Composition of delta-decalactone

Abstract

The invention relates to a composition of δ-decalactone comprising at least one compound chosen from the group A and / or at least one compound chosen from the group B, said composition comprising an excess of enantiomer (R) over enantiomer (S), the enantiomeric excess of enantiomer (R) over enantiomer (S) being at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and more preferably at least 99%.

Description

COMPOSITION OF DELTA-DECALACTONEFIELD OF THE INVENTION

[0001] The present invention relates to the field of biotechnology. It relates, more particularly, to a novel composition of natural delta-decalactone. Such natural delta- decalactone may be obtained by a method for producing 13-hydroxy-9(Z)-octadecenoic acid (13 -HOD) with microorganisms from a mixture of fatty acids comprising linoleic acid (LA), the 13-HOD being then converted to delta-decalactone.PRIOR ART

[0002] Lactones derived from hydroxy fatty acids are substances very widely found in nature. They are found in animals, yeasts, and moulds, and represent important aromatic components in fruits or milk and butter. Yeast biologically converts fatty acids hydroxylated at different positions to mainly produce two types of lactones: gamma (y) or delta (5). In particular 5-decalactone and 5-dodecalactone are highly appreciated as flavoring compounds in the food industry. They occur, in low quantities, in milk products, like butter, and they bring about an important contribution to the typical taste of these products. As a consequence of their low natural occurrence and their highly appreciated organoleptic characteristics, which are in particular searched for to confer the natural taste of butter and other milk products to margarine and yoghurt, there exists a strong need for processes capable of providing industrial quantities of lactones, and in a quality which is in conformity with the legislations relative to food products.

[0003] Different methods have been proposed for the biosynthesis of lactones. Known processes include for example methods, which are based on the microbial bioconversion of hydrated fatty acids (hydroxy-derivatives of fatty acids). For example, hydroxy fatty acids such as 13-hydroxyoctadecenoic acid can be converted into 5- decalactone.

[0004] 5-decalactone can also directly be obtained from massoia lactone by enzymatic reduction, itself extracted from massoia bark oil. The extraction of massoia bark oil from massoia trees is however associated with deforestation issues.

[0005] Chemical synthesis is an effective solution and is commonly adopted for lactones intended for the cosmetic industry. For lactones intended for food use, this solution has the drawback that the aromas using lactones produced with this technique cannot be defined as "natural flavors" according to the laws in force in most industrialized countries. Taking into account the fact that consumers show that they prefer naturalproducts to synthetic ones, the search for microbiological processes for the production of lactones with high yields and low production costs is very important for the industry.

[0006] 5-decalactone (DDL), which exhibits a coconut, peach or milky scent and which is a raw material put into food products can be mass-produced at a high yield by means of a production method using the cell’s own enzymatic reaction to produce 5- lactone. DDL can be produced by converting pure linoleic acid (LA) to the intermediate hydroxy fatty acid 13-hydroxy-9(Z)-octadecenoic acid (13-HOD) which is then further converted into DDL. The method for producing DDL via 13-HOD is a known method, which is environmentally friendly and exhibits high specificity compared to existing chemical production methods. It requires, however, the use of pure LA, which is not widely available on industrial scale, to purify the enzymes produced by the microorganisms before use.

[0007] The prior art describes various methods for the production of DDL using pure linoleic acid or 13-HOD as starting material. For example, Korean patent applications KR20150098497 and KR20150108809 suggest bioconversion of pure LA to DDL via 13- HOD, performed by enzymes isolated and purified from microorganisms. Similarly, the US 2021 / 0071210 Al proposes methods for the bioconversion of 13-HOD to DDL using specific microorganisms. Kang et al. Biotechnol Lett (2016) 38:817-823 describes the production of DDL from LA via 13-HOD by one-pot reaction using linoleate 13 -hydratase and whole Yarrowia lipolytica cells. Of note, in all proposed processes, isolated or pure LA is used as starting material for the bioconversion process.

[0008] In methods for preparing 13-HOD it would be advantageous to use a different starting material, to avoid the need for purification or isolation of LA. The reference IT201900015713 discloses a process for the production of natural dodecalactones and decalactones using a hydrolysate of vegetable oil as substrate. The fatty acids in the hydrolysate are hydrated by a probiotic microorganism selected from the microorganisms belonging to the genera Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Pediococcus and Leuconostoc. The hydration product is then converted to lactones by microbiological degradation using yeasts. The reactions, however, are not selective for LA. As a consequence, multiple fatty acids are converted in the hydration reaction, and a mixture of reaction products is obtained. This suggests that a hydrolysate of vegetable oil is not a good starting material.SUMMARY OF THE INVENTION

[0009] The present invention aims to overcome the aforementioned drawback.

[0010] The invention especially aims to propose a composition of 5-decalactone obtained through an alternative route from a mixture of fatty acids for selectively producing 5-decalactone.

[0011] The inventors surprisingly found that LA is selectively converted into 13- HOD from a hydrolysate of a vegetable oil, more precisely a mixture of fatty acids, when the hydrolysate is contacted with E. coli cells expressing the enzyme linoleate 13- hydratase from Lactobacillus acidophilus. Of note, only LA is metabolized, thus allowing a good yield and conversion.

[0012] To this end, the invention proposes a composition of 5-decalactone comprising at least one compound chosen from the group A consisting of:Compound (III) Compound (IV)Compound (VII) Compound (VIII)Compound (IX) Compound (X)Compound (XI) Compound (XII) and / or at least one compound chosen from the group B consisting of: linoleic acid, oleic acid, stearic acid, palmitic acid, and linolenic acid, said composition comprising an excess of enantiomer (R) over enantiomer (S), the enantiomeric excess of enantiomer (R) over enantiomer (S) being at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and more preferably at least 99%.Preferably the composition of the invention comprises at least one compound chosen from the group A consisting of compounds (I) to (XII) and one compound chosen from the group B consisting of: linoleic acid, oleic acid, stearic acid, palmitic acid, and linolenic acid.

