or a mixture of homofarnesol isomers containing EEH (preferably a mixture of EE:EZ isomers in an 80:20 weight ratio) is in the range of about 0.5-2:1, or about 0.25-2:1, or about 0.1-2:1, or about 1:1, or about 0.5:1.

[0426] 67. The process according to paragraph 66, wherein the weight ratio of SHC / HAC enzymes to EEH or a mixture of homofarnesol isomers containing EEH (preferably a mixture of EE:EZ isomers in an 80:20 weight ratio) is in the range of about 1:1, or about 0.5:1, or about 0.1:1.

[0427] The following numbered paragraphs define additional aspects of the present disclosure: 1. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, comprising: wherein EE-homofarnesol (EEH) or a mixture of isomers containing EE-homofarnesol (EEH) is enzymatically converted to (-)-ambrox or a mixture containing (-)-ambrox; wherein the enzymatic conversion is carried out using a squalenehopene cyclase / homofarnesol ambrox cyclase (SHC / HAC) biocatalyst having a polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, 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% identity to SEQ ID NO:20 (ApaSHC1), SEQ ID NO:19 (TelSHC), SEQ ID NO:14 (GmoSHC), SEQ ID NO:28 (BmeSHC), SEQ ID NO:29 (SalSHC), and / or SEQ ID NO:30 (ApaSHCA), under reaction conditions suitable for the production of (-)-ambrox; and wherein the mixture of isomers containing EEH is selected from one or more of the group consisting of: [(3E,7E) and [(3Z,7E)], and / or [(3E,7E) and (3E,7Z)], and / or [(3Z,7E), (3E,7E), and (3E,7Z)] (also designated as [EE:EZ], and / or [EE:ZE], and / or [EE:EZ:ZE], respectively).

[0428] 2. The process according to paragraph 1, wherein the process is carried out using a SHC / HAC biocatalyst having at least 70%, at least 75%, at least 80%, at least 85%, 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% identity to SEQ ID NO:20 (ApaSHC1) or SEQ ID NO:28 (BmeSHC).

[0429] 3. The process according to paragraph 2, wherein the conversion of EEH or a mixture of isomers containing EEH to (-)-ambrox occurs at a temperature ranging from about 30°C to about 50°C, e.g., from about 35°C to about 50°C, and at a pH ranging from about 5 to about 7.

[0430] 4. The process according to paragraph 1 or paragraph 2, wherein the process is carried out in the presence of a solubilizing agent, e.g., SDS.

[0431] 5. The process according to any one of paragraphs 1-4, comprising (a) culturing one or more recombinant host cells expressing an SHC / HAC enzyme under conditions that allow for the production of a WT SHC / HAC biocatalyst prior to conversion of EEH or a mixture of isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox.

[0432] 6. The process according to paragraph 5, wherein the culturing step and the subsequent converting step optionally occur in the same reaction vessel under different reaction conditions.

[0433] 7. The process according to paragraph 6, wherein the incubation step is in the pH range of 6 to 7 and the EEH to (-)-ambrox step is in the pH range of 4.8 to 5.5.

[0434] 8. The process according to any one of paragraphs 1 to 7, wherein the mixture of isomers comprising EEH comprises a mixture of EE:EZ isomers.

[0435] 9. The process according to paragraph 8, wherein the mixture of EE:EZ isomers is in a weight ratio selected from the group consisting of EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; and EE:EZ 66:34, or wherein the mixture of EE:EZ isomers is in a weight ratio of EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; and / or EE:EZ 66:34.

[0436] 10. The process of paragraph 9, wherein the mixture of EE:EZ isomers is in an 80:20 weight ratio.

[0437] 11. The process of any one of paragraphs 1-10, wherein the weight ratio of SHC / HAC biocatalyst to EEH or a mixture of isomers containing EEH (preferably a mixture of EE:EZ isomers in an 80:20 weight ratio) is in the range of about 0.5-2:1, or about 0.25-2:1, or about 0.1-2:1, or about 1:1, or about 0.5:1.

[0438] 12. The process according to paragraph 11, wherein the weight ratio of SHC / HAC enzymes to EEH or a mixture of isomers containing EEH (preferably a mixture of EE:EZ isomers in an 80:20 weight ratio) is about 1:1, or about 0.5:1, or about 0.1:1.

[0439] 13. The process of any one of paragraphs 1 through 12, wherein (-)-ambrox is produced in admixture with one or more of by-products (II), (IV), and / or (III).

[0440] 14. The process of paragraph 13, in which (-)-ambrox is separated from the reaction medium.

[0441] 15. The process of paragraph 14, wherein the (-)-ambrox is separated from the reaction medium using a filtration step, a decantation step, or a combination of filtration and decantation steps.

[0442] 16. The process of paragraph 15, wherein the filtration step is a belt filtration or rotary filtration step.

[0443] 17. The process of any one of paragraphs 13-16, wherein prior to the separation step, (-)-ambrox is heated to a maximum temperature of 55°C.

[0444] 18. The process of paragraph 17, wherein the recovered (-)-ambrox is solubilized in a solvent. 19. The process of paragraph 18, wherein the solubilized (-)-ambrox is filtered.

[0445] 20. The process of paragraph 18 or 19, wherein (-)-ambrox is recovered in solid form by removing the solvent by evaporation.

[0446] 21. The process of any one of paragraphs 14 through 20, wherein (-)-Ambrox is substantially free of by-products (II), (IV), and / or (III).

[0447] 22. A reaction product comprising (-)-ambrox, obtainable by the process of any one of paragraphs 1 to 21.

[0448] 23. (-)-Ambrox is the reaction product of paragraph 22 in solid form. 24. (-)-Ambrox is the reaction product of paragraph 23 in amorphous or crystalline form.

[0449] 25. A method for producing a product containing (-)-ambrox, comprising incorporating the reaction product of any one of paragraphs 22-24 into the product.

[0450] 26. The method of paragraph 25, wherein the product is a fragrance product, a cosmetic product, a cleaning product, a detergent product, and / or a soap product.

[0451] 27. A fragrance or cosmetic, or consumer care product, comprising the reaction product of any one of paragraphs 22 to 24.

[0452] 28. A fragrance or cosmetic or consumer care composition comprising the reaction product of any one of paragraphs 22-24 and an additional component.

[0453] 29. Use of the reaction product of any one of paragraphs 22 to 24 as part of a fragrance product or a cosmetic or consumer care product.

[0454] Ambrox and its uses Further provided herein are reaction products made by the processes described herein. The reaction products may, for example, comprise, consist essentially of, or consist of (-)-ambrox and one or more additional compounds, such as one or more of a compound represented by formula (II), a compound represented by formula (III), and a compound represented by formula (IV).

[0455] As used herein, the term "ambrox" refers to (-)-ambrox, represented by formula (I) below, as well as isomerically pure ambrox, or isomers of ambrox represented by formulas (II), (III), and / or (IV) below: [ka] and (-)-ambrox in admixture with one or more molecules represented by the formula:

[0456] The nomenclature for the reaction products represented by formulas (I), (II), (III), and (IV) is given below. Table 2. Nomenclature for reaction products represented by formulas (I), (II), (III), and (IV). [Table 3-1]

[0457] [Table 3-2]

[0458] (-)-Ambrox is commercially known as Ambrox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlyn (Quest), Cetalox Laevo (Firmenich), Ambermor (Aromor), and / or Norambrenolide Ether (Pacific).

