Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01295—Momilactone-A synthase (1.1.1.295)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01331—Secoisolariciresinol dehydrogenase (1.1.1.331)

Definitions

• the present invention provides novel processes for the alcohol dehydrogenase- catalyzed production of drimane aldehydes by the oxidation of the respective drimane alcohol precursors performed in vitro or in vivo.
• the present invention also relates to the identification of enzymes having corresponding alcohol dehydrogenase activity from different microbial and plant sources.
• a further aspect of the present invention relates to the provision of corresponding coding sequences of such enzymes, recombinant vectors, and recombinant host cells suitable for the production of such alcohol dehydrogenase.
• Another aspect of the invention relates to the use of such drimane aldehydes, as obtained according to the present invention, as intermediates for the production of odorant, flavor or fragrance or insect/pest control ingredients.
• Terpenes are found in most organisms (microorganisms, animals and plants). These compounds are made up of five carbon units called isoprene units and are classified by the number of these units present in their structure, which may comprise cyclic structural elements. Thus monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms, respectively. Sesquiterpenes, for example, are widely found in the plant kingdom. Many sesquiterpene molecules are known for their flavor and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Numerous sesquiterpene hydrocarbons and sesquiterpenoids have been identified. Chemical synthesis approaches have been developed but are still complex and not always cost-effective.
• terpene synthases There are numerous sesquiterpene synthases present in the plant kingdom, all using the same substrate (farnesyl diphosphate, FPP), but having different product profiles. Genes and cDNAs encoding sesquiterpene synthases have been cloned and the corresponding recombinant enzymes characterized.
• drimane aldehydes are plants or microorganisms naturally containing the sesquiterpene; however, the content of drimane aldehydes sesquiterpenes in these natural sources can be low. There still remains a need for the provision of novel processes of producing drimane aldehydes.
• the present invention provides novel processes of producing drimane aldehydes in particular methods which may be implemented into the fully biochemical synthesis of drimane aldehydes in an aqueous environment, as for example in a host cell-based process.
• a first aspect of the invention provides a process for the preparation of a drimane aldehyde of formula (I) in the form of any one of its stereoisomers or a mixture thereof, and wherein n is 0 and one dotted line is a carbon-carbon double bond and the others are a carbon-carbon single bond; or n is 1 and all dotted lines are a carbon-carbon single bond, comprising contacting a drimane alcohol of formula (II) in the form of any one of its stereoisomers or a mixture thereof, and wherein n is 0 and one dotted line is a carbon-carbon double bond and the others are a carbon-carbon single bond; or n is 1 and all dotted lines are a carbon-carbon single bond, with a polypeptide having oxidoreductase enzyme activity; and optionally isolating a drimane aldehyde from the reaction.
• An embodiment of the invention is wherein the oxidoreductase enzyme is alcohol dehydrogenase (ADH) enzyme.
• ADH alcohol dehydrogenase
• An embodiment of the 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 of SEQ ID NOs: 1 to 33.
• an embodiment of the invention is wherein the ADH enzyme is a momilactone A synthase.
• the momilactone A synthase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NO: 1 , 2, 6, 12, 21 or 33.
• an embodiment of the invention is wherein the ADH enzyme is a secoisolariciresinol dehydrogenase.
• the secoisolariciresinol dehydrogenase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NO: 3, 7, 8, 10, 16 or 17.
• the ADH enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 6.
• a further aspect of the invention provides an isolated polypeptide having ADH activity comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 33 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 33.
• polypeptides having ADH activity comprising an amino acid sequence of any of SEQ ID NOs: 1 to 6 are capable of producing a drimane aldehyde of formula (I) at more than 10 mg/L.
• the isolated polypeptide having ADH activity comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 6 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 6.
• An embodiment of the invention is wherein the step of contacting a drimane alcohol with a polypeptide having ADH activity is performed in the presence of a co-factor; preferably the co-factor is NAD + or NAD(P) + .
• the co-factor optionally regenerated using a cofactor regeneration system.
• a co-factor regenerating system can push the equilibrium of the process of the invention towards the generation of the desired product, for example drimane aldehyde. In this way the process of the invention can be more optimized and efficient in terms of reagents used therefore more timely and cost effective than without the use of a co-factor regenerating system.
• an embodiment of the invention is wherein the process of the invention is performed in the presence of a co-factor regeneration system.
• the drimane aldehyde is selected of a group consisting of: drimenal, albicanal, beta-bicyclofarnesal, or 8-hydroxy-11-drimanal, each in stereoisomerically pure form or as a mixture of at least two stereoisomers thereof, or combinations thereof comprising at least two members of said group.
• drimane alcohol is selected from the group consisting of drimenol, albicanol, beta-bicyclofarnesol, drimane-8a,11-diol, each in stereoisomerically pure form or as a mixture of at least two stereoisomers thereof, or combinations thereof comprising at least two members of said group.
• An embodiment of the invention is wherein the process is performed in vivo in cell culture or in vitro in a liquid reaction medium, under conditions conducive to the production of drimane aldehyde.
• An embodiment of the invention is wherein said process 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 enzyme activity, and optionally (ii) at least one polypeptide having the ability to convert the non-cyclic sesquiterpene precursor FPP to at least one drimane alcohol of formula (II).
• An embodiment of the invention is wherein said non-human host cell or host organism is selected from a prokaryotic or eukaryotic microorganism, or a cell derived therefrom; in particular, wherein said non-human host cell or host organism is selected from bacterial, fungal and plant cells or plants.
• An embodiment of the invention is wherein the process further comprises oxidizing the drimane aldehyde of formula (I) using chemical or biocatalytic synthesis or a combination of both.
• a further aspect of the invention provides a polypeptide having ADH activity has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of S EQ ID NOs: 1 to 33.
• a further aspect of the invention provides the use of a polypeptide having ADH activity for the preparation of a compound of formula (I).
• a further aspect of the invention provides a compound of formula (I) obtained or obtainable by the process of any of the previous claims.
• a further aspect of the invention provides a recombinant host cell or a recombinant non-human host organism comprising a compound of formula (I) and/or a compound of formula (II).
• a further aspect of the invention provides the of a compound of formula (I) as defined in anyone of the preceding claims, for preparing odorants, flavour or fragrance ingredients; or as insect/pest control.
• Figure 1 GC-MS analysis of albicanal produced in E. coli DP1205 when coexpressing the albicanol synthase LoTpsI with either the SEQ ID NO: 1 (A) or the alcohol dehydrogenase SEQ ID NO: 2 (B).
• the MS spectra of the produced albicanal by the alcohol dehydrogenase SEQ ID NO: 2 from (B) is shown in (C) and it is similar to the MS spectra of reference albicanal (D).
• Figure 2 Structures of drimenal, albicanal, beta-bicyclofarnesal, and 8-hydroxy-11- drimanal.
• RNA ribonucleic acid mRNA messenger ribonucleic acid miRNA micro RNA siRNA small interfering RNA rRNA ribosomal RNA tRNA transfer RNA
• radical naming conventions can include either a mono-radical or a di-radical, depending on the context.
• a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a di-radical.
• a substituent identified as alkyl that requires two points of attachment includes di-radicals such as -CH2-, - CH2CH2-, -CH2CH(CH3)CH2-, and the like.
• a substituent is depicted as a di-radical (i.e. , has two points of attachment to the rest of the molecule), it is to be understood that the substituent can be attached in any directional configuration unless otherwise indicated.
• a substituent depicted as -AE- or " A ⁇ E includes the substituent being oriented such that the A is attached at the leftmost attachment point of the molecule as well as the case in which A is attached at the rightmost attachment point of the molecule.
• the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
• reference to “a substituent” encompasses a single substituent as well as two or more substituents, and the like.
• “comprise” or “comprises” or “comprising” or “comprised of” refer to groups that are open, meaning that the group can include additional members in addition to those expressly recited.
• the phrase, “comprises A” means that A must be present, but that other members can be present too.