[0013] In compounds (I)-(XII), “n” is an integer which possible value is indicated in each formula.

[0014] “5 -decalactone” (herein referred to as DDL) is a compound of formula (A). In the following of the text, 5-decalactone will be referred to, indifferently, as “5- decalactone” or “delta- decalactone” or “DDL”.

[0015] DDL may be then enantiomer (R) and enantiomer (S).

[0016] “5 -HOD” refers to 5 -hydroxydecanoic acid represented in formula (B).

[0017] In the composition of 5-decalactone of the invention, either one or more of the compounds of group A, or one or more of the compounds of group B, or one or more of both the compounds of group A and group B, are impurities that are found in said composition when it is made, and which are characteristic of the mixture of fatty acids used as a starting material in the bioconversion of linoleic acid present in said mixture by a microorganism having linoleate 13-hydratase activity.

[0018] In other words, the mixture of fatty acids comprises linoleic acid and further comprises oleic acid and / or stearic acid and / or palmitic acid and / or linolenic acid. And despite the presence of at least one of these fatty acids in addition to linoleic acid in the starting mixture, the composition of 5-decalactone according to the invention, recovered after bioconversion, demonstrate an unexpectedly high selectivity of the hydration for linoleic acid. This unlock the possibility of using industrial oil instead of pure linoleic acid, thereby strongly reducing the industrial costs and making the preparation of the 5- decalactone composition more readily.

[0019] In the present text, the compounds of groups A and B may be referred to as “impurities” of these groups.

[0020] Advantageously, the bioconversion process of the invention is highly enantioselective, thus leading to a composition comprising a large enantiomeric excess in favor of enantiomer (R).

[0021] According to other optional features of the composition of 5-decalactone of the invention: the composition of 5-decalactone comprises at least one compound chosen from the group A consisting of: compound (I. a), and compound (V.a) as represented below:Compound (V.a) the concentration of 5-decalactone in the composition is at least 90% by weight, preferably at least 95% by weight, more preferably at least 96% by weight, more preferably at least 99% by weight, more preferably at least 99.5% by weight, most preferably at least 99.9% by weight, based on the total weight of the composition; the concentration of each of the compounds from group A is inferior or equal to 3% by weight, preferably inferior or equal to 1% by weight, more preferably inferior or equal to 0.5% by weight, more preferably inferior or equal to 0.05% by weight based on the total weight of the composition; the concentration of each of the compounds from group B is inferior or equal to 5% by weight, preferably inferior or equal to 3% by weight, more preferablyinferior or equal to 1% by weight, preferably inferior or equal to 0.1% by weight, based on the total weight of the composition; the 5-decalactone displays a mean isotopic13C deviation ranging from -40%o to - 28%o ; the enantiomeric excess is 100% of enantiomer (R); the composition is obtained by bioconversion of linoleic acid in a mixture of said linoleic acid and at least one fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, by a microorganism having linoleate 13 -hydratase activity; the composition is obtained by bioconversion of linoleic acid in a mixture of said linoleic acid and at least one fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, by a microorganism expressing a protein having linoleate 13-hydratase activity (13 HOD) and a yeast (DDL); the microorganism expresses a recombinant protein; the microorganism is a microorganism of the genus Escherichia; the yeast is either wild type or genetically modified (= genetically engineered); the composition is obtained by the following method:• (A) culturing a microorganism of the genus Escherichia expressing a recombinant protein having linoleate 13-hydratase activity, wherein said recombinant protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence according to SEQ ID NO: 1;• (B) adding linoleic acid and at least fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, to the composition obtained in step (A);• (C) incubating the composition obtained in step (B) to obtain a composition comprising 13-hydroxyoctadecenoic acid;• (D) adding a microorganism of the genus Yarrowia or Lipomyces. capable of degrading 13-hydroxyoctadecenoic acid, to the composition obtained in step (C);• (E) incubating the composition obtained in step (D); and• (F) recovering the 5-decalactone.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the biopathway overview from linoleic acid (LA) to 5-HOD and / or 5-decalactone (DDL) in a 2-step bioconversion;

[0023] Figure 2 shows a schematic description of an embodiment of a one-pot bioconversion of LA to DDL;

[0024] Figure 3 shows chromatograms of a broth sample and a commercial sample (racemic) of DDL in chiral analysis.DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a composition of 5-decalactone comprising at least one compound chosen from the group A consisting of:Compound (III) Compound (IV)Compound (VII) Compound (VIII)Compound (XI) Compound (XII)and / or at least one compound chosen from the group B consisting of: linoleic acid, oleic acid, stearic acid, palmitic acid, and linolenic acid, said composition comprising an excess of enantiomer (R) over enantiomer (S), the enantiomeric excess of enantiomer (R) over enantiomer (S) being at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and more preferably at least 99%.

[0026] In the composition of 5-decalactone of the invention, the compounds of groupsA and B, namely compounds (I) to (XII), and oleic acid, stearic acid, palmitic acid, and / or linolenic acid, are impurities that are found in said composition when it is made, and which are characteristic of the mixture of fatty acids used as a starting material in the bioconversion of linoleic acid present in said mixture by a microorganism having linoleate 13-hydratase activity. In other words, the mixture of fatty acids comprises linoleic acid and further comprises oleic acid and / or stearic acid and / or palmitic acid and / or linolenic acid.

