Recombinant production of Santarene

Abstract

The present invention relates to the field of recombinant production of santalene and related products. In particular, the present invention relates to a method for producing a composition comprising at least one santalene, comprising a step of converting farnesyl pyrophosphate to at least one santalene, said conversion being carried out by a polypeptide exhibiting santalene synthase activity. Furthermore, the present invention contemplates a composition comprising a mixture of β-santalene and α-santalene, comprising an excess of β-santalene, obtainable by the method of the present invention. The present invention also relates to the use of a santalene synthase polypeptide, a heterologous polynucleotide encoding it, a vector or genetic construct comprising said polynucleotide, a host cell or a non-human transgenic organism comprising said genetic construct or vector, for producing a composition comprising at least one santalene, preferably β-santalene, more preferably a mixture of β-santalene and α-santalene. The present invention further relates to a method for producing a composition comprising at least one santalol, preferably β-santalol, comprising producing a composition comprising at least one santalene by the above-mentioned method of the present invention and oxidizing said at least one santalene, preferably β-santalene, to the respective alcohol to produce a composition comprising at least one santalol, preferably β-santalol.The present invention further relates to a kit for producing a composition comprising at least one santalene, comprising the above-mentioned polypeptide, heterologous polynucleotide, vector or genetic construct, host cell or non-human transgenic organism, and to a non-human host cell or non-human transgenic organism expressing a polypeptide exhibiting santalene synthase activity from the above-mentioned heterologous polynucleotide, vector or genetic construct.

Description

[Technical field]

[0001] The present invention relates to the field of recombinant production of santalene and related products. In particular, the present invention relates to a method for producing a composition comprising at least one santalene, comprising a step of converting farnesyl pyrophosphate to at least one santalene, said conversion being carried out by a polypeptide exhibiting santalene synthase activity. Furthermore, the present invention contemplates a composition comprising a mixture of β-santalene and α-santalene, comprising an excess of β-santalene, obtainable by the method of the present invention. The present invention also relates to the use of a santalene synthase polypeptide, a heterologous polynucleotide encoding it, a vector or genetic construct comprising said polynucleotide, a host cell or a non-human transgenic organism comprising said genetic construct or vector, for producing a composition comprising at least one santalene, preferably β-santalene, more preferably a mixture of β-santalene and α-santalene. The present invention further relates to a method for producing a composition comprising at least one santalol, preferably β-santalol, comprising producing a composition comprising at least one santalene by the above-mentioned method of the present invention and oxidizing said at least one santalene, preferably β-santalene, to the respective alcohol to produce a composition comprising at least one santalol, preferably β-santalol.The present invention further relates to a kit for producing a composition comprising at least one santalene, comprising the above-mentioned polypeptide, heterologous polynucleotide, vector or genetic construct, host cell or non-human transgenic organism, and to a non-human host cell or non-human transgenic organism expressing a polypeptide exhibiting santalene synthase activity from the above-mentioned heterologous polynucleotide, vector or genetic construct. [Background technology]

[0002] Sandalwood oil is a major perfume ingredient. It also serves many aromatherapy applications. The major components of sandalwood oil include α-santalol and β-santalol. β-santalol is the major fragrance-affecting component of sandalwood oil. Sandalwood oil is in great shortage due to the difficulty of cultivating the sandalwood plant. β-santalol is made from β-santalene. In sandalwood plants, the production of santalol is mediated by sesquiterpene synthase.

[0003] Sesquiterpene synthases cyclize the ubiquitous precursor farnesyl pyrophosphate (FPP) through a unique proton transfer cascade. The outcome of the cyclization reaction depends on the identity of the terpene synthase. Genes encoding plant terpene synthases can be deployed for microbial production of terpenes. Microbial production of santalene, including α-santalene, trans-α-bergamotene, and β-santalene, has been demonstrated by using santalene synthase from camphor tree (Cinnamomum Camphora) or sandalwood (Santalum album) (US Patent Publication No. 2020 / 0010822A1). However, such microorganisms produce a terpene mixture consisting mostly of α-santalene (50%) and only a small amount of β-santalene (20%).

[0004] Preparations with higher β-santalene concentrations would be useful, for example, to give a sandalwood impression to perfume materials. Because α-santalene and β-santalene have very similar physicochemical properties, separation techniques are difficult to use to obtain preparations with high concentrations of β-santalene. To produce β-santalene, a terpene synthase that produces more than 50% β-santalene is required.

[0005] Furthermore, there is a constant need for fragrance compositions that have new or improved olfactory characteristics. Summary of the Invention [Problem to be solved by the invention]

[0006] The technical problem underlying the present invention shall be understood as the provision of means and methods that meet the above-mentioned needs. This technical problem is solved by the embodiments characterized in the claims and in the following specification. [Means for solving the problem]

[0007] The present invention relates to a method for producing a composition comprising at least one santalene, comprising the step of converting farnesyl pyrophosphate to at least one santalene, said conversion being carried out by at least one polypeptide exhibiting santalene synthase activity, said at least one polypeptide being (i) a) an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 18, or 21-28; b) an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NOs: 1, 18, or 21-28; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 2 or 3 or 19; d) an amino acid sequence encoded by a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid sequence as set forth in SEQ ID NO: 2 or 3 or 19; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting santalene synthase activity. or an amino acid sequence selected from the group consisting of (ii) a) an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 17; b) an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in SEQ ID NOs: 4 to 17; and c) the amino acid sequence of a fragment of (a) or (d), which fragment encodes a polypeptide exhibiting santalene synthase activity. The present invention relates to a method comprising administering to a subject a nucleic acid sequence comprising an amino acid sequence selected from the group consisting of:

[0008] The composition produced by the process of the present invention comprises at least one santalene as defined herein, preferably β-santalene, or a mixture of both α-santalene and β-santalene. When both α-santalene and β-santalene are present, it is preferred that the composition comprises an excess of β-santalene over α-santalene.

[0009] In one preferred embodiment of the method of the present invention, the composition comprising at least one santalene is substantially free of any one or all of the following: cis-α-bergamotene (AS No. 18252-46-5), (E)-β-farnesene (CAS No. 18794-84-8), trans-β-bergamotene (CAS No. 15438-94-5), and β-bisabolene (CAS No. 495-61-4). By substantially free, it is meant that the composition does not contain detectable amounts of cis-α-bergamotene (AS No. 18252-46-5), (E)-β-farnesene (CAS No. 18794-84-8), trans-β-bergamotene (CAS No. 15438-94-5), and / or β-bisabolene (CAS No. 495-61-4).

[0010] In another preferred embodiment of the method of the invention, the composition comprising at least one santalene further comprises a detectable amount of sesquithujene (CAS No. 58319-06-5).Preferably, a detectable amount of sesquithujene (CAS No. 58319-06-5) means 0.01% to 3% (mol / mol).

[0011] In yet another preferred embodiment of the method of the present invention, the composition comprising at least one santalene further comprises a detectable amount of sesquithujene (CAS No. 58319-06-5), and the composition is substantially free of any one or all of the following: cis-α-bergamotene ((AS No. 18252-46-5), (E)-β-farnesene (CAS No. 18794-84-8), trans-β-bergamotene (CAS No. 15438-94-5), and β-bisabolene (CAS No. 495-61-4).

[0012] "%" in the context of solution concentrations referred to herein means percent (mol / mol) unless otherwise indicated.

[0013] It should be understood that, in this specification and claims, "a" or "an" may refer to one or more of the items referenced below, depending on the context in which it is used. Thus, for example, a reference to "an" item may mean that at least one of the items mentioned is available.

[0014] As used below, the terms "having", "including" or "comprising" are intended to have either an open-ended or closed-ended meaning. Thus, having these terms with a closed-ended meaning may refer to a situation in which the described embodiment does not have other features than those introduced by these terms, i.e., the terms have a closed-ended meaning in the sense of "consisting of" or "consisting essentially of". When having a closed-ended meaning, the terms refer to a situation in which the described embodiment has one or more other features than those introduced by these terms.

[0015] Furthermore, as used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "typically" and "more typically" are used in conjunction with features to indicate that these features are preferred features, i.e., this term is intended to indicate that alternative features may also be envisaged in accordance with the present invention.

[0016] Furthermore, it will be understood that the term "at least one" as used herein means that one or more of the items mentioned following the term may be used in accordance with the present invention. For example, if the term indicates that at least one item must be used, this may be understood as one item or more than one item, i.e., two, three, four, five, or any other number of items. Depending on the item to which the term refers, a person skilled in the art will understand what upper limit, if any, the term may refer to.

[0017] The process according to the invention may either consist of the steps mentioned above or may include additional steps, which may be pretreatment steps or steps required for the preparation of at least one santalene-containing composition, such as purification steps.

[0018] The term "production" as used herein refers to the production of a composition comprising at least one santalene, in particular a composition comprising β-santalene, more preferably a composition comprising a mixture of α-santalene and β-santalene, and most preferably a composition comprising an excess of β-santalene. This production allows for the production of said at least one santalene in any degree of purity in the composition. The higher the degree of purity envisaged, the more additional purification may be required. The method may be carried out ex vivo, for example in one or more reaction vials. Alternatively, the method may be carried out in whole or in part in an organism, such as a non-human transgenic organism, including a plant or a microorganism, including the host cells mentioned elsewhere herein, preferably a non-vertebrate transgenic organism.

[0019] The term "santalene" as used in accordance with the present invention refers to α-santalene (CAS number 512-61-8; 6,7-dimethyl-7-(4-methylpent-3-enyl)-2,3,4,5-tetrahydro-1H-tricyclo[2.2.1.0 2,6 ]Heptane; molecular formula C 15 H 24 ), β-santalene (CAS number 511-59-1, (1R,3R,4S)-3-methyl-2-methylidene-3-(4-methylpent-3-enyl) bicycle[2.2.1]heptane; molecular formula C 15 H 24 ), and epi-β-santalene (CAS number 25532-78-9; (3S)-3-methyl-2-methylidene-3-(4-methylpent-3-enyl) bicycle [2.2.1] heptane; molecular formula C15H24), and trans-a-bergamotene (CAS number 13474-59-4; hereinafter also referred to as trans-α-bergamotene or shortened to bergamotene). Preferably, said santalene is β-santalene (CAS number 511-59-1, molecular formula C15H24). [ka]

[0020] Formula I represents (-)-β-santalene (CAS number 511-59-1, hereafter referred to as β-santalene).

