Method for producing zizyphus compound

Abstract

The present application relates to a method for producing succinyl sesquiterpenes such as succinyl alcohol compounds and / or their derivatives by contacting at least one polypeptide with farnesyl diphosphate (FPP) and a polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily, which can be obtained from plants of the genus Dryopteris, in particular plants of the species Dryopteris fragrans. The method can be carried out in vitro or in vivo. Also provided are the amino acid sequences of polypeptides useful in the method and nucleic acids encoding the polypeptides. The present invention further provides host cells or organisms, which are genetically modified to express the polypeptides and can be used to produce succinyl sesquiterpenes such as succinyl alcohol compounds.

Description

Technical Field

[0001] Provided herein are biochemical methods for producing albicanol and related compounds, such as phosphorylated albicanol compounds and derivatives thereof, which methods comprise the use of novel polypeptides having albicanyl diphosphate synthase activity. Background Art

[0002] Terpenes are present in most organisms (microorganisms, animals and plants). These compounds are composed of a five-carbon unit called an isoprene unit and are classified by the number of these units present in their structure. Therefore, monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms respectively. For example, sesquiterpenes are widely present in the plant kingdom. Many sesquiterpene molecules are well known because of their flavor and aromatic properties and their beauty, medical and antibacterial effects. Many sesquiterpene hydrocarbons and sesquiterpene compounds have been identified. Chemical synthesis approaches have been developed, but are still very complicated and not always cost-effective.

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

[0004] Many major sources of sesquiterpenes, such as compounds with a drimane structure, such as drimane compounds or drimane compounds, are plants that naturally contain sesquiterpenes. However, the content of sesquiterpenes in these natural sources may be low. There is still a need to discover new terpene synthases and more economical and efficient methods to produce sesquiterpenes, such as drimane compounds, which are potential basic materials for preparing high-value flavor ingredients (such as Ambrox). Summary of the invention

[0005] The above problems can be solved by providing a novel class of enzymes that exhibit pyrocatechol diphosphate synthase activity and produce pyrocatechol diphosphate with unexpectedly high selectivity from FPP. In particular, a novel pyrocatechol diphosphate synthase gene and two natural variants thereof were identified from the fern Dryopteris fragrans.

[0006] The novel synthase (DfHAD) and its variants have only ≤23% homology with the fungal synthases CvTPS1 and LoTPS1 or other bifunctional terpene synthases, the latter as previously determined by the applicant and described in EP application number 17174399.0 (see also the comparison of Figure 9). However, more importantly, the synthase of the present invention also contains class I motifs and class II motifs. The motifs are characteristic of terpene synthases and are one of the basic characteristics of the hyssop synthase described in EP application number 17174399.0. Class I is mainly found in monoterpene or sesquiterpene synthases, while Class II is mainly found in diterpene synthases.

[0007] Since the fern DfHADs of the present invention contain a slightly modified class I motif and a slightly modified class II motif, they can be further distinguished from the earlier described fungal pyrocalyx synthases.

[0008] The pyrrolidone diphosphate synthase and variants thereof of the present invention may also be considered as "haloacid-dehalogenase-like hydrolases" ("HAD-like hydrolases") for the following reasons:

[0009] In the Pfam search, a haloacid dehalogenase-like hydrolase domain (HAD_2, PF13419.5) ("Domain 1") at positions 86 to 195 (of SEQ ID NO: 2) and a haloacid dehalogenase-like hydrolase domain (PF00702.25) ("Domain 3") at positions 40 to 187 (of SEQ ID NO: 2) with a domain E value of less than 0.005 can be identified. These HAD-like hydrolase domains are found in an enzyme superfamily that includes enzymes with phosphatase activity. These domains can be found in the N-terminal half of DfHAD.

[0010] By Pfam search analysis, the highest identity found in NCBI using blastp was 29% (99% coverage) to a protein belonging to fungi that contained a putative HAD-like domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : The structures of buburane, (+)-buburane and (-)-buburane.

[0012] Figure 2 : GC / MS chromatogram of a dichloromethane extract obtained from the leaves of Dryopteris fragrans (only the region showing sesquiterpenes). The arrow indicates the pyrocalyx alcohol peak.

[0013] Figure 3 : Figure 2 Mass spectrum of the fennel peak in

[0014] Figure 4 : GC / MS chromatogram of the broth extract of E. coli (expressing DfHAD) (only the region showing sesquiterpenes). The arrow indicates the fennel peak.

[0015] Figure 5 : Figure 4 Mass spectrum of the zygomorphol peak in .

[0016] Figure 6 : GC / MS chromatogram of dichloromethane extract of transfected tobacco leaves (only the region showing sesquiterpenes). The arrow indicates the peak of fennel.

[0017] Figure 7 : Figure 6 Mass spectrum of the fennel peak in

[0018] Figure 8 : GC chromatogram of samples from in vitro biochemical assay of DfHAD

[0019] FIG9 : Alignment of amino acid sequences of putative terpene synthases containing class I and class II motifs: DfHAD of the present invention (SEQ ID NO: 2) DfHAD-9 (V274A) (SEQ ID NO: 6) DfHAD-8 (K532R) (SEQ ID NO: 7) NO:4); CvTps1, LoTps1, OCH93767.1, EMD37666.1, EMD37666-B, XP_001217376.1, OJJ98394.1, GAO87501.1, XP_008034151.1, XP_007369631.1, ACg006372, KIA75676.1, XP_001820867.2, CEN60542.1, XP_009547469.1, KLO09124.1 and OJI95797.1 as previously described. The solid line is Pfam domain 1 and the dotted line is Pfam domain 3. The boxes are modified class I and class II motifs and other motifs III and IV.

[0020] Fig.10 : List of engineered mutations in the class I and class II motifs of DfHAD.

[0021] Fig.11 : Chromatograms of sesquiterpene diphosphate (upper) and monophosphate 301.1574 (lower) of extracted ions 381.1237 from E. coli extract.

[0022] Fig.12 : Chromatograms of sesquiterpene diphosphate (upper) and monophosphate 301.1574 (lower) of extracted ions 381.1237 from a purified DfHAD sample mixed with FPP. DETAILED DESCRIPTION

[0023] Abbreviations used

[0024] bp base pair

[0025] BAP bacterial alkaline phosphatase

[0026] kb kilobase

[0027] DNA deoxyribonucleic acid

[0028] cDNA complementary DNA

[0029] DTT Dithiothreitol

[0030] FPP Farnesyl diphosphate

[0031] GC Gas Chromatography

[0032] HAD Haloacid Dehalogenase

[0033] MS Mass Spectrometer / Mass Spectrometry

[0034] MVA Mevalonic Acid

[0035] PCR polymerase chain reaction

[0036] RNA Ribonucleic acid

[0037] mRNA messenger RNA

[0038] miRNA

[0039] siRNA Small interfering RNA

[0040] rRNA ribosomal RNA

[0041] tRNA transfer RNA

[0042] definition

[0043] For the purpose of this application, "fennel" relates to (+)-fennel (CAS: 54632-04-1).

[0044] For the purposes of this application, "buminol" relates to (-)-buminol (CAS: 468-68-8).

[0045] For the purposes of this application, "Ambrox" relates to the IUPAC name: (-)-(3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan (CAS: 6790-58-5).

[0046] The term "bifunctional terpene synthase" or "polypeptide having bifunctional terpene synthase activity" relates to a polypeptide comprising class I and class II synthase motifs and having bifunctional terpene activity of protonation-initiated cyclization and ionization-initiated cyclization catalytic activity. (J. Schrader & J. Bohlman. Biotechnology of isoprenoids. Adv. Biochem. Eng. Biot., 148, 1-475. (DOI: 10.1007 / 978-3-319-20107-8)) Bifunctional terpene synthase belongs to the haloacid dehalogenase-like (HAD-like) hydrolase superfamily, which comprises domains corresponding to Pfam domains PF13419.5 (domain 1) and PF00702.25 (domain 3).

[0047] The term "terpenoid sesquiterpenes" relates to terpenoids having Figure 1 The terpenes with the thiazane-like carbon skeleton structure shown.

[0048] The term "class I synthase" relates to terpene synthases which catalyse reactions initiated by ionisation, such as monoterpene and sesquiterpene synthases.

[0049] The term "class I synthase motif" relates to the active site of a terpene synthase comprising a conserved DDxx(D / E) motif. The aspartate residue of the class I motif binds a divalent metal ion (most commonly Mg) such as in association with the binding of a diphosphate group. 2+ ), and catalyzes the ionization and cleavage of the allyl diphosphate bond of the substrate.

[0050] The term "modified class I synthase motif" relates to the active site of a terpene synthase comprising the conserved DSFDxx(D / E) (SEQ ID NO: 11), in particular DSFDSLE (SEQ ID NO: 13) motif.

[0051] The term "class II synthase" relates to a terpene synthase that catalyzes a cyclization reaction initiated by protonation, such as is typically involved in the biosynthesis of triterpenes and labdane diterpenes. In class II terpene synthases, the reaction initiated by protonation may involve, for example, an acidic amino acid donating a proton to a terminal double bond.

[0052] The term "class II synthase motif" relates to the active site of a terpene synthase comprising the conserved DxDD or DxD(T / S)T motif.

[0053] The term "modified class II synthase motif" relates to the active site of a terpene synthase comprising the conserved HDxD(T / S) (SEQ ID NO: 12), in particular the HDLDT (SEQ ID NO: 14) motif or the motif DLDTTS (SEQ ID NO: 23).

[0054] The term "fenacetyl diphosphate synthase" or "polypeptide having fenacetyl diphosphate synthase activity" or "fenacetyl diphosphate synthase protein" or "having the ability to produce fenacetyl diphosphate" relates to a polypeptide capable of catalyzing the synthesis of fenacetyl diphosphate in the form of any stereoisomer or a mixture thereof starting from an acyclic terpene pyrophosphate, in particular farnesyl diphosphate (FPP). Ffenacetyl diphosphate may be the sole product or may be part of a sesquiterpene mixture. The mixture may contain fenacetyl monophosphate and / or fenacetol in any proportion, in particular in a major proportion, and more particularly in a substantial proportion as defined below.

[0055] As described below, "Folding pyridine diphosphate synthase activity" is determined under "standard conditions": they can be determined using recombinant pyridine diphosphate synthase expressing host cells, disrupted pyridine diphosphate synthase expressing cells, their fractions or enriched or purified pyridine diphosphate synthase enzymes, at a temperature of about 20 to 45°C, such as about 25 to 40°C, preferably 25 to 32°C, in a culture medium or reaction medium (preferably buffered) with a pH value of 6 to 11, preferably 7 to 9, and in the presence of a reference substrate (especially FPP), added at an initial concentration of 1 to 100 μM, preferably 5 to 50 μM, especially 30 to 40 μM, or produced endogenously by the cell host. The conversion reaction to form pyridine diphosphate is carried out for 10 minutes to 5 hours, preferably about 1 to 2 hours. If there is no endogenous alkaline phosphatase, one or more exogenous phosphatases are added to the reaction mixture to convert the pyridine diphosphate formed by the synthase. Zingiol can then be determined by conventional methods, for example after extraction with an organic solvent such as ethyl acetate. Specific examples of suitable standard conditions are applied in the experimental part below, such as in Example 5, which conditions should also form part of the general disclosure of the present invention.

[0056] The terms "biological function", "function", "biological activity" or "activity" refer to the ability of a terpene synthase as described herein to catalyze the formation of: fennel diphosphate and / or fennel alcohol or a mixture of compounds comprising fennel diphosphate, and / or fennel monophosphate and / or fennel alcohol and / or one or more other terpenes, in particular fennel diphosphate.

[0057] The term "phosphatase" or "polypeptide with phosphatase activity" relates to a polypeptide that can catalyze the cleavage of an ester of phosphoric acid into a phosphate radical and a corresponding alcohol. Depending on the respective optimal pH, the enzyme can be an alkaline phosphatase (EC 3.1.3.1) or an acid phosphatase (EC 3.1.3.2). In particular, according to the present invention, alkaline phosphatases of different sources, more particularly bacterial origin, can be used. The phosphatase used should be able to produce an alcohol of thiamethoxam from the corresponding monophosphate or diphosphate, such as thiamethoxam and / or thiamethoxam.

[0058] The term "mixture of terpenes" or "mixture of sesquiterpenes" refers to a mixture of terpenes or sesquiterpenes which comprises fennel diphosphate and / or fennel monophosphate and / or fennel alcohol and may further comprise one or more additional terpenes or one or more additional sesquiterpenes.

[0059] The "mevalonate pathway" (also known as the "isoprene pathway" or "HMG-CoA reductase pathway") is an essential metabolic pathway in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five-carbon building blocks called 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 pyrophosphate isomerase (idi). Combining the mevalonate pathway with enzyme activities to produce the terpene precursors GPP, FPP, or GGPP, particularly FPP synthase (ERG20), allows recombinant cells to produce terpenes.

[0060] As used herein, the term "host cell" or "transformed cell" refers to a cell (or organism) that has been altered to harbor at least one nucleic acid molecule, for example, a recombinant gene encoding a desired protein or nucleic acid sequence, which, when transcribed, produces a functional polypeptide of the present invention, i.e., a fennel diphosphate synthase protein, which can be used to produce fennel diphosphate and / or fennel monophosphate and / or fennel alcohol or a corresponding terpene mixture containing fennel diphosphate and / or fennel monophosphate and / or fennel alcohol. The host cell is particularly a bacterial cell, a fungal cell or a plant cell or plant. The host cell may contain a recombinant gene that has been integrated into the nuclear or organelle genome of the host cell. Alternatively, the host may contain the recombinant gene outside the chromosome.

[0061] The term "organism" refers to any non-human multicellular or unicellular organism, such as a plant or a microorganism. In particular, the microorganism is a bacterium, a yeast, an algae or a fungus.

[0062] The term "plant" is used interchangeably to include plant cells, including plant protoplasts, plant tissues, plant cell tissue cultures that produce regenerated plants or plant parts, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits, etc. Any plant can be used to practice the methods of the embodiments herein.

[0063] "Capable of producing FPP" is meant when a particular organism or cell naturally produces FPP or when it does not naturally produce FPP but is transformed with a nucleic acid as described herein to produce FPP. An organism or cell transformed to produce a higher amount of FPP than the naturally occurring organism or cell is also encompassed as "an organism or cell capable of producing FPP".

[0064] A particular organism or cell is "capable of producing fennel diphosphate" when it naturally produces fennel diphosphate, or when it does not naturally produce fennel diphosphate but is transformed with a nucleic acid described herein to produce fennel diphosphate. An organism or cell that has been transformed to produce a higher amount of fennel diphosphate than the naturally occurring organism or cell is also encompassed as an "organism or cell capable of producing fennel diphosphate."

[0065] When a particular organism or cell naturally produces zirconia, or when it does not naturally produce zirconia but is transformed with a nucleic acid described herein to produce zirconia diphosphate, and optionally further transformed with a nucleic acid to produce an enzyme activity that converts zirconia diphosphate to zirconia, then the particular organism or cell is "capable of producing zirconia." An organism or cell that has been transformed to produce a higher amount of zirconia than a naturally occurring organism or cell is also encompassed as an "organism or cell capable of producing zirconia."

[0066] For purposes of the specification and appended claims herein, the use of "or" means "and / or" unless stated otherwise. Similarly, the various tenses of "comprising," "containing," "including," and "comprising" are interchangeable and not limiting.

[0067] It should be further understood that where the term “comprising” is used in the description of various embodiments, those skilled in the art will appreciate that in some specific cases, the language “consisting essentially of” or “consisting of” may be used to alternatively describe the embodiments.

[0068] As used herein, the terms "purified", "substantially purified" and "isolated" refer to a state free of other different compounds with which the compounds of the invention are normally associated in their natural state, so that "purified", "substantially purified" and "isolated" articles account for at least 0.5%, 1%, 5%, 10% or 20% by weight, or at least 50% or 75% by weight of a given sample mass. In one embodiment, these terms refer to a compound of the invention accounting for at least 95%, 96%, 97%, 98%, 99% or 100% by weight of a given sample mass. As used herein, when referring to nucleic acids or proteins, the terms "purified", "substantially purified" and "isolated" of nucleic acids or proteins also refer to a purified or concentrated state that is different from the state naturally present in, for example, a prokaryotic or eukaryotic environment, such as in a bacterial or fungal cell, or in a mammal, especially a human body. Any degree of purification or concentration, as long as it is greater than the degree of purification or concentration found in nature, including (1) purification from other related structures or compounds, or (2) association with structures or compounds with which they are not normally associated in the prokaryotic or eukaryotic environment, is within the meaning of "isolated." The nucleic acids, proteins, or classes of nucleic acids or proteins described herein can be isolated or otherwise associated with structures or compounds with which they are not normally associated in nature according to various methods and processes known to those skilled in the art.

[0069] In the context of the specification provided herein and the appended claims, the use of "or" means "and / or" unless stated otherwise.

[0070] Similarly, the various tenses of "comprising," "including," "comprising," and "including" are interchangeable and not limiting.

[0071] The term "about" indicates a possible variation of ±25%, in particular ±15%, ±10%, more in particular ±5%, ±2% or ±1% of the stated value.

[0072] The term "substantially" describes a value range of about 80 to 100%, such as 85 to 99.9%, particularly 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9%, especially 99 to 99.9%.

[0073] “Predominantly” means a proportion in the range of greater than 50%, for example in the range of 51% to 100%, in particular in the range of 75% to 99.9%; especially 85 to 98.5%, for example 95 to 99%.

[0074] In the context of the present invention, "main product" means a single compound or a group of at least 2 compounds, such as 2, 3, 4, 5 or more, in particular 2 or 3 compounds, which is "mainly" prepared by the reaction described herein and is contained in the reaction in a major proportion based on the total amount of the components of the product formed by the reaction. The proportion can be a molar ratio, a weight ratio, or, preferably based on chromatographic analysis, an area ratio calculated from the corresponding chromatogram of the reaction products.

[0075] In the context of the present invention, "by-product" means a single compound or a group of at least 2 compounds, such as 2, 3, 4, 5 or more, in particular 2 or 3 compounds, which single compound or group of compounds is not "primarily" prepared by the reaction described herein.

[0076] Due to the reversibility of enzymatic reactions, unless otherwise stated, the present invention relates to the enzymatic or biocatalytic reactions described herein in both reaction directions.

[0077] "Functional mutants" of the polypeptides described herein include "functional equivalents" of such polypeptides as defined below.

[0078] The term "stereoisomer" specifically includes conformational isomers.

[0079] According to the present invention, all "stereoisomeric forms" of the compounds described herein are generally included, such as structural isomers, especially stereoisomers and mixtures thereof, such as optical isomers or geometric isomers, such as E and Z isomers, and combinations thereof. If several asymmetric centers are present in a molecule, the present invention includes all combinations of different conformations of these asymmetric centers, such as enantiomeric pairs.

[0080] "Stereoselectivity" describes the ability to produce a specific stereoisomer of a compound in stereoisomerically pure form or the ability to specifically convert a specific stereoisomer from multiple stereoisomers in an enzymatic process as described herein. More specifically, this means that the product of the invention is enriched with respect to a specific stereoisomer, or the educt may be depleted with respect to a specific stereoisomer. This can be quantified by the purity %ee parameter calculated according to the following formula:

[0081] %ee=[X A -X B ] / [X A +X B ]*100,

[0082] Where X A and X B represents the molar ratio of stereoisomers A and B (Molenbruch).

[0083] The term "selectively converting" or "increasing selectivity" generally means that during the entire course of the reaction (i.e., between the start and the end of the reaction), at a certain point in time in the reaction, or during a "period" of the reaction, a specific stereoisomeric form, such as the E-form of an unsaturated hydrocarbon, is converted in a higher proportion or amount (compared on a molar basis) than the corresponding other stereoisomeric forms, such as the Z-form. In particular, during a "period" the selectivity can be observed to correspond to a conversion rate of 1 to 99%, 2 to 95%, 3 to 90%, 5 to 85%, 10 to 80%, 15 to 75%, 20 to 70%, 25 to 65%, 30 to 60%, or 40 to 50% of the initial amount of substrate. The higher proportion or amount can be expressed, for example, in the following manner:

[0084] - a higher maximum isomer yield observed during the entire reaction process or during said period thereof;

[0085] - a higher relative content of the isomer at a determined percentage value of substrate conversion; and / or

[0086] - The same relative content of isomers at higher percentage conversion values;

[0087] Each of these is preferably viewed relative to a reference method performed under otherwise identical conditions using a known chemical or biochemical method.

[0088] According to the present invention, all "isomeric forms" of the compounds described herein are generally included, such as structural isomers, especially stereoisomers and mixtures thereof, such as optical isomers or geometric isomers, such as E and Z isomers, and combinations thereof. If several asymmetric centers are present in a molecule, the present invention includes all combinations of different conformations of these asymmetric centers, such as enantiomeric pairs, or any mixture of stereoisomeric forms.

[0089] 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 under "standard conditions" as defined herein.

[0090] The various yield parameters ("yield" or YP / S; "specific productivity yield"; or space-time yield (STY)) are well known in the art and are determined as described in the literature.

[0091] "Yield" and "YP / S" (both expressed as mass of product produced / mass of material consumed) are used as synonyms herein.

[0092] The specific productivity-yield describes the amount of product produced per gram of biomass per liter of fermentation broth per hour. The wet cell weight expressed as WCW describes the number of biologically active microorganisms in the biochemical reaction. The value is given as g of product per g WCW per hour (i.e. g / g WCW). -1 h -1 Alternatively, the amount of biomass can also be expressed as the amount of dry cell weight, expressed as DCW. In addition, by measuring 600nm (OD 600 ) and using experimentally determined correlation factors to estimate the corresponding wet cell or dry cell weight, respectively.

[0093] The terms "fermentative production" or "fermentation" refer to the ability of a microorganism (aided by enzyme activities contained in or produced by said microorganism) to produce a compound in a cell culture using at least one carbon source added to the incubation.

[0094] The term "fermentation broth" is understood to mean a liquid, in particular an aqueous solution or an aqueous / organic solution, which is based on a fermentation process and which has been subjected to no or a work up such as described herein.

[0095] "Enzymatic" or "biocatalytic" methods refer to methods that are carried out under the catalytic action of an enzyme (including enzyme mutants as defined herein). Thus, the method can be carried out in the presence of the enzyme in isolated form (purified, enriched) or crude form, or in the presence of a cell system, in particular a natural or recombinant microbial cell that contains the enzyme in active form and has the ability to catalyze the conversion reaction as disclosed herein.

[0096] If the present disclosure refers to features, parameters and ranges thereof with different degrees of preference (including generic, non-explicitly preferred features, parameters and ranges thereof), then, unless otherwise stated, any combination of two or more of these features, parameters and ranges is included in the disclosure of the present invention regardless of their respective degrees of preference.

[0097] Detailed description

[0098] a. Specific embodiments of the present invention

[0099] The present invention specifically relates to the following embodiments:

[0100] 1. An isolated polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily, or a variant or mutant thereof, having cyclic terpene synthase activity, wherein the polypeptide comprises:

[0101] a. a modified class I synthase motif (DSFDxx(D / E)) as shown in SEQ ID NO: 11, which corresponds to sequence positions 174 to 180 of SEQ ID NO: 2, wherein x can be any naturally occurring amino acid residue, and is preferably selected from S and L, in particular the motif of SEQ ID NO: 13; and

[0102] b. the C-terminus of the modified class I motif, the modified class II synthase motif (HDxD(T / S) shown in SEQ ID NO: 12, which corresponds to sequence positions 366 to 370 of SEQ ID NO: 2, wherein x can be any naturally occurring amino acid residue, and preferably represents L, in particular the motif of SEQ ID NO: 14; and

[0103] c. Optionally, at least one other sequence motif selected from the group consisting of:

[0104] i. a partial sequence from positions 86 to 195 of SEQ ID NO: 2; and

[0105] ii. The partial sequence from positions 40 to 187 of SEQ ID NO:2.

[0106] In a specific embodiment, an isolated polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily, or a variant or mutant thereof, having cyclic terpene synthase activity is provided, wherein the polypeptide comprises:

[0107] a. a modified class I synthase motif as shown in SEQ ID NO: 13, which corresponds to sequence positions 174 to 180 of SEQ ID NO: 2, and

[0108] b. the C-terminus of the modified class I motif, the modified class II synthase motif shown in SEQ ID NO: 14, corresponding to sequence positions 366 to 370 of SEQ ID NO: 2, and

[0109] c. at least one other sequence motif selected from the group consisting of:

[0110] i. a partial sequence from positions 86 to 195 of SEQ ID NO: 2; and

[0111] ii. The partial sequence from positions 40 to 187 of SEQ ID NO:2.

[0112] In an alternative to the above embodiment, the modified class II synthase motif may also be the motif of SEQ ID NO:23 (ie, DLDTTS), which corresponds to sequence positions 367 to 372 of SEQ ID NO:2.

[0113] Alternatively or in addition to one or more of the above sequence motifs, the isolated polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily or a variant or mutant thereof having cyclic terpene synthase activity may also comprise:

[0114] - sequence motif III: QCKSKGCW (SEQ ID NO: 24), which corresponds to sequence positions 542 to 549 of SEQ ID NO: 2, and / or

[0115] - Sequence motif IV: DGILQVYFDVERPRIDPVVVAN (SEQ ID NO:25), which corresponds to sequence positions 404 to 424 of SEQ ID NO:2.