[0027] Preferably, the concentration of 5-decalactone in the composition is at least 90% by weight, preferably at least 95% by weight, more preferably at least 96% by weight, more preferably at least 99% by weight, more preferably at least 99.5% by weight, most preferably at least 99.9% by weight, based on the total weight of the composition.

[0028] The purity of 5-decalactone is measured by gas chromatography (GC), by measuring the area in % under the curves obtained, using several reference solutions prepared in ethyl acetate. This method is well-known by the skilled person.

[0029] Linoleic acid is also referred to as cis, cis-9,12-Octadecadienoic acid according to IUPAC, and presents the following formula: H3C(CH2)4CH=CHCH2CH=CH(CH2)7COOH.

[0030] Oleic acid is also referred to as (Z)-octadec-9-enoic acid according to IUPAC, and presents the following formula: CH3(CH2)7CH=CH(CH2)7COOH.

[0031] Stearic acid is also referred to as octadecanoic acid according to IUPAC, and presents the following formula: CHTC^li COOH.

[0032] Palmitic acid is also referred to as hexadecanoic acid according to IUPAC, and presents the following formula: CHdC^luCOOH.

[0033] Linolenic acid is also referred to as (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid according to IUPAC, and presents the following formula: H3CCH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH.

[0034] The composition of 5-decalactone may comprise compound (I.a) and / or and compound (V.a) represented below, which are both impurities of group A.

[0035] Compound (I.a) is a specific compound (I) with n=3, and compound (V.a) is a specific compound (V) with n=l.Compound (I.a)

[0036] Compound (I.a) presents the following formula:H3C(CH2)4COH(CH2)2CH=CH(CH2)3COOH.Compound (V.a)

[0037] Compound (V.a) presents the following formula: H3C(CH2)4COH(CH2)2CH=CHCH2COHCH2COOH.

[0038] The impurities of groups A and B have been identified by gas chromatography / mass spectroscopy (acronym GC / MS).

[0039] According to an optional feature of the invention, the 5-decalactone displays a mean isotopic13C deviation ranging from -40%o to -28%o. The mean isotopic13C deviation may also be referred to as “S13C”.

[0040] During photosynthesis, the assimilation of carbonic gas by plants occurs according to three principle types of metabolism: metabolism C3, metabolism C4 and metabolism CAM. The three photosynthetic processes from C3, C4 or CAM plants will generate isotopic effects, in particular the13C isotopic effect, which helps traceability of the botanic origins. Away from industrial activity, atmospheric carbon dioxide displays a mean isotopic deviation of about 513C = -8%o all over the world. The effect of CO2integration by the plant leads to a decrease of13C isotopic ratio in plants. However, the C3 photosynthetic pathway is very discriminative toward 13C, whereas C4 plant discrimination toward13C is lower. As a consequence, 513C isotopic deviation of plants will vary depending of the photosynthetic mechanism. Plants with a photosynthetic metabolism of the C3 type, such as rice and wheat, display a mean isotopic deviation 513C of about -28%o ± 6%o. Meanwhile, plants with a C4 photosynthetic mechanism, such as maize, will display a mean isotopic deviation of about 513C = -14%o ± 6%o. These ranges of 513C are typically measured when the plant itself is analysed, the result of the measure being an average value of the 513C of the constituents of the plant. Hence, molecules extracted from such plants usually have a 513C value within the same range than the plants.

[0041] According to an embodiment of the composition of the invention, the enantiomeric excess is 100% of enantiomer (R).

[0042] For the sake of clarity, “enantiomeric excess” is a measurement of purity used for chiral substances. It is defined as the absolute difference between the mole fraction of each enantiomer, and reflects the degree to which a sample contains one enantiomer in greater amounts than the other. A racemic mixture has an enantiomeric excess of 0%, while a single completely pure enantiomer has an enantiomeric excess of 100% like the present embodiment.

[0043] According to a preferred embodiment, the composition of 5-decalactone is obtained by bioconversion of linoleic acid in a mixture of said linoleic acid and at least one fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, by a microorganism having linoleate 13- hydratase activity.

[0044] According to another preferred embodiment, the composition of 5- decalactone is obtained by bioconversion of linoleic acid in a mixture of said linoleic acid and at least one fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, by a microorganism expressing a protein having linoleate 13 -hydratase activity (13 HOD), and a yeast (DDL).

[0045] The microorganism preferably expresses a recombinant protein.

[0046] The microorganism preferably is a microorganism of the genus Escherichia.

[0047] The yeast may be either wild type or genetically modified / engineered.

[0048] According to another preferred embodiment, the composition of 5- decalactone is obtained by the following method:- (A) culturing a microorganism of the genus Escherichia expressing a recombinant protein having linoleate 13-hydratase activity, wherein said recombinant protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence according to SEQ ID NO: 1;- (B) adding linoleic acid and at least fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, to the composition obtained in step (A);- (C) incubating the composition obtained in step (B) to obtain a composition comprising 13 -hydroxyoctadecenoic acid;- (D) adding a microorganism of the genus Yarrowia or Lipomyces, capable of degrading 13 -hydroxyoctadecenoic acid, to the composition obtained in step (C);- (E) incubating the composition obtained in step (D); and- (F) recovering the 5-decalactone.

[0049] The biopathway overview from linoleic acid (LA) to 5 -HOD and / or 5- decalactone (DDL) in a 2-step bioconversion is illustrated in Figure 1.

[0050] 13-hydroxy-9(Z)-octadecenoic acid (referred to herein as “13 -HOD”) is a compound of formula (III).

