Method for producing drimanyl acetate compounds

Abstract

To provide novel methods for producing drimanyl acetate compounds, in particular methods for achieving the fully biochemical synthesis of drimanyl acetates in an aqueous environment.SOLUTION: The above-mentioned problem can be solved by providing a new class of enzymes which show acetyl transferase activity and produce a drimanyl acetate, like albicanyl acetate or drimenyl acetate, from respective drimane alcohol precursors, like albicanol or drimenol via acetylation and using acetyl-CoA as acetyl group donor. Due to their physicochemical properties, in particular as they are liquid at an ambient temperature, acetylated derivatives of drimanyl alcohols can serve as more appropriate materials.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides a novel method for the production of drimanyl acetate compounds using acetyltransferase catalysis by acetylation of the respective drimanyl alcohol source carried out in vitro or in vivo. The present invention also relates to the identification of enzymes with corresponding acetyltransferase activity from different microbial and plant sources. The present invention also relates to the provision of enzyme variants derived from said newly identified enzymes. A further aspect of the present invention relates to the provision of the corresponding coding sequences of such enzymes and variants, recombinant vectors, and recombinant host cells suitable for the production of such acetyltransferases and variants and suitable for carrying out the novel method for the production of drimanyl acetate compounds. Another aspect of the present invention relates to the use of such drimanyl acetates obtained according to the present invention as intermediates for the manufacture of odorants, flavors or fragrances, or insect / pest control ingredients.

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

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

[0004] The main sources of sesquiterpenes, for example, drimanyl alcohols, especially many of the compounds with drimane structure, such as arbikanol or drimenol, are plants or microorganisms that naturally contain sesquiterpenes.However, the content of sesquiterpenes in these natural sources may be low.Even if available, such drimanyl alcohols are difficult to handle during their further processing, mainly in light of the fact that they are solid at ambient temperature.Providing derivatives that are easy to handle would be an improved approach and would simplify the further processing of drimanyl alcohols.

[0005] Akita, H. et al., Tetrahedron: Asymmetry 11 (2000). 1375-1388, describe the lipase-catalyzed asymmetric synthesis of (+)-arvikanyl acetate. This prior art approach requires at least eight chemical synthesis steps and two sequential reactions catalyzed by lipases to obtain enantiomerically pure arvikanyl acetate. Furthermore, the acetylation reaction was carried out at 33° C. in the presence of diisopropyl ether and isopropenyl acetate, conditions that are incompatible with the physiological conditions required for the biosynthetic pathway. Moreover, under physiological conditions, to the best of our knowledge, lipases are not capable of catalyzing the required transesterification reaction. Although ester formation by lipases is possible, this activity is strongly dependent on the water content present. Thus, in an aqueous environment such as the in vivo setting of the present invention, lipases catalyze the hydrolysis of ester bonds rather than acetylation (Jaeger K. et al; FEMS Microbiology Reviews, 1994, 15:1 pp29-63).

[0006] There remains a need to provide novel methods for producing drimanyl acetate compounds, particularly methods that can be implemented for the complete biochemical synthesis of drimanyl acetate in an aqueous environment, such as, for example, a host cell-based process that provides drimanyl alcohol precursors by metabolism of a sugar substrate.

[0007] overview The above problems can be solved by providing a new class of enzymes that exhibit acetyltransferase activity and produce drimanyl acetates, such as arbikanyl acetate or drimenol acetate, from the respective drimanyl alcohol precursors, such as arbikanol or drimenol, by acetylation using acetyl-CoA as acetyl group donor.Due to their physicochemical properties, the acetylated derivatives of drimanyl alcohols can serve as more suitable materials, especially since they are liquid at ambient temperature. [Brief description of the drawings]

[0008] [Figure 1a] The structures of (+)-albicanol, (-)-drimenol and bicyclofarnesol are shown, as well as the structure of the drimane moiety, which is more specific than the drimane structure of Figure 1c. [Figure 1b] FIG. 1 shows a reaction scheme illustrating the cell biological production of arbikanyl acetate via the acyclic sesquiterpene precursors FPP and arbicanol. [Figure 1c] The more general "drimane structure" is shown, indicating the potential positions of the C=C double bond. [Diagram 2] (A / B) GC-FID analysis of arbicanonyl acetate produced using engineered S. cerevisiae strain YST069 co-expressing arbicanonyl synthase XP_007369631.1 with either acetyltransferase CrDAT (A) or acetyltransferase FgaAT (B). The MS spectrum of arbicanonyl acetate produced by acetyltransferase CrDAT from (A) is shown in (C), which is identical to the MS spectrum from an arbicanonyl acetate standard. [Diagram 3] FIG. 1 shows the relative amounts of arbicanyl acetate produced by nine acetyltransferases (CrDAT, FgaAT, OAH94415.1, TcTAT, CrMAT, LiAAT-4, GAO81666.1, CfACT1-6 and CfACT1-8) that were found to be active on arbicanol (as described in Example 2). [Figure 4] The relative amounts of drimenyl acetate produced by nine acetyltransferases (CrDAT, FgaAT, OAH94415.1, TcTAT, CrMAT, LiAAT-4, GAO81666.1, CfACT1-6 and CfACT1-8) that were also found to be active on arbicanol (as described in Example 2) are shown. [Diagram 5]The relative amounts of arbikanyl acetate produced by S. cerevisiae cells expressing CrDAT, FgaAT, OAH94415.1, TcTAT, CrMAT, LiAAT-4, GAO81666.1, CfACT1-6, CfACT1-8, ERR364415-1_contig_8546 and DfATC13 acetyltransferases are shown. [Figure 6] The relative amounts of drimenyl acetate produced by S. cerevisiae cells expressing CrDAT, FgaAT, OAH94415.1, TcTAT, GAO81666.1, CfACT1-6, CfACT1-8, XP_001258079.1, ERR364415-1_contig_8546 and DfATC13 acetyltransferases are shown. [Figure 7] FIG. 7A shows the MS spectrum of drimenyl acetate produced by acetyltransferase CrDAT, and FIG. 7B shows that this MS spectrum is identical to the MS spectrum from a drimenyl acetate standard. [Figure 8] FIG. 8A shows the MS spectrum of bicyclofarnesyl acetate produced by acetyltransferase CrDAT, and FIG. 8B shows that this MS spectrum is identical to the MS spectrum from a bicyclofarnesyl acetate standard. [Figure 9] The relative amounts of bicyclofarnesyl acetate produced by S. cerevisiae cells expressing CrDAT, FgaAT, TcTAT, CrMAT, GAO81666.1, CfACT1-6, CfACT1-8, BAU61551.1, PsSalAT, XP_001217250.1, ERR364415-1_contig_8546, PYI04555.1, and DfACT13 acetyltransferases are shown.

[0009] Abbreviations used bp base pair kb kilobase CoA Coenzyme A DNA deoxyribonucleic acid cDNA Complementary DNA DTT Dithiothreitol FPP Farnesyl diphosphate GC Gas chromatograph MS mass spectrometer / mass spectrometry MVA Mevalonic acid PCR Polymerase Chain Reaction RNA Ribonucleic acid mRNA Messenger Ribonucleic Acid miRNA MicroRNA siRNA Small interfering RNA rRNA ribosomal RNA tRNA transcription RNA

[0010] definition Unless otherwise stated, the following definitions of technical terms shall apply: For the purposes of the present invention, "acetyltransferase" or "polypeptide having acetyltransferase activity" or "polypeptide capable of transferring an acetyl group" refers more generally to an enzyme of the class of acyltransferases EC 2.3.1, and in particular to acetyl-CoA:alcohol O-acetyltransferases EC 2.3.1.84. It exhibits the ability to acetylate at least one drimanyl alcohol selected from albikano, drimenol and bicyclofarnesol using acetyl-CoA as the acetyl group donor. The drimanyl acetate can be produced in any of its stereoisomeric forms or as a mixture thereof. The albikano acetate, drimenyl acetate or bicyclofarnesyl acetate can be the sole product when the corresponding alcohol precursor is present as the sole acetyl group acceptor, or can be part of a mixture of two or more drimenyl acetates when a mixture of two or more drimenyl alcohols is provided and the acetyltransferase is not substrate specific. In the case of increased selectivity, the acetyltransferase may form predominantly a single drimanyl acetate. The acetyltransferases as described herein may show the same or different preferences or specificities for different drimanyl alcohols as substrates. For example, a first type of acetyltransferase may acetylate predominantly albicanole, a second type of acetyltransferase may acetylate predominantly drimenol, and a third type of acetyltransferase may acetylate predominantly bicyclofarnesol. In such a case, when a mixture of such drimanyl alcohols is used as a substrate, albicanole acetate, drimenol acetate, or bicyclofarnesyl acetate will be formed as the respective major products. In the case of substrate specificity, the acetyltransferase may selectively form a single drimanyl acetate even when a mixture of such drimanyl alcohols is used as a substrate. In particular, the acetylation is carried out with the retention of the respective stereochemical configurations of the drimanyl alcohol substrates.

[0011] "Acetyl group donor" refers to a chemical entity or molecule that acts as a source for the enzymatic transfer of an acetyl group from a donor to an acceptor molecule, such as a molecule with a functional hydroxyl group, which can also react with the acetyl group to form the corresponding acetate ester. A particular acetyl group donor is acetyl-coenzyme A (acetyl-CoA).

[0012] The term "drimane sesquiterpene" or "drimane" refers to a cyclic terpene having a drimane-like carbon skeleton structure as depicted in Figure 1a, or more specifically the more general structure in Figure 1c, where the potential location of the optional C=C double bond is indicated by a dotted line.

[0013] The term "drimanyl alcohol" refers to hydroxylated derivatives of "drimane sesquiterpenes" or "drimanes". Examples include albicanoll, drimenol and bicyclofarnesol in any stereoisomeric form.

[0014] The term "dorimanyl acetate" relates to acetyl ester derivatives of such dorimanyl alcohols, such as arbikanyl acetate, dorimenyl acetate, and bicyclofarnesyl acetate.

[0015] For the purposes of this application "arbicanol" relates in particular to (+)-arbicanol (CAS: 54632-04-1).

[0016] For the purposes of this application "Drimenol" relates in particular to (-)-Drimenol (CAS: 468-68-8).

[0017] For the purposes of this application, "bicyclofarnesol" specifically refers to (+)-bicyclofarnesol or [(4aS,8aS)-2,5,5,8a-tetramethyl-3,4,4a,5,6,7,8,8a-octahydro-1-naphthalenyl]methanol (IUPAC name).

[0018] "Farnesyl diphosphate" refers to (2E,6E)-3,7,11-trimethyldodeca-2,6,10-triene-1-pyrophosphate (FPP).

[0019] For the purposes of this application, "Ambrox" refers to the IUPAC name: (-)-(3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan (CAS: 6790-58-5).

[0020] "Terpene synthase" or "sesquiterpene synthase" or "drimane sesquiterpene synthase" are used interchangeably herein.

[0021] The term "bifunctional terpene synthase" or "polypeptide having bifunctional terpene synthase activity" relates to a polypeptide as further defined in International Application PCT / EP2018 / 064344, filed May 31, 2018.

[0022] The term "arbicanyl diphosphate synthase" or "polypeptide having arbicanyl diphosphate synthase activity" or "arbicanyl diphosphate synthase protein" or "capable of producing arbicanyl diphosphate" relates to a polypeptide capable of catalyzing the synthesis of arbicanyl diphosphate, in the form of any of its stereoisomers or mixtures thereof, starting from acyclic terpene pyrophosphate, in particular farnesyl diphosphate (FPP). Arbicanyl diphosphate may be the only product or may be part of a mixture of sesquiterpenes. The mixture may contain arbicanyl monophosphate and / or arbicanol. Such polypeptides are described, for example, in the international application PCT / CN2018 / 088902, filed May 29, 2018.

[0023] "Arbikanyl diphosphate synthase activity" is determined under "standard conditions" as described in International Application PCT / CN2018 / 088902.

[0024] The term "arbicanol synthase" or "polypeptide having arbicanol synthase activity" or "arbicanol synthase protein" relates to a polypeptide capable of catalyzing the synthesis of arbicanol, in the form of any of its stereoisomers or mixtures thereof, starting from an acyclic terpene pyrophosphate, in particular farnesyl diphosphate (FPP). Arbicanol may be the sole product or may be part of a mixture of two or more sesquiterpenes.

[0025] The term "drimenol synthase" or "polypeptide having drimenol synthase activity" or "drimenol synthase protein" relates to a polypeptide capable of catalyzing the synthesis of drimenol, in the form of any of its stereoisomers or mixtures thereof, starting from an acyclic terpene pyrophosphate, in particular farnesyl diphosphate (FPP). Drimenols may be the sole product or may be part of a mixture of two or more sesquiterpenes.

[0026] "Arbicanol synthase activity" and drimenol synthase activity are determined, for example, as described in International Application PCT / EP2018 / 0643444, WO 2015 / 169871 or WO 2015 / 176959.

[0027] The "phosphatase" enzymes used in the present invention have the ability to convert orthophosphate esters to the respective alcohols and orthophosphoric acid under the consumption of water. Included herein are acid phosphatases (EC 3.1.3.2 with acidic reaction optimum) and alkaline phosphatases (EC 3.1.3.1 with alkaline reaction optimum).

[0028] The terms "biological function", "function", "biological activity" or "activity" refer to the ability of a terpene synthase as described herein to a) catalyze the formation of arbicanyl diphosphate and / or arbicanol, hereinafter: arbicanyl diphosphate, and / or arbicanyl monophosphate and / or arbicanol and / or one or more other terpenes, in particular arbicanyl diphosphate, or b) catalyze the formation of drimanyl alcohol or a mixture of two or more drimanyl alcohols and optionally one or more other terpenes.

[0029] The terms "biological function," "function," "biological activity," or "activity" refer to the ability of an acetyltransferase, as described herein, to catalyze the formation of drimanyl acetate or a mixture of two or more drimanyl acetates, optionally with one or more other acetylated compounds.

[0030] The term "mixture of terpenes" or "mixture of sesquiterpenes" refers to a mixture of terpenes or sesquiterpenes that includes at least one of arbicanol, drimenol, and bicyclofarnesol, and may include one or more additional terpenes and / or one or more additional sesquiterpenes.

[0031] The "mevalonate pathway", also known as the "isoprenoid pathway" or "HMG-CoA reductase pathway", is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The mevalonate pathway starts with acetyl-CoA and produces two 5-carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The key enzymes are acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and isopentenyl diphosphate isomerase. Combining the mevalonate pathway with enzyme activities, especially FPP synthase, to produce the terpene precursors GPP, FPP, or GGPP, allows recombinant cell production of terpenes.

[0032] As used herein, the term "host cell" or "transformed cell" refers to a cell (or organism) that has been modified to contain at least one nucleic acid molecule, e.g., a recombinant gene encoding a desired protein or nucleic acid sequence, which upon transcription produces at least one functional polypeptide of the invention required to perform a biocatalytic method or other recombinant method as described herein. In particular, such host cells or transformed cells provide an acetyltransferase useful for preparing at least one drimanyl acetate from the corresponding drimanyl alcohol. They may also provide other enzymes, such as arbikanyl diphosphate synthase proteins, useful for producing arbikanyl diphosphate and / or arbikanyl monophosphate and / or arbikanol, or the corresponding mixture of terpenes containing arbikanyl diphosphate and / or arbikanyl monophosphate and / or arbikanol. They may also provide a terpene synthase useful for preparing at least one drimanyl alcohol. The host cell is in particular a bacterial cell, a fungal cell, or a plant cell or plant. The host cell may contain the recombinant gene integrated into the nuclear or organellar genome of the host cell, or alternatively, the host may contain the recombinant gene extrachromosomally.

[0033] The term "organism" refers to a non-human multicellular or unicellular organism, such as a plant, or a microorganism. In particular, the microorganism is a bacterium, a yeast, an algae, or a fungus.

[0034] The term "plant" is used interchangeably to include plant cells, including plant cell tissue cultures giving rise to plant protoplasts, plant tissues, regenerated plants, or parts of plants, or plant organs such as roots, stems, leaves, flowers, pollen, ovaries, embryos, fruits, etc. Any plant can be used to practice the methods of one embodiment herein.

[0035] A particular organism or cell is meant to be "capable of producing FPP" if it naturally produces FPP, or if it does not naturally produce FPP but has been transformed with a nucleic acid as described herein to produce FPP. Organisms or cells that have been transformed to produce greater amounts of FPP than naturally occurring organisms or cells are also encompassed by "organisms or cells capable of producing FPP."

[0036] A particular organism or cell is "capable of producing drimanyl acetate" if it naturally produces drimanyl acetate, or if it does not naturally produce drimanyl acetate but has been transformed with a nucleic acid as described herein to produce drimanyl acetate. Organisms or cells that have been transformed to produce greater amounts of drimanyl acetate than naturally occurring organisms or cells are also encompassed by "organisms or cells capable of producing drimanyl acetate."

[0037] A particular organism or cell is meant to be "capable of producing drimanyl alcohol" if it naturally produces drimanyl alcohol, or if it does not naturally produce drimanyl alcohol, but has been transformed to produce drimanyl diphosphate, and optionally further transformed with a nucleic acid to produce an enzymatic activity that converts drimanyl diphosphate to drimanyl alcohol. Organisms or cells transformed to produce greater amounts of drimanyl alcohol than naturally occurring organisms or cells are also encompassed by "organisms or cells capable of producing drimanyl alcohol."

