process

Abstract

Enzyme-mediated method for producing (+)-amber ketal.

Description

[Technical Field]

[0001] Technical Field The present invention generally relates to methods for producing amber ketals using the enzyme squalene-hopene cyclase (SHC). The invention further relates to compositions produced by the methods, various uses of the compositions, and consumer products containing the compositions.

[0002] background Amber ketal, alone or in combination with other woody or amber components, provides a powerful, persistent amber and woody scent useful in fragrance compositions. Amber ketal is traditionally prepared from manool through numerous chemical transformations. However, the supply of naturally occurring manool is limited. For this reason, new routes have been developed in recent years. It is known from WO 2021 / 209482 that amber ketal can be biocatalytically prepared from 6-(hydroxymethyl)-10-methyl-5,9-undecadien-2-one (CAS 606493-86-1). Nevertheless, it would be desirable to provide a new, efficient, and cost-effective route to obtain amber ketal. Summary of the Invention

[0003] overview According to a first aspect of the present invention, there is provided a method for producing (+)-amber ketal (compound of formula (II)) from acyclic acetate of formula (I) via an SHC (squalene-hopene cyclase) mediated process.

[0004] In one specific embodiment, a method for preparing a compound of formula (II) is provided, [ka]

[0005] wherein the method comprises contacting a compound of formula (I) with a squalene-hopene cyclase (SHC) enzyme to obtain a compound of formula (III): [ka] [ka] Hydrolysis follows.

[0006] In a further specific embodiment, a method of making a compound of formula (II) is provided, [ka]

[0007] wherein the method comprises reacting a compound of formula (I) [ka] with a qualene-hopene cyclase (SHC) enzyme and an exogenous hydrolase.

[0008] In certain embodiments of the first aspect of the present invention, the compound of formula (I) is a compound in which the double bond between C-8 and C-9 is in the E-configuration. In certain embodiments, the compound of formula (I) is a compound in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration.

[0009] According to a second aspect of the invention there is provided a compound or composition obtainable or obtainable by a method of the first or second aspect of the invention.

[0010] Particular embodiments of the present invention may provide one or more of the following advantages: · Biocatalytic route for the preparation of (+)-amber ketal; · milder reaction conditions (e.g. lower temperatures); High selectivity; Use of alternative raw materials

[0011] The details, examples, and preferences provided in connection with any particular one or more aspects of the invention are further described herein and apply equally to all aspects of the invention. All possible combinations of the embodiments, examples, and preferences described herein are encompassed by the present invention unless otherwise indicated herein or clearly contradicted by context. [Brief explanation of the drawings]

[0012] Brief description of the diagram [Figure 1] FIG. 1 shows the conversion of 6,10-dimethyl-2-(4-oxopentylidene)undeca-5,9-dien-1-yl acetate (E,Z-AFA) to the compound of formula (II) ((+)-amberketal) in the presence of SHC and lipase. [Figure 2] FIG. 2 shows the conversion of 6,10-dimethyl-2-(4-oxopentylidene)undec-5,9-dien-1-yl acetate (E,Z-AFA) to the compound of formula (III) (AEE) in the presence of SHC.

[0013] Array Overview SEQ ID NO: 1 is the amino acid sequence of wild-type Alicyclobacillus acidocaldarius SHC (AacSHC). SEQ ID NO: 2 is the amino acid sequence of Alicyclobacillus acidocaldarius SHC enzyme variant #65 (SHC#65). SEQ ID NO: 3 is the amino acid sequence of wild-type Gluconobacter morbifer SHC (GmoSHC). SEQ ID NO: 4 is the amino acid sequence of wild-type Acetobacter pasteurianus SHCA (ApaSHCA). SEQ ID NO: 5 is the amino acid sequence of wild-type Acetobacter pasteurianus SHC1 (ApaSHC1). SEQ ID NO: 6 is the amino acid sequence of wild-type Bradyrhizobium japonicum SHC (BjaSHC). SEQ ID NO: 7 is the amino acid sequence of wild-type Bacillus megaterium SHC (BmeSHC). SEQ ID NO: 8 is the amino acid sequence of wild-type Thermosynecochoccus elongatus SHC (TelSHC1). SEQ ID NO: 9 is the amino acid sequence of wild-type Zymomonas mobilis SHC 1 (ZmoSHC1). SEQ ID NO: 10 is the amino acid sequence of wild-type Zymomonas mobilis SHC2 (ZmoSHC2).

[0014] Detailed explanation The present invention is based, at least in part, on the surprising discovery that the (+) amber ketal can be prepared from the acyclic acetate of formula (I) by an SHC (squalene-hopene cyclase) mediated process.

[0015] Thus, in a first aspect of the present invention there is provided a process for producing a compound of formula (II) from a compound of formula (I) in the presence of the enzyme squalene-hopene cyclase (SHC).

[0016] In certain embodiments, a method for preparing a compound of formula (II) is provided, [ka]

[0017] wherein the method comprises contacting a compound of formula (I) with squalene-hopene cyclase (SHC) to obtain a compound of formula (III): [ka] [ka] Hydrolysis follows.

[0018] In certain embodiments, the compound of formula (I) is a compound in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (an E,Z-compound of formula (I)).

[0019] In certain embodiments, the hydrolysis is carried out under acidic conditions, and the acid is selected from aqueous mineral acids, including, for example, HCl, H2SO4, and HClO4; aqueous sulfonic acids, including p-toluenesulfonic acid (PTSA); aqueous sulfonic acids, including pyridinium p-toluenesulfonic acid (PPTS), camphorsulfonic acid (CSA), methanesulfonic acid (MsOH); aqueous phosphoric and phosphonic acids, including H3PO4, PhPO(OH)2; and aqueous carboxylic acids, including AcOH, HC0H, oxalic acid, and benzoic acid.

[0020] In certain embodiments, the acid can be selected from the group consisting of acidic alumina and SiO2.

[0021] The hydrolysis of compounds of formula (III) under acidic conditions is known in the art and is described, for example, by Grant et al. (Australian Journal of Chemistry, 1994 Vol 47, 71-90).

[0022] The acid may be added to the reaction mixture after about 80 wt % or more of the compound of formula (I) has been converted to the compound of formula (II). Adding the acid earlier will result in a lower yield of the compound of formula (II).

[0023] In certain embodiments, the acidic hydrolysis is carried out in situ. In certain embodiments, hydrolysis is carried out via transesterification in the presence of a Bronsted or Lewis acid and an alcohol, where the acid can be selected from Bronsted or Lewis acids (e.g., Al(Oi-Pr)3), and the alcohol ROH can be selected from, for example, MeOH, EtOH, etc.

[0024] As described herein, instead of the acidic hydrolysis of a compound of formula (III) obtained when a compound of formula (I) is contacted with a squalene-hopene cyclase (SHC) enzyme, the compound of formula (I) can be contacted with a mixture comprising a squalene-hopene cyclase (SHC) enzyme and an exogenous hydrolase.

[0025] Without wishing to be bound by theory, it is believed that the compound of formula (I) is converted in a first step to 6-(hydroxymethyl)-10,14-dimethylpentadeca-5,9,13-trien-2-one (HFA), which is then cyclized to the compound of formula (II).

