Lipoxygenase catalyzed production of unsaturated C10-aldehydes from polyunsaturated fatty acids (PUFA)

Abstract

The present invention provides a novel process for the production of aliphatic unsaturated C10-aldehyde compounds catalyzed by lipoxygenase (LOX) from a polyunsaturated fatty acid (PUFA) source. The invention also relates to the isolation and characterization of novel, preferably bifunctional LOXs from different algae sources, and the identification of structural and / or functional related LOXs from different bacterial sources. The invention also relates to the provision of enzyme mutants derived from said newly identified enzymes. Another aspect of the invention relates to the corresponding coding sequences of said enzymes, recombinant vectors and recombinant host cells, which are suitable for the production of such LOXs and for carrying out a new process for the production of aliphatic unsaturated C10-aldehyde compounds. Another aspect of the invention relates to the use of a specific aldehyde or aldehyde mixture obtained according to the invention as a flavoring ingredient or an ingredient of a food or feed composition.

Description

[0001] The present invention is a divisional application of invention patent application No. 201980068076.9 filed on October 18, 2019, entitled “Lipoxygenase-catalyzed production of unsaturated C10-aldehydes from polyunsaturated fatty acids (PUFA)”. Technical Field

[0002] The present invention provides a method for producing aliphatic unsaturated C from a polyunsaturated fatty acid (PUFA) source catalyzed by lipoxygenase (LOX). 10 The present invention also relates to the isolation and characterization of novel, preferably bifunctional LOX from different algal sources, and the identification of structurally and / or functionally related LOX from different bacterial sources. The present invention also relates to the provision of enzyme mutants derived from the newly identified enzymes. Another aspect of the present invention relates to the corresponding coding sequences of the enzymes, recombinant vectors and recombinant host cells, which are suitable for producing such LOX and for carrying out the decomposition of aliphatic unsaturated C 10 - A novel method for producing aldehyde compounds. Another aspect of the present invention relates to the use of a specific aldehyde or aldehyde mixture obtained according to the present invention as a flavoring ingredient or as a component of a food or feed composition. Background Art

[0003] Unsaturated C 10 -aldehydes decadienal and decatrienal are important ingredients in chicken and citrus seasonings (food flavors). Despite the high production cost and low yield, flavorists cannot replace them with other ingredients because of their unique olfactory properties. More than 200 commercial formulas contain C 10 -aldehyde.

[0004] C 6 and C 9 Aldehydes are usually biosynthesized by plant defense systems through a two-step enzymatic reaction starting from polyunsaturated fatty acids (PUFA) (see Scheme 1 below). First, LOX converts fatty acids into fatty acid hydroperoxides (HPO). Subsequently, lipid hydroperoxide lyases (HPLs) break down HPO into metabolites, including aldehydes and alcohols. C 6 and C 9 The components are well known. 6 and C 9 The industrial route of aldehyde seasoning ingredients is relatively mature and the product quality is stable. Therefore, the price is still lower than C 10 Prices for similar products.

[0005] With C 6 and C 9 Compared with the analogs, the C 10The industrial process for the aldehyde component is more challenging (see Scheme 1 below, right half). It starts with the peroxidation of linoleic acid and α-linolenic acid catalyzed by 9-LOX. 9-LOX is obtained from a plant source (potato). Considering that there is no available HPL that can cleave the 9-HPO intermediate to C 10 fragments, so the typical current method relies on thermal degradation of 9-HPO. In general, this method has two disadvantages. One is the product variation problem caused by the variation in the quality of potato extracts from different suppliers, that is, the yield obtained in each production batch is different due to the different enzyme content in the potato.

[0006] Another is the low yield of the thermal cracking step, resulting in high production costs.

[0007]

[0008] Solution 1.C 6 ~C 10 Current industrial production routes for aldehyde components.

[0009] Alsufyani, T. et al., in Chemistry and Physics of Lipids 183 (2014) 100-109, describe several seaweeds, including Ulva, that can produce decadienal and decatrienal via the conventional LOX / HPL pathway. This prior art document does not identify any gene sequence, coding sequence, or protein sequence involved in the bioconversion, nor does it identify any key amino acid residues that determine high LOX activity.

[0010] Lee, J. et al., Environmental Pollution 227 (2017) 252-262, provide a review of algal and bacterial odor problems that have been published over the past fifty years. Two species of Microcystis (cyanobacteria) are reported to produce decatrienal. However, the prior art focuses on odor pollution in water and does not provide specific teachings on genes, coding sequences, or protein sequences that lead to the formation of the decatrienal.

[0011] Zhu, ZJ. et al. further studied the bioconversion of fatty acids into mainly C based on LOX activity and HPL activity in Journal of Agriculture and Food Chemistry. (2018) 66(5): 1233-1241. 8 -C 9A multifunctional LOX in the aldehyde step, PhLOX from the seaweed Pyropia haitanensis (also described by Chen, Hai-min et al. in Algal Research, 12, (2015) 316-327). The multifunctional LOX is said to show LOX, HPL and allene oxide synthase (AOS) activities. The production of 2E,4Z-decadienal by-product was observed only by feeding hydrolyzed fish oil, without feeding many other test substrates such as ALA, ARA, EPA and DHA. No decatrienal was observed. In the prior art, γ-linolenic acid was not used as a substrate. The productivity of the decatrienal by-product is very low and has no industrial value.

[0012] Another multifunctional LOX, PhLOX2, from the seaweed Pyropia haitanensis was described by Zhu et al. in PLoS One. (2015) 10(2): e0117351. EPA, ARA, GLA, and DHA were used as substrates; no unsaturated C 10 aldehyde.

[0013] Chinese patent application CN 104293805 describes a multifunctional LOX protein sequence from the seaweed Pyropia haitanensis (PhLOX), which is also expressed in E. coli. The LOX species does not produce decadienal and decatrienal when fed with fatty acid substrates. It only produces short-chain aldehydes.

[0014] Chinese patent application CN 104293837 A describes another multifunctional LOX from the seaweed Pyropia haitanensis (PhLOX) expressed in E. coli. 10 -Evidence of aldehydes, especially decadienal and decatrienal.

[0015] WO2008056291 and EP-A-1 921 134 describe a cyanobacterium LOX, WP_012407347.1, and propose its use in the production of fatty acid hydroperoxides, but do not provide a method for producing unsaturated C 10 -Evidence for aldehydes such as decadienal.

[0016] Although the unsaturated C 10 There are different reports on the biocatalytic synthesis of C-aldehydes, but the enzyme systems described in the prior art still suffer from the problem of low productivity and, therefore, have not been developed for the industrial-scale production of C 10 -aldehydes provide a suitable base.

[0017] Therefore, the problem to be solved by the present invention is to provide an improved biocatalytic method for producing unsaturated C 10 Another problem to be solved by the present invention is to provide a method suitable for completely biosynthesizing unsaturated C 10 - Novel biocatalysts for the production of aldehydes, in particular decadienal and / or decatrienal. Summary of the invention

[0018] Surprisingly, the above problems can be solved by providing unique and superior LOX from new sources. In particular, the present inventors have succeeded in isolating novel bifunctional LOX from the seaweed source Cladophora oligoclara, which produces large amounts of decadienal and / or decatrienal from different PUFA substrates. The present inventors have also succeeded in isolating novel bifunctional LOX from the seaweed Ulva fasciata, which also produces large amounts of decadienal and / or decatrienal from different PUFA substrates.

[0019] Based on the sequence information from the novel LOX, the inventors have also surprisingly succeeded in identifying LOX from bacterial sources (mainly cyanobacteria) with the desired catalytic LOX activity.

[0020] Based on sequence comparisons between the newly identified enzymes, the inventors were able to characterize the unsaturated C 10 The inventors systematically investigated the structure and function of suitable bifunctional LOXs that showed excellent productivity and / or specificity for C-aldehyde compounds, particularly decadienal and / or decatrienal, more particularly decadienal. For several bacterial LOXs, productivity enhancement was observed. Based on such studies, the inventors were able to 10 - Further improve the LOX productivity in the industrial production of aldehydes.

[0021] The newly identified protein sequence can be functionally expressed in a bacterial host such as E. coli. Surprisingly, a culture with a high cell density can be obtained, which has improved enzymatic capacity and can be used for the C 10 Industrial-scale production of -aldehydes. This recombinant E. coli host has high productivity in different decadienals and / or decatrienals if fed with specific fatty acids as substrates.

[0022] The new method allows for a more cost-effective approach to the complete biocatalytic production of decadienal and / or decatrienal.

[0023] If desired, the aldehydes can be converted into suitable derivatives, in particular the corresponding alcohols, by chemical or in particular biochemical conversion, for example by using conventional enzymes such as alcohol dehydrogenases (ADH). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .Unsaturated C 10 Structural formulas of the aldehyde stereoisomers 2E,4Z,7Z-decatrienal, 2E,4E,7Z-decatrienal, 2E,4Z-decadienal and 2E,4E-decadienal.

[0025] Figure 2 .SPME / GC / MS chromatogram of fresh Ulva fasciata sample.

[0026] Figure 3 .SPME / GC / MS chromatogram of fresh C. oligoclara sample.

[0027] Figure 4 .MS spectrum of 2E,4Z-decadienal.

[0028] Figure 5 .MS spectrum of 2E,4E-decadienal.

[0029] Figure 6 .MS spectrum of 2E,4Z,7Z-decatrienal.

[0030] Figure 7 .MS spectrum of 2E,4E,7Z-decatrienal.

[0031] Figure 8 Results of feeding CoLOX of the present invention with γ-linolenic acid; compared with negative control (BL21 = untransformed E. coli cells; pETDuet = BL21 transformed with an empty vector);

[0032] Fig. 9 Results of feeding CoLOXs of the invention with a mixture of α-linolenic acid and linoleic acid compared to negative controls (BL21 = untransformed E. coli cells; empty vector = E. coli cells transformed with pETDuet-1).

[0033] Fig.10 Results of CoLOXs of the invention fed with fish oil compared to negative controls (BL21 = untransformed E. coli cells; empty vector = E. coli cells transformed with pETDuet-1).

[0034] Figure 11. Sequence alignment of UfLOX2 and bacterial LOX to mine key amino acid residues.

[0035] Fig.12 .Results of UfLOX2 mutagenesis studies.

[0036] Fig.13.Effects of different cofactors on UfLOX2 activity.

[0037] Fig.14 . Alignment of different CoLOX amino acid sequences to generate the consensus sequence of SEQ ID NO:51.

[0038] Fig.15 .Alignment of different bacterial LOX amino acid sequences to generate the consensus sequence of SEQ ID NO:52.

[0039] Fig.16 .Alignment of UfLOX2 and different bacterial LOX amino acid sequences to generate the consensus sequence of SEQ ID NO:53.

[0040] Fig.17 . Alignment of different CoLOX, UfLOX2 and different bacterial LOX amino acid sequences to generate the consensus sequence of SEQ ID NO:54.

[0041] Fig.18 . Average productivity of bacterial LOX mutants (black) compared to the native sequence (grey). DETAILED DESCRIPTION

[0042] Abbreviations used

[0043] AOS allene oxide synthase

[0044] bp base pair

[0045] kb kilobase

[0046] DNA deoxyribonucleic acid

[0047] cDNA complementary DNA

[0048] GC Gas Chromatography

[0049] HPO Hydroperoxide

[0050] HPL Lipid hydroperoxide lyase

[0051] LOX Lipoxygenase

[0052] MS Mass Spectrometer / Mass Spectrometry

[0053] PUFA Polyunsaturated fatty acids

[0054] PCR polymerase chain reaction

[0055] RNA Ribonucleic acid

[0056] mRNA messenger RNA

[0057] miRNA

[0058] siRNA Small interfering RNA

[0059] rRNA ribosomal RNA

[0060] tRNA Unless otherwise specified, tRNA transfer RNA Xaa (or X) used herein or in the sequence listing attached to the specification refers to any known natural amino acid residue or chemical bond.

[0061] The specific PUFAs (PUFA substrates) specifically mentioned herein are selected from the following polyunsaturated omega-3 and omega-6 fatty acids and natural or synthetic mixtures of at least two thereof:

[0062] Omega-3 fatty acids

[0063]

[0064] Omega-6 fatty acids

[0065]

[0066] Non-limiting examples of specific PUFA mixtures specifically mentioned herein are selected from the group consisting of fish oil, flaxseed oil, arachidonic acid oil, linseed oil, evening primrose oil, echium oil, microalgae oil and borage oil.

[0067] definition

[0068] "Lipoxygenases" (LOX) (also called linoleate:oxygen oxidoreductases, EC 1.13.11.12) constitute a large gene family of non-heme iron-containing fatty acid dioxygenases that are ubiquitous in plants and animals. LOX catalyze the regio- and stereospecific dioxygenation of PUFAs containing at least one (1Z,4Z)-pentadiene system. Substrates for LOX are therefore, for example, linoleic acid (LA), α-linolenic acid (ALA) or arachidonic acid (ARA).

[0069] As used herein, the term "LOX" specifically refers to a PUFA degrading enzyme that has the ability to initiate a dioxygenation step at an appropriate chain position of the PUFA molecule, which ultimately results in the removal of at least one unsaturated C 10 - the formation of aldehyde fragments, in particular at least one decadienal and / or decatrienal compound, as a result of this oxidative degradation reaction. 10 Compounds can react with other oxidation products of different chain lengths, such as shorter chain lengths, such as C 6 - or C 9 Unsaturated aldehydes are produced as by-products, but especially the C10 Compounds may be produced as major products, i.e., with other oxidation products of different chain lengths, e.g., shorter chain lengths, such as C 6 - or C 9 unsaturated aldehyde, in excess of the molar ratio, or more particularly, the C 10 Compounds may be produced as a single product species.

[0070] The "LOX / HPL pathway" or "LOX / HPL pathway" refers to the classic two-step enzymatic reaction for the oxidative degradation of polyunsaturated fatty acid molecules. First, LOXs (LOX) convert the fatty acids into fatty acid hydroperoxides (HPOs). Subsequently, HPLs (HPL) break down the HPOs into metabolites, including aldehydes and alcohols.

[0071] "Bifunctional" LOX refers herein to a single enzyme molecule that exhibits both LOX and HPL activities required for the oxidative degradation of polyunsaturated fatty acid molecules (regardless of the specific enzymatic mechanism). In a specific embodiment, such bifunctional LOX may substantially not exhibit AOS activity, and more particularly such AOS activity may be absent. As shown in the experimental part, such bifunctional LOX not only form fatty acid hydroperoxide intermediates, but if used as synthetic artificial substrates, they also exhibit the ability to degrade such fatty acid hydroperoxide compounds. "Bifunctional" LOX refers herein in particular to a single enzyme molecule that exhibits both LOX and HPL activities required for the oxidative degradation of polyunsaturated fatty acid molecules (regardless of the specific enzymatic mechanism). Therefore, as a result of this oxidative degradation reaction, the bifunctional LOX catalyzes the formation of at least one unsaturated C 10 - an aldehyde fragment, in particular at least one decadienal and / or decatrienal compound. 10 Compounds can react with other oxidation products of different chain lengths, such as shorter chain lengths, such as C 6 - or C 9 Unsaturated aldehydes are produced as by-products, but especially the C 10 Compounds may be produced as major products, i.e., with other oxidation products of different chain lengths, e.g., shorter chain lengths, such as C 6 - or C 9 The unsaturated aldehyde is produced in an excess molar ratio, or more particularly, the C 10 Compounds may be produced as a single product species.

[0072] Without being bound by any mechanistic considerations, the HLP activity of the "bifunctional LOX" of the present invention can be further described as the ability to cleave the hydroperoxide intermediate of the PUFA substrate exclusively or preferentially at the CC bond on the carboxyl terminal side (relative to its HOO-group). This also distinguishes the enzyme of the present invention from plant-derived LOX / HLP enzyme systems, such as shown in Scheme 1 above. 18 -PUFAs), the bifunctional LOX of the present invention can be considered to encompass both 9-LOX activity and 9-HPL activity. In contrast to the prior art 9-HLP of rice plants, the 9-HPL activity of the bifunctional LOX of the present invention results in the cleavage of a hydroperoxide intermediate on the opposite (carboxyl terminal) side of the HOO-group of the intermediate. 10 -aldehyde, an additional double bond in the β-position relative to the HOO-group appears to be favorable or necessary, so that carbon chain cleavage between the C atom bearing the HOO-group and its α-position carbon atom will occur. As a result, C 10 -aldehydes instead of C in the case of plant enzymes 9 -aldehyde. This is illustrated in Scheme 2 below using GLA as an example.

[0073]

[0074] Scheme 2. LOX enzymes of the present invention (resulting in C 10 Comparison of the cleavage sites of the enzymes of the prior art (resulting in C9 aldehyde formation) and the prior art enzymes (resulting in C9 aldehyde formation).

[0075] It is obvious from the above scheme 2 that the "bifunctional LOX" of the present invention is used to generate unsaturated C 10 -aldehyde, a specific PUFA substrate is used. Basically, the preferred PUFA substrate should contain a cis double bond between the omega-9 and 10 carbon atoms (i.e., between positions (C-9) and (C-10) in C18 fatty acids and between positions (C-11) and (C-12) in C20 fatty acids), and between omega-12 and 13 carbon atoms (i.e., between positions (C-6) and (C-7) in C18 fatty acids and between positions (C-8) and (C-9) in C20 fatty acids). For example, in the case of C18 fatty acids, fatty acids containing two cis double bonds in the all-cis 6,9 configuration (see GLA and SDA) are preferred substrates, and in the case of C20 fatty acids, fatty acids containing two cis double bonds in the all-cis 8,11 configuration (see EPA or ARA) are preferred substrates. These preferred PUFA substrates can also be considered "reference substrates". In order to qualify as a "bifunctional LOX of the present invention", as long as the LOX is able to convert at least one such "reference substrate" into an unsaturated C 10-aldehyde, in particular at least one selected from (2E,4Z)-2,4-decadienal, (2E,4E)-2,4-decadienal, (2E,4Z,7Z)-2,4,7-decatrienal and (2E,4E,7Z)-2,4,7-decatrienal, is sufficient.

[0076] "Unsaturated C 10 "-aldehyde" encompasses any mono-, di- or tri-unsaturated straight chain aliphatic aldehyde having ten carbon atoms in its hydrocarbyl chain. It includes such compounds in any stereoisomerically pure form or in the form of a mixture of at least two different stereoisomers. Specific non-limiting examples of such aldehydes are decadienal and decatrienal.

[0077] "Decadienal" encompasses such compounds in any stereoisomerically pure form or in the form of a mixture of at least two different stereoisomers. Typical examples are 2E,4Z-decadienal and 2E,4E-decadienal and mixtures thereof.

[0078] "Decatrienal" encompasses such compounds in any stereoisomerically pure form or in the form of a mixture of at least two different stereoisomers. Typical examples are 2E,4Z,7Z-decatrienal, 2E,4E,7Z-decatrienal and mixtures thereof.

[0079] As used herein, the term "PUFA" must be understood in a broad sense. In particular, it encompasses a single "pure" or "substantially pure" type of PUFA molecule (such as HTA, ALA, SDA, EPA, LA, GLA or ARA) or any mixture comprising at least two different types of PUFA. PUFA substrates also encompass natural products containing at least one PUFA in mixture with other natural or synthetic ingredients, such as

[0080] a) Borage oil (contains a high percentage of GLA)

[0081] b) Evening primrose oil (contains a high percentage of GLA)

[0082] c) Arachidonic acid oil (contains a high percentage of ARA)

[0083] d) Echium seed oil (contains a high percentage of SDA)

[0084] e) Fish oil (contains high ratios of EPA and DHA)

[0085] f) Flaxseed oil (contains a high percentage of ALA)

[0086] g) Microalgae oil (contains a high percentage of DHA)

[0087] The "bifunctional LOX activity" was determined under "standard conditions" as described in the experimental section. Typically, the LOX product GLA-HPO and HPL products of hexanal and decadienal were quantified by peak area by GC-MS and LC-UV. To infer that the bifunctional LOX activity produced decadienal, we can calculate the peak area ratio of decadienal to GLA-HPO based on the LC-UV data, as shown in Table 9.

[0088] The terms "biological function," "function," "biological activity," or "activity" of LOX refer to the ability of LOX as described herein to catalyze the formation of at least one unsaturated C10 aldehyde from at least one type of PUFA molecule.

[0089] 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 at least one functional polypeptide of the invention, in particular a LOX or a bifunctional LOX as defined above. The host cell is in particular a bacterial cell, a fungal cell or a plant cell or plant. The host cell may comprise a recombinant gene or several genes, for example genes organized as an operon, which have been integrated into the nuclear or organelle genome of the host cell. Alternatively, the host may comprise the recombinant gene extrachromosomally.

