Enzymes and their applications

Abstract

To provide a process for preparing (-)-Ambrox or an isomer mixture of (-)-Ambrox.SOLUTION: In a process, (3E,7E)-homofarnesol or an isomer mixture of (3E,7E)-homofarnesol is converted enzymatically to give (-)-Ambrox or an isomer mixture of (-)-Ambrox, wherein the enzymatic conversion using an SHC / HAC enzyme having a specific structure is carried out under reaction conditions suitable for the production of (-)-Ambrox.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a squalene-hopencyclase / homofarnesol-ambrox cyclase (SHC / HAC) derivative enzyme modified with respect to a reference SHC / HAC protein, an amino acid sequence comprising the SHC / HAC derivative enzyme, a nucleotide sequence encoding the SHC / HAC derivative, a vector comprising the nucleotide sequence encoding the SHC / HAC derivative, and recombinant host cells comprising the nucleotide sequence encoding the SHC / HAC derivative. The present invention also relates to means for functionally expressing the nucleotide sequence encoding the SHC / HAC derivative, and to a method using recombinant microorganisms comprising the SHC / HAC derivative and the nucleotide sequence encoding WT SHC / HAC to produce ambrox, preferably (-)-ambrox. [Background technology]

[0002] Squalene-hoppe cyclase (SHC, EC 5.4.99.17) is a membrane-bound prokaryotic enzyme that acts as a biocatalyst for the cyclization of the linear triterpenoid squalene to hopen and hopanol. Early SHC research focused on the characteristics of SHC in the thermophilic, acidophilic bacterium Alicyclobacillus acidocaldarius (formerly Bacillus acidocaldarius) (see Neumann & Simon 1986, Biol Chem Hoppe-Seyler 367, 723-729, Seckler & Poralla 1986, Biochem Biophys Act 356-363, and Ochs et al 1990, J Bacteriol 174, 298-302). However, more recently, other SHCs from Zymomonas mobilis and Bradyrhizobium japonicum have been purified and characterized in terms of their natural (e.g., squalene) and non-natural substrates (e.g., homofarnesol and citral) (see, e.g., WO2010 / 139710, WO2012 / 066059, and Seitz et al 2012, J. Molecular Catalysis B: Enzymatic 84, 72-77).

[0003] Early studies by Neumann and Simon (1986 - ibid.) revealed that homofarnesol is an additional substrate of Alicyclobacillus acidocardarius SHC (AacSHC). However, the cyclization rate of non-natural homofarnesol by purified AacSHC, as taught by Neumann and Simon (1986), was reported to be only 3% of that of the natural substrate squalene. The formation rate of ambrox (product 2b) increased at homofarnesol (product 1b) concentrations from 0.25 mM to 2.0 mM and decreased slightly in the presence of 4 mM product 1b. The difference in cyclization rates can be partly attributed to the fact that the natural SHC substrate squalene is twice the size of non-natural homofarnesol (a C30-carbon compound), which is a 16-carbon compound.

[0004] (JP2009060799 - Kao) also discloses a method for producing ambrox from homofarnesol using SHC from A. acidocardarius. While JP2009060799 teaches the possibility of using microorganisms containing SHC for ambrox synthesis, it only discloses ambrox production from homofarnesol using an SHC extract prepared from recombinant microorganisms expressing the SHC gene, rather than by means of recombinant whole cells expressing the SHC gene. The conversion percentage from homofarnesol to ambrox using the SHC extract was reported to be 17.5% when carried out at 60°C and pH 5.2-6.0 for 14 hours, but only 6.8% when carried out at pH 6.6. The conversion rate from 3E,7E-homofarnesol to ambrox using SHC extract at 60°C and pH 5.6 for 64 hours was reported to be 63% when a substrate concentration of 0.2% homofarnesol (2 g / l) was used.

[0005] WO2010 / 139719A2 and its US equivalent (US2012 / 0135477A1) describe at least three SHC enzyme extracts possessing cyclase activity from homofarnesol to ambrox. Dimomonas movilis (Zmo) SHC and Bradyrhizobium japonica (Bjp) SHC enzymes have been reported to exhibit homofarnesol conversion rates of 41% and 22%, respectively, after a 16-hour reaction when a homofarnesol concentration of 10 mM (2.36 g / l) was used, while the conversion rate of AacSHC was reported to be only 1.2% (presumably at the same homofarnesol concentration), although experimental details are not provided. ZmoSHC and BjpSHC enzyme extracts were prepared from recombinant microorganisms expressing the SHC gene by lysing E. coli host cells producing the SHC enzyme and separating the soluble SHC fraction.

[0006] Seitz et al (2012 - ibid.) reported on the functional expression and biochemical characteristics of three SHC enzymes, two derived from Z. movilis (ZmoSHC1 and ZmoSHC2) and one from A. acidocardarius. They reported that when a 10 mM (2.36 g / l) homofarnesol concentration was used, an "efficient" conversion from homofarnesol to ambrox (22.95%) was observed using wild-type ZmoSHC1, no conversion from homofarnesol to ambrox was observed using WT ZmoSHC2, and a relatively low conversion from homofarnesol to ambrox (3.4%) was found for AacSHC. The observed trend regarding the relatively low conversion from homofarnesol to ambrox for AacSHC is consistent with the results of Neumann and Simon (1986 - ibid.), which are also disclosed in WO2010 / 139719A2, as discussed above. The three SHC enzymes were used in cell suspension format (by partial disruption of host E. coli cells using a freeze-thaw cycle) and as partially purified membrane-bound fractions.

[0007] WO2012 / 066059 discloses mutants with cyclase activity, as well as its use for methods of biocatalytic cyclization of terpenes, particularly for producing isopulegol by cyclization of citronellal, for producing menthol, and for the biocatalytic conversion of other compounds having terpene-type structural motifs. Sequence alignment of various SHCs identified phenylalanine 486 (F486) as a strongly conserved amino acid residue, and a series of substitutional variants of the dimomonas movilis SHC enzyme were constructed. Some of these substitutions led to loss of activity, while others resulted in the formation of novel terpenoid products (isoplegol) from terpene substrates such as citronellal.

[0008] A 2012 PhD dissertation by Seitz (http: / elib.uni-stuttgart.de / handle / 11682 / 1400) reported that the F486Y mutation in ZmoSHC1 provided a reduced biotransformation rate of homofarnesol, approximately 1.5 times lower, from 34.8% (WT ZmoSHC1) to 23.9% (mutant ZmoSHC1 F486Y). When the mutant equivalent (Y420C) in AacSHC was tested, it was hypothesized that the enzyme activity for larger substrates would decrease, while the activity for smaller substrates would increase. When the mutant was tested under the same conditions as the wild type and its enzyme activity was compared, it was observed that the mutant showed no transformation of homofarnesol substrates at all. Therefore, it was concluded that the Y420 amino acid residue is critical for the activity of AacSHC for all substrates.

[0009] Other site-directed mutagenesis studies of SHCs in this field (e.g., Hoshino and Sato 2002, Chem Commun 291-301) have focused on the effects of mutations in highly conserved regions (e.g., F601) and their effects on natural substrates (i.e., squalene or squalene analogs) rather than non-natural substrates such as homofarnesol.

[0010] In summary, the limited disclosure in this art regarding the biotransformation process for the successful conversion of homofarnesol to ambrox relates only to relatively low concentrations / volume of homofarnesol substrate (in the range of 0.25 mM to 2 mM to 10 mM, or roughly 0.06 g / l to 2.36 g / l) using wild-type SHC polypeptides with homofarnesol-ambrox cyclase (HAC) activity. SHC enzymes possessing HAC activity were either (i) extracts prepared by either disrupting E. coli host cells containing the SHC enzyme and separating the insoluble and soluble SHC liquid fractions, (ii) partially purified membrane fractions, or (iii) recombinant whole cells expressing the WT SHC gene and producing the SHC enzyme for use in a reaction to bioconvert homofarnesol to ambrox using a solubilizer containing either (i) Triton X-100 (Neumann and Simon 1986 ibid., Seitz et al 2012 ibid., see JP2009060799) or (ii) taurodeoxycholic acid (disclosed in US2012 / 0135477A1) in the reaction mixture.

[0011] Using these WT SHC extracts and / or whole cells of recombinant microorganisms expressing the SHC gene, the resulting homofarnesol to ambrox conversion rates were found to vary depending on the source of the SHC enzyme used, the amount of homofarnesol starting material, and the reaction conditions. To date, 100% conversion rates from homofarnesol to ambrox using wild-type SHC enzymes have not been achieved at the reported inclusion concentrations (0.06–2.36 g / l). In addition, preliminary studies using SHC derivatives prepared using site-directed mutagenesis studies have yielded only negative results (i.e., reduced homofarnesol conversion rates) rather than positive results (i.e., improved conversion rates). Furthermore, purified SHC enzyme extracts or SHC membrane-bound fractions have only been used in published studies, or whole cells of recombinant microorganisms expressing the WT SHC gene have only been used under specific reaction conditions with solubilizers such as Triton X-100 or taurodeoxycholic acid. There is no evidence that recombinant microorganisms containing either WT or mutant SHC can provide a more efficient and cost-effective bioconversion from homofarnesol to ambrox using optimized reaction conditions. Therefore, it is desirable to improve the cited known processes for preparing ambrox from homofarnesol by improving reaction rate, specificity, yield, and productivity, and by reducing costs (for example, by simplifying the process either by using whole cells of the recombinant microorganism or by using a “one-pot” process that combines both the biocatalytic production and bioconversion steps). [Overview of the project]

[0012] In various aspects, the present invention provides an application of recombinant host cells containing either an SHC / HAC derivative enzyme or a WT SHC / HAC enzyme when used in a method for preparing an ambrox material containing an ambrox isomer referred to as (-)-ambrox and an ambrox-like molecule (as a byproduct) under specific reaction conditions. Unlike the disclosures in the art relating to AacSHC, the applicant is the first to demonstrate that a whole recombinant microorganism expressing an SHC derivative gene can be used to bioconvert homofarnesol to ambrox. In addition, whole recombinant microorganisms expressing the WT SHC gene and / or producing the SHC enzyme may be used to bioconvert homofarnesol to ambrox under specific reaction conditions not disclosed in this art.

[0013] Surprisingly, it was also found that introducing up to five amino acid mutations into the amino acid sequence of the WT SHC / HAC reference sequence as disclosed herein yields SHC / HAC derivative enzymes with significantly improved homofarnesol to ambrox conversion rates compared to the unmodified SHC reference enzyme as disclosed herein. These novel SHC / HAC derivative enzymes are useful, both individually and in combination, for the production of ambrox material from homofarnesol substrates, particularly (-)-ambrox.

[0014] A further surprising discovery is that, apart from one variant (F601Y), the SHC derivative enzymes disclosed herein typically contain non-conservative substitutions at amino acid residue positions in non-conserved portions of the reference SHC polypeptide sequence. This is an unexpected discovery because changes in conserved regions of an enzyme are more likely to impair the function of the enzyme (at least in relation to its native substrate) than changes in non-conserved regions of the protein.

[0015] A further surprising discovery is that the characterized SHC derivative enzymes of the present disclosure work optimally (for non-native substrates such as homofarnesol) at about 35 °C rather than at about 60 °C, which is the normal reaction temperature of thermophilic microorganisms such as AacSHC. The application of the SHC derivatives of the present disclosure to methods for preparing ambrox from homofarnesol at a lower reaction temperature has significant cost advantages for industrial-scale ambrox production cycles.

[0016] Another advantage of the present invention is that the SHC derivative enzymes of this disclosure catalyze an efficient bioconversion process, which can achieve 100% conversion of homofarnesol substrate when optimized for relatively high (e.g., about 50 times) homofarnesol substrate concentrations compared to concentrations previously described in the prior art (e.g., 125 g / l EEH), whereas the reference WT SHC protein converts only about 10% of the same substrate, even at high enzyme / cell concentrations. All cited prior art disclosures relate to the use of purified membrane extracts containing SHC or purified SHC extracts (prepared from microorganisms expressing the SHC gene), or the use of recombinant microorganisms expressing the WT SHC gene under specific bioconversion reaction conditions (e.g., using specific solubilizers). Even then, 100% homofarnesol conversion at much lower EEH concentrations has not been reported. Nor has a "one-pot" reaction been reported in which recombinant cells proliferate in a first step, produce the SHC enzyme, and subsequently convert EEH to (-)-ambrox in the same vessel. A further advantage of the present invention is that recombinant host cells producing SHC derivative enzymes exhibit a high initial reaction rate, which allows for the production of high quantities of the product in a relatively short period of time, while using only relatively low amounts of biocatalyst. In short, the selection and efficient expression of recombinant microorganisms containing either WT SHC / HAC or specific SHC / HAC derivative enzymes, as well as their application under specific bioconversion reaction conditions, leads to a more efficient bioconversion process. The final product ((-)-Amblox) can be separated and easily purified. Unlike the cited techniques, the SHC / HAC derivative enzyme is not used as a pure enzyme but in a whole-cellular context (as a biocatalyst), which is a more cost-effective and user- and environmentally friendly approach as it does not require additional enzyme purification and isolation steps.

[0017] In summary, the present disclosure provides a bioconversion / in vivo conversion method for making ambrox in recombinant microbial strains, which is (i) economically attractive, (ii) environmentally friendly, and (iii) results in the selective production of (-)-ambrox as the main compound, which is effectively separated from other by-products that do not contribute to the olfactory quality of the final product under selective crystallization conditions.

[0018] Detailed description of the invention As used herein, the term "SHC" means the squalene-hopene cyclase enzyme from any of the sources listed in Tables 10-12. In a preferred embodiment, the term SHC includes the Dimomonas mobilis SHC enzyme and the Alicyclobacillus acidocaldarius SHC enzyme disclosed in BASF's WO2010 / 139719, US2012 / 01345477A1, Seitz et al (2012 ibid), and Seitz (2012 PhD thesis, ibid). For convenience of reference, the designation "AacSHC" is used for Alicyclobacillus acidocaldarius SHC, the designation "ZmoSHC" is used for Dimomonas mobilis SHC, and the designation "BjpSHC" is used for Bradyrhizobium japonicum SHC. The percent sequence identity of WT AacSHC and their sequences relative to each other (which can vary depending on the algorithm used) are set forth in Tables 18 and 19.

[0019] Alignment of WT SHC sequences prepared by Hoshino and Sato (2002 ibid.) shows that multiple motifs were detected in all four sequences, consisting of the core sequence Gln-XXX-Gly-X-Trp, which was found six times in both the Z·Mobilis and A·Acidocardarius SHC sequences (see Figure 3 in Reipen et al 1995, Microbiology 141, 155-161). Hoshino and Sato (2002 ibid.) reported that aromatic amino acids were more abundant than usual in SHCs and that they focused on two characteristic motifs in SHCs. 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 on the X-ray structural analysis of A·acidocaldarius SHC. The DXDDTA motif appears to correlate with the SHC active site. Exemplary sequence alignments from prior art show multiple cyclic motifs provided herein in Figure 2 (from Hoshino and Sato (2002 ibid.)) and Figure 3 (from Seitz PhD dissertation (2012)).

[0020] The reference (or wild-type) AacSHC protein as used herein refers to the AacSHC protein disclosed in Sequence ID No. 1. The reference AacSHC enzyme of this disclosure has homofarnesol-ambrox cyclase (HAC) activity useful for the biocatalytic reaction of SHC with a homofarnesol substrate to produce ambrox derivatives. The main reaction of reference AacSHC is the cyclization of a linear or non-linear substrate, such as homofarnesol, to produce ambrox.

[0021] Ambrox As used herein, the term "ambilox" includes (-)-ambilox of formula (I), as well as (-)-ambilox in a stereoisomerically pure form or as a mixture with at least one of the following molecules of formula (II), (IV), and / or (III). [ka]

[0022] (-)- Amblox (-)-Amblox is commercially known as Amblox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlyn (Quest), Cetalox Laevo (Firmenich), Ambermor (Aromor), and / or Norambrenolide Ether (Pacific).

[0023] (-)-Amblox is an industrially important aromatic compound and has long been used in the fragrance industry. The particular desirable sensory benefits of (-)-Amblox come from the (-) stereoisomer rather than the (+) stereoisomer. The scent of the (-) stereoisomer is described as musk-like, woody, warm, or amber-like, while the (+)-Amblox enantiomer has a relatively weak scent note. The scents and scent thresholds of Amblox-like products also differ. Although various concentrated (-)-Amblox materials are commercially available, it is desirable to produce highly concentrated (-)-Amblox material, ideally pure (-)-Amblox.

[0024] (-)-Amblox production (-)-Amblox can be produced from sclareol according to the production process described below. Sclareol is a product extracted from the natural plant clary sage. However, since natural starting materials are used in this process, it involves a multi-step reaction, the process is roundabout, the supply and stability of the starting materials may not always be sufficient, and there are potential problems that the reaction may not be environmentally friendly because oxidizing agents such as chromic acid or permanganic acid are used in the oxidative decomposition step of (+)-sclareol. [ka]

[0025] (-)-Amblox can also be synthesized from homofarnesol using different routes. For example, homofarnesol can be obtained by brominating, cyanating, and hydrolyzing nerolidol to homofarnesylic acid, followed by reduction. Alternatively, homofarnesol may be obtained from farnesol, farnesyl loride, beta-farnesene, or other substrates. Beta-farnesene can be converted directly to E,E-homofarnesol (EEH), or indirectly to EEH via E,E-homofarnesylic acid esters that are subsequently converted to EEH. An overview of (-)-ambax production from different substrates can be found in US2012 / 0135477A1, WO2010 / 139719, US2013.0273619A1, WO2013 / 156398A1, as well as Seitz's PhD dissertation (2012 ibid.) and Schaefer 2011 (Chemie Unserer Zeit 45, 374-388).

[0026] Homofarnesol can exist as a mixture of four isomers: (3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E), whereas literature suggests that (-)-ambrox can only be obtained from (3E,7E) homofarnesol (see Neumann and Simon (1986), ibid.). In this specification, references to (3E,7E) homofarnesol refer to E,E-homofarnesol, which is also known as EEH.

[0027] US2012 / 0135477A1 reports the conversion of (3Z,7E) to (-)-ambrox using ZmoSHC (SEQ ID NO: 2) (see Examples 2-4), but according to the disclosure of Schaefer (2011) (ibid.), (7E,3Z) is converted only to 9b-epi-ambrox (i.e., compound III) and not to (-)-ambrox, as outlined above. In this specification, references to (3Z,7E) homofarnesol refer to E,Z-homofarnesol, which is also called EZH.

[0028] In some embodiments, preferably, the homofarnesol starting material comprises a mixture of (3E,7E) and (3Z,7E), referred herein to as the EE:EZ stereoisomer mixture (in particular, see examples and Table 20).

[0029] The EE:EZ stereoisomer mixture of homofarnesol has the CAS number 35826-67-6. [ka]

[0030] As the examples show (see, for example, Examples 5, 7, 9, 10, 11, 18, 19, and 20), in certain embodiments, the homofarnesol starting material is a mixture of isomers.

[0031] Accordingly, in some embodiments, the homofarnesol starting material may also include a mixture of four isomers, EE:EZ:ZZ:ZE, corresponding to (3E,7E) and (3Z,7E), (3Z,7Z), and 3E,7Z.

[0032] In some embodiments, preferably, the homofarnesol starting material is selected from one or more of the following groups: [(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)].

[0033] Preferably, the homofarnesol starting material is selected from one or more of the following groups: [(3E,7E), (3Z,7E)] and / or [(3Z,7E), (3E / 7E), and (3E,7Z)].

[0034] Therefore, in certain embodiments, 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.

[0035] In some embodiments, the homofarnesol starting material preferably contains >90% E,E-homofarnesol (EEH).

[0036] In another embodiment, the homofarnesol starting material contains an EE:EZ weight ratio of 86:14.

[0037] In certain embodiments, homofarnesol starting materials include an EE:EZ weight-to-weight ratio of 80:20.

[0038] In certain embodiments, homofarnesol starting materials include an EE:EZ weight ratio of 70:30.

[0039] In a further embodiment, the homofarnesol starting material contains an EE:EZ weight ratio of 69:31.

[0040] In some embodiments, the homofarnesol starting material consists of or essentially consists of a mixture of four isomers EE:EZ:ZZ:ZE corresponding to (3E,7E) and (3Z,7E), (3Z,7Z) and (3E,7Z).

[0041] In some embodiments, preferably, the homofarnesol starting material consists of or essentially consists of a mixture of isomers selected from one or more of the following groups: [(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)].

[0042] Preferably, the homofarnesol starting material consists of or essentially consists of a mixture of isomers selected from one or more of the following groups: q[(3E,7E), (3Z,7E)] and / or [(3Z,7E), (3E,7E), and (3E,7Z)].

[0043] Therefore, in certain embodiments, the EEH:EZH isomer ratios are 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, 75:2 5, 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 an EEH:EZH ratio of approximately 50:50, or essentially consisting of such a ratio.

[0044] In some embodiments, the homofarnesol starting material preferably consists of or is essentially E,E-homofarnesol (EEH) >90%.

[0045] In other embodiments, the homofarnesol starting material consists of or essentially consists of an EE:EZ weight ratio of 86:14.

[0046] In certain embodiments, the homofarnesol starting material consists of or essentially consists of an EE:EZ weight ratio of 80:20.

[0047] In certain embodiments, the homofarnesol starting material consists of or essentially consists of a 70:30 EE:EZ weight ratio.

[0048] In a further embodiment, the homofarnesol starting material consists of or essentially consists of an EE:EZ weight ratio of 69:31.

[0049] In aspects of this disclosure, Ambrox is produced using an SHC / HAC derivative enzyme.

[0050] SHC / HAC derivative As used herein, the term "SHC / HAC derivative" means that the amino acid sequence of an SHC / HAC derivative is modified, altered, or variant amino acid sequence that is changed compared to the amino acid sequence of a reference (or wild-type) SHC sequence that conforms to at least SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Generally, an SHC / HAC derivative includes an altered form of SHC having at least one modification that alters (e.g., increases) the enzyme activity of its substrate (e.g., EEH).

[0051] The SHC / HAC derivatives of this disclosure are tested for their homofarnesol-ambrox cyclase activity. As a result, these SHC / HAC derivatives that convert homofarnesol to ambrox are referred to herein as HAC derivatives and SHC derivatives. While exemplary SHC / HAC derivatives are provided for enzymes derived from the microbial strain sources Alicyclobacillus acidocardarius, Dimomonas movilis, and Bradyrhizobium japonicum, this disclosure also covers equivalent SHC / HAC derivatives from other microbial strain sources, including, but not limited to, SHC / HAC enzymes derived from Methylococcus capsulatus, Frankia arni, Acetobacter pasteurianum, and Tetrahymena pyriformis (see, e.g., WO2010 / 139719, US2012 / 01345477, WO2012 / 066059, and Tables 10–12).

[0052] As used herein, the term “amino acid modification” means the insertion of one or more amino acids between two amino acids, the deletion of one or more amino acids, or the substitution of one or more amino acids with one or more different amino acids (which may be conserved or non-conservative) of the amino acid sequence of a reference amino acid sequence (e.g., the wild-type (WT) amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4). Amino acid modifications can be readily identified by comparing the amino acid sequence of the SHC / HAC derivative amino acid sequence with the amino acid sequence of the reference amino acid sequence (e.g., the wild-type (WT) amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4). Exemplary WT SHC amino acid sequence alignments are provided in Figures 1-4 and Tables 18 and 19.

[0053] Conservative amino acid substitutions can be made, for example, based on the similarity of the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the amino acid residues to be included. The 20 naturally occurring amino acids outlined above can be grouped into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile, (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basicity: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe.

[0054] Accordingly, the term “conservative substitution” as used herein means the exchange of one amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the exchange of Asp with Glu retains one negative charge in the thus modified polypeptide. In addition, glycine and proline can be substituted for each other based on their ability to disrupt the alpha-helix. Some preferred conservative substitutions among the six groups above are those in the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (ii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. In light of known genetic codes and recombinant and synthetic DNA techniques, skilled scientists can readily construct DNA encoding conservative amino acid variants.

[0055] As used herein, “non-conservative substitution” or “non-conservative amino acid exchange” is defined as the exchange of one amino acid with another amino acid listed in the six standard amino acid groups (1) through (6) shown above.

[0056] Typically, the SHC / HAC derivatives of this disclosure are prepared using non-conservative substitutions that alter the biological function (e.g., HAC activity) of the disclosed SHC / HAC derivative.

[0057] For convenience of reference, the single-letter amino acid symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission are shown below. Three-letter codes are also provided for reference purposes. [Table 1]

[0058] Amino acid modifications, such as amino acid substitutions, can be introduced using known protocols of recombinant gene technology, including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in vitro transcription. These can be used to introduce such changes into WT SHC sequences and yield SHC / HAC derivative enzymes. The derivatives can then be screened for the functional activity of SHC / HAC.

[0059] SHC / HAC derivative enzyme The present invention provides an SHC / HAC derivative and describes an enzyme having homofarnesol-ambrox cyclase (HAC) activity comprising an amino acid sequence having about 1 to about 50 mutations independently selected from substitutions, deletions, or insertions to the amino acid sequence of a reference (or wild-type) SHC sequence according to at least SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0060] In various embodiments, mutations or combinations of mutations enhance the activity of an SHC / HAC derivative for converting homofarnesol to ambrox compared to a reference SHC enzyme that does not exhibit this deletion / addition. Protein modeling described herein may be used to guide such substitutions, deletions, or insertions in the SHC reference sequence. For example, a structural model of the SHC amino acid sequence may be constructed using AacSHC coordinates (shown, e.g., in Figures 19 and 20). As shown herein, such homology models are useful for directing improvements to the SHC enzyme for converting homofarnesol to (-)-ambrox.

[0061] Therefore, in various embodiments, SHC / HAC derivatives may have approximately 1 to approximately 45 mutations, approximately 1 to approximately 40 mutations, approximately 1 to approximately 35 mutations, approximately 1 to approximately 30 mutations, approximately 1 to approximately 25 mutations, approximately 1 to approximately 20 mutations, approximately 1 to approximately 15 mutations, approximately 1 to approximately 10 mutations, or approximately 1 to approximately 5 mutations relative to the amino acid sequence of a reference (or wild-type) SHC sequence according to at least SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0062] In various embodiments, the SHC / HAC derivative comprises a sequence having at least 5 or at least 10 mutations, but not more than about 20 or 30 mutations, relative to the amino acid sequence of a reference (or wild-type) SHC sequence that conforms to at least SEQ ID NO: 1, 2, 3, or 4. In various embodiments, the SHC derivative comprises a sequence having about 1 mutation, about 2 mutations, about 3 mutations, about 4 mutations, about 5 mutations, about 6 mutations, about 7 mutations, about 8 mutations, about 9 mutations, about 10 mutations, about 11 mutations, about 12 mutations, about 13 mutations, about 14 mutations, about 15 mutations, about 16 mutations, about 17 mutations, about 18 mutations, about 19 mutations, about 20 mutations, about 21 mutations, about 22 mutations, or about 23 mutations relative to a reference SHC (e.g., SEQ ID NO: 1, 2, 3, or 4). It may have approximately 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mutations.

[0063] In these and other embodiments, the SHC / HAC derivative exhibits at least approximately 50% sequence identity, at least approximately 55% sequence identity, at least approximately 60% sequence identity, at least approximately 65% ​​sequence identity, at least approximately 70% sequence identity, at least approximately 75% sequence identity, and at least approximately 80% sequence identity between a WT SHC (e.g., SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4) and a reference sequence (e.g., AacSHC (SEQ ID NO: 1) and other SHC sequences (e.g., see Tables 18 and 19, where at least 34-52% identity is shown between ZmoSHC of WO2010 / 139719) and a reference sequence (e.g., AacSHC (SEQ ID NO: 1) and other SHC sequences (e.g., ZmoSHC of WO2010 / 139719)). It may contain amino acid sequences having column identity, at least about 85% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity.

[0064] In various embodiments, the SHC variant has higher activity for converting homofarnesol to ambrox than the wild-type enzyme, for example, higher production of (-)-ambrox upon contact with a homofarnesol substrate than the reference wild-type enzyme (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4).

[0065] For example, SHC / HAC derivatives exhibit at least approximately 50% identity, approximately 51% identity, approximately 52% identity, approximately 53% identity, approximately 54% identity, approximately 55% identity, approximately 56% identity, approximately 57% identity, approximately 58% identity, approximately 59% identity, approximately 60% identity, approximately 61% identity, approximately 62% identity, approximately 63% identity, approximately 64% identity, approximately 65% ​​identity, approximately 66% identity, approximately 67% identity, and approximately 68% identity between a reference SHC (e.g., SEQ ID NO: 1 or 2 or 3 or 4) or a reference sequence (e.g., AacSHC (SEQ ID NO: 1) and other SHC sequences (e.g., ZmoSHC of WO2010 / 139719)) and a reference sequence (see Tables 18 and 19, where at least 34-52% identity is shown between AacSHC (SEQ ID NO: 1) and other SHC sequences (e.g., ZmoSHC of WO2010 / 139719)). % identity, approximately 69% identity, approximately 70% identity, approximately 71% identity, approximately 72% identity, approximately 73% identity, approximately 74% identity, approximately 75% identity, approximately 76% identity, approximately 77% identity, approximately 78% identity, approximately 79% identity, approximately 80% identity, approximately 81% identity, approximately 82% identity, approximately 83% identity, approximately 84% identity, approximately 85% identity, approximately It may contain amino acid sequences having 86% identity, approximately 87% identity, approximately 88% identity, approximately 89% identity, approximately 90% identity, approximately 91% sequence identity, approximately 92% sequence identity, approximately 93% sequence identity, approximately 94% sequence identity, approximately 95% sequence identity, approximately 96% sequence identity, approximately 97% sequence identity, approximately 98% sequence identity, or approximately 99% sequence identity.

