Method for preparing drimane aldehydes

Abstract

The invention provides a novel method for producing drimane aldehydes through catalysis of alcohol dehydrogenase. The method is realized by oxidizing a corresponding drimane alcohol precursor in vitro or in vivo. The invention also relates to the identification of enzymes having corresponding alcohol dehydrogenase activity from different microbial and plant sources. Further forms of the invention relate to providing corresponding coding sequences of such enzymes, recombinant vectors and recombinant host cells suitable for producing such alcohol dehydrogenases. Another form of the invention relates to the use of such dripane aldehydes obtained according to the invention as intermediates for the production of odorants, flavors or fragrances or insect / pest control ingredients.

Description

Technical Field

[0001] The present invention provides a novel method for producing drimane (bicyclofarnes) aldehydes catalyzed by alcohol dehydrogenases, by oxidizing the corresponding drimane alcohol precursor in vitro or in vivo. The present invention also relates to the identification of enzymes having corresponding alcohol dehydrogenase activity from various microbial and plant sources. A further aspect of the present invention relates to providing corresponding coding sequences for such enzymes, recombinant vectors, and recombinant host cells suitable for producing such alcohol dehydrogenases. Another aspect of the present invention relates to the use of the drimane aldehydes obtained according to the present invention as intermediates for producing odorants, flavors, fragrances, or insect / pest control ingredients. Background Art

[0002] Terpenes are found in most organisms (microorganisms, animals, and plants). These compounds consist of five-carbon units (called isoprene units) and are classified according to the number of these units present in their structure, which may include cyclic structural elements. Thus, monoterpenes, sesquiterpenes, and diterpenes refer to terpenes containing 10, 15, and 20 carbon atoms, respectively. For example, sesquiterpenes are widely found in the plant kingdom. Many sesquiterpenoid molecules are known for their flavor and aroma properties, as well as for their cosmetic, medicinal, and antimicrobial effects. A wide variety of sesquiterpenoid hydrocarbons and sesquiterpenoids have been identified. Although chemical synthesis methods have been developed, they remain complex and not always cost-effective.

[0003] The biosynthetic production of terpenes involves enzymes called terpene synthases. Numerous terpene synthases exist in the plant kingdom, all of which utilize the same substrate (farnesyl diphosphate, FPP) but produce different product structures. Genes and cDNAs encoding terpene synthases have been cloned, and the corresponding recombinant enzymes have been characterized.

[0004] Many major sources of sesquiterpenes, such as compounds with a thiamin structure, particularly thiamin aldehydes, are plants or microorganisms that naturally contain sesquiterpenes; however, the content of thiamin aldehyde sesquiterpenes in these natural sources may be low. There is still a need to provide new methods for producing thiamin aldehydes. Summary of the Invention

[0005] The present invention provides a novel method for producing salbutamol aldehydes. Specifically, the method can implement the complete biochemical synthesis of salbutamol aldehydes in an aqueous environment, such as in a host cell-based process.

[0006] The first aspect of the present invention provides a method for preparing an alkane aldehyde of formula (I):

[0007]

[0008] The aldehyde is in the form of any one of its stereoisomers or a mixture thereof,

[0009] Where n is 0, and one dotted line is a carbon-carbon double bond, and the other dotted lines are carbon-carbon single bonds; or n is 1, and all dotted lines are carbon-carbon single bonds,

[0010] The method comprises contacting an alkanol of formula (II) with a polypeptide having oxidoreductase activity:

[0011]

[0012] The alcohol is in the form of any one of its stereoisomers or a mixture thereof,

[0013] wherein n is 0 and one dashed line is a carbon-carbon double bond and the other dashed lines are carbon-carbon single bonds; or n is 1 and all dashed lines are carbon-carbon single bonds; and

[0014] Optionally, the hydroxyaniline aldehyde is isolated from the reaction.

[0015] One embodiment of the present invention is wherein the oxidoreductase is an alcohol dehydrogenase (ADH) enzyme.

[0016] One embodiment of the present invention is 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 one of SEQ ID NOs: 1 to 33.

[0017] One embodiment of the present invention is wherein 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 one of SEQ ID NO: 1, 2, 6, 12, 21 or 33.

[0018] One embodiment of the present invention is wherein the ADH enzyme is a 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 one of SEQ ID NO: 3, 7, 8, 10, 16 or 17.

[0019] One embodiment of the present invention is 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 one of SEQ ID NOs: 1 to 6.

[0020] 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 one of SEQ ID NOs: 1 to 33, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 33.

[0021] As shown in the accompanying examples, a polypeptide having ADH activity comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 6 is capable of producing the anthracene aldehyde of formula (I) at a yield of more than 10 mg / L. Therefore, a preferred embodiment of all aspects of the invention described herein is wherein 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 one of SEQ ID NOs: 1 to 6, or comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 6.

[0022] One embodiment of the present invention is wherein the step of contacting the alkanol with the polypeptide having ADH activity is carried out in the presence of a cofactor; preferably, the cofactor is NAD + or NAD(P) + .

[0023] The cofactor is optionally regenerated using a cofactor regeneration system. One advantage of using a cofactor regeneration system is that it can drive the equilibrium of the method of the present invention toward the production of the desired product (e.g., an alkane aldehyde). In this way, the method of the present invention can be more optimized and efficient in terms of the reagents used, and thus more timely and economical than when not using a cofactor regeneration system.

[0024] Therefore, one embodiment of the present invention is wherein the method of the present invention is performed in the presence of a cofactor regeneration system.

[0025] One embodiment of the present invention is wherein the drimanoid aldehyde is selected from the group consisting of drimenal, albicanal, β-bicyclofarnesal or 8-hydroxy-11-drimanal, each in stereoisomerically pure form or a mixture of at least two stereoisomers thereof, or a combination comprising at least two members of said group.

[0026] One embodiment of the present invention is wherein the drimenol is selected from the group consisting of drimenol, albicanol, β-bicyclofarnesol, drimenol-8α,11-diol, each in stereoisomerically pure form or a mixture of at least two stereoisomers thereof, or a combination comprising at least two members of said group.

[0027] An embodiment of the present invention is wherein the method is performed in vivo in a cell culture environment or in vitro in a liquid reaction medium under conditions favorable for the production of the analkanal.

[0028] One embodiment of the present invention is that the method is performed in a recombinant host cell or a recombinant non-human host organism, which is capable of functionally expressing (I) at least one polypeptide having oxidoreductase activity, and optionally (II) at least one polypeptide having the ability to convert an acyclic sesquiterpene precursor FPP into at least one fumaranol of formula (II).

[0029] One embodiment of the present invention is wherein the non-human host cell or host organism is selected from prokaryotic or eukaryotic microorganisms, or cells derived therefrom; in particular, wherein the non-human host cell or host organism is selected from bacteria, fungi and plant cells or plants.

[0030] One embodiment of the present invention is wherein the method further comprises oxidizing the anthracene aldehyde of formula (I) using chemical or biocatalytic synthesis or a combination of both.

[0031] A further aspect of the present invention provides 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 any one of SEQ ID NOs: 1 to 33.

[0032] A further aspect of the present invention provides use of a polypeptide having ADH activity in preparing a compound of formula (I).

[0033] A further aspect of the present invention provides a compound of formula (I) obtained or obtainable by a process as claimed in any one of the preceding claims.

[0034] A further aspect of the present 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).

[0035] A further aspect of the invention provides the use of a compound of formula (I) as defined in any one of the preceding claims for the preparation of an odorant, flavor or fragrance ingredient; or for insect / pest control. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 GC-MS analysis of zirconia aldehyde produced when the zirconia aldehyde synthase LoTps1 was co-expressed with either SEQ ID NO: 1 (A) or the alcohol dehydrogenase SEQ ID NO: 2 (B) in E. coli DP1205. The MS spectrum of zirconia aldehyde produced by the alcohol dehydrogenase SEQ ID NO: 2 in (B) is shown in (C), which is similar to the MS spectrum of the reference zirconia aldehyde.

[0037] Figure 2 : The structure of buxinal, folding leaf moss aldehyde, β-bicyclofarnesaldehyde or 8-hydroxy-11-buxinal. DETAILED DESCRIPTION

[0038] The following detailed description describes various aspects and embodiments provided herein. This description should be read from the perspective of one of ordinary skill in the relevant art. Therefore, information known to such persons of ordinary skill is not necessarily included.

[0039] bp——base pair

[0040] kb - kilobase

[0041] DNA - deoxyribonucleic acid

[0042] cDNA – complementary DNA

[0043] DTT - dithiothreitol

[0044] GC – Gas Chromatography

[0045] IPTG——Isopropyl-D-thiogalactopyranoside

[0046] LB - Lysogeny Broth

[0047] MS – Mass Spectrometer / Mass Spectrometry

[0048] PCR — polymerase chain reaction

[0049] RNA – Ribonucleic Acid

[0050] mRNA – messenger ribonucleic acid

[0051] miRNA – microRNA

[0052] siRNA – small interfering RNA

[0053] rRNA – ribosomal RNA

[0054] tRNA – transfer RNA

[0055] definition

[0056] It should be understood that, depending on the context, certain naming conventions for radicals may include monoradicals or diradicals. For example, when 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 identified as an alkyl group requiring two points of attachment includes a diradical, such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc.

[0057] Where a substituent is described as 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, a substituent represented by -AE- or Substituents of include those oriented such that A is attached at the leftmost point of attachment in the molecule as well as those where A is attached at the rightmost point of attachment in the molecule.

[0058] 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" encompasses a single substituent as well as two or more substituents, and so forth.

[0059] As used herein, "for example," "such as," "such as," or "including" are intended to introduce examples that further illustrate a more general subject matter. Unless expressly stated otherwise, such examples are provided merely to aid in understanding the embodiments shown in the present disclosure and are not meant to be limiting in any way. These phrases also do not indicate any preference for the disclosed embodiments.

[0060] As used herein, "include," "comprising," "containing," and "comprised of" refer to open groups, meaning that the group may include other members in addition to those explicitly stated. For example, the phrase "comprising A" means that A must be present, but other members may also be present. The terms "including," "having," and "composed of" and their grammatical variations have the same meaning. In contrast, "consist of," "composed of," or "composed of" refer to closed groups. For example, the phrase "consisting of A" means that only A is present.

[0061] 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 does occur one or more times.

[0062] As used herein, "or" is to be given its broadest reasonable interpretation and is not limited to an "either / or" construction. 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. Furthermore, for example, if A defines a class that may have multiple members (e.g., A1 and A2), then one or more members of the class may be present at the same time.

[0063] As used herein, certain monovalent or multivalent groups having only a single atom may be referred to by the name of the atom. For example, in some cases, the substituent "-H" may be referred to as "hydrogen" or "hydrogen atom," or 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."

[0064] The point of attachment of a group is usually indicated by a dash (-) or an asterisk (*). For example, groups such as *-CH2-CH3 or -CH2-CH3 both represent ethyl groups.

[0065] Chemical structures are often displayed using a "skeleton" format, so that no carbon atoms are shown explicitly and hydrogen atoms attached to them are omitted entirely. For example, the structure represents butane (i.e., n-butane). In addition, aromatic groups such as benzene are represented by showing a contributing resonance structure. For example, the structure Represents toluene.

