Squalene hopene cyclase (SHC) variant

Abstract

Squalene hopene cyclase (SHC) enzyme and its variants and their use in the preparation of (-)-ambromide from bis- ...

Description

[0001] This application is a divisional application, with its parent application having application number 202080094930.1, an application date of December 3, 2020, and an invention titled "Squalene Hobene Cycloylase (SHC) Variant". Technical Field

[0002] This invention generally relates to SHC / HAC enzymes and their variants. It also relates to various uses of SHC / HAC enzymes and their variants, such as the enzymatic conversion of (3E,7E)-homofarnesol (EEH) to (-)-ambrox or the enzymatic conversion of E,E-bisomofarnesol (BisEEH) to ambra oxide. Furthermore, this invention relates to the products of the enzymatic reactions, such as (-)-ambrox or ambra oxide prepared using SHC / HAC enzymes and their variants, and various uses of said products. Background Technology

[0003] Squalene-hope cyclase (SHC) is a membrane-bound enzyme that acts as a biocatalyst to cyclize linear triterpenoid squalene into hope and hope alcohol.

[0004] Numerous wild-type and variant SHC enzymes from various bacteria have been shown to be useful for converting (3E,7E)-mefenoxuron to (-)-ambroxol (see, for example, 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; WO2010 / 139719; US 8759043; WO 2012 / 066059; Seitz et al. 2012, J Molecular Catalysis B: Enzymatic 84, 72-77; and Seitz et al. 2012, J Molecular Catalysis B: Enzymatic 84, 72-77; and Seitz et al. 2012). 2012 Doctoral Dissertation ( http: / / elib.uni-stuttgart.de / handle / 1 1682 / 1400 (The contents of which are incorporated herein by reference). New and improved methods for preparing (-)-ambroxan are needed, for example, using novel SHC enzymes or enzyme variants. New and improved methods for cyclizing other substrates are also desired, for example, to form compounds that can be used or used as fragrances. Summary of the Invention

[0005] According to a first aspect of the invention, a method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol is provided, the method comprising enzymatically converting a mixture of (3E,7E)-mifetalol (EEH) or mifetalol isomers containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol isomerics using an SHC / HAC enzyme variant.

[0006] The SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% identical to SEQ ID NO: 1.

[0007] The SHC / HAC enzyme variants have amino acid changes relative to SEQ ID NO: 1 at positions 132, 224, and 432, corresponding to positions M132R, A224V, and I432T, respectively.

[0008] The SHC / HAC enzyme variant has an amino acid sequence change relative to SEQ ID NO: 1 at position 557 corresponding to SEQ ID NO: 1 and at least one position corresponding to position 81, 431 or 613 corresponding to SEQ ID NO: 1.

[0009] According to a second aspect of the invention, (-)-ambroxanone, which is obtained by or can be obtained by the method of the first aspect of the invention, is provided, including any embodiment thereof.

[0010] According to a third aspect of the invention, the use of (-)-ambroxan (including any embodiment thereof) of the second aspect of the invention is provided as part of a fragrance, cosmetic or consumer product.

[0011] According to a fourth aspect of the invention, fragrances, cosmetics, or consumer products comprising (-)-ambroxol (including any embodiment thereof) of the second aspect of the invention are provided.

[0012] According to a fifth aspect of the invention, an SHC / HAC enzyme variant is provided, the amino acid sequence of which has at least about 70.0% identity with SEQ ID NO: 1.

[0013] The SHC / HAC enzyme variants have amino acid changes relative to SEQ ID NO: 1 at positions 132, 224, and 432, corresponding to positions M132R, A224V, and I432T, respectively.

[0014] The SHC / HAC enzyme variant has an amino acid sequence change relative to SEQ ID NO: 1 at position 557 corresponding to SEQ ID NO: 1 and at least one position corresponding to position 81, 431 or 613 corresponding to SEQ ID NO: 1.

[0015] According to a sixth aspect of the invention, a nucleic acid sequence encoding an SHC / HAC enzyme variant of the fifth aspect of the invention is provided, including any embodiment thereof.

[0016] According to a seventh aspect of the invention, a construct comprising the nucleic acid sequence of the sixth aspect of the invention is provided, including any embodiment thereof.

[0017] According to an eighth aspect of the invention, a carrier comprising an embodiment of the seventh aspect of the invention is provided, including any embodiment thereof.

[0018] According to a ninth aspect of the invention, a recombinant host cell comprising the nucleic acid sequence of the sixth aspect of the invention, the construct of the seventh aspect of the invention, or the vector of the eighth aspect of the invention, including any embodiment thereof, is provided.

[0019] According to a tenth aspect of the invention, a method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol is provided, the method comprising enzymatically converting a mixture of (3E,7E)-mifetalol (EEH) or mifetalol isomers containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol isomers using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least about 70.0% identity with the amino acid sequence of the wild-type SHC / HAC enzyme, and wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 557, 81, 431 and 613 corresponding to SEQ ID NO: 1.

[0020] According to an eleventh aspect of the invention, an SHC / HAC enzyme variant is provided, 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 the wild-type SHC / HAC enzyme, and wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 557, 81, 431 and 613 corresponding to SEQ ID NO: 1.

[0021] According to a twelfth aspect of the invention, a method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol is provided, the method comprising enzymatically converting a mixture of (3E,7E)-mifetalol (EEH) or mifetalol isomers containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol is contained in an SHC / HAC enzyme or an SHC / HAC enzyme variant thereof, wherein the SHC / HAC enzyme or the SHC / HAC enzyme variant has an amino acid sequence having at least about 70.0% identity with the amino acid sequence of the wild-type SHC / HAC enzyme.

[0022] According to a thirteenth aspect of the invention, an SHC / HAC enzyme or a variant of the SHC / HAC enzyme is provided, wherein the SHC / HAC enzyme or the variant of the SHC / HAC enzyme has an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of the wild-type SHC / HAC enzyme.

[0023] According to a fourteenth aspect of the invention, a method for preparing ambergris oxide is provided, the method comprising enzymatically converting E,E-bisimilar farnesol or a mixture of bisimilar farnesol isomers containing E,E-bisimilar farnesol to ambergris oxide or a mixture containing ambergris oxide using an SHC / HAC enzyme or an SHC / HAC enzyme variant thereof. For example, the SHC / HAC enzyme or an SHC / HAC enzyme variant may be used according to any aspect of the invention.

[0024] According to the fifteenth aspect of the invention, ambergris oxides are provided that are obtained by the method of the fourteenth aspect of the invention, or that can be obtained by the method of the fourteenth aspect of the invention, including any embodiments thereof.

[0025] According to the sixteenth aspect of the invention, the use of ambergris oxide (including any embodiment thereof) of the fifteenth aspect of the invention is provided as part of a fragrance, cosmetic, or consumer product.

[0026] According to the seventeenth aspect of the invention, a fragrance, cosmetic, or consumer product comprising ambergris oxide of the fifteenth aspect of the invention is provided, including any embodiment thereof.

[0027] 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.

[0028] In certain 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.

[0029] Certain embodiments of the present invention may provide one or more of the following advantages:

[0030] Improved EEH conversion rate, especially in the first 12 hours or the first 6 hours;

[0031] Cycling of new substrates and / or identification of new products;

[0032] Increased productivity (g / L / hour or g / L / hour / g biocatalyst);

[0033] An industrial-scale method for preparing final products such as (-)-ambroside and ambroxol oxide and / or mixtures of its isomers;

[0034] Compared with other isomers of cyclohexane, it exhibits improved selectivity for EEH.

[0035] Details, examples, and preferences provided relating to any particular aspect or one of the described aspects of the invention will be further described herein and are equally applicable to all aspects of the invention. Unless otherwise stated herein or clearly contradicted by the context, any combination of all possible variations of the embodiments, examples, and preferences described herein is included in the invention. Attached Figure Description

[0036] Figure 1 The relative activities of the SHC variants under initial and optimized reaction conditions are shown. Reactions were carried out at 4 g / L EEH under initial conditions (35°C, pH 5.4, 0.070% SDS) or at T, pH, and [SDS] set as optimal for each variant (optimized conditions), with the biocatalyst loaded to 10.0 OD. 650nm .

[0037] Figure 2 The relative activities of the SHC variant compared to the parental 215G2 SHC enzyme were shown under optimized conditions. The reaction was carried out at 4 g / L EEH, with the biocatalyst loaded to an OD of 10.0. 650nm T, pH, and [SDS] are set to conditions defined as optimal for each variant.

[0038] Figure 3 The conversion of EEH to (-)-ambroxol via SHC / HAC enzyme variants was demonstrated under optimal conditions compared to the 215G2 parental SHC / HAC enzyme. Reactions were carried out at 125 g / L EEH and 250 g / L biocatalyst, in the presence of 1.3% SDS, and at T and pH conditions defined as optimal for each variant.

[0039] Figure 4 The relative improvement in EEH conversion using the optimal SHC / HAC enzyme variant is shown compared to the reference 215G2 SHC / HAC parent enzyme. Reactions were carried out at 125 g / L EEH and 250 g / L biocatalyst, in the presence of 1.3% SDS, and at T and pH conditions defined as optimal for each variant. At all time points, the EEH conversion obtained using 215G2 SHC was set as 100% (reference).

[0040] Figure 5 The relative activities of the SHC / HAC variant compared to the parental (reference) 215G2 SHC / HAC enzyme were shown under optimal conditions. The reaction was carried out at 4 g / L BisEEH with the biocatalyst loaded to an OD of 10.0. 650nm T, pH, and [SDS] are set to conditions defined as optimal for each variant.

[0041] Figure 6 The bihomogeneous cyclization of farnesol using parental (reference) 215G2 SHC / HAC enzyme and novel variant SHC / HAC enzyme is shown. The reaction was carried out at 125 g / L BisEEH and 250 g / L biocatalyst, in the presence of 1.3% SDS, and at 35°C and pH 5.4 (all SHC enzymes).

[0042] Figure 7 Amino acid sequence alignments are shown 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).

[0043] Figure 8 The amino acid sequence alignment is shown 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.

[0044] Figure 9A The amino acid sequence alignments using CLUSTAL O (1.2.4) are shown 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). Amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted with white letters on a black background.

[0045] Figure 9B The amino acid sequence alignments using CLUSTAL O (1.2.4) are shown 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). Amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted with white letters on a black background.

[0046] Figure 10 It is a reaction scheme used to produce a starting mixture of E,E / Z-bihomonol.

[0047] Figure 11 This is a schematic diagram illustrating the cyclization reaction of bisimilar farnesol using the SHC enzyme. The bisimilar farnesol used consists of a mixture of isomers.

[0048] Figure 12 The conversion of EEH to (-)-ambroxol via SHC / HAC enzyme variants was demonstrated under optimal conditions compared to the 215G2 parental SHC / HAC enzyme. Reactions were carried out at 125 g / L EEH and 250 g / L biocatalyst, in the presence of 1.3% SDS, and at T and pH conditions defined as optimal for each variant.

[0049] Figure 13 The conversion of EEH to (-)-ambroxol via SHC / HAC enzyme variants was demonstrated under optimal conditions compared to the 215G2 parental SHC / HAC enzyme. Reactions were carried out at 125 g / L EEH and 125 g / L biocatalyst, in the presence of 0.65% SDS, and at T and pH conditions defined as optimal for each variant.

[0050] Figure 14 The conversion of EEH to (-)-ambroxol via the SHC / HAC enzyme variant SHC#65 under optimal conditions was demonstrated compared to the 215G2 parental SHC / HAC enzyme. Reactions were carried out at 125 g / L to 300 g / L EEH and 250 g / L biocatalyst or a [EEH]:[cell] ratio of 1. SDS was provided at a constant [SDS]:[cell] ratio of 0.052. Reactions were carried out under T and pH conditions defined as optimal for the SHC variant SHC#65.

[0051] Figure 15 The conversion of EEH to (-)-ambroxol was demonstrated 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). All listed wild-type SHC enzymes were reacted at 4 g / L EEH, and all other listed wild-type SHC enzymes except for 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) were reacted at 125 g / L EEH. Reactions were carried out at T and pH conditions and SDS concentrations defined as optimal for each listed wild-type SHC enzyme. The SDS concentration was adjusted in the 125 g / L EEH biotransformation to allow for optimal biocatalyst activity.

[0052] Figure 16The conversion of EEH to (-)-ambroxol via SHC / HAC is shown. Biotransformation was carried out using SHC#65 biocatalyst. The reaction was conducted at 125 g / L EEH using 150 g / L cells, at 45°C and pH 5.6, and in the presence of 0.060 to 0.090% SDS corresponding to an [SDS]:[cell] ratio of 0.040 to 0.060. The EEH conversion values ​​are shown at 72 hours of reaction.

[0053] Sequence Overview

[0054] SEQ ID NO: 1 is wild-type Cyclopyralid (a type of bacteria). Alicyclobacillus acidocaldarius )(Aac)SHC amino acid sequence.

[0055] SEQ ID NO:2 corresponds to SEQ ID NO:1 having substitutions for M132R, A224V, I432T, A557T, and H431L, and may be referred to herein as SHC / HAC enzyme variant #49.

[0056] SEQ ID NO:3 corresponds to SEQ ID NO:1 having substitutions for M132R, A224V, I432T, A557T and R613S, and may be referred to herein as SHC / HAC enzyme variant #65.

[0057] SEQ ID NO:4 corresponds to SEQ ID NO:1 having the substitutions M132R, A224V, I432T, Y81H, A557T and R613S, and may be referred to herein as SHC / HAC enzyme variant #66.

[0058] SEQ ID NO: 5 corresponds to SEQ ID NO: 1 having substitutions M132R, A224V, I432T, Y81H, H431L and A557T, and may be referred to herein as SHC / HAC enzyme variant #110B8.

[0059] SEQ ID NO: 6 is the nucleic acid sequence encoding the polypeptide (SHC / HAC enzyme variant #49) of SEQ ID NO: 2.

[0060] SEQ ID NO: 7 is the nucleic acid sequence encoding the polypeptide (SHC / HAC enzyme variant #65) of SEQ ID NO: 3.

[0061] SEQ ID NO: 8 is the nucleic acid sequence encoding the polypeptide (SHC / HAC enzyme variant #66) of SEQ ID NO: 4.

[0062] SEQ ID NO: 9 is the nucleic acid sequence encoding the polypeptide (SHC / HAC enzyme variant #110B8) of SEQ ID NO: 5.

[0063] SEQ ID NO: 10 can be called 215G2 and corresponds to the wild-type AacSHC amino acid sequence with mutations M132R, A224V and I432T.

[0064] SEQ ID NO: 11 is the wild-type amino acid sequence of ZmoSHC1.

[0065] SEQ ID NO: 12 is the wild-type amino acid sequence of ZmoSHC2.

[0066] SEQ ID NO: 13 is the wild-type amino acid sequence of BjpSHC / BjaSHC.

[0067] SEQ ID NO: 14 is the wild-type amino acid sequence of GmoSHC.

[0068] SEQ ID NO: 15 is the nucleotide sequence encoding wild-type AacSHC.

[0069] SEQ ID NO: 16 is the nucleotide sequence encoding 215G2 SHC.

[0070] SEQ ID NO: 17 corresponds to SEQ ID NO: 1 having substitutions for M132R, A224V, I432T, T90A and R613S, and may be referred to herein as SHC / HAC enzyme variant #90C7.

[0071] SEQ ID NO: 18 corresponds to SEQ ID NO: 1 having substitutions for M132R, A224V, I432T, A172T and M277K, and may be referred to herein as SHC / HAC enzyme variant #115A7.

[0072] SEQ ID NO: 19 is the wild-type amino acid sequence of TelSHC.

[0073] SEQ ID NO: 20 is the wild-type amino acid sequence of ApaSHC1.

[0074] SEQ ID NO: 21 is a GmoSHC variant.

[0075] SEQ ID NO: 22 is the nucleotide sequence encoding the polypeptide (SHC / HAC enzyme variant #90C7) of SEQ ID NO: 17.

[0076] SEQ ID NO: 23 is the nucleotide sequence encoding the polypeptide (SHC / HAC enzyme variant #115A7) of SEQ ID NO: 18.

[0077] SEQ ID NO: 24 is the amino acid sequence of the SHC / HAC variant 215G2 SHC with the additional mutation L37Q.

[0078] SEQ ID NO: 25 is the amino acid sequence of the SHC / HAC variant 215G2 SHC with the additional mutation V174I.

[0079] SEQ ID NO: 26 is the amino acid sequence of the SHC / HAC variant 215G2 SHC with additional mutations V174I and F601Y.

[0080] SEQ ID NO: 27 is the amino acid sequence of the SHC / HAC variant 215G2 SHC with additional mutations L37Q, V174I and F601Y.

[0081] SEQ ID NO: 28 is the wild-type amino acid sequence of BmeSHC.

[0082] SEQ ID NO: 29 is the wild-type amino acid sequence of SalSHC.

[0083] SEQ ID NO: 30 is the wild-type amino acid sequence of ApaSHCA. Summary of the Invention

[0084] SHC / HAC enzymes and their variants

[0085] As used herein, the term "SHC enzyme" refers to wild-type (WT) squalene-hope cyclases naturally found in, for example, thermophilic bacteria such as *Acidobacterium oxyphylla*. SHCs that function in the cyclization of cyclohexane to ambroxol can also be called cyclohexane-ambroxol cyclases (HACs). Therefore, the term "SHC / HAC enzyme" may be used herein.

[0086] As used herein, the term "variant" should be understood as a polypeptide that differs from the polypeptide from which it is derived by one or more variations in its amino acid sequence. A polypeptide from which a variant is derived is also called a parent or reference polypeptide. Typically, variants are artificially constructed, preferably by genetic technology. Typically, the polypeptide from which a variant is derived is a wild-type protein or a wild-type protein domain. However, variants that can be used in this 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. Variations in the amino acid sequence can be amino acid exchanges (substitutions), insertions, deletions, N-terminal truncation, or C-terminal truncation, or any combination of these variations, which may occur at one or more sites.

[0087] As used herein, the term "SHC / HAC enzyme variant" refers to an enzyme derived from a wild-type SHC enzyme but with one or more amino acid alterations compared to the wild-type SHC enzyme and therefore not naturally occurring in prokaryotes. One or more amino acid alterations can, for example, alter (e.g., increase) the enzyme activity of a substrate (e.g., EEH).

[0088] 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 SHC / HAC enzymes and / or SHC / HAC enzyme variants can be determined by incubating purified SHC / HAC enzymes or enzyme variants, or extracts from host cells or intact recombinant host organisms that produce SHC / HAC enzymes or enzyme variants, with a suitable substrate under appropriate conditions, and analyzing the reaction products (e.g., by gas chromatography (GC) or HPLC). Further details regarding the assays of SHC / HAC enzyme and / or SHC / HAC enzyme variant activity and the analysis of reaction products are provided in the examples. These assays involve the production of SHC / HAC enzyme variants in recombinant host cells (e.g., *Escherichia coli*).

[0089] 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 in so-called activity tests used to monitor the formation of the desired product. The SHC / HAC enzyme derivatives of this disclosure are characterized by their ability to cyclize bisimifarnesol (e.g., EEH) to (-)-ambroside and exhibit biological activities, such as HAC activity. The SHC / HAC enzyme derivatives of this disclosure are also characterized by their ability to cyclize bimifarnesol (e.g., E,E-bisimifarnesol) to ambergris oxide.

[0090] As used herein, “bioactivity” means 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, particularly to a receptor, or to a nucleic acid); inhibitory activity (e.g., enzyme inhibitory activity); activating activity (e.g., enzyme activating activity); or toxicity. It is not required that the variant exhibit the same degree of such activity as the parent or wild-type polypeptide. A variant is considered a variant in the context of this application if it exhibits relevant activity to the extent that it is at least 10% of the parent polypeptide activity. Similarly, a variant is considered a variant in the context of this application if it exhibits relevant bioactivity to the extent that it is at least 10% of the parent polypeptide activity (since the terms derivative and variant are used interchangeably throughout this disclosure). In other embodiments, the SHC / HAC enzyme variants of this disclosure exhibit better yields than reference SHC proteins (e.g., wild-type SHC / HAC enzymes or known SHC / HAC enzyme variants). The term “yield” refers to the number of grams of recyclable product per gram of feedstock (which can be calculated as a percentage of molar conversion). In another embodiment, the SHC / HAC enzyme variants of this disclosure exhibit altered (e.g., increased) productivity relative to a reference SHC protein (e.g., wild-type AacSHC or 215G2 AacSHC). The term "productivity" refers to the amount of recyclable product in grams per liter of reaction capacity per hour of bioconversion time (i.e., time after substrate addition). The term "productivity" also refers to the amount of recyclable product in grams per liter of reaction capacity per hour of bioconversion time (i.e., time after substrate addition) per gram of biocatalyst used in the reaction.

[0091] In a further embodiment, the SHC / HAC enzyme variant of this disclosure shows improved 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 product concentration obtained in the reaction medium and the concentration of the SHC / HAC variant enzyme (e.g., a purified SHC / HAC enzyme variant or an extract from a recombinant host cell that produces the SHC / HAC enzyme variant). In various embodiments, the SHC / HAC enzyme variant of this disclosure exhibits a modified (e.g., increased) fold of enzyme activity (e.g., modified / increased simifanol-ambroxol cyclase (HAC) activity) relative 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 can 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 times.

[0092] As used herein, the term "amino acid alteration" 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 different amino acids in the amino acid sequence relative to a reference amino acid sequence (which may be conserved or non-conserved). Substitution replaces the amino acids in the reference sequence with the same number of amino acids in the variant sequence. For example, the reference amino acid sequence may be 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, for example, may be an SHC / HAC enzyme variant sequence (e.g., Aac 215G2 variant – SEQ ID NO: 10).

[0093] Amino acid alterations can be easily identified by comparing the amino acid sequence of the SHC / HAC enzyme variant with that of a reference amino acid sequence.

[0094] For example, conserved amino acid substitutions can be performed based on the similarity of the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the amino acid residues involved. The aforementioned 20 naturally occurring amino acids can be divided into the following six standard amino acid groups:

[0095] (1) Hydrophobicity: Met, Ala, Val, Leu, Ile;

[0096] (2) Neutral hydrophilicity: Cys, Ser, Thr; Asn, Gln;

[0097] (3) Acidity: Asp, Glu;

[0098] (4) Alkaline: His, Lys, Arg;

[0099] (5) Residues affecting chain orientation: Gly, Pro; and

[0100] (6) Aromatics: Trp, Tyr, Phe.

[0101] Therefore, as used herein, the term "conservative substitution" refers to the substitution of an amino acid by another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the exchange of Glu for Asp retains a negative charge in such a modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt the α-helix. Some preferred conserved substitutions in the six groups mentioned above are exchanges in the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (iii) 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 readily construct DNA encoding conserved amino acid variants.

[0102] As used herein, “non-conservative substitution” or “non-conservative amino acid exchange” is defined as the exchange of an amino acid for another amino acid listed in one of the different groups of the six standard amino acid groups (1) to (6) shown 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 single-letter amino acid symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee are shown below. Three-letter codes are also provided for reference.

[0103]

[0104] Amino acid alterations can be introduced using known protocols of recombinant gene technology, such as amino acid substitution, including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in vitro transcription. These alterations can be used to introduce these changes into a reference sequence, generating SHC / HAC enzyme variants. The SHC / HAC functional activity of the enzyme variants can then be screened.

[0105] Suitable sources of SHC / HAC enzymes include, for example, *Acidithiobacillus thermophilus* (Aac) and *Morphospira hygroscopica* (Morphospira hygroscopica). Zymomonas mobilis )(Zmo), Bradyrhizobium japonicum ( Bradyrhizobium japonicum (Bjp), Glucosamine mobius ( Gluconobacter morbifer (Gmo), Burkholderia bifidum ( Burkholderia ambifaria Anthrax ( Bacillus anthracis ), capsular methylcoccus ( Methylococcus capsulatus ), Franklinella ( Frankia alni ), Pasteurella multocida ( Acetobacter pasteurianus (Apa), Slender thermophilic Synechococcus ( Thermosynechococcus elongatus (Tel), Sky Blue Streptomyces ( Streptomyces coelicolor (Sco), Rhodopseudomonas palustris ( Rhodopseudomonas palustris(Rpa), Terediella ( Teredinibacter turnerae (Ttu), Bacillus anthracis ( Pelobacter carbinolicus (Pca), Bacillus megaterium ( Bacillus megaterium (Bme), Streptomyces albopictus ( Streptomyces albolongus (Sal) and Tetrahymena piriformis ( Tetrahymena pyriformis (See, for example, WO2010 / 139719, US2012 / 01345477, WO2012 / 066059, the contents of which are incorporated herein by reference).

[0106] Specifically, the SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) can be *Aac* SHC / HAC enzyme, *ZmoSHC1* SHC / HAC enzyme, *Bjp* or *Bja* SHC / HAC enzyme, *Gmo* SHC / HAC enzyme, *ApaSHC1* SHC / HAC enzyme, or *Bme* SHC / HAC enzyme. Specifically, the SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) can be *Aac* SHC / HAC enzyme.

[0107] For ease of reference, the names "AacSHC" can be used to refer to Aac (Aac) SHC / HAC enzyme, "ZmoSHC" can be used to refer to Zmo (Zmo) SHC / HAC enzyme, "BjpSHC" or "BjaSHC" can be used to refer to Bjp (Bjp) SHC / HAC enzyme, "ApaSHC" can be used to refer to Apa (Apa) SHC / HAC enzyme, "BmeSHC" can be used to refer to Bme (Bme) SHC / HAC enzyme, "SalSHC" can be used to refer to Streptomyces (Sal) SHC / HAC enzyme, and "GmoSHC" can be used to refer to Glucosobacterium mobius (Gmo) SHC / HAC enzyme.