[0038] In the description and appended claims, the use of "or" means "and / or" unless specifically stated otherwise. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0039] It is further understood that where the descriptions of various embodiments use the term "comprising," those of ordinary skill in the art will understand that in some specific instances the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0040] The terms "purified", "substantially purified" and "isolated" as used herein refer to a state in which the compound of the invention is free of other heterogeneous compounds that are normally associated with it in its natural state, and thus "purified", "substantially purified" and "isolated" refer to at least 0.5%, 1%, 5%, 10% or 20% by weight of the mass of a given sample, or at least 50% or 75% by weight of the mass of a given sample. In one embodiment, these terms refer to a compound of the invention that comprises at least 95%, 96%, 97%, 98%, 99% or 100% by weight of the mass of a given sample. As used herein, the terms "purified", "substantially purified" and "isolated" of a nucleic acid or protein when referring to a nucleic acid or protein also refer to a state of purification or concentration that is different from that which naturally occurs in an environment such as, for example, a prokaryotic or eukaryotic environment, such as a bacterial or fungal cell, or a mammalian organism, particularly the human body. Any degree of purification or concentration greater than that found in nature, including (1) purification from other associated structures or compounds, or (2) association with structures or compounds not normally associated in said prokaryotic or eukaryotic environment, is within the meaning of "isolated." The nucleic acids or proteins or classes of nucleic acids or proteins described herein may be isolated or otherwise associated with structures or compounds not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.

[0041] The term "about" indicates a potential variation of ±25%, particularly ±15%, ±10%, and more particularly ±5%, ±2% or ±1% of the stated value.

[0042] The term "substantially" describes values ​​in the range of about 80-100%, such as, for example, 85-99.9%, particularly 90-99.9%, more particularly 95-99.9%, or 98-99.9%, particularly 99-99.9%.

[0043] "Predominantly" refers to a percentage in the range of more than 50%, for example, in the range of 51-100%, particularly 75-99.99%, more particularly 85-99.5%, for example, in the range of 95-99%.

[0044] "Major product" in the context of the present invention denotes a single compound or a group of at least two compounds, for example two, three, four, five or more, in particular two or three compounds, which is "predominantly" prepared by a reaction as described herein and which is present in said reaction in a predominant proportion based on the total amount of the constituents of the product formed by said reaction. Said proportion may be a molar ratio, a weight ratio, or preferably an area ratio calculated from the corresponding chromatograms of the reaction products based on chromatographic analysis.

[0045] A "by-product" in the context of the present invention denotes a single compound or a group of at least two compounds, e.g. two, three, four, five or more, in particular two or three compounds, which single compound or group of compounds is not "predominantly" prepared by a reaction as described herein.

[0046] Because enzymatic reactions are reversible, the present invention relates to both directions of the enzymatic or biocatalytic reactions described herein, unless otherwise stated.

[0047] "Functional variants" of the polypeptides described herein include "functional equivalents" of the polypeptides as defined below.

[0048] The term "stereoisomer" specifically includes conformational isomers.

[0049] In general, the present invention includes all "stereoisomeric forms" of the compounds described herein, such as structural isomers, particularly stereoisomers, and mixtures thereof, such as optical isomers, or geometric isomers, such as E-isomers and Z-isomers, and combinations thereof. When multiple asymmetric centers are present in a molecule, the present invention encompasses all combinations of the different conformations of these asymmetric centers, such as enantiomeric pairs.

[0050] "Stereoselectivity" describes the ability to produce a particular stereoisomer of a compound in stereoisomerically pure form or to specifically convert a particular stereoisomer from multiple stereoisomers in an enzyme-catalyzed process as described herein. More specifically, this means that the product of the present invention is enriched with respect to a particular stereoisomer or that the extract is diluted with respect to a particular stereoisomer. This can be quantified via the purity %ee parameter, calculated according to the following formula: %ee=[X A -X B ] / [X A +X B ] * 100 In the formula, X A and X B represents the molar ratio (molar fraction) of stereoisomers A and B.

[0051] The term "selectively converting" or "enhancing selectivity" generally means that a particular stereoisomeric form of an unsaturated hydrocarbon, such as the E form, is converted in a higher percentage or amount (compared on a molar basis) than the corresponding other stereoisomeric form, such as the Z form, either during the entire course of the reaction (i.e., between the start and end of the reaction), at a particular time point in the reaction, or during an "interval" of the reaction. In particular, the selectivity may be observed during an "interval" corresponding to a conversion rate of 1-99%, 2-95%, 3-90%, 5-85%, 10-80%, 15-75%, 20-70%, 25-65%, 30-60%, or 40-50% of the initial amount of substrate. The higher percentage or amount may be expressed, for example, in terms of: - a higher maximum yield of the isomer observed during the entire course of the reaction or during said interval; - a higher relative amount of an isomer at a defined percentage of the conversion of the substrate; and / or - Identical relative amounts of isomers at higher % conversion values and each is preferably observed in comparison to a reference method, said reference method being a known chemical or biochemical means performed under otherwise identical conditions.

[0052] In general, in accordance with the present invention, all "isomeric forms" of the compounds described herein are included, such as structural isomers, particularly stereoisomers, and mixtures thereof, such as optical isomers, or geometric isomers, such as E-isomers and Z-isomers, as well as combinations thereof. When multiple asymmetric centers are present in a molecule, the present invention includes all combinations of the different conformations of these asymmetric centers, such as pairs of enantiomers, or any mixtures of stereoisomeric forms.

[0053] The "yield" and / or "conversion" of the reaction according to the invention is determined over a defined period of time, for example 4, 6, 8, 10, 12, 16, 20, 24, 36 or 48 hours, during which the reaction is carried out. In particular, the reaction is carried out under precisely defined conditions, for example "standard conditions" as defined herein.

[0054] Different yield parameters ("yield" or Y P / S "specific yield"; or "space-time yield (STY)") is well known in the art and is determined as described in the literature.

[0055] "Yield" and "Y P / S " (expressed as mass of product produced / mass of material consumed, respectively) are used synonymously herein.

[0056] Specific productivity yield describes the amount of product produced per gram of biomass per hour and per L of fermentation broth. The amount of wet cell weight, described as WCW, describes the amount of biologically active microorganisms in a biochemical reaction. This value is expressed as grams of product per gram of WCW per hour (i.e., g / gWCW). -1 h -1 Alternatively, the amount of biomass can be expressed as the amount of dry cell weight, described as DCW. Furthermore, the biomass concentration is given as the optical density at 600 nm (OD 600 ) and use an experimentally determined correlation coefficient to estimate the corresponding wet or dry cell weight, respectively.

[0057] The term "fermentative production" or "fermentation" refers to the ability of a microorganism (assisted by enzymatic activity contained in or caused by said microorganism) to produce a compound in cell culture utilizing at least one carbon source added to the incubation.

[0058] The term "fermentation broth" is understood to mean a liquid, in particular an aqueous or aqueous / organic solution, that is based on a fermentation process and that is untreated or, for example, treated as described herein.

[0059] An "enzymatically catalyzed" or "biocatalytic" process means that the process is carried out under the catalysis of an enzyme, including an enzyme variant, as defined herein. Thus, the process can be carried out either in the presence of said enzyme in isolated (purified, concentrated) or crude form, or in the presence of a cellular system, in particular a natural or recombinant microbial cell, that contains said enzyme in active form and has the ability to catalyze a conversion reaction as disclosed herein.

[0060] Where the present disclosure refers to features, parameters and ranges thereof (including general, explicitly non-preferred features, parameters and ranges) of differing degrees of preference, unless otherwise stated, combinations of two or more of such features, parameters and ranges are encompassed by the disclosure herein, regardless of their respective degrees of preference.

[0061] Detailed Description a. Specific embodiments of the present invention 1. A method for the biocatalytic production of at least one, in particular one, two or three, in particular one or two, drimanyl acetate compounds, comprising: (1) contacting at least one, particularly one, drimanyl alcohol in stereoisomerically pure form or in the form of a mixture of stereoisomers in the presence of an acetyl group donor with at least one, particularly one, polypeptide having an acetyltransferase activity capable of transferring an acetyl group from said acetyl group donor to at least one, particularly one, drimanyl alcohol, to obtain at least one drimanyl acetate as main product, particularly one drimanyl acetate as main product; (2) optionally isolating at least one, and in particular one, drimanyl acetate compound from the reaction product of step (1); A method comprising: If multiple drimanyl acetates are formed, the mixture may be further separated and the individual acetates purified.

[0062] 2. The method of embodiment 1, wherein the dorimanyl acetate compound is selected from the group consisting of arbikanil acetate, dorimenyl acetate, and bicyclofarnesyl acetate, each of which is present in a stereoisomerically pure form, or as a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of the acetate group. In certain embodiments, only one dorimanyl alcohol is used as a substrate, and only one dorimanyl acetate is obtained as a product, in a stereoisomerically pure form, or as a mixture of at least two stereoisomers thereof, particularly in a stereoisomerically pure form.

[0063] 3. The method of embodiment 1 or 2, wherein the drimanyl alcohol is selected from the group consisting of arbicanol, particularly (+)-arbicanol, drimenol, particularly (-)-drimenol, and bicyclofarnesol, particularly (+)-bicyclofarnesol, each present in stereoisomerically pure form or as a mixture of at least two stereoisomers thereof, or a combination thereof comprising at least two members of said group of alcohols. In certain embodiments, only one drimanyl alcohol, particularly one in stereoisomerically pure form, is used as the substrate.

[0064] 4. The method of any one of the preceding embodiments, wherein the acetyl group donor is acetyl-coenzyme A (acetyl-CoA). The donor may be exogenously added to the reaction mixture, for example in an in vitro process applying an isolated, enriched or purified enzyme, or more specifically may be present endogenously, for example in an in vivo process applying a host cell system that produces acetyl-CoA as a metabolite and expresses a polypeptide or polypeptides required to perform the intended acetylation or a more complex multi-step process involving said acetylation as one step.

[0065] 5. The acetyltransferase is a) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 118, 121, 124, 127, 130, 133, 136, 143 and 144, and b) a polypeptide having acetyltransferase activity and comprising an amino acid sequence exhibiting a degree of sequence identity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with at least one of the amino acid sequences of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 118, 121, 124, 127, 130, 133, 136, 143, and 144. 3. The method of any one of the preceding embodiments, selected from:

[0066] The ability of certain acetyltransferases of the invention to produce one or more drimenyl acetate compounds selected from the group consisting of arbikanyl acetate, drimenyl acetate, and bicyclofarnesyl acetate is illustrated by the following list: [Table 1]

[0067] 6. The method of any one of the preceding embodiments, further comprising biocatalytically forming said at least one, particularly one, drimanyl alcohol compound prior to step 1).

[0068] 7. The method of embodiment 6, wherein the drimanyl alcohol compound may be present endogenously in a reaction mixture, for example in an in vivo process employing a host cell system expressing a polypeptide or polypeptides required to produce the drimanyl acetate compound as a metabolite and to carry out the intended drimanyl alcohol synthesis or a more complex multi-step process involving the drimanyl alcohol synthesis as one step, whereby the drimanyl alcohol is enzymatically synthesized from an acyclic sesquiterpene precursor.

[0069] Alternatively, the drimanyl alcohol compound may be produced chemically or enzymatically and added exogenously to the reaction mixture, for example in an in vitro process applying isolated, enriched or purified synthase enzymes required for its formation, as defined below.

[0070] 8. The method of embodiment 7, wherein said acyclic sesquiterpene precursor is farnesyl pyrophosphate (FPP).

[0071] 9. The method of embodiment 7 or 8, wherein the enzymatic synthesis of drimanyl alcohol is catalyzed by one or more polypeptides capable of converting the acyclic sesquiterpene precursor in one or more enzymatic steps to at least one drimanyl alcohol, in particular one drimanyl alcohol, in particular as the main product.

[0072] 10. The method of any one of embodiments 6 to 9, wherein said at least one drimanyl alcohol is produced from FPP in one or more, in particular two, enzymatic steps.

[0073] 11. The at least one drimanyl alcohol is a) a polypeptide having drimanyl sesquiterpene synthase activity that forms said drimanyl alcohol (one-step biosynthesis); or b) a polypeptide having drimanyl phosphate synthase activity that forms at least one drimanyl phosphate intermediate and a polypeptide having phosphatase activity that converts said at least one drimanyl phosphate (monophosphate and / or diphosphate) intermediate into at least one drimanyl alcohol (two-step biosynthesis) 11. The method of embodiment 10, wherein the FPP is produced by enzymatic conversion of FPP catalyzed by

[0074] 12. a) said polypeptide having drimane sesquiterpene synthase activity is selected from arbikanol synthase activity, drimenol synthase activity, bicyclofarnesol synthase activity or any combination of such activities, in particular exhibiting preferentially one of said activities, more particularly exhibiting specifically one of said activities; and b) the polypeptide combination comprises a drimanyl diphosphate synthase activity, in particular an arbikanyl diphosphate synthase activity, and a phosphatase enzyme, such as a bacterial alkaline phosphatase; The method of embodiment 11.

[0075] 13. a) the polypeptide having drimane sesquiterpene synthase activity is selected from drimane synthases as described in PCT / EP2018 / 064344 (filed May 31, 2018) and drimenol synthases as described in WO 2015 / 169871 and WO 2015 / 176959, b) The polypeptide having drimanyl phosphate synthase activity is an arbikanyl diphosphate synthase as described in the specification of International Application PCT / CN2018 / 088902 filed on May 29, 2018, which comprises the ability to produce an arbikanyl phosphate derivative such as monophosphate, more specifically an arbikanyl diphosphate derivative from farnesyl diphosphate (FPP) as a substrate. The method of embodiment 12.

[0076] Albikanyl diphosphate synthase as described in International Application PCT / CN2018 / 088902 (filed May 29, 2018) is DfHAD, DfHAD-9(V274A), DfHAD-His_GST, and DfHAD-8(K532R) from Dryopteris fragrans and polypeptides derived therefrom having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. It is.

[0077] Drimane synthase (i.e., albicanol synthase or drimenol synthase) as described in International Application PCT / EP2018 / 064344 (filed May 31, 2018) is [Table 2] and polypeptides derived therefrom having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. It is.

[0078] Dorimenol synthase as described in WO 2015 / 169871 is [Table 3] and polypeptides derived therefrom having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. It is.

[0079] The dorimenol synthase as described in WO 2015 / 176959 is VaTPS3 of Valeriana amurensis and polypeptides derived therefrom having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. It is.

[0080] The SEQ ID NOs for the amino acid and nucleotide sequences of each of the above synthase enzymes are listed at the end of this specification. These polypeptides and nucleic acids, as well as polypeptides and nucleic acids derived therefrom having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of these sequences, are also part of the disclosure.

[0081] 14. The drimane sesquiterpene synthase is a) a polypeptide having (bifunctional) arbicanol synthase activity and comprising the amino acid sequence of SEQ ID NO:5, or a variant or variant polypeptide having arbicanol synthase activity and comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:5, as described in International Application PCT / EP2018 / 064344; b) A polypeptide having (bifunctional) drimenol synthase activity and comprising the amino acid sequence of SEQ ID NO: 7, or a variant or variant polypeptide having drimenol synthase activity and comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7, as described in International Application PCT / EP2018 / 064344. 14. The method of embodiment 13, wherein said method is selected from the group consisting of

[0082] 15. The method of any one of the preceding embodiments, carried out in vivo in a host cell culture, or in vitro in a host cell lysate or a liquid reaction medium containing concentrated or isolated polypeptides required to produce at least one drimanyl acetate, each under conditions conducive to the production of at least one drimanyl acetate.

[0083] In particular, the reaction is carried out in the presence of endogenously formed or exogenously added acetyl-CoA as an acetyl group donor. In particular, the reaction is carried out in the presence of endogenously formed or exogenously added FPP. In particular, endogenously formed FPP is the result of metabolism of at least one carbon source, such as a sugar substrate, that is bioconvertible to FPP. Of particular interest are in vivo methods within cells in which FPP and acetyl-CoA are endogenously formed.

[0084] Some of these host cells or organisms do not naturally produce FPP.Suitable for carrying out the method of the embodiment as described herein, an organism or cell that does not naturally produce acyclic terpene pyrophosphate precursor, such as FPP, is genetically modified to produce said precursor.The organism or cell can be so transformed, for example, before or simultaneously with modification with the nucleic acid described according to any of the above embodiments.Methods for transforming an organism to produce acyclic terpene pyrophosphate precursor, such as FPP, are already known in the art.For example, introducing the enzyme activity of the mevalonate pathway is a suitable strategy for making an organism produce FPP.

[0085] 16. a) at least one acetyltransferase as defined in embodiment 5; optionally b) at least one polypeptide capable of converting the acyclic sesquiterpene precursor FPP into at least one drimanyl alcohol as defined in any one of embodiments 9 to 14; and optionally c) at least one enzyme selected from the enzymes involved in the mevalonate pathway defined above 16. The method of embodiment 15, wherein the method is carried out in a recombinant non-human host cell or organism capable of functionally expressing

[0086] In a particular embodiment, the enzymes a) and b) or the enzymes a), b) and c) are functionally expressed by a cell line as applied in the in vivo method of the invention.