[0066] Various SHC / HAC derivatives tested for SHC enzyme activity are listed in one or more of Tables 1-9. Thus, in various embodiments, SHC / HAC derivatives may have at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 mutations selected from one or more of Tables 1-9. In some embodiments, the SHC / HAC derivative is a modified SHC polypeptide having up to 4 mutations compared to the wild-type / reference amino acid sequence according to SEQ ID NO: 1, and comprising an amino acid sequence containing at least one substitution F601Y or M132R in combination with at least one of F129L and / or I432T relative to SEQ ID NO: 1, and optionally including a leader sequence that assists expression and activity in E. coli.

[0067] In another embodiment, the SHC / HAC derivative is a modified SHC polypeptide having up to eight mutations compared to the wild-type / reference amino acid sequence (or its modified counterpart for expression by E. coli) according to SEQ ID NO: 1, and comprising an amino acid sequence with one or more single-amino acid changes at positions selected from the group consisting of positions 77, 92, 129, 132, 224, 432, 579, 601, and 605 relative to SEQ ID NO: 1, wherein the SHC / HAC derivative has modified (e.g., increased) enzyme activity relative to SEQ ID NO: 1.

[0068] In one embodiment, the SHC derivative comprises one or more substitutions selected from the group of variants consisting of T77X, I92X, F129X, M132X, A224X, I432X, Q579X, F601Y, and F605W relative to SEQ ID NO: 1. T77X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. I92X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F129X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. M132X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. A224X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. I432X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. Q579X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F601X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F605X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y.

[0069] In one embodiment, the SHC derivative comprises one or more substitutions selected from the group of variants consisting of T77A, I92V, F129L, M132R, A224V, I432T, Q579H, F601Y, and F605W relative to SEQ ID NO: 1.

[0070] In another embodiment, the SHC derivative comprises one or more substitutions selected from the group of variants consisting of S129X, V145X, F182X, Y185X, G282X, I498X, H646X, and F698X relative to SEQ ID NO: 2. S129X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. V145X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F182X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. Y185X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. G282X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. I498X has X selected from A, B, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. H646X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F668X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F698X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y.

[0071] In one embodiment, the SHC derivative includes one or more substitutions selected from the group of variants consisting of S129A, V145V, F182L, Y185R, G282V, I498T, H646H, F668Y, and F698X relative to SEQ ID NO: 2, as shown in Table 2.

[0072] In a further embodiment, the SHC derivative comprises one or more substitutions selected from the group of variants consisting of G85X, V100X, F137X, I140X, V233X, I450X, N598X, F620X, and F624X relative to SEQ ID NO: 3. G85X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. V100X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F137X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. I140X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. V233X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. I450X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. N598X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F620X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F624X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y.

[0073] In one embodiment, the SHC derivative includes one or more substitutions selected from the group of variants consisting of G85A, V100V, F137L, I140R, V233V, I450T, N598H, F620Y, and F624W relative to SEQ ID NO: 3, as shown in Tables 3 and 3a.

[0074] In a further embodiment, the SHC derivative comprises one or more substitutions selected from the group of variants consisting of A88X, V104X, F141X, Y144X, V241X, I459X, M607X, F628X, and F658X relative to SEQ ID NO: 4. A88X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. V104X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F141X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. Y144X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. V241X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. I459X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. M607X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F628X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. F658X has X selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y.

[0075] In a further embodiment, the SHC derivative includes one or more substitutions selected from the group consisting of A88A, V104V, F141L, Y144R, V241V, I459T, M607H, F628Y, and F658W for SEQ ID NO: 4, as shown in Table 4.

[0076] Combination of SHC derivatives In one embodiment, the SHC derivative includes one or more substitutions selected from the group of variants consisting of T77A, F129L, M132R, I92V, A224V, I432T, Q579H, and F601Y relative to SEQ ID NO: 1, as shown in Table 5.

[0077] In one embodiment, the SHC derivative includes one or more substitutions selected from the group of variants consisting of S129A, V145V, F182L, Y185R, G282V, I498T, H646H, and F668Y relative to SEQ ID NO: 2, as shown in Table 6.

[0078] In one embodiment, the SHC derivative includes one or more substitutions selected from the group of variants consisting of G85A, V100V, F137L, I140R, V233V, I450T, N598H, and F620Y for SEQ ID NO: 3, as shown in Table 7.

[0079] In a further embodiment, the SHC derivative includes one or more substitutions selected from the group consisting of A88A, V104V, F141L, Y144R, V241V, I459T, M607H, and F628Y for SEQ ID NO: 4, as shown in Table 8. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

[0080] In a preferred embodiment, the SHC derivative comprises at least one substituted F601Y or M132R in combination with at least one of F129L and / or I432T for SEQ ID NO: 1.

[0081] The SHC derivative provided in this disclosure, called SHC3, contains the following substitution F601Y compared to the reference SHC protein (SEQ ID NO: 1).

[0082] Hoshino and Sato (2002 ibid.) identified F601 as a highly conserved amino acid residue among prokaryotes and eukaryotes. It has been reported that the SHC derivative F601Y showed a greatly increased Vmax for oxide squalene substrates (not squalene). However, when squalene is used, F601Y shows a decrease in affinity to WT AacSHC (i.e., a higher K M ) and catalytic efficiency / activity (Kcat / K M This shows a decrease in ). Data on the efficacy of AacSHC when homofarnesol is used as the enzyme substrate in the F601Y mutant are not provided in Hoshino and Sato (2002 ibid.).

[0083] The SHC derivative provided in this disclosure, called SHC10, contains the following substitution F129L compared to the reference SHC protein (SEQ ID NO: 1).

[0084] The SHC derivative provided in this disclosure, referred to as SHC30, includes the following substitutions F601Y and F129L compared to the reference SHC protein (SEQ ID NO: 1).

[0085] The SHC derivative provided in this disclosure, referred to as SHC26, includes the following substitutions M132R and I432T compared to the reference SHC protein (SEQ ID NO: 1).

[0086] The SHC derivative referred to as 215G2 provided in this disclosure includes the following substitutions M132R, I432T, and A224V compared to the reference SHC protein (SEQ ID NO: 1).

[0087] The SHC derivatives provided in this disclosure, referred to as SHC32, include the following substitutions F601Y, M132R, and I432T compared to the reference SHC protein (SEQ ID NO: 1).

[0088] The SHC derivatives provided in this disclosure, referred to as SHC31, include the following substitutions F129L, M132R, and I432T compared to the reference SHC protein (SEQ ID NO: 1).

[0089] The SHC derivatives provided in this disclosure, referred to as SHC33, include the following substitutions F601Y, F129L, M132R, and I432T compared to the reference SHC protein (SEQ ID NO: 1).

[0090] The SHC derivative referred to as 101A10 provided in this disclosure includes the following substitutions F601Y and Q579H compared to the reference SHC protein (SEQ ID NO: 1).

[0091] The SHC derivative provided in this disclosure, referred to as 111C8, includes the following substitutions T77A+I92V and F129L compared to the reference SHC protein (SEQ ID NO: 1).

[0092] In a preferred embodiment, the SHC derivative includes at least one substituted F668Y or Y185R in combination with at least one of F182L and / or I498T for SEQ ID NO: 2.

[0093] The SHC derivative provided in this disclosure, called SHC3ZM1, contains the following substitution F668Y compared to the reference SHC protein (SEQ ID NO: 2).

[0094] Hoshino and Sato (2002 ibid.) identified F601 as a highly conserved amino acid residue among prokaryotes and eukaryotes. It has been reported that the SHC derivative F601Y showed a greatly increased Vmax for oxide squalene substrates (not squalene). However, when squalene is used, F601Y shows a decrease in affinity to WT AacSHC (i.e., a higher K M ) and catalytic efficiency / activity (Kcat / K M This shows a decrease in ( ). Data on the efficacy of AacSHC when homofarnesol is used as an enzyme substrate in the F601Y mutant are not provided by Hoshino and Sato. In ZmoSHC1, F668Y is an SHC derivative equivalent to F601Y.

[0095] The SHC derivative provided in this disclosure, called SHC10ZM1, contains the following substitution F182L compared to the reference SHC protein (SEQ ID NO: 2).

[0096] The SHC derivative provided in this disclosure, called SHC30ZM1, includes the following substitutions F668Y and F182L compared to the reference SHC protein (SEQ ID NO: 2).

[0097] The SHC derivative provided in this disclosure, called SHC26ZM1, contains the following substitutions Y185R and I498T compared to the reference SHC protein (SEQ ID NO: 2).

[0098] The SHC derivative provided in this disclosure, referred to as 215G2ZM1, includes the following substitutions Y185R, I498T, and G282V compared to the reference SHC protein (SEQ ID NO: 2).

[0099] The SHC derivative provided in this disclosure, called SHC32ZM1, includes the following substitutions F668Y, Y185R, and I498T compared to the reference SHC protein (SEQ ID NO: 2).

[0100] The SHC derivative provided in this disclosure, called SHC31ZM1, includes the following substitutions F182L, Y185R, and I498T compared to the reference SHC protein (SEQ ID NO: 2).

[0101] The SHC derivative provided in this disclosure, called SHC33ZM1, includes the following substitutions F668Y, F182L, Y185R, and I498T compared to the reference SHC protein (SEQ ID NO: 2).

[0102] The SHC derivative provided in this disclosure, referred to as 101A10ZM1, contains the following substitutions F668Y and H646H compared to the reference SHC protein (SEQ ID NO: 2).

[0103] The SHC derivative provided in this disclosure, referred to as 111C8ZM1, includes the following substitutions S129A+V145V and F182L compared to the reference SHC protein (SEQ ID NO: 2).

[0104] In a preferred embodiment, the SHC derivative comprises at least substituted F620Y or I140R in combination with at least one of F137L and / or I450T for SEQ ID NO: 3.

[0105] The SHC derivative provided in this disclosure, called SHC3ZM2, contains the following substitution F620Y compared to the reference SHC protein (SEQ ID NO: 3).

[0106] Hoshino and Sato (2002 ibid.) identified F601 as a highly conserved amino acid residue among prokaryotic and eukaryotic SHC species. It has been reported that the AacSHC derivative F601Y showed a greatly increased Vmax for oxide squalene substrates (rather than squalene). However, when squalene is used, F601Y shows a decrease in affinity to WT AacSHC (i.e., a higher K). M ) and catalytic efficiency / activity (Kcat / K MThis shows a decrease in ). Data on the efficacy of AacSHC when homofarnesol is used as an enzyme substrate in the F601Y mutant are not provided in Hoshino and Sato (2002). F620Y is an SHC derivative equivalent to F601Y in ZmoSHC2.

[0107] The SHC derivative provided in this disclosure, called SHC10ZM2, contains the following substitution F137L compared to the reference SHC protein (SEQ ID NO: 3).

[0108] The SHC derivative provided in this disclosure, called SHC30ZM2, contains the following substitutions F620Y and F137L compared to the reference SHC protein (SEQ ID NO: 3).

[0109] The SHC derivative provided in this disclosure, called SHC26ZM2, contains the following substitutions I140R and I450T compared to the reference SHC protein (SEQ ID NO: 3).

[0110] The SHC derivative provided in this disclosure, referred to as 215G2ZM2, includes the following substitutions I140R, I450T, and V233V compared to the reference SHC protein (SEQ ID NO: 3).

[0111] The SHC derivative provided in this disclosure, called SHC32ZM2, includes the following substitutions F620Y, I140R, and I450T compared to the reference SHC protein (SEQ ID NO: 3).

[0112] The SHC derivative provided in this disclosure, called SHC31ZM2, includes the following substitutions F137L, I140R, and I450T compared to the reference SHC protein (SEQ ID NO: 3).

[0113] The SHC derivatives provided in this disclosure, referred to as SHC33ZM2, include the following substitutions F620Y, F137L, I140R, and I450T compared to the reference SHC protein (SEQ ID NO: 3).

[0114] The SHC derivative provided in this disclosure, referred to as 101A10ZM2, contains the following substitutions F620Y and N598H compared to the reference SHC protein (SEQ ID NO: 3).

[0115] The SHC derivative provided in this disclosure, referred to as 111C8ZM2, contains the following substitutions G85A+V100V and F137L compared to the reference SHC protein (SEQ ID NO: 3).

[0116] In a preferred embodiment, the SHC derivative comprises at least substituted F628Y or Y144R in combination with at least one of F141L and / or I459T for SEQ ID NO: 4.

[0117] The SHC derivative provided in this disclosure, called SHC3Bjp, contains the following substitution F628Y compared to the reference SHC protein (SEQ ID NO: 4).

[0118] Hoshino and Sato (2002 ibid.) identified F601 as a highly conserved amino acid residue among prokaryotes and eukaryotes. It has been reported that the SHC derivative F601Y showed a greatly increased Vmax for oxide squalene substrates (not squalene). However, when squalene is used, F601Y shows a decrease in affinity to WT AacSHC (i.e., a higher K M ) and catalytic efficiency / activity (Kcat / K M This shows a decrease in ( ). Data on the efficacy of AacSHC when homofarnesol is used as the enzyme substrate for the F601Y mutant are not provided by Hoshino and Sato. F628Y is an SHC derivative equivalent to F601Y in BjpSHC.

[0119] The SHC derivative provided in this disclosure, called SHC10Bjp, contains the following substitution F141L compared to the reference SHC protein (SEQ ID NO: 4).

[0120] The SHC derivative provided in this disclosure, called SHC30Bjp, includes the following substitutions F628Y and F141L compared to the reference SHC protein (SEQ ID NO: 4).

[0121] The SHC derivative provided in this disclosure, called SHC26Bjp, contains the following substitutions Y144R and I459T compared to the reference SHC protein (SEQ ID NO: 4).

[0122] The SHC derivative provided in this disclosure, referred to as 215G2Bjp, includes the following substitutions Y144R, I459T, and V241V compared to the reference SHC protein (SEQ ID NO: 4).

[0123] The SHC derivative provided in this disclosure, called SHC32Bjp, includes the following substitutions F628Y, Y144R, and I459T compared to the reference SHC protein (SEQ ID NO: 4).

[0124] The SHC derivative provided in this disclosure, called SHC31Bjp, includes the following substitutions F141L, Y144R, and I459T compared to the reference SHC protein (SEQ ID NO: 4).

[0125] The SHC derivatives provided in this disclosure, referred to as SHC33Bjp, include the following substitutions F628Y, F141L, Y144R, and I459T compared to the reference SHC protein (SEQ ID NO: 4).

[0126] The SHC derivative provided in this disclosure, referred to as 101A10Bjp, contains the following substitutions F628Y and M607H compared to the reference SHC protein (SEQ ID NO: 4).

[0127] The SHC derivative provided in this disclosure, called 111C8Bjp, includes the following substitutions A88A+V104V and F141L compared to the reference SHC protein (SEQ ID NO: 4).

[0128] amino acid sequence In some aspects, the AacSHC / HAC derivative comprises one or more polypeptides described in one or more of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and / or 171.

[0129] Preferably, the AacSHC / HAC derivatives of this disclosure have an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, and / or SEQ ID NO: 171.

[0130] In another embodiment, the ZmoSHC1 / HAC derivative comprises one or more polypeptides described in one or more of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and / or 173.

[0131] Preferably, the ZmoSHC1 / HAC derivative of the present disclosure has an amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, and / or SEQ ID NO: 173.

[0132] In a further embodiment, the ZmoSHC2 / HAC derivative comprises one or more polypeptides described in one or more of SEQ ID NOs: 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and / or 175.

[0133] In an additional embodiment, the BjpSHC / HAC derivative comprises one or more polypeptides listed in one or more of SEQ ID NOs: 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, and / or 177.

[0134] Array alignment For example, due to the different lengths of SHC reference sequences such as AacSHC, ZmoSHC1, ZmoSHC2, and BjpSHC polypeptide sequences, the amino acid residue at position X of the reference AacSHC sequence (SEQ ID NO: 1) corresponds to a different amino acid position B on the ZmoSHC1 reference sequence (SEQ ID NO: 2), a different amino acid position J on the ZmoSHC2 reference sequence (SEQ ID NO: 3), and a different amino acid position Z on the BjpSHC reference sequence (SEQ ID NO: 4). In addition, modification of the SHC reference sequence can also modify the SHC derivative sequence relative to the reference SHC sequence.

[0135] The term "position" refers to a specific amino acid residue present in a reference SHC protein, identified by its specific numbering. Modifications to an SHC reference protein, whether through amino acid insertion or deletion, result in different numbering between the reference SHC amino acid sequence and the SHC derivative amino acid sequence. For example, if an amino acid is inserted between amino acids 509 and 510 in the reference SHC protein, the amino acid following the insertion will have numbering 511 in the SHC derivative protein, while it retains numbering 510 in the SHC reference protein.

[0136] Assay for determining the activity of WT SHC / HAC and SHC / HAC derivatives Assays for determining and quantifying WT SHC / HAC and / or SHC / HAC derivative enzyme activity are described herein and are known in the art. For example, WT SHC / HAC and / or SHC / HAC derivative activity can be determined by incubating purified SHC / HAC enzyme or extract from host cells, or a complete recombinant host organism producing SHC / HAC enzyme, with a suitable substrate under suitable conditions, and by analyzing the reaction product (e.g., by gas chromatography (GC) or HPLC analysis). Further details of SHC / HAC and / or SHC / HAC enzyme activity assays and analysis of reaction products are provided in the examples. These assays involve the production of SHC derivatives by recombinant host cells (e.g., Escherichia coli).

[0137] As used herein, the term "activity" refers to the ability of an enzyme to react with a substrate to provide a target product. Activity can be determined, in known activity tests, by the increase of the target product as a function of time, the decrease of the substrate (or starting material), or a combination of these parameters. The SHC / HAC derivatives of this disclosure are characterized by their ability to bioconvert homofarnesol to (-)-ambrox and exhibit biological activity such as HAC activity.

[0138] As used herein, “biological activity” means any activity that a polypeptide may exhibit, and without limitation includes, 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 toxic activity. It is not required that a variant or derivative exhibit such activity to the same degree as the parent polypeptide. In the context of this application, a variant is considered to be one in which it exhibits activity relating to at least 10% of the activity of the parent polypeptide. Similarly, in the context of this application, a derivative is considered to be one in which it exhibits biological activity relating to at least 10% of the activity of the parent polypeptide (so that the terms derivative and variant are used interchangeably in this disclosure).

[0139] In other embodiments, the SHC / HAC derivatives of this disclosure exhibit better target yields than the reference SHC protein. The term “target yield” means grams of recoverable product per gram of raw material (which can be calculated as a percentage of molar conversion).

[0140] In additional embodiments, the SHC / HAC derivatives of the present disclosure exhibit modified (e.g., increased) target productivity relative to a reference SHC protein. The term “target productivity” means the amount of recoverable target product in grams per liter of fermentation capacity per hour of bioconversion time (i.e., time after substrate addition).

[0141] In a further embodiment, the SHC / HAC derivatives of this disclosure exhibit a modified target yield coefficient compared to the reference SHC protein. The term “target yield coefficient” refers to the ratio between the concentration of the product obtained in the reaction medium and the concentration of the SHC derivative (e.g., purified SHC enzyme or extract from recombinant host cells expressing the SHC enzyme).

[0142] In various embodiments, the SHC derivatives of the present disclosure exhibit a modified (e.g., increased) ~-fold increase in enzymatic activity (e.g., modified / increased homofarnesol-ambrox cyclase (HAC) activity) compared to a reference SHC protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4). This increase in activity is at least by 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 the coefficient.

[0143] Nucleotide sequence This disclosure further relates to isolated nucleic acid molecules comprising nucleotide sequences encoding SHC derivatives described herein.

[0144] As used herein, the term “nucleic acid molecule” specifically refers to the polynucleotides of this disclosure, which may be DNA, cDNA, genomic DNA, synthetic DNA, or RNA, and may be double-stranded or single-stranded, sense and / or antisense strands. The term “nucleic acid molecule” applies particularly to polynucleotides (one or more) as used herein, for example, as full-length nucleotide sequences or fragments or parts thereof, each encoding an enzymatic polypeptide, such as an enzyme in a metabolic pathway, or a fragment or part thereof.

[0145] The term also encompasses other molecules, such as cDNA whose corresponding genomic DNA has introns and therefore a different sequence, genomic fragments lacking at least one adjacent gene, cDNA or genomic DNA fragments produced by polymerase chain reaction (PCR) lacking at least one adjacent gene, restriction fragments lacking at least one adjacent gene, fusion proteins (e.g., His tags), mutains, or DNA encoding proteins that do not exist in nature, and nucleic acids that are degenerate variants of cDNA or naturally occurring nucleic acids. In addition, it includes hybrid genes, i.e., recombinant nucleotide sequences that are part of genes encoding fusion proteins that do not exist in nature. Fusion proteins can have one or more amino acids (such as, but not limited to, histidine (His)) added to a protein, which is usually added to the N-terminus of the protein, but also at the C-terminus or fused within the protein region. Such fusion proteins or fusion vectors encoding such proteins typically serve three purposes: (i) to increase the production of recombinant proteins, (ii) to increase the solubility of recombinant proteins, and (iii) to assist in the purification of recombinant proteins by providing ligands for affinity purification. The term “nucleic acid molecule” also encompasses codon-optimized sequences suitable for expression by specific microbial host cells (e.g., E. coli host cells). As used herein, “codon-optimized” means a protein-coding sequence of nucleic acid adapted for expression by prokaryotic or eukaryotic host cells, particularly bacterial host cells such as E. coli host cells, by substitution of one or more, preferably a significant number, codons with codons frequently used in bacterial (e.g., E. coli) host cell genes. In this regard, the nucleotide sequences encoding reference sequence numbers 1, 2, 3, and / or 4, as well as all their variants / derivatives, may be the original ones found in the source (e.g., AacSHC, ZmoSHC1, ZmoSHC2, or BjpSHC, respectively), or the genes may be codon-optimized for a selected host organism, such as E. coli.

[0146] 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. Segments of nucleic acid molecules, in particular partial genes, can be referred to as DNA fragments of genes. Fragments may also contain several open reading frames (ORFs), either repeats of the same ORF or different ORFs. The term specifically refers to coding nucleotide sequences, but also includes non-coding sequences, such as non-transcribed or non-translated sequences, or nucleotide sequences that code for a polypeptide in whole or in part. For example, genes used herein for assembly, diversification, or recombination may be non-coding sequences, sequences that code for a polypeptide, or protein-coding sequences, or parts or fragments thereof, and have a sequence length sufficient for the desired recombination event. More specifically, such genes have a minimum length of 3 bp, preferably at least 100 bp, and more preferably at least 300 bp.

[0147] It should be clear from the foregoing that the reference to isolated DNA does not mean, for example, DNA present among hundreds or millions of other DNA molecules in a cDNA or genomic DNA library or in a restriction digest mixture or genomic DNA restriction digest in an electrophoretic gel slice. The isolated nucleic acid molecules of this disclosure include segments that are not found in their natural state.

[0148] As used herein, the term “isolated DNA” may refer to (1) DNA containing a sequence that is not identical to any naturally occurring sequence, a polynucleotide or nucleic acid that does not exist naturally (e.g., created by the artificial combination of two otherwise separated sequence segments by human intervention (e.g., artificial manipulation of isolated segments of nucleic acids by genetic engineering techniques)), or (2) DNA having a naturally occurring sequence (e.g., cDNA or genomic DNA) that does not contain at least one gene adjacent to the gene containing the target DNA in the genome of an organism in which the gene containing the target DNA exists naturally.

[0149] Specifically, the term “isolated DNA” as used herein with respect to nucleic acid sequences may also refer to nucleic acids or polynucleotides produced by recombinant DNA technology, such as DNA constructs containing polynucleotides heterogeneous to a host cell and optionally incorporated into a host cell. Chimeric nucleotide sequences may specifically be produced as recombinant molecules. The term “recombination” applies specifically to the assembly of polynucleotides, linking such polynucleotides or portions thereof with or without recombination to achieve crossover or genetic mosaicism. For example, this is carried out to link nucleic acid segments of desired function to produce a desired combination of function. Recombinant genes encoding polypeptides described herein include the coding sequence of the polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for the expression of the polypeptide. Since many microorganisms can express multiple gene products from polycistron mRNA, multiple polypeptides may, if desired, be expressed under the control of a single regulatory region for those microorganisms. The code sequence and the control region are considered to be operably linked when the control region and the code sequence are positioned such that the control region is effective in controlling the transcription or translation of the sequence.

[0150] As used herein, the term “recombinant” specifically refers to enzymes produced by recombinant DNA technology, i.e., enzymes produced from cells transformed with an exogenous DNA construct encoding the desired enzyme. “Synthetic” enzymes are those prepared by chemical synthesis. Chimeric enzymes can specifically be produced as recombinant molecules. Therefore, the term “recombinant DNA” includes recombinant DNA incorporated into vectors, self-replicating plasmids or viruses, or into the genomic DNA of prokaryotes or eukaryotes (or the genome of homologous cells at locations other than their natural chromosomal locations).

[0151] In a further aspect, the nucleic acid molecules(s) of this disclosure are operationally ligated to expression control sequences that enable expression by prokaryotic and / or eukaryotic host cells. As used herein, “operationally ligated” means that the expression control sequence is incorporated into the gene construct to effectively control the expression of the coding sequence in question. The transcription / translation control elements mentioned above include, but are not limited to, inducible and non-inducible, constitutive, cell cycle-controlled, and metabolically controlled promoters, enhancers, operators, silencers, repressors, and other elements known to those skilled in the art that drive or otherwise control gene expression. Such control elements include, but are not limited to, control elements that direct to constitutive expression or enable inducible expression, such as the CUP-1 promoter, tet repressors employed in tet-on or tet-off systems, lac systems, and trp system control elements. For 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 mimetic of allolactose, a lactose metabolite that induces transcription of the lac operon, and is therefore used to induce gene expression when the gene is under the control of the lac operator. Another example of a regulatory element that induces gene expression is lactose.

[0152] Similarly, the nucleic acid molecules(s) of the Disclosure may form part of a hybrid gene encoding additional polypeptide sequences, such as sequences that function as markers or reporters. Examples of marker and reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine-guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures relating to the implementation of the Disclosure, those skilled in the art will know of additional useful reagents, such as additional sequences that can function as markers or reporters.

[0153] In some embodiments, this disclosure provides recombinant polynucleotides encoding WT SHC or the above SHC / HAC derivatives, which can be inserted into vectors for expression and optional purification. One type of vector is a plasmid, which represents a circular double-stranded DNA loop in which additional DNA segments are ligated. Certain types of vectors can control the expression of genes in which they are functionally linked. These vectors are called “expression vectors.” Typically, expression vectors preferred for DNA recombination techniques are plasmids. Typically, expression vectors contain genes such as WT SHC or SHC / HAC variants described herein. Since plasmids are the most commonly used vector type, the terms “plasmid” and “vector” are used interchangeably herein.

[0154] Such vectors may include, but are not limited to, DNA sequences that do not naturally exist in host cells, DNA sequences that are not normally transcribed to RNA or translated to proteins ("expressed"), and other genes or DNA sequences that you wish to introduce into a non-recombinant host. Typically, it will be understood that the genome of the recombinant host described herein is extended by the stable introduction of one or more recombinant genes. However, self- or replicating plasmids or vectors may also be used within the scope of this disclosure. Furthermore, this disclosure may be carried out using low copy number, e.g., single copy, or high copy number (as illustrated herein) plasmids or vectors.

[0155] In a preferred embodiment, the vectors of the Disclosure comprise plasmids, phagemids, phages, cosmids, artificial bacterial and artificial yeast chromosomes, knockouts or knock-in constructs, synthetic nucleic acid sequences, or cassettes, some of which may be produced in the form of linear polynucleotides, plasmids, megaplasmids, synthetic or artificial chromosomes, such as plant, bacterial, mammalian, or yeast artificial chromosomes.

[0156] The protein encoded by the introduced polynucleotide is preferably expressed in the cell at the time of vector introduction. A variety of gene substrates can be incorporated into the plasmid. The plasmid is often a standard cloning vector, such as a bacterial multicopy plasmid. The substrates can be incorporated into the same or different plasmids. Often, at least two different types of plasmids with different types of selectable markers are used to allow selection of cells containing at least two types of the vector.

[0157] Typically, bacterial or yeast cells can be transformed by one or more of the following nucleotide sequences, as is well known in the art. For in vivo recombination, the genes to be recombined with the genome or other genes are used to transform the host using standard transformation techniques. In a preferred embodiment, DNA providing an origin of replication is incorporated into the construct. The origin of replication can be suitably selected by those skilled in the art. Depending on the nature of the genes, supplemental origins of replication may not be required if sequences capable of functioning as origins of replication already exist in the gene or genome.