[0066] Other terms are defined in other parts of this specification even if not included in this section.

[0067] The term "polypeptide" refers to a continuous polymer of amino acid residues, such as an amino acid sequence of at least 15 residues, at least 30 residues, or at least 50 residues. In some embodiments herein, the polypeptide comprises an amino acid sequence that is an enzyme, or a fragment thereof, or a variant or mutant thereof.

[0068] The term "protein" refers to an amino acid sequence of any length in which the amino acids are linked by covalent peptide bonds, and includes oligopeptides, peptides, polypeptides and full-length proteins, whether naturally occurring or synthetic.

[0069] The term "isolated" polypeptide refers to an amino acid sequence that has been removed from its natural environment by any method known in the art, or a combination of methods, including recombinant, biochemical, and synthetic methods.

[0070] The terms "biological function," "function," "biological activity," or "activity" refer to the ability of a bismulol synthase to catalyze the formation of bismulol or a mixture of compounds comprising bismulol and one or more terpenes.

[0071] The terms "nucleic acid sequence," "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably to refer to a sequence of nucleotides. A nucleic acid sequence can be single-stranded or double-stranded deoxyribonucleotides or ribonucleotides of any length and includes 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 naturally occurring DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers, and nucleic acid probes. The skilled artisan understands that the nucleic acid sequence of RNA is identical to the sequence of DNA, except that thymine (T) is replaced by uracil (U). The term "nucleotide sequence" should also be understood to include polynucleotide molecules or oligonucleotide molecules, either in the form of individual fragments or as components of a larger nucleic acid.

[0072] "Isolated nucleic acid" or "isolated nucleic acid sequence" refers to a nucleic acid or nucleic acid sequence that is found in an environment that is different from that in which it occurs in nature, and can include those that are substantially free of contaminating endogenous materials. As used herein, the term "naturally occurring" as applied to nucleic acids refers to a nucleic acid that is found in the cells of an organism in nature and has not been intentionally modified by man in a laboratory.

[0073] A "recombinant nucleic acid sequence" is a nucleic acid sequence produced by combining genetic material from more than one source using laboratory methods (e.g., molecular cloning), thereby creating or modifying a nucleic acid sequence that does not occur in nature and is not otherwise found in a biological organism.

[0074] "Recombinant DNA technology" refers to molecular biology methods for preparing recombinant nucleic acid sequences as described, for example, in Laboratory Manuals, ed., Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0075] The term "gene" refers to a DNA sequence that includes a region that is transcribed into an RNA molecule, such as mRNA, in a cell and is operably linked to an appropriate regulatory region (e.g., a promoter). Thus, a gene may include multiple operably linked sequences, such as a promoter, a 5' leader sequence (including, for example, sequences involved in translation initiation), a coding region of cDNA or genomic DNA, introns, exons, and / or a 3' untranslated sequence (including, for example, a transcription termination site).

[0076] "Chimeric gene" refers to any gene that can not usually be found in species in nature, particularly a gene in which one or more parts of the nucleic acid sequence are not associated with each other in nature. For example, a promoter is not associated with part or all of a transcription region or with another regulatory region in nature. The term "chimeric gene" should be understood to include expression constructs in which a promoter or transcriptional regulatory sequence is operably linked to one or more coding sequences or antisense (i.e., the reverse complementary strand of the sense strand) or inverted repeats (sense and antisense, whereby RNA transcripts form double-stranded RNA after transcription). The term "chimeric gene" also includes genes obtained by combining parts of one or more coding sequences to produce new genes.

[0077] "3' URT" or "3' untranslated sequence" (also called "3' untranslated region" or "3' end") refers to a nucleic acid sequence found downstream of the coding sequence of a gene that contains, for example, a transcription termination site and (in most but not all eukaryotic mRNAs) a polyadenylation signal, such as AAUAAA or a variant thereof. Following transcription termination, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which participates in the transport of the mRNA to the site of translation, such as the cytoplasm.

[0078] "Gene expression" encompasses "heterologous expression" and "overexpression" and involves the transcription of a gene and the translation of mRNA into protein. Overexpression refers to the production of a gene product in a transgenic cell or organism, as measured by mRNA, polypeptide, and / or enzyme activity levels, that exceeds the level of production in a non-transformed cell or organism of a similar genetic background.

[0079] As used herein, "expression vector" refers to a nucleic acid molecule that is engineered using molecular biology methods and recombinant DNA technology to deliver foreign or exogenous DNA into a host cell. An expression vector typically includes sequences required for the correct transcription of the nucleotide sequence. The coding region usually encodes a protein of interest, but may also encode RNA, such as antisense RNA, siRNA, etc.

[0080] As used herein, "expression vector" includes any linear or circular recombinant vector, including but not limited to viral vectors, phages and plasmids. The skilled person can select a suitable vector according to the expression system. In one embodiment, the expression vector includes a nucleic acid of the embodiments herein, which is operably linked to at least one "regulatory sequence" that controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosome binding site, and optionally includes at least one selection marker. When a regulatory sequence is functionally related to a nucleic acid of the embodiments herein, the nucleotide sequence is "operably linked".

[0081] "Regulatory sequence" refers to a nucleic acid sequence that determines the expression level of the nucleic acid sequence of the embodiments herein and is capable of regulating the transcription rate of the nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences include promoters, enhancers, transcription factors, promoter elements, etc.

[0082] "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 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". The promoter regulatory sequence may include upstream and downstream elements that may affect the stability of transcription, RNA processing or the associated coding nucleic acid sequence. Promoters include sequences of natural origin and synthesis. The coding nucleic acid sequence is usually located downstream of the promoter relative to the transcription direction starting with the transcription start site.

[0083] The term "constitutive promoter" refers to an unregulated promoter that allows for continuous transcription of a nucleic acid sequence to which it is operably linked.

[0084] As used herein, the term "operably connected" refers to the connection of a polynucleotide element in a functional relationship. When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably connected". For example, if a promoter or transcriptional regulatory sequence can affect the transcription of a coding sequence, the promoter or transcriptional regulatory sequence is operably connected to the coding sequence. Operably connected means that the DNA sequence being connected is usually adjacent. The nucleotide sequence associated with the promoter sequence can be of homologous or heterologous origin relative to the plant to be transformed. The sequence can also be fully or partially synthesized. Regardless of the source, the nucleic acid sequence associated with the promoter sequence will be expressed or silenced according to the promoter properties connected after being attached to the polypeptide of this paper's embodiment. The relevant nucleic acid can encode the protein that needs to be expressed or suppressed at all times or alternatively at a specific time in the whole organism or in a specific tissue, cell or cell chamber. This nucleotide sequence encodes the protein that the desired phenotypic traits are given to the host cell or organism that is changed or transformed by it. More particularly, the nucleotide sequence of interest results in the production of buminol or a mixture comprising buminol and one or more terpenes in a cell or organism. In particular, the nucleotide sequence encodes a polypeptide having buminol synthase activity.

[0085] "Target peptide" refers to an amino acid sequence that targets a protein or polypeptide to an intracellular organelle (i.e., mitochondria or plastids) or the extracellular space (secretion signal peptide). The nucleic acid sequence encoding the target peptide can be fused to the nucleic acid sequence encoding the amino terminus (e.g., N-terminus) of the protein or polypeptide, or can be used to replace the native targeting polypeptide.

[0086] 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 complementary to the template.

[0087] As used herein, the term "host cell" or "transformed cell" refers to a cell (or organism) that has been altered to harbor at least one nucleic acid molecule, for example, a recombinant gene encoding a desired protein or nucleic acid sequence, which, after transcription, can produce a buminol synthase protein for producing buminol or a mixture comprising buminol and one or more terpenes. The host cell is specifically a bacterial cell, a fungal cell, or a plant cell. The host cell may contain a recombinant gene that has been integrated into the host cell's nuclear or organelle genome. Alternatively, the host cell may also contain a recombinant gene outside the chromosome.

[0088] Homologous sequences include orthologous or paralogous sequences. Methods for identifying orthologs or paralogs include phylogenetic methods, sequence similarity and hybridization methods known in the art and described herein.

[0089] Paralogs are derived from gene duplication, which produces two or more genes with similar sequences and similar functions. Paralogs are typically clustered together and formed by duplication of genes in related plant species. Paralogs are found in groups of similar genes using paired blast analysis or using programs such as CLUSTAL during phylogenetic analysis of gene families. Among paralogs, a consensus sequence can be identified as a sequence that is characterized by being in a related gene and having a similar function to that of a gene.

[0090] Orthologs or orthologous sequences are sequences that are similar to each other because they are found in species passed down by a common ancestor. For example, plant species known to have a common ancestor contain many enzymes with similar sequences and functions. For example, by using CLUSTAL or BLAST programs to construct a phylogenetic tree of the gene family of a species, technicians can identify orthologous sequences and predict the functions of orthologs. 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 gene knockout / knockdown) related polypeptides. Technicians can understand that genes with similar transcript profiles (having a common transcript greater than 50% regulation, or having a common transcript greater than 70% regulation, or a common transcript greater than 90% regulation) can have similar functions. Homologs, paralogs, orthologs, and any other variants of the sequences described herein are expected to work in a similar manner by causing host cells, organisms (such as plants) to produce buprenorphine synthase proteins.

[0091] The term "selectable marker" refers to any gene that, after expression, can be used to select for one or more cells containing the selectable marker. Examples of selectable markers are described below. Those skilled in the art will appreciate that different antibiotic, fungicide, auxotrophic or herbicide selectable markers may be suitable for use with different target species.

[0092] The term "organism" refers to any non-human multicellular or unicellular organism, such as a plant or a microorganism. In particular, a microorganism refers to a bacterium, yeast, algae or fungus.

[0093] The term "plant" is used interchangeably to include plant cells, including plant protoplasts, plant tissues, plant cell tissue cultures to produce regenerated plants, or parts of plants, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits, etc. Any plant can be used to practice the methods of the embodiments herein.

[0094] For purposes of the description and appended claims provided herein, the use of "or" means "and / or" unless stated otherwise. Similarly, the various tenses of "including," "comprising," "comprising," and "comprising" are interchangeable and not limiting.

[0095] It should be further understood that while the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific cases, the language "consisting essentially of" or "consisting of" may be used alternatively to describe the embodiment.

[0096] Compounds of the Invention

[0097] The present invention provides a method for preparing an alkane aldehyde of formula (I):

[0098]

[0099] The aldehyde is in the form of any one of its stereoisomers or a mixture thereof,

[0100] Where n is 0, and one dashed line is a carbon-carbon double bond, and the other dashed lines are carbon-carbon single bonds; or n is 1, and all dashed lines are carbon-carbon single bonds.

[0101] In some embodiments, the benzophenone aldehyde is selected from the group consisting of benzophenone, cyclopentane aldehyde, β-bicyclofarnesaldehyde, or 8-hydroxy-11-benzophenone, each in stereoisomerically pure form or a mixture of at least two stereoisomers thereof, or a combination comprising at least two members of said group.