[0026] Thus, in a further aspect, there is provided a method of preparing a compound of formula (II): [ka]

[0027] wherein the method comprises reacting a compound of formula (I) [ka] This involves contacting the enzyme with squalene-hopene cyclase (SHC) and an exogenous hydrolase.

[0028] Compounds of formula (I) exist in four different stereoisomeric forms, for example, compounds of formula (I) having the E,E- or E,Z-configuration.

[0029] In certain embodiments, the compound of formula (I) is a compound in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration (an E,Z-compound of formula (I)).

[0030] In certain embodiments, the method comprises contacting an E,Z-compound of formula (I) with a squalenehopene cyclase (SHC) enzyme in the absence of other stereoisomers of formula (I).

[0031] In other embodiments, the compound of formula (I) may be, for example, a mixture of stereoisomers. In certain embodiments, the mixture comprises an E,E-compound of formula (I) and one or more other stereoisomers of formula (I). In certain embodiments, the mixture comprises an E,Z-compound of formula (I) and one or more other stereoisomers of formula (I).

[0032] In certain embodiments, the method comprises contacting a mixture comprising, consisting essentially of, or consisting of an E,E-compound of Formula (I) and an E,Z-compound of Formula (I) with an SHC enzyme. In certain embodiments, the composition is free of other stereoisomers of Formula (I).

[0033] The weight ratio of the E,Z-compounds of Formula (I) to the sum of the other stereoisomers of Formula (I) may be, for example, about 10:90 or greater. For example, the weight ratio of the E,Z-compounds of Formula (I) to the sum of the other stereoisomers of Formula (I) may be about 20:80 or greater, or about 30:70 or greater, or about 40:60 or greater, or about 50:50 or greater, or about 60:40 or greater, or about 70:30 or greater, or about 80:20 or greater, or about 90:10 or greater, or about 95:5 or greater, or about 99:1 or greater.

[0034] The weight ratio of the E,Z-compounds of Formula (I) to the sum of the other stereoisomers of Formula (I) may be, for example, about 99: 1 or less. For example, the weight ratio of the E,Z-compounds of Formula (I) to the sum of the other stereoisomers of Formula (I) may be about 95:5 or less, or about 90:10 or less, or about 85:15 or less, or about 80:20 or less, or about 60:40 or less.

[0035] For example, the weight ratio of the E,Z-compound of Formula (I) to the sum of the other stereoisomers of Formula (I) may range from about 10:90 to about 99:1, or from about 10:90 to about 90:10, or from about 20:80 to about 80:20, or from about 50:50 to about 80:20, or from about 60:40 to about 80:20.

[0036] The weight ratio of the E,Z-compound of formula (I) to the E,E-compound of formula (I) may be, for example, about 10:90 or greater. For example, the weight ratio of the E,Z-compound of formula (I) to the E,E-compound of formula (II) may be about 20:80 or greater, or about 30:70 or greater, or about 40:60 or greater, or about 50:50 or greater, or about 60:40 or greater, or about 70:30 or greater, or about 80:20 or greater, or about 90:10 or greater, or about 95:5 or greater, or about 99:1 or greater.

[0037] The weight ratio of the E,Z-compound of formula (I) to the E,E-compound of formula (I) may be, for example, about 99.5:0.5 or less. For example, the weight ratio of the E,Z-compound of formula (I) to the E,E-compound of formula (I) may be about 99:1 or less, or about 95:5 or less, or about 90:10 or less, or about 85:15 or less, or about 80:20 or less, or about 70:30 or less, or about 60:40 or less.

[0038] For example, the weight ratio of the E,Z-compound of formula (I) to the E,E-compound of formula (I) may range from about 10:90 to about 99:1, or from about 10:90 to about 90:10, or from about 20:80 to about 80:20, or from about 50:50 to about 80:20, or from about 60:40 to about 80:20.

[0039] The amount of each stereoisomer in a mixture of stereoisomers can be identified, for example, by gas chromatography or NMR spectroscopy.

[0040] The number of stereoisomers of the compound of Formula (I) present can affect the reaction rate. The SHC enzyme may be capable of converting the E,Z-compound of Formula (I) from a complex mixture of stereoisomers of the compound of Formula (I). The mixture may contain only two of the stereoisomers, e.g., the E,Z-compound and the E,E-compound of Formula (I), three stereoisomers (i.e., the E,Z-compound, the E,E-compound, and the Z,E-compound or the Z,Z-compound of Formula (I)), or all four stereoisomers. However, low conversion rates may be observed, which is consistent with the notion that other stereoisomers compete with the E,Z-compound of Formula (I) for access to the SHC enzyme and may therefore act as competitive inhibitors and / or alternative substrates for the conversion of the E,Z-compound of Formula (I). Thus, the substrate of the compound of Formula (I) may consist of an isomeric mixture of two to four isomers, preferably two isomers. Preferably, the substrate of the compound of formula (I) comprises, consists essentially of, or consists of an isomeric mixture of the E,Z- and E,E-compounds of formula (I).

[0041] To date, compounds of formula (I) have not yet been described in the literature. Thus, in a second aspect of the present invention there is provided a compound of formula (I) and its use as a starting material for the preparation of a compound of formula (II).

[0042] The compound of formula (I) can be synthesized according to acylation conditions known to those skilled in the art, as exemplified below: As an acetylating agent, acetyl chloride (AcCl) or acetic anhydride (AcO) can be used in the presence of a base (e.g., an amine, such as 4-(dimethylamino)pyridine (DMAP), a trialkylamine (e.g., trimethylamine), tetrahydrofuran (THF), 2-methyltetrahydrofuran (MeTHF), cyclohexane, PhMe, trimethylamine, or an organic solvent such as dichloromethane (DCM).

[0043] Compound of formula (II) The compounds of formula (II) produced by the methods described herein can be isolated by steam extraction / distillation or organic solvent extraction with a water-immiscible solvent (to separate the reaction product and unreacted substrate from the biocatalyst, which remains in the aqueous phase), followed by evaporation of the solvent to obtain the crude reaction product as determined by gas chromatography (GC) analysis. Steam extraction / distillation and organic solvent extraction methods are known to those skilled in the art.

[0044] For example, the resulting compound of formula (II) can be extracted from the entire reaction mixture using an organic solvent, such as a water-immiscible solvent (e.g., toluene). Alternatively, the resulting compound of formula (II) can be extracted from the solid phase of the reaction mixture (obtained, for example, by centrifugation or filtration) using a water-miscible solvent (e.g., ethanol) or a water-immiscible solvent (e.g., toluene). As a further example, the compound of formula (II) can exist in the solid phase as a crystal or in an amorphous form and can be separated from the remaining solid phase (cellular material or debris) and liquid phase by filtration. As a further example, at temperatures above the melting point of the compound of formula (II), the compound of formula (II) can form an oil layer on the aqueous phase, which can be removed and recovered. After removing the oil layer, to ensure complete recovery of the compound of formula (II), an organic solvent can be added to the aqueous phase containing the biomass to extract the remaining compound of formula (II) contained in, on, or around the biomass. The organic layer can be combined with the oil layer, and the whole can then be further processed to isolate and purify the compound of formula (II). The compound of formula (II) can be further selectively crystallized to remove by-products and unreacted compounds of formula (I) from the final product. The term "selective crystallization" refers to a process step in which the compound of formula (II) is crystallized from a solvent while the by-products remain dissolved in the crystallization solvent, so that the isolated crystalline material contains only the compound of formula (II), or only olfactorily acceptable amounts of by-products. A water-miscible solvent, such as ethanol, can be used in the selective crystallization step. Selective crystallization of the compound of formula (II) can be affected by the presence of unreacted compounds of formula (I) and the ratio of the compound of formula (II) to other detectable by-products. Selective crystallization of the compound of formula (I) is possible even when the conversion of the compound of formula (I) to the compound of formula (II) is only 10%.