[0116] The sequence motif III can be further modified to the motif: QCEDGGW (SEQ ID NO: 27), wherein the N-terminal partial sequence motif QCE can be modified to: QCQ, QCK, QCA, QCD, QCG, QQN, QDE, QED or QET, and independently of this, the C-terminal partial sequence motif "GGW" can be changed to GGF or GSW.

[0117] 2. The polypeptide according to embodiment 1, which has the ability to produce succinyl sesquiterpenes and / or phosphate derivatives thereof, such as monophosphates, more particularly diphosphate derivatives thereof, particularly from farnesyl diphosphate (FPP) as a substrate.

[0118] 3. The polypeptide according to embodiment 2, which has the ability to produce a fennel phosphate derivative, such as a monophosphate, more particularly a fennel diphosphate, from farnesyl diphosphate (FPP) as a substrate.

[0119] 4. An isolated polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily having cyclic terpene synthase activity, wherein the polypeptide is DfHAD comprising the amino acid sequence of SEQ ID NO:2, or a mutant or natural variant thereof comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2 and retaining the terpene synthase activity.

[0120] 5. The polypeptide according to embodiment 4, which has the ability to produce succinyl sesquiterpenes and / or phosphate derivatives thereof, such as monophosphates, more particularly diphosphate derivatives thereof, particularly from farnesyl diphosphate (FPP) as a substrate.

[0121] 6. The polypeptide according to embodiment 5, which has the ability to produce a fennel phosphate derivative, such as a monophosphate, more particularly a fennel diphosphate, from farnesyl diphosphate (FPP) as a substrate.

[0122] 7. The polypeptide according to any of the preceding embodiments, which is isolated from a plant of the genus Dryopteris, in particular a plant of the species Dryopteris fragrans or other plant or fern species.

[0123] 8. An isolated polypeptide, in particular a natural variant of DfHAD according to any one of embodiments 1 to 4, selected from:

[0124] a. DfHAD-9(V274A) comprising an amino acid substitution (V274A) at position 274 of SEQ ID NO:2, or in an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2; and

[0125] b. DfHAD-8(K532R), comprising an amino acid substitution (K532R) at position 532 of SEQ ID NO:2, or in an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2.

[0126] 9. The polypeptide according to any one of embodiments 4 to 8, further comprising:

[0127] a. a modified class I synthase motif (DSFDxx(D / E)) as shown in SEQ ID NO: 11, which corresponds to sequence positions 174 to 180 of SEQ ID NO: 2, wherein x can be any naturally occurring amino acid residue, and is preferably selected from S and L, in particular the motif of SEQ ID NO: 13; and

[0128] b. the C-terminus of the modified class I synthase motif I, the modified class II synthase motif (HDxD(T / S) shown in SEQ ID NO: 12, which corresponds to sequence positions 366 to 370 of SEQ ID NO: 2, wherein x can be any naturally occurring amino acid residue, and preferably represents L, in particular the motif of SEQ ID NO: 14; and

[0129] c. and optionally, at least one other sequence motif selected from:

[0130] i. a partial sequence from positions 86 to 195 of SEQ ID NO: 2; and

[0131] ii. The partial sequence from positions 40 to 187 of SEQ ID NO:2.

[0132] In a specific embodiment, an isolated polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily, or a variant or mutant thereof, having cyclic terpene synthase activity is provided, wherein the polypeptide further comprises:

[0133] a. a modified class I synthase motif as shown in SEQ ID NO: 13, which corresponds to sequence positions 174 to 180 of SEQ ID NO: 2, and

[0134] b. the C-terminus of the modified class I motif, the modified class II synthase motif shown in SEQ ID NO: 14, corresponding to sequence positions 366 to 370 of SEQ ID NO: 2, and

[0135] c. at least one other sequence motif selected from the group consisting of:

[0136] i. a partial sequence from positions 86 to 195 of SEQ ID NO: 2; and

[0137] ii. The partial sequence from positions 40 to 187 of SEQ ID NO:2.

[0138] In an alternative to the above embodiment, the modified class II synthase motif may also be the motif of SEQ ID NO:23 (ie, DLDTTS), which corresponds to sequence positions 367 to 372 of SEQ ID NO:2.

[0139] Alternatively or in addition to one or more of the above sequence motifs, the isolated polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily or a variant or mutant thereof having cyclic terpene synthase activity may also comprise:

[0140] - sequence motif III: QCKSKGCW (SEQ ID NO: 24), which corresponds to sequence positions 542 to 549 of SEQ ID NO: 2, and / or

[0141] - Sequence motif IV: DGILQVYFDVERPRIDPVVVAN (SEQ ID NO:25), which corresponds to sequence positions 404 to 424 of SEQ ID NO:2.

[0142] The sequence motif III can be further modified to the motif: QCEDGGW (SEQ ID NO: 27), wherein the N-terminal partial sequence motif QCE can be modified to: QCQ, QCK, QCA, QCD, QCG, QQN, QDE, QED or QET, and independently of this, the C-terminal partial sequence motif "GGW" can be changed to GGF or GSW.

[0143] 10. The polypeptide according to one of embodiments 8 and 9, which is selected from:

[0144] a. DfHAD-9 (V274A), comprising the amino acid sequence of SEQ ID NO: 6;

[0145] b. DfHAD-8(K532R), comprising the amino acid sequence of SEQ ID NO: 4; and

[0146] c. DfHAD-QW fungus comprising the amino acid sequence of SEQ ID NO:19.

[0147] 11. A polypeptide according to any of the preceding embodiments, which catalyzes the conversion of (acyclic) farnesyl diphosphate (particularly (2E,6E)-3,7,11-trimethyldodeca-2,6,10-triene-1-pyrophosphate; FPP) to produce farnesyl phosphate derivatives, such as monophosphates, more particularly farnesyl diphosphate, preferably with a selectivity of >50%.

[0148] 12. The polypeptide according to any one of the preceding embodiments,

[0149] a. comprising an amino acid sequence selected from SEQ ID NO: 2, 4 and 6; or

[0150] b. Encoded by a nucleic acid molecule comprising a coding nucleotide sequence selected from SEQ ID NO: 1, 3, 5, 7, 8, 9 and 10.

[0151] 13. An isolated nucleic acid molecule,

[0152] a. comprising a nucleotide sequence encoding a polypeptide according to any one of the preceding embodiments; or

[0153] b. comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 8, 9 and 10, or having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 8, 9 or 10, and encoding a polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily having terpene synthase activity, the polypeptide particularly having the ability to produce succinyl sesquiterpenes and / or phosphate derivatives thereof, such as monophosphates, more particularly diphosphate derivatives thereof, more particularly having the ability to produce succinyl phosphate derivatives, such as monophosphates, more particularly succinyl diphosphate, from farnesyl diphosphate (FPP) as a substrate, or

[0154] c. comprising a nucleotide sequence comprising a sequence complementary to one of the sequences of b.; or

[0155] d. comprises a nucleotide sequence that hybridizes to the nucleotide sequence of a., b. or c. under stringent conditions.

[0156] In a specific embodiment, the nucleic acid may be naturally present in a plant of the genus Dryopteris, such as a plant of the species Dryopteris fragrans or other plant or fern species, or obtained by modifying SEQ ID NO: 1, 3, 5, 7, 8, 9 or 10 or its reverse complement.

[0157] In another embodiment, the nucleic acid is isolated or derived from a plant of the genus Dryopteris, such as a species of Dryopteris fragrans.

[0158] 14. An expression construct comprising at least one nucleic acid molecule according to embodiment 13.

[0159] 15. A vector comprising at least one nucleic acid molecule according to embodiment 13 or at least one expression construct according to embodiment 14.

[0160] 16. The vector of embodiment 15, wherein the vector is a prokaryotic, viral or eukaryotic vector.

[0161] 17. The vector of embodiment 15 or 16, wherein the vector is an expression vector.

[0162] 18. The vector according to any one of embodiments 15 to 17, which is a plasmid vector.

[0163] 19. A recombinant host cell or a recombinant non-human host organism comprising:

[0164] a. at least one isolated nucleic acid molecule of embodiment 13, which is optionally stably integrated into the genome; or

[0165] b. at least one expression construct of embodiment 14, which is optionally stably integrated into the genome; or

[0166] c. At least one vector according to any one of embodiments 15 to 18.

[0167] 20. The host cell or host organism according to embodiment 19, which is selected from prokaryotic or eukaryotic microorganisms or cells derived therefrom.

[0168] 21. The host cell or host organism according to embodiment 20, which is selected from bacterial cells, fungal cells and plant cells, or a plant.

[0169] 22. The host cell or host organism according to embodiment 21, wherein the fungal cell is a yeast cell.

[0170] 23. The host cell or host organism of embodiment 21, wherein the bacterial cell is selected from the genus Escherichia, in particular the species E. coli, and the yeast is selected from the genus Saccharomyces or Pichia, in particular from the species Saccharomyces cerevisiae or Pichia pastoris.

[0171] Some of these host cells or organisms do not naturally produce FPP. In order to be suitable for implementing the methods of the embodiments described herein, organisms or cells that do not naturally produce acyclic terpene pyrophosphate precursors such as FPP are genetically modified to produce the precursor. For example, they can be so transformed before or simultaneously with the modification of the nucleic acid according to any of the above embodiments. Methods for transforming organisms so that they produce acyclic terpene pyrophosphate precursors such as FPP are known in the prior art. For example, introducing enzyme activity of the mevalonate pathway is a suitable strategy for making an organism produce FPP.

[0172] 24. A method of producing at least one catalytically active polypeptide according to any one of embodiments 1 to 12, the method comprising:

[0173] a. Cultivating the non-human host organism or host cell of embodiment 19 to express or overexpress at least one polypeptide according to any one of embodiments 1 to 12; and

[0174] b. Optionally, isolating the polypeptide from the non-human host cell or organism cultured in step a.

[0175] 25. The method of embodiment 24, further comprising, before step a), transforming a non-human host organism or cell with at least one nucleic acid according to embodiment 13, at least one construct according to embodiment 14, or at least one vector according to any one of embodiments 15 to 18, so that it expresses or overexpresses the polypeptide according to any one of embodiments 1 to 12.

[0176] 26. A method for preparing a mutant polypeptide having terpene synthase activity, the mutant polypeptide particularly having the ability to produce succinyl sesquiterpenes and / or phosphate derivatives thereof, such as monophosphates, more particularly diphosphate derivatives thereof, and more particularly having the ability to produce fennel phosphate derivatives, such as monophosphates, more particularly fennel diphosphate, from farnesyl diphosphate (FPP) as a substrate, the method comprising the following steps:

[0177] a. Selecting a nucleic acid molecule according to embodiment 13;

[0178] b. modifying the selected nucleic acid molecule to obtain at least one mutant nucleic acid molecule;

[0179] c. transforming a host cell or a unicellular host organism with the mutant nucleic acid sequence to express a polypeptide encoded by the mutant nucleic acid sequence;

[0180] d. screening the expression products for at least one mutant comprising terpene synthase activity, in particular the ability to produce terpenoid sesquiterpenes and / or phosphate derivatives thereof, such as monophosphates, more particularly diphosphate derivatives thereof, and more particularly the ability to produce fennel phosphate derivatives, such as monophosphates, more particularly fennel diphosphate, from farnesyl diphosphate (FPP) as a substrate; and

[0181] e. Optionally, if the polypeptide does not have the desired mutant activity, repeating steps a. to d. until a polypeptide having the desired mutant activity is obtained; and

[0182] f. Optionally, if a polypeptide having the desired mutant activity is identified in step d., isolating the corresponding mutant nucleic acid obtained in step c.

[0183] 27. A method for producing succinyl sesquiterpenes, especially succinyl alcohol, comprising:

[0184] a. contacting farnesyl diphosphate (FPP) with a polypeptide as defined in any one of embodiments 1 to 12, with a polypeptide prepared according to embodiment 24 or 25, or with a mutant polypeptide prepared according to embodiment 26, thereby obtaining at least one phosphate, in particular a diphosphate of a sesquiterpene, in particular folding phosphate, more particularly folding phosphate;

[0185] b. chemically or enzymatically cleaving the phosphate portion of the product, particularly the diphosphate portion; and

[0186] c. Optionally, isolating the succinyl sesquiterpenes, in particular succinyl alcohol.

[0187] In a particular embodiment, the phosphate moiety can be enzymatically cleaved by applying a phosphatase, more particularly an acid or alkaline phosphatase. Alkaline phosphatases of different origins are preferred, such as bacterial enzymes. Suitable phosphatases are commercially available enzymes.

[0188] 28. The method of embodiment 27, wherein the succinyl sesquiterpene comprises succinyl alcohol, preferably as the major product.

[0189] 29. The method according to any one of embodiments 27 and 28, comprising providing, in particular transforming, a non-human host organism or a host cell with at least one nucleic acid according to embodiment 13, at least one construct according to embodiment 14 or at least one vector according to any one of embodiments 15 to 16, such that it expresses or overexpresses a polypeptide according to any one of embodiments 1 to 12.

[0190] 30. The method according to any one of embodiments 27 to 29, wherein the FPP is contacted with a non-human host organism or a host cell, with a cell lysate thereof, or with a culture medium containing host cells of said non-human host organism, and / or with a polypeptide as defined in any one of embodiments 1 to 12, which polypeptide is isolated from a non-human host organism or a host cell, a cell lysate or a culture medium.

[0191] 31. The method of embodiment 30, wherein the succinyl sesquiterpene is produced by the non-human host organism or host cell by fermentation.

[0192] 32. The method according to embodiment 30, wherein the succinyl sesquiterpene is produced by an enzymatic process comprising converting FPP with the isolated polypeptide according to any one of embodiments 1 to 12, optionally in the presence of other adjuvants.

[0193] 33. Use of a polypeptide as defined in any one of embodiments 1 to 12 for the preparation of an odorant, flavoring or fragrance ingredient, in particular Ambrox.

[0194] 34. Use of the succinyl sesquiterpene prepared according to any one of embodiments 27 to 32 in the preparation of odorants, flavoring agents or fragrance ingredients, especially Ambrox.

[0195] 35. A method for producing Ambrox, the method comprising:

[0196] a. providing zizyphus alcohol by the method of any one of the embodiments 27 to 32,

[0197] b. Optionally, separating the folded phytol produced in step a.; and

[0198] c. Conversion of fennel to Ambrox in a manner known per se, for example as reported in Tetrahedron: Asymmetry 11 (2000) 1375-1388.

[0199] 36. A composition comprising a substance prepared according to embodiment 34 or 35.

[0200] 37. The composition according to embodiment 36, which is selected from the group consisting of:

[0201] a. Body care compositions

[0202] b. Home Care Compositions

[0203] c. Fragrance compositions.

[0204] Unless otherwise indicated, within the range of "at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity," particular values ​​are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, while values ​​of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity are even more particular.

[0205] b. Polypeptides suitable for use according to the present invention

[0206] In the context of this article, the following definitions apply:

[0207] The general terms "polypeptide" or "peptide", which are used interchangeably, refer to a linear chain or sequence of amino acid residues linked in a peptide manner, whether natural or synthetic, containing from about 10 to more than 1000 residues. Short-chain polypeptides of up to 30 residues are also referred to as "oligopeptides".

[0208] The term "protein" refers to a macromolecular structure composed of one or more polypeptides. The amino acid sequence of its polypeptides represents the "primary structure" of the protein. The amino acid sequence also predetermines the "secondary structure" of the protein by forming special structural elements (such as α-helices and β-pleated structures formed in polypeptide chains). The arrangement of multiple such secondary structural elements defines the "tertiary structure" or spatial arrangement of the protein. If a protein contains more than one polypeptide chain, the chains are arranged in space to form the "quaternary structure" of the protein. The correct spatial arrangement or "folding" of the protein is a prerequisite for protein function. Denaturation or unfolding will destroy protein function. If this destruction is reversible, the protein function can be restored by refolding.

[0209] The typical protein function referred to herein is "enzyme function", i.e. the protein acts as a biocatalyst on a substrate, such as a compound, and catalyzes the conversion of the substrate to a product. Enzymes may display a high or low degree of substrate and / or product specificity.

[0210] Thus, a "polypeptide" referred to herein as having a particular "activity" implicitly refers to a correctly folded protein that exhibits the indicated activity, such as a particular enzymatic activity.

[0211] Thus, unless otherwise stated, the term "polypeptide" also encompasses the terms "protein" and "enzyme".

[0212] Similarly, the term "polypeptide fragment" encompasses the terms "protein fragment" and "enzyme fragment".

[0213] The term "isolated polypeptide" refers to an amino acid sequence removed from its natural environment by any method known in the art, or a combination of methods, including recombinant, biochemical, and synthetic methods.

[0214] "Target peptide" refers to an amino acid sequence that targets a protein or polypeptide to an intracellular organelle (i.e., mitochondria or plastids) or an extracellular space (secretion signal peptide). A nucleic acid sequence encoding a target peptide can be fused to a nucleic acid sequence encoding the amino terminus (e.g., N-terminus) of a protein or polypeptide, or can be used to replace a natural targeting polypeptide.

[0215] The present invention also relates to "functional equivalents" (also called "analogs" or "functional mutants") of the polypeptides specifically described herein.

[0216] For example, a "functional equivalent" refers to a polypeptide which, in a test for determining the activity of the enzymatic pyrrolidone diphosphate synthase, exhibits at least 1 to 10% higher or lower, or at least 20%, or at least 50%, or at least 75%, or at least 90% higher or lower pyrrolidone diphosphate synthase activity compared to the polypeptide specifically described herein.

[0217] According to the invention, "functional equivalents" also encompass specific mutants which have an amino acid different from the specifically stated amino acid in at least one sequence position of the amino acid sequences described herein, but which still have one of the above-mentioned biological activities, such as enzymatic activity. Thus, "functional equivalents" include mutants obtainable by addition, substitution, in particular conservative substitution, deletion and / or inversion of one or more, for example 1 to 20, 1 to 15 or 5 to 10 amino acids, wherein the changes may occur at any sequence position, as long as they result in a mutant having the profile of the properties of the invention. Functional equivalence is also provided in particular if the activity pattern qualitatively overlaps between the mutant and the unchanged polypeptide, i.e. if, for example, an interaction with the same agonist or antagonist or substrate is observed, but at a different rate (i.e., by EC 50 or IC 50 The following table shows examples of suitable (conservative) amino acid substitutions:

[0218]

[0219] "Functional equivalents" in the above sense are also "precursors" of the polypeptides described herein, as well as "functional derivatives" and "salts" of the polypeptides described herein.

[0220] In this context, a "precursor" is a natural or synthetic precursor of a polypeptide that may or may not have the desired biological activity.

[0221] The expression "salts" refers to salts of carboxyl groups and acid addition salts of amino groups of the protein molecules according to the invention. Salts of carboxyl groups can be produced in a known manner and include inorganic salts, such as sodium, calcium, ammonium, iron and zinc salts, and salts formed with organic bases such as amines, such as triethanolamine, arginine, lysine, piperidine, etc. Acid addition salts, such as salts formed with inorganic acids such as hydrochloric acid or sulfuric acid, and salts formed with organic acids such as acetic acid and oxalic acid, are also encompassed by the present invention.

[0222] The "functional derivatives" of the polypeptides according to the present invention can also be produced at the functional amino acid side groups or their N-termini or C-termini using known techniques. Such derivatives include, for example: aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, which can be obtained by reacting with ammonia or with primary or secondary amines; N-acyl derivatives of free amino groups, which are generated by reacting with acyl groups; or O-acyl derivatives of free hydroxyl groups, which are generated by reacting with acyl groups.

[0223] "Functional equivalents" naturally also include polypeptides obtainable from other organisms and naturally occurring variants. For example, the area of ​​homologous sequence regions can be determined by sequence comparison, and equivalent polypeptides can be determined based on the specific parameters of the present invention.

[0224] "Functional equivalents" also include "fragments" of polypeptides according to the present invention, such as single domains or sequence motifs, or N-terminal and / or C-terminal truncated forms, which may or may not show the desired biological function. Preferably, such "fragments" at least qualitatively retain the desired biological function.

[0225] Furthermore, a "functional equivalent" is a fusion protein having one of the polypeptide sequences described herein or a functional equivalent derived therefrom and at least one further functionally different heterologous sequence in functional N-terminal or C-terminal association (i.e., without substantial mutual functional impairment of the fusion protein parts). Non-limiting examples of these heterologous sequences are, for example, signal peptides, histidine anchors or enzymes.

[0226] Also included according to the invention are "functional equivalents" homologous to the specifically disclosed polypeptides. They have at least 60%, preferably at least 75%, in particular at least 80 or 85%, for example 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homology (or identity) with 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 homology or identity expressed as a percentage of a homologous polypeptide according to the invention refers in particular to the identity expressed as a percentage of amino acid residues based on the total length of one of the amino acid sequences specifically described herein.

[0227] Identity data expressed as a percentage can also be determined with the aid of a BLAST alignment, the algorithm blastp (protein-protein BLAST) or by applying the Clustal settings as described in detail herein below.

[0228] In case of possible protein glycosylation, "functional equivalents" according to the invention include the polypeptides described herein in deglycosylated or glycosylated form, as well as modified forms which can be obtained by altering the glycosylation pattern.

[0229] Functional equivalents or homologues of the polypeptides according to the invention can be produced by mutagenesis, for example by point mutations, lengthening or shortening the protein or as described in more detail below.

[0230] Functional equivalents or homologues of polypeptides according to the present invention can be identified by screening mutants such as a combined database of shortened mutants. For example, the diversity database of protein variants can be produced by the combined mutagenesis at the nucleic acid level, for example, by the enzymatic connection of a synthetic oligonucleotide mixture. There are many methods that can be used to produce a database of potential homologues from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and then the synthetic gene can be connected to a suitable expression vector. The use of a degenerate genome makes it possible to provide all sequences in a mixture, which encodes a potential protein sequence set required. The synthetic method of a degenerate oligonucleotide is well known to those skilled in the art.

[0231] In the prior art, several techniques are known for screening gene products of combinatorial databases produced by point mutation or shortening, and for screening cDNA libraries of gene products with selected properties. These techniques can be applied to rapidly screen gene libraries produced by combinatorial mutagenesis of homologues according to the present invention. The most commonly used techniques for screening large gene libraries based on high-throughput analysis include cloning the gene library in a reproducible expression vector, transforming suitable cells with the resulting vector database, and expressing the combinatorial genes under specific conditions, under which the detection of the desired activity promotes the separation of the vector encoding the gene (whose product is detected). Recursive integration mutagenesis (REM) is a technique that increases the frequency of functional mutants in a database, which can be used in conjunction with screening tests to identify homologues.

[0232] Embodiments provided herein provide orthologues and paralogues of polypeptides disclosed herein, and methods for identifying and separating such orthologues and paralogues. The definition of term "orthologue" and "paralogue" is given below, and is applicable to amino acid and nucleic acid sequences.

[0233] c. Encoding nucleic acid sequences suitable for use according to the present invention

[0234] In the context of this article, the following definitions apply:

[0235] The terms "nucleic acid sequence", "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a sequence of nucleotides. Nucleic acid sequences can be single-stranded 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. The skilled artisan understands that the nucleic acid sequence of RNA is the same as the DNA sequence, with the difference that thymine (T) is replaced by uracil (U). The term "nucleotide sequence" should also be understood to include polynucleotide molecules or oligonucleotide molecules in separate fragment forms or as components of larger nucleic acids.

[0236] "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 that in which the nucleic acid or nucleic acid sequence is found in nature, and can include those that are substantially free of contaminating endogenous materials.

[0237] As used herein, the term "naturally-occurring" as applied to nucleic acids refers to a nucleic acid that is found in the cells of an organism in nature and has not been intentionally modified by man in a laboratory.

[0238] A "fragment" of a polynucleotide or nucleic acid sequence refers to a continuous nucleotide, which is particularly at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp and / or at least 60 bp of the length of a polynucleotide of one embodiment herein. In particular, a fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 continuous nucleotides of a polynucleotide of one embodiment herein. Without limitation, a fragment of a polynucleotide herein can be used as a PCR primer and / or probe, or for antisense gene silencing or RNAi.

[0239] As used herein, the term "hybridization" or hybridization under certain conditions is intended to describe the conditions of hybridization and washing, under which the nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions can make at least about 70%, for example, at least about 80%, and for example, at least about 85%, 90% or 95% homogeneity sequences remain bound to each other. The definition of low stringency, medium and high stringency hybridization conditions is provided below. Those skilled in the art can select suitable hybridization conditions with minimal experiments as illustrated by, for example, Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). In addition, stringent conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0240] A "recombinant nucleic acid sequence" is a nucleic acid sequence produced by combining genetic material from more than one source using laboratory methods (e.g., molecular cloning), thereby creating or modifying a nucleic acid sequence that does not occur in nature and cannot otherwise be found in a biological organism.

[0241] "Recombinant DNA technology" refers to molecular biology methods 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.

