Method for producing drimanaldehyde

Abstract

The present invention provides a novel method for the alcohol dehydrogenase-catalyzed production of drimanaldehyde by oxidation of the respective drimane alcohol precursor, carried out in vitro or in vivo. The present invention also relates to the identification of enzymes with corresponding alcohol dehydrogenase activity from different microbial and plant sources. A further aspect of the present invention relates to the provision of corresponding coding sequences for such enzymes, recombinant vectors, and recombinant host cells suitable for the production of such alcohol dehydrogenases. Another aspect of the present invention relates to the use of such drimanaldehydes obtained according to the present invention as intermediates for the production of odorants, flavor or fragrance components, or pest control components.

Description

[Technical Field]

[0001] The present invention provides a novel method for the alcohol dehydrogenase-catalyzed production of drimanaldehyde by oxidation of the respective drimane alcohol precursor, carried out in vitro or in vivo. The present invention also relates to the identification of enzymes with corresponding alcohol dehydrogenase activity from different microbial and plant sources. A further aspect of the present invention relates to the provision of corresponding coding sequences for such enzymes, recombinant vectors, and recombinant host cells suitable for the production of such alcohol dehydrogenases. Another aspect of the present invention relates to the use of such drimanaldehydes obtained according to the present invention as intermediates for the production of odorants, flavor or fragrance components, or pest control components.

[0002] Terpenes are present in most living organisms (microorganisms, animals, and plants). These compounds are composed of five carbon units called isoprene units and are classified by the number of these units present in the structure, which may include cyclic structural elements. Thus, monoterpenes, sesquiterpenes, and diterpenes are terpenes containing 10, 15, and 20 carbon atoms, respectively. For example, sesquiterpenes are widespread in the plant kingdom. Many sesquiterpene molecules are known for their flavor and aroma properties, as well as their effectiveness as cosmetic, pharmaceutical, and antibacterial agents. Numerous sesquiterpene hydrocarbons and sesquiterpenoids have been identified. Chemical synthesis approaches have been developed but are still complex and not always cost-effective.

[0003] The biosynthetic production of terpenes involves enzymes called terpene synthases. There are numerous sesquiterpene synthases in the plant kingdom, all of which use the same substrate (farnesyl diphosphate, FPP) but have different product profiles. Genes and cDNAs encoding sesquiterpene synthases have been cloned, and the corresponding recombinant enzymes have been characterized.

[0004] Many of the major sources of sesquiterpenes, such as compounds having a drimane structure, especially drimanaldehyde, are plants or microorganisms that naturally contain sesquiterpenes, but the content of drimanaldehyde sesquiterpene in these natural sources may be low. There remains a need to provide a new method for producing drimanaldehyde.

[0005] overview The present invention provides novel methods for producing drimanaldehyde, particularly methods that can be incorporated into a completely biochemical synthesis of drimanaldehyde in an aqueous environment, such as a host cell-based process.

[0006] The first aspect of the present invention is a compound of formula (I) [ka] wherein n is 0 and one of the dotted lines is a carbon-carbon double bond and the other is a carbon-carbon single bond, or n is 1 and all of the dotted lines are carbon-carbon single bonds. in the form of any one of its stereoisomers or a mixture thereof, comprising Formula (II) [ka] wherein n is 0 and one of the dotted lines is a carbon-carbon double bond and the other is a carbon-carbon single bond, or n is 1 and all of the dotted lines are carbon-carbon single bonds. contacting the drimane alcohol of formula (I) in the form of any one of its stereoisomers or a mixture thereof with a polypeptide having oxidoreductase enzymatic activity; Optionally, isolating the drimanaldehyde from the reaction; The present invention provides a method comprising:

[0007] One embodiment of the present invention is one in which the oxidoreductase enzyme is an alcohol dehydrogenase (ADH) enzyme.

[0008] In one embodiment of the present invention, the ADH enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1-33.

[0009] One embodiment of the present invention is one in which the ADH enzyme is momilactone A synthase. Preferably, the momilactone A synthase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1, 2, 6, 12, 21 or 33.

[0010] In one embodiment of the present invention, the ADH enzyme is secoisolariciresinol dehydrogenase. Preferably, the secoisolariciresinol dehydrogenase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 3, 7, 8, 10, 16 or 17.

[0011] In one embodiment of the present invention, the ADH enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1-6.

[0012] A further aspect of the present invention provides an isolated polypeptide having ADH activity, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1 to 33, or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 33.

[0013] As shown in the accompanying Examples, a polypeptide having ADH activity comprising the amino acid sequence of any of SEQ ID NOs: 1-6 is capable of producing more than 10 mg / L of drimanaldehyde of formula (I). Accordingly, a preferred embodiment of all aspects of the invention described herein is an isolated polypeptide having ADH activity, which comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-6, or which comprises the amino acid sequence of any of SEQ ID NOs: 1-6.

[0014] In one embodiment of the present invention, the step of contacting the drimane alcohol with the polypeptide having ADH activity is carried out in the presence of a cofactor, preferably NAD + or NAD(P) + It is what it is.

[0015] The cofactor is optionally regenerated using a cofactor regeneration system. One advantage of using a cofactor regeneration system is that such a system can shift the equilibrium of the method of the present invention toward the production of the desired product, such as drimanaldehyde. In this way, the method of the present invention can be more optimized and efficient in terms of the reagents used, and therefore more time- and cost-effective than when a cofactor regeneration system is not used.

[0016] Thus, one embodiment of the present invention is one in which the method of the present invention is carried out in the presence of a cofactor regenerating system.

[0017] One embodiment of the present invention is one in which the drimanaldehyde is selected from the group consisting of drimenal, albicanal, β-bicyclofarnesal or 8-hydroxy-11-drimanal, each in stereomerically pure form or a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of the above group.

[0018] One embodiment of the present invention is one in which the drimane alcohol is selected from the group consisting of drimenol, arbicanol, β-bicyclofarnesol, drimane-8α,11-diol, each in stereomerically pure form or a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of the above group.

[0019] In one embodiment of the present invention, the method is carried out in vivo in a cell culture or in vitro in a liquid reaction medium under conditions conducive to the production of drimanaldehyde.

[0020] In one embodiment of the present invention, the method is carried out in a recombinant host cell or a recombinant non-human host organism capable of functionally expressing (i) at least one polypeptide having oxidoreductase enzymatic activity and, optionally, (ii) at least one polypeptide having the ability to convert the acyclic sesquiterpene precursor FPP to at least one drimane alcohol of formula (II).

[0021] One embodiment of the present invention is one in which the non-human host cell or host organism is selected from a prokaryotic or eukaryotic microorganism or a cell derived therefrom, in particular one in which said non-human host cell or host organism is selected from a bacterium, a fungus, and a plant cell or plant.

[0022] An embodiment of the present invention is one in which the method further comprises oxidizing the drimanaldehyde of formula (I) using chemical synthesis or biocatalytic synthesis, or a combination of both.

[0023] A further aspect of the present invention provides a polypeptide having ADH activity, which has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1 to 33.

[0024] A further aspect of the present invention provides the use of a polypeptide having ADH activity for the preparation of a compound of formula (I).

[0025] A further aspect of the present invention provides a compound of formula (I) obtained or obtainable by any of the above processes.

[0026] A further aspect of the invention provides a recombinant host cell or a recombinant non-human host organism comprising a compound of formula (I) and / or a compound of formula (II).

[0027] A further aspect of the present invention provides a compound of formula (I) as defined in any one of the above for preparing an odorant, flavouring or fragrance ingredient, or as a pest control. [Brief explanation of the drawings]

[0028] [Figure 1] 1 shows GC-MS analysis of albicanal produced in E. coli DP1205 upon co-expression of albicanol synthase LoTpsl with either the alcohol dehydrogenase of SEQ ID NO: 1 (A) or SEQ ID NO: 2 (B). The MS spectrum of albicanal produced by the alcohol dehydrogenase of SEQ ID NO: 2 in (B) is shown in (C) and is similar to the MS spectrum of reference albicanal (D). [Figure 2] FIG. 1 shows the structures of drimenal, albicanal, β-bicyclofarnesal, and 8-hydroxy-11-drimanal.

[0029] Detailed Description The following detailed description describes various aspects and embodiments provided herein. This description should be read from the perspective of one skilled in the relevant art. As such, it may not necessarily include information that is known to such skilled artisans. bp base pair kb kilobase DNA deoxyribonucleic acid cDNA complementary DNA DTT Dithiothreitol GC Gas Chromatography IPTG Isopropyl-D-thiogalacto-pyranoside LB lysogeny medium MS mass spectrometer / mass spectrometry PCR polymerase chain reaction RNA ribonucleic acid mRNA messenger ribonucleic acid miRNA microRNA siRNA small interfering RNA rRNA ribosomal RNA tRNA transfer RNA

[0030] definition It is understood that certain radical naming conventions can include either monoradicals or diradicals depending on the context. For example, if a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a diradical. For example, a substituent specified as alkyl requiring two points of attachment includes diradicals such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc.

[0031] Wherever a substituent is depicted as a diradical (i.e., having two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any orientation unless otherwise indicated. Thus, for example, -AE- or [ka] Substituents depicted as include those where A is attached to the point of attachment at the right end of the molecule, as well as those where A is oriented so that it is attached to the point of attachment at the left end of the molecule.

[0032] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "substituent" includes one substituent as well as two or more substituents, and the like.

[0033] As used herein, the terms "for example," "for instance," "such as," or "including" are meant to introduce examples that further clarify a more general subject matter. Unless otherwise specified, such examples are provided merely as an aid in understanding the embodiments set forth in this disclosure and are not meant to be limiting in any way. Furthermore, these terms do not imply any kind of preference over the disclosed embodiments.

[0034] As used herein, "comprise" or "comprises" or "comprising" or "comprised of" refers to an open group, meaning that the group may include additional members in addition to those explicitly listed. For example, the phrase "comprising A" means that A must be present, but other members may also be present. The terms "include," "have," and "composed of," and their grammatical variations, have the same meaning. In contrast, "consist of" or "consists of" or "consisting of" refers to a closed group. For example, the phrase "consisting of A" means that A and only A are present.

[0035] As used herein, "optionally" means that the subsequently described event may or may not occur. In some embodiments, the optional event does not occur. In some other embodiments, the optional event occurs one or more times.

[0036] As used herein, "or" should be given its broadest reasonable interpretation and is not limited to an either / or configuration. Thus, the phrase "comprising A or B" means that A may be present and B may not be present, or B may be present and A may not be present, or both A and B may be present. Further, for example, if A defines a class that can have multiple members, e.g., A1 and A2, then one or more members of the class may be present at the same time.

[0037] As used herein, certain substituents or linking groups having only a single atom may be referred to by the name of that atom. For example, in some cases, the substituent "-H" may be referred to as "hydrogen" or "hydrogen atom," the substituent "-F" may be referred to as "fluorine" or "fluorine atom," and the linking group "-O-" may be referred to as "oxygen" or "oxygen atom."

[0038] The point of attachment of the group is generally indicated by a terminal dash (-) or asterisk ( * ) For example, * Groups such as -CH2-CH3 or -CH2-CH3 all represent ethyl groups.

[0039] Chemical structures are often shown using a "skeletal" form in which the carbon atoms are not explicitly shown and the hydrogen atoms attached to them are omitted entirely. For example, the structure [ka] represents butane (i.e., n-butane). Additionally, aromatic groups such as benzene are represented by showing one of the contributing resonance structures. For example, the structure [ka] represents toluene.

[0040] Other terms not included in this subsection are defined elsewhere in this specification.

[0041] The term "polypeptide" refers to an amino acid sequence of contiguous polymerized amino acid residues, e.g., at least 15 residues, at least 30 residues, at least 50 residues. In some embodiments herein, a polypeptide comprises an amino acid sequence that is an enzyme or a fragment or variant thereof.

[0042] The term "protein" refers to an amino acid sequence of any length in which the amino acids are joined by covalent peptide bonds, whether natural or synthetic, and includes oligopeptides, peptides, polypeptides and full-length proteins.

[0043] The term "isolated" polypeptide is known in the art and refers to an amino acid sequence that has been removed from its natural environment by any method or combination of methods, including recombinant, biochemical, and synthetic methods.

[0044] The terms "biological function," "function," "biological activity," or "activity" refer to the ability of drimenol synthase to catalyze the formation of drimenol or a mixture of compounds comprising drimenol and one or more terpenes.

[0045] The terms "nucleic acid sequence," "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably and refer to a sequence of nucleotides. A nucleic acid sequence can be single- or double-stranded deoxyribonucleotides or ribonucleotides of any length and can include coding and non-coding sequences of genes, exons, introns, sense and antisense complementary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified natural DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers, and nucleic acid probes. Those skilled in the art will recognize that the nucleic acid sequence of RNA is identical to the DNA sequence, except that thymine (T) is replaced by uracil (U). It should also be understood that the term "nucleotide sequence" includes polynucleotide or oligonucleotide molecules in the form of separate fragments or as components of a larger nucleic acid.

[0046] An "isolated nucleic acid" or "isolated nucleic acid sequence" refers to a nucleic acid or nucleic acid sequence that is in an environment different from that in which it naturally occurs, and may include being substantially free of endogenous contaminants. As used herein, the term "naturally occurring," when applied to nucleic acids, refers to a nucleic acid that is contained in the cells of an organism in nature and that has not been intentionally modified by man in the laboratory.

