Recombinant production of Zizaene and other sesquiterpenes formed by conversion of bisabolol cations

Abstract

The present invention relates to a method for producing a composition comprising zizaene, the method comprising the following subsequent steps: i) providing farnesyl pyrophosphate and at least one polypeptide exhibiting zizaene synthase activity under conditions suitable for the polypeptide to convert at least a portion of the farnesyl pyrophosphate to zizaene; ii) converting at least a portion of the farnesyl pyrophosphate to a bisabolol cation with the polypeptide; and iii) producing a composition containing zizaene by converting the bisabolol cation to zizaene and optionally at least one additional compound with the polypeptide, wherein the at least one polypeptide comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence as set forth in SEQ ID NO: 1; b) an amino acid sequence which is at least 55% identical to the amino acid sequence as shown in SEQ ID NO: 1; c) an amino acid sequence encoded by a nucleic acid sequence represented by any one of SEQ ID NO: 2, 3 or 5; d) an amino acid sequence encoded by a nucleic acid sequence at least 55% identical to the nucleic acid sequence as shown in any one of SEQ ID NO: 2, 3 or 5; and e) an amino acid sequence that is a fragment of any sequence listed in a) to d). Furthermore, the present invention relates, inter alia, to a zizaene-containing composition obtainable by said method as well as to a corresponding non-human host cell and to a corresponding polypeptide exhibiting zizaene synthase activity.

Description

Technical Field:

[0001] The present invention relates to the field of recombinant production of zizaene and other sesquiterpenes and related compounds formed by the conversion of myrrh-based cations. In particular, the present invention relates to a method for producing a composition containing zizaene, in which farnesyl pyrophosphate is converted to zizaene by at least one polypeptide exhibiting zizaene synthase activity, and to a composition obtainable by said method. Furthermore, the present invention relates to a method for producing a composition containing at least one oxidation product of zizaene, and to a corresponding non-human host cell or transgenic organism and a corresponding polypeptide exhibiting zizaene synthase activity. Background Art:

[0002] Vetiver oil is used in fine fragrances and has a persistent and precious woody fragrance. Vetiver oil is extracted from Vetiveria zizanoides, which is mainly produced in Indonesia, and contains more than 150 sesquiterpene-derived components. The harvesting of vetiver aims to collect the roots deep in the soil. Therefore, this requires a large amount of labor and causes serious erosion, making the demand for alternatives to vetiver oil increasing.

[0003] Historically, there has been some debate about the key sensory components of vetiver oil. However, nowadays, the literature generally agrees on the importance of zizaene (see formula I below; CAS: 18444-94-5) and its oxidation products zizanal (see formula II; CAS: 82509-29-3), khusimol (see formula III; CAS: 16223-63-5) and khusimone (see formula IV; CAS: 30557-76-7) for the properties of vetiver oil (Ouyang Angew. Chem. Int. Ed. [Angewandte Chemie International Edition] 2021, 60, 5666–5672; Sell - A Fragrant Introduction to Terpenoid Chemistry [A Fragrant Introduction to Terpenoid Chemistry]; Panten - Flavors and Fragrances [Flavors and Fragrances], 4. Natural Raw Materials [Natural Raw Materials]).

[0004]

[0005] For biotechnologically produced alternatives to vetiver oil, enzymes for producing the key sensory components, in particular zizaene, will be required. However, the only zizaene synthase known to date is the zizaene synthase from vetiver (VzZIS protein AJL25242.1, see WO 2010 / 134004 A1 from Firmenich SA).

[0006] The zizaene synthase from vetiver not only produces zizaene from farnesyl pyrophosphate, but also produces specific minor products in specific ratios, and the minor products and their oxidation products also affect the sensory properties of vetiver oil.

[0007] However, for use in fine fragrances, a zizaene synthase can be expected that produces different ratios of minor products or even different minor products - to give the composition improved sensory properties. Detailed description:

[0008] Accordingly, the fundamental problem of the present invention is to identify alternative zizaene synthases that are suitable for recombinantly producing zizaene and related products, and preferably, producing a composition containing zizaene that has different sensory characteristics compared to the composition obtained with the known zizaene synthase from vetiver.

[0009] This problem is surprisingly solved by a method for producing a composition containing zizaene, which method comprises the following successive steps:

[0010] i) providing farnesyl pyrophosphate and at least one polypeptide exhibiting zizaene synthase activity, under conditions suitable for the polypeptide to convert at least a portion of the farnesyl pyrophosphate into zizaene;

[0011] ii) converting at least a portion of the farnesyl pyrophosphate into bisabolyl cation with the polypeptide;

[0012] iii) producing a composition containing zizaene by converting the bisabolyl cation into zizaene and optionally at least one additional compound with the polypeptide; and

[0013] iv) optionally, removing any remaining farnesyl pyrophosphate and / or the at least one polypeptide exhibiting zizaene synthase activity from the composition containing zizaene to a large extent,

[0014] wherein the at least one polypeptide comprises or consists of the following: 1) an amino acid sequence selected from the group consisting of:

[0015] a.1) The amino acid sequence as shown in SEQ ID NO:1;

[0016] b.1) An amino acid sequence that is at least 55%, preferably at least 60%, more preferably at least 65%, at least 70%, at least 75%, at least 80% or at least 85%, even more preferably at least 90%, at least 91%, at least 92%, at least 93% or at least 94%, and most preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence as shown in SEQ ID NO:1;

[0017] c.1) An amino acid sequence encoded by a nucleic acid sequence as shown in any one of SEQ ID NO:2, 3 or 5;

[0018] d.1) An amino acid sequence encoded by a nucleic acid sequence that is at least 55%, preferably at least 60%, more preferably at least 65%, at least 70%, at least 75%, at least 80% or at least 85%, even more preferably at least 90%, at least 91%, at least 92%, at least 93% or at least 94%, and most preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequence as shown in any one of SEQ ID NO:2, 3 or 5; and

[0019] e.1) An amino acid sequence that is a fragment of any of the sequences listed in a.1) to d.1), and the fragment exhibits zizaene synthase activity;

[0020] Or 2) An amino acid sequence selected from the group consisting of:

[0021] a.2) The amino acid sequence as shown in any one of SEQ ID NO:7 to 28, preferably 7 to 24 or 28;

[0022] b.2) An amino acid sequence that is at least 55%, preferably at least 60%, more preferably at least 65%, at least 70%, at least 75%, at least 80% or at least 85%, even more preferably at least 90%, at least 91%, at least 92%, at least 93% or at least 94%, and most preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence as shown in any one of SEQ ID NO:7 to 28, preferably 7 to 24 or 28; and

[0023] c.2) An amino acid sequence that is a fragment of any of the sequences listed in a.2) and b.2), and the fragment exhibits zizaene synthase activity.

[0024] Definition:

[0025] As used herein, the term "zizaene-containing composition" is defined as zizaene formed by the conversion of bisabolyl cations or preferably a mixture of zizaene and one or more other terpenes and / or terpenoid compounds (see formula VII), where the zizaene or the mixture can be dissolved or dispersed in the respective culture medium and / or mixed with other components. Preferably, the zizaene-containing composition comprises zizaene and at least one additional compound selected from the group consisting of: α-cadinene, unknown terpene X, β-cadinene, unknown terpene Y, precocene, cis-muurola-4(15),5-diene, β-acoradiene, α-neocallitropsene, and acoranol.

[0026] As used herein, the term "polypeptide" is defined as a continuous sequence of amino acids that are interconnected with each other by peptide bonds. The polypeptides according to the invention typically comprise at least 50, at least 100, or at least 200 amino acid residues in length such that the amino acid chain can form the three-dimensional structure required to exert zizaene synthase activity. The term "protein" is used interchangeably herein.

[0027] The formulations "polypeptide(s) exhibiting zizaene synthase activity" and "at least one polypeptide(s) exhibiting zizaene synthase activity" are used synonymously herein.

[0028] As used herein, the term "zizaene synthase activity" is defined as follows:

[0029] A polypeptide exhibiting such activity has the ability to convert at least a portion of the provided farnesyl pyrophosphate into zizaene (see formula I above), and preferably belongs to the enzyme class (EC) 4.2.3.X, such as EC 4.2.3.37 or EC 4.2.3.B17.

[0030] Preferably, the corresponding polypeptide converts farnesyl pyrophosphate into at least one, preferably at least two additional compounds (sesquiterpenes or sesquiterpenoid compounds other than zizaene) in at least one side reaction, and these additional compounds are selected from the group consisting of: α-cadinene, unknown terpene X, β-cadinene, unknown terpene Y, precocene, cis-muurola-4(15),5-diene, β-acoradiene, α-neocallitropsene, and acoranol, preferably consisting of α-cadinene, β-cadinene, unknown terpene Y, and β-acoradiene, more preferably consisting of α-cadinene and β-acoradiene.

[0031] In the case of zizaene, the additional compound is formed by conversion of the bisabolyl cation (see formula VII), where the bisabolyl cation is formed by isomerization of the nerolidol cation (see formula VI), and the nerolidol cation is formed by isomerization of the farnesyl cation (see formula V), which is formed by cleavage of the pyrophosphate of the polypeptide reaction center.

