Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D9/00—Crystallisation
  • B01D9/005—Selection of auxiliary, e.g. for control of crystallisation nuclei, of crystal growth, of adherence to walls; Arrangements for introduction thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B57/00—Separation of optically-active compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B63/00—Purification; Separation; Stabilisation; Use of additives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
  • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
  • C07D307/92—Naphthofurans; Hydrogenated naphthofurans
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
  • C11B9/00—Essential oils; Perfumes
  • C11B9/0069—Heterocyclic compounds
  • C11B9/0073—Heterocyclic compounds containing only O or S as heteroatoms
  • C11B9/0076—Heterocyclic compounds containing only O or S as heteroatoms the hetero rings containing less than six atoms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
  • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y504/00—Intramolecular transferases (5.4)
  • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
  • C12Y504/99017—Squalene--hopene cyclase (5.4.99.17)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/09—Geometrical isomers

Definitions

• the present invention is concerned with a method of preparing, isolating and purifying the isomer (-)-Ambrox. More particularly, the invention is concerned with a method of preparing (-)-Ambrox by means of a bioconversion, as well as to a method of its recovery and purification from a reaction mixture.
• AMBROFIXTM is the proprietary Givaudan trade name of the enantiomerically pure compound (-)-Ambrox, which has the general formula (I).
• AMBROFIX is a very important molecule in the perfumers' palette of ingredients. It delivers a highly powerful, highly substantive and highly stable ambery note for use in all perfumery applications.
• AMBROFIXTM available from Givaudan, is the most suitable material for obtaining an authentic ambergris odour note.
• AMBROFIXTM is produced synthetically from starting materials of natural origin.
• the availability and quality of certain starting materials are dependent on climatic conditions, as well as socio-economic factors.
• starting materials may be extracted from natural resources, with modest yields, they are available at prices that will, in all likelihood, increasingly render their use uneconomical on an industrial scale. Accordingly, if commercial industrial supplies of AMBROFIXTM are to continue to be available at a reasonable cost, there is a need for a more cost-effective process of production and purification, which is capable of industrialization.
• AMBROFIXTM An industrially scalable biotechnological route into AMBROFIXTM would be attractive because it is potentially less complex and less polluting than fully synthetic procedures.
• a potentially useful substrate on which to attempt a bioconversion to provide (-)-Ambrox is homofarnesol.
• Neumann et al Biol. Chem. Hoppe-Seyler Vol. 367 pp 723-726 (1986)
• SHC Squalene Hopene Cyclase
• the homofarnesol employed was a mixture of the four geometric isomers of this molecule. Of the four isomers, only the 7E,3E geometric isomer (using conventional nomenclature) could be cyclized, and then only with very low yield of the desired (-)-Ambrox.
• JP 2009-60799 discloses a synthesis whereby 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 homofarnesol extracts containing SHC. The homofarnesol mixture is converted to
• (-)-Ambrox and its 9-epi stereoisomer, and purification can be carried out by distillation or by column chromatography.
• Kao does not describe a process whereby homofarnesol is converted into (-)-Ambrox using intact microorganisms producing SHC, and furthermore, it does not provide any technical teaching related to the downstream processing of complex reaction mixtures obtained by such processes that can yield (-)-Ambrox in olfactively pure form.
• 7E,3E/Z-homofarnesol mixtures 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 (-)-Ambrox, to yield a reaction mixture from which (-)-Ambrox can be isolated in olfactively pure form with surprisingly facile downstream processing.
• SHC Squalene Hopene Cyclase
• I n one aspect of the invention there is provided the enzyme-catalyzed cyclisation of homofarnesol to provide a reaction mixture comprising (-)-Ambrox, wherein the homofarnesol comprises a mixture of 7E,3E/Z-geometric isomers of homofarnesol, and wherein the reaction is carried out in the presence of a recombinant microorganism producing the enzyme, more particularly an intact recombinant microorganism producing the enzyme.
• 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 a variant enzyme or is a microorganism expressing a gene encoding the SHC enzyme, preferably a recombinant E. coli microorganism.
• the SHC biocatalyst can be used in any form such as but not limited to a purified SHC enzyme, a crude extract containing an SHC enzyme or an immobilised SHC enzyme (e.g. on a carrier), or the biocatalyst can be a microorganism having produced or producing the SHC enzyme, such as an intact recombinant whole cell and/or fragmented cell or a membrane fraction containing the SHC enzyme.
• the homofarnesol mixture is enriched in the 7E,3E-geometric isomer.
• the homofarnesol mixture is at least 55/45 by weight 7E,3E/7E,3Z.