[0158] Bacterial or yeast cells can be transformed with exogenous or heterologous DNA when such DNA is introduced into the cell. The transformed DNA may or may not be integrated, i.e., covalently linked into the cell's genome. In prokaryotes and yeast, for example, the transformed DNA may be maintained as an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transfected cell is one in which the transfected DNA is integrated into the chromosome so that it is inherited by daughter cells through chromosomal replication. This stability is demonstrated by the ability of the eukaryotic cell to establish a cell line or clone consisting of a population of daughter cells containing the transformed DNA.

[0159] Generally, the introduced DNA is not originally endogenous in the host that receives the DNA. However, within the scope of this disclosure is the isolation of a DNA segment from a given host and subsequent introduction of one or more additional copies of that DNA into the same host to, for example, enhance the production of a gene product or alter the gene expression pattern. In some cases, the introduced DNA will modify or even replace endogenous genes or DNA sequences, for example, by homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms, plant cells, and plants.

[0160] This disclosure also features recombinant hosts. The term “recombinant host” is also referred to as “genetically modified host cell” or “transgenic cell” and refers to a host cell that contains heterologous nucleic acids or whose genome has been extended by at least one incorporated DNA sequence. The host cells of this disclosure may be genetically modified by polynucleotides or vectors as outlined above.

[0161] Host cells that may 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) that can be transformed by recombinant bacteriophage DNA, plasmid DNA, bacterial artificial chromosomes, or cosmid DNA expression vectors containing the polynucleotide molecules of this disclosure, and simple eukaryotic cells such as yeast (e.g., Saccharomyces and Pichia) that can be transformed by recombinant yeast expression vectors containing the polynucleotide molecules of this disclosure. Depending on the host cell used to introduce the polynucleotides of this disclosure and the respective vectors, the polynucleotides may be integrated, for example, into chromosomes or mitochondrial DNA, or maintained outside of chromosomes, such as in episomes, or transiently contained within the cell.

[0162] In particular, the terms “cell” or “producing cell” as used herein in reference to genetic engineering and the introduction of one or more genes or clusters of genes into a cell are understood to mean either a prokaryotic or eukaryotic cell. Both prokaryotic and eukaryotic host cells intended for use in accordance with this disclosure include bacterial host cells such as Escherichia coli or Bacillus species, yeast host cells such as S. cerevisiae, insect host cells such as Spodoptera frugiperda, or human host cells such as HeLa and Jurkat.

[0163] Specifically, the cells are eukaryotic cells, preferably fungal, mammalian, or plant cells, or prokaryotic cells. Suitable eukaryotic cells include, for example, mammalian cells, yeast cells, or insect cells (including Sf9), amphibian cells (including melanocytes), or helminthic cells including Caenorhabditis cells (including Caenorhabditis elegans), without limitation. Suitable mammalian cells include, for example, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 cells, HEK293T cells, HEK293 T-Rex™ cells, or other transfectable eukaryotic cell lines, without limitation. Suitable bacterial cells include, without limitation, Escherichia coli.

[0164] Preferably, prokaryotes such as Escherichia coli, Bacillus, and Streptomyces, or mammalian cells such as HeLa cells or Jurkat cells, or plant cells such as Arabidopsis may be used.

[0165] Preferably, the cells are Aspergillus sp. or fungal cells, which may be selected from the group consisting of the genera Saccharomyces, Candida, Kluyveromyces, Hansenula, Schizosaccharomyces, Yarrowia, Pichia, and Aspergillus.

[0166] Preferably, the E. coli host cell is an E. coli host cell recognized by industry and regulatory authorities (including, but not limited to, E. coli K12 host cells or E. coli BL21 host cells as shown in the example).

[0167] One preferred host cell for use in this disclosure is Escherichia coli, which can be recombinantly prepared as described herein. Therefore, the recombinant host may be a recombinant E. coli host cell. A library of available mutants, plasmids, detailed metabolic computer models, and other information for E. coli is available, enabling the rational design of various modules for enhancing product yield. Methods similar to those described above can be used to create recombinant E. coli microorganisms for Saccharomyces.

[0168] In one embodiment, recombinant Escherichia coli microorganisms include, but are not limited to, nucleotide sequences encoding SHC genes disclosed in one or more of Tables 10, 11, and 12 of this Spec, or their variants, homologs, variants, derivatives, or fragments; and functional equivalents / homologies thereof.

[0169] Preferably, the recombinant E. coli microorganism includes the vector construct provided in Figures 5 and 21.

[0170] In another preferred embodiment, recombinant Escherichia coli microorganisms include nucleotide sequences encoding WT SHC / HAC and WT SHC / HAC derivative genes, or functional equivalents / homologies thereof, including but not limited to variants, homologs, variants, derivatives, or fragments thereof as listed in one or more of Tables 13, 14, 15, 16, 17, and / or Table 4a.

[0171] Another preferred host cell for use in this disclosure is S. cerevisiae, which is widely used as a chassis organism in synthetic biology. Therefore, the recombinant host can be S. cerevisiae. A library of available mutants, plasmids, detailed computer models of metabolism, and other information about S. cerevisiae is available, enabling the rational design of various modules to enhance product yield. Methods for producing recombinant S. cerevisiae microorganisms are known.

[0172] Cell culture is carried out in a conventional manner. The culture medium contains a carbon source, at least one nitrogen source, and inorganic salts, to which vitamins are added. The components of this medium may be those conventionally used for culturing the microbial species in question.

[0173] The carbon sources used in this method include any molecules that can be metabolized by recombinant host cells to promote proliferation and / or the production of (-)-ambax. Examples of suitable carbon sources include, but are not limited to, sucrose (found in molasses, for example), fructose, xylose, glycerol, glucose, cellulose, starch, cellobiose, or other glucose-containing polymers.

[0174] In embodiments employing yeast as the host, suitable carbon sources include, for example, sucrose, fructose, xylose, ethanol, glycerol, and glucose. The carbon source can be supplied to the host organism throughout the culture period, or alternatively, the organism may be grown for a period in the presence of another energy source, such as protein, and then supplied with a carbon source only during the fed-batch phase.

[0175] The suitability of recombinant host cell microorganisms for use in the methods of this disclosure can be determined by simple test procedures using well-known methods. For example, the microorganism to be tested can be grown in a nutrient-rich medium (e.g., LB medium, bactotryptone yeast extract medium, nutrient medium, etc.) under pH, temperature, and aeration conditions commonly used for microbial growth. Once recombinant microorganisms (i.e., recombinant host cells) that produce the desired product of bioconversion are selected, the product is typically produced on a large scale by a producing host cell line, by a suitable expression system and fermentation, for example, by microbial production in cell cultures.

[0176] In one embodiment of this disclosure, a limited minimum medium such as M9A is used for cell culture.

[0177] The components of M9A medium include 14 g / l KH2PO4, 16 g / l K2HPO4, 1 g / l sodium citrate 3.2H2O, 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.

[0178] In another aspect of this disclosure, a nutrient-rich medium such as LB was used. The LB medium consists of 10 g / l tryptone, 5 g / l yeast extract, and 5 g / l NaCl.

[0179] Other examples of mineral media and M9 mineral media are disclosed, for example, in US6524831B2 and US2003 / 0092143A1.

[0180] Recombinant microorganisms can be grown in batches, fed batches, continuous processes, or a combination thereof. Typically, recombinant microorganisms are grown in a fermentor at a specified temperature(s) for a desired period in the presence of suitable nutrients, such as a carbon source, to produce sufficient enzymes to bioconvert homofarnesol to ambrox and to produce a desired amount of ambrox containing (-)-ambrox.

[0181] Recombinant host cells can be cultured in any preferred manner, for example, by batch culture or fed-batch culture.

[0182] As used herein, the term "batch culture" refers to a culture method in which the culture medium is neither added to nor removed during the culture process.

[0183] As used herein, the term "federation" refers to a culture method in which culture medium is added during the culture process, but the culture medium is not removed.

[0184] One aspect of this disclosure provides a method for producing ambrox in a cell system, comprising: expressing WT SHC or an SHC / HAC derivative in the cell system under preferred conditions; feeding homofarnesol to the cell system; converting the homofarnesol to ambrox using the SHC or SHC / HAC derivative produced using the cell system; collecting ambrox from the cell system; and optionally isolating (-)-ambrox material from the system. Expression of other nucleotide sequences may be useful to enhance the method. The bioconversion method may include the additional expression of other nucleotide sequences by the cell system. Expression of other nucleotide sequences may enhance the bioconversion pathway for producing (-)-ambrox.

[0185] Further aspects of the present disclosure are bioconversion methods for producing (-)-ambax, comprising: growing host cells containing a WT SHC / HAC or SHC / HAC derivative gene; producing WT SHC / HAC or an SHC / HAC derivative in the host cells; supplying homofarnesol (e.g., EEH) to the host cells; incubating the host cells under conditions of pH, temperature, and solubilizer suitable to promote the conversion from homofarnesol to ambrox; and collecting (-)-ambax. The production of WT SHC / HAC and / or SHC / HAC derivatives by the host cells provides a method for producing (-)-ambax when homofarnesol is added to the host cells under suitable reaction conditions. The conversion achieved may be enhanced by adding more biocatalysts and SDS to the reaction mixture.

[0186] Recombinant host cell microorganisms can be cultured in several ways to provide a suitable amount of cells expressing WT SHC or SHC / HAC derivatives for subsequent bioconversion steps. Since the applicable microorganisms for the bioconversion steps vary considerably (e.g., yeast, bacteria, and fungi), the culture conditions are naturally tailored to the specific requirements of each species, and these conditions are well known and documented. Any method known in the art for growing cells of a recombinant host cell microorganism can be used to produce cells available for the subsequent bioconversion steps of this disclosure. Typically, the cells are grown to a specific density (measurable as optical density (OD)) to produce sufficient biomass for the bioconversion reaction.

[0187] The selected culture conditions not only affect the quantity of cells (biomass) obtained, but the quality of the culture conditions also affects how the biomass becomes a biocatalyst. Recombinant host cell microorganisms that express WT SHC or SHC / HAC derivative genes and produce WT SHC or SHC / HAC derivative enzymes are called biocatalysts suitable for use in biotransformation reactions. In some embodiments, the biocatalyst is recombinant whole cells producing WT SHC or SHC / HAC derivatives, which may be in suspension or immobilized format. In other embodiments, the biocatalyst is a membrane fraction or liquid fraction prepared from recombinant whole cells producing WT SHC or SHC / HAC derivatives (e.g., Seitz et al 2012 - disclosed above).

[0188] Recombinant whole cells producing WT SHC or SHC / HAC derivatives include whole cells collected from a fermenter (for a bioconversion reaction) or cells within the fermenter (which are then used in a one-pot reaction). Recombinant whole cells producing WT SHC or SHC / HAC derivatives may include intact recombinant whole cells and / or cell debris. In any case, WT SHC or SHC / HAC derivatives are somehow bound to a membrane (such as a cell membrane) to receive and / or interact with a substrate (e.g., homofarnesol), and this membrane (such as a cell membrane) may be part of or contain whole cells (e.g., recombinant whole cells). WT SHC or SHC / HAC derivatives may also be in an immobilized form (e.g., bound to an enzyme carrier) that allows them to interact with a substrate (e.g., homofarnesol). WT SHC or SHC / HAC derivatives may also be used in a soluble form.

[0189] In one embodiment, the biocatalyst is produced in sufficient quantities (to produce sufficient biomass), harvested, washed before the bioconversion step (and optionally stored (e.g., frozen or freeze-dried)).

[0190] In a further embodiment, cells are produced in sufficient quantities (to produce a sufficient biocatalyst), the reaction conditions are then adjusted, and there is no need to harvest and wash the biocatalyst for the biotransformation reaction. This one-step (or "one-pot") method is advantageous because it simplifies the process while reducing costs. The culture medium used to grow the cells is also suitable for use in the biotransformation reaction, provided that the reaction conditions are adjusted to facilitate the biotransformation reaction.

[0191] The optimal pH for cell proliferation is within the range of 6.0 to 7.0. The optimal pH for the bioconversion reaction depends on the type of SHC / HAC enzyme used in the reaction. pH is controlled using techniques well known to those skilled in the art.

[0192] As shown in Example 9, a "one-pot" method is used to bioconvert homofarnesol to (-)-ambrox with a conversion rate of 100%. As shown in Example 18, a "one-pot" method is used to bioconvert homofarnesol to (-)-ambrox with a conversion rate of 99%.

[0193] Any references herein to the 99% / 100% conversion rate from homofarnesol substrates to (-)-ambrox refer to the 99% / 100% conversion of homofarnesol isomers (i.e., EEH) that can be converted to (-)-ambrox using WT SHC / HAC or SHC / HAC derivative enzymes.

[0194] While the terms “mixture” or “reaction mixture” may be used interchangeably with the term “culture medium” in this disclosure (especially when relating to a “one-pot” reaction), it should be noted that growing cells to produce sufficient biomass requires cell culture / fermentation medium, but medium is not required for the bioconversion step, as a reaction buffer of a suitable pH will suffice.

[0195] The bioconversion method described herein is carried out under conditions of time, temperature, pH, and solubilizer to result in the conversion of homofarnesol raw material stock to (-)-ambrox. The pH of the reaction mixture may be in the range of 4 to 8, preferably 5 to 6.5, more preferably 4.8 to 6.0 for SHC / HAC derivative enzymes, and in the range of about pH 5.0 to about pH 7.0 for WT SHC enzymes, and can be maintained by adding buffer to the reaction mixture. An exemplary buffer for this purpose is citrate buffer. The preferred temperature is between about 15°C and about 45°C, preferably between about 20°C and about 40°C, but this is higher for thermophilic organisms, especially when WT enzymes from thermophilic microorganisms (e.g., WT SHC / HAC) are used, and can be up to 55°C. The temperature may be kept constant or changed during the bioconversion process.

[0196] The applicant has shown that it may be useful to include solubilizers (e.g., surfactants, detergents, solubility enhancers, water-miscible organic solvents, etc.) in biotransformation reactions. As used herein, the term “surfactant” means a component that reduces the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants can act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. Examples of surfactants include, but are not limited to, Triton X-100, Tween 80, taurodeoxycholic acid, sodium taurodeoxycholate, sodium dodecyl sulfate (SDS), and / or sodium lauryl sulfate (SLS).

[0197] Triton X-100 can be used to partially purify WT SHC / HAC or SHC / HAC derivative enzymes (in soluble or membrane fraction / suspension form), while it can also be used in biotransformation reactions (see, for example, disclosures by Seitz (2012 PhD dissertation, ibid.) and Neumann and Simon (1986 - ibid.) and JP2009060799).

[0198] However, surprisingly, as Example 14 demonstrates, the applicant selected and identified SDS as a particularly useful solubilizer from a long list of other less useful solubilizers. In particular, the applicant identified SDS as a significantly superior solubilizer to, for example, Triton X-100 in terms of reaction rate and yield of the bioconversion reaction from homofarnesol to (-)-ambrox (both when EEH is used at 4 g / l and 125 g / l). As shown by the comparative data in Example 12, the applicant demonstrated that for at least one SHC / HAC derivative enzyme, the best bioconversion activity from homofarnesol to (-)-ambrox using Triton X-100 (in the concentration range of about 0.005% to 0.48%) during the reaction was only about 20% of the activity obtained with SDS (at a concentration of about 0.07%).

[0199] While we do not wish to be constrained by theory, the use of SDS in recombinant microbial host cells may be advantageous because SDS can favorably interact with the host cell membrane to make the SHC enzyme (which is a membrane-bound enzyme) more accessible to the homofarnesol substrate. In addition, the inclusion of a suitable level of SDS in the reaction mixture may improve the properties of the emulsion (homofarnesol in water) and / or improve access of the homofarnesol substrate to the SHC enzyme within the host cell, while simultaneously preventing disruption (e.g., denaturation of the SHC (WT or SHC / HAC derivative) enzyme).

[0200] The concentration of solubilizers (e.g., SDS) used in bioconversion reactions is influenced by the amount of biomass and the substrate (EEH) concentration. That is, there is a degree of interdependence between the solubilizer (e.g., SDS) concentration, the amount of biomass, and the substrate (EEH) concentration. For example, as the concentration of homofarnesol substrate increases, sufficient amounts of biocatalyst and solubilizer (e.g., SDS) are required for efficient bioconversion. If the solubilizer (e.g., SDS) concentration is too low, suboptimal homofarnesol conversion may be observed. On the other hand, if the solubilizer (e.g., SDS) concentration is too high, there is a risk that the biocatalyst may be affected by the destruction of intact microbial cells and / or the denaturation / inactivation of SHC / HAC enzymes.

[0201] The selection of a suitable SDS concentration in relation to biomass quantity and substrate (EEH) concentration is within the scope of knowledge of those skilled in the art. For example, predictive models for determining suitable SDS, substrate (EEH), and biomass concentrations are available to those skilled in the art. As a further example, Example 3 shows that when a biocatalyst with 4 g / l EEH and 10.0 OD (650 nm) is used, an SDS concentration in the range of 0.010 to 0.075% is suitable. Example 7 shows that when 125 g / l EEH is used in 2 × biomass wet weight, a adjusted SDS concentration (1.55%) is suitable. However, studies of EEH conversion percentages to (-)-Amblox using different SDS / cell ratio values ​​demonstrate that the correct selection of ratios for biocatalysts, homofarnesol substrates, and solubilizers (e.g., SDS) facilitates the development of robust bioconversion reaction systems, which show some tolerance for certain ranges of SDS concentration (see, e.g., Figure 17) and pH range (see, e.g., Figure 15, Figure 18).

[0202] The bioconversion reaction temperature for WT SHC enzymes (e.g., AacSHC) is approximately 45-60°C, preferably 55°C.

[0203] The pH range for the bioconversion reaction of WT SHC enzymes (e.g., AacSHC) is approximately 5.0 to 7.0, more preferably approximately 5.6 to approximately 6.2, and even more preferably approximately 6.0.

[0204] The temperature for the bioconversion reaction of SHC / HAC derivative enzymes is approximately 34°C to 50°C, preferably 35°C.

[0205] The pH of the bioconversion reaction of SHC / HAC derivative enzymes is approximately 4.8 to 6.4, preferably approximately 5.2 to 6.0.

[0206] Preferably, the solubilizer used in the biotransformation reaction is SDS.

[0207] The SDS concentration used in the biotransformation reaction of WT SHC enzymes (e.g., AacSHC) is in the range of approximately 0.010 to 0.075%, preferably approximately 0.030%, when approximately 4 g / l of EEH is used.

[0208] The SDS concentration used in the bioconversion reaction of SHC / HAC derivative enzymes is in the range of approximately 0.0025 to 0.090%, preferably approximately 0.050%, when approximately 4 g / l of EEH is used.

[0209] The biocatalyst is introduced into the reaction at an OD of 10.0 (650 nm) when homofarnesol is introduced into the reaction at an EEH concentration of approximately 4 g / l.

[0210] The [SDS] / [cell] ratio is in the range of about 10:1 to 20:1, preferably about 15:1 to 18:1, and preferably about 16:1, when the ratio of biocatalyst to EEH homofarnesol is about 2:1.

[0211] In the biotransformation reaction of SHC variant enzymes, the SDS concentration is in the range of about 1-2%, preferably in the range of about 1.4-1.7%, and more preferably in the range of about 1.5%, when the homofarnesol concentration is about 125 g / l of EEH and the biocatalyst concentration is 250 g / l (corresponding to an OD of about 175 (650 nm)).

[0212] The ratio of the biocatalyst to the EEH homofarnesol substrate is in the range of approximately 0.5:1 to 2:1, and in some embodiments, 2:1, preferably about 1:1 or 0.5:1.

[0213] In some embodiments, ambrox is produced using a biocatalyst to which a homofarnesol substrate is added. The substrate can be added by supplying it using known means (e.g., peristaltic pump, injection syringe, etc.). Homofarnesol is an oil-soluble compound and is supplied in oil format. Given that the biocatalyst (microbial cells such as intact recombinant whole cells and / or cell fragments and / or immobilized enzymes) is present in the aqueous phase, the bioconversion reaction can be considered a three-phase system (including aqueous, solid, and oil phases) when homofarnesol is added to the bioconversion reaction mixture. This is true even when SDS is present. For clarification, when a soluble WT SHC or SHC / HAC derivative is used as the biocatalyst, this can be considered a two-phase system.

[0214] The number of homofarnesol isomers present can affect the reaction rate. As Example 11 shows, SHC / HAC derivative enzymes can bioconvert E,E-homofarnesol from a complex mixture of homofarnesol isomers (e.g., EE:EZ:ZE:ZZ) to (-)-ambrox. However, lower conversion rates are typically observed, which is consistent with the view that homofarnesol isomers other than EEH may compete with EEH for access to the SHC / HAC derivative enzyme and therefore may act as competitive inhibitors of the conversion from EEH to (-)-ambrox and / or also as alternative substrates.

[0215] Therefore, preferably, the homofarnesol substrate contains 2 to 4 isomers, preferably a mixture of stereoisomers of 2 isomers.

[0216] Therefore, preferably, the homofarnesol substrate consists of 2 to 4 isomers, preferably a mixture of stereoisomers of 2 isomers, or essentially consists of such a mixture.

[0217] Preferably, the homofarnesol substrate contains a mixture of EE:EZ stereoisomers.

[0218] Preferably, the homofarnesol substrate consists of or is essentially a mixture of EE:EZ stereoisomers.

[0219] As shown in Example 9, a 100% conversion of EE:EZ in a weight ratio of 87:13 was observed in a "one-pot" fermentation and bioconversion reaction carried out over a period of 22.5 days. Approximately 10 g of EEH was converted during this period.

[0220] As described in detail in Example 7, in a preferred embodiment, recombinant host cells expressing SHC / HAC derivative genes and producing active SHC / HAC derivative enzymes are grown in the same fermenter vessel used to convert a homofarnesol source into (-)-ambrox as a mixture with one or more by-products (II), (IV), and / or (III, disclosed, e.g., Figure 12) to a sufficient biomass concentration suitable for use as a biocatalyst. The resulting (-)-ambrox may be isolated by steam extraction / distillation or 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 evaporation of the solvent, to obtain the crude reaction product determined by gas chromatography (GC) analysis. Steam extraction / distillation and organic solvent extraction methods are known to those skilled in the art.

[0221] As an example, the obtained (-)-ambrox can be extracted from the entire reaction mixture using an organic solvent such as a non-aqueous miscible solvent (e.g., toluene). Alternatively, the obtained (-)-ambrox can be extracted from the solid phase of the reaction mixture (e.g., obtained by centrifugation or filtration) using a water-miscible solvent (e.g., ethanol) or a non-aqueous miscible solvent (e.g., toluene). As a further example, (-)-ambrox exists in the solid phase as crystals or in amorphous form and can also be separated from the remaining solid phase (cellular material or its fragments) and from the liquid phase by means of filtration. As a further example, at temperatures above the melting point of (-)-ambrox (approximately 75°C), (-)-ambrox may form an oily layer on the aqueous phase, which can be removed and collected. To ensure the complete recovery of (-)-ambrox after the oily layer has been removed, an organic solvent may be added to the aqueous phase containing the biomass to extract any residual (-)-ambrox contained in, on, or around the biomass. The organic layer can be combined with the oil layer before the entire layer is further processed to isolate and purify (-)-ambrox.

[0222] (-)-Amblox may be further selectively crystallized to remove by-products (II), (III), and (IV), as well as any unreacted homofarnesol substrates, from the final (-)-Amblox product. The term “selective crystallization” refers to a process step in which (-)-Amblox crystallizes from the solvent, while compounds (II), (III), and (IV) remain dissolved in the crystallization solvent to such an extent that the isolated crystalline material contains only the (-)-Amblox product, or, if it contains any of the other compounds (II), (III), or (IV), they are present only in olfactory-acceptable amounts.

[0223] The selective crystallization step may use a water-miscible solvent, such as ethanol. The olfactory purity of the final (-)-ambrox product is determined by testing the crystalline material with a 10% ethanol extract in water or by testing the crystalline material itself. The final (-)-ambrox product is tested for its olfactory purity, quality, and perceptual profile against a commercially available reference (-)-ambrox product. The (-)-ambrox material is also tested in expert application studies to determine whether the material meets the standards with respect to its perceptual stimulation profile. The various applications of Ambrox include, but are not limited to, fine fragrances or consumer products such as fabric care, toiletries, beauty, and cleaning products, and essentially encompass all products in which Ambrox ingredients are commercially used and are currently available, including, but are not limited to, Ambrox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlyn (Quest), Cetalox Laevo (Firmenich), Ambermor (Aromor), and Norambrenolide Ether (Pacific) products.

[0224] The selective crystallization of (-)-ambrox can also be influenced by the presence of unreacted homofarnesol substrate, as well as the ratio of (-)-ambrox to other detectable byproducts (II), (III), and / or (IV). Even when only 10% conversion from homofarnesol substrate to (-)-ambrox is obtained (as shown in Example 7 using the WT SHC / HAC enzyme), selective crystallization of (-)-ambrox is still possible.

[0225] Suitable water-miscible and water-miscible organic solvents suitable for use in the extraction and / or selective crystallization of (-)-ambrox include, but are not limited to, aliphatic hydrogen carbons, preferably having 5 to 8 carbon atoms, e.g., pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; halogenated aliphatic hydrogen carbons, preferably having 1 or 2 carbon atoms, e.g., dichloromethane, chloroform, carbon tetrachloride, dichloroethane, or tetrachloroethane; aromatic hydrocarbons, e.g., benzene, toluene, xylene, chlorobenzene, or dichlorobenzene; aliphatic acyclic and cyclic ethers or alcohols, preferably having 4 to 8 carbon atoms, e.g., ethanol, isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran; or esters, e.g., ethyl acetate or n-butyl acetate; or ketones, e.g., 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, tertiary butyl methyl ether, and tBME), diisopropyl ether, tetrahydrofuran, ethyl acetate, and / or mixtures thereof.

[0226] Preferably, a water-miscible solvent such as ethanol is used for the extraction of (-)-ambrox 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.

[0227] As used herein, the term “isolated” refers to biotransformation products, such as (-)-ambrox, that have been separated or purified from their associated components. Substances produced by cellular systems different from their naturally occurring sources are “isolated” because they will inevitably not contain their naturally occurring components. The degree of isolation or purity can be measured by any suitable method, such as gas chromatography (GC), HPLC, or NMR analysis.

[0228] In some embodiments, the final product ((-)-ambrox) is isolated and purified until it is homogeneous (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).

[0229] Preferably, the amount of (-)-ambrox produced may be about 1 mg / l to about 20,000 mg / l (20 g / l) or more, for example, about 20 g / l to about 200 g / l or 100 to 200 g / l, preferably about 125 g / l or 150 g / l or about 188 g / l.

[0230] As shown in Example 7, at least 125 g / l of (-)-ambrox can be produced by a bioconversion reaction using recombinant E. coli host cells that produce SHC / HAC derivative enzymes over approximately two days.

[0231] As Example 19 demonstrates, bioconversion can be carried out at EEH levels of 188 g / l or higher if efficient mixing is achieved, because stirring efficiency appears to be the only limitation of the system. In addition, biocatalysts with improved activity (for example, in terms of SHC variants with further improved activity, or in terms of increased SHC enzyme production) can improve or maintain productivity with less biomass, which is advantageous with respect to mixing efficiency.

[0232] For example, (-)-Ambrox in concentrations of approximately 1 to 100 mg / l, approximately 30 to 100 mg / l, approximately 50 to 200 mg / l, approximately 100 to 500 mg / l, approximately 100 to 1,000 mg / l, approximately 250 to 5,000 mg / l, approximately 1,000 (1 g / l) to 15,000 mg / l (15 g / l), or approximately 2,000 (2 g / l) to 10,000 mg / l (10 g / l), or approximately 2,000 (2 g / l) to 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, 300 g / l, 400 g / l, or 500 g / l are produced.

[0233] Preferably, (-)-Amblox at a concentration of at least 100 g / l is produced within a period of 48 to 72 hours.

[0234] Preferably, (-)-Amblox at a concentration of about 150 g / l is produced within a period of about 48 to 72 hours.

[0235] Preferably, (-)-Amblox at a concentration of about 200 g / l is produced within a period of about 48 to 72 hours.

[0236] Preferably, (-)-Amblox at a concentration of about 250 g / l is produced within a period of about 48 to 72 hours.

[0237] Those skilled in the art will understand that higher cumulative productivity can be achieved by performing a continuous process, such as removing the product, adding substrate raw materials, and adding or (partially) replacing biomass.

[0238] Preferably, the bioconversion from EEH to (-)-Amblox in the presence of recombinant host cells containing WT SHC / HAC or SHC / HAC derivatives is given in mol percent and based on the mol of EEH employed: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 This produces Ambrox yields of 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100. Particularly preferred are yields between 5 and 100, 10 and 100, 25 and 100, 30 and 100, 35 and 100, especially between 40 and 100, 45 and 100, 50 and 100, 60 and 100, and 70 and 100 mol percent.

