Preparation of terpenoid compounds

Abstract

The present invention provides a method for the preparation of a compound of the formula (A), (B), (C) and / or (D). Said compound may be used as a perfumery, flavor or aroma ingredient, and / or as a precursor thereof. Included in this invention is an in vivo process for the preparation of said compound in recombinant cells. The present invention also provides recombinant cells which may be used in said method. Novel compounds are also part of the invention.

Description

[0001] PREPARATION OF TERPENOID COMPOUNDS

[0002] Technical field

[0003] The present invention relates to the field of biosynthesis. More specifically, it concerns a method for preparing a compound of the formula (A), (B), (C) and / or (D) starting from compound of the formula (I). The compounds of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’4), (C’5), (D’1 b), (A’1-1) and (A’1-2) are also part of the invention. Said compounds may be used as a perfumery, flavor or aroma ingredient and / or a precursor thereof.

[0004] Background

[0005] In the perfumery, flavor or aroma industry, there is a constant need to provide new compounds and / or new methods for the preparation thereof. Key amongst such compounds are terpene compounds which are naturally found in most organisms (microorganisms, animals and plants). These compounds are made up of five carbon units called isoprene units and are classified by the number of these units present in their structure. Thus monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms respectively. Sesquiterpenes, for example, are widely found in the plant kingdom. Many sesquiterpene molecules are known for their flavor and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Numerous sesquiterpene hydrocarbons and sesquiterpenoids have been identified. Diterpenes are formed from four isoprene units, 20 carbons, and are secondary metabolites identified in plants and fungi. Although most diterpenes are non-volatile and some are present in essential oils.

[0006] Biosynthetic production of terpene compounds usually involves enzymes called terpene synthases. These enzymes convert an acyclic terpene precursor in one or more terpene products. This cyclization process is a key step in the biosynthetic pathways that lead to the formation of a vast array of terpenoid compounds. Terpene cyclases operate through different mechanisms to achieve the cyclization of their substrates. Based on these mechanisms, they are broadly classified into two main classes: Type I (Class I) and Type II (Class II).

[0007] The Type I terpene cyclase enzymes catalyze cyclization through the formation of carbocation intermediates. The reaction is initiated by the release of the diphosphate group from the substrate, generating a highly reactive carbocation that undergoes a series of intramolecular cyclizations and rearrangements to form the final product.

[0008] The Type II terpene cyclase enzymes employ a protonation-initiated mechanism. A conserved acidic residue in the enzyme protonates a double bond in the substrate, leading to the formation of a carbocation intermediate. This intermediate then undergoes cyclization and rearrangement to yield the final cyclic product.

[0009] Despite the improvements that have been made in this field, there nevertheless remains a need for new methods of producing terpene compounds which are building blocks for the preparation of highly valuable perfumery, flavor or aroma ingredients.

[0010] This problem is addressed by the present invention which provides a novel method comprising an enzymatic step for preparing a compound of the formula (A), (B), (C) and / or (D) starting from compound of the formula (I).

[0011] Summary The present invention is based on the surprising finding of the inventors that meroterpenoid cyclases and squalene cyclases can be used in the cyclisation of linear terpenoid compounds to produce a compound of the formula (A), (B), (C) and / or (D). Accordingly, the invention provides a new method for the preparation of a compound of the formula (A), (B), (C) and / or (D). This aspect of the invention and the compound of the formula (A), (B), (C) and / or (D) enable new routes toward the preparation of perfumery, flavor or aroma compounds.

[0012] A first aspect of the invention provides a method for preparing a compound of the formula (A), (B), (C) and / or (D), wherein

[0013] R° represents either H or a C1-4 alkyl group, preferably ethyl or H; and wherein each R2represents independently from each other either H or an alcohol protecting group, particularly , preferably H,

[0014] R3represents H or a C1-4 alkyl group, preferably CH3 ; and n independently from each other represents 1 or 2; wherein any dotted line represents the bond by which the substituent is bound to the rest of the molecule; and any wavy line represents independently from each other a carbon-carbon bond which when linked to the carbon-carbon double bond is either in the Z and / or in the E-configuration, preferably in the E-configuration, wherein the method comprises:

[0015] (a) contacting a compound of the formula (I) with a terpene cyclase enzyme under conditions suitable for the terpene cyclase enzyme to produce the compound of the formula (A), (B), (C) and / or (D).

[0016] An embodiment of the invention is wherein the compound of the formula (A) is a compound of the formula (A’), the compound of the formula (B) is a compound of the formula (B’), the compound of the formula (C) is a compound of the formula (C’) and / or the compound of the formula (D) is a compound of the formula (D’)

[0017]

[0018] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’); preferably, of the formula (A’1); preferably, of the formula (A’1 a) or (A'1 b), more preferably of the formula (A’1-1 a) or (A'1 -1 b)

[0019]

[0020] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’); preferably, of the formula (A’3); preferably, of the formula (A’3a); more preferably, of the formula (A’3-1a) or (A'3-2a)

[0021] (A’3-2a).

[0022] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’); preferably, of the formula (A’5); preferably, of the formula (A’5a); more preferably, of the formula (A’5-1a) or (A'5-2a)

[0023]

[0024] (A’5-2a).

[0025] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (B’); preferably, of the formula (B’3); more preferably, of the formula (B’3a)

[0026] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (B’); preferably, of the formula (B’5); more preferably, of the formula (B’5a)

[0027]

[0028] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (C’); preferably, of the formula (C’3); more preferably, of the formula (C’3a)

[0029] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (C’); preferably, of the formula (C’5); more preferably, of the formula (C’5a)

[0030]

[0031] (C’5a).

[0032] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (D’); preferably, of the formula (D’1); preferably, of the formula (D’1 a); more preferably, of the formula (D’1 ab)

[0033] An embodiment of the invention is wherein the compound the formula (I) is compound of the formula (lb) and the compound of the formula (A) is a compound of the formula (A’1 a), the compound of the formula (B) is a compound ofthe formula (B’1), the compound of the formula (C) is a compound of the formula (C’1) and / or the compound of the formula (D) is a compound of the formula (D’1 a)

[0034] An embodiment of the invention is wherein the compound the formula (I) is compound of the formula (li), and the compound of the formula (A) is a compound of the formula (A’3a), the compound of the formula (B) is a compound of the formula (B’3a), the compound of the formula (C) is a compound of the formula (C’3a) and / or the compound of the formula (D) is a compound of the formula (D’1 a)

[0035] (H),

[0036] An embodiment of the invention is wherein the compound the formula (I) is compound of the formula (If), and the compound of the formula (A) is a compound of the formula (A’5a), the compound of the formula (B) is a compound of the formula (B’5a) and / or the compound of the formula (C) is a compound of the formula (C’5a)

[0037] An embodiment of the invention is wherein the terpene cyclase enzyme is a meroterpenoid cyclase enzyme and / or a squalene cyclase enzyme.

[0038] An embodiment of the invention is wherein the terpene cyclase enzyme is a bacterial membrane- integrated MeroTPS, a fungal membrane-integrated MeroTPS and / or a bacterial soluble MeroTPS. More in particular, the polypeptide having MeroTPS enzyme activity is a bacterial membrane-integrated MeroTPS.

[0039] An embodiment of the invention is wherein the method futher comprises one or more steps prior to step (a), said step(s) comprising:

[0040] (i) preparing compound of the formula (la) (namely, beta-farnesene) from farnesyl diphosphate (FPP) using a beta-farnesene synthase; preferably, an E-beta-farnesene synthase (EC 4.2.3.47); and / or

[0041] (ii) preparing FPP from isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) using one or more enzymes having prenyltransferase activity; in particular, a FPP synthase; preferably, an EE-FPP synthase (EC 2.5.1.10).

[0042] A further embodiment of the invention is wherein the method is an in vivo or a bioconversion process.

[0043] A further embodiment of the invention is wherein said method is performed in a recombinant cell capable of functionally expressing the terpene cyclase enzyme as defined herein above.

[0044] A futher aspect of the invention provides a compound selected from the group consisting of the compound of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’4), (C’5), (D’1 b), (A’1-1) and (A’1-2).

[0045] A further aspect of the invention provides a recombinant cell comprising, capable of producing or producing a compound of the formula (A), (B), (C) and / or (D); preferably, a compound of the formula (A’), (B’), (C’) and / or (D’); more preferably, a compound of the formula (A’1), (A’2), (A’3), (A’4), (A’5), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’3), (C’4), (C’5), (D’1a) and / or (D’1 b); more preferably, a compound of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’4), (C’5), (D’1 b), (A’1-1) and (A’1- 2).

[0046] A further aspect of the invention provides the use of the compound of the invention as a perfumery, flavor or aroma ingredient, or as a precursor thereof.

[0047] A further aspect of the invention provides the use of a terpene cyclase enzyme, preferably a meroterpenoid cyclase enzyme, to produce a compound of the invention.

[0048] of the

[0049] Figure 1 : Representative Electron impact (El) mass spectrum (MS) from compound of the formula (A’1-1 a) (A), (A’1-2a) (B), (B’1) (C), (C’1) (D) and (D’1 a) (E) produced by bioconversion of (E)-p-Farnesene (lb).

[0050] Figure 2: GC-MS chromatogram from the in vivo cyclisation of (E)-p-Farnesene (lb) to compound of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a) and (D’1 a) by the bacterial membrane-integrated meroterpenoid cyclase WP_318017018.1 (SEQ ID NO: 73) in E. coli DP1205.

[0051] Figure 3: Overlay of the GC-MS chromatograms from the in vivo cyclisation of (E)-p-Farnesene (lb) to compound of the formula (B’1), (C’1), (A’1-1 a) and (D’1 a) by the bacterial membrane-integrated meroterpenoid cyclases WP_229232892.1 (Seq ID NO: 55) (A), WP_033281172.1 (Seq ID NO: 29) (B) and RLD81128.1 (Seq ID NO: 20) (C) in E.coli DP1205.

[0052] Figure 4: (A) GC-MS chromatogram from bioconversion cyclisation of (E)-p-Farnesene (lb) to compound of the formula (B’1), (C’1), (A’1-1 a), (A’1-1 b), (A’1-2a) and (D’1 a) by the bacterial membrane-integrated meroterpenoid cyclase WSW93534.1 (SEQ ID NO: 74) expressed in E. coli C43(DE3) in the presence of ethanol as co-solvent. (B) Representative Electron impact (El) mass spectrum (MS) from compound of the formula (A’1-1 b).

[0053] Figure 5: Overlay of the GC-MS chromatograms from the in vivo cyclisation of (E)-p-Farnesene (lb) to compound of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a) and (D’1 a) by the bacterial membrane-integrated meroterpenoid cyclases WP_033281172.1 (Seq ID NO: 29) (A) and mutant variant WP_033281172.1 S9M (Seq ID NO: 80) (B) in E.coli DP1205.

[0054] Figure 6: GC-MS chromatogram from the in vivo cyclisation of (E)-p-Farnesene (lb) to compound of the formula (A’1-1 a), (A’1-2a) and (D’1 a) by the squalene cyclase BmeSHC_G595M (SEQ ID NO: 144) in E. coli DP1205.

[0055] Figure 7: GC-MS chromatogram from bioconversion cyclisation of compound of the formula (If) to compound of the formula (B’5a), (C’5a) and (A’5-2a) by the bacterial membrane-integrated meroterpenoid cyclase WP_317769678.1 (SEQ ID NO: 71) expressed in E. coli C43(DE3).

[0056] Figure 8: Representative Electron impact (El) mass spectrum (MS) from compound of the formula (B’5a) (A), (C’5a) (B) and (A’5-2a) (C) produced by the bioconversion of compound of the formula (If).

[0057] Figure 9: GC-MS chromatogram from bioconversion cyclisation of compound of the formula (li) to the compound of the formula (B’3a), (C’3a), (A’3-1 a) and (D’1 a) by the bacterial membrane-integrated meroterpenoid cyclase WP_318017018.1 (SEQ ID NO: 73) expressed in E. coli C43(DE3).

[0058] Figure 10: Electron impact (El) mass spectrum (MS) from compound of the formula (B’3a) (A), (C’3a) (B) and (A’3-1 a) (C) produced by the bioconversion of compound of the formula (li). Description of the Sequences

[0059]

[0060]

[0061] Abbreviations

[0062] ADH alcohol dehydrogenase BVMO Baeyer-Villiger Monooxygenase bp base pair kb kilo base DNA deoxyribonucleic acid cDNA complementary DNA DMAPP dimethylallyl diphosphate FMO Flavin Monooxygenase FPP farnesyl diphosphate GPP geranyldiphosphate GGPP geranylgeranyl diphosphate GGPS geranylgeranyl diphosphate synthase GC gas chromatograph IPP isopentenyl diphosphate iMS mass spectrometer / mass spectrometry MVA mevalonic acid PP diphosphate, pyrophosphate PCR polymerase chain reaction RNA ribonucleic acid SHC squalene cyclase MeroTPS meroterpenoid cyclase mRNA messenger ribonucleic acid miRNA micro RNA siRNA small interfering RNA rRNA ribosomal RNA tRNA transfer RNA TPP terpenyl diphosphate

[0063] Definitions

[0064] General terms

[0065] For the descriptions herein and the appended claims, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are interchangeable and not intended to be limiting.

[0066] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of or "consisting of.

[0067] The term “about” indicates a potential variation of ± 25% of the stated value, in particular ± 15%, ± 10 %, more particularly ± 5%, ± 2% or ± 1 %.

[0068] The term "substantially" describes a range of values of from about 80 to 100%, such as, for example, 85-99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9% and especially 99 to 99.9%.

[0069] “Predominantly” refers to a proportion in the range of above 50%, as for example in the range of 51 to 100%, particularly in the range of 60 to 100%, particularly in the range of 70 to 100%, particularly in the range of 75 to 100%, particularly in the range of 80 to 100%, particularly in the range of 85 to 100%, particularly in the range of 90 to 100%, particularly in the range of 95 to 100%, particularly in the range of 96 to 100%, particularly in the range of 97 to 100%, particularly in the range of 98 to 100%, particularly in the range of 99 to 100%, particularly in the range of 99,5 to 100%, particularly in the range of 99,8 to 100%; more in particular, in the range of 51 to 99,9%, particularly in the range of 60 to 99,9%, particularly in the range of 70 to 99,9%, particularly in the range of 75 to 99,9%, particularly in the range of 80 to 99,9%, particularly in the range of 85 to 99,9%, particularly in the range of 90 to 99,9%, particularly in the range of 95 to 99,9%, particularly in the range of 96 to 99,9%, particularly in the range of 97 to 99,9%, particularly in the range of 98 to 99,9%, particularly in the range of 99 to 99,9%, particularly in the range of 99,5 to 99,9%.

[0070] A “main product” in the context of the present invention designates a single compound or a group of at least 2 compounds, like 2, 3, 4, 5 or more, particularly 2 or 3 compounds, which single compound or group of compounds is “predominantly” prepared by a reaction as described herein, and is contained in said reaction in a predominant proportion (or “relative ratio”) based on the total amount of the product compounds formed by said reaction. Said proportion may be a molar proportion, a weight proportion or, preferably based on chromatographic analytics, an area proportion calculated from the corresponding chromatogram of the reaction products.

[0071] In the context of the invention, the term “relative ratio” (expressed in %) refers to the proportion of a given compound based on the total quantified product compound (A), (B), (C) and / or (D) in a reaction. This proportion corresponds to the area ratio derived from the corresponding GC-FID chromatogram. The calculation considers only those product compounds (A), (B), (C), and / or (D) that were quantifiable (and therefore quantified). Compounds below the limit of quantification (designated as “not quantified” or “n.q” in the Examples) were excluded from the determination of the relative ratio (%).

[0072] A “side product” in the context of the present invention designates a single compound or a group of at least 2 compounds, like 2, 3, 4, 5 or more, particularly 2 or 3 compounds, which single compound or group of compounds is not “predominantly” prepared by a reaction as described herein.

[0073] In the context of the invention, the term “relative enzyme activity” (expressed in %) refers to the activity of a given enzyme compared to the best-performing enzyme (defined as 100%) under same conditions. The relative enzyme activity (%) is calculated as the proportion of the total quantified product compounds (A), (B), (C), and / or (D) obtained with the given enzyme relative to the total quantified product compounds (A), (B), (C), and / or (D) obtained with the best-performing enzyme. Compounds below the limit of quantification (designated as “not quantified” or “n.q” in the Examples) were excluded from this calculation. This proportion corresponds to the area ratio derived from the corresponding GC-FID chromatogram.

[0074] The term "stereoisomers" includes conformational isomers and in particular configuration isomers.

[0075] Included in general are, according to the invention, all “stereoisomeric forms” of the compounds described herein, such as “constitutional isomers” and “stereoisomers”.

[0076] “Stereoisomeric forms” encompass in particular, “stereoisomers” and mixtures thereof, e.g. configuration isomers (optical isomers), such as enantiomers, or geometric isomers (diastereomers), such as E- and Z-isomers, and combinations thereof. If one or more asymmetric centers are present in one molecule, the invention encompasses all combinations of different conformations of these asymmetry centers, e.g. enantiomeric pairs.

[0077] “Stereoselectivity” describes the ability to produce a particular stereoisomer of a compound in a stereoisomerically pure form or to specifically convert a particular stereoisomer in an enzyme catalyzed method as described herein out of a plurality of stereoisomers. More specifically, this means that a product of the invention is enriched with respect to a specific stereoisomer, or an educt may be depleted with respect to a particular stereoisomer. This may be quantified via the purity %ee-parameter calculated according to the formula:

[0078] %ee = [XA-XB] / [ XA+XB]*100, wherein XA and XB represent the molar ratio (Molenbruch) of the stereoisomers A and B.

[0079] The term “selectively converting” or “increasing the selectivity” in general means that a particular stereoisomeric form, as for example the E-form, of an unsaturated hydrocarbon, is converted in a higher proportion or amount (compared on a molar basis) than the corresponding other stereoisomeric form, as for example Z-form, either during the entire course of said reaction (i.e. between initiation and termination of the reaction), at a certain point of time of said reaction, orduring an “interval” of said reaction. In particular, said selectivity may be observed during an “interval” corresponding 1 to 99%, 2 to 95%, 3 to 90%, 5 to 85%, 10 to 80%, 15 to 75%, 20 to 70%, 25 to 65%, 30 to 60%, or 40 to 50% conversion of the initial amount of the substrate. Said higher proportion or amount may, for example, be expressed in terms of:

[0080] . a higher maximum yield of an isomer observed during the entire course of the reaction or said interval thereof;

[0081] . a higher relative amount of an isomer at a defined % degree of conversion value of the substrate; and / or

[0082] . an identical relative amount of an isomer at a higher % degree of conversion value; each of which preferably being observed relative to a reference method, said reference method being performed under otherwise identical conditions with known chemical or biochemical means.

[0083] Generally, also comprised in accordance with the invention are all “isomeric forms” of the compounds described herein, such as constitutional isomers and in particular stereoisomers and mixtures of these, such as, for example, optical isomers or geometric isomers, such as E- and Z-isomers, and combinations of these. If several centers of asymmetry are present in a molecule, then the invention comprises all combinations of different conformations of these centers of asymmetry, such as, for example, pairs of enantiomers, or any mixtures of stereoisomeric forms.

[0084] “Yield" and I or the "conversion rate" of a reaction according to the invention is determined over a defined period of, for example, 4, 6, 8, 10, 12, 16, 20, 24, 36 or 48 hours, in which the reaction takes place. In particular, the reaction is carried out under precisely defined conditions, for example at “standard conditions” as herein defined.

[0085] The different yield parameters ("Yield" or Yp / s; " Specific Productivity Yield"; or Space-Time- Yield (STY)) are well known in the art and are determined as described in the literature.

[0086] "Yield" and "Yp / s" (each expressed in mass of product produced / mass of material consumed) are herein used as synonyms.

[0087] The specific productivity-yield describes the amount of a product that is produced per h and L fermentation broth per g of biomass. The amount of wet cell weight stated as WCW describes the quantity of biologically active microorganism in a biochemical reaction. The value is given as g product per g WCW per h (i.e. g / gWCW-1h-1). Alternatively, the quantity of biomass can also be expressed as the amount of dry cell weight stated as DCW. Furthermore, the biomass concentration can be more easily determined by measuring the optical density at 600 nm (ODeoo) and by using an experimentally determined correlation factor for estimating the corresponding wet cell or dry cell weight, respectively.

[0088] The terms "purified", "substantially purified", and "isolated" as used herein refer to the state of being free of other, dissimilar compounds with which a compound of the invention is normally associated in its natural state, so that the "purified", "substantially purified", and "isolated" subject comprises at least 0.5%, 1 %, 5%, 10%, or 20%, or at least 50% or 75% of the mass, by weight, of a given sample. In one embodiment, these terms refer to the compound of the invention comprising at least 95, 96, 97, 98, 99 or 100%, of the mass, by weight, of a given sample. As used herein, the terms "purified," "substantially purified," and "isolated" when referring to a nucleic acid or protein, or nucleic acids or proteins, also refers to a state of purification or concentration different than that which occurs naturally, for example in an prokaryotic or eukaryotic environment, like, for example in a bacterial or fungal cell, or in the mammalian organism, especially human body. Any degree of purification or concentration greater than that which occurs naturally, including (a) the purification from other associated structures or compounds or (b) the association with structures or compounds to which it is not normally associated in said prokaryotic or eukaryotic environment, are within the meaning of "isolated”. The nucleic acid or protein or classes of nucleic acids or proteins, described herein, may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.

[0089] Biochemical and biological terms

[0090] The term “enzymatically catalyzed” or “biocatalytic” method means that said method is performed under the catalytic action of an enzyme, including enzyme mutants. Thus, the method can either be performed in the presence of said enzyme in isolated (purified, enriched) or crude form or in the presence of a cellular system, in particular, natural or recombinant microbial cells containing said enzyme in active form, and having the ability to catalyze the conversion reaction as disclosed herein.

[0091] The term "domain" refers to a set of amino acids or a partial sequence of amino acids residues conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between protein homologues, amino acids that are highly conserved at specific positions of such domain indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.

[0092] The term "motif" or consensus sequence" or "signature" refers to a short-conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains but may also include only part of the domain. Signatures are predictive models which describe protein families, domains or sites.

[0093] The sequences of motifs can be described using the standard IUPAC one-letter codes forthe amino acids. Ambiguities are indicated by listing the acceptable amino acids for a given position between brackets. For example, [LWI] stands for L (Leucine), W (Tryptophan) or I (Isoleucine). X represent positions where independently of each other any natural amino acid residue is present.

[0094] A “protein family” is defined as a group of proteins that share a common evolutionary origin reflected by their related functions, similarities in sequence, or similar primary, secondary or tertiary structure. Proteins within protein families are usually homologous and have similar structure of conserved functional domains and motifs.

[0095] Specialist databases exist for the identification of protein domains, for example, SMART (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi?GENOMIC=1) (Schultz et al., (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al., (2020) Nucleic Acids Res 49, D458-D460), InterPro (Paysan- Lafosse et al, Nucleic Acids Research, Nov 2022; Mulder et al., (2003) Nucl. Acids. Res. 31 , 315-318), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)).

[0096] Useful tools to search or predict protein domains or protein family signatures in protein sequence are for example the NCBI conserved domain search tool (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) or the InterProScan tool (http: / / www.ebi.ac.uk / interpro / search / sequence / ). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.

[0097] The term "Pfam" refers to a large collection of protein domains and protein families maintained by the Pfam Consortium and available at several sponsored world wide web sites, such as the InterPro consortium web site https: / / www.ebi.ac.uk / interpro / (European Molecular Biology Laboratory- Europe an Bioinformatics Institute (EMBL_EBI). The latest release of Pfam is Pfam 35.0 (November 2021), based on the UniProt Reference Proteomes (El-Gebali S. et al, 2019, Nucleic Acids Res. 47, Database issue D427- D432). Pfam domains and families are identified using multiple sequence alignments and hidden Markov models (HMMs). Pfam-A family or domain assignments, are high quality assignments generated by a curated seed alignment using representative members of a protein family and profile hidden Markov models based on the seed alignment (Unless otherwise specified, matches of a queried protein to a Pfam domain or family are Pfam-A matches). All identified sequences belonging to the family are then used to automatically generate a full alignment for the family (Sonnhammer (1998) Nucleic Acids Research 26, 320-322; Bateman (2000) Nucleic Acids Research 26, 263-266; Bateman (2004) Nucleic Acids Research 32, Database Issue, D138-D141 ; Finn (2006) Nucleic Acids Research Database Issue 34, D247-251 ; Finn (2010) Nucleic Acids Research Database Issue 38, D211-222). By accessing the Pfam database, for example, using any of the above- reference websites, protein sequences can be queried against the HMMs using HMMER homology search software (e.g., HMMER2, HMMER3, or a higher version, hmmer.janelia.org / ). Significant matches that identify a queried protein as being in a pfam family (or as having a particular Pfam domain) are those in which the bit score is greater than or equal to the gathering threshold for the Pfam domain. Expectation values (E-values) can also be used as a criterion for inclusion of a queried protein in a Pfam or for determining whether a queried protein has a particular Pfam domain, where low e-values, much less than 1 .0, for example less than 0.1 , or less.

[0098] The term “InterPro” refers to a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites (Paysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, Bileschi ML, Bork P, Bridge A, Colwell L, Gough J, Haft DH, Letunic I, Marchler-Bauer A, Mi H, Natale DA, Orengo CA, Pandurangan AP, Rivoire C, Sigrist CJA, Sillitoe I, Thanki N, Thomas PD, Tosatto SCE, Wu CH, Bateman A. InterPro in 2022. Nucleic Acids Research, Nov 2022, (doi: 10.1093 / nar / gkac993)). To classify proteins in this way, InterPro uses predictive models, known as signatures, provided by several collaborating databases (referred to as member databases) that collectively make up the InterPro consortium. A key value of InterPro is that it combines protein signatures from these member databases into a single searchable resource, capitalising on their individual strengths to produce a powerful integrated database and diagnostic tool. Additionally, InterPro adds value to the entries by providing detailed functional annotation as well as adding relevant GO terms that enable automatic annotation of millions of GO terms across the protein sequence databases. InterPro integrates signatures from the following 13 member databases: OATH, ODD, HAMAP, MobiDB Lite, Panther, Pfam, PIRSF, PRINTS, Prosite, SFLD, SMART, SUPERFAMILY and NCBIfam. The member databases use a variety of different methods to classify proteins. Each of the databases has a particular focus (e.g. protein domains defined from structure, or full-length protein families with shared function). InterPro integrates the signatures from the member databases into InterPro entries and identifies where different member database entries are the same entity. InterPro website (https: / / www.ebi.ac.uk / interpro / ) can be used to obtain information about individual protein families, domains, important sites, perform a sequence search or browse through InterPro annotations. InterPro is updated approximately every 8 weeks. InterPro website also provides InterProScan tool (https: / / www.ebi.ac.uk / interpro / about / interproscan / ), a software package that allows sequences to be scanned against InterPro's member database signatures. Users who have novel nucleotide or protein sequences that they wish to functionally characterise can use InterProScan to run the scanning algorithms against the InterPro database in an integrated way. In InterPro, a protein family is defined as a group of proteins that share a common evolutionary origin reflected by their related functions, similarities in sequence, or similar primary, secondary or tertiary structure. A match to an InterPro, or InterPro ID, entry of this type indicates membership of a protein family.

[0099] The “E-value” (expectation value) is the number of hits that would be expected to have a score equal to or better than this value, by chance alone. This means that a good E-value which gives a confident prediction is much less than 1. E-values around 1 is what is expected by chance. Thus, the lower the E- value, the more specific the search for domains will be. Only positive numbers are allowed.

[0100] A “precursor” compound or molecule of a target compound or molecule as described herein is converted to said target compound, preferably through the enzymatic action of a suitable polypeptide performing at least one structural or functional change on said precursor molecule. For example, a “non- cyclic precursor” (like a “non-cyclic terpenyl precursor”) may be converted to the cyclic target molecule (like a cyclic terpene compound) through the action of a cyclase or synthase enzyme, irrespective of the particular enzymatic mechanism of such enzyme, in one or more steps.

[0101] The enzyme nomenclature or enzyme classification (EC) established by the International Union of Biochemistry and Molecular Biology (IUBMB) is a system of naming and categorizing enzymes based on their catalytic activity and biochemical properties. The enzyme nomenclature is widely used in biochemistry to classify and categorize based on their function. The E.C. classification assigns each enzyme a number reflecting the reaction or the type of reaction catalyzed by this enzyme. The enzyme classification can be explored using the ‘ExplorEnz’ database (https: / / www. enzymedatabase. org / ) or International Union of Biochemistry and Molecular Biology (IUBMB) web site (https: / / iubmb.qmul.ac.uk). Information can be found about the classification and nomenclature of enzymes, their functions and properties. The database can be searched to find information for a specific enzyme family or enzyme.

[0102] The term “biological function,” “function”, “biological activity” or “activity” of a terpenyl-disphosphate synthase refers to the ability of a terpenyl-diphosphate synthase as described herein to catalyze the formation of at least one terpenyl diphosphate compound from the corresponding precursor terpene.

[0103] The term “biological function,” “function”, “biological activity” or “activity” of a terpenyl-diphosphate phosphatase refers to the ability of the terpenyl-diphosphate phosphatase as described herein to catalyze the removal of a diphosphate group from said terpenyl disphophate compound (precursor) to form the corresponding terpene alcohol.

[0104] The term "homologous" or “endogenous” when used to indicate the relation between a given (recombinant) polynucleotide (such as DNA or RNA) or polypeptide and a given host organism or host cell such as the cell as disclosed herein, is understood to mean that in nature the polynucleotide or polypeptide molecule is produced by a host cell or organism of the same species, such as of the same variety or strain.