[0102] The present invention provides a method for preparing a thiazolidine aldehyde of formula (I), comprising contacting a thiazolidine alcohol of formula (II) with a polypeptide having oxidoreductase activity:

[0103]

[0104] The alcohol is in the form of any one of its stereoisomers or a mixture thereof, wherein

[0105] n is 0, and one dashed line is a carbon-carbon double bond, and the other dashed lines are carbon-carbon single bonds; or n is 1, and all dashed lines are carbon-carbon single bonds.

[0106] In some embodiments, the squalane alcohol is selected from the group consisting of squalane, zephyrol, β-bicyclofarnesol, squalane-8α,11-diol, each in stereoisomerically pure form or a mixture of at least two stereoisomers thereof, or a combination comprising at least two members of said group.

[0107] Preferably, the thiazolin alcohol is zephyrol.

[0108] In some embodiments, the benzophenone aldehyde is selected from the group consisting of benzophenone, cyclopentane aldehyde, β-bicyclofarnesaldehyde, or 8-hydroxy-11-benzophenone, each in stereoisomerically pure form or a mixture of at least two stereoisomers thereof, or a combination comprising at least two members of said group.

[0109] Preferably, the anthranil aldehyde is fenvalerate.

[0110] A preferred embodiment of the present invention is wherein the thiamin alcohol is thiaminol and the thiamin aldehyde is thiamin aldehyde.

[0111] If the compounds disclosed herein have at least one chiral center that is not explicitly indicated in the formula, the compounds of formula (I) and formula (II) may be in the form of any one of their stereoisomers or mixtures thereof. For the sake of clarity, by stating "any one of their stereoisomers or mixtures thereof" or similar expressions, it is meant the normal meaning understood by those skilled in the art, i.e., compounds of formula (I) and formula (II) may be pure enantiomers or diastereomers. In other words, compounds of formula (I) and formula (II) have multiple stereo centers, and each of the stereo centers may have two different stereochemistries (e.g., R or S). Compounds of formula (I) and formula (II) may even be in the form of pure enantiomers, or in the form of a mixture of multiple enantiomers or multiple diastereomers. Compounds of formula (I) and formula (II) may be racemic or non-racemic (scalemic, ratio optical rotation) forms. Therefore, compounds of formula (I) and formula (II) may be a stereoisomer, or in the form of a composition of matter comprising or consisting of various stereoisomers.

[0112] The separation of each isomer or the selective synthesis of each isomer is accomplished by applying various methods well known to those skilled in the art. Unless otherwise indicated (e.g., in the case of clearly indicating the stereochemistry of a chiral center), all such isomers and mixtures thereof are included within the scope of the compounds disclosed herein. In addition, the compounds disclosed herein may exist in one or more crystalline or amorphous forms. Unless otherwise indicated, all such forms are included within the scope of the compounds disclosed herein, including any polymorphic forms.

[0113] Isotopes may be present in the described compounds. Each chemical element represented in a compound structure may include any isotope of that element. For example, in a compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in the compound. Any position in the compound where a hydrogen atom may be present may include any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Therefore, the compounds mentioned herein encompass all possible isotopic forms unless the context clearly indicates otherwise.

[0114] The method of the present invention uses alkanol as a starting material.

[0115] In one embodiment of the present invention, the salutidine compound may be present endogenously in the reaction mixture, for example in an in vivo method utilizing a host cell system that produces the salutidine compound as a metabolite and expresses the desired polypeptide(s) for the desired salutidine synthesis, or in a more complex multi-step method comprising salutidine synthesis as a step whereby the salutidine is enzymatically synthesized from an acyclic sesquiterpene precursor.

[0116] Alternatively, the alkane alcohol compound may be produced chemically or enzymatically and added exogenously to the reaction mixture, for example in an in vitro method using an isolated, enriched or purified synthase required for its formation as defined below.

[0117] In one embodiment of the present invention, the method is carried out in a recombinant host cell or a recombinant non-human host organism, which is capable of functionally expressing (I) at least one polypeptide having oxidoreductase activity, and optionally (II) at least one polypeptide having the ability to convert the acyclic sesquiterpene precursor FPP into at least one fumaranol of formula (II).

[0118] Examples of polypeptides capable of converting the acyclic sesquiterpene precursor FPP into at least one fumaranol of formula (II) are known in the art. For example, many examples of such enzymes are disclosed in WO2018220113 and WO2020078871.

[0119] A preferred embodiment of the present invention is wherein the fusan alcohol of formula (II) is fennel alcohol and the fusan aldehyde is fennel aldehyde. In this preferred embodiment of the present invention, the oxidoreductase is an alcohol dehydrogenase (ADH). 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 one of SEQ ID NOs: 1 to 33, or comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 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 one of SEQ ID NOs: 1 to 6, or comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 6.

[0120] Enzyme of the present invention

[0121] The present invention provides a method for preparing a hydroxyanisole aldehyde of formula (I) by contacting a hydroxyanisole alcohol of formula (II) with a polypeptide having oxidoreductase activity.

[0122] Oxidoreductases are enzymes that catalyze the transfer of electrons from one molecule (reductant, also known as electron donor) to another (oxidant, also known as electron acceptor). Oxidoreductases comprise a broad class of enzymes that catalyze biological oxidation / reduction reactions. Because many chemical and biochemical transformations involve oxidation / reduction processes, oxidoreductases have important applications in developing biotechnological methods for synthesizing desired compounds.

[0123] There are several different classes of oxidoreductases, defined primarily by their substrates and / or mode of action. Examples include alcohol dehydrogenases, ketoreductases, peroxidases, hydroxylases, oxygenases, and reductases.

[0124] The present inventors sought to determine whether oxidoreductases could be used to reduce compounds of formula (II) to produce compounds of formula (I). Surprisingly, they identified several enzymes that could be used for this purpose, as shown in the accompanying examples. To the best of the present inventors' knowledge, this is the first time that oxidoreductases, specifically alcohol dehydrogenases (ADHs), have been used in this reaction. Prior to the present invention, ADH enzymes had not been shown to accept compounds of formula (I), particularly zizol, as substrates.

[0125] "Alcohol dehydrogenase" and "ADH" enzyme are used interchangeably herein to refer to polypeptides that have the enzymatic ability to oxidize primary and secondary alcohols to aldehydes and ketones.

[0126] The present invention encompasses all enzymes belonging to the EC class 1.1.1. These enzymes include the following EC numbers and categories:

[0127] 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. 9D-xylulose reductase, 1.1.1.10L-xylulose reductase, 1.1.1.11D-arabinitol 4-dehydrogenase, 1.1.1.12L-arabinitol 4-dehydrogenase, 1.1.1.13L-arabinitol 2-dehydrogenase, 1.1.1.14L-iditol 2-dehydrogenase, 1.1.1.15D-iditol 2-dehydrogenase, 1.1.1.16galactitol 2-dehydrogenase, 1.1.1.17mannitol-1-phosphate 5-dehydrogenase, 1.1.1.18inositol 2-dehydrogenase, 1.1.1.19glucuronide reductase, 1.1.1.20glucuronolactone reductase, 1.1.1.21aldose reductase, 1.1.1.22UDP-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 Mevalonate reductase, 1.1.1.33 Mevalonate 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, decarboxylating), 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-hydroxycholanoate 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 Fructose uronate reductase, 1.1.1.58 Tadgalactone 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 Transfer entry: 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 Keto-acid reductoisomerase (NADP(+)), 1.1.1.87 Homoisocitrate dehydrogenase, 1.1.1.88 Hydroxymethylglutaryl Coenzyme A reductase, 1.1.1.90 Aryl alcohol dehydrogenase, 1.1.1.91 Aryl alcohol dehydrogenase (NADP(+)), 1.1.1.92 Oxaloylglycolate reductase (decarboxylation), 1.1.1.93 Tartaric acid 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-Hydroxyfatty acid dehydrogenase, 1.1.1.99 (S)-2-Hydroxyfatty acid dehydrogenase, 1.1.1.100 3-Oxoacyl-[acyl carrier protein] reductase, 1.1.1.101 Acylglycerol ketone-phosphate reductase, 1.1.1.102 3-Dehydrosphingosine 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 enzyme (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-threoaldose 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-gulonate 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,

