Recombinant production of C20 terpenoid alcohols

Abstract

A method for producing at least one C20 terpenoid alcohol is disclosed, comprising converting geranylgeranyl pyrophosphate to copalyl diphosphate (CPP) or labd-13-en-8-ol diphosphate (LPP) and converting the CPP or LPP to at least one C20 terpenoid alcohol, said conversion being carried out by a polypeptide exhibiting diterpene alcohol synthase activity capable of converting the CPP to manool, the LPP to sclareol, and / or the LPP to abienol, said polypeptide comprising an amino acid sequence as defined in the claims. The present invention further relates to the above-mentioned polypeptides exhibiting diterpene alcohol synthase activity and fusion proteins comprising said polypeptides, polynucleotides encoding same, vectors or gene constructs comprising said polynucleotides, host cells comprising said vectors or gene constructs, non-human transgenic organisms comprising the polynucleotides, vectors, gene constructs or host cells, and their use for producing at least one C20 terpenoid alcohol.

Description

[Technical field]

[0001] The present invention relates to the field of recombinant production of C20 terpenoid alcohols. In particular, the present invention relates to a method for producing at least one C20 terpenoid alcohol, comprising the steps of converting geranylgeranyl pyrophosphate to copalyl diphosphate (CPP) or labd-13-en-8-ol diphosphate (LPP) and converting the CPP or LPP to at least one C20 terpenoid alcohol, said conversion being performed by a polypeptide exhibiting diterpene alcohol synthase activity, said diterpene alcohol synthase activity being capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol, said polypeptide having a) an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7 or 34; b) an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7 or 34 that is at least 60%, at least 70%, at least 80%, at least 85%, a) an amino acid sequence that is 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:1, or 2, or 35; b) an amino acid sequence that is 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:1, or 2, or 35; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO:1, or 2, or 35; 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:1, or 2, or 35; and e) an amino acid sequence selected from the group consisting of a fragment of any one of (a) to (d), wherein the fragment encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol.The present invention further relates to the above-mentioned polypeptides exhibiting diterpene alcohol synthase activity and fusion proteins comprising said polypeptides, polynucleotides encoding same, vectors or gene constructs comprising said polynucleotides, host cells comprising said vectors or gene constructs, non-human transgenic organisms comprising said polynucleotides, vectors, gene constructs or host cells.Furthermore, the present invention contemplates the use of said polypeptides, fusion polypeptides, polynucleotides, vectors or gene constructs, host cells or non-human transgenic organisms for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.Furthermore, the present invention encompasses a kit for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol. [Background technology]

[0002] Sclareol ((+)-sclareol), abienol (Z-abienol) and manool ((+)-manool) are members of the labdane diterpenes. Diterpenes are C20 terpenes that occur naturally in plants and microorganisms. These labdane diterpene molecules have commercial value because they can be converted into amber notes that are applied in the fragrance industry. Examples of amber notes include amber ketal, manool ketone, ambroxide and sclareolide. Several chemical or biocatalytic pathways have been disclosed for converting diterpene molecules into amber notes. Sclareol can be converted into ambroxide, e.g., Barrero et al. 1993, tetrahedron 49, 10405-10412; Farbood, European Patent Application No. 0 204 009 B1) or sclareolide (Farbood, European Patent Application No. 0 419 026 A1). Manool can be converted to amber ketals, e.g., U.S. Pat. No. 7,294,492 (Cryptococcus) or manool ketones (EP 1 688 501 B1), and abienol can be converted to ambroxides (e.g., Barrero et al. 1993, tetrahedron 49, 10405-10412) or sclareolides (U.S. Pat. No. 5,525,728).

[0003] Plant sources of these compounds include Salvia sclarea and Nicotiana glutinosa for sclareol, Halocarpus biformis (pink or yellow pine) for manool, and balsam fir (Abies balsamea) for abienol.

[0004] Genes encoding terpene cyclases that produce diterpenes have been extensively described (Zerbe, Trends Biotechnol 2015 Jul;33(7):419-28.), and microbial production of these compounds has been demonstrated (e.g., Schalk J. Am. Chem. Soc. 2012,134,18900-18903). Diterpene biosynthesis begins with geranylgeranyl pyrophosphate (GGPP). GGPP is widely present in nature as a precursor of carotenoids, plant hormones, etc. GGPP synthases are widely known, for example from Synechococcus sp. PCC 7002, Saccharomyces cerevisiae, Mentha piperita, and Arabidopsis thaliana, such as crtE (Feng Front. Plant Sci., 25 May 2020 and references therein), but also from Corynebacterium glutamicum, such as the idsA gene (Heider FEBS Journal 281 (2014) 4906-4920).

[0005] Starting from GGPP, the biosynthesis of diterpenes is usually mediated by two steps: step 1 to cyclize diphosphates (e.g., labd-13-en-8-ol diphosphate, or LPP, copalyl-PP, or CPP) and step 2 to convert this substrate to the final product. Usually, step 1 is performed by type II diterpene synthases, whereas step 2 is performed by type I diterpene synthases. There are type II synthases known to perform both steps, e.g., abienol synthase from Abies balsamea (Zerbe JOURNAL OF BIOLOGICAL CHEMISTRY VOL.287,NO.15,pp.12121-12131,April 6,2012). Usually, the enzymes of step I are αβγ domain proteins, characterized by the presence of a DXDD motif in the γ domain. The enzyme of step 2 can be an αβγ domain protein or an αβ domain protein, characterized by the presence of a DDXXD motif in the β domain. A review of diterpene synthases can be found in Zerbe et al., Trends in Biotechnology, 2015, 33(7), 419-428.

[0006] For the biosynthesis of related diterpenes the following genes have been described: for sclareol, LPP synthase (LPPS) and sclareol synthase (SS) from Salvia sclarea (Caniard et al. BMC Plant Biology 2012,12:119; Schalk WO 2009 / 101126), where LPPS is an αβγ protein (type II) and SS is an αβ protein (type I); Ignea et al (Metabolic Engineering 27(2015),65-75) have demonstrated the synthesis of sclareol in yeast exclusively with LPPS from Nicotiana glutinosa and similar enzymes (Julien, WO 2014 / 022434 A1).

[0007] For abienols, LPPS and ABS from Nicotiana tabacum (Salaud, The Plant Journal (2012) 72, 1-17; WO 2008 / 07031 A1), ABS from Abies balsamea (Zerbe JOURNAL OF BIOLOGICAL CHEMISTRY VOL.287, NO.15, pp.12121-12131, April 6, 2012) and ABS from Abies and ABS from Nicotiana or SS from Salvia (WO 2016 / 94178 A1) can be carried out in either step.

[0008] For Manool, CPPS in step 1 derived from Triticum aestivum, or Salvia Miltiorrhiza, or Talaromyces verruculosus, or Coleus Forskohlii, Marrubium vulgare, or Rosmarinus officinale; SS in step 2 derived from Salvia (US Patent No. 2019 / 0352673); CPPS in step 1 derived from Coleus forskohlii; OmTPS4 in step 2 derived from Origanum majorana (Johnson J. Biol. Chem. (2019) 294(4) 1349-1362; International Publication No. 2020 / 028795).

[0009] A genetically engineered microorganism for producing sclareol, manool or abienol is described, which includes the introduction of the following genetic elements:

[0010] The GGPP synthase was selected from the group of GGPP synthases described, for example, in Feng Front.Plant Sci.,25 May 2020. For the purpose of producing GGPP, microbial enzymes of the CrtE type have also been employed, for example crtE from Pantoea agglomerans (AAA24819) (Schalk J.Am.Chem.Soc.2012,134,18900-18903). IdsA from Corynebacterium has been shown to have extremely high catalytic efficiency (Heider FEBS Journal 281(2014)4906-4920).

[0011] The genes in step 1 leading to LPP or (+)-CPP were selected from different sources in the prior art: LPPS from Salvia sclarea (Caniard et al. BMC Plant Biology 2012,12:119; Schalk, WO 2009 / 101126), LPPS from Nicotiana glutinosa (Allylix, WO 2014 / 022434), CfLPPS from Coleus forskohlii (Pateraki Plant Physiol.,164,1222-1236; WO 2015 / 091943), NtLPPS from Nicotiana tabacum (Salaud, The Plant Journal (2012) 72, 1-17; International Publication No. WO 2008 / 07031A1), GhLPPS from Grindelia hirsutula, TwLPPS from Tripterygium wilfordii, and CcLPPS from Cistus creticus (Falara, Plant Physiology, 2010, Vol. 154, pp. 301-310). CPPS derived from Triticum aestivum, Salvia Miltiorrhiza, Talaromyces verruculosus, Coleus Forskohlii, Marrubium vulgare, Rosmarinus officinale (US Patent No. 2019 / 0352673) are also used.

[0012] Ma and coworkers described the biochemical characterization of diterpene synthases from Taiwania cryptomerioides (Ma Li-Ting et al., The Plant Journal, vol. 100, no. 6, 1254-1272). Specifically, they characterized five monofunctional diTPS functions not previously observed in gymnosperms, including a monofunctional class II enzyme that forms labd-13-en-8-ol diphosphate (LPP, TcCPS2) and (+)-copalyl diphosphate (CPP, TcCPS4), and three class I diTPSs that generate biholmene (TcKSL1), levopimaradiene (TcKSL3), and phyllocladanol (TcKSL5), respectively. Furthermore, none of these diterpene synthases showed diterpene alcohol synthase activity, let alone the production of sclareol, manool, or abienol.