[0105] The term "heterologous" when used with respect to a polynucleotide (such as DNA or RNA) or a polypeptide refers to a polynucleotide or polypeptide that does not occur naturally as part of the host cell such as the cell as disclosed herein. In other words, heterologous polynucleotides or polypeptides are not endogenous to the cell into which they are introduced but have been obtained from another cell or synthetically or recombinantly produced.

[0106] As used herein, the term “host cell”, “recombinant cell” or “transformed cell” refers to a cell (or organism) altered to harbor at least one nucleic acid molecule, for instance, a recombinant gene encoding a desired protein or nucleic acid sequence which upon transcription yields at least one functional polypeptide of the present invention. The host cell is particularly a bacterial cell, a fungal cell or a plant cell or plants. The host cell may contain a recombinant gene or several genes, as for example organized as an operon, which has been integrated into the nuclear organelle genomes of the host cell. Alternatively, the host may contain the recombinant gene extra-chromosomally. Methods of introducing recombinant nucleic acid sequences into such host cells are well known in the art and constitute routine laboratory methodologies which do not need to be further described herein.

[0107] The term “organism” refers to any non-human multicellular or unicellular organism such as a plant, or a microorganism. Particularly, a micro-organism is a bacterium, a yeast, an algae or a fungus.

[0108] The term “plant” is used interchangeably to include plant cells including plant protoplasts, plant tissues, plant cell tissue cultures giving rise to regenerated plants, or parts of plants, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits and the like. Any plant can be used to carry out the methods of an embodiment herein.

[0109] The “mevalonate pathway” also known as the “isoprenoid pathway” or “HMG-CoA reductase pathway” is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five-carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP). Key enzymes are acetoacetyl-CoA thiolase (atoB), HMG-CoA synthase (mvaS), HMG-CoA reductase (mvaA), mevalonate kinase (MvaK1), phosphomevalonate kinase (MvaK2), a mevalonate diphosphate decarboxylase (MvaD), and an isopentenyl diphosphate isomerase (idi). Combining the mevalonate pathway with enzyme (prenyltransferase) activity to generate the terpene precursors GPP, FPP or GGPP, like in particular FPP synthase (ERG20), allows the recombinant cellular production of terpenes.

[0110] The term “prenyltransferase”, “prenyltransferase enzyme” or “polypeptide having prenyltransferase activity” represents a group of enzymes having the ability to condense successively five-carbon units such as isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form non-cyclic (linear) terpenyl-diphosphate compounds such as geranyl-diphosphate (GPP), farnesyl-diphosphate (FPP) or geranylgeranyl-diphosphate (GGPP) containing 10, 15 and 20 carbons, respectively. Some prenyltransferases can add 5-carbon units to linear terpenyl-diphosphate compounds thereby extending the carbon chain length. An example of prenyl transferase are farnesyl diphosphate (FPP) synthases (FPP synthases; EC 2.5.1.10) having the ability of producing FPP from IPP and DMAPP. Another example of prenyl transferase are geranyl-diphosphate synthases (GGPP synthases; EC 2.5.1 .29) having the ability of producing GGPP from IPP and DMAPP or by adding 5 carbons to FPP.

[0111] The term “beta-farnesene synthase”, “beta-farnesene synthase enzyme” or “polypeptide having beta-farnesene synthase activity” (EC 4.2.3.47) represents a group of enzymes having the ability to cleave the diphosphate moiety and a subsequent rearrangement of the farnesyl carbocation intermediate, resulting in the formation of beta-farnesene. Structurally, beta-farnesene synthase belongs to the family of terpene synthases, characterized by conserved motifs such as the DDXXD and NSE / DTE triads, which coordinate divalent metal ions necessary for enzymatic activity.

[0112] The term “terpene cyclase”, “terpene cyclase enzyme” or “polypeptide having terpene cyclase activity” represents a group of enzymes that catalyze the cylization of terpene precursors into cyclic terpene compounds. Terpene cyclases are divided into two categories depending on the way the initial carbocation is generated. In class I (or type I) terpene cyclase, the diphosphate group of the linear terpenoid precursor is abstracted to form an allylic carbocation on the terpene moiety. In class II, the initial carbocation is formed by protonation of a double bond or epoxy group in the terpene carbon chain. Thus, class I cyclase necessarily use substrates with a diphosphate group, while class II cyclase (since they do not need a diphosphate group for the generation of the initial carbocation) can use terpenoids as substrates.

[0113] In the context of the invention, the term “squalene cyclase (SHC)”, “SHC enzyme”, or “polypeptide having squalene cyclase activity” relates to a polypeptide having terpene cyclase activity wherein the substrate does not contain a diphosphate functional group. The squalene cyclase enzyme family comprises squalene cyclases and 2,3-oxidosqualene cyclases and enzymes catalyzing mechanistically related cyclization reactions. Squalene cyclases catalyze a protonation-initiated cyclization cascade of a linear terpene to a cyclic compound. The squalene cyclase enzyme family includes for example squalene-hopene cyclases catalyzing the cyclization of squalene to hopene (EC 5.4.99.17) and squalene-hopanol cyclases catalyzing the cyclization of squalene to hopan-22-ol (EC 4.2.1.129). Tetraprenyl-p-curcumene-sporulenol cyclases catalyze similar cyclization of linear terpene substrate (EC 4.2.1 .137). It was shown that tetraprenyl-p-curcumene-sporulenol cyclase can also catalyze the cyclization of squalene (Sato, T., et al. (2011). Journal of the American Chemical Society 133(44): 17540-17543), thus tetraprenyl-p-curcumene- sporulenol cyclases are also members of the squalene cyclase family.

[0114] The enzymatic activity of a squalene cyclase is determined under “standard conditions” as for example described in the Examples section.

[0115] In the context of the invention, the term “meroterpenoid cyclase (MeroTPS)”, ‘MeroTPS enzyme” or “polypeptide having meroterpenoid cyclase activity” relates to a polypeptide capable of catalyzing the cyclization of meroterpenoids, which are a class of natural products that consist of a terpenoid moiety combined with a non-terpenoid (often aromatic) moiety. Said meroterpenoid cyclases have been described in PCT / EP2024 / 066253 and were categorized into 3 categories, i.e. membrane-integrated meroterpenoid cyclase of bacterial origin, membrane-integrated meroterpenoid cyclase of fungal origin, and soluble meroterpenoid cyclases of bacterial origin.

[0116] The enzymatic activity of a meroterpenoid cyclase is determined under “standard conditions” as for example described in the Examples section.

[0117] Chemical terms:

[0118] “Diphosphate” and “pyrophosphate” as used herein are synonyms.

[0119] “Terpenes” are a large and diverse class of organic compounds, produced by a variety of plants, particularly conifers, and by some insects. Terpenes are hydrocarbons. Although sometimes used interchangeably with "terpenes", “terpenoids” or “isoprenoids” are modified terpenes as they contain additional functional groups, usually oxygen-containing.

[0120] “Terpenoids” (“isoprenoids”) are a large and diverse class of naturally occurring organic chemicals derived from terpenes. Although sometimes used interchangeably with the term “terpenes”, “terpenoids” contain additional functional groups, usually O-containing groups, like for example hydroxyl, carbonyl or carboxyl groups. Most are multicyclic structures with oxygen-containing functional groups. Unless stated otherwise, in the context of the present description the term “terpene” and the term “terpenoid” may be used interchangeably.

[0121] Terpenes (and terpenoids) may be classified by the number of isoprene units in the molecule; a prefix in the name indicates the number of terpene units needed to assemble the molecule. Hemiterpenes consist of a single isoprene unit. Monoterpenes consist of two isoprene units and have the molecular formula C10H16. Sesquiterpenes consist of three isoprene units and have the molecular formula C15H24. Diterpenes are composed of four isoprene units and have the molecular formula C20H32.

[0122] “Terpenyl” designates noncyclic and cyclic chemical hydrocarbyl residues which are derived from the C5 building block isoprene and in particular contain one or more such building blocks.

[0123] “Cyclic terpene” or cyclic terpenyl” or “cyclic diterpene” or cyclic diterpenyl” relates to a terpene compound or terpenyl residue which comprises in its structure at lest on, as for example 1 , 2, 3, 4 or 5 carbocyclic condensed and / or non-condensed rings, preferably two carbocyclic condensed rings.

[0124] “Bicyclic terpene” or bicyclic terpenyl” or “bicyclic diterpene” or bicyclic diterpenyl” relates to a terpene compound or terpenyl residue which comprises in its structure two carbocyclic rings, preferably two carbocyclic condensed rings.

[0125] “Derivatives of terpenes” or “derivatives of terpenoids” in the context of the present invention in particular refer to such chemical compounds which are obtained from a terpene or terpenoid by chemical and / or enzymatic modification.

[0126] A “hydrocarbyl” residue is a chemical group which essentially is composed of carbon and hydrogen atoms and may be a non-cyclic, linear or branched, saturated or unsaturated moiety, or a cyclic saturated or unsaturated moiety, aromatic or non-aromatic moiety. A hydrocarbyl residue comprises 1 to 30, 1 to 25,

[0127] 1 to 20, 1 to 15 or 1 to 10 or 1 to 5 carbon atoms in the case of a non-cyclic structure. It comprises 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10 or in particular 4, 5, 6 or 7 carbon atoms in the case of a cyclic structure. Said hydrocarbyl residues may be non-substituted or may carry at least one, like 1 to 5, preferably 0, 1 or

[0128] 2 substituents.

[0129] Particular examples of such hydrocarbyl residues are non-cyclic linear or branched alkyl or alkenyl residues; or mono- or polycyclic, in particular mono- or bicyclic, saturated or unsaturated, nonaromatic moieties, as for example found in cyclic (for example bicyclic) or non-cyclic terpene type compound, and labdane-type compounds.

[0130] In the present document, a “Cx-y-alkyl” group is an alkyl group comprising x to y carbon atoms, i.e., for example, a Ci-3-alkyl group is an alkyl group comprising 1 to 3 carbon atoms. The alkyl group can be linear or branched. For example -CH(CH3)-CH2-CH3 is considered as a C4-alkyl group.

[0131] An “alkenyl” residue represents linear or branched, mono- or polyunsaturated hydrocarbon residues. It comprises 2 to 30, 2 to 25, 2 to 20, 2 to 15 or 2 to 10 or 2 to 7, 2 to 6, 2 to 5, or 2 to 4 carbon atoms. I may have up to 10, like 1 , 2, 3, 4 or 5 C=C double bonds.

[0132] An "alkylene" represents straight-chain or singly or multiply branched hydrocarbon bridging groups having 1 to 10 carbon atoms, for example Ci-C -alkylene groups selected from -CH2-, -(CH2)2-, -(CH2)3-,- (CH2)4-, -(CH2)2-CH(CH3)-, -CH2-CH(CH3)-CH2-, (CH2)4-, -(CH2)5-, -(CH2)6, -(CH2)7-, -CH(CH3)-CH2-CH2- CH(CH3)- or -CH(CH3)-CH2-CH2-CH2-CH(CH3)-, and in particular Ci-C4-alkylene groups selected from - CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)2-CH(CH3)-, -CH2-CH(CH3)-CH2-.

[0133] An “alkylidene” group represents a straight chain or branched hydrocarbon substituent linked via a double bond to the body of the molecule. It comprises 1 to 6 carbon atoms. As examples of such “Ci-Ce- alkylidenes” there may be mentioned methylidene (=CH2) ethylidene, (=CH-CH2), n- propylidene, n- butylidene, n-pentlyiden, n-hexylidene and the constitutional isomers thereof, as for example iso- propylidene.

[0134] An “alkenylidene” represents the mono-unsaturated analogue of the above mentioned alkylidenes with more than 2 carbon atoms and may be called “Cs-Ce-alkenylidenes”. n- propenylidene, n-butenylidene, n-pentenlyiden, and n-hexenylidene may be mentioned as examples.

[0135] In case identical labels for symbols or groups are present in several formulae, in the present document, the definition of said group or symbol made in the context of one specific formula applies also to other formulae which comprises the same said label.

[0136] The term “independently from each other” in this document means, in the context of substituents, moieties, or groups, that identically designated substituents, moieties, or groups can occur simultaneously with a different meaning in the same molecule.

[0137] Any single dotted line in any formulae represents the bond by which said substituent is bound to the rest of a molecule.

[0138] Any wavy line in any formula of this document represents a carbon-carbon bond which when linked to the carbon-carbon double bond is either in the Z- or in the E-configuration.

[0139] The “substituent” of the above-mentioned residues contains one hetero atom, like O or N. Preferably the substituents are independently selected from -OH, C=O, or - COOH. Most preferably said substituent is -OH.

[0140] A “mono- or polycyclic hydrocarbyl residue” comprise 1 , 2 or 3 condensed (anellated) or noncondensed, optionally substituted, saturated or unsaturated hydrocarbon ring groups (or “carbocyclic” groups). Each cycle may comprise independently of each other 3 to 8, in particular 5 to 7, more particularly 6 ring carbon atoms. As examples of monocyclic residues there may be mentioned "cycloalkyl" groups which are carbocyclic radicals having 3 to 7 ring carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl; and the corresponding “cycloalkenyl” groups. Cycloalkenyl" (or "mono- or polyunsaturated cycloalkyl") represents, in particular, monocyclic, mono- or polyunsaturated carbocyclic groups having 5 to 8, preferably up to 6, carbon ring members, for example monounsaturated cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenylradicals.

[0141] As examples of polycyclic residues there may be mentioned groups wherein 1 , 2 or 3 of such cycloalkyl and / or cycloalkenyl are linked together, as for example anellated, in order to form a polycyclic cycloalkyl or cycloalkenyl ring. As non-limiting example the bicyclic decalinyl residue composed of two anellated 6-membered carbon rings may be mentioned.

[0142] The number of substituents in such mono- or polycyclic hydrocarbyl residues may vary from 1 to 10, in particular 1 to 5 substituents. Suitable substituents of such cyclic residues are selected from lower alkyl, lower alkenyl, alkylidene, alkenylidene, or residues containing one hetero atom, like O or N as for example -OH or - COOH. In particular, the substituents are independently selected from -OH, - COOH, methyl and methylidene.

[0143] Unsaturated cyclic groups may contain 1 or more, as for example 1 , 2 or 3 C=C bonds and are aromatic, or in particular nonaromatic.

[0144] The above-mentioned mono- or polycyclic saturated or unsaturated groups may also contain at least one, like 1 , 2, 3 or 4 ring heteroatoms, such as O, N or S.

[0145] The term "alcohol protecting group" in this document means a group which protects the hydroxyl in any of the formulas in this document and which can be easily removed, (i.e. deprotected), by state-of-the- art methods, resulting to the respective compound with the free hydroxyl group.

[0146] The alcohol protecting group is introduced by a chemical reaction of the compound of the respective formula having OH with a protecting agent.

[0147] The protecting agents leading to the corresponding alcohol protecting groups are known to the person skilled in the art, as well as the chemical process and conditions for this reaction.

[0148] If, for example, the alcohol protecting group forms an ester with the rest of the molecule, the suitable protecting agent is for example an acid, an anhydride, or an acyl halide.

[0149] If the alcohol protecting group forms an acetal or a ketal with the rest of the molecule, the suitable protecting agent is an aldehyde or a ketone.

[0150] If the alcohol protecting group forms an ether with the rest of the molecule, the suitable protecting agent is an alkyl halide, e.g. MeO(CH2)2OCH2CI, or an enol ether, e.g. 3,4-dihydro-2 / 7-pyran.

[0151] The preferred alcohol protecting group is an acyl group, particularly a group of the formula defined later on in this document.

[0152] If the present disclosure refers to features, parameters and ranges thereof of different degree of preference (including general, not explicitly preferred features, parameters and ranges thereof) then, unless otherwise stated, any combination of two or more of such features, parameters and ranges thereof, irrespective of their respective degree of preference, is encompassed by the disclosure of the present description. Detailed Description

[0153] The present inventors sought to identity new methods and compounds toward the preparation of perfumery, flavor or aroma compounds. In this context, the present inventors surprisingly found that meroterpenoid cyclases and squalene cyclases could be used in the cyclisation of linear terpenoid compounds to produce a compound of the formula (A), (B), (C) and / or (D).

[0154] Accordingly, the present invention provides a novel method for preparing a compound of the formula (A), (B), (C) and / or (D) starting from compound of the formula (I). The compounds of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5),(C’1), (C’2), (C’4), (C’5), (D’1 b), (A’1-1) and (A’1-2) are also part of the invention. Said compounds may be used as a perfumery, flavor or aroma ingredient and / or a precursor thereof.

[0155] Method of the invention

[0156] A first aspect of the invention provides a method for preparing a compound of the formula (A), (B), (C) and / or (D), wherein

[0157] R° represents either H or a C1-4 alkyl group, preferably ethyl or H; and wherein each R2represents independently from each other either H or an alcohol protecting group, particularly , preferably H,

[0158] R3represents H or a C1-4 alkyl group, preferably CH3 ; and n independently from each other represents 1 or 2; wherein any dotted line represents the bond by which the substituent is bound to the rest of the molecule; and any wavy line represents independently from each other a carbon-carbon bond which when linked to the carbon-carbon double bond is either in the Z and / or in the E-configuration, preferably in the E-configuration, wherein the method comprises:

[0159] (a) contacting a compound of the formula (I) with a terpene cyclase enzyme under conditions suitable for the terpene cyclase enzyme to produce the compound of the formula (A), (B), (C) and / or (D).

[0160] It is important to realize that by starting from a specific starting material compound of the formula (I), the process yields one or several product compounds of the formula (A), (B), (C) and / or (D).

[0161] The starting material compounds of the formula (I) for the present method of preparing a compound of the formula (A), (B), (C) and / or (D) are the compounds selected from the group consisting of

[0162]

[0163] An embodiment of the invention is wherein the configuration of the carbon-carbon double of the compound of the formula (I) is in the E-configuration.

[0164] An embodiment of the invention is wherein R1represents with n being preferably 1 and / or R2being H or either H or an alcohol protecting group, particularly R3being a C1-4 alkyl group, preferably CH3; preferably, R1represents An embodiment of the invention is wherein the compound of the formula (A) is a compound of the formula (A’), the compound of the formula (B) is a compound of the formula (B’), the compound of the formula (C) is a compound of the formula (C’) and / or the compound of the formula (D) is a compound of the formula (D’)

[0165] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound selected of the group consisting of

[0166]

[0167] (D’1); or a derivative thereof; wherein each R2represents independently from each other either H or an alcohol protecting group, particularly being a C1-4 alkyl group, preferably CH3. An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’), preferably of the formula (A’1), preferably of the formula (A’1 a) or (A'1 b), more preferably of the formula (A’1-1a) or (A'1 -1 b) An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’), preferably of the formula (A’3), preferably of the formula (A’3a), more preferably of the formula (A’3-1a) or (A'3-2a) An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’), preferably of the formula (A’5), preferably of the formula (A’5a), more preferably of the formula (A’5-1a) or (A'5-2a)

[0168] (A’5-2a). An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (B’), preferably of the formula (B’3), preferably of the formula (B’3a) An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (B’), preferably of the formula (B’5), preferably of the formula (B’5a) An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (C’), preferably of the formula (C’3), preferably of the formula (C’3a)

[0169] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (C’), preferably of the formula (C’5), preferably of the formula (C’5a)

[0170]

[0171] (C’5a).

[0172] An embodiment of the invention is wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (D’), preferably of the formula (D’1), preferably of the formula (D’1 a), more preferably of the formula (D’1 ab)

[0173] An embodiment of the invention is wherein the compound the formula (I) is compound of the formula (la), particularly of the formula (lb), and the compound of the formula (A) is a compound of the formula (A’1 a), the compound of the formula (B) is a compound of the formula (B’1), the compound of the formula

[0174] (C) is a compound ofthe formula (C’1) and / or the compound of the formula (D) is a compound of the formula (D’1a)

[0175] In said embodiment, the compound of the formula (A’1 a) may be a compound of the formula (A’1-a) or (A’1-2a); preferably, a compound of the formula (A’1-1 a) An embodiment of the invention is wherein the compound the formula (I) is compound of the formula (li), and the compound of the formula (A) is a compound of the formula (A’3a), the compound of the formula (B) is a compound of the formula (B’3a), the compound of the formula (C) is a compound of the formula (C’3a) and / or the compound of the formula (D) is a compound of the formula (D’1 a)

[0176] In said embodiment, the compound of the formula (A’3a) may be a compound of the formula (A’3- An embodiment of the invention is wherein the compound the formula (I) is compound of the formula (If), and the compound of the formula (A) is a compound of the formula (A’5a), the compound of the formula (B) is a compound of the formula (B’5a) and / or the compound of the formula (C) is a compound of the formula (C’5a)

[0177] In said embodiment, the compound of the formula (A’5a) may be a compound of the formula (A’5- 1 a) or (A’5-2a); preferably, a compound of the formula (A’5-2a)

[0178] Particularly, it has been found that the process provides specific product compounds of the formula (A), (B), (C) and / or (D) from the specific starting material compounds of the formula (I). Said specific starting material compounds of the formula (I) and said specific product compounds of the formula (A), (B), (C) and (D) are listed herein below in Table A and Table B. In said Tables, the “Rx” groups correspond to the groups identified elsewhere in the description. b. Compounds of the formula (A), (B), (C) and (D)

[0179]

[0180]

[0181] Terpene cyclases in the method of the invention

[0182] Terpene cyclases are divided into two categories depending on the way the initial carbocation is generated. In class I (or type I) terpene cyclase, the diphosphate group of the linear terpenoid precursor is abstracted to form an allylic carbocation on the terpene moiety. In class II, the initial carbocation is formed by protonation of a double bond or epoxy group in the terpene carbon chain. Thus, class I cyclase necessarily use substrates with a diphosphate group, while class II cyclase (since they do not need a diphosphate group for the generation of the initial carbocation) can use terpenoids as substrates. For all terpene cyclases, the generated reactive carbocation species triggers the subsequent cascade reaction including carbocation reactions with double bonds, alkyl-shifts, hydride shifts or carboncarbon bound formation. The reaction can be terminated by deprotonation of a carbon atom adjacent to the carbocation or by quenching of the carbocation with a hydroxyl group or molecule of water.

[0183] The type II activity in terpene cyclases is associated with aspartate-rich conserved motifs. Typical examples of class II terpene cyclases are the class II diterpene cyclases catalyzing the protonation-initiated cyclization of geranylgeranyl-diphosphate into for example, labdadienyl-diphosphate intermediates or other cyclic diphosphate intermediates (Peters, R. J. (2010). Nat. Prod. Rep. 27, 1521 — 1530; Zerbe, P. et a / (2015). Plant J. 83, 783-793).

[0184] Squalene cyclases (SHCs) constitute a classical example of class II terpene cyclases where the substrate does not contain a diphosphate functional group. The squalene cyclase enzyme family comprise squalene cyclases and 2,3-oxidosqualene cyclases and enzymes catalyzing mechanistically related cyclization reactions. Squalene cyclases catalyze a protonation-initiated cyclization cascade of a linear terpene to a cyclic compound. Thus, squalene cyclases are class II terpene cyclases. The squalene family includes for example squalene-hopene cyclases catalyzing the cyclization of squalene to hopene (EC 5.4.99.17) and squalene-hopanol cyclases catalyzing the cyclization of squalene to hopan-22-ol (EC 4.2.1.129). Tetraprenyl-p-curcumene-sporulenol cyclases catalyze similar class II cyclization of linear terpene substrate (EC 4.2.1 .137). It was shown that tetraprenyl-p-curcumene-sporulenol cyclase can also catalyze the cyclization of squalene (Sato, T., et al. (2011). Journal of the American Chemical Society 133(44): 17540-17543), thus tetraprenyl-p-curcumene-sporulenol cyclases are also members of the squalene cyclase family.

[0185] Squalene cyclase polypeptides have typically a length between 600 and 800 amino acids and are membrane-associated proteins. They bind to the surface of cellular membranes but do not contain a transmembrane region. Squalene cyclases are classified in the IPR018333 family of the InterPro protein sequence classification database (https: / / www.ebi.ac.uk / interpro / entry / lnterPro / IPR018333 / ) (InterPro release 93.0, 2nd March 2023). The structure of squalene cyclases is organized in two domains comprising several alpha-helices, recognized as the p-domain and y-domain or the py-domain architecture (Christianson DW, Chem. Rev, 2017, 117, 11570-11648). The two domains have characteristic sequence signatures as described in the Pfam database under the Pfam Squalene-hopene cyclase N-terminal domain (PF13249) and Squalene-hopene cyclase C-terminal domain and (PF13243) (Pfam 35.0 released, 19 November 2021). The presence of the IPR018333, PF13249 or PF13243 protein sequences signatures can be predicted using the NCBI conserved domain search tool (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) or the InterProScan tool (http: / / www.ebi.ac.uk / interpro / search / sequence / ).

[0186] The squalene cyclase polypeptide contains characteristic conserved amino acid motifs located along the sequence and associated with the protein architecture and enzymatic reaction. In particular, the squalene cyclase contains at least one or more amino acid motifs selected from:

[0187] ■ [SP][TP][VIL]WDTx[LWI] (SEQ ID NO: 205),

[0188] . PGG[WF][GYA]F (SEQ ID NO: 206),

[0189] . PDxDD[TAS][TIAS] (SEQ ID NO: 207),

[0190] . [MIL]QxxxG[GA][WF]x[AS][FY] (SEQ ID NO: 208),

[0191] . Qxxx[GH]xWxG[RK]WGxx[YF]xYG (SEQ ID NO: 209),

[0192] . Qxx[DN]G[GS][WF][GS]ExxxS (SEQ ID NO: 210), and

[0193] . [STA]xx[SFN][QC]T[AGT]W[AS][LIV]xx[LQ] (SEQ ID NO: 211). The motif sequences are described using the standard IUPAC one-letter codes for the amino acids. Ambiguities are indicated by listing the acceptable amino acids for a given position between brackets. For example, [SP] or [S or P] stands for S (serine), or P (proline). The “x” represents positions where independently of each other any natural amino acid residue is present. The function of the square brackets has been described above.

[0194] Meroterpenoids are hybrid secondary metabolites derived from mixed biosynthetic pathways and are partially derived from a terpenoid co-substrate (Cornforth, J.W. Terpenoid biosynthesis. Chem. Br. 1968, 4, 102-106). The non-terpenoid part can originate for example from polyketides, alkaloids, phenols, or amino acids biosynthetic pathway. Large chemical diversity is found among meroterpenoids, in particular in bacteria and in fungi.

[0195] The meroterpenoids biosynthetic pathways follow several modular biosynthetic steps. In the first step, the building blocks are generated from the corresponding biosynthetic pathway (e.g. terpenoids, polyketides). The terpenoid and non-terpenoid parts are assembled by prenyltransferases. The precursors of the terpenoid parts are generally linear terpenoid-diphosphates such as geranyl-diphosphate, farnesyl- diphosphate or geranylgeranyl-diphosphate.

[0196] In the following step, the linear polyene terpenoid part of the hybrid precursor is cyclized to form a monocyclic or polycyclic structure. This cyclization is catalyzed by a specific class of non-canonical class II terpene cyclases named meroterpenoid cyclase first discovered in fungi. The first discovered representative meroterpenoid cyclase is Pyr4 from Aspergillus fumigatus Af293 (Itoh, T., et al. (2010). Nature Chemistry 2(10): 858-864).

[0197] In many meroterpenoids, the linear terpenoid precursor is first activated by a stereoselective epoxidation by a monooxygenase of one of the double-bonds. The meroterpenoid cyclases catalyze then the protonation of the epoxide moiety generating a reactive carbocation species and triggering a subsequent cascade reaction similar to other terpene cyclases. Some meroterpenoid cyclases can convert the isoprenic precursors to cyclized products without the involvement of a prior epoxidation step. These meroterpenoid cyclases are able to directly protonate the terminal double bond generating a reactive carbocation and catalyzing a cyclization. For example, MacJ from the fungi Penicillium terrestry w as the first identified fungi meroterpenoid cyclase using a type II double-bond protonation initiations reaction (Tang, M.-C., etal. (2017). Organic Letters 19(19): 5376-5379). Another example of meroterpenoid cyclase which initiates polyene cyclization by direct double bond protonation is DmtA1 from bacteria (Streptomyces youssoufiensis OUC68199) (Yao et al, Nat. Commun., 2018, 9, 4091).

[0198] Like other class II terpene cyclases, the carbocation generated by meroterpenoid cyclases triggers a cascade reaction generally starting by the attack of a double bond and generating monocyclic or polycyclic structure with a tertiary carbocation. The reaction is terminated either by deprotonation to form a double bond or by reacting with a water molecule to generate a tertiary alcohol. Typical cyclic structures found in meroterpenoids compounds contain drimane or labdane scafolds.

[0199] The largest group of meroterpenoid cyclases are compact membrane-integrated proteins containing several (generally seven) transmembrane helices. This protein architecture based on transmembrane helices can easily be predicted using for example the TMHMM 2.0 server available at https: / / dtu.biolib.com / DeepTMHMM (Krogh, A., et al. (2001) J Mol Biol 305(3): 567-580.). In addition to the protein architecture, meroterpenoid cyclases differ from other class II cyclases such as the squalene cyclases by their smaller polypeptide size. The bacterial and fungal meroterpenoid cyclase polypetides have a length ranging from 150 to 550 residues. The transmembrane helices are located over a portion of the polypetide covering 180 to 300 amino acid and carry the catalytic domains.

[0200] Recently meroterpenoid cyclases having a protein architecture different from the membrane- integrated meroterpenoid cyclases were described. For example, MstE from the bacteria Scytonema sp. PCC 1002 is a soluble cyclase having a structure similar to canonical cyclases such as diterpene synthases and squalene cyclases, but nevertheless different, since it is a monodomain protein with only an a-domain (Moosmann, P., et al. (2020). Nat Chem 12(10): 968-972). Soluble bacterial meroterpenoid cyclase polypetides have length ranging from 150 to 550.