[0459] (-)-Ambrox is an industrially important aroma compound that has long been used in the fragrance industry. The particularly desirable sensory benefits from (-)-ambrox come from the (-)-isomer, rather than the (+)-isomer. The odor of the (-)-isomer has been described as musky, woody, warm, or ambery, while the enantiomer of (+)-ambrox has a relatively weak odor note. The odors and odor thresholds of ambrox-like products also differ. While various (-)-ambrox-enriched materials are commercially available, it is desirable to produce highly concentrated (-)-ambrox materials, ideally pure (-)-ambrox. The process described herein may produce (-)-ambrox, represented by formula (I), alone or in mixtures with by-products such as the compounds represented by formulas (II), (III), and / or (IV) above.

[0460] (-)-Ambrox can be produced from sclareolide according to the production process described below. Sclareol is a natural product extracted from the plant clary sage. However, because natural starting materials are used in this process, it involves multi-step reactions, and there are potential problems in that the operation is tedious, the amount and stability of the supply of starting materials are not always satisfactory, and the reaction is not environmentally friendly because oxidizing agents such as chromic acid or permanganate are used in the oxidative decomposition step of (+)-sclareol.

[0461] [ka]

[0462] (-)-Ambrox can also be synthesized from homofarnesol using various routes. For example, homofarnesol can be obtained by bromination, cyanation, and hydrolysis of nerolidol to give homofarnesylic acid, which can then be reduced. Alternatively, homofarnesol can be obtained from farnesol, farnesyl chloride, beta-farnesene, or other substrates.

[0463] The processes described herein may produce (-)-ambrox of formula (I), either alone or in admixture with by-products such as compounds shown in formulas (II), (III), and / or (IV) above. For example, other stereoisomers of formula (I) may also be produced by the processes described herein.

[0464] Thus, provided herein is a compound of formula (I) or a composition comprising a compound of formula (I) obtained by or obtainable by the process described herein (including all aspects thereof).

[0465] In some embodiments, not all of the homofarnesol (e.g., EEH) is converted to (-)-ambrox or a by-product of the reaction. Thus, the compositions described herein, e.g., compositions obtained by or obtainable by the processes described herein, may contain homofarnesol (e.g., in addition to EEH, e.g., a compound represented by Formula (I), and / or a compound represented by Formulas (II), (III), and / or (IV)). Any remaining homofarnesol may be separated from the other reaction products such that the (-)-ambrox product does not contain homofarnesol. In other embodiments, all of the homofarnesol starting material is converted to (-)-ambrox represented by Formula (I) or a by-product of the reaction by the processes described herein.

[0466] Thus, the compositions described herein may comprise, consist essentially of, or consist of one or more of a compound represented by formula (I), a compound represented by formula (II), a compound represented by formula (III), a compound represented by formula (IV), a homofarnesol starting material (e.g., EEH), and other stereoisomers of a compound represented by formula (I). For example, the compositions described herein may comprise, consist essentially of, or consist of a compound represented by formula (I) and one or more of a compound represented by formula (II), a compound represented by formula (III), and a compound represented by formula (IV).

[0467] Thus, the compositions described herein may contain greater than or equal to about 50 wt % of the compound of formula (I), based on the total weight of the compound of formula (I), the compound of formula (II), the compound of formula (III), and the compound of formula (IV). For example, the compositions described herein may comprise, based on the total weight of the compound represented by Formula (I), the compound represented by Formula (II), the compound represented by Formula (III), and the compound represented by Formula (IV), equal to or greater than about 55 wt%, or equal to or greater than about 60 wt%, or equal to or greater than about 65 wt%, or equal to or greater than about 70 wt%, or equal to or greater than about 75 wt%, or equal to or greater than about 80 wt%, or equal to or greater than about 85 wt%, or equal to or greater than about 90 wt%, or equal to or greater than about 95 wt% of the compound represented by Formula (I). The compositions described herein may contain, for example, about 100 wt% or less of the compound represented by formula (I), based on the total weight of the compound represented by formula (I), the compound represented by formula (II), the compound represented by formula (III), and the compound represented by formula (IV). For example, the mixture may contain about 99 wt% or less of the compound represented by formula (I), or about 98 wt% or less of the compound represented by formula (III), or about 97 wt% or less of the compound represented by formula (I), based on the total weight of the compound represented by formula (I), the compound represented by formula (II), the compound represented by formula (III), and the compound represented by formula (IV).For example, the compositions described herein may contain from about 50 wt % to about 100 wt %, or from about 60 wt % to about 99 wt %, or from about 70 wt % to about 98 wt %, or from about 80 wt % to about 97 wt %, or from about 90 wt % to about 97 wt % of the compound of formula (I), based on the total weight of the compound of formula (I), the compound of formula (II), the compound of formula (III), and the compound of formula (IV).

[0468] The weight ratio of the compound of Formula (I) to the total weight of the compound of Formula (II), the compound of Formula (III), and the compound of Formula (IV) in the compositions described herein can range, for example, from about 60:40 to about 99: 1. For example, the weight ratio of the compound of Formula (I) to the total weight of the compound of Formula (II), the compound of Formula (III), and the compound of Formula (IV) can range from about 65:35 to about 99: 1, or from about 70:30 to about 99: 1, or from about 75:25 to about 99: 1, or from about 80:20 to about 99: 1, or from about 85:15 to about 99: 1, or from about 90:10 to about 99: 1, or from about 95:5 to about 99: 1. For example, the weight ratio of the compound of Formula (I) to the total weight of the compound of Formula (II), the compound of Formula (III), and the compound of Formula (IV) can range from about 65:35 to about 98:2, or from about 70:30 to about 97:3, or from about 75:25 to about 96:4, or from about 80:20 to about 95:5, or from about 85:15 to about 90:10.

[0469] The weight ratio of the compound of Formula (I) to homofarnesol (e.g., EEH) in the compositions described herein can range, for example, from about 90:10 to about 100:0. For example, the weight ratio of the compound of Formula (I) to homofarnesol (e.g., EEH) in the compositions described herein can range from about 92:8 to about 100:0, or from about 94:6 to about 100:0, or from about 95:5 to about 100:0, or from about 96:4 to about 99.5:0.5, or from about 97:3 to about 99.0:1.0, or from about 98:2 to about 99.0:1.0.

[0470] The amounts of the compound of formula (I), the compound of formula (II), the compound of formula (III), and the compound of formula (IV) in the mixture of stereoisomers may be quantified by, for example, gas chromatography and / or identified by spectroscopy.

[0471] (-)-Ambrox as synthesized by the processes described herein (e.g., using an SHC / HAC enzyme or variant thereof, and optionally, a recombinant host cell) may be, for example, in amorphous or crystalline form.

[0472] (-)-Ambrox produced by the methods described herein (e.g., using an SHC / HAC enzyme or variant thereof, and, optionally, a recombinant host cell) may be isolated by steam extraction / distillation using a water-immiscible solvent or organic solvent extraction (to separate the reaction product and unreacted substrate from the biocatalyst, which remains in the aqueous phase), followed by evaporation of the solvent to obtain the crude reaction product as determined by gas chromatographic (GC) analysis. Methods for steam extraction / distillation and organic solvent extraction are known to those skilled in the art.