[0013] In fact, genes for step 2 leading to sclareol, manool or abienol are rare. Sclareol synthase from Salvia sclarea is known to produce manool when combined with CPPS (US Patent Application Publication No. 2019 / 0352673). OmTPS4 from Origanum majorana is a manool synthase with CPPS, but when combined with LPPS, it does not produce sclareol but produces manoyl oxide (Johnson 2019). Jia (ACS Catal. 2018, 8, 3133-3137) discloses that Salvia sclareol synthase can be converted to isoabienol synthase by mutating residue N431 to I, D, or E, and that 13R-sclareol synthase can be changed to 13S-sclareol synthase by mutating N431Q. Jia et al. claim that sclareol synthase exceptionally has an asparagine (N431) in the product outcome determining region around the residue that is important for adding water to labdanoyl-PP to form sclareol. N. tabacum abienol synthase produces Z-biformeol by CPPS from Salvia fruticosa.Jia et al. identified sclareol synthase from Salvia sclarea and numerous step 2 diterpene synthases from different species, such as manoyl oxide synthase from Coleus forskohlii (GenBank accession: KF444508); 1 IrMS, mirtiradiene synthase from Isodon rubescens (KX831652); CfMS, mirtiradiene synthase from C. forskohlii (KF444509); RoMS1, mirtiradiene synthase 1 from Rosemarius officinalis (KF805858); SmMS, Salvia miltiorhiza Miltiorrhiza (ABV08817); RoMS1, mirtiradiene synthase from Rosemarius officinalis (KF805859); SfMS, mirtiradiene synthase from Salvia fruticosa (KP091841); MvELS, 9,13-epoxy-labd-14-ene synthase from Marrubium vulgare (KJ584454), where it has been reported that residue N438 of SsSS determines the ability to generate 13-hydroxylated labdane diterpenes such as sclareol or manool. Summary of the Invention [Problem to be solved by the invention]

[0014] Although various step 2 enzyme-encoding genes have been reported in the prior art, nevertheless, there exists a need for highly efficient enzymes that can be applied to catalyze the step 2 reaction in the production of C20 terpenoid alcohols, particularly abienol, sclareol and / or manool. Furthermore, it is desirable to have an enzyme that is not limited to the production of only one C20 terpenoid alcohol. [Means for solving the problem]

[0015] 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.

[0016] Accordingly, the present invention provides a method for producing at least one C20 terpenoid alcohol, comprising: a) converting geranylgeranyl pyrophosphate to copalyl diphosphate (CPP) or labd-13-en-8-ol diphosphate (LPP); and b) converting the CPP or LPP to at least one C20 terpenoid alcohol, said conversion being carried out by a polypeptide exhibiting diterpene alcohol synthase activity, said diterpene alcohol synthase activity being capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol. wherein the polypeptide comprises a) an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; 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 one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 2, 16, 17, 18 or SEQ ID NO: 35; 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:1, 2, 16, 17, 18 or SEQ ID NO:35; and e) an amino acid sequence of a fragment of any one of (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol. 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:

[0017] 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 item is available.

[0018] As used below, the terms "having", "including" or "comprising" are intended to have 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.

[0019] 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.

[0020] 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.

[0021] The process according to the invention may consist of steps (a) and (b) mentioned above or may comprise additional steps, which may be pre-treatment steps or steps required for the production of C20 terpenoid alcohols, such as purification steps.

[0022] The term "production" as used herein refers to the production of at least one C20 terpenoid alcohol, particularly cyclic C20 terpenoid alcohol, more preferably manool, sclareol and / or abienol, from CPP or LPP (CAS number 1000876-36-7). This production can provide said at least one C20 terpenoid alcohol with any degree of purity. 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 microorganism or a plant, including a host cell as referred to elsewhere herein.

[0023] The term "C20 terpenoid alcohol" as used according to the present invention relates to a C20 terpenoid comprising an alcohol moiety. Terpenes are isoprenes of polymeric nature. Terpenoids may further comprise functional chemical moieties. C20 terpenoids are also referred to as diterpenoids or diterpenes. Preferably, said at least one C20 terpenoid alcohol referred to according to the present invention is a cyclic C20 terpenoid alcohol. More preferably, manool (CAS number 596-85-0, molecular formula C 20 H 34 O), sclareol (CAS number 515-03-7, molecular formula C 20 H 36 O2) or abienol (CAS number 17990-16-8, molecular formula C 20 H 34 O).

[0024] 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.

[0025] The term "diterpene alcohol synthase activity" as used herein refers to the activity of an enzyme capable of converting a starting material such as LPP or CPP into a C20 terpenoid alcohol. Diterpene synthases undergo complex electrophilic ring formations and / or rearrangements resulting in diverse backbone structures. Diterpene synthases can be classified as class I enzymes that use terpene diphosphate as a substrate, which is generated from geranylgeranyl phosphate from class II enzymes. The polypeptides having diterpene alcohol synthase activity referred to above are typically type I enzymes. Preferably, the diterpene alcohol synthase activity can convert copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) (CAS number 1000876-36-7) to sclareol, and / or LPP (CAS number 1000876-36-7) to abienol. Preferably, the polypeptide having diterpene alcohol synthase activity according to the present invention comprises a conserved region as shown in SEQ ID NO:24 or a sequence having one or several amino acid mutations in SEQ ID NO:24, wherein the serine at position 4 of SEQ ID NO:24 is converted or substituted with a threonine, preferably the serine at this position is converted.

[0026] In addition, the polypeptides having diterpene alcohol synthase activity according to the present invention are Pfam domains PF01397.23 (terpene synthase, N-terminal domain), PF03936.18 (terpene synthase family, metal binding domain) and PF19086.2 (terpene synthase family 2, C-terminal metal binding) (PFAM version 35.0); Pfam: The protein families database in 2021; J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, GA Salazar, ELL Sonnhammer, SCE Tosato, L. Paladin, S. Raj, LJ Richardson, RD Finn, A. Bateman Nucleic Acids Research (2020) doi; 10.1093 / nar / gkaa913.

[0027] A polypeptide exhibiting diterpene alcohol synthase activity according to the present invention, wherein the diterpene alcohol synthase activity is capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol, the polypeptide comprising: a) an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; 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 one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 2, 16, 17, 18 or 35; 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: 1, 2, 16, 17, 18 or 35; and e) an amino acid sequence of a fragment of any one of (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol. The amino acid sequence is selected from the group consisting of:

[0028] Preferably, the polypeptide exhibiting diterpene alcohol synthase activity is capable of converting CPP to manool and LPP to sclareol. More preferably, the polypeptide a) an amino acid sequence as set forth in SEQ ID NO: 4, 6, 7, 9, 10 or SEQ ID NO: 34; 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 NO: 4, 6, 7, 9, 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 2, 16, 17 or 35; 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, 16, 17 or 35; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting CPP to manool and LPP to sclareol. The amino acid sequence is selected from the group consisting of:

[0029] Preferably, the polypeptide exhibiting diterpene alcohol synthase activity is also capable of converting LPP to abienol. a) an amino acid sequence as set forth in SEQ ID NO: 3, 5 or 8; 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 NO: 3, 5 or 8; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1 or 18; 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: 1 or 18; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting LPP to abienol. The amino acid sequence is selected from the group consisting of:

[0030] The sequence identity 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 only with parts of 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 one of SEQ ID NOs: 3-7 or SEQ ID NO: 34 or a nucleic acid sequence as shown in SEQ ID NO: 1 or 2 or 35. 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.

[0031] Sequence alignments can be generated using many 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 for example incorporated in the "NEEDLE" program which performs a global alignment of two sequences. The NEEDLE program is for example included 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) - typically 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 many BLOSUMs is "BLOSUM62", which is often the "default" setting for many programs when aligning protein sequences. BLAST (Basic Local Alignment Search Tool) - consists of several individual programs (BlastP, BlastN) that are primarily used to search for similar sequences in large sequence databases. 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, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (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.

[0032] Sequence identity as used herein is preferably the value 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.

[0033] 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., improving expression of an enzyme or increasing the enzymatic activity of an enzyme), for example, by biological techniques known to those of skill 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-specific mutagenesis, directed evolution, genetic recombination, etc.).

[0034] Variant nucleic acid sequences encoded by nucleic acid sequences as set forth in SEQ ID NO: 1, 2, 16, 17, 18 or 35, or amino acid sequences that are 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 nucleic acid sequences as set forth in SEQ ID NO: 1, 2, 16, 17, 18 or 35, may differ from the nucleic acid sequence set forth in SEQ ID NO: 1, 2, 16, 17, 18 or 35 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 are preferably capable of hybridizing to each other 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 washing steps in 0.2x SSC, 0.1% SDS at 50-65°C. The skilled artisan knows that these hybridization conditions, with respect to the temperature and concentration of the buffer, for example when organic solvents are present, 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 0.1-5x SSC (pH 7.2). When 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, in 0.1x SSC. The hybridization conditions for DNA:RNA hybrids are preferably 0.1×SSC at 30° C. to 55° C., preferably 45° C. to 55° C. The above mentioned hybridization temperatures are determined, for example, for a nucleic acid 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 such as that shown in SEQ ID NO:1, 2, 16, 17, 18 or 35, 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 such as that shown in SEQ ID NO:1, 2, 16, 17, 18 or 35.

[0035] In a further embodiment, the polypeptides of the invention comprise conserved amino acids at the positions shown in Figures 5 or 6, preferably those shown in Figure 6. The conserved amino acid positions are shown in Figures 5 and 6 as white font letters on a black background.

[0036] It has been found that the polypeptides exhibiting diterpene alcohol synthase activity of the present invention typically comprise a stretch of amino acids in the N-terminal region which in one letter code is EKKSFGSMCI (SEQ ID NO: 56), or ENKSFGSMCI (SEQ ID NO: 58), or ENNSFGSMCI (SEQ ID NO: 55), or EKNSFGSMCI (SEQ ID NO: 57). Preferably, the polypeptides of the present invention comprise a sequence as shown in SEQ ID NO: 56 or 58. Substitution of the first lysine within this stretch of sequence with asparagine, or substitution of the asparagine within this stretch of sequence with lysine, respectively, did not significantly affect the performance of the enzyme in producing at least one C20 terpenoid alcohol referred to herein.