[0201] Meroterpenoid cyclase polypeptides contain characteristic conserved amino acid motifs located along the sequence and associated with the protein architecture or enzymatic reaction as follows:

[0202] Membrane-integrated meroterpenoid cyclase of bacterial origin containing at least one or more amino acid motifs selected from:

[0203] . [W]xxx[D]xx[ILVMN] (SEQ ID NO: 212),

[0204] . PxxAxxxNxxWE (SEQ ID NO: 213),

[0205] . MxxxFxxMLxxR (SEQ ID NO: 214),

[0206] . RxxxxGQS (SEQ ID NO: 215), and

[0207] . NxxMS (SEQ ID NO: 216).

[0208] Membrane-integrated meroterpenoid cyclase of fungal origin containing a least one or more amino acid motifs selected from:

[0209] . [WY]Exx[YFW] (SEQ ID NO: 217), and

[0210] . [DNE]xSYxxP (SEQ ID NO: 218).

[0211] Soluble meroterpenoid cyclases of bacterial origin containing a least one or more amino acid motifs selected from:

[0212] . GxWxxxW[WG]xxxxY (SEQ ID NO: 219),

[0213] . WxxxHxxV[TSA] (SEQ ID NO: 220), and

[0214] . GxWxD[FY] (SEQ ID NO: 221).

[0215] The motif sequences are described using the standard IUPAC one-letter codes for the amino acids. Residues x represent independently of each other any natural amino acid residue, and wherein optionally in each of the above motifs, 1 , 2, 3 or 4 amino acid residues different from the x residues may be modified, for example by amino acid substitution, in particular by conservative substitutions, provided that the enzyme retains, at least to analytically detectable extent, its enzyme activity. The function of the square brackets has been described above. Particular examples of suitable standard conditions for each of the above-described enzyme activities may be taken from the Examples section below.

[0216] An embodiment of the invention is wherein the terpene cyclase enzyme is a meroterpenoid cyclase (MeroTPS) enzyme and / or a squalene cyclase (SHC) enzyme.

[0217] For the avoidance of doubt, SHCs and meroterpenoid cyclases are distinct classes of enzymes which can be distinguished by physical characteristics. Furthermore, meroterpenoid cyclases can be classified as (i) bacterial membrane-integrated meroterpenoid cyclases; (ii) fungal membrane-integrated meroterpenoid cyclases; (iii) bacterial soluble meroterpenoid cyclases.

[0218] Table A below outlines the differences between SHCs and the different types of meroterpenoid cyclases.

[0219] Table A: enzyme characteristics

[0220] Hence the skilled person can, from the information provided herein, readily identify whether an enzyme is a SHC enzyme or a class of meroterpenoid cyclase enzyme.

[0221] Several meroterpenoid cyclases catalyze reactions of cyclisation of the terpenoid part of the meroterpenoid hybrid precursor to labdane cyclic structures. However, for the first time, the present inventors surprisingly found that meroterpenoid cyclases could be used for the cyclization of a linear terpenoid (such as a compound of the formula (I)) to a compound of the formula (A), (B), (C) and / or (D).

[0222] Accordingly, an embodiment of the method of the invention is characterized in that the terpene cyclase enzyme is a meroterpenoid cyclase enzyme.

[0223] In a particular embodiment of the invention, the terpene cyclase enzyme is a bacterial membrane- integrated meroterpenoid cyclase enzyme, a fungal membrane-integrated meroterpenoid cyclase enzyme and / or a bacterial soluble meroterpenoid cyclase enzyme. More in particular, the terpene cyclase enzyme is a bacterial membrane-integrated meroterpenoid cyclase enzyme. Accordingly, a preferred embodiment of the invention is wherein the terpene cyclase enzyme is a bacterial membrane-integrated meroterpenoid cyclase enzyme comprising at least one or more amino acid motifs selected from:

[0224] . [W]xxx[D]xx[ILVMN] (SEQ ID NO: 212),

[0225] . PxxAxxxNxxWE (SEQ ID NO: 213),

[0226] . MxxxFxxMLxxR (SEQ ID NO: 214),

[0227] . RxxxxGQS (SEQ ID NO: 215), and

[0228] . NxxMS (SEQ ID NO: 216); wherein residues x represent independently of each other any natural amino acid residue.

[0229] Accordingly, in a further preferred embodiment of the invention, the terpene cyclase enzyme is a meroterpenoid cyclase enzyme, preferably a bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204. In one embodiment, said enzyme has the amino acid sequence of any one of SEQ ID NOs: 1 to 141 and 162 to 204.

[0230] Accordingly, in yet a further preferred embodiment of the invention, the terpene cyclase enzyme is a meroterpenoid cyclase enzyme, preferably a bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 96 and 181 to 204. In one embodiment, said enzyme has the amino acid sequence of any one of SEQ ID NOs: 1 to 96 and 181 to 204.

[0231] Accordingly, in yet a further preferred embodiment of the invention, the terpene cyclase enzyme is a meroterpenoid cyclase enzyme, preferably a bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 80 and 181 to 204. In one embodiment, said enzyme has the amino acid sequence of any one of SEQ ID NOs: 1 to 80 and 181 to 204.

[0232] In an embodiment of the invention, the terpene cyclase enzyme is a meroterpenoid cyclase enzyme, preferably a bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181-204; preferably, to any one of SEQ ID NOs: 74 and 190-192. In one embodiment, said enzyme has the amino acid sequence of any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181-204; preferably, of any one of SEQ ID NOs: 74 and 190-192.

[0233] Furthermore, the present inventors identified a mutation at amino acid position 9 relative to SEQ ID NO: 29 which surprisingly shifts the relative ratios of the product compounds produced in the method of the invention. In particular, the inventors demonstrated that the mutant enzyme represented by SEQ ID NO: 80 was able to produce a significantly higher ratio of compound of the formula B’1 from compound of the formula (1 b) when compared to the wild-type enzyme not having the mutation at amino acid position 9 (SEQ ID NO: 29). Furthermore, when compared with the wild-type enzyme, the compound of the formula (D’1 a) was not detected (i.e. below limit of quantification).

[0234] Accordingly, in yet a further embodiment of the invention, the terpene cyclase enzyme is a mutant meroterpenoid cyclase enzyme, preferably a mutant bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204; preferably, to any one of SEQ ID NOs: 1 to 96 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29.

[0235] Preferably, the terpene cyclase enzyme is a mutant meroterpenoid cyclase enzyme, preferably a mutant bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 80 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29. In one embodiment, the mutant meroterpenoid cyclase enzyme has the amino acid sequence provided in SEQ ID NO: 80.

[0236] More preferably, the terpene cyclase enzyme is a mutant meroterpenoid cyclase enzyme, preferably a mutant bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181-204; preferably, to any one of SEQ ID NOs: 74 and 190-192; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29.

[0237] With reference to the above embodiments, the substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29 may be a substitution to cysteine, methionine or threonine.

[0238] Furthermore, the present inventors identified a mutation at amino acid position 123, 126 or 165 relative to SEQ ID NO: 74 which surprisingly shifts the relative ratios and / or the titers of the product compounds produced in the method of the invention, when compared to the wild-type enzyme not having any of the mutations at amino acid position 123, 126 and 165 (SEQ ID NO: 74).

[0239] Accordingly, in an alternative or yet a further embodiment of the invention, the terpene cyclase enzyme is a mutant meroterpenoid cyclase enzyme, preferably a mutant bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204; preferably, to any one of SEQ ID NOs: 1 to 96 and 181 to 204 ; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74.

[0240] Preferably, the terpene cyclase enzyme is a mutant meroterpenoid cyclase enzyme, preferably a mutant bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 80 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74. In one embodiment, the mutant meroterpenoid cyclase enzyme has the amino acid sequence provided in any of SEQ ID NOs: 181 to 204.

[0241] More preferably, the terpene cyclase enzyme is a mutant meroterpenoid cyclase enzyme, preferably a mutant bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181-204; preferably, to any one of SEQ ID NOs: 74 and 190-192; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74. In one embodiment, the mutant meroterpenoid cyclase enzyme has the amino acid sequence provided in any one of SEQ ID NOs: 190 to 192.

[0242] With reference to the above embodiments, the substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74 may be a substitution to methionine, serine or glycine.

[0243] Also, surprisingly, the present inventors found for the first time that squalene cyclases could be used for the cyclization of a linear terpenoid (such as a compound of the formula (I)) to a compound of the formula (A), (B), (C) and / or (D).

[0244] Accordingly, in another embodiment of the method of the invention, the terpene cyclase enzyme is a squalene cyclase enzyme.

[0245] Accordingly, another preferred embodiment of the invention is wherein the terpene cyclase enzyme is a squalene cyclase enzyme, comprising at least one one or more amino acid motifs selected from:

[0246] ■ [SP][TP][VIL]WDTx[LWI] (SEQ ID NO: 205),

[0247] . PGG[WF][GYA]F (SEQ ID NO: 206),

[0248] . PDxDD[TAS][TIAS] (SEQ ID NO: 207),

[0249] . [MIL]QxxxG[GA][WF]x[AS][FY] (SEQ ID NO: 208),

[0250] . Qxxx[GH]xWxG[RK]WGxx[YF]xYG (SEQ ID NO: 209),

[0251] . Qxx[DN]G[GS][WF][GS]ExxxS (SEQ ID NO: 210), and

[0252] . [STA]xx[SFN][QC]T[AGT]W[AS][LIV]xx[LQ] (SEQ ID NO: 211); wherein, residues x represent independently of each other any natural amino acid residue.

[0253] Accordingly, in a further preferred embodiment of the invention, the terpene cyclase enzyme is squalene cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 142 to 148. In one embodiment, said enzyme has the amino acid sequence of any one of SEQ ID NOs: 142 to 148.

[0254] Further aspects of the menthod of the invention

[0255] A further aspect of the invention provides a method for preparing a compound of the formula (A’1 a), (B’1), (C’1) and / or (D’1 a), wherein the method comprises contacting a compound of the formula (lb) with a terpene cyclase enzyme under conditions suitable for the terpene cyclase enzyme to produce the compound of the formula (A’1 a), (B’1), (C’1) and / or (D’1 a). In said aspect of the invention, the compound of the formula (A’1 a) may be a compound of the formula (A’1-1 a) or (A’1-2a); preferably, a compound of the formula (A’1-1 a).

[0256] In one embodiment of this aspect of the invention, the compound of the formula (A’1-1 a), (B’1) and / or (C’1) is the main product as defined in the Section “Definitions”. In particular, the relative ratio (%) of the compound of the formula (A’1-1 a), (B’1) and / or (C’1) based on the total quantified product compound (A), (B), (C) and / or (D) (namely, (A’1 a), (B’1), (C’1) and / or (D’1 a)) is in the range of 51 to 100%, particularly in the range of 60 to 100%, particularly in the range of 70 to 100%, particularly in the range of 75 to 100%, particularly in the range of 80 to 100%, particularly in the range of 85 to 100%, particularly in the range of 90 to 100%, particularly in the range of 95 to 100%; more in particular, in the range of 96 to 100%, particularly in the range of 97 to 100%, particularly in the range of 98 to 100%, particularly in the range of 99 to 100%, particularly in the range of 99,5 to 100%, particularly in the range of 99,8 to 100%.

[0257] In another embodiment of said aspect of the invention, the compound of the formula (A’1-1 a) is the main product as defined in the Section “Definitions”. In particular, the relative ratio (%) of compound of the formula (A’1-1 a) based on the total quantified product compound (A), (B), (C) and / or (D) (namely, (A’1 a), (B’1), (C’1) and / or (D’1 a)) is in the range of 51 to 100%, particularly in the range of 60 to 100%, particularly in the range of 70 to 100%, particularly in the range of 75 to 100%, particularly in the range of 80 to 100%, particularly in the range of 85 to 100%, particularly in the range of 90 to 100%, particularly in the range of 95 to 100%; more in particular, in the range of 96 to 100%, particularly in the range of 97 to 100%, particularly in the range of 98 to 100%, particularly in the range of 99 to 100%, particularly in the range of 99,5 to 100%, particularly in the range of 99,8 to 100%.

[0258] In said above embodiments, with the exception of SEQ ID NO: 144, the terpene cyclase enzyme may be any of the meroterpenoid cyclase enzymes and the squalene cyclase enzymes as defined in the Section “Terpene cyclases in the method of the invention”.

[0259] In another embodiment of said aspect of the invention, the compound of the formula (D’1 a) is the main product as defined in the Section “Definitions”. In particular, the relative ratio (%) of compound of the formula (D’1 a) based on the total quantified product compound (A), (B), (C) and / or (D) (namely, (A’1 a), (B’1), (C’1) and / or (D’1 a)) is in the range of 51 to 100%, particularly in the range of 60 to 100%, particularly in the range of 70 to 100%, particularly in the range of 75 to 100%, particularly in the range of 80 to 100%, particularly in the range of 85 to 100%, particularly in the range of 90 to 100%, particularly in the range of 95 to 100%; more in particular, in the range of 96 to 100%, particularly in the range of 97 to 100%, particularly in the range of 98 to 100%, particularly in the range of 99 to 100%, particularly in the range of 99,5 to 100%, particularly in the range of 99,8 to 100%.

[0260] In said above embodiment, the terpene cyclase enzyme is squalene cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 144. In one embodiment, said enzyme has the amino acid sequence of SEQ ID NO: 144.

[0261] Another further aspect of the invention provides a method for preparing a compound of the formula (A’3a), (B’3a), (C’3a) and / or (D’1 a), wherein the method comprises contacting a compound of the formula (li) with a terpene cyclase enzyme under conditions suitable for the terpene cyclase enzyme to produce the compound of the formula (A’3a), (B’3a), (C’3a) and / or (D’1 a). In said aspect of the invention, the compound of the formula (A’3a) may be a compound of the formula (A’3-1 a) or (A’3-2a); preferably, a compound of the formula (A’3-1 a).

[0262] In one embodiment of this aspect of the invention, the compound ofthe formula (B’3a) and / or (C’3a) is the main product as defined in the Section “Definitions”. In particular, the relative ratio (%) of the compound of the formula (B’3a) and / or (C’3a) based on the total quantified product compound (A), (B), (C) and / or (D) (namely, (A’3a), (B’3a), (C’3a) and / or (D’1 a)) is in the range of 51 to 100%, particularly in the range of 60 to 100%, particularly in the range of 70 to 100%, particularly in the range of 75 to 100%, particularly in the range of 80 to 100%, particularly in the range of 85 to 100%, particularly in the range of 90 to 100%, particularly in the range of 95 to 100%; more in particular, in the range of 96 to 100%, particularly in the range of 97 to 100%, particularly in the range of 98 to 100%, particularly in the range of 99 to 100%, particularly in the range of 99,5 to 100%, particularly in the range of 99,8 to 100%.

[0263] In said above embodiment, the terpene cyclase enzyme may be any of the meroterpenoid cyclase enzymes and the squalene cyclase enzymes as defined in the Section “Terpene cyclases in the method of the invention”. Preferably, the terpene cyclase is a meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 69, 71 , 73, 74, 75, 76, 77, 78, 100, 173, 177, 181 , 182, 184, 185, 188, 191 , 194, 197, 200, 202 and 203. In one embodiment, the meroterpenoid cyclase enzyme has the amino acid sequence provided in any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 69, 71 , 73, 74, 75, 76, 77, 78, 100, 173, 177, 181 , 182, 184, 185, 188, 191 , 194, 197, 200, 202 and 203.

[0264] Another further aspect of the invention provides a method for preparing a compound of the formula (A’5a), (B’5a) and / or (C’5a), wherein the method comprises contacting a compound of the formula (li) with a terpene cyclase enzyme under conditions suitable for the terpene cyclase enzyme to produce the compound of the formula (A’5a), (B’5a) and / or (C’5a). In said aspect of the invention, the compound of the formula (A’5a) may be a compound of the formula (A’5-1 a) or (A’5-2a); preferably, a compound of the formula (A’5-2a).

[0265] In one embodiment of this aspect of the invention, the compound ofthe formula (B’5a) and / or (C’5a) is the main product as defined in the Section “Definitions”. In particular, the relative ratio (%) of the compound of the formula (B’5a) and / or (C’5a) based on the total quantified product compound (A), (B), (C) and / or (D) (namely, (A’5a), (B’5a) and / or (C’5a)) is in the range of 51 to 100%, particularly in the range of 60 to 100%, particularly in the range of 70 to 100%, particularly in the range of 75 to 100%, particularly in the range of 80 to 100%, particularly in the range of 85 to 100%, particularly in the range of 90 to 100%, particularly in the range of 95 to 100%; more in particular, in the range of 96 to 100%, particularly in the range of 97 to 100%, particularly in the range of 98 to 100%, particularly in the range of 99 to 100%, particularly in the range of 99,5 to 100%, particularly in the range of 99,8 to 100%.

[0266] In said above embodiment, the terpene cyclase enzyme may be any of the meroterpenoid cyclase enzymes and the squalene cyclase enzymes as defined in the Section “Terpene cyclases in the method of the invention”. Preferably, the terpene cyclase is a meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 20, 29, 46, 48, 69, 71 , 73, 74, 75, 76, 77 and 78. In one embodiment, the meroterpenoid cyclase enzyme has the amino acid sequence provided in any one of SEQ ID NOs: 20, 29, 46, 48, 69, 71 , 73, 74, 75, 76, 77 and 78.

[0267] Obtention of compound ofthe formula (I) in the method ofthe invention

[0268] The person skilled in the art is aware that the compounds used as starting material, i.e. of the compound of the formula (I), are either commercially available (e.g. beta-farnesene) or can be synthesized by known methods.

[0269] Methods for the chemical production are for example described in e.g. Lee, Y., et al., (1982). Tetrahedron Lett., 23: 2671-2672.

[0270] Methods for the enzymatic production leverage metabolic engineering and biocatalysis to produce the compound of the formula (I); in particular, the compound of the formula (lb).

[0271] Accordingly, a further embodiment of this aspect of the invention is wherein the method futher comprises one or more steps prior to step (a), said step(s) comprising:

[0272] (i) preparing compound of the formula (la) from farnesyl diphosphate (FPP) using a beta-farnesene synthase; preferably, a E-beta-farnesene synthase (EC 4.2.3.47); and / or

[0273] (ii) preparing FPP from isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) using one or more enzymes having prenyltransferase activity; in particular, a FPP synthase; preferably, a EE-FPP synthase (EC 2.5.1.10). Examples of beta-farnesene synthase enzymes that can be used in step (i) of the method of the invention are well known in the art (see e.g. Tang, X., et al. (2025). Phytochemistry, 229: 114304). Such example is, but not limited to, the trans-beta-farnesene synthase (AAX39387.1) represented by SEQ ID NO: 149 in the present invention.

[0274] Examples of FPP synthase enzymes that can be used in step (ii) of the method of the invention are well known in the art (see e.g. Wang C. et al. (2017). Bioresource Technology, 241 : 430).

[0275] In this embodiment of the invention, the precursor compounds to FPP are provided from IPP and DMAPP.

[0276] Accordingly, a further embodiment of this aspect of the invention is wherein the method further comprises the steps of preparing IPP and DMAPP as described herein below.

[0277] One means for the preparation of IPP and DMAPP is via the “mevalonate pathway”. The “mevalonate pathway” also known as the “isoprenoid pathway” or “HMG-CoA reductase pathway” is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five-carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP). Combining the mevalonate pathway with enzyme activity to generate the terpene precursors GPP, FPP or GGPP allows the recombinant cellular production of terpenes. The pathway is well known in the art. The list of enzymes required for the conversion of acetyl- CoA to IPP and DMAPP is provided below:

[0278] . Acetyl-CoA acetyltransferase (ACAT),

[0279] . 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase),

[0280] . 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase),

[0281] . Mevalonate kinase,

[0282] . Phosphomevalonate kinase,

[0283] . Mevalonate diphosphate decarboxylase,

[0284] . Isopentenyl diphosphate isomerase.

[0285] An alternative means for the preparation of IPP, and DMAPP is via the methylerythritol phosphate (MEP). The pathway is well known in the art. The list of enzymes required for the conversion of glyceraldehyde 3-phosphate (GAP) and pyruvate to IPP and DMAPP is provided below:

[0286] . 1-Deoxy-D-xylulose 5-phosphate synthase (DXS),

[0287] . 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR),

[0288] . 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT, IspD),

[0289] . 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK, IspE),

[0290] . 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS, IspF),

[0291] . 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS, IDS),

[0292] . 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR). Further alternative pathways to the preparation of IPP and DMAPP are known, see for example: Rinaldi, M. A., et al. (2022). Natural Product Reports 39(1): 90-118. https: / / doi.org / 10.1039 / D1 NP00025J (see part 3 of this article).

[0293] Reaction conditions in the method of the invention

[0294] The method of the present invention may be an in vivo process or a bioconversion process.

[0295] The term in vivo process (or whole-cell production, or in-vivo production, or in-vivo biosynthesis) refers to a process of using a metabolically active cell where the primary metabolism is active to produce the precursors for the processes of the invention (preferably a microbial cell) to convert a carbon source to a new compound, such as the conversion of a carbon source to a terpene or terpene-derived compound.

[0296] Preferred sources of carbon are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add mixtures of various sources of carbon. Other possible sources of carbon are oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid or linoleic acid, alcohols such as glycerol, methanol or ethanol and organic acids such as acetic acid or lactic acid.

[0297] The cells thus contain all enzymes of one or more biosynthetic pathways. At least some of the enzymes involved in the method are part of the cell's primary metabolism. For example, the cells may contain the enzymes of a pathway to convert a carbon source (e.g., glucose, glycerol, isoprenol, prenol, CO2) to terpenoid precursors (e.g., IPP, DMAPP, FPP) and a pathway converting the terpene precursor to a terpene or terpene derived molecule such as a compound of the formula (I), (A), (B), (C) and / or (D). The enzymes may be present naturally in the cell or the cells can be transformed to produce the enzymes.

[0298] Alternatively, the methods of the present invention may be performed under bioconversion, also known as biotransformation conditions. Bioconversion processes refer to processes of conversion of compounds to different products using a biological process or agent such enzymes or whole cells (preferably a microbial cell). Bioconversion does not include the use of a cell's primary metabolism (as defined above) to produce the precursors for the methods of the invention. A bioconversion process can comprise multistep reactions each performed by a different enzyme. The compounds used in bioconversion process can be extracted from a natural source or produced using a separate chemical or a biochemical process.

[0299] The at least one polypeptide / enzyme which is present during the bioconversion method of the invention or an individual step of the multistep method as defined herein above, can be present in living cells naturally or recombinantly producing the enzyme or enzymes, in harvested cells, dead cells, in permeabilized cells, in crude cell extracts, in purified extracts, or in essentially pure or completely pure form, i.e. under bioconversion conditions. Such extracts may comprise membrane fraction or a liquid fraction prepared from the recombinant host cell that expresses at least one polypeptide / enzyme. The cells may be immobilized on a suitable substrate as is known in the art. At least one polypeptide / enzyme may be present in solution or as an enzyme immobilized on a carrier. One or several enzymes may simultaneously be present in soluble and / or immobilized forms.

[0300] It can be understood by the skilled person that there may be advantages for the use of an in vivo process.

[0301] In particular, a bioconversion process involves multiple steps, typically:

[0302] - Preparation or isolation of the starting compound to be transformed. The compound can be prepared using a chemical or biochemical process or by extraction from a natural source.

[0303] - Production of the enzymes or (living) cells used for the bioconversion.

[0304] - Biotransformation reaction by contacting the compound with the enzymes or (living) cells.

[0305] - Product Recovery and Refinement.

[0306] In comparison, an in vivo process requires a limited number of steps, generally limited to:

[0307] - The cultivation of the microorganism under conditions suitable for the production of the desired compound.

[0308] - Harvesting the cells or growing medium and purification of the desired compound.

[0309] Therefore, the in vivo process is usually more efficient and cost-effective than a bioconversion process.

[0310] Laboratory methods that can be used in in vivo and bioconversion processes of the invention are well known in the art. There follows a discussion on some of the methods that can be used.

[0311] The bioconversion processes according to the invention can be performed in common reactors, which are known to those skilled in the art, and in different ranges of scale, e.g. from a laboratory scale (few milliliters to dozens of liters of reaction volume) to an industrial scale (several liters to thousands of cubic meters of reaction volume). If the polypeptide is used in a form encapsulated by non-living, optionally permeabilized cells, in the form of a more or less purified cell extract or in purified form, a chemical reactor can be used. The chemical reactor usually allows controlling the amount of at least one enzyme, the amount of at least one substrate, the pH, the temperature and the circulation of the reaction medium.

[0312] Where the method of the invention is in vivo then it is preferred that the reaction is performed in a fermenter, where parameters necessary for suitable living conditions forthe living cells (e.g. culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, and the like) can be controlled.

[0313] The term "fermentative production" or "fermentation" refers to the ability of a microorganism (assisted by enzyme activity contained in or generated by said microorganism) to produce a chemical compound in cell culture utilizing at least one carbon source added to the incubation.

[0314] The term "fermentation broth" or "fermentation medium" is understood to mean a liquid, particularly aqueous or aqueous / organic solution which is based on a fermentative process and has not been worked up or has been worked up, for example, as described herein.

[0315] Those skilled in the art are familiar with chemical reactors or bioreactors, e.g. with procedures for up-scaling chemical or biotechnological methods from laboratory scale to industrial scale, or for optimizing process parameters, which are also extensively described in the literature (for biotechnological methods see e.g. Crueger und Crueger, Biotechnologie - Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Munchen, Wien, 1984).

[0316] The culture medium that is to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D. C., USA, 1981).

[0317] These media that can be used according to the invention may comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and / or trace elements.

[0318] Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials containing these compounds. Examples of sources of nitrogen include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex sources of nitrogen, such as corn-steep liquor, soybean flour, soy-bean protein, yeast extract, meat extract and others. The sources of nitrogen can be used separately or as a mixture.

[0319] Inorganic salt compounds that may be present in the media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0320] Inorganic sulfur-containing compounds, for example sulfates, sulfites, di-thionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as sources of sulfur. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used as sources of phosphorus.

[0321] Chelating agents can be added to the medium, in orderto keep the metal ions in solution. Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0322] The fermentation media used according to the invention may also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from complex components of the media, such as yeast extract, molasses, corn-steep liquor and the like. In addition, suitable precursors can be added to the culture medium. The precise composition of the compounds in the medium is strongly dependent on the particular experiment and must be decided individually for each specific case. Information on media optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (1997) Growing media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.

[0323] All components of the medium are sterilized, either by heating (20 min at 1 .5 bar and 121 °C) or by sterile filtration. The components can be sterilized either together or if necessary, separately. All the components of the medium can be present at the start of growing, or optionally can be added continuously or by batch feed.

[0324] The temperature of the culture is normally between 15 °C and 45 °C, preferably 25 °C to 40 °C and can be kept constant or can be varied during the experiment. The pH value of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH value for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, e.g. fatty acid polyglycol esters, can be used for controlling foaming. To maintain the stability of plasmids, suitable substances with selective action, e.g. antibiotics, can be added to the medium. Oxygen or oxygen-containing gas mixtures, e.g. the ambient air, are fed into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20 °C to 45 °C. Culture is continued until a maximum of the desired product has formed. This is normally achieved within 1 hour to 160 hours.

[0325] Where the method of the invention is a bioconversion, cells containing the at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or radiofrequency pulses, French presses, or chemical means, such as hypotonic media, lytic enzymes and detergents present in the medium, or combination of such methods. Examples for detergents are SDS, digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween ® 20, deoxycholate, CHAPS (3-[(3-

[0326] Cholamidopropyl)dimethylammonio]-1-propansulfonate), Nonidet ® P40 (Ethylphenolpoly(ethyleneglycolether), and the like. As stated above, where the method of the invention is an in vivo process, then a detergent is not required for the reasons stated herein.

[0327] The conversion reaction can be carried out batch wise, semi-batch wise or continuously. Reactants (and optionally nutrients) can be supplied at the start of reaction or can be supplied subsequently, either semi-continuously or continuously.

[0328] The bioconversion reaction of the invention, depending on the particular reaction type, may be performed in an aqueous, aqueous-organic or non-aqueous reaction medium.

[0329] An aqueous or aqueous-organic medium may contain a suitable buffer in order to adjust the pH to a value in the range of 5 to 1 1 , like 6 to 10.

[0330] In an aqueous-organic medium an organic solvent miscible, partly miscible or immiscible with water may be applied. Non-limiting examples of suitable organic solvents are listed below. Further examples are mono- or polyhydric, aromatic or aliphatic alcohols, in particular monohydric aliphatic alcohols, most preferably ethanol.

[0331] The non-aqueous medium may contain is substantially free of water, i.e. will contain less than about 1 wt.-% or 0.5 wt.-% of water.

[0332] Bioconversion methods may also be performed in an organic non-aqueous medium. As suitable organic solvents there may be mentioned aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; aromatic carbohydrates, like benzene, toluene, xylenes, chlorobenzene or dichlorobenzene, aliphatic acyclic and ethers, like diethylether, methyl-tert. -butylether, ethyl-tert. -butylether, dipropylether, diisopropylether, dibutylether; or mixtures thereof.

[0333] The concentration of the reactants / substrates may be adapted to the optimum bioconversion reaction conditions, which may depend on the specific enzyme applied. For example, the initial substrate concentration may be in the 0,1 to 0,5 M, as for example 10 to 100 mM.

[0334] The bioconversion reaction temperature may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the reaction may be performed at a temperature in a range of from 0 to 70 °C, as for example 20 to 50 or 25 to 40°C. Examples for reaction temperatures are about 30 °C, about 35 °C, about 37 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C and about 60 °C.

[0335] The bioconversion may proceed until equilibrium between the substrate and then produces) is achieved, but may be stopped earlier. Usual process times are in the range from 1 minute to 25 hours, in particular 10 min to 6 hours, as for example in the range from 1 hour to 4 hours, in particular 1 .5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.