[0047] A "recombinant nucleic acid sequence" is a nucleic acid sequence obtained by combining genetic material from two or more sources using laboratory methods (e.g., molecular cloning) to create or modify a nucleic acid sequence that does not occur in nature and is not otherwise found in a living organism.

[0048] "Recombinant DNA technology" refers to molecular biological techniques for producing recombinant nucleic acid sequences, such as those described in, for example, 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.

[0049] The term "gene" refers to a DNA sequence comprising a region that is transcribed in a cell into an RNA molecule, e.g., mRNA, operably linked to a suitable regulatory region, e.g., a promoter. Thus, a gene may comprise several operably linked sequences, such as a promoter, a 5' leader sequence including, e.g., sequences involved in translation initiation, a coding region of cDNA or genomic DNA, introns, exons, and / or 3' untranslated sequences including, e.g., a transcription termination site.

[0050] "Chimeric gene" refers to any gene that is not normally found in a species in nature, particularly a gene in which one or more portions of a nucleic acid sequence are not naturally linked to each other. For example, a promoter is not naturally linked to part or all of the transcribed region or another regulatory region. The term "chimeric gene" is understood to include an expression construct in which a promoter or transcriptional regulatory sequence is operably linked to one or more coding sequences, or antisense, i.e., the reverse complement of the sense strand, or inverted repeat sequences (sense and antisense, whereby the RNA transcript forms double-stranded RNA upon transcription). The term "chimeric gene" also includes genes obtained by combining portions of one or more coding sequences to generate a new gene.

[0051] "3'UTR" or "3' untranslated sequence" (also referred to as "3' untranslated region" or "3' end") refers to nucleic acid sequences found downstream of the coding sequence of a gene, including, for example, transcription termination sites and (in most, but not all, eukaryotic mRNAs) polyadenylation signals, such as AAUAAA or variants thereof. After transcription is terminated, 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 translation site, e.g., the cytoplasm.

[0052] "Gene expression" encompasses "heterologous expression" and "overexpression" and involves transcription of a gene and translation of mRNA into protein. Overexpression refers to the production of a gene product, as measured by levels of mRNA, polypeptide, and / or enzymatic activity, in a transgenic cell or organism that exceeds production levels in a non-transformed cell or organism of a similar genetic background.

[0053] "Expression vector" as used herein refers to a nucleic acid molecule designed using molecular biology methods and recombinant DNA technology to deliver foreign or exogenous DNA into host cells. Expression vectors typically contain sequences required for proper transcription of nucleotide sequences. The coding region usually codes for a protein of interest, but may also code for RNA, such as antisense RNA, siRNA, etc.

[0054] As used herein, the term "expression vector" includes any linear or circular recombinant vector, including, but not limited to, viral vectors, bacteriophages, and plasmids. Those skilled in the art can select an appropriate vector depending on the expression system. In one embodiment, the expression vector comprises a nucleic acid of an embodiment herein operably linked to at least one regulatory sequence controlling transcription, translation, initiation, and termination, such as a transcription promoter, operator, or enhancer, or an mRNA ribosomal binding site, and optionally including at least one selectable marker. A nucleotide sequence is considered "operably linked" when the regulatory sequence is functionally related to the nucleic acid of an embodiment herein.

[0055] "Regulatory sequence" refers to a nucleic acid sequence that determines the expression level of a nucleic acid sequence in one embodiment of the present specification and can regulate the transcription rate of a nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences include promoters, enhancers, transcription factors, promoter elements, etc.

[0056] A "promoter" 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, but not limited to, transcription factor binding sites, repressor and activator protein binding sites. The term promoter also includes the term "promoter regulatory sequence." Promoter regulatory sequences may include upstream and downstream elements that can affect the transcription, RNA processing, or stability of the associated coding nucleic acid sequence. Promoters include naturally occurring and synthetic sequences. The coding nucleic acid sequence is usually located downstream of the promoter relative to the direction of transcription starting from the transcription start site.

[0057] The term "constitutive promoter" refers to an unregulated promoter that allows for continuous transcription of an operably linked nucleic acid sequence.

[0058] As used herein, the term "operably linked" refers to the 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 example, a promoter or transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operatively linked typically means that the linked DNA sequences are contiguous. The nucleotide sequence linked to the promoter sequence can be of homologous or heterologous origin to the plant to be transformed. The sequence may be fully or partially synthetic. Regardless of origin, the nucleic acid sequence linked to the promoter sequence is expressed or silenced depending on the properties of the linked promoter after being linked to a polypeptide of one embodiment of the present invention. The linked nucleic acid may encode a protein that is desired to be expressed or suppressed consistently throughout the entire organism, or alternatively, at a specific time or in a specific tissue, cell, or cell compartment. Such a nucleotide sequence specifically encodes a protein that confers a desired phenotypic trait on a host cell or organism modified or transformed therewith. More specifically, the linked nucleotide sequence results in the production of drimenol or a mixture comprising drimenol and one or more terpenes in a cell or organism. In particular, the nucleotide sequence encodes a polypeptide having drimenol synthase activity.

[0059] A "targeting peptide" refers to an amino acid sequence that targets a protein or polypeptide to an intracellular organelle, i.e., mitochondria or plastid, or to the extracellular space (secretory signal peptide). A nucleic acid sequence encoding a targeting peptide can be fused to a nucleic acid sequence encoding the amino terminus, e.g., the N-terminus, of a protein or polypeptide, or can be used to replace a native targeting polypeptide.

[0060] The term "primer" refers to a short nucleic acid sequence that hybridizes to a template nucleic acid sequence and is used to polymerize a nucleic acid sequence that is complementary to the template.

[0061] As used herein, the term "host cell" or "transformed cell" refers to a cell (or organism) that has been modified to carry at least one nucleic acid molecule, e.g., a recombinant gene encoding a desired protein or nucleic acid sequence that, upon transcription, produces a drimenol synthase protein useful for producing drimenol or a mixture comprising drimenol and one or more terpenes. Host cells are particularly bacterial, fungal, or plant cells. A host cell may contain a recombinant gene integrated into the nuclear genome or organelle genome of the host cell. Alternatively, the host may contain the recombinant gene extrachromosomally.

[0062] Homologous sequences include orthologous or paralogous sequences. Methods for identifying orthologs or paralogs, including phylogenetic, sequence similarity, and hybridization methods, are known in the art and are described herein.

[0063] Paralogs result from gene duplication, resulting in two or more genes with similar sequences and similar functions. Paralogs are usually clustered and formed by gene duplication within related plant species. Paralogs are found in groups of similar genes using pairwise Blast analysis or during phylogenetic analysis of gene families using programs such as CLUSTAL. In paralogs, sequences characteristic of related genes can be identified, and consensus sequences with similar gene functions can be identified.

[0064] Orthologs or orthologous sequences are sequences similar to each other because they are found in species derived from a common ancestor. For example, plant species with a common ancestor are known to contain many enzymes with similar sequences and functions. Those skilled in the art can identify orthologous sequences and predict the function of orthologs by constructing a polygenic tree of a gene family in a species using, for example, CLUSTAL or BLAST programs. A method for identifying or confirming similar functions between homologous sequences is by comparing the transcript profiles in host cells or organisms, such as plants, that overexpress or lack (in knockout / knockdown) related polypeptides. Those skilled in the art will understand that genes with similar transcript profiles, where more than 50% of the regulated transcripts are in common, or more than 70% of the regulated transcripts are in common, or more than 90% of the regulated transcripts are in common, have similar functions. Homologs, paralogs, orthologs, and any other variants of the sequences herein are expected to function similarly by creating host cells, organisms, such as plants, that produce drimenol synthase protein.

[0065] The term "selectable marker" refers to any gene that, upon expression, can be used to select for a cell or cells containing the selectable marker. Examples of selectable markers are listed below. Those skilled in the art will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.

[0066] The term "organism" refers to any non-human multicellular or unicellular organism, such as a plant or a microorganism. In particular, the microorganism is a bacterium, yeast, algae or fungus.

[0067] 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 plant parts or plant organs, such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits, etc. Any plant can be used to practice the methods of one embodiment herein.

[0068] With respect to this description 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 are not intended to be limiting.

[0069] It should be further understood that when the term "comprising" is used in describing various embodiments, those skilled in the art will understand that in some specific instances, the embodiments may alternatively be described using the words "consisting essentially of" or "consisting of."

[0070] Compounds of the Method of the Invention The present invention relates to a compound of formula (I) [ka] wherein n is 0 and one of the dotted lines is a carbon-carbon double bond and the other is a carbon-carbon single bond, or n is 1 and all of the dotted lines are carbon-carbon single bonds. in the form of any one of its stereoisomers or a mixture thereof.

[0071] In some embodiments, the drimanaldehyde is selected from the group consisting of drimenal, albicanal, β-bicyclofarnesal, or 8-hydroxy-11-drimanal, each in a stereomerically pure form or a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of the above group.

[0072] The present invention provides a method for preparing drimanaldehyde of formula (I), comprising the step of: [ka] wherein n is 0 and one of the dotted lines is a carbon-carbon double bond and the other is a carbon-carbon single bond, or n is 1 and all of the dotted lines are carbon-carbon single bonds. in the form of any one of its stereoisomers or a mixture thereof with a polypeptide having oxidoreductase enzyme activity.

[0073] In some embodiments, the drimane alcohol is selected from the group consisting of drimenol, arbicanol, β-bicyclofarnesol, drimane-8α,11-diol, each in a stereomerically pure form or a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of the above group.

[0074] Preferably, the drimane alcohol is arbicanol.

[0075] In some embodiments, the drimanaldehyde is selected from the group consisting of drimenal, albicanal, β-bicyclofarnesal, or 8-hydroxy-11-drimanal, each in a stereomerically pure form or a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of the above group.

[0076] Preferably, the drimanaldehyde is albicanal.

[0077] A preferred embodiment of the invention is one in which the drimane alcohol is arbicanol and the drimane aldehyde is arbicanal.

[0078] When the compounds disclosed herein have at least one asymmetric center not specifically shown in the formula, the compounds of formulas (I) and (II) may be in the form of any one of the stereoisomers or a mixture thereof. For clarity, by "any one of the stereoisomers or a mixture thereof" or similar expressions, it is intended the ordinary meaning understood by those skilled in the art, i.e., that the compounds of formulas (I) and (II) may be pure enantiomers or diastereomers. In other words, the compounds of formulas (I) and (II) have several stereocenters, each of which may have two different stereochemistries (e.g., R or S). The compounds of formulas (I) and (II) may be in the form of a pure enantiomer or a mixture of enantiomers or diastereoisomers. The compounds of formulas (I) and (II) may be in racemic or scalemic form. Thus, the compounds of formulas (I) and (II) may be in the form of a single stereoisomer or a composition of matter comprising or consisting of various stereoisomers.

[0079] Separation of individual isomers or selective synthesis of individual isomers can be achieved by applying various methods known to those skilled in the art. Unless otherwise indicated (e.g., when the stereochemistry of an asymmetric center is explicitly indicated), all such isomers and mixtures thereof are included within the scope of the compounds disclosed herein. Furthermore, the compounds disclosed herein may exist in one or more crystalline or amorphous forms. Unless otherwise indicated, all such forms, including any polymorphic forms, are included within the scope of the compounds disclosed herein.

[0080] Isotopes may be present in the compounds described. Each chemical element represented in a compound structure may include any isotope of the element. For example, in a compound structure, it may be explicitly disclosed or understood that a hydrogen atom is present in the compound. At any position in the compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including, but not limited to, hydrogen-1 (protium) and hydrogen-2 (deuterium). Therefore, reference to a compound herein encompasses all possible isotopic forms unless the context clearly dictates otherwise.

[0081] The process of the present invention starts with drimane alcohol.

[0082] In one embodiment of the invention, the drimane alcohol compounds may be present endogenously in a reaction mixture, for example in an in vivo process employing a host cell system that produces the drimane alcohol compound as a metabolic product and expresses one or more polypeptides required to carry out the intended drimane alcohol synthesis, or in a more complex multi-step process that includes the drimane alcohol synthesis as one step in the enzymatic synthesis of the drimane alcohol from an acyclic sesquiterpene precursor.

[0083] Alternatively, the drimane alcohol compounds are produced chemically or enzymatically and added exogenously to the reaction mixture, for example in an in vitro process applying isolated, enriched or purified synthase enzymes required for their formation as defined below.

[0084] In one embodiment of the present invention, the method is carried out in a recombinant host cell or a recombinant non-human host organism capable of functionally expressing (i) at least one polypeptide having oxidoreductase enzyme activity, and optionally (ii) at least one polypeptide having the ability to convert the acyclic sesquiterpene precursor FPP to at least one drimane alcohol of formula (II).

[0085] Examples of polypeptides capable of converting the acyclic sesquiterpene precursor FPP to at least one drimane alcohol of formula (II) are known in the art. For example, WO2018220113 and WO2020078871 disclose many examples of such enzymes.

[0086] A preferred embodiment of the present invention is one in which the drimane alcohol of formula (II) is alkanaol and the drimane aldehyde is alkanaal. In this preferred embodiment of the present invention, the oxidoreductase is an alcohol dehydrogenase (ADH) enzyme. More preferably, the ADH comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-33, or comprises the amino acid sequence of any of SEQ ID NOs: 1-33. Most preferably, the ADH comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-6, or comprises the amino acid sequence of any of SEQ ID NOs: 1-6.