• the homofarnesol mixture is at least 70/30 by weight 7E,3E/7E,3Z. In a still more particular embodiment, the homofarnesol mixture is at least 80/20 by weight 7E,3E/7E,3Z
• the homofarnesol mixture is at least 90/10 by weight 7E,3E/7E,3Z. In a still more particular embodiment, the homofarnesol mixture is at least 95/5 by weight 7E,3E/7E,3Z.
• the homofarnesol mixture consists of 7E,3E/Z -geometric isomers and no other geometric isomers of homofarnesol.
• 7E, 7Z, 3E or 3Z used in connection with homofarnesol refers respectively to the orientation of the double bond at the 7-position and 3-position of homofarnesol.
• the 7E,3E-homofarnesol compound has the CAS No. 459- 89-2, whereas the 7E,3Z-homofarnesol compound has the CAS No. 138152-06-4.
• the use of the term 7E, 3E/Z-homofarnesol refers to a mixture of the compounds.
• PCT2014/EP/072882 (published as WO2015/059290) referred to above, which are hereby incorporated by reference in their entirety.
• they describe a synthesis of homofarnesol mixtures that proceeds by converting farnesene, more particularly alpha- farnesene and/or beta-farnesene, to its corresponding cyclopropanated farnesene derivative, using an organic solution of an N-alkyl-N-nitroso urea.
• the cyclopropanated derivative then undergoes ring-opening and rearrangement reactions in the presence of a Bronsted acid to afford the homofarnesol mixture, which is selective for the 7E,3E geometric isomer.
• Using farnesene, as a starting material is particularly preferred because it ensures that the E-configuration of the double bond at the 7 position of homofarnesol is fixed.
• SHC Squalene Hopene Cyclase
• 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 set forth in Example 3b of US20120135477A1).
• the enzyme can also be produced by recombinant means, using techniques that are generally known in the art.
• recombinant as used with respect to the production of enzymes shall refer to enzymes produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous DNA construct encoding the desired enzyme.
• the term “recombinant DNA” therefore includes a recombinant DNA incorporated into a vector into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote (or the genome of a homologous cell, at a position other than the natural chromosomal location).
• Nucleic acid molecule(s) is/are operatively linked to expression control sequences allowing expression in prokaryotic and/or eukaryotic host cells.
• operatively linked means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
• transcriptional/translational regulatory elements referred to above include but are not limited to inducible and non-inducible, constitutive, cell cycle regulated, metabolically regulated promoters, enhancers, operators, silencers, repressors and other elements that are known to those skilled in the art and that drive or otherwise regulate gene expression.
• regulatory elements include but are not limited to regulatory elements directing constitutive expression or which allow inducible expression like, for example, CUP-1 promoter, the tet-repressor as employed, for example, in the tet-on or tet-off systems, the lac system, the trp system regulatory elements.
• Isopropyl ⁇ -D-l- thiogalactopyranoside is an effective inducer of protein expression in the concentration range of 100 ⁇ to 1.0 mM.
• This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is therefore used to induce protein expression where the gene is under the control of the lac operator.
• nucleic acid molecule(s) can form part of a hybrid gene encoding additional polypeptide sequences, for example, a sequence that functions as a marker or reporter.
• marker and reporter genes including 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
• CAT chloramphenicol acetyltransferase
• ADA adenosine deaminase
• DHFR aminoglycoside phosphotransferase dihydrofolate reductase
• HPH hygromycin-B-phosphotransferase
• XGPRT phosphoribosyltransferase
• Recombinant polynucleotides can encode SHC enzymes such as the wild type SHC or a variant thereof, which may be inserted into a vector for expression and optional purification.
• SHC enzymes such as the wild type SHC or a variant thereof
• One type of vector is a plasmid representing 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 functionally linked. These vectors are called "expression vectors".
• expression vectors suitable for DNA recombination techniques are of the plasmid type.
• an expression vector comprises a gene such as the wild type SHC or a variant thereof.
• plasmid and vector are used
• the plasmid is the vector type most often used.
• Such vectors can include DNA sequences which include but are 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 a protein ("expressed") and other genes or DNA sequences which one desires to introduce into the non-recombinant host. It will be appreciated that typically the genome of a recombinant host is augmented through the stable introduction of one or more recombinant genes.
• autonomous or replicative plasmids or vectors can also be used within the scope of this disclosure.
• the present disclosure can be practiced using a low copy number, e.g., a single copy, or high copy number plasmid or vector.
• the vector of the present disclosure comprises plasmids, phagemids, phages, cosmids, artificial bacterial and artificial yeast chromosomes, knockout or knock-in constructs, Synthetic nucleic acid sequences or cassettes and subsets may be produced in the form of linear polynucleotides, plasmids, megaplasmids, synthetic or artificial chromosomes, such as plant, bacterial, mammalian or yeast artificial
• the proteins encoded by the introduced polynucleotide are produced within the cell upon introduction of the vector.