[0087] 17. The method of embodiment 16, wherein said non-human host cell or host organism is selected from a prokaryotic or eukaryotic microorganism, or a cell derived therefrom.

[0088] 18. The method of embodiment 17, wherein said non-human host cell or host organism is selected from bacterial, fungal and plant cells or plants.

[0089] 19. The method of embodiment 18, wherein the fungal cell is a yeast cell, in particular a yeast cell selected from the genera Saccharomyces, Pichia or Yarrowia, in particular from the species Saccharomyces cerevisiae (budding yeast), Pichia pastori (Pichia yeast) or Yarrowia lipolytica (alkane-utilizing yeast).

[0090] 20. The method of embodiment 18, wherein said bacterial cell is selected from the genus Rhodococcus, Pseudomonas, Bacillus, or Escherichia, in particular Escherichia coli.

[0091] 21. The method of any one of the preceding embodiments, further comprising, as step (3), treating at least one drimanyl acetate of step (1) or step (2) to obtain a derivative using chemical synthesis or biocatalytic synthesis or a combination of both.

[0092] 22. The method of embodiment 21, wherein the derivative is a hydrocarbon, alcohol, diol, triol, acetal, ketal, aldehyde, acid, ether, amide, ketone, lactone, epoxide, acetate, glycoside, ester and / or polycyclic compound.

[0093] 23. The method of any one of the preceding embodiments, wherein the drimanyl acetate comprises arbikanyl acetate, drimenyl acetate or bicyclofarnesyl acetate, particularly arbikanyl acetate or drimenyl acetate, as the predominant drimenyl alcohol product or particularly as the sole drimenyl alcohol product.

[0094] 24. The method, in particular, comprises the steps of: a) at least one nucleic acid, expression construct or vector comprising a nucleic acid sequence encoding at least one polypeptide having acetyltransferase activity capable of transferring an acetyl group from an acetyl group donor to a drimanyl alcohol, optionally stably integrated into the genome; b) at least one nucleic acid, expression construct or vector comprising a nucleic acid sequence encoding at least one polypeptide having drimanyl alcohol synthase activity capable of producing drimanyl alcohol from an acyclic sesquiterpene precursor, optionally stably integrated into the genome; c) at least one nucleic acid, expression construct or vector comprising a nucleic acid sequence encoding at least one polypeptide involved in a biosynthetic pathway for producing said acyclic sesquiterpene precursor, optionally stably integrated into the genome; 3. The method of any one of the preceding embodiments, comprising transforming with

[0095] In a particular embodiment, the non-human host organism or host cell is transformed with a) and b), or a), b) and c), and more particularly comprises said nucleic acids stably integrated into the genome. Said nucleic acids a), b) and / or c) may be located on the same or on two or more different vectors.

[0096] 25. A polypeptide having acetyltransferase activity and capable of transferring an acetyl group from an acetyl group donor to a drimanyl alcohol to produce drimanyl acetate, the polypeptide comprising an amino acid sequence having 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more but less than 100% sequence identity to at least one amino acid sequence selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 118, 121, 124, 127, 130, 133, 136, 143, and 144.

[0097] 26. An isolated nucleic acid molecule comprising: a) comprises a nucleotide sequence encoding the polypeptide of embodiment 5; or b) comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% but less than 100% sequence identity to a nucleotide sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 116, 117, 119, 120, 122, 123, 125, 126, 128, 129, 131, 132, 134, and 135; or c) comprises a nucleotide sequence that comprises a sequence complementary to one of the sequences in a) or b); or d) comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of a), b) or c); An isolated nucleic acid molecule.

[0098] 27. An expression construct comprising at least one nucleic acid molecule of embodiment 26.

[0099] 28. A vector comprising at least one nucleic acid molecule of embodiment 25 or at least one expression construct of embodiment 26.

[0100] 29. The vector of embodiment 28, wherein the vector is a prokaryotic, viral or eukaryotic vector.

[0101] 30. The vector of embodiment 28 or 29, wherein the vector is an expression vector.

[0102] 31. The vector of any one of embodiments 28 to 30, wherein the vector is a plasmid vector.

[0103] 32. A recombinant host cell or a recombinant non-human host organism, comprising: a) at least one isolated nucleic acid molecule of embodiment 26, optionally stably integrated into the genome; or b) at least one expression construct according to embodiment 27, optionally stably integrated into the genome; or c) At least one vector according to any one of embodiments 28 to 31. A recombinant host cell or a recombinant non-human host organism comprising:

[0104] In a particular embodiment, the non-human host organism or host cell is transformed with a) and b), or a), b) and c), and more particularly comprises said nucleic acid stably integrated into the genome.

[0105] 33. The host cell or host organism of embodiment 32, selected from a prokaryotic or eukaryotic microorganism, or a cell derived therefrom.

[0106] 34. The host cell or host organism of embodiment 33, selected from bacterial, fungal and plant cells or plants.

[0107] 35. The host cell or host organism of embodiment 34, wherein the fungal cell is a yeast cell.

[0108] 36. The host cell or host organism of embodiment 35, wherein the bacterial cell is selected from the genus Escherichia, in particular from the species Escherichia coli, and the yeast cell is selected from the genus Saccharomyces, Pichia, or Yarrowia, in particular from the species Saccharomyces cerevisiae, Pichia pastoris, or Yarrowia lipolytica.

[0109] 37. A method for producing at least one catalytically active polypeptide according to embodiment 25, comprising: a) culturing a non-human host organism or host cell according to any one of embodiments 32 to 34 to express or overexpress at least one polypeptide according to embodiment 25; b) optionally isolating the polypeptide from the non-human host cell or organism cultured in step a); A method comprising:

[0110] 38. The method of embodiment 37, further comprising, prior to step a), a step of providing that a non-human host organism or cell is transformed with at least one nucleic acid according to claim 26, or at least one construct according to claim 27, or at least one vector according to any one of claims 28 to 31, in particular such that the non-human host organism or cell expresses or overexpresses the polypeptide according to claim 25.

[0111] 39. A method for preparing a variant polypeptide comprising an acetyltransferase activity and capable of transferring an acetyl group from an acetyl group donor to at least one, particularly one, drimanyl alcohol to produce at least one, particularly one, drimanyl acetate, the method comprising: a) selecting a nucleic acid molecule encoding a polypeptide selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 118, 121, 124, 127, 130, 133, 133, 136, 143, and 144; b) modifying the selected nucleic acid molecule to obtain at least one mutant nucleic acid molecule; c) transforming a host cell or a unicellular host organism with the mutant nucleic acid sequence to express the polypeptide encoded by the mutant nucleic acid sequence; d) screening the expression products for at least one variant that contains acetyltransferase activity; e) optionally, if the polypeptide does not have the desired mutant activity, repeating process steps a) to d) until a polypeptide having the desired mutant activity is obtained; f) optionally, if a polypeptide having the desired mutant activity is identified in step d), isolating the corresponding mutant nucleic acid obtained in step c); A method comprising:

[0112] 40. Use of an acetyltransferase as defined in any one of the preceding embodiments for preparing an odorant, flavor or fragrance ingredient or an insect / pest control ingredient, for example for use in the preparation of a composition selected from a body care, home care, or fragrance composition.

[0113] b. Polypeptides applicable according to the present invention In this context, the following definitions apply: The general terms "polypeptide" or "peptide" may be used interchangeably and refer to natural or synthetic linear chains or sequences of peptide-linked contiguous amino acid residues, including from about 10 to more than about 1,000 residues. Shorter polypeptides having up to 30 residues are also referred to as "oligopeptides."

[0114] The term "protein" refers to a macromolecular structure comprising one or more polypeptides. The amino acid sequence of the polypeptide(s) represents the "primary structure" of the protein. The amino acid sequence also determines the "secondary structure" of the protein by the formation of special structural elements such as α-helical and β-sheet structures formed within the polypeptide chain. The arrangement of multiple such secondary structural elements defines the "tertiary structure" or spatial arrangement of the protein. When a protein comprises multiple polypeptide chains, said chains are arranged spatially to form the "quaternary structure" of the protein. Correct spatial arrangement or "folding" of a protein is a prerequisite for the function of the protein. Denaturation or unfolding destroys the function of the protein. If such destruction is reversible, the function of the protein can be restored by refolding.

[0115] A typical protein function referred to herein is an "enzymatic function", i.e., the protein acts as a biocatalyst on a substrate, e.g., a compound, and catalyzes the conversion of said substrate to a product. Enzymes may exhibit a high or low degree of substrate and / or product specificity.

[0116] Thus, a "polypeptide" referred to herein as having a particular "activity" implicitly refers to a correctly folded protein that exhibits the indicated activity, such as a particular enzymatic activity.

[0117] Thus, unless otherwise indicated, the term "polypeptide" also encompasses the terms "protein" and "enzyme."

[0118] Similarly, the term "polypeptide fragment" encompasses the terms "protein fragment" and "enzyme fragment."

[0119] The term "isolated polypeptide" refers to an amino acid sequence that has been removed from its natural environment by any method or combination of methods known in the art, including recombinant, biochemical and synthetic methods.

[0120] "Targeting peptide" refers to an amino acid sequence that targets a protein or polypeptide to an intracellular organelle, i.e., mitochondria or plastids, or to the extracellular space (secretory signal peptide). The nucleic acid sequence encoding the targeting peptide can be fused to a nucleic acid sequence encoding the amino terminus, e.g., the N-terminus, of the protein or polypeptide, or can be used in place of the native targeting polypeptide.

[0121] The present invention also relates to "functional equivalents" (also called "analogs" or "functional variants") of the polypeptides specifically described herein.

[0122] For example, a "functional equivalent" refers to a polypeptide that exhibits an enzymatic activity that is at least 1-10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% greater or less than the enzymatic activity of a polypeptide specifically described herein in a test used to determine the enzymatic activity and that serves as a basis for said comparison.

[0123] "Functional equivalents" also refer according to the invention to specific variants which, at at least one sequence position of the amino acid sequences described herein, have an amino acid different from the specifically mentioned amino acid, but which nevertheless have one of the above mentioned biological activities, such as for example enzymatic activity. Thus, "functional equivalents" include variants obtained by addition, substitution, in particular conservative substitution (i.e. so that the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility), deletion and / or inversion of one or more, for example 1 to 20, in particular 1 to 15 or 5 to 10, amino acids, where the described changes can occur at any sequence position, provided that they result in a variant with a profile of properties according to the invention. Functional equivalence is observed when the activity pattern is qualitatively consistent between the variant and the unchanged polypeptide, i.e. for example when interaction with the same agonist or antagonist or substrate is observed, but at different rates (i.e. EC50 or IC 50 Conservative amino acid substitutions (wherein the substitutions are expressed by a value of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6

[0124] [Table 4]

[0125] "Functional equivalents" in the above sense are also "precursors" of the polypeptides described herein, as well as "functional derivatives" and "salts" of the polypeptides.

[0126] A "precursor" in this case is a natural or synthetic precursor of a polypeptide, with or without the desired biological activity.

[0127] The expression "salts" refers to salts of carboxyl groups as well as acid addition salts of amino groups of the protein molecules according to the invention. The salts of carboxyl groups can be produced in a known manner and include inorganic salts, such as sodium, calcium, ammonium, iron and zinc salts, as well as salts with organic bases, such as amines, for example triethanolamine, arginine, lysine, piperidine, etc. Acid addition salts, such as salts with inorganic acids, for example hydrochloric acid and sulfuric acid, as well as salts with organic acids, such as acetic acid and oxalic acid, are also within the scope of the present invention.

[0128] "Functional derivatives" of the polypeptides according to the invention can also be produced on functional amino acid side groups or at their N- or C-termini using known techniques. Such derivatives include, for example, aliphatic esters of carboxylic acid groups, obtained by reaction with ammonia or with primary or secondary amines, amides of carboxylic acid groups, N-acyl derivatives of free amino groups produced by reaction with acyl groups, or O-acyl derivatives of free hydroxyl groups produced by reaction with acyl groups.

[0129] "Functional equivalents" of course also include naturally occurring variants as well as polypeptides obtainable from other organisms. For example, the extent of homologous sequence regions can be established by sequence comparison and equivalent polypeptides can be determined based on the specific parameters of the invention.

[0130] "Functional equivalents" also include "fragments" such as individual domains or sequence motifs of the polypeptides according to the invention, or N- and / or C-terminal truncated forms, which may or may not exhibit a desired biological function. Preferably, such "fragments" retain at least a qualitatively desired biological function.

[0131] "Functional equivalents" are also fusion proteins that have one of the polypeptide sequences described herein or functional equivalents derived therefrom functionally associated at the N-terminus or C-terminus with at least one additional, functionally different, heterologous sequence (i.e., without substantial mutual dysfunction of the fusion protein moieties). Non-limiting examples of these heterologous sequences are, for example, signal peptides, histidine anchors or enzymes.

[0132] "Functional equivalents" also included according to the present invention are homologues to the specifically disclosed polypeptides. These have a homology (or identity) of at least 60%, preferably at least 75%, in particular at least 80 or 85%, such as 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, with one of the specifically disclosed amino acid sequences, calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448. The homology or identity (expressed as a percentage) of the homologous polypeptide according to the present invention means in particular the identity (expressed as a percentage) of the amino acid residues based on the full length of one of the specifically described amino acid sequences.

[0133] Identity data expressed as a percentage may also be determined by BLAST alignment, using the algorithm blastp (protein-protein BLAST) or by applying the Clustal settings described below.

[0134] In the case of possible protein glycosylation, "functional equivalents" according to the invention include not only the polypeptides as described herein in deglycosylated or glycosylated form, but also modified forms which can be obtained by altering the glycosylation pattern.

[0135] Functional equivalents or homologues of the polypeptides according to the invention can be generated by mutagenesis, for example by point mutation, lengthening or shortening of the protein, or as explained in more detail below.

[0136] Functional equivalents or homologs of the polypeptides according to the invention can be identified by screening a combinatorial database of mutants, such as truncation mutants. For example, a diverse database of protein variants can be created by combinatorial mutagenesis at the nucleic acid level, for example by enzymatic ligation of a mixture of synthetic oligonucleotides. Numerous methods can be used to create a database of potential homologs from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automatic DNA synthesizer, and the synthetic genes can then be ligated with a suitable expression vector. The use of degenerate genomes makes it possible to provide all sequences in a mixture, which code for the desired set of potential protein sequences. Methods for the synthesis of degenerate oligonucleotides are known to those skilled in the art.

[0137] In the prior art, several techniques are known for screening gene products of combinatorial databases created by point mutation or truncation, and for screening cDNA libraries for gene products with selected properties. These techniques can be adapted for rapid screening of gene banks produced by combinatorial mutagenesis of homologs according to the present invention. The most frequently used technique for screening large gene banks based on high-throughput analysis involves cloning the gene bank in replicable expression vectors, transformation of suitable cells with the resulting vector database, and expression of the combinatorial genes under conditions in which detection of the desired activity facilitates the isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM) is a technique that increases the frequency of functional variants in a database and can be used in combination with screening tests to identify homologs.

[0138] Embodiments provided herein provide orthologs and paralogs of the polypeptides disclosed herein, as well as methods for identifying and isolating such orthologs and paralogs. Definitions of the terms "ortholog" and "paralog" are provided below and apply to amino acid and nucleic acid sequences.

[0139] c. Coding nucleic acid sequences applicable according to the present invention In this context, the following definitions apply: The terms "nucleic acid sequence", "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a sequence of nucleotides. Nucleic acid sequences can be single- or double-stranded deoxyribonucleotides or ribonucleotides of any length, and include coding and non-coding sequences of genes, exons, introns, sense and antisense complementary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers and nucleic acid probes. Those skilled in the art will appreciate that the nucleic acid sequence of RNA is identical to the DNA sequence, with the difference being that uracil (U) is used instead of thymine (T). The term "nucleic acid sequence" should also be understood as including polynucleotide or oligonucleotide molecules in the form of separate fragments or as components of a larger nucleic acid.

[0140] An "isolated nucleic acid" or "isolated nucleic acid sequence" refers to a nucleic acid or nucleic acid sequence that is in an environment other than that in which it naturally occurs, and may be substantially free of contaminating endogenous material.

[0141] The term "naturally-occurring" as applied herein to nucleic acids refers to nucleic acids that are found within the cells of organisms in nature and have not been intentionally modified by man in the laboratory.

[0142] A "fragment" of a polynucleotide or nucleic acid sequence refers to a contiguous nucleotide of the polynucleotide of the embodiment herein, particularly at least 15bp, at least 30bp, at least 40bp, at least 50bp and / or at least 60bp in length.In particular, a 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 the embodiment herein.Without being limited thereto, a fragment of a polynucleotide of the embodiment herein can be used as a PCR primer and / or as a probe, or for antisense gene silencing or RNAi.

[0143] As used herein, the term "hybridization" or hybridizing under certain conditions is intended to describe hybridization and washing conditions under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions may be such that sequences with at least about 70%, such as at least about 80%, such as at least about 85%, 90%, or 95% identity remain bound to each other. Definitions of low stringency, medium stringency, and high stringency hybridization conditions are provided herein below. Appropriate hybridization conditions can be selected by those skilled in the art with minimal experimentation, as exemplified by Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Further stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0144] A "recombinant nucleic acid sequence" is a nucleic acid sequence that results from the use of laboratory methods (e.g., molecular cloning) to combine genetic material from more than a source to create or modify a nucleic acid sequence that does not occur in nature and is not otherwise found in an organism.