[0021] The "at least one santalene" referred to in accordance with the present invention is preferably β-santalene. More preferably, it is a mixture of β-santalene and α-santalene, with an excess of β-santalene. More preferably, said β-santalene is present in the composition in a relative amount of at least about 50%, at least about 60%, at least about 70%, preferably about 50% to about 80%, about 60% to about 75%, about 65% to about 70%, more preferably at least about 67% relative to the total santalene. The total santalene referred to herein includes all santalenes found in the composition. Preferably, these are α-santalene, β-santalene, and bergamotene. Small amounts of other santalenes may occur as well.

[0022] More preferably, the ratio of bergamotene to β-santalene is less than 1.0, such as 0.9 or less, 0.8 or less, or 0.7 or less, such as 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or even 0.1 or less. In another aspect of the invention, the total santalene contains only 10% or less bergamotene and an excess of β-santalene over α-santalene. In a further aspect of the invention, the combined percentage (mol / mol) of α-santalene and bergamotene is less than the percentage (mol / mol) of β-santalene of total santalene.

[0023] In a further aspect of the invention, the composition comprises at least one santalene, preferably β-santalene in excess of α-santalene, and the composition further comprises: a) a detectable amount, preferably 0.01% to 3% (mol / mol), of sesquithujene (CAS No. 58319-06-5); b) substantially free of any or all of the following: cis-α-bergamotene ((AS No. 18252-46-5), (E)-β-farnesene (β-farnesene, CAS No. 18794-84-8), trans-β-bergamotene (CAS No. 15438-94-5), and β-bisabolene (CAS No. 495-61-4), or a combination of a) and b).

[0024] The absence, or at least a significantly reduced amount, of (E)-β-farnesene is useful, for example, when a composition containing santalene is used to produce santalol, an oxidation reaction in which the presence of (E)-β-farnesene is less desirable.

[0025] The term "polypeptide" as used in accordance with the present invention refers to a continuous sequence of amino acids linked together by peptide bonds. A polypeptide according to the present invention is typically composed of at least 50, at least 100 or at least 200 amino acids in length, such that the amino acid chain can form the three-dimensional structure required to exert the enzymatic activity referred to elsewhere herein. The term "protein" may be used interchangeably herein.

[0026] The term "santalene synthase activity" as used herein refers to the activity of an enzyme that can convert farnesyl pyrophosphate as starting material to at least one santalene. Typically, santalene synthase can catalyze the production of sesquiterpenoids from (2E,6E)-farnesyl diphosphate, such as α-santalene, β-santalene, epi-β-santalene, and trans-α-bergamotene, as well as a mixture of trace amounts of α-farnesene and β-farnesene. They may also use (Z,Z)-farnesyl diphosphate isomers for the conversion to α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene (EC 4.2.3.81).

[0027] Preferably, a polypeptide having santalene synthase activity according to the invention comprises: a) an amino acid sequence as set forth in any of SEQ ID NOs: 1, 18, or 21-28; b) an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NOs: 1, 18, or 21-28; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 2 or 3 or 19; d) an amino acid sequence encoded by a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid sequence as set forth in SEQ ID NO: 2 or 3 or 19; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting santalene synthase activity. The amino acid sequence is selected from the group consisting of:

[0028] In one aspect of the invention, the polypeptide is a dual function synthase, ie, the polypeptide is a santalene synthase and also a sesquithudiene synthase (EC 4.2.3.102 (2E,6E)-farnesyl-diphosphate diphosphate-lyase (sesquithudiene-forming)).

[0029] In a further aspect of the invention, the polypeptides of the invention do not produce substantial amounts of β-farnesene when santalene and / or sesquithujene are produced.

[0030] Preferably, the polypeptide having santalene synthase activity is derived from or based on a polypeptide from the genus Oryza, including, but not limited to, polypeptides from O. meridionalis, O. glumipatula, O. sativa, O. rufipogon, O. glaberrima, O. nivara, O. barthii, O. punctata.

[0031] The polypeptides having santalene synthase activity according to the present invention preferably produce β-santalene and α-santalene in a ratio of 1 or more, preferably at least 1.1, more preferably at least 1.2, and even more preferably 1.3, under conditions suitable for the production of these santalenes.

[0032] These santalene synthases of the invention produce β-santalene and α-santalene with a molar ratio of β-santalene to α-santalene of 1 or more, preferably as measured by GC-FID; for example, the ratio is at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or at least 2. The ratio of β-santalene to α-santalene can be at least 3:1, preferably at least 4:1, more preferably at least 5:1, even more preferably 6:1, even more preferably at least 7:1, most preferably at least 8:1, or even at least 9:1. In one embodiment of the invention, the ratio is 100:1 or less.

[0033] Yet another aspect of the present invention relates to a polypeptide having santalene synthase activity as defined herein, which produces β-santalene in excess over α-santalene, which is in excess of trans-α-bergamotene. Typically, santalene synthases known in the art produce significant amounts of bergamotene, which is undesirable santalene in some applications. Surprisingly, the polypeptide having santalene synthase activity of the present invention has been shown to produce only small amounts of bergamotene (less than 10% of total santalene) and produce β-santalene in excess over α-santalene.

[0034] "Sequence identity" as referred to herein above defines the relationship between amino acid or nucleic acid sequences and can be determined by comparing these sequences. Usually, sequence identity is determined by comparing two sequences over the entire length of the sequences, but it can also be compared over only parts of the sequences aligned with each other. Preferably, sequence identity is compared herein over the entire length of the sequences. Sequence identity refers to the degree of relatedness between polypeptide or nucleic acid sequences. Sequence identity is expressed as the percentage of identical amino acids or nucleotides in two sequences compared with each other. Thus, when two sequences are aligned, the number of amino acids or nucleotides that match between the sequences is generally determined and related to the total number of amino acids or nucleotides in the aligned sequence or part of the sequence. For example, variant sequences can be defined by their sequence identity when compared to a parent sequence, i.e., an amino acid sequence as shown in any two of SEQ ID NOs: 1, 18, or 21-28, or a nucleic acid sequence as shown in SEQ ID NOs: 2 or 3 or 19. To determine the percent identity between two sequences, the first step is to generate a pairwise sequence alignment between these two sequences, aligning the two sequences over their entire, total or full length (i.e., pairwise global alignment). The alignment is generated using the programs or software described herein. The preferred alignment for the purposes of the present invention is the alignment that allows the maximum sequence identity to be determined.

[0035] Sequence alignments are generated using various software tools such as the 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. 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, within 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) - is usually generated based on alignment of conserved regions of protein domains, for example (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 mainly used to search for similar sequences in large sequence databases. The BLAST programs also generate local alignments.Typically, the "BLAST" interface provided by NCBI (National Center for Biotechnology Information) is used, as is an improved version ("BLAST2"). "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.

[0036] 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 can 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). In such a case, the identity 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.

[0037] The variant amino acid or nucleic acid sequences referred to herein may be naturally occurring variants, such as allelic variants or orthologous, paralogous or homologous variants. Alternatively, such sequences may be artificially generated to improve the properties of an enzyme or nucleic acid (e.g., improve the expression of an enzyme or increase the enzymatic activity of an enzyme), for example by biological techniques known to those 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.). Typically, variants of polypeptides having santalene synthase activity according to the invention are polypeptides having one or several amino acid substitutions compared to the amino acid sequence of any of SEQ ID NOs: 1, 18, or 21-28, preferably the artificial amino acid sequence.

[0038] Variant nucleic acid sequences encoded by an amino acid sequence as set forth in SEQ ID NO: 2 or 3 or 19, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid sequence as set forth in SEQ ID NO: 2 or 3 or 19, may differ from the nucleic acid sequence set forth in SEQ ID NO: 2 or 3 or 19 by at least one nucleotide substitution, addition and / or deletion for reasons described elsewhere herein. It will be understood that polynucleotides comprising such variant nucleic acid sequences referred to herein can hybridize to each other, preferably under stringent hybridization conditions. Stringent hybridization conditions referred to herein are preferably 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more wash steps in 0.2x SSC, 0.1% SDS at 50-65°C. The skilled artisan knows that these hybridization conditions, with regard to the temperature and concentration of the buffer, for example in the presence of an organic solvent, vary depending on the type of nucleic acid. For example, under "standard hybridization conditions", the temperature varies depending on the type of nucleic acid and is 42°C to 58°C in an aqueous buffer with a concentration of 0.1 to 5×SSC (pH 7.2). If an organic solvent is present in the above-mentioned buffer, for example 50% formamide, the temperature under standard conditions is about 42°C. Hybridization conditions for DNA:DNA hybrids are preferably 20°C to 45°C, preferably 30°C to 45°C, with 0.1×SSC. Hybridization conditions for DNA:RNA hybrids are preferably 30°C to 55°C, preferably 45°C to 55°C, with 0.1×SSC. The above-mentioned hybridization temperatures are determined, for example, for nucleic acids of about 100 bp (= base pairs) in length and 50% G+C content in the absence of formamide.Those skilled in the art will know how to determine the required hybridization conditions by referring to the above textbooks or the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Thus, a variant nucleic acid sequence can be derived from a polynucleotide that can hybridize under stringent hybridization conditions to a nucleic acid sequence that encodes an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO: 2 or 3 or 19, or an amino acid sequence encoded by a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid sequence as shown in SEQ ID NO: 2 or 3 or 19.

[0039] The present invention further provides a method for preparing a variant polypeptide having santalene synthase activity, comprising the steps of: (a) selecting a nucleic acid set forth in any one of SEQ ID NOs: 2, 3, or 19; (b) modifying the selected nucleic acids to obtain at least one mutant nucleic acid; (c) transforming a host cell or a unicellular organism as defined herein with the mutated nucleic acid sequence to express the polypeptide encoded by the mutated nucleic acid sequence; (d) screening the polypeptide for at least one altered property; (e) optionally, if the polypeptide does not have the desired variant santalene synthase activity, repeating process steps (a)-(d) until a polypeptide having the desired variant santalene synthase activity as defined herein is obtained; (f) optionally, if a polypeptide having the desired variant santalene synthase activity is identified in step (d), isolating the corresponding mutant nucleic acid obtained in step (c).