[0473] As an example, the resulting (-)-ambrox may be extracted from the total reaction mixture using an organic solvent, such as a water-immiscible solvent (e.g., toluene). Alternatively, the resulting (-)-ambrox may be extracted from the solid-state 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). Alternatively, the resulting (-)-ambrox may be extracted from the solid-state reaction mixture using a mixture of solvents. As a further example, (-)-ambrox may exist in the solid phase as crystals or in amorphous form and may be separated from the remaining solid phase (cellular material or debris) and liquid phase using filtration. As a further example, at temperatures above the melting point of (-)-ambrox (approximately 75°C), (-)-ambrox may form an oily layer on top of the aqueous phase, which may be removed and collected. To ensure complete recovery of (-)-ambrox after the oil layer is removed, an organic solvent may be added to the aqueous phase containing the biomass to extract any remaining (-)-ambrox contained in, on, or around the biomass. The organic layer may be combined with the oil layer before being further processed in its entirety to isolate and purify the (-)-ambrox. The (-)-ambrox may also be selectively crystallized to remove by-products (II), (IV), and (III) and any unreacted homofarnesol substrate from the final (-)-ambrox product. The term "selective crystallization" refers to a processing step in which (-)-ambrox is crystallized from the crystallization solvent to the extent that the isolated crystalline material contains only the (-)-ambrox product (or, if it contains any other compounds (II), (III), or (IV), then these are present only in olfactory-acceptable amounts), while compounds (II), (III), and (IV) remain dissolved in the solvent. The (-)-ambrox may, for example, be free or substantially free of by-products (II), (III), and (IV). The selective crystallization step may use a water-miscible solvent such as ethanol or the like.Selective crystallization of (-)-ambrox can be affected by the presence of unreacted homofarnesol substrate and also by the ratio of (-)-ambrox to other detectable by-products (II), (III), and / or (IV). Even if only 10% conversion of the homofarnesol substrate to (-)-ambrox is obtained, selective crystallization of (-)-ambrox is still feasible.

[0474] The olfactory purity of the final (-)-ambrox product may be determined using a 10% ethanol extract in water or by testing the crystalline material. The final (-)-ambrox product is tested for its olfactory purity, quality, and its sensory profile against a commercially available (-)-ambrox product reference. The (-)-ambrox material is also tested in expert applied studies to determine if the material meets specifications for its organoleptic profile.

[0475] Examples of suitable water-miscible and water-immiscible organic solvents suitable for use in the extraction and / or selective crystallization of (-)-ambrox include, but are not limited to, aliphatic hydrocarbons, preferably having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; halogenated aliphatic hydrocarbons, preferably having 1 or 2 carbon atoms, such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, or tetrachloroethane; aromatic hydrocarbons, such as benzene, 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, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran, or esters, such as ethyl acetate or n-butyl acetate, or ketones, such as methyl isobutyl ketone, or dioxane, or mixtures thereof. Particularly preferred solvents are the above-mentioned heptane, methyl tert-butyl ether (also known as MTBE, tert-butyl methyl ether, tertiary butyl methyl ether, and tBME), diisopropyl ether, tetrahydrofuran, ethyl acetate, and / or mixtures thereof. Preferably, a water-miscible solvent such as ethanol is used for the extraction of (-)-ambrox from the solid-phase reaction mixture. The use of ethanol is advantageous because it is easy to handle, non-toxic, and environmentally friendly.

[0476] The term "isolated," as used herein, refers to a biotransformation product, such as (-)-ambrox, that has been separated or purified from components that accompany the biotransformation product. An entity is "isolated" when produced in a cellular system that is different from the source from which it naturally originates, because the cellular system will not necessarily be free of components that naturally accompany the entity. The degree of isolation or purity can be measured by any appropriate method, such as gas chromatography (GC), HPLC, or NMR analysis.

[0477] In some embodiments, the final product ((-)-ambrox) is isolated and purified to homogeneity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 89.5% pure, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% pure).

[0478] Desirably, the amount of (-)-ambrox produced may be from about 1 mg / L to about 20,000 mg / L (20 g / L), or even more, such as from about 20 g / L to about 200 g / L, or 100-200 g / L, preferably about 125 g / L or 150 g / L or about 188 g / L.

[0479] At least 125 g / L (-)-ambrox may be produced over approximately two days in bioconversion reactions using recombinant E. coli host cells producing SHC / HAC enzymes or enzyme variants. While stirring efficiency may be the only limitation of the system, bioconversions can be performed at 188 g / L EEH or higher if efficient mixing is achieved. Additionally, biocatalysts with improved activity (e.g., in terms of SHC variants with further improved activity or increased SHC enzyme production) may improve or maintain productivity by using less biomass or increased substrate concentrations, which are advantageous in terms of mixing efficiency.

[0480] For example, about 1 to about 100 mg / L, about 30 to about 100 mg / L, about 50 to about 200 mg / L, about 100 to about 500 mg / L, about 100 to about 1,000 mg / L, about 250 to about 5,000 mg / L, about 1,000 (1 g / L) to about 15,000 mg / L (15 g / L), or about 2,000 (2 g / L) to about 10,000 mg / L (10 g / L), or about 2,000 (2 g / L) to about 25,000 mg / L (25 g / L), or about 2,000 (2 g / L) to about 25,000 mg / L (25 g / L), 26,000 mg / L (26 g / L), 27,000 mg / L (27 g / L), 28,000 mg / L (28 g / L), 29,000 mg / L (29 g / L), 30,000 mg / L (30 g / L), 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 125 g / L, 130 g / L, 140 g / L, 150 g / L, 160 g / L, 170 g / L, 180 g / L, 190 g / L, or 200 g / L, or 300 g / L, or 400 g / L, or 500 g / L of (-)-ambrox is produced.

[0481] Preferably, (-)-ambrox at a concentration of at least 100 g / l is produced within a period of 48 to 72 hours. Preferably, (-)-ambrox at a concentration of about 150 g / l is produced within a period of 48 to 72 hours, and preferably, (-)-ambrox at a concentration of about 200 g / l is produced within a period of 48 to 72 hours. Preferably, (-)-ambrox at a concentration of about 250 g / l is produced within a period of 48 to 72 hours.

[0482] The bioconversion of homofarnesol to (-)-ambrox according to the present disclosure produces (-)-ambrox as the primary compound, but may also produce compounds other than (-)-ambrox, which may or may not impart pleasant olfactory notes to the bioconversion mixture and thus may contribute in a positive or negative way to the sensory characteristics of the (-)-ambrox end product. Consequently, sensory analysis is performed using well-established sensory tests utilized by skilled experts (e.g., perfumers) so that the test can help determine whether a chemically related product is also an olfactorily related end product compared to a reference product. Removal of one or more by-product compounds from (-)-ambrox may improve the odor of a mixture of reaction products containing (-)-ambrox, even if the removed compound is actually an odorless compound per se. That is, odor enhancement of (-)-ambrox may be observed in the absence of compounds II, III, and IV.

[0483] Various uses of (-)-Ambrox include, but are not limited to, fine fragrance or consumer products such as fabric care, toiletries, beauty care and cleaning products, detergent products, and soap products, including essentially all products in which currently available Ambrox ingredients are used commercially, including, but not limited to, the following products: Ambrox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlin (Quest), Cetalox Levo (Firmenich), Ambermor (Aromor), and Norambrenolide Ether (Pacific).

[0484] Thus, the use of (-)-ambrox obtained by or obtainable from the processes described herein as part of a fragrance, cosmetic, or consumer product is further provided herein. Also provided herein is a product comprising (-)-ambrox obtained by or obtainable from the processes described herein. The product may be, for example, a fragrance, cosmetic, or consumer product.

[0485] Ambroxide and its uses Further provided herein are reaction products made by the processes described herein.The reaction products may, for example, comprise, consist essentially of, or consist of amberoxide and one or more additional compounds, such as one or more of a compound represented by formula (II), a compound represented by formula (III), and a compound represented by formula (IV).

[0486] As used herein, the term "ambroxide" refers to ambroxide represented by formula (X) below, as well as isomerically pure forms thereof, or the isomers represented by formulas (XI), (XII), and / or (XIII): [ka] The present invention also includes an amber oxide of formula (X) in admixture with one or more molecules of formula (X).