[0051] The “Escherichia” genus according to the present invention refers to the genus of gram-negative rod-shaped bacteria of the family Enterobacteriaceae with active movement (peritrich flagellated) or unflagellated without active movement. Related thereto, the microorganisms of the genus Escherichia according to the present invention include, but are not limited to Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, Escherichia senegalensis, Escherichia vulneris. Preferably, the microorganism of the genus Escherichia is Escherichia coli.

[0052] In general the microorganism of the genus Escherichia used in the method for preparing 13 -HOD may be used as a whole cell.

[0053] According to the present invention, the microorganism of the genus Escherichia expresses a recombinant protein having linoleate 13 -hydratase activity, comprising an amino acid sequence having at least 80% identity to the amino acid sequence as shown in SEQ ID NO: 1.

[0054] SEQ ID NO: 1 is the amino acid sequence of the enzyme linoleate 13- hydratase from Lactobacillus acidophilus.

[0055] SEQ ID NO: 1 reads as follows:HYSSGNYEAFVNASKPKDVDQKSAYLVGSGLASLASAVFLIRDGHMKGDRIHILEELSL PGGSMDGIYNKQKESYI IRGGREMEAHFECLWDLFRSIPSAENKDESVLDEFYRLNRKD PSFAKTRVIVNRGHELPTDGQLLLTPKAVKEIIDLCLTPEKDLQNKKINEVFSKEFFES NFWLYWSTMFAFEPWASAMEMRRYLMRFVQHVSTLKNLSSLRFTKYNQYESLILPMVKY LKDRGVQFHYNTVVDNI FVNRSNGEKIAKQILLTENGEKKSIDLTENDLVFVTNGSITE STTYGDNLHPASEEHKLGATWKLWQNLAAQDDDFGHPDVFCKDIPKANWVMSATITFKN NDIVPFIEAVNKKDPHSGSIVTSGPTTIKDSNWLLGYSISRQPHFEAQKPNELIVWLYG LFSDTKGNYVEKTMPDCNGIELCEEWLYHMGVPEERIPEMASAATTIPAHMPYITSYFM PRALGDRPKVVPDHSKNLAFIGNFAETPRDTVFTTEYSVRTAMEAVYTLLNIDRGVPEV FASAFDVRMLMNAMYYLNDQKKLEDLDLPIAEKLAIKGMLKKVKGTYIEELLKKYKLV*

[0056] In one embodiment, the recombinant protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence as shown in SEQ ID NO: 1. In another embodiment, the recombinant protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence as shown in SEQ ID NO: 1. In other embodiments, the recombinant protein comprises an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO: 1.

[0057] In another embodiment, the recombinant protein consists of an amino acid sequence having at least 85% identity to the amino acid sequence as shown in SEQ ID NO: 1. In another embodiment, the recombinant protein consists of an amino acid sequence having at least 90% identity to the amino acid sequence as shown in SEQ ID NO: 1. In yet other embodiments, the recombinant protein consists of an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence as shown in SEQ ID NO: 1.

[0058] In the most preferred embodiment, the recombinant protein comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 1.

[0059] The microorganism used in the method of the present invention is usually a transformed microorganism. The transformed microorganism can be obtained by introducing a nucleic acid encoding the recombinant protein. Suitable nucleic acid sequences include, but are not limited to, the nucleotide sequence as shown in SEQ ID NO:2.

[0060] SEQ ID NO:2 reads as follows:1 atgcattata gtagtggtaa ttatgaagct tttgtaaacg caagtaaacc taaggatgtc61 gatcagaagt ccgcatatct tgttggttca ggtttggcat cgcttgctag tgctgtattt121 ttaattcgtg atggtcacat gaagggtgat agaattcata tccttgaaga attgagcctt 181 ccaggtggtt caatggatgg gatctataat aagcaaaaag aaagctacat cattcgtggt 242 ggtcgtgaaa tggaagccca ttttgaatgc ttgtgggact tgtttagatc gattccatca 301 gctgaaaata aagatgaatc ggtcctggat gaattttacc gtttaaatag aaaagatcca 361 agtttcgcaa agactcgtgt cattgttaac cgcggacatg aacttccaac tgacggtcaa 421 ttacttctta ctcccaaggc tgttaaagaa attattgatc tttgcttaac tcctgaaaaa481 gatttacaaa ataaaaaaat taatgaagtc tttagtaaag aattttttga atcaaacttc 541 tggctttact ggtcaacgat gtttgccttt gagccatggg caagtgcgat ggaaatgcgt 601 cgttacttaa tgcgttttgt tcaacacgtt tctacactta agaatttatc atcactacgc 661 tttactaagt ataaccaata tgaatcatta attttaccaa tggttaaata cttgaaagat 721 cgcggcgtgc aattccatta caacaccgtt gttgataata tctttgttaa ccgttcaaat 781 ggtgaaaaga ttgctaagca aattctttta actgaaaacg gtgaaaaaaa gagcatcgat841 ttaacagaaa atgacctcgt cttcgttact aacggttcaa ttactgaaag tacaacttat901 ggtgataact tgcacccagc ttctgaggaa cataaattag gtgctacttg gaaattatgg 961 caaaacttgg cagcgcaaga tgatgacttc ggtcacccag atgtcttctg caaggatatt 1021 ccaaaggcta actgggtaat gtctgctaca attactttta agaataatga tattgtgcca 1081 ttcattgaag cagttaataa gaaggatcca cacagcggct caattgtaac tagtgggcct 1141 actacgatta aggattctaa ctggctactt ggttattcaa tcagtcgtca gcctcacttt 1201 gaagcacaaa agcctaacga attgattgta tggctttatg gtttgttctc agacaccaaa 1261 ggtaactatg ttgaaaagac tatgcctgac tgtaacggta ttgaattatg tgaagaatgg 1321 ctttaccaca tgggtgttcc tgaagaaaga atcccagaaa tggcttcagc tgctacgact 1381 attccagcac acatgccata tattacttca tacttcatgc caagagcatt aggcgacaga 1441 cccaaggttg tgccagacca ctcaaagaac ttggccttca ttggtaactt tgctgaaacg 1501 ccaagagaca ctgtctttac cactgaatac tctgtcagaa ctgcgatgga agctgtatac 1561 accttgctta acattgatcg tggtgtgcca gaagtatttg catctgcctt cgatgtcaga 1621 atgctcatga acgcaatgta ctacttgaat gatcaaaaga agcttgaaga tcttgatttg 1681 cctattgctg aaaagttggc aattaagggg atgctcaaga aagttaaggg cacttatata 1741 gaggaattgc ttaagaagta taagttggtt tag