[0242] The term "gene" refers to a DNA sequence comprising a region that is transcribed into an RNA molecule (e.g., mRNA in a cell) operably linked to an appropriate regulatory region (e.g., a promoter). Thus, a gene may comprise several operably linked sequences, such as a promoter, a 5' leader sequence (comprising, for example, sequences involved in translation initiation), a coding region of a cDNA or genomic DNA, introns, exons, and / or a 3' non-translated sequence (comprising, for example, a transcription termination site).

[0243] "Polycistronic" refers to a nucleic acid molecule, particularly mRNA, that can encode more than one polypeptide in the same nucleic acid molecule.

[0244] "Chimeric gene" refers to any gene that is not usually found in species in nature, particularly a gene in which one or more parts of the nucleic acid sequence are not associated with each other in nature. For example, a promoter is not associated with part or all of a transcription region or with another regulatory region in nature. The term "chimeric gene" should be understood to include expression constructs in which a promoter or transcriptional regulatory sequence is operably linked to one or more coding sequences or antisense (i.e., the reverse complementary strand of the sense strand) or inverted repeats (sense and antisense, whereby RNA transcripts form double-stranded RNA after transcription). The term "chimeric gene" also includes genes obtained by combining parts of one or more coding sequences to produce new genes.

[0245] "3'URT" or "3' untranslated sequence" (also called "3' untranslated region" or "3' end") refers to a nucleic acid sequence found downstream of a gene coding sequence that contains, for example, a transcription termination site and (in most but not all eukaryotic mRNAs) a polyadenylation signal, such as AAUAAA or a variant thereof. After transcription termination, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly (A) tail may be added, which is involved in the transport of the mRNA to the site of translation, such as the cytoplasm.

[0246] The term "primer" refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and used to polymerize a nucleic acid sequence that is complementary to the template.

[0247] The term "selectable marker" refers to any gene that can be used to select one or more cells that include the selectable marker after expression. Examples of selectable markers are described below. Those skilled in the art will appreciate that different antibiotic, fungicide, auxotrophic or herbicide selectable markers may be applicable to different target species.

[0248] The present invention also relates to nucleic acid sequences encoding the polypeptides as defined herein.

[0249] In particular, the invention also relates to nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, such as cDNA, genomic DNA and mRNA) encoding one of the above polypeptides and functional equivalents thereof, which can be obtained, for example, by using artificial nucleotide analogs.

[0250] The present invention relates both to isolated nucleic acid molecules encoding polypeptides according to the invention or biologically active segments thereof, and to nucleic acid fragments which can be used, for example, as hybridization probes or primers for identifying or amplifying encoding nucleic acids according to the invention.

[0251] The invention also relates to nucleic acids having a certain degree of "identity" to the sequences specifically disclosed herein. "Identity" between two nucleic acids refers in each case to the identity of the nucleotides over the entire length of the nucleic acid.

[0252] "Identity" between two nucleotide sequences (also applicable to peptides or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues), or the number of identical residues in the two sequences, when the comparison of the two sequences is generated. Identical residues are defined as identical residues in the two sequences at a given position of the comparison. The percentage of sequence identity used herein is calculated from the best comparison by dividing the number of identical residues between the two sequences by the total number of residues in the shortest sequence and multiplying by 100. The best comparison is the comparison with the highest probability of the percentage of identity. Rooms can be introduced into one or more positions of the comparison in one or two sequences to obtain the best comparison. These rooms are then considered to be non-identical residues for calculating the percentage of sequence identity. The comparison for determining the percentage of amino acid or nucleic acid sequence identity can be implemented in a variety of ways using computer programs and computer programs publicly available, for example, on the Internet.

[0253] In particular, the BLAST program (Tatiana et al., FEMS Microbiol Lett., 1999, 174: 247-250, 1999) set to default parameters, which is available from the National Center for Biotechnology Information (NCBI) at http: / / www.ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi, can be used to obtain optimal alignment of protein or nucleic acid sequences and calculate the percentage of sequence identity.

[0254] In another example, the identity can be calculated by the VectorNTI Suite 7.1 program of Informax Corporation (USA) using the Clustal method (Higgins DG, Sharp PM. ((1989))) with the following settings:

[0255] Multiple alignment parameters:

[0256] Gap opening deduction 10 points

[0257] Gap extension deduction 10 points

[0258] Gap separation deduction range 8

[0259] Gap separation deduction

[0260] Alignment delay identity percentage 40

[0261] Residue-specific gap

[0262] Hydrophilic residue gap

[0263] Transition weight 0

[0264] Pairwise comparison parameters:

[0265] FAST algorithm

[0266] K-tuple size 1

[0267] Gap deduction 3

[0268] Window size 5

[0269] Optimal diagonal number 5

[0270] Alternatively, identity can be determined according to the method of Chenna et al. (2003), web page: http: / / www.ebi.ac.uk / Tools / clustalw / index.html# and the following settings:

[0271]

[0272] All nucleic acid sequences mentioned herein (single-stranded and double-stranded DNA and RNA sequences, such as cDNA and mRNA) can be produced from nucleotide building blocks by chemical synthesis in a known manner, for example, by condensation of fragments of complementary nucleic acid building blocks of each overlap of a double helix. The chemical synthesis of oligonucleotides can be carried out in a known manner, for example, by the phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides, and the filling of vacancies by means of Klenow fragments of DNA polymerase and ligation reactions, and general cloning techniques are described in Sambrook et al. (1989), see below.

[0273] Furthermore, the nucleic acid molecules according to the invention may additionally comprise untranslated sequences from the 3' and / or 5' end of the coding genetic region.

[0274] The invention further relates to nucleic acid molecules which are complementary to the specifically described nucleotide sequences or to segments thereof.

[0275] The nucleotide sequences according to the invention make it possible to generate probes and primers that can be used to identify and / or clone homologous sequences in other cell types and organisms. Such probes or primers typically contain a nucleotide sequence region that hybridizes under "stringent" conditions (as defined elsewhere herein) to at least about 12, preferably at least about 25, for example about 40, 50 or 75 consecutive nucleotides of the sense strand or corresponding antisense strand of the nucleic acid sequence according to the invention.

[0276] "Homologous" sequences include orthologous or paralogous sequences. Methods for identifying orthologs or paralogs include phylogenetic methods, sequence similarity and hybridization methods known in the art and described herein.

[0277] "Paralogs" or paralogous sequences are derived from gene duplications, which produce two or more genes with similar sequences and similar functions. Paralogs are often clustered together and formed by duplication of genes within related plant species. Paralogs are found in groups of similar genes using Paired Blast analysis or using programs such as CLUSTAL during phylogenetic analysis of gene families. Among paralogs, consensus sequences can be identified as sequences that are characteristic of related genes and have similar functions of the genes.

[0278] "Orthologs" or orthologous sequences are sequences that are similar to each other because they are found in species passed down by a common ancestor. For example, plant species with a common ancestor are known to contain many enzymes with similar sequences and functions. For example, by using the CLUSTAL or BLAST program to construct a phylogenetic tree of a species' gene family, a technician can identify orthologous sequences and predict the functions of orthologs. A method for identifying or confirming similar functions between homologous sequences is by comparing transcript profiles in host cells or organisms (such as plants or microorganisms) that overexpress or lack (in gene knockout / knockdown) related polypeptides. The technician can understand that genes with similar transcript profiles (having a common transcript greater than 50% regulation, or having a common transcript greater than 70% regulation, or a common transcript greater than 90% regulation) will have similar functions. By causing a host cell, an organism such as a plant or a microorganism to produce a terpene synthase protein, homologs, paralogs, orthologs, and any other variants of the sequences described herein are expected to function in a similar manner.

[0279] The term "selectable marker" refers to any gene that can be used to select one or more cells that include the selectable marker after expression. Examples of selectable markers are described below. Those skilled in the art will appreciate that different antibiotic, fungicide, auxotrophic or herbicide selectable markers may be applicable to different target species.

[0280] An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid, and may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or free of chemical precursors or other chemicals if chemically synthesized.

[0281] Can be separated according to the nucleic acid molecules of the present invention by means of standard techniques of molecular biology and the sequence information provided according to the present invention.For example, can use one of concrete disclosed complete sequence or its fragment as hybridization probe and standard hybridization technique (for example, be described in Sambrook, (1989)) from suitable cDNA library separation cDNA.

[0282] In addition, the nucleic acid molecules that comprise one of disclosed sequence or its segment can use the oligonucleotide primers based on this sequence structure, separate by polymerase chain reaction.The nucleic acid increased in this way can be cloned in the suitable vector, and can characterize by dna sequencing.Oligonucleotide according to the present invention can also be by the synthetic method of standard, for example use automatic DNA synthesizer to prepare.

[0283] According to nucleic acid sequences of the present invention or derivatives thereof, homologues or parts of these sequences can be separated from other bacteria, for example, by conventional hybridization techniques or PCR techniques, for example by genome or cDNA libraries. These DNA sequences hybridize with sequences according to the present invention under standard conditions.

[0284] "Hybridization" refers to the ability of a polynucleotide or oligonucleotide to bind to nearly complementary sequences under standard conditions, without nonspecific binding between non-complementary pairs occurring under these conditions. For this purpose, the sequences may be 90-100% complementary. The property of complementary sequences to be able to bind specifically to each other is used, for example, in Northern or Southern blotting or in primer binding in PCR or RT-PCR.

[0285] Short oligonucleotides in conserved regions are advantageously used for hybridization. However, longer nucleic acid fragments or complete sequences of the invention may also be used for hybridization. These "standard conditions" vary depending on the nucleic acid used (oligonucleotides, longer fragments or complete sequences) or the type of nucleic acid used for hybridization (DNA or RNA). For example, the melting temperature of DNA:DNA hybrids is about 10°C lower than that of DNA:RNA hybrids of the same length.

[0286] For example, depending on the specific nucleic acid, standard conditions refer to a temperature of 42 to 58° C. in a buffered aqueous solution with a concentration of 0.1 to 5×SSC (1×SSC=0.15M NaCl, 15mM sodium citrate, pH 7.2), or additionally in the presence of 50% formamide (e.g., 42° C., 5×SSC, 50% formamide). Advantageously, the hybridization conditions for DNA:DNA hybrids are 0.1×SSC at a temperature of about 20° C. to 45° C., preferably about 30° C. to 45° C. For DNA:RNA hybrids, the hybridization conditions are advantageously 0.1×SSC and a temperature of about 30° C. to 55° C., preferably about 45° C. to 55° C. These stated hybridization temperatures are examples of calculated melting temperature values ​​for a nucleic acid of about 100 nucleotides in length and a G+C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization have been described in relevant genetics textbooks (e.g., Sambrook et al., 1989) and can be calculated using formulas known to those skilled in the art, for example depending on the length of the nucleic acid, the type of hybrid or the G+C content. Those skilled in the art can obtain more information about hybridization from the following textbooks: Ausubel et al. (eds), (1985), Brown (ed) (1991).

[0287] "Hybridization" can be performed in particular under stringent conditions. Such hybridization conditions are described, for example, in Sambrook (1989), or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

[0288] As used herein, the term "hybridizes or hybridizes under certain conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions can be such that sequences of at least about 70%, such as at least about 80%, and such as at least about 85%, 90%, or 95% identity remain bound to each other. Definitions of low stringency, medium, and high stringency hybridization conditions are provided herein.

[0289] Those skilled in the art can select appropriate hybridization conditions with minimal experimentation as illustrated by, for example, Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). In addition, stringent conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0290] As used herein, the defined low stringency conditions are as follows. The filter containing DNA is pretreated at 40°C for 6 hours in a solution containing 35% formamide, 5xSSC, 50mM Tris-HCl (pH 7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and 5-20x10 6 32P-labeled probe. Incubate the filter in the hybridization mixture at 40°C for 18-20 hours, then wash at 55°C for 1.5 hours. In a solution containing 2x SSC, 25mM Tris-HCl (pH 7.4), 5mM EDTA and 0.1% SDS. Replace the wash solution with fresh solution and incubate at 60°C for another 1.5 hours. Blot the filter dry and perform autoradiography.

[0291] As used herein, the defined medium stringency conditions are as follows. The filter containing DNA is pretreated at 50°C for 7 hours in a solution containing 35% formamide, 5x SSC, 50mM Tris-HCl (pH 7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg / ml denatured salmon sperm DNA. Hybridization is performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and 5-20x10 632P-labeled probe. The filter was incubated in hybridization mixture at 50°C for 30 hours and then washed at 55°C for 1.5 hours. In a solution containing 2x SSC, 25mM Tris-HCl (pH 7.4), 5mM EDTA and 0.1% SDS. The wash solution was replaced with fresh solution and incubated at 60°C for another 1.5 hours. The filter was blotted dry and subjected to autoradiography.

[0292] As used herein, the defined high stringency conditions are as follows. The DNA-containing filter is prehybridized at 65°C for 8 hours to overnight 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. 6 The filters were hybridized at 65°C for 48 hours in a prehybridization mixture containing cpm 32P-labeled probe. The filters were washed at 37°C for 1 hour in a solution containing 2xSSC, 0.01% PVP, 0.01% Ficoll and 0.01% BSA. They were then washed in 0.1x SSC at 50°C for 45 minutes.

[0293] If the above conditions are not suitable (eg, as for interspecies hybridization), other low, medium, and high stringency conditions well known in the art (eg, for interspecies hybridization) can be used.

[0294] A test kit for a nucleic acid sequence encoding a polypeptide of the present invention may include primers and / or probes specific to the nucleic acid sequence encoding the polypeptide, and protocols for using the primers and / or probes to detect the nucleic acid sequence encoding the polypeptide in a sample. Such a test kit may be used to determine whether a plant, organism, microorganism or cell has been modified, i.e., whether it has been transformed with a sequence encoding a polypeptide.

[0295] To test the functionality of a variant DNA sequence according to one embodiment herein, the target sequence is operably linked to a selectable or screenable marker gene and the expression of the reporter gene is tested in transient expression assays using microorganisms or protoplasts or in stably transformed plants.

[0296] The invention also relates to derivatives of the specifically disclosed or derivable nucleic acid sequences.

[0297] Thus, further nucleic acid sequences according to the invention may be derived from the sequences specifically disclosed herein and may differ therefrom by one or more, e.g. 1 to 20, in particular 1 to 15 or 5 to 10 additions, substitutions, insertions or deletions of one or several (e.g. 1 to 10) nucleotides and still encode a polypeptide having the desired properties.

[0298] The invention also encompasses nucleic acid sequences which comprise so-called silent mutations or which have been altered compared to the specifically stated sequences depending on the codon usage of the particular native or host organism.

[0299] According to a specific embodiment of the present invention, variant nucleic acid can be prepared to adapt its nucleotide sequence to a specific expression system. For example, if the amino acid is encoded by a specific codon, then a known bacterial expression system can more effectively express the polypeptide. Due to the degeneracy of the genetic code, more than one codon can encode the same amino acid sequence, and a plurality of nucleic acid sequences can encode the same protein or polypeptide, all of which DNA sequences are encompassed in one embodiment of this paper. Where appropriate, the nucleic acid sequence encoding the polypeptide described herein can be optimized to increase expression in a host cell. For example, a nucleic acid of one embodiment of this paper can be synthesized using host-specific codons to improve expression.

[0300] The invention also encompasses naturally occurring variants of the sequences described herein, such as splice variants or allelic variants.

[0301] Allelic variants have at least 60% homology, preferably at least 80% homology, very particularly preferably at least 90% homology over the entire amino acid range at the derived amino acid level (regarding the homology at the amino acid level, reference should be made to the detailed information given above for the polypeptides). Advantageously, the homology may be higher over partial regions of the sequence.

[0302] The invention also relates to sequences obtainable by conservative nucleotide substitutions, ie sequences in which the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility.

[0303] The present invention also relates to molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. Due to natural allelic variation, such genetic polymorphisms may be present in cells from different populations or in cells within a population. Allelic variants may also include functional equivalents. These natural variations will typically produce 1 to 5% changes in the nucleotide sequence of a gene. The polymorphisms may result in changes in the amino acid sequence of the polypeptides disclosed herein. Allelic variants may also include functional equivalents.

[0304] Furthermore, derivatives are also to be understood as homologues of the nucleic acid sequences according to the invention, for example homologues of animals, plants, fungi or bacteria, shortened sequences, single-stranded DNA or RNA of coding and non-coding DNA sequences. For example, at the DNA level, homologues have at least 40%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80% homology over a given entire DNA region in the sequences specifically disclosed herein.

[0305] Furthermore, derivatives are to be understood as fusions, for example, with promoters. The promoters added to the nucleotide sequence may be modified by at least one nucleotide exchange, at least one insertion, inversion and / or deletion, although without impairing the function or efficacy of the promoter. Furthermore, the efficacy of promoters may be increased by changing their sequence, or may be completely exchanged with more effective promoters or even promoters of organisms of different genera.

[0306] d. Generation of functional polypeptide mutants

[0307] In addition, those skilled in the art are familiar with methods for generating functional mutants, that is, a nucleotide sequence encoding a polypeptide 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 SEQ ID NOs disclosed herein with respect to amino acids; and / or encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 70% sequence identity to any of the SEQ ID NOs disclosed herein with respect to nucleotides.

[0308] Depending on the technology used, those skilled in the art can introduce completely random or more targeted mutations into genes or non-coding nucleic acid regions (e.g., important for regulating expression), and subsequently generate genetic libraries. The molecular biology methods required for this purpose are known to the skilled person 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 thereby have long been known to those skilled in the art, for example:

[0310] - site-specific mutagenesis, in which single or multiple nucleotides of a gene are replaced in a targeted manner (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey),

[0311] - saturation mutagenesis, in which the codon for any amino acid can be exchanged or added at any site in the gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22: 1593; Barettino D, Feigenbutz M, Valcárel R, Stunnenberg HG (1994) Nucleic Acids Res 22: 541; Barik S (1995) Mol Biotechnol 3: 1),

[0312] - Error-prone polymerase chain reaction, in which the nucleotide sequence is mutated by an error-prone DNA polymerase (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18: 3739);

[0313] - SeSaM method (sequence saturation method), in which preferred exchange is prevented by the polymerase. Schenk et al., Biospektrum, Vol. 3, 2006, 277-279,

[0314] - gene propagation in mutant strains in which the mutation rate of the nucleotide sequence is increased, 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

[0315] -DNA shuffling, in which a group of closely related genes is created and digested, and these fragments are used as templates for polymerase chain reactions, in which, through repeated strand separation and recombination, the full-length mosaic gene is ultimately generated (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747).

[0316] 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), a skilled worker can produce functional mutants in a directed manner on a large scale. To this end, in a first step, first, a gene library of the respective polypeptide is generated, for example using the methods given above. The gene library is expressed in a suitable manner, for example by means of a bacterial or phage display system.

[0317] The relevant gene of the host organism expressing the functional mutant (its function corresponds to the desired characteristic to a large extent) can be submitted to another mutation cycle. The step of mutation and selection or screening can be repeated iteratively until the functional mutant of the present invention has the desired characteristic to a sufficient degree. Using this iterative process, a limited number of mutations, such as 1, 2, 3, 4 or 5 mutations, can be carried out in stages, and their impact on the studied activity can be evaluated and selected. Then the selected mutant can be subjected to further mutation steps in the same manner. In this way, the number of single mutants to be studied can be significantly reduced.

[0318] The results according to the invention also provide important information about the structure and sequence of related polypeptides, which is necessary for producing other polypeptides with desired modified properties in a targeted manner. In particular, so-called "hot spots" can be defined, i.e. sequence segments that are potentially suitable for modifying properties by introducing targeted mutations.

[0319] Information can also be derived regarding positions in the amino acid sequence where mutations may occur that may have little effect on activity and which can be designated as potential "silent mutations."

[0320] e. Constructs expressing the polypeptides of the present invention

[0321] In the context of this article, the following definitions apply:

[0322] "Gene expression" encompasses "heterologous expression" and "overexpression" and involves transcription of genes and translation of mRNA into proteins. Overexpression refers to the production of gene products measured at the level of mRNA, polypeptide and / or enzyme activity in transgenic cells or organisms that exceeds the production level in non-transformed cells or organisms of similar genetic background.

[0323] As used herein, "expression vector" refers to a nucleic acid molecule that is engineered using molecular biology methods and recombinant DNA technology to deliver foreign or exogenous DNA to a host cell. An expression vector typically includes sequences required for the correct transcription of a nucleotide sequence. The coding region usually encodes a target protein, but may also encode RNA, such as antisense RNA, siRNA, etc.

[0324] As used herein, "expression vector" includes any linear or circular recombinant vector, including but not limited to viral vectors, phages and plasmids. The technician can select a suitable vector according to the expression system. In one embodiment, the expression vector includes the nucleic acid of the embodiments herein, which is operably connected to at least one "regulatory sequence" that controls transcription, translation, initiation and termination, such as a transcriptional promoter, an operator or an enhancer, or an mRNA ribosome binding site, and optionally includes at least one selection marker. When the regulatory sequence is functionally related to the nucleic acid of the embodiments herein, the nucleotide sequence is "operably connected".

[0325] As used herein, "expression system" encompasses any combination of nucleic acid molecules required for co-expression of two or more polypeptides in vivo or in vitro in a given expression host. The respective coding sequences can be located on a single nucleic acid molecule or vector, such as a vector comprising multiple cloning sites, or on a polycistronic nucleic acid, or can be distributed on two or more physically different vectors.

[0326] As used herein, the terms "amplifying" and "amplification" refer to the use of any suitable amplification method for producing or detecting recombinant naturally expressed nucleic acids, as described in detail below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo dT primers) for amplifying (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic DNA or mRNA) or recombinant nucleic acids (e.g., cDNA) of the invention in vivo, ex vivo or in vitro.

[0327] "Regulatory sequence" refers to a nucleic acid sequence that determines the expression level of the nucleic acid sequence of the embodiments herein and is capable of regulating the transcription rate of the nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences include promoters, enhancers, transcription factors, promoter elements, etc.

[0328] According to the present invention, "promoter", "nucleic acid with promoter activity" or "promoter sequence" is understood to refer to a nucleic acid that, when functionally linked to a nucleic acid to be transcribed, regulates the transcription of the nucleic acid. "Promoter" refers in particular to a nucleic acid sequence that controls the expression of a coding sequence by providing a binding site for RNA polymerase and other factors required for transcription, including but not limited to transcription factor binding sites, inhibitors and activator protein binding sites. The term promoter also includes the term "promoter regulatory sequence". The promoter regulatory sequence may include upstream and downstream elements that may affect the stability of transcription, RNA processing or the associated coding nucleic acid sequence. Promoters include sequences of natural origin and synthesis. The coding nucleic acid sequence is usually located downstream of the promoter relative to the transcription direction starting with the transcription start site.

[0329] In the present context, "functional" or "operably" connected is understood to refer to, for example, the sequential arrangement of one of the nucleic acids with a regulatory sequence. For example, a sequence with promoter activity, and a nucleic acid sequence to be transcribed and optional other regulatory elements (for example, a nucleic acid sequence that ensures nucleic acid transcription) and, for example, a terminator, are arranged in such a way that each regulatory element can perform its function after the nucleic acid sequence is transcribed. This does not necessarily require a direct connection in a chemical sense. Genetic control sequences, such as enhancer sequences, can even exert effects on the target sequence from a more distant position or even from other DNA molecules. A preferred arrangement is such that the nucleic acid sequence to be transcribed is located downstream (i.e., 3' end) of the promoter sequence, so that the two sequences are covalently linked together. The distance between the promoter sequence and the nucleic acid sequence to be recombinantly expressed can be less than 200 base pairs, or less than 100 base pairs or less than 50 base pairs.

[0330] Besides promoters and terminators, the following may be mentioned as examples of further regulatory elements: targeting sequences, enhancers, polyadenylation signals, selection 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).

[0331] 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.

[0332] As used herein, the term "operably connected" refers to the connection of polynucleotide elements in a functional relationship. When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably connected". For example, if a promoter or transcriptional regulatory sequence can affect the transcription of a coding sequence, the promoter or transcriptional regulatory sequence is operably connected to the coding sequence. Operably connected means that the DNA sequence connected is usually adjacent. The nucleotide sequence associated with the promoter sequence can be of homologous or heterologous origin relative to the plant to be transformed. The sequence can also be fully or partially synthesized. Regardless of the source, the nucleic acid sequence associated with the promoter sequence will be expressed or silenced according to the promoter properties connected after being attached to the polypeptide of the present embodiment. The relevant nucleic acid can encode the protein that needs to be expressed or suppressed in the whole organism or in a specific tissue, cell or cell chamber at all times or alternatively at a specific time. This kind of nucleotide sequence particularly encodes the protein that the desired phenotypic traits are given to the host cell or organism changed or transformed by it. More particularly, the relevant nucleotide sequence causes one or more target products as defined herein to be produced in a cell or organism. In particular, the nucleotide sequence encodes a polypeptide having enzymatic activity as defined herein.

[0333] The nucleotide sequence as described herein above may be part of an "expression cassette". The terms "expression cassette" and "expression construct" are used synonymously. A (preferably recombinant) expression construct comprises a nucleotide sequence encoding a polypeptide according to the invention and under the genetic control of a regulatory nucleic acid sequence.