[0032]

[0033] As used herein, the term "sequence identity" or "sequence identical to" defines the relationship between amino acid sequences or nucleic acid sequences and can be determined by comparing these sequences. Generally, sequence identity is determined by comparing two sequences over the entire length of the sequences, but it is also possible to compare only a part of the sequences aligned with each other. Preferably, sequence identity is compared over the entire length of the sequences herein. Sequence identity refers to the degree of relatedness between polypeptide sequences or nucleic acid sequences. It will be expressed as the percentage of identical amino acids or nucleotides in the two sequences compared to each other. Thus, when aligning two sequences, the number of matching amino acids or nucleotides between these sequences is usually determined and related to the total number of amino acids or nucleotides in the aligned sequences or sequence parts. For example, when compared to a parent sequence (i.e., an amino acid sequence as shown in any of SEQ ID NO:1, or a nucleic acid sequence as shown in SEQ ID NO:2, 3 or 5), a variant sequence can be defined by its sequence identity. To determine the percentage of identity between two sequences in a first step, a pairwise sequence alignment is generated between these two sequences, where the two sequences are aligned over their complete, entire or full length (i.e., pairwise global alignment). The alignment is generated with the programs or software described herein. The preferred alignment for the purposes of the present invention is the alignment from which the highest sequence identity can be determined.

[0034] Sequence alignments can be generated with many software tools, such as the Needleman and Wunsch algorithm - Needleman, Saul B. and 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. For example, this algorithm is implemented in the “NEEDLE” program, which performs a global alignment of two sequences. The NEEDLE program is included in, for example, 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).

[0035] BLOSUM (BLOcks Substitution Matrix) - typically generated based on alignments of conserved regions (e.g., protein domains) (Henikoff S, Henikoff JG: “Amino acid substitution matrices from protein blocks”, Proceedings of the National Academy of Sciences of the USA; November 15, 1992; 89(22):10915-9). One of many BLOSUMs is “BLOSUM62”, which is typically the “default” setting in many programs when aligning protein sequences.

[0036] "BLAST" (Basic Local Alignment Search Tool) consists of several individual programs (BlastP, BlastN), which are mainly used to search for similar sequences in large sequence databases. The BLAST program also creates local alignments. Typically, the BLAST interface provided by NCBI (National Centre for Biotechnology Information) is used, which is an improved version ("BLAST2"). "Original" BLAST: Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and 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.

[0037] As used herein, "sequence identity" is preferably a value determined by the EMBOSS pairwise alignment algorithm "Needle". In particular, the NEEDLE program in the EMBOSS package (version 2.8.0 or later, EMBOSS: The European Molecular Biology Open Software Suite – Rice, P., et al., Trends in Genetics (2000) 16:276-277; http: / / emboss.bioinformatics.nl) can be used to calculate the "longest-identity" using the NOBRIEF option (with NO's 'Simplified identity and similarity'). Identity between two aligned sequences is calculated in the following situation: the number of corresponding positions in the alignment that show the same amino acids in the two sequences is divided by the total length of the alignment minus 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 the alignment of nucleic acid sequences, the default parameters are: matrix = DNAfull; open gap penalty = 10.0; gap extension penalty = 0.5.

[0038] As used herein, the term "zizaene" is defined as the compound shown in Formula I above (CAS: 18444-94-5) and all of its stereoisomeric forms, in particular its enantiomeric forms, and all possible mixtures of the compound and its stereoisomeric forms. Preferably, however, "zizaene" should be understood to mean only the compound shown in Formula I.

[0039] As used herein, the term "wild rice aldehyde" is defined as the compound shown in Formula II above (CAS: 82509-29-3) and all of its stereoisomeric forms, in particular its enantiomeric forms, and all possible mixtures of the compound and its stereoisomeric forms. Preferably, however, "wild rice aldehyde" should be understood to mean only the compound shown in Formula II.

[0040] As used herein, the term "humulene alcohol" is defined as the compound shown in Formula III above (CAS: 16223-63-5) and all of its stereoisomeric forms, in particular its enantiomeric forms, and all possible mixtures of the compound and its stereoisomeric forms. Preferably, however, "wild rice aldehyde" should be understood to mean only the compound shown in Formula III.

[0041] As used herein, the term "kusenone" is defined as the compound shown in Formula IV above (CAS: 30557-76-7) and all of its stereoisomeric forms, in particular its enantiomeric forms, as well as all possible mixtures of said compound and its stereoisomeric forms. Preferably, however, "kusenone" should be understood to mean only the compound shown in Formula IV.

[0042] As used herein, the term "unknown terpene X" is defined as a compound or mixture of compounds that exhibits Figure 4 the mass spectrum shown, preferably a terpene and / or terpene compound, wherein the peaks are at least at m / z = 91 (highest peak), 108, 117, 133, and 204, preferably at least at m / z = 41, 55, 65, 77, 91 (highest peak), 105, 108, 117, 133, 145, 189, and 204, wherein "unknown" should be understood to mean that the chemical structure has not been determined.

[0043] As used herein, the term "unknown terpene Y" is defined as a compound or mixture of compounds that exhibits a mass spectrum as shown in Figure 5 wherein the peaks are at least at m / z = 91, 105, 133 (highest peak), 134, and 204, preferably at least at m / z = 41, 55, 63, 70, 77, 83, 91, 105, 117, 133 (highest peak), 134, 141, 157, and 204, wherein "unknown" should be understood to mean that the chemical structure has not been determined.

[0044] As used herein, "% of total" means the area percentage of a given compound in the corresponding gas chromatogram (GC) obtained by subjecting a zizaene-containing composition to GC-FID (gas chromatography using a flame ionization detector), and preferably corresponds to mol% in the zizaene-containing composition.

[0045] As used herein, "CrZIS" means zizaene synthase from Capsella rubella according to the present invention, while "VzZIS" refers to the known zizaene synthase from Vetiveria zizanioides.

[0046] ***

[0047] Preferably, the polypeptide exhibiting zizaene synthase activity is or is based on a polypeptide from the taxonomic families Camelineae or Brassicaceae, more preferably from the genus Capsella (in particular Capsella rubella), the genus Arabidopsis (in particular Arabidopsis thaliana or Arabidopsis lyrata), the genus Brassica (in particular Brassica campestris, Brassica rapa, Brassica napus, Brassica oleracea or Brassica cretica), the genus Eutrema (in particular Eutrema salsugineum), the genus Noccaea (in particular Noccaea caerulescens) or the genus Raphanus (in particular Raphanus sativus), with the genera Capsella, Arabidopsis and Brassica being particularly preferred.

[0048] The variant amino acid or nucleic acid sequences of the sequence of SEQ ID NO:1 or the sequences of SEQ ID NOs:2, 3 and 5 can be naturally occurring variants, such as allelic variants or orthologous, paralogous or homologous variants, respectively. Alternatively, such sequences can be artificially generated, for example by biotechnologies known to those skilled in the art (such as, for example, molecular evolution or rational design) or by using mutagenesis techniques known in the art or described elsewhere herein (random mutagenesis, site-directed mutagenesis, directed evolution, gene recombination, etc.) to attempt to improve the properties of the enzyme or nucleic acid (for example, improving the expression of the enzyme or increasing the enzyme activity of the enzyme). Typically, a variant of the polypeptide having zizaene synthase activity according to the invention is a polypeptide having one or several amino acid substitutions compared to the amino acid sequence of SEQ ID NO:1 (preferably an artificial amino acid sequence).

[0049] The variant nucleic acid sequence may differ from the sequences of SEQ ID NOs:2, 3 and 5 due to at least one nucleotide substitution, addition and / or deletion. It should be understood that the polynucleotide comprising such a variant nucleic acid sequence as mentioned herein is preferably capable of hybridizing with the nucleic acid sequences as shown in SEQ ID NOs:2, 3 and 5 under stringent hybridization conditions.

[0050] The 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 at 50 °C to 65 °C in 0.2x SSC, 0.1% SDS. Those skilled in the art know that these hybridization conditions vary according to the type of nucleic acid and, for example, the temperature and concentration of the buffer when organic solvents are present. For example, under "standard hybridization conditions", in an aqueous buffer at a concentration of 0.1 to 5x SSC (pH 7.2), the temperature varies according to the type of nucleic acid and is between 42 °C and 58 °C. If an organic solvent, such as 50% formamide, is present in the above buffer, the temperature under standard conditions is about 42 °C. The hybridization conditions for DNA:DNA hybrids are preferably 0.1x SSC and between 20 °C and 45 °C, preferably between 30 °C and 45 °C. The above hybridization temperatures are determined, for example, for nucleic acids of about 100 bp (= base pairs) in length and a G+C content of 50% in the absence of formamide.

[0051] Those skilled in the art know how to determine the required hybridization conditions by referring to the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989; Hames and Higgins (eds.) 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, variant nucleic acid sequences can be derived that are capable of hybridizing with the nucleic acid sequences shown in SEQ ID NO: 2, 3, or 5 under stringent hybridization conditions.

[0052] Typically, the amino acid sequence of a fragment of any of the sequences listed in a) to d) of the method of the present invention consists 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 from the above sequences or sequence variants to exhibit zizaene synthase activity.

[0053] Preferably, the amino acid sequence listed in a) to e) of the methods of the present invention or a fragment thereof comprises at least one, preferably at least two, more preferably three Pfam domains. More preferably, the Pfam domain is selected from the group consisting of: terpene synthase N-terminal domain (PF01397.23), terpene synthase family metal binding domain (PF03936.18) and terpene synthase family 2 C-terminal metal binding (PF19086.2). The Pfam domains mentioned in this article will be analyzed using PFAM (version 35.0). For detailed information about PFAM, see: Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, G. A. Salazar, E. L. Sonnhammer, S. C. E. Satto, L. Paladin, S. Raj, L. J. Richardson, R. D. Finn, A. Bateman Nucleic Acids Research (2020) doi:10.1093 / nar / gkaa913, http: / / pfam.xfam.org / or https: / / doi.org / 10.1093 / nar / gkaa913.