[0487] The nomenclature for the reaction products represented by formulas (X), (XI), (XII), and (XIII) is given below. Table 3. Nomenclature for reaction products represented by formulas (X), (XI), (XII), and (XIII). [Table 4-1]

[0488] [Table 4-2]

[0489] Ambroxide can be produced from (+)-larixol as described in Bolster et al., Tetrahedron, 2002, 58(26), pages 5275-5285. However, it would be desirable to provide an alternative or improved method for producing ambroxide.

[0490] The processes described herein may produce the ambiroxide of formula (X), either alone or in admixture with by-products such as compounds of formula (XI), (XII), and / or (XIII). For example, other stereoisomers of formula (X) may also be produced by the processes described herein.

[0491] Accordingly, provided herein is a compound of formula (X), or a composition comprising a compound of formula (X), obtained by or obtainable by the processes described herein (including all aspects thereof).

[0492] In some embodiments, not all of the bishomofarnesol (e.g., bis-EEH) is converted to amber oxide or a by-product of the reaction. Thus, the compositions described herein, e.g., compositions obtained by or obtainable from the processes described herein, may contain amber oxide (e.g., in addition to bis-EEH, e.g., compounds represented by formula (X) and / or compounds represented by formulas (XI), (XII), and / or (XIII)). Any remaining amber oxide may be separated from the other reaction products so that the amber oxide product does not contain amber oxide. In other embodiments, all of the bishomofarnesol starting material is converted to amber oxide represented by formula (X) or a by-product of the reaction by the processes described herein.

[0493] Thus, the compositions described herein may comprise, consist essentially of, or consist of one or more of a compound represented by formula (X), a compound represented by formula (XI), a compound represented by formula (XII), a compound represented by formula (XIII), a bishomofarnesol starting material (e.g., bisEEH), and other stereoisomers of a compound represented by formula (X). For example, the compositions described herein may comprise, consist essentially of, or consist of a compound represented by formula (X) and one or more of a compound represented by formula (XI), a compound represented by formula (XII), and a compound represented by formula (XIII).

[0494] Thus, the compositions described herein may comprise greater than or equal to about 50 wt % of the compound of formula (X), based on the total weight of the compound of formula (X), the compound of formula (XI), the compound of formula (XII), and the compound of formula (XIII). For example, the compositions described herein may comprise, based on the total weight of the compound represented by Formula (X), the compound represented by Formula (XI), the compound represented by Formula (XII), and the compound represented by Formula (XIII), equal to or greater than about 55 wt%, or equal to or greater than about 60 wt%, or equal to or greater than about 65 wt%, or equal to or greater than about 70 wt%, or equal to or greater than about 75 wt%, or equal to or greater than about 80 wt%, or equal to or greater than about 85 wt%, or equal to or greater than about 90 wt%, or equal to or greater than about 95 wt% of the compound represented by Formula (X). The compositions described herein may contain, for example, about 100 wt% or less of the compound represented by formula (X), based on the total weight of the compound represented by formula (X), the compound represented by formula (XI), the compound represented by formula (XII), and the compound represented by formula (XIII). For example, the mixture may contain about 99 wt% or less of the compound represented by formula (X), or about 98 wt% or less of the compound represented by formula (X), or about 97 wt% or less of the compound represented by formula (X), based on the total weight of the compound represented by formula (X), the compound represented by formula (XI), the compound represented by formula (XII), and the compound represented by formula (XIII).For example, the compositions described herein may contain from about 50 wt % to about 100 wt %, or from about 60 wt % to about 99 wt %, or from about 70 wt % to about 98 wt %, or from about 80 wt % to about 97 wt %, or from about 90 wt % to about 97 wt % of the compound of Formula (X), based on the total weight of the compound of Formula (X), the compound of Formula (XI), the compound of Formula (XII), and the compound of Formula (XIII).

[0495] The weight ratio of the compound of Formula (X) to the total weight of the compound of Formula (XI), the compound of Formula (XII), and the compound of Formula (XIII) in the compositions described herein can range, for example, from about 60:40 to about 99: 1. For example, the weight ratio of the compound of Formula (X) to the total weight of the compound of Formula (XI), the compound of Formula (XII), and the compound of Formula (XIII) can range from about 65:35 to about 99: 1, or from about 70:30 to about 99: 1, or from about 75:25 to about 99: 1, or from about 80:20 to about 99: 1, or from about 85:15 to about 99: 1, or from about 90:10 to about 99: 1, or from about 95:5 to about 99: 1. For example, the weight ratio of the compound of Formula (X) to the total weight of the compound of Formula (XI), the compound of Formula (XII), and the compound of Formula (XIII) can range from about 65:35 to about 98:2, or from about 70:30 to about 97:3, or from about 75:25 to about 96:4, or from about 80:20 to about 95:5, or from about 85:15 to about 90:10.

[0496] The weight ratio of the compound of Formula (X) to bishomofarnesol (e.g., bisEEH) in the compositions described herein can range, for example, from about 90:10 to about 100:0. For example, the weight ratio of the compound of Formula (X) to bishomofarnesol (e.g., bisEEH) in the compositions described herein can range from about 92:8 to about 100:0, or from about 94:6 to about 100:0, or from about 95:5 to about 100:0, or from about 96:4 to about 99.5:0.5, or from about 97:3 to about 99.0:1.0, or from about 98:2 to about 99.0:1.0.

[0497] The amounts of the compound of formula (X), the compound of formula (XI), the compound of formula (XII), and the compound of formula (XIII) in the mixture of stereoisomers may be quantified, for example, by gas chromatography and / or identified by spectroscopy.

[0498] Ambroxide as synthesized by the processes described herein (e.g., using an SHC / HAC enzyme or variant thereof, and optionally, a recombinant host cell) may be, for example, in amorphous or crystalline form.

[0499] Ambroxides produced by the methods described herein (e.g., using an SHC / HAC enzyme or variant thereof, and, optionally, a recombinant host cell) may be isolated by steam extraction / distillation using a water-immiscible solvent or organic solvent extraction (to separate the reaction product and unreacted substrate from the biocatalyst, which remains in the aqueous phase), followed by evaporation of the solvent to obtain a crude reaction product as determined by gas chromatographic (GC) analysis. Methods for steam extraction / distillation and organic solvent extraction are known to those skilled in the art.

[0500] As an example, the resulting ambroxide may be extracted from the total reaction mixture using an organic solvent, such as a water-immiscible solvent (e.g., toluene). Alternatively, the resulting ambroxide 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 ambroxide may exist in the solid phase as a crystal or in an amorphous form and may be separated from the remaining solid phase (cell material or its debris) and liquid phase using filtration.

[0501] As a further example, at temperatures above the melting point of amburoxide, amburoxide may form an oil layer on top of the aqueous phase, which can be removed and collected.To ensure complete recovery of amburoxide after the oil layer is removed, an organic solvent can be added to the aqueous phase containing the biomass to extract any remaining amburoxide contained in, on, or around the biomass.The organic layer can be combined with the oil layer before being further processed in its entirety to isolate and purify amburoxide.Amburoxide can also be selectively crystallized to remove by-products (XI), (XII), and (XIII) and any unreacted bishomofarnesol substrate from the final amburoxide product. The term "selective crystallization" refers to a processing step in which ambroxide is crystallized from the solvent to such an extent that the isolated crystalline material contains only the ambroxide product (or, if it contains any other compounds (X), (XII), or (XIII), these are present only in olfactory-acceptable amounts), while compounds (XI), (XII), and (XIII) remain dissolved in the crystallization solvent. The ambroxide may, for example, be free or substantially free of by-products (XI), (XII), and (XIII). The selective crystallization step may use a water-miscible solvent such as ethanol or the like. The selective crystallization of ambroxide may be affected by the presence of unreacted homofarnesol substrate and by the ratio of ambroxide to other detectable by-products (XI), (XII), and / or (XIII). Even if only 10% conversion of the homofarnesol substrate to amburoxide was obtained, selective crystallization of amburoxide is still feasible.