[0087] Enzymes of the Invention The present invention provides a method for preparing a drimane aldehyde of formula (I) by contacting a drimane alcohol of formula (II) with a polypeptide having oxidoreductase enzyme activity.

[0088] Oxidoreductases are enzymes that catalyze the transfer of electrons from one molecule, a reducing agent, also called an electron donor, to another molecule, an oxidizing agent, also called an electron acceptor. Oxidoreductases constitute a large class of enzymes that catalyze biological oxidation / reduction reactions. Because many chemical and biochemical transformations involve oxidation / reduction processes, oxidoreductases are highly useful in developing biotechnological methods to synthesize desirable compounds.

[0089] There are several different classes of oxidoreductases, defined primarily by their substrate and / or mode of action, such as alcohol dehydrogenases, ketoreductases, peroxidases, hydroxylases, and oxygenases and reductases.

[0090] The present inventors sought to identify whether oxidoreductases could be used to reduce compounds of formula (II) to form compounds of formula (I). Surprisingly, several such enzymes have been identified that can be used for this purpose, as shown in the accompanying examples. To the best of the inventors' knowledge, this is the first time that an oxidoreductase, particularly an alcohol dehydrogenase (ADH) enzyme, has been used for this reaction. Prior to the present invention, it had not been demonstrated that the ADH enzyme would accept compounds of formula (I), particularly albicanol, as a substrate.

[0091] "Alcohol dehydrogenase" and "ADH" enzyme are used interchangeably herein to refer to polypeptides having the enzymatic ability to oxidize primary and secondary alcohols to aldehydes and ketones.