• the diverse gene substrates may be incorporated into plasmids.
• the plasmids are often standard cloning vectors, e.g., bacterial multicopy plasmids.
• the substrates can be incorporated into the same or different plasmids. Often at least two different types of plasmid having different types of selectable markers are used to allow selection for cells containing at least two types of vectors.
• bacterial or yeast cells may be transformed with any one or more of the following nucleotide sequences as is well known in the art.
• the gene to be recombined with the genome or other genes is used to transform the host using standard transforming techniques.
• DNA providing an origin of replication is included in the construct.
• the origin of replication may be suitably selected by the skilled person.
• a supplemental origin of replication may not be required if sequences are already present with the genes or genome that are operable as origins of replication themselves.
• a bacterial or yeast cell may be transformed by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
• the transforming DNA may or may not be integrated, i.e. covalently linked into the genome of the cell.
• the transforming DNA may be maintained on an episomal element such as a plasmid.
• a stably transformed cell is one in which the transfected DNA has become 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 cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
• the introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of the disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene.
• the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis.
• Suitable recombinant hosts include microorganisms, plant cells, and plants.
• the present disclosure also features recombinant hosts.
• recombinant host also referred to as a “genetically modified host cell” or a “transgenic cell” denotes a host cell that comprises a heterologous nucleic acid or the genome of which has been augmented by at least one incorporated DNA sequence.
• a host cell of the present disclosure may be genetically engineered with the polynucleotide or the vector as outlined above.
• the host cells that may be used for purposes of the disclosure include but are not limited to prokaryotic cells such as bacteria (for example, E. coli and B. subtilis), which can be transformed with, for example, recombinant bacteriophage DNA, plasmid DNA, bacterial artificial chromosome, or cosmid DNA expression vectors containing the polynucleotide molecules of the disclosure; simple eukaryotic cells like yeast (for example, Saccharomyces and Pichia), which can be transformed with, for example, recombinant yeast expression vectors containing the polynucleotide molecule of the disclosure.
• prokaryotic cells such as bacteria (for example, E. coli and B. subtilis), which can be transformed with, for example, recombinant bacteriophage DNA, plasmid DNA, bacterial artificial chromosome, or cosmid DNA expression vectors containing the polynucleotide molecules of the disclosure
• simple eukaryotic cells like yeast (for example, Saccharo
• the polynucleotide can integrate, for example, into the chromosome or the mitochondrial DNA or can be maintained extrachromosomally like, for example, episomally or can be only transiently comprised in the cells.
• the term "cell" as used herein in particular with reference to genetic engineering and introducing one or more genes or an assembled cluster of genes into a cell, or a production cell is understood to refer to any prokaryotic or eukaryotic cell.
• Prokaryotic and eukaryotic host cells are both contemplated for use according to the disclosure, including bacterial host cells like E. coli or Bacillus sp, yeast host cells, such as 5. cerevisiae, nsect host cells, such as Spodoptora frugiperda or human host cells, such as HeLa and
• cerevisiae There are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant S. cerevisiae microorganisms. Culturing of cells is performed, in a conventional manner.
• the culture medium contains a carbon source, at least one nitrogen source and inorganic salts, and vitamins are added to it.
• the constituents of this medium can be the ones which are conventionally used for culturing the species of microorganism in question.
• Carbon sources of use in the instant method include any molecule that can be
• suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, glycerol, glucose, cellulose, starch, cellobiose or other glucose containing polymer.
• I n em bodiments employing yeast as a 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 throughout the cultivation 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 a source of carbon only during the fed-batch phase.
• a recombinant host cell microorganism for use in the methods of the present disclosure may be determined by simple test procedures using well known methods.
• the microorganism to be tested may be propagated in a rich medium (e.g., LB-medium, Bacto-tryptone yeast extract medium, nutrient medium and the like) at a pH, temperature and under aeration conditions commonly used for propagation of the microorganism.
• a rich medium e.g., LB-medium, Bacto-tryptone yeast extract medium, nutrient medium and the like
• the products are typically produced by a production host cell line on the large scale by suitable expression systems and fermentations, e.g. by microbial production in cell culture.
• a defined minimal medium such as M9A is used for cell cultivation.
• the components of M9A medium comprise: 14 g/L KH 2 P0 4 , 16 g/L K 2 HP0 4 , 1 g/L Na 3 Citrate.2H 2 0, 7.5 g/L (NH 4 ) 2 S0 4 , 0.25 g/L MgS0 4 .7H 2 0, 0.015 g/L CaCI 2 .2H 2 0, 5 g/L of glucose and 1.25 g/L yeast extract).
• nutrient rich medium such as LB (Luria- Bertani) was used.
• the components of LB comprise: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCI).
