Formation of novel aroma compounds by ionylideneethane synthase.

Abstract

The present invention relates to the use of α-ionylideneethane as an aroma compound and to the use of α-ionylideneethane synthase in the production of one or more aroma compounds. The method of the present invention for preparing one or more aroma compounds comprises the steps of: a) providing farnesyl diphosphate and α-ionylideneethane synthase as defined herein under suitable conditions for the α-ionylideneethane synthase to produce α-ionylideneethane, b) converting the farnesyl diphosphate in vitro or in a host cell, c) optionally converting the α-ionylideneethane to one or more further aroma compounds, d) isolating the α-ionylideneethane and optionally the one or more further aroma compounds, and e) optionally purifying the α-ionylideneethane and optionally the one or more further aroma compounds. The present invention also relates to a method for perfuming a product, in particular a method for imparting and / or enhancing an odor or flavor, in which at least one α-ionylideneethane synthase is used. Additionally, the present invention provides a fragrance compound or composition and / or a fragrance composition and / or a perfumed or scented product, which i) comprises at least one α-ionylideneethane. Furthermore, the present invention encompasses a perfumed or scented product comprising at least one α-ionylideneethane. The present invention further relates to a method for producing α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), comprising, in the following order: a) contacting farnesyl diphosphate with at least one α-ionylideneethane synthase under conditions suitable for producing at least one α-ionylideneethane; b) producing at least one α-ionylideneethane; c) exposing the at least one α-ionylideneethane produced in step b) to conditions suitable for oxidative cleavage of the α-ionylideneethane to produce α-ionone; and d) optionally isolating the α-ionone produced in step c).The present invention also relates to a host cell for producing α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), the host cell comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylideneethane synthase, and capable of oxidative cleavage of α-ionylideneethane to produce α-ionone. Finally, the present invention relates to the use of a host cell comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylideneethane synthase for (i) producing α-ionylideneethane; (ii) producing α-ionone; (iii) producing vitamin A; (iv) converting α-ionylideneethane to α-ionone; (v) converting α-ionylideneethane to vitamin A; (vi) for heterologous reconstitution of terpenes or terpenoids; (vii) producing industrial products; (viii) for fermentation production systems for producing sesquiterpenes.

Description

[Technical field]

[0001] The present invention relates to a method for preparing one or more aroma compounds using α-ionylideneethane synthase, as well as to the use of such enzyme for the preparation of aroma compounds and aroma compositions and fragrances. Furthermore, the present invention relates to the production of α-ionone using this enzyme, and also to the novel use of α-ionylideneethane (E,Z α-ionylideneethane = 1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene) as an aroma compound.

[0002] In particular, the present invention relates to the use of α-ionylideneethane as a fragrance compound and to the use of α-ionylideneethane synthase in the production of one or more fragrance compounds. The method of the invention for preparing one or more aroma compounds comprises the steps of: a) providing farnesyl diphosphate and an α-ionylideneethane synthase as defined herein, preferably α-ionylideneethane as defined in claims 3, 4, 5, under suitable conditions for the α-ionylideneethane synthase to produce α-ionylideneethane; b) converting the farnesyl diphosphate in vitro or in a host cell; c) optionally converting the α-ionylideneethane to one or more further aroma compounds; d) isolating the α-ionylideneethane and optionally the one or more further aroma compounds; and e) optionally purifying the α-ionylideneethane and optionally the one or more further aroma compounds. The present invention also relates to a method for perfuming a product, in particular a method for imparting and / or enhancing an odor or flavor, in which at least one α-ionylidene ethane is used.The present invention also provides a fragrance compound or composition and / or a fragrance composition and / or a perfumed or scented product, comprising i) at least one α-ionylidene ethane as defined in claim 1 or 2; ii) optionally at least one further fragrance compound different from i) and iii) optionally at least one diluent.Furthermore, the present invention encompasses perfumed or scented products comprising at least one α-ionylidene ethane as defined herein.The present invention further relates to a method for producing α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), comprising, in the following order: a) contacting farnesyl diphosphate with at least one α-ionylideneethane synthase under conditions suitable for producing at least one α-ionylideneethane; b) producing at least one α-ionylideneethane; c) exposing the at least one α-ionylideneethane produced in step b) to conditions suitable for oxidative cleavage of the α-ionylideneethane to produce α-ionone; and d) optionally isolating the α-ionone produced in step c). The present invention also relates to a host cell for producing α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), the host cell comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylideneethane synthase, the host cell being preferably a bacterial cell, a yeast cell, a fungal cell, an algae cell, a blue-green algae cell, a non-human animal cell, a non-human mammalian cell, or a plant cell, and the host cell being suitable for the oxidative cleavage of α-ionylideneethane to produce α-ionone.Finally, the present invention relates to the use of a host cell comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylideneethane synthase as defined herein, (i) for producing α-ionylideneethane, preferably 2Z,4E-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene), preferably as an aroma component or aroma compound that is a precursor of an aroma substance, or as a precursor of vitamin A; (ii) α-ionone, preferably The present invention relates to the use of a fermentation system for producing R-α-ionone, (iii) for producing vitamin A, (iv) for converting α-ionylideneethane to α-ionone, (v) for converting α-ionylideneethane to vitamin A, (vi) for heterologous reconstitution of terpenes or terpenoids, (vii) for producing an industrial product, preferably a fragrance composition, a flavor or fragrance, a pharmaceutical composition, an agricultural composition, an animal feed, a human nutrition product, a cosmetic, a colorant (carotenoid), or a radical scavenger, and / or (viii) for producing sesquiterpenes. Preferably, the host cell is transgenic for a nucleic acid encoding an α-ionylideneethane synthase and comprises an active form of the α-ionylideneethane synthase as defined herein.

[0003] The present invention also relates to a method for preparing α-ionone (E-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), comprising the step of converting α-ionylideneethane to α-ionone in the presence of farnesyl diphosphate and α-ionylideneethane synthase in vitro or in a host cell. Additionally, the present invention relates to a method for preparing an aroma composition, flavor, fragrance or perfume, comprising: a) producing α-ionylideneethane according to the method for preparing α-ionylideneethane of the present invention; and / or producing α-ionone according to the method for preparing α-ionone of the present invention; b) isolating and optionally purifying the α-ionylideneethane and / or α-ionone of step a); and c) adding the isolated and optionally purified α-ionylideneethane and / or α-ionone of step b) as an ingredient to an aroma chemical composition of the present invention as described herein below, such as an aroma composition, flavor, fragrance or perfume conveying any one of the following olfactory notes: floral-violet or woody-orris (iris) root in the case of α-ionylideneethane, floral-violet in the case of α-ionone. The present invention further provides a host cell for preparing α-ionylideneethane and / or α-ionone, the host cell comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylideneethane synthase. The present invention also contemplates compositions comprising (i) the host cell of the present invention, α-ionylideneethane and / or α-ionone, or (ii) the α-ionylideneethane synthase, α-ionylideneethane and / or α-ionone as defined herein, as well as kits comprising the host cell of the present invention, or the composition of the present invention.Finally, the present invention relates to a) the use of a host cell of the invention: (i) for producing α-ionylideneethane, preferably 2Z,4E-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene), preferably as an aroma component, which is a precursor of an aroma substance, or as a precursor of vitamin A; (ii) α-ionone, preferably The present invention relates to the use of the host cells of the present invention for the production of R-α-ionone;(iii) for the production of vitamin A;(iv) for the conversion of α-ionylideneethane to α-ionone;(v) for the conversion of α-ionylideneethane to vitamin A;(vi) for the heterologous reconstruction of terpenes or terpenoids;(vii) for the production of industrial products, preferably aroma compositions, flavors or fragrances, pharmaceutical compositions, agricultural compositions, animal feed, human nutritional products, cosmetics, colorants (carotenoids), or radical scavengers;(viii) for fermentation production systems, preferably for the production of sesquiterpenes in the host cells of the present invention. The present invention also relates to the use of α-ionylideneethane as an aroma chemical or compound. [Background technology]

[0004] During the past decades, intensive scientific research has focused on terpenes, the most abundant secondary metabolites in all living organisms. There are more than 55,000 terpenoid substances widely distributed among different families of natural products found in all kingdoms of life.

[0005] Many terpenoids are generally secondary metabolites, since they are not primarily essential for the growth, development or reproduction of any organism. However, this classification does not extend to the broader additional effects of these secondary metabolites, which maintain ecosystem function. These substances may play important roles and provide plants with an evolutionary advantage in terms of their distinct chemical sensitivity properties, such as smell. Thus, among other things, they may exert insecticidal effects that protect plants and crops from parasites and pathogens, or act as pollinator attractants in the reproductive process.

[0006] Many terpenoids are well known for their economic importance, being widely used as basic structural moieties in the production of drugs, flavors, fragrances, pigments and disinfectants. For example, α-ionone is used as a fragrance in perfumes, cosmetics and personal care products, as well as in household cleaning agents and detergents. The monoterpene alcohol linalool, a component of the main essential oil of rosewood, Aniba rosaeodora, is the most frequently used component, especially in the production of perfumes. Furthermore, the sesquiterpene lactone, artemisinin, is extracted from the shrub Artemisia annua and used in the first-line treatment of malaria. Taxol, a tricyclic diterpene isolated from the bark of the Pacific yew, Taxus brevifolia, and its structural analogues are used as anticancer drugs.

[0007] Terpenes are mainly synthesized in plants through a common biosynthetic pathway. Regardless of their diverse structures and functions, all terpenes are constructed from isoprene units (five carbon atoms) according to the isoprene rule. According to the number of isoprene units in their structure that are linked through head-tail addition, terpenes are classified according to the number of their carbon atoms or sesquiterpenoid moieties, respectively: monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) or polyterpenes with up to 30,000 linked isoprene units. Similar to terpenes, terpenoids are also classified according to the number of isoprene units, which are organized as in monoterpenoids (C10) or sesquiterpenoids (C15) and further named with the suffix "-oids" (like ~).

[0008] Isopentyl diphosphate (IPP) and its electrophilic isomer, dimethylallyl diphosphate (DMAPP), are universal precursors in the biosynthesis of terpenes. Starting from these two building blocks, linear prenyl diphosphates are synthesized by a group of enzymes belonging to the prenyltransferases. IPP and DMAPP are condensed by the catalytic effect of the prenyltransferase geranyl diphosphate synthase to give C10 geranyl diphosphate (GPP), an intermediate that can be converted into cyclic or linear end products that represent the group of monoterpenes.

[0009] Similarly, sesquiterpenes are produced via the addition of a third isoprene unit to GPP to form C15 farnesyl diphosphate, also known as farnesyl pyrophosphate (FPP), the biosynthetic precursor of common sesquiterpenes. Further polymerization of IPP and DMAPP produces longer prenyl diphosphates that form different classes of terpenes named according to the number of isoprene units they contain.

[0010] IPP and DMAPP biosynthesis are accomplished via two independent pathways: the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although the MVA pathway was considered a universal pathway in the synthesis of terpenes, it has been found to be less prominent in plant secondary metabolites than the MEP pathway during the past decade. MVA is predominant in the cytoplasm and mitochondria of most eukaryotes, archaea, some eubacteria, and plants, generating precursors to multiple analogs such as sesquiterpenes (C15) and triterpenes (C30) in the cytoplasm. Meanwhile, the MEP pathway is the main pathway in the chloroplasts of higher plants, cyanobacteria, eubacteria, and algae. Due to its location of biosynthesis in plastids, MEP leads to monoterpenes (C10), diterpenes (C20), and carotenoids (C40).

[0011] The mevalonate pathway (MVA) pathway, also known as the mevalonate pathway, isoprenoid pathway, or 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase pathway, was discovered in yeast and animals in the 1950s. The MVA pathway begins with the Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA through the catalysis of the enzyme acetoacetyl-CoA transferase. Acetoacetyl-CoA is converted to HMG-CoA by HMG synthase through an aldol reaction with another acetyl-CoA. In the next two reduction steps, two nicotinamide adenine dinucleotide phosphate molecules are required to convert HMG-CoA to mevalonate (MVA) using HMG-CoA reductase. Subsequent phosphorylation of MVA gives mevalonate 5-diphosphate (MVAPP) through two reactions catalyzed by mevalonate kinase (MK) and phosphomevalonate kinase (PMK), respectively. Finally, IPP is generated from the decarboxylation of MVAPP by an ATP-coupled decarboxylation reaction catalyzed by mevalonate 5-bisphosphate decarboxylase (MVD).IPP:DMAPP isomerase (IDI) then catalyzes the interconversion between IPP and DMAPP.

[0012] The methylerythritol phosphate pathway (MEP) or MVA-independent pathway was discovered in bacteria and green algae and in the chloroplasts of higher plants in the late 1990s and early 2000s. This pathway starts with two different precursors, pyruvate and D-glyceraldehyde 3-phosphate (G3P). Both molecules undergo condensation catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS) using thiamine pyrophosphate as a cofactor to yield 1-deoxy-D-xylulose 5-phosphate (DXP). In the next step, DXP ​​is isomerized to MEP by DXP reductoisomerase (DXR). 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) synthase consequently catalyzes the coupling between MEP and cytidine triphosphate (CTP) to produce methylerythritol cytidyl diphosphate (CDP-ME). In an ATP-dependent reaction, CDP-ME kinase phosphorylates CDP-ME to 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP). The latter then undergoes cyclization to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) in a reaction catalyzed by MEcPP synthase, releasing cytidine monophosphate (CMP). This pathway is completed by the ring-opening of cyclic pyrophosphate and reductive dehydration of MEcPP to 4-hydroxy-3-methylbut-2-enyl-diphosphate (HMBPP) catalyzed by HMBPP synthase. HMBPP is ultimately converted to a mixture of IPP and DMAPP by HMBPP reductase.

[0013] Abscisic acid (ABA) is an isoprenoid plant hormone that is synthesized in the plastid MEP pathway (Abscisic Acid: Metabolism, transport and signalling. Da-Peng Zhan-Editor. Springer 2014). The sesquiterpenoid abscisic acid is primarily known for controlling developmental processes and abiotic stress responses in higher plants. Recent studies have shown that abscisic acid also exhibits various pharmacological activities. However, plants are not the only organisms that produce and utilize abscisic acid. For example, abscisic acid production has been confirmed in plant pathogenic fungi such as Botrytis cinerea, blue-green algae, the animal parasite Toxoplasma gondii, and mammals, including humans. Unlike structurally related sesquiterpenes formed from the mevalonic acid-derived precursor farnesyl diphosphate, in plants the C15 backbone of abscisic acid is formed after cleavage of the C40 carotenoid of MEP.

[0014] Abscisic acid is produced by plants through the carotenoid pathway, but a few plant pathogenic fungi can also produce this sesquiterpene, but they use a unique pathway that begins with the cyclization of farnesyl diphosphate to 2Z,4E-α-ionylideneethane, which then undergoes several oxidation steps by oxidoreductases.

[0015] α α-Ionylideneethane and α-ionylideneethane synthase are known from studies on the production of the plant hormone abscisic acid, but have not been linked to the use as a fragrance compound or to the production of fragrance compounds or compositions, respectively.

[0016] In contrast to α-ionylideneethane, α-ionone is a known aroma compound. As stated, α-ionone is a highly valuable aroma chemical that conveys floral notes (Panten, J. and Surburg, H., Ullmann's Encyclopedia of Industrial Chemistry, 2000). Although technical synthesis of α-ionone is carried out, for example, by acid-catalyzed cyclization of pseudo-ionones obtained from the condensation of citral and acetone, natural α-ionone is generally believed to be biosynthesized by oxidative degradation of carotenoids in vivo. Therefore, there is a need for further means and methods for the synthesis of natural α-ionone. Prior to the present invention, there was no known precedent for the oxidative degradation of α-ionylideneethane to α-ionone.

[0017] In recent years, many terpenes, including monoterpenes, sesquiterpenes and their alcohols, have been produced in microbial systems to provide alternatives to terpenes from plant sources.Most commercially available terpenes are produced by chemical synthesis or by extraction from plant materials.Plant sources often suffer from low concentrations, harvesting dependency, the presence of pesticides and / or extinction risk of plant species.Biotechnological production of terpenes can provide a sustainable and economically viable alternative to plant sources.

[0018] Terpenes consist of over 30,000 compounds and are primarily produced by plants. Considering this, there is a need for additional production systems for terpenes as alternatives to plant sources.

[0019] Recently, Otto et al. (Microb Cell Fact (2019) 18:205) established a multi-step metabolic pathway for the production of abscisic acid in the yeast S. cerevisiae. In another study, a biosynthetic pathway to abscisic acid via ionylideneethane was described in the fungus Botrytis cinerea in a study by Inomata and coworkers (Phytochemistry. 2004 Oct;65(19):2667-78.doi:10.1016 / j.phytochem.2004.08.025.). However, this pathway to abscisic acid has not been used to produce ionylideneethane and α-ionone for industrial applications. Ionylideneethane has not been considered as an aroma compound so far, and the use of the abscisic acid synthesis pathway for the production of aroma compounds has not been reported by these authors. Furthermore, it was not known that ionylideneethane may also be a useful precursor for vitamin A production. Summary of the Invention [Problem to be solved by the invention]

[0020] The object of the present invention is to provide novel aroma chemicals. These should have pleasant organoleptic properties. A further object of the present invention is to provide substances that can be used as aroma chemicals in ready-to-use compositions. In particular, there is a need for strong odorants with a pleasant odor. Furthermore, they should be capable of creating new advantageous sensory profiles by combining with other aroma chemicals. In addition, these aroma chemicals should be obtainable from readily available starting materials, allowing their rapid and economical production. [Means for solving the problem]

[0021] The technical problem underlying the present invention may therefore be seen as the provision of means and methods meeting the above-mentioned needs. The technical problem is solved by the embodiments characterized in the claims, herein below and in the examples.

[0022] The present invention relates to a method for preparing one or more fragrance compounds, comprising: a) providing farnesyl diphosphate and α-ionylideneethane synthase under conditions suitable for the α-ionylideneethane synthase to produce α-ionylideneethane; b) converting farnesyl diphosphate to α-ionylideneethane in vitro or in a host cell; c) optionally converting the α-ionylideneethane to one or more further aroma compounds; d) isolating the α-ionylideneethane and optionally one or more further aroma compounds; e) optionally purifying the α-ionylideneethane and optionally one or more further aroma compounds; The present invention relates to a method comprising the steps of:

[0023] One aspect of the invention relates to a method for preparing one or more aroma compounds, the method comprising the steps of providing farnesyl diphosphate and α-ionylideneethane synthase, converting the farnesyl diphosphate to α-ionylideneethane in the presence of the farnesyl diphosphate and α-ionylideneethane synthase in vitro or in a host cell, optionally converting all or a portion of the α-ionylideneethane to one or more further aroma compounds, isolating the α-ionylideneethane and optionally the one or more further aroma compounds, and optionally purifying the α-ionylideneethane and optionally the one or more further aroma compounds. Preferably, at least one aroma compound is α-ionylideneethane, more preferably the α-ionylideneethane is 2Z,4E-α-ionylideneethane (E,Z α-ionylideneethane=1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene). Preferably, at least one of the further aroma compounds is an α-ionone, preferably R-α-ionone, more preferably the process of the invention is a process for the preparation of α-ionylideneethane and α-ionone, and optionally one or more aroma compounds other than α-ionylideneethane and α-ionone.

[0024] In a preferred embodiment of the method of the present invention for preparing one or more aroma compounds, the method further comprises the steps of exposing all or a portion of the at least one α-ionylideneethane produced to conditions suitable for the oxidative cleavage of the α-ionylideneethane to produce at least one α-ionone, preferably R-α-ionone, and preferably converting all or a portion of the at least one α-ionylideneethane into an α-ionone, preferably R-α-ionone, by chemical or enzymatic oxidative cleavage of the α-ionylideneethane.

[0025] The method of the present invention for preparing one or more aroma compounds, such as the aroma compound α-ionylideneethane, can be carried out in vitro or in a host cell. It includes providing farnesyl diphosphate and one or more α-ionylideneethane synthases as defined herein. The farnesyl diphosphate is provided as a substrate for one or more α-ionylideneethane synthases. The method further includes converting the farnesyl diphosphate to α-ionylideneethane by the one or more α-ionylideneethane synthases. The α-ionylideneethane thus produced is isolated and, optionally, purified.

[0026] Thanks to the inventors, it has been possible to identify ionylideneethane as a fragrance compound. This discovery was unexpected, since ionylideneethane was not previously considered a fragrance compound. Surprisingly, it has been found by the inventors that α-ionylideneethane can be used to prepare one or more fragrance compounds that convey floral-violet and / or woody-orris / iris root notes to perfumes, fragrances or fragrances.

[0027] In addition, the inventors have found that ionylideneethane may also be a useful precursor for the production of vitamin A, which has not yet been reported in the prior art. Moreover, the inventors have found that this portion of the abscisic acid synthesis pathway can advantageously be used for industrial scale production of aroma compounds for the aroma chemical compositions of the present invention, which is also a novel and surprising discovery.

[0028] α-Ionylideneethane is a sesquiterpenoid.

[0029] Details of the compound 2E,4E-α-ionylideneethane can be found, for example, at https: / / pubchem.ncbi.nlm.nih.gov / compound / 101359914. 2Z,4E-α-ionylideneethane is described, for example, at https: / / pubchem.ncbi.nlm.nih.gov / compound / 101760128 and https: / / www.biocyc.org / compound?orgid=META&id=CPD-20099.

[0030] As shown in the following examples, α-ionylideneethane synthase (IES) from the plant pathogenic fungus Botrytis cinerea, having the amino acid sequence shown in SEQ ID NO: 1, has been successfully cloned and expressed by the present inventors in Rhodobacter sphaeroides to produce 2Z,4E-α-ionylideneethane as a novel aroma compound, a precursor of aroma compounds, and a potential precursor of vitamin A. After scaling up the production of 2Z,4E-α-ionylideneethane from shake flasks to fermenters in the DASGIP laboratory, a novel compound was detected in the dodecane phase of the fermentation broth, which could be unexpectedly identified as R-α-ionone. Isolation and identification of this compound are also shown in the examples.

[0031] The present invention therefore also relates to a novel method for producing α-ionone and mixtures of aroma compounds comprising α-ionone and / or α-ionylideneethane.

[0032] The method of the present invention for preparing one or more aroma compounds, such as the aroma compound α-ionylideneethane, can be carried out in vitro or in a host cell as defined herein. Preferably, the method for preparing the aroma compound α-ionylideneethane is carried out in a host cell as defined herein.

[0033] When the method for preparing one or more aroma compounds, such as α-ionylideneethane, is carried out in vitro, farnesyl diphosphate is provided as substrate in solution, for example in a suitable reaction buffer.For the conversion of farnesyl diphosphate to α-ionylideneethane, a suitable enzyme is used in the in vitro method.A non-limiting example for such an enzyme is α-ionylideneethane synthase (IES), which belongs to the subclass of carbon-oxygen lyase acting on phosphate (EC 4.2.3). α-Ionylideneethane synthase catalyzes the reaction of the substrate farnesyl diphosphate to the product α-ionylideneethane, possibly via a three-step reaction mechanism involving two neutral intermediates, β-farnesene and allofarnesene, in fungi (Takino et al., BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY 2019, VOL. 83, NO. 9, 1642-1649). The sequence of α-ionylideneethane synthase is disclosed elsewhere herein. Tests for measuring the activity of α-ionylideneethane synthase as defined herein are well known in the literature (see, for example, Takino et al., 2019, loc.cit.). Suitable tests for measuring the activity of α-ionylideneethane synthase as defined herein are also shown in the examples below.

[0034] Farnesyl diphosphate (FDP), also known as farnesyl pyrophosphate (FPP), is an intermediate in both the mevalonate and non-mevalonate pathways used by organisms in the biosynthesis of terpenes, terpenoids, and sterols. Details of the compound farnesyl diphosphate can be found, for example, at https: / / pubchem.ncbi.nlm.nih.gov / compound / Farnesyl-diphosphate.

