Process for isolating and purifying ambrox

Abstract

To provide a method of isolating and purifying (-)-Ambrox from a reaction mixture comprising (-)-Ambrox.SOLUTION: There is provided a method of isolating and purifying (-)-Ambrox from a reaction mixture, comprising one or more of the following compounds (II), (III) and (IV), wherein the mixture is formed as a result of an enzyme-catalyzed cyclization of homofarnesol comprising a mixture of 7E, 3E of homofarnesol and 7E, 3Z homofarnesol geometric isomers, wherein the reaction is carried out in the presence of a recombinant microorganism expressing the gene encoding the enzyme, and wherein the method comprises a step of selectively crystallizing (-)-Ambrox from a mixture containing one or more of the compounds (II), (III) or (IV).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for preparing, isolating and purifying the isomer (-)-Ambrox. More specifically, the present invention relates to a method for preparing (-)-Ambrox by bioconversion, as well as a method for recovering and purifying it from the reaction mixture.

Background Art

[0002] AMBROFIX (trademark) is the trade name of Givaudan, the owner of the enantiomerically pure compound (-)-Ambrox having the general formula (I).

Chemical formula

[0003] An industrially expandable biotechnological route to AMBROFIX (trademark) would be attractive because it would potentially be less complex and less polluting than a completely synthetic procedure.

[0004] An industrially expandable biotechnological route to AMBROFIX (trademark) would be attractive because it would potentially be less complex and less polluting than a completely synthetic procedure. (-)-Ambrox can potentially be produced by bioconversion using substrates such as homofarnesol. In the influential paper by Neumann et al. (Biol. Chem. Hoppe-Seyler Vol. 367 pp 723-726 (1986)), the feasibility of the conversion of homofarnesol to (-)-ambrox under enzymatic catalysis using the enzyme squalene-hopene cyclase (SHC) was discussed. The homofarnesol used was a mixture of four geometric isomers of this molecule. Only the 7E,3E geometric isomer (using the normal nomenclature) of the four isomers can be cyclized, so only a very low yield of the desired (-)-ambrox is obtained.

[0005] JP 2009-60799 (Kao) discloses the synthesis by which SHC acts on a homofarnesol substrate to produce (-)-ambrox. The substrate is a mixture of all four geometric isomers (3Z,7Z; 3E,7Z; 3Z,7E; and 3E,7E). The document only discloses the preparation of (-)-ambrox from a homofarnesol extract containing SHC. The homofarnesol mixture is converted to (-)-ambrox and its 9-epi stereoisomer, and purification can be carried out by distillation or column chromatography. Kao does not describe a process for converting homofarnesol to (-)-ambrox using the native microorganism that produces SHC, and furthermore, provides no technical teaching regarding the downstream processing of the complex mixture obtained by such a process by which (-)-ambrox can be obtained in an olfactory pure form.

[0006] As far as the applicant is aware, the prior art does not describe any practicable and industrially scalable process for providing (-)-ambrox in an olfactory pure form, including the SHC-catalyzed bioconversion of homofarnesol. Furthermore, even if the bioconversion of homofarnesol is achieved on an industrial scale, a cost-effective source of highly pure 3E,7E-homofarnesol must be available. However, while the synthetic pathway of homofarnesol is described in the literature (see, for example, US 2013 / 0273619), to the applicant's knowledge, there is no currently available industrial-scale source of cost-effective, pure 7E,3E-homofarnesol.

[0007] There is a need to provide an economically feasible and industrially scalable route to the valuable fragrance ingredient (-)-ambrein. In the co-pending patent applications PCT / EP2014 / 072891 (published as WO2015 / 059293) and PCT2014 / EP / 072882 (published as WO2015 / 059290), the applicant has described an efficient method for preparing a 7E,3E / Z-homofarnesol mixture rich in the 7E,3E geometric isomer. The 7E,3E / Z-homofarnesol mixture is prepared from beta-farnesene, and the isomer information contained in this starting material is preserved such that the homofarnesol double bond at the 7-position is fixed in the E-configuration. However, this sophisticated chemistry still results in a 3E / Z isomer mixture. Pure 7E,3E-homofarnesol remains synthetically challenging and can only be achieved by the economically disadvantageous purification of the isomer mixture.

[0008] Surprisingly, the applicant has found that the 7E,3E / Z-homofarnesol mixture can undergo a bioconversion process, whereby the homofarnesol mixture is enzymatically cyclized in the presence of a recombinant microorganism expressing an enzyme, in particular a squalene-hopene cyclase (SHC) biocatalyst, capable of bioconverting homofarnesol to (-)-ambrein, to produce a reaction mixture from which (-)-ambrein can be isolated in an olfactory pure form by surprisingly easy downstream processing.

[0009] In one aspect of the present invention, there is provided an enzymatic cyclization of homofarnesol to provide a reaction mixture containing (-)-ambrox, wherein the homofarnesol comprises a mixture of 7E,3E / Z-geometric isomers of homofarnesol, and the reaction is carried out in the presence of a recombinant microorganism that produces the enzyme, more specifically, an intact recombinant microorganism that produces the enzyme.

[0010] In an embodiment of the present invention, the cyclization reaction is carried out in the presence of an SHC biocatalyst capable of bioconverting homofarnesol to (-)-ambrox. The SHC biocatalyst is a wild-type or mutant enzyme, or a microorganism that expresses a gene encoding the SHC enzyme, preferably a recombinant Escherichia coli microorganism. The SHC biocatalyst can be used in any form, including but not limited to a purified SHC enzyme, a crude extract containing the SHC enzyme, or an immobilized SHC enzyme (e.g., on a carrier), or the biocatalyst can be an intact recombinant whole cell and / or fragmented cell or membrane fraction containing the SHC enzyme, which is a microorganism that produces or produces the SHC.

[0011] In a specific embodiment of the present invention, the homofarnesol mixture is rich in the 7E,3E-geometric isomer. In a more specific embodiment, the homofarnesol mixture is at least 55 / 45 by weight of 7E,3E / 7E,3Z. In a more specific embodiment, the homofarnesol mixture is at least 70 / 30 by weight of 7E,3E / 7E,3Z. In an even more specific embodiment, the homofarnesol mixture is at least 80 / 20 by weight of 7E,3E / 7E,3Z. In an even more specific embodiment, the homofarnesol mixture is at least 90 / 10 by weight of 7E,3E / 7E,3Z. In an even more specific embodiment, the homofarnesol mixture is at least 95 / 5 by weight of 7E,3E / 7E,3Z. In a specific embodiment of the present invention, the homofarnesol mixture consists of the 7E,3E / Z-geometric isomers of homofarnesol and does not contain other geometric isomers.