[0145] "Recombinant DNA techniques" refers to molecular biology procedures for preparing recombinant nucleic acid sequences, e.g., as described in Laboratory Manuals edited by Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.

[0146] The term "gene" refers to a DNA sequence that comprises an RNA molecule, e.g., a region that is transcribed into an mRNA in a cell, operably linked to an appropriate regulatory region, e.g., a promoter. Thus, a gene may comprise multiple operably linked sequences, such as a promoter, e.g., a sequence involved in translation initiation, a coding region of cDNA or genomic DNA, introns, 5' leader sequences including exons, and / or 3' untranslated sequences, e.g., including a transcription termination site.

[0147] "Polycistronic" refers to a nucleic acid molecule, particularly an mRNA, that can separately encode two or more polypeptides within the same nucleic acid molecule.

[0148] "Chimeric gene" refers to any gene that is not normally found in nature in a species, particularly a gene in which there are one or more portions of nucleic acid sequences that are not naturally associated with each other. For example, a promoter is not naturally associated with part or all of the transcription region or with another regulatory region. The term "chimeric gene" is understood to include an expression construct in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or antisense, i.e., the reverse complement of the sense strand, or inverted repeat sequences (sense and antisense, whereby the RNA transcript forms a double-stranded RNA upon transcription). The term "chimeric gene" also includes genes obtained by combining parts of one or more coding sequences to produce a new gene.

[0149] "3'UTR" or "3' untranslated sequence" (also called "3' untranslated region" or "3' end") refers to nucleic acid sequences found downstream of the coding sequence of a gene, including, for example, a transcription termination site and (in most, but not all, eukaryotic mRNAs) a polyadenylation signal, such as AAUAAA or a variant thereof. After transcription termination, 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., the cytoplasm.

[0150] The term "primer" refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and is used to polymerize a nucleic acid sequence complementary to the template.

[0151] The term "selectable marker" refers to any gene that, upon expression, can be used to select a cell or cells that contain the selectable marker. Examples of selectable markers are described below. Those skilled in the art will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.

[0152] The present invention also relates to nucleic acid sequences which code for the polypeptides defined herein.

[0153] In particular, the present invention also relates to nucleic acid sequences (single- and double-stranded DNA and RNA sequences, e.g. cDNA, genomic DNA and mRNA) encoding one of the above-mentioned polypeptides and their functional equivalents, which can be obtained, for example, using artificial nucleotide analogues.

[0154] The present invention relates both to isolated nucleic acid molecules encoding a polypeptide according to the invention or a biologically active segment thereof, and to nucleic acid fragments which can be used, for example, as hybridization probes or primers to identify or amplify the encoding nucleic acid according to the invention.

[0155] The present invention also relates to nucleic acids that have a degree of "identity" with the sequences specifically disclosed herein. "Identity" between two nucleic acids means in each case nucleotide identity over the entire length of the nucleic acid.

[0156] The "identity" between two nucleotide sequences (also for peptide or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues) that are identical in the two sequences when an alignment of these two sequences is generated. An identical residue is defined as a residue that is identical in the two sequences at a given position in the alignment. As used herein, the percentage of sequence identity is calculated from the optimal alignment by taking the number of identical residues between the 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. To obtain an optimal alignment, gaps may be introduced in one or both sequences at one or more positions in the alignment. These gaps are then considered as non-identical residues for the purposes of calculating the percentage of sequence identity. Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be accomplished in a variety of ways using computer programs, for example, publicly available computer programs available on the World Wide Web.

[0157] In particular, the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999), available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi) set to default parameters, can be used to obtain optimal alignments of protein or nucleic acid sequences and to calculate the percentage of sequence identity.

[0158] In another example, identity can be calculated using the Clustal Method (Higgins DG, Sharp PM. (1989)) with the following settings using Vector NTI Suite 7.1 from Informax, Inc. (USA): Multiple alignment parameters Gap Opening Penalty 10 Gap extension penalty 10 Gap Separation Penalty Range 8 Gap Separation Penalty Off % identity for alignment delay 40 Residue Specificity Gap Off Hydrophilic Residue Gap Off Transition weighting: 0 Pairwise alignment parameters: FAST algorithm on K tuple size 1 Gap Penalty 3 Window Size 5 Best diagonal number 5

[0159] Alternatively, identity may be determined according to Chenna, et al. (2003), web page: http: / / www.ebi.ac.uk / Tools / clustalw / index.html#, and the following settings: DNA Gap Open Penalty 15.0 DNA Gap Extension Penalty 6.66 DNA Matrix Identity Protein Gap Open Penalty 10.0 Protein extension penalty 0.2 Protein Matrix Gonnet Protein / DNA ENDGAP -1 Protein / DNA GAPDIST 4

[0160] All nucleic acid sequences described herein (single-stranded and double-stranded DNA and RNA sequences, e.g., cDNA and mRNA) can be produced by known methods by chemical synthesis from nucleotide building blocks, e.g., fragment condensation of individual overlapping complementary nucleic acid building blocks of double helix. Chemical synthesis of oligonucleotides can be carried out by known methods, e.g., by the phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation and gap filling of synthetic oligonucleotides by Klenow fragment of DNA polymerase and ligation reaction, as well as general cloning techniques, are described in Sambrook et al. (1989), see below.

[0161] A nucleic acid molecule according to the present invention may further comprise untranslated sequences from the 3' and / or 5' end of the coding gene region.

[0162] The present invention further relates to nucleic acid molecules which are complementary to the specifically described nucleotide sequences or segments thereof.

[0163] The nucleotide sequences according to the invention allow the generation of probes and primers that can be used for the identification and / or cloning of homologous sequences in other cell types and organisms. Such probes or primers generally comprise a nucleotide sequence region that hybridizes under "stringent" conditions (as defined elsewhere herein) on at least about 12, preferably at least about 25, e.g. about 40, 50 or 75 consecutive nucleotides of the sense strand or the corresponding antisense strand of the nucleic acid sequence according to the invention.

[0164] "Homologous" sequences include ortholog or paralog sequences. Methods for identifying orthologs or paralogs, including phylogenetic analysis, sequence similarity and hybridization methods, are known in the art and described herein.

[0165] "Paralogue" results from gene duplication, which results in two or more genes with similar sequences and similar functions. Paralogs are typically formed by gene duplication within plant species that cluster together and are related. Paralogs are found within groups of similar genes using pairwise Blast analysis, or during phylogenetic analysis of gene families using programs such as CLUSTAL. In paralogs, consensus sequences that are characteristic of sequences within related genes and have similar functions of genes can be identified.

[0166] "Orthologs" or orthologous sequences are sequences that are similar to each other because they are found in species that descend from a common ancestor. For example, it is known that plant species that share a common ancestor contain many enzymes with similar sequences and functions. Those skilled in the art can identify orthologous sequences and predict the function of orthologs by constructing a polygene tree of a certain gene family, for example, using CLUSTAL or BLAST programs. A method for identifying or confirming similar functions between homologous sequences is by comparing the transcript profiles of related polypeptides overexpressed and deleted (in knockout / knockdown) in a host cell or organism, such as a plant or microorganism. Those skilled in the art will understand that genes with similar transcript profiles, genes with more than 50% regulated transcripts in common, or genes with more than 70% regulated transcripts in common, or genes with more than 90% regulated transcripts in common have similar functions. Homologs, paralogs, orthologs and any other variants of the sequences herein are expected to function in a similar manner by generating host cells, organisms, such as plants or microorganisms, that produce terpene synthase proteins.

[0167] The term "selectable marker" refers to any gene that, upon expression, can be used to select a cell or cells that contain the selectable marker. Examples of selectable markers are described below. Those skilled in the art will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.

[0168] An "isolated" nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid, and further can be substantially free of other cellular material or culture medium if it is produced by recombinant techniques, and can be free of chemical precursors or other chemicals if it is chemically synthesized.

[0169] The nucleic acid molecules according to the invention can be isolated by standard techniques of molecular biology and the sequence information provided by the invention. For example, one of the specifically disclosed complete sequences or a segment thereof can be used as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, (1989)) to isolate cDNA from an appropriate cDNA library.

[0170] Furthermore, the nucleic acid molecule comprising one of the disclosed sequences or a segment thereof can be isolated by polymerase chain reaction using the oligonucleotide primer constructed based on this sequence.The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequencing.The oligonucleotide according to the present invention can also be produced by standard synthesis methods, for example, using an automatic DNA synthesizer.

[0171] The nucleic acid sequences according to the invention or derivatives thereof, homologues or parts of these sequences can be isolated from other bacteria, for example via genomic or cDNA libraries, by conventional hybridization or PCR techniques, which DNA sequences hybridize under standard conditions with the sequences according to the invention.

[0172] "Hybridize" refers to the ability of a polynucleotide or oligonucleotide to bind to a nearly complementary sequence under standard conditions, but under these conditions non-specific binding does not occur between non-complementary partners. For this, the sequences can be 90-100% complementary. The property of complementary sequences to be able to bind specifically to each other is exploited, for example, in Northern or Southern blotting, or in primer binding in PCR or RT-PCR.

[0173] Short oligonucleotides of the conserved regions are advantageously used for hybridization.However, it is also possible to use longer fragments or complete sequences of the nucleic acid according to the present invention for hybridization.These "standard conditions" vary depending on the nucleic acid (oligonucleotide, longer fragment, or complete sequence) used, or the type of nucleic acid (DNA or RNA) used for hybridization.For example, the melting temperature of DNA:DNA hybrid is about 10°C lower than that of DNA:RNA hybrid of the same length.

[0174] For example, depending on the particular nucleic acid, standard conditions mean a temperature of 42-58°C in an aqueous buffer solution with a concentration of 0.1-5xSSC (1xSSC = 0.15M NaCl, 15mM sodium citrate, pH 7.2) or even in the presence of 50% formamide, for example 42°C in 5xSSC, 50% formamide. Advantageously, the hybridization conditions in the case of DNA:DNA hybrids are 0.1xSSC and the temperature is about 20°C-45°C, preferably about 30°C-45°C. In the case of DNA:RNA hybrids, the hybridization conditions are advantageously 0.1xSSC and the temperature is about 30°C-55°C, preferably about 45°C-55°C. These stated temperatures for hybridization are examples of values ​​of melting temperature calculated for a nucleic acid with a length of about 100 nucleotides and a G+C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant genetics textbooks, for example, Sambrook et al., 1989, and can be calculated using formulas known to those skilled in the art, for example, depending on the length of the nucleic acid, the type of hybrid, or the G+C content. Those skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), (1985), Brown (ed) (1991).

[0175] "Hybridization" can be carried out under particularly stringent conditions, such as those described in Sambrook (1989) or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

[0176] As used herein, the term hybridization or hybridize under certain conditions is intended to describe the conditions of hybridization and washing under which the nucleotide sequences that are significantly identical or homologous to each other remain bound to each other.The conditions may be such that the sequences that have at least about 70%, for example at least about 80%, for example at least about 85%, 90% or 95% identity remain bound to each other.The definitions of low stringency, medium stringency and high stringency hybridization conditions are provided herein below.

[0177] Appropriate hybridization conditions can be selected by one of skill in the art with minimal experimentation, as exemplified by Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Further stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0178] As used herein, the defined conditions of low stringency are as follows: Filters containing DNA are pretreated for 6 hours at 40°C in a solution containing 35% formamide, 5x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (w / v) dextran sulfate, and 5-20x10 6Use 32P-labeled probes of 1000 ng / mL. Filters are incubated in hybridization mixture at 40°C for 18-20 h, then washed at 55°C for 1.5 h in a solution containing 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated at 60°C for an additional 1.5 h. Filters are blotted dry and exposed for autoradiography.

[0179] As used herein, the defined conditions of medium stringency are as follows: Filters containing DNA are pretreated for 7 hours at 50°C in a solution containing 35% formamide, 5x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (w / v) dextran sulfate, and 5-20x10 6 A 32P-labeled probe of 10 ...

[0180] As used herein, the defined conditions of high stringency are as follows: Prehybridization of DNA-containing filters is performed for 8 hours to overnight at 65°C in a buffer consisting of 6x SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg / ml denatured salmon sperm DNA. Prehybridization of 100 μg / ml denatured salmon sperm DNA and 5-20×10 6The filters are hybridized in a prehybridization mixture containing a 32P-labeled probe of cpm at 65° C. for 48 hours. The filters are washed in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA at 37° C. for 1 hour, followed by a wash in 0.1×SSC at 50° C. for 45 minutes.

[0181] Other conditions of low, medium, and high stringency well known in the art (e.g., conditions used for cross-species hybridizations) can be used when the above conditions are inappropriate (e.g., conditions used for cross-species hybridizations).

[0182] A detection kit for a nucleic acid sequence encoding a polypeptide of the present invention may include primers and / or probes specific for the nucleic acid sequence encoding the polypeptide, and an associated protocol for detecting the nucleic acid sequence encoding the polypeptide in a sample using the primers and / or probes. Such a detection kit can be used to determine whether a plant, organism, microorganism or cell has been modified, i.e., transformed with a sequence encoding the polypeptide.

[0183] To test the functionality of a variant DNA sequence according to embodiments herein, the sequence of interest is operably linked to a selectable or screenable marker gene and expression of said reporter gene is tested in transient expression assays, for example using microorganisms, or using protoplasts, or using stably transformed plants.

[0184] The present invention also relates to derivatives of the specifically disclosed or derivable nucleic acid sequences.

[0185] Thus, further nucleic acid sequences according to the invention may be derived from the sequences specifically disclosed herein and may differ therefrom by the addition, substitution, insertion or deletion of one or more (such as for example 1-10) nucleotides, for example 1-20, in particular 1-15 or 5-10 amino acids, and may further encode a polypeptide having the desired property profile.

[0186] The present invention also encompasses nucleic acid sequences which contain so-called silent mutations or which are modified compared to the specifically described sequences according to the codon usage of a particular source or host organism.

[0187] According to certain embodiments of the present invention, a variant nucleic acid can be prepared to adapt its nucleotide sequence to a particular expression system. For example, bacterial expression systems are known to express polypeptides more efficiently if the amino acids are encoded by specific codons. Due to the degeneracy of the genetic code, two or more codons may code for the same amino acid sequence, and multiple nucleic acid sequences may code for the same protein or polypeptide, and all of these DNA sequences are encompassed by the embodiments of the present specification. Where appropriate, the nucleic acid sequences encoding the polypeptides described herein may be optimized for increased expression in a host cell. For example, the nucleic acids of the embodiments of the present specification may be synthesized with host-specific codons to improve expression.

[0188] The present invention also encompasses naturally occurring variants of the sequences described herein, such as splice variants or allelic variants.

[0189] Allelic variants may have at least 60% homology at the derived amino acid level, preferably at least 80% homology, particularly preferably at least 90% homology over the entire sequence range (with regard to homology at the amino acid level, see details above for polypeptides). Advantageously, the homology may be higher over subregions of the sequence.

[0190] The present invention also relates to sequences resulting from conservative nucleotide substitutions, ie, whereby the subject amino acid is replaced with an amino acid of the same charge, size, polarity and / or solubility.

[0191] The present invention also relates to molecules derived from the specifically disclosed nucleic acids due to sequence polymorphisms. Such genetic polymorphisms may exist in cells or populations from different populations due to natural allelic variations. Allelic variants may include functional equivalents. These natural variations usually result in 1-5% variability in the nucleotide sequence of a gene. Said polymorphisms may result in changes in the amino acid sequence of the polypeptides disclosed herein. Allelic variants may also include functional equivalents.

[0192] Furthermore, derivatives should also be understood as homologues of the nucleic acid sequences according to the invention, such as animal, plant, fungal or bacterial homologues, truncated sequences, single-stranded DNA or RNA of coding and non-coding DNA sequences. For example, homologues have at the DNA level at least 40%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80% homology over the entire DNA region given in the sequences specifically disclosed herein.

[0193] Furthermore, derivatives should be understood to be, for example, fusions with promoters. The promoters added to the described nucleotide sequences can be modified by at least one nucleotide exchange, at least one insertion, inversion and / or deletion, but the functionality or effectiveness of the promoter is not impaired. Furthermore, the effectiveness of promoters can be increased by changing their sequences, or even completely replaced with more effective promoters, even in organisms of different genera.

[0194] d. Generation of Functional Polypeptide Variants Furthermore, those skilled in the art are familiar with methods for generating functional variants, i.e., nucleotide sequences encoding polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the amino acid related SEQ ID NOs as disclosed herein, and / or polypeptides encoded by nucleic acid molecules comprising a nucleotide sequence having at least 70% sequence identity to any one of the nucleotide related SEQ ID NOs as disclosed herein.

[0195] Depending on the technique used, the skilled artisan can introduce completely random or more directed mutations into genes or non-coding nucleic acid regions (which regions are important, for example, for regulating expression) and then generate gene libraries. The molecular biology methods required for this purpose are known to the skilled artisan and are described, for example, in Sambrook and Russell, Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001.