[0035] In plants, farnesyl diphosphate is converted to abscisic acid by the oxidative cleavage of β-carotene. Abscisic acid is one of the important plant hormones and is known as a signaling molecule for plant abiotic stress and as a regulator of plant dormancy and germination. On the other hand, farnesyl diphosphate can be directly cyclized to α-ionylideneethane, which is oxidized to obtain abscisic acid. In 2006, a putative biosynthetic gene cluster for abscisic acid was identified. Gene disruption experiments suggested that two cytochromes, P450 (BcABA1,2) and a short-chain dehydrogenase / reductase (BcABA4), are involved in the five-step oxidative modification of α-ionylideneethane to abscisic acid. BcABA3 has been identified as a novel terpene synthase that catalyzes the cyclization of farnesyl diphosphate to α-ionylideneethane, and heterologous production of abscisic acid was achieved by utilizing four bcABA genes from Aspergillus oryzae (Takino et al., 2018, J. Am. Chem. Soc., 140, 12392-12395). BcABA3-catalyzed cyclizations include (1) ionization-initiated cyclization of farnesyl diphosphate to β-farnesene, (2) isomerization of β-farnesene to allofarnesene, and (3) protonation-initiated cyclization of allofarnesene to give α-ionylideneethane.

[0036] In one embodiment of an in vitro method for preparing one or more aroma compounds of the present invention, farnesyl diphosphate may be biocatalytically converted to α-ionylideneethane using a crude protein extract or an isolated enzyme, whereby the conversion of farnesyl diphosphate to α-ionylideneethane is catalyzed by an α-ionylideneethane synthase as defined herein.

[0037] Suitable conditions for carrying out the method for preparing one or more aroma compounds of the present invention in vitro are described in the literature.In addition, methods for isolating and purifying α-ionylideneethane and methods for formulating said compounds are described in the art; see, for example, the supporting information for the publication by Takino et al., J.Am.Chem.Soc.2018,140,39,12392-12395.A brief summary of the procedure used by the present inventors includes, for example, the extraction of fermentation broth with tBME and distillation of the solvent.The distillation fraction is purified by column chromatography.

[0038] As one of skill in the art will appreciate, the produced α-ionylideneethane, after isolation and / or purification, can also be chemically treated or subjected to one or more chemical reactions to obtain a desired product, such as α-ionone or vitamin A or a precursor of vitamin A.

[0039] Alternatively, the method for preparing one or more aroma compounds of the present invention may be carried out in a host cell as defined herein. The host cell preferably produces or contains farnesyl diphosphate as a substrate. The host cell further comprises a nucleic acid encoding an enzymatically active α-ionylideneethane synthase for converting farnesyl diphosphate to α-ionylideneethane. The nucleic acid encoding an enzymatically active α-ionylideneethane synthase for converting farnesyl diphosphate to α-ionylideneethane is preferably a heterologous nucleic acid.

[0040] In accordance with the present invention, α-ionylideneethane production in a host cell can be modulated by modifying the expression or activity of one or more proteins involved in α-ionylideneethane biosynthesis. It may be desirable to utilize an organism as a host cell that naturally produces one or more α-ionylideneethane compounds. Alternatively, it may be desirable to generate production of α-ionylideneethane that is not naturally produced by the host cell.

[0041] It may be desirable to introduce one or more heterologous α-ionylideneethane-synthetic polypeptides into the host cell. One example of a heterologous α-ionylideneethane-synthetic polypeptide is α-ionylideneethane synthase. As will be apparent to one of skill in the art, any of a variety of heterologous polypeptides, such as those disclosed herein, may be used. The selection takes into consideration, for example, the particular α-ionylideneethane compound, such as E,Z-α-ionylideneethane, whose production is to be enhanced. The present disclosure contemplates the introduction of heterologous α-ionylideneethane-synthetic polypeptides, such as those set forth in SEQ ID NOs: 1-17 and 19-33 and variants thereof, as well as the modulation of expression or activity levels of heterologous α-ionylideneethane-synthetic polypeptides, including altering constitutive or inducible expression patterns, for example, as described elsewhere herein.

[0042] The α-ionylideneethane produced is isolated and purified from the host cells by methods described in the art, which can then be used to produce compositions such as those disclosed herein, such as fragrance compositions, flavors or fragrances, animal feed, human nutritional products, cosmetics, colorants (carotenoids), radical scavengers, pharmaceutical compositions, or compounds for the crop protection industry.

[0043] The produced α-ionylideneethane can also be used in the host cell as a precursor for a biosynthetic pathway, such as a biosynthetic pathway for producing α-ionone, or a biosynthetic pathway for producing a precursor for the synthesis of vitamin A. To this end, the host cell can contain additional nucleic acids, preferably heterologous nucleic acids, for example encoding one, two, three or even more, or preferably all, of the enzymes of the mevalonate pathway. Such enzymes include acetyl-CoA C-acetyltransferase, hydroxymethylglutaryl-CoA synthase, (2E,6E)-farnesyl diphosphate synthase, isopentenyl-diphosphate DELTA-isomerase, hydroxymethylglutaryl-CoA reductase, diphosphomevalonate decarboxylase, mevalonate kinase, and phosphomevalonate kinase, all of which are well known in the art (see, e.g., Goldstein and Brown, Nature. 1990 Feb 1;343(6257):425-30. doi:10.1038 / 343425a0). Corresponding sequences for enzymes involved in the mevalonate pathway are available, for example, under EC numbers 2.3.1.9, 2.3.3.10, 2.5.1.10, 5.3.3.2, 1.1.1.88, 4.1.1.33, 2.7.1.36, and 2.7.4.2.

[0044] Alternatively or in addition to one, two, three, or even more, or preferably all, enzymes of the mevalonate pathway, the host cell may comprise a nucleic acid, preferably a heterologous nucleic acid encoding, for example, one, two, three, or more, or preferably all, enzymes of the deoxyxylulose phosphate (DXP or DOXP) pathway, also known as the non-mevalonate pathway, mevalonate-independent pathway, or MEP pathway, such as 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, (E)-4-his(III)-phosphate synthase, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase, 4-(cytidine 5'-diphospho)- ... 2,4-cyclodiphosphate synthase, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol Examples of such enzymes include hydroxy-3-methylbut-2-enyl-diphosphate synthase (ferredoxin), (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (flavodoxin), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase, and isopentenyl-diphosphate DELTA-isomerase (see, e.g., Rohmer, Nat Prod Rep. 1999 Oct;16(5):565-74. doi:10.1039 / a709175c). Corresponding sequences for enzymes involved in the deoxyxylulose phosphate pathway are available, for example, under the EC numbers 2.2.1.7, 1.1.1.267, 2.7.7.60, 2.7.1.148, 4.6.1.12, 1.17.7.1, 1.17.7.3, 1.17.1.2, 1.17.7.4, and 5.3.3.2.

[0045] Alternatively or in addition to one, two, three or even more, or preferably all, enzymes of the mevalonate and / or deoxyxylulose phosphate pathway, the host cell may contain one or more nucleic acids encoding oxidative enzymes, preferably one or more nucleic acids encoding carotene dioxygenases and / or peroxidases that catalyze oxidation reactions. The latter oxidative enzymes are known and described in the literature (Menzel, MS, P., in "Flavours and Fragrances", Berger, RG (ed.), Springer, Berlin, 2007; Zelena, K. et al., J. Agric. Food Chem, 2009, 57, 9951; Rajagopalan, A. et al., Adv. Synth. Catal, 2013, 355, 3321).

[0046] The host cells according to the present disclosure or the present invention can be produced based on standard genetic and molecular biology techniques generally known in the art, which also apply to suitable cell culture conditions for carrying out the above-mentioned methods in the host cells. In addition, methods for isolating and purifying α-ionylideneethane from host cells (see, for example, Sambrook et al., Molecular cloning: a laboratory manual / Sambrook, Joseph; Russell, David W.--. 3rd ed.-- New York: Cold Spring Harbor Laboratory, 2001; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994)) can be used.

[0047] As will be appreciated by one of skill in the art, the produced α-ionylideneethane may be isolated and / or purified from or within the host cell and then chemically treated or subjected to one or more chemical reactions to obtain a desired product.

[0048] In a preferred embodiment of the method for preparing one or more aroma compounds of the present invention in vitro or in a host cell, the α-ionylideneethane synthase is a fungal or bacterial α-ionylideneethane synthase.

[0049] As mentioned in the introduction, E,Z-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene; E,Z-IE, (1)) is the first cyclic intermediate in the biosynthesis of fungal abscisic acid (2). The specific sesquiterpene synthase that converts farnesyl pyrophosphate (3) to E,Z-α-ionylideneethane is α-ionylideneethane synthase (IE synthase); see Figure 1.

[0050] The amino acid sequence of α-ionylideneethane synthase has been described in the art (Takino, J. et al., J. Am. Chem. Soc., 2018, 140, 12392 Fig. S1) and is available under the database accession numbers and SEQ ID NOs shown in Table 1 below. Table 1 above further includes the organism from which the sequence originates.

[0051] [Table 1]

[0052] In addition, the present inventors have engineered synthetic α-ionylideneethane synthase sequences, which are shown in the sequence listing as SEQ ID NOs: 20-33.

[0053] Preferably, the α-ionylideneethane synthase is a) an amino acid sequence represented by SEQ ID NO: 1 to 17 or 19 to 33; b) an amino acid sequence having at least 40% sequence identity at the amino acid level with any one of SEQ ID NOs: 1 to 17 or 19 to 33 and having α-ionylideneethane synthase activity; c) a fragment of the amino acid sequence of a) or b) that has enzymatic activity; The amino acid sequence is selected from the group consisting of:

[0054] Preferably, the α-ionylideneethane synthase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 66%, 70%, 71%, 75%, 76%, 80%, 81%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity at the amino acid level to any of SEQ ID NOs: 1-17 or 19-33, preferably to SEQ ID NO: 1 or 19, and having α-ionylideneethane synthase activity.

[0055] In one embodiment, α-ionylideneethane synthases useful in the methods, host cells and uses of the invention are shown in FIG. 7 with conserved amino acids in white font on a black background.

[0056] In another embodiment, an α-ionylideneethane synthase useful in the methods, host cells, and uses of the invention preferably comprises the Pfam domains DUF1175 (PF06672) and GATA (PF00320) (PFAM version 35.0); see protein family database in Pfam:2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, GA Salazar, ELL Sonnhammer, SCE Tosato, L. Paladin, S. Raj, LJ Richardson, RD Finn, A. Bateman Nucleic Acids Research (2020) doi:10.1093 / nar / gkaa913.

[0057] The α-ionylideneethane synthases defined herein may be produced by chemical synthesis or recombinant molecular biology techniques well known to those skilled in the art, as also shown in the Examples below, which apply mutatis mutandis to the isolation of α-ionylideneethane synthases from host cells or supernatants; see, e.g., Sambrook et al., Molecular cloning: a laboratory manual / Sambrook, Joseph; Russell, David W.--. 3rd ed.--New York: Cold Spring Harbor Laboratory, 2001; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).

[0058] In an even further preferred embodiment of the method for preparing one or more aroma compounds of the present invention, the host cell comprises nucleic acids encoding one, two, three or more, or preferably all, of the enzymes of the mevalonate pathway and / or nucleic acids encoding one, two, three or more, or preferably all, of the enzymes of the deoxyxylulose phosphate (DOXP) pathway to provide farnesyl diphosphate as a substrate for producing α-ionylideneethane.

[0059] In another embodiment, it is contemplated that host cells are supplied with farnesol, which is then pyrophosphorylated under appropriate cell culture conditions to provide farnesyl diphosphate / pyrophosphate.

[0060] In another preferred embodiment of the method for preparing one or more aroma compounds of the present invention, the host cell further comprises one or more nucleic acids encoding oxidative enzymes, preferably one or more nucleic acids encoding carotene dioxygenases and / or peroxidases, said enzymes catalyzing oxidation reactions and may, for example, support the synthesis of α-ionone in the host cell, as described elsewhere herein.

[0061] One potential candidate could be, for example, a gene from Pseudocercospora pini-densiflorae CBS 125139. This microorganism is thought to produce abscisic acid via α-ionylidene ethanol, as described in Okamoto, M. et al., Phytochemistry, 1988, 27, 3465. When blasting the sequence of α-ionylidene ethane synthase from Botrytis to the whole organism, an open reading frame of 1140 bp is found, which could be the terpene synthase mentioned in the paper by Okamato, M. et al. of 1988. So far, we have no experimental evidence that Pseudocercospora pini-densiflorae actually converts farnesyl diphosphate to α-ionylidene ethanol.

[0062] A variety of oxidase enzymes are thought to be used to convert α-ionylideneethane into the oxidized precursor for the chemical synthesis of vitamin A. Reasonable candidates include P450 monooxygenases and laccases.

[0063] Direct biosynthesis of vitamin A via α-ionylideneethane is highly unlikely.

[0064] Preferably, the one or more aroma compounds produced by the method of the present invention are or comprise α-ionylideneethane, more preferably the α-ionylideneethane is 2Z,4E-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene). 2Z,4E-α-ionylideneethane is also referred to herein as E,Z-α-ionylideneethane. In this disclosure, both terms are used interchangeably.

[0065] Advantageously, the methods of the present invention can be used for the large-scale production of E,Z-α-ionylideneethane in vitro or in a host cell, thereby enabling, for example, the production of novel odor compounds or other compositions disclosed herein.

[0066] Additionally, E,Z-α-ionylideneethane can be used in the synthesis of α-ionone or vitamin A as disclosed herein.

[0067] The present invention also relates to a method for preparing α-ionone, comprising the step of converting α-ionylideneethane to α-ionone in the presence of farnesyl diphosphate and α-ionylideneethane synthase in vitro or in a host cell.

[0068] The present invention further provides a method for preparing α-ionone, the method comprising the steps of: a) providing farnesyl diphosphate and α-ionylideneethane synthase; b) converting the farnesyl diphosphate to α-ionylideneethane; and c) converting the α-ionylideneethane to α-ionone in vitro or in a host cell.

[0069] The present invention also includes a method for preparing α-ionone or a mixture of α-ionylideneethane and α-ionone comprising the steps of: a) providing farnesyl diphosphate and α-ionylideneethane synthase; and b) contacting α-ionylideneethane with the α-ionylideneethane synthase in vitro or in a host cell under conditions that allow for the production of α-ionone or a mixture of α-ionylideneethane and α-ionone.

[0070] Specifically, the present invention relates to a method for producing α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), comprising the steps of: a) contacting farnesyl diphosphate as defined herein with at least one α-ionylideneethane synthase, preferably as defined in claim 3, 4 or 5, under conditions suitable for producing at least one α-ionylideneethane; b) producing at least α-ionylideneethane; c) exposing at least one α-ionylideneethane produced in step b) to conditions suitable for the oxidative cleavage of the α-ionylideneethane to produce α-ionone; d) optionally isolating the α-ionone produced in step c).

[0071] In a preferred embodiment of the method for preparing α-ionone of the present invention, the method comprises the step of converting farnesyl diphosphate to α-ionylideneethane by an α-ionylideneethane synthase disclosed herein.

[0072] In another preferred embodiment of the method for preparing α-ionone of the present invention, α-ionylideneethane is converted to α-ionone, preferably by oxidative cleavage. The oxidative cleavage can be carried out chemically or enzymatically.

[0073] "Oxidative cleavage" refers to a reaction in which a carbon-carbon bond is broken and the carbon that formed the carbon-carbon bond is simultaneously oxidized.

[0074] The oxidative cleavage to α-ionone can be accomplished by various means known in the art for the oxidation of molecules. Oxygen from air can be used, as well as oxygen from oxygen donors (such as, but not limited to, hydrogen peroxide or other peroxides, ozone), as well as enzymes that provide oxygen to the reaction. As shown by the inventors, the oxidative cleavage can be carried out under conditions that allow for the production of α-ionylideneethane as well as α-ionone, and does not require sophisticated technology, provided that at least some α-ionylideneethane is produced.

[0075] The enzymatic oxidative cleavage can be carried out using oxidative enzymes such as carotene dioxygenase or peroxidase, or a combination thereof, the use of which can result in an improved bioconversion step in the production process of natural α-ionone by the host cells disclosed herein or in vitro.

[0076] Preferably, the α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one) prepared by the method of the present invention is R-α-ionone.

[0077] Although α-ionone is generally believed to be biosynthesized in vivo by the oxidative degradation of carotenoids, our preliminary literature search has not shown any precedent for the oxidative degradation of α-ionylideneethane to α-ionone.

[0078] Advantageously, the following examples show a new access to natural R-α-ionone based on microbial systems. It is shown that R-α-ionone (R-4) is probably formed by oxidative cleavage of α-ionylideneethane (1); see FIG. 5.

[0079] In the method of preparing α-ionone of the present invention, α-ionylideneethane is converted to α-ionone, thereby producing α-ionone. The synthesis method can be carried out in vitro or in a host cell, preferably a host cell of the present invention.

[0080] In a preferred embodiment of the method for preparing α-ionone of the present invention, α-ionylideneethane is converted to α-ionone by chemical and / or enzymatic oxidative cleavage. The conversion can be for a portion of the α-ionylideneethane, a substantial portion thereof, or more or less all of the α-ionylideneethane present. It is envisioned that the use of oxidizing enzymes, such as carotene dioxygenases or peroxidases, can result in an improvement of the bioconversion step in the process of producing natural R-α-ionone by the host cells disclosed herein.

[0081] Preferably, the α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one) is R-α-ionone.

[0082] The present invention further relates to a host cell for producing α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylideneethane synthase as defined herein, preferably as defined in claim 3, 4 or 5, preferably a bacterial cell, a yeast cell, a fungal cell, an algae cell, a cyanobacterial cell, a non-human animal cell, a non-human mammalian cell or a plant cell, suitable for the oxidative cleavage of α-ionylideneethane to produce α-ionone, in one aspect the host cell is capable of oxidatively cleaving α-ionylideneethane. In a preferred embodiment, (i) an α-ionylideneethane synthase as defined in claim 3, 4 or 5 converts farnesyl diphosphate to α-ionylideneethane in a host cell of the invention; and / or (ii) α-ionylideneethane is partially or totally converted to α-ionone in a host cell of the invention by chemical or enzymatic oxidative cleavage.

[0083] α-ionone is a colorless to slightly yellow liquid that is moderately water soluble. α-ionone occurs naturally in plants such as violets, blackberries, and plums. α-ionone is also found in tobacco and tobacco smoke. It is an aromatic ketone responsible for the scent of violets. It has a sweet odor like violets, and a woody, berry, floral taste. Thus, α-ionone is used as a fragrance in perfumes, cosmetics, and personal care products, as well as household cleaning agents and detergents (Lalko et al., Food Chem Toxicol. 2007;45 Suppl 1:S235-40. doi:10.1016 / j.fct.2007.09.046). It is also utilized as a food flavoring, such as in beverages, ice cream, baked goods, and candies. α-ionone is a component of bitter orange extract and is widely used in dietary supplements. Alpha-ionone is used as an ingredient in cat and dog repellents applied to lawns, plants, and outdoor furniture, and as a beetle attractant on roses.

[0084] According to the present invention, α-ionone compound production in a host cell can be modulated by modifying the expression or activity of one or more proteins involved in α-ionone biosynthesis. It may be desirable to utilize an organism as a host cell that naturally produces one or more ionone compounds. Alternatively, it may be desirable to generate production of α-ionone that is not naturally produced by the host cell.

[0085] It may be desirable to introduce one or more heterologous α-ionone-synthetic polypeptides into the host cell. As will be apparent to one of skill in the art, any of a variety of heterologous polypeptides as disclosed herein can be used. In one example, farnesyl diphosphate can be converted to α-ionylideneethane by α-ionylideneethane synthase as disclosed herein, and then α-ionylideneethane can be converted to α-ionone by an enzyme that catalyzes oxidative cleavage, such as carotene dioxygenase or peroxidase, as disclosed herein. Selection takes into account, for example, the particular ionone compound whose production is to be enhanced, e.g., α-ionone or R-α-ionone. The present disclosure contemplates not only the introduction of heterologous α-ionone-synthetic polypeptides, but also the modulation of expression or activity levels of heterologous α-ionone-synthetic polypeptides, including, for example, altering constitutive or inducible expression patterns, as described elsewhere herein.

[0086] In the past, the extraction of fragrance compounds from flowers and other plants was the only source of raw materials for products such as perfumes. For example, it has been shown recently that the biodegradation of carotenoids is an important pathway for the formation of apocarotenoids. However, it is now more economical to synthesize these compounds in the laboratory. Advantageously, the method of the present invention can be used for the large-scale production of E,α-ionone in vitro or in a host cell, which allows, for example, the production of novel odor compounds or other compositions disclosed herein.

[0087] For the determination of the content of α-ionylideneethane and / or α-ionone produced by the method of the present invention, several useful techniques may be used, such as gas chromatography-flame ionization detector (GC-FID), gas chromatography-mass selective detector (GC-MSD), high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), high performance liquid chromatography-refractive index detector (HPLC-RID), high performance liquid chromatography-variable wavelength detector (HPLC-VWD), gel permeation chromatography (GPC), high performance thin layer chromatography (HPTLC), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA) and infrared spectroscopy (IR), which are known in the art; see for example the review by Jiang et al, Curr Protoc Plant Biol. 2016;1:345-358. doi:10.1002 / cppb.20024. Further methods for the extraction, purification, and analysis of α-ionylideneethane and / or α-ionone are provided in the Examples.

[0088] In a preferred embodiment, the process of the present invention produces ionylideneethane with α-ionone in a ratio of about 8:1 or less, preferably about 5:1, 4:1, 3:1, 2:1, 1:1, or 0.5:1, or even 0.1:1.

[0089] In another preferred embodiment, at least 10%, preferably at least 20%, 30%, 40%, 50%, 70%, 80%, 90%, 95%, or 99% of the α-ionylideneethane is converted to α-ionone in the process of the invention.

[0090] The present invention further relates to the use of α-ionylideneethane as a fragrance compound.

[0091] Preferably, the α-ionylideneethane has floral-violet and / or woody-orris / iris root notes. The present invention also relates to the use of α-ionylideneethane synthase in the production of one or more aroma compounds. Preferably, the α-ionylideneethane synthase is a) α-ionylideneethane synthase (EC 4.2.3), which belongs to the subclass of carbon-oxygen lyases acting on phosphate; b) an α-ionylideneethane synthase that is a fungal or bacterial α-ionylideneethane synthase; c) i) an amino acid sequence represented by SEQ ID NO: 1 to 17 or 19 to 33; ii) an amino acid sequence having at least 40% sequence identity at the amino acid level with any one of SEQ ID NOs: 1 to 17 or 19 to 33 and having α-ionylideneethane synthase activity; and iii) an enzymatically active fragment of the amino acid sequence of a) or b) having α-ionylideneethane synthase activity; the α-ionylideneethane synthase comprising an amino acid sequence selected from the group consisting of: and d) any combination of the above a) to c).

[0092] In one embodiment, the α-ionylideneethane synthase is for preparing one or more aroma compounds imparting floral-violet and / or woody-orris / iris root notes to a perfume, fragrance or fragrance. In another embodiment, the α-ionylideneethane is produced by an α-ionylideneethane synthase disclosed herein, preferably an α-ionylideneethane synthase as defined in claim 3, 4 or 5. The definitions, explanations and embodiments relating to the method of the invention apply mutatis mutandis to the use of the invention.

[0093] The present invention further relates to a method for preparing vitamin A, preferably in vitro or in a host cell, comprising the step of converting α-ionylideneethane to vitamin A, which comprises chemically or enzymatically converting α-ionylideneethane to (2E,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-2-en-1-yl)penta-2,4-dien-1-ol via the respective alcohol, followed by Wittig salt formation under isomerization ([(2E,4E)-3-methyl-5-(2,6,6-trimethylcyclohexen-1-yl)penta-2,4-dienyl]-triphenyl-phosphonium), and preparation of vitamin A by Wittig reaction with C5-aldehyde [(E)-3-methyl-4-oxo-but-2-enyl]acetate; see also FIG. 6.