[0012] Those skilled in the art will understand that the terms 7E, 7Z, 3E or 3Z used in relation to homofarnesol refer to the orientation of the double bonds at the 7- and 3-positions of homofarnesol, respectively. The 7E,3E-homofarnesol compound has CAS No. 459-89-2, while the 7E,3Z-homofarnesol compound has CAS No. 138152-06-4. The use of the term 7E,3E / Z-homofarnesol refers to a mixture of these compounds.

[0013] According to the method of the present invention, a method for obtaining a homofarnesol mixture useful as a substrate in a cyclization reaction is described in the co-pending applications PCT / EP2014 / 072891 (published as WO2015 / 059293) and PCT2014 / EP / 072882 (published as WO2015 / 059290) mentioned above, which are hereby incorporated by reference in their entirety. Generally speaking, these describe the synthesis of a homofarnesol mixture that proceeds by converting farnesene, more specifically, alpha-farnesene and / or beta-farnesene, to the corresponding cyclopropanated farnesene derivative using an organic solution of N-alkyl-N-nitrosourea. The cyclopropanated derivative then undergoes a ring-opening and rearrangement reaction in the presence of a Bronsted acid that provides a homofarnesol mixture selective for the 7E,3E geometric isomer. Using farnesene as the starting material is particularly preferred to ensure that the E-structure of the double bond at the 7-position of homofarnesol is fixed.

[0014] The special reaction conditions forming a particular embodiment of the present invention are described in the co-pending applications as well as in the examples below in this specification and will not be elaborated further here. The cyclization of homofarnesol to provide a reaction mixture containing (-)-ambrox can be catalyzed by squalene-hopene cyclase (SHC). SHC may be a wild-type enzyme (e.g., SEQ ID NO: 1) or a variant thereof (e.g., SEQ ID NO: 2 or SEQ ID NO: 4). SHC can be obtained from Alicyclobacillus acidocaldarius (Bacillus acidocaldarius), Zymomonas mobilis, or Bradyrhizobium japonicum (as described in Example 3b of US20120135477A1).

[0015] However, the enzyme can also be produced by recombinant means using techniques commonly known in the art. The term "recombinant" as used with respect to an enzyme refers to an enzyme produced by recombinant DNA technology, i.e., produced from a cell transformed with an exogenous DNA construct encoding the desired enzyme. Thus, the term "recombinant DNA" encompasses recombinant DNA incorporated into a vector, a self-replicating plasmid or virus, or the genomic DNA of a prokaryote or eukaryote (or the genome of a homologous cell at a location other than its natural chromosomal site).

[0016] The nucleic acid molecule is operably linked to an expression control sequence that enables expression in a prokaryotic and / or eukaryotic host cell. As used herein, "operably linked" means that the expression control sequence is incorporated into the gene construct such that it effectively controls the expression of the coding sequence of interest. The transcription / translation control elements mentioned above include, but are not limited to, inducible and non-inducible, constitutive, cell cycle-regulated, metabolism-regulated 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 can be directed towards constitutive expression or can enable inducible expression, for example, like the CUP-1 promoter, the tet repressor employed in, for example, the tet on or tet off systems, the lac system, the trp system control elements, etc., but are not limited thereto. By way of example, isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effective inducer of protein expression within a concentration range of 100 μM to 1.0 mM. This compound is a molecular mimic of allolactose, a lactose metabolite that induces transcription of the lac operon, and is therefore used to induce protein expression when the gene is under the control of the lac operator.

[0017] Similarly, the nucleic acid molecule can form part of a hybrid gene encoding an additional polypeptide sequence, such as a sequence that functions as a marker or reporter. 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 relevant to the practice of the present disclosure, those of skill in the art will be aware of additional useful reagents, such as additional sequences that can serve as markers or reporters.

[0018] The recombinant polynucleotide can encode an SHC enzyme, such as wild-type SHC or a variant thereof, which can be inserted into a vector for expression and optional purification. Certain vectors are plasmids, which represent circular double-stranded DNA loops into which additional DNA segments are ligated. Some 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 recombination techniques are plasmid type. Typically, an expression vector contains a gene such as wild-type SHC or a variant thereof. Since plasmids are the most commonly used vector type, the terms "plasmid" and "vector" are used interchangeably herein.

[0019] Such vectors can include, but are not limited to, DNA sequences that do not naturally exist in the host cell, DNA sequences that are usually not transcribed into RNA or translated into proteins ("expressed"), and other genes or DNA sequences that one wishes to introduce into the non-recombinant host. Typically, it will be understood that the genome of the recombinant host is expanded by the stable introduction of one or more recombinant genes. However, self or replicating plasmids or vectors can also be used within the scope of the present disclosure. Further, the present disclosure can be practiced using plasmids or vectors that are of low copy number, such as single copy, or high copy number.

[0020] In preferred embodiments, the vectors of the present disclosure include plasmids, phagemids, phages, cosmids, artificial bacterial and yeast chromosomes, knockout or knock-in constructs, synthetic nucleic acid sequences, or cassettes, some of which can be produced in the form of linear polynucleotides, plasmids, megaplasmids, synthetic or artificial chromosomes such as plant, bacterial, mammalian, or yeast artificial chromosomes.

[0021] The protein encoded by the introduced polynucleotide is preferably produced intracellularly upon introduction of the vector. A variety of genetic substrates can be incorporated into the plasmid. Plasmids are often standard cloning vectors, such as bacterial multicopy plasmids. 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 enable the selection of cells containing at least two types of vectors.

[0022] Typically, bacterial or yeast cells can be transformed by 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, DNA providing an origin of replication is included in the construct. The origin of replication can be appropriately selected by those skilled in the art. Depending on the nature of the gene, if the sequences are already present in a gene or genome that is operable as an origin of replication by itself, a supplementary origin of replication may not be required.

[0023] Bacterial or yeast cells can be transformed by foreign or heterologous DNA when such DNA is introduced into the cell. The transformed DNA may be integrated, i.e., covalently linked into the genome of the cell, or it may not be integrated. In prokaryotes and yeast, for example, the transformed DNA can be maintained as an episomal element such as a plasmid. For eukaryotic cells, stably transfected cells are those in which the transfected DNA has been integrated into the chromosome such that it is inherited by daughter cells via chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones composed of a population of daughter cells containing the transformed DNA.

[0024] Generally, the DNA introduced 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 the production of a gene product or to change 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.

[0025] The present disclosure also features a recombinant host. The term "recombinant host" is also sometimes referred to as a "genetically modified host cell" or "transgenic cell" and refers to a host cell that contains a heterologous nucleic acid or whose genome is extended by at least one integrated DNA sequence. The host cells of the present disclosure can be genetically modified by polynucleotides or vectors as outlined above.