[0196] Methods for modifying genes, and thus the polypeptides encoded by them, have long been known to those skilled in the art and include, for example: - site-directed mutagenesis, in which individual nucleotides or multiple nucleotides of a gene are replaced in a directed manner (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey), - saturation mutagenesis, which allows the exchange or addition of codons for any amino acid at any point in a gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22:1593; Barettino D, Feigenbutz M, Valcarel R, Stunnenberg HG (1994) Nucleic Acids Res 22:541; Barik S (1995) Mol Biotechnol 3:1), - the error-prone polymerase chain reaction, in which the nucleotide sequence is mutated by an error-prone DNA polymerase (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18:3739); - the preferred exchange is prevented by the polymerase, SeSaM method (sequence saturation method) Schenk et al., Biospektrum, Vol. 3, 2006, 277-279; - passage of genes in mutator strains, for example, where an increased mutation rate of nucleotide sequences occurs due to defects in DNA repair mechanisms (Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E. coli mutator strain. In: Trower MK (Ed.) In vitro mutagenesis protocols. Humana Press, New Jersey), or -DNA shuffling, in which a pool of closely related genes is formed, digested, and the fragments are used as templates for polymerase chain reaction, ultimately generating a full-length mosaic gene by repeated strand separation and reannealing (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747).

[0197] Using so-called directed evolution (described inter alia in Reetz MT and Jaeger KE (1999), Topics Curr Chem 200:31; Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods for optimizing industrial polypeptides by directed evolution, In: Demain AL, Davies JE (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), the skilled person can generate functional variants in a directed manner on a large scale. For this, in a first step, a gene library of the respective polypeptide is first created, for example using the methods indicated above. This gene library is expressed in an appropriate manner, for example by means of a bacterial or phage display system.

[0198] The relevant gene of the host organism expressing the functional mutant with the characteristic roughly corresponding to the desired characteristic can be subjected to another mutation cycle. The mutation and selection or screening steps can be repeated iteratively until the functional mutants present have the desired characteristic to a sufficient degree. Using this iterative procedure, a limited number of mutations, for example 1, 2, 3, 4 or 5 mutations, can be carried out stepwise, and evaluated and selected for their effect on the activity of interest. The selected mutants can then be subjected to further mutation steps in a similar manner. In this way, the number of individual mutants to be investigated can be significantly reduced.

[0199] The results according to the invention also provide important information regarding the structure and sequence of related polypeptides, which is required for the targeted generation of further polypeptides with desired modified properties. In particular, it is possible to define so-called "hot spots", i.e. sequence segments that are potentially suitable for modifying properties by introducing targeted mutations.

[0200] It is also possible to derive information regarding positions in the amino acid sequence that may be surrounded by mutations that are expected to have little effect on activity; such mutations may be referred to as potential "silent mutations."

[0201] e. Constructs for expressing the polypeptides of the invention In this context, the following definitions apply: "Expression of a gene" encompasses "heterologous expression" and "overexpression" and includes transcription of a gene and translation of mRNA into a protein. Overexpression refers to the production of a gene product, as measured by levels of mRNA, polypeptide and / or enzyme activity, in a transgenic cell or organism that exceeds the level of production in a non-transformed cell or organism with a similar genetic background.

[0202] As used herein, "expression vector" refers to a nucleic acid molecule that is engineered using molecular biology methods and recombinant DNA technology to deliver foreign or exogenous DNA to a host cell. Expression vectors typically contain sequences required for proper transcription of nucleotide sequences. The coding region usually codes for a protein of interest, but may also code for RNA, such as antisense RNA, siRNA, etc.

[0203] As used herein, "expression vector" includes any linear or circular recombinant vector, including but not limited to viral vectors, bacteriophages and plasmids. Those skilled in the art can select an appropriate vector depending on the expression system. In one embodiment, the expression vector comprises a nucleic acid of an embodiment herein operably linked to at least one "regulatory sequence" that controls transcription, translation, initiation and termination, such as a transcription promoter, operator or enhancer, or mRNA ribosome binding site, and optionally includes at least one selectable marker. A nucleotide sequence is "operably linked" when the regulatory sequence is functionally related to the nucleic acid of an embodiment herein.

[0204] As used herein, an "expression system" encompasses any combination of nucleic acid molecules required for the expression or co-expression of two or more polypeptides in a given expression host, either in vivo or in vitro. Each coding sequence may be located on a single nucleic acid molecule or vector, e.g., a vector containing a multiple cloning site, or on a polycistronic nucleic acid, or may be distributed on two or more physically distinct vectors.

[0205] As used herein, the terms "amplify" and "amplification" refer to the use of any suitable amplification method to generate or detect recombinant forms of naturally expressed nucleic acids, as described in detail below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo dT primers) for amplifying naturally expressed (e.g., genomic DNA or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention in vivo, ex vivo, or in vitro (e.g., by polymerase chain reaction, PCR).

[0206] "Regulatory sequence" refers to a nucleic acid sequence that determines the expression level of a nucleic acid sequence of the embodiments herein and can regulate the rate of transcription of a nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences include promoters, enhancers, transcription factors, promoter elements, and the like.

[0207] A "promoter", "nucleic acid having promoter activity" or "promoter sequence" is understood according to the present invention to mean a nucleic acid that, when functionally linked to a nucleic acid to be transcribed, regulates the transcription of said nucleic acid. A "promoter" refers in particular to a nucleic acid sequence that controls the expression of a coding sequence by providing binding sites for RNA polymerase and other factors necessary for proper transcription, including but not limited to 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 affect the transcription, RNA processing, or stability of the associated coding nucleic acid sequence. Promoters include naturally occurring and synthetic sequences. The coding nucleic acid sequence is usually located downstream of the promoter with respect to the direction of transcription starting from the transcription start site.

[0208] In this context, a "functional" or "operable" linkage is understood to mean, for example, a contiguous sequence of one of the nucleic acids and a regulatory sequence. For example, a sequence having promoter activity, a sequence of the nucleic acid sequence to be transcribed, and optionally further regulatory elements, such as a nucleic acid sequence ensuring the transcription of the nucleic acid, for example a terminator, are linked in such a way that each of the regulatory elements can perform its function during the transcription of the nucleic acid sequence. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as enhancer sequences, can also exert their function on target sequences from more distant locations or from other DNA molecules. A preferred sequence is one in which the nucleic acid sequence to be transcribed is located behind the promoter sequence (i.e. at the 3' end) so that the two sequences are covalently linked. The distance between the promoter sequence and the nucleic acid sequence to be recombinantly expressed can be less than 200 base pairs, or less than 100 base pairs, or less than 50 base pairs.

[0209] In addition to promoters and terminators, examples of other regulatory elements can include targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, replication origins, etc. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0210] The term "constitutive promoter" refers to a non-regulated promoter that allows for continuous transcription of a nucleic acid sequence to which it is operably linked.

[0211] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter, or rather a transcriptional regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operatively linked means that the DNA sequences 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. This sequence may also be wholly or partially synthetic. Regardless of origin, the nucleic acid sequence associated with the promoter sequence, after binding to the polypeptide of an embodiment herein, is expressed or silenced according to the properties of the promoter to which it is linked. The associated nucleic acid may code for a protein that is desired to be expressed or suppressed throughout the organism at all times, or at a particular time, or in a particular tissue, cell, or cell compartment. Such nucleotide sequences code for proteins that, in particular, confer desired phenotypic traits to the host cell or organism modified or transformed thereby. More specifically, the associated nucleotide sequence results in the production of a product or products of interest, as defined herein, in the cell or organism. In particular, the nucleotide sequence encodes a polypeptide having an enzymatic activity as defined herein.

[0212] The nucleotide sequence as described herein may be part of an "expression cassette". The terms "expression cassette" and "expression construct" are used synonymously. A (preferably recombinant) expression construct comprises a nucleotide sequence encoding a polypeptide according to the invention, said nucleotide sequence being under the genetic control of a regulatory nucleic acid sequence.

[0213] In the process applied according to the present invention, the expression cassette may be part of an "expression vector", in particular a recombinant expression vector.

[0214] An "expression unit" is understood according to the invention to mean an expression-active nucleic acid which comprises a promoter as defined herein and which, after functional linkage with a nucleic acid or gene to be expressed, regulates the expression, i.e. regulates the transcription and translation of said nucleic acid or gene. In this context, therefore, an "expression unit" is also called a "regulatory nucleic acid sequence". In addition to the promoter, other regulatory elements may also be present, such as enhancers.

[0215] "Expression cassette" or "expression construct" is understood according to the present invention to mean an expression unit functionally linked to the nucleic acid to be expressed or to the gene to be expressed. Thus, an expression cassette, in contrast to an expression unit, contains not only nucleic acid sequences that regulate transcription and translation, but also nucleic acid sequences that are expressed as a protein as a result of transcription and translation.

[0216] The term "expression" or "overexpression" in the context of the present invention describes the production or increase of intracellular activity of one or more polypeptides in a microorganism encoded by corresponding DNA.For this purpose, it is possible, for example, to introduce a gene into an organism, to replace an existing gene with another gene, to increase the copy number of a gene, to use a strong promoter, or to use a gene that codes for a corresponding polypeptide with high activity, and these means can be combined in any way.

[0217] Preferably, such constructs according to the invention contain a promoter 5' upstream and a terminator sequence 3' downstream of the respective coding sequence, and optionally other conventional regulatory elements, in each case operably linked to the coding sequence.

[0218] Nucleic acid constructs according to the invention particularly comprise sequences encoding polypeptides derived from, for example, amino acid related SEQ ID NOs as described herein or reverse complements thereof, or derivatives and homologues thereof, advantageously operably or functionally linked to one or more regulatory signals for controlling, e.g., increasing, gene expression.

[0219] In addition to these regulatory sequences, the natural regulation of these sequences may still be present in front of the actual structural gene, and may optionally be genetically modified so that the natural regulation is switched off and the expression of the gene is enhanced. However, the nucleic acid construct may also be a simpler construct, i.e., no additional regulatory signals are inserted in front of the coding sequence and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that it is no longer regulated and gene expression is increased.

[0220] A preferred nucleic acid construct also advantageously comprises one or more of the above-mentioned "enhancer" sequences operably linked to the promoter, which allows enhanced expression of the nucleic acid sequence. Additional advantageous sequences may also be inserted at the 3' end of the DNA sequence, such as further regulatory elements or terminators. One or more copies of the nucleic acid according to the invention may be present in the construct. In the construct, other markers, for example genes that complement auxotrophy or antibiotic resistance, may also be optionally present to select the construct.

[0221] Examples of suitable regulatory sequences are promoters, e.g., cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI. q , T7, T5, T3, gal, trc, ara, rhaP(rhaP BAD )SP6, lambda-P R , or lambda-P Lpromoters, which are advantageously used in gram-negative bacteria. Further advantageous regulatory sequences are found, for example, in the gram-positive promoters amy and SPO2, yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. Artificial promoters can also be used for regulation.

[0222] For expression in the host organism, the nucleic acid construct is advantageously inserted into a vector, such as a plasmid or a phage, which allows optimal expression of the gene in the host.Vector is also understood to mean, in addition to plasmids and phages, all other vectors known to those skilled in the art, i.e. viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids and linear or circular DNA or artificial chromosomes.These vectors can replicate autonomously or chromosomally in the host organism.These vectors are further developments of the present invention.Binary vectors or cpo-integrating vectors are also applicable.

[0223] Suitable plasmids include, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III in E. coli. 113 -B1, λgt11 or pBdCI, pIJ101, pIJ364, pIJ702 or pIJ361 in Streptomyces, pUB110, pC194 or pBD214 in Bacillus, pSA77 or pAJ667 in Corynebacterium, pALS1, pIL2 or pBB116 in fungi, 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 in yeast, or pLGV23, pGHlac in plants. +, pBIN19, pAK2004 or pDH51. The above-mentioned plasmids are only a small selection of possible plasmids. Further plasmids are well known to those skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0224] In the further development of the vector, the nucleic acid construct according to the invention or the vector comprising the nucleic acid according to the invention can also advantageously be introduced into the microorganism in the form of linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination. This linear DNA can consist of a linearized vector such as a plasmid or exclusively of the nucleic acid construct or nucleic acid according to the invention.

[0225] For optimal expression of a heterologous gene in an organism, it is advantageous to modify the nucleic acid sequence to conform to the specific "codon usage" used in that organism, which can be readily determined by computer evaluation of other known genes of the organism of interest.

[0226] The expression cassette according to the invention is produced by fusing a suitable promoter to a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Conventional recombination and cloning techniques for this purpose are described, for example, in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0227] For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector that allows optimal expression of the gene in the host. Vectors are well known to those skilled in the art and can be found, for example, in "cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0228] Alternative embodiments of the present disclosure provide methods of "modifying gene expression" in a host cell. For example, the polynucleotides of the present disclosure may be enhanced, overexpressed, or induced in a host cell or host organism under certain circumstances (e.g., upon exposure to certain temperatures or culture conditions).

[0229] The modification of expression of the polynucleotide provided herein can also result in ectopic expression, which is a different expression pattern between modified organisms and control or wild-type organisms.The modification of expression occurs from the interaction of the polypeptide of the embodiment of the present specification with exogenous or endogenous modulators, or as a result of chemical modification of the polypeptide.This term also refers to the modified expression pattern of the polynucleotide of the embodiment of the present specification, which is modified below detection level or completely suppressed activity.

[0230] Also provided herein, in one embodiment, is an isolated, recombinant, or synthetic polynucleotide that encodes a polypeptide or a variant polypeptide provided herein.

[0231] In one embodiment, several polypeptides encoding nucleic acid sequences are co-expressed in a single host, particularly under the control of different promoters.In another embodiment, several polypeptides encoding nucleic acid sequences can be present on a single transformation vector, or can be co-transformed simultaneously using separate vectors and selecting transformants containing both chimeric genes.Similarly, one or polypeptides encoding genes can be expressed together with other chimeric genes in a single plant, cell, microorganism or organism.

[0232] f. Host Applicable to the Present Invention Depending on the context, the term "host" can refer to a wild-type host or a genetically modified, recombinant host, or both.

[0233] In principle, all prokaryotic or eukaryotic organisms can be considered as hosts or recombinant host organisms for the nucleic acids or nucleic acid constructs according to the invention.

[0234] The vectors according to the invention can be used to produce recombinant hosts, which can be transformed, for example, with at least one vector according to the invention and used to produce the polypeptides according to the invention. Advantageously, the above-mentioned recombinant constructs according to the invention are introduced into a suitable host system and expressed. Preferably, common cloning and transfection methods known to those skilled in the art are used to express the described nucleic acids in the respective expression systems, such as co-precipitation, protoplast fusion, electroporation, retroviral transfection. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0235] Advantageously, microorganisms such as bacteria, fungi or yeasts are used as host organisms. Advantageously, gram-positive or gram-negative bacteria are used, preferably bacteria from the family Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, Streptococcaceae or Nocardiaceae, particularly preferably bacteria from the genera Escherichia, Pseudomonas, Streptomyces, Lactococcus, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus. The genus and species of Escherichia coli are highly preferred. Furthermore, other advantageous bacteria can be found in the group consisting of the α-proteobacteria, β-proteobacteria or γ-proteobacteria. Advantageously, yeasts of the families Saccharomyces or Pichia are also suitable hosts.

[0236] Alternatively, whole plants or plant cells can serve as natural or recombinant hosts. Non-limiting examples include plants or cells derived therefrom of the genus Nicotiana, particularly Nicotiana benthamiana and Nicotiana tabacum (tobacco); and the genus Arabidopsis, particularly Arabidopsis thaliana.

[0237] Depending on the host organism, the organisms used in the process according to the invention are grown or cultured in a manner known by those skilled in the art. The culture can be batch, semi-batch or continuous. Nutrients can be present at the beginning of the fermentation or can be fed later semi-continuously or continuously. This is also explained in more detail below.

[0238] g. Recombinant Production of Polypeptides According to the Invention The present invention further relates to a method for the recombinant production of a polypeptide according to the invention or a functional, biologically active fragment thereof, comprising culturing a polypeptide-producing microorganism, optionally inducing expression of the polypeptide by applying at least one inducer having gene expression and isolating the expressed polypeptide from the culture. The polypeptide can also be produced in this way on an industrial scale, if desired.

[0239] The microorganisms produced according to the invention may be cultivated continuously or discontinuously in a batch process or in a fed-batch process or in repeated fed-batch processes. Overviews of known cultivation methods can be found in the following textbooks: Chmiel (Bioprozesstechnik 1. Einfuehrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0240] The culture medium used must be adequate to meet the requirements of the respective strain. Descriptions of media for various microorganisms can be found in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington, DC, USA, 1981).

[0241] These media that can be used according to the invention usually contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0242] Preferred carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars may also be added to the medium via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add a mixture of different carbon sources. Other possible carbon sources are fats and oils, 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.

[0243] The nitrogen source is usually an organic or inorganic nitrogen compound or a material containing these compounds. Examples of nitrogen sources include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources, such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources may be used alone or in mixtures.

[0244] Inorganic salt compounds which may be present in the medium include chlorides, phosphates or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0245] As sulfur sources, inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur-containing compounds such as mercaptans and thiols can be used.

[0246] As phosphorus source phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used.

[0247] Chelating agents may be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuic acid, or organic acids, such as citric acid.

[0248] The fermentation medium used according to the invention usually contains other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid and pyridoxine. Growth factors and salts often come from components of complex media, such as yeast extract, molasses, corn steep liquor, etc. Furthermore, suitable precursors may be added to the culture medium. The exact composition of compounds in the medium is highly dependent on each experiment and is determined individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed. PM Rhodes, PF 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).

