Enzymes and applications thereof

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 squalene-hopene cyclase / homofarnesol-ambrox cyclase (SHC / HAC) derivative enzymes modified with respect to a reference SHC / HAC protein, amino acid sequences comprising the SHC / HAC derivative enzymes, nucleotide sequences encoding SHC / HAC derivatives, vectors comprising nucleotide sequences encoding SHC / HAC derivatives, and recombinant host cells comprising nucleotide sequences encoding SHC / HAC derivatives. The present invention also relates to means for functionally expressing the nucleotide sequences encoding SHC / HAC derivatives, and methods of using recombinant microorganisms comprising nucleotide sequences encoding SHC / HAC derivatives and WT SHC / HAC to make ambrox, preferably (-)-ambrox. [Background technology]

[0002] Squalene-hopene 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 hopene and hopanol. Early SHC studies focused on the properties of the SHC of 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). More recently, however, other SHCs from Zymomonas mobilis and Bradyrhizobium japonicum have been purified and characterized in terms of their natural (e.g., squalene) and unnatural 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.) disclosed that homofarnesol is an additional substrate for Alicyclobacillus acidocaldarius SHC (AacSHC). However, the cyclization rate of unnatural homofarnesol by the purified AacSHC 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 with homofarnesol (product 1b) concentrations from 0.25 mM to 2.0 mM and decreased somewhat in the presence of 4 mM product 1b. The difference in cyclization rate may be partially attributed to the fact that the natural SHC substrate squalene is twice the size (C30 carbon compound) of unnatural homofarnesol, which is a 16-carbon compound.

[0004] (JP2009060799 - Kao) also discloses a method for producing ambrox from homofarnesol using SHC from A. acidocaldarius. While JP2009060799 teaches the possibility of using microorganisms containing SHC for the synthesis of ambrox, JP2009060799 only discloses the production of ambrox from homofarnesol using SHC extract prepared from a recombinant microorganism expressing the SHC gene, not by means of recombinant microbial whole cells expressing the SHC gene. The conversion percentage of homofarnesol to ambrox using SHC extract was reported to be 17.5% when carried out at a temperature of 60°C and pH 5.2-6.0 for 14 hours, but only 6.8% when carried out at a pH of 6.6. The percent conversion of 3E,7E-homofarnesol to ambrox using SHC extract at 60°C and pH 5.6 for 64 h 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 with homofarnesol to ambrox cyclase activity. Zymomonas mobilis (Zmo) SHC and Bradyrhizobium japonicum (Bjp) SHC enzymes were reported to show 41% and 22% homofarnesol conversion rates, respectively, in a 16 h reaction when a homofarnesol concentration of 10 mM (2.36 g / l) was used, whereas the conversion rate of AacSHC was reported to be only 1.2% (presumably at the same homofarnesol concentration), although no experimental details were provided. ZmoSHC and BjpSHC enzyme extracts were prepared from recombinant microorganisms expressing the SHC gene by disrupting E. coli host cells producing the SHC enzyme and isolating the soluble SHC fraction.

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

[0007] WO2012 / 066059 discloses mutants with cyclase activity and their use in methods for the biocatalytic cyclization of terpenes, in particular for the production of isopulegol by cyclization of citronellal, for the production of menthol and for the biocatalytic transformation of other compounds with terpene-type structural motifs. Sequence alignment of various SHCs identified phenylalanine 486 (F486) as a strongly conserved amino acid residue, and a series of substitution variants of the Zymomonas mobilis SHC enzyme were generated. Some of these substitutions led to loss of activity, while others led to the formation of a novel terpenoid product (isopulegol) from terpene substrates such as citronellal.

[0008] A report in a PhD thesis by Seitz in 2012 (http: / / elib.uni-stuttgart.de / handle / 11682 / 1400) shows that the F486Y mutation in ZmoSHC1 provided a reduced biotransformation rate of homofarnesol by approximately 1.5-fold, from 34.8% (WT ZmoSHC1) to 23.9% (mutant ZmoSHC1 F486Y). When the mutant equivalent in AacSHC (Y420C) was tested, it was speculated that the enzyme activity towards larger substrates would be reduced and the activity towards smaller substrates would be increased. When the mutant was tested under the same conditions as the wild type and the enzyme activity was compared, it was observed that the mutant did not show any conversion of homofarnesol substrate at all. Therefore, it was concluded that the Y420 amino acid residue is critical for the activity of AacSHC towards all substrates.

[0009] Other site-directed mutagenesis studies of SHC in the 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 analogues) rather than unnatural substrates such as homofarnesol.

[0010] In summary, the limited disclosure in the art of bioconversion processes for the successful conversion of homofarnesol to ambrox relates only to relatively low concentrations / volumes of homofarnesol substrate (within the concentration range of 0.25 mM to 2 mM to 10 mM, or approximately 0.06 g / l to 2.36 g / l) using wild-type SHC polypeptides having homofarnesol-ambrox cyclase (HAC) activity. The SHC enzyme with HAC activity was either (i) an extract prepared by either disrupting E. coli host cells containing the SHC enzyme and separating insoluble and soluble SHC liquid fractions, (ii) a partially purified membrane fraction, 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 solubilizing agent that included either (i) Triton X-100 (see Neumann and Simon 1986 ibid., Seitz et al. 2012 ibid., 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 obtained conversion rate of homofarnesol to ambrox was found to vary depending on the source of SHC enzyme used, the amount of homofarnesol starting material, and the reaction conditions. So far, 100% conversion percentage of homofarnesol to ambrox using wild-type SHC enzyme has not been achieved at the reported concentrations covered (0.06-2.36 g / l). In addition, preliminary investigations using SHC derivatives prepared using site-directed mutagenesis studies provided only negative (i.e., reduced homofarnesol conversion) results rather than positive (i.e., improved conversion). In addition, only purified SHC enzyme extracts or SHC membrane-bound fractions have been used in published studies, or whole cells of recombinant microorganisms expressing the WT SHC gene under specific reaction conditions using solubilizing agents such as Triton X-100 or taurodeoxycholic acid. There is no evidence that recombinant microorganisms containing either the WT or mutant SHC can provide a more efficient and cost-effective bioconversion of homofarnesol to ambrox using optimized reaction conditions. Therefore, it is desirable to improve the cited known processes for preparing ambrox from homofarnesol, at least by improving the reaction rate, specificity, yield, productivity, and reducing costs (e.g., by simplifying the process either by using whole cells of recombinant microorganisms or by using a "one-pot" process that combines both biocatalytic production and bioconversion steps). Summary of the Invention

[0012] In various aspects, the present invention provides SHC / HAC derivatives, amino acid sequences comprising SHC / HAC derivative enzymes, nucleotide sequences encoding SHC / HAC derivative enzymes, vectors comprising nucleotide sequences encoding SHC / HAC derivative enzymes, recombinant host cells comprising vectors having nucleotide sequences encoding SHC / HAC derivative enzymes, and the application of recombinant host cells comprising either SHC / HAC derivative enzymes or WT SHC / HAC enzymes when used in a process for preparing ambrox materials containing an ambrox isomer designated (-)-ambrox and ambrox-like molecules (as by-products) under certain reaction conditions. Unlike disclosures in the art regarding AacSHC, applicants have shown for the first time that whole recombinant microorganisms expressing SHC derivative genes can be used to bioconvert homofarnesol to ambrox. In addition, whole recombinant microorganisms expressing the WT SHC gene and / or producing the SHC enzyme can be used to bioconvert homofarnesol to ambrox under specific reaction conditions not disclosed in the art.

[0013] It has also been surprisingly discovered that introducing up to five amino acid mutations into the amino acid sequence of the WT SHC / HAC reference sequence as disclosed herein results in SHC / HAC derivative enzymes that have significantly improved homofarnesol to ambrox conversion rates as compared to the unmodified SHC reference enzyme as disclosed herein. These novel SHC / HAC derivative enzymes, alone and in combination, are useful for the production of ambrox materials, particularly (-)-ambrox, from homofarnesol substrates.

[0014] An additional surprising finding is that, apart from one mutation (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 finding, since changes in conserved regions of an enzyme are more likely to impair the function of the enzyme (at least in relation to its natural substrates) 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 operate optimally (for unnatural substrates such as homofarnesol) at about 35° C. rather than the typical reaction temperature of about 60° C. for thermophilic microorganisms such as AacSHC. Application of the SHC derivatives of the present disclosure to a process for preparing ambrox from homofarnesol at lower reaction temperatures has significant cost advantages for industrial-scale ambrox production cycles.

[0016] Another advantage of the present invention is that the SHC derivative enzymes of the present disclosure catalyze an efficient bioconversion process, which can lead to 100% conversion of homofarnesol substrate when optimized for a relatively high (e.g., about 50-fold) homofarnesol substrate concentration compared to concentrations previously described in the prior art (e.g., 125 g / l EEH), while the reference WT SHC protein only converts about 10% of the same substrate even at high enzyme / cell concentrations. All of the cited prior art disclosures relate to the use of purified membrane extracts or purified SHC extracts (prepared from microorganisms expressing the SHC gene) containing SHC, or the use of recombinant microorganisms expressing the WT SHC gene under specific bioconversion reaction conditions (e.g., using specific solubilizing agents). Even so, 100% homofarnesol conversion at much lower EEH concentrations has not been reported. Also, a "one-pot" reaction in which recombinant cells grow and produce SHC enzyme in a first step, followed by conversion of EEH to (-)-ambrox in the same vessel, has not been reported. A further advantage of the present invention is that the recombinant host cells producing the SHC derivative enzymes exhibit high initial reaction rates, which allows for the production of high amounts of product within a relatively short period of time, while using only relatively low amounts of biocatalyst. In summary, the selection and efficient expression of recombinant microorganisms containing either WT SHC / HAC or specific SHC / HAC derivative enzymes, and their application under specific bioconversion reaction conditions, leads to a more efficient bioconversion process. The final product ((-)-ambrox) can be separated and easily purified. Unlike the cited techniques, the SHC / HAC derivative enzymes are not used as pure enzymes, but in whole-cell content (as biocatalysts), which is a more cost-effective and more user- and environmentally friendly approach since no additional enzyme purification and isolation steps are required.

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

[0018] Detailed Description of the Invention The term "SHC" as used herein refers to a squalene-hopene cyclase enzyme from any of the sources listed in Tables 10-12. In preferred embodiments, the term SHC encompasses the Zymomonas 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 dissertation, ibid). For ease of reference, the designation "AacSHC" is used for Alicyclobacillus acidocaldarius SHC, the designation "ZmoSHC" is used for Zymomonas mobilis SHC, and the designation "BjpSHC" is used for Bradyrhizobium japonicum SHC. The percent sequence identity of these sequences relative to WT AacSHC and each other (which may vary depending on the algorithm used) is given in Tables 18 and 19.

[0019] An alignment of the WT SHC sequences prepared by Hoshino and Sato (2002 ibid.) showed that multiple motifs were detected in all four sequences, consisting of a core sequence Gln-XXX-Gly-X-Trp, which is found six times in the SHC sequences of both Z. mobilis and A. acidocaldarius (see Fig. 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 the SHC and noted two characteristic motifs in the SHC: 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 DXDDTA motif. Wendt et al (1997, Science 277, 1811-1815 and 1999, J Mol Biol 286, 175-187) reported the X-ray structure of A. acidocaldarius SHC. The DXDDTA motif appears to correlate with the SHC active site. Exemplary sequence alignments from the prior art show multiple cyclic motifs, provided herein in Figure 2 (from Hoshino and Sato (2002 ibid.)) and Figure 3 (from Seitz PhD thesis (2012)).

[0020] As used herein, a reference (or wild-type) AacSHC protein refers to the AacSHC protein disclosed in SEQ ID NO: 1. The reference AacSHC enzyme of the present disclosure has the activity of a homofarnesol-ambrox cyclase (HAC), which is useful for the production of ambrox derivatives by the biocatalytic reaction of SHC with a homofarnesol substrate. The primary reaction of the reference AacSHC is the cyclization of linear or non-linear substrates, such as homofarnesol, to produce ambrox.

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

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

[0023] (-)-Ambrox is an industrially important aroma compound and has been used for a long time in the fragrance industry. The particular desirable sensory benefits of (-)-Ambrox come from the (-) stereoisomer rather than the (+) one. The odor of the (-) stereoisomer is described as musky, woody, warm, or ambery, while the (+)-Ambrox enantiomer has a relatively weak odor note. The odors and odor thresholds of ambrox-like products also differ. Although various (-)-Ambrox concentrated materials are commercially available, it is desirable to produce highly concentrated (-)-Ambrox materials, ideally pure (-)-Ambrox.

[0024] Production of (-)-ambrox (-)-Ambrox can be produced from sclareolide according to the production process as follows. Sclareol is a product extracted from the natural plant clary sage. However, since natural starting materials are used in this process, it involves multi-step reactions, the process is circuitous, the amount and stability of the supply of 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 step of oxidative decomposition of (+)-sclareol. [ka]

[0025] (-)-Ambrox is also synthesized from homofarnesol using different routes. For example, homofarnesol can be obtained by bromination, cyanation, and hydrolysis of nerolidol to give homofarnesylic acid, followed by reduction. Alternatively, homofarnesol may be obtained from farnesol, farnesyl chloride, 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 ester, which is then converted to EEH. Overviews of the production of (-)-ambrox from different substrates can be found in US2012 / 0135477A1, WO2010 / 139719, US2013.0273619A1, WO2013 / 156398A1, as well as in the PhD dissertation of Seitz (2012 ibid.) and Schaefer 2011 (Chemie Unserer Zeit 45, 374-388).

[0026] While homofarnesol can exist as a mixture of four isomers, the (3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E) isomers, from the literature it appears that (-)-ambrox can only be derived from (3E,7E) homofarnesol (see Neumann and Simon (1986) supra). References to (3E,7E) homofarnesol as used herein are references to E,E-homofarnesol, which is also referred to as EEH.

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

[0028] In some embodiments, preferably, the homofarnesol starting material comprises a mixture of (3E,7E) and (3Z,7E), referred to herein as the EE:EZ stereoisomeric mixture (especially with reference to the Examples and Table 20).

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

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

[0031] Thus, in some embodiments, the homofarnesol starting material can also include a mixture of four isomers EE:EZ:ZZ:ZE corresponding to (3E,7E), (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 group: [(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] Thus, in certain embodiments, the ratio of EEH:EZH is about 100:00, 99:01, 98:02, 97:03, 96:04, 95:05, 94:06, 93:07, 92:08, 91:09, 90:10, 89:11, 88:12, 87:13, 86:14, 85:15, 84:16, 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, preferably the homofarnesol starting material contains >90% E,E-homofarnesol (EEH).

[0036] In other embodiments, the homofarnesol starting material contains a weight ratio of EE:EZ of 86:14.

[0037] In certain embodiments, the homofarnesol starting material comprises an EE:EZ weight ratio of 80:20.

[0038] In certain embodiments, the homofarnesol starting material comprises a weight ratio of EE:EZ of 70:30.

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

[0040] In some embodiments, the homofarnesol starting material consists or consists essentially 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 or consists essentially of a mixture of isomers selected from one or more of the following group: [(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 consists essentially of a mixture of isomers selected from one or more of the following group: q[(3E,7E), (3Z,7E)], and / or [(3Z,7E), (3E,7E), and (3E,7Z)].

[0043] Thus, in certain embodiments, the ratio of EEH:EZH isomers is about 100:00, 99:01, 98:02, 97:03, 96:04, 95:05, 94:06, 93:07, 92:08, 91:09, 90:10, 89:11, 88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, 81:19, 80:20, 79:21, 78:22, 77:23, 76:24, 75:2 50:50, 74:26, 73:27, 72:28, 71:29, 70:30, 69:31, 68:32, 67:33, 66:34, 65:35, 64:36, 63:37, 62:38, 61:39, 60:40, 59:41, 58:42, 57:43, 56:44, 55:45, 54:46, 53:47, 52:48, 51:49, or about 50:50 EEH:EZH ratio.

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

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

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

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

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

[0049] In an embodiment of the present disclosure, ambrox is produced using SHC / HAC derivative enzymes.

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

[0051] The SHC / HAC derivatives of the present disclosure are tested for their homofarnesol-ambrox cyclase activity. As a result, those 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 Alicyclobacillus acidocaldarius, Zymomonas mobilis, and Bradyrhizobium japonicum microbial strain sources, the present disclosure is also directed to equivalent SHC / HAC derivatives from other microbial strain sources, including, but not limited to, SHC / HAC enzymes from Methylococcus capsulatus, Frankia arnii, 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" refers to an insertion of one or more amino acids between two amino acids, a deletion of one or more amino acids, or a substitution of one or more amino acids with one or more different amino acids (which may be conservative or non-conservative) relative to the amino acid sequence of a reference amino acid sequence (such as, for example, the wild-type (WT) amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4). Amino acid modifications can be readily identified by comparison of the amino acid sequence of the SHC / HAC derivative amino acid sequence to the amino acid sequence of the reference amino acid sequence (such as, for example, the wild-type (WT) amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or 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, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The twenty naturally occurring amino acids outlined above can be grouped into six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile, (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that influence chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe.

[0054] Thus, the term "conservative substitution" as used herein refers to the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the replacement of Asp with Glu retains one negative charge in the polypeptide so modified. In addition, glycine and proline can be substituted for each other based on their ability to break alpha-helix. Some preferred conservative substitutions within the above six groups are the replacements within the following subgroups: (i) Ala, Val, Leu, and Ile, (ii) Ser and Thr, (ii) Asn and Gln, (iv) Lys and Arg, and (v) Tyr and Phe. In view of the known genetic code and recombinant and synthetic DNA techniques, skilled scientists can easily construct DNA encoding conservative amino acid variants.

[0055] As used herein, a "non-conservative substitution" or "non-conservative amino acid exchange" is defined as the replacement of one amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

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

[0057] For ease of reference, the one-letter amino acid symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission are shown as follows: Three-letter codes are also provided for reference purposes. [Table 1]

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

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

[0060] In various embodiments, the mutation or combination of mutations enhances the activity of the SHC / HAC derivative for converting homofarnesol to ambrox, as compared to a reference SHC enzyme that does not exhibit this deletion / addition. Protein modeling, as described herein, can 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 can be created using the coordinates of AacSHC (e.g., as shown in Figures 19 and 20). As shown herein, such homology models are useful for directing improvements in SHC enzymes for converting homofarnesol to (-)-ambrox.

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

[0062] In various embodiments, the SHC / HAC derivatives include sequences that have 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 according to at least SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4. In various embodiments, the SHC derivatives have 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, about 23 mutations, or more relative to a reference SHC (such as, for example, SEQ ID NO: 1 or 2 or 3 or 4). The polypeptide may have about 24 mutations, about 25 mutations, about 26 mutations, about 27 mutations, about 28 mutations, about 29 mutations, about 30 mutations, about 31 mutations, about 32 mutations, about 33 mutations, about 34 mutations, about 35 mutations, about 36 mutations, about 37 mutations, about 38 mutations, about 39 mutations, about 40 mutations, about 41 mutations, about 42 mutations, about 43 mutations, about 44 mutations, about 45 mutations, about 46 mutations, about 47 mutations, about 48 mutations, about 49 mutations, or about 50 mutations.

[0063] In these and other embodiments, the SHC / HAC derivatives have at least about 50% sequence identity, at least about 55% sequence identity, at least about 60% sequence identity, at least about 65% sequence identity, at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, or at least about 90% sequence identity between WT SHC (e.g., SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4) or between a reference sequence (e.g., AacSHC (SEQ ID NO:1) and another SHC sequence (see, e.g., Tables 18 and 19 in WO 2010 / 139719, which show at least 34-52% identity between AacSHC (SEQ ID NO:1) and ZmoSHC). The amino acid sequence may comprise an amino acid sequence having 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 greater activity for converting homofarnesol to ambrox than the wild-type enzyme, e.g., greater production of (-)-ambrox upon contact with a homofarnesol substrate than a reference wild-type enzyme (such as, e.g., SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4).

[0065] For example, an SHC / HAC derivative may have at least about 50% identity, about 51% identity, about 52% identity, about 53% identity, about 54% identity, about 55% identity, about 56% identity, about 57% identity, about 58% identity, about 59% identity, about 60% identity, about 61% identity, about 62% identity, about 63% identity, about 64% identity, about 65% identity, about 66% identity, about 67% identity, about 68% identity, about 70% identity, about 72% identity, about 74% identity, about 76% identity, about 78% identity, about 79% identity, about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, about 100% identity, about 101% identity, about 102% identity, about 103% identity, about 104% identity, about 105% identity, about 106% identity, about 107% identity, about 108% identity, about 109% identity, about 110% identity, about 111% identity, about 112% identity, about 113% identity, about 114% identity, about 115% identity, about 116% identity, about 117% identity, about 118% identity, about 119% identity, about 120% identity, about 125% identity, about 126% % identity, about 69% identity, about 70% identity, about 71% identity, about 72% identity, about 73% identity, about 74% identity, about 75% identity, about 76% identity, about 77% identity, about 78% identity, about 79% identity, about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 84% identity, about 85% identity, about It may comprise an amino acid sequence having 86% identity, about 87% identity, about 88% identity, about 89% identity, about 90% identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, or about 99% sequence identity.

[0066] Various SHC / HAC derivatives that have been tested for SHC enzymatic activity are listed in one or more of Tables 1-9. Thus, in various embodiments, the SHC / HAC derivative 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 four mutations compared to the wild-type / reference amino acid sequence according to SEQ ID NO:1, and comprising an amino acid sequence comprising at least the substitutions F601Y or M132R in combination with at least any one or more of F129L and / or I432T relative to SEQ ID NO:1, and optionally comprising a leader sequence that aids in expression and activity in E. coli.

[0067] In other embodiments, the SHC / HAC derivative is a modified SHC polypeptide and comprises an amino acid sequence having up to eight mutations compared to the wild-type / reference amino acid sequence according to SEQ ID NO:1 (or its counterpart modified for expression in E. coli) and including one or more single amino acid alterations 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, and the SHC / HAC derivative has modified (e.g., increased) enzymatic activity relative to SEQ ID NO:1.