[0026] Host cells that can be used for the purposes of the present disclosure include, for example, prokaryotic cells such as bacteria (e.g., Escherichia coli and Bacillus subtilis) that can be transformed by recombinant bacteriophage DNA, plasmid DNA, bacterial artificial chromosomes, or cosmid DNA expression vectors containing the polynucleotide molecules of the present disclosure; 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, but are not limited thereto. 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, as an episome, or can be contained intracellularly only transiently.

[0027] As used herein, the term "cell" or production cell, particularly in reference to genetic engineering and introducing one or more genes or a set of gene clusters 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 and include bacterial host cells such as Escherichia 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.

[0028] 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 insect cells (including Sf9), amphibian cells (including melanocyte cells), or worm cells including cells of 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, Escherichia coli.

[0029] Preferably, prokaryotes such as Escherichia coli, Bacillus, Streptomyces, or mammalian cells such as HeLa cells or Jurkat cells, or plant cells such as Arabidopsis can be used. Preferably, the cell is an Aspergillus species or a fungal cell, preferably selected from the group consisting of the genera Saccharomyces, Candida, Kluyveromyces, Hansenula, Schizosaccharomyces, Yarrowia, Pichia, and Aspergillus. Preferably, the Escherichia coli host cell is an Escherichia coli host cell recognized by the industry and regulatory authorities (including, but not limited to, the Escherichia coli K12 host cell or the Escherichia coli BL21 host cell shown in the examples).

[0030] 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. For E. coli, there are libraries of available mutants, plasmids, detailed computer models of metabolism, and other information, enabling the rational design of various modules for enhancing product yields. A method similar to that described above for Saccharomyces can be used to create recombinant E. coli microorganisms. In one embodiment, the recombinant E. coli microorganism comprises a nucleotide sequence encoding the SHC gene, or a functional equivalent / homologue thereof, including but not limited to its variants, homologues, mutants, derivatives, or fragments.

[0031] 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. For S. cerevisiae, there are libraries of available mutants, plasmids, detailed computer models of metabolism, and other information, enabling the rational design of various modules for enhancing product yields. Methods for creating recombinant S. cerevisiae microorganisms are known.

[0032] Cell culture is carried out in a conventional manner. The culture medium contains a carbon source, at least one nitrogen source, and inorganic salts, to which vitamins are added. The components of this medium can be those conventionally used for culturing the microorganism species. The carbon source used in the present method includes any molecule that can be metabolized by the recombinant host cell to promote growth and / or the production of (-)-ambrox. Examples of suitable carbon sources include, but are not limited to, sucrose, fructose, xylose, glycerol, glucose, cellulose, starch, cellobiose, or other glucose-containing polymers (such as those found in molasses).

[0033] In embodiments where yeast is employed as the host, for example, carbon sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The carbon source can be provided to the host organism over the course of the culture period, or alternatively, the organism can be grown for a period in the presence of another energy source, such as protein, and then the carbon source can be provided only during the fed-batch phase.

[0034] The suitability of recombinant host cell microorganisms for use in the methods of the present disclosure can be determined by simple test procedures 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.) under the pH, temperature, and aeration conditions commonly used for the growth of the microorganism. Once a recombinant microorganism (i.e., a recombinant host cell) that produces the desired bioconversion product is selected, the product is typically produced on a large scale by the production host cell strain, using a suitable expression system and fermentation, such as by microbial production in a cell culture.

[0035] In one embodiment of the present disclosure, a minimal defined medium such as M9A is used for cell culture. The components of the M9A medium are KH 2 PO 4 14 g / L, K 2 HPO 4 16 g / L, sodium citrate 3 .2H 2 O 1 g / L, (NH 4 ) 2 SO 4 7.5 g / L, MgSO 4 .7H 2 O 0.25 g / L, CaCl 2 .2H 2 O 0.015 g / L, glucose 5 g / L, and yeast extract 1.25 g / L). In another aspect of the present disclosure, an enriched medium such as LB was used. The composition of the LB (Luria - Bertani) medium includes 10 g / L of tryptone, 5 g / L of yeast extract, and 5 g / L of NaCl.

[0036] Other examples of mineral media and M9 mineral media are disclosed, for example, in US6524831B2 and US2003 / 0092143A1. The recombinant microorganism can be grown by a batch, fed - batch, or continuous process, or a combination thereof. Typically, the recombinant microorganism is grown for a desired period in the presence of a suitable nutrient source, such as a carbon source, at a defined temperature in a fermentor, and bioconverts homofarnesol to (-)-ambrinol in a desired amount.

[0037] The recombinant host cell can be cultured in any suitable manner, for example, by batch culture or fed - batch culture. As used herein, the term "batch culture" is a culture method in which the culture medium is neither added nor removed during the culture. As used herein, the term "fed - batch" means a culture method in which the culture medium is added during the culture, but the culture medium is not removed.

[0038] One aspect of the present disclosure provides a method for producing (-)-ambrinol in a cell - based system, the method comprising producing wild - type SHC or a mutant thereof in a cell - based system under suitable conditions, feeding homofarnesol to the cell - based system, converting homofarnesol to (-)-ambrinol using the wild - type SHC or a mutant thereof produced using the cell - based system, recovering ambrinol from the cell - based system, and isolating (-)-ambrinol from the system. The expression of other nucleotide sequences serves to enhance the bioconversion pathway for making (-)-ambrinol.

[0039] A further aspect of the disclosure is a bioconversion method for making (-)-ambrox, the method comprising growing a host cell comprising a wild-type SHC or a mutant thereof gene, producing the wild-type SHC or its mutant enzyme in the host cell, supplying homofarnesol (e.g., EEH) to the host cell, incubating the host cell under conditions of appropriate pH, temperature, and solubilizing agent to facilitate the conversion of homofarnesol to ambrox, and recovering (-)-ambrox. Production of the wild-type SHC or its mutant enzyme in 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.

[0040] Recombinant host cell microorganisms can be cultured in a number of ways to provide a suitable amount of cells that express the wild-type SHC or mutant enzyme for subsequent bioconversion steps. Since the applicable microorganisms for the bioconversion step vary widely (e.g., yeast, bacteria, and fungi), it is natural that the culture conditions are adjusted to the specific requirements of each species, and those conditions are well known and documented. Any method known in the art for growing cells of recombinant host cell microorganisms can be used to produce cells available for the subsequent bioconversion step of the disclosure. Typically, the cells are grown to a specific density (as measurable as optical density (OD)) to produce sufficient biomass for the bioconversion reaction. The culture conditions chosen not only affect the amount of cells (biomass) obtained but also how the quality of the culture conditions affects how the biomass becomes a biocatalyst. Recombinant host cell microorganisms that express the wild-type SHC or mutant gene and produce the wild-type SHC or mutant enzyme are called biocatalysts suitable for use in the bioconversion reaction. In some embodiments, the biocatalyst is a recombinant whole cell that produces the wild-type SHC or mutant, or it can be in a suspension or immobilized format.