• the recombinant microorganism may be grown in a batch, fed batch or continuous process or combinations thereof.
• the recombinant microorganism is grown in a fermentor at a defined temperature in the presence of a suitable nutrient source, e.g., a carbon source, for a desired period of time to bioconvert homofarnesol to (-)-Ambrox in a desired amount.
• a suitable nutrient source e.g., a carbon source
• the recombinant host cells may be cultivated in any suitable manner, for example by batch cultivation or fed-batch cultivation.
• batch cultivation is a cultivation method in which culture medium is neither added nor withdrawn during the cultivation.
• fed-batch means a cultivation method in which culture medium is added during the cultivation but no culture medium is withdrawn.
• One embodiment of the present disclosure provides a method of producing (-)-Ambrox in a cellular system comprising producing wild type SHC or variants thereof under suitable conditions in a cellular system, feeding homofarnesol to the cellular system, converting the homofarnesol to (-)-Ambrox using the SHC or variants produced using the cellular system, collecting (-)-Ambrox from cellular system and isolating the (-)-Ambrox from the system.
• Expression of other nucleotide sequences may serve to enhance the method.
• the bioconversion method can include the additional expression of other nucleotide sequences in the cellular system. The expression of other nucleotide sequences may enhance the bioconversion pathway for making (-)-Ambrox.
• a further embodiment of the present disclosure is a bioconversion method of making (-)-Ambrox comprising growing host cells comprising wild type SHC or variant genes, producing wild type SHC or variant enzymes in the host cells, feeding homofarnesol (e.g. EEH) to the host cells, incubating the host cells under conditions of pH, temperature and solubilizing agent suitable to promote the conversion of homofarnesol to Ambrox and collecting (-)-Ambrox.
• homofarnesol e.g. EEH
• the production of the wild type SHC or variant enzymes in the host cells provides a method of making (-)-Ambrox when homofarnesol is added to the host cells under suitable reaction conditions. Achieved conversion may be enhanced by adding more biocatalyst and SDS to the reaction mixture.
• the recombinant host cell microorganism may be cultured in a number of ways in order to provide cells in suitable amounts expressing the wild type SHC or variant enzymes for the subsequent bioconversion step. Since the microorganisms applicable for the
• bioconversion step vary broadly (e.g. yeasts, bacteria and fungi), culturing conditions are, of course, adjusted to the specific requirements of each species and these conditions are well known and documented. Any of the art known methods for growing cells of recombinant host cell microorganisms may be used to produce the cells utilizable in the subsequent bioconversion step of the present disclosure. Typically the cells are grown to a particular density (measurably as optical density (OD)) to produce a sufficient biomass for the bioconversion reaction.
• OD optical density
• the cultivation conditions chosen influence not only the amount of cells obtained (the biomass) but the quality of the cultivation conditions also influences how the biomass becomes a biocatalyst.
• biocatalyst which is suitable for use in a bioconversion reaction.
• the biocatalyst is a recombinant whole cell producing wild type SHC or variant enzymes or it may be in suspension or an immobilized format.
• the biocatalyst is produced in sufficient amounts (to create a sufficient biomass), harvested and washed (and optionally stored (e.g. frozen or lyophilized)) before the bioconversion step.
• the cells are produced in sufficient amounts (to create a sufficient biocatalyst) and the reaction conditions are then 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 provided that the reaction conditions are adjusted to facilitate the bioconversion reaction.
• bioconversion methods of the present disclosure are carried out under conditions of time, temperature, pH and solubilizing agent to provide for conversion of the
• 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 variant enzymes and in the range of from about pH 5.0 to about pH 7.0 for the wild type SHC enzyme and can be maintained by the addition of buffers to the reaction mixture.
• An exemplary buffer for this purpose is a citric acid buffer.
• the preferred temperature is between from about 15°C and about 45°C, preferably about 20°C and about 40°C although it can be higher, up to 55°C for thermophilic organisms especially if the wild type enzyme from a thermophilic microorganism is used. The temperature can be kept constant or can be altered during the bioconversion process.
• a solubilizing agent e.g. a surfactant, detergent, solubility enhancer, water miscible organic solvent and the like
• surfactants include but are not limited to Triton X-100, Tween 80, taurodeoxycholate, Sodium taurodeoxycholate, Sodium dodecyl sulfate (SDS), and/or sodium lauryl sulfate (SLS).
• the Applicant has selected and identified SDS as a particularly useful solubilizing agent from a long list of other less useful solubilizing agents.
• the Applicant identified SDS as a remarkably better solubilizing agent than e.g. Triton X-100 in terms of reaction velocity and yield for the homofarnesol to (-)-Ambrox bioconversion reaction.