[0068] In one embodiment, the SHC derivative comprises one or more substitutions selected from the group of mutants 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 mutants 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 comprises one or more substitutions selected from the group of mutants consisting of S129A, V145V, F182L, Y185R, G282V, I498T, H646H, F668Y, and F698X relative to SEQ ID NO:2, as set forth 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 comprises one or more substitutions selected from the group of mutants consisting of G85A, V100V, F137L, I140R, V233V, I450T, N598H, F620Y, and F624W relative to SEQ ID NO:3, as set forth in Table 3 and Table 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 further embodiments, the SHC derivative comprises one or more substitutions selected from the group consisting of A88A, V104V, F141L, Y144R, V241V, I459T, M607H, F628Y, and F658W relative to SEQ ID NO:4, as set forth in Table 4.

[0076] SHC Derivative Combinations In one embodiment, the SHC derivative comprises one or more substitutions selected from the group of mutants consisting of T77A, F129L, M132R, I92V, A224V, I432T, Q579H, and F601Y relative to SEQ ID NO:1, as set forth in Table 5.

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

[0078] In one embodiment, the SHC derivative comprises one or more substitutions selected from the group of mutants consisting of G85A, V100V, F137L, I140R, V233V, I450T, N598H, and F620Y relative to SEQ ID NO:3, as set forth in Table 7.

[0079] In further embodiments, the SHC derivatives include one or more substitutions selected from the group consisting of A88A, V104V, F141L, Y144R, V241V, I459T, M607H, and F628Y relative to SEQ ID NO:4, as set forth 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 the substitutions F601Y or M132R in combination with at least any one or more of F129L and / or I432T relative to SEQ ID NO:1.

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

[0082] Hoshino and Sato (2002 ibid.) identified F601 as a highly conserved amino acid residue among prokaryotic and eukaryotic species. It was reported that the SHC derivative F601Y showed a greatly increased Vmax with oxidosqualene (but not squalene) substrate. However, F601Y showed a decreased affinity (i.e., a higher K) relative to WT AacSHC when squalene was used. M ) and catalytic efficiency / activity (Kcat / K M ) is shown to be decreased. No data on AacSHC efficacy when homofarnesol is used as the enzyme substrate in the F601Y mutant are provided in Hoshino and Sato (2002 ibid).

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

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

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

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

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

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

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

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

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

[0092] In a preferred embodiment, the SHC derivative comprises at least the substitutions F668Y or Y185R in combination with at least any one or more of F182L and / or I498T relative to SEQ ID NO:2.

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

[0094] Hoshino and Sato (2002 ibid.) identified F601 as a highly conserved amino acid residue among prokaryotic and eukaryotic species. It was reported that the SHC derivative F601Y showed a greatly increased Vmax with oxidosqualene (but not squalene) substrate. However, F601Y showed a decreased affinity (i.e., a higher K) relative to WT AacSHC when squalene was used. M ) and catalytic efficiency / activity (Kcat / K M ) is shown. No data on AacSHC efficacy when homofarnesol is used as the enzyme substrate for the F601Y mutant is provided in Hoshino and Sato. The equivalent SHC derivative to F601Y in ZmoSHC1 is F668Y.

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

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

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

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

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

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

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

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

[0103] The SHC derivative provided in this disclosure, designated 111C8ZM1, contains 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, relative to SEQ ID NO:3, at least the substitutions F620Y or I140R in combination with at least any one or more of F137L and / or I450T.

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

[0106] Hoshino and Sato (2002 ibid.) identified F601 as a highly conserved amino acid residue among prokaryotic and eukaryotic SHC species. It was reported that the AacSHC derivative F601Y showed a greatly increased Vmax with oxidosqualene (but not squalene) substrate. However, F601Y showed reduced affinity (i.e., a higher K) relative to WT AacSHC when squalene was used. M ) and catalytic efficiency / activity (Kcat / K M) is decreased. No data on AacSHC efficacy when homofarnesol is used as the enzyme substrate for the F601Y mutant are provided in Hoshino and Sato (2002). The equivalent SHC derivative to F601Y in ZmoSHC2 is F620Y.

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

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

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

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

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

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

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

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

[0115] The SHC derivative provided in this disclosure, designated 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 the substitutions F628Y or Y144R in combination with at least any one or more of F141L and / or I459T relative to SEQ ID NO:4.

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

[0118] Hoshino and Sato (2002 ibid.) identified F601 as a highly conserved amino acid residue among prokaryotic and eukaryotic species. It was reported that the SHC derivative F601Y showed a greatly increased Vmax with oxidosqualene (but not squalene) substrate. However, F601Y showed a decreased affinity (i.e., a higher K) relative to WT AacSHC when squalene was used. M ) and catalytic efficiency / activity (Kcat / K M ) is shown. No data on AacSHC efficacy when homofarnesol is used as the enzyme substrate for the F601Y mutant is provided in Hoshino and Sato. The equivalent SHC derivative to F601Y in BjpSHC is F628Y.

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

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

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

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

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

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

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

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

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

[0128] Amino acid sequence In some embodiments, the AacSHC / HAC derivatives comprise one or more of the polypeptides set forth 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 the present 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 other embodiments, a ZmoSHC1 / HAC derivative comprises one or more of the polypeptides set forth 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 derivatives of the present disclosure have 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 further embodiments, the ZmoSHC2 / HAC derivative comprises one or more of the polypeptides set forth in one or more of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, and / or SEQ ID NO:175.

[0133] In additional embodiments, the BjpSHC / HAC derivative comprises one or more of the polypeptides set forth in one or more of SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, and / or SEQ ID NO:177.

[0134] Sequence alignment Due to the different lengths of SHC reference sequences, e.g., the AacSHC, ZmoSHC1, ZmoSHC2, and BjpSHC polypeptide sequences, an 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, alterations of the SHC reference sequences can also alter the SHC derivative sequence relative to the reference SHC sequence.

[0135] The term "position" refers to a particular amino acid residue present in a reference SHC protein, identified by a particular numbering of the amino acid. Alteration of the SHC reference protein, either by insertion or deletion of an amino acid, will result in different numbering between the reference SHC amino acid sequence and the SHC derivative amino acid sequence. As an example, if an amino acid is inserted between amino acids 509 and 510 of a reference SHC protein, the amino acid next to the insertion will have the number 511 in the SHC derivative protein, while it retains the number 510 in the SHC reference protein.

[0136] Assays to determine 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. By way of example, WT SHC / HAC and / or SHC / HAC derivative activity can be determined by incubating purified SHC / HAC enzyme or extract from a host cell, or a whole recombinant host organism that produced the SHC / HAC enzyme, with an appropriate substrate under appropriate conditions and analyzing the reaction products (e.g., by gas chromatography (GC) or HPLC analysis). Further details of SHC / HAC and / or SHC / HAC enzyme activity assays and analysis of the reaction products are provided in the Examples. These assays involve producing SHC derivatives by recombinant host cells (e.g., E. coli).

[0137] The term "activity" as used herein refers to the ability of an enzyme to react with a substrate to provide a target product. Activity may be determined by the increase in target product as a function of time, the decrease in substrate (or starting material), or a combination of these parameters, in what is known as an activity test. The SHC / HAC derivatives of the present 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" refers to any activity that a polypeptide may exhibit, including, without limitation, enzymatic activity, binding activity to another compound (e.g., binding to another polypeptide, particularly binding to a receptor, or binding to a nucleic acid), inhibitory activity (e.g., enzyme inhibitory activity), activating activity (e.g., enzyme activating activity), or toxic effect. There is no requirement that a variant or derivative exhibit such activity to the same extent as the parent polypeptide. A variant is considered to be a variant in the context of this application if it exhibits an activity related to at least 10% of the activity of the parent polypeptide. Similarly, a derivative is considered to be a derivative in the context of this application if it exhibits a biological activity related to at least 10% of the activity of the parent polypeptide (as the terms derivative and variant are used interchangeably in this disclosure).

[0139] In other embodiments, the SHC / HAC derivatives of the present disclosure exhibit better target yields than the reference SHC protein. The term "target yield" refers to grams of recoverable product per gram of feedstock (which may be calculated as a percent molar conversion).

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

[0141] In further embodiments, the SHC / HAC derivatives of the present disclosure exhibit an altered target yield factor relative to a reference SHC protein. The term "target yield factor" refers to the ratio between the resulting product concentration in the reaction medium and the concentration of the SHC derivative (e.g., purified SHC enzyme, or an extract from a recombinant host cell expressing the SHC enzyme).

[0142] In various embodiments, the SHC derivatives of the disclosure exhibit an altered (e.g., increased) fold increase in enzymatic activity (e.g., altered / increased homofarnesol-ambrox cyclase (HAC) activity) relative 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 by at least a factor of 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.

[0143] Nucleotide sequence The present disclosure further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an SHC derivative described herein.

[0144] The term "nucleic acid molecule" as used herein specifically refers to a polynucleotide of the present disclosure, which may be DNA, cDNA, genomic DNA, synthetic DNA, or RNA, and may be double-stranded or single-stranded, sense and / or antisense strand. The term "nucleic acid molecule" specifically applies to a polynucleotide or polynucleotides as used herein, for example, as a full-length nucleotide sequence or a fragment or portion thereof, each of which encodes a polypeptide having enzymatic activity, such as an enzyme of a metabolic pathway, or a fragment or portion thereof.

[0145] The term also encompasses other molecules, such as cDNAs in which the corresponding genomic DNA contains introns and therefore a different sequence, genomic fragments lacking at least one of the flanking genes, fragments of cDNAs or genomic DNAs produced by polymerase chain reaction (PCR) and lacking at least one of the flanking genes, restriction fragments lacking at least one of the flanking genes, DNAs encoding non-naturally occurring proteins such as fusion proteins (e.g. His tags), muteins, or fragments of a given protein, and nucleic acids that are degenerate variants of cDNAs or naturally occurring nucleic acids. In addition, it encompasses hybrid genes, i.e. recombinant nucleotide sequences that are part of a gene that encodes a non-naturally occurring fusion protein. Fusion proteins can add one or more amino acids to a protein (such as, but not limited to, histidine (His)), usually at the N-terminus of the protein, but also at the C-terminus or fused within a region of the protein. Such fusion proteins or fusion vectors encoding such proteins typically serve three purposes: (i) to increase the production of the recombinant protein, (ii) to increase the solubility of the recombinant protein, and (iii) to aid in the purification of the recombinant protein by providing a ligand for affinity purification. The term "nucleic acid molecule" also encompasses codon-optimized sequences suitable for expression by a particular microbial host cell, such as an E. coli host cell. As used herein, the term "codon-optimized" refers to a nucleic acid protein-coding sequence adapted for expression by a prokaryotic or eukaryotic host cell, particularly a bacterial host cell, such as an E. coli host cell, by the replacement of one or more, or preferably a substantial number of, codons with codons frequently used in bacterial (e.g. E. coli) host cell genes. In this regard, the nucleotide sequences encoding the reference sequences SEQ ID NO:1, 2, 3, and / or 4 and all variants / derivatives thereof may be those originally 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, for example, E. coli.

[0146] Ribonucleic acid (RNA) molecules may be produced by in vitro transcription. Segments of DNA molecules are also contemplated within the scope of the present disclosure, and may be produced, for example, by polymerase chain reaction (PCR) or by treatment with one or more restriction endonucleases. Segments of nucleic acid molecules, particularly those that are partial genes, may be referred to as DNA fragments of genes. Fragments may also contain several open reading frames (ORFs), either the same ORF or repeats of different ORFs. The term is intended to specifically refer to coding nucleotide sequences, but also to encompass nucleotide sequences that are non-coding, e.g., non-transcribed or non-translated sequences, or that code for a polypeptide in whole or in part. Genes as used herein, for example for assembly, diversification, or recombination, may be non-coding sequences, or 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 events. More specifically, said genes have a minimum length of 3 bp, preferably at least 100 bp, more preferably at least 300 bp.

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

[0148] The term "isolated DNA" as used herein can refer to (1) DNA that contains a sequence that is not identical to that of any naturally occurring sequence, a non-naturally occurring polynucleotide or nucleic acid (e.g., created by the artificial combination of two otherwise separated sequence segments by human intervention (e.g., the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques)), or (2) DNA that, in the context of a naturally occurring sequence (e.g., cDNA or genomic DNA), does not contain at least one of the genes that flank the gene containing the DNA of interest in the genome of the organism in which the gene containing the DNA of interest naturally occurs.

[0149] The term "isolated DNA" as used herein specifically with respect to nucleic acid sequences may also refer to nucleic acids or polynucleotides produced by recombinant DNA technology, such as DNA constructs that contain polynucleotides heterologous to a host cell and are optionally integrated into the host cell. Chimeric nucleotide sequences may specifically be produced as recombinant molecules. The term "recombination" specifically refers to the assembly of polynucleotides, joining such polynucleotides or portions thereof with or without recombination to achieve crossover or genetic mosaicism. For example, this is performed to join nucleic acid segments of desired functions to produce a desired combination of functions. A recombinant gene encoding a polypeptide as described herein includes the coding sequence of that polypeptide operably linked in a sense orientation to one or more control regions suitable for expressing the polypeptide. Since many microorganisms are capable of expressing multiple gene products from polycistronic mRNA, multiple polypeptides may be expressed under the control of a single control region for those microorganisms, if desired. A coding sequence and a control region are considered to be operably linked when the control region and coding sequence are positioned so that the control region is effective to control the transcription or translation of the sequence.

[0150] The term "recombinant" as used herein, specifically with respect to enzymes, refers to enzymes produced by recombinant DNA techniques, i.e., produced from cells transformed with an exogenous DNA construct encoding the desired enzyme. "Synthetic" enzymes are those prepared by chemical synthesis. Chimeric enzymes may specifically be produced as recombinant molecules. Thus, the term "recombinant DNA" encompasses recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote (or into the genome of a homologous cell at a location other than its natural chromosomal location).

[0151] In a further aspect, the nucleic acid molecule(s) of the present disclosure are operatively linked to an expression control sequence that allows expression by a prokaryotic and / or eukaryotic host cell. As used herein, "operatively linked" means that the expression control sequence is incorporated into a genetic construct to effectively control the expression of the coding sequence of interest. The transcription / translation control elements referred to above include, but are not limited to, inducible and non-inducible, constitutive, cell cycle-controlled, 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 constitutive expression or allow inducible expression, such as, for example, the CUP-1 promoter, the tet repressor employed in, for example, the tet on or tet off system, the lac system, the trp system control elements. As an example, isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effective inducer of gene expression in the concentration range of 100 μM to 1.0 mM. This compound is a molecular mimic of the lactose metabolite allolactose, which 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 control element that induces gene expression is lactose.

[0152] Similarly, the nucleic acid molecule(s) of the present 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 involved in the practice of the present disclosure, those skilled in the art will be aware of additional useful reagents, such as additional sequences that may function as markers or reporters.

[0153] In some embodiments, the present disclosure provides recombinant polynucleotides encoding WT SHC or the SHC / HAC derivatives described above, which can be inserted into a vector for expression and optional purification. One type of vector is a plasmid, which represents a circular double-stranded DNA loop into which additional DNA segments are ligated. Certain vectors can control the expression of genes to which they are operably linked. These vectors are called "expression vectors". Usually, expression vectors suitable for DNA recombinant technology are of the plasmid type. Typically, expression vectors contain genes such as the 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 DNA sequences, including but not limited to DNA sequences that are not naturally present in the host cell, DNA sequences that are not normally transcribed into RNA or translated into protein ("expressed"), and other genes or DNA sequences that one wishes to introduce into a non-recombinant host. Typically, it will be understood that the genome of the recombinant host described herein is expanded by stable introduction of one or more recombinant genes. However, self- or replicating plasmids or vectors may also be used within the scope of the present disclosure. Additionally, the present disclosure may be practiced using low copy number, e.g., single copy, or high copy number (as exemplified herein) plasmids or vectors.

[0155] In preferred embodiments, vectors of the present disclosure include plasmids, phagemids, phages, cosmids, artificial bacterial and artificial yeast chromosomes, knock-out 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, e.g., plant, bacterial, mammalian, or yeast artificial chromosomes.

[0156] The protein encoded by the introduced polynucleotide is preferably expressed in the cell upon introduction of the vector. The various genetic substrates can be incorporated into a 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 for the selection of cells containing at least two types of vectors.

[0157] Typically, bacteria or yeast cells can be transformed with any one or more of the following nucleotide sequences as is well known in the art. For in vivo recombination, the gene to be recombined with the genome or other genes is used to transform the host using standard transformation techniques. In a preferred embodiment, a DNA that provides an origin of replication is included in the construct. The origin of replication can be suitably selected by those skilled in the art. Depending on the nature of the gene, if a sequence that can act as an origin of replication by itself already exists in the gene or genome, a supplementary origin of replication may not be required.

[0158] Bacteria or yeast cells can be transformed by exogenous or heterologous DNA when such DNA is introduced into the cell. The transforming DNA may or may not be integrated, i.e., covalently linked into the genome of the cell. In prokaryotes and yeast, for example, the transforming 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 a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish a cell line or clone that is composed of a population of daughter cells that contain the transforming DNA.

[0159] Generally, the introduced DNA is not originally endogenous to the host that is the recipient of the DNA, but it is within the scope of the present disclosure to isolate a DNA segment from a given host and subsequently introduce one or more additional copies of that DNA into the same host, for example to enhance production of a gene product or to alter the expression pattern of a gene. In some cases, the introduced DNA will modify or even replace an endogenous gene or DNA sequence, for example, by homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms, plant cells, and plants.

[0160] The present disclosure also features recombinant host. The term "recombinant host", also referred to as "genetically modified host cell" or "transgenic cell", refers to a host cell that contains heterologous nucleic acid or whose genome is expanded by at least one integrated DNA sequence. The host cell of the present disclosure can be genetically modified by polynucleotide or vector as outlined above.

[0161] Host cells that can be used for the purposes of the present 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 chromosome, or cosmid DNA expression vectors containing the polynucleotide molecules of the present 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 the present disclosure. Depending on the host cell and the respective vector used to introduce the polynucleotide of the present disclosure, the polynucleotide can be integrated, for example, into chromosomal or mitochondrial DNA, or can be maintained extrachromosomally, for example, in an episome, or can only be transiently contained within the cell.

[0162] The term "cell" or production cell, as used herein, particularly with reference to genetic engineering and the introduction of one or more genes or clusters of genes into a cell, is understood to refer to any prokaryotic or eukaryotic cell. Both prokaryotic and eukaryotic host cells are contemplated for use in accordance with the present disclosure, including bacterial host cells such as E. coli or Bacillus species, yeast host cells such as S. cerevisiae, insect host cells such as Spodoptora frugiperda, or human host cells such as HeLa and Jurkat.

[0163] Specifically, the cell is a eukaryotic cell, preferably a fungal, mammalian or plant cell, or a prokaryotic cell. Suitable eukaryotic cells include, for example, without limitation, mammalian cells, yeast cells, or helminth cells, including insect cells (including Sf9), amphibian cells (including melanophore cells), or Caenorhabditis cells (including Caenorhabditis elegans). Suitable mammalian cells include, for example, without limitation, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 cells, HEK293T cells, HEK293 T-RexTM cells, or other transfectable eukaryotic cell lines. Suitable bacterial cells include, without limitation, E. coli.

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

[0165] Preferably, the cell is an Aspergillus sp. or a fungal cell, which may preferably 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 agencies (including, but not limited to, an E. coli K12 host cell or, as shown in the examples, an E. coli BL21 host cell).

[0167] One preferred host cell for use in the present disclosure is E. coli, which can be recombinantly prepared as described herein.Therefore, the recombinant host can be a recombinant E. coli host cell.There is a library of mutants, plasmids, detailed computer models of metabolism, and other information available for E. coli, allowing rational design of various modules to enhance product yield.A method similar to that described above for Saccharomyces can be used to make recombinant E. coli microorganisms.

[0168] In one embodiment, the recombinant E. coli microorganism comprises a nucleotide sequence encoding an SHC gene (e.g., as disclosed in any one or more of Tables 10, 11, and 12 herein, or a functional equivalent / homolog thereof, including but not limited to a variant, homolog, mutant, derivative, or fragment thereof.

[0169] Preferably, the recombinant E. coli microorganism comprises the vector construct provided in FIGS.

[0170] In another preferred embodiment, the recombinant E. coli microorganism comprises a nucleotide sequence encoding a WT SHC / HAC and WT SHC / HAC derivative gene, or a functional equivalent / homolog thereof, including but not limited to a variant, homolog, mutant, derivative, or fragment thereof, as set forth in any one or more of Table 13, Table 14, Table 15, Table 16, Table 17, and / or Table 4a.

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

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

[0173] Carbon sources for use in the present methods include any molecule that can be metabolized by the recombinant host cell to promote growth and / or production of (-)-ambrox. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., found in molasses), 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 provided to the host organism throughout the culture period, or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with the carbon source only during the fed-batch phase.

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

[0176] In one embodiment of the present disclosure, a defined minimal medium such as M9A is used for cell culture.

[0177] The components of M9A medium are 14 g / l KH 2 PO 4 , 16g / l K 2 HPO 4 , 1g / l sodium citrate 3 .2H 2 O, 7.5 g / l (NH 4 ) 2 SO 4 , 0.25 g / l MgSO 4 .7H 2 O, 0.015 g / l CaCl 2 .2H 2 O, 5 g / l glucose, and 1.25 g / l yeast extract).

[0178] In another embodiment of the present disclosure, a rich medium such as LB was used. The components of LB medium include 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] The recombinant microorganism can be grown in a batch, fed batch, or continuous process, or a combination thereof. Typically, the recombinant microorganism is grown in a fermentor at a defined temperature(s) in the presence of a suitable nutrient source, e.g., a carbon source, for a desired period of time to produce sufficient enzymes to bioconvert homofarnesol to ambrox and to produce the desired amount of ambrox, including (-)-ambrox.

[0181] The recombinant host cells can be cultured in any suitable manner, such as by batch or fed-batch culture.

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

[0183] The term "fed-batch" as used herein refers to a culture method in which culture medium is added during the culture, but the culture medium is not removed.

[0184] One embodiment of the present disclosure provides a method for producing ambrox in a cell-based system, comprising expressing WT SHC or SHC / HAC derivatives in the cell-based system under suitable conditions, feeding homofarnesol to the cell-based system, converting homofarnesol to ambrox using the SHC or SHC / HAC derivatives produced using the cell-based system, collecting ambrox from the cell-based system, and optionally isolating the (-)-ambrox material from the system. Expression of other nucleotide sequences serves to enhance the method. The bioconversion method may include additional expression of other nucleotide sequences by the cell-based system. Expression of other nucleotide sequences may enhance the bioconversion pathway to make (-)-ambrox.