[0336] In a preferred embodiment of the present invention, the method is performed in a recombinant (host) cell capable of functionally expressing the terpene cyclase enzyme as defined herein above.

[0337] Advantageously, microorganisms such as bacteria, fungi or yeasts are used as host cells. Advantageously, gram-positive or gram-negative bacteria are used, preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, Streptococcaceae or Nocardiaceae, especially preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Lactococcus, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus. The genus and species Escherichia coll is quite especially preferred. Furthermore, other advantageous bacteria are to be found in the group of alpha-Proteobacteria, beta-Proteobacteria or gamma-Proteobacteria. Advantageously also yeasts of families like Saccharomyces or Pichia are suitable hosts.

[0338] Preferably, the cell is a bacterium or a fungal cell, in particular a yeast cell. Preferably, the cell is a unicellular organism, a cultured cell derived from a multi-cellular organism, a cell present in a cultured tissue derived from a multicellular organism, or a cell present in a living multicellular organism. Preferably, the cell is a bacterial cell of the genus Escherichia, preferably E. coll, or a yeast cell of the genus Saccharomyces, preferably S. cerevisiae, of the genus Yarrowia, preferably Y. lipolytica, or of the genus Pichia, preferably P. pastoris.

[0339] Alternatively, entire plants or plant cells may serve as natural or recombinant host. As non-limiting examples, the following plants or cells derived therefrom may be mentioned: the genera Nicotiana, in particular Nicotiana benthamiana and Nicotiana tabacum (tobacco); as well as Arabidopsis, in particular Arabidopsis thaliana.

[0340] Product isolation in the method of the invention

[0341] The methodology of the present invention can further include a step of recovering an end product or an intermediate product, optionally in stereoisomerically or enantiomerically substantially pure form. The term “recovering” includes extracting, harvesting, isolating or purifying the compound from culture or reaction media. Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like. Identity and purity of the isolated product may be determined by known techniques, like High Performance Liquid Chromatography (HPLC), gas chromatography (GC), Spektroskopy (like IR, UV, NMR), Colouring methods, TLC, NIRS, enzymatic or microbial assays, (see for example: Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; und Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S. 89-90, S. 521-540, S. 540-547, S. 559-566, 575-581 und S. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.).

[0342] The compounds produced in any of the methods described herein can be converted to derivatives ("derivatives thereof) such as, but not limited to hydrocarbons, esters, amides, glycosides, ethers, epoxides, aldehydes, ketones, alcohols, diols, acetals or ketals. The terpene compound derivatives can be obtained by a chemical method or a biochemical (enzymatic) method. The biochemical conversion can be performed in vivo or in vitro using isolated enzymes, enzymes from lysed cells or bioconversion using whole cells.

[0343] Recombinant cells of the invention

[0344] A further aspect of the invention provides a recombinant cell comprising, capable of producing or producing a compound of the formula (A), (B), (C) and / or (D); preferably, a compound of the formula (A’), (B’), (C’) and / or (D’); more preferably, a compound selected from the group consisting of the compound of the formula (A’1), (A’2), (A’3), (A’4), (A’5), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’3), (C’4), (C’5), (D’1 a) and / or (D’1 b); more preferably, a compound selected from the group consisting of the compound of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’4), (C’5), (D’1 b), (A’1-1) and (A’ 1-2).

[0345] In a further preferred embodiment, the compound of the formula (A’1-1) is a compound of the formula (A’1 -1 a) or (A’1-1 b); preferably, of the formula (A’1 -1 a); the compound of the formula (A’1 -2) is a compound of the formula (A’1-2a); the compound of the formula (B’3) is a compound of the formula (B’3a); the compound of the formula (B’5) is a compound of the formula (B’5a); and the compound of the formula (C’5) is a compound of the formula of the formula (C’5a).

[0346] Methods for preparing a recombinant cell comprising, capable of producing or producing said compound are provided herein.

[0347] In an embodiment of said aspect of the invention, the cell comprises, is capable of expressing or expresses a terpene cyclase enzyme, preferably a meroterpenoid cyclase enzyme, more preferably a bacterial membrane-integrated meroterpenoid cyclase enzyme. Said meroterpenoid cyclase enzyme preferably comprises at least one or more amino acid motifs selected from: . [W]xxx[D]xx[ILVMN] (SEQ ID NO: 212),

[0348] . PxxAxxxNxxWE (SEQ ID NO: 213),

[0349] . MxxxFxxMLxxR (SEQ ID NO: 214),

[0350] . RxxxxGQS (SEQ ID NO: 215), and

[0351] . NxxMS (SEQ ID NO: 216); wherein residues x represent independently of each other any natural amino acid residue.

[0352] In a preferred embodiment of the invention, the cell comprises, is capable of expressing or expresses a meroterpenoid cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204, preferably to any one of SEQ ID NOs: 1 to 96 and 181 to 204, more preferably to any one of SEQ ID NOs: 1 to 80 and 181 to 204. In a further embodiment, the cell comprises a meroterpenoid cyclase enzyme having the amino acid sequence of any one of SEQ ID NOs: 1 to 141 and 162 to 204, preferably to any one of SEQ ID NOs: 1 to 96 and 181 to 204, more preferably of any one of SEQ ID NOs: 1 to 80 and 181 to 204. In said embodiments, said meroterpenoid cyclase enzyme may be a mutant meroterpenoid cyclase enzyme having an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29. In a specific embodiment, the mutant meroterpenoid cyclase enzyme is represented by SEQ ID NO: 80. In alternative or further embodiments, said meroterpenoid cyclase enzyme may be a mutant meroterpenoid cyclase enzyme having an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74. In a specific embodiment, the mutant meroterpenoid cyclase enzyme is represented by any one of SEQ ID NOs: 181 to 204.

[0353] In another preferred embodiment of the invention, the cell comprises, is capable of expressing or expresses a meroterpenoid cyclase enzyme, preferably a bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181-204; preferably, to any one of SEQ ID NOs: 74 and 190-192. In a further embodiment, the cell comprises a meroterpnoid cyclase enzyme having the amino acid sequence of any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181-204; preferably, to any one of SEQ ID NOs: 74 and 190-192. In said embodiments, said meroterpenoid cyclase enzyme may be a mutant meroterpenoid cyclase enzyme having an amino acid substitution at amino acid position 9 relative to sequence provided in SEQ ID NO: 29. In a specific embodiment, the mutant meroterpenoid cyclase enzyme is represented by SEQ ID NO: 80. In alternative or further embodiments, said meroterpenoid cyclase enzyme may be a mutant meroterpenoid cyclase enzyme having an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74. In a specific embodiment, the mutant meroterpenoid cyclase enzyme is represented by any one of SEQ ID NOs: 190 to 192.

[0354] With reference to the above embodiments, the substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29 may be a substitution to cysteine, methionine or threonine. With reference to the above embodiments, the substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74 may be a substitution to methionine, serine or glycine.

[0355] In another embodiment of said aspect of the invention, the cell comprises, is capable of expressing or expresses a squalene cyclase enzyme. Said squalene cyclase enzyme preferably comprises at least one or more amino acid motifs selected from:

[0356] ■ [SP][TP][VIL]WDTx[LWI] (SEQ ID NO: 205),

[0357] . PGG[WF][GYA]F (SEQ ID NO: 206),

[0358] . PDxDD[TAS][TIAS] (SEQ ID NO: 207),

[0359] . [MIL]QxxxG[GA][WF]x[AS][FY] (SEQ ID NO: 208),

[0360] . Qxxx[GH]xWxG[RK]WGxx[YF]xYG (SEQ ID NO: 209),

[0361] . Qxx[DN]G[GS][WF][GS]ExxxS (SEQ ID NO: 210), and

[0362] . [STA]xx[SFN][QC]T[AGT]W[AS][LIV]xx[LQ] (SEQ ID NO: 211); wherein, residues x represent independently of each other any natural amino acid residue.

[0363] In a preferred embodiment, the cell comprises, is capable of expressing or expresses a squalene cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 142 to 148. In a further embodiment, the cell comprises a squalene cyclase enzyme having the sequence of any one of SEQ ID NOs: 142 to 148.

[0364] The recombinant cell may be any such cell suitable for the production of a compound of the formula (A), (B), (C) and / or (D).

[0365] A list of suitable cells for the production of a compound of the formula (A). (B), (C) and / or (D) is provided above in relation to the method of the invention and are also cells for this aspect of the invention.

[0366] Preferably, the cell is a bacterium or a fungal cell, in particular a yeast. Preferably, the cell is a unicellular organism, a cultured cell derived from a multi-cellular organism, a cell present in a cultured tissue derived from a multicellular organism, or a cell present in a living multicellular organism. Preferably, the cell is a bacterial cell of the genus Escherichia, preferably E. coli, of the genus Pseudomonas, preferably Pseudomonas alloputida, or a yeast cell of the genus Saccharomyces, preferably S. cerevisiae, of the genus Yarrowia, preferably Y. lipolytica, or of the genus Pichia, preferably P. pastoris.

[0367] Methods of introducing recombinant nucleic acid sequences into such host cells are well known in the art and constitute routine laboratory methodologies which do not need to be further described herein.

[0368] An embodiment of this aspect of the invention is wherein the cell further comprises, is capable of expressing or expresses one or more prenyltransferase enzyme(s); in particular, one or more FPP synthase enzymes (EC 2.5.1.10). Preferably, the cell further comprises one or more beta-farnesene synthase enzymes (EC 4.2.3.47).

[0369] A further embodiment of this aspect of the invention is wherein the cell of the invention comprises, is capable of expressing or expresses enzymes for providing IPP and DMAPP. Said compound may be provided via the “mevalonate pathway”, methylerythritol phosphate (MEP) pathway or alternative pathways for the preparation of IPP and DMAPP.

[0370] In one embodiment of the invention, the cell comprises, is capable of expressing or expresses enzymes of the mevalonate pathway:

[0371] . Acetyl-CoA acetyltransferase (ACAT)

[0372] . 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)

[0373] . 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase)

[0374] . Mevalonate kinase

[0375] . Phosphomevalonate kinase

[0376] . Mevalonate diphosphate decarboxylase

[0377] . Isopentenyl diphosphate isomerase

[0378] . Dimethylallyl diphosphate synthase

[0379] In a further embodiment of the invention, the cell comprises, is capable of expressing or expresses enzymes of the MEP pathway:

[0380] . 1-Deoxy-D-xylulose 5-phosphate synthase (DXS)

[0381] . 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR)

[0382] . 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT, IspD)

[0383] . 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK, IspE)

[0384] . 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS, IspF)

[0385] . 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS, IDS)

[0386] . 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR)

[0387] Cell culture fermentation media of the invention

[0388] A further aspect of the invention provides a cell culture fermentation medium comprising a compound of the formula (A), (B), (C) and / or (D); preferably, a compound of the formula (A’), (B’), (C’) and / or (D’); more preferably, a compound of the formula (A’1), (A’2), (A’3), (A’4), (A’5), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’3), (C’4), (C’5), (D’1 a) and / or (D’1 b); more preferably, a compound selected from the group consisting of the compound of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’4), (C’5), (D’1 b), (A’1-1) and (A’1-2).

[0389] In a further preferred embodiment, the compound of the formula (A’1-1) is a compound of the formula (A’1 -1 a) or (A’1-1 b); preferably, of the formula (A’1 -1 a); the compound of the formula (A’1-2) is a compound of the formula (A’1-2a); the compound of the formula (B’3) is a compound of the formula (B’3a); the compound of the formula (B’5) is a compound of the formula (B’5a); and the compound of the formula (C’5) is a compound of the formula of the formula (C’5a).

[0390] Accordingly, a further aspect of the invention provides a cell culture fermentation medium comprising the recombinant cell of the invention as described herein above.

[0391] The cell culture fermentation media can be a nutrient rich broth for the growth and maintenance of the cells during the production phase. Yeast culture conditions for maintaining and propagating various strains can require specific formulations of complex media for use in cloning and protein expression, and can be appreciated by those of skill in the art. Commercially available culture media can be used from ThermoFisher for example. The media can be YPD broth or can have a yeast nitrogen base. Yeast can be grown in YPD or synthetic media at 30 °C.

[0392] Lysogeny broth (LB) is typically used for bacterial cells. The bacterial cells can have antibiotic resistance to prevent the growth of other cells in the culture media and contamination.

[0393] Reaction mixture of the invention

[0394] The method of the invention may be a bioconversion process.

[0395] Accordingly, an aspect of the present invention is a reaction mixture comprising a compound of the formula (A), (B), (C) and / or (D); preferably, a compound of the formula (A’), (B’), (C’) and / or (D’); more preferably, a compound of the formula (A’1), (A’2), (A’3), (A’4), (A’5), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’3), (C’4), (C’5), (D’1 a) and / or (D’1 b); more preferably, a compound selected from the group consisting of the compound of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’4), (C’5), (D’1 b), (A’1-1) and (A’1-2).

[0396] In a further preferred embodiment, the compound of the formula (A’1-1) is a compound of the formula (A’1 -1 a) or (A’1-1 b); preferably, of the formula (A’1 -1 a); the compound of the formula (A’1-2) is a compound of the formula (A’1-2a); the compound of the formula (B’3) is a compound of the formula (B’3a); the compound of the formula (B’5) is a compound of the formula (B’5a); and the compound of the formula (C’5) is a compound of the formula of the formula (C’5a).

[0397] Additional components of the reaction mixture may include detergents, co-factors, cells, cell-debris, cell culture media, and other such components well known to the person skilled in the art.

[0398] New compounds of the invention

[0399] A further aspect of the invention provides a compound obtained or obtainable by a method of the invention or from a recombinant cell of the invention or from a cell culture fermentation medium of the invention or from a reaction mixture of the invention as described herein above. Preferably, a futher aspect of the invention provides a compound selected from the group consisting of the compound of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5), (C’1), (C’2), (C’4), (C’5), (D’1 b), (A’1-1) and (A’1-2).

[0400] In a further preferred embodiment, the compound of the formula (A’1-1) is a compound of the formula (A’1-1 a) or (A’1-1 b); preferably, of the formula (A’1-1 a); the compound of the formula (A’1-2) is a compound of the formula (A’1-2a); the compound of the formula (B’3) is a compound of the formula (B’3a); the compound of the formula (B’5) is a compound of the formula (B’5a); and the compound of the formula (C’5) is a compound of the formula of the formula (C’5a).

[0401] Use of compounds of the invention

[0402] A further aspect of the invention comprises the use of a compound according to any of the previous embodiments of the invention as a perfumery, flavor or aroma ingredient, or as a precursor thereof.

[0403] As mentioned above, the invention comprises the use of a compound of the invention as a perfuming ingredient, or as a precursor thereof. In other words, it concerns a method or a process to confer, enhance, improve or modify the odor properties of a perfuming composition or of a perfumed article or of a surface, which method comprises adding to said composition or article an effective amount of at least a compound of the invention, e.g. to impart its typical note. Understood that the final hedonic effect may depend on the precise dosage and on the organoleptic properties of the invention’s compound, but anyway the addition of the invention’s compound will impart to the final product its typical touch in the form of a note, touch or aspect depending on the dosage.

[0404] By “use of a compound of the invention” it has to be understood here also the use of any composition containing a compound of the invention and which can be advantageously employed in the perfumery industry.

[0405] Said compositions, which in fact can be advantageously employed as perfuming ingredients, are also an object of the present invention.

[0406] Therefore, another object of the present invention is a perfuming composition comprising: i) as a perfuming ingredient, at least one invention’s compound as defined above; ii) at least one ingredient selected from the group consisting of a perfumery carrier and a perfumery base; and iii) optionally at least one perfumery adjuvant. By “perfumery carrier” it is meant here a material which is practically neutral from a perfumery point of view, i.e. that does not significantly alter the organoleptic properties of perfuming ingredients. Said carrier may be a liquid or a solid.

[0407] As liquid carrier one may cite, as non-limiting examples, an emulsifying system, i.e. a solvent and a surfactant system, or a solvent commonly used in perfumery. A detailed description of the nature and type of solvents commonly used in perfumery cannot be exhaustive. However, one can cite as non-limiting examples, solvents such as butylene or propylene glycol, glycerol, dipropyleneglycol and its monoether, 1 ,2,3-propanetriyl triacetate, dimethyl glutarate, dimethyl adipate 1 ,3-diacetyloxypropan-2-yl acetate, diethyl phthalate, isopropyl myristate, benzyl benzoate, benzyl alcohol, 2-(2-ethoxyethoxy)-1 -ethanol, triethyl citrate or mixtures thereof, which are the most commonly used. For the compositions which comprise both a perfumery carrier and a perfumery base, other suitable perfumery carriers than those previously specified, can be also ethanol, water / ethanol mixtures, limonene or other terpenes, isoparaffins such as those known under the trademark Isopar™ (origin: Exxon Chemical) or glycol ethers and glycol ether esters such as those known under the trademark Dowanol™ (origin: Dow Chemical Company), or hydrogenated castors oils such as those known under the trademark Cremophor® RH 40 (origin: BASF).

[0408] Solid carrier is meant to designate a material to which the perfuming composition or some element of the perfuming composition can be chemically or physically bound. In general, such solid carriers are employed either to stabilize the composition, or to control the rate of evaporation of the compositions or of some ingredients. Solid carriers are of current use in the art and a person skilled in the art knows how to reach the desired effect. However, by way of non-limiting examples of solid carriers, one may cite absorbing gums or polymers or inorganic materials, such as porous polymers, cyclodextrins, wood-based materials, organic or inorganic gels, clays, gypsum talc or zeolites.

[0409] As other non-limiting examples of solid carriers, one may cite encapsulating materials. Examples of such materials may comprise wall-forming and plasticizing materials, such as mono, di- or trisaccharides, natural or modified starches, hydrocolloids, cellulose derivatives, polyvinyl acetates, polyvinylalcohols, proteins or pectins, or yet the materials cited in reference texts such as H. Scherz, Hydrokolloide: Stabilisatoren, Dickungs- und Geliermittel in Lebensmitteln, Band 2 der Schriftenreihe Lebensmittelchemie, Lebensmittelqualitat, Behr's Verlag GmbH & Co., Hamburg, 1996. The encapsulation is a well-known process to a person skilled in the art, and may be performed, for instance, by using techniques such as spray-drying, agglomeration or yet extrusion; or consists of a coating encapsulation, including coacervation and complex coacervation techniques.

[0410] As non-limiting examples of solid carriers, one may cite in particular the core-shell capsules with resins of aminoplast, polyamide, polyester, polyurea or polyurethane type or a mixture thereof (all of said resins are well known to a person skilled in the art) using techniques like phase separation process induced by polymerization, interfacial polymerization, coacervation or altogether (all of said techniques have been described in the prior art), optionally in the presence of a polymeric stabilizer or of a cationic copolymer. Resins may be produced by the polycondensation of an aldehyde (e.g. formaldehyde, 2,2- dimethoxyethanal, glyoxal, glyoxylic acid or glycolaldehyde and mixtures thereof) with an amine such as urea, benzoguanamine, glycoluryl, melamine, methylol melamine, methylated methylol melamine, guanazole and the like, as well as mixtures thereof. Alternatively, one may use preformed resins alkylolated polyamines such as those commercially available under the trademark Urac® (origin: Cytec Technology Corp.), Cymel® (origin: Cytec Technology Corp.), Urecoll® or Luracoll® (origin: BASF).

[0411] Other resins are the ones produced by the polycondensation of an a polyol, like glycerol, and a polyisocyanate, like a trimer of hexamethylene diisocyanate, a trimer of isophorone diisocyanate or xylylene diisocyanate or a Biuret of hexamethylene diisocyanate or a trimer of xylylene diisocyanate with trimethylolpropane (known with the tradename of Takenate®, origin: Mitsui Chemicals), among which a trimer of xylylene diisocyanate with trimethylolpropane and a Biuret of hexamethylene diisocyanate are preferred.

[0412] Some of the seminal literature related to the encapsulation of perfumes by polycondensation of amino resins, namely melamine-based resins with aldehydes includes articles such as those published by K. Dietrich et al. Acta Polymerica, 1989, vol. 40, pages 243, 325 and 683, as well as 1990, vol. 41 , page 91 . Such articles already describe the various parameters affecting the preparation of such core-shell microcapsules following prior art methods that are also further detailed and exemplified in the patent literature. US 4’396'670, to the Wiggins Teape Group Limited is a pertinent early example of the latter. Since then, many other authors have enriched the literature in this field and it would be impossible to cover all published developments here, but the general knowledge in encapsulation technology is very significant. More recent publications of pertinence, which disclose suitable uses of such microcapsules, are represented for example by the article of K. Bruyninckx and M. Dusselier, ACS Sustainable Chemistry & Engineering, 2019, vol. 7, pages 8041-8054.

[0413] By “perfumery base” what is meant here is a composition comprising at least one perfuming coingredient.

[0414] Said perfuming co-ingredient is not a compound of the invention. Moreover, by “perfuming coingredient” it is meant here a compound, which is used in a perfuming preparation or a composition to impart a hedonic effect. In other words such a co-ingredient, to be considered as being a perfuming one, must be recognized by a person skilled in the art as being able to impart or modify in a positive or pleasant way the odor of a composition, and not just as having an odor. The perfuming ingredient may impart an additional benefit beyond that of modifying or imparting an odor, such as long-lasting, blooming, malodour counteraction, antimicrobial effect, antiviral effect, microbial stability, or pest control.

[0415] The nature and type of the perfuming co-ingredients present in the base do not warrant a more detailed description here, which in any case would not be exhaustive, the skilled person being able to select them on the basis of his general knowledge and according to the intended use or application and the desired organoleptic effect. In general terms, these perfuming co-ingredients belong to chemical classes as varied as alcohols, lactones, aldehydes, ketones, esters, ethers, acetates, nitriles, terpenoids, nitrogenous or sulphurous heterocyclic compounds and essential oils, and said perfuming co-ingredients can be of natural or synthetic origin.

[0416] In particular one may cite perfuming co-ingredients which are commonly used in perfume formulations, such as:

[0417] Aldehydic ingredients: decanal, dodecanal, 2-methyl-undecanal, 10-undecenal, octanal, nonanal and / or nonenal;

[0418] Aromatic-herbal ingredients: eucalyptus oil, camphor, eucalyptol, 5- methyltricyclo[6.2.1 ,0~2,7~]undecan-4-one, 1-methoxy-3-hexanethiol, 2-ethyl-4,4-dimethyl-1 ,3-oxathiane, 2,2,7 / 8,9 / 10-Tetramethylspiro[5.5]undec-8-en-1-one, menthol and / or alpha-pinene;

[0419] Balsamic ingredients: coumarin, ethylvanillin and / or vanillin;

[0420] Citrus ingredients: dihydromyrcenol, citral, orange oil, linalyl acetate, citronellyl nitrile, orange terpenes, limonene, 1-p-menthen-8-yl acetate and / or 1 ,4(8)-p-menthadiene;

[0421] Floral ingredients:methyl dihydrojasmonate, linalool, citronellol, phenylethanol, 3-(4-tert- butylphenyl)-2-methylpropanal, hexylcinnamic aldehyde, benzyl acetate, benzyl salicylate, tetrahydro-2- isobutyl-4-methyl-4(2H)-pyranol, beta ionone, methyl 2-(methylamino)benzoate, (E)-3-methyl-4-(2,6,6- trimethyl-2-cyclohexen-1-yl)-3-buten-2-one, (1 E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-1-penten-3-one, 1- (2,6,6-trimethyl-1 ,3-cyclohexadien-1 -yl)-2-buten-1 -one, (2E)-1 -(2,6,6-trimethyl-2-cyclohexen-1 -yl)-2- buten-1 -one, (2E)-1 -[2,6,6-trimethyl-3-cyclohexen-1 -yl]-2-buten-1 -one, (2E)-1 -(2,6,6-trimethyl-1 - cyclohexen-1 -yl)-2-buten-1 -one, 2,5-dimethyl-2-indanmethanol, 2,6,6-trimethyl-3-cyclohexene-1 - carboxylate, 3-(4,4-dimethyl-1 -cyclohexen-1 -yl)propanal, 3-(3,3 / 1 ,1-dimethyl-5-indanyl)propanal, hexyl salicylate, 3,7-dimethyl-1 ,6-nonadien-3-ol, 3-(4-isopropylphenyl)-2-methylpropanal, verdyl acetate, geraniol, p-menth-1-en-8-ol, 4-(1 ,1-dimethylethyl)-1-cyclohexyle acetate, 1 ,1-dimethyl-2-phenylethyl acetate, 4-cyclohexyl-2-methyl-2-butanol, amyl salicylate , high cis methyl dihydrojasmonate, 3-methyl-5- phenyl-1 -pentanol, verdyl proprionate, geranyl acetate, tetrahydro linalool, cis-7-p-menthanol, propyl (S)- 2-(1 ,1-dimethylpropoxy)propanoate, 2-methoxynaphthalene, 2, 2, 2-trichloro-1 -phenylethyl acetate, 4 / 3-(4- hydroxy-4-methylpentyl)-3-cyclohexene-1-carbaldehyde, amylcinnamic aldehyde, 8-decen-5-olide, 4- phenyl-2-butanone, isononyle acetate, 4-(1 , 1 -dimethylethyl)-1 -cyclohexyl acetate, verdyl isobutyrate and / or mixture of methylionones isomers;

[0422] Fruity ingredients: gamma-undecalactone, 2,2,5-trimethyl-5-pentylcyclopentanone, 2-methyl-4- propyl-1 ,3-oxathiane, 4-decanolide, ethyl 2-methyl-pentanoate, hexyl acetate, ethyl 2-methylbutanoate, gamma-nonalactone, allyl heptanoate, 2-phenoxyethyl isobutyrate, ethyl 2-methyl-1 ,3-dioxolane-2-acetate, diethyl 1 ,4-cyclohexanedicarboxylate, 3-methyl-2-hexen-1-yl acetate, 1-[3,3-dimethylcyclohexyl]ethyl [3- ethyl-2-oxiranyl]acetate and / or diethyl 1 ,4-cyclohexane dicarboxylate;

[0423] Green ingredients: 2-methyl-3-hexanone (E)-oxime, 2,4-dimethyl-3-cyclohexene-1-carbaldehyde, 2-tert-butyl-1 -cyclohexyl acetate, styrallyl acetate, allyl (2-methylbutoxy)acetate, 4-methyl-3-decen-5-ol, diphenyl ether, (Z)-3-hexen-1-ol and / or 1 -(5, 5-dimethyl-1 -cyclohexen-1 -yl)-4-penten-1 -one; Musk ingredients: 1 ,4-dioxa-5,17-cycloheptadecanedione, (Z)-4-cyclopentadecen-1-one, 3- methylcyclopentadecanone, 1-oxa-12-cyclohexadecen-2-one, 1-oxa-13-cyclohexadecen-2-one, (9Z)-9- cycloheptadecen-1-one, 2-{(1 S)-1 -[(1 R)-3,3-dimethylcyclohexyl]ethoxy}-2-oxoethyl propionate, 3-methyl- 5-cyclopentadecen-1-one, 4,6,6,7,8,8-hexamethyl-1 ,3,4,6,7,8-hexahydrocyclopenta[g]isochromene, (1 S,1 'R)-2-[1-(3',3'-dimethyl-1 '-cyclohexyl)ethoxy]-2-methylpropyl propanoate, oxacyclohexadecan-2-one and / or (1 S,1 'R)-[1-(3',3'-dimethyl-1 '-cyclohexyl)ethoxycarbonyl]methyl propanoate;

[0424] Woody ingredients: 1-[(1 RS,6SR)-2,2,6-trimethylcyclohexyl]-3-hexanol, 3,3-dimethyl-5-[(1 R)- 2,2,3-trimethyl-3-cyclopenten-1-yl]-4-penten-2-ol, 3,4'-dimethylspiro[oxirane-2,9'- tricyclo[6.2.1 ,02,7]undec[4]ene, (l-ethoxyethoxy)cyclododecane, 2,2,9, 11-tetramethylspiro[5.5]undec-8- en-1-yl acetate, 1 -(octahydro-2, 3, 8, 8-tetramethyl-2-naphtalenyl)-1 -ethanone, patchouli oil, terpenes fractions of patchouli oil, Clearwood®, (1 'R,E)-2-ethyl-4-(2',2',3'-trimethyl-3'-cyclopenten-1 '-yl)-2-buten-1- ol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol, methyl cedryl ketone, 5-(2,2,3-trimethyl-3- cyclopentenyl)-3-methylpentan-2-ol, 1-(2,3,8,8-tetramethyl-1 ,2,3,4,6,7,8,8a-octahydronaphthalen-2- yl)ethan-1-one and / or isobornyl acetate;

[0425] Other ingredients (e.g. amber, powdery spicy or watery): dodecahydro-3a,6,6,9a-tetramethyl- naphtho[2,1-b]furan and any of its stereoisomers, heliotropin, anisic aldehyde, eugenol, cinnamic aldehyde, clove oil, 3-(1 ,3-benzodioxol-5-yl)-2-methylpropanal, 7-methyl-2H-1 ,5-benzodioxepin-3(4H)-one, 2,5,5- trimethyl-1 ,2,3,4,4a,5,6,7-octahydro-2-naphthalenol, 1-phenylvinyl acetate, 6-methyl-7-oxa-1-thia-4- azaspiro[4.4]nonane and / or 3-(3-isopropyl-1-phenyl)butanal.