[0041] In one embodiment, the biocatalyst is produced, harvested in sufficient quantity (to create sufficient biomass), washed (and optionally stored (e.g., frozen or lyophilized)) prior to the bioconversion step. In a further embodiment, cells are produced in sufficient quantity (to create sufficient biocatalyst), and then the reaction conditions are adjusted without the need to harvest and wash the biocatalyst for the bioconversion reaction. This one-step (or "one-pot") method is advantageous as it simplifies the process while reducing costs. The culture medium used to grow the cells is also suitable for use in the bioconversion reaction if the reaction conditions are adjusted such that they promote the bioconversion reaction.

[0042] The bioconversion method of the present disclosure is carried out under conditions of time, temperature, pH, and solubilizing agent that result in the conversion of a homofarnesol feedstock to (-)-ambrinol. 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 the SHC mutant enzyme, and in the range of about pH 5.0 to about pH 7.0 for the wild-type SHC enzyme, and can be maintained by the addition of a buffer to the reaction mixture. An exemplary buffer for this purpose is a 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, up to 55°C, for thermophilic organisms, particularly when a wild-type enzyme from a thermophilic microorganism is used. The temperature may be kept constant or changed during the bioconversion process.

[0043] Applicants have shown that it may be useful to include a solubilizing agent (e.g., surfactant, detergent, solubility enhancer, water-miscible organic solvent, etc.) during the bioconversion reaction. 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). The applicant selected and identified SDS as a particularly useful solubilizer from a long list of other less useful solubilizers. In particular, the applicant identified SDS as a solubilizer that is significantly superior to, for example, Triton X-100, from the perspective of reaction rate and yield for the bioconversion reaction of homofarnesol to (-)-ambrinol.

[0044] While not wishing to be bound by theory, the use of SDS in recombinant microbial host cells may be advantageous because SDS can advantageously interact with the host cell membrane to make the SHC enzyme, which is a membrane-bound enzyme, more accessible to the homofarnesol substrate. In addition, the inclusion of SDS at a suitable level in the reaction mixture can improve the properties of the emulsion (homofarnesol in water) and / or improve the access of the homofarnesol substrate to the SHC enzyme within the host cell, while at the same time preventing disruption (e.g., denaturation / inactivation of wild-type SHC or mutant enzymes).

[0045] The concentration of the solubilizer (e.g., SDS) used in the bioconversion reaction is affected by the biomass amount and the substrate (EEH) concentration. That is, there is a certain degree of interdependence among the solubilizer (e.g., SDS) concentration, the biomass amount, and the substrate (EEH) concentration. As an example, as the concentration of the homofarnesol substrate increases, a sufficient amount of biocatalyst and solubilizer (e.g., SDS) is required for an 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 is affected by either the disruption of intact microbial cells and / or the denaturation / inactivation of the SHC / HAC enzyme.

[0046] In relation to the biomass amount and the substrate (EEH) concentration, the selection of a suitable concentration of SDS is within the knowledge of those skilled in the art. As an example, predictive models for determining suitable SDS, substrate (EEH), and biomass concentrations are available to those skilled in the art. The temperature of the bioconversion reaction for the wild-type SHC enzyme is about 45 to 60 °C, preferably 55 °C. The pH range of the bioconversion reaction for the wild-type SHC enzyme is from about 5.0 to 7.0, more preferably from about 5.6 to about 6.2, and even more preferably about 6.0. The temperature of the bioconversion reaction for the SHC mutant enzyme is from about 34 °C to about 50 °C, preferably about 35 °C. The pH of the bioconversion reaction for the SHC mutant enzyme is about 4.8 - 6.4, preferably about 5.2 - 6.0.

[0047] Preferably, the solubilizing agent used in the bioconversion reaction is SDS. The [SDS] / [cell] ratio is in the range of about 10:1 to 20:1, preferably about 15:1 to 18:1, preferably about 16:1 when the ratio of the biocatalyst to EEH homofarnesol is about 2:1. The SDS concentration in the bioconversion reaction for the SHC mutant 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 of EEH and the biocatalyst concentration is 250 g / l (corresponding to an OD of about 175 at 650 nm). The ratio of the biocatalyst to the EEH homofarnesol substrate is in the range of about 0.5:1 to 2:1, 2:1 in some embodiments, preferably about 1:1 or 0.5:1.

[0048] In some embodiments, (-)-ambrinol is produced using a biocatalyst to which the homofarnesol substrate is added. The substrate can be added by supplying it using known means (e.g., peristaltic pump, syringe for injection, etc.). Homofarnesol is an oil-soluble compound and is provided in an oil format. In view of the biocatalyst being present in the aqueous phase, the bioconversion reaction can be regarded as a two-phase system when homofarnesol is added to the bioconversion reaction mixture. This is true even when a solubilizing agent (e.g., SDS) is present. Further details of a suitable bioconversion process are disclosed in the examples below in this specification.

[0049] The bioconversion process according to the present invention produces a reaction mixture that also contains the desired (-)-ambrox and also a number of by-products. More specifically, the reaction mixture contains, in addition to (-)-ambrox, novel structural isomers of (-)-ambrox as described in formula (II), as well as known stereoisomers of (-)-ambrox as described in formulas (III) and (IV), a complex mixture of by-products.

Chemical formula

[0050] The applicant does not intend to be bound by any particular theory, but believes that the compound of formula (II) is formed by cyclization of the 7E,3Z-geometric isomer of homofarnesol. This has a detection threshold of >500 ng / l and is described as being practically odorless. As described above, the applicant believes that the compound of formula (II) is a novel molecule, and as such this compound forms a further aspect of the present invention.

[0051] A perfume ingredient and perfume composition consisting of or containing compound (II), and an aromatic article containing the same, form an additional aspect of the present invention. Use of the compound of formula (II) as a perfume ingredient in perfume applications such as high-grade perfumes or functional perfume compositions such as personal care, household care and fabric care compositions forms a further additional aspect of the present invention. A mixture of (-)-ambrox and an olfactorily acceptable amount of compound (II) forms another aspect of the present invention.

[0052] In connection with the compound of formula (II), or any of the other by-products (III) or (IV), or any substance present as an impurity in (-)-ambrox actually formed according to the process of the present invention, the term "olfactorily acceptable amount" as used herein means that the compound or substance is present in a mixture with (-)-ambrox in an amount below its odor detection threshold or in an amount that does not contribute to its olfactory characteristics so as to affect the olfactory properties of (-)-ambrox. (-)-Ambrox containing any such compound or substance in an olfactorily acceptable amount will be identifiable to a skilled perfumer as having the odor characteristics of a commercial grade of (-)-ambrox, such as AMBROFIX (trademark), obtained by a synthetic procedure from ex-sclareol and available from Givaudan.