[0185] A further aspect of the disclosure is a bioconversion method for making (-)-ambrox, comprising growing a host cell containing a WT SHC / HAC or SHC / HAC derivative gene, producing WT SHC / HAC or SHC / HAC derivative in the host cell, feeding homofarnesol (e.g., EEH) to the host cell, incubating the host cell under suitable pH, temperature, and solubilizing agent conditions to promote conversion of homofarnesol to ambrox, and collecting (-)-ambrox. The production of WT SHC / HAC and / or SHC / HAC derivative by the host cell provides a method for making (-)-ambrox when homofarnesol is added to the host cell under suitable reaction conditions. The conversion achieved can be enhanced by adding more biocatalyst and SDS to the reaction mixture.

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

[0187] The culture conditions chosen not only affect the amount of cells (biomass) obtained, but also the quality of the culture conditions affect how the biomass becomes a biocatalyst. A recombinant host cell microorganism expressing the WT SHC or SHC / HAC derivative gene and producing the WT SHC or SHC / HAC derivative enzyme is called a biocatalyst that is suitable for use in bioconversion reactions. In some embodiments, the biocatalyst is a recombinant whole cell producing the WT SHC or SHC / HAC derivative, or it can be in suspension or immobilized format. In other embodiments, the biocatalyst is a membrane fraction or liquid fraction prepared from a recombinant whole cell producing the WT SHC or SHC / HAC derivative (e.g., as disclosed in Seitz et al. 2012 - supra).

[0188] Recombinant whole cells producing WT SHC or SHC / HAC derivatives include whole cells harvested from a fermenter (for a bioconversion reaction) or cells in a 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, the WT SHC or SHC / HAC derivatives are associated in some manner with 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 comprise a whole cell (e.g., recombinant whole cell). The WT SHC or SHC / HAC derivatives may be in an immobilized form (e.g., associated with an enzyme carrier) that allows the WT SHC or SHC / HAC derivatives to interact with a substrate (e.g., homofarnesol). The WT SHC or SHC / HAC derivatives may be used in a soluble form.

[0189] In one embodiment, the biocatalyst is produced in sufficient quantities (to generate sufficient biomass), harvested, and washed (and optionally stored (e.g., frozen or lyophilized)) prior to the bioconversion step.

[0190] In a further embodiment, the cells are produced in sufficient quantities (to generate sufficient biocatalyst) and then the reaction conditions are adjusted, eliminating the need to harvest and wash the biocatalyst for the bioconversion 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 bioconversion reactions, provided that the reaction conditions are adjusted to promote the bioconversion reaction.

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

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

[0193] As used herein, any reference herein to a 99% / 100% conversion rate of a homofarnesol substrate to (-)-ambrox is a reference to 99% / 100% conversion of the homofarnesol isomer (i.e., EEH) that can be converted to (-)-ambrox using the WT SHC / HAC or SHC / HAC derivative enzymes.

[0194] It should be noted that while the term "mixture" or "reaction mixture" may be used interchangeably with the term "medium" in this disclosure (especially as it pertains to "one-pot" reactions), while growing cells to generate sufficient biomass requires cell culture / fermentation medium, medium is not required for the bioconversion step, since a reaction buffer at a suitable pH will be sufficient.

[0195] The bioconversion method of the present disclosure is carried out under time, temperature, pH, and solubilizing agent conditions to effect the conversion of homofarnesol feedstock to (-)-ambrox. The pH of the reaction mixture may be in the range of 4-8, preferably 5 to 6.5, more preferably 4.8-6.0 for SHC / HAC derivative enzymes, and in the range of about pH 5.0 to about pH 7.0 for WT SHC enzymes, and may be maintained by the addition of a 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, although this may be higher in thermophilic organisms, up to 55°C, especially when WT enzymes from thermophilic microorganisms (e.g., WT SHC / HAC) are used. The temperature may be kept constant or may be varied during the bioconversion process.

[0196] Applicants have indicated that it may be useful to include a solubilizing agent (e.g., a surfactant, detergent, solubility enhancer, water-miscible organic solvent, etc.) in a bioconversion reaction. As used herein, the term "surfactant" refers to a component that reduces the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and 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] While Triton X-100 can be used to partially purify WT SHC / HAC or SHC / HAC derivative enzymes (in soluble or membrane fraction / suspension form), it can also be used in bioconversion reactions (see, e.g., the disclosures of Seitz (2012 PhD thesis, supra) and Neumann and Simon (1986 - supra) and JP2009060799).

[0198] Surprisingly, however, Applicants have selected and identified SDS as a particularly useful solubilizing agent from a long list of other less useful solubilizing agents, as shown in Example 14. In particular, Applicants have identified SDS as a significantly better solubilizing agent than, for example, Triton X-100 in terms of reaction rate and yield of the homofarnesol to (-)-ambrox bioconversion reaction (both when EEH is used at 4 g / l and 125 g / l). As shown by the comparative data in Example 12, Applicants have shown that for at least one SHC / HAC derivative enzyme, the highest homofarnesol to (-)-ambrox bioconversion activity using Triton X-100 (at a concentration range of about 0.005% to 0.48%) in the reaction was only approximately 20% of the activity obtained with SDS (at a concentration of about 0.07%).

[0199] Without wishing to be bound by theory, the use of SDS in recombinant microbial host cells can 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, inclusion of a suitable level of SDS in the reaction mixture can 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 at the same time preventing disruption (e.g., denaturation of the SHC (WT or SHC / HAC derivative) enzyme).

[0200] The concentration of solubilizer (e.g., SDS) used in the bioconversion reaction is influenced by the amount of biomass and the concentration of substrate (EEH). That is, there is a certain degree of interdependence between the solubilizer (e.g., SDS) concentration, the amount of biomass, and the concentration of substrate (EEH). For example, as the concentration of homofarnesol substrate increases, sufficient amounts of biocatalyst and solubilizer (e.g., SDS) are required for efficient bioconversion reaction to occur. For example, 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 may be a risk that the biocatalyst will be affected by either destruction of intact microbial cells and / or denaturation / inactivation of SHC / HAC enzymes.

[0201] Selection of a suitable concentration of SDS in the context of biomass amount and substrate (EEH) concentration is within the knowledge of one of skill in the art. By way of example, predictive models for determining suitable SDS, substrate (EEH), and biomass concentrations are available to one of skill in the art. As a further example, Example 3 shows that when 4 g / l EEH and a biocatalyst with an OD (650 nm) of 10.0 are used, an SDS in the range of 0.010-0.075% is appropriate. Example 7 shows that an adjusted SDS concentration (1.55%) is appropriate when 125 g / l EEH is used at 2× wet weight of biomass. However, examination of the percentage EEH conversion to (-)-ambrox using different SDS / cell ratio values ​​indicates that the correct selection of the ratio of biocatalyst, homofarnesol substrate, and solubilizing agent (e.g., SDS) facilitates the development of a robust biotransformation reaction system, which shows some tolerance to a range of SDS concentrations (see, e.g., FIG. 17) and pH range (see, Example 15, FIG. 18).

[0202] The temperature of the bioconversion reaction for WT SHC enzyme (eg, AacSHC) is about 45-60°C, preferably 55°C.

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

[0204] The temperature of the SHC / HAC derivative enzyme bioconversion reaction is from about 34°C to about 50°C, preferably about 35°C.

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

[0206] Preferably, the solubilizing agent used in the bioconversion reaction is SDS.

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

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

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

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

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

[0212] The ratio of biocatalyst to EEH homofarnesol substrate is in the range of about 0.5:1 to 2:1, 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 homofarnesol substrate is added. Substrate can be added by feeding using known means (e.g. peristaltic pump, injection syringe, etc.). Homofarnesol is an oil-soluble compound and is provided in oil format. Given that the biocatalyst (microbial cells such as intact recombinant whole cells and / or cell debris and / or immobilized enzymes) is present in the aqueous phase, the bioconversion reaction can be considered as a three-phase system (including aqueous phase, solid phase, and oil phase) when homofarnesol is added to the bioconversion reaction mixture. This is true even when SDS is present. For clarity, when soluble WT SHC or SHC / HAC derivatives are used as biocatalysts, this is considered as a two-phase system.

[0214] The number of homofarnesol isomers present can affect the speed of the reaction. As Example 11 shows, the SHC / HAC derivative enzymes are capable of bioconverting 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 notion that homofarnesol isomers other than EEH can compete with EEH for access to the SHC / HAC derivative enzymes and therefore may act as competitive inhibitors of the conversion of EEH to (-)-ambrox and / or may also act as alternative substrates.

[0215] Thus, preferably, the homofarnesol substrate comprises a stereoisomeric mixture of two to four isomers, preferably two isomers.

[0216] Thus, preferably, the homofarnesol substrate consists or consists essentially of a stereoisomeric mixture of two to four isomers, preferably two isomers.

[0217] Preferably, the homofarnesol substrate comprises an EE:EZ stereoisomer mixture.

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

[0219] As shown in Example 9, 100% conversion of EE:EZ at a weight ratio of 87:13 was observed in a "one-pot" fermentation and bioconversion reaction carried out for 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, a fermentor is used to grow recombinant host cells expressing SHC / HAC derivative genes and producing active SHC / HAC derivative enzymes in the same fermentor vessel used to convert a homofarnesol source to (-)-ambrox in admixture with one or more of by-products (II), (IV), and / or (III, e.g., as disclosed in 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 water-immiscible solvent (to separate the reaction products and unreacted substrate from the biocatalyst which remains in the aqueous phase), followed by evaporation of the solvent to obtain a crude reaction product as determined by gas 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 water-immiscible solvent (e.g., toluene). Alternatively, the obtained (-)-ambrox can be extracted from the solid phase of the reaction mixture (obtained, for example, by centrifugation or filtration) using a water-miscible solvent (e.g., ethanol) or a water-immiscible solvent (e.g., toluene). As a further example, (-)-ambrox can be present in the solid phase as crystals or in amorphous form and separated from the remaining solid phase (cell material or its debris) and also from the liquid phase by means of filtration. As a further example, at temperatures above the melting point of (-)-ambrox (approximately 75° C.), (-)-ambrox can form an oil layer on top of the aqueous phase, which can be removed and collected. To ensure complete recovery of (-)-ambrox after the oil layer has been removed, an organic solvent can 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 two are further processed to isolate and purify the (-)-ambrox.

[0222] (-)-Ambrox may further be selectively crystallized to remove by-products (II), (III), and (IV), as well as any unreacted homofarnesol substrate, from the final (-)-ambrox product. The term "selective crystallization" refers to a process step whereby (-)-ambrox crystallizes from the solvent while compounds (II), (III), and (IV) remain dissolved in the crystallization solvent to the extent that the isolated crystalline material contains only the (-)-ambrox 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 using a 10% ethanol extract in water or by testing the crystalline material. The final (-)-Ambrox product is tested for its olfactory purity, quality, and its sensory profile against a commercial reference of (-)-Ambrox product. The (-)-Ambrox material is also tested in an expert application study to determine if the material meets specifications for its sensory irritation profile. Various applications of (-)-Ambrox include, but are not limited to, fine fragrance or consumer products, such as fabric care, toiletry, beauty, and cleaning products, essentially all products in which currently available Ambrox ingredients are commercially used, including, but not limited to, Ambrox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlyn (Quest), Cetalox Laevo (Firmenich), Ambermor (Aromor), and Norambrenolide Ether (Pacific) products.

[0224] 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 by-products (II), (III), and / or (IV). Even when only 10% conversion of the homofarnesol substrate to (-)-ambrox is obtained (as shown with the WT SHC / HAC enzyme in Example 7), selective crystallization of (-)-ambrox is still possible.

[0225] Examples of suitable water-miscible and water-immiscible organic solvents suitable for use in the extraction and / or selective crystallization of (-)-ambrox include, but are not limited to, aliphatic hydrocarbons, preferably having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane, halogenated aliphatic hydrocarbons, preferably having 1 or 2 carbon atoms, such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, or tetrachloroethane, aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene, or dichlorobenzene, aliphatic acyclic and cyclic ethers or alcohols, preferably having 4 to 8 carbon atoms, such as ethanol, isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran, or esters, such as ethyl acetate or n-butyl acetate, or ketones, such as methyl isobutyl ketone, or dioxane, or mixtures thereof. Particularly preferably used solvents are the abovementioned 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 to extract (-)-ambrox from the solid phase of the reaction mixture. The use of ethanol is advantageous because it is easy to handle, it is non-toxic, and it is environmentally friendly.

[0227] As used herein, the term "isolated" refers to a biotransformation product, such as (-)-ambrox, that has been separated or purified from components that accompany it. An entity that is produced by a cell-based system that is different from the source from which it naturally originates is "isolated" because it will not necessarily contain components that naturally accompany it. 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 to homogeneity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 89.5% pure, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% pure).

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

[0230] As Example 7 shows, at least 125 g / L of (-)-ambrox is produced by biotransformation using recombinant E. coli host cells producing the SHC / HAC derivative enzymes over a period of about 2 days.

[0231] As Example 19 shows, if efficient mixing is achieved, it is possible to carry out bioconversions at 188 g / l EEH or higher, since mixing efficiency appears to be the only limitation of the system. In addition, biocatalysts with improved activity (e.g., in terms of SHC variants with further improved activity or in terms of increased SHC enzyme production) can improve or maintain productivity using less biomass, which is advantageous in terms of mixing efficiency.

[0232] For example, about 1 to about 100 mg / l, about 30 to about 100 mg / l, about 50 to about 200 mg / l, about 100 to about 500 mg / l, about 100 to about 1,000 mg / l, about 250 to about 5,000 mg / l, about 1,000 (1 g / l) to about 15,000 mg / l (15 g / l), or about 2,000 (2 g / l) to about 10,000 mg / l (10 g / l) or about 2,000 (2 g / l) to about 25,000 mg / l (25 g / l), 26,000mg / l (26g / l), 27,000mg / l (27g / l), 28,000mg / l (28g / l), 29,000mg / l (29g / l), 30,000mg / l (30g / l), 40g / l, 50g / l, 60g / l, 70g / l, 80g / l, 90g / l, 100g / l, 110g / l, 120g / l, 125g / l, 130g / l, 140g / l, 150g / l, 160g / l, 170g / l, 180g / l, 190g / l, or 200g / l or 300g / l or 400g / l or 500g / l are produced.

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

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

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

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

[0237] Those skilled in the art will appreciate that higher cumulative production titers can be achieved by implementing continuous processes, such as removing product, substrate feed, and biomass addition or (partial) replacement.

[0238] Preferably, the bioconversion of EEH to (-)-ambrox in the presence of recombinant host cells containing WT SHC / HAC or SHC / HAC derivatives is given in mol percent and is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 125, 126, 1 , 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 yields of ambrox. Particularly preferred are yields between 5 and 100, 10 and 100, 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, 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 starting material remaining))×100. Preferably, the bioconversion of EEH to (−)-ambrox in the presence of WT SHC or SHC / HAC derivative enzymes is given in mol percent and based on the moles of EEH employed. , 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% yield of (-)-ambrox. Particularly preferably, the yield is between 5 and 100, 10 and 100, 20 and 100, 25 and 100, 30 and 100, 35 and 100, in particular between 40 and 100, 45 and 100, 50 and 100, 60 and 100, 70 and 100.

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

[0241] In a further embodiment of the invention, a recombinant host cell comprising a nucleotide sequence encoding an SHC / HAC derivative is characterized in that it exhibits a greater than 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 1 , 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 yield and / or reaction rate, where the term conditions relates to reaction conditions such as substrate concentration, enzyme concentration, reaction duration, and / or temperature.

[0242] The desired development of a bioconversion process to make (-)-ambrox from homofarnesol by a recombinant strain of E. coli containing a nucleotide sequence encoding the WT / reference SHC or SHC / HAC derivative may provide a low-cost and industrially economical process for the production of (-)-ambrox.

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

[0244] Functional homologues of the WT reference SHC / HAC or SHC / HAC derivative polypeptides described herein are also suitable for use in producing ambrox by a recombinant host. Thus, a recombinant host can include one or more heterologous nucleic acid(s) encoding a functional homologue of the polypeptides described above and / or a heterologous nucleic acid encoding a 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 functions 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 evolution 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 mutants of wild-type coding sequences, 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 coding sequences of different naturally occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding functional homologs described herein are known and include, among others, evolutionary engineering, site-directed mutagenesis, and random mutagenesis techniques, and may be useful to increase the specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide:polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to nucleic acids that encode functionally homologous polypeptides.

[0246] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database 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 as a measure of homology between two nucleic acid sequences. A nucleic acid sequence encoding any of the proteins disclosed herein, or a portion thereof, can be used as a hybridization probe according to standard hybridization techniques. Hybridization of the probe to DNA or RNA from a test source (e.g., a mammalian cell) is indicative of the presence of the related DNA or RNA in the test source. Hybridization conditions are known to those of skill 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 equivalent to hybridization in 2x sodium chloride / sodium citrate (SSC) at 30°C, followed by washing in 1x SSC, 0.1% SDS at 50°C. High stringency conditions are defined as equivalent to hybridization in 6x sodium chloride / sodium citrate (SSC) at 45°C, followed by a wash in 0.2x SSC, 0.1% SDS at 65°C.

[0248] Sequence analysis to identify functional homologs may also include BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using related amino acid sequences as reference sequences. Amino acid sequences are in some cases predicted from nucleotide sequences. Polypeptides in the database with greater than 40% sequence identity are candidates for further evaluation for suitability for use in SHC / HAC bioconversion reactions. Amino acid sequence similarity allows for conservative amino acid substitutions, such as the substitution of one hydrophobic residue for another or one polar residue for another. If desired, manual inspection of such candidates may be performed to narrow down the number of candidates to be further evaluated. Manual inspection may be performed, for example, by selecting candidates that appear to have conserved functional domains.

[0249] Typically, polypeptides that exhibit at least about 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%, or 69% amino acid sequence identity. In some embodiments, the conserved regions exhibit at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity. Sequence identity may be determined as shown above and below.

[0250] The WT SHC and / or SHC / HAC derivatives produced are based on amino acid sequence SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, or a variant, homologue, mutant, derivative, or fragment thereof.

[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%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... Based on an amino acid sequence 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 an amino acid sequence produced from E. coli.

[0253] "Percent identity" is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to the nucleotides in the DNA sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, with respect to the nucleotide sequence of the gene, and does not take into account any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent identity of nucleotide sequences can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve the best alignment over the entire length of the sequences to be compared.

[0254] The terms "polypeptide" and "protein" are used interchangeably herein to refer to any peptide-linked chain of amino acids, regardless of length or post-translational modification.

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

[0256] The term "variant" as used herein should be understood as a polypeptide that differs by one or more changes in amino acid sequence compared to the polypeptide from which it is derived. The polypeptide from which the variant is derived is also known as the parent or reference polypeptide. Typically, the variant is constructed artificially, preferably by genetic technology means. Typically, the polypeptide from which the variant is derived is a wild-type protein or wild-type protein domain. However, variants usable in the present disclosure can also be derived from a homolog, ortholog, or paralog of the parent polypeptide, or from an artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent polypeptide. The change in amino acid sequence can be an amino acid exchange, an insertion, a deletion, an N-terminal truncation, or a C-terminal truncation, or any combination of these changes, which can occur at one or several sites.

[0257] In preferred embodiments, variants usable in the present disclosure exhibit a total number of changes in amino acid sequence (i.e., exchanges, insertions, deletions, N-terminal truncations, and / or C-terminal truncations) 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). The amino acid exchanges can be conservative and / or non-conservative. In a preferred embodiment, the variants usable in the present disclosure differ from the protein or domain from which it is derived by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid exchanges, preferably conservative amino acid changes. The variants may additionally or alternatively contain amino acid deletions, which may be N-terminal truncations, C-terminal truncations, or internal deletions, or any combination thereof. Such variants containing N-terminal truncations, C-terminal truncations, and / or internal deletions are referred to as "deletion variants" or "fragments" in the context of this application. The terms "deletion variants" and "fragments" are used interchangeably herein. Deletion variants may be naturally occurring (e.g., splice variants) or they may be constructed artificially, preferably by genetic technology means. Typically, the protein or protein domain from which the deletion variant is derived is a wild-type protein. However, the deletion variants of the present disclosure may also be derived from a homolog, ortholog, or paralog of the parent polypeptide, or may also be derived from an artificially constructed variant, provided that the deletion variant exhibits at least one biological activity of the parent polypeptide. Preferably, the deletion variant (or fragment) has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids at its N-terminus and / or at its C-terminus and / or internally, compared to the parent polypeptide.

[0258] As used herein, a "variant" may alternatively or additionally be characterized by a degree of sequence identity to the parent polypeptide from which it is derived. A variant or SHC / HAC derivative of a WT / reference SHC / HAC of the present disclosure has at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... The sequence identity may be 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] The expression "at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least "83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity" is used herein with respect to polypeptide and polynucleotide sequence comparisons.

[0260] A polynucleotide belonging to any of the families of enzymes or proteins disclosed herein can be identified based on its similarity to related genes or proteins, respectively. For example, identification can be based on sequence identity. In certain preferred embodiments, the disclosure provides nucleic acid molecules that include (a) nucleic acid molecules encoding the polypeptides of SEQ ID NOs: 5-163 (see Tables 14-17 and Table 4a provided herein), (b) the nucleotide sequences of SEQ ID NOs: 6-168, 169, 170, 172, 174, and 176 (see Tables 14-17 and Table 4a provided herein), and (c) nucleic acid molecules that include a segment of at least 30 (e.g., at least 30, 40, 50, 60, 80, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 850, 900, 950, 1000, or 1010) nucleotides of SEQ ID NOs: 6-168, 169, 170, 172, 174, and 176 (see Tables 14-17 and Table 4a provided herein). 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152 The invention features 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 to the sequence.

[0261] Preferably, the subject polypeptide and the reference polypeptide show the sequence identity shown over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. Preferably, the subject polynucleotide and the reference polynucleotide show the sequence identity shown over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides. When the reference sequence to which the two sequences are compared and to which the sequence identity percentage is to be calculated is not specified, unless specifically indicated otherwise, the sequence identity should be calculated with reference to the longer of the two sequences to be compared. When the reference sequence is shown, unless specifically indicated otherwise, the sequence identity is determined based on the full length of the reference sequence shown by SEQ ID NO: 1, 2, 3, and / or 4.