[0054] Preferably, the amino acid sequence of the present invention or a fragment thereof comprises Figure 7 At least 30, at least 50, at least 65 or all conserved residues are shown in white letters on a black background (each letter represents a specific amino acid). Figure 7 The sequences are indicated by consecutive white letters (each letter represents a specific amino acid) on a black background. Preferably, the conserved segments consist of at least 2, at least 3 or at least 4 amino acids. Particularly preferred conserved segments are those corresponding to the following amino acid positions: SEQ ID NO: 1: 119 to 123, 141 to 144, 159 to 161, 218 to 221, 223 to 226, 238 to 240, 253 to 255 and 440 to 442. The amino acid sequence or fragment thereof preferably contains at least 4, more preferably at least 6 and most preferably all of these particularly preferred conserved segments.

[0055] More preferably, the amino acid sequence of the present invention or a fragment thereof comprises Figure 6 At least 50, at least 70, at least 90, at least 105 or all conserved residues are shown in white letters on a black background (each letter represents a specific amino acid). Figure 6indicated by a sequence of contiguous white letters (each letter representing a specific amino acid) on a black background. Preferably, the conserved segment consists of at least 3, at least 5 or at least 7 amino acids. Particularly preferred conserved segments are those corresponding to the following amino acid positions: SEQ ID NO:1: 117 to 121, 139 to 143, 214 to 219, 242 to 246, 272 to 276, 286 to 293, 328 to 334, 336 to 340, 343 to 349, 371 to 380 and 414 to 418. The amino acid sequence or a fragment thereof preferably contains at least 5, more preferably at least 8 and most preferably all of these particularly preferred conserved segments.

[0056] Preferably, the amino acid sequence or a fragment thereof exhibits the SDVFX1X2F motif (see Figure 6 amino acid positions 129 to 135 of SEQ ID NO:1 in

[0057] where S = serine, D = aspartic acid, V = valine, F = phenylalanine, X1 = any amino acid, preferably asparagine (N), lysine (K) or aspartic acid (D), and X2 = any amino acid, preferably arginine (R), isoleucine (I) or lysine (K). Surprisingly, this motif is also present in the known zizaene synthase (VzZIS) from Vetiveria zizanioides - although the latter has a very low overall sequence identity compared to SEQ ID NO:1.

[0058] The method of the present invention also relates to the conversion of farnesyl pyrophosphate to zizaene by an enzyme comprising a first segment containing a tag peptide and a second segment containing a polypeptide exhibiting zizaene synthase activity. The enzyme comprising the first segment and the second segment may be referred to herein as a "tagged enzyme".

[0059] The tag peptide is preferably selected from the group consisting of nitrogen utilization protein (NusA), thioredoxin (Trx), maltose binding protein (MBP), glutathione S-transferase (GST), small ubiquitin-like modifier (SUMO), or calcium binding protein (Fh8) and functional homologs thereof. As used herein, a functional homolog of a tag peptide is a tag peptide that has at least about the same effect on the solubility of a tagged enzyme as compared to the untagged enzyme. Typically, homologs differ in that one or more amino acids have been inserted, substituted, deleted, or extended in the peptide of which it is a homolog. Homologs can particularly 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. Homologs can particularly have a sequence identity of at least 40%, more particularly at least 50%, preferably at least 55%, more preferably at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the sequence of NusA, Trx, MBP, GST, SUMO, or Fh8.

[0060] Particularly suitable is maltose binding protein from Escherichia coli or a functional homolog thereof. The use of the tagged enzyme according to the invention is particularly advantageous because it can contribute to an increase in the production of terpenoids or terpenes (such as zizaene), in particular an increase in the cellular production of terpenoids or terpenes.

[0061] To improve the solubility of the tagged enzyme (compared to the enzyme without a tag), the first segment of the enzyme preferably binds to the N-terminus of the second segment at its C-terminus. Alternatively, the first segment of the tagged enzyme binds to the C-terminus of the second segment at its N-terminus.

[0062] In addition, the invention relates to a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a first segment containing a tag peptide (preferably an MBP, NusA, Trx, GST, SUMO, or Fh8 tag or a functional homolog of any of these), and a second segment exhibiting zizaene synthase activity.

[0063] In addition, the invention relates to a host cell comprising the nucleic acid encoding the tagged zizaene synthase. The host cell can particularly include a gene containing any one of these sequences or a functional homolog thereof.

[0064] The method according to the invention may consist of steps i) to iii) or may include one or more additional steps. Such additional steps may be pre-treatment steps or steps required to obtain a zizaene-containing composition, such as purification steps, in which, for example, the untransformed portion of farnesyl pyrophosphate and / or at least one polypeptide exhibiting zizaene synthase activity may be removed from the zizaene-containing composition. The method may also include one or more additional steps in which the zizaene and / or at least one additional compound provided in step iii) is further transformed (e.g., oxidized).

[0065] The method may be carried out in vitro, for example in one or more reaction vials. Alternatively, the method may be carried out, in whole or in part, in the non-human host cells or transgenic organisms mentioned below.

[0066] The above-mentioned transformation step may be carried out in vitro, i.e., in one or more suitable reaction vials containing all the components required for the transformation. Those skilled in the art are well aware of how to adjust the reaction conditions in order to carry out the reaction effectively. For example, a suitable buffer may be used to provide the components in an environment with a suitable pH and a suitable salt concentration. A suitable temperature may also be applied in such cases, which will not be elaborated on.

[0067] Alternatively, the transformation step may be carried out in a host cell, as described below. It should be understood that the host cell should be capable of producing zizaene. If necessary, the host cell needs to be genetically modified to express a polypeptide exhibiting zizaene synthase activity. The culture conditions and time of the host cell should be sufficient to allow the expression of the polypeptide and the conversion of farnesyl pyrophosphate to zizaene. Particularly preferred conditions are also described in the accompanying examples below or are known to those skilled in the art.

[0068] However, the transformation step of the method according to the invention may also be carried out in an organism (typically a multicellular organism, such as the transgenic non-human organisms mentioned below). Typically, the organism is genetically modified to express the polypeptide required to convert farnesyl pyrophosphate to zizaene. However, depending on the choice of a given non-human transgenic organism, those skilled in the art are well aware of which conditions need to be applied.

[0069] If the method according to the invention is carried out in vivo (i.e., in a host cell or a non-human transgenic organism), it should be understood that the host cell or non-human transgenic organism should express a polypeptide exhibiting zizaene synthase activity such that the conversion of farnesyl pyrophosphate to zizaene can be carried out in the host cell or non-human transgenic organism.

[0070] In the method of the present invention, the polypeptide exhibiting zizaene synthase activity is preferably encoded by a heterologous polynucleotide, which is preferably contained in a vector or a gene construct.

[0071] In this context, the term "heterologous polynucleotide" means that the polynucleotide encoding the polypeptide exhibiting zizaene synthase activity is not naturally present in the host cell or organism into which it is introduced. Thus, the heterologous polynucleotide is derived from a first species or is a polynucleotide that has been artificially modified, while the host cell or non-human transgenic organism is from a second species different from the first species. The heterologous polynucleotide can be contained in a vector or a gene construct as specified below. Alternatively, the heterologous polynucleotide can be introduced into the genome of the host cell or non-human transgenic organism such that, upon integration into the genome, the polypeptide exhibiting zizaene synthase activity encoded by the heterologous polynucleotide will be expressed. Typically, the heterologous polynucleotide should be integrated into the genome of the host cell or non-human transgenic organism at a locus that permits expression of the heterologous polynucleotide (e.g., near an endogenous promoter).

[0072] The term "vector" preferably encompasses phages, plasmids, cosmids, viral vectors, and artificial chromosomes, such as bacterial or yeast artificial chromosomes (YACs). Vectors encompassing the polynucleotides of the present invention preferably further contain a selectable marker for propagation and / or selection in the host. The vector can be incorporated into the host cell by a variety of techniques well known in the art. If introduced into the host cell, the vector may be present in the cytoplasm or may be incorporated into the genome. In the latter case, it should be understood that the vector may further contain nucleic acid sequences that permit homologous recombination or heterologous insertion.

[0073] Vectors can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. The terms "transformation" and "transfection", conjugation and transduction as used in the context of the present invention are intended to encompass a variety of existing methods for introducing foreign nucleic acids (such as DNA) into a host cell, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, f-mating, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for transforming or transfecting host cells (including plant cells) can be found in: Sambrook et al. (cited above) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Volume 44, Agrobacterium protocols, edited by Gartland and Davey, Humana Press, Totowa, NJ. Alternatively, plasmid vectors can be introduced by heat shock or electroporation techniques. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line before being applied to the host cell.

[0074] Preferably, the vectors mentioned herein are suitable as cloning vectors, i.e., they can be replicated in a microbial system. Such vectors ensure efficient cloning in bacteria and preferably yeast or fungi and enable stable transformation of plants. In particular, those vectors that must be mentioned are the various binary and co-integrate vector systems suitable for T-DNA-mediated transformation. Such vector systems generally have the following characteristics: they contain at least the vir genes required for Agrobacterium-mediated transformation, as well as sequences that define the T-DNA (T-DNA borders). These vector systems preferably also contain additional cis-regulatory regions, such as promoters and terminators and / or selectable markers, using which suitable transformed host cells or organisms can be identified. While co-integrate vector systems arrange the vir genes and T-DNA sequences on the same vector, binary systems are based on at least two vectors, one of which carries the vir genes but not the T-DNA, while the second carries the T-DNA but not the vir genes.