[0045] Examples of water-miscible and water-immiscible organic solvents suitable for use in the extraction and / or selective crystallization of the compound of formula (I) are aliphatic hydrocarbons, preferably those having 5 to 8 carbon atoms, such as pentane, cyclopentane, cyclohexane, heptane, octane or cyclooctane, aromatic hydrocarbons, such as toluene, xylene, chlorobenzene or 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, methyl-tetrahydrofuran or esters, such as ethyl acetate, n-butyl acetate or ketones, such as methyl isobutyl ketone, or mixtures thereof. Particularly preferred solvents are heptane, methyl tert-butyl ether (MTBE, also known as tert-butyl methyl ether, tertiary butyl methyl ether, tBME), diisopropyl ether, methyl tetrahydrofuran, ethyl acetate, and / or mixtures thereof. Preferably, a water-miscible solvent such as ethanol is used to extract the compound of formula (II) from the solid phase of the reaction mixture. The use of ethanol has the advantages of being easy to handle, non-toxic, and environmentally friendly.

[0046] As used herein, the term "isolated" refers to a bioconversion product, such as a compound of Formula (II), that has been separated or purified from components that accompany it. An entity produced in a cellular system different from its naturally occurring source is "isolated" because it necessarily does not contain components that naturally accompany it. The degree of separation or purity can be measured by any appropriate method, such as gas chromatography (GC), HPLC, or NMR analysis.

[0047] In some embodiments, the compound of Formula (II) is isolated from the resulting crude product and purified (e.g., to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity).

[0048] Desirably, the concentration of the compound of Formula (II) in the reaction broth obtained by the methods described herein can be from about 1 mg / L to about 20,000 mg / L (20 g / L) or more, for example, from about 20 g / L to about 200 g / L or 100 to 500 g / L (including 150 g / L, 250 g / L, 300 g / L, 350 g / L, 400 g / L, or 450 g / L).

[0049] The compounds of formula (II) obtained by the processes described herein may be, for example, in amorphous or crystalline form.

[0050] The compound of formula (II) contains a number of asymmetric carbon atoms, and therefore, one or more stereoisomers of the compound of formula (II), including enantiomers and diastereomers, may exist. In addition to the compound of formula (II), the product produced by the method described herein may contain one or more stereoisomers of the compound of formula (II). The resulting stereoisomer may depend on the stereoisomer of the compound of formula (I) used.

[0051] For example, in addition to the compound of formula (II), the compound of formula (IV) (also known as (-)-epi-8-ambeketal) can also be made. [ka]

[0052] In certain embodiments, other stereoisomers of the compound of formula (II) are not made by the method or are not present in the product of the method, for example, in certain embodiments, the compound of formula (IV) is not made by the method or is not present in the product of the method.

[0053] The methods described herein can produce, for example, a compound of formula (II) and one or more other stereoisomers of the compound of formula (II) (e.g., a compound of formula (IV)). Thus, the compositions described herein, such as compositions obtained by the methods described herein or compositions obtainable by the methods described herein, can include a compound of formula (II) and one or more stereoisomers of the compound of formula (II) (e.g., a compound of formula (IV)).

[0054] As used herein, "making a compound of Formula (II)" can also refer to "making" or "obtaining" the respective compound, or to "making" or "obtaining" a mixture comprising, consisting essentially of, or consisting of the respective compound.

[0055] SHC enzyme In the methods described herein, the compound of formula (I) is enzymatically converted using an SHC enzyme. As used herein, the term "SHC enzyme" refers to both the wild-type squalene hopene cyclase (SHC) enzyme that occurs naturally in bacteria, and to mutants of this SHC enzyme.

[0056] The term "variant" is understood as a polypeptide that differs from the polypeptide from which it is derived by one or more changes in the amino acid sequence. A polypeptide derived from a variant is also known as a parent polypeptide. Typically, variants are produced artificially, preferably by genetic engineering. Typically, the polypeptide from which a variant is derived is a wild-type enzyme. However, variants usable in the present disclosure may also be derived from a homolog, ortholog, or paralog of the parent polypeptide, or from an artificially constructed variant. The change in amino acid sequence may be an amino acid exchange (substitution), insertion, deletion, N-terminal truncation, C-terminal truncation, or a combination thereof, and these changes may occur at one or several positions.

[0057] Generally, polypeptides exhibiting at least about 30% amino acid sequence identity are useful for identifying conserved regions. Conserved regions of related polypeptides exhibit at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% amino acid sequence identity. In some embodiments, the conserved regions have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity. Determining sequence identity is well known to those skilled in the art.

[0058] In one specific embodiment, the wild-type squalene hopene cyclase enzyme is isolated from the thermophilic bacterium Alicyclobacillus acidocaldarius.

[0059] In certain embodiments, the SHC enzyme (e.g., from which the SHC enzyme may be derived—wild-type or mutant thereof) may be an Alicyclobacillus acidocaldarius (Aac) SHC enzyme, an Acetobacter pasteurianus (Apa) SHC enzyme, a Zymomonas mobilis (Zmo) SHC enzyme, a Bradyrhizobium japonicum (Bja) SHC enzyme, a Bacillus megaterium (Bme) SHC enzyme, a Thermosynecochoccus elongatus (TelSHC1) SHC enzyme, or a Gluconobacter morbifer (Gmo) SHC enzyme. In certain embodiments, the SHC enzyme (e.g., from which the SHC enzyme may be derived—wild-type or mutant thereof) may be a Bacillus megaterium SHC enzyme. In certain embodiments, the SHC enzyme (e.g., from which the SHC enzyme may be derived—wild-type or mutant thereof) may be an Alicyclobacillus acidocaldarius SHC enzyme. In a further embodiment, the SHC enzyme (eg, from which the SHC enzyme may be derived - wild-type or a mutant thereof) may be a Bradyrhizobium japonicum SHC enzyme.

[0060] For ease of reference, "AacSHC" may be used to refer to the Alicyclobacillus acidocaldarius (Aac) SHC enzyme, "ApaSHC" may be used to refer to the Acetobacter pasteurianus (Apa) SHC enzyme, "ZmoSHC" may be used to refer to the Zymomonas mobilis (Zmo) SHC enzyme, "BmeSHC" may be used to refer to the Bacillus megaterium (Bme) SHC enzyme, "BjaSHC" may be used to refer to the Bradyrhizobium japonicum (Bja) SHC enzyme, "TelSHC1" may be used to refer to the Thermosynecochoccus elongatus (TelSHC1) SHC enzyme, and "GmoSHC" may be used to refer to the Gluconobacter morbifer (Gmo) SHC enzyme.