• the use of SDS with recombinant microbial host cells may be advantageous as the SDS may interact advantageously with the host cell membrane in order to make the SHC enzyme (which is a membrane bound enzyme) more accessible to the homofarnesol substrate.
• the inclusion of SDS at a suitable level in the reaction mixture may 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 the disruption (e.g.
• the concentration of the solubilising agent (e.g. SDS) used in the bioconversion reaction is influenced by the biomass amount and the substrate (EEH) concentration. That is, there is a degree of interdependency between the solubilising agent (e.g. SDS) concentration, the biomass amount and the substrate (EEH) concentration.
• concentration of homofarnesol substrate increases, sufficient amounts of biocatalyst and solubilising agent (e.g. SDS) are required for an efficient bioconversion reaction to take place. If, for example, the solubilising agent (e.g. SDS) concentration is too low, a suboptimal homofarnesol conversion may be observed.
• the solubilising agent e.g. SDS
• concentration is too high, then there may be a risk that the biocatalyst is affected through either the disruption of the intact microbial cell and/or an denaturation/inactivation of the SHC/HAC enzyme.
• the selection of a suitable concentration of SDS in the context of the biomass amount and substrate (EEH) concentration is within the knowledge of the Skilled Person.
• a predictive model is available to the Skilled Person to determine the suitable SDS, substrate (EEH) and biomass concentrations.
• the temperature of the bioconversion reaction for a wild type SHC enzyme is from about 45-60°C, preferably 55°C.
• the pH range of the bioconversion reaction for a wild type SHC enzyme is from about 5.0 to 7.0, more preferably from about 5.6 to about 6.2, even more preferably about 6.0.
• the temperature of the bioconversion reaction for a SHC variant enzyme is about 34°C to about 50°C, preferably about 35°C.
• the pH of the bioconversion reaction for a SHC variant enzyme is about 4.8-6.4, preferably about 5.2-6.0.
• the solubilising agent used in the bioconversion reaction is SDS.
• the [SDS]/[cells] ratio is in the range of about, 10:1-20:1, preferably about 15:1-18:1, preferably about 16:1 when the ratio of biocatalyst to EEH homofarnesol is about 2:1
• the SDS concentration in the bioconversion reaction for a SHC variant enzyme is in the range of about 1-2%, preferably in the range of about 1.4-1.7%, even more preferably about 1.5% when the homofarnesol concentration is about 125g/l EEH and the biocatalyst concentration is 250g/l (corresponding to an OD of about 175 (650nm)).
• the ratio of biocatalyst to EEH homofarnesol substrate is in the range of about 0.5:1-2:1, in some embodiments 2:1, preferably about 1:1 or 0.5:1.
• (-)-Ambrox is produced using a biocatalyst to which the
• homofarnesol substrate is added. It is possible to add the substrate by feeding using known means (e.g. peristaltic pump, infusion syringe and the like).
• Homofarnesol is an oil soluble compound and is provided in an oil format. Given that the biocatalyst is present in an aqueous phase, the bioconversion reaction may be regarded as a two phase system when homofarnesol is added to the bioconversion reaction mixture. This is the case even when a solubilizing agent (e.g. SDS) is present.
• a solubilizing agent e.g. SDS
• the bioconversion process according to the present invention produces a reaction mixture containing the desired (-)-Ambrox, and also a number of by-products. More particularly, the reaction mixture contains, in addition to (-)-Ambrox a complex mixture of by-products, including a novel constitutional isomer of (-)-Ambrox according to the formula (II), as well as known stereo isomers of (-)-Ambrox according to the formulae (III) and (IV)
• the compound of formula (II) is formed by the cyclization of the 7E,3Z-geometric isomer of homofarnesol. It has been described as practically odourless, with a detection threshold of > 500 ng/l.
• olfactory acceptable amount as used herein in relation to the compound of formula (II), or any of the other by-products (III) or (IV), or indeed, any material that may be present as an impurity in (-)-Ambrox formed in accordance with a method of the present invention, is understood to mean that the compound or material is present in a mixture with (-)-Ambrox in an amount below its odour detection threshold, or in an amount at which it will not contribute its olfactory characteristics in a way that will affect the olfactory character of (-)-Ambrox.
• (-)-Ambrox containing an olfactory acceptable amount of any such compound or material would be identifiable to a skilled perfumer as possessing the odour character of commercial grades of (-)-Ambrox, such as AMBROFIXTM obtained by a synthetic procedure ex-sclareol, and available from Givaudan.
• the reaction mixture contains no, or substantially no, unreacted homofarnesol.
• homofarnesol was a powerful solvent for (-)-Ambrox as well as for the aforementioned by-products of the bioconversion process.
• (-)-Ambrox and the by-products remain dissolved together in a crude, intractable mixture, from which it is difficult, protracted and costly to separate and ultimately isolate (-)-Ambrox in olfactively pure form.