• the terms “include,” “have,” and “composed of” and their grammatical variants have the same meaning.
• “consist of” or “consists of” or “consisting of” refer to groups that are closed.
• the phrase “consists of A” means that A and only A is present.
• optional event means that the subsequently described event(s) 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.
• a or B is to be given its broadest reasonable interpretation, and is not to be limited to an either/or construction.
• the phrase “comprising A or B” means that A can be present and not B, or that B is present and not A, or that A and B are both present.
• A for example, defines a class that can have multiple members, e.g., Ai and A2, then one or more members of the class can be present concurrently.
• substituents or linking groups having only a single atom may be referred to by the name of the atom.
• substituent “-H” may be referred to as “hydrogen” or “a hydrogen atom”
• substituent “-F” may be referred to as “fluorine” or “a fluorine atom”
• linking group “-O-” may be referred to as “oxygen” or “an oxygen atom”.
• Points of attachment for groups are generally indicated by a terminal dash (-) or by an asterisk (*).
• a group such as *-CH2-CHs or -CH2-CH3 both represent an ethyl group.
• Chemical structures are often shown using the “skeletal” format, such that carbon atoms are not explicitly shown, and hydrogen atoms attached to carbon atoms are omitted entirely.
• the structure represents butane (i.e., n- butane).
• aromatic groups such as benzene, are represented by showing one of the contributing resonance structures.
• the structure represents toluene.
• polypeptide means an amino acid sequence of consecutively polymerized amino acid residues, for instance, at least 15 residues, at least 30 residues, at least 50 residues.
• a polypeptide comprises an amino acid sequence that is an enzyme, or a fragment, or a variant thereof.
• protein refers to an amino acid sequence of any length wherein amino acids are linked by covalent peptide bonds, and includes oligopeptide, peptide, polypeptide and full length protein whether naturally occurring or synthetic.
• isolated polypeptide refers to an amino acid sequence that is removed from its natural environment by any method or combination of methods known in the art and includes recombinant, biochemical and synthetic methods.
• biological function refers to the ability of the drimenol synthase to catalyze the formation of drimenol or a mixture of compounds comprising drimenol and one or more terpenes.
• nucleic acid sequence refers to the sequence of nucleotides.
• a nucleic acid sequence may be a single-stranded or double-stranded deoxyribonucleotide, or ribonucleotide of any length, and include coding and noncoding sequences of a gene, exons, introns, sense and anti-sense complimentary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and/or RNA sequences, synthetic DNA and RNA sequences, fragments, primers and nucleic acid probes.
• nucleic acid sequences of RNA are identical to the DNA sequences with the difference of thymine (T) being replaced by uracil (U).
• nucleotide sequence should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid.
• isolated nucleic acid or “isolated nucleic acid sequence” relates to a nucleic acid or nucleic acid sequence that is in an environment different from that in which the nucleic acid or nucleic acid sequence naturally occurs and can include those that are substantially free from contaminating endogenous material.
• naturally- occurring as used herein as applied to a nucleic acid refers to a nucleic acid that is found in a cell of an organism in nature and which has not been intentionally modified by a human in the laboratory.
• Recombinant nucleic acid sequences are nucleic acid sequences that result from the use of laboratory methods (for example, molecular cloning) to bring together genetic material from more than on source, creating or modifying a nucleic acid sequence that does not occur naturally and would not be otherwise found in biological organisms.
• “Recombinant DNA technology” refers to molecular biology procedures to prepare a recombinant nucleic acid sequence as described, for instance, in Laboratory Manuals edited by Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook etal., 1989, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.
• the term “gene” means a DNA sequence comprising a region, which is transcribed into a RNA molecule, e.g., an mRNA in a cell, operably linked to suitable regulatory regions, e.g., a promoter.
• a gene may thus comprise several operably linked sequences, such as a promoter, a 5’ leader sequence comprising, e.g., sequences involved in translation initiation, a coding region of cDNA or genomic DNA, introns, exons, and/or a 3’non-translated sequence comprising, e.g., transcription termination sites.
• a promoter e.g., a promoter, a 5’ leader sequence comprising, e.g., sequences involved in translation initiation, a coding region of cDNA or genomic DNA, introns, exons, and/or a 3’non-translated sequence comprising, e.g., transcription termination sites.
• a “chimeric gene” refers to any gene which is not normally found in nature in a species, in particular, a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature.
• the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region.
• the term “chimeric gene” is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or to an antisense, i.e., reverse complement of the sense strand, or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription).
• the term “chimeric gene” also includes genes obtained through the combination of portions of one or more coding sequences to produce a new gene.
• a “3’ UTR” or “3’ non-translated sequence” refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal such as AAUAAA or variants thereof. After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the site of translation, e.g., cytoplasm.
• “Expression of a gene” encompasses “heterologous expression” and “overexpression” and involves transcription of the gene and translation of the mRNA into a protein. Overexpression refers to the production of the gene product as measured by levels of mRNA, polypeptide and/or enzyme activity in transgenic cells or organisms that exceeds levels of production in non-transformed cells or organisms of a similar genetic background.
• “Expression vector” as used herein means a nucleic acid molecule engineered using molecular biology methods and recombinant DNA technology for delivery of foreign or exogenous DNA into a host cell.
• the expression vector typically includes sequences required for proper transcription of the nucleotide sequence.
• the coding region usually codes for a protein of interest but may also code for an RNA, e.g., an antisense RNA, siRNA and the like.
• an “expression vector” as used herein includes any linear or circular recombinant vector including but not limited to viral vectors, bacteriophages and plasmids. The skilled person is capable of selecting a suitable vector according to the expression system.
• the expression vector includes the nucleic acid of an embodiment herein operably linked to at least one regulatory sequence, which controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosomal binding site and, optionally, including at least one selection marker.
• Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the nucleic acid of an embodiment herein.
• regulatory sequence refers to a nucleic acid sequence that determines expression level of the nucleic acid sequences of an embodiment herein and is capable of regulating the rate of transcription of the nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences comprise promoters, enhancers, transcription factors, promoter elements and the like.
• Promoter refers to a nucleic acid sequence that controls the expression of a coding sequence by providing a binding site for RNA polymerase and other factors required for proper transcription including without limitation transcription factor binding sites, repressor and activator protein binding sites.
• the meaning of the term promoter also includes the term “promoter regulatory sequence”.
• Promoter regulatory sequences may include upstream and downstream elements that may influences transcription, RNA processing or stability of the associated coding nucleic acid sequence. Promoters include naturally-derived and synthetic sequences.
• the coding nucleic acid sequences is usually located downstream of the promoter with respect to the direction of the transcription starting at the transcription initiation site.
• constitutitutive promoter refers to an unregulated promoter that allows for continual transcription of the nucleic acid sequence it is operably linked to.
• operably linked refers to a linkage of polynucleotide elements in a functional relationship.
• a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence.
• Operably linked means that the DNA sequences being linked are typically contiguous.
• the nucleotide sequence associated with the promoter sequence may be of homologous or heterologous origin with respect to the plant to be transformed. The sequence also may be entirely or partially synthetic.
• the nucleic acid sequence associated with the promoter sequence will be expressed or silenced in accordance with promoter properties to which it is linked after binding to the polypeptide of an embodiment herein.
• the associated nucleic acid may code for a protein that is desired to be expressed or suppressed throughout the organism at all times or, alternatively, at a specific time or in specific tissues, cells, or cell compartment.
• Such nucleotide sequences particularly encode proteins conferring desirable phenotypic traits to the host cells or organism altered or transformed therewith. More particularly, the associated nucleotide sequence leads to the production of drimenol or a mixture comprising drimenol and one or more terpenes in the cell or organism. Particularly, the nucleotide sequence encodes a polypeptide having drimenol synthase activity.
• Target peptide refers to an amino acid sequence which targets a protein, or polypeptide to intracellular organelles, i.e., mitochondria, or plastids, or to the extracellular space (secretion signal peptide).