[0037] A fragment of a polypeptide exhibiting diterpene alcohol synthase activity according to the invention may be a polypeptide consisting of any of the amino acid sequences of the above mentioned sequences and sequence variants, having a length sufficient to exhibit the diterpene alcohol synthase activity as specified above. In this context, a conserved region of the above mentioned polypeptide has been identified according to the invention. This region is shown in SEQ ID NO: 24 or in a sequence with one or several amino acid mutations in SEQ ID NO: 24, in which the serine at position 4 of SEQ ID NO: 24 is converted or replaced by threonine (preferably said serine is converted) and is located at amino acids 486 to 497 of SEQ ID NO: 3 or at amino acids 486 to 497 of SEQ ID NO: 4. This region in the polypeptide according to the invention exhibiting diterpene alcohol synthase activity differs from the product determining region in other homologous synthases, in particular from the known Salvia sclareol synthase. It is therefore envisaged, preferably, that the above mentioned biologically active fragment of the polypeptide comprises the amino acid sequence of the conserved product outcome determining region as specified above. Typically, the fragment comprises or consists of a stretch 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 derived from the above sequences or sequence variants of the invention and provides diterpene alcohol synthase activity.

[0038] The above-mentioned polypeptides exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol can 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 a polypeptide exhibiting diterpene alcohol synthase activity. The additional amino acid sequence can be, for example, a polypeptide having other enzymatic activity, such as type II diterpene synthase activity for catalysis of step 1, a polypeptide having a support function for the functioning of a polypeptide exhibiting diterpene synthase activity, or a polypeptide or peptide having a marker or labeling function, for example, for monitoring the appropriate expression or for purification purposes, such as a tag (e.g., MYC tag, FLAG tag, His tag, etc.) or a fluorescent protein (e.g., GFP, BFP, YFP, or CFP).

[0039] The present disclosure further relates to a method for preparing a C20 terpenoid alcohol, preferably manool, sclareol and / or abienol, comprising converting copalyl diphosphate (CPP) and / or labd-13-en-8-ol diphosphate (LPP) to a C20 terpenoid alcohol, preferably manool, sclareol and / or abienol, respectively, in the presence of an enzyme, the enzyme comprising a first segment comprising a tag peptide and a second segment comprising a diterpene alcohol synthase according to the present invention. The enzyme comprising the first segment and the second segment may be referred to herein as a "tagged enzyme".

[0040] The tag peptide is preferably selected from the group of nitrogen utilization protein (NusA), thioredoxin (Trx), maltose binding protein (MBP), glutathione S-transferase (GST), ubiquitin-like factor (SUMO) or calcium binding protein (Fh8) and functional homologues thereof. As used herein, a functional homologue 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 non-tagged enzyme. Typically, a homologue differs in that the peptide of the homologue has one or more amino acids inserted, substituted, deleted or extended. A homologue 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.

[0041] Particularly preferred is the maltose binding protein from Escherichia coli or a functional homologue thereof.

[0042] The use of tagged enzymes according to the present invention is particularly advantageous in that it may contribute to increased product, in particular increased cellular production of terpenoids or terpenes, such as C20 terpenoid alcohols, preferably manool, sclareol and / or abienol.

[0043] To improve the solubility of the tagged enzyme (compared to the untagged enzyme), it is preferred that the first segment of the enzyme is linked at its C-terminus to the N-terminus of the second segment, alternatively, the first segment of the tagged enzyme is linked at its N-terminus to the C-terminus of the second segment.

[0044] 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 a MBP, NusA, Trx, GST, SUMO or Fh8-tag or a functional homologue of any of these, and a second segment comprising a diterpene alcohol synthase. The second segment may comprise, for example, an amino acid sequence as set forth in any one of SEQ ID NOs: 3-7, 28-30, 34 or 40-54, or a functional analogue thereof.

[0045] Furthermore, the present invention relates to a host cell comprising said nucleic acid encoding said tagged diterpene alcohol synthase. Particular nucleic acids according to the invention encoding the tagged enzyme are shown in any one of SEQ ID NO: 8 to SEQ ID NO: 10 and SEQ ID NO: 28 to 30. The host cell may in particular comprise a gene comprising any of these sequences or a functional analogue thereof.

[0046] Furthermore, the present invention relates to an enzyme comprising a first segment comprising a tag peptide and a second segment comprising a polypeptide having an enzymatic activity for converting a polyprenyl diphosphate to a terpene, in particular a diterpene alcohol synthase, wherein the tag peptide is preferably selected from the group of MBP, NusA, Trx or SET. Specific enzymes comprising the tagged enzyme according to the present invention are shown in any one of SEQ ID NOs: 8 to 10 and 28 to 30.

[0047] Preferably, the fusion protein further comprises a polypeptide exhibiting the enzymatic activity of a type II diterpene synthase. The conversion in step a) is carried out by a further polypeptide exhibiting the enzymatic activity of a type II diterpene synthase which converts geranylgeranyl pyrophosphate (GGP) to LPPs and / or CPPs. Thus, the polypeptide exhibiting diterpene synthase activity is preferably comprised in a fusion polypeptide comprising at least one further polypeptide which exhibits the enzymatic activity of a type II diterpene synthase which converts geranylgeranyl pyrophosphate (GGP) to LPPs and / or CPPs, has maltose binding properties or is a thioredoxin or a thioredoxin fusion protein. More preferably, the further polypeptide is preferably an LPP synthase from Coleus forskohlii (CfLPPS) (Pateraki, Plant Physiol., 164, 1222-1236 (2014); WO 2015 / 091943) or an LPP synthase from Nicotiana tabacum (NtLPPS) (Salaud, The Plant Journal (2012) 72, 1-17; WO 200807031 A1), ... forskohlii CPP synthase (CfCPPS) (Johnson, J. Biol. Chem. (2019) 294(4) 1349-1362; WO2020028795), thioredoxin and maltose binding protein (MBP).

[0048] In step a) of the method of the present invention, geranylgeranyl pyrophosphate is converted into copalyl diphosphate (CPP) or labd-13-en-8-ol diphosphate (LPP). The conversion is typically carried out enzymatically. Enzymes capable of converting geranylgeranyl phosphate into CPP or LPP are well known in the art. Preferably, the conversion is carried out by a polypeptide exhibiting the enzyme activity of a type II diterpene synthase, more preferably an LPP synthase, which converts geranylgeranyl pyrophosphate (GGP) into LPP and / or CPP, preferably an LPP synthase from Coleus forskohlii (CfLPPS) or an LPP synthase from Nicotiana tabacum (NtLPPS), preferably a CPP synthase from Coleus forskohlii (CfCPPS). It will be understood that a polypeptide exhibiting type II diterpene synthase activity is included in a fusion polypeptide together with a polypeptide exhibiting diterpene alcohol synthase activity of the invention, as described in more detail elsewhere herein.

[0049] The above step a) 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, suitable buffers can be used to provide the components in an environment with a suitable pH and suitable salt concentration. The suitable temperature in such a setting can also be applied as is.

[0050] Alternatively, step a) may be carried out in a host cell as described elsewhere herein. It is to be understood that the host cell shall be capable of producing GGP as well as the type II conversion enzyme as specified above. If necessary, the host cell must be genetically modified to express such type II enzyme or other enzymes or proteins required for GGP synthesis. The host cell shall be cultured under conditions and for a time sufficient to allow the expression of the above-mentioned enzymes and to convert GGP to CPPs and / or LPPs. Particularly preferred conditions are also described in the attached examples below.

[0051] Furthermore, step a) of the method of the 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 which is genetically modified to express the type II enzymes required for the conversion of GGP to CPPs and / or LPPs.

[0052] In step b) of the method of the present invention, the CPP or LPP is converted to at least one C20 terpenoid alcohol, said conversion being carried out by a polypeptide exhibiting diterpene alcohol synthase activity, said diterpene alcohol synthase activity being preferably capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol and / or LPP to abienol by the diterpene alcohol synthase of the present invention.

[0053] Step b) of the method of the invention can also be carried out in vitro or in a host cell or organism as specified above in step a), particularly preferred conditions being described in the appended examples below.

[0054] Preferably, said step b) or said steps a) and b) are performed in a host cell or a non-human transgenic organism. More preferably, said host cell or non-human transgenic organism is a host cell or a non-human transgenic organism of the invention as described in more detail elsewhere herein. It will be understood that the conditions that need to be applied to perform step b) or steps a) and b) in a host cell or non-human transgenic organism are dependent on said host cell or non-human transgenic organism. However, the skilled person is well aware of what conditions need to be applied depending on the choice of a given host cell or non-human transgenic organism.

[0055] Preferably, the method of the present invention includes a step of obtaining the at least one C20 terpenoid alcohol produced.

[0056] The term "obtained" as used herein refers to providing at least one C20 terpenoid alcohol with any degree of purity after step b). Thus, the at least one C20 terpenoid alcohol may be provided in essentially pure form or as a composition with additional components. Thus, the method of the present invention may include one or more purification steps after step b) is completed. The purification techniques that need to be applied depend on how steps a) and / or b) of the method of the present invention are performed. For example, it will be understood that less purification will be required to obtain, for example, essentially pure at least one C20 terpenoid alcohol if these steps are performed 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 steps a) and b) are performed in vivo, i.e. in a host cell as defined elsewhere herein, further purification and pretreatment steps may be required. Typically, the host cells that need to be harvested and the harvested cells may have to be lysed to release the C20 terpenoid alcohol from the cells. Subsequent purification steps must also remove cell debris in order to purify the C20 terpenoid alcohol from the remaining components. Furthermore, when the steps are carried out in vivo in animals or plants, further pretreatment and / or purification steps may be necessary to obtain at least one C20 terpenoid alcohol. The skilled person is well aware of suitable pretreatment and / or purification steps for a given situation in which steps a) and b) are carried out. Conceivable purification techniques may be extraction techniques, chromatography such as LC, GC or HPLC, size exclusion chromatography, affinity chromatography, distillation, centrifugation, filtration, etc. Conceivable pretreatment steps may be recovery, heat treatment, sonication, chemical and / or enzymatic treatment, etc. Particularly preferred treatments are described in the attached examples below.