• Reducing the level of unreacted homofarnesol in admixture with (-)-Ambrox and the compounds (II), (III) and (IV) was found to considerably facilitate downstream processing and isolation/purification of (-)-Ambrox.
• Downstream processing is a critical operation in the manufacture of useful compounds formed by bioconversion processes. As part of the synthesis of a compound, it can affect the compound's physical properties. In the case of the preparation of perfume ingredients by biotech methods, it is desirable that a target compound can be separated from a reaction mixture in olfactively pure form in order that the desired odour characteristics of the target compound are not distorted by odour contributions of the complex mixture of contaminants and by-products that may be present in the fermentation medium or the biocatalyst.
• the invention provides in another of its aspects a method of isolating and purifying (-)-Ambrox from a reaction mixture, comprising one or more of the compounds (II), (III) and (IV).
• (-)-Ambrox either does not contain any of the compounds (II), (III) or (IV), or if it does contain any of said compounds, then each should be present in an olfactory acceptable amount.
• the reaction mixture obtained from the bioconversion process generally comprises a solid phase containing crude (-)-Ambrox and one or more of the by-products (II), (III) and (IV), as well as cellular material and/or debris thereof; and a liquid phase or liquid phases comprising water and/or any unreacted homofarnesol.
• the solid phase may be separated from the liquid phase or phases by filtration or centrifugation. Furthermore, by selecting a filter with an appropriate pore size, it is also possible to effect separation of the crude (-)-Ambrox, from the cellular material and/or debris. Once the crude (-)-Ambrox is separated from cellular material and/or debris thereof, it may be washed, before being subjected to further work-up procedures to isolate (-)-Ambrox from compounds (II), (III) and (IV). Alternatively, instead of filtration or centrifugation, the reaction mixture can be warmed to a temperature above the melting point of (-)-Ambrox, whereupon (-)-Ambrox forms an oil phase above an aqueous phase containing the cellular material and debris.
• the aqueous and cellular material can be washed with a water-immiscible organic solvent (such as toluene) to remove any residual (-)-Ambrox that may have been entrained in the aqueous phase, and these washings can be combined with the oil phase.
• the oil phase can thereafter be concentrated by evaporation to provide a crude mixture comprising (-)-Ambrox and one or more of the compounds (II), (III) and (IV),which mixture can then subjected to further work-up procedures to isolate and purify (-)-Ambrox.
• reaction mixture instead of warming the reaction mixture to form a (-)-Ambrox- containing oil phase, the reaction mixture can be extracted with a suitable water- immiscible organic solvent (such as toluene) to form an organic phase containing
• a suitable water- immiscible organic solvent such as toluene
• the organic phase can be concentrated by evaporation to provide a crude mixture comprising (-)-Ambrox and one or more of the compounds (II), (III) and (IV), which can be subjected to further workup procedures to isolate and purify (-)-Ambrox.
• the reaction mixture can be steam distilled to remove the distillate from the cellular material and debris.
• the distillate can be collected as a biphasic mixture, before the oil phase of the biphasic mixture comprising a mixture of (-)- Ambrox and one or more of the compounds (II), (III) and (IV) is separated from the aqueous phase, and then subjected to further work-up procedures to isolate and purify (- )-Ambrox.
• said method of isolating and purifying (-)-Ambrox comprises the step of selectively crystallizing (-)-Ambrox from a mixture containing one or more of the compounds (II), (III) or (IV).
• selective crystallizing refers to a process step whereby (-)-Ambrox is caused to crystallize from a solvent, whilst the compounds (II), (III) and (IV) remain dissolved in the crystallizing solvent, to such an extent that isolated crystalline material contains only (-)-Ambrox, or if it contains any of the compounds (II), (III) or (IV), then they are present only in olfactory acceptable amounts.
• Crystallization may be carried out in a suitable organic solvent.
• the choice of solvent is based on considerations, such as solubility differences at room temperature and at high temperatures, or in boiling solvent; and for the need of an abundance of crystals recoverable in cool solvent.
• a compound to be separated is dissolved in a relatively polar solvent and then a relatively less polar solvent can be added to bring the dissolved compound to its solubility limit, whereupon it will start to crystallize.
• 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. Pairs of solvents may also be employed.
• selective crystallization is undertaken by dissolving the mixture containing (-)-Ambrox and one or more of the compounds (II), (III) and (IV) in warm ethanol and allowing (-)-Ambrox to selectively crystallize by slowly adding a non-solvent, such as water, to the cooling ethanolic solution.
• a non-solvent such as water
• (-)-Ambrox in olfactively pure form contains less than 5 % by weight of any of the compounds (II), (III) or (IV).