[0061] The AacSHC, ZmoSHC, and BjaSHC enzyme sequences are disclosed in BASF WO 2010 / 139719, US 2012 / 01345477A1, Seitz et al (2012) J. Molecular Catalysis B. Enzymatic 84: 72-77, and Seitz (2012 PhD thesis, https: / / elib.uni-stuttgart.de / handle / 11682 / 1400.ZmoSHC). Two different sequences for ZmoSHC, designated ZmoSHC1 and ZmoSHC2, have been disclosed. The GmoSHC enzyme sequence is disclosed in WO 2018 / 157021. Table 1 shows the sources and accession numbers of the wild-type SHC enzymes.

[0062] [Table 1]

[0063] The processes for making compounds of formula (II) described herein are carried out under conditions of time, temperature, pH and solubilizing agent (if used) that provide for the conversion of a compound of formula (I) to a compound of formula (II).

[0064] The process for making the compounds of formula (II) may be carried out at the optimum temperature range or optimum temperature and / or optimum pH range or optimum pH and / or optimum concentration range or optimum solubilizing agent concentration (if used) for the particular enzyme used.

[0065] The pH of the reaction mixture for the SHC wild-type enzyme or the SHC enzyme variant under consideration is in the range of 4 to 8, preferably 4.5 to 6.5, and more preferably 4.5 to 6.5, and can be maintained by performing the reaction in an appropriate buffer or by adjusting the pH over the course of the reaction. Exemplary buffers for this purpose are citrate buffer or succinate buffer.

[0066] For SHC enzymes, the temperature can be between about 15° C. and about 60° C., e.g., about 15° C. to about 50° C., or about 15° C. to about 45° C., or about 30° C. to about 60° C., or about 35° C. to about 55° C. The temperature can be kept constant or can be varied during the bioconversion process.

[0067] In certain embodiments, once the hydrolysis under acidic conditions is initiated, the temperature is increased, for example, from an initial reaction temperature of about 30°C to 40°C to about 50°C to 70°C.

[0068] It may be useful to include a solubilizing agent(s) in the bioconversion reaction (e.g., surfactants, detergents, solubility enhancers, water-miscible organic solvents, etc.). Examples of surfactants include Triton (R) X-100, Tween (R)80, taurodeoxycholic acid, sodium taurodeoxycholate, sodium dodecyl sulfate (SDS), and / or sodium lauryl sulfate (SLS). In a specific embodiment, SDS is used as the solubilizing agent.

[0069] The methods for making compounds of formula (III) disclosed herein can be carried out at the optimum temperature range or optimum temperature and / or optimum pH range or optimum pH and / or optimum solubilizing agent (if used) concentration range or optimum solubilizing agent concentration for the particular enzyme used.

[0070] The pH of the reaction mixture for the SHC wild-type enzyme or the SHC enzyme variant under consideration is in the range of 4 to 8, preferably 4.5 to 6.5, and more preferably 4.5 to 6.5, and can be maintained by performing the reaction in an appropriate buffer or by adjusting the pH over the course of the reaction. Exemplary buffers for this purpose are citrate buffer or succinate buffer.

[0071] For SHC enzymes, the temperature can be between about 15° C. and about 60° C., e.g., about 15° C. to about 50° C., or about 15° C. to about 45° C., or about 30° C. to about 60° C., or about 35° C. to about 55° C. The temperature can be kept constant or can be varied during the bioconversion process.

[0072] It may be useful to include a solubilizing agent(s) in the bioconversion reaction (e.g., surfactants, detergents, solubility enhancers, water-miscible organic solvents, etc.). Examples of surfactants include Triton (R) X-100, Tween (R) 80, taurodeoxycholic acid, sodium taurodeoxycholate, sodium dodecyl sulfate (SDS), and / or sodium lauryl sulfate (SLS). In a specific embodiment, SDS is used as the solubilizing agent.

[0073] In one specific embodiment, the methods described herein are carried out in the presence of an SHC enzyme and an exogenous hydrolase.

[0074] It should be noted that the SHC-mediated processes described herein are carried out using whole cells that produce the respective squalene hopene cyclase. The reaction mixture containing whole cells also contains lipase- and / or esterase-type hydrolase enzymes, which are produced by the cells to ensure their own metabolic purposes. While these endogenous hydrolases may hydrolyze, to a lesser extent, the ester groups of compounds that serve as substrates for the SHC enzyme (e.g., compound I) or compounds resulting from the SHC cyclization reaction itself (e.g., compound III), these compounds are not substrates recognized by endogenous hydrolases in their primary metabolic functions.

[0075] Therefore, exogenous hydrolytic enzymes (lipases or esterases) are required to induce hydrolysis or to significantly increase the level of hydrolysis by enzymes naturally present in cells.

[0076] As used herein, the terms "exogenous," e.g., "exogenous hydrolase" and "exogenous lipase," refer to the active inclusion of a hydrolase, e.g., a lipase. Thus, the term "exogenous" explicitly excludes hydrolases formed in whole cells producing squalenehopene cyclase. Alternatively, those skilled in the art can engineer the respective SHC-producing strain to additionally produce hydrolases. Such hydrolases are considered equivalent to "exogenous hydrolases" within the meaning of the present invention.

[0077] When the method for making (+)-amber ketal (a compound of formula (II) described herein) is carried out in the presence of an exogenous hydrolase, the term "hydrolase" refers to an enzyme that catalyzes the hydrolysis. The hydrolase can be any type of hydrolase in the EC 3.1 class (e.g., the EC 3.1.1 class). In one particular embodiment, the exogenous hydrolase is a lipase.

[0078] Lipases are polypeptides with lipase activity. Their physiological function is to hydrolyze triglycerides into di- or monoglycerides, fatty acids, and glycerol. For example, lipases are those disclosed in Table 5. EC numbers refer to the Enzyme Nomenclature 1992 of the NC-IUBMB, Academic Press, San Diego, Calif., including supplements 1-5 in Eur. J. Biochem., 1994, 223:1-5; Eur. J. Biochem., 1995, 232:1-6; Eur. J. Biochem., 1996, 237:1-5; Eur. J. Biochem., 1997, 250:1-6; and Eur. J. Biochem., 1999, 264:610-650, respectively. The nomenclature is regularly supplemented and updated; see, for example, https: / / iubmb.qmul.ac.uk / .

[0079] Lipases can be obtained by culturing suitable microorganisms and then isolating the lipase expressed by the microorganisms. Microorganisms suitable for this purpose belong to the genera Mucor, Aspergillus, Rhizopus, Rhizomucor, Pseudomonas, Candida, Humicola, Thermomyces, Burkholderia, and Penicillium. Preferred species are Pseudomonas cepacia and Pseudomonas fluorescens. Lipases obtained by culturing suitable microorganisms and then isolating the lipase expressed by the microorganisms may also be referred to as microbial lipases herein.

[0080] However, lipases are also commercially available from sources such as Sigma Aldrich and Biocatalysts Limited (UK). In one embodiment, the lipase belongs to the genus Pseudomonas, for example Pseudomonas cepacia or Pseudomonas fluorescens.

[0081] In another embodiment, the lipase can be immobilized ("immobilized lipase"). An immobilized lipase is a lipase adsorbed onto a carrier. A carrier is an inert solid material. The immobilized lipase can be maintained in suspension in the phase in which the reactions described herein are carried out, for example, via agitation.