[0249] All medium components are sterilized by heating (20 min at 1.5 bar and 121° C.) or by sterile filtration. The components may be sterilized together or separately if necessary. All medium components may be present at the start of the culture or may be added continuously or batchwise.

[0250] The temperature of the culture is usually between 15°C and 45°C, preferably between 25°C and 40°C, and may be varied or kept constant during the experiment. The pH of the medium is desirably in the range of 5 to 8.5, preferably around 7.0. The pH for growth can be controlled during growth by adding basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds, such as phosphoric acid or sulfuric acid. To control foaming, antifoaming agents, such as fatty acid polyglycol esters, may be used. To maintain the stability of the plasmid, suitable selectively acting substances, such as antibiotics, may be added to the medium. To maintain aerobic conditions, oxygen or a mixture of oxygen-containing gases, such as ambient air, is passed through the culture. The temperature of the culture is usually in the range of 20°C and 45°C. The culture is continued until a maximum of the desired product is formed. This goal is usually achieved within 10 hours to 160 hours.

[0251] The fermentation broth is then further processed. If desired, the biomass can be completely or partially removed from the fermentation broth by separation techniques, such as centrifugation, filtration, decantation, or a combination of these methods, or can be left entirely in the fermentation broth.

[0252] If the polypeptide is not secreted into the culture medium, the cells can be lysed and the product obtained from the lysate by known protein isolation methods. The cells can be optionally disrupted by high frequency ultrasound, high pressure, e.g., French press, osmotic lysis, the action of detergents, lytic enzymes or organic solvents, homogenizers, or by some combination of the aforementioned methods.

[0253] Polypeptides can be purified using known chromatographic techniques such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other conventional techniques such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, TG, Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0254] To isolate recombinant proteins, it may be advantageous to use vector systems or oligonucleotides that extend the cDNA with a defined nucleotide sequence and thus code for modified polypeptides or fusion proteins that facilitate, for example, purification. Suitable modifications of this type are, for example, anchors, such as the modification known as hexahistidine anchors, or so-called "tags" that function as epitopes that can be recognized as antigens for antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press). These anchors can serve to bind proteins to solid supports, for example polymer matrices that can be used as packings in chromatography columns or on microtiter plates or some other carriers.

[0255] At the same time, these anchors can also be used for protein recognition. For protein recognition, it is also possible to use conventional markers, such as fluorescent dyes, enzymatic markers, or radioactive markers, which form detectable reaction products after reacting with substrates, either alone or in combination with the anchors for protein derivatization.

[0256] h. Polypeptide immobilization The enzyme or polypeptide according to the invention can be used in the methods described herein in free or immobilized form. An immobilized enzyme is an enzyme immobilized on an inert carrier. Suitable carrier materials and enzymes immobilized thereon are known from EP 1149849, EP 1069183 and German Patent Application DE-OS100193773 and the references cited therein. In this regard, the disclosures of these documents are incorporated herein by reference 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, polyolefins such as polyurethane, polyethylene and polypropylene. To prepare the supported enzyme, the carrier material is usually used in finely divided granular form, preferably in porous form. The particle size of the carrier material is usually less than 5 mm, in particular less than 2 mm (particle size distribution curve). Similarly, when using dehydrogenases as whole cell catalysts, free or immobilized forms can be selected. Carrier materials are, for example, calcium alginate and carrageenan. Enzymes as well as cells can also be directly crosslinked with glutaraldehyde (crosslinking to CLEA). Corresponding other immobilization techniques are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes", K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out the method according to the invention is also given, for example, in Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol 3, Chapter 17, VCH, Weinheim.

[0257] i. Reaction conditions for the biocatalytic process of the present invention The reactions of the present invention can be carried out under in vivo or in vitro conditions.

[0258] The at least one polypeptide / enzyme present during the individual steps of the method of the invention or of the multi-step method as defined above may be present in living cells which naturally or recombinantly produce 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 support. One or more enzymes may be present simultaneously in soluble and / or immobilized form.

[0259] The method according to the invention can be carried out in common reactors known to the skilled person, in a different range of scales, for example from laboratory scale (reaction volume of a few milliliters to tens of liters) to industrial scale (reaction volume of a few liters to several thousand cubic meters). If the polypeptide is used in the form of a more or less purified cell extract or in purified form, encapsulated by non-living, optionally permeabilized cells, a chemical reactor can be used. A chemical reactor usually makes it possible to control the amount of at least one enzyme, the amount of at least one substrate, the pH, the temperature and the circulation of the reaction medium. If at least one polypeptide / enzyme is present in living cells, the process is fermentation. In this case, the biocatalytic production is carried out in a bioreactor (fermenter), in which the parameters necessary for suitable survival conditions for the living cells (for example nutrients, temperature, aeration, the presence or absence of oxygen or other gases, culture medium with antibiotics, etc.) can be controlled. The skilled person is familiar with chemical reactors or bioreactors, for example, as well as procedures for scaling up chemical or biotechnological procedures from laboratory to industrial scale or for optimizing process parameters, which are also widely described in the literature (for biotechnological procedures see, for example, Krueger und Crueger, Biotechnologie - Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Muenchen, Wien, 1984).

[0260] Cells containing at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or high frequency pulses, French press, or chemical means, such as hypotonic medium, lytic enzymes present in the medium and detergents, or a combination of such methods. Examples of detergents are digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween® 20, deoxycholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), Nonidet® P40 (ethylphenol poly(ethylene glycol ether)), etc.

[0261] Instead of living cells, biomass of non-viable cells containing the required biocatalyst may also be applied in the biotransformation reactions of the present invention.

[0262] When the at least one enzyme is immobilized, it is bound to an inert support as described above.

[0263] The conversion reaction can be carried out batchwise, semi-batchwise or continuously. The reactants (and optionally nutrients) can be fed at the beginning of the reaction or later on in a semi-continuous or continuous manner.

[0264] The reactions of the present invention can be carried out in aqueous, aqueous-organic, or non-aqueous reaction media, depending on the particular reaction type.

[0265] The aqueous or aqueous-organic medium may contain a suitable buffer to adjust the pH to a value in the range of 5 to 11, for example 6 to 10.

[0266] In the aqueous-organic medium, organic solvents can be applied that are miscible, partially miscible or immiscible with water. Non-limiting examples of suitable organic solvents are listed below. Further examples are monohydric or polyhydric, aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols such as glycerol.

[0267] The non-aqueous medium may be substantially free of water, that is, it will contain less than about 1% or about 0.5% water by weight.

[0268] The biocatalytic process may also be carried out in an organic non-aqueous medium. Suitable organic solvents include, for example, aliphatic hydrocarbons having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene or dichlorobenzene; aliphatic acyclic and ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether; or mixtures thereof.

[0269] The reactant / substrate concentrations can be adapted to optimal reaction conditions, which may depend on the particular enzyme applied. For example, the initial substrate concentration can be in the range of 0.1-0.5 M, such as 10-100 mM.

[0270] The reaction temperature can be adapted to the optimal reaction conditions, which may depend on the particular enzyme applied. For example, the reaction can be carried out at a temperature ranging from 0 to 70°C, such as from 20 to 50°C or from 25 to 40°C. Examples of reaction temperatures are about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C and about 60°C.

[0271] The process may proceed until equilibrium between the substrate and subsequent products is reached, but may be stopped earlier. Typical process times range from 1 minute to 25 hours, particularly from 10 minutes to 6 hours, for example from 1 hour to 4 hours, particularly from 1.5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.

[0272] Where the host is a transgenic plant, optimal growth conditions can be provided, such as optimal light, water, nutrient conditions, etc.

[0273] The specific reaction conditions for carrying out the preparation of the dorimanyl acetate compound are as follows: in an aqueous environment incubated at 20-35°C and pH 4-7, the acetyltransferase enzyme can be present as a purified polypeptide or in a whole cell system. The substrate concentration can vary between 10-100 mM.

[0274] k. Product isolation The method of the present invention may further include a step of recovering the final product or intermediate product, optionally in a substantially stereoisomerically or enantiomerically pure form. The term "recovering" includes extracting, harvesting, isolating or purifying the compound from the culture or reaction medium. The recovery of the compound can be carried out according to any conventional isolation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, non-ionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH change, solvent extraction (e.g., with conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0275] The identity and purity of the isolated products can be determined by known techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopy (IR, UV, NMR, etc.), colorimetry, TLC, NIRS, enzymatic or microbial assays (see, for example, Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.).

[0276] The cyclic terpene compounds produced by any of the methods 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. Terpene compound derivatives can be obtained by chemical methods such as, but not limited to, oxidation, reduction, alkylation, acylation and / or rearrangement. Alternatively, terpene compound derivatives can be obtained using biochemical methods by contacting terpene compounds with enzymes such as, but not limited to, oxidoreductases, monooxygenases, dioxygenases, transferases, etc. Biochemical conversion can be performed in vitro using isolated enzymes, enzymes from lysed cells, or in vivo using whole cells.

[0277] Fermentative production of l. dorimenyl acetate The present invention also relates to a method for the fermentative production of drimanyl acetate.

[0278] Fermentation as used according to the invention can be carried out, for example, in stirred fermenters, bubble columns and loop reactors. A comprehensive overview of the types of possible methods, including the type and geometric design of the stirrers, can be found in "Chmiel: Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Band 1". In the process of the invention, typical variants that can be used are, for example, the following variants known to the skilled artisan or described, for example, in "Chmiel, Hammes and Bailey: Biochemical Engineering", for example, cultivated by batch, fed-batch, repeated fed-batch or continuous fermentation, with or without biomass recycling. Depending on the production strain, sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen or a suitable gas mixture may be carried out in order to obtain a good yield (YP / S).

[0279] The culture medium used must be adequate to meet the requirements of the particular strain. Descriptions of media for various microorganisms can be found in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington, DC, USA, 1981).

[0280] These media that can be used according to the invention usually contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0281] Preferred carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars may also be added to the medium via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add a mixture of different carbon sources. Other possible carbon sources are fats and oils, 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.

[0282] The nitrogen source is usually an organic or inorganic nitrogen compound or a material containing these compounds. Examples of nitrogen sources include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources, such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources may be used alone or in mixtures.

[0283] Inorganic salt compounds which may be present in the medium include chlorides, phosphates or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0284] As sulfur sources, inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides as well as organic sulfur-containing compounds such as mercaptans and thiols can be used.

[0285] As phosphorus source phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used.

[0286] Chelating agents may be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuic acid, or organic acids, such as citric acid.

[0287] The fermentation medium used according to the invention contains other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid and pyridoxine. Growth factors and salts are often derived from components of complex media, such as yeast extract, molasses, corn steep liquor, etc. In addition, suitable precursors may be added to the culture medium. The exact composition of compounds in the medium is highly dependent on the specific experiment and must be determined individually for each specific case. Information regarding the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (1997). Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO).

[0288] All medium components are sterilized by heating (20 min at 1.5 bar and 121° C.) or by sterile filtration. The components may be sterilized together or separately if necessary. All medium components may be present at the start of the culture or may be added continuously or batchwise, as desired.

[0289] The temperature of the culture is usually 15°C to 45°C, preferably 25°C to 40°C, and may be varied or kept constant during the experiment. The pH of the medium is desirably in the range of 5 to 8.5, preferably around 7.0. The pH for growth can be controlled during growth by adding basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds, such as phosphoric acid or sulfuric acid. To control foaming, antifoaming agents, such as fatty acid polyglycol esters, may be used. To maintain the stability of the plasmid, suitable selectively acting substances, such as antibiotics, may be added to the medium. To maintain aerobic conditions, oxygen or a mixture of oxygen-containing gases, such as ambient air, is passed through the culture. The temperature of the culture is usually in the range of 20°C to 45°C. The culture is continued until a maximum of the desired product is formed. This is usually achieved within 1 hour to 160 hours.

[0290] The method of the present invention may further comprise the step of recovering said drimanyl acetate.

[0291] The term "recovering" includes extracting, harvesting, isolating or purifying a compound from a culture medium. Recovery of a compound can be carried out according to any conventional isolation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, non-ionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH change, solvent extraction (e.g., with conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0292] The biomass of the broth can be removed before the intended separation. Processes for removing the biomass, such as filtration, sedimentation and flotation, are known to those skilled in the art. The biomass can then be removed, for example, using centrifuges, separators, decanters, filters or flotation separators. To maximize the recovery of valuable products, washing of the biomass is often recommended, for example in the form of diafiltration. The choice of method also depends on the biomass content and characteristics of the biomass in the fermenter broth, as well as the interaction of the biomass with the valuable products.

[0293] In one embodiment, the fermentation broth can be sterilized or pasteurized. In a further embodiment, the fermentation broth is concentrated. Depending on the requirements, this concentration can be done batchwise or continuously. Pressure and temperature ranges should be selected first to avoid product damage and second to minimize equipment and energy usage. Skillful selection of pressure and temperature levels, especially for multi-stage evaporation, can allow energy savings.

[0294] The following examples are illustrative only and are not intended to limit the scope of the claims and embodiments described herein.

[0295] Also included within the scope of the present invention are numerous possible variations that will become immediately apparent to those of ordinary skill in the art after reviewing the disclosure provided herein.

[0296] Experimental Department material: Unless otherwise stated, all chemical and biochemical materials and microorganisms or cells used herein are commercially available.

[0297] Unless otherwise specified, recombinant proteins are cloned and expressed by standard methods, e.g., as described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0298] Gas chromatography-mass spectrometry (GC-MS) Agilent Intuvo 9000 series GC system equipped with a custom DB-5MS UI column (film thickness 10 m × 0.25 mm × 0.25 μm) (Agilent Technologies Inc, Santa Clara, CA). The GC was coupled to two detectors by a 1:1 detector splitter chip (G4588-60502, Agilent Technologies Inc, Santa Clara, CA). The first detector was an Agilent 5977B series mass spectrometer and the second detector was a standard Intuvo 9000 flame ionization detector (FID). Helium was used as the carrier gas at a constant flow rate of 2.5 ml / min. Injections were performed in split (1:100) mode with the injector temperature set at 240 °C. The oven temperature was programmed from 150° C. (held for 0.1 min) to 240° C. at 40° C. / min, then to 325° C. at 180° C. / min and held for 0.5 min.

[0299] Example 1 Selection of candidate acetyltransferases for converting drimane sesquiterpenes to drimanyl acetylated sesquiterpenes Acetyltransferases constitute a genetically diverse class of enzymes with over 8,000 known representatives (PFAM database: PF02458 transferase family). Although the repertoire of molecules that acetyltransferases accept as substrates is vast, none have been reported to accept sesquiterpene alcohols as substrates. To identify acetyltransferases capable of acetylating drimane-type sesquiterpene alcohols, 54 of the thousands of known acetyltransferases from plants, fungi and bacteria (Table 1) were selected on the following grounds:

[0300] Two of the five clades known from the plant acetyltransferase BAHD family, clades 3 and 5, have members identified for the acetylation of alkaloids and terpenoids with acetyl-CoA as the acyl donor (Curr Opin Plant Biol. 2006, 9(3):331-40). Furthermore, some of the substrates used by acetyltransferases from clades 3 and 5 are bulky, polycyclic, and have sterically hindered alcohol groups as the acyl acceptor (BMC Genomics 2011, 12:236; Curr Opin Plant Biol. 2006, 9:331-40; Elife. 2017 Mar 14;6: e23001; Planta. 2015, 242:709-19). On this basis, 21 candidates from plant BAHD family clades 3 and 5 were selected, including an identified acetyltransferase involved in the biosynthesis of the polycyclic diterpene paclitaxel (Taxol) (Proc Natl Acad Sci U S A. 2000, 18; 97(2): 583-587) and two acetyltransferases from Coleus forskohlii (Plectranthus barbatus) involved in the biosynthesis of the bicyclic labdane diterpene forskolin (Elife 2017, 14; 6: e23001).

[0301] Similar to the plant candidates, seven fungal acetyltransferases were selected based on their ability to accept acetyl-CoA as the acyl donor and the structural features of their substrates: bulky polycyclic compounds with sterically hindered alcohol groups (FEMS Microbiol Lett. 2005, 251:193-201; Chembiochem. 2009, 10:2325-8; Biotechnol Equip. 2014, 28(5):818-826; Nat Chem. 2010, 2:858-64). Among these, we selected the protein AstG (from Aspergillus oryzae, NCBI accession number XP_023091083.1), which is involved in the biosynthesis of aryl acid esterified drimane-type sesquiterpene lactones, asterolides (Sci Rep. 2016, 6:32865).

[0302] In addition, 21 fungal acetyltransferases were searched from the NCBI protein database based on sequence similarity to AstG using a PSI Blast search (Trends Biochem Sci. 2002, 27:161-4) (standard parameters, two replicates). Results were visualized on a phylogenetic tree using the NCBI Blast Tree View function to show the homology between AstG and 500 PSI-Blast derived sequences. Candidates were selected by homology with the query sequence derived from a single branch of the phylogenetic tree.