[0092] All enzymes belonging to class EC 1.1.1 are within the scope of the present invention. These include the following EC numbers and classes: 1.1.1.1 Alcohol dehydrogenase, 1.1.1.2 Alcohol dehydrogenase (NADP(+)), 1.1.1.3 Homoserine dehydrogenase, 1.1.1.4 (R,R)-butanediol dehydrogenase, 1.1.1.6 Glycerol dehydrogenase, 1.1.1.7 Propanediol phosphate dehydrogenase, 1.1.1.8 Glycerol-3-phosphate dehydrogenase (NAD(+)), 1.1.1.9 D-xylulose reductase, 1.1.1.10 L-xylulose reductase, 1.1.1.11 D-arabinitol 4-dehydrogenase, 1.1.1.12 L-arabinitol 4-dehydrogenase, 1.1.1.13 L-arabinitol 2-dehydrogenase, 1.1.1.14 L-iditol 2-dehydrogenase, 1.1.1.15 D-iditol 2-dehydrogenase, 1.1.1.16 Galactitol 2-dehydrogenase, 1.1.1.17 Mannitol-1-phosphate 5-dehydrogenase, 1.1.1.18 Inositol 2-dehydrogenase, 1.1.1.19 Glucuronic acid reductase, 1.1.1.20 Glucuronolactone reductase, 1.1.1.21 Aldose reductase, 1.1.1.22 UDP-glucose 6-dehydrogenase, 1.1.1.23 Histidinol dehydrogenase, 1.1.1.24 Quinate / shikimate dehydrogenase (NAD(+)), 1.1.1.25 Shikimate dehydrogenase (NADP(+)), 1.1.1.26 Glyoxylate reductase, 1.1.1.27 L-lactate dehydrogenase, 1.1.1.28 D-lactate dehydrogenase, 1.1.1.29 Glycerate dehydrogenase, 1.1.1.30 3-hydroxybutyrate dehydrogenase, 1.1.1.31 3-hydroxyisobutyrate dehydrogenase, 1.1.1.32 Mevaldate reductase, 1.1.1.33 Mevaldate reductase (NADPH), 1.1.1.34 Hydroxymethylglutaryl-CoA reductase (NADPH), 1.1.1.35 3-hydroxyacyl-CoA dehydrogenase, 1.1.1.36 Acetoacetyl-CoA reductase, 1.1.1.37 Malate dehydrogenase, 1.1.1.38 Malate dehydrogenase (oxaloacetate decarboxylation), 1.1.1.39 Malate dehydrogenase (decarboxylation), 1.1.1.40 Malate dehydrogenase (oxaloacetate decarboxylation) (NADP(+)), 1.1.1.41 Isocitrate dehydrogenase (NAD(+)), 1.1.1.42 Isocitrate dehydrogenase (NADP(+)), 1.1.1.43 Phosphogluconate 2-dehydrogenase, 1.1.1.44 Phosphogluconate dehydrogenase (NADP(+)-dependent, decarboxylation), 1.1.1.45 L-gulonic acid 3-dehydrogenase, 1.1.1.46 L-arabinose 1-dehydrogenase, 1.1.1.47 Glucose 1-dehydrogenase [NAD(P)(+)], 1.1.1.48 D-galactose 1-dehydrogenase, 1.1.1.49 Glucose-6-phosphate dehydrogenase (NADP(+)), 1.1.1.50 3α-hydroxysteroid 3-dehydrogenase (Si-specific), 1.1.1.51 3(or 17)β-hydroxysteroid dehydrogenase, 1.1.1.52 3α-hydroxycholanic acid dehydrogenase (NAD(+)), 1.1.1.53 3α(or 20β)-hydroxysteroid dehydrogenase, 1.1.1.54 Allyl alcohol dehydrogenase, 1.1.1.55 Lactaldehyde reductase (NADPH), 1.1.1.56 Ribitol 2-dehydrogenase, 1.1.1.57 Fructuronate reductase, 1.1.1.58 Tagaturonate reductase, 1.1.1.59 3-Hydroxypropionate dehydrogenase, 1.1.1.60 2-hydroxy-3-oxopropionate reductase, 1.1.1.61 4-hydroxybutyrate dehydrogenase, 1.1.1.62 17β-estradiol 17-dehydrogenase, 1.1.1.64 Testosterone 17β-dehydrogenase (NADP(+)), 1.1.1.65 Pyridoxine 4-dehydrogenase, 1.1.1.66 ω-hydroxydecanoate dehydrogenase, 1.1.1.67 Mannitol 2-dehydrogenase, 1.1.1.68 Entry transfer: 1.5.1.20, 1.1.1.69 Gluconate 5-dehydrogenase, 1.1.1.71 Alcohol dehydrogenase [NAD(P)(+)], 1.1.1.72 Glycerol dehydrogenase (NADP(+)), 1.1.1.73 Octanol dehydrogenase, 1.1.1.75 (R)-aminopropanol dehydrogenase, 1.1.1.76 (S,S)-butanediol dehydrogenase, 1.1.1.77 Lactaldehyde reductase, 1.1.1.78 Methylglyoxal reductase (NADH), 1.1.1.79 Glyoxylate reductase (NADP(+)), 1.1.1.80 Isopropanol dehydrogenase (NADP(+)), 1.1.1.81 Hydroxypyruvate reductase, 1.1.1.82 Malate dehydrogenase (NADP(+)), 1.1.1.83 D-Malate dehydrogenase (decarboxylation), 1.1.1.84 Dimethylmalate dehydrogenase, 1.1.1.85 3-Isopropylmalate dehydrogenase, 1.1.1.86 Ketol-acid reductoisomerase (NADP(+)), 1.1.1.87 Homoisocitrate dehydrogenase, 1.1.1.88 Hydroxymethylglutaryl-CoA reductase, 1.1.1.90 Aryl alcohol dehydrogenase, 1.1.1.91 Aryl alcohol dehydrogenase (NADP(+)), 1.1.1.92 Oxaloglycolate reductase (decarboxylation), 1.1.1.93 Tartrate dehydrogenase, 1.1.1.94 Glycerol-3-phosphate dehydrogenase [NAD(P)(+)], 1.1.1.95 Phosphoglycerate dehydrogenase, 1.1.1.96 Diiodophenylpyruvate reductase, 1.1.1.97 3-hydroxybenzyl alcohol dehydrogenase, 1.1.1.98 (R)-2-hydroxy fatty acid dehydrogenase, 1.1.1.99 (S)-2-hydroxy fatty acid dehydrogenase, 1.1.1.100 3-oxoacyl-[acyl carrier protein] reductase, 1.1.1.101 acylglycerol phosphate reductase, 1.1.1.102 3-dehydrosphinganine reductase, 1.1.1.103 L-threonine 3-dehydrogenase, 1.1.1.104 4-oxoproline reductase, 1.1.1.105 all-trans-retinol dehydrogenase (NAD(+)), 1.1.1.106 Pantoate 4-dehydrogenase, 1.1.1.107 Pyridoxal 4-dehydrogenase, 1.1.1.108 Carnitine 3-dehydrogenase, 1.1.1.110 Aromatic 2-oxoacid reductase, 1.1.1.111 3-(imidazol-5-yl)lactate dehydrogenase, 1.1.1.112 Indanol dehydrogenase, 1.1.1.113 L-xylose 1-dehydrogenase, 1.1.1.114 Apiose 1-reductase, 1.1.1.115 Ribose 1-dehydrogenase (NADP(+)), 1.1.1.116 D-arabinose 1-dehydrogenase (NAD(+)), 1.1.1.117 D-arabinose 1-dehydrogenase [NAD(P)(+)], 1.1.1.118 Glucose 1-dehydrogenase (NAD(+)), 1.1.1.119 Glucose 1-dehydrogenase (NADP(+)), 1.1.1.120 Galactose 1-dehydrogenase (NADP(+)), 1.1.1.121 Aldose 1-dehydrogenase (NAD(+)), 1.1.1.122 D-threo-aldose 1-dehydrogenase, 1.1.1.123 Sorbose 5-dehydrogenase (NADP(+)), 1.1.1.124 Fructose 5-dehydrogenase (NADP(+)), 1.1.1.125 2-Deoxy-D-gluconate 3-dehydrogenase, 1.1.1.126 2-Dehydro-3-deoxy-D-gluconate 6-dehydrogenase, 1.1.1.127 2-Dehydro-3-deoxy-D-gluconate 5-dehydrogenase, 1.1.1.129 L-Threonate 3-dehydrogenase, 1.1.1.130 3-Dehydro-L-gulonic acid 2-dehydrogenase, 1.1.1.131 Mannuronate reductase, 1.1.1.132 GDP-mannose 6-dehydrogenase, 1.1.1.133 dTDP-4-dehydrorhamnose reductase, 1.1.1.134 dTDP-6-deoxy-L-talose 4-dehydrogenase, 1.1.1.135 GDP-6-deoxy-D-talose 4-dehydrogenase, 1.1.1.136 UDP-N-acetylglucosamine 6-dehydrogenase, 1.1.1.137 Ribitol-5-phosphate 2-dehydrogenase, 1.1.1.138 Mannitol 2-dehydrogenase (NADP(+)), 1.1.1.140 Sorbitol-6-phosphate 2-dehydrogenase, 1.1.1.141 15-hydroxyprostaglandin dehydrogenase (NAD(+)), 1.1.1.142 D-pinitol dehydrogenase, 1.1.1.143 Sequoyitol dehydrogenase, 1.1.1.144 Perillyl alcohol dehydrogenase, 1.1.1.145 3β-hydroxy-Δ(5)-steroid dehydrogenase, 1.1.1.146 11β-hydroxysteroid dehydrogenase, 1.1.1.147 16α-hydroxysteroid dehydrogenase, 1.1.1.148 Estradiol 17α-dehydrogenase. 1.1.1.149 20α-hydroxysteroid dehydrogenase, 1.1.1.150 21-hydroxysteroid dehydrogenase (NAD(+)), 1.1.1.151 21-hydroxysteroid dehydrogenase (NADP(+)), 1.1.1.152 3α-hydroxy-5β-androstan-17-one 3α-dehydrogenase, 1.1.1.153 Sepiapterin reductase (L-erythro-7,8-dihydrobiopterin formation), 1.1.1.154 Ureidoglycolate dehydrogenase, 1.1.1.156 Glycerol 2-dehydrogenase (NADP(+)), 1.1.1.157 3-hydroxybutyryl-CoA dehydrogenase, 1.1.1.159 7α-Hydroxysteroid dehydrogenase, 1.1.1.160 Dihydrobunolol dehydrogenase, 1.1.1.162 Erythrulose reductase, 1.1.1.163 Cyclopentanol dehydrogenase, 1.1.1.164 Hexadecanol dehydrogenase, 1.1.1.165 2-Alkyne-1-ol dehydrogenase, 1.1.1.166 Hydroxycyclohexanecarboxylic acid dehydrogenase, 1.1.1.167 Hydroxymalonate dehydrogenase, 1.1.1.168 2-Dehydropantolactone reductase (Re-specific), 1.1.1.169 2-Dehydropantoate 2-reductase, 1.1.1.170 3β-Hydroxysteroid-4α-carboxylic acid 3-dehydrogenase (decarboxylation), 1.1.1.172 2-oxoadipate reductase, 1.1.1.173 L-rhamnose 1-dehydrogenase, 1.1.1.174 cyclohexane-1,2-diol dehydrogenase, 1.1.1.175 D-xylose 1-dehydrogenase, 1.1.1.176 12α-hydroxysteroid dehydrogenase, 1.1.1.177 glycerol-3-phosphate 1-dehydrogenase (NADP(+)), 1.1.1.178 3-hydroxy-2-methylbutyryl-CoA dehydrogenase, 1.1.1.179 D-xylose 1-dehydrogenase (NADP(+), D-xylono-1,5-lactone formation), 1.1.1.181 cholest-5-ene-3β,7α-diol 3β-dehydrogenase, 1.1.1.183 Geraniol dehydrogenase (NADP(+)), 1.1.1.184 Carbonyl reductase (NADPH), 1.1.1.185 L-glycol dehydrogenase, 1.1.1.186 dTDP-galactose 6-dehydrogenase, 1.1.1.187 GDP-4-dehydro-D-rhamnose reductase, 1.1.1.188 Prostaglandin F synthase, 1.1.1.189 Prostaglandin E2 9-reductase, 1.1.1.190 Indole-3-acetaldehyde reductase (NADH), 1.1.1.191 Indole-3-acetaldehyde reductase (NADPH), 1.1.1.192 Long-chain alcohol dehydrogenase, 1.1.1.193 5-Amino-6-(5-phosphoribosylamino)uracil reductase, 1.1.1.194 Coniferyl alcohol dehydrogenase, 1.1.1.195 Cinnamyl alcohol dehydrogenase, 1.1.1.196 15-hydroxyprostaglandin-D dehydrogenase (NADP(+)), 1.1.1.197 15-hydroxyprostaglandin dehydrogenase (NADP(+)), 1.1.1.198 (+)-Borneol dehydrogenase, 1.1.1.199 (S)-usnic acid reductase, 1.1.1.200 Aldose-6-phosphate reductase (NADPH), 1.1.1.201 7β-hydroxysteroid dehydrogenase (NADP(+)), 1.1.1.202 1,3-Propanediol dehydrogenase, 1.1.1.203 Uronic acid dehydrogenase, 1.1.1.205 IMP dehydrogenase, 1.1.1.206 Tropinone reductase I, 1.1.1.207 (-)-Menthol dehydrogenase, 1.1.1.208 (+)-Neomenthol dehydrogenase, 1.1.1.209 3(or 17)α-Hydroxysteroid dehydrogenase, 1.1.1.210 3β-(or 20α)-Hydroxysteroid dehydrogenase, 1.1.1.211 Long-chain 3-hydroxyacyl-CoA dehydrogenase, 1.1.1.212 3-oxoacyl-[acyl carrier protein] reductase (NADH), 1.1.1.213 3α-Hydroxysteroid dehydrogenase (Re specific), 1.1.1.214 2-Dehydropantolactone reductase (Si-specific), 1.1.1.215 Gluconate 2-dehydrogenase, 1.1.1.216 Farnesol dehydrogenase, 1.1.1.217 Benzyl-2-methyl-hydroxybutyrate dehydrogenase, 1.1.1.218 Morphine 6-dehydrogenase, 1.1.1.219 Dihydroflavanol 4-reductase, 1.1.1.220 6-pyruvoyltetrahydropterin 2'-reductase, 1.1.1.221 Vomifoliol dehydrogenase, 1.1.1.223 Isopiperitenol dehydrogenase, 1.1.1.224 Mannose-6-phosphate 6-reductase, 1.1.1.225 Chlordecone reductase, 1.1.1.226 4-hydroxycyclohexanecarboxylate dehydrogenase, 1.1.1.227 (-)-borneol dehydrogenase, 1.1.1.228 (+)-sabinol dehydrogenase, 1.1.1.229 diethyl 2-methyl-3-oxosuccinate reductase, 1.1.1.230 3α-hydroxyglycyrrhetinate dehydrogenase, 1.1.1.231 15-hydroxyprostaglandin-I dehydrogenase (NADP(+)), 1.1.1.232 15-hydroxyicosatetraenoic acid dehydrogenase, 1.1.1.233 N-acylmannosamine 1-dehydrogenase, 1.1.1.234 Flavanone 4-reductase, 1.1.1.235 8-Oxocoformycin reductase, 1.1.1.236 Tropinone reductase II, 1.1.1.237 Hydroxyphenylpyruvate reductase, 1.1.1.238 12β-hydroxysteroid dehydrogenase, 1.1.1.239 3α-(17β)-hydroxysteroid dehydrogenase (NAD(+)), 1.1.1.240 N-acetylhexosamine 1-dehydrogenase, 1.1.1.241 6-endo-hydroxycineole dehydrogenase, 1.1.1.243 Carveol dehydrogenase, 1.1.1.244 Methanol dehydrogenase, 1.1.1.245 Cyclohexanol dehydrogenase, 1.1.1.247 Codeinone reductase (NADPH), 1.1.1.248 Salutaridine reductase (NADPH), 1.1.1.250 D-arabinitol 2-dehydrogenase, 1.1.1.251 Galactitol-1-phosphate 5-dehydrogenase, 1.1.1.252 Tetrahydroxynaphthalene reductase, 1.1.1.254 (S)-carnitine 3-dehydrogenase, 1.1.1.255 Mannitol dehydrogenase, 1.1.1.256 Fluoren-9-ol dehydrogenase, 1.1.1.257 4-(hydroxymethyl)benzenesulfonate dehydrogenase, 1.1.1.258 6-hydroxyhexanoate dehydrogenase, 1.1.1.259 3-hydroxypimeloyl-CoA dehydrogenase, 1.1.1.260 Sulcatone reductase, 1.1.1.261 sn-glycerol-1-phosphate dehydrogenase, 1.1.1.262 4-Hydroxythreonine-4-phosphate dehydrogenase, 1.1.1.263 1,5-anhydro-D-fructose reductase, 1.1.1.264 L-idonic acid 5-dehydrogenase [NAD(P)(+)], 1.1.1.265 3-methylbutanal reductase, 1.1.1.266 dTDP-4-dehydro-6-deoxyglucose reductase, 1.1.1.267 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 1.1.1.268 2-(R)-hydroxypropyl-CoM dehydrogenase, 1.1.1.269 2-(S)-hydroxypropyl-CoM dehydrogenase, 1.1.1.270 3β-hydroxysteroid 3-dehydrogenase, 1.1.1.271 GDP-L-fucose synthase, 1.1.1.272 D-2-hydroxyacid dehydrogenase (NADP(+)), 1.1.1.273 Verosimin dehydrogenase, 1.1.1.274 2,5-didehydrogluconate reductase (2-dehydro-D-gluconic acid formation), 1.1.1.275 (+)-trans-carveol dehydrogenase, 1.1.1.276 Serine 3-dehydrogenase (NADP(+)), 1.1.1.277 3β-hydroxy-5β-steroid dehydrogenase, 1.1.1.278 3β-hydroxy-5α-steroid dehydrogenase, 1.1.1.279 (R)-3-hydroxyacid ester dehydrogenase, 1.1.1.280 (S)-3-hydroxyacid-ester dehydrogenase, 1.1.1.281 GDP-4-dehydro-6-deoxy-D-mannose reductase, 1.1.1.282 Quinate / shikimate dehydrogenase [NAD(P)(+)], 1.1.1.283 Methylglyoxal reductase (NADPH), 1.1.1.284 S-(hydroxymethyl)glutathione dehydrogenase, 1.1.1.285 3''-deamino-3''-oxonicotianamine reductase, 1.1.1.286 Isocitrate-homoisocitrate dehydrogenase, 1.1.1.287 D-arabinitol dehydrogenase (NADP(+)), 1.1.1.288 Xanthoxin dehydrogenase, 1.1.1.289 Sorbose reductase, 1.1.1.290 4-phosphoerythronic acid dehydrogenase, 1.1.1.291 2-hydroxymethylglutarate dehydrogenase, 1.1.1.292 1,5-anhydro-D-fructose reductase (1,5-anhydro-D-mannitol formation), 1.1.1.294 chlorophyll(ide) b reductase, 1.1.1.295 momilactone A synthase, 1.1.1.296 dihydrocarveol dehydrogenase, 1.1.1.297 limonene-1,2-diol dehydrogenase, 1.1.1.298 3-hydroxypropionic acid dehydrogenase (NADP(+)), 1.1.1.299 Malate dehydrogenase [NAD(P)(+)], 1.1.1.300 NADP-retinol dehydrogenase, 1.1.1.301 D-arabitol-phosphate dehydrogenase, 1.1.1.302 2,5-diamino-6-(ribosylamino)-4(3H)-pyrimidinone 5'-phosphate reductase, 1.1.1.303 Diacetyl reductase [(R)-acetoin formation], 1.1.1.304 Diacetyl reductase [(S)-acetoin formation], 1.1.1.305 UDP-glucuronate oxidase (UDP-4-keto-hexouronic acid decarboxylation), 1.1.1.306 S-(hydroxymethyl)mycothiol dehydrogenase, 1.1.1.307 D-xylose reductase [NAD(P)H], 1.1.1.308 Sulfopropanediol 3-dehydrogenase, 1.1.1.309 Phosphonoacetaldehyde reductase (NADH), 1.1.1.310 (S)-sulfolactate dehydrogenase, 1.1.1.311 (S)-1-phenylethanol dehydrogenase, 1.1.1.312 2-hydroxy-4-carboxymuconic acid semialdehyde hemiacetal dehydrogenase, 1.1.1.313 Sulfoacetaldehyde reductase (NADPH), 1.1.1.315 11-cis-retinol dehydrogenase, 1.1.1.316 L-galactose 1-dehydrogenase, 1.1.1.317 Perakin reductase, 1.1.1.318 Eugenol synthase, 1.1.1.319 Isoeugenol synthase, 1.1.1.320 Benzyl reductase [(S)-benzoin formation], 1.1.1.321 Benzyl reductase [(R)-benzoin formation], 1.1.1.322 (-)-endo-fenchol dehydrogenase, 1.1.1.323 (+)-thujone-3-ol dehydrogenase, 1.1.1.324 8-hydroxygeraniol dehydrogenase, 1.1.1.325 Sepiapterin reductase (L-threo-7,8-dihydrobiopterin formation), 1.1.1.326 Zerumbone synthase, 1.1.1.327 5-exo-hydroxycamphor dehydrogenase, 1.1.1.328 Nicotine blue oxidoreductase, 1.1.1.329 2-Deoxy-scyllo-inosamine dehydrogenase, 1.1.1.330 Very long chain 3-oxoacyl-CoA reductase, 1.1.1.331 Secoisolariciresinol dehydrogenase, 1.1.1.332 Chanoclavine-I dehydrogenase, 1.1.1.333 Decaprenylphospho-β-D-erythro-pentofuranoside-2-ulose 2-reductase, 1.1.1.334 Methylecgonone reductase, 1.1.1.335 UDP-N-acetyl-2-amino-2-deoxyglucuronate dehydrogenase, 1.1.1.336 UDP-N-acetyl-D-mannosamine dehydrogenase, 1.1.1.337 L-2-hydroxycarboxylic acid dehydrogenase (NAD(+)), 1.1.1.338 (2R)-3-sulfolactate dehydrogenase (NADP(+)), 1.1.1.339 dTDP-6-deoxy-L-talose 4-dehydrogenase (NAD(+)), 1.1.1.340 1-deoxy-11β-hydroxypentalenoate dehydrogenase, 1.1.1.341 CDP-abequase synthase, 1.1.1.342 CDP-paratose synthase, 1.1.1.343 Phosphogluconate dehydrogenase (NAD(+)-dependent, decarboxylation), 1.1.1.344 dTDP-6-deoxy-L-talose 4-dehydrogenase [NAD(P)(+)], 1.1.1.345 D-2-hydroxy acid dehydrogenase (NAD(+)), 1.1.1.346 2,5-didehydrogluconate reductase (2-dehydro-L-gulonic acid formation), 1.1.1.347 geraniol dehydrogenase (NAD(+)), 1.1.1.348 (3R)-2'-hydroxyisoflavanone reductase, 1.1.1.349 norsolonate ketoreductase, 1.1.1.350 ureidoglycolate dehydrogenase (NAD(+)), 1.1.1.351 phosphogluconate dehydrogenase [NAD(P)(+)-dependent, decarboxylation], 1.1.1.352 5'-hydroxyavelantin dehydrogenase, 1.1.1.353 versiconal hemiacetal acetate reductase, 1.1.1.354 Farnesol dehydrogenase (NAD(+)), 1.1.1.355 2'-dehydrokanamycin reductase, 1.1.1.356 GDP-L-coritose synthase, 1.1.1.357 3α-hydroxysteroid 3-dehydrogenase, 1.1.1.358 2-dehydropantolactone reductase, 1.1.1.359 Aldose 1-dehydrogenase [NAD(P)(+)], 1.1.1.360 Glucose / galactose 1-dehydrogenase, 1.1.1.361 Glucose-6-phosphate 3-dehydrogenase, 1.1.1.362 Aklaviketone reductase, 1.1.1.363 Glucose-6-phosphate dehydrogenase [NAD(P)(+)], 1.1.1.364 dTDP-4-dehydro-6-deoxy-α-D-gulose 4-ketoreductase, 1.1.1.365 D-galacturonate reductase, 1.1.1.366 L-idonic acid 5-dehydrogenase (NAD(+)), 1.1.1.367 UDP-2-acetamido-2,6-β-L-arabino-hexulo-4-ose reductase, 1.1.1.368 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase, 1.1.1.369 D-chiro-inositol 1-dehydrogenase, 1.1.1.370 scyllo-inositol 2-dehydrogenase (NAD(+)), 1.1.1.371 scyllo-inositol 2-dehydrogenase (NADP(+)), 1.1.1.372 D / L-glyceraldehyde reductase, 1.1.1.373 Sulfolactaldehyde 3-reductase, 1.1.1.374 UDP-N-acetylglucosamine 3-dehydrogenase, 1.1.1.375 L-2-hydroxycarboxylic acid dehydrogenase [NAD(P)(+)], 1.1.1.376 L-arabinose 1-dehydrogenase [NAD(P)(+)], 1.1.1.377 L-rhamnose 1-dehydrogenase (NADP(+)), 1.1.1.378 L-rhamnose 1-dehydrogenase [NAD(P)(+)], 1.1.1.379 (R)-mandelate dehydrogenase, 1.1.1.380 L-gulonic acid 5-dehydrogenase, 1.1.1.381 3-hydroxyacid dehydrogenase, 1.1.1.382 Ketol-acid reductoisomerase (NAD(+)), 1.1.1.383 Ketol-acid reductoisomerase [NAD(P)(+)], 1.1.1.384 dTDP-3,4-didehydro-2,6-dideoxy-α-D-glucose 3-reductase, 1.1.1.385 Dihydroanticapsin 7-dehydrogenase, 1.1.1.386 Ipsdienol dehydrogenase, 1.1.1.387 L-serine 3-dehydrogenase (NAD(+)), 1.1.1.388 Glucose-6-phosphate dehydrogenase (NAD(+)), 1.1.1.389 2-dehydro-3-deoxy-L-galactonate 5-dehydrogenase, 1.1.1.390 Sulfoquinovose 1-dehydrogenase, 1.1.1.391 3β-hydroxycholanic acid 3-dehydrogenase (NAD(+)), 1.1.1.392 3α-Hydroxycholanic acid dehydrogenase (NADP(+)), 1.1.1.393 3β-Hydroxycholanic acid 3-dehydrogenase (NADP(+)), 1.1.1.394 Aurakin B dehydrogenase, 1.1.1.395 3α-Hydroxybile acid CoA 3-dehydrogenase, 1.1.1.396 Bacteriochlorophyllide-a dehydrogenase, 1.1.1.397 β-Methylindole-3-pyruvate reductase, 1.1.1.398 2-Glutathionyl-2-methylbut-3-en-1-ol dehydrogenase, 1.1.1.399 2-Oxoglutarate reductase, 1.1.1.400 2-Methyl-1,2-propanediol dehydrogenase, 1.1.1.401 2-Dehydro-3-deoxy-L-rhamnoate dehydrogenase (NAD(+)), 1.1.1.402 D-erythritol 1-phosphate dehydrogenase, 1.1.1.403 D-threitol dehydrogenase (NAD(+)), 1.1.1.404 Tetrachlorobenzoquinone reductase, 1.1.1.405 Ribitol-5-phosphate 2-dehydrogenase (NADP(+)), 1.1.1.406 Galactitol 2-dehydrogenase (L-tagatose formation), 1.1.1.407 D-altritol 5-dehydrogenase, 1.1.1.408 4-phospho-D-threonate 3-dehydrogenase, 1.1.1.409 4-phospho-D-erythronic acid 3-dehydrogenase, 1.1.1.410 D-erythronic acid 2-dehydrogenase, 1.1.1.411 L-threonate 2-dehydrogenase, 1.1.1.412 2-Alkyl-3-oxoalkanoate reductase, 1.1.1.413 A-factor γ-butyrolactone 1'-reductase (1S formation), 1.1.1.414 L-galactonate 5-dehydrogenase, 1.1.1.415 Noscapine synthase, 1.1.1.416 Isopyridoxal dehydrogenase (5-pyridoxolactone formation), 1.1.1.417 3β-hydroxysteroid-4β-carboxylic acid 3-dehydrogenase (decarboxylation), 1.1.1.418 Plant 3β-hydroxysteroid-4α-carboxylic acid 3-dehydrogenase (decarboxylation), 1.1.1.419 Nepetalactol dehydrogenase, 1.1.1.420 D-apiose dehydrogenase, 1.1.1.421 D-Apionate oxidoisomerase, 1.1.1.422 Pseudoephedrine dehydrogenase, 1.1.1.423 (1R,2S)-Ephedrine 1-dehydrogenase, 1.1.1.424 D-Xylose 1-dehydrogenase (NADP(+), D-xylono-1,4-lactone formation), 1.1.1.425 Levoglucosane dehydrogenase, 1.1.1.426 UDP-N-acetyl-α-D-quinovosamine dehydrogenase, 1.1.1.427 D-Arabinose 1-dehydrogenase (NADP(+)), 1.1.1.428 4-methylthio 2-oxobutanoate reductase (NADH), 1.1.1.429 (2S)-[(R)-hydroxy(phenyl)methyl]succinyl-CoA dehydrogenase.