[0040] The above-mentioned fragments may be polypeptides consisting of any of the above-mentioned sequences and sequence variants, which have a length sufficient to exhibit the above-specified santalene synthase activity.It is therefore envisaged that the above-mentioned biologically active fragments of the polypeptides preferably contain the amino acid sequence of the catalytically active region of santalene synthase.Typically, the fragments consist of at least 20, at least 30, at least 40, at least 50, at least 100, at least 150 or at least 200 consecutive amino acids in length from the above-mentioned sequences or sequence variants.

[0041] Preferably, a fragment of a polypeptide as referred to herein or a variant polypeptide as referred to herein comprises at least one, preferably at least two, more preferably three Pfam domains.Typically, the Pfam domains referred to according to the invention are the N-terminal domain of a terpene synthase (PF01397.21), the metal binding domain of a terpene synthase (PF03936.16) and the Pfam domain of the trichodiene synthase TRI5 (PF06330.11). Pfam domains referred to herein were analyzed using PFAM version 32.0, for more information on PFAM please see "The Pfam protein families database in 2019: S. El-Gebali, J. Mistry, A. Bateman, S.R.Addy, A. Luciani, S.C.Totter, M. Qureshi, L.J.Richardson, G.A.Salazar, A.Smart, E.L.S. Sonnhammer, L.Hirsh, L.Paladin, D.Piovesan, S.C.E.Tosatto, R.D.Finn Nucleic Acids Research (2019) and http: / / pfam.xfam.org / .

[0042] Preferably, a polypeptide or fragment having santalene synthase activity according to the invention comprises at least one, preferably all, of the conserved domains shown in Figure 4. The conserved domains are shown in Figure 4 in light text on a black background. Preferably, the conserved domains consist of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 amino acids. Particularly preferred conserved domains to be mentioned according to the invention are those having the following amino acids of SEQ ID NO: 1: amino acids 82-96, 133-146, 148-156, 163-185, 195-205, 214-225, 227-233, 235-242, 345-352, 375-382, 400-409, and 411-419.

[0043] In another embodiment, the santalene synthase of the present invention comprises a GRXCX4W motif (SEQ ID NO: 20) in the N-terminal region, characterized by the amino acid glycine followed by arginine, followed by any amino acid, followed by cysteine, followed by four amino acids of any type, and finally tryptophan.

[0044] In a preferred embodiment, the santalene synthase or fragment thereof of the invention has an amino acid stretch corresponding to amino acid positions 281 to 341 of SEQ ID NO: 1 with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 changes compared to the amino acid stretch of SEQ ID NO: 1. In a further aspect of the invention, the changes in said amino acid stretch corresponding to positions 281 to 341 of SEQ ID NO: 1 are either in amino acids corresponding to positions 293 to 295 and / or 317 of SEQ ID NO: 1.

[0045] Preferably, a polypeptide having santalene synthase activity according to the invention comprises: a) an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 17; b) an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in SEQ ID NOs: 4 to 17; c) the amino acid sequence of a fragment of (a) or (d), which fragment encodes a polypeptide exhibiting santalene synthase activity. The amino acid sequence may further comprise an amino acid sequence selected from the group consisting of:

[0046] The above-mentioned polypeptides exhibiting santalene synthase activity may also be included in fusion polypeptides. Such fusion polypeptides contain one or more additional amino acid sequences in addition to the amino acid sequence of the polypeptide exhibiting santalene synthase activity. The additional amino acid sequence may be a polypeptide with other enzymatic activity, such as farnesyl pyrophosphate synthase or cytochrome P450 monooxygenase, as specified elsewhere herein, or a polypeptide or peptide with a marker or labeling function, such as a tag (e.g., MYC tag, FLAG tag, His tag, etc.) or a fluorescent protein (e.g., GFP, BFP, YFP or CFP), for example, to monitor appropriate expression or for purification purposes.

[0047] The present disclosure further relates to a method for preparing santalene and santalol, comprising converting santalene to farnesyl diphosphate (FPP) in the presence of an enzyme comprising a first segment comprising a tag peptide and a second segment comprising a santalene synthase according to the present invention, the enzyme comprising said first segment and said second segment being referred to herein as a "tagged enzyme."

[0048] 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 the peptide of the homolog has one or more amino acid residues inserted, substituted, deleted or extended. A homolog may in particular include one or more substitutions of a hydrophilic amino acid for another hydrophilic amino acid or one or more substitutions of a hydrophobic amino acid for another hydrophobic amino acid. A homologue may in particular have at least 40%, more particularly at least 50%, preferably at least 55%, more preferably at least 60% sequence identity, 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.

[0049] Particularly preferred is the maltose binding protein from Escherichia coli or a functional homologue thereof.

[0050] Use of tagged enzymes according to the invention is particularly advantageous in that it may contribute to increased product, in particular increased cellular production of terpenoids or terpenes, such as santalene and / or sesquithujenes.

[0051] 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.

[0052] The present invention further relates to a nucleic acid comprising a nucleotide sequence encoding a polypeptide, the polypeptide comprising a first segment comprising a tag peptide, preferably MBP, NusA, Trx, GST, SUMO or Fh8-tag or a functional homologue of any of these, and a second segment comprising santalene synthase. The second segment may for example comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 4-17 or a functional analogue thereof. An example is a synthetic fusion protein of SEQ ID NO: 18.

[0053] Furthermore, the present invention relates to a host cell comprising said nucleic acid encoding said tagged santalene synthase.

[0054] A particular nucleic acid according to the invention encoding a tagged enzyme is shown in SEQ ID NO: 19. The host cell may in particular comprise a gene comprising any of those sequences or a functional analogue thereof.

[0055] In the methods of the present invention, farnesyl pyrophosphate is enzymatically converted to at least one santalene by a santalene synthase identified herein.

[0056] The above-mentioned conversion step can be carried out in vitro, i.e. in a suitable reaction vial containing all the components required for the conversion as described above. Those skilled in the art are well aware of how to adjust the reaction conditions so that the reaction is carried out efficiently. For example, a suitable buffer can be used to provide the components in an environment with a suitable pH and a suitable salt concentration. The suitable temperature in such a setting can also be applied as is.

[0057] Alternatively, the conversion step may be carried out in a host cell as described elsewhere herein. Preferably, the host cell is selected from the group consisting of bacterial cells, yeast cells, fungal cells, algae cells or cyanobacterial cells, non-human animal cells or non-human mammalian cells and plant cells. It should be understood that the host cell shall be capable of producing santalene. If necessary, the host cell must be genetically modified to express the enzymes or proteins required for santalene synthesis, including the santalene synthase described above. The host cell shall be cultured under conditions and for a time sufficient to allow the expression of the enzymes described above and to convert farnesyl pyrophosphate to at least one santalene. Particularly preferred conditions are also described in the accompanying examples below or are known to those skilled in the art.

[0058] Furthermore, the conversion step of the method of the present invention can also be carried out in an organism, typically a multicellular organism, such as a transgenic non-human organism as mentioned elsewhere herein. Typically, said organism is genetically modified so that the enzyme required for converting farnesyl pyrophosphate to at least one santalene is expressed. However, the skilled artisan is well aware of what conditions need to be applied depending on the choice of a given non-human transgenic organism.

[0059] When the method of the invention is carried out in vivo, i.e. in a host cell or a non-human transgenic organism, said host cell or non-human transgenic organism will be understood to express a polypeptide exhibiting santalene synthase activity as specified above, such that the conversion of at least one of farnesyl pyrophosphate to santalene can be carried out in said host cell or non-human transgenic organism. Preferably, said polypeptide exhibiting santalene synthase activity is encoded by a heterologous polynucleotide, vector or genetic construct.

[0060] The term "heterologous polypeptide" in the present context means that the polynucleotide encoding a polypeptide exhibiting santalene synthase activity does not naturally occur in the host cell or organism into which it is introduced. Thus, a heterologous polynucleotide is a polynucleotide that originates from a first species or that has been artificially modified, while the host cell or non-human transgenic organism originates from a second species different from said first species. The heterologous polynucleotide may be comprised in a vector or a genetic construct as specified herein below. Alternatively, the heterologous polynucleotide may be introduced into the genome of the host cell or non-human transgenic organism such that upon integration into the genome, the polypeptide exhibiting santalene synthase activity encoded by said heterologous polynucleotide is expressed. Typically, the heterologous polynucleotide shall be integrated into the genome of the host cell or non-human transgenic organism at a locus that allows expression of the heterologous polynucleotide, for example in the vicinity of an endogenous promoter.

[0061] The term "vector" preferably includes phages, plasmids, cosmids, viral vectors and artificial chromosomes such as bacterial or yeast artificial chromosomes (YACs). The vectors including the polynucleotides of the present invention preferably further include a selection marker for propagation and / or selection in a host. The vectors can be integrated into the host cell by various techniques well known in the art. When introduced into the host cell, the vectors can be present in the cytoplasm or integrated into the genome. In the latter case, it is understood that the vectors can further include nucleic acid sequences that allow for homologous recombination or heterologous insertion. The vectors can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. When used in this context, the terms "transformation" and "transfection", conjugation and transduction are intended to include multiple prior art processes for introducing foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, f-factor conjugation, natural competence, carbon-based cluster, chemically mediated introduction, electroporation or particle bombardment. Suitable methods for transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (loc.cit.) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa, New Jersey. Alternatively, a plasmid vector can be introduced by heat shock or electroporation techniques. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line before application to the host cell.