[0334] In the methods used according to the invention, the expression cassette may be part of an "expression vector", in particular a recombinant expression vector.

[0335] According to the invention, an "expression unit" is understood to mean a nucleic acid with expression activity, which comprises a promoter as defined herein and, after functional connection with a nucleic acid or gene to be expressed, regulates expression, i.e. the transcription and translation of said nucleic acid or said gene. It is therefore also referred to in this context as a "regulatory nucleic acid sequence". In addition to the promoter, other regulatory elements, such as enhancers, may also be present.

[0336] According to the invention, an "expression cassette" or "expression construct" is understood to be an expression unit which is functionally linked to a nucleic acid to be expressed or a gene to be expressed. Thus, in contrast to an expression unit, an expression cassette contains not only nucleic acid sequences which regulate transcription and translation, but also nucleic acid sequences which are expressed as a protein as a result of transcription and translation.

[0337] In the context of the present invention, the term "expression" or "overexpression" describes the production or increase of the intracellular activity of one or more polypeptides encoded by the corresponding DNA in a microorganism. To this end, for example, the gene can be introduced into the organism, an existing gene can be replaced by another gene, the copy number of the gene can be increased, a strong promoter can be used or a gene encoding a highly active corresponding polypeptide can be used. Alternatively, these measures can be combined.

[0338] Preferably, such constructs according to the invention comprise a promoter 5' upstream and a terminator sequence 3' downstream of the respective coding sequence and optionally further customary regulatory elements, in each case operably linked to the coding sequence.

[0339] The nucleic acid construct according to the invention comprises in particular a sequence encoding a polypeptide, which is derived, for example, from an amino acid related SEQ ID NO as described herein or its reverse complement, or derivatives and homologues thereof, and has been operably or functionally linked to one or more regulatory signals for advantageously controlling, for example increasing, gene expression.

[0340] In addition to these regulatory sequences, the natural regulation of these sequences may still be present before the actual structural gene and may optionally have been genetically modified so that the natural regulation has been turned off and the expression of the gene has been enhanced. However, the nucleic acid construct may also have a simpler construction, i.e., no additional regulatory signals are inserted before the coding sequence and the natural promoter with regulatory effect has not been removed. Instead, the natural regulatory sequences are mutated so that regulation no longer occurs and gene expression is increased.

[0341] Preferred nucleic acid constructs advantageously also comprise one or more " enhancer " sequences already mentioned that are functionally connected to the promoter, and this sequence makes it possible to enhance the expression of the nucleic acid sequence. Other advantageous sequences, such as other regulatory elements or terminators, can also be inserted at the 3 ' end of the DNA sequence. One or more copies of nucleic acid according to the present invention can be present in the construct. In this construct, other markers can also be present optionally, such as genes complementary to auxotrophy or antibiotic resistance, so that this construct can be selected.

[0342] Examples of suitable regulatory sequences are found in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, rhaP (rhaP BAD )SP6, lambda-P R or lambda-P LIn the promoters, they are advantageously used in Gram-negative bacteria. Other advantageous regulatory sequences are present, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. Artificial promoters can also be used for regulation.

[0343] In order to express in the host organism, the nucleic acid construct is advantageously inserted into a carrier, for example a plasmid or a phage, which makes it possible for the optimal expression of the gene in the host. Except for plasmids and phages, carriers are also understood to be all other carriers known to those skilled in the art, i.e., for example viruses, for example SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids and linear or circular DNA or artificial chromosomes. These carriers can replicate autonomously or by chromosome replication in the host organism. These carriers are further developments of the present invention. Binary or cpo integration vectors are also applicable.

[0344] Suitable plasmids are, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113 In pB1, λgt11 or pBdCI, in pIJ101, pIJ364, pIJ702 or pIJ361 in Streptomyces, in pUB110, pC194 or pBD214 in Bacillus, in pSA77 or pAJ667 in Corynebacterium, in pALS1, pIL2 or pBB116 in fungi, in pAG-1, YEp6, YEp13 or pEMBLYe23 in yeast, or in pLGV23, pGHlac+, pBIN19, pAK2004 or pDH51 in plants. The above plasmids are a small selection of possible plasmids. Further plasmids are well known to the skilled worker 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).

[0345] In the further development of the vector, the vector comprising nucleic acid construct of the present invention or nucleic acid of the present invention can also be advantageously introduced into the microorganism in the form of linear DNA and integrated into the genome of the host organism by heterologous or homologous recombination. This linear DNA can be composed of linearized vectors such as plasmids, or only consists of nucleic acid construct of the present invention or nucleic acid.

[0346] For optimal expression of heterologous genes in an organism, it is advantageous to modify the nucleic acid sequence to match the specific "codon usage" used in the organism. "Codon usage" can be readily determined by computer evaluation of other known genes of the organism in question.

[0347] The expression cassette according to the present invention is produced by fusing a suitable promoter to a suitable coding nucleotide sequence and terminator or polyadenylation signal. Conventional recombination and cloning techniques are used for this purpose, for example as described in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and T. J Silhavy, ML Berman and LV Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0348] For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector, which enables optimal expression of the gene in the host. Vectors are well known to the skilled person and can be found, for example, in "cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0349] An alternative embodiment of the embodiments herein provides a method of "changing gene expression in a host cell". For example, under certain contexts (e.g., exposure to a certain temperature or culture conditions), the polynucleotides of the embodiments herein can be enhanced or overexpressed or induced in a host cell or host organism.

[0350] The change of expression of the polynucleotides provided herein can also produce ectopic expression, which is a different expression pattern in the altered and control or wild-type organisms. The change of expression occurs by contact of a polypeptide of an embodiment of the present invention with an exogenous or endogenous regulator or is caused by chemical modification of the polypeptide. The term also refers to the changed expression pattern of the polynucleotides of the embodiments of the present invention, which is changed to below the detection level or completely inhibited activity.

[0351] In one embodiment, also provided herein is an isolated, recombinant or synthetic polynucleotide encoding a polypeptide or variant polypeptide provided herein.

[0352] In one embodiment, multiple nucleic acid sequences encoding polypeptides are co-expressed in a single host, particularly under the control of different promoters. In another embodiment, multiple nucleic acid sequences encoding polypeptides can be present on a single transformation vector, or a separate vector can be used and transformants containing two chimeric genes can be selected to be co-transformed simultaneously. Similarly, one or more polypeptide encoding genes can be expressed in a single plant, cell, microorganism or organism together with other chimeric genes.

[0353] f. Hosts suitable for the present invention

[0354] Depending on the context, the term "host" may refer to a wild-type host or a genetically altered recombinant host or both.

[0355] 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.

[0356] Using the vector according to the present invention, a recombinant host can be produced, which can be transformed with at least one vector according to the present invention, and can be used to produce a polypeptide according to the present invention. Advantageously, the recombinant construct according to the present invention as described above is introduced into a suitable host system and expressed. Preferably, common cloning and transfection methods known to those skilled in the art, such as coprecipitation, protoplast fusion, electroporation, retroviral transfection, etc., are used to express the nucleic acid in respective expression systems. Suitable systems are described 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.

[0357] Advantageously, microorganisms such as bacteria, fungi or yeast are used as host organisms. Advantageously, Gram-positive or Gram-negative bacteria are used, preferably bacteria of the family Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferably Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus. Escherichia coli genus and species are very particularly preferred. Furthermore, other advantageous bacteria are found in the alpha-Proteobacteria, beta-Proteobacteria or gamma-Proteobacteria groups. Advantageously, yeasts such as Saccharomyces cerevisiae or Pichia family are also suitable hosts.

[0358] Alternatively, whole plants or plant cells can be used as natural or recombinant hosts. As non-limiting examples, the following plants or cells derived therefrom may be mentioned: Nicotiana, in particular Nicotiana benthamiana and Nicotiana tabacum (tobacco); and Arabidopsis, in particular Arabidopsis thaliana.

[0359] Depending on the host organism, the organism used in the method according to the invention is grown or cultured in a manner known to those skilled in the art. Cultivation can be carried out in batches, semi-batch or continuously. Nutrients can be given at the beginning of fermentation or can be provided later, semi-continuously or continuously. This is also described in more detail below.

[0360] g. Recombinant production of the polypeptide according to the invention

[0361] The present invention further relates to a method for recombinantly producing a polypeptide according to the invention or a functional biologically active fragment thereof, wherein a microorganism producing the polypeptide is cultivated, expression of the polypeptide is induced optionally by applying at least one inducing agent for inducing gene expression, and the polypeptide is isolated from the culture. If desired, the polypeptide can also be produced on an industrial scale in this way.

[0362] The microorganism produced according to the present invention can be cultivated continuously or discontinuously by batch process or fed-batch process or repeated fed-batch process. An overview of known culture methods can be found in the textbook (Bioprozesstechnik 1.Einführungin die Bioverfahrenstechnik [Bioprocess technology 1.Introduction tobioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) of Chmiel or in the textbook (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheralequipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) of Storhas.

[0363] The culture medium used must suitably meet the requirements of the individual strains. Descriptions of culture media for various microorganisms are given in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981).

[0364] The culture media which can be used according to the invention generally comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0365] Preferred carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be added to the culture medium by other by-products of complex compounds (such as molasses) or sugar refining. It is also advantageous to add a mixture of different carbon sources. Other possible carbon sources are oils and fats, such as soybean oil, sunflower seed 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.

[0366] The nitrogen source is generally an organic or inorganic nitrogen compound or a material containing such compounds. Examples of nitrogen sources include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or composite nitrogen sources, such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or in combination.

[0367] Inorganic salt compounds that may be present in the culture medium include chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0368] Inorganic sulfur-containing compounds, such as sulfates, sulfites, dithionites, tetrasulfates, thiosulfates, sulfides, and organic sulfur compounds, such as mercaptans and thiols, can be used as sulfur sources.

[0369] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.

[0370] Chelating agents may be added to the culture medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0371] The fermentation medium used according to the present invention also generally comprises 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 generally derived from the components of complex culture media, such as yeast extract, molasses, corn steep liquor, etc. In addition, suitable precursors can be added to the culture medium. The exact composition of the compound in the culture medium depends to a large extent on the corresponding experiment, and is determined respectively for each specific case. Information about culture medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed. PM Rhodes, PF Stanbury, IRL Press (1997) p.53-73, ISBN 0 19 963577 3). Growth medium can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) etc.

[0372] All components of the medium are sterilized by heating (20 minutes at 1.5 bar and 121°C) or by sterile filtration. The components can be sterilized together or individually as required. All components of the medium can be given at the beginning of the culture or added continuously or in batches.

[0373] The temperature of the culture is usually between 15°C and 45°C, preferably 25°C to 40°C, and can be changed or kept constant during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH value during growth can be controlled by adding alkaline compounds (such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water) or acidic compounds (such as phosphoric acid or sulfuric acid). Defoamers such as fatty acid polyethylene glycol esters can be used to control foaming. In order to maintain the stability of the plasmid, suitable selective substances such as antibiotics can be added to the culture medium. In order to maintain aerobic conditions, oxygen or an oxygen-containing gas mixture (such as ambient air) is supplied to the culture. The temperature of the culture is usually in the range of 20°C to 45°C. Continue to cultivate until the maximum amount of the desired product is formed. This goal will usually be achieved within 10 to 160 hours.

[0374] 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 remain completely therein.

[0375] If the polypeptide is not secreted in the culture medium, the cells can also be lysed and the product can be obtained from the lysate by known methods for isolating proteins. The cells can be disrupted optionally by high-frequency ultrasound, high pressure, for example in a high-pressure cell lyser (French press), by osmosis, by the action of detergents, lytic enzymes or organic solvents, by a homogenizer or by a combination of several of the above methods.

[0376] The polypeptide can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), such as Q-agarose chromatography, ion exchange chromatography and hydrophobic chromatography, and other conventional techniques, such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, TG, Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, New York, or Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0377] In order to separate recombinant proteins, it may be advantageous to use a carrier system or oligonucleotides that extend the cDNA by a limited nucleotide sequence, and thus encode a changed polypeptide or fusion protein, which is, for example, used for easier purification. Suitable modifications of this type are, for example, so-called "tags" that serve as anchors, such as modifications of six-histidine anchors or epitopes that can be identified as antibody antigens (e.g., described in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press). These anchors can be used to connect proteins to solid supports, such as polymer matrices, which can, for example, be used as fillers in chromatographic columns, or can be used on microtiter plates or other carriers.

[0378] At the same time, these anchors can also be used to identify proteins. In order to identify proteins, common labels such as fluorescent dyes, enzyme labels (forming a detectable reaction product after reacting with a substrate), or radioactive labels can also be used, alone or in combination with anchors to derivatize proteins.

[0379] h. Immobilization of Peptides

[0380] Enzyme or polypeptide according to the present invention can be used in free form or immobilized in the method described herein.Immobilized enzyme is an enzyme fixed on an inert carrier. Suitable carrier materials and enzymes fixed thereon are known from EP-A-1149849, EP-A-1069183 and DE-OS100193773 and from the references cited therein. In this respect, the full disclosure of these documents is referred to. Suitable carrier materials include, for example, clay, clay minerals, such as kaolinite, diatomaceous earth, perlite, silicon dioxide, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, synthetic polymers, such as polystyrene, acrylic resin, phenolic resin, polyurethane and polyolefin, such as polyethylene and polypropylene. In order to prepare the enzyme of load, carrier materials are usually used in the form of subdivided particles, preferably in a porous form. The particle diameter of the carrier material is usually not more than 5mm, particularly not more than 2mm (particle size distribution curve). Similarly, when using dehydrogenase as a whole cell catalyst, free form or fixed form can be selected. Carrier materials are, for example, calcium alginate and carrageenan. Enzymes and cells can also be directly crosslinked with glutaraldehyde (crosslinked with CLEAs). Corresponding and other immobilization techniques are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol 3, Chapter 17, VCH, Weinheim gives further information on bioconversion and bioreactors for carrying out the method according to the invention.

[0381] i. Reaction conditions of the biocatalytic production method of the present invention

[0382] The reaction of the present invention can be carried out under in vivo or in vitro conditions.

[0383] The at least one polypeptide / enzyme present in the method of the invention or in a single step of the multi-step method defined above may be naturally present in living cells, or in harvested cells (i.e. under in vivo conditions), dead cells, permeabilized cells, crude cell extracts, purified extracts, or in substantially pure or completely pure form (i.e. under in vitro conditions), recombinantly produced one or more enzymes. The at least one enzyme may be present in solution or in the form of an enzyme immobilized on a support. One or more enzymes may be present in both soluble and / or immobilized form.

[0384] The method according to the invention can be carried out in common reactors known to those skilled in the art and can be carried out in different scales, for example from laboratory scale (a few milliliters to tens of liters of reaction volume) to industrial scale (a few liters to thousands of cubic meters of reaction volume). If the polypeptide is used in the form of encapsulation by inanimate, optionally permeabilized cells, in the form of more or less purified cell extracts or in purified form, a chemical reactor can be used. Chemical reactors generally allow the control of the amount of at least one enzyme, the amount of at least one substrate, pH, temperature and the circulation of the reaction medium. When at least one polypeptide / enzyme is present in living cells, the process will be fermentation. In this case, biocatalytic production will be carried out in a bioreactor (fermenter), where the parameters necessary for the suitable living conditions of living cells (e.g., culture medium with nutrients, temperature, aeration, aerobic or anaerobic or other gases, antibiotics, etc.) can be controlled. The person skilled in the art is familiar with chemical reactors or bioreactors, for example using procedures for scaling up chemical or biotechnological methods from the laboratory scale to the industrial scale or for optimizing process parameters, which are also widely described in the literature (for biotechnological methods, see, for example, Crueger und Crueger, Biotechnologie—Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, München, Wien, 1984).

[0385] The cells containing at least one enzyme can be permeabilized by physical or mechanical means such as ultrasound or radio frequency pulses, high pressure cell lysers (French press) or chemical means such as the presence of a hypotonic medium, a lysing enzyme and a detergent in the culture medium or a combination of these methods. Examples of detergents are digitoxin, n-dodecyl maltoside, octyl glucoside, X-100, 20, deoxycholate, CHAPS (3-[(3-chloroamidopropyl) dimethylammonium]-1-propane sulfonate), P40 (ethylphenol poly (ethylene glycol ether)) and the like.

[0386] Instead of living cells, non-living cell biomass containing the desired biocatalyst can also be applied to the bioconversion reaction of the present invention.

[0387] If at least one enzyme is immobilized, it is attached to an inert support as described above.

[0388] The conversion reaction can be carried out batchwise, semi-batchwise or continuously. The reactants (and optionally nutrients) can be provided at the start of the reaction, or can be provided subsequently semi-continuously or continuously.

[0389] Depending on the particular reaction type, the reactions of the present invention can be carried out in aqueous, aqueous-organic or non-aqueous reaction media.

[0390] The aqueous or aqueous-organic medium may contain a suitable buffer to adjust the pH to a value between 5 and 11, such as between 6 and 10.

[0391] In aqueous-organic media, organic solvents that are miscible, partially miscible or immiscible with water may be used. Non-limiting examples of suitable organic solvents are listed below. Further examples are monobasic or polybasic, aromatic or aliphatic alcohols, particularly polybasic aliphatic alcohols, such as glycerol.

[0392] The non-aqueous medium may comprise substantially no water, ie, will comprise less than about 1 wt% or 0.5 wt% water.

[0393] The biocatalytic process can also be carried out in an organic non-aqueous medium. As suitable organic solvents, mention may be made of 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 and ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether; or mixtures thereof.

[0394] The concentration of reactants / substrates may be adapted to the optimal reaction conditions, which may depend on the specific enzyme applied. For example, the initial substrate concentration may be 0.1 to 0.5 M, such as 10 to 100 mM.

[0395] The reaction temperature can be adapted to the optimal reaction conditions, which may depend on the specific enzyme being applied. For example, the reaction can be carried out at a temperature of 0 to 70° C., such as 20 to 50 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.

[0396] The process can be continued until equilibrium is reached between the substrate and the subsequent product, but can be stopped earlier. Common process times are from 1 minute to 25 hours, particularly from 10 minutes to 6 hours, for example from 1 hour to 4 hours, particularly from 1.5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.

[0397] If the host is a transgenic plant, optimal growth conditions can be provided, such as optimal light, water, and nutrient conditions.

[0398] k. Product separation

[0399] The method of the present invention may further include the step of recovering the final product or intermediate product, which is optionally in the substantially pure form of a stereoisomer or enantiomer. The term "recovery" includes extracting, harvesting, separating or purifying the compound from a culture medium or reaction medium. The recovery of the compound can be carried out according to any conventional separation or purification method known in the art, including but not limited to treatment with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, aluminum oxide, etc.), changes in pH, solvent extraction (e.g., using conventional solvents, such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, freeze drying, etc.

[0400] 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 (e.g. IR, UV, NMR), staining methods, TLC, NIRS, enzyme or microbiological assays (see, for example: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 1127-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S. 89-90, S. 521-540, S. 540-547, S. 559-566, 575-581 and S.581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.).

[0401] The cyclic terpene compounds produced by any of the methods described herein can be converted into derivatives such as, but not limited to, hydrocarbons, esters, amides, glycosides, ethers, epoxides, aldehydes, ketones, alcohols, diols, acetals or ketals. Terpene compound derivatives can be obtained by chemical methods such as, but not limited to, oxidation, reduction, alkylation, acylation and / or rearrangement. Alternatively, terpene compound derivatives can be obtained by contacting the terpene compound with an enzyme using a biochemical method, such as, but not limited to, an oxidoreductase, a monooxygenase, a dioxygenase, a transferase. Biochemical transformations can be performed in vitro using isolated enzymes, enzymes from lysed cells, or in vivo using whole cells or organisms.

[0402] 1. Fermentation production of fennel diphosphate and / or fennel alcohol

[0403] The present invention also relates to a method for producing zirconia diphosphate and / or zirconia alcohol by fermentation.

[0404] Fermentation used according to the invention can be carried out, for example, in stirred fermenters, bubble columns and loop reactors. For a comprehensive overview of possible process types, including agitator types and geometric designs, see "Chmiel: Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Band 1". In the method of the invention, available typical variants are the following variants known to those skilled in the art or explained, for example, in "Chmiel, Hammes and Bailey: Biochemical Engineering", such as batch, fed-batch, repeated fed-batch or continuous fermentation, with or without recovery of biomass. Depending on the production strain, injection of air, oxygen, carbon dioxide, hydrogen, nitrogen or a suitable gas mixture can be carried out to achieve a good yield (YP / S).

[0405] The culture medium to be used must meet the requirements of the specific strain in an appropriate manner. Descriptions of culture media for various microorganisms are given in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981).

[0406] The culture media which can be used according to the invention generally comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0407] Preferred carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be added to the culture medium by other by-products of complex compounds (such as molasses) or sugar refining. It is also advantageous to add a mixture of various carbon sources. Other possible sources of carbon are oils and fats, such as soybean oil, sunflower seed 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.

[0408] The nitrogen source is generally an organic or inorganic nitrogen compound or a material containing such compounds. Examples of nitrogen sources include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or composite nitrogen sources, such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or in combination.

[0409] Inorganic salt compounds that may be present in the culture medium include chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0410] Inorganic sulfur-containing compounds, such as sulfates, sulfites, dithionites, tetrasulfates, thiosulfates, sulfides, and organic sulfur compounds, such as mercaptans and thiols, can be used as sulfur sources.

[0411] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.

[0412] Chelating agents may be added to the culture medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0413] The fermentation medium used according to the present invention may also contain other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, niacin, pantothenic acid and pyridoxine. Growth factors and salts are usually derived from complex components of the culture medium, such as yeast extract, molasses, corn steep liquor, etc. In addition, suitable precursors may be added to the culture medium. The precise composition of the compounds in the culture medium depends largely on the specific experiment and must be determined separately for each specific case. Information on culture medium optimization can be found in the textbook "Applied Microbiol. Physiology, APractical Approach" (1997). Growth medium can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO), etc. .

[0414] All components of the medium are sterilized by heating (20 minutes at 1.5 bar and 121°C) or by sterile filtration. The components can be sterilized together or separately as required. All components of the medium can be given at the beginning of growth, or you can choose to add them continuously or in batches.

[0415] The temperature of the culture is usually between 15°C and 45°C, preferably 25°C to 40°C, and can be kept constant or can be changed during the experiment. The pH value of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH value during growth can be controlled by adding alkaline compounds (such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water) or acidic compounds (such as phosphoric acid or sulfuric acid). Defoamers such as fatty acid polyethylene glycol esters can be used to control foaming. In order to maintain the stability of the plasmid, suitable substances such as antibiotics with selective effects can be added to the culture medium. In order to maintain aerobic conditions, oxygen or an oxygen-containing gas mixture (such as ambient air) is supplied to the culture. The temperature of the culture is usually 20°C to 45°C. Continue to cultivate until the maximum amount of the desired product is formed. This goal will usually be achieved within 1 to 160 hours.

[0416] The method of the present invention may further include a step of recovering fennel diphosphate and / or fennel alcohol.

[0417] The term "recovery" includes extracting, harvesting, isolating or purifying compounds from a culture medium. The recovery of compounds can be carried out according to any conventional separation or purification method known in the art, including but not limited to treatment with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), pH changes, solvent extraction (e.g., using conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, freeze drying, etc.

[0418] Before the expected separation, the biomass of the fermentation liquid can be removed. The method of removing the biomass is well known to those skilled in the art, such as filtering, sedimentation and flotation. Therefore, the biomass can be removed, for example, by a centrifuge, a separator, a decanter, a filter or in a flotation device. In order to reclaim the valuable product to the greatest extent, it is usually recommended to wash the biomass, for example in the form of diafiltration. The selection of the method depends on the content of the biomass in the fermentation liquid and the character of the biomass, and the interaction of the biomass and the valuable product.

[0419] In one embodiment, the fermented liquid can be sterilized or pasteurized. In another embodiment, the fermented liquid is concentrated. Depending on the need, the concentration can be carried out in batches or continuously. The pressure and temperature range should be selected so that firstly product damage does not occur and secondly the use of equipment and energy needs to be minimized. The skilled selection of the pressure and temperature levels of multi-stage evaporation can especially save energy.

[0420] The following examples are illustrative only and are not meant to limit the scope of the embodiments and claims described herein.

[0421] Numerous possible variations that will become immediately apparent to those skilled in the art after considering the disclosure provided herein also fall within the scope of the present invention.

[0422] Experimental Section

[0423] The present invention will now be described in more detail by way of the following examples.

[0424] Material:

[0425] Unless otherwise specified, all chemical and biochemical materials and microorganisms or cells used herein are commercially available products.

[0426] Unless otherwise indicated, recombinant proteins were cloned and expressed by standard methods, such as those described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0427] method:

[0428] Standard assay for measuring pyrrolidone diphosphate synthase activity (in vitro)

[0429] Suitable methods are described in Examples 5 and 7 below.

[0430] Gas chromatography-mass spectrometry (GC-MS)

[0431] An Agilent 6890 series gas chromatograph system was used, equipped with a DB1-ms column, 30m×0.25mm×0.25μm film thickness (P / N 122-0132, J&W scientific Inc., Folsom, CA), and connected to a 5975 series mass spectrometer. The carrier gas was helium with a flow rate of 0.7mL / min. The injector was injected in split (1:25) mode, and the injector temperature was set to 250°C. The oven temperature was programmed from 50°C (held for 5 minutes) to 300°C at a rate of 5°C / min, then to 340°C at a rate of 50°C / min and held for 3 minutes.