[0502] The olfactory purity of the final Ambroxide product may be determined using a 10% ethanol extract in water or by testing the crystalline material. The final Ambroxide product is tested for its olfactory purity, quality, and its sensory profile against a commercially available Ambroxide product reference. Ambroxide materials are also tested in expert applied studies to determine if the material meets specifications for its organoleptic profile.

[0503] Examples of suitable water-miscible and water-immiscible organic solvents suitable for use in the extraction and / or selective crystallization of amber oxide include, but are not limited to, aliphatic hydrocarbons, preferably having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; halogenated aliphatic hydrocarbons, preferably having 1 or 2 carbon atoms, such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, or tetrachloroethane; aromatic hydrocarbons, such as benzene, 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, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran, or esters, such as ethyl acetate or n-butyl acetate, or ketones, such as methyl isobutyl ketone, or dioxane, or mixtures thereof. Particularly preferred solvents are the above-mentioned heptane, methyl tert-butyl ether (also known as MTBE, tert-butyl methyl ether, tertiary butyl methyl ether, and tBME), diisopropyl ether, tetrahydrofuran, ethyl acetate, and / or mixtures thereof. Preferably, a water-miscible solvent such as ethanol is used for the extraction of amber oxide from the solid-phase reaction mixture. The use of ethanol is advantageous because it is easy to handle, non-toxic, and environmentally friendly.

[0504] The term "isolated," as used herein, refers to a biotransformation product, such as ambroxide, that has been separated or purified from components that accompany the biotransformation product. An entity is "isolated" when produced in a cellular system different from the source from which it naturally originates, because the cellular system will not necessarily be free of components that naturally accompany the entity. The degree of isolation or purity can be measured by any appropriate method, such as gas chromatography (GC), HPLC, or NMR analysis.

[0505] In some embodiments, the final product (Ambroxide) is isolated and purified to homogeneity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 89.5% pure, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% pure).

[0506] Desirably, the amount of ambroxide produced may be from about 1 mg / L to about 20,000 mg / L (20 g / L), or even more, such as from about 20 g / L to about 200 g / L, or 100-200 g / L, preferably about 125 g / L or 150 g / L or about 188 g / L.

[0507] For example, about 1 to about 100 mg / L, about 30 to about 100 mg / L, about 50 to about 200 mg / L, about 100 to about 500 mg / L, about 100 to about 1,000 mg / L, about 250 to about 5,000 mg / L, about 1,000 (1 g / L) to about 15,000 mg / L (15 g / L), or about 2,000 (2 g / L) to about 10,000 mg / L (10 g / L), or about 2,000 (2 g / L) to about 25,000 mg / L (25 g / L), or about 2,000 (2 g / L) to about 25,000 mg / L (25 g / L), 26,000 mg / L (26 g / L), ), 27,000 mg / L (27 g / L), 28,000 mg / L (28 g / L), 29,000 mg / L (29 g / L), 30,000 mg / L (30 g / L), 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 125 g / L, 130 g / L, 140 g / L, 150 g / L, 160 g / L, 170 g / L, 180 g / L, 190 g / L, or 200 g / L, or 300 g / L, or 400 g / L, or 500 g / L of amber oxide is produced.

[0508] Preferably, ambroxide at a concentration of at least 100 g / l is produced within a period of 48 to 72 hours. Preferably, a concentration of about 150 g / l of ambroxide is produced within a period of 48 to 72 hours. Preferably, a concentration of about 200 g / l of ambroxide is produced within a period of 48 to 72 hours. Preferably, ambroxide at a concentration of about 250 g / l is produced within a period of 48 to 72 hours.

[0509] The bioconversion of bishomofarnesol to ambroxide according to the present disclosure produces (-)-ambrox as the main compound, but compounds other than ambroxide may also be produced, which may or may not impart a pleasant olfactory note to the bioconversion mixture and thus may contribute positively or negatively to the sensory characteristics of the ambroxide end product. Consequently, sensory analysis is performed using well-established sensory tests utilized by skilled experts (e.g., perfumers), so that the test can help determine whether a chemically related product is also an olfactorily related end product compared to a reference product. Removal of one or more by-product compounds from ambroxide can improve the odor of the remaining compound (ambroxide), even if the removed compound is actually an odorless compound itself. That is, the odor enhancement of ambroxide may be observed in the absence of compounds XI, XII, and XIII.

[0510] Various uses of Ambroxide include, but are not limited to, fine fragrance or consumer products such as fabric treatments, toiletries, beauty and cleaning products, detergent products, and soap products, which encompass essentially all products in which currently available Ambroxide ingredients are used commercially.

[0511] Thus, the use of the ambroxide obtained by or obtainable from the process described herein as part of a fragrance or cosmetic or consumer product is further provided herein.A product comprising the ambroxide obtained by or obtainable from the process described herein is also provided herein.The product may be, for example, a fragrance or cosmetic or consumer product.

[0512] Fragrance Composition Further provided herein is the use of the compounds and compositions described herein as or in fragrance compositions. Thus, also provided herein are fragrance compositions comprising one or more compounds of formula (I) or (X). A "fragrance composition" can be, for example, any composition comprising one or more compounds of formula (I) or (X) and a substrate.

[0513] As used herein, "substrate" encompasses all known fragrance ingredients selected from the wide range of natural products and synthetic molecules currently available, such as essential oils, alcohols, aldehydes and ketones, ethers and acetals, esters and lactones, macrocycles and heterocycles, and / or the like, in admixture with one or more ingredients or excipients conventionally used in conjunction with odorants in fragrance compositions (e.g., carrier materials, diluents, and other adjuvants commonly used in the art).

[0514] Fragrance ingredients known in the art are readily available commercially from major fragrance manufacturers. Non-limiting examples of such ingredients include: essential oils and extracts, such as bead onion, costus root oil, oakmoss 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;

[0515] 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™ ((E)-3-methyl-5-(2,2,3-trimethylcyclopenta[4-methyl-2 ... -3-en-1-yl)pent-4-en-2-ol; eugenol (4-allyl-2-methoxyphenol); ethyl linalool ((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); Phenylethyiol (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;

[0516] - 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 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;

[0517] 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]);

[0518] macrocycles, such as ambrettolide ((Z)-oxacycloheptadecan-10-en-2-one); ethylene brassylate (1,4-dioxacycloheptadecan-5,17-dione); and / or Exaltolide® (16-oxacyclohexadecan-1-one); and - heterocycles, e.g., isobutylquinoline (2-isobutylquinoline) Includes.

[0519] As used herein, "carrier material" means a material that is practically neutral from the odorant's point of view, ie, a material that does not significantly modify the odorant's organoleptic properties.

[0520] By "diluent" is intended any diluent 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).

[0521] The term "adjuvant" refers to an ingredient that may be employed in a fragrance composition for reasons not specifically related to the olfactory performance of the composition. For example, an adjuvant, such as an antioxidant adjuvant, may be an ingredient that acts as an aid in processing the fragrance ingredient(s) or compositions containing said ingredient(s), or may improve the handling or storage of the fragrance ingredient(s) or compositions containing 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).

[0522] It may also be an ingredient that provides additional benefits such as imparting color or texture, or that imparts lightfastness or chemical stability to one or more ingredients contained in the fragrance composition.