• (-)-Ambrox in olfactively pure form contains less than 4 %, less than 3 %, less than 2 %, less than 1 %, less than 0.9 %, less than 0.8 %, less than 0.7 %, less than 0.6 %, less than 0.5 %, less than 0.4 %, less than 0.3 %, less than 0.2 %, less than 0.1 %, or less than 0.05 % by weight of each of the compounds (II), (III) or (IV).
• the quality of separation of (-)-Ambrox from the mixture of the compounds (II), (III) and/or (IV) by selective crystallization may be influenced by the composition of the mixture from which it is separated. More particularly, the quality of the separation of (-)-Ambrox from a mixture of compounds (II), (III) and/or (IV) by crystallization was improved when the weight ratio of (-)-Ambrox to the other compounds (II), (III) and (IV) in the mixture was greater than 70:30, more particularly 80:20, more particularly 90:10, still more particularly 95:5, and more particularly still 97:3.
• the quality of the separation of (-)-Ambrox by crystallization may be influenced by the amount of unreacted homofarnesol present in the mixture from which it is separated. More particularly, the quality of separation is improved when the level of unreacted homofarnesol is less than 30 wt % by weight, more particularly less than 20 wt %, more particularly less than 10 % by weight, more particularly still less than 5 wt % and still more particularly less than 3 % by weight, still more particularly less than 2 % by weight, and more particularly still less than 1 % by weight, based on the weight of the mixture from which (-)-Ambrox is crystallized.
• the reagents and reaction conditions employed in the bioconversion process of the present invention are such that the reaction proceeds with 100 % conversion of homofarnesol, or substantially so, thus leaving no unreacted homofarnesol in the reaction mixture.
• unreacted homofarnesol is present, although economically disadvantageous, it can be separated from (-)-Ambrox and other by-products by distillation, for example.
• a method of isolating and purifying (-)-Ambrox from a mixture comprising one or more of the compounds (II), (III) and (IV), which mixture is free, or substantially free, of homofarnesol are provided.
• the isolation and purification of (-)-Ambrox from a mixture comprising one or more of the compounds (II), (III) and (IV), and free or substantially free of homofarnesol is achieved by the selective crystallization of
• (-)-Ambrox obtained according to a method of the present invention is obtained in olfactively pure form.
• Olfactively pure (-)-Ambrox forms another aspect of the present invention.
• (-)-Ambrox in crystalline form forms yet another aspect of the present invention.
• (-)-Ambrox formed in accordance with the method of the present invention may be mixed with one or more additional perfume ingredients to form perfume compositions that find use in perfumery applications, including use in fine perfumery, as well as use in consumer products, such as personal care, fabric care and household care applications. Accordingly, the invention provides in another of its aspects a perfume composition comprising (-)-Ambrox and at least one other perfume ingredient, wherein said perfume composition contains olfactory acceptable amounts of one or more of the compounds (II), (III) or (IV).
• 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 0.22 mm x 0.25 mm x 12m.
• Carrier Gas Helium.
• Injector temperature 230 °C.
• Split 1 50.
• Flow 1.0 ml/min. Transfer line: 250 °C.
• MS-quadrupol 106 °C.
• MS-source 230 °C.
• phase separation After phase separation the water phase is extracted twice with THF (2 x 1 1). This gives 1100 ml of phase B and 1075 of phase C. Whereas phase A gives a 51% conversion of a terminal alkene to a cyclopropane in a subsequent cyclopropanation reaction, phase B gives ⁇ 0.5% cyclopropane and phase C gives no detectable conversion.
• > 99% MNU is extracted after the first phase separation. Usually the water phase is therefore discarded after the first phase separation (from organic phase A) after treatment with cone, aqueous KOH and acetic acid.
• N-Methyl-N-nitroso urea 1.35 M in THF (136 ml, 184 mmol) is added dropwise at 0 °C to a rapidly stirred mixture of E-beta-Farnesene (CAS 18794-84-8) (25 g, 122 mmol) and 5 aqueous KOH (50 ml, 40%) at 0 - 5 °C.
• E-beta-Farnesene CAS 18794-84-8
• 5 aqueous KOH 50 ml, 40%
• Pd(acac)2 7.4 mg, 0.024 mmol, 0.02%
• the remaining MNU solution is added over 4 h at 0 - 5 °C.
• the desired compound could be further isolated by distillative purification.
• the gene encoding Alicyclobaci ⁇ ⁇ 'us acidocaldarius squalene hopene cyclase (AacSHC) (GenBank M73834, Swissprot P33247) was inserted into plasmid pET-28a(+), where it is under the control of an I PTG inducible T7-promotor for protein production in Escherichia coli.
• the plasmid was transformed into E. coli strain BL21(DE3) using a standard heat- shock transformation protocol.