[0062] Preferably, the vectors mentioned herein are suitable as cloning vectors, i.e. cloning vectors capable of replicating in microbial systems. Such vectors ensure efficient cloning in bacteria, preferably yeast or fungi, and allow stable transformation of plants. Vectors to be mentioned are in particular the various binary and co-integrating vector systems suitable for T DNA mediated transformation. Such vector systems are usually characterized by comprising at least the vir genes (required for Agrobacterium mediated transformation) and sequences that delimit the T-DNA (T-DNA borders). These vector systems also preferably comprise further cis-regulatory regions such as promoters and terminators and / or selection markers making it possible to identify suitable transformed host cells or organisms. Co-integrating vector systems are those in which the vir genes and the T DNA sequences are located on the same vector, while binary systems are based on at least two vectors, one of which carries the vir genes but lacks T-DNA and the second of which carries the T DNA but lacks the vir genes. As a result, the latter mentioned vectors are relatively small, easy to manipulate and replicable in both E. coli and Agrobacterium. These binary vectors include the pBIB-HYG, pPZP, pBecks, pGreen series of vectors. Preferably, those used according to the present invention are Bin19, pBI101, pBinAR, pGPTV and pCAMBIA. An overview of binary vectors and their uses can be found in Hellens et al, Trends in Plant Science (2000) 5, 446-451.Furthermore, by using an appropriate cloning vector, it is possible to introduce the polynucleotide into a host cell or an organism such as a plant or an animal, thereby enabling the transfer of the polynucleotide to a host cell or an organism such as a plant or an animal. Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), chapter 6 / 7, pp.71-119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in; Transgenic Plants, vol.1, Engineering and Utilization, Ed.; Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in; Transgenic Plants, vol.1, Engineering and Utilization, Ed.; Kung and R. Wu, Academic Press (1993), 128-143; Potrykus 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 205 225, can be used for plant transformation.

[0063] More preferably, the vector of the present invention is an expression vector. In such an expression vector, i.e., the vector containing the polynucleotide of the present invention, a nucleic acid sequence operably linked to an expression control sequence (also called "expression cassette") capable of being expressed in a prokaryotic or eukaryotic cell or an isolated fraction thereof. Suitable expression vectors are known in the art, such as Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen) or pSPORT1 (GIBCO BRL). Further exemplary fusion expression vectors are pGEX (Pharmacia Biotech Inc; Smith 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), in which glutathione S-transferase (GST), maltose E-binding protein and protein A are fused to the recombinant target protein, respectively. Examples of suitable inducible non-fusogenic E. coli expression vectors are, inter alia, pTrc (Amann 1988, Gene 69:301-315) and pET 11d (Studier 1990, Methods in Enzymology 185, 60-89). The expression of the target gene in the pTrc vector is based on transcription from a hybrid trp-lac fusion promoter by the host RNA polymerase. The expression of the target gene in the pET 11d vector is based on transcription from a T7-gn10-lac fusion promoter, which is mediated by a coexpressed viral RNA polymerase (T7 gn1). The viral polymerase is provided by the host strains BL21(DE3) or HMS174(DE3), which are derived from a resident lambda prophage carrying a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.The skilled artisan is familiar with other vectors suitable for prokaryotes, such as, for example, pLG338, pACYC184, pBR series, such as pBR322, pUC series, such as pUC18 or pUC19, M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, lambdagt11 or pBdCl for E. coli, plJ101, plJ364, plJ702 or plJ361 for Streptomyces, pUB110, pC194 or pBD214 for Bacillus, and pSA77 or pAJ667 for Corynebacterium. Examples of vectors for expression in the yeast S. cerevisiae include pYepSec1 (Baldari 1987, Embo J. 6:229-234), pMFa (Kurjan 1982, Cell 30:933-943), pJRY88 (Schultz 1987, Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and processes for constructing vectors suitable for use in other fungi, such as filamentous fungi, include those described in detail in van den Hondel, C.A.M.J., & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in; Applied Molecular Genetics of fungi, J.F. Peberdy et al., Ed., pp. 1-28, Cambridge University Press; Cambridge or More Gene Manipulations in Fungi (J.W.Bennett & L.L. Lasure, Ed., pp. 396-428; Academic Press; San Diego). Further suitable yeast vectors are, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.Alternatively, the polynucleotides of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expressing proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow 1989, Virology 170:31-39).

[0064] Furthermore, the vector can be an integrating vector. An integrating vector refers to a linear or circular DNA molecule that can be integrated into the genome of a microorganism, such as the genome of a bacteria, to provide stable inheritance of the gene encoding the polypeptide of interest, such as the santalene synthase of the present invention. An integrating vector generally comprises one or more segments that include the gene sequence encoding the polypeptide of interest under the control (i.e., operably linked) of an additional nucleic acid segment that provides its transcription.

[0065] Such further segments may include promoter and termination sequences as well as one or more segments that induce the integration of the gene of interest into the genome of the target cell, usually by a process of homologous recombination. Typically, an integrating vector is a vector that can be introduced into a target cell, but has a replicon that is not functional in that organism. Integration of a segment containing a 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 the host cell of the invention can be incorporated into the (expression) vector. For example, a DNA sequence of a signal peptide leader can be fused in frame to the nucleic acid of the invention, such that the santalene synthase of the invention is initially translated as a fusion protein containing the signal peptide. Depending on the nature of the signal peptide, the expressed polypeptide undergoes different targeting. A secretory signal peptide that is functional in the intended host cell promotes, for example, extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptide to specific organelles, such as chloroplasts, mitochondria and peroxisomes. The signal peptide can be cleaved from the polypeptide upon transport to the intended organelle or upon transport out of the cell. At the amino or carboxyl terminus of the polypeptide, fusion of additional peptide sequences can occur.

[0066] The term "genetic construct" as used herein refers to a polynucleotide comprising the polynucleotide of the present invention and additional functional nucleic acid sequences. The genetic construct according to the present invention is preferably a linear DNA molecule. Typically, the genetic construct according to the present invention can be a targeting construct that allows random or site-specific integration of the targeting construct into genomic DNA. Such a targeting construct preferably contains DNA of sufficient length to perform either homologous or heterologous recombination, as described in detail below. In either case, the construct should preferably be complete, with structures for controlling gene expression, such as a promoter, a transcription initiation site, a polyadenylation site, and a transcription termination site.

[0067] Preferably, the method of the present invention comprises the step of obtaining at least one santalene-containing composition as prepared above.

[0068] The term "obtaining" as used herein refers to providing a composition comprising at least one santalene of any purity. Thus, the composition may consist essentially of at least one santalene in essentially pure form, or it may be a mixture comprising additional components other than at least one santalene. Thus, the method of the present invention may include one or more purification steps. The purification technique that needs to be applied depends on how the method of the present invention is carried out. For example, it will be understood that less purification will be required if the method is carried out in vitro, i.e. in a reaction vial using isolated components, such as isolated enzymes, additives and auxiliary components, such as reaction buffers. However, if the method is carried out in vivo, i.e. in a host cell as defined elsewhere herein, further purification and pretreatment steps may be required. Typically, the host cells need to be harvested and the harvested cells are lysed to release the composition comprising at least one santalene from said cells. In the subsequent purification steps, cell debris must be removed as well, with the aim of purifying at least one santalene from the remaining components. Furthermore, if the process is carried out in vivo in an animal or plant, more further pretreatment and / or purification steps may be required. The skilled person is well aware of suitable pretreatment and / or purification steps depending on the given situation in which the method may be carried out. Contemplated purification techniques may be extraction techniques, chromatography such as LC, GC or HPLC, size exclusion chromatography, affinity chromatography, distillation, centrifugation, filtration, etc. Contemplated pretreatment steps may be recovery, heat treatment, sonication, treatment with chemicals and / or enzymes, etc. Particularly preferred treatments are described in the attached examples below.

[0069] Advantageously, the research underlying the present invention has revealed that a putative rice β-santalene synthase can be synthesized by expression in bacteria, resulting in the synthesis of an excess of a mixture of santalene, including α-santalene and β-santalene. Previously, it was reported that santalene synthase mainly produces α-santalene from farnesyl pyrophosphate, which contains only a small amount of β-santalene. Furthermore, the purification or enrichment of β-santalene from a composition essentially containing α-santalene is cumbersome. Thanks to the surprising properties of the present invention and the polypeptides exhibiting santalene synthase activity found according to the present invention, santalene, and preferably compositions containing β-santalene, can be produced more efficiently, especially in recombinant production approaches.

[0070] The definitions and explanations of terms made in the above specification apply mutatis mutandis to the following embodiments of the present invention, unless otherwise specified.

[0071] In a preferred embodiment of the method of the present invention, the step of converting farnesyl pyrophosphate to at least one santalene is carried out in a host cell.

[0072] The term "host cell" as used herein refers to a prokaryotic or eukaryotic cell capable of converting farnesyl pyrophosphate to at least one santalene, said conversion being carried out by a polypeptide exhibiting santalene synthase activity. Thus, the host cell of the present invention is capable of expressing a polypeptide exhibiting santalene synthase activity. Preferably, said polypeptide exhibiting santalene synthase activity can be encoded by a heterologous polynucleotide or vector or genetic construct of the present invention. The host cell is typically transformed with said heterologous polynucleotide, vector or genetic construct so that the polypeptide exhibiting santalene synthase activity as specified above can be expressed. The transformed vector or genetic construct can be maintained as a non-integrated vector, e.g. a plasmid, or alternatively can be integrated into the host cell genome as specified in more detail elsewhere herein.

[0073] In one aspect of the invention, a host cell of the invention is a transgenic cell, i.e. a cell that is transgenic for a nucleic acid encoding a santalene synthase of the invention, preferably a transgenic non-plant cell such as a transgenic microbial cell.

[0074] Host cells according to the invention 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.

[0075] Preferably, said host cell is selected from the group consisting of 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 and a plant cell. More preferably, the host cell may be selected from any one of the following organisms:

[0076] Bacteria: The bacterial host cell may be selected from the group consisting of, for example, Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, Lactococcus, Ensifer, or Pantoea.

[0077] Gram positive: Bacillus, Streptomyces: 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 The most preferred prokaryotic organisms include, but are not limited to, Bacillus licheniformis, Bacillus megaterium, 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.