• a nucleic acid sequence encoding a target peptide may be fused to the nucleic acid sequence encoding the amino terminal end, e.g., N-terminal end, of the protein or polypeptide, or may be used to replace a native targeting polypeptide.
• the term “primer” refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and is used for polymerization of a nucleic acid sequence complementary to the template.
• the term “host cell” or “transformed cell” refers to a cell (or organism) altered to harbor at least one nucleic acid molecule, for instance, a recombinant gene encoding a desired protein or nucleic acid sequence which upon transcription yields a drimenol synthase protein useful to produce drimenol ora mixture comprising drimenol and one or more terpenes.
• the host cell is particularly a bacterial cell, a fungal cell or a plant cell.
• the host cell may contain a recombinant gene which has been integrated into the nuclear or organelle genomes of the host cell. Alternatively, the host may contain the recombinant gene extra-chromosomally.
• Homologous sequences include orthologous or paralogous sequences. Methods of identifying orthologs or paralogs including phylogenetic methods, sequence similarity and hybridization methods are known in the art and are described herein.
• Paralogs result from gene duplication that gives rise to two or more genes with similar sequences and similar functions. Paralogs typically cluster together and are formed by duplications of genes within related plant species. Paralogs are found in groups of similar genes using pair-wise Blast analysis or during phylogenetic analysis of gene families using programs such as CLUSTAL. In paralogs, consensus sequences can be identified characteristic to sequences within related genes and having similar functions of the genes.
• Orthologs are sequences similar to each other because they are found in species that descended from a common ancestor. For instance, plant species that have common ancestors are known to contain many enzymes that have similar sequences and functions. The skilled artisan can identify orthologous sequences and predict the functions of the orthologs, for example, by constructing a polygenic tree for a gene family of one species using CLUSTAL or BLAST programs. A method for identifying or confirming similar functions among homologous sequences is by comparing of the transcript profiles in host cells or organisms, such as plants, overexpressing or lacking (in knockouts/knockdowns) related polypeptides.
• genes having similar transcript profiles with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or greater than 90% regulated transcripts in common will have similar functions.
• Homologs, paralogs, orthologs and any other variants of the sequences herein are expected to function in a similar manner by making the host cells, organism such as plants producing drimenol synthase proteins.
• selectable marker refers to any gene which upon expression may be used to select a cell or cells that include the selectable marker. Examples of selectable markers are described below. The skilled artisan will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.
• organism refers to any non-human multicellular or unicellular organisms such as a plant, or a microorganism. Particularly, a micro-organism is a bacterium, a yeast, an algae or a fungus.
• plant is used interchangeably to include plant cells including plant protoplasts, plant tissues, plant cell tissue cultures giving rise to regenerated plants, or parts of plants, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits and the like. Any plant can be used to carry out the methods of an embodiment herein.
• the present invention provides a process for the preparation of a drimane aldehyde of formula (I) in the form of any one of its stereoisomers or a mixture thereof, and wherein n is 0 and one dotted line is a carbon-carbon double bond and the others are a carbon-carbon single bond; or n is 1 and all dotted lines are a carbon-carbon single bond.
• the drimane aldehyde is selected of a group consisting of: drimenal, albicanal, beta-bicyclofarnesal, or 8-hydroxy-11-drimanal, each in stereoisomerically pure form or as a mixture of at least two stereoisomers thereof, or combinations thereof comprising at least two members of said group.
• the present invention provides a process for the preparation of a drimane aldehyde of formula (I) comprising contacting a drimane alcohol of formula (II) in the form of any one of its stereoisomers or a mixture thereof and wherein n is 0 and one dotted line is a carbon-carbon double bond and the others are a carboncarbon single bond; or n is 1 and all dotted lines are a carbon-carbon single bond, with a polypeptide having oxidoreductase enzyme activity.
• the drimane alcohol is selected from the group consisting of drimenol, albicanol, beta-bicyclofarnesol, drimane-8a,11-diol, each in stereoisomerically pure form or as a mixture of at least two stereoisomers thereof, or combinations thereof comprising at least two members of said group.
• the drimane alcohol is albicanol.
• the drimane aldehyde is selected from the group consisting of: drimenal, albicanal, beta-bicyclofarnesal, or 8-hydroxy-11-drimanal, each in stereoisomerically pure form or as a mixture of at least two stereoisomers thereof, or combinations thereof comprising at least two members of said group.
• the drimane aldehyde is albicanal.
• a preferred embodiment of the invention is wherein the drimane alcohol is albicanol and the drimane aldehyde is albicanal.
• said compounds of formula (I) and (II) may be in a form of any one of its stereoisomers or a mixture thereof.
• any one of its stereoisomers or a mixture thereof or the similar, it is meant the normal meaning understood by a person skilled in the art, i.e. that the compounds of formula (I) and (II) can be a pure enantiomer or diastereomer.
• the compounds of formula (I) and (II) possess several stereocenters and each of said stereocenter can have two different stereochemistries (e.g. R or S).
• the compounds of formula (I) and (II) may even be in the form of a pure enantiomer or in the form of a mixture of enantiomers or diastereoisomers.
• the compounds of formula (I) and (II) can be in a racemic form or scalemic form. Therefore, the compounds of formula (I) and (II) can be one stereoisomer or in the form of a composition of matter comprising, or consisting of, various stereoisomers.
• Isotopes may be present in the compounds described. Each chemical element as represented in a compound structure may include any isotope of said element.
• a hydrogen atom may be explicitly disclosed or understood to be present in the compound.
• the hydrogen atom can be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium).
• reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise.
• the process of the invention has a drimane alcohol as a starting material.
• the drimane alcohol compound may endogenously be present in the reaction mixture, for example in an in vivo process applying a host cell system producing said drimane alcohol compound as metabolite and expressing the required polypeptide or polypeptides for performing the intended drimane alcohol synthesis or a more complex, multistep process encompassing said drimane alcohol synthesis as one step whereby said drimane alcohol is enzymatically synthesized from a non-cyclic sesquiterpene precursor.
• said drimane alcohol compound is either chemically or enzymatically produced, and is exogenously added to the reaction mixture, for example in an in vitro process applying an isolated, enriched or purified synthase enzyme required for its formation as defined below.
• the process 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 enzyme activity, and optionally (ii) at least one polypeptide having the ability to convert the non-cyclic sesquiterpene precursor FPP to at least one drimane alcohol of formula (II).
• polypeptide having the ability to convert the non-cyclic sesquiterpene precursor FPP to at least one drimane alcohol of formula (II) are known in the art.
• WO2018220113 and W02020078871 discloses therein many examples of such enzymes.
• a preferred embodiment of the invention is where in the drimane alcohol of formula (II) is albicanol and the drimane aldehyde is albicanal.
• the oxidoreductase is an alcohol dehydrogenase (ADH) enzyme. More preferably the ADH comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 33 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 33.
• the ADH comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 6 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 6.
• the present invention provides a process for the preparation of a drimane aldehyde of formula (I) by contacting a drimane alcohol of formula (II) with a polypeptide having oxidoreductase enzyme activity.
• Oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor.
• Oxidoreductases comprise the large class of enzymes that catalyze biological oxidation/reduction reactions. Because many chemical and biochemical transformations involve oxidation/reduction processes, oxidoreductases have much utility in the development of biotech methods of synthesis of desirable compounds.
• oxidoreductases There are several different classes of oxidoreductases which are primarily defined according to their substrate and/or mode of action. For example; alcohol dehydrogenases, ketoreductases, peroxidases, hydroxylases and oxygenases, and reductases.
• the present inventors sought to identify whether an oxidoreductase could be used to reduce the compound of formula (II) to form a compound of formula (I). Surprisingly they identified several such enzymes which can be used for this purpose, as shown in the accompanying examples. To the best knowledge of the inventors this is the first time that an oxidoreductase, particularly an alcohol dehydrogenase (ADH) enzyme, has been used for this reaction. Until the present invention it has not been demonstrated that ADH enzymes accept compounds of formula (I), in particular albicanol, as a substrate.