[0432] Example 1

[0433] Procurement of plant material and leaf transcriptome sequencing.

[0434] Plant material from the genus Dryopteris was collected from northern China. To determine whether Dryopteris fragrans contains fennel, fresh leaves were extracted with dichloromethane for chemical analysis. The extracts were analyzed by GC / MS with the above parameters. The identification of the products was based on the mass spectra and the retention index ( Figure 2 and Figure 3 ).

[0435] Dryopteris odoratus samples with IDs PNLI20141074 and YGY-15-2706 were used for transcriptome analysis. Total RNA from Dryopteris odoratus was extracted using RNeasy Plant Mini Kit (50) 74904 from QIAGEN.

[0436] Use the Total RNA sample PNLI20141074 was processed with UltraTM RNA library preparation kit (NEB, USA) and TruSeqPE cluster kit (Illumina, USA) and then sequenced on an Illumina Miseq sequencer. 20.88 million 2x 350bp paired end reads were generated. These reads were assembled together using Trinity (http: / / trinityrnaseq.sf.net / ) software to obtain 85753 contigs with an N50 of 1373. Contigs were analyzed using EMBOSS software (http: / / emboss.sourceforge.net / ) to obtain protein sequences. The sequence of AstC from the prior art [Y. Shinohara, S. Takahashi, H. Osada & Y. Koyama (2016) Identification of a novel sesquiterpene biosynthetic machinery involved in astellolide biosynthesis. Sci. Rep. 6: 32865 (DOI: 10.1038 / srep32865).] was used to search for potential sesquiterpene synthases in the protein sequence data of Dryopteris odorifera. This approach provided DfHAD, a 5' sequence of an atypical haloacid-dehalogenase-like hydrolase, which was highly expressed in the transcriptome by CLC genomics workbench analysis.

[0437] In addition to PNLI20141074, also use SMRTbell TM Template preparation kit (PacBio, USA) and Total RNA sample YGY-15-2706 was treated with DNA / polymerase kit (PacBio, USA) and then sequenced on PacBio RS II sequencer. 960,000 reads with an average length of 2302 bp were generated. These reads were analyzed by Smrtanalysis (v_2.3.0) (http: / / www.pacb.com) and 24,848 contigs with N50 of 1448 were obtained, which contained the 3' sequence of DfHAD. By combining these two methods, the putative full length of DfHAD was assembled and used for gene cloning.

[0438] First use SMARTer TMThe total RNA sample of YGY-15-2706 was reverse transcribed to cDNA by RACE cDNA amplification kit (Clontech). The product was then used as a template for gene cloning. DfHAD and its variants DfHAD-8 (K532R) and DfHAD-9 (V274A) were amplified from cDNA using forward primer (5'-ATGGAGTTCTCTGCCTCTG-3') (SEQ ID NO: 15) and reverse primer (5'-GGTTTGGCTTATGGAAGGT-3') (SEQ ID NO: 16). The enzymatic activity of DfHAD and DfHAD-8 (K532R) and DfHAD-9 (V274A) was assessed as described in Examples 2 and 3.

[0439] Example 2

[0440] Functional expression and characterization of DfHAD in an E. coli expression system.

[0441] The sequences of DfHAD and its variants DfHAD-8 (K532R) and DfHAD-9 (V274A) were optimized by following the Genscript genetic codon frequency of E. coli. They were then synthesized in vitro and subcloned into pETDuet-1 plasmid (Novagen) or pJ401 plasmid (DNA 2.0) for subsequent expression in E. coli.

[0442] BL21 (DE3) E. coli cells (Tiangen) were co-transformed with plasmid pACYC-29258-4506, which contains genes encoding a heterologous mevalonate pathway, and plasmids pETDuet-DfHAD or pETDuet-DfHAD-8 (K532R) or pETDuet-DfHAD-9 (V274A). To increase the productivity of the cells, the heterologous FPP synthase and enzymes from the complete heterologous mevalonate (MVA) pathway were expressed in the same cells. The construction of an expression plasmid comprising a FPP synthase gene and genes for a complete MVA pathway is described in patent WO2013064411 or M. Schalk, L. Pastore, M. A. Mirata, S. Khim, M. Schouwey, F. Deguerry, V. Pineda, L. Rocci & L. Daviet (2012) Toward a biosynthetic route to sclareol and amber odorants. J. Am. Chem. Soc. 134, 18900-3 (DOI: 10.1021 / ja307404u). In short, an expression plasmid was prepared comprising two operons consisting of genes encoding enzymes for a complete mevalonate pathway. The first synthetic operon consisted of the Escherichia coli acetoacetyl-CoA thiolase (atoB), Staphylococcus aureus HMG-CoA synthase (mvaS), Staphylococcus aureus HMG-CoA reductase (mvaA), and Saccharomyces cerevisiae FPP synthase (ERG20) genes, which were synthesized in vitro (DNA2.0, Menlo Park, CA, USA) and ligated into the NcoI-BamHI-digested pACYCDuet-1 vector (Invitrogen) to generate pACYC-29258. A second operon containing mevalonate kinase (MvaK1), phosphomevalonate kinase (MvaK2), mevalonate diphosphate decarboxylase (MvaD) and isopentenyl diphosphate isomerase (idi) was amplified from genomic DNA of Streptococcus pneumoniae (ATCC BAA-334) and ligated into the second multiple cloning site of pACYC-29258, providing plasmid pACYC-29258-4506. Thus, this plasmid contains genes encoding all enzymes of the biosynthetic pathway from acetyl-CoA to FPP.

[0443] BL21(DE3) E. coli cells (Tiangen) were co-transformed with plasmid pACYC / ScMVA, which contains genes encoding the heterologous mevalonate pathway, and plasmid pETDuet-DfHAD or pETDuet-DfHAD-8(K532R) or pETDuet-DfHAD-9(V274A).

[0444] To construct the pACYC / ScMVA plasmid, we grouped the eight biosynthetic genes into two synthetic operons, termed the "upper" and "lower" mevalonate (MVA) pathways. As the upper MVA pathway, we created a synthetic operon consisting of acetoacetyl-CoA thiolase from Escherichia coli encoded by atoB, HMG-CoA synthase from Saccharomyces cerevisiae encoded by ERG13 and ERG19, respectively, and a truncated form of HMG-CoA reductase. This operon converts the major metabolite acetyl-CoA into (R)-mevalonate. As the "lower" mevalonate pathway, we created a second synthetic operon encoding mevalonate kinase (ERG12, Saccharomyces cerevisiae), phosphomevalonate kinase (ERG8, Saccharomyces cerevisiae), phosphomevalonate decarboxylase (MVD1, Saccharomyces cerevisiae), isopentenyl diphosphate isomerase (idi, E. coli), and farnesyl pyrophosphate (FPP) synthase (IspA, E. coli). Finally, a second FPP synthase (ERG20) from Saccharomyces cerevisiae was introduced into the upper pathway operon to improve the conversion of isoprenoid C5 units (IPP and DMAPP) to farnesyl pyrophosphate (FPP). Each operon was subcloned into one of the multiple cloning sites of a low-copy expression plasmid under the control of the bacteriophage T7 promoter (pACYCDuet-1, Invitrogen). Thus, this plasmid contains genes encoding all enzymes of the biosynthetic pathway from acetyl-CoA to FPP.

[0445] The co-transformed cells were selected on LB agar plates containing kanamycin (final concentration of 50 μg / mL) or ampicillin (final concentration of 50 μg / mL) (optional) and chloramphenicol (final concentration of 35 μg / mL). A single colony was used to inoculate 5 mL of liquid LB medium supplemented with the same antibiotics. The culture was incubated overnight at 37°C with 200 rpm shaking. The next day, 0.2 mL of overnight culture was inoculated into 2 mL of TB medium supplemented with the same antibiotics and glycerol (final content of 3% w / v). After incubation at 37°C for 5 hours and shaking at 200 rpm, the culture was cooled to 25°C, shaken at 200 rpm for 1 hour, and then IPTG (final 0.1 mM) was added to each test tube and covered with 200 μL of decane. The culture was incubated at 25°C for another 48 hours at 200 rpm. The culture was then extracted twice with 1 volume of ethyl acetate. 50 μl of 2 mg / mL of isolongifolene was added to the organic phase as an internal standard, and the samples were then analyzed by GC / MS. The GC / MS analysis used the same system as described in Example 1. The carrier gas was helium with a constant flow rate of 0.7 mL / min. The injector was in split (1:25) mode, and the injector temperature was set to 250°C. The oven temperature was programmed to heat from 50°C (held for 5 minutes) to 300°C at a rate of 5°C / min, and then to 340°C at 50°C / min and held for 3 minutes. The identification of the products was based on mass spectrometry and retention indices. GC / MS analysis showed that the major product produced by recombinant cells expressing DfHAD, DfHAD-8(K532R) and DfHAD-9(V274A) was pyrocatechol, with titers ranging from 0.2 mg / L to 3 mg / L ( Figure 4 and Figure 5 ).

[0446] Example 3

[0447] Functional expression and characterization of DfHAD in a transient tobacco expression system.

[0448] DfHAD was integrated into the modified binary vector pMTPS1 to form pMTPS1-DfHAD. pMTPS1 is an engineered vector based on the commercial plasmid pCAMBIA2300 (Cambia). The Cauliflower Mosaic Virus promoter (CaMV35S) was inserted between the EcoRI and KpnI restriction enzyme sites. The octopine synthase (OCS) terminator was inserted between PstI and HindIII. The NPTII selection marker in the left and right borders of the transfer DNA (T-Border) of pCAMBIA2300 was replaced by the codon-optimized Gallus gallus farnesyl diphosphate synthase (GgFPS) using the XhoI restriction endonuclease, and then the direction was confirmed; the box of the RNA silencing inhibitor P19 of the tomato bush stunt virus (Tomato bush stunt virus) controlled by the cauliflower mosaic virus 35S promoter and terminator was assembled into the HindIII site to deliver pMTPS1. Finally, the codon-optimized DfHAD was inserted between SalI and PstI. Thus, the binary vector pMTPS1 was formed to produce the substrate farnesyl pyrophosphate (FPP), which was then converted into folded leaf moss alcohol.

[0449] 300ng plasmid pMTPS1-DfHAD is transformed into Agrobacterium tumefaciens EHA105. On LB-agarose plates containing kanamycin (50 μg / mL) and rifampicin (25 μg / mL), the transformed cells are selected and incubated at 28°C for 2 days. Single colony is inoculated into 50mL LB with the same antibiotics and incubated overnight at 28°C in a shaker at a speed of 200rpm. When OD600 reaches 1.0, the culture is centrifuged at room temperature at 5000rpm for 10 minutes. Cell pellet is resuspended in 20mL of MgSO4 (10mM). Centrifuge the suspension again, and resuspend the cell pellet with acetosyringone (AS) solution (765 μM) to adjust OD600 to 0.5. The resuspension was infiltrated into the intercellular space from the back of tobacco leaves (Nicotiana benthamiana). The tobacco plants had previously been grown for 4 weeks at 28°C, 12 hours of light / 12 hours of darkness. Three days after infection, the infected leaves were fed with mevalonic acid (25mM). After 8 hours, the leaf samples were harvested, and about 500-1000mg of leaves were weighed into 10mL Eppendorf tubes and immediately frozen in liquid nitrogen. The samples were ground into powder and extracted overnight with 2mL of ethyl acetate containing 20μg / ml dodecane as an internal standard. Each sample was filtered, and the filtrate was dried with anhydrous Na2SO4 and then analyzed by GC / MS. The tobacco samples were extracted with 4ml ethyl acetate (3.4ppm of dodecane was added). The extracts were analyzed by GC / MS with the above parameters. Samples were injected in split (1:5) mode, and the injector temperature was set to 250°C.

[0450] In this tobacco transient transformation experiment, infected plants expressing DfHAD produced pyrocatechol as the major peak based on the mass spectra and retention indices in GC / MS analysis ( Figure 6 and Figure 7 ). In this experiment, the yield of zizyphus mol was calculated based on the internal standard, and each gram of fresh tobacco leaves contained 3.75 μg of zizyphus mol.

[0451] Example 4

[0452] Functional expression and characterization of mutant DfHAD in an Escherichia coli expression system.

[0453] A series of DfHAD mutants were constructed to confirm the importance of the identified class I and class II motifs for its terpene synthase activity (see Fig.10 The corresponding genes were inserted into pJ401 and the activity was evaluated as described in Example 2.

[0454] The importance of the atypical class I insertion SF and the atypical class II motif H was highlighted in this experiment:

[0455] - 90% loss of activity after SF removal

[0456] - When the basic residue H was replaced by the acidic residue D, the activity was lost by 47%

[0457] -100% activity loss when removing SF and replacing H with D

[0458] Perhaps more surprisingly, replacement of the acidic residue E with the functional equivalent D resulted in an 85% loss of activity. Double mutations of D to E and E to D resulted in a 99% loss of activity.

[0459] Example 5

[0460] Characterization of purified DfHAD in in vitro systems.

[0461] To purify DfHAD to evaluate its in vitro activity, its coding sequence (optimized for E. coli expression) was subcloned into the pGS-21a plasmid (Genscript). The expressed protein was fused with an N-terminal 6xHis-GST-tag (SEQ ID NO: 18, encoded by SEQ ID NO: 17). After transformation in BL21 (DE3) E. coli cells and selection on LB-agar plates containing ampicillin (final concentration of 50 μg / mL), a single colony was inoculated into 40 mL LB medium supplemented with the same antibiotics, and the culture was incubated at 37 ° C at 180 rpm overnight. The next day, 5-10 mL of culture was inoculated into 500 mL of 2X YT medium containing 50 mg / L ampicillin and incubated at 37 ° C at 150 rpm for about 3 hours (OD600 = 0.7-1.2). The culture was then cooled to 25°C for 1.5 hours, and 0.25 mL of 1 M IPTG was added, followed by incubation at 25°C for 18 hours at 150 rpm. The culture was harvested by centrifugation at 3500 g for 15 minutes at 4°C, and the precipitate was washed twice with cooling buffer A (50 mM Na2HPO4, 10 mM imidazole, 500 mM NaCl, pH 7.8). The precipitate was resuspended in buffer A (without SigmaFast EDTA, Sigma) containing protease inhibitors, and the volume was adjusted to 25 mL. 25 mg lysozyme, 25 μL DNase I, 125 μL MgCl2 and 60 μL 0.2 M PMSF were added to the suspension. After incubation on ice for 30 minutes, followed by ultrasonic treatment (open for 3 seconds, close for 12 seconds, a total of 3 minutes, repeated once), the supernatant was collected by centrifugation at 4°C and 8000 g for 10 minutes. The protein was purified by a batch process on glutathione-agarose (Sigma G4510) according to the manufacturer's protocol for GST-tagged proteins. The molecular weight of the protein was estimated by SDS-PAGE and compared with a molecular weight standard (PAGE-MASTER Plus, Genescript), corresponding to the molecular weight calculated from the amino acid sequence of DfHAD (65 kDa) plus the amino acid sequence of GST-Tag (21 kDa) (i.e. 91 kDa).

[0462] For in vitro biochemical assay of DfHAD protein, 1.5 μM of purified recombinant protein was added to 2 ml reaction mixture containing 10 mM MgCl2, 5 mM DTT and 29 μM FPP in 50 mM Tris-HCl pH 8.0 buffer. The sample was incubated at 30 ° C at 50 rpm for 2 hours. The sample was then divided into two parts, and 20 μL of bacterial alkaline phosphatase (BAP) (Sangon-B004081-100) was added to one of them, and the reaction was continued at 25 ° C for 1 hour. The BAP enzyme EC3.1.3.1 is derived from Escherichia coli, and its sequence information can be obtained from Genbank accession number M13345. A control without added protein was also run under the same conditions. For GC / MS analysis, 10 μL of 2 mg / mL of isolongifolene (internal standard) was added to a 0.5 mL aliquot as an internal standard. The reactants were then extracted with 0.25 mL of ethyl acetate and GC / MS was performed as described above.

[0463] After GC-MS analysis, Figure 8 As shown in Figure 2, we were unable to detect any pyrocatechol. Since the previous in vivo assay was successful and the bacterial extract contained a phosphatase from E. coli, we speculated that this might be due to the enzyme forming pyrocatechol instead of pyrocatechol, which is comparable to the catalytic performance of AstC. Therefore, we performed the same experiment in the presence of bacterial alkaline phosphatase (BAP) ( Figure 8 Indeed, the addition of this commercial phosphatase allowed us to detect fennel in our samples, as well as the formation of farnesol from FPP, suggesting that fennel diphosphate is formed via a protonation-initiated mechanism.

[0464] Example 6

[0465] Synthesis of pyrocatechol diphosphate and monophosphate

[0466] These syntheses were based on two previous publications: Lira LM et al (2013), Tetrahedron Lett, 54, 1690-1692; Thulasiram HV et al (2006), J Org Chem, 71, 1739-1741.

[0467] Preparation of pyrocatechol diphosphate

[0468] To a solution of fennel (64 mg, 0.288 mmol) in 2,2,2-trichloroacetonitrile (0.721 ml, 7.20 mmol) was added 0.70 mL of TEAP (bistriethylammonium phosphate, see Note 1) at room temperature. Two 0.720 mL of TEAP were added at 5 minute intervals. The resulting mixture was stirred for 3 hours. It was then loaded onto a silica gel column and eluted with 150 mL of 100% ethyl acetate and 90 mL of a mixture of isopropanol: NH4OH: H2O (6.0:2.5:0.5) to give fraction 1 (discarded) and fraction 2, respectively. Fraction 2 was concentrated under reduced pressure and diluted with 10 mL of a 10% MeOH / H2O mixture. The resulting mixture was loaded onto a pre-conditioned (see Note 2) Waters SPE: Oasis MCX 6cc (500 mg) LP extraction column and eluted with 20 mL of 10% MeOH / H2O to obtain fractions 1 and 2, and then eluted with 20 mL of 50% MeOH / H2O to obtain fractions 3 and 4. Fractions 3 and 4 were combined and lyophilized to obtain off-white solid fennel diphosphate (35 mg, 0.092 mmol, 31%).

[0469] Note:

[0470] 1) TEAP was prepared by mixing 0.91 mL of a solution of 2 mL phosphoric acid in 7.82 mL acetonitrile and 1.5 mL of a solution of 4.4 mL triethylamine in 4.0 mL acetonitrile (J Org Chem, 71, 1739-1741).

[0471] 2) The column was first eluted with 5 mL MeOH and then with 5 mL H2O.

[0472] Preparation of pyrocatechol monophosphate

[0473] Tetrabutylammonium dihydrogen phosphate (133 mg, 0.391 mmol) was added to a round-bottom flask containing pyrrol (87 mg, 0.391 mmol) and 2,2,2-trichloroacetonitrile (1 ml, 9.97 mmol). The resulting mixture was stirred for 48 hours. It was then loaded onto a silica gel column and eluted with 150 mL of ethyl acetate and 90 mL of a mixture of isopropanol: NH4OH: H2O (6.0:2.5:0.5) to give fractions 1 (discarded) and 2, respectively. Fraction 2 was concentrated under reduced pressure (until about 12 mL of liquid remained), loaded onto a pre-adjusted (see Note 1) DOWEX50WX8-2 column, and washed with 100 mL of a 0.025 M NH4HCO3 solution. The resulting filtrate was collected and lyophilized to give pyrrol monophosphate (see Note 2) (9 mg, 0.025 mmol, 7%) as a "sticky" white solid.

[0474] Note:

[0475] 1) A chromatography column (21 cm high, 3.5 cm diameter) was filled with Dowex 50WX8 ion exchange beads to a depth of 5 cm and flushed first with NH4OH / H2O 3:1 mixture (60 mL, CAUTION: exothermic) and then with 0.025 M NH4HCO3 solution (120 mL or until the pH of the filtrate is between 8.5 and 9.5).

[0476] 2) Contaminated with 14% pyrocatechol diphosphate.

[0477] Example 7

[0478] High-resolution mass spectrometry detection of pyrocatechol diphosphate and monophosphate as DfHAD enzyme products

[0479] Preparation of Escherichia coli extracts from culture broth

[0480] The sequence of DfHAD is optimized by the genetic codon frequency of Escherichia coli. It is then synthesized in vitro and subcloned into the pETDuet-1 plasmid (Novagen), and then expressed in Escherichia coli BL21 (DE3). Escherichia coli cells (Tiangen) are also co-transformed with plasmid pACYC / ScMVA (comprising genes encoding heterologous mevalonate pathways) and plasmid pETDuet-DfHAD. Co-transformed cells are selected on LB agar plates containing ampicillin (final concentration of 50 μg / mL) and chloramphenicol (final concentration of 34 μg / mL). 5mL liquid LB culture medium supplemented with the same antibiotics is inoculated using a single colony. The culture is incubated overnight at 37°C with 200rpm shaking. The next day, 2mL overnight culture is inoculated into 200mL AM+5Y culture medium supplemented with the same antibiotics and glycerol (final concentration of 3% w / v). After incubation at 37°C for 5 hours and shaking at 200 rpm, the cultures were cooled to 25°C and shaken at 200 rpm for another hour before IPTG (final 0.1 mM) was added to each flask. The cultures were incubated at 25°C at 200 rpm for another 48 hours.

[0481] The culture was sonicated and centrifuged. The pH of the culture supernatant was adjusted to 8.0 with ammonia before loading onto the SPE column. A Sep-Pak C18 column (6cc / 500mg, Waters, MA) was conditioned with 10mL MeOH and 15mL 10mM NH4HCO3, and 40mL of the culture sample was loaded onto the SPE column. The column was then eluted with 3mL MeOH / 10mM NH4HCO3 (90 / 10, v / v). The eluate was filtered with a 0.22μm hydrophilic PTFE syringe filter for UHPLC Q-Exactive MS analysis.

[0482] Analytical method: UHPLC Q-Exactive MS analysis

[0483] A Vanquish ultra-high performance liquid chromatograph coupled to a Q Exactive plus hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) was used for the analysis.

[0484] UHPLC was performed on a Luna 3μm C18 (100mm×2mm) column (Phenomenex, USA) with a flow rate of 0.3mL / min. The mobile phase consisted of A: 5mM HCOONH4 (adjustable to pH 8.0 by ammonia) and B acetonitrile. A linear gradient was used for elution: 0-0.5 min, 15% B; 0.5-7 min, 15%-80% B; 7.01-10.5 min, 98% B, 10.6-13.0 min, 98%-15%. The column temperature was set at 35°C and the injection volume was 1μL. The Q-Exactive MS system was operated in negative ionization mode (ESI-) using a HESI source and targeted SIM-ddMS. 2 The acquisition was performed in HESI mode or targeted SIM mode. The parameters for HESI were as follows: sheath gas flow rate 48, auxiliary gas flow rate 11, spray voltage 3.8KV, capillary temperature 320°C and auxiliary heater temperature 300°C. The parameters for targeted SIM mode were as follows: resolution 35,000, AGC target 5e4, maximum IT 100 ms, cycle count 5, isolation window 4.0 m / z. The inclusion mass of sesquiterpene diphosphate and monophosphate was set to 381.1237 [MH] - and 301.1574[MH] - In dd-MS 2 In the settings, the resolution is 17,500 and the stepping is 20, 35, 50.

[0485] The sesquiterpene monophosphate and diphosphate were semi-quantified by calculation of the EIC 381.1237 or 301.1574 area. The FPP standard curve was drawn using the EIC 381.1237 area and the FPP standard concentration. Since low concentrations of FPP are unstable, the FPP standard solution was freshly prepared on the same day as the sample analysis.

[0486] Analysis results of E. coli extracts

[0487] In the fermentation of DfHAD expressing E. coli strain, fennel diphosphate, fennel monophosphate, FPP (farnesyl diphosphate) and FMP (farnesyl monophosphate) were identified by accurate molecular weight, characteristic fragment ions and synthetic standards. Ffennel diphosphate (52.7 ng / mL) and FPP (48.3 ng / mL) were at trace levels, while fennel monophosphate (2.9 μg / mL) and FMP (4.8 μg / mL) were dominant, similar to fennel alcohol (2.1 μg / mL) and farnesol (10.5 μg / mL) ( Fig.12 ).

[0488] In vitro biochemical assay to detect pyrrolidine diphosphate and monophosphate

[0489] To purify DfHAD, its coding sequence (optimized for E. coli expression) was subcloned into a modified plasmid pGS-21a (Genscript). The plasmid was modified to allow the gene to be inserted between the KpnI and NdeI restriction sites. The expressed protein was fused with an N-terminal 6xHis-GST-tag. After transformation in BL21 (DE3) E. coli cells and selection on LB agar plates containing ampicillin (final concentration of 50 μg / mL), a single colony was inoculated into 40 mL LB medium supplemented with the same antibiotics, and the culture was incubated at 37 ° C at 180 rpm overnight. The next day, 5-10 mL of culture was inoculated into 500 mL of 2X YT medium containing 50 mg / L ampicillin and incubated at 37 ° C at 150 rpm for about 3 hours (OD600 = 0.7-1.2). The culture was then cooled to 25°C for 1.5 hours, and 0.25 mL of 1 M IPTG was added, followed by incubation at 25°C for 18 hours at 150 rpm. The culture was harvested by centrifugation at 3500 g for 15 minutes at 4°C, and the precipitate was washed twice with cooling buffer A (50 mM Na2HPO4, 10 mM imidazole, 500 mM NaCl, pH 7.8). The precipitate was resuspended in buffer A (without SigmaFast EDTA, Sigma) containing protease inhibitors, and the volume was adjusted to 25 mL. 25 mg lysozyme, 25 μL DNase I, 125 μL MgCl2 and 60 μL 0.2 M PMSF were added to the suspension. After incubation on ice for 30 minutes, followed by ultrasonic treatment (open for 3 seconds, close for 12 seconds, a total of 3 minutes, repeated once), the supernatant was collected by centrifugation at 4°C and 8000 g for 10 minutes. The protein was purified by a batch process on glutathione-agarose (Sigma G4510) according to the manufacturer's protocol for GST-tagged proteins.

[0490] For in vitro biochemical assay of DfHAD protein, 1.5 μM of purified recombinant protein was added to 2 ml reaction mixture containing 10 mM MgCl2, 5 mM DTT and 128 μM FPP in 50 mM Tris-HCl pH 8.0 buffer. The sample was incubated at 30°C at 50 rpm for 2 hours. The sample was then divided into two parts and 20 μL of bacterial alkaline phosphatase (Sangon-B004081-100) was added to one of them and the reaction was continued at 25°C for 1 hour. A control without added protein was also run under the same conditions.

[0491] Analytical results of DfHAD in vitro assay

[0492] Both fennel diphosphate and fennel monophosphate were identified in the in vitro assay (Figure 13). A 20:1 ratio of fennel diphosphate:fennel monophosphate was observed, as well as FPP and farnesyl monophosphate (FMP), while fennel alcohol was not detected (Table 1). Sesquiterpene diphosphates such as fennel diphosphate and FPP are unstable and easily degraded to their monophosphates. Ffennel monophosphate and FMP in the in vitro biochemical assay are likely degradation products of their respective diphosphate precursors.

[0493] After the addition of the phosphatase BAP, no sesquiterpene phosphates were observed, while fennel and farnesol were detected. Therefore, the major product of DfHAD is fennel diphosphate, and fennel is produced from fennel diphosphate by one or two phosphatases.

[0494] Table 1. Concentrations of sesquiterpenoid phosphates and sesquiterpenoids (μg / mL)

[0495]

[0496] a: Not detected

[0497] Example 8

[0498] Preparation of DfHAD enzyme mutants and determination of their in vitro activity

[0499] The amino acid sequence of native DfHAD (SEQ ID NO: 2) was modified to specific conserved amino acid sequence motifs to determine the criticality of these motifs for enzyme activity.

[0500] To this end, synthetic genes of 4 different mutants of the native DfHAD enzyme of SEQ ID NO: 2 were prepared by a commercial supplier. The genes were expressed in vitro and then assayed for activity as described in Example 2 above. In particular, the concentration of fennel was determined by GC / MS (mg / L / OD). The results are summarized in the following Table 2.

[0501] Table 2: DfHAD enzyme mutants and their enzyme activities

[0502]

[0503] Natural motifs: DSFDSLE (SEQ ID NO: 13); QCKSKGCW (SEQ ID NO: 24) and DGILQVYFDVERPRIDPVVVAN (SEQ ID NO: 25)

[0504] The experimental results clearly show that each of the above sequence motifs is essential for the same activity. Changing the modified Class I motif (SEQ ID NO: 14) to SEQ ID NO: 29 can lead to almost complete loss of activity. This method of changing the native synthase motif III (SEQ ID NO: 24) to SEQ ID NO: 26 significantly reduced the enzyme activity, while the exchange of the respective conserved fungal motif SEQ ID NO: 27 led to a doubling of the enzyme activity. Mutating the native synthase motif IV (SEQ ID NO: 25) may lead to a complete loss of activity.

[0505] The contents of documents cross-referenced herein are incorporated herein by reference.

[0506] Table 3: The sequences referred to herein are:

[0507]

[0508] NA = Nucleic Acid

[0509] AA = amino acid

[0510] sequence

[0511] Coding sequence for DfHAD (SEQ ID NO: 1)

[0512]

[0513] Amino acid sequence for DfHAD (SEQ ID NO: 2)

[0514] MEFSASAPPPRLASVIILEPLGFLLTPHYSSQLPKKLLRRLLCTRIWHRYQRGRLRLRDAAMLLAQLPFLAVSDHPWALDNLASLLRPTAVRAVPWMLLLLDFLRDELHLKVVCATNSSPEELQELRHQFPALFAKVDATVSSGEEGVGKPSVRFLQAALDKAGVHAQQTLYLDSFDSLETIMAARSLGMHALSVEPCHIDELTARASSGQLRDAQLIRRIVCAMHGPAVSAVVSGSITSSGPQTAKIEELPTAADSHLRSAALTSAQQFFLKVIAPHRPEKPFVQLPSLTSEGIRIYDTFAQFVIADLLDDTRFLPMQSPPPNGLITFVNPSAYLADDIKNGNSHIVPGVQFYASDACTLIDIPHDLDTTSVGLSVLHKFGKVDKDTLNKVLDRMLEQVSEDDGILQVYFDVERPRIDPVVVANTVFLFHLGKRGHEVARSEKFVESVLLQRAYEEGTLYYNLGEAFLVSVARLVHEFKEHFTRSGMRRALEERLRERARAGMQERDDALALAMRIRACALCGLAGEGLTKAAEQELLRLQCKSKGCWGCHPFYRNGSNVLSWIGSEALTTAYAIAALQPIDI

[0515] Coding sequence for DfHAD-8 (K532R) (SEQ ID NO: 3)

[0516]

[0517] For the amino acid sequence of DfHAD-8(K532R) (SEQ ID NO:4)

[0518] MEFSASAPPPRLASVIILEPLGFLLTPHYSSQLPKKLLRRLLCTRIWHRYQRGRLRLRDAAMLLAQLPFLAVSDHPWALDNLASLLRPTAVRAVPWMLLLLDFLRDELHLKVVCATNSSPEELQELRHQFPALFAKVDATVSSGEEGVGKPSVRFLQAALDKAGVHAQQTLYLDSFDSLETIMAARSLGMHALSVEPCHIDELTARASSGQLRDAQLIRRIVCAMHGPAVSAVVSGSITSSGPQTAKIEELPTAADSHLRSAALTSAQQFFLKVIAPHRPEKPFVQLPSLTSEGIRIYDTFAQFVIADLLDDTRFLPMQSPPPNGLITFVNPSAYLADDIKNGNSHIVPGVQFYASDACTLIDIPHDLDTTSVGLSVLHKFGKVDKDTLNKVLDRMLEQVSEDDGILQVYFDVERPRIDPVVVANTVFLFHLGKRGHEVARSEKFVESVLLQRAYEEGTLYYNLGEAFLVSVARLVHEFKEHFTRSGMRRALEERLRERARAGMQERDDALALAMRIRACALCGLAGEGLTRAAEQELLRLQCKSKGCWGCHPFYRNGSNVLSWIGSEALTTAYAIAALQPIDI

[0519] For the coding sequence of DfHAD-9(V274A) (SEQ ID NO:5)

[0520]

[0521] Amino acid sequence for DfHAD-9 (V274A) (SEQ ID NO: 6)

[0522] MEFSASAPPPRLASVIILEPLGFLLTPHYSSQLPKKLLRRLLLCTRIWHRYQRGRLRLRDAAMLLAQLPFLAVSDHPWALDNLASLLRPTAVRAVPWMLLLLDFLRDELHLKVVCATNSSPEELQELRHQFPALFAKVDATVSSGEE GVGKPSVRFLQAALDKAGVHAQQTLYLDSFDSLETIMAARSLGMHALSVEPCHIDELTARASSGQLRDAQLIRRIVCAMHGPAVSAVVSGSITSSGPQTAKIEELPTAADSHLRSAALTSAQQFFLKAIAPHRPEKPFVQLPSLTS EGIRIYDTFAQFVIADLLDDTRFLPMQSPPPNGLITFVNPSAYLADDIKNGNSHIVPGVQFYASDACTLIDIPHDLDTTSVGLSVLHKFGKVDKDTLNKVLDRMLEQVSEDDGILQVYFDVERPRIDPVVVANTVFLFHLGKRGHE VARSEKFVESVLLQRAYEEGTLYYNLGEAFLVSVARLVHEFKEHFTRSGMRRALEERLRERARAGMQERDDALALAMRIRACALCGLAGEGLTKAAEQELLRLQCKSKGCWGCHPFYRNGSNVLSWIGSEALTTAYAIAALQPIDI

[0523] Sequence of DfHAD codon optimized by Genscript genetic codon frequency of E. coli (SEQ ID NO: 7)

[0524]

[0525] Sequence of DfHAD codon optimized by the Genscript genetic codon frequency of tobacco (SEQ ID NO: 8)

[0526]

[0527] Sequence of DfHAD-8 (K532R) codon optimized by Genscript genetic codon frequency of E. coli (SEQ ID NO: 9)

[0528]

[0529] Sequence of DfHAD-9 (V274A) codon optimized by Genscript genetic codon frequency of E. coli (SEQ ID NO: 10)

[0530]

[0531] Modified Class I Motif: D SF Dxx(D / E) (SEQ ID NO:11)

[0532] Modified Class II Motif: H DxD(T / S)T (SEQ ID NO: 12)

[0533] Modified Class I Motif: D SF DSLE (SEQ ID NO: 13)

[0534] Modified Class II Motif: H DLDT (SEQ ID NO: 14)

[0535] Forward primer (5'-ATGGAGTTCTCTGCCTCTG-3') (SEQ ID NO: 15)

[0536] Reverse primer (5'-GGTTTGGCTTATGGAAGGT-3'). (SEQ ID NO: 16)

[0537] Codon optimization of DfHAD-6His-GST using the Genscript genetic codon frequency of E. coli

[0538] Sequence (SEQ ID NO:17)

[0539]

[0540] For the amino acid sequence of DfHAD-6His–GST (SEQ ID NO:18)

[0541] MSGSHHHHHHSSGMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLGHTGHRSDDDDKHMEFSASAPPPRLASVIILEPLGFLLTPHYSSQLPKKLLRRLLCTRIWHRYQRGRLRLRDAAMLLAQLPFLAVSDHPWALDNLASLLRPTAVRAVPWMLLLLDFLRDELHLKVVCATNSSPEELQELRHQFPALFAKVDATVSSGEEGVGKPSVRFLQAALDKAGVHAQQTLYLDSFDSLETIMAARSLGMHALSVEPCHIDELTARASSGQLRDAQLIRRIVCAMHGPAVSAVVSGSITSSGPQTAKIEELPTAADSHLRSAALTSAQQFFLKVIAPHRPEKPFVQLPSLTSEGIRIYDTFAQFVIADLLDDTRFLPMQSPPPNGLITFVNPSAYLADDIKNGNSHIVPGVQFYASDACTLIDIPHDLDTTSVGLSVLHKFGKVDKDTLNKVLDRMLEQVSEDDGILQVYFDVERPRIDPVVVANTVFLFHLGKRGHEVARSEKFVESVLLQRAYEEGTLYYNLGEAFLVSVARLVHEFKEHFTRSGMRRALEERLRERARAGMQERDDALALAMRIRACALCGLAGEGLTKAAEQELLRLQCKSKGCWGCHPFYRNGSNVLSWIGSEALTTAYAIAALQPIDI

[0542] For the amino acid sequence of the mutant DfHAD-QW fungus (SEQ ID NO:19)

[0543] MEFSASAPPPRLASVIILEPLGFLLTPHYSSQLPKKLLRRLLCTRIWHRYQRGRLRLRDAAMLLAQLPFLAVSDHPWALDNLASLLRPTAVRAVPWMLLLLDFLRDELHLKVVCATNSSPEELQELRHQFPALFAKVDATVSSGEEGVGKPSVRFLQAALDKAGVHAQQTLYLDSFDSLETIMAARSLGMHALSVEPCHIDELTARASSGQLRDAQLIRRIVCAMHGPAVSAVVSGSITSSGPQTAKIEELPTAADSHLRSAALTSAQQFFLKVIAPHRPEKPFVQLPSLTSEGIRIYDTFAQFVIADLLDDTRFLPMQSPPPNGLITFVNPSAYLADDIKNGNSHIVPGVQFYASDACTLIDIPHDLDTTSVGLSVLHKFGKVDKDTLNKVLDRMLEQVSEDDGILQVYFDVERPRIDPVVVANTVFLFHLGKRGHEVARSEKFVESVLLQRAYEEGTLYYNLGEAFLVSVARLVHEFKEHFTRSGMRRALEERLRERARAGMQERDDALALAMRIRACALCGLAGEGLTKAAEQELLRLQCEDGGWGCHPFYRNGSNVLSWIGSEALTTAYAIAALQPIDI*

[0544] Amino acid sequence for mutant DfHADQW mut (SEQ ID NO:20)

[0545] MEFSASAPPPRLASVIILEPLGFLLTPHYSSQLPKKLLRRLLCTRIWHRYQRGRLRLRDAAMLLAQLPFLAVSDHPWALDNLASLLRPTAVRAVPWMLLLLDFLRDELHLKVVCATNSSPEELQELRHQFPALFAKVDATVSSGEEGVGKPSVRFLQAALDKAGVHAQQTLYLDSFDSLETIMAARSLGMHALSVEPCHIDELTARASSGQLRDAQLIRRIVCAMHGPAVSAVVSGSITSSGPQTAKIEELPTAADSHLRSAALTSAQQFFLKVIAPHRPEKPFVQLPSLTSEGIRIYDTFAQFVIADLLDDTRFLPMQSPPPNGLITFVNPSAYLADDIKNGNSHIVPGVQFYASDACTLIDIPHDLDTTSVGLSVLHKFGKVDKDTLNKVLDRMLEQVSEDDGILQVYFDVERPRIDPVVVANTVFLFHLGKRGHEVARSEKFVESVLLQRAYEEGTLYYNLGEAFLVSVARLVHEFKEHFTRSGMRRALEERLRERARAGMQERDDALALAMRIRACALCGLAGEGLTKAAEQELLRLQCAAAGCWGCHPFYRNGSNVLSWIGSEALTTAYAIAALQPIDI*

[0546] Amino acid sequence (SEQ ID NO: 21) of the mutant DfHAD-SEQ64 motif: MEFSASAPPPRLASVIILEPLGFLLTPHYSSQLPKKLLRRLLCTRIWHRYQRGRLRLRDAAMLLAQLPFLAVSDHPWALDNLASLLRPTAVRAVPWMLLLLDFLRDELHLKVVCATNSSPEELQELRHQFPALFAKVDATVSSGEEGVGKPSVRFLQAALDKAGVHAQQTLYLDSFDPLETIMAARSLGMHALSVEPCHIDELTARASSGQLRDAQLIRRIVCAMHGPAVSAVVSGSITSSGPQTAKIEELPTAADSHLRSAALTSAQQFFLKVIAPHRPEKPFVQLPSLTSEGIRIYDTFAQFVIADLLDDTRFLPMQSPPPNGLITFVNPSAYLADDIKNGNSHIVPGVQFYASDACTLIDIPHDLDTTSVGLSVLHKFGKVDKDTLNKVLDRMLEQVSEDDGILQVYFDVERPRIDPVVVANTVFLFHLGKRGHEVARSEKFVESVLLQRAYEEGTLYYNLGEAFLVSVARLVHEFKEHFTRSGMRRALEERLRERARAGMQERDDALALAMRIRACALCGLAGEGLTKAAEQELLRLQCKSKGCWGCHPFYRNGSNVLSWIGSEALTTAYAIAALQPIDI*

[0547] Amino acid sequence (SEQ ID NO: 22) of the mutant DfHAD-MOS motif

[0548] MEFSASAPPPRLASVIILEPLGFLLTPHYSSQLPKKLLRRLLCTRIWHRYQRGRLRLRDAAMLLAQLPFLAVSDHPWALDNLASLLRPTAVRAVPWMLLLLDFLRDELHLKVVCATNSSPEELQELRHQFPALFAKVDATVSSGEEGVGKPSVRFLQAALDKAGVHAQQTLYLDSFDSLETIMAARSLGMHALSVEPCHIDELTARASSGQLRDAQLIRRIVCAMHGPAVSAVVSGSITSSGPQTAKIEELPTAADSHLRSAALTSAQQFFLKVIAPHRPEKPFVQLPSLTSEGIRIYDTFAQFVIADLLDDTRFLPMQSPPPNGLITFVNPSAYLADDIKNGNSHIVPGVQFYASDACTLIDIPHDLDTTSVGLSVLHKFGKVDKDTLNKVLDRMLEQVSEDDGILQVAAAVERPRIDPVVVANTVFLFHLGKRGHEVARSEKFVESVLLQRAYEEGTLYYNLGEAFLVSVARLVHEFKEHFTRSGMRRALEERLRERARAGMQERDDALALAMRIRACALCGLAGEGLTKAAEQELLRLQCKSKGCWGCHPFYRNGSNVLSWIGSEALTTAYAIAALQPIDI*

[0549] Alternative class II motif: DLDTTS (SEQ ID NO:23)

[0550] Motif III: QCKSKGCW (SEQ ID NO:24)

[0551] Motif IV: DGILQVYFDVERPRIDPVVVAN (SEQ ID NO:25)

[0552] Modified motif III: QCAAAGCW (SEQ ID NO:26)

[0553] Modified motif III: QCEDGGW (SEQ ID NO:27)

[0554] Modified motif IV: DGILQVAAAVERPRIDPVVVAN (SEQ ID NO:28)

[0555] Modified Class I motif mutated: DSFDPLE (SEQ ID NO: 29). Sequence Listing <110> Firmenich SA <120> pyrol synthase <130> 10860 / WO <160> 29 <170> PatentIn version 3.5 <210> 1 <211> 1755 <212> DNA <213> DfHAD_Dryopteris fragrans <220> <221> CDS <222> (1)..(1755) <400> 1 atg gag ttc tct gcc tct gct cct cct cct agg cta gcc agt gtc ata 48 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 ata ttg gag cct ctc ggc ttc ctc ctc aca cca cac tac tcc tct cag 96 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 ctt ccc aaa aag ctg ctc cgt cgc ctg ttg tgc act aga atc ​​tgg cac 144 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 agg tat cag cga ggc cgc ctt cgc ctg cgt gac gct gct atg ctg ctc 192 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 gcc cag ctc cca ttc cta gct gtg tct gat cac ccc tgg gct ctg gac 240 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 aat ctc gca agc ctg ctc cgc ccc aca gct gtg cgt gcg gtg cca tgg 288 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 atg ctg ctg ctg ctc gac ttc cta cga gac gag ctc cat ctg aag gta 336 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 gtc tgc gcg acc aac tcc tcc cca gaa gag ctg caa gag ctg cgc cac 384 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 cag ttt ccg gcc ctc ttt gcc aag gtc gat gcc acc gtt tct tca ggc 432 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 gag gag ggc gtg ggc aag ccg tcc gtg cgc ttc ctg cag gct gcg ttg 480 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 gac aaa gcc ggt gtc cac gcg cag caa acc ttg tat ctt gac tct ttt 528 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 gac agc ttg gag acc atc atg gct gca cgc tct ctt ggc atg cat gca 576 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 cta tct gta gag cca tgc cac att gat gag ctc acc gcc agg gcc tct 624 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 tcc ggc cag cta aga gat gca cag ctt ata agg cgt att gtg tgc gcc 672 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 atg cac ggg cca gca gta tct gca gtt gtg tcg ggc agt atc aca tcg 720 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 tcc ggc cca cag aca gca aag atc gag gaa ttg cca aca gct gct gat 768 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 agt cat ctc cgc agc gca gct ctc act tct gct cag cag ttt ttc ctc 816 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 aaa gtt att gct cca cat cgt cct gag aag cca ttc gtc cag ctt cca 864 Lys Val Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 tct ctc acc tcg gag ggc atc cga ata tac gac acc ttt gca cag ttt 912 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 gtc ata gcc gac ctg ctc gac gac acc cgc ttc cta ccc atg caa tct 960 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 cct cct ccc aat ggg ctc atc acc ttt gtt aac cca agc gcg tac ctt 1008 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 gct gat gat ata aag aat ggc aac agc cat att gtc ccg ggt gtg caa 1056 Only Asp Asp Ile Lys Asn Gly Asn Served His Ile Val Pro Gly Val Gln 340 345 350 ttt tac gca tcc gat gcg tgc act ctc atc gac atc cca cat gac cta 1104 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 gac acc acc tcc gtt gc tg tca gta ctg cac aag ttt gga aag gtg 1152 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 Gac Aag Gac Aca CTC AAC AAA GTG CTA GAC AGA ATG CTTC GAG CA GTG 1200 Asp Lys Asp Thr With Asn Lys Val With Asp Arg With Glu Gln Val 385 390 395 400 agt gaa gac gac ggc att ctg cag gtg tat ttt gat gtg gag cgt ccg 1248 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 cgc atc gat cca gtt gtg gtg gca aac acg gtg ttt ctg ttc cac ttg 1296 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 gga aag aga ggg cat gag gtg gcg agg agt gag aag ttt gtg gag agt 1344 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 gtg ctg ctg cag agg gca tac gaa gaa ggg acg ttg tat tac aac ctg 1392 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 ggg gaa gca ttt ttg gtg agt gtg gcg agg ctg gtg cac gag ttt aag 1440 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 gag cac ttt aca agg agc ggc atg agg agg gca ctg gag gag agg cta 1488 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 aga gag cgg gca agg gcg ggc atg caa gag agg gat gat gcg ctg gcg 1536 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 cta gcc atg cgc att cgt gca tgc gct ttg tgt ggc ctg gcc gga gag 1584 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 ggc ctc aca aaa gca gca gag cag gag ctt ttg cgc ctg cag tgc aag 1632 Gly Leu Thr Lys Ala Ala Glu Gln Glu Leu Leu Arg Leu Gln Cys Lys 530 535 540 tcc aag ggc tgt tgg ggg tgc cac cct ttc tat cgc aat ggc agt aat 1680 Ser Lys Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 gtg ctc agc tgg atc ggc agt gag gcc ctt acc act gct tac gct att 1728 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 gct gcg cta cag ccc att gat att taa 1755 Ala Ala Leu Gln Pro Ile Asp Ile 580 <210> 2 <211> 584 <212> PRT <213> Dryopteris fragrans <400> 2 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 Lys Val Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 Ala Asp Asp Ile Lys Asn Gly Asn Ser His Ile Val Pro Gly Val Gln 340 345 350 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 Gly Leu Thr Lys Pathway Glu Gln Glu Leu Leu Arg Leu Gln Cys Lys 530 535 540 Ser Lys Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 Pathway Leu Gln Pro Ile Asp Ile 580 <210> 3 <211> 1755 <212> DNA <213> DfHAD-8(K532R)_Dryopteris fragrans <220> <221> CDS <222> (1)..(1755) <400> 3 atg gag ttc tct gcc tct gct cct cct cct agg cta gcc agt gtc ata 48 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 ata ttg gag cct ctc ggc ttc ctc ctc aca cca cac tac tcc tct cag 96 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 ctt ccc aaa aag ctg ctc cgt cgc ctg ttg tgc act aga atc tgg cac 144 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 agg tat cag cga ggc cgc ctt cgc ctg cgt gac gct gct atg ctg ctc 192 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 gcc cag ctc cca ttc cta gct gtg tct gat cac ccc tgg gct ctg gac 240 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 aat ctc gca agc ctg ctc cgc ccc aca gct gtg cgt gcg gtg cca tgg 288 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 atg ctg ctg ctg ctc gac ttc cta cga gac gag ctc cat ctg aag gta 336 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 gtc tgc gcg acc aac tcc tcc cca gaa gag ctg caa gag ctg cgc cac 384 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 cag ttt ccg gcc ctc ttt gcc aag gtc gat gcc acc gtt tct tca ggc 432 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 gag gag ggc gtg ggc aag ccg tcc gtg cgc ttc ctg cag gct gcg ttg 480 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 gac aaa gcc ggt gtc cac gcg cag caa acc ttg tat ctt gac tct ttt 528 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 gac agc ttg gag acc atc atg gct gca cgc tct ctt ggc atg cat gca 576 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 cta tct gta gag cca tgc cac att gat gag ctc acc gcc agg gcc tct 624 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 tcc ggc cag cta aga gat gca cag ctt ata agg cgt att gtg tgc gcc 672 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 atg cac ggg cca gca gta tct gca gtt gtg tcg ggc agt atc aca tcg 720 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 tcc ggc cca cag aca gca aag atc gag gaa ttg cca aca gct gct gat 768 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 agt cat ctc cgc agc gca gct ctc act tct gct cag cag ttt ttc ctc 816 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 aaa gtt att gct cca cat cgt cct gag aag cca ttc gtc cag ctt cca 864 Lys Val Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 tct ctc acc tcg gag ggc atc cga ata tac gac acc ttt gca cag ttt 912 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 gtc ata gcc gac ctg ctc gac gac acc cgc ttc cta ccc atg caa tct 960 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 cct cct ccc aat ggg ctc atc acc ttt gtt aac cca agc gcg tac ctt 1008 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 gct gat gat ata aag aat ggc aac agc cat att gtc ccg ggt gtg caa 1056 Ala Asp Asp Ile Lys Asn Gly Asn Ser His Ile Val Pro Gly Val Gln 340 345 350 ttt tac gca tcc gat gcg tgc act ctc atc gac atc cca cat gac cta 1104 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 gac acc acc tcc gtt ggc ttg tca gta ctg cac aag ttt gga aag gtg 1152 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 gac aag gac aca ctc aac aaa gtg cta gac aga atg ctc gag caa gtg 1200 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 agt gaa gac gac ggc att ctg cag gtg tat ttt gat gtg gag cgt ccg 1248 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 cgc atc gat cca gtt gtg gtg gca aac acg gtg ttt ctg ttc cac ttg 1296 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 gga aag aga ggg cat gag gtg gcg agg agt gag aag ttt gtg gag agt 1344 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 gtg ctg ctg cag agg gca tac gaa gaa ggg acg ttg tat tac aac ctg 1392 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 ggg gaa gca ttt ttg gtg agt gtg gcg agg ctg gtg cac gag ttt aag 1440 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 gag cac ttt aca agg agc ggc atg agg agg gca ctg gag gag agg cta 1488 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 aga gag cgg gca agg gcg ggc atg caa gag agg gat gat gcg ctg gcg 1536 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 ctg gcc atg cgc att cgt gca tgc gct ttg tgt ggc ctg gcc gga gag 1584 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 ggc ctc aca aga gca gca gag cag gag cta ctg cgc ctg cag tgc aag 1632 Gly Leu Thr Arg Ala Ala Glu Gln Glu Leu Leu Arg Leu Gln Cys Lys 530 535 540 tcc aag ggc tgt tgg ggg tgc cac cct ttc tat cgc aat ggc agt aat 1680 Ser Lys Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 gtg ctc agc tgg atc ggc agt gag gcc ctt acc act gct tac gct att 1728 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 gct gcg cta cag ccc att gat att taa 1755 Ala Ala Leu Gln Pro Ile Asp Ile 580 <210> 4 <211> 584 <212> PRT <213> DfHAD-8(K532R)_Dryopteris fragrans <400> 4 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 Lys Val Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 Ala Asp Asp Ile Lys Asn Gly Asn Ser His Ile Val Pro Gly Val Gln 340 345 350 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 Gly Leu Thr Arg Ala Ala Glu Gln Glu Leu Leu Arg Leu Gln Cys Lys 530 535 540 Ser Lys Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 Ala Ala Leu Gln Pro Ile Asp Ile 580 <210> 5 <211> 1755 <212> DNA <213> DfHAD-9(V274A)_Dryopteris fragrans <220> <221> CDS <222> (1)..(1755) <400> 5 atg gag ttc tct gcc tct gct cct cct cct agg cta gcc agt gtc ata 48 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 ata ttg gag cct ctc ggc ttc ctc ctc aca cca cac tac tcc tct cag 96 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 ctt ccc aaa aag ctg ctc cgt cgc ctg ttg tgc act aga atc tgg cac 144 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 agg tat cag cga ggc cgc ctt cgc ctg cgt gac gct gct atg ctg ctc 192 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 gcc cag ctc cca ttc cta gct gtg tct gat cac ccc tgg gct ctg gac 240 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 aat ctc gca agc ctg ctc cgc ccc aca gct gtg cgt gcg gtg cca tgg 288 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 atg ctg ctg ctg ctc gac ttc cta cga gac gag ctc cat ctg aag gta 336 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 gtc tgc gcg acc aac tcc tcc cca gaa gag ctg caa gag ctg cgc cac 384 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 cag ttt ccg gcc ctc ttt gcc aag gtc gat gcc acc gtt tct tca ggc 432 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 gag gag ggc gtg ggc aag ccg tcc gtg cgc ttc ctg cag gct gcg ttg 480 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 gac aaa gcc ggt gtc cac gcg cag caa acc ttg tat ctt gac tct ttt 528 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 gac agc ttg gag acc atc atg gct gca cgc tct ctt ggc atg cat gca 576 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 cta tct gta gag cca tgc cac att gat gag ctc acc gcc agg gcc tct 624 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 tcc ggc cag cta aga gat gca cag ctt ata agg cgt att gtg tgc gcc 672 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 atg cac ggg cca gca gta tct gca gtt gtg tcg ggc agt atc aca tcg 720 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 tcc ggc cca cag aca gca aag atc gag gaa ttg cca aca gct gct gat 768 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 agt cat ctc cgc agc gca gct ctc act tct gct cag cag ttt ttc ctc 816 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 aaa gct att gct cat cgt cct gag aag cca ttc gtc cag ctt ca 864 Lys Ala Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 tct ctc acc tcg gag ggc atc cga ata tac gac acc ttt gca cag ttt 912 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290,295,300 gtc ata gcc gac ctg ctc gac gac acc cgc ttc cta ccc atg caa tct 960 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 cct cct ccc aat ggg ctc atc acc ttt gtt aac cca agc gcg tac ctt 1008 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 gct gat gat ata aag aat ggc aac agc cat att gtc ccg ggt gtg caa 1056 Only Asp Asp Ile Lys Asn Gly Asn Served His Ile Val Pro Gly Val Gln 340 345 350 ttt tac gca tct gat gcg tgc act ctc atc gac atc cca cat gac cta 1104 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 gac acc acc tcc gtt ggc ttg tca gta ctg cac aag ttt gga aag gtg 1152 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 gac aag gac aca ctc aac aaa gtg cta gac aga atg ctg gag caa gtg 1200 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 agt gaa gac gac ggc att ctc cag gtg tat ttt gat gtg gag cgt ccg 1248 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 cgc atc gat cca gtt gtg gtg gca aac acg gtg ttt ctg ttc cac ttg 1296 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 gga aag aga ggg cat gag gtg gcg agg agt gag aag ttt gtg gag agt 1344 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 gtg ctg ctg cag agg gca tac gaa gaa ggg acg ttg tat tac aac ctg 1392 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 ggg gaa gca ttt ttg gtg agt gtg gcg agg ctg gtg cac gag ttt aag 1440 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 gag cac ttt aca agg agc ggc atg agg agg gca ctg gag gag agg cta 1488 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 aga gag cgg gca agg gcg ggc atg caa gag agg gat gat gcg ctg gcg 1536 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 ctg gcc atg cgc att cgt gca tgc gct ttg tgt ggc ctg gcc gga gag 1584 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 ggc ctc aca aaa gca gca gag cag gag cta ctg cgc ctg cag tgc aag 1632 Gly Leu Thr Lys Ala Ala Glu Gln Glu Leu Leu Arg Leu Gln Cys Lys 530 535 540 tcc aag ggc tgt tgg ggg tgc cac cct ttc tat cgc aat ggc agt aat 1680 Ser Lys Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 gtg ctc agc tgg atc ggc agt gag gcc ctt acc act gct tac gct att 1728 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 gct gcg cta cag ccc att gat att taa 1755 Ala Ala Leu Gln Pro Ile Asp Ile 580 <210> 6 <211> 584 <212> PRT <213> DfHAD-9(V274A)_Dryopteris fragrans <400> 6 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 Lys Ala Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 Ala Asp Asp Ile Lys Asn Gly Asn Ser His Ile Val Pro Gly Val Gln 340 345 350 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 Gly Leu Thr Lys Ala Ala Glu Gln Glu Leu Leu Arg Leu Gln Cys Lys 530 535 540 Ser Lys Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 Ala Ala Leu Gln Pro Ile Asp Ile 580 <210> 7 <211> 1755 <212> DNA <213> Codon-optimized sequence of DfHAD using Genscript genetic codon frequencies from E. coli <400> 7 atggagttca gcgcgagcgc tccgccgccg cgtctggcga gcgtgatcat tctggaaccg 60 ctgggttttc tgctgacccc gcactacagc agccagctgc cgaagaaact gctgcgtcgt 120 ctgctgtgca cccgtatctg gcaccgttat cagcgtggcc gtctgcgtct gcgtgacgcg 180 gcgatgctgc tggcgcaact gccgttcctg gcggttagcg accacccgtg ggcgctggat 240 aacctggcga gcctgctgcg tccgaccgcg gttcgtgcgg tgccgtggat gctgctgctg 300 ctggactttc tgcgtgatga gctgcacctg aaagtggttt gcgcgaccaa cagcagcccg 360 gaggaactgc aggaactgcg tcaccaattc ccggcgctgt ttgcgaaggt tgacgcgacc 420 gtgagcagcg gcgaggaagg tgttggcaaa ccgagcgtgc gtttcctgca agcggcgctg 480 gataaggcgg gcgtgcacgc gcagcaaacc ctgtacctgg acagctttga tagcctggag 540 accatcatgg cggcgcgtag cctgggtatg cacgcgctga gcgttgagcc gtgccacatt 600 gacgaactga ccgcgcgtgc gagcagcggt cagctgcgtg atgcgcaact gatccgtcgt 660 attgtttgcg cgatgcacgg tccggctgtg agcgcggtgg ttagcggtag catcaccagc 720 agcggtccgc agaccgcgaa aattgaggaa ctgccgaccg cggcggacag ccacctgcgt 780 agcgcggcgc tgaccagcgc gcagcaattc tttctgaaag tgattgcgcc gcaccgtccg 840 gagaagccgt tcgttcaact gccgagcctg accagcgaag gtatccgtat ttatgacacc 900 ttcgcgcagt ttgtgatcgc ggatctgctg gacgataccc gtttcctgcc gatgcaaagc 960 ccgccgccga acggcctgat tacctttgtt aacccgagcg cgtacctggc ggacgatatc 1020 aaaaacggta acagccacat tgttccgggc gtgcagttct atgcgagcga cgcgtgcacc 1080 ctgatcgata ttccgcacga cctggatacc accagcgttg gtctgagcgt gctgcacaag 1140 tttggcaaag ttgacaagga taccctgaac aaggtgctgg atcgtatgct ggagcaagtt 1200 agcgaagacg atggtatcct gcaagtttac tttgacgtgg agcgtccgcg tattgatccg 1260 gtggttgtgg cgaacaccgt gttcctgttt cacctgggta aacgtggcca cgaagttgcg 1320 cgtagcgaga agttcgttga aagcgtgctg ctgcagcgtg cgtacgagga aggcaccctg 1380 tactataacc tgggcgaagc gtttctggtt agcgtggcgc gtctggtgca cgagttcaaa 1440 gaacacttta cccgtagcgg tatgcgtcgt gcgctggagg aacgtctgcg tgagcgtgcg 1500 cgtgcgggta tgcaagaacg tgacgatgcg ctggcgctgg cgatgcgtat ccgtgcgtgc 1560 gcgctgtgcg gtctggcggg cgagggtctg accaaggcgg cggagcagga actgctgcgt 1620 ctgcaatgca agagcaaagg ttgctggggc tgccacccgt tctaccgtaa cggtagcaac 1680 gttctgagct ggatcggcag cgaagcgctg accaccgcgt atgcgattgc ggcgctgcag 1740 ccgatcgaca tttaa 1755 <210> 8 <211> 1755 <212> DNA <213> Codon-optimized sequence of DfHAD using the tobacco Genscript genetic codon frequencies <400> 8 atggaatttt ctgcttcagc tccacctcca agacttgctt cagttattat tcttgagcct 60 ttgggatttc ttttgactcc acattactct tcacaattgc ctaagaaact tttgagaagg 120 cttttgtgta caagaatttg gcataggtac caaaggggta ggcttagatt gagggatgct 180 gctatgcttt tggctcaact tccatttttg gctgtttcag atcatccttg ggctcttgat 240 aatttggctt ctcttttgag accaactgct gttagggctg ttccttggat gcttttgctt 300 ttggattttc ttagagatga acttcatttg aaggttgttt gcgctactaa ttcttcacca 360 gaagagcttc aagagttgag gcatcaattt cctgctttgt ttgctaaggt tgatgctaca 420 gtttcttcag gagaagaggg agttggtaaa ccatctgtta gatttcttca agctgctttg 480 gataaggctg gtgttcatgc tcaacaaact ctttatttgg attctttcga ttcacttgaa 540 acaattatgg ctgctaggtc attgggaatg catgctcttt ctgttgaacc atgtcatatt 600 gatgagttga ctgctagagc ttctcagga cattgaggg atgctcaact tattagaagg 660 attgtttgcg ctatgcatgg tcctgctgtt tcagctgttg tttctgtc aattacttct 720 tcaggtccac aacagctaa aattgagag cttcctactg ctgctgattc tcatttgaga 780 tcagctgctc ttacatctgc tcacaattt ttcctaag ttattgctcc acatagacct 840 gaaaagccat ttgttcaact tccttctttg acttcagagg gatcaggat ctatgataca 900 ttcgctcaat tcgttatcgc tgatctttg gatgacta gtttttgcc aatgcaatca 960 cctccaccta atggtcttat cacattcgtt aacccttctg cttattggc tgatgatatt 1020 aaaaatgta actcacatat tgttcaggt gttcatttt acgctctga tgcttgtact 1080 ttgattgata ttcctcatga tctgatact acatctgttg gacttcagt ttgcataag 1140 ttcggtaaag ttgatagga tacacttaat aaggttttgg atagaatgct tgaacaagtt 1200 tcagaggatg atggaatcct tcaagtttac ttcgatgttg aaagacctag gattgatcca 1260 gttgttgttg ctacactgt ttttctttc catttggga aaagaggtca tgaggttgct 1320 agatcagaaa agtttgttga gtctgttctt ttgcaaagag cttacgaaga gggaactttg 1380 tattacaatc ttggtgaagc ttttcttgtt tctgttgcta gacttgttca tgagtttaag 1440 gagcatttta caaggtctgg aatgagaagg gctttggaag agagacttag ggaaagagct 1500 agggctggta tgcaagagag agatgatgct cttgctttgg ctatgagaat tagggcttgt 1560 gctctttgcg gtttggctgg agaaggtctt acaaaggctg ctgaacaaga gcttttgaga 1620 ttgcaatgca agtctaaagg atgttggggt tgccatccat tctacaggaa tggttctaac 1680 gttttgtcat ggattggttc tgaggctctt actacagctt acgctattgc tgctcttcaa 1740 cctattgata tttga 1755 <210> 9 <211> 1755 <212> DNA <213> Sequence of DfHAD-8(K532R) codon-optimized by Genscript genetic codon frequency in Escherichia coli <400> 9 atggagttca gcgcgagcgc tccgccgccg cgtctggcga gcgtgatcat tctggaaccg 60 ctgggttttc tgctgacccc gcactacagc agccagctgc cgaagaaact gctgcgtcgt 120 ctgctgtgca cccgtatctg gcaccgttat cagcgtggcc gtctgcgtct gcgtgacgcg 180 gcgatgctgc tggcgcaact gccgttcctg gcggttagcg accacccgtg ggcgctggat 240 aacctggcga gcctgctgcg tccgaccgcg gttcgtgcgg tgccgtggat gctgctgctg 300 ctggactttc tgcgtgatga gctgcacctg aaagtggttt gcgcgaccaa cagcagcccg 360 gaggaactgc aggaactgcg tcaccaattc ccggcgctgt ttgcgaaggt tgacgcgacc 420 gtgagcagcg gcgaggaagg tgttggcaaa ccgagcgtgc gtttcctgca agcggcgctg 480 gataaggcgg gcgtgcacgc gcagcaaacc ctgtacctgg acagctttga tagcctggag 540 accatcatgg cggcgcgtag cctgggtatg cacgcgctga gcgttgagcc gtgccacatt 600 gacgaactga ccgcgcgtgc gagcagcggt cagctgcgtg atgcgcaact gatccgtcgt 660 attgtttgcg cgatgcacgg tccggctgtg agcgcggtgg ttagcggtag catcaccagc 720 agcggtccgc agaccgcgaa aattgaggaa ctgccgaccg cggcggacag ccacctgcgt 780 agcgcggcgc tgaccagcgc gcagcaattc tttctgaaag tgattgcgcc gcaccgtccg 840 gagaagccgt tcgttcaact gccgagcctg accagcgaag gtatccgtat ttatgacacc 900 ttcgcgcagt ttgtgatcgc ggatctgctg gacgataccc gtttcctgcc gatgcaaagc 960 ccgccgccga acggcctgat tacctttgtt aacccgagcg cgtacctggc ggacgatatc 1020 aaaaacggta acagccacat tgttccgggc gtgcagttct atgcgagcga cgcgtgcacc 1080 ctgatcgata ttccgcacga cctggatacc accagcgttg gtctgagcgt gctgcacaag 1140 tttggcaaag ttgacaagga taccctgaac aaggtgctgg atcgtatgct ggagcaagtt 1200 agcgaagacg atggtatcct gcaagtttac tttgacgtgg agcgtccgcg tattgatccg 1260 gtggttgtgg cgaacaccgt gttcctgttt cacctgggta aacgtggcca cgaagttgcg 1320 cgtagcgaga agttcgttga aagcgtgctg ctgcagcgtg cgtacgagga aggcaccctg 1380 tactataacc tgggcgaagc gtttctggtt agcgtggcgc gtctggtgca cgagttcaaa 1440 gaacacttta cccgtagcgg tatgcgtcgt gcgctggagg aacgtctgcg tgagcgtgcg 1500 cgtgcgggta tgcaagaacg tgacgatgcg ctggcgctgg cgatgcgtat ccgtgcgtgc 1560 gcgctgtgcg gtctggcggg cgagggtctg acccgggcgg cggagcagga actgctgcgt 1620 ctgcaatgca agagcaaagg ttgctggggc tgccacccgt tctaccgtaa cggtagcaac 1680 gttctgagct ggatcggcag cgaagcgctg accaccgcgt atgcgattgc ggcgctgcag 1740 ccgatcgaca tttaa 1755 <210> 10 <211> 1755 <212> DNA <213> Sequence of DfHAD-9(V274A) codon-optimized by Genscript genetic codon frequency in Escherichia coli <400> 10 atggagttca gcgcgagcgc tccgccgccg cgtctggcga gcgtgatcat tctggaaccg 60 ctgggttttc tgctgacccc gcactacagc agccagctgc cgaagaaact gctgcgtcgt 120 ctgctgtgca cccgtatctg gcaccgttat cagcgtggcc gtctgcgtct gcgtgacgcg 180 gcgatgctgc tggcgcaact gccgttcctg gcggttagcg accacccgtg ggcgctggat 240 aacctggcga gcctgctgcg tccgaccgcg gttcgtgcgg tgccgtggat gctgctgctg 300 ctggactttc tgcgtgatga gctgcacctg aaagtggttt gcgcgaccaa cagcagcccg 360 gaggaactgc aggaactgcg tcaccaattc ccggcgctgt ttgcgaaggt tgacgcgacc 420 gtgagcagcg gcgaggaagg tgttggcaaa ccgagcgtgc gtttcctgca agcggcgctg 480 gataaggcgg gcgtgcacgc gcagcaaacc ctgtacctgg acagctttga tagcctggag 540 accatcatgg cggcgcgtag cctgggtatg cacgcgctga gcgttgagcc gtgccacatt 600 gacgaactga ccgcgcgtgc gagcagcggt cagctgcgtg atgcgcaact gatccgtcgt 660 attgtttgcg cgatgcacgg tccggctgtg agcgcggtgg ttagcggtag catcaccagc 720 agcggtccgc agaccgcgaa aattgaggaa ctgccgaccg cggcggacag ccacctgcgt 780 agcgcggcgc tgaccagcgc gcagcaattc tttctgaaag cgattgcgcc gcaccgtccg 840 gagaagccgt tcgttcaact gccgagcctg accagcgaag gtatccgtat ttatgacacc 900 ttcgcgcagt ttgtgatcgc ggatctgctg gacgataccc gtttcctgcc gatgcaaagc 960 ccgccgccga acggcctgat tacctttgtt aacccgagcg cgtacctggc ggacgatatc 1020 aaaaacggta acagccacat tgttccgggc gtgcagttct atgcgagcga cgcgtgcacc 1080 ctgatcgata ttccgcacga cctggatacc accagcgttg gtctgagcgt gctgcacaag 1140 tttggcaaag ttgacaagga taccctgaac aaggtgctgg atcgtatgct ggagcaagtt 1200 agcgaagacg atggtatcct gcaagtttac tttgacgtgg agcgtccgcg tattgatccg 1260 gtggttgtgg cgaacaccgt gttcctgttt cacctgggta aacgtggcca cgaagttgcg 1320 cgtagcgaga agttcgttga aagcgtgctg ctgcagcgtg cgtacgagga aggcaccctg 1380 tactataacc tgggcgaagc gtttctggtt agcgtggcgc gtctggtgca cgagttcaaa 1440 gaacacttta cccgtagcgg tatgcgtcgt gcgctggagg aacgtctgcg tgagcgtgcg 1500 cgtgcgggta tgcaagaacg tgacgatgcg ctggcgctgg cgatgcgtat ccgtgcgtgc 1560 gcgctgtgcg gtctggcggg cgagggtctg accaaggcgg cggagcagga actgctgcgt 1620 ctgcaatgca agagcaaagg ttgctggggc tgccacccgt tctaccgtaa cggtagcaac 1680 gttctgagct ggatcggcag cgaagcgctg accaccgcgt atgcgattgc ggcgctgcag 1740 ccgatcgaca tttaa 1755 <210> 11 <211> 7 <212> PRT <213> Modified class I motif: DSFDxx(D / E) <220> <221> MISC_FEATURE <222> (5)..(6) <223> Xaa can be any natural amino acid <220> <221> MISC_FEATURE <222> (7) <223> Xaa can be D or E <400> 11 Asp Ser Phe Asp Xaa Xaa Xaa 1 5 <210> 12 <211> 6 <212> PRT <213> Modified class II motif: HDxD(T / S)T <220> <221> MISC_FEATURE <222> (3) <223> Xaa can be any natural amino acid <220> <221> MISC_FEATURE <222> (5) <223> Xaa can be T or S <400> 12 His Asp Xaa Asp Xaa Thr 1 5 <210> 13 <211> 7 <212> PRT <213> Modified class I motif: DSFDSLE <400> 13 Asp Ser Phe Asp Ser Leu Glu 1 5 <210> 14 <211> 5 <212> PRT <213> Modified class II motif: HDLDT <400> 14 His Asp Leu Asp Thr 1 5 <210> 15 <211> 19 <212> DNA <213> Forward Primer 5' - 3' <400> 15 atggagttct ctgcctctg 19 <210> 16 <211> 19 <212> DNA <213> Reverse primer 5' - 3' <400> 16 ggtttggctt atggaaggt 19 <210> 17 <211> 2496 <212> DNA <213> Sequence of DfHAD-6His-GST codon optimized by Genscript genetic codon frequency of E. coli <220> <221> CDS <222> (1)..(2496) <400> 17 atg tct ggt tct cat cat cat cat cat cat agc agc ggt atg tcc cct 48 Met Ser Gly Ser His His His His His Ser Ser Gly Met Ser Pro 1 5 10 15 ata cta ggt tat tgg aaa att aag ggc ctt gtg caa ccc act cga ctt 96 Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu 20 25 30 ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag cat ttg tat gag cgc 144 Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg 35 40 45 gat gaa ggt gat aaa tgg cga aac aaa aag ttt gaa ttg ggt ttg gag 192 Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu 50 55 60 ttt ccc aat ctt cct tat tat att gat ggt gat gtt aaa tta aca cag 240 Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln 65 70 75 80 tct atg gcc atc ata cgt tat ata gct gac aag cac aac atg ttg ggt 288 Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly 85 90 95 ggt tgt cca aaa gag cgt gca gag att tca atg ctt gaa gga gcg gtt 336 Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val 100 105 110 ttg gat to spank tac ggt gtt tcg spank to gca tat agt aaa gac ttt 384 Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe 115 120 125 gaa act ctc aaa gtt gat ttt ctt agc aag cta cct gaa atg ctg aaa 432 Glu Thr Leu Lys Val Asp Phe Leu Ser Leu Lys Pro Glu Met Leu Lys 130 135 140 atg ttc gaa gat cgt tta tgt cat aaa aca tat tta aat ggt gat cat 480 Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His 145 150 155 160 gta acc cat cct gac ttc atg ttg tat gac gct ctt gat gtt gtt tta 528 Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu 165 170 175 tac atg gac cca atg tgc ctg gat gcg ttc cca aaa tta gtt tgt ttt 576 Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe 180 185 190 aaa aaa cgt att gaa gct atc cca caa att gat aag tac ttg aaa tcc 624 Lys Arg Lys Glu Ala Lys Pro Gln Lys Asp Lys Tyr Leu Lys Ser 195 200 205 agc aag tat ata gca tgg cct ttg cag ggc tgg caa gcc acg ttt ggt 672 Serving Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly 210 215 220 ggt ggc gac cat cct cca aaa tcg gat ctg ggc cac aca ggc cat aga 720 Gly Gly Asp His Pro Pro Lys Ser Asp Leu Gly His Thr Gly His Arg 225 230 235 240 tct gac gac gac gac aag cat atg gag ttc agc gcg agc gct ccg ccg 768 Ser Asp Asp Asp Asp Lys His Met Glu Phe Ser Ala Ser Ala Pro Pro 245 250 255 ccg cgt ctg gcg agc gtg atc att ctg gaa ccg ctg ggt ttt ctg ctg 816 Pro Arg Leu Ala Ser Val Ile Ile Leu Glu Pro Leu Gly Phe Leu Leu 260 265 270 acc ccg cac tac agc agc cag ctg ccg aag aaa ctg ctg cgt cgt ctg 864 Thr Pro His Tyr Ser Ser Gln Leu Pro Lys Lys Leu Leu Arg Arg Leu 275 280 285 ctg tgc acc cgt atc tgg cac cgt tat cag cgt ggc cgt ctg cgt ctg 912 Leu Cys Thr Arg Ile Trp His Arg Tyr Gln Arg Gly Arg Leu Arg Leu 290 295 300 cgt gac gcg gcg atg ctg ctg gcg caa ctg ccg ttc ctg gcg gtt agc 960 Arg Asp Ala Ala Met Leu Leu Ala Gln Leu Pro Phe Leu Ala Val Ser 305 310 315 320 gac cac ccg tgg gcg ctg gat aac ctg gcg agc ctg ctg cgt ccg acc 1008 Asp His Pro Trp Ala Leu Asp Asn Leu Ala Ser Leu Leu Arg Pro Thr 325 330 335 gcg gtt cgt gcg gtg ccg tgg atg ctg ctg ctg ctg gac ttt ctg cgt 1056 Ala Val Arg Ala Val Pro Trp Met Leu Leu Leu Leu Asp Phe Leu Arg 340 345 350 gat gag ctg cac ctg aaa gtg gtt tgc gcg acc aac agc agc ccg gag 1104 Asp Glu Leu His Leu Lys Val Val Cys Ala Thr Asn Ser Ser Pro Glu 355 360 365 gaa ctg cag gaa ctg cgt cac caa ttc ccg gcg ctg ttt gcg aag gtt 1152 Glu Leu Gln Glu Leu Arg His Gln Phe Pro Ala Leu Phe Ala Lys Val 370 375 380 gac gcg acc gtg agc agc ggc gag gaa ggt gtt ggc aaa ccg agc gtg 1200 Asp Ala Thr Val Ser Ser Gly Glu Glu Gly Val Gly Lys Pro Ser Val 385 390 395 400 cgt ttc ctg caa gcg gcg ctg gat aag gcg ggc gtg cac gcg cag caa 1248 Arg Phe Leu Gln Ala Ala Leu Asp Lys Ala Gly Val His Ala Gln Gln 405 410 415 acc ctg tac ctg gac agc ttt gat agc ctg gag acc atc atg gcg gcg 1296 Thr Leu Tyr Leu Asp Ser Phe Asp Ser Leu Glu Thr Ile Met Ala Ala 420 425 430 cgt agc ctg ggt atg cac gcg ctg agc gtt gag ccg tgc cac att gac 1344 Arg Ser Leu Gly Met His Ala Leu Ser Val Glu Pro Cys His Ile Asp 435 440 445 gaa ctg acc gcg cgt gcg agc agc ggt cag ctg cgt gat gcg caa ctg 1392 Glu Leu Thr Ala Arg Ala Ser Ser Gly Gln Leu Arg Asp Ala Gln Leu 450 455 460 atc cgt cgt att gtt tgc gcg atg cac ggt ccg gct gtg agc gcg gtg 1440 Ile Arg Arg Ile Val Cys Ala Met His Gly Pro Ala Val Ser Ala Val 465 470 475 480 gtt agc ggt agc atc acc agc agc ggt ccg cag acc gcg aaa att gag 1488 Val Ser Gly Ser Ile Thr Ser Ser Gly Pro Gln Thr Ala Lys Ile Glu 485 490 495 gaa ctg ccg acc gcg gcg gac agc cac ctg cgt agc gcg gcg ctg acc 1536 Glu Leu Pro Thr Ala Ala Asp Ser His Leu Arg Ser Ala Ala Leu Thr 500 505 510 agc gcg cag caa ttc ttt ctg aaa gtg att gcg ccg cac cgt ccg gag 1584 Ser Ala Gln Gln Phe Phe Leu Lys Val Ile Ala Pro His Arg Pro Glu 515 520 525 aag ccg ttc gtt caa ctg ccg agc ctg acc agc gaa ggt atc cgt att 1632 Lys Pro Phe Val Gln Leu Pro Ser Leu Thr Ser Glu Gly Ile Arg Ile 530 535 540 tat gac acc ttc gcg cag ttt gtg atc gcg gat ctg ctg gac gat acc 1680 Tyr Asp Thr Phe Ala Gln Phe Val Ile Ala Asp Leu Leu Asp Asp Thr 545 550 555 560 cgt ttc ctg ccg atg caa agc ccg ccg ccg aac ggc ctg att acc ttt 1728 Arg Phe Leu Pro Met Gln Ser Pro Pro Pro Asn Gly Leu Ile Thr Phe 565 570 575 gtt aac ccg agc gcg tac ctg gcg gac gat atc aaa aac ggt aac agc 1776 Val Asn Pro Ser Ala Tyr Leu Ala Asp Asp Ile Lys Asn Gly Asn Ser 580 585 590 cac to gtt ccg ggc gtg cag ttc tat gcg agc gac gcg tgc acc ctg 1824 His Ile Val Pro Gly Val Gln Phe Tyr Ala Ser Asp Ala Cys Thr Leu 595 600 605 atc gat att ccg cac gac ctg gat acc acc agc gtt ggt ctg agc gtg 1872 Ile Asp Ile Pro His Asp Leu Asp Thr Thr Ser Val Gly Leu Ser Val 610 615 620 ctg cac aag ttt ggc aaa gtt gac aag gat acc ctg aac aag gtg ctg 1920 Leu His Lys Phe Gly Lys Val Asp Lys Asp Thr Leu Asn Lys Val Leu 625 630 635 640 gat cgt atg ctg gag caa gtt agc gaa gac gat ggt atc ctg caa gtt 1968 Asp Arg Met Leu Glu Gln Val Ser Glu Asp Asp Gly Ile Leu Gln Val 645 650 655 tac ttt gac gtg gag cgt ccg cgt att gat ccg gtg gtt gtg gcg aac 2016 Tyr Phe Asp Val Glu Arg Pro Arg Ile Asp Pro Val Val Val Ala Asn 660 665 670 acc gtg ttc ctg ttt cac cctg ggt aaa cgt ggc cac gaa gtt gcg cgt 2064 Thr Val Phe Leu Phe His Leu Gly Lys Arg Gly His Glu Val Ala Arg 675 680 685 agc gag aag ttc gtt gaa agc gtg ctg ctg cag cgt gcg tac gag gaa 2112 Ser Glu Lys Phe Val Glu Ser Val Leu Leu Gln Arg Ala Tyr Glu Glu 690,695,700 ggc acc ctg tac tat aac ctg ggc gaa gcg ttt ctg gtt agc gtg gcg 2160 Gly Thr Leu Tyr Tyr Asn Leu Gly Glu Ala Phe Leu Val Ser Val Ala 705,710,715,720 cgt ctg gtg cac gag ttc aaa gaa cac ttt acc cgt agc ggt atg cgt 2208 Arg Leu Val His Glu Phe Lys Glu His Phe Thr Arg Ser Gly Met Arg 725 730 735 cgt gcg ctg gag gaa cgt cgt cgt gag cgt gcg cgt gcg ggt atg caa 2256 Arg Ala Leu Glu Glu Arg Leu Arg Glu Arg Ala Arg Ala Gly Met Gln 740,745,750 gaa cgt gac gat gcg ctg gcg ctg gcg atg cgt atc cgt gcg tgc gcg 2304 Glu Arg Asp Asp Ala Leu Ala Leu Ala Met Arg Ile Arg Ala Cys Ala 755 760 765 ctg tgc ggt ctg gcg ggc gag ggt ctg acc aag gcg gcg gag cag gaa 2352 Leu Cys Gly Leu Ala Gly Glu Gly Leu Thr Lys Ala Ala Glu Gln Glu 770 775 780 ctg ctg cgt ctg caa tgc aag agc aaa ggt tgc tgg ggc tgc cac ccg 2400 Leu Leu Arg Leu Gln Cys Lys Ser Lys Gly Cys Trp Gly Cys His Pro 785 790 795 800 ttc tac cgt aac ggt agc aac gtt ctg agc tgg atc ggc agc gaa gcg 2448 Phe Tyr Arg Asn Gly Ser Asn Val Leu Ser Trp Ile Gly Ser Glu Ala 805 810 815 ctg acc acc gcg tat gcg att gcg gcg ctg cag ccg atc gac att taa 2496 Leu Thr Thr Ala Tyr Ala Ile Ala Ala Leu Gln Pro Ile Asp Ile 820 825 830 <210> 18 <211> 831 <212> PRT <213> Sequence of DfHAD-6His-GST codon optimized by Genscript genetic codon frequency of E. coli <400> 18 Met Ser Gly Ser His His His His His Ser Ser Gly Met Ser Pro 1 5 10 15 Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu 20 25 30 Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg 35 40 45 Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu 50 55 60 Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln 65 70 75 80 Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly 85 90 95 Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val 100 105 110 Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe 115 120 125 Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys 130 135 140 Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His 145 150 155 160 Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu 165 170 175 Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe 180 185 190 Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser 195 200 205 Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly 210 215 220 Gly Gly Asp His Pro Pro Lys Ser Asp Leu Gly His Thr Gly His Arg 225 230 235 240 Ser Asp Asp Asp Asp Lys His Met Glu Phe Ser Ala Ser Ala Pro Pro 245 250 255 Pro Arg Leu Ala Ser Val Ile Ile Leu Glu Pro Leu Gly Phe Leu Leu 260 265 270 Thr Pro His Tyr Ser Ser Gln Leu Pro Lys Lys Leu Leu Arg Arg Leu 275 280 285 Leu Cys Thr Arg Ile Trp His Arg Tyr Gln Arg Gly Arg Leu Arg Leu 290 295 300 Arg Asp Ala Ala Met Leu Leu Ala Gln Leu Pro Phe Leu Ala Val Ser 305 310 315 320 Asp His Pro Trp Ala Leu Asp Asn Leu Ala Ser Leu Leu Arg Pro Thr 325 330 335 Ala Val Arg Ala Val Pro Trp Met Leu Leu Leu Leu Asp Phe Leu Arg 340 345 350 Asp Glu Leu His Leu Lys Val Val Cys Ala Thr Asn Ser Ser Pro Glu 355 360 365 Glu Leu Gln Glu Leu Arg His Gln Phe Pro Ala Leu Phe Ala Lys Val 370 375 380 Asp Ala Thr Val Ser Ser Gly Glu Glu Gly Val Gly Lys Pro Ser Val 385 390 395 400 Arg Phe Leu Gln Ala Ala Leu Asp Lys Ala Gly Val His Ala Gln Gln 405 410 415 Thr Leu Tyr Leu Asp Ser Phe Asp Ser Leu Glu Thr Ile Met Ala Ala 420 425 430 Arg Ser Leu Gly Met His Ala Leu Ser Val Glu Pro Cys His Ile Asp 435 440 445 Glu Leu Thr Ala Arg Ala Ser Ser Gly Gln Leu Arg Asp Ala Gln Leu 450 455 460 Ile Arg Arg Ile Val Cys Ala Met His Gly Pro Ala Val Ser Ala Val 465 470 475 480 Val Ser Gly Ser Ile Thr Ser Ser Gly Pro Gln Thr Ala Lys Ile Glu 485 490 495 Glu Leu Pro Thr Ala Ala Asp Ser His Leu Arg Ser Ala Ala Leu Thr 500 505 510 Ser Ala Gln Gln Phe Phe Leu Lys Val Ile Ala Pro His Arg Pro Glu 515 520 525 Lys Pro Phe Val Gln Leu Pro Ser Leu Thr Ser Glu Gly Ile Arg Ile 530 535 540 Tyr Asp Thr Phe Ala Gln Phe Val Ile Ala Asp Leu Leu Asp Asp Thr 545 550 555 560 Arg Phe Leu Pro Met Gln Ser Pro Pro Pro Asn Gly Leu Ile Thr Phe 565 570 575 Val Asn Pro Ser Ala Tyr Leu Ala Asp Asp Ile Lys Asn Gly Asn Ser 580 585 590 His Ile Val Pro Gly Val Gln Phe Tyr Ala Ser Asp Ala Cys Thr Leu 595 600 605 Ile Asp Ile Pro His Asp Leu Asp Thr Thr Ser Val Gly Leu Ser Val 610 615 620 Leu His Lys Phe Gly Lys Val Asp Lys Asp Thr Leu Asn Lys Val Leu 625 630 635 640 Asp Arg Met Leu Glu Gln Val Ser Glu Asp Asp Gly Ile Leu Gln Val 645 650 655 Tyr Phe Asp Val Glu Arg Pro Arg Ile Asp Pro Val Val Val Ala Asn 660 665 670 Thr Val Phe Leu Phe His Leu Gly Lys Arg Gly His Glu Val Ala Arg 675 680 685 Ser Glu Lys Phe Val Glu Ser Val Leu Leu Gln Arg Ala Tyr Glu Glu 690 695 700 Gly Thr Leu Tyr Tyr Asn Leu Gly Glu Ala Phe Leu Val Ser Val Ala 705 710 715 720 Arg Leu Val His Glu Phe Lys Glu His Phe Thr Arg Ser Gly Met Arg 725 730 735 Arg Ala Leu Glu Glu Arg Leu Arg Glu Arg Ala Arg Ala Gly Met Gln 740 745 750 Glu Arg Asp Asp Ala Leu Ala Leu Ala Met Arg Ile Arg Ala Cys Ala 755 760 765 Leu Cys Gly Leu Ala Gly Glu Gly Leu Thr Lys Ala Ala Glu Gln Glu 770 775 780 Leu Leu Arg Leu Gln Cys Lys Ser Lys Gly Cys Trp Gly Cys His Pro 785 790 795 800 Phe Tyr Arg Asn Gly Ser Asn Val Leu Ser Trp Ile Gly Ser Glu Ala 805 810 815 Leu Thr Thr Ala Tyr Ala Ile Ala Ala Leu Gln Pro Ile Asp Ile 820 825 830 <210> 19 <211> 583 <212> PRT <213> DfHAD-QW <400> 19 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 Lys Val Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 Ala Asp Asp Ile Lys Asn Gly Asn Ser His Ile Val Pro Gly Val Gln 340 345 350 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 Gly Leu Thr Lys Ala Ala Glu Gln Glu Leu Leu Arg Leu Gln Cys Glu 530 535 540 Asp Gly Gly Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn Val 545 550 555 560 Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile Ala 565 570 575 Ala Leu Gln Pro Ile Asp Ile 580 <210> 20 <211> 584 <212> PRT <213> DfHAD-QW mute <400> 20 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 Lys Val Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 Ala Asp Asp Ile Lys Asn Gly Asn Ser His Ile Val Pro Gly Val Gln 340 345 350 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 Gly Leu Thr Lys Ala Ala Glu Gln Glu Leu Leu Arg Leu Gln Cys Ala 530 535 540 Ala Ala Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 Ala Ala Leu Gln Pro Ile Asp Ile 580 <210> 21 <211> 584 <212> PRT <213> DfHAD-SEQ64 motif <400> 21 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 Asp Pro Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 Lys Val Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 Ala Asp Asp Ile Lys Asn Gly Asn Ser His Ile Val Pro Gly Val Gln 340 345 350 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 Ser Glu Asp Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro 405 410 415 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 Gly Leu Thr Lys Pathway Glu Gln Glu Leu Leu Arg Leu Gln Cys Lys 530 535 540 Ser Lys Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 Pathway Leu Gln Pro Ile Asp Ile 580 <210> 22 <211> 584 <212> PRT <213> DfHAD-MOS motif <400> 22 Met Glu Phe Ser Ala Ser Ala Pro Pro Pro Arg Leu Ala Ser Val Ile 1 5 10 15 Ile Leu Glu Pro Leu Gly Phe Leu Leu Thr Pro His Tyr Ser Ser Gln 20 25 30 Leu Pro Lys Lys Leu Leu Arg Arg Leu Leu Cys Thr Arg Ile Trp His 35 40 45 Arg Tyr Gln Arg Gly Arg Leu Arg Leu Arg Asp Ala Ala Met Leu Leu 50 55 60 Ala Gln Leu Pro Phe Leu Ala Val Ser Asp His Pro Trp Ala Leu Asp 65 70 75 80 Asn Leu Ala Ser Leu Leu Arg Pro Thr Ala Val Arg Ala Val Pro Trp 85 90 95 Met Leu Leu Leu Leu Asp Phe Leu Arg Asp Glu Leu His Leu Lys Val 100 105 110 Val Cys Ala Thr Asn Ser Ser Pro Glu Glu Leu Gln Glu Leu Arg His 115 120 125 Gln Phe Pro Ala Leu Phe Ala Lys Val Asp Ala Thr Val Ser Ser Gly 130 135 140 Glu Glu Gly Val Gly Lys Pro Ser Val Arg Phe Leu Gln Ala Ala Leu 145 150 155 160 Asp Lys Ala Gly Val His Ala Gln Gln Thr Leu Tyr Leu Asp Ser Phe 165 170 175 Asp Ser Leu Glu Thr Ile Met Ala Ala Arg Ser Leu Gly Met His Ala 180 185 190 Leu Ser Val Glu Pro Cys His Ile Asp Glu Leu Thr Ala Arg Ala Ser 195 200 205 Ser Gly Gln Leu Arg Asp Ala Gln Leu Ile Arg Arg Ile Val Cys Ala 210 215 220 Met His Gly Pro Ala Val Ser Ala Val Val Ser Gly Ser Ile Thr Ser 225 230 235 240 Ser Gly Pro Gln Thr Ala Lys Ile Glu Glu Leu Pro Thr Ala Ala Asp 245 250 255 Ser His Leu Arg Ser Ala Ala Leu Thr Ser Ala Gln Gln Phe Phe Leu 260 265 270 Lys Val Ile Ala Pro His Arg Pro Glu Lys Pro Phe Val Gln Leu Pro 275 280 285 Ser Leu Thr Ser Glu Gly Ile Arg Ile Tyr Asp Thr Phe Ala Gln Phe 290 295 300 Val Ile Ala Asp Leu Leu Asp Asp Thr Arg Phe Leu Pro Met Gln Ser 305 310 315 320 Pro Pro Pro Asn Gly Leu Ile Thr Phe Val Asn Pro Ser Ala Tyr Leu 325 330 335 Ala Asp Asp Ile Lys Asn Gly Asn Ser His Ile Val Pro Gly Val Gln 340 345 350 Phe Tyr Ala Ser Asp Ala Cys Thr Leu Ile Asp Ile Pro His Asp Leu 355 360 365 Asp Thr Thr Ser Val Gly Leu Ser Val Leu His Lys Phe Gly Lys Val 370 375 380 Asp Lys Asp Thr Leu Asn Lys Val Leu Asp Arg Met Leu Glu Gln Val 385 390 395 400 Ser Glu Asp Asp Gly Ile Leu Gln Val Ala Ala Ala Val Glu Arg Pro 405 410 415 Arg Ile Asp Pro Val Val Val Ala Asn Thr Val Phe Leu Phe His Leu 420 425 430 Gly Lys Arg Gly His Glu Val Ala Arg Ser Glu Lys Phe Val Glu Ser 435 440 445 Val Leu Leu Gln Arg Ala Tyr Glu Glu Gly Thr Leu Tyr Tyr Asn Leu 450 455 460 Gly Glu Ala Phe Leu Val Ser Val Ala Arg Leu Val His Glu Phe Lys 465 470 475 480 Glu His Phe Thr Arg Ser Gly Met Arg Arg Ala Leu Glu Glu Arg Leu 485 490 495 Arg Glu Arg Ala Arg Ala Gly Met Gln Glu Arg Asp Asp Ala Leu Ala 500 505 510 Leu Ala Met Arg Ile Arg Ala Cys Ala Leu Cys Gly Leu Ala Gly Glu 515 520 525 Gly Leu Thr Lys Ala Ala Glu Gln Glu Leu Leu Arg Leu Gln Cys Lys 530 535 540 Ser Lys Gly Cys Trp Gly Cys His Pro Phe Tyr Arg Asn Gly Ser Asn 545 550 555 560 Val Leu Ser Trp Ile Gly Ser Glu Ala Leu Thr Thr Ala Tyr Ala Ile 565 570 575 Ala Ala Leu Gln Pro Ile Asp Ile 580 <210> 23 <211> 6 <212> PRT <213> Alternative class II motif: DLDTTS <400> 23 Asp Leu Asp Thr Thr Ser 1 5 <210> 24 <211> 8 <212> PRT <213> Motif III: QCKSKGCW <400> 24 Gln Cys Lys Ser Lys Gly Cys Trp 1 5 <210> 25 <211> twenty two <212> PRT <213> Motif IV: DGILQVYFDVERPRIDPVVVAN <400> 25 Asp Gly Ile Leu Gln Val Tyr Phe Asp Val Glu Arg Pro Arg Ile Asp 1 5 10 15 Pro Val Val Val Ala Asn 20 <210> 26 <211> 8 <212> PRT <213> Modified motif III <400> 26 Gln Cys Ala Ala Ala Gly Cys Trp 1 5 <210> 27 <211> 7 <212> PRT <213> Modified motif III <400> 27 Gln Cys Glu Asp Gly Gly Trp 1 5 <210> 28 <211> twenty two <212> PRT <213> Modified Motif IV <400> 28 Asp Gly Ile Leu Gln Val Ala Ala Ala Val Glu Arg Pro Arg Ile Asp 1 5 10 15 Pro Val Val Val Ala Asn 20 <210> 29 <211> 7 <212> PRT <213> Modified Class I Motif-Mutated <400> 29 Asp Ser Phe Asp Pro Leu Glu 1 5

Claims

1. An isolated polypeptide of the haloacid dehalogenase-like (HAD-like) hydrolase superfamily having terpene synthase activity, wherein the polypeptide is DfHAD having an amino acid sequence of SEQ ID NO:

2.

2. An isolated polypeptide which is a variant of the DfHAD of claim 1 selected from the group consisting of: a. DfHAD-9 (V274A) having the amino acid sequence of SEQ ID NO: 6; and b. DfHAD-8 (K532R) of the amino acid sequence of SEQ ID NO:

4.

3. An isolated nucleic acid molecule, the nucleotide sequence of which is SEQ ID NO:

1.

4. An expression construct comprising at least one nucleic acid molecule according to claim 3.

5. A vector comprising at least one nucleic acid molecule according to claim 3 or at least one expression construct according to claim 4.

6. A recombinant non-human host organism comprising: a. at least one isolated nucleic acid molecule according to claim 3, which is optionally stably integrated into the genome; or b. at least one expression construct according to claim 4, which is optionally stably integrated into the genome; or c. at least one vector according to claim 5, The recombinant non-human host organism is a prokaryotic microorganism or a eukaryotic microorganism, or a bacterial cell or a fungal cell derived therefrom.

7. A method for producing at least one polypeptide according to claim 1 or 2, the method comprising: a. Cultivating the non-human host organism of claim 6 to express or overexpress at least one polypeptide according to claim 1 or 2; and b. Optionally, isolating the polypeptide from the non-human host organism cultured in step a.

8. A method for preparing a mutant polypeptide having terpene synthase activity, particularly terpene diphosphate synthase activity, the method comprising the following steps: a. selecting the nucleic acid molecule according to claim 3; b. modifying the selected nucleic acid molecule to obtain at least one mutant nucleic acid molecule; c. transforming a host cell or a unicellular host organism with the mutant nucleic acid sequence to express a polypeptide encoded by the mutant nucleic acid sequence; d. screening the expression products for at least one mutant comprising terpene synthase activity, particularly folded styryl diphosphate synthase activity; and, e. Optionally, if the polypeptide does not have the desired mutation activity, repeat steps a. to d. until a polypeptide having the desired mutation activity is obtained; and f. Optionally, if a polypeptide having the desired mutant activity is identified in step d., isolating the corresponding mutant nucleic acid obtained in step c.

9. A method for producing succinyl sesquiterpenes, especially succinyl alcohol, comprising: a. contacting farnesyl diphosphate (FPP) with a polypeptide as defined in claim 1 or 2, with a polypeptide prepared according to claim 7, or with a mutant polypeptide prepared according to claim 8, thereby obtaining at least one diphosphate of sesquiterpene, in particular folded moss base diphosphate; b. chemically or enzymatically cleaving the diphosphate portion of the product; and c. Optionally, isolating the succinyl sesquiterpenes, in particular succinyl alcohol.

10. Use of the polypeptide defined in claim 1 or 2 in the preparation of a flavoring or fragrance ingredient.

11. Use of the polypeptide defined in claim 1 or 2 in the preparation of Ambrox.

12. A method for producing Ambrox, the method comprising: a. providing folded moss alcohol by the method described in claim 9; b. Optionally, separating the folded phytol produced in step a.; and c. The conversion of fennel to Ambrox is carried out in a manner known per se.

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