[0426] A perfumery base according to the invention may not be limited to the above mentioned perfuming co-ingredients, and many other of these co-ingredients are in any case listed in reference texts such as the book by S. Arctander, Perfume and Flavor Chemicals, 1969, Montclair, New Jersey, USA, or its more recent versions, or in other works of a similar nature, as well as in the abundant patent literature in the field of perfumery. It is also understood that said co-ingredients may also be compounds known to release in a controlled manner various types of perfuming compounds also known as properfume or profragrance. Nonlimiting examples of suitable properfume may include 4-(dodecylthio)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)- 2-butanone, 4-(dodecylthio)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-butanone, trans-3-(dodecylthio)-1- (2,6,6-trimethyl-3-cyclohexen-1-yl)-1-butanone, 2-(dodecylthio)octan-4-one, 2-phenylethyl oxo(phenyl)acetate, 3,7-dimethylocta-2,6-dien-1-yl oxo(phenyl)acetate, (Z)-hex-3-en-1-yl oxo(phenyl)acetate, 3,7-dimethyl-2,6-octadien-1 -yl hexadecanoate, bis(3,7-dimethylocta-2,6-dien-1 -yl) succinate (2-((2-methylundec-1 -en-1 -yl)oxy)ethyl)benzene, 1 -methoxy-4-(3-methyl-4-phenethoxybut-3-en- 1 -yl)benzene, (3-methyl-4-phenethoxybut-3-en-1 -yl)benzene, 1 -(((Z)-hex-3-en-1 -yl)oxy)-2-methylundec-1 - ene, (2-((2-methylundec-1-en-1-yl)oxy)ethoxy)benzene, 2-methyl-1-(octan-3-yloxy)undec-1-ene, 1- methoxy-4-(1 -phenethoxyprop-1 -en-2-yl)benzene, 1 -methyl-4-(1 -phenethoxyprop-1 -en-2-yl)benzene, 2- (1 -phenethoxyprop-1 -en-2-yl)naphthalene, (2-phenethoxyvinyl)benzene, 2-(1 -((3,7-dimethyloct-6-en-1 - yl)oxy)prop-1-en-2-yl)naphthalene, (2-((2-pentylcyclopentylidene)methoxy)ethyl)benzene, 4-allyl-2- methoxy-1 -((2-methoxy-2-phenylvinyl)oxy)benzene, (2-((2- pentylcyclopentylidene)methoxy)ethyl)benzene, (2-((2-heptylcyclopentylidene)methoxy)ethyl)benzene, 1- isopropyl-4-methyl-2-((2-pentylcyclopentylidene)methoxy)benzene, 2-methoxy-1-((2- pentylcyclopentylidene)methoxy)-4-propylbenzene, 3-methoxy-4-((2-methoxy-2- phenylvinyl)oxy)benzaldehyde, 4-((2-(hexyloxy)-2-phenylvinyl)oxy)-3-methoxybenzaldehyde or a mixture thereof.

[0427] By “perfumery adjuvant”, it is meant here an ingredient capable of imparting additional added benefit such as a color, a particular light resistance, chemical stability, etc. A detailed description of the nature and type of adjuvant commonly used in perfuming composition cannot be exhaustive, but it has to be mentioned that said ingredients are well known to a person skilled in the art. One may cite as specific non-limiting examples the following: viscosity agents (e.g. surfactants, thickeners, gelling and / or rheology modifiers), stabilizing agents (e.g. preservatives, antioxidant, heat / light and or buffers or chelating agents, such as BHT), coloring agents (e.g. dyes and / or pigments), preservatives (e.g. antibacterial or antimicrobial or antifungal or anti irritant agents), abrasives, skin cooling agents, fixatives, insect repellants, ointments, vitamins and mixtures thereof.

[0428] It is understood that a person skilled in the art is perfectly able to design optimal formulations for the desired effect by admixing the above-mentioned components of a perfuming composition, simply by applying the standard knowledge of the art as well as by trial and error methodologies.

[0429] An invention’s composition consisting of at least one compound of of the invention and at least one perfumery carrier consists of a particular embodiment of the invention as well as a perfuming composition comprising at least one compound of the invention, at least one perfumery carrier, at least one perfumery base, and optionally at least one perfumery adjuvant.

[0430] According to a particular embodiment, the compositions mentioned above, comprise more than one compound of the invention and enable the perfumer to prepare accords or perfumes possessing the odor tonality of various compounds of the invention, creating thus new building block for creation purposes.

[0431] For the sake of clarity, it is also understood that any mixture resulting directly from a chemical synthesis, e.g. a reaction medium without an adequate purification, in which the compound of the invention would be involved as a starting, intermediate or end-product could not be considered as a perfuming composition according to the invention as far as said mixture does not provide the inventive compound in a suitable form for perfumery. Thus, unpurified reaction mixtures are generally excluded from the present invention unless otherwise specified.

[0432] The invention’s compound can also be advantageously used in all the fields of modern perfumery, i.e. fine or functional perfumery, to positively impart or modify the odor of a consumer product into which said compound (I) is added. Consequently, another object of the present invention consists of a perfumed consumer product comprising, as a perfuming ingredient, at least one compound of the invention, as defined above. The invention’s compound can be added as such or as part of an invention’s perfuming composition.

[0433] For the sake of clarity, “perfumed consumer product” is meant to designate a consumer product which delivers at least a pleasant perfuming effect to the surface or space to which it is applied (e.g. skin, hair, textile, or home surface). In other words, a perfumed consumer product according to the invention is a perfumed consumer product which comprises a functional formulation, as well as optionally additional benefit agents, corresponding to the desired consumer product, and an olfactive effective amount of at least one invention’s compound. For the sake of clarity, said perfumed consumer product is a non-edible product.

[0434] The nature and type of the constituents of the perfumed consumer product do not warrant a more detailed description here, which in any case would not be exhaustive, the skilled person being able to select them on the basis of his general knowledge and according to the nature and the desired effect of said product.

[0435] Non-limiting examples of suitable perfumed consumer products include a perfume, such as a fine perfume, a splash or eau de parfum, a cologne or a shave or after-shave lotion; a fabric care product, such as a liquid or solid detergent, a fabric softener, a liquid or solid scent booster, a fabric refresher, an ironing water, a paper, a bleach, a carpet cleaner, a curtain-care product; a body-care product, such as a hair care product (e.g. a shampoo, a coloring preparation or a hairspray, a color-care product, a hair shaping product, a dental care product), a disinfectant, an intimate care product; a cosmetic preparation (e.g. a skin cream or lotion, a vanishing cream or a deodorant or antiperspirant (e.g. a spray or roll on), a hair remover, a tanning or sun or after sun product, a nail product, a skin cleansing, a makeup); or a skin-care product (e.g. a soap, a shower or bath mousse, oil or gel, or a hygiene product or a foot / hand care products); an air care product, such as an air freshener or a “ready to use” powdered air freshener which can be used in the home space (rooms, refrigerators, cupboards, shoes or car) and / or in a public space (halls, hotels, malls, etc..); or a home care product, such as a mold remover, a furnisher care product, a wipe, a dish detergent or a hard-surface (e.g. a floor, bath, sanitary or a window-cleaning) detergent; a leather care product; a car care product, such as a polish, a wax or a plastic cleaner.

[0436] Some of the above-mentioned perfumed consumer products may represent an aggressive medium for the invention’s compounds, so that it may be necessary to protect the latter from premature decomposition, for example by encapsulation or by chemically binding it to another chemical which is suitable to release the invention’s ingredient upon a suitable external stimulus, such as an enzyme, light, heat or a change of pH.

[0437] The proportions in which the compounds according to the invention can be incorporated into the various aforementioned products or compositions vary within a wide range of values. These values are dependent on the nature of the article to be perfumed and on the desired organoleptic effect as well as on the nature of the co-ingredients in a given base when the compounds according to the invention are mixed with perfuming co-ingredients, solvents or additives commonly used in the art.

[0438] For example, in the case of perfuming compositions, typical concentrations are in the order of 0.001 % to 10 % by weight, or even more, of the compounds of the invention based on the weight of the composition into which they are incorporated. In the case of perfumed consumer product, typical concentrations are in the order of 0.01 % to 1 % by weight, or even more, of the compounds of the invention based on the weight of the consumer product into which they are incorporated.

[0439] Additionally, the intermediates compounds produced in any of the embodiments described herein can be converted to derivatives such as, but not limited to hydrocarbons, alcohols, diols, triols, acetals, ketals, aldehydes, acids, ethers, amides, ketones, lactones, epoxides, acetates, glycosides and / or an esters. These derivatives can be obtained by a chemical method such as, but not limited to oxidation, reduction, alkylation, acylation and / or rearrangement. Alternatively, the derivatives can be obtained using a biochemical method by contacting the terpene compound with an enzyme such as, but not limited to an oxidoreductase, a monooxygenase, a dioxygenase, a transferase. The biochemical conversion can be performed in-vitro using isolated enzymes, enzymes from lysed cells or in vivo using whole cells. The conversion can be a cyclization reaction realized by chemical or biochemical method. The derivatives can be used as perfumery, flavor or aroma ingredients.

[0440] Use of a terpene cyclase enzyme to produce a compound of the invention

[0441] A further aspect of the invention provides the use of a terpene cyclase enzyme, preferably a meroterpenoid cyclase enzyme, to produce a compound according to any of the previous embodiments.

[0442] A list of suitable terpene cyclase enzymes, preferably meroterpenoid cyclase enzymes, for use in the production of a compound according to any of the previous embodiments, such as a compound of the formula (A), (B), (C) and / or (D), is provided above in relation to the method of the invention and are also enzymes for this aspect of the invention.

[0443] New terpene cyclase enzyme of the invention

[0444] A further aspect of the invention provides new mutant meroterpenoid cyclase enzymes which were found to surprisingly shift the relative ratios of the product compounds and / or the titers of the product compounds produced in the method of the invention, when compared to the wild-type enzyme.

[0445] Accordingly, the invention provides a mutant meroterpenoid cyclase enzyme, preferably a mutant bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204; preferably, to any one of SEQ ID NOs: 1 to 96 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29.

[0446] Preferably, the mutant meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 80 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29. In one embodiment, the mutant meroterpenoid cyclase enzyme has the amino acid sequence provided in SEQ ID NO: 80.

[0447] More preferably, the mutant meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181 -204; preferably, to any one of SEQ ID NOs: 74 and 190-192; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29.

[0448] With reference to the above embodiments, the substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29 may be a substitution to cysteine, methionine or threonine.

[0449] In an alternative or yet a further embodiment of the invention, the invention provides a mutant meroterpenoid cyclase enzyme, preferably a mutant bacterial membrane-integrated meroterpenoid cyclase enzyme, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204; preferably, to any one of SEQ ID NOs: 1 to 96 and 181 to 204 ; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74.

[0450] Preferably, the mutant meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 80 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74. In one embodiment, the mutant meroterpenoid cyclase enzyme has the amino acid sequence provided in any of SEQ ID NOs: 181 to 204.

[0451] More preferably, the mutant meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181 -204; preferably, to any one of SEQ ID NOs: 74 and 190-192; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74. In one embodiment, the mutant meroterpenoid cyclase enzyme has the amino acid sequence provided in any one of SEQ ID NOs: 190 to 192. With reference to the above embodiments, the substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74 may be a substitution to methionine, serine or glycine.

[0452] Polypeptides and nucleic acids of the invention or used in the method of the invention

[0453] The generic terms “polypeptide” or “peptide”, which may be used interchangeably, refer to a natural or synthetic linear chain or sequence of consecutive, peptidically linked amino acid residues, comprising about 10 to up to more than 1.000 residues. Short chain polypeptides with up to 30 residues are also designated as “oligopeptides”.

[0454] The term “protein” refers to a macromolecular structure consisting of one or more polypeptides. The amino acid sequence of its polypeptide(s) represents the “primary structure” of the protein. The amino acid sequence also predetermines the “secondary structure” of the protein by the formation of special structural elements, such as alpha-helical and beta-sheet structures formed within a polypeptide chain. The arrangement of a plurality of such secondary structural elements defines the “tertiary structure” or spatial arrangement of the protein. If a protein comprises more than one polypeptide chains said chains are spatially arranged forming the “quaternary structure” of the protein. A correct spatial arrangement or “folding” of the protein is prerequisite of protein function. Denaturation or unfolding destroys protein function. If such destruction is reversible, protein function may be restored by refolding.

[0455] A typical protein function referred to herein is an “enzyme function”, i.e. the protein acts as biocatalyst on a substrate, for example a chemical compound, and catalyzes the conversion of said substrate to a product. An enzyme may show a high or low degree of substrate and / or product specificity.

[0456] A “polypeptide” referred to herein as having a particular “activity” thus implicitly refers to a correctly folded protein showing the indicated activity, as for example a specific enzyme activity.

[0457] Thus, unless otherwise indicated the term “polypeptide” also encompasses the terms “protein” and “enzyme”.

[0458] Similarly, the term “polypeptide fragment” encompasses the terms “protein fragment" and “enzyme fragment”.

[0459] The term “isolated polypeptide” refers to an amino acid sequence that is removed from its natural environment by any method or combination of methods known in the art and includes recombinant, biochemical and synthetic methods. “Target peptide” refers to an amino acid sequence which targets a protein, or polypeptide to intracellular organelles, i.e., mitochondria, or plastids, or to the extracellular space (secretion signal peptide). A nucleic acid sequence encoding a target peptide may be fused to the nucleic acid sequence encoding the amino terminal end, e.g., N-terminal end, of the protein or polypeptide, or may be used to replace a native targeting polypeptide.

[0460] The present invention also relates to "functional equivalents" (also designated as “analogs” or “functional mutations”) of the polypeptides specifically described herein.

[0461] For example, "functional equivalents" refer to polypeptides which, in a test used for determining enzymatic activity display at least a 1 to 10 %, or at least 20 %, or at least 50 %, or at least 75 %, or at least 90 % higher or lower activity, as that of the polypeptides specifically described herein.

[0462] "Functional equivalents”, according to the invention, also cover particular mutants, which, in at least one sequence position of an amino acid sequences stated herein, have an amino acid that is different from that concretely stated one, but nevertheless possess one of the aforementioned biological activities, as for example enzyme activity. "Functional equivalents" thus comprise mutants obtainable by one or more, like 1 to 20, in particular 1 to 15 or 5 to 10 amino acid additions, substitutions, in particular conservative substitutions, deletions and / or inversions, where the stated changes can occur in any sequence position, provided they lead to a mutant with the profile of properties according to the invention. Functional equivalence is in particular also provided if the activity patterns coincide qualitatively between the mutant and the unchanged polypeptide, i.e. if, for example, interaction with the same agonist or antagonist or substrate, however at a different rate, (i.e. expressed by a EC50 or IC50 value or any other parameter suitable in the present technical field) is observed. Examples of suitable (conservative) amino acid substitutions are shown in the following table:

[0463] Original residue Examples of substitution

[0464] Ala Ser

[0465] Arg Lys

[0466] Asn Gin; His

[0467] Asp Glu

[0468] Cys Ser

[0469] Gin Asn

[0470] Glu Asp

[0471] Gly Pro

[0472] His Asn ; Gin

[0473] He Leu; Vai

[0474] Leu He; Vai

[0475] Lys Arg ; Gin ; Glu

[0476] Met Leu ; He Phe Met ; Leu ; Tyr

[0477] Ser Thr

[0478] Thr Ser

[0479] Trp Tyr

[0480] Tyr Trp ; Phe

[0481] Vai He; Leu

[0482] "Functional equivalents" in the above sense are also "precursors" of the polypeptides described herein, as well as "functional derivatives" and "salts" of the polypeptides.

[0483] "Precursors" are in that case natural or synthetic precursors of the polypeptides with or without the desired biological activity.

[0484] The expression "salts" means salts of carboxyl groups as well as salts of acid addition of amino groups of the protein molecules. Salts of carboxyl groups can be produced in a known way and comprise inorganic salts, for example sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases, for example amines, such as triethanolamine, arginine, lysine, piperidine and the like. Salts of acid addition, for example salts with inorganic acids, such as hydrochloric acid or sulfuric acid and salts with organic acids, such as acetic acid and oxalic acid, are also covered by the invention.

[0485] "Functional derivatives" of polypeptides according to the invention can also be produced on functional amino acid side groups or at their N-terminal or C-terminal end using known techniques. Such derivatives comprise for example aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, produced by reaction with acyl groups; or O-acyl derivatives of free hydroxyl groups, produced by reaction with acyl groups.

[0486] ’’Functional equivalents” naturally also comprise polypeptides that can be obtained from other organisms, as well as naturally occurring variants. For example, areas of homologous sequence regions can be established by sequence comparison, and equivalent polypeptides can be determined on the basis of the concrete parameters of the invention.

[0487] "Functional equivalents" also comprise “fragments”, like individual domains or sequence motifs, of the polypeptides according to the invention, or N- and or C-terminally truncated forms, which may or may not display the desired biological function. Preferably such “fragments” retain the desired biological function at least qualitatively.

[0488] "Functional equivalents" are, moreover, fusion proteins, which have one of the polypeptide sequences stated herein or functional equivalents derived there from and at least one further, functionally different, heterologous sequence in functional N-terminal or C-terminal association (i.e. without substantial mutual functional impairment of the fusion protein parts). Non-limiting examples of these heterologous sequences are e.g. signal peptides, histidine anchors or enzymes.

[0489] “Functional equivalents” which are also comprised in accordance with the invention are homologs to the specifically disclosed polypeptides. These have at least 60%, preferably at least 75%, in particular at least 80 or 85%, such as, for example, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, homology (or identity) to one of the specifically disclosed amino acid sequences, calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448. A homology or identity, expressed as a percentage, of a homologous polypeptide according to the invention means in particular an identity, expressed as a percentage, of the amino acid residues based on the total length of one of the amino acid sequences described specifically herein.

[0490] The identity data, expressed as a percentage, may also be determined with the aid of BLAST alignments, algorithm blastp (protein-protein BLAST), or by applying the Clustal settings specified herein below.

[0491] In the case of a possible protein glycosylation, "functional equivalents" according to the invention comprise polypeptides as described herein in deglycosylated or glycosylated form as well as modified forms that can be obtained by altering the glycosylation pattern.

[0492] Functional equivalents or homologues of the polypeptides according to the invention can be produced by mutagenesis, e.g. by point mutation, lengthening or shortening of the protein or as described in more detail below.

[0493] Functional equivalents or homologs of the polypeptides according to the invention can be identified by screening combinatorial databases of mutants, for example shortening mutants. For example, a variegated database of protein variants can be produced by combinatorial mutagenesis at the nucleic acid level, e.g. by enzymatic ligation of a mixture of synthetic oligonucleotides. There are a great many methods that can be used for the production of databases of potential homologues from a degenerated oligonucleotide sequence. Chemical synthesis of a degenerated gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene can then be ligated in a suitable expression vector. The use of a degenerated genome makes it possible to supply all sequences in a mixture, which code for the desired set of potential protein sequences. Methods of synthesis of degenerated oligonucleotides are known to a person skilled in the art.

[0494] In the prior art, several techniques are known for the screening of gene products of combinatorial databases, which were produced by point mutations or shortening, and for the screening of cDNA libraries for gene products with a selected property. These techniques can be adapted for the rapid screening of the gene banks that were produced by combinatorial mutagenesis of homologues according to the invention. The techniques most frequently used for the screening of large gene banks, which are based on a high- throughput analysis, comprise cloning of the gene bank in expression vectors that can be replicated, transformation of the suitable cells with the resultant vector database and expression of the combinatorial genes in conditions in which detection of the desired activity facilitates isolation of the vector that codes for the gene whose product was detected. Recursive Ensemble Mutagenesis (REM), a technique that increases the frequency of functional mutants in the databases, can be used in combination with the screening tests, in order to identify homologues.

[0495] An embodiment provided herein provides orthologs and paralogs of polypeptides disclosed herein as well as methods for identifying and isolating such orthologs and paralogs. A definition of the terms “ortholog” and “paralog” is given below and applies to amino acid and nucleic acid sequences.

[0496] The polypeptides of the invention include all active forms, including active subsequences, e.g., catalytic domains or active sites, of an enzyme of the invention. In one aspect, the invention provides catalytic domains or active sites as set forth below. In one aspect, the invention provides a peptide or polypeptide comprising or consisting of an active site domain as predicted through use of a database such as Pfam (http: / / pfam.wustl.edu / hmmsearch.shtml) (which is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, The Pfam protein families database, A. Bateman, E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. L. Sonnhammer, Nucleic Acids Research, 30(1):276-280, 2002) or equivalent, as for example InterPro and SMART databases (http: / / www.ebi.ac.uk / interpro / scan.html, http: / / smart.embl-heidelberg.de / ).

[0497] The invention also encompasses “polypeptide variant” having the desired activity, wherein the variant polypeptide is selected from an amino acid sequence having at least 40%, 45%, 50%. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a specific, in particular natural, amino acid sequence as referred to by a specific SEQ ID NO and contains at least one substitution modification relative to said SEQ ID NO.

[0498] Coding nucleic acid sequences applicable according to the invention

[0499] In this context the following definitions apply:

[0500] The terms “nucleic acid sequence,” “nucleic acid,” “nucleic acid molecule” and “polynucleotide” are used interchangeably meaning a sequence of nucleotides. A nucleic acid sequence may be a singlestranded or double-stranded deoxyribonucleotide, or ribonucleotide of any length, and include coding and non-coding sequences of a gene, exons, introns, sense and anti-sense complimentary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers and nucleic acid probes. The skilled artisan is aware that the nucleic acid sequences of RNA are identical to the DNA sequences with the difference of thymine (T) being replaced by uracil (U). The term “nucleotide sequence” should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid.

[0501] An “isolated nucleic acid” or “isolated nucleic acid sequence” relates to a nucleic acid or nucleic acid sequence that is in an environment different from that in which the nucleic acid or nucleic acid sequence naturally occurs and can include those that are substantially free from contaminating endogenous material.

[0502] The term “naturally-occurring” as used herein as applied to a nucleic acid refers to a nucleic acid that is found in a cell of an organism in nature and which has not been intentionally modified by a human in the laboratory.

[0503] A “fragment” of a polynucleotide or nucleic acid sequence refers to contiguous nucleotides that is particularly at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp and / or at least 60 bp in length of the polynucleotide of an embodiment herein. Particularly the fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 contiguous nucleotides of the polynucleotide of an embodiment herein. Without being limited, the fragment of the polynucleotides herein may be used as a PCR primer, and / or as a probe, or for anti-sense gene silencing or RNAi.

[0504] “Recombinant nucleic acid sequences” are nucleic acid sequences that result from the use of laboratory methods (for example, molecular cloning) to bring together genetic material from more than on source, creating or modifying a nucleic acid sequence that does not occur naturally and would not be otherwise found in biological organisms.

[0505] “Recombinant DNA technology” refers to molecular biology procedures to prepare a recombinant nucleic acid sequence as described, for instance, in Laboratory Manuals edited by Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.

[0506] The term “gene” means a DNA sequence comprising a region, which is transcribed into a RNA molecule, e.g., an mRNA in a cell, operably linked to suitable regulatory regions, e.g., a promoter. A gene may thus comprise several operably linked sequences, such as a promoter, a 5’ leader sequence comprising, e.g., sequences involved in translation initiation, a coding region of cDNA or genomic DNA, introns, exons, and / or a 3’non-translated sequence comprising, e.g., transcription termination sites. “Polycistronic” refers to nucleic acid molecules, in particular mRNAs, that can encode more than one polypeptide separately within the same nucleic acid molecule.

[0507] A “chimeric gene” refers to any gene which is not normally found in nature in a species, in particular, a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature. For example, the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region. The term “chimeric gene” is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or to an antisense, i.e., reverse complement of the sense strand, or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription). The term "chimeric gene" also includes genes obtained through the combination of portions of one or more coding sequences to produce a new gene.

[0508] A “3’ UTR” or “3’ non-translated sequence” (also referred to as “3’ untranslated region,” or “3’end”) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal such as AAUAAA or variants thereof. After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the site of translation, e.g., cytoplasm.

[0509] The term “primer” refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and is used for polymerization of a nucleic acid sequence complementary to the template.

[0510] The term “selectable marker” refers to any gene which upon expression may be used to select a cell or cells that include the selectable marker. Examples of selectable markers are described below. The skilled artisan will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.

[0511] The invention also relates to nucleic acid sequences that code for polypeptides as defined herein.

[0512] In particular, the invention also relates to nucleic acid sequences (single-stranded and doublestranded DNA and RNA sequences, e.g. cDNA, genomic DNA and mRNA), coding for one of the above polypeptides and their functional equivalents, which can be obtained for example using artificial nucleotide analogs.

[0513] The invention relates both to isolated nucleic acid molecules, which code for polypeptides according to the invention or biologically active segments thereof, and to nucleic acid fragments, which can be used for example as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the invention. The present invention also relates to nucleic acids with a certain degree of “identity” to the sequences specifically disclosed herein. "Identity" between two nucleic acids means identity of the nucleotides, in each case over the entire length of the nucleic acid.

[0514] The “identity” between two nucleotide sequences (the same applies to peptide or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues) or that are identical in the two sequences when an alignment of these two sequences has been generated. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment. The percentage of sequence identity, as used herein, is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as nonidentical residues for the calculation of the percentage of sequence identity. Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs and for instance publicly available computer programs available on the world wide web.

[0515] Particularly, the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi, can be used to obtain an optimal alignment of protein or nucleic acid sequences and to calculate the percentage of sequence identity.

[0516] Alternatively, the identity may be determined according to Chenna, et al. (2003), the web page: http: / / www.ebi.ac.Uk / Tools / clustalw / index.html# and the following settings:

[0517] DNA Gap Open Penalty 15.0

[0518] DNA Gap Extension Penalty 6.66

[0519] DNA Matrix Identity

[0520] Protein Gap Open Penalty 10.0

[0521] Protein Gap Extension Penalty 0.2

[0522] Protein matrix Gonnet

[0523] Protein / DNA ENDGAP -1

[0524] Protein / DNA GAPDIST 4

[0525] All the nucleic acid sequences mentioned herein (single-stranded and double-stranded DNA and RNA sequences, for example cDNA and mRNA) can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. Chemical synthesis of oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2ndedition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989), see below.

[0526] The nucleic acid molecules according to the invention can in addition contain non-translated sequences from the 3’ and / or 5’ end of the coding genetic region.

[0527] The invention further relates to the nucleic acid molecules that are complementary to the concretely described nucleotide sequences or a segment thereof.

[0528] The nucleotide sequences according to the invention make possible the production of probes and primers that can be used for the identification and / or cloning of homologous sequences in other cellular types and organisms. Such probes or primers generally comprise a nucleotide sequence region which hybridizes under “stringent” conditions (as defined herein elsewhere) on at least about 12, preferably at least about 25, for example about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or of a corresponding antisense strand.

[0529] “Homologous” sequences include orthologous or paralogous sequences. Methods of identifying orthologs or paralogs including phylogenetic methods, sequence similarity and hybridization methods are known in the art and are described herein.

[0530] “Paralogs” result from gene duplication that gives rise to two or more genes with similar sequences and similar functions. Paralogs typically cluster together and are formed by duplications of genes within related plant species. Paralogs are found in groups of similar genes using pair-wise Blast analysis or during phylogenetic analysis of gene families using programs such as CLUSTAL. In paralogs, consensus sequences can be identified characteristic to sequences within related genes and having similar functions of the genes.

[0531] “Orthologs”, or orthologous sequences, are sequences similar to each other because they are found in species that descended from a common ancestor. For instance, plant species that have common ancestors are known to contain many enzymes that have similar sequences and functions. The skilled artisan can identify orthologous sequences and predict the functions of the orthologs, for example, by constructing a polygenic tree for a gene family of one species using CLUSTAL or BLAST programs. A method for identifying or confirming similar functions among homologous sequences is by comparing of the transcript profiles in host cells or organisms, such as plants or microorganisms, overexpressing or lacking (in knockouts / knockdowns) related polypeptides. The skilled person will understand that genes having similar transcript profiles, with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or greater than 90% regulated transcripts in common will have similar functions. Homologs, paralogs, orthologs and any other variants of the sequences herein are expected to function in a similar manner by making the host cells, organism such as plants or microorganisms producing terpene cyclase proteins.

[0532] A nucleic acid molecule according to the invention can be recovered by means of standard techniques of molecular biology and the sequence information supplied according to the invention. For example, cDNA can be isolated from a suitable cDNA library, using one of the concretely disclosed complete sequences or a segment thereof as hybridization probe and standard hybridization techniques (as described for example in Sambrook, (1989)).

[0533] In addition, a nucleic acid molecule comprising one of the disclosed sequences or a segment thereof, can be isolated by the polymerase chain reaction, using the oligonucleotide primers that were constructed on the basis of this sequence. The nucleic acid amplified in this way can be cloned in a suitable vector and can be characterized by DNA sequencing. The oligonucleotides according to the invention can also be produced by standard methods of synthesis, e.g. using an automatic DNA synthesizer.

[0534] To test a function of variant DNA sequences according to an embodiment herein, the sequence of interest is operably linked to a selectable or screenable marker gene and expression of said reporter gene is tested in transient expression assays, for example, with microorganisms or with protoplasts or in stably transformed plants.

[0535] The invention also relates to derivatives of the concretely disclosed or derivable nucleic acid sequences.

[0536] Thus, further nucleic acid sequences according to the invention can be derived from the sequences specifically disclosed herein and can differ from it by one or more, like 1 to 20, in particular 1 to 15 or 5 to 10 additions, substitutions, insertions or deletions of one or several (like for example 1 to 10) nucleotides, and furthermore code for polypeptides with the desired profile of properties.

[0537] The invention also encompasses nucleic acid sequences that comprise so-called silent mutations or have been altered, in comparison with a concretely stated sequence, according to the codon usage of a special original or host organism.

[0538] According to a particular embodiment of the invention variant nucleic acids may be prepared in order to adapt its nucleotide sequence to a specific expression system. For example, bacterial expression systems are known to more efficiently express polypeptides if amino acids are encoded by particular codons. Due to the degeneracy of the genetic code, more than one codon may encode the same amino acid sequence, multiple nucleic acid sequences can code for the same protein or polypeptide, all these DNA sequences being encompassed by an embodiment herein. Where appropriate, the nucleic acid sequences encoding the polypeptides described herein may be optimized for increased expression in the host cell. For example, nucleic acids of an embodiment herein may be synthesized using codons particular to a host for improved expression.