[0053] In a preferred embodiment of the invention, the reaction mixture contains no or substantially no unreacted homofarnesol. The Applicant has discovered that homofarnesol is a strong solvent for (-)-ambrox as well as for the above by-products of the bioconversion process. Thus, in the presence of a significant amount of homofarnesol, (-)-ambrox and the by-products remain dissolved together in a difficult-to-handle crude mixture from which it is difficult and time-consuming to separate and ultimately isolate (-)-ambrox in an olfactorily pure form. It has been found that reducing the level of unreacted homofarnesol in the mixture of (-)-ambrox with compounds (II), (III) and (IV) considerably facilitates downstream processing and the isolation / purification of (-)-ambrox.

[0054] Downstream processing is an important step in the production of useful compounds formed by bioconversion processes, as understood by those skilled in the art. As part of compound synthesis, it can affect the physical properties of the compound. In the case of the preparation of fragrance components by biotechnological methods, it is desirable that the target compound can be separated from the reaction mixture in an olfactorily pure form so that the desired odor properties of the target compound are not distorted by the odor contributions of complex mixtures of impurities and by-products that may be present in the fermentation medium or the biocatalyst.

[0055] Accordingly, in another aspect, the present invention provides a method for isolating and purifying (-)-ambrox from a reaction mixture comprising one or more of compounds (II), (III) and (IV). In yet another aspect of the present invention, there is provided a method for improving or enhancing the odor of (-)-ambrox, comprising the step of separating and purifying (-)-ambrox from a reaction mixture containing one or more of compounds (II), (III) and (IV).

[0056] In the isolated and purified form, (-)-ambrox should not contain any of compounds (II), (III) or (IV), or, if it contains any of said compounds, each should be present in an olfactorily acceptable amount. Reaction mixtures obtained from bioconversion processes such as the above-described processes herein generally contain crude (-)-ambrox as well as one or more of by-products (II), (III) and (IV), and a solid phase containing cell material and / or fragments thereof; and a liquid phase or liquid phases containing water and / or some unreacted homofarnesol.

[0057] The solid phase may be separated from the liquid phase(s) by filtration or centrifugation. Further, by selecting a filter with an appropriate pore size, it is also possible to achieve the separation of crude (-)-ambrox from the cell material and / or fragments. Once the crude (-)-ambrox is separated from the cell material and / or its fragments, it may be washed before undergoing further procedures to isolate (-)-ambrox from compounds (II), (III), and (IV).

[0058] Alternatively, instead of filtration or centrifugation, the reaction mixture is warmed to a temperature above the melting point of (-)-ambrox, whereupon the reaction mixture forms an oil phase on top of the aqueous phase containing the cell material and fragments. Optionally, and to ensure complete recovery of (-)-ambrox, the aqueous cell material may be washed with a water-immiscible organic solvent (such as toluene) to remove any residual (-)-ambrox that may be mixed in the aqueous phase, and these washings can be combined with the oil phase. Thereafter, the oil phase can be concentrated by evaporation to provide a crude mixture containing (-)-ambrox as well as one or more of by-products (II), (III), and (IV), and this mixture can then undergo further procedures for isolating and purifying (-)-ambrox.

[0059] In another embodiment, instead of warming the reaction mixture to form an oil phase containing (-)-ambrox, the reaction mixture is extracted with a suitable water-immiscible organic solvent (such as toluene) to form an organic phase containing (-)-ambrox as well as one or more of compounds (II), (III), and (IV), which can be separated from the aqueous phase containing the cell material and fragments. The organic phase can be concentrated by evaporation to provide a crude mixture containing (-)-ambrox as well as one or more of compounds (II), (III), and (IV), and this can undergo further procedures for isolating and purifying (-)-ambrox.

[0060] In yet another alternative method, the reaction mixture can be steam distilled to remove distillates from the cell material and fragments. The distillates can be collected as a biphasic mixture that separates the oil phase of the biphasic mixture containing (-)-ambrox as well as one or more of compounds (II), (III), and (IV) from the aqueous phase and then undergoes further processing steps to isolate and purify (-)-ambrox.

[0061] In a specific embodiment of the present invention, the method for isolating and purifying (-)-ambrox includes the step of selectively crystallizing (-)-ambrox from a mixture containing one or more of compounds (II), (III), or (IV). The term "selectively crystallize" means that thereby (-)-ambrox crystallizes from the solvent while compounds (II), (III), and (IV) dissolve and remain in the crystallization solvent to such an extent that the isolated crystalline material contains only (-)-ambrox or, if it contains any of compounds (II), (III), or (IV), they are present only in an olfactorily acceptable amount.

[0062] Crystallization can be carried out in a suitable organic solvent. The choice of solvent is based on considerations such as differences in solubility at room temperature and high temperature or in boiling solvents; and the need for the amount of recoverable crystals in the cooled solvent. Usually, the compound to be separated is dissolved in a relatively polar solvent, and then a relatively less polar solvent can be added to bring the solubility limit of the dissolved compound, thereby starting crystallization. Also, in an industrial process, it is related not only to cost but also to the issues of handling safety. Suitable solvents include, but are not limited to, methanol, acetone, petroleum ether, hexane, t-butyl methyl ether, THF, and ethyl acetate. Preferred solvents include toluene or ethyl alcohol. Combinations of solvents can also be employed.

[0063] In a particularly preferred embodiment of the present invention, the selective crystallization is carried out by dissolving a mixture containing (-)-ambrox and one or more of compounds (II), (III) and (IV) in warm ethanol and slowly adding a non-solvent such as water to the ethanol solution during cooling to selectively crystallize (-)-ambrox. Considering the close structural relationship between (-)-ambrox and the by-product compounds (II), (III) and (IV), each of which is a structural isomer and two stereoisomers of (-)-ambrox, it was remarkable that (-)-ambrox could be selectively crystallized from such a mixture, resulting in (-)-ambrox in a high yield and in an odor-pure form. One of ordinary skill in the art would naturally expect that the compounds would co-crystallize with (-)-ambrox and make the downstream processing much more complex, time-consuming and costly than actually found.

[0064] The surprisingly easy way in which (-)-ambrox is separated from a mixture containing compounds (II), (III) and / or (IV) by crystallization shows a prominent advantage of the present invention. The ease with which (-)-ambrox can be separated by crystallization may be in contrast to the observation that (-)-ambrox cannot be recovered from a mixture containing (II), (III) and / or (IV) by other purification techniques such as distillation because the boiling points of (-)-ambrox and the by-products (II), (III) and (IV) are very close and in such an easy way and in such a high yield.

[0065] The term "odor-pure" as used in the context of (-)-ambrox is intended to mean that (-)-ambrox does not contain compound (II), (III) or (IV), or any other material found in the reaction mixture, or, if such compounds or materials are present, that they are present in an odor-acceptable amount as defined herein. In an embodiment of the present invention, (-)-ambrinol in an olfactorily pure form contains less than 5% by weight of any of compounds (II), (III) or (IV).