[0061] The skilled person is aware, however, that other nucleotide sequences may be used, e.g. due to degeneracy of the genetic code, or codon optimization with respect to the host microorganism. Methods of introducing nucleic acids or plasmids into microorganisms are known to one of skill in the art.

[0062] ‘Linoleate 13 -hydratase activity” refers to capability of performing hydration of LA to obtain 13 -HOD. Linoleate 13 -hydratase, is, for example, naturally expressed by Lactobacillus acidophilus and is capable of converting LA to 13 -HOD. Linoleate 13- hydratase activity may be determined in an assay as described in Kim et al. (2015) Biotechnology and Bioengineering, Vol. 112, No. 11, pages 2206-2213. A suitable assay for determination of linoleate 13-hydratase activity can be performed in 50 mM citratephosphate buffer (pH 5.0) containing 5 mM linoleic acid, 0. 1 mg / mL purified enzyme, and 4% (v / v) ethanol at 35°C in a 2 mL-tube for 10 min. Conversion of LA into 13-HOD can be detected by gas chromatography.

[0063] In particular embodiments, the linoleate 13-hydratase activity of the recombinant protein is higher or equal than that of wildtype linoleate 13-hydratase from Lactobacillus acidophilus (SEQ ID NO: 1). In one embodiment, the linoleate 13-hydratase activity of the recombinant protein exceeds that of the wildtype enzyme by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, or by at least 100%. In other embodiments, the ratio of the linoleate 13-hydratase activity of the recombinant protein to the linoleate 13-hydratase activity of the wild-type enzyme ranges from 1 to 1.5, or from 1 to 1.4, or from 1 to 1.3, or from 1 to 1.2, or from 1 to 1.1.

[0064] Step (A) of the method of the invention comprises culturing the microorganism. Typically, the culturing conditions are suitable to allow expression of the recombinant protein. The culturing conditions are not particularly limited, and standard cell culture conditions can be used, as they are known in the art. The culture temperature may range from about 25 °C to about 40°C or from about 30°C to about 39°C, or from about 35°C to about 38°C. Most preferably, the microorganisms are cultured at a temperature of about 37°C. The pH value may range from about 5 to about 9, or from about 6 to about 8. Also, the culture can be performed under an aerobic, anoxic, or anaerobic condition depending on nature of a host cell. Any appropriate methods can be used as a culturing method. Such a culture method includes, for example, a batch culture method, a feeding culture method, and a continuous culture method. When the expression of the recombinant protein produced by the transformed microorganism is under the control of an inducible promoter such as lac promoter, the expression of the protein may be induced by adding an inducer such as IPTG (isopropyl-P -thiogalactopyranoside) to the culture medium. Cultivation can also be carried out on a particularly large scale. Accordingly, step (A) may include adding an inducer such as IPTG to the culture.

[0065] Step (B) of the method of the invention comprises adding LA and at least one fatty acid different from LA to the composition obtained from incubation step (A). The “at least one fatty acid different from LA” is also referred to as “at least one other fatty acid” hereinafter. The LA and the at least one other fatty acid may be added simultaneously to the culture. Therefore, step (B) refers to the addition of a mixture of fatty acids comprising LA and at least one fatty acid different from LA. Most preferably, the LA and the at least one other fatty acid are added as a mixture, i.e. they are comprised in the same composition. In preferred embodiments the mixture comprises, or consists of, a hydrolysate of a vegetable oil.

[0066] “Hydrolysate” according to the present invention refers to a composition obtainable by hydrolyzing fatty acid esters. Hydrolysis of fatty acid esters may be effected by contacting a fatty acid ester with a lipase. It is preferred that the hydrolysate is obtained by hydrolyzing vegetable oil. In accordance with the present invention, the hydrolysate comprises LA and at least one fatty acid other than LA.

[0067] “Fatty acids” according to the present invention, unless otherwise specified, include all common fatty acids known in the art. Fatty acids include saturated and unsaturated fatty acids. Known unsaturated fatty acids include, but are not limited to, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid,erucic acid, and docosahexaenoic acid, palmitic acid, oleic acid, or stearic acid; and / or combinations thereof. Known saturated fatty acids include, but are not limited to, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, and behenic acid.

[0068] In one embodiment, the hydrolysate is a hydrolyzed vegetable oil. In one embodiment, the method according to the present invention may comprise adding a hydrolyzed vegetable oil, comprising linoleic acid and at least one further fatty acid. “Vegetable oils” according to the present invention include all common vegetable oils known in the art. In one embodiment, the hydrolyzed vegetable oil is selected from the group consisting of hydrolyzed palm oil, hydrolyzed com oil, hydrolyzed soybean oil, hydrolyzed olive oil, hydrolyzed linseed oil, hydrolyzed canola oil, hydrolyzed safflower oil, hydrolyzed peanut oil or hydrolyzed sunflower oil, hydrolyzed tall oil, and combinations thereof. In preferred embodiments, the hydrolyzed vegetable oil is hydrolyzed sunflower or soybean oil.