[0090] 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, yeast, algae or fungus.

[0091] 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.

[0092] "Capable of producing" an unsaturated C aldehyde is meant when the particular organism or cell naturally produces such an aldehyde or when it does not naturally produce such an aldehyde but is transformed with a nucleic acid described herein to produce such an aldehyde. 10 An organism or cell that has been transformed to produce an aldehyde in higher amounts than the naturally occurring organism or cell is also referred to as "capable of producing an unsaturated C 10 aldehyde”.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] The term "about" indicates a possible variation of ±25%, in particular ±15%, ±10%, more in particular ±5%, ±2% or ±1% of the stated value.

[0097] 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%.

[0098] “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%.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] "Functional mutants" of the polypeptides described herein include "functional equivalents" of such polypeptides as defined below.

[0103] The term "stereoisomer" specifically includes conformational isomers.

[0104] 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.

[0105] "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:

[0106] %ee=[X A -X B ] / [X A +X B ]*100,

[0107] Where X A and X B represents the molar ratio of stereoisomers A and B (Molenbruch).

[0108] 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:

[0109] - a higher maximum isomer yield observed during the entire reaction process or during said period thereof;

[0110] - a higher relative content of the isomer at a determined percentage value of substrate conversion; and / or

[0111] - The same relative content of isomers at higher percentage conversion values;

[0112] Each of these is preferably viewed relative to a reference method performed under otherwise identical conditions using a known chemical or biochemical method.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] "Yield" and "YP / S" (both expressed as mass of product produced / mass of material consumed) are used as synonyms herein.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] "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.

[0121] 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.

[0122] Detailed description

[0123] a. Specific embodiments of the present invention

[0124] The present invention relates to the following specific embodiments:

[0125] 1. A polypeptide having the enzymatic activity of a lipoxygenase, in particular a bifunctional LOX, whose amino acid sequence comprises a consensus sequence pattern selected from SEQ ID NO: 54; or comprises at least one partial consensus sequence pattern of SEQ ID NO: 54 selected from the following:

[0126] a)AKxxxxxADxxxxxxxxHxxxxHxxxxPxA (SEQ ID NO:240),

[0127] b)VxGxxxxxxxxxxLxxxxxxxxxxxxxxHxxxNxxQxxYxxxxxN (SEQ ID NO: 241),

[0128] and

[0129] c) LxxxxxxIxxxNxxxxxxYxxxxPxxxxxSI (SEQ ID NO: 242);

[0130] d) or any combination of a), b) and c), in particular a combination of a), b) and c).

[0131] Each amino acid residue x can be independently selected from any natural amino acid residue.

[0132] 2. The polypeptide of embodiment 1, which comprises the enzymatic activity of a lipoxygenase, in particular a bifunctional LOX, and whose amino acid sequence comprises a consensus sequence pattern selected from SEQ ID NO: 53; or comprises at least one partial consensus sequence pattern of SEQ ID NO: 53 selected from the following:

[0133] a)LxxxxxYxxxxxX1xxxxxxX 2 GxxxxxxxKxLPxPxxxFxWxxxX 3 xxxPxxI (SEQ ID NO: 243)

[0134] b) WxxAKxCxQxADxxHxExxxHxxxxHxxMxPxA (SEQ ID NO: 244);

[0135] c) GxVxGxxxxxxxxxxLxxxxxxxxxxCxPxHxxxNxxQxxYxxxxxNMPxAxY (SEQ ID NO: 245),

[0136] d)QxxxxxxLxxxxxDxxGxYxxxX 4 F (SEQ ID NO: 246),

[0137] e) QxxLxxxxxxIxxxNxxRxxxYxxxxxxxxxNSI (SEQ ID NO: 247),

[0138] f) or any combination from a) to e), in particular a combination of b), c) and e), or a combination of a) to e),

[0139] in

[0140] Each amino acid residue x can be independently selected from any natural amino acid residue,

[0141] X 1 represents 0 to 7 identical or different natural amino acid residues,

[0142] X 2 represents 0 or 1 natural amino acid residue,

[0143] X 3 represents 0 to 7 identical or different natural amino acid residues, and

[0144] X 4 represents 0 to 8 identical or different natural amino acid residues.

[0145] 3. The polypeptide of embodiment 1, which comprises the enzymatic activity of a lipoxygenase, in particular a bifunctional LOX, and whose amino acid sequence comprises a consensus sequence pattern selected from SEQ ID NO: 52; or comprises at least one partial consensus sequence pattern of SEQ ID NO: 52 selected from the following:

[0146] a)LxxxxxYxxxxxX 1 xxxxxxX 2 GGxxxxxxKxLPxPxAxFxWxxxX 3 xxxPxxI (SEQ ID NO: 248),

[0147] b)WxxAKxCxQxADxNHxExxxHxxxTHxVMxPxAxxT (SEQ ID NO: 249),

[0148] c) GxVxGxxxxxxxxxxLxxxxxxxxxxCxPxHxxxNxxQxxYxxxxxNMPxAxY (SEQ ID NO: 250),

[0149] d)QxxxxxxLxxxxYDxLGxYxxxX 4 F (SEQ ID NO: 251),

[0150] e) FQxxLxxxxxxIxxxNxxRxxxYxxxxPxxxxNSI (SEQ ID NO: 252),

[0151] g) or any combination from a) to e), in particular a combination of b), c) and e); or a) to e),

[0152] f)

[0153] in

[0154] Each amino acid residue x can be independently selected from any natural amino acid residue,

[0155] X 1 represents 0 to 7 identical or different natural amino acid residues,

[0156] X 2 represents 0 or 1 natural amino acid residue,

[0157] X 3 represents 0 to 6 identical or different naturally occurring amino acid residues, and

[0158] X 4 represents 0 to 8 identical or different natural amino acid residues.

[0159] The present invention also relates to several groups of polypeptides which have the enzymatic activity of lipoxygenase, in particular bifunctional LOX, and which may not show at least one of the above sequence patterns of embodiments 1, 2 and 3 in the same way, or may show a sequence pattern similar to at least one of the above patterns, but not showing a complete match therewith.

[0160] 4. Therefore, another embodiment of the present invention relates to a polypeptide having the enzymatic activity of lipoxygenase, especially bifunctional LOX, which optionally satisfies any one of the preceding embodiments and comprises an amino acid sequence selected from the group consisting of:

[0161] a) SEQ ID NO: 3, 6, 9, 12 or 15;

[0162] b) SEQ ID NO: 18

[0163] c) SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50

[0164] and

[0165] d) an amino acid sequence having at least 40% sequence identity with at least one of a), b) or c) and retaining said enzymatic activity of lipoxygenase.

[0166] Therefore, the polypeptide of this embodiment may or may not satisfy the limitations of any of Embodiments 1, 2 and 3.

[0167] The first group of specific polypeptides comprises an amino acid sequence selected from the group consisting of:

[0168] SEQ ID NO:3, 6, 9, 12 or 15; (CoLOXs) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to at least one of these sequences and retaining the bifunctional LOX activity, and which may not meet the limitations of any of embodiments 1, 2 and 3;

[0169] Or choose from:

[0170] SEQ ID NO:3, 6, 9, 12 or 15; (CoLOXs) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to at least one of these sequences and retaining the bifunctional LOX activity, and which meet the limitations of any of embodiments 1, 2 and 3.

[0171] A second specific group of polypeptides comprises an amino acid sequence selected from the group consisting of:

[0172] SEQ ID NO: 18 (UfLOX2) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and retaining the bifunctional LOX activity, and which may not meet the limitations of any of embodiments 1, 2 and 3; or selected from the following:

[0173] SEQ ID NO:18 (UfLOX2) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and retaining the bifunctional LOX activity, and which meet the limitations of any one of embodiments 1, 2 and 3;

[0174] A third group of specific polypeptides comprises an amino acid sequence selected from the group consisting of:

[0175] SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 (bacterial LOX) and 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 at least one of these sequences and retaining the bifunctional LOX activity, and which may not meet the limitations of any of embodiments 1, 2 and 3; or selected from the following:

[0176] SEQ ID NO:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 (bacterial LOX) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to at least one of these sequences and retaining the bifunctional LOX activity, and which meet the limitations of any of embodiments 1, 2 and 3.

[0177] A specific subset of the third group of polypeptides relates to SEQ ID NOs: 20 and 26, and amino acid sequences that have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to at least one of these sequences and retain the bifunctional LOX activity.

[0178] 5. A polypeptide comprising the enzymatic activity of a lipoxygenase, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 171 72, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238 or 239; and an amino acid sequence having at least 40% sequence identity to at least one of the sequences and retaining the enzymatic activity of lipoxygenase.

[0179] A fourth specific group of polypeptides comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186 , 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238 and 239, and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to at least one of the sequences and retaining the bifunctional LOX activity.

[0180] 6. A polypeptide as defined in any of the preceding embodiments, which has LOX activity (preferably bifunctional) and mutants thereof.

[0181] Specific examples of suitable mutants of UfLOX 2 (SEQ ID NO: 18) are:

[0182] - a mutant of SEQ ID NO: 18, wherein one or more, e.g. 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid mutations are made at sequence positions different from potential critical positions (e.g. C7, D134, R136, C161, A219, S256, C278, S305, C409 and G526 of SEQ ID NO: 18) (generating a mutation profile), and the mutant provides a bifunctional LOX mutant with a characteristic profile, e.g. an unsaturated C 10 -aldehyde productivity, unsaturated C 10 - an aldehyde product spectrum, a substrate spectrum, a by-product spectrum or a combination thereof, which is substantially identical if compared to the non-mutated parent enzyme; and other mutants derived from the mutant, which have a degree of sequence identity to SEQ ID NO: 18 of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, retaining the mutation spectrum and preferably still showing substantially the same characteristic spectrum as the non-mutated enzyme. In particular, such single or multiple mutants can be obtained by making so-called conservative mutations, for example, conservative amino acid substitutions as defined herein below.

[0183] - a mutant of SEQ ID NO: 18, wherein one or more, e.g. 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid mutations are made at potential critical sequence positions selected from C7, D134, R136, C161, A219, S256, C278, S305, C409 and G526 of SEQ ID NO: 18 (generating another mutation spectrum), having a different characteristic spectrum compared to the non-mutated parent enzyme, e.g. increased unsaturated C 10 -aldehyde productivity, different unsaturated C 10 - an aldehyde product spectrum, a different PUFA substrate spectrum, less by-product production or a combination thereof; and other mutants derived therefrom, which have a degree of sequence identity to SEQ ID NO: 18 of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and which retain the mutation spectrum at the critical positions and preferably still show the modified functional spectrum. In particular, such single or multiple mutants in critical positions can be obtained by making so-called non-conservative mutations.

[0184] Based on the sequence alignments provided herein (see Figures 11, 14, 15, 16 and 17), the results of mutation experiments performed using a specific LOX (such as UfLOX2) can be similarly transferred to the corresponding amino acid residue positions of another LOX enzyme described herein, thereby evaluating the corresponding mutations in the other enzyme, and further obtaining a suitable bifunctional LOX enzyme suitable for preparing at least one unsaturated C-1-6-phosphate dehydrogenase from at least one PUFA substrate. 10 -aldehyde.

[0185] Specific examples of suitable mutants of bacterial LOX are:

[0186] Single and multiple mutants of any of the polypeptides of SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50, which retain the enzymatic activity of lipoxygenase, especially bifunctional LOX, these mutants are particularly selected from the following mutants: mutants comprising an amino acid sequence selected from SEQ ID NO: 254, 256, 258, 260, 262, 264, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290; or mutants encoded by a nucleotide sequence, which encodes a nucleotide sequence that retains the enzymatic activity of lipoxygenase, particularly selected from SEQ ID NO: NO: 253, 255, 257, 259, 261, 263, 265, 267, 269, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287 and 289.

[0187] If compared to the non-mutated parent enzyme, such bifunctional LOX mutants may display different characteristic profiles, such as increased unsaturated C 10 -aldehyde productivity, different unsaturated C 10 - aldehyde product profile, different PUFA substrate profile, less by-product production or a combination thereof;

[0188] Also provided are mutants derived from SEQ ID NO: 254, 256, 258, 260, 262, 264, 266, 268, 270, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290 and having a degree of sequence identity to the respective native bacterial LOX amino acid sequence of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, while retaining the mutational spectrum at the critical positions and preferably still showing the modified functional spectrum. In particular, such single or multiple mutants in critical positions can be obtained by making so-called conservative mutations.

[0189] Based on the specific mutants disclosed, the skilled person will be able to generate such further functional mutants.For example, in this regard, conservative amino acid substitutions may be made in one or more of the mutation positions listed in the table below.

[0190]

[0191]

[0192] Non-limiting examples of possible conservative amino acid residue substitutions are provided in the subsequent sections of the specification.

[0193] 7. The polypeptide according to any one of embodiments 1 to 6, which has the enzymatic activity of a bifunctional LOX, in particular a combination of LOX and HPL activities.

[0194] 8. The polypeptide according to any one of embodiments 1 to 7, which has the ability to convert at least one polyunsaturated fatty acid (PUFA), in particular at least one selected from omega-3 and omega-6 PUFA, into at least one mono- or polyunsaturated aliphatic aldehyde.

[0195] 9. The polypeptide of embodiment 8, which has the ability to convert at least one PUFA into at least one polyunsaturated aliphatic C 10 -aldehyde capacity.

[0196] 10. The polypeptide of embodiment 9, which has the ability to convert at least one PUFA into at least one polyunsaturated aliphatic C selected from decadienal and decatrienal. 10 -aldehyde capacity, the polyunsaturated aliphatic C 10 - the aldehyde is in the form of a substantially pure stereoisomer or in the form of a mixture of at least two stereoisomers, preferably selected from 2E,4Z-decadienal, 2E,4E-decadienal, 2E,4Z,7Z-decatrienal, 2E,4E,7Z-decatrienal and mixtures thereof.

[0197] 11. The polypeptide according to any one of embodiments 7 to 10, wherein the PUFA is selected from C 16 -C 22 -, in particular selected from C 16 -C 20 -PUFA, more particularly selected from omega-3C 16 -C 20 -PUFA and omega-6C 16 -C 20 -PUFA.

[0198] 12. The polypeptide of embodiment 11, wherein the PUFA is selected from the group consisting of:

[0199] a)C 16 -PUFA hexadecatrienoic acid (HTA),

[0200] b)C 18 -PUFAs linoleic acid (LA), alpha-linolenic acid (ALA) and gamma-linolenic acid (GLA), stearidonic acid (SDA);

[0201] c)C 20 -PUFAs arachidonic acid (ARA) and eicosapentaenoic acid (EPA)

[0202] d) C 22 -PUFA Docosahexaenoic acid (DHA)

[0203] 13. A nucleic acid encoding the polypeptide of any one of embodiments 1 to 12 or a complementary sequence thereof.

[0204] 14. The nucleic acid of embodiment 13, comprising a coding nucleotide selected from the group consisting of:

[0205] a) SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13 and 14 (CoLOX sequences);

[0206] b) SEQ ID NO: 16 and 17 (UfLOX2 sequence);

[0207] c) codon-optimized coding sequences according to SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and native coding sequences according to SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 and 74;

[0208] d) a nucleotide sequence encoding a single or multiple mutants of any sequence c), which sequence c) encodes a polypeptide retaining the enzymatic activity of lipoxygenase, in particular selected from SEQ ID NO: 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287 and 289;

[0209] e)SEQ ID NO:75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 1 19, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 1 59, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, and 237;

[0210] f) a sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to at least one of the sequences of a), b), c) or d); or

[0211] g) the complementary sequence of any one of sequences a), b), c), d), e) and f).

[0212] 15. An expression vector comprising the encoding nucleic acid of any one of embodiments 13 and 14.

[0213] 16. The expression vector of embodiment 15, which is in the form of a viral vector, a bacteriophage or a plasmid.

[0214] 17. The expression vector of embodiment 15 or 16, wherein the encoding nucleic acid is linked to at least one regulatory sequence and, optionally, comprises at least one selectable marker.

[0215] 18. A recombinant non-human host organism or cell carrying at least one nucleic acid according to any one of embodiments 13 and 14, or comprising at least one expression vector according to one of embodiments 15 to 17.

[0216] 19. The non-human host organism of embodiment 18, wherein the non-human host organism is a eukaryote or a prokaryote, in particular a plant, a bacterium or a fungus, more in particular a bacterium or a yeast.

[0217] 20. The non-human host organism of embodiment 19, wherein the bacterium is Escherichia or Bacillus, in particular E. coli, and the yeast is Saccharomyces, Yarrowia or Pichia, in particular S. cerevisiae, Y. lipolytica or P. pastoris.

[0218] 21. The non-human host cell of embodiment 20, which is a plant cell, algae or seaweed.

[0219] 22. A method for producing at least one polypeptide according to any one of embodiments 1 to 12, the method comprising:

[0220] a) cultivating a non-human host organism or a cell carrying at least one nucleic acid according to any of embodiments 13 and 14 and expressing or overexpressing at least one polypeptide according to any of embodiments 1 to 12;

[0221] b) Optionally, isolating the polypeptide from the non-human host organism or cell cultured in step a).

[0222] 23. The method of embodiment 22, further comprising, before step a), providing at least one nucleic acid according to any one of embodiments 13 or 14 to a non-human host organism or cell, so that it expresses or overexpresses a polypeptide according to any one of embodiments 1 to 12.

[0223] 24. A method for preparing a mutant polypeptide capable of converting at least one polyunsaturated fatty acid (PUFA), particularly omega-3 or omega-6 PUFA, into at least one mono- or polyunsaturated aliphatic aldehyde, the method comprising the steps of:

[0224] a) selecting a nucleic acid according to any one of embodiments 13 and 14;

[0225] b) modifying the selected nucleic acid to obtain at least one mutant nucleic acid;

[0226] c) providing the mutant nucleic acid sequence to a host cell or a unicellular organism to express a polypeptide encoded by the mutant nucleic acid sequence;

[0227] d) screening for at least one mutant polypeptide having the activity of converting at least one polyunsaturated fatty acid (PUFA), in particular omega-3 or omega-6 PUFA, into at least one mono- or polyunsaturated aliphatic aldehyde;

[0228] e) optionally, if the mutant polypeptide does not have the desired activity, repeating the process of steps a) to d) until a polypeptide having the desired activity is obtained; and,

[0229] f) Alternatively, if a mutant polypeptide having the desired activity is identified in step d) or e), the corresponding mutant nucleic acid is isolated.

[0230] 25. A method for preparing at least one mono- or polyunsaturated aliphatic aldehyde, the method comprising:

[0231] a) contacting at least one PUFA substrate with a polypeptide as defined in any one of embodiments 1 to 12 or a polypeptide encoded by a nucleic acid as defined in any one of embodiments 13 and 14, thereby converting the at least one PUFA compound into a reaction product comprising at least one mono- or polyunsaturated aliphatic aldehyde; and

[0232] b) Optionally, isolating the at least one mono- or polyunsaturated aliphatic aldehyde obtained in step a).

[0233] 26. The method of embodiment 25, wherein step a) is performed in vivo in a cell culture in the presence of oxygen, or in vitro in a liquid reaction medium in the presence of oxygen.

[0234] If performed in vivo, the method comprises, prior to step a), introducing into a non-human host organism or cell, and optionally stably integrating into the respective genome, a nucleic acid molecule encoding one or more polypeptides having the enzymatic activity required to carry out the corresponding one or more biocatalytic conversion steps.

[0235] 27. The method according to any one of embodiments 25 and 26, wherein step a) is carried out by culturing a non-human host organism or cell expressing at least one polypeptide having enzymatic activity, preferably a bifunctional LOX, in the presence of a PUFA substrate under conditions favouring peroxidation and subsequent cleavage of at least one PUFA.

[0236] 28. The method according to embodiment 25, wherein the at least one mono- or polyunsaturated aliphatic aldehyde is selected from decadienal and decatrienal.

[0237] 29. The method of embodiment 28, wherein the decadienal is selected from 2E,4E-decadienal and 2E,4Z-decadienal and mixtures thereof; and wherein the decatrienal is selected from 2E,4E,7Z-decatrienal and 2E,4Z,7Z-decatrienal and mixtures thereof.

[0238] 30. The method according to any one of embodiments 25 to 29, wherein the PUFA substrate is an isolated substantially pure PUFA compound, or a natural or synthetic composition comprising at least one PUFA convertible by the preferably bifunctional LOX.