[0128] 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 Sequoiadole 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-androstane-17-one 3α-dehydrogenase, 1.1.1.153 Septopterin reductase (L-erythro-7,8-dihydrobiopterin forming), 1.1.1.154 Ureaglycolate dehydrogenase, 1.1.1.156 Glycerol 2-dehydrogenase (NADP(+)), 1.1.1.157 3-Hydroxybutyryl-CoA dehydrogenase, 1.1.1.1597 α-Hydroxysteroid dehydrogenase, 1.1.1.160 Dihydrobuprofen dehydrogenase, 1.1.1.162 Erythrulose reductase, 1.1.1.163 Cyclopentanol dehydrogenase, 1.1.1.164 Hexadecanol dehydrogenase, 1.1.1.165 2-Alkyn-1-ol dehydrogenase, 1.1.1.166 Hydroxycyclohexanecarboxylate dehydrogenase, 1.1.1.167 Hydroxymalonate dehydrogenase, 1.1.1.168 2-Dehydropantoate lactone reductase (Re-specific), 1.1.1.169 2-Dehydropantoate 2-reductase, 1.1.1.170 3β-Hydroxysteroid-4α-carboxylate 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, 1.1.1.17612 α-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-xylan-1,5-lactone forming), 1.1.1.181 Cholester-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-ethylene 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-D dehydrogenase (NADP(+)), 1.1.1.198 (+)-Borneol dehydrogenase, 1.1.1.199 (S)-Usnate 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 (+)-Neomethol dehydrogenase, 1.1.1.209 3(or 17)α-Hydroxysteroid dehydrogenase, 1.1.1.2103 β-(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-methylhydroxybutyrate dehydrogenase, 1.1.1.218 Morphine 6-dehydrogenase, 1.1.1.219 Dihydroflavanol 4-reductase, 1.1.1.220 6-Pyruvyltetrahydropterin 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(-)-Borneolate dehydrogenase, 1.1.1.228 (+)-Sabinol dehydrogenase, 1.1.1.229 2-methyl-3-oxosuccinate diethyl ester reductase, 1.1.1.230 3α-hydroxyglycyrrhetinic acid dehydrogenase, 1.1.1.231 15-Hydroxyprostaglandin-I dehydrogenase (NADP(+)), 1.1.1.232 15-Hydroxyeicosatetraenoate 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 (NADP(+)), 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 Methylheptenone (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-Methylbutyraldehyde 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-CoA dehydrogenase, 1.1.1.269 2-(S)-hydroxypropyl-CoA 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 Vellosimine dehydrogenase, 1.1.1.274 2,5-Didehydroglucose reductase (2-dehydro-D-gluconic acid forming), 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-hydroxyester dehydrogenase, 1.1.1.280 (S)-3-hydroxyester 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'-oxonicotinamine reductase, 1.1.1.286 Isocitrate-homoisocitrate dehydrogenase, 1.1.1.287 D-arabinitol dehydrogenase (NADP(+)), 1.1.1.288 Flavoxin dehydrogenase, 1.1.1.289 Sorbitol reductase, 1.1.1.290 4-phosphoerythritol dehydrogenase, 1.1.1.291 2-Hydroxymethylglutarate dehydrogenase, 1.1.1.292 1,5-Anhydro-D-fructose reductase (1,5-anhydro-D-mannitol forming), 1.1.1.294 Chlorophyll (ester) b reductase, 1.1.1.295 Coumarin lactone-A synthase, 1.1.1.296 Dihydrocarveol dehydrogenase, 1.1.1.297 Limonene-1,2-diol dehydrogenase, 1.1.1.298 3-Hydroxypropionate dehydrogenase (NADP(+)), 1.1.1.299 Malate dehydrogenase [NAD(P)(+)], 1.1.1.300 NADP-retinol dehydrogenase, 1.1.1.301 D-arabinitol 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 forming], 1.1.1.304 Diacetyl reductase [(S)-acetoin forming], 1.1.1.305 UDP-glucuronide oxidase (UDP-4-keto-hexaaldehyde decarboxylation), 1.1.1.306 S-(hydroxymethyl)actiothiol 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)-Sulfilate dehydrogenase, 1.1.1.311 (S)-1-phenylethanol dehydrogenase, 1.1.1.312 2-Hydroxy-4-carboxymuconate 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 perakine reductase, 1.1.1.318 eugenol synthase, 1.1.1.319 isoeugenol synthase, 1.1.1.320 benzoyl reductase [(S)-benzil forming], 1.1.1.321 benzoyl reductase [(R)-benzil forming], 1.1.1.322 (-)-endo-fenchol dehydrogenase, 1.1.1.323 (+)-orientalis-3-ol dehydrogenase, 1.1.1.324 8-Hydroxygeraniol dehydrogenase, 1.1.1.325 Biopterin reductase (L-threo-7,8-dihydrobiopterin forming), 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-deoxyscyl myosamine dehydrogenase, 1.1.1.330 very long chain 3-oxoacyl-CoA reductase, 1.1.1.331 secoisolariciresinol dehydrogenase, 1.1.1.332 ergotamine-I dehydrogenase, 1.1.1.333 decaprenyl phosphate-β-D-erythro-pentofuranoside-2-ketose 2-reductase, 1.1.1.334 methylecgonine (ecgonone) reductase, 1.1.1.335 UDP- N-acetyl-2-amino-2-deoxyglucuronic acid dehydrogenase, 1.1.1.336 UDP-N-acetyl-D-mannosamine dehydrogenase, 1.1.1.337 L-2-hydroxycarboxylate 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-hydroxypentalenate dehydrogenase, 1.1.1.341 CDP-abetonose synthase, 1.1.1.342 CDP-paratose synthase, 1.1.1.343 phosphogluconate dehydrogenase (NAD(+)-dependent, decarboxylating), 1.1.1.344 dTDP-6-deoxy-L-talose 4-dehydrogenase [NAD(P)(+)], 1.1.1.345 D-2-hydroxyacid dehydrogenase (NAD(+)), 1.1.1.346 2,5-didehydroglucose reductase (2-dehydro-L-gulonic acid forming), 1.1.1.347 geraniol dehydrogenase (NAD(+)), 1.1.1.348 (3R)-2'-hydroxyisoflavanone reductase, 1.1.1.349 norsolorinic acid ketoreductase, 1.1.1.350 ureidoglycolate dehydrogenase (NAD(+)), 1.1.1.351 phosphogluconate dehydrogenase [NAD(P)(+)-dependent, decarboxylating], 1.1.1.352 5'-hydroxyavermectin 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-colitose 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) (+)], 1.1.1.364dTDP-4-dehydro-6-deoxy-α-D-gulose 4-ketoreductase, 1.1.1.365D-galacturonate reductase, 1.1.1.366L-iduronate 5-dehydrogenase (NAD(+)), 1.1.1.367UDP-2-acetylamino-2,6-β-L-arabino-hexano-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 Sulfilactaldehyde 3-reductase, 1.1.1.374 UDP-N-acetylglucosamine 3-dehydrogenase, 1.1.1 .375 L-2-hydroxycarboxylate 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 Keto-acid reductoisomerase (NAD(+)), 1.1.1.383 Keto-acid reductoisomerase [NAD(P)(+)], 1.1.1.384dTDP-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-hydroxycholanoic acid 3-dehydrogenase (NAD(+)), 1.1.1.392 3α-Hydroxycholanoic acid dehydrogenase (NADP(+)), 1.1.1.393 3β-Hydroxycholanoic acid 3-dehydrogenase (NADP(+)), 1.1.1.394 Aurachin 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-Glutathione-2-methylbut-3-en-1-ol dehydrogenase, 1.1.1.399 2-Ketoglutarate reductase, 1.1.1.400 2-Methyl-1,2-propanediol dehydrogenase, 1.1.1.401 2-dehydro-3-deoxy-L-rhamnolate dehydrogenase (NAD(+)), 1.1.1.402 D-erythritol 1-phosphate dehydrogenase, 1.1.1.403 D-threitol dehydrogenase (NAD(+)), 1.1.1.404 Chlorobenzoquinone reductase, 1.1.1.405 Ribitol 5-phosphate 2-dehydrogenase (NADP(+)), 1.1.1.406 Galactitol 2-dehydrogenase (L-tagatose forming), 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-erythronate 3-dehydrogenase, 1.1.1.410 D-erythronate 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 type γ-butyrolactone 1'-reductase (1S forming), 1.1.1.414 L-galactonate 5-dehydrogenase, 1.1.1.415 Noscapine synthase, 1.1.1.416 Isopyridoxal dehydrogenase (5-pyridoxolactone forming), 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-apiate 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-xylonic acid-1,4-lactone forming), 1.1.1.425 levoglucosan dehydrogenase, 1.1.1.426 UDP-N-acetyl-α-D-quinolamine 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.

[0129] It is known to those skilled in the art that ADH reactions generally require cofactors. The reduction reactions catalyzed by the ADH enzymes described herein generally also require cofactors. As used herein, the term "cofactor" refers to a non-protein compound that binds to and acts on the ADH enzyme. Cofactors suitable for use in the ADH enzymes of the present invention described herein include, but are not limited to, NAD(P) + (nicotinamide adenine dinucleotide phosphate), NAD(P)H(NAD(P) + reduced form), NAD + (nicotinamide adenine dinucleotide) and NADH (NAD + Typically, the reduced form of the cofactor is added to the reaction mixture.

[0130] Therefore, a preferred embodiment of the present invention is wherein the process of the present invention is carried out in the presence of a cofactor; preferably, the cofactor is NAD or NAD(P) + .

[0131] The cofactor is optionally regenerated using a cofactor regeneration system. One advantage of using a cofactor regeneration system is that it can drive the equilibrium of the method of the present invention toward the production of the desired product (e.g., an alkane aldehyde). In this way, the method of the present invention can be more optimized and efficient in terms of the reagents used, and thus more timely and economical than when not using a cofactor regeneration system.

[0132] Therefore, one embodiment of the present invention is wherein the method of the present invention is performed in the presence of a cofactor regeneration system.

[0133] For the avoidance of doubt, the aspects of the invention described below relating to polypeptides, nucleic acids, recombinant cells, expression vectors, etc. can be used in the method of the first aspect of the invention to prepare the anthracene aldehyde of formula (I).

[0134] As mentioned above, the present inventors sought to determine whether ADH could be used to oxidize an isoflurane alcohol of formula (II) to form an isoflurane aldehyde of formula (I). Several enzymes that could be used for this purpose are shown in the accompanying Examples.

[0135] To the surprise of the inventors, they identified several members of the oxadiazole lactone A synthase class of ADH enzymes that were able to oxidize the oxadiazole alcohol of formula (II) to the oxadiazole aldehyde of formula (I). Therefore, a preferred embodiment of the present invention is that the ADH is oxadiazole lactone A synthase. Oxadiazole lactone A synthase is a known ADH enzyme, which has been assigned a classification number of EC1.1.1.295. Preferably, the oxadiazole lactone A synthase used in the method of the present invention has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO: 1, 2, 6, 12, 21 or 33.

[0136] Furthermore, to the inventors' surprise, they identified members of the secoisolariciresinol dehydrogenase class of ADH enzymes capable of oxidizing the fusan alcohol of formula (II) to the fusan aldehyde of formula (I). Therefore, a preferred embodiment of the present invention is wherein the ADH is a secoisolariciresinol dehydrogenase. Secoisolariciresinol dehydrogenases are a known class of ADH enzymes, which have been assigned the classification number EC 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.

[0137] 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 one of SEQ ID NOs: 1 to 33, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 33.

[0138] As shown in the accompanying examples, a polypeptide having ADH activity comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 25 is capable of producing an aldehyde of formula (I) at a concentration of greater than 2 mg / L. Therefore, a preferred embodiment of all aspects of the invention described herein is one 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 one of SEQ ID NOs: 1 to 25, or comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 25.

[0139] As shown in the accompanying examples, a polypeptide having ADH activity comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 13 is capable of producing the anthracene aldehyde of formula (I) at a yield of more than 5 mg / L. Therefore, a preferred embodiment of all aspects of the invention described herein is wherein 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 one of SEQ ID NOs: 1 to 13, or comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 13.

[0140] As shown in the accompanying examples, a polypeptide having ADH activity comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 6 is capable of producing the anthracene aldehyde of formula (I) at a yield of more than 10 mg / L. Therefore, a preferred embodiment of all aspects of the invention described herein is wherein 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 one of SEQ ID NOs: 1 to 6, or comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 6.

[0141] In addition to the ADH polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 33, Table 1 of the accompanying Examples shows that enzymes 34 to 71 can also be used to prepare the anthracene aldehydes of formula (I).

[0142] Thus, one embodiment of the present invention is wherein the method of the present invention uses an ADH polypeptide of any one of enzymes 34 to 71.

[0143] A further aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having ADH activity, and the polypeptide 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 one of SEQ ID NOs: 1 to 33, or comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 33.

[0144] A further aspect of the 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 one of SEQ ID NOs: 34 to 66, or a nucleotide sequence comprising any one of SEQ ID NOs: 34 to 66 or its reverse complement.

[0145] Another aspect of the present invention provides a nucleic acid molecule encoding the polypeptide provided herein.

[0146] In one embodiment, the present invention provides a vector comprising a nucleic acid molecule described herein. In another embodiment, the vector is an expression vector. In yet another embodiment, the vector is a prokaryotic vector, a viral vector, or a eukaryotic vector.

[0147] The present invention also provides a non-human host organism or host cell comprising (1) the above-mentioned nucleic acid molecule, or (2) an expression vector comprising the nucleic acid molecule. In one embodiment, the non-human organism or host cell is a prokaryotic cell or a eukaryotic cell. In another embodiment, the host cell is a bacterial cell, a plant cell, a fungal cell, or a yeast cell. In yet another embodiment, the bacterial cell is Escherichia coli and the yeast cell is Saccharomyces cerevisiae.

[0148] Further provided is the use of a polypeptide as described herein for producing a compound of formula (I).

[0149] Further provided are nucleotide sequences obtained by modifying any one of SEQ ID NOs: 34 to 66 or its reverse complement, which encompass any sequence obtained by modifying any one of SEQ ID NOs: 34 to 66 or its reverse complement using any method known in the art, such as by introducing any type of mutation, such as deletion, insertion and / or substitution mutation.