[0069] The amount or concentration of linoleic acid in the mixture or hydrolysate to be added to the culture can vary. For example, the concentration of linoleic acid in the mixture or hydrolysate to be added to the culture may range from about 20 wt.% to about 90 wt.%, with respect to the total weight of the mixture or hydrolysate, respectively. In other embodiment, the concentration of linoleic acid in the hydrolysate is from about 20 wt.% to about 80 wt.%, or about 30 wt.% to about 75 wt.%, or from about 35 wt.% to about 70 wt.%, or from about 40 wt.% to about 65 wt.%, or from about 45 wt.% to about 60 wt. %, with respect to the total weight of the mixture or hydrolysate . In one embodiment, the concentration of linoleic acid in the hydrolysate is between about 40 wt.% and 50 wt.%, with respect to the total weight of the mixture or hydrolysate. In another embodiment, the concentration of linoleic acid in the hydrolysate is between about 50 wt.% and 60 wt.%, with respect to the total weight of the mixture or hydrolysate. In one embodiment, the concentration of linoleic acid in the hydrolysate is between about 60 wt.% and 70 wt.%, with respect to the total weight of the mixture or hydrolysate. In one embodiment, the concentration of linoleic acid in the hydrolysate is between about 70 wt.% and 80 wt.%, with respect to the total weight of the mixture or hydrolysate. Of note, the concentration of linoleic acid in the hydrolysate can also be above 80 wt.% with respect to the total weight of the mixture or hydrolysate, such as for example 90 wt.% with respect to the total amount of the hydrolysate, or for example 95 wt.% with respect to the total weight of themixture or hydrolysate. In one embodiment, the concentration of linoleic acid in the hydrolysate can also be below 30 wt.% with respect to the total weight of the mixture or hydrolysate, such as for example 20 wt.% with respect to the total weight of the mixture or hydrolysate, or for example 10 wt.% with respect to the total weight of the mixture or hydrolysate.

[0070] Upon addition of the mixture or hydrolysate the resulting culture is typically further cultured or incubated to allow the hydration reaction to proceed. The culturing conditions after addition of the substrate may be similar to those in step (A). In certain embodiments the temperature is increased to about 40°C after addition of the substrate.

[0071] The composition obtained from step (B) is preferably incubated for about 1 to 8 hours, or for about 2 to 7 hours, or for about 3 to 6 hours, or for about 4 to 5 hours. The preferred pH ranges from about 5 to about 9, preferably from about 6 to about 8, e.g. about 6.5 or 7 or 7.5. The pH may regulated or not, preferably the pH is regulated.

[0072] In a particular embodiment, linoleic acid is selectively converted to 13 -HOD. “Selectively converted to 13-hydroxyoctadecenoic acid” according to the present invention means, that only linoleic acid is converted, wherein other fatty acids are substantially not converted.

[0073] The selectivity (in %) according to the invention, is defined as Yield / Conversion Rate = (number of moles of 13-HOD obtained) / (number of moles of LA converted).

[0074] In this regard, a particular aspect of the invention relates to the selectivity for the hydration of linoleic acid (LA) to 13-hydroxyoctadecenoic acid (13-HOD). Accordingly, in one embodiment of the invention, the selectivity for the hydration of linoleic acid to 13-hydroxyoctadecenoic acid is at least 70%. That is, in one embodiment of the invention, the selectivity for the hydration of linoleic acid to 13- hydroxyoctadecenoic acid is at least 75%. In another embodiment, the selectivity for the hydration of linoleic acid to 13-hydroxyoctadecenoic acid is at least 80%. In another embodiment, the selectivity for the hydration of linoleic acid to 13-hydroxyoctadecenoic acid is at least 85%. In another embodiment, the selectivity for the hydration of linoleic acid to 13-hydroxyoctadecenoic acid is at least 90%. In another embodiment, the selectivity for the hydration of linoleic acid to 13-hydroxyoctadecenoic acid is at least 95%. In another embodiment, the selectivity for the hydration of linoleic acid to 13- hydroxyoctadecenoic acid is more than 95%. Also included are embodiments, wherein the selectivity for the hydration of linoleic acid to 13-hydroxyoctadecenoic acid is below 70%.

[0075] According to the present invention the term “about” means ±10% of the specified numeric value, preferably ±5% and most preferably ±2%.

[0076] The composition obtained in step (D) comprising said microorganism and 13- HOD according to the first aspect and the microorganism of the genus Yarrowia or Lipomyces may then be incubated for about 1 hour to about 30 hours. The composition may be incubated for 1 hour to 30 hours, or for 15 hours to 25 hours, or for 20 hours to 25 hours, or for 25 hours to 30 hours. In one embodiment, the composition is incubated for more than 30 hours. In a preferred embodiment, the composition is incubated for about 21 hours. The preferred incubation temperature is about 30°C.

[0077] ‘A microorganism of the genus Yarrowia" comprises all species of Yarrowia as known in the prior art. The microorganism of the genus Yarrowia includes, but is not limited to, Yarrowia brassicae, Yarrowia bubula, Yarrowia deformans, Yarrowia divulgata, Yarrowia keelungensis, Yarrowia lipolytica, Yarrowia parophoni, Yarrowia porcina, or Yarrowia yakushimensis . In a preferred embodiment, the microorganism of the genus Yarrowia is Yarrowia lipolytica.