[0075] Thus, the last-mentioned vectors are relatively small, easy to manipulate, and can replicate in both Escherichia coli and Agrobacterium species. These binary vectors include vectors from the pBIB-HYG, pPZP, pBecks, pGreen series. Preferably, Bin19, pBI101, pBinAR, pGPTV, and pCAMBIA are used according to the invention. An overview of binary vectors and their use can be found in Hellens et al., Trends in Plant Science (2000) 5, 446–451. Additionally, by using appropriate cloning vectors, polynucleotides can be introduced into host cells or organisms (such as plants or animals) and thus used for the transformation of plants such as those disclosed and cited in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapters 6 / 7, pp. 71-119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants, in: Transgenic Plants, Volume 1, Engineering and Utilization, edited by Kung and R.W. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Volume 1, Engineering and Utilization, edited by Kung and R.W. Wu, Academic Press, (1993), 128-143; Potrykus 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 205 225.

[0076] More preferably, the vector of the present invention is an expression vector. In such an expression vector, that is, a vector containing the polynucleotide of the present invention, the nucleic acid sequence is operably linked to an expression control sequence (also referred to as an "expression cassette"), allowing expression in prokaryotic or eukaryotic cells or their isolated components. Suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen) or pSPORT1 (GIBCO BRL). Additional examples of typical fusion expression vectors are pGEX (Pharmacia Biotech Inc; Smith 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, Massachusetts) and pRIT5 (Pharmacia, Piscataway, New Jersey), in which glutathione S-transferase (GST), maltose E binding protein and protein A are fused to the recombinant target protein, respectively.

[0077] Examples of suitable inducible non-fusion E. coli expression vectors are in particular pTrc (Amann 1988, Gene 69:301-315) and pET 11d (Studier 1990, Methods in Enzymology 185, 60-89). Expression of the target gene from the pTrc vector is based on transcription from a hybrid trp-lac fusion promoter by the host RNA polymerase. Expression of the target gene from the pET 11d vector is based on transcription of a T7-gn10-lac fusion promoter, which is mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is provided by the host strains BL21(DE3) or HMS174(DE3), from a resident λ prophage carrying the T7 gn1 gene under the transcriptional control of the lacUV 5 promoter. Persons skilled in the art are familiar with other vectors suitable for prokaryotic organisms; these vectors are, for example in E. coli, pLG338, pACYC184, pACYCDuet-1 (Novagen, Merck, Germany), the pBR series (e.g. pBR322), the pUC series (e.g. pUC18 or pUC19), the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, lambdagt11 or pBdCl; in Streptomyces, plJ101, plJ364, plJ702 or plJ361; in Bacillus, pUB110, pC194 or pBD214; in Corynebacterium, pSA77 or pAJ667.

[0078] Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYep Sec1 (Baldari 1987, Embo J. [Journal of the European Molecular Biology Organization] 6:229-234), pMFa (Kurjan 1982, Cell [Cell] 30:933-943), pJRY88 (Schultz 1987, Gene [Gene] 54:113-123) and pYES2 (Invitrogen, San Diego, California). Vectors and procedures for constructing vectors suitable for other fungi, such as filamentous fungi, include those detailed in Applied Molecular Genetics of fungi [Applied Molecular Genetics of Fungi], J.F. Peberdy et al., editors, pages 1-28, Cambridge University Press [Cambridge University Press]: Cambridge [Cambridge] or in More Gene Manipulations in Fungi [More Gene Manipulations in Fungi] (J.W. Bennett & L.L. Lasure, editors, pages 396-428: Academic Press [Academic Press]: San Diego) by van den Hondel, C.A.M.J.J., and Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi" [Gene Transfer Systems and Vector Development for Filamentous Fungi]. Additional suitable yeast vectors are, for example, pAG-1, YEp6, YEp13 or pEMBLYe23. Alternatively, the polynucleotides of the present invention can also be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors that can be used to express proteins in cultured insect cells, such as Sf9 cells, include the pAc series (Smith 1983, Mol. Cell Biol [Molecular and Cellular Biology]. 3:2156-2165) and the pVL series (Lucklow 1989, Virology [Virology] 170:31-39).

[0079] However, the vector can be an integrating vector. An integrating vector refers to a linear or circular DNA molecule that can integrate into the genome of, for example, a microorganism (such as the genome of a bacterium) and provide stable inheritance of a gene encoding a polypeptide of interest (such as the zizaene synthase of the present invention). Integrating vectors typically contain one or more segments that contain a gene sequence encoding the polypeptide of interest, which is under the control (i.e., operably linked) of additional nucleic acid segments that provide for its transcription.

[0080] Such additional segments can include promoter and terminator sequences, and one or more segments that typically drive the incorporation of a gene of interest into the genome of a target cell by a homologous recombination process. Typically, the integration vector will be a vector that can be transferred into a target cell, but has a replicon that is non-functional in that organism. If the segment containing the gene of interest contains an appropriate marker, integration of that segment can be selected for. One or more nucleic acid sequences encoding an appropriate signal peptide that is not naturally associated with the polypeptide to be expressed in the host cell of the present invention can be integrated into the (expression) vector.

[0081] For example, the DNA sequence of a signal peptide leader sequence can be fused in-frame to the nucleic acid of the present invention such that the zizaene synthase of the present invention is initially translated as a fusion protein containing the signal peptide. Depending on the nature of the signal peptide, the expressed polypeptide will be targeted differently. For example, a secretory signal peptide that functions in the intended host cell enhances the extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptide to certain organelles such as chloroplasts, mitochondria, and peroxisomes. The signal peptide can be cleaved from the polypeptide either during transport to the intended organelle or from the cell. Fusions providing additional peptide sequences at the amino or carboxy terminus of the polypeptide are possible.

[0082] As used herein, the term "gene construct" refers to a polynucleotide that contains the polynucleotide of the present invention and additional functional nucleic acid sequences. The gene construct according to the present invention is preferably a linear DNA molecule. Typically, the gene construct according to the present invention can be a targeting construct that allows the targeting construct to integrate randomly or site-specifically into genomic DNA. Such a targeting construct preferably contains DNA of sufficient length for homologous or heterologous recombination, as detailed below. In both cases, the construct must preferably be perfect, having structures that control gene expression, such as a promoter, a transcription start site, a polyadenylation site, and a transcription termination site.

[0083] Preferably, the method of the present invention includes the step of obtaining the composition containing zizaene. As used herein, the term "obtaining" means providing a composition containing zizaene in any purity. Thus, the method of the present invention may encompass one or more purification steps. The purification techniques to be applied depend on how the method of the present invention is implemented. For example, if the method is implemented in vitro, i.e., in a reaction vial using isolated components (such as isolated enzymes), adducts, and auxiliary components (such as reaction buffers), it should be understood that less purification is required. However, if the method is implemented in vivo, i.e., in a non-human host cell or transgenic organism as defined herein, further purification and pretreatment steps may be required. Typically, the host cells need to be harvested and the harvested cells lysed to release the composition containing zizaene from the cells. Subsequent purification steps should remove cell debris and aim to purify the composition containing zizaene from the remaining components.

[0084] Furthermore, if these steps are implemented in an animal or a plant, even further pretreatment and / or purification steps may be required. Depending on the given circumstances in which the method can be implemented, those skilled in the art are well aware of the appropriate pretreatment and / or purification steps. The purification techniques envisaged may be extraction techniques, chromatography (such as LC, GC, or HPLC), size exclusion chromatography, affinity chromatography, distillation, centrifugation, filtration, etc. The pretreatment steps envisaged may be harvesting, heat treatment, sonication, treatment with chemicals and / or enzymes, etc. Particularly preferred measures are described in the following accompanying examples.

[0085] The method of the present invention is preferably implemented in non-human host cells.

[0086] As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell capable of converting farnesyl pyrophosphate into zizaene, wherein the conversion is carried out by a polypeptide exhibiting zizaene synthase activity. Thus, the host cells of the present invention are capable of expressing a polypeptide exhibiting zizaene synthase activity. Preferably, the polypeptide exhibiting zizaene synthase activity may be encoded by a heterologous polynucleotide, vector, or gene construct of the present invention. The host cells are typically transformed with the heterologous polynucleotide, vector, or gene construct such that the polypeptide exhibiting zizaene synthase activity as specified above can be expressed. The transformed vector or gene construct may be maintained as a non-integrating vector, such as a plasmid, or alternatively, may be integrated into the host cell genome, as specified in more detail herein.

[0087] In one aspect of the present invention, the host cells of the present invention are transgenic cells that are transgenic for the nucleic acid encoding the zizaene synthase of the present invention, preferably transgenic non-plant cells, such as transgenic microbial cells.

[0088] Host cells according to the present invention can be produced based on standard genetic and molecular biological techniques well known in the art, such as those described in the following: Sambrook, J., and Russell, D. W. “Molecular Cloning: A Laboratory Manual” 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (2001); and F. M. Ausubel et al. eds., “Current protocols in molecular biology”, John Wiley and Sons, Inc., New York (1987), and its later supplements.

[0089] Preferably, the host cell is a non - human host cell selected from the group consisting of: bacterial cells (such as cyanobacterial cells), fungal cells (such as yeast cells), plant cells (such as algal cells), and non - human animal cells (such as non - human mammalian cells). More preferably, the host cell can be selected from any one of the following organisms:

[0090] Bacteria: Bacterial host cells can be selected, for example, from the group consisting of: Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, Lactococcus, Ensifer, or Pantoea.

[0091] Gram-positive: Bacillus, Streptomyces: Useful Gram-positive bacterial host cells include, but are not limited to, Bacillus cells, e.g., Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus Jautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. The most preferred prokaryotes are Bacillus cells, preferably Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis or Bacillus lentus cells.

[0092] Some other preferred bacteria include strains of the order Actinomycetales, preferably Streptomyces, preferably Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382), Streptomyces lividans, or Streptomyces murinus, or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, Streptococcus lactis. Further preferred bacteria include strains belonging to the genus Myxococcus, e.g., Myxococcus virescens.