[0239] The activity of the SHC / HAC enzyme is defined by the reaction rate in mol percent (amount of product / (amount of product + amount of remaining starting material)) × 100). Preferably, the bioconversion from EEH to (-)-ambrox in the presence of WT SHC or SHC / HAC derivative enzyme is given in mol percent and based on the mol of EEH employed: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 It produces (-)-ambrox yields of 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100. Particularly preferred are yields between 5 and 100, 10 and 100, 20 and 100, 25 and 100, 30 and 100, 35 and 100, and especially between 40 and 100, 45 and 100, 50 and 100, 60 and 100, and 70 and 100.

[0240] In a preferred embodiment of the present invention, the yield and / or reaction rate is determined over a specified period of time, for example, 4, 6, 8, 10, 12, 16, 20, 24, 36, or 48 hours, during which time EEH is converted to (-)-ambaxil by recombinant host cells containing a nucleotide sequence encoding a WT SHC or SHC / HAC derivative enzyme according to the present disclosure. In further variants, the reaction is carried out under finely defined conditions, for example, 25°C, 30°C, 40°C, 50°C, or 60°C. In particular, the yield and / or reaction rate is determined by carrying out a reaction in which EEH is converted to (-)-ambaxil by an SHC / HAC derivative enzyme according to the present invention at 35°C over a period of time of 24 to 72 hours.

[0241] In a further aspect of the invention, a recombinant host cell comprising a nucleotide sequence encoding an SHC / HAC derivative is characterized in that it exhibits a yield and / or reaction rate of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 500, 1000 times or more higher than that in the reaction of homofarnesol to give (-)-ambrinol compared to the WT SHC or SHC / HAC derivative enzyme under the same conditions. Here, the term conditions relates to reaction conditions such as substrate concentration, enzyme concentration, reaction period, and / or temperature.

[0242] The desired development of a bioconversion process for making (-)-ambrinol from homofarnesol by a recombinant strain of Escherichia coli comprising a nucleotide sequence encoding WT / reference SHC or an SHC / HAC derivative can provide a low-cost and industrially economical process for (-)-ambrinol production.

[0243] As shown in Example 7, the present disclosure results in 100% conversion of E,E-homofarnesol (125 g / l) to (-)-ambrinol after 48 hours of incubation with the optimized SHC / HAC derivative, with an 8-fold improvement in yield when the AacSHC derivative is used compared to the WT AacSHC enzyme (see Figure 11).

[0244] Functional homologs of the WT reference SHC / HAC or SHC / HAC derivative polypeptides described herein are also suitable for use in producing amblox by a recombinant host. Therefore, the recombinant host may comprise one or more heterologous nucleic acid(s) encoding a functional homolog of the polypeptide described above and / or a heterologous nucleic acid encoding the SHC / HAC derivative enzyme described herein.

[0245] A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide and has one or more of the biochemical or physiological function(s) of the reference polypeptide. The functional homolog and the reference polypeptide may be naturally occurring polypeptides, and the sequence similarity may be due to convergent or divergent evolutionary events. Thus, functional homologs are sometimes referred to in the literature as homologs or orthologs or paralogs. Variants of naturally occurring functional homologs, such as polypeptides encoded by variants of the wild-type coding sequence, may themselves be functional homologs. Functional homologs can also be created by site-directed mutagenesis of the coding sequence of a polypeptide or by combining domains from the coding sequences of different naturally occurring polypeptides (“domain swapping”). Techniques for modifying genes encoding functional homologs described herein are known and include, inter alia, evolutionary engineering techniques, site-directed mutagenesis techniques, and random mutagenesis techniques, which may be useful for increasing the specific activity of a polypeptide, altering substrate specificity, changing expression levels, changing intracellular location, or modifying polypeptide:polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term “functional homolog” is sometimes applied to nucleic acids encoding functionally homologous polypeptides.

[0246] Functional homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, querying databases of nucleotide or polypeptide sequences can identify homologs of nucleic acid sequences encoding SHC derivative polypeptides, etc.

[0247] Hybridization can also be used to identify functional homologs and / or to measure homology between two nucleic acid sequences. A nucleic acid sequence or portion encoding any of the proteins disclosed herein may be used as a hybridization probe according to standard hybridization techniques. Hybridization of a probe to DNA or RNA from a test source (e.g., mammalian cells) is an indicator of the presence of the relevant DNA or RNA in the test source. Hybridization conditions are known to those skilled in the art and can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY, 6.3.1-6.3.6, 1991. Mild hybridization conditions are defined as hybridization in 2× sodium chloride / sodium citrate (SSC) at 30°C, followed by washing in 1× SSC, 0.1% SDS at 50°C. Highly stringent conditions are defined as hybridization in 6 × sodium chloride / sodium citrate (SSC) at 45°C, followed by washing in 0.2 × SSC and 0.1% SDS at 65°C.

[0248] Sequence analysis for identifying functional homologs may also include BLAST, Reciprocal BLAST, or PSI-BLAST analysis of essential (non-redundant) databases using the relevant amino acid sequence as a reference sequence. In some cases, the amino acid sequence is inferred from the nucleotide sequence. Polypeptides in the database with more than 40% sequence identity are candidates for further evaluation of their suitability for use in SHC / HAC bioconversion reactions. Amino acid sequence similarity allows for conserved amino acid substitutions, such as substitution of one by one hydrophobic residue or substitution of another by one polar residue. Where desirable, manual examination of such candidates may be performed to narrow down the number of candidates to be further evaluated. Manual examination may be performed, for example, by selecting candidates that appear to have conserved functional domains.

[0249] Typically, polypeptides exhibiting at least approximately 30% amino acid sequence identity are useful for identifying conserved regions. Conserved regions of closely 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%, and 69% amino acid sequence identity. In some embodiments, the conserved region exhibits at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity. Sequence identity can be determined as shown above and below.

[0250] The resulting WT SHC and / or SHC / HAC derivatives are based on amino acid sequence numbers 1, 2, 3, or 4, or their variants, homologs, variants, derivatives, or fragments.

[0251] The SHC produced is 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%, 7 Based on amino acid sequences having 8%, 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.

[0252] In addition, the reference SHC produced is based on the amino acid sequence produced from E. coli.

[0253] "Identity Percentage (%)" is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to nucleotides in a DNA sequence, after the nucleotide sequence has been aligned and gaps introduced where necessary to achieve the maximum possible sequence identity percentage, without considering any conservative substitutions as part of sequence identity. Alignment for the purpose of determining the identity percentage of a nucleotide sequence can be achieved in various ways within the scope of the skills of the art, for example, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, which include any algorithm required to achieve the best possible alignment over the full length of the sequences to be compared.

[0254] The terms “polypeptide” and “protein” are used interchangeably herein and refer to amino acid chains linked by either peptide, regardless of length or post-translational modification.

[0255] As used herein, the term "derivative" includes, but is not limited to, "variant." The terms "derivative" and "variant" are interchangeable as used herein.

[0256] As used herein, the term “variant” should be understood as a polypeptide that differs from the polypeptide from which it is derived by only one or more changes in its amino acid sequence. The polypeptide from which the variant is derived is also known as the parent or reference polypeptide. Typically, variants are constructed artificially, preferably by means of genetic technology. Typically, the polypeptide from which the variant is derived is a wild-type protein or wild-type protein domain. However, variants available for use in this disclosure may also be derived from homologs, orthologs, or paralogs of the parent polypeptide, or from artificially constructed variants if the variant exhibits at least one of the biological activities of the parent polypeptide. Changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal shortening, or C-terminal shortening, or any combination thereof, and may occur at one or more sites.

[0257] In a preferred embodiment, variants available for use in this disclosure represent a total number of 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) changes in the amino acid sequence (i.e., exchanges, insertions, deletions, N-terminal shortenings, and / or C-terminal shortenings). Amino acid exchanges may be conservative and / or non-conservative. In a preferred embodiment, variants available for use in this disclosure differ from the protein or domain from which they originate from 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 only conservative amino acid changes. The variant may also or alternatively include amino acid deletions, which may be N-terminal shortening, C-terminal shortening, or internal deletion, or any combination thereof. Such variants including N-terminal shortening, C-terminal shortening, and / or internal deletion are referred to in the context of this application as “deletion variants” or “fragments.” The terms “deletion variant” and “fragments” are interchangeable herein. Deletion variants may exist naturally (e.g., splice variants) or they may be constructed artificially, preferably by genetic technology. Typically, the protein or protein domain from which the deletion variant is derived is a wild-type protein. However, the deletion variants of this disclosure may be derived from homologs, orthologs, or paralogs of the parent polypeptide, or from artificially constructed variants if 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 acid deletions at its N-terminus and / or C-terminus and / or internally, compared to the parent polypeptide.

[0258] As used herein, “variant” may be characterized by, alternatively or in addition, a certain degree of sequence identity thereto to the parent polypeptide from which it is derived. The WT / reference SHC / HAC variants or SHC / HAC derivatives of this disclosure are characterized by 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%, Sequences may have sequence identity of 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.

[0259] Expressions such as "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 "Sequence identity of 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%" is used herein in reference to polypeptide and polynucleotide sequence comparisons.

[0260] Polynucleotides belonging to any of the enzyme or protein families disclosed herein can be identified based on their similarity to the respective gene or protein. For example, identification may be based on sequence identity. In certain preferred embodiments, the Disclosure relates to nucleic acid molecules comprising (a) nucleic acid molecules encoding polypeptides of SEQ ID NOs. 5-163 (see Tables 14-17 and 4a provided herein), (b) nucleotide sequences of SEQ ID NOs. 6-168, 169, 170, 172, 174, and 176 (see Tables 14-17 and 4a provided herein), and (c) nucleic acid molecules comprising 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 SEQ ID NOs. 6-168, 169, 170, 172, 174, and 176 (see Tables 14-17 and 4a provided herein) and at least 3 0%, 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 Characterized by isolated nucleic acid molecules that are 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.

[0261] Preferably, the polypeptide and the reference polypeptide exhibit the indicated sequence identity over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. Preferably, the polynucleotide and the reference polynucleotide exhibit the indicated sequence identity over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides. If two sequences are compared and no reference sequence is specified to which the sequence identity percentage should be calculated, unless otherwise specifically indicated, the sequence identity should be calculated by reference to the longer of the two sequences to be compared. If a reference sequence is provided, unless otherwise specifically indicated, the sequence identity is determined based on the full length of the reference sequence indicated by SEQ ID NOs: 1, 2, 3, and / or 4.

[0262] For example, a peptide sequence consisting of 130 amino acids compared to the full length of a reference SHC with 631 amino acid residues may show a maximum sequence identity percentage of 20.6% (130 / 631 × 100), while a sequence with a length of 300 amino acids may show a maximum sequence identity percentage of 47.5% (300 / 631 × 100). The similarity between nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. Such alignment can be performed by several algorithms known in the art, preferably by the calculation algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), by hmmalign (HMMER package, http: / hmmer.wustl.edu / ), or for example, http: / www.ebi.ac.uk / Tools / clustalw / or http: / www.ebi.ac.uk / Tools / clustalw2 / index.html or by the CLUSTAL algorithm available at http: / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80), or by the GAP program (a computational algorithm of the University of Iowa) used for the sequence alignment against WT SHC provided in Table 18 herein, or by the computational algorithm of Myers and Miller (1989 - Cabios 4: 11-17) disclosed and used in the WT SHC sequence alignment of Table 19 provided herein.

[0263] The preferred parameters used are http: / www.ebi.ac.uk / Tools / clustalw / or http: / www.ebi.ac.uk / Tools / clustalw2 / index.html the default parameters set therein.

[0264] The grade of sequence identity (sequence match) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated in the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215, 403-410. The BLAST nucleotide search is performed by the BLASTN program, score = 100, word length = 12, to obtain a nucleotide sequence homologous to the nucleic acid encoding the relevant protein.

[0265] BLAST protein search is performed using the BLASTP program, score=50, word length=3 to obtain amino acid sequences homologous to the SrKO polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Sequence matching analysis may be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: 154-162) or Markov random fields. Where sequence identity percentages are referenced in this application, those percentages are calculated in relation to the full length of the longer sequence unless otherwise specifically indicated.

[0266] perception The bioconversion from homofarnesol to (-)-ambrox according to this disclosure produces (-)-ambrox as the major compound, but may also produce other compounds that may or may not impart a pleasant olfactory note to the bioconversion mixture, and thus may contribute positively or negatively to the perceptual properties of the (-)-ambrox final product. Therefore, perceptual analysis should be performed using well-established perceptual tests available to skilled professionals (e.g., perfumers) so that the test can help determine whether a chemically related target product is also an olfactory related final product to a reference product. As the perceptual analysis of Example 22 shows, the removal of one or more by-product compounds from (-)-ambrox may improve the odor of the remaining compound ((-)-ambrox), even if the removed compound itself is actually odorless. That is, an enhancement of the odor of (-)-ambrox was observed in the absence of compounds II, III, and IV. [Modes for carrying out the invention]

[0267] Aspects of the present invention 1. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture of stereoisomers containing EEH is enzymatically converted to (-)-ambrox or a mixture containing (-)-ambrox, the enzymatic conversion being carried out using an SHC / HAC enzyme under reaction conditions suitable for the production of (-)-ambrox, and the mixture of stereoisomers containing EEH essentially consists of homofarnesol isomers selected from the group consisting of [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E), and (3E,7Z)], respectively, also referred to as [EE:EZ], [EE:ZE], and [EE:EZ:ZE]. 2. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture of stereoisomers containing EEH is enzymatically converted to give (-)-ambrox or a mixture containing (-)-ambrox, and the enzymatic conversion using an SHC / HAC enzyme is carried out under reaction conditions suitable for the production of (-)-ambrox, and the reaction is carried out in the presence of a solubilizer, wherein Triton X-100 or taurodeoxycholic acid is not used in combination with the wild-type SHC / HAC enzyme. 3. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture of stereoisomers containing EEH is enzymatically converted to (-)-ambrox or a mixture containing (-)-ambrox, the enzymatic conversion being carried out using an SHC / HAC enzyme under reaction conditions suitable for the production of (-)-ambrox, the mixture of stereoisomers containing EEH essentially consists of homofarnesol isomers selected from the group consisting of [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E), and (3E,7Z)], respectively, and the reaction occurring in a three-phase system comprising an aqueous phase, a solid phase, and an oil phase. 4. A process according to paragraph 1, paragraph 2, or paragraph 3, wherein the process is an SHC / HAC enzyme polypeptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or an SH selected from Table 1, Table 5, Table 2, Table 6, Table 3, Table 7, Table 4, Table 8, or Table 13, Table 14, or from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 171, 173, 175, 177, and / or 178. The process is carried out using a C / HAC derivative or a sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 5. One of the processes described in paragraphs 1-4, wherein the process uses recombinant host cells that produce SHC / HAC enzymes. 6. A process according to paragraph 4 or paragraph 5, wherein the nucleotide sequence encoding the SHC / HAC enzyme is selected from the group consisting of SEQ ID NOs: 165, 166, 167, 168, 169, or SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and / or 170, 172, 174 and / or 176. 7. A process according to any one of paragraphs 1-6, in which the conversion from homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C and a pH in the range of approximately 4-8. 8. A process according to any one of paragraphs 1 to 7, wherein the conversion from homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for wild-type SHC / HAC or SHC / HAC derivative enzymes listed in Table 24 or Table 24a, preferably in a pH range of 5.0 to 6.2, and preferably at a temperature of 35°C. 9. A process according to any one of paragraphs 3 to 8, wherein the SDS / cell ratio is in the range of 10:1 to 20:1, preferably 16:1, when the biocatalyst to EEH ratio is approximately 2:1. 10. A process according to any one of paragraphs 3 to 9, wherein the weight ratio of biocatalyst to homofarnesol is in the range of about 0.5 to 2:1, preferably about 1:1 or 0.5:1. 11. A process that follows any one of paragraphs 3-10, wherein the cell proliferation and biotransformation reaction steps are carried out in the same reaction vessel. 12. A process according to paragraph 2, wherein the homofarnesol substrate comprises one or more homofarnesol stereoisomers. 13. A process according to paragraph 12, wherein the homofarnesol substrate contains or essentially consists of two homofarnesol stereoisomers. 14. A process according to paragraph 13, wherein the homofarnesol substrate contains or essentially consists of EE:EZ stereoisomers. 15. A process in which homo farnesol follows any one of paragraph 14, where homo farnesol is 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 The mixture contains or essentially consists of an EE:EZ stereoisomer mixture in a weight ratio selected from the group consisting of 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, and 60:40. 16. A process according to paragraph 15, wherein homofarnesol contains or essentially consists of a mixture of EE:EZ stereoisomers in a weight ratio selected from the group consisting of EE:EZ92:8, EE:EZ90:10, EE:EZ80:20, EE:EZ86:14, EE:EZ70:30, EE:EZ69:31, and EE:EZ66:34. 17. A process according to paragraph 15 or paragraph 16, wherein homofarnesol contains or essentially consists of an EE:EZ stereoisomer mixture in an 80:20 weight ratio. 18. A process according to any one of paragraphs 1 to 17, wherein (-)-ambrox is produced as a mixture with at least one of the by-products (II), (IV), and / or (III). 19. A process according to any one of paragraphs 1-18, wherein (-)-ambrox is isolated from the biotransformation reaction mixture using an organic solvent or steam extraction / distillation step or filtration. 20. A process according to any one of paragraphs 1-19, wherein (-)-ambrox is isolated from the solid phase of the biotransformation reaction mixture using an organic solvent or a steam extraction / distillation step. 21. A process according to paragraph 19 or paragraph 20, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent. 22. A process according to paragraph 21, wherein (-)-ambrox is isolated from the reaction mixture using ethanol or toluene. 23. A process according to any one of paragraphs 19-22, wherein (-)-ambrox is selectively crystallized using an organic solvent. 24. A process according to paragraph 23, wherein (-)-ambrox substantially does not contain by-products (II), (IV), and / or (III). 25. A process following any one of paragraphs 1 to 24, which produces (-)-ambrox in a concentration range of approximately 125 to 200 g / l. 26. (-)-Amblox that can be obtained by any one of the methods of paragraphs 1 to 25, wherein the (-)-Amblox has an odor threshold of about 0.1 to about 0.5 ng / l. 27. (-)-Amblocs of paragraph 26, in solid form, preferably amorphous or crystalline. 28. A process for making a product containing (-)-Amblox, comprising incorporating into the product one of the (-)-Amblox described in either paragraph 26 or paragraph 27. 29. The process described in paragraph 28, wherein the product is a fragrance product, cosmetic product, cleaning product, detergent product, or soap product. 30. A fragrance, cosmetic, or consumer care product comprising (-)-Amblox as defined in either paragraph 26 or paragraph 27. 31. A fragrance, cosmetic, or consumer care composition comprising (-)-Amblox of paragraph 26 or paragraph 27 and one or more additional components. 32. Use of (-)-Amblox as part of a fragrance, cosmetic, or consumer product, e.g., fabric care, toiletries, beauty, and / or cleaning product, as described in paragraph 26 or paragraph 27. 33. A process for expanding, enhancing, or imparting the fragrance of a fragrance composition, (a) A step of preparing a reaction mixture comprising (-)-ambrox as a mixture of one or more by-product compounds (II), (III), or (IV), [ka] (b) A step of extracting (-)-ambrox as a mixture with one or more of the by-product compounds (II), (III), or (IV), (c) A step of selectively crystallizing (-)-ambrox from the extract mixture, The process includes the step of mixing an aromatic extension or enhancement product, produced according to a process including the above, with the fragrance composition. (-)-Amblox is prepared by enzymatic conversion of (3E,7E)-homofarnesol (EEH) or a mixture of stereoisomers containing EEH, using SHC / HAC enzymes under reaction conditions suitable for the production of (-)-Amblox. A mixture of stereoisomers containing EEH essentially consists of homofarnesol isomers selected from the group consisting of [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E), and (3E,7Z)], respectively, also referred to as [EE:EZ], [EE:ZE], and [EE:EZ:ZE]. 34. The process described in paragraph 33, wherein the reaction occurs in a three-phase system comprising an aqueous phase, a solid phase, and an oil phase. 35. A process according to paragraph 33 or paragraph 34, wherein the process is an SHC / HAC enzyme polypeptide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or an SHC / HAC derivative selected from Table 1, Table 5, Table 2, Table 6, Table 3, Table 7, Table 4, Table 8, or Table 14, or selected from SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 171, 173, 175, and / or 177. or using sequences that have at least 30% identity, at least 40% identity, at least 50% identity, at least 60% identity, at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with sequence number 1, sequence number 2, sequence number 3, or sequence number 4. 36. Any one of the processes described in paragraphs 33-35, wherein the process uses recombinant host cells that produce SHC / HAC enzymes. 37. A process according to paragraph 35 or paragraph 36, wherein the nucleotide sequence encoding the SHC / HAC enzyme is selected from the group consisting of SEQ ID NOs: 165, 166, 167, 168, 169, or SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and / or 170, 172, 174, and / or 176. 38. A process according to any one of paragraphs 33-37, in which the conversion from homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C and a pH in the range of approximately 4-8. 39. A process according to any one of paragraphs 33-38, wherein the conversion of homofarnesol to (-)-ambrox occurs using one or more reaction conditions for wild-type SHC / HAC or SHC / HAC derivative enzymes listed in Table 24 or Table 24a, preferably at a pH range of 5.0 to 6.2 and preferably at a temperature of 35°C. 40. A process according to any one of paragraphs 34-39, wherein the SDS / cell ratio is in the range of 10:1 to 20:1, preferably 16:1, when the ratio of biocatalyst to EEH is approximately 2:1. 41. A process according to any one of paragraphs 34-40, wherein the weight ratio of biocatalyst to homofarnesol is in the range of about 0.5-2:1, preferably about 1:1 or 0.5:1. 42. A process that follows any one of paragraphs 34-41, wherein the cell proliferation and biotransformation reaction steps are carried out in the same reaction vessel. 43. A process that follows any one of paragraphs 33-42, in which homofarnesol is 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, 7 The mixture contains or essentially consists of an EE:EZ stereoisomer mixture in a weight ratio selected from the group consisting of 9:21, 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, and 60:40. 44. A process according to paragraph 43, wherein homofarnesol contains or essentially consists of a mixture of EE:EZ stereoisomers in a weight ratio selected from the group consisting of EE:EZ92:08, EE:EZ90:10, EE:EZ80:20, EE:EZ86:14, EE:EZ70:30, EE:EZ69:31, and EE:EZ66:34. 45. A process according to paragraph 43 or paragraph 44, wherein homofarnesol contains or essentially consists of an EE:EZ stereoisomer mixture in an 80:20 weight ratio. 46. ​​A process according to any one of paragraphs 33-45, wherein (-)-ambrox is produced as a mixture with at least one of the by-products (II), (IV), and / or (III). 47. A process according to any one of paragraphs 33-46, wherein (-)-ambrox is isolated from the biotransformation reaction mixture using an organic solvent or steam extraction / distillation step or filtration. 48. A process according to any one of paragraphs 33-47, wherein (-)-ambrox is isolated from the solid phase of the biotransformation reaction mixture using an organic solvent or a steam extraction / distillation step. 49. A process according to paragraph 47 or paragraph 48, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent. 50. A process according to paragraph 49, wherein (-)-ambrox is isolated from the reaction mixture using ethanol or toluene. 51. A process according to any one of paragraphs 47-49, wherein (-)-ambrox is selectively crystallized using an organic solvent. 52. A process according to paragraph 51, wherein (-)-ambrox substantially does not contain by-products (II), (IV), and / or (III). 53. A process following any one of paragraphs 33-52, which produces (-)-ambrox in a concentration range of approximately 125-200 g / l. 54. A process that follows any one of paragraphs 33-53, wherein (-)-Amblox has an odor threshold of approximately 0.1 to approximately 0.5 ng / l.