[0093] As known to those skilled in the art, the ADH reaction typically requires a cofactor. The reduction reaction catalyzed by the ADH enzyme described herein also typically requires a cofactor. As used herein, the term "cofactor" refers to a non-protein compound that acts in conjunction with the ADH enzyme. Suitable cofactors for use with the ADH enzyme in the methods of the invention described herein include NAD(P). + (nicotinamide adenine dinucleotide phosphate), NAD(P)H(NAD(P) + reduced form), NAD + (nicotinamide adenine dinucleotide) and NADH (NAD+ Generally, the reduced form of the cofactor is added to the reaction mixture.

[0094] Therefore, a preferred embodiment of the present invention is to carry out the method of the present invention in the presence of a cofactor, preferably NAD or NAD(P). + is.

[0095] The cofactor is optionally regenerated using a cofactor regeneration system. One advantage of using a cofactor regeneration system is that such a system can shift the equilibrium of the method of the present invention toward the production of the desired product, such as drimanaldehyde. In this way, the method of the present invention can be more optimized and efficient in terms of the reagents used, and therefore more time- and cost-effective than when a cofactor regeneration system is not used.

[0096] Thus, one embodiment of the present invention is to carry out the method of the present invention in the presence of a cofactor regenerating system.

[0097] For the avoidance of doubt, the aspects of the invention described below in relation to polypeptides, nucleic acids, recombinant cells, expression vectors etc. may be used to prepare drimanaldehyde of formula (I) in the method of the first aspect of the invention.

[0098] As mentioned above, the inventors sought to identify whether ADHs could be used to oxidize drimane alcohols of formula (II) to form drimane aldehydes of formula (I). Several such enzymes that can be used for this purpose are shown in the accompanying examples.

[0099] The inventors were surprised to find that some members of the momilactone A synthase class of ADH enzymes can oxidize drimane alcohol of formula (II) to form drimane aldehyde of formula (I). Accordingly, a preferred embodiment of the present invention is one in which the ADH is a momilactone A synthase. Momilactone A synthase is a known class of ADH enzymes assigned to EC classification 1.1.1.295. Preferably, the momilactone A synthase used in the methods of the present invention has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1, 2, 6, 12, 21 or 33.

[0100] Furthermore, the inventors were surprised to discover that members of the secoisolariciresinol dehydrogenase class of ADH enzymes can oxidize drimane alcohol (II) to form drimane aldehyde (I). Accordingly, a preferred embodiment of the present invention is one in which the ADH is a secoisolariciresinol dehydrogenase. Secoisolariciresinol dehydrogenases are a known class of ADH enzymes assigned to EC classification 1.1.1.331. Preferably, the secoisolariciresinol dehydrogenase used in the methods of the present invention has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3, 7, 8, 10, 16, or 17.

[0101] A further aspect of the present invention provides an isolated polypeptide having ADH activity, which comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1 to 33, or comprises the amino acid sequence of any of SEQ ID NOs: 1 to 33.

[0102] As shown in the accompanying Examples, a polypeptide having ADH activity comprising the amino acid sequence of any of SEQ ID NOs: 1-25 is capable of producing greater than 2 mg / L of drimanaldehyde of formula (I). Accordingly, preferred embodiments of all aspects of the invention described herein are those in which the isolated polypeptide having ADH activity comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1-25, or comprises the amino acid sequence of any of SEQ ID NOs: 1-25.

[0103] As shown in the accompanying Examples, a polypeptide having ADH activity comprising the amino acid sequence of any of SEQ ID NOs: 1-13 is capable of producing greater than 5 mg / L of drimanaldehyde of formula (I). Accordingly, preferred embodiments of all aspects of the invention described herein are those in which the isolated polypeptide having ADH activity comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-13, or comprises the amino acid sequence of any of SEQ ID NOs: 1-13.

[0104] As shown in the accompanying Examples, a polypeptide having ADH activity comprising the amino acid sequence of any of SEQ ID NOs: 1-6 is capable of producing greater than 10 mg / L of drimanaldehyde of formula (I). Accordingly, preferred embodiments of all aspects of the invention described herein are those in which the isolated polypeptide having ADH activity comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-6, or comprises the amino acid sequence of any of SEQ ID NOs: 1-6.

[0105] In addition to the ADH polypeptide comprising any of the amino acid sequences of SEQ ID NOs: 1 to 33, Table 1 in the accompanying Examples indicates that enzymes 34 to 71 can also be used to prepare drimanaldehyde of formula (I).

[0106] Thus, one embodiment of the present invention is to use an ADH polypeptide of any one of enzymes 34 to 71 in the method of the present invention.

[0107] A further aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having ADH activity, wherein the isolated nucleic acid molecule comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-33, or comprises the amino acid sequence of any of SEQ ID NOs: 1-33.

[0108] A further aspect of the present invention provides an isolated nucleic acid comprising a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 34 to 66, or comprising the nucleotide sequence of any of SEQ ID NOs: 34 to 66 or its reverse complement.

[0109] A further aspect of the present invention provides nucleic acid molecules encoding the polypeptides provided herein.

[0110] In one aspect, provided herein is a vector comprising a nucleic acid molecule described herein. In another aspect, the vector is an expression vector. In a further aspect, the vector is a prokaryotic vector, a viral vector, or a eukaryotic vector.

[0111] Also provided are non-human host organisms or host cells comprising (1) the nucleic acid molecules described above, or (2) expression vectors comprising the nucleic acid molecules. In one embodiment, the non-human organism or host cell is a prokaryotic or eukaryotic cell. In another embodiment, the host cell is a bacterial cell, a plant cell, a fungal cell, or a yeast cell. In a further embodiment, the bacterial cell is E. coli, and the yeast cell is Saccharomyces cerevisiae.

[0112] Further provided is the use of a polypeptide described herein to produce a compound of formula (I).

[0113] Further provided are nucleotide sequences obtained by modifying any of SEQ ID NOs: 34-66, or their reverse complements, including any sequence obtained by modifying any of SEQ ID NOs: 34-66, or their reverse complements, by introducing any type of mutation, such as, for example, deletion, insertion and / or substitution mutations, using any method known in the art.

[0114] Nucleic acids comprising sequences obtained by mutation of any of SEQ ID NOS: 34-66 or their reverse complements are encompassed in embodiments herein, so long as the sequences they comprise share at least the specified sequence identity with any of SEQ ID NOS: 34-66 or their reverse complements, and encode a polypeptide having ADH activity as defined in any of the above embodiments. Mutations can be any type of mutation in these nucleic acids, for example, point mutations, deletion mutations, insertion mutations, and / or frameshift mutations of one or more nucleotides in the DNA sequence of any of SEQ ID NOS: 34-66. In one embodiment, the nucleic acids of one embodiment herein may be truncated as long as they encode a polypeptide described herein.

[0115] Variant nucleic acids can be generated to adapt their nucleotide sequences to particular expression systems, for example, bacterial expression systems are known to express polypeptides more efficiently if amino acids are encoded by particular codons.

[0116] Due to the degeneracy of genetic code, two or more codons may code for the same amino acid sequence, and multiple nucleic acid sequences may code for the same protein or polypeptide, and all of these DNA sequences are included in the embodiments of the present specification.If necessary, the nucleic acid sequence encoding ADH can be optimized to increase expression in host cells.For example, the nucleotide of one embodiment of the present specification can be synthesized using codons specific to the host to improve expression.

[0117] In one embodiment, provided herein is an isolated, recombinant, or synthetic nucleic acid sequence of any of SEQ ID NOs: 34-66, or a fragment thereof, encoding a polypeptide having ADH activity comprising the amino acid sequence of any of SEQ ID NOs: 1-33, which catalyzes the production of drimanaldehyde of formula (I).

[0118] cDNA, genomic DNA and RNA sequences are also provided herein. Any nucleic acid sequence encoding ADH or a variant thereof is also referred to herein as an ADH coding sequence.

[0119] According to one embodiment, the nucleic acid of any one of SEQ ID NOs: 34 to 66 is a coding sequence of the ADH gene encoding ADH obtained as described in the Examples.

[0120] A fragment of a polynucleotide of any one of SEQ ID NOs: 34-66 particularly refers to a contiguous nucleotide of 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 one embodiment of the present specification. In particular, a polynucleotide fragment comprises at least 25, particularly at least 50, particularly at least 75, particularly at least 100, particularly at least 150, particularly at least 200, particularly at least 300, particularly at least 400, particularly at least 500, particularly at least 600, particularly at least 700, particularly at least 800, particularly at least 900, particularly at least 1000 contiguous nucleotides of the polynucleotide of one embodiment of the present specification. Without limitation, the polynucleotide fragment of the present specification can be used as a PCR primer and / or probe, or for antisense gene silencing or RNAi.