[0539] The invention also encompasses naturally occurring variants, e.g. splicing variants or allelic variants, of the sequences described therein.

[0540] Allelic variants may have at least 60 % homology at the level of the derived amino acid, preferably at least 80 % homology, quite especially preferably at least 90 % homology over the entire sequence range (regarding homology at the amino acid level, reference should be made to the details given above for the polypeptides). Advantageously, the homologies can be higher over partial regions of the sequences.

[0541] The invention also relates to sequences that can be obtained by conservative nucleotide substitutions (i.e. as a result thereof the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility).

[0542] The invention also relates to the molecules derived from the concretely disclosed nucleic acids by sequence polymorphisms. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents. These natural variations usually produce a variance of 1 to 5 % in the nucleotide sequence of a gene. Said polymorphisms may lead to changes in the amino acid sequence of the polypeptides disclosed herein. Allelic variants may also include functional equivalents.

[0543] Furthermore, derivatives are also to be understood to be homologs of the nucleic acid sequences according to the invention, for example animal, plant, fungal or bacterial homologs, shortened sequences, single-stranded DNA or RNA of the coding and noncoding DNA sequence. For example, homologs have, at the DNA level, a homology of at least 40 %, preferably of at least 60 %, especially preferably of at least 70 %, quite especially preferably of at least 80 % overthe entire DNA region given in a sequence specifically disclosed herein.

[0544] Moreover, derivatives are to be understood to be, for example, fusions with promoters. The promoters that are added to the stated nucleotide sequences can be modified by at least one nucleotide exchange, at least one insertion, inversion and / or deletion, though without impairing the functionality or efficacy of the promoters. Moreover, the efficacy of the promoters can be increased by altering their sequence or can be exchanged completely with more effective promoters even of organisms of a different genus.

[0545] Generation of functional polypeptide mutants

[0546] Moreover, a person skilled in the art is familiar with methods for generating functional mutants, that is to say nucleotide sequences which code for a polypeptide with at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to anyone of amino acid related to SEQ ID NOs as disclosed herein and / or encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 50% sequence identity to anyone of the nucleotide related to SEQ ID NOs as disclosed herein.

[0547] Depending on the technique used, a person skilled in the art can introduce entirely random or else more directed mutations into genes or else noncoding nucleic acid regions (which are for example important for regulating expression) and subsequently generate genetic libraries. The methods of molecular biology required for this purpose are known to the skilled worker and for example described in Sambrook and Russell, Molecular Cloning.3rd Edition, Cold Spring Harbor Laboratory Press 2001 .

[0548] Methods for modifying genes and thus for modifying the polypeptide encoded by them have been known to the skilled worker for a long time, such as, for example:

[0549] - site-specific mutagenesis, where individual or several nucleotides of a gene are replaced in a directed fashion (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey),

[0550] - saturation mutagenesis, in which a codon for any amino acid can be exchanged or added at any point of a gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22:1593; Barettino D, Feigenbutz M, Valcarel R, Stunnenberg HG (1994) Nucleic Acids Res 22:541 ; Barik S (1995) Mol Biotechnol 3:1),

[0551] - error-prone polymerase chain reaction, where nucleotide sequences are mutated by error-prone DNA polymerases (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18:3739);

[0552] - the SeSaM method (sequence saturation method), in which preferred exchanges are prevented by the polymerase. Schenk et al., Biospektrum, Vol. 3, 2006, 277-279

[0553] - the passaging of genes in mutator strains, in which, for example owing to defective DNA repair mechanisms, there is an increased mutation rate of nucleotide sequences (Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E.coli mutator strain. In: Trower MK (Ed.) In vitro mutagenesis protocols. Humana Press, New Jersey), or

[0554] - DNA shuffling, in which a pool of closely related genes is formed and digested and the fragments are used as templates for a polymerase chain reaction in which, by repeated strand separation and reassociation, full-length mosaic genes are ultimately generated (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91 :10747).

[0555] Using so-called directed evolution (described, inter alia, in Reetz MT and Jaeger K-E (1999), Topics Curr Chem 200:31 ; Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods for optimizing industrial polypeptides by directed evolution, In: Demain AL, Davies JE (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), a skilled worker can produce functional mutants in a directed manner and on a large scale. To this end, in a first step, gene libraries of the respective polypeptides are first produced, for example using the methods given above. The gene libraries are expressed in a suitable way, for example by bacteria or by phage display systems. The relevant genes of host organisms which express functional mutants with properties that largely correspond to the desired properties can be submitted to another mutation cycle. The steps of the mutation and selection or screening can be repeated iteratively until the present functional mutants have the desired properties to a sufficient extent. Using this iterative procedure, a limited number of mutations, for example 1 , 2, 3, 4 or 5 mutations, can be performed in stages and assessed and selected for their influence on the activity in question. The selected mutant can then be submitted to a further mutation step in the same way. In this way, the number of individual mutants to be investigated can be reduced significantly.

[0556] The results according to the invention also provide important information relating to structure and sequence of the relevant polypeptides, which is required for generating, in a targeted fashion, further polypeptides with desired modified properties. In particular, it is possible to define so-called “hot spots”, i.e. sequence segments that are potentially suitable for modifying a property by introducing targeted mutations.

[0557] Information can also be deduced regarding amino acid sequence positions, in the region of which mutations can be affected that should probably have little effect on the activity and can be designated as potential “silent mutations”.

[0558] Constructs for expressing polypeptides of the invention and / or used in the method of the invention

[0559] In this context the following definitions apply:

[0560] “Expression of a gene” encompasses “heterologous expression” and “over-expression” and involves transcription of the gene and translation of the mRNA into a protein. Overexpression refers to the production of the gene product as measured by levels of mRNA, polypeptide and / or enzyme activity in transgenic cells or organisms that exceeds levels of production in non-transformed cells or organisms of a similar genetic background.

[0561] “Expression vector” as used herein means a nucleic acid molecule engineered using molecular biology methods and recombinant DNA technology for delivery of foreign or exogenous DNA into a host cell. The expression vector typically includes sequences required for proper transcription of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for an RNA, e.g., an antisense RNA, siRNA and the like.

[0562] An “expression vector” as used herein includes any linear or circular recombinant vector including but not limited to viral vectors, bacteriophages and plasmids. The skilled person is capable of selecting a suitable vector according to the expression system. In one embodiment, the expression vector includes the nucleic acid of an embodiment herein operably linked to at least one “regulatory sequence”, which controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosomal binding site and, optionally, including at least one selection marker. Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the nucleic acid of an embodiment herein.

[0563] An “expression system” as used herein encompasses any combination of nucleic acid molecules required for the expression of one, or the co-expression of two or more polypeptides either in vivo of a given expression host, or in vitro. The respective coding sequences may either be located on a single nucleic acid molecule or vector, as for example a vector containing multiple cloning sites, or on a polycistronic nucleic acid, or may be distributed over two or more physically distinct vectors. As a particular example there may be mentioned an operon comprising a promotor sequence, one or more operator sequences and one or more structural genes each encoding an enzyme as described herein.

[0564] As used herein, the terms "amplifying" and "amplification" refer to the use of any suitable amplification methodology for generating or detecting recombinant of naturally expressed nucleic acid, as described in detail, below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo dT primer) for amplifying (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic DNA or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention in vivo, ex vivo or in vitro.

[0565] “Regulatory sequence” refers to a nucleic acid sequence that determines expression level of the nucleic acid sequences of an embodiment herein and is capable of regulating the rate of transcription of the nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences comprise promoters, enhancers, transcription factors, promoter elements and the like.

[0566] A “promoter”, a “nucleic acid with promoter activity” or a “promoter sequence” is understood as meaning, in accordance with the invention, a nucleic acid which, when functionally linked to a nucleic acid to be transcribed, regulates the transcription of said nucleic acid. “Promoter” in particular refers to a nucleic acid sequence that controls the expression of a coding sequence by providing a binding site for RNA polymerase and other factors required for proper transcription including without limitation transcription factor binding sites, repressor and activator protein binding sites. The meaning of the term promoter also includes the term “promoter regulatory sequence”. Promoter regulatory sequences may include upstream and downstream elements that may influences transcription, RNA processing or stability of the associated coding nucleic acid sequence. Promoters include naturally-derived and synthetic sequences. The coding nucleic acid sequences is usually located downstream of the promoter with respect to the direction of the transcription starting at the transcription initiation site.

[0567] In this context, a “functional” or “operative” linkage is understood as meaning for example the sequential arrangement of one of the nucleic acids with a regulatory sequence. For example the sequence with promoter activity and of a nucleic acid sequence to be transcribed and optionally further regulatory elements, for example nucleic acid sequences which ensure the transcription of nucleic acids, and for example a terminator, are linked in such a way that each of the regulatory elements can perform its function upon transcription of the nucleic acid sequence. This does not necessarily require a direct linkage in the chemical sense. Genetic control sequences, for example enhancer sequences, can even exert their function on the target sequence from more remote positions or even from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be transcribed is positioned behind (i.e. at the 3’-end of) the promoter sequence so that the two sequences are joined together covalently. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly can be smaller than 200 base pairs, or smaller than 100 base pairs or smaller than 50 base pairs.

[0568] In addition to promoters and terminator, the following may be mentioned as examples of other regulatory elements: targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, replication origins and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0569] The term “constitutive promoter” refers to an unregulated promoter that allows for continual transcription of the nucleic acid sequence it is operably linked to.

[0570] As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous. The nucleotide sequence associated with the promoter sequence may be of homologous or heterologous origin with respect to the plant to be transformed. The sequence also may be entirely or partially synthetic. Regardless of the origin, the nucleic acid sequence associated with the promoter sequence will be expressed or silenced in accordance with promoter properties to which it is linked after binding to the polypeptide of an embodiment herein. The associated nucleic acid may code for a protein that is desired to be expressed or suppressed throughout the organism at all times or, alternatively, at a specific time or in specific tissues, cells, or cell compartment. Such nucleotide sequences particularly encode proteins conferring desirable phenotypic traits to the host cells or organism altered or transformed therewith. More particularly, the associated nucleotide sequence leads to the production of the product or products of interest as herein defined in the cell or organism. Particularly, the nucleotide sequence encodes a polypeptide having an enzyme activity as herein defined.

[0571] The nucleotide sequence as described herein above may be part of an “expression cassette”. The terms “expression cassette” and “expression construct” are used synonymously. The (preferably recombinant) expression construct contains a nucleotide sequence which encodes a polypeptide according to the invention and which is under genetic control of regulatory nucleic acid sequences.

[0572] In a method applied according to the invention, the expression cassette may be part of an “expression vector”, in particular of a recombinant expression vector. An “expression unit” is understood as meaning, in accordance with the invention, a nucleic acid with expression activity which comprises a promoter as defined herein and, after functional linkage with a nucleic acid to be expressed or a gene, regulates the expression, i.e. the transcription and the translation of said nucleic acid or said gene. It is therefore in this connection also referred to as a “regulatory nucleic acid sequence”. In addition to the promoter, other regulatory elements, for example enhancers, can also be present.

[0573] An “expression cassette” or “expression construct” is understood as meaning, in accordance with the invention, an expression unit which is functionally linked to the nucleic acid to be expressed or the gene to be expressed. In contrast to an expression unit, an expression cassette therefore comprises not only nucleic acid sequences which regulate transcription and translation, but also the nucleic acid sequences that are to be expressed as protein as a result of transcription and translation.

[0574] The terms “expression” or “overexpression” describe, in the context of the invention, the production or increase in intracellular activity of one or more polypeptides in a microorganism, which are encoded by the corresponding DNA. To this end, it is possible for example to introduce a gene into an organism, replace an existing gene with another gene, increase the copy number of the gene(s), use a strong promoter or use a gene which encodes for a corresponding polypeptide with a high activity; optionally, these measures can be combined.

[0575] Preferably such constructs according to the invention comprise a promoter 5’-upstream of the respective coding sequence and a terminator sequence 3’-downstream and optionally other usual regulatory elements, in each case in operative linkage with the coding sequence.

[0576] Nucleic acid constructs according to the invention comprise in particular a sequence coding for a polypeptide for example derived from the amino acid related SEQ ID NOs as described therein or the reverse complement thereof, or derivatives and homologs thereof and which have been linked operatively or functionally with one or more regulatory signals, advantageously for controlling, for example increasing, gene expression.

[0577] In addition to these regulatory sequences, the natural regulation of these sequences may still be present before the actual structural genes and optionally may have been genetically modified so that the natural regulation has been switched off and expression of the genes has been enhanced. The nucleic acid construct may, however, also be of simpler construction, i.e. no additional regulatory signals have been inserted before the coding sequence and the natural promoter, with its regulation, has not been removed. Instead, the natural regulatory sequence is mutated such that regulation no longer takes place and the gene expression is increased. A preferred nucleic acid construct advantageously also comprises one or more of the already mentioned “enhancer” sequences in functional linkage with the promoter, which sequences make possible an enhanced expression of the nucleic acid sequence. Additional advantageous sequences may also be inserted at the 3’-end of the DNA sequences, such as further regulatory elements or terminators. One or more copies of the nucleic acids according to the invention may be present in a construct. In the construct, other markers, such as genes which complement auxotrophisms or antibiotic resistances, may also optionally be present so as to select for the construct.

[0578] Examples of suitable regulatory sequences are present in promoters such as cos, tac, trp, tet, trp- tet, Ipp, lac, Ipp-lac, laclq, T7, T5, T3, gal, trc, ara, rhaP (rhaPBAD)SP6, lambda-PR or in the lambda-PL promoter, and these are advantageously employed in Gram-negative bacteria. Further advantageous regulatory sequences are present for example in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1 , MFalpha, AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH. Artificial promoters may also be used for regulation.

[0579] For expression in a host organism, the nucleic acid construct is inserted advantageously into a vector such as, for example, a plasmid or a phage, which makes possible optimal expression of the genes in the host. Vectors are also understood as meaning, in addition to plasmids and phages, all the other vectors which are known to the skilled worker, that is to say for example viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids and linear or circular DNA or artificial chromosomes. These vectors are capable of replicating autonomously in the host organism or else chromosomally. These vectors are a further development of the invention. Binary or cpo-integration vectors are also applicable.

[0580] Suitable plasmids are, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-lll113-B1 , Agt11 or pBdCI, in Streptomyces pl J101 , plJ364, plJ702 or pl J361 , in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1 , plL2 or pBB116, in yeasts 2alphaM, pAG-1 , YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHIac+, pBIN19, pAK2004 or pDH51 . The abovementioned plasmids are a small selection of the plasmids which are possible. Further plasmids are well known to the skilled worker and can be found for example in the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0581] In a further development of the vector, the vector which comprises the nucleic acid construct according to the invention or the nucleic acid according to the invention can advantageously also be introduced into the microorganisms in the form of a linear DNA and integrated into the host organism’s genome via heterologous or homologous recombination. This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or the nucleic acid according to the invention. For optimal expression of heterologous genes in organisms, it is advantageous to modify the nucleic acid sequences to match the specific “codon usage” used in the organism. The “codon usage” can be determined readily by computer evaluations of other, known genes of the organism in question.

[0582] An expression cassette according to the invention is generated by fusing a suitable promoter to a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Customary recombination and cloning techniques are used for this purpose, as are described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc, and Wiley Interscience (1987).

[0583] For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which makes possible optimal expression of the genes in the host. Vectors are well known to the skilled worker and can be found for example in “cloning vectors” (Pouwels P. H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0584] An alternative embodiment of an embodiment herein provides a method to “alter gene expression” in a host cell. For instance, the polynucleotide of an embodiment herein may be enhanced or overexpressed or induced in certain contexts (e.g. upon exposure to certain temperatures or culture conditions) in a host cell or host organism.

[0585] Alteration of expression of a polynucleotide provided herein may also result in ectopic expression which is a different expression pattern in an altered and in a control or wild-type organism. Alteration of expression occurs from interactions of polypeptide of an embodiment herein with exogenous or endogenous modulators, or as a result of chemical modification of the polypeptide. The term also refers to an altered expression pattern of the polynucleotide of an embodiment herein which is altered below the detection level or completely suppressed activity.

[0586] In one embodiment, provided herein is also an isolated, recombinant or synthetic polynucleotide encoding a polypeptide or variant polypeptide provided herein.

[0587] In one embodiment, several polypeptide encoding nucleic acid sequences are co-expressed in a single host, particularly under control of different promoters. In another embodiment, several polypeptide encoding nucleic acid sequences can be present on a single transformation vector or be co-transformed at the same time using separate vectors and selecting transformants comprising both chimeric genes. Similarly, one or polypeptide encoding genes may be expressed in a single plant, cell, microorganism or organism together with other chimeric genes.

[0588] Recombinant production of polypeptides according to the invention The invention further relates to methods for recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultured, optionally the expression of the polypeptides is induced by applying at least one inducer inducing gene expression and the expressed polypeptides are isolated from the culture. The polypeptides can also be produced in this way on an industrial scale, if desired.

[0589] The microorganisms produced according to the invention can be cultured continuously or discontinuously in the batch method or in the fed-batch method or repeated fed-batch method. A summary of known cultivation methods can be found in the textbook by Chmiel (Bioprozesstechnik 1 . Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1 . Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)). Standard laboratory methods can be used for this purpose and are known in the art and also further described herein.

[0590] If the polypeptides are not secreted in the culture medium, the cells can also be lysed and the product can be obtained from the lysate by known methods for isolation of proteins. The cells can optionally be disrupted with high-frequency ultrasound, high pressure, for example in a French press, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of several of the aforementioned methods.

[0591] The polypeptides can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), such as Q-sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and with other usual techniques such as ultrafiltration, crystallization, saltingout, dialysis and native gel electrophoresis. Suitable methods are described for example in Cooper, T. G., Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0592] For isolating the recombinant protein, it can be advantageous to use vector systems or oligonucleotides, which lengthen the cDNA by defined nucleotide sequences and therefore code for altered polypeptides or fusion proteins, which for example serve for easier purification. Suitable modifications of this type are for example so-called "tags" functioning as anchors, for example the modification known as hexa-histidine anchor or epitopes that can be recognized as antigens of antibodies (described for example in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). These anchors can serve for attaching the proteins to a solid carrier, for example a polymer matrix, which can for example be used as packing in a chromatography column, or can be used on a microtiter plate or on some other carrier.

[0593] At the same time these anchors can also be used for recognition of the proteins. For recognition of the proteins, it is moreover also possible to use usual markers, such as fluorescent dyes, enzyme markers, which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors for derivatization of the proteins.

[0594] The numerous possible variations that will become immediately evident to a person skilled in the art after having considered the disclosure provided herein also fall within the scope of the invention.

[0595] A non-limiting list of embodiments according to the invention is displayed hereafter.

[0596] Embodiments according to the invention

[0597] 1 . A method for preparing a compound of the formula (A), (B), (C) and / or (D), or a derivative thereof, wherein

[0598] R° represents either H or a C1-4 alkyl group, preferably ethyl or H; and wherein each R2represents independently from each other either H or an alcohol protecting group, particularly , preferably H,

[0599] R3represents H or a C1-4 alkyl group, preferably CH3; and n independently from each other represents 1 or 2; wherein any dotted line represents the bond by which the substituent is bound to the rest of the molecule; and any wavy line represents independently from each other a carbon-carbon bond which when linked to the carbon-carbon double bond is either in the Z or in the E-configuration, preferably in the E- configuration, wherein the method comprises:

[0600] (a) contacting a compound of the formula (I) with a terpene cyclase enzyme under conditions suitable for the terpene cyclase enzyme to produce the compound of the formula (A), (B), (C) and / or (D). The method according to embodiment 1 , wherein the configuration of the carbon-carbon double bond of the compound of the formula (I) is in the E-configuration. The method according to embodiment 1 or 2, wherein R1represents with n being preferably 1 and / or R2being H or an alcohol protecting group, particularly R3being a C1-4 alkyl group, preferably CH3; preferably, R1represents

[0601] 4. The method according to any of the preceding embodiments, wherein the compound of the formula (A) is a compound of the formula (A’), the compound of the formula (B) is a compound of the formula (B’), the compound of the formula (C) is a compound of the formula (C’) and / or the compound of the formula (D) is a compound of the formula (D’) 5. The method according to any of the preceding embodiments, wherein the compound of the formula

[0602] (A), (B), (C) and / or (D) is a compound selected of the group consisting of wherein each R2represents independently from each other either H or an alcohol protecting group, particularly being a C1-4 alkyl group, preferably CH3. The method according to any of the preceding embodiments, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’), preferably of the formula (A’1), preferably of the formula (A’1 a) or (A'1 b), more preferably of the formula (A’1-1 a) or (A'1 -1 b) The method according to any of embodiments 1 to 5, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’), preferably of the formula (A’3), preferably of the formula (A’3a), more preferably of the formula (A’3-1 a) or (A'3-2a)

[0603] The method according to any of embodiments 1 to 5, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’), preferably of the formula (A’5), preferably of the formula (A’5a), more preferably of the formula (A’5-1 a) or (A'5-2a)

[0604] (A’5-2a). 9. The method according to any of embodiments 1 to 5, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (B’), preferably of the formula (B’3), preferably of the formula (B’3a) 10. The method according to any of embodiments 1 to 5, wherein the compound of the formula (A), (B),

[0605] (C) and / or (D) is a compound of the formula (B’), preferably of the formula (B’5), preferably of the

[0606] 11. The method according to any of embodiments 1 to 5, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (C’), preferably of the formula (C’3), preferably of the formula (C’3a)

[0607] The method according to any of embodiments 1 to 5, wherein the compound of the formula (A), (B),

[0608] (C) and / or (D) is a compound of the formula (C’), preferably of the formula (C’5), preferably of the formula (C’5a) The method according to any of embodiments 1 to 5, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (D’), preferably of the formula (D’1), preferably of the formula (D’1a), more preferably of the formula (D’1 ab)

[0609] The method according to any of embodiments 1 to 5, wherein the compound of the formula (I) is a compound of the formula (lb) and the compound of the formula (A) is a compound of the formula (A’1 a), the compound of the formula (B) is a compound of the formula (B’1), the compound of the formula (C) is a compound of the formula (C’1) and / or the compound of the formula (D) is a compound of the formula (D’1 a)

[0610] 15. The method according to embodiment 14, wherein the compound of the formula (A’1 a) is a compound of the formula (A’1-1 a) or (A’1-2a); preferably, a compound of the formula (A’1-1 a). 16. The method according to any of embodiments 14 and 15, wherein the compound of the formula (A’1-

[0611] 1 a), (B’1) and / or (C’1) is the main product (as defined in the Section “Definitions”).

[0612] 17. The method according to any of embodiments 14 and 16, wherein the compound of the formula (A’1- 1 a) is the main product (as defined in the Section “Definitions”).

[0613] 18. The method according to any of embodiments 1 to 5, wherein the compound the formula (I) is compound of the formula (li), and the compound of the formula (A) is a compound of the formula (A’3a), the compound of the formula (B) is a compound of the formula (B’3a), the compound of the formula (C) is a compound of the formula (C’3a) and / or the compound of the formula (D) is a compound of the formula (D’1 a)

[0614] 19. The method according to embodiment 18, wherein the compound of the formula (A’3a) is a compound of the formula (A’3-1 a) or (A’3-2a); preferably, a compound of the formula (A’3-1 a). 20. The method according to any of embodiments 18 and 19, wherein the compound of the formula (B’3a) and / or (C’3a) is the main product (as defined in the Section “Definitions”).

[0615] 21. The method according to any of embodiments 1 to 5, wherein the compound the formula (I) is compound of the formula (If), and the compound of the formula (A) is a compound of the formula (A’5a), the compound of the formula (B) is a compound of the formula (B’5a) and / or the compound of the formula (C) is a compound of the formula (C’5a)

[0616] 22. The method according to embodiment 21 , wherein the compound of the formula (A’5a) is a compound of the formula (A’5-1 a) or (A’5-2a); preferably, a compound of the formula (A’5-2a).

[0617] 23. The method according to any of embodiments 21 and 22, wherein the compound of the formula (B’5a) and / or (C’5a) is the main product (as defined in the Section “Definitions”).

[0618] 24. The method according to any of the preceding embodiments, wherein the terpene cyclase enzyme is a meroterpenoid cyclase enzyme and / or a squalene cyclase enzyme, preferably the enzyme is a meroterpenoid cyclase enzyme.

[0619] 25. The method according to embodiment 24, wherein the meroterpenoid cyclase enzyme is a bacterial membrane-integrated meroterpenoid cyclase enzyme, preferably comprising at least one or more amino acid motifs selected from:

[0620] [W]xxx[D]xx[ILVMN] (SEQ ID NO: 212),

[0621] PxxAxxxNxxWE (SEQ ID NO: 213),

[0622] MxxxFxxMLxxR (SEQ ID NO: 214),

[0623] RxxxxGQS (SEQ ID NO: 215), and

[0624] NxxMS (SEQ ID NO: 216); wherein residues x represent independently of each other any natural amino acid residue.

[0625] 26. The method according to any of embodiments 24 and 25, wherein the meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204; preferably, to any one of SEQ ID NOs: 1 to 96 and 181 to 204; more preferably, to any one of SEQ ID NOs: 1 to 80 and 181 to 204.

[0626] 27. The method according to any of embodiments 24 to 26, wherein the meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181-204. The method according to any of embodiments 18 to 20 and 24 to 27, wherein the the terpene cyclase enzyme is a meroterpenoid cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 69, 71 , 73, 74, 75, 76, 77, 78, 100, 173, 177, 181 , 182, 184, 185, 188, 191 , 194, 197, 200, 202 and 203. The method according to any of embodiments 21 to 27, wherein the the terpene cyclase enzyme is a meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 20, 29, 46, 48, 69, 71 , 73, 74, 75, 76, 77 and 78. The method according to any of embodiments 24 to 29, wherein the meroterpenoid cyclase has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29. The method according to any of embodiments 24 to 30, wherein the meroterpenoid cyclase has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74. The method according to embodiment 24, wherein the squalene cyclase enzyme comprises at least one or more amino acid motifs selected from:

[0627] ■ [SP][TP][VIL]WDTx[LWI] (SEQ ID NO: 205),

[0628] . PGG[WF][GYA]F (SEQ ID NO: 206),

[0629] . PDxDD[TAS][TIAS] (SEQ ID NO: 207),

[0630] . [MIL]QxxxG[GA][WF]x[AS][FY] (SEQ ID NO: 208),

[0631] . Qxxx[GH]xWxG[RK]WGxx[YF]xYG (SEQ ID NO: 209),

[0632] . Qxx[DN]G[GS][WF][GS]ExxxS (SEQ ID NO: 210), and

[0633] . [STA]xx[SFN][QC]T[AGT]W[AS][LIV]xx[LQ] (SEQ ID NO: 211); wherein residues x represent independently of each other any natural amino acid residue. The method according to any of embodiments 24 and 32, wherein the squalene cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 142 to 148; preferably, to any one of SEQ ID NOs: 142, 143, 145 to 148. The method according to any of embodiments 14, 15, 24 and 32, wherein the compound of the formula (D’1 a) is the main product (as defined in the Section “Definitions”) and wherein the terpene cyclase enzyme is a squalene cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 144; preferably, the squalene cyclase enzyme has the amino acid sequence of SEQ ID NO: 144 The method according to any of the preceding embodiments, wherein the process is an in vivo or a bioconversion process. The method according to any of the preceding embodiments, wherein the process is performed in a recombinant cell capable of functionally expressing the terpene cyclase enzyme; preferably, said recombinant cell is a bacterial cell, a plant cell, a fungal cell such as a yeast cell; more preferably, said recombinant cell is of the genus Escherichia, Pseudomonas, Saccharomyces, Yarrowia or Pichia. A compound of the formula (A), (B), (C) and / or (D) obtained or obtainable by the method of any one of embodiments 1 to 36. A compound selected from the group consisting of the compound of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5),(C’1), (C’2), (C’4), (C’5), (D’1b), (A’1-1) and (A’1-2)

[0634] or a derivative thereof; wherein

[0635] R° represents either H or a C1-4 alkyl group, preferably ethyl or H;

[0636] R2represents either H or an alcohol protecting group, particularly , preferably H, R3represents a C1-4 alkyl group, preferably CH3; and any dotted line represents the bond by which the substituent is bound to the rest of the molecule; and any wavy line represents independently from each other a carbon-carbon bond which when linked to the carbon-carbon double bond is either in the Z or in the E-configuration, preferably in the E- configuration. The compound according to claim 38, wherein the compound of the formula (A’1-1) is a compound of the formula (A’1-1 a) or (A’1-1 b), the compound of the formula (A’1-2) is a compound of the formula (A’1-2a), the compound of the formula (B’3) is a compound of the formula (B’3a), the compound of the formula (B’5) is a compound of the formula (B’5a) and the compound of the formula (C’5) is a compound of the formula of the formula (C’5a)

[0637] (A’1-1 a), (A’1-2a),

[0638] (B’5a), (C’5a). A recombinant cell comprising the compound according to any of embodiments 37 to 39. The recombinant cell according to embodiment 40, wherein the cell comprises the terpene cyclase enzyme according to any of embodiments 24 to 34. A cell culture fermentation medium comprising the recombinant cell according to any of embodiments 40 and 41 . A reaction mixture comprising the compound according to any of embodiments 37 to 39. Use of the compound according to any of embodiments 37 to 39 as a perfumery, flavor or aroma ingredient, or as a precursor thereof. Use of a terpene cyclase enzyme, preferably a meroterpenoid cyclase enzyme, to produce the compound according to any of embodiments 37 to 39 or as defined in any of embodiments 1 to 23 and 37 to 39. A mutant meroterpenoid cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204; preferably, to any one of SEQ ID NOs: 1 to 96 and 181 to 204; more preferably, to any one of SEQ ID NOs: 1 to 80 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29; preferably, the mutant meroterpenoid cyclase enzyme has the amino acid sequence provided in SEQ ID NO: 80. A mutant meroterpenoid cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204; preferably, to any one of SEQ ID NOs: 1 to 96 and 181 to 204; more preferably, to any one of SEQ ID NOs: 1 to 80 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74; preferably, the mutant meroterpenoid cyclase enzyme has the amino acid sequence provided in any of SEQ ID NOs: 181 to 204.