[0066] In a more specific embodiment, (-)-ambrinol in an olfactorily pure form contains less than 4% by weight, less than 3% by weight, less than 2% by weight, less than 1% by weight, less than 0.9% by weight, less than 0.8% by weight, less than 0.7% by weight, less than 0.6% by weight, less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight or less than 0.05% by weight of each of compounds (II), (III) or (IV). The quality of the separation of (-)-ambrinol by selective crystallization from a mixture containing compounds (II), (III) and / or (IV) can be affected by the composition of the mixture from which it is separated. More specifically, the quality of the separation of (-)-ambrinol by crystallization from a mixture of compounds (II), (III) and / or (IV) is improved when the weight ratio of (-)-ambrinol to the other compounds (II), (III) and / or (IV) in the mixture is greater than 70:30, more specifically 80:20, more specifically 90:10, even more specifically 95:5, and even more specifically 97:3.

[0067] Furthermore, the quality of the separation of (-)-ambrinol by crystallization can be affected by the amount of unreacted homofarnesol present in the mixture from which it is separated. More specifically, the quality of the separation is improved when the level of unreacted homofarnesol is less than 30% by weight, more specifically less than 20% by weight, more specifically less than 10% by weight, even more specifically less than 5% by weight, and even more specifically less than 3% by weight, even more specifically less than 2% by weight, and even more specifically less than 1% by weight, based on the weight of the mixture from which (-)-ambrinol is crystallized and separated.

[0068] Preferably, the reagents and reaction conditions used in the bioconversion process of the present invention are such that the reaction proceeds or substantially proceeds to 100% conversion of homofarnesol, and thus no unreacted homofarnesol remains in the reaction mixture. However, if unreacted homofarnesol is present, although it is economically disadvantageous, it can be separated from (-)-ambrinol and other by-products, for example, by distillation.

[0069] Accordingly, in a specific embodiment of the present invention, there is provided a method for isolating and purifying (-)-ambrinol from a mixture containing one or more of compounds (II), (III) and (IV), wherein the mixture does not contain or substantially does not contain homofarnesol. In a more specific embodiment, the isolation and purification of (-)-ambrinol from a mixture containing one or more of compounds (II), (III) and (IV) and not containing or substantially not containing homofarnesol is achieved by the selective crystallization of (-)-ambrinol.

[0070] The (-)-ambrinol obtained by the method of the present invention is obtained in an olfactorily pure form. Olfactorily pure (-)-ambrinol forms another aspect of the present invention. The crystalline form of (-)-ambrinol forms yet another aspect of the present invention. The (-)-ambrinol formed according to the method of the present invention may be mixed with one or more additional fragrance components to form a fragrance composition suitable for use in fragrance products, including use in high-quality perfumes and in consumer products such as personal care, fabric care and household care products.

[0071] Accordingly, in another aspect thereof, the present invention provides a fragrance composition comprising (-)-ambrinol and at least one other fragrance component, wherein the fragrance composition contains an olfactorily acceptable amount of one or more of compounds (II), (III) or (IV). The present invention will be further described with reference to the following examples.

[0072] Example Example 1: Preparation of Homofarnesol General analysis conditions: Non-polar GC / MS: 50°C / 2 minutes, 20°C / min to 200°C, 35°C / min to 270°C. GC / MS Agilent 5975C MSD with HP 7890A series GC system. Non-polar column: BPX5 from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22 mm × 0.25 mm × 12 m. Carrier gas: helium. Injector temperature: 230°C. Split 1:50. Flow: 1.0 ml / min. Transfer line: 250°C. MS - quadrupole type: 106°C. MS source: 230°C.

[0073] 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) is heated to reflux (105°C) with stirring for 3.5 hours. At 40°C, NaNO2 (101 g, 1.45 mol) dissolved in water (200 ml) is added. After 15 minutes, THF (1000 ml) is added, which results in a clear biphasic mixture. Concentrated H2SO4 (110 g, 1.1 mol) is added at 0 - 5°C and stirred within 1.5 hours. After an additional 0.5 hour at 0 - 5°C, two clear phases separate at 25°C. The organic phase (A) (1065 ml, theoretically 1.35 M) is stored at 0 - 5°C for several days or immediately advanced to the cyclopropanation reaction vessel.

[0074] After phase separation, the aqueous phase is extracted twice with THF (2×1L). This gives 1100 ml of phase B and 1075 of phase C. Phase A gives a 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. Thus, typically the aqueous phase is discarded after the first phase separation (from organic phase A) after treatment with aqueous concentrated KOH and acetic acid.

[0075] B) Preparation of E-Δ-Farnesene Using MNU in THF [Chemical formula] 1.35 M N-methyl-N-nitrosourea in THF (136 ml, 184 mmol) is added dropwise at 0 °C to a vigorously stirred mixture of E-beta-farnesene (CAS 18794-84-8) (25 g, 122 mmol) and aqueous KOH solution (50 ml, 40%) at 0 - 5 °C. After addition of 4 ml of the MNU solution, Pd(acac)2 (7.4 mg, 0.024 mmol, 0.02%) pre-dissolved in 0.5 ml dichloromethane is added. The remaining MNU solution is added at 0 - 5 °C over 4 h. GC at this stage shows 28% unreacted E-beta-farnesene, 65% of the desired monocyclopropane (shown above), and 3% of the biscyclopropanated compound 5. After 16 h at 25 °C, acetic acid (100 ml) is added at 0 - 5 °C, then tert-butyl methyl ether (250 ml) is added. 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 (2×100 ml), 10% aqueous NaOH (2×100 ml), and water (2×100 ml), dried over MgSO4, filtered, and concentrated to give 26.9 g of a slightly yellow liquid. This contains 9% E-beta-farnesene, 82% of the desired monocyclopropane compound, and 6% of the biscyclopropanated by-product.

[0076] The desired compound can be further isolated by distillation purification. 1 g of K 2 CO 3 (1 g) addition and distillation by a 30 cm steel coil column at 40 - 60 mbar gives 147 g of the monocyclopropane compound (68% corr) at 135 - 145 °C. The fractions are pooled to give 92 g of the monocyclopropane compound with 100% purity.

[0077] Analysis data of E-Δ-farnesene: 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). Calculated for C16H26: C, 88.00; H, 12.00. Results: C, 87.80; H, 12.01.

[0078] 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-Δ-farnesene) (1 g, 4.6 mmol), dodecane (0.2 g, 1.15 mmol, internal standard), and L-(+)-tartaric acid (1 g, 6.9 mmol) in a pressure tube is heated at 150 °C with stirring. After 18 h and complete conversion (by 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 Na 2 CO 3 aqueous solution (50 ml) and concentrated NaCl (2 × 50 ml), dried over 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 the internal standard. E / Z ratio 68:22. The analytical data for the E isomer are in agreement with those from the literature. See, for example, P. Kocienski, S. Wadman J. Org. Chem. 54, 1215 (1989).