[0121] It will be apparent to those skilled in the art that a gene comprising a polynucleotide according to one embodiment of the present disclosure can be cloned by methods known in the art based on available nucleotide sequence information, such as that found in the attached sequence listing. These methods include, for example, designing DNA primers representing the flanking sequences of the gene, one of which is generated in the sense orientation to initiate synthesis of the sense strand, and the other is made in reverse complementarity to generate the antisense strand. Thermostable DNA polymerases, such as those used in the polymerase chain reaction, are commonly used to perform such experiments. Alternatively, a DNA sequence representing the gene can be chemically synthesized and then introduced into a DNA vector molecule that can be propagated, for example, by a suitable bacterium, such as E. coli.

[0122] Related embodiments provided herein provide PCR primers and / or probes for detecting nucleic acid sequences encoding ADH. Those skilled in the art will know how to synthesize degenerate or specific PCR primer pairs for amplifying nucleic acid sequences encoding ADH or fragments thereof based on any of SEQ ID NOS: 34-66. A detection kit for a nucleic acid sequence encoding ADH may include primers and / or probes specific to the nucleic acid sequence encoding ADH and an associated protocol for detecting the nucleic acid sequence encoding ADH in a sample using the primers and / or probes. Such a detection kit can be used to determine whether a plant, organism, or cell has been modified, i.e., transformed, with a sequence encoding ADH.

[0123] To test the functionality of a mutant DNA sequence according to one embodiment of the present disclosure, the sequence of interest is operably linked to a selectable or screenable marker gene, and reporter gene expression is tested in transient expression assays using protoplasts or in stably transformed plants. Those skilled in the art will recognize that DNA sequences capable of driving expression are constructed modularly. Thus, expression levels from shorter DNA fragments may differ from those from the longest fragment and may differ from each other. Functional equivalents of the nucleic acid sequences encoding the ADH proteins provided herein, i.e., nucleotide sequences that hybridize under stringent conditions to any of the nucleic acid sequences set forth in SEQ ID NOs: 34-66, are also provided herein.

[0124] Those skilled in the art will know how to identify homologous sequences in other organisms and how to determine the percentage of sequence identity between homologous sequences. Such newly identified DNA molecules can then be sequenced and the sequence compared to the nucleic acid sequences of any of SEQ ID NOs: 34-66.

[0125] The percentage of identity between two peptide or nucleotide sequences is a function of the number of identical amino acid or nucleotide residues in the two sequences when these two sequences are aligned. An identical residue is defined as the same residue in the two sequences at a given position in the alignment. As used herein, the percentage of sequence identity is calculated from the optimal alignment by dividing the number of identical residues between two sequences by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment with the highest possible percentage of identity. Gaps can be introduced into one or both sequences at one or more positions in the alignment to obtain optimal alignment. These gaps are then considered as non-identical residues in 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, for example, public computer programs available on the World Wide Web. Preferably, the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to default parameters and available from the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi can be used to obtain optimal alignment of protein or nucleic acid sequences and to calculate the percentage of sequence identity.

[0126] Related embodiments provided herein provide nucleic acid sequences complementary to a nucleic acid sequence according to any of SEQ ID NOS: 34-66, such as inhibitory RNA, or nucleic acid sequences that hybridize under stringent conditions with at least a portion of a nucleotide sequence according to any of SEQ ID NOS: 34-66. An alternative embodiment of an embodiment herein provides a method of modifying gene expression in a host cell. For example, a polynucleotide of an embodiment herein can be enhanced, overexpressed, or induced in a host cell or host organism under certain circumstances (e.g., upon exposure to certain temperatures or culture conditions).

[0127] The modification of the expression of the polynucleotide provided herein can result in ectopic expression, which is the expression pattern that is different between modified organisms and control or wild-type organisms.The change in expression occurs from the interaction of the polypeptide of one embodiment of the present invention with exogenous or endogenous modulators, or as a result of chemical modification of the polypeptide.This term also refers to the modification of the expression pattern of the polynucleotide of one embodiment of the present invention, which is modified below the detection level or completely suppressed activity.

[0128] In one embodiment, provided herein is an isolated, recombinant, or synthetic polynucleotide that encodes a polypeptide or variant polypeptide provided herein.

[0129] In one embodiment, an isolated nucleic acid molecule is provided that encodes a polypeptide having ADH activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1-33, or comprising the amino acid sequence of any of SEQ ID NOs: 1-33.

[0130] In one embodiment, provided herein is an isolated polypeptide having ADH activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1-33, or comprising the amino acid sequence of any of SEQ ID NOs: 1-33.

[0131] According to one embodiment, the polypeptide consists of the amino acid sequence of any of SEQ ID NOs: 1-33.

[0132] In one embodiment, at least one polypeptide having ADH activity used in any of the embodiments described herein, or encoded by a nucleic acid used in any of the embodiments described herein, comprises an amino acid sequence that is a genetically engineered variant of any of SEQ ID NOs: 1-33. In one embodiment, the polypeptide comprises an amino acid sequence encoded by a nucleotide sequence obtained by modification of any of SEQ ID NOs: 34-66, or its reverse complement.

[0133] The polypeptide is also intended to include mutants and truncated polypeptides, so long as they have ADH activity.

[0134] According to another embodiment, at least one polypeptide having ADH activity used in any of the embodiments described herein or encoded by a nucleic acid used in any of the embodiments described herein comprises an amino acid sequence that is a variant of any of SEQ ID NOs: 1-33 obtained by genetic engineering, so long as said variant has ADH activity and has the required percentage of identity to any of SEQ ID NOs: 1-33 described herein.

[0135] According to another embodiment, at least one polypeptide having ADH activity used in any of the embodiments described herein or encoded by a nucleic acid used in any of the embodiments described herein is a variant of any of SEQ ID NOs: 1-33 that may be naturally found in other organisms, so long as it has ADH activity. As used herein, polypeptide includes not only polypeptide or peptide fragments that encompass the amino acid sequences identified herein, but also truncated or variant polypeptides, so long as they have ADH activity and share at least the specified percentage of identity with the corresponding fragment of any of SEQ ID NOs: 1-33.

[0136] Examples of mutant polypeptides are naturally occurring proteins that are produced by alternative mRNA splicing events or by proteolytic cleavage of the polypeptides described herein.Proteolytic modifications include, for example, differences in N-terminus or C-terminus when expressed in different types of host cells, due to the proteolytic removal of one or more terminal amino acids from the polypeptide of one embodiment of the present invention.The polypeptides encoded by nucleic acids obtained by natural or artificial mutation of the nucleic acid of one embodiment of the present invention as described below are also encompassed in the present embodiment.

[0137] Polypeptide variants obtained by fusion of additional peptide sequences at the amino and carboxyl termini can also be used in the methods of one embodiment of the present specification. In particular, such fusions can enhance polypeptide expression, be useful for protein purification, or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences can be, for example, signal peptides. Another aspect includes methods using variant polypeptides, such as those obtained by fusion with other oligopeptides or polypeptides and / or those linked to signal peptides. Polypeptides obtained by fusion with another functional protein can also be advantageously used in the methods of one embodiment of the present specification.

[0138] Variants may differ from the polypeptides of one embodiment herein by the incorporation of modifying groups covalently or non-covalently linked to the polypeptide backbone. Variants also include polypeptides that differ from the polypeptides provided herein by the introduction of N-linked or O-linked glycosylation sites and / or the addition of cysteine ​​residues. Those skilled in the art will recognize methods for modifying amino acid sequences and maintaining biological activity.

[0139] It is clear to those skilled in the art that in addition to the gene sequences shown in the sequences disclosed herein, DNA sequence polymorphisms may exist within a given population, which may result in changes in the amino acid sequences of the polypeptides disclosed herein.Such genetic polymorphisms may exist in cells from different populations or may exist within a population due to natural allelic variation.Allelic variants may also include functional equivalents.

[0140] Further embodiments also relate to molecules derived from the specifically disclosed nucleic acids by such sequence polymorphisms. These natural variations typically result in about 1-5% variability in the nucleotide sequence of a gene or amino acid sequence of a polypeptide disclosed herein. As noted above, nucleic acids encoding the polypeptides of one embodiment herein, or variants thereof, are useful tools for modifying non-human host organisms or cells, and for use in the methods described herein.

[0141] One embodiment provided herein provides the amino acid sequence of ADH protein, including orthologs and paralogs, and methods for identifying and isolating orthologs and paralogs of ADH in other organisms. In particular, the orthologs and paralogs of ADH so identified are capable of producing compounds of Formula (I).

[0142] ADH polypeptide can be obtained by extracting it from any organism that expresses it using standard protein or enzyme extraction techniques.When host organism is a single-cell organism or cell that releases the polypeptide of one embodiment of the present invention into culture medium, polypeptide can be simply recovered from culture medium by, for example, centrifugation, optionally followed by washing step and resuspension in suitable buffer.When organism or cell accumulates polypeptide in its cell, polypeptide can be obtained by disrupting or lysing cell, and optionally further extracting polypeptide from cell lysate.

[0143] According to another embodiment, at least one polypeptide having ADH may be used in the method of the present invention.

[0144] The functionality or activity of any ADH protein, variant, or fragment can be determined using various methods. For example, transient or stable overexpression in plant, bacterial, or yeast cells can be used to test whether the protein is active, i.e., produces the compound of formula (I). ADH activity can be assessed in the assays described in the Examples herein to demonstrate functionality. A variant or derivative of the ADH polypeptide of one embodiment herein retains the ability to produce the compound of formula (I). The amino acid sequence variants of ADH provided herein can have additional desirable biological functions, including, for example, changes in substrate utilization, reaction kinetics, product distribution, or other changes.

[0145] Further provided is at least one vector comprising a nucleic acid molecule described herein.

[0146] Also provided herein are vectors selected from the group of prokaryotic vectors, viral vectors and eukaryotic vectors.

[0147] Further provided herein are vectors that are expression vectors.

[0148] The nucleic acid sequence of one embodiment of the present specification that encodes an ADH protein can be inserted into an expression vector and / or contained in a chimeric gene inserted into an expression vector, thereby producing the ADH protein in a host cell or a non-human host organism.Vectors for inserting transgenes into the genome of a host cell are known in the art, and include plasmids, viruses, cosmids, and artificial chromosomes.Binary vectors or cointegration vectors into which the chimeric gene is inserted can also be used to transform host cells.

[0149] One embodiment provided herein provides a recombinant expression vector comprising the nucleic acid sequence of an ADH gene or a chimeric gene comprising the nucleic acid sequence of an ADH gene operably linked to a related nucleic acid sequence, such as a promoter sequence. For example, a chimeric gene comprising the nucleic acid sequence of any of SEQ ID NOS: 34-66 or a variant thereof can be operably linked to a promoter sequence suitable for expression in plant, bacterial, or fungal cells, and can optionally be linked to a 3' untranslated nucleic acid sequence.

[0150] Alternatively, a promoter sequence may already be present in the vector, such that the nucleic acid sequence to be transcribed is inserted into the vector downstream of the promoter sequence. Vectors may be engineered to have an origin of replication, a multiple cloning site, and a selectable marker.

[0151] In one embodiment, an expression vector comprising a nucleic acid described herein can be used as a tool to transform a non-human host organism or host cell suitable for practicing the methods of one embodiment herein in vivo.

[0152] The expression vectors provided herein can be used in methods for producing genetically transformed non-human host organisms and / or host cells, non-human host organisms and / or host cells harboring a nucleic acid of one embodiment herein, and methods for producing polypeptides having ADH activity as described herein.

[0153] Recombinant non-human host organisms and host cells that harbor at least one nucleic acid of an embodiment herein and are transformed to heterologously express or overexpress at least one polypeptide of an embodiment herein are also very useful tools for carrying out the method of an embodiment herein. Accordingly, such non-human host organisms and host cells are provided herein.

[0154] In one embodiment, a host cell or non-human host organism is provided that comprises at least one of the nucleic acid molecules described herein or that comprises at least one vector that comprises at least one of the nucleic acid molecules.

[0155] Nucleic acids according to any of the above embodiments can be used to transform non-human host organisms and cells, and the expressed polypeptides can be any of the above polypeptides.

[0156] In one embodiment, the non-human host organism or host cell is a prokaryotic cell. In another embodiment, the non-human host organism or host cell is a bacterial cell. In a further embodiment, the non-human host organism or host cell is Escherichia coli.

[0157] In one embodiment, the non-human host organism or host cell is a eukaryotic cell. In another embodiment, the non-human host organism or host cell is a yeast cell. In a further embodiment, the non-human host organism or cell is Saccharomyces cerevisiae.

[0158] In one embodiment, a non-human host organism or host cell expresses a polypeptide, provided that the organism or cell has been transformed to harbor nucleic acid encoding said polypeptide, which nucleic acid is transcribed into mRNA, and the polypeptide is found in the host organism or cell.

[0159] Suitable methods for transforming non-human host organisms or host cells have been previously described and are also provided herein.

[0160] To carry out one embodiment of the present invention in vivo, a host organism or host cell is cultured under conditions that promote the production of the compound of formula (I). When the host is a unicellular organism, the conditions that promote the production of the compound of formula (I) may include the addition of suitable cofactors to the culture medium of the host. In addition, the culture medium may be selected to maximize the synthesis of the compound of formula (I). Examples of optimal culture conditions are described in more detail in the Examples.

[0161] A non-human host organism suitable for carrying out the method of one embodiment herein in vivo can be any non-human multicellular or unicellular organism. In one embodiment, the non-human host organism used to carry out the method of one embodiment herein in vivo is a plant, prokaryote, or fungus. Any plant, prokaryote, or fungus can be used. In another embodiment, the non-human host organism used to carry out the method of one embodiment herein in vivo is a microorganism. Any microorganism can be used; for example, the microorganism can be a bacterium or yeast, such as E. coli or Saccharomyces cerevisiae.