[0093] Gram-negative: species of Escherichia, Pseudomonas, Rhodobacter, Paracoccus, Ensifer or Pantoea: Preferred Gram-negative bacteria are Escherichia coli, species of Pseudomonas, preferably Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11) or Pseudomonas denitrificans, Rhodobacter capsulatus or Rhodobacter sphaeroides, Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens, Pantoea ananatis, or Sinorhizobium meliloti (also known as Ensifer meliloti).

[0094] Fungi:Aspergillus, Fusarium, Trichoderma: The host cell can be a fungal cell. As used herein, "fungus" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as the Oomycota and Deuteromycotina, and all mitosporic fungi. Representative groups of Ascomycota include, for example, Neurospora, Eupenicillium (= Penicillium), Emericella (= Aspergillus), Eurotium (= Aspergillus), and true yeasts listed below. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, for example, Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, for example, Saprolegniomycetous aquatic fungi (water molds), such as Achlya. Examples of mitosporic 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 Deuteromycotina, Hyphomycetes, such as the genera Fusarium, Humicola, Trichoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, especially Fusarium oxysporum (DSM2672), Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113), Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli or Dreschlera halodes. Other preferred fungi include strains belonging to Basidiomycotina, Basidiomycetes, such as the genera Coprinus, Phanerochaete, Coriolus or Trametes, especially Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g., NA-12) or Trametes (formerly called Polyporus), such as Trametes versicolor (e.g., PR4 28-A). Further preferred fungi include strains belonging to Zygomycotina, Mycorycetes, such as the genera Rhizopus or Mucor, especially Mucor hiemalis.

[0096] Yeasts: Pichia, Saccharomyces: The fungal host cell can be a yeast cell. As used herein, yeast includes ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the Fungilmperfecti (Blastomycetes). Ascosporogenous yeasts are divided into the Spermophthoraceae and the Saccharomycetaceae. The latter consists of four subfamilies: the Schizosaccharomycoideae (e.g., Schizosaccharomyces), the Nadsonioideae, the Lipomycoideae, and the Saccharomycoideae (e.g., Kluyveromyces, Pichia, and Saccharomyces). Basidiosporogenous yeasts include Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungilmperfecti are divided into two families: the Sporobolomycetaceae (e.g., Sporobolomyces and Bullera) and the Cryptococcaceae (e.g., Candida).

[0097] In addition to fungi, the non-human eukaryotic host cells further include, but are not limited to, non-human animal cells, particularly non-human mammalian cells, avian cells, reptilian cells, or insect cells, or plant cells.

[0098] Most preferably, the host cell is a bacterial host cell, particularly Rhodobacter, preferably Rhodobacter sphaeroides host cell, or Escherichia, preferably Escherichia coli host cell, and particularly preferably Rhodobacter, preferably Rhodobacter sphaeroides host cell.

[0099] Alternatively, but also preferably, the method of the present invention is carried out in a non-human transgenic organism.

[0100] As used herein, the term "genetically modified non-human organism" or "non-human genetically modified organism" refers to an organism that has been genetically modified to contain a polynucleotide, vector or gene construct of the present invention. The genetic modification may be the result of any type of homologous or heterologous recombination event, mutagenesis or gene editing process. Thus, a genetically modified non-human organism differs from its non-genetically modified counterpart in that it contains a polynucleotide, vector or gene construct that is not naturally occurring (i.e., heterologous) in its genome. The non-human organism contemplated as a genetically modified non-human organism according to the present invention is preferably a multicellular organism. In addition, the non-human organism is preferably an animal or a plant. Preferred animals are mammals, especially laboratory animals such as rodents, e.g., mice, rats, rabbits, etc., or farm animals such as sheep, goats, cows, horses, etc. Preferred plants are crop plants or vegetables, especially selected from the group consisting of: Arabidopsis species, Nicotiana spp., Cichorium intybus, Lactuca sativa, Mentha spp., Artemisia annua, tuber-forming plants, oil crops (such as Brassica species or Brassica napus), fruit-bearing flowering plants (angiosperms) and trees.

[0101] Methods for generating genetically modified 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 [Transgenic Plants: Gene constructs, vector and transformation method]. 2018. DOI. 10.5772 / intechopen.79369; Pinkert, C.A. (ed.) 1994. Transgenic animal technology: A laboratory handbook. [Transgenic animal technology: A laboratory handbook] Academic Press, Inc. [Academic Press Inc.], San Diego, California; Monastersky G.M. and Robl, J.M. (eds.) (1995) Strategies in Transgenic Animal Science. [Strategies in Transgenic Animal Science] ASM Press. [ASM Press] Washington, D.C.; Sambrook, supra, Ausubel, supra).

[0102] Preferably, the non-human genetically modified organism is a microorganism, a plant or a non-human animal to be sacrificed, preferably an invertebrate. Thus, according to the latter, methods of treating animals are not covered by the methods of the present invention.

[0103] The present invention also relates to a composition containing zizaene obtainable by the method of the present invention as described above.

[0104] In addition to zizaene, the composition containing zizaene preferably comprises at least one, preferably at least two, further compounds selected from the group consisting of: α - cedrene, unknown terpene X, β - cedrene, unknown terpene Y, pre - polyene, cis - mollerene - 4(15),5 - diene, β - acoradiene, α - neocopacene, and acoranol, preferably consisting of α - cedrene, β - cedrene, unknown terpene Y, and β - acoradiene, more preferably consisting of α - cedrene and β - acoradiene.

[0105] Preferably, in the composition containing zizaene, the content of zizaene is at least 15 mol%, preferably at least 25 mol%, the content of α - cedrene is at least 10 mol%, preferably at least 15 mol%, and the content of β - acoradiene is at least 10 mol%, preferably at least 15 mol%.

[0106] In addition to zizaene, the composition containing zizaene preferably comprises at least one further compound selected from the group consisting of: unknown terpene X, unknown terpene Y, cis - mollerene - 4(15),5 - diene, α - neocopacene, and acoranol, preferably unknown terpene Y.

[0107] Preferably, in the composition containing zizaene, the total content of β - caryophyllene and acora - 3,9 - diene is 0.5 mol% or less, preferably 0.3 mol% or less, more preferably 0.1 mol% or less.

[0108] The present invention also relates to a method for producing at least one terpene and / or terpene compound, the method comprising the following subsequent steps:

[0109] i) Providing farnesyl pyrophosphate and at least one polypeptide under conditions suitable for the polypeptide to convert at least a portion of the farnesyl pyrophosphate into the bisabolyl cation (see formula VII above);

[0110] ii) Converting at least a portion of the farnesyl pyrophosphate into the bisabolyl cation with the polypeptide;

[0111] iii) Converting the bisabolyl cation into at least one terpene and / or terpene compound with the polypeptide; and

[0112] iv) Optionally purifying one or more of the at least one terpene and / or terpene compound from the mixture,

[0113] wherein the at least one polypeptide is capable of converting at least a portion of farnesyl pyrophosphate to bisabolyl cation and comprises an amino acid sequence selected from the group consisting of or consisting of:

[0114] a) the amino acid sequence as set forth in SEQ ID NO:1;

[0115] b) an amino acid sequence that is at least 55% identical to the amino acid sequence as set forth in SEQ ID NO:1;

[0116] c) an amino acid sequence encoded by a nucleic acid sequence as set forth in any one of SEQ ID NO:2, 3 or 5;

[0117] d) an amino acid sequence encoded by a nucleic acid sequence that is at least 55% identical to the nucleic acid sequence as set forth in any one of SEQ ID NO:2, 3 or 5; and

[0118] e) an amino acid sequence that is a fragment of any of the sequences listed in a) to d), said fragment being capable of converting at least a portion of farnesyl pyrophosphate to bisabolyl cation.

[0119] And the step ii) of generating bisabolyl cation from farnesyl pyrophosphate preferably includes the formation of an intermediate of farnesyl cation (see formula V above) and / or the formation of an intermediate of nerolidol cation (see formula VI above), preferably the formation of both intermediates.

[0120] The method may further comprise an additional step v): converting at least one terpene produced in steps i) to iv) to a terpenoid, preferably by an oxidation reaction. Non-limiting examples of such terpenoids that can be produced by the method are zizaene aldehyde (see formula II above), selinaol (see formula III above) and selinone (see formula IV above).

[0121] Furthermore, the present invention relates to the use of a polypeptide exhibiting zizaene synthase activity as described above, a non-human host cell or transgenic organism as described above, or a heterologous polynucleotide, vector or gene construct as described above for producing a composition containing zizaene, preferably a composition containing zizaene as described above. The composition containing zizaene obtainable by the method of the present invention can be used in perfume or fragrance applications, for cosmetic uses, for pharmaceuticals, as an insect repellent or insect attractant, for agriculture (e.g., for crop protection or animal feeding), or for antimicrobial applications.

[0122] Furthermore, it is possible to provide a kit for producing a composition containing zizaene, the kit comprising the following components: i) a polypeptide exhibiting zizaene synthase activity as described above, ii) a non-human host cell or transgenic organism as described above and / or iii) a heterologous polynucleotide, vector or gene construct as described above.

[0123] As used herein, the term "kit" refers to a collection of components required to carry out the method of the present invention for producing a composition containing zizaene. The kit shall include any of the foregoing components i) to iii) as a single component or any combination thereof. Typically, the components of the kit are provided in separate containers or within a single container. The container also typically contains instructions for carrying out the method of the present invention for producing a composition containing zizaene. In addition, the kit may preferably contain 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 for purifying the composition containing zizaene.

[0124] Furthermore, the present invention relates to a method for producing a composition comprising at least one, preferably at least two, more preferably all of the oxidation products of zizaene selected from the group consisting of: zizaenal (see formula II), selina-4-en-7-ol (see formula III) and selin-4-en-7-one (see formula IV), wherein:

[0125] a) a composition containing zizaene is provided by carrying out the method of the present invention as described above; and

[0126] b) the composition containing zizaene provided in step a) is enzymatically and / or chemically oxidized, preferably enzymatically oxidized, such that at least a portion of the contained zizaene is converted to zizaenal, selina-4-en-7-ol and / or selin-4-en-7-one.