[0108] The sequences of AacSHC, ZmoSHC, and BjpSHC enzymes are published in BASF WO2010 / 139719, US2012 / 01345477A1, Seitz et al. (as cited above), and Seitz (as cited above in his 2012 doctoral dissertation). Two different sequences for ZmoSHC are published, designated ZmoSHC1 and ZmoSHC2. The sequence of GmoSHC / HAC enzyme is published in WO2018 / 157021. The SalSHC enzyme is published in Liu et al. (2020): A Novel Soluble Squalene-HopeneCyclase 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.

[0109] Table 1 discloses the source and accession number of wild-type SHC enzymes.

[0110] Table 1. Sources and accession numbers of wild-type (WT) SHC enzymes.

[0111]

[0112]

[0113] The sequences of wild-type AacSHC, wild-type ZmoSHC1, wild-type ZmoSHC2, wild-type BjpSHC, wild-type GmoSHC, wild-type TelSHC, wild-type ApaSHC1, wild-type BmeSHC, wild-type SalSHC, and wild-type ApaSHCA are also disclosed in this paper (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).

[0114] Alignment of the WT SHC sequences prepared by Hoshino and Sato (2002, as described above) revealed multiple motifs detected in all four sequences, and these motifs consisted of a core sequence Gln-XXX-Gly-X-Trp found six times in the SHC sequences of *Cyclopyralidus motilityis* and *Cyclopyralidus tumefaciens* (see Reipen et al. 1995, *Microbiology* 141, 155-161). Figure 3 Hoshino and Sato (2002, as described above) reported an unusual abundance of aromatic amino acids in SHCs and noted two characteristic motifs: one is the 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 is the DXDDTA motif. Wendt et al. (1997, Science 277, 1811-1815 and 1999, J Mol Biol 286, 175-187) reported X-ray structural analysis of *Acidothermia esculenta*. The DXDDTA motif appears to be associated with the active site of SHCs.

[0115] The reference AacSHC protein used herein may refer to the wild-type AacSHC protein disclosed in SEQ ID NO: 1. AacSHC possesses cyclohexane cyclase (HAC) activity, which can be used to produce ambroxol derivatives via a biocatalytic reaction of SHC with a cyclohexane substrate. The primary reaction of AacSHC is the cyclization of linear or nonlinear substrates (e.g., cyclohexane) to produce ambroxol.

[0116] The functional homologs of wild-type SHC / HAC enzymes or SHC / HAC enzyme variants described herein are also suitable for cyclization reactions, such as for the production of (-)-ambroside, for example, in a recombinant host. Therefore, a recombinant host may include one or more heteronucleotides encoding the functional homologs of the aforementioned polypeptides and / or heteronucleotides encoding SHC / HAC-derived enzymes as described herein.

[0117] A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide and performs one or more biochemical or physiological functions of the reference polypeptide. Both the functional homolog and the reference polypeptide can be naturally occurring polypeptides, and the sequence similarity can be due to convergent or divergent evolutionary events. Therefore, functional homologs are sometimes referred to in the literature as homologs, orthologs, or paralogs. Variants of naturally occurring functional homologs, such as polypeptides encoded by mutants of wild-type coding sequences, can themselves be functional homologs. Functional homologs can also be generated by site-directed mutagenesis of the coding sequence of a polypeptide or by combining domains of coding sequences from different naturally occurring polypeptides (“domain exchange”). Techniques for modifying genes encoding the functional homologs described herein are known and, in particular, include directed evolution, site-directed mutagenesis, and random mutagenesis, and can be used to increase the specific activity of the polypeptide, alter substrate specificity, change expression levels, alter subcellular locations, 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 functional homologous polypeptides.

[0118] Functional homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, searching databases of nucleotide or polypeptide sequences can identify homologs of nucleic acid sequences encoding SHC-derived polypeptides, etc.

[0119] Hybridization can also be used to identify functional homologs and / or as a measure of homology between two nucleic acid sequences. Nucleic acid sequences encoding any protein or portion thereof disclosed herein can be used as hybridization probes according to standard hybridization techniques. Hybridization of the probe with DNA or RNA from a test source (e.g., mammalian cells) indicates 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 at 30°C in 2x sodium chloride / sodium citrate (SSC), followed by washing at 50°C in 1x SSC, 0.1% SDS. Highly stringent conditions are defined as equivalent to hybridization at 45°C in 6x sodium chloride / sodium citrate (SSC), followed by washing at 65°C in 0.2x SSC, 0.1% SDS. Sequence analysis for identifying functional homologs can also involve BLAST, Reciprocal BLAST, or PSI-BLAST analyses using a non-redundant database of relevant amino acid sequences as reference sequences. In some cases, the amino acid sequences are deduced from the nucleotide sequences. Peptides with greater than 40% sequence identity in the database are candidates for further evaluation of their suitability for SHC / HAC biotransformation. Amino acid sequence similarity allows for conserved amino acid substitutions, such as replacing one hydrophobic residue with another or one polar residue with another. Such candidates can be manually examined if needed to narrow down the number of candidates for further evaluation. Manual examination can be performed by selecting candidates that appear to have, for example, conserved functional domains.

[0120] Typically, peptides exhibiting at least approximately 30% amino acid sequence identity can be used to identify conserved regions. The conserved regions of the relevant peptides 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%, and 69% amino acid sequence identity. In some implementations, the conserved regions exhibit amino acid sequence identity of 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%. Sequence identity can be determined as described above and below.

[0121] The SHC / HAC enzyme or enzyme variant described herein and used in the methods described herein may, for example, be based on 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 variants, homologs, mutants, derivatives, or fragments thereof. The SHC / HAC enzyme or enzyme variant may, for example, have the same characteristics as 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. NO: 30 has an amino acid sequence with 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%, 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.

[0122] In addition, the generated reference SHC can be based on the amino acid sequence generated from E. coli.

[0123] The "percentage (%) identity" of a gene's nucleotide sequence is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to nucleotides in the DNA sequence after alignment of sequences and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignments used to determine percentage nucleotide sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. The terms "peptide" and "protein" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modifications.

[0124] As used herein, the term "derivative" includes, but is not limited to, variants. The terms "derivative" and "variant" are used interchangeably herein.

[0125] In a preferred embodiment, the variant enzymes usable in this disclosure exhibit up to 200 (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) variations (i.e., exchanges, insertions, deletions, N-terminal truncation, and / or C-terminal truncation) in the amino acid sequence. Amino acid exchanges can be conserved and / or non-conserved. In a preferred embodiment, variants usable in this disclosure differ from the derived protein or domain 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 conserved amino acid changes. Variants may additionally or alternatively include amino acid deletions, which may be N-terminal truncation, C-terminal truncation, or internal deletions, or any combination thereof. Such variants comprising N-terminal truncation, C-terminal truncation, and / or internal deletions are referred to in the context of this application as “deletion variants” or “fragments.” The terms “deletion variant” and “fragment” are used interchangeably herein. Deletion variants may be naturally occurring (e.g., splicing variants) or may be artificially constructed, preferably by genetic engineering means. Typically, the protein or protein domain that produces the deletion variant is a wild-type protein. However, the deletion variants of this disclosure may also be derived from homologs, orthologs, or paralogs of the parent polypeptide, or from artificially constructed variants, provided that the deletion variant exhibits at least one biological activity of the parent polypeptide. Preferably, the deletion variant (or fragment) has 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 deleted from its N-terminus and / or its C-terminus and / or interior compared to the parent polypeptide.

[0126] In some implementations, the SHC / HAC enzyme variants described herein include only substitutions and not any deletions or insertions.

[0127] As used herein, a “variant” may alternatively or additionally be characterized by a certain 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 this disclosure may have sequence identity with their respective reference polypeptides or respective reference polynucleotides having 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%, 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.

[0128] The statement “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%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity” is used throughout the specification for peptide and polynucleotide sequence comparisons. Polynucleotides or proteins belonging to any enzyme family disclosed herein can be identified based on their similarity to related genes or proteins. For example, identification can be based on sequence identity.In certain preferred embodiments, this disclosure is characterized by an isolated nucleic acid molecule that, together with (a) a nucleic acid molecule encoding a polypeptide (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) of the wild-type SHC / HAC enzyme disclosed herein, (b) a nucleotide sequence SEQ ID NO: 15, and (c) includes SEQ ID NO: 15. The nucleic acid molecule containing 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) nucleotide segments of NO: 15 has a content of at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 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% identity.

[0129] Preferably, the polypeptide in question and the reference polypeptide exhibit indicated sequence identity over consecutive segments 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 indicated sequence identity over consecutive segments of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300, or more nucleotides. When comparing two sequences and no reference sequence is specified for comparison to calculate the percentage of sequence identity, unless otherwise specified, sequence identity will be calculated with reference to the longer of the two sequences to be compared. If a reference sequence is specified, 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 stated.

[0130] For example, compared to the amino acids of the full-length wild-type AacSHC with 631 amino acid residues, a peptide sequence consisting of 130 amino acids may exhibit a maximum sequence identity percentage of 20.6% (130 / 631x100), while a sequence with a length of 300 amino acids may exhibit a maximum sequence identity percentage of 47.5% (300 / 631x100).

[0131] The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. This alignment can be performed using several algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER software package, http: / / hmmer.wustl.edu / ), or using, for example, methods available from [source missing]. https: / / www.ebi.ac.uk / Tools / msa / clustalo / The preferred parameters used are: CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), or GAP program (mathematical algorithm from the University of Iowa), or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17), or CloneManager9. https: / / www.ebi.ac.uk / Tools / msa / clustalo / The default parameters set above.

[0132] The level of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms were incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. The BLASTN program (score=100, word length=12) was used to perform a BLAST polynucleotide search to obtain polynucleotide sequences homologous to those nucleic acids encoding the relevant protein.

[0133] BLAST protein search was performed using the BLASTP program (score=50, word length=3) to obtain amino acid sequences homologous to the SHC peptide. For vacancy alignments used for comparative purposes, vacancy BLAST was used as described in Altschul et al. (1997) NucleicAcids Res. 25, 3389-3402. When using the BLAST and vacancy BLAST programs, the default parameters for each program were used. Sequence matching analysis could 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, unless otherwise specified, these percentages are calculated relative to the full length of longer sequences.

[0134] In a particular implementation, CLUSTAL O (version 1.2.4) is used to determine the % identity between two sequences.

[0135] The specific SHC / HAC enzymes and enzyme variants that can be used in the methods described herein are further described below.

[0136] Aac 215G2 SHC / HAC variant with novel mutation

[0137] Surprisingly, it was found that the SHC / HAC enzyme variant derived from the Aac SHC / HAC enzyme variant (215G2SHC / HAC enzyme variant) disclosed in WO2016 / 170099 provided improved enzymatic activity for the conversion of EEH to (-)-ambroxol. Even more surprisingly, it was found that the SHC / HAC enzyme variant derived from 215G2 provided improved enzymatic activity for the conversion of E,E-bisimilar farnesol to ambergris oxide.

[0138] In addition to the amino acid substitutions already present in the 215G2 SHC / HAC enzyme variants, the new SHC / HAC enzyme variants have two or three amino acid changes.

[0139] Therefore, this paper provides a method for preparing (-)-ambroxol by enzymatically converting EEH to (-)-ambroxol. This paper also provides a method for preparing ambergris oxide by enzymatically converting E,E-bisimilar farnesol to ambergris oxide. These processes can use any wild-type SHC / HAC enzyme or enzyme variant described herein, particularly the Aac 215G2 SHC / HAC variant described herein.

[0140] The SHC / HAC enzyme variant of Aac 215G2 SHC / HAC has an amino acid sequence that is at least about 70.0% identical to SEQ ID NO: 1. For example, the SHC / HAC enzyme variant 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 with SEQ ID NO: 1.

[0141] The enzyme variant of Aac 215G2 SHC / HAC has less than 100% identity with SEQ ID NO: 1. For example, the SHC / HAC enzyme variant may have equal or less than about 99.5% or equal or less than about 99.0% identity with SEQ ID NO: 1.

[0142] For example, an enzyme variant of Aac 215G2 SHC / HAC may have about 70.0% to about 99.5%, about 80.0% to about 99.0%, about 85.0% to about 98.5%, or about 90.0% to about 98.0% identity with SEQ ID NO: 1.

[0143] The "percentage (%) identity" of a polypeptide or nucleotide sequence is defined, respectively, as the percentage of amino acids or nucleotides in a candidate sequence that are identical to those in a reference sequence after alignment of sequences and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignment for determining the percentage of sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modifications.

[0144] The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. Such alignment can be performed using several algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER software package, http: / / hmmer.wustl.edu / ), or using, for example, methods available from [source missing]. https: / / www.ebi.ac.uk / Tools / msa / clustalo / The preferred parameters used are the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), or the GAP procedure (a mathematical algorithm from the University of Iowa), or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17). https: / / www.ebi.ac.uk / Tools / msa / clustalo / The default parameters set above.

[0145] Sequence identity percentages can be calculated using algorithms such as BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms were incorporated into the BLASTN and BLASTP programs in Altschul et al. (1990) J. Mol. Biol. 215, 403-410. A BLAST polynucleotide search can be performed using the BLASTN program (score=100, word length=12) to obtain polynucleotide sequences homologous to nucleic acids encoding related proteins. A BLAST protein search can be performed using the BLASTP program (score=50, word length=3) to obtain amino acid sequences homologous to peptides.

[0146] To obtain vacancy alignments for comparative purposes, vacancy BLAST is used, as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and vacancy BLAST procedures, the default parameters of each procedure are used. Sequence matching analysis can 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, unless otherwise specified, these percentages are calculated relative to the full length of longer sequences.

[0147] In a particular implementation, CLUSTAL O (version 1.2.4) is used to determine the % identity between two sequences.

[0148] In some embodiments, the SHC / HAC enzyme variant may have equal to or less than about 30 amino acid changes compared to SEQ ID NO: 1. For example, the SHC / HAC enzyme variant may have equal to or less than about 25, equal to or less than about 20, equal to or less than about 15, equal to or less than about 10, equal to or less than about 9, equal to or less than about 8, equal to or less than about 7, or equal to or less than about 6 amino acid changes 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 changes compared to SEQ ID NO: 1. Amino acid changes may be, for example, insertions, deletions, and / or substitutions as described above.

[0149] In some embodiments, the only amino acid change in the SHC / HAC enzyme variant compared to SEQ ID NO: 1 is substitution (i.e., no insertion or deletion).

[0150] Amino acid changes are defined relative to a reference sequence. An amino acid change relative to a reference sequence means that the amino acid sequence of the variant sequence differs from the reference sequence.

[0151] Amino acids in the reference sequence and variant sequences can be assigned numbers, with the numbering starting from the N-terminal amino acid of the polypeptide (i.e., the N-terminal amino acid of the polypeptide is numbered 1, the next amino acid is numbered 2, and so on). The "position" of the reference sequence refers to the specific amino acid residue present in the reference sequence, as identified by its specific number. The "position" of the variant sequence refers to the specific amino acid residue present in the variant sequence, as identified by its specific number.

[0152] Because variant sequences can include deletions or insertions compared to the reference sequence, the amino acid numbers in the variant sequence may differ from those in the reference sequence. For example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO: 1, the amino acid following the insertion will have number 133 in the variant sequence, while it retains number 132 in the reference sequence. In this example, the position in the variant sequence corresponding to position 132 in the reference sequence is position 133. Therefore, the amino acid retained from the reference sequence in the variant sequence can be defined with reference to the “corresponding position” in the reference sequence. In other words, the “position” in the variant sequence can be defined by referring to the “corresponding position” in the reference sequence. In particular, substitutions in the variant sequence compared to the reference sequence can be defined by referring to the “corresponding position” in the reference sequence, even if any insertions and / or deletions are present in the reference sequence. In the case of deletions of amino acids in the reference sequence, there is no “corresponding position” in the variant sequence. If there are no insertions or deletions compared to the reference sequence (i.e., only substitutions), the “corresponding position” in the reference sequence will be the same as the position in the variant sequence.

[0153] The original amino acid changes in the 215G2 SHC / HAC enzyme variant (compared to wild-type Aac SHC) are preserved in the novel Aac 215G2 SHC / HAC enzyme variant described herein. The original amino acid changes in the 215G2 SHC / HAC enzyme variant compared to wild-type Aac SHC are the substitutions of M132R, A224V, and I432T (i.e., the M residue at position 132 is replaced by an R residue, the A residue at position 224 is replaced by a V residue, and the I residue at position 432 is replaced by a T residue).

[0154] Therefore, the novel Aac 215G2 SHC / HAC enzyme variants have amino acid changes relative to SEQ ID NO: 1 at positions 132, 224, and 432, respectively, namely M132R, A224V, and I432T. The numbers "132" in "M132R", "224" in "A224V", and "432" in "I432T" refer to the number of SEQ ID NO: 1 and, as mentioned above, do not necessarily correspond to variant sequence numbers due to optional other insertions and / or deletions.

[0155] The novel Aac 215G2 SHC / HAC enzyme variant also has amino acid changes relative to SEQ ID NO: 1 at position 557 and at least one position corresponding to positions 81, 431, or 613 of SEQ ID NO: 1. For example, these amino acid changes can be substitutions, such as non-conservative substitutions.

[0156] For example, the amino acid sequence of the SHC / HAC enzyme variant may have an amino acid change relative to SEQ ID NO: 1 at position 557 and at one of positions 81, 431 or 613 corresponding to SEQ ID NO: 1. These amino acid changes may be, for example, substitutions, such as non-conservative substitutions.

[0157] For example, the amino acid sequence of the SHC / HAC enzyme variant may have amino acid changes relative to SEQ ID NO: 1 at position 557 corresponding to SEQ ID NO: 1 and at two positions selected from positions 81, 431, and 613 corresponding to SEQ ID NO: 1. These amino acid changes may be, for example, substitutions, such as non-conservative substitutions.

[0158] For example, the SHC / HAC enzyme variant may have amino acid changes relative to SEQ ID NO: 1 at position 557 and at all positions 81, 431, and 613 corresponding to SEQ ID NO: 1. These amino acid changes may be substitutions, such as non-conservative substitutions.

[0159] For example, the SHC / HAC enzyme variant may have amino acid changes relative to SEQ ID NO: 1 at positions 431 and 557 corresponding to SEQ ID NO: 1. These amino acid changes may be, for example, substitutions, such as non-conserved substitutions. In some embodiments, the SHC / HAC enzyme variant has the sequence of SEQ ID NO: 2.

[0160] For example, the SHC / HAC enzyme variant may have amino acid changes relative to SEQ ID NO: 1 at positions 557 and 613 corresponding to positions 557 and 613. These amino acid changes may be, for example, substitutions, such as non-conserved substitutions. In some embodiments, the SHC / HAC enzyme variant has the sequence of SEQ ID NO: 3.

[0161] For example, the SHC / HAC enzyme variant may have amino acid changes relative to SEQ ID NO: 1 at positions 81, 557, and 613 corresponding to positions 81, 557, and 613. These amino acid changes may be, for example, substitutions, such as non-conserved substitutions. In some embodiments, the SHC / HAC enzyme variant has the sequence of SEQ ID NO: 4.

[0162] For example, the SHC / HAC enzyme variant may have amino acid changes relative to SEQ ID NO: 1 at positions 81, 431, and 557 corresponding to SEQ ID NO: 1. These amino acid changes may be, for example, substitutions, such as non-conserved substitutions. In some embodiments, the SHC / HAC enzyme variant has the sequence of SEQ ID NO: 5.

[0163] The amino acid change corresponding to position 557 of SEQ ID NO: 1 can be, for example, A557X. This means that amino acid A at position 557 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 557.

[0164] The new amino acid (X) 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 amino acid change corresponding to position 557 of SEQ ID NO: 1 can replace the amino acid (i.e., A) of SEQ ID NO: 1 with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the amino acid change corresponding to position 557 of SEQ ID NO: 1 can replace the amino acid (i.e., A) of SEQ ID NO: 1 with threonine (i.e., the amino acid change corresponding to position 557 of SEQ ID NO: 1 is A557T).

[0165] The amino acid change corresponding to position 81 of SEQ ID NO: 1 can be, for example, Y81X. This means that amino acid Y at position 81 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 81.

[0166] The new amino acid (X) 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 change corresponding to position 81 of SEQ ID NO: 1 can replace the amino acid (i.e., Y) of SEQ ID NO: 1 with a basic amino acid (i.e., His, Lys, or Arg). For example, the amino acid change corresponding to position 81 of SEQ ID NO: 1 can replace the amino acid (i.e., Y) of SEQ ID NO: 1 with histidine (i.e., the amino acid change corresponding to position 81 of SEQ ID NO: 1 is Y81H).

[0167] The amino acid change corresponding to position 431 of SEQ ID NO: 1 can be, for example, H431X. This means that amino acid H at position 431 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 431.

[0168] The new amino acid (X) 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 amino acid change corresponding to position 431 of SEQ ID NO: 1 can replace the amino acid (i.e., H) of SEQ ID NO: 1 with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the amino acid change corresponding to position 431 of SEQ ID NO: 1 can replace the amino acid (i.e., H) of SEQ ID NO: 1 with leucine (i.e., the amino acid change corresponding to position 431 of SEQ ID NO: 1 is H431L).

[0169] The amino acid change corresponding to position 613 of SEQ ID NO: 1 can be, for example, R613X. This means that the amino acid R at position 613 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 613.

[0170] The new amino acid (X) corresponding to position 631 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 amino acid change corresponding to position 613 of SEQ ID NO: 1 can replace the amino acid (i.e., R) of SEQ ID NO: 1 with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the amino acid change corresponding to position 613 of SEQ ID NO: 1 can replace the amino acid (i.e., R) of SEQ ID NO: 1 with serine (i.e., the amino acid change corresponding to position 613 of SEQ ID NO: 1 is R613S).

[0171] In some embodiments, the novel Aac 215G2 SHC variant is identical to SEQ ID NO: 1, except for the following amino acid substitutions:

[0172] (i) M132R, A224V, I432T, A557T, and H431L (SEQ ID NO: 2); or

[0173] (ii) M132R, A224V, I432T, A557T, and R613S (SEQ ID NO: 3); or

[0174] (iii) M132R, A224V, I432T, A557T, Y81H, and R613S (SEQ ID NO: 4); or

[0175] (iv) M132R, A224V, I432T, A557T, Y81H and H431L (SEQ ID NO: 5).

[0176] SHC / HAC enzyme variants may, for example, have one or more further amino acid changes at positions 90, 172 and / or 277 corresponding to SEQ ID NO: 1.

[0177] The amino acid change corresponding to position 90 of SEQ ID NO: 1 can be, for example, T90X. This means that amino acid T at position 90 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 90.

[0178] The new amino acid (X) 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 amino acid change at position 90 of SEQ ID NO: 1 can replace the amino acid (i.e., T) of SEQ ID NO: 1 with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, Ile). For example, the amino acid change at position 90 of SEQ ID NO: 1 can replace the amino acid (i.e., T) of SEQ ID NO: 1 with alanine (i.e., the amino acid change at position 90 of SEQ ID NO: 1 is T90A).

[0179] The amino acid change corresponding to position 172 of SEQ ID NO: 1 can be, for example, A172X. This means that amino acid T at position 172 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 172.

[0180] The new amino acid (X) 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 amino acid change corresponding to position 172 of SEQ ID NO: 1 can replace the amino acid (i.e., A) of SEQ ID NO: 1 with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, Gln). For example, the amino acid change corresponding to position 172 of SEQ ID NO: 1 can replace the amino acid (i.e., A) of SEQ ID NO: 1 with threonine (i.e., the amino acid change corresponding to position 172 of SEQ ID NO: 1 is A172T).

[0181] The amino acid change corresponding to position 277 of SEQ ID NO: 1 can be, for example, M277X. This means that amino acid M at position 277 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 277.

[0182] The new amino acid (X) 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 amino acid change at position 277 of SEQ ID NO: 1 can replace the amino acid (i.e., M) of SEQ ID NO: 1 with a basic amino acid (i.e., His, Lys, Arg). For example, the amino acid change at position 277 of SEQ ID NO: 1 can replace the amino acid (i.e., M) of SEQ ID NO: 1 with lysine (i.e., the amino acid change at position 277 of SEQ ID NO: 1 is M277K).

[0183] For example, the SHC / HAC enzyme variant may have one or more further amino acid changes at positions 37, 174, and / or 601 corresponding to SEQ ID NO: 1. For example, the SHC / HAC enzyme variant may have one or more amino acid substitutions (e.g., conserved or non-conserved substitutions) at positions 37, 174, and / or 601 corresponding to SEQ ID NO: 1.

[0184] The amino acid change corresponding to position 37 of SEQ ID NO: 1 can be, for example, L37X. This means that amino acid L at position 37 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 37.

[0185] The new amino acid (X) 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 amino acid change corresponding to position 37 of SEQ ID NO: 1 can replace the amino acid (i.e., L) of SEQ ID NO: 1 with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln). For example, the amino acid change corresponding to position 37 of SEQ ID NO: 1 can replace the amino acid (i.e., L) of SEQ ID NO: 1 with glutamine (i.e., the amino acid change corresponding to position 37 of SEQ ID NO: 1 is L37Q).

[0186] The amino acid change corresponding to position 174 of SEQ ID NO: 1 could be, for example, V174X. This means that amino acid V at position 174 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 174.

[0187] The new amino acid (X) 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. The new amino acid (X) corresponding to position 174 of SEQ ID NO: 1 can be, for example, a hydrophobic amino acid (i.e., Met, Ala, Leu, or Ile). For example, the amino acid change corresponding to position 174 of SEQ ID NO: 1 can replace the amino acid (i.e., V) of SEQ ID NO: 1 with isoleucine (i.e., the amino acid change corresponding to position 174 of SEQ ID NO: 1 is V174I).

[0188] The amino acid change corresponding to position 601 of SEQ ID NO: 1 can be, for example, F601X. This means that amino acid F at position 601 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 601.

[0189] The new amino acid (X) 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) 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 change corresponding to position 601 of SEQ ID NO: 1 can replace the amino acid (i.e., F) of SEQ ID NO: 1 with tyrosine (i.e., the amino acid change corresponding to position 601 of SEQ ID NO: 1 is F601Y).