[0239] 31. The method of embodiment 30, wherein the natural PUFA composition is selected from:

[0240] a) Borage oil (contains a high percentage of GLA),

[0241] b) Arachidonic acid oil (contains a high proportion of ARA),

[0242] c) Fish oil (containing a high proportion of EPA),

[0243] d) Flaxseed oil

[0244] e) Echium oil

[0245] f) the corresponding oil hydrolysates of a) to e);

[0246] g) a mixture of LA and ALA; and

[0247] h) A mixture comprising at least two of a) to g).

[0248] 32. The method of embodiment 30 or 31, wherein a preferably bifunctional LOX is applied, which comprises the amino acid sequence of SEQ ID NO: 3, 6, 9, 12 or 15; (CoLOX) or a sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the substrate is selected from:

[0249] h) Borage oil (containing a high proportion of GLA) in order to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0250] i) Evening primrose oil (containing a high proportion of GLA) in order to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0251] j) Arachidonic acid oil (containing a high proportion of ARA) to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0252] k) Echium seed oil (containing a high proportion of SDA) to produce 2E,4Z,7Z-decatrienal and / or 2E,4E,7Z-decatrienal as the main product

[0253] l) Fish oil (containing a high proportion of EPA) in order to produce 2E,4Z,7Z-decatrienal and / or 2E,4E,7Z-decatrienal as the main product

[0254] m) linseed oil (containing a high proportion of ALA) to produce 2E,4Z,7Z-decatrienal and / or 2E,4E,7Z-decatrienal as the main product

[0255] n) microalgae oil (containing a high proportion of DHA) to produce 2E,4Z-decadienal, 2E,4E-decadienal, 2E,4Z,7Z-decatrienal and / or 2E,4E,7Z-decatrienal as the main product

[0256] o) LA, so as to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0257] p) GLA, so as to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0258] q) ARA, so as to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0259] r) EPA, so as to produce 2E,4Z,7Z-decatrienal and / or 2E,4E,7Z-decatrienal as the main product.

[0260] 33. The method of embodiment 30 or 31, wherein a preferably bifunctional LOX is applied, which comprises the amino acid sequence SEQ ID NO: 18 (UfLOX2) or a sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the substrate is selected from:

[0261] a) Borage oil (containing a high proportion of GLA) in order to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0262] b) Evening primrose oil (containing a high proportion of GLA) to produce 2E,4Z-decadienal and 2E,4E-decadienal as the main products

[0263] c) Arachidonic acid oil (containing a high proportion of ARA) to produce 2E,4Z-decadienal and 2E,4E-decadienal as the main products

[0264] d) Echium seed oil (containing a high proportion of SDA) to produce 2E,4Z,7Z-decatrienal and 2E,4E,7Z-decatrienal as the main products

[0265] e) Fish oil (containing a high proportion of EPA) to produce 2E,4Z,7Z-decatrienal and 2E,4E,7Z-decatrienal as the main products

[0266] f) linseed oil (containing a high proportion of ALA) to produce 2E,4Z,7Z-decatrienal and 2E,4E,7Z-decatrienal as the main products

[0267] g) microalgae oil (containing a high proportion of DHA oil in steps a) to e) so as to produce 2E,4Z-decadienal, 2E,4E-decadienal, 2E,4Z,7Z-decatrienal and 2E,4E,7Z-decatrienal as the main products,

[0268] h) LA, so as to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0269] i) GLA, so as to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0270] j) ARA to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product

[0271] k) EPA, so as to produce 2E,4Z,7Z-decatrienal and / or 2E,4E,7Z-decatrienal as the main product.

[0272] 34. The method according to any one of embodiments 25 to 31 or 33, wherein a crude or partially purified homogenate of Ulva fasciata containing said preferably bifunctional LOX activity is applied.

[0273] 35. The method of embodiment 30 or 31, wherein a preferably bifunctional LOX is applied, which comprises the amino acid sequence SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 (bacterial LOX) or a sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the substrate is selected from:

[0274] a) GLA, so as to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product, and

[0275] b) ARA, so as to produce 2E,4Z-decadienal and / or 2E,4E-decadienal as the main product.

[0276] 36. The method of embodiments 25 to 35, further comprising chemical or enzymatic isomerization of the obtained mono- or poly-unsaturated aliphatic aldehyde; or chemical or enzymatic conversion of the obtained mono- or poly-unsaturated aliphatic aldehyde into the corresponding alcohol or hydrocarbyl ester.

[0277] 37. The process according to any one of embodiments 25 to 36, wherein the conversion of the PUFA substrate is carried out in a liquid reaction medium supplemented with at least one cofactor selected from metal salts soluble in the liquid reaction medium, such as in particular divalent or polyvalent metal salts. Specific salts are halide salts, such as chloride, bromide or fluoride salts. As examples of metal ions, mention may be made of divalent or polyvalent metal cations or alkaline earth metal cations, more particularly divalent or polyvalent cations derived from Mg, Mn and Fe, such as Mg 2+ , Mn 2+ and Fe 2+ or Fe 3+ .

[0278] Optionally, the method of any of the preceding embodiments further comprises processing the obtained aldehyde into a corresponding derivative using chemical or biocatalytic synthesis or a combination of the two. For example, such derivatives can be selected from hydrocarbons, alcohols, diols, triols, acetals, ketals, acids, ethers, amides, ketones, lactones, epoxides, acetates, glycosides and / or esters.

[0279] 38. At least two unsaturated C selected from 2E,4Z-decadienal, 2E,4E-decadienal, 2E,4Z,7Z-decadienal and 2E,4E,7Z-decadienal 10- A combination of aldehyde isomers, wherein the specific ratio between 2E,4E-decadienal and 2E,4Z-decadienal is 3:1 to 1:9, and the specific ratio between 2E,4Z,7Z-decatrienal and 2E,4E,7Z-decatrienal is 3:1 to 1:9.

[0280] 39. Use of a mono- or polyunsaturated aliphatic aldehyde or a mixture of at least two such aldehydes and / or the corresponding conversion products obtained by the process according to any one of embodiments 25 to 37 and mixtures thereof or the isomeric combinations according to embodiment 38 as a flavoring ingredient for the manufacture of a food or feed composition.

[0281] 40. A food or feed composition supplemented with at least one flavoring ingredient as defined in embodiment 39.

[0282] 41. Use of a polypeptide comprising the enzymatic activity of a lipoxygenase as defined in any one of claims 1 to 12 or encoded by a nucleotide sequence as defined in any one of claims 13 and 14 for the preparation of at least one mono- or polyunsaturated aliphatic aldehyde, in particular by a process as defined in any one of claims 25 to 37.

[0283] b. Polypeptides suitable for use according to the present invention

[0284] In the context of this article, the following definitions apply:

[0285] 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".

[0286] 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.

[0287] 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.

[0288] 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.

[0289] Thus, unless otherwise stated, the term "polypeptide" also encompasses the terms "protein" and "enzyme".

[0290] Similarly, the term "polypeptide fragment" encompasses the terms "protein fragment" and "enzyme fragment".

[0291] 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.

[0292] "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.

[0293] The present invention also relates to "functional equivalents" (also called "analogs" or "functional mutants") of the polypeptides specifically described herein.

[0294] For example, a "functional equivalent" refers to a polypeptide which, in a test for determining the activity of the enzyme LOX, exhibits at least 1 to 10% higher or lower enzymatic activity than the polypeptide specifically described herein, or at least 20%, or at least 50%, or at least 75%, or at least 90% higher or lower.

[0295] According to the invention, "functional equivalents" also encompass specific mutants which have in at least one sequence position of the amino acid sequences described herein an amino acid different from the specifically stated amino acid, but which nevertheless have one of the above-mentioned biological activities, such as enzymatic activity. Thus, "functional equivalents" include mutants obtainable by additions, substitutions, in particular conservative substitutions (i.e., as a result of which the amino acid in question is replaced by an amino acid with the same charge, size, polarity and / or solubility), deletions and / or inversions of one or more, such as 1 to 20, 1 to 15 or 5 to 10 amino acids, wherein said 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 coincides 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 different rates (i.e., by EC 50 or IC 50The following table shows examples of suitable (conservative) amino acid substitutions:

[0296]

[0297] "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.

[0298] In this context, a "precursor" is a natural or synthetic precursor of a polypeptide that may or may not have the desired biological activity.

[0299] 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.

[0300] 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.

[0301] "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.

[0302] "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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] c. Encoding nucleic acid sequences suitable for use according to the present invention

[0312] In the context of this article, the following definitions apply:

[0313] 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.

[0314] "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.

[0315] 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.

[0316] 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.

[0317] 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).

[0318] 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.

[0319] "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.

[0320] 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).

[0321] "Polycistronic" refers to a nucleic acid molecule, particularly mRNA, that can encode more than one polypeptide in the same nucleic acid molecule.

[0322] "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.

[0323] "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.

[0324] 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.

[0325] 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.

[0326] The present invention also relates to nucleic acid sequences encoding the polypeptides as defined herein.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] "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.

[0331] 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.

[0332] In another example, the identity can be calculated by the Vector NTI Suite 7.1 program of Informax Corporation (USA) using the Clustal method (Higgins DG, Sharp PM. ((1989))) with the following settings:

[0333] Multiple alignment parameters:

[0334]

[0335] Pairwise comparison parameters:

[0336]

[0337] 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:

[0338]

[0339]

[0340] 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.

[0341] 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.

[0342] The invention further relates to nucleic acid molecules which are complementary to the specifically described nucleotide sequences or to segments thereof.

[0343] 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.

[0344] "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.

[0345] "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.

[0346] "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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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.

[0352] "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.

[0353] 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.

[0354] 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).

[0355] "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.

[0356] 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.

[0357] 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).

[0358] 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 and 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.

[0359] 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.

[0360] 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. The DNA is then prehybridized in a buffer containing 100 μg / ml denatured salmon sperm DNA and 5 to 20 x 10 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, and then washed in 0.1xSSC at 50°C for 45 minutes.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] The invention also relates to derivatives of the specifically disclosed or derivable nucleic acid sequences.

[0365] 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.

[0366] The invention also encompasses nucleic acid sequences which contain so-called silent mutations or which have been altered compared to the specifically stated sequences according to the codon usage of the particular origin or host organism.

[0367] 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.

[0368] The invention also encompasses naturally occurring variants of the sequences described herein, such as splice variants or allelic variants.

[0369] 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.

[0370] The invention also relates to sequences obtainable by conservative nucleotide substitutions, ie as a result of which the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility.

[0371] 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.

[0372] 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.

[0373] 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.

[0374] d. Generation of functional polypeptide mutants

[0375] 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.

[0376] 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.

[0377] Methods for modifying genes and thereby modifying the polypeptides encoded thereby have long been known to those skilled in the art, for example:

[0378] - using different methods to directly synthesize the entire coding sequence (Sriram Kosuri and George M. Church, 2014, Nature Methods, 11: 499-507),

[0379] - 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),

[0380] - 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),

[0381] - 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);

[0382] - SeSaM method (sequence saturation method), in which preferred exchange is prevented by the polymerase. Schenk et al., Biospektrum, Vol. 3, 2006, 277-279,

[0383] - 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

[0384] -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).

[0385] 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.

[0386] The relevant gene of the host organism expressing the functional mutant (whose 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.

[0387] 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.

[0388] 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."

[0389] e. Constructs expressing the polypeptides of the present invention

[0390] In the context of this article, the following definitions apply:

[0391] "Gene expression" encompasses "heterologous expression" and "overexpression" and involves the transcription of a gene and the translation of mRNA into protein. Overexpression refers to the production of a gene product measured at the level of mRNA, polypeptide and / or enzyme activity in a transgenic cell or organism that exceeds the level of production in a non-transformed cell or organism of a similar genetic background.

[0392] 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.

[0393] 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".

[0394] 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 may 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 may be distributed on two or more physically different vectors. As a specific example, an operon may be mentioned, which comprises a promoter sequence, one or more operator sequences and one or more structural genes, each gene encoding an enzyme as described herein.

[0395] 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.

[0396] "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.

[0397] 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.

[0398] 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.

[0399] 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).

[0400] 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.

[0401] 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.

[0402] 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.

[0403] In the methods used according to the invention, the expression cassette may be part of an "expression vector", in particular a recombinant expression vector.

[0404] 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.

[0405] 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.

[0406] 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.

[0407] 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.

[0408] 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.

[0409] 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.

[0410] 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.

[0411] Examples of suitable regulatory sequences are found in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI q , T7, T5, T3, gal, trc, ara, rhaP(rhaP BAD )SP6, lambda-P R , or in lambda-P L In 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.

[0412] 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.

[0413] 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-B1, λgt11, or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702, or pIJ361, in Bacillus pUB110, pC194, or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL2, or pBB116, in yeast 2alphaM, pAG-1, YEp6, YEp13, or pEMBLYe23, or in plants pLGV23, pGHlac + , pBIN19, pAK2004 or pDH51. The above plasmids are a small selection of possible plasmids. Other plasmids are well known to the skilled person 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).

[0414] 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.

[0415] 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.

[0416] 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).

[0417] 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 in, for example, "cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0418] 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.

[0419] 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.

[0420] Also provided herein is an embodiment an isolated, recombinant or synthetic polynucleotide encoding a polypeptide or variant polypeptide provided herein.

[0421] 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.

[0422] f. Hosts suitable for the present invention

[0423] Depending on the context, the term "host" may refer to a wild-type host or a genetically altered recombinant host or both.

[0424] 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.

[0425] Using the vector according to the present invention, a recombinant host can be produced, which can, for example, 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 a respective expression system. 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.

[0426] Advantageously, microorganisms such as bacteria, fungi or yeast are used as host organisms. Advantageously, Gram-positive or Gram-negative bacteria are used, preferably Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, Streptococcaceae or Nocardiaceae, particularly preferably Escherichia, Pseudomonas, Streptomyces, Lactococcus, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus. The genus and species of Escherichia coli are very particularly preferred. In addition, other advantageous bacteria are found in the alpha-Proteobacteria, beta-Proteobacteria or gamma-Proteobacteria groups. Advantageously, yeasts such as the genus Saccharomyces or Pichia family are also suitable hosts.

[0427] 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.

[0428] Depending on the host organism, the organism used in the method according to the invention is grown or cultivated 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 they can be provided semi-continuously or continuously later. This is also described in more detail below.

[0429] g. Recombinant production of the polypeptide according to the invention

[0430] 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.

[0431] 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.

[0432] 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).

[0433] 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.

[0434] 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.

[0435] 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.

[0436] 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.

[0437] 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.

[0438] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.

[0439] 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.

[0440] 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.

[0441] 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.

[0442] 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.

[0443] 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.

[0444] 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.

[0445] 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.

[0446] In order to separate the recombinant protein, it may be advantageous to use a carrier system or oligonucleotide that extends the cDNA by a limited nucleotide sequence, and thus encodes a changed polypeptide or fusion protein, which is, for example, used for easier purification. Suitable modifications of this type are, for example, so-called "tags" 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.

[0447] 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.

[0448] h. Immobilization of Peptides

[0449] 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.

[0450] i. Reaction conditions of the biocatalytic production method of the present invention

[0451] The reaction of the present invention can be carried out under in vivo or in vitro conditions.

[0452] 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.

[0453] 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).

[0454] 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.

[0455] Instead of living cells, non-living cell biomass containing the desired biocatalyst can also be applied to the bioconversion reaction of the present invention.

[0456] If at least one enzyme is immobilized, it is attached to an inert support as described above.

[0457] The conversion reaction can be carried out batchwise, semi-batchwise or continuously. The reactants (and optional nutrients) can be provided at the start of the reaction, or can be provided subsequently semi-continuously or continuously.

[0458] 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.

[0459] 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.

[0460] 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.

[0461] The non-aqueous medium may comprise substantially no water, ie, will comprise less than about 1 wt% or 0.5 wt% water.

[0462] 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.

[0463] 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.

[0464] 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.

[0465] 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.

[0466] If the host is a transgenic plant, optimal growth conditions can be provided, such as optimal light, water, and nutrient conditions.

[0467] k. Product Isolation and Derivatization

[0468] 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.

[0469] The identity and purity of the isolated product can be determined by known techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopy (e.g. IR, UV, NMR), 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 11 27-32; 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.).

[0470] The unsaturated C 10 The aldehyde compound is converted into a derivative such as, but not limited to, a hydrocarbon, an ester, an amide, a glycoside, an ether, an epoxide, an aldehyde, a ketone, an alcohol, a diol, an acetal or a ketal.10 Aldehyde derivatives can be obtained by chemical methods such as, but not limited to, oxidation, reduction, alkylation, acylation and / or rearrangement. 10 Aldehyde derivatives can be obtained by biochemical methods by making unsaturated C 10 The aldehyde is obtained by contacting with an enzyme, such as, but not limited to, an oxidoreductase, a monooxygenase, a dioxygenase, a transferase. The biochemical transformation can be performed in vitro using isolated enzymes, enzymes from lysed cells, or in vivo using whole cells.

[0471] l. Unsaturated C 10 - Fermentation production of aldehydes

[0472] The present invention also relates to a method for producing unsaturated C 10 Aldehyde method.

[0473] 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).

[0474] 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).

[0475] 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.

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 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.

[0480] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.

[0481] 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.

[0482] 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, nicotinic acid, 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 compound in the culture medium depends largely on the specific experiment and must be determined separately for each specific case. Information about culture medium optimization can be found in the textbook "Applied Microbiol. Physiology, APractical Approach" (1997). Growth medium may also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO), etc.

[0483] 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.

[0484] 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.

[0485] The method of the present invention may further comprise recovering the one or more unsaturated C 10 Aldehyde step.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] The following examples are illustrative only and are not intended to limit the scope of the invention described in the Summary, Detailed Description, or Claims.

[0490] 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.

[0491] Experimental Section

[0492] Material:

[0493] Unless otherwise specified, all chemical and biochemical materials and microorganisms or cells used herein are commercially available products.

[0494] 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.

[0495] method:

[0496] Functional expression of lipoxygenase

[0497] The coding sequence of lipoxygenase (LOX) was optimized by following the genetic codon frequency of E. coli, synthesized, and then subcloned into pETDuet-1 (Novagen, Merck KGaA, Germany) plasmid for subsequent expression in E. coli. BL21 E. coli cells (Tiangen, China) were transformed with plasmid pETDuet-LOX. Transformed cells were selected on LB agar plates containing ampicillin (final 50 μg / mL). Single colonies were used to inoculate 25 mL of liquid LB medium containing ampicillin (final 50 μg / mL). The culture was incubated at 37 ° C and 200 rpm shaking. After incubation for 4 hours, the culture was cooled to 20 ° C for 1.5 hours, and IPTG (final 0.016 mM) was added to induce protein expression. In order to express the protein, the culture was incubated for another 16 hours at 20 ° C and 200 rpm shaking. The culture was spun and resuspended in 3 mL of reaction buffer (25 mM Tris-HCl pH 7.5) and then sonicated to prepare a protein solution. The protein solution was transferred to a 20 mL SPME vial, and 30 μL of fatty acid substrate and 10 μL of internal standard (80 ppm of α-ionone in ethanol) were added to the vial. After incubation for 10 minutes, the following SPME-GC-MS method was used for the analysis of decadienal and decatrienal.

[0498] Solid phase microextraction gas chromatography mass spectrometry (SPME-GC-MS)

[0499] The reaction mixture was concentrated on a solid phase microextraction (SPME) fiber assembly polydimethylsiloxane / Carboxen / divinylbenzene (57329-U, SUPELCO). The extraction was performed in headspace mode at 40°C for 20 minutes. After extraction, the SPME fiber was introduced into the GC-MS inlet and maintained at 250°C for 5 minutes, and then the products were analyzed on an Agilent 6890 series GC system equipped with a DB1-ms column 30m×0.25mm×0.25μm film thickness (P / N 122-0132, J&W Scientific Inc., Folsom, CA) and combined with a 5975 series mass spectrometer (Agilent, USA). The carrier gas was helium with a flow rate of 0.7mL / min. The injection was performed in splitless mode with the injector temperature set to 250°C. The oven temperature was programmed from 50°C (held for 5 minutes) to 250°C at 15°C / min (held for 5 minutes). Identification of products was based on mass spectra and retention indices along with respective product standards.

[0500] Liquid chromatography coupled with UV detection and mass spectrometry (LC-UV / MS)

[0501] 200 μL of the reaction mixture was diluted with 800 μL of acetonitrile and then placed on ice for 30 minutes. The mixture was filtered with 0.2 μL of regenerated cellulose membrane (5190-5108, Agilent) to remove protein precipitates. 1 μL of the sample was injected into a liquid chromatograph to quantitatively determine decadienal and byproducts.

[0502] Part A:

[0503] Isolation and characterization of UfLOX

[0504] Example 1: Analysis of seaweed sources and aromatic aldehydes

[0505] Plant material of Ulva fasciata (sample ID: PA-2017-0012) was collected from Nan'ao City, Guangdong Province, China. 1 gram of crushed sample was placed in a 20 mL vial for further SPME-GC-MS analysis.