[0162] Isolated higher eukaryotic cells can also be used in place of whole organisms as hosts for practicing the methods of one embodiment herein in vivo. Suitable eukaryotic cells can be any non-human cell, such as a plant or fungal cell.

[0163] Further provided herein are methods comprising transforming a host cell or a non-human host organism with a nucleic acid encoding a polypeptide having ADH activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1-33, or comprising the amino acid sequence of any of SEQ ID NOs: 1-33.

[0164] In one embodiment, the methods provided herein comprise culturing a non-human host organism or host cell transformed to express a polypeptide under conditions that allow for the production of the polypeptide, wherein the polypeptide comprises a sequence of amino acids having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 1-33.

[0165] The non-human host organisms or host cells transformed to express the polypeptides according to the invention may contain additional exogenous polypeptide sequences.

[0166] For example, the method of the invention comprises contacting a drimane alcohol of formula (II) with a polypeptide having oxidoreductase enzyme activity.

[0167] The drimane alcohol of formula (II) can be supplied to a host organism or host cell transformed to express a polypeptide according to the invention by adding it to the culture medium (or other such growth substrate known to those skilled in the art).

[0168] Alternatively, another embodiment of the present invention is to transform the host organism or host cell with one or more additional polypeptides capable of producing the drimane alcohol of formula (II).

[0169] Thus, the method of the present invention for providing drimanaldehyde of formula (I) may be a multi-step enzymatic process, as will be appreciated by those skilled in the art.

[0170] Examples of polypeptides capable of producing drimane alcohols of formula (II) are known in the art.

[0171] For example, WO2018220113 (incorporated herein by reference) discloses methods and enzymes for producing arbicanol and / or drimenol, the starting materials for the methods of the present invention, from acyclic farnesyl diphosphate precursor molecules (FPP). In particular, the polypeptides encoded by SEQ ID NO: 1 and SEQ ID NO: 29 of WO2018220113 can be used in this reaction.

[0172] Similarly, WO2019229064 (incorporated herein by reference) discloses methods and enzymes for producing arbicanol, the starting material for the methods of the present invention, from the acyclic farnesyl diphosphate precursor molecule (FPP).

[0173] The following examples are illustrative only and are not intended to limit the scope of the claims and embodiments described herein.

[0174] Recombinant production of polypeptides according to the invention The present invention further relates to a method for the recombinant production of a polypeptide according to the invention or a functional, biologically active fragment thereof, which comprises culturing a polypeptide-producing microorganism, optionally inducing expression of the polypeptide by applying at least one inducer that induces gene expression, and isolating the expressed polypeptide from the culture. In this way, the polypeptide can also be produced on an industrial scale, if desired.

[0175] The microorganisms produced according to the invention can be cultivated continuously or discontinuously in a batch process, or in a fed-batch or repeated fed-batch process. A summary of known cultivation methods can be found in the textbooks by Chmiel (Bioprozesstechnik 1. Einfuehrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0176] The culture medium used must be appropriate to the requirements of each strain. Culture media for various microorganisms are described in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981).

[0177] These media that can be used according to the present invention usually contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0178] Preferred carbon sources are sugars, such as monosaccharides, disaccharides, or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose. Sugars can also be added to the medium via complex compounds such as molasses or other by-products of sugar refining. It can also be advantageous to add a mixture of different carbon sources. Other possible carbon sources 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.

[0179] The nitrogen source is usually an organic or inorganic nitrogen compound or a substance containing such a compound. Examples of nitrogen sources 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 nitrogen sources such as corn steep liquor, soybean flour, soybean protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or in mixtures.

[0180] Inorganic salt compounds that may be present in the medium include chlorides, phosphorus or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0181] Inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur compounds such as mercaptans and thiols can be used as sulfur sources.

[0182] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts can be used as the phosphorus source.

[0183] Chelating agents can be added to the medium to keep metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuic acid, or organic acids such as citric acid.

[0184] The fermentation medium used in accordance with the present invention usually also contains other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid, and pyridoxine. Growth factors and salts are often derived from components of complex media, such as yeast extract, molasses, corn steep liquor, etc. Additionally, suitable precursors can be added to the culture medium. The exact composition of compounds in the medium is highly experimentally dependent and must be determined individually for each specific case. Information regarding medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO).

[0185] All components of the medium are sterilized either by heating (20 minutes at 1.5 bar and 121°C) or by sterile filtration. Components can be sterilized together or separately as needed. All components of the medium can be present at the start of the culture or added either continuously or batchwise.

[0186] The culture temperature is usually 15°C to 45°C, preferably 25°C to 40°C, and can be varied or kept constant during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The growth pH can be controlled during growth by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, such as fatty acid polyglycol esters, can be used to control foaming. To maintain plasmid stability, suitable selective substances, such as antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or an oxygen-containing gas mixture, such as ambient air, is supplied to the medium. The culture temperature is usually in the range of 20°C to 45°C. The culture is continued until the desired product is formed to the maximum extent. This goal is usually achieved within 10 to 160 hours.

[0187] The fermentation broth is then further processed. Depending on the requirements, the biomass can be completely or partially removed from the fermentation broth by separation techniques such as centrifugation, filtration, decanting or a combination of these methods, or can be left entirely in the fermentation broth.

[0188] If the polypeptide is not secreted into the culture medium, the cells may be lysed and the product obtained from the lysate by known methods for isolating proteins. Cells can optionally be disrupted by high frequency ultrasound, high pressure, for example in a French press, by osmotic lysis, by the action of detergents, lytic enzymes or organic solvents, using a homogenizer, or by some combination of the above methods.

[0189] Polypeptides can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), e.g., Q-Sepharose chromatography, ion exchange chromatography, and hydrophobic chromatography, as well as other conventional techniques, such as ultrafiltration, crystallization, salting out, 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 Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0190] To isolate recombinant proteins, it may be advantageous to use vector systems or oligonucleotides that extend cDNA by a defined nucleotide sequence, thus encoding modified polypeptides or fusion proteins, which, for example, facilitate easier purification.Suitable modifications of this type are, for example, so-called "tags" that function as anchors, such as modifications known as hexahistidine anchors, or epitopes that can be recognized as antigens by antibodies (e.g., as described in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press).These anchors can be useful for binding proteins to solid supports, such as polymer matrices, and can be used, for example, as packing for chromatography columns, or on microtiter plates or some other supports.

[0191] At the same time, these anchors can also be used for protein recognition, for which purpose it is also possible to use conventional markers, such as fluorescent dyes, enzymatic markers which form detectable reaction products after reaction with a substrate, or radioactive markers, either alone or in combination with anchors for protein derivatization.

[0192] Polypeptide immobilization The enzymes or polypeptides according to the present invention can be used in the methods described herein in a free or immobilized state. An immobilized enzyme is an enzyme fixed to an inert support. Suitable support materials and enzymes immobilized thereon are known from EP 1149849, EP 1069183, and DE-OS 100193773, as well as the references cited therein. Reference is made to the full disclosure of these documents in this regard. Suitable support materials include, for example, clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, synthetic polymers such as polystyrene, acrylic resins, phenol-formaldehyde resins, polyurethanes, and polyolefins such as polyethylene and polypropylene. To prepare supported enzymes, the support material is usually used in a fine particulate form, with a porous form being preferred. The particle size of the support material is usually 5 mm or less, particularly 2 mm or less (particle size distribution curve). Similarly, when dehydrogenases are used as whole-cell catalysts, free or immobilized forms can be selected. Carrier materials are, for example, calcium alginate and carrageenan. Enzymes and cells can also be directly cross-linked with glutaraldehyde (cross-linking to CLEA). Corresponding and other immobilization techniques are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out the method according to the invention can also be found, for example, in Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol. 3, Chapter 17, VCH, Weinheim.

[0193] Reaction conditions for the biocatalyst production method of the present invention The reaction of the present invention can be carried out under in vivo or in vitro conditions.

[0194] The at least one polypeptide / enzyme present in the method of the invention or in the individual steps of the multi-step method defined herein above may be present in living cells, harvested cells, i.e., under in vivo conditions, which naturally or recombinantly produce the enzyme(s), or in dead cells, permeabilized cells, crude cell extracts, purified extracts, or in substantially pure or completely pure form, i.e., under in vitro conditions. The at least one enzyme may be present in solution or as an enzyme immobilized on a support. One or several enzymes may simultaneously be present in soluble and / or immobilized form.

[0195] The method according to the present invention can be carried out in a variety of scales, from laboratory scale (reaction volumes of several milliliters to several tens of liters) to industrial scale (reaction volumes of several liters to several thousand cubic meters), in conventional reactors known to those skilled in the art. Chemical reactors can be used when polypeptides are used in the form of encapsulated in non-viable, optionally permeabilized cells, in the form of a substantially purified cell extract, or in a purified form. Chemical reactors typically allow for control of the amount of at least one enzyme, the amount of at least one substrate, pH, temperature, and circulation of the reaction medium. When at least one polypeptide / enzyme is present in living cells, the process is fermentation. In this case, biocatalytic production is carried out in a bioreactor (fermenter), where the parameters required for suitable survival conditions for living cells (e.g., culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, etc.) can be controlled. Those skilled in the art are familiar with chemical reactors or bioreactors, procedures for scaling up, for example, chemical or biotechnological processes from laboratory scale to industrial scale, or procedures for optimizing process parameters, which are also well documented in the literature (for biotechnological processes, see, for example, Krueger und Krueger, Biotechnologie - Lehrbuch der angewandten Mikrobiologie, 2nd Ed., R. Oldenbourg Verlag, München, Wien, 1984).

[0196] Cells containing at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or high-frequency pulses, French press, or chemical means, such as hypotonic medium, lytic enzymes and detergents present in the medium, or a combination of such methods. Examples of detergents include digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween® 20, deoxycholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), Nonidet® P40 (ethylphenol poly(ethylene glycol ether)), etc.

[0197] Instead of living cells, non-living cell biomass containing the required biocatalyst can also be applied in the biotransformation reactions of the present invention.

[0198] When the at least one enzyme is immobilized, it is bound to an inert support as described above.

[0199] The conversion reaction can be carried out batchwise, semi-batchwise, or continuously. The reactants (and optionally nutrients) can be fed at the beginning of the reaction or subsequently fed semi-continuously or continuously.

[0200] The reactions of the present invention can be carried out in aqueous, aqueous-organic, or non-aqueous reaction media, depending on the particular reaction type.

[0201] The aqueous or aqueous-organic medium may contain a suitable buffer to adjust the pH to a value in the range of 5 to 11, for example 6 to 10.

[0202] The aqueous organic medium may contain organic solvents that are miscible, partially miscible, or immiscible with water. Non-limiting examples of suitable organic solvents are listed below. Further examples are monohydric or polyhydric aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols such as glycerol.

[0203] The non-aqueous medium is substantially free of water, ie, contains less than about 1% or 0.5% water by weight.

[0204] The biocatalytic process can also be carried out in an organic, non-aqueous medium. Suitable organic solvents include, for example, aliphatic hydrocarbons having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene, or dichlorobenzene; aliphatic acyclic ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, or mixtures thereof.

[0205] The reactant / substrate concentrations can be adapted to the optimal reaction conditions, which may depend on the particular enzyme being applied. For example, the initial substrate concentration can be 0.1-0.5 M, e.g., 10-100 mM.

[0206] The reaction temperature can be adapted to the optimal reaction conditions, which may depend on the particular enzyme being used. For example, the reaction can be carried out at a temperature ranging from 0 to 70°C, such as 20 to 50°C or 25 to 40°C. Examples of 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.

[0207] The process can proceed until equilibrium between the substrate and the subsequent product is reached, but can be stopped earlier. Typical process times are in the range of 1 minute to 25 hours, particularly 10 minutes to 6 hours, for example 1 hour to 4 hours, particularly 1.5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.

[0208] If the host is a genetically modified plant, optimal growth conditions can be provided, such as optimal light, water and nutrient conditions.

[0209] Specific reaction conditions for preparing the drimanaldehyde compound are as follows: ADH enzyme can be present as a purified polypeptide or in a whole cell system in an aqueous environment incubated at 20-37°C and pH 4-7. Substrate concentrations can vary from 0.1 mM to 100 mM.

[0210] Product isolation The methods of the present invention may further include recovering the final product or intermediate product, optionally in substantially stereoisomerically or enantiomerically pure form. The term "recovering" includes extracting, harvesting, isolating, or purifying the compound from the culture medium or reaction medium. Recovery of the compound can be carried out according to any conventional isolation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, non-ionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH change, solvent extraction (e.g., with conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0211] The identity and purity of the isolated products can be determined by known techniques such as high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopy (IR, UV, NMR, etc.), colorimetry, TLC, NIRS, enzymatic assays or microbial assays. (e.g. Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140, Malakhova et al. (1996) Biotekhnologiya 11 27-32, and (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.)

[0212] Cyclic terpene compounds produced by any of the methods described herein can be converted into derivatives such as, but not limited to, hydrocarbons, esters, amides, glycosides, ethers, epoxides, aldehydes, ketones, alcohols, diols, acetals, or ketals. Terpene compound derivatives can be obtained by chemical methods such as, but not limited to, oxidation, reduction, alkylation, acylation, and / or rearrangement. Alternatively, terpene compound derivatives can be obtained using biochemical methods by contacting terpene compounds with enzymes such as, but not limited to, oxidoreductases, monooxygenases, dioxygenases, and transferases. Biochemical conversions can be performed in vitro using isolated enzymes, enzymes from lysed cells, or in vivo using whole cells.