[0127] Preferably, the method may further include the step of obtaining the composition comprising at least one oxidation product of zizaene, i.e., the corresponding pretreatment and / or purification step.

[0128] In step b) of the method, the oxidation of the zizaene contained therein is preferably carried out enzymatically. Suitable enzymes capable of oxidizing zizaene and thus converting it into zizanal, selina-4-en-1-ol and / or selinone are well known in the art. Preferably, cytochrome P450 monooxygenase (CYP) or laccase is used to oxidize zizaene into zizanal, selina-4-en-1-ol and / or selinone, in particular those described in international patent application WO 2013 / 064411 A1 (Firmenich SA) or patent US 6,200,786 B1 (Givaudan S.A.), respectively. However, the oxidation can also be carried out chemically.

[0129] Step b) of the method can also be carried out in vivo or in vitro, typically depending on how step a) is carried out. Thus, if step a) is carried out in vivo, for example in a non-human host cell or a transgenic organism as specified herein, it is preferably contemplated that step b) is also carried out in vivo, and preferably in the same host cell or transgenic organism. Typically, the non-human host cell or transgenic organism should also be able to oxidize zizaene into zizanal, selina-4-en-1-ol and / or selinone. Thus, preferably, the host cell or transgenic organism can express the CYP as specified above. For this purpose, a heterologous polynucleotide encoding the CYP or a vector or gene construct containing such a polynucleotide can be present in the non-human host cell or transgenic organism. How such a heterologous polynucleotide, vector or gene construct can be introduced into the host cell or non-human transgenic organism is well known in the art and is described in detail elsewhere herein.

[0130] Alternatively, step b) of the method for producing a composition containing at least one oxidation product of zizaene is carried out by a chemical method rather than an enzymatic method. Preferably, the chemical oxidation of zizaene is carried out as disclosed, for example, in A. Denicourt-Nowicki et al., “Catalytic Oxidation Processes for the Upgrading of Terpenes”, Catalysts 2019, 9(11), 893.

[0131] Preferably, in the method, in addition to zizaene, the zizaene-containing composition provided in step a) further contains at least one, preferably at least two additional compounds selected from the group consisting of: α-cedrene, unknown terpene X, β-cedrene, unknown terpene Y, pre-polyene, cis-muurola-4(15),5-diene, β-acoradiene, α-neoclovene, and acoranol, preferably consisting of α-cedrene, β-cedrene, unknown terpene Y, and β-acoradiene, more preferably consisting of α-cedrene and β-acoradiene, and at least one, preferably at least two of the compounds are oxidized in step b).

[0132] The method may further include one or more purification steps after step b), such as distillation to remove other compounds, and if necessary, the ratio of these compounds can be changed by distillation.

[0133] The present invention relates to an oxidized composition, which contains at least one, preferably at least two, more preferably all oxidation products of zizaene selected from the group consisting of: wild rice aldehyde, selinaol, and selinone, and the oxidized composition can also be obtained by the method.

[0134] The present invention also relates to a non-human host cell as described above, which expresses a polypeptide exhibiting zizaene synthase activity as described above from a heterologous polynucleotide, vector, or gene construct as described above, wherein the non-human host cell is transgenic for the polynucleotide encoding the polypeptide. Preferably, the non-human host cell produces a composition containing zizaene or a composition containing at least one, preferably at least two, more preferably all oxidation products of zizaene selected from the group consisting of: wild rice aldehyde, selinaol, and selinone.

[0135] In addition, the present invention relates to a non-human transgenic organism as described above, which expresses a polypeptide exhibiting zizaene synthase activity as described above from a heterologous polynucleotide, vector, or gene construct as described above, wherein the non-human transgenic organism is transgenic for the polynucleotide encoding the polypeptide. Preferably, the non-human transgenic organism produces a composition containing zizaene or a composition containing at least one, preferably at least two, more preferably all oxidation products of zizaene selected from the group consisting of: wild rice aldehyde, selinaol, and selinone.

[0136] The non-human host cell, non-human transgenic organism, and method of the present invention may include the polypeptide exhibiting zizaene synthase activity of the present invention, as well as one or more additional polypeptides exhibiting zizaene synthase activity, including the known zizaene synthase (VzZIS) protein from vetiver.

[0137] Finally, but not least, the present invention relates to a polypeptide that exhibits zizaene synthase activity and comprises an amino acid sequence that is less than 100%, but at least 85%, preferably at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% identical to the amino acid sequence shown in SEQ ID NO:1 or a fragment thereof that exhibits zizaene synthase activity. Preferably, the polypeptide comprises the conserved regions and / or the SDVFX1X2F motif as defined above in the description of the method of the present invention.

[0138] Additional preferred features and embodiments of the polypeptide of the present invention are also described in the description of the method of the present invention above.

[0139] All references cited throughout this specification are incorporated herein by reference in their entirety or for the specific disclosures mentioned. Description of the Drawings:

[0140] Figure 1 : Schematic diagram of the vector p-mev-SPppa-MBP-CrZIS for cloning and expressing CrZIS (= zizaene synthase from Capsella bursa-pastoris) in Rhodobacter sphaeroides; mob = mobilization sequence, kan = kanamycin resistance gene, mvaA = 3-hydroxy-3-methylglutaryl-CoA reductase, idi = isopentenyl-diphosphate delta-isomerase, hcs = 3-hydroxy-3-methylglutaryl-CoA synthase, mvk = mevalonate kinase, pmk = phosphomevalonate kinase, mvd = mevalonate diphosphate decarboxylase, MBP = maltose binding protein, rep = origin of replication

[0141] Figure 2 : Schematic diagram of the vector p-mev-SPppa-MBP-VzZIS for cloning and expressing VzZIS (= zizaene synthase from Vetiveria zizanioides) in Rhodobacter sphaeroides; abbreviations see Figure 1

[0142] Figure 3 : Gas chromatography (GC) of the zizaene-containing composition obtained with Rhodobacter sphaeroides expressing CrZIS or VzVIS respectively

[0143] Figure 4 : Mass spectrometry (MS) of the unknown terpene X obtained with Rhodobacter sphaeroides expressing CrZIS – GC retention time (RT) = 16.54 min

[0144] Figure 5 : MS of the unknown terpene Y obtained with Rhodobacter sphaeroides expressing CrZIS – RT = 17.16 min

[0145] Figure 6 : Amino acid alignment of CrZIS with SEQ ID NO:1 and proteins from different species. The alignment was performed using MUSCLE (Multiple Sequence Comparison by Log-Expectation) with standard parameters (numbering taken from SEQ ID NO:1).

[0146] Figure 7 : Identical to Figure 6 except that three additional proteins with lower sequence identity compared to SEQ ID NO:1 were aligned.

[0147] ***

[0148] The following sequences are referred to throughout the specification and the accompanying sequence listing:

[0149] SEQ ID NO:1: Capsella rubella zizaene synthase (CrZIS) protein

[0150] SEQ ID NO:2: Nucleotide sequence encoding the CrZIS protein in Capsella rubella

[0151] SEQ ID NO:3: Synthetic coding sequence for expressing CrZIS in Escherichia coli

[0152] SEQ ID NO:4: Vetiver zizaene synthase (VzZIS) protein

[0153] SEQ ID NO:5: Synthetic coding sequence for expressing CrZIS in Rhodobacter sphaeroides

[0154] SEQ ID NO:6: Synthetic coding sequence for expressing VzZIS in Rhodobacter sphaeroides

[0155] SEQ ID NO:7: Synthetic amino acid sequence that is 95% identical to variant number 1 of SEQ ID NO:1

[0156] SEQ ID NO:8: Synthetic amino acid sequence that is 95% identical to variant number 2 of SEQ ID NO:1

[0157] SEQ ID NO:9: Synthetic amino acid sequence that is 90% identical to variant number 1 of SEQ ID NO:1

[0158] SEQ ID NO:10: Synthetic amino acid sequence that is 90% identical to variant number 2 of SEQ ID NO:1

[0159] SEQ ID NO:11: A synthetic amino acid sequence that is 85% identical to Variant No. 1 of SEQ ID NO:1

[0160] SEQ ID NO:12: A synthetic amino acid sequence that is 85% identical to Variant No. 2 of SEQ ID NO:1 SEQ IDNO:13: Uncharacterized protein of R0G872 Capsella bursa-pastoris

[0161] (83% identical to SEQ ID NO:1)

[0162] SEQ ID NO:14: Uncharacterized protein (fragment) of R0F0Z3 Capsella bursa-pastoris

[0163] (83% identical to SEQ ID NO:1)

[0164] SEQ ID NO:15: Q4KSH9 α - amyrin synthase Arabidopsis thaliana

[0165] (82% identical to SEQ ID NO:1)

[0166] SEQ ID NO:16: Uncharacterized protein of A0A178UD70 Arabidopsis thaliana

[0167] (82% identical to SEQ ID NO:1)

[0168] SEQ ID NO:17: D7MLJ7 terpene synthase / cyclase family protein Arabidopsis lyrata

[0169] Lyrata subsp. (81% identical to SEQ ID NO:1)

[0170] SEQ ID NO:18: Uncharacterized protein of A0A5S9YBB7 Arabidopsis thaliana

[0171] (81% identical to SEQ ID NO:1)

[0172] SEQ ID NO:19: A synthetic amino acid sequence that is 80% identical to Variant No. 1 of SEQ ID NO:1