[0079] Example 2 SHC Plasmid Preparation and Biocatalyst Production SHC Plasmid Preparation The gene encoding Alicyclobacillus acidocaldarius squalene-hopene cyclase (AacSHC) (GenBank M73834, Swissprot P33247) was inserted into plasmid pET-28a(+). There, it is under the control of the IPTG-inducible T7 promoter for protein production in E. coli. The plasmid was transformed into the E. coli strain BL21(DE3) using a standard heat shock transformation protocol.

[0080] Erlenmeyer Flask Culture For protein production, either rich medium (LB medium) or minimal medium was used. M9 is an example of minimal medium and was used successfully.

[0081] Culture Medium Preparation The minimal medium selected by default was prepared as follows for 350 ml of culture: 35 ml of citrate / phosphate stock (KH 2 PO 4 133 g / l, (NH 4 ) 2 HPO 4 40 g / l, citric acid.H 2 O 17 g / g, pH adjusted to 6.3) with H 2 O 307 ml was added and the pH was adjusted to 6.8 with 32% NaOH as needed. 0.850 ml of 50% MgSO 4 was autoclaved and then 0.035 ml of trace element solution (composition in the next section), 0.035 ml of thiamine solution, and 7 ml of 20% glucose were added.

[0082] SHC Biocatalyst Production (Biocatalyst Production) For small-scale biocatalyst production (wild-type SHC or SHC mutant), 350 ml of culture (supplemented with 50 μg / ml kanamycin in the medium) was inoculated from a preculture of Escherichia coli strain BL21(DE3) containing the SHC production plasmid. The cells were grown at 37 °C with constant stirring (250 rpm) to an optical density (OD 650nm ) of approximately 0.5. Protein production was then induced by adding IPTG to a concentration of 300 μM, followed by further incubation for 5 - 6 hours with constant shaking. The resulting biomass was finally collected by centrifugation and washed with 50 mM Tris-HCl buffer pH 7.5. The cells were stored as pellets at 4 °C or -20 °C until further use. Generally, regardless of the medium used, 2.5 to 4 grams of cells (wet weight) were obtained from 1 liter of culture.

[0083] Fermentation was prepared and carried out in a 750 ml Infors HT reaction vessel. 168 ml of deionized water was added to the fermentation vessel. The reaction vessel was equipped with all the necessary probes (pO 2 , pH, sampling, antifoaming agent), C+N feed and a bottle of sodium hydroxide, and autoclaved. After autoclaving, the following components were added to the reaction vessel: 20 ml of 10× phosphate / citrate buffer 14 ml of 50% glucose 0.53 ml of MgSO 4 solution 2 ml of (NH 4 ) 2 SO 4 solution 0.020 ml of trace element solution 0.400 ml of thiamine solution 0.200 ml of kanamycin stock.

[0084] The reaction conditions were set as follows: pH = 6.95, pO 2 = 40%, T = 30 °C, stirred at 300 rpm. Cascade: rpm set point at 300, minimum 300, maximum 1000, flow L / min set point 0.1, minimum 0, maximum 0.6. Antifoaming agent control: 1:9. From the seed culture, the fermenter was inoculated to an OD 650nm of 0.4 - 0.5. This seed culture was grown in LB medium (+ kanamycin) at 37 °C and 220 rpm for 8 hours. The fermentation was first carried out in batch mode for 11.5 hours, and then the C+N feed was started with a feed solution (sterile glucose solution (143 ml H 2 O + 35 g glucose), to which, after sterilization, 17.5 ml of (NH 4 ) 2 SO 4 solution, 1.8 ml of MgSO 4 solution, 0.018 ml of trace element solution, 0.360 ml of thiamine solution, and 0.180 ml of kanamycin stock were added). The feed was carried out at a constant flow rate of approximately 4.2 ml / hour. Glucose and NH 4 +Measurements were made externally to assess the availability of C and N sources in the cultures. Usually, the glucose level remains very low.

[0085] The cultures were grown for a total of approximately 25 hours, where they typically reached an OD of 40 - 45. 650nm Then, IPTG was added to the fermenter (either as an IPTG pulse or over 3 - 4 hours using a syringe) to a final concentration of approximately 1 mM, and SHC production was initiated by setting the temperature at 40°C and pO 2 at 20%. The induction of SHC production continued for 16 hours at 40°C. At the end of induction, the 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.

[0086] Result 1a Generally, the specific activity of the produced biocatalyst was higher when using minimal medium compared to rich medium, with all other conditions remaining unchanged. Induction was successful at 30 or 37°C. It was noted that when induction was carried out at 40 - 43°C, a biocatalyst with higher specific activity was obtained.

[0087] Result 1b Table 1 below shows, for two examples, the culture volume, optical density, and amount of cells at both the start and end of induction, and the amount of biomass collected (wet weight).

Table 1

[0088] Wild - type SHC amino acid sequence (SEQ ID NO: 1) (GenBank M73834, Swissprot P33247) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR

[0089] Variant F601Y SHC amino acid sequence (SEQ ID NO: 2) - variant with respect to SEQ ID NO: 1 MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR

[0090] Variant F605W SHC nucleotide sequence (SEQ ID NO: 3)

[0091] Variant F605W SHC amino acid sequence (SEQ ID NO: 4) - variant with respect to SEQ ID NO: 1 MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR

[0092] Example 3 Bioconversion of 7E,3E / Z-homofarnesol mixture Bioconversion was carried out using the following reaction conditions: The reaction (total volume 150.1 g) was carried out in an Infors HT 750 ml fermenter containing a total of 146 g / l of homofarnesol, in 0.1 M citric acid / sodium citrate buffer pH 5.4, with a homofarnesol substrate that is a mixture of 86:14 7E,3E:7E,3Z, 250 g / l of cells (formed according to the method of Example 2, fermentation), and 1.55% SDS. The reaction was carried out at 35 °C with constant stirring (900 rpm), and pH control was carried out using 10 to 40% citric acid in water. The reaction mixture was subjected to the isolation and purification steps described in Example 4 below.

[0093] Example 4 Downstream Processing Procedure The reaction mixture formed from the bioconversion of 7E,3E / Z-homofarnesol (86:14 3E:3Z) was subjected to steam distillation. The 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 2 below (see "crude"). The organic phase was then concentrated by drying. Thereafter, ethanol was added to the crude dried product and the mixture was warmed until the product dissolved. Room temperature water was slowly added, stirred occasionally, and (-)-ambrinol was crystallized while cooling in an ice bath.