[0057] Advantageously, the research underlying the present invention has revealed that the family of step 2 enzymes from Cupressa gigantea, namely Cup2v1 and Cup2v2b, can efficiently convert CPPs and LPPs to the C20 terpenoid alcohols manool, sclareol and / or abienol. In particular, the Cup2v1 and Cup2v2b enzymes, when expressed, for example, in Rhodobacter, have been found to be particularly efficient for recombinantly producing C20 terpenoid alcohols, as described in the attached Examples below. Furthermore, it has been found that the Cup2v2a and Cup2v2b enzymes, i.e., polypeptides having an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 6, 7, 9, 10 or 34 as specified elsewhere herein, or variants thereof, can produce two C20 terpenoid alcohols, namely manool and sclareol. Cup2v1, a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3, 5 or 8 as specified elsewhere herein, or a variant thereof, was efficient in producing abienol.

[0058] The present invention makes it possible to produce C20 terpenoid alcohols more efficiently, particularly in recombinant production techniques.

[0059] In one embodiment, an enzyme is considered useful in the method of the present invention if it preferentially produces C20 terpenoid alcohols. In a further embodiment, preferential production of C20 terpenoid alcohols is understood to mean that when the enzyme is provided with a wide variety of substrates under conditions suitable for the enzyme to be active among the products produced by the enzyme, C20 terpenoid alcohols predominate. For example, from all molecules produced by the enzyme, more than 50% of the molecules are C20 terpenoid alcohols.

[0060] In another embodiment, a polypeptide of the invention exhibiting diterpene alcohol synthase activity is characterized by the fact that it preferentially produces manool from the CPP and / or sclareol from the LPP and / or abienol from the LPP.

[0061] In a further embodiment, preferential production of manool, sclareol and / or abienol is understood to mean that manool, sclareol and / or abienol predominate among the products produced by the enzyme when a suitable substrate for the enzyme, such as LPP or CPP, is provided under conditions suitable for the enzyme to be active, e.g., from all molecules produced by the enzyme, more than 50% of the molecules are any one of manool, sclareol or abienol.

[0062] The present invention relates to a) producing one or more C20 terpenoid alcohols, preferably abienol, manool and / or sclareol, according to the method of the invention, preferably according to any one of claims 1 to 5; b) optionally purifying the one or more C20 terpenoid alcohols; and c) preparing and formulating a fragrance composition with said one or more C20 terpenoid alcohols; The present invention further relates to a method for producing a fragrance composition comprising:

[0063] A fragrance composition as used herein may be, for example, a flavor, fragrance or fragrance, see for example Chemistry and Technology of Flavors and Fragrances, Editor(s); David J. Rowe, First published; 26 October 2004, Print ISBN: 9781405114509 | Online ISBN: 9781444305517 | DOI: 10.1002 / 9781444305517, Blackwell Publishing Ltd.

[0064] 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.

[0065] The present invention also provides a composition or fragrance composition comprising said at least one C20 terpenoid alcohol, preferably manool, sclareol and / or abienol, obtainable by the process of the present invention.

[0066] Additionally, the present invention relates to a composition comprising a host cell or a non-human transgenic organism and said at least one C20 terpenoid alcohol, preferably manool, sclareol and / or abienol, obtainable by the method of the invention, preferably the method according to any one of claims 1 to 5, wherein the host cell or the non-human transgenic organism is recombinantly a) an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; 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 one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 2, 16, 17, 18 or 35; 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: 1, 2, 16, 17, 18 or 35; and e) an amino acid sequence of a fragment of any one of (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol. The present invention relates to a composition comprising at least one polypeptide exhibiting diterpene alcohol synthase activity, the polypeptide having the following structure:

[0067] The present invention further relates to a polypeptide exhibiting diterpene alcohol synthase activity, the diterpene alcohol synthase activity being capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol, the polypeptide comprising: a) an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; 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 one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 2, 16, 17, 18 or 35; 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: 1, 2, 16, 17, 18 or 35; and e) an amino acid sequence of a fragment of any one of (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol. The present invention also relates to a polypeptide having an amino acid sequence selected from the group consisting of:

[0068] Preferably, the diterpene alcohol synthase activity is capable of converting CPP to manool and LPP to sclareol. a) an amino acid sequence as set forth in SEQ ID NO: 4, 6, 7, 9, 10 or 34; 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 NO: 4, 6, 7, 9, 10 or 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 2, 16, 17 or 35; 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, 16, 17 or 35; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting CPP to manool and LPP to sclareol. The amino acid sequence is selected from the group consisting of:

[0069] Preferably, the diterpene alcohol synthase activity is also capable of converting LPP to abienol. More preferably, the polypeptide is a) an amino acid sequence as set forth in SEQ ID NO: 3, 5 or 8; 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 NO: 3, 5 or 8; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1 or 18; 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: 1 or 18; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting LPP to abienol. The amino acid sequence is selected from the group consisting of:

[0070] The present invention also contemplates a fusion polypeptide comprising a polypeptide of the invention and at least one further polypeptide, (i) preferably exhibiting the enzymatic activity of a type II diterpene synthase converting geranylgeranyl pyrophosphate (GGP) to LPP and / or CPP, (ii) having maltose binding properties, or (iii) being a thioredoxin or a thioredoxin fusion protein. More preferably, said further polypeptide is preferably selected from the group consisting of LPP synthase from Coleus forskohlii (CfLPPS) or LPP synthase from Nicotiana tabacum (NtLPPS), preferably CPP synthase from Coleus forskohlii (CfCPPS), thioredoxin and maltose binding protein (MBP).

[0071] The present invention also provides a method for producing a polypeptide having diterpene alcohol synthase activity of the invention, comprising the steps of: (a) transforming a host cell or a unicellular organism with a nucleic acid sequence of the invention to express a polypeptide having diterpene alcohol synthase activity; (b) obtaining or isolating the polypeptide having diterpene alcohol synthase activity from the host cell of step (a); and (c) optionally purifying the polypeptide having diterpene alcohol synthase activity. The present invention also relates to a method comprising the steps of:

[0072] The present invention relates to a) selecting a nucleic acid of the invention or a nucleic acid encoding a polypeptide of the invention; b) modifying the selected nucleic acids to obtain at least one mutant nucleic acid; c) transforming a host cell or a unicellular organism with the mutant nucleic acid sequence to express the polypeptide encoded by the mutant nucleic acid sequence; d) screening the polypeptides for at least one altered property and diterpene alcohol synthase activity; and e) optionally, if the polypeptide does not have the desired variant diterpene alcohol synthase activity, repeating process steps (a)-(d) until a polypeptide having the desired variant diterpene alcohol synthase activity is obtained; f) optionally, if a polypeptide having the desired variant diterpene alcohol synthase activity is identified in step (d), isolating the corresponding mutant nucleic acid obtained in step (c). The present invention further relates to a method for preparing a variant polypeptide having diterpene alcohol synthase activity, comprising:

[0073] The present invention relates to a polynucleotide encoding a polypeptide of the invention or a fusion polypeptide of the invention or the reverse complement or complementary sequence thereof.

[0074] The term "polynucleotide" as used in accordance with this specification refers to a deoxyribonucleotide or ribonucleotide polymer in either single-stranded or double-stranded form, and includes known analogs that have the basic properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids), unless otherwise limited. The term as used herein includes the sequences set forth herein as well as their complementary or reverse complementary sequences. Thus, the term includes DNAs or RNAs with backbones that have been modified for stability or other reasons. Additionally, DNAs or RNAs that contain unusual bases such as inosine or modified bases such as tritylated bases, to name just two examples, are also included as polynucleotides. It will be understood that a wide variety of modifications have been made to DNAs and RNAs that serve many useful purposes known to those of skill in the art. All nucleic acid sequences herein that code for a particular polypeptide of the invention may have silent mutations due to the degeneracy of the genetic code. The degeneracy of the genetic code results in a large number of functionally identical polynucleotides that code for the same polypeptide. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such a nucleic acid variation is a silent mutation.

[0075] The polynucleotide of the present invention is intended to code for a polypeptide of the present invention, i.e. to comprise a nucleic acid sequence encoding said polypeptide of the present invention. In addition, the polynucleotide of the present invention may comprise additional nucleic acid sequences. Preferably, the polynucleotide of the present invention may comprise, in addition to the open reading frame, further untranslated sequences at the 3' and 5' ends of the coding gene region, i.e. at least 500, preferably 200, more preferably 100 nucleotides of the sequence upstream of the 5' end of the coding region and at least 100, preferably 50, more preferably 20 nucleotides of the sequence downstream of the 3' end of the coding gene region.

[0076] The polynucleotides of the invention are preferably provided as isolated polynucleotides (i.e., purified or at least isolated from its natural context, such as its natural locus) or in a genetically modified or exogenously (i.e., artificially) engineered form. An isolated polynucleotide may, for example, comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flanks the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. The polynucleotides are preferably provided in the form of double-stranded or single-stranded molecules. It will be understood that by referring to any of the foregoing polynucleotides of the invention, the invention also refers to the complement or reverse complement of the particular sequence previously referred to or variants thereof. Polynucleotides encompass DNA polynucleotides, including cDNA and genomic DNA, or RNA polynucleotides.

[0077] However, the present invention also relates to polynucleotide variants derived from the polynucleotides of the present invention and capable of disrupting the transcription or translation of the polynucleotides of the present invention. Such variant polynucleotides include antisense nucleic acids, ribozymes, siRNA molecules, morpholino nucleic acids (phosphorodiamidate morpholino oligos), triplex-forming oligonucleotides, inhibitory oligonucleotides or microRNA molecules, all of which specifically recognize the polynucleotides of the present invention due to the presence of complementary or substantially complementary sequences. These techniques are well known to those skilled in the art. Suitable variant polynucleotides of the above-mentioned types can be easily designed based on the structure of the polynucleotides of the present invention.

[0078] Additionally, chemically modified polynucleotides are included, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides.

[0079] The present invention also relates to a vector or genetic construct comprising a polynucleotide of the present invention.

[0080] 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 a number of 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 clusters, chemically mediated transfer, electroporation or particle bombardment. Methods suitable for transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning; A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) 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, plasmid vectors may be introduced by heat shock or electroporation techniques. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.