[0506] To determine whether U. fasciata contained decadienal or decatrienal, fresh samples were analyzed by SPME-GC-MS as described in the “Methods” section.

[0507] One gram of crushed ulvae sample was placed in a 20 mL vial containing 3 mL Tris-HCl buffer (pH = 7.5). 30 μL fatty acid substrates (30 μL of LA, ALA, GLA, EPA, ARA, borage oil hydrolysate, arachidonic acid oil hydrolysate, linseed oil hydrolysate or fish oil hydrolysate in 1 ml of ethanol, respectively) and 10 μL of internal standard (80 ppm of α-ionone in ethanol) were added to the vial for incubation. After incubation at room temperature for 10 minutes, the SPME-GC-MS method described in the "Methods" section was used for the analysis of decadienal and decatrienal.

[0508] GC-MS analysis showed that in Ulva lobata, 2E,4Z-decadienal (retention time 13.0 min) and 2E,4E-decadienal (retention time 13.25 min) were present in limited amounts ( Figure 2 , Figure 4 and Figure 5 ). Its content increased significantly when fed with γ-linolenic acid (Table 1).

[0509] Table 1. SPME-GC-MS analysis of Ulva lobata before and after feeding with gamma-linolenic acid (GLA)

[0510]

[0511] Example 2: Transcriptome analysis and identification of UfLOX protein

[0512] Total RNA from Ulva lobata was extracted using the RNeasy Plant Mini Kit (Qiagen, Germany). Total RNA samples were processed with UltraTM RNA library preparation kit (NEB, USA) and TruSeq PE cluster kit (Illumina, USA), and then sequenced on Illumina HiSeq 2500 System. 38 million 2x150bp paired end reads were generated. Trinity (http: / / trinityrnaseq.sf.net / ) software was used to process the reads, and 91564 transcripts with N50 of 2262 were obtained. The obtained transcripts were translated into protein sequences, and then the NCBI non-redundant protein sequence database was searched using the tblastx algorithm for functional annotation. A candidate protein sequence of LOX was mined by Pfam search and relative expression level.

[0513] First use SMARTer TM RACE cDNA amplification kit (Clontech, Takara, Japan) reverse transcribed the total RNA sample of Ulva lobata into cDNA. The product was then used as a template for gene cloning. The coding sequence of UfLOX2 (SEQ ID NO: 18) was amplified from cDNA using a forward primer (5'-TCGTCCAACAGGTTCTCTT-3') (SEQ ID NO: 57) and a reverse primer (5'-TTCTTTCCACTCACCGCCA-3') (SEQ ID NO: 58).

[0514] Example 3: Functional Characterization of UfLOX2

[0515] The coding sequence of UfLOX2 was optimized by following the frequency of the genetic code of E. coli, synthesized, and then subcloned into the pETDuet-1 plasmid for subsequent expression in E. coli. The following codon-optimized sequence was applied: UfLOX2 (SEQ ID NO: 17), and the plasmid pETDuet-UfLOX2 was obtained.

[0516] Functional expression of the gene was performed as described above in the "Methods" section to produce protein solutions. The enzymatic activity of UfLOX2 was assessed as follows:

[0517] a) UfLOX2 (SEQ ID NO: 18) was tested by feeding fatty acid substrates including gamma-linolenic acid (GLA), alpha-linolenic acid (ALA), linoleic acid (LA) and arachidonic acid (ARA) as follows:

[0518] The E. coli protein solution (3 mL) containing UfLOX2 was placed in a 20 mL SPME vial, and 30 μL of fatty acid substrate (30 μL of LA, ALA, GLA, EPA, ARA, borage oil, arachidonic acid oil, linseed oil or fish oil in 1 mL of ethanol, respectively) and 10 μL of internal standard (80 ppm of α-ionone in ethanol) were added to the vial for incubation. After 10 minutes at room temperature, the SPME-GC-MS method described in the “Methods” section was used for the analysis of decadienal and decatrienal.

[0519] When fed with specific substrates, UfLOX2 showed the ability to produce decadienal (retention times 12.60 and 12.80 min) (Table 2)

[0520] Table 2. SPME-GC-MS analysis of UfLOX2 before and after feeding with GLA and arachidonic acid (ARA)

[0521]

[0522] b) To demonstrate the lytic activity of UfLOX2, fatty acid hydroperoxide feeding experiments were performed.

[0523] To test HPL activity, UfLOX2 was produced in E. coli, and cell lysates containing UfLOX2 were prepared to test its HPL activity. An aliquot of UfLOX2 was fed with GLA as a positive control for the preparation of decadienal. The second and third aliquots of UfLOX2 were denatured (boiled at 100°C for 20 minutes), and GLA or GLA hydroperoxide (GLA-HPO) were added as negative controls, respectively, to exclude the production of decadienal by UfLOX2 and to show the function of converting GLA-HPO to decadienal in a non-UfLOX2 manner. The fourth aliquot of UfLOX2 was fed with GLA hydroperoxide (GLA-HPO) to demonstrate its HPL activity compared to the third aliquot (i.e., non-UfLOX2 conversion of GLA-HPO to decadienal). Additionally, the buffer used to prepare the aliquots was included as a negative control to demonstrate the non-UfLOX2 conversion of GLA-HPO to decadienal.

[0524] In order to prepare the GLA hydroperoxide (GLA-HPO) intermediate, 50 mL of UfLOX2 protein solution was incubated with 0.5 mL of GLA (60 mg / mL) and stored at room temperature for 10 minutes. The reaction mixture was then loaded on an HLB column (Waters, US Part No. 186000118). The column was eluted with 10 mL of methanol to obtain GLA-HPO. After incubation for 1 hour, the reaction mixture was checked with LC-MS.

[0525] The results are summarized in Table 3 below.

[0526] Table 3. Decadienal peak areas produced by feeding heat-treated or untreated UfLOX2 together with γ-linolenic acid hydroperoxide intermediates

[0527]

[0528] Part B:

[0529] CoLOX isolation and characterization

[0530] Example 4: Source of seaweed and analysis of aromatic aldehydes

[0531] Plant material of Cladophora oligoclada (sample ID: AVLH2012-011) was collected from Qingdao, Shandong Province, China. 1 gram of the crushed sample was placed in a 20 mL vial for further SPME-GC-MS analysis.

[0532] The identification of the peaks was based on the comparison of their mass spectra and retention indices with those in the in-house library. GC-MS analysis revealed four major components of C. oligoclada as shown in Tables 4 and Figures 3 to 7 As shown:

[0533] Table 4. Flavor aldehydes identified from Cladophora spp.

[0534] Components Retention time Peak area% 2E,4Z-Decadienal 22.0 minutes 12.1％ 2E,4E-Decadienal 22.6 minutes 11.9％ 2E,4Z,7Z-Decatrienal 21.8 minutes 21.7％ 2E,4E,7Z-Decatrienal 22.5 minutes 12.2％

[0535] Example 5: Transcriptome analysis and identification of CoLOX proteins

[0536] Fresh samples from Cladophora microphylla were extracted by MiniBest Plant RNA Extraction Kit to extract total RNA according to Protocol I (Cat.#9769v201309Da, Takara, Japan) provided by the kit. Total RNA samples were processed using TruSeq PE ClusterKit (Illumina, USA) and then sequenced on the Illumina MiSeq system. 14 million paired-end reads of 2x251bp were generated. Reads were processed using Trinity (http: / / trinityrnaseq.sf.net / ) software to obtain 225,917 transcripts with an N50 of 676. The obtained transcripts were translated into protein sequences and then the NCBI non-redundant protein sequence database was searched using the tblastx algorithm for functional annotation. A candidate protein sequence of LOX was mined by Pfam search and relative expression level.

[0537] First use SMARTer TM The total RNA sample of Cladophora microcladae (sample ID: PA-2017-0028) was reverse transcribed into cDNA using the RACE cDNA amplification kit (Clontech, Takara, Japan). The product was then used as a template for gene cloning. Several coding sequences of LOX were amplified from cDNA using a forward primer (5'-CTCTCTCTCTTTCTCTCTGTGTTCT-3' (SEQ ID NO: 55) and a reverse primer (5'-CTCGTTCCCTTACCGTCT-3') (SEQ ID NO: 56), named CoLOX-3 (SEQ ID NO: 3) (and its variants) CoLOX-0317 (SEQ ID NO: 6), CoLOX-19 (SEQ ID NO: 9), CoLOX-22 (SEQ ID NO: 12) and CoLOX-d4 (SEQ ID NO: 15).

[0538] Example 6: Functional Characterization of CoLOX Protein

[0539] The nucleic acid sequences of CoLOX-3 and its variants CoLOX-0317, CoLOX-19, CoLOX-22 and CoLOX-d4 were codon-optimized by following the genetic codon frequency of E. coli and then subcloned into pETDuet-1 between the NdeI and KpnI sites (Novagen, Merck KGaA, Germany), respectively, for subsequent expression in E. coli. The following codon optimized sequences were used: CoLOX-3 (SEQ ID NO: 2), CoLOX-0317 (SEQ ID NO: 5), CoLOX-19 (SEQ ID NO: 8), CoLOX-22 (SEQ ID NO: 11) and CoLOX-d4 (SEQ ID NO: 14), and the following plasmids were prepared: pETDuet-CoLOX-3, pETDuet-CoLOX-0317, pETDuet-CoLOX-19, pETDuet-CoLOX-22 and pETDuet-CoLOX-d4. Functional expression of genes was performed as described in the "Methods" section above. The culture was centrifuged and resuspended in 3 mL of buffer (25 mM Tris-HCl pH 7.5, 0.2 mM CaCl 2 ), followed by ultrasonic treatment to prepare the corresponding protein solution.

[0540] The crude protein solutions (3 mL) of CoLOX-3, CoLOX-0317, CoLOX-19, CoLOX-22 and CoLOX-d4 were placed in 20 mL SPME vials, and 30 μL fatty acid substrates (30 μL LA, ALA, GLA, EPA, ARA, borage oil, arachidonic acid oil, linseed oil or fish oil in 1 mL ethanol, respectively) and 10 μL internal standard (80 ppm of α-ionone in ethanol) were added to the vials for incubation. After 10 minutes at room temperature, the SPME-GC-MS method described in the "Methods" section was used for the analysis of decadienal and decatrienal. A mixture of buffer plus fatty acid plus internal standard was used as a control.

[0541] All five proteins showed the ability to produce decadienal and / or decatrienal when fed with the specified substrates (see Tables 5 and 6 below and Figure 8 , Fig. 9 and Fig.10 ).

[0542] Table 5. Peak ratio of decadienal / internal standard after GLA feeding (normalized by protein concentration)

[0543] The ratio of 2E,4Z-decadienal The ratio of 2E,4E-decadienal BL21 0.023±0.023 0.049±0.049 Empty vector 0.000±0.000 0.000±0.000 CoLOX-3 2.866±1.824 7.712±2.633 CoLOX-d4 0.917±0.631 1.931±0.329 CoLOX-19 0.340±0.200 1.113±0.325 CoLOX-22 1.729±0.933 6.019±1.422 CoLOX-0317 0.207±0.096 0.888±0.262

[0544] Table 6. Peak ratios of decadienal / internal standard after feeding fish oil hydrolysate (normalized by protein concentration)

[0545]

[0546] Part C:

[0547] Generate C in public database 10 Mining and characterization of aldehyde LOX

[0548] Example 7: Mining and selecting LOX by sequence analysis

[0549] Due to its activity in producing decadienal and decatrienal, UfLOX2 was used to search for more LOX from GenBank by using BLASTP 2.8.0+ (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). A total of 188 LOX were found by this method, of which 181 LOX were from cyanobacteria, 5 LOX were from Proteobacteria, and 2 LOX were from Planctomycetes, with sequence identities less than 42% with UfLOX2. As shown in Table 7, 16 LOX were selected as representatives with relatively high sequence identities with UfLOX2 and representing their own homologs, as shown in Table 7. Two known LOX from red algae were listed and used for comparison. The remaining 83 LOX with relatively high identity to UfLOX2 are listed in the attached sequence listing as SEQ ID NO: 75 to 239 (amino acid and nucleic acid sequences. When necessary, the start codon was set to ATG.

[0550] Table 7. List of dual-function LOX

[0551]

[0552] Note: a: CoLOX-3 of the present invention; b: UfLOX2 of the present invention; c: AFQ59981.1 (PhLOX), for example, was described by Jechan Lee et al. in Environmental Pollution 227 (2017) 252-262; d: AGN54275.1 (PhLOX2), was described by Zhujun Zhu et al. in PLoS One (2015) 10 (2): e0117351.

[0553] The amino acid sequence identities and the number of different residues are summarized in Table 8. The upper right box shows the number of mismatched amino acids and the lower left box shows the sequence identity. The sequence identity between bacterial LOX and UfLOX2 ranges from 32% to 42%. The sequence identity between bacterial LOX and CoLOX-3 ranges from 13% to 16%. The sequence identity between bacterial LOX and red algae LOX is less than 15%.

[0554]

[0555] Example 8: Expression and functional characterization of the mined bacterial LOX

[0556] The coding sequence of the bifunctional LOX was optimized by following the genetic code frequency of E. coli, synthesized, and then subcloned into the pETDuet-1 plasmid for subsequent expression in E. coli.

[0557] Functional expression of the mined LOX was performed as described in the "Methods" section above. Different LOX proteins expressed by E. coli were released by ultrasonic treatment in 25mM Tris-HCl buffer (pH7.5) to deliver LOX protein solutions separately. Each LOX protein solution was transferred to a 20mL SPME vial, and 30μL GLA and 10μL internal standard were added to the vial. After incubation for 10 minutes, SPME-GC-MS was used for the analysis of decadienal, decatrienal and hexanal, while LC-UV was used for the analysis of decadienal, decatrienal and GLA-HPO (an intermediate between γ-linolenic acid and decadienal). SPME-GC-MS was performed as described in the "Methods" section above. GC-MS analysis showed that there were 2E, 4Z-decadienal (retention time 13.0 minutes), 2E, 4E-decadienal (retention time 13.25) and hexanal in the reaction of each LOX, but the levels were different. LC-UV showed 2E,4Z-decadienal (retention time at 280 nm, 6.61 minutes), 2E,4E-decadienal (retention time at 280 nm, 6.62 minutes), and GLA-HPO (retention time at 235 nm, 6.90 minutes).

[0558] The selectivity, bifunctionality and productivity of LOX for the decadienal end product from the GLA substrate were calculated and are shown in Table 9 below (comparisons involving UfLOX2 and CoLOX-3). 10 ) and hexanal (C 6 ) to deduce the selectivity. The productivity can be derived from the peak area of ​​decadienal. 10 ) to GLA-HPO (intermediate) to deduce bifunctionality. In this comparison, UfLOX2 is still the best bifunctional LOX, followed by cyanobacterial bifunctional LOX WP_002738122.1 (from Microcystis aeruginosa) and WP_015204462.1 (from Crinalium epipsammum). Compared with CoLOX-3, there are still some cyanobacterial LOXs with similar activities, such as WP_039200563.1, WP_073641301.1.

[0559]

[0560] Part D:

[0561] Further characterization of LOX of the present invention

[0562] Example 9: Characterization of key amino acids in high performance LOX

[0563] Experiment 1:

[0564] In the alignment view, high-performance LOX, UfLOX2 and WP_002738122.1 and WP_015204462.1 were compared with other less active LOX (see Figure 11). To mine potential key amino acid residues of high-activity LOX, multiple potential positions were selected and marked with asterisks (indicating potential key positions) and dots (indicating other potential positions).

[0565] The importance of some of the identified conserved residues was explored by mutagenesis studies. The results are summarized in Table 10.

[0566] Table 10. Modified amino acids of UfLOX2 used for functional studies.

[0567]

[0568] 1) Double mutation in positions 134 and 136

[0569] 2) Numbering relative to SEQ ID NO: 18

[0570] In a first series of mutagenesis studies, some UfLOX2 mutants showed reduced activity, see Fig.12 .

[0571] Based on these data, the following conclusions can be drawn:

[0572] 1) D142 / M143, N150, C174, K209, C268 and A331 are not critical for this activity;

[0573] 2) C7, D134 / R136, C161, A219, S256, C278, S305, C409 and G526 are critical for this activity, as the corresponding mutants showed reduced activity at different levels.

[0574] Experiment 2:

[0575] The residues identified in Experiment 1 were introduced into bacterial LOX along with several other residues that are conserved in bacterial LOX to improve productivity. The designed sequences are shown in Table 11.

[0576] Table 11. Modified amino acids of LOX mutants.

[0577]

[0578] The coding sequences of bacterial LOX mutants were optimized by following the genetic codon frequency of E. coli, synthesized, and then subcloned into the pETDuet-1 plasmid for subsequent expression in E. coli.

[0579] Functional expression of bacterial LOXs mutants was performed as described in the “Methods” section above. Different LOX proteins expressed by E. coli were released by sonication in 25 mM Tris-HCl buffer (pH 7.5) to deliver LOX protein solutions separately. Each LOX protein solution was transferred to a 20 mL SPME vial, and 30 μL of GLA and 10 μL of internal standard were added to the vial. After incubation for 10 minutes, LC-UV was used for the analysis of decadienal. The productivity of the LOX mutants of the decadienal end product was calculated and is shown in Fig.18 (Their natural counterparts were used for comparison). WP_002738122.1mut, WP_002738122.1mut2, WP_015204462.1mut, WP_015204462.1mut2, WP_015204462.1mut3, WP_015178512.1mut, WP_006635899.1mut and WP_099099431.1mut all showed higher productivity compared to their natural counterparts.

[0580] Example 10: Characterization of LOX cofactors

[0581] Previous studies have shown that five essential conserved amino acid residues in the active site are involved in cofactor binding, as described by Toralf Senger et al. in J. Biol. Chem. 2005, 280: 7588-7596 (where the residues cited are His-585, His-590, His-774, Asn-778 and Ile-899). Iron and manganese are both reported to be cofactors, as described by Alexandra Andreou et al. in J. Biol. Chem. 2010. Algal LOX and bacterial LOX also have these five conserved residues, as shown in the alignment in Figure 11, indicating that the addition of iron and manganese can improve the activity of LOX. Therefore, we tested the importance of iron and manganese on the activity of UfLOX2. The observed results clearly demonstrate the importance of adding manganese (and magnesium to a lesser extent) to the reaction for enhancing enzymatic activity. Therefore, manganese is important for enabling / improving LOX activity. The results are summarized in Fig.13 We also tested iron in our tests, but the effect was not as significant as with manganese (data not shown).

[0582] Example 11: Downstream product profiling

[0583] In the case of decadienal prepared using UfLOX2 and γ-linolenic acid, the molar yield of total decadienal (including 2E,4Z-decadienal and 2E,4E-decadienal) was approximately 30% to 40% based on LC-UV / MS quantification with external calibration as described in the Methods section above. However, the total percentage of decadienal was higher than 90% based on total volatiles.

[0584] To obtain information on other downstream byproducts, UfLOX2 was produced in E. coli. Cell lysate (20 mL) containing UfLOX2 was fed with GLA at room temperature. 200 μl sample aliquots were collected and mixed with 800 μl acetonitrile for further analysis by LC-UV / MS as described in the Methods section above. Nine byproducts were proposed based on the observed mass spectra and comparison with the literature (see Table 12).

[0585] Table 12. Byproducts

[0586]

[0587] All publications mentioned in this application are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0588] Sequence Listing

[0589] Table 13. Sequences described and used in this article

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603] NA = Nucleic acid sequence

[0604] AA = amino acid sequence

[0605] Notes to the above sequence listing:

[0606] SEQ ID NO:59-74 refers to the corresponding natural coding sequences of SEQ ID NO:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50

[0607] SEQ ID NOs: 75-238 are paired representations of the corresponding putative coding sequences (for sequences without "ATG", the start codon was changed to "ATG"; sequences were not codon-optimized and therefore considered "native" except for the start codon) and the amino acid sequences of the mined LOX mined from NCBI

[0608] ·SEQ ID NO: 239—Amino acid sequence of 5MEE_A mined from NCBI

[0609] · SEQ ID NO: 253-290 refers to mutants of bacterial LOX:

[0610] Included within the general disclosure of the present specification are any encoding nucleic acids described herein that lack a 5'-terminal start codon triplet or have an artificial or natural start codon triplet.