[0173] SEQ ID NO:20: A synthetic amino acid sequence that is 80% identical to Variant No. 2 of SEQ ID NO:1

[0174] SEQ ID NO:21: A synthetic amino acid sequence that is 75% identical to SEQ ID NO:1

[0175] SEQ ID NO:22: Uncharacterized protein of M4CD84 Brassica rapa subsp. pekinensis

[0176] (70% identical to SEQ ID NO:1)

[0177] SEQ ID NO:23: Uncharacterized protein Thellungiella salsuginea

[0178] (70% identical to SEQ ID NO:1)

[0179] SEQ ID NO:24: Uncharacterized protein Brassica rapa

[0180] (70% identical to SEQ ID NO:1)

[0181] SEQ ID NO:25: A0A1J3JN53 α - amorphene synthase Thlaspi caerulescens

[0182] (65% identical to SEQ ID NO:1)

[0183] SEQ ID NO:26: Uncharacterized protein Arabidopsis thaliana

[0184] (55% identical to SEQ ID NO:1)

[0185] SEQ ID NO:27: Q9T0K1 (Z) - γ - bisabolene synthase 2 Arabidopsis thaliana

[0186] (55% identical to SEQ ID NO:1)

[0187] SEQ ID NO:28: Capsella bursa - pastoris zizaene synthase (CbpZIS) protein

[0188] (95% identical to SEQ ID NO:1)

[0189] SEQ ID NO:29: Synthetic coding sequence example for expressing CbpZIS in Rhodobacter sphaeroides

[0190] The examples are for illustrative purposes only. In no event should they be construed as limiting the scope. Example 1: Identification of zizaene synthase from Capsella rubella

[0191] An amino acid sequence (see SEQ ID NO:1) was extracted from the publicly available Capsella rubella genome, which is shown in the UniProt database under the accession number R0GUH0 (https: / / www.uniprot.org / uniprotkb / R0GUH0 / entry).

[0192] Capsella bursa-pastoris genome sequencing citation: Slotte, T., Hazzouri, K., J. et al., “The Capsella rubella genome and the genomic consequences of rapid mating system evolution,” Nature Genetics 45, 831–835 (2013), https: / / doi.org / 10.1038 / ng.2669.

[0193] In the BLASTP analysis (nr database), the best search hits (83% identity) were two sequences each annotated as uncharacterized proteins from Capsella tatarinowii. Four other sequences were within 80% identity, and these sequences were found in Arabidopsis. A MUSCLE alignment of SEQ ID NO: 1 and related sequences is shown in Figure 6 and Figure 7 middle.

[0194] The sequence identity with the known zizaene synthase protein from Vetiver (GenBank accession number AJQ30127) is only 29%. Shepherd's purse is a plant that is not related to Vetiver, and it is unknown whether it contains zizaene or zizaene-related metabolites.

[0195] The amino acid sequence is encoded by a nucleic acid sequence annotated as α-bapasite synthase in Capsella tataricus (RefSeq No. XP_006281945.1; see SEQ ID NO: 2) based on homology to the bapasite synthase from Arabidopsis thaliana (GenBank Accession No. AY876386).

[0196] Example 2: Cloning for expression in E. coli

[0197] To test whether the above amino acid sequence SEQ ID NO:1 has zizaene synthase activity, it was expressed in Escherichia coli. For this purpose, a nucleic acid sequence encoding R0GUH0 for Escherichia coli expression and having the corresponding linker sequence (see SEQ ID NO:3) was synthesized using a standard sequence service provider (Genscript BiotechCorp., USA), and it was cloned into the expression vector pACYCDuet-1 (Novagen, Merck KGaA, Germany) using the BamHI and NotI restriction sites. The resulting plasmid was labeled pAC-CrZIS.

[0198] The plasmid was introduced into the Escherichia coli strain BL21 DE3 carrying pMEV, which has been described in Schmidt et al. 2017 (Scientific Reports | 7:862 | DOI: 10.1038 / s41598-017-00893-3). Transformants were selected on LB-agar plates + chloramphenicol (30 μg / ml) + kanamycin (30 μg / ml) + 1% glucose. Positive transformants (tested by miniprep and restriction digestion) were labeled Escherichia coli BL21-DE3-pMEV-pAC-CrZIS. Using the same method, a control strain carrying pMEV and pACYCDuet-1 was generated and labeled BL21-DE3-pMEV-pACYC-DUET-1.

[0199] Example 3: Production of zizaene in Escherichia coli

[0200] Escherichia coli BL21-DE3-pMEV-pAC-CrZIS and BL21-DE3-pMEV-pACYC-DUET-1 were each inoculated into 5 ml of LB liquid medium + chloramphenicol (30 μg / ml) + kanamycin (30 μg / ml) + 1% glucose and incubated overnight at 37 °C and 250 rpm. The next day, the cultures were diluted 1:25 into 10 ml of 2xYT medium + chloramphenicol (30 μg / ml) + kanamycin (30 μg / ml) and allowed to grow at 37 °C and 250 rpm until the A600 was 0.5. Subsequently, 1 mM IPTG was added as an inducer, 1 ml of n-dodecane was added to capture the product, and the cultures were further incubated at 28 °C and 250 rpm for 24 hours.

[0201] For GC-MS analysis, dodecane was separated from the culture by centrifugation and diluted 200-fold with ethyl acetate. 2 μL was analyzed by gas chromatography (GC) via GC-MS as detailed by Cankar et al. (FEBS Letters 588 (2014) 1001-1007).

[0202] Surprisingly, the BL21-DE3-pMEV-pAC-CrZIS strain produced zizaene when compared to BL21-DE3-pMEV-pACYC-DUET-1.

[0203] Example 4: Constructs for expression in Rhodobacter sphaeroides

[0204] Zizaene synthase from Lobularia maritima (see SEQ ID NO:1; hereinafter "CrZIS") and zizaene synthase from Vetiveria zizanioides (GenBank accession number AJQ30127; see SEQ ID NO:4; hereinafter "VzZIS") were expressed in Rhodobacter sphaeroides.

[0205] For this purpose, synthetic codon-optimized nucleic acid sequences for the expression of CrZIS or VzZIS in Rhodobacter sphaeroides, respectively (see SEQ ID NO:5 or SEQ ID NO:6, respectively), were ordered from a standard sequence provider (GenScript Biotech Corporation, USA).

[0206] Each sequence was cloned into plasmid SPppa-MBP using BamHI and HindIII restriction sites as described in WO2019 / 045568 A1 (Isobionics B.V.), and also included the mevalonate pathway as described in WO 2019 / 045568 A1. The resulting ligation mixture was transformed into Escherichia coli S17-1 cells. p-mev-SPppa-MBP-CrZIS (see Figure 1 ) or p-mev-SPppa-MBP-VzZIS (see Figure 2 ) was transferred from Escherichia coli S17-1 to Rhodobacter sphaeroides strain Rs265-9c by conjugation using standard procedures (see US 9,260,709B2, Isobionics B.V.).

[0207] Example 5: Production of zizaene in Rhodobacter sphaeroides

[0208] In a 100 ml shake flask without baffles, seed cultures of Rs265-9c / p-mev-SPppa-MBP-CrZIS or Rs265-9c / p-mev-SPppa-MBP-VzZIS were carried out with 20 ml of RS102 medium (containing 100 mg / L neomycin) and a loopful of glycerol stock solution, respectively. The seed culture flasks were incubated at 30 °C for 72 h in an orbital shaker incubator at 110 rpm with an orbit of 50 mm.

[0209] Flask generation experiments were carried out in 300 ml shake flasks with 2 bottom baffles. 20 ml of RS102 medium (As described in US 2020 / 0010822A1, Isobionix Biotechnology) and neomycin at a final concentration of 100 mg / L were added to the shake flasks together with 2 ml of sterile dodecane. The volume of the inoculum was adjusted to obtain a final OD600 value of 0.05 in 20 ml of medium. The shake flasks were maintained at 30 °C for 72 h in an orbital shaker incubator at 110 rpm with an orbit of 50 mm. The flask experiments were carried out in duplicate.

[0210] Next, for GC-MS analysis, dodecane was separated from the culture by centrifugation and diluted 10-fold with acetone.

[0211] Gas chromatography (GC) was performed on a Shimadzu GC2010Plus equipped with a Restek RTX-5Sil MS capillary column (30 m × 0.25 mm, 0.5 μm). The syringe and FID detector temperatures were set at 280 °C and 300 °C, respectively. The gas flow rate through the column was set at 40 ml / min. The initial oven temperature was 160 °C, which was increased to 180 °C at a rate of 2 °C / min and further increased to 300 °C at a rate of 50 °C / min and held at this temperature for 3 min. The injected sample volume was 1 μL, with a split ratio of 1:50 and a nitrogen makeup flow rate of 30 ml / min.

[0212] Compounds found in the dodecane layer from the cultures of Rs265-9c / p-mev-SPppa-MBP-CrZIS or Rs265-9c / p-mev-SPppa-MBP-VzZIS were identified based on their retention times (RT) and quantified by integrating their peak areas in the gas chromatography (see Figure 3 ). Peak area analysis revealed that both strains produced zizaene as the main product (see Table 1), as confirmed by mass spectrometry.

[0213] Each strain also produces by-products, with 4-α-cadinene, β-cadinene, pre-polyene, and β-acoradiene being produced by both, and several others being specific to one of the strains. In particular, Rs265-9c / p-mev-SPppa-MBP-CrZIS (see the "CrZIS" column in Table 1) produces a large amount of an unknown terpene Y (its retention index (RI) = 1434 (mass spectrum see Figure 5 )) and a small amount of another unknown terpene X (its RI = 1409 (mass spectrum see Figure 4 ))), while Rs265-9c / p-mev-SPppa-MBP-VzZIS (see the "VzZIS" column in Table 1) does not produce these.