[0190] SHC / HAC enzyme variants may have one or more further amino acid changes, for example, at positions 77, 92, 129, 579, 601, and / or 605 corresponding to SEQ ID NO: 1. For example, SHC / HAC enzyme variants may have one or more amino acid substitutions (e.g., conserved or non-conserved substitutions) at positions 77, 92, 129, 579, 601, and / or 605 corresponding to SEQ ID NO: 1.

[0191] The amino acid change corresponding to position 77 of SEQ ID NO: 1 can be, for example, T77X. This means that amino acid T at position 77 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 77.

[0192] The new amino acid (X) 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 amino acid change corresponding to position 77 of SEQ ID NO: 1 can replace the amino acid (i.e., T) of SEQ ID NO: 1 with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the amino acid change corresponding to position 77 of SEQ ID NO: 1 can replace the amino acid (i.e., T) of SEQ ID NO: 1 with alanine (i.e., the amino acid change corresponding to position 77 of SEQ ID NO: 1 is T77A).

[0193] The amino acid change corresponding to position 92 of SEQ ID NO: 1 can be, for example, I92X. This means that amino acid I at position 92 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 92.

[0194] The new amino acid (X) 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 amino acid change corresponding to position 92 of SEQ ID NO: 1 can replace the amino acid (i.e., I) of SEQ ID NO: 1 with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the amino acid change corresponding to position 92 of SEQ ID NO: 1 can replace the amino acid (i.e., I) of SEQ ID NO: 1 with valine (i.e., the amino acid change corresponding to position 92 of SEQ ID NO: 1 is I92V).

[0195] The amino acid change corresponding to position 129 of SEQ ID NO: 1 can be, for example, F129X. This means that amino acid F at position 129 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 129.

[0196] The new amino acid (X) 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 amino acid change corresponding to position 129 of SEQ ID NO: 1 can replace the amino acid (i.e., F) of SEQ ID NO: 1 with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile). For example, the amino acid change corresponding to position 129 of SEQ ID NO: 1 can replace the amino acid (i.e., F) of SEQ ID NO: 1 with leucine (i.e., the amino acid change corresponding to position 129 of SEQ ID NO: 1 is F129L).

[0197] The amino acid change corresponding to position 579 of SEQ ID NO: 1 can be, for example, Q579X. This means that amino acid Q at position 579 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 579.

[0198] The new amino acid (X) 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 amino acid change corresponding to position 579 of SEQ ID NO: 1 can replace the amino acid (i.e., Q) of SEQ ID NO: 1 with a basic amino acid (i.e., His, Lys, or Arg). For example, the amino acid change corresponding to position 579 of SEQ ID NO: 1 can replace the amino acid (i.e., Q) of SEQ ID NO: 1 with histidine (i.e., the amino acid change corresponding to position 579 of SEQ ID NO: 1 is Q579H).

[0199] The amino acid change corresponding to position 601 of SEQ ID NO: 1 can be, for example, F601X. This means that amino acid F at position 601 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 601.

[0200] The new amino acid (X) 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) 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 change corresponding to position 601 of SEQ ID NO: 1 can replace the amino acid (i.e., F) of SEQ ID NO: 1 with histidine (i.e., the amino acid change corresponding to position 601 of SEQ ID NO: 1 is F601Y).

[0201] The amino acid change corresponding to position 605 of SEQ ID NO: 1 can be, for example, F605X. This means that amino acid F at position 605 of SEQ ID NO: 1 is replaced by any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 605.

[0202] The new amino acid (X) 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) 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 change corresponding to position 605 of SEQ ID NO: 1 can replace the amino acid (i.e., F) of SEQ ID NO: 1 with tryptophan (i.e., the amino acid change corresponding to position 601 of SEQ ID NO: 1 is F605W).

[0203] For example, SHC / HAC enzyme variants may have amino acid changes (e.g., substitutions) at positions 132 and 432 corresponding to SEQ ID NO: 1.

[0204] For example, SHC / HAC enzyme variants may have an amino acid change (e.g., substitution) at position 601 corresponding to SEQ ID NO: 1.

[0205] For example, SHC / HAC enzyme variants may have amino acid changes (e.g., substitutions) at positions 77, 92, and 129 corresponding to SEQ ID NO: 1.

[0206] For example, SHC / HAC enzyme variants may have amino acid changes (e.g., substitutions) at positions 579 and 601 corresponding to SEQ ID NO: 1.

[0207] For example, SHC / HAC enzyme variants may have amino acid changes (e.g., substitutions) at positions 129, 132, and 432 corresponding to SEQ ID NO: 1.

[0208] For example, SHC / HAC enzyme variants may have amino acid changes (e.g., substitutions) at positions 132, 432, and 601 corresponding to SEQ ID NO: 1.

[0209] For example, SHC / HAC enzyme variants may have amino acid changes (e.g., substitutions) at positions 129, 132, 432, and 601 corresponding to SEQ ID NO: 1.

[0210] Compared to the SHC / HAC enzyme variant of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10, the new SHC / HAC enzyme variant may, for example, have increased enzyme activity for converting EEH to (-)-ambroside or BisEEH to ambergris oxide. Increased enzyme activity can refer to any aspect of the enzymatic conversion of EEH to (-)-ambroside or BisEEH to ambergris oxide, including, for example, increased total conversion of EEH or BisEEH, increased conversion rate of EEH or BisEEH (e.g., in the first 6 or 12 hours of the reaction), increased yield of (-)-ambroside or ambergris oxide, and reduced yield of byproducts. Increased enzyme activity can generally be defined by increased productivity, which can be defined based on the production of (-)-ambroside or ambergris oxide per gram of biocatalyst, per hour, and per liter of reaction.

[0211] Compared to 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, the new SHC / HAC enzyme variant can, for example, provide increased EEH or E,E-bi-dimethylfarnesol (BisEEH) conversion. Therefore, the method described herein can have increased EEH or BisEEH conversion levels compared to the method 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 variant can, for example, provide 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. Therefore, the method described herein can 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 novel SHC / HAC enzyme variant can, for example, provide increased EEH or BisEEH conversion rates during the first 4, 6, 8, 12, or 24 hours of the reaction compared to the wild-type SHC / HAC enzyme of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10. Therefore, the method described herein can have increased EEH or BisEEH conversion rates during the first 4, 6, 8, 12, or 24 hours of the reaction compared to the wild-type SHC / HAC enzyme of SEQ ID NO: 1 and / or the SHC / HAC enzyme variant of SEQ ID NO: 10. This can be compared with using two 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 with using each enzyme under its respective optimized reaction conditions (e.g., optimized pH and temperature) (which may differ from each other).

[0212] For example, a novel SHC / HAC enzyme variant may be available within the first 12 hours of the reaction, or the method may achieve at least about 40% conversion of EEH or BisEEH within the first 12 hours of the reaction. Alternatively, a novel SHC / HAC enzyme variant may be available within the first 12 hours of the reaction, or the method may achieve at least about 45%, at least about 50%, at least about 55%, or at least about 60% conversion of EEH or BisEEH within the first 12 hours of the reaction. Alternatively, a novel SHC / HAC enzyme variant may be available within the first 6 hours of the reaction, or the method may achieve at least about 30% conversion of EEH or BisEEH within the first 6 hours of the reaction. Finally, a novel SHC / HAC enzyme variant may be available within the first 12 hours of the reaction, or the method may achieve at least about 35%, at least about 45%, at least about 50%, or at least about 55% conversion of EEH or BisEEH within the first 12 hours of the reaction. This can be compared with the use of two 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 with the use of each enzyme under their respective optimized reaction conditions (e.g., optimized pH and temperature) (which may differ from each other).

[0213] The conversion of EEH to (-)-ambroside or BisEEH to ambergris oxide can be determined, for example, by using the activity assays described above, and can be calculated as the number of grams of recyclable product per gram of feedstock (which can be calculated as a percentage of molar conversion).

[0214] As used herein, the 99% / 100% conversion of jofarne alcohol substrate to (-)-ambroxol as mentioned herein refers to the 99% / 100% conversion to the jofarne alcohol isomer (i.e., EEH) of (-)-ambroxol using SHC / HAC enzymes or enzyme variants.

[0215] As used herein, the 99% / 100% conversion of bis-average farnesol substrate to ambergris oxide refers to the 99% / 100% conversion of the bis-average farnesol isomer (i.e., BisEEH) to ambergris oxide using SHC / HAC enzymes or enzyme variants.

[0216] The optimal temperature for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC can be, for example, equal to or greater than about 3°C. C. For example, the optimal temperature range for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC can be from about 40°C to about 50°C, such as from about 42°C to about 48°C or from about 44°C to about 46°C. For example, the optimal temperature for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC can be from about 40°C to about 50°C. C. The method for preparing (-)-ambroside or ambergris oxide disclosed herein can be carried out at the optimal temperature of the SHC / HAC enzyme variant.

[0217] The optimal pH for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC can be, for example, equal to or greater than about 5.4. For example, the optimal pH range for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC can be from about 5.2 to about 6.0, for example from about 5.4 to about 5.8, for example from about 5.6 to about 5.8. For example, the optimal pH for the SHC / HAC enzyme variant of Aac 215G2 SHC / HAC can be about 5.6 or about 5.8. The methods disclosed herein for preparing (-)-ambroxol or ambergris oxide can be carried out at the optimal pH of the SHC / HAC enzyme variant.

[0218] The optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the method disclosed herein for preparing (-)-ambroside or ambergris oxide can be, for example, from about 0.010 w / w% to about 0.10 w / w%. For example, when the substrate (e.g., EEH or BisEEH) is used at 4 g / L and the cells reach an OD of 10, the optimal concentration of SDS is achieved. 650nm When using a substrate (e.g., EEH or BisEEH) at 125 g / L with 250 g / L cells, the optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the method disclosed herein for preparing (-)-ambroxol or ambergris oxide can be, for example, from about 1.0 w / L to about 1.5 w / L. For example, when using a substrate (e.g., EEH or BisEEH) at 125 g / L with 250 g / L cells, the optimal concentration of SDS can be from about 1.2 w / L to about 1.4 w / L.

[0219] The methods disclosed herein for preparing (-)-ambroxan or ambergris oxide can be carried out at the optimal temperature range or optimal temperature and / or optimal pH range or optimal pH and / or optimal SDS concentration range or optimal SDS concentration for the specific enzyme used, as listed in Tables 7, 9 or 11 in the examples below.

[0220] Other variants with novel mutations at positions 81, 90, 172, 277, 431, 557 and / or 613 corresponding to SEQ ID NO: 1

[0221] As discussed above, it was surprising to find that the SHC / HAC enzyme variant (215G2 SHC / HAC enzyme variant), derived from the Aac SHC / HAC enzyme variant disclosed in WO2016 / 170099, provides improved enzymatic activity for the conversion of EEH to (-)-ambroxol and BisEEH to ambergris oxide. In addition to the amino acid substitutions already present in the 215G2 SHC / HAC enzyme variant, the new SHC / HAC enzyme variant also exhibits two or three amino acid changes.

[0222] Other SHC / HAC enzyme variants with one or more novel mutations identified at positions 81, 90, 172, 277, 431, 557 and 613 of SEQ ID NO: 1 are also expected to provide enzymatic activity (e.g., improved enzymatic activity) for converting EEH to (-)-ambroxol or BisEEH to ambergris oxide.

[0223] Specifically, SHC / HAC enzyme variants derived from other non-Aac species but having one or more novel amino acid alterations identified at positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO: 1 are also expected to provide enzymatic activity for converting EEH to (-)-ambroxol or BisEEH to ambergris oxide. Specifically, enzyme variants derived from ZmoSHC1, ZmoSHC2, BjpSHC, GmoSHC, TelSHC, ApaSHC1, BmeSHC, SalSHC, or ApaSHCA having one or more novel amino acid alterations identified at positions 81, 90, 172, 277, 431, 557, and 613 of SEQ ID NO: 1 are also expected to provide enzymatic activity for converting EEH to (-)-ambroxol or BisEEH to ambergris oxide.

[0224] Therefore, this paper provides a method for preparing (-)-ambroxol by enzymatically converting EEH to (-)-ambroxol. This paper also provides a method for preparing ambergris oxide by enzymatically converting E,E-bisimilar farnesol to ambergris oxide. These processes can use any wild-type SHC / HAC enzyme or enzyme variant described herein.

[0225] In addition, this article provides SHC / HAC enzyme variants that have at least about 70.0% identity with the amino acid sequence of wild-type SHC / HAC enzymes, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid changes relative to wild-type SHC / HAC enzymes at positions selected from positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1.

[0226] Specifically, this document provides a method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol, the method comprising enzymatically converting EEH or a mixture containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol using an SHC / HAC variant having at least about 70.0% identity with the amino acid sequence of the wild-type SHC / HAC enzyme, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1.

[0227] Specifically, this document provides a method for preparing ambergris oxide or a mixture containing ambergris oxide, the method comprising enzymatically converting BisEEH or a mixture containing BisEEH into ambergris oxide or a mixture containing ambergris oxide using an SHC / HAC variant having at least about 70.0% identity with the amino acid sequence of the wild-type SHC / HAC enzyme, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1.

[0228] SHC / HAC enzyme variants may, for example, have an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of wild-type SHC / HAC enzymes. For instance, SHC / HAC enzyme variants may have an amino acid sequence that is at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 95.5%, at least about 96.5%, at least about 97.0%, at least about 97.5%, at least about 98.0%, at least about 98.5%, or at least about 99.0% identical to the amino acid sequence of wild-type SHC / HAC enzymes.

[0229] For example, SHC / HAC enzyme variants may have amino acid sequences that are less than 100% identical to wild-type SHC / HAC enzyme sequences, such as being equal to or less than about 99.5% or equal to or less than about 99.0% identical.

[0230] For example, the amino acid sequence of the SHC / HAC enzyme variant may have about 70.0% to about 99.5%, about 80.0% to about 99.0%, about 85.0% to about 98.5%, or about 90.0% to about 98.0%.

[0231] The amino acid sequence of the wild-type SHC / HAC enzyme can be, for example, the amino acid sequence 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 can be the amino acid sequence of AacSHC (SEQ ID NO: 1).

[0232] Therefore, in some embodiments, the SHC / HAC enzyme variant may have an amino acid sequence that is at least about 70.0% 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 or SEQ ID NO: 20. For example, the amino acid sequence of the SHC / HAC enzyme variant has 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% of the same amino acid sequence as 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.

[0233] For example, SHC / HAC enzyme variants may have amino acid sequences that are less than 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, for example, equal to or less than about 99.5% or equal to or less than about 99.0% identical.

[0234] For example, the SHC / HAC enzyme variant may have about 70.0% to about 99.5%, about 80.0% to about 99.0%, about 85.0% to about 98.5%, or about 90.0% to about 98.0% of the identity with SEQ ID NO: 1, 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.

[0235] The "percentage (%) identity" of a polypeptide or nucleotide sequence is defined, respectively, as the percentage of amino acids or nucleotides in a candidate sequence that are identical to those in a reference sequence after alignment of sequences and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignment for determining the percentage of sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modifications.

[0236] The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. Such alignment can be performed using several algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER software package, http: / / hmmer.wustl.edu / ), or using, for example, methods available from [source missing]. https: / / www.ebi.ac.uk / Tools / msa / clustalo / The preferred parameters used are the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), or the GAP procedure (a mathematical algorithm from the University of Iowa), or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17). https: / / www.ebi.ac.uk / Tools / msa / clustalo / The default parameters set above.

[0237] Sequence identity percentages can be calculated using algorithms such as BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms were incorporated into the BLASTN and BLASTP programs in Altschul et al. (1990) J. Mol. Biol. 215, 403-410. A BLAST polynucleotide search can be performed using the BLASTN program (score=100, word length=12) to obtain polynucleotide sequences homologous to nucleic acids encoding related proteins. A BLAST protein search can be performed using the BLASTP program (score=50, word length=3) to obtain amino acid sequences homologous to peptides.

[0238] To obtain vacancy alignments for comparative purposes, vacancy BLAST can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and vacancy BLAST procedures, the default parameters of each procedure are used. Sequence matching analysis can 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, unless otherwise specified, these percentages are calculated relative to the full length of longer sequences.

[0239] In a particular implementation, CLUSTAL O (version 1.2.4) is used to determine the % identity between two sequences.

[0240] In some embodiments, the SHC / HAC enzyme variant may have an amino acid change equal to or less than about 200 compared to the wild-type SHC / HAC enzyme, for example, 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. For example, compared to the wild-type SHC / HAC enzyme, for example, 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. Compared to NO:30, the SHC / HAC enzyme variants can have amino acid changes of equal to or less than about 150 or equal to or less than about 120 or equal to or less than about 100 or equal to or less than about 95 or equal to or less than about 90 or equal to or less than about 85 or equal to or less than about 80 or equal to or less than about 75 or equal to or less than about 70 or equal to or less than about 65 or equal to or less than about 60 or equal to or less than about 55 or equal to or less than about 50 or equal to or less than about 45 or equal to or less than about 40 or equal to or less than about 35 or equal to or less than about 30 or equal to or less than about 25 or equal to or less than about 20 or equal to or less than about 15 or equal to or less than about 10.

[0241] For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have at least about 1 or at least about 2 or at least about 3 or at least about 4 or at least about 5 or at least about 6 amino acid changes.

[0242] For example, compared to wild-type SHC / HAC enzymes, such as SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 20, SHC / HAC enzyme variants may have about 1 to about 30 amino acid changes. For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 2 to about 25 amino acid changes. For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 3 to about 20 amino acid changes. For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 4 to about 15 amino acid changes. For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 5 to about 10 amino acid changes.

[0243] For example, amino acid changes can be insertions, deletions, and / or substitutions as described above. For example, amino acid changes can be substitutions, such as non-conservative substitutions.

[0244] In some embodiments, the only amino acid change compared to wild-type SHC / HAC enzymes (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) is substitution (i.e. no insertion or deletion).

[0245] Amino acid changes are defined relative to a reference sequence. An amino acid change relative to a reference sequence means that the amino acid sequence of the variant sequence differs from the reference sequence.

[0246] Amino acids in the reference sequence and variant sequences can be assigned numbers, with the numbering starting from the N-terminal amino acid of the polypeptide (i.e., the N-terminal amino acid of the polypeptide is numbered 1, the next amino acid is numbered 2, and so on). The "position" of the reference sequence refers to the specific amino acid residue present in the reference sequence, as identified by its specific number. The "position" of the variant sequence refers to the specific amino acid residue present in the variant sequence, as identified by its specific number.

[0247] Because variant sequences can include deletions or insertions compared to the reference sequence, the amino acid numbers in the variant sequence may differ from those in the reference sequence. For example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO: 1, the amino acid following the insertion will have number 133 in the variant sequence, while it retains number 132 in the reference sequence. In this example, the position in the variant sequence corresponding to position 132 in the reference sequence is position 133. Therefore, the amino acid retained from the reference sequence in the variant sequence can be defined with reference to the “corresponding position” in the reference sequence. In other words, the “position” in the variant sequence can be defined by referring to the “corresponding position” in the reference sequence. In particular, substitutions in the variant sequence compared to the reference sequence can be defined by referring to the “corresponding position” in the reference sequence, even if any insertions and / or deletions are present in the reference sequence. In the case of deletions of amino acids in the reference sequence, there is no “corresponding position” in the variant sequence. If there are no insertions or deletions compared to the reference sequence (i.e., only substitutions), the “corresponding position” in the reference sequence will be the same as the position in the variant sequence.

[0248] Wild-type SHC / HAC enzymes from different species have different polypeptide lengths. Wild-type sequences can be compared using the algorithm described above to identify “corresponding positions” in two different wild-type SHC / HAC enzymes. Therefore, the amino acid at the position in the variant sequence corresponding to the position in the reference sequence can be, for example, a different amino acid residue and / or can have a different number than the reference sequence number. For example, amino acid M at position 132 of AacSHC (SEQ ID NO: 1) can correspond to amino acid Y at position 185 of ZmoSHC1 (SEQ ID NO: 11).

[0249] Therefore, amino acid changes can be defined relative to two different reference sequences. For example, an amino acid change can be a change compared to a first reference sequence (e.g., the wild-type SHC / HAC enzyme sequence of a derived variant), and the position of the amino acid change in the variant sequence can be defined by referring to a second reference sequence (e.g., AacSHC (SEQ ID NO: 1)). Thus, an amino acid change in an SHC / HAC enzyme variant can be located relative to the first wild-type SHC / HAC enzyme at a position defined by referring to the second wild-type SHC / HAC enzyme.

[0250] The SHC / HAC enzyme variant amino acid sequence has one or more amino acid changes relative to the wild-type SHC / HAC enzyme amino acid sequence at positions selected from positions 81, 90, 172, 277, 431, 557, and 613 corresponding to SEQ ID NO: 1. For example, the amino acid change may be at one or more positions selected from positions 81, 431, 557, and 613 corresponding to SEQ ID NO: 1. The amino acid change may be, for example, a substitution, such as a non-conservative substitution. The wild-type SHC / HAC enzyme amino acid sequence 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.

[0251] In some embodiments, the amino acid sequence of the SHC / HAC enzyme variant, relative to the wild-type SHC / HAC enzyme amino acid sequence, has an amino acid change at at least one position corresponding to position 557 of SEQ ID NO: 1 and positions 81, 431, or 613 of SEQ ID NO: 1. The amino acid change can be, for example, substitution, such as a non-conservative substitution. The wild-type SHC / HAC enzyme amino acid sequence can 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 can be SEQ ID NO: 1.

[0252] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence may have an amino acid change at position 557 corresponding to SEQ ID NO: 1 and at one of positions 81, 431, or 613 corresponding to SEQ ID NO: 1. The amino acid change may be, for example, substitution, such as non-conservative substitution. The wild-type SHC / HAC enzyme amino acid sequence 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.

[0253] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence may have amino acid changes at two positions: position 557 corresponding to SEQ ID NO: 1 and positions selected from positions 81, 431, and 613 corresponding to SEQ ID NO: 1. Amino acid changes may be, for example, substitutions, such as non-conservative substitutions. The wild-type SHC / HAC enzyme amino acid sequence 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.

[0254] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence may have amino acid changes at position 557 corresponding to SEQ ID NO: 1 and at all positions 81, 431, and 613 corresponding to SEQ ID NO: 1. Amino acid changes may be, for example, substitutions, such as non-conservative substitutions. The wild-type SHC / HAC enzyme amino acid sequence 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.

[0255] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence has amino acid changes at positions 90 and 613 corresponding to SEQ ID NO: 1. These amino acid changes can be, for example, substitutions, such as non-conservative substitutions. The wild-type SHC / HAC enzyme amino acid sequence can 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 can be SEQ ID NO: 1.

[0256] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence has amino acid changes at positions 172 and 277 corresponding to SEQ ID NO: 1. These amino acid changes can be, for example, substitutions, such as non-conservative substitutions. The wild-type SHC / HAC enzyme amino acid sequence can 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 can be SEQ ID NO: 1.

[0257] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence has amino acid changes at positions 557 and 431 corresponding to SEQ ID NO: 1. These amino acid changes can be, for example, substitutions, such as non-conservative substitutions. The wild-type SHC / HAC enzyme amino acid sequence can 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 can be SEQ ID NO: 1.

[0258] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence has amino acid changes at positions 557 and 613 corresponding to SEQ ID NO: 1. These amino acid changes can be, for example, substitutions, such as non-conserved substitutions. The wild-type SHC / HAC enzyme amino acid sequence can 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 can be SEQ ID NO: 1.

[0259] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence has amino acid changes at positions 81, 557, and 613 corresponding to SEQ ID NO: 1. These amino acid changes can be, for example, substitutions, such as non-conservative substitutions. The wild-type SHC / HAC enzyme amino acid sequence can 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 can be SEQ ID NO: 1.

[0260] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence has amino acid changes at positions 81, 431, and 557 corresponding to SEQ ID NO: 1. These amino acid changes can be, for example, substitutions, such as non-conservative substitutions. The wild-type SHC / HAC enzyme amino acid sequence can 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 can be SEQ ID NO: 1.

[0261] The amino acid change at position 557 of SEQ ID NO: 1 relative to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 557.

[0262] The novel amino acid (X) at position 557 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be threonine.

[0263] The amino acid change at position 81 corresponding to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) may not be 81.

[0264] The novel amino acid (X) at position 81 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel amino acid (X) in the SHC / HAC enzyme variant can be a basic amino acid (i.e., His, Lys, or Arg). For example, the novel amino acid in the SHC / HAC enzyme variant can be histidine.

[0265] The amino acid change at position 90 corresponding to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid for a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 90.

[0266] The novel amino acid (X) at position 90 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be alanine.

[0267] The amino acid change at position 172 of SEQ ID NO: 1 relative to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 172.

[0268] The novel amino acid (X) at position 172 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be threonine.

[0269] The amino acid change at position 277 of SEQ ID NO: 1 relative to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 277.

[0270] The new amino acid (X) at position 277 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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.

[0271] The amino acid change at position 431 of SEQ ID NO: 1 relative to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid for a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) may not be 431.

[0272] The novel amino acid (X) at position 431 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be leucine.

[0273] The amino acid change at position 613 of SEQ ID NO: 1 relative to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1 and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) may not be 613.

[0274] The novel amino acid (X) at position 613 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be serine.

[0275] The amino acids and positions of the wild-type ZmoSHC1, ZmoSHC2, BjpSHC, GmoSHC, TelSHC, and ApaSHC1 sequences (SEQ ID NO: 11, 12, 13, 14, 19, and 20, respectively) corresponding to AacSHC (SEQ ID NO: 1) (e.g., amino acids at positions 81, 431, 557, and 613 of AacSHC) are as follows: Figure 9A As shown. The amino acid sequences and positions of the wild-type ZmoSHC1, ZmoSHC2, BjpSHC, GmoSHC, TelSHC, ApaSHC1, BmeSHC, SalSHC, and ApaSHCA sequences (SEQ ID NO: 11, 12, 13, 14, 19, 20, 28, 29, and 30, respectively) corresponding to AacSHC (SEQ ID NO: 1) (e.g., amino acids at positions 81, 431, 557, and 613 of AacSHC) are as follows: Figure 9B As shown.