[0611] 1. CoLOX

[0612] CoLOX-3 coding sequence - SEQ ID NO: 1

[0613]

[0614] Codon-optimized coding sequence of CoLOX-3 using Genscript genetic codon frequency of E. coli - SEQ ID NO: 2

[0615]

[0616] Amino acid sequence of CoLOX-3 – SEQ ID NO:3

[0617] MTSSPTVRSMVMLAVLAVSALESAPCASAFATLPRALVRPQAALKYRAEDKNDVDVAPAGSTASDVSKPEGKATAVAKGTVNAPIEEAWKVFRSFSNMNQWMPVYGEWEATGDSVGDTRTFNFKDQPTFFTTERLVGLDDSQYKMKYTLVNCKGSPVPIESIDTIVTFTANDDVTEVDWRSWTKSPMVDLIKGRQAAGYAGGIAALDRYLNPSLGTVDVTIKSADNLDGDFLSSSYATLMVTDADPEQVHAKEWGTSPEFDAKPVQFSLLKPDSKLYMSVMLTKCGVDAPVGYAVFDIQKSLKSGETVTETFQLEGSNDATLTVEMELNLRQGSILPQSKAQKNLATLVALQQSVERVRDRIVTIGKLAGEPEKSVWEYERKSGLPKSVKGLPRSEVLPPHKIALMVDAIAEYAYTQFQLVQRLLPVRNSYDRYAAYFAPEGEEYVPIPQILKDMTWSTDDEFIRQIFAGLNPLQVEVVKNKAGLPSKLQELKARDGSDVDKLISEGRLYVLDYSVLKDLDLNRNGVTLYAPTMLIYRTGGDKLDVLGIMLEPRRDDAPVYTPDSETPNKFLLAKCHVACADNQVHQFTYHLGYAHLATEPLAIASHNVLEKNSHPLGMFLKPHFRDNIGINYLARQTLVADEDAITDHTFATGTAQGVSMVVDAFKSYNFLESGLPDELRRRGFERSDDLKVYRYRDDGWLVWDTLWKYAEDMVNELYGTDNDVTADKVVQEWAREASGSDTADVQGFPESITTKYILTKVLTTIIWQASALHSALNYIQYPYTATPINRAASIFGPVPDGEADITEQDILDVIPGGLDDENNRGLTLSIFQGLLSWLLRTPENPTLDEVGSPIPNRNNPIEWVEFRSKYPQVYYNLDQNLAVVEKIIEERNKGLASPYEVLLPSHIAASINI

[0618] Coding sequence of CoLOX-0317—SEQ ID NO: 4

[0619]

[0620]

[0621] Amino acid sequence of CoLOX-0317 – SEQ ID NO:6

[0622] MTSSPTVRSMVMLAVLAVYALESTPCASAFATLPRALVRPQAALKYRAEDKNDVDVAPAGSTASDVSKPEGKATAVAKGTVNAPIEEAWKVFRSFSNMNQWMPVYGEWEATGDSVGDTRTFNFKDQPTFFTTERLVGLDDSQYKMKYTLVNCKGSPVPIESIDTIVTFTANDDVTEVDWRSWTKSPMVDLIKGRQAAGYAGGIAALDRYLNPSLGTVDVTIKSADNLDGNFLSSSYATLMVTDADPEQVHAKEWGTSPEFDAKPVQFSLLKPDSKLYMSVMLTKYGVDTPVGYAVFDIQKSLKSGETVTETFQLEGSNDATLTVEMELNLRQGSVLPQSKAQKNLATLVALQQSVERVRDRIVTIGKLAGEPEKSVWEYERKSGLPKSVKGLPRSEVLPPHKIALMVDAIAEYAYTQFQLVQRLLPVRNSYDRYAAYFAPEGEEYVPIPQILKDMTWSTDDEFIRQIFAGLNPLQVEVVKNKAGLPSKLQELKAKDGSDVDKLISEGRLYVLDYSVLKDLDLDRNGVTLYAPTMLIYRTGGDKLDVLGIMLEPRRDDAPVYTPDSETPNKFLLAKCHVACADNQVHQFTYHLGYAHLATEPLAIASHNVLEKNSHPLGMFLKPHFRDNIGINYLARQTLVADEDAITDHTFATGTAQGVSMVVDAFKSYNFLESGLPDELRRRGFERSDDLKVYRYRDDGWLIWDTLWKYAEDMVNELYGTDNDVAADKVVQEWAKEASGSDTADVQGFPESITTKYILTKVLTTIIWQASALHSALNYIQYPYTATPINRAASIFGPVPDGEADITEQDILDVIPGGLDDENNRGLTLSIFQGLLSWLLRTPENPTLDEVGSPIPNRNNPIEWVEFRSKYPQVYYNLDQNLAVVEKIIEERNKGLASPYEVLLPSHIAASINI

[0623] CoLOX-19 coding sequence – SEQ ID NO: 7

[0624]

[0625] Codon-optimized coding sequence of CoLOX-19 using Genscript genetic codon frequencies of E. coli - SEQ ID NO: 8

[0626]

[0627] Amino acid sequence of CoLOX-19 – SEQ ID NO:9

[0628] MTSSPTVRSMVMLAVLAVSALESAPCASAFATLPRALVRPQAALKYRAEDKNDVDVAPAGSTASDVSKPEGKATAVAKGTVNAPIEEAWKVFRSFSNMDQWMPVYGEWEATGDSVGDTRTFNFKDQPTFFTTERLVGLDDSQYKMKYTLVDCKGSPVPIESIDTIVTFTANDDVTEVDWRSWTKSPMVDLIKGRQAAGYAGGIAALDRYLNPSLGTVDVTIKSADNLDGDFLSSSYATLMVTDADPEQVHAKEWGTSPEFDAKPVQFSLLKPDSKLYMNVMLTKYGVDTPVGYAVFDIQKSLKSGETVTETFQLEGSNDATLTVEMELNLRQGSVLPQSKAQKNLATLVALQQSVERVRDRIVTIGKLAGEPEKSVWEYERKSGLPKSVKGLPRSEVLPPHKIALMVDAIAEYAYTQFQLVQRLLPVRNSYDRYAAYFAPEGEEYVPIPQILKDMTWSTDDEFIRQIFAGLNPLQVEVVKNKAGLPSKLQELKAKDGSDVDKLISEGRLYVLDYSVLKDLDLNRNGVTLYAPTMLIYRTGGDKLDVLGIMLEPRRDDAPVYTPDSETPNKFLLAKCHVACADNQVHQFTYHLGYAHLATEPLAIASHNVLEKNSHPLGMFLKPHLRDNIGINYLARQTLVADEDAITDHTFATGTAQGVSMVVDAFKSYNFLESGLPDELRRRGFERSDDLKVYRYRDDGWLVWDTLWKYAEDMVNELYGTDNDVAADKVVQEWAREASGSDTADVQGFPESITTKYILTKVLTTIIWQASALHSALNYIQYPYTATPINRAASIFGPVPDGEADITEQDILDVIPGGLDDENNRGLTLSIFQGLLSWLLRTPENPTLDEVGSPIPNRNNPIEWVEFRSKYPQVYYNLDQNLAVVEKIIEERNKGLASPYEVLLPSHIAASINI

[0629] CoLOX-22 coding sequence - SEQ ID NO: 10

[0630]

[0631] Codon-optimized coding sequence of CoLOX-22 using Genscript genetic codon frequency of E. coli - SEQ ID NO: 11

[0632]

[0633] Amino acid sequence of CoLOX-22 – SEQ ID NO:12

[0634] MTSSPTVRSMVMLAVLAVSALESAPCASAFATLPRALVRPQAALKYRAEDKNDVDVAPAGSTASDVSKPEGKATAVAKGTVNAPIEEAWKVFRSFSNMNQWMPVYGEWEATGDSVGDTRTFNFKDQPTFFTTERLVGLDDSQYKMKYTLVDCKGSPVPIESIDTIVTFTANDDVTEVDWRSWTKSPMVDLIKGRQAAGYAGGIAALDRYLNPSLGTVDVTIKSADNLDGDFLSSSYATLMVTDADPEQVHAKEWGTSPEFDAKPVQFSLLKPDSKLYMSVMLTKCGVDAPVGYAVFDIQKSLKSGETVTETFQLEGSNDATLTVEMELNLRQGSILPQSKAQKNLATLVALQQSVERVRDRIVTIGKLAGEPEKSVWEYERKSGLPKSVKGLPRSEVLPPHKIALMVDAIAEYAYTQFQLVQRLLPVRNSYDRYAAYFAPEGEEYVPIPQILKDMTWSTDDEFIRQIFAGLNPLQVEVVKNKAGLPSKLQELKAKDGSDVDKLISEGRLYVLDYSVLKDLDLNRNGVTLYAPTMLIYRTGGDKLDVLGIMLEPRRDDAPVYTPDSETPNKFLLAKCHVACADNQVHQFTYHLGYAHLATEPLAIASHNVLEKNSHPLGMFLKPHLRDNIGINYLARQTLVADEDAITDHTFATGTAQGVSMVVDAFKSYNFLESGLPDELRRRGFERSDDLKVYRYRDDGWLVWDTLWKYAEDMVNELYGTDNDVAADKVVQEWAREASGSDTADVQGFPESITTKYILTKVLTTIIWQASALHSALNYIQYPYTATPINRAASIFGPVPDGEADITEQDILDVIPGGLGDENNRGLTLSIFQGLLSWLLRTPENPTLDEVGSPIPNRNNPIEWVEFRSKYPQVYYNLDQNLAVVEKIIEERNKGLASPYEVLLPSHIAASINI

[0635] CoLOX-d4 coding sequence - SEQ ID NO: 13

[0636]

[0637] Codon-optimized coding sequence of CoLOX-d4 using Genscript genetic codon frequency of E. coli - SEQ ID NO: 14

[0638]

[0639] Amino acid sequence of CoLOX-d4 – SEQ ID NO:15

[0640] MTSSPTVRSMVMLAVLAVSALESAPCASAFATLPRALVRPQAALKYRAEDKNDVDVAPAGSTASDVSKPEGKATAVAKGTVNAPIEEAWKVFRSFSNMDQWMPVYGEWEATGDSVGDTRTFNFKDQPTFFTTERLVGLDDSQYKMKYTLVDCKGSPVPIESIDTIVTFTANDDVTEVDWRSWTKSPMVDLIKGRQAAGYAGGIAALDRYLNPSLGTVDVTIKSADNLDGDFLSSSYATLMVTDADPEQVHAKEWGTSPEFDAKPVQFSLLKPDSKLYMNVMLTKYGVDTPVGYAVFDIQKSLKSGETVTETFQLEGSNDATLTVEMELNLRQGSVLPQSKAQKNLATLVALQQSVERVRDRIVTIGKLAGEPEKSVWEYERKSGLPKSVKGLPRSEVLPPHKIALMVDAIAEYAYTQFQLVQRLLPVRNSYDRYAAYFAPEGEEYVPIPQILKDMTWSTDDEFIRQIFAGLNPLQVEVVKNKAGLPSKLQELKAKDGSDVDKLISEGRLYVLDYSVLKDLDLNRNGVTLYAPTMLIYRTGGDKLDVLGIMLEPRRDDAPVYTPDSETPNKFLLAKCHVACADNQVHQFTYHLGYAHLATEPLAIASHNVLEKNSHPLGMFLKPHFRDNIGINYLARQTLVADEDAITDHTFATGTAQGVSMVVDAFKSYNFLESGLPDELRRRGFERSDDLKVYRYRDDGWLVWDTLWKYAEDMVNELYGTDNDVTADKVVQEWAREASGSDTADVQGFPESITTKYILTKVLTTIIWQASALHSALNYIQYPYTATPINRAASIFGPVPDGEADITEQDILDVIPGGLDDENNRGLTLSIFQGLLSWLLRTPENPTLDEVGSPIPNRNNPIEWVEFRSKYPQVYYNLDQNLAVVEKIIEERNKGLASPYEVLLPSHIAASINI

[0641] 2.UfLOX

[0642]

[0643] Codon-optimized coding sequence of UfLOX2 using Genscript genetic codon frequency in E. coli - SEQ ID NO: 17

[0644]

[0645] Amino acid sequence of UfLOX2 – SEQ ID NO:18

[0646] MPSIKPCLPGDSANSAARTASIKEKRAQIGYDYKMLPKLALASAPPAKFVELSDAYMAERIGETAKFFKNKEMTKARRMFDVVNRMEDFNDYFILPPVIAPEHAKGKWMEDDFFAEQRLSGANPLVLAKLDRDDARAEILEDMNLDFSVNSELSRGNIYVCDYTGTDPTYRGPCMVTGGENNSGKKKWLPKPLSWFRWIEDDKNKVGGKLVPVAIQLDASEDPVNYVRKDSRVYTPNEEHEYDWLFAKICVQVADSLHHEMGSHLARCHFTMEPIAVCVHRTMAEEHPIALLLNLHMRFHIANDSVAAYTLIGPSGNVDDLMPGTLRESMALLTESYDKWDLIGTNFENDLFNREVNDDERLPHYPYRDDGKLIWKIIEDWVEKYVNAFYDNDDEVEGDPELQAFAKECKDKKEGGRVKGMPETIRSRSMLVEILTSIIFVCGPGHGAINFSQYDYMSFVPNMPLAIYEDIQLLADQKEPVTEAQLMSILPDGETAARQLEIVYNLTAYKFDKFGDYDRTFKEWYGETFEAHFKDYPLVIQGYRQLQVALRQSEVEIKKRNAKRPNNYPYMQQSEMLNSISI

[0647] 3. Bacterial LOX

[0648] Codon-optimized coding sequence of WP_002738122.1 – SEQ ID NO:19

[0649]

[0650] Amino acid sequence of WP_002738122.1 – SEQ ID NO:20

[0651] MVNTPPPTPCLPQNEPDANRRADSLNLQRQAYRYDYQYLPPLVLMESVPAAENFSFQYITERLAATAELPANMLAVKVKSFLDPLDELQDYEDFFAIIPLPKIAKVYQTNDAFAEQRLSGANPLVLHLLKPGDARAQVLNQIPSSKTDFEPLFQVNQELAAGNIYIADYTGTDINYLGPSLIQGGTHAKGRKYLPKPRAFFWWRKSGIRDRGKLVPIAIQFGENAEKLYTPFEKNPLAWLFAKICVQVADSNHHEMNSHLCRTHFVMEPIAIGTARQLAENHPLSLLLKPHLRFMLTNNHLGQERLINPGGPVDELLAGTLGESMALVKDAYANWNLRDFAFPKEISNRGMDDTERLPHYPYRDDGMLVWQSINQFVSDYLHYFYPNPQDITNDQELQAWAGELSNSAADQGGNVKGMPANFTDVEDLIEVVTTIIFICGPLHSAVNYGQYDYMTFAANMPLAAYCDLPEAIKDTTGSIIGDARGSITEKDILQLLPPYKKAADQLQSLFTLSDYRYDQLGYYDKAFRELYGRKFEEVFAEGDQATITGFLRQFQQNLNMNEQEIDANNQKRIVPYTYLKPSLILNSISI

[0652] Codon-optimized coding sequence of WP_006635899.1 – SEQ ID NO:21

[0653]

[0654] Amino acid sequence of WP_006635899.1 – SEQ ID NO:22

[0655] MVDNMKPLLPQDDPNPEQRHDSLNRQQQAYQFDYESLSPLALLKDVPAVENFSSKYLAERILATSELPANMLAADSRTFLDPLDELQDYEDFFTWLPLPGVAKIYQTDRSFAEQRLSGANPMVLRLLHQEDSRAETLAQLCCLQPLFDLRKELQDKNIYIADYTGTDEHYRGPAKVAGGTYEKGRKYLPKPRAFFAWRWTGIRDRGEMTPIAIQLDPKPGSHLYTPFDPPIDWLYAKLCVQVADANHHEMSSHLGRTHLVMEPIAIVTARQLAKNHPLSLLLKPHFRFMLTNNDLARSHLIAPGGPVDELLGGTLAETMELTREACSTWSLDEFALPAELKNRGMDDPNQLPHYPYRDDGLLLWDAIETFVSGYLKFFYPTNEGIVQDVELQTWAKELASDDGGKVKGMPHHIDTVEQLIAIVTTVIFTCGPQHSAVNFPQYDYMSFAANMPLAAYRDIPGITASGHLEVITENDILRLLPPYKRAADQLQILFILSAYRYDRLGYYDKSFRELYRMSFDEVFAGTPIQLLARQFQQNLNMAEQKIDANNQKRVIPYFALKPSLVLNSISM

[0656] Codon-optimized coding sequence of WP_015178512.1 – SEQ ID NO:23

[0657]

[0658] Amino acid sequence of WP_015178512.1 – SEQ ID NO:24

[0659] MVDNMKPSLPQDDPNQEQRKDSLNRQQQAYQFDYESLSPLALLKNVPAVENFSSKYIGERILATSELPANMLAADSRTFLDPLDELQDYEDFFTLLPLPAVAKIYQTDRSFAEQRLSGANPMVLRLLDAGDPRAQTLAQISSFHPLFDLGQELQQKNIYVADYTGTDEHYRAPSKIGGGSYEKGRKFLPKPRAFFAWRWTGIRDRGEMTPIAIQLDPTPDSHVYTPFDPPVDWLFAKLCVQVADANHHEMSSHLGRTHLVMEPIAIVTARQLAQNHPLSLLLKPHFRFMLTNNELARSYLIAPGGPVDELLGGTLPETMEIAREACSTWSLDEFALPAELKNRGMDDTNQLPHYPYRDDGLLLWDAIETFVSGYLKFFYPTEIAIVQDVELQTWAQELASDRGGKVKGMPPRINTVEQLIKIVTTIIFTCGPQHSAVNFPQYEYMSFAANMPLAAYRDIPKITASGNLEVITEKDILRLLPPYKRAADQLKILFTLSAYRYDRLGYYDKSFRELYRMSFDEVFAGTPIQLLARQFQQNLNMAEQKIDANNQKRVIPYIALKPSLVINSISM

[0660] Codon-optimized coding sequence of WP_015204462.1 – SEQ ID NO:25

[0661]

[0662] Amino acid sequence of WP_015204462.1 – SEQ ID NO:26

[0663] MPQPYLPQNEPNPEKRNNDLSDQQQAYEYDYKYLPPLVLLKKIPAFENFSAQYIAERVVATSELVPNMLAAKARSFLDPLDDIKDYEDLFTLLPLPEVAKVYQTNNSFAEQRLSGANPFVIRLLDEDDPRSQVLEQIPSFKDDFEPLFDVRKELAAGNIYITDYTGTDEYYRGPSMVQGGTYEKGRKYLPKPLAFFWWQRTGISDRGKLVPIAIQLDASKNSKVYTPTNSKVYTPFEQNPLDWLFAKLCVQIADGNHHEMSSHLCRTHFVMEPIAIGTAHQLAENHPLSLLLRPHFLFMLTNNHLGQQRLINPGGPVDELLAGTLPESMELVKDAYEGWNIKEFAFPTEIKNRGMDNTERLPHYPYRDDGMLVWKAIHTFVSDYVNHFYPTPEDITGDTELQAWAKELSDQSAQTNGGKVKGMPTSFTTVQELIEIVTTIIFICGPQHSAVNYAQDGYMTFAANMPLAAYRDIPKQSHKPQDQPTATPSVAVQTTAEQTTAEQTKAVEITADKATLDQNTVLQKRAVQTTTVEIPEDQITEEQILKLLPPYKRTADQLQSLFVLSAYQYDRLGYYEKAFQQLYNDKFEDVFKDDNNQAIIAIVRQFQQNLNMVEQEIDANNKKRVVPYLYLKPSLILNSISI

[0664] Codon-optimized coding sequence of WP_028091425.1 – SEQ ID NO:27

[0665]

[0666] Amino acid sequence of WP_028091425.1 – SEQ ID NO:28

[0667] MQPFLPQNDPNPSQRQSSLEKGRKEYQFMYDFLPPMAMIKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQDKFGSSINLIERLATGNLYVADYRSLAFIQGGTYAKGKKYLPAPLAFFCWRTSGFQDRGQLVPVAIQINPKAGKASPLLTPFDDPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLKPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVNDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYQSSADLKADAELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQPIQQKGDIKDRQALIDFLPPAKPTSTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELNMVQRKIELNNKRRLVNYKYLQPRLILNSISI

[0668] Codon-optimized coding sequence of OBQ01436.1 – SEQ ID NO:29

[0669]

[0670] Amino acid sequence of OBQ01436.1 – SEQ ID NO:30

[0671] MQPFLPQNDPNPAQRQSCLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPILQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQAKFGNSINLIERLATGNLYVADYRSLAFIQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKASPLLTPFDDPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLRPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQEIQQNGDIEDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELSVVQRKIELNNKGRLVNYEYLQPGLILNSISI