[0094] Specifically, the present invention provides a method for preparing vitamin A comprising: a) contacting farnesyl diphosphate with one or more α-ionylideneethane synthases as defined herein, preferably with one or more α-ionylideneethane synthases as defined in claim 3, 4 or 5, under conditions suitable for producing at least one α-ionylideneethane; b) producing α-ionylideneethane; c) chemical or enzymatic conversion of α-ionylideneethane to (2E,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-2-en-1-yl)penta-2,4-dien-1-ol via the respective alcohol, followed by Wittig salt formation under isomerization ([(2E,4E)-3-methyl-5-(2,6,6-trimethylcyclohexen-1-yl)penta-2,4-dienyl]-triphenyl-phosphonium) and preparation of vitamin A by Wittig reaction with C5-aldehyde [(E)-3-methyl-4-oxo-but-2-enyl]acetate.

[0095] Preferably, at least one, more preferably two, even more preferably all of the method steps of the method for preparing vitamin A of the present invention are carried out in vitro. In another embodiment of the method of the present invention, the method comprises using a host cell comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylidene ethane synthase as defined herein, preferably an α-ionylidene ethane synthase as defined in claim 3, 4 or 5. Preferably, the host cell is a bacterial cell, a yeast cell, a fungal cell, an algae cell, a blue-green algae cell, a non-human animal cell, a non-human mammalian cell or a plant cell, more preferably a bacterial cell or a yeast cell. Preferably, the host cell is used in fermentation.

[0096] The synthesis of vitamin A and / or similar carotenoids may not be exclusively biocatalytic / enzymatic: chemoenzymatic conversions are also possible: the biomoiety is constituted by glucose via farnesyl diphosphate to form α-ionylideneethane or the respective alcohol (α-ionylideneethanol), followed by a purely chemically catalyzed conversion of α-ionylideneethane / α-ionylideneethanol to vitamin A.

[0097] Although the position of the cyclohexene double bond in E,Z-α-ionylideneethane is different from that in vitamin A, E,Z-α-ionylideneethane may be an interesting precursor of vitamin A.

[0098] One goal of the present invention was to provide proof of concept for α-ionylideneethane synthesis in Rhodobacter. To assess whether α-ionylideneethane is a reasonable starting point for a hybrid biochemical synthesis of vitamin A, sufficient starting material can be obtained, for example, but not limited to, by fermentation of the newly constructed Rhodobacter strain ROB034, as described in the Examples below.

[0099] In particular, in the present context, the functionalization of the terminal methyl group at C-11 of α-ionylideneethane to accommodate carbon chain elongation and the migration of the cyclohexene double bond from C-5 to C-4 are important milestones.

[0100] Furthermore, the present invention should also be able to provide materials for evaluating the chemical or biocatalytic conversion of E,Z-α-ionylideneethane to more direct precursors of vitamin A.

[0101] Methods for extracting and measuring vitamin A are well described in the art; see, for example, the review by Zhang et al., Molecules. 2018 Jun;23(6):1484. Published online 2018 Jun 19. doi:10.3390 / molecules23061484.

[0102] The present invention further relates to a method for perfuming a product, in particular for imparting and / or enhancing an odor or flavor, in which at least one α-ionylideneethane as defined herein is used, preferably an α-ionylideneethane with floral-violet and / or woody-orris / iris root notes, more preferably 2Z,4E-α-ionylideneethane.

[0103] The present invention further relates to a method for perfuming a product, in particular a method for imparting and / or enhancing an odor or flavor, in which at least one α-ionylideneethane synthase as defined herein is used, comprising a step of preparing one or more aroma compounds according to the method of the invention, optionally followed by a step of purifying the one or more aroma compounds, and then a step of perfuming a product with the one or more aroma compounds.

[0104] A further aspect of the present invention relates to a method for modifying the aroma of a ready-to-use composition, said method comprising the step of incorporating α-ionylideneethane and / or α-ionone, the latter preferably produced by the method of the present invention, into the ready-to-use composition to obtain an aroma-modified ready-to-use composition.

[0105] The compounds of the present invention and their aroma chemical compositions have advantageous organoleptic properties, in particular a pleasant aroma impression.Therefore, they can be preferably used as aroma ingredients in perfume compositions, body care compositions (including cosmetic compositions and oral and dental hygiene products), hygiene products, cleaning compositions (including dishwashing compositions), textile detergent compositions, compositions for scent dispensers, foods, dietary supplements, pharmaceutical compositions, crop protection compositions and other ready-to-use compositions.

[0106] The pleasant odor, low volatility and good solubility of α-ionylideneethane and / or α-ionone (the latter preferably produced by the method of the present invention) make them suitable ingredients for compositions where a pleasant odor is desired. Thanks to their physical properties, α-ionylideneethane and / or α-ionone can be successfully combined with other odor chemicals and conventional ingredients, in particular in aromatized ready-to-use compositions such as perfume compositions. This allows, for example, the creation of odor compositions, in particular perfume compositions, with novel and advantageous sensory profiles.

[0107] Furthermore, the α-ionylideneethane and / or α-ionone produced by the method of the present invention can be produced in good yield and purity by simple synthesis starting from readily available starting materials. Thus, the α-ionylideneethane and / or α-ionone produced by the method of the present invention can be produced on a large scale in a simple and cost-effective manner.

[0108] Additionally, the present invention provides a method for preparing an aroma chemical composition of the present invention, such as, but not limited to, a fragrance composition, flavor, fragrance, or perfume, comprising: a) producing α-ionylideneethane according to the method for preparing α-ionylideneethane of the present invention; b) isolating and optionally purifying the α-ionylideneethane of step a); c) adding the isolated and optionally purified α-ionylideneethane of step b) as an ingredient to an aroma chemical composition of the invention as described herein, such as an aroma composition, flavor, fragrance or perfume conveying one of the following olfactory notes: floral-violet and / or woody-orris root in the case of α-ionylideneethane, and floral-violet in the case of α-ionone; The present invention provides a method comprising:

[0109] Orris root (rhizoma iridis) is the root of Iris germanica, Iris pallida, and Iris florentina. The most valuable component of orris root is orris oil (0.1-0.2%), a yellow-white mass containing myristic acid. Once important in Western herbal medicine, it is now used mainly as a fixative and base note in perfumes; see for example John Charles Sawer, Odorographia a natural history of raw materials and drugs used in the perfume industry intended to serve growers, manufacturers and consumers. The odor profile of orris root is a powdery, earthy, rooty scent with woody, violet floral notes. The expressions woody-orris (iris) root or woody-orris / iris root should be understood to refer to these typical notes of orris root or iris root.

[0110] Additionally, the present invention also relates to a method for preparing a fragrance composition, flavor, fragrance, or perfume, comprising: a) producing α-ionone according to any one of the methods for preparing α-ionone of the present invention; b) isolating and optionally purifying the α-ionone of step a); and c) adding the isolated and optionally purified α-ionone of step b) as an ingredient to a chemical composition of the invention, such as, but not limited to, an aroma composition, flavor, fragrance, or perfume that conveys one of the following olfactory notes: floral-violet or woody-orris (iris) root in the case of α-ionylideneethane, and floral-violet in the case of α-ionone.

[0111] As one of skill in the art will appreciate, the latter process may also include, as additional process steps, the production, isolation, and optional purification of α-ionylideneethane.

[0112] The present invention therefore also relates to a method for preparing a fragrance composition, flavour, fragrance or perfume, comprising: a) producing α-ionylideneethane according to the process for preparing α-ionylideneethane of the present invention; and / or b) producing α-ionone according to any one of the methods for preparing α-ionone of the present invention; c) isolating and, optionally, purifying the α-ionylideneethane of step a) and / or the α-ionone of step b); d) adding the isolated and optionally purified α-ionylideneethane and / or α-ionone of step c) as an ingredient to an aroma chemical composition of the invention, such as, but not limited to, an aroma composition, flavor, fragrance or perfume conveying any one of the following olfactory notes: floral-violet or woody-orris (iris) root in the case of α-ionylideneethane, and floral-violet in the case of α-ionone.

[0113] Monoterpenes and sesquiterpenes are used industrially as ingredients of flavors, fragrances and cosmetics. Fragrances and aromas are used as important additives to promote the final quality of food and beverages, as well as body care and other hygiene products. However, recently, there has been an increasing demand for products of natural origin. Natural flavor compounds that can improve the sensory appeal of these products have therefore become more valuable and more expensive than their artificial counterparts. α-Ionylideneethane and / or α-ionone, the latter preferably produced by the method of the present invention, can be advantageously used to produce fragrance compositions, flavors, fragrances, or perfumes, or any other product disclosed herein.

[0114] Means and methods for preparing aroma compounds or compositions, flavors, fragrances, or perfumes are well known in the art; see, for example, Flavors and Fragrances: Chemistry, Bioprocessing and Sustainability RG Berger; Black et al., EP2897465B1, Chromatography of Aroma Compounds and Fragrances, Cserhati, T. (2010).

[0115] α-Ionylideneethane and / or α-ionone, the latter preferably produced by the method of the present invention, can generally be used in ready-to-use compositions, in particular in aromatized ready-to-use compositions. As used herein, "aromatized ready-to-use composition" refers to a ready-to-use composition that induces a primarily pleasant odor and / or taste impression. In a preferred embodiment, the aromatized ready-to-use composition is a perfumed ready-to-use composition, i.e., induces a pleasant odor.

[0116] Perfumed ready-to-use compositions are, for example, compositions used in personal care, home care, industrial applications and other applications such as pharmaceutical or crop protection compositions.

[0117] Preferably, α-ionylideneethane and / or α-ionone, the latter preferably produced by the process of the present invention, is used in a composition selected from the group consisting of perfume compositions, body care compositions (including cosmetic compositions and oral and dental hygiene products), hygiene products, cleaning compositions (including dishwashing compositions), textile detergent compositions, compositions for scent dispensers, foods, dietary supplements, pharmaceutical compositions and crop protection compositions. α-ionylideneethane and / or α-ionone, the latter preferably produced by the process of the present invention, is used as aroma chemical, preferably as fragrance, in said compositions.

[0118] In particular, α-ionylideneethane and / or α-ionone are used to impart sweet, floral, violet, orris, rooty and / or woody notes; or α-ionylideneethane and / or α-ionone are used to create a scent in the composition reminiscent of floral and / or woody elements.

[0119] Details of the above listed compositions are given below.

[0120] Similarly, α-ionylideneethane and / or α-ionone (the latter preferably produced by the method of the present invention) can improve the sensory profile of an aroma chemical composition as a result of a synergistic effect with other aroma chemicals (e.g. other fragrances) contained in the composition, meaning that this compound can provide a potentiator effect to said other aroma chemicals, and thus this compound is suitable as an enhancer of other aroma chemicals.

[0121] The present invention therefore also relates to the use of α-ionylideneethane, alone or in combination with α-ionone, to modify the fragrance properties (e.g., scented) of aromatized (e.g., perfumed) compositions; in particular as an enhancer of other fragrance chemicals.

[0122] The enhancer effect of a substance means that the substance, in an aromachemical formulation (such as a perfume formulation), improves and intensifies the overall sensory (e.g. olfactory) impression of the formulation. For example, in the mint field, menthyl methyl ether is known to enhance the perfume or taste mixture of peppermint oil, particularly considerably strengthening the top note and resulting in a more complex perception, even though the ether itself does not emit a particularly strong odor by itself. In fragrance applications, Hedione® (methyl dihydrojasmonate) only exhibits a light jasmine floral odor note by itself, but as an odor enhancer, it enhances the diffusion, freshness and richness of the volume of the perfume composition. The enhancer effect is particularly desired when an application characterized by a top note is required, in which case the odor impression is to be transmitted particularly quickly and intensively, for example in deodorants, deodorants or in the taste field of chewing gum.

[0123] To achieve such an enhancer effect, α-ionylideneethane and / or α-ionone may be used, for example, in an amount of 0.001 to 10% by weight (wt %), for example, in an amount of 0.01 to 2 wt %, preferably in an amount of 0.05 to 1 wt %, in particular in an amount of 0.1 to 0.5 wt %, based on the total weight of the resulting aromachemical composition.

[0124] Additionally, α-ionylideneethane alone or in combination with α-ionone may provide additional positive effects to the composition in which it is used, for example, the compound may improve the overall performance of the composition in which it is incorporated, such as the stability, e.g., formulation stability, spreadability, or retention of the composition.

[0125] In one embodiment, the present invention provides an aroma chemical composition comprising α-ionylideneethane without or with α-ionone and: (i) at least one further aroma chemical; or (ii) at least one non-aromatic chemical carrier; or (iii) a composition comprising both (i) and (ii).

[0126] As used herein, the term "fragrance composition" or "aroma chemical composition" refers to a composition that induces a pleasant fragrance, e.g., a pleasant odor impression. Unless otherwise specified, both terms are used interchangeably.

[0127] The non-fragrance chemical carriers in the fragrance chemical compositions of the present invention may be selected from surfactants, oil components and solvents, among others.

[0128] In one embodiment, the additional fragrance chemical is different from α-ionylideneethane or α-ionone, i.e., is not a stereoisomer of α-ionylideneethane or α-ionone, or a mixture of two or more stereoisomers of α-ionylideneethane or α-ionone.

[0129] Thanks to their physical properties, the α-ionylideneethane and / or α-ionone produced by the method of the present invention can be successfully combined with other aroma chemicals (e.g., other fragrances) and other conventional ingredients, in particular in aromatized (e.g., scented) ready-to-use compositions, such as perfume compositions. This allows, for example, the production of aroma compositions (e.g., perfume compositions) with novel and advantageous sensory profiles. In particular, as already explained above, the compounds can provide an enhancer effect for other aroma chemicals (such as other fragrances).

[0130] Thus, in one preferred embodiment, the aroma chemical composition comprises α-ionylideneethane without or with α-ionone as defined herein and at least one further aroma chemical different from α-ionylideneethane or α-ionone.

[0131] The further fragrance chemicals may for example be one, preferably two, three, four, five, six, seven, eight or further fragrance chemicals selected from the group consisting of: Geranyl acetate, α-hexylcinnamaldehyde, 2-phenoxyethyl isobutyrate, dihydromyrcenol, methyl dihydrojasmonate, 4,6,6,7,8,8-hexamethyl-1,3,4,6,7,8-hexahydrocyclopenta[g]benzopyran, tetrahydrolinalool, ethyl linalool, benzyl salicylate, 2-methyl-3-(4-tert-butylphenyl)propanal, cinnamyl alcohol, 4,7-methano-3a,4,5,6,7,7a-hexahydro-5-indenyl acetate and / or 4,7-methano-3a,4,5,6,7,7a-hexahydro-6-indenyl acetate, citronellol, citronellyl acetate, tetrahydrogeraniol, vanillin, linalyl acetate, styrolyl acetate, octahydro-2,3,8,8-tetramethyl-2-acetonaphthone and / or 2-acetyl-1,2,3,4,6,7,8-octahydro-2,3,8,8-tetramethylnaphthalene, hexyl salicylate, 4-tert-butylcyclohexyl acetate, 2-tert-butylcyclohexyl acetate, cyclohexyl acetate, alpha-ionone, n-alpha-methyl ionone, alpha-isomethyl ionone, coumarin, terpinyl acetate, 2-phenylethyl alcohol, 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-carboxaldehyde, alpha-amylcinnamaldehyde, ethylene brassylate, (E)- and / or (Z)-3-methylcyclopentadeca-5-enone, 15-pentadeca-11-enolide and / or 15-pentadeca-12-enolide, 15-cyclopentadeca-nolide, 1-(5,6,7, 8-Tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthalenyl)ethanone, 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol, cis-3-hexenyl acetate, trans-3-hexenyl acetate, trans-2 / cis-6-nonadienol, 2,4-dimethyl-3-cyclohexenecarboxaldehyde, 2,4,4,7-tetramethyloct-6-en-3-one, 2,6-Dimethyl-5-hepten-1-al, borneol, 3-(3-isopropylphenyl)butanal, 2-methyl-3-(3,4-methylenedioxyphenyl)-propanal, 3-(4-ethylphenyl)-2,2-dimethylpropanal, 7-methyl-2H-1,5-benzodioxepin-3(4H)-one, 3,3,5-trimethylcyclohexyl acetate, 2,5,5-trimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene -2-ol, 3-(4-tert-butylphenyl)-propanal, ethyl 2-methylpentanoate, ethoxymethoxycyclododecane, 2,4-dimethyl-4,4a,5,9b-tetrahydroindeno[1,2-d][1,3]dioxine, (2-tert-butylcyclohexyl)acetate, and 3-[5,5,6-trimethylbicyclo[2.2.1]hept-2-yl]cyclohexan-1-ol, 2,4-diethylocta-2,6-dienal.

[0132] In yet another preferred embodiment, the at least one aroma chemical (i) is selected from the group consisting of methyl benzoate, benzyl acetate, geranyl acetate, 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol, linalool, 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol and methyl benzoate.

[0133] In yet another preferred embodiment, the at least one aroma chemical (i) is selected from the group consisting of ethyl vanillin, vanillin, 2,5-dimethyl-4-hydroxy-2H-furan-3-one (furaneol) or 3-hydroxy-2-methyl-4H-pyran-4-one (maltol).

[0134] Further fragrance chemicals with which the compounds of formula (1) and / or (4) can be combined to give compositions according to the presently claimed invention can be found, for example, in S. Arctander, Perfume and Flavor Chemicals, Vol. I and II, Montclair, NJ, 1969, self-published or K. Bauer, D. Garbe and H. Surburg, Common Fragrance and Flavor Materials, 4th Ed., Wiley-VCH, Weinheim 2001. In particular, the following may be mentioned: Extracts from natural sources, for example essential oils, concretes, absolutes, resins, resinous substances, balsams, tinctures, for example ambergris tincture, amyris oil, angelica seed oil, angelica root oil, anise oil, valerian oil, basil oil, tree moss absolute, bay oil, wormwood oil, benzoin resin, bergamot oil, beeswax absolute, birch tar oil, bitter almond oil, savory oil, butucan leaf oil, cabreva oil, cade oil, chalmers oil, cananga oil, cardamom oil, cascarilla oil, cassia oil, cassia absolute, castoreum absolute absolute), cedar leaf oil, cedarwood oil, cistus oil, citronella oil, lemon oil, copaiba balsam, copaiba balsam oil, coriander oil, costus root oil, cumin oil, cypress oil, davana oil, dill weed oil, dill seed oil, eau de brouts absolute, oak moss absolute, elemi oil, tarragon oil, eucalyptus citriodora oil, eucalyptus oil, fennel oil, pine needle oil, galbanum oil, galbanum resin, geranium oil, grapefruit oil, guaiacwood oil, gurjan balsam, gurjan balsam oil, helichrysum absoluteabsolute), helichrysum oil, ginger oil, iris root absolute, iris root oil, jasmine absolute, chamus oil, chamomile oil blue, roman chamomile oil, carrot seed oil, cascarilla oil, pine needle oil, spearmint oil, caraway oil, labdanum oil, labdanum absolute, labdanum resin, lavandin absolute, lavandin oil, lavender absolute, lavender oil, lemongrass oil, lovage oil, lime oil distilled, lime oil pressed, linalool oil, litsea cubeba oil, bay leaf oil, mace oil, marjoram oil, mandarin oil, massoia bark bark oil, mimosa absoluteabsolute), Musk Seed Oil, Mus Tinker, Clary Sage Oil, Nutmeg Oil, Myrrh Absolute, Myrrh Oil, Myrtle Oil, Clove Leaf Oil, Clove Flower Oil, Neroli Oil, Olibanum Absolute, Olibanum Oil, Opopanax Oil, Orange Blossom Absolute, Orange Oil, Origanum Oil, Palmarosa Oil, Patchouli Oil, Parilla Oil, Balsam of Peru Oil, Parsley Leaf Oil, Parsley Seed Oil, Petitgrain Oil, Peppermint Oil, Pepper Oil, Pimento Oil, Pine Oil, Pennyroyal Oil ole, rose absolute, rosewood oil, rose oil, rosemary oil, dalmatian sage oil, Spanish sage oil, sandalwood oil, celery seed oil, spike lavender oil, star anise oil, styrax oil, tagetes oil, fir needle oil, tea tree oil, turpentine oil, thyme oil, tolu balsam, tonka absolute, tuberose absolute, vanilla extract, violet leaf absolute, verbena oil, vetiver oil, juniper berry oil, wine lees oil, absinthe oil, wintergreen oil, hyssop oil, civet absolute, cinnamon leaf oil, cinnamon bark oil, and fractions thereof or components isolated therefrom;

[0135] Individual fragrances from the group of hydrocarbons, such as, for example, 3-carene, α-pinene, β-pinene, α-terpinene, γ-terpinene, p-cymene, bisabolene, camphene, caryophyllene, cedrene, farnesene, limonene, longifolene, myrcene, ocimene, valencene, (E,Z)-1,3,5-undecatriene, styrene, diphenylmethane, etc.;

[0136] Aliphatic alcohols, such as hexanol, octanol, 3-octanol, 2,6-dimethylheptanol, 2-methyl-2-heptanol, 2-methyl-2-octanol, (E)-2-hexenol, (E)- and (Z)-3-hexenol, 1-octen-3-ol, a mixture of 3,4,5,6,6-pentamethyl-3 / 4-hepten-2-ol and 3,5,6,6-tetramethyl-4-methyleneheptan-2-ol, (E,Z)-2,6-nonadienol, 3,7-dimethyl-7-methoxyoctan-2-ol, 9-decenol, 10-undecenol, 4-methyl-3-decen-5-ol, etc.;

[0137] Aliphatic aldehydes and their acetals, such as hexanal, heptanal, octanal, nonanal, decanal, undecanal, dodecanal, tridecanal, 2-methyloctanal, 2-methylnonanal, (E)-2-hexenal, (Z)-4-heptenal, 2,6-dimethyl-5-heptenal, 10-undecenal, (E)-4-decenal, 2-dodecenal, 2,6,10-trimethyl-9-undecenal, 2,6,10-trimethyl-5,9-undecadienal; aliphatic ketones and their oximes, such as 2-heptanone, 2-octanone, 3-octanone, 2-nonanone, 5-methyl-3-heptanone, 5-methyl-3-heptanone oxime, 2,4,4,7-tetramethyl-6-octen-3-one, 6-methyl-5-hepten-2-one, etc. Aliphatic sulfur-containing compounds, such as 3-methylthiohexanol, 3-methylthiohexyl acetate, 3-mercaptohexanol, 3-mercaptohexyl acetate, 3-mercaptohexyl butyrate, 3-acetylthiohexyl acetate, 1-menthene-8-thiol, and the like;

[0138] Aliphatic nitriles, such as 2-nonenenitrile, 2-undecenenitrile, 2-tridecenenitrile, 3,12-tridecadienenitrile, 3,7-dimethyl-2,6-octadienenitrile, 3,7-dimethyl-6-octenenitrile, etc.;

[0139] Esters of aliphatic carboxylic acids, for example (E) and (Z)-3-hexenyl formate, ethyl acetoacetate, isoamyl acetate, hexyl acetate, 3,5,5-trimethylhexyl acetate, 3-methyl-2-butenyl acetate, (E)-2-hexenyl acetate, (E) and (Z)-3-hexenyl acetate, octyl acetate, 3-octyl acetate, 1-octen-3-yl acetate, ethyl butyrate, butyl butyrate, isoamyl butyrate, hexyl butyrate, (E) and (Z)-3-hexenyl isobutyrate, hexyl crotonate, ethyl sovalerate, ethyl 2-methylpentanoate, ethyl hexanoate, allyl hexanoate, ethyl heptanoate, allyl heptanoate, ethyl octanoate, ethyl (E,Z)-2,4-decadienoate, methyl 2-octynate, methyl 2-nonynate, allyl 2-isoamyloxyacetate, methyl-3,7-dimethyl-2,6-octadienoate, 4-methyl-2-pentyl crotonate, and the like;