[0639] The invention will now be described in further details by way of the following Examples. Said Examples are illustrative only and are not intended to limit the scope of the embodiments as described herein.

[0640] EXAMPLES

[0641] Materials and Methods

[0642] Unless otherwise stated, all chemical and biochemical materials and microorganisms or cells employed herein are commercially available products. (E)-p-Farnesene, compound of the formula (lb), was purchased from Sigma-Aldrich (73492, >90% purity).

[0643] Unless otherwise specified, recombinant proteins are cloned and expressed by standard methods, such as, for example, as described by Sambrook, J., Fritsch, E.F and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0644] Cultivation of engineered bacteria cells under conditions enabling production of terpene compounds

[0645] The DP1205 E. co / / cells engineered to produce increased levels of the terpenoid precursor farnesyl diphosphate (FPP) (as described in WO / 2021 / 005097) were transformed with one or two expression plasmids carrying genes encoding for enzymes from linear terpene biosynthetic pathways and / or for meroterpenoid cyclases. The transformed cells were cultured with the appropriate antibiotics (kanamycin (50 pg / mL) and / or carbenicillin (50 pg / mL) and / or chloramphenicol (34 pg / mL)) and / or streptomycin (50 pg / mL)) on LB-agarose plates.

[0646] Single colonies were selected to inoculate 0.5 mL liquid LB medium supplemented with the same antibiotic(s) and 1 % glucose in 2 mL polypropylene deep-well plates (Thermo Fisher Scientific, Massachusetts, USA) and grown overnight at 30 °C, 1000 rpm in a Multitron Pro incubation shaker (Infers HT, Basel, Switzerland). The following day, 0.5 mL of AM medium (as described in PCT / EP2024 / 066253) per well containing a 1 % mineral oil (w / v) (2705-01 , VWR International, LLC.) / 0.1 % (w / v) Tween 80 (Sigma-Aldrich, Missouri, USA) emulsion with 0.1 mM IPTG and the same antibiotic(s) was inoculated with 25 pL of the overnight culture and grown at 25 °C, 1000 rpm for 72h. After incubation, the cultures were extracted with 1.5 volume of ethyl acetate containing an internal standard (1 -undecanol) for quantification. The organic phase from each well was then analyzed using GC-MS / FID as described below.

[0647] GC-MS / FID analysis methods and compound identification

[0648] Samples were analyzed using an Agilent 8890 GC system coupled with a 5977B series Mass Selective Detector (MSD) and a flame ionization detector (FID). The instrument is equipped with a split / splitless injector (Agilent Technologies, CA) and a CombiPAL autosampler (PAL LSI 85 autosampler, Agilent Technologies, CA) injection system. The GC inlet temperature was set to 260 °C and 1 .0 pL of sample was injected in split mode with a ratio of 25:1 (23.304 PSI) and analyzed on a DB-5ms capillary column (30 m x 0.25 mm inner diameter x 0.25 pm film thickness; Agilent J&W) using helium as a carrier gas at a constant flow of 2.5 mL / min. The initial temperature of the oven was set at 80 °C and was programmed to 260 °C (20 °C / min) and then to 300 °C (40 °C / min; hold 1 min). The identification of known compounds was achieved by comparing mass spectrometry (MS) data and linear retention index (LRI) values with those of internally available standards. Quantification was done by the flame ionization detector (FID). An internal standard was used.

[0649] Example 1 : In vivo production of compound of the formula (B’1 ), (C’1 ), (A’1-1a), (A’1-2a) and / or (D’1a) in bacterial cells of E. coli engineered to produce (E)-P-Farnesene (lb) and expressing a wildtype bacterial membrane-integrated meroterpenoid cyclase.

[0650] In this example, putative bacterial membrane-integrated meroterpenoid cyclases were tested in vivo in bacterial cells for their ability to cyclize (E)-p-Farnesene (lb). Enzyme candidates were selected based on a BLAST search using standard parameters with the protein sequence of the bacterial membrane- integrated meroterpenoid cyclase DmtA1 (Nat Commun 9, 4091 (2018). https: / / doi.org / 10.1038 / s41467- 018-06411-x). Enzymes candidates having high to low protein sequence similarity with DmtA1 were randomly picked. The DNA fragments coding for enzyme candidates were codon optimized and cloned into an expression vector containing the clodfl 3 origin, the streptomycin resistance (SmR), T5 promoter, the RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 222), lambda TO terminator and lac operatorto control transcription as well as the lactose operon repressor (lacl).

[0651] The DNA sequence encoding the (E)-p-Farnesene synthase AaBFS (SEQ ID NO: 149) was codon optimized and cloned into the expression plasmid pD444 (ATUM, Newark, California) resulting in the plasmid pD444-AaBFS. Bacterial membrane-integrated meroterpenoid cyclase encoding plasmids were co-transformed with pD444-AaBFS into the FPP producing E. coli strain DP1205, as described in W02021 / 005097. The resulting strains were cultivated in deep-well plates under conditions enabling the production of terpene compounds and subsequently analysed by GC-MS / FID.

[0652] Surprisingly, 79 bacterial membrane-integrated meroterpenoid cyclases (SEQ ID NO: 1-79) were able to cyclise (E)-p-Farnesene to five new products which were identified as compounds of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a) and (D’1 a). The MS spectra of the corresponding compounds are shown in Figure 1 (A)-(E). The ratios of the cyclisation products are shown in Table 1 . Interestingly, for most cyclases, compound of the formula (A’1-1 a) was produced with the highest abundance, followed by compound of the formula (C’1) and (B’1), while compound of the formula (D’1 a) and (A’1-2a) were only produced in small amounts. The highest cyclisation rate of (E)-p-Farnesene was observed when using E. coli co-expressing the bacterial membrane-integrated meroterpenoid cyclase WP_318017018.1 (SEQ ID NO: 73) and AaBFS (Figure 2). As shown in Figure 2, (E)-p-Farnesene was converted to the new compounds of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a) and (D’1 a). The ratios of the cyclisation products from (E)-p-Farnesene were 70 % for compound of the formula (A’1-1 a), 12 % for compound of the formula (B’1), 17 % for compound of the formula (C’1), ~1 % for compound of the formula (A’1-2a) and ~1 % for compound of the formula (D’1 a).

[0653] As shown in Table 1 , it was surprisingly found that the ratios of the cyclisation products are not constant but depend significantly on the bacterial membrane-integrated meroterpenoid cyclase used. This finding is examplified in Figure 3 by showing an overlay of the chromatograms of the in vivo cyclisation of (E)-p-farnesene by the bacterial membrane-integrated meroterpenoid cyclases WP_033281172.1 (SEQ ID NO: 29), WP_229232892.1 (SEQ ID NO: 55)) and RLD81 128.1 (SEQ ID NO: 20). Depending on the bacterial membrane-integrated meroterpenoid cyclase used, the major cyclisation product can either be compound of the formula (A’1-1 a) (WP_033281172.1 and WP_229232892.1) or compound of the formula (C’1) (RLD81128.1) Interestingly, the meroterpene cyclase RLD81128.1 also produces a significantly higher ratio of compound of the formula (D’1 a) compared to WP_033281172.1 and WP_229232892.1.

[0654] Table 1 : in vivo production of compound of the formula (A’1-1 a), (B’1), (C’1), (A’1-2a) and / or (D’1 a) from (E)-p-Farnesene (lb). The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis and excluding compounds designated as “n.q.”; n.q. = not quantified as below limit of quantification.

[0655] Example 2: Bioconversion of (E)-P-Farnesene (lb) to compound of the formula (B’1), (C’1 ), (A’1-1a), (A’1-1 b), (A’1-2a) and / or (D’1a) using bacterial cells of E. coli expressing a bacterial membrane- integrated meroterpenoid cyclase.

[0656] In this example the production of compounds of the formula (B’1), (C’1), (A’1-1 a), (A’1-1 b), (A’1- 2a) and / or (D’1 a) from (E)-p-Farnesene (lb) is demonstrated by the bioconversion of (E)-p-Farnesene (lb) with bacterial cells expressing bacterial membrane-integrated meroterpenoid cyclases. The bioconversion was tested with the 79 bacterial membrane-integrated meroterpenoid cyclases as listed in Table 1 . For this, as described in Example 1 , the polynucleotides encoding the bacterial membrane-integrated meroterpenoid cyclase were codon optimized and cloned into an expression vector containing the clodfl 3 origin, the streptomycin resistance (SmR), T5 promoter, the RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 222), the lambda TO terminator and lac operator to control transcription as well as the lactose operon repressor (lacl). Each of the vectors encoding for a bacterial membrane-integrated meroterpenoid cyclase was transformed in E. coli C43(DE3) (Sigma-Aldrich, Missouri, USA).

[0657] The resulting strains were cultivated in 2 mL polypropylene deep-well plates (DWP) (Thermo Fisher Scientific, Massachusetts, USA) in 0.5 mL LB medium at 37°C, 1000 rpm overnight. 25 pL of each culture was used to inoculate other 2 mL polypropylene deep-well plate containing 0.5 mL AM medium with 0.1 mM IPTG, 50 ug / mL streptomycin and 6 pL of (E)-p-Farnesene 50% w / v in ethanol). (E)-p-Farnesene was obtained from Sigma-Aldrich, Missouri, USA. The DWP were closed with an air permeable membrane and incubated for 72h at 25 °C, 1000 rpm in a Multitron Pro incubation shaker (Infers HT, Basel, Switzerland). After incubation, the plate was extracted with ethyl acetate containing an internal standard for quantification. The organic phase of each well was analyzed by GC-MS / FID.

[0658] It was found that the 79 bacterial membrane-integrated meroterpenoid cyclases that could convert (E)-p-Farnesene in v / vo to compound of the formula (B’1), (C’1), (A’1-1a), (A’1-2a) and / or (D’1a) (Example 1), were also able to do so by bioconversion.

[0659] Surprisingly, in comparison to the in vivo cyclisation of (E)-p-Farnesene from Example 1 , a new product was formed, which was identified as compound of the formula (A’1-1 b). The MS spectrum of compound of the formula (A’1-1 b) is shown in Figure 4 (B). Compound of the formula (A’1-1 b) is an ethyl ether of compound of the formula (A’1-1a) and is most likely formed when the carbocation during the cyclisation reaction is quenched with ethanol. The fact that ethanol was added as a co-solvent for (E)-p- Farnesene for the bioconversion and is not present in the in vivo conditions explains that this product was not detected in the in vivo screening described in Example 1 .

[0660] The (E)-p-Farnesene bioconversion results of the eight most active cyclases are shown in Table 2. When the bioconversion cyclisation titers were compared to the in vivo cyclisation of (E)-p-Farnesene from Example 1 (Table 1), it was found that there was a strong correlation. Consequently, bacterial membrane- integrated meroterpenoid cyclases with high in vivo cyclization activity, were similarly active when they were used in bioconversion.

[0661] Table 2: Bioconversion of (E)-p-Farnesene (l-b) to compound of the formula (A’1-1 a), (B’1), (C’1), (A’1-2a), (D’1 a) and / or (A’1-1 b). The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis.

[0662] Under the screening conditions, the bacterial membrane-integrated meroterpenoid cyclase WSW93534.1 (SEQ ID NO: 74) was the most active enzyme. The corresponding chromatogram of the GC- MS / FID analysis is shown in Figure 4 (A) and shows that (E)-p-Farnesene is converted to compounds of the formula (B’1), (C’1), (A’1-1a), (A’1-1 b), (A’1-2a) and (D’1a).

[0663] Example 3: In vivo production of different ratios of compound of the formula (B’1 ), (C’1 ), (A’1-1a), (A’1-2a) and / or (D’1a) in bacterial cells of E. coli engineered to produce (E)-P-Farnesene (lb) and expressing a mutant bacterial membrane-integrated meroterpenoid cyclase.

[0664] In this example, the ratio between the compounds of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a) and (D’1 a) produced by the cyclisation of (E)-p-Farnesene (lb) by a bacterial membrane-integrated meroterpenoid cyclase are changed by protein engineering. Therefore, mutant variants of the bacterial membrane-integrated meroterpenoid cyclase WP_033281172.1 (SEQ ID NO: 29) were created. Mutant variants were designed by a structure guided approach that was based on a protein structure model of WP_033281172.1 (SEQ ID NO: 29), which was built using ESMFold (Zeming et al. Evolutionary-scale prediction of atomic level protein structure with a language model. bioRxiv 2022.07.20.500902). End to end atomic level structure prediction from the protein sequence was based on the pretrained neural network model esm. pretrained. esmfold_v1 (https: / / github.eom / facebookresearch / esm#esmfold). Extracted representations of the protein sequence are derived from the protein language model ESM-2.

[0665] The protein model of WP_033281172.1 adopts a pore like structure consisting of 7 helices. This finding was in agreement with the transmembrane helice prediction software TMHMM 2.0 (TMHMM 2.0 server available at https: / / dtu.biolib.com / DeepTMHMM (Krogh, A., et al. (2001) J Mol Biol 305(3)), which also predicted the presence of 7 transmembrane helices.

[0666] It was assumed that the active site is oriented inside the pore-like structure. For the creation of the mutant enzyme library, nine amino acids residues located inside and at the entrance of the active site of WP_033281172.1 (SEQ ID NO: 29) were then selected as target amino acids for the creation of a series of single point mutation variants.

[0667] Codon optimized DNA sequences encoding said mutant variants were ordered and cloned into an expression vector as described above. The vectors containing the genes of the mutant enzymes were then each transformed into DP1205 E. co / / harboring the vector pD444-AaBFS (as described in Example 1). The resulting strains were tested for the in vivo production of compounds of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a) and (D’1 a) as previously described. Among the mutants tested, WP_033281172.1 S9M (SEQ ID NO: 80) was able to significantly shift the ratios between the (E)-p-Farnesene cyclisation products. A comparison of the compounds of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a) and (D’1 a) produced by the wild-type enzyme (SEQ ID NO: 29) and the mutant enzyme (SEQ ID NO: 80) is shown in Figure 5. Interestingly, WP_033281172.1 S9M (SEQ ID NO: 80) is able to produce a significantly higher ratio of compound of the formula (B’1) compared to the wild-type enzyme. Furthermore, in comparison to the wildtype enzyme, compound of the formula (D’1 a) was not detected in this mutant.

[0668] Example 4: In vivo production of compound of the formula (A’1-1a), (B’1 ), (C’1), (A’1-2a) and / or (D’1a) in bacterial cells of E. coli engineered to produce (E)-P-Farnesene (lb) and expressing a squalene cyclase.

[0669] In this example, squalene cyclase candidates were tested in vivo to cyclise (E)-p-Farnesene (lb) to compound of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a) and / or (D’1 a). Therefore, the polynucleotides encoding the squalene cyclase candidates were codon optimized for expression in E. coli and cloned into the expression plasmid pD424 (ATUM, Newark, California). The polynucleoide encoding the (E)-p- Farnesene synthase AaBFS (SEQ ID NO: 149) was codon optimized and cloned into the expression plasmid pJ401 (ATUM, Newark, California) resulting in the plasmid pJ401 -AaBFS. Squalene cyclase encoding plasmids were co-transformed with pJ401 -AaBFS into the FPP producing E. coli strain DP1205.

[0670] The resulting strains were cultivated in deep-well plates under conditions enabling the production of terpenoid compounds and were subsequently analyzed. Table 3 shows the results for seven squalene cyclase enzymes. Under these conditions, they were found to cyclize (E)-p-Farnesene to compound of the formula (A’1-1 a) and (D’1 a) with compound of the formula (B’1), (C’1) and / or (A’1-2a) being not quantified as below the limit of quantification.

[0671] Table 3: in vivo production of compound of the formula (A’1-1 a), (B’1), (C’1), (A’1-2a) and / or (D’1 a) from (E)-p-Farnesene (l-b). The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis and excluding compounds designated as “n.q.”; n.q. = not quantified as below limit of quantification.

[0672] Under the screening conditions, the squalene cyclase BmeSHC_G595M (SEQ ID NO: 144) was found to have the highest cyclisation activity. Compared to the other squalene cyclases, it was surprisingly found that BmeSHC_G595M (SEQ ID NO: 144) produced compound of the formula (D’1 a) in higher titer than compound of the formula (A’1-1 a).

[0673] Subsequently, the above-described E.coli strain co-expressing the (E)-p-Farnesene synthase AaBFS (SEQ ID NO: 149) and the squalene cyclase BmeSHC_G595M (SEQ ID NO: 144), was further investigated under larger scale culture conditions. For this, the strain was cultivated in a shake flask culture. For the cultivation, a 2 mL over-night culture (LB medium containing 1 % of glucose and the corresponding antibiotics) was used to inoculated 50 mL AM medium (containing the corresponding antibiotics and 0.1 mM IPTG) in a shake flask culture. The shake flask culture was incubated in a multiron incubator (Infors HT) at 130 rpm shaking at 25°C for 3 days. After the cultivation, terpene compounds were extracted by ethyl acetate. The combined organic extracts were dried by anhydrous MgSO4 and concentrated before analysis by GC-MS / FID. The corresponding chromatogram is shown on Figure 6. Under the shake flask cultivation conditions, the compound of the formula (A’1-2a) could be additionally detected next to the compounds of the formula (A’1-1 a) and (D’1 a) which were already detected under DWP culture conditions above.

[0674] Example 5: Production and isolation of compounds of the formula (B’1 ), (C’1 ), (A’1-1a), (A’1-1 b), (A’1-2a), (D’1aa) and (D’1ab) for structure elucidation and characterization.

[0675] To isolate pure compounds of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a), (D’1 aa) and (D’1 ab) for structure elucidation and characterization, the bacterial strain described in Example 1 expressing the meroterpenoid cyclase WP_318017018.1 (SEQ ID NO: 73) was cultivated in shake flask cultures. Therefore, 50 mL overnight shake flask cultures (LB medium containing 1 % of glucose and the corresponding antibiotics) were used to inoculate 500 mL shake flask cultures containing AM medium, the corresponding antibiotics, 0.1 mM IPTG and 5 % (v / v) n-Decane. The flask cultures were inoculated in a multiron incubator (Infors HT) at 130 rpm shaking at 25°C for 3 days. After the cultivation, terpene compounds were extracted by ethyl acetate. The combined organic extracts were dried by anhydrous MgSO4 and concentrated. Pure compounds were isolated by column chromatography (SiO2, pentane / Et2O 100 / 0 -> pentane / Et2O 60 / 40).

[0676] The following analytical data was obtained for compounds of the formula (B’1), (C’1), (A’1-1 a), (A’1-2a), (D’1 aa) and (D’1 ab):

[0677] Compound of the formula (A’1-1a):

[0678] (2R,4aS,8aR)-5,5,8a-trimethyl-2-vinyldecahydronaphthalen-2-ol:

[0679] 1H-NMR (600 MHz, CDCb): 0.76 (s, 3H), 0.87 (s, 3H), 0.89 (s, 3H), 0.96 (dd, 1 H, J = 12.3 Hz, J = 2.4 Hz), 1.06 (td, 1 H, J = 13.1 Hz, J = 3.6 Hz), 1.18 (td, 1 H, J = 13.6 Hz, J = 4.7 Hz), 1 .28 (qd, 1 H, J = 13.6 Hz, J = 3.3 Hz), 1 .35-1.42 (m, 4H), 1.44 (d, 1 H, J = 12.7 Hz), 1.50 (td, 1 H, J = 13.3 Hz, J = 4.3 Hz), 1 .57 (dd, 1 H, J = 12.6 Hz, J = 2.5 Hz), 1 .57-1.68 (m, 2H), 2.17 (dq, 1 H, J = 13.0 Hz, 3.0 Hz), 5.09 (d, 1 H, J = 10.7 Hz), 5.30 (d, 1 H, J = 17.6 Hz), 6.11 (dd, 1 H, J = 17.5 Hz, J = 10.8 Hz).

[0680] 13C NMR (150 MHz, CDCb): 6 145.26, 112.32, 71.72, 58.30, 53.96, 42.44, 42.37, 39.00, 35.21 , 33.29, 32.93, 21.26, 21.05, 20.41 , 18.53.

[0681] Melting point: 62°C, [a]D-60.5° (c 1 .6, CHCb).

[0682] Compound of the formula (A’1-2a):

[0683] (2S,4aS,8aR)-5,5,8a-trimethyl-2-vinyldecahydronaphthalen-2-ol

[0684] 1H-NMR (600 MHz, CDCb): 0.84 (s, 3H), 0.86-0.90 (m, 1 H), 0.88 (s, 3H), 0.99 (td, 1 H, J = 13.6 Hz, J = 4.0 Hz), 1.13-1.21 (m, 1 H), 1.16 (s, 3H), 1.24-1.45 (m, 5H), 1.45-1.58 (m, 2H), 1.61-1.73 (m, 3H), 4.95 (dd, 1 H, J = 10.6 Hz, J = 1 .3 Hz), 5.19 (dd, 1 H, J = 17.3 Hz, J = 1 .4 Hz), 5.86 (dd, 1 H, J = 17.3 Hz, J = 10.7 Hz).

[0685] 13C NMR (125 MHz, CDCb): 6 147.81 , 110.37, 73.35, 55.19, 54.14, 42.52, 42.44, 39.70, 34.58, 33.21 , 33.00, 21.34, 21.07, 18.36, 18.24.

[0686] 2-((4aS,8aR,E)-5,5,8a-trimethyloctahydronaphthalen-2(1 H)-ylidene)ethan-1-ol13C NMR (90 MHz, CDCb): 6 141 .61 , 121 .81 , 58.41 , 55.37, 53.76, 42.55, 41 .96, 36.23, 33.19, 33.11 , 29.34, 23.02, 21.42, 19.23, 19.08.

[0687] To isolate pure compounds of the formula (A’1-1 b) for the structure elucidation and characterization, the bacterial strain described in Example 2 expressing the meroterpenoid cyclase WSW93534.1 (SEQ ID NO: 74) was used. The strain was cultivated in a 50 mL overnight shake flask culture (LB medium containing 1 % of glycose and the corresponding antibiotic) and was used to inoculate a 500 mL shake flask culture containing AM medium, the corresponding antibiotic, 0.1 mM IPTG. The flask cultures were inoculated in a multiron incubator (Infors HT) at 130 rpm shaking at 25°C for 24 hours. The cells were concentrated to an OD600 of 20 and transferred to a 500 mL round-bottom flask. (E)-p- Farnesene (Sigma-Aldrich, Missouri, USA) in 50% (w / in ethanol) was added to a final concentration of 10 g / L and the suspension was strongly agitated by a magnetic bar and a magnetic mixer at 25°C. After 3 days, the bioconversion products were extracted by ethyl acetate. The combined organic extracts were dried by anhydrous MgSC and concentrated. The pure compound was isolated by column chromatography (SiC>2, pentane / Et2O 100 / 0 -> pentane / Et2O 60 / 40).

[0688] The following analytical data was obtained for compound of the formula (A’1-1 b):

[0689] Compound of the formula (A’1-1 b):

[0690] (4aR,6R,8aS)-6-ethoxy-1 ,1 ,4a-trimethyl-6-vinyldecahydronaphthalene

[0691] 1H NMR (600 MHz, CDCb) 6 0.75 (s, 3H), 0.87 (d, 6H, J = 13.9 Hz), 0.97 (dd, 1 H, J = 12.3 Hz, 2.5 Hz), 1.04 (td, 1 H, J = 13.8 Hz, 4.3 Hz), 1.08 (t, 3H, J = 7.0 Hz), 1.17 (td, 1 H, J = 13.7 Hz, 4.3 Hz), 1.24 (qd, 1 H, J = 13.5 Hz, J = 3.2 Hz), 1 .33-1.42 (m, 4H), 1 .45 (d, 1 H, J = 12.7 Hz), 1 .56-1.70 (m, 3H), 2.35 (dq, 1 H, J = 12.8 Hz, J = 3.1 Hz), 3.27 (qd, 2H, J = 7.0 Hz, J = 2.3 Hz), 5.20-5.25 (m, 2H), 5.73 (ddd, 1 H, J = 17.7 Hz, J = 1 1.1 Hz, J = 1.2 Hz).

[0692] 13C NMR (151 MHz, CDCb) 6 142.10, 114.57, 75.66, 57.63, 55.64, 54.09, 42.55, 42.42, 35.17, 33.98, 33.30, 32.89, 21.27, 21 .25, 20.15, 18.51 , 16.05.

[0693] Example 6: Bioconversion of compound of the formula (If) to compound of the formula (B’5a), (C’5a) and / or (A’5-2a) using bacterial cells of E. coli expressing a bacterial membrane-integrated meroterpenoid cyclase.

[0694] In this example, the production of compound of the formula (B’5a), (C’5a) and / or (A’5-2a) from compound of the formula (If) is demonstrated by the bioconversion of compound of the formula (If) with bacterial cells expressing a bacterial membrane-integrated meroterpenoid cyclase. The bioconversion was tested with twelve bacterial membrane-integrated meroterpenoid cyclases as listed in Table 4. For this, as described in Example 1 , the polynucleotides encoding the bacterial membrane-integrated meroterpenoid cyclase were codon optimized and cloned into an expression vector containing the clodfl 3 origin, the streptomycin resistance (SmR), T5 promoter, the RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 222), the lambda TO terminator and lac operator to control transcription as well as the lactose operon repressor (lacl). Each of the vectors encoding for a bacterial membrane-integrated meroterpenoid cyclase was transformed in E. coli C43(DE3) (Sigma-Aldrich, Missouri, USA).

[0695] The resulting strains were cultivated in 2 mL polypropylene deep-well plates (DWP) (Thermo Fisher Scientific, Massachusetts, USA) in 0.5 mL LB medium at 37°C, 1000 rpm overnight. 25 pL of each culture was used to inoculate other 2 mL polypropylene deep-well plate containing 0.5 mL AM medium with 0.1 mM IPTG, 50 pg / mL streptomycin and 6 pL of a 200 mg / mL ethanolic solution of compound of the formula (If). Compound of the formula (If) was internally synthesized following the procedure described in WO2017 / 009205. The DWP were closed with an air permeable membrane and incubated for 72h at 25 °C, 1000 rpm in a Multitron Pro incubation shaker (Infors HT, Basel, Switzerland). After incubation, the plates were extracted with ethyl acetate containing an internal standard for quantification. The organic phase of each well was analyzed by GC-MS / FID. The results are shown in Table 4.

[0696] All tested meroterpenoid cyclase enzymes were found to cyclise compound of the formula (If) into compound of the formula (B’5a), (C’5a) and / or (A’5-2a). The MS spectra of the corresponding compounds are shown in Figure 8 (A)-(C). Under the screening conditions, the bacterial membrane-integrated meroterpenoid cyclase WP_317769678.1 (SEQ ID NO: 71) was the most active enzyme. The corresponding chromatogram of the GC-MS / FID analysis is shown in Figure 7 and shows the conversion of compound of the formula (If) to compound of the formula (B’5a), (C’5a) and (A’5-2a).

[0697] Table 4: Bioconversion of compound of the formula (If) to compound of the formula (B’5a), (C’5a) and / or (A’5-2a). The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis and excluding compounds designated as “n.q.”; n.q. = not quantified as below limit of quantification. Example 7: Production and isolation of compounds of the formula (B’5a), (C’5a) and (A’5-2a) for structure elucidation and characterization.

[0698] To isolate pure compounds of the formula (B’5a), (C’5a) and (A’5-2a) for structure elucidation and characterization, the bioconversion of compound of the formula (If) was scaled up from a deep-well plate culture to a shake flask experiment. Therefore, the bacterial cells expressing the bacterial meroterpenoid cyclase WP_317769678.1 (SEQ ID NO: 71) - as described in Example 6 - was used.

[0699] An overnight preculture of the strain was cultivated in a shake flask culture containing 30 mL of LB medium, supplemented with 1 % of glucose and the corresponding antibiotic. The preculture was then used to inoculate 400 mL AM medium in a shake flask culture containing the corresponding antibiotic and 0.1 mM IPTG. The flask cultures were incubated in a Multitron incubator (Infers HT), with shaking at 150 rpm at 25°C for 5 hours before the compound of the formula (If) (synthesized as described in WO2017009205A1 from (E)-p-Farnesene) was added as an ethanolic solution (200 mg / mL) to reach a final concentration of 2 g / L. The shake flask culture was then further incubated at 150 rpm shaking and 25°C for 3 days. Bioconversion products were extracted using ethyl acetate. The combined organic extracts were dried over anhydrous MgSC and then concentrated. Pure compounds were isolated by reverse-phase chromatography, utilizing a water and acetonitrile solvent system with a gradient from 70% to 90% acetonitrile over 30 minutes.

[0700] The following analytical data were obtained for compounds of the formula (B’5a), (C’5a) and (A’5-1 a).

[0701] Compound of the formula (B’5a): 3-((4aS,8aR)-5,5,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalen-2-yl)propan-1-ol

[0702] 13C NMR (CDCb, 151 MHz) 6 18.99, 19.09, 21.59, 21 .31 , 30.15, 30.66, 32.98, 33.02, 33.53, 35.12, 40.04, 42.27, 51.49, 63.02, 133.20, 136.10.

[0703] Compound of the formula (C’5a):

[0704] 3-((4aS,8aR)-5,5,8a-trimethyl-1 ,4,4a,5,6,7,8,8a-octahydronaphthalen-2-yl)propan-1-ol

[0705] 13C NMR (CDCb, 151 MHz) 6 18.91 , 19.19, 21.43, 23.72, 30.54, 32.76, 32.85, 32.90, 33.98, 41.99, 42.85, 48.55, 48.83, 62.92, 120.72, 135.26.