[0303] Finally, chloramphenicol acetyltransferases are known to be promiscuous enzymes and can accommodate bulkier substrates than chloramphenicol (Protein Sci. 2012, 21(4): 520-530). Thus, five bacterial chloramphenicol acetyltransferases from class 1 to class 3 were selected as putative chloramphenicol acetyltransferases from the NCBI protein database or from the literature (Biochem J. 1990, 272:505-10). [Table 5-1] [Table 5-2]

[0304] Example 2 In vivo production of arbikanyl acetate in Saccharomyces cerevisiae co-expressing arbikanol synthase and different putative acetyltransferases Each of the enzyme candidates was screened for in vivo bioconversion of arbicanol to arbicanyl acetate by coexpression with the gene encoding arbicanol synthase XP_007369631.1 (NCBI accession no. XP_007369631.1) from Dichomitus squalens in an engineered Saccharomyces cerevisiae strain with increased levels of endogenous farnesyl diphosphate (FPP).

[0305] To increase the level of the endogenous FPP pool in S. cerevisiae, extra copies of all yeast endogenous genes involved in the mevalonate pathway, from ERG10 encoding acetyl-CoA C-acetyltransferase to ERG20 encoding FPP synthase, were integrated into the genome of S. cerevisiae strain CEN.PK2-1C (Euroscarf, Frankfurt, Germany) under the control of a galactose-inducible promoter, similar to that described in Paddon et al., Nature, 2013, 496:528-532. Briefly, the three cassettes were integrated into the LEU2, TRP1 and URA3 loci, respectively. The first cassette, containing the genes ERG20 and truncated HMG1 (tHMG1 as described in Proc Natl Acad Sci USA, 1997, 109:E111-8) under the control of the bidirectional promoter of GAL10 / GAL1, and the genes ERG19 and ERG13, also under the control of the GAL10 / GAL1 promoter, was flanked by two 100-nucleotide regions corresponding to the upstream and downstream sections of LEU2. In the second cassette, in which the genes IDI1 and tHMG1 were under the control of the GAL10 / GAL1 promoter and the gene ERG13 was under the control of the GAL7 promoter region, this cassette was flanked by two 100-nucleotide regions corresponding to the upstream and downstream sections of TRP1. The third cassette, containing the genes ERG10, ERG12, tHMG1 and ERG8, all under the control of the GAL10 / GAL1 promoter, was flanked by two 100-nucleotide regions corresponding to the upstream and downstream sections of URA3. All genes in the three cassettes contained 200 nucleotides of their own terminator region. Also, an extra copy of GAL4 under the control of a mutant form of its own promoter was integrated upstream of the ERG9 promoter region as described in Proc Natl Acad Sci USA, 1991, 88:8597-8601. Furthermore, the expression of ERG9 was modified by promoter exchange.The GAL7, GAL10 and GAL1 genes were deleted using a cassette containing the HIS3 gene with its own promoter and terminator. The resulting strain was mated with strain CEN.PK2-1D (Euroscarf, Frankfurt, Germany) to obtain a diploid strain called YST045 that was induced for sporulation according to Solis-Escalante et al, FEMS Yeast Res, 2015, 15:2. Spore isolation was achieved by resuspending asci in 200 μL of 0.5 M sorbitol containing 2 μL of zymolyase (1000 UmL-1, Zymo research, Irvine, CA) and incubating at 37 °C for 20 min. Then, plating on a medium containing 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose and 20 g / L agar, one germinated spore was isolated and called YST069.

[0306] To express XP_007369631.1 and the evaluated acetyltransferases in YST069, a plasmid was constructed in vivo using yeast endogenous homologous recombination as previously described in Kuijpers et al., Microb Cell Fact., 2013, 12:47. The plasmid is composed of four DNA fragments that were used to co-transform S. cerevisiae. The fragments were as follows: a) Plasmid pF167 (SEQ ID NO: 1) linearized by enzymatic restriction with BsmBI. pF167 was previously constructed by in vivo assembly in yeast and contains the yeast marker LEU2 with its own promoter and terminator, the E. coli marker AmpR, the 2μ yeast replication origin, the E. coli pUC replication origin and the nucleotides 5′-GCACTTGCTACACTGTCAGGATAGCTTCCGTCACATGGTGGCGATCACCGTACATCTGAG-3′ (SEQ ID NO: 2) and 5′-AGGTGCAGTTCGCGTGCAATTATAACGTCGTGGCAACTGTTATCAGTCGTACCGCGCCAT-3′ (SEQ ID NO: 3) for homologous recombination; b) A fragment composed of 5'-GCACTTGCTACACTGTCAGGATAGCTTCCGTCACATGGTGGCGATCACCGTACATCTGAG-3' (SEQ ID NO: 2), the terminator region of the yeast gene PGK1 and the sesquiterpene synthase XP_007369631.1 DNA sequence codons optimized for its expression in S. cerevisiae (SEQ ID NO: 4), which was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025). The bidirectional GAL1 / GAL10 promoter region from yeast was added to this fragment by PCR overlap extension. (Yolov and Shabarova., Nucleic Acids Res. 1990, 18(13):3983-6); c) a fragment consisting of 60 bp corresponding to the first nucleotide of the yeast GAL10 promoter region and 60 bp of the yeast CYC1 terminator region, one of the acetyltransferase DNA coding sequences to be evaluated (codon optimized for its expression in S. cerevisiae), which was obtained by DNA synthesis (ATUM, Menlo Park, Calif. 94025); and d) A fragment composed of the terminator region of the yeast gene CYC1 and the sequence 5'-AGGTGCAGTTCGCGTGCAATTATAACGTCGTGGCAACTGTTATCAGTCGTACCGCGCCAT-3' (SEQ ID NO: 3), which was obtained by DNA synthesis (ATUM, Menlo Park, Calif. 94025).

[0307] YST069 was transformed with the fragments required for plasmid assembly in vivo. Yeast transformation was performed using the lithium acetate protocol as described by Gietz and Woods, Methods Enzymol., 2002, 350:87-96. The transformation mixture was plated on SmLeu-medium containing 6.7 g / L nitrogen base for yeast culture without amino acids (BD Difco, NJ, USA), 1.6 g / L dropout supplement without leucine (Sigma Aldrich, MO, USA), 20 g / L glucose and 20 g / L agar. Plates were incubated at 30 °C for 3-4 days. Individual colonies were used to produce arbicanol and arbicanil acetate in deep-well plates containing 250 μL of medium as described in Westfall et al., Proc Natl Acad Sci USA, 2012, 109:E111-118, and 50 μL of adipic acid diisodecyl ester (abcr GmbH, Germany) as an organic overlay. The deep-well plates were incubated at 30°C in a plate incubator for 3 days. To extract arbicanol and arbicanil acetate produced by yeast cells, each well of the deep-well plate was extracted with 700 μL of ethyl acetate containing an internal standard. The production of arbicanol and arbicanil acetate was identified using GC-MS analysis and quantified by GC-FID using the internal standard described above.

[0308] Under these experimental conditions, arbicanyl acetate was detected in nine combinations of arbicanol synthase and acetyltransferase (Table 2). Surprisingly, the two most active acetyltransferases (CrDAT and FgaAT) produced titers of arbicanyl acetate of more than 150 mg / L under non-optimized screening conditions. The GC-FID chromatograms are shown in Figure 2. Furthermore, Figure 2 shows that the MS spectrum of yeast-derived arbicanyl acetate is identical to that of the authentic arbicanyl acetate.

[0309] Interestingly, the most active acetyltransferase in our screen, CrDAT, is a member of clade 3 of the plant BAHD family and is involved in plant alkaloid biosynthesis, whereas the other tested acetyltransferases from the same family and clade, PsSalAT and RsVISY, are involved in plant alkaloid biosynthesis (thebaine and vinorine, respectively), but were not active when arbicanol was presented as a substrate. Furthermore, FgaAT from Aspergillus fumigatus (active against fumigalacubin B), also involved in alkaloid biosynthesis, produced substantial amounts of arbicanyl acetate in our screen, whereas the putative fumigalacubin B O-acetyltransferase GAO81666.1 surprisingly produced an order of magnitude less arbicanyl acetate than FgaAT.

[0310] It is noteworthy that neither the acetyltransferase AstG nor any of the proteins selected on the basis of similarity to AstG (except for GAO81666.1, see above) produced the drimanyly acetylated sesquiterpene arbikanyl acetate, despite the fact that AstG is involved in the biosynthesis of modified drimane-type sesquiterpene asterolides.

[0311] The only identified acetyltransferases in diterpene biosynthesis are those involved in the production of paclitaxel (Taxol) and forskolin. Of the eight acetyltransferases tested from C. forskohlii, only CfACT1-6 and CfACT1-8 were able to acetylate arbicanol. Although the TcTAT and TcDBAT genes can acetylate structurally similar intermediates in the biosynthesis of taxol (taxa-4(20),11(12)-dien-5a-yl acetate and baccatin III, respectively), only TcTAT produced arbicanyl acetate.

[0312] Adding to the currently documented complexity of identifying an acetyltransferase capable of using arbicanol as the acyl acceptor, of five tested substrate-versatile chloramphenicol acetyltransferases, only one was able to convert arbicanol to arbicanyl acetate.

[0313] [Table 6]

[0314] Example 3 In vivo production of drimenyl acetate in Saccharomyces cerevisiae co-expressing drimenol synthase and selected acetyltransferase candidates For the production of drimenyl acetate in Saccharomyces cerevisiae, the conversion of drimenol to drimenyl acetate was evaluated using nine selected acetyltransferases (CrDAT, FgaAT, OAH94415.1, TcTAT, CrMAT, LiAAT-4, GAO81666.1, CfACT1-6, CfACT1-8) capable of converting arbicannol to arbicannyl acetate, as shown in Example 2.

[0315] Dorimenyl acetate was produced in vivo by co-expressing each of the selected acetyltransferase enzyme candidates with the gene encoding dorimenol synthase XP_006461126 (NCBI accession no. XP_006461126) from Agaricus bisporus in the engineered S. cerevisiae strain YST069 (see Example 2), which has increased levels of endogenous FPP.

[0316] To express XP_006461126 and selected acetyltransferases (CrDAT, FgaAT, OAH94415.1, TcTAT, CrMAT, LiAAT-4, GAO81666.1, CfACT1-6 and CfACT1-8) in YST069, plasmids were constructed in vivo using yeast endogenous homologous recombination as previously described in Kuijpers et al., Microb Cell Fact., 2013, 12:47. The plasmids consist of four DNA fragments that were used to co-transform S. cerevisiae. The fragments were as follows: a) Plasmid pF167 (SEQ ID NO: 1) linearized by enzymatic restriction with BsmBI. pF167 was previously constructed by in vivo assembly in yeast and contains the yeast marker LEU2 with its own promoter and terminator, the E. coli marker AmpR, the 2μ yeast replication origin, the E. coli pUC replication origin and the nucleotides 5′-GCACTTGCTACACTGTCAGGATAGCTTCCGTCACATGGTGGCGATCACCGTACATCTGAG-3′ (SEQ ID NO: 2) and 5′-AGGTGCAGTTCGCGTGCAATTATAACGTCGTGGCAACTGTTATCAGTCGTACCGCGCCAT-3′ (SEQ ID NO: 3) for homologous recombination; b) A fragment composed of 5'-GCACTTGCTACACTGTCAGGATAGCTTCCGTCACATGGTGGCGATCACCGTACATCTGAG-3' (SEQ ID NO: 2), the terminator region of the yeast gene PGK1 and the sesquiterpene synthase XP_006461126 DNA sequence codons optimized for its expression in S. cerevisiae (SEQ ID NO: 6), which was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025). The bidirectional GAL1 / GAL10 promoter region from yeast was added to this fragment by PCR overlap extension. (Yolov and Shabarova., Nucleic Acids Res. 1990, 18(13):3983-6); c) a fragment consisting of 60 bp corresponding to the first nucleotide of the yeast GAL10 promoter region and 60 bp of the yeast CYC1 terminator region, one of the acetyltransferase DNA coding sequences to be evaluated (codon optimized for its expression in S. cerevisiae), which was obtained by DNA synthesis (ATUM, Menlo Park, Calif. 94025); and d) A fragment composed of the terminator region of the yeast gene CYC1 and the sequence 5'-AGGTGCAGTTCGCGTGCAATTATAACGTCGTGGCAACTGTTATCAGTCGTACCGCGCCAT-3' (SEQ ID NO: 3), which was obtained by DNA synthesis (ATUM, Menlo Park, Calif. 94025).

[0317] Yeast transformation, screening conditions, and quantification of drimenol and drimenyl acetate were performed as described in Example 2 for the production of arbicanol and arbicanyl acetate.

[0318] The relative amounts of arbicanil acetate and drimenyl acetate (from Example 2) are shown in Figures 3 and 4, respectively. From the nine enzyme candidates tested, seven enzymes (CrDAT, FgaAT, OAH94415.1, TcTAT, GAO81666.1, CfACT1-6 and CfACT1-8) produced drimenyl acetate. Due to the high structural similarity of arbicanol and drimenol, it was expected that similar relative conversion rates would be found.

[0319] Unexpectedly, the conversion of drimenol to the corresponding acetate was found to be significantly altered. In particular, CrDAT, which was found to produce the highest relative amount of arbikanyl acetate, was found to be one of the least active producers of drimenol. In contrast, FgaAT and CfACT1-8 were found to produce the highest relative amount of ofd acetate but were less active towards arbikanol. These surprising findings illustrate the difficulty of identifying suitable acetyltransferase enzyme candidates that accept non-physiological substrates.

[0320] Example 4 Selection of candidate acetyltransferases for converting drimane sesquiterpenes to acetylated drimanyl sesquiterpenes based on acetyltransferases active against drimenol and albicanol a) Experiment 1 To further identify acetyltransferases capable of acetylating drimane-type sesquiterpene alcohols, NCBI Protein Blast searches were performed to find similar protein sequence homologs using the amino acid sequences of the acetyltransferases CrDAT and FgaAT (shown in Examples 2 and 3) that have activity against arbicanol and drimenol. Protein Blast searches were performed using default parameters (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999).

[0321] The plant acetyltransferase XP_008340165.2 homolog to CrDAT and four fungal acetyltransferase homologs to FgaAT (KEY80391, PYI04555.1, XP_001276734.1, XP_024709055.1) were searched by Blast search and are listed in Table 3.

[0322] [Table 7]

[0323] b) Experiment 2 In addition, additional acetyltransferase candidates were searched for in the transcriptomes of the liverwort Bazzania trilobata and the fern Dryopteris fragrans. The liverwort Bazzania is rich in terpenoids, including the drimane sesquiterpene, and albicanylic acetate and albicanylic caffeate have been reported as natural products from liverworts in the genus Bazzania (Asakawa et al, Phytochemistry, Volume 30, Issue 9, 1991, Pages 3037-3040). Similarly, several different natural products have been reported in the genus Dryopteris, including albicanil acetate (Hideyuki Ito et al. Chem. Pharm. Bull. 48(8) 1190-1195 (2000); Froissard D et al. Nat Prod Commun. 2014 Jan;9(1):137-40.).

[0324] The transcriptome of Bazzania trilobata (NCBI accession number ER364415) was assembled using CLC Genomic Workbench (Qiagen), resulting in a total of 22083 contigs with an average length of 1,225 base pairs. The CrDAT amino acid sequence was used to search for homologous sequences in the Bazzania trilobata transcriptome. For this search, the tBlastn algorithm (Altschul et al. 1990, J. Mol. Biol. 215, 403-410) was used with default parameters. Transcripts with an E-value of more than 0.001 were considered. Ten transcripts belonging to the plant acetyltransferase BAHD family with low amino acid sequence homology of 20% to CrDAT were selected.

[0325] Plant material from D. fragrans was collected from northern China. Fresh leaves of D. fragrans (sample ID PNLI20141074) were used for transcriptome analysis. Total RNA of D. fragrans was extracted using QIAGEN's RNeasy Plant Mini Kit (catalog no. 74904). Total RNA sample PNLI20141074 was processed using NEBNext® UltraTM RNA Library Prep Kit for Illumina (NEB, USA) and TruSeq PE Cluster Kit (Illumina, USA) and then sequenced on an Illumina Miseq sequencer. 20.88 million paired-end reads of 2 × 350 bp were generated. The reads were assembled using Trinity (http: / / trinityrnaseq.sf.net / ) software, resulting in 85753 contigs with an N50 of 1373 bp. The contigs were analyzed using EMBOSS software (http: / / emboss.sourceforge.net / ) to obtain the protein sequences. The amino acid sequence (SEQ ID NO: 144) of the acetyltransferase ERR364415-1_contig_8546 (obtained from the Bazzania trilobata (NCBI accession number ERR364415) transcriptome as described above) was used to search for homologous sequences in the D. fragrans transcriptome. For this search, the tBlastn algorithm was used with standard parameters (Altschul et al 1990, J. Mol. Biol. 215, 403-410). This approach provided 20 transcripts belonging to the plant acetyltransferase BAHD family.

[0326] Example 5 In vivo production of drimanyl acetate in Saccharomyces cerevisiae by co-expressing enzymes for producing either arbikanol, drimenol or bicyclofarnesol with selected acetyltransferase candidates. An extensive screen for acetyltransferases for converting albikano, drimenol and bicyclofarnesol to their corresponding acetate derivatives was performed. A total of 89 acetyltransferases, as described in Table 1 of Example 1 and in Example 4, were screened in vivo in engineered Saccharomyces cerevisiae cells. The screening data generated for a preliminary number of acetyltransferases as described in Examples 2 and 3 were not repeated. Therefore, only new combinations of acetyltransferases with Saccharomyces cerevisiae cells producing either albikano, drimenol or bicyclofarnesol that were not tested in Examples 2 and 3 were screened.