[0140] Acyclic terpene alcohols, such as geraniol, nerol, linalool, lavandulol, nerolidol, farnesol, tetrahydrolinalool, 2,6-dimethyl-7-octen-2-ol, 2,6-dimethyloctan-2-ol, 2-methyl-6-methylene-7-octen-2-ol, 2,6-dimethyl-5,7-octadien-2-ol, 2,6-dimethyl-3,5-octadien- 2-ol, 3,7-dimethyl-4,6-octadien-3-ol, 3,7-dimethyl-1,5,7-octatrien-3-ol, 2,6-dimethyl-2,5,7-octatrien-1-ol, and their formates, acetates, propionates, isobutyrates, butyrates, isovalerates, pentanoates, hexanoates, crotonates, tiglinates, and 3-methyl-2-butenoates;

[0141] Acyclic terpene aldehydes and ketones, such as geranial, neral, citronellal, 7-hydroxy-3,7-dimethyloctanal, 7-methoxy-3,7-dimethyloctanal, 2,6,10-trimethyl-9 undecenal, geranyl acetone, and the dimethyl and diethyl acetals of geranial, neral, and 7-hydroxy-3,7-dimethyloctanal; cyclic terpene alcohols, such as menthol, isopulegol, α-tert-butyl ether, methyl ... Pineol, terpin-4-ol, menthan-8-ol, menthan-1-ol, menthan-7-ol, borneol, isoborneol, linalool oxide, nopol, cedrol, ambrinol, vethyrol, guajole, and their formates, acetates, propionates, isobutyrates, butyrates, isovalerates, pentanoates, hexanoates, crotonates, tiglinates, and 3-methyl-2-butenoates;

[0142] Cyclic terpene aldehydes and ketones, such as menthone, isomenthone, 8-mercaptomenthan-3-one, carvone, camphor, fenchone, α-ionone, β-ionone, α-n-methylionone, β-n-methylionone, α-isomethylionone, β-isomethylionone, α-irone, α-damascone, β-damascone, β-damascenone, δ-damascone, γ-damascone, 1-(2,4,4-trimethyl-2-cyclohexen-1-yl)- 2-Buten-1-one, 1,3,4,6,7,8a-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methano-naphthalen-8(5H)-one, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-butenal, nootkatone, dihydronootkatone, 4,6,8-megastigmatrien-3-one, α-sinensal, β-sinensal, acetylated cedarwood oil (methyl cedryl ketone), etc;

[0143] Cyclic alcohols, such as 4-tert-butylcyclohexanol, 3,3,5-trimethylcyclohexanol, 3-isocamphylcyclohexanol, 2,6,9-trimethyl-Z2,Z5,E9-cyclododecatrien-1-ol, 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol, etc.;

[0144] Alicyclic alcohols, such as α-3,3-trimethylcyclohexylmethanol, 1(4-isopropylcyclohexyl)ethanol, 2-methyl-4-(2,2,3-trimethyl-3-cyclopent-1-yl)butanol, 2-methyl-4-(2,2,3-trimethyl-3-cyclopent-1-yl)-2-buten-1-ol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopent-1-yl)-2-buten-1-ol, 3-methyl-5- (2,2,3-trimethyl-3-cyclopent-1-yl)pentan-2-ol, 3-methyl-5-(2,2,3-trimethyl-3-cyclopent-1-yl)-4-penten-2-ol, 3,3-dimethyl-5-(2,2,3-trimethyl-3-cyclopent-1-yl)-4-penten-2-ol, 1-(2,2,6-trimethylcyclohexyl)pentan-3-ol, 1-(2,2,6-trimethylcyclohexyl)hexan-3-ol, etc.;

[0145] Cyclic and alicyclic ethers, such as cineol, cedryl methyl ether, cyclododecyl methyl ether, 1,1-dimethoxycyclododecane, (ethoxymethoxy)cyclododecane, α-cedrene epoxide, 3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan, 3a-ethyl-6,6,9a-trimethyldodecahydro-naphtho[2,1-b]furan, 1,5,9-trimethyl-13-oxabicyclo-[10.1.0]trideca-4,8-diene, rose oxide, 2-(2,4-dimethyl-3-cyclohexen-1-yl)-5-methyl-5-(1-methylpropyl)-1,3-dioxane, and the like;

[0146] Cyclic and macrocyclic ketones, such as 4-tert-butylcyclohexanone, 2,2,5-trimethyl-5-pentylcyclopentanone, 2-heptylcyclopentanone, 2-pentylcyclopentanone, 2-hydroxy-3-methyl-2-cyclopenten-1-one, 3-methyl-cis-2-penten-1-yl-2-cyclopenten-1-one, 3-methyl-2-pentyl-2-cyclopenten-1-one, 3-methyl-4-cyclopentadecenone, 3-methyl-5-cyclopentadecenone, 3 -Methylcyclopentadecanone, 4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone, 4-tert-pentylcyclohexanone, 5-cyclohexadecen-1-one, 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone, 8-cyclohexadecen-1-one, 7-cyclohexadecen-1-one, (7 / 8)-cyclohexadecen-1-one, 9-cycloheptadecen-1-one, cyclopentadecanone, cyclohexadecanone, etc.;

[0147] Alicyclic aldehydes, such as 2,4-dimethyl-3-cyclohexenecarbaldehyde, 2-methyl-4-(2,2,6-trimethylcyclohexen-1-yl)-2-butenal, 4-(4-hydroxy-4-methylpentyl)-3-cyclohexenecarbaldehyde, 4-(4-methyl-3-penten-1-yl)-3-cyclohexenecarbaldehyde, etc.;

[0148] Alicyclic ketones, such as 1-(3,3-dimethylcyclohexyl)-4-penten-1-one, 2,2-dimethyl-1-(2,4-dimethyl-3-cyclohexen-1-yl)-1-propanone, 1-(5,5-dimethyl-1-cyclohexen-1-yl)-4-penten-1-one, 2,3,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydro-2-naphthalenyl methyl ketone, methyl 2,6,10-trimethyl-2,5,9-cyclododecatrienyl ketone, tert-butyl(2,4-dimethyl-3-cyclohexen-1-yl)ketone, etc.;

[0149] Esters of cyclic alcohols, such as 2-tert-butylcyclohexyl acetate, 4-tert-butylcyclohexyl acetate, 2-tert-pentylcyclohexyl acetate, 4-tert-pentylcyclohexyl acetate, 3,3,5-trimethylcyclohexyl acetate, decahydro-2-naphthyl acetate, 2-cyclopentylcyclopentyl crotonate, 3-pentyltetrahydro-2H-pyran-4-yl acetate. 4,7-methano-3a,4,5,6,7,7a-hexahydro-5 or 6-indenyl acetate, 4,7-methano-3a,4,5,6,7,7a-hexahydro-5 or 6-indenyl propionate, 4,7-methano-3a,4,5,6,7,7a-hexahydro-5 or 6-indenyl isobutyrate, 4,7-methanooctahydro-5 or 6-indenyl acetate, etc.;

[0150] Esters of alicyclic alcohols, such as 1-cyclohexylethyl crotonate;

[0151] Esters of alicyclic carboxylic acids, such as allyl 3-cyclohexylpropionate, allyl cyclohexyloxyacetate, cis- and trans-methyl dihydrojasmonate, cis- and trans-methyl jasmonate, methyl 2-hexyl-3-oxocyclopentanecarboxylate, ethyl 2-ethyl-6,6 dimethyl-2-cyclohexenecarboxylate, ethyl 2,3,6,6-tetramethyl-2-cyclohexenecarboxylate, ethyl 2-methyl-1,3-dioxolane-2-acetate, and the like;

[0152] Aromatic aliphatic alcohols, such as benzyl alcohol, 1-phenylethyl alcohol, 2-phenylethyl alcohol, 3-phenylpropanol, 2-phenylpropanol, 2-phenoxyethanol, 2,2-dimethyl-3-phenylpropanol, 2,2-dimethyl-3-(3-methylphenyl)propanol, 1,1-dimethyl-2-phenylethyl alcohol, 1,1-dimethyl-3-phenylpropanol, 1-ethyl-1-methyl-3-phenylpropanol, 2-methyl-5-phenylpentanol, 3-methyl-5-phenylpentanol, 3-phenyl-2-propen-1-ol, 4-methoxybenzyl alcohol, 1-(4-isopropylphenyl)ethanol, etc.;

[0153] Esters of aromatic aliphatic alcohols and aliphatic carboxylic acids, such as benzyl acetate, benzyl propionate, benzyl isobutyrate, benzyl isovalerate, 2-phenylethyl acetate, 2-phenylethyl propionate, 2-phenylethyl isobutyrate, 2-phenylethyl isovalerate, 1-phenylethyl acetate, α-trichloromethylbenzyl acetate, α,α-dimethylphenylethyl acetate, α,α-dimethylphenylethyl butyrate, cinnamyl acetate, 2-phenoxyethyl isobutyrate, 4-methoxybenzyl acetate, etc.;

[0154] Aromatic aliphatic ethers, such as 2-phenylethyl methyl ether, 2-phenylethyl isoamyl ether, 2-phenylethyl 1-ethoxyethyl ether, phenylacetaldehyde dimethyl acetal, phenylacetaldehyde diethyl acetal, hydratropaldehyde dimethyl acetal, phenylacetaldehyde glycerol acetal, 2,4,6-trimethyl-4-phenyl-1,3-dioxane, 4,4a,5,9b-tetrahydroindeno[1,2-d]-m-dioxine, 4,4a,5,9b-tetrahydro-2,4-dimethylindeno[1,2-d]-m-dioxine, etc.;

[0155] Aromatic and araliphatic aldehydes, such as benzaldehyde, phenylacetaldehyde, 3-phenylpropanal, hydratropaldehyde, 4-methylbenzaldehyde, 4-methylphenylacetaldehyde, 3-(4-ethylphenyl)-2,2-dimethylpropanal, 2-methyl-3-(4-isopropylphenyl)propanal, 2-methyl-3-(4-tert-butylphenyl)propanal, 2-methyl-3-(4-isobutylphenyl)propanal, 3-(4-tert-butylphenyl)propanal; cinnamaldehyde, α-butylcinnamaldehyde, α-amylcinnamaldehyde, α-hexylcinnamaldehyde, 3-methyl-5-phenylpentanal, 4-methoxybenzaldehyde, 4-hydroxy-3-methoxy-benzaldehyde, 4-hydroxy-3-ethoxybenzaldehyde, 3,4-methylenedioxybenzaldehyde, 3,4-dimethoxybenzaldehyde, 2-methyl-3-(4-methoxyphenyl)propanal, 2-methyl-3-(4-methylenedioxyphenyl)propanal, etc.;

[0156] Aromatic and araliphatic ketones, such as acetophenone, 4-methylacetophenone, 4-methoxyacetophenone, 4-tert-butyl-2,6-dimethylacetophenone, 4-phenyl-2-butanone, 4-(4-hydroxyphenyl)-2-butanone, 1-(2-naphthalenyl)-ethanone, 2-benzofuranyl ethanone, (3-methyl-2-benzofuranyl) ethanone, benzophenone, 1,1,2,3,3,6-hexamethyl-5-indanyl methyl ketone, 6-tert-butyl-1,1 dimethyl-4 indanyl methyl ketone, 1-[2,3-dihydro-1,1,2,6-tetramethyl-3-(1-methylethyl)-1H-5 indenyl] ethanone, 5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-acetonaphthone, etc.;

[0157] Aromatic and aliphatic carboxylic acids and their esters, such as benzoic acid, phenylacetic acid, methyl benzoate, ethyl benzoate, hexyl benzoate, benzyl benzoate, methyl phenyl acetate, ethyl phenyl acetate, geranyl phenyl acetate, phenylethyl phenyl acetate, methyl cinnamate, ethyl cinnamate, benzyl cinnamate, phenylethyl cinnamate, cinnamyl cinnamate, allyl phenoxy acetate, methyl salicylate, isoamyl salicylate, hexyl salicylate, cyclohexyl salicylate, cis-3-hexenyl salicylate, benzyl salicylate, phenylethyl salicylate, methyl 2,4-dihydroxy-3,6-dimethylbenzoate, ethyl 3-phenylglycidate, ethyl 3-methyl-3-phenylglycidate, etc.;

[0158] Nitrogen-containing aromatic compounds, such as 2,4,6-trinitro-1,3-dimethyl-5-tert-butylbenzene, 3,5-dinitro-2,6-dimethyl-4-tert-butylacetophenone, cinnamonitrile, 3-methyl-5-phenyl-2-pentenonitrile, 3-methyl-5-phenylpentanonitrile, methyl anthranilate, methyl-N-methyl anthranilate, 7-hydroxy-3,7-dimethyloctanal, 2-methyl-3-(4-tert-butylphenyl)propanal or the Schiff base of methyl anthranilate with 2,4-dimethyl-3-cyclohexenecarbaldehyde, 6-isopropylquinoline, 6-isobutylquinoline, 6-sec-butylquinoline, 2-(3-phenylpropyl)pyridine, indole, skatole, 2-methoxy-3-isopropyl-pyrazine, 2-isobutyl-3-methoxypyrazine, etc.;

[0159] Phenols, phenyl ethers and phenyl esters, such as estragole, anethole, eugenol, eugenyl methyl ether, isoeugenol, isoeugenyl methyl ether, thymol, carvacrol, diphenyl ether, β-naphthyl methyl ether, β-naphthyl ethyl ether, β-naphthyl isobutyl ether, 1,4-dimethoxybenzene, eugenyl acetate, 2-methoxy-4-methylphenol, 2-ethoxy-5-(1-propenyl)phenol, p-cresyl phenyl acetate, etc.;

[0160] Heterocyclic compounds, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one, 2-ethyl-4-hydroxy-5-methyl-2H-furan-3-one, 3-hydroxy-2-methyl-4H-pyran-4-one, 2-ethyl-3-hydroxy-4H-pyran-4-one, etc.;

[0161] Lactones, for example 1,4-octanolide, 3-methyl-1,4-octanolide, 1,4-nonanolide, 1,4-decanolide, 8-decen-1,4-olide, 1,4-undecanolide, 1,4-dodecanolide, 1,5-decanolide, 1,5-dodecanolide, 4-methyl-1,4-decanolide, 1,15-pentadecanolide, cis and trans-11-pentadecanolide, cis and trans-1 2-Pentadecen-1,15-olide, 1,16-hexadecanolide, 9-hexadecen-1,16-olide, 10-oxa-1,16-hexadecanolide, 11-oxa-1,16-hexadecanolide, 12-oxa-1,16-hexadecanolide, ethylene 1,12-dodecanedioate, ethylene 1,13-tridecanedioate, coumarin, 2,3-dihydrocoumarin, octahydrocoumarin, and the like.

[0162] In a preferred embodiment, the at least one non-fragrance chemical carrier (ii) is selected from the group consisting of surfactants, oil components, antioxidants, deodorant actives and solvents.

[0163] In the context of the presently claimed invention, a "solvent" serves to dilute the compounds of formula (1) and / or (4) used in accordance with the present invention and / or any further components of the composition that do not have a fragrance of their own.

[0164] The amount of solvent is selected depending on the composition.

[0165] In yet another preferred embodiment, the solvent is selected from the group consisting of ethanol, isopropanol, diethylene glycol monoethyl ether, glycerol, propylene glycol, 1,2-butylene glycol, dipropylene glycol, triethyl citrate, and isopropyl myristate.

[0166] In yet another preferred embodiment, the solvent is present in the composition in an amount of 0.01% to 99.0% by weight, more preferably in an amount of 0.05% to 95.0% by weight, even more preferably in an amount of 0.1% to 80.0% by weight, most preferably in an amount of 0.1% to 70.0% by weight, especially 0.1% to 60.0% by weight, based on the total weight of the composition.

[0167] In yet another preferred embodiment of the present invention, the composition comprises 0.05% to 10% by weight, more preferably 0.1% to 5% by weight, even more preferably 0.2% to 3% by weight of the solvent based on the total weight of the composition. In yet another preferred embodiment of the present invention, the composition comprises 20% to 70% by weight, more preferably 25% to 50% by weight of the solvent based on the total weight of the composition.

[0168] One embodiment of the present invention is directed to a composition comprising a compound of formula (1) and / or (4) and at least one oil component.

[0169] In a preferred embodiment, the oil component is present in an amount of 0.1 to 80 wt.-%, more preferably 0.5 to 70 wt.-%, even more preferably 1 to 60 wt.-%, even more preferably 1 to 50 wt.-%, in particular 1 to 40 wt.-%, more particularly 5 to 25 wt.-%, in particular 5 to 15 wt.-%, based on the total weight of the composition.

[0170] The oil component may be, for example, Guerbet alcohols, based on aliphatic alcohols containing 6 to 18, preferably 8 to 10, carbon atoms, and other further esters, such as, for example, myristyl myristate, myristyl palmitate, myristyl stearate, myristyl isostearate, myristyl oleate, myristyl behenate, myristyl erucate, cetyl myristate, cetyl palmitate, cetyl stearate, cetyl isostearate, cetyl oleate, cetyl behenate, cetyl erucate, stearyl myristate, stearyl palmitate, stearyl stearate, stearyl isostearate, stearyl oleate, stearyl behenate, stearyl erucate, isostearyl myristate, isostearate ... The oleyl ester may be selected from tearyl palmitate, isostearyl stearate, isostearyl isostearate, isostearyl oleate, isostearyl behenate, isostearyl oleate, oleyl myristate, oleyl palmitate, oleyl stearate, oleyl isostearate, oleyl oleate, oleyl behenate, oleyl erucate, behenyl myristate, behenyl palmitate, behenyl stearate, behenyl isostearate, behenyl oleate, behenyl behenate, behenyl erucate, erucyl myristate, erucyl palmitate, erucyl stearate, erucyl isostearate, erucyl oleate, erucyl behenate, and erucyl erucate.Also included are esters of C18 to C38 alkyl-hydroxycarboxylic acids with linear or branched C6 to C22 fatty alcohols, more specifically dioctyl malate, esters of linear and / or branched fatty acids with polyhydric alcohols (for example propylene glycol, dimer diols or trimer triols), triglycerides based on C6 to C10 fatty acids, liquid mono-, di- and triglyceride mixtures based on C6 to C18 fatty acids, esters of C6 to C22 fatty alcohols and / or Guerbet alcohols with aromatic carboxylic acids, more specifically benzoic acid, esters of dicarboxylic acids with polyols containing 2 to 10 carbon atoms and 2 to 6 hydroxyl groups, vegetable oils, branched primary alkyl esters, Suitable are choline, substituted cyclohexanes, linear and branched C6-C22 fatty alcohol carbonates, for example dicaprylyl carbonate (Cetiol® CC), Guerbet carbonates based on fatty alcohols containing 6 to 18, preferably 8 to 10, carbon atoms, esters of benzoic acid with linear and / or branched C6-C22 alcohols (for example Finsolv® TN), linear or branched, symmetrical or asymmetrical dialkyl ethers containing 6 to 22 carbon atoms per alkyl group, for example dicaprylyl ether (Cetiol® OE), ring-opening products of epoxidized fatty acid esters with polyols and hydrocarbons or mixtures thereof.

[0171] It should be understood that antioxidants can inhibit or prevent undesirable changes in the composition to be protected caused by oxygen effects and other oxidation processes. The effect of antioxidants is in most cases to act as free radical scavengers for the free radicals generated during autoxidation.

[0172] In a preferred embodiment, the antioxidant is selected from the group consisting of: Amino acids (e.g., glycine, alanine, arginine, serine, threonine, histidine, tyrosine, tryptophan, etc.) and their derivatives, Imidazoles (e.g. urocanic acid) and their derivatives, Peptides such as D,L-carnosine, D-carnosine, L-carnosine (=β-alanyl-L-histidine) and their derivatives, Carotenoids, carotenes (e.g. α-carotene, β-carotene, lycopene, lutein) or their derivatives, Chlorogenic acid and its derivatives, Lipoic acid and its derivatives (e.g. dihydrolipoic acid), aurothioglucose, propylthiouracil and other thiols (e.g. thioredoxin, glutathione, cysteine, cystine, cystamine and their glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, gamma-linoleyl, cholesteryl and glyceryl esters) and their salts, Dilauryl thiodipropionate, distearyl thiodipropionate, thiodipropionic acid and their derivatives (esters, ethers, peptides, lipids, nucleotides, nucleosides and salts), Sulfoximine compounds (e.g. buthionine sulfoximine, homocysteine ​​sulfoximine, buthionine sulfone, penta-, hexa-, and heptathionine sulfoximine), (Metal) chelating agents (e.g. alpha-hydroxy fatty acids, palmitic acid, phytic acid, lactoferrin), α-hydroxy acids (e.g. citric acid, lactic acid, malic acid), Humic acid, bile acids, bile extract, bilirubin, biliverdin, boldin (= alkaloids from the plant, Peumus boldus, boldo extract, EDTA, EGTA and their derivatives, Unsaturated fatty acids and their derivatives (e.g., gamma-linolenic acid, linoleic acid, oleic acid), Folic acid and its derivatives, Ubiquinone and ubiquinol and their derivatives, Vitamin C and derivatives (e.g., ascorbyl palmitate, Mg Ascorbyl phosphate, Ascorbyl acetate), Tocopherol and derivatives (e.g. vitamin E acetate), Vitamin A and derivatives (e.g. vitamin A palmitate), Coniferyl benzoate of gum benzoin, rutinic acid and its derivatives, α-glycosyl rutin, ferulic acid, furfurylidene glucitol, Butylated hydroxytoluene (BHT), Butylated hydroxyanisole (BHA), Nordihydroguaiacic acid, nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid and their derivatives, mannose and its derivatives, Superoxide dismutase, Zinc and its derivatives (e.g. ZnO, ZnSO4), Selenium and its derivatives (e.g. selenomethionine) Stilbene and its derivatives (e.g. stilbene oxide, trans-stilbene oxide).

[0173] In a preferred embodiment, the antioxidant is selected from the group consisting of pentaerythrityl tetradibutylhydroxyhydrocinnamate, nordihydroguaiaretic acid, ferulic acid, resveratrol, propyl gallate, butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), ascorbyl palmitate, and tocopherol.

[0174] In yet another preferred embodiment, the composition according to the currently claimed invention may comprise the antioxidant in an amount of 0.001 to 25 wt. %, preferably 0.005 to 10 wt. %, more preferably 0.01 to 8 wt. %, even more preferably 0.025 to 7 wt. %, and even more preferably 0.05 to 5 wt. %, based on the total weight of the composition.

[0175] Deodorant compositions (deodorants and antiperspirants) neutralize, mask or eliminate body odor, which is formed by the action of skin bacteria on apocrine sweat, resulting in the formation of unpleasant-smelling decomposition products.

[0176] Thus, one embodiment of the present invention is directed to a composition comprising a compound of formula (1) and / or (4) and at least one deodorant active, in a preferred embodiment, the deodorant active is selected from the group consisting of antiperspirants, esterase inhibitors, and antimicrobial agents.

[0177] Suitable antiperspirants are selected from the group consisting of aluminum, zirconium or zinc salts. Examples are aluminum chloride, aluminum chlorohydrate, aluminum dichlorohydrate, aluminum sesquichlorohydrate and their complexes, for example with 1,2-propylene glycol, aluminum hydroxyalantoinate, aluminum chloride tartrate, aluminum zirconium trichlorohydrate, aluminum zirconium tetrachlorohydrate, aluminum zirconium pentachlorohydrate and their complexes, for example with amino acids, such as glycine. Aluminum chlorohydrate, aluminum zirconium tetrachlorohydrate, aluminum zirconium pentachlorohydrate and their complexes are preferably used.

[0178] In preferred embodiments, the antiperspirant is selected from the group consisting of aluminum chloride, aluminum chlorohydrate, aluminum dichlorohydrate, aluminum sesquichlorohydrate, aluminum hydroxyalantoinate, aluminum chloride tartrate, aluminum zirconium trichlorohydrate, aluminum zirconium tetrachlorohydrate, and aluminum zirconium pentachlorohydrate.