[0094] Table 2 shows the GC analysis results for the crystallized product. The data show a concentrated (-)-ambrinol that does not actually contain the by-products (a), (b), or (c) found in the crystallized sample. It should be noted that "a", "b", and "c" in Table 2 refer to compound (II), compound (IV), and compound (III), respectively. "EZH" and "EEH" refer to 7E,3Z-homofarnesol and 7E,3E-homofarnesol, respectively.

Table 2

[0095] Example 5 Extraction of the solid phase of the reaction broth: Considering that (-)-ambrox is not soluble in water and is not liquid at temperatures below approximately 75°C, these properties were considered potential advantages for extracting the product from the solid phase of biotransformation using either a water-miscible solvent (e.g., ethanol) or a water-immiscible solvent (e.g., toluene).

[0096] 200 ml of the reaction broth was centrifuged to separate the solid from the liquid (water) phase (Sorvall GS3, 5000 rpm, 10 min, 10°C). As a result, approximately 80 ml of solid pellet was separated from approximately 120 ml of the liquid phase. Analysis of the aqueous phase after MTBE extraction (gas chromatography) showed that it contained approximately 0.3% or less of the (-)-ambrox initially present in 200 ml of the reaction broth. Toluene and 99% ethanol were used to extract (-)-ambrox from the solid phase.

[0097] Toluene extraction: The 80 ml of the solid phase was extracted 6 times with 45 ml of toluene (approximately 1 / 2 of the solid phase volume, with vigorous shaking for 30 seconds and centrifugation (Sorvall GS3, 5000 rpm, 10 min, 10°C)). The solvent phase was analyzed by GC for its (-)-ambrox content. 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 1.35 × the initial total reaction broth volume (200 ml) or 3.4 × the solid phase volume.

[0098] Ethanol extraction: 80 ml of the solid phase was extracted with approximately 160 ml (two volumes) of 99% ethanol (Infors Multifors HT, 35 °C, 1000 rpm, 30 min) and then centrifuged. (-)-Ambrox did not crystallize during the extraction procedure. After four washes (a total of 640 ml of ethanol, i.e., 3.2 × the total initial reaction broth volume or 8 × the volume of the solid phase), approximately 99% of the ambrox initially present in the reaction broth was recovered. Sufficient ethanol is required to prevent crystallization of (-)-ambrox in the first extraction step (soluble in ethanol). When only 1 or 1 / 2 volume of the solid phase was used in the first extraction step, a sticky paste was obtained, which was difficult to handle, and (-)-ambrox crystallized as needles on the pellet during centrifugation. The temperature did not seem to be a factor causing this crystallization (extraction and centrifugation were tested at room temperature and approximately 35 °C to 40 °C).

[0099] The (-)-ambrox concentration in the ethanol phase and the ethanol / water ratio of the liquid phase (residual moisture of the solid phase) seemed to be the cause of crystal formation. However, it was noted that it was possible to reduce the volume of ethanol to 1 volume of the solid phase.

[0100] (-)-Ambrox does not exist in the liquid phase at room temperature, so it can be separated with the biomass and extracted with an organic solvent (e.g., a water-miscible solvent such as ethanol or a water-immiscible solvent such as toluene). The centrifugation step that separates (-)-ambrox into the solid phase of the reaction mixture is advantageous because it reduces the amount of solvent required to extract (-)-ambrox.

[0101] Example 6 Functional analysis Objective: To perform a functional analysis of (-)-ambrox and compounds (II), (III), and (IV) formed in the "crude" material and the "crystallized" material. The in vivo conversion of E,E-homofarnesol results in (-)-ambrox and compound (IV). The in vivo conversion of E,Z-homofarnesol results in the macrocyclic ether compound (II) and the epi-ambrox compound (III). The crude mixture of (-)-ambrox contains the desired (-)-ambrox, compound (II), (III) and (IV) present in amounts of 87.1 wt%, 2.8 wt%, 2.5 wt%, and 7.6 wt%, respectively.

[0102] When the crude mixture was selectively crystallized (laboratory scale), the crystallized material had the same constituents as the crude mixture but was present in amounts of 99.1 wt%, 0.1 wt%, 0.1 wt%, and 0.7 wt%, respectively. The results of the sensory analysis were as follows: (-)-Ambrox: odor threshold 0.2 ng / l. Compound (IV): weak, IsoE, woody, GC detection threshold 5 - 10 ng. Compound (II): "odorless" (GC threshold > 500 ng). Compound (III): GC threshold approximately 10× higher than (-)-ambrox (approx. 2 ng).

[0103] Conclusion The sensory analysis of the three by-products (compounds II, III, and IV) shows a weaker odor than that from (-)-ambrox. Indeed, the odor of epi-ambrox (compound III) is approximately 10 times weaker than that of (-)-ambrox, suggesting that it is essentially odorless.

[0104] Example 7 Recovery of ambrox by steam extraction Purities obtained for the crude (-)-ambrox and the crystallized (-)-ambrox (steam-extracted) EE:EZ-Homofarnesol 86:14's in vivo conversion resulted in a reaction mixture that was steam-extracted. The steam distillate was collected as a biphasic 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 the table below (see "crude"). The organic phase was then concentrated to a dry state. Next, ethanol was added to the crude dry product and the mixture was warmed until the product dissolved. Water was slowly added at room temperature, stirring occasionally, and (-)-ambrinol was crystallized while cooling in an ice bath.

[0105] The table data also show the GC analysis results for the product obtained after the steam extraction / distillation step ("crude") and the crystallized product ((-)-ambrinol). References to "EZH" and "EEH" in the table refer to (3Z,7E)-homofarnesol and 7E,3E-homofarnesol, respectively. The following table data show that a specific starting material (EEH:EZH 86:14) produces a very specific mixture of the desired final product (-)-ambrinol and by-products (II, IV, and III) using WT SHC or SHC derivatives. The data for selective crystallization show a concentrated (-)-ambrinol in a state where by-products (II), (IV), or (III) are not actually found in the crystallized samples. Thus, this EE:EZ mixture provides an olfactory pure (-)-ambrinol product that is selectively crystallized in a relatively simple, straightforward, and cost-effective manner.

[0106]

Table 3

[0107] Steam extraction / filtration is an environmentally friendly method for isolating (-)-ambrinol. This is because it provides a convenient, solvent-free isolation of (-)-ambrinol and involves the inactivation of the biocatalyst. The (-)-ambrox produced using a 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 a solid phase (e.g., using a water-miscible solvent) using methods known to those skilled in the art.

Claims

Claim 1 A fragrance ingredient comprising (-)-ambrox and less than 5% by weight of any one of compounds (II), (III) or (IV). 【Chemical 1】 Claim 2 The fragrance ingredient according to claim 1, comprising crystalline (-)-ambrox. Claim 3 A fragrance composition comprising the fragrance ingredient according to claim 1 or 2 and at least one other fragrance ingredient. ​

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