[0268] Additional Aspects of the Invention 1. A squalene-hopencyclase (SHC) / homofarnesol-ambrox cyclase (HAC) derivative comprising an amino acid sequence having 1 to 50 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO: 1. 2. An SHC / HAC derivative according to paragraph 1, wherein the SHC derivative comprises an amino acid sequence having 1 to 40 mutations, 1 to 30 mutations, 1 to 20 mutations, 1 to 10 mutations, or 1 to 6 mutations relative to SEQ ID NO: 1. 3. An SHC / HAC derivative according to paragraph 1 or paragraph 2, wherein the SHC / HAC derivative comprises an amino acid sequence having at least 40% identity, at least 50% identity, or at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 90% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with respect to SEQ ID NO: 1. 4. An SHC / HAC derivative according to paragraph 3, wherein the SHC variant contains an amino acid sequence having at least 95% identity with SEQ ID NO: 1. 5. An SHC / HAC derivative comprising 1 to 10 mutations independently selected from substitution, deletion, or insertion relative to SEQ ID NO: 1, wherein one or more mutations other than SHC active site mutations are located within domain 2 of the SHC enzyme (Figure 19 and / or 20). 6. An SHC / HAC derivative that follows any one of paragraphs 1 to 5, in which one or more mutations are selected from Table 1 for SEQ ID NO: 1, and if only one mutation is selected, it is not F601Y. 7. An SHC / HAC derivative according to paragraph 6, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations are selected from Table 1 or Table 5. 8. An SHC / HAC derivative of paragraph 2 having up to six mutations relative to SEQ ID NO: 1, and containing an amino acid sequence that includes at least one substitution F601Y or M132R in combination with at least one of F129L and / or I432T. 9. An SHC / HAC derivative of paragraph 7 having up to eight amino acid changes compared to SEQ ID NO: 1, and comprising an amino acid sequence having one or more amino acid changes at positions selected from the group consisting of positions 77, 129, 132, 192, 224, 432, 579, 601, and 605 compared to SEQ ID NO: 1, wherein the SHC / HAC derivative has increased HAC enzyme activity compared to SEQ ID NO: 1. 10. An SHC / HAC derivative according to paragraph 9, comprising one or more substitutions selected from the group consisting of T77A, F129L, M132R, I92V, A224V, I432T, Q579H, F601Y, and / or F605W for SEQ ID NO: 1. 11. An SHC / HAC derivative according to paragraph 10, comprising F601Y. 12. An SHC / HAC derivative according to paragraph 10, comprising F129L. 13. SHC / HAC derivatives according to paragraph 10, comprising F601Y and F129L. 14. SHC / HAC derivatives according to paragraph 10, comprising M132R and I432T. 15. An SHC / HAC derivative according to paragraph 14, further comprising the amino acid substitution A224V. 16. An SHC / HAC derivative according to paragraph 14, further comprising F601Y. 17. An SHC / HAC derivative according to paragraph 14, further comprising F129L. 18. An SHC / HAC derivative according to paragraph 17, further comprising F601Y. 19. An SHC / HAC derivative according to paragraph 11, further comprising Q579H. 20. SHC derivatives according to paragraph 10, comprising T77A, I92V, and F129L. 21. An SHC / HAC derivative that follows any one of the preceding paragraphs and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and / or 171. 22. An isolated nucleotide sequence encoding an SHC derivative that follows any one of paragraphs 1-21. 23. An isolated nucleotide sequence according to paragraph 22, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and / or 170. 24. A construct containing the nucleotide sequence of paragraph 22 or paragraph 23. 25. A construct that conforms to paragraph 24 and includes a promoter functionally ligated to a nucleotide sequence of paragraph 22 or 23. 26. The construct of paragraph 25, wherein the promoter is an inductive or constitutive promoter. 27. A vector containing a construct that follows one of paragraphs 24-26. 28. The vector in paragraph 27 is a plasmid. 29. A vector that conforms to paragraph 28 and can be directed to expression by a host cell selected from prokaryotes, yeasts, plants, and insect host cells. 30. A vector that conforms to any one construct from paragraphs 24-26 or any one from paragraphs 27-29, wherein the construct or vector is capable of integration into the genome of a host cell selected from prokaryotes, yeasts, plants, and insect host cells. 31. Recombinant host cells comprising a nucleotide sequence following paragraph 22 or 23, a construct following any one of paragraphs 24-26 or 30, or a vector following any one of paragraphs 27-30. 32. Recombinant host cells according to paragraph 31, wherein the host cells are selected from a group of prokaryotic host cells consisting of bacteria of the genera Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. 33. The recombinant host cell described in paragraph 32, wherein the host cell is an E. coli (E. coli) host cell. 34. Recombinant host cells as described in paragraph 33, wherein the host cells overexpress genes encoding SHC / HAC derivatives. 35. A method for preparing an SHC / HAC derivative according to any one of paragraphs 1 to 21, comprising the step of culturing one or more recombinant host cells according to any one of paragraphs 31 to 34 under conditions that allow for the production of an SHC / HAC derivative enzyme. 36. The method of paragraph 35, wherein cell culture occurs under conditions favorable for biocatalyst production. 37. A method for preparing (-)-ambax, comprising converting homofarnesol to (-)-ambax by using recombinant host cells according to any one of paragraphs 31-34, or by using recombinant host cells comprising SEQ ID NO: 169 or SEQ ID NO: 165 encoding WT SHC / HAC, wherein, if WT SHC / HAC is used, the bioconversion from homofarnesol to (-)-ambax is carried out by a solubilizer other than Triton X-100 or taurodeoxycholic acid. 38. A method according to paragraph 37, wherein the conversion from homofarnesol to (-)-ambrox is performed under bioconversion reaction conditions suitable for WT SHC / HAC or SHC / HAC derivative enzymes. 39. A method according to paragraph 37 or 38, wherein the conversion from homofarnesol to (-)-ambrox occurs at a pH, temperature, and solubilizer concentration suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 40. A method according to paragraph 39, wherein the conversion from homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C and a pH in the range of approximately 4 to 8, in the presence of a solubilizer other than Triton X-100 or taurodeoxycholic acid for WT SHC / HAC enzymes. 41. A method according to any one of paragraphs 37-40, wherein the conversion from homofarnesol to (-)-ambrox occurs using one or more reaction conditions for WT SHC / HAC or SHC / HAC derivative enzymes listed in Table 24 or Table 24a. 42. Any one of the methods described in paragraphs 37 to 41, wherein the weight ratio of the biocatalyst to homofarnesol is in the range of about 0.5:1 to 2:1, preferably about 1:1 or 0.5:1. 43. A method according to any one of paragraphs 37-42, wherein the cell proliferation and biotransformation reaction steps are carried out in the same reaction vessel. 44. A method according to any one of paragraphs 37-43, wherein the homofarnesol substrate comprises one or more homofarnesol stereoisomers. 45. The method of paragraph 44, wherein the homofarnesol substrate comprises two homofarnesol stereoisomers. 46. ​​The method of paragraph 45, wherein the homofarnesol substrate contains the EE:EZ stereoisomer. 47. A method according to any one of paragraphs 44-46, wherein homofarnesol contains an EE:EZ stereoisomer mixture in a weight ratio selected from the group consisting of 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, and 70:30. 48. The method of paragraph 47, wherein homofarnesol comprises a mixture of EE:EZ stereoisomers in a weight ratio selected from the group consisting of EE:EZ90:10, EE:EZ80:20, EE:EZ86:14, EE:EZ70:30, EE:EZ69:31, and EE:EZ66:34. 49. The method of paragraph 35 or 36, wherein homofarnesol comprises an EE:EZ stereoisomer mixture in a weight ratio of 80:20. 50. One of the methods described in paragraphs 37–49, wherein (-)-ambrox is produced as a mixture with one or more of the by-products (II), (IV), and / or (III). 51. One of the methods described in paragraphs 37-50, wherein (-)-ambrox is isolated from the biotransformation reaction mixture using an organic solvent or a vapor extraction / distillation step, or (-)-ambrox crystals are isolated directly from the biotransformation reaction mixture by means of filtration. 52. A method according to paragraph 51, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent. 53. The method of paragraph 52, wherein (-)-ambrox is selectively crystallized using an organic solvent. 54. The method of paragraph 52 or 53, wherein (-)-ambrox substantially does not contain by-products (II), (IV), and / or (III). 55. (-)-Amblocs that can be obtained by any one of the methods described in paragraphs 51-54. 56. (-)-Amblocs of paragraph 55, in solid form, preferably amorphous or crystalline. 57. A method for making a product containing (-)-Amblox, comprising incorporating (-)-Amblox as defined in paragraph 55 or 56 into a product, preferably a fragrance product, a cosmetic product, a cleaning product, a detergent product, or a soap product. 58. A fragrance, cosmetic, or consumer care product comprising (-)-Amblox as defined in paragraph 55 or 56. 59. A fragrance, cosmetic, or consumer care composition comprising (-)-Amblox of paragraph 55 or 56 and one or more additional components. 60. Use of (-)-Amblocs as part of a fragrance, cosmetic, or consumer product, e.g., fabric care, toiletries, beauty, and / or cleaning product, as described in paragraph 55 or 56. 61. Use of an SHC / HAC derivative enzyme following any one of paragraphs 1-21, a nucleotide sequence following paragraph 22 or 23, a construct following any one of paragraphs 24-26 or 30, a vector following any one of paragraphs 27-30, or a recombinant host cell following any one of paragraphs 31-34, or a recombinant host cell expressing WT SHC / HAC, for the bioconversion from homofarnesol to (-)-ambrox, wherein the WT SHC / HAC enzyme is used in the bioconversion reaction with a solubilizer other than Triton X-100. 62. A method for preparing (-)-ambrox or a stereoisomer mixture of (-)-ambrox, wherein (3E,7E)-homofarnesol or a stereoisomer mixture of (3E,7E)-homofarnesol is enzymatically converted to give (-)-ambrox or a stereoisomer mixture of (-)-ambrox, and the enzymatic conversion using an SHC / HAC enzyme is carried out under reaction conditions suitable for the production of (-)-ambrox, and the reaction is carried out in the presence of a solubilizer, in which case Triton X-100 is not used in combination with the WT SHC / HAC enzyme. 63. A process according to paragraph 62, wherein the process is carried out using an SHC / HAC enzyme selected from the group consisting of AacSHC (SEQ ID NO: 1), ZmoSHC1 (SEQ ID NO: 2), ZmoSHC2 (SEQ ID NO: 3), and BjpSHC (SEQ ID NO: 4), an SHC / HAC derivative selected from Tables 1, 5, 2, 6, 3, 7, 4, and / or 8, or a sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with respect to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and / or SEQ ID NO: 4. 64. A process according to paragraph 63, wherein the conversion from homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C, at a pH in the range of 4 to 8, and in the presence of a solubilizer other than Triton X-100 for WT SHC. 65. The process follows paragraph 64, using the reaction conditions for WT SHC / HAC or their respective SHC / HAC derivative enzymes as described in Table 24 or Table 24a. 66. A process according to any one of paragraphs 62-65, wherein the process includes (a) culturing one or more recombinant host cells expressing WT SHC or SHC derivative enzymes under conditions that allow for the expression of WT SHC or SHC / HAC derivative polypeptides, prior to the conversion of E,E-homofarnesol to (-)-ambrox. 67. A process according to paragraph 66, wherein the culture step and the subsequent conversion step occur in the same reaction vessel under different reaction conditions. 68. A process according to paragraph 67, wherein the culture step is in a pH range of approximately 6 to approximately 7, and the homofarnesol to (-)-ambrox step is in a pH range of approximately 4.8 to 5.5. 69. A process according to any one of paragraphs 62-68, wherein the homofarnesol substrate contains the EE:EZ stereoisomer. 70. A process according to paragraph 69, wherein homofarnesol contains a mixture of EE:EZ stereoisomers in a weight ratio selected from the group consisting of EE:EZ90:10, EE:EZ80:20, EE:EZ86:14, EE:EZ70:30, EE:EZ69:31, and EE:EZ66:34. 71. The process of paragraph 70, wherein homofarnesol contains EE:EZ in a weight ratio of 80:20. 72. One of the methods described in paragraphs 62-71, wherein (-)-ambrox is produced as a mixture with one or more of the by-products (II), (IV), and / or (III). 73. (-)-Ambloc is isolated from the reaction mixture by any one of the methods described in paragraphs 62-72, using an organic solvent or steam extraction / distillation step or filtration. 74. A method according to paragraph 73, in which (-)-ambrox is isolated from the reaction mixture using an organic solvent. 75. The method of paragraph 74, wherein (-)-ambrox is selectively crystallized using an organic solvent. 76. The method of paragraph 74 or 75, wherein (-)-ambrox is substantially free of by-products (II), (IV), and / or (III). 77. (-)-Amblocs can be obtained by any one of the methods described in paragraphs 72-76. 78. (-)-Amblocs of paragraph 26, in solid form, preferably amorphous or crystalline. 79. A method for making a product, comprising incorporating the (-)-Unbrox of paragraph 77 or 78 into the product. 80. The method of paragraph 79, wherein the product is a fragrance product, cosmetic product, cleaning product, detergent product, or soap product. 81. A fragrance, cosmetic, or consumer care product containing (-)-Amblox as defined in paragraph 77 or 78. 82. A fragrance, cosmetic, or consumer care composition comprising (-)-Amblox of paragraph 77 or 78 and additional components. 83. Use of (-)-Amblox as part of a fragrance or cosmetic consumer care product, as described in paragraph 77 or 78.

[0269] Additional aspects of the present invention (ZmoSHC1) 1. A squalene-hopencyclase (SHC) / homofarnesol-ambrox cyclase (HAC) derivative comprising an amino acid sequence having 1 to 50 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO: 2. 2. An SHC / HAC derivative according to paragraph 1, wherein the SHC derivative comprises an amino acid sequence having 1 to 40 mutations, 1 to 30 mutations, 1 to 20 mutations, 1 to 10 mutations, or 1 to 6 mutations relative to SEQ ID NO: 2. 3. An SHC / HAC derivative according to paragraph 1 or paragraph 2, wherein the SHC / HAC derivative comprises an amino acid sequence having at least 40% identity, at least 50% identity, or at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 90% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with respect to SEQ ID NO: 2. 4. An SHC / HAC derivative according to paragraph 3, wherein the SHC variant contains an amino acid sequence having at least 95% identity with SEQ ID NO: 2. 5. An SHC / HAC derivative comprising 1 to 10 mutations independently selected from substitution, deletion, or insertion relative to SEQ ID NO: 2, wherein one or more mutations other than SHC active site mutations are located within domain 2 of the SHC enzyme (Figure 19 and / or 20). 6. An SHC / HAC derivative that follows any one of paragraphs 1-5, in which one or more mutations are selected from Table 2 for SEQ ID NO: 2, and if only one mutation is selected, it is not F668Y. 7. An SHC / HAC derivative from paragraph 6, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations are selected from Table 2 and / or Table 6. 8. An SHC / HAC derivative of paragraph 2 having up to six mutations relative to SEQ ID NO: 2, and containing an amino acid sequence that includes at least one of F182L and / or I498T, with substitution F668Y or Y185R. 9. An SHC / HAC derivative of paragraph 7 having up to eight amino acid changes compared to SEQ ID NO: 2, and comprising an amino acid sequence that includes one or more amino acid changes at positions selected from the group consisting of positions 129, 145, 182, 185, 282, 498, 647, and 668 compared to SEQ ID NO: 2, wherein the SHC / HAC derivative has increased HAC enzyme activity compared to SEQ ID NO: 2. 10. An SHC / HAC derivative according to paragraph 9, comprising one or more substitutions selected from the group consisting of S129A, V145V, F182L, Y185R, G282V, I498T, H646H, and F668Y for SEQ ID NO: 2. 11. An SHC / HAC derivative according to paragraph 10, comprising F668Y. 12. An SHC / HAC derivative according to paragraph 10, comprising F182L. 13. SHC / HAC derivatives according to paragraph 10, comprising F668Y and F182L. 14. SHC / HAC derivatives according to paragraph 10, comprising Y185R and I498T. 15. An SHC / HAC derivative according to paragraph 14, further comprising G282V. 16. An SHC / HAC derivative according to paragraph 14, further comprising F668Y. 17. An SHC / HAC derivative according to paragraph 14, further comprising F182L. 18. An SHC / HAC derivative according to paragraph 17, further comprising F668Y. 19. An SHC / HAC derivative according to paragraph 11, further comprising H646H. 20. SHC derivatives according to paragraph 10, comprising S129A, V145V, and F182L. 21. An SHC / HAC derivative that follows any one of the preceding paragraphs and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and / or 75. 22. An isolated nucleotide sequence encoding an SHC derivative that follows any one of paragraphs 1-21. 23. An isolated nucleotide sequence according to paragraph 22, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, and / or 76. 24. A construct containing the nucleotide sequence of paragraph 22 or paragraph 23. 25. A construct that conforms to paragraph 24 and includes a promoter functionally ligated to a nucleotide sequence of paragraph 22 or 23. 26. The construct of paragraph 25, wherein the promoter is an inductive or constitutive promoter. 27. A vector containing a construct that follows one of paragraphs 24-26. 28. The vector in paragraph 27 is a plasmid. 29. A vector that conforms to paragraph 28 and can be directed to expression by a host cell selected from prokaryotes, yeasts, plants, and insect host cells. 30. A vector that conforms to any one construct from paragraphs 24-26 or any one from paragraphs 27-29, wherein the construct or vector is capable of integration into the genome of a host cell selected from prokaryotes, yeasts, plants, and insect host cells. 31. Recombinant host cells comprising a nucleotide sequence following paragraph 22 or 23, a construct following any one of paragraphs 24-26 or 30, or a vector following any one of paragraphs 27-30. 32. Recombinant host cells according to paragraph 31, wherein the host cells are selected from a group of prokaryotic host cells consisting of bacteria of the genera Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. 33. The recombinant host cell described in paragraph 32, wherein the host cell is an E. coli host cell. 34. Recombinant host cells as described in paragraph 33, wherein the host cells overexpress genes encoding SHC / HAC derivatives. 35. A method for preparing an SHC / HAC derivative according to any one of paragraphs 1 to 21, comprising the step of culturing one or more recombinant host cells according to any one of paragraphs 31 to 34 under conditions that allow for the production of an SHC / HAC derivative enzyme. 36. The method of paragraph 35, wherein cell culture occurs under conditions favorable for biocatalyst production. 37. A method for preparing (-)-ambax, comprising converting homofarnesol to (-)-ambax by using recombinant host cells according to any one of paragraphs 31-34, or by using recombinant host cells containing sequence number 166 encoding WT SHC / HAC, wherein, if WT SHC / HAC is used, the bioconversion from homofarnesol to (-)-ambax is carried out by a solubilizer other than Triton X-100. 38. A method according to paragraph 37, wherein the conversion from homofarnesol to (-)-ambrox is performed under bioconversion reaction conditions suitable for WT SHC / HAC or SHC / HAC derivative enzymes. 39. A method according to paragraph 37 or 38, wherein the conversion from homofarnesol to (-)-ambrox occurs at a pH, temperature, and solubilizer concentration suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 40. A method according to paragraph 39, wherein the conversion from homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C and a pH in the range of approximately 4 to 8, in the presence of a solubilizer other than Triton X-100 for WT SHC / HAC enzymes. 41. A method according to any one of paragraphs 37-40, wherein the conversion from homofarnesol to (-)-ambrox occurs using one or more reaction conditions for WT SHC / HAC or SHC / HAC derivative enzymes listed in Table 24 or Table 24a. 42. Any one of the methods described in paragraphs 37 to 41, wherein the weight ratio of the biocatalyst to homofarnesol is in the range of about 0.5:1 to 2:1, preferably about 1:1 or 0.5:1. 43. A method according to any one of paragraphs 37-42, wherein the cell proliferation and biotransformation reaction steps are carried out in the same reaction vessel. 44. A method according to any one of paragraphs 37-43, wherein the homofarnesol substrate comprises one or more homofarnesol stereoisomers. 45. The method of paragraph 44, wherein the homofarnesol substrate comprises two homofarnesol stereoisomers. 46. ​​The method of paragraph 45, wherein the homofarnesol substrate contains the EE:EZ stereoisomer. 47. A method according to any one of paragraphs 44-46, wherein homofarnesol contains an EE:EZ stereoisomer mixture in a weight ratio selected from the group consisting of 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, and 70:30. 48. The method of paragraph 47, wherein homofarnesol comprises a mixture of EE:EZ stereoisomers in a weight ratio selected from the group consisting of EE:EZ90:10, EE:EZ80:20, EE:EZ86:14, EE:EZ70:30, EE:EZ69:31, and EE:EZ66:34. 49. The method of paragraph 35 or 36, wherein homofarnesol comprises an EE:EZ stereoisomer mixture in a weight ratio of 80:20. 50. One of the methods described in paragraphs 37–49, wherein (-)-ambrox is produced as a mixture with one or more of the by-products (II), (IV), and / or (III). 51. (-)-Amblocx is isolated from the biotransformation reaction mixture by any one of the methods described in paragraphs 37-50, using an organic solvent or steam extraction / distillation step or filtration. 52. A method according to paragraph 51, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent. 53. The method of paragraph 52, wherein (-)-ambrox is selectively crystallized from (-)-ambrox using an organic solvent. 54. The method of paragraph 52 or 53, wherein (-)-ambrox substantially does not contain by-products (II), (IV), and / or (III). 55. (-)-Amblocs that can be obtained by any one of the methods described in paragraphs 51-54. 56. (-)-Amblocs of paragraph 55, in solid form, preferably amorphous or crystalline. 57. A method for making a product containing (-)-Amblox, comprising incorporating (-)-Amblox as defined in paragraph 55 or 56 into a product, preferably a fragrance product, a cosmetic product, a cleaning product, a detergent product, or a soap product. 58. A fragrance, cosmetic, or consumer care product comprising (-)-Amblox as defined in paragraph 55 or 56. 59. A fragrance, cosmetic, or consumer care composition comprising (-)-Amblox of paragraph 55 or 56 and one or more additional components. 60. Use of (-)-Amblocs as part of a fragrance, cosmetic, or consumer product, e.g., fabric care, toiletries, beauty, and / or cleaning product, as described in paragraph 55 or 56. 61. Use of an SHC / HAC derivative enzyme following any one of paragraphs 1-21, a nucleotide sequence following paragraph 22 or 23, a construct following any one of paragraphs 24-26 or 30, a vector following any one of paragraphs 27-30, or a recombinant host cell following any one of paragraphs 31-34, or a recombinant host cell expressing WT SHC / HAC, for the bioconversion from homofarnesol to (-)-ambrox, wherein the WT SHC / HAC enzyme is used in the bioconversion reaction with a solubilizer other than Triton X-100.

[0270] Further aspects of the present invention (ZmoSHC2) 1. A squalene-hopencyclase (SHC) / homofarnesol-ambrox cyclase (HAC) derivative comprising an amino acid sequence having 1 to 50 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO: 3. 2. An SHC / HAC derivative according to paragraph 1, wherein the SHC derivative comprises an amino acid sequence having 1 to 40 mutations, 1 to 30 mutations, 1 to 20 mutations, 1 to 10 mutations, or 1 to 6 mutations relative to SEQ ID NO: 3. 3. An SHC / HAC derivative according to paragraph 1 or paragraph 2, wherein the SHC / HAC derivative comprises an amino acid sequence having at least 40% identity, at least 50% identity, or at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 90% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with respect to SEQ ID NO: 3. 4. An SHC / HAC derivative according to paragraph 3, wherein the SHC variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 3. 5. An SHC / HAC derivative comprising 1 to 10 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO: 3, wherein one or more mutations other than SHC active site mutations are located within domain 2 of the SHC enzyme (Figure 19 and / or 20). 6. An SHC / HAC derivative that follows any one of paragraphs 1-5, in which one or more mutations are selected from Table 3 for SEQ ID NO: 3, and in which case only one mutation is selected, is not F620Y. 7. An SHC / HAC derivative from paragraph 6, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations are selected from Table 3 and / or Table 7. 8. An SHC / HAC derivative of paragraph 2 having up to six mutations relative to SEQ ID NO: 3, and containing an amino acid sequence that includes at least one of F137L and / or I450T, with substitution F620Y or I140R. 9. An SHC / HAC derivative of paragraph 7 having up to eight amino acid changes compared to SEQ ID NO: 3, and comprising an amino acid sequence having one or more amino acid changes at positions selected from the group consisting of positions 85, 100, 137, 140, 233, 450, 598, and 620 compared to SEQ ID NO: 3, wherein the SHC / HAC derivative has increased HAC enzyme activity compared to SEQ ID NO: 3. 10. An SHC / HAC derivative according to paragraph 9, comprising one or more substitutions selected from the group consisting of G85A, V100V, F137L, I140R, V233V, I450T, N598H, and F620Y for SEQ ID NO: 3. 11. An SHC / HAC derivative according to paragraph 10, comprising F620Y. 12. An SHC / HAC derivative according to paragraph 10, comprising F137L. 13. SHC / HAC derivatives according to paragraph 10, comprising F620Y and F137L. 14. SHC / HAC derivatives according to paragraph 10, comprising I140R and I450T. 15. An SHC / HAC derivative according to paragraph 14, further comprising V233V. 16. An SHC / HAC derivative according to paragraph 14, further comprising F620Y. 17. An SHC / HAC derivative according to paragraph 14, further comprising F137L. 18. An SHC / HAC derivative according to paragraph 17, further comprising F620Y. 19. An SHC / HAC derivative according to paragraph 11, further comprising N598H. 20. SHC derivatives according to paragraph 10, comprising G85A, V100V, and F137L. 21. An SHC / HAC derivative that follows any one of the preceding paragraphs and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, and / or 111. 22. An isolated nucleotide sequence encoding an SHC derivative that follows any one of paragraphs 1 to 21. 23. An isolated nucleotide sequence according to paragraph 22, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, and / or 112. 24. A construct containing the nucleotide sequence of paragraph 22 or paragraph 23. 25. A construct that conforms to paragraph 24 and includes a promoter functionally ligated to a nucleotide sequence of paragraph 22 or 23. 26. The construct of paragraph 25, wherein the promoter is an inductive or constitutive promoter. 27. A vector containing a construct that follows one of paragraphs 24-26. 28. The vector in paragraph 27 is a plasmid. 29. A vector that conforms to paragraph 28 and can be directed to expression in host cells selected from prokaryotes, yeasts, plants, and insect host cells. 30. A vector that conforms to any one construct from paragraphs 24-26 or any one from paragraphs 27-29, wherein the construct or vector is capable of integration into the genome of a host cell selected from prokaryotes, yeasts, plants, and insect host cells. 31. Recombinant host cells comprising a nucleotide sequence following paragraph 22 or 23, a construct following any one of paragraphs 24-26 or 30, or a vector following any one of paragraphs 27-30. 32. Recombinant host cells according to paragraph 31, wherein the host cells are selected from a group of prokaryotic host cells consisting of bacteria of the genera Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. 33. The recombinant host cell described in paragraph 32, wherein the host cell is an E. coli host cell. 34. Recombinant host cells as described in paragraph 33, wherein the host cells overexpress genes encoding SHC / HAC derivatives. 35. A method for preparing an SHC / HAC derivative according to any one of paragraphs 1 to 21, comprising the step of (a) culturing one or more recombinant host cells according to any one of paragraphs 31 to 34 under conditions that allow for the production of an SHC / HAC derivative enzyme. 36. The method of paragraph 35, wherein cell culture occurs under conditions favorable for biocatalyst production. 37. A method for preparing (-)-ambax, comprising converting homofarnesol to (-)-ambax by using recombinant host cells according to any one of paragraphs 31-34, or by using recombinant host cells containing sequence number 167 encoding WT SHC / HAC, wherein, if WT SHC / HAC is used, the bioconversion from homofarnesol to (-)-ambax is carried out by a solubilizer other than Triton X-100. 38. A method according to paragraph 37, wherein the conversion from homofarnesol to (-)-ambrox is performed under bioconversion reaction conditions suitable for WT SHC / HAC or SHC / HAC derivative enzymes. 39. A method according to paragraph 37 or 38, wherein the conversion from homofarnesol to (-)-ambrox occurs at a pH, temperature, and solubilizer concentration suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 40. The method described in paragraph 39, wherein the conversion from homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C, at a pH in the range of approximately 4 to 8, in the presence of a solubilizer other than Triton X-100 for WT SHC / HAC enzymes. 41. A method according to any one of paragraphs 37-40, wherein the conversion from homofarnesol to (-)-ambrox occurs using one or more reaction conditions for WT SHC / HAC or SHC / HAC derivative enzymes listed in Table 24 or Table 24a. 42. Any one of the methods described in paragraphs 37 to 41, wherein the weight ratio of the biocatalyst to homofarnesol is in the range of about 0.5:1 to 2:1, preferably about 1:1 or 0.5:1. 43. A method according to any one of paragraphs 37-42, wherein the cell proliferation and biotransformation reaction steps are carried out in the same reaction vessel. 44. A method according to any one of paragraphs 37-43, wherein the homofarnesol substrate comprises one or more homofarnesol stereoisomers. 45. The method of paragraph 44, wherein the homofarnesol substrate comprises two homofarnesol stereoisomers. 46. ​​The method of paragraph 45, wherein the homofarnesol substrate contains the EE:EZ stereoisomer. 47. A method according to any one of paragraphs 44-46, wherein homofarnesol contains an EE:EZ stereoisomer mixture in a weight ratio selected from the group consisting of 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, and 70:30. 48. The method of paragraph 47, wherein homofarnesol comprises a mixture of EE:EZ stereoisomers in a weight ratio selected from the group consisting of EE:EZ90:10, EE:EZ80:20, EE:EZ86:14, EE:EZ70:30, EE:EZ69:31, and EE:EZ66:34. 49. The method of paragraph 35 or 36, wherein homofarnesol comprises an EE:EZ stereoisomer mixture in a weight ratio of 80:20. 50. One of the methods described in paragraphs 37–49, wherein (-)-ambrox is produced as a mixture with one or more of the by-products (II), (IV), and / or (III). 51. (-)-Amblocx is isolated from the biotransformation reaction mixture by any one of the methods described in paragraphs 37-50, using an organic solvent or steam extraction / distillation step or filtration. 52. A method according to paragraph 51, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent. 53. The method of paragraph 52, wherein (-)-ambrox is selectively crystallized from (-)-ambrox using an organic solvent. 54. The method of paragraph 52 or 53, wherein (-)-ambrox substantially does not contain by-products (II), (IV), and / or (III). 55. (-)-Amblocs that can be obtained by any one of the methods described in paragraphs 51-54. 56. (-)-Amblocs of paragraph 55, in solid form, preferably amorphous or crystalline. 57. A method for making a product containing (-)-Amblox, comprising incorporating (-)-Amblox as defined in paragraph 55 or 56 into a product, preferably a fragrance product, a cosmetic product, a cleaning product, a detergent product, or a soap product. 58. A fragrance, cosmetic, or consumer care product comprising (-)-Amblox as defined in paragraph 55 or 56. 59. A fragrance, cosmetic, or consumer care composition comprising (-)-Amblox of paragraph 55 or 56 and one or more additional components. 60. Use of (-)-Amblocs as part of a fragrance, cosmetic, or consumer product, e.g., fabric care, toiletries, beauty, and / or cleaning product, as described in paragraph 55 or 56. 61. Use of an SHC / HAC derivative enzyme following any one of paragraphs 1-21, a nucleotide sequence following paragraph 22 or 23, a construct following any one of paragraphs 24-26 or 30, a vector following any one of paragraphs 27-30, or a recombinant host cell following any one of paragraphs 31-34, or a recombinant host cell expressing WT SHC / HAC, for the bioconversion from homofarnesol to (-)-ambrox, wherein the WT SHC / HAC enzyme is used in the bioconversion reaction with a solubilizer other than Triton X-100.