[0179] When sweat is present in the armpit area, extracellular enzymes-esterases, mainly proteases and / or lipases, break down the esters formed by bacteria and present in sweat, releasing odor in the process.Suitable esterase inhibitors are, for example, trialkyl citrates, such as trimethyl citrate, tripropyl citrate, triisopropyl citrate, tributyl citrate, and especially triethyl citrate.Esterase inhibitors inhibit enzyme activity and thus reduce odor generation.Free acid is probably released by cleavage of citrate esters, reducing the pH value of the skin to such an extent that enzymes are inactivated by acylation. Other esterase inhibitors are, for example, sterol sulfates or phosphates, such as the sulfates or phosphates of lanosterol, cholesterol, campesterol, stigmasterol and sitosterol, dicarboxylic acids and their esters, for example glutaric acid, glutaric acid monoethyl ester, glutaric acid diethyl ester, adipic acid, adipic acid monoethyl ester, adipic acid diethyl ester, malonic acid and malonic acid diethyl ester, hydroxycarboxylic acids and their esters, for example citric acid, malic acid, tartaric acid or tartaric acid diethyl ester and zinc glycine.

[0180] In a preferred embodiment, the esterase inhibitor is selected from the group consisting of trimethyl citrate, tripropyl citrate, triisopropyl citrate, tributyl citrate, triethyl citrate, lanosterol, cholesterol, campesterol, stigmasterol, sitosterol sulfate, sitosterol phosphate, glutaric acid, glutaric acid monoethyl ester, glutaric acid diethyl ester, adipic acid, adipic acid monoethyl ester, adipic acid diethyl ester, malonic acid, malonic acid diethyl ester, citric acid, malic acid, tartaric acid, tartaric acid diethyl ester, and zinc glycinate.

[0181] The compositions according to the presently claimed invention contain an esterase inhibitor in the range of 0.01 to 20% by weight, preferably 0.1 to 10% by weight, and more specifically 0.5 to 5% by weight, based on the total weight of the composition.

[0182] The term "antimicrobial agent" as used herein includes substances that have bactericidal and / or bacteriostatic properties.Typically, these substances include, for example, 4-hydroxybenzoic acid and its salts and esters, N-(4-chlorophenyl)-N'-(3,4-dichlorophenyl)-urea, 2,4,4'-trichloro-2'-hydroxydiphenyl ether (triclosan), 4-chloro-3,5-dimethylphenol, 2,2'-methylene-bis-(6-bromo-4-chlorophenol), 3-methyl-4-(1-methylethyl)-phenol, 2-benzyl-4-chlorophenol, 3-(4-chlorophenoxy)-4-hydroxybenzoic acid, ... -propane-1,2-diol, 3-iodo-2-propynyl butylcarbamate, chlorhexidine, 3,4,4'-trichlorocarbanilide (TTC), phenoxyethanol, glycerol monocaprate, glycerol monocaprylate, glycerol monolaurate (GML), diglycerol monocaprate (DMC), and salicylic acid-N-alkylamides, such as salicylic acid-n-octylamide or salicylic acid-n-decylamide, act against gram-positive bacteria.

[0183] In a preferred embodiment, the antimicrobial agent is chitosan, phenoxyethanol, 5-chloro-2-(2,4-dichlorophenoxy)-phenol, 4-hydroxybenzoic acid and its salts and esters, N-(4-chlorophenyl)-N'-(3,4-dichlorophenyl)-urea, 2,4,4'-trichloro-2'-hydroxydiphenyl ether (triclosan), 4-chloro-3,5-dimethylphenol, 2,2'-methylene-bis-(6-bromo-4-chlorophenol), 3-methyl-4-(1-methylphenyl)-2-(2 ... The glycerol monocaprate is selected from the group consisting of glycerol monocaprylate, glycerol monolaurate (GML), diglycerol monocaprate (DMC), and salicylic acid-N-alkylamide. Compositions according to the presently claimed invention contain the antimicrobial agent in the range of 0.01 to 5% by weight, preferably 0.1 to 2% by weight, based on the total weight of the composition.

[0184] In a preferred embodiment, the composition preferably comprises a surfactant.Due to the characteristic fragrance properties of the compounds of formula (1) and / or (4) and their substantivity, tenacity and stability, they can be particularly used to impart odor, preferably fragrance or fragrance impression to surfactant-containing compositions, such as detergents (especially laundry care products and multi-purpose detergents).They can be preferably used to impart persistent floral and / or green and / or sweet notes and / or woody notes and / or root notes and / or violet notes odor impression to surfactant-containing compositions.

[0185] In a preferred embodiment, the surfactant is selected from the group consisting of anionic, nonionic, cationic, amphoteric, and zwitterionic surfactants, hi yet another preferred embodiment, the surfactant is an anionic surfactant.

[0186] Thus, compositions according to the presently claimed invention may preferably comprise at least one surfactant. The surfactant may be selected from anionic, nonionic, cationic and / or amphoteric or zwitterionic surfactants. Surfactant-containing compositions, such as shower gels, foam baths, shampoos, etc., preferably comprise at least one anionic surfactant.

[0187] The compositions according to the invention usually contain surfactants in an amount of 0-40% by weight, preferably 0-20% by weight, more preferably 0.1-15% by weight, especially 0.1-10% by weight, in the aggregate, based on the total weight of the composition. Typical examples of non-ionic surfactants are fatty alcohol polyglycol ethers, alkylphenol polyglycol ethers, fatty acid polyglycol esters, fatty acid amide polyglycol ethers, fatty amine polyglycol ethers, alkoxylated triglycerides, mixed ethers and mixed formals, optionally partially oxidized alkyl(enyl) oligoglycosides or glucuronic acid derivatives, fatty acid-N-alkylglucamides, protein hydrolysates (especially wheat-based vegetable products), polyol fatty acid esters, sugar esters, sorbitan esters, polysorbates and amine oxides. When the non-ionic surfactants contain polyglycol ether chains, they may have a conventional homolog distribution, but preferably have a narrow range homolog distribution.

[0188] Zwitterionic surfactants are surface-active compounds that contain at least one quaternary ammonium group and at least one COO(-) or SO3(-) group in the molecule. Particularly suitable zwitterionic surfactants are so-called betaines, such as N-alkyl-N,N-dimethylammonium glycinates, such as cocoalkyldimethylammonium glycinate, N-acylaminopropyl-N,N-dimethylammonium glycinates, such as cocoacylaminopropyldimethylammonium glycinate, and 2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines containing 8 to 18 carbon atoms in the alkyl or acyl group, and cocoacylaminoethylhydroxyethylcarboxymethylglycinate. Particularly preferred is the fatty acid amide derivative known under the CTFA name of cocamidopropyl betaine.

[0189] Also suitable as cosurfactants are amphoteric surfactants. Amphoteric surfactants are surface-active compounds that contain at least one free amino group and at least one -COOH or SO3H group in the molecule in addition to a C8-C18 alkyl or acyl group and can form an intramolecular salt. Examples of suitable amphoteric surfactants are N-alkyl glycines, N-alkyl propionic acids, N-alkyl amino butyric acids, N-alkyl imino dipropionic acids, N-hydroxyethyl-N-alkyl amidopropyl glycines, N-alkyl taurines, N-alkyl sarcosines, 2-alkyl amino propionic acids, and alkyl amino acetic acids, with the alkyl groups having about 8 to 18 carbon atoms. Particularly preferred amphoteric surfactants are N-cocoa alkyl amino propionates, cocoa acyl amino ethyl amino propionates, and acyl sarcosines.

[0190] Anionic surfactants are characterized by a water-soluble anionic group, for example a carboxylate, sulfate, sulfonate or phosphate group, and a lipophilic group. Dermatologically safe anionic surfactants are known to many practitioners from the relevant textbooks and are commercially available. They are in particular alkyl sulfates in the form of their alkali metal, ammonium or alkanolammonium salts, alkyl ether sulfates, alkyl ether carboxylates, acyl isethionates, acyl sarcosinates, acyltaurines containing linear C12-C18 alkyl or acyl groups, and sulfosuccinates, and acyl glutamates in the form of their alkali metal or ammonium salts.

[0191] Particularly suitable cationic surfactants are quaternary ammonium compounds, preferably ammonium halides, more particularly chlorides and bromides such as alkyltrimethylammonium chloride, dialkyldimethylammonium chloride and trialkylmethylammonium chloride, for example cetyltrimethylammonium chloride, stearyltrimethylammonium chloride, distearyldimethylammonium chloride, lauryldimethylammonium chloride, lauryldimethylbenzylammonium chloride and tricetylmethylammonium chloride. In addition, easily biodegradable quaternary ester compounds, such as dialkylammonium methosulfate and methylhydroxyalkyldialkyloxyalkylammonium methosulfate, commercially available under the name Stepantexe, and the corresponding products of the Dehyquart® series, can be used as cationic surfactants. "Esterquats" are generally understood to be quaternized fatty acid triethanolamine ester salts. They can impart a certain softness to the composition. They are known substances that are prepared by the relevant methods of organic chemistry. Other cationic surfactants suitable for use according to the invention are the quaternized protein hydrolysates.

[0192] One embodiment of the currently claimed invention is directed to a composition selected from the group consisting of a perfume composition, a body care composition, a hygiene product, a cleaning composition, a fabric cleaning composition, a fragrance dispenser composition, a food product, a dietary supplement, a pharmaceutical composition, and a crop protection composition.

[0193] The aforementioned compositions are preferably aroma chemical compositions, more preferably fragrance compositions.

[0194] Suitable compositions are, for example, perfume compositions, body care compositions (including cosmetic compositions and oral and dental hygiene products), hygiene products, cleaning compositions (including dishwashing compositions), textile detergent compositions, compositions for scent dispensers, foods, dietary supplements, pharmaceutical compositions and crop protection compositions.

[0195] The perfume compositions may be selected from fine fragrances, deodorants in liquid, gel or applied to a solid carrier, aerosol sprays, scented cleansers, perfumed candles, and oils such as lamp oils or massage oils.

[0196] Examples of fine fragrances are perfume extracts, Eau de Parfum, Eau de Toilette, Eau de Cologne, Eau de Solide, and Extrait Parfum.

[0197] Body care compositions include cosmetic compositions and oral and dental hygiene products, such as aftershave, preshave products, splash colognes, bar and liquid soaps, shower gels, shampoos, shaving soaps, shaving foams, bath oils, oil-in-water, water-in-oil and water-in-oil-in-water type cosmetic emulsions, such as skin creams and lotions, face creams and lotions, sunscreen creams and lotions, after-sun creams and lotions, hand creams and lotions, foot creams and lotions, depilatory creams and lotions, aftershave creams and lotions, sun protection creams and lotions, hair care products, such as hairsprays, hair gels, setting hair lotions, hair conditioners, hair shampoos, permanent and semi-permanent hair colorants, kohls, and the like. hair shaping compositions such as waving and hair smoothing compositions, hair tonics, hair creams and hair lotions, deodorants and antiperspirants such as underarm sprays, roll-ons, deodorant sticks and deodorant creams, decorative cosmetic products such as eyeliners, eye shadows, nail polishes, make-up products, lipsticks and mascaras, and oral and dental hygiene products such as toothpastes, dental floss, mouthwashes, breath fresheners, dental foams, dental gels, and dental strips.

[0198] The hygiene products may be selected from incense sticks, insecticides, repellents, propellants, rust removers, perfumed freshening wipes, underarm pads, baby diapers, sanitary napkins, toilet paper, cosmetic wipes, pocket tissues, dish detergents, and deodorants.

[0199] Cleaning compositions such as cleaners for solid surfaces may be selected from perfumed, acidic, alkaline and neutral cleaners, e.g. floor cleaners, window cleaners, dishwashing compositions both for hand and machine washing, bath and sanitary cleaners, scouring milk, solid and liquid toilet cleaners, powder and foam carpet cleaners, waxes and polishes such as furniture polishes, floor waxes, shoe creams, disinfectants, surface disinfectants and sanitary cleaners, brake cleaners, pipe cleaners, limescale removers, grill and oven cleaners, algae and moss removers, mould removers, facade cleaners.

[0200] The textile detergent composition may be selected from liquid detergents, powder detergents, laundry pre-treatments such as bleaches, soaking agents and stain removers, fabric softeners, laundry soaps, laundry tablets.

[0201] Food means any raw, cooked, or processed edible substance, ice, beverage, or composition used or intended for use, in whole or in part, for human consumption, or chewing gum, gummies, jellies, and confectioneries.

[0202] A dietary supplement is a product intended for ingestion that contains dietary ingredients intended to add additional nutritional value to the diet. The dietary ingredients can be one or any combination of the following substances: vitamins, minerals, herbs or other botanicals, amino acids, dietary substances that people use to supplement their diet by increasing their total dietary intake, concentrates, metabolites, ingredients, or extracts. Dietary supplements can be found in many forms, such as tablets, capsules, softgels, gelcaps, liquids, or powders.

[0203] Pharmaceutical compositions include compositions intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, as well as articles (other than food) intended to affect the structure or any function of the body of humans or other animals.

[0204] Crop protection compositions include compositions intended for the management of plant diseases, weeds and other pests (both vertebrate and invertebrate) which damage agricultural crops and forests.

[0205] In a preferred embodiment, the composition comprises an antiseptic, an abrasive, an anti-acne agent, an agent for combating skin aging, an anti-cellulite agent, an anti-dandruff agent, an anti-inflammatory agent, an inflammation suppressing agent, an irritation soothing agent, an astringent, an antiperspirant, an antiseptic, an antistatic agent, a binder, a buffering agent, a carrier material, a chelating agent, a cell stimulant, a care agent, a depilatory agent, an emulsifier, an enzyme, an essential oil, a fiber, a film-forming agent, a fixing agent, a foaming agent, a foam stabilizing agent, an antifoaming agent, a foaming accelerator, a disinfectant, a gelling agent, a gel-forming agent, a hair care agent, a hair shaping agent, a hair styling agent, a moisture donating agent, a moisturizing agent, a humectant, a bleaching agent, a strengthening agent, a stain remover, an optical brightener, an impregnating agent, an antifouling agent, a friction reducing agent, a lubricant, a moisturizing cream, an ointment, an opacifying agent, a plasticizer, a coating In some embodiments, the composition further comprises at least one auxiliary selected from the group consisting of: agents, polishing agents, gloss agents, polymers, powders, proteins, refatting agents, exfoliants, silicones, skin soothing agents, skin cleansing agents, skin care agents, skin healing agents, skin whitening agents, skin protection agents, skin emollients, cooling agents, skin cooling agents, warming agents, skin warming agents, stabilizers, UV absorbers, UV filters, softeners, suspending agents, skin sunscreens, thickeners, vitamins, waxes, fats, phospholipids, saturated fatty acids, mono- or polyunsaturated fatty acids, hydroxy acids, polyhydroxy fatty acids, liquefying agents, dyes, color protection agents, pigments, corrosion inhibitors, polyols, electrolytes, and silicone derivatives.

[0206] For example, the method may involve reacting α-ionylideneethane without or with α-ionone: (i) at least one further aroma chemical different from α-ionylideneethane or α-ionone, or (ii) at least one non-aromatic chemical carrier; or (iii) may be carried out by mixing both (i) and (ii).

[0207] The present invention is also directed to a method for modifying the fragrance properties (e.g., odor properties) of an aroma chemical composition, such as, for example, a perfumed composition, in particular a perfumed ready-to-use composition, comprising the step of introducing α-ionylideneethane, without or with α-ionone, into an aroma chemical composition, such as, for example, a perfumed composition, in particular a perfumed ready-to-use composition.

[0208] In particular, the present invention is directed to a method for preparing a perfume composition, a body care composition, a hygiene product, a cleaning composition, a textile detergent composition, a composition for a scent dispenser, a food product, a dietary supplement, a pharmaceutical composition or a crop protection composition comprising including α-ionylideneethane without or with α-ionone in the perfume composition, the body care composition, the hygiene product, the cleaning composition, the textile detergent composition, a composition for a scent dispenser, a food product, a dietary supplement, a pharmaceutical composition or a crop protection composition.

[0209] In one embodiment, the present invention is directed to a method for imparting sweet, floral, violet, orris, rooty, and / or woody reminiscent notes to a perfume composition, a body care composition, a hygiene product, a cleaning composition, a textile detergent composition, a fragrance dispenser composition, a food product, a dietary supplement, a pharmaceutical composition, or a crop protection composition, comprising including α-ionylideneethane without or with α-ionone in the perfume composition, the body care composition, the hygiene product, the cleaning composition, the textile detergent composition, the fragrance dispenser composition, a food product, a dietary supplement, a pharmaceutical composition, or a crop protection composition.

[0210] Preferably the process of the present invention is or comprises a fermentation process.

[0211] Additionally, the present invention relates to a fragrance compound and / or fragrance composition and / or perfumed or scented product comprising: i) at least one α-ionylideneethane as defined herein, preferably an α-ionylideneethane as defined in claim 1 or 2; ii) optionally at least one further aroma compound different from i); iii) optionally at least one diluent; The present invention relates to a fragrance compound and / or a fragrance composition and / or a perfume scented or scented product comprising:

[0212] Preferably, the fragrance compound and / or fragrance composition and / or perfumed or scented product of the present invention comprises i) and ii), or i) and iii), more preferably i), ii) and iii).

[0213] The present invention also relates to a perfumed or scented product comprising at least one α-ionylideneethane as defined herein, preferably an α-ionylideneethane having floral-violet and / or woody-orris / iris root notes, more preferably 2Z,4E-α-ionylideneethane.

[0214] For example, the α-ionylideneethanes as defined herein, preferably the α-ionylideneethanes as defined herein, can be used in compositions selected from perfumes, detergent and cleaning compositions, cosmetics, body care products, hygiene products, oral and dental hygiene products, scent dispensers, and other compositions and products as defined herein.

[0215] As used herein, the singular forms "a," "an," and "the" include both singular and plural references unless the context clearly dictates otherwise. By way of example, "a cell" refers to one or more cells.

[0216] As used herein, the term "about," when quantifying the value of a referenced item, number, percentage, or term, refers to a range of plus or minus 10 percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the value of the referenced item, number, percentage, or term. A range of plus or minus 10 percent is preferred.

[0217] As used herein, the terms "comprising", "comprises" and "comprised of" are synonymous with "including", "includes" or "containing" or "contains" and are inclusive or open-ended and do not exclude additional, unrecited components, elements or method steps. Clearly, the term "comprising" encompasses the term "consisting of". More specifically, the term "comprise", as used herein, means that the claim includes all the recited elements or method steps, but may also include additional unspecified elements or method steps. For example, a method comprising steps a), b) and c) encompasses, in the narrowest sense, a method consisting of steps a), b) and c). The phrase "consisting of" means that the composition (or kit or method) has the recited elements (or steps) and nothing else. In contrast, the term "comprises" may also encompass a method comprising further steps, such as steps a), b) and c) as well as steps d) and e).

[0218] For clarity, when numerical ranges are used herein, such as "a concentration of 1 to 5 micromolar," the range includes not only 1 and 5 micromolar, but also all values ​​between 1 and 5 micromolar, for example, 2, 3 and 4 micromolar.

[0219] By definition, the term "in vitro" refers to outside of a living body and an artificial environment. Thus, the term "in vitro" as used herein refers to outside or outside of an animal or human body. The term "in vitro" as used herein should be understood to include "ex vivo." The term "ex vivo" generally refers to tissues or cells that are removed from an animal or human body and maintained or grown outside the body, for example in a culture vessel. The term "in vivo" as used herein refers to inside or inside of an animal or human body.

[0220] By definition, the term "terpene" includes only hydrocarbons composed of carbon and hydrogen. In contrast, the term "terpenoid" refers to terpenes that contain further functional groups that give rise to derivatives such as alcohols, aldehydes, ketones and acids; see e.g. Flavors and Fragrances: Chemistry, Bioprocessing and Sustainability RG Berger; Black et al., Terpenoids and their role in wine flavour: recent advances. Australian Journal of Grape and Wine Research 21, 582-600, 2015; Zhou & Pichersky, More is better: the diversity of terpene metabolism in plants. Current Opinion in Plant Biology 2020, 55:1-10; Degenhardt J, Kollner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70(15):1621-1637). In the scientific literature, the term terpene is often used interchangeably with the term terpenoid, although they have different meanings. As used herein, the term "terpene" includes both hydrocarbons and their functional derivatives.

[0221] Sesquiterpenes are C15 terpenoids composed of three isoprene units. Like monoterpenes, sesquiterpenes can be acyclic or contain rings with many unique combinations. They are found in many other biological systems, especially in higher plants and marine organisms and fungi. Naturally, they occur as hydrocarbons or in oxygenated forms such as lactones, alcohols, acids, aldehydes, and ketones. Sesquiterpenes also include essential oils and aromatic compounds that have some pharmacological activity.

[0222] "Fragrance compounds", also known as fragrances, fragrances, odorants, or flavors - or their sensory components - are chemicals that have sensory properties that indicate a wide variety of odors. They include many classes of volatile chemical compounds, such as alcohols, aldehydes, ketones, acids, esters, lactones, and terpenes, and are widely used in the food, detergent, cosmetic, and pharmaceutical industries. For an individual chemical or class of chemicals to impart an odor or scent, ideally it must be volatile enough to be transmitted through the air to the olfactory system at the top of the nose. Primarily, the sensory properties are important, i.e. the compound must have advantageous odor (olfactory) or taste properties. Furthermore, the fragrance compound must also have further positive secondary properties, such as, for example, an efficient method of preparation, the possibility of providing a better sensory profile as a result of synergy with other fragrances, higher stability under certain application conditions, higher extendibility, better and longer lasting properties, etc. As described elsewhere herein, the inventors have been able to identify ionylidene ethane as a fragrance compound. This discovery was unexpected since ionylideneethane has not previously been considered a fragrance compound. It has further been found by the inventors that α-ionylideneethane may be used to prepare one or more fragrance compounds that impart floral-violet and / or woody-orris / iris root notes to a perfume, fragrance or fragrance.

[0223] As used herein, "fragrance compound" includes at least one fragrance compound, but may also include 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more fragrance compounds. The fragrance compound may further include one or more diluents or other ingredients, such as ingredients defined herein.

[0224] Fragrance compositions and ingredients are well known in the art (see, e.g., Fundamentals of Fragrance Chemistry, Charles S. Sell, John Wiley & Sons (2019)) and are also illustrated in the examples below.

[0225] A "perfumed or scented product" is a product that contains at least one fragrance compound, such as α-ionylideneethane and / or α-ionone, and can include, for example, consumer products such as fine fragrances, personal care products, home care products, and air care products, preferably fine fragrances such as parfum, extrait de parfum, eau de parfum, millesime, parfum de toilette, eau de toilette, Preferably the personal care product is selected from lotions, creams, moisturizers, body washes, hand soaps, shampoos, conditioners, and soaps; preferably the home care product is selected from fabric conditioners, fabric softeners, laundry detergents, laundry additives, rinse additives, bleach, dryer sheets, perfume beads, car care products, dishwashing detergents, and hard surface cleaners; preferably the air care product is selected from candles, aerosols, air fresheners, liquid electric deodorants, air freshener diffusers, gel deodorants, plug-in deodorants, plug-in oils, and wax melts; see, e.g., EP 3468527 B1.

[0226] The terms "protein" or "polypeptide" or "(poly)peptide" or "peptide" (all terms used interchangeably unless otherwise indicated) as used herein encompass isolated and / or purified and / or recombinant (poly)peptides that are essentially free from other host cell polypeptides. The term "peptide", as referred to herein, comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or even more amino acid residues, wherein the alpha carboxyl group of one amino acid residue is bonded to the alpha amino group of another amino acid residue. A post-translational modification of a protein or peptide as used and contemplated herein is a modification of a newly formed protein or peptide and may include deletion, substitution or addition of amino acids, chemical modification of certain amino acids, such as amidation, acetylation, phosphorylation, glycosylation, formation of pyroglutamic acid, oxidation / reduction of the sulfa group at methionine or addition of similar small molecules to certain amino acids.

[0227] As is well known to those skilled in the art, enzymes are proteins. Enzymes bind their substrates and convert them into products. A plot of the initial reaction velocity versus substrate concentration shows a rectangular hyperbola. The reaction velocity (v) is equal to (Vmax[A]) / (Km+[A]) as described by the Michaelis-Menten equation, where Vmax is the maximum velocity, [A] is the substrate concentration, and Km is the Michaelis constant or substrate concentration at half-maximal velocity. Steady-state enzyme kinetics is used to determine Km values ​​for substrates, Vmax values ​​for enzymes, and Ki values ​​for various inhibitors, including drugs.