[0262] For example, a peptide sequence consisting of 130 amino acids compared to the full length amino acid sequence of a reference SHC having 631 amino acid residues may show a maximum percentage of sequence identity of 20.6% (130 / 631×100), while a sequence having a length of 300 amino acids may show a maximum percentage of sequence identity of 47.5% (300 / 631×100). The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined by sequence alignment. Such alignment can be performed by several algorithms known in the art, preferably by the computational 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 by, for example, http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or or by the GAP program (University of Iowa computational algorithm) utilized in the sequence alignment to WT SHC provided in Table 18 herein; or by the computational algorithm of Myers and Miller (1989 - Cabios 4: 11-17) disclosed and utilized in the WT SHC sequence alignment in 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 These are the default parameters set in

[0264] The sequence identity grade (sequence match) can be calculated, for example, using BLAST, BLAT, or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score=100, wordlength=12, to obtain polynucleotide sequences that are homologous to nucleic acids encoding related proteins.

[0265] BLAST protein searches are performed with 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 BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence match analysis can be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: 154-162) or Markov Random Field. When percentages of sequence identity are referred to in this application, these percentages are calculated relative to the total length of the longer sequence, unless specifically indicated otherwise.

[0266] Perception Although the bioconversion of homofarnesol to (-)-ambrox according to the present disclosure produces (-)-ambrox as the main compound, compounds other than (-)-ambrox may also be produced, which may or may not impart a pleasant olfactory note to the bioconversion mixture and thus may contribute in a positive or negative manner to the sensory properties of the (-)-ambrox end product. Thus, a sensory analysis is performed using well-established sensory tests utilized by trained experts (e.g., perfumers) so that the test can help determine whether a chemically related target product is also an olfactorily related end product to a reference product. As the sensory analysis of Example 22 shows, the removal of one or more by-product compounds from (-)-ambrox can improve the odor of the remaining compound ((-)-ambrox), even when the removed compound itself is actually an odorless compound. That is, an enhancement of the odor of (-)-ambrox was observed in the absence of compounds II, III, and IV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0267] Aspects of the invention 1. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture of EEH-containing stereoisomers is enzymatically converted to (-)-ambrox or a mixture containing (-)-ambrox, wherein the enzymatic conversion is carried out using SHC / HAC enzymes under reaction conditions suitable for the production of (-)-ambrox, and wherein the mixture of EEH-containing stereoisomers consists essentially 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)], also referred to as [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively. 2. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture of EEH-containing stereoisomers is enzymatically converted to provide (-)-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 wherein Triton X-100 or taurodeoxycholic acid is not used in combination with the wild-type SHC / HAC enzyme when the reaction is carried out in the presence of a solubilizing agent. 3. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, wherein (3E,7E)-homofarnesol (EEH) or a mixture of EEH-containing stereoisomers is enzymatically converted to (-)-ambrox or a mixture containing (-)-ambrox, the enzymatic conversion being carried out using SHC / HAC enzymes under reaction conditions suitable for the production of (-)-ambrox, the mixture of EEH-containing stereoisomers essentially consisting 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)], also referred to as [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively, and the reaction occurring in a three-phase system comprising an aqueous phase, a solid phase, and an oil phase. 4. The process according to paragraph 1 or paragraph 2 or paragraph 3, wherein the process comprises selecting 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 enzyme polypeptide sequence selected from Table 1, Table 5, Table 2, Table 6, Table 3, Table 7, Table 4, Table 8, or Table 13, 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, 177, and / or 178. C / HAC derivatives or sequences having at least 30% identity, 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 to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 5. The process of any one of paragraphs 1-4, wherein the process employs a recombinant host cell that produces the SHC / HAC enzyme. 6. The 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 NO: 165, 166, 167, 168, 169, or SEQ ID NO: 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. The process according to any one of paragraphs 1-6, wherein the conversion of homofarnesol to (-)-ambrox occurs at a temperature in the range of 30° C. to 60° C. and at a pH in the range of about 4 to 8. 8. The process according to any one of paragraphs 1-7, wherein the conversion of homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for the wild-type SHC / HAC or SHC / HAC derivative enzymes set forth in Table 24 or Table 24a, preferably in the pH range of 5.0 to 6.2, and preferably at a temperature of 35° C. 9. The 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 about 2:1. 10. The process according to any one of paragraphs 3-9, wherein the weight ratio of biocatalyst to homofarnesol is within the range of about 0.5 to 2:1, preferably about 1:1 or 0.5:1. 11. The process according to any one of paragraphs 3 to 10, wherein the cell growth and bioconversion reaction steps are carried out in the same reaction vessel. 12. The process according to paragraph 2, wherein the homofarnesol substrate comprises one or more homofarnesol stereoisomers. 13. The process according to paragraph 12, wherein the homofarnesol substrate comprises or consists essentially of two homofarnesol stereoisomers. 14. The process according to paragraph 13, wherein the homofarnesol substrate comprises or consists essentially of EE:EZ stereoisomers. 15. The process according to any one of paragraphs 14, wherein 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, 79 69:31, 68:32, 67:33, 66:34, 65:35, 64:36, 63:37, 62:38, 61:39, and 60:40. 16. The process according to paragraph 15, wherein the homofarnesol comprises or consists essentially of an EE:EZ stereoisomeric mixture in a weight ratio selected from the group consisting of EE:EZ 92:8, EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and EE:EZ 66:34. 17. The process according to paragraph 15 or paragraph 16, wherein the homofarnesol comprises or consists essentially of a mixture of EE:EZ stereoisomers in a weight ratio of 80:20. 18. The process according to any one of paragraphs 1 to 17, wherein (-)-ambrox is produced in admixture with at least one or more of by-products (II), (IV), and / or (III). 19. The process according to any one of paragraphs 1-18, wherein (-)-ambrox is isolated from the bioconversion reaction mixture using an organic solvent or steam extraction / distillation step or filtration. 20. The process according to any one of paragraphs 1-19, wherein (-)-ambrox is isolated from the solid phase of the bioconversion reaction mixture using an organic solvent or a steam extraction / distillation step. 21. The process according to paragraph 19 or paragraph 20, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent. 22. The process according to paragraph 21, wherein (-)-ambrox is isolated from the reaction mixture using ethanol or toluene. 23. The process according to any one of paragraphs 19 to 22, wherein (-)-ambrox is selectively crystallized using an organic solvent. 24. The process according to paragraph 23, wherein (-)-ambrox is substantially free of by-products (II), (IV), and / or (III). 25. The process according to any one of paragraphs 1-24, wherein (-)-ambrox is produced in a concentration range of about 125-200 g / l. 26. (-)-Ambrox, obtainable by the method of any one of paragraphs 1 to 25, wherein the (-)-ambrox has an odor threshold of about 0.1 to about 0.5 ng / l. 27. (-)-Ambrox of paragraph 26 in a solid form, preferably in amorphous or crystalline form. 28. A process for making a product containing (-)-ambrox, comprising incorporating into the product the (-)-ambrox of any one of paragraphs 26 or 27. 29. The process of paragraph 28, wherein the product is a fragrance product, a cosmetic product, a cleaning product, a detergent product, or a soap product. 30. A fragrance or cosmetic or consumer care product containing (-)-ambrox according to any one of paragraphs 26 or 27. 31. A fragrance or cosmetic or consumer care composition comprising (-)-ambrox of paragraph 26 or paragraph 27 and one or more additional components. 32. Use of (-)-ambrox according to paragraph 26 or 27 as part of a fragrance or cosmetic or consumer product, such as a fabric care, toiletry, beauty and / or cleaning product. 33. A process for extending, enhancing, or imparting the aroma of a fragrance composition, comprising: (a) preparing a reaction mixture comprising (−)-ambrox in admixture with one or more of by-product compounds (II), (III), or (IV); [ka] (b) extracting (-)-ambrox in admixture with one or more of by-product compounds (II), (III), or (IV); (c) selectively crystallizing (-)-ambrox from the extraction mixture; mixing an aroma-enhanced or -enhanced product produced according to a process comprising the steps of: (-)-Ambrox 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 (-)-ambrox; The mixture of stereoisomers containing EEH consists essentially 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)], which are also designated [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively. 34. The process of paragraph 33, wherein the reaction occurs in a three-phase system including an aqueous phase, a solid phase, and an oil phase. 35. The process according to paragraph 33 or paragraph 34, wherein the process comprises the step of: a 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 a 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 with a sequence having at least 30% identity, 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 to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 36. The process of any one of paragraphs 33-35, wherein the process employs a recombinant host cell that produces the SHC / HAC enzyme. 37. The 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 NO: 165, 166, 167, 168, 169, or SEQ ID NO: 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. The process according to any one of paragraphs 33-37, wherein the conversion of homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C and at a pH in the range of about 4 to 8. 39. The process according to any one of paragraphs 33 to 38, wherein the conversion of homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for the wild-type SHC / HAC or SHC / HAC derivative enzyme set forth in Table 24 or Table 24a, preferably in the pH range of 5.0 to 6.2, and preferably at a temperature of 35°C. 40. The process according to any one of paragraphs 34 to 39, 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 about 2:1. 41. The process according to any one of paragraphs 34-40, wherein the weight ratio of biocatalyst to homofarnesol is within the range of about 0.5 to 2:1, preferably about 1:1 or 0.5:1. 42. A process according to any one of paragraphs 34 to 41, wherein the cell growth and bioconversion reaction steps are carried out in the same reaction vessel. 43. The process according to any one of paragraphs 33 to 42, wherein 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 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. The process according to paragraph 43, wherein the homofarnesol comprises or consists essentially of an EE:EZ stereoisomeric mixture in a weight ratio selected from the group consisting of EE:EZ 92:08, EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and EE:EZ 66:34. 45. The process according to paragraph 43 or paragraph 44, wherein the homofarnesol comprises or consists essentially of a mixture of EE:EZ stereoisomers in a weight ratio of 80:20. 46. ​​The process according to any one of paragraphs 33 to 45, wherein (-)-ambrox is produced in a mixture with at least one or more of by-products (II), (IV), and / or (III). 47. The process according to any one of paragraphs 33-46, wherein (-)-ambrox is isolated from the bioconversion reaction mixture using an organic solvent or steam extraction / distillation step or filtration. 48. The process according to any one of paragraphs 33-47, wherein (-)-ambrox is isolated from the solid phase of the bioconversion reaction mixture using an organic solvent or a steam extraction / distillation step. 49. The process according to paragraph 47 or paragraph 48, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent. 50. The process according to paragraph 49, wherein (-)-ambrox is isolated from the reaction mixture using ethanol or toluene. 51. The process according to any one of paragraphs 47 to 49, wherein (-)-ambrox is selectively crystallized using an organic solvent. 52. The process according to paragraph 51, wherein (-)-ambrox is substantially free of by-products (II), (IV), and / or (III). 53. The process according to any one of paragraphs 33-52, wherein (-)-ambrox is produced in a concentration range of about 125-200 g / l. 54. The process according to any one of paragraphs 33 to 53, wherein (-)-ambrox has an odor threshold of about 0.1 to about 0.5 ng / l.

[0268] Additional Aspects of the Invention 1. A squalene-hopene cyclase (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, 1-30 mutations, 1-20 mutations, 1-10 mutations, or 1-6 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, or 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 to SEQ ID NO:1. 4. An SHC / HAC derivative according to paragraph 3, wherein the SHC variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO:1. 5. An SHC / HAC derivative, comprising 1-10 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO:1, wherein one or more mutations other than an SHC active site mutation are located within domain 2 of the SHC enzyme (Figures 19 and / or 20). 6. An SHC / HAC derivative according to any one of paragraphs 1 to 5, in which one or more mutations are selected from Table 1 relative to 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. The SHC / HAC derivative of paragraph 2, comprising an amino acid sequence having up to six mutations relative to SEQ ID NO:1 and including at least the substitutions F601Y or M132R in combination with at least any one or more of F129L and / or I432T. 9. The SHC / HAC derivative of paragraph 7, comprising an amino acid sequence having up to eight amino acid alterations relative to SEQ ID NO:1 and including one or more amino acid alterations at positions selected from the group consisting of positions 77, 129, 132, 192, 224, 432, 579, 601, and 605 relative to SEQ ID NO:1, wherein the SHC / HAC derivative has increased HAC enzymatic activity relative to SEQ ID NO:1. 10. An SHC / HAC derivative according to paragraph 9, which comprises one or more substitutions relative to SEQ ID NO:1 selected from the group consisting of T77A, F129L, M132R, I92V, A224V, I432T, Q579H, F601Y, and / or F605W. 11. SHC / HAC derivatives according to paragraph 10, including F601Y. 12. An SHC / HAC derivative according to paragraph 10, comprising F129L. 13. SHC / HAC derivatives according to paragraph 10, including F601Y and F129L. 14. An SHC / HAC derivative 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, including T77A and I92V and F129L. 21. An SHC / HAC derivative according to any one of the preceding paragraphs, having 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 according to 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: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and / or 170. 24. A construct comprising the nucleotide sequence of paragraph 22 or paragraph 23. 25. A construct according to paragraph 24, comprising a promoter operably linked to the nucleotide sequence of paragraph 22 or 23. 26. The construct of paragraph 25, wherein the promoter is an inducible or constitutive promoter. 27. A vector comprising a construct according to any one of paragraphs 24 to 26. 28. The vector of paragraph 27, wherein the vector is a plasmid. 29. A vector according to paragraph 28, capable of directing expression by a host cell selected from prokaryotic, yeast, plant, and insect host cells. 30. A construct according to any one of paragraphs 24 to 26 or a vector according to any one of paragraphs 27 to 29, wherein the construct or vector is capable of integration into the genome of a host cell selected from a prokaryotic, yeast, plant, and insect host cell. 31. A recombinant host cell comprising a nucleotide sequence according to paragraph 22 or 23, or a construct according to any one of paragraphs 24 to 26 or 30, or a vector according to any one of paragraphs 27 to 30. 32. A recombinant host cell according to paragraph 31, wherein the host cell is selected from the group of prokaryotic host cells consisting of bacteria of the genera Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. 33. The recombinant host cell of paragraph 32, wherein the host cell is an E. coli host cell. 34. A recombinant host cell according to paragraph 33, wherein the host cell overexpresses a gene encoding an SHC / HAC derivative. 35. A method for preparing an SHC / HAC derivative according to any one of paragraphs 1 to 21, comprising culturing one or more recombinant host cells according to any one of paragraphs 31 to 34 under conditions permissive for production of an SHC / HAC derivative enzyme. 36. The method of paragraph 35, wherein the cell culture occurs under conditions suitable for biocatalyst production. 37. A method for preparing (-)-ambrox, comprising converting homofarnesol to (-)-ambrox by using a recombinant host cell according to any one of paragraphs 31-34, or by using a recombinant host cell comprising SEQ ID NO: 169 or SEQ ID NO: 165 encoding a WT SHC / HAC, wherein when a WT SHC / HAC is used, the bioconversion of homofarnesol to (-)-ambrox is carried out by a solubilizing agent other than Triton X-100 or taurodeoxycholic acid. 38. The method according to paragraph 37, wherein the conversion of homofarnesol to (-)-ambrox is under biotransformation conditions suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 39. The method according to paragraph 37 or 38, wherein the conversion of homofarnesol to (-)-ambrox occurs at a pH, temperature and solubilizing agent concentration suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 40. The method according to paragraph 39, wherein the conversion of homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C, at a pH in the range of about 4 to 8, and in the presence of a solubilizing agent other than Triton X-100 or taurodeoxycholic acid for the WT SHC / HAC enzyme. 41. The method according to any one of paragraphs 37-40, wherein the conversion of homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for the WT SHC / HAC or SHC / HAC derivative enzymes set forth in Table 24 or Table 24a. 42. The process of any one of paragraphs 37-41, wherein the weight ratio of biocatalyst to homofarnesol is within the range of about 0.5:1 to 2:1, preferably about 1:1 or 0.5:1. 43. The method according to any one of paragraphs 37 to 42, wherein the cell growth and bioconversion reaction steps are carried out in the same reaction vessel. 44. The 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 comprises an EE:EZ stereoisomer. 47. The method according to any one of paragraphs 44-46, wherein the homofarnesol comprises an EE:EZ stereoisomeric 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 the homofarnesol comprises an EE:EZ stereoisomer mixture in a weight ratio selected from the group consisting of EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and EE:EZ 66:34. 49. The method of paragraph 35 or 36, wherein the homofarnesol comprises a mixture of EE:EZ stereoisomers in a weight ratio of 80:20. 50. The process of any one of paragraphs 37-49, wherein (-)-ambrox is produced in a mixture with one or more of by-products (II), (IV), and / or (III). 51. The process of any one of paragraphs 37-50, wherein (-)-ambrox is isolated from the bioconversion reaction mixture using an organic solvent or a steam extraction / distillation step, or (-)-ambrox crystals are isolated directly from the bioconversion reaction mixture by means of filtration. 52. The process 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 process of paragraph 52 or 53, wherein the (-)-ambrox is substantially free of by-products (II), (IV), and / or (III). 55. (-)-Ambrox, obtainable by the process of any one of paragraphs 51 to 54. 56. (-)-Ambrox of paragraph 55 in a solid form, preferably in amorphous or crystalline form. 57. A method for making a product containing (-)-ambrox, comprising incorporating the (-)-ambrox of 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. Fragrances or cosmetics or consumer care products containing (-)-ambrox of paragraph 55 or 56. 59. A fragrance or cosmetic or consumer care composition comprising (-)-ambrox of paragraph 55 or 56 and one or more additional components. 60. Use of (-)-ambrox of paragraph 55 or 56 as part of a fragrance or cosmetic or consumer product, such as a fabric care, toiletry, beauty and / or cleaning product. 61. Use of an SHC / HAC derivative enzyme according to any one of paragraphs 1 to 21, a nucleotide sequence according to paragraph 22 or 23, a construct according to any one of paragraphs 24 to 26 or 30, a vector according to any one of paragraphs 27 to 30, or a recombinant host cell according to any one of paragraphs 31 to 34, or a recombinant host cell expressing a WT SHC / HAC, for the bioconversion of homofarnesol to (-)-ambrox, wherein the WT SHC / HAC enzyme is used in the bioconversion reaction with a solubilizing agent other than Triton X-100. 62. A method for preparing (-)-ambrox or a stereoisomeric mixture of (-)-ambrox, wherein (3E,7E)-homofarnesol or a stereoisomeric mixture of (3E,7E)-homofarnesol is enzymatically converted to provide (-)-ambrox or a stereoisomeric mixture of (-)-ambrox, and the enzymatic conversion using an SHC / HAC enzyme is performed under reaction conditions suitable for the production of (-)-ambrox, and wherein Triton X-100 is not used in combination with the WT SHC / HAC enzyme when the reaction is performed in the presence of a solubilizing agent. 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), BjpSHC (SEQ ID NO:4), an SHC / HAC derivative selected from Table 1, Table 5, Table 2, Table 6, Table 3, Table 7, Table 4, and / or Table 8, or a sequence having at least 30% identity, 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 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 of 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 solubilizing agent other than Triton X-100 for WT SHC. 65. A process according to paragraph 64, wherein the reaction conditions for WT SHC / HAC or the respective SHC / HAC derivative enzymes set out in Table 24 or Table 24a are used. 66. The process according to any one of paragraphs 62-65, wherein the process comprises, prior to conversion of E,E-homofarnesol to (-)-ambrox, (a) culturing one or more recombinant host cells expressing a WT SHC or SHC derivative enzyme under conditions permissive for expression of the WT SHC or SHC / HAC derivative polypeptide. 67. A process according to paragraph 66, wherein the culturing step and the subsequent converting step occur in the same reaction vessel but under different reaction conditions. 68. The process according to paragraph 67, wherein the culturing step is at a pH range of about 6 to about 7 and the homofarnesol to (-)-ambrox step is at a pH range of about 4.8 to 5.5. 69. The process according to any one of paragraphs 62-68, wherein the homofarnesol substrate comprises an EE:EZ stereoisomer. 70. The process according to paragraph 69, wherein the homofarnesol comprises an EE:EZ stereoisomeric mixture in a weight ratio selected from the group consisting of EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and EE:EZ 66:34. 71. The process of paragraph 70, wherein the homofarnesol comprises EE:EZ in a weight ratio of 80:20. 72. The process of any one of paragraphs 62-71, wherein (-)-ambrox is produced in a mixture with one or more of by-products (II), (IV), and / or (III). 73. The process of any one of paragraphs 62-72, wherein (-)-ambrox is isolated from the reaction mixture using an organic solvent or a steam extraction / distillation step or filtration. 74. The process according to paragraph 73, wherein (-)-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 process of paragraph 74 or 75, wherein the (-)-ambrox is substantially free of by-products (II), (IV), and / or (III). 77. (-)-Ambrox, obtainable by the process of any one of paragraphs 72 to 76. 78. (-)-Ambrox of paragraph 26 in a solid form, preferably in amorphous or crystalline form. 79. A process for making a product, comprising incorporating (-)-ambrox of paragraph 77 or 78 into the product. 80. The method of paragraph 79, wherein the product is a fragrance product, a cosmetic product, a cleaning product, a detergent product, or a soap product. 81. Fragrances or cosmetics or consumer care products containing (-)-ambrox of paragraph 77 or 78. 82. A fragrance or cosmetic or consumer care composition comprising (-)-ambrox of paragraph 77 or 78 and an additional component. 83. Use of (-)-ambrox of paragraph 77 or 78 as part of a fragrance or cosmetic consumer care product.