[0672] Codon-optimized coding sequence of OBQ25779.1 – SEQ ID NO:31

[0673]

[0674] Amino acid sequence of OBQ25779.1 – SEQ ID NO:32

[0675] MINIMQPFLPQNDPNPGQRQSSLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQAKFGNSINLIERLATGNLYVADYRSLAFIQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGQASPLLTPFDKPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLKPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQAIQQKGDIKDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELNVVQRKIELNNKGRLVNYEYLQPRLILNSISI

[0676] Codon-optimized coding sequence of WP_039200563.1 – SEQ ID NO:33

[0677]

[0678] Amino acid sequence of WP_039200563.1 – SEQ ID NO:34

[0679] MKPFLPQNDPNPTQRQSSLEKGRKEYEFRYDFLPPMAMLKNVPPSENFSTKYIAERTIETAELPSNMMAVKAHAMWDPLDELQDYEDFFPVLQKPNVMKNYETDDSFAEQRLCGVNPVVLCQIKQMPANFAFTIEELQAKFGNSIDLRERLATGNLYVADYRPLAFIRGGTFAKGKKYLPAPLAFFCWRSSGFQDRGQLVPIAIQINPKEGKASPLLTPFDDSSTWFYAKSCVQIADANHHEMSSHLCRTHFVMEPFAVVTPRQLAQNHPLRILLKPHFRFMLANNDLGRQRLVNRGGPVDELLAGTLQESLQIVVDAYTDWRLDQFALPTELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLELYYKSPADLTADVELQAWARELVAQDGGRVKGMSDRIDTLKQLVEIVTTIIYTCGPLHSAVNFPQYEYMGFIPNMPLAAYQPIKKEGVCTRKELIDFLPAAKPTSSQLTTLFTLSAYRYDRLGYYEEEEFEDPNADDVVNKFQQELNVVQRKIELSNKGRLVNYEYLQPRLILNSISI

[0680] Codon-optimized coding sequence of WP_012407347.1 – SEQ ID NO:35

[0681]

[0682] Amino acid sequence of WP_012407347.1 – SEQ ID NO:36

[0683] MKPYLPQNDPDPTKRQILLERNQGEYEFDYDFLVPMAMLKNVPSIENFSTKYIAERTLETAELPINMLAVKTRSLWDPLDELQDYEDYFPVLPKPNIIKTYQSDDSFCEQRLCGANPFVLRRIEQMPDGFAFTILELQEKFGDSINLVEKLANGNLYVADYRALAFVKGGSYERGKKFLPTPIAFFCWRSSGFSDRGQLVPIVIQINPADGKQSQLITPFDDPLTWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAIVTARQLAENHPLSLLLKPHFRFMLANNDLARKRLISRGGPVDELLAGTLQESLQIVVNAYTEWSLDQFSLPTELKNRGMDDPDNLPHYPYRDDGLLLWNAIKKFVSEYLQIYYKTPQDLAEDLELQSWVQELVSQSGGRVKGISDRINTLDQLVDIATAVIFTCGPQHAAVNYSQYEYMTFMPNMPLAAYKQMTSEGTIPDRKSLLSFLPPSKQTADQLSILFILSAYRYDRLGYYDDKFLDPEAQDVLAKFQQELNEAEREIELNNKSRLINYNYLKPRLVTNSISV

[0684] Codon-optimized coding sequence of WP_027843955.1 – SEQ ID NO:37

[0685]

[0686] Amino acid sequence of WP_027843955.1 – SEQ ID NO:38

[0687] MKPYLPQNDPNPEKRKDWLNKNREEYQFNFNYLSPLPLIDDVPNNEAFSPKYLAERLPLTFGKLSANTLGIRLRSFWDPFDEFQDYEDFFPVLPTPELLKTYQNDEYFAEQRLSGVNPMVIRSIKELPPHFAFSIRDLQAEFGTSLNLEQELNNGNLYIADYTSLSFVRGGSYLRGRKSLPAPIALFCWRNSGYCDRGELTPIAIQLVPELGTGSRILTPFDSHLNWLYAKICMQIADANHHEMSSHLCHTHLVMEPFAVVTARQLAENHPLGLLLRPHFRFMLHNNELARKNLINQGGYVDNLLGGTLRESLQIVRDAYFKNAEEFWSLDEFALPKEIANRGLDDTDRLPHYPYRDDGMLLWNAIEKFVSNYLSIYYPNPGDIKDDRELQAWAAELVAADGGRVKGVPSQFENLQQLIDVVTGIIFTCGPQHSAVNYPQYEYMAFVPNMPLAGYQAVDSNPNMDLKSLMAFLPPPNQTADQLQIIYGLSAYRYDRLGYYDREFSDPHAEEVVRLFQQDLNQVERKIELRNKNRLVEYNFLKPSLVLNSISI

[0688] Codon-optimized coding sequence of WP_073641301.1 – SEQ ID NO:39

[0689]

[0690] Amino acid sequence of WP_073641301.1 – SEQ ID NO:40

[0691] MKPYLPQNDPDPIKRKYSLEHKKEEYEFDHDFLSPMAMLKDVPAVENFSTRYIAERTVETAELPINMLAVKTRALWDPLDELQDYEDYFPVLPKPNVIKTYQTDDSFCEQRLCGANPMALQQIKEMPLGFEFTIEELQEKFGESINLVEKLADGNLYVTDYRPLSFVKGGTYERGKKYLPTPLAFFCWRSSGFSDRGQLVPIAIQLNPAVGRQSQLITPFDDPLTWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAIVTARQLADNHPLNLLLKPHFRFMLANNDLGRKRLVNRGGPVDELLAGTLQESLQIVVNAYKEWSLDEFALPTEIKNRGMDDKLKLPHYPYRDDGMLLWNAIKKFVSEYLKLYYKTPQDLTADLELQAWAQELVSESGGRVKGVPSRIEKLEQLVDIATAVIFTCGPQHAAVNYSQYEYMTFMPNMPLAAYKQMTAEGTIADRKSLLSFLPPSKQTADQLSILFILSAYRYDRLGYYDDKFADPEAQDILVTFQQDLNEVERKIELNNKSRLIKYNYLKPRLVTNSISV

[0692] Codon-optimized coding sequence of WP_096647440.1 – SEQ ID NO:41

[0693]

[0694] Amino acid sequence of WP_096647440.1 – SEQ ID NO:42

[0695] MKPYLPQNDPEPTQRKNFLERKQGEYEFDHKFLKPMAMLKNVPSIENFSTKYIAERTVETAELPLNMLAVKTRSLWDPLDELQDYEDYFPVLPKPNVIKTYQTDNSFCEQRLCGANPLVLRQIQQMPDGFAFTISELQEKFGDSIDLEERLKTGNLYVADYRALAFVKGGTYERGKKYLPTPIAFFCWRSSGFSDRGQLVPIAIQINPTDGKQSQLITPFDEPLVWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAIVTARQLADNHPLNLLLKPHFRFMLANNELGRQRLVNRGGPVDELLAGTLQESLQIVVNAYKEWSLDQFSLPTELKNRGMDNSDKLPHYPYRDDGLLLWNAIKKFVSEYLKLYYKTPQDLTADFELQSWAQELVSQSGGRVKGVSDRITTLDQLIDIATAVIFTCGPQHAAVNYSQYEYMTFIPNMPLAAYKQITSEGNIPDRKSLLSFLPPSKQTADQLSILFILSAYRYDRLGYYDDKFLDPEAQEILVTFQQELNEAERQIELNNKSRLINYDYLKPRLVTNSISV

[0696] Codon-optimized coding sequence of WP_099099431.1 – SEQ ID NO:43

[0697]

[0698] Amino acid sequence of WP_099099431.1 – SEQ ID NO:44

[0699] MKPYLPQKDPDVKVRINWLDKNREEYKFNYDYLAPLPVIDKVPHKEIFSAEYTTKRLASMASLAPNMLAAKARNFLDPLDELEEYEELLSLLPKPDVIKNYKTDSCFAEQRLSGANPLAIQKIDVLPDNFAVTDAHFQKVAGTEFTLEKALKEGKLYFLDYPLLSDIKGGVYNNVKKYLPKPQALFYWQSNDSPNGGSLVPVAIQINHDSGGKSVIYTPDDPHLDWFLAKTCVQIADGNHQELGSHFAYTHAVMAPFAIVTARQLAENHPIALLLKPHFRFMLFDNDLGRTQFLQPGGPVDEFMAGSLAESLGFVAKVYEEWSVEKFTFPRLIKSRRTDDPEILPHFPFRDDGMLIWNAVEKFVYEYLQLYYKTSQDLIDDYELQNWARELVAQDGGRVKGMPAKIETLEQLIEIISVVVFTCAPLHSALNFSQYEYMAFVPNMPYAAYHPIPETKGVDLETIMKILPPFKQAADQVMWTEILTSYHYDKLGFYDEEFADPLAQEIVVQFQQNLHEIERQIDIRNQTRPIPYNYFKPSQIINSINT

[0700] Codon-optimized coding sequence of WP_052672367.1 – SEQ ID NO:45

[0701]

[0702] Amino acid sequence of WP_052672367.1 – SEQ ID NO:46

[0703] MKPYLPQHEPDAIARQNRLIKNRADYVLDYNYLPPIPLQTPVPQQERFSAEYTARRLASFANLVPNMLMARARNAFDPLDTLEEYADLLPVLPKPNVIKNYQADWCFAEQRLSGINPPAIRRIDALPENLPISNSSFQHSVGAEHNLEQALKEGKLYCLDYPLLSGIGGGNYQNLPKYLPKPQALFYWRSDNSKIGGSLVPVAIKILNELGGKNLVYTPNDAPLDWFLAKTCVQMADANHQELGTHFAKTHAVMAPIAAITARELGENHPLTLLLKPHFRFMLFDNELGRTQFLQPTGPTEELLAGTLEESVQLVVQAYEEWSIDTTFPLELQQRQMHDPEILPHYPFRDDGILVWNAIHQFVTEYLQIYYHTPQDISADYEVQNWARELVDSGRVKGMPESIDTLAQLIDIIAVVIFTCAPLHSCLNLAQYEYMTFVPNMPYAAYHPIPTTKGVDMATIVKIMPPFQRAIDQILWTDILSAFQYDKLGFYEEDFADPKAQEVLQRFQDNLQQVEEKIEMHNQIRPIPYNYLKPSRIMNSINT

[0704] Codon-optimized coding sequence of WP_073631249.1 – SEQ ID NO:47

[0705]

[0706] Amino acid sequence of WP_073631249.1 – SEQ ID NO:48

[0707] MKPYLPQHDPNPEARRNWLEQNREDYKFDHNYLAPIPILDKVPHKELFSPQYTAKRLASMADLVPNMLAAKARNFFDPLDELEEYEALLSILPKPSVIKNYKTDSCFAEQRLSGANPMAMHRIDELPEKFPVTNDHFQKAVGAEHNLEAALKEGKLYLLDYPLLFDIKGGTYQNIKKYLPKPQALFYWQSNGNKNSGSLVPIAIQIHNDTGGDSLIYTPDDPHLDWFLAKTCVQIADANHQELGSHFARTHAVMAPFAIVTARQLGENHPLALLLKPHFRFMLYDNDLGRTHFLQAGGPVDEFMAGTLQESLGFVAKAYEEWSLDNAVFPTEVKNRKMDDPDILPHYPFRDDGMLLWDAVKKFVTEYLQLYYKTPQDLSEDYELQNWARELAAQDGGCVKGMPEKIETIEQLIHVVTVVVFTCAPLHSALNFSQYEYMAFVPNMPYAAYYPVPETKGVDMQTIMKMLPPFKQAADQVMWSDILTSFHYDKLGHYDEEFANPMAQAILLQFQQNLHEVERQIEIKNQSRPIPYNYLKPSEIINSINT

[0708] Codon-optimized coding sequence of WP_013220336.1 – SEQ ID NO:49

[0709]

[0710] Amino acid sequence of WP_013220336.1 – SEQ ID NO:50

[0711] MNTSLPQNDSDPQGRKDRLERRRALYVFNYDYVPPIPMIDKVPHEEYFSPKYTAERLASMAKLAPNMLAAKTKRLFDPLDELNEYDEMFIFLDKPGIVRGYRTDESFGEQRLSGVNPMSIRRLDKLPEDFPIMDEYLEQSLGSPHTLAQALQEGRLYFLEFPQLAHVKEGGLYRGRKKYLPKPRALFCWDGNHLQPVAIQISGQPGGRLFIPRDSDLDWFVAKLCVQIADANHQELGTHFARTHVVMAPFAVVTHRQLAENHPLHILLRPHFRFMLYDNDLGRTRFIQPDGPVEHMMAGTLEESIGISAAFYKEWRLDEAAFPIEIARRKMDDPEVLPHYPFRDDGMLLWDGIQKFVKEYLALYYQSPEDLVQDQELRNWARELTANDGGRVAGMPGRIETVDQLTSILSTVIYTCAPLHSALNFAQYEYIGYVPNMPYAAYHPIPEEGGVDMETLMKILPPYEQAALQLKWTEILTSYHYDRLGHYDEKFEDPQAQAVVEQFQQELAAVEQEIDQRNQDRPLAYTYLKPSEIINSINT

[0712] 4. Consensus sequence

[0713] Consensus sequence of CoLox – SEQ ID NO:51

[0714] M x S x PTVRSMVMLAVLAV x ALES x PCASAFATLPRALVRPQAALKYRAEDKNDVDVAPAGSTASDVSKPEGKATAVAKGTVNAPIEEAWKVFRSFSNM x QWMPVYGEWEATGDSVGDTRTFNFKDQPTFFTTERLVGLDDSQYKMKYTLV xCKGSPVPIESIDTIVTFTANDDVTEVDWRSWTKSPMVDLIKGRQAAGYAGGIAALDRYLNPSLGTVDVTIKSADNLDG x FLSSSYATLMVTDADPEQVHAKEWGTSPEFDAKPVQFSLLKPDSKLYM x VMLTK x GVD x PVGYAVFDIQKSLKSGETVTETFQLEGSNDATLTVEMELNLRQGS x LPQSKAQKNLATLVALQQSVERVRDRIVTIGKLAGEPEKSVWEYERKSGLPKSVKGLPRSEVLPPHKIALMVDAIAEYAYTQFQLVQRLLPVRNSYDRYAAYFAPEGEEYVPIPQILKDMTWSTDDEFIRQIFAGLNPLQVEVVKNKAGLPSKLQELKA x DGSDVDKLISEGRLYVLDYSVLKDLDL x RNGVTLYAPTMLIYRTGGDKLDVLGIMLEPRRDDAPVYTPDSETPNKFLLAKCHVACADNQVHQFTYHLGYAHLATEPLAIASHNVLEKNSHPLGMFLKPH x RDNIGINYLARQTLVADEDAITDHTFATGTAQGVSMVVDAFKSYNFLESGLPDELRRRGFERSDDLKVYRYRDDGWL x WDTLWKYAEDMVNELYGTDNDV x ADKVVQEWA x EASGSDTADVQGFPESITTKYILTKVLTTIIWQASALHSALNYIQYPYTATPINRAASIFGPVPDGEADITEQDILDVIPGGL x Consensus sequence of the protein sequence of bacterial LOX – SEQ ID NO:52

[0715]

[0716] The five essential conserved amino acid residues of the active site putatively involved in cofactor binding are shown in enlarged bold.

[0717] Consensus sequence of bacterial LOX and UfLOX2 protein sequences – SEQ ID NO:53

[0718]

[0719] The five essential conserved amino acid residues of the active site putatively involved in cofactor binding are shown in enlarged bold.

[0720] Consensus sequence of bacterial LOX, CoLOXs and UfLOX2 protein sequences – SEQ ID NO:54

[0721] The five essential conserved amino acid residues of the active site putatively involved in cofactor binding are shown in enlarged bold.

[0722] 5. Others

[0723] CoLOX forward primer (5'-CTCTCTCTCTTTCTCTCTGTTCT-3') (SEQ ID NO: 55)

[0724] CoLOX reverse primer (5'-CTCGTTCCCTTACCGTCT-3') (SEQ ID NO: 56)

[0725] UfLOX2 forward primer (5'-TCGTCCAACAGGTTCTCTT-3') (SEQ ID NO: 57)

[0726] UfLOX2 reverse primer (5'-TTCTTTCCACTCACCGCCA-3'). (SEQ ID NO: 58)

[0727] 6. The corresponding natural coding sequences of SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50

[0728]

[0729] Coding sequence of WP_006635899.1 - SEQ ID NO: 60

[0730]

[0731] Coding sequence of WP_015178512.1 - SEQ ID NO: 61

[0732]

[0733] Coding sequence of WP_015204462.1 - SEQ ID NO: 62

[0734]

[0735] Coding sequence of WP_028091425.1 - SEQ ID NO: 63

[0736]

[0737] Coding sequence of OBQ01436.1 - SEQ ID NO:64

[0738]

[0739] Coding sequence of OBQ25779.1 - SEQ ID NO:65

[0740]

[0741] Coding sequence of WP_039200563.1 - SEQ ID NO: 66

[0742]

[0743] Coding sequence of WP_012407347.1 - SEQ ID NO: 67

[0744]

[0745] Coding sequence of WP_027843955.1 - SEQ ID NO: 68

[0746]

[0747] Coding sequence of WP_073641301.1 - SEQ ID NO: 69

[0748]

[0749] Coding sequence of WP_096647440.1 - SEQ ID NO: 70

[0750]

[0751] Coding sequence of WP_099099431.1 - SEQ ID NO: 71

[0752]

[0753] Coding sequence of WP_052672367.1 - SEQ ID NO: 72

[0754]

[0755] Coding sequence of WP_073631249.1 - SEQ ID NO: 73

[0756]

[0757] Coding sequence of WP_013220336.1 - SEQ ID NO: 74

[0758]

[0759] 7. Coding sequence (start codon changed to ATG) and amino acid sequence mined from NCBI

[0760] Coding sequence of WP_108935963.1 - SEQ ID NO: 75

[0761]

[0762] Amino acid sequence of WP_108935963.1 – SEQ ID NO:76

[0763] MVNTPPPTPCLPQNEPDANRRADSLNLQRQAYRYDYQYLPPLVLMESVPAAENFSLQYITERLAATAELPANMLAVKVKSFLDPLDELQDYEDFFAIIPLPKIAKVYQTNDAFAEQRLSGANPLVLRLLKPGDAGAQVLNQIPSSKTDFEPLFQVNQELAAGNIYIADYTGTDANYLGPSFVQGGTHAKGRKYLPKPRAFFWWRKSGIRDRGKLVPIAIQFGENAEKLYTPFEKNPLAWLFAKICVQVADSNHHEMNSHLCRTHFVMEPIAIGTARQLAENHPLSLLLKPHLRFMLTNNHLGQERLINPGGPVDELLAGTLGESMALVKDAYANWNLRDFAFPKEISNRGMDDTERLPHYPYRDDGMLVWQSINQFVSDYLHYFYPNPQDITNDQELQAWARELSNSAADQGGNVKGMPANFTDVEDLIEVVTTIIFICGPLHSAVNYGQYDYMTFAANMPLAAYCDLPEAIKDTTGSIIGDARGSITEKDILQLLPPYKKAADQLQSLFTLSDYRYDRLGYYDKAFRELYGRKFEEVFAEGDQATITGFLRQFQQNLNMNEQEIDANNQKRIVPYTYLKPSLILNSISI

[0764] Coding sequence of WP_110985169.1 – SEQ ID NO:77

[0765]

[0766] Amino acid sequence of WP_110985169.1 – SEQ ID NO:78

[0767] MPSLPQNDPDLQARQALLKQQQERYQFNFEYLAPLAMLDEVPKDENFSGAYLAERLTRAADLPVNMLAAKAHSLLDPLDRLEDYDDLFTLLPKPAIANTFQTDEVFAEQRLSGANPMAIRRLDPSNPPSAYLNIKQQLATKGKTLVERNLYYVDYSELSFIQGGTYAKGKKYLPTPFALFSWQSMGYRDHKTSDHGELLPIAIQIQQNNSGRVYTPRDAHLDWLFAKLCVQIADGNHHEMSSHLCRTHFVMEPIAVVTARQLAEDHPLYILLQPHFRFMLANNELGRKQLIQHGGPVDKLLAGTLAESLQVVKNSFESWSLDQFSFPTEVRNRGMDSPDLPHFPYRDDGQLVWDAIYKFVTDYLRLFYADSDALKNDEELQSWLKELRDPQGGRIKGVPEHIQALEPLVEMVTTIIFTCGPQHCAVNYTQYEYMALASNIPLAAYQDLTGLENGSETKPAITDEAHLMQYLPPYQQAAGQLQIMNILTDYRYDKLGYYDRTFKDAFAGSSFDTAVDAVVEQFKQNLRVVETEIDLDNRKRVIEYPYLKPSLILNSISI