[0078] Some other preferred bacteria include strains of the order Actinomycetales, preferably the genus Streptomyces, preferably Streptomyces spheroides (ATTC23965), Streptomyces thermoviolaceus (IFO12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include strains belonging to the genus Myxococcus, such as M. virescens.

[0079] Gram-negative: Escherichia, Pseudomonas, Rhodobacter, Paracoccus, Ensifer or Pantoea species; preferred Gram-negative bacteria are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC15958) or Pseudomonas fluorescens (NRRL B-11) or Pseudomonas denitrificans, Rhodobacter capsulatus or Rhodobacter sphaeroides. The most common fungus is Sinorhizobium meliloti, also known as Paracoccus sphaeroides, Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens, Pantoea ananatis, or Ensifer meliloti.

[0080] fungi: The host cell may be a fungal cell: Aspergillus, Fusarium, Trichoderma. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and Deuteromycotina, and all vegetative spore-forming fungi. Representative groups of the Ascomycota include, for example, the genera Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus) and the true yeasts listed below. Examples of the Basidiomycota include mushrooms, rusts and smuts. Representative groups of the Chytridiomycota include, for example, the genera Allomyces, Blastocladiella, Coelomomyces and aquatic fungi. Representative groups of Oomycota include Saprolegniomycetous aquatic fungi (water molds), such as Achlya. Examples of vegetative spore-forming fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of Zygomycota include Rhizopus and Mucor.

[0081] 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.

[0082] Yeast, Pichia, Saccharomyces: 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).

[0083] 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.

[0084] Most preferably, the host cell is a bacterial host cell, in particular a Rhodobacter host cell.

[0085] In another preferred embodiment of the method of the present invention, the step of converting farnesyl pyrophosphate to at least one santalene is carried out in a non-human transgenic organism as defined hereinbelow, or in a non-vertebrate transgenic organism, such as a plant or a microorganism.

[0086] The term "transgenic non-human organism" as used herein refers to an organism genetically modified to contain a polynucleotide, vector or gene construct of the present invention. Said genetic modification may be the result of any kind of homologous or heterologous recombination event, mutagenesis or gene editing process. Thus, a transgenic non-human organism shall differ from its non-transgenic counterpart in that it contains a non-naturally occurring (i.e. heterologous) polynucleotide, vector or gene construct in its genome. The non-human organism envisaged as a transgenic non-human organism according to the present invention is preferably a multicellular organism. Moreover, the non-human organism is preferably an animal or a plant. Preferred animals are mammals, particularly rodents, e.g. laboratory animals such as mice, rats, rabbits, etc. or livestock animals such as sheep, goats, cows, horses, etc. Preferred plants are in particular crops or vegetables selected from the group consisting of Arabidopsis spp., Nicotiana spp., Cichorum intybus, Lactuca sativa, Mentha spp., Artemisia annua, tuber-forming plants, oil crops such as Brassica spp. or Brassica napus, fruit-bearing flowering plants (angiosperms) and trees.

[0087] Methods for generating transgenic non-human organisms are well known in the art, see, e.g., Lee-Yoon Low et al., Transgenic Plants; Gene constructs, vector and transformation methods. 2018. DOI.10.5772 / intechopen.79369; Pinkert, CA (ed.) 1994. Transgenic animal technology; A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky GM and Robl, JM (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press. Washington DC); Sambrook, loc.cit, Ausubel, loc.cit).

[0088] Preferably, said non-human transgenic organism is a plant or a sacrificed non-human animal, therefore, according to the latter, methods of treating animals are not included in the methods of the invention.

[0089] The present invention also relates to a composition comprising a mixture of β-santalene and α-santalene, containing an excess of β-santalene, obtainable by the method of the present invention. Preferably, said β-santalene is present in the composition in a relative amount of at least about 50%, at least about 60%, at least about 70%, preferably about 50% to about 80%, about 60% to about 75%, about 65% to about 70%, more preferably at least about 67% of the total santalene. Preferably, the composition is substantially free of β-farnesene. The composition of the present invention is preferably a lipophilic composition.

[0090] In another embodiment of the present invention, the composition of the above invention further comprises a sesquithugene, preferably in an amount of at least 0.01%.

[0091] Surprisingly, it has been found that the presence of sesquithugene in the santalene composition of the present invention improves the olfactory properties of the santalene composition of the present invention.Compared to typical santalene compositions, the composition of the present invention has a stronger woody note.So far, sesquithugene has not been reported to affect the olfactory properties.

[0092] In yet another aspect of the present invention, the ratio of bergamotene, preferably trans-α-bergamotene, to β-santalene in the composition produced with the aid of the santalene synthase of the present invention is less than 1:2, 1:3, 1:4, 1:5, or 1:10.

[0093] In another aspect of the invention, the composition produced is substantially free of any one of cis-α-bergamotene ((AS No. 18252-46-5), (E)-β-farnesene (CAS No. 18794-84-8), trans-β-bergamotene (CAS No. 15438-94-5), or β-bisabolene (CAS No. 495-61-4), or substantially free of all of these, preferably substantially free of (E)-β-farnesene (CAS No. 18794-84-8).

[0094] In one embodiment, the composition of the present invention consists essentially of β-santalene, α-santalene, trans-α-bergamotene, sesquithujene, and epi-β-santalene, preferably β-santalene, α-santalene, trans-α-bergamotene, and sesquithujene.

[0095] The present invention relates to the use of the above-mentioned polypeptides, heterologous polynucleotides, vectors or genetic constructs, host cells, or non-human transgenic organisms for the manufacture of a composition comprising at least one santalene, preferably β-santalene, more preferably a mixture of β-santalene and α-santalene, even more preferably a mixture of β-santalene, α-santalene and sesquithujene. The β-santalene, α-santalene, sesquithujene, or bergamotene produced by the method of the invention, or the composition of the invention, may be used in flavor or fragrance applications, cosmetic applications, pharmaceuticals, as insect repellents or attractants, or in agriculture, such as for crop protection or animal husbandry.

[0096] Furthermore, the present invention relates to a method for preparing a composition comprising at least one santalol, preferably β-santalol, comprising: a) producing at least one composition comprising santalene by the method of the present invention described above; b) oxidizing at least one santalene, preferably β-santalene, to its respective alcohol to produce a composition comprising at least one santalol, preferably β-santalol; The present invention relates to a method comprising the steps of:

[0097] Preferably, the composition produced in step a) of the method of the present invention for producing a composition comprising at least one santalol, preferably β-santalol, is substantially free of (E)-β-farnesene or at least has a significantly reduced amount thereof.

[0098] Preferably, the method further comprises the step of obtaining said composition comprising at least one santalol, more preferably, said composition comprises a mixture of α-santalene and β-santalene with an excess of β-santalene.

[0099] The term "santalol" as used herein refers to an alcoholic sesquiterpene. The term includes alpha santalol, (Z)-5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol; CAS number: 115-71-9; molecular formula C 15 H 24 O) and β-santalol, (2Z)-2-methyl-5-[(1S,2R,4R)-2-methyl-3-methylidenebicyclo[2.2.1]heptan-2-yl]pent-2-en-1-ol; CAS number: 77-42-9; molecular formula C 15 H 24 O).

[0100] In the above-mentioned method of the present invention, a composition comprising at least one santalene is produced in a first step, step (a), as described by the method of the present invention elsewhere herein. In a subsequent step, step (b), said at least one santalene contained in the composition is oxidized to produce a composition comprising at least one santalol as mentioned above. In one aspect of the present invention, the oxidation of at least one santalene is preferably performed enzymatically. Suitable enzymes capable of oxidizing santalene to santalol are well known in the art. Preferably, a cytochrome P450 monooxygenase (CYP) is used to oxidize santalene to santalol. More preferably, the CYP is selected from the group consisting of: CYP76F37v1, CYP76F37v2, CYP76F38v1, CYP76F38v2, CYP76F39v1, CYP76F41, CYP76F42, CYP76F39v2, CYP76F40, and CYP736A167. Additionally, the oxidation can be carried out chemically.

[0101] Step (b) of the above method can also be carried out in vivo or ex vivo, typically depending on how step (a) is carried out. Thus, if step (a) is carried out in vivo, for example in a host cell or non-human transgenic organism as specified elsewhere herein, it is preferably envisaged that step (b) is also carried out in vivo, preferably in the same host cell or non-human transgenic organism. Typically, the host cell or non-human transgenic organism shall be capable of oxidizing santalene to santalol. Preferably, said host cell or non-human transgenic organism is therefore capable of expressing a CYP as specified above. For this purpose, a heterologous polynucleotide encoding said CYP, or a vector or gene construct comprising such a polynucleotide, may be present in the host cell or non-human transgenic organism. How such a heterologous polynucleotide, vector or gene construct can be introduced into said host cell or non-human transgenic organism is well known in the art and is described in detail elsewhere herein.

[0102] Alternatively, step b) of the method for producing a composition comprising at least one santalol is carried out by a chemical process rather than an enzymatic process. Preferably, the chemical oxidation to santalol is carried out as disclosed in WO 2021 / 063831.

[0103] The present invention also relates to compositions comprising β-santalol and α-santalol produced from a precursor composition comprising both β-santalene and α-santalene produced by the method of the present invention, wherein the β-santalol herein is present in a greater amount on a w / w basis than the α-santalol due to the excess β-santalene content in the precursor composition. The ratio of β-santalol to α-santalol in these compositions is greater than 1, preferably the ratio is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or at least 2. The ratio of β-santalol to α-santalol can be at least 3:1, preferably at least 4:1, more preferably at least 5:1, even more preferably 6:1, even more preferably at least 7:1, most preferably at least 8:1, or even at least 9:1. In one embodiment of the present invention, the ratio is 100:1 or less.

[0104] Thus, the present invention also contemplates a method for producing a composition comprising β-santalol in excess of α-santalol, without the need for a) reducing the α-santalene content before conversion to α-santalol, and / or b) increasing the β-santalol content after conversion from santalene by distillation or other means, the method comprising the steps of producing a composition comprising β-santalene in excess of α-santalene by the method of the present invention, and then, in one or more steps, oxidizing β-santalene to β-santalol and oxidizing α-santalene to α-santalol.