[0269] Additional Aspects of the Invention (ZmoSHC1) 1. A squalene-hopene cyclase (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, 1-30 mutations, 1-20 mutations, 1-10 mutations, or 1-6 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, or 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 to SEQ ID NO:2. 4. An SHC / HAC derivative according to paragraph 3, wherein the SHC variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO:2. 5. An SHC / HAC derivative, comprising 1-10 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO:2, wherein one or more mutations other than an SHC active site mutation are located within domain 2 of the SHC enzyme (Figures 19 and / or 20). 6. An SHC / HAC derivative according to any one of paragraphs 1 to 5, in which one or more mutations are selected from Table 2 relative to SEQ ID NO:2, and if only one mutation is selected it is not F668Y. 7. The SHC / HAC derivative of 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, comprising an amino acid sequence having up to six mutations relative to SEQ ID NO:2 and including at least the substitutions F668Y or Y185R in combination with at least any one or more of F182L and / or I498T. 9. The SHC / HAC derivative of paragraph 7, comprising an amino acid sequence having up to eight amino acid alterations relative to SEQ ID NO:2 and including one or more amino acid alterations at positions selected from the group consisting of positions 129, 145, 182, 185, 282, 498, 647, and 668 relative to SEQ ID NO:2, wherein the SHC / HAC derivative has increased HAC enzymatic activity relative to SEQ ID NO:2. 10. An SHC / HAC derivative according to paragraph 9, which comprises one or more substitutions selected from the group consisting of S129A, V145V, F182L, Y185R, G282V, I498T, H646H, and F668Y relative to SEQ ID NO:2. 11. SHC / HAC derivatives according to paragraph 10, including F668Y. 12. An SHC / HAC derivative according to paragraph 10, comprising F182L. 13. SHC / HAC derivatives according to paragraph 10, including 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. SHC / HAC derivatives according to paragraph 11, further comprising H646H. 20. SHC derivatives according to paragraph 10, comprising S129A and V145V and F182L. 21. An SHC / HAC derivative according to any one of the preceding paragraphs, having 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 according to 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: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, and / or 76. 24. A construct comprising the nucleotide sequence of paragraph 22 or paragraph 23. 25. A construct according to paragraph 24, comprising a promoter operably linked to the nucleotide sequence of paragraph 22 or 23. 26. The construct of paragraph 25, wherein the promoter is an inducible or constitutive promoter. 27. A vector comprising a construct according to any one of paragraphs 24 to 26. 28. The vector of paragraph 27, wherein the vector is a plasmid. 29. A vector according to paragraph 28, capable of directing expression by a host cell selected from prokaryotic, yeast, plant, and insect host cells. 30. A construct according to any one of paragraphs 24 to 26 or a vector according to any one of paragraphs 27 to 29, wherein the construct or vector is capable of integration into the genome of a host cell selected from a prokaryotic, yeast, plant, and insect host cell. 31. A recombinant host cell comprising a nucleotide sequence according to paragraph 22 or 23, or a construct according to any one of paragraphs 24 to 26 or 30, or a vector according to any one of paragraphs 27 to 30. 32. A recombinant host cell according to paragraph 31, wherein the host cell is selected from the group of prokaryotic host cells consisting of bacteria of the genera Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. 33. The recombinant host cell of paragraph 32, wherein the host cell is an E. coli host cell. 34. A recombinant host cell according to paragraph 33, wherein the host cell overexpresses a gene encoding an SHC / HAC derivative. 35. A method for preparing an SHC / HAC derivative according to any one of paragraphs 1 to 21, comprising culturing one or more recombinant host cells according to any one of paragraphs 31 to 34 under conditions permissive for production of an SHC / HAC derivative enzyme. 36. The method of paragraph 35, wherein the cell culture occurs under conditions suitable for biocatalyst production. 37. A method for preparing (-)-ambrox, comprising converting homofarnesol to (-)-ambrox by using a recombinant host cell according to any one of paragraphs 31-34, or by using a recombinant host cell comprising SEQ ID NO: 166 encoding a WT SHC / HAC, wherein when a WT SHC / HAC is used, the bioconversion of homofarnesol to (-)-ambrox is carried out by a solubilizing agent other than Triton X-100. 38. The method according to paragraph 37, wherein the conversion of homofarnesol to (-)-ambrox is under biotransformation conditions suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 39. The method according to paragraph 37 or 38, wherein the conversion of homofarnesol to (-)-ambrox occurs at a pH, temperature and solubilizing agent concentration suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 40. The method according to paragraph 39, wherein the conversion of homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C, at a pH in the range of about 4 to 8, and in the presence of a solubilizing agent other than Triton X-100 for WT SHC / HAC enzyme. 41. The method according to any one of paragraphs 37-40, wherein the conversion of homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for the WT SHC / HAC or SHC / HAC derivative enzymes set forth in Table 24 or Table 24a. 42. The process of any one of paragraphs 37-41, wherein the weight ratio of biocatalyst to homofarnesol is within the range of about 0.5:1 to 2:1, preferably about 1:1 or 0.5:1. 43. The method according to any one of paragraphs 37 to 42, wherein the cell growth and bioconversion reaction steps are carried out in the same reaction vessel. 44. The 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 comprises an EE:EZ stereoisomer. 47. The method according to any one of paragraphs 44-46, wherein the homofarnesol comprises an EE:EZ stereoisomeric 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 the homofarnesol comprises an EE:EZ stereoisomeric mixture in a weight ratio selected from the group consisting of EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and EE:EZ 66:34. 49. The method of paragraph 35 or 36, wherein the homofarnesol comprises a mixture of EE:EZ stereoisomers in a weight ratio of 80:20. 50. The process of any one of paragraphs 37-49, wherein (-)-ambrox is produced in a mixture with one or more of by-products (II), (IV), and / or (III). 51. The process of any one of paragraphs 37-50, wherein (-)-ambrox is isolated from the bioconversion reaction mixture using an organic solvent or steam extraction / distillation step or filtration. 52. The process 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 process of paragraph 52 or 53, wherein the (-)-ambrox is substantially free of by-products (II), (IV), and / or (III). 55. (-)-Ambrox, obtainable by the process of any one of paragraphs 51 to 54. 56. (-)-Ambrox of paragraph 55 in a solid form, preferably in amorphous or crystalline form. 57. A method for making a product containing (-)-ambrox, comprising incorporating the (-)-ambrox of 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. Fragrances or cosmetics or consumer care products containing (-)-ambrox of paragraph 55 or 56. 59. A fragrance or cosmetic or consumer care composition comprising (-)-ambrox of paragraph 55 or 56 and one or more additional components. 60. Use of (-)-ambrox of paragraph 55 or 56 as part of a fragrance or cosmetic or consumer product, such as a fabric care, toiletry, beauty and / or cleaning product. 61. Use of an SHC / HAC derivative enzyme according to any one of paragraphs 1 to 21, a nucleotide sequence according to paragraphs 22 or 23, a construct according to any one of paragraphs 24 to 26 or 30, a vector according to any one of paragraphs 27 to 30, or a recombinant host cell according to any one of paragraphs 31 to 34, or a recombinant host cell expressing a WT SHC / HAC, for the bioconversion of homofarnesol to (-)-ambrox, wherein the WT SHC / HAC enzyme is used in the bioconversion reaction with a solubilizing agent other than Triton X-100.

[0270] Further aspects of the invention (ZmoSHC2) 1. A squalene-hopene cyclase (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, 1-30 mutations, 1-20 mutations, 1-10 mutations, or 1-6 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, or 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 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 to SEQ ID NO:3. 5. An SHC / HAC derivative, comprising 1-10 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO:3, wherein one or more mutations other than an SHC active site mutation are located within domain 2 of the SHC enzyme (Figures 19 and / or 20). 6. An SHC / HAC derivative according to any one of paragraphs 1 to 5, in which one or more mutations are selected from Table 3 relative to SEQ ID NO:3, and if only one mutation is selected it is not F620Y. 7. The SHC / HAC derivative of 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, comprising an amino acid sequence having up to six mutations relative to SEQ ID NO:3 and including at least the substitutions F620Y or I140R in combination with at least any one or more of F137L and / or I450T. 9. The SHC / HAC derivative of paragraph 7, comprising an amino acid sequence having up to eight amino acid alterations relative to SEQ ID NO:3 and including one or more amino acid alterations at a position selected from the group consisting of positions 85, 100, 137, 140, 233, 450, 598, and 620 relative to SEQ ID NO:3, wherein the SHC / HAC derivative has increased HAC enzymatic activity relative to SEQ ID NO:3. 10. An SHC / HAC derivative according to paragraph 9, which comprises one or more substitutions selected from the group consisting of G85A, V100V, F137L, I140R, V233V, I450T, N598H, and F620Y relative to SEQ ID NO:3. 11. SHC / HAC derivatives according to paragraph 10, including F620Y. 12. An SHC / HAC derivative according to paragraph 10, comprising F137L. 13. SHC / HAC derivatives according to paragraph 10, including F620Y and F137L. 14. SHC / HAC derivatives according to paragraph 10, including 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. SHC / HAC derivatives according to paragraph 11, further comprising N598H. 20. SHC derivatives according to paragraph 10, including G85A and V100V and F137L. 21. An SHC / HAC derivative according to any one of the preceding paragraphs, having 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 according to 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 comprising the nucleotide sequence of paragraph 22 or paragraph 23. 25. A construct according to paragraph 24, comprising a promoter operably linked to the nucleotide sequence of paragraph 22 or 23. 26. The construct of paragraph 25, wherein the promoter is an inducible or constitutive promoter. 27. A vector comprising a construct according to any one of paragraphs 24 to 26. 28. The vector of paragraph 27, wherein the vector is a plasmid. 29. A vector according to paragraph 28, capable of directing expression in a host cell selected from a prokaryotic, yeast, plant, and insect host cell. 30. A construct according to any one of paragraphs 24 to 26 or a vector according to any one of paragraphs 27 to 29, wherein the construct or vector is capable of integration into the genome of a host cell selected from a prokaryotic, yeast, plant, and insect host cell. 31. A recombinant host cell comprising a nucleotide sequence according to paragraph 22 or 23, or a construct according to any one of paragraphs 24 to 26 or 30, or a vector according to any one of paragraphs 27 to 30. 32. A recombinant host cell according to paragraph 31, wherein the host cell is selected from the group of prokaryotic host cells consisting of bacteria of the genera Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. 33. The recombinant host cell of paragraph 32, wherein the host cell is an E. coli host cell. 34. A recombinant host cell according to paragraph 33, wherein the host cell overexpresses a gene encoding an SHC / HAC derivative. 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 permissive for production of an SHC / HAC derivative enzyme. 36. The method of paragraph 35, wherein the cell culture occurs under conditions suitable for biocatalyst production. 37. A method for preparing (-)-ambrox, comprising converting homofarnesol to (-)-ambrox by using a recombinant host cell according to any one of paragraphs 31-34, or by using a recombinant host cell comprising SEQ ID NO: 167 encoding a WT SHC / HAC, wherein when a WT SHC / HAC is used, the bioconversion of homofarnesol to (-)-ambrox is carried out by a solubilizing agent other than Triton X-100. 38. The method according to paragraph 37, wherein the conversion of homofarnesol to (-)-ambrox is under biotransformation conditions suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 39. The method according to paragraph 37 or 38, wherein the conversion of homofarnesol to (-)-ambrox occurs at a pH, temperature and solubilizing agent concentration suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 40. The method according to paragraph 39, wherein the conversion of homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C, at a pH in the range of about 4 to 8, and in the presence of a solubilizing agent other than Triton X-100 for WT SHC / HAC enzymes. 41. The method according to any one of paragraphs 37-40, wherein the conversion of homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for the WT SHC / HAC or SHC / HAC derivative enzymes set forth in Table 24 or Table 24a. 42. The process of any one of paragraphs 37-41, wherein the weight ratio of biocatalyst to homofarnesol is within the range of about 0.5:1 to 2:1, preferably about 1:1 or 0.5:1. 43. The method according to any one of paragraphs 37 to 42, wherein the cell growth and bioconversion reaction steps are carried out in the same reaction vessel. 44. The 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 comprises an EE:EZ stereoisomer. 47. The method according to any one of paragraphs 44-46, wherein the homofarnesol comprises an EE:EZ stereoisomeric 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 the homofarnesol comprises an EE:EZ stereoisomeric mixture in a weight ratio selected from the group consisting of EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and EE:EZ 66:34. 49. The method of paragraph 35 or 36, wherein the homofarnesol comprises a mixture of EE:EZ stereoisomers in a weight ratio of 80:20. 50. The process of any one of paragraphs 37-49, wherein (-)-ambrox is produced in a mixture with one or more of by-products (II), (IV), and / or (III). 51. The process of any one of paragraphs 37-50, wherein (-)-ambrox is isolated from the bioconversion reaction mixture using an organic solvent or steam extraction / distillation step or filtration. 52. The process 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 process of paragraph 52 or 53, wherein the (-)-ambrox is substantially free of by-products (II), (IV), and / or (III). 55. (-)-Ambrox, obtainable by the process of any one of paragraphs 51 to 54. 56. (-)-Ambrox of paragraph 55 in a solid form, preferably in amorphous or crystalline form. 57. A method for making a product containing (-)-ambrox, comprising incorporating the (-)-ambrox of 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. Fragrances or cosmetics or consumer care products containing (-)-ambrox of paragraph 55 or 56. 59. A fragrance or cosmetic or consumer care composition comprising (-)-ambrox of paragraph 55 or 56 and one or more additional components. 60. Use of (-)-ambrox of paragraph 55 or 56 as part of a fragrance or cosmetic or consumer product, such as a fabric care, toiletry, beauty and / or cleaning product. 61. Use of an SHC / HAC derivative enzyme according to any one of paragraphs 1 to 21, a nucleotide sequence according to paragraphs 22 or 23, a construct according to any one of paragraphs 24 to 26 or 30, a vector according to any one of paragraphs 27 to 30, or a recombinant host cell according to any one of paragraphs 31 to 34, or a recombinant host cell expressing a WT SHC / HAC, for the bioconversion of homofarnesol to (-)-ambrox, wherein the WT SHC / HAC enzyme is used in the bioconversion reaction with a solubilizing agent other than Triton X-100.

[0271] Additional aspects of the invention (BJpSHC) 1. A squalene-hopene cyclase (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, 1-30 mutations, 1-20 mutations, 1-10 mutations, or 1-6 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, or 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 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 to SEQ ID NO:4. 5. An SHC / HAC derivative, comprising 1-10 mutations independently selected from substitutions, deletions, or insertions relative to SEQ ID NO:4, wherein one or more mutations other than an SHC active site mutation are located within domain 2 of the SHC enzyme (Figures 19 and / or 20). 6. An SHC / HAC derivative according to any one of paragraphs 1 to 5, in which one or more mutations are selected from Table 4 relative to SEQ ID NO:4, and if only one mutation is selected it is not F628Y. 7. The SHC / HAC derivative of 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, comprising an amino acid sequence having up to six mutations relative to SEQ ID NO:4 and including at least the substitutions F628Y or I140R in combination with at least any one or more of F137L and / or I450T. 9. The SHC / HAC derivative of paragraph 7, comprising an amino acid sequence having up to eight amino acid alterations relative to SEQ ID NO:4 and including one or more amino acid alterations 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 enzymatic activity relative to SEQ ID NO:4. 10. An SHC / HAC derivative according to paragraph 9, which comprises one or more substitutions selected from the group consisting of A88A, V104V, F141L, Y144R, V241V, I459T, M607H, and F628Y relative to SEQ ID NO:4. 11. SHC / HAC derivatives according to paragraph 10, including F628Y. 12. An SHC / HAC derivative according to paragraph 10, comprising F141L. 13. SHC / HAC derivatives according to paragraph 10, including F628Y and F141L. 14. An SHC / HAC derivative 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 and V145V and F182L. 21. An SHC / HAC derivative according to any one of the preceding paragraphs, having 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 according to 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: 114, 116, 118, 120, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, and / or 148. 24. A construct comprising the nucleotide sequence of paragraph 22 or paragraph 23. 25. A construct according to paragraph 24, comprising a promoter operably linked to the nucleotide sequence of paragraph 22 or 23. 26. The construct of paragraph 25, wherein the promoter is an inducible or constitutive promoter. 27. A vector comprising a construct according to any one of paragraphs 24 to 26. 28. The vector of paragraph 27, wherein the vector is a plasmid. 29. A vector according to paragraph 28, capable of directing expression by a host cell selected from prokaryotic, yeast, plant, and insect host cells. 30. A construct according to any one of paragraphs 24 to 26 or a vector according to any one of paragraphs 27 to 29, wherein the construct or vector is capable of integration into the genome of a host cell selected from a prokaryotic, yeast, plant, and insect host cell. 31. A recombinant host cell comprising a nucleotide sequence according to paragraph 22 or 23, or a construct according to any one of paragraphs 24 to 26 or 30, or a vector according to any one of paragraphs 27 to 30. 32. A recombinant host cell according to paragraph 31, wherein the host cell is selected from the group of prokaryotic host cells consisting of bacteria of the genera Escherichia, Streptomyces, Bacillus, Pseudomonas, Lactobacillus, and Lactococcus. 33. The recombinant host cell of paragraph 32, wherein the host cell is an E. coli host cell. 34. A recombinant host cell according to paragraph 33, wherein the host cell overexpresses a gene encoding an SHC / HAC derivative. 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 permissive for production of an SHC / HAC derivative enzyme. 36. The method of paragraph 35, wherein the cell culture occurs under conditions suitable for biocatalyst production. 37. A method for preparing (-)-ambrox, comprising converting homofarnesol to (-)-ambrox by using a recombinant host cell according to any one of paragraphs 31-34, or by using a recombinant host cell comprising SEQ ID NO: 168 encoding a WT SHC / HAC, wherein when a WT SHC / HAC is used, the bioconversion of homofarnesol to (-)-ambrox is carried out by a solubilizing agent other than Triton X-100. 38. The method according to paragraph 37, wherein the conversion of homofarnesol to (-)-ambrox is under biotransformation conditions suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 39. The method according to paragraph 37 or 38, wherein the conversion of homofarnesol to (-)-ambrox occurs at a pH, temperature and solubilizing agent concentration suitable for the WT SHC / HAC or SHC / HAC derivative enzyme. 40. The method according to paragraph 39, wherein the conversion of homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C, at a pH in the range of about 4 to 8, and in the presence of a solubilizing agent other than Triton X-100 for WT SHC / HAC enzymes. 41. The method according to any one of paragraphs 37-40, wherein the conversion of homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for the WT SHC / HAC or SHC / HAC derivative enzymes set forth in Table 24 or Table 24a. 42. The process of any one of paragraphs 37-41, wherein the weight ratio of biocatalyst to homofarnesol is within the range of about 0.5:1 to 2:1, preferably about 1:1 or 0.5:1. 43. The method according to any one of paragraphs 37 to 42, wherein the cell growth and bioconversion reaction steps are carried out in the same reaction vessel. 44. The 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 comprises an EE:EZ stereoisomer. 47. The method according to any one of paragraphs 44-46, wherein the homofarnesol comprises an EE:EZ stereoisomeric 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 the homofarnesol comprises an EE:EZ stereoisomeric mixture in a weight ratio selected from the group consisting of EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and EE:EZ 66:34. 49. The method of paragraph 35 or 36, wherein the homofarnesol comprises a mixture of EE:EZ stereoisomers in a weight ratio of 80:20. 50. The process of any one of paragraphs 37-49, wherein (-)-ambrox is produced in a mixture with one or more of by-products (II), (IV), and / or (III). 51. The process of any one of paragraphs 37-50, wherein (-)-ambrox is isolated from the bioconversion reaction mixture using an organic solvent or steam extraction / distillation step or filtration. 52. The process 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 process of paragraph 52 or 53, wherein the (-)-ambrox is substantially free of by-products (II), (IV), and / or (III). 55. (-)-Ambrox, obtainable by the process of any one of paragraphs 51 to 54. 56. (-)-Ambrox of paragraph 55 in a solid form, preferably in amorphous or crystalline form. 57. A method for making a product containing (-)-ambrox, comprising incorporating the (-)-ambrox of 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. Fragrances or cosmetics or consumer care products containing (-)-ambrox of paragraph 55 or 56. 59. A fragrance or cosmetic or consumer care composition comprising (-)-ambrox of paragraph 55 or 56 and one or more additional components. 60. Use of (-)-ambrox of paragraph 55 or 56 as part of a fragrance or cosmetic or consumer product, such as a fabric care, toiletry, beauty and / or cleaning product. 61. Use of an SHC / HAC derivative enzyme according to any one of paragraphs 1 to 21, a nucleotide sequence according to paragraph 22 or 23, a construct according to any one of paragraphs 24 to 26 or 30, a vector according to any one of paragraphs 27 to 30, or a recombinant host cell according to any one of paragraphs 31 to 34, or a recombinant host cell expressing a WT SHC / HAC, for the bioconversion of homofarnesol to (-)-ambrox, wherein the WT SHC / HAC enzyme is used in the bioconversion reaction with a solubilizing agent 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 according to the amino acid sequence of SHC or a derivative described herein. The CMS of SHC includes a squalene / homofarnesol binding pocket domain (SHBD) that includes a squalene / homofarnesol binding pocket (SHBP) and a squalene / homofarnesol substrate bound to the SBD (see, e.g., Figures 19 and 20). This SHC crystal model structure (CMS) facilitates in silico testing of potential SHC / HAC derivative enzyme candidates.

[0273] Thus, in yet other embodiments, the disclosure provides methods for screening for an enzyme capable of binding to an SHBP (e.g., an SHC / HAC derivative), the method comprising use of a CMS of an SHC / HAC. In another aspect, the disclosure provides methods for screening for an enzyme capable of binding to an SHBP (e.g., a reference SHC or an SHC / HAC derivative), the method comprising contacting an 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 for test compounds (e.g., modulators) useful in modulating the activity of an SHC derivative enzyme.

[0274] In another aspect, the disclosure provides a method for predicting, simulating or modeling the molecular features and / or intermolecular interactions of a reference SHC and / or SHC / HAC derivative having a squalene / homofarnesol binding domain (SHBD), comprising the use of a computer model that 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 displaying 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 recited integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. The term "comprise" also means "comprises" and "consists of." For example, a composition "comprising" X may consist solely of X, or may include something additional, e.g., X+Y. It should also be noted that the singular forms "a," "an," and "the" used in this specification and the appended claims include plural referents unless the content clearly dictates otherwise. For example, a reference to a "gene" or an "enzyme" is a reference to "one or more genes" or "one or more enzymes."

[0276] It should be understood that the present disclosure is not limited to the specific methodology, protocol, and reagent described herein, as they may vary.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.In accordance with the present disclosure, conventional molecular biology, microbiology, and recombinant DNA techniques may be employed, which are within the skill of the art.

[0277] The present disclosure is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways. The phraseology and terminology used herein is also for the purpose of description and should not be regarded as limiting.

[0278] Preferably, the terms used herein are defined as set forth 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's specifications, instructions, GenBank accession numbers, sequence submissions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety.