[0150] Nucleic acids comprising sequences obtained by mutation of any one of SEQ ID NOs: 34 to 66 or its reverse complement are included in one embodiment herein, provided that the sequence they comprise has at least the sequence identity defined with any one of SEQ ID NOs: 34 to 66 or its reverse complement, and provided that they encode a polypeptide having ADH activity as defined in any of the above embodiments. The mutation may be any type of mutation of these nucleic acids, for example, a point mutation, a deletion mutation, an insertion mutation, and / or a frameshift mutation of one or more nucleotides of the DNA sequence of any one of SEQ ID NOs: 34 to 66. In one embodiment, the nucleic acids of the embodiments herein may be truncated, provided that they encode a polypeptide as described herein.

[0151] Variant nucleic acids can be prepared to adapt their nucleotide sequence to a particular expression system. For example, bacterial and yeast expression systems are known to express polypeptides more efficiently if the amino acids are encoded by specific codons.

[0152] Due to the degeneracy of the genetic code, more than one codon may encode the same amino acid sequence, and multiple nucleic acid sequences may also encode the same protein or polypeptide, all of which DNA sequences are included in one embodiment of the present invention. Where appropriate, the nucleic acid sequence encoding ADH can be optimized to improve its expression in the host cell. For example, host-specific codons can be used to synthesize the nucleotides in one embodiment of the present invention to improve expression.

[0153] In one embodiment, provided herein is an isolated, recombinant or synthetic nucleic acid sequence of any one of SEQ ID NOs: 34 to 66, encoding a polypeptide having ADH activity comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 33 or a fragment thereof, which catalyzes the production of an alkane aldehyde of formula (I):

[0154] 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.

[0155] According to one embodiment, the nucleic acid of any one of SEQ ID NOs: 34 to 66 is the coding sequence of the ADH gene encoding the ADH obtained as described in the Examples.

[0156] SEQ ID NO:34 to 66 any one of the polynucleotide fragment refers to continuous nucleotides, its length is particularly at least 15bp, at least 30bp, at least 40bp, at least 50bp and / or at least 60bp of the polynucleotide of this paper embodiment. Specifically, the fragment of the polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 continuous nucleotides of the polynucleotide of this paper embodiment. Without limitation, the polynucleotide fragments herein can be used as PCR primers and / or probes, or for antisense gene silencing or RNAi.

[0157] It will be clear to those skilled in the art that genes, including the polynucleotides described herein, can be cloned by methods known in the art based on available nucleotide sequence information (e.g., information in the attached sequence listing). These methods include, for example, designing DNA primers representing sequences flanking the gene, wherein one primer is generated in the forward direction to initiate synthesis of the forward strand; and another primer is generated in the reverse complementary direction to generate the antisense strand. Such experiments are typically performed using a thermostable DNA polymerase (e.g., an enzyme for the polymerase chain reaction). Alternatively, a DNA sequence representing a gene can be chemically synthesized and then introduced into a DNA vector molecule that can be propagated, for example, by compatible bacteria (e.g., E. coli) or yeast cells.

[0158] In related embodiments provided herein, PCR primers and / or probes for detecting nucleic acid sequences encoding acyltransferases are provided. Those skilled in the art will understand how to synthesize degenerate or specific PCR primer pairs to amplify nucleic acid sequences encoding ADH or fragments thereof based on any one of SEQ ID NOs: 34 to 66. A kit for detecting nucleic acid sequences encoding ADH may include primers and / or probes specific for nucleic acid sequences encoding ADH, and protocols for detecting nucleic acid sequences encoding ADH in a sample using the primers and / or probes. Such detection kits can be used to determine whether a plant, organism, or cell has been modified, i.e., whether it has been transformed by a sequence encoding ADH.

[0159] In order to test the function of the variant DNA sequence, according to one embodiment of the present invention, the target sequence is operably linked to a selectable or screenable marker gene, and a transient expression analysis is performed in protoplasts or stably transformed plants to test the expression of the reporter gene. It will be appreciated by those skilled in the art that the DNA sequence capable of driving expression is constructed as a module. Therefore, the expression level of a shorter DNA fragment may be different from the expression level of the longest fragment, and may also be different from each other. Also provided herein are functional equivalents of the nucleic acid sequences encoding the ADH proteins provided herein, i.e., nucleotide sequences that hybridize under stringent conditions with the nucleic acid sequences of any one of SEQ ID NOs: 34 to 66.

[0160] Those skilled in the art will appreciate that in other organisms, the method for identifying homologous sequences, and the method for determining the sequence identity percentage between the homologous sequences. These newly identified DNA molecules can be checked order subsequently, and with the nucleotide sequence of any one compared among SEQ ID NO:34 to 66.

[0161] The percent identity between two peptides or nucleotide sequences is a function of the number of identical amino acids or nucleotide residues in the two sequences after the alignment is generated. Identical residues are defined as identical residues in the two sequences at a given position in the alignment. The percent identity of the sequence used herein is calculated from the best alignment by dividing the number of identical residues between the two sequences by the total number of residues in the shortest sequence and multiplying by 100. The best alignment is the alignment with the highest probability of percent identity. Rooms can be introduced into one or more positions of the alignment in one or two sequences to obtain the best alignment. These rooms are then considered to be non-identical residues for calculating sequence identity percentages. Alignment for determining amino acid or nucleotide sequence identity percentages can be achieved in a variety of ways using computer programs and, for example, publicly available computer programs on the internet. Preferably, the BLAST program (Tatiana et al., FEMS Microbiol Lett., 1999, 174: 247-250, 1999), available from the National Center for Biotechnology Information (NCBI) at http: / / www.ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi, set to default parameters can be used to obtain optimal alignment of protein or nucleic acid sequences and calculate percentages of sequence identity.

[0162] Related embodiments provided herein provide a nucleic acid sequence that is complementary to the nucleic acid sequence of any one of SEQ ID NOs: 34 to 66, such as an inhibitory RNA, or a nucleic acid sequence that hybridizes under stringent conditions to at least a portion of the nucleotide sequence of any one of SEQ ID NOs: 34 to 66. An alternative embodiment of the embodiments herein provides a method for altering gene expression in a host cell. For example, the polynucleotides of the embodiments herein can be enhanced, overexpressed, or induced in a host cell or host organism under certain circumstances (e.g., upon exposure to a specific temperature or culture conditions).

[0163] Alteration of expression of the polynucleotides provided herein can also result in ectopic expression, which is a different expression pattern in the altered and control or wild-type organisms. Alteration of expression occurs due to the interaction of a polypeptide of one embodiment herein with an exogenous or endogenous modulator or due to chemical modification of the polypeptide. The term also refers to an altered expression pattern of a polynucleotide of the embodiments herein, i.e., altered to below the level of detection or completely inhibited activity.

[0164] In one embodiment, also provided herein are isolated, recombinant, or synthetic polynucleotides encoding the polypeptides or variant polypeptides provided herein.

[0165] 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 one of SEQ ID NOs: 1 to 33, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 33.

[0166] 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 one of SEQ ID NOs: 1 to 33, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 33.

[0167] According to one embodiment, the polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 1 to 33.

[0168] In one embodiment, at least one polypeptide having ADH activity used in any embodiment described herein or a polypeptide encoded by a nucleic acid according to any embodiment described herein comprises a variant amino acid sequence of any one of SEQ ID NOs: 1 to 33 obtained by genetic engineering. In one embodiment, the polypeptide comprises an amino acid sequence encoded by a nucleotide sequence obtained by modifying any one of SEQ ID NOs: 34 to 66 or its reverse complement.

[0169] Polypeptides are also meant to include variants and truncated polypeptides, provided they have ADH activity.

[0170] According to another embodiment, at least one polypeptide having ADH activity for use in any embodiment described herein or a polypeptide encoded by a nucleic acid according to any embodiment described herein comprises a variant amino acid sequence of any one of SEQ ID NOs: 1 to 33 obtained by genetic engineering, provided that the variant has ADH activity and has the required percentage identity with any one of SEQ ID NOs: 1 to 33 described herein.

[0171] According to another embodiment, at least one polypeptide having ADH activity used in any embodiment described herein or a polypeptide encoded by a nucleic acid according to any embodiment described herein is a variant of any one of SEQ ID NOs: 1 to 33, which variant may naturally occur in other organisms, provided that it has ADH activity. The polypeptides used herein include polypeptides or peptide fragments comprising the amino acid sequences identified herein, as well as truncated or variant polypeptides, provided that they have ADH activity and have at least a specified percentage identity with the corresponding fragment of any one of SEQ ID NOs: 1 to 33.

[0172] Examples of variant polypeptides are naturally occurring proteins produced by alternative mRNA splicing events or proteolytic cleavage of polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N-terminus or C-terminus when expressed in different types of host cells due to proteolytic removal of one or more terminal amino acids in the polypeptides of the embodiments described herein. Polypeptides encoded by nucleic acids obtained by natural or artificial mutations of the nucleic acids of the embodiments described herein (as described below) are also encompassed in the embodiments described herein.

[0173] Polypeptide variants produced by fusing additional peptide sequences at the amino and carboxyl termini can also be used in the methods of the embodiments herein. Specifically, such fusions can enhance the expression of the polypeptide, aid in the purification of the protein, or improve the enzymatic activity of the polypeptide in a desired environment or expression system. For example, such additional peptide sequences can be signal peptides. Another aspect encompasses methods using variant polypeptides, such as polypeptides obtained by fusion with other oligopeptides or polypeptides and / or polypeptides linked to a signal peptide. Polypeptides produced by fusion with another functional protein can also be advantageously used in the methods of the embodiments herein.

[0174] Variants can also differ from the polypeptides of the embodiments herein by the addition of modifying groups covalently or non-covalently attached 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 understand how to modify an amino acid sequence while retaining its biological activity.

[0175] In addition to the gene sequences shown in the sequences disclosed herein, those skilled in the art will appreciate that DNA sequence polymorphisms may exist within a particular population, which may result in variations in the amino acid sequences of the polypeptides disclosed herein. Such gene polymorphisms may exist in cells from different populations or within the same population due to natural allelic variation. Allelic variants may also include functional equivalents.

[0176] Further embodiments also relate to molecules derived from the specifically disclosed nucleic acid sequence polymorphisms. These natural variations typically result in about 1% to 5% variation in the nucleotide sequence of a gene or the amino acid sequence of a polypeptide disclosed herein. As described above, nucleic acids encoding the polypeptides of the embodiments herein or variants thereof are useful tools for modifying non-human host organisms or cells, as well as for modifying non-human host organisms or cells intended for use in the methods described herein.

[0177] The embodiments provided herein provide amino acid sequences of ADH proteins, including orthologs and paralogs, and methods for identifying and isolating the ADH orthologs and paralogs in other organisms. Specifically, the ADH orthologs and paralogs identified herein are capable of producing compounds of formula (I).

[0178] ADH polypeptides can be obtained by extraction from any organism expressing the polypeptide using standard protein or enzyme extraction techniques. If the host organism is a unicellular organism or cell and the polypeptide of the embodiments herein is released into the culture medium, the polypeptide can be simply collected from the culture medium, for example by centrifugation, optionally followed by a washing step, and resuspended in a suitable buffer. If the organism or cell accumulates the polypeptide intracellularly, the polypeptide can be obtained by disrupting or lysing the cells and optionally further extracting the polypeptide from the cell lysate.