[0082] The following are examples of the methods and other aspects described herein, and therefore should not be construed as limitations on the disclosure, but are merely provided to teach how to make examples of the disclosure.

[0083] example The abbreviations used in the examples below and in Figures 1 and 2 have the following meanings:

[0084] AFA: 6,10-dimethyl-2-(4-oxopentylidene)undeca-5,9-dien-1-yl acetate (compound of formula (I)) (E,Z)-AFA: (2Z,5E)-6,10-dimethyl-2-(4-oxopentylidene)undeca-5,9-dien-1-yl acetate HFA: 6-(hydroxymethyl)-10,14-dimethylpentadeca-5,9,13-trien-2-one E,Z-HFA: (5Z,9E)-6-(hydroxymethyl)-10,14-dimethylpentadeca-5,9,13-trien-2-one AEE: ((4aR,5aS,10aS,10bR)-(3,7,7,10a-tetramethyl-1,5,6,6a,7,8,9,10,10a,10b-decahydro-4aH-benzo[f]chromen-4a-yl)methyl acetate (compound of formula (III))

[0085] Example 1: (2Z,5E)-6,10-dimethyl-2-(4-oxopentylidene)undeca-5,9-dien-1-yl acetate(E,Z-Acyloxyfarnesylacetone = E,Z-AFA) A solution of (5Z,9E)-6-(hydroxymethyl)-10,14-dimethylpentadeca-5,9,13-trien-2-one (1 g, 3.3 mmol) in dichloromethane (5 mL) was treated with triethylamine (0.38 g, 3.8 mmol) and acetyl chloride (0.27 mL, 3.8 mmol) at room temperature. The resulting mixture was stirred at room temperature for 2.5 hours, treated with water (10 mL) and MTBE (10 mL), and the aqueous phase was extracted with MTBE (2 × 10 mL). The combined organic phases were washed with saturated aqueous NaCl (10 ml), dried (MgSO), filtered, and concentrated under reduced pressure to give 1.03 g of a crude oil, which was purified by filtration over SiO (eluting with MTBE) to give (2Z,5E)-6,10-dimethyl-2-(4-oxopentylidene)undeca-5,9-dien-1-yl acetate (0.94 g, 88% yield).

[0086] 1 H NMR (400 MHz, CDCl3) δ ppm 5.36 (t, J = 7.3, 1H), 5.11-5.03 (m, 2H), 4.60 (s, 2H), 2.48 (t, J = 7.1, 2H), 2.35 (dt, J = 7.1, 7.3, 2H), 2.12 (s, 3H), 2.05 (s, 3H), 2.10-2.00 (m, 6H), 1.99-1.92 (m, 2H), 1.67 (br. s, 3H), 1.59 (br. s, 3H), 1.58 (br. s, 3H). 13C NMR (100 MHz, CDCl3) δ ppm 207.87 (s, 1 C), 170.95 (s, 1 C), 135.44 (s, 1 C), 134.71 (s, 1 C), 131.25 (s, 1 C), 128.91 (d, 1 C), 124.21 (d, 1 C), 123.50 (d, 1 C), 61.74 (t, 1 C), 43.44 (t, 1 C), 39.63 (t, 1 C), 35.16 (t, 1 C), 29.86 (q, 1 C), 26.67 (t, 1 C), 26.52 (t, 1 C), 25.63 (q, 1 C), 22.01 (t, 1 C), 20.93 (q, 1 C), 17.62 (q, 1 C), 15.97 (q, 1 C). GC-MS (EI) m / z 320 (1), 278 (1), 260 (1), 245 (1), 227 (1), 217 (3), 202 (3), 187 (2), 175 (5), 159 (6), 149 (3), 137 (4), 133 (28), 123 (11), 109 (8), 107 (10), 105 (11), 95 (10), 93 (16), 91 (10), 81 (39), 79 (11), 69 (87), 67 (13), 60 (1), 55 (11), 53 (10), 43 (100), 41 (52), 29 (3).

[0087] Example 2: Biocatalyst production To produce squalene hopene cyclase in Escherichia coli, the gene encoding the desired squalene hopene cyclase (wt or mutant) was inserted into the plasmid pET-28a(+) under the control of an IPTG-inducible T7 promoter. This plasmid was transformed into E. coli BL21(DE3) using standard heat shock transformation methods.

[0088] Culture medium The minimal medium used as default for biocatalyst production contained: 10% 10x citric acid / phosphate buffer (133g / l KH2PO4, 40g / l (NH4)2HPO4, 17g / l citric acid.H2O in deionized water, pH adjusted to 6.8 with 32% NaOH), · 0.01% trace element solution (50g / l Na2EDTA.2H2O, 20g / l FeSO4.7H2O, 3g / l H3BO3, 0.9g / l MnSO4.2H2O, 1.1g / l CoCl2, 80g / l CuCl2, 240g / l NiSO4.7H2O, 100g / l in deionized water KI, 1.4g / l (NH4)6Mo7O 24 .4H2O, 1g / l ZnSO4.7H2O), 0.01% thiamine solution (2.25 g / l thiamine hydrochloride in deionized water), 2% glucose solution (20% w / v glucose in deionized water).

[0089] The citrate / phosphate buffer solution was first autoclaved, and the other components were added from sterile solutions that had been autoclaved or filter-sterilized (0.2 μm).

[0090] fermentation Fermentation was carried out in a 750 ml InforsHT reactor. 168 ml of deionized water was added to the fermentation vessel. The reactor was equipped with all necessary probes (pO2, pH, sampling, antifoam), C+N feed and sodium hydroxide bottles and autoclaved. After autoclaving, the reactor was added: 20ml 10x phosphate / citrate buffer 14ml 50% glucose 0.53ml MgSO4 solution 2 ml (NH4)2SO4 solution (50% (w / v) (NH4)2SO4 in deionized water) 0.020ml trace element solution 0.400ml thiamine solution 0.200ml Kanamycin solution (50mg / ml)

[0091] Run parameters were: pH = 6.95, pO2 = 40%, T = 30°C, 300 rpm. Cascade: rmp set point 300, min 300, max 1000, flow (l / min) set point 0.1, min 0, max 0.6. Antifoam control: 1:9.

[0092] The seed culture was cultivated in LB medium (plus kanamycin) at 37°C, 220 rpm for 8 hours. Fermenters were inoculated with this seed culture to an OD650nm of 0.4-0.5. Fermentation was first carried out in a batch mode for 11.5 hours, and then The C+N feed was started with the feed solution (sterilized glucose solution (143 ml H2O + 35 g glucose), to which, after sterilization, the following was added: 17.5 ml (NH4)2SO4 solution, 1.8 ml MgSO4 solution, 0.018 ml trace element solution, 0.360 ml thiamine solution, 0.180 ml kanamycin solution. The feed was run at a constant flow rate of approximately 4.2 ml / h. The glucose and NH4 + Measurements of were performed externally to assess the availability of C- and N-sources during the culture.

[0093] The culture was grown for a total of approximately 25 hours, typically reaching an OD of 40-45. SHC production was then induced by adding IPTG to the fermenter at a concentration of 1 mM and continued for approximately 16 hours at 30°C and pO2 = 20%. At the end of induction, the cells were collected by centrifugation, washed with citrate / sodium phosphate buffer, pH 5.6, and stored as pellets at 4°C or -20°C until further use.