[0276] Amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC are highlighted with white letters on a black background. Therefore, the amino acids directly above or below the highlighted amino acids are those in ZmoSHC2, BjaSHC, GmoSHC, ApaSHC1, ApaSHC1, ZmoSHC1, and TelSHC corresponding to positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 of AacSHC (SEQ ID NO: 1). For example, the amino acid corresponding to amino acid Y at position 81 of AacSHC (SEQ ID NO: 1) in BjaSHC, GmoSHC, ApaSHC1, and ZmoSHC1 is Y. The amino acid corresponding to amino acid Y at position 81 of AacSHC (SEQ ID NO: 1) in ZmoSHC2 and TelSHC is F. Position 84 of TelSHC is the position of TelSHC corresponding to position 81 of AacSHC (SEQ ID NO: 1).

[0277] In some embodiments, one or more original amino acid changes in the 215G2 SHC / HAC enzyme variant (compared to wild-type AacSHC (SEQ ID NO: 1)) can be retained in the novel SHC / HAC enzyme variant described herein. The amino acid changes in the 215G2 SHC / HAC enzyme variant compared to wild-type AacSHC are the substitutions of M132R, A224V, and I432T (i.e., the M residue at position 132 is replaced with an R residue, the A residue at position 224 is replaced with a V residue, and the I residue at position 432 is replaced with a T residue).

[0278] Therefore, compared to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence may have one or more amino acid changes at positions 132, 224, and 432 corresponding to SEQ ID NO: 1. For example, the amino acid change may be a substitution.

[0279] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence may have one, two, or three amino acid changes at positions 132, 224, and 432, which correspond to SEQ ID NO: 1.

[0280] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence may have amino acid changes at positions 132 and 432 corresponding to SEQ ID NO: 1.

[0281] For example, relative to the wild-type SHC / HAC enzyme amino acid sequence, the SHC / HAC enzyme variant amino acid sequence may have amino acid changes at positions 132, 224, and 432 corresponding to SEQ ID NO: 1.

[0282] The amino acid change at position 132 of SEQ ID NO: 1 relative to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 132.

[0283] The novel amino acid (X) at position 132 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel amino acid (X) in the SHC / HAC enzyme variant can be a basic amino acid (i.e., His, Lys, or Arg). For example, the novel amino acid in the SHC / HAC enzyme variant can be arginine.

[0284] The amino acid change at position 224 of SEQ ID NO: 1 relative to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 224.

[0285] The novel amino acid (X) at position 224 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be valine.

[0286] The amino acid change at position 432 of SEQ ID NO: 1 relative to the wild-type SHC / HAC enzyme amino acid sequence can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 432.

[0287] The novel amino acid (X) at position 432 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be threonine.

[0288] The amino acids and positions of the amino acids corresponding to AacSHC (SEQ ID NO: 1) in the wild-type ZmoSHC1, ZmoSHC2, BjpSHC, GmoSHC, TelSHC, and ApaSHC1 sequences (SEQ ID NO: 11, 12, 13, 14, 19, and 20, respectively) (e.g., at positions 132, 224, and 432 in AacSHC) are shown in Figure 9.

[0289] For example, the SHC / HAC enzyme variants described herein may have one or more additional amino acid alterations (e.g., substitutions) at other positions of AacSHC identified in WO2016 / 170099.

[0290] Therefore, compared to the wild-type SHC / HAC enzyme, the SHC / HAC enzyme variants described herein may have one or more further amino acid changes at positions 77, 92, 129, 579, 601, and / or 605 corresponding to SEQ ID NO: 1. For example, relative to the wild-type SHC / HAC enzyme, the SHC / HAC enzyme variants may have one or more amino acid substitutions (e.g., conserved or non-conserved substitutions) at positions 77, 92, 129, 579, 601, and / or 605 corresponding to SEQ ID NO: 1.

[0291] The amino acid change at position 77 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 77.

[0292] The novel amino acid (X) at position 77 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be alanine.

[0293] The amino acid change at position 92 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 could be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid for a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 92.

[0294] The novel amino acid (X) at position 92 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be valine.

[0295] The amino acid change at position 129 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 129.

[0296] The novel amino acid (X) at position 129 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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 novel amino acid in the SHC / HAC enzyme variant can be leucine.

[0297] The amino acid change at position 579 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 could be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid for a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 579.

[0298] The novel amino acid (X) at position 579 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel amino acid (X) in the SHC / HAC enzyme variant can be a basic amino acid (i.e., His, Lys, Arg). For example, the novel amino acid in the SHC / HAC enzyme variant can be histidine.

[0299] The amino acid change at position 601 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 601.

[0300] The novel amino acid (X) at position 601 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel amino acid (X) in the SHC / HAC enzyme variant can be an aromatic amino acid (i.e., Trp, Tyr, Phe). For example, the novel amino acid in the SHC / HAC enzyme variant can be tyrosine.

[0301] The amino acid change at position 605 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 could be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) in the variant sequence may not be 605.

[0302] The novel amino acid (X) at position 605 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel amino acid (X) in the SHC / HAC enzyme variant can be an aromatic amino acid (i.e., Trp, Tyr, Phe). For example, the novel amino acid in the SHC / HAC enzyme variant can be tryptophan.

[0303] The amino acid positions corresponding to amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in the wild-type AacSHC 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) are shown in the sequence. Figure 9A In the wild-type AacSHC (SEQ ID NO: 1), it is highlighted with white letters on a black background.

[0304] The amino acid positions corresponding to amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC (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) in wild-type AacSHC are shown in the figure. Figure 9B In the wild-type AacSHC (SEQ ID NO: 1), it is highlighted with white letters on a black background.

[0305] SHC / HAC enzyme variants may, for example, have one or more further amino acid changes at positions 37, 174, and / or 601 corresponding to the wild-type SHC / HAC enzyme amino acid sequence. For example, the SHC / HAC enzyme variants may have one or more amino acid substitutions (e.g., conserved or non-conserved substitutions) at positions 37, 174, and / or 601 corresponding to the wild-type SHC / HAC enzyme amino acid sequence.

[0306] The amino acid change at position 37 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1, and since variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) may not be 37.

[0307] The novel amino acid (X) at position 37 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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.

[0308] The amino acid change at position 174 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 could be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1 and since variants may additionally contain insertions and / or deletions, the new amino acid (X) may not be numbered 174.

[0309] The novel amino acid (X) at position 174 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel 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.

[0310] The amino acid change at position 601 of the wild-type SHC / HAC enzyme amino acid sequence relative to SEQ ID NO: 1 can be, for example, the substitution of the wild-type SHC / HAC enzyme amino acid with a different amino acid (X). As mentioned above, since the wild-type sequence may have a different length than SEQ ID NO: 1 and since the variant may additionally contain insertions and / or deletions, the number of the new amino acid (X) may not be 601.

[0311] The novel amino acid (X) at position 601 of SEQ ID NO: 1 in the amino acid sequence of the SHC / HAC enzyme variant 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 novel amino acid (X) in the SHC / HAC enzyme variant can be an aromatic amino acid (i.e., Trp, Tyr, Phe), such as tyrosine.

[0312] The amino acid positions corresponding to amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in the wild-type AacSHC 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) are shown in the sequence. Figure 9A In the wild-type AacSHC (SEQ ID NO: 1), it is highlighted with white letters on a black background.

[0313] The amino acid positions corresponding to amino acid positions 81, 90, 132, 224, 172, 277, 431, 432, 557, and 613 in wild-type AacSHC (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) in wild-type AacSHC are shown in the figure. Figure 9B In the wild-type AacSHC (SEQ ID NO: 1), it is highlighted with white letters on a black background.

[0314] Any combination of amino acid alterations described herein is envisioned. Specifically, combinations of amino acid alterations at positions corresponding to the combinations identified in AacSHC and WO2016 / 17009 herein are envisioned.

[0315] In some embodiments, the SHC / HAC enzyme variant is identical to SEQ ID NO: 1, except for the following amino acid substitutions:

[0316] (i) M132R, A224V, I432T, A557T, and H4331L (SEQ ID NO: 2); or

[0317] (ii) M132R, A224V, I432T, A557T, and R613S (SEQ ID NO: 3); or

[0318] (iii) M132R, A224V, I432T, A557T, Y81H, and R613S (SEQ ID NO: 4); or

[0319] (iv) M132R, A224V, I432T, A557T, Y81H, and H431L (SEQ ID NO: 5); or

[0320] (v) M132R, A224V, I432T, T90A, and R613S (SEQ ID NO: 17); or

[0321] (vi) M132R, A224V, I432T, A172T and M277K (SEQ ID NO: 18).

[0322] For example, compared to the 215G2 SHC enzyme, the new SHC / HAC enzyme variant may have increased enzymatic activity for converting EEH to (-)-ambroside or BisEEH to ambergris oxide. Increased enzymatic activity can refer to any aspect of the enzymatic conversion of EEH to (-)-ambroside or BisEEH to ambergris oxide, including, for example, increased total conversion of EEH or BisEEH, increased conversion rate of EEH or BisEEH (e.g., in the first 6 or 12 hours of the reaction), increased yield of (-)-ambroside or ambergris oxide, and reduced yield of byproducts. Increased enzymatic activity can generally be translated into increased productivity, which can be defined based on (-)-ambroside or ambergris oxide produced per liter of reaction capacity and per hour of bioconversion time, or per liter of reaction capacity per hour of reaction time (i.e., time after substrate addition) and per gram of biocatalyst used in the reaction.

[0323] For example, the new SHC / HAC enzyme variant can provide increased EEH or BisEEH conversion compared to the 215G2 SHC enzyme. Therefore, the method described herein can have increased EEH or BisEEH conversion levels compared to the method using the 215G2 SHC enzyme. For example, the new SHC / HAC enzyme variant can provide increased EEH or BisEEH conversion rates compared to the 215G2 SHC enzyme. Therefore, the method described herein can have increased EEH or BisEEH conversion rates compared to the 215G2 SHC enzyme.

[0324] For example, the novel SHC / HAC enzyme variant may provide increased EEH or BisEEH conversion rates in the first 4, 6, 8, 12, or 24 hours of the reaction compared to the 215G2 SHC enzyme. Therefore, the methods described herein may have increased EEH or BisEEH conversion rates in the first 4, 6, 8, 12, or 24 hours of the reaction compared to the 215G2 SHC enzyme. This can be compared with using both enzymes (i.e., the novel SHC / HAC enzyme variant and the 215G2 SHC enzyme) under the same reaction conditions (e.g., the same pH and temperature) or with using each enzyme under its respective optimized reaction conditions (e.g., optimized pH and temperature) (which may differ from each other).

[0325] For example, a novel SHC / HAC enzyme variant can convert at least about 40% of EEH or BisEEH within the first 12 hours of the reaction, or the method can allow for at least about 40% conversion of EEH or BisEEH within the first 12 hours of the reaction. For example, a novel SHC / HAC enzyme variant can convert at least about 45%, at least about 50%, at least about 55%, or at least about 60% of EEH or BisEEH within the first 12 hours of the reaction, or the method can allow for at least about 45%, at least about 50%, at least about 55%, or at least about 60% conversion of EEH or BisEEH within the first 12 hours of the reaction. For example, a novel SHC / HAC enzyme variant can convert at least about 30% of EEH or BisEEH within the first 6 hours of the reaction, or the method can allow for at least about 30% conversion of EEH or BisEEH within the first 6 hours of the reaction. For example, the new SHC / HAC enzyme variant can convert at least about 35%, or at least about 45%, or at least about 50%, or at least about 55% of EEH or BisEEH within the first 12 hours of the reaction, or the method can allow for at least about 35%, or at least about 45%, or at least about 50%, or at least about 55% of EEH or BisEEH conversion within the first 12 hours of the reaction. This can be compared with 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 with using each enzyme under its respective optimized reaction conditions (e.g., optimized pH and temperature) (which may differ from each other).

[0326] The conversion of EEH to (-)-ambroside or BisEEH to ambergris oxide can be determined, for example, by the activity assay described above, and can be calculated as the number of grams of recyclable product per gram of feedstock (which can be calculated as a percentage molar conversion).

[0327] As used herein, the 99% / 100% conversion of cyclofarnesol substrate to (-)-ambrosyl or biscyclofarnesol substrate to ambergris oxide refers to the ability to convert to isomers of (-)-ambrosyl or ambergris oxide using SHC / HAC enzymes or enzyme variants.

[0328] The optimal temperature for SHC / HAC enzyme variants can be, for example, equal to or greater than about 35°C. For example, the optimal temperature range for SHC / HAC enzyme variants can be from about 40°C to about 50°C, such as from about 42°C to about 48°C or from about 44°C to about 46°C. For example, the optimal temperature for SHC / HAC enzyme variants can be from about 4... C. The method for preparing (-)-ambroside or ambergris oxide disclosed herein can be carried out at the optimal temperature of the SHC / HAC enzyme variant.

[0329] The optimal pH for the SHC / HAC enzyme variant may be, for example, equal to or greater than about 5.4. For example, the optimal pH for the SHC / HAC enzyme variant may be from about 5.2 to about 6.0, for example from about 5.4 to about 5.8, for example from about 5.6 to about 5.8. For example, the optimal pH for the SHC / HAC enzyme variant may be about 5.6 or about 5.8. The methods disclosed herein for preparing (-)-ambroxol or ambergris oxide can be carried out at the optimal pH of the SHC / HAC enzyme variant.

[0330] The optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium for the method disclosed herein for preparing (-)-ambroside or ambergris oxide is 4 g / L with an OD of 10. 650nm When used with substrates (e.g., EEH or BisEEH) in cells, the concentration can be, for example, from about 0.010 w / w% to about 0.10 w / w%. For example, the optimal concentration of SDS is 4 g / L with 10 OD. 650nm When used with a substrate (e.g., EEH or BisEEH) in the same manner as cells, the concentration can be from about 0.040 w / w% to about 0.080 w / w, for example, about 0.050 w / w. The optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the method disclosed herein for preparing (-)-ambroside or ambergris oxide can be, for example, from about 1.0 w / w% to about 1.5 w / w when used with a substrate (e.g., EEH or BisEEH) at 125 g / l and 250 g / l cells. The optimal concentration of sodium dodecyl sulfate (SDS) in the reaction medium of the method disclosed herein for preparing (-)-ambroside or ambergris oxide can be, for example, from about 0.45 w / w% to about 0.85 w / w, for example, about 0.65 w / w, when used with a substrate (e.g., EEH or BisEEH) at 125 g / l and 125 g / l cells. For example, when using a substrate (e.g., EEH or BisEEH) at 125 g / L with 250 g / L of cells, the optimal concentration of SDS can be from about 1.2 w / w% to about 1.4 w / w, for example, about 1.3 w / w.

[0331] The methods disclosed herein for preparing (-)-ambroxan or ambergris oxide can be carried out at the optimal temperature range or optimal temperature and / or optimal pH range or optimal pH and / or optimal SDS concentration range or optimal SDS concentration for the specific enzyme used, as shown in Tables 7, 9 or 11 of the examples below.

[0332] The following numbered paragraphs define further aspects of this disclosure.

[0333] 1. A method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol, said method comprising enzymatically converting (3E,7E)-mifetalol (EEH) or a mixture of mifetalol isomers containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol using an SHC / HAC enzyme variant.

[0334] The SHC / HAC enzyme variants have an amino acid sequence that is at least about 70.0% identical to the wild-type SHC / HAC enzyme sequence.

[0335] The SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1.

[0336] 2. A method for preparing ambergris oxide or a mixture containing ambergris oxide, said method comprising enzymatically converting E,E-bis ...

[0337] The SHC / HAC enzyme variants have an amino acid sequence that is at least about 70.0% identical to the wild-type SHC / HAC enzyme sequence.

[0338] The SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1.

[0339] 3. The method in any of the preceding paragraphs, 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.

[0340] 4. The method of any of the preceding paragraphs, 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 the wild-type SHC / HAC enzyme.

[0341] 5. The method of any one of paragraphs 1 to 4, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at positions 90 and 613 corresponding to SEQ ID NO: 1.

[0342] 6. The method of any one of paragraphs 1 to 4, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at positions 172 and 277 corresponding to SEQ ID NO: 1.

[0343] 7. The method of any one of paragraphs 1 to 4, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at at least one 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.

[0344] 8. The method of paragraph 7, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid alteration relative to the wild-type SHC / HAC enzyme at positions 557 and 431 corresponding to SEQ ID NO: 1.

[0345] 9. The method of paragraph 7, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid alteration relative to the wild-type SHC / HAC enzyme at positions 557 and 613 corresponding to SEQ ID NO: 1.

[0346] 10. The method of paragraph 8 or 9, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at position 81 corresponding to SEQ ID NO: 1.

[0347] 11. The method of any of the preceding paragraphs, wherein one or more, for example, all amino acid changes at positions 81, 90, 172, 277, 431, 557 and 613 are substitutions, for example, non-conservative substitutions.

[0348] 12. Any of the methods described in the preceding paragraphs, wherein:

[0349] The amino acid change at position 81 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, such as histidine; and / or

[0350] The amino acid change at position 90 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, such as alanine; and / or

[0351] The amino acid change corresponding to position 172 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as threonine; and / or

[0352] The amino acid change corresponding to position 277 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, such as lysine; and / or

[0353] The amino acid change at position 431 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, such as leucine; and / or

[0354] The amino acid change corresponding to position 557 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as threonine; and / or

[0355] The amino acid change at position 613 corresponding to SEQ ID NO: 1 replaces the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as serine.

[0356] 13. Any of the methods described in the preceding paragraph, wherein the SHC / HAC enzyme variant amino acid sequence has one or more further amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0357] The amino acid change corresponds to position 132 of SEQ ID NO: 1;

[0358] The amino acid change corresponding to position 224 of SEQ ID NO: 1; and

[0359] The amino acid change corresponds to position 432 of SEQ ID NO: 1.

[0360] 14. The method in paragraph 13, in which:

[0361] The amino acid change corresponding to position 132 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a basic amino acid, such as arginine; and / or

[0362] The amino acid change corresponding to position 224 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as valine; and / or

[0363] The amino acid change at position 432 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a neutral hydrophilic amino acid, such as threonine.

[0364] 15. Any of the methods described in the preceding paragraph, wherein the SHC / HAC enzyme variant amino acid sequence has one or more further amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0365] The amino acid change corresponding to position 77 of SEQ ID NO: 1; and / or

[0366] The amino acid change corresponding to position 92 of SEQ ID NO: 1; and / or

[0367] The amino acid change corresponding to position 129 of SEQ ID NO: 1; and / or

[0368] The amino acid change corresponding to position 579 of SEQ ID NO: 1; and / or

[0369] The amino acid change corresponding to position 601 of SEQ ID NO: 1; and / or

[0370] The amino acid change corresponds to position 605 of SEQ ID NO: 1.

[0371] 16. The method in paragraph 15, in which:

[0372] The amino acid change at position 77 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as alanine; and / or

[0373] The amino acid change at position 92 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as valine; and / or

[0374] The amino acid change corresponding to position 129 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as leucine; and / or

[0375] The amino acid change corresponding to position 579 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a basic amino acid, such as histidine; and / or

[0376] The amino acid change at position 601 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tyrosine; and / or

[0377] The amino acid change at position 605 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tryptophan.

[0378] 17. Any of the methods described in the preceding paragraph, wherein the SHC / HAC enzyme variant amino acid sequence has one or more further amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0379] The amino acid change corresponding to position 37 of SEQ ID NO: 1; and / or

[0380] The amino acid change corresponding to position 174 of SEQ ID NO: 1; and / or

[0381] The amino acid change corresponds to position 601 of SEQ ID NO: 1.

[0382] 18. The method in paragraph 17, in which:

[0383] The amino acid change corresponding to position 37 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a neutral hydrophilic amino acid, such as glutamine; and / or

[0384] The amino acid change corresponding to position 174 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as isoleucine; and / or

[0385] The amino acid change at position 601 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tyrosine.

[0386] 19. The method of any of the preceding paragraphs, 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.

[0387] 20. Any of the methods described in the preceding paragraph, wherein the method comprises culturing recombinant host cells that produce SHC / HAC enzyme variants.

[0388] 21. The method of paragraph 20, wherein the recombinant host cell comprises a nucleic acid sequence encoding an SHC / HAC enzyme, for example 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.

[0389] 22. The method of any of the preceding paragraphs, wherein (-)-ambroxan is produced by mixing with at least one or more of the byproducts (II), (III) or (IV).

[0390] 23. A method for preparing (-)-ambroxol or a reaction mixture containing (-)-ambroxol according to any one of the preceding claims, said method comprising enzymatically converting a mixture of isomers of (3E,7E)-mifetalol (EEH) or mifetalol containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol using an SHC / HAC enzyme variant.

[0391] The SHC / HAC enzyme variants have an amino acid sequence that is at least about 70.0% 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, or SEQ ID NO: 20.

[0392] The SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 81, 90, 172, 277, 431, 557, and 613 corresponding to SEQ ID NO: 1.

[0393] The mixture of isomers containing EEH is selected from one or more of the following group: [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E) and (3E,7Z)], also named [EE:EZ], [EE:ZE] and [EE:EZ:ZE], respectively.

[0394] 24. A method for preparing ambergris oxide or a mixture containing ambergris oxide according to any one of the preceding claims, said method comprising enzymatically converting a mixture of isomers of (E,E)-bisimilar farnesol (EEH) or BisEEH-containing bisimilar farnesol using an SHC / HAC enzyme variant to ambergris oxide or a mixture containing ambergris oxide.

[0395] The SHC / HAC enzyme variants have an amino acid sequence that is at least about 70.0% 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, or SEQ ID NO: 20.

[0396] The SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 81, 90, 172, 277, 431, 557, and 613 corresponding to SEQ ID NO: 1.

[0397] The mixture of isomers containing BisEEH is selected from one or more of the following group: [(E,E) and [(Z,E)] and / or [(E,E) and (E,Z)] and / or [(Z,E), (E,E) and (E,Z)], also named [EE:EZ], [EE:ZE] and [EE:EZ:ZE], respectively.

[0398] 25. A (-)-ambroxan, which is obtained by any of the methods described in the preceding paragraph or may be obtained by any of the methods described in the preceding paragraph, for example, in amorphous or crystalline form.

[0399] 26. An ambergris oxide, which is obtained by any of the methods described in the foregoing paragraphs or may be obtained by any of the methods described in the foregoing paragraphs, for example, in an amorphous or crystalline form.

[0400] 27. Use of (-)-ambroside of paragraph 25 and / or ambergris oxide of paragraph 26 as part of a fragrance, cosmetic or consumer product.

[0401] 28. Fragrances, cosmetics or consumer products containing (-)-ambroside of paragraph 25 and / or ambergris oxide of paragraph 26.

[0402] 29. An 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 changes relative to the wild-type SHC / HAC enzyme at positions selected from positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1.

[0403] 30. The SHC / HAC enzyme variants 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.

[0404] 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 the wild-type SHC / HAC enzyme.

[0405] 32. A variant of the SHC / HAC enzyme from any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at positions 90 and 613 corresponding to SEQ ID NO: 1.

[0406] 33. A variant of the SHC / HAC enzyme from any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at positions 172 and 277 corresponding to SEQ ID NO: 1.

[0407] 34. The SHC / HAC enzyme variant of any one of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at position 557 corresponding to SEQ ID NO: 1 and at least one position corresponding to position 81, 431 or 613 corresponding to SEQ ID NO: 1.

[0408] 35. A variant of the SHC / HAC enzyme from any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at positions 557 and 431 corresponding to SEQ ID NO: 1.

[0409] 36. A variant of the SHC / HAC enzyme from any of paragraphs 29 to 31, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at positions 557 and 613 corresponding to SEQ ID NO: 1.

[0410] 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 change relative to the wild-type SHC / HAC enzyme at position 81 corresponding to SEQ ID NO: 1.

[0411] 38. Any SHC / HAC enzyme variant in paragraphs 29 to 37, wherein one or more of the amino acid changes at positions 81, 90, 172, 277, 431, 557, or 613 are, for example, all of them, substitutions, such as non-conserved substitutions.

[0412] 39. Any of the SHC / HAC enzyme variants in paragraphs 29 to 38, wherein:

[0413] The amino acid change at position 81 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, such as histidine; and / or

[0414] The amino acid change at position 90 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, such as alanine; and / or

[0415] The amino acid change corresponding to position 172 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as threonine; and / or

[0416] The amino acid change at position 277 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, such as lysine; and / or

[0417] The amino acid change at position 431 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, such as leucine; and / or

[0418] The amino acid change corresponding to position 557 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as threonine; and / or

[0419] The amino acid change at position 613 corresponding to SEQ ID NO: 1 replaces the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as serine.

[0420] 40. An SHC / HAC enzyme variant of any one of paragraphs 29 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more further amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0421] The amino acid change corresponds to position 132 of SEQ ID NO: 1;

[0422] The amino acid change corresponding to position 224 of SEQ ID NO: 1; and

[0423] The amino acid change corresponds to position 432 of SEQ ID NO: 1.

[0424] 41. The SHC / HAC enzyme variant in paragraph 40, wherein:

[0425] The amino acid change corresponding to position 132 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a basic amino acid, such as arginine; and / or

[0426] The amino acid change corresponding to position 224 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as valine; and / or

[0427] The amino acid change at position 432 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a neutral hydrophilic amino acid, such as threonine.

[0428] 42. An SHC / HAC enzyme variant of any one of paragraphs 29 to 41, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more further amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0429] The amino acid change corresponding to position 77 of SEQ ID NO: 1; and / or

[0430] The amino acid change corresponding to position 92 of SEQ ID NO: 1; and / or

[0431] The amino acid change corresponding to position 129 of SEQ ID NO: 1; and / or

[0432] The amino acid change corresponding to position 579 of SEQ ID NO: 1; and / or

[0433] The amino acid change corresponding to position 601 of SEQ ID NO: 1; and / or

[0434] The amino acid change corresponds to position 605 of SEQ ID NO: 1.