[0214] The presence of these new, hitherto unknown terpenes - in particular, unknown terpene Y - in the zizaene-containing compositions obtained with CrZIS results in different sensory characteristics compared to the compositions obtained with VzZIS.

[0215] Example 6: Production of zizaene in Rhodobacter sphaeroides by zizaene synthase from Sison amomum

[0216] The amino acid sequence (see SEQ ID NO: 28) was retrieved from the Sison amomum genomic sequence (DOI: 10.1111 / tpj.13563) by TBLASTN search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Citation for Sison amomum genome sequencing: Kasianov et al., Plant J [Plant Journal] July 2017; 91(2): 278-291.

[0217] As determined in a BLASTP analysis (nr database), the sequence identity with zizaene synthase from Sison arvensis (see SEQ ID NO: 1) is 95%.

[0218] Zizaene synthase from Sison amomum (see SEQ ID NO: 28; hereinafter "CbpZIS") and zizaene synthase from Vetiveria zizanioides (GenBank accession number AJQ30127; see SEQ ID NO: 4; hereinafter "VzZIS") were expressed in Rhodobacter sphaeroides.

[0219] To this end, synthetic codon-optimized nucleic acid sequences for the expression of CbpZIS or VzZIS in Rhodobacter sphaeroides, respectively, were ordered from a standard sequence provider (GenScript Biotech Corporation, USA) (see SEQ ID NO:29 or SEQ ID NO:6, respectively).

[0220] Each sequence was cloned into the plasmid SPppa-MBP using the BamHI and HindIII restriction sites as described in WO2019 / 045568 A1 (Isobionix Biotechnology Corporation), which also includes the mevalonate pathway as described in WO2019 / 045568 A1. The resulting ligation mixture was transformed into Escherichia coli S17-1 cells. p-mev-SPppa-MBP-CbpZIS or p-mev-SPppa-MBP-VzZIS was transferred from Escherichia coli S17-1 to the Rhodobacter sphaeroides strain Rs265-9c by conjugation using standard procedures (see US 9,260,709 B2, Isobionix Biotechnology Corporation).

[0221] Seed cultures, corresponding shake flask production experiments, and GC-MS analyses of Rs265-9c / p-mev-SPppa-MBP-CbpZIS or Rs265-9c / p-mev-SPppa-MBP-VzZIS were performed as in Example 5 above.

[0222] Compounds found in the dodecane layer from cultures of Rs265-9c / p-mev-SPppa-MBP-CbpZIS or Rs265-9c / p-mev-SPppa-MBP-VzZIS were identified based on their retention times (RT), and quantified by integrating their peak areas in the gas chromatogram (data not shown). Peak area analysis revealed that both strains produced zizaene as the major product, as confirmed by mass spectrometry (data not shown).

[0223] Table 1: Analysis of zizaene-containing compositions by GC-MS

[0224]

[0225]

[0226] RT = retention time, (Lit.) RI = (literature) retention index, *) from NIST database compared to 7, **) area percentage in GC-FID.

Claims

1. A method for producing at least one terpene and / or terpenoid compound, characterized in that The method comprises the following subsequent steps: i) providing farnesyl pyrophosphate and at least one polypeptide, under conditions suitable for the polypeptide to convert at least a portion of the farnesyl pyrophosphate into the bisabolyl cation; ii) converting at least a portion of the farnesyl pyrophosphate into the bisabolyl cation with the polypeptide; iii) converting the bisabolyl cation into at least one terpene and / or terpenoid compound with the polypeptide; and iv) optionally purifying one or more of the at least one terpene and / or terpenoid compound from the mixture, wherein the at least one polypeptide is capable of converting at least a portion of farnesyl pyrophosphate into the bisabolyl cation and comprises an amino acid sequence selected from the group consisting of or consisting of: a) the amino acid sequence as shown in SEQ ID NO:1; b) an amino acid sequence that is at least 55% identical to the amino acid sequence as shown in SEQ ID NO:1; c) an amino acid sequence encoded by a nucleic acid sequence as shown in any one of SEQ ID NO:2, 3 or 5; d) an amino acid sequence encoded by a nucleic acid sequence that is at least 55% identical to the nucleic acid sequence as shown in any one of SEQ ID NO:2, 3 or 5; and e) an amino acid sequence that is a fragment of any of the sequences listed in a) to d), said fragment being capable of converting at least a portion of farnesyl pyrophosphate into the bisabolyl cation.

2. A method for producing a composition containing zizaene, characterized in that The method comprises the following subsequent steps: i) providing farnesyl pyrophosphate and at least one polypeptide exhibiting zizaene synthase activity, under conditions suitable for the polypeptide to convert at least a portion of the farnesyl pyrophosphate into zizaene; ii) converting at least a portion of the farnesyl pyrophosphate into the bisabolyl cation with the polypeptide; and iii) producing a zizaene-containing composition by converting the bisabolyl cation into zizaene and optionally at least one additional compound with the polypeptide, wherein the at least one polypeptide comprises an amino acid sequence selected from the group consisting of: a) the amino acid sequence as shown in SEQ ID NO:1; b) an amino acid sequence that is at least 55% identical to the amino acid sequence as shown in SEQ ID NO:1; c) an amino acid sequence encoded by a nucleic acid sequence as shown in any one of SEQ ID NO:2, 3 or 5; d) an amino acid sequence encoded by a nucleic acid sequence that is at least 55% identical to the nucleic acid sequence as shown in any one of SEQ ID NO:2, 3 or 5; and e) an amino acid sequence that is a fragment of any of the sequences listed in a) to d), said fragment exhibiting zizaene synthase activity.

3. The method according to claim 1 or 2, characterized in that The zizaene-containing composition or the at least one terpene and / or terpenoid compound produced in step iii) respectively comprises at least one, preferably at least two additional compounds selected from the group consisting of: α-cadinene, unknown terpene X, β-cadinene, unknown terpene Y, prepolylene, cis-muurola-4(15),5-diene, β-acoradiene, α-neocopadiene and acoranol, preferably consisting of α-cadinene, β-cadinene, unknown terpene Y and β-acoradiene, more preferably consisting of α-cadinene and β-acoradiene.

4. The method according to any one of claims 1 to 3, characterized in that In the zizaene-containing composition or the at least one terpene and / or terpenoid compound produced in step iii) respectively, the content of zizaene is at least 15 mol%, preferably at least 25 mol%, the content of α-cadinene is at least 10 mol%, preferably at least 15 mol%, and the content of β-acoradiene is at least 10 mol%, preferably at least 15 mol%.

5. The method according to any one of the preceding claims, characterized in that The zizaene-containing composition or the at least one terpene and / or terpenoid compound produced in step iii) respectively comprises at least one additional compound selected from the group consisting of: unknown terpene X, unknown terpene Y, cis-muurola-4(15),5-diene, α-neocopadiene and acoranol, preferably unknown terpene Y.

6. The method according to any one of the preceding claims, characterized in that In the zizaene-containing composition or the at least one terpene and / or terpenoid compound produced in step iii) respectively, the total content of β-cubebene and acora-3,9-diene is 0.5 mol% or lower, preferably 0.3 mol% or lower, more preferably 0.1 mol% or lower.

7. The method according to any one of the preceding claims, characterized in that The method is implemented in non-human host cells, which are preferably selected from the group consisting of: bacterial cells such as cyanobacterial cells, particularly Escherichia coli or Rhodobacter sphaeroides cells, fungal cells such as yeast cells, plant cells such as algal cells, and non-human animal cells such as non-human mammalian cells, or non-human transgenic organisms, preferably non-vertebrate animals, plants or microorganisms to be sacrificed.

8. The method according to any one of the preceding claims, wherein The polypeptide is encoded by a heterologous polynucleotide, which is preferably contained in a vector or gene construct.

9. A zizaene-containing composition obtainable by the method according to any one of claims 2 to 8, preferably further characterized as described in any one of claims 3 to 6.

10. Use of the polypeptide characterized in claim 2, the host cell or transgenic organism characterized in claim 7 or the heterologous polynucleotide, vector or gene construct characterized in claim 8 for producing a zizaene-containing composition.

11. A method for producing a composition comprising at least one, preferably at least two, more preferably all oxidation products of zizaene selected from the group consisting of: zizanal, germacrenol and germacrone, characterized in that: a) Providing a zizaene-containing composition by carrying out the method according to any one of claims 2 to 8; and, b) The zizaene-containing composition provided in step a) is enzymatically and / or chemically oxidized, preferably enzymatically oxidized, such that at least a part of the contained zizaene is converted into zizanial, selina-4(15),5-dien-8-ol and / or selinone.

12. The method according to claim 11, wherein In addition to zizaene, the zizaene-containing composition provided in step a) further contains at least one, preferably at least two, other compounds selected from the group consisting of: α-cadinene, unknown terpene X, β-cadinene, unknown terpene Y, prepolypene, cis-muurola-4(15),5-diene, β-acoradiene, α-neocopadiene and acoranol, preferably consisting of α-cadinene, β-cadinene, unknown terpene Y and β-acoradiene, more preferably consisting of α-cadinene and β-acoradiene, and at least one, preferably at least two, of said compounds are oxidized in step b).

13. An oxidized composition obtainable by the method according to claim 11 or 12.

14. A non-human host cell expressing a polypeptide exhibiting zizaene synthase activity as characterized in claim 2 from a heterologous polynucleotide, vector or gene construct as characterized in claim 8, wherein the non-human host cell is transgenic for the polynucleotide encoding said polypeptide.

15. A polypeptide exhibiting zizaene synthase activity and comprising an amino acid sequence that is less than 100% but at least 85% identical to the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof exhibiting zizaene synthase activity.

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