[0094] Example 3: Screening for squalenehopene cyclase The SHC enzyme was screened for its ability to cyclize E,Z-AFA (Example 1) in reactions containing 1 g / L substrate, cells at OD 650 nm, and sodium dodecyl sulfate (SDS) as a surfactant. Reactions (1 ml volume) were carried out in a citrate / sodium phosphate buffer at the pH, temperature, and SDS concentrations shown in Table 2. In negative control reactions, cells not producing the SHC enzyme were used as the biocatalyst. Reactions were incubated in a Heidolph Synthesis 1L / 2L apparatus for 22 hours under constant agitation (800 rpm) and thoroughly extracted with MTBE before being assayed for substrate and product content (GC-FID).

[0095] [Table 2]

[0096] As illustrated in Example 5, methyl (3,7,7,10a-tetramethyl-1,5,6,6a,7,8,9,10,10a,10b-decahydro-4aH-benzo[f]chromen-4a-yl)acetate (AEE) was identified as the cyclization product of E,Z-AFA. E,Z-hydroxyfarnesylacetone (E,Z-HFA) and (+)-amber ketal were also detected as reaction products, albeit in trace amounts (Table 3). In a negative control reaction using cells that do not produce SHC, E,Z-HFA was observed as a hydrolysis product of E,Z-AFA.

[0097] [Table 3]

[0098] Example 4: Lipase screening Lipase enzymes were screened to identify an enzyme that showed high conversion of E,Z-AFA to E,Z-HFA. Reactions (total volume 1 ml) containing 5 g / L substrate and approximately 1.25 g / L lipase were carried out in potassium phosphate buffer, pH 7.5, at 35°C in a Heidolph Synthesis 1L / 2L apparatus under constant stirring (800 rpm). After 24 hours, the reaction mixture was extracted with MTBE and analyzed for E,Z-HFA content (GC-FID). The results are shown in Table 4 below. The extent of hydrolysis varied depending on the lipase used.

[0099] [Table 4]

[0100] Example 5: (4aR,5aS,10aS,10bR)-(3,7,7,10a-tetramethyl-1,5,6,6a,7,8,9,10,10a,10b-decahydro-4aH-benzofchromen-4a-yl)methyl acetate (AEE; compound of formula (III)). 40 mg of E,Z-AFA (Example 1) was cyclized in a preparative-scale reaction. The reaction mixture contained 2 g / L E,Z-AFA and 330 g / L wild-type BmeSHC-producing cells, and was run at 45 °C in a citrate / Na phosphate buffer, pH 5.6, in the presence of 0.050% SDS. Complete conversion occurred in approximately 20 h. The reaction mixture was extracted with MTBE (3x), and the MTBE fractions were pooled, dried over MgSO, filtered, and the solvent evaporated to give 0.188 g of crude product, which was purified by microflash chromatography on SiO (MTBE / heptane) to give the title compound (6.5 mg, 16% yield).

[0101] 1 H NMR (600 MHz, C6D6) δ ppm 4.55-4.49 (m, 2H), 4.23 (d, J = 11.7, 1H), 2.44 (dt, J = 3.0, 12.8, 1H), 1.79 (br. s, 3H), 1.78 (s, 3H), 1.73-0.78 (m, 12H), 0.77 (s, 3H), 0.70 (s, 3H), 0.65 (s, 3H), 0.67-0.60 (m, 1H). 13C NMR (150 MHz, C6D6) δ ppm 170.21 (s, 1 C), 148.32 (s, 1 C), 95.24 (d, 1 C), 76.70 (s, 1 C), 60.28 (t, 1 C), 56.34 (d, 1 C), 52.68 (d, 1 C), 41.94 (t, 1 C), 39.26 (t, 1 C), 36.70 (s, 1 C), 35.09 (t, 1 C), 33.39 (q, 1 C), 33.14 (s, 1 C), 21.56 (q, 1 C), 20.54 (q, 1 C), 20.30 (q, 1 C), 19.44 (t, 1 C), 18.87 (t, 1 C), 18.24 (t, 1 C), 15.60 (q, 1 C). GC-MS (EI) m / z 320 (6), 302 (1), 287 (1), 278 (1), 260 (8), 247 (1), 245 (12), 217 (17), 202 (3), 189 (9), 175 (6), 161 (5), 149 (10), 137 (21), 135 (11), 133 (11), 123 (28), 122 (30), 121 (22), 109 (20), 107 (16), 105 (14), 95 (21), 93 (22), 91 (15), 81 (24), 79 (17), 69 (22), 67 (14), 61 (1), 55 (25), 43 (100), 41 (27), 29 (5).

[0102] Example 6: (+)-Amber ketal from E,Z-AFA Reactions were performed using E. coli cells producing E,Z-AFA and BmeSHC with or without the addition of Pseudomonas fluorescens lipase. Reactions (3 ml volume) contained 2 g / L E,Z-AFA, cells to an OD650nm of 50, and 0.125 g / L SDS. Reactions were performed in citrate / sodium phosphate buffer, pH 5.6, at 45°C with constant agitation (350 rpm on a Radleys Carousel 12 Plus). 0.5 g / L Pseudomonas fluorescens lipase was added per reaction.

[0103] Samples were collected over time, extracted with MTBE, and analyzed for substrate (E,Z-AFA) and products (AEE, E,Z-HFA, (+)-amber ketal) by GC-FID. In the absence of exogenous lipase, E,Z-AFA was converted to AEE, but because whole cells were used, background lipase hydrolysis activity was low and only traces of E,Z-HFA and (+)-amber ketal were observed. The conversion rate was approximately 20% in 22 h (Figure 2). When lipase was added to the reaction, complete conversion of E,Z-AFA was obtained within 4–6 h. (+)-Amber ketal appeared as the major reaction product, and E,Z-HFA was observed at low levels (Figure 1).

[0104] Example 7: Preparation of (+)-amber ketal from E,Z-AFA The reaction was carried out as in Example 5. Complete conversion of E,Z-AFA was obtained in approximately 20 hours. The reaction was then acidified to pH 2.2 by adding phosphoric acid. The amount of AEE decreased over time, with a concomitant increase in (+)-amber ketal. Further acidification (pH 2.0) allowed for the complete conversion of AEE to (+)-amber ketal.