• ADH alcohol dehydrogenase
• Alcohol dehydrogenase and “ADH” enzymes are used interchangeably herein to refer to a polypeptide having an enzymatic capability of the oxidation of primary and secondary alcohols to aldehydes and ketones.
• NADP(+), D-xylono-1 ,5-lactone-forming 1.1.1.181 cholest-5-ene-3beta,7alpha- diol 3beta-dehydrogenase, 1 .1 .1 .183 geraniol dehydrogenase (NADP(+)), 1 .1 .1 .184 carbonyl reductase (NADPH), 1 .1 .1 .185 L-glycol dehydrogenase, 1 .1 .1 .186 dTDP- galactose 6-dehydrogenase, 1.1.1.187 GDP-4-dehydro-D-rhamnose reductase, 1.1.1.188 prostaglandin-F synthase, 1.1.1.189 prostaglandin-E2 9-reductase, 1.1.1.190 indole-3-acetaldehyde reductase (NADH), 1.1.1.191 indole-3- acetaldehy
• ADH reactions typically require a cofactor.
• Reduction reactions catalyzed by the ADH enzymes as described herein also typically require a cofactor.
• the term "cofactor" refers to a non-protein compound that operates in combination with a ADH enzyme.
• Cofactors suitable for use with the ADH enzymes in the processes of the invention described herein include, but are not limited to, NAD(P) + (nicotinamide adenine dinucleotide phosphate), NAD(P)H (the reduced form of NAD(P) + ), NAD + (nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD + ).
• NAD(P) + nicotinamide adenine dinucleotide phosphate
• NAD(P)H the reduced form of NAD(P) +
• NAD + nicotinamide adenine dinucleotide
• NADH the reduced form of NAD +
• a preferred embodiment of the invention is wherein the process of the invention is performed in the presence of a co-factor; preferably the co-factor is NAD or NAD(P) + .
• the co-factor is optionally regenerated using a cofactor regeneration system.
• a co-factor regenerating system can push the equilibrium of the process of the invention towards the generation of the desired product, for example drimane aldehyde. In this way the process of the invention can be more optimized and efficient in terms of reagents used therefore more timely and cost effective than without the use of a co-factor regenerating system.
• an embodiment of the invention is wherein the process of the invention is performed in the presence of a co-factor regeneration system.
• momilactone A synthase class of ADH enzymes can oxidize a drimane alcohol of formula (II) to form a drimane aldehyde of formula (I).
• ADH is a momilactone A synthase.
• Momilactone A synthases are a known class of ADH enzymes with the allocated classification EC 1.1.1 .295.
• the momilactone A synthase used in the process of the invention has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NO: 1 , 2, 6, 12, 21 or 33.
• ADH secoisolariciresinol dehydrogenase
• secoisolariciresinol dehydrogenase are a known class of ADH enzymes with the allocated classification EC 1.1.1.331.
• the secoisolariciresinol dehydrogenase used in the process of the 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.
• a further aspect of the invention provides an isolated polypeptide having ADH activity comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 33 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 33.
• polypeptides having ADH activity comprising an amino acid sequence of any of SEQ ID NOs: 1 to 25 are capable of producing a drimane aldehyde of formula (I) at more than 2 mg/L.
• the isolated polypeptide having ADH activity comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 25 or comprises the amino acid sequence of any of SEQ ID NOs: 1 to 25.
• polypeptides having ADH activity comprising an amino acid sequence of any of SEQ ID NOs: 1 to 13 are capable of producing a drimane aldehyde of formula (I) at more than 5 mg/L.
• the isolated polypeptide having ADH activity comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 13 or comprises the amino acid sequence of any of SEQ I D NOs: 1 to 13.
• polypeptides having ADH activity comprising an amino acid sequence of any of SEQ ID NOs: 1 to 6 are capable of producing a drimane aldehyde of formula (I) at more than 10 mg/L.
• the isolated polypeptide having ADH activity comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 1 to 6 or comprises the amino acid sequence of any of SEQ ID NOs: 1 to 6.
• table 1 of the accompanying examples shows that enzymes 34 to 71 can also be used to prepare drimane aldehyde of formula (I).
• an embodiment of the invention is wherein the process of the invention uses an ADH polypeptides of any of enzymes 34 to 71 .
• a further aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having ADH activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1 to 33 or comprising the amino acid sequence any of SEQ ID NOs: 1 to 33.
• 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 of SEQ ID NOs: 34 to 66 or comprising a nucleotide sequence any of SEQ ID NOs: 34 to 66 or the reverse complement thereof.
• a further aspect of the invention provides a nucleic acid molecule encoding a polypeptide provided herein.
• a vector comprising the nucleic acid molecules described herein.
• the vector is an expression vector.
• the vector is a prokaryotic vector, viral vector or a eukaryotic vector.
• non-human host organism or a host cell comprising (1) a nucleic acid molecule described above, or (2) an expression vector comprising said nucleic acid molecule.
• the non-human organism or host cell is a prokaryotic or eukaryotic cell.
• the host cell is a bacterial cell, a plant cell, a fungal cell or a yeast.
• the bacterial cell is E. coli and the yeast cell is Saccharomyces cerevisiae.
• nucleotide sequence obtained by modifying any of SEQ ID NOs: 34 to 66 or the reverse complement thereof which encompasses any sequence that has been obtained by modifying the sequence of any of SEQ ID NOs: 34 to 66, or of the reverse complement thereof using any method known in the art, for example, by introducing any type of mutations such as deletion, insertion and/or substitution mutations.
• nucleic acids comprising a sequence obtained by mutation of any of SEQ ID NOs: 34 to 66 or the reverse complement thereof are encompassed by an embodiment herein, provided that the sequences they comprise share at least the defined sequence identity of any of SEQ ID NOs: 34 to 66 or the reverse complement thereof and provided that they encode a polypeptide having ADH activity, as defined in any of the above embodiments.
• Mutations may be any kind of mutations of these nucleic acids, for example, point mutations, deletion mutations, insertion mutations and/or frame shift mutations of one or more nucleotides of the DNA sequence of any of SEQ ID NOs: 34 to 66.
• the nucleic acid of an embodiment herein may be truncated provided that it encodes a polypeptide as described herein.
• a variant nucleic acid may be prepared in order to adapt its nucleotide sequence to a specific expression system.
• bacterial expression systems are known to more efficiently express polypeptides if amino acids are encoded by particular codons.
• nucleic acid sequences encoding the ADH may be optimized for increased expression in the host cell.
• nucleotides of an embodiment herein may be synthesized using codons particular to a host for improved expression.
• an isolated, recombinant or synthetic nucleic acid sequence of any of SEQ ID NOs: 34 to 66 encoding for a polypeptide having ADH activity comprising the amino acid sequence of any of SEQ ID NOs: 1 to 33 or fragments thereof that catalyze production of a drimane aldehyde of formula (I):
• nucleic acid sequence encoding the ADH or variants thereof is also referred herein as a ADH encoding sequence.
• nucleic acid of any of SEQ ID NOs: 34 to 66 is the coding sequence of an ADH gene encoding an ADH obtained as described in the examples.
• a fragment of a polynucleotide of any of SEQ ID NOs: 34 to 66 refers to contiguous nucleotides that is particularly at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp and/or at least 60 bp in length of the polynucleotide of an embodiment herein.
• the fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 contiguous nucleotides of the polynucleotide of an embodiment herein.
• the fragment of the polynucleotides herein may be used as a PCR primer, and/or as a probe, or for anti-sense gene silencing or RNAi.
• genes including the polynucleotides of an embodiment herein, can be cloned on basis of the available nucleotide sequence information, such as found in the attached sequence listing, by methods known in the art. These include e.g. the design of DNA primers representing the flanking sequences of such gene of which one is generated in sense orientations and which initiates synthesis of the sense strand and the other is created in reverse complementary fashion and generates the antisense strand. Thermo stable DNA polymerases such as those used in polymerase chain reaction are commonly used to carry out such experiments. Alternatively, DNA sequences representing genes can be chemically synthesized and subsequently introduced in DNA vector molecules that can be multiplied by e.g. compatible bacteria such as e.g. E. coli.