[0768] Coding sequence of WP_053540410.1 – SEQ ID NO:79

[0769]

[0770] Amino acid sequence of WP_053540410.1 – SEQ ID NO:80

[0771] MQPFLPQNDPNPAQRQSSLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDTLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQDKFGNSINLIERLATGNLYVADYRSLAFIQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKVSPLLTPFDDPLTWFYAKSCVQIADGNHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLKPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQEIQQKGDIEDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELNVVQRKIELNNKGRLVNYEYLQPRLILNSISI

[0772] Coding sequence of WP_035367771.1 – SEQ ID NO:81

[0773]

[0774] Amino acid sequence of WP_035367771.1 – SEQ ID NO:82

[0775] MINIMQPFLPQNDPNPAQRQSCLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQDKFGSSINLIERLATGNLYVADYRSLAFIQGGTYAKGKKYLPAPLAFFCWRTSGFQDRGQLVPVAIQIAPKAGKVSPLLTPFDDPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLKPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVNDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYQSSADLKADAELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQPIQQKGDIKDRKALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELNMVQRKIELNNKGRLVNYEYLQPRLILNSISI

[0776] Coding sequence of OBQ35765.1 – SEQ ID NO:83

[0777]

[0778] Amino acid sequence of OBQ35765.1 – SEQ ID NO:84

[0779] MKPFLPQNDPNPGQRQSSLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEVAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQDKFGNSINLIERLATGNLYVADYRSLAFVQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKVSPLLTPFDDPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLKPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYRSSADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQAIQQKGDIKDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELNVVQRKIELNNKGRLVNYEYLQPRLILNSISI

[0780] Coding sequence of OBQ09764.1 – SEQ ID NO:85

[0781]

[0782] Amino acid sequence of OBQ09764.1 – SEQ ID NO:86

[0783] MQPFLPQNDPNPAQRQSCLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPILQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQAKFGNSINLIERLATGNLYVADYRSLAFVQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKASPLLTPFDDPLTWFYAKSCVQIADGNHHEMSSHLCRTHFVMEPFAVVTPRQLAENHPLRILLKPHFRFMLANNDLGRQRLVNRGGIVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIITTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQEIQQKGDIKDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFADPNADQVVNKFQQELSVVQRKIELNNRGRLVNYEYLQPGLILNSISI

[0784] Coding sequence of OBQ23315.1 – SEQ ID NO:87

[0785]

[0786] Amino acid sequence of OBQ23315.1 – SEQ ID NO:88

[0787] MQPFLPQNDPNPAQRQSSLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDTLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQDKFGNSINLIERLATGNLYVADYRSLAFIQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKVSPLLTPFDDPLTWFYAKSCVQIADANHHEMNSHLCRTHLVMEPFAVVTPRQLAENHPLRILLRPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSSADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQAIQQKGDIKDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDRNADQVVNKFQQELNVVQRKIELNNKGRLVNYEYLQPRLILNSISI

[0788] Coding sequence of OBQ30848.1 – SEQ ID NO:89

[0789]

[0790] Amino acid sequence of OBQ30848.1 – SEQ ID NO:90

[0791] MQPFLPQNDPNPAQRQSCLEKGRKEYKFMYDFLPPMAMIKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQDKFGNSINLIERLATGNLYVADYRSLAFVQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKVSPLLTPFDDPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLKPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQEIQQKGDIKDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELNVVQRKIELNNKGRLVNYEYLQPRLILNSISI

[0792] Coding sequence of OBQ23778.1 – SEQ ID NO:91

[0793]

[0794] Amino acid sequence of OBQ23778.1 – SEQ ID NO:92

[0795] MQPFLPQNDPNPAQRQSSLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQDKFGNSINLIERLATGNLYVADYRSLAFIQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKASPLLTPFDDPLTWFYAKSCVQIADGNHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLRPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQAIQEKGDIKDRQALIDFLPLAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELSVVQRKIELNNKGRLVNYEYLQPRLILNSISI

[0796] Coding sequence of WP_015083575.1 – SEQ ID NO:93

[0797]

[0798] Amino acid sequence of WP_015083575.1 – SEQ ID NO:94

[0799] MQPFLPQNDPNPAQRQSCLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMPANFAFTIEELQDKFGNSINLIERLATGNLYVADYRSLAFVQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKASPLLTPFDDPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVVTPRQLAENHPLRILLRPHFRFMLANNDLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARELVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQEIQQKGDIEDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFADPNADKVVNKFQQELSVVQRKIELNNKGRLVNYEYLQPRLILNSISI

[0800] Coding sequence of WP_027404620.1 – SEQ ID NO:95

[0801]

[0802] Amino acid sequence of WP_027404620.1 – SEQ ID NO:96

[0803] MKPFLPQNDPNPTQRQSSLEKGRKEYEFRYDFLPPMAMLKNVPPSENFSTKYIAERTIETAELPSNMMAVKAHAMWDPLDELQDYEDFFPVLQKPNVMKNYETDDSFAEQRLCGVNPVVLRQIKQMPVNFAFTIEELQAKFGNSIDLRERLATGNLYVADYRPLAFIRGGTFAKGKKYLPAPLAFFCWRSSGFQDRGQLVPIAIQINPKEGKASPLLTPFDDSSTWFYAKSCVQIADANHHEMSSHLCRTHFVMEPFAVVTPRQLAQNHPLRILLKPHFRFMLANNDLGRQRLVNRGGPVDELLAGTLQESLQIVLDAYTDWRLDQFALPTELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLELYYKSPADLTADVELQAWARELVAQDGGRVKGMSDRIDTLKQLVEIVTTIIYTCGPLHSAVNFPQYEYMGFIPNMPLAAYQPIKKEGVCTRKELIDFLPAAKPTSSQLTTVFTLSAYRYDRLGYYEEEEFEDPNADDVVNKFQQELNVVQRKIELSNKGRLVNYEYLQPRLILNSISI

[0804] Coding sequence of WP_114084873.1 – SEQ ID NO:97

[0805]

[0806] Amino acid sequence of WP_114084873.1 – SEQ ID NO:98

[0807] MKPYLPQNDPDPTKRKILLERNQGEYEFDYDFLTPMAMLKNVPSIENFSTKYIAERTLETAELPINMLAVKTRSLWDPLDELQDYEDYFPVLPKPNVIKTYQTDDSFCEQRLCGANPFVLRRIEKMPDGFAFTILELQEKFGDSINLVDKLTNGNLYVADYRALAFVKGGTYERGKKYLPTPIAFFCWRSSGFSDRGQLVPIVIQINPTDGKQSQLITPFDDPLTWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAIVTARQLAENHPLSLLLKPHFRFMLANNDLARKRLISRGGPVDELLAGTLQESLQIVVNAYQEWSLDQFSLPTELKNRGMDDPNNLPHYPYRDDGLLLWNAIKKFVSEYLQIYYKTPQDLAADLELQSWAQELVSQSGGRVKGISNRIDTLDQLVDIATAVIFTCGPQHAAVNYSQYEYMTFMPNMPLAAYKQMTSEGTIPDRKSLLSFLPPSKQTADQLSILFILSAYRYDRLGYYDDKFVDPEAQDVLAKFQQDLNEAEREIELNNKSRLINYNYLKPRLVTNSISV

[0808] Coding sequence of WP_096538768.1 – SEQ ID NO:99

[0809]

[0810] Amino acid sequence of WP_096538768.1 – SEQ ID NO:100

[0811] MKPYLPQNDPDPTKRKSFLERKQEEYEFDYDFLPPMAMLKDVPAVENFSTKYIAERAVETAELPINMLAVKTHTLWDPLDELQDYEDYFPVLPKPTVIKTYQTDDSFCEQRLCGSNPMALRQIKEMPLDFEFTIQELQRKFGESINLAEKLANGNLYITDYRSLSFVKGGTYERGRKYLPTPLAFFCWRSSGFSDRGQLVPIAIQLNPAAGKQSQLITPFDDPLAWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAIVTARQLADNHPLNLLLKPHFRFMLANNDLGRKRLVNRGGPVDELLAGTLQESLQIVVNAYKEWSLDKFALPTEIKNRGVDDPQKLPHYPYRDDGMLLWNAIKKFVSEYLNLYYKTPEDLTADFELQAWAQELVSQSGGRVKGVPDRIEKLEQLIDIATAVIFTCGPQHAAVNYPQYEYMTFMPNMPLAGYKQMTSEGTIADRKSLLSFLPPPKQTADQLSILFILSAYRYDRLGYYDDKFADPEAEDIVATFQQDLNEVDREIELNNKSRLIKYNYLKPRLVTNSIGI

[0812] Coding sequence of RCJ25669.1 – SEQ ID NO:101

[0813]

[0814] Amino acid sequence of RCJ25669.1 – SEQ ID NO:102

[0815] MNPYLPQNDPDPTKRKFSLERKLEEYEFDYNFLPPMAMLKDVPAVENFSTKYIAERAVETAELPLNMLAVKTRSLWDPLDELQDYEDYFPVLPKPDVIKTYQTDDSFCEQRLCGANPMALRQIKEMPLGFEFTIQELQEKFGESINLAEKLANGNLYITDYRPLSFVKGGTYERGKKYLPTPLAFFCWRSSGFSDRGQLVPIAIQLNPALGKQSQLITPFDDPLTWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAIVTARQLADNHPLNILLKPHFRFMLANNDLGRKRLVNRGGPVDELLAGTLQESLQIVVNAYKEWSLDQFALPTEIKNRGVDNPDNLPHYPYRDDGMLLWNAIKKFVSEYLKLYYKTPEDLTADFELQAWAQELVSQSGGRVKGVPSRIEKLEQLVDITTAVIFTCGPQHAAVNYPQYEYMTFMPNMPLAGYKQMTSEGTIPDRKSLLSFLPPPKQTADQLSILFILSAYRYDRLGYYDDKFADSEAEQILVTFHQDLTEVEREIELNNKSRLIKYDYLKPRLVTNSISI

[0816] Coding sequence of WP_017318478.1 – SEQ ID NO:103

[0817]

[0818] Amino acid sequence of WP_017318478.1 – SEQ ID NO:104

[0819] MKPNLPQHEPNPEARRNWLEQNREDYKFDHNYLAPIPILDKVPHQELFSPKYTAKRLASMANLVPNMLAAKARNFFDPLDELEEYEDLLPILPKPSVIKNYKTDSCFAEQRLSGANPMAMHRIDALPENFPVTNDHFQKAVGAAHDLEAALKEGKLYLLDYPLLFDIKGGTYQNIKKYLPKPQALFYWQSNGNKNSGSLMPIAIQLHNDTDGDSLIYTPDDPHLDWFLAKTCVQMADGNHQELGSHFARTHAVMGPFAVVTARQLGENHPLSLLLRPHFRFMLYDNDLGRTHFLQPGGPVDEFMAGTLQESLGFVGKAYEEWSLDNAVFATEIKNRKMDDPEILPHYPFRDDGMLVWDAVKKFVTEYIQLYYKTPQDLSEDYELQNWARELAAQDGGRVKGMPEKIETIEQLIDIVTVVVFTCAPLHSALNFSQYEYMAFVPNMPYAAYHPVPETKGVDMQTIMKMLPPFKHAADQVMWSDILTSFHYDKLGHYDEEFADPIAQEILVQFQQNLHEVERQIEIKNQSRPIPYNYLKPSEIINSINT

[0820] Coding sequence of KJH71567.1 – SEQ ID NO:105

[0821]

[0822] Amino acid sequence of KJH71567.1 – SEQ ID NO:106

[0823] MIKPYLPQHEPDAIARQNRLIKNRADYVLDYNYLPPIPLQTPVPQQERFSAEYTARRLASFANLVPNMLMARARNAFDPLDTLEEYADLLPVLPKPNVIKNYQADWCFAEQRLSGINPPAIRRIDALPENLPISNSSFQHSVGAEHNLEQALKEGKLYCLDYPLLSGIGGGNYQNLPKYLPKPQALFYWRSDNSKIGGSLVPVAIKILNELGGKNLVYTPNDAPLDWFLAKTCVQMADANHQELGTHFAKTHAVMAPIAAITARELGENHPLTLLLKPHFRFMLFDNELGRTQFLQPTGPTEELLAGTLEESVQLVVQAYEEWSIDTTFPLELQQRQMHDPEILPHYPFRDDGILVWNAIHQFVTEYLQIYYHTPQDISADYEVQNWARELVDSGRVKGMPESIDTLAQLIDIIAVVIFTCAPLHSCLNLAQYEYMTFVPNMPYAAYHPIPTTKGVDMATIVKIMPPFQRAIDQILWTDILSAFQYDKLGFYEEDFADPKAQEVLQRFQDNLQQVEEKIEMHNQIRPIPYNYLKPSRIMNSINT

[0824] Coding sequence of WP_017327314.1 – SEQ ID NO:107

[0825]

[0826] Amino acid sequence of WP_017327314.1 – SEQ ID NO:108

[0827] MNTAVRPSLPQKDPNSNKRNDYLERNREDYQFDRSLLPPLPFMQKVPKREYFSPEYTAKRLASMANLPANMLAAKAKRFLDPLDSLEEYEELIPLLSKPNLLKNYRTDEFFGEQRLSGANAMATRRLAKLPSDFAVDNALFQQVLETDGTLDAALAEGRLYFLEHPYLNRIKGGESEYGRKYMPKTRSLFYWKSDDSPVGGALLPVAIELKSEATNTPIVYTPKDAPLDWLFAKLCVQVADANHQELGSHFAFTHTAMGPFAMVTARQLAENHPVSLLLEPHFQFMLFDNDLGRAQFLNPGGPVDRFLAGTLEETLTFVVDTLDRWSIDTFDFPSIIERQNMDDPEVLPHYPFRDDGMLIWDAVKEFITNYLSIYYKTPEDIREDYELQNWAKELAAFDSGRVKGMPETIESLQQLIDILSVVIFTCAPLHSNLNFTQYEYMIFVPNMPYAAYHPVPEQKGIDMETILKFLPPYKQAADQVYWTMVLTSYHHDKLGFYEDDFADPLAQDALVQFQQNLADIERKIEIENQHRPVPYQYFLPSEIINSINT

[0828] Coding sequence of WP_100898502.1 – SEQ ID NO:109

[0829]

[0830] Amino acid sequence of WP_100898502.1 – SEQ ID NO:110

[0831] MKPYLPQNDPNGNYRASWLDKNREEYNFNYDYLAPLPVIDKVPHKEIFSAEYTAKRLASMATLAPNMLAAKARNFLDPLDELEEYEELLALLPKPDVIKNYKTDSCFAEQRLSGANPLAIRRINVLPDNFAVTDYHFQKIAGAEFTLEKALKEGKLYFLDYPLLSDIQGGVYNNVKKYLPKPQALFYWQSNDSFNGGSLVPVAIQINHDSGANSLYTPDDPHLDWFLAKTCVQIADGNHQELGSHFSYTHAVMAPFAIVTARQLAENHPIALLLKPHFRFMLFDNDLGRTQFLQPGGPVDEFMAGSLAESVGFVAKTYEEWSVEKFTFPRLIKSRQTDDPEILPHFPFRDDGILIWNAIEKFVAEYLQLYYKTSQDLSDDYELQNWARELVAQDGGRVKGMPAKIETLEQLIEIISVVVFTCAPLHSALNFSQYEYMAFVPNMPYAAYHPIPETKGVDLETIMKILPPFKQAADQVMWTEILTSFHYDKLGFYDEEFADPLAQEIVVQFQHNLHQIERQIDIRNQTRPIPYNYLKPSQIINSINT

[0832] Coding sequence of RCJ35150.1 – SEQ ID NO:111

[0833]

[0834] Amino acid sequence of RCJ35150.1 – SEQ ID NO:112

[0835] MVKPYLPQKDPDVNVRINWLDKNREEYKFNYDYLAPLPVIDKVPHKEIFSAEYTAKRLASMATLAPNMLAAKARNFLDPLNELEEYEELLSLLPKPDVIKNYKTDSCFAEQRLSGANPLAIQKIDVLPDNFAVTDAHFQKVAGTEFTLEKALKEGKLYFLDYPLLSDIQGGIYENVKKYLPKPQALFYWQSNDSSNGGSLVPVAIQINHDSGAKSVIYTPDDPHLDWFLAKTCVQIADGNHQELGSHFAYTHAVMAPFAIVTARQLAENHPIALLLKPHFRFMLFDNDLGRTQFLQPGGPVDEFMAGSLAESLGFVAKVYEEWSVEKFTFPRLIKSRRTDDPEILPHFPFRDDGILIWNAVEKFVYEYLQLYYKTSQDLIDDYELQNWARELVAQDGGKVKGMPAKIETLEQLIEIISVVVFTCAPLHSALNFSQYEYMAFVPNMPYAAYHPIPETKGVDLETIMKILPPFKQAADQVMWTEILTSYHYDKLGFYDEEFADPLAQEIVVQFQQNLHEIERQIDIKNQTRPIPYNYFKPSQIINSINT

[0836] Coding sequence of WP_094352972.1 – SEQ ID NO:113

[0837]

[0838] Amino acid sequence of WP_094352972.1 – SEQ ID NO:114

[0839] MKPYLPQKDPDVNVRINWLDRNREEYKFNYDYLAPLPVIDKVPHKEIFSAEYTAKRLASMASLAPNMLAAKARNFLDPLDELEEYEELLSLLPKPDVIKNYKTDSCFAEQRLSGANPLAIQKIDVLPDNFAVTDAHFQKVAGTEFTLQKALKEGKLYFLDYPLLSDIKGGVYDNVKKYLPKPQALFYWQSNDSSNGGSLVPVAIQINHDSGGKSVIYTPDDPHLDWFLAKTCVQIADGNHQELGSHFAYTHAVMAPFAIVTARQLAENHPIALLLKPHFRFMLFDNDLGRTQFLQPGGPVDQFMAGSLAESLGFVAKVYEEWSVEKFTFPRLIKSRRTDNPEILPHFPFRDDGILIWNAVEKFVAEYLQLYYKTSQDISDDYELQNWARELVAQDGGKVKGMPAKIETLEQLIEIISVVVFTCAPLHSALNFSQYEYMAFVPNMPYAAYHPIPETKGVDLETIMKILPPFKQAADQVMWTEILTSYHYDKLGFYDEEFADSLAQEIVVQFQQNLHEIERQIDIRNQTRPIPYNYFKPSEIINSINT

[0840] Coding sequence of WP_104909167.1 – SEQ ID NO:115

[0841]

[0842] Amino acid sequence of WP_104909167.1 – SEQ ID NO:116

[0843] MKPYLPQKDPDVNVRINWLDKNREEYKFNYNYLAPLPIIDKVPHKEIFSAEYTAKRLASMATLAPNMLAAKARNFLDPLDELEEYEELLSLLPKPDVIKNYKTDSCFAEQRLSGANPLAIQRIDVLPDNFAVTDSHFQKVAGTKLTLEKALKEGKLYFLDYPLLSDIQGGVYDNIQKYLPKPQALFYWQSNDSSNGGSLVPVAIQINHDSGAKSVIYTPDDPHLDWFLAKTCVQIADGNHQELGSHFAYTHAVMAPFAIVTARQLAENHPIALLLKPHFRFMLFDNDLGRTQFLQPGGPVDEFMAGSLAESLGFVAKVYEEWSVEKFTFPRLIKSRRTDDPEILPHFPFRDDGILIWNAVEKFVAEYLQLYYKTSQELIDDYELQNWARELVAQDGGKVKGMPDKIETLEQLIEIISVVVFTCAPLHSALNFSQYEYMAFVPNMPYAAYHPIPEIKGVDLETIMKILPPFKQAADQVMWTEILTSYHYDKLGFYDEEFADPLAQEIVVQFQQNLHEIERQIDIRNQTRPIPYNYFKPSQIINSINT

[0844] Coding sequence of WP_106217928.1 – SEQ ID NO:117

[0845]