[0435] 43. The SHC / HAC enzyme variant in paragraph 42, wherein:

[0436] The amino acid change at position 77 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as alanine; and / or

[0437] The amino acid change at position 92 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as valine; and / or

[0438] The amino acid change corresponding to position 129 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as leucine; and / or

[0439] The amino acid change corresponding to position 579 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a basic amino acid, such as histidine; and / or

[0440] The amino acid change at position 601 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tyrosine; and / or

[0441] The amino acid change at position 605 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tryptophan.

[0442] 44. An SHC / HAC enzyme variant of any one of paragraphs 29 to 43, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more further amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0443] The amino acid change corresponding to position 37 of SEQ ID NO: 1; and / or

[0444] The amino acid change corresponding to position 174 of SEQ ID NO: 1; and / or

[0445] The amino acid change corresponds to position 601 of SEQ ID NO: 1.

[0446] 45. The SHC / HAC enzyme variant in paragraph 44, wherein:

[0447] The amino acid change corresponding to position 37 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a neutral hydrophilic amino acid, such as glutamine; and / or

[0448] The amino acid change corresponding to position 174 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as isoleucine; and / or

[0449] The amino acid change at position 601 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tyrosine.

[0450] 46. ​​An SHC / HAC enzyme variant of any one 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.

[0451] 47. The nucleic acid sequence of any one of the SHC / HAC enzyme variants in paragraphs 29 to 46.

[0452] 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.

[0453] 49. A construct containing the nucleic acid sequence of paragraph 47 or 48.

[0454] 50. The carrier of the construct containing paragraph 49.

[0455] 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.

[0456] 52. The recombinant host cell of paragraph 51, wherein the construct is integrated into the genome of the host cell.

[0457] 53. The recombinant host cell of paragraph 51 or 52, wherein the recombinant host cell is selected from prokaryotic, yeast, plant and / or insect host cells.

[0458] 54. A recombinant host cell of any one of paragraphs 51 to 53, wherein the recombinant host cell is a cell selected from the genus Escherichia (…). Escherichia Streptomyces ( Streptomyces ), Bacillus spp. Bacillus ), Pseudomonas spp. Pseudomonas Lactobacillus () Lactobacillus ) and Lactococcus spp. Lactococcus Bacteria of the genus *Escherichia coli*, for example, the recombinant host cell described therein is *Escherichia coli*. E. coli. ).

[0459] 55. The method of any one of paragraphs 1-24, wherein a mixture of homofarin alcohol isomers comprising EEH includes a mixture of EE:EZ isomers.

[0460] 56. The method of paragraph 55, wherein the weight ratio of the EE:EZ isomer mixture 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; or the EE:EZ isomer mixture is selected from: 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.

[0461] 57. The method in paragraph 56, wherein the weight ratio of the EE:EZ isomer mixture is 80:20.

[0462] 58. The method of any one of paragraphs 1-24 or any one of paragraphs 55-57, wherein the weight ratio of the SHC / HAC biocatalyst to EEH or a mixture of EEH-containing isomers (preferably an EE:EZ isomer mixture in a weight ratio of 80:20) is in the range of about 0.5-2:1, about 0.25-2:1, about 0.1-2:1, about 1:1, or about 0.5:1.

[0463] 59. The method of paragraph 58, wherein the weight ratio of the SHC / HAC biocatalyst to EEH or a mixture of EEH-containing isomers of eugenol (preferably a mixture of EE:EZ isomers in a weight ratio of 80:20) is in the range of about 1:1 or about 0.5:1 or about 0.1:1.

[0464] Other SHC / HAC enzymes and enzyme variants

[0465] Further surprising findings revealed that the SHC / HAC enzyme variant also acts on other substrates, such as E,E-bis-farnesol, to produce products such as ambergris oxide.

[0466] Furthermore, it was surprisingly found that some wild-type SHC enzymes offered improved (i.e., higher) selectivity for EEH relative to other isomers of equimolarin compared to WT AacSHC.

[0467] Therefore, it is expected that other wild-type SHC / HAC enzymes and other variants of wild-type SHC / HAC enzymes will also provide enzymatic activity (e.g., improved enzymatic activity) for converting EEH to (-)-ambroside and / or BisEEH to ambergris oxide.

[0468] Therefore, this paper provides a method for preparing (-)-ambroside by enzymatically converting EEH to (-)-ambroside. This paper also provides a method for preparing ambergris oxide by enzymatically converting E,E-bisimilar farnesol to ambergris oxide. These methods can use any wild-type SHC / HAC enzyme or enzyme variant described herein.

[0469] In addition, this article provides SHC / HAC enzymes or SHC / HAC enzyme variants that have at least about 70.0% identity with the amino acid sequence of wild-type SHC / HAC enzymes.

[0470] In particular, this article provides a method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol, the method comprising enzymatically converting a mixture of EEH or a imipenem containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol using an SHC / HAC enzyme or a variant of an SHC / HAC enzyme having at least about 70.0% identity with the amino acid sequence of a wild-type SHC / HAC enzyme.

[0471] In particular, this article provides a method for preparing ambergris oxide or a mixture containing ambergris oxide, the method comprising enzymatically converting BisEEH or a mixture of bis-mimic farnesol isomers containing BisEEH to ambergris oxide or a mixture containing ambergris oxide using an SHC / HAC enzyme or a variant of an SHC / HAC enzyme having at least about 70.0% identity with the amino acid sequence of a wild-type SHC / HAC enzyme.

[0472] The “selectivity” of an enzyme or enzyme variant refers to its ability to react with a particular substrate compared to another substrate. For example, a WT SHC enzyme or a variant of WT SHC that is selective for EEH relative to other isomers of homofarin or for BisEEH relative to other isomers of bihomfarin means that the WT SHC enzyme or the variant of WT SHC is more likely to convert EEH than other isomers of homofarin or more likely to convert BisEEH than other isomers of bihomfarin.

[0473] For example, the wt% of the total product formed by the reaction of WT SHC enzyme or a variant of WT SHC with EEH may be at least about 1 percentage point higher than the wt% of the total product formed by the reaction of WT AacSHC with EEH. For example, the wt% of the total product formed by the reaction of WT SHC enzyme or a variant of WT SHC with EEH may be at least about 2, at least about 3, or at least about 4 percentage points higher than the wt% of the total product formed by the reaction of WT AacSHC with EEH. For example, the wt% of the total product formed by the reaction of WT SHC enzyme or a variant of WT SHC with EEH may be as high as about 40, as high as about 30, as high as about 20, as high as about 15, or as high as about 10 percentage points higher than the wt% of the total product formed by the reaction of WT AacSHC with EEH. For example, the wt% of the total product formed by the reaction of WT SHC enzyme or a variant of WT SHC with EEH may be about 1 to about 40, or about 2 to about 30, or about 3 to about 20, or about 4 to about 10 percentage points higher than the wt% of the total product formed by the reaction of WT AacSHC with EEH. The total product formed by the reaction of WT SHC enzyme or a variant of WT SHC or WT AacSHC, when EEH is used as a substrate, may, for example, contain, consist substantially of, or consist of compounds of formula (I) ((-)-ambroxol) and formula (IV) described herein. The total product formed by the reaction of WT SHC enzyme or a variant of WT SHC or a variant of WT AacSHC, when bisEEH is used as a substrate, may, for example, contain, consist substantially of, or consist of compounds of formula (X) and / or formula (XII) described herein.

[0474] For example, the wt% of (-)-ambroxol formed using WT SHC enzyme or an enzyme variant of WT SHC can be, for example, at least about 1 percentage point higher than the wt% of (-)-ambroxol formed due to the reaction of WT AacSHC with EEH. For example, the wt% of (-)-ambroxol formed due to the reaction of WT SHC enzyme or an enzyme variant of WT SHC with EEH can be at least about 2, at least about 3, or at least about 4 percentage points higher than the wt% of (-)-ambroxol formed due to the reaction of WT AacSHC with EEH. For example, the wt% of (-)-ambroxol formed due to the reaction of WT SHC enzyme or an enzyme variant of WT SHC with EEH can be as high as about 40, as high as about 30, as high as about 20, as high as about 15, or as high as about 10 percentage points higher than the wt% of (-)-ambroxol formed due to the reaction of WT AacSHC with EEH. For example, the wt% of (-)-ambroxol formed by the reaction of WT SHC enzyme or an enzyme variant of WT SHC with EEH can be about 1 to about 40, or about 2 to about 30, or about 3 to about 20, or about 4 to about 10 percentage points higher than the wt% of (-)-ambroxol formed by the reaction of WT AacSHC with EEH.

[0475] The selectivity of WT SHC enzymes or WT SHC enzyme variants can also be compared with the selectivity of WT AacSHC or WT AacSHC variants by comparing the EEH:EZH conversion rate (i.e., % conversion of EEH:% conversion of EZH) or the bisEEH:bisEZH conversion rate (i.e., % conversion of bisEEH:% conversion of bisEZH) of the reaction using each enzyme. This can be determined by measuring the amount of EEH and EZH or bisEEH and bisEZH remaining in the reaction mixture at the end of the reaction. Alternatively, the selectivity of WT SHC enzymes or WT SHC enzyme variants can also be compared with the selectivity of WT AacSHC or WT AacSHC variants by comparing the ratio of products generated by conversion with EEH (compounds of formulas I and IV) and EZH (compounds of formulas II and III) or by conversion with bisEEH (compounds of formulas X and XII) and bisEZH (compounds of formulas XI and XIII), respectively.

[0476] SHC enzymes or WT SHC enzyme variants can, for example, provide an EEH:EZH conversion rate of at least about 2.0 in the preparation of (-)-ambroxol from a mixture containing EEH and EZH. For example, WT SHC enzymes or WT SHC enzyme variants can provide an EEH:EZH conversion rate of at least about 2.5 or at least about 3.0 or at least about 3.5 in the preparation of (-)-ambroxol from a mixture containing EEH and EZH. For example, WT SHC enzymes or WT SHC enzyme variants can provide an EEH:EZH conversion rate of up to about 5.0 or up to about 4.5 or up to about 4.0 in the preparation of (-)-ambroxol from a mixture containing EEH and EZH. For example, WT SHC enzymes or enzyme variants of WT SHC can provide an EEH:EZH conversion rate in the range of about 2.0 to about 5.0, about 2.5 to about 4.5, or about 3.0 to about 4.0 in the preparation of (-)-ambroxol from a mixture containing EEH and EZH. This may, for example, contrast with the conversion rate provided by AacSHC in the preparation of (-)-ambroxol from a mixture containing EEH and EZH, which may be, for example, less than about 2.0.

[0477] Wild-type SHC / HAC enzymes (e.g., SHC / HAC enzyme variants from which SHC / HAC enzymes may be derived) can be, for example, SHCs obtained from Acidothermia cyclophosphamide (Aac), Zmo, Bj. japonicus (Bjp), Gmo, Burkholderia diversicolor, Bacillus anthracis, Methylcoccus capsulatum, Frankerella, Acetobacter pastoris (Apa), Synechococcus thermophilus (Tel), Streptomyces coli (Sco), Rhodopseudomonas palustris (Rpa), Treddy bacteria (Ttu), Bacillus anthracis (Pca), Tetrahymena piriformis (Tpy), Bacillus megaterium (Bme), or Streptomyces alba (Sal) (see, for example, WO2010 / 139719, US2012 / 01345477, WO2012 / 066059, the contents of which are incorporated herein by reference).

[0478] Specifically, wild-type SHC / HAC enzymes (e.g., SHC / HAC enzyme variants from which can be derived) can be *Aac* SHC / HAC enzymes, *Zmo* SHC / HAC enzymes, *Bjp / Bja* SHC / HAC enzymes, *Apa* SHC / HAC enzymes, *Bme* SHC / HAC enzymes, or *Gmo* SHC / HAC enzymes. Specifically, wild-type SHC / HAC enzymes (e.g., SHC / HAC enzyme variants from which can be derived) can be *Aac* SHC / HAC enzymes.

[0479] When a wild-type SHC enzyme or a variant of WT SHC exhibits higher selectivity for EEH relative to other isomers of cyclofarnesol compared to variants of AacSHC and / or AacSHC, the wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) is not an Aac. Specifically, when a wild-type SHC enzyme or a variant of WT SHC exhibits higher selectivity for EEH relative to other isomers of cyclofarnesol compared to variants of WT AacSHC and / or WT AacSHC, the wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) may be selected from TelSHC1, ApaSHC1, ZmoSHC1, ZmoSHC2, BjaSHC, GmoSHC, BmeSHC, SalSHC, and ApaSHCA. For example, when a wild-type SHC enzyme or a variant of WT SHC has higher selectivity for EEH relative to other isomers of sine acephate compared to variants of WT AacSHC and / or WTAacSHC, a wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) may be selected from ZmoSHC1, BjaSHC, GmoSHC, ApaSHC1, and BmeSHC.

[0480] For ease of reference, the names "AacSHC" can be used to refer to Aac (Aac) SHC / HAC enzyme, "ZmoSHC" can be used to refer to Zmo (Zmo) SHC / HAC enzyme, "BjpSHC" or "BjaSHC" can be used to refer to Bjp (Bjp) SHC / HAC enzyme, "GmoSHC" can be used to refer to Gmo (Gmo) SHC / HAC enzyme, and "BmeSHC" can be used to refer to Bacillus megaterium SHC / HAC enzyme.

[0481] SHC / HAC enzymes or SHC / HAC enzyme variants may, for example, have an amino acid sequence that is at least about 70.0% identical to the amino acid sequence of wild-type SHC / HAC enzymes. For instance, SHC / HAC enzymes or SHC / HAC enzyme variants may have an amino acid sequence that is at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 95.5%, at least about 96.5%, at least about 97.0%, at least about 97.5%, at least about 98.0%, at least about 98.5%, or at least about 99.0% identical to the amino acid sequence of wild-type SHC / HAC enzymes.

[0482] SHC / HAC enzymes have an amino acid sequence that is 100% identical to that of wild-type SHC / HAC enzymes.

[0483] SHC / HAC enzyme variants have amino acid sequences that are less than 100% identical to wild-type SHC / HAC enzyme sequences, for example, equal to or less than about 99.5% or equal to or less than about 99.0% identical.

[0484] For example, the amino acid sequence of the SHC / HAC enzyme variant may have about 70.0% to about 99.5%, about 80.0% to about 99.0%, about 85.0% to about 98.5%, or about 90.0% to about 98.0%.

[0485] The amino acid sequence of the wild-type SHC / HAC enzyme can 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 can be AacSHC (SEQ ID NO: 1).

[0486] Therefore, in some embodiments, the SHC / HAC enzyme or SHC / HAC enzyme variant may have an amino acid sequence that is at least about 70.0% 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. For example, an SHC / HAC enzyme or an SHC / HAC enzyme variant 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% of the same amino acid sequence as 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.

[0487] For example, SHC / HAC enzymes may have amino acid sequences that are 100% identical to those of 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.

[0488] For example, SHC / HAC enzyme variants may have amino acid sequences that are less than 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, for example, equal to or less than about 99.5% or equal to or less than about 99.0% identical.

[0489] For example, the SHC / HAC enzyme variant may have about 70.0% to about 99.5%, about 80.0% to about 99.0%, about 85.0% to about 98.5%, or about 90.0% to about 98.0% of the identity with SEQ ID NO: 1, 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.

[0490] The "percentage (%) identity" of a polypeptide or nucleotide sequence is defined, respectively, as the percentage of amino acids or nucleotides in a candidate sequence that are identical to those in a reference sequence after alignment of sequences and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignment for determining the percentage of sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modifications.

[0491] The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. Such alignment can be performed using several algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER software package, http: / / hmmer.wustl.edu / ), or using, for example, methods available from [source missing]. https: / / www.ebi.ac.uk / Tools / msa / clustalo / The preferred parameters used are the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), or the GAP procedure (a mathematical algorithm from the University of Iowa), or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17). https: / / www.ebi.ac.uk / Tools / msa / clustalo / The default parameters set above.

[0492] Sequence identity percentages can be calculated using algorithms such as BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms were incorporated into the BLASTN and BLASTP programs in Altschul et al. (1990) J. Mol. Biol. 215, 403-410. A BLAST polynucleotide search can be performed using the BLASTN program (score=100, word length=12) to obtain polynucleotide sequences homologous to nucleic acids encoding related proteins. A BLAST protein search can be performed using the BLASTP program (score=50, word length=3) to obtain amino acid sequences homologous to peptides.

[0493] To obtain vacancy alignments for comparative purposes, vacancy BLAST is used, as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and vacancy BLAST procedures, the default parameters of each procedure are used. Sequence matching analysis can 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, unless otherwise specified, these percentages are calculated relative to the full length of longer sequences.

[0494] In a particular implementation, CLUSTAL O (version 1.2.4) is used to determine the % identity between two sequences.

[0495] In some embodiments, the SHC / HAC enzyme variant may have an amino acid change equal to or less than about 200 compared to the wild-type SHC / HAC enzyme, for example, 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. For example, compared to the wild-type SHC / HAC enzyme, for example, 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. Compared to NO:30, the SHC / HAC enzyme variants can have amino acid changes of equal to or less than about 150 or equal to or less than about 120 or equal to or less than about 100 or equal to or less than about 95 or equal to or less than about 90 or equal to or less than about 85 or equal to or less than about 80 or equal to or less than about 75 or equal to or less than about 70 or equal to or less than about 65 or equal to or less than about 60 or equal to or less than about 55 or equal to or less than about 50 or equal to or less than about 45 or equal to or less than about 40 or equal to or less than about 35 or equal to or less than about 30 or equal to or less than about 25 or equal to or less than about 20 or equal to or less than about 15 or equal to or less than about 10.

[0496] For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have at least about 1 or at least about 2 or at least about 3 or at least about 4 or at least about 5 or at least about 6 amino acid changes.

[0497] For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 1 to about 30 amino acid changes. For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 2 to about 25 amino acid changes. For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 3 to about 20 amino acid changes. For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 4 to about 15 amino acid changes. For example, compared to wild-type SHC / HAC enzymes, such as 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, SHC / HAC enzyme variants may have about 5 to about 10 amino acid changes.

[0498] For example, amino acid changes can be insertions, deletions, and / or substitutions as described above. For example, amino acid changes can be substitutions, such as non-conservative substitutions.

[0499] In some embodiments, the only amino acid change compared to wild-type SHC / HAC enzymes (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) is substitution (i.e. no insertion or deletion).

[0500] Amino acid changes are defined relative to a reference sequence. An amino acid change relative to a reference sequence means that the amino acid sequence of the variant sequence differs from the reference sequence.

[0501] Amino acids in the reference sequence and variant sequences can be assigned numbers, with the numbering starting from the N-terminal amino acid of the polypeptide (i.e., the N-terminal amino acid of the polypeptide is numbered 1, the next amino acid is numbered 2, and so on). The "position" of the reference sequence refers to the specific amino acid residue present in the reference sequence, as identified by its specific number. The "position" of the variant sequence refers to the specific amino acid residue present in the variant sequence, as identified by its specific number.

[0502] Because variant sequences can include deletions or insertions compared to the reference sequence, the amino acid numbers in the variant sequence may differ from those in the reference sequence. For example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO: 1, the amino acid following the insertion will have number 133 in the variant sequence, while it retains number 132 in the reference sequence. In this example, the position in the variant sequence corresponding to position 132 in the reference sequence is position 133. Therefore, the amino acid retained from the reference sequence in the variant sequence can be defined with reference to the “corresponding position” in the reference sequence. In other words, the “position” in the variant sequence can be defined by referring to the “corresponding position” in the reference sequence. In particular, substitutions in the variant sequence compared to the reference sequence can be defined by referring to the “corresponding position” in the reference sequence, even if any insertions and / or deletions are present in the reference sequence. In the case of deletions of amino acids in the reference sequence, there is no “corresponding position” in the variant sequence. If there are no insertions or deletions compared to the reference sequence (i.e., only substitutions), the “corresponding position” in the reference sequence will be the same as the position in the variant sequence.

[0503] Wild-type SHC / HAC enzymes from different species have different polypeptide lengths. Wild-type sequences can be compared using the algorithm described above to identify “corresponding positions” in two different wild-type SHC / HAC enzymes. Therefore, the amino acid at the position in the variant sequence corresponding to the position in the reference sequence can be, for example, a different amino acid residue and / or can have a different number than the reference sequence number. For example, amino acid M at position 132 of AacSHC (SEQ ID NO: 1) can correspond to amino acid Y at position 185 of ZmoSHC1 (SEQ ID NO: 11).

[0504] Therefore, amino acid changes can be defined relative to two different reference sequences. For example, an amino acid change can be a change compared to a first reference sequence (e.g., the wild-type SHC / HAC enzyme sequence of a derived variant), and the position of the amino acid change in the variant sequence can be defined by referring to a second reference sequence (e.g., AacSHC (SEQ ID NO: 1)). Thus, an amino acid change in an SHC / HAC enzyme variant can be located relative to the first wild-type SHC / HAC enzyme at a position defined by referring to the second wild-type SHC / HAC enzyme.

[0505] SHC / HAC enzyme variants may, for example, have one or more specific substitutions or combinations of substitutions as defined in reference SEQ ID NO: 1 above.

[0506] In particular, the SHC / HAC enzyme variant may have one or more 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.

[0507] For example, each of these substitutions can be independently defined as described above in the subsections entitled “Aac 215G2 variants” and “other variants with novel mutations at positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1”.

[0508] In particular, SHC / HAC enzyme variants may have one or more of the following substitution combinations:

[0509] (i) Substitutions at positions 132, 224 and 432 corresponding to SEQ ID NO: 1;

[0510] (ii) Substitutions at positions 132, 224, 432, 557 and 431 corresponding to SEQ ID NO: 1;

[0511] (iii) Substitutions at positions 132, 224, 432, 557 and 613 corresponding to SEQ ID NO: 1;

[0512] (iv) Substitutions at positions 132, 224, 432, 557, 81 and 613 corresponding to SEQ ID NO: 1;

[0513] (v) Substitutions at positions 132, 224, 432, 557, 81 and 431 corresponding to SEQ ID NO: 1;

[0514] (vi) Substitutions at positions 132, 224, 432, 90 and 613 corresponding to SEQ ID NO: 1;

[0515] (vii) Substitutions at positions 132, 224, 432, 172 and 277 corresponding to SEQ ID NO: 1;

[0516] (viii) Substitution at positions 132, 224, 432 and 37 corresponding to SEQ ID NO: 1;

[0517] (ix) Substitutions at positions 132, 224, 432 and 174 corresponding to SEQ ID NO: 1;

[0518] (x) Substitutions at positions 132, 224, 432, 174 and 601 corresponding to SEQ ID NO: 1;

[0519] (xi) The substitutions at positions 132, 224, 432, 37, 174 and 601 corresponding to SEQ ID NO: 1.

[0520] In some embodiments, the SHC / HAC enzyme variant is identical to SEQ ID NO: 1, except for the following amino acid substitutions:

[0521] (i) M132R, A224V, I432T, A557T, and H431L (SEQ ID NO: 2); or

[0522] (ii) M132R, A224V, I432T, A557T, and R613S (SEQ ID NO: 3); or

[0523] (iii) M132R, A224V, I432T, A557T, Y81H, and R613S (SEQ ID NO: 4); or

[0524] (iv) M132R, A224V, I432T, A557T, Y81H, and H431L (SEQ ID NO: 5); or

[0525] (v) M132R, A224V, I432T, T90A, and R613S (SEQ ID NO: 17); or

[0526] (vi) M132R, A224V, I432T, A172T, and M277K (SEQ ID NO: 18); or

[0527] (vii) M132R, A224V, I432T and L37Q (SEQ ID NO: 24); or

[0528] (viii) M132R, A224V, I432T, V174I (SEQ ID NO: 25); or

[0529] (ix)M132R, A224V, I432T, V174I, and F601Y (SEQ ID NO: 26); or

[0530] (x)M132R, A224V, I432T, L37Q, V174I and F601Y (SEQ ID NO: 27).

[0531] The following numbered paragraphs define other aspects of this disclosure:

[0532] 1. A method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol, said method comprising enzymatically converting a mixture of (3E,7E)-mifetalol (EEH) or mifetalol isomers containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol using an SHC / HAC enzyme or an SHC / HAC enzyme variant,

[0533] The SHC / HAC enzyme or its variants have an amino acid sequence that is at least about 70.0% identical to the wild-type SHC / HAC enzyme.

[0534] Among them, the WT SHC / HAC enzyme has higher selectivity for EEH compared to other isomers of cyclophosphamide.

[0535] 2. A method for preparing ambergris oxide or a mixture containing ambergris oxide, said method comprising enzymatically converting E,E-bis ...

[0536] The SHC / HAC enzyme or its variants have an amino acid sequence that is at least about 70.0% identical to the wild-type SHC / HAC enzyme.

[0537] 3. A method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol, said method comprising enzymatically converting 3E,7E-mifetalol (EEH) or a mixture of mifetalol isomers containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol using an SHC / HAC enzyme or an SHC / HAC enzyme variant.

[0538] The SHC / HAC enzyme or SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% 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; and

[0539] The mixture of isomers containing EEH is selected from one or more of the following groups: [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E) and (3E,7Z)], also named [EE:EZ], [EE:ZE] and [EE:EZ:ZE], respectively.

[0540] 4. A method for preparing ambergris oxide or a mixture containing ambergris oxide, said method comprising enzymatically converting E,E-bis ...

[0541] The SHC / HAC enzyme or SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% 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; and

[0542] The mixture of isomers containing EEH is selected from one or more of the following groups: [(E,E) and [(Z,E)] and / or [(E,E) and (E,Z)] and / or [(Z,E), (E,E) and (E,Z)], also named [EE:EZ], [EE:ZE] and [EE:EZ:ZE], respectively.

[0543] 5. The method in paragraph 1 or 3, wherein the SHC / HAC enzyme has higher selectivity for EEH relative to other isomers of equimolar ol compared to WT AacSHC.