• PCR primers and/or probes for detecting nucleic acid sequences encoding a ADH are provided.
• the skilled artisan will be aware of methods to synthesize degenerate or specific PCR primer pairs to amplify a nucleic acid sequence encoding the ADH or fragments thereof, based on any of SEQ ID NOs: 34 to 66.
• a detection kit for nucleic acid sequences encoding the ADH may include primers and/or probes specific for nucleic acid sequences encoding the ADH, and an associated protocol to use the primers and/or probes to detect nucleic acid sequences encoding the ADH in a sample. Such detection kits may be used to determine whether a plant, organism or cell has been modified, i.e., transformed with a sequence encoding the ADH.
• the sequence of interest is operably linked to a selectable or screenable marker gene and expression of the reporter gene is tested in transient expression assays with protoplasts or in stably transformed plants.
• DNA sequences capable of driving expression are built as modules. Accordingly, expression levels from shorter DNA fragments may be different than the one from the longest fragment and may be different from each other.
• nucleic acid sequence coding the ADH proteins provided herein, i.e., nucleotide sequences that hybridize under stringent conditions to the nucleic acid sequence of any of SEQ ID NOs: 34 to 66.
• the percentage of identity between two peptide or nucleotide sequences is a function of the number of amino acids or nucleotide residues that are identical in the two sequences when an alignment of these two sequences has been generated. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment.
• the percentage of sequence identity is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100.
• the optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment.
• Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs and for instance publicly available computer programs available on the world wide web.
• the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain an optimal alignment of protein or nucleic acid sequences and to calculate the percentage of sequence identity.
• a related embodiment provided herein provides a nucleic acid sequence which is complementary to the nucleic acid sequence according to any of SEQ ID NOs: 34 to 66 such as inhibitory RNAs, or nucleic acid sequence which hybridizes under stringent conditions to at least part of the nucleotide sequence according to any of SEQ ID NOs: 34 to 66.
• An alternative embodiment of an embodiment herein provides a method to alter gene expression in a host cell. For instance, the polynucleotide of an embodiment herein may be enhanced or overexpressed or induced in certain contexts (e.g. upon exposure to a certain temperature or culture conditions) in a host cell or host organism.
• Alteration of expression of a polynucleotide provided herein may also result in ectopic expression which is a different expression pattern in an altered and in a control or wildtype organism. Alteration of expression occurs from interactions of polypeptide of an embodiment herein with exogenous or endogenous modulators, or as a result of chemical modification of the polypeptide. The term also refers to an altered expression pattern of the polynucleotide of an embodiment herein which is altered below the detection level or completely suppressed activity.
• provided herein is also an isolated, recombinant or synthetic polynucleotide encoding a polypeptide or variant polypeptide provided herein.
• an isolated nucleic acid molecule encoding a polypeptide having ADH activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1 to 33 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 33.
• an isolated polypeptide having ADH activity comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1 to 33 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 33.
• the polypeptide consists of the amino acid sequence of any of SEQ ID NOs: 1 to 33.
• the at least one polypeptide having ADH activity used in any of the herein-described embodiments or encoded by the nucleic acid used in any of the herein-described embodiments comprises an amino acid sequence that is a variant of any of SEQ ID NOs: 1 to 33, obtained by genetic engineering.
• the polypeptide comprises an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying any of SEQ ID NOs: 34 to 66 or the reverse complement thereof.
• Polypeptides are also meant to include variants and truncated polypeptides provided that they have ADH activity.
• the at least one polypeptide having a ADH activity used in any of the herein-described embodiments or encoded by the nucleic acid used in any of the herein-described embodiments comprises an amino acid sequence that is a variant of any of SEQ ID NOs: 1 to 33, obtained by genetic engineering, provided that said variant has ADH activity and has the required percentage of identity to any of SEQ ID NOs: 1 to 33 as described herein.
• the at least one polypeptide having a ADH activity used in any of the herein-described embodiments or encoded by the nucleic acid used in any of the herein-described embodiments is a variant of any of SEQ ID NOs: 1 to 33 that can be found naturally in other organisms provided that it has a ADH activity.
• the polypeptide includes a polypeptide or peptide fragment that encompasses the amino acid sequences identified herein, as well as truncated or variant polypeptides provided that they have ADH activity and that they share at least the defined percentage of identity with the corresponding fragment of any of SEQ ID NOs: 1 to 33.
• variant polypeptides are naturally occurring proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- or C- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides of an embodiment herein. Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of an embodiment herein, as described thereafter, are also encompassed by an embodiment herein.
• Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends can also be used in the methods of an embodiment herein.
• a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system.
• additional peptide sequences may be signal peptides, for example.
• Another aspect encompasses methods using variant polypeptides, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides.
• Polypeptides resulting from a fusion with another functional protein can also be advantageously used in the methods of an embodiment herein.
• a variant may also differ from the polypeptide of an embodiment herein by attachment of modifying groups which are covalently or non-covalently linked to the polypeptide backbone.
• the variant also includes a polypeptide which differs from the polypeptide provided herein by introduced N-linked or O-linked glycosylation sites, and/or an addition of cysteine residues. The skilled artisan will recognize how to modify an amino acid sequence and preserve biological activity.
• DNA sequence polymorphisms may exist within a given population, which may lead to changes in the amino acid sequence of the polypeptides disclosed herein.
• Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents.
• nucleic acid encoding the polypeptide or variants thereof of an embodiment herein is a useful tool to modify non-human host organisms or cells and to modify nonhuman host organisms or cells intended to be used in the methods described herein.
• An embodiment provided herein provides amino acid sequences of ADH proteins including orthologs and paralogs as well as methods for identifying and isolating orthologs and paralogs of the ADH in other organisms. Particularly, so identified orthologs and paralogs of the ADH and are capable of producing a compound of formula (I).
• the ADH polypeptide can be obtained by extraction from any organism expressing it, using standard protein or enzyme extraction technologies. If the host organism is an unicellular organism or cell releasing the polypeptide of an embodiment herein into the culture medium, the polypeptide may simply be collected from the culture medium, for example by centrifugation, optionally followed by washing steps and re-suspension in suitable buffer solutions. If the organism or cell accumulates the polypeptide within its cells, the polypeptide may be obtained by disruption or lysis of the cells and optionally further extraction of the polypeptide from the cell lysate.
• the at least one polypeptide having a ADH can be used in the processes of the invention.
• ADH protein, variant or fragment may be determined using various methods. For example, transient or stable overexpression in plant, bacterial or yeast cells can be used to test whether the protein has activity, i.e. , produces a compound of formula (I). ADH activity may be assessed in assay described in the examples herein, indicating functionality. A variant or derivative of a ADH polypeptide of an embodiment herein retains an ability to produce a compound of formula (I). Amino acid sequence variants of the ADH provided herein may have additional desirable biological functions including, e.g., altered substrate utilization, reaction kinetics, product distribution or other alterations.
• At least one vector comprising the nucleic acid molecules described herein.
• Also provided herein is a vector selected from the group of a prokaryotic vector, viral vector and a eukaryotic vector.
• a vector that is an expression vector is an expression vector.
• the nucleic acid sequences of an embodiment herein encoding ADH proteins can be inserted in expression vectors and/or be contained in chimeric genes inserted in expression vectors, to produce ADH proteins in a host cell or non-human host organism.
• the vectors for inserting transgenes into the genome of host cells are well known in the art and include plasmids, viruses, cosmids and artificial chromosomes. Binary or co-integration vectors into which a chimeric gene is inserted can also be used for transforming host cells.
• An embodiment provided herein provides recombinant expression vectors comprising a nucleic acid sequence of a ADH gene, or a chimeric gene comprising a nucleic acid sequence of a ADH gene, operably linked to associated nucleic acid sequences such as, for instance, promoter sequences.