[0523] It is not possible to give an exhaustive description of the nature and type of adjuvants commonly used in fragrance compositions containing them, but it should be mentioned that said ingredients are well known to those skilled in the art.

[0524] Also provided herein is a consumer product comprising a compound or composition, or a fragrance composition (including any embodiment thereof) as described herein. 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.

[0525] Homofarnesol Homofarnesol may have isomerism as shown below. Table 4. Homofarnesol isomers. [Table 5-1]

[0526] [Table 5-2]

[0527] Beta-farnesene can be converted directly to E,E-homofarnesol (EEH) or indirectly to EEH via E,E-homofarnesate. E,E-homofarnesate is then converted to EEH. Summary information on the production of (-)-ambrox from various substrates can be found in US2012 / 0135477A1, WO 2010 / 139719, US2013.0273619A1, WO 2013 / 156398A1, as well as Seitz PhD thesis (2012, as cited above) and Schaefer 2011 (Chemie Unserer Zeit 45, 374-388), the contents of which are incorporated herein by reference.

[0528] US 2012 / 0135477 A1 reports the conversion of (3Z,7E) to (-)-ambrox according to the present disclosure in Schaefer (2011) (cited above), but using ZmoSHC, where (7E,3Z) is not converted to (-)-ambrox, but only to 9b-epi-ambrox (i.e., compound (III) as outlined above). As used herein, reference to (3Z,7E) homofarnesol is a reference to E,Z-homofarnesol, which is also designated EZH.

[0529] Homofarnesol may be a mixture of four isomers, the (3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E) isomers, although from the literature it appears that (-)-ambrox can only be derived from (3E,7E) homofarnesol (see Neumann and Simon (1986), cited above). As used herein, reference to (3E,7E)-homofarnesol is a reference to E,E-homofarnesol, which is also designated EEH.

[0530] The starting material for the processes described herein for preparing (-)-ambrox may be, for example, (3E,7E)-homofarnesol or a mixture containing (3E,7E)-homofarnesol, e.g., a mixture of homofarnesol isomers containing (3E,7E)-homofarnesol.

[0531] Preferably, the homofarnesol starting material comprises a mixture of (3E,7E) and (3Z,7E), referred to herein as a mixture of EE:EZ isomers. The EE:EZ isomeric mixture of homofarnesol has a CAS number of 35826-67-6. [ka]

[0532] The homofarnesol raw material / starting material may be a mixture of isomers. Consequently, the homofarnesol starting material may also contain a mixture of the four isomers EE:EZ:ZZ:ZE, corresponding to (3E,7E), (3Z,7E), (3Z,7Z), and (3E,7Z). Preferably, the homofarnesol starting material is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)], and / or [(3E,7E) and (3E,7Z)].

[0533] Preferably, the homofarnesol starting material is selected from one or more of the following mixtures: [(3E,7E), (3Z,7E)] and / or [(3Z,7E), (3E / 7E), and (3E,7Z)], which are also designated [EE:EZ] and [EE:EZ:ZE], respectively.

[0534] Consequently, in some embodiments, the ratio of EEH:EZH is about 100:00; 99:01; 98:02; 97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09; 90:10; 89:11; 88:12; 87:13; 86:14; 85:15; 84:16; 3:17;82:18;81:19;80:20;79:21;78:22;77:23;76:24;75:25;74:26;73:27;72:28;71:29;70:30;69:31;68:32;67:33;66:34;65:35;64:36;63:37;62:38: 61:39;60:40;59:41;58:42;57:43;56:44;55:45;54:46;53:47;52:48;51:49;or about 50:50. For example, the ratio of EEH:EZH can range from about 50:50 to about 100:00, or from about 50:50 to about 99:01, or from about 60:40 to about 99:1, or from about 70:30 to about 95:5, or from about 80:20 to about 95:5.

[0535] In some embodiments, preferably the homofarnesol starting material contains >90% E,E-homofarnesol (EEH). In other embodiments, the homofarnesol starting material comprises an EE:EZ weight ratio of 86:14. In some embodiments, the homofarnesol starting material comprises an EE:EZ weight ratio of 80:20. In some embodiments, the homofarnesol starting material comprises a 70:30 EE:EZ weight ratio. In a further embodiment, the homofarnesol starting material comprises an EE:EZ weight ratio of 69:31.

[0536] The number of homofarnesol isomers present may also affect the speed of the reaction. An SHC / HAC enzyme or enzyme variant may be capable of converting E,E-homofarnesol to (-)-ambrox from a complex mixture of homofarnesol isomers (e.g., EE:EZ:ZE:ZZ). However, lower conversion rates may be observed, consistent with the notion that homofarnesol isomers other than EEH may compete with EEH for access to the SHC / HAC derivative enzyme, and thus may act as competitive inhibitors of EEH conversion to (-)-ambrox and / or as alternative substrates (see, e.g., Eichhorn et al. (2018) Adv. Synth. Catal. 360:2339-2351, the contents of which are incorporated herein by reference). Consequently, the homofarnesol substrate may contain an isomeric mixture of two to four isomers, preferably two isomers.

[0537] Consequently, the homofarnesol substrate may consist of, or consist essentially of, an isomeric mixture of two to four isomers, preferably two isomers. Preferably, the homofarnesol substrate comprises a mixture of EE:EZ isomers. Preferably, the homofarnesol substrate consists of or consists essentially of a mixture of EE:EZ isomers.

[0538] For example, if a mixture of EE:EZ isomers is used, then compounds other than (-)-ambrox (e.g., compounds II, III, and IV as set forth in Table 2) are in an "oily" form (rather than a solid form) which facilitates stirring of the reaction mixture and the resulting bioconversion process.

[0539] Bishomofarnesol Bishomofarnesol may have isomerism as shown in Table 5 below. Table 5. Bishomofarnesol isomers. [Table 6]

[0540] Bishomofarnesol may be produced from E-nerolidol as described in the examples below. For example, bishomofarnesol may be produced as a mixture of two or more isomers (e.g., a mixture of E,E-bishomofarnesol and E,Z-bishomofarnesol). Although bishomofarnesol can exist as a mixture of four isomers (the (Z,Z), (E,Z), (Z,E), and (E,E) isomers), ambroxide appears to be derived only from E,E-bishomofarnesol.

[0541] The starting material for the processes described herein for preparing amberoxide may be, for example, E,E-bishomofarnesol, or a mixture comprising E,E-bishomofarnesol, e.g., a mixture of bishomofarnesol isomers comprising E,E-bishomofarnesol.

[0542] Preferably, the bishomofarnesol starting material comprises a mixture of (bisEEH) and (bisEZH), referred to herein as a mixture of EE:EZ isomers. The bishomofarnesol raw material / starting material may be a mixture of isomers. Consequently, the bishomofarnesol starting material may also contain a mixture of the four isomers EE:EZ:ZZ:ZE.

[0543] Consequently, in some embodiments, the ratio of bisEEH:bisEZH is about 100:00; 99:01; 98:02; 97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09; 90:10; 89:11; 88:12; 87:13; 86:14; 85:15; 84:16 ;83:17;82:18;81:19;80:20;79:21;78:22;77:23;76:24;75:25;74:26;73:27;72:28;71:29;70:30;69:31;68:32;67:33;66:34;65:35;64:36;63:37;62:38: 61:39;60:40;59:41;58:42;57:43;56:44;55:45;54:46;53:47;52:48;51:49;or about 50:50. For example, the ratio of bisEEH:bisEZH may range from about 50:50 to about 100:00, or from about 50:50 to about 99:01, or from about 60:40 to about 99:1, or from about 70:30 to about 95:5, or from about 80:20 to about 95:5.