[0271] Additional Aspects of the Invention (BJpSHC) 1. A squalene-hopencyclase (SHC) / homofarnesol-ambrox cyclase (HAC) derivative comprising an amino acid sequence having 1 to 50 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO: 4. 2. An SHC / HAC derivative according to paragraph 1, wherein the SHC derivative comprises an amino acid sequence having 1 to 40 mutations, 1 to 30 mutations, 1 to 20 mutations, 1 to 10 mutations, or 1 to 6 mutations relative to SEQ ID NO: 4. 3. An SHC / HAC derivative according to paragraph 1 or paragraph 2, wherein the SHC / HAC derivative comprises an amino acid sequence having at least 40% identity, at least 50% identity, or at least 60% identity, or at least 70% identity, or at least 80% identity, or at least 90% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with respect to SEQ ID NO: 4. 4. An SHC / HAC derivative according to paragraph 3, wherein the SHC variant comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 4. 5. An SHC / HAC derivative comprising 1 to 10 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO: 4, wherein one or more mutations other than SHC active site mutations are located within domain 2 of the SHC enzyme (Figure 19 and / or 20). 6. An SHC / HAC derivative that follows any one of paragraphs 1-5, in which one or more mutations are selected from Table 4 for SEQ ID NO: 4, and in which case only one mutation is selected, is not F628Y. 7. An SHC / HAC derivative from paragraph 6, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations are selected from Table 4 and / or Table 8. 8. An SHC / HAC derivative of paragraph 2 having up to six mutations relative to SEQ ID NO: 4, and containing an amino acid sequence that includes at least one of F137L and / or I450T, with substitution F628Y or I140R. 9. An SHC / HAC derivative of paragraph 7 having up to eight amino acid changes relative to SEQ ID NO: 4, and comprising an amino acid sequence having one or more amino acid changes at positions selected from the group consisting of positions 88, 104, 141, 144, 241, 459, 607, and 628 relative to SEQ ID NO: 4, wherein the SHC / HAC derivative has increased HAC enzyme activity relative to SEQ ID NO: 4. 10. An SHC / HAC derivative according to paragraph 9, comprising one or more substitutions selected from the group consisting of A88A, V104V, F141L, Y144R, V241V, I459T, M607H, and F628Y for SEQ ID NO: 4. 11. An SHC / HAC derivative according to paragraph 10, comprising F628Y. 12. An SHC / HAC derivative according to paragraph 10, comprising F141L. 13. SHC / HAC derivatives according to paragraph 10, comprising F628Y and F141L. 14. SHC / HAC derivatives according to paragraph 10, comprising Y144R and I459T. 15. An SHC / HAC derivative according to paragraph 14, further comprising V241V. 16. An SHC / HAC derivative according to paragraph 14, further comprising F628Y. 17. An SHC / HAC derivative according to paragraph 14, further comprising F141L. 18. An SHC / HAC derivative according to paragraph 17, further comprising F628Y. 19. An SHC / HAC derivative according to paragraph 11, further comprising M607H. 20. SHC derivatives according to paragraph 10, comprising S129A, V145V, and F182L. 21. An SHC / HAC derivative that follows any one of the preceding paragraphs and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, and / or 147. 22. An isolated nucleotide sequence encoding an SHC derivative that follows any one of paragraphs 1-21. 23. An isolated nucleotide sequence according to paragraph 22, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, and / or 148. 24. A construct containing the nucleotide sequence of paragraph 22 or paragraph 23. 25. A construct that conforms to paragraph 24 and includes a promoter functionally ligated to a nucleotide sequence of paragraph 22 or 23. 26. The construct of paragraph 25, wherein the promoter is an inductive or constitutive promoter. 27. A vector containing a construct that follows one of paragraphs 24-26. 28. The vector in paragraph 27 is a plasmid. 29. A vector that conforms to paragraph 28 and can be directed to expression by a host cell selected from prokaryotes, yeasts, plants, and insect host cells. 30. A vector that conforms to any one construct from paragraphs 24-26 or any one from paragraphs 27-29, wherein the construct or vector is capable of integration into the genome of a host cell selected from prokaryotes, yeasts, plants, and insect host cells. 31. Recombinant host cells comprising a nucleotide sequence following paragraph 22 or 23, a construct following any one of paragraphs 24-26 or 30, or a vector following any one of paragraphs 27-30. 32. Recombinant host cells according to paragraph 31, wherein the host cells are selected from a group of prokaryotic host cells consisting of bacteria of the genera Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. 33. The recombinant host cell described in paragraph 32, wherein the host cell is an E. coli host cell. 34. Recombinant host cells as described in paragraph 33, wherein the host cells overexpress genes encoding SHC / HAC derivatives. 35. A method for preparing an SHC / HAC derivative according to any one of paragraphs 1 to 21, comprising the step of (a) culturing one or more recombinant host cells according to any one of paragraphs 31 to 34 under conditions that allow for the production of an SHC / HAC derivative enzyme. 36. The method of paragraph 35, wherein cell culture occurs under conditions favorable for biocatalyst production. 37. A method for preparing (-)-ambax, comprising converting homofarnesol to (-)-ambax by using recombinant host cells according to any one of paragraphs 31-34, or by using recombinant host cells containing sequence number 168 encoding WT SHC / HAC, wherein, if WT SHC / HAC is used, the bioconversion from homofarnesol to (-)-ambax is carried out by a solubilizer other than Triton X-100. 38. A method according to paragraph 37, wherein the conversion from homofarnesol to (-)-ambrox is performed under bioconversion reaction conditions suitable for WT SHC / HAC or SHC / HAC derivative enzymes. 39. A method according to paragraph 37 or 38, wherein the conversion from homofarnesol to (-)-ambrox occurs at a pH, temperature, and solubilizer concentration suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 40. The method described in paragraph 39, wherein the conversion from homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C, at a pH in the range of approximately 4 to 8, in the presence of a solubilizer other than Triton X-100 for WT SHC / HAC enzymes. 41. A method according to any one of paragraphs 37-40, wherein the conversion from homofarnesol to (-)-ambrox occurs using one or more reaction conditions for WT SHC / HAC or SHC / HAC derivative enzymes listed in Table 24 or Table 24a. 42. Any one of the methods described in paragraphs 37 to 41, wherein the weight ratio of the biocatalyst to homofarnesol is in the range of about 0.5:1 to 2:1, preferably about 1:1 or 0.5:1. 43. A method according to any one of paragraphs 37-42, wherein the cell proliferation and biotransformation reaction steps are carried out in the same reaction vessel. 44. A method according to any one of paragraphs 27-31, wherein the homofarnesol substrate comprises one or more homofarnesol stereoisomers. 45. The method of paragraph 44, wherein the homofarnesol substrate comprises two homofarnesol stereoisomers. 46. ​​The method of paragraph 45, wherein the homofarnesol substrate contains the EE:EZ stereoisomer. 47. A method according to any one of paragraphs 44-46, wherein homofarnesol contains an EE:EZ stereoisomer mixture in a weight ratio selected from the group consisting of 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, and 70:30. 48. The method of paragraph 47, wherein homofarnesol comprises a mixture of EE:EZ stereoisomers in a weight ratio selected from the group consisting of EE:EZ90:10, EE:EZ80:20, EE:EZ86:14, EE:EZ70:30, EE:EZ69:31, and EE:EZ66:34. 49. The method of paragraph 35 or 36, wherein homofarnesol comprises an EE:EZ stereoisomer mixture in a weight ratio of 80:20. 50. One of the methods described in paragraphs 37–49, wherein (-)-ambrox is produced as a mixture with one or more of the by-products (II), (IV), and / or (III). 51. (-)-Amblocx is isolated from the biotransformation reaction mixture by any one of the methods described in paragraphs 37-50, using an organic solvent or steam extraction / distillation step or filtration. 52. A method according to paragraph 51, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent. 53. The method of paragraph 52, wherein (-)-ambrox is selectively crystallized from (-)-ambrox using an organic solvent. 54. The method of paragraph 52 or 53, wherein (-)-ambrox substantially does not contain by-products (II), (IV), and / or (III). 55. (-)-Amblocs that can be obtained by any one of the methods described in paragraphs 51-54. 56. (-)-Amblocs of paragraph 55, in solid form, preferably amorphous or crystalline. 57. A method for making a product containing (-)-Amblox, comprising incorporating (-)-Amblox as defined in paragraph 55 or 56 into a product, preferably a fragrance product, a cosmetic product, a cleaning product, a detergent product, or a soap product. 58. A fragrance, cosmetic, or consumer care product comprising (-)-Amblox as defined in paragraph 55 or 56. 59. A fragrance, cosmetic, or consumer care composition comprising (-)-Amblox of paragraph 55 or 56 and one or more additional components. 60. Use of (-)-Amblocs as part of a fragrance, cosmetic, or consumer product, e.g., fabric care, toiletries, beauty, and / or cleaning product, as described in paragraph 55 or 56. 61. Use of an SHC / HAC derivative enzyme following any one of paragraphs 1-21, a nucleotide sequence following paragraph 22 or 23, a construct following any one of paragraphs 24-26 or 30, a vector following any one of paragraphs 27-30, or a recombinant host cell following any one of paragraphs 31-34, or a recombinant host cell expressing WT SHC / HAC, for the bioconversion from homofarnesol to (-)-ambrox, wherein the WT SHC / HAC enzyme is used in the bioconversion reaction with a solubilizer other than Triton X-100.

[0272] In another aspect, a crystal model structure (CMS) of SHC is provided based on the structural coordinates of SHC by the amino acid sequence of SHC or derivatives described herein. The CMS of SHC comprises a squalene / homofarnesol binding pocket domain (SHBD) containing a squalene / homofarnesol binding pocket (SHBP) and a squalene / homofarnesol substrate bound to the SBD (see, for example, Figures 19 and 20). This SHC crystal model structure (CMS) facilitates in silico testing of potential SHC / HAC derivative enzyme candidates.

[0273] Therefore, in other embodiments, the Disclosure provides a method for screening enzymes (e.g., SHC / HAC derivatives) capable of binding to SHBD, the method comprising the use of CMS of SHC / HAC. In another aspect, the Disclosure provides a method for screening enzymes (e.g., reference SHC or SHC / HAC derivatives) capable of binding to SHBP, the method comprising contacting SHBP with a test compound (e.g., an SHC derivative) and determining whether the test compound binds to the SHBP. In some embodiments, the method is to screen test compounds (e.g., modulators) useful for modulating the activity of SHC derivative enzymes.

[0274] In another aspect, the Disclosure provides a method for predicting, simulating, or modeling the molecular characteristics and / or intermolecular interactions of reference SHCs and / or SHC / HAC derivatives having a squalene / homofarnesol-binding domain (SHBD), which includes the use of a computer model, which includes, uses, or draws the structural coordinates of the squalene / homofarnesol-binding domain as defined above, to provide an image of the ligand-binding domain, and optionally displays the image.

[0275] In this specification and the following claims, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” are understood to imply the inclusion of the integer or step or group of integers or steps described, rather than the exclusion of any other integer or step or group of integers or steps. The term “comprise” also means “inclusion” and “consisting of.” For example, a composition “comprises” X may consist solely of X or may include something additional, such as X + Y. It should also be noted that the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include multiple references unless the content is explicitly stated otherwise. For example, a reference to “gene” or “enzyme” is a reference to “one or more genes” or “one or more enzymes.”

[0276] It should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, which may vary. It should also be understood that the technical terms used herein are for the purpose of describing specific aspects only and are not intended to limit the scope of this disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. Conventional molecular biology, microbiology, and recombinant DNA techniques may be employed in accordance with this disclosure, which are within the realm of the art.

[0277] This disclosure is not limited in its application to the details of the construction and arrangement of the components described below or illustrated in the drawings. Other embodiments of this disclosure are possible and can be implemented or performed in various ways. The language and terminology used herein are also for illustrative purposes only and should not be considered limiting.

[0278] Preferably, terms used herein are defined as those described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0279] Several documents are cited in the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, GenBank accession numbers, sequence submissions, etc.) is incorporated herein by reference in whole, whether above or below.

[0280] The examples described herein are illustrative and not intended to be an limitation of the disclosure. Different aspects of the disclosure have been described in accordance with the disclosure. Many modifications and variations may become the techniques described and illustrated herein without deviating from the spirit and scope of the disclosure. It should be understood that the examples are merely illustrative and not an limitation of the scope of the disclosure. [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20]

[0281] Sequence ID 1 (Alicyclobacillus acidocaldarius), AacSHC MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Sequence ID 2 (Zymomonas mobilis), ZmoSHC1 MGIDRMNSLSRLLMKKIFGAEKTSYKPASDTIIGTDTLKRPNRRPEPTAKVDKTIFKTMGNSLNNTLVSACDWLIGQQKPDGHWVGAVESNASMEAEWCLALWFLGLEDHPLRPRLGNALLEMQREDGSWGVYFGAGNGDINATVEAYAALRSLGYSADNPVLKKAAAWIAEKGGLKNIRV FTRYWLALIGEWPWEKTPNLPPEIIWFPDNFVFSIYNFAQWARATMVPIAILSARRPSRPLRPQDRLDELFPEGRARFDYELPKKEGIDLWSQFFRTTDRGLHWVQSNLLKRNSLREAAIRHVLEWIIRHQDADGGWGGIQPPWVYGLMALHGEGYQLYHPVMAKALSALDDPGWRHDRGE SSWIQATNSPVWDTMLALMALKDAKAEDRFTPEMDKAADWLLARQVKVKGDWSIKLPDVEPGGWAFEYANDRYPDTDDTAVALIALSSYRDKEEWQKKGVEDAITRGVNWLIAMQSECGGWGAFDKDNNRSILSKIPFCDFGESIDPPSVDVTAHVLEAFGTLGLSRDMPVIQKAIDYVRS EQEAEGAWFGRWGVNYIYGTGAVLPALAAIGEDMTQPYITKACDWLVAHQQEDGGWGESCSSYMEIDSIGKGPTTPSQTAWALMGLIAANRPEDYEAIAKGCHYLIDRQEQDGSWKEEEFTGTGFPGYGVGQTIKLDDPALSKRLLQGAELSRAFMLRYDFYRQFFPIMALSRAERLIDLNN Sequence ID 3 (Zymomonas mobilis), ZmoSHC2 MTVSTSSAFHHSPLSDDVEPIIQKATRALLEKQQQDGHWVFELEADATIPAEYILLKHYLGEPEDLEIEAKIGRYLRRIQGEHGGWSLFYGGDLDLSATVKAYFALKMIGDSPDAPHMLRARNEILARGGAMRANVFTRIQLALFGAMSWEHVPQMPVELMLMP EWFPVHINKMAYWARTVLVPLLVLQALKPVARNRRGILVDELFVPDVLPTLQESGDPIWRRFFSALDKVLHKVEPYWPKNMRAKAIHSCVHFVTERLNGEDGLGAIYPAIANSVMMYDALGYPENHPERAIARRAVEKLMVLDGTEDQGDKEVYCQPCLSPIWDT ALVAHAMLEVGGDEAEKSAISALSWLKPQQILDVKGDWAWRRPDLRPGGWAFQYRNDYYPDVDDTAVVTMAMDRAAKLSDLHDDFEESKARAMEWTIGMQSDNGGWGAFDANNSYTYLNNIPFADHGALLDPPTVDVSARCVSMMAQAGISITDPKMKAAVDYL LKEQEEDGSWFGRWGVNYIYGTWSALCALNVAALPHDHLAVQKAVAWLKTIQNEDGGWGENCDSYALDYSGYEPMDSTASQTAWALLGLMAVGEANSEAVTKGINWLAQNQDEEGLWKEDYYSGGGFPRVFYLRYHGYSKYFPLWALARYRNLKKANQPIVHYGM Sequence ID No. 4 (Bradyrhizobium japonicum), BjpSHC MTVTSSASARATRDPGNYQTALQSTVRAAADWLIANQKPDGHWVGRAESNACMEAQWCLALWFMGLEDHPLRKRLGQSLLDSQRPDGAWQVYFGAPNGDINATVEAYAALRSLGFRDDEPAVRRAREWIEAKGGLRNIRVFTRYWLALIGEWPWEKTPNIPPEVIWFPLWF PFSIYNFAQWARATLMPIAVLSARRPSRPLPPENRLDALFPHGRKAFDYELPVKAGAGGWDRFFRGADKVLHKLQNLGNRLNLGLFRPAATSRVLEWMIRHQDFDGAWGGIQPPWIYGLMALYAEGYPLNHPVLAKGLDALNDPGWRVDVGDATYIQATNSPVWDTILTLL AFDDAGVLGDYPEAVDKAVDWVLQRQVRVPGDWSMKLPHVKPGGWAFEYANNYYPDTDDTAVALIALAPLRHDPKWKAKGIDEAIQLGVDWLIGMQSQGGGWGAFDKDNNQKILTKIPFCDYGEALDPPSVDVTAHIIEAFGKLGISRNNHPSMVQALDYIRREQEPSGPWF GRWGVNYVYGTGAVLPALAAIGEDMTQPYIGRACDWLVAHQQADGGWGESCASYMDVSAVGRGTTTASQTAWALMALLAANRPQDKDAIERGCMWLVERQSAGTWDEPEFTGTGFPGYGVGQTIKLNDPALSQRLMQGPELSRAFMLRYGMYRHYFPLMALGRALRPQSHS Sequence ID 149 (Burkholderia ambifaria) MNDLTEMATLSAGTVPAGLDAAVASATDALLAAQNADGHWVYELEADSTIPAEYVLLVHYLGETPNLELEQKIGRYLRRVQQADGGWPLFTDGAPNISASVKAYFALKVIGDDENAEHMQRARRAIQAMGGAEMSNVFTRIQLALYGAIPWRAVPMMPVEIMLL PQWFPFHLSKVSYWARTVIVPLLVLNAKRPIAKNPRGVRIDELFVDPPVNAGLLPRQGHQSPGWFAFFRVVDHALRAADGLFPNYTRERAIRQAVSFVDERLNGEDGLGAIYPAMANAVMMYDVLGYAEDHPNRAIARKSIEKLLVVQEDEAYCQPCLSPVWDT SLAAHALLETGDARAEAVIRGLEWLRPLQILDVRGDWISRRPHVRPGGWAFQYANPHYPDVDDTAVVAVAMDRVQKLKHNDAFRDSIARAREWVVGMQSSDGGWGAFEPENTQYYLNNIPFSDHGALLDPPTADVSGRCLSMLAQLGETPLNSEPARRALDYM LKEQEPDGSWYGRWGMNYVYGTWTALCALNAAGLTPDDPRVKRGAQWLLSIQNKDGGWGEDGDSYKLNYRGFEQAPSTASQTAWALLGLMAAGEVNNPAVARGVEYLIAEQKEHGLWDETRFTATGFPRVFYLRYHGYRKFFPLWALARYRNLKRNNATRVTGL Sequence ID 151 (Burkholderia ambifaria) MIRRMNKSGPSPWSALDAAIARGRDALMRLQQPDGSWCFELESDATITAEYILMMHFMDKIDDARQEKMARYLRAIQRLDTHGGWDLYVDGDPDVSCSVKAYFALKAAGDSEHAPHMVRARDAILELGGAARSNVFTRILLATFGQVPWRATPFMPIEFVLFPKWVPISM YKVAYWARTTMVPLLVLCKARARNPRNIAIPELFVTPPDQERQYFPPARGMRRAFLALDRVVRHVEPLLPKRLRQRAIRHAQAWCAERMNGEDGLGGIFPPIVYSYQMMDVLGYPDDHPLRRDCENALEKLLVTRPDGSMYCQPCLSPVWDTAWSTMALEQARGVAVPE AGAPASALDELDARIARAYDWLAERQVNDLRGDWIENAPADTQPGGWAFQYANPYYPDIDDSAVVTAMLDRRGRTHRNADGSHPYAARVARALDWMRGLQSRNGGFAAFDADCDRLYLNAIPFADHHALLDPPTEDVSGRVLLCFGVTKRADDRASLARAIDYVKRTQQP DGSWWGRWGTNYLYGTWSVLAGLALAGEDPSQPYIARALAWLRARQHADGGWGETNDSYIDPALAGTNAGESTSNCTAWALLAQMAFGDGESESVRRGIAYLQSVQQDDGFWWHRSHNAPGFPRIFYLKYHGYTAYFPLWALARYRRLAGGVSAAGAHAVPASTGADAALA Sequence ID 153 (Bacillus anthracis) MLLYEKAHEEIVRRATALQTMQWQDGTWRFCFEGAPLTDCHMIFLLKLLGRDKEIEPFVERVASLQTNEGTWKLHEDEVGGNLSATIQSYAALLASKKYTCEDANMKRAENFIQERGGVARAHFMTKFLLAIHGEYEYPSLFHLPTPIMFLQND SPFSIFELSSSARIHLIPMMLCLNKRFRVGKKLLPNLNHIAGGGGEWFREDRSPVFQTLLSDVKQIISYPLSLHHKGYEEIERFMKERIDENGTLYSYATASFYMIYALLALGHSLQSSMIQKAIAGITSYIWKMERGNHLQNSPSTVWDTALL SYALQEAQVSKDNKMIQNATAYLLKKQHTKKADWSVHAPALTPGGWGFSDVNTTIPDIDDTTAVLRALARSRGNKNIDNAWKKGGNWIKGLQNNDGGWGAFEKGVTSKLLAKLPIENASDMITDPSTPDITGRVLEFFGTYAQNELPEKQIQRA INWLMNVQEENGSWYGKWGICYLYGTWAVMTGLRSLGIPSSNPSLTRAASWLEHIQHEDGGWGESCHSSVEKRFVTLPFSTPSQTAWALDALISYYDTETPAIRKGVSYLLSNPYVNERYPTGTGLPGAFYIRYHSYAHIYPLLTLAHYIKKYRK Sequence ID 155 (Frankia alni) MPAGVGVLVWLDQRLRAMGRPDLVTTTGGAEIPFVLVAATASTVGVALALRRPRHPVGWLFLALGGVLLLSGGTQGYAAYGAVARPGRLPAADLVAIYADAGFIPWLVLVALILHLTPTGRPLSARWGRIALATAVAGGLWLLVGLVTTETMQPPFQSVTNPLLIGGPLPLLVARRVLGLATGAGVVLAAVSLIVRFRRSVDVERRQLLWVAAVPLPVLMAASFAASYAGNNTAAGLAAATLIGLLAIGAGLAIGQYHLYDVEEILSRAVTYLLVSGLLAASYATVVIVVGQSLAGRTGRSQISAVLATLAAVAVTAPAYRKIQEGVDRRFSRRRFETLQVIRRYLRDPDPDVAVEE VLRRALGDPTLAVAYLVDDRRQWVSADGQPANPGNSFMAAVEVYRRGRPIARVTFDRGRAQPGLVRAAATAATAELDNAGLRAAVALQLVEVRQSRTRIAAAQFAERRTIERNLHDGAQQRLLALALQLRAVQLGGDEASLRQAISTGIDQLQAAVVELRELANGLHPAVLADGGLAAALDDVAARTPVPIKISAPDRRYPPDLEAAAWFIACEAMANAVKHAHPTTIAVDVSAPDGQLIVEVRDDGIGGAQPSGPGLRGIADRAEAFGGSLTVHTDPGTGTTIRALLHRRSPLSSGRRSVMIEGCVDVVAVRRFRCRSSRGSGSRRRSSWRCGGICGSRCTGMMSRSCSRNAASKLIT SEQ ID NO:157(ロドシュードモナス·パレント(Rhodopseudomonas patent)) MDSILAPRADAPRNIDGALRESVQQAADWLVANQKPDGHWVGRAETNATMEAQWCLALWFLGLEDHPLRVRLGRALLDTQRPDGAWHVFYGAPNGDINATVEAYAALRSLGHRDDEEPLRKARDWILSKGGLANIRVFTRYWLALIGEWPWEKTPNILPEVIWLPTWFPFS IYNFAQWARATLMPIAVLSAHRPSRPLAPQDRLDALFPQGRDSFNYDLPARLGAGVWDVIFRKIDTILHRLQDWGARRGPHGIMRRGAIDHVLQWIIRHQDYDGSWGGIQPPWIYGLMALHTEGYAMTHPVMAKALDALNEPGWRIDIGDATFIQATNSPVWDTMLSLLAF DDAGLGERYPEQVERAVRWVLKRQVLVPGDWSVKLPDVKPGGWAFEYANNFYPDTDDTTSVALMALAPFRHDPKWQAEGIEDAIQRGIDWLVAMQCKEGGWGAFDKDNDKKILAKIPFCDFGEALDPPSADVTAHIIEFAKVGLDRNHPSIVRALDYLKREQEPEGPWFGR WGVNYVYGTGAVLPALAAIGEDMRQPYIARACDWLIARQQANGGWGESCVSYMDAKQAGEGTATASQTAWALMALIAADRPQDRDAIERGCLYLTETQRDGTWQEVHYTGTGFPGYGVGQTIKLNDPLLSKRLMQGPELSRSFMLRYDLYRHYFPMMAIGRVLRQRGDRSGH Sequence ID 159 (Streptomyces coelicolor) MTATTDGSTGASLRPLAASASDTDITIPAAAAGVPEAAARATRRATDFLLAKQDAEGWWKGDLETNVTMDAEDLLLRQFLGIQDEETTRAAALFIRGEQREDGTWATFYGGPGELSTTIEAYVALRLAGDSPEAPHMARAAEWIRSRGGIASARVFTRIWLALFGWWKW DDLPELPPELIYFPTWVPLNIYDFGCWARQTIVPLTIVSAKRPVRPAPFLDELHTDPARPNPPRPLAPVASWDGAFQRIDKALHAYRKVAPRRLRRAAMNSAARWIIERQENDGCWGGIQPPAVYSVIALYLLGYDLEHPVMRAGLESLDRFAVWREDGARMIEACQSP VWDTCLATIALADAGVPEDHPQLVKASDWMLGEQIVRPGDWSVKRPGPPGGWAFEFHNDNYPDIDDTAEVVLALRRVRHHDPERVEKAIGRGVRWNLGMQSKNGAWGAFDVDNTSAFPNRLPFCDFGEVIDPPSADVTAHVVEMLAVEGLAHDPRTRRGIQWLLDAQETD GSWFGRWGVNYVYGTGSVIPALTAAAGLPTSHPAIRRAVRWLESVQNEDGGWGEDLRSYRYVREWSGRGASTASQTGWALMALLAAGERDSKAVERGVAWLAATQREDGSWDEPYFTGTGFPWDFSINYNLYRQVFPLTALGRYVHGEPFAKKPRAADAPAEAAPAEVKGS Sequence ID 169 Variant 101A10 (Sequence ID 30) Variant 101A10 (Sequence ID 29) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVHYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant 111C8 ((Sequence ID 28) Variant 111C8 (Sequence ID 27) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGAWALYPGGPPDLDTTVEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC215G2 (Sequence ID 22) Variant SHC215G2 (SEQ ID NO: 21) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRRWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRVLHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC3 (SEQ ID NO: 26) Variant SHC3 (SEQ ID NO: 25) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC10 (SEQ ID NO: 32) Variant SHC10 (SEQ ID NO: 31) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC26 (SEQ ID NO: 24) Variant SHC26 (SEQ ID NO: 23) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRRWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC30 (SEQ ID NO: 34) Variant SHC30 (SEQ ID NO: 33) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC31 (SEQ ID NO: 36) Variant SHC31 (SEQ ID NO: 35) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRRWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC32 (SEQ ID NO: 38) Variant SHC32 (SEQ ID NO: 37) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRRWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC33 (SEQ ID NO: 40) Variant SHC33 (SEQ ID NO: 39) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRRWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHTPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant F605W (Sequence ID 170) Variant F605W (Sequence ID 171) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR Sequence ID 166 (ZmoSHC1) [Table 21] [Table 22] [Table 23] [Table 24] [Brief explanation of the drawing]

[0282] For a better understanding of this disclosure, please refer to the accompanying drawings. [Figure 1-4] Figures 1-4 show the sequence alignment of selected AacSHC derivatives relative to SEQ ID NO: 1 of AacSHC. In descending order, the SEQ ID NOs in Figure 1 are SEQ ID NO: 1, SEQ ID NO: 29, SEQ ID NO: 27, SEQ ID NO: 21, SEQ ID NO: 19, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39. [Figure 5] Figure 5 shows the plasmid map. [Figure 6] Figure 6 shows the relative HAC activity of wild-type AacSHC and AacSHC derivatives listed in Table 24 under standard conditions (pH 6.0, 55°C, 0.050% SDS, cells with OD 650nm = 10). [Figure 7] Figure 7a shows the HAC activity profile of AacSHC derivatives relative to WT AacSHC, using homofarnesol quality EEH:EZH87:13 and homofarnesol purity 96% (determined by NMR). Figure 7b shows the relative improvement of AacSHC derivatives relative to WT SHC (yield at 4h (initial rate) and 22h), using homofarnesol quality EEH:EZH87:13 and homofarnesol purity 96% (determined by NMR). [Figure 8]Figure 8a shows the HAC activity profiles of AacSHC derivatives compared to WT AacSHC, using homofarnesol quality EEH:EZH92:08 and homofarnesol purity 100% (determined by NMR). Figure 8b shows the relative improvement of AacSHC derivatives compared to WT SHC (yield at 4h (initial rate) and 22h), using homofarnesol quality EEH:EZH92:08 and homofarnesol purity 100% (determined by NMR). [Figure 9] Figure 9a shows the HAC activity profiles of AacSHC derivatives listed in Table 24 relative to WT AacSHC, using homofarnesol quality EEH:EZH66:33 and at 76% purity of homofarnesol (determined by NMR). Figure 9b shows the relative improvement of AacSHC derivatives relative to WT SHC (yield at 4h (initial rate) and 22h), using homofarnesol quality EEH:EZH66:33 and at 76% purity of homofarnesol (determined by NMR). [Figure 10] Figure 10 shows the HAC activity results for three SHC derivatives that show approximately 10-fold (215G2), 7-fold (SHC26), and 6-fold (SHC32) improvements compared to the wild-type AacSHC / HAC enzyme. [Figure 11] Figure 11 shows the conversion of E,E-homofarnesol to ambrox observed by the SHC / HAC derivative (215G2 SHC) and WT AacSHC. After 7 hours of reaction (estimated initial reaction rate), the conversion by variant 215G2 SHC was 13 times higher than that achieved by wild-type SHC. After 48 hours of reaction, the conversion by the variant was approximately 8 times higher than that of the wild-type enzyme. [Figure 12]Figure 12 shows the reaction products (ambix and product (IV)) produced when EEH is used as a starting material (for bioconversion with WT SHC and / or SHC / HAC derivatives), and the reaction products ((-)-ambix (I) and products (II), (IV), and (III) (see Table 21)) produced when EE:EZ is used as a starting material. For convenience of reference, compounds I-IV can be identified as follows: I: (3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan(-)-ambrox II: (7aS,11aS,Z)-5,8,8,11a-tetramethyl-2,3,6,7,7a,8,9,10,11,11a-decahydrobenzo[b]oxonin IV: (3aS,5aS,9aS,9bS)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan III: (3aRS,5aSR,9aSR,9bSR)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan 9-epi-ambrox. [Figure 13] Figure 13 shows the GC analysis of the reaction products for Ambrox and products (II), (IV), and (III) in Table 25. [Figure 14] Figure 14 shows the GC analysis of the reaction products for Ambrox and products (II), (IV), and (III) in Table 25. [Figure 15] Figure 15 provides comparative data on 215G2 SHC variant activity in a whole-cell biotransformation assay in the presence of either Triton X-100 or SDS. [Figure 16] Figure 16 shows the percentage of converted EEH for different SDS / cell ratios. [Figure 17] Figure 17 shows the percentage of EEH conversion in a standard bioconversion reaction (described in Example 7) for three different SDS concentrations. [Figure 18] Figure 18 shows the percentage of EEH conversion in a standard bioconversion reaction (described in Example 7) for three different pH values. [Figure 19] Figure 19 shows the locations of identified mutations in SHC / HAC variants 101A10, 111C8, and 215G2 on the SHC crystal structure (in color). Variant 215G2 is shown in red, variant 101A10 in purple (wine red), and variant 111C8 in green. Amino acids identified as contributing to increased activity are highlighted in yellow in the substrate analogs with co-crystallized side chains. Other mutations in the identified variants that do not exhibit improved activity are marked in blue. It is noteworthy that the blue mutations are scattered approximately equally (i.e., 50:50) across the two domains of the enzyme, while most of the identified beneficial AacSHC mutations are located within domain 2 (with one exception). The only exception is mutation F601Y, which is located near the active site. [Figure 20] Figure 20 shows the following mutations (black and white). Mutations that do not have a beneficial effect on SHC / HAC activity are shown in black and are scattered across the two domains of the SHC enzyme. Identified mutations in the SHC variants exhibiting improved SHC / HAC activity (101A10, 111C8, and 215G2) are shown in gray and, with only one exception, are located within domain 2 of the SHC enzyme. The side chains of mutations contributing to the improved activity of the variants are highlighted. [Figure 21] Figure 21 shows the cloning and expression regions of plasmid pET-28a(+). The sequence numbers in Figure 21 are as follows: pET28a (nucleotide sequence): SEQ ID NO: 179, pET28a (amino acid sequence): SEQ ID NO: 180, pET28b (nucleotide sequence): SEQ ID NO: 181, pET28b (amino acid sequence): SEQ ID NO: 182, pET28c (nucleotide sequence): SEQ ID NO: 183, and pET28c (amino acid sequence): SEQ ID NO: 184. [Figure 22]Figure 22 shows the volume-based productivity of a 1.5× enriched EEH bioconversion reaction containing 375 g / l cells, 188 g / l EEH, and 2.33% SDS, compared to the volume-based productivity of a standard bioconversion (Example 7) carried out in parallel with 125 g / l EEH, 250 g / l cells, and 1.55% SDS. [Figure 23] Figure 23 shows a standard bioconversion (EEH 125 g / l, cells 250 g / l, SDS 1.55%) carried out as described in Example 7. The citrate buffer pH 5.4 was replaced with either 0.5% or 0.9% NaCl, but all other reaction parameters remained unchanged. Bioconversion in citrate buffer was carried out in parallel as a control. [Figure 24] Figure 24 shows the progress of solid-phase extraction of (-)-ambrox by toluene washing, expressed as the percentage of (-)-ambrox initially present in 200 ml of total reaction broth (due to the volume ratio of broth / toluene, the percentage in the first extract exceeds 100%). [Figure 25] Figure 25 shows the progress of solid-phase extraction of (-)-ambrox by ethanol washing, expressed as a percentage of the initial amount of (-)-ambrox. After four washes (totaling 640 ml of EtOH, i.e., 3.2 × initial total reaction broth volume or 8 × solid phase volume), approximately 99% of the (-)-ambrox initially present in the reaction broth was recovered.