[0105] Array List SEQ ID NO: 1 (amino acid sequence of wild-type Alicyclobacillus acidocaldarius SHC) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR

[0106] SEQ ID NO: 2 (amino acid sequence of Alicyclobacillus acidocaldarius SHC enzyme variant #65) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRRWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRVLHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTTWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYSHVFPTLALGRYKQAIERR*

[0107] SEQ ID NO: 3 (amino acid sequence of wild-type Gluconobacter morbifer SHC) MSPADISTKSSSFQRLDNMLPEAVSSACDWLIDQQKPDGHWVGPVESNACMEAQWCLALWFLGQEDHPLRPRLAQALLEMQREDGSWGIYVGADHGDINTTVEAYAALRSMGYAADMPIMAKSAAWIQQKGGLRNVRVFTRYWLALIGEWPWDKTPNLPPEIIWLPDNFIFSIYNFAQWARATMMPLTILSARRPSRPLLPENRLDGLFPEGRENFDYELPVKGEEDLWGRFFRAADKGLHSLQSFPVRRFVPREAAIRHVIEWIIRHQDADGGWGGIQPPWIYGLMALSVEGYPLHHPVLAKAMDALNDPGWRRDKGDASWIQATNSPVWDTMLAVLALHDAGAEDRYSPQMDKAIGWLLDRQVRVKGDWSIKLPDTEPGGWAFEYANDKYPDTDDTAVALIALAGCRHRPEWRERDIEGAISRGVNWLLAMQSSSGGWGAFDKDNNRSILTKIPFCDFGEALDPPSVDVTAHVLEAFGLLGISRNHPSVQKALAYIRSEQERNGAWFGRWGVNYVYGTGAVLPALAAIGEDMTQPYIVRACDWLMSVQQENGGWGESCASYMDINAVGHGVATASQTAWALIGLLAAKRPKDREAIARGCQFLIERQEDGSWTEEEYTGTGFPGYGVGQAIKLDDPSLPDRLLQGAELSRAFMLRYDLYRQYFPVMALSRARRMMKEDASAAA

[0108] SEQ ID NO: 4 (amino acid sequence of wild-type Acetobacter pasteurianus SHC-A) MAADGSALSESRLSSEALDRAVLSAHTALSQAQQDDGHWVYELEADATIPAEYILLEHFMDRIDDALEQKIAIYLRRIQSEEHGGWPLYHNGKFDLSATVKAYFALKAVGDDINAPHMQRAREAILDHGGAERSNVFTRSQLALFGEVPWRATPVMPVELMLLPAKAFFSVWNMSYWSRTVIAPLLVLAALRPVAANPRQVHVRELFVTPPEKVQDWIRGPYRSAWGYVFKGLDSVLRPVVPFIPEKTHKKAIQAALDFIEPRLNGKDGLGAIYPAMANVVMMYRAMGVPDEDPRAKTAWEAVQALIVEKDDEAYCQPCVSPIWDTGLSGHAMIEAASGPNGIAPEKTVAELKKASAWLRSKQILNVKGDWAVRNPNLAPGGWAFQYGNDYYPDVDDTAVVGMLLHREGDPTNAEAIERARTWIVGMQSTDGGWGAFDIDNNKDVLNHIPFADHGALLDPPTADVTARCISFLAQLRNPEDEPVIQRGLEYLRKEQEKDGSWFGRWGTNYIYGTWSALCALNAAGVSHDDPAVVKAVEWLRSVQRADGGWGEGCESYEGGPHGTYGESLPSQTAWAVLGLMAAGRRDDPAVTRGIAWLADQQDANGEWHEDPYNAVGFPKVFYLRYHGYKQFFPLMALARYRNLESSNTRRVSFGF

[0109] SEQ ID NO: 5 (amino acid sequence of wild-type Acetobacter pasteurianus SHC-1) MNMASRFSLKKILRSGSDTQGTNVNTLIQSGTSDIVRQKPAPQEPADLSALKAMGNSLTHTLSSACEWLMKQQKPDGHWVGSVGSNASMEAEWCLALWFLGLEDHPLRPRLGKALLEMQRPDGSWGTYYGAGSGDINATVESYAALRSLGYAEDDPAVSKAAAWIISKGGLKNVRVFTRYWLALIGEWPWEKTPNLPPEIIWFPDNFVFSIYNFAQWARATMMPLAILSARRPSRPLRPQDRLDALFPGGRANFDYELPTKEGRDVIADFFRLADKGLHWLQSSFLKRAPSREAAIKYVLEWIIWHQDADGGWGGIQPPWVYGLMALHGEGYQFHHPVMAKALDALNDPGWRHDKGDASWIQATNSPVWDTMLSLMALHDANAEERFTPEMDKALDWLLSRQVRVKGDWSVKLPNTEPGGWAFEYANDRYPDTDDTAVALIAIASCRNRPEWQAKGVEEAIGRGVRWLVAMQSSCGGWGAFDKDNNKSILAKIPFCDFGEALDPPSVDVTAHVLEAFGLLGLPRDLPCIQRGLAYIRKEQDPTGPWFGRWGVNYLYGTGAVLPALAALGEDMTQPYISKACDWLINCQQENGGWGESCASYMEVSSIGHGATTPSQTAWALMGLIAANRPQDYEAIAKGCRYLIDLQEEDGSWNEEEFTGTGFPGYGVGQTIKLDDPAISKRLMQGAELSRAFMLRYDLYRQLFPIIALSRASRLIKLGN

[0110] SEQ ID NO: 6 (amino acid sequence of wild-type Bradyrhizobium japonicum SHC) MTVTSSASARATRDPGNYQTALQSTVRAAADWLIANQKPDGHWVGRAESNACMEAQWCLALWFMGLEDHPLRKRLGQSLLDSQRPDGAWQVYFGAPNGDINATVEAYAALRSLGFRDDEPAVRRAREWIEAKGGLRNIRVFTRYWLALIGEWPWEKTPNIPPEVIWFPLWFPFSIYNFAQWARATLMPIAVLSARRPSRPLPPENRLDALFPHGRKAFDYELPVKAGAGGWDRFFRGADKVLHKLQNLGNRLNLGLFRPAATSRVLEWMIRHQDFDGAWGGIQPPWIYGLMALYAEGYPLNHPVLAKGLDALNDPGWRVDVGDATYIQATNSPVWDTILTLLAFDDAGVLGDYPEAVDKAVDWVLQRQVRVPGDWSMKLPHVKPGGWAFEYANNYYPDTDDTAVALIALAPLRHDPKWKAKGIDEAIQLGVDWLIGMQSQGGGWGAFDKDNNQKILTKIPFCDYGEALDPPSVDVTAHIIEAFGKLGISRNHPSMVQALDYIRREQEPSGPWFGRWGVNYVYGTGAVLPALAAIGEDMTQPYIGRACDWLVAHQQADGGWGESCASYMDVSAVGRGTTTASQTAWALMALLAANRPQDKDAIERGCMWLVERQSAGTWDEPEFTGTGFPGYGVGQTIKLNDPALSQRLMQGPELSRAFMLRYGMYRHYFPLMALGRALRPQSHS

[0111] SEQ ID NO: 7 (amino acid sequence of wild-type Bacillus megaterium SHC) MIILLKEVQLEIQRRIAYLRPTQKNDGSFRYCFETGVMPDAFLIMLLRTFDLDKEVLIKQLTERIVSLQNEDGLWTLFDDEEHNLSATIQAYTALLYSGYYQKNDRILRKAERYIIDSGGISRAHFLTRWMLSVNGLYEWPKLFYLPLSLLLVPTYVPLNFYELSTYARIHFVPMMVAGNKKFSLTSRHTPSLSHLDVREQKQESEETTQESRASIFLVDHLKQLASLPSYIHKLGYQAAERYMLERIEKDGTLYSYATSTFFMIYGLLALGYKKDSFVIQKAIDGICSLLSTCSGHVHVENSTSTVWDTALLSYALQEAGVPQQDPMIKGTTRYLKKRQHTKLGDWQFHNPNTAPGGWGFSDINTNNPDLDDTSAAIRALSRRAQTDTDYLESWQRGINWLLSMQNKDGGFAAFEKNTDSILFTYLPLENAKDAATDPATADLTGRVLECLGNFAGMNKSHPSIKAAVKWLFDHQLDNGSWYGRWGVCYIYGTWAAITGLRAVGVSASDPRIIKAINWLKSIQQEDGGFGESCYSASLKKYVPLSFSTPSQTAWALDALMTICPLKDQSVEKGIKFLLNPNLTEQQTHYPTGIGLPGQFYIQYHSYNDIFPLLALAHYAKKHSS