[0081] 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.

[0082] 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 (Invitrogene) 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 rhabditis 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, CAM JJ, & Punt, PJ (1991) "Gene transfer systems and vector development for filamentous fungi, in; Applied Molecular Genetics of fungi, JF 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).

[0083] Furthermore, the vector may 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, resulting in stable inheritance of a gene encoding a polypeptide of interest, such as the alcohol acyltransferase of the present invention. An integrating vector generally includes one or more segments that include a gene sequence encoding a polypeptide of interest under the control (i.e., operably linked) of an additional nucleic acid segment that provides for its transcription.

[0084] 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 alcohol acyltransferase 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.

[0085] 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.

[0086] Furthermore, the present invention relates to a host cell which contains a vector or a genetic construct of the invention.

[0087] The host cell of the present invention is capable of expressing the polypeptide of the present invention contained in a vector or genetic construct of the present invention. The host cell is typically transformed with a vector or genetic construct such that the polypeptide of the present invention can be expressed from said vector or genetic construct. The transformed vector or genetic construct may be maintained as a non-integrated vector, for example a plasmid, or alternatively, may be integrated into the host cell genome, as specified in more detail elsewhere herein.

[0088] 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.

[0089] Preferably, the 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:

[0090] Bacteria: The bacterial host cell may, for example, be selected from the group consisting of Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, Lactococcus, or Pantoea.

[0091] 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 prokaryotic organisms include, but are not limited to, 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.

[0092] 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.

[0093] Gram-negative: E. coli, Pseudomonas, Rhodobacter, Paracoccus: Preferred Gram-negative bacteria are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC15958) or Pseudomonas fluorescens (NRRL B-11), Rhodobacter capsulatus or Rhodobacter sphaeroides, Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens ... zeaxanthinifaciens or Pantoea ananatis.

[0094] fungi: The host cell may be a fungal cell, such as Aspergillus, Fusarium, Trichoderma. As used herein, "fungi" includes fungi of 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, for example, Saprolegniomycetous aquatic fungi (water molds), such as Achlya. Examples of vegetative spore-forming fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of Zygomycota include, for example, Rhizopus and Mucor.

[0095] Some preferred fungi include strains belonging to the Deuteromycotina, the Hyphomycetes, such as the genera Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM2672), Humicola insolens, Trichoderma resii, and the like. 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 strains belonging to the subdivision Basidiomycotina, 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 (formerly called Polyporus), such as T. versicolor (e.g. PR4 28-A).

[0096] Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, such as the genera Rhizopus and Mucor, in particular Mucor hiemalis.

[0097] Yeast, Pichia, Saccharomyces: The fungal host cell may be a yeast cell. As used herein, yeast includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Ascosporogenous yeast Yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is composed of four subfamilies, Schizosaccharomycoideae (e.g., Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., Kluyveromyces, Pichia, and Saccharomyces). Basidiosporogenous yeasts include the genera Leucosporidium and Saccharomyces. Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium and Filobasidiella. Yeasts belonging to the Fungi lmperfecti are divided into two families, Sporobolomycetaceae (e.g., Sporobolomyces and Bullera) and Cryptococcaceae (e.g., Candida).

[0098] 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.

[0099] Most preferably, the host cell is a bacterial host cell, in particular a Rhodobacter host cell.

[0100] The present invention also relates to a transgenic non-human organism comprising a polynucleotide of the invention, a vector or a genetic construct of the invention or a host cell of the invention.

[0101] 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.

[0102] In one embodiment the non-human transgenic organism is a non-human transgenic organism transgenic for a polypeptide of the invention, a fusion protein comprising said polypeptide, a polynucleotide encoding same, a vector or a gene construct comprising said polynucleotide.

[0103] In one embodiment, the host cell is a non-human cell in vitro, for example in cell culture.

[0104] In another embodiment, the term "non-human" will be understood to refer to organisms other than humans that are not animals (e.g., plants, fungi, or microorganisms) or animals other than mammals, preferably non-vertebrate animals.

[0105] Methods for producing 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 method. 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).

[0106] The present invention generally contemplates the use of a polypeptide of the invention or a fusion polypeptide of the invention, a polynucleotide of the invention, a vector or genetic construct of the invention, a host cell of the invention or a non-human transgenic organism of the invention for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

[0107] The C20 terpenoid alcohols produced according to the present invention may have various uses in different industrial fields. In particular, the C20 terpenoid alcohols are used to produce flavors, pesticides, fragrances, pharmaceutical compositions, cosmetics or chemical components.

[0108] Furthermore, the present invention also relates to a kit for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol, comprising a polypeptide of the present invention or a fusion polypeptide of the present invention, a polynucleotide of the present invention, a vector or genetic construct of the present invention, a host cell of the present invention or a non-human transgenic organism of the present invention.

[0109] The term "kit" as used herein refers to a collection of components required to carry out the method of the present invention for producing at least one C20 terpenoid alcohol. 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 at least one C20 terpenoid alcohol. In addition, the kit may preferably include additional 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 purification of at least one C20 terpenoid alcohol.

[0110] 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.

[0111] Embodiment 1: A method for producing at least one C20 terpenoid alcohol, comprising: a) converting geranylgeranyl pyrophosphate to copalyl diphosphate (CPP) or labd-13-en-8-ol diphosphate (LPP); and b) converting the CPP or LPP to at least one C20 terpenoid alcohol, said conversion being carried out by a polypeptide exhibiting diterpene alcohol synthase activity, said diterpene alcohol synthase activity being capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol. wherein the polypeptide comprises a) an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; 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 one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 2, 16, 17, 18 or 35; 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: 1, 2, 16, 17, 18 or 35; and e) an amino acid sequence of a fragment of any one of (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol. The method of claim 1, comprising administering to said patient an amino acid sequence selected from the group consisting of:

[0112] Embodiment 2: The method of claim 1, wherein the polypeptide comprises an amino acid sequence of the conserved region as shown in SEQ ID NO:24.

[0113] Embodiment 3: The method of embodiment 1 or 2, wherein the at least one C20 terpenoid alcohol is a cyclic C20 terpenoid alcohol.

[0114] Embodiment 4: The method of any one of embodiments 1 to 3, wherein the at least one C20 terpenoid alcohol is manool, sclareol or abienol.

[0115] Embodiment 5: The method of any one of embodiments 1 to 4, wherein the polypeptide exhibiting diterpene alcohol synthase activity is capable of converting CPP to manool and LPP to sclareol.

[0116] Embodiment 6: The polypeptide comprises: a) an amino acid sequence as set forth in SEQ ID NO: 4, 6, 7, 9, 10 or 34; 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 NO: 4, 6, 7, 9, 10 or 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 2, 16, 17 or 35; 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, 16, 17 or 35; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting CPP to manool and LPP to sclareol. 6. The method of embodiment 5, comprising an amino acid sequence selected from the group consisting of:

[0117] Embodiment 7: The method of any one of embodiments 1 to 4, wherein the polypeptide exhibiting diterpene alcohol synthase activity is capable of converting LPP to abienol.

[0118] Embodiment 8: The polypeptide comprises: a) an amino acid sequence as set forth in SEQ ID NO: 3, 5 or 8; 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 NO: 3, 5 or 8; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1 or 18; 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: 1 or 18; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting LPP to abienol. 8. The method of embodiment 7, comprising an amino acid sequence selected from the group consisting of:

[0119] Embodiment 9: The method according to any one of embodiments 1 to 8, wherein the conversion in step a) is carried out by a further polypeptide exhibiting the enzymatic activity of a type II diterpene synthase that converts geranylgeranyl pyrophosphate (GGP) to LPPs and / or CPPs.

[0120] Embodiment 10: The method according to any one of embodiments 1 to 9, wherein the polypeptide exhibiting diterpene synthase activity preferably exhibits the enzymatic activity of a type II diterpene synthase converting geranylgeranyl pyrophosphate (GGP) to LPPs and / or CPPs, has maltose binding properties, or is comprised in a fusion polypeptide comprising at least one further polypeptide which is a thioredoxin or a thioredoxin fusion protein.

[0121] Embodiment 11: The method according to embodiment 10, wherein the further polypeptide is preferably selected from the group consisting of LPP synthase from Coleus forskohlii (CfLPPS) or LPP synthase from Nicotiana tabacum (NtLPPS), preferably CPP synthase from Coleus forskohlii (CfCPPS), thioredoxin and maltose binding protein (MBP).

[0122] Embodiment 12: The method according to any one of embodiments 1 to 12, wherein step b) or steps a) and b) are carried out in a host cell or a non-human transgenic organism.

[0123] Embodiment 13: The method of any one of embodiments 1 to 12, further comprising obtaining the at least one C20 terpenoid alcohol produced.

[0124] Embodiment 14: A composition comprising at least one C20 terpenoid alcohol, preferably manool, sclareol and / or abienol, obtainable by the method according to any one of embodiments 1 to 14.

[0125] Embodiment 15: A polypeptide exhibiting diterpene alcohol synthase activity, the diterpene alcohol synthase activity being capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol, the polypeptide comprising: a) an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; 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 one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 2, 16, 17, 18 or 35; 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: 1, 2, 16, 17, 18 or 35; and e) an amino acid sequence of a fragment of any one of (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labd-13-en-8-ol diphosphate (LPP) to sclareol, and / or LPP to abienol. A polypeptide having an amino acid sequence selected from the group consisting of:

[0126] Embodiment 16: The polypeptide of embodiment 15, comprising an amino acid sequence of the conserved region as shown in SEQ ID NO:24.

[0127] Embodiment 17: The polypeptide of embodiment 15 or 16, wherein the diterpene alcohol synthase activity is capable of converting CPP to manool and LPP to sclareol.

[0128] Embodiment 18: a) an amino acid sequence as set forth in SEQ ID NO: 4, 6, 7, 9, 10 or 34; 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 NO: 4, 6, 7, 9, 10 or 34; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 2, 16, 17 or 35; 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, 16, 17 or 35; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting CPP to manool and LPP to sclareol. 18. The polypeptide of embodiment 17, comprising an amino acid sequence selected from the group consisting of:

[0129] Embodiment 19: The polypeptide of embodiment 15 or 16, wherein the diterpene alcohol synthase activity is capable of converting LPP to abienol.