[0179] According to another embodiment, at least one polypeptide having ADH can be used in the methods of the present invention.

[0180] The functionality or activity of ADH proteins, variants or fragments can be determined by a variety of methods. For example, it can be transiently or stably overexpressed in plants, bacteria or yeast cells to detect whether the protein is active, that is, whether the compound of formula (I) is produced. ADH activity can be evaluated by the assay method described in the embodiments herein to indicate its functionality. The variants or derivatives of the ADH polypeptides in the embodiments herein retain the ability to produce compounds of formula (I). The ADH amino acid sequence variants provided herein may have other desired biological functions, such as altered substrate utilization, reaction kinetics, product distribution or other changes.

[0181] Further provided is at least one vector comprising a nucleic acid molecule described herein.

[0182] Also provided herein are vectors selected from the group consisting of prokaryotic vectors, viral vectors, and eukaryotic vectors.

[0183] This article also provides a vector, which is an expression vector.

[0184] In the embodiments herein, the nucleic acid sequence encoding the ADH protein can be inserted into an expression vector and / or contained in a chimeric gene inserted into an expression vector to produce the ADH protein in a host cell or non-human host organism. Vectors for inserting transgenes into the host cell genome are well known in the art and include plasmids, viruses, cosmids, and artificial chromosomes. Binary vectors or co-integration vectors into which chimeric genes are inserted can also be used to transform host cells.

[0185] One embodiment provided herein provides a recombinant expression vector comprising a nucleic acid sequence of an ADH gene, or a chimeric gene comprising a nucleic acid sequence of an ADH gene, and operably linked to a related nucleic acid sequence, such as a promoter sequence. For example, a chimeric gene comprising a nucleic acid sequence of any one of SEQ ID NOs: 34 to 66 or a variant thereof can be operably linked to a promoter sequence suitable for expression in a plant cell, a bacterial cell, or a fungal cell, and the promoter sequence is optionally linked to a 3' non-translated nucleic acid sequence.

[0186] Alternatively, the promoter sequence may already be present in the vector, and the nucleic acid sequence to be transcribed is inserted downstream of the promoter sequence in the vector. The vector may be engineered to have an origin of replication, a multiple cloning site, and a selectable marker.

[0187] In one embodiment, expression vectors comprising the nucleic acids described herein can be used as a means to transform a non-human host organism or host cell suitable for performing the methods of the embodiments herein in vivo.

[0188] The expression vectors provided herein can be used in methods for preparing genetically transformed non-human host organisms and / or host cells, in non-human host organisms and / or host cells carrying nucleic acids of the embodiments herein, and in methods for preparing polypeptides having ADH activity as described herein.

[0189] Recombinant non-human host organisms and host cells, which have been transformed to carry at least one nucleic acid according to the present invention, to heterologously express or overexpress at least one polypeptide according to the present invention, are also very useful tools for practicing the methods of the present invention. Accordingly, such non-human host organisms and host cells are provided herein.

[0190] In one embodiment, a host cell or non-human host organism comprising at least one nucleic acid molecule described herein or comprising at least one vector comprising at least one nucleic acid molecule is provided.

[0191] The nucleic acid according to any of the above embodiments may be used to transform non-human host organisms and cells, and the expressed polypeptide may be any of the polypeptides described above.

[0192] 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.

[0193] 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 host cell is Saccharomyces cerevisiae.

[0194] In one embodiment, the non-human host organism or host cell expresses a polypeptide if the organism or cell is transformed to contain a nucleic acid encoding the polypeptide, the nucleic acid is transcribed into mRNA and the polypeptide is present in the host organism or cell.

[0195] Suitable methods for transforming non-human host organisms or host cells have been previously described and are provided herein.

[0196] In order to implement the embodiments herein in vivo, the host organism or host cell is cultured under conditions that are conducive to the production of the compound of formula (I). If the host is a unicellular organism, the conditions that are conducive to the production of the compound of formula (I) may include adding suitable cofactors to the host culture medium. In addition, culture medium may be selected to maximize the synthesis of the compound of formula (I). Examples of optimal culture conditions will be described in more detail in the Examples.

[0197] The non-human host organism for implementing the methods of the embodiments herein in vivo can be any non-human multicellular or unicellular organism. In one embodiment, the non-human host organism for implementing the methods of the embodiments herein in vivo is a plant, a prokaryotic organism, or a fungus. Any plant, prokaryotic organism, or fungus can be used. In another embodiment, the non-human host organism for implementing the methods of the embodiments herein in vivo is a microorganism. Any microorganism can be used, for example, the microorganism can be a bacterium or a yeast, such as Escherichia coli or Saccharomyces cerevisiae.

[0198] Isolated higher eukaryotic cells can also serve as hosts in place of intact organisms to practice the methods of the embodiments herein in vivo. Suitable eukaryotic cells can be any non-human cell, such as a plant cell or a fungal cell.

[0199] Also provided herein is a method comprising transforming a host cell or a non-human host organism with a nucleic acid encoding a polypeptide having ADH activity, wherein the polypeptide 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 one of SEQ ID NOs: 1 to 33, or comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 33.

[0200] In one embodiment, the methods provided herein comprise culturing a non-human host organism or host cell transformed to express a polypeptide under conditions permitting production of the polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1 to 33.

[0201] The non-human host organism or host cell transformed to express the polypeptide of the present invention may contain additional exogenous polypeptide sequences.

[0202] For example, the methods of the present invention comprise contacting an isanol of formula (II) with a polypeptide having oxidoreductase activity.

[0203] The alkanol of formula (II) may be provided to a host organism or host cell transformed to express a polypeptide of the invention by addition to the culture medium (or other growth substrate known to those skilled in the art).

[0204] Alternatively, another embodiment of the present invention is wherein the host organism or host cell is further transformed with one or more additional polypeptides capable of producing the thiazolidine alcohol of formula (II).

[0205] Therefore, as will be appreciated by those skilled in the art, the method of the present invention for providing the anthranil aldehyde of formula (I) may be a multi-step enzymatic method.

[0206] Examples of polypeptides capable of producing the alkane alcohols of formula (II) are known in the art.

[0207] For example, WO2018220113 (incorporated herein by reference) discloses methods and enzymes for producing zirconia and / or bucatinol (which are starting materials for the methods of the present invention) from an acyclic farnesyl diphosphate precursor molecule (FPP). In particular, the polypeptides encoded by SEQ ID NO: 1 and SEQ ID NO: 29 in WO2018220113 can be used in this reaction.

[0208] Similarly, WO2019229064 (incorporated herein by reference) discloses methods and enzymes for producing pyrrol (the starting material for the methods of the present invention) from an acyclic farnesyl diphosphate precursor molecule (FPP).

[0209] The following examples are for illustration only and are not intended to limit the scope of the claims and embodiments described herein.

[0210] Recombinant production of the polypeptide of the present invention

[0211] 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, wherein a microorganism producing the polypeptide is cultured, expression of the polypeptide is optionally induced by administering at least one inducer of gene expression, and the polypeptide is isolated from the culture. If desired, the polypeptide can also be produced on an industrial scale in this manner.

[0212] The microorganism that produces according to the present invention can be cultivated continuously or discontinuously by batch process or fed-batch process or repeated fed-batch process.The general introduction of known culture method can be in the textbook (Bioprozesstechnik 1.Einführungin die Bioverfahrenstechnik [Bioprocess technology 1.Introduction tobioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) of Chmiel or in the textbook (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheralequipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) of Storhas.

[0213] The culture medium used must be appropriately adapted to the requirements of the respective strain. Descriptions of culture media for various microorganisms are given in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981).

[0214] The culture media which can be used according to the invention generally comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0215] The preferred carbon source is sugar, for example monose, disaccharide or polysaccharide. Good carbon source is for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar also can be added in the substratum by other by-products of complicated compound (for example molasses) or sugar refining. It is also favourable to add the mixture of different carbon sources. Other possible carbon sources are oil and fat, for example soybean oil, sunflower seed oil, peanut oil and coconut oil, fatty acid for example palmitic acid, stearic acid or linoleic acid, alcohol for example glycerol, methanol or ethanol, and organic acid, for example acetic acid or lactic acid.

[0216] The nitrogen source is generally an organic or inorganic nitrogen compound or a material 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, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or as a mixture.

[0217] Inorganic salt compounds that may be present in the culture medium include chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0218] 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.

[0219] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.

[0220] Chelating agents may be added to the culture medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0221] The fermentation medium used according to the present invention generally also includes other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid and pyridoxine. Growth factors and salts are generally derived from the components of complex culture media, such as yeast extract, molasses, corn steep liquor, etc. In addition, suitable precursors can be added to the culture medium. The exact composition of the compound in the culture medium depends to a large extent on the corresponding experiment and is determined respectively for each specific case. Information about culture medium optimization can be found in the textbook "Applied Microbiol.Physiology,APractical Approach" (Ed.PM Rhodes,PF Stanbury,IRL Press (1997) p.53-73, ISBN 019 963577 3). Growth medium can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) etc.

[0222] All components of the culture medium are sterilized by heating (1.5 bar and 121°C for 20 minutes) or by sterile filtration. These components can be sterilized together or separately as needed. All components of the culture medium can be given at the beginning of the culture or added continuously or in batches.

[0223] The culture temperature is usually between 15°C and 45°C, preferably 25°C to 40°C, and can be changed 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 pH value during growth can be controlled by adding alkaline compounds (such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water) or acidic compounds (such as phosphoric acid or sulfuric acid). Defoaming agents such as fatty acid polyethylene glycol esters can be used to control foaming. In order to maintain the stability of the plasmid, suitable selective substances such as antibiotics can be added to the culture medium. In order to maintain aerobic conditions, oxygen or an oxygen-containing gas mixture (such as ambient air) is supplied to the culture. The temperature of the culture is usually in the range of 20°C to 45°C. Continue to cultivate until the maximum amount of the desired product is formed. This goal will usually be achieved within 10 to 160 hours.

[0224] The fermentation broth is then processed further. Depending on requirements, the biomass can be completely or partially removed from the fermentation broth by separation techniques such as centrifugation, filtration, decantation or a combination of these methods, or can remain completely therein.

[0225] If the polypeptide is not secreted in the culture medium, the cells can also be lysed and the product can be obtained from the lysate by known methods for isolating proteins. The cells can alternatively be disrupted by high-frequency ultrasound, high pressure, for example in a high-pressure cell lyser (French press), by osmosis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by a combination of several of the above methods.

[0226] The polypeptide can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), e.g., Q-agarose chromatography, ion exchange chromatography and hydrophobic chromatography, and other conventional techniques, such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, TG, Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, New York, or Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0227] In order to separate the recombinant protein, it may be advantageous to use a carrier system or oligonucleotide that extends the cDNA by the nucleotide sequence of limitation, and therefore encodes a changed polypeptide or fusion protein, which is for example used for easier purification. Suitable modifications of this type are, for example, so-called "tags" serving as anchors, such as modifications of hexa-histidine anchors or epi-positions that can be identified as antibody antigens (e.g., described in Harlow, E. and Lane, D., 1988, Antibodies:A Laboratory Manual. Cold Spring Harbor (NY) Press). These anchors can be used to connect the protein to a solid support, such as a polymer matrix, which can, for example, be used as the filler in a chromatographic column, or can be used in microtiter plates or other carriers.