[0213] In one embodiment of the present invention, the method further comprises oxidizing the drimanaldehyde of formula (I) using chemical synthesis or biocatalytic synthesis, or a combination of both.

[0214] Fermentative production of drimanaldehyde The present invention also relates to a method for the fermentative production of drimanaldehyde.

[0215] The fermentation used according to the invention can be carried out, for example, in stirred fermenters, bubble columns and loop reactors. A comprehensive overview of possible process types, including the type and geometric design of the agitator, can be found in "Chmiel: Bioprozesstechnik: Einführung in die Bioverfahrenstechnik, Band 1". In the process of the invention, typical variants that can be used are the following variants known to those skilled in the art or described, for example, in "Chmiel, Hammes and Bailey: Biochemical Engineering", such as batch, fed-batch, repeated fed-batch or continuous fermentation with and without biomass recirculation. Depending on the production strain, sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen or a suitable gas mixture can be carried out to achieve a good yield (YP / S).

[0216] The culture medium used must adequately meet the requirements of the particular strain. Culture media for various microorganisms are described in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington, DC, USA, 1981). These media that can be used according to the present invention contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0217] Preferred carbon sources are sugars, such as monosaccharides, disaccharides, or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose. Sugars can also be added to the medium via complex compounds such as molasses or other by-products of sugar refining. It can also be advantageous to add a mixture of various carbon sources. Other possible carbon sources 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.

[0218] The nitrogen source is usually an organic or inorganic nitrogen compound or a substance containing such a compound. Examples of nitrogen sources 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 nitrogen sources such as corn steep liquor, soybean flour, soybean protein, yeast extract, meat extract, etc. The nitrogen sources can be used individually or as a mixture.

[0219] Inorganic salt compounds that may be present in the medium include chlorides, phosphates or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0220] Inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, as well as organic sulfur compounds such as mercaptans and thiols can be used as sulfur sources.

[0221] Phosphoric acid, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts, can be used as the phosphorus source. Chelating agents can be added to the medium to keep metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuic acid, or organic acids such as citric acid.

[0222] The fermentation medium used in accordance with the present invention may also contain other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid, and pyridoxine. Growth factors and salts are often derived from the complex components of the medium, such as yeast extract, molasses, corn steep liquor, etc. Additionally, suitable precursors can be added to the culture medium. The exact composition of compounds in the medium is highly dependent on the specific experiment and must be determined individually for each specific case. Information regarding medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (1997), and growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO).

[0223] All components of the medium are sterilized either by heat (20 minutes at 1.5 bar and 121°C) or by sterile filtration. Components can be sterilized together or separately as needed. All components of the medium can be present at the start of growth or added continuously or by batch feeding.

[0224] The culture temperature is usually 15°C to 45°C, preferably 25°C to 40°C, and can be kept constant or varied during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH for growth can be controlled during growth by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, such as fatty acid polyglycol esters, can be used to control foaming. To maintain plasmid stability, suitable substances with selective activity, such as antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or an oxygen-containing gas mixture, such as ambient air, is supplied to the medium. The culture temperature is usually 20°C to 45°C. The culture is continued until the desired product is formed to the maximum extent, which is usually achieved within 1 to 160 hours.

[0225] The method of the present invention may further comprise the step of recovering the drimanaldehyde.

[0226] The term "recovering" includes extracting, harvesting, isolating, or purifying a compound from a culture medium. Recovery of a compound can be performed according to any conventional isolation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, non-ionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH change, solvent extraction (e.g., with conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0227] Prior to the intended isolation, the biomass of the broth can be removed. Processes for removing biomass, such as filtration, sedimentation, and flotation, are known to those skilled in the art. As a result, the biomass can be removed, for example, using a centrifuge, separator, decanter, filter, or flotation device. To maximize the recovery of useful products, washing of the biomass, for example, in the form of diafiltration, is often desirable. The choice of method depends on the biomass content and characteristics in the fermenter broth, as well as the interaction of the biomass with the useful products.

[0228] In one embodiment, the fermentation broth can be sterilized or pasteurized. In a further embodiment, the fermentation broth is concentrated. Depending on the requirements, this concentration can be performed batchwise or continuously. The pressure and temperature ranges should be selected so that, firstly, damage to the product does not occur and, secondly, the use of equipment and energy is minimized. A judicious selection of pressure and temperature levels for the multi-stage evaporation allows for particular energy savings.

[0229] A further aspect of the present invention provides a recombinant host cell or recombinant non-human host organism comprising a compound of formula (I) and / or a compound of formula (II). Examples of such host cells are provided in the accompanying Examples section of this application.

[0230] Further conversion of drimanaldehyde to other compounds The process of the present invention relates to the preparation of drimanaldehyde of formula (I).

[0231] In a preferred embodiment of the present invention, drimanaldehyde is converted to drimanic acid using chemical or biocatalytic synthesis, or a combination of both.

[0232] An example of the method of the present invention is one in which the drimanic acid is albicanic acid.

[0233] Albicanic acid can then be used as a starting material for the synthesis of perfume materials, including polywood. The structure and synthesis of polywood via drimanaldehyde of formula (I) are known and can be carried out without inventive input from those skilled in the art.

[0234] The following examples are illustrative only and are not intended to limit the claims and embodiments described herein.

[0235] Also within the scope of the present invention are numerous possible variations that will be readily apparent to those of ordinary skill in the art after reviewing the disclosure provided herein.

[0236] Example Example 1: Oxidoreductase-catalyzed oxidation of arbicanol to arbicanal Alcohol dehydrogenases are nicotinamide adenine dinucleotide (NAD+)- or (NADP+)-dependent oxidoreductases. They are one of the most abundant enzyme classes and catalyze the reversible reduction of aldehydes and ketones to their corresponding alcohols. The equilibrium of this reversible reaction is highly dependent on ADH but can shift when certain substrates are enzymatically modified and no longer available to ADH. To date, no ADH is known to accept albicanol as a substrate.

[0237] To identify ADHs capable of oxidizing arbicanol, 269 ADHs were selected from public libraries and transcriptomes and tested for promiscuity in accepting arbicanol and catalyzing its conversion to arbicanal.

[0238] Two batches of 269 alcohol dehydrogenase candidates were screened. The first batch contained 122 alcohol dehydrogenase candidates obtained from transcriptomes derived from the organisms Bazzania trilobata, Polytrichum species, Laricifomes officinalis, Antrodia cinnamomea, and Porella navicularis. This batch was screened in Saccharomyces cerevisiae. Screening was performed as described in WO 2020078871. Under the screening conditions used, conversion of albicanol to albicanal by any of the 122 alcohol dehydrogenase candidates was not detectable.

[0239] A second batch of alcohol dehydrogenase candidates, including 149 ADH candidates obtained from publications, the NCBI protein database, the Phytozome database (DOE Joint Genome Institute), and transcriptomes derived from the Polytrichum species (WO 2021 / 105236), was screened in E. coli. Therefore, an E. coli strain producing the sesquiterpene albicanol was constructed. For this purpose, the farnesyl pyrophosphate (FPP)-overexpressing E. coli strain DP1205, recently described in WO 2021005097, was used as the base strain. DP1205 was transformed with the expression plasmid pJ424 (ATUM, Newark, CA) containing the E. coli codon-optimized gene version of the arbicanol synthase LoTpsl described in WO2018220113 to obtain E. coli strain DP1205 pJ424(LoTpsl).

[0240] Various alcohol dehydrogenase candidates were ordered from TWIST Bioscience (San Francisco, CA) as E. coli codon-optimized genes cloned into the expression vector pET29a and added to DP1205 pJ424(LoTpsl). Transformants were selected on LB medium plates supplemented with the appropriate antibiotic. Single colonies from each transformation were initially grown overnight at 37°C in deep-well plates in 0.5 mL of LB medium supplemented with 1% glucose and the appropriate antibiotic. The next day, 0.5 mL of medium supplemented with the same antibiotics, 0.1 mM IPTG, and 100 μL of mineral oil / water emulsion (10% oil / water emulsion containing 0.1% (w / v) Tween 80) was added to a deep-well plate and inoculated with 20 μL of the overnight culture, as described by Tsuruta et al. (Tsuruta H, Paddon CJ, Eng D, Lenihan JR, Horning T, et al. (2009 PLoS ONE 4(2): e4489 doi:10.1371 / journal.pone.0004489)). The plate was incubated at 25 °C for 72 h. To obtain arbicanol and arbicanal produced by E. coli cells, each well of the deep-well plate was extracted with 700 μL of ethyl acetate containing an internal standard. The production of arbicanol and arbicanal was identified using GC-MS analysis and quantified by GC-FID using the internal standards described above.

[0241] The amount of albicanal produced under these conditions is shown in Table 1. From the 149 ADHs tested, six candidates: BAG99023.1, AJP06249.1, XP_002446247.1, XP_008669542.1, BAV31336.1, and XP_004494228.1, produced more than 10 mg / L of albicanal. Under these conditions, the momilactone A synthases XP_002446247.1 and XP_008669542.1 produced the highest titers of albicanal at 58 mg / L and 41 mg / L, respectively. GC-MS chromatograms of E. coli-based production of functional ADHs are shown in Figure 1. Furthermore, Figure 1 shows that the MS spectrum of albicanal from E. coli is similar to that of the reference albicanal.

[0242] A total of 71 ADHs were able to produce albicanal. Of these, 33 ADHs produced more than 1 mg / L of the compound, and 6 produced more than 10 mg / L. This is the first time that it has been demonstrated that an ADH enzyme can accept albicanol as a substrate and produce albicanal in quantities suitable for further commercial development.

[0243] The amino acid sequences of enzymes 1 to 33 are disclosed in SEQ ID NOs: 1 to 33.

[0244] Similarly, the accession numbers of the amino acid sequences of enzymes 34 to 71 capable of producing albicanal are shown in Table 2.

[0245] [Table 1]

[0246] [Table 2]

[0247] [Table 3-1] Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18

Claims

1. Formula (I) 【Chemistry 1】 wherein n is 0 and one of the dotted lines is a carbon-carbon double bond and the other is a carbon-carbon single bond, or n is 1 and all of the dotted lines are carbon-carbon single bonds. in the form of any one of its stereoisomers or a mixture thereof, comprising Formula (II) 【Chemistry 2】 wherein n is 0 and one of the dotted lines is a carbon-carbon double bond and the other is a carbon-carbon single bond, or n is 1 and all of the dotted lines are carbon-carbon single bonds. in the form of any one of its stereoisomers or a mixture thereof with a polypeptide having oxidoreductase enzymatic activity; optionally isolating the drimanaldehyde from the reaction; A method comprising:

2. 2. The method of claim 1, wherein the oxidoreductase enzyme is an alcohol dehydrogenase (ADH) enzyme.

3. 3. The method of claim 1 or 2, wherein the ADH enzyme is momilactone A synthase or secoisolariciresinol dehydrogenase.

4. 3. The method of claim 1 or 2, wherein the ADH enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 1-33.

5. 5. The method of claim 4, wherein the ADH enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1-6.

6. The step of contacting the drimane alcohol with the polypeptide having ADH activity is carried out in the presence of a cofactor, and preferably the cofactor is NAD ＋ or NAD(P) ＋ 6. The method according to claim 1, wherein

7. 7. The method of any one of claims 1 to 6, wherein the drimanaldehyde is selected from the group consisting of drimenal, albicanal, β-bicyclofarnesal, or 8-hydroxy-11-drimanal, each in a stereomerically pure form or a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of said group.

8. 8. The method of any one of claims 1 to 7, wherein the drimane alcohol is selected from the group consisting of drimenol, albikanol, β-bicyclofarnesol, drimane-8α,11-diol, each in stereomerically pure form or a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of said group.

9. 9. A method according to any one of claims 1 to 8, carried out in vivo in a cell culture or in vitro in a liquid reaction medium under conditions favouring the production of drimanaldehyde.

10. 10. The method of claim 9, wherein the method is carried out in a recombinant host cell or a recombinant non-human host organism capable of functionally expressing (i) at least one polypeptide having oxidoreductase enzyme activity and, optionally, (ii) at least one polypeptide having the ability to convert the acyclic sesquiterpene precursor FPP to at least one drimane alcohol of formula (II).

11. 11. The method of claim 10, wherein the non-human host cell or host organism is selected from prokaryotic or eukaryotic microorganisms or cells derived therefrom, in particular the non-human host cell or host organism is selected from bacteria, fungi, and plant cells or plants.

12. 12. The method of any one of claims 1 to 11, further comprising oxidizing said drimanaldehyde of formula (I) using chemical synthesis or biocatalytic synthesis, or a combination of both.

13. A polypeptide having ADH activity, wherein the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 1-33.

14. Use of a polypeptide having ADH activity for preparing a compound of formula (I).

15. A compound of formula (I) obtained or obtainable by a process according to any one of claims 1 to 11.

16. A recombinant host cell or a recombinant non-human host organism comprising a compound of formula (I) and / or a compound of formula (II).

17. 17. Use of a compound of formula (I) as defined in any one of claims 1 to 16 for preparing an odorant, flavouring or fragrance ingredient or as a pest control.

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