[0130] Embodiment 20: a) an amino acid sequence as set forth in SEQ ID NO: 3, 5 or 8; 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 NO: 3, 5 or 8; c) an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1 or 18; 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: 1 or 18; and e) An amino acid sequence of any one of fragments (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting LPP to abienol. 20. The polypeptide of embodiment 19, comprising an amino acid sequence selected from the group consisting of:

[0131] Embodiment 21: A fusion polypeptide comprising a polypeptide according to any one of embodiments 15 to 20 and at least one further polypeptide, (i) exhibiting the enzymatic activity of a type II diterpene synthase, preferably converting geranylgeranyl pyrophosphate (GGP) to LPPs and / or CPPs, (ii) having maltose binding properties, or (iii) being a thioredoxin or a thioredoxin fusion protein.

[0132] Embodiment 22: The fusion polypeptide according to embodiment 21, wherein the further polypeptide is preferably selected from the group consisting of LPP synthase from Coleus forskohlii (CfLPPS) or LPP synthase from Nicotiana tabacum (NtLPPS), preferably CPP synthase from Coleus forskohlii (CfCPPS), thioredoxin and maltose binding protein (MBP).

[0133] Embodiment 23: A polynucleotide encoding a polypeptide according to any one of embodiments 15 to 20 or a fusion polypeptide according to embodiment 21 or 22 or the reverse complement or the complementary sequence thereof.

[0134] Embodiment 24: A vector or genetic construct comprising a polynucleotide according to embodiment 23.

[0135] Embodiment 25: A host cell comprising the vector or genetic construct of embodiment 24.

[0136] Embodiment 26: The host cell according to embodiment 25, which 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.

[0137] Embodiment 27: A transgenic non-human organism comprising a polynucleotide according to embodiment 23, a vector or a gene construct according to embodiment 24, or a host cell according to embodiment 25 or 26.

[0138] Embodiment 28: Use of a polypeptide according to any one of embodiments 15 to 20 or a fusion polypeptide according to embodiment 21 or 22, a polynucleotide according to embodiment 23, a vector or genetic construct according to embodiment 24, a host cell according to embodiment 25 or 26 or a non-human transgenic organism according to embodiment 27 for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

[0139] Embodiment 29: The use of embodiment 28, wherein the C20 terpenoid alcohol is used to produce a flavoring, pesticide, fragrance, drug, cosmetic or chemical component.

[0140] Embodiment 30: A kit for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol, comprising a polypeptide according to any one of embodiments 15 to 20 or a fusion polypeptide according to embodiment 21 or 22, a polynucleotide according to embodiment 23, a vector or genetic construct according to embodiment 24, a host cell according to embodiment 25 or 26 or a non-human transgenic organism according to embodiment 27.

[0141] 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]

[0142] [Figure 1] GC MS analysis of dichloromethane extract from Cupressus gigantea. A clear peak of manool was observed at 19.7 min, which corresponds to the Rt of manool standard. [Figure 2a] GC analysis of strains pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v1-Prplm-CgIsdA and pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v1-Prplm-CgIsdA. a) and b); analysis revealed a compound eluting at 13.61 min (subsequently identified as abienol); c, d, e are results from constructs expressing Cup2v2a and Cup2v2b in combination with LPP synthase. The analysis revealed a new compound eluting at 14.03 min (subsequently identified as sclareol); f) GC analysis of strain pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA produced a compound eluting at 13.29 min (subsequently identified as manool). [Figure 2b] This is a continuation of Figure 2a. [Figure 2c] This is a continuation of Figure 2b. [Figure 2d] This is a continuation of Figure 2c. [Figure 2e] This is a continuation of Figure 2d. [Figure 2f] Continued from Figure 2e. [Figure 3a] GC MS analysis of the strains. a) GC MS analysis of pBBR-MEV-PcrtE-TrxCfLPPS-mbpCupr2v1-Prplm-CgIsdA confirmed that the compound corresponds to abienol; b) GC MS analysis of pBBR-MEV-PcrtE-TrxCfLPPS-mbpCupr2v2b-Prplm-CgIsdA confirmed that the compound corresponds to sclareol; c) GC MS analysis of pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA revealed that the compound corresponds to manool. [Figure 3b] This is a continuation of Figure 3a. [Figure 3c] This is a continuation of Figure 3b. [Figure 4]Alignment of product determining regions. CfMS, manoyl oxide synthase from Coleus forskohlii (GenBank accession: KF444508); IrMS, mirtiradiene synthase from Isodon rubescens (KX831652); CfMS, mirtiradiene synthase from C. forskohlii (KF444509); RoMS1, mirtiradiene synthase 1 from Rosemarius officinalis (KF805858); SmMS, mirtiradiene synthase from Salvia Miltiorrhiza (ABV08817); RoMS1, mirtiradiene synthase from Rosemarius officinalis (KF805858). officinalis (KF805859); SfMS, miltiradiene synthase from Salvia fruticosa (KP091841); MvELS, 9,13-epoxy-labd-14-ene synthase from Marrubium vulgare (KJ584454); SsSS Salvia sclarea sclareol synthase (JN133922); SsSS-iAS variant of SsSS, an isoabienol synthase (Jia et al ACS Catal. 2018,8,3133-3137). [Figure 5-1] Cup2v2b (SEQ ID NO: 4), Cup2v2a (SEQ ID NO: 34), Cup2v1 (SEQ ID NO: 3) and TcKSL1, TcKSL2 and TcKSL8 found in the National Center for Biotechnology Information (NCBI) database under accession numbers KT588484, KT588485 and KT588489, respectively; furthermore, a protein alignment of the sequence of ScSS as found in SEQ ID NO: 3 of WO2009101126. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 6-1]Protein alignment of Cup2v2b (SEQ ID NO: 4), Cup2v2a (SEQ ID NO: 34), Cup2v1 (SEQ ID NO: 3) and TcKSL1, TcKSL2 and TcKSL8 found in the National Center for Biotechnology Information (NCBI) database under accession numbers KT588484, KT588485 and KT588489, respectively. [Figure 6-2] This is a continuation of Figure 6-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0143] The following sequences are referenced throughout the specification and in the accompanying sequence protocols. SEQ ID NO:1: Cup2v1 cDNA sequence SEQ ID NO:2: Cup2v2b cDNA sequence SEQ ID NO: 3: Cup2v1 protein SEQ ID NO: 4: Cup2v2b protein SEQ ID NO: 5: Truncated Cup2v1 protein SEQ ID NO: 6: Truncated Cup2v2a protein SEQ ID NO: 7: Truncated Cup2v2b protein SEQ ID NO: 8: MBP-cleaved Cup2v1 protein SEQ ID NO: 9: MBP-cleaved Cup2v2a protein SEQ ID NO: 10: MBP truncated cup2v2b protein SEQ ID NO: 11: SsSS cleavage protein SEQ ID NO: 12: Trx-CfCPS protein SEQ ID NO: 13: Trx-CfLPPS protein SEQ ID NO: 14: Trx-NtLPPS protein SEQ ID NO: 15: CgIdsA protein SEQ ID NO: 16: MBP-Cup2v2b DNA SEQ ID NO: 17: MBP-Cup2v2a DNA SEQ ID NO: 18: MBP-Cup2v1 DNA SEQ ID NO: 19: SsSS cDNA SEQ ID NO: 20: Trx-CfLPPS DNA SEQ ID NO: 21: Trx-CfCPS DNA SEQ ID NO: 22: CgidsA cDNA SEQ ID NO: 23: Trx-NtLPPS DNA SEQ ID NO: 24: Conserved regions of Cup2v1, Cup2v2a and Cup2v2b proteins SEQ ID NO: 25: Product determining region of CfMOS protein; Product determining region of IrMS protein; Product determining region of CfMS protein; Product determining region of CRoMS1 protein; Product determining region of SmMS protein; Product determining region of RoMS2 protein; Product determining region of SfMS protein; Product determining region of MvELS protein SEQ ID NO: 26: Product determining region of SsSS protein SEQ ID NO: 27: Product determining region of SsSS-iAS protein SEQ ID NO: 28: MBP-Cupr2v2b-2 polypeptide SEQ ID NO:29 MBP-Cupr2v2b-3 polypeptide SEQ ID NO: 30 MBP-Cupr2v2b-4 polypeptide SEQ ID NO: 31 MBP-Cupr2v2b-2 DNA SEQ ID NO: 32 MBP-Cupr2v2b-3 DNA SEQ ID NO: 33 MBP-Cupr2v2b-4 DNA SEQ ID NO:34 Cup2v2a protein SEQ ID NO:35 Cup2v2a DNA SEQ ID NO: 36 Truncated Cup2v1 DNA SEQ ID NO: 37 Truncated Cup2v2b DNA SEQ ID NO:38 Truncated Cup2v2a DNA SEQ ID NO: 39 DNA C-terminal and N-terminal double truncation Cup2v1 SEQ ID NO: 40 Protein C-terminal and N-terminal double truncation Cup2v1 SEQ ID NO: 41 Variant 1 protein SEQ ID NO: 42 Variant 2 protein SEQ ID NO: 43 Variant 3 protein SEQ ID NO: 44 Variant 4 protein SEQ ID NO: 45 Variant 5 protein SEQ ID NO: 46 Variant 6 protein SEQ ID NO: 47 Variant 7 protein SEQ ID NO: 48 Variant 8 protein SEQ ID NO: 49 Variant 9 protein SEQ ID NO:50 Variant 10 protein SEQ ID NO:51 Variant 11 protein SEQ ID NO:52 Variant 12 protein SEQ ID NO:53 Variant 13 protein SEQ ID NO:54 Variant 14 protein SEQ ID NO: 55 Cup motif ENNSFGSMCI SEQ ID NO: 56 Cup motif EKKSFGSMCI SEQ ID NO: 57 Cup motif EKNSFGSMCI SEQ ID NO:58 Cup motif ENKSFGSMCI

[0144] Furthermore, the following polypeptides with the given single amino acid substitutions are also polypeptides according to the invention: In SEQ ID NO: 4, Lys at position 84 may be replaced by Asn; In SEQ ID NO: 6, Asn at position 3 may be replaced by Lys; In SEQ ID NO: 7, Lys at position 3 may be replaced by Asn; In SEQ ID NO: 9, Asn at position 375 may be replaced by Lys; In SEQ ID NO: 10, Asn at position 375 may be replaced by Lys; In SEQ ID NO: 3, position 398 is filled with Ile or Thr; In SEQ ID NO: 5, position 317 is filled with Ile or Thr. EXAMPLES

[0145] The examples are intended to be merely illustrative of the invention and should not be construed as limiting the scope in any way.