[0280] The examples described herein are illustrative of the present disclosure and are not intended to be limiting thereof. Different aspects of the present disclosure have been described according to the present disclosure. Many modifications and variations can be made to the techniques described and illustrated herein without departing from the spirit and scope of the present disclosure. Therefore, it should be understood that the examples are merely illustrative and are not limiting of the scope of the present disclosure. [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20]

[0281] SEQ ID NO:1 (Alicyclobacillus acidocaldarius), AacSHC MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR SEQ ID NO:2 (Zymomonas mobilis), ZmoSHC1 MGIDRMNSLSRLLMKKIFGAEKTSYKPASDTIIGTDTLKRPNRRPEPTAKVDKTIFKTMGNSLNNTLVSACDWLIGQQKPDGHWVGAVESNASMEAEWCLALWFLGLEDHPLRPRLGNALLEMQREDGSWGVYFGAGNGDINATVEAYAALRSLGYSADNPVLKKAAAWIAEKGGLKNIRV FTRYWLALIGEWPWEKTPNLPPEIIWFPDNFVFSIYNFAQWARATMVPIAILSARRPSRPLRPQDRLDELFPEGRARFDYELPKKEGIDLWSQFFRTTDRGLHWVQSNLLKRNSLREAAIRHVLEWIIRHQDADGGWGGIQPPWVYGLMALHGEGYQLYHPVMAKALSALDDPGWRHDRGE SSWIQATNSPVWDTMLALMALKDAKAEDRFTPEMDKAADWLLARQVKVKGDWSIKLPDVEPGGWAFEYANDRYPDTDDTAVALIALSSYRDKEEWQKKGVEDAITRGVNWLIAMQSECGGWGAFDKDNNRSILSKIPFCDFGESIDPPSVDVTAHVLEAFGTLGLSRDMPVIQKAIDYVRS EQEAEGAWFGRWGVNYIYGTGAVLPALAAIGEDMTQPYITKACDWLVAHQQEDGGWGESCSSYMEIDSIGKGPTTPSQTAWALMGLIAANRPEDYEAIAKGCHYLIDRQEQDGSWKEEEFTGTGFPGYGVGQTIKLDDPALSKRLLQGAELSRAFMLRYDFYRQFFPIMALSRAERLIDLNN SEQ ID NO:3 (Zymomonas mobilis), ZmoSHC2 MTVSTSSAFHHSPLSDDVEPIIQKATRALLEKQQQDGHWVFELEADATIPAEYILLKHYLGEPEDLEIEAKIGRYLRRIQGEHGGWSLFYGGDLDLSATVKAYFALKMIGDSPDAPHMLRARNEILARGGAMRANVFTRIQLALFGAMSWEHVPQMPVELMLMP EWFPVHINKMAYWARTVLVPLLVLQALKPVARNRRGILVDELFVPDVLPTLQESGDPIWRRFFSALDKVLHKVEPYWPKNMRAKAIHSCVHFVTERLNGEDGLGAIYPAIANSVMMYDALGYPENHPERAIARRAVEKLMVLDGTEDQGDKEVYCQPCLSPIWDT ALVAHAMLEVGGDEAEKSAISALSWLKPQQILDVKGDWAWRRPDLRPGGWAFQYRNDYYPDVDDTAVVTMAMDRAAKLSDLHDDFEESKARAMEWTIGMQSDNGGWGAFDANNSYTYLNNIPFADHGALLDPPTVDVSARCVSMMAQAGISITDPKMKAAVDYL LKEQEEDGSWFGRWGVNYIYGTWSALCALNVAALPHDHLAVQKAVAWLKTIQNEDGGWGENCDSYALDYSGYEPMDSTASQTAWALLGLMAVGEANSEAVTKGINWLAQNQDEEGLWKEDYYSGGGFPRVFYLRYHGYSKYFPLWALARYRNLKKANQPIVHYGM SEQ ID NO:4 (Bradyrhizobium japonicum), BjpSHC MTVTSSASARATRDPGNYQTALQSTVRAAADWLIANQKPDGHWVGRAESNACMEAQWCLALWFMGLEDHPLRKRLGQSLLDSQRPDGAWQVYFGAPNGDINATVEAYAALRSLGFRDDEPAVRRAREWIEAKGGLRNIRVFTRYWLALIGEWPWEKTPNIPPEVIWFPLWF PFSIYNFAQWARATLMPIAVLSARRPSRPLPPENRLDALFPHGRKAFDYELPVKAGAGGWDRFFRGADKVLHKLQNLGNRLNLGLFRPAATSRVLEWMIRHQDFDGAWGGIQPPWIYGLMALYAEGYPLNHPVLAKGLDALNDPGWRVDVGDATYIQATNSPVWDTILTLL AFDDAGVLGDYPEAVDKAVDWVLQRQVRVPGDWSMKLPHVKPGGWAFEYANNYYPDTDDTAVALIALAPLRHDPKWKAKGIDEAIQLGVDWLIGMQSQGGGWGAFDKDNNQKILTKIPFCDYGEALDPPSVDVTAHIIEAFGKLGISRNNHPSMVQALDYIRREQEPSGPWF GRWGVNYVYGTGAVLPALAAIGEDMTQPYIGRACDWLVAHQQADGGWGESCASYMDVSAVGRGTTTASQTAWALMALLAANRPQDKDAIERGCMWLVERQSAGTWDEPEFTGTGFPGYGVGQTIKLNDPALSQRLMQGPELSRAFMLRYGMYRHYFPLMALGRALRPQSHS SEQ ID NO:149 (Burkholderia ambifaria) MNDLTEMATLSAGTVPAGLDAAVASATDALLAAQNADGHWVYELEADSTIPAEYVLLVHYLGETPNLELEQKIGRYLRRVQQADGGWPLFTDGAPNISASVKAYFALKVIGDDENAEHMQRARRAIQAMGGAEMSNVFTRIQLALYGAIPWRAVPMMPVEIMLL PQWFPFHLSKVSYWARTVIVPLLVLNAKRPIAKNPRGVRIDELFVDPPVNAGLLPRQGHQSPGWFAFFRVVDHALRAADGLFPNYTRERAIRQAVSFVDERLNGEDGLGAIYPAMANAVMMYDVLGYAEDHPNRAIARKSIEKLLVVQEDEAYCQPCLSPVWDT SLAAHALLETGDARAEAVIRGLEWLRPLQILDVRGDWISRRPHVRPGGWAFQYANPHYPDVDDTAVVAVAMDRVQKLKHNDAFRDSIARAREWVVGMQSSDGGWGAFEPENTQYYLNNIPFSDHGALLDPPTADVSGRCLSMLAQLGETPLNSEPARRALDYM LKEQEPDGSWYGRWGMNYVYGTWTALCALNAAGLTPDDPRVKRGAQWLLSIQNKDGGWGEDGDSYKLNYRGFEQAPSTASQTAWALLGLMAAGEVNNPAVARGVEYLIAEQKEHGLWDETRFTATGFPRVFYLRYHGYRKFFPLWALARYRNLKRNNATRVTGL SEQ ID NO:151 (Burkholderia ambifaria) MIRRMNKSGPSPWSALDAAIARGRDALMRLQQPDGSWCFELESDATITAEYILMMHFMDKIDDARQEKMARYLRAIQRLDTHGGWDLYVDGDPDVSCSVKAYFALKAAGDSEHAPHMVRARDAILELGGAARSNVFTRILLATFGQVPWRATPFMPIEFVLFPKWVPISM YKVAYWARTTMVPLLVLCKARARNPRNIAIPELFVTPPDQERQYFPPARGMRRAFLALDRVVRHVEPLLPKRLRQRAIRHAQAWCAERMNGEDGLGGIFPPIVYSYQMMDVLGYPDDHPLRRDCENALEKLLVTRPDGSMYCQPCLSPVWDTAWSTMALEQARGVAVPE AGAPASALDELDARIARAYDWLAERQVNDLRGDWIENAPADTQPGGWAFQYANPYYPDIDDSAVVTAMLDRRGRTHRNADGSHPYAARVARALDWMRGLQSRNGGFAAFDADCDRLYLNAIPFADHHALLDPPTEDVSGRVLLCFGVTKRADDRASLARAIDYVKRTQQP DGSWWGRWGTNYLYGTWSVLAGLALAGEDPSQPYIARALAWLRARQHADGGWGETNDSYIDPALAGTNAGESTSNCTAWALLAQMAFGDGESESVRRGIAYLQSVQQDDGFWWHRSHNAPGFPRIFYLKYHGYTAYFPLWALARYRRLAGGVSAAGAHAVPASTGADAALA SEQ ID NO: 153 (Bacillus anthracis) MLLYEKAHEEIVRRATALQTMQWQDGTWRFCFEGAPLTDCHMIFLLKLLGRDKEIEPFVERVASLQTNEGTWKLHEDEVGGNLSATIQSYAALLASKKYTCEDANMKRAENFIQERGGVARAHFMTKFLLAIHGEYEYPSLFHLPTPIMFLQND SPFSIFELSSSARIHLIPMMLCLNKRFRVGKKLLPNLNHIAGGGGEWFREDRSPVFQTLLSDVKQIISYPLSLHHKGYEEIERFMKERIDENGTLYSYATASFYMIYALLALGHSLQSSMIQKAIAGITSYIWKMERGNHLQNSPSTVWDTALL SYALQEAQVSKDNKMIQNATAYLLKKQHTKKADWSVHAPALTPGGWGFSDVNTTIPDIDDTTAVLRALARSRGNKNIDNAWKKGGNWIKGLQNNDGGWGAFEKGVTSKLLAKLPIENASDMITDPSTPDITGRVLEFFGTYAQNELPEKQIQRA INWLMNVQEENGSWYGKWGICYLYGTWAVMTGLRSLGIPSSNPSLTRAASWLEHIQHEDGGWGESCHSSVEKRFVTLPFSTPSQTAWALDALISYYDTETPAIRKGVSYLLSNPYVNERYPTGTGLPGAFYIRYHSYAHIYPLLTLAHYIKKYRK SEQ ID NO: 155 (Frankia alni) MPAGVGVLVWLDQRLRAMGRPDLVTTTGGAEIPFVLVAATASTVGVALALRRPRHPVGWLFLALGGVLLLSGGTQGYAAYGAVARPGRLPAADLVAIYADAGFIPWLVLVALILHLTPTGRPLSARWGRIALATAVAGGLWLLVGLVTTETMQPPFQSVTNPLLIGGPLPLLVARRVLGLATGAGVVLAAVSLIVRFRRSVDVERRQLLWVAAVPLPVLMAASFAASYAGNNTAAGLAAATLIGLLAIGAGLAIGQYHLYDVEEILSRAVTYLLVSGLLAASYATVVIVVGQSLAGRTGRSQISAVLATLAAVAVTAPAYRKIQEGVDRRFSRRRFETLQVIRRYLRDPDPDVAVEE VLRRALGDPTLAVAYLVDDRRQWVSADGQPANPGNSFMAAVEVYRRGRPIARVTFDRGRAQPGLVRAAATAATAELDNAGLRAAVALQLVEVRQSRTRIAAAQFAERRTIERNLHDGAQQRLLALALQLRAVQLGGDEASLRQAISTGIDQLQAAVVELRELANGLHPAVLADGGLAAALDDVAARTPVPIKISAPDRRYPPDLEAAAWFIACEAMANAVKHAHPTTIAVDVSAPDGQLIVEVRDDGIGGAQPSGPGLRGIADRAEAFGGSLTVHTDPGTGTTIRALLHRRSPLSSGRRSVMIEGCVDVVAVRRFRCRSSRGSGSRRRSSWRCGGICGSRCTGMMSRSCSRNAASKLIT SEQ ID NO:157(ロドシュドモナス·パレント(Rhodopseudomonas patent)) MDSILAPRADAPRNIDGALRESVQQAADWLVANQKPDGHWVGRAETNATMEAQWCLALWFLGLEDHPLRVRLGRALLDTQRPDGAWHVFYGAPNGDINATVEAYAALRSLGHRDDEEPLRKARDWILSKGGLANIRVFTRYWLALIGEWPWEKTPNILPEVIWLPTWFPFS IYNFAQWARATLMPIAVLSAHRPSRPLAPQDRLDALFPQGRDSFNYDLPARLGAGVWDVIFRKIDTILHRLQDWGARRGPHGIMRRGAIDHVLQWIIRHQDYDGSWGGIQPPWIYGLMALHTEGYAMTHPVMAKALDALNEPGWRIDIGDATFIQATNSPVWDTMLSLLAF DDAGLGERYPEQVERAVRWVLKRQVLVPGDWSVKLPDVKPGGWAFEYANNFYPDTDDTTSVALMALAPFRHDPKWQAEGIEDAIQRGIDWLVAMQCKEGGWGAFDKDNDKKILAKIPFCDFGEALDPPSADVTAHIIEFAKVGLDRNHPSIVRALDYLKREQEPEGPWFGR WGVNYVYGTGAVLPALAAIGEDMRQPYIARACDWLIARQQANGGWGESCVSYMDAKQAGEGTATASQTAWALMALIAADRPQDRDAIERGCLYLTETQRDGTWQEVHYTGTGFPGYGVGQTIKLNDPLLSKRLMQGPELSRSFMLRYDLYRHYFPMMAIGRVLRQRGDRSGH SEQ ID NO:159 (Streptomyces coelicolor) MTATTDGSTGASLRPLAASASDTDITIPAAAAGVPEAAARATRRATDFLLAKQDAEGWWKGDLETNVTMDAEDLLLRQFLGIQDEETTRAAALFIRGEQREDGTWATFYGGPGELSTTIEAYVALRLAGDSPEAPHMARAAEWIRSRGGIASARVFTRIWLALFGWWKW DDLPELPPELIYFPTWVPLNIYDFGCWARQTIVPLTIVSAKRPVRPAPFLDELHTDPARPNPPRPLAPVASWDGAFQRIDKALHAYRKVAPRRLRRAAMNSAARWIIERQENDGCWGGIQPPAVYSVIALYLLGYDLEHPVMRAGLESLDRFAVWREDGARMIEACQSP VWDTCLATIALADAGVPEDHPQLVKASDWMLGEQIVRPGDWSVKRPGPPGGWAFEFHNDNYPDIDDTAEVVLALRRVRHHDPERVEKAIGRGVRWNLGMQSKNGAWGAFDVDNTSAFPNRLPFCDFGEVIDPPSADVTAHVVEMLAVEGLAHDPRTRRGIQWLLDAQETD GSWFGRWGVNYVYGTGSVIPALTAAAGLPTSHPAIRRAVRWLESVQNEDGGWGEDLRSYRYVREWSGRGASTASQTGWALMALLAAGERDSKAVERGVAWLAATQREDGSWDEPYFTGTGFPWDFSINYNLYRQVFPLTALGRYVHGEPFAKKPRAADAPAEAAPAEVKGS SEQ ID NO:169 Variant 101A10 (SEQ ID NO: 30) Variant 101A10 (SEQ ID NO: 29) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVHYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant 111C8 ((SEQ ID NO: 28) Variant 111C8 (SEQ ID NO: 27) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGAWALYPGGPPDLDTTVEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVLTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR Variant SHC215G2 (SEQ ID NO: 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 (SEQ ID NO: 170) Variant F605W (SEQ ID NO: 171) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR SEQ ID NO: 166 (ZmoSHC1) [Table 21] [Table 22] [Table 23] [Table 24] [Brief description of the drawings]

[0282] For a better understanding of the present disclosure, reference is made to the accompanying drawings. [Figure 1-4] Figures 1-4 show sequence alignments of selected AacSHC derivatives to AacSHC SEQ ID NO: 1. In descending order of listing, 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. [Diagram 5] FIG. 5 shows the plasmid map. [Figure 6] FIG. 6 shows the relative HAC activity of wild-type AacSHC and the AacSHC derivatives listed in Table 24 under standard conditions (pH 6.0, 55° C., 0.050% SDS, cells with an OD650 nm of 10). [Figure 7] Figure 7a shows the HAC activity profile of AacSHC derivatives versus WT AacSHC, using homofarnesol quality EEH:EZH87:13 and homofarnesol purity 96% (determined using NMR). Figure 7b shows the relative improvement (4h (initial rate) and 22h yield) of AacSHC derivatives versus WT SHC, using homofarnesol quality EEH:EZH87:13 and homofarnesol purity 96% (determined using NMR). [Figure 8]Figure 8a shows the HAC activity profile of AacSHC derivatives versus WT AacSHC, using homofarnesol quality EEH:EZH92:08 and homofarnesol purity 100% (determined using NMR). Figure 8b shows the relative improvement (4h (initial rate) and 22h yield) of AacSHC derivatives versus WT SHC, using homofarnesol quality EEH:EZH92:08 and homofarnesol purity 100% (determined using NMR). [Figure 9] Figure 9a shows the HAC activity profile of the AacSHC derivatives listed in Table 24 versus WT AacSHC, using homofarnesol quality EEH:EZH66:33 at 76% purity of homofarnesol (determined using NMR). Figure 9b shows the relative improvement (4h (initial rate) and 22h yield) of the AacSHC derivatives versus WT SHC, using homofarnesol quality EEH:EZH66:33 at 76% purity of homofarnesol (determined using NMR). [Figure 10] FIG. 10 shows the HAC activity results for three SHC derivatives that show approximately 10-fold (215G2), 7-fold (SHC26), and 6-fold (SHC32) improvements over the wild-type AacSHC / HAC enzyme. [Figure 11] 11 shows E,E-homofarnesol conversion to ambrox observed with SHC / HAC derivatives (215G2 SHC) and WT AacSHC. In 7-hour reactions (estimate of initial reaction rates), conversion with variant 215G2 SHC was 13-fold higher than that achieved with wild-type SHC. In 48-hour reactions, conversion with the variant was approximately 8-fold that of the wild-type enzyme. [Figure 12]12 shows the reaction products produced when EEH is used as the starting material (ambrox and product (IV)) and when EE:EZ is used as the starting material ((-)-ambrox (I) and products (II), (IV), and (III) (see Table 21)). For ease of reference, compounds I-IV may 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]oxonine 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] FIG. 13 shows the GC analysis of the reaction products for Ambrox and products (II), (IV), and (III) in Table 25. [Figure 14] FIG. 14 shows the GC analysis of the reaction products for Ambrox and products (II), (IV), and (III) in Table 25. [Figure 15] FIG. 15 provides comparative data on 215G2 SHC variant activity by whole cell biotransformation assay in the presence of either Triton X-100 or SDS. [Figure 16] FIG. 16 shows the percentage of EEH converted for different SDS / cell ratios. [Figure 17] FIG. 17 shows the % of EEH conversion in a standard bioconversion reaction (described in Example 7) for three different SDS concentrations. [Figure 18] FIG. 18 shows the % of EEH conversion in a standard bioconversion reaction (described in Example 7) for three different pH values. [Figure 19] FIG. 19 shows the location of the identified mutations of SHC / HAC variants 101A10, 111C8, and 215G2 on the SHC crystal structure (colored). Variant 215G2 is in red, variant 101A10 in purple (wine red), and variant 111C8 in green. Amino acids identified as responsible for increased activity are highlighted in yellow on the substrate analogue with which their side chains were co-crystallized. Other mutations in the identified variants with unimproved activity are marked in blue. It is noteworthy that the blue mutations are scattered approximately 50:50 over the two domains of the enzyme, while the identified beneficial AacSHC mutations are mostly located within domain 2 (apart from one). The only exception is mutation F601Y, which is in the vicinity of 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. Mutations identified in SHC variants (101A10, 111C8, and 215G2) that show improved SHC / HAC activity are shown in grey and, with only one exception, are located within domain 2 of the SHC enzyme. The side chains of the mutations that contribute to the improved activity of the variants are highlighted. [Figure 21] Figure 21 shows the cloning and expression region of plasmid pET-28a(+). The SEQ ID NOs in the sequence of 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]FIG. 22 shows the volumetric productivity of a 1.5× concentrated EEH bioconversion reaction containing 375 g / l cells, 188 g / l EEH, 2.33% SDS compared to the volumetric productivity of a standard bioconversion (Example 7) carried out in parallel at 125 g / l EEH, 250 g / l cells, 1.55% SDS. [Figure 23] Figure 23 shows a standard biotransformation (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 by either 0.5% or 0.9% NaCl, while all other reaction parameters remained unchanged. Biotransformations in citrate buffer were carried out in parallel as controls. [Figure 24] FIG. 24 shows the progress of solid-phase extraction of (-)-ambrox with toluene washes as a % of the amount of (-)-ambrox initially present in the 200 ml total reaction broth (the % in the first extract exceeds 100% due to the volume ratio broth / toluene). [Diagram 25] Figure 25 shows the progress of solid-phase extraction of (-)-ambrox with ethanol washes as a percentage of the amount of (-)-ambrox initially present. After four washes (a total of 640 ml EtOH, i.e., 3.2x the total initial reaction broth volume or 8x the solid phase volume), approximately 99% of the (-)-ambrox initially present in the reaction broth was recovered.

[0283] For the avoidance of doubt, all references to WT SHC and SHC variants are to WT AacSHC (SEQ ID NO:1) and variants thereof (eg, 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(+), where it is under the control of an IPTG-inducible T7 promoter for protein production by E. coli (see Figures 5 and 21). The plasmid was transformed into E. coli strain BL21(DE3) using a standard heat shock transformation protocol.

[0285] Erlenmeyer flask culture For protein production, either complex (LB) or minimal media was used. M9 is an example of a minimal medium that has been used successfully.

[0286] Medium preparation The default minimal medium was prepared as follows for a 350 ml culture: 35 ml citrate / phosphate stock (133 g / l KH 2 PO 4 , 40g / l (NH 4 ) 2 HPO 4 , 17g / l citric acid.H 2 307 ml of HO (pH adjusted to 6.3) 2 2O was added and the pH was adjusted to 6.8 with 32% NaOH as needed. 0.850 ml of 50% MgSO 4 After autoclaving, 0.035 ml of trace element solution (composition in 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 variants), 350 ml cultures (medium supplemented with 50 μg / ml kanamycin) were inoculated with a preculture of E. coli strain BL21(DE3) containing the SHC production plasmid. Cells were grown at 37 °C with constant agitation (250 rpm) to an optical density (OD ) of approximately 0.5. 650nm ) was grown. Protein production was then induced by the addition of IPTG at a concentration of 300 μM, followed by an additional 5-6 h 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. Typically, 2.5 to 4 grams of cells (wet weight) were obtained from 1 liter of culture, independent of the medium used. Fermentations were prepared and carried out in a 750 ml InforsHT reactor. 168 ml of deionized water was added to the fermentation vessel. The reactor contained all the necessary probes (pO 2 , pH, sampling, antifoaming agent), C+N feed and sodium hydroxide bottles were prepared and autoclaved. After autoclaving, the reaction vessel was 20ml 10x phosphate / citrate buffer 14ml 50% glucose 0.53ml MgSO 4 solution 2ml (NH 4 ) 2 SO 4 solution 0.020ml trace element solution 0.400ml thiamine solution 0.200ml Kanamycin Stock Added. The operating parameters were as follows: pH = 6.95, pO 2 = 40%, T = 30°C, stirring at 300 rpm. Cascade: rpm set point at 300, min 300, max 1000, flow l / min set point 0.1, min 0, max 0.6. Antifoam control: 1:9. From the seed culture, the fermenter was incubated at an OD of 0.4-0.5. 650nm This seed culture was grown in LB medium (+kanamycin) at 37°C and 220 rpm for 8 h. The fermentation was first carried out in batch mode for 11.5 h, after which the feed solution (sterile glucose solution (143 ml H 2A C+N feed of 17.5 ml (NH 0 + 35 g glucose) was started after sterilization. 4 ) 2 SO 4 Solution, 1.8 ml MgSO 4 solution, 0.018 ml trace element solution, 0.360 ml thiamine solution, 0.180 ml kanamycin stock were added. The feed was performed at a constant flow rate of approximately 4.2 ml / h. Glucose and NH 4 + Measurements were made externally to assess the availability of C and N sources in the culture. Normally, glucose levels remained very low. Cultures were grown for a total of approximately 25 hours, at which point they typically reached an OD of 40-45. 650nm IPTG was then added to the fermenter (either as an IPTG pulse or using an injection syringe over a period of 3-4 hours) to a concentration of approximately 1 mM, the temperature was raised to 40°C, and the pO 2 SHC production was started by setting the pH at 20%. Induction of SHC production continued for 16 h at 40° C. At the end of induction, cells were collected by centrifugation, washed with 0.1 M citric acid / sodium citrate buffer pH 5.4, and stored as pellets at 4° C. or −20° C. until further use.