[0544] In some embodiments, preferably the bishomofarnesol starting material contains >90% E,E-bishomofarnesol (EEH). In other embodiments, the bishomofarnesol starting material comprises a bisEEH:bisEZH weight ratio of 86:14. In some embodiments, the bishomofarnesol starting material comprises an 80:20 weight ratio of bisEEH:bisEZH. In some embodiments, the bishomofarnesol starting material comprises a 70:30 weight ratio of bisEEH:bisEZH. In a further embodiment, the bishomofarnesol starting material comprises a 69:31 weight ratio of bisEEH:bisEZH.

[0545] The number of bishomofarnesol isomers present may also affect the speed of the reaction. The SHC / HAC enzyme or enzyme variant may be capable of converting E,E-bishomofarnesol to ambroxide from a complex mixture of bishomofarnesol isomers (e.g., EE, EZ, ZE, ZZ). However, lower conversion rates may be observed, consistent with the notion that bishomofarnesol isomers other than bisEEH may compete with bisEEH for access to the SHC / HAC derivative enzyme, and thus may act as competitive inhibitors and / or alternative substrates for the conversion of bisEEH to ambroxide. Consequently, the bishomofarnesol substrate may contain an isomeric mixture of two to four isomers, preferably two isomers.

[0546] Consequently, the bishomofarnesol substrate may consist of, or consist essentially of, an isomeric mixture of two to four isomers, preferably two isomers. Preferably, the bishomofarnesol substrate comprises a mixture of EE:EZ isomers. Preferably, the bishomofarnesol substrate consists of or consists essentially of a mixture of EE:EZ isomers.

[0547] Nucleic acids and methods for making nucleic acids Further provided herein is a nucleic acid encoding an SHC / HAC enzyme or SHC / HAC enzyme variant as described herein. The nucleic acid may be, for example, an isolated nucleic acid.

[0548] In particular, provided herein are constructs comprising nucleic acid sequences encoding SHC / HAC enzymes or enzyme variants as described herein. As used herein, a "construct" is an artificially created segment of nucleic acid to be transfected into a target cell. The construct may comprise a nucleic acid encoding the SHC / HAC enzyme or enzyme variant and a controller of expression (e.g., a promoter).

[0549] Further provided herein is a vector comprising the construct as described herein.As used herein, "vector" refers to a DNA molecule that is used as a vehicle to artificially transport foreign genetic material into cells, so that it can replicate and / or express in cells.Vector can be, for example, a plasmid, a virus vector, a cosmid, or an artificial chromosome.

[0550] The terms "construct" and "vector" may be overlapping, for example, when the construct is a plasmid. In particular, provided herein are nucleic acids encoding the amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0551] In particular, provided herein are nucleic acids having the sequences of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:22, and SEQ ID NO:23, which may be comprised in, for example, a construct or vector as described herein.

[0552] The term "nucleic acid" or "nucleic acid molecule," as used herein, specifically refers 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, sense and / or antisense strand. The term "nucleic acid" or "nucleic acid molecule," as used herein, specifically applies to a polynucleotide(s), e.g., a full-length nucleotide sequence or a fragment or portion thereof, each of which encodes a polypeptide having an enzymatic activity, e.g., an enzyme of a metabolic pathway, or a fragment or portion thereof.

[0553] The term also encompasses separate molecules, and therefore distinct sequences, such as cDNA (where the corresponding genomic DNA has introns); genomic fragments lacking at least one of the flanking genes; cDNA or genomic DNA fragments 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; DNA encoding non-naturally occurring proteins, such as fusion proteins (e.g., His tags), muteins, or fragments of a given protein; and nucleic acids that are degenerate variants of cDNA or naturally occurring nucleic acids. In addition, it encompasses hybrid genes, i.e., recombinant nucleotide sequences that are part of a gene encoding a non-naturally occurring fusion protein. Fusion proteins can add one or more amino acids (such as, but not limited to, histidine (His)) to a protein, often at the N-terminus, but also at the C-terminus, or can be fused within a protein domain. Such fusion proteins or fusion vectors 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 purification of the recombinant protein by providing an affinity purification ligand.

[0554] The term "nucleic acid" or "nucleic acid molecule" also encompasses codon-optimized sequences suitable for expression in a particular microbial host cell (e.g., an E. coli host cell). As used herein, the term "codon-optimized" refers to a nucleic acid protein-coding sequence that has been adapted for expression in a prokaryotic or eukaryotic host cell, particularly a bacterial host cell such as an E. coli host cell, by substitution of one or more, or preferably a significant number of, codons with codons more frequently used in the genes of the bacterial (e.g., E. coli) host cell.

[0555] In this regard, the nucleotide sequence encoding the reference amino acid sequence (SEQ ID NO: 1 or SEQ ID NO: 10) and variants / derivatives thereof may be the original sequence found in the source (e.g., SEQ ID NO: 1 found in AacSHC), or the gene may be codon-optimized for a selected host organism, such as, for example, E. coli.

[0556] Ribonucleic acid (RNA) molecules can be produced by in vitro transcription. Segments of DNA molecules are also considered within the scope of this disclosure and can be produced, for example, by polymerase chain reaction (PCR) or by treatment with one or more restriction endonucleases. A segment of a nucleic acid molecule may also be referred to as a DNA fragment of a gene, particularly a partial DNA fragment of a gene. A fragment can also contain several open reading frames (ORFs), either repeats of the same ORF or repeats of different ORFs. The term specifically refers to a coding nucleotide sequence, but also encompasses non-coding, e.g., non-transcribed or non-translated, nucleotide sequences, or nucleotide sequences that encode a polypeptide in whole or in part. As used herein, a gene, e.g., a gene for assembly, diversification, or recombination, can be a non-coding sequence, or a polypeptide-encoding sequence or a protein-encoding sequence, or a portion or fragment thereof, having a sequence length sufficient to allow successful recombination events. More specifically, the gene has a length of at least 3 bp, preferably at least 100 bp, and more preferably at least 300 bp. It will be clear from the above that reference to isolated DNA does not refer to DNA present among hundreds to millions of other DNA molecules, e.g., in a cDNA or genomic DNA library or genomic DNA restriction digest, e.g., in a restriction digest reaction mixture or electrophoretic gel slice. The isolated nucleic acid molecules of the present disclosure encompass segments not found in the natural state by themselves.

[0557] As used herein, the term "isolated DNA" can refer to (1) DNA containing a sequence that is not identical to any naturally occurring sequence, nor to a non-naturally occurring polynucleotide, nucleic acid sequence (e.g., created by the artificial combination of two otherwise separated segments of sequence through human intervention (e.g., the artificial manipulation of segments of isolated nucleic acids, e.g., by genetic engineering techniques)), or (2) in the context of DNA with a naturally occurring sequence (e.g., cDNA or genomic DNA), DNA that is free of at least one gene flanking the gene containing the DNA of interest in the genome of the organism in which the gene containing the DNA of interest naturally occurs.

[0558] The term "isolated DNA," as used herein, specifically with respect to nucleic acid sequences, may also refer to nucleic acids or polynucleotides produced by recombinant DNA techniques, e.g., DNA constructs containing polynucleotides heterologous to a host cell (which are optionally integrated into the host cell). Chimeric nucleotide sequences may specifically be produced as recombinant molecules. The term "recombinant" specifically applies to the assembly of polynucleotides in which polynucleotides or portions thereof are joined together, with or without recombination to achieve crossover or genetic mosaicism. For example, joining nucleic acid segments of desired functions together is performed to generate a desired combination of functions. A recombinant gene encoding a polypeptide described herein may include a sequence encoding the polypeptide operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from polycistronic mRNAs, multiple polypeptides can be expressed in those microorganisms under the control of a single regulatory region, if desired. A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective to regulate the transcription or translation of the sequence.