[0146] Example 1: Cloning of Cup2v1, Cup2v2a and Cup2v2b Analysis of Cupressus gigantea terpenes. Cupressus gigantea trees were obtained from Esveld (Boskoop). Extracts were prepared from the stem cortex by grinding the cortex material under liquid nitrogen to a fine powder and extracting 100 mg of this powder with 1 ml of dichloromethane. The dichloromethane phase was analyzed by GC MS. A clear peak for manool was observed at 19.7 min, corresponding to the Rt of manool standard.

[0147] RNA was extracted and sequenced from cDNA from Cupressus tissues. Approximately 15 mL of extraction buffer (2% hexadecyltrimethylammonium bromide, 2% polyvinylpyrrolidinone K30, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0 M NaCl, 0.5 g / L spermidine, and 2% β-mercaptoethanol) was warmed to 65°C, and then 3 g of ground cortical tissue was added and mixed. The mixture was extracted twice with an equal volume of chloroform:isoamyl alcohol (1:24), and a quarter volume of 10 M LiCl was added to the supernatant and mixed. RNA was precipitated overnight at 4°C and collected by centrifugation at 10,000 g for 20 min. The pellet was dissolved in 500 μL of SSTE [1.0 M NaCl, 0.5% SDS, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)] and extracted once with an equal volume of chloroform:isoamyl alcohol. Two volumes of ethanol were added to the supernatant, incubated at -20°C for at least 2 h, and centrifuged at 13000 g to remove the supernatant. The pellet was air-dried and resuspended in water. Total RNA (60 μg) was sent to Vertis Biotechnology AG (Freising, Germany). PolyA+RNA was isolated and random primed cDNA was synthesized using randomized N6 adapter primers and M-MLV H-reverse transcriptase. The cDNA was sheared to fragment and 500 bp size fragments were used for further analysis. The cDNA has adapter sequences A and B ligated to their 5' and 3' ends as defined by Illumina. This material was then analyzed on an Illumina MiSeq sequencing instrument. The MiSeq generated a total of 19,608,859 sequence reads. Sequences from Illumina sequencing adapters were trimmed using Trimmomatic-0.32, paired-end sequences were overlapped using Seqprep, and phiX contamination was removed using bowtie2 (version 2.2.1) (phiX DNA was used as a spike-in control and is typically present at <1%). Paired-end and single reads were used in Trinity assembly (trinityrnaseq-2.0.2).A total of 88,667 contigs were assembled by Trinity.

[0148] To identify sesquiterpene synthases, the C. gigantea contigs were used to generate a database of cDNA sequences, in which the TBLASTN program was used to identify cDNA sequences encoding proteins that showed identity to the protein sequences of sesquiterpene synthases, including kaurene synthase from Arabidopsis thaliana (Q9SAK2), sclareol synthase from Salvia sclarea (AET21246.1), abienol synthase from Abies balsamifera (H8ZM73.1), and 13-labdene-8,15-diol pyrophosphate synthase from Salvia sclarea (AET21248.1). A total of 184 contigs in the cDNA database of C. gigantea were identified as having significant homology with sesquiterpene synthases. The contigs were classified into 68 groups according to overlap in the sequences. These 68 contigs were further characterized by analyzing them using the BLASTX program, which aligned them to protein sequences present in the UniProt database (downloaded on August 28, 2015). We manually identified 12 of them as putative diterpene synthase sequences according to their homology with terpene synthase sequences present in UniProt and their characteristics.

[0149] Identification of Cup2v1, Cup2v2a, and Cup2v2b Based on thorough analysis of the characteristics of the cDNA sequences, three of them were selected by the present inventors as the most promising candidate genes. The cDNA sequences shown in SEQ ID NO: 1 and 2 were identified as Cup2v1 and Cup2v2b, respectively. The Cup2v1 protein is shown as SEQ ID NO: 3, and the Cup2v2b protein is shown as SEQ ID NO: 4. The Cup2v1 and Cup2v2b proteins are 93.8% identical to each other at the amino acid level.

[0150] The third cDNA sequence was similar to Cup2v2b and was designated Cup2v2a.

[0151] We generated artificially shortened versions of the sequence, whereby the plastid targeting signal was removed and the N-terminus was altered. These truncated amino acid sequences, designated trcup2v1, trcup2v2a and trcup2v2b, are shown in SEQ ID NOs: 5-7, respectively. The full-length Cup2v2a protein is shown in SEQ ID NO: 34, and its cDNA sequence is shown in SEQ ID NO: 35.

[0152] Of the known Salvia sclareol synthase (SsSS), a truncated version was generated as a control (trSsSS).

[0153] BLAST of the NCBI nr protein database revealed that the closest homologue of these proteins is a diterpene synthase (AOG18231.1) of unknown product specificity from Taiwania cryptomerioides with 67.6% amino acid identity. BLAST of the uniprot database of characterized proteins revealed an ent-kaurene synthase from Vitex agnuscastus with 39.1% amino acid identity.

[0154] [Table 1]

[0155] The inventors have identified Cup2v1, Cup2v2a and Cup2v2b proteins as candidates for step 2 diterpene alcohol synthases to produce abienol, manool and / or sclareol. The inventors have identified a region that is essentially conserved between Cup2v1, Cup2v2a and Cup2v2b (see alignment FIG. 4). This region in the synthase is in a position that corresponds to the product determining region of other synthases, but is different from the product determining region of said other synthases, such as the product determining region in the known Salvia sclareol synthase. Although Cup2v1, Cup2v2a and Cup2v2b have different product specificities (see below), the regions typically involved in determining product specificity in other known diterpene synthases are highly divergent yet conserved among the Cup proteins.

[0156] Example 2: Construction of plasmids for expressing step 1 and step 2 genes in Rhodobacter For expression in Rhodobacter, fusion proteins were designed containing truncated versions of Cup2v1, Cup2v2a, Cup2v2b containing the maltose binding protein (designated mbpCup2v1, mbpCup2v2a and mbpCup2v2b, see SEQ ID NOs: 8-10, respectively) and fusion proteins of a number of step 1 genes, CfLPPS, CfCPPS and NtLPPS with thioredoxin Trx (see SEQ ID NOs: 12-14). For comparison, constructs were also prepared in which CfLPPS was expressed in combination with a truncated version of sclareol synthase (SsSS) from Salvia sclarea. This truncated version corresponds to SsSS as published in Schalk J. Am. Chem. Soc. 2012, 134, 18900-18903.

[0157] A construct was made in which the mevalonate operon from Paracoccus zeaxanthinifaciens was expressed from its native promoter as described in EP 2 336 310 A1 together with CgIdsA expressed from the Lppa promoter as described in WO 2018 / 160066 A1 and an operon consisting of the crtE promoter followed by the trx-step 1 gene, the ribosome binding site and the mbp-step 2 gene.

[0158] The following series of constructs were prepared: a.pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v1-Prplm-CgIsdA b.pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v1-Prplm-CgIsdA c.pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v2a-Prplm-CgIsdA d.pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2a-Prplm-CgIsdA e.pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2b-Prplm-CgIsdA f.pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA g.pBBR-MEV-PcrtE-TrxCfLPPS-SsSS-Prplm-CgIsdA

[0159] These constructs were introduced into E. coli S17-1 and the resulting strains were used to conjugate into Rhodobacter sphaeroides Rs265-9c using standard procedures. The resulting strains were named after their plasmids.

[0160] Example 3: Small-scale recombinant production of C20 terpenoid alcohols Each strain was used in a small-scale production trial essentially as described in US Patent Publication No. 2020 / 0010822A1. To this end, seed cultures were made in unbaffled 100 ml shake flasks containing 20 ml of RS102 medium with 100 mg / L neomycin and one loopful of glycerol stock. Seed culture flasks were incubated at 30° C. for 72 hours in a shaking incubator with 50 mm orbit at 110 rpm.

[0161] At the end of 72 hours, the OD600 of the cultures was assessed to calculate the exact volume of culture and transferred to a larger flask.

[0162] Shake flask experiments were performed in 300 ml shake flasks with two bottom baffles. 20 ml of RS102 medium and neomycin to a final concentration of 100 mg / L were added to the flask along with 2 ml of sterile n-dodecane. The inoculum volume was adjusted to give a final OD600 value of 0.05 in 20 ml of medium.

[0163] The flasks were kept at 30°C in a shaking incubator with 50 mm orbit at 110 rpm for 72 hours. Afterwards, the cultures were collected in pre-weighed 50 ml PP tubes and then centrifuged at 4500 x g for 20 minutes. The n-dodecane layer was transferred to a microcentrifuge tube for subsequent GC analysis.

[0164] Ten microliters of ethyl laurate was weighed into a 10 ml glass vial, to which 800 μl of the isolated dodecane solution was added and weighed. Then, 8 ml of acetone was added to the vial to dilute the concentration of dodecane for more accurate GC analysis. Approximately 1.5 ml of the acetone solution of terpene-containing dodecane was transferred to a chromatography vial. Each sample was analyzed by gas chromatography as described in US Patent Application Publication No. 2020 / 0010822A1. To identify the compounds, approximately 2 μL was analyzed by GC / MS using a gas chromatograph as described in detail in Cankar et al. (2015). The products were identified by comparing the retention times and mass spectra with standards of sclareol, manool, and abienol (Sigma-Aldrich).