[0228] At the same time, these anchors can also be used to identify proteins. In order to identify proteins, conventional labels such as fluorescent dyes, enzyme labels (which react with substrates to form detectable reaction products), or radioactive labels can also be used, alone or in combination with anchors to derivatize proteins.

[0229] Immobilization of peptides

[0230] Enzyme or polypeptide according to the present invention can be used in free form or immobilized in the methods described herein.Immobilized enzyme refers to an enzyme fixed on an inert carrier. Suitable carrier materials and enzymes fixed thereon are known from EP-A-1149849, EP-A-1069183 and DE-OS100193773 and from the references cited therein. In this respect, the full disclosures of these files are referred to. Suitable carrier materials include, for example, clay, clay minerals (for example kaolinite, diatomaceous earth, perlite), silicon dioxide, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchange materials, synthetic polymers (for example polystyrene, acrylic resin, phenolic resin, polyurethane) and polyolefins (for example polyethylene and polypropylene). In order to prepare loaded enzymes, carrier materials are usually in the form of finely dispersed particles, preferably porous forms. The particle diameter of the carrier material is usually no more than 5mm, especially no more than 2mm (particle size distribution curve). Similarly, when using dehydrogenase as a full-cell catalyst, free form or immobilized form can be selected. Carrier materials are, for example, calcium alginate and carrageenan. Enzymes and cells can also be cross-linked directly with glutaraldehyde (cross-linking with CLEAs). 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 is given in Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol. 3, Chapter 17, VCH, Weinheim.

[0231] Reaction conditions of the biocatalytic production method of the present invention

[0232] The reaction of the present invention can be carried out under in vivo or in vitro conditions.

[0233] The at least one polypeptide / enzyme present in a single step of the method of the present invention or the multi-step method defined herein can be present in living cells that naturally or recombinantly produce the one or more enzymes, in harvested cells (i.e., under in vivo conditions), in dead cells, in permeabilized cells, in a crude cell extract, in a purified extract, or in a substantially pure or completely pure form (i.e., under in vitro conditions). The at least one enzyme can be present in solution or as an enzyme immobilized on a support or encapsulated. The one or more enzymes can be present in both soluble and / or immobilized form.

[0234] The method according to the present invention can be carried out in common reactors well known to those skilled in the art, and can be carried out in different scale ranges, for example, from laboratory scale (several milliliters to tens of liters of reaction volume) to industrial scale (several liters to thousands of cubic meters of reaction volume). If polypeptide is in the form of a cell encapsulation by inanimate, alternatively permeabilized, in the form of a more or less purified cell extract or in the form of purification, then a chemical reactor can be used. Chemical reactors generally allow the amount of control at least one enzyme, the amount of at least one substrate, pH, temperature and the circulation of the reaction medium. When there is at least one polypeptide / enzyme in living cells, the process will be fermentation. In this case, biocatalytic production will be carried out in a bioreactor (fermenter), wherein the parameters necessary for the living conditions suitable for living cells (for example, culture medium, temperature, ventilation, aerobic or anaerobic or other gases, antibiotics, etc. with nutrition) can be controlled. The person skilled in the art is familiar with chemical reactors or bioreactors, for example using procedures for scaling up chemical or biotechnological processes from the laboratory scale to the industrial scale or for optimizing process parameters, which are also widely described in the literature (for biotechnological processes, see, for example, Crueger und Crueger, Biotechnologie—Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, München, Wien, 1984).

[0235] The cells containing at least one enzyme can be permeabilized by physical or mechanical means such as ultrasound or radiofrequency pulses, high pressure cell lysers (French press) or chemical means such as the presence of a hypotonic medium, a lytic enzyme and a detergent in the culture medium or a combination of these methods. Examples of detergents include digitonin, n-dodecyl maltoside, octyl glucoside, X-100, 20, deoxycholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate), P40 (ethylphenol poly (ethylene glycol ether)), etc.

[0236] Instead of living cells, non-living cell biomass containing the desired biocatalyst can also be used in the bioconversion reaction of the present invention.

[0237] If at least one enzyme is immobilized, it is attached to an inert support as described above.

[0238] The conversion reaction can be carried out batchwise, semi-batchwise or continuously. The reactants (and optionally nutrients) can be provided at the start of the reaction, or can be provided subsequently semi-continuously or continuously.

[0239] Depending on the particular reaction type, the reactions of the present invention can be carried out in aqueous, aqueous-organic or non-aqueous reaction media.

[0240] The aqueous or aqueous-organic medium may contain a suitable buffer to adjust the pH to 5 to 11, for example 6 to 10.

[0241] In aqueous-organic media, organic solvents that are miscible, partially miscible or immiscible with water can be used. Non-limiting examples of suitable organic solvents are listed below. Further examples are monobasic or polybasic, aromatic or aliphatic alcohols, particularly polybasic aliphatic alcohols, such as glycerol.

[0242] The non-aqueous medium may comprise substantially no water, ie, may comprise less than about 1% or 0.5% by weight water.

[0243] 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 hydrocarbons; ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether; or mixtures thereof.

[0244] The concentrations of reactants / substrates can be adjusted to optimize the reaction conditions, depending on the specific circumstances of the enzyme used. For example, the initial substrate concentration can be 0.1 to 0.5 M, such as 10 to 100 mM.

[0245] The reaction temperature can be adjusted according to the optimal reaction conditions, depending on the specific conditions of the enzyme used. For example, the reaction can be carried out at a temperature of 0 to 70°C (e.g., 20 to 50°C or 25 to 40°C). Examples of reaction temperatures include 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.

[0246] The process can be continued until the substrate and product reach equilibrium, but can also be stopped early. Typical process times are 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 only non-limiting examples of suitable process conditions.

[0247] If the host is a transgenic plant, optimal growing conditions can be provided, such as optimal light, water, and nutrient conditions.

[0248] The specific reaction conditions for preparing aniline aldehydes are as follows. The reaction is incubated in an aqueous environment at 20 to 37°C and a pH of 4 to 7. The ADH enzyme can be present as a purified polypeptide or a whole-cell system. The substrate concentration can vary from 0.1 to 100 mM.

[0249] Product separation

[0250] The method of the present invention may further include the step of reclaiming the final product or intermediate product, which is optionally a substantially pure form of a stereoisomer or enantiomer. The term "recovery" includes extracting, gathering in the crops, separating or purifying the compound from a culture medium or reaction medium. The recovery of the compound can be carried out according to any conventional separation or purification method known in the art, including but not limited to: processing with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), processing with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, aluminum oxide, etc.), changing the pH value, solvent extraction (e.g., with conventional solvents, such as alcohol, ethyl acetate, hexane, etc.), processing, distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, freeze-drying, etc.

[0251] The identity and purity of the isolated product can be determined by known techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopy (e.g., IR, UV, NMR), colorimetric methods, TLC, NIRS, enzyme or microbiological assays (see, for example: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 1127-32; and 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 and 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.).

[0252] The 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 also be obtained by biochemical methods, i.e., by contacting the terpene compound with an enzyme such as, but not limited to, an oxidoreductase, a monooxygenase, a dioxygenase, or a transferase. Biochemical conversions can be performed in vitro using isolated enzymes, enzymes from lysed cells, or in vivo using whole cells.

[0253] In one embodiment of the present invention, the method further comprises oxidizing the anthracene aldehyde of formula (I) using chemical or biocatalytic synthesis or a combination of both.

[0254] Fermentation production of alkane aldehydes

[0255] The present invention also relates to a fermentation production method of alkane aldehydes.

[0256] Fermentation used according to the present invention can be carried out, for example, in stirred fermentor tanks, bubble columns and loop reactors. For a comprehensive overview of possible method types, including agitator types and geometric designs, see "Chmiel: Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Band 1". In the method of the present invention, available typical variations are those known to those skilled in the art or, for example, explained in "Chmiel, Hammes and Bailey: Biochemical Engineering", such as batch, fed-batch, repeated fed-batch or continuous fermentation with or without biomass recovery. Depending on the production strain, injection of air, oxygen, carbon dioxide, hydrogen, nitrogen or a suitable gas mixture can be performed to achieve a good yield (YP / S).

[0257] The culture medium to be used must meet the requirements of the specific strain in an appropriate manner. Descriptions of culture media for various microorganisms are given in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981).

[0258] The culture media which can be used according to the invention generally comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0259] The preferred carbon source is sugar, for example monose, disaccharide or polysaccharide. Very good carbon source is for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar also can be added in the substratum by other by-products of complicated compound (for example molasses) or sugar refining. It is also favourable to add the mixture of various carbon sources. Other possible sources of carbon are oil and fat, for example soybean oil, sunflower seed oil, peanut oil and coconut oil, fatty acid for example palmitic acid, stearic acid or linoleic acid, alcohol for example glycerol, methanol or ethanol, and organic acid, for example acetic acid or lactic acid.

[0260] The nitrogen source is generally an organic or inorganic nitrogen compound or a material 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, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or as a mixture.

[0261] Inorganic salt compounds that may be present in the culture medium include chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0262] Inorganic sulfur-containing compounds, such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur compounds, such as mercaptans and sulfhydryls, can be used as sulfur sources.

[0263] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.

[0264] Chelating agents may be added to the culture medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0265] The fermentation medium used according to the present invention may also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, niacin, pantothenic acid and pyridoxine. Growth factors and salts are usually derived from complex components of the culture medium, such as yeast extract, molasses, corn steep liquor, etc. In addition, suitable precursors can be added to the culture medium. The precise composition of the compounds in the culture medium depends largely on the specific experiment and must be determined separately for each specific case. Information about culture medium optimization can be found in the textbook "Applied Microbiol. Physiology, APractical Approach" (1997). Growth medium can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO), etc.

[0266] All components of the culture medium are sterilized by heating (1.5 bar and 121°C for 20 minutes) or by sterile filtration. These components can be sterilized together or separately as needed. All components of the culture medium can be given at the beginning of the culture or added continuously or in batches.

[0267] The culture temperature is usually between 15°C and 45°C, preferably 25°C to 40°C, and can be kept constant or changed during the experiment. The pH value of the culture medium should be in the range of 5 to 8.5, preferably around 7.0. The pH value during growth can be controlled by adding alkaline compounds (such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water) or acidic compounds (such as phosphoric acid or sulfuric acid). Defoaming agents such as fatty acid polyethylene glycol esters can be used to control foaming. In order to maintain the stability of the plasmid, suitable selective substances such as antibiotics can be added to the culture medium. In order to maintain aerobic conditions, oxygen or an oxygen-containing gas mixture (such as ambient air) is supplied to the culture. The temperature of the culture is usually in the range of 20°C to 45°C. Continue to cultivate until the maximum amount of the desired product is formed. This goal will usually be achieved within 1 to 160 hours.

[0268] The method of the present invention may further comprise a step of recovering the anthranil aldehyde.