• a chimeric gene comprising a nucleic acid sequence of any of SEQ ID NOs: 34 to 66 or a variant thereof may be operably linked to a promoter sequence suitable for expression in plant cells, bacterial cells or fungal cells, optionally linked to a 3’ non-translated nucleic acid sequence.
• the promoter sequence may already be present in a vector so that the nucleic acid sequence which is to be transcribed is inserted into the vector downstream of the promoter sequence.
• Vectors can be engineered to have an origin of replication, a multiple cloning site, and a selectable marker.
• an expression vector comprising a nucleic acid as described herein can be used as a tool for transforming non-human host organisms or host cells suitable to carry out the method of an embodiment herein in vivo.
• the expression vectors provided herein may be used in the methods for preparing a genetically transformed non-human host organism and/or host cell, in non-human host organisms and/or host cells harboring the nucleic acids of an embodiment herein and in the methods for making polypeptides having a ADH activity, as described herein.
• Recombinant non-human host organisms and host cells transformed to harbor at least one nucleic acid of an embodiment herein so that it heterologously expresses or overexpresses at least one polypeptide of an embodiment herein are also very useful tools to carry out the method of an embodiment herein. Such non-human host organisms and host cells are therefore provided herein.
• a host cell or non-human host organism comprising at least one of the nucleic acid molecules described herein or comprising at least one vector comprising at least one of the nucleic acid molecules.
• a nucleic acid according to any of the above-described embodiments can be used to transform the non-human host organisms and cells and the expressed polypeptide can be any of the above-described polypeptides.
• 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.
• the non-human host organism or host cell is a eukaryotic cell. In another embodiment, the non-human host organism or host cell is a yeast cell. In a further embodiment, the non-human host organism or cell is Saccharomyces cerevisiae. In one embodiment the non-human host organism or host cell expresses a polypeptide, provided that the organism or cell is transformed to harbor a nucleic acid encoding said polypeptide, this nucleic acid is transcribed to mRNA and the polypeptide is found in the host organism or cell.
• Suitable methods to transform a non-human host organism or a host cell have been previously described and are also provided herein.
• the host organism or host cell is cultivated under conditions conducive to the production of a compound of formula (I).
• conditions conducive to the production of a compound of formula (I) may comprise addition of suitable cofactors to the culture medium of the host.
• a culture medium may be selected, so as to maximize a compound of formula (I) synthesis. Examples of optimal culture conditions are described in a more detailed manner in the examples.
• Non-human host organisms suitable to carry out the method of an embodiment herein in vivo may be any non-human multicellular or unicellular organisms.
• the non-human host organism used to carry out an embodiment herein in vivo is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus can be used.
• the non-human host organism used to carry out the method of an embodiment herein in vivo is a microorganism. Any microorganism can be used, for example, the microorganism can be a bacteria or yeast, such as E. coli or Saccharomyces cerevisiae.
• Isolated higher eukaryotic cells can also be used, instead of complete organisms, as hosts to carry out the method of an embodiment herein in vivo.
• Suitable eukaryotic cells may be any non-human cell, such as plant or fungal cells.
• a method comprising transforming a host cell or a non-human host organism with a nucleic acid encoding a polypeptide having ADH activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1 to 33 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 33.
• a method provided herein comprises cultivating a non-human host organism or a host cell transformed to express a polypeptide wherein the polypeptide comprises a sequence of amino acids that has at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 1 to 33 under conditions that allow for the production of the polypeptide.
• the non-human host organism or a host cell transformed to express a polypeptide according to the invention may comprise additional exogenous polypeptide sequences.
• the process of the invention comprises contacting a drimane alcohol of formula (II) with a polypeptide having oxidoreductase enzyme activity.
• the drimane alcohol of formula (II) can be supplied to the host organism or a host cell transformed to express a polypeptide according to the invention by means of addition to the culture medium (or other such growth substrate as will be known to the skilled person).
• the host organism or a host cell is also transformed with one or more additional polypeptides which can produce the drimane alcohol of formula (II).
• the process of the invention to provide a drimane aldehyde of formula (I) can be a multi-step enzymatic process, as can be appreciate by the skilled person.
• polypeptides which can produce the drimane alcohol of formula (II) are known in the art.
• WQ2018220113 discloses methods and enzymes for the production of albicanol and/or drimenol, which are starting materials for the process of the invention, from acyclic farnesyl diphosphate precursor molecules (FPP).
• FPP farnesyl diphosphate precursor molecules
• polypeptides encoded by SEQ ID NO: 1 and SEQ ID NO: 29 of W02018220113 can be used for this reaction.
• WO2019229064 discloses methods and enzymes for the production of albicanol, a starting material for the process of the invention, from acyclic farnesyl diphosphate precursor molecules (FPP).
• FPP farnesyl diphosphate precursor molecules
• the invention further relates to methods for recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultured, optionally the expression of the polypeptides is induced by applying at least one inducer inducing gene expression and the expressed polypeptides are isolated from the culture.
• the polypeptides can also be produced in this way on an industrial scale, if desired.
• the microorganisms produced according to the invention can be cultured continuously or discontinuously in the batch method or in the fed-batch method or repeated fed- batch method.
• a summary of known cultivation methods can be found in the textbook by Chmiel (Bioreatechnik 1. Einfiihrung in die Biovonstechnik [Bioprocess technology 1 . Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere bamboo [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
• the culture medium to be used must suitably meet the requirements of the respective strains. 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 D. C., USA, 1981). These media usable according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
• Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Very good carbon sources are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other byproducts of sugar refining. It can also be advantageous to add mixtures of different carbon sources.
• oils and fats for example soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, for example palmitic acid, stearic acid or linoleic acid, alcohols, for example glycerol, methanol or ethanol and organic acids, for example acetic acid or lactic acid.
• Nitrogen sources are usually organic or inorganic nitrogen compounds or materials that contain these compounds.
• nitrogen sources comprise 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, soya flour, soya protein, yeast extract, meat extract and others.
• the nitrogen sources can be used alone or as a mixture.
• Inorganic salt compounds that can be present in the media comprise the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
• Inorganic sulfur-containing compounds for example sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, as well as organic sulfur compounds, such as mercaptans and thiols, can be used as the sulfur source.
• Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
• Chelating agents can be added to the medium, in order to keep the metal ions in solution.
• suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
• the fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
• growth factors and salts often originate from the components of complex media, such as yeast extract, molasses, corn-steep liquor and the like.
• suitable precursors can be added to the culture medium.
• the exact composition of the compounds in the medium is strongly dependent on the respective experiment and is decided for each specific case individually. Information on media optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach” (Ed. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3).
• Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
• All components of the medium are sterilized, either by heat (20 min at 1.5 bar and 121 °C) or by sterile filtration.
• the components can either be sterilized together, or separately if necessary.
• All components of the medium can be present at the start of culture or can be added either continuously or batchwise.
• the culture temperature is normally between 15°C and 45°C, preferably 25°C to 40°C and can be varied or kept constant during the experiment.
• the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
• the pH for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid.
• Antifoaming agents for example fatty acid polyglycol esters, can be used for controlling foaming.
• suitable selective substances for example antibiotics, can be added to the medium.
• oxygen or oxygen-containing gas mixtures for example ambient air, are fed into the culture.
• the temperature of the culture is normally in the range from 20°C to 45°C.
• the culture is continued until a maximum of the desired product has formed. This target is normally reached within 10 hours to 160 hours.
• the fermentation broth is then processed further.
• the biomass can be removed from the fermentation broth completely or partially by separation techniques, for example centrifugation, filtration, decanting or a combination of these methods or can be left in it completely.
• the cells can also be lysed and the product can be obtained from the lysate by known methods for isolation of proteins.
• the cells can optionally be disrupted with high-frequency ultrasound, high pressure, for example in a French press, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of several of the aforementioned methods.