[0228] The "turnover number" (kcat or catalytic rate constant) of an enzyme is the maximum number of molecules of substrate that can be converted to product per active site per unit time for several different substrates to different products. The kcat / Km values ​​or specificity constants of various substrates can be compared. The substrate with the largest value is the best substrate for the enzyme, explaining the name specificity constant. The rate of any reaction is limited by the rate at which reactant molecules collide. Diffusion limited rates for biomolecular reactions are around 10 8 ~10 9 M -1 s -1 The ratio kcat / Km is the first-order rate constant. The product of kcat / Km and the substrate concentration (at subsaturating levels) gives the rate of the enzyme-catalyzed reaction. This ratio is proportional to the substrate concentration and is therefore specified as first order. 10 8 ~10 9 M -1 s -1 Enzymes with a ratio of kcat / Km in the vicinity (close to the maximum allowed by the diffusion rate) achieve full catalysis. For example, triosephosphate isomerase (EC 5.3.1.1), an enzyme in the glycolytic pathway, is an enzyme with this property. However, most enzymes have specificity constants orders of magnitude below this value. Methods for determining the turnover number of an enzyme are well known in the art; see, for example, https: / / doi.org / 10.1016 / B978-0-12-801238-3.05143-6 or Heckmann et al., PNAS September 15, 2020 117(37)23182-23190; https: / / doi.org / 10.1073 / pnas.2001562117.

[0229] Sequence identity, homology or similarity is defined herein as the relationship between two or more amino acid sequences or two or more nucleic acid sequences as determined by comparing these sequences. Usually, sequence identity or similarity is compared over the entire length of the sequences, but it may also be compared over only a portion of the alignment of the sequences with each other. Preferably, sequence identity or similarity is compared herein over the entire length of the sequences. In the art, "identity" or "similarity" also means the degree of sequence relatedness between polypeptide sequences or nucleic acid sequences, as the case may be, as determined by the match between such sequences.

[0230] Sequence alignments can be generated using several software tools, such as: -Needleman and Wunsch Algorithm -Needleman, Saul B. & Wunsch, Christian D. (1970). “A general method applicable to the search for similarities in the amino acid sequence of two proteins”. Journal of Molecular Biology 48(3):443-453.

[0231] This algorithm is incorporated, for example, in the "NEEDLE" program, which performs a global alignment of two sequences. The NEEDLE program is contained, for example, in the European Molecular Biology Open Software Suite (EMBOSS). -EMBOSS - a collection of various programs:The European Molecular Biology Open Software Suite (EMBOSS), Trends in Genetics 16(6), 276(2000). -BLOSUM (BLOcks SUbstitution Matrix) - typically generated based on alignment of conserved regions of e.g. protein domains (Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the USA. 1992 Nov 15; 89(22): 10915-9). One of the many BLOSUMs is "BLOSUM62", which is often the "default" setting for many programs when aligning protein sequences. -BLAST (Basic Local Alignment Search Tool) - consists of several individual programs (BlastP, BlastN) that are primarily used to search for similar sequences in large sequence databases. BLAST programs also generate local alignments. An improved version ("BLAST2"), the "BLAST" interface provided by NCBI (National Center for Biotechnology Information), is usually used. “Original” BLAST: Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) “Basic local alignment search tool.” J.Mol.Biol.215:403-410;BLAST2: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402.

[0232] Sequence identity, as used herein, is preferably the value as determined by the EMBOSS pairwise alignment algorithm "Needle". In particular, the NEEDLE program from the EMBOSS package may be used, using the NOBRIEF option ('Brief identity and similarity' to NO), which calculates the "longest identity" (version 2.8.0 and later, EMBOSS: The European Molecular Biology Open Software Suite - Rice, P., et al. Trends in Genetics (2000) 16: 276-277; http: / / emboss.bioinformatics.nl). The identity, homology or similarity between two aligned sequences is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids in both sequences is divided by the total length of the alignment after subtracting the total number of gaps in the alignment. For the alignment of amino acid sequences, the default parameters are: matrix=Blosum62; open gap penalty=10.0; gap extension penalty=0.5. For alignment of nucleic acid sequences, the default parameters are: matrix=DNAfull; open gap penalty=10.0; gap extension penalty=0.5.

[0233] As used herein, the term "α-ionylideneethane synthase" refers to a sesquiterpene synthase capable of converting farnesyl diphosphate to α-ionylideneethane. Thus, as used herein, the term "α-ionylideneethane synthase activity" refers to an enzyme activity that catalyzes the conversion of farnesyl diphosphate to α-ionylideneethane, preferably via cyclization of farnesyl diphosphate to α-ionylideneethane. α-Ionylideneethane synthase genes have been found in microorganisms, including fungi as well as bacteria, and have been well described in the art (Takino et al., J. Am. Chem. Soc. 2018, 140, 39, 12392-12395; Siewers et al., Appl. Environ. Microbiol. 72:4619-4626(2006); Otto et al., Microb Cell Fact(2019)18:205; Inomata et al., Bioscience, Biotechnology, and Biochemistry, Volume 68, Issue 12, 1 January 2004, Pages 2571-2580, https: / / doi.org / 10.1271 / bbb.68.2571; Takino et al., 2019, Bioscience Biotechnology and Biochemistry, 83(9), 1642-1649). For example, Takino et al., 2019, described the cyclization of farnesyl diphosphate to α-ionylideneethane catalyzed by a novel sesquiterpene synthase BcABA3, which shows low amino acid sequence identity with sesquiterpene synthases, in the abscisic acid biosynthetic pathway in plant pathogenic fungi. Another version of the BcABA3 enzyme and its use in abscisic acid production are reported in Chinese patent application CN108753744.

[0234] "Homolog" means bacterial, fungal, plant or animal homologs, preferably plant homologs, of the proteins or enzymes referred to herein, such as α-ionylideneethane synthase as defined herein, but also includes truncated sequences, single stranded DNA or RNA of coding and non-coding DNA sequences.

[0235] An enzyme variant may be defined by its sequence identity when compared to a parent protein or enzyme, such as an α-ionylideneethane synthase having an amino acid sequence set forth in any one of SEQ ID NOs: 1-17 or 19-33.

[0236] Sequence identity is usually provided as "% sequence identity" or "% identity". In a first step, to determine the percent identity between two amino acid sequences, a pairwise sequence alignment is made between the two sequences, aligning the two sequences over their entire, total or full length (i.e., pairwise global alignment). The alignment is generated using a program or software described herein. A preferred alignment for the purposes of the present invention is one in which the maximum sequence identity can be determined.

[0237] As used herein, "protein" or "polypeptide" or "peptide" encompasses peptidomimetics of proteins or enzymes referred to herein, such as α-ionylideneethane synthase as defined herein. As known in the art, peptidomimetics are compounds whose essential elements (pharmacophores) mimic natural peptides or proteins in 3D space, retain the ability to interact with biological targets (such as enzyme substrates), and produce the same biological effect (such as α-ionylideneethane synthase activity), see for example the review by Vagner et al. 2008, Current Opinion in Chemical Biology 12, Pages 292-296. Peptidomimetics are designed to avoid some of the problems associated with natural polypeptides, such as proteolytic stability (duration of biological activity) and poor bioavailability. Often certain other properties, such as selectivity for biological targets or substrates or potency of biological activity, such as the biological activities mentioned above, can be substantially improved.

[0238] Discrepancies between the nucleic acid or amino acid sequences of a protein or enzyme referred to herein, such as the α-ionylideneethane synthase as defined herein, and the nucleic acid or amino acid sequences of functional homologues of said enzymes, may in particular be the result of modifications made to improve the properties of the enzyme or nucleic acid (e.g. increasing the expression of the enzyme or increasing the enzymatic activity of the enzyme), by biological techniques known to the person skilled in the art, such as molecular evolution or rational design, or by using mutagenesis techniques known in the art and described elsewhere herein (random mutagenesis, site-directed mutagenesis, directed evolution, genetic recombination, etc.).

[0239] The sequence of the enzyme or nucleic acid may be altered as a result of one or more natural variations. Examples of such natural modifications or variations are glycosylation differences (more broadly defined as "post-translational modifications"), alternative splicing differences and single nucleic acid polymorphisms (SNPs). Nucleic acids may be modified to encode polypeptides that differ by at least one amino acid or by 2, 3, 4, 5, 6 or even more amino acids, which may encode polypeptides that contain one or more amino acid substitutions, deletions and / or insertions, which polypeptides still have biological or enzymatic activity, such as α-ionylideneethane synthase activity, as defined herein. Additionally, artificial gene synthesis (synthetic DNA), codon optimization or codon pair optimization may be used, for example based on methods as described in WO 2008 / 000632 or as provided by commercial DNA synthesis companies such as DNA2.0, Geneart and GenScript.

[0240] The sequence of the enzyme or the sequence of the nucleic acid can be modified by gene editing. Gene editing or genome editing can be performed using various techniques such as "gene shuffling" or "directed evolution" consisting of repeated DNA shuffling, in which DNA is inserted, replaced or removed from the genome, followed by appropriate screening and / or selection to generate variants of the nucleic acid or part thereof encoding a protein with modified biological activity (Castle et al., (2004) Science 304(5674):1151-4; US Pat. Nos. 5,811,238 and 6,395,547), or by "T-DNA activation" tagging (Hayashi et al. Science (1992) 1350-1353) (the resulting transgenic organisms show a dominant phenotype due to the modification of the expression of the gene close to the introduced promoter), or by "TILLING" (Targeted Induced Local Lesions Induced) TILLING is a type of genetic modification that can be obtained by TILLING (Tilling Genomes), and refers to a mutagenesis technique useful for creating and / or identifying nucleic acids that code for proteins with altered expression and / or activity. TILLING also allows for the selection of organisms carrying such mutant variants. Methods for TILLING are well known in the art (reviewed by McCallum et al., (2000) Nat Biotechnol 18:455-457; Stemple (2004) Nat Rev Genet 5(2):145-50). Another technique uses artificially engineered nucleases such as zinc finger nucleases, transcription activator-like effector nucleases (TALENs), CRISPR / Cas systems and engineered meganucleases, e.g. engineered homing endonucleases (Esvelt, KM.; Wang, HH. (2013), Mol Syst Biol 9(1):641; Tan, WS. et al. (2012), Adv Genet 80:37-97; Puchta, H.; Fauser, F. (2013), Int. J. Dev. Biol 57:629-637).

[0241] Derivatives of proteins or enzymes referred to herein, such as α-ionylideneethane synthase as defined herein, include functional, i.e. enzymatically active variants, which may be obtained by deletion, insertion or substitution of amino acid residues from / to the amino acid sequence. The modification or mutation may be the substitution of an amino acid residue by a different one, the deletion of an amino acid residue or the insertion of an amino acid residue. For example, an amino acid residue involved in substrate binding may be modified or mutated. To provide a specific example, the modified or mutated amino acid sequence preferably has improved, e.g. increased, α-ionylideneethane synthase activity compared to the unmodified amino acid sequence shown in any one of SEQ ID NOs: 1-17 or 19-33. To this end, for example, site-directed mutagenesis of the above-mentioned α-ionylideneethane synthase may focus on amino acid residues found in highly conserved motifs between homologues, allowing the identification of mutants that generate intermediates of the α-ionylideneethane and / or α-ionone synthesis reaction, or a more detailed elucidation of the cyclization mechanism. Preferably, said homologue, variant, derivative or peptidomimetic of a protein or enzyme referred to herein, such as an α-ionylidene ethane synthase as defined herein, has at least 50%, 60%, 70%, 80%, 90% or even 100% of the biological or enzymatic activity of the unmodified or unmutated protein or enzyme, for example at least 50%, 60%, 70%, 80%, 90% or even 100% of the α-ionylidene ethane synthase activity of the unmodified or unmutated α-ionylidene ethane synthase of any one of the amino acid sequences of SEQ ID NOs: 1-17 or 19-33. Said homologue, variant, derivative or peptidomimetic preferably also maintains the substrate specificity and / or substrate preference of the unmodified or unmutated protein or enzyme, for example the substrate specificity and / or substrate preference of the α-ionylidene ethane synthase of any one of SEQ ID NOs: 1-17 or 19-33. For example, a homolog, variant, derivative, or peptidomimetic of an α-ionylideneethane synthase of any one of SEQ ID NOs: 1-17 or 19-33 allows for the conversion of farnesyl diphosphate to α-ionylideneethane, as described elsewhere herein. In a preferred embodiment, the homolog, variant, derivative, or peptidomimetic has a turnover number that is at least 90% of the turnover number of an α-ionylideneethane synthase of any one of the amino acid sequences of SEQ ID NOs: 1-17 or 19-33.

[0242] DNA and the proteins they encode can be modified using a variety of techniques known in molecular biology to generate variant proteins or enzymes with new or altered properties (see, e.g., Sambrook; Ausubel, cited elsewhere herein).

[0243] Random PCR mutagenesis is described, for example, in Rice (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471, and combinatorial multiple cassette mutagenesis is described, for example, in Crameri (1995) Biotechniques 18:194-196.

[0244] Alternatively, nucleic acids, e.g., genes, can be reconstituted after random or "stochastic" fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; and 5,605,793.

[0245] Alternatively, modifications, additions or deletions are introduced by error-prone PCR, shuffling, site-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis (phage-assisted continuous evolution, in vivo continuous evolution), cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-directed mutagenesis, gene reassembly, gene site saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch modification mutagenesis, modified deletion host strain mutagenesis, chemical mutagenesis, radiation-induced mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer generation and / or combinations thereof and other methods.

[0246] Alternatively, "gene site saturation mutagenesis" or "GSSM" includes methods that use degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, as detailed in U.S. Pat. Nos. 6,171,820 and 6,764,835.

[0247] Alternatively, synthetic ligation reassembly (SLR) involves a method of non-stochastically ligating oligonucleotide building blocks together, for example as disclosed in US Pat. No. 6,537,776.

[0248] Alternatively, tailored multiple site combinatorial assembly ("TMSCA") is a method for generating multiple progeny polynucleotides with different combinations of mutations at multiple sites by using at least two mutagenic non-overlapping oligonucleotide primers in one reaction. Such methods are described, for example, in WO 2009 / 018449.

[0249] The proteins or enzymes referred to herein, such as the α-ionylideneethane synthase as defined herein, may also be fusion proteins. The term "fusion protein" as used herein refers to a chimeric protein (literally made from parts from different sources) created through the joining of two or more genes that originally code for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties from each of the original proteins. For example, a fusion protein as defined herein may include an affinity tag for protein purification (His tag, FLAG tag, etc., see e.g. Kimple et al., 2015, Curr Protoc Protein Sci.; 73:Unit-9.9. doi:10.1002 / 0471140864.ps0909s73) or a label for detection. A "label" as referred to herein is a detectable compound or composition that is directly or indirectly conjugated to another molecule, such as the α-ionylideneethane synthase as defined herein, to facilitate detection of that molecule. Specific non-limiting examples of labels include fluorescent tags, enzyme linkages and radioactive isotopes, which are well known in the art. In one embodiment, a protease cleavage site and / or linker (i.e. a protease cleavage site; or a linker; or both a protease cleavage site and a linker; or a linker includes a protease cleavage site) may be present between the protein or enzyme referred to herein, such as the α-ionylideneethane synthase defined herein, and the label or purification tag. For example, the protease cleavage site may be used to cleave the purification tag by treatment with a protease, such as enterokinase or thrombin, as needed. For example, a His tag may be used as a tag for expression and purification, while the protein or enzyme referred to herein, such as the α-ionylideneethane synthase defined herein, may be isolated after cleavage by a protease.As known by those skilled in the art, besides the basic role in linking together functional domains (in the case of flexible and rigid linkers), linkers can provide many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression levels, and achieving desired pharmacokinetic profiles. The linker can be, for example, a protein / peptide linker, such as a polyglycine linker or other linkers known in the art (see, for example, Chen et al., Adv Drug Deliv Rev. 2013; 65(10): 1357-1369). Obviously, the linker can be designed so that it contains a protease cleavage site. In another embodiment, the fusion protein can carry a signal peptide for targeting the expressed polypeptide, for example to a specific organelle, as described elsewhere herein.

[0250] Fusion proteins as defined herein may be produced by chemical synthesis or recombinant molecular biology techniques well known to those skilled in the art, which apply mutatis mutandis to the isolation of fusion proteins from host cells or supernatants; see, e.g., Sambrook et al., Molecular cloning: a laboratory manual / Sambrook, Joseph; Russell, David W.--. 3rd ed.--New York: Cold Spring Harbor Laboratory, 2001; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).

[0251] The term "nucleic acid" as used herein includes reference to deoxyribonucleotide or ribonucleotide polymers, i.e., polynucleotides, in either single-stranded or double-stranded form, and encompasses known analogs that have the basic properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in the same manner as natural nucleotides (e.g., peptide nucleic acids), unless otherwise limited. Polynucleotides can be full-length or subsequences of native or heterologous structures or control genes. Unless otherwise indicated, the term includes reference to the designated sequence as well as its complementary sequence. Thus, DNA or RNA with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Additionally, DNA or RNA containing unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are "polynucleotides" as the term is used herein. It will be understood that a wide variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide" as used herein includes such chemically, enzymatically or metabolically modified forms of polynucleotides as well as chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, among others. All nucleic acid sequences herein that code for a polypeptide or enzyme, such as α-ionylideneethane synthase as defined herein, also by reference to the genetic code, describe all possible silent variations of the nucleic acid. The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, the term "conservatively modified variants" refers to nucleic acids that code for identical or conservatively modified variants of amino acid sequences due to the degeneracy of the genetic code. The term "degeneracy of the genetic code" refers to the fact that a large number of functionally identical nucleic acids code for a particular protein. For example, the codons GCA, GCC, GCG and GCU all code for the amino acid alanine.Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one type of conservatively modified variation. The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. An "enzymatically active fragment of an amino acid sequence" of a protein or enzyme referred to herein, such as an α-ionylideneethane synthase as defined herein, refers to a stretch of at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, or 250 amino acid residues having a biological or enzymatic activity referred to herein, such as an α-ionylideneethane synthase activity as defined herein. The terms "polypeptide", "peptide" and "protein" apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. A necessary property of such analogs of natural amino acids is that, when incorporated into a protein, the protein is specifically reactive with antibodies elicited against the same protein but made entirely of natural amino acids. The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Within the context of this application, oligomers (oligonucleotides, oligopeptides, etc.) are considered as a type of group of polymers. Oligomers have a relatively small number of monomeric units, generally 2-100, particularly 6-100, including, for example, primer sequences such as those used for cloning the α-ionylideneethane synthase used in the examples.

[0252] The term "heterologous" when used in connection with a nucleic acid (DNA or RNA) or protein or enzyme of the present disclosure, such as α-ionylidene ethane synthase as defined herein, refers to a nucleic acid or protein that does not naturally occur as part of the organism, cell, genome or DNA or RNA sequence in which it is found, or that is found at one or more locations in a cell or genome or DNA or RNA sequence that is different from the one in which it is found in nature. Heterologous nucleic acids or proteins or enzymes of the present disclosure, such as α-ionylidene ethane synthase as defined herein, are not endogenous to the cell in which they are introduced, but have been obtained from another cell or have been synthetically or recombinantly produced. Generally, but not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is expressed. Genes that are endogenous to a particular host cell, but have been modified from their natural form, for example through the use of DNA shuffling, are also referred to as heterologous. The term "heterologous" also includes non-natural multiple copies of naturally occurring DNA sequences. Thus, the term "heterologous" can refer to a DNA segment that is foreign or heterologous to the cell, or that is homologous to the cell but at a location and / or number within the host cell nucleic acid where the segment is not normally found. An exogenous DNA segment is expressed to give rise to an exogenous polypeptide.

[0253] As used herein, a "homologous" DNA sequence is a DNA sequence that is naturally associated with the host cell into which it is introduced. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is encompassed herein by the term heterologous nucleic acid or protein.

[0254] The terms "modified", "modification", "mutated" or "mutation", when used herein in reference to a protein or polypeptide compared to another protein or polypeptide (e.g. compared to an α-ionylideneethane synthase as defined herein comprising or consisting of the amino acid sequence of SEQ ID NO: 1-17 or 19-33), apply mutatis mutandis to a nucleotide or nucleic acid sequence. The terms referred to are used to contemplate that a modified nucleotide or nucleic acid sequence encoding a protein or polypeptide having biological or enzymatic activity, such as α-ionylideneethane synthase activity, has at least one difference in the nucleotide or nucleic acid sequence compared to the nucleotide or nucleic acid sequence of the protein or polypeptide to which it is compared, e.g. the amino acid sequence of any one of SEQ ID NO: 1-17 or 19-33. The terms are used regardless of whether the modified or mutated protein has in fact been obtained by mutagenesis of the nucleic acid encoding these amino acids or modification of the polypeptide or protein, or in another way, e.g. using artificial gene synthesis methods. Mutagenesis is a method well known in the art and includes site-directed mutagenesis via PCR or oligonucleotide-mediated mutagenesis, e.g., as described in Sambrook, J., and Russell, DW Molecular Cloning: A Laboratory Manual. 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001).The terms "modified", "modification", "mutated" or "mutation", when used herein in reference to a gene, are used to intend that at least one nucleotide in the nucleotide sequence of that gene or its regulatory sequence differs from the nucleotide sequence to which it is compared, for example the nucleotide sequence encoding the amino acid sequence of any of SEQ ID NOs: 1-17 or 19-33. The modification or mutation may in particular be the substitution of a nucleotide by a different one, the deletion of a nucleotide or the insertion of a nucleotide.

[0255] The nucleic acid encoding the protein or enzyme referred to herein, such as the α-ionylideneethane synthase defined herein, is operably linked to an expression control sequence that allows expression in a prokaryotic or eukaryotic host cell, or an isolated fraction thereof, in a vector or genetic construct. Thus, in one embodiment, the vector is an expression vector. Expression of the nucleic acid encoding the protein or enzyme referred to herein, such as the α-ionylideneethane synthase defined herein, comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in prokaryotic or eukaryotic host cells are well known in the art. In one embodiment, these include regulatory sequences ensuring initiation of transcription and / or polyA signals ensuring termination and stabilization of transcription. Further regulatory elements may comprise transcriptional as well as translational enhancers. Possible regulatory elements allowing expression in prokaryotic host cells include, for example, the lac-, trp-, or tac-promoter of E. coli or the Rhodobacter promoter (https: / / doi.org / 10.1073 / pnas.2010087117), and examples of regulatory elements allowing expression in eukaryotic host cells are the AOX1- or GAL1-promoter in yeast, or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer, or globin intron in mammalian and other animal cells. Plant promoters are described, for example, in Plant Biotechnology: Principles and Applications, pp117-172, 2017. Furthermore, inducible expression control sequences can be used in the expression vector. Such inducible vectors can include tet or lac operator sequences or sequences that are inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. In addition to elements involved in transcription initiation, such control elements may also include transcription termination signals downstream of the polynucleotide, such as the SV40-poly-A site or the tk-poly-A site.In the present context, suitable expression vectors are known in the art, such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen) or pSPORT1 (Invitrogen). Expression vectors derived from viruses, such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses or bovine papilloma viruses, may be used for delivery of polynucleotides or vectors to targeted cell populations.

[0256] Methods well known to those skilled in the art can be used to construct vectors or genetic constructs containing nucleic acids encoding the proteins or enzymes referred to herein, such as the α-ionylideneethane synthase defined herein; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001) NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).

[0257] The term "gene" as used herein is used broadly to refer to any segment of nucleic acid associated with a biological function, such as a nucleic acid encoding an enzymatically active α-ionylideneethane synthase as defined herein. Thus, genes include coding sequences and / or regulatory sequences required for their expression. For example, genes refer to nucleic acid fragments that express mRNA or functional RNA or encode specific proteins, including regulatory sequences. Genes also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and can include sequences that are designed to have desired parameters.