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

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

[0290] Example 2 Preparation and activity screening of SHC variants Method 2 For the avoidance of doubt, EE corresponds to (3E,7E), the EZ mixture corresponds to (3Z,7E), ZE corresponds to (7Z,3E), ZZ corresponds to (7Z,3Z) and EEH corresponds to (3E,7E). An enzyme evolution program was performed using the wild-type (WT) Alicyclobacillus acidocaldarius SHC (AacSHC) gene as template (GenBank M73834, Swissprot P33247). A library of approximately 10,500 SHC variants was generated and screened for variants that exhibited increased EEH cyclization ability. Screening was performed in reactions in citrate buffer pH 6.0 (0.150 ml) containing 4 g / l EEH and 0.050% SDS at 55 °C under constant stirring. Depending on the hits selected for validation, standard tests were performed using 4 g / l EEH, 0.050% SDS, and OD of 10.0. 600nm The reaction was carried out in a citrate buffer pH 6.0 containing cells expressing the SHC variant of ambrox. 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 the activity profile (EEH conversion to (-)-ambrox) to be studied, as determined by gas chromatography analysis (see analytical methods below). From this validation round, three variants (101A10, 111C8, and 215G2) with improved EEH cyclization activity were obtained, and a total of eight mutations were then identified in those three variants. Mutational studies were then performed to identify which of those 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 650nmwas 10.0 cells, 0.05% and 0.1% SDS (two concentrations), and the reaction was carried out at 55° C. under constant agitation.

[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 of 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 the plasmids for the production of the individual variants, cultivated in Erlenmeyer flasks, and induced for SHC production as described above. In this way, it was ensured that all cultures contained the same or very similar amounts of SHC. The 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 SHC. Table 24 below shows the optimal reaction conditions for the wild type and each of the variants taking into account the characteristics of each SHC / HAC derivative enzyme. [Table 27]

[0295] Consideration 3 Example 3 shows the noted differences in reaction conditions for SHC derivatives compared to WT SHC. Considerable deviations from wild-type SHC for temperature, pH, and SDS concentration optima were observed for the SHC variants. Only a few mutations have a significant effect on optimal bioconversion reaction conditions. For determination of the individual reaction conditions for the selected SHC variants, reactions were run at a substrate input of 4 g / l of EEH and an optical density OD of 10.0. 650nm The experiment was carried out in cells producing wild-type or SHC derivatives of SHC. temperature The data in Table 24 show the surprising discovery that while the WT SHC enzyme has optimal activity at 55° C. (within the range of 45-60° C.), several SHC derivatives have optimal activity at 35° C. (34-50° C.). Application of the SHC derivatives of the present disclosure to the process for preparing (-)-ambrox from E,E-homofarnesol at lower reaction temperatures has significant cost advantages for the production of (-)-ambrox on an industrial scale. Solubilizer SDS was selected and identified from a long list of potential solubilizing agents that were not useful in biotransformation reactions (see Example 14 for more information). SDS is better than, for example, Triton X-100 in terms of reaction rate and yield (both in tests with EEH 4 g / l and when EEH 125 g / l is used as provided in Example 7).

[0296] Example 4 Testing the activity of SHC variants compared to the WT SHC enzyme under standard conditions Method 4 To compare the relative activity of the biocatalysts, the production of the variants (listed in Table 24) is described as follows. E. coli cells were transformed with the plasmid for the production of one of the SHC variants, and then the E. coli cells were cultured in LB medium at 37° C. and 280 rpm to reach an OD of 0.50. 650nmThe cells were grown to 30° C. and enzyme production was induced by addition of IPTG. Induction continued for 5.5 hours at 37° C. and 280 rpm. Cells were collected by centrifugation, washed with 0.1 M citric acid pH 6.0, and stored at −20° C. until further use. When comparing the activity of SHC variants (see FIG. 6), samples of the reaction mixtures were loaded onto an SDS-PAGE gel to analyze the SHC content of the reactions. This analysis confirmed that all reactions contained the same amount of SHC enzyme.

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

[0298] Result 4b With the 215G2 SHC variant, no notable effect on the activity of this variant was observed when NaCl was added to the reaction (concentrations tested were 5 to 100 mM (only)). In addition, addition of NaCl up to 100 mM or up to 154 mM (0.9% NaCl) did not show a negative effect on the SHC activity of variant 215G2. These findings suggest that biotransformation reactions can be carried out in the absence of buffers, in the presence of physiological NaCl solutions (0.9%), etc., provided that the pH is maintained at an appropriate value (e.g., about 5.4 (5.2-5.6)).

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

[0300] Example 5 Activity profile of WT SHC and SHC derivatives Method 5 Activity tests were performed in a Heidolph Synthesis 1 apparatus in 5 ml volume in 0.1 M citrate buffer under constant shaking at 900 rpm. The pH of the buffer used, the temperature at which the reaction was carried out, and the SDS (sodium dodecyl sulfate) concentration in the reaction depended on the SHC variant used (wild type or variant). The optimal conditions for each of the variants tested are summarized in Table 24 above. A homofarnesol starting material of 96% purity and a homofarnesol substrate with an EEH:EZH ratio of 87:13 were used. For the avoidance of doubt, 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 performed under optimized conditions are shown in FIG. 7B (activity profile of SHC derivatives relative to WT SHC) and FIG. 7A showing the relative activity improvement of SHC derivatives relative to WT SHC (initial rate at 4 h and yield at 22 h). Homofarnesol used: EEH:EZH92:08, purity (NMR): 100% The results of standard tests performed under optimized conditions are shown in FIG. 8B (activity profiles of AacSHC derivatives relative to WT AacSHC) and FIG. 8A showing the relative improvement of AacSHC derivatives relative to WT SHC (initial rate at 4 h and yield at 22 h). Homofarnesol used: EEH:EZH66:33, purity (NMR): 76% The results of standard tests performed under optimized conditions are shown in Figure 9B (activity profiles of the AacSHC derivatives listed in Table 24 relative to WT AacSHC) and Figure 9A showing the relative improvement of the AacSHC derivatives relative to WT SHC (initial rate at 4 h and yield at 22 h).

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

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

[0304] Result 6 The results in FIG. 10 showed that for the substrates used, the three best variants were identified and observed to show approximately 10-fold (215G2), 7-fold (SHC26), and 6-fold (SHC32) improvements over the wild-type SHC enzyme.

[0305] Example 7 Biotransformation performance at 125 g / l E,E-homofarnesol (EEH) Method 7 The 215G2 SHC variant was used to address the goal of increasing volumetric productivity. A design of series of experiments (DOE) study was conducted to optimize test reaction conditions including parameters pH, cell concentration, and SDS concentration. The reaction conditions were 125 g / l EEH (from homofarnesol of EE:EZ86:14), 250 g / l cells, 1.55% SDS, and the reaction was carried out at 35° C. in 0.1 M citrate buffer pH 5.4. A typical reaction (total volume of 150 g) is set up in a 0.75 liter Infors fermenter as follows: The reaction vessel is charged with the appropriate amount of homofarnesol corresponding to 18.75 g of EEH. 2.33 g of SDS are 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 that produced the 215G2 SHC variant by suspending the cells in 0.1 M citrate buffer pH 5.4. After determination of the cell wet weight of this suspension by centrifugation at 10° C. and 17210 g for 10 min, an appropriate volume of cells is added to the reaction vessel in order to introduce 37.5 g of cells into the reaction. The volume of the reaction is adjusted to 150 g by the required amount of reaction buffer. The reaction is carried out at 900 rpm at 37° C. under constant stirring. pH control is performed with 40% citric acid in water. Reactions were sampled over time (1 ml) and extracted with 5 volumes of MTBE / tBME (5 ml). After clarification of the solvent phase by centrifugation (tabletop centrifuge, 13000 rpm, 2 min) and 10-fold dilution with MTBE / tBME, the homofarnesol and ambrox content of the reactions was determined by GC analysis. The same reaction was performed with E. coli cells that produced wild-type SHC enzyme. In this case, the reaction was carried out in 0.1 M citrate buffer pH 6.0 at 55° C. A summary of the reaction conditions for this example is provided in the second line of Table 24a below. The reaction conditions shown in the first line of Table 24a below are taken from previous examples (e.g., Examples 3-5). [Table 28]

[0306] Result 7 Figure 11 shows the observed EEH conversion to ambrox by the two enzymes. In the 7 hour reaction (estimate of initial reaction rates), conversion by variant 215G2 SHC was 13-fold higher than that achieved by wild-type SHC. In the 48 hour reaction, conversion by the variant was approximately 8-fold that of the wild-type enzyme.

[0307] General Comment 7 cell concentration All concentrations (g / l) of cells in the reactions described in this example are given as wet weight of cells. The concentration of cells as wet weight (g / l) of a cell suspension is determined after centrifuging a sample of this cell suspension at 17210 g and 4° C. for 10 min. Cell g / l and OD 650nm Correlation between With bioconversion of 125 g / l of EEH by 215G2 SHC or WT SHC, 250 g / l of cells in this reaction resulted in an OD of approximately 172 in the reaction. 650nm When different biocatalyst preparations were tested, the OD 650nm Variations in the ratio of biocatalyst to the amount of biocatalyst were observed. The biocatalyst was used in the standard test at 4 g / l EEH but at an OD of 10.0. 650nm When cells were applied, the OD 650nm It was estimated that 10.0 is equivalent to 1.45 g / l of cells.

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

[0309] Example 8 GC analysis Method 8 For the quantification of their EEH and ambrox content, 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 analysis by gas chromatography. 1 μl of the solvent phase was injected onto a 30 m × 0.32 mm × 0.25 μm Zebron ZB-5 column (split ratio 3). The column was run at a constant flow (4 ml / min H 2 ) and developed with a temperature gradient of 100°C, 15°C / min to 200°C, 120°C / min to 240°C, 4 min at 240°C, which resulted in the separation of Ambrox, EEH, and EZH. Inlet temperature was 200°C, detector temperature: 300°C. The EEH conversion was calculated from the peak areas corresponding to ambrox and EEH using the following formula: 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: 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 steam extraction. Solvents used were, for example, MTBE or hexane:isopropanol (3:2). Reactions were repeatedly extracted with equal volumes of solvent and the solvent fractions were analyzed by GC until substrate or product was no longer detectable. Typically, 5 to 6 washes were sufficient. Alternatively, extraction of reaction products was done by steam extraction.

[0311] Example 9 One-pot reaction Method 9 A 200 ml fermentation was carried out with E. coli BL21(DE3) transformed with pET28a(+) 215G2 SHC plasmid for the production of 215G2 SHC with an 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 citric acid, and the agitator speed to 500 rpm. The culture volume was estimated from all additions made during the culture growth (feed and basal consumption). 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 with 700 μl 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 added repeatedly.

[0312] Result 9 Upon reaching completion, 10.6 g of EEH had been cyclized to ambrox. The reaction product (structure provided below) was extracted by steam extraction and quantitatively recovered from the reaction mixture. [ka] Notes on reaction products When homofarnesol EE:EZ 87:13 is converted by SHC, the reaction products ambrox, (II), (IV), and (III) are produced, as depicted in FIG. 12, reflecting the EE:EZ ratio of the starting material. When EEH is used as the 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 produced. However, when a mixture of EEH and EZH is used, ambrox (I) and products (II), (IV), and (III) are produced. If 100% conversion of EE:EZ66:34 occurs, this will provide 66%:34% ((Ambrox+(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 gives ambrox of 99% purity (GC) in at least 70% yield.

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

[0314] Example 10 Conversion of the EE:EZ homofarnesol mixture. For the avoidance of doubt, EE corresponds to (3E,7E), the 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 146 g / l total homofarnesol, 250 g / l cells and 1.55% SDS. EE:EZ 86:14 (highest EEH content in this example), EE:EZ 69:31 (lowest EEH content in this example), EE:EZ 80:20 EH:EZH 70:30 Biotransformation of the 7E,3E / 7E,3Z homofarnesol mixture. The bioconversion was initiated using the following reaction conditions: The reaction (150.1 g total volume) was carried out in an InforsHT 750 ml fermenter in 0.1 M citric acid / sodium citrate buffer pH 5.4, containing 146 g / l total homofarnesol, with homofarnesol substrate being a mixture of 7E,3E:7E,3Z in 86:14, 250 g / l 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 with 10 to 40% citric acid in water. The reaction mixture was sampled over time, and samples were solvent extracted for GC analysis. It was noted that homofarnesol conversion occurred equally quickly for the two species of homofarnesol (EE:EZ 86:14 and EE:EZ 69:31).

[0316] Result 10 During bioconversion of 125 g / l of E,E-homofarnesol from EEH:EZH86:14 material with 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 acidocaldarius 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 produced 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 Bioconversion of ambrox from homofarnesol according to the present disclosure produces (-)-ambrox as the major compound, but may also produce compounds other than (-)-ambrox (e.g., compounds (II), (IV), and (III)) as identified above, which may or may not impart 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)). Thus, selective separation of products (II), (IV), and (III) from the (-)-ambrox end product increases its value as a fragrance or flavor or cosmetic or consumer care product, in cases where the products negatively contribute to the sensory properties of the ambrox end product. Sensory analysis is performed using well-established sensory tests utilized by skilled perfumers. The purity of the (-)-Ambrox final product may be an indicator of the olfactory quality of the product if the product alone is the major contributor to the desired sensory profile.

[0318] Example 11 Conversion of EEH 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 with 215G2 SHC. For comparison purposes, other homofarnesols of EE:EZ 2:1 or 93:07 were also used. The conversion of the EE:EZ:ZE:ZZ-homofarnesol mixture was investigated with the 215G2 SHC variant, but not under optimized conditions. The reaction conditions were pH 5.8, 0.10% SDS in 100 mM citrate buffer, 40° C. The following EEH conversion was observed, and all reactions were performed at a constant 2 g / l EEH (hence varying total homofarnesol concentration).

[0319] Result 11 The following homofarnesol isomer mixture conversions 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 the complex EE:EZ:ZE:ZZ homofarnesol mixture. As expected, the observed lower conversion rate results from a lower ambrox yield. This result is consistent with the notion that homofarnesol isomers other than EEH may compete with EEH for access to the SHC / HAC derivative enzymes and therefore may act as competitive inhibitors and / or alternative substrates for the conversion of EEH to (-)-ambrox.

[0321] Example 12 Comparative data on whole-cell biotransformations using Triton X-100 and SDS. Method 12 E. coli host cells were grown according to the protocol of Method 4 in Example 4. Bioconversion reactions using 215G2 SHC variants were carried out according to the standard test in Example 4. OD in 0.1 M citrate / sodium phosphate buffer, pH 5.4 was 1.0. 650nm 10.0 cells and 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 FIG. 15 provides a comparison of the activity of 215G2 SHC variants in a whole cell biotransformation assay using Triton X-100 concentrations ranging from 0.005% to 0.48% and SDS at a concentration of 0.07%.

[0323] Consideration 12 The data show that the maximal activity with Triton X-100 was only approximately 20% of the activity obtained with SDS.

[0324] Example 13 SDS / cell ratio Method 13 EEH substrate 4g / l, OD produced by 215G2 SHC derivative enzyme 650nm A bioconversion reaction was set up according to Method 4 of Example 4 using 5.0 cells.

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

[0326] Consideration 13 The results show that there is some 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 reaction to occur.

[0327] Example 14 Examination of potential solubilizing agents for use in biotransformation reactions. Method 14 Various solubilizing agents (outlined in Table 26 below) were subjected to EEH cyclization of 215G2 SHC using the same conditions as in the standard test (4 g / l EEH, OD of 10.0 650nm SDS, a soluble solubilizer, was tested as a possible substitute for SDS (0.060-0.070%) in cells of the rat. The possibility of enhancing activity (cumulative effect) by combining SDS at its optimal concentration (0.060-0.070%) with the other solubilizers used (at concentrations determined to be individually optimal from the screening done with 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 improved activity compared to the control reaction performed with SDS at concentrations ranging from 0.060 to 0.070%. The activity observed with the compounds used alone at the concentration defined as optimal was only about 20% of that obtained in the control reaction with SDS. It was noted that when no solubilizer was added, 20% EEH conversion was achieved. When SDS was used and an additional solubilizer was added (at the concentration defined as optimal in the study), no synergistic effect was observed. Rather, a decrease in the EEH conversion percentage was observed. It can be concluded from the study that under the conditions tested, the compounds do not improve EEH conversion at all, but rather have a detrimental effect on the cyclization, and that SDS is the most useful of the solubilizers studied. Additionally, tests with "deep eutectic solvents" and ionic liquids, which are known to help solubilize water-insoluble compounds, did not produce positive results. [Table 29]

[0329] Consideration 14 Applicants selected and identified SDS as a useful solubilizing agent from a long list of other solubilizing agents that had been shown to be not useful in the homofarnesol to (-)-ambrox bioconversion reaction of the present disclosure.

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

[0331] Result 15 The data in FIG. 17 show that the bioconversion reaction proceeded at 4 g / l EEH and OD 650nm These results indicate that HAC activity appears to be less sensitive to changes in SDS concentration than when tested by the standard test on cells applied at 10.0. The data in FIG. 18 show that when biotransformation is applied, the system has a HAC activity of 4 g / L EEH and an OD 650nm These results indicate that the β-lactamase appears to be less sensitive to pH fluctuations than when tested by the standard test with cells applied at 10.0.

[0332] Consideration 15 The data show robustness of the bioconversion reaction at 125 g / l EEH and 250 g / l cells over the range of SDS concentrations and pH tested.

[0333] Example 16 Location of identified SHC / HAC mutations on the crystal structure The locations of the identified mutations in the AacSHC / HAC variants are marked in Figure 19 as follows: variant 215G2 is in red, variant 101A10 is in purple (wine red), and variant 111C8 is in green. For the amino acids identified as responsible for the increased activity, their side chains are highlighted in yellow on the co-crystallized substrate analog. Other mutations in the identified variants that do not have improved activity are marked in blue. It is noted that the blue mutations are scattered approximately half and half (i.e., 50:50) over the two domains of the enzyme, while the identified beneficial AacSHC mutations are mostly located (with the exception of one) in domain 2. The only exception is the mutation F601Y, which is in the vicinity of the active site. When considering only both SHC / HAC derivative enzymes 215G2 and 111C8, all of the mutations are located in domain 2. Figure 20 provides the same information in black and white.

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

[0335] Consideration 16 The crystal structure is useful for identifying SHC / HAC derivatives with desirable structure / activity relationships, particularly with respect to the conversion of homofarnesol to (-)-ambrox. A useful preselection step might be to limit 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 analytical conditions Non-polar 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. Non-polar column: BPX5 from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22mm x 0.25mm x 12m. Carrier gas: Helium. Injector temperature: 230°C. Split 1:50. Flow: 1.0ml / min. Transfer line: 250°C. MS-quadrupole: 106°C. MS source: 230°C.

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

[0338] B) Preparation of E-Δ Farnesene using MNU in THF [ka] N-Methyl-N-nitrosourea 1.35 M in THF (136 ml, 184 mmol) is added dropwise to a vigorously stirred mixture of E-beta-farnesene (CAS18794-84-8) (25 g, 122 mmol) and aqueous KOH (50 ml, 40%) at 0° C.-5° C. After addition of 4 ml of MNU solution, Pd(acac)2 (7.4 mg, 0.024 mmol, 0.02%) predissolved in 0.5 ml dichloromethane is added. The remaining MNU solution is added over 4 h at 0-5 °C. GC at this stage showed 28% unconverted E-β-farnesene, 65% desired monocyclopropane (shown above), and 3% biscyclopropanated compound 5. After 16 h at 25 °C, acetic acid (100 ml) is added at 0-5 °C followed by tert-butyl methyl ether (250 ml). After phase separation, the organic phase is washed with 2 M HCl (250 ml) and the aqueous phase is extracted with tert-butyl methyl ether (250 ml). The combined organic layers are washed with water (2 x 100 ml), 10% aqueous NaOH (2 x 100 ml), and water (2 x 100 ml), dried with MgSO4, filtered, and concentrated to give 26.9 g of a slightly yellow liquid, which contains 9% E-β-farnesene, 82% desired monocyclopropane compound, and 6% biscyclopropanated by-product. The desired compound can be further isolated by distillation purification. 2 CO 3 Addition of (1 g) and distillation through a 30 cm steel coil column at 40-60 mbar gives 147 g of monocyclopropane compound (68% corr) at 135-145° C. Pooling of fractions gives 92 g monocyclopropane compound with 100% purity.

[0339] Analytical data for 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] C) 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 under stirring at 150° C. After 18 h and complete conversion (according to GC), the mixture is poured into water (50 ml) and toluene (50 ml). The phases are separated and the aqueous phase is extracted with toluene (50 ml). The combined organic layers are washed with concentrated aqueous Na2CO3 (50 ml) and concentrated NaCl (2 x 50 ml), dried with MgSO4, filtered and evaporated under reduced pressure to give a brownish resin (1.35 g). This is mixed with 30% aqueous KOH (4.3 ml) and stirred at 25°C for 2 h. GC analysis reveals the formation of 96% (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol according to an internal standard. E / Z ratio 68:22. Analytical data for the E isomer are consistent with those from the literature. See, for example, P. Kocienski, S. Wadman J. Org. Chem. 54, 1215 (1989).