[0078] “A microorganism of the genus Lipomyces” comprises all species of Lipomyces (also known as Waltomyces) as known in the prior art. The microorganism of the genus Lipomyces includes, but is not limited to, Lipomyces arxii, Lipomyces chichibuensis, Lipomyces doorenjongii, Lipomyces japonicas, Lipomyces kalimantanensis, Lipomyces kockii, Lipomyces kononenkoae, Lipomyces lipofer, Lipomyces maratuensis, Lipomyces mesembrius, Lipomyces okinawensis, Lipomyces oligophaga, Lipomyces orientalisn, Lipomyces spencermartinsiae, Lipomyces starkeyi, Lipomyces tetrasporus, Lipomyces tropi calls, Lipomyces yamadae, Lipomyces yamanashiensis, Lipomyces yarrowii. In a preferred embodiment, the microorganism of the genus Lipomyces is Lypomyces lipofer.

[0079] “Capable of degrading 13 -hydroxyoctadecenoic acid” according to the present invention relates to -oxidation of 13-hydroxyoctadecenoic acid to generate 5 -HOD and is a process well known in the art.

[0080] The process may comprise an additional optional step wherein the pH is adjusted, that is, acidified, in order for the 5-HOD to be under its acidic form. The pH adjustment can be performed before or after step (F). In one embodiment, the pH adjustment step may be performed before step (F). In this embodiment step (F) relates mainly to the recovery of 5-HOD which is converted in DDL.

[0081] In another embodiment, wherein the pH adjustment step is performed after step (F), step (F) relates mainly to the recovery of 5-HOD, which is then converted in DDL.

[0082] The recovery of DDL and / or 5-HOD can include a purification step.

[0083] ‘Purification” according to the present invention includes all purification methods known in the art. Preferred purification methods include, but are not limited to those, chromatography, distillation, liquid / liquid extraction, adsorption or any combination thereof.

[0084] Preferably, the DDL obtained and / or purified according to the present invention can be used in the flavor and or fragrance industry.

[0085] In particular embodiments, steps ( A)-(F) can be performed successively or in parallel in a so-called “one-pot bioconversion”.

[0086] In a particular embodiment, the steps (A) to (F) can be performed in a “One- pot bioconversion” described in Figure 2. Accordingly, the microorganisms used in steps (A) and (D) are cultured separately, preferably in parallel, preferably in 2 bioreactors. The bioconversion of step (A) is then performed in another reactor or bioreactor in the presence of LA and at least one other fatty acid. At the end of the bioconversion, the cultured microorganism of step (D) is then added in the same reactor or bioreactor. In other words, the cultured microorganism of step (D) is added to a mixture comprising notably 13 -HOD, the microorganism of step (A), the bioconversion medium of step (A), and the at least one other fatty acid which was substantially not degraded by the microorganism of step (A).

[0087] This set-up is particularly advantageous as it avoids several manipulation steps of separation and recovery of 13 -HOD, thus also reducing the risk of loss of yield of 13 -HOD. Further at least one other fatty acid originating from the mixture comprising LA may enter the beta-oxidation cycle of the microorganism used in step (D), thereby facilitating the purification of 5-HOD and / or DDL.

[0088] Alternatively, the steps (A) to (F) can be performed sequentially. Accordingly, the microorganisms used in step (A) and (D) are cultured separately, preferably in parallel, preferably in 2 bioreactors. The bioconversion of step (A) is then performed in another reactor or bioreactor in the presence of LA and at least one other fatty acid. At the end of the bioconversion, the 13-HOD is isolated from the fermentation broth. The 13-HOD obtained is then used for the preparation of DDL or 5-HOD.EXAMPLES - METHODS OF ANALYSIS1) Analytical method for monitoring and quantifying DDL and impurities.

[0089] The analytical method for the quantification of productsby GC-FID is the following.

[0090] In a 15-mL centrifuge tube, remove 1 mL of broth (“mout” in French) using a P100-1000 pipette. Add 100 pL of 37% HC1 using a P10-100 pipette. Shake lightly then add 4 mL of AcOEt using a P5000 pipette. Make a second independent preparation. Vortex 5 min, 2000 rpm. Centrifugation 4 min x 4000 rpm.

[0091] In a 4 mL tube, remove the supernatant and dry with MgSO4 (0.5 g, i.e. approximately 5 mm high in the flask). Either allow to settle for at least 20 min or filter the dried AcOEt phase through 0.2pm hydrophobic PTFE.

[0092] In a 1.5mL vial, take 250pL of the dried AcOEt phase then add 250pL of MSTFA using a P250 pipette. Vortex 10 sec then place the vials on a hot plate for 20 min at 60°C. Allow to return to room temperature before GC / FID injection.

[0093] The analytical conditions are summarized in Table 1 below.Table 12) Identification of compounds of groups A and B by gas chromatography / mass spectroscopy (acronym GC / MS).

[0094] The monitoring an quantification of DDL is carried out by GC / MS

[0095] The analytical conditions are summarized in Table 2 below.Table 23) Purity of the sample (%GC area)

[0096] The purity of 5-decalactone is measured by gas chromatography (GC), by measuring the area in % under the curves obtained (%GC area), using several reference solutions prepared in ethyl acetate. This method is well-known by the skilled person.