• the polypeptides can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), such as Q-sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and with other usual techniques such as ultrafiltration, crystallization, salting-out, dialysis and native gel electrophoresis. Suitable methods are described for example in Cooper, T. G., Biochemische Anlagenmann, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
• vector systems or oligonucleotides which lengthen the cDNA by defined nucleotide sequences and therefore code for altered polypeptides or fusion proteins, which for example serve for easier purification.
• Suitable modifications of this type are for example so-called "tags" functioning as anchors, for example the modification known as hexa-histidine anchor or epitopes that can be recognized as antigens of antibodies (described for example in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press).
• anchors can serve for attaching the proteins to a solid carrier, for example a polymer matrix, which can for example be used as packing in a chromatography column, or can be used on a microtiter plate or on some other carrier.
• a solid carrier for example a polymer matrix
• these anchors can also be used for recognition of the proteins.
• markers such as fluorescent dyes, enzyme markers, which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors for derivatization of the proteins.
• the enzymes or polypeptides according to the invention can be used free or immobilized in the method described herein.
• An immobilized enzyme is an enzyme that is fixed to an inert carrier. Suitable carrier materials and the enzymes immobilized thereon are known from EP-A-1149849, EP-A-1 069 183 and DE-OS 100193773 and from the references cited therein. Reference is made in this respect to the disclosure of these documents in their entirety.
• Suitable carrier materials include for example clays, clay minerals, such as kaolinite, diatomaceous earth, perlite, silica, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, synthetic polymers, such as polystyrene, acrylic resins, phenol formaldehyde resins, polyurethanes and polyolefins, such as polyethylene and polypropylene.
• the carrier materials are usually employed in a finely-divided, particulate form, porous forms being preferred.
• the particle size of the carrier material is usually not more than 5 mm, in particular not more than 2 mm (particle-size distribution curve).
• Carrier materials are e.g. Ca-alginate, and carrageenan.
• Enzymes as well as cells can also be crosslinked directly with glutaraldehyde (cross-linking to CLEAs).
• G. Drauz and H. Waldmann Enzyme Catalysis in Organic Synthesis 2002, Vol. Ill, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out methods according to the invention are also given for example in Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol 3, Chapter 17, VCH, Weinheim.
• reaction conditions for biocatalytic production methods of the invention may be performed under in vivo or in vitro conditions.
• the at least one polypeptide/enzyme which is present during a method of the invention or an individual step of a multistep-method as defined herein above, can be present in living cells naturally or recombinantly producing the enzyme or enzymes, in harvested cells, i.e. under in vivo conditions, or, in dead cells, in permeabilized cells, in crude cell extracts, in purified extracts, or in essentially pure or completely pure form, i.e. under in vitro conditions.
• the at least one enzyme may be present in solution or as an enzyme immobilized on a carrier. One or several enzymes may simultaneously be present in soluble and/or immobilised form.
• the processes according to the invention can be performed in common reactors, which are known to those skilled in the art, and in different ranges of scale, e.g. from a laboratory scale (few millilitres to dozens of litres of reaction volume) to an industrial scale (several litres to thousands of cubic meters of reaction volume).
• a chemical reactor can be used.
• the chemical reactor usually allows controlling the amount of the at least one enzyme, the amount of the at least one substrate, the pH, the temperature and the circulation of the reaction medium.
• the process will be a fermentation.
• the biocatalytic production will take place in a bioreactor (fermenter), where parameters necessary for suitable living conditions for the living cells (e.g. culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, and the like) can be controlled.
• a bioreactor e.g. with procedures for up-scaling chemical or biotechnological methods from laboratory scale to industrial scale, or for optimizing process parameters, which are also extensively described in the literature (for biotechnological methods see e.g. Crueger und Crueger, Biotechnologie - Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Miinchen, Wien, 1984).
• Cells containing the at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or radiofrequency pulses, French presses, or chemical means, such as hypotonic media, lytic enzymes and detergents present in the medium, or combination of such methods.
• detergents are digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween ® 20, deoxycholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propansulfonate), Nonidet ® P40 (Ethylphenolpoly(ethyleneglycolether), and the like.
• the at least one enzyme is immobilised, it is attached to an inert carrier as described above.
• the conversion reaction can be carried out batch wise, semi-batch wise or continuously.
• Reactants and optionally nutrients
• reaction of the invention may be performed in an aqueous, aqueous-organic or non-aqueous reaction medium.
• An aqueous or aqueous-organic medium may contain a suitable buffer in order to adjust the pH to a value in the range of 5 to 11 , like 6 to 10.
• an organic solvent miscible, partly miscible or immiscible with water may be applied.
• suitable organic solvents are listed below.
• Further examples are mono- or polyhydric, aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols like glycerol.
• the non-aqueous medium may contain is substantially free of water, i.e. will contain less that about 1 wt.-% or 0.5 wt.-% of water.
• Biocatalytic methods may also be performed in an organic non-aqueous medium.
• organic solvents there may be mentioned aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; aromatic carbohydrates, like benzene, toluene, xylenes, chlorobenzene or dichlorobenzene, aliphatic acyclic and ethers, like diethylether, methyl-tert.-butylether, ethyl-tert.-butylether, dipropylether, diisopropylether, dibutylether; or mixtures thereof.
• the concentration of the reactants/substrates may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied.
• the initial substrate concentration may be in the 0,1 to 0,5 M, as for example 10 to 100 mM.
• the reaction temperature may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied.
• the reaction may be performed at a temperature in a range of from 0 to 70 °C, as for example 20 to 50 or 25 to 40 °C.
• Examples for reaction temperatures are about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C and about 60°C.
• the process may proceed until equilibrium between the substrate and then product(s) is achieved, but may be stopped earlier.
• Usual process times are in the range from 1 minute to 25 hours, in particular 10 min to 6 hours, as for example in the range from 1 hour to 4 hours, in particular 1 .5 hours to 3.5 hours.. These parameters are non-limiting examples of suitable process conditions.
• optimal growth conditions can be provided, such as optimal light, water and nutrient conditions, for example.
• reaction conditions for performing the preparation of drimane aldehyde compound are as follows.
• the ADH enzyme can be present as a purified polypeptide or in a wholecell system.
• Substrate concentration might vary between 0.1 and 100 mM.
• the process of the present invention can further include a step of recovering an end or intermediate product, optionally in stereoisomerically or enantiomerically substantially pure form.
• recovery includes extracting, harvesting, isolating or purifying the compound from culture or reaction media.
• Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like.
• a conventional resin e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.
• a conventional adsorbent e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.
• solvent extraction e.
• the cyclic terpene compound produced in any of the processes described herein can be converted to derivatives such as, but not limited to hydrocarbons, esters, amides, glycosides, ethers, epoxides, aldehydes, ketones, alcohols, diols, acetals or ketals.
• the terpene compound derivatives can be obtained by a chemical method such as, but not limited to oxidation, reduction, alkylation, acylation and/or rearrangement.
• the terpene compound derivatives can be obtained using a biochemical method by contacting the terpene compound with an enzyme such as, but not limited to an oxidoreductase, a monooxygenase, a dioxygenase, a transferase.
• an enzyme such as, but not limited to an oxidoreductase, a monooxygenase, a dioxygenase, a transferase.
• the biochemical conversion can be performed in-vitro using isolated enzymes, enzymes from lysed cells or in-vivo using whole cells.
• the process further comprises oxidizing the drimane aldehyde of formula (I) using chemical or biocatalytic synthesis or a combination of both. Fermentative production of a drimane aldehyde
• the invention also relates to processes for the fermentative production of a drimane aldehyde.
• a fermentation as used according to the present invention can, for example, be performed in stirred fermenters, bubble columns and loop reactors.
• stirred fermenters for example, be performed in stirred fermenters, bubble columns and loop reactors.
• a comprehensive overview of the possible method types including stirrer types and geometric designs can be found in "Chmiel: Bioreatechnik: Einbowung in die Biovonstechnik, Band 1 ".