[0105] This conversion of santalene to the respective alcohol can be performed biosynthetically and / or chemically. Following the conversion to santalol, purification steps such as distillation to remove other compounds may be included, and the ratio of β-santalol to α-santalol may be altered by distillation if necessary, but a composition containing more β-santalol than α-santalol can be achieved without further altering the ratio of β-santalol to α-santalol after providing a composition containing β-santalene in excess of α-santalene by using an improved β-santalene synthase. Thus, one aspect of the present invention is a method for producing a composition containing β-santalol in excess of α-santalol, the method comprising the steps of producing a composition containing β-santalene in excess of α-santalene by the method of the present invention, followed in one or more steps by oxidizing β-santalene to β-santalol and α-santalene to α-santalol, wherein the oxidation of santalene is followed by distillation of santalol for purification of santalol without substantially increasing the β-santalol content over the α-santalol content.

[0106] The present invention also relates to a composition comprising β-santalol in excess of α-santalol produced by any of the methods of the present invention using the improved β-santalene synthase of the present invention or the host cell of the present invention, optionally with a total of less than 10% (w / w), or even less than 8% (w / w) of bergamotol in the composition. In another aspect, the composition of the present invention comprising β-santalol in excess of α-santalol produced by any of the methods of the present invention using the improved β-santalene synthase of the present invention or the host cell of the present invention comprises less than 3% epi-β-santalol.

[0107] The use of the present composition comprising santalene and sesquithujene in the production of santalol has an additional advantage: since the santalol composition comprising sesquithujene has improved olfactory properties, the olfactory properties of the resulting santalol composition are also improved.

[0108] One aspect of the present invention relates to polypeptides having santalene synthase activity of the present invention that produce β-santalol in excess of α-santalol, comprising β-santalene and α-santalene and / or β-santalol and α-santalol, with the ratio of β-santalene to α-santalene or the ratio of β-santalol to α-santalol being at least 1.3 or more, 1.5 or more, or 2 or more, respectively, nucleic acids encoding such polypeptides, expression cassettes comprising such nucleic acids, host cells comprising such expression cassettes, methods of the present invention, and compositions produced by the enzymes and methods of the present invention.

[0109] The present invention also provides a method for producing a sesquithugene or a composition comprising a sesquithugene, comprising: a) producing a composition comprising at least one santalene and a sesquithujene by the method of the present invention; and optionally b) isolating the sesquithugene from the composition. The present invention relates to a method comprising the steps of:

[0110] The present invention also relates to a kit for producing a composition comprising at least one santalene comprising the above-mentioned polypeptide, heterologous polynucleotide, vector or genetic construct, host cell, or non-human transgenic organism, wherein the composition is preferably substantially free of β-farnesene and / or comprises a sesquithugene.

[0111] The term "kit" as used herein refers to a collection of components required to carry out the method of the present invention for producing a composition comprising at least one santalene. The kit is intended to include any of the above-mentioned components, either as a single component or any combination thereof. Typically, the components of the kit are provided in separate containers or in a single container. The container typically also includes instructions for carrying out the method of the present invention for producing a composition comprising at least one santalene. Furthermore, the kit may preferably include further components required to carry out the method of the present invention, such as incubation reagents, culture media, washing solutions, solvents and / or reagents or means required for the purification of the composition comprising at least one santalene.

[0112] The present invention also contemplates a non-human host cell expressing a polypeptide exhibiting santalene synthase activity from the heterologous polynucleotide, vector or genetic construct described above. Preferably, said host cell is selected from the group consisting of 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 and a plant cell. More preferably, said non-human host cell produces at least one santalene, preferably β-santalene.

[0113] The present invention relates to a non-human transgenic organism expressing a polypeptide exhibiting santalene synthase activity from a heterologous polynucleotide, vector or genetic construct as mentioned above. Preferably, said non-human transgenic organism is a plant or a non-human animal. More preferably, said non-human host cell produces at least one santalene, preferably β-santalene.

[0114] The non-human host cells, non-human transgenic organisms, and methods of the invention may comprise a polypeptide exhibiting santalene synthase activity of the invention, and one or more further polypeptides exhibiting santalene synthase activity, including those known in the art under the following names: CiCaSSy (QNV69588) or Santalum album santalene synthase (E3W202), and / or those santalene synthases contained in the International Patent Application having Application No. PCT / EP2021 / 064642.

[0115] Finally, the present invention relates to at least one composition according to claim 10, or a composition according to claim 11, or any of the methods according to claims 1 to 8, and (ii) to (iv): (ii) at least one aroma chemical (X) other than a compound selected from the group consisting of β-santalene, α-santalene, trans-α-bergamotene, sesquithujene, and epi-β-santalene; or (iii) at least one non-aromatic chemical carrier; or (iv) A composition comprising both (ii) and (iii). The present invention relates to a fragrance composition comprising a composition produced by any one of the methods described above.

[0116] In a preferred embodiment, at least one composition according to claim 10 or a composition as defined in claim 11 or produced according to any of claims 1 to 8 is present in an amount ranging from 0.01% by weight to 70.0% by weight, based on the total weight of the fragrance composition.

[0117] In another preferred embodiment, the at least one non-fragrance chemical carrier (iii) is selected from a surfactant, an oil component, an antioxidant, a deodorant active, or a solvent.

[0118] In yet a further preferred embodiment, the composition is selected from a perfume composition, a body care composition, a hygiene product, a cleaning composition, a fabric detergent composition, a composition for a scent dispenser, a food product, a dietary supplement, a pharmaceutical composition, or a crop protection composition.

[0119] The following embodiments are particularly preferred embodiments contemplated in accordance with the present invention: All definitions and explanations of terms made above apply mutatis mutandis.

[0120] Embodiment 1. A method for producing a composition comprising at least one santalene, comprising converting farnesyl pyrophosphate to at least one santalene, said conversion being carried out by a polypeptide exhibiting santalene synthase activity, said polypeptide comprising: (ii) a) an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 18, or 21-28; b) an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NOs: 1, 18, or 21-28; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOs: 2 or 3 or 19; d) an amino acid sequence encoded by a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid sequence as set forth in any one of SEQ ID NOs: 2 or 3 or 19; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting santalene synthase activity. or (ii) a) an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 17; b) an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in SEQ ID NOs: 4 to 17; c) the amino acid sequence of a fragment of (a) or (d), which fragment encodes a polypeptide exhibiting santalene synthase activity. The method of claim 1, comprising administering to said patient an amino acid sequence selected from the group consisting of:

[0121] Embodiment 2: The method of embodiment 1, wherein the at least one santalene is β-santalene.

[0122] Embodiment 3: The method of embodiment 1 or 2, wherein the at least one santalene is a mixture of β-santalene and α-santalene with an excess of β-santalene, preferably wherein the composition also includes a sesquithujene.

[0123] Embodiment 4: The method of embodiment 3, wherein the β-santalene is present in the composition in a relative amount of at least about 50%, at least about 60%, at least about 70%, preferably about 50% to about 80%, about 60% to about 75%, about 65% to about 70%, more preferably at least about 67% relative to the total santalene.

[0124] Embodiment 5: The method of any one of embodiments 1 to 4, wherein the step of converting farnesyl pyrophosphate to at least one santalene is carried out in a host cell.

[0125] Embodiment 6: The host cell of embodiment 5, wherein said host cell is selected from the group consisting of 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 and a plant cell.

[0126] Embodiment 7: The method of any one of embodiments 1 to 4, wherein the step of converting farnesyl pyrophosphate to at least one santalene is carried out in a non-human transgenic organism.

[0127] Embodiment 8: The method of embodiment 7, wherein said non-human transgenic organism is a plant or a sacrificed non-human animal.

[0128] Embodiment 9: The method according to any one of embodiments 5 to 8, wherein said polypeptide exhibiting santalene synthase activity is encoded by a heterologous polynucleotide, vector or genetic construct.

[0129] Embodiment 10: The method of any one of embodiments 1 to 9, further comprising obtaining a composition comprising at least one santalene.

[0130] Embodiment 11: A composition comprising a mixture of β-santalene and α-santalene, containing an excess of β-santalene, preferably also comprising a sesquithujene obtainable by the method according to any one of embodiments 1 to 10.

[0131] Embodiment 12: The composition of embodiment 11, wherein the β-santalene is present in the composition in a relative amount of at least about 50%, at least about 60%, at least about 70%, preferably about 50% to about 80%, about 60% to about 75%, about 65% to about 70%, more preferably at least about 67% relative to the total santalene.

[0132] Embodiment 13: Use of a polypeptide according to any one of embodiments 1 to 4, a heterologous polynucleotide, vector or gene construct according to embodiment 9, a host cell according to embodiment 5 or 6 or a non-human transgenic organism according to embodiment 7 or 8 for the manufacture of a composition comprising at least one santalene, preferably β-santalene, more preferably a mixture of β-santalene and α-santalene, even more preferably a mixture of β-santalene, α-santalene and sesquithujene, most preferably as defined in embodiment 11 or 12.

[0133] Embodiment 14: The use according to embodiment 13, wherein said santalene is used to produce a flavor, fragrance, drug, or cosmetic.

[0134] Embodiment 15: A method for producing a composition comprising at least one santalol, preferably β-santalol, comprising: a) producing at least one composition comprising santalene by a method according to any one of embodiments 1 to 10; b) oxidizing said at least one santalene, preferably β-santalene, to the respective alcohol to produce a composition comprising at least one santalol, preferably β-santalol.

[0135] Embodiment 16: The method of embodiment 15, further comprising obtaining said composition comprising at least one santalol.

[0136] Embodiment 17: The method of embodiment 15 or 16, wherein the composition comprises a mixture of α-santalene and β-santalene, with an excess of β-santalene.

[0137] Embodiment 18: A kit for producing a composition comprising at least one santalene comprising a polypeptide according to embodiment 1, a heterologous polynucleotide, vector or genetic construct according to embodiment 9, a host cell according to embodiment 5 or 6, or a non-human transgenic organism according to embodiment 7 or 8, wherein the composition is substantially free of β-farnesene, preferably comprising a sesquithugene.