[0283] To avoid any ambiguity, all references to WT SHC and SHC variants refer to WT AacSHC (SEQ ID NO: 1) and its variants (e.g., those listed in Table 23 and / or Table 24).

[0284] Example 1 Biocatalyst production Method 1 SHC plasmid preparation The gene encoding Alicyclobacillus acidocaldarius squalene-hopene cyclase (AacSHC) was inserted into plasmid pET-28a(+). Thus, it is under the control of the IPTG-inducible T7 promoter for protein production in Escherichia coli (see Figures 5 and 21). The plasmid was transformed into the E. coli strain BL21(DE3) using a standard heat shock transformation protocol.

[0285] Erlenmeyer flask culture For protein production, either complex (LB) or minimal medium was used. M9 is one example of minimal medium and was used successfully.

[0286] Culture medium preparation The minimal medium selected by default was prepared as follows for a 350 ml culture. 307 ml of H2O was added to 35 ml of citrate / phosphate stock (133 g / l KH2PO4, 40 g / l (NH4)2HPO4, 17 g / l citric acid.H2O, pH adjusted to 6.3), and the pH was adjusted to 6.8 with 32% NaOH as needed. After autoclaving 0.850 ml of 50% MgSO4, 0.035 ml of trace element solution (composition in the next section), 0.035 ml of thiamine solution, and 7 ml of 20% glucose were added.

[0287] SHC biocatalyst production (biocatalyst production) For small-scale biocatalyst production (wild-type SHC or SHC variant), a 350 ml culture (supplemented with 50 μg / ml kanamycin in the medium) was inoculated with a preculture of the E. coli strain BL21(DE3) containing the SHC production plasmid. The cells were grown at 37 °C with constant agitation (250 rpm) to an optical density (OD 650nm ) of approximately 0.5. Next, protein production was induced by adding 300 μM IPTG, followed by a further 5-6 hours of incubation with constant shaking. The resulting biomass was finally collected by centrifugation and washed with 50 mM Tris-HCl buffer pH 7.5. The cells were stored as pellets at 4°C or -20°C until further use. Generally, 2.5 to 4 grams of cells (wet weight) were obtained from 1 liter of culture, independently of the culture medium used. Fermentation was prepared and carried out in a 750 ml InforsHT reaction vessel. 168 ml of deionized water was added to the fermentation vessel. Bottles of all necessary probes (pO2, pH, sampling, defoamer), C+N feed, and sodium hydroxide were prepared in the reaction vessel and autoclaved. After autoclaving, the reaction vessel was prepared as follows: 20 ml 10x phosphate / citrate buffer 14ml 50% glucose 0.53ml MgSO4 solution 2ml (NH4)2SO4 solution 0.020ml trace element solution 0.400 ml thiamine solution 0.200ml Kanamycin Stock Added. The parameters used were as follows: pH=6.95, pO2=40%, T=30℃, stirring at 300 rpm. Cascade: rpm setpoint at 300, min 300, max 1000, flow l / min setpoint 0.1, min 0, max 0.6. Defoamer control: 1:9. From seed culture, transfer to fermentation tank with an OD of 0.4-0.5 650nmThe cells were inoculated to achieve the desired result. This seed culture was grown in LB medium (+ kanamycin) at 37°C and 220 rpm for 8 hours. Fermentation was initially carried out in batch mode for 11.5 hours, after which a C+N feed was started using a feed solution (sterile glucose solution (143 ml H2O + 35 g glucose)). After sterilization, 17.5 ml of (NH4)2SO4 solution, 1.8 ml of MgSO4 solution, 0.018 ml of trace element solution, 0.360 ml of thiamine solution, and 0.180 ml of kanamycin stock were added. The feed was carried out at a constant flow rate of approximately 4.2 ml / h. + External measurements were taken to assess the availability of C and N sources in the culture. Typically, glucose levels remain very low. The culture was allowed to grow for a total of approximately 25 hours, at which point they typically reached an OD of 40-45. 650nm The cell count then increased. SHC production was then initiated by adding IPTG to the fermenter at a concentration of approximately 1 mM (either as an IPTG pulse or over a period of 3–4 hours using an injection syringe), setting the temperature to 40°C, and the pO2 to 20%. The induction of SHC production continued for 16 hours at 40°C. At the end of the induction, the cells were collected by centrifugation, washed with 0.1 M citrate / sodium citrate buffer pH 5.4, and stored as pellets at 4°C or -20°C until further use.

[0288] Result 1a Generally, with all other conditions unchanged, the specific activity of the produced biocatalysts was higher when minimal media were used compared to complex media. Induction was successful at 30 or 37°C. It was noted that when induction was performed at 40–43°C, biocatalysts with higher specific activity were obtained.

[0289] Result 1b Table 22 below shows the culture volume, optical density, and cell volume at both the start and end of induction, as well as the amount of biomass collected (wet weight), for two examples. [Table 25]

[0290] Example 2 Preparation and activity screening of SHC variants Method 2 To avoid any ambiguity, EE corresponds to (3E,7E), EZ mixture corresponds to (3Z,7E), ZE corresponds to (7Z,3E), ZZ corresponds to (7Z,3Z), and EEH corresponds to (3E,7E). The enzyme evolution program was performed using the wild-type (WT) Alicyclobacillus acidocardarius SHC (AacSHC) gene as a template (GenBank M73834, Swissprot P33247). A library of approximately 10,500 SHC variants was produced, and variants showing increased EEH cyclization ability were screened. Screening was carried out at 55°C under constant stirring in citrate buffer pH 6.0 (0.150 ml) containing 4 g / l EEH and 0.050% SDS. The selected hits for verification were tested using standard methods: 4 g / l EEH, 0.050% SDS, 10.0 OD. 600nm The reaction was carried out in citrate buffer pH 6.0 containing cells expressing the SHC variant. The final volume was 1 ml, and the reaction was incubated at 55°C and vigorously stirred on a magnetic stirrer. Sampling of the reaction over time allowed for examination of the activity profile (EEH conversion to (-)-Amblox) determined by gas chromatography analysis (see analytical methods below). From this validation round, three variants with improved EEH cyclization activity (101A10, 111C8, and 215G2) were obtained, and a total of eight mutations were identified within these three variants. Mutation studies were then conducted to identify which of these mutations were beneficial for EEH cyclization to Ambrox. In addition to this AacSHC derivative, another AacSHC variant was constructed that contained all of the identified beneficial mutations (SHC33, outlined in Table 23 below). Screening conditions were 4 g / l EEH, OD 650nmThe cells were 10.0, and SDS was 0.05% and 0.1% (two concentrations). The reaction was carried out at 55°C under constant stirring.

[0291] Result 2a [Table 26]

[0292] Result 2b Of the three selected mutations (101A10, 111C8, and 215G2), 215G2 showed the best activity.

[0293] Example 3 Optimized reaction conditions for SHC variants Reaction parameters examined: temperature, SDS concentration, and pH Method 3 The reaction conditions for the SHC variant derivatives identified in Table 23 were individually optimized with respect to temperature, pH, and SDS concentration. For this purpose, *E. coli* cells were transformed with plasmids for the production of individual variants, cultured in Erlenmeyer flasks, and SHC production was induced as described above. This method ensured that all cultures contained the same or very similar amounts of SHC. Cells were collected by centrifugation, washed with 0.1 M citrate buffer (pH 6.0), and stored at -20°C until further use.

[0294] Result 3 The results of this optimization study are summarized in the table below. Optimization rounds were also performed on wild-type SHCs. Table 24 below shows the optimal reaction conditions for the wild type and for each variant, taking into account the characteristics of each SHC / HAC derivative enzyme. [Table 27]

[0295] Consideration 3 Example 3 illustrates the notable differences in reaction conditions for SHC derivatives compared to wild-type SHC. Significant deviations from wild-type SHC were observed in SHC variants for optimal temperature, pH, and SDS concentration. Only a few mutations have a significant effect on the optimal bioconversion reaction conditions. To determine the individual reaction conditions for selected SHC variants, the reaction was performed with the addition of 4 g / l of EEH substrate and an optical density of 10.0 OD. 650nm The tests were performed using cells that produced wild-type or SHC derivatives. temperature The data in Table 24 show the surprising finding that while the WT SHC enzyme exhibits optimal activity at 55°C (within the range of 45–60°C), several SHC derivatives exhibit optimal activity at 35°C (34–50°C). The application of the SHC derivatives of this disclosure to methods for preparing (-)-ambrox from E,E-homofarnesol at lower reaction temperatures offers a significant cost advantage for industrial-scale (-)-ambrox production. Solubilizer From a long list of potential solubilizers that were not useful in the biotransformation reaction, SDS was selected and identified (see Example 14 for more information). SDS is better than, for example, Triton X-100 in terms of reaction rate and yield (both in the test with EEH 4 g / l and when EEH 125 g / l is used as provided in Example 7).

[0296] Example 4 SHC variant activity testing compared to WT SHC enzyme under standard conditions. Method 4 To compare the relative activity of the biocatalysts, the production of variants (listed in Table 24) is described below. E. coli cells were transformed with a plasmid for the production of one type of SHC variant, and then the E. coli cells were cultured in LB medium at 37°C and 280 rpm, with an OD of 0.50. 650nmCells were grown to a certain size, and enzyme production was induced by the addition of IPTG. Induction was carried out at 37°C and 280 rpm for 5.5 hours. Cells were collected by centrifugation, washed with 0.1 M citrate pH 6.0, and stored at -20°C until further use. When comparing the activity of SHC variants (see Figure 6), a sample of the reaction mixture was loaded onto an SDS-PAGE gel to analyze the SHC content of the reaction. This analysis confirmed that all reactions contained the same amount of SHC enzyme.

[0297] Result 4a Figure 6 shows the relative activity of wild-type and SHC variant under standard conditions (pH 6.0, 55°C, 0.050% SDS, OD 650nm (10 cells). It was also noted that wild-type SHC and SHC variants tested according to the examples of this disclosure are solvent-tolerant. This means that selected water-immiscible solvents (up to nearly 100%) can be added to the biotransformation reaction.

[0298] Result 4b When the 215G2 SHC variant was used, no notable effect on the variant's activity was observed when NaCl was added to the reaction (tested concentrations ranged from 5 to 100 mM only). In addition, the addition of NaCl up to 100 mM or up to 154 mM (0.9% NaCl) did not negatively affect the SHC activity of variant 215G2. These findings suggest that the bioconversion reaction can occur in the presence of physiological NaCl solution, etc., but not in the presence of buffer, provided that the reaction is carried out in a physiological NaCl solution (0.9%), etc., and the pH is maintained at an appropriate value (e.g., approximately 5.4 (5.2-5.6)).

[0299] Consideration 4 Figure 6 illustrates the ranking of the activity of selected variants and wild-type SHC enzymes in terms of EEH conversion to (-)-ambrox.

[0300] Example 5 Activity profiles of WT SHC and SHC derivatives Method 5 Activity tests were performed in a Heidolph Synthesis 1 apparatus at 900 rpm with constant shaking in a 5 ml volume of 0.1 M citrate buffer. The pH of the buffer used, the reaction temperature, and the SDS (sodium dodecyl sulfate) concentration during the reaction were determined according to the SHC variant used (wild type or variant). The optimal conditions for each tested variant are summarized in Table 24 above. A homofarnesol starting material with a purity of 96% and a homofarnesol substrate having an EEH:EZH ratio of 87:13 were used. To avoid any ambiguity, the EE:EZ mixture is a mixture of ((3E,7E) and (3Z,7E) isomers.

[0301] Result 5 Homofarnesol used: EEH:EZH87:13, purity (NMR): 96%. The results of standard tests conducted under optimized conditions are shown in Figure 7B (activity profile of SHC derivatives relative to WT SHC) and Figure 7A (yield at 4h (initial rate) and 22h), showing the relative improvement in the activity of SHC derivatives relative to WT SHC. Homofarnesol used: EEH:EZH92:08, purity (NMR): 100% The results of standard tests conducted under optimized conditions are shown in Figure 8B (activity profile of AacSHC derivatives relative to WT AacSHC) and Figure 8A (yield at 4h (initial rate) and 22h), showing the relative improvement of AacSHC derivatives relative to WT SHC. Homofarnesol used: EEH:EZH66:33, purity (NMR): 76% The results of standard tests conducted under optimized conditions are shown in Figure 9B (activity profiles of AacSHC derivatives listed in Table 24 relative to WT AacSHC) and Figure 9A (yields at 4h (initial rate) and 22h), showing the relative improvement of AacSHC derivatives relative to WT SHC.

[0302] Consideration 5 The main conclusion was that, independently of the quality of the homofarnesol substrate used, the four best SHC derivative enzymes were ranked as follows: 215G SHC, SHC26, SHC32, and SHC3.

[0303] Example 6 Determine the mass balance from reactions completely extracted by the solvent. Method 6 All conditions were kept constant, and two reactions were carried out for each variant. Homofarnesol was used as the substrate. After incubation for 4 hours and 22 hours, the reaction products and unreacted substrates were completely extracted for each variant by a total of six washes with equal volumes of tert-butyl methyl ether (MTBE / tBME). The homofarnesol and ambrox content of each wash was determined by GC analysis. The total amount of ambrox formed and the remaining homofarnesol was calculated from a calibration curve created using a standard ambrox and homofarnesol solution.

[0304] Result 6 The results in Figure 10 show that, with the substrates used, the three best variants demonstrated improvements of approximately 10 times (215G2), 7 times (SHC26), and 6 times (SHC32) compared to the wild-type SHC enzyme, and these improvements were observed.

[0305] Example 7 Performance of biotransformation in E,E-homofarnesol (EEH) at 125 g / l Method 7 Using the 215G2 SHC variant, we aimed to increase productivity per unit volume. We designed a series of experimental (DOE) studies and optimized the test reaction conditions, including parameters such as pH, cell concentration, and SDS concentration. The reaction conditions were 125 g / l of EEH (from homofarnesol of EE:EZ86:14), 250 g / l of cells, and 1.55% SDS, and the reaction was carried out at 35°C in 0.1 M citrate buffer at pH 5.4. A typical reaction (total volume of 150 g) is set up in a 0.75 liter Infors fermenter as follows: An appropriate amount of homofarnesol corresponding to 18.75 g of EEH is added to the reaction vessel. 2.33 g of SDS is added from a 15.5% (w / w) solution prepared in 0.1 M citrate buffer pH 5.4. A cell suspension is prepared from E. coli cells producing the 215G2 SHC variant by suspending the cells in 0.1 M citrate buffer pH 5.4. After determining the wet weight of the cells in this suspension by centrifugation at 10°C and 17210 g for 10 minutes, an appropriate volume of cells is added to the reaction vessel to introduce 37.5 g of cells into the reaction. The reaction volume is adjusted to 150 g according to the required amount of reaction buffer. The reaction is carried out at 37°C with constant stirring at 900 rpm. pH control is performed using 40% citrate in water. The reaction was sampled at various time points (1 ml) and extracted with 5 times the volume of MTBE / tBME (5 ml). After clarification of the solvent phase by centrifugation (benchtop centrifuge, 13000 rpm, 2 min) and 10-fold dilution with MTBE / tBME, the homofarnesol and ambrox content of the reaction was determined by GC analysis. The same reaction was carried out using E. coli cells producing wild-type SHC enzyme. In this case, the reaction was carried out at 55°C in 0.1 M citrate buffer, pH 6.0. A summary of the reaction conditions for this example is provided in the second row of Table 24a below. The reaction conditions shown in the first row of Table 24a below are taken from previous examples (e.g., Examples 3-5). [Table 28]

[0306] Result 7 Figure 11 shows the observed conversion of EEH to Ambrox by the two enzymes. At 7 hours of reaction (estimated initial reaction rate), the conversion by variant 215G2 SHC was 13 times higher than that achieved by wild-type SHC. At 48 hours of reaction, the conversion by the variant was approximately 8 times higher than that by the wild-type enzyme.

[0307] 7 general comments cell concentration All cell concentrations (g / l) in the reaction described in this example are given in terms of wet weight of the cells. The concentration of cells in a cell suspension as wet weight (g / l) is determined after centrifugation of a sample of this cell suspension at 17210 g and 4°C for 10 minutes. Cellular g / l and OD 650nm correlation between Using 215G2 SHC or WT SHC bioconversion with EEH 125g / l, 250g / l cells in this reaction yield approximately 172 OD in that reaction. 650nm Corresponds to OD. When different biocatalyst preparations are tested, OD 650nm A variation in the ratio of biocatalyst to biocatalyst amount was observed. The biocatalyst is used in standard tests with an EEH of 4 g / l, but the OD of 10.0 650nm When the cells were applied, OD 650nm It was estimated that 10.0 is equivalent to 1.45 g / l of cells.

[0308] Consideration 7 The data demonstrate the development of an optimized and efficient HAC bioconversion process using a relatively high EEH substrate concentration (125 g / l) compared to disclosures in this field, where only homofarnesol substrate concentrations ranging from approximately 0.2 g / l (see JP2009060799) to about 2.36 g / l (10 mM) have been disclosed in this field (see WO2010 / 139719A2, US2012 / 0135477A1 and Seitz et al (2012) ibid.).

[0309] Example 8 GC analysis Method 8 To quantify the EEH and ambrox content, the samples were extracted with an appropriate volume of tert-butyl methyl ether (MBTE / tBME). The solvent fraction was separated from the aqueous phase by centrifugation prior to gas chromatography analysis. 1 μl of the solvent phase was injected into a 30 m × 0.32 mm × 0.25 μm Zebron ZB-5 column (split ratio 3). The column was developed with a constant flow (4 ml / min H2) over a temperature gradient of 100°C, 15°C / min up to 200°C, 120°C / min up to 240°C, and 4 minutes at 240°C, which resulted in the separation of ambrox, EEH, and EZH. The inlet temperature was 200°C, and the detector temperature was 300°C. The EEH conversion is performed using the following formula, derived from the peak areas corresponding to Ambrox and EEH: Conversion (%) = 100 × (Area) アンブロックス_ピーク / (area アンブロックス_ピーク +Area E,E-ホモファルネソールピーク )) It was calculated by [method]. The identity of the reaction product, ambrox, was confirmed by GC-MS (recorded values ​​and intensities: m / z 221 (100%), m / z 97 (40%), m / z 137 (3.3%), m / z 43 (2.6%), m / z 41 (2.5%), m / z 55 (2.4%), m / z 95 (1.9%), m / z 67 (1.8%), m / z 81 (138%), m / z 222 (1.7%)).

[0310] Consideration 8 Product recovery was performed by either solvent extraction or vapor extraction. The solvents used were, for example, MTBE or hexane:isopropanol (3:2). The reaction was repeatedly extracted with equal volumes of solvent until the substrate or product was no longer detectable, and the solvent fraction was analyzed by GC. Generally, 5 to 6 washes were sufficient. Alternatively, the extraction of the reaction product was performed by vapor extraction.

[0311] Example 9 One-pot reaction Method 9 Fermentation of 200 ml was carried out with E. coli BL21(DE3) transformed with the pET28a(+) 215G2 SHC plasmid for the production of 215G2 SHC with N-terminal HisTag, using the standard growth and induction protocol described above. At the end of the induction phase, aeration was turned off, the temperature was set to 35°C, the pH to 5.5 with citrate, and the stirrer speed to 500 rpm. The volume of the culture was estimated from all additions (feed and basal consumption) made during culture growth. According to this volume and the OD of the culture, an appropriate amount of SDS was added to the fermenter. EEH was added up to 4 g / l. The reaction was sampled over time, and samples (150–300 μl) were extracted by 700 μl of MTBE for GC analysis. EEH was directly converted to ambrox in the culture broth. The reaction was carried out for a total of 22.5 days, during which EEH was repeatedly added.

[0312] Result 9 Upon completion, 10.6 g of EEH had been cyclized to ambrox. The reaction product (structure provided below) was extracted by vapor extraction and quantitatively recovered from the reaction mixture. [ka] Notes on reaction products When homofarnesol EE:EZ 87:13 is converted by SHC, reaction products ambrox, (II), (IV), and (III) are produced, as shown in Figure 12, reflecting the EE:EZ ratio of the starting materials. When EEH is used as a starting material, only (-)-ambrox(I) and product(IV) are produced. When EZH(3Z,7E) is used as the starting material, only products (II) and (III) are formed. However, when a mixture of EEH and EZH is used, ambrox (I) and products (II), (IV), and (III) are produced. If a 100% conversion of EE:EZ66:34 occurs, this will provide a 66%:34% ((Unbrox+(IV)):((II)+(III)). When steam extraction is performed, it extracts all four products, ambrox, as well as products (II), (IV), and (III), and the crystallization step yields ambrox with a purity of 99% (GC) in a yield of at least 70%.

[0313] Consideration 9 The data indicate that (-)-ambrox production is possible via bioconversion or "one-pot" reaction systems, and that selective enrichment of ambrox is achieved after vapor extraction and / or crystallization. When the homofarnesol starting material is a mixture of EE and EZ (e.g., 86:14) isomers, two products are derived from each of those isomers (four in total), with (-)-ambrox being the overwhelmingly dominant component in the crude product and the major component in the crystallized material (99.1% purity). (+)-ambrox was not detected.

[0314] Example 10 EE: Conversion of EZ Homofarnesol Mixture To avoid any ambiguity, EE corresponds to (3E,7E), EZ mixture corresponds to (3Z,7E), ZE corresponds to (7Z,3E), ZZ corresponds to (7Z,3Z), EEH corresponds to (3E,7E), and EZH corresponds to (3Z,7E).

[0315] Method 10 The EE:EZ mixture was bioconverted under the following reaction conditions using the following homofarnesol substrate (EE:EZ homofarnesol mixture) at a total homofarnesol concentration of 146 g / l, cell concentration of 250 g / l, and SDS 1.55%. EE:EZ 86:14 (highest EEH content in this example), EE:EZ 69:31 (the lowest EEH content in this example), EE:EZ 80:20 EEH:EZH 70:30 Bioconversion of 7E,3E / 7E,3Z homofarnesol mixture The biotransformation was initiated using the following reaction conditions: The reaction (total volume of 150.1 g) was carried out in a 750 ml InforsHT fermenter in 0.1 M citrate / sodium citrate buffer, pH 5.4, containing a total of 146 g / l of homofarnesol. The reaction was performed using a homofarnesol substrate (a mixture of 86:14 7E,3E:7E,3Z), 250 g / l of cells (produced according to the method of Example 1), and 1.55% SDS. The reaction was carried out at 35°C with constant stirring (800 rpm), and pH control was performed using 10-40% citrate in water. The reaction mixture was sampled at different time points, and the samples were solvent-extracted for GC analysis. It was noted that homofarnesol conversion occurred equally rapidly in both homofarnesol types (EE:EZ86:14 and EE:EZ69:31).

[0316] Result 10 When bioconversion of 125 g / l of E,E-homofarnesol was performed from EEH:EZH86:14 material using WT SHC and one specific SHC derivative (215G2 SHC), conversion of both E,E- and E,Z-homofarnesol was observed. That is, the wild-type SHC enzyme from Alicyclobacillus acidocardarius produced the same reaction products (i.e., ambrox, products (II), (IV), and (III)) from EEH:EZH86:14 material as the SHC variants from Table 23 produce from the EEH:EZH mixture. Figures 13 and 14 provide GC analysis of the reaction products for ambrox and products (II), (IV), and (III).

[0317] Consideration 10 The bioconversion of homofarnesol to ambrox according to this disclosure produces (-)-ambrox as the major compound, but may also produce other compounds (e.g., compounds (II), (IV), and (III)) as identified above, which may or may not confer a pleasant olfactory note to the (-)-ambrox product. As shown above, under selective crystallization conditions, ambrox is separable from other by-products ((II), (III), and (IV)). Therefore, if the products contribute negatively to the perceptual properties of the final ambrox product, selective separation of products (II), (IV), and (III) from the final (-)-ambrox product increases its value as a fragrance or flavor or cosmetic or consumer care product. Perceptual analysis is performed using well-established perceptual tests available to skilled perfumers. The purity of the (-)-Amblox final product can be an indicator of the olfactory quality of the product if the product itself is the primary factor in the desired perceptual profile.

[0318] Example 11 EEH conversion from EE:EZ:ZE:ZZ-homofarnesol mixture Method 11 EE:EZ:ZE:ZZ-homofarnesol 40:26:20:14 was used as a substrate for EEH conversion using 215G2 SHC. For comparison, other homofarnesols of EE:EZ2:1 or 93:07 were also used. The conversion of the EE:EZ:ZE:ZZ-homofarnesol mixture was investigated using the 215G2 SHC variant, but not under optimized conditions. Reaction conditions were pH 5.8 in 100 mM citrate buffer, 0.10% SDS, and 40°C. The following EEH conversion was observed, and all reactions were carried out at a constant 2 g / l EEH concentration (and therefore, at various total homofarnesol concentrations).

[0319] Result 11 The following conversion rates for homofarnesol isomer mixtures were observed. EE:EZ 2:1 50~55% EE:EZ 93:7 78% EE:EZ:ZE:ZZ 40:26:20:14 6%

[0320] Consideration 11 In addition to the observed yields, the data indicate that the 215G2 SHC variant is capable of converting EEH to ambrox from a complex EE:EZ:ZE:ZZ homofarnesol mixture. As expected, lower conversion rates were observed to result in lower ambrox yields. This result is consistent with the view that homofarnesol isomers other than EEH may compete with EEH for access to the SHC / HAC derivative enzyme and therefore may act as competitive inhibitors and / or alternative substrates for the conversion of EEH to (-)-ambrox.

[0321] Example 12 Comparative data of whole-cell biotransformation using Triton X-100 and SDS Method 12 E. coli host cells were grown according to the protocol of Method 4 in Example 4. The biotransformation reaction using the 215G2 SHC variant was performed according to the standard test in Example 4. OD in 0.1M citrate / sodium phosphate buffer, pH 5.4 650nm 10.0 cells, 4 g / l homofarnesol substrate, 35°C, and 0.07% SDS were selected as the most suitable reaction conditions for the 215G2 SHC variant.

[0322] Result 12 Figure 15 provides a comparison of the activity of the 215G2 SHC variant in a whole-cell bioconversion assay using Triton X-100 in a concentration range of 0.005% to 0.48% and SDS at a concentration of 0.07%.