[0269] The term "recovery" includes extracting, harvesting, isolating or purifying the compound from the culture medium. The recovery of the compound can be carried out according to any conventional separation or purification method known in the art, including but not limited to: treatment with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, aluminum oxide, etc.), changing the pH value, solvent extraction (e.g., with conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0270] Before the separation of expectation, the biomass of fermented liquid can be removed.The method of removing biomass is well known to those skilled in the art, for example, filtration, sedimentation and flotation.Therefore, can for example, remove biomass by centrifuge, separator, decanter, filter or in flotation equipment.In order to reclaim valuable product to the greatest extent, it is usually recommended to wash biomass, for example, in the form of diafiltration.The selection of method depends on the content of biomass and the character of biomass in the fermented liquid, and the interaction of biomass and valuable product.

[0271] In one embodiment, the fermentation broth can be sterilized or pasteurized. In another embodiment, the fermentation broth is concentrated. This concentration can be performed batchwise or continuously, as desired. The pressure and temperature ranges should be selected to prevent product damage and minimize equipment and energy use. Skilled selection of pressure and temperature levels for multi-stage evaporation can particularly save energy.

[0272] A further aspect of the present 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). Examples of such host cells are given in the Examples section of this application.

[0273] Further transformation of paraffin aldehydes to other compounds

[0274] The process of the present invention relates to the preparation of paraffin aldehydes of formula (I).

[0275] In a preferred embodiment of the present invention, the salbutamol aldehydes are converted to salbutamol acids using chemical or biocatalytic synthesis or a combination of both.

[0276] An embodiment of the method of the present invention is wherein the albicanic acid is albicanic acid.

[0277] Subsequently, hyssoic acid can be used as a starting material for the synthesis of perfumery materials, including polywood. The structure of polywood and its synthesis from paraffin aldehydes of formula (I) are known and can be carried out without inventive step by a person skilled in the art.

[0278] The following examples are for illustration only and are not intended to limit the scope of the claims and embodiments described herein.

[0279] Numerous possible variations that will become immediately apparent to those skilled in the art after considering the disclosure provided herein also fall within the scope of the invention.

[0280] Example

[0281] Example 1: Oxidoreductase-catalyzed oxidation of zirconia to zirconia aldehyde

[0282] Alcohol dehydrogenases are nicotinamide adenine dinucleotide (NAD+)- or (NADP+)-dependent oxidoreductases. As one of the most abundant enzymes, they catalyze the reversible reduction of aldehydes and ketones to their corresponding alcohols. The equilibrium of this reversible reaction is strongly dependent on ADH, but this equilibrium shifts when a substrate is enzymatically modified, making it no longer accessible to ADH. To date, no ADH is known to accept zizyphodol as a substrate.

[0283] To identify ADHs capable of oxidizing zizol, 269 ADHs were selected from public libraries and transcriptomes and tested for their substrate promiscuity in accepting zizol and catalyzing its conversion to zizaldehyde.

[0284] 269 ​​alcohol dehydrogenase candidates were screened in two batches. The first batch contained 122 alcohol dehydrogenase candidates, derived from the transcriptomes of organisms such as Bazzania trilobata, Bazzania sp., Laricifomes officinalis, Antrodia cinnamomea and Porella navicularis. This batch was screened in Saccharomyces cerevisiae. The screening process was carried out as described in WO2020078871A1. Under the screening conditions used, it was not possible to detect any of the above 122 alcohol dehydrogenase candidates that could convert fennel ethanol to fennel aldehyde.

[0285] The second batch of alcohol dehydrogenase candidates contained 149 ADH candidates derived from publications, the NCBI protein database, the plant genome database (DOE Joint Genome Institute), and the transcriptome from the genus Trichoderma (WO2021 / 105236) and were screened in Escherichia coli. Therefore, a strain of Escherichia coli producing the sesquiterpenoid cyclopentyl isothiocyanate was constructed. To this end, the Escherichia coli strain DP1205 (recently described in WO2021005097) overexpressing farnesyl pyrophosphate (FPP) was used as the base strain. DP1205 was transformed with the expression plasmid pJ424 (ATUM, Newark, California), which contains a codon-optimized version of the cyclopentyl isothiocyanate synthase LoTps1 gene of Escherichia coli, as described in WO2018220113A1, to produce the E. coli strain DP1205 pJ424 (LoTps1).

[0286] Different alcohol dehydrogenase candidates were ordered from TWIST Bioscience (San Francisco, California) and cloned into the expression vector pET29a as codon-optimized genes in E. coli and added to DP1205 pJ424 (LoTps1). Transformed cells were screened in LB medium supplemented with appropriate antibiotics. Each single transformed colony was first cultured overnight in a deep-well plate at 37°C in 0.5 mL of LB medium supplemented with 1% glucose and the appropriate antibiotics. The next day, 0.5 mL of the culture medium 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)) (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. The culture plate was incubated at 25°C for 72 hours. To obtain fennel and fennel produced by E. coli cells, each well of the deep-well plate was extracted with 700 μL of ethyl acetate containing an internal standard. fennel and fennel were identified by GC-MS analysis and quantified by GC-FID using the above-mentioned internal standard.

[0287] The amount of calciferol produced under these conditions is shown in Table 1. Of the 149 ADHs tested, six candidate enzymes—BAG99023.1, AJP06249.1, XP_002446247.1, XP_008669542.1, BAV31336.1, and XP_004494228.1—produced calciferol production exceeding 10 mg / L. Under these conditions, oxadiazolidine synthases XP_002446247.1 and XP_008669542.1 produced the highest calciferol titers of 58 mg / L and 41 mg / L, respectively. Figure 1 The GC-MS chromatogram of the functional ADH produced by E. coli is shown. Figure 1 The MS spectrum of E. coli-derived zirconia aldehyde was similar to that of the reference zirconia aldehyde.

[0288] A total of 71 ADHs were found to produce calciferol. Of these, 33 produced yields exceeding 1 mg / L, and six produced yields exceeding 10 mg / L. This is the first demonstration that an ADH enzyme can accept calciferol as a substrate and produce large quantities of calciferol suitable for further commercial development.

[0289] The amino acid sequences of enzymes 1 to 33 are disclosed in SEQ ID NOs: 1 to 33.

[0290] For enzymes 34 to 71 that also produce calciferol, Table 2 provides the accession numbers of the amino acid sequences.

[0291] Table 1:

[0292] Enzyme number Fennel aldehyde (mg / L) Enzyme number Fennel aldehyde (mg / L) Enzyme number Fennel aldehyde (mg / L) 1 58.11 25 2.00 49 0.43 2 41.17 26 1.94 50 0.42 3 24.49 27 1.68 51 0.42 4 21.11 28 1.62 52 0.39 5 18.77 29 1.59 53 0.38 6 11.31 30 1.51 54 0.37 7 9.55 31 1.43 55 0.35 8 6.85 32 1.29 56 0.33 9 6.23 33 1.22 57 0.33 10 5.84 34 1.00 58 0.30 11 5.82 35 0.97 59 0.29 12 5.31 36 0.73 60 0.28 13 5.01 37 0.68 61 0.28 14 4.86 38 0.68 62 0.28 15 4.37 39 0.66 63 0.28 16 4.16 40 0.63 64 0.28 17 3.68 41 0.62 65 0.27 18 3.55 42 0.58 66 0.26 19 3.44 43 0.58 67 0.25 20 3.44 44 0.57 68 0.25 21 2.36 45 0.49 69 0.19 22 2.30 46 0.47 70 0.18 23 2.17 47 0.47 71 0.17 24 2.04 48 0.43

[0293] Table 2:

[0294] Enzyme number Accession number / enzyme name Enzyme number Accession number / enzyme name Enzyme number Accession number / enzyme name 34 OCH84571.1 49 PNX90329.1 64 AAEL017320-PA 35 AKS44056.1 50 QCD88014.1 65 OIT37090.1 36 EXC01952.1 51 XP_013613370.1 66 ABK24336.1 37 BAG96093.1 52 XP_002533854.1 67 QHO42451.1 38 MG677124.1 53 XP_006395524.1 68 ABR18410.1 39 PHU20197.1 54 P10807.1 69 XP_012074416.2 40 TbDH 55 AORIB40_NS.05917 70 TcADH2 41 KOM39082.1 56 XP_033420033.1 71 XP_010917565.1 42 XP_002967778.2 57 XP_008462605.1 43 BBM89983.1 58 BAG97977.1 44 SsADH 59 XP_002460956.1 45 CmADH 60 AAEL001461-PA 46 NP_001239862.1 61 XP_006598999.1 47 KAF3773316.1 62 LqCy 48 KHN02191.1 63 XP_012435076.1 . Sequence Listing:

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

Claims

1. A method for preparing an alkane aldehyde of formula (I): The aldehyde is in the form of any one of its stereoisomers or a mixture thereof, Where n is 0, and one dotted line is a carbon-carbon double bond, and the other dotted lines are carbon-carbon single bonds; or n is 1, and all dotted lines are carbon-carbon single bonds, The method comprises contacting an alkanol of formula (II) with a polypeptide having oxidoreductase activity: The alcohol is in the form of any stereoisomer or mixture thereof, wherein n is 0 and one dashed line is a carbon-carbon double bond and the other dashed lines are carbon-carbon single bonds; or n is 1 and all dashed lines are carbon-carbon single bonds; and Optionally, the hydroxyaniline aldehyde is isolated from the reaction.

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

3. The method according to claim 1 or 2, wherein the ADH enzyme is oxadiazolone A synthase or secoisolariciresinol dehydrogenase.

4. 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 to 33.

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 one of SEQ ID NOs: 1 to 6.

6. A method according to any one of the preceding claims, wherein The step of contacting the alkanol with the polypeptide having ADH activity is carried out in the presence of a cofactor; preferably, the cofactor is NAD + or NAD(P) + .

7. The method according to any one of the preceding claims, wherein the benzophenone aldehyde is selected from the group consisting of benzophenone, cyclopentane aldehyde, β-bicyclofarnesaldehyde or 8-hydroxy-11-benzophenone, each in stereoisomerically pure form or a mixture of at least two stereoisomers thereof, or a combination comprising at least two members of said group.

8. The method according to any one of the preceding claims, wherein the buburan alcohol is selected from the group consisting of buburanol, zephyrol, β-bicyclofarnesol, buburan-8α,11-diol, each in stereoisomerically pure form or a mixture of at least two stereoisomers thereof, or a combination comprising at least two members of said group.

9. A method according to any one of the preceding claims, wherein the method is carried out in vivo in a cell culture environment or in vitro in a liquid reaction medium under conditions favourable for the production of the analkanoid aldehyde.

10. The method according to claim 9, wherein the method is performed in a recombinant host cell or a recombinant non-human host organism capable of functionally expressing (I) at least one polypeptide having oxidoreductase activity, and optionally (II) at least one polypeptide having the ability to convert an acyclic sesquiterpene precursor FPP into at least one fumaranol of formula (II).

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

12. The method according to any one of the preceding claims, wherein the method further comprises oxidizing the anthranil aldehyde of formula (I) using chemical 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 NO: 1 to 33.

14. Use of a polypeptide having ADH activity in the preparation of a compound of formula (I).

15. A compound of formula (I) obtained or obtainable by a process as claimed in any one of the preceding claims.

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. Use of a compound of formula (I) as defined in any one of the preceding claims for the preparation of an odorant, flavor or fragrance ingredient; or for insect / pest control.

Images