[0112] SEQ ID NO: 8 (amino acid sequence of wild-type Thermosynecochoccus elongates SHC) MPTSLATAIDPKQLQQAIRASQDFLFSQQYAEGYWWAELESNVTMTAEVILLHKIWGTEQRLPLAKAEQYLRNHQRDHGGWELFYGDGGDLSTSVEAYMGLRLLGVPETDPALVKARQFILARGGISTRIFTKLHLALIGCYDWRGIPSLPPWIMLLPE GSPFTIYEMSSWARSSTVPLLIVMDRKPVYGMDPPITLDELYSEGRANVVWELPRQGDWRDVFIGLDRVFKLFETLNIHPLREQGLKAAEEWVLERQEASGDWGGIIPAMLNSLLALRALDYAVDDPIVQRGMAAVDRFAIETETEYRVQPCVSPVWDTAL VMRAMVDSGVAPDHPALVKAGEWLLSKQILDYGDWHIKNKKGRPGGWAFEFENRFYPDVDDTAVVVMALHAVTLPNENLKRRAIERAVAWIASMQCRPGGWAAFDVDNDQDWLNGIPYGDLKAMIDPNTADVTARVLEMVGRCQLAFDRVALDRALAYLRNEQEPEGCWFGRWGVNYLYGTSGVLTALSLVAPRYDRWRIRRAAEWLMQCQNADGGWGETCWSYHDPSLKGKGDSTASQTAWAIIGLLAAGDATGDYATEAIERGIAYLLETQRPDGTWHEDYFTGTGFPCHFYLKYHYYQQHFPLTALGRYARWRNLLAT

[0113] SEQ ID NO: 9 (amino acid sequence of wild-type Zymomonas mobilis SHC1) MGIDRMNSLSRLLMKKIFGAEKTSYKPASDTIIGTDTLKRPNRRPEPTAKVDKTIFKTMGNSLNTLVSACDWLIGQQKPDGHWVGAVESNASMEAEWCLALWFLGLEDHPLRPRLGNALLEMQREDGSWGVYFGAGNGDINATVEAYAALRSLGYSADNPVLKKAAAWIAEKGGLKNIRV FTRYWLALIGEWPWEKTPNLPPEIIWFPDNFVFSIYNFAQWARATMVPIAILSARRPSRPLRPQDRLDELFPEGRARFDYELPKKEGIDLWSQFFRTTDRGLHWVQSNLLKRNSLREAAIRHVLEWIIRHQDADGGWGGIQPPWVYGLMALHGEGYQLYHPVMAKALSALDDPGWRHDRGE SSWIQATNSPVWDTMLALMALKDAKAEDRFTPEMDKAADWLLARQVKVKGDWSIKLPDVEPGWAFEYANDRYPDTDDTAVALIALSSYRDKEEWQKKGVEDAITRGVNWLIAMQSECGGWGAFDKDNNRSILSKIPFCDFGESIDPPSVDVTAHVLEAFGTLGLSRDMPVIQKAIDYVRSEQEAEGAWFGRWGVNYIYGTGAVLPALAAIGEDMTQPYITKACDWLVAHQQEDGGWGESCSSYMEIDSIGKGPTTPSQTAWALMGLIAANRPEDYEAIAKGCHYLIDRQEQDGSWKEEEFTGTGFPGYGVGQTIKLDDPALSKRLLQGAELSRAFMLRYDFYRQFFPIMALSRAERLIDLNN

[0114] SEQ ID NO: 10 (amino acid sequence of wild-type Zymomonas mobilis SHC2) MTVSTSSAFHHSPLSDDVEPIIQKATRALLEKQQQDGHWVFELEADATIPAEYILLKHYLGEPEDLEIEAKIGRYLRRIQGEHGGWSLFYGGDLDLSATVKAYFALKMIGDSPDAPHMLRARNEILARGGAMRANVFTRIQLALFGAMSWEHVPQMPVELMLMPEWFPVHINKMAYWARTVLVPLLVLQALKPVARNRRGILVDELFVPDVLPTLQESGDPIWRRFFSALDKVLHKVEPYWPKNMRAKAIHSCVHFVTERLNGEDGLGAIYPAIANSVMMYDALGYPENHPERAIARRAVEKLMVLDGTEDQGDKEVYCQPCLSPIWDTALVAHAMLEVGGDEAEKSAISALSWLKPQQILDVKGDWAWRRPDLRPGGWAFQYRNDYYPDVDDTAVVTMAMDRAAKLSDLHDDFEESKARAMEWTIGMQSDNGGWGAFDANNSYTYLNNIPFADHGALLDPPTVDVSARCVSMMAQAGISITDPKMKAAVDYLLKEQEEDGSWFGRWGVNYIYGTWSALCALNVAALPHDHLAVQKAVAWLKTIQNEDGGWGENCDSYALDYSGYEPMDSTASQTAWALLGLMAVGEANSEAVTKGINWLAQNQDEEGLWKEDYYSGGGFPRVFYLRYHGYSKYFPLWALARYRNLKKANQPIVHYGM

Claims

1. Formula (I) 【Chemistry 1】 6,10-dimethyl-2-(4-oxopentylidene)undeca-5,9-dien-1-yl acetate.

2. The compound of claim 1, wherein the double bond between C-8 and C-9 is in the E-configuration.

3. The compound according to claim 1 or claim 2, wherein the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration.

4. Acyclic acetate of formula (I) is converted to formula (II) by a process mediated by SHC (squalene-hopene cyclase). 【Chemistry 2】 How to make a compound.

5. Formula (II) 【Transformation 3】 5. The method of claim 4, wherein the compound of formula The method comprises: 【Chemistry 4】 contacting a compound of formula (III) with squalene-hopene cyclase (SHC) 【Transformation 5】 obtaining a compound of formula followed by hydrolysis.

6. 6. The method of claim 5, wherein the hydrolysis occurs under acidic conditions.

7. Hydrolysis may be performed using aqueous mineral acids, aqueous sulfonic acids, aqueous phosphoric and phosphonic acids, aqueous carboxylic acids, acidic alumina and SiO ２ 7. The process of claim 5 or claim 6, wherein the process occurs under acidic conditions obtained by adding an acid selected from:

8. Formula (II) 【Transformation 6】 5. The method of claim 4 for producing a compound of the formula: The method comprises: 【Transformation 7】 contacting the compound of formula (I) with a squalene-hopene cyclase (SHC) enzyme and an exogenous hydrolase.

9. 9. The method of claim 8, wherein the exogenous hydrolase is a lipase.

10. The method according to any one of claims 4 to 9, wherein the compound of formula (I) is a compound in which the double bond between C-8 and C-9 is in the E-configuration and the double bond between C-4 and C-5 is in the Z-configuration.

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