[0846] Amino acid sequence of WP_106217928.1 – SEQ ID NO:118

[0847] MNVIQPSSAQIERETRQFLPDRDQYKFDYDFLKPLALLQPVVPALPTPPGYPRVPGSSTFSPYYVFTRSSLPNTLDPFDGLQAFDDFFPAQGKPEVSKIYQSDRSFAEQRLSGVNPMVLHRIVQIPPQSSVTYEELQLACPHLRLDMALANGNIYVADYSGLGFVQGGTFKDLKKYLPTPVAFFYFDETQQELIPIAIQVQPKPGGAIFTPQDTPLDWLVAKMCVQIADANHHEMGAHLCWTHFVMEPFAISTPRQLAINHPVHLLLAPHLRFLLAINDQGRQLLVNPYVDGQVGGHVDRIMAGTLEESLEIVKHTYSEWSLDKFAFPQEIQNRGLEDANKLPHFPYRDDGLLLWNAIHKFVSGYLKYCYPTPADIQADRELQAWAQELASPDGGRVKGMPCSFSTVEQLIEVIANVIFTCGPQHAAVNYSQFDYMAYIPNMPHAAYVNITGKGMIPDEKALMKFLPPRDQAEAQIKIVTYLSFYRHDRLGYYDRAFNLTFRETPVKMMVQQFQQELNEIEQRIDTRNRQRFVPYPYLKPSLVPNSFSA

[0848] Coding sequence of WP_019498926.1 – SEQ ID NO:119

[0849]

[0850] Amino acid sequence of WP_019498926.1 – SEQ ID NO:120

[0851] MNAYNLDLDPTYIKYKTILTENRNEYEFDLSDRDLAPIPMLKGNLPRSENFSIDYLGRVAAPMAKLAANTLAVKLKSAWDPLDELQDYEDFFQVLEKPKVISTYQSDKAFAEQRLSGPNPLVLKRVDDLAQYFQSSDIAEIETKLGDSIDLTDNLYVADYTELLPIPSGTFDRGRTYLPRPIALFSWRSEASSDRGQLVPVAIKLDVPLKDKTILTPEDESLDWLYAKTCVQIADGNYHELMSHLCRTHFVMEPFAIATGQHLPETHHLGALLRQHFKFMLALSKFARKTLIASGGSIDRILAGELSGSLEIIRQAFRTWRFDSFSFPQAIAARGMDDAQKLPHYPYRDDGKLVWDAIWQFVSAYLGLHYHTADSISSDRALQDWAQKLHLVFSIAGGDGKGMPAQIDTLEQLVEVVTTIVFTCGPQHAAVNFPQYEYMTFAPNMPLSSYREFAGAAEFTQKDFMRFLPPSQQAAGQLSTTFLLSSFRYDRLGHYDPSFFEAFADGMQDKVKTVVTAFQQQLDVVEAEIDRRNQNRTVPYPYLKPSLIPNSISI

[0852] Coding sequence of WP_103124384.1 – SEQ ID NO:121

[0853]

[0854] Amino acid sequence of WP_103124384.1 – SEQ ID NO:122

[0855] MKPYLPQVDPNPNIRKDELVKNQADYKFNHNYLAPIPVIDKVPHQELFSAEYTAKRLASMANLAPNMLAAKARNFLDPLDELEEYEELLTLLPKPAVMNNYKTDSCFAEQRLSGANPLAIQRIENLPENIGVTNAHFQKAVGTESSLEAALKEGKLYLLDYPTLFDIKGGTSQNLRKYLPKPQALFYWQSNGLPNGGSLRPVAIKLNNDAGTDGLIYTPDDPYLDWFLAKTSVQIADGNHQELGSHFAYTHAVMAPFCIATARQLAANHPIALLLKPHFRFMLFDNDLGRTHFLQPGGPVDEFMAGSLQESLTFVVKTYQEWSVEKFVFPTLMRNQNMDDPEILPHFPFRDDGILIWDAIQKFVTDYLQLYYQTSQDLSEDYELQNWARELVAQDGGRVKGMPEKIETIDQLIQIITVVIFTCAPFHSALNFSQYEYMAFVPNMPYAAYHPTPEKKGVDMQTIMKILPPFKQAADQVMWTHILTSYHHDKLGYYDEEFSDPLAQELVMQFQQNLHDIERKIDIRNQTRPIPYNYLKPSQIINSINT

[0856] Coding sequence of BBD59026.1 – SEQ ID NO:123

[0857]

[0858] Amino acid sequence of BBD59026.1 – SEQ ID NO:124

[0859] MKPYLPQVDPNPNIRKDELVKNQTDYKFNHNYLAPIPVIDKVPHQELFSAEYTAKRLASMANLAPNMLAAKARNFLDPLDELEEYEELLTLLPKPAVMNNYKTDSCFAEQRLSGANPLAIQRIDSLPEKLGITNAHFQKSVGTESSLEAALKEGKLYLLDYPTLFDIKGGISQNLRKYLPKPQALFYWQSNGLPNGGSLRPVAIKLNNDPGTDGLIYTPDDPYLDWFLAKTSVQIADGNHQELGSHFAYTHAVMAPFCIATARQLAANHPIALLLKPHFRFMLFDNDLGRTHFLQPGGPVDEFMAGSLQESLTFVVKTYQEWSVEKFVFPTLMRNQNMDDPEILPHFPFRDDGILIWDAIQKFVTDYLQLYYQTSQDLSEDYELQNWARELVAQDGGRVKGMPEKIETVDQLIQIITVVIFTCAPFHSALNFSQYEYMAFVPNMPYAAYHPTPEKKGVDMQTIMKILPPFKQAADQVMWTHILTSYHHDKLGYYDEEFADPLAQELVVQFQQNLHDIERKIDIRNQTRPIPYDYLKPSQIINSINT

[0860] Coding sequence of WP_096579406.1 – SEQ ID NO:125

[0861]

[0862] Amino acid sequence of WP_096579406.1 – SEQ ID NO:126

[0863] MKPYLPQVDPNPNIRKDELFKNQTDYKFNHNYLAPIPVIDKVPHQELFSAEYTAKRLASMANLAPNMLAAKARNFLDPLDELEEYEELLTLLPKPAVMNNYKTDSCFAEQRLSGANPLAIQRIENLPENIGVTNAHFQKAVGTESSLEAALKEGKLYLLDYPTLFDIKGGISQNLRKYLPKPQALFYWQSNGLPNGGSLRPVAIKLNNDAGTDGLIYTPDDPYLDWFLAKTSVQIADGNHQELGSHFAYTHAVMAPFCIATARQLAANHPIALLLKPHFRFMLFDNDLGRTHFLQPGGPVDEFMAGSLQESLTFVVKTYQEWSVEKFVFPTLMKNQNMDDPEILPHFPFRDDGILIWDAIQKFVTEYLQLYYQTSQDLSEDYELQNWARELVAQDGGRVQGMPEKIEAVDQLIQIITVVIFTCAPFHSALNFSQYEYMAFVPNMPYAAYHPTPEKKGVDMQTIMKILPPFKQAADQVMWTHILTSYHHDKLGYYDEEFADPLAQELVVQFQQNLHDIERKIDIRNQTRPIPYNYLKPSQIINSINT

[0864] Coding sequence of WP_019504688.1 – SEQ ID NO:127

[0865]

[0866] Amino acid sequence of WP_019504688.1 – SEQ ID NO:128

[0867] MKNKSKTNVGEKMAIFSPALSEDELAQRTQYLKFQQQEYEFTHEYVEGLSLFKEVPVQEGFSTAYLADREFQLSAISINMLAVEPRPFLDPLETLGDYENFYKIIRKPGVANIYQTDRAFAEQRLSGVNPLVIKKFTEMPAGVDISLQDLGQETQVLFSSSATNLQAEIQRGHIFVADYTESLSFVEGGTYEKGRKYLPKPIAFFWWRKDGIKDRGELVPIAIAIELNTADKKWKILIPRDKDLHWTAAKLCVQIADANHHEMSTHLGRTHLVMEPFAVSTARQLAKNHPLGLLLRQHFRFMIAINDMARRELINPGGFVEAALAGTLPESLRIVKNACVSWNIKDFAFPTELKNRGMDEKDDRDNYKLPHYPYRDDGLMLWNAIEDFVTGYLKIFYPKPEDIQSDRELQQWAAELASADGGKVAKMPEKISDIEELIEIITTIIFICGPQHSAVNFPQYEYIGFIPNMPLAAYQEITGAEDQFKEERDLLQLLPPLKQTATQLLTMYNLSTYHYDRLGYYDEEFENTVKGTDIEPIVAKFKQDLNQIEVEIDNKNKDRTIPYPFLKPSLVLNSICI

[0868] Coding sequence of OCQ98836.1 – SEQ ID NO:129

[0869]

[0870] Amino acid sequence of OCQ98836.1 – SEQ ID NO:130

[0871] MKPYLPQVDPNPNIRKDELVKNREDYKFNHDYLAPIPVIDKVPHKELFSAEYTAKRLASMANLAPNMLAAKARNFLDPLDELEEYEELLTLLPKPAVMNNYKTDSCFAEQRLSGANPLAIRRIDSLPANLGITNAHFQKSVGTESNLEAALKEGKLYLLDYPTLFDIKGGTSQNVRKYLPKPQALFYWQSNGVANGGSLRPVAIKLNNDAGTDGLIYTPDDPYLDWFLAKTSVQIADGNHQELGSHFAYTHAVMAPFCIATARQLAANHPIALLLRPHFRFMLFDNDLGRTHFLQPGGPVDEFMAGSLEESLTFVVKTYQEWSVDKFVFPTLMKSQNMDDPDILPHFPFRDDGILIWNAIHKFVTDYLQLYYKTPQDLSEDYELQNWARELVAQDGGRVKGMPEKIETIDQLIQVITVIVFTCAPFHSALNFAQYEYMAFVPNMPYAAYHPTPESKGVDMQTIMKLLPPFKQAADQVMWTHILTSYHYDKLGYYDEEFADPLAQELVVQFQQNLHDIERQIDIRNQTRPIPYNFLKPSQIINSINT

[0872] Coding sequence of WP_062293357.1 – SEQ ID NO:131

[0873]

[0874] Amino acid sequence of WP_062293357.1 – SEQ ID NO:132

[0875] MKPYLPQVDPNPNIRKDELVKNREDYKFNHDYLAPIPVIDKVPHQELFSAEYTAKRLASMANLAPNMLAAKARNFLDPLDELEEYEELLTLLPKPAVMNNYKTDSCFAEQRLSGANPLAIRRIDSLPANLGITNAHFQKSVGTESNLEAALKEGKLYLLDYPALFDIKGGTSQNVRKYLPKPQALFYWQSNGVANGGSLHPVAIKLNNDAGTDGLIYTPDDPYLDWFLAKTSVQIADGNHQELGSHFAYTHAVMAPFCIATARQLAANHPIALLLKPHFRFMLFDNDLGRTHFLQPGGPVDEFMAGSLEESLTFVVKTYQEWSVDKFVFPTLMKSQNMDDPDVLPHFPFRDDGMLIWNAIHKFVTDYLQLYYKTSQDLSEDYELQNWARELVAQDGGRVKGMPDKIETIDQLIQIITVVVFTCAPFHSALNFSQYEYMAFVPNMPYAAYHPTPESKGVDMQTIMKILPPFKQAADQVMWTHILTSYHYDKLGYYDEEFADPLAQELVVQFQQNLHDIERKIDIRNQTRPIPYNFLKPSQIINSINT

[0876] Coding sequence of WP_104398120.1 – SEQ ID NO:133

[0877]

[0878] Amino acid sequence of WP_104398120.1 – SEQ ID NO:134

[0879] MLTPSLPKNDSDPVKRQDLLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRIENVFDPFDKLEDYEELFPILPKPTSIKTWQSNTGFAYQRLAGANPMVIRGISSLPNNFPVSDTIFQKAMGPDKTIASEAAKGNLFLADYAPLNNLTLGSYQRGMKAVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTQPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKELALRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNGDTELQAWARKLMSPEGGGIKKLVFDGQLDTLAKLVEVVTQIIFVAGPQHAAVNYPQYDYLAFCPNIPLAGYQSPPKAAEEVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRTYPILAVFQAKLKAIENQIDRRNLTRFTPYIFLKPSRIPNSINI

[0880] Coding sequence of WP_002758835.1 – SEQ ID NO:135

[0881]

[0882] Amino acid sequence of WP_002758835.1 – SEQ ID NO:136

[0883] MLTPSLPKNDPDPVKRQDLLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRVENIFDPFDTLEDYEELFPILPKPTSIKTWQSNTGFAYQRLAGANPLVIRGISSLPDNFPVSDAIFQKAMGPDKTIDSEAAKGNLFLADYAPLNNLTLGSYQKGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTQPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLDTATELAINHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIEIIKTSYRQRLDNFADYTLPKQLAFRQVDDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNGDTELQAWVRKLMSPEGGGIKKLVSDGELDTLAKLVEVVTQIIFVAGPQHAAVNYPQYDYLAFCPNIPLAGYQSPPKAAEEVDIDYILRLLPPQSQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRTYPILAVFQAKLKAIENEIDRRNLTRFTPYIFLKPSRIPNSINI

[0884] Coding sequence of WP_072927101.1 – SEQ ID NO:137

[0885]

[0886] Amino acid sequence of WP_072927101.1 – SEQ ID NO:138

[0887] MLTPSLPQNDPDPAKRQELLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRIENVFDPFDKLEDYEELFPILPQPTSIKTWQSNTSFAYQRLAGANPMVIRGISSLPDNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLNNLTLGSYQKGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTQPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKQLELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWVRTLMSPEGGGIKKLVSEGELDTLAKLIEVVTQIIFVAGPQHAAVNYPQYDYLAFCPNIPLAGYQSPPKAAEQVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRTYPILAVFQAKLKAIENQIDRRNLTRFTPYIFLKPSRIPNSINI

[0888] Coding sequence of WP_110578596.1 – SEQ ID NO:139

[0889]

[0890] Amino acid sequence of WP_110578596.1 – SEQ ID NO:140

[0891] MLTPSLPKNDPDPVKRQDLLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRIENVFDPFDKLEDYEELFPILPKPTSIKTWQSNTGFAYQRLAGANPMVIRGISSLPDNFPVSDAIFQKAMGPDKTIASEAARGNLFLADYAPLNNLTLGNYQRGMKAVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTQPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKQLELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWVRKLMSPEGGGIKKLVSDGQLDTLAKLIEVVTQIIFVAGPQHAAVNYSQYDYLAFCPNIPLAGYQSPPKAAEEVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRTYPILAVFQAKLKAIENEIDRRNLTRFTPYIFLKPSRIPNSINI

[0892] Coding sequence of WP_045360762.1 – SEQ ID NO:141

[0893]

[0894] Amino acid sequence of WP_045360762.1 – SEQ ID NO:142

[0895] MLTPSLPQNDPDPAKRQDLLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRVENIFDPFDKLEDYEELFPLLPQPTSIKNWQSNTSFAYQRLAGANPMVIRGISSLPDNFPVTDAIFQKAMGPDKTIASEAAKGNLFLADYATLHHLTLGSYQRGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTLPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLTHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINTLAESELISPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKQLELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNGDTELQAWVRKLMSPEGGGIKKLVSDGQLDTLAKLIEVVTQIIFVAGPQHAAVNYPQYDYLAFCPNIPLAGYQSPPKAAEEVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRTYPILAVFQAKLKAIENEIDRRNLTRFTPYIFLKPSRIPNSINI

[0896] Coding sequence of REJ48186.1 – SEQ ID NO:143

[0897]

[0898] Amino acid sequence of REJ48186.1 – SEQ ID NO:144

[0899] MLTPSLPKNDPDPVKRQELLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRVENIFDPFDKLEDYEELFPILPQPTSIKTWQSNTSFAYQRLAGANPMVIRGISSLPNNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLHHLTLGSYQRGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTLPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKQLELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWARKLMSSEGGGIKKLVSDGELDTLAKLVEVVTQIIFVAGPQHAAVNYPQYDYLAFSPNIPLAGYQSPPKAAEEVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRAYPILAVFQAKLKAIENEIDRRNLTRFTPYIFLKPSRIPNSINI

[0900] Coding sequence of REJ50596.1 – SEQ ID NO:145

[0901]

[0902] Amino acid sequence of REJ50596.1 – SEQ ID NO:146

[0903] MLTPSLPQNDPDPAKRQDLLRRQKQVYVYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRVENIFDPFDKLEDYEELFPILPKPTSIKTWQSNTSFAYQRLAGANPMVIRGISSLPDNFPVTDAIFQKAMGPDKTIASEAAKGNLFLADYAPLHHLTLGSYQKGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTQPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIEIIKTSYRQRLDNFADYTLPKQLAFRQVDDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNKDTELQAWVRKLMSPEGGGIKKLVSDGKLDTLAKLIEVVTQIIFIAGPQHAAVNYSQYDYLAFCANIPLAGYQSPPKASEEVDMDYILRLLPPQAQATYQLEIMHTLTAFQFNRFGYPSRNDFPDQRTYPILAVFQAKLKAIENEIDRRNSTRITPYIFLKPSRIPNSINI

[0904] Coding sequence of WP_041804209.1 – SEQ ID NO:147

[0905]

[0906] Amino acid sequence of WP_041804209.1 – SEQ ID NO:148

[0907] MLTPSLPKNDPDPVKRQDLLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRVENIFDPFDKLEDYEELFPILPQPTSIKTWQSNTSFAYQRLAGANPMVIRGISSLPNNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLHHLTLGSYQRGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTLPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKQLELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWARKLMSSEGGGIKKLVSDGELDTLAKLVEVVTQIIFVAGPQHAAVNYPQYDYLAFSPNIPLAGYQSPPKAAEEVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRAYPILAVFQAKLKAIENEIDRRNLTRFTPYIFLKPSRIPNSINI

[0908] Coding sequence of WP_004162848.1 – SEQ ID NO:149

[0909]

[0910] Amino acid sequence of WP_004162848.1 – SEQ ID NO:150

[0911] MLTPSLPKNDPDPVKRQDLLRRQKQVYIYDSVNGITLVKDLPPHENFSISYQVMRGKGFSALIANGVATRVENIFDPFDKLEDYEELFPILPQPTSIKTWQSNTSFAYQRLAGANPMVIRGISSLPNNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLHHLTLGSYQRGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTLPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKQLELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWARKLMSSEGGGIKKLVSDGELDTLAKLVEVVTQIIFVAGPQHAAVNYPQYDYLAFSPNIPLAGYQSPPKAAEEVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRAYPILAVFQAKLKAIENEIDRRNLTRFTPYIFLKPSRIPNSINI

[0912] Coding sequence of BAG04096.1 – SEQ ID NO:151

[0913]

[0914] Amino acid sequence of BAG04096.1 – SEQ ID NO:152

[0915] MYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRVENIFDPFDKLEDYEELFPILPQPTSIKTWQSNTSFAYQRLAGANPMVIRGISSLPNNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLHHLTLGSYQRGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTLPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKQLELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWARKLMSSEGGGIKKLVSDGELDTLAKLVEVVTQIIFVAGPQHAAVNYPQYDYLAFSPNIPLAGYQSPPKAAEEVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRAYPILAVFQAKLKAIENEIDRRNLTRFTPYIFLKPSRIPNSINI

[0916] Coding sequence of WP_002786802.1 – SEQ ID NO:153

[0917]

[0918] Amino acid sequence of WP_002786802.1 – SEQ ID NO:154

[0919] MLTPSLPQNDPDPAKRQELLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRIENVFDPFDKLEDYEELFPILPQPTSIKTWQSNTSFAYQRLAGANPMVIRGISSLPDNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLNNLTLGSYQRGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQEPTLPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYALPKQLELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWVRTLMSPEGGGIKKLVSEGELDTLAKLIEVVTQIIFVAGPQHAAVNYPQYDYLAFCPNIPLAGYQSPPKAAEQVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRTYPILAVFQAKLKAIENQIDRRNLTRFTPYIFLKPSRIPNSINI

[0920] Coding sequence of WP_002800102.1 – SEQ ID NO:155

[0921]

[0922] Amino acid sequence of WP_002800102.1 – SEQ ID NO:156

[0923] MIPSLPQNDADSIKRQELLQRQKQVYIYDSVSGITLVKDLPAQENFSISYQLMLRKGLSALIANSVATKIENVFDPFDKLEDYEQLFPLLPKPTSIKTWQSNTSFAYQRLAGLNPMVIRGISSIPDNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLNNLTLGSYQRGMKTATAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTVPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLKPHFQFTLAINTLAESELISPGGFVDRLLAGTLEASIEIIKTSYRQRLDNFADYTLPKQLAFRQVDDTSRLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWVRKLMSPEGGGIKKLVSDGKLDTLAKLIEVVTQIIFIAGPQHAAVNYSQYDYLAFCANIPLAGYQSPPKASEEVDMDYILRLLPPQAQATYQLEIMHTLTAFQFNRFGYPSRNDFPDQRTYPILAVFQAKLKAIENEIDRRNