Biocatalytic methods for the controlled degradation of terpene compounds.

Abstract

Provided herein are biocatalytic methods for producing terpene degradation products useful as starting materials for the production of perfume ingredients such as ambrox. In particular, novel terpene-degrading polypeptides (enal-cleaving polypeptides) and novel peptides (oxygenases) that convert terpene compounds to oxygenated derivatives, as well as mutants and variants derived therefrom, are provided, which can be applied to a novel type of fully enzymatic multistep degradation pathway that allows for the controlled stepwise conversion and degradation of linear or cyclic terpene substrates. The aforementioned novel biosynthetic strategies enable the complete biochemical synthesis of valuable terpene-derived compounds, such as manooloxy or gamma-ambrol. The present invention also provides recombinant host organisms that contain the necessary genetic information for functional expression of the set of enzymes required to catalyze the combined enzymatic conversion and degradation steps.

Description

[Technical Field]

[0001] Provided herein are biocatalytic methods for producing terpene degradation products useful as starting materials for the production of perfume ingredients such as ambrox. In particular, novel terpene-degrading polypeptides (enal-cleaving polypeptides) and novel peptides (oxygenases) that convert terpene compounds to oxygenated derivatives, as well as mutants and variants derived therefrom, are provided, which can be applied to a novel type of fully enzymatic multistep degradation pathway that allows for the controlled stepwise conversion and degradation of linear or cyclic terpene substrates. The novel biosynthetic strategies described above enable the complete biochemical synthesis of valuable terpene-derived compounds, such as manooloxy or gamma ambrol. The present invention also provides recombinant host organisms that contain the necessary genetic information for functional expression of the set of enzymes required to catalyze the combined enzymatic conversion and degradation steps.

[0002] Background of the Invention Terpenes are found in most living organisms (microorganisms, animals, and plants). These compounds are composed of five-carbon units, known as isoprene units, and are classified according to the number of these units present in the isoprene structure. Thus, hemiterpenes, monoterpenes, sesquiterpenes, and diterpenes are terpenes containing 5, 10, 15, and 20 carbon atoms (i.e., 1, 2, 3, and 4 isoprene units), respectively. For example, sesquiterpenes are widespread in the plant kingdom. Many sesquiterpene molecules are known for their flavor and fragrance properties, as well as cosmetic, medicinal, and antibacterial activities. Numerous sesquiterpene hydrocarbons and sesquiterpenoids have been identified.

[0003] The biosynthetic production of terpenes involves enzymes called terpene synthases. These enzymes convert acyclic terpene precursors into one or more terpene products. In particular, diterpene synthases cyclize the precursor geranylgeranyl diphosphate (GGPP) to produce diterpenes. The GGPP cyclization reaction often requires the combined function of two enzyme polypeptides, type I and type II diterpene synthases, in two sequential enzymatic reactions. Type II diterpene synthases catalyze the cyclization / rearrangement of GGPP initiated by protonation of the terminal double bond of GGPP, resulting in a cyclic diterpene diphosphate intermediate. This intermediate is then further transformed by type I diterpene synthases, which catalyze an ionization-initiated cyclization.

[0004] Diterpene synthases exist in plants and other organisms and use substrates such as GGPP but have different product profiles. Genes and cDNAs encoding diterpene synthases have been cloned and the corresponding recombinant enzymes have been characterized.

[0005] Enzymes that catalyze the specific or preferential cleavage or removal of diphosphate groups from terpene diphosphate intermediates, particularly cyclic terpene diphosphate intermediates such as the diterpenes copalyl diphosphate (CPP) or labdanediol diphosphate (LPP), have recently been described in a previous European patent application (EP Application No. 18182783.3). The enzymes do not change the number or carbon atoms of the terpene diphosphate.

[0006] However, there is a need for terpene-derived compounds that can be considered as degradation products of terpene precursors, such as acyclic or cyclic sesquiterpenes or diterpenes, which can be further converted chemically and / or enzymatically into final products and applied, for example, as fragrance ingredients.

[0007] The problem to be solved by the present invention is to provide polypeptides that exhibit enzymatic terpene-degrading activity or that convert such terpenes into degradable derivatives.

[0008] Another problem to be solved by the present invention is to establish a novel, fully biocatalytic degradation pathway to produce defined terpene degradation products.

[0009] Summary of the Invention Surprisingly, the above-mentioned problems have been solved by providing a new class of polypeptides with enal cleavage activity that, for the first time, enables the specific synthesis of carbonyl-functionalized terpene compounds specifically shortened by two carbon atoms and the respective biocatalytic processes. For example, this new class of enzymes enables the conversion of copalal, a labdane-type compound containing a diterpene carbon skeleton and a terminal aldehyde group, to the respective dinor-labdane-type compound manooloxy, which maintains the carbon skeleton shortened by two carbon atoms, i.e., consisting of 18 carbon atoms.

[0010] Surprisingly, we have overcome the aforementioned challenges of alternative approaches by providing a new class of polypeptides with Baeyer-Villiger monooxygenase (BVMO) activity that enables the specific oxidation of terpene compounds to esters (Baeyer-Villiger oxidation) and the respective biocatalytic processes. For example, a novel class of BVMOs enables the conversion of coparal, a labdane-type compound with a diterpene carbon skeleton and a terminal aldehyde group, to its respective norlabdane formate esters. By the aforementioned Baeyer-Villiger oxidation, labdane-type compounds can be easily converted to their respective norlabdanes by the action of polypeptides with esterase activity. This step results in a shortening of one carbon atom. If the terminal aldehyde group is replaced with a terminal keto group, the same shortening can be achieved, but this time by one or more carbonates. Repeated combinations of BVMO-catalyzed oxygenation and esterase-catalyzed cleavage steps enable the stepwise shortening of the hydrocarbon chain of terpene molecules.

[0011] Combining the degradation steps catalyzed by the enal-cleavage enzymes and BVMO enzymes described above allows the construction of an entirely new biochemical degradation pathway that can be applied to a wider variety of carbonyl-functionalized compounds, particularly cyclic or acyclic terpenes or terpenoids.

[0012] The aforementioned biocatalytic steps may be combined with several other preceding (upstream) or subsequent (downstream) enzymatic steps, making it possible to provide a biocatalytic multi-step process for the complete enzymatic synthesis of many valuable complex terpene molecules from their respective precursors.

[0013] The following schemes show two specific embodiments of the two alternative pathways of the present invention (the "enal-cleaving polypeptide pathway" and the "BMVO pathway") that allow the decomposition of the coparal of labdane-type aldehydes to mannooloxy, which are described in more detail in subsequent sections of this specification. The schemes also show the decomposition of mannooloxy to γ-ambrool by applying an additional BMVO-based decomposition step.

[0014] [ka]

[0015] In full analogy with the reaction sequence exemplified above, this basic biosynthetic strategy can be applied to any other isomer of copalol or any other labdane-type aldehyde to provide manooloxy, γ-ambryl acetate, a structurally related isomer of γ-ambrol.

[0016] It may also be applied to structurally different monocyclic or acyclic carbonyl compounds, as described in more detail below. [Brief explanation of the drawings]

[0017] [Figure 1]Figure 1 shows a schematic diagram of the chromosomal integration of genes encoding mevalonate pathway enzymes and the construction of two synthetic gene operons (the gene "mvaK1" encoding mevalonate kinase from S. pneumoniae; the gene "mvaD" encoding phosphomevalonate decarboxylase from S. pneumoniae; the gene "mvaK2" encoding phosphomevalonate kinase from S. pneumoniae; the gene "fni" encoding isopentenyl diphosphate isomerase from S. pneumoniae; the gene "mvaA" encoding HMG-CoA synthase from S. aureus; the gene "mvaS" encoding HMG-CoA reductase from S. aureus; the gene "atoB" encoding acetoacetyl-CoA thiolase from E. coli; and the gene "ERG20" encoding FPP synthase from S. cerevisiae). [Figure 2] FIG. 1 shows the conversion of manooloxy to γ-ambryl acetate using BVMOs in a whole-cell bioconversion assay. GC-MS analysis of the products formed during the bioconversion of manooloxy with different BVMOs: SCH23-BVMO1, SCH24-BVMO1, and SCH46-BVMO1. The top chromatogram shows the GC-MS analysis of manooloxy, and the bottom chromatogram shows the GC-MS analysis of bioconversion using control cells not expressing recombinant BVMO. [Figure 3] Figure 1 shows the conversion of coparal using BVMOs in a whole-cell bioconversion assay. GC-MS analysis of the products (compounds 3a, 3b, 4a, and 4b described in the experimental section) formed during the bioconversion of cis-coparal and trans-coparal by different BVMOs: SCH23-BVMO1, SCH24-BVMO1, and SCH46-BVMO1. The top chromatogram shows the GC-MS analysis of bioconversion using control cells not expressing recombinant BVMO. [Figure 4]Figure 1 shows the kinetics of coparal conversion using SCH23-BVMO1 in a whole-cell bioconversion assay, with GC-MS analysis of the products formed during the bioconversion of cis-coparal and trans-coparal by SCH23-BVMO1 (compounds 1a, 1b, 3a, 3b, 4a, 4b described in the experimental section) after 0, 18, and 42 hours of incubation. [Figure 5] FIG. 1 shows the in vitro conversion of manooloxy using BVMO. GC-MS analysis of the conversion of manooloxy using SCH23-BVMO1 and SCH24-BVMO1 shows the formation of γ-ambrolic acetate. The top chromatogram shows GC-MS analysis of the conversion using a control protein that does not contain recombinant BVMO. [Figure 6] FIG. 1 shows the in vitro conversion of manooloxy using BVMO and esterase. GC-MS analysis of the conversion of manooloxy using SCH23-BVMO1, SCH23-EST, and a combination of SCH23-BVMO1 and SCH23-EST shows the formation of γ-ambrol. The top chromatogram shows GC-MS analysis of the conversion using a control protein that does not contain the recombinant enzyme. [Figure 7] FIG. 1 shows the in vitro conversion of manooloxy using BVMO and esterase. GC-MS analysis of the conversion of manooloxy using SCH24-BVMO1, SCH24-EST, and a combination of SCH24-BVMO1 and SCH24-EST shows the formation of γ-ambrol. The top chromatogram shows GC-MS analysis of the conversion using a control protein that does not contain the recombinant enzyme. [Figure 8] FIG. 1 shows the in vitro conversion of compounds 4a and 4b to compounds 5a and 5b using esterases. GC-MS analysis of the in vitro conversion of compounds 4a and 4b with SCH23-EST1, SCH24-EST1, and SCH25-EST1 shows the formation of compounds 5a and 5b. [Figure 9]Figure 1 shows the in vitro conversion of coparal to compounds 5a and 5b using SCH23-BVMO1 and esterases. GC-MS analysis of the in vitro conversion of cis-coparal and trans-coparal by SCH23-BVMO in combination with SCH23-EST1, SCH24-EST1, and SCH25-EST1 showed the formation of compounds 5a and 5b, marked with *. The peak at 11.95 min corresponds to γ-ambryl acetate; the observation of this compound in samples incubated with BVMO alone is due to the presence of small amounts of ambryl acetate in the coparal mixture used in these assays. [Figure 10] Figure 1 shows the in vitro conversion of coparal to compounds 5a and 5b using SCH24-BVMO1 and esterase. GC-MS analysis of the in vitro conversion of cis-coparal and trans-coparal by SCH23-BVMO in combination with SCH23-EST1 and SCH25-EST1 showed the formation of compounds 5a and 5b, marked with *. The peak at a retention time of 11.95 min corresponds to γ-ambryl acetate; the observation of this compound in samples incubated with BVMO alone is due to the presence of small amounts of ambryl acetate in the mixture of coparal used in these assays. [Figure 11] FIG. 1 shows the biochemical production of 14,15-dinor-labdane compounds 5a, 5b and biosynthetic intermediates in engineered bacterial cells expressing BVMO and an esterase. The top chromatogram shows GC-MS analysis of compounds produced by E. coli cells transformed with the pJ401-CPAL-1 plasmid, which expresses enzymes in the coparal biosynthetic pathway. The bottom chromatogram shows GC-MS analysis of cells further transformed with a second plasmid carrying a nucleotide sequence encoding the BVMO enzyme or the BVMO enzyme together with an esterase. [Figure 12]FIG. 1 shows GC-MS analysis of the products of biotransformation of compounds 5a and 5b by E. coli cells expressing various alcohol dehydrogenases. The top chromatogram shows GC-MS analysis of the bioconversion using control cells that do not express recombinant alcohol dehydrogenase, and the bottom chromatogram shows GC-MS analysis of the conversion using cells expressing recombinant RrhSecADH, SCH80-00043, SCH80-04254, SCH80-06135, or SCH80-06582 proteins. [Figure 13] FIG. 1 shows the biochemical production of γ-ambryl acetate and biosynthetic intermediates in engineered bacterial cells expressing BVMO, esterase, and alcohol dehydrogenase. The top chromatogram shows GC-MS analysis of compounds produced by E. coli cells transformed with the pJ401-CPAL-1 plasmid, which expresses enzymes in the coparal biosynthetic pathway. The middle chromatogram shows GC-MS analysis of cells further transformed with a second plasmid carrying sequences encoding SCH-BVMO1 and SCH24-EST. The bottom chromatogram shows GC-MS analysis of cells transformed with pJ401-CPAL-1 and the pJ423-secADH-23BVMO-EST plasmid, which expresses RrhSecADH, SCH23-BVMO1, and SCH23-EST. [Figure 14]A) GC-MS analysis of terpenes and derivatives produced using engineered Saccharomyces cerevisiae strains expressing GGPP synthase carG, CPP synthase SmCPS2, CPP phosphatase TalVeTPP, and either SCH23-ADH1, SCH23-BVMO1, SCH23-EST1, and SCH23-ADH2 (YST120 w / plasmid) or SCH24-ADH1a, SCH24-BVMO1, SCH24-EST1, and SCH24-ADH2a (YST121 w / plasmid) (the control strain was YST075, which expresses only the copalol biosynthetic pathway). B) GC-MS analysis of the region where farnesal was identified (mass spectrum of farnesal is shown). C) GC-MS analysis of the region where manooloxy was identified (mass spectrum of manooloxy is shown). [Figure 15] Figure 1 shows GC-MS analysis of manooloxy produced using engineered budding yeast strains expressing GGPP synthase carG, CPP synthase SmCPS2, CPP phosphatase TalVeTPP, and either SCH23-ADH1, SCH23-BVMO1, and SCH23-EST1 (YST177) or SCH24-ADH1a, SCH24-BVMO1, and SCH24-EST1 (YST178). The control strain was YST075, which expresses only the copalol biosynthetic pathway. The mass spectrum of manooloxy is shown. [Figure 16]FIG. 1 shows GC-MS analysis of diterpenes and derivatives produced using E. coli cells expressing CPP synthase, phosphatase, alcohol dehydrogenase, and / or SCH94-3944. The top chromatogram shows the diterpene region of the GC-MS analysis of compounds produced by E. coli cells transformed with the pJ401-CPOL-4 plasmid, which expresses enzymes in the copalol biosynthetic pathway. The bottom chromatogram shows the GC-MS analysis of compounds produced by the same E. coli cells further transformed with plasmids pJ423-SCH94-3945, pJ423-SCH94-3944, or pJ423-SCH94-3944-3945, which express SCH94-3945, SCH94-3944, or a combination of SCH94-3944 and SCH94-3945. [Figure 17] FIG. 1 shows GC-MS analysis of sesquiterpenes and derivatives produced using E. coli cells expressing phosphatase, alcohol dehydrogenase, and SCH94-3944. The top chromatogram shows GC-MS analysis of compounds produced by E. coli cells transformed with the pJ401-FAL-1 plasmid, which expresses enzymes in the farnesal biosynthetic pathway. The bottom chromatogram shows GC-MS analysis of compounds produced by the same E. coli cells further transformed with the pJ423-SCH94-3944 plasmid, which expresses the SCH94-3944 protein. [Figure 18] FIG. 1 shows GC-MS analysis of the products of citral, citronellal, and (E)-2-dodecanal biotransformation by E. coli cells expressing SCH94-3944. For each compound, GC-MS analysis of transformations using control E. coli cells and cells transformed to express the SCH94-3944 protein is shown. [Figure 19]FIG. 1 shows GC-MS analysis of sesquiterpenes and diterpenes produced using E. coli cells expressing CPP synthase, phosphatase, and alcohol dehydrogenase. The chromatogram shows GC-MS analysis of compounds produced by E. coli cells transformed with the pJ401-CPAL-1 plasmid, which expresses enzymes in the coparal biosynthetic pathway. [Figure 20] FIG. 1 shows GC-MS analysis of diterpenes and derivatives produced using E. coli cells expressing CPP synthase, phosphatase, alcohol dehydrogenase, and SCH80-05241, SCH94-3944, PdigitDUF4334, PitalDUF4334-1, or AspWeDUF4334. The top chromatogram shows the diterpene region of the GC-MS analysis of compounds produced by E. coli DP1205 cells transformed with the pJ401-CPAL-1 plasmid expressing enzymes of the copral biosynthetic pathway. The bottom chromatogram shows the GC-MS analysis of compounds produced by the same E. coli cells further transformed with a second plasmid expressing the recombinant protein of SCH80-05241, SCH94-3944, PdigitDUF4334, PitalDUF4334-1, or AspWeDUF4334. [Figure 21]FIG. 1 shows GC-MS analysis of diterpenes and derivatives produced using E. coli cells expressing CPP synthase, phosphatase, alcohol dehydrogenase, and CnecaDUF4334, Rins-DUF4334, RhoagDUF4334-2, RhoagDUF4334-3, RhoagDUF4334-4, CgatDUF4334, GclavDUF4334, TcurvaDUF4334, or PprotDUF4334. The upper chromatogram shows the results of the pJ401-CPAL-1 plasmid expressing enzymes in the coparal biosynthetic pathway. The first chromatogram shows the diterpene region of a GC-MS analysis of compounds produced by E. coli DP1205 cells transformed with DUF4334, and the second chromatogram shows a GC-MS analysis of compounds produced by the same E. coli cells further transformed with a second plasmid expressing recombinant proteins of CnecaDUF4334, Rins-DUF4334, RhoagDUF4334-2, RhoagDUF4334-3, RhoagDUF4334-4, CgatDUF4334, GclavDUF4334, TcurvaDUF4334, or PprotDUF4334. [Figure 22] FIG. 1 shows GC-MS analysis of sesquiterpenes and derivatives produced using E. coli cells expressing phosphatase, alcohol dehydrogenase, and SCH80-05241, SCH94-3944, PdigitDUF4334, PitalDUF4334-1, or AspWeDUF4334. The top chromatogram shows the sesquiterpene region of the GC-MS analysis of compounds produced by E. coli DP1205 cells transformed with the pJ401-CPAL-1 plasmid expressing enzymes of the coparal biosynthetic pathway. The bottom chromatogram shows the GC-MS analysis of compounds produced by the same E. coli cells further transformed with a second plasmid expressing recombinant protein of SCH80-05241, SCH94-3944, PdigitDUF4334, PitalDUF4334-1, or AspWeDUF4334. [Figure 23]FIG. 1 shows GC-MS analysis of sesquiterpenes and derivatives produced using E. coli cells expressing phosphatase, alcohol dehydrogenase, and CnecaDUF4334, Rins-DUF4334, RhoagDUF4334-2, RhoagDUF4334-3, RhoagDUF4334-4, CgatDUF4334, GclavDUF4334, TcurvaDUF4334, or PprotDUF4334. The upper chromatogram shows the ... the pJ401-CPAL-1 plasmid expressing enzymes of the coparal biosynthetic pathway. The sesquiterpene region of GC-MS analysis of compounds produced by transformed E. coli cells is shown, and the subsequent chromatogram shows GC-MS analysis of compounds produced by the same E. coli cells further transformed with a second plasmid expressing the recombinant protein CnecaDUF4334, Rins-DUF4334, RhoagDUF4334-2, RhoagDUF4334-3, RhoagDUF4334-4, CgatDUF4334, GclavDUF4334, TcurvaDUF4334, or PprotDUF4334. [Figure 24] FIG. 1 shows the alignment and conserved amino acids of GXWXG and DUF4334 domain-containing proteins that catalyze enal cleavage, with the predicted location of each protein family domain boxed. [Figure 25] FIG. 1 shows the farnesal and coparal conversion activities of single amino acid variants of SCH94-3944, where the activities are expressed as a percentage of the wild-type enzyme activity as the total amount of mannoyloxy and geranylacetone produced. [Figure 26]FIG. 1 shows GC-MS analysis of the biochemical production of coparal and γ-ambryl acetate by E. coli cells expressing CPP synthase, phosphatase, alcohol dehydrogenase, enal cleavage enzyme, and BVMO. The top chromatogram shows the diterpene region of the GC-MS analysis of compounds produced by E. coli DP1205 cells transformed with the pJ401-CPAL-1 plasmid expressing enzymes in the coparal biosynthetic pathway, and the bottom chromatogram shows the diterpene region of the GC-MS analysis of compounds produced by E. coli DP1205 cells transformed with the pJ401-CPAL-1 plasmid expressing enzymes in the coparal biosynthetic pathway. The chromatograms in Figure 1 show GC-MS analysis of compounds produced by the same E. coli cells additionally transformed with a second plasmid expressing AspWeBVMO, SCH94-3944, the combination of SCH94-3944 and AspWeBVMO, the combination of SCH94-3944 and SCH23-BVMO1, the combination of SCH94-3944 and SCH24-BVMO1, and the combination of SCH94-3944 and SCH46-BVMO1. [Figure 27] FIG. 1 shows GC-MS analysis of terpenes and derivatives produced using engineered budding yeast strains expressing GGPP synthase carG, CPP synthase SmCPS2, CPP phosphatase TalVeTPP, alcohol dehydrogenase SCH23-ADH1, and either AspWeDUF4334 (YST184), CnecaDUF4334 (YST185), Pdigit7033 (YST186), SCH94-3944 (YST187), or SCH80-05241 (YST188). [Figure 28] A) Percentage of identified terpenes produced by YST184, YST185, YST186, YST187, and YST188. B) Total amount of identified terpenes (SumT) produced by YST184, YST185, YST186, YST187, and YST188 relative to the amount of identified terpenes in the control (SumT-C). The control strain was YST075, which expresses the copalol biosynthetic pathway. [Figure 29]FIG. 1 shows GC-MS analysis of terpenes and derivatives produced using engineered Saccharomyces cerevisiae strains expressing GGPP synthase carG, CPP synthase SmCPS2, CPP phosphatase TalVeTPP, alcohol dehydrogenase SCH23-ADH, enal cleavage polypeptide AspWeDUF4334, and either SCH23-BVMO1(YST190), SCH24-BVMO1(YST191), or AspWeBVMO(YST192). [Figure 30] A) The total amount of identified terpenes (SumT) produced by YST190, YST191 and YST192 relative to the amount of identified terpenes in YST184 (SumT-C), and B) the percentage of identified terpenes produced by YST190, YST191 and YST192. [Figure 31] FIG. 1 shows GC-MS analysis of diterpenes and diterpene derivatives produced using E. coli cells expressing LPP synthase, phosphatase, alcohol dehydrogenase, and an enal-cleaving polypeptide. The top chromatogram shows GC-MS analysis of compounds produced by E. coli DP1205 cells transformed with the pJ401-LOH-2 vector, which expresses enzymes in the labdanediol biosynthetic pathway. The bottom chromatogram shows GC-MS analysis of compounds produced by the same E. coli cells further transformed with a second plasmid expressing either AzeTolADH1 alcohol dehydrogenase or SCH94-3945 alcohol dehydrogenase together with the SCH94-3944 enal-cleaving polypeptide. [Figure 32] FIG. 1 shows the alignment and conserved amino acids of FMO-like domain-containing proteins with BVMO activity, with the predicted location of each protein family domain indicated within the box. [Figure 33]Figure 1 shows GC-MS / FID analysis of terpenes and derivatives produced using engineered Saccharomyces cerevisiae strains expressing bifunctional PvCPS, CPP phosphatase TalVeTPP, alcohol dehydrogenase SCH23-ADH, enal-cleaving polypeptide AspWeDUF4334, Baeyer-Villiger monooxygenase SCH23-BVMO1, and either esterase SCH23-EST(YST257) or esterase SCH24-EST(YST258). [Figure 34] Figure 1 shows GC-MS analysis of the biochemical production of γ-ambrol by E. coli cells expressing CPP synthase, phosphatase, alcohol dehydrogenase, enal cleavage enzyme, BVMO, and esterase. A. GC-MS analysis of compounds produced by E. coli DP1205 cells transformed with the pJ401-Mnoxy plasmid expressing enzymes of the manooloxy biosynthetic pathway. B. GC-MS analysis of compounds produced by the same E. coli cells additionally expressing BVMO (SCH24-BVMO). C. GC-MS analysis of compounds produced by the same E. coli cells additionally expressing BVMO (SCH24-BVMO) and esterase (SCH24-EST).

[0018] Abbreviations used ADH alcohol dehydrogenase BVMO Baeyer-Villiger monooxygenase bp base pair kb kilobase CPP copalyl diphosphate CPS copalyl diphosphate synthase DNA deoxyribonucleic acid cDNA complementary DNA DMAPP dimethylallyl diphosphate DTT Dithiothreitol FMO flavin monooxygenase FPP Farnesyl diphosphate GPP geranyl diphosphate GGPP Geranylgeranyl diphosphate GGPS Geranylgeranyl diphosphate synthase GC gas chromatograph IPP Isopentenyl diphosphate LPP labdanediol diphosphate LPS labdanediol diphosphate synthase MS mass spectrometer / mass spectrometry MVA Mevalonic acid PP diphosphate, pyrophosphate PCR polymerase chain reaction RNA ribonucleic acid mRNA messenger ribonucleic acid miRNA microRNA siRNA small interfering RNA rRNA ribosomal RNA tRNA transfer RNA TPP Terpenyl diphosphate definition a) General terms In this description and the appended claims, the use of "or" means "and / or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0019] Furthermore, when the term "comprising" is used in describing various embodiments, those skilled in the art will understand that in some specific instances, the embodiments can alternatively be described using the phrases "consisting essentially of" or "consisting of."

[0020] As used herein, the terms "purified," "substantially purified," and "isolated" refer to a state free from other heterogeneous compounds with which the compounds of the invention are normally associated in their natural state, such that the "purified," "substantially purified," and "isolated" subjects comprise at least 0.5%, 1%, 5%, 10%, or 20% by weight, or at least 50% or 75% by weight, of the mass of a given sample. In one embodiment, these terms refer to compounds of the invention comprising 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" when referring to nucleic acids or proteins, or nucleic acids or proteins, also refer to a state of purification or enrichment that differs from that found in nature, e.g., in a prokaryotic or eukaryotic environment, e.g., in a bacterial or fungal cell, or in a mammalian organism, particularly the human body. Any degree of purity or concentration greater than that found in nature, including (1) purification from other related structures or compounds, or (2) association with structures or compounds with which it is not normally associated in the aforementioned 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 with which it is not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.

[0021] The term "about" indicates a potential variation of ±25%, particularly ±15%, ±10%, and more particularly ±5%, ±2% or ±1% of the stated value.

[0022] The term "substantially" denotes a value in the range of approximately 80 to 100%, for example 85 to 99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9%, in particular 99 to 99.9%.

[0023] "Predominantly" refers to a percentage in the range of more than 50%, for example, 51 to 100%, particularly 75 to 99.9%, more particularly 85 to 98.5%, for example, 95 to 99%.

[0024] In the context of the present invention, a "major product" refers to a single compound or a group of at least two compounds, e.g., two, three, four, five or more compounds, particularly two or three compounds, which is "predominantly" prepared by the reaction described herein and is included in said reaction in a predominant proportion based on the total amount of the components 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 chromatogram of the reaction products based on chromatographic analysis.

[0025] A "by-product" in the context of the present invention refers to a single compound or a group of at least two compounds, e.g., 2, 3, 4, 5 or more, particularly 2 or 3 compounds, which single compound or group of compounds is not "predominantly" prepared by the reaction described herein.

[0026] Because enzymatic reactions are reversible, the present invention relates to both directions of the enzymatic or biocatalytic reactions described herein, unless otherwise specified.

[0027] "Functional variants" of the polypeptides described herein include "functional equivalents" of said polypeptides as defined below.

[0028] "Stereoisomer" includes conformational isomers, particularly configurational isomers.

[0029] Generally included, according to the present invention, are all "stereoisomeric forms," ​​eg, "conformational isomers" and "stereoisomers," of the compounds described herein.

[0030] "Stereoisomeric forms" especially include "stereoisomers" and mixtures thereof, e.g., configurational isomers (optical isomers) such as enantiomers, or geometric isomers (diastereomers) such as E- and Z-isomers, and combinations thereof. When one or more asymmetric centers are present in a molecule, the present invention includes all combinations of the different conformations of these asymmetric centers, e.g., enantiomeric pairs.

[0031] "Stereoselectivity" refers to the ability to produce a particular stereoisomer of a compound in stereomerically pure form, or to specifically convert a particular stereoisomer from among multiple stereoisomers in the enzyme-catalyzed methods described herein. More specifically, this means that the product of the invention may be enriched with respect to a particular stereoisomer, or the starting material (e.g., e ... %ee=[X A -X B ] / [X A +X B ]*100 In the formula, X A and X B represents the molar ratio of stereoisomers A and B (Molenbruch).

[0032] The terms "selectively convert" or "enhance selectivity" generally mean that a particular stereoisomeric form of an unsaturated hydrocarbon, e.g., the E-form, is converted at a higher rate or amount (compared on a molar basis) than the corresponding other stereoisomeric form, e.g., the Z-form, over the entire course of the reaction (i.e., from the beginning to the end of the reaction), at a certain point in the reaction, or during any "interval" of the reaction. In particular, the selectivity may be observed during "intervals" corresponding to conversion 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 rate 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 the aforementioned interval; - a higher relative amount of the isomer at a defined degree (%) of conversion of the substrate; and / or - Identical relative amounts of isomers at higher degrees of conversion (%). Any of these are preferably observed in comparison to a reference method, which is carried out under otherwise identical conditions using known chemical or biochemical means.

[0033] In general, the present invention includes all "isomeric forms" of the compounds described herein, such as structural isomers, particularly stereoisomers, and mixtures thereof, such as optical or geometric isomers, e.g., E- and Z-isomers, and combinations thereof. When several asymmetric centers are present in a molecule, the present invention includes all combinations of the different conformations of these asymmetric centers, e.g., pairs of enantiomers, or any mixture of stereoisomeric forms.

[0034] 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.

[0035] Different yield parameters ("yield" or Y P / S "Specific Productivity Yield"; or space-time yield (STY)) is well known in the art and is determined as described in the literature.

[0036] As used herein, the terms "yield" and "Y P / S ” (expressed as mass of product produced / mass of material consumed, respectively) are used synonymously.

[0037] Specific productivity yield represents the amount of product produced per gram of biomass per hour and per L of fermentation broth. Wet cell weight, denoted WCW, represents the amount of biologically active microorganisms in a biochemical reaction. This value is expressed as gram of product per gram of WCW per hour (i.e., g / gWCW). -1 h -1 Alternatively, the amount of biomass can be expressed as dry cell weight, denoted DCW. Furthermore, the biomass concentration can be calculated by 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.

[0038] Where the present disclosure refers to features, parameters and ranges thereof of different priority (including general, non-explicitly preferred features, parameters and ranges thereof), unless otherwise stated, any combination of two or more of such features, parameters and ranges thereof is encompassed by the disclosure herein, regardless of their respective priorities.

[0039] b) Biochemical terminology The term "domain" refers to a set of amino acids or a partial sequence of amino acid residues that are conserved at specific positions along an alignment of the sequences of evolutionarily related proteins. While amino acids at other positions may differ between protein homologs, highly conserved amino acids at specific positions in a domain indicate amino acids that are likely to be essential for the structure, stability, or function of the protein. Domains can be identified by the high degree of amino acid conservation in the aligned sequences of a family of protein homologs and can be used as identifiers to determine whether any polypeptide in question belongs to a previously identified polypeptide family.

[0040] The term "motif" or "consensus sequence" or "signature" refers to a short, conserved region in the sequences of evolutionarily related proteins. A motif is often highly conserved as part of a domain, but may also comprise only a portion of a domain.

[0041] A "protein family" is defined as a group of proteins with a common evolutionary origin, reflected by their related functions, sequence similarity, or similar primary, secondary, or tertiary structure. Proteins within a protein family are usually homologous and have similar structures of conserved functional domains and motifs.

[0042] Specialized databases for identifying domains exist, such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequence motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). Domains or motifs may be identified using routine techniques, such as by sequence alignment.

[0043] The term "Pfam" refers to the large collection of protein domains and protein families maintained by the Pfam Consortium and available at several sponsored world-wide websites, such as http: / / pfam.xfam.org / (European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL_EBI)). The latest release of Pfam is Pfam 32.0 (September 2018), which is part of the UniProt Reference Proteomes (El-Gebali S. et al, 2019, Nucleic Acids Res. 47, Database issue Pfam domains and families are identified using multiple sequence alignments and hidden Markov models (HMMs). Pfam-A family or domain assignments are high-quality assignments generated by probing seed alignments using representative members of the protein family and profile hidden Markov models based on the seed alignments (unless otherwise specified, the match between the query protein and the Pfam domain or family is a Pfam-A match). A full family alignment is then automatically generated using all identified sequences belonging to the family (Sonnhammer (1998) Nucleic Acids Research 26, 320-322; Bateman (2000) Nucleic Acids Research 26, 263-266; Bateman (2004) Nucleic Acids Research 32, Database Issue, D138-D141; Finn (2006) Nucleic Acids Research Database Issue 34, D247-251; Finn (2010) Nucleic Acids Research Database Issue 38, D211-222).For example, once the Pfam database is accessed using one of the above-referenced websites, protein sequences can be queried against the HMM using HMMER homology search software (e.g., HMMER2, HMMER3, or higher versions (hmmer.janelia.org / )). Significant matches that identify a queried protein as belonging to a pfam family (or having a particular Pfam domain) are those whose bit score is equal to or greater than the collection threshold for the Pfam domain. Expectation values ​​(e-values) can also be used as criteria for including a queried protein in a Pfam or determining whether a queried protein has a particular Pfam domain, where a low e-value is one much lower than 1.0, e.g., less than or equal to 0.1.

[0044] The "E-value" (expected value) is the expected number of hits that would have a score equal to or higher than this value by pure chance. That is, a good E-value, where predictions are reliable, is much lower than 1. An E-value around 1 is what you would expect by chance. Therefore, the lower the E-value, the more specific the domain search. Only positive numbers are allowed (defined by Pfam).

[0045] "Precursor" molecules of the target compounds described herein are preferably converted to said target compounds by the enzymatic action of an appropriate polypeptide that performs at least one structural change on said precursor molecule. For example, a "diphosphate precursor" (such as a "terpenyl diphosphate precursor") is converted to said target compound (such as a terpene alcohol) via enzymatic removal of the diphosphate moiety, e.g., by removal of a monophosphate or diphosphate group by a phosphatase enzyme. For example, a "non-cyclic precursor" (such as an non-cyclic terpenyl precursor) can be converted to a cyclic target molecule (such as a cyclic terpene compound) in one or more steps via the action of a cyclic enzyme or synthase, regardless of the specific enzymatic mechanism of such enzyme.

[0046] The term "protein tyrosine phosphatase" refers to a group of enzymes generally known to remove phosphate groups from phosphorylated tyrosine residues on proteins. A particular subgroup of this family described herein is an enzyme useful for dephosphorylating phosphorylated terpene molecules.

[0047] "Terpene synthase" refers to a polypeptide that converts a terpene precursor molecule into a terpene target molecule, particularly a processed target terpene alcohol or terpene hydrocarbon. Non-limiting examples of such terpene precursor molecules are acyclic compounds selected from farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), or a mixture of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). If the resulting terpene contains a diphosphate moiety, the synthase is called a "terpenyl diphosphate synthase."

[0048] The terms "terpenyl diphosphate synthase" or "polypeptide with terpenyl diphosphate synthase activity" or "terpenyl diphosphate synthase protein" or "ability to produce terpenyl diphosphate" refer to a polypeptide capable of catalyzing the synthesis of terpenyl diphosphate in any of its stereoisomers or a mixture thereof, starting from acyclic terpene pyrophosphates, particularly GPP, FPP, GGPP, or IPP in combination with DMAPP. Terpenyl diphosphate may be the sole product or part of a mixture of terpenyl phosphates. The mixture may also contain terpenyl monophosphate and / or terpene alcohols. The above definition also applies to the group of "bicyclic terpenyl diphosphate synthases" that produce bicyclic terpenyl diphosphates, such as CPP or LPP. An example of such a "terpenyl diphosphate synthase" is copalyl diphosphate synthase (CPS). Copalyl diphosphate may be the sole product or part of a mixture of copalyl phosphates. The mixture may contain copalyl monophosphate and / or other terpenyl diphosphates. Another example of such a "terpenyl diphosphate synthase" is labdanediol diphosphate synthase (LPS). Labdanediol diphosphate may be the only product or may be part of a mixture of labdanediol phosphates. The mixture may contain labdanediol monophosphate and / or terpenyl diphosphate.

[0049] The terms "terpenyl diphosphate phosphatase" or "polypeptide having terpenyl diphosphate phosphatase activity" or "terpenyl diphosphate phosphatase protein" or "capable of producing terpene alcohols" refer to polypeptides capable of catalyzing the removal of diphosphate or monophosphate moieties (regardless of the specific enzymatic mechanism) to form dephosphorylated compounds, particularly alcohol compounds corresponding to the aforementioned terpenyl moiety. The terpene alcohol may be present in the product in any of its stereoisomers or as a mixture thereof. The terpene alcohol may be the sole product or part of a mixture with other terpene compounds, such as dephosphorylated analogs of the respective (e.g., acyclic) precursors of the aforementioned terpenyl diphosphates. The above definition also applies to the group of "bicyclic terpenyl diphosphate phosphatases" that produce bicyclic terpene alcohols, such as copalol or labdanediol.

[0050] An example of such a "terpenyl diphosphate phosphatase" enzyme is copalyl diphosphate phosphatase (CPP phosphatase). Copalol may be the sole product or may be part of a mixture with dephosphorylated precursors, such as farnesol and / or geranylgeraniol; and / or by-products resulting from secondary enzyme activities in the reaction mixture, such as esters or aldehydes of such alcohols or other cyclic or acyclic diterpenes. Another example of such a "terpenyl diphosphate phosphatase" enzyme is labdanediol diphosphate phosphatase (LPP phosphatase). Labdanediol may be the sole product or may be part of a mixture with dephosphorylated precursors, such as farnesol and / or geranylgeraniol; and / or by-products resulting from secondary enzyme activities in the reaction mixture, such as esters or aldehydes of such alcohols or other cyclic or acyclic diterpenes.

[0051] An "enal cleavage enzyme" or "enal cleavage protein" or "enal cleavage polypeptide" in the context of the present invention refers to an "α,β-unsaturated aldehyde carbon-carbon double bond cleavage enzyme," which may also be referred to as an "α,β-unsaturated aldehyde C═C bond cleavage enzyme" or "α,β-unsaturated aldehyde C═C cleavage enzyme" or "enal C═C cleavage enzyme." The enal cleavage proteins of the present invention may also be designated as members of the "DUF4334 protein family" and / or as members of the "GXWXG protein family," based on the domain organization of the proteins.

[0052] More specifically, the enal-cleaving enzyme of the present invention has the ability to cleave labdan-type carbonyl compounds, such as labdan aldehyde, particularly coparal, into the respective dinorlabdan carbonyl compounds. "Baeyer-Villiger monooxygenases" (BVMOs) are flavoenzymes, a class of polypeptides with oxidoreductase activity (EC 1.14.13.X). These enzymes catalyze the oxidation of linear and cyclic (aromatic or nonaromatic) aldehydes or ketones to their corresponding esters or lactones, a process very similar to the chemical Baeyer-Villiger oxidation. During enzymatic oxidation, one atom of molecular oxygen is incorporated into the carbon-carbon bond of an unactivated carbonyl compound. BVMOs require NADPH or NADH as cofactors, or accept both. BVMOs also require molecular oxygen as a cosubstrate. More specifically, the BVMO of the present invention has the ability to oxidize terpene-derived aldehydes or ketones, such as labdane-type carbonyl compounds, e.g., labdane aldehyde, particularly coparal and / or manooloxy, to their respective carbonyl esters.

[0053] The term "esterase" refers to a polypeptide having hydrolase activity that decomposes esters into acids and alcohols in a chemical reaction with water (hydrolysis). Esterases in the context of the present invention are selected from the class of carboxylic acid ester hydrolases (EC 3.1.1.-) and cleave acyl groups, such as acetyl or formyl groups, from the respective ester substrates. More specifically, the esterases of the present invention have the ability to cleave labdan ester compounds, such as γ-ambryl acetate, to form the respective labdan alcohols, such as γ-ambrol.

[0054] "Alcohol dehydrogenase" (ADH) in the context of the present invention refers to an enzyme that binds NAD as a cofactor. + or NADP + These enzymes are members of the EC family 1.1.1.1 (NAD + dependent) or 1.1.1.2 (NADP + The ADH of the present invention is a member of the oxidative oxidation enzyme family (dependent on the oxidation of labdanes). More specifically, the ADH of the present invention has the ability to oxidize labdan alcohols to the respective labdan carbonyl compounds (aldehydes or ketones), such as oxidizing copalol to copal and / or labdanediol to the respective aldehydes or other labdan derivatives of copalol or labdanediol, e.g., the respective nor- or dinor-labdan derivatives of copalol or labdanediol. The ADH as used herein may be endogenous to the respective biocatalytic process or may be exogenous.

[0055] "Enal cleavage activity" can be determined under "standard conditions" as described later in this specification. Enal cleavage activity can be determined using host cells expressing recombinant enal-cleaving polypeptides, disrupted cells expressing enal-cleaving polypeptides, fractions thereof, or concentrated or purified enal-cleaving polypeptides in a preferably buffered culture or reaction medium having a pH ranging from 6 to 11, preferably 7 to 9, at a temperature of about 20 to 45°C, e.g., about 25 to 40°C, preferably 25 to 32°C, in the presence of a reference substrate, particularly copalal, either added at an initial concentration of 1 to 100 μM mg / ml, preferably 5 to 50 μM, particularly 30 to 40 μM, or endogenously produced by the host cells. The conversion reaction to form the respective cleavage product, such as copalal, is carried out for 10 minutes to 5 hours, preferably about 1 to 2 hours. The cleavage product can then be extracted with an organic solvent, e.g., ethyl acetate, and then conventionally determined.

[0056] "BVMO activity" can be determined under "standard conditions," as described later in this specification. BVMO activity can be determined using host cells expressing recombinant BVMO, cells expressing disrupted BVMO, fractions thereof, or concentrated or purified BVMO enzyme in a preferably buffered culture or reaction medium having a pH ranging from 6 to 11, preferably 7 to 9, at a temperature of about 20 to 45°C, e.g., about 25 to 40°C, preferably 25 to 32°C, at an initial concentration of 1 to 100 μM mg / ml, preferably 5 to 50 μM, particularly 30 to 40 μM, or in the presence of a reference substrate, particularly coparal and / or mano-oloxy, endogenously produced by the host cells and in the presence of molecular oxygen. In in vitro assays, a coenzyme selected from NADH and NADPH must be added at an appropriate concentration range that is easy to determine. The conversion reaction to form the respective cleavage products, such as formyl esters 1a and / or 1b in the case of coparal and γ-ambryl acetate in the case of manooloxy, is carried out for 10 minutes to 5 hours, preferably about 1 to 2 hours. The oxidation products can then be extracted with an organic solvent, such as ethyl acetate, and then determined by conventional methods.

[0057] "Terpenyl diphosphate synthase activity" (e.g., CPS or LPS activity) is determined under "standard conditions" as described later in this specification. Terpenyl diphosphate synthase activity can be determined using recombinant host cells expressing terpenyl diphosphate synthase, disrupted cells expressing terpenyl diphosphate synthase, fractions thereof, or concentrated or purified terpenyl diphosphate synthase in a preferably buffered culture or reaction medium having a pH ranging from 6 to 11, preferably 7 to 9, at a temperature of about 20 to 45°C, e.g., about 25 to 40°C, preferably 25 to 32°C, at an initial concentration of 1 to 100 μM mg / ml, preferably 5 to 50 μM, particularly 30 to 40 μM, or in the presence of a reference substrate, particularly GGPP, endogenously produced by the host cells. The conversion reaction to form terpenyl diphosphate is carried out for 10 minutes to 5 hours, preferably about 1 to 2 hours. In the absence of endogenous phosphatases, one or more exogenous phosphatases, such as alkaline phosphatases, are added to the reaction mixture to convert the terpenyl diphosphates formed by the synthases to their respective terpene alcohols, which can then be determined by conventional methods after extraction with an organic solvent, such as ethyl acetate.

[0058] "Terpenyl diphosphate phosphataseThe activity (e.g., CPP or LPP phosphatase activity) is determined under "standard conditions" as described later in this specification. Terpenyl diphosphate phosphatase activity can be determined using host cells expressing recombinant terpenyl diphosphate phosphatase, disrupted cells expressing terpenyl diphosphate phosphatase, fractions thereof, or concentrated or purified terpenyl diphosphate phosphatase enzyme in a preferably buffered culture or reaction medium having a pH in the range of 6 to 11, preferably 7 to 9, in the presence of a reference substrate, here, e.g., CPP or LPP, added at an initial concentration of 1 to 100 μM mg / ml, preferably 5 to 50 μM, and particularly 30 to 40 μM, or endogenously produced by the host cells. The conversion reaction to form terpenyl diphosphate is carried out for 10 minutes to 5 hours, preferably about 1 to 2 hours. The terpene alcohol can then be determined by conventional methods after extraction with an organic solvent such as ethyl acetate.

[0059] Specific examples of standard conditions suitable for each of the above mentioned enzymatic activities can be found in the "Experimental Part" below.

[0060] The terms "biological function," "function," "biological activity," or "activity" of a terpenyl synthase refer to the ability of a terpenyl diphosphate synthase described herein to catalyze the formation of at least one terpenyl diphosphate from a corresponding precursor terpene.

[0061] The terms "biological function," "function," "biological activity," or "activity" of a terpenyl diphosphate phosphatase refer to the ability of the terpenyl diphosphate phosphatase described herein to catalyze the removal of a diphosphate group from the aforementioned terpenyl compound to form the corresponding terpene alcohol.

[0062] The mevalonate pathway, also known as the isoprenoid pathway or HMG-CoA reductase pathway, is an essential metabolic pathway present in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and generates two five-carbon building blocks, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The key enzymes are acetoacetyl-CoA thiolase (atoB), HMG-CoA synthase (mvaS), HMG-CoA reductase (mvaA), mevalonate kinase (MvaK1), phosphomevalonate kinase (MvaK2), mevalonate diphosphate decarboxylase (MvaD), and isopentenyl diphosphate isomerase (idi). Combining the mevalonate pathway with enzymes that generate the terpene precursors GPP, FPP, or GGPP, particularly FPP synthase (ERG20), enables recombinant production of terpenes.

[0063] As used herein, the term "host cell" or "transformed cell" refers to a cell (or organism) that has been modified to carry at least one nucleic acid molecule, e.g., a recombinant gene encoding a desired protein or a nucleic acid sequence that, upon transcription, results in at least one functional polypeptide of the invention, particularly a terpenyl diphosphate synthase protein or a terpenyl diphosphate phosphatase enzyme as defined hereinabove. Host cells are, in particular, bacterial, fungal, or plant cells or plants. A host cell may contain a recombinant gene or multiple genes integrated into the host cell's nucleus or organelle genome, for example, organized as an operon. Alternatively, the host may contain the recombinant gene extrachromosomally.

[0064] "Organism" refers to any multicellular or unicellular organism other than a human, such as a plant, or a microorganism. In particular, a microorganism is a bacterium, yeast, algae, or fungus.

[0065] The term "plant" is used interchangeably to include plant cells, including plant protoplasts, plant tissues, plant cell tissue cultures that give rise to regenerated plants, or plant parts or plant organs, such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits, etc. Any plant can be used to carry out the methods of the embodiments herein.

[0066] A particular organism or cell is "capable of producing FPP" if it naturally produces FPP, or if it does not naturally produce FPP but has been transformed to produce FPP using a nucleic acid described herein. 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."

[0067] A particular organism or cell is "capable of producing GGPP" if it naturally produces GGPP, or if it does not naturally produce GGPP but has been transformed to produce GGPP using a nucleic acid described herein. Organisms or cells transformed to produce greater amounts of GGPP than naturally occurring organisms or cells are also encompassed by "organisms or cells capable of producing GGPP."

[0068] A particular organism or cell is "capable of producing terpenyl diphosphate" if it naturally produces a terpenyl diphosphate as defined herein, or if it does not naturally produce said diphosphate but has been transformed to produce said diphosphate using a nucleic acid described herein. Organisms or cells transformed to produce greater amounts of terpenyl diphosphate than naturally occurring organisms or cells are also encompassed by "organisms or cells capable of producing terpenyl diphosphate."

[0069] A particular organism or cell is "capable of producing terpene alcohols" if it naturally produces the terpene alcohols defined herein, or if it does not naturally produce the alcohols but is transformed to produce the alcohols using the nucleic acids described herein. Organisms or cells that are transformed to produce greater amounts of terpene alcohols than naturally occurring organisms or cells are also encompassed by "organisms or cells capable of producing terpene alcohols." The same applies to certain organisms that are "capable of producing labdane alcohols."

[0070] A particular organism or cell is "capable of producing an ester" if it naturally produces an ester as defined herein, or if it does not naturally produce said ester but has been transformed with a nucleic acid described herein to produce said ester. Organisms or cells transformed to produce greater amounts of an ester than naturally occurring organisms or cells are also encompassed by "organisms or cells capable of producing an ester."

[0071] A particular organism or cell is "capable of producing a target product" if it naturally produces a target product (e.g., an ester, alcohol, or carbonyl compound, more particularly a labdan-type compound) as defined herein, or if it does not naturally produce the target product but is transformed to produce the target product using a nucleic acid described herein. Organisms or cells that are transformed to produce a target product in greater amounts than naturally occurring organisms or cells are also included in the term "organisms or cells that can produce a target product."

[0072] The term "fermentative production" or "fermentation" refers to the ability of a microorganism (assisted by enzymatic activities contained in or produced by said microorganism) to produce a compound in cell culture utilizing at least one carbon source added to the incubation.

[0073] 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 either unprocessed or processed, for example as described herein.

[0074] An "enzyme-catalyzed" or "biocatalytic" process, as defined herein, means that the process is carried out under the catalytic action of an enzyme (including variants of the enzyme). Thus, the process can be carried out in the presence of the enzyme in isolated (purified, concentrated) or crude form, or in the presence of a cellular system, particularly a natural or recombinant microbial cell, that contains the enzyme in active form and has the ability to catalyze the conversion reactions disclosed herein.

[0075] c) Chemical terminology: The term "α,β-unsaturated carbonyl" compounds refers to compounds of the general formula R a R b C=C(R c represents an organic molecule containing an aldehyde or keto group of —C═O, wherein the C═C bond may be in any stereoisomeric configuration, and wherein the residue R a , R b and R c may be the same or different and may have the meanings defined below for the particular α,β-unsaturated carbonyl compound.

[0076] The "labdane" compounds in the context of the present invention exhibit the following basic structure, whose carbon skeleton consists of 20 carbon atoms: The depicted carbon atom numbering is applied to further define specific positions within said carbon skeleton.

[0077] [ka]

[0078] The term "labdan" comes from this basic C 20The present invention encompasses any compound of the structure in any stereoisomeric form, and includes variants of this structure containing one or more unsaturated C-C bonds, particularly one or more C=C bonds, at any position within the carbocyclic ring and / or side chain. Also included are variants thereof containing one or more substituents, such as a substituent selected from the group: -OH, =O, -O-CO_R, where R may be linear or branched alkyl, particularly lower alkyl, more particularly C1-C4 alkyl, such as methyl, ethyl, n- or i-propyl, or n-, i- or t-butyl, and where -COOH may be present at any of the primary, secondary or tertiary C atoms shown.

[0079] Such "labdane" "labdane-derived" compounds have basic C 20 These include compounds in which the carbon skeleton has been modified by the deletion of one or more carbon atoms. Examples include: Norlabdan (C 19 Skeleton), Dinorlabdan (C 18 skeleton), trinorlabdane (C 17 skeleton), tetranorlabdane (C 16 The position of the missing carbon atom is indicated by the carbon number. For example, norlabdane, which is missing the 15th carbon, is written as "15-norlabdane."

[0080] Such "labdane" or "labdane-derived" compounds include those that incorporate the basic C structure by inserting a heteroatom between the two C atoms of the labdane skeleton. 20 Also included are compounds in which the carbon skeleton has been modified, for example, by inserting an ether bridge between positions 14 and 15, converting labdane to norlabdane, particularly a norlabdane ester.

[0081] Non-limiting examples of substituted labdanes or substituted labdanes derived structures are shown below: [ka]

[0082] As used herein, "diphosphate" and "pyrophosphate" are synonymous terms.

[0083] "Terpenes" are a diverse class of organic compounds produced by a variety of plants, especially conifers, and some insects. Terpenes are hydrocarbons. Although sometimes used interchangeably with "terpenes," "terpenoids" or "isoprenoids" are modified terpenes because they contain additional functional groups, usually oxygen-containing.

[0084] "Terpenoids" ("isoprenoids") are a broad and diverse class of naturally occurring organic chemicals derived from terpenes. Although sometimes used interchangeably with the term "terpenes," "terpenoids" typically contain additional O-containing functional groups, such as hydroxyl, carbonyl, or carboxyl groups. Most are polycyclic structures with oxygen-containing functional groups. Unless otherwise specified, the terms "terpene" and "terpenoid" may be used interchangeably in the context of this specification.

[0085] Terpenes (and terpenoids) can be classified by the number of isoprene units in the molecule, with the prefix in the name indicating the number of terpene units required to assemble the molecule. Hemiterpenes consist of one isoprene unit. Monoterpenes consist of two isoprene units and have the molecular formula C 10 H 16 Sesquiterpenes consist of three isoprene units and have the molecular formula C 15 H 24 Diterpenes consist of four isoprene units and have the molecular formula C 20 H 32 is.

[0086] "Terpenyls" refers to acyclic and cyclic chemical hydrocarbyl residues derived from the C5 building block isoprene, and specifically contains one or more such building blocks.

[0087] "Cyclic terpene" or "cyclic terpenyl" or "cyclic diterpene" or "cyclic diterpenyl" refers to a terpene compound or terpenyl residue that contains at least one, e.g., 1, 2, 3, 4 or 5, carbocyclic fused and / or unfused rings in its structure, preferably 2, carbocyclic fused rings.

[0088] "Bicyclic terpene" or "bicyclic terpenyl" or "bicyclic diterpene" or "bicyclic bicycloditerpenyl" refers to a terpene compound or terpenyl residue that contains two carbocyclic rings in its structure, preferably two fused carbocyclic rings.

[0089] "Derivatives of terpenes" or "derivatives of terpenoids" in the context of the present invention refer in particular to compounds obtained from terpenes or terpenoids by chemical and / or enzymatic modification. More particularly, such derivatives include "hydrocarbon chain degraded" derivatives.

[0090] A "hydrocarbon chain degraded" terpene or terpenoid differs from its undegraded precursor in that the precursor carbon skeleton has a reduced number of carbon atoms.

[0091] A "hydrocarbyl" residue is a chemical group consisting essentially of carbon and hydrogen atoms and may be acyclic, linear or branched, saturated or unsaturated, or cyclic, saturated or unsaturated, aromatic or non-aromatic. If the hydrocarbyl residue is acyclic, it contains 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 carbon atoms. If the hydrocarbyl residue is cyclic, it contains 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, or especially 4, 5, 6, or 7 carbon atoms.

[0092] The aforementioned hydrocarbyl residue may be unsubstituted or may have at least one, for example 1 to 5, preferably 0, 1 or 2, substituents.

[0093] Particular examples of such hydrocarbyl residues are acyclic linear or branched alkyl or alkenyl residues as defined below; or monocyclic or polycyclic, particularly monocyclic or bicyclic, saturated or unsaturated non-aromatic moieties, such as found in cyclic (e.g., bicyclic) or acyclic terpene-type compounds and labdan-type compounds as defined herein.

[0094] An "alkyl" residue represents a linear or branched saturated hydrocarbon residue containing 1 to 30, 1 to 25, 1 to 20, 1 to 15, or 1 to 10, or 1 to 7, 1 to 6, 1 to 5, or 1 to 4 carbon atoms.

[0095] An "alkenyl" residue represents a linear or branched, monovalent or polyvalent unsaturated hydrocarbon residue. The "alkenyl" residue contains 2 to 30, 2 to 25, 2 to 20, 2 to 15, or 2 to 10, or 2 to 7, 2 to 6, 2 to 5, or 2 to 4 carbon atoms. The "alkenyl" residue may have up to 10, for example 1, 2, 3, 4, or 5 C=C double bonds.

[0096] The term "lower alkyl" or "short chain alkyl" refers to a saturated straight or branched chain hydrocarbon radical having 1 to 4, 1 to 5, 1 to 6, or 1 to 7, especially 1 to 4, carbon atoms. Examples that may be mentioned include: methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl; as well as n-heptyl, their mono- or poly-branched analogs.

[0097] "Long chain alkyl" refers to a saturated straight or branched hydrocarbyl radical having, for example, 8 to 30, e.g., 8 to 20 or 8 to 15, carbon atoms, such as octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, hencosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, squaryl, structural isomers, particularly mono- or poly-branched isomers thereof.

[0098] "Long chain alkenyl" refers to the mono- or polyunsaturated analog of the "long chain alkyl" groups described above.

[0099] "Short-chain alkenyl" (or "lower alkenyl") denotes a mono- or polyunsaturated, especially monounsaturated, straight-chain or branched hydrocarbon radical having 2 to 4, 2 to 6, or 2 to 7 carbon atoms and one double bond in any position, examples being C2-C6-alkenyl, such as ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl. , 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, Examples include 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl and 1-ethyl-2-methyl-2-propenyl.

[0100] "Alkylene" refers to a linear or mono- or poly-branched hydrocarbon bridge group having 1 to 10 carbon atoms, such as a C1-C7 alkylene group selected from -CH2, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)2-CH(CH3)-, -CH2-CH(CH3)-CH2-, (CH2)4-, -(CH2)5-, -(CH2)6, -(CH2)7-, -CH(CH3)-CH2-CH2-CH2-CH(CH3)- or -CH(CH3)-CH2-CH2-CH2-CH2-CH(CH3)-, in particular a C1-C4 alkylene group selected from -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)2-CH(CH3)-, -CH2-CH(CH3)-CH2-.

[0101] An "alkylidene" group represents a straight or branched hydrocarbon substituent attached to the molecular body via a double bond. An "alkylidene" group contains 1 to 6 carbon atoms. Examples of such "C1-C6-alkylidene" include methylidene (=CH2), ethylidene, (=CH-CH2), n-propylidene, n-butylidene, n-pentylidene, n-hexylidene, and structural isomers thereof, such as isopropylidene.

[0102] "Alkenylidene" refers to the monounsaturated analog of the above alkylidene having more than two carbon atoms, and is sometimes referred to as "C3-C6-alkenylidene." Examples include n-propenylidene, n-butenylidene, n-pentenylidene, and n-hexenylidene.

[0103] The "substituents" of the above-mentioned residues contain one heteroatom such as O or N. Preferably, the substituents are independently selected from -OH, C=O, or -COOH. Most preferably, the aforementioned substituent is -OH.

[0104] A "monocyclic or polycyclic hydrocarbyl residue" includes one, two, or three fused (annelated) or non-fused, optionally substituted, saturated or unsaturated hydrocarbon ring groups (or "carbocyclic" groups). Each ring may, independently of the others, contain 3 to 8, particularly 5 to 7, and more particularly 6, ring carbon atoms. Examples of monocyclic residues include "cycloalkyl" groups, which are carbocyclic radicals having 3 to 7 carbon ring atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl; and the corresponding "cycloalkenyl" groups. "Cycloalkenyl" (or "mono- or polyunsaturated cycloalkyl") refers in particular to monocyclic, mono-, or polyunsaturated carbocyclic groups having 5 to 8, preferably up to 6, carbon ring members, such as monounsaturated cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl radicals.

[0105] Examples of polycyclic residues include groups in which one, two, or three such cycloalkyl and / or cycloalkenyl groups are bonded together, e.g., by annelation, to form a polycyclic cycloalkyl or cycloalkenyl ring. A non-limiting example is the bicyclic decalinyl residue, which is composed of two annelated six-membered carbocyclic rings.

[0106] The number of substituents on such monocyclic or polycyclic hydrocarbyl residues may vary from 1 to 10, especially from 1 to 5 substituents. Suitable substituents on such cyclic residues are selected from lower alkyl, lower alkenyl, alkylidene, alkenylidene, or residues containing one heteroatom such as O or N, e.g., —OH or —COOH. In particular, the substituents are independently selected from —OH, —COOH, methyl, and methylidene.

[0107] Unsaturated cyclic groups may contain one or more, for example 1, 2 or 3, C=C bonds and may be aromatic or, especially, non-aromatic.

[0108] The monocyclic or polycyclic saturated or unsaturated groups mentioned above may contain at least one, for example 1, 2, 3 or 4, ring heteroatoms such as O, N or S.

[0109] [Table 1]

[0110] [Table 2-1]

[0111] [Table 2-2]

[0112] [Table 2-3]

[0113] [Table 2-4]

[0114] [Table 2-5]

[0115] [Table 2-6]

[0116] [Table 2-7]

[0117] [Table 2-8]

[0118] [Table 2-9]

[0119] [Table 2-10]

[0120] [Table 2-11]

[0121] [Table 2-12]

[0122] [Table 2-13]

[0123] Detailed Description a. Specific Embodiments of the Invention i) The present invention relates to the following specific embodiments of biocatalytic methods involving the use of polypeptides with BMVO activity: 1. A biocatalytic method for preparing an ester compound, comprising: (1) General formula I [ka] [In the formula, "a" represents a single or double bond; When "a" represents a double bond, "x" is the integer 1, and when "a" represents a single bond, "x" is the integer 2; R 1 represent, independently of one another, H or lower alkyl, such as C1-C4-alkyl, in particular H or methyl, R 2 represents H, a linear or branched, saturated or unsaturated, optionally substituted hydrocarbyl residue, in particular one having from 2 to 20, more particularly from 5 to 15, carbon atoms, or the group Cyc-A-, where: Cyc represents an optionally substituted, saturated or unsaturated, monocyclic or polycyclic hydrocarbyl residue, A represents a chemical bond or an optionally substituted linear or branched alkylene bridge, in particular methylene; R 3 are independently H or C1-C 30 , C1~C 20 , especially C1~C 15 or a lower alkyl group such as C1-C4-alkyl, in particular H or methyl, more particularly H, When "a" represents a single bond, Z represents a hydrocarbyl residue containing a carbonyl group, in particular an aldehyde or keto group, or when "a" represents a double bond, Z together with the carbon atom to which it is attached forms a carbonyl group (C=O, in particular an aldehyde or keto group) or an alkylidene residue, in particular a C1-C6-alkylidene residue, having a terminal carbonyl group, in particular an aldehyde or keto group; When "a" represents a double bond and Z, together with the carbon atom to which it is attached, forms a carbonyl group (C=O), R 2 and R 1 may together with the carbon atoms to which they are attached form a cyclic, especially a monocyclic, saturated or unsaturated, optionally substituted carbocyclic ring group, especially a 5- to 7-membered ring; wherein the carbonyl compound of the general formula I is provided in stereoisomerically pure form or as a mixture of stereoisomers, contacting the precursor with a natural or recombinant polypeptide having Baeyer-Villiger monooxygenase (BVMO) (EC 1.13.14.-) activity to form the respective carbonyl ester product, in particular by introducing an oxygen atom between the carbonyl group and the α-carbon atom of the precursor; (2) optionally separating the carbonyl ester formed in step (1), wherein said carbonyl ester compound is obtained in stereoisomerically pure form or as a mixture of stereoisomers; A biocatalytic method comprising:

[0124] 2. In carbonyl compounds of general formula I, "a" represents a chemical double bond, Z is =O (see manooloxy) or =C (R 4 )-C(R 5 )=O (see Coparal), or "a" represents a single chemical bond, and Z is -C(R 5 )=O (see norlabdane compounds 3a and 3b), where: R 4 and R 5 2. A biocatalytic method according to embodiment 1, wherein, independently of one another, represent H or lower alkyl, such as C1-C4-alkyl, in particular H or methyl.

[0125] 3. A biocatalytic method according to any one of the preceding embodiments, wherein the carbonyl compound of general formula I has a labdane-type structure, in particular a labdane, norlabdane or dinorlabdane structure.

[0126] 4. The carbonyl ester formed is of formula II [ka] [In the formula, R 2and R 3 is as defined above, E represents a hydrocarbyl residue containing a carbonyl ester group as described above, or E and R 2 and together with the carbon atom to which they are attached form a cyclic ester group.

[0127] 5. The carbonyl ester group E is -OC(O)-R 1 , -C(R 1 )2-OC(O)R 5 , -C(R 1 )=C(R 4 )-OC(R 5 )=O; and E and R 2 and the carbon atom to which they are attached, the cyclic ester ring being, for example, a group represented by the formula IIa and IIb [ka] [In the formula, R 1 , R 3 , R 4 and R 5 is as defined above], and a cyclic ester group representing a 5- to 7-membered ring, particularly a 6-membered ring, such as an ester of 5. The biocatalytic method of embodiment 4, wherein the biocatalytic method is selected from:

[0128] 6.R 2represents the group Cyc-A-, where A represents a linear or branched C1-C4-alkylene bridge, in particular methylene, and Cyc represents a monocyclic or polycyclic, in particular bicyclic, saturated or unsaturated hydrocarbyl residue, in particular a bicyclic annelated hydrocarbyl residue comprising 5 to 7, in particular 6, ring atoms per ring, wherein Cyc is optionally substituted with 1 to 10, in particular 1 to 5, substituents, in particular said substituents may be independently selected from C1-C4-alkyl, C1-C4-alkylidene, C3-C6-alkenylidene, C2-C4-alkenyl, oxo (=O), hydroxy, or amino, in particular C1-C4-alkyl such as methyl, and C1-C4-alkylidene such as methylidene.

[0129] 7.R 2 The method of any one of the preceding embodiments, wherein the Cyc residue in is obtained by terpene cyclization to form an optionally substituted decalinyl residue, in particular a bicyclic residue.

[0130] 8. Cyc-A is a compound of formula IIIa, IIIb or IIIc [ka] 8. The method of embodiment 7, wherein R represents a bicyclic residue having 15 carbon atoms.

[0131] 9. A polypeptide having BVMO activity, (1) A group of polypeptides that contain within their amino acid sequence a flavin-containing monooxygenase (FMO) protein family domain having Pfam ID number PF00743; or a domain that retains at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with PF00743; In particular, the polypeptide of the present invention having BVMO activity is -5 Less than or equal to 1 x 10 -10 Less than or equal to 1 x 10 -15 or less, or 1×10 -18 Below, especially 1×10-10 ~1×10 -18 in the range of 1×10 -14 ~1×10 -17 If the sequence matches the domain PF00743 with an e value in the range of 0.01 to 0.01, the protein is identified as a member of the FMO protein family containing the domain. The sequence of a polypeptide having BVMO activity is applied as the query sequence. For example, the following websites can be used to search and calculate such e-values: http: / / pfam.xfam.org / , http: / / www.ebi.ac.uk / Tools / hmmer / search / hmmscan or http: / / www.ebi.ac.uk / Tools / pfa / pfamscan / and / or (2) Selected from the group of polypeptides containing at least one, two, three, four, five, six, seven, or all of the sequence motifs / domains selected from the following: GAGxSGL as set forth in SEQ ID NO: 197; EKNxxxxGTWxENRYPGCACDVPxHxYXXSFE as set forth in SEQ ID NO: 198; or any submotif thereof comprising up to 15, up to 10, or up to 5 consecutive amino acid residues, e.g., corresponding to residues 1-10, 11-20, or 21-32 of SEQ ID NO: 198; LxNAxGILNxWxxPxIPG shown in SEQ ID NO: 199 or any submotif thereof comprising up to 15, up to 10, or up to 5 consecutive amino acid residues, e.g., corresponding to residues 1-10 or 11-18 of SEQ ID NO: 199; LxxKxVxxIGxGSSGIQIxPxI as shown in SEQ ID NO: 200; or any submotif thereof comprising up to 15, up to 10, or up to 5 consecutive amino acid residues, e.g., corresponding to residues 1-10 or 11-18 of SEQ ID NO: 200; GCRRxTPGxxYLExL shown in SEQ ID NO: 201 or any submotif thereof comprising up to 15, up to 10, or up to 5 consecutive amino acid residues, e.g., corresponding to residues 1-10 or 11-15 of SEQ ID NO: 201; CATGFDxxxxPRFxxxG as shown in SEQ ID NO: 202 or any submotif thereof comprising up to 15, up to 10, or up to 5 consecutive amino acid residues, e.g., corresponding to residues 1-10 or 11-17 of SEQ ID NO: 202; PNxFxxxGPNxPxxNGxV shown in SEQ ID NO: 203 or any submotif thereof comprising up to 15, up to 10, or up to 5 consecutive amino acid residues, e.g., corresponding to residues 1-10 or 11-18 of SEQ ID NO: 203; AxWPGSxLHYxEAxxxPRxED shown in SEQ ID NO: 204 or any submotif thereof comprising up to 15, up to 10, or up to 5 consecutive amino acid residues, e.g., corresponding to residues 1-10 or 11-21 of SEQ ID NO: 204, where: The motif residues x independently represent any naturally occurring amino acid residue, and optionally, in each of the motifs 1 to 5, for example, 1, 2, 3, 4 or 5 of the conserved amino acid residues (i.e., different from the x residues) may be modified, for example, by amino acid substitution, particularly by conservative substitution, provided that the BVMO enzymatic activity is retained, at least analytically detectable. and / or (3) A group of polypeptides consisting of: (a) a polypeptide comprising the amino acid sequence of SCH23-BVMO1 shown in SEQ ID NO: 2; (b) a polypeptide comprising the amino acid sequence of SCH24-BVMO1 set forth in SEQ ID NO:6; (c) a polypeptide comprising the amino acid sequence of SCH25-BVMO1 set forth in SEQ ID NO: 10; (d) a polypeptide comprising the amino acid sequence of SCH46-BVMO1 set forth in SEQ ID NO: 13; (e) a polypeptide comprising the amino acid sequence of AspWeBVMO set forth in SEQ ID NO: 16 (preferential substrate manooloxy and its isomers); (f) A polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the amino acid sequences of a) to e). 2. The method of any one of the preceding embodiments, wherein the method is selected from:

[0132] In the five specific reference BVMO polypeptides described above, the protein family domain having Pfam ID number PF00743 can be located at the amino acid residue positions shown in the table below (see also the alignment depicted in Figure 32 and the framed sequence portion therein).

[0133] [Table 3]

[0134] The numbering of amino acid residues refers to the residue number in each SEQ ID NO of each protein sequence in the attached sequence listing.

[0135] Another specific embodiment refers to polypeptide variants of the novel polypeptides of the present invention having BVMO activity identified above by any of the specific amino acid sequences of SEQ ID NOs: 2, 6, 10, and 13, wherein the polypeptide variant is selected from amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 2, 6, 10, 13, and 16, and contains at least one substitution modification relative to unmodified any of SEQ ID NOs: 2, 6, 10, 13, and 16.

[0136] 10. The method of any one of the preceding embodiments, wherein the method is performed in vitro or in vitro.

[0137] 11. The method of embodiment 10, carried out in vivo, comprising, prior to step (1), recombinantly expressing, in particular in a non-human host cell, one or more polypeptides having the enzymatic activity required to carry out the BVMO-catalyzed enzymatic step.

[0138] 12. The method of embodiment 11, wherein the non-human host cell is transformed with a nucleic acid encoding at least one polypeptide having BVMO activity.

[0139] 13. The method of embodiment 11 or 12, wherein said non-human host cell is a eukaryotic or prokaryotic cell, in particular a plant cell, a bacterial cell or a fungal cell, in particular a yeast cell.

[0140] 14. The method of any one of embodiments 11 to 13, wherein the non-human host cell is a unicellular organism, a cultured cell derived from a multicellular organism, a cell present in a cultured tissue derived from a multicellular organism, or a cell present in a living multicellular organism.

[0141] 15. The method of any one of embodiments 10 to 13, wherein the non-human host cell is a bacterium of the genus Escherichia, in particular Escherichia coli, and the yeast is a yeast of the genus Saccharomyces or Pichia, in particular Saccharomyces cerevisiae, or a plant cell.

[0142] 16. The carbonyl compound of general formula I is a) labdan aldehydes, in particular coparal (or any of their stereoisomeric forms, for example the cis or trans forms or mixtures of the cis and trans forms), which can be converted by the aforementioned BVMOs into the respective norlabdane formates, in particular (5S,9S,10S)-15-norlabda-8(20),13-dien-14-yl formate or any of its stereoisomeric forms; b) dinorlabdane ketones, in particular manooloxy or any of its stereoisomeric forms, which can be converted by the aforementioned BVMO to γ-ambryl acetate or any of its stereoisomeric forms; or c) norlabdane aldehyde, in particular the C1-degraded analogue of copal or its stereoisomerically different human forms, in particular of the formula [ka] or any stereoisomeric form thereof, according to the aforementioned BVMO, in particular the formula [ka] or any of the stereoisomeric forms thereof is a labdan-type compound selected from The method of any one of the preceding embodiments, wherein optionally the resulting product is isolated in essentially stereoisomerically pure form or as a mixture of stereoisomers.

[0143] Further specific inventive examples of BVMO-catalyzed conversion of carbonyl compounds to their respective esters are summarized in the following schematic overview: [ka] wherein the parameter "n" is an integer between 1 and 20, between 1 and 15, between 1 and 10, or 1, 2, 3, 4, or 5.

[0144] 17. The method according to embodiment 16, a), further comprising, before step (1): Biocatalytic oxidation of labdane alcohol to labdane aldehyde, especially the oxidation of copalol to copalal, The labdane alcohol is optionally formed by biocatalytic conversion of at least one terpenyl diphosphate precursor selected from IPP, DMAPP, FPP and GGPP, in particular in one step or a combination of at least two steps known in the prior art.

[0145] The aforementioned labdane alcohol can be produced, for example, by using a biocatalyst: a) can be produced in one step from geranylgeranyl diphosphate (GGPP) in a cyclization / dephosphorylation reaction; b) can be produced in two steps from GGPP by cyclization to form a labdan diphosphate, e.g., copalyl diphosphate (CPP), which is then dephosphorylated to give a labdan alcohol; c) can be produced from IPP and DMAPP by the action of bifunctional GGPP synthase / CPP synthase directly to labdan diphosphate, e.g., CPP, which is then dephosphorylated; The GGPP used in these steps can also be provided by different biocatalytic steps: d) a GGPP synthase is available that produces GGPP directly from IPP and DMAPP; or e) GGPP can be provided from IPP and DMAPP via FPP by the action of FPP synthase, and then FPP is converted to GGPP by the action of GGPP synthase.

[0146] 18. The biocatalytic oxidation of labdane alcohols, in particular copalol, to copalal is catalyzed by an exogenous or endogenous polypeptide having alcohol dehydrogenase (ADH) (EC 1.1.1.-) activity; and / or The aforementioned biocatalytic formation of labdane alcohols is i) the biocatalytic dephosphorylation of labdane diphosphate to labdane alcohol, in particular copalyl diphosphate (CPP) to copalol, catalyzed by polypeptides with terpenyl diphosphate (TPP) phosphatase activity, and / or ii) the biocatalytic cyclization of a terpenyl diphosphate precursor, e.g., geranylgeranyl diphosphate (GGPP), to a CPP, catalyzed by a polypeptide having CPP synthase activity, such as SmCPS2 (SEQ ID NO: 185); or the biocatalytic cyclization of IPP and DMAPP to a CPP, catalyzed by a bifunctional polypeptide having prenyltransferase and copalyl diphosphate synthase activity, such as PvCPS, and / or iii) The biocatalytic formation of GGPP from FPP or from IPP and DMAPP, each catalyzed by a polypeptide having GGPP synthase activity. 18. The method of embodiment 17, comprising at least one step selected from:

[0147] 19. The aforementioned biocatalytic oxidations, particularly the oxidation of copalol to copalal, a) A polypeptide comprising the amino acid sequence of SCH23-ADH1_wt shown in SEQ ID NO: 134 b) a polypeptide comprising the amino acid sequence of SCH24-ADH1_wt shown in SEQ ID NO: 140 c) a polypeptide comprising the amino acid sequence of SCH94-3945_wt shown in SEQ ID NO: 161 d) A polypeptide comprising the amino acid sequence of SCH80-0540_wt shown in SEQ ID NO: 164 e) a polypeptide comprising the amino acid sequence of AzTolADH1_wt shown in SEQ ID NO: 167 f) a polypeptide comprising the amino acid sequence of CdGeoA_wt shown in SEQ ID NO: 179 g) A polypeptide having ADH activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the amino acid sequences of a) to f). The enzyme is catalyzed by a polypeptide having alcohol dehydrogenase (ADH) activity selected from and / or The aforementioned biocatalytic dephosphorylation, specifically the dephosphorylation of copalyl diphosphate (CPP) to copalol, a) a polypeptide comprising the amino acid sequence of AspWE TPP set forth in SEQ ID NO: 170, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; b) a polypeptide comprising the amino acid sequence of TalCeTPP set forth in SEQ ID NO: 176, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and c) a polypeptide comprising the amino acid sequence of TalVeTPP set forth in SEQ ID NO: 194, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; catalyzed by a polypeptide having terpenyl diphosphate (TPP) phosphatase activity selected from Further suitable phosphatases are also disclosed in the applicant's earlier EP application no. 18182783.3, which is incorporated by reference; and / or The aforementioned biocatalytic cyclization, particularly the cyclization of geranylgeranyl diphosphate (GGPP) to CPP, A polypeptide having copalyl diphosphate synthase activity comprising the amino acid sequence of SmCPS2 set forth in SEQ ID NO: 185, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. is catalyzed by a polypeptide selected from The aforementioned biocatalytic cyclizations, particularly the cyclization of IPP and DNMAPP to CPP, A polypeptide having prenyltransferase activity and copalyl diphosphate synthase activity, comprising the amino acid sequence of PvCPS set forth in SEQ ID NO: 173, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. is catalyzed by a polypeptide selected from and / or The biocatalytic formation of GGPP is catalyzed by a polypeptide having GGPP synthase activity, a) a polypeptide comprising the amino acid sequence of carG set forth in SEQ ID NO: 182, or a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; b) a polypeptide comprising the amino acid sequence of CrtE set forth in SEQ ID NO: 191, or an amino acid sequence having at least 70, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; c) A polypeptide comprising the amino acid sequence of PvCPS set forth in SEQ ID NO: 173, or an amino acid sequence having at least 70, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 20. The method of embodiment 18, wherein the method is selected from:

[0148] 20. The method of any of the preceding embodiments, further comprising, as step (3), treating the carbonyl ester formed in step (1) or isolated in step (2) to obtain a derivative thereof using chemical synthesis or biocatalytic synthesis, or a combination of both, and optionally isolating the derivative of step (3), wherein said derivative may be selected from, inter alia, hydrocarbons, alcohols, diols, triols, acetals, ketals, aldehydes, acids, ethers, amides, ketones, lactones, epoxides, acetates, glycosides, and / or esters.

[0149] 21. The method of embodiment 20, wherein step (3) comprises hydrolyzing the carbonyl ester compound with an esterase activity EC 3.1.1 (carboxylic acid ester hydrolase) to obtain the corresponding de-esterified product (which may be an alcohol or its isomerization product), and optionally isolating the derivative of step (3).

[0150] 22. The method of embodiment 21, wherein the deesterified product of step (3) is subjected to an enzymatic redox reaction in a further step (4), wherein the redox reaction comprises oxidizing the alcohol group formed in step (3) to the corresponding keto group by the enzymatic action of an exogenous or endogenous alcohol dehydrogenase (ADH) (EC 1.1.1.-).

[0151] 23. Esterase is a) a polypeptide comprising the amino acid sequence of SCH23-esterase shown in SEQ ID NO: 20; b) a polypeptide comprising the amino acid sequence of SCH24-esterase as set forth in SEQ ID NO: 24; c) a polypeptide comprising the amino acid sequence of SCH25-esterase shown in SEQ ID NO: 28; d) a polypeptide comprising the amino acid sequence of SCH46-esterase set forth in SEQ ID NO: 31; or e) A polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the amino acid sequences of a) to d) and having esterase activity. 22. The method of embodiment 21, wherein the method is selected from the group consisting of:

[0152] 24. a) Norlabdane esters, in particular norlabdane formates, are deesterified by the aforementioned esterases to give norlabdane carbonyl compounds, in particular compounds of the formula [ka] Carbonyl compounds or to obtain the respective enol, which is converted via isomerization to the aforementioned carbonyl compound; or b) deesterification of tetranorlabdane esters, in particular γ-ambryl acetate, with the aforementioned esterases to give tetranorlabdane, in particular γ-ambrol; or c) dinorlabdane formate esters, especially those of the formula [ka] Formate ester of or any stereoisomeric form thereof, can be deesterified with the aforementioned esterase to give the corresponding dinorlabdane alcohol, in particular the dinorlabdane alcohol of the formula [ka] Alcohol compounds of or any stereoisomeric form thereof, 24. The process of embodiment 23, wherein, optionally, the product obtained is isolated in essentially stereoisomerically pure form or as a mixture of stereoisomers.

[0153] 25. ADH a) A polypeptide comprising the amino acid sequence of SCH23-ADH2_wt shown in SEQ ID NO: 137 b) a polypeptide comprising the amino acid sequence of SCH24-ADH2_wt shown in SEQ ID NO: 143 c) a polypeptide comprising the amino acid sequence of RrhSecADH_wt shown in SEQ ID NO: 146 d) A polypeptide comprising the amino acid sequence of SCH80-06135_wt shown in SEQ ID NO: 155 e) A polypeptide having ADH activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the amino acid sequences of a) to d). 23. The method of embodiment 22, wherein the compound is selected from the group consisting of:

[0154] 26. The dinorlabdane alcohol obtained, in particular the alcohol of the formula [ka] of alcohol or stereoisomeric forms thereof, by oxidation with the aforementioned ADH to the corresponding dinorlabdane carbonyl compounds, in particular manooloxy; 25. The process of embodiment 24, wherein, optionally, the product obtained is isolated in essentially stereoisomerically pure form or as a mixture of stereoisomers.

[0155] ii) The present invention relates to the following specific embodiments of biocatalytic methods involving the use of polypeptides with enal dissociating activity: 27.General formula IV [ka] [In the formula, R 1 represents H or lower alkyl, especially methyl; R 2 represents H, a linear or branched, saturated or unsaturated, optionally substituted hydrocarbyl group, in particular an alkyl or alkenyl group, in particular a group having up to 30, up to 20, up to 15 or up to 10 carbon atoms, or the residue Cyc-A-, where: Cyc represents an optionally substituted, saturated or unsaturated, especially non-aromatic, monocyclic or polycyclic, especially monocyclic or bicyclic, especially a hydrocarbyl residue having 5 to 7 ring carbon atoms, A represents a chemical bond or an optionally substituted linear or branched alkylene bridge, in particular methylene; R 3 represent, independently of one another, H or lower alkyl such as C1-C4-alkyl, in particular H or methyl, and more particularly each is H. 1. A biocatalytic method for preparing a compound of formula (I), comprising: (1) General formula V [ka] [In the formula, R 1 , R 2 and R 3 is as defined above, R 4 represents H or lower alkyl, in particular H or methyl; R 5 represents H or lower alkyl, especially H] the corresponding undecomposed precursor of contacting the enzyme with a natural or recombinant polypeptide having enal cleavage activity, particularly a polypeptide having α,β-unsaturated aldehyde C═C bond cleavage activity, wherein the compound may be present in essentially stereoisomerically pure form (e.g., E or Z form) or as a mixture of stereoisomers; (2) optionally isolating the decomposition product of formula IV obtained in step (1), wherein the compound of general formula IV can be obtained in stereoisomerically pure form or as a mixture of stereoisomers; A method comprising:

[0156] 28. The polypeptide having enal cleavage activity as described above, a) at least one DUF4334 protein family domain having Pfam ID number PF14232 (particularly within the C-terminal region of their amino acid sequence); and / or b) at least one GXWXG protein family domain having Pfam ID number PF14231 (particularly within the N-terminal region of their amino acid sequence); or c) a domain that retains at least 90% sequence identity with PF14232 or PF14231 28. The method of embodiment 27, wherein the polypeptide is selected from the group of polypeptides comprising:

[0157] In particular, the polypeptide of the present invention having enal cleavage activity is capable of cleaving 1×10 -5 Less than or equal to 1 x 10 -10 Less than or equal to 1 x 10 -15 Less than or equal to 1 x 10 -20 Less than or equal to 1 x 10 -25 Less than or equal to 1 x 10 -30 Less than or equal to 1 x 10-35 The following e values, especially 1×10 -20 ~1×10 -32 , more specifically 1×10 -25 ~1×10 -31 If the protein matches the aforementioned domain with an e value in the range of 0.1 to 1.2, it is identified as a member of the DUF4334 protein family, which includes the aforementioned domain PF14232. For example, the following websites can be used to search and calculate such e-values: http: / / pfam.xfam.org / , http: / / www.ebi.ac.uk / Tools / hmmer / search / hmmscan or http: / / www.ebi.ac.uk / Tools / pfa / pfamscan / In particular, the polypeptide of the present invention having enal cleavage activity is capable of cleaving 1×10 -5 Less than 1×10 -10 Less than or equal to 1 x 10 -15 Less than or equal to 1 x 10 -20 Less than or equal to 1 x 10 -25 Less than or equal to 1 x 10 -30 Less than or equal to 1 x 10 -35 The following e values, especially 1×10 -20 ~1×10 -30 If the e-value is in the range of 0.01, the protein is identified as a member of the GXWXG protein family containing the aforementioned domain PF14232. As a query sequence, the sequence of a polypeptide having enal cleavage activity is applied. For example, the following websites can be used to search and calculate such e-values: http: / / pfam.xfam.org / , http: / / www.ebi.ac.uk / Tools / hmmer / search / hmmscan or http: / / www.ebi.ac.uk / Tools / pfa / pfamscan / and / or The polypeptide having the enal cleavage activity is G-[Y or "-"]-xWxGxx-[F, L or I]-x-[T, S or R]-G-[H or D] as shown in SEQ ID NO: 205; or any submotif thereof comprising up to 10 or up to 5 consecutive amino acid residues, such as, for example, corresponding to residues 1-8 or 9-13 of SEQ ID NO: 205; W-[Y, A or V]-GKx-[F or Y]-x-[S or D] shown in SEQ ID NO: 206; or any submotif thereof comprising up to four consecutive amino acid residues, such as, for example, those corresponding to residues 1-4 or 5-8 of SEQ ID NO: 206; [G or S]-x-[A or G]-x-[L or V]-xxxx-[F,Y or L]-RGxV as set forth in SEQ ID NO: 207; or any submotif thereof comprising up to 10 or up to 5 consecutive amino acid residues, such as, for example, corresponding to residues 1-8 or 9-14 of SEQ ID NO: 207; [M or L]-[V or I]-YDxxP-[I or V]-xD-[H or S]-[F or L] shown in SEQ ID NO: 208; or any submotif thereof comprising up to 10 or up to 5 consecutive amino acid residues, such as those corresponding to residues 1-6 or 7-12 of SEQ ID NO: 208. and wherein the polypeptide comprises at least one sequence motif / domain selected from the group consisting of: where: The motif residues x may independently represent any naturally occurring amino acid residue, and optionally, in each of the motifs 1 to 5, for example, 1, 2, 3, 4 or 5 amino acid residues different from the x residues may be modified, for example by amino acid substitution, in particular by conservative substitution, provided that the enzyme is retained at least analytically detectably. and / or The polypeptide having the enal cleavage activity may be a polypeptide of the group consisting of the following polypeptides each comprising an amino acid sequence: a) SCH94-3944 as shown in SEQ ID NO: 34 b) SCH80-05241 as shown in SEQ ID NO: 38 c) Pdigit7033 shown in SEQ ID NO: 42 d) PitalDUF4334-1 shown in SEQ ID NO: 46 e) AspWeDUF4334 as shown in SEQ ID NO: 49 f) RhoagDUF4334-2 as shown in SEQ ID NO: 53 g) RhoagDUF4334-3 as shown in SEQ ID NO: 56 h) RhoagDUF4334-4 as shown in SEQ ID NO: 59 i) CnecaDUF4334 as shown in SEQ ID NO: 62 j) Rins-DUF4334 as shown in SEQ ID NO: 69 k) CgatDUF4334 shown in SEQ ID NO: 72 l) GclavDUF4334 shown in SEQ ID NO: 75 m) TcurvaDUF4334 as shown in SEQ ID NO: 81 n) PprotDUF4334 as set forth in SEQ ID NO: 87, and o) A polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences a) to n), and retaining the enzyme activity of degrading the terpene precursor of formula (1). 28. The method of embodiment 27, wherein the method is selected from:

[0158] Another specific embodiment refers to polypeptide variants of the novel polypeptides of the present invention having enal dissociating activity identified above by any of the specific amino acid sequences of SEQ ID NOs: 34, 38, 42, 46, 49, 53, 56, 59, 62, 69, 72, 75, 81, and 87, wherein the polypeptide variant is selected from amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 34, 38, 42, 46, 49, 53, 56, 59, 62, 69, 72, 75, 81, and 87 and including at least one substitution modification relative to any of SEQ ID NOs: 34, 38, 42, 46, 49, 53, 56, 59, 62, 69, 72, 75, 81, and 87.

[0159] In the 14 specific reference enal-cleaving polypeptides described above, the protein family domains having Pfam ID numbers PF14232 and PF14231 can be located at the amino acid residue positions shown in the table below (see also the alignment depicted in Figure 31 and the framed sequence portions therein).

[0160] [Table 4]

[0161] Amino acid residue numbering refers to the residue number in each SEQ ID NO of each protein sequence in the attached sequence listing.

[0162] 29. The following group of variants, wherein the enal-cleaving polypeptide consists of the following polypeptides and contains the respective amino acid sequences: a) SCH94-3944-T51A_variant as shown in SEQ ID NO: 91 b) SCH94-3944-H53A_variant as shown in SEQ ID NO: 93 c) SCH94-3944-L59A_variant as set forth in SEQ ID NO: 95 d) SCH94-3944-W64A_variant as set forth in SEQ ID NO: 97 e) SCH94-3944-S71A_variant as set forth in SEQ ID NO: 101 f) SCH94-3944-R106A_variant as shown in SEQ ID NO: 103 g) SCH94-3944-Y115A_variant as shown in SEQ ID NO: 105 h) SCH94-3944-D116A_variant as shown in SEQ ID NO: 107 i) SCH94-3944-M136A_variant as shown in SEQ ID NO: 111 j) SCH94-3944-K139A_variant as shown in SEQ ID NO: 113 k) SCH94-3944-R156A_variant as set forth in SEQ ID NO: 119, and l) A polypeptide comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences a) to l), which retains the enzyme activity for degrading the terpene precursor of formula (1) and retains the position of the mutated amino acid sequence. 29. The method of embodiment 28, wherein the method is selected from:

[0163] 30. Compounds of formula V, for example terpene-type compounds, are applied, wherein R 1 represents H or methyl, R 2 is H or a) an acyclic, linear or branched, saturated or unsaturated hydrocarbyl residue having 1 to 20, in particular 1 to 10, 1 to 15 or 1 to 20, carbon atoms; or b) the group Cyc-A-, in which A represents a linear or branched C1-C4-alkylene bridge, in particular methylene, and Cyc represents a monocyclic or polycyclic, in particular bicyclic, saturated or unsaturated hydrocarbyl residue, in particular a bicyclic hydrocarbyl residue containing 5 to 7, in particular 6, ring atoms per ring, optionally substituted by 1 to 10, in particular 1 to 5, substituents independently selected from C1-C4-alkyl, C1-C4-alkylidene, C2-C4-alkenyl, oxo, hydroxy, or amino, in particular C1-C4-alkyl, such as methyl, and C1-C4-alkylidene, such as methylidene, Each R 3 represents H R 4 represents H or methyl, R 5 represents H or methyl, 30. The method of any of embodiments 27 to 29.

[0164] 31. The compound of general formula V has a labdan structure and / or Cyc-A is a compound of formula IIIa, IIIb or IIIc [ka] 31. The method of embodiment 30, wherein the residue represents:

[0165] 32. The precursor of formula (V) is selected from farnesal, geranylgeranial, citral, dodecanal, labdane-type compounds such as 8-hydroxy-labd-13-en-15-al, and coparal; The method of any one of embodiments 27 to 31, wherein each is in the form of a mixture of its stereoisomers or in its stereoisomerically pure form.

[0166] 33. The method of any one of embodiments 27 to 32, wherein the decomposition products of formula (IV) are selected from geranylacetone, farnesylacetone, methylheptenone, decanal; or manooloxy, or 8-hydroxy-14,15-dinorlabdan-13-one, each in the form of a mixture of its stereoisomers or in its stereoisomerically pure form.

[0167] Further specific inventive examples of enal-cleavage enzyme-catalyzed conversion of carbonyl compounds to their respective cleavage products are summarized in the following schematic overview: [ka] wherein the parameter "n" is an integer between 1 and 20, between 1 and 15, between 1 and 10, or 1, 2, 3, 4, or 5.

[0168] 34. The method of any one of embodiments 27 to 33, which is performed in vitro or in vitro.

[0169] 35. The method of embodiment 34, carried out in vivo, comprising, prior to step (1), recombinantly expressing, in particular in a non-human host cell, one or more polypeptides having the enzymatic activity necessary to carry out the chain degradation step.

[0170] 36. The method of embodiment 35, wherein the non-human host cell is transformed with a nucleic acid encoding at least one polypeptide having enal cleavage activity.

[0171] 37. The method of embodiment 35 or 36, wherein said non-human host cell is a eukaryotic or prokaryotic cell, in particular a plant cell, a bacterial cell or a fungal cell, in particular a yeast cell.

[0172] 38. The method of any one of embodiments 35 to 37, wherein the non-human host cell is a unicellular organism, a cultured cell derived from a multicellular organism, a cell present in a cultured tissue derived from a multicellular organism, or a cell present in a living multicellular organism.

[0173] 39. The method of any one of embodiments 35 to 38, wherein the non-human host cell is a bacterium of the genus Escherichia, preferably Escherichia coli, and the yeast is a yeast of the genus Saccharomyces or Pichia, preferably Saccharomyces cerevisiae, or a plant cell.

[0174] 40. The method of any of embodiments 27 to 39, comprising, as step (3), treating the compound of formula IV formed in step (1) or isolated in step (2) to obtain a derivative thereof using chemical synthesis or biocatalytic synthesis, or a combination of both, and optionally isolating the derivative of step (3), wherein said derivative may be selected from, inter alia, hydrocarbons, alcohols, diols, triols, acetals, ketals, aldehydes, acids, ethers, amides, ketones, lactones, epoxides, acetates, glycosides and / or esters.

[0175] 41. The method of embodiment 40, wherein step (3) comprises treating the compound of formula IV formed in step (1) or isolated in step (2) with a polypeptide having Baeyer-Villiger monooxygenase (BVMO) activity to form the respective carbonyl ester.

[0176] 42. The method of embodiment 41, further comprising hydrolyzing the carbonyl ester compound with an esterase to obtain the corresponding de-esterified product, which may be an alcohol or its isomerization product, and optionally isolating the derivative of step (3).

[0177] 43. The method of embodiment 41, wherein the polypeptide having BVMO activity is as defined above in embodiment 9.

[0178] 44. Esterase is a) a polypeptide comprising the amino acid sequence of SCH23-esterase shown in SEQ ID NO: 20; b) a polypeptide comprising the amino acid sequence of SCH24-esterase as set forth in SEQ ID NO: 24; c) a polypeptide comprising the amino acid sequence of SCH25-esterase shown in SEQ ID NO: 28; d) a polypeptide comprising the amino acid sequence of SCH46-esterase set forth in SEQ ID NO: 31; or e) A polypeptide having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the amino acid sequences of a) to d) and having esterase activity. 43. The method of embodiment 42, wherein the compound is selected from the group consisting of:

[0179] 45. The method of any one of embodiments 41 to 44, wherein the carbonyl compound is a dinorlabdanyl ketone, in particular a manooloxy, which is converted by the aforementioned BVMO to the respective tetranorlabdanyl acetate, in particular γ-ambryl acetate.

[0180] 46. ​​The method according to embodiment 45, wherein tetranorlabdanyl acetate, in particular γ-ambryl acetate, is de-esterified by said esterase to form the respective tetranorlabdan, in particular γ-ambrol.

[0181] 47. The method of any one of the preceding embodiments 27 to 46, wherein prior to step (1): Biocatalytic oxidation of labdane alcohol to labdane aldehyde, especially the oxidation of copalol to copalal, The labdane alcohol is optionally formed by biocatalytic conversion of at least one terpene diphosphate precursor selected from IPP, DMAPP, FPP and GGPP, in particular in a single step or a combination of at least two steps known in the prior art.

[0182] The aforementioned labdane alcohol can be produced, for example, by using a biocatalyst: a) can be produced in one step from geranylgeranyl diphosphate (GGPP) in a cyclization / dephosphorylation reaction; b) can be produced in two steps from GGPP by cyclization to form a labdan diphosphate, e.g., copalyl diphosphate (CPP), which is then dephosphorylated to give a labdan alcohol; c) can be produced from IPP and DMAPP by the action of bifunctional GGPP synthase / CPP synthase directly to labdan diphosphate, e.g., CPP, which is then dephosphorylated; The GGPP used in these steps can also be provided by different biocatalytic steps: d) a GGPP synthase is available that produces GGPP directly from IPP and DMAPP; or e) GGPP can be provided from IPP and DMAPP via FPP by the action of FPP synthase, and then FPP is converted to GGPP by the action of GGPP synthase.

[0183] 48. The biocatalytic oxidation of labdane alcohols, in particular copalol, to copalal is catalyzed by an exogenous or endogenous polypeptide having alcohol dehydrogenase (ADH) (EC 1.1.1.-) activity; and / or The aforementioned biocatalytic formation of labdane alcohols is i) the biocatalytic dephosphorylation of labdan diphosphate to labdan alcohol, in particular copalyl diphosphate (CPP) to copalol, catalyzed by polypeptides with terpenyl diphosphate (TPP) phosphatase activity, and / or ii) the biocatalytic cyclization of a terpenyl diphosphate precursor, e.g., geranylgeranyl diphosphate (GGPP), to a CPP, catalyzed by a polypeptide having CPP synthase activity, such as SmCPS2 (SEQ ID NO: 185); or the biocatalytic cyclization of IPP and DMAPP to a CPP, catalyzed by a bifunctional polypeptide having prenyltransferase and copalyl diphosphate synthase activity, such as PvCPS, and / or iii) The biocatalytic formation of GGPP from FPP or from IPP and DMAPP, each catalyzed by a polypeptide having GGPP synthase activity. 48. The method of embodiment 47, comprising at least one step selected from:

[0184] 49. The aforementioned biocatalytic oxidation, particularly the oxidation of copalol to copalal, a) A polypeptide comprising the amino acid sequence of SCH23-ADH1_wt shown in SEQ ID NO: 134 b) a polypeptide comprising the amino acid sequence of SCH24-ADH1_wt shown in SEQ ID NO: 140 c) a polypeptide comprising the amino acid sequence of SCH94-3945_wt shown in SEQ ID NO: 161 d) A polypeptide comprising the amino acid sequence of SCH80-0540_wt shown in SEQ ID NO: 164 e) a polypeptide comprising the amino acid sequence of AzTolADH1_wt shown in SEQ ID NO: 167 f) a polypeptide comprising the amino acid sequence of CdGeoA_wt shown in SEQ ID NO: 179 g) A polypeptide having ADH activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the amino acid sequences of a) to f). The enzyme is catalyzed by a polypeptide having alcohol dehydrogenase (ADH) activity selected from and / or The aforementioned biocatalytic dephosphorylation, specifically the dephosphorylation of copalyl diphosphate (CPP) to copalol, d) a polypeptide comprising the amino acid sequence of AspWE TPP set forth in SEQ ID NO: 170, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; e) a polypeptide comprising the amino acid sequence of TalCeTPP set forth in SEQ ID NO: 176, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and f) a polypeptide comprising the amino acid sequence of TalVeTPP set forth in SEQ ID NO: 194, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; catalyzed by a polypeptide having terpenyl diphosphate (TPP) phosphatase activity selected from Further suitable phosphatases are also disclosed in the applicant's earlier EP application no. 18182783.3, which is incorporated by reference; and / or The aforementioned biocatalytic cyclization, particularly the cyclization of geranylgeranyl diphosphate (GGPP) to CPP, A polypeptide having copalyl diphosphate synthase activity comprising the amino acid sequence of SmCPS2 set forth in SEQ ID NO: 185, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. is catalyzed by a polypeptide selected from The aforementioned biocatalytic cyclizations, particularly the cyclization of IPP and DNMAPP to CPP, A polypeptide having prenyltransferase activity and copalyl diphosphate synthase activity, comprising the amino acid sequence of PvCPS set forth in SEQ ID NO: 173, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. is catalyzed by a polypeptide selected from and / or The biocatalytic formation of GGPP is catalyzed by a polypeptide having GGPP synthase activity, d) a polypeptide comprising the amino acid sequence of carG set forth in SEQ ID NO: 182, or a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; e) a polypeptide comprising the amino acid sequence of CrtE set forth in SEQ ID NO: 191, or an amino acid sequence having at least 70, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; f) A polypeptide comprising the amino acid sequence of PvCPS set forth in SEQ ID NO: 173, or an amino acid sequence having at least 70, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. 49. The method of embodiment 48, wherein the method is selected from:

[0185] iii) The present invention relates to the following specific embodiments of enal-dissociating enzymes and corresponding coding sequences: 50. An isolated polypeptide having enal cleavage activity, in particular the activity of an α,β-unsaturated aldehyde C═C bond cleavage enzyme, as defined in any of embodiments 28 and 29. Polypeptides of the invention include all active forms, including active subsequences, such as catalytic domains or active sites, of enzymes having enal cleavage activity.

[0186] 51. A nucleic acid sequence encoding the polypeptide of embodiment 50, in particular a nucleic acid sequence selected from SEQ ID NOs: 33, 35, 36, 37, 39, 40, 41, 43, 44, 45, 47, 48, 50, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 68, 70, 71, 73, 74, 76, 80, 82, 86, 88, 92, 94, 96, 98, 102, 104, 106, 108, 112, and 120, as well as SEQ ID NOs: 33, 35, 36, 37, 39, 40, 41, 43, 44, 45, 47, 48, 50, 51, 52, 54, 55, 57, 58, 60, 61, 63, 64, 68, 70, 71, 73, 74, 76, 80, 82, 86, 88, 92, 94, 96, 98, 102, 104, 106, 108, 112, and 120. An isolated nucleic acid molecule comprising a nucleic acid sequence having a degree of sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to any one of the foregoing sequences.

[0187] 52. An expression cassette comprising the nucleotide sequence of at least one nucleic acid molecule of embodiment 50.

[0188] 53. An expression vector comprising the nucleotide sequence of at least one nucleic acid molecule according to embodiment 51, or at least one expression cassette according to embodiment 52.

[0189] 54. The expression vector of embodiment 53, wherein the vector is a prokaryotic vector, a viral vector, or a eukaryotic vector.

[0190] 55. The expression vector of embodiment 53 or 54, which is a plasmid or a combination of two or more plasmids.

[0191] 56. A recombinant non-human host cell comprising at least one nucleic acid molecule as defined in embodiment 51, or at least one expression cassette as defined in embodiment 52, or an expression vector as defined in any one of embodiments 53 to 55.

[0192] 57. The host cell of embodiment 56, wherein at least one nucleic acid molecule or at least one expression cassette is stably integrated into the genome of the cell.

[0193] 58. The host cell according to embodiment 56 or 57, which is a prokaryotic or eukaryotic cell, in particular a plant cell, a bacterial or fungal cell, in particular a yeast.

[0194] 59. The host cell of any of embodiments 56 to 58, which is a unicellular organism, a cultured cell derived from a multicellular organism, a cell present in a cultured tissue derived from a multicellular organism, or a cell present in a living multicellular organism.

[0195] 60. The host cell of embodiment 59, which is a bacterium of the genus Escherichia, preferably Escherichia coli, or a yeast cell of the genus Saccharomyces, preferably Saccharomyces cerevisiae, or a yeast cell of the genus Pichia, preferably P. pastoris.

[0196] 61. A method for producing at least one polypeptide having enal cleavage activity according to embodiment 51, the method comprising: (i) expressing said at least one polypeptide in a non-human host cell according to any one of embodiments 57 to 60; (ii) optionally isolating said at least one polypeptide from the non-human host cell used in step (i); A method comprising:

[0197] 62. The method of embodiment 61, further comprising, prior to step (i): preparing a non-human host cell used in step (i) by introducing at least one nucleic acid molecule as defined in embodiment 51, or at least one expression cassette as defined in embodiment 52, or at least one expression vector as defined in any one of embodiments 53 to 55 into the non-human cell, thus obtaining a host cell capable of expressing or overexpressing at least one polypeptide having enal cleavage activity according to embodiment 50.

[0198] 63. A method for preparing a mutant polypeptide having enal cleavage activity, the method comprising: (i) providing a nucleic acid molecule according to embodiment 51; (ii) modifying the nucleotide sequence of said nucleic acid molecule, in particular the nucleotide sequence encoding the polypeptide of embodiment 50, to obtain at least one variant nucleic acid molecule; (iii) recombinantly expressing said mutant nucleic acid molecule in a non-human host cell; (iv) screening the expression products obtained in step (iii) for at least one mutant polypeptide having enal cleavage activity; (v) optionally repeating steps (ii) through (iv) with the mutant nucleic acid molecule until the expression product comprises a mutant polypeptide having the desired enal cleavage activity; (vi) optionally isolating the mutant polypeptide having the desired enal cleavage activity; A method comprising:

[0199] iv) The present invention relates to the following specific embodiments of BVMO enzymes and corresponding coding sequences: 64. An isolated polypeptide having BVMO activity as defined in embodiment 9. Polypeptides of the invention include all active forms, including active subsequences, eg, catalytic domains or active sites, of enzymes having BVMO activity.

[0200] 65. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide according to embodiment 64, in particular a nucleic acid sequence selected from SEQ ID NOs: 1, 3, 4, 5, 7, 8, 9, 11, 12, 14, 15, 17 and 18, and a nucleic acid sequence having a degree of sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with any one of the aforementioned sequences of SEQ ID NOs: 1, 3, 4, 5, 7, 8, 9, 11, 12, 14, 15, 17 and 18.

[0201] 66. An expression cassette comprising the nucleotide sequence of at least one nucleic acid molecule according to embodiment 65.

[0202] 67. An expression vector comprising the nucleotide sequence of at least one nucleic acid molecule according to embodiment 65, or at least one expression cassette according to embodiment 66.

[0203] 68. The expression vector of embodiment 67, wherein the vector is a prokaryotic vector, a viral vector, or a eukaryotic vector.

[0204] 69. The expression vector of embodiment 67 or 68, which is a plasmid or a combination of two or more plasmids.

[0205] 70. A recombinant non-human host cell comprising at least one nucleic acid molecule as defined in embodiment 65, or at least one expression cassette as defined in embodiment 66, or at least one expression vector as defined in any one of embodiments 67 to 69.

[0206] 71. The host cell of embodiment 70, wherein at least one nucleic acid molecule or at least one expression cassette is stably integrated into the genome of the cell.

[0207] 72. The host cell according to embodiment 70 or 71, which is a prokaryotic or eukaryotic cell, in particular a plant cell, a bacterial or fungal cell, in particular a yeast.

[0208] 73. The host cell of any one of embodiments 70 to 72, which is a unicellular organism, a cultured cell derived from a multicellular organism, a cell present in a cultured tissue derived from a multicellular organism, or a cell present in a living multicellular organism.

[0209] 74. The host cell of embodiment 72, which is a bacterium of the genus Escherichia, preferably Escherichia coli, or a yeast cell of the genus Saccharomyces, preferably Saccharomyces cerevisiae, or a yeast cell of the genus Pichia, preferably Pichia yeast.

[0210] 75. A method for producing at least one polypeptide having BVMO activity according to embodiment 64, comprising: (i) expressing said at least one polypeptide in a non-human host cell according to any one of embodiments 70 to 74; (ii) optionally isolating said at least one polypeptide from the non-human host cell used in step (i); A method comprising:

[0211] 76. The method of embodiment 75, further comprising, prior to step (i): preparing a non-human host cell used in step (i) by introducing at least one nucleic acid molecule as defined in embodiment 65, or at least one expression cassette as defined in embodiment 66, or at least one expression vector as defined in any one of embodiments 67 to 69 into the non-human cell, thus obtaining a host cell capable of expressing or overexpressing at least one polypeptide having BVMO activity according to embodiment 64.

[0212] 77. A method for preparing a mutant polypeptide having BVMO activity, the method comprising: (i) providing a nucleic acid molecule according to embodiment 65; (ii) modifying the nucleotide sequence of said nucleic acid molecule, in particular the nucleotide sequence encoding the polypeptide of embodiment 64, to obtain at least one variant nucleic acid molecule; (iii) recombinantly expressing said mutant nucleic acid molecule in a non-human host cell; (iv) screening the expression products obtained in step (iii) for at least one mutant polypeptide having BVMO activity; (v) optionally, repeating steps (ii) through (iv) with the mutant nucleic acid molecule until the expression product comprises a mutant polypeptide having the desired BVMO activity; (vi) optionally isolating the mutant polypeptide having the desired BVMO activity; A method comprising:

[0213] v) The present invention relates to the following specific embodiments relating to a biocatalytic multistep in vivo method for converting labdane compounds by applying polypeptides with enal cleavage activity and / or BVMO activity: 78. An in vivo method for preparing labdane-type terpenes, comprising: The method comprises the following series of reaction steps: (1) optionally converting labdane alcohol, particularly coparol, to the respective labdane aldehyde, particularly coparal, by the enzymatic action of an exogenous or endogenous ADH polypeptide, particularly an ADH as defined in any of embodiments 19 or 49; (2) converting the aforementioned labdan aldehyde, in particular coparal, of step (1) into the respective dinorlabdane carbonyl compound, in particular manooloxy, by the action of a polypeptide having enal cleavage activity, in particular a polypeptide as defined in any of embodiments 28 and 29; (3) optionally, converting said dinorlabdane carbonyl compounds, in particular manooloxy, of step (2) into the respective tetranorlabdanyl acetates, in particular γ-ambryl acetates, by the action of a polypeptide having BVMO activity, in particular a BVMO as defined in embodiment 9; (4) optionally, converting said tetranorlabdanyl acetate, in particular γ-ambryl acetate, of step (3) into the respective tetranorlabdane alcohol, in particular γ-ambrol, by the action of a polypeptide having esterase activity, in particular an esterase as defined in any of embodiments 23 and 44; and optionally, (5) isolating the product of step (2), (3), or (4). providing a recombinant host expressing a set of polypeptides having the enzymatic activities necessary to catalyze

[0214] 79. An in vivo method for preparing labdane-type cycloterpenes, comprising: The method comprises the following series of reaction steps: (1) optionally converting labdane alcohol, particularly coparol, to the respective labdane aldehyde, particularly coparal, by the enzymatic action of an exogenous or endogenous ADH polypeptide, particularly an ADH as defined in any of embodiments 19 or 49; (2) converting the aforementioned labdan aldehydes, in particular coparal, of step (1) into the respective norlabdan ester compounds, in particular [4-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylene-decalin-1-yl]-2-methyl-but-1-enyl]formate (compounds 1a, 1b), by the action of a polypeptide having BVMO activity, in particular a BVMO as defined in any of embodiments 9; (3) converting the aforementioned labdane ester compounds of step (2), in particular [4-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylene-decalin-1-yl]-2-methyl-but-1-enyl] formates (compounds 1a, 1b), into the respective norlabdane aldehydes, in particular 4-[(1S,8aS)-5,5,8a-trimethyl-2-methylene-decalin-1-yl]-2-methyl-butanal (compounds 3a, 3b), optionally by the action of a polypeptide having esterase activity, in particular an esterase as defined in either embodiment 23 or 44; (4) converting the aforementioned norlabdane aldehydes of step (3), in particular 4-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylene-decalin-1-yl]-2-methyl-butanal (compounds 3a, 3b), into the respective dinorlabdane esters, in particular [3-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylene-decalin-1-yl]-1-methyl-propyl]formate (compounds 4a, 4b), by the action of a polypeptide having BVMO activity, in particular a BVMO as defined in any of embodiments 9; (5) converting the aforementioned dinorlabdane esters of step (4), in particular [3-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylene-decalin-1-yl]-1-methyl-propyl] formates (compounds 4a, 4b) into the respective dinorlabdane alcohols, in particular 4-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylene-decalin-1-yl]butan-2-ol (compounds 5a, 5b), by the action of a polypeptide having esterase activity, in particular an esterase as defined in either embodiment 23 or 44; (6) optionally, converting the aforementioned dinorlabdane alcohols of step (5), in particular 4-[(1S,8aS)-5,5,8a-trimethyl-2-methylene-decalin-1-yl]butan-2-ol (compounds 5a, 5b), into the respective dinorlabdane carbonyl compounds, in particular mannooloxy, by the action of an exogenous or endogenous polypeptide having ADH activity, in particular an ADH as defined in either embodiment 19 or 49; (7) optionally, converting said dinorlabdane carbonyl compounds, in particular manooloxy, of step (6) into the respective tetranorlabdanyl acetates, in particular γ-ambryl acetates, by the action of a polypeptide having BVMO activity, in particular a BVMO as defined in any of embodiments 9; (8) converting the aforementioned tetranorlabdanyl acetate, in particular γ-ambryl acetate, of step (7) into the respective tetranorlabdane alcohol, in particular γ-ambrol, by the action of a polypeptide having esterase activity, in particular an esterase as defined in any of embodiments 23 or 44; and optionally, (9) isolating the product of step (5), (6), (7) or (8); providing a recombinant host expressing a set of polypeptides having the enzymatic activities necessary to catalyze

[0215] 80. The ADHs applied in steps (1) and (6) are the same or different, exogenous or endogenous, and / or The BVMOs applied in steps (2), (4) and (7) are the same or different, and / or The esterases applied in steps (3), (5) and (8) are the same or different; The method of embodiment 79.

[0216] 81. The recombinant host is subjected to the following series of reaction steps prior to step (1): (i) biocatalytic formation of geranylgeranyl diphosphate (GGPP) by the action of a polypeptide having GGPP synthase activity, in particular a GGPP synthase as defined in any of embodiments 19 and 49; (ii) biocatalytic cyclization of GGPP to said labdan diphosphate, in particular copalyl diphosphate (CPP), by the action of a polypeptide having labdan diphosphate synthase activity, in particular a polypeptide comprising the CPP synthase activity as defined in any of embodiments 19 and 49; (iii) biocatalytic dephosphorylation of said labdan diphosphate to said labdan alcohol, in particular CPP to copalol, by the action of a polypeptide having labdan diphosphate phosphatase activity, in particular a polypeptide having TPP phosphatase activity as defined in any of embodiments 19 and 49; 81. The method of any one of embodiments 78 to 80, wherein a series of polypeptides having the enzymatic activity required to catalyze the above are additionally expressed and applied.

[0217] 82. The method of any one of embodiments 78 to 81, wherein the recombinant host is applied additionally expressing at least one polypeptide that catalyzes an enzymatic step of the mevalonate pathway or the MEP pathway.

[0218] 83. The method according to any one of embodiments 78 to 82, wherein a recombinant host is applied in which the coding sequence of each catalytically active polypeptide is carried on one or more expression vectors and / or stably integrated into the genome of the host.

[0219] 84. The method of any one of embodiments 1 to 49 and 78 to 83, carried out in vivo, wherein the method comprises, prior to step (1), introducing into the non-human host organism or cell, and optionally stably integrating into the respective genome, one or more nucleic acid molecules encoding one or more polypeptides having the enzymatic activity required to carry out the respective step or steps of the biocatalytic transformation.

[0220] 85. The method of any one of embodiments 1 to 49 and 78 to 83, wherein the method is carried out by applying a non-human host organism or cell that endogenously produces FPP and / or GGPP; or a mixture of IPP and DMAPP; or a non-human host organism that has been genetically modified to produce increased amounts of FPP and / or GGPP and / or a mixture of IPP and DMAPP.

[0221] Among these host cells or organisms applicable to the present invention, some do not naturally produce FPP or GGPP, or a mixture of IPP and DMAPP. Such organisms or cells that do not naturally produce acyclic terpene pyrophosphate precursors, such as FPP or GGPP, or a mixture of IPP and DMAPP, can be genetically modified to produce the precursors. Such organisms or cells can be transformed in this manner, for example, before modification with the nucleic acids described herein. Methods for transforming organisms to produce acyclic terpene pyrophosphate precursors, such as FPP or GGPP, or a mixture of IPP and DMAPP, are already known in the art. For example, introducing the enzyme activity of the mevalonate pathway is a suitable strategy for allowing organisms to produce FPP or GGPP, or a mixture of IPP and DMAPP.

[0222] 86. A recombinant microorganism as defined in any of embodiments 78 to 85.

[0223] vi) The present invention relates to the following specific embodiments relating to the further conversion of intermediate chemical compounds obtained by the biocatalytic methods described herein into additional end products of particular interest: 87. A method for preparing an epoxy-tetranorlabdane compound, in particular Ambrox, comprising the steps of: (1) providing a tetranorlabdane alcohol, particularly γ-ambrole, or a tetranorlabdane acetate, particularly γ-ambryl acetate, or a dinorlabdane carbonyl compound, particularly manooloxy; by applying a biocatalytic process comprising one or more process steps as defined in any of claims 1 to 49 or 78 to 83, and optionally isolating said product; (2) converting the aforementioned product of step (1) into an epoxy-tetranorlabdane, in particular ambrox, by applying one or more chemical and / or biochemical transformation steps; A method comprising:

[0224] 88. A method for preparing diepoxy-dinorlabdane, in particular Z11, comprising the steps of: (1) providing a dinorlabdane carbonyl compound, in particular manooloxy, by applying a process which results in the formation of the aforementioned dinorlabdane carbonyl compound, in particular manooloxy, comprising one or more process steps as defined in any of claims 1 to 49 or 78 to 84, and optionally isolating the dinorlabdane carbonyl compound, in particular manooloxy; (2) converting the aforementioned dinorlabdane carbonyl compound, particularly manooloxy, of step (1) into the aforementioned diepoxy-dinorlabdane, particularly Z-11, by applying one or more chemical and / or biochemical transformation steps; A method comprising:

[0225] b. Polypeptides applicable according to the present invention In this regard, the following definitions apply: The general terms "polypeptide" or "peptide" may be used interchangeably and refer to a linear chain or sequence of naturally occurring or synthetic contiguous, peptidically linked amino acid residues, containing from about 10 residues up to over 1,000 residues. Short polypeptides of up to 30 residues are also referred to as "oligopeptides."

[0226] The term "protein" refers to a polymeric structure consisting of one or more polypeptides. The amino acid sequence of the polypeptide(s) represents the "primary structure" of the protein. The amino acid sequence also predetermines the "secondary structure" of the protein through the formation of specific structural elements, such as α-helical and β-sheet structures, within the polypeptide chain. The arrangement of these secondary structural elements defines the "tertiary structure" or spatial arrangement of the protein. When a protein contains two or more polypeptide chains, the spatial arrangement of these chains forms the "quaternary structure" of the protein. The correct spatial arrangement of a protein, i.e., "folding," is a prerequisite for protein function. Denaturation or unfolding destroys the function of the protein. If such destruction is reversible, refolding can restore the protein's function.

[0227] A typical protein function referred to herein is an "enzymatic function," i.e., a protein acts as a biocatalyst on a substrate, e.g., a compound, to catalyze the conversion of said substrate to a product. Enzymes may have a high or low degree of specificity for the substrate and / or product.

[0228] Thus, a "polypeptide" referred to herein as having a particular "activity" implicitly refers to a correctly folded protein that exhibits the specified activity, e.g., a particular enzymatic activity.

[0229] Thus, unless otherwise specified, the term "polypeptide" also encompasses the terms "protein" and "enzyme."

[0230] Similarly, the term "polypeptide fragment" encompasses the terms "protein fragment" and "enzyme fragment."

[0231] 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.

[0232] A "targeting peptide" refers to an amino acid sequence that targets a protein or polypeptide to an intracellular organelle, i.e., mitochondria, or plastid, or to the extracellular space (secretory signal peptide). A nucleic acid sequence encoding a targeting peptide may be fused to a nucleic acid sequence encoding the amino terminus, e.g., the N-terminus, of a protein or polypeptide, or may be used to replace a native targeting polypeptide.

[0233] The present invention also relates to "functional equivalents" (also called "analogs" or "functional variants") of the polypeptides specifically described herein.

[0234] For example, a "functional equivalent" refers to a polypeptide that exhibits at least 1-10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% greater or less activity in an assay used to determine enzymatic terpenyl diphosphate synthase activity or terpenyl diphosphate phosphatase activity compared to the activity of a polypeptide specifically described herein.

[0235] According to the present invention, "functional equivalents" also encompass specific variants of the amino acid sequences described herein, which have an amino acid different from that specifically described, at least in one sequence position, but which nevertheless possess one of the aforementioned biological activities, e.g., enzymatic activity. Thus, "functional equivalents" are variants that can be obtained by addition, substitution, especially conservative substitution, deletion and / or inversion of one or more, e.g., 1 to 20, in particular 1 to 15 or 5 to 10, amino acids, where the described changes may be made at any sequence position, provided that they result in a variant with a profile of properties according to the invention. In particular, a qualitative agreement of the activity pattern between the variant and the unchanged polypeptide, i.e., for example, interactions with the same agonist or antagonist or substrate, but at different rates (i.e., EC 50 Or IC 50 Functional equivalence is also provided when a substitution is observed (expressed as a function of the amino acid sequence, or any other parameter appropriate in the current art). Examples of suitable (conservative) amino acid substitutions are shown in the table below.

[0236] [Table 5]

[0237] "Functional equivalents" in the above sense are also "precursors" of the polypeptides described herein, as well as "functional derivatives" and "salts" of the polypeptides.

[0238] A "precursor" in this case is a natural or synthetic precursor of a polypeptide, which may or may not have the desired biological activity.

[0239] The term "salts" refers not only to acid addition salts of amino groups of the protein molecule according to the present invention, but also to salts of carboxyl groups. Salts of carboxyl groups can be prepared by known methods and include inorganic salts such as sodium, calcium, ammonium, iron, and zinc salts, as well as salts with organic bases, such as amines such as triethanolamine, arginine, lysine, and piperidine. Acid addition salts, such as salts with inorganic acids such as hydrochloric acid or sulfuric acid, and salts with organic acids such as acetic acid and oxalic acid, are also included in the scope of the present invention.

[0240] "Functional derivatives" of the polypeptides of the present 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, amides of carboxylic acid groups obtainable by reaction with ammonia or primary or secondary amines, 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.

[0241] "Functional equivalents" naturally include naturally occurring variants as well as polypeptides obtainable from other organisms. For example, areas of homologous sequence regions can be established by sequence comparison and equivalent polypeptides can be determined based on the specific parameters of the invention.

[0242] "Functional equivalents" also include "fragments," such as individual domains or sequence motifs of the polypeptides according to the invention, or N- and C-terminally truncated forms, which may or may not exhibit the desired biological function. Preferably, such "fragments" retain at least qualitatively the desired biological function.

[0243] A "functional equivalent" is also a fusion protein that functionally joins, at its N- or C-terminus, one of the polypeptide sequences described herein or functional equivalents derived therefrom with at least one additional, functionally distinct, 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.

[0244] Also included in the present invention are "functional equivalents" that are homologs of the specifically disclosed polypeptides, which have at least 60%, preferably at least 75%, in particular at least 80 or 85%, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology (or identity) with one of the specifically disclosed amino acid sequences, as calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85(8), 1988, 2444-2448. The percentage homology or identity of a homologous polypeptide according to the present invention refers in particular to the identity expressed as a percentage of amino acid residues based on the full length of one of the amino acid sequences specifically described herein.

[0245] Identity data, expressed as a percentage, can be determined using BLAST alignment, the algorithm blastp (protein-protein BLAST), or by applying the Clustal settings defined later in this specification.

[0246] In the case of possible glycosylation of proteins, "functional equivalents" according to the present invention include the polypeptides described herein in deglycosylated or glycosylated form, as well as modified forms that can be obtained by altering the glycosylation pattern.

[0247] Functional equivalents or homologues of the polypeptides according to the invention can be generated by mutagenesis, for example by point mutation, by lengthening or shortening the protein, or as explained in more detail below.

[0248] Functional equivalents or homologs of the polypeptides of the present invention can be identified by screening combinatorial databases of mutants, such as truncated mutants. For example, combinatorial mutagenesis at the nucleic acid level, for example, by enzymatic ligation of a mixture of synthetic oligonucleotides, can be used to create a rich database of protein variants. There are numerous methods that can be used to create a database of potential homologies from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed using an automatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate expression vector. The use of degenerate genomes makes it possible to provide a mixture of all sequences encoding the desired set of potential protein sequences. Methods for synthesizing degenerate oligonucleotides are known to those skilled in the art.

[0249] Several techniques are known in the prior art for screening gene products from combinatorial databases generated 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 techniques for screening large gene banks are based on high-throughput analysis and involve cloning the gene bank in replicable expression vectors, transforming appropriate cells with the resulting vector database, and expressing the combinatorial genes under conditions that facilitate the isolation of vectors encoding the genes for the detected products upon detection of the desired activity. Recursive ensemble mutagenesis (REM), a technique for increasing the frequency of functional variants in a database, can be used in conjunction with screening tests to identify homologs.

[0250] Embodiments provided herein provide orthologs and paralogs of the polypeptides disclosed herein and 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.

[0251] Polypeptides of the present invention include all active forms, including active subsequences of the enzymes of the present invention, such as catalytic domains or active sites. In one aspect, the present invention provides catalytic domains or active sites as described below. In one aspect, the present invention provides protein families searchable from databases such as Pfam (http: / / pfam.wustl.edu / hmmsearch.shtml) (The Pfam protein families database, A. Bateman, E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. Sonnhammer, Nucleic Acids Research, 30(1):276-280, 2002), or equivalent databases, such as the InterPro and SMART databases ( http: / / www.ebi.ac.uk / interpro / scan.html , http: / / smart.embl-heidelberg.de / ) is provided.

[0252] The present invention also encompasses "polypeptide variants" having a desired activity, wherein the variant polypeptide is selected from amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a specific, particularly naturally occurring, amino acid sequence referenced by a particular SEQ ID NO, and contains at least one substitution modification relative to said SEQ ID NO.

[0253] c. Coding nucleic acid sequences applicable according to the present invention In this regard, the following definitions apply: The terms "nucleic acid sequence," "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably to 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 recognize that a nucleic acid sequence of RNA is identical to a DNA sequence, with the difference being the substitution of thymine (T) for uracil (U). The term "nucleic acid sequence" should also be understood to include polynucleotide or oligonucleotide molecules, either in the form of separate fragments or as components of a larger nucleic acid.

[0254] An "isolated nucleic acid" or "isolated nucleic acid sequence" refers to a nucleic acid or nucleic acid sequence that is in an environment that is different from the environment in which the nucleic acid or nucleic acid sequence naturally occurs, and can include being substantially free of contaminating endogenous material.

[0255] The term "naturally occurring," as used herein as applied to nucleic acids, refers to nucleic acids that are found within the cells of organisms in nature and that have not been intentionally modified by man in the laboratory.

[0256] The "fragment" of a polynucleotide or nucleic acid sequence refers to a contiguous nucleotide, particularly a polynucleotide of the present embodiment having a length of at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp, and / or at least 60 bp. In particular, a polynucleotide fragment 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, and more particularly at least 1000 contiguous nucleotides of the polynucleotide of the present embodiment. Without limitation, the polynucleotide fragment of the present invention can be used as a PCR primer and / or a probe, or for antisense gene silencing or RNAi.

[0257] 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. These conditions may be conditions under which sequences with at least about 70%, e.g., at least about 80%, e.g., at least about 85%, 90%, or 95% identity remain bound to each other. Definitions of low, moderate, and high stringency hybridization conditions are provided later in this specification. 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).

[0258] A "recombinant nucleic acid sequence" is a nucleic acid sequence that results from combining genetic material from multiple sources using laboratory methods (e.g., molecular cloning) to create or modify a nucleic acid sequence that does not occur in nature and would not otherwise be found in an organism.

[0259] "Recombinant DNA techniques" refers to molecular biology procedures for preparing recombinant nucleic acid sequences, as described, for example, 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.

[0260] The term "gene" refers to a DNA sequence comprising a region that is transcribed into an RNA molecule such as mRNA in a cell, and is operably linked to an appropriate regulatory region, e.g., a promoter. Thus, a gene may comprise multiple operably linked sequences, such as a promoter, a 5' leader sequence comprising, e.g., a sequence involved in translation initiation, a coding region of cDNA or genomic DNA, introns, exons, and / or a 3' untranslated sequence comprising, e.g., a transcription termination site.

[0261] "Polycistronic" refers to a nucleic acid molecule, particularly an mRNA, that can separately encode two or more polypeptides within the same nucleic acid molecule.

[0262] "Chimeric gene" refers to any gene not normally found in a given species of nature, particularly a gene in which one or more portions of nucleic acid sequence are present that are not naturally associated with each other. For example, a promoter is not naturally associated with part or all of the transcribed region or another regulatory region. The term "chimeric gene" is understood to include an expression construct in which a promoter or transcriptional regulatory sequence is operably linked to one or more coding sequences or antisense, i.e., the reverse complement of the sense strand, or inverted repeat sequences (sense and antisense, whereby the RNA transcript forms double-stranded RNA upon transcription). The term "chimeric gene" also includes genes obtained by combining portions of one or more coding sequences to generate a new gene.

[0263] "3'UTR" or "3' untranslated sequence" (also called "3' untranslated region" or "3' end") refers to a nucleic acid sequence found downstream of the coding sequence of a gene, which includes, 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 is completed, the mRNA transcript can be cleaved downstream of the polyadenylation signal, and a poly(A) tail can be added, which is involved in transport of the mRNA to the site of translation, e.g., the cytoplasm.

[0264] The term "primer" refers to a short nucleic acid sequence that hybridizes to a template nucleic acid sequence and is used to polymerize a nucleic acid sequence that is complementary to the template.

[0265] The term "selectable marker" refers to any gene that, upon expression, can be used to select cells containing the selectable marker. Examples of selectable markers are provided below. Those skilled in the art will know that different antibiotic, fungicide, supplemental or herbicide selectable markers are applicable to different target species.

[0266] The present invention also relates to nucleic acid sequences that encode the polypeptides defined herein.

[0267] 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 functional equivalents thereof, which can be obtained, for example, using artificial nucleotide analogues.

[0268] 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 that can be used, for example, as hybridization probes or primers to identify or amplify the encoding nucleic acid according to the invention.

[0269] The present invention also relates to nucleic acids that have a degree of "identity" to the sequences specifically disclosed herein. "Identity" between two nucleic acids means, in each case, nucleotide identity over the entire length of the nucleic acid.

[0270] "Identity" between two nucleotide sequences (the same applies to peptide or amino acid sequences) refers to the number or number of nucleotide residues (or amino acid residues) that are identical in the two sequences when the two sequences are aligned. Identical residues are defined as residues that are identical in the two sequences at a given position in the alignment. As used herein, percentage sequence identity is calculated by taking the number of identical residues between the two sequences from the optimal alignment, dividing it by the number of residues in the shortest sequence, and multiplying by 100. The optimal alignment is the alignment that results in the highest percentage identity. To achieve optimal alignment, gaps may be introduced into one or both sequences at one or more positions in the alignment. These gaps are considered non-identical residues in calculating the percentage sequence identity. Alignment to determine the percentage identity of amino acid or nucleic acid sequences can be achieved in a variety of ways using computer programs, such as publicly available computer programs available on the World Wide Web.

[0271] In particular, the BLAST program (Tatiana et al., FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to default parameters, available from the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi), can be used to obtain optimal alignments of protein or nucleic acid sequences and to calculate the percentage of sequence identity.

[0272] In another example, identity can be determined using the Vector NTI Suite 7.1 program from Informax (USA) using the Clustal method (Higgins DG, Sharp PM. ((1989))) with the following settings:

[0273] 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 specific gaps off Hydrophilic residue gap off Transition weighing 0 Pairwise alignment parameters: FAST algorithm on K-tuple size 1 Gap penalty 3 Window size 5 Number of best diagonals: 5 Alternatively, identity may be determined according to the web page of Chenna, et al. (2003): http: / / www.ebi.ac.uk / Tools / clustalw / index.html# and the following settings:

[0274] DNA Gap Open Penalty 15.0 DNA Gap Extension Penalty 6.66 DNA Matrix Identity Protein Gap Open Penalty 10.0 Protein Gap Extension Penalty 0.2 Protein matrix Gonnet Protein / DNA ENDGAP -1 Protein / DNA GAPDIST 4 All nucleic acid sequences (single- and double-stranded DNA and RNA sequences, e.g., cDNA and mRNA) described herein can be produced by chemical synthesis from nucleotide building blocks, for example, by fragment condensation of individual overlapping complementary nucleic acid building blocks of a double helix, in a known manner. Chemical synthesis of oligonucleotides can be carried out by known methods, for example, by the phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and the gap-filling and ligation reactions using the Klenow fragment of DNA polymerase, as well as general cloning techniques, are described in Sambrook et al. (1989) (see below).

[0275] Nucleic acid molecules according to the present invention can further include untranslated sequences from the 3' and / or 5' ends of the coding gene region.

[0276] The present invention further relates to nucleic acid molecules that are complementary to the specifically described nucleotide sequences or segments thereof.

[0277] The nucleotide sequences of the present invention allow for the generation of probes and primers that can be used to identify and / or clone 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) over 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 a nucleic acid sequence of the present invention.

[0278] "Homologous" sequences include orthologous or paralogous sequences. Methods for identifying orthologs or paralogs, including phylogenetic analysis, sequence similarity, and hybridization methods, are known in the art and described herein.

[0279] "Paralogues" result from gene duplication, resulting in two or more genes with similar sequences and similar functions. Paralogs are typically formed by gene duplication within related plant species and cluster together. Paralogs are found in groups of similar genes using pairwise Blast analysis or during phylogenetic analysis of gene families using programs such as CLUSTAL. In paralogs, consensus sequences can be identified that are characteristic of sequences within related genes and have similar gene functions.

[0280] "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, plant species that share a common ancestor are known to contain many enzymes with similar sequences and functions. One skilled in the art can identify orthologous sequences and predict the function of orthologs by constructing a phylogenetic tree of a certain gene family using, for example, CLUSTAL or BLAST programs. One method for identifying or confirming similar functions between homologous sequences includes comparing transcription profiles in which related polypeptides are overexpressed or deleted (knocked out / knocked down) in host cells or organisms, such as plants or microorganisms. One skilled in the art will understand that genes with similar transcription profiles and more than 50% of regulated transcripts in common, more than 70% of regulated transcripts in common, or more than 90% of regulated transcripts in common have similar functions. Homologs, paralogs, orthologs and any other variants of the sequences herein are expected to function similarly by causing an organism, such as a host cell, plant or microorganism, to produce a terpene synthase protein.

[0281] The term "selectable marker" refers to any gene that, upon expression, can be used to select a cell or cells containing the selectable marker. Examples of selectable markers are provided below. One skilled in the art will know that different antibiotic, fungicide, supplemental or herbicide selectable markers are applicable to different target species.

[0282] Nucleic acid molecules according to the invention can be recovered by standard techniques of molecular biology and the sequence information provided by the invention. For example, cDNA can be isolated from an appropriate cDNA library using one of the specifically disclosed complete sequences, or a segment thereof, as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, (1989)).

[0283] Furthermore, nucleic acid molecules comprising one of the disclosed sequences or a segment thereof can be isolated by polymerase chain reaction using oligonucleotide primers constructed based on this sequence.The nucleic acid thus amplified can be cloned into an appropriate vector and characterized by DNA sequencing.The oligonucleotides according to the present invention can also be produced by standard synthesis methods, for example, using an automatic DNA synthesizer.

[0284] The nucleic acid sequences according to the invention or derivatives, homologues or parts of these sequences can be isolated from other bacteria, for example via genomic or cDNA libraries, for example by conventional hybridization or PCR techniques, which DNA sequences hybridize under standard conditions with the sequences according to the invention.

[0285] "Hybridize" refers to the ability of a polynucleotide or oligonucleotide to bind to a nearly complementary sequence under standard conditions, while non-specific binding between non-complementary partners does not occur under these conditions. Thus, sequences can be 90-100% complementary. The ability of complementary sequences to bind specifically to each other is utilized, for example, in Northern or Southern blotting, or for primer binding in PCR or RT-PCR.

[0286] For hybridization, short oligonucleotides of the conserved region are advantageously used. However, it is also possible to use longer fragments or the complete sequence 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 on which type of nucleic acid - DNA or RNA - is used for hybridization. For example, the melting temperature of DNA:DNA hybrids is about 10°C lower than that of DNA:RNA hybrids of the same length.

[0287] For example, depending on the specific nucleic acid, standard conditions refer to temperatures between 42°C and 58°C in an aqueous buffer solution having a concentration of 0.1 to 5xSSC (1xSSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2), or even in the presence of 50% formamide, e.g., 42°C and 50% formamide in 5xSSC. Advantageously, hybridization conditions for DNA:DNA hybrids are 0.1xSSC and temperatures between about 20°C and 45°C, preferably between about 30°C and 45°C. Hybridization conditions for DNA:RNA hybrids are advantageously 0.1xSSC and temperatures between about 30°C and 55°C, preferably between about 45°C and 55°C. The stated temperatures for these hybridizations are examples of melting temperatures calculated in the absence of formamide for a nucleic acid of about 100 nucleotides in length and 50% G+C content. The experimental conditions for DNA hybridization are described in relevant genetic textbooks, such as Sambrook et al., 1989, and can be calculated using formulas known to those skilled in the art, depending on, for example, 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).

[0288] "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.

[0289] As used herein, the term hybridization or hybridizing under certain conditions is intended to describe the conditions of hybridization and washing in which the nucleotide sequences that are significantly identical or homologous to each other remain bound to each other.This condition may be the conditions in which 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 and high stringency hybridization conditions are provided herein.

[0290] Appropriate hybridization conditions can be selected by one of ordinary 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).

[0291] As used herein, low stringency conditions are defined as follows: DNA-containing filters 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 using 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 5-20 x 106 cpm of 32P-labeled probe. Filters are incubated in the hybridization mixture for 18-20 hours at 40°C, then washed for 1.5 hours at 55°C. The filters were washed in a solution containing 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution was replaced with fresh solution and the filters were further incubated at 60°C for 1.5 hours. The filters were blotted dry and subjected to autoradiography.

[0292] As used herein, the defined conditions for moderate stringency are as follows: DNA-containing filters 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 using 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 5–20 x 106 cpm of 32P-labeled probe. Filters are incubated in the hybridization mixture at 50°C for 30 hours, then washed at 55°C for 1.5 hours. The filters were washed in a solution containing 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution was replaced with fresh solution and the filters were further incubated at 60°C for 1.5 hours. The filters were blotted dry and subjected to autoradiography.

[0293] As used herein, high stringency conditions are defined as follows: DNA-containing filters are prehybridized 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. Filters are hybridized for 48 hours at 65°C in a prehybridization mixture containing 100 μg / ml denatured salmon sperm DNA and 5-20 x 10 cpm of 32P-labeled probe. Filters are washed for 1 hour at 37°C in a solution containing 2x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a 45-minute wash in 0.1x SSC at 50°C.

[0294] If the above conditions are inappropriate (e.g., conditions employed for cross-species hybridizations), other conditions of low, medium, and high stringency well known in the art (e.g., conditions employed for cross-species hybridizations) may be used.

[0295] Kits for detecting nucleic acid sequences encoding polypeptides of the present invention may include primers and / or probes specific for the nucleic acid sequences encoding the polypeptides and associated protocols for detecting the nucleic acid sequences encoding the polypeptides in a sample using the primers and / or probes. Such detection kits may be used to determine whether a plant, organism, microorganism, or cell has been modified, i.e., transformed, with a sequence encoding the polypeptide.

[0296] 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.

[0297] The present invention also relates to derivatives of the specifically disclosed nucleic acid sequences or nucleic acid sequences which can be derivatized.

[0298] 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 1 to 10) nucleotides, for example 1 to 20, in particular 1 to 15 or 5 to 10 nucleotides, and may further encode a polypeptide having a desired profile of properties.

[0299] The present invention also encompasses nucleic acid sequences which contain so-called silent mutations, or which have been altered in accordance with the codon usage of a particular primary or host organism compared to the specifically described sequences.

[0300] According to certain embodiments of the present invention, variant nucleic acids may be prepared to adapt their nucleotide sequences to specific expression systems. For example, bacterial expression systems are known to express polypeptides more efficiently when amino acids are encoded by specific codons. Due to the degeneracy of the genetic code, multiple codons may encode the same amino acid sequence, and multiple nucleic acid sequences may encode the same protein or polypeptide; all of these DNA sequences are encompassed by the embodiments herein. Where appropriate, nucleic acid sequences encoding the polypeptides described herein may be optimized for enhanced expression in host cells. For example, the nucleic acids of the embodiments herein may be synthesized using codons specific to the host to improve expression.

[0301] The present invention also encompasses naturally occurring variants of the sequences described herein, such as splice variants or allelic variants.

[0302] Allelic variants may have at least 60% homology, preferably at least 80% homology, particularly preferably at least 90% homology over the entire sequence range at the derived amino acid level (see details above for polypeptides regarding homology at the amino acid level). Advantageously, the homology may be higher over partial regions of the sequence.

[0303] The invention also relates to sequences that can be obtained by conservative nucleotide substitutions, ie, so that the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility.

[0304] The present invention also relates to molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. Such genetic polymorphisms may exist in cells or populations from different populations due to natural allelic variation. Allelic variants may also include functional equivalents. These natural variations typically result in 1-5% variance in the nucleotide sequence of a gene. Such polymorphisms may result in changes in the amino acid sequence of the polypeptides disclosed herein. Allelic variants may also include functional equivalents.

[0305] Furthermore, derivatives are also to be understood as homologs of the nucleic acid sequences according to the invention, such as animal, plant, fungal or bacterial homologs, truncated sequences, single-stranded DNA or RNA coding and non-coding DNA sequences, for example homologs which, at the DNA level, have 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.

[0306] 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 without impairing the functionality or effectiveness of the promoter. Furthermore, the effectiveness of promoters can be increased by modifying their sequences, or even completely replaced with more effective promoters, even in organisms of different genus.

[0307] d. Generation of Functional Polypeptide Variants Furthermore, one of skill in the art is familiar with methods for generating functional variants, i.e., nucleotide sequences encoded by a nucleic acid molecule comprising a nucleotide sequence 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 of the amino acid related SEQ ID NOs disclosed herein and / or at least 70% sequence identity to any of the amino acid related SEQ ID NOs disclosed herein.

[0308] Depending on the technique used, one skilled in the art can introduce completely random mutations or other more directed mutations into genes or other non-coding nucleic acid regions (e.g., those important for regulating expression), and then generate a genetic library. The molecular biology methods required for this purpose are known to those skilled in the art and are described, for example, in Sambrook and Russell, Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001.

[0309] Methods for modifying genes and thereby modifying the polypeptides encoded by said genes have long been known to those skilled in the art, and include, for example, the following. - site-directed mutagenesis, a method for the directed replacement of individual or several nucleotides in a gene (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey); - saturation mutagenesis, a method that allows the exchange or addition of codons for any amino acid at any position in a gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22:1593; Barettino D, Feigenbutz M, Valca'rel R, Stunnenberg HG (1994) Nucleic Acids Res 22:541; Barik S (1995) Mol Biotechnol 3:1), - error-prone polymerase chain reaction, a method in which nucleotide sequences are mutated by error-prone DNA polymerases (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18:3739); - SeSaM (Sequence Saturation Mutagenesis) method, which prevents preferential exchanges by polymerases (Schenk et al., Biospektrum, Vol. 3, 2006, 277-279), - passage of genes in mutant strains with an increased rate of mutation of nucleotide sequences, for example 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, a method in which pools of closely related genes are formed and digested, and the fragments are used as templates for polymerase chain reaction, with repeated strand separation and recombination ultimately producing a full-length mosaic gene (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747).

[0310] Using so-called directed evolution (described in particular 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), those skilled in the art can generate functional variants in a directed and large-scale manner. For this purpose, in a first step, a gene library for each polypeptide is first generated, for example, using the methods described above. The gene library is expressed in a suitable manner, for example, by a bacterial or phage display system.

[0311] The relevant gene in the host organism that expresses a functional mutant with properties that closely correspond to the desired property can be subjected to another mutation cycle. The mutation and selection or screening steps can be repeated iteratively until a sufficient number of functional mutants are found to possess the desired property. This iterative procedure can be used to incrementally introduce a limited number of mutations, e.g., 1, 2, 3, 4, or 5 mutations, evaluate their effects on the activity in question, and select. The selected mutants can then be subjected to further mutation steps in the same manner. In this way, the number of individual mutants to be investigated can be significantly reduced.

[0312] The results according to the present invention also provide important information about the structure and sequence of related polypeptides, which is necessary for further generating polypeptides with desired altered properties in a targeted manner. In particular, it is possible to define so-called "hot spots", i.e., sequence segments that are potentially suitable for altering properties by introducing targeted mutations.

[0313] Information can also be obtained regarding the location of the amino acid sequence in which mutations can be made that will likely have little effect on activity and can be referred to as potential "silent mutations."

[0314] e. Constructs for expressing the polypeptides of the present invention In this regard, the following definitions apply: "Gene expression" 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 transfected cell or organism that exceeds production levels in a non-transformed cell or organism of a similar genetic background.

[0315] As used herein, "expression vector" refers to a nucleic acid molecule designed using molecular biology and recombinant DNA technology to deliver foreign or exogenous DNA into host cells.Expression vectors typically contain the sequences required for proper transcription of nucleotide sequences.The coding region usually codes for the protein of interest, but may also code for RNA, such as antisense RNA, siRNA, etc.

[0316] 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 an mRNA ribosomal 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.

[0317] As used herein, "expression system" encompasses any combination of nucleic acid molecules necessary for the expression of a polypeptide or the 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, on a polycistronic nucleic acid, or distributed on two or more physically distinct vectors. A specific example is an operon containing a promoter sequence, one or more operator sequences, and one or more structural genes, each encoding an enzyme described herein.

[0318] 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 more 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).

[0319] "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.

[0320] According to the present invention, a "promoter," "nucleic acid having promoter activity," or "promoter sequence" is understood to mean a nucleic acid that, when operably linked to a nucleic acid to be transcribed, regulates the transcription of said nucleic acid. In particular, a "promoter" refers 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, binding sites for transcription factors, repressor and activator proteins. The term promoter also includes the term "promoter regulatory sequence." A promoter regulatory sequence may contain 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. A coding nucleic acid sequence is usually located downstream of a promoter in the direction of transcription starting from the transcription start site.

[0321] In this context, "functional" or "operably" linked refers to, for example, the contiguous arrangement of one of the nucleic acids with 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 transcription of the nucleic acid, and, for example, a terminator, are linked so that each of the regulatory elements can perform its function during transcription of the nucleic acid sequence. This does not necessarily require direct linkage in the chemical sense. Gene control sequences, such as enhancer sequences, can even exert their function on the target sequence from more distant locations or even from other DNA molecules. A preferred arrangement is one in which the nucleic acid sequence to be transcribed is located behind the promoter sequence (i.e., at the 3' end), and 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, less than 100 base pairs, or less than 50 base pairs.

[0322] In addition to promoters and terminators, examples of other regulatory elements 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).

[0323] The term "constitutive promoter" refers to an unregulated promoter that allows for continuous transcription of a nucleic acid sequence to which it is operably linked.

[0324] As used herein, the term "operably linked" refers to the linking of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter, or rather a transcriptional regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operatively linked typically means that the linked DNA sequences are contiguous. The nucleotide sequence 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, once bound to a polypeptide of an embodiment herein, the nucleic acid sequence associated with the promoter sequence is expressed or silenced according to the characteristics of the promoter to which it is linked. The associated nucleic acid may encode a protein that is desired to be expressed or suppressed throughout the entire organism, at a specific time, or in a specific tissue, cell, or cellular compartment. Such a nucleotide sequence specifically encodes a protein that confers a desired phenotype on a host cell or organism altered or transformed therewith. More specifically, the associated nucleotide sequence results in the production of a desired product or products as defined herein in the cell or organism. In particular, the nucleotide sequence encodes a polypeptide having an enzymatic activity as defined herein.

[0325] The nucleotide sequences described herein may be part of an "expression cassette." The terms "expression cassette" and "expression construct" are used interchangeably. A (preferably recombinant) expression construct comprises a nucleotide sequence that encodes a polypeptide according to the invention and is under the genetic control of a regulatory nucleic acid sequence.

[0326] In the processes applied according to the present invention, the expression cassette may be part of an "expression vector", in particular part of a recombinant expression vector.

[0327] "Expression unit" is understood to mean, according to the present invention, a nucleic acid which comprises a promoter as defined herein and which, after being functionally linked to a nucleic acid or gene to be expressed, has expression activity which regulates the expression, i.e., transcription and translation, of said nucleic acid or said gene. Expression units are therefore also called "regulatory nucleic acid sequences" in this context. In addition to the promoter, other regulatory elements may also be present, such as enhancers.

[0328] "Expression cassette" or "expression construct" is understood according to the present invention to mean an expression unit operably linked to a nucleic acid to be expressed or a gene to be expressed. Thus, in contrast to an expression unit, an expression cassette not only comprises nucleic acid sequences that regulate transcription and translation, but also nucleic acid sequences that are to be expressed as a protein as a result of transcription and translation.

[0329] In the context of the present invention, the terms "expression" or "overexpression" refer to the production in a microorganism of one or more polypeptides encoded by the corresponding DNA or to the enhancement of their intracellular activity. This can be achieved, for example, by introducing a gene into the organism, replacing an existing gene with another gene, increasing the copy number of the gene, using a strong promoter, using a gene encoding a corresponding polypeptide with increased activity, or any combination of these means.

[0330] Preferably, such constructs according to the invention comprise a promoter 5' upstream of the respective coding sequence, a terminator sequence 3' downstream, and optionally other conventional regulatory elements, in each case operably linked to the coding sequence.

[0331] Nucleic acid constructs according to the present invention particularly comprise sequences encoding polypeptides derived, for example, from the amino acid-related SEQ ID NOs described herein, or reverse complements thereof, or derivatives and homologs thereof, advantageously operatively or functionally linked to one or more regulatory signals for controlling, e.g., increasing, gene expression.

[0332] In addition to these regulatory sequences, the natural regulation of these sequences may still exist before the actual structural gene, and can be optionally genetically altered so that the natural regulation is switched off and gene expression is enhanced.However, the nucleic acid construct may also have a simpler structure, that is, no additional regulatory signal is inserted before 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 under control and gene expression is increased.

[0333] A preferred nucleic acid construct advantageously also contains one or more of the previously described "enhancer" sequences operably linked to the promoter, which allow for enhanced expression of the nucleic acid sequence. Additional regulatory elements or advantageous sequences, such as terminators, may be inserted at the 3' end of the DNA sequence. One or more copies of the nucleic acid according to the invention may be present in the construct. Optionally, other markers, such as genes that complement auxotrophy or antibiotic resistance, may be present in the construct for selection of the construct.

[0334] 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 LThese regulatory sequences are found in promoters, which are preferably used in Gram-negative bacteria. Further advantageous regulatory sequences are found, for example, in the promoters amy and SPO2 of Gram-positive bacteria, and in the promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, and ADH of yeast or fungi. Regulation can also be achieved using artificial promoters.

[0335] For expression in a host organism, the nucleic acid construct is advantageously inserted into a vector, such as a plasmid or phage, to allow optimal expression of the gene in the host.Vector is understood to mean not only plasmids and phages, but also all other vectors known to those skilled in the art, namely, 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 in the host organism, or alternatively, can replicate chromosomally.These vectors are a further development of the present invention.Binary vectors or cpo-integrating vectors are also applicable.

[0336] Suitable plasmids include, for example, E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113 -B1, λgt11 or pBdCI, pIJ101, pIJ364, pIJ702 or pIJ361 of Streptomyces, pUB110, pC194 or pBD214 of Bacillus, pSA77 or pAJ667 of Corynebacterium, pALS1, pIL2 or pBB116 of fungi, 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 of yeast, or pLGV23, pGHlac of plants +, pBIN19, pAK2004 or pDH51. The above-mentioned plasmids are only a small selection of the 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).

[0337] As a 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 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 can consist solely of the nucleic acid construct or nucleic acid according to the invention.

[0338] For optimal expression of a heterologous gene in an organism, it is advantageous to modify the nucleic acid sequence to match the particular "codon usage" used by the organism, which can be readily determined by computer evaluation of other known genes of the organism in question.

[0339] The expression cassette according to the present invention is produced by fusing a suitable promoter to a suitable coding nucleotide sequence and a terminator or polyadenylation signal. For this purpose, conventional recombinant and cloning techniques are used, for example, as described in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984); Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0340] 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).

[0341] Alternative embodiments of the present disclosure provide methods of "altering 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).

[0342] The expression of the polynucleotide provided herein may be changed, which is the expression pattern that is different between the changed organism and the control or wild-type organism, ectopic expression.The change in expression occurs from the interaction of the polypeptide of the embodiment herein with exogenous or endogenous modulator, or as a result of chemical modification of the polypeptide.This term also refers to the expression pattern of the polynucleotide of the embodiment herein being changed to below detection level, or activity being completely suppressed.

[0343] In one embodiment, also provided herein are isolated, recombinant, or synthetic polynucleotides that encode the polypeptides or variant polypeptides provided herein.

[0344] 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 then select transformants containing both chimeric genes.Similarly, one or polypeptide encoding genes can be expressed together with other chimeric genes in a single plant, cell, microorganism or organism.

[0345] f. Hosts 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.

[0346] 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.

[0347] The vectors of the present invention can be used to produce recombinant hosts that can be transformed with at least one vector of the present invention and used to produce the polypeptides of the present invention. Advantageously, the recombinant constructs of the present invention are introduced into a suitable host system and expressed. Preferably, the nucleic acids described are expressed in the respective expression systems using common cloning and transfection methods known to those skilled in the art, such as co-precipitation, protoplast fusion, electroporation, retroviral transfection, etc. 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.

[0348] 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 species Escherichia coli are highly preferred. Furthermore, other advantageous bacteria can be found in the groups of the Alphaproteobacteria, Betaproteobacteria or Gammaproteobacteria. Advantageously, yeasts such as Saccharomyces or Pichia are also suitable hosts.

[0349] Alternatively, whole plants or plant cells can be used as natural or recombinant hosts. Non-limiting examples include the following plants or cells derived therefrom: Nicotiana, particularly Nicotiana benthamiana and Nicotiana tabacum, and Arabidopsis, particularly Arabidopsis thaliana.

[0350] Depending on the host organism, the organisms used in the process according to the invention are grown or cultured in a manner known to those skilled in the art. The culture may be batch, semi-batch or continuous. Nutrients may be present at the start of the fermentation or may be fed later in a semi-continuous or continuous manner, as will also be explained in more detail below.

[0351] g. Recombinant Production of Polypeptides of the Invention The present invention further relates to a method for recombinantly producing a polypeptide according to the invention or a functional, biologically active fragment thereof, comprising culturing a microorganism that produces the polypeptide, optionally inducing expression of the polypeptide by applying at least one inducer that induces gene expression, and isolating the expressed polypeptide from the culture. The polypeptide can also be produced on an industrial scale in this way, if desired.

[0352] The microorganisms produced according to the invention can be cultivated continuously or discontinuously in batch, fed-batch or repeated fed-batch processes. Overviews of known cultivation methods can be found in the textbooks by Chmiel (Bioprozesstechnik 1. Einfuehrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas (Bioreactors and peripheral equipment [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0353] The culture medium to be used must be appropriate to the requirements of each strain. The American Society for Microbiology's "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981) describes culture media for various microorganisms.

[0354] These media that can be used according to the present invention usually contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0355] Preferred carbon sources are sugars, such as monosaccharides, disaccharides, or polysaccharides. Excellent carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose. Sugars can also be added to the medium through complex compounds such as molasses, or by-products of sugar refining. It is also 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.

[0356] The nitrogen source is usually an organic or inorganic nitrogen compound or a material containing such a compound. Examples of nitrogen sources include ammonia gas or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean flour, soybean protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or in combination.

[0357] Inorganic salt compounds that may be present in the medium include chlorides, phosphates or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0358] As sulfur sources, sulfur-containing inorganic compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur compounds such as mercaptans and thiols can be used.

[0359] As the phosphorus source, phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts thereof can be used.

[0360] Chelating agents can be added to the medium to keep metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuate, or organic acids such as citric acid.

[0361] Fermentation media used in accordance with the present invention typically also contain other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid, and pyridoxine. Growth factors and salts are often derived from components of complex media, such as yeast extract, molasses, and corn steep liquor. Additionally, appropriate precursors can be added to the culture medium. The exact composition of compounds in the medium is highly experimentally dependent and must be determined individually for each specific case. Information regarding medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can be obtained from commercial sources, such as Standard 1 (Merck) or BHI (Brain Heart Fusion Medium, DIFCO).

[0362] All components of the medium are sterilized by heat (20 minutes at 1.5 bar and 121°C) or sterile filtration. The components can be sterilized together or separately if necessary. All components of the medium can be present at the start of the culture or can be added continuously or batchwise.

[0363] The culture temperature is usually between 15°C and 45°C, preferably between 25°C and 40°C, and can be varied or kept constant during the experiment. The pH of the medium is between 5 and 8.5, preferably around 7.0. The pH during growth can be controlled by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid. Antifoaming agents such as fatty acid polyglycol esters can be used to control foaming. To maintain plasmid stability, appropriate selective substances, such as antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or an oxygen-containing gas mixture, such as ambient air, is supplied to the culture. The culture temperature is usually between 20°C and 45°C. The culture is continued until the desired product is maximally formed. This goal is usually achieved within 10 to 160 hours.

[0364] The fermentation broth is then further processed. Depending on the requirements, the biomass can be completely or partially removed from the fermentation broth by separation techniques such as centrifugation, filtration, decantation or a combination of these methods, or can be left entirely in the fermentation broth.

[0365] If the polypeptide is not secreted into the culture medium, the cells can be lysed and the product obtained from the lysate by known methods for isolating proteins. Optionally, the cells can be disrupted by high frequency ultrasound, by high pressure, e.g., in a French press, by osmotic lysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers, or by some combination of the aforementioned methods.

[0366] Polypeptides can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), e.g., Q-sepharose chromatography, ion exchange chromatography, and hydrophobic chromatography, as well as other conventional techniques, such as ultrafiltration, crystallization, salting out, dialysis, and native gel electrophoresis. Suitable methods are described, for example, in Cooper, T.G., Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, New York, or Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0367] To isolate recombinant proteins, it may be advantageous to use vector systems or oligonucleotides, which extend the cDNA by a defined nucleotide sequence and thus encode modified polypeptides or fusion proteins, which, for example, allow for easier purification. Suitable modifications of this type are, for example, so-called "tags" that function as anchors, such as the modification known as a hexahistidine anchor or epitopes that can be recognized as antigens by antibodies (see, for example, Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press). These anchors can serve to attach proteins to solid supports, such as polymer matrices, which can be used, for example, as packing for chromatography columns or on microtiter plates or other supports.

[0368] At the same time, these anchors can also be used for protein recognition, which can be achieved by using conventional markers, such as fluorescent dyes, enzymatic markers that form detectable reaction products after reaction with a substrate, or radioactive markers, alone or in combination with anchors for protein derivatization.

[0369] h. Polypeptide immobilization The enzymes or polypeptides of the present invention can be used in the methods described herein in a free or immobilized form. An immobilized enzyme refers to an enzyme immobilized on an inert support. Suitable support materials and enzymes immobilized thereon are known from EP 1149849, EP 1069183, DE-OS 100193773, and the documents cited therein. Reference is made in this regard to the disclosures of these documents in their entirety. Suitable support materials include, for example, clay, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, synthetic polymers such as polystyrene, acrylic resins, phenol-formaldehyde resins, polyurethanes, and polyolefins such as polyethylene and polypropylene. For the production of supported enzymes, the support material is usually employed in the form of finely divided particles, with porous forms being preferred. The particle size of the support material is usually 5 mm or less, especially 2 mm or less (particle size distribution curve). Similarly, when dehydrogenase is used as a whole cell catalyst, either free or immobilized form can be selected. Carrier materials include, for example, calcium alginate and carrageenan. Both enzymes and cells can also be directly cross-linked with glutaraldehyde (cross-linking to CLEA). Other corresponding immobilization techniques are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out the method according to the invention can also be found, for example, in Rehm et al. (eds.) Biotechnology, 2nd Edn, Vol. 3, Chapter 17, VCH, Weinheim.

[0370] i. Reaction conditions for the biocatalyst production method of the present invention The reaction of the present invention can be carried out under in vivo or in vitro conditions.

[0371] The at least one polypeptide / enzyme present during the individual steps of the method of the present invention or the multi-step method defined herein above may be present in living cells that 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 simultaneously be present in soluble and / or immobilized form.

[0372] The method according to the present invention can be carried out in conventional reactors known to those skilled in the art, ranging in scale from laboratory scale (reaction volumes of several milliliters to several tens of liters) to industrial scale (reaction volumes of several liters to several thousand cubic meters). When the polypeptide is used in a form encapsulated in non-living, optionally permeabilized cells, in the form of a more or less purified cell extract, or in a purified form, a chemical reactor can be used. A chemical reactor typically allows control of the amount of at least one enzyme, the amount of at least one substrate, pH, temperature, and circulation of the reaction medium. When at least one polypeptide / enzyme is present in living cells, the process is fermentation. In this case, biocatalyst production is carried out in a bioreactor (fermenter), where the parameters required for the proper survival of the living cells (e.g., culture medium with nutrients, temperature, aeration, the presence or absence of oxygen or other gases, antibiotics, etc.) can be controlled. Those skilled in the art are familiar with chemical reactors or bioreactors, and procedures for scaling up chemical or biotechnological processes from laboratory scale to industrial scale or for optimizing process parameters are also well described in the literature (for biotechnological methods see, for example, Krueger und Krueger, Biotechnologie-Lehrbuch der angewandtenMikrobiologie, 2nd Ed., R. Oldenbourg Verlag, München, Wien, 1984).

[0373] Cells containing at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or radiofrequency pulses, French press, or chemical means, such as hypotonic solutions, lysing enzymes, and detergents present in the medium, or a combination thereof. Examples of detergents include digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween® 20, deoxycholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), Nonidet® P40 (ethylphenol poly(ethylene glycol ether)), etc.

[0374] Instead of living cells, biomass of non-viable cells containing the necessary biocatalyst may be applied to the bioconversion reactions of the present invention.

[0375] When the at least one enzyme is immobilized, it is attached to an inert support as described above.

[0376] 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 fed thereafter semi-continuously or continuously.

[0377] 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.

[0378] 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.

[0379] The aqueous organic medium may be applied with organic solvents that are miscible, partially miscible, or immiscible with water. Non-limiting examples of suitable organic solvents are given below. Further examples are monohydric or polyhydric, aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols such as glycerol.

[0380] A non-aqueous medium is substantially free of water, ie, contains less than about 1% or 0.5% water by weight.

[0381] The biocatalytic process may be carried out in an organic, non-aqueous medium. Suitable organic solvents include aliphatic hydrocarbons having, for example, 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene, or dichlorobenzene; aliphatic acyclic ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, or dibutyl ether; or mixtures thereof.

[0382] The reactant / substrate concentrations can be adjusted to optimal reaction conditions, which depend on the specific enzyme being applied. For example, the initial substrate concentration can be 0.1-0.5 M, e.g., 10-100 mM.

[0383] The reaction temperature may be adjusted to optimal reaction conditions, which may depend on the particular enzyme being used. For example, the reaction may be carried out at a temperature ranging from 0 to 70°C, such as 20 to 50°C or 25 to 40°C. Examples of reaction temperatures are about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C, and about 60°C.

[0384] The process may proceed until equilibrium between the substrate and the product is reached, but may be stopped earlier. Typical process times are in the range of 1 minute to 25 hours, particularly in the range of 10 minutes to 6 hours, for example in the range of 1 hour to 4 hours, particularly in the range of 1.5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.

[0385] If the host is a transgenic plant, optimal growth conditions can be provided, for example, optimal light, water and nutrient conditions.

[0386] k. Product isolation The methods of the present invention can further include 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 a compound from a culture or reaction medium. Recovery of the compound can be performed according to any conventional isolation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH change, solvent extraction (e.g., treatment with common solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0387] 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, 521-540, 540-547, 559-566, 575-581 and pp. 111-112). 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.).

[0388] Cyclic terpene compounds produced by any of the methods described herein can be converted into derivatives, including, 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, including, but not limited to, oxidation, reduction, alkylation, acylation, and / or rearrangement. Alternatively, terpene compound derivatives can be obtained using biochemical methods by contacting the terpene compound with enzymes, including, but not limited to, oxidoreductases, monooxygenases, dioxygenases, and transferases. Biochemical conversions can be performed in vitro using isolated enzymes, enzymes from lysed cells, or in vivo using whole cells.

[0389] l. Fermentative production of terpene / terpenoid compounds, e.g., labdane-type compounds The present invention also relates to methods for the fermentative production of terpene / terpenoid compounds, such as labdane-type compounds.

[0390] The fermentation used according to the invention can be carried out, for example, in stirred fermenters, bubble columns, and loop reactors. A comprehensive overview of possible process types, including the type and geometric design of the agitator, can be found in "Chmiel: Bioprozesstechnik: Einfuehrung in die Bioverfahrenstechnik, Band 1." Typical variants that can be used in the process of the invention are those known to those skilled in the art or described, for example, in "Chmiel, Hammes, and Bailey: Biochemical Engineering," such as batch, fed-batch, repeated fed-batch, or other continuous fermentations with and without biomass recycling. Depending on the production strain, sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen, or a suitable gas mixture can be performed to achieve a good yield (YP / S).

[0391] The medium to be used must meet the requirements of the particular strain in an appropriate manner. The American Society for Microbiology's handbook "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981) describes culture media for a variety of microorganisms.

[0392] These media that can be used according to the present invention may contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0393] Preferred carbon sources are sugars, such as monosaccharides, disaccharides, or polysaccharides. Excellent carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose. Sugars can also be added to the medium through complex compounds such as molasses, or by-products of sugar refining. It is also 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.

[0394] The nitrogen source is usually an organic or inorganic nitrogen compound or a material containing such a compound. Examples of nitrogen sources include ammonia gas or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean flour, soybean protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or in combination.

[0395] Inorganic salt compounds that may be present in the medium include chlorides, phosphates or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0396] As sulfur sources, sulfur-containing inorganic compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and also organic sulfur compounds such as mercaptans and thiols can be used.

[0397] As the phosphorus source, phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts thereof can be used.

[0398] Chelating agents can be added to the medium to keep metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuate, or organic acids such as citric acid.

[0399] The fermentation medium used in accordance with the present invention may also contain other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid, and pyridoxine. Growth factors and salts are often derived from components of complex media, such as yeast extract, molasses, and corn steep liquor. Additionally, appropriate precursors can be added to the culture medium. The exact composition of compounds in the medium is highly experimentally dependent and must be determined individually for each specific case. Information on medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (1997). Growth media can be obtained from commercial sources, such as Standard 1 (Merck) or BHI (Brain Heart Fusion Medium, DIFCO).

[0400] All components of the medium are sterilized by heat (20 minutes at 1.5 bar and 121°C) or sterile filtration. The components can be sterilized together or separately if necessary. All components of the medium can be present at the start of the culture or optionally added continuously or batchwise.

[0401] The culture temperature is usually between 15°C and 45°C, preferably between 25°C and 40°C, and can be varied or kept constant during the experiment. The pH of the medium is between 5 and 8.5, preferably around 7.0. The pH during growth can be controlled by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid. Antifoaming agents such as fatty acid polyglycol esters can be used to control foaming. To maintain plasmid stability, appropriate selective substances, such as antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or an oxygen-containing gas mixture, such as ambient air, is supplied to the culture. The culture temperature is usually between 20°C and 45°C. The culture is continued until the desired product is maximally formed. This goal is usually achieved within 1 to 160 hours.

[0402] The method of the present invention may further comprise the step of recovering said terpene alcohol.

[0403] The term "recovery" includes extracting, harvesting, isolating, or purifying a compound from a culture medium. Recovery of a compound can be performed according to any conventional isolation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, non-ionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH alteration, solvent extraction (e.g., treatment with conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0404] Before the desired isolation, the biomass of the broth can be removed. Processes for removing biomass are known to those skilled in the art and include, for example, filtration, sedimentation, and flotation. As a result, the biomass can be removed, for example, using a centrifuge, separator, decanter, filter, or flotation device. In order to maximize the recovery of valuable products, it is often desirable to wash the biomass, for example, in the form of diafiltration. The choice of method depends on the content and properties of the biomass in the fermentation broth, and the interaction between the biomass and the valuable product.

[0405] In one embodiment, the fermentation broth can be sterilized or pasteurized. In a further embodiment, the fermentation broth is concentrated. Depending on the requirements, this concentration can be performed batchwise or continuously. The pressure and temperature ranges should be selected so that, firstly, damage to the product does not occur, and, secondly, equipment and energy usage is minimized. A judicious selection of pressure and temperature levels, especially in the case of multi-stage evaporation, allows for energy savings.

[0406] The following examples are illustrative only and are not intended to limit the scope of the embodiments described herein.

[0407] Also included within the scope of the present invention are numerous possible variations that will become readily apparent to those of ordinary skill in the art after reviewing the disclosure provided herein.

[0408] Experimental part The present invention will now be described in more detail with reference to the following examples.

[0409] a) Material: Unless otherwise specified, all chemicals and biochemicals and microorganisms or cells used herein are commercially available.

[0410] Unless otherwise specified, recombinant proteins are cloned and expressed according to 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.

[0411] b) General method Preparation of cell-free protein fraction The expression vector was transformed into Escherichia coli KRX cells (Promega Corporation, Madison, WI, USA), and transformants were selected on LB plates supplemented with the appropriate antibiotic. The cells were then cultured in 25 mL of liquid LB medium supplemented with the appropriate antibiotic at 37°C until the OD reached 1. Recombinant protein expression was induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside and 0.1% (w / v) L-rhamnose monohydrate, and the cells were incubated at 25°C for 24 hours with moderate shaking.

[0412] Bacterial cells were harvested by centrifugation (5000g, 12 min) and disrupted on ice in 1.8 mL of 50 mM MOPSO buffer, pH 7.4, containing 15% glycerol by sonication (Sonics, Vibracell X 130 sonicator with a 6 mm diameter tip microprobe; three 20-second, 20 kHz pulses at 80% of maximum power). The lysate was cleared by centrifugation (3500g, 8 min, 4°C), and the resulting supernatant was frozen and used as the enzyme source for in vitro assays.

[0413] In vitro enzyme assay Protein fractions containing one of the recombinant proteins were incubated in an assay consisting of 20 μl of cell-free extract, 160–320 mg / L substrate (using a 40 g / L substrate stock solution in DMSO), 1 mM coenzyme as needed, and 50 mM MOPSO (pH 7.4) in a final volume of 0.5–1 mL in a borosilicate glass and PTFE-sealed screw-cap tube (11 mL capacity) (Wheaton, Millville, NJ 08332, USA) for 4 h at 24°C with shaking at 230 rpm. The assay was extracted with 1 volume of methyl tert-butyl ether (MTBE) and analyzed by GC-MS as described below.

[0414] Whole-cell bioconversion assay Bioconversion of compounds was performed using E. coli cells expressing the recombinant enzyme. The expression vector was transformed into E. coli KRX cells (Promega Corporation, Madison, WI, USA), and transformed cells were selected on LB broth plates supplemented with the appropriate antibiotic. 5 mL of LB broth supplemented with 1% glucose and the appropriate antibiotic was cultured overnight at 30°C. The next day, 20 mL of Terrific Broth supplemented with the appropriate antibiotic was inoculated to an initial optical density of 0.2–0.75. This culture was grown in a shake flask at 37°C until an optical density of 1–4 was reached. Recombinant protein expression was induced by adding 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) and 0.1% rhamnose. The culture was then dispensed in 0.5–1 mL aliquots into 12 mL glass tubes and incubated at 20°C with moderate shaking.

[0415] Ninety minutes after induction of recombinant protein expression, substrate was added to each tube. Substrate was added to a final concentration of 0.25–1 g / L using a 40 g / L stock solution in DMSO. Alternatively, an emulsion was prepared by dissolving 150 mg / mL Tween® 80 (Sigma-Aldrich) and 300 mg / mL substrate in water, and added to the assay to reach a final substrate concentration of 12 mg / mL.

[0416] After 8–48 h of incubation, the cultures were extracted with one volume of MTBE and analyzed by GC-MS as described below.

[0417] Cultivation of engineered bacterial cells under conditions that allow for the production of terpene compounds DP1205 E. coli cells were transformed with one or two expression plasmids carrying terpene biosynthetic genes and / or terpene-modifying enzymes, and the transformed cells were cultured on LB-agarose plates with the appropriate antibiotics (kanamycin (50 μg / mL) and / or chloramphenicol (34 μg / mL)). A single colony was used to inoculate 5 mL of liquid LB medium supplemented with the same antibiotic, 4 g / L glucose, and 10% (v / v) dodecane. The next day, 0.2 mL of the overnight culture was inoculated into 2 mL of TB medium supplemented with the same antibiotic and 10% (v / v) dodecane. The culture was incubated at 37 °C until an optical density of 3 was reached. Then, 1 mM IPTG was added to induce recombinant protein expression, and the culture was incubated at 20 °C for 72 h.

[0418] The culture was then extracted with one volume of MTBE, and the composition of the organic phase was analyzed by GC-MS as described below. For quantification, an internal standard (α-longipinene (Aldrich)) was added to the extract before GC-MS analysis, and the concentrations of the components were estimated based on comparison of peak areas.

[0419] GC-MS analysis method Whole cell bioconversion assay samples were analyzed using an Agilent 7890A GC system (Agilent Technologies, CA) coupled to a 5975C series mass selective detector (MSD) and equipped with a split / splitless injector.

[0420] The GC inlet temperature was set to 230 °C, and 1.0 μL of sample was injected in split mode (split ratio 20:1). The analysis was performed on a DB-5ms capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent J&W) using helium as the carrier gas at a constant flow rate of 1 mL / min. The initial oven temperature was set to 80 °C and programmed to 240 °C (10 °C / min; hold 1 min), then 300 °C (20 °C / min; hold 1 min).

[0421] In vitro assay samples were analyzed using an Agilent 6890N GC system coupled to a 5975 series mass selective detector (MSD) and equipped with a split / splitless injector (Agilent Technologies, CA) and a CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland) injection system. The GC inlet temperature was set to 250 °C, and 1.0 μL of sample was injected in pulsed splitless mode (pulse pressure 1.56 bar, pulse time 0.6 min). The samples were analyzed on a DB-1ms capillary column (30 m x 0.25 mm i.d. x 0.25 μm film thickness, Agilent J&W) at a constant flow rate of 1.2 mL / min using helium as the carrier gas. The initial oven temperature was 100 °C (hold 1 min), programmed to 260 °C (10–20 °C / min), then 300 °C (30 °C / min; hold 1 min). For compounds with low molecular weight, the analysis was carried out under the same conditions except that the initial oven temperature was lowered to 80°C.

[0422] Engineering recombinant strains that degrade terpene compounds Recombinant strains capable of producing or converting compounds were engineered by introducing nucleotide sequences encoding one or more of the following proteins: - Baeyer-Villiger monooxygenase (BVMO) selected from the following: SCH23-BVMO1 (SEQ ID NO: 2) from Hyphozyma roseonigra; SCH24-BVMO1 (SEQ ID NO: 6) from Filobasidium magnum, SCH25-BVMO1 (SEQ ID NO: 10) from Papiliotrema laurentii and SCH46-BVMO1 (SEQ ID NO: 13) from Bensingtonia ciliata ; - an esterase selected from the following: SCH23-EST (SEQ ID NO: 20) from Hyphozyma roseonigra; SCH24-EST (SEQ ID NO: 24) from Filobasidium magnum, SCH25-EST (SEQ ID NO: 28) from Papiliotrema laurentii; and - an enal-cleaving enzyme (lyase) selected from the following: SCH94-3944 Rhodococcus erythropolis (SEQ ID NO: 34), SCH80-05241 Rhodococcus rhodochrous (SEQ ID NO: 38), Pdigit7033 Penicillium digitatum (SEQ ID NO: 42), PitalDUF4334-1 Penicillium italicum (SEQ ID NO: 46), AspWeDUF4334 Aspergillus wentii (SEQ ID NO: 49), RhoagDUF4334-2 Rhodococcus hoagii strain PAM2288 (SEQ ID NO: 53), RhoagDUF4334-3 Rhodococcus hoagii strain N128 (SEQ ID NO: 56), RhoagDUF4334-4 Rhodococcus hoagii NBRC10125 (SEQ ID NO: 59), CnecaDUF4334 Cupriavidus necator (SEQ ID NO: 62), Rins-DUF4334 Ralstonia insidiosa (SEQ ID NO: 69), CgatDUF4334 Cryptococcus gattii EJB2 (SEQ ID NO: 72), GclavDUF4334 Grosmannia clavigera kw1407 (SEQ ID NO: 75), TcurvaDUF4334 Thermomonospora curvata (SEQ ID NO: 81), PprotDUF4334 Pseudomonas protegens (SEQ ID NO: 87).

[0423] Bacterial host cells for in vitro enzyme assays or whole-cell bioconversion assays were selected from E. coli KRX cells (Promega Corporation, Madison, Wisconsin, USA) and E. coli BL21 Star™ (DE3) cells (ThermoFisher).

[0424] To biochemically produce terpene compounds using one or more enzymes selected from the above enzymes, host cells were engineered to increase production of farnesyl pyrophosphate (FPP) using the mevalonate enzyme pathway and further transformed to express sesquiterpene or diterpene biosynthetic enzymes.

[0425] Engineering recombinant Escherichia coli strains for FPP production by chromosomal integration of genes encoding mevalonate pathway enzymes An E. coli strain was engineered to produce farnesyl pyrophosphate (FPP) by chromosomal integration of recombinant genes encoding mevalonate pathway enzymes. See also the structural scheme and recombination events depicted in Figure 1.

[0426] An upper pathway operon (acetyl-CoA to mevalonate operon 1) was designed, consisting of the atoB gene encoding acetoacetyl-CoA thiolase from Escherichia coli and the mbaA and mbaS genes encoding HMG-CoA synthase and HMG-CoA reductase from Staphylococcus aureus.

[0427] As the operon of the lower mevalonate pathway (operon 2 from mevalonate to farnesyl pyrophosphate), we selected a native operon from the Gram-negative bacterium Streptococcus pneumoniae encoding mevalonate kinase (mvaK1), phosphorymevalonate kinase (mvaK2), phosphorymevalonate decarboxylase (mvaD), and isopentenyl diphosphate isomerase (fni).

[0428] To convert isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to FPP, a codon-optimized S. cerevisiae FPP synthase-encoding gene (ERG20) was introduced into the 3' end of the upper pathway operon.

[0429] The above operon was synthesized using DNA2.0 and integrated into the araA gene of E. coli strain BL21(DE3). The heterologous pathway was introduced using the CRISPR / Cas9 genome editing system in two separate recombination steps. The first operon (lower pathway; operon 2) contained a spectinomycin (Spec) marker, which was used to screen for Spec-resistant candidate integrants. The second operon was designed to replace the Spec marker of the previously integrated operon, and after the second recombination event, Spec-resistant candidate integrants were screened (see Figure 1). A guide RNA expression vector targeting the araA gene was designed and synthesized using DNA2.0. To verify operon integration using PCR, PCR primers were designed to amplify the integration target of the araA gene and the recombination junction of the integrant. One clone yielding the correct PCR result was then fully sequenced and stored as strain DP1205.

[0430] Engineering recombinant bacterial cells to produce copalol An operon was constructed containing two cDNAs encoding: - AspWeTPP (SEQ ID NO: 170), a protein with terpenyl diphosphate phosphatase activity from Aspergillus wentii, capable of dephosphorylating terpenyl diphosphate compounds such as copalyl PP (GenBank accession number OJJ34585.1); and - PvCPS (SEQ ID NO: 173), a protein with prenyltransferase and copalyl diphosphate synthase activity from Talaromyces verruculosus (GenBank Accession No. BBF88128.1), which catalyzes the production of copalyl PP from IPP and DMAPP.

[0431] The cDNAs encoding AspWeTPP and PvCPS were codon-optimized (SEQ ID NOs: 171 and 174). An operon containing these two cDNAs and an RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 196) located upstream of the cDNA was designed. This operon was synthesized and cloned into the pJ401 expression plasmid (ATUM, Newark, CA) to obtain plasmid pJ401-CPOL-4.

[0432] By transforming E. coli cells, such as DP1205 E. coli, with this plasmid pJ401-CPOL-4, recombinant cells capable of producing copalol can be obtained when cultured under conditions that allow the production of terpene compounds.

[0433] Engineering recombinant bacterial cells for the production of coparal An operon was constructed containing three cDNAs encoding: - AspWeTPP (GenBank accession number OJJ34585.1), a protein with terpenyl diphosphate phosphatase activity from Aspergillus wentii (SEQ ID NO: 170), which has the ability to dephosphorylate terpenyl diphosphate compounds such as copalyl PP; AzTolADH1 (SEQ ID NO: 167), a protein with alcohol dehydrogenase (ADH) activity from Azoarcus toluclasticus, capable of oxidizing terpene alcohols, such as copalol, to the respective carbonyl compounds, such as copalal (GenBank accession number WP_018990713.1); and - PvCPS (SEQ ID NO: 173), a protein with prenyltransferase and copalyl diphosphate synthase activity from Talaromyces verruculosus, which has the ability to produce cyclic terpenyl diphosphate compounds, such as copalyl diphosphate, from IPP and DMAPP (GenBank Accession No. BBF88128.1).

[0434] The cDNAs encoding AspWeTPP, AzTolADH1, and PvCPS were codon-optimized (SEQ ID NOs: 171, 168, and 174). An operon containing these three cDNAs in succession and an RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 196) located upstream of each cDNA was designed. This operon was synthesized and cloned into the pJ401 expression plasmid (ATUM, Newark, CA) to obtain the plasmid pJ401-CPAL-1.

[0435] By transforming E. coli cells, such as DP1205 E. coli, with this plasmid pJ401-CPOL-4, recombinant cells capable of producing copalol can be obtained when cultured under conditions that allow the production of terpene compounds.

[0436] Engineering recombinant bacterial cells to produce farnesal An operon was constructed containing two cDNAs encoding: TalCeTPP (SEQ ID NO: 176), a protein with terpenyl diphosphate phosphatase activity from Talaromyces cellulolyticus, which has the ability to dephosphorylate terpenyl diphosphate compounds such as farnesyl diphosphate (GenBank: GAM42000.1); and - CdGeoA (NCBI accession number WP_043683915.1) (SEQ ID NO: 179), a protein with alcohol dehydrogenase (ADH) activity from Castellaniella defragrans, which has the ability to oxidize terpene alcohols, such as farnesol, to their respective carbonyl compounds, such as farnesal.

[0437] The cDNAs encoding TalCeTPP and CdGeoA were codon-optimized (SEQ ID NOs: 177 and 180). An operon was designed containing two cDNAs in succession, each with an RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 196) located upstream of the other cDNA. This operon was synthesized and cloned into the pJ401 expression plasmid (ATUM, Newark, CA) to obtain the plasmid pJ401-FAL-1.

[0438] By transforming E. coli cells, such as DP1205 E. coli, with this plasmid pJ401-FAL-1, recombinant cells capable of producing farnesal can be obtained when cultured under conditions that allow the production of terpene compounds.

[0439] Engineering recombinant bacterial cells to produce labdanediol An operon was constructed containing three cDNAs encoding: - TalVeTPP (Genbank accession number KUL89334.1) (SEQ ID NO: 194), a protein with terpenyl diphosphate phosphatase activity from Talaromyces verruculosus, which has the ability to dephosphorylate terpenyl diphosphate compounds such as labdanediol PP; - SsLPS (Genbank accession number AET21247.1) (SEQ ID NO: 188), a protein with labdanediol-phyllophosphate (LPP) synthase activity from Salvia sclarea, which has the ability to generate cyclic terpene diphosphate compounds such as labdanediol diphosphate from GGPP; and - CrtE, a geranylgeranyl diphosphate synthase from Pantoea agglomerans (GenBank accession number AAA24819.1) (SEQ ID NO: 191), which has the ability to produce GGPP from FPP.

[0440] The cDNAs encoding TalVeTPP, SsLPS, and CrtE were codon-optimized (SEQ ID NOs: 195, 189, and 192). An operon containing these three cDNAs in succession and an RBS sequence (AAGGAGGTAAAAAA) (SEQ ID NO: 196) located upstream of each cDNA was designed. This operon was synthesized and cloned into the pJ401 expression plasmid (ATUM, Newark, CA) to obtain the plasmid pJ401-LOH-2.

[0441] By transforming E. coli cells, such as DP1205 E. coli, with this plasmid pJ401-LOH-2, recombinant cells capable of producing labdenediol can be obtained when cultured under conditions that allow the production of terpene compounds.

[0442] Transformation, selection and cultivation of yeast cells All yeast transformations were performed using the lithium acetate protocol described by Gietz and Woods, Methods Enzymol., 2002, 350:87-96. The transformation mixture was plated on SmUra- or SmLeu-medium plates containing 6.7 g / L Yeast Nitrogen Base without amino acids (BD Difco, New Jersey, USA), 1.92 g / L Dropout Supplement without uracil (Sigma-Aldrich, Missouri, USA) or 1.6 g / L Dropout Supplement without leucine (Sigma-Aldrich, Missouri, USA), 20 g / L glucose, and 20 g / L agar. Plates were incubated at 30°C for 3–4 days.

[0443] Engineering yeast cells to increase endogenous farnesyl diphosphate levels To increase the endogenous farnesyl diphosphate (FPP) pool in S. cerevisiae cells, extra copies of the endogenous yeast genes involved in the mevalonate pathway, from ERG10, encoding acetyl-CoA C-acetyltransferase, to ERG20, encoding FPP synthase, were integrated into the genome of the S. cerevisiae strain CEN.PK2-1C (Euroscarf, Frankfurt, Germany) under the control of a galactose-inducible promoter, as described by Paddon et al., Nature, 2013, 496:528-532. Briefly, three cassettes were integrated into the LEU2, TRP1, and URA3 loci, respectively. The first cassette contained the genes ERG20 and truncated HMG1 (tHMG1, described in Donald et al., Proc Natl Acad Sci USA, 1997, 109:E111-8) under the control of the GAL10 / GAL1 bidirectional promoter, and the genes ERG19 and ERG13, also under the control of the GAL10 / GAL1 promoter. This cassette was flanked by two 100-nucleotide regions corresponding to the upstream and downstream regions of LEU2. The second cassette contained the genes IDI1 and tHMG1 under the control of the GAL10 / GAL1 promoter, and the gene ERG13 under the control of the GAL7 promoter region. This cassette was flanked by two 100-nucleotide regions corresponding to the upstream and downstream regions of TRP1. The third cassette contained the genes ERG10, ERG12, tHMG1, and ERG8, all under the control of the GAL10 / GAL1 promoter. This cassette was flanked by two 100-nucleotide regions corresponding to the upstream and downstream regions of URA3. All genes in the three cassettes contained their own 200-nucleotide terminator regions. An extra copy of GAL4 under the control of a mutated version of its own promoter was also integrated upstream of the ERG9 promoter region, as described by Griggs and Johnston, Proc Natl Acad Sci USA, 1991, 88:8597-8601. Furthermore, ERG9 expression was altered 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, designated YST045, which was induced for sporulation according to Solis-Escalante et al., FEMS Yeast Res, 2015, 15:2. Spore isolation was performed using 2 μL of zymolyase (1000 U mL). -1 This was achieved by resuspending the asci in 200 μL of 0.5 M sorbitol containing 1% ethanol (Zymo Research, Irvine, CA) and incubating at 37°C for 20 minutes. The asci were then plated onto a medium containing 20 g / L peptone, 10 g / L yeast extract, and 20 g / L agar, and a single germinated spore was isolated and designated YST075.

[0444] Engineering recombinant yeast cells for the production of copalol. For copalol production, expression of the GGPP synthase carG (from Blakeslea trispora, NCBI accession number JQ289995.1) (SEQ ID NO: 182), copalyl-pyrophosphate synthase SmCPS2 (from Salvia miltiorrhiza, NCBI accession number ABV57835.1) (SEQ ID NO: 185), and copalyl-pyrophosphate phosphatase TalVeTPP (from Talaromyces verruculosus, NCBI accession number KUL89334.1) (SEQ ID NO: 194) in different engineered yeast cells was achieved using a plasmid system 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 six DNA fragments that were used to cotransform Saccharomyces cerevisiae. The fragments were as follows: a) Primers using the plasmid pESC-LEU (Agilent Technologies, California, USA) as a template [ka] (SEQ ID NO: 124) and [ka] (SEQ ID NO: 125) The LUE2 yeast marker was constructed by PCR using b) Primers using the plasmid pESC-URA as a template [ka] (SEQ ID NO: 126) and [ka] (SEQ ID NO: 127) constructed by PCR using the Amp E. coli marker; c) Primers using pESC-URA as a template [ka] (SEQ ID NO: 128) and [ka] (SEQ ID NO: 129) yeast replication origin, obtained by PCR using; d) Primers using pESC-URA as a template [ka] (SEQ ID NO: 130) and [ka] (SEQ ID NO: 131) The E. coli origin of replication was obtained by PCR using; e) The last 60 nucleotides of fragment "d", 200 nucleotides downstream of the stop codon of the yeast gene PGK1, the GGPP synthase coding sequence carG, the bidirectional yeast promoter for GAL10 / GAL1, the coding sequence for TalVeTPP, 200 nucleotides downstream of the stop codon of the yeast gene CYC1 and the sequence [ka] (SEQ ID NO: 132), which fragment was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025); and f) A fragment consisting of the last 60 nucleotides of fragment "e", 200 nucleotides downstream of the stop codon of the yeast gene CYC1, the SmCPS2 copalyl pyrophosphate synthase coding sequence, the bidirectional yeast promoter GAL10 / GAL1, and 60 nucleotides corresponding to the start of fragment "a", this fragment was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025).

[0445] Optionally, the GGPP synthase carG and copalyl pyrophosphate synthase were replaced by the bifunctional PvCPS.

[0446] Engineering recombinant yeast cells for the production of manooloxy To degrade copalol to mannanoloxy, we integrated different alcohol dehydrogenases (ADHs), Baeyer-Villiger monooxygenases (BVMOs), and esterases (ESTs) into the genome of strain YST075. Each integrated cassette was composed of four fragments: 1) 658 bp corresponding to the upstream region of the NDT80 gene and sequence [ka] (SEQ ID NO: 121), which was obtained by PCR using the genomic DNA of the YST075 strain as a template; 2) Array [ka] (SEQ ID NO: 121), the CYC1 terminator region, one of the genes encoding BVMO, the intergenic region between the GAL1 gene and the GAL10 gene, one of the genes encoding esterase, the terminator region of the ADH1 gene and the sequence [ka] (SEQ ID NO: 122), which was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025); 3) Array [ka] (SEQ ID NO: 122), the PGK1 terminator region, one of the genes encoding alcohol dehydrogenase, the promoter regions of the GAL1 gene and the GAL10 gene, one of the genes encoding alcohol dehydrogenase, the CYC1 terminator region and sequence [ka] (SEQ ID NO: 123), which may contain one or two alcohol dehydrogenases depending on the experiment performed. They were obtained by DNA synthesis (ATUM, Menlo Park, CA 94025); and 4) Array [ka] (SEQ ID NO: 123), and a 405 bp fragment corresponding to the NDT80 gene, which was obtained by PCR using the genomic DNA of the YST075 strain as a template.

[0447] Engineering recombinant yeast cells to degrade copalol to manooloxy. To degrade copalol to mannooloxy, we integrated alcohol dehydrogenase and different enal-cleaving polypeptides into the genome of strain YST075. Each integrated cassette was composed of three fragments: 1) 658 bp corresponding to the upstream region of the NDT80 gene and sequence [ka] (SEQ ID NO: 121), which was obtained by PCR using the genomic DNA of the YST075 strain as a template; 2) Array [ka] (SEQ ID NO: 121), the intergenic region between the GAL1 and GAL10 genes, one of the genes encoding enal cleavage polypeptides, the terminator region of the ADH1 gene and the sequence [ka] (SEQ ID NO: 122), which fragment was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025); and 3) Array [ka] (SEQ ID NO: 122), the PGK1 terminator region, the promoter region of the gene encoding alcohol dehydrogenase, the GAL1 gene and the GAL10 gene, the sequence [ka] (SEQ ID NO: 123) and a 405 bp fragment corresponding to the NDT80 gene. This fragment was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025).

[0448] In both cases, copalol production was achieved by expressing the biosynthetic pathway in a plasmid system as described above.

[0449] Engineering recombinant yeast cells for the production of γ-ambryl acetate To degrade copalol to γ-ambryl acetate, we performed genome integration of strain YST075 using alcohol dehydrogenase, enal cleavage polypeptide, and different Baeyer-Villiger monooxygenases (BVMOs). Each integration cassette was formed with three fragments: 1) 658 bp corresponding to the upstream region of the NDT80 gene and sequence [ka] (SEQ ID NO: 121), which was obtained by PCR using the genomic DNA of the YST075 strain as a template; 2) Array [ka] (SEQ ID NO: 121), the terminator region of the CYC1 gene, one of the genes encoding the tested BVMOs, the intergenic region between the GAL1 and GAL10 genes, the gene encoding the enal cleavage polypeptide, the terminator region of the ADH1 gene and the sequence [ka] (SEQ ID NO: 122), which fragment was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025); and 3) Array [ka] (SEQ ID NO: 122), the PGK1 terminator region, the promoter region of the gene encoding alcohol dehydrogenase, the GAL1 gene and the GAL10 gene, the sequence [ka] (SEQ ID NO: 123) and a 405 bp fragment corresponding to the NDT80 gene. This fragment was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025).

[0450] In both cases, copalol production was achieved by expressing the biosynthetic pathway in a plasmid system as described above.

[0451] Engineering recombinant yeast cells for γ-ambrol production To degrade copalol to γ-ambrol, we performed genome integration of strain YST075 using alcohol dehydrogenase, enal cleavage polypeptide, Baeyer-Villiger monooxygenase (BVMO), and different esterases (ESTs). Each integration cassette was formed by four overlapping fragments: 1) A fragment containing at least 300 bp of upstream sequence of the BUD9 gene and at least 60 bp of overlapping sequence for in vivo assembly, obtained by PCR using the genomic DNA of the YST075 strain as a template; 2) A fragment containing the terminator region of the ADH1 gene, one of the genes encoding the tested esterases, and the intergenic region between the GAL1 and GAL10 genes, flanked by sequences that allow in vivo assembly. This fragment was obtained by DNA synthesis (ATUM, Menlo Park, CA 94025); 3) a fragment containing the URA3 yeast marker with its own promoter and terminator flanked by sequences allowing homologous recombination, this fragment being obtained by PCR; and 4) A fragment of at least 300 bp corresponding to the downstream region of the BUD9 gene and containing at least 60 bp of overlapping sequence that allows in vivo assembly. This fragment was obtained by PCR using the genomic DNA of the YST075 strain as a template.

[0452] In both cases, copalol production was achieved by expressing the biosynthetic pathway in a plasmid system as described above.

[0453] Cultivation of artificial yeast cells under conditions enabling the production of terpene compounds and GC-MS analysis The cells were cultured under conditions similar to those described in Westfall et al., Proc Natl Acad Sci USA, 2012, 109:E111-118, and the production of terpenes and their derivatives from the artificial yeast cells was evaluated using 10% dodecane or 10% isopropyl myristate (IPM) as an organic overlay. The culture was then extracted with two volumes of MTBE, and the composition of the organic phase was analyzed by GC-MS using an Agilent 7890A GC system equipped with a 5975C Series mass selective detector (MSD) and a split / splitless injector and a GC Injector 80 injection system (Agilent Technologies, CA). The GC inlet temperature was set to 260°C, and 1.0 μl of sample was injected in splitless mode and analyzed on an HP-5 GC column (30 m × 0.25 mm × 0.25 μm; Agilent J&W) at a constant flow rate of 1.2 mL / min using helium as the carrier gas. The initial oven temperature was set to 100°C and programmed to 300°C (10°C / min).

[0454] c) Example Example 1 In vivo conversion of ambryloxy to γ-ambryl acetate using BVMO Codon-optimized cDNAs encoding SCH23-BVMO1 (SEQ ID NO: 2) from Hyphozyma roseonigra, SCH24-BVMO1 (SEQ ID NO: 6) from Filobasidium magnum, and SCH46-BVMO1 (SEQ ID NO: 13) from Bensingtonia ciliata were synthesized and cloned into the pJ414 expression plasmid (ATUM, Newark, CA, USA), resulting in plasmids pJ414-SCH23-BVMO1, pJ414-SCH24-BVMO1, and pJ414-SCH46-BVMO1. These expression plasmids were transformed into KRX E. coli cells (Promega Corporation, Madison, WI, USA). Transformed cells were grown and used in the whole-cell bioconversion assay described above using mannooloxy as a substrate. A negative control consisting of cells transformed with the empty plasmid was also included. In the presence of SCH23-BVMO1, SCH24-BVMO1, or SCH46-BVMO1 recombinant proteins, the conversion of ambroxyloxy to γ-ambryl acetate was observed (Figure 2). No conversion was observed in the negative control. This experience indicates that SCH23-BVMO1, SCH24-BVMO1, or SCH46-BVMO1 can catalyze the following conversions: [ka]

[0455] These results indicate that SCH23-BVMO1, SCH24-BVMO1, and SCH46-BVMO1 catalyze the Baeyer-Villiger oxidation of mannooloxyl.

[0456] Example 2 In vivo conversion of coparal to compound 4 using BVMO Codon-optimized cDNAs encoding SCH23-BVMO1 (SEQ ID NO: 3) from Hyphozyma roseonigra, SCH24-BVMO1 (SEQ ID NO: 7) from Filobasidium magnum, and SCH46-BVMO1 (SEQ ID NO: 14) from Bensingtonia ciliata were synthesized and cloned into the pJ414 expression plasmid (ATUM, Newark, CA, USA), resulting in plasmids pJ414-SCH23-BVMO1, pJ414-SCH24-BVMO1, and pJ414-SCH46-BVMO1. These expression plasmids were transformed into KRX Escherichia coli cells (Promega Corporation, Madison, WI, USA). The cells were cultured and used in the whole-cell bioconversion assay described above, using a mixture of cis-copalal and trans-copalal as substrates. A negative control consisting of cells transformed with the empty plasmid was also included. In the presence of recombinant SCH23-BVMO1, SCH24-BVMO1, or SCH46-BVMO1 proteins, conversion of cis-coparal and trans-coparal was observed. GC-MS analysis of the bioconversion products after 42 h of incubation (Figure 3) shows the formation of four major products: two stereoisomers 3a and 3b and two stereoisomers 4a and 4b.

[0457] Time-point measurements of bioconversion show the formation of compounds 1a and 1b as intermediate products. Figure 4 compares GC-MS analysis of the conversion of cis-coparal and trans-coparal by SCH23-BVMO1 at different times. Similar product profile evolution is observed for SCH24-BVMO1 and SCH46-BVMO1. The sequential formation of these compounds indicates that trans-coparal and cis-coparal are converted to compounds 4a and 4b in several steps. Compounds 1a and 1b and compounds 4a and 4b are formate esters. Such functional groups can be formed from aldehyde compounds by Baeyer-Villiger monooxygenase. Therefore, the following reaction scheme, including enzymatic and non-enzymatic (chemical) reactions, can be depicted to explain the conversion of trans-coparal by SCH23-BVMO1, SCH24-BVMO1, or SCH46-BVMO1.

[0458] [ka]

[0459] In this scheme, the recombinant enzyme catalyzes two Baeyer-Villiger oxidations of two different aldehydes. First, the α,β-unsaturated aldehyde group of trans-coparal is oxidized by the recombinant enzyme in the first Baeyer-Villiger oxidation to form compound 1a. The enol formate functional group of compound 1a is unstable under the experimental conditions and is partially hydrolyzed to form compound 2a. This latter compound is rapidly converted to compounds 3a and 3b via keto-enol tautomerization and is therefore not detected by GC-MS analysis. Compounds 3a and 3b are substrates for the same enzyme, which catalyzes a second Baeyer-Villiger oxidation to form compounds 4a and 4b.

[0460] The reaction scheme below depicts a similar reaction for the conversion of cis-copalal with SCH23-BVMO1, SCH24-BVMO1, or SCH46-BVMO1.

[0461] [ka]

[0462] These results indicate that SCH23-BVMO1, SCH24-BVMO1, and SCH46-BVMO1 catalyze the Baeyer-Villiger oxidation of labdan aldehyde compounds.

[0463] Example 3 In vitro conversion of mannooloxyl using BVMO and esterase For this experiment, the following recombinant proteins were used: SCH23-BVMO1 (SEQ ID NO: 2) from Hyphozyma roseonigra, SCH24-BVMO1 (SEQ ID NO: 6) from Filobasidium magnum, SCH23-EST (SEQ ID NO: 20) from Hyphozyma roseonigra, and SCH24-EST (SEQ ID NO: 24) from Filobasidium magnum. Codon-optimized cDNAs encoding SCH23-BVMO1 (SEQ ID NO: 3) and SCH24-BVMO1 (SEQ ID NO: 7) were synthesized and cloned into the pJ414 expression plasmid (ATUM, Newark, CA), resulting in plasmids pJ414-SCH23-BVMO1 and pJ414-SCH24-BVMO1. Codon-optimized cDNAs encoding SCH23-EST (SEQ ID NO: 21) and SCH24-EST (SEQ ID NO: 25) were synthesized and cloned into the pJ431 expression plasmid (ATUM, Newark, CA) to generate plasmids pJ414-SCH23-EST and pJ414-SCH24-EST.

[0464] KRX Escherichia coli cells (Promega Corporation, Madison, WI, USA) were transformed with each of these expression plasmids. Transformed cells were grown, and cell-free lysates were prepared as described. In vitro enzyme assays were performed using either of these protein fractions, or a combination of two of these protein fractions. In vitro assay conditions were as described above, supplemented with 160 mg / L mannooloxy, 60 μM flavin adenine dinucleotide (FAD), and 500 μM reduced β-nicotinamide adenine dinucleotide phosphate (NADPH).

[0465] Using crude fractions containing the recombinant SCH23-BVMO1 and SCH24-BVMO1 proteins, the conversion of ambroxol to γ-ambroxol acetate was observed. No conversion was detected when using control lysates obtained from E. coli cells transformed with an empty plasmid (Figure 5). From these experiments, the following enzymatic reactions can be deduced:

[0466] [ka]

[0467] In vitro enzyme assays were also performed using protein fractions containing recombinant esterase enzymes and combinations of protein fractions containing recombinant BVMO and recombinant esterase enzymes. These assays were performed as described above using manooloxy as the substrate. GC-MS analysis of the formed products (Figures 6 and 7) shows the conversion of manooloxy to γ-ambryl acetate in the presence of the BVMO enzyme (SCH23-BVMO1 or SCH24-BVMO1), and further conversion of γ-ambryl acetate to γ-ambrool when the esterase enzyme (SCH23-EST or SCH24-EST) was present in the assay. When the esterase was used in the absence of BVMO, no substrate conversion was observed (Figures 6 and 7).

[0468] This experiment shows that in the presence of BVMO and esterase, manooloxy is converted to γ-ambrol according to the reaction scheme depicted below.

[0469] [ka]

[0470] Example 4 In vitro conversion of compounds 4a and 4b to compounds 5a and 5b using esterases Codon-optimized cDNAs encoding the SCH23-EST (SEQ ID NO: 21) from Hyphozyma roseonigra, the SCH24-EST (SEQ ID NO: 25) from Filobasidium magnum, and the SCH46-EST (SEQ ID NO: 32) from Bensingtonia ciliata were synthesized and cloned into the pJ414 expression plasmid (ATUM, Newark, CA) to generate plasmids pJ414-SCH23-EST1, pJ414-SCH24-EST1, and pJ414-SCH46-EST1. These expression plasmids were transformed into KRX E. coli cells (Promega Corporation, Madison, WI, USA). Transformed cells were grown, and cell-free lysates were prepared as described. These protein fractions were used in in vitro enzyme assays according to the conditions described above.

[0471] As shown in Figure 8, conversion of the two stereoisomers 4a and 4b to compounds 5a and 5b was observed using crude fractions containing recombinant SCH23-EST1, SCH24-EST1, and SCH25-EST1 proteins. In contrast, no conversion was detected when lysates containing recombinant BVMO enzymes were used (these proteins were therefore used as control reactions in this series of experiments). Under these conditions, the enzymatic activity of SCH23-EST1 and SCH25-EST1 was higher than that of SCH24-EST1.

[0472] From these experiments, the following enzymatic reactions can be deduced: [ka]

[0473] Example 5 In vitro conversion of coparal using BVMO and esterase For this experiment, the following recombinant proteins were used: SCH23-BVMO1 (SEQ ID NO: 2) from Hyphozyma roseonigra, SCH24-BVMO1 (SEQ ID NO: 6) from Filobasidium magnum, SCH25-BVMO1 (SEQ ID NO: 10) from Papiliotrema laurentii, SCH23-EST (SEQ ID NO: 20) from Hyphozyma roseonigra, SCH24-EST (SEQ ID NO: 24) from Filobasidium magnum, SCH25-EST (SEQ ID NO: 28) from Papiliotrema laurentii.

[0474] Codon-optimized cDNAs encoding SCH23-BVMO1 (SEQ ID NO:3), SCH24-BVMO1 (SEQ ID NO:7), and SCH25-BVMO1 (SEQ ID NO:11) were synthesized and cloned into the pJ414 expression plasmid (ATUM, Newark, CA) to generate plasmids pJ414-SCH23-BVMO1, pJ414-SCH24-BVMO1, and pJ414-SCH25-BVMO1. Codon-optimized cDNAs encoding SCH23-EST (SEQ ID NO:21), SCH24-EST (SEQ ID NO:25), and SCH25-EST (SEQ ID NO:29) were synthesized and cloned into the pJ431 expression plasmid (ATUM, Newark, CA) to generate plasmids pJ414-SCH23-EST and pJ414-SCH25-EST.

[0475] KRX E. coli cells (Promega Corporation, Madison, WI, USA) were transformed with these expression plasmids. Transformed cells were grown, and cell-free lysates were prepared as described. In vitro enzyme assays were performed using protein fractions containing the recombinant BVMO enzyme or the recombinant esterase enzyme, or by combining protein fractions containing the recombinant BVMO enzyme and the esterase enzyme. Assays were performed as described above, supplemented with a mixture of 320 mg / L cis-coparal and trans-coparal, 60 μM flavin adenine dinucleotide (FAD), and 500 μM reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) as substrates.

[0476] Figure 9 compares the products from the conversion of coparal in the presence of SCH23-BVMO1 alone and in combination with different esterase enzymes. In the presence of SCH23-BVMO1, the major products are formate compounds 1a, 1b, and 4a, 4b. When assays were performed in the presence of SCH23-EST or SCH25-EST, the major products of the conversion were compounds 5a and 5b, indicating that these two esterase enzymes can efficiently hydrolyze the formate intermediates produced by the BVMO enzymes. In the presence of SCH24-EST, hydrolysis of the same intermediates (1a, 1b, and 4a, 4b) was observed; however, with this enzyme, hydrolysis of intermediates 1a and 1b by SCH24-EST appeared to be more efficient than hydrolysis of intermediates 4a and 4b.

[0477] Similar conversion of cis- and trans-coparal was observed when SCH24-BVMO1 was combined with the esterases SCH23-EST or SCH24-EST (Figure 10). In control experiments, no conversion was observed when coparal was incubated with the esterases alone.

[0478] From these experiments, the following enzymatic pathway can be deduced:

[0479] [ka]

[0480] Example 6 In vivo production of 14,15-dinor-labdane compounds 5a and 5b and biosynthetic intermediates in engineered bacterial cells expressing BVMO and esterase In this experiment, E. coli cells were transformed with the plasmid pJ401-CPAL-1 (described previously) to produce copalal as described in the experimental section. When DP1205 E. coli was transformed and grown under the conditions described in the experimental section, the formation of trans-copalal and cis-copalal was confirmed (Figure 11, top chromatogram). The two double-bond isomers of copalal were detected because of the relative ease of isomerization of (E)-α,β-unsaturated aldehydes (Konning et al., Org. Lett., 2012, 14 (20), pp 5258-5261). The additional detection of labd-8(20)-en-15-ol was due to the endogenous enoate reductase activity of E. coli.

[0481] Bacterial cells were then transformed with a second expression plasmid carrying the codon-optimized cDNA encoding SCH24-BVMO1 (SEQ ID NO: 7) from Filobasidium magnum (ATCC® 20918™) or SCH46-BVMO1 (SEQ ID NO: 14) from Bensingtonia ciliata. These plasmids were prepared by cloning the optimized cDNA into the pJ423 expression plasmid (ATUM, Newark, CA), resulting in plasmids pJ423-SCH23-BVMO and pJ423-SCH46-BVMO, respectively. Cells transformed with the two plasmids were cultured and analyzed for production of terpene compounds and terpene derivatives using the conditions described in the Experimental Section. Under these conditions, compounds 1a and 1b, 3a and 3b, and 4a and 4b were detected in the solvent extract of the culture broth (Figure 11). These results demonstrate that the combination of these enzymes can be used to introduce into recombinant cells the biosynthesis of labdane diterpenes such as copalol and the sequential enzymatic cleavage of two carbon-carbon bonds in the side chain.

[0482] Similarly, bacterial cells were cotransformed with plasmid pJ401-CPAL-1 and a second plasmid carrying a gene encoding BVMO and a gene encoding an esterase: pJ423-SCH24-BVMO-SCH24-EST, which was prepared by inserting a synthetic operon consisting of the codon-optimized cDNA encoding SCH24-BVMO1 (SEQ ID NO: 7) and the codon-optimized cDNA encoding SCH24-EST (SEQ ID NO: 25) into the pJ423 expression plasmid (ATUM, Newark, CA); or pJ423-SCH46-BVMO-SCH46-EST, which was prepared by inserting a synthetic operon consisting of the codon-optimized cDNA encoding SCH46-BVMO (SEQ ID NO: 14) and the codon-optimized cDNA encoding SCH46-EST (SEQ ID NO: 32) into the pJ423 expression plasmid (ATUM, Newark, CA). The cells were cultured and analyzed for production of terpene compounds and terpene derivatives under the conditions described in the experimental section. Under these conditions, compounds 5a and 5b were detected, and the amounts of pathway intermediates (compounds 1a, 1b, 3a, 3b, 4a, and 4b) were confirmed to be reduced.

[0483] This series of experiments demonstrates that the following biosynthetic pathway can be introduced into host cells transformed to express diterpene biosynthetic enzymes in combination with BVMO and esterase.

[0484] [ka]

[0485] Example 7 : In vivo conversion of compounds 5a and 5b to manooloxy using alcohol dehydrogenase.

[0486] For this experiment, the following alcohol dehydrogenases were evaluated for the oxidation of compounds 5a and 5b to manooloxy: RrhSecADH from Rhodococcus rhodochrous (SEQ ID NO: 146), SCH80-00043 (SEQ ID NO: 149) from Rhodococcus rhodochrous; SCH80-04254 (SEQ ID NO: 152) from Rhodococcus rhodochrous; SCH80-06135 (SEQ ID NO: 155) from Rhodococcus rhodochrous; SCH80-06582 (SEQ ID NO: 158) from Rhodococcus rhodochrous. (See also WO 2005 / 026338; the ADHs listed above are merely non-limiting examples and other known ADHs may be substituted).

[0487] Codon-optimized cDNAs encoding each of these proteins were synthesized and cloned into the vector pJ401, resulting in plasmids pJ401-RrhSecADH, pJ401-SCH80-00043, pJ401-SCH80-04254, pJ401-SCH80-06135, and pJ401-SCH80-06582 (ATUM, Newark, CA).

[0488] These expression plasmids were transformed into KRX E. coli cells (Promega Corporation, Madison, WI, USA). Transformed cells were cultured and used in the whole-cell bioconversion assay described above, using a mixture of compounds 5a and 5b as substrate. Five hours after inducing recombinant protein expression, the substrate was added to a final concentration of 0.55 mg / mL using an emulsion of 50 mg / mL Tween 80 and 25 mg / mL substrate in water. A negative control consisting of cells transformed with an empty plasmid was also included. Oxidation reactions were observed only in the presence of recombinant proteins of SCH80-06135 and RrhSecADH (Figure 12), indicating that these enzymes can catalyze the following reactions:

[0489] [ka]

[0490] Example 8 : In vivo production of the tetranor-labdane compound γ-ambrol and biosynthetic intermediates in engineered bacterial cells expressing BVMO, esterase, and alcohol dehydrogenase.

[0491] In this experiment, the plasmid pJ401-CPAL-1 (described above) was used to transform DP1205 E. coli cells, which generated a background strain producing copral (cis- and trans-isomers) as described in the previous section.

[0492] This strain was then cotransformed with the plasmid pJ423-SCH24-BVMO-SCH24-EST (described previously) to further express BVMO and the esterase in the same cell. The recombinant organism produced 14,15-dinorlabdane compounds, similar to the observations made in the previous section.

[0493] To continue the degradation of the side chains to form the tetranorlabdane derivative, the secondary alcohol group of compounds 5a and 5b must be oxidized to the corresponding ketone. Therefore, a plasmid containing nucleotide sequences encoding BVMO, an esterase, and an appropriate alcohol dehydrogenase (identified in Example 7) was constructed. For the alcohol dehydrogenase, a codon-optimized cDNA encoding RrhSecADH from Rhodococcus sp. (Accession No. WP_043801412.1) (SEQ ID NO: 147) was synthesized, and a synthetic operon was designed combining the RrhSecADH cDNA with the cDNAs encoding SCH24-BVMO and SCH24-EST. This operon was cloned into the pJ423 expression plasmid, resulting in the pJ423-secADH-23BVMO-EST plasmid. When DP1205 E. coli cotransformed with vector pJ401-CPAL-1 and vector pJ423-secADH-23BVMO-EST was cultured under the above conditions, γ-ambrol was detected by GC-MS analysis of the culture broth (Figure 13). These data indicate that when compounds 5 (5a and 5b) are oxidized to mannooloxy in the presence of an appropriate ADH, BVMO can catalyze the following pathway step to provide γ-ambrol.

[0494] This series of experiments demonstrates that the following biosynthetic pathways can be introduced into recombinant host cells:

[0495] [ka]

[0496] Example 9 In vivo production of monooxygenases in budding yeast cells using alcohol dehydrogenase (ADH), Baeyer-Villiger monooxygenase (BVMO), and esterases (EST) from Hyphozyma roseonigra or Cryptococcus albidus For the production of mannooloxy, genes encoding GGPP synthase carG (from Blakeslea trispora, NCBI accession number JQ289995.1) (SEQ ID NO: 182), copalyl-pyrophosphate synthase SmCPS2 (from Salvia miltiorrhiza, NCBI accession number ABV57835.1) (SEQ ID NO: 185), copalyl-pyrophosphate phosphatase TalVeTPP (from Talaromyces verruculosus, NCBI accession number KUL89334.1) (SEQ ID NO: 194), and alcohol dehydrogenase SCH23-ADH1 (SEQ ID NO: 134), Baeyer-Villiger monooxygenase SCH23-BVMO1 (SEQ ID NO: 2), esterase SCH23-EST (SEQ ID NO: 20), and alcohol dehydrogenase SCH23-ADH2 (from Hyphozyma Genes encoding either the alcohol dehydrogenase SCH24-ADH1 (SEQ ID NO: 140), the Baeyer-Villiger monooxygenase SCH24-BVMO1 (SEQ ID NO: 6), the esterase SCH24-EST1 (SEQ ID NO: 24) and the alcohol dehydrogenase SCH24-ADH2 (from Filobasidium magnum) (SEQ ID NO: 143) were expressed in the engineered Saccharomyces cerevisiae strain YST075 as described in the General Methods section above. All genes were codon-optimized for their expression in S. cerevisiae (SCH23-ADH1, SEQ ID NO: 135; SCH23-BVMO1, SEQ ID NO: 4; SCH23-EST, SEQ ID NO: 22; SCH23-ADH2, SEQ ID NO: 138; SCH24-ADH1, SEQ ID NO: 141; SCH24-BVMO1, SEQ ID NO: 8; SCH24-EST, SEQ ID NO: 26; SCH24-ADH2, SEQ ID NO: 144; carG, SEQ ID NO: 183; SmCPS2, SEQ ID NO: 186; and TalVeTPP, SEQ ID NO: 195).

[0497] YST120 (containing SCH23-ADH1, SCH23-BVMO1, SCH23-EST, and SCH23-ADH2) and YST121 (containing SCH24-ADH1a, SCH24-BVMO1, SCH24-EST, and SCH24-ADH2), which also carry the plasmid system for copalol biosynthesis, were obtained and cultured under the conditions described in the General Methods section above.

[0498] Under these conditions, copalol was detected in all cultures. Only strains containing SCH23-ADH1 or SCH24-ADH1 were able to convert copalol to copalal (Figure 14A). Furthermore, farnesal was detected in the cultures expressing alcohol dehydrogenase (Figure 14B). Accumulation of nerolidol and farnesol was detected in all cultures (Figure 14A).

[0499] Furthermore, manooloxy was identified in cultures containing strains YST120 and YST121 carrying a plasmid carrying the copalol biosynthesis genes (Figure 14C). Neither γ-ambryl acetate nor γ-ambrol was identified. However, the presence of manooloxy suggests that the BVMO, EST, and ADH were functionally expressed in the engineered yeast cells. We speculate that the amount of manooloxy available may have been limiting for BVMO to catalyze its conversion to γ-ambryl acetate.

[0500] Example 10 In vivo production of monooxygenases in budding yeast cells using alcohol dehydrogenase (ADH), Baeyer-Villiger monooxygenase (BVMO), and esterases (EST) from Hyphozyma roseonigra or Cryptococcus albidus For the production of manooloxy, we used GGPP synthase carG (from Blakeslea trispora, NCBI accession number JQ289995.1), copalyl-pyrophosphate synthase SmCPS2 (from Salvia miltiorrhiza, NCBI accession number ABV57835.1), copalyl-pyrophosphate phosphatase TalVeTPP (from Talaromyces verruculosus, NCBI accession number KUL89334.1), alcohol dehydrogenase SCH23-ADH1, and Baeyer-Villiger monooxygenase SCH23-BVMO1 and esterase SCH23-EST (from Hyphozyma roseonigra) or Baeyer-Villiger monooxygenase SCH24-BVMO1 and esterase SCH24-EST (from Cryptococcus Genes encoding either the serovar 1 or serovar 2 (derived from T. albidus) were expressed in the artificial S. cerevisiae strain YST075 as described in General Methods.

[0501] The resulting strains, designated YST177 (containing carG, SmCPS2, TalVeTPP, SCH23-ADH1, SCH23-BVMO1, and SCH23-EST) and YST178 (containing carG, SmCPS2, TalVeTPP, SCH23-ADH1, SCH24-BVMO1, and SCH24-EST), were grown as described in the General Methods section above and analyzed by GC-MS as described above.

[0502] Coparol, copral, nerolidol, farnesol, and farnesal were identified in the culture after extraction. It was expected that engineered cells lacking the alcohol dehydrogenases SCH23-ADH2 or SCH24-ADH2 would accumulate intermediate 5a (or 5b) and would be unable to produce mano-ol-oxy. Interestingly, mano-ol-oxy was identified (Figure 15), but molecule 5a (or 5b) was not detected. These results suggest that SCH23-ADH2 and SCH24-ADH2 may contribute to the production of mano-ol-oxy in yeast cells, but are not essential for mano-ol-oxy production under the conditions tested. We speculate that endogenous alcohol dehydrogenase activity in yeast is involved in this conversion.

[0503] Example 11 Characterization of SCH94-3944, an enzyme from Rhodococcus erytheropolis with carbon-carbon bond cleavage activity In this experiment, the plasmid pJ401-CPOL-4 (described previously) was transformed into DP1205 E. coli cells to create a background strain that produced copalol. This transformed strain produced copalol as the major product in the culture medium at concentrations up to 500 mg / L in tube assays (Figure 16).

[0504] This strain was then further transformed with a second plasmid carrying one or more E. coli codon-optimized cDNAs from R. erytheropolis. Two cDNAs were selected: - SCH94-3945 (SEQ ID NO: 161), encoding a putative alcohol dehydrogenase, - SCH94-3944 (SEQ ID NO: 34), which encodes a 157 amino acid protein containing two protein family domains: the "GXWXG" protein domain (pfam14231, http: / / pfam.xfam.org / ) and the domain "DUF4334" of unknown function (pfam14232, http: / / pfam.xfam.org / ).

[0505] Expression vectors were prepared using pJ423 as the background, containing either a codon-optimized cDNA encoding SCH94-3945 (pJ423-SCH94-3945), a codon-optimized cDNA encoding SCH94-3944 (pJ423-SCH94-3944), or a bicistronic operon composed of optimized cDNAs encoding SCH94-3945 and SCH94-3944 (pJ423-SCH94-3944-3945).

[0506] When cells were transformed with vector pJ401-CPOL-4 and vector pJ423-SCH94-3944, no difference was observed compared to cells transformed with pJ401-CPOL-4 alone, indicating that the SCH94-3944 recombinant protein did not convert copalol.When cells were transformed with vector pJ401-CPOL-4 and vector pJ423-SCH94-3945, the formation of cis-copalal and trans-copalal was observed, indicating that SCH94-3945 is an alcohol dehydrogenase capable of oxidizing copalol to copalal (Figure 16).

[0507] When cells were transformed with vector pJ401-CPOL-4 and vector pJ423-SCH94-3944-3945, the formation of copalal was observed as the major product in tube assays at concentrations up to 1 g / L in the cultures, and under these assay conditions, the conversion of cis- and trans-copalal was nearly complete (Figure 16).

[0508] This experiment demonstrates that the SCH94-3944 enzyme can cleave the α-β carbon-carbon double bond of coparal and catalyze the direct conversion of cis-coparal and trans-coparal to the 14,15-dinor-labdane compound manooloxy, as shown in the following scheme:

[0509] [ka]

[0510] Example 12 In vivo conversion of cis- and trans-farnesal using an enal-cleaving polypeptide from Rhodococcus erythropolis In this experiment, plasmid pJ401-FAL-1 (described previously) was used to transform DP1205 E. coli cells to generate a background strain that produced cis- and trans-farnesal as major products at concentrations of up to 500 mg / L in culture under tube assay conditions (Figure 17).

[0511] This strain was then further transformed with the plasmid pJ423-SCH94-3944, which contains the cDNA encoding SCH94-3944 from R. erytheropolis. Analysis of the compounds produced by the cells by GC-MS showed the formation of geranylacetone (Figure 17). Therefore, this experiment demonstrates that the SCH94-3944 enzyme can cleave the α-β carbon-carbon double bond of the acyclic compound farnesal and catalyze the direct conversion of cis-farnesal and trans-farnesal to geranylacetone, as shown in the following scheme:

[0512] [ka]

[0513] Under the test conditions applied, no conversion to farnesol was observed.

[0514] Example 13 In vivo conversion of citral using an enal-cleaving polypeptide from Rhodococcus erythropolis Bioconversion of compounds was performed using E. coli KRX (Promega) cells transformed with the plasmid pJ423-SCH94-3944, thereby overexpressing the recombinant SCH94-3944 protein. Substrate was added to the cell culture at a final concentration of 12 g / L using a 2:1 substrate:Tween 80 emulsion. Bioconversion was performed as described in the experimental section. A negative control was performed using cells transformed with the pJ423 expression plasmid without an insert. Several substrates were tested: citral (a mixture composed of geranial and neral), citronellal (2,3-dihydrocitral), and (E)-2-dodecanal. Cells were incubated in the presence of various compounds for 24 h, and the conversion products were analyzed as described in the experimental section.

[0515] In the presence of SCH94-3944 recombinant protein, both geranial and neral were converted to methylheptenone (Figure 18), indicating that this enzyme can cleave the α-β carbon-carbon double bond of acyclic monoterpene aldehydes, as shown in the scheme below.

[0516] [ka]

[0517] formula [ka] of citronellal, no conversion was obtained in the presence of SCH94-3944 recombinant protein (Figure 18), indicating that unsaturation of the α,β-carbon bond is required for catalysis.

[0518] (E)-2-Dodecanal [ka] When SCH94-3944 was used, conversion to decanal was observed. However, the conversion yield was significantly lower than that of citral (Figure 18). This observation suggests that the absence of a 3-methyl group adversely affects the enzymatic conversion by the SCH94-3944 protein.

[0519] Example 14 In vivo conversion of coparal and farnesal using GXWXG and DUF4334 domain-containing proteins from other organisms The SCH94-3944 protein sequence contains the GXWXG protein family domain and the DUF4334 protein family domain. Proteins with similar domain structures were searched for in other organisms to test whether the enzymatic activity associated with SCH94-3944 could also be associated with these homologous enzymes.

[0520] In this experiment, DP1205 E. coli cells were transformed with the plasmid pJ401-CPAL-1 (described previously) to generate a background strain producing coparal (cis- and trans-isomers) as described in the previous section. In this strain, the FPP synthase is expressed from a genomic operon. Because the terpenyl phosphatase AspWeTPP can dephosphorylate FPP in addition to GGPP and AzeTolADH1 can also oxidize farnesol, large amounts of trans-farnesol were detected in addition to coparal when DP1205 cells were transformed with pJ401-CPAL-1 (Figure 19).

[0521] This strain was then co-transformed with a second plasmid carrying genes encoding proteins containing the GXWXG protein family domain and the DUF4334 protein family domain. Several proteins were selected: SCH80-05241 (SEQ ID NO: 38) from Rhodococcus rhodochrous (® ATCC 12674™), Pdigit7033 (SEQ ID NO: 42) from Penicillium digitatum, PitalDUF3443-1 (SEQ ID NO: 46) from Penicillium italicum, AspWeDUF3443 (SEQ ID NO: 49) from Aspergillus wentii, - RhoagDUF4334-2 (SEQ ID NO: 53) from Rhodococcus hoagii strain PAM2288, - RhoagDUF4334-3 (SEQ ID NO: 56) from Rhodococcus hoagii strain N128, - RhoagDUF4334-4 (SEQ ID NO: 59) from Rhodococcus hoagii NBRC 10125, CnecaDUF4334 (SEQ ID NO: 62) from Cupriavidus necator, - Rins-DUF4334 (SEQ ID NO: 69) from Ralstonia insidiosa, CgatDUF4334 (SEQ ID NO: 72) from Cryptococcus gattii EJB2, GclavDUF4334 (SEQ ID NO: 75) from Grosmannia clavigera kw1407, TcurvaDUF4334 (SEQ ID NO: 81) from Thermomonospora curvata, and - PprotDUF4334 (SEQ ID NO: 87) from Pseudomonas protegens.

[0522] Codon-optimized cDNAs encoding each of these proteins were designed and cloned into the pJ423 expression plasmid (ATUM, Newark, CA). DP1205 E. coli cells were cotransformed with one of these plasmids and the plasmid pJ401-CPAL-1. Figures 20 and 21 show the conversion of cis-copalal and trans-copalal to mannooloxy in the presence of recombinant proteins containing the GXWXG and DUF4334 domains, respectively. Under the assay conditions, copalal conversion was nearly complete with each recombinant enzyme, except for the GclavDUF4334 enzyme, where only minor conversion was observed. Figures 22 and 23 show the conversion of cis-farnesal and trans-farnesal to geranylacetone. Farnesal conversion was also complete with each enzyme, except for GclavDUF4334, where only approximately 50% of the farnesal was converted.

[0523] This experiment demonstrates that proteins containing a GXWXG protein family domain in their N-terminal region and a DUF4334 protein family domain in their C-terminal region can catalyze enal cleavage activity toward copalal and farnesal, as shown in the scheme below.

[0524] [ka]

[0525] Example 15 : Variants of SCH94-3944 with a single amino acid modification Alignment of the amino acid sequences of GXWXG- and DUF4334-domain-containing proteins with enal cleavage activity revealed conserved amino acids along the amino acid sequence and within the two protein domains (Figure 24). Conserved residues within a protein family are often important for enzymatic activity.

[0526] To assess the contribution of conserved residues in the enzymes containing the GXWXG and DUF4334 domains to enzymatic activity, we designed artificial mutants of the SCH94-3944 protein in which the conserved residues were individually substituted with alanine residues: W44, T51, H53, L59, W64, K67, S71, R106, Y115, D116, D122, M136, K139, F152, L154, and R156. The modified proteins were SCH94-3944-W44A, SCH94-3944-T51A, SCH94-3944-H53A, SCH94-3944-L59A, SCH94-3944-W64A, SCH94-3944-K67A, SCH94-3944-S71A, SCH94-3944-R106A, S They were designated CH94-3944-Y115A, SCH94-3944-D116A, SCH94-3944-D122A, SCH94-3944-M136A, SCH94-3944-K139A, SCH94-3944-F152A, SCH94-3944-L154A, and SCH94-3944-R156A.

[0527] Codon-optimized cDNAs encoding each of these proteins were designed and cloned into the pJ423 expression plasmid (ATUM, Newark, CA). DP1205 E. coli cells were cotransformed with one of these plasmids and the plasmid pJ401-CPAL-1. No conversion of coparal or farnesal was observed in the presence of recombinant proteins SCH94-3944-W44A, SCH94-3944-K67A, SCH94-3944-D122A, SCH94-3944-F152A, or SCH94-3944-L154A. Conversion of coparal and farnesal was observed with the SCH94-3944-T51A, SCH94-3944-H53A, SCH94-3944-L59A, SCH94-3944-W64A, SCH94-3944-S71, SCH94-3944-R106A, SCH94-3944-Y115A, SCH94-3944-D116A, SCH94-3944-M136A, SCH94-3944-K139A, and SCH94-3944-R156A enzymes, but at lower efficiency than the wild-type SCH94-3944 protein. Figure 25 shows the activity of each single amino acid variant enzyme relative to wild-type SCH94-3944.

[0528] Example 16 In vivo production of γ-ambryl acetate by combining enal cleavage activity and BVMO activity in Escherichia coli cells In this experiment, the plasmid pJ401-CPAL-1 (described previously) was used to transform DP1205 E. coli cells to generate a background strain producing copal (cis- and trans-isomers) as described above.

[0529] This strain was then cotransformed with a second plasmid carrying a codon-optimized nucleotide sequence encoding either an enzyme with enal cleavage activity or an enzyme with BVMO activity, or a second vector carrying an operon composed of a codon-optimized cDNA encoding an enal cleavage polypeptide and a codon-optimized cDNA encoding a BVMO: - pJ423-AspWeBVMO, which contains the optimized DNA sequence encoding AspWeBVMO (SEQ ID NO: 17); - pJ423-SCH94-3944, which contains the optimized DNA sequence encoding SCH94-3944 (SEQ ID NO: 35); - pJ423-SCH94-3944-SCH23-BVMO, which contains the optimized DNA sequences encoding SCH94-3944 and SCH23-BVMO1 (SEQ ID NOs: 35 and 3); - pJ423-SCH94-3944-SCH24-BVMO containing the optimized DNA sequences encoding SCH94-3944 and SCH23-BVMO1 (SEQ ID NOs: 35 and 7); - pJ423-SCH94-3944-SCH46-BVMO, which contains the optimized DNA sequences encoding SCH94-3944 and SCH46-BVMO1 (SEQ ID NOs: 35 and 14).

[0530] The transformed cells were cultivated and the formation of terpene derivatives was analyzed by GC-MS as described above.

[0531] When cells were transformed with vector pJ401-CPAL-1 and either the empty pJ423 vector or pJ423-AspWeBVMO, the formation of only cis-copalal and trans-copalal was observed (FIG. 26).

[0532] When cells were transformed with vectors pJ401-CPAL-1 and pJ423-SCH94-3944, the formation of mannooloxyl accompanied by complete conversion of copalal was observed (Figure 26). When cells were transformed with vector pJ401-CPAL-1 and vector pJ423, which coexpresses an enal-cleaving polypeptide and BVMO, the formation of γ-ambryl acetate in addition to mannooloxyl was observed. Different ratios of mannooloxyl to γ-ambryl acetate were observed depending on the BVMO enzyme.

[0533] This experiment demonstrates that γ-ambryl acetate can be produced by introducing the following pathway into host cells:

[0534] [ka]

[0535] Example 17 In vivo mannoaloxy production in budding yeast cells using SCH23-ADH1 and different enal-cleaving polypeptides from Hyphozyma roseonigra For the production of manooloxy, genes encoding the GGPP synthase carG (from Blakeslea trispora, NCBI accession number JQ289995.1), the copalyl-pyrophosphate synthase SmCPS2 (from Salvia miltiorrhiza, NCBI accession number ABV57835.1), the copalyl-pyrophosphate phosphatase TalVeTPP (from Talaromyces verruculosus, NCBI accession number KUL89334.1), the alcohol dehydrogenase SCH23-ADH1 (from Hyphozyma roseonigra), and one of the tested enal-cleaving polypeptides were expressed in the artificial Saccharomyces cerevisiae strain YST075 as described in the General Methods section. Five enal-cleaving polypeptides were evaluated: - AspWeDUF4334 (from Aspergillus wentii; GenBank accession number OJJ34591.1) (SEQ ID NO: 49). - CnecaDUF4334 (from Cupriavidus necator; GenBank accession number WP_049800708.1) (SEQ ID NO: 62). - Pdigit7033 (from Penicillium digitatum) (SEQ ID NO: 42). - SCH94-3944 (from Rhodococcus erytheropolis) (SEQ ID NO: 34). - SCH80-05241 (derived from Rhodococcus rhodochrous).

[0536] All genes were codon-optimized for their expression in S. cerevisiae (AspWeDUF4334, SEQ ID NO: 51; CnecaDUF4334, SEQ ID NO: 64; Pdigit7033, SEQ ID NO: 44; SCH94-03944, SEQ ID NO: 36; and SCH80-05241, SEQ ID NO: 40).

[0537] The constructed strains were designated YST184 (containing AspWeDUF4334), YST185 (containing CnecaDUF4334), YST186 (containing Pdigit7033), YST187 (containing SCH94-03944), and YST188 (containing SCH80-05241). These strains were cultured as described in the General Methods section above. The production levels of mannitol and other compounds were determined using GC-MS analysis.

[0538] Under the conditions tested, coparal, nerolidol, farnesal, geranylacetone, and manooloxy were identified in all cultures in which enal-cleaving polypeptides were expressed (Figure 27). As expected, all tested enal-cleaving polypeptides were able to produce geranylacetone and manooloxy using farnesal or coparal as substrates, respectively. In cultures YST184, YST185, YST186, YST187, and YST188, manooloxy accounted for 37%, 1%, 54%, 22%, and 52% of the total terpenes identified, respectively (Figure 28A).

[0539] Interestingly, in cultures of strains containing alcohol dehydrogenase and different enal-cleaving polypeptides, the total amount of identified terpenes was 2- to 4-fold higher than in control cultures (Figure 28B).

[0540] Example 18: In vivo production of γ-ambrylacetate in Saccharomyces cerevisiae cells using SCH23-ADH1 from Hyphozyma roseonigra, AspWeDUF4334 from Aspergillus wentii, and different Baeyer-Villiger monooxygenases (BVMOs) For the production of γ-ambrylacetate, GGPP synthase carG (from Blakeslea trispora, NCBI accession number JQ289995.1), copalyl-pyrophosphate synthase SmCPS2 (from Salvia miltiorrhiza, NCBI accession number ABV57835.1), copalyl-pyrophosphate phosphatase TalVeTPP (from Talaromyces verruculosus, NCBI accession number KUL89334.1), alcohol dehydrogenase SCH23-ADH1 (from Hyphozyma roseonigra), and enal cleavage polypeptide AspWeDUF4334 (from Aspergillus The gene encoding Saccharomyces cerevisiae (Saccharomyces cerevisiae) from Saccharomyces cerevisiae (Saccharomyces cerevisiae), derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae), and one of the tested Baeyer-Villiger monooxygenases (BVMOs), was expressed in the artificial Saccharomyces cerevisiae strain YST075 as described in General Methods.

[0541] Three BVMOs were evaluated: - SCH23-BVMO1 (from Hyphozyma roseonigra) (SEQ ID NO: 2). - SCH24-BVMO1 (from Filobasidum magnum) (SEQ ID NO: 6). - AspWeBVMO (from Aspergillus wentii; GenBank accession number OJJ34587.1) (SEQ ID NO: 16).

[0542] All genes were codon-optimized for their expression in S. cerevisiae (SCH23-BVMO1, SEQ ID NO: 4; SCH24-BVMO1, SEQ ID NO: 8; and AspWeBVMO, SEQ ID NO: 18).

[0543] The resulting strains were designated YST190 (containing SCH23-BVMO1), YST191 (containing SCH24-BVMO1), and YST192 (containing AspWeBVMO). These strains were cultured as described in the General Methods section above, and the amounts of mannooloxy and other compounds produced were identified by GC-MS analysis.

[0544] Under the conditions tested, copalol, nerolidol, farnesol, geranylacetone, mannooloxy, and γ-ambryl acetate were identified in all cultures (Figure 29). Interestingly, unlike previous experiments, the conversion of copalol to copalal was not complete. Furthermore, the total amount of terpenes produced was lower compared to strains without BVMO (Figure 30A). In cultures YST190, YST191, and YST192, γ-ambryl acetate accounted for 37%, 27%, and 20% of the total terpenes identified, respectively (Figure 30B).

[0545] Example 19 : Labdanediol biosynthetic pathway and in vivo production of sclareol oxide using carbon-carbon bond enal cleavage polypeptides In this experiment, plasmid pJ401-LOH-2 (described previously) was used to transform DP1205 E. coli cells to generate a background strain producing labdanediol ((13E)-13-labdane-8,15-diol) as described above.

[0546] This strain was then co-transformed with a second plasmid carrying a codon-optimized nucleotide sequence encoding an enzyme with alcohol dehydrogenase and enal cleaving polypeptide activity: pJ423-AzetolADH1, which contains an optimized DNA sequence encoding the alcohol dehydrogenase AzetolADH1; and - pJ423-SCH94-3944-3945, which contains the optimized DNA sequences encoding the alcohol dehydrogenase SCH94-3944 and the enal cleavage polypeptide SCH94-3945.

[0547] The transformed cells were cultivated and the formation of terpene derivatives was analyzed by GC-MS as described above.

[0548] When cells were transformed with the pJ401-LOH-2 vector and the empty pJ423 vector, the formation of labdanediol was observed (FIG. 31).

[0549] When cells were transformed with the pJ401-LOH-2 vector and pJ423-AzetolADH1, which coexpresses alcohol dehydrogenase, the formation of two new products was confirmed (Figure 31). NMR analysis revealed that the two compounds were two isomers of (+)-8,13-epoxy-labdan-15-al (compounds 7a and 7b), as shown in the following scheme. These two compounds arise from the instability of 8-hydroxy-labd-13-en-15-al (6), generated by oxidation of labdanediol. The proposed mechanism for the dehydration and rearrangement of compound 6 to compounds 7a and 7b is shown in the following scheme.

[0550] When cells were transformed with the pJ401-LOH-2 vector and pJ423-SCH9...

Claims

1. General formula IV 【Chemistry 1】 [In the formula, R １ represents H or lower alkyl; R ２ represents H, a linear or branched, saturated or unsaturated, substituted or unsubstituted hydrocarbyl residue, or the residue Cyc-A-, Where: Cyc represents a substituted or unsubstituted, saturated or unsaturated, monocyclic or polycyclic hydrocarbyl residue, A represents a chemical bond or a substituted or unsubstituted, linear or branched alkylene bridge; R ３ are each independently H or lower alkyl, the method comprising the steps of: (1) General formula V 【Chemistry 2】 [In the formula, R １ , R ２ and R ３ is as defined above, R ４ represents H or lower alkyl; R ５ represents H or lower alkyl. the corresponding undecomposed precursor of contacting a polypeptide having enal cleavage activity, wherein said compound of formula V may be present in essentially stereoisomerically pure form or as a mixture of stereoisomers, The group of polypeptides, wherein the polypeptide having enal cleavage activity comprises an amino acid sequence selected from the following: a) SCH94-3944 as depicted in SEQ ID NO: 34; b) SCH80-05241 as set forth in SEQ ID NO: 38; c) Pdigit7033 as set forth in SEQ ID NO:42; d) PitalDUF4334-1 as set forth in SEQ ID NO: 46; e) AspWeDUF4334 as set forth in SEQ ID NO: 49; f) RhoagDUF4334-2 as set forth in SEQ ID NO:53; g) RhoagDUF4334-3 as set forth in SEQ ID NO:56; h) RhoagDUF4334-4 as set forth in SEQ ID NO:59; i) CnecaDUF4334 as set forth in SEQ ID NO:62; j) Rins-DUF4334 as set forth in SEQ ID NO: 69; k) CgatDUF4334 as set forth in SEQ ID NO: 72; m) TcurvaDUF4334 as set forth in SEQ ID NO: 81; n) PprotDUF4334 as set forth in SEQ ID NO: 87; and o) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with any one of the amino acid sequences of a) to k), m) and n), and retaining the enzyme activity of degrading the terpene precursor of formula V. The method comprises the steps selected from the following:

2. The method of claim 1, further comprising, as step (2), isolating the compound of formula IV obtained in step (1), wherein the compound of formula IV is obtained in stereoisomerically pure form or as a mixture of stereoisomers.

3. Applying a terpene precursor of formula V, Where: R １ represents H or methyl, R ２ is H or an acyclic, linear or branched, saturated or unsaturated hydrocarbyl residue having 1 to 20 carbon atoms; or The cyclic group Cyc-A-, Where: A is a linear or branched C １ ~C ４ -alkylene bridge, Cyc is a monocyclic or polycyclic, saturated or unsaturated hydrocarbyl residue, optionally substituted with 1 to 10 substituents, the substituents being C １ ~C ４ -Alkyl, C １ ~C ４ -Alkylidene, C ２ ~C ４ - independently selected from alkenyl, oxo, hydroxy, or amino; Each R ３ represents H R ４ represents H or methyl, R ５ represents H or methyl, 3. The method according to claim 1 or 2.

4. said compound of general formula IV has a labdan type structure and / or Cyc-A has the formula IIIa, IIIb or IIIc 【Chemistry 3】 The method according to any one of claims 1 to 3, wherein the amino acid sequence represents one of the residues:

5. 5. The method of any one of claims 1 to 4, further comprising, as step (3), treating the compound of formula IV formed in step (1) or isolated in step (2) to obtain a derivative thereof using chemical synthesis or biocatalytic synthesis or a combination of both, wherein the derivative may be selected from a hydrocarbon, an alcohol, a diol, a triol, an acetal, a ketal, an aldehyde, an acid, an ether, an amide, a ketone, a lactone, an epoxide, an acetate, a glycoside and / or an ester.

6. Step (3) comprises treating said compound of formula IV formed in step (1) or isolated in step (2) with a polypeptide having Baeyer-Villiger monooxygenase (BVMO) activity (EC.1.13.14.-) to form the respective carbonyl ester compound, wherein said polypeptide having Baeyer-Villiger monooxygenase (BVMO) activity (EC.1.13.14.-) is selected from the group of polypeptides consisting of: (a) a polypeptide comprising the amino acid sequence of SCH23-BVMO1 set forth in SEQ ID NO:2; (b) a polypeptide comprising the amino acid sequence of SCH24-BVMO1 set forth in SEQ ID NO:6; (d) a polypeptide comprising the amino acid sequence of SCH46-BVMO1 set forth in SEQ ID NO: 13; and (f) a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of any one of (a), (b) and (d) and which retains Baeyer-Villiger monooxygenase (BVMO) activity that converts the compound of formula IV to its respective carbonyl ester compound.

7. The method of claim 6, further comprising hydrolyzing the carbonyl ester compound with an esterase (EC 3.1.1) in step (3) to obtain a corresponding de-esterified product.

8. The method of claim 6 or 7, further comprising, as step (3), treating the carbonyl ester compound and / or the corresponding deesterification product to obtain a derivative thereof using chemical synthesis or biocatalytic synthesis, or a combination of both, wherein the derivative may be selected from hydrocarbons, alcohols, diols, triols, acetals, ketals, aldehydes, acids, ethers, amides, ketones, lactones, epoxides, acetates, glycosides and / or esters.

9. 1. An in vivo method for preparing labdane-type terpenes comprising: The method comprises the following series of reaction steps: (1) converting labdan alcohols to their respective labdan aldehydes by the enzymatic action of an ADH polypeptide; (2) converting the labdane aldehyde of step (1) into the respective dinorlabdane carbonyl compound by the method according to any one of claims 1 to 4; (3) converting the dinorlabdane carbonyl compounds of step (2) to their respective tetranorlabdanyl acetates by the method according to any one of claims 5 to 8; (4) converting the tetranorlabdanyl acetate of step (3) into the respective tetranorlabdanyl alcohol by the action of a polypeptide having esterase activity; and (5) isolating the tetranorlabdanyl alcohol of step (4). The method comprises providing a recombinant host expressing a set of polypeptides having the enzymatic activities necessary to catalyze

10. 1. A method for preparing an epoxy-tetranorlabdane compound, the method comprising: (1) providing tetranorlabdane alcohol or tetranorlabdane acetate, or a dinorlabdane carbonyl compound by applying a biocatalytic process comprising one or more steps according to any one of claims 1 to 8; (2) converting the tetranorlabdane alcohol or tetranorlabdane acetate, or dinorlabdane carbonyl compound of step (1) into an epoxy-tetranorlabdane by applying one or more chemical and / or biochemical conversion steps; A method comprising:

11. 1. A method for preparing a diepoxy-dinorlabdane, the method comprising: (1) Providing a dinorlabdane carbonyl compound by applying a method comprising one or more steps according to any one of claims 1 to 9; (2) converting said dinorlabdane carbonyl compound to said diepoxy-dinorlabdane by applying one or more chemical and / or biochemical conversion steps; A method comprising:

12. 1. A method for preparing an epoxy-tetranorlabdane compound, the method comprising: (1) providing tetranorlabdane alcohol, by applying a biocatalytic method comprising one or more steps according to any one of claims 1 to 10; (2) converting the tetranorlabdane alcohol of step (1) into epoxy-tetranorlabdane by applying one or more chemical and / or biochemical conversion steps; A method comprising:

13. A method for preparing a diepoxy-dinorlabdane, the method comprising the steps of: (1) providing a dinorlabdane carbonyl compound by applying a method resulting in the formation of said dinorlabdane carbonyl compound, the method comprising one or more steps according to any one of claims 1 to 10; (2) converting the dinorlabdane carbonyl compound of step (1) into the diepoxy-dinorlabdane by applying one or more chemical and / or biochemical conversion steps; A method comprising:

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