[0165] GC analysis of strains pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v1-Prplm-CgIsdA and pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v1-Prplm-CgIsdA revealed a compound eluting at 13.61 min (Fig. 2a, b). GC MS analysis confirmed that this compound corresponds to abienol (Fig. 3a). The following titers (g / kg n-dodecane) were found for abienol with the constructs: 1.9 for pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v1-Prplm-CgIsdA and 3.5 for pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v1-Prplm-CgIsdA.

[0166] GC analysis of strain pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA (Fig. 2f) produced a compound eluting at 13.29 min. GC MS analysis revealed that this compound corresponds to manool (Fig. 3c). The following titers (g / kg n-dodecane) were found for manool by the constructs: 1.5 for pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA; 1.5 for pBBR-MEV-PcrtE-TrxCfCPPS-mbpCup2v2a-Prplm-CgIsdA;

[0167] GC analysis of strains pBBR-MEV-PcrtE-TrxNtLPPS-mbpCup2v2a-Prplm-CgIsdA, pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2a-Prplm-CgIsdA, pBBR-MEV-PcrtE-TrxCfLPPS-SsSS-Prplm-CgIsdA and pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2b-Prplm-CgIsdA revealed a new compound eluting at 14.03 min (Fig. 2c, d, e). GC MS analysis confirmed that this compound corresponds to sclareol (Fig. 3b). Quantitative analysis of sclareol by the different constructs is shown in the table below.

[0168] [Table 2]

[0169] Further sequence variants of Cup2v2b with additional sequence at the N-terminus compared to SEQ ID NO:7 were also tested in a similar setup as fusion proteins with MBP at the N-terminus (SEQ ID NOs:28-30). All three showed similar levels of sclareol production, as shown by pBBR-MEV-PcrtE-TrxCfLPPS-mbpCup2v2b-Prplm-CgIsdA in the fourth row of Table 1 above.

Claims

1. A method for producing at least one C20 terpenoid alcohol, comprising: a) converting geranylgeranyl pyrophosphate to copalyl diphosphate (CPP) or labda-13-ene-8-ol diphosphate (LPP); and b) converting CPP or LPP to at least one C20 terpenoid alcohol, said conversion being carried out by a polypeptide exhibiting diterpene alcohol synthase activity, said diterpene alcohol synthase activity being capable of converting copalyl diphosphate (CPP) to manool, labda-13-ene-8-ol diphosphate (LPP) to sclareneol, and / or LPP to abienol, wherein said polypeptide is selected from the group consisting of: a) an amino acid sequence as shown in any one of SEQ ID NOs: 3-10 or SEQ ID NO: 34; 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 the amino acid sequence as shown in any one of SEQ ID NOs: 3-10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NOs: 1, 2, 16, 17, 18 or 35; 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 the nucleic acid sequence as shown in SEQ ID NOs: 1, 2, 16, 17, 18 or 35; and e) a fragment of any one of (a)-(d) that encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) to manool, labda-13-ene-8-ol diphosphate (LPP) to sclareneol, and / or LPP to abienol A method comprising an amino acid sequence selected from the group consisting of.

2. Said polypeptide exhibiting diterpene alcohol synthase activity is capable of converting CPP to manool and LPP to sclareneol, and preferably a) an amino acid sequence as shown in SEQ ID NOs: 4, 6, 7, 9, 10 or 34; 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 the amino acid sequence as shown in SEQ ID NO: 4, 6, 7, 9, 10 or 34; c) an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO: 2, 16, 17 or 35; 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 the nucleic acid sequence as shown in SEQ ID NO: 2, 16, 17 or 35; and e) a fragment of any one of (a) to (d), which encodes a polypeptide showing diterpene alcohol synthase activity capable of converting CPP to manool and LPP to sclareneol, and the amino acid sequence of the fragment The method according to claim 1, comprising an amino acid sequence selected from the group consisting of **Claim 3** The polypeptide showing diterpene alcohol synthase activity can convert LPP to abienol, and preferably, a) an amino acid sequence as shown in SEQ ID NO: 3, 5 or 8; 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 the amino acid sequence as shown in SEQ ID NO: 3, 5 or 8; c) an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO: 1 or 18; 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 the nucleic acid sequence as shown in SEQ ID NO: 1 or 18; and e) a fragment of any one of (a) to (d), which encodes a polypeptide showing diterpene alcohol synthase activity capable of converting LPP to abienol, and the amino acid sequence of the fragment The method according to claim 1, comprising an amino acid sequence selected from the group consisting of **Claim 4** The method according to claim 1, wherein the conversion in step a) is carried out by a further polypeptide exhibiting the enzymatic activity of a type II diterpene synthase that converts geranylgeranyl pyrophosphate (GGPP) into LPP and / or CPP.

5. The method according to claim 1, wherein step b) or steps a) and b) are carried out in a host cell or a non-human transgenic organism.

6. A composition comprising a host cell or a non-human transgenic organism and at least one C20 terpenoid alcohol obtainable by the method according to any one of claims 1 to 5, preferably manool, sclareol and / or abienol, wherein the host cell or non-human transgenic organism is recombinantly a) an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; 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 the amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NOs: 1, 2, 16, 17, 18 or 35; 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 the nucleic acid sequence as shown in SEQ ID NOs: 1, 2, 16, 17, 18 or 35; and e) a fragment of any one of (a) to (d) that encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyl diphosphate (CPP) into manool, labda-13-ene-8-ol diphosphate (LPP) into sclareol, and / or LPP into abienol A composition comprising at least one polypeptide exhibiting diterpene alcohol synthase activity.

7. A polypeptide exhibiting diterpene alcohol synthase activity, wherein the diterpene alcohol synthase activity can convert copalyldiphosphate (CPP) to manool, labda-13-ene-8-ol diphosphate (LPP) to sclaroleol, and / or LPP to abienol, and the polypeptide is a) an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; 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 the amino acid sequence as shown in any one of SEQ ID NOs: 3 to 10 or SEQ ID NO: 34; c) an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO: 1, 2, 16, 17, 18 or 35; 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 the nucleic acid sequence as shown in SEQ ID NO: 1, 2, 16, 17, 18 or 35; and e) a fragment of any one of (a) to (d), which encodes a polypeptide exhibiting diterpene alcohol synthase activity capable of converting copalyldiphosphate (CPP) to manool, labda-13-ene-8-ol diphosphate (LPP) to sclaroleol, and / or LPP to abienol A polypeptide having an amino acid sequence selected from the group consisting of **Claim 8** The diterpene alcohol synthase activity can convert CPP to manool and LPP to sclaroleol, and preferably a) an amino acid sequence as shown in SEQ ID NOs: 4, 6, 7, 9, 10 or 34; 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 the amino acid sequence as shown in SEQ ID NOs: 4, 6, 7, 9, 10 or 34; c) an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO: 2, 16, 17 or 35; 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 the nucleic acid sequence as shown in SEQ ID NO: 2, 16, 17 or 35; and e) an amino acid sequence of a fragment of any one of (a) to (d), which encodes a polypeptide showing diterpene alcohol synthase activity capable of converting CPP to manool and LPP to sclareneol The polypeptide according to claim 7, comprising an amino acid sequence selected from the group consisting of.

9. The diterpene alcohol synthase activity can convert LPP to abienol, and preferably a) an amino acid sequence as shown in SEQ ID NO: 3, 5 or 8; 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 the amino acid sequence as shown in SEQ ID NO: 3, 5 or 8; c) an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO: 1 or 18; 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 the nucleic acid sequence as shown in SEQ ID NO: 1 or 18; and e) an amino acid sequence of a fragment of any one of (a) to (d), which encodes a polypeptide showing diterpene alcohol synthase activity capable of converting LPP to abienol The polypeptide according to claim 7, comprising an amino acid sequence selected from the group consisting of.

10. A fusion polypeptide comprising the polypeptide according to claim 7 and at least one further polypeptide that (i) preferably exhibits the enzyme activity of type II diterpene synthase that converts geranylgeranyl pyrophosphate (GGPP) to LPP and / or CPP, (ii) has maltose-binding properties, or (iii) is thioredoxin or a thioredoxin fusion protein.

11. A polynucleotide encoding the polypeptide according to claim 7, the fusion polypeptide according to claim 10, or its reverse complementary sequence or complementary sequence.

12. A vector or gene construct comprising the polynucleotide according to claim 11.

13. A host cell comprising the vector or gene construct according to claim 12.

14. A transgenic non-human organism comprising the polynucleotide according to claim 11.

15. A transgenic non-human organism comprising the vector or gene construct according to claim 12.

16. A transgenic non-human organism comprising the host cell according to claim 13.

17. Use of the polypeptide according to claim 7 or the fusion polypeptide according to claim 10 for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

18. Use of the polynucleotide according to claim 11 for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

19. Use of the vector or gene construct according to claim 12 for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

20. Use of the host cell according to claim 13 for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

21. Use of the non-human transgenic organism according to claim 14 for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

22. Use of the non-human transgenic organism according to claim 15 for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

23. Use of the non-human transgenic organism according to claim 16 for producing at least one C20 terpenoid alcohol, preferably abienol, manool and / or sclareol.

24. A method for preparing a variant polypeptide having diterpene alcohol synthase activity, comprising a) a step of selecting the nucleic acid according to claim 11; b) a step of modifying the selected nucleic acid to obtain at least one mutant nucleic acid; c) a step of transforming a host cell or a unicellular organism with the mutant nucleic acid sequence to express the polypeptide encoded by the mutant nucleic acid sequence; d) a step of screening the polypeptide for at least one modified property and diterpene alcohol synthase activity; and e) optionally, if the polypeptide does not have the desired variant diterpene alcohol synthase activity, repeating process steps (a) to (d) until a polypeptide having the desired variant diterpene alcohol synthase activity is obtained; f) optionally, if a polypeptide having the desired variant diterpene alcohol synthase activity is identified in step (d), a step of isolating the corresponding mutant nucleic acid obtained in step (c) A method comprising.

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