[0323] Consideration 12 The data shows that the highest activity achieved with Triton X-100 is only about 20% of the activity obtained by SDS.

[0324] Example 13 SDS / cell ratio Method 13 OD produced 4g / l of EEH substrate and 215G2 SHC derivative enzyme. 650nm Using cells from 5.0, the biotransformation reaction was set up according to Method 4 of Example 4.

[0325] Result 13 The results are shown in Figure 16, which displays the converted EEH percentage for different SDS / cell ratios. Figure 16 shows that the percentage of EEH conversion to (-)-Amblox depends on the SDS / cell ratio, using different SDS / cell ratio values. This ratio must be carefully set to achieve the maximum conversion. For example, if the SDS concentration is too low, near-optimal homofarnesol conversion may be observed. On the other hand, if the SDS concentration is too high, for example, there is a risk that the biocatalyst may be affected by either the destruction of intact microbial cells and / or the denaturation / inactivation of SHC / HAC enzymes. When the bioconversion reaction was carried out according to Method 7 of Example 7 using 125 g / l EEH and 250 g / l biocatalyst, the best bioconversion protocol exhibits a [SDS] / [cell] ratio of 16:1.

[0326] Consideration 13 The results indicate a degree of interdependence between solubilizer (SDS) concentration, biomass amount, and substrate (EEH) concentration. For example, as the concentration of homofarnesol substrate increases, sufficient amounts of biocatalyst and solubilizer (SDS) are required for efficient bioconversion reactions to occur.

[0327] Example 14 Testing of possible solubilizers for use in biotransformation reactions Method 14 Various solubilizers (outlined in Table 26 below) were subjected to the EEH cyclization reaction of 215G2 SHC under the same conditions as the standard test (4 g / l EEH, 10.0 OD). 650nm (of cells), was tested as a possible substitute for SDS. The possibility of enhancing activity (cumulative effect) by combining SDS at its optimal concentration (0.060–0.070%) with other solubilizers used (at concentrations individually determined to be optimal from screening performed on those compounds (see Table 26 below)) was also tested using standard tests. In addition, several "deep eutectic solvents" and ionic liquids known to help solubilize water-insoluble compounds were also tested.

[0328] Result 14 Table 26 below summarizes which solubilizers (e.g., surfactants, detergents, solubility enhancers, etc.) have been tested so far in the EEH cyclization reaction of 215G2 SHC. In no case was there any improved activity compared to the control reaction performed using SDS at concentrations in the range of 0.060–0.070%. The activity observed with these compounds used alone at the concentration defined as optimal was only about 20% of that obtained in the control reaction with SDS. It was noteworthy that 20% EEH conversion was achieved when no solubilizer was added at all. No synergistic effect was observed when SDS was used and additional solubilizers were added (at the concentration defined as optimal in the test). Rather, a decrease in the EEH conversion percentage was observed. Under the conditions tested, the study can be concluded that the compounds do not improve EEH conversion at all, and in fact have an adverse effect on cyclization, and that SDS is the most useful of the solubilizers studied. In addition, no positive results were obtained from tests using "deep eutectic solvents" and ionic liquids, which are known to help solubilize water-insoluble compounds. [Table 29]

[0329] Consideration 14 The applicant selected and identified SDS as a useful solubilizer from a long list of other solubilizers that had been shown not to be useful in the bioconversion reaction from homofarnesol to (-)-ambrox as described herein.

[0330] Example 15 Sensitivity to SDS concentration in biotransformation reactions Method 15 The applicable conditions are those for standard bioconversion with 250 g / l biocatalyst and 1.55% SDS at 125 g / l (described in Example 7). Two other SDS concentrations (1.40% and 1.70% SDS) were also tested. All SDS concentrations are expressed as weight / weight %. Standard biotransformation reaction conditions (described in Example 7) using 250 g / l biocatalyst and 1.55% SDS at 125 g / l were also used to test different pH values. Controls were carried out at pH 5.4 in 0.1 M citrate buffer. Reactions at lower pH levels were carried out in 0.1 M acetate buffer.

[0331] Result 15 The data in Figure 17 shows that the biotransformation reaction involved EEH 4g / l and OD 650nm Standard tests on cells treated with 10.0 suggest that HAC activity is less sensitive to changes in SDS concentration than when it was tested. The data in Figure 18 shows that when the biotransformation reaction is applied, the system has HAC activity at EEH 4g / l and OD 650nm This indicates that the cells appear less sensitive to pH fluctuations than when tested by standard tests with cells treated at 10.0.

[0332] Consideration 15 The data demonstrate the certainty of the biotransformation reaction at EEH 125 g / l and 250 g / l cells within the tested SDS concentration and pH ranges.

[0333] Example 16 Location of identified SHC / HAC mutations in the crystal structure The locations of the identified mutations in the AacSHC / HAC variants are marked in Figure 19 as follows: Variant 215G2 is red, variant 101A10 is purple (wine red), and variant 111C8 is green. Amino acids identified as contributing to increased activity are highlighted in yellow in the substrate analog with co-crystallized side chains. Other mutations in the identified variants that do not exhibit improved activity are marked in blue. It is noteworthy that the blue mutations are scattered approximately equally (i.e., 50:50) across the two domains of the enzyme, while most of the identified beneficial AacSHC mutations are located within domain 2 (with one exception). The only exception is mutation F601Y, which is located near the active site. When considering only the SHC / HAC derivative enzymes 215G2 and 111C8, all mutants are located within domain 2. Figure 20 provides the same information in black and white.

[0334] Result 16 All beneficial mutants (red / green / purple) corresponding to 215G2, 111C8, and 101A10 are largely located within domain 2 of the SHC crystal structure (provided in Figure 19), with the exception of one mutant, F601Y (Wendt et al (1997) Science 277: 1811). The beneficial mutant combinations of SHC are numbered according to the wild-type AacSHC (SEQ ID NO: 1).

[0335] Consideration 16 Crystal structures are useful for identifying SHC / HAC derivatives with desirable structure / activity relationships, particularly in relation to the conversion from homofarnesol to (-)-ambrox. A useful preliminary selection step may be to limit the selection to amino acid residues located within domain 2 of the SHC / HAC crystal structure (see Figures 19 and 20).

[0336] Example 17 Preparation of homofarnesol Method 17 General analysis conditions Nonpolar GC / MS: 50°C / 2 min, 20°C / min 200°C, 35°C / min 270°C. GC / MS Agilent 5975C MSD with HP 7890A series GC system. Nonpolar column: BPX5 from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22 mm × 0.25 mm × 12 m. Carrier gas: Helium. Injector temperature: 230°C. Split: 1:50. Flow rate: 1.0 ml / min. Transfer line temperature: 250°C. MS quadrupole type temperature: 106°C. MS source temperature: 230°C.

[0337] A) Preparation of MNU in THF A solution (400 ml) of urea (175 g, 2.9 mol) and methylamine hydrochloride (198 g, 2.9 mol) in water was heated under reflux (105°C) for 3.5 hours with stirring. At 40°C, NaNO2 (101 g, 1.45 mol) dissolved in water (200 ml) was added. After 15 minutes, THF (1000 ml) was added, which yielded a clear two-phase mixture. Concentrated H2SO4 (110 g, 1.1 mol) was added at 0-5°C and stirred within 1.5 hours. After a further 0.5 hours at 0-5°C, the two clear phases separated at 25°C. Organic phase (A) (1065 ml, theoretically 1.35 M) was stored at 0-5°C for several days or immediately proceeded to the cyclopropanation reactor. Following phase separation, the aqueous phase is extracted twice with THF (2 × 1:1). This yields 1100 ml of phase B and 1075 ml of phase C. Phase A yields 51% conversion from terminal alkenes to cyclopropane in the subsequent cyclopropanation reaction, while phase B yields <0.5% cyclopropane, and phase C yields no detectable conversion. We conclude that >99% of MNU is extracted after the initial phase separation. Therefore, typically, the aqueous phase is discarded after treatment with concentrated aqueous KOH and acetic acid following the initial phase separation (from organic phase A).

[0338] B) Preparation of E-Δfarnesene using MNU in THF [ka] 1.35 M N-methyl-N-nitrosourea in THF (136 ml, 184 mmol) is added dropwise at 0°C to a vigorously stirred mixture of E-beta-farnesene (CAS 18794-84-8) (25 g, 122 mmol) and KOH aqueous solution (50 ml, 40%) at 0-5°C. After adding 4 ml of MNU solution, Pd(acac)2 (7.4 mg, 0.024 mmol, 0.02%) pre-dissolved in 0.5 ml of dichloromethane is added. The remaining MNU solution was added over 4 hours at 0–5°C. GC at this stage showed 28% unconverted E-β-farnesene, 65% the desired monocyclopropane (shown above), and 3% biscyclopropanated compound 5. After 16 hours at 25°C, acetic acid (100 ml) was added at 0–5°C, followed by tert-butyl methyl ether (250 ml). After phase separation, the organic phase was washed with 2 M HCl (250 ml), and the aqueous phase was extracted with tert-butyl methyl ether (250 ml). The combined organic layers were washed with water (2 × 100 ml), 10% NaOH aqueous solution (2 × 100 ml), and water (2 × 100 ml), dried over MgSO4, filtered, and concentrated to obtain 26.9 g of a slightly yellowish liquid. This contained 9% E-β-farnesene, 82% the desired monocyclopropane compound, and 6% biscyclopropanated byproduct. The desired compound can be further isolated by distillation. Addition of 1 g of K2CO3 (1 g) and distillation using a 30 cm steel coil column at 40–60 mbar yields 147 g of monocyclopropane compound (68% corr) at 135–145°C. The fraction is pooled to obtain 92 g of 100% pure monocyclopropane compound.

[0339] Analysis data of E-Δfarnesene: 1H-NMR (CDCl3, 400 MHz): 5.1 (2 m, 2 H), 4.6 (2 H), 2.2 (2 H), 2.1 (4 H), 2.0 (2 H), 1.7 (s, 3 H), 1.6 (2 s, 6 H), 1.3 (1 H), 0.6 (2 H), 0.45 (2 H) ppm. 13C-NMR (CDCl3, 400 MHz): 150.9 (s), 135.1 (s), 131.2 (s), 124.4 (d), 124.1 (d), 106.0 (t), 39.7 (t), 35.9 (t), 26.7 (t), 25.7 (q), 17.7 (q), 16.0 (d), 6.0 (t) ppm. GC / MS: 218 (2%, M+), 203 (5%, [M - 15]+), 175 (11%), 147 (31%), 134 (15%), 133 (20%), 121 (12%), 107 (55%), 95 (16%), 93 (30%), 91 (20%), 82 (11%), 81 (33%), 79 (42%), 69 (100%), 67 (22%), 55 (20%), 53 (21%), 41 (75%). IR (film): 3081 (w), 2967 (m), 2915 (m), 2854 (m), 1642 (m), 1439 (m), 1377 (m), 1107 (w), 1047 (w), 1018 (m), 875 (s), 819 (m), 629 (w). Anal, calcd. for C16H26: C, 88.00; H, 12.00. Found: C, 87.80; H, 12.01.

[0340] Preparation of (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol ((7E)-homofarnesol) A mixture of (E)-(6,10-dimethylundeca-1,5,9-trien-2-yl)cyclopropane(E-Δfarnesene) (1 g, 4.6 mmol), dodecane (0.2 g, 1.15 mmol, internal standard), and L-(+)-tartaric acid (1 g, 6.9 mmol) in a pressure tube is heated at 150°C with stirring. After 18 hours and complete conversion (according to GC), the mixture is poured into water (50 ml) and toluene (50 ml). The phases were separated, and the aqueous phase was extracted with toluene (50 ml). The combined organic layers were washed with concentrated Na2CO3 aqueous solution (50 ml) and concentrated NaCl (2 × 50 ml), dried over MgSO4, filtered, and evaporated under reduced pressure to obtain a brownish resin (1.35 g). This was mixed with 30% KOH aqueous solution (4.3 ml) and stirred at 25°C for 2 hours. GC analysis revealed the formation of 96% (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol according to an internal standard. The E / Z ratio was 68:22. The analytical data for the E isomer is consistent with those from the literature. See, for example, P. Kocienski, S. Wadman J. Org. Chem. 54, 1215 (1989).

[0341] Result 17 The data demonstrate the preparation of homofarnesol suitable for bioconversion to (-)-ambrox.

[0342] Consideration 17 This process for preparing homofarnesol is also described in two concurrently pending patent applications PCT / EP2014 / 072882 (WO2015 / 059290) and PCT / EP2014 / 072891 (WO2015 / 059293), the overall contents of which are incorporated herein by reference.

[0343] Example 18 One-pot reaction In this experiment, (i) fermentation was performed using an E. coli strain producing the 215G2 SHC variant (as described in Example 1, e.g.), followed by (ii) direct EEH conversion in the fermentation broth. Since the three parameters [cells], [EEH], and [SDS] (g / l) are related, it was necessary to adjust the parameters [EEH] and [SDS] in the available volume of fermentation broth according to the cell concentration (g / l) obtained at the end of fermentation. The goal was to convert 125 g / l of EEH at a 1.55% SDS concentration using 250 g / l of cells. To enable reasonable bioconversion, the cells had to be in a resting, glucose-depleted state. Aeration was turned off.

[0344] Method 18 fermentation: To enable highly accurate determination of the volume of fermentation broth in the reaction vessel at the end of fermentation, the volume of the sample taken out and all volumes of additions made to the fermenter (feed, base, acid, etc.) were recorded.

[0345] Determining the cell concentration in fermented broth: To determine the wet weight (g / l) of cells, a sample (5-10 ml) of fermentation broth was withdrawn under constant agitation and placed in a centrifuge tube. The mass of the sample was recorded. The sample was centrifuged at 17210 g and 4°C for 10 minutes (e.g., 12000 rpm, SS-34 rotor, Sorvall RC3B centrifuge). The supernatant was carefully removed by pipetting, and the mass of the pellet was recorded. The wet weight concentration of cells was recorded in g. 細胞 / l ブロス or g 細胞 / g ブロス This will be decided. The volume of fermentation broth in the fermenter was determined according to all additions and removals. If the fermenter was on a scale, the mass of the fermentation broth was determined by weighing; otherwise, it was assumed that 1 ml = 1 g.

[0346] Determining the required amounts of homofarnesol and SDS: According to the determined cell concentration and volume of the fermentation broth, the amounts of E,E-homofarnesol and SDS to be added to the reaction vessel were determined to maintain the same ratio between the three, EEH 125 g / l, cell 250 g / l, and 1.55% SDS, as described in the bioconversion described in Example 9.

[0347] Set up biotransformation: 1. The temperature was set to 35°C. Ventilation was turned off. 2. A calculated amount of homofarnesol was added to the fermented broth. 3. The required amount of SDS was carefully added from a stock solution of 15.5% SDS aqueous solution. 4. The reaction mixture was thoroughly mixed at 800 rpm for approximately 15 minutes. 5. The pH of the reaction was recorded (internal pH electrode). 6. The sample (approximately 1 ml) was withdrawn into a 15 ml Falcon tube. Approximately 5 ml of deionized water was added, and after thorough mixing, the pH was recorded using an externally calibrated electrode. 7. The pH inside the reaction vessel was gradually set to 5.4 using 85% H3PO4 (value measured with an externally calibrated electrode). Meanwhile, the pH was periodically controlled with an external electrode as described above (6.). 8. pH was controlled during bioconversion using, for example, 10-25% H3PO4 and 32% NaOH. 9. Sampling of the reaction: Approximately 1 ml of the reaction mixture was placed in a 15 ml Falcon tube. Approximately 5 ml of MTBE was added. The sample was extracted by vigorous shaking. 10. The dispensed samples were centrifuged at maximum speed for 1 minute using a benchtop centrifuge (Eppendorf tubes). 100 μl of the solvent phase was added to a GC vial containing 900 μl of MTBE. During the first day of bioconversion, samples were taken every 1 to 1.5 hours. From the following day onward, only three samples were taken per day. 11. One μl of the solvent phase was analyzed for its ambrox and EEH content as described in Example 8. 12. Convert EEH (%) to 100 × (Unbrox面積 / ( Unblocks 面積 +EEH 面積 )) is used for calculation.

[0348] Result 18 The results indicate that one-pot fermentation + EEH conversion was carried out on a 1.9-liter scale in a KLF2000 reaction vessel (Bioengineering). Cells at 251 g / l allowed for the conversion of 238 g of EEH (251 g / l cells) to ≥93% in 47 hours. When measured 93 hours after the start, the conversion was 99%. A similar one-pot experiment was performed in an Infors HT 0.751 reaction vessel. Following fermentation using a standard protocol (Example 1), cells from a reactor collected from another fermentation performed in parallel using the same protocol were added. The resulting broth volume was 479 g. The cell concentration was determined to be 313.7 g / l, which was 1.25 × the standard cell concentration for bioconversion (250 g / l cells). Therefore, EEH and SDS were added to the reaction vessel. 75.1 g of EEH (equivalent to 157 g / l of EEH in this example) was converted to 98% in less than 90 hours. This result indicates that one-pot fermentation + EEH conversion is possible with EEH ≥ 125 g / l, as long as the fermentation performed provides cells producing the 215G2 SHC variant at a sufficiently high cell density.

[0349] Consideration 18 Advantageously, a 99% conversion of the substrate was achieved. This is commercially very useful when expensive starting materials (e.g., EEH) are used.

[0350] Example 19 To increase productivity per unit volume. Method 19 To further increase productivity per unit volume, a 1.5× concentrated bioconversion was performed containing 375 g / l of cells, 188 g / l of EEH, and 2.33% of SDS. A standard bioconversion was performed in parallel with 125 g / l of EEH, 250 g / l of cells, and 1.55% of SDS (Example 7). The two reactions were carried out in an Infors HT 0.7501 reaction vessel with all other parameters unchanged.

[0351] Result 19 The results in Figure 22 show that the conversion percentage after 75 hours was 88% for 1.5× bioconversion versus 95% for standard bioconversion. After 96 hours, the percentage conversion was 93% for EEH conversion in 1.5× bioconversion versus 97% for standard bioconversion. The percentage conversion with 1.5× bioconversion was 96% of that obtained with standard bioconversion. It was noted that stirring in 1.5× bioconversion became more difficult over time as the oily homofarnesol disappeared and was replaced by solid reaction products. This may explain the somewhat lower conversion level of 1.5× bioconversion. Using a reaction vessel equipped with better mixing equipment may improve EEH conversion with 1.5× bioconversion. The results show that bioconversion is possible at EEH levels of 188 g / l or higher when efficient mixing is achieved. Stirring efficiency appears to be the only limitation of the system. (-)- Unbrox productivity "(-)-Amblox productivity" refers to the amount of recoverable (-)-ambaxil in grams per liter of bioconversion time (i.e., time after substrate addition). Referring to Figure 22, (-)-ambax productivity is calculated as follows: Bioconversion of EEH at 125 g / l (cells 250 g / l) 1.25h productivity: 10.3 grams per liter per hour 8.25h productivity: 6.3 grams per liter per hour 21.25 hours of productivity: 4.1 grams per liter per hour Bioconversion of EEH at 187.5 g / l (375 g / l cells) 1.25h productivity: 12.2 grams per liter per hour 8.25h productivity: 8.2 grams per liter per hour 21.25 hours of productivity: 5.5 grams per liter per hour. The productivity calculated approximately 6-8 hours after the start can be considered to correspond to the initial response rate, and this best represents the system's best conversion rate. A typical bioconversion using 125 g / l of EEH in 250 g / l of cells shows Ambrox productivity between 6.3 and 8.5 grams per hour and liter per liter after approximately 6–8 hours (corresponding to the initial reaction rate).

[0352] Example 20 Replacing the reaction buffer with NaCl solution method 20 A standard bioconversion (EEH 125 g / l, cells 250 g / l, SDS 1.55%) was performed as described in Example 7, but the citrate buffer pH 5.4 was replaced with either 0.5% or 0.9% NaCl, and all other reaction parameters remained unchanged. Bioconversion in citrate buffer was performed in parallel as a control.

[0353] Result 20 The results in Figure 23 show that the EEH conversion rate was the same as in the reactions performed with buffer and 0.9% NaCl. The conversion rate was lower in the reaction performed with 0.5% NaCl alone. The results suggest the possibility of bioconversion in the absence of buffer, provided that precise pH control and sufficient ionic strength are ensured.

[0354] Example 21 Extraction of the solid phase of reaction broth Given that (-)-Amblox is not soluble in water and is not liquid at temperatures below approximately 75°C, these properties were considered a potential advantage for extracting products from the solid phase of biotransformations using either water-miscible (e.g., ethanol) or water-immiscible (e.g., toluene) solvents.

[0355] Method 21 200 ml of the reaction broth was centrifuged to separate the solid from the liquid (aqueous) phase (Sorvall GS3, 5000 rpm, 10 min, 10°C). This separated approximately 80 ml of solid pellet from approximately 120 ml of the liquid phase. Analysis of the aqueous phase after MTBE extraction (gas chromatography, Example 8) showed that it contained approximately 0.3% or less of the (-)-ambrofix initially present in the 200 ml reaction broth. Toluene and 99% ethanol were used to extract Ambrofix from the solid phase.

[0356] Result 21 Toluene extraction: 80 ml of the solid phase was extracted 6 times by 45 ml of toluene (approximately half the solid phase volume), vigorous shaking for 30 seconds, and centrifugation (Sorvall GS3, 5000 rpm, 10 min, 10°C). The solvent phase was analyzed by GC for its (-)-ambrox content. Over 99.5% of the (-)-ambrox initially present in the reaction broth was extracted by six extractions, each corresponding to a total toluene volume of 1.35 × initial total reaction broth volume (200 ml) or 3.4 × solid phase volume. The graph in Figure 24 shows the extraction progress by toluene washing as a percentage of the amount of (-)-ambrox initially present in 200 ml of total reaction broth (due to the volume ratio of broth / toluene, the percentage in the first extract exceeds 100%). Ethanol extraction: 80 ml of solid phase was extracted with approximately 160 ml (2x volume) of 99% ethanol (Infors Multifors HT, 35°C, 1000 rpm, 30 minutes), followed by centrifugation. Ambrox did not crystallize during the extraction procedure. The graph in Figure 25 shows that after four washes (a total of 640 ml of EtOH, i.e., 3.2 × total initial reaction broth volume or 8 × solid phase volume), approximately 99% of the ambrox initially present in the reaction broth was recovered. Sufficient ethanol is required in the first extraction step to prevent ambrox crystallization (it is soluble in ethanol). When only 1 or 1 / 2x volume of solid phase was used in the first extraction step, a sticky paste was obtained, which was difficult to handle, and (-)-ambrox crystallized as needle-like pellets during centrifugation. Temperature did not appear to be a contributing factor to this crystallization (extraction and centrifugation were tested at room temperature and approximately 35°C–40°C). The (-)-ambrox concentration in the EtOH phase and the EtOH / water ratio in the liquid phase (residual moisture in the solid phase) appeared to be factors in crystal formation. However, it was noted that it was possible to reduce the volume of ethanol to one-fold the volume of the solid phase.

[0357] Comment 21 Since (-)-ambrox does not exist in the liquid phase at room temperature, it can be separated with the biomass and extracted with an organic solvent (e.g., a water-miscible solvent (e.g., ethanol) or a water-miscible solvent (e.g., toluene)). The centrifugation step to separate (-)-ambrox into the solid phase of the reaction mixture is advantageous because it reduces the amount of solvent required to extract (-)-ambrox.

[0358] Example 22 Perceptual Analysis Objective: To perform sensory analysis of the (-)-ambrox and by-products (compounds II, III, and IV) formed in the "crude" and "crystallized" extracts.

[0359] Result 22(a) The conversion of EEH yields (-)-ambrox (compound I) and (-)-ambrox isomers (compound IV). Result 22(b) Bioconversion of EZH yields macrocyclic ethers (compound II) and 9b-epi-ambrox (compound III). Result 22(c) The crude composition of (-)-Amblox contains compounds I, II, III, and IV, with each compound present in amounts of 87.1%, 2.8%, 2.5%, and 7.6%, respectively. Result 22(d) The compositions of selectively crystallized materials (laboratory scale) have the same constituent components present in amounts of 99.1%, 0.1%, 0.1%, and 0.7%, respectively. The results of the perceptual analysis were as follows: (-)-Amblox: OTH 0.2 ng / l (OTH is the odor threshold). Compound IV from EEH: weak, IsoE, woody, GC-TH 5-10 ng. Compound II from EZH: "Odorless" (GC-TH > 500 ng) (GC-TH is the detection threshold). Compound III from EZH: Approximately 10× higher GC-TH than Ambrox (approximately 2 ng).

[0360] conclusion The combined percentage of each of the three by-products (compounds II, III, and IV) in the "crude" extract is approximately 3%. The combined percentage of each of the three by-products (compounds II, III, and IV) in the "crystallized" extract is approximately 1% (laboratory scale). Sensory analysis of the three by-products (compounds II, III, and IV) shows a weaker odor than that from (-)-Amblox. In fact, the odor of 9b-epi-ambrox (compound III) is about 10 times weaker than that of (-)-ambrox, suggesting that it is essentially odorless. Perceptual analysis showed that the removal of one or more by-product compounds from (-)-ambrox can improve the odor of the remaining compound (i.e., (-)-ambrox), even if the removed compound itself is actually odorless. Specifically, an enhancement of the ambrox odor was observed in the absence of compounds II, III, and IV.

[0361] Example 23 Amblox recovery by steam extraction Method 23 Purity obtained for crude (-)-ambrox (vapor extracted) and crystallized (-)-ambrox The bioconversion reaction of EE:EZ86:14 was steam extracted, and the reaction product was crystallized as follows: The steam distillate was collected as a two-phase mixture. The organic phase was retained, and the aqueous phase was discarded. The composition of the organic phase was analyzed by GC, and the results are shown in Table 25 below (see "crude"). The organic phase was then concentrated to a dry state. Next, ethanol was added to the dried crude product, and the mixture was heated until the product dissolved. At room temperature, water was slowly added, and (-)-ambrox crystallized under occasional stirring and cooling in an ice bath.

[0362] Result 23 Table 25 below also shows the GC analysis results of the ("crude") product and the crystallized product ((-)-ambrox) obtained after the steam extraction / distillation step. The references to "EZH" and "EEH" in Table 25 refer to (3Z,7E) homofarnesol and 7E,3E homofarnesol, respectively. Table 25 shows that a specific starting material (EEH:EZH86:14) produces a highly specific mixture (II, IV, and III) of the desired final product (-)-ambrox and by-products using WT SHC or SHC derivatives. Selective crystallization data show a strong enrichment of (-)-ambrox (I), with virtually no by-products (II), (IV), or (III) found in the crystallized samples. Thus, this EE:EZ mixture provides an olfactory-pure (-)-ambrox product that crystallizes selectively in a relatively simple, straightforward, and cost-effective manner. [Table 30]

[0363] Consideration 23 Vapor extraction / filtration is an environmentally friendly method for isolating ambrox because it provides convenient solvent-free isolation of ambrox and is accompanied by the secondary inactivation of its biocatalyst.

[0364] Summary 23 (-)-Amblocx produced by the bioconversion reaction can be extracted from the entire reaction mixture using a solvent (e.g., by a non-aqueous miscible solvent, or by vapor extraction / distillation, or by filtration) or from the solid phase (e.g., by a water-miscible solvent) using methods known to those skilled in the art.

Claims

1. A biotransformation reaction mixture comprising an aqueous phase, a solid phase, and an oil phase, wherein the solid phase contains (-)-ambrox, which is a reaction product, in solid form. Here, the reaction product (-)-ambrox is the by-product (II), (IV), and / or (III): 【Chemistry 1】 The reaction mixture is free from or contains by-products (II), (IV), and / or (III) in olfactory-tolerable amounts only.

2. A reaction product comprising (-)-ambrox in solid form as described in claim 1, wherein the reaction product comprises 80% to 97% (-)-ambrox.

3. A reaction product isolated from the biotransformation reaction mixture described in claim 1, wherein the reaction product comprises 80% to 97% (-)-ambrox.

4. The reaction mixture or reaction product according to any one of claims 1 to 3, wherein (-)-Amblocx is in an amorphous form.

5. The reaction mixture or reaction product according to any one of claims 1 to 3, wherein (-)-ambrox is in crystalline form.

6. The reaction mixture or reaction product according to any one of claims 1 to 5, wherein the reaction product comprises 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% (-)-ambrox.

7. The reaction mixture or reaction product according to any one of claims 1 to 6, wherein the (-)-ambrox content is determined by the peak area as measured by gas chromatography.

8. The reaction mixture or reaction product according to any one of claims 1 to 7, wherein by-products (II), (IV), and (III) are not found in solid form.

9. The reaction mixture or reaction product according to any one of claims 1 to 8, wherein the amount of (-)-ambrox product is 20 g / l to 200 g / l.

10. A method for producing a product containing (-)-ambrox, the method comprising incorporating a reaction product or reaction mixture according to any one of claims 1 to 9 into the product.

11. The method according to claim 10, wherein the product is a fragrance product, a cosmetic product, a detergent product, or a soap product.

12. Use of the reaction product or reaction mixture according to any one of claims 1 to 9 as part of a fragrance, cosmetic, or consumer care product.

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