• the minimal medium chosen as default was prepared as follows for 350 ml culture : to 35 ml citric acid/phosphate stock (133g/i KH 2 P0 4 , 40g/l (N H 4 ) 2 HP0 4 , 17g/g citric acid.H 2 0 with pH adjusted to 6.3) was added 307 ml H 2 0 ; the pH adjusted to 6.8 with 32% NaOH as required. After autoclaving 0.850 ml 50% MgS0 4 , 0.035 ml trace elements solution (composition in next section) solution, 0.035 ml Thiamin solution and 7 ml 20% glucose were added.
• Protein production was then induced by the addition of I PTG to a concentration of 300 ⁇ followed by incubation for a further 5-6 hours with constant shaking. The resulting biomass was finally collected by centrifugation, washed with 50 mM Tris-HCI buffer pH 7.5. The cells were stored as pellets at 4°C or -20°C until further use. I n general 2.5 to 4 grams of cells (wet weight) were obtained from 1 litre of culture, independently of the medium used.
• the fermentation was prepared and run in 750 ml InforsHT reactors. To the fermentation vessel was added 168 ml deionized water. The reaction vessel was equipped with all required probes (p0 2 , pH, sampling, antifoam), C + N feed and sodium hydroxide bottles and autoclaved. After autoclaving, the following ingredients are added to the reactor:-
• the fermenter was inoculated from a seed culture to an OD 6 50nm of 0.4-0.5.
• This seed culture was grown in LB medium (+ Kanamycin) at 37°C, 220 rpm for 8 h.
• the fermentation was run first in batch mode for 11.5h, where after was started the C+ N feed with a feed solution (sterilized glucose solution (143 ml H 2 0+ 35g glucose) to which had been added after sterilization : 17.5 ml (NH 4 ) 2 S0 4 solution, 1.8 ml MgS0 4 solution, 0.018 ml trace elements solution, 0.360 ml Thiamine solution, 0.180 ml kanamycin stock.
• the feed was run at a constant flow rate of approx. 4.2 ml/h.
• Glucose and NH 4 + measurements were done externally to evaluate availability of the C- and N-sources in the culture. Usually glucose levels stay very low.
• Induction of SHC production lasted for 16 h 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.
• Table 1 shows for two examples the culture volume, optical density a amount of cells both at induction start and induction end as well as the amount of biomass collected (wet weight).
• variant F601Y SHC amino acid sequence (SEQ ID No. 2) - variant with respect to SEQ ID No. 1 MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALY PGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLN IYEFGSWARATVVALS IVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIR ALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQAS ISPVWDTGLAVLALRAA GLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVWWALNTLRLPDER
• Bioconversion was undertaken using the following reaction conditions: The reaction (150.1 g total volume) run in 0.1 M citric acid/sodium citrate buffer pH 5.4 in an InforsHT 750 ml fermenter contained 146g/l total homofarnesol using a homofarnesol substrate, which was a mixture of 7E,3E:7E,3Z of 86:14, 250g/l cells (formed in accordance with the method of Example 2, fermentation) and 1.55% SDS. The reaction was run at 35°C with constant stirring (900 rpm), pH control was done using 10 to 40% citric acid in water.
• the reaction mixture was subjected to isolation and purification steps as set forth in Example 4, below.
• Table 1 shows the GC analytics results for the crystallized product.
• the data show a strong enrichment of (-)-Ambrox, with practically no by-products (a), (b) or (c) being found in the crystallized sample.
• a”, “b” and “c” refer to compound (I I), compound (IV) and compound (I II) respectively.
• EZH” and “EEH” refer to 7E,3Z-homofarnesol and 7E,3E- homofarnesol respectively.
• Biotransformation of ⁇ , ⁇ -homofarnesol results in the macrocyclic ether compound (II) and epi-Ambrox compound (I II).
• a crude mixture of (-)-Ambrox comprises the desired (-)-Ambrox, compound (II), (III) and (IV) present in an amount of 87.1 wt %, 2.8 wt %, 2.5 wt % and 7.6 wt % respectively.
• the crystallised material has the same components as the crude mixture, but they are present in an amount of 99.1 wt %, 0.1 wt %, 0.1 wt % and 0.7 wt % respectively.
• Compound (IV) weak, IsoE, woody, GC-detection threshold 5-10ng.
• the tabulated data also shows the GC analytics results for products obtained after the steam extraction/distillation step ("crude”) and the crystallized product ((-)-Ambrox).
• the references in the Table to "EZH” and “EEH” refer to (3Z,7E)-homofarnesol and 7E,3E- homofarnesol respectively.
• Table shows the GC analytics results for the crystallized product.
• the (-)-Ambrox produced using the bioconversion reaction may be extracted using solvent from the whole reaction mixture (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 which are known to those skilled in the art.

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