[0138] Embodiment 19: A non-human host cell expressing a polypeptide exhibiting santalene synthase activity from a heterologous polynucleotide, vector or genetic construct according to embodiment 9.

[0139] Embodiment 20: The non-human host cell according to embodiment 19, wherein said host cell is selected from the group consisting of: 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 and a plant cell.

[0140] Embodiment 21: A non-human host cell according to embodiment 19 or 20, wherein said non-human host cell produces at least one santalene, preferably β-santalene.

[0141] Embodiment 22: A non-human transgenic organism expressing a polypeptide exhibiting santalene synthase activity from a heterologous polynucleotide, vector or gene construct according to embodiment 9.

[0142] Embodiment 23: A non-human transgenic organism according to embodiment 22, wherein said non-human transgenic organism is a plant or a non-human animal.

[0143] Embodiment 24: A non-human transgenic organism according to embodiment 22 or 23, wherein said non-human host cell produces at least one santalene, preferably β-santalene.

[0144] All references cited throughout this specification are incorporated herein by reference in their entirety or with respect to the disclosure content specifically mentioned. [Brief description of the drawings]

[0145] [Figure 1] Schematic diagram of vector pm-SPppa-MBP-OmBSS-mpmii alt. [Figure 2-1] β-Santalene production in E. coli. GC-MS analysis of the dodecane phase of BL21-DE3-pMEV-pAC-OmBSS (A) and BL21-DE3-pMEV-pACYC-DUET-1 (B). [Figure 2-2] This is a continuation of Figure 2-1. [Diagram 3] Production of β-santalene in E. coli. GC analysis of the dodecane phase of Rs265-9c / pm-SPppa-OmBSS-mpmii alt. Percentage of total santalene: α-santalene: 25.7%; β-santalene: 67.3%; trans-α-bergamotene 7.0%. [Figure 4-1]Amino acid alignment of the Oryza santalene synthase with SEQ ID NO: 1 and proteins from other Oryza species. The alignment was performed using MUSCLE (Multiple Sequence Comparison by Log-Expectation) and standard parameters. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0146] The following sequences are referenced throughout the specification and in the accompanying sequence protocols. SEQ ID NO:1: Putative Oryza meridionalis β-santalene synthase (OmBSS) protein SEQ ID NO:2: E. coli synthetic coding sequence OmBSS SEQ ID NO: 3: Synthetic coding sequence OmBSS of Rhodobacter SEQ ID NO: 4: NM49164 Oryza sativa amino acid sequence SEQ ID NO:5: A0A0D9Z7P2_9ORYZ Uncharacterized Oryza glumipatula protein SEQ ID NO:6: A2XGY8_ORYSI Uncharacterized Oryza sativa subsp. indica protein SEQ ID NO:7: A0A0E0NVD9_ORYRU Uncharacterized Oryza rufipogon protein SEQ ID NO:8: A0A0N7KHA7|A0A0N7KHA7_ORYSJ Os03g0361700 Oryza sativa subsp. japonica protein (fragment) SEQ ID NO:9: Q10L24_ORYSJ Terpene synthase family, metal binding domain containing protein, expressed from Oryza sativa subsp. japonica SEQ ID NO: 10: I1PBH4_ORYGL Uncharacterized Oryza glaberrima protein SEQ ID NO:11: C7J0Q6_ORYSJ Os03g0361700 Oryza sativa subsp. japonica protein SEQ ID NO:12: A0A0E0GMM5_ORYNI Uncharacterized Oryza nivara protein SEQ ID NO:13: A0A0D3FIR1_9ORYZ Uncharacterized Oryza barthii protein SEQ ID NO:14: A3AI64_ORYSJ Uncharacterized Oryza sativa subsp. japonica protein SEQ ID NO: 15: A0A0E0NVE4_ORYRU Uncharacterized Oryza rufipogon protein SEQ ID NO:16: A0A0E0D0X2_9ORYZ Uncharacterized Oryza meridionalis protein SEQ ID NO:17: A0A0D3FIR4_9ORYZ Uncharacterized Oryza barthii protein SEQ ID NO: 18: Synthetic fusion protein of maltose binding protein MBP and Oryza meridionalis β-santalene synthase (OmBSS) SEQ ID NO: 19: Synthetic coding sequence encoding a synthetic fusion protein of maltose binding protein and Oryza meridionalis β-santalene synthase (OmBSS). SEQ ID NO: 20: GRXCX4W motif SEQ ID NO: 21: Synthetic santalene synthase SynBSS1 SEQ ID NO: 22: Synthetic santalene synthase SynBSS2 SEQ ID NO: 23: Synthetic santalene synthase SynBSS3 SEQ ID NO: 24: Synthetic santalene synthase SynBSS4 SEQ ID NO: 25: Synthetic santalene synthase SynBSS5 SEQ ID NO: 26: Synthetic santalene synthase SynBSS6 SEQ ID NO: 27: Synthetic santalene synthase SynBSS7 SEQ ID NO: 28: Synthetic santalene synthase SynBSS8 EXAMPLES

[0147] The examples are intended to be merely illustrative of the invention and should not be construed as limiting the scope in any way.

[0148] Example 1: Identifying rice santalene synthase The protein sequence represented by UniProt accession number A0A0E0D0X4 (SEQ ID NO: 1) was extracted from the Oryza meridionalis genome (https: / / www.genome.jp / dbget-bin / www_bget?uniprot:A0A0E0D0X4_9ORYZ). A0A0E0D0X4: [ka]

[0149] In a BLASTP analysis (nr database), the best blast hit (98.4% identical) has a sequence annotated as (+)-germacrene D synthase from Oryza sativa japonica. + / - 7 other sequences map within 90% identity and these sequences are found in other rice variants (e.g. Oryza sativa indica). A MUSCLE alignment of SEQ ID NO:1 and related sequences is shown in Figure 4. The best hit was an Oryza glumipatula protein that is 95% uncharacterized.

[0150] However, the sequence identity with known santalene synthase proteins is very limited: CiCaSSy (QNV69588): 30.9% Santalum album santalene synthase (E3W202): 27.4%.

[0151] To test the use of this protein, it was expressed in Escherichia coli and Rhodobacter sphaeroides.

[0152] Example 2: Cloning for expression in E. coli. The following sequence was synthesized using a standard sequence service provider and cloned into the expression vector pACYC-Duet-1 (Novagen) using the BamHI and NotI restriction sites. The plasmid was labeled pAC-OsBSS. Synthetic sequence encoding A0A0E0D0X4 for expression in E. coli (SEQ ID NO:2): [ka]

[0153] This plasmid was introduced into E. coli BL21 DE3 carrying pMEV as described in Schmidt et al. 2017 (Scientific Reports|7:862|DOI:10.1038 / s41598-017-00893-3). Transformants were selected on LB agar plates + chloramphenicol (30 μg / mL) + kanamycin (30 μg / mL) + 1% glucose. A positive transformant (tested by miniprep and restriction digest) was labeled E. coli BL21-DE3-pMEV-pAC-OmBSS. Using the same method, a control strain containing pMEV and pACYC-DUET-1 was created and labeled BL21-DE3-pMEV-pACYC-DUET-1.

[0154] Example 3: Production of β-santalene in E. coli Strains BL21-DE3-pMEV-pAC-OmBSS and BL21-DE3-pMEV-pACYC-DUET-1 were inoculated into 5 mL of LB liquid medium + chloramphenicol (30 μg / mL) + kanamycin (30 μg / mL) + 1% glucose and incubated overnight at 37° C. and 250 rpm. The next day, the culture was diluted 1:25 into 10 mL of 2xYT medium + chloramphenicol (30 μg / mL) + kanamycin (30 μg / mL) and grown at 37° C. and 250 rpm until the A600 was 0.5. Subsequently, 1 mM IPTG was added as an inducer, 1 mL of n-dodecane was added to capture the product, and the culture was incubated at 28° C. and 250 rpm for another 24 hours.

[0155] For GC-MS analysis, dodecane was separated from the cultures by centrifugation and diluted 200-fold with ethyl acetate. 2 μL was analyzed by GC / MS using a gas chromatograph as described in detail in Cankar et al. (2015).

[0156] Surprisingly, when compared with BL21-DE3-pMEV-pACYC-DUET-1, BL21-DE3-pMEV-pAC-OmBSS produced α-santalene, trans-α-bergamotene, and β-santalene, and the major santalene product of OmBSS in this system was found to be β-santalene (Figure 2).

[0157] Example 4: Constructs for expressing OmBSS in Rhodobacter sphaeroides. The following synthetic DNA was ordered from Genscript: Synthetic gene for expression of Rhodobacter OmBSS (SEQ ID NO:3): [ka]

[0158] This fragment was cloned into plasmid pm-SPppa-MBP-CiCaSSy-mpmii-alt from US20200010822A1 using HindIII and BamHI restriction sites. The ligation mixture was transformed into E. coli S17-1 cells. Transfer of pm-SPppa-MBP-OmBSS-mpmii alt from S17-1 to R. sphaeroides Rs265-9c by conjugation (Figure 1) was performed using standard procedures (US9,260,709B2). The expressed fusion protein is shown in SEQ ID NO: 18.

[0159] Example 5: Production of β-santalene in Rhodobacter sphaeroides Seed culture of Rs265-9c / pm-SPppa-MBP-OmBSS-mpmii alt was performed in a 100 mL shake flask without baffles using 20 mL of RS102 medium containing 100 mg / L neomycin and a loop of glycerol stock. The seed culture flask was incubated at 30°C for 72 hours in a shaking incubator with 50 mm orbit at 110 rpm.

[0160] Shake flask production experiments were performed in 300 mL shake flasks with two bottom baffles. 20 mL of RS102 medium (described in US Patent Publication No. 20200010822A1) and neomycin to a final concentration of 100 mg / L were added to the flask along with 2 mL of sterile n-dodecane. The amount of inoculum was adjusted to give a final OD600 value of 0.05 in 20 mL of medium. The flasks were kept at 30°C in a shaking incubator with 50 mm orbit at 110 rpm for 72 hours. Shake flask experiments were performed in duplicate.