[0258] The term "chimeric gene," as used herein, refers to any gene that contains 1) a DNA sequence that includes regulatory and coding sequences that are not found together in nature, or 2) a sequence that encodes portions of a protein that are not naturally contiguous, or 3) a portion of a promoter that is not naturally contiguous. Thus, a chimeric gene may contain regulatory and coding sequences that are derived from different sources, or it may contain regulatory and coding sequences that are derived from the same source but that are organized differently than found in nature.

[0259] "Genetic constructs" as used herein can vary in complexity according to the insertion of interest. The constructs can be designed to be randomly inserted into the genome of an organism, called genetic recombination by addition, or can be designed to be inserted into the genome at a specific targeted site at the correct location of a determined chromosome, called genetic recombination by homologous recombination. In both cases, the construct must be complete, with structures to control gene expression, such as promoters, transcription start sites, polyadenylation sites, and transcription termination sites. That is, the information being inserted into the recipient genome has a beginning, middle, and end, thus avoiding the problem of uncontrolled expression in the host cell or organism.

[0260] The terms "open reading frame" and "ORF" as used herein refer to the amino acid sequence encoded between the translation initiation and termination codons of a coding sequence. The terms "initiation codon" and "termination codon" refer to the unit of three contiguous nucleotides ("codons") in a coding sequence that specify the initiation and chain termination of protein synthesis (mRNA translation), respectively.

[0261] As used herein, "coding sequence" refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes non-coding sequences. It may constitute an "uninterrupted coding sequence," i.e., lacking introns, as in cDNA, or it may contain one or more introns bounded by the appropriate splice junctions. An "intron" is a sequence of RNA that is contained in the primary transcript but is removed through cleavage and religation of the RNA within the cell to produce a mature mRNA that can be translated into a protein.

[0262] "Regulatory sequence" as used herein refers to a nucleotide sequence located upstream (5' non-coding sequences) or within or downstream (3' non-coding sequences) of a coding sequence that affects the transcription, RNA processing or stability or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns and polyadenylation signal sequences. These include sequences that may be natural and synthetic sequences and combinations of synthetic and natural sequences. As noted above, the term "suitable regulatory sequence" is not limited to promoters. Examples of regulatory sequences include promoters (transcriptional promoters, constitutive promoters, inducible promoters), operators, enhancers, mRNA ribosomal binding sites and appropriate sequences that control transcription and translation initiation and termination. A nucleic acid sequence is "operably linked" when the regulatory sequence functionally relates to the DNA or cDNA sequence of the present disclosure. As used herein, the terms "operably linked" or "operably linked" refer to a juxtaposition in which the components so described are in a relationship that permits them to function in their intended manner. A control sequence "operably linked" to another control sequence and / or to a coding sequence is ligated in such a way that transcription and / or expression of the coding sequence is achieved under conditions compatible with the control sequences. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to link two protein coding regions, contiguous and in the same reading frame. Each of the regulatory sequences can be independently selected from heterologous and homologous regulatory sequences.

[0263] "Promoter" as used herein refers to a nucleotide sequence that is usually upstream (5') to its coding sequence and controls the expression of said coding sequence by providing recognition to RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter, which is a short DNA sequence consisting of a TATA box and other sequences that serve to specify the transcription start site, to which control elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that contains a minimal promoter plus control elements that are capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often called enhancers. Thus, an "enhancer" is a DNA sequence that can stimulate promoter activity and can be a native element of the promoter or a heterologous element inserted to increase the level or tissue specificity of the promoter. It can operate in both orientations (normal or inverted) and can function when moved either upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from native genes, or may be composed of different elements from different promoters found in nature, or even composed of synthetic DNA segments. Promoters may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0264] "Expression cassette" as used herein means a DNA sequence capable of directing the expression of a particular nucleotide sequence, for example a nucleotide sequence encoding an α-ionylideneethane synthase as defined herein, in a suitable host cell as defined herein, comprising a promoter operably linked to a nucleotide sequence of interest operably linked to a termination signal. The coding region usually encodes a protein of interest, but may also encode a functional RNA of interest, for example an antisense RNA or a non-translated RNA, in the sense or antisense orientation. An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. An expression cassette may be naturally occurring, but also one that has been obtained in a recombinant form useful for heterologous expression. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of a multicellular organism, the promoter may also be specific to a particular tissue or organ or to a developmental stage in, for example, plant development.

[0265] The term "vector" as used herein refers to a construct composed of genetic material designed to direct the transformation of a targeted cell. A vector contains multiple genetic elements that are oriented positionally and sequentially, i.e. operably linked with other necessary elements, so that the nucleic acid in the nucleic acid cassette can be transcribed and, if necessary, translated in the transformed cell. In particular, the vector may be selected from the group of viral vectors, (bacteri)phages, cosmids or plasmids. The vector may also be a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or an Agrobacterium binary vector. The vector may or may not be self-transmissible or mobilizable, and may be in double-stranded or single-stranded, linear or circular form capable of transforming a host organism, such as, for example, Rhodobacter, either by integration into the cell genome or by being present extrachromosomally (e.g. an autonomously replicating plasmid with an origin of replication). In particular, shuttle vectors are included, meaning DNA vehicles capable of replicating naturally or by design in two different host organisms as defined herein. Preferably, the nucleic acid in the vector is under the control of and operably linked to a promoter or other regulatory element suitable for transcription in a host cell as specified herein. The vector may be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, it may contain its own promoter or other regulatory element, and in the case of cDNA, it may be under the control of a promoter or other regulatory element suitable for expression in a host cell. The vector containing the nucleic acid may be prepared based on methods known in the art. For example, a cDNA sequence encoding the α-ionylideneethane synthase defined herein operably linked to a suitable regulatory element, such as a transcriptional or translational regulatory nucleic acid sequence, may be used.

[0266] The term "vector", as used herein, includes reference to vectors for standard cloning procedures ("cloning vectors"), as well as to more specialized types of vectors such as (autosomal) expression vectors and cloning vectors that are used for integration into a host cell chromosome ("integrating vectors").

[0267] A "cloning vector" generally contains one or a few restriction endonuclease recognition sites into which foreign DNA sequences may be inserted in a determinable manner without eliminating an essential biological function of the vector, as well as a marker gene that is suitable for use in identifying and selecting cells transformed with the cloning vector.

[0268] The term "expression vector" as used herein refers to a linear or circular DNA molecule that contains a segment encoding a polypeptide of interest under the control (i.e., operably linked) of an additional nucleic acid segment that provides for its transcription. Such additional segments may include promoter and termination sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. In particular, an expression vector comprises a nucleotide sequence that includes, in a 5' to 3' direction, (a) a transcriptional and translational initiation region recognized by the host organism, (b) a coding sequence for a polypeptide of interest, and (c) a transcriptional and translational termination region recognized by the host organism and operably linked thereto. A "plasmid" refers to an autonomously replicating extrachromosomal DNA that is not integrated into the genome of a microorganism and is usually circular in nature.

[0269] "Integrating vector" refers to a DNA molecule, linear or circular, that can be integrated, for example, into the genome of a microorganism, e.g., a bacterial genome, resulting in stable genetic inheritance of a gene encoding a polypeptide of interest, such as an α-ionylideneethane synthase, as defined herein. Integrating vectors generally contain one or more segments that include a genetic sequence encoding a polypeptide of interest under the control of (i.e., operably linked to) an additional nucleic acid segment that provides for its transcription.

[0270] Such further segments may include promoter and termination sequences and one or more segments that drive the integration of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. In general, an integrating vector is one that can be transferred to a target cell, but has a non-functional replicon in that organism. Integration of the segment containing the gene of interest can be selected if an appropriate marker is included within the segment. One or more nucleic acid sequences encoding suitable signal peptides that are not naturally associated with the polypeptide to be expressed in a host cell as defined herein, preferably a host cell of the invention, can be incorporated into the (expression) vector. For example, a DNA sequence for a signal peptide leader can be fused in frame to a nucleic acid of the present disclosure such that a protein or enzyme referred to herein, such as an α-ionylideneethane synthase as defined herein, is initially translated as a fusion protein containing the signal peptide. Depending on the nature of the signal peptide, the expressed polypeptides are targeted differently. Secretory signal peptides that are functional in the intended host cell, for example, promote the extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptide to certain organelles, such as chloroplasts, mitochondria and peroxisomes. The signal peptide may be cleaved from the polypeptide upon transport to the organelle of interest or upon transport out of the cell. It is possible to provide for the fusion of further peptide sequences to the amino or carboxyl terminus of the polypeptide.

[0271] The host cell is transformed with the vector or genetic construct disclosed herein. Those skilled in the art are well aware of the genetic elements that must be present on the genetic construct to successfully transform, select and grow host cells containing the vector or genetic construct disclosed herein. The host cell can express the polypeptides or enzymes referred to herein, such as proteins having α-ionylideneethane synthase activity, contained in the vector or genetic construct of the present disclosure. The host cell also contains farnesyl diphosphate as a substrate for the expressed enzymatically active α-ionylideneethane synthase.

[0272] "Transformation" and "transforming", as used herein, refer to the introduction of a heterologous nucleotide sequence, such as a nucleotide sequence encoding a protein or enzyme referred to herein, such as α-ionylideneethane synthase as defined herein, into a host cell, regardless of the method used for insertion, such as direct uptake, transduction, conjugation, f-mating, or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or alternatively may be integrated into the host cell genome.

[0273] Host cells according to the present disclosure can be produced according to standard genetic and molecular biology techniques generally known in the art, for example as described in Sambrook, J., and Russell, DW "Molecular Cloning: A Laboratory Manual" 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001); and FM Ausubel et al, eds., "Current protocols in molecular biology", John Wiley and Sons, Inc., New York (1987) and later supplements thereto.

[0274] The host cell may be any cell selected from a microbial cell, such as a bacterial cell, an archaeal cell, a fungal cell, such as a yeast cell, and a protist cell. The host cell may also be an algal or cyanobacterial cell, a non-human animal cell or a mammalian cell, or a plant cell.

[0275] Specifically, the host cell may be selected from any one of the following organisms:

[0276] bacteria The bacterial host cell may, for example, be selected from the group consisting of the genera Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, or Lactococcus.

[0277] Gram positive: Bacillus, Streptomyces:

[0278] Useful gram-positive bacterial host cells include Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus Jautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, and the like. The most preferred prokaryotic organisms include, but are not limited to, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis. The most preferred prokaryotic organisms are Bacillus cells, preferably Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis or Bacillus lentus Bacillus cells.

[0279] Some other preferred bacteria include the species of the order Actinomycetales, preferably the genus Streptomyces, preferably Streptomyces spheroides (ATTC23965), Streptomyces thermoviolaceus (IFO12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillium verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include species belonging to the genus Myxococcus, such as M. virescens.

[0280] Gram-negative: E. coli, Pseudomonas, Rhodobacter, Paracoccus Preferred gram-negative bacteria are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11), Rhodobacter capsulatus or Rhodobacter sphaeroides, Paracoccus carotinifaciens or Paracoccus zeaxanthinifaciens.

[0281] fungi Aspergillus, Fusarium, Trichoderma

[0282] The host cell may be a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota as well as the phyla Oomycota and Deuteromycotina and all vegetative spore-forming fungi. Representative groups of the phylum Ascomycota include, for example, the genera Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus) and the true yeasts listed below. Examples of Basidiomycota include mushrooms, rusts and smuts. Representative groups of Chytridiomycota include, for example, Allomyces, Blastocladiella, Coelomomyces and aquatic fungi. Representative groups of Oomycota include, for example, Saprolegniomycetous aquatic fungi (water molds), such as Achlya. Examples of vegetative spore-forming fungi include Aspergillus, Penicillium, Candida and Alternaria. Representative groups of the phylum Zygomycota include, for example, the genera Rhizopus and Mucor.

[0283] Some preferred fungi include species belonging to the subdivision Deuteromycotina, the class Hyphomycetes, such as the genera Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM2672), Humicola insolens, and the like. insolens, Trichoderma resii, Myrothecium verrucana (IFO6113), Verticillium alboatrum, Verticillium dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli or Dreschlera halodes.Other preferred fungi include species belonging to the subdivision Basidiomycotina, the class Basidiomycetes, such as the genera Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus), such as T. versicolor (e.g. PR4 28-A). Further preferred fungi include species belonging to the subdivision Zygomycotina, the class Mycoraceae, such as the genera Rhizopus and Mucor, in particular Mucor hiemalis.

[0284] yeast Genus Pichia Saccharomyces

[0285] The fungal host cell may be a yeast cell. Yeast, as used herein, includes ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes) genus. Ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is composed of four subfamilies, Schizosaccharomycoideae (e.g. the genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae (e.g. the genera Kluyveromyces, Pichia and Saccharomyces). Basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., Sporobolomyces and Bullera) and Cryptococcaceae (e.g., Candida).

[0286] eukaryotes Eukaryotic host cells further include, but are not limited to, non-human animal cells, non-human mammalian cells, avian cells, reptilian cells, insect cells or plant cells.

[0287] In a preferred embodiment, the host cell is a host cell selected from the following: a) bacterial cells of the group of gram-negative bacteria, such as the genus Rhodobacter (e.g. Rhodobacter sphaeroides or Rhodobacter capsulatus), the genus Paracoccus (e.g. P. carotinifaciens, P. zeaxanthinifaciens), the genus Escherichia or the genus Pseudomonas; b) bacterial cells selected from the group of gram-positive bacteria, such as Bacillus, Corynebacterium, Brevibacterium, Amycolatopis; c) fungal cells selected from the group of Aspergillus, Blakeslea, Peniciliium, Phaffia (Xanthophyllomyces), Pichia, Saccharamoyces, Kluyveromyces, Yarrowia and Hansenula; d) a transgenic plant cell or a culture comprising a transgenic plant cell, the cell being of a transgenic plant selected from Arabidopsis spp., Nicotiana spp., Cichorum intybus, lacuca sativa, Mentha spp., Artemisia annua, tuber-forming plants, oil crops such as Brassica spp. or Brassica napus, fruit-producing flowering plants (angiosperms) and trees; or e) A culture comprising a transgenic mushroom or a transgenic mushroom cell, the microorganism being selected from the genera Schizophyllum, Agaricus and Pleurotisi.

[0288] More preferred host cells from organisms are microorganisms belonging to the genera Escherichia, Saccharomyces, Pichia, Rhodobacter, Pseudomonas or Paracoccus, (e.g. Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens) and even more preferred are E. coli, S. cerevisae, Rhodobacter sphaeroides, Rhodobacter capsulatus, and the like. capsulatus or Amycolatopis sp.

[0289] Particularly preferred are host cells of the genus Rhodobacter selected from the group Rhodobacter capsulatus and Rhodobacter sphaeroides.

[0290] The present invention also provides a fermentation composition comprising: (a) a genetically modified microbial host cell cultured in a culture medium, the microbial host cell being a microbial host cell of the invention; (b) α-ionylideneethane and / or α-ionone produced by the microbial host cell of the present invention; The present invention relates to a fermentation composition comprising:

[0291] The present invention further provides a host cell for preparing α-ionone, the host cell comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylideneethane synthase.

[0292] The definitions and explanations relating to the terms "host cell", "host cell disclosed herein" or "host cell referred to herein" apply mutatis mutandis to the host cell of the present invention.

[0293] The host cells of the invention comprise a heterologous nucleic acid encoding an α-ionylideneethane synthase as disclosed herein and farnesyl diphosphate as a substrate for the α-ionylideneethane synthase.

[0294] Advantageously, the host cells of the invention may be used for the production of α-ionylideneethane, as shown in the examples below. Preferably, the α-ionylideneethane is E,Z-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene).

[0295] The host cells of the invention may further be used to produce α-ionone, as shown in the examples below. The host cells of the invention are suitable for converting α-ionylideneethane to α-ionone. Preferably, the α-ionone is R-α-ionone.

[0296] Preferably, α-ionylideneethane and / or α-ionone are used in the host cell of the invention as a precursor of and / or for the synthesis of vitamin A. Thus, said host cell is capable of converting α-ionylideneethane into vitamin A.

[0297] The host cells may also be used for the heterologous reconstitution of terpenes or terpenoids.

[0298] The host cells may further be utilized to produce industrial products, preferably fragrance compositions, flavors or fragrances, animal feed, human nutritional products, cosmetics, colorants (carotenoids), or radical scavengers.

[0299] The host cells of the present invention are capable of functioning as a fermentation production system to produce sesquiterpenes, as defined herein.

[0300] In a preferred embodiment of the host cell of the invention, α-ionylideneethane synthase converts farnesyl diphosphate to α-ionylideneethane.

[0301] In another preferred embodiment of the host cell of the invention, at least a portion of the produced α-ionylideneethane is converted to α-ionone by chemical or enzymatic oxidative cleavage.

[0302] In yet a further preferred embodiment of the present invention, the α-ionylideneethane synthase is a fungal or bacterial α-ionylideneethane synthase. In a preferred embodiment, the α-ionylideneethane synthase is from a fungus of the phylum Ascomyta, preferably from the subphylum Pezizomycotina. In one embodiment, the fungus is from the family Sclerotiniaceae or Rutstroemiaceae, for example Botrytis species or Rutstroemia species.

[0303] In a further preferred embodiment of the host cell of the invention, the α-ionylideneethane synthase is: a) an amino acid sequence shown in any one of SEQ ID NOs: 1 to 17 or 19 to 33; b) an amino acid sequence having at least 40%, 50%, 55%, 60%, 65%, 66%, 70%, 71%, 75%, 80%, 81%, 85%, 86%, 90%, or 95% sequence identity at the amino acid level to any one of SEQ ID NOs: 1 to 17 or 19 to 33, and having α-ionylideneethane synthase activity; c) an enzymatically active fragment of the amino acid sequence of a) or b) having α-ionylideneethane synthase activity; The amino acid sequence is selected from the group consisting of:

[0304] In yet a further preferred embodiment of the host cell of the invention, the host cell (i) one or more nucleic acids encoding an enzyme of the mevalonate pathway, and / or one or more nucleic acids encoding an enzyme of the deoxyxylulose phosphate (DXP) pathway; and / or (ii) one or more nucleic acids encoding oxidases, preferably one or more nucleic acids encoding carotene dioxygenases and / or peroxidases; and / or (iii) further comprising one or more nucleic acids encoding an enzyme for the synthesis of vitamin A.

[0305] In another preferred embodiment of the host cell of the present invention, the host cell is a bacterial cell, a yeast cell, a fungal cell, an algae cell or a blue-green algae cell, a non-human animal cell or a non-human mammalian cell, a non-vertebrate animal cell or a plant cell, preferably a bacterial cell or a yeast cell. In one embodiment, the host cell is an isolated cell, i.e., it is not within a multicellular organism. More preferably, the host cell is a Saccharomyces cerevisiae host cell or a Rhodobacter host cell, even more preferably a Rhodobacter sphaeroides host cell.

[0306] Additionally, the present invention relates to a composition comprising (i) a host cell of the invention, α-ionylideneethane and / or α-ionone, or (ii) an α-ionylideneethane synthase, α-ionylideneethane and / or α-ionone as defined herein. Preferably, the α-ionylideneethane and / or α-ionone is produced by the method of the invention.

[0307] The present invention also relates to a kit comprising a host cell of the invention, or an aroma compound or composition of the invention.

[0308] The present invention further relates to a method for preparing α-ionone, comprising the step of converting farnesyl diphosphate to α-ionylideneethane in the presence of an enzyme comprising a first segment comprising a tag peptide and a second segment comprising an α-ionylideneethane synthase, preferably comprising any of SEQ ID NOs: 1-17 or 19-33, preferably SEQ ID NO: 1 and an amino acid sequence having at least 50%, 55%, 60%, 65%, 66%, 70%, 71%, 75%, 76%, 80%, 81%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity at the amino acid level. The enzyme comprising said first segment and said second segment is referred to herein as a "tagged enzyme".

[0309] The present invention also relates to the use of such tagged enzyme versions of α-ionylideneethane synthase having at least 50%, 55%, 60%, 65%, 66%, 70%, 71%, 75%, 76%, 80%, 81%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity at the amino acid level to any of SEQ ID NOs: 1-17 or 19-33, and preferably to SEQ ID NO: 1, in the production of one or more aroma compounds.

[0310] Additionally, a tagged enzyme version of α-ionylideneethane synthase having at least 50%, 55%, 60%, 65%, 66%, 70%, 71%, 75%, 76%, 80%, 81%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity at the amino acid level to any of SEQ ID NOs: 1-17 or 19-33, and preferably SEQ ID NO: 1, may be used in a method of the invention for preparing α-ionone and / or α-ionylideneethane in the presence of an enzyme comprising a first segment comprising a tag peptide and a second segment comprising an α-ionylideneethane synthase as described herein, the method comprising the step of converting farnesyl diphosphate to α-ionylideneethane.

[0311] The tag peptide is preferably selected from the group of nitrogen utilization protein (NusA), thioredoxin (Trx), maltose binding protein (MBP), glutathione S-transferase (GST), small ubiquitin-like modifier (SUMO), or calcium binding protein (Fh8), and functional homologs thereof. As used herein, a functional homolog of a tag peptide is a tag peptide that has at least about the same effect on the solubility of the tagged enzyme compared to the untagged enzyme. Typically, a homolog differs in that one or more amino acids are inserted, substituted, deleted, or extended in the peptide to which it is a homolog. A homolog may in particular include one or more substitutions of a hydrophilic amino acid for another hydrophilic amino acid, or a hydrophobic amino acid for another hydrophobic amino acid. A homologue may in particular have at least 40% sequence identity, more particularly at least 50%, preferably at least 55%, more preferably at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the sequence of NusA, Trx, MBP, GST, SUMO or Fh8.

[0312] Particularly preferred is the maltose binding protein from Escherichia coli or a functional homologue thereof.

[0313] The use of tagged enzymes according to the invention is particularly advantageous in that it may contribute to increased production of α-ionylideneethane and / or α-ionone, in particular increased cellular production.

[0314] To improve the solubility of the tagged enzyme (compared to the untagged enzyme), the first segment of the enzyme is preferably linked by its C-terminus to the N-terminus of the second segment, or alternatively, the first segment of the tagged enzyme is linked by its N-terminus to the C-terminus of the second segment.

[0315] Furthermore, the present invention relates to an enzyme comprising a first segment comprising a tag peptide and a polypeptide having an enzymatic activity of converting farnesyl diphosphate to α-ionylideneethane, in particular a second segment comprising α-ionylideneethane synthase, preferably a tag peptide selected from the group MBP, NusA, Trx or SET, as well as nucleic acids encoding same, and a host cell carrying said nucleic acid and producing said tagged enzyme.

[0316] Finally, the present invention relates to a) a host cell of the invention, (i) for producing α-ionylideneethane, preferably E,Z-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene), preferably as an aroma component, which is a precursor of aroma substances, or as a precursor of vitamin A; (ii) for producing α-ionone, preferably R-α-ionone; (iii) for producing vitamin A; (iv) for converting α-ionylideneethane to α-ionone; (v) for converting α-ionylideneethane to vitamin A; (vi) for heterologous reconstruction of terpenes or terpenoids; (vii) for producing industrial products, preferably fragrance compositions, flavours or fragrances, pharmaceutical compositions, agricultural compositions, animal feed, human nutritional products, cosmetics, colourants (carotenoids), or radical scavengers; (viii) for a fermentation production system for producing sesquiterpenes, preferably in a host cell as defined herein, i.e. a bacterial cell, a yeast cell, a fungal cell, an algae cell or a cyanobacterial cell, a non-human animal cell or a non-human mammalian cell, or a plant cell, more preferably a bacterial cell, or a yeast cell; A host cell; b) The use of α-ionylideneethane as a fragrance chemical or compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0317] 1. Production of α-ionylideneethane, preferably E,Z-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene) and / or α-ionone by the process of the present invention.

[0318] 2. A compound of embodiment 1, (i) at least one further aroma chemical different from α-ionylideneethane or α-ionone, or (ii) at least one non-aromatic chemical carrier; or (iii) a mixture of (i) and (ii).