[0327] The engineered S. cerevisiae strain YST069 was used for arbikanil acetate and drimenyl acetate production screening as described in Examples 2 and 3.

[0328] Bicyclofarnesyl acetate was produced in vivo by co-expressing each of the selected acetyltransferase enzyme candidates together with the enzymes AstC (SEQ ID NO: 138), AstI (SEQ ID NO: 140) and AstK (SEQ ID NO: 142), which are responsible for the production of bicyclofarnesol from asterolide biosynthesis, in the engineered S. cerevisiae strain YST069 (Yasutomo Shinohara et al. Sci Rep. 2016, 6: 32865).

[0329] For the simultaneous expression of AstC and AstI, an expression cassette containing the S. cerevisiae-type optimized codons of the AstC-encoding gene (SEQ ID NO: 138) and the AstI-encoding gene (SEQ ID NO: 140) as well as the bidirectional GAL1 / GAL10 promoter was constructed and integrated into the genome of YST069, resulting in a new strain called YST216. The codon-optimized DNA sequences of AstC and AstI were obtained by DNA synthesis (ATUM, Menlo Park, CA 94025). The bidirectional GAL1 / GAL10 promoter region from yeast was added to these genes by PCR overlap extension (Yolov and Shabarova., Nucleic Acids Res. 1990, 18(13):3983-6).

[0330] To express AstK and the evaluated acetyltransferases in YST216, plasmids were constructed in vivo using yeast endogenous homologous recombination as previously described in Kuijpers et al., Microb Cell Fact., 2013, 12:47. The plasmids consist of four DNA fragments that were used to co-transform S. cerevisiae. The fragments were as follows: a) Plasmid pF167 (SEQ ID NO: 1) linearized by enzymatic restriction with BsmBI; b) A fragment composed of 5'-GCACTTGCTACACTGTCAGGATAGCTTCCGTCACATGGTGGCGATCACCGTACATCTGAG-3' (SEQ ID NO: 2), the terminator region of the yeast gene PGK1 and the AstK DNA sequence codons optimized for its expression in S. cerevisiae (SEQ ID NO: 141), which was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025). The bidirectional GAL1 / GAL10 promoter region from yeast was added to this fragment by PCR overlap extension. (Yolov and Shabarova., Nucleic Acids Res. 1990, 18(13):3983-6); c) a fragment consisting of 60 bp corresponding to the first nucleotide of the yeast GAL10 promoter region and 60 bp of the yeast CYC1 terminator region, one of the acetyltransferase DNA coding sequences to be evaluated (codon optimized for its expression in S. cerevisiae), which was obtained by DNA synthesis (ATUM, Menlo Park, Calif. 94025); and d) A fragment composed of the terminator region of the yeast gene CYC1 and the sequence 5'-AGGTGCAGTTCGCGTGCAATTATAACGTCGTGGCAACTGTTATCAGTCGTACCGCGCCAT-3' (SEQ ID NO: 3), which was obtained by DNA synthesis (ATUM, Menlo Park, Calif. 94025).

[0331] YST216 was transformed with the fragments required for plasmid assembly in vivo. Yeast transformation was performed as described in Example 2. Screening conditions and quantification of bicyclofarnesol and bicyclofarnesyl acetate were identical to those described in Example 2, except that 25 μL of mineral oil (2705-01, VWR International, LLC.) was used as the organic overlay in the screening procedure.

[0332] result: In addition to the nine enzymes previously described (CrDAT, FgaAT, OAH94415.1, TcTAT, CrMAT, LiAAT-4, GAO81666.1, CfACT1-6, CfACT1-8) with activity against arbikanol (see Example 2), arbikanyl acetate was detected under these conditions when using the acetyltransferase ERR364415-1_contig_8546 from Bazzania trilobata (SEQ ID NO: 124 or 144) and DfATC13 from Dryopteris fragrans (SEQ ID NO: 118). However, their activities were lower than those of CrDAT and FgaAT. The relative amounts of arbikanyl acetate are shown in FIG. 5.

[0333] In addition to the seven enzyme candidates (CrDAT, FgaAT, OAH94415.1, TcTAT, GAO81666.1, CfACT1-6 and CfACT1-8) that can convert drimenol to drimenyl acetate (see Example 3), three additional acetyltransferases, XP_001258079.1 (SEQ ID NO: 127) from Aspergillus fischeri, ERR364415-1_contig_8546 (SEQ ID NO: 124 or 144) from Bazzania trilobata and DfATC13 (SEQ ID NO: 118) from Dryopteris fragrans, were found to produce drimenyl acetate from drimenol in the extended screen. The relative amount of drimenyl acetate obtained is shown in FIG. 6. As shown in FIG. 7, the MS spectrum of drimenyl acetate from yeast is found to be identical to the MS spectrum of authentic drimenyl acetate. Interestingly, under these experimental conditions, ERR364415-1_contig_8546 and DfATC13 showed the highest activity among all acetyltransferases tested.

[0334] Bicyclofarnesyl acetate was detected for 13 combinations of AstC, AstI and AstK with 89 acetyltransferases tested. Figure 8 shows that the MS spectrum of bicyclofarnesyl acetate from yeast is identical to that of the authentic bicyclofarnesyl acetate. The relative amounts of bicyclofarnesyl acetate are shown in Figure 9. The acetyltransferases FgaAT from Aspergillus fumigatus and ERR364415-1_contig_8546 from Bazzania trilobata showed the highest acetylation activity with bicyclofarnesol. Of these 13 discovered acetyltransferases that have activity against bicyclofarnesol, namely, CrDAT, FgaAT, TcTAT, CrMAT, GAO81666.1, CfACT1-6, CfACT1-8, and BAU61551.1 (SEQ ID NO: 133), PsSalAT (SEQ ID NO: 136), XP_001217250.1 (SEQ ID NO: 130), ERR364415-1_contig, as described in Example 2 above, Of the 13 contigs, nine, namely CrDAT, FgaAT, TcTAT, CrMAT, GAO81666.1, CfACT1-6, CfACT1-8, ERR364415-1_contig_8546 (SEQ ID NO: 124 or 144), PYI04555.1 (SEQ ID NO: 121 or 143), and DfACT13 (SEQ ID NO: 118), were also found to have activity against arbikanol and drimenol. It is worth noting that the acetyltransferase AstG, involved in the biosynthesis of arylate-esterified drimane-type sesquiterpene lactones, asterolides (Sci Rep. 2016, 6:32865), did not have activity against any of the drimane-type sesquiterpene alcohols tested. This reiterates the unpredictability and complexity of identifying acetyltransferases that can use arbicanol, drimenol, or bicyclofarnesol as acyl acceptors.

[0335] The contents of the cross-referenced documents are incorporated by reference.

[0336] [Table 8-1] [Table 8-2] [Table 8-3]

[0337] Sequence Listing SEQ ID NO:1 Plasmid pF167 [ka] [ka] [ka]

[0338] SEQ ID NO:2 Sequence for homologous recombination [ka]

[0339] SEQ ID NO:3 Sequence for homologous recombination [ka]

[0340] SEQ ID NO:4 XP_007369631.1 DNA sequence codon optimized for its expression in S. cerevisiae [ka] [ka]

[0341] SEQ ID NO:5 XP_007369631.1 Amino acid sequence of Dichomitus squalens albicanol synthase [ka]

[0342] SEQ ID NO:6 XP_006461126 Agaricus bisporus drimenol synthase DNA sequence codon optimized for its expression in S. cerevisiae [ka] [ka]

[0343] SEQ ID NO:7 XP_006461126 protein sequence of Agaricus bisporus drimenol synthase [ka]

[0344] SEQ ID NO:8 CrDAT DNA sequence codon optimized for its expression in S. cerevisiae [ka] [ka]

[0345] SEQ ID NO:9 CrDAT protein sequence [ka]

[0346] SEQ ID NO:10 TcTAT DNA sequence codon optimized for its expression in S. cerevisiae [ka] [ka]

[0347] SEQ ID NO:11 TcTAT protein sequence [ka]

[0348] SEQ ID NO:12 CrMAT DNA sequence codon optimized for its expression in S. cerevisiae [ka]

[0349] SEQ ID NO:13 CrMAT protein sequence [ka]

[0350] SEQ ID NO:14 LiAAT-4 DNA sequence codon optimized for its expression in S. cerevisiae [ka]

[0351] SEQ ID NO:15 LiAAT-4 protein sequence [ka] [ka]

[0352] SEQ ID NO:16 FgaAT DNA sequence codon optimized for its expression in S. cerevisiae [ka]

[0353] SEQ ID NO:17 FgaAT protein sequence [ka]

[0354] SEQ ID NO:18 GAO81666.1 DNA sequence codon optimized for its expression in S. cerevisiae [ka]

[0355] SEQ ID NO:19 GAO81666.1 protein sequence [ka]

[0356] SEQ ID NO:20 CfACT1-6 DNA sequence codon optimized for its expression in S. cerevisiae [ka]

[0357] SEQ ID NO:21 CfACT1-6 protein sequence [ka]

[0358] SEQ ID NO:22 CfACT1-8 DNA sequence codon optimized for its expression in S. cerevisiae [ka] [ka]

[0359] SEQ ID NO:23 CfACT1-8 protein sequence [ka] Optionally, the C-terminal "T" may be missing.

[0360] SEQ ID NO:24 OAH94415.1 DNA sequence codon optimized for its expression in S. cerevisiae [ka] [ka]

[0361] SEQ ID NO:25 OAH94415.1 protein sequence [ka]

[0362] SEQ ID NO:116 DfACT13 native nucleotide sequence [ka]

[0363] SEQ ID NO:117 DfACT13 nucleotide sequence codon optimized for its expression in S. cerevisiae [ka]

[0364] SEQ ID NO:118 DfACT13 protein sequence [ka]

[0365] SEQ ID NO:119 PYI04555.1 native nucleotide sequence [ka] [ka]

[0366] SEQ ID NO:120 PYI04555.1 nucleotide sequence codons optimized for its expression in S. cerevisiae, encoding the C-terminal extended protein variant of SEQ ID NO: 143 [ka] [ka]

[0367] SEQ ID NO:121 PYI04555.1 protein sequence C-terminal extended protein variant of SEQ ID NO: 143 [ka]

[0368] SEQ ID NO:122 ERR364415-1_contig_8546 native nucleotide sequence [ka] [ka]

[0369] SEQ ID NO:123 ERR364415-1_contig_8546 nucleotide sequence codons encoding the C-terminal extended protein variant of SEQ ID NO: 144, optimized for its expression in S. cerevisiae [ka]

[0370] SEQ ID NO:124 ERR364415-1_contig_8546 protein sequence C-terminal extended protein variant of SEQ ID NO: 144 [ka]

[0371] SEQ ID NO:125 XP_001258079.1 Native nucleotide sequence [ka] [ka]

[0372] SEQ ID NO:126 XP_001258079.1 nucleotide sequence codon optimized for its expression in S. cerevisiae [ka]

[0373] SEQ ID NO:127 XP_001258079.1 Protein sequence [ka] [ka]

[0374] SEQ ID NO:128 XP_001217250.1 Native nucleotide sequence [ka]

[0375] SEQ ID NO:129 XP_001217250.1 nucleotide sequence codon optimized for its expression in S. cerevisiae [ka] [ka]

[0376] SEQ ID NO:130 XP_001217250.1 Protein sequence [ka]

[0377] SEQ ID NO:131 BAU61551.1 native nucleotide sequence [ka] [ka]

[0378] SEQ ID NO:132 BAU61551.1 nucleotide sequence codon optimized for its expression in S. cerevisiae [ka] [ka]

[0379] SEQ ID NO:133 BAU61551.1 protein sequence [ka]

[0380] SEQ ID NO:134 The native nucleotide sequence of PsSalAT [ka] [ka]

[0381] SEQ ID NO:135 PsSalAT nucleotide sequence codon optimized for its expression in S. cerevisiae [ka]

[0382] SEQ ID NO:136 PsSalAT protein sequence [ka] [ka]

[0383] SEQ ID NO:137 AstC nucleotide sequence codon optimized for its expression in S. cerevisiae [ka]

[0384] SEQ ID NO:138 AstC protein sequence [ka] [ka]

[0385] SEQ ID NO:139 AstI nucleotide sequence codon optimized for its expression in S. cerevisiae [ka]

[0386] SEQ ID NO:140 AstI protein sequence [ka]

[0387] SEQ ID NO:141 AstK nucleotide sequence codon optimized for its expression in S. cerevisiae [ka]

[0388] SEQ ID NO:142 AstK protein sequence [ka]

[0389] SEQ ID NO:143 PYI04555.1 protein sequence without C-terminal extension [ka]

[0390] SEQ ID NO:144 ERR364415-1_contig_8546 protein sequence without C-terminal extension [ka]

[0391] Further sequences applicable in the present invention are listed below. The "previous" SEQ ID NOs refer to those used in the respective patent documents. [Table 9] [Table 10] [Table 11] [Table 12]

Claims

1. 1. A method for biocatalytically producing a drimanyl acetate compound, comprising: (1) contacting a drimanyl alcohol with a polypeptide having acetyltransferase activity of the enzyme class EC 2.3.1 capable of transferring an acetyl group from the acetyl donor to the drimanyl alcohol in the presence of an acetyl donor to obtain drimanyl acetate; the acetyl group donor is acetyl-coenzyme A (acetyl-CoA); The acetyltransferase is a) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 9, 17, 118, 124, 144, 23, 21, 11, 19, 13, 15, 25, 121, 143, 127, 130, 133, or 136, and b) a polypeptide having acetyltransferase activity and comprising an amino acid sequence exhibiting a degree of sequence identity of at least 90% with at least one of the amino acid sequences of SEQ ID NOs: 9, 17, 118, 124, 144, 23, 21, 11, 19, 13, 15, 25, 121, 143, 127, 130, 133 or 136. is selected from The method wherein said drimanyl alcohol is arbikanol.

2. 2. The method of claim 1, wherein the drimanyl acetate compound is arbikanil acetate.

3. 3. The method of claim 1 or 2, further comprising enzymatic synthesis of the drimanyl alcohol from farnesyl pyrophosphate (FPP) prior to step (1).

4. The method of claim 3, wherein the enzymatic synthesis of the drimanyl alcohol is catalyzed by one or more polypeptides capable of converting the farnesyl pyrophosphate (FPP) into at least one drimanyl alcohol in one or more enzymatic steps.

5. 5. The method of claim 4, wherein the drimanyl alcohol is produced from FPP in a single enzymatic step or in two or more enzymatic steps.

6. The dorimanyl alcohol is a) a polypeptide having a drimanyl sesquiterpene synthase activity that forms the drimanyl alcohol; or b) a polypeptide having drimanyl phosphate synthase activity that forms at least one drimanyl phosphate intermediate, in combination with a polypeptide having phosphatase activity that converts said at least one drimanyl phosphate intermediate into at least one drimanyl alcohol; The method of claim 5, wherein the FPP is produced by enzymatic conversion catalyzed by

7. a) the polypeptide having drimane sesquiterpene synthase activity is selected from an arbikanol synthase activity; and b) the polypeptide combination comprises a drimanyl diphosphate synthase activity and a phosphatase; The method of claim 6.

8. The drimane sesquiterpene synthase is a) a polypeptide having arbicanol synthase activity and comprising the amino acid sequence of SEQ ID NO:5, or a mutant or variant polypeptide having arbicanol synthase activity and comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

5. The method of claim 7, wherein the compound is selected from the group consisting of

9. 9. The method of any one of claims 1 to 8, carried out in vivo in a cell culture or in vitro in a liquid reaction medium under conditions conducive to the production of said drimanyl acetate.

10. a) at least one acetyltransferase as defined in claim 1; b) at least one polypeptide capable of converting FPP into at least one drimanyl alcohol as defined in any one of claims 4 to 8, and / or c) at least one enzyme selected from enzymes involved in the mevalonate pathway; The method of claim 9, wherein the method is carried out in a recombinant host cell or a recombinant non-human host organism capable of functionally expressing

11. The method of claim 10 , wherein the non-human host cell or organism is selected from bacterial, fungal and plant cells or plants.

12. A method according to any one of claims 1 to 11, further comprising, as step (2), isolating the drimanyl acetate from the reaction product of step (1).

13. 13. The method of any one of claims 1 to 12, further comprising, as step (3), treating the drimanyl acetate of step (1) or step (2) to obtain a derivative using chemical synthesis or biocatalytic synthesis or a combination of both.

14. Providing a non-human host organism or host cell, a) at least one nucleic acid comprising a nucleic acid sequence encoding at least one polypeptide having acetyltransferase activity capable of transferring an acetyl group from an acetyl donor to a drimanyl alcohol; and / or b) at least one nucleic acid comprising a nucleic acid sequence encoding at least one polypeptide having drimanyl alcohol synthase activity capable of producing drimanyl alcohol from FPP; and / or c) at least one nucleic acid comprising a nucleic acid sequence encoding at least one polypeptide involved in a biosynthetic pathway for producing said FPP; 14. The method according to any one of claims 1 to 13, comprising transforming with

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