[0161] For GC-MS analysis, dodecane was separated from the culture by centrifugation and diluted 10-fold with acetone.

[0162] Gas chromatography was performed on a Shimadzu GC2010 Plus equipped with a Restek RTX-5Sil MS capillary column (30 m×0.25 mm, 0.5 μm). The injector and FID detector temperatures were set at 280° C. and 300° C., respectively. The gas flow rate through the column was set at 40 mL / min. The initial oven temperature was 160° C., which was increased to 180° C. at a rate of 2° C. / min and then to 300° C. at a rate of 50° C. / min and held at that temperature for 3 min. The injected sample volume was 1 μL, with a split ratio of 1:50 and a nitrogen make-up flow rate of 30 mL / min.

[0163] Compounds found in the dodecane layer of Rs265-9c / pm-SPppa-MBP-OmBSS-mpmii alt culture were identified according to their retention times and quantified by integrating the peak areas of the chromatograms (Figure 3). Analysis of the peak areas revealed that 26% of the total santalene was α-santalene, 7% was trans-α-bergamotene, and 67% was β-santalene.

[0164] A second experiment was conducted to analyze in more detail the terpene materials produced, particularly those terpenes known from other santalene synthases. The results of the GC analysis of the compounds found in the dodecane layer are shown in Table I. Surprisingly, some of the terpenes known to be produced by known santalene synthases were not produced, and the santalene synthase of the present invention was found to have a novel and very different product profile, including the production of sesquithujenes.

[0165] [Table 1]

[0166] Substances in italics were predicted to be present but were not produced by OmBSS, and other known santalene synthases were confirmed to produce detectable amounts of these in the same setup.

[0167] RT = retention time, Found RI = experimental RI, Lit. RI = RI from published sources, ND not detected.

[0168] Example 6: Olfactory comparison of the novel santalene compositions of the present invention with known santalene compositions A santalene composition comprising santalene and sesquithujene was produced as described in the previous examples. For comparison, a state-of-the-art santalene composition was produced as described in WO 2018 / 160066.

[0169] When the Santarene compositions of the present invention were smelled in comparison to known Santarene compositions, the compositions of the present invention had improved olfactory properties with increased woody notes.

[0170] Compositions containing santalene and sesquithujene can be used in fragrance compositions in the same manner that known compositions containing santalene are used, and compositions of the present invention containing santalol can be used in fragrance compositions in the same manner that known santalol compositions are used.

[0171] Suitable fragrance compositions are, for example, but not limited to, 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.

[0172] The perfume compositions may be selected from fine fragrances, room fresheners in liquid form, in gel form or applied to a solid carrier, aerosol sprays, scented cleaners, perfumed candles and oils such as lamp oils or massage oils.

[0173] Examples of fine fragrances are perfume extracts, Eau de Parfum, Eau de Toilette, Eau de Cologne, Eau de Solide and Extrait Parfum.

[0174] The compositions of the present invention may also be used for flavoring purposes.

[0175] Advantageous Composition A composition of the present invention containing a mixture of santalene and sesquithujene was formulated into a perfume composition according to Tables II and III and designated Compound A.

[0176] The mixtures of santalene and sesquithujene of the present invention were formulated into compositions according to Tables II and III. The mixtures shown in Table I are designated "Compound A" in Tables II and III.

[0177] [Table 2]

[0178] [Table 3]

[0179] The compositions according to Table II and Table III, namely 1A, 1B, 2A, 2B, are Deo Pump Spray, Clean Hair Conditioner, Face Wash Gel, Foam Bath Concentrate, Hair Gel, Self-Forming Body Wash, Spray-type Sun Care Emulsion, Spray-type Sun Protection Emulsion, Emollient Facial Gel, Two-phase Oil Foam Bath, Shampoo, Shower Bath, Hydroalcoholic AP / Deo Pump Spray, Aerosol, Hydroalcoholic AP / Deo Roll-on, Styling Gel Type "Out of Bed" The composition may be included in various compositions selected from the group consisting of "Baby Shampoo for Sensitive Skin", "Baby Shampoo for Sensitive Skin", "Skin Care Shampoo for Sensitive Skin", "Shampoo for Sensitive Skin", "Bed", "Shaving Foam", "Baby Shampoo for Sensitive Skin", "Body Wash for Sensitive Skin", "Gloss Enhancing Shampoo for Sensitive Scalp", "Deodorant Stick", "Baby Wipes", "After Shave Balm", "Face Gel", "Face Day Care Cream", "Face Cleanser", "Body Lotion", "Sun Care SPF50+", "Spray Lotion", "Hand Dish Cleaner (Regular), Hand Dish Cleaner (Concentrate), Sanitary Cleaner (Concentrate), All-Purpose Cleaner, Antibacterial Fabric Softener, Detergent Composition, Powder Detergent Composition and Liquid Detergent Composition.

[0180] Those skilled in the art will be familiar with the various general formulations of the above mentioned products.

[0181] Compositions 1A, 1B, 2A and 2B can be combined in specific formulations, for example, as disclosed in IP.com Number: IPCOM000258614D, entitled New Aroma Chemicals, pages 6-46, Tables 1-D13, where "Fragrance Composition 1A" is replaced with an equal amount of Composition 1A, 1B, 2A, or 2B.

Claims

1. 1. A method for producing a composition comprising at least one santalene, comprising converting farnesyl pyrophosphate to at least one santalene, said conversion being carried out by at least one polypeptide exhibiting santalene synthase activity, said at least one polypeptide comprising: a) an amino acid sequence as set forth in any of SEQ ID NOs: 1, 18, or 21-28; b) an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NOs: 1, 18, or 21-28; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOs: 2 or 3 or 19; d) an amino acid sequence encoded by a nucleic acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a polypeptide encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOs: 2 or 3 or 19; and e) The amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting santalene synthase activity. The method of claim 1, wherein the amino acid sequence is selected from the group consisting of:

2. 10. The method of claim 1, wherein the at least one santalene is β-santalene.

3. 2. The method of claim 1, wherein the at least one santalene is a mixture of β-santalene and α-santalene with an excess of β-santalene, and preferably also includes a sesquithujene.

4. 4. The method of claim 3, wherein the β-santalene is present in the composition in a relative amount of at least about 50%, at least about 60%, at least about 70%, preferably from about 50% to about 80%, from about 60% to about 75%, from about 65% to about 70%, more preferably at least about 67%, based on total santalene.

5. 2. The method of claim 1, wherein the step of converting farnesyl pyrophosphate to at least one santalene is carried out in a host cell.

6. 2. The method of claim 1, wherein the step of converting farnesyl pyrophosphate to at least one santalene is carried out in a non-vertebrate transgenic organism, preferably a plant or a microorganism.

7. 10. The method of claim 1, wherein the composition further comprises a detectable amount of sesquithujene and is substantially free of any or all of cis-α-bergamotene ((AS No. 18252-46-5), (E)-β-farnesene (CAS No. 18794-84-8), trans-β-bergamotene (CAS No. 15438-94-5), or β-bisabolene (CAS No. 495-61-4), preferably substantially free of (E)-β-farnesene (CAS No. 18794-84-8).

8. The method of claim 1, wherein the polypeptide exhibiting santalene synthase activity is encoded by a heterologous polynucleotide, vector, or gene construct.

9. 1. A composition comprising a mixture of β-santalene and α-santalene containing an excess of β-santalene obtainable by the method of claim 1, further comprising a detectable amount of sesquithujene, and being substantially free of any or all of cis-α-bergamotene (AS No. 18252-46-5), (E)-β-farnesene (CAS No. 18794-84-8), trans-β-bergamotene (CAS No. 15438-94-5), or β-bisabolene (CAS No. 495-61-4), preferably substantially free of (E)-β-farnesene (CAS No. 18794-84-8).

10. 10. A composition comprising a mixture of β-santalene and α-santalene containing an excess of β-santalene obtainable by the method of claim 1, wherein the β-santalene is present in the composition in a relative amount of at least about 50%, at least about 60%, at least about 70%, preferably about 50% to about 80%, about 60% to about 75%, about 65% to about 70%, more preferably at least about 67%, based on the total santalene, and the composition is substantially free of β-farnesene and / or comprises sesquithujene.

11. Use of a polypeptide according to claim 1, a heterologous polynucleotide, vector or gene construct according to claim 8, a host cell according to claim 5 or a non-vertebrate transgenic organism according to claim 6 for the manufacture of a composition comprising at least one santalene, preferably β-santalene, more preferably a mixture of β-santalene and α-santalene, and most preferably also a sesquithugene.

12. 1. A method for producing a composition comprising at least one santalol, preferably β-santalol, comprising: a) producing a composition comprising at least one santalene by the method of claim 1 or preparing a composition according to claim 10; b) oxidizing at least one santalene, preferably β-santalene, to the respective alcohol to produce a composition comprising at least one santalol, preferably β-santalol.

13. A kit for producing a composition comprising at least one santalene, comprising a polypeptide according to claim 1, a heterologous polynucleotide, vector, or gene construct according to claim 8, a host cell according to claim 5, or a non-vertebrate transgenic organism according to claim 6.

14. A non-human host cell that expresses a polypeptide exhibiting santalene synthase activity from a heterologous polynucleotide, vector, or gene construct described in claim 8, wherein the non-human host cell is transgenic for a nucleic acid encoding the santalene synthase described in claim 1.

15. A non-human or non-vertebrate transgenic organism that expresses a polypeptide exhibiting santalene synthase activity from a heterologous polynucleotide, vector, or gene construct described in claim 8, and that is transgenic for a nucleic acid encoding the santalene synthase described in claim 1.

16. 1. A fragrance composition comprising: i) at least one composition according to claim 10 or the method according to claim 1, and (ii) to (iv): ii) at least one aroma chemical (X) other than a compound selected from the group consisting of β-santalene, α-santalene, trans-α-bergamotene, sesquithujene, and epi-β-santalene; or iii) at least one non-fragrance chemical carrier; or iv) a composition comprising both (ii) and (iii). A fragrance composition comprising a composition produced by any of the methods described above.

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