[0319] 3. At least one fragrance chemical different from α-ionylideneethane or α-ionone is selected from the group consisting of geranyl acetate, α-hexylcinnamaldehyde, 2-phenoxyethyl isobutyrate, dihydromyrcenol, methyl dihydrojasmonate, 4,6,6,7,8,8-hexamethyl-1,3,4,6,7,8-hexahydrocyclopenta[g]benzopyran, tetrahydrolinalool, ethyl linalool, benzyl salicylate, 2-methyl-3-(4-tert-butylphenyl)propanal, cinnamyl alcohol, 4,7-methano- 3a,4,5,6,7,7a-Hexahydro-5-indenyl acetate and / or 4,7-methano-3a,4,5,6,7,7a-hexahydro-6-indenyl acetate, citronellol, citronellyl acetate, tetrahydrogeraniol, vanillin, linalyl acetate, styrolyl acetate, octahydro-2,3,8,8-tetramethyl-2-acetonaphthone and / or 2-acetyl-1,2,3,4,6,7,8-octahydro-2,3,8,8-tetramethylnaphthalene, hexyl salicylate, 4-tert-butylcyclohexane Hexyl acetate, 2-tert-butylcyclohexyl acetate, α-ionone, α-methylionone, α-isomethylionone, coumarin, terpinyl acetate, 2-phenylethyl alcohol, 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-carboxaldehyde, α-amylcinnamaldehyde, ethylene brassylate, (E)- and / or (Z)-3-methylcyclopentadeca-5-enone, 15-pentadeca-11-enolide and / or 15-pentadeca-12-enolide, 15-cyclopentadeca-nolide , 1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthalenyl)ethanone, 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol, cis-3-hexenyl acetate, trans-3-hexenyl acetate, trans-2 / cis-6-nonadienol, 2,4-dimethyl-3-cyclohexenecarboxaldehyde, 2,4,4,7-tetramethyloct-6-en-3-one, 2,6-Dimethyl-5-hepten-1-al, borneol, 3-(3-isopropylphenyl)butanal, 2-methyl-3-(3,4-methylenedioxyphenyl)-propanal, 3-(4-ethylphenyl)-2,2-dimethylpropanal, 7-methyl-2H-1,5-benzodioxepin-3(4H)-one, 3,3,5-trimethylcyclohexyl acetate, 2,5,5-trimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalene-2 3. The composition of embodiment 2, wherein the aryl ester is selected from the group consisting of 2-(4-tert-butylphenyl)-propanal, 3-(4-tert-butylphenyl)-propanal, ethyl 2-methylpentanoate, ethoxymethoxycyclododecane, 2,4-dimethyl-4,4a,5,9b-tetrahydroindeno[1,2-d][1,3]dioxine, (2-tert-butylcyclohexyl)acetate, and 3-[5,5,6-trimethylbicyclo[2.2.1]hept-2-yl]cyclohexan-1-ol.

[0320] 4. The composition of embodiment 2 or 3, wherein the at least one non-fragrance chemical carrier (ii) is selected from the group consisting of surfactants, oil components, antioxidants, deodorant actives, and solvents.

[0321] 5. The composition of embodiment 4, wherein the solvent is selected from the group consisting of ethanol, isopropanol, diethylene glycol monoethyl ether, glycerol, propylene glycol, 1,2-butylene glycol, dipropylene glycol, triethyl citrate, and isopropyl myristate.

[0322] 6. The composition of embodiment 5, wherein the at least one solvent is present in the composition in an amount of 0.01% by weight to 99.0% by weight, based on the total weight of the composition.

[0323] 7. The composition of embodiment 5, wherein the at least one deodorant active is selected from the group consisting of antiperspirants, esterase inhibitors, and antimicrobial agents.

[0324] 8. The composition of embodiment 5, wherein the at least one surfactant is selected from the group consisting of anionic, nonionic, cationic, amphoteric, and zwitterionic surfactants.

[0325] 9. The aromachemical composition of any one of embodiments 2-8, which is an aromatized ready-to-use composition.

[0326] 10. The aromachemical composition according to embodiment 9, wherein the aromatized ready-to-use composition is selected from perfume compositions, body care compositions, hygiene products, cleaning compositions, textile detergent compositions, compositions for scent dispensers, foods, dietary supplements, pharmaceutical compositions, and crop protection compositions.

[0327] 11. Use of a compound according to embodiment 1 as a fragrance chemical.

[0328] 12. Use of a compound according to embodiment 1 for preparing an aroma chemical composition.

[0329] 13. Use of a compound as defined in embodiment 1 for modifying the fragrance characteristics of an aroma chemical composition.

[0330] 14. The use according to any one of embodiments 11 to 13, wherein the aroma chemical composition is an aromatized ready-to-use composition.

[0331] 15. The use according to embodiment 14, wherein the aromatized ready-to-use composition is selected from perfume compositions, body care compositions, hygiene products, cleaning compositions, textile detergent compositions, compositions for scent dispensers, food products, dietary supplements, pharmaceutical compositions, and crop protection compositions.

[0332] array SEQ ID NOs: 1 to 17 and 19 correspond to the amino acid sequences of α-ionylideneethane synthases shown in Table 1.

[0333] SEQ ID NO:18 corresponds to a Rhodobacter codon-optimized DNA encoding the amino acid sequence of SEQ ID NO:1.

[0334] SEQ ID NOs: 20-33 correspond to synthetic α-ionylideneethane synthases made by the present inventors in an inventive manner. [Brief description of the drawings]

[0335] [Figure 1] E,Z-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene; E,Z-IE,1) is the first cyclic intermediate in the biosynthesis of fungal abscisic acid (2). It is formed from farnesyl pyrophosphate (3) by α-ionylideneethane synthase (IE synthase). [Diagram 2] There are reports in the literature claiming that, contrary to the enzyme used in the examples below, an α-ionylideneethane synthase cyclizes farnesyl diphosphate to the cyclohexenepentadienol derivative 4; (see Okamoto et al., Phytochemistry, Volume 27, Issue 11, 1988, Pages 3465-3469). [Figure 3A] GC traces of t-BME extract from Rhodobacter ROB034 from a DASGIP fermenter (A) and shake flask culture (B), respectively. The peak at retention time 6.4 min was identified as α-ionone. [Figure 3B] GC traces of t-BME extract from Rhodobacter ROB034 from a DASGIP fermenter (A) and shake flask culture (B), respectively. The peak at retention time 6.4 min was identified as α-ionone. [Figure 4] α-Ionone (4) = (E)-4-((2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one. [Diagram 5] It is shown that R-α-ionone (R-4) is probably formed by oxidative cleavage of α-ionylideneethane (1). [Figure 6]A method for preparing vitamin A is presented which involves the conversion of α-ionylideneethane via the respective alcohol to (2E,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-2-en-1-yl)penta-2,4-dien-1-ol, followed by Wittig salt formation and Wittig reaction with a C5-aldehyde. [Figure 7-1] FIG. 1 shows an alignment of the α-ionylideneethane synthase of SEQ ID NO: 1 with other α-ionylideneethane synthases, with conserved amino acids shown in white font on a black background. [Figure 7-2] FIG. 1 shows an alignment of the α-ionylideneethane synthase of SEQ ID NO: 1 with other α-ionylideneethane synthases, with conserved amino acids shown in white font on a black background. EXAMPLES

[0336] The present invention will now be illustrated by the following examples which should not be construed as limiting the scope of the invention.

[0337] overview E,Z-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene; E,Z-IE, 1) is the first cyclic intermediate in the biosynthesis of fungal abscisic acid (2). It is formed from farnesyl pyrophosphate (3) by a specific sesquiterpene synthase; see Figure 1.

[0338] α-Ionylideneethane synthase (IES) (SEQ ID NO:1) from Botrytis cinerea was successfully cloned and expressed in Rhodobacter sphaeroides to evaluate the production of 1 as a potential precursor of vitamin A.

[0339] After scaling up the production of 1 from shake flasks to DASGIP laboratory fermenters (~1 liter working volume), a novel compound, namely α-ionone, was detected in the dodecane phase of the fermentation broth. The isolation and identification of this compound are summarized below.

[0340] Example 1: Gene expression of α-ionylideneethane synthase (IES) from Botrytis cinerea in Rhodobacter 1.1 Configuration of the production system The DNA sequence of α-ionylideneethane synthase was derived from the transcript Bcin08g03880.1 of Botrytis cinerea B05.10 (ASM83294v1). The respective gene (Bcin08g03880) is located at 1,491,127–1,494,679 on chromosome 8. The data was extracted from the Ensembl Fungi database (Ensembl Genomes 2020-enabling non-vertebrate genomic research, Nucleic Acids Research, 2019, [doi.org / 10.1093 / nar / gkz890]) and used as a template for the custom synthesis of the α-ionylideneethane synthase gene with codon usage adapted to Rhodobacter sphaeroides (BioCat, Heidelberg) (SEQ ID NO: 18). The α-ionylideneethane synthase gene was cloned into the position of the santalene synthase gene of the plasmid pm-SPppa-MBP-CiCaSSy-mpmii alt, known from WO 2018160066. The newly constructed plasmid was named pROB018. Like the santalene synthase in the template plasmid, the α-ionylideneethane synthase protein is generated as an N-terminal fusion to the maltose binding protein from E. coli. Additionally, the plasmid contains all the genes of the mevalonate pathway that ultimately delivers farnesyl diphosphate as a substrate for α-ionylideneethane synthase. In addition, Rhodobacter also contains the deoxyxylulose phosphate (DXP) pathway as a supplemental source of farnesyl diphosphate on its chromosome.

[0341] Transfer of the plasmid into Rhodobacter was performed using standard procedures (see, for example, U.S. Pat. No. 260709B2, WO 2014014339, and WO 2011074954). The plasmid was transformed into E. coli 17 and then transferred by conjugation into Rhodobacter ROB002. Cultivation on malic acid medium eliminates E. coli contamination. Absence of E. coli contamination was demonstrated by PCR amplification using E. coli-lacZ specific oligonucleotides known in the art.

[0342] 1.2 Cultivation of ROB034 Rhodobacter ROB034 carrying the α-ionylideneethane synthase gene from Botrytis cinerea on the plasmid pROB018 was cultivated in the DASGIP system according to known methods as described in WO2018160066. A preculture of 250 mL mROB002 medium in a 1 liter unbaffled Erlenmeyer flask was inoculated with 1.5 mL of cold stock culture. After 26 h of incubation at 30 °C (250 rpm, 5 cm amplitude), 69 mL of preculture medium was used to inoculate the main culture. The main culture was started with 0.6 L of mROB001 medium supplemented with 10% (w / w) dodecane and fed with a total of 646 mL of feed solution according to standard procedures. After 141 h, the fermentation was terminated.

[0343] Example 2: Isolation of terpenes 2.1 Work-up 1225 g of fermentation broth was extracted with 800 g of t-BME by stirring for 30 min. No obvious phase separation was observed, so 25 mL of DMSO and 100 g of NaCl were added. Phase separation was further improved by centrifugation at 5000×g for 15 min. The organic layer (653 g) was decanted and the aqueous layer (1301 g) was discarded. The clear organic layer was dried over Na2SO4 and concentrated by rotary evaporation.

[0344] 2.2 Purification of α-ionylideneethane From the 89.6 g crude reaction extract (35 GC-a % IE, 2.6 GC-a % α-ionone, 57 GC-a % dodecane), dodecane was removed by distillation (250 mL distillation apparatus equipped with a distillation bridge): T bath = max. 128℃, T in = 92 to 103°C, T dist = 89-94 °C, p = 7-10 mbar. The sump obtained by the removal of dodecane was heated at 2 mbar and T using the "Pilot-Dist Spaltrohrkolonne" (M311 L4-06). head = 80° C. Further purification of the distilled fractions was carried out by column chromatography (cyclohexane:ethyl acetate).

[0345] 2.3 Purification of α-ionone Dodecane was removed by distillation from 49.8 g of the crude reaction mixture (5.2 GC-a% α-ionone, 1.7 GC-a% α-ionylideneethane, 81 GC-a% dodecane) using a "split tube distillation column" (30 mbar and Thead = 106 ° C.); α-ionylideneethane as well as α-ionone were already evaporated at 2 mbar and Thead = about 81 ° C.).

[0346] 2.4 Analysis GC analysis Preparation of GC-samples from fermentation broth NaCl is added to the sample to improve phase separation. The sample is mixed on a vortex shaker until all salts are dissolved. The solids (i.e., biomass) are removed by centrifugation (20 min, 15° C., 4500×g) and the upper liquid dodecane layer is removed. 100 μL of dodecane was mixed with 900 μL of acetone internal standard solution and the samples were analyzed by GC (Method: GC107B_0672_b-Bisabolene, A030_GC107B_0672 lsobionics_qual, Column: Optima35 MS, 30m*0.25mm*0.25μm) or by RCS / ON-M311 (Method: GC610, CP-SIL 50m; 0.32mm ID; 1.2μm FD; 80℃-8min; -250℃-34min; Tinjection=250℃, Tdetection=280℃).

[0347] GC-MS and NMR For the analysis of broth, GC-MSyobNMR was performed.

[0348] polarization analysis Specific rotations were measured on a Jasco P2000 polarimeter equipped with a sodium vapor lamp and a 1 dm quartz cuvette. Samples were dissolved in chloroform and measured at room temperature.

[0349] 2.5 Results 2.5.1 Identification of α-ionone When recombinant Rhodobacter expressing the B. cinerea α-ionylideneethane synthase was grown in a DASGIP fermentor, the formation of an additional compound was observed in the gas chromatogram of the fermentation broth, which was barely detectable when the strain was grown in shake flask cultures.

[0350] [Table 2]

[0351] The new peak was analyzed by GC-MS, which showed a mass of 192 g / mol. Interpretation of the mass spectrum suggested that the compound was α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one) or its isomer (2,6,6-trimethylcyclohex-2-en-1-ylidene)butan-2-one). GC analysis with authentic α-ionone showed identical retention times for the new compound.

[0352] FIG. 2 shows the formula for α-ionone (4) = (E)-4-((2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one.

[0353] 2.5.2 Purification of terpenes from fermentation broth Terpenes were isolated from the fermentation broth to confirm the structures of the molecules and for further characterization.

[0354] α-Ionylideneethane Workup of the whole fermentation broth with tBME extraction gave 89.6 g of a clear dark brown solution. The crude reaction mixture was purified by distillation.

[0355] The resulting distillation sump (32 g) contained 70 GC-a% α-ionylideneethane and 7 GC-a% dodecane. Losses of α-ionylideneethane occurred within the two collected distillates (12 and 28 GC-a% for distillates 1 and 2). Based on GC-a%, a loss of α-ionylideneethane of about 30% is considered, which needs to be optimized either by distillation conditions and / or by another second stage during fermentation: it is preferable to evaporate the terpene product rather than the co-solvent (i.e. dodecane), so a high boiling solvent should be used as co-solvent instead of dodecane.

[0356] DMSO was added in the first extraction step to facilitate phase separation.

[0357] Subsequent "Pilot-Dist" sump distillation gave a total of 7.5 g (6 fractions) of α-ionylideneethane with a purity of 87-89 GC-a%. The major by-product was α-ionone (8-9 GC-a%).

[0358] The following α-ionylideneethane samples were obtained by a final purification step using column chromatography with cyclohexane:ethyl acetate as the eluent:

[0359] [Table 3]

[0360] α-Ionone Distillation of 49.8 g of crude reaction extract gave 6.8 g of sump (55.4 GC-a% α-ionone). After purification by column chromatography (cyclohexane:ethyl acetate). Fraction BOH-L-42 Fr. 36-42 was further purified by a second column chromatography (cyclohexane:ethyl acetate) to give BOH-L-47 Fr. 34-42. Similarly, BOH-L-52 Fr. 42-56 was purified from another fermentation run.

[0361] The following α-ionone fractions were obtained:

[0362] [Table 4]

[0363] In this study, a total of two purification experiments were carried out: after removing tBME, in the subsequent step, dodecane was removed from the crude fermentation product by distillation, and column chromatography was applied to determine the yield. a) a 6.3 g α-ionylideneethane sump of 97 GC-a% 0.09 GC-a% α-ionone purity from a 32 g distillation sump; and b) 0.82 g of α-ionone could be isolated with a purity of 98 GC-a% (different by-products) from a 6.8 g sump.

[0364] 13 C-NMR confirmed the following structure:

[0365] [ka]

[0366] 2.5.3 Stereochemistry of isolated terpenes The α-ionone isolated from the fermentation broth is also found to be nearly optically pure: this material shows only a single peak on chiral GC, with the same retention time as one of the two peaks from the racemic standard. The α-ionone isolated from the fermentation broth was further analyzed by polarimetry, with a specific rotation of +388° [α]. D 20 (c 0.75, CHCl3). This value is in good agreement with literature data for the R-enantiomer. Similarly, α-ionylideneethane has a specific rotation of +441° [α] D (c0.762, CHCl3).

[0367] 2.5.4 Olfactory notes Olfactory assessment: A 1% by weight solution of the α-ionylideneethane obtained in Example 2.5.2 in triethyl citrate was prepared and evaluated by a panel of four expert perfumers using freshly soaked blotter paper at room temperature of about 20° C. The olfactory notes were ranked from 1 (very weak) to 9 (strong).

[0368] [Table 5]

[0369] Advantageous perfume ingredients The α-ionylideneethane or α-ionone is formed in the perfume composition according to the two tables; compound A is to be understood as being α-ionylideneethane or α-ionone.

[0370] [Table 6]

[0371] [Table 7]

[0372] The olfactory examples of the table: olfactory notes of the olfactory evaluation, as well as the fragrance compositions according to the table: fragrance compositions 1A and 1B, as well as the fragrance compositions according to the table: fragrance compositions 2A and 2B, i.e. 1A, 1B, 2A, 2B, may be contained in various compositions listed below: Deo pump spray ·Clean Hair Conditioner ·Face wash gel ·Foam bath concentrate Hair gel Self-foaming body wash Sprayable sunscreen emulsion Sprayable sun protection emulsion Emollient facial gel · Two-phase oil foam bath ·shampoo Shower bath Hydroalcohol AP / Deo pump spray ·aerosol ·Water-based / alcohol-based AP / Deo roll-on Styling gel type "Out of Bed" Shaving foam · Baby shampoo for sensitive skin Body wash for sensitive skin Gloss Enhancing Shampoo for Sensitive Scalps Deo Stick Baby wipes Aftershave balm Face gel ·Face day care cream ·Face cleanser Body lotion Sun care SPF50+, sprayable lotion Hand dish cleaner, regular Hand dish cleaner, concentrate Sanitary cleaners, concentrates ·Multipurpose cleaner ·Antibacterial fabric softener Detergent composition Powder detergent composition Liquid detergent compositions

[0373] Those skilled in the art will be familiar with the various general formulations of the above mentioned products.

[0374] Perfume Oil Compositions 1A, 1B, 2A, and 2B can be formulated in specific formulations, for example, as disclosed in IP.com Number: IPCOM000258614D, entitled New Arroma Chemicals, pages 6-46, Tables 1-D13, where "Fragrance Composition 1A" is replaced by an equal amount of Perfume Oil Composition 1A, 1B, 2A, or 2B.

Claims

1. Use of α-ionylideneethane as a fragrance compound.

2. 2. Use according to claim 1 as a fragrance compound with floral-violet and / or woody-orris / iris root notes.

3. Use of an α-ionylideneethane synthase in the production of one or more aroma compounds.

4. The α-ionylideneethane synthase a) said α-ionylideneethane synthase (EC 4.2.3) belonging to the subclass of carbon-oxygen lyases acting on phosphate; b) the α-ionylideneethane synthase, which is a fungal or bacterial α-ionylideneethane synthase; c) i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 17 or 19 to 33; ii) an amino acid sequence having at least 40% sequence identity at the amino acid level with any of SEQ ID NOs: 1 to 17 or 19 to 33, and having α-ionylideneethane synthase activity; and iii) an enzymatically active fragment of the amino acid sequence of a) or b) having α-ionylideneethane synthase activity; the α-ionylideneethane synthase comprising an amino acid sequence selected from the group consisting of: c) any combination of a) to c) above; The use according to claim 3, selected from the group consisting of:

5. 4. The use according to claim 3, wherein the α-ionylideneethane synthase is for preparing one or more aroma compounds that impart floral-violet and / or woody-orris / iris root notes to perfumes, fragrances or fragrances.

6. 3. The use according to claim 1 or 2, wherein the α-ionylideneethane is produced by an α-ionylideneethane synthase as defined in claim 3, 4 or 5.

7. 1. A method for preparing one or more fragrance compounds, comprising: a) providing farnesyl diphosphate and an α-ionylideneethane synthase, preferably an α-ionylideneethane synthase as defined in claim 3, under conditions suitable for said α-ionylideneethane synthase to produce α-ionylideneethane; b) converting farnesyl diphosphate to α-ionylideneethane in vitro or in a host cell; c) optionally converting the α-ionylideneethane to one or more further aroma compounds; d) isolating the α-ionylideneethane and / or, optionally, one or more further aroma compounds; e) optionally purifying the α-ionylideneethane and / or, optionally, one or more further aroma compounds; A method comprising:

8. The method comprises: f) exposing the α-ionylideneethane to conditions suitable for oxidative cleavage of the α-ionylideneethane to produce α-ionone; g) converting α-ionylideneethane to α-ionone, preferably R-α-ionone; h) optionally purifying the α-ionone; The method of claim 7 further comprising:

9. 10. A method for perfuming a product, in particular a method for imparting and / or enhancing an odor or flavor, in which at least one α-ionylideneethane synthase as defined in claim 3, 4 or 5 is used, said method comprising the steps of preparing one or more aroma compounds according to the method of claim 7 or 8, optionally followed by a step of purifying the one or more aroma compounds, and then a step of perfuming a product with said one or more aroma compounds.

10. i) at least one α-ionylideneethane as defined in claim 1 or 2; ii) optionally at least one further aroma compound different from i); iii) optionally at least one diluent; and 1. A fragrance compound or composition and / or fragrance composition and / or perfume scented or scented product comprising:

11. A perfumed or scented product comprising at least one α-ionylideneethane as defined in claim 1 or 2.

12. 1. A process for producing α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), comprising the steps of: a) contacting farnesyl diphosphate with at least one α-ionylideneethane synthase as defined in claim 3, 4, or 5 under conditions suitable to produce at least one α-ionylideneethane; b) producing said at least one α-ionylideneethane; c) exposing the at least one α-ionylideneethane produced in step b) to conditions suitable for oxidative cleavage of the α-ionylideneethane to produce α-ionone; d) optionally isolating the α-ionone produced in step c); A method comprising:

13. 10. A host cell for producing α-ionone (4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one), the host cell comprising farnesyl diphosphate and a heterologous α-ionylideneethane synthase as defined in claim 3, 4, or 5, the host cell being preferably a bacterial cell, a yeast cell, a fungal cell, an algae cell, a cyanobacterium cell, a non-human animal cell, a non-human mammalian cell, or a plant cell, the host cell being suitable for the oxidative cleavage of α-ionone to produce α-ionone.

14. (i) the α-ionylideneethane synthase as defined in claim 3, 4 or 5 converts farnesyl diphosphate to α-ionylideneethane; and / or (ii) α-ionylideneethane is converted chemically and / or enzymatically to α-ionone by oxidative cleavage; The host cell of claim 13.

15. 10. Use of a host cell comprising farnesyl diphosphate and a heterologous nucleic acid encoding an α-ionylideneethane synthase as defined in claim 3, 4 or 5, (i) for producing α-ionylideneethane, preferably 2Z,4E-α-ionylideneethane (1,5,5-trimethyl-6-[(1E,3Z)-3-methyl-penta-1,3-dienyl]cyclohexene), preferably as a fragrance ingredient or compound that is a precursor of a fragrance substance, or as a precursor of vitamin A, (ii) to produce α-ionone, preferably R-α-ionone; (iii) for producing vitamin A; (iv) to convert α-ionylideneethane to α-ionone; (v) for converting α-ionylideneethane to vitamin A; (vi) for heterologous reconstitution of terpenes or terpenoids; (vii) for producing industrial products, preferably fragrance compositions, flavors or fragrances, pharmaceutical compositions, agricultural compositions, animal feed, human nutrition products, cosmetics, colorants (carotenoids) or radical scavengers, and / or (viii) for a fermentation production system for producing sesquiterpenes; use.

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