[0559] The term "recombinant," as used herein, with particular reference to enzymes, refers to enzymes produced by recombinant DNA techniques, i.e., enzymes produced from cells transformed with an exogenous DNA construct en...

Claims

1. 1. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme variant; wherein the SHC / HAC enzyme variant has at least about 70.0% identity to SEQ ID NO:1; wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications, relative to SEQ ID NO:1, at positions corresponding to positions 132, 224, and 432 of SEQ ID NO:1, which are M132R, A224V, and I432T, respectively; and wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, compared to SEQ ID NO:1, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to position 81, 431, or 613 of SEQ ID NO:

1.

2. 1. A process for preparing amburoxide or a mixture containing amburoxide, the process comprising enzymatically converting (E,E)-bishomofarnesol (bisEEH) or a mixture of bishomofarnesol isomers containing bisEEH using an SHC / HAC enzyme variant to amburoxide or a mixture containing amburoxide; wherein the SHC / HAC enzyme variant has at least about 70.0% identity to SEQ ID NO:1; wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications, relative to SEQ ID NO:1, at positions corresponding to positions 132, 224, and 432 of SEQ ID NO:1, which are M132R, A224V, and I432T, respectively; and wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, compared to SEQ ID NO:1, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to position 81, 431, or 613 of SEQ ID NO:

1.

3. 1. A SHC / HAC enzyme variant having an amino acid sequence having at least about 70.0% identity to SEQ ID NO:1, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications, relative to SEQ ID NO:1, at positions corresponding to positions 132, 224, and 432 of SEQ ID NO:1, which are M132R, A224V, and I432T, respectively; and wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification, compared to SEQ ID NO:1, at a position corresponding to position 557 of SEQ ID NO:1 and at least one position corresponding to position 81, 431, or 613 of SEQ ID NO:

1.

4. 4. The process of claim 1 or 2 or the SHC / HAC enzyme variant of claim 3, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least about 90.0% identity to SEQ ID NO:

1.

5. 5. The process or SHC / HAC enzyme variant of any one of claims 1 to 4, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications compared to SEQ ID NO: 1 at positions corresponding to positions 557 and 431 of SEQ ID NO:

1.

6. 6. The process or SHC / HAC enzyme variant of any one of claims 1 to 5, wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications compared to SEQ ID NO: 1 at positions corresponding to positions 557 and 613 of SEQ ID NO:

1.

7. 7. The process or SHC / HAC enzyme variant of claim 5 or 6, wherein the amino acid sequence of the SHC / HAC enzyme variant further comprises an amino acid modification, compared to SEQ ID NO: 1, at a position corresponding to position 81 of SEQ ID NO:

1.

8. 8. The process or SHC / HAC enzyme variant of any one of claims 1 to 7, wherein one or more of the amino acid modifications at positions 81, 431, 557, or 613 is a substitution, such as a non-conservative substitution.

9. the amino acid modification at the position corresponding to position 557 of SEQ ID NO: 1 replaces that amino acid in SEQ ID NO: 1 with a neutral hydrophilic amino acid, e.g., threonine; and / or the amino acid modification at the position corresponding to position 81 of SEQ ID NO:1 is a substitution of that amino acid in SEQ ID NO:1 with a basic amino acid, e.g., histidine; and / or the amino acid modification at the position corresponding to position 431 of SEQ ID NO: 1 is a substitution of that amino acid in SEQ ID NO: 1 with a hydrophobic amino acid, e.g., leucine; and / or the amino acid modification at a position corresponding to position 613 of SEQ ID NO: 1 is a substitution of that amino acid in SEQ ID NO: 1 with a neutral hydrophilic amino acid, e.g., serine; 9. The process or SHC / HAC enzyme variant according to any one of claims 1 to 8.

10. 10. The process or SHC / HAC enzyme variant of any one of claims 1 to 9, wherein the SHC / HAC enzyme variant has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 (e.g., an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5).

11. 11. The process of any one of claims 1 to 10, wherein the enzymatic conversion occurs at a temperature ranging from about 30°C to about 50°C (e.g., from about 40° to about 50°C) and / or at a pH ranging from about 5 to about 6.

12. 12. The process of any one of claims 1 to 11, wherein the process comprises culturing a recombinant host cell that produces the SHC / HAC enzyme variant.

13. The process of claim 12, wherein the recombinant host cell comprises a nucleic acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 (e.g., a nucleic acid sequence selected from SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9).

14. (-)-Ambrox gives by-products (II), (III), and (IV), 【Chemistry 1】 The process according to any one of claims 1 to 13, wherein the product is produced in admixture with at least one of:

15. 15. The process or SHC / HAC enzyme variant of any one of claims 1 to 14, wherein the SHC / HAC enzyme variant has increased enzymatic activity compared to SEQ ID NO:

1.

16. 16. The process or SHC / HAC enzyme variant of any one of claims 1 to 15, wherein the SHC / HAC enzyme variant provides an increased EEH conversion rate over the first 6 hours, or over the first 8 hours, or over the first 12 hours of the reaction compared to SEQ ID NO:1 and / or SEQ ID NO:

10.

17. (-)-Ambrox obtained or obtainable by a process according to any one of claims 1 to 16, for example in amorphous or crystalline form.

18. 18. Use of (-)-ambrox according to claim 17 as part of a fragrance or cosmetic or consumer product, such as a fabric treatment, toiletry product, beauty product, cleaning product, detergent product, and / or soap product.

19. A fragrance or cosmetic or consumer product comprising (-)-ambrox according to claim 17.

20. A nucleic acid sequence encoding the SHC / HAC enzyme variant of any one of claims 1 to 19.

21. 21. The nucleic acid sequence of claim 20, wherein the nucleic acid sequence is selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 (e.g., the nucleic acid sequence is selected from SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9).

22. A construct comprising the nucleic acid sequence of claim 20 or 21.

23. A vector comprising the construct of claim 22.

24. 24. A recombinant host cell comprising a nucleic acid sequence according to claim 20 or 21, a construct according to claim 22, or a vector according to claim 23.

25. 25. The recombinant host cell of claim 24, wherein the recombinant host cell is a bacterium having a genus selected from Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus (e.g., the recombinant host cell is E. coli).

26. 17. The process of any one of claims 1 to 16, wherein the mixture of isomers comprising EEH is an EE:EZ isomer mixture.

27. the homofarnesol EE:EZ isomer mixture is in a weight ratio selected from the group consisting of: EE:EZ 90:10; EE:EZ 80:20; EE:EZ 86:14; EE:EZ 70:30; EE:EZ 69:31; and EE:EZ 66:34; or 27. The process of claim 26, wherein the homofarnesol EE:EZ isomer mixture is in a weight ratio of 90:10 EE:EZ; 80:20 EE:EZ; 86:14 EE:EZ; 70:30 EE:EZ; 69:31 EE:EZ; or 66:34 EE:EZ.

28. 28. The process of claim 27, wherein the EE:EZ isomer mixture is in a weight ratio of 80:

20.

29. 29. The process of any one of claims 1-16 or 26-28, wherein the weight ratio of SHC / HAC biocatalyst to EEH or a mixture of homofarnesol isomers containing EEH is in the range of about 0.5-2:1, or about 0.25-2:1, or about 0.1-2:1, or about 1:1, or about 0.5:

1.

30. 30. The process of claim 29, wherein the weight ratio of SHC / HAC biocatalyst to EEH or the mixture of homofarnesol isomers containing EEH in the 80:20 weight ratio EE:EZ isomer mixture is about 1:1, or about 0.5:1, or about 0.1:1.

Images