• typical variants available are the following variants known to those skilled in the art or explained, for example, in “Chmiel, Hammes and Bailey: Biochemical Engineering", such as batch, fed-batch, repeated fed-batch or else continuous fermentation with and without recycling of the biomass.
• sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen or appropriate gas mixtures may be effected in order to achieve good yield (YP/S).
• the culture medium that is to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the handbook "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D. C., USA, 1981). These media that can be used according to the invention may comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and/or trace elements.
• Preferred sources of carbon are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add mixtures of various sources of carbon.
• oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid or linoleic acid, alcohols such as glycerol, methanol or ethanol and organic acids such as acetic acid or lactic acid.
• Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials containing these compounds.
• sources of nitrogen include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex sources of nitrogen, such as corn-steep liquor, soybean flour, soy-bean protein, yeast extract, meat extract and others.
• the sources of nitrogen can be used separately or as a mixture.
• Inorganic salt compounds that may be present in the media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
• Inorganic sulfur-containing compounds for example sulfates, sulfites, di-thionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as sources of sulfur.
• Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used as sources of phosphorus.
• Chelating agents can be added to the medium, in order to keep the metal ions in solution.
• suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
• the fermentation media used according to the invention may also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
• Growth factors and salts often come from complex components of the media, such as yeast extract, molasses, corn-steep liquor and the like.
• suitable precursors can be added to the culture medium.
• the precise composition of the compounds in the medium is strongly dependent on the particular experiment and must be decided individually for each specific case. Information on media optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach” (1997) Growing media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.
• All components of the medium are sterilized, either by heating (20 min at 1 .5 bar and 121 °C) or by sterile filtration.
• the components can be sterilized either together, or if necessary separately.
• All the components of the medium can be present at the start of growing, or optionally can be added continuously or by batch feed.
• the temperature of the culture is normally between 15 °C and 45 °C, preferably 25 °C to 40 °C and can be kept constant or can be varied during the experiment.
• the pH value of the medium should be in the range from 5 to 8.5, preferably around 7.0.
• the pH value for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid.
• Antifoaming agents e.g. fatty acid polyglycol esters, can be used for controlling foaming.
• suitable substances with selective action e.g. antibiotics, can be added to the medium.
• Oxygen or oxygen-containing gas mixtures e.g. the ambient air, are fed into the culture in order to maintain aerobic conditions.
• the temperature of the culture is normally from 20 °C to 45 °C. Culture is continued until a maximum of the desired product has formed. This is normally achieved within 1 hour to 160 hours.
• the process of the present invention can further include a step of recovering said drimane aldehyde.
• the term “recovering” includes extracting, harvesting, isolating or purifying the compound from culture media.
• Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like.
• a conventional resin e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.
• a conventional adsorbent e.g., activate
• biomass of the broth Before the intended isolation the biomass of the broth can be removed. Processes for removing the biomass are known to those skilled in the art, for example filtration, sedimentation and flotation. Consequently, the biomass can be removed, for example, with centrifuges, separators, decanters, filters or in flotation apparatus. For maximum recovery of the product of value, washing of the biomass is often advisable, for example in the form of a diafiltration. The selection of the method is dependent upon the biomass content in the fermenter broth and the properties of the biomass, and also the interaction of the biomass with the product of value.
• the fermentation broth can be sterilized or pasteurized.
• the fermentation broth is concentrated. Depending on the requirement, this concentration can be done batch wise or continuously.
• the pressure and temperature range should be selected such that firstly no product damage occurs, and secondly minimal use of apparatus and energy is necessary. The skillful selection of pressure and temperature levels for a multistage evaporation in particular enables saving of energy.
• a further aspect of the invention provides a recombinant host cell or a recombinant non-human host organism comprising a compound of formula (I) and/or a compound of formula (II). Examples of such host cells are shown in the accompanying example section of this application.
• the process of the invention relates to the preparation of a drimane aldehyde of formula (I).
• the drimane aldehyde is converted to a drimane acid using chemical or biocatalytic synthesis or a combination of both.
• drimane acid is albicanic acid.
• Albicanic acid can subsequently be used as staring material for the synthesis of perfuming materials, including polywood.
• the structure and synthesis of polywood via drimane aldehydes of formula (I) is known and can be performed with no inventive input from the skilled person.
• EXAMPLE 1 Oxidoreductase catalysed oxidation of albicanol to albicanal
• Alcohol dehydrogenases are nicotinamide adenine dinucleotide (NAD+)- or (NADP+)- dependent oxidoreductases. As one of the most abundant enzyme class, they catalyze the reversible reduction of aldehydes and ketones to their corresponding alcohols. The equilibrium of this reversable reaction is strongly ADH depended but can be shifted when one substrate is enzymatically modified and thereby is no longer accessible to the ADH. To date, there is no known ADH that accepts albicanol as substrate.
• ADHs capable of oxidizing albicanol 269 ADHs from public libraries and transcriptomes, were selected and tested for their promiscuity to accept albicanol and catalyze its conversion to albicanal.
• the 269 alcohol dehydrogenases candidates were screened in two batches.
• the first batch contained the 122 Alcohol dehydrogenases candidates, derived from transcriptomes coming from the organisms Bazzania trilobata, Bazzania sp., Laricifomes officinalis, Antrodia cinnamomea and Porella navicularis.
• This batch was screened in Saccharomyces cerevisiae. The screening was done as described in W02020078871 A1 . Under the screening conditions used, it was not possible to detect the conversion of albicanol to albicanal by any of the above mentioned 122 alcohol dehydrogenase candidate.
• a second batch of alcohol dehydrogenases candidates which contained 149 ADH candidates derived from publications, the NCBI protein database, Phytozome database (DOE Joint Genome Institute) and a transcriptome coming from the organism Bazzania sp. (WO2021/105236) were screened in E.coli. Therefore an E.coli strain producing the sesquiterpene albicanol was constructed. For this purpose the farnesyl-pyrophosphate (FPP) overexpressing E.coli strain DP1205, recently described in WO2021005097, was used as base strain.
• FPP farnesyl-pyrophosphate
• DP1205 was transformed with the expression plasmid pJ424 (ATUM, Newark, California), which contained an E.coli codon optimized gene version of the albicanol synthase LoTpsI, described in WO2018220113 A1 to yield the E.coli Strain DP1205 pJ424(LoTpsl).
• the different alcohol dehydrogenase candidates were ordered at TWIST Bioscience (San Francisco, California) as E.coli codon optimized genes cloned in the expression vector pET29a and were added into DP1205 pJ424(LoTpsl).
• the transformed cells were selected on LB medium plates supplemented with the appropriate antibiotics. Single colonies of each transformation were first cultivated overnight in Deep Well Plates at 37°C in 0.5 mL LB medium supplemented with 1 % glucose and with the appropriate antibiotics. The next day, 0.5 mL of medium as described in Tsuruta et al (Tsuruta H, Paddon CJ, Eng D, Lenihan JR, Horning T, et al.
• the quantity of albicanal produced under these conditions are shown on T able 1 .
• 6 candidates: BAG99023.1 , AJP06249.1 , XP_002446247.1 , XP_008669542.1 , BAV31336.1 and XP_004494228.1 produced more than 10 mg/L of albicanal.
• the momilactone A synthases XP_002446247.1 and XP_008669542.1 produced the highest titer of albicanal with 58 mg/L and 41 mg/L, respectively.
• the GC-MS chromatograms of the E.co//-based production of functioning ADHs are displayed in Figure 1. Further, Figure 1 shows that the MS spectrum of the E.co//-derived albicanal is similar to a reference albicanal MS spectrum.
• ADHs were able to produce albicanal. Of these, 33 ADHs produced more than 1 mg/L of the compound, and 6 produced more than 10 mg/L. This is the first time it has been demonstrated that ADH enzymes can accept abicanol as a substrate and produce quantities of albicanal suitable for further commercialization development.
• amino acid sequence of enzymes 1 to 33 are disclosed in SEQ ID NOs: 1 to 33.
• accession numbers for the amino acid sequence are provided in Table 2.

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