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

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

[0343] Example 18 One-pot reaction In this experiment, (i) fermentation with an E. coli strain producing the 215G2 SHC variant (as described for example in Example 1) was performed, followed by (ii) EEH conversion directly 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 depending on the concentration of cells (g / l) obtained at the end of the fermentation. The goal was to convert 125 g / l of EEH by 250 g / l of cells at an SDS concentration of 1.55%. To allow a reasonable bioconversion, the cells must be in a quiescent, glucose-starved state. Aeration was switched off.

[0344] Method 18 fermentation: To allow a very accurate determination of the volume of fermentation broth in the reaction vessel at the end of the fermentation, the volumes of the samples taken and all the addition volumes made to the fermenter (feed, base, acid...) were recorded.

[0345] Determination of cell concentration in fermentation broth: For the determination of the wet weight of cells (g / l), a sample (5-10 ml) of the fermentation broth was withdrawn under constant stirring and placed in a centrifuge tube. The mass of the sample was recorded. The sample was centrifuged for 10 min at 17210 g and 4°C (e.g. 12000 rpm, SS-34 rotor, Sorvall RC3B centrifuge). The supernatant was removed by careful pipetting and the mass of the pellet was recorded. The wet weight concentration of cells was determined in g 細胞 / l ブロス or g 細胞 / g ブロス It is determined as follows. The volume of fermentation broth in the fermenter was determined according to all additions and withdrawals. 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 amount of homofarnesol and SDS needed: 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, 125 g / l EEH, 250 g / l cells, 1.55% SDS, as described in the bioconversion described in Example 9.

[0347] Set up the biotransformation: 1. Temperature set at 35°C. Ventilation turned off. 2. A calculated amount of homofarnesol was added to the fermentation broth. 3. The required amount of SDS was carefully added from a stock solution of 15.5% SDS in water. 4. The reaction was mixed well at 800 rpm for approximately 15 minutes. 5. The pH of the reaction was recorded (internal pH electrode). 6. Samples (approximately 1 ml) were drawn into 15 ml Falcon tubes. Approximately 5 ml of deionized water was added and after thorough mixing, the pH was recorded with an external calibrated electrode. 7. The pH in the reaction vessel is 85%H 3 PO 4 The pH was gradually adjusted to 5.4 (measured with an external calibrated electrode) using the pH-adjustment method, while the pH was periodically controlled with an external electrode as described above (6.). 8. The pH should be adjusted to, for example, 10-25% H during the bioconversion. 3 PO 4 and 32% NaOH was used as a control. 9. Sampling 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 aliquots were spun for 1 min at maximum speed in a tabletop 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 biotransformation, samples were taken every 1-1.5 hours. From the following days, only 3 samples were taken per day. 11. 1 μl of the solvent phase was analyzed for its ambrox and EEH content as described in Example 8. 12. EEH conversion (%) = 100 × (Ambrox 面積 / (Ambrox 面積 +EEH 面積 )) is calculated.

[0348] Result 18 Results show that one-pot fermentation + EEH conversion was performed at 1.9 liter scale in a KLF2000 reactor (Bioengineering). 251 g / l cells allowed conversion of 238 g EEH (251 g / l cells) to ≧93% in 47 hours. Conversion was 99% when measured 93 h after initiation. A similar one-pot experiment was carried out in an Infors HT 0.751 reactor. After the fermentation following the standard protocol (example 1), cells from the reactor pooled from another fermentation carried out in parallel with 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 times the cell concentration of the standard bioconversion (250 g / l cells). EEH and SDS were therefore added to the reactor. 75.1 g of EEH (equivalent to 157 g / l EEH in this example) were converted to 98% in less than 90 hours. This result shows that it is possible to carry out a one-pot fermentation + EEH conversion at ≥ 125 g / l EEH, as long as the fermentation carried out provides cells that produced the 215G2 SHC variant at a sufficiently high cell density.

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

[0350] Example 19 Increase productivity per volume. Method 19 To further increase the volumetric productivity, a 1.5x concentrated bioconversion was carried out containing 375 g / l cells, 188 g / l EEH, 2.33% SDS. A standard bioconversion was carried out in parallel with 125 g / l EEH, 250 g / l cells, 1.55% SDS (Example 7). Two reactions were carried out in an Infors HT 0.7501 reaction vessel with all other parameters remaining constant.

[0351] Result 19 The results in FIG. 22 show that the percent conversion after 75 hours from start was 88% for the 1.5× bioconversion vs. 95% for the standard bioconversion. The percent conversion after 96 hours from start was 93% EEH convert for the 1.5× bioconversion vs. 97% for the standard bioconversion. The percent conversion from the 1.5× bioconversion was 96% of that obtained from the standard bioconversion. It was noted that stirring in the 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 the 1.5× bioconversion. Using a reaction vessel equipped with better mixing equipment may improve the EEH conversion from the 1.5× bioconversion. The results show that it is possible to perform bioconversion at EEH 188 g / L or more if efficient mixing is achieved. The stirring efficiency appears to be the only limitation of the system. (-)-Ambrox Productivity "(-)-Ambrox productivity" refers to the amount of recoverable (-)-ambrox in grams per liter of biotransformation per hour of biotransformation time (i.e., time after substrate is added). In this regard, with reference to Figure 22, (-)-ambrox productivity is calculated as follows: Bioconversion of 125g / l EEH (cells 250g / l) Productivity in 1.25h: 10.3 grams per liter per hour Productivity in 8.25h: 6.3 grams per liter per hour Productivity in 21.25h: 4.1 grams per liter per hour Bioconversion of 187.5g / l EEH (375g / l cells) Productivity in 1.25h: 12.2 grams per liter per hour Productivity in 8.25h: 8.2 grams per liter per hour Productivity in 21.25h: 5.5 grams per liter per hour The productivities calculated approximately 6-8 hours after initiation can be considered to correspond to the initial rate of reaction, which best represents the maximum conversion of the system. A typical bioconversion using 250 g / l cells and 125 g / l EEH shows an ambrox productivity of between 6.3 and 8.5 grams per liter per hour after approximately 6-8 hours (corresponding to the initial rate of reaction).

[0352] Example 20 Replacement of reaction buffer with NaCl solution method 20 Standard biotransformations (EEH 125 g / l, cells 250 g / l, SDS 1.55%) were carried out as described in Example 7, except that citrate buffer pH 5.4 was replaced by either 0.5% or 0.9% NaCl, and all other reaction parameters remained unchanged. Biotransformations in citrate buffer were carried out in parallel as controls.

[0353] Result 20 The results in Figure 23 show that the EEH conversion rate was the same for reactions performed in buffer and 0.9% NaCl. The conversion rate was lower for reactions performed in 0.5% NaCl only. The results demonstrate the feasibility of performing bioconversions in the absence of buffers, provided that precise pH control and sufficient ionic strength are ensured.

[0354] Example 21 Extraction of the solid phase of the reaction broth Given that (-)-ambrox is neither soluble in water nor liquid at temperatures below approximately 75°C, these properties have been made a potential advantage for extracting the product from the solid phase of biotransformation using either water-miscible (e.g., ethanol) and 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 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 (-)-ambrox initially present in the 200 ml reaction broth. Toluene and ethanol 99% were used to extract the Ambrofix from the solid phase.

[0356] Result 21 Toluene Extraction: 80 ml of the solid phase were extracted 6x with 45 ml toluene (approximately 1 / 2 the solid phase volume, 30 s vigorous shaking, centrifugation (Sorvall GS3, 5000 rpm, 10 min, 10°C). The solvent phase was analyzed by GC for its (-)-ambrox content. More than 99.5% of the (-)-ambrox initially present in the reaction broth was extracted by 6 extractions corresponding to a total toluene volume of 1.35x the initial total reaction broth volume (200 ml) or 3.4x the solid phase volume. The graph in Figure 24 shows the progress of the extraction with toluene washes as % of the (-)-ambrox amount initially present in the 200 ml total reaction broth (% in the first extract exceeds 100% due to the volume ratio broth / toluene). Ethanol Extraction: 80 ml of the solid phase was extracted with approximately 160 ml (2 volumes) of 99% ethanol (Infors Multifors HT, 35° C., 1000 rpm, 30 min) and then centrifuged. Ambrox did not crystallize during the extraction procedure. The graph in FIG. 25 shows that after 4 washes (total of 640 ml EtOH, i.e. 3.2× total initial reaction broth volume or 8× volume of solid phase), approximately 99% of the ambrox initially present in the reaction broth was recovered. Sufficient ethanol is required to prevent ambrox crystallization in the first extraction step (soluble in ethanol). When only 1 or 1 / 2 volumes of the solid phase were used in the first extraction step, a sticky paste was obtained that was difficult to handle, and (−)-ambrox crystallized as needles on the pellet during centrifugation. Temperature did not appear to be a factor in this crystallization (extractions and centrifugations were tested at room temperature and approximately 35° C.-40° C.). The concentration of (-)-Ambrox in the EtOH phase and the EtOH / water ratio of the liquid phase (residual water in the solid phase) appeared to be factors in crystal formation, however, it was noted that the volume of ethanol could be reduced to 1 volume of the solid phase.

[0357] Comments 21 Since (-)-ambrox is not present in the liquid phase at room temperature, it separates with the biomass and can be extracted with an organic solvent, such as a water-miscible solvent (e.g., ethanol) or a water-immiscible solvent (e.g., toluene). The centrifugation step, which separates (-)-ambrox into the solid phase of the reaction mixture, is advantageous as it reduces the amount of solvent needed to extract (-)-ambrox.

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

[0359] Result 22(a) Conversion of EEH results in (-)-ambrox (compound I) and the (-)-ambrox isomer (compound IV). Result 22(b) Biotransformation of EZH leads to a macrocyclic ether (compound II) and 9b-epi-ambrox (compound III). Result 22(c) The crude composition of (-)-ambrox contained 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) Compositions of the selectively crystallized material (laboratory scale) have the same components present in amounts of 99.1, 0.1, 0.1, and 0.7%, respectively. The results of the perception analysis were as follows: (-)-Ambrox: OTH 0.2ng / 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 at the detection threshold). Compound III from EZH: approx. 10× higher GC-TH than ambrox (approx. 2 ng).

[0360] conclusion The total percentage of each of the three by-products (compounds II, III, and IV) in the "crude" extract is approximately 3%. The total 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 (-)-ambrox. 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. Sensory analysis showed that removal of one or more by-product compounds from (-)-ambrox can improve the odor of the remaining compound (i.e., (-)-ambrox) even when the removed compound itself is in fact an odorless compound: an odor enhancement of ambrox was observed in the absence of compounds II, III, and IV.

[0361] Example 23 Ambrox recovery by steam extraction Method 23 Purities obtained for crude (-)-ambrox (vapor extracted) and crystallized (-)-ambrox The EE:EZ86:14 biotransformation reaction mixture 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 dryness. Ethanol was then added to the dried crude product and the mixture was warmed until the product was dissolved. At room temperature, water was slowly added and (-)-ambrox was crystallized with occasional stirring and cooling in an ice bath.

[0362] Result 23 Table 25 below also shows the GC analysis of the product obtained after the steam extraction / distillation step ("crude") and the crystallized product ((-)-ambrox). References to "EZH" and "EEH" in Table 25 refer to (3Z,7E) homofarnesol and 7E,3E homofarnesol, respectively. Table 25 shows that a particular starting material (EEH:EZH 86:14) produces the desired end product (-)-ambrox and a highly specific mixture of by-products (II, IV, and III) using WT SHC or SHC derivatives. Selective crystallization data shows 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 olfactorily pure (-)-ambrox product that is selectively crystallized in a relatively straightforward and cost-effective manner. [Table 30]

[0363] Consideration 23 Steam extraction / filtration is an environmentally friendly method for isolating ambrox because it provides a convenient, solvent-free isolation of ambrox with no collateral deactivation of the biocatalyst.

[0364] Summary 23 The (-)-ambrox produced using the bioconversion reaction can be extracted from the total reaction mixture using a solvent (e.g., using a water-immiscible solvent or by steam extraction / distillation or by filtration) or from the solid phase (e.g., using a water-miscible solvent) using methods known to those of skill in the art.

Claims

1. (3E,7E)-homofarnesol (EEH) or a mixture of EEH-containing stereoisomers is enzymatically converted to (-)-ambrox or a mixture containing (-)-ambrox; the enzymatic conversion is carried out using SHC / HAC enzymes under reaction conditions suitable for the production of (-)-ambrox; the mixture of stereoisomers comprising EEH consists essentially of homofarnesol isomers selected from one or more of the group consisting of [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E), and (3E,7Z)], which are also designated [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively; A process for preparing (-)-ambrox or a mixture containing (-)-ambrox.

2. (3E,7E)-homofarnesol (EEH) or a mixture of EEH-containing stereoisomers is enzymatically converted to give (-)-ambrox or a mixture containing (-)-ambrox; an enzymatic conversion using an SHC / HAC enzyme is carried out under reaction conditions suitable for the production of (-)-ambrox; When the reaction is carried out in the presence of a solubilizing agent, Triton X-100 or taurodeoxycholic acid is not used in combination with the wild-type SHC / HAC enzyme; A process for preparing (-)-ambrox or a mixture containing (-)-ambrox.

3. (3E,7E)-homofarnesol (EEH) or a mixture of EEH-containing stereoisomers is enzymatically converted to (-)-ambrox or a mixture containing (-)-ambrox; the enzymatic conversion is carried out using SHC / HAC enzymes under reaction conditions suitable for the production of (-)-ambrox; the mixture of stereoisomers comprising EEH consists essentially of homofarnesol isomers selected from one or more of the group consisting of [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E), and (3E,7Z)], which are also designated [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively; The reaction takes place in a three-phase system including an aqueous phase, a solid phase, and an oil phase; A process for preparing (-)-ambrox or a mixture containing (-)-ambrox.

4. The process comprises combining 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 13, or Table 14, or selected from SEQ ID NO: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, or SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 with at least at least) 30%, 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, 4. The process according to claim 1 or 2 or 3.

5. the process employs recombinant host cells that produce the SHC / HAC enzymes; The process according to any one of claims 1 to 4.

6. the nucleotide sequence encoding the SHC / HAC enzyme is selected from the group consisting of SEQ ID NO: 165, 166, 167, 168, 169, or SEQ ID NO: 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; 6. The process according to claim 4 or 5.

7. The conversion of homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C and at a pH in the range of about 4 to 8. The process according to any one of claims 1 to 6.

8. The conversion of homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for the wild-type SHC / HAC or SHC / HAC derivative enzymes set forth in Table 24 or Table 24a, preferably in the pH range of 5.0 to 6.2, and preferably at a temperature of 35°C. The process according to any one of claims 1 to 7.

9. When the biocatalyst to EEH ratio is about 2:1, the SDS / cell ratio is in the range of 10:1 to 20:1, preferably 16:1; The process according to any one of claims 3 to 8.

10. the weight ratio of biocatalyst to homofarnesol is within the range of about 0.5 to 2:1, preferably about 1:1 or 0.5:1; The process according to any one of claims 3 to 9.

11. The cell growth and bioconversion steps are carried out in the same reaction vessel; The process according to any one of claims 3 to 10.

12. The homofarnesol substrate comprises one or more homofarnesol stereoisomers.

3. The process of claim 2.

13. the homofarnesol substrate comprises or consists essentially of two homofarnesol stereoisomers; 13. The process of claim 12.

14. the homofarnesol substrate comprises or consists essentially of the EE:EZ stereoisomer; 14. The process of claim 13.

15. Homofarnesol: 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, and / or 60:40, 15. The process according to any one of claims 14.

16. the homofarnesol comprises or consists essentially of an EE:EZ stereoisomeric mixture in a weight ratio selected from the group consisting of EE:EZ 92:08, EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and / or EE:EZ 66:34; 16. The process of claim 15.

17. homofarnesol comprises or consists essentially of a mixture of EE:EZ stereoisomers in a weight ratio of 80:20; 17. The process according to claim 15 or 16.

18. (-)-Ambrox is produced in admixture with at least one or more of by-products (II), (IV), and / or (III); The process according to any one of claims 1 to 17.

19. (-)-Ambrox is isolated from the bioconversion reaction mixture using an organic solvent or steam extraction / distillation step or filtration; The process according to any one of claims 1 to 18.

20. (-)-Ambrox is isolated from the solid phase of the bioconversion reaction mixture using an organic solvent or a steam extraction / distillation step; The process according to any one of claims 1 to 19.

21. (-)-Ambrox is isolated from the reaction mixture using an organic solvent; 21. The process according to claim 19 or 20.

22. (-)-Ambrox is isolated from the reaction mixture using ethanol or toluene.

22. The process of claim 21.

23. (-)-Ambrox is selectively crystallized using an organic solvent; The process according to any one of claims 19 to 22.

24. (-)-Ambrox is substantially free of by-products (II), (IV), and / or (III); 24. The process of claim 23.

25. (-)-Ambrox is produced in a concentration range of about 125-200 g / l. The process according to any one of claims 1 to 24.

26. (-)-Ambrox has an odor threshold of about 0.1 to about 0.5 ng / l; (-)-Ambrox obtainable by the method according to any one of claims 1 to 25.

27. (-)-Ambrox according to claim 26 in solid form, preferably in amorphous or crystalline form.

28. The method comprises incorporating (-)-Ambrox according to any one of claims 26 or 27 into a product. (-)-A process for making products containing ambrox.

29. the product is a fragrance product, a cosmetic product, a cleaning product, a detergent product, or a soap product; 29. The process of claim 28.

30. A fragrance or cosmetic or consumer care product comprising (-)-ambrox according to any one of claims 26 or 27.

31. A fragrance or cosmetic or consumer care composition comprising (-)-ambrox according to claim 26 or 27 and one or more additional components.

32. 28. Use of (-)-Ambrox according to claim 26 or 27 as part of a fragrance or cosmetic or consumer product, such as a fabric care, toiletry, beauty and / or cleaning product.

33. (a) preparing a reaction mixture comprising (−)-ambrox in admixture with one or more of by-product compounds (II), (III), or (IV); 【Chemistry 1】 (b) extracting (−)-ambrox in admixture with one or more of by-product compounds (II), (III), or (IV); (c) selectively crystallizing (-)-ambrox from the extraction mixture; mixing an aroma-enhanced or -enhanced product produced according to a process comprising the steps of: (-)-Ambrox 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 (-)-ambrox; the mixture of stereoisomers comprising EEH consists essentially of homofarnesol isomers selected from one or more of the group consisting of [(3E,7E) and [(3Z,7E)] and / or [(3E,7E) and (3E,7Z)] and / or [(3Z,7E), (3E,7E), and (3E,7Z)], which are also designated [EE:EZ], [EE:ZE], and [EE:EZ:ZE], respectively; A process for extending, enhancing or imparting the aroma of a fragrance composition.

34. The reaction takes place in a three-phase system including an aqueous phase, a solid phase, and an oil phase; 34. The process of claim 33.

35. The process comprises using 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 NO: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, or an SHC / HAC enzyme polypeptide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, 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 NO: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, with a sequence having at least 30% identity, 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 to sequence number 2, SEQ ID NO:3, or SEQ ID NO:4, 35. The process of claim 33 or 34.

36. the process employs recombinant host cells that produce the SHC / HAC enzymes; The process according to any one of claims 33 to 35.

37. the nucleotide sequence encoding the SHC / HAC enzyme is selected from the group consisting of SEQ ID NO: 165, 166, 167, 168, 169, or SEQ ID NO: 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; 37. The process of claim 35 or 36.

38. The conversion of homofarnesol to (-)-ambrox occurs at a temperature in the range of 30°C to 60°C and at a pH in the range of about 4 to 8. The process according to any one of claims 33 to 37.

39. The conversion of homofarnesol to (-)-ambrox occurs using one or more of the reaction conditions for the wild-type SHC / HAC or SHC / HAC derivative enzymes set forth in Table 24 or Table 24a, preferably in the pH range of 5.0 to 6.2, and preferably at a temperature of 35°C. The process according to any one of claims 33 to 38.

40. When the biocatalyst to EEH ratio is about 2:1, the SDS / cell ratio is in the range of 10:1 to 20:1, preferably 16:1; 40. The process according to any one of claims 34 to 39.

41. the weight ratio of biocatalyst to homofarnesol is within the range of about 0.5 to 2:1, preferably about 1:1 or 0.5:1; The process according to any one of claims 34 to 40.

42. The cell growth and bioconversion steps are carried out in the same reaction vessel; The process according to any one of claims 34 to 41.

43. Homofarnesol: 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, and / or 60:40, 43. The process according to any one of claims 33 to 42.

44. the homofarnesol comprises or consists essentially of an EE:EZ stereoisomeric mixture in a weight ratio selected from the group consisting of EE:EZ 92:08, EE:EZ 90:10, EE:EZ 80:20, EE:EZ 86:14, EE:EZ 70:30, EE:EZ 69:31, and / or EE:EZ 66:34; 44. The process of claim 43.

45. homofarnesol comprises or consists essentially of a mixture of EE:EZ stereoisomers in a weight ratio of 80:20; 45. The process of claim 43 or 44.

46. (-)-Ambrox is produced in admixture with at least one of by-products (II), (IV), and / or (III); The process according to any one of claims 33 to 45.

47. (-)-Ambrox is isolated from the bioconversion reaction mixture using an organic solvent or steam extraction / distillation step or filtration; The process according to any one of claims 33 to 46.

48. (-)-Ambrox is isolated from the solid phase of the bioconversion reaction mixture using an organic solvent or a steam extraction / distillation step; 48. The process according to any one of claims 33 to 47.

49. (-)-Ambrox is isolated from the reaction mixture using an organic solvent; 49. The process of claim 47 or 48.

50. (-)-Ambrox is isolated from the reaction mixture using ethanol or toluene.

50. The process of claim 49.

51. (-)-Ambrox is selectively crystallized using an organic solvent; 50. The process according to any one of claims 47 to 49.

52. (-)-Ambrox is substantially free of by-products (II), (IV), and / or (III); 52. The process of claim 51.

53. (-)-Ambrox is produced in a concentration range of about 125-200 g / liter.

53. The process according to any one of claims 33 to 52.

54. (-)-Ambrox has an odor threshold of about 0.1 to about 0.5 ng / l; 54. The process according to any one of claims 33 to 53.

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