[0544] 6. The method of any one of paragraphs 1, 3 or 5, wherein the wt% of the total product formed by the reaction of SHC / HAC enzyme or enzyme variant with EEH is at least about 1 percentage point higher than the wt% of the total product formed by the reaction of AacSHC with EEH, for example at least about 2 percentage points or at least about 3 percentage points higher.

[0545] 7. The method of any one of paragraphs 1, 3, 5 or 6, wherein the EEH:EZH conversion rate is at least about 2.0, for example at least about 2.5 or at least about 3.0.

[0546] 8. The method of any one of paragraphs 1 to 7, wherein the wild-type SHC / HAC enzyme is obtained from Acid-Temperature Cyclocarya (Aac), Zmo, Slow-Rhizobium japonicum (Bjp), Gluconobacterium mobius (Gmo), Burkholderia diversicolor, Bacillus anthracis, Methylcoccus capsulatum, Franckella, Acetobacter pastoris (Apa), Synechococcus thermophilus (Tel), Streptomyces coli (Sco), Rhodopseudomonas palustris (Rpa), Terediella (Ttu), Bacillus anthracis (Pca), Tetrahymena piriformis, Bacillus megaterium, or Streptomyces albus.

[0547] 9. The method of any one of paragraphs 1 to 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, for example wherein the wild-type SHC / HAC enzyme is SEQ ID NO: 1.

[0548] 10. The method of paragraph 1, wherein the mixture of isomers of cyclophosphamide 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)].

[0549] 11. The method in any of the preceding paragraphs, wherein the method uses a solubilizer selected from Triton X-100, Tween 80, taurine deoxycholate, sodium taurine deoxycholate, sodium dodecyl sulfate (SDS) and / or sodium lauryl sulfate (SLS).

[0550] 12. The method of any one of paragraphs 1 to 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 the wild-type SHC / HAC enzyme.

[0551] 13. The method of any of the preceding paragraphs, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid alteration relative to the wild-type SHC / HAC enzyme at one or more positions selected from positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1.

[0552] 14. The method of any one of paragraphs 1 to 13, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change at positions 90 and 613 corresponding to SEQ ID NO: 1.

[0553] 15. The method of any one of paragraphs 1 to 13, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change at positions 172 and 277 corresponding to SEQ ID NO: 1.

[0554] 16. The method of any one of paragraphs 1 to 13, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at position 557 corresponding to SEQ ID NO: 1 and at least one position corresponding to position 81, 431 or 613 corresponding to SEQ ID NO: 1.

[0555] 17. The method of paragraph 16, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid alteration relative to the wild-type SHC / HAC enzyme at positions 557 and 431 corresponding to SEQ ID NO: 1.

[0556] 18. The method of paragraph 16, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid alteration relative to the wild-type SHC / HAC enzyme at positions 557 and 613 corresponding to SEQ ID NO: 1.

[0557] 19. The method of paragraph 17 or 18, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at position 81 corresponding to SEQ ID NO: 1.

[0558] 20. The method of any of the preceding paragraphs, wherein one or more of the amino acid changes at positions 81, 90, 172, 277, 431, 557 or 613 are, for example, all of them, substitutions, such as non-conservative substitutions.

[0559] 21. Any of the methods described in the preceding paragraphs, wherein:

[0560] The amino acid change at position 81 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, such as histidine; and / or

[0561] The amino acid change at position 90 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, such as alanine; and / or

[0562] The amino acid change corresponding to position 172 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as threonine; and / or

[0563] The amino acid change at position 277 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, such as lysine; and / or

[0564] The amino acid change at position 431 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, such as leucine; and / or

[0565] The amino acid change corresponding to position 557 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as threonine; and / or

[0566] The amino acid change at position 613 corresponding to SEQ ID NO: 1 replaces the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as serine.

[0567] 22. Any of the methods described in the preceding paragraph, wherein the SHC / HAC enzyme variant amino acid sequence has one or more amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0568] The amino acid change corresponds to position 132 of SEQ ID NO: 1;

[0569] The amino acid change corresponding to position 224 of SEQ ID NO: 1; and

[0570] The amino acid change corresponds to position 432 of SEQ ID NO: 1.

[0571] 23. The method in paragraph 22, among which:

[0572] The amino acid change corresponding to position 132 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a basic amino acid, such as arginine; and / or

[0573] The amino acid change corresponding to position 224 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as valine; and / or

[0574] The amino acid change at position 432 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a neutral hydrophilic amino acid, such as threonine.

[0575] 24. Any method described in the preceding paragraph, wherein the SHC / HAC enzyme variant amino acid sequence has one or more amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0576] The amino acid change corresponding to position 77 of SEQ ID NO: 1; and / or

[0577] The amino acid change corresponding to position 92 of SEQ ID NO: 1; and / or

[0578] The amino acid change corresponding to position 129 of SEQ ID NO: 1; and / or

[0579] The amino acid change corresponding to position 579 of SEQ ID NO: 1; and / or

[0580] The amino acid change corresponding to position 601 of SEQ ID NO: 1; and / or

[0581] The amino acid change corresponds to position 605 of SEQ ID NO: 1.

[0582] 25. The method in paragraph 24, among which:

[0583] The amino acid change at position 77 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as alanine; and / or

[0584] The amino acid change at position 92 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as valine; and / or

[0585] The amino acid change corresponding to position 129 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as leucine; and / or

[0586] The amino acid change corresponding to position 579 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a basic amino acid, such as histidine; and / or

[0587] The amino acid change at position 601 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tyrosine; and / or

[0588] The amino acid change at position 605 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tryptophan.

[0589] 26. Any method described in the preceding paragraph, wherein the SHC / HAC enzyme variant amino acid sequence has one or more further amino acid changes selected from the wild-type SHC / HAC amino acid sequence:

[0590] The amino acid change corresponding to position 37 of SEQ ID NO: 1; and / or

[0591] The amino acid change corresponding to position 174 of SEQ ID NO: 1; and / or

[0592] The amino acid change at position 601 corresponding to SEQ ID NO: 1.

[0593] 27. The method in paragraph 26, among which:

[0594] The amino acid change corresponding to position 37 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a neutral hydrophilic amino acid, such as glutamine; and / or

[0595] The amino acid change corresponding to position 174 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as isoleucine; and / or

[0596] The amino acid change at position 601 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tyrosine.

[0597] 28. The method of any of the preceding paragraphs, 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.

[0598] 29. Any of the methods described in the preceding paragraph, wherein the method comprises culturing recombinant host cells that produce SHC / HAC enzyme variants.

[0599] 30. The method of paragraph 29, wherein the recombinant host cell comprises a nucleic acid sequence encoding an SHC / HAC enzyme, for example 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.

[0600] 31. The method of any of the preceding paragraphs, wherein (-)-ambroxan is produced by mixing with at least one or more of the byproducts (II), (III) or (IV).

[0601] 32. A (-)-ambroxan, which is obtained by any of the methods described in the preceding paragraph or may be obtained by any of the methods described in the preceding paragraph, for example, in amorphous or crystalline form.

[0602] 33. An ambergris oxide, which is obtained by any of the methods described in the foregoing paragraphs or may be obtained by any of the methods described in the foregoing paragraphs, for example, in an amorphous or crystalline form.

[0603] 34. Use of (-)-ambroside of paragraph 32 and / or ambergris oxide of paragraph 33 as part of a fragrance, cosmetic or consumer product.

[0604] 35. Fragrances, cosmetics or consumer products containing (-)-ambroside of paragraph 32 and / or ambergris oxide of paragraph 33.

[0605] 36. An SHC / HAC enzyme or a variant of an SHC / HAC enzyme 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.

[0606] 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, such as SEQ ID NO: 1.

[0607] 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 the wild-type SHC / HAC enzyme.

[0608] 39. The SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 38, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at one or more positions selected from positions 81, 90, 172, 277, 431, 557 and 613 corresponding to SEQ ID NO: 1.

[0609] 40. The SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change at positions 90 and 613 corresponding to SEQ ID NO: 1.

[0610] 41. The SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change at positions 172 and 277 corresponding to SEQ ID NO: 1.

[0611] 42. The SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at at least one position corresponding to position 557 of SEQ ID NO: 1 and at at least one position corresponding to position 81, 431 or 613 of SEQ ID NO: 1.

[0612] 43. The SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at positions 557 and 431 corresponding to SEQ ID NO: 1.

[0613] 44. The SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 39, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the wild-type SHC / HAC enzyme at positions 557 and 613 corresponding to SEQ ID NO: 1.

[0614] 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 change relative to the wild-type SHC / HAC enzyme at position 81 corresponding to SEQ ID NO: 1.

[0615] 46. ​​An SHC / HAC enzyme or enzyme variant of any of paragraphs 36 to 45, wherein one or more, for example all, amino acid changes at positions 81, 90, 172, 277, 431, 557, or 613 are substitutions, for example, non-conserved substitutions.

[0616] 47. Any of the SHC / HAC enzymes or enzyme variants listed in paragraphs 36 to 46, wherein:

[0617] The amino acid change at position 81 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, such as histidine; and / or

[0618] The amino acid change at position 90 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, such as alanine; and / or

[0619] The amino acid change corresponding to position 172 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as threonine; and / or

[0620] The amino acid change corresponding to position 277 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a basic amino acid, such as lysine; and / or

[0621] The amino acid change at position 431 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a hydrophobic amino acid, such as leucine; and / or

[0622] The amino acid change corresponding to position 557 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as threonine; and / or

[0623] The amino acid change at position 613 corresponding to SEQ ID NO: 1 replaces the amino acid in the wild-type SHC / HAC amino acid sequence with a neutral hydrophilic amino acid, such as serine.

[0624] 48. An SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 47, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC amino acid sequence, and is selected from:

[0625] The amino acid change corresponds to position 132 of SEQ ID NO: 1;

[0626] The amino acid change corresponding to position 224 of SEQ ID NO: 1; and

[0627] The amino acid change corresponds to position 432 of SEQ ID NO: 1.

[0628] 49. The SHC / HAC enzyme or enzyme variant of paragraph 48, wherein:

[0629] The amino acid change corresponding to position 132 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a basic amino acid, such as arginine; and / or

[0630] The amino acid change corresponding to position 224 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as valine; and / or

[0631] The amino acid change at position 432 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a neutral hydrophilic amino acid, such as threonine.

[0632] 50. An SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 49, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC amino acid sequence, and is selected from:

[0633] The amino acid change corresponding to position 77 of SEQ ID NO: 1; and / or

[0634] The amino acid change corresponding to position 92 of SEQ ID NO: 1; and / or

[0635] The amino acid change corresponding to position 129 of SEQ ID NO: 1; and / or

[0636] The amino acid change corresponding to position 579 of SEQ ID NO: 1; and / or

[0637] The amino acid change corresponding to position 601 of SEQ ID NO: 1; and / or

[0638] The amino acid change corresponds to position 605 of SEQ ID NO: 1.

[0639] 51. The SHC / HAC enzyme or enzyme variant of paragraph 50, wherein:

[0640] The amino acid change at position 77 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as alanine; and / or

[0641] The amino acid change at position 92 of SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as valine; and / or

[0642] The amino acid change corresponding to position 129 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as leucine; and / or

[0643] The amino acid change corresponding to position 579 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a basic amino acid, such as histidine; and / or

[0644] The amino acid change at position 601 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tyrosine; and / or

[0645] The amino acid change at position 605 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tryptophan.

[0646] 52. An SHC / HAC enzyme or enzyme variant thereof, as described in paragraphs 36 to 51, wherein the amino acid sequence of the SHC / HAC enzyme variant has one or more amino acid changes relative to the wild-type SHC / HAC amino acid sequence, and is selected from:

[0647] The amino acid change corresponding to position 37 of SEQ ID NO: 1; and / or

[0648] The amino acid change corresponding to position 174 of SEQ ID NO: 1; and / or

[0649] The amino acid change at position 601 corresponding to SEQ ID NO: 1.

[0650] 53. The SHC / HAC enzyme or enzyme variant of paragraph 52, wherein:

[0651] The amino acid change corresponding to position 37 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a neutral hydrophilic amino acid, such as glutamine; and / or

[0652] The amino acid change corresponding to position 174 of SEQ ID NO: 1 involves replacing an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with a hydrophobic amino acid, such as isoleucine; and / or

[0653] The amino acid change at position 601 corresponding to SEQ ID NO: 1 replaces an amino acid in the wild-type SHC / HAC enzyme amino acid sequence with an aromatic amino acid, such as tyrosine.

[0654] 54. The SHC / HAC enzyme or enzyme variant of any one 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.

[0655] 55. The nucleic acid sequence of the SHC / HAC enzyme or enzyme variant of any one of paragraphs 36 to 54.

[0656] 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.

[0657] 57. A construct containing the nucleic acid sequence of paragraph 55 or 56.

[0658] 58. The carrier of the construct containing paragraph 57.

[0659] 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.

[0660] 60. Recombinant host cells of paragraph 59, wherein the construct is integrated into the genome of the host cell.

[0661] 61. The recombinant host cell of paragraph 59 or 60, wherein the recombinant host cell is selected from prokaryotic, yeast, plant and / or insect host cells.

[0662] 62. The recombinant host cell of any one 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, wherein the recombinant host cell is Escherichia coli.

[0663] 63. The method of any one of paragraphs 1-31, wherein a mixture of homofarin alcohol isomers comprising EEH includes a mixture of EE:EZ isomers.

[0664] 64. The method of paragraph 63, wherein the EE:EZ isomer mixture is selected from: 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 EE:EZ isomer mixture 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.

[0665] 65. The method in paragraph 64, wherein the weight ratio of the EE:EZ isomer mixture is 80:20.

[0666] 66. The method of any one of paragraphs 1-31 or 63-65, wherein the weight ratio of the SHC / HAC enzyme to EEH or a mixture of EEH-containing isomers (preferably a mixture of EE:EZ isomers in a weight ratio of 80:20) is in the range of about 0.5-2:1, about 0.25-2:1, about 0.1-2:1, about 1:1, or about 0.5:1.

[0667] 67. The method of paragraph 66, wherein the weight ratio of the SHC / HAC enzyme to EEH or a mixture of EEH-containing isomers (preferably a mixture of EE:EZ isomers in a weight ratio of 80:20) is in the range of about 1:1 or about 0.5:1 or about 0.1:1.

[0668] The following numbered paragraphs define additional aspects of this disclosure.

[0669] 1. A method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol, wherein EE-mefenoxuron (EEH) or a mixture of isomers containing EE-mefenoxuron (EEH) is enzymatically converted to (-)-ambroxol or a mixture containing (-)-ambroxol, wherein the enzymatic conversion uses enzymes having the same characteristics as 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 ...29 (SalSHC) and / or SEQ ID NO: NO: 30 (ApaSHCA) is a squalene hopene cyclase / mesofarethol ambroxol cyclase (SHC / HAC) biocatalyst having a polypeptide sequence with 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%, and at least 99% identity. The reaction is carried out under reaction conditions suitable for producing (-)-ambroxol, and the isomer mixture containing EEH is selected from one or more of the following group: [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E) and (3E,7Z)], also named [EE:EZ] and / or [EE:ZE] and / or [EE:EZ:ZE].

[0670] 2. The method of paragraph 1, wherein the method is carried out using an 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%, or at least 99% identity with SEQ ID NO: 20 (ApaSHC1) or SEQ ID NO: 28 (BmeSHC).

[0671] 3. The method of paragraph 2, wherein the conversion of EEH or a mixture of isomers containing EEH to (-)-ambroxan is carried out at a temperature of about 30°C to about 50°C, for example, about 35°C to about 50°C, at a pH of about 5 to about 7.

[0672] 4. The method of paragraph 1 or paragraph 2, wherein the method is carried out in the presence of a solubilizer such as SDS.

[0673] 5. The method of any one of paragraphs 1-4, wherein the method comprises (a) culturing one or more recombinant host cells expressing SHC / HAC enzymes under conditions that allow for the production of WT SHC / HAC biocatalysts prior to converting EEH or a mixture of isomers containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol.

[0674] 6. According to the method in paragraph 5, the cultivation step and the subsequent transformation step are optionally carried out in the same reaction vessel under different reaction conditions.

[0675] 7. The method in paragraph 6, wherein the pH range of the culture step is 6-7, and the pH range of the EEH to (-)-ambroxan step is 4.8-5.5.

[0676] 8. The method of any one of paragraphs 1-7, wherein the isomer mixture containing EEH includes the EE:EZ isomer mixture.

[0677] 9. The method of paragraph 8, wherein the weight ratio of the EE:EZ isomer mixture is selected from: 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 weight ratio of the EE:EZ isomer mixture is: 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.

[0678] 10. The method in paragraph 9, wherein the weight ratio of the EE:EZ isomer mixture is 80:20.

[0679] 11. The method of any one of paragraphs 1-10, wherein the weight ratio of the SHC / HAC biocatalyst to EEH or a mixture of isomers containing EEH (preferably an EE:EZ isomer mixture in a weight ratio of 80:20) is about 0.5-2:1, about 0.25-2:1, about 0.1-2:1, about 1:1, or about 0.5:1.

[0680] 12. The method of paragraph 11, wherein the weight ratio of SHC / HAC enzyme to EEH or a mixture of isomers containing EEH (preferably a mixture of EE:EZ isomers in a weight ratio of 80:20) is about 1:1 or about 0.5:1 or about 0.1:1.

[0681] 13. The method of any one of paragraphs 1-12, wherein (-)-ambroxan is produced by mixing with one or more of byproducts (II), (IV) and / or (III).

[0682] 14. The method in paragraph 13, wherein (-)-ambroxan is separated from the reaction medium.

[0683] 15. The method of paragraph 14, wherein (-)-ambroxan is separated from the reaction medium using a filtration step, a decantation step, or a combination of filtration and decantation steps.

[0684] 16. The method in paragraph 15, wherein the filtering step is a belt filtering or a rotary filtering step.

[0685] 17. The method of any one of paragraphs 13-16, wherein (-)-ambroxan is heated to a maximum temperature of 55°C prior to the separation step.

[0686] 18. The method in paragraph 17, wherein the recovered (-)-ambroxan is dissolved in a solvent.

[0687] 19. The method in paragraph 18, in which the dissolved (-)-ambroxan is filtered.

[0688] 20. The method of paragraph 18 or 19, wherein (-)-ambroxan is recovered in solid form by removing the solvent through evaporation.

[0689] 21. The method of any one of paragraphs 14-20, wherein (-)-ambroxan is substantially free of byproducts (II), (IV) and / or (III).

[0690] 22. A reaction product comprising (-)-ambroxan, which can be obtained by any of the methods described in paragraphs 1-21.

[0691] 23. The reaction products in paragraph 22, of which (-)-ambroxan is in solid form.

[0692] 24. The reaction products in paragraph 23, wherein (-)-ambromide is in amorphous or crystalline form.

[0693] 25. A method for preparing a product containing (-)-ambroxan, comprising incorporating the reaction product of any one of paragraphs 22-24 into the product.

[0694] 26. The method of paragraph 25, wherein the product is a fragrance product, cosmetic product, cleaning product, detergent product and / or soap product.

[0695] 27. Fragrances, cosmetics, or consumer care products that contain the reaction product of any one of paragraphs 22-24.

[0696] 28. A fragrance, cosmetic, or consumer care composition comprising a reaction product and additional components of any one of paragraphs 22-24.

[0697] 29. Use of the reaction product of any one of paragraphs 22-24 as part of a fragrance or cosmetic consumer care product.

[0698] Ambroxol and its uses

[0699] This document further provides reaction products prepared by the methods described herein. For example, the reaction products may comprise (-)-ambroxan and one or more other compounds (e.g., one or more of compounds of formula (II), formula (III), and formula (IV), substantially composed of or consisting of those compounds.

[0700] As used herein, the term "ambroxan" includes (-)-ambroxan of formula (I) and (-)-ambroxan in pure isomer form or mixed with one or more of the following molecules of formulas (II), (III) and / or (IV).

[0701]

[0702] (I)

[0703]

[0704] The names of the reaction products of formulas (I), (II), (III) and (IV) are as follows.

[0705] Table 2. Nomenclature of the reaction products of formulas (I), (II), (III) and (IV).

[0706]

[0707] (-)-Ambroxol is commercially known as Ambrox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlyn (Quest), Cetalox Laevo (Firmenich), Ambermor (Aromor), and / or Norambrenolide Ether (Pacific).

[0708] (-)-Ambroxol is an industrially important aromatic compound and has long been used in the fragrance industry. The specific desired sensory benefits of (-)-ambroxol come from the (-) isomer, rather than the (+) isomer. The odor of the (-) isomer is described as musky, woody, warm, or amber, while the (+)-ambroxol enantiomer has a relatively weak odor. The odor and odor threshold of ambroxol products also differ. Although various materials rich in (-)-ambroxol are commercially available, it is desirable to produce highly concentrated (-)-ambroxol materials, ideally pure (-)-ambroxol. The methods described herein can be used to prepare (-)-ambroxol of formula (I) alone or in combination with byproducts such as compounds of formulas (II), (III), and / or (IV) above.

[0709] (-)-Ambroxol can be produced from sclareolide using the following process. Sclareolide is a product extracted from the natural plant sclareolide. However, this process has potential problems due to the use of natural starting materials, as it involves a multi-stage reaction, its operation is circuitous, the supply and stability of the starting materials may not always be satisfactory, and the reaction may not be environmentally friendly because an oxidizing agent, such as chromic acid or permanganate, is used in the oxidative degradation step of (+)-sclareolide.

[0710]

[0711] (-)-Ambroxol can also be synthesized from farnesol using different routes. For example, farnesol can be obtained by bromination, cyanation, and hydrolysis of nerolidol to produce farnesol, followed by reduction. Alternatively, farnesol can be obtained from farnesol, farnesyl chloride, β-farnesene, or other substrates.

[0712] The methods described herein can be used to prepare (-)-ambroxan of formula (I) alone or in combination with byproducts such as compounds of formulas (II), (III) and / or (IV). For example, other stereoisomers of formula (I) can be prepared by the methods described herein.

[0713] Therefore, this document provides compounds of formula (I) or compositions comprising compounds of formula (I), which are obtained by or may be obtained by the methods described herein, including all embodiments thereof.

[0714] In some embodiments, not all of the homofaryl alcohol (e.g., EEH) is converted to (-)-ambroside or a byproduct of the reaction. Therefore, the compositions described herein, such as those obtained by the methods described herein or those obtainable by the methods described herein, may contain homofaryl alcohol (e.g., EEH, such as compounds other than those of formula (I) and / or those of formulas (II), (III), and / or (IV)). Any remaining homofaryl alcohol can be separated from the other reaction products such that the (-)-ambroside product does not contain homofaryl alcohol. In other embodiments, all homofaryl alcohol starting materials are converted to (-)-ambroside of formula (I) or a byproduct of the reaction by the methods described herein.

[0715] Therefore, the compositions described herein may comprise, substantially consist of, or consist of one or more of the compounds of formula (I), formula (II), formula (III), formula (IV), a homofarenoic alcohol starting material (e.g., EEH), and other stereoisomers of the compounds of formula (I). For example, the compositions described herein may comprise, substantially consist of, or consist of one or more of the compounds of formula (I) and formula (II), formula (III), and formula (IV).

[0716] Therefore, based on the total weight of the compounds of formula (I), formula (II), formula (III), and formula (IV), the composition described herein may contain equal to or greater than about 50 wt% of the compound of formula (I). For example, based on the total weight of the compounds of formula (I), formula (II), formula (III), and formula (IV), the composition described herein may contain 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 of formula (I). Based on the total weight of the compounds of formula (I), formula (II), formula (III), and formula (IV), the composition described herein may, for example, contain equal to or less than about 100 wt% of the compound of formula (I). For example, based on the total weight of the compounds of formula (I), formula (II), formula (III), and formula (IV), the mixture may contain equal to or less than about 99 wt%, or equal to or less than about 98 wt%, or equal to or less than about 97 wt% of the compound of formula (I). For example, based on the total weight of the compounds of formula (I), formula (II), formula (III), and formula (IV), the composition described herein may contain about 50 wt% to about 100 wt%, or about 60 wt% to about 99 wt%, or about 70 wt% to about 98 wt%, or about 80 wt% to about 97 wt%, or about 90 wt% to about 97 wt% of the compound of formula (I).

[0717] In the compositions described herein, the weight ratio of the compound of formula (I) to the total weight of the compounds of formula (II), (III), and (IV) can be, 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 compounds of formula (II), (III), and (IV) can be 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) may be 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.

[0718] In the compositions described herein, the weight ratio of the compound of formula (I) to homofarinol (e.g., EEH) can be, for example, from about 90:10 to about 100:0. For example, the weight ratio of the compound of formula (I) to homofarinol (e.g., EEH) in the compositions described herein can be 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.

[0719] The amounts of compounds of formula (I), formula (II), formula (III) and formula (IV) in a mixture of stereoisomers can be quantified, for example, by gas chromatography and / or identified by NMR spectroscopy.

[0720] (-)-Ambroxol synthesized by the methods described herein (e.g., using SHC / HAC enzymes or variants thereof and optionally recombinant host cells) can be, for example, in amorphous or crystalline form.

[0721] (-)-Ambroxol produced by the methods described herein (e.g., using SHC / HAC enzymes or variants thereof and optionally recombinant host cells) can be separated by steam extraction / distillation or by organic solvent extraction using a non-aqueous miscible solvent (to separate the reaction product and unreacted substrate from the biocatalyst remaining in the aqueous phase) followed by solvent evaporation to obtain a crude reaction product as determined by gas chromatography (GC) analysis. Steam extraction / distillation and organic solvent extraction methods are known to those skilled in the art.

[0722] For example, the obtained (-)-ambroside can be extracted from the entire reaction mixture using an organic solvent such as an aqueous immiscible solvent (e.g., toluene). Alternatively, the obtained (-)-ambroside can be extracted from the solid phase of the reaction mixture (by means of centrifugation or filtration) using an aqueous immiscible solvent (e.g., ethanol) or an aqueous immiscible solvent (e.g., toluene). Alternatively, the obtained (-)-ambroside can be extracted from the solid phase of the reaction mixture using a solvent mixture. As a further example, (-)-ambroside exists in the solid phase in crystalline or amorphous form and can also be separated from the remaining solid phase (cell material or fragments thereof) and liquid phase by filtration. As a further example, at a temperature above the melting point of (-)-ambroside (approximately 75°C), an oil layer can form on top of the aqueous phase, which can be removed and collected. To ensure complete recovery of (-)-ambroxol after oil layer removal, an organic solvent can be added to the aqueous phase containing biomass to extract any residual (-)-ambroxol contained in, on, or around the biomass. The organic layer can be combined with the oil layer and then processed as a whole for further separation and purification of (-)-ambroxol. (-)-ambroxol can be further selectively crystallized to remove byproducts (II), (IV), and (III) and any unreacted cyclofarnesol substrate from the final (-)-ambroxol product. The term "selective crystallization" refers to a process step that crystallizes (-)-ambroxol from a solvent while compounds (II), (III), and (IV) remain dissolved in the crystallization solvent to achieve separation of crystalline material containing only (-)-ambroxol product or, if it contains any of the other compounds (II), (III), or (IV), they are present only in olfactoryly acceptable amounts. (-)-Ambroxol may be, for example, free of or substantially free of byproducts (II), (III), and (IV). The selective crystallization step can be performed using a water-miscible solvent, such as ethanol. Selective crystallization of (-)-Ambroxol may be affected by the presence of unreacted cyclofarnesol substrate and the ratio of (-)-Ambroxol to other detectable byproducts (II), (III), and / or (IV). Selective crystallization of (-)-Ambroxol is still possible even with only a 10% conversion of the cyclofarnesol substrate to (-)-Ambroxol.

[0723] The olfactory purity of the final (-)-ambroside product can be determined using a 10% ethanol extract in water or by testing the crystalline material. The final (-)-ambroside product underwent olfactory purity, quality, and sensory characteristics testing against commercially available references for (-)-ambroside products. The (-)-ambroside material was also tested by experts in application studies to determine whether the material met specifications regarding its sensory characteristics.

[0724] Examples of suitable water-miscible and non-water-miscible organic solvents for the extraction and / or selective crystallization of (-)-ambroxan include, but are not limited to, aliphatic hydrocarbons, preferably those having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; halogenated aliphatic hydrocarbons, preferably those having one or two 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 those 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 heptane, methyl tert-butyl ether (also known as MTBE, tert-butyl methyl ether, tert-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 (-)-ambroxol from the solid phase of the reaction mixture. The use of ethanol is advantageous because it is easy to handle, non-toxic, and environmentally friendly.

[0725] As used herein, the term "isolated" refers to a biotransformation product that has been isolated or purified from its accompanying components, such as (-)-ambroside. An entity produced in a cellular system different from its natural origin is "isolated" because it will necessarily be free of its naturally occurring accompanying components. Resolution or purity can be measured by any suitable method, such as gas chromatography (GC), HPLC, or NMR analysis.

[0726] In some embodiments, the final product ((-)-ambroxan) 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).

[0727] Ideally, the amount of (-)-ambroxan produced can be from about 1 mg / l to about 20,000 mg / l (20 g / l) or higher, for example 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.

[0728] Using recombinant *E. coli* host cells that produce SHC / HAC enzymes or enzyme variants, at least 125 g / L (-)-ambroxol can be produced in the biotransformation reaction within approximately 2 days. Biotransformation can be carried out at concentrations of 188 g / L EEH or higher, provided efficient mixing is achieved, as stirring efficiency may be the only limiting factor for the system. Furthermore, biocatalysts with improved activity (e.g., in relation to SHC variants with further improved activity or in relation to increased SHC enzyme production) can improve or maintain productivity using less biomass or increased substrate concentrations, which is advantageous for mixing efficiency.

[0729] For example, it produces 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 (-)-ambroxol.

[0730] Preferably, (-)-ambroxan at a concentration of at least 100 g / L is produced over a period of 48 to 72 hours.

[0731] Preferably, (-)-ambroxol at a concentration of about 150 g / L is produced over a period of about 48 to 72 hours. Preferably, (-)-ambroxol at a concentration of about 200 g / L is produced over a period of about 48 to 72 hours.

[0732] Preferably, (-)-ambroxan at a concentration of about 250 g / L is produced over a period of about 48 to 72 hours.

[0733] The biotransformation of cyclohexane to (-)-ambroside according to this disclosure produces (-)-ambroside as the main compound, but may also produce compounds other than (-)-ambroside, which may or may not impart a pleasant olfactory odor to the biotransformation mixture and thus may have a positive or negative impact on the sensory properties of the (-)-ambroside final product. Therefore, sensory analysis is performed using sophisticated sensory testing employed by trained experts (e.g., perfumers) so that the testing can help determine whether the chemically relevant product is also an olfactorily relevant final product relative to a reference product. Removal of one or more byproduct compounds from (-)-ambroside can improve the odor of the reaction product mixture containing (-)-ambroside, even if the removed compound is actually odorless in itself. That is, an enhanced odor of (-)-ambroside can be observed in the absence of compounds II, III, and IV.

[0734] The various applications of ambroxol include, but are not limited to, refined fragrances or consumer products such as fabric care, toiletries, beauty care and cleaning products, detergent products and soap products, essentially including all currently available ambroxol ingredients used commercially, including but not limited to: Ambrox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlyn (Quest), Cetalox Laevo (Firmenich), Ambermor (Aromor) and Norambrenolide Ether (Pacific) products.

[0735] Therefore, this document further provides the use of (-)-ambroxol, obtained by or obtainable by the methods described herein, as part of a fragrance, cosmetic, or consumer product. This document also provides a product comprising (-)-ambroxol, obtained by or obtainable by the methods described herein. For example, this product may be a fragrance, cosmetic, or consumer product.

[0736] Ambergris oxides and their uses

[0737] This document further provides reaction products prepared by the methods described herein. The reaction products may, for example, comprise, ambergris oxide and one or more other compounds (e.g., compounds of formula (XI), (XII), and (XIII), substantially composed of or consisting of these compounds.

[0738] As used herein, the term "ambergris oxide" includes ambergris oxide of formula (X) and ambergris oxide in pure isomer form or mixed with one or more of the following molecules of formula (XI), (XII) and / or (XIII).

[0739]

[0740]

[0741] The names of the reaction products of formulas (X), (XI), (XII) and (XIII) are as follows.

[0742] Table 3. Nomenclature of the reaction products of formulas (X), (XI), (XII) and (XIII).

[0743]

[0744] As described by Bolster et al., Tetrahedron, 2002, 58(26), pp. 5275-5285, ambergris oxide can be produced from (+)-larixol. However, it is desirable to provide alternative or improved methods for producing ambergris oxide.

[0745] The methods described herein can be used to prepare ambergris oxides of formula (X) alone or in mixture with byproducts such as compounds of formula (XI), (XII), and / or (XIII). Other stereoisomers of formula (X), for example, can be prepared by the methods described herein.

[0746] Therefore, this document provides compounds of formula (X) or compositions comprising compounds of formula (X), which are obtained by or may be obtained by the methods described herein, including all embodiments thereof.

[0747] In some embodiments, not all bisimertinol (e.g., BisEEH) is converted to ambergris oxide or a reaction byproduct. Therefore, compositions described herein, such as those obtained by or achievable through the methods described herein, may contain bisimertinol (e.g., BisEEH, such as compounds of formula (X) and / or compounds of formulas (XI), (XII), and / or (XIII)). Any remaining bisimertinol can be separated from other reaction products such that the ambergris oxide product does not contain bisimertinol. In other embodiments, all bisimertinol starting materials are converted to ambergris oxide of formula (X) or a reaction byproduct by the methods described herein.

[0748] Therefore, the compositions described herein may comprise, consist substantially of, or consist of one or more of the compounds of formula (X), formula (XI), formula (XII), formula (XIII), bi-dimethyl farnesol starting materials (e.g., bisEEH), and other stereoisomers of the compound of formula (X). For example, the compositions described herein may comprise, consist substantially of, or consist of one or more of the compounds of formula (X) and formula (XI), formula (XII), and formula (XIII).

[0749] Therefore, based on the total weight of the compounds of formula (X), formula (XI), formula (XII), and formula (XIII), the compositions described herein may contain equal to or greater than about 50 wt% of the compound of formula (X). For example, based on the total weight of the compounds of formula (X), formula (XI), formula (XII), and formula (XIII), the compositions described herein may contain 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 of formula (X). Based on the total weight of the compounds of formula (X), formula (XI), formula (XII), and formula (XIII), the compositions described herein may, for example, contain equal to or less than about 100 wt% of the compound of formula (X). For example, based on the total weight of compounds of formula (X), (XI), (XII), and (XIII), the mixture may contain equal to or less than about 99 wt%, or equal to or less than about 98 wt%, or equal to or less than about 97 wt% of compounds of formula (X). For example, based on the total weight of compounds of formula (X), (XI), (XII), and (XIII), the compositions described herein may contain about 50 wt% to about 100 wt%, or about 60 wt% to about 99 wt%, or about 70 wt% to about 98 wt%, or about 80 wt% to about 97 wt%, or about 90 wt% to about 97 wt% of compounds of formula (X).

[0750] In the compositions described herein, the weight ratio of the compound of formula (X) to the total weight of the compounds of formula (XI), (XII), and (XIII) can be, 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 compounds of formula (XI), (XII), and (XIII) can be 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) may be 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.

[0751] In the compositions described herein, the weight ratio of the compound of formula (X) to bi-average farnesol (e.g., bisEEH) can be, for example, from about 90:10 to about 100:0. For example, the weight ratio of the compound of formula (X) to bi-average farnesol (e.g., bisEEH) in the compositions described herein can be 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.

[0752] The amounts of compounds of formula (X), formula (XI), formula (XII), and formula (XIII) in a mixture of stereoisomers can be quantified, for example, by gas chromatography and / or identified by NMR spectroscopy.

[0753] Ambergris oxide synthesized by the methods described herein (e.g., using SHC / HAC enzymes or variants thereof and optionally recombinant host cells) can be, for example, in amorphous or crystalline form.

[0754] Ambergris oxides produced by the methods described herein (e.g., using SHC / HAC enzymes or variants thereof and recombinant host cells) can be separated by steam extraction / distillation or by organic solvent extraction using a non-aqueous miscible solvent (to separate the reaction products and unreacted substrates from the biocatalyst remaining in the aqueous phase) followed by solvent evaporation to obtain crude reaction products as determined by gas chromatography (GC) analysis. Steam extraction / distillation and organic solvent extraction methods are known to those skilled in the art.

[0755] For example, the obtained ambergris oxide can be extracted from the entire reaction mixture using an organic solvent such as an aqueous immiscible solvent (e.g., toluene). Alternatively, the obtained ambergris oxide can be extracted from the solid phase of the reaction mixture (by, for example, centrifugation or filtration) using an aqueous immiscible solvent (e.g., ethanol) or an aqueous immiscible solvent (e.g., toluene). As a further example, the ambergris oxide exists in the solid phase in crystalline or amorphous form and can also be separated from the remaining solid phase (cellular material or fragments thereof) and liquid phase by filtration.

[0756] As a further example, at temperatures above the melting point of ambergris oxide, an oil layer can form on top of the aqueous phase, which can be removed and collected. To ensure complete recovery of the ambergris oxide after removing the oil layer, an organic solvent can be added to the aqueous phase containing biomass to extract any residual ambergris oxide contained in, on, or around the biomass. The organic layer can be combined with the oil layer and then processed as a whole for further separation and purification of the ambergris oxide. The ambergris oxide can be further selectively crystallized to remove byproducts (XI), (XII), and (XIII) and any unreacted bi-dimethyl farnesol substrate from the final ambergris oxide product. The term "selective crystallization" refers to a process step that crystallizes ambergris oxide from a solvent while compounds (XI), (XII), and (XIII) remain dissolved in the crystallization solvent to achieve separation of crystalline material containing only the ambergris oxide product or, if it contains any of the other compounds (X), (XII), or (XIII), they are present only in olfactorily acceptable amounts. For example, ambergris oxide may be free of or substantially free of byproducts (XI), (XII), and (XIII). The selective crystallization step can be performed using a water-miscible solvent, such as ethanol. Selective crystallization of ambergris oxide can be affected by the presence of unreacted cyclofarnesol substrate and the ratio of ambergris oxide to other detectable byproducts (XI), (XII), and / or (XIII). Selective crystallization of ambergris oxide is still possible even with only a 10% conversion of the cyclofarnesol substrate to ambergris oxide.

[0757] The olfactory purity of the final ambergris oxide product can be determined using a 10% ethanol extract in water or by testing the crystalline material. The final ambergris oxide product was tested against commercially available references for ambergris oxide to assess its olfactory purity, quality, and sensory properties. The ambergris oxide material was also tested by experts in application studies to determine if it met specifications regarding its sensory properties.

[0758] Examples of suitable water-miscible and non-water-miscible organic solvents for the extraction and / or selective crystallization of ambergris oxides include, but are not limited to, aliphatic hydrocarbons, preferably those having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; halogenated aliphatic hydrocarbons, preferably those having one or two carbon atoms, such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, or tetrachloroethane; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene, or dichlorobenzene; and aliphatic acyclic and cyclic ethers or alcohols, preferably those 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 heptane, methyl tert-butyl ether (also known as MTBE, tert-butyl methyl ether, tert-butyl methyl ether, and tBME), diisopropyl ether, tetrahydrofuran, ethyl acetate, and / or mixtures thereof. Preferably, a water-miscible solvent such as ethanol is used to extract ambergris oxide from the solid phase of the reaction mixture. The use of ethanol is advantageous because it is easy to handle, non-toxic, and environmentally friendly.

[0759] As used herein, the term "isolated" refers to a biotransformation product that has been separated or purified from its accompanying components, such as ambergris oxide. An entity produced in a cellular system different from its naturally occurring source is "isolated" because it will necessarily be free of the naturally occurring components it accompanies. Separation or purity can be measured by any suitable method, such as gas chromatography (GC), HPLC, or NMR analysis.

[0760] In some embodiments, the final product (ambergris oxide) is separated 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).

[0761] Ideally, the amount of ambergris oxide produced can be from about 1 mg / l to about 20,000 mg / l (20 g / l) or higher, for example 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.

[0762] For example, it produces 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 Ambergris oxides in the following concentrations: (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, 200 g / l, 300 g / l, 400 g / l, or 500 g / l.

[0763] Preferably, ambergris oxide at a concentration of at least 100 g / L is produced over a period of 48 to 72 hours.

[0764] Preferably, ambergris oxide at a concentration of about 150 g / L is produced over a period of about 48 to 72 hours. Preferably, ambergris oxide at a concentration of about 200 g / L is produced over a period of about 48 to 72 hours.

[0765] Preferably, ambergris oxide at a concentration of about 250 g / L is produced over a period of about 48 to 72 hours.

[0766] The biotransformation of ambergris oxide from bi-average farnesol according to this disclosure produces ambergris oxide as the main compound, but may also produce compounds other than ambergris oxide, which may or may not impart a pleasant olfactory odor to the biotransformation mixture and thus may have a positive or negative impact on the sensory properties of the ambergris oxide final product. Therefore, sensory analysis is performed using sophisticated sensory tests employed by trained experts (e.g., perfumers) so that the tests can help determine whether the chemically relevant product is also an olfactorily relevant final product relative to a reference product. Removal of one or more byproduct compounds from ambergris oxide can improve the odor of the remaining compound (ambergris oxide), even if the removed compound is actually odorless in itself. That is, an enhancement of the ambergris oxide odor can be observed in the absence of compounds XI, XII, and XIII.

[0767] The various applications of ambergris oxide include, but are not limited to, refined fragrances or consumer products such as fabric care, toiletries, beauty care and cleaning products, detergents and soaps, essentially encompassing all products currently available for the commercial use of ambergris oxide ingredients.

[0768] Therefore, this document further provides the use of ambergris oxide obtained by or obtainable through the methods described herein as part of fragrances, cosmetics, or consumer products. This document also provides products comprising ambergris oxide obtained by or obtainable through the methods described herein. For example, such products may be fragrances, cosmetics, or consumer products.

[0769] Flavoring composition

[0770] This document further provides information on the use of the compounds and compositions described herein as flavoring compositions or in flavoring compositions.

[0771] Therefore, this document also provides fragrance compositions comprising one or more compounds of formula (I) or (X). For example, a "fragrance composition" can be any composition comprising one or more compounds of formula (I) or (X) and a base material.

[0772] As used herein, “base material” includes all known fragrance ingredients selected from a wide range of natural products and currently available synthetic molecules (e.g., essential oils, alcohols, aldehydes and ketones, ethers and acetals, esters and lactones, macrocyclic compounds and heterocyclic compounds) and / or mixed with one or more ingredients or excipients (e.g., carrier materials, diluents and other auxiliaries commonly used in the art) that are typically used in combination with odorants in fragrance compositions.

[0773] Fragrance ingredients known in the art are readily available commercially from major fragrance manufacturers. Non-limiting examples of such ingredients include:

[0774] - Essential oils and extracts, such as castoreum, hibiscus oil, oakmoss oil, geranium oil, tree moss oil, basil oil, fruit oils such as bergamot and mandarin oil, myrtle oil, palm sugar oil, patchouli oil, petitgrain oil, jasmine oil, rose oil, sandalwood oil, wormwood oil, lavender oil and / or ylang-ylang oil;

[0775] - Alcohols, such as cinnamyl alcohol ((E)-3-phenylpropyl-2-en-1-ol); cis-3-hexenol ((Z)-hexa-3-en-1-ol); citronellol (3,7-dimethyloct-6-en-1-ol); dihydrolaurol (2,6-dimethyloct-7-en-2-ol); Ebanol TM ((E)-3-methyl-5-(2,2,3-trimethylcyclopentan-3-en-1-yl)pentan-4-en-2-ol); eugenol (4-allyl-2-methoxyphenol); ethyl linalool ((E)-3,7-dimethylnon-1,6-dien-3-ol); farnesol ((2E,6Z)-3,7,11-trimethyldodecyl-2,6,10-trien-1-ol); geraniol ((E)-3,7-dimethyloctyl-2,6-dien-1-ol); SuperMuguet TM (E)-6-ethyl-3-methyloct-6-en-1-ol; linalool (3,7-dimethyloct-1,6-dien-3-ol); menthol (2-isopropyl-5-methylcyclohexanol); nerolidol (3,7-dimethyl-2,6-octadien-1-ol); phenethyl alcohol (2-phenethyl alcohol); Rhodinol TM (3,7-Dimethyloct-6-en-1-ol); Sandalore TM (3-Methyl-5-(2,2,3-trimethylcyclopentan-3-en-1-yl)pentan-2-ol); terpineol (2-(4-methylcyclohexane-3-en-1-yl)propan-2-ol); or Timberol TM (1-(2,2,6-trimethylcyclohexane)hex-3-ol); 2,4,7-trimethyloct-2,6-dien-1-ol; and / or [1-methyl-2-(5-methylhex-4-en-2-yl)cyclopropyl]-methanol;

[0776] - Aldehydes and ketones, such as anisaldehyde (4-methoxybenzaldehyde); α-pentylcinnamaldehyde (2-benzylidene heptanal); Georgywood TM (1-(1,2,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthol-2-yl)ethanecone); hydroxycitronellol (7-hydroxy-3,7-dimethyloctanal); Iso E Super (1-(2,3,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthol-2-yl)ethanecone); 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 cypressone; methyl ionone; verbenatone; and / or vanillin;

[0777] – Ethers and acetals, such as ambroxan® (3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro-1H-benzo[e][1]benzofuran); geraniol methyl ether ((2E)-1-methoxy-3,7-dimethyloctyl-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]);

[0778] – Macrocyclic compounds, such as Ambretolide ((Z)-oxetane-10-en-2-one); ethyl bainite (1,4-dioxetane-5,17-dione); and / or Exaltolide® (16-oxetane-1-one); and

[0779] - Heterocyclic compounds, such as isobutylquinoline (2-isobutylquinoline).

[0780] As used in this article, “carrier material” refers to a material that is actually neutral from the perspective of the odorant, that is, a material that does not significantly alter the sensory properties of the odorant.

[0781] "Diluent" refers to any diluent that is typically used in conjunction with odorants, such as diethyl phthalate (DEP), dipropylene glycol (DPG), isopropyl myristate (IPM), triethyl citrate (TEC), and alcohols (e.g., ethanol).

[0782] The term "auxiliary agent" refers to an ingredient that may be used in a fragrance composition for reasons not particularly related to the olfactory properties of the fragrance composition. For example, an auxiliary agent may be an ingredient that assists in the processing of one or more fragrance components or a composition containing said one or more components, or it may improve the handling or storage of aromatic components or compositions containing them, such as an antioxidant auxiliary agent. 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 related phenols, hydroquinone (CAS 121-31-9).

[0783] It can also be an ingredient that provides additional benefits, such as imparting color or texture. It can also be an ingredient that imparts light resistance or chemical stability to one or more ingredients contained in a fragrance composition.

[0784] A detailed description of the properties and types of auxiliaries commonly used in fragrance compositions containing auxiliaries is not exhaustive, but it must be mentioned that the ingredients are well known to those skilled in the art.

[0785] This document also provides consumer products comprising compounds or compositions or fragrance compositions as described herein, including any embodiments thereof. For example, a consumer product may be a cosmetic (e.g., eau de toilette or fragrance), a cleaning product, a stain remover product, or a soap product.

[0786] All-method nitrocellulose

[0787] Alloferne alcohol may have the following isomerism.

[0788] Table 4. Isomers of fenofiol.

[0789]

[0790] β-farnesene can be directly converted to E,E-metafarnesol (EEH) or indirectly via E,E-metafarnesol ester, followed by conversion to EEH. Reviews on the production of (-)-ambroside from different substrates can be found in US2012 / 0135477A1, WO2010 / 139719, US2013.0273619A1, WO2013 / 156398A1, and Seitz's doctoral dissertation (2012, as cited above) and Schaefer 2011 (Chemie Unserer Zeit 45, 374-388), the contents of which are incorporated herein by reference.

[0791] US2012 / 0135477A1 reports the conversion of (3Z,7E) to (-)-ambroxol using ZmoSHC, but according to the disclosure in Schaefer (2011) (as described above), (7E,3Z) is converted only to 9b-epi-ambroxol (i.e., compound (III) outlined above) and not (-)-ambroxol. As used herein, reference to (3Z,7E) cyclofarnesol refers to E,Z-cyclofarnesol, also known as EZH.

[0792] Although cyclofarnesol can be a mixture of four isomers (i.e., (3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E) isomers), according to the literature it appears that (-)-ambroside can only be obtained from (3E,7E)cyclofarnesol (see Neumann and Simon (1986) cited above). As used herein, reference to (3E,7E)-cyclofarnesol refers to E,E-cyclofarnesol, which is also known as EEH.

[0793] The starting material used in the method for preparing (-)-ambroxan described herein can be, for example, (3E,7E)-homologne or a mixture containing (3E,7E)-homologne, such as a mixture of isomers of homologne containing (3E,7E)-homologne.

[0794] Preferably, the starting material for homofarenoic alcohol comprises a mixture of (3E,7E) and (3Z,7E), referred herein to as the EE:EZ isomer mixture. The CAS number for the EE:EZ isomer mixture of homofarenoic alcohol is 35826-67-6.

[0795]

[0796] The homofarin stock solution / starting material can be a mixture of isomers. Therefore, the homofarin starting material can also comprise a mixture of four isomers EE:EZ:ZZ:ZE, corresponding to (3E,7E), (3Z,7E), (3Z,7Z), and (3E,7Z). Preferably, the homofarin 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)].

[0797] Preferably, the starting material for cyclofarnesol is selected from one or more of the following mixtures: [(3E,7E), (3Z,7E)] and / or [(3Z,7E), (3E / 7E) and (3E,7Z)], also named [EE:EZ] and [EE:EZ:ZE], respectively.

[0798] Therefore, in some implementations, the EEH:EZH ratio is approximately 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 approximately 50:50. For example, the ratio of EEH:EZH can be approximately 50:50 to approximately 100:00, approximately 50:50 to approximately 99:01, approximately 60:40 to approximately 99:1, approximately 70:30 to approximately 95:5, or approximately 80:20 to approximately 95:5.

[0799] In some embodiments, preferably, the equimolar alcohol starting material comprises >90% E,E-equimolar alcohol (EEH).

[0800] In other embodiments, the cyclophosphamide starting material comprises an EE:EZ weight ratio of 86:14.

[0801] In some embodiments, the cyclophosphamide starting material comprises an EE:EZ weight ratio of 80:20.

[0802] In some embodiments, the cyclophosphamide starting material comprises an EE:EZ weight ratio of 70:30.

[0803] In a further embodiment, the cyclophosphamide starting material comprises an EE:EZ weight ratio of 69:31.

[0804] The number of isomers of zefarin present can affect the reaction rate. SHC / HAC enzymes or enzyme variants may be able to convert E,E-zefarin to (-)-ambroxol from complex mixtures of zefarin isomers (e.g., EE:EZ:ZE:ZZ). However, lower conversion rates may be observed, consistent with the view that zefarin isomers other than EEH may compete with EEH for proximity to SHC / HAC-derived enzymes and thus may act as competitive inhibitors of EEH to (-)-ambroxol and / or also as alternative substrates (see, for example, Eichhorn et al. (2018) Adv. Synth. Catal. 360: 2339-2351, the contents of which are incorporated herein by reference). Therefore, zefarin substrates can contain 2-4 isomers, preferably a mixture of two isomers.

[0805] Therefore, the cyclophosphamide substrate can consist of 2-4 isomers, preferably a mixture of two isomers, or essentially consist of them.

[0806] Preferably, the homofarin substrate comprises an EE:EZ isomer mixture.

[0807] Preferably, the homofarin substrate consists of or is substantially composed of an EE:EZ isomer mixture.