[0706] Compound of the formula (A’5-2a): (2R,4aS,8aR)-2-(3-hydroxypropyl)-5,5,8a-trimethyldecahydronaphthalen-2-ol

[0707] 13C NMR (CDCb, 151 MHz) 6 18.34, 21 .01 , 21 .31 , 26.40, 33.00, 33.19, 34.56, 39.59, 42.42, 42.51 , 42.58, 54.48, 55.52, 63.48, 72.39. The relative configurations were determined by NMR, whereas the absolute configurations were inferred based on the cyclization mechanism of analogous compounds with established absolute configurations.

[0708] Example 8: Bioconversion of (E)-P-Farnesene (lb) to compound of the formula (B’1), (C’1 ), (A’1-1a), (A’1-2a) and / or (D’1a) in bacterial cells of E. coli expressing a mutant bacterial membrane-integrated meroterpenoid cyclase.

[0709] In this example, mutants of the bacterial membrane-integrated meroterpenoid cyclase WSW93534.1 (SEQ ID NO: 74) were created and screened. For this, a protein structure model of WSW93534.1 (SEQ ID NO: 74) was created as described in Example 3. This model was used to identify amino acid positions in the active site that were selected as target amino acids for the creation of a series of single point mutation variants.

[0710] The polynucleotides encoding the above mutants of the bacterial membrane-integrated meroterpenoid cyclase WSW93534.1 (SEQ ID NO: 74) were codon optimized and cloned into an expression vector containing the clodfl 3 origin, the streptomycin resistance (SmR), T5 promoter, the RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 222), the lambda TO terminator and lac operator to control transcription as well as the lactose operon repressor (lacl). The expression vectors coding the mutants of the bacterial membrane-integrated meroterpenoid cyclase were transformed into E. coli C43(DE3) (Sigma- Aldrich, Missouri, USA). The resulting strains were tested forthe bioconversion of (E)-p-Farnesene in deepwell plates as described in Example 2. The substrate (E)-p-Farnesene was added as pure compound to each well (2 pL / well). After incubation, the plates were extracted with ethyl acetate containing an internal standard for quantification. The organic phase of each well was analyzed by GC-MS / FID.

[0711] Among the mutants tested from the bacterial membrane-integrated meroterpenoid cyclase WSW93534.1 (SEQ ID NO: 74), the mutants WSW93534.1_A126S (SEQ ID NO: 190), WSW93534.1_G123M (SEQ ID NO: 191) and WSW93534.1_P165G (SEQ ID NO: 192) were found to significantly shift the ratios between the (E)-p-Farnesene cyclisation products as well as their titers compared to the wild-type enzyme WSW93534.1. The results are shown in Table 5. The mutation in WSW93534.1_G123M increased the overall cyclisation titer by 23% compared to the wild-type enzyme, while increasing the ratios of compounds of the formula (B’1) and (C’1) over (A’1-1 a). The mutation in WSW93534.1_A126S was found to have the reverse effect by increasing the ratio of compound of the formula (A’1-1 a) over (B’1) and (C’1). Surprisingly, the mutation in WSW93534.1_P165G significantly increased the ratio of compound of the formula (D’1 a) over (A’1-1 a), (B’1) and (C’1).

[0712] Subsequently, the mutants of WP_200714303.1 (SEQ ID NO: 48), WP_317769678.1 (SEQ ID NO: 71), WP_318017018.1 (SEQ ID NO: 73), WSX12386.1 (SEQ ID NO: 75), WSY54005.1 (SEQ ID NO: 76), WTE43000.1 (SEQ ID NO: 77) and WTK72062.1 (SEQ ID NO: 78) were generated to contain the mutations identified above at the corresponding amino acid position 123, 126 or 165 relative to SEQ ID NO: 74. This 2024P0179WQ resulted in SEQ ID NOs: 181-183, SEQ ID NOs: 184-186, SEQ ID NOs: 187-189, SEQ ID NOs: 190-192, SEQ ID NOs: 193-195, SEQ ID NOs: 196-198, SEQ ID NOs: 199-201 , 202-204; respectively. Protein sequence alignment was used to identify the corresponding amino acid positions relative to SEQ ID NO: 74. The mutants were tested as described above and the results are shown in Table 5. It was found that these amino acid positions are functionally conserved in all bacterial membrane-integrated meroterpenoid cyclases tested. By transposing the mutations of the WSW93534.1 mutants, it was possible to obtain a similar shift in the ratio of compound of the formula (B’1), (C’1), (A’1-1 a) and / or (D’1 a) as described above. Surprisingly, it was found that the mutation at the amino acid position 126 relative to SEQ ID NO: 74 (e.g. A126S) generally led to significantly higher cyclisation activity of (E)-p-Farnesene (lb) and / or a preferred ratio of compound of the formula (A’1 -1 a).

[0713] Table 5: Bioconversion of compound of the formula (lb) to compound of the formula (A’1-1 a), (B’1), (C’1), (A’1-2a) and / or (D’1 a) by a wild-type or a mutant bacterial membrane-integrated meroterpenoid cyclases. The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis and excluding compounds designated as “n.q.”; n.q. = not quantified as below limit of quantification.

[0714] Example 9: Bioconversion of (E)-P-Farnesene (lb) to compound of the formula (B’1), (C’1 ), (A’1-1a), (A’1-2a) and / or (D’1a) using bacterial cells of Pseudomonas alloputida expressing a bacterial membrane-integrated meroterpenoid cyclase.

[0715] In this example, the production of compound of the formula (B’1), (C’1), (A’1-1 a), (A’1- 2a) and / or (D’1 a) from (E)-p-Farnesene (lb) is demonstrated by the bioconversion of (E)-p-Farnesene with bacterial cells of Pseudomonas alloputida (DSM 6125) expressing a wild-type or a mutant bacterial membrane- integrated meroterpenoid cyclase. The bioconversion was tested with seven bacterial membrane- integrated meroterpenoid cyclases as listed in Table 6. For this, the polynucleotides were codon optimized for expression in Pseudomonas alloputida and cloned into the vector pGingerBK-LacUV5 (JPUB_020837; Microbiol Spectr. 2023 Jun 15;11 (3):e0037323) using standard cloning techniques.

[0716] The vectors encoding for bacterial membrane-integrated meroterpenoid cyclases were then transformed into Pseudomonas alloputida (DSM 6125) as previously described (Bioscience, Biotechnology, and Biochemistry, Volume 58, Issue 5, 1 January 1994, Pages 851-854). The resulting strains were cultivated overnight in 2 mL polypropylene deep-well plates (DWP) (Thermo Fisher Scientific, Massachusetts, USA) in 0.5 mL LB medium containing 50 pg / mL kanamycin at 30°C, 1000 rpm. 25 pL of each culture was used to inoculate a new 2 mL polypropylene deep-well plate containing 0.5 mL AM medium with 1 mM IPTG, 50 pg / mL kanamycin and 2 pL of pure (E)-p-Farnesene. (E)-p-Farnesene was obtained from Sigma-Aldrich, Missouri, USA. The DWP were closed with an air permeable membrane and incubated for 72h at 25°C, 1000 rpm in a Multitron Pro incubation shaker (Infers HT, Basel, Switzerland). After incubation, the plates were extracted with ethyl acetate containing an internal standard for quantification. The organic phase of each well was analyzed by GC-MS / FID. The results are shown in Table 6. Under the screening conditions, the bacterial membrane-integrated meroterpenoid cyclase mutant WSW93534.1_G123M (SEQ ID NO: 191) was the most active enzyme in Pseudomonas alloputida. Interestingly, it was found that the mutant WSW93534.1_G123M is four times more active than the wildtype enzyme. The second most active bacterial membrane-integrated meroterpenoid cyclase in Pseudomonas alloputida was found to be WTE43000.1 (SEQ ID NO: 77).

[0717] Table 6: Bioconversion cyclisation of compound of the formula (lb) to compound of the formula (A’1-1a), (B’1), (C’1), (A’1-2a) and / or (D’1a) by a bacterial membrane-integrated meroterpenoid cyclase. The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis and excluding compounds designated as “n.q.”; n.q. = not quantified as below limit of quantification.

[0718] Example 10: Bioconversion of (E)-P-Farnesene (lb) to compound of the formula (B’1), (C’1), (A’1-1a), (A’1-1b), (A’1-2a) and / or (D’1a) using yeast cells of Saccharomyces cerevisiae expressing a bacterial membrane-integrated meroterpenoid cyclase.

[0719] In this example, the production of compound of the formula (B’1), (C’1), (A’1-1a), (A’1-1 b), (A’1-2a) and / or (D’1a) from (E)-p-Farnesene (lb) is demonstrated by the bioconversion of compound of the formula (lb) with yeast cells of Saccharomyces cerevisiae expressing a bacterial membrane-integrated meroterpenoid cyclase.

[0720] S. cerevisiae strain construction

[0721] The polynucleotides encoding the bacterial membrane-integrated meroterpenoid cyclase WP_033281172.1 (SEQ ID NO 29), WP_200714303.1 (SEQ ID NO 48), WP_317769678.1 (SEQ ID NO 71), WP_318017018.1 (SEQ ID NO 73), WSX12386.1 (SEQ ID NO 75), WSY54005.1 (SEQ ID NO 76), WTE43000.1 (SEQ ID NO 77) and WTK72062.1 (SEQ ID NO 78) were codon optimized for expression in S. cerevisiae, placed under control of promoter KI_TDH2.pro (Kluyveromyces lactis promoter of KLLA0F20988g, SEQ ID NO: 223) and flanked on the 3’ side by Sc_RPL15A.term terminator (S. cerevisiae terminator, SEQ ID NO: 224). Each of the resulting expression cassettes encoding for a bacterial meroterpenoid cyclase was then integrated into a non-coding region of the genome of S. cerevisiae SHK001 (Moreno-Paz et al. (2024). ACS Synth Biol. 13: 1312-1322).

[0722] Media

[0723] S. cerevisiae strains were grown at 30°C in Yeast Extract Phytone Dextrose media for transformations and precultures (YEPhD, 2% Difco phytone peptone (211906, Gibco BRL), 1% Bacto Yeast extract (212750, Gibco BRL), and 2% d-glucose (G7528, Sigma-Aldrich).

[0724] In deep-well plates (DWP), 0.5 YEPhD (1 :1 YEPhD to milliQ) was used for pre-culture of the S. cerevisiae strains. To avoid ethanol formation by the S. cerevisiae strains in the main culture, a production medium with a continuous glucose feed by glucoamylase (GLA)-catalyzed glucose release from maltodextrin (GLA- glucose release medium) was applied (Kemmer, A et al. (2024). Bioengineering, 11 : 107). (E)-p-Farnesene (P3500-95, BedoukianBio) was mixed with ethanol abs. (83672, VWR) in a 1 : 2 ratio. 3 pL of said mixture was added to each well of the DWP. Thereafter, 600 pL of the production medium (GLA-glucose release medium) was added.

[0725] Cultivation conditions in deep-well plates (DWP)

[0726] Individual strains of S. cerevisiae, each harboring a bacterial membrane-integrated meroterpenoid cyclase, were cultivated in 250 pL 0.5 YephD in 96-well half deep-well plates (736-0198, VWR) for 48h at 30°C, 750 rpm and 80% humidity in an Infers HT incubator. 6 pL of this pre-culture was used for inoculation of 600 pL of the production medium (GLA-glucose release medium) supplemented with (E)-p-Farnesene in 96-well 2 mL square well deep-well plates (DWP) (186002482, Waters). Main cultures were incubated for 5 days at 30°C, 1000 rpm, 80% humidity. Deep-well plates were sealed to prevent contamination and evaporation. On day 5, 800 pL ethyl acetate (L10925, Thermo Fisher Scientific) supplemented with 5 ppm internal standard, tetradecane (87140, Sigma-Aldrich), was added to 600 pL whole broth to completely stop the reaction and extract all molecules formed. The deep-well plates were sealed and shaken vigorously. Subsequently, the samples were centrifuged for 30 minutes at 20°C and 2000 rpm and used directly for GC-MS analysis.

[0727] Results

[0728] The results are shown in Table 7. Under the screening conditions described above, the bacterial membrane-integrated meroterpenoid cyclase WP_317769678.1 (SEQ ID NO: 71) was the most active enzyme in S. cerevisiae.

[0729] Table 7: Bioconversion cyclisation of compound of the formula (lb) to compound of the formula (B’1), (C’1), (A’1-1a), (A’1-1 b), (A’1-2a) and / or (D’1a) by a bacterial membrane-integrated meroterpenoid cyclases expressed in S. cerevisiae cells. The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis and excluding compounds designated as “n.q.”; n.q. = not quantified as below limit of quantification. Example 11 : Bioconversion of (E)-P-Farnesene (lb) to compound of the formula (B’1), (C’1), (A’1-1a), (A’1-1 b), (A’1-2a) and / or (D’1a) using yeast cells of Yarrowia lipolytica expressing a bacterial membrane-integrated meroterpenoid cyclase.

[0730] In this example, the production of compound of the formula (B’1), (C’1), (A’1-1 a), (A’1-1 b), (A’1-2a) and / or (D’1 a) from (E)-p-Farnesene (lb) is demonstrated by the bioconversion of compound of the formula (lb) with yeast cells of Yarrowia lipolytica expressing a bacterial membrane-integrated meroterpenoid cyclase.

[0731] Yarrowia lipolytica strain construction

[0732] The polynucleotides encoding the bacterial membrane-integrated meroterpenoid cyclase WP_200714303.1 (SEQ ID NO 48), WP_267502276.1 (SEQ ID NO 69), WP_317769678.1 (SEQ ID NO 71), WP_318017018.1 (SEQ ID NO 73), WSX12386.1 (SEQ ID NO 75), WSY54005.1 (SEQ ID NO 76) and WTE43000.1 (SEQ ID NO 77) were codon optimized for expression in Y. lipolytica, placed under control of promoter YI_HYPO. pro (Y. lipolytica promoter of YALI0D09889g, SEQ ID NO: 225) and flanked on the 3’ side by YI_YT013.term terminator (adjusted Y. lipolytica terminator of YALI0E23584g, SEQ ID NO: 226). Each of the resulting expression cassettes encoding for a bacterial meroterpenoid cyclase was then integrated into a non-coding region of the genome of Yarrowia lipolytica strain ML324 (MAT a; deposited under number ATCC18943).

[0733] Media

[0734] Y. lipolytica strains were grown at 28°C in Yeast Extract Phytone Dextrose media for transformations and precultures (YEPhD, 2% Difco phytone peptone (211906, Gibco BRL), 1 % Bacto Yeast extract (212750, Gibco BRL), and 2% d-glucose (G7528, Sigma-Aldrich).

[0735] In deep-well plates (DWP), 0.5 YEPhD (1 :1 YEPhD to milliQ) was used for pre-culture of the Y. lipolytica strains. The production medium used for the main cultures is based on Verduyn et al. (Verduyn C et al. Yeast (1992), 8:501-517) with modifications as described in WQ2015007748, using 20 g / L KH2PO4 (P5655, Sigma-AIrdich), 6 g / L urea (1.08487, Merck), 33 g / L glucose. aq (1.08342, Merck), 1.5 g / L MgSO4.7H2O (1.05886, Sigma-Aldrich) and 70 g / L MES hydrate (M2933, Sigma-Aldrich). (E)-p-Farnesene (P3500-95, BedoukianBio) is mixed with ethanol abs. (83672, VWR) in a 1 : 2 ratio. 3 .L of said mixture was added to each well of the DWP. Thereafter, 600 .L production medium is added.

[0736] Cultivation conditions in deep-well plates (DWP)

[0737] Individual transformants of Y. lipolytica, each harboring a bacterial membrane-integrated meroterpenoid cyclase, were cultivated in 250 pL 0.5 YephD in 96-well half deep-well plates (736-0198, VWR) for 48h at 28°C, 800 rpm and 80% humidity in an Infers HT incubator. 6 pL of this pre-culture was used for inoculation of 600 pL of the production medium supplemented with (E)-p-Farnesene in 96-well 2 mL square well deep-well plates (186002482, Waters). Main cultures were incubated for 5 days at 28°C, 1000 rpm, 80% humidity. All deep-well plates were sealed to prevent contamination and evaporation. On day 5, 800 pL ethyl acetate (L10925, Thermo Fisher Scientific) supplemented with 5 ppm internal standard, tetradecane (87140, Sigma-Aldrich), was added to 600 pL whole broth to completely stop the reaction and extract all molecules formed. The deep-well plates were sealed and shaken vigorously. Subsequently, the samples were centrifuged for 30 minutes at 20°C and 2000 rpm and used directly for GC-MS analysis.

[0738] Results

[0739] The results are shown in Table 8. Under the screening conditions described above, the bacterial membrane-integrated meroterpenoid cyclase WP_200714303.1 (SEQ ID NO: 48) was the most active enzyme in Y. lipolytica.

[0740] Table 8: Bioconversion cyclisation of compound of the formula (lb) to compound of the formula (B’1), (C’1), (A’1-1 a), (A’1-1 b), (A’1-2a) and / or (D’1 a) by a bacterial membrane-integrated meroterpenoid cyclases expressed in Y. lipolytica cells. The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis and excluding compounds designated as “n.q.”; n.q. = not quantified as below limit of quantification.

[0741] Example 12: Bioconversion of compound of the formula (li) to compound of the formula (B’3a), (C’3a), (A’3-1a) and / or (D’1a) using bacterial cells of E. coli expressing a bacterial membrane- integrated meroterpenoid cyclase.

[0742] In this example, the production of compound of the formula (B’3a), (C’3a), (A’3-1 a) and / or (D’1 a) from compound of the formula (li) is demonstrated by the bioconversion of compound of the formula (li) with bacterial cells expressing a bacterial membrane-integrated meroterpenoid cyclase. The bioconversion was tested with twenty-eight bacterial membrane-integrated meroterpenoid cyclases as listed in Table 9. For this, as described in Example 1 , the polynucleotides encoding the bacterial membrane-integrated meroterpenoid cyclase were codon optimized and cloned into an expression vector containing the clodfl 3 origin, the streptomycin resistance (SmR), T5 promoter, the RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 222), the lambda TO terminator and lac operatorto control transcription as well as the lactose operon repressor (lacl). Each of the vectors encoding for a bacterial membrane-integrated meroterpenoid cyclase was transformed in E. coli C43(DE3) (Sigma-Aldrich, Missouri, USA). The resulting strains were cultivated in 2 mL polypropylene deep-well plates (DWP) (Thermo Fisher Scientific, Massachusetts, USA) in 0.5 mL LB medium at 37°C, 1000 rpm overnight. 25 pL of each culture was used to inoculate other 2 mL polypropylene deep-well plate containing 0.5 mL AM medium with 0.1 mM IPTG, 50 pg / mL streptomycin and 6 pL of a 200 mg / mL compound of the formula (li) ethanolic solution. Compound of the formula (li) was synthesized as described in Fiorito et al. (Fiorito et al., ACS Catalysis (2018) 8: 9382-9387). The DWP was closed with an air permeable membrane and incubated for 72h at 25 °C, 1000 rpm in a Multitron Pro incubation shaker (Infers HT, Basel, Switzerland). After incubation, the plate was extracted with ethyl acetate containing an internal standard for quantification. The organic phase of each well was analyzed by GC-MS / FID. The results are shown in Table 9.

[0743] All tested meroterpenoid cyclase enzymes were found to cyclise compound of the formula (li) into compound of the formula (B’3a), (C’3a), (A’3-1 a) and / or (D’1 a). The MS spectra of the corresponding compounds (B’3a), (C’3a) and (A’3-1 a) are shown in Figure 10 (A)-(C). A representative GC-MS / FID chromatogram of a bacterial membrane-integrated meroterpenoid cyclase from this screening, WP_318017018.1 (SEQ ID NO: 73), is shown in Figure 9. It shows that compound of the formula (li) was converted to compound of the formula (B’3a), (C’3a), (A’3-1 a) and (D’1 a).

[0744] Table 9: Bioconversion of compound of the formula (li) to compound of the formula (B’3a), (C’3a), (A’3-1 a) and (D’1 a). The relative ratio (%) of each compound and the relative enzyme activity (%) of each enzyme (with the best-performing enzyme defined as 100%) are determined according to the definitions provided in the Definitions section, based on GC-FID chromatogram analysis and excluding compounds designated as “n.q.”; n.q. = not quantified as below limit of quantification.

[0745] Each compound of the formula (B'3a), (C’3a) and (A’3-1 a) was isolated as pure compound for NMR structure elucidation from a shake flask culture similarly as described in Example 7. Therefore, the bacterial strain expressing the meroterpenoid cyclase WP_318017018.1 (SEQ ID NO: 73) was cultivated in a 400 mL flask culture and compound of the formula (li) was added as an ethanolic solution (200 mg / mL) to reach a final concentration of 2 g / L. Compound of the formula (A'3-1 a) was previously described by B. Winter, Helv. Chim. Acta (2004) 87, 1616-1627 and compound of the formula (C'3a) was described in Russian chemical bulletin (1994) 43.1 : 153-160. Compound of the formula (B’3a) was characterized as shown below. of the formula (B’3a)

[0746] 2-((4aS,8aR)-5,5,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalen-2-yl)ethan-1-ol

[0747] 1H-NMR (600 MHz, CDCb): 5.21 (s, 1 H, HC=C).

[0748] MS spectrum:

[0749] EI-MS (70 eV): 222 (M+, 44), 207 (100), 189 (42), 177 (69), 163 (53), 137 (44), 107 (75), 93 (62), 81 (54), 69 (52).

Claims

CLAIMS1 . A method for preparing a compound of the formula (A), (B), (C) and / or (D),or a derivative thereof, whereinR° represents either H or a C1-4 alkyl group, preferably ethyl or H;and wherein each R2represents independently from each other either H or an alcohol protecting group, particularly, preferably H,R3represents H or a C1-4 alkyl group, preferably CH3; and n independently from each other represents 1 or 2; wherein any dotted line represents the bond by which the substituent is bound to the rest of the molecule; and any wavy line represents independently from each other a carbon-carbon bond which when linked to the carbon-carbon double bond is either in the Z or in the E-configuration, preferably in the E- configuration, wherein the method comprises:(a) contacting a compound of the formula (I)with a terpene cyclase enzyme under conditions suitable for the terpene cyclase enzyme to produce the compound of the formula (A), (B), (C) and / or (D).

2. The method according to claim 1 , wherein the configuration of the carbon-carbon double bond of the compound of the formula (I) is in the E-configuration.

3. The method according to claim 1 or 2, wherein R1representswith n being preferably 1 and / or R2being H or an alcohol protecting group, particularlyR3being a C1-4 alkyl group, preferably CH3;preferably, R1represents4. The method according to any of the preceding claims, wherein the compound of the formula (A) is a compound of the formula (A’), the compound of the formula (B) is a compound of the formula (B’), the compound of the formula (C) is a compound of the formula (C’) and / or the compound of the formula (D) is a compound of the formula (D’)5. The method according to any of the preceding claims, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound selected of the group consisting of147(D’1); or a derivative thereof;wherein each R2represents independently from each other either H or an alcohol protecting group, particularly, R3being a C1-4 alkyl group, preferably CH3.

6. The method according to any of the preceding claims, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (A’), preferably of the formula (A’1), preferably of the formula (A’1 a) or (A'1 b), more preferably of the formula (A’1-1 a) or (A'1 -1 b)7. The method according to any of the preceding claims, wherein the compound of the formula (A), (B), (C) and / or (D) is a compound of the formula (D’), preferably of the formula (D’1), preferably of the formula (D’1 a), more preferably of the formula (D’1 ab)8. The method according to any of claims 1 to 5, wherein the compound of the formula (I) is a compound of the formula (lb) and the compound of the formula (A) is a compound of the formula (A’1a), the compound of the formula (B) is a compound of the formula (B’1), the compound of the formula (C) is a compound of the formula (C’1) and / or the compound of the formula (D) is a compound of the formula (D’1a)9. The method according to any of claims 1 to 5, wherein the compound the formula (I) is a compound of the formula (li), and the compound of the formula (A) is a compound of the formula (A’3a), the compound of the formula (B) is a compound of the formula (B’3a), the compound of the formula (C) is a compound of the formula (C’3a) and / orthe compound of the formula (D) is a compound of the formula (D’1a)15110. The method according to any of claims 1 to 5, wherein the compound the formula (I) is a compound of the formula (If), and the compound of the formula (A) is a compound of the formula (A’5a), the compound of the formula (B) is a compound of the formula (B’5a) and / or the compound of the formula (C) is a compound of the formula (C’5a)11 . The method according to any of the preceding claims, wherein the terpene cyclase enzyme is a meroterpenoid cyclase enzyme and / or a squalene cyclase enzyme, preferably the enzyme is a meroterpenoid cyclase enzyme.

12. The method according to claim 11 , wherein the meroterpenoid cyclase enzyme is a bacterial membrane-integrated meroterpenoid cyclase enzyme, preferably comprising at least one or more amino acid motifs selected from:[W]xxx[D]xx[ILVMN] (SEQ ID NO: 212),PxxAxxxNxxWE (SEQ ID NO: 213),MxxxFxxMLxxR (SEQ ID NO: 214),RxxxxGQS (SEQ ID NO: 215), andNxxMS (SEQ ID NO: 216); wherein residues x represent independently of each other any natural amino acid residue.15213. The method according to any of claims 11 and 12, wherein the meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 141 and 162 to 204; more preferably, to any one of SEQ ID NOs: 1 to 96 and 181 to 204.

14. The method according to any of claims 11 to 13, wherein the meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 55, 69, 71 , 73, 74, 75, 76, 77, 78, 79, 100, 173, 177 and 181-204.

15. The method according to any of claims 9, 11 to 14, wherein the terpene cyclase enzyme is a meroterpenoid cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 15, 20, 21 , 29, 46, 48, 69, 71 , 73, 74, 75, 76, 77, 78, 100, 173, 177, 181 , 182, 184, 185, 188, 191 , 194, 197, 200, 202 and 203.

16. The method according to any of claims 10 to 14, wherein the terpene cyclase enzyme is a meroterpenoid cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 20, 29, 46, 48, 69, 71 , 73, 74, 75, 76, 77 and 78.

17. The method according to any of claims 11 to 16, wherein the meroterpenoid cyclase has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29.

18. The method according to any of claims 11 to 17, wherein the meroterpenoid cyclase has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74.

19. The method according to claim 11 , wherein the squalene cyclase enzyme comprises at least one or more amino acid motifs selected from:■ [SP][TP][VIL]WDTx[LWI] (SEQ ID NO: 205),. PGG[WF][GYA]F (SEQ ID NO: 206),. PDxDD[TAS][TIAS] (SEQ ID NO: 207),. [MIL]QxxxG[GA][WF]x[AS][FY] (SEQ ID NO: 208),. Qxxx[GH]xWxG[RK]WGxx[YF]xYG (SEQ ID NO: 209),. Qxx[DN]G[GS][WF][GS]ExxxS (SEQ ID NO: 210), and. [STA]xx[SFN][QC]T[AGT]W[AS][LIV]xx[LQ] (SEQ ID NO: 211); wherein residues x represent independently of each other any natural amino acid residue.15320. The method according to any of claims 11 and 19, wherein the squalene cyclase enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 142 to 148.

21. The method according to any of the preceding claims, wherein the process is an in vivo or a bioconversion process.

22. The method according to any of the preceding claims, wherein the process is performed in a recombinant cell capable of functionally expressing the terpene cyclase enzyme; preferably, said recombinant cell is a bacterial cell, a plant cell, a fungal cell such as a yeast cell; more preferably, said recombinant cell is of the genus Escherichia, Pseudomonas, Saccharomyces, Yarrowia or Pichia.

23. A compound selected from the group consisting of the compound of the formula (A’1), (A’2), (A’4), (B’1), (B’2), (B’3), (B’4), (B’5),(C’1), (C’2), (C’4), (C’5), (D’1b), (A’1-1) and (A’1-2)154or a derivative thereof; whereinR° represents either H or a C1-4 alkyl group, preferably ethyl or H;R2represents either H or an alcohol protecting group, particularly, preferably H, R3represents a C1-4 alkyl group, preferably CH3; and any dotted line represents the bond by which the substituent is bound to the rest of the molecule; and any wavy line represents independently from each other a carbon-carbon bond which when linked to the carbon-carbon double bond is either in the Z or in the E-configuration, preferably in the E- configuration.

24. The compound according to claim 23 wherein the compound of the formula (A’1-1) is a compound of the formula (A’1-1 a) or (A’1-1 b), the compound of the formula (A’1-2) is a compound of the formula (A’1-2a), the compound of the formula (B’3) is a compound of the formula (B’3a), the compound of the formula (B’5) is a compound of the formula (B’5a) and the compound of the formula (C’5) is a compound of the formula of the formula (C’5a)(A’1-1 a), (A’1-2a),15525. A recombinant cell comprising a compound according to any of claims 23 and 24.

26. The recombinant cell according to claim 25, wherein the cell comprises the terpene cyclase enzyme as defined in claims 11 to 20.

27. A cell culture fermentation medium comprising the recombinant cell according to any of claims 25 and 26.

28. A reaction mixture comprising the compound according to any of claims 23 and 24.

29. Use of the compound according to any of claims 23 and 24 as a perfumery, flavor or aroma ingredient, or as a precursor thereof.

30. Use of a terpene cyclase enzyme, preferably a meroterpenoid cyclase enzyme, to produce the compound as defined in any of claims 1 to 10, 23 and 24.31 . A mutant meroterpenoid cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 80 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 9 relative to the sequence provided in SEQ ID NO: 29.

32. A mutant meroterpenoid cyclase enzyme having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOs: 1 to 80 and 181 to 204; wherein the mutant meroterpenoid cyclase enzyme has an amino acid substitution at amino acid position 123, 126 and / or 165 relative to the sequence provided in SEQ ID NO: 74.156

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