[0097] The analytical conditions are summarized in Table 3 below.Table 3Compounds of Groups A and B have been identified as follows:- 1 g / L (0.1 % by weight) of compound (I.a) (Retention time at 20.9 mins),-0.2 g / L (0.02 % by weight) of compound (V.a) (Retention time at 22.4 mins),- 0.5 g / 1 (0.0.05 % by weight) of linoleic acid (Retention time at 22.7 mins),- 5 g / 1 (0.5 % by weight) of oleic acid (Retention time at 22.8 mins),-0.7 g / 1 (0.07 % by weight) of stearic acid (Retention time at 23 mins), and - 1 g / 1 (0.1 % by weight) of palmitic acid (Retention time at 21.3 mins).4) Chiral analysis : enantiomeric excess and enantiomeric product

[0098] The sample used for absolute configuration is a broth sample. For comparison with DDL extracted from broth sample, a commercial sample of DDL is used.

[0099] The preparation of the sample is the following.

[0100] In a centrifuge glass tube, add 1 mL of broth and 100 pL HC1 37% v / v. Agitate, then add 4 mL of Ethyl acetate (EtOAc), vortex for 4 min and centrifuge for 4 min at around 3000g. Take the supernatant (EtOAc phase) and dry with around 0.5g of MgSO4. In a 4 mL vial, take 1 mL of dried EtOAc, add 100 pL of formic acid and dd around 0.5g MgSO4. Heat at 77°C for 30 min under stirring. Filter on hydrophobic PTFE before injection.

[0101] For the commercial sample: prepare a solution at around 1000 ppm in Ethyl acetate.

[0102] The analytical conditions are summarized in Table 4 below.Table 4

[0103] The chromatograms of both samples are shown in Figure 3.

[0104] The enantiomeric excess is calculated with the relative surface areas in %area of the 2 enantiomers (R) and (S) according to the formula %ee = ((R - S) / (R + S))* 100.

[0105] In figure 3, chromatogram (a) is the broth sample, and chromatogram (b) is the commercial racemic DDL.

[0106] The retention times of both enantiomers are 20.2 min and 20.5 min, as summarized in Table 5 below, which correspond to (S) and (R) enantiomers respectively based on the elution order.

[0107] This shows a large excess of enantiomer (R) over enantiomer (S), with an enantiomeric excess in the broth sample of 92.1%.Table 5

Claims

CLAIMS1. Composition of 5-decalactone comprising at least one compound chosen from the group A consisting of:Compound (IX) Compound (X)Compound (XI) Compound (XII) and / or at least one compound chosen from the group B consisting of: linoleic acid, oleic acid, stearic acid, palmitic acid, and linolenic acid,said composition comprising an excess of enantiomer (R) over enantiomer (S), the enantiomeric excess of enantiomer (R) over enantiomer (S) being at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and more preferably at least 99%.

2. Composition of 5-decalactone according to claim 1, comprising at least one compound chosen from the group A consisting of: compound (I.a), and compound (V.a) as represented below:Compound (V.a)3. Composition of 5-decalactone according to claim 1 or claim 2, wherein the concentration of 5-decalactone in the composition is at least 90% by weight, preferably at least 95% by weight, more preferably at least 96% by weight, more preferably at least 99% by weight, more preferably at least 99.5% by weight, most preferably at least 99.9% by weight, based on the total weight of the composition.

4. Composition of 5-decalactone according to any of the preceding claims, wherein the concentration of each of the compounds of group A is inferior or equal to 3% by weight, preferably inferior or equal to 1% by weight, more preferably inferior or equal to 0.5% by weight, more preferably inferior or equal to 0.05% by weight, based on the total weight of the composition.

5. Composition of 5-decalactone according to any of the preceding claims, wherein the concentration of each of the compounds of group B is inferior or equal to 5% by weight, preferably inferior or equal to 3% by weight, more preferably inferior or equal to 1% by weight, preferably inferior or equal to 0.1% by weight, based on the total weight of the composition.

6. Composition of 5-decalactone according to any of the preceding claims, wherein the 5- decalactone displays a mean isotopic13C deviation ranging from -40%o to -28%o.

7. Composition of 5-decalactone according to any of the preceding claims, wherein the enantiomeric excess is 100% of enantiomer (R).

8. Composition of 5-decalactone according to any of the preceding claims, said composition being obtained by bioconversion of linoleic acid in a mixture of said linoleic acid and at least one fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, by a microorganism having linoleate 13 -hydratase activity.

9. Composition of 5-decalactone according to any of claims 1 to 7, said composition being obtained by bioconversion of linoleic acid in a mixture of said linoleic acid and at least one fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, by a microorganism expressing a protein having linoleate 13 -hydratase activity, and a yeast.

10. Composition of 5-decalactone according to claim 9, wherein the microorganism expresses a recombinant protein.

11. Composition of 5-decalactone according to claim 9 or claim 10, wherein the microorganism is a microorganism of the genus Escherichia.

12. Composition of 5-decalactone according to any of claims 9 to 11, wherein the yeast is either wild type or genetically modified.

13. Composition of 5-decalactone according to any of claims 1 to 7, said composition being obtained by the following method:- (A) culturing a microorganism of the genus Escherichia expressing a recombinant protein having linoleate 13-hydratase activity, wherein said recombinant protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence according to SEQ ID NO: 1;- (B) adding linoleic acid and at least fatty acid different from linoleic acid chosen from the group consisting of oleic acid, stearic acid, palmitic acid, and linolenic acid, to the composition obtained in step (A);- (C) incubating the composition obtained in step (B) to obtain a composition comprising 13 -hydroxyoctadecenoic acid;- (D) adding a microorganism of the genus Yarrowia or Lipomyces, capable of degrading 13 -hydroxyoctadecenoic acid, to the composition obtained in step (C);- (E) incubating the composition obtained in step (D); and- (F) recovering the 5-decalactone.

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