[0808] For example, if an EE:EZ isomer mixture is used, compounds other than (-)-ambroxol (such as compounds II, III and IV listed in Table 2) are in an "oily" form (rather than a solid form), which promotes the stirring of the reaction mixture and the resulting biotransformation process.

[0809] Double-average method for nitrocellulose

[0810] Bis-average nitrocellulose may have the isomerism shown in Table 5 below.

[0811] Table 5. Bis-average isomers of nitroglycerin.

[0812]

[0813] Bisimilar farnesol can be produced from E-nerolidol, as described in the examples below. For example, bisimilar farnesol can be produced as a mixture of two or more isomers (e.g., a mixture of E,E-bisimilar farnesol and E,Z-bisimilar farnesol).

[0814] Although bisimertinol may exist in the form of a mixture of four isomers ((Z,Z), (E,Z), (Z,E) and (E,E) isomers), it appears that ambergris oxide can only be obtained from E,E-bisimertinol.

[0815] The starting material used in the method for preparing ambergris oxide described herein can be, for example, E,E-bisimifarnesol or a mixture containing E,E-bisimifarnesol, such as a mixture of bisimifarnesol isomers containing E,E-bisimifarnesol.

[0816] Preferably, the bi-homogeneol starting material comprises a mixture of (BisEEH) and (BisEZH), referred herein as the EE:EZ isomer mixture.

[0817] The bi-homogeneous nitrocellulose stock solution / starting material can be a mixture of isomers. Therefore, the bi-homogeneous nitrocellulose starting material can also contain a mixture of four isomers:EE:EZ:ZZ:ZE.

[0818] Therefore, in some implementations, the ratio of BisEEH:BisEZH is approximately 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 approximately 50:50. For example, the ratio of BisEEH:BisEZH can be about 50:50 to about 100:00 or about 50:50 to about 99:01 or about 60:40 to about 99:1 or about 70:30 to about 95:5 or about 80:20 to about 95:5.

[0819] In some embodiments, preferably, the bi-dimethyl farnesol starting material comprises >90% E,E-bi-dimethyl farnesol (EEH).

[0820] In other embodiments, the bi-average farnesol starting material comprises a BisEEH:BisEZH weight ratio of 86:14. In some embodiments, the bi-average farnesol starting material comprises a BisEEH:BisEZH weight ratio of 80:20.

[0821] In some embodiments, the bi-average farnesol starting material comprises a BisEEH:BisEZH weight ratio of 70:30.

[0822] In a further embodiment, the bi-average farnesol starting material comprises a BisEEH:BisEZH weight ratio of 69:31.

[0823] The number of bisimilatory farnesol isomers present can affect the reaction rate. SHC / HAC enzymes or enzyme variants may be able to convert E,E-bissimilatory farnesol to ambergris oxide from complex mixtures of bisimilatory farnesol isomers (e.g., EE:EZ:ZE:ZZ). However, lower conversion rates may be observed, consistent with the view that bisimilatory farnesol isomers other than BisEEH may compete with BisEEH for proximity to SHC / HAC enzymes or enzyme variants and thus may act as competitive inhibitors of BisEEH to ambergris oxide and / or also as alternative substrates. Therefore, bisimilatory farnesol substrates can contain 2-4 isomers, preferably a mixture of two isomers.

[0824] Therefore, the bi-average farnesol substrate can consist of 2-4 isomers, preferably a mixture of two isomers, or essentially consist of them.

[0825] Preferably, the bi-homogeneous phenoxyethanol substrate comprises a mixture of EE:EZ isomers.

[0826] Preferably, the bi-homogeneous phenoxyethanol substrate consists of or is substantially composed of a mixture of EE:EZ isomers.

[0827] Nucleic acids and methods for preparing nucleic acids

[0828] This article further provides nucleic acids encoding SHC / HAC enzymes or SHC / HAC enzyme variants as described herein. For example, the nucleic acid can be an isolated nucleic acid.

[0829] Specifically, this article provides constructs comprising nucleic acid sequences encoding SHC / HAC enzymes or enzyme variants as described herein. As used herein, a “construct” is an artificially generated segment of nucleic acid to be transfected into target cells. Constructs may contain nucleic acids encoding SHC / HAC enzymes or enzyme variants and expression controllers (e.g., promoters).

[0830] This article further provides vectors that contain the constructs described herein. As used herein, a "vector" is a DNA molecule used as a medium to artificially carry foreign genetic material into a cell in which it can be replicated and / or expressed. For example, a vector can be a plasmid, a viral vector, a granulosome, or an artificial chromosome.

[0831] The terms “construction” and “vector” can overlap, for example, when the construct is a plasmid.

[0832] Specifically, this article provides nucleic acids encoding the amino acid sequences 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.

[0833] Specifically, this document provides nucleic acids having 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, for example, be contained in constructs or vectors as described herein.

[0834] As used herein, the term "nucleic acid" or "nucleic acid molecule" should specifically refer to the polynucleotides of this disclosure, which can be DNA, cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded, with a sense strand and / or antisense strand. The term "nucleic acid" or "nucleic acid molecule" should be particularly applied to one or more polynucleotides as used herein, for example as a full-length nucleotide sequence or a fragment or portion thereof, which encode a polypeptide or a fragment or portion thereof having enzymatic activity (e.g., an enzyme in a metabolic pathway).

[0835] The term also includes individual molecules, such as cDNA, where the corresponding genomic DNA has introns and therefore has a distinct sequence; genomic fragments lacking at least one flanking gene; cDNA or genomic DNA fragments produced by polymerase chain reaction (PCR) and lacking at least one flanking gene; restriction fragments lacking at least one flanking gene; DNA encoding non-naturally occurring proteins (e.g., fusion proteins (e.g., His tags), mutant proteins, or fragments of a given protein); and nucleic acids as degenerate variants of naturally occurring nucleic acids or cDNA. Furthermore, it includes recombinant nucleotide sequences as part of a heterozygous gene, i.e., a gene encoding a non-naturally occurring fusion protein. Fusion proteins can be added to a protein by one or more amino acids (e.g., but not limited to histidine (His)), typically at the N-terminus of the protein but also at the C-terminus or fused within a region of the protein. Such fusion proteins or fusion vectors encoding such proteins are typically used for three purposes: (i) increasing the yield of recombinant proteins; (ii) increasing the solubility of recombinant proteins; and (iii) aiding in the purification of recombinant proteins by providing ligands for affinity purification.

[0836] The term “nucleic acid” or “nucleic acid molecule” also includes codon-optimized sequences suitable for expression in specific microbial host cells (e.g., *E. coli* host cells). As used herein, the term “codon-optimized” refers to a nucleic acid protein-coding sequence adapted for expression in prokaryotic or eukaryotic host cells (particularly bacterial host cells such as *E. coli* host cells) by replacing one or more, or preferably a considerable number, of codons with codons more frequently used in bacterial (e.g., *E. coli*) host cell genes.

[0837] In this regard, the nucleotide sequence encoding the reference amino acid sequence (SEQ ID NO: 1 or SEQ ID NO: 10) and its variants / derivatives can be the original sequence found in the source (e.g., SEQ ID NO: 1 found in AacSHC) or the gene can be codon-optimized for a selected host organism such as Escherichia coli.

[0838] 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. Fragments of nucleic acid molecules may be referred to as DNA segments of genes, particularly segments of partial genes. A fragment may also contain several open reading frames (ORFs), which are either repeats of the same ORF or different ORFs. This term should specifically refer to coding nucleotide sequences, but should also include non-coding nucleotide sequences, such as wholly or partially untranscribed or untranslated sequences, or sequences encoding polypeptides. Genes as used herein (e.g., for assembly, diversification, or recombination) can be non-coding sequences or sequences encoding polypeptides or sequences encoding proteins, or portions or fragments thereof, having a sufficient sequence length for successful recombination events. More specifically, the gene has a minimum length of 3 bp, preferably at least 100 bp, more preferably at least 300 bp. As will be apparent from the foregoing, references to isolated DNA do not imply DNA present in hundreds to millions of other DNA molecules, such as cDNA or genomic DNA libraries, or in restriction digests such as restriction digestion reaction mixtures or electrophoresis gel slices. Isolated nucleic acid molecules in this disclosure include segments not found in their natural state.

[0839] As used herein, the term “isolated DNA” can mean (1) DNA containing a sequence different from any naturally occurring sequence, non-naturally occurring polynucleotides or nucleic acids (e.g., produced by artificial combination (e.g., by artificial manipulation of isolated nucleic acid segments by, for example, genetic engineering techniques)) or (2) in the case of DNA having a naturally occurring sequence (e.g., cDNA or genomic DNA), but not containing DNA flanking at least one gene in the genome of an organism containing the DNA of interest.

[0840] As used herein, the term "isolated DNA" (particularly relating to nucleic acid sequences) can also refer to nucleic acids or polynucleotides produced through recombinant DNA technology, such as DNA constructs containing polynucleotides heterologous to the host cell, which are optionally incorporated into the host cell. Chimeric nucleotide sequences can be specifically produced as recombinant molecules. The term "recombination" should be particularly applied to assembling polynucleotides, linking such polynucleotides or portions thereof together, with or without recombination, to achieve cross-interchange or gene mosaicism. For example, linking nucleic acid segments with desired functions together to produce the desired functional combination. Recombinant genes encoding the polypeptides described herein may include the coding sequence of the polypeptide operatively ligated in a meaningful direction to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region in these microorganisms if desired. The coding sequence and the regulatory region are considered operatively ligated when the regulatory region is positioned such that the regulatory region is effective for transcription or translation of the regulatory sequence.

[0841] As used herein, the term "recombinant" (especially in relation to enzymes) refers to enzymes produced through recombinant DNA technology, i.e., produced by cells transformed from exogenous DNA constructs encoding the desired enzyme. "Synthetic" enzymes are those prepared through chemical synthesis. Chimeric enzymes can be specifically produced as recombinant molecules. Therefore, the term "recombinant DNA" includes recombinant DNA inserted into vectors, integrated into autonomously replicating plasmids or viruses, or integrated into the genomic DNA of prokaryotes or eukaryotes (or the genome of homologous cells, at a location other than the natural chromosome).

[0842] On the other hand, one or more nucleic acid molecules of this disclosure are operatively linked to an expression control sequence that allows expression in prokaryotic and / or eukaryotic host cells. As used herein, “operatively linked” means integrated into a genetic construct such that the expression control sequence effectively controls the expression of a target coding sequence. The aforementioned transcriptional / translational regulatory elements include, but are not limited to, inducible and non-inducible, constitutive, cell cycle-regulated, metabolically regulated promoters, enhancers, operons, silencers, repressors, and other elements known to those skilled in the art that drive or otherwise regulate gene expression. Such regulatory elements include, but are not limited to, regulatory elements that direct constitutive expression or allow inducible expression, such as the CUP-1 promoter, the tet-repressor employed (e.g., used in tet-on or tet-off systems), lac system, and trp system regulatory elements. As an example, isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effective inducer of gene expression in the concentration range of 100 μM to 1.0 mM. This compound is a molecular mimic of allolactose (a lactose metabolite that triggers transcription by the lac operon), and is therefore used to induce gene expression when a gene is under the control of the lac operon. Another example of a regulatory element that induces gene expression is lactose. Similarly, one or more nucleic acid molecules of this disclosure can form part of a heterozygous gene encoding an additional polypeptide sequence (e.g., a sequence used as a marker or reporter). Examples of markers and reporter genes include β-lactamases, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin-β-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine-guanine phosphoribosyltransferase (XGPRT). As with many standard procedures associated with the practice of this disclosure, those skilled in the art will recognize other useful agents, such as other sequences that can function as markers or reporters.

[0843] In some embodiments, this disclosure provides recombinant polynucleotides encoding SHC / HAC enzymes or variants thereof, which can be inserted into vectors for expression and optional purification. One type of vector is a plasmid representing a circular double-stranded DNA loop linking to another DNA segment. Certain vectors control the expression of genes functionally linked to them. These vectors are called “expression vectors.” Expression vectors suitable for recombinant DNA technologies are typically plasmid-type. Typically, expression vectors contain genes, such as the SHC / HAC enzymes or variants thereof described herein. In this specification, the terms “plasmid” and “vector” are used interchangeably because plasmids are the most commonly used vectors.

[0844] Such vectors may include DNA sequences, including but not limited to DNA sequences that are not naturally present in host cells, DNA sequences that are not normally transcribed into RNA or translated into protein (“expressed”), and other genes or DNA sequences desired to be introduced into a non-recombinant host. It should be understood that the genome of the recombinant host described herein is typically enhanced by the stable introduction of one or more recombinant genes. However, autonomous or replicative plasmids or vectors may also be used within the scope of this disclosure. Furthermore, this disclosure may be implemented using low copy number (e.g., single copy) or high copy number (as illustrated herein) plasmids or vectors.

[0845] In a preferred embodiment, the vector of this disclosure comprises plasmids, phage particles, phages, granules, artificial bacterial and yeast chromosomes, knockout or knock-in constructs, synthetic nucleic acid sequences or cassettes, and can generate subgroups in the form of linear polynucleotides, plasmids, giant plasmids, synthetic or artificial chromosomes (e.g., plant, bacterial, mammalian or yeast artificial chromosomes).

[0846] Preferably, after the vector is introduced, the protein encoded by the introduced polynucleotide is expressed within the cell. Multiple gene substrates can be incorporated into the plasmid. The plasmid is typically a standard cloning vector, such as a bacterial multicopy plasmid. The substrate can be incorporated into the same or different plasmids. Typically, at least two different types of plasmids with different types of selection markers are used to allow selection of cells containing at least two types of vectors.

[0847] Typically, bacterial or yeast cells can be transformed using any one or more nucleotide sequences known in the art. For in vivo recombination, the gene to be recombinated with the genome or other genes is used to transform the host using standard transformation techniques. In a suitable embodiment, DNA providing the origin of replication is included in the construct. The origin of replication can be appropriately selected by those skilled in the art. Depending on the nature of the gene, if a sequence already exists in the gene or genome that can function as an origin of replication, a supplementary origin of replication may not be necessary.

[0848] Host cells, methods for preparing host cells, and methods for preparing ambroxol and ambergris oxide using host cells.

[0849] This article further provides recombinant host cells comprising nucleic acid sequences, constructs, or vectors as described herein. This article further provides recombinant host cells that produce SHC / HAC enzymes or enzyme variants as described herein.

[0850] The methods described herein for producing (-)-ambroxol or ambergris oxide may, for example, involve culturing recombinant host cells as described herein. As used herein, the term "culturing" refers to the process of producing living cells that generate SHC / HAC enzymes or enzyme variants as described herein, which can be used in the methods described herein for producing (-)-ambroxol or ambergris oxide. Cells do not necessarily need to divide and replicate themselves, although this is not excluded.

[0851] When such DNA has been introduced into a cell, bacterial or yeast cells can be transformed by exogenous or heterologous DNA. The transformed DNA may or may not integrate, meaning it is covalently linked to the cell's genome. For example, in prokaryotes and yeast, the transformed DNA can be retained on accessory elements such as plasmids. For eukaryotic cells, stably transfected cells are those in which the transfected DNA has integrated into the chromosome, allowing it to be inherited by daughter cells through chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones consisting of populations of daughter cells containing the transformed DNA.

[0852] Generally, the introduced DNA is not initially present in the host that acts as the recipient of the DNA. However, isolating a DNA segment from a given host and subsequently introducing one or more additional copies of that DNA into the same host to, for example, increase the production of gene products or alter gene expression patterns, is also within the scope of this disclosure. In some cases, the introduced DNA will modify or even replace endogenous genes or DNA sequences through, for example, homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms, plant cells, and plants.

[0853] This disclosure also characterizes recombinant hosts. The term "recombinant host" (also known as "genetically modified host cell" or "transgenic cell") refers to a host cell containing heterologous nucleic acids or whose genome has been enhanced by at least one integrated DNA sequence. The host cells of this disclosure can be genetically engineered using the polynucleotides or vectors described above.

[0854] Host cells that can be used for the purposes of this disclosure include, but are not limited to, prokaryotic cells such as bacteria (e.g., Escherichia coli and Bacillus subtilis). B. subtilis It can be transformed, for example, with recombinant phage DNA, plasmid DNA, bacterial artificial chromosome, or copious DNA expression vectors containing polynucleotide molecules of the present disclosure; simple eukaryotic cells such as yeast (e.g., *Saccharomyces* and *Pichia pastoris*). PichiaThis can be achieved, for example, by transformation with a recombinant yeast expression vector containing, for example, a polynucleotide molecule containing the present disclosure. Depending on the host cell and the appropriate vector used to introduce the polynucleotide, the polynucleotide can be integrated into, for example, chromosomal or mitochondrial DNA, or can be maintained extrachromosomally (e.g., via episomes), or may be contained only temporarily within the cell.

[0855] As used herein, the term "cell," particularly when referring to genetic engineering and the introduction of one or more genes or assembled gene clusters into a cell, or the generation of a cell, is understood to mean any prokaryotic or eukaryotic cell. Both prokaryotic and eukaryotic host cells are intended to be used in accordance with this disclosure, including bacterial host cells such as species of *Escherichia coli* or *Bacillus*, and yeast host cells such as *Saccharomyces cerevisiae*. S. cerevisiae Insect host cells, such as those of the meadow moth, or human host cells, such as HeLa and Jurkat cells.

[0856] Specifically, the cells are eukaryotic cells, preferably fungal, mammalian, or plant cells or prokaryotic cells. Suitable eukaryotic cells include, for example, but not limited to, mammalian cells, yeast cells, or insect cells (including Sf9), amphibian cells (including melanocytes), or include neobacus nematodes (including Caenorhabditis elegans). Caenorhabditis elegans The cells of worms. Suitable mammalian cells include, for example, but not limited to, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 cells, HEK293T cells, HEK293T-RexTM cells, or other transfectable eukaryotic cell lines. Suitable bacterial cells include, but are not limited to, Escherichia coli.

[0857] Preferably, prokaryotes such as Escherichia coli, Bacillus, or Streptomyces can be used, or mammalian cells such as HeLa cells or Jurkat cells, or plant cells such as Arabidopsis thaliana.

[0858] For example, cells can be selected from prokaryotic, yeast, plant and / or insect host cells.

[0859] Preferably, the cells are species of the genus Aspergillus (…). Aspergillus sp) or fungal cells, preferably selected from the genera *Saccharomyces* and *Candida*. Candida ), Kluyveromyces ( Kluyveromyces ), *Hansenula*, *Schizosaccharomyces*, *Yersinia* ( Yarrowia ), Pichia pastoris and Aspergillus.

[0860] Preferably, the cell is a bacterial cell, for example, having a genus selected from Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. For example, the bacteria may be Escherichia coli.

[0861] Preferably, the Escherichia coli host cell is an Escherichia coli host cell recognized by industry and regulatory agencies (including but not limited to Escherichia coli K12 host cells or Escherichia coli BL21 host cells).

[0862] A preferred host cell for use with this disclosure is *Escherichia coli*, which can be recombinantly prepared as described herein. Therefore, the recombinant host can be a recombinant *E. coli* host cell. Mutant libraries, plasmids, detailed metabolic computer models, and other information available for use with *E. coli* allow for the rational design of various modules to increase product yield. Recombinant *E. coli* microorganisms can be prepared using methods similar to those described above for *Yeast*.

[0863] In one embodiment, the recombinant Escherichia coli microorganism contains a nucleotide sequence encoding an SHC / HAC enzyme or an enzyme variant gene.

[0864] Preferably, the recombinant Escherichia coli microorganism comprises a vector construct as described herein. In another preferred embodiment, the recombinant Escherichia coli microorganism comprises a nucleotide sequence encoding the SHC / HAC enzyme and enzyme variants disclosed herein.

[0865] Another preferred host cell used in conjunction with this disclosure is *Saccharomyces cerevisiae*, a fundamental organism widely used in synthetic biology. Therefore, *Saccharomyces cerevisiae* can be a recombinant host. Mutant libraries, plasmids, detailed metabolic computer models, and other information available for use with *Saccharomyces cerevisiae* allow for the rational design of various modules to increase product yield. Methods for preparing recombinant *Saccharomyces cerevisiae* microorganisms are known.

[0866] Cell culture can be performed in a conventional manner. The culture medium may contain a carbon source, at least one nitrogen source, and inorganic salts, with vitamins added. The components of the culture medium may be those commonly used to culture the target microbial species. Carbon sources used in this method include any molecule that can be metabolized by recombinant host cells to promote growth and / or produce (-)-ambroxol or ambergris oxide. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, glycerol, glucose, cellulose, starch, cellobiose, or other glucose-containing polymers.

[0867] In implementations using yeast as the host, carbon sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The host organism can be provided with a carbon source throughout the culture period; alternatively, the organism can be allowed to grow for a period in the presence of another energy source, such as protein, and then the carbon source can be provided only during fed-batch stages.

[0868] The suitability of the recombinant host cell microorganisms used in the methods of this disclosure can be determined using well-known methods through simple testing procedures. For example, the microorganism to be tested can be propagated in a rich culture medium (e.g., LB medium, Bacto-tryptone yeast extract medium, nutrient medium, etc.) under pH, temperature, and reaction conditions typically used for propagating microorganisms. Once a recombinant microorganism (i.e., a recombinant host cell) producing the desired biotransformation product has been selected, the product is typically produced on a large scale by the producing host cell line via a suitable expression system and fermentation (e.g., by microorganisms in cell culture). In one embodiment of this disclosure, a well-defined basic culture medium, such as M9A, is used for cell culture.

[0869] The components of M9A medium include: 14 g / L KH2PO4, 16 g / L K2HPO4, 1 g / L Na3Citrate·2H2O, 7.5 g / L (NH4)2SO4, 0.25 g / L MgSO4·7H2O, 0.015 g / L CaCl2·2H2O, 5 g / L glucose, and 1.25 g / L yeast extract.

[0870] In another embodiment of this disclosure, a nutrient-rich culture medium, such as LB, is used. The components of LB medium include: 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl. Other examples of mineral media and M9 mineral media are disclosed, for example, in US 6524831B2 and US 2003 / 0092143A1.

[0871] Another example of a basic culture medium can be prepared as follows: For a 350 ml culture: Add 307 ml of H2O to 35 ml of citric acid / pho...

Claims

1. A method for preparing (-)-ambroxol or a mixture containing (-)-ambroxol, said method comprising enzymatically converting (3E,7E)-mifetalol (EEH) or a mixture of mifetalol isomers containing EEH to (-)-ambroxol or a mixture containing (-)-ambroxol using an SHC / HAC enzyme variant. The SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% identical to SEQ ID NO:

1. The SHC / HAC enzyme variants have amino acid changes relative to SEQ ID NO: 1 at positions 132, 224, and 432, corresponding to positions M132R, A224V, and I432T, respectively. The SHC / HAC enzyme variant has an amino acid sequence change relative to SEQ ID NO: 1 at position 557 corresponding to SEQ ID NO: 1 and at least one position corresponding to position 81, 431 or 613 corresponding to SEQ ID NO:

1.

2. A method for preparing ambergris oxide or a mixture containing ambergris oxide, said method comprising enzymatically converting (E,E)-bis-dimethylfarnesol (BisEEH) or a mixture of bis-dimethylfarnesol isomers containing BisEEH to ambergris oxide or a mixture containing ambergris oxide using an SHC / HAC enzyme variant. The SHC / HAC enzyme variant has an amino acid sequence that is at least about 70.0% identical to SEQ ID NO:

1. The SHC / HAC enzyme variants have amino acid changes relative to SEQ ID NO: 1 at positions 132, 224, and 432, corresponding to positions M132R, A224V, and I432T, respectively. The SHC / HAC enzyme variant has an amino acid sequence change relative to SEQ ID NO: 1 at position 557 corresponding to SEQ ID NO: 1 and at least one position corresponding to position 81, 431 or 613 corresponding to SEQ ID NO:

1.

3. An SHC / HAC enzyme variant having an amino acid sequence having at least about 70.0% identity with SEQ ID NO:

1. The SHC / HAC enzyme variants have amino acid changes relative to SEQ ID NO: 1 at positions 132, 224, and 432, corresponding to positions M132R, A224V, and I432T, respectively. The SHC / HAC enzyme variant has an amino acid sequence change relative to SEQ ID NO: 1 at position 557 corresponding to SEQ ID NO: 1 and at least one position corresponding to position 81, 431 or 613 corresponding to SEQ ID NO:

1.

4. The method 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 that is at least about 90.0% identical to SEQ ID NO:

1.

5. The method or SHC / HAC enzyme variant of any of the preceding claims, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to SEQ ID NO: 1 at positions 557 and 431 corresponding to positions 557 and 431 of SEQ ID NO:

1.

6. The method or SHC / HAC enzyme variant of any of the preceding claims, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to SEQ ID NO: 1 at positions 557 and 613 corresponding to positions 557 and 613 of SEQ ID NO:

1.

7. The method of claim 5 or 6 or the SHC / HAC enzyme variant, wherein the amino acid sequence of the SHC / HAC enzyme variant has a further amino acid change relative to SEQ ID NO: 1 at position 81 corresponding to position 81.

8. The method or SHC / HAC enzyme variant of any of the preceding claims, wherein one or more of the amino acid changes at positions 81, 431, 557 or 613 are substitutions, such as non-conservative substitutions.

9. The method or SHC / HAC enzyme variant of any of the preceding claims, wherein: The amino acid change corresponding to position 557 of SEQ ID NO: 1 is to replace the amino acid in SEQ ID NO: 1 with a neutral hydrophilic amino acid, such as threonine; and / or The amino acid change corresponding to position 81 of SEQ ID NO: 1 is to replace the amino acid of SEQ ID NO: 1 with a basic amino acid, such as histidine; and / or The amino acid change corresponding to position 431 of SEQ ID NO: 1 is to replace the amino acid of SEQ ID NO: 1 with a hydrophobic amino acid, such as leucine; and / or The amino acid change corresponding to position 613 of SEQ ID NO: 1 is to replace the amino acid in SEQ ID NO: 1 with a neutral hydrophilic amino acid, such as serine.

10. The method or SHC / HAC enzyme variant of any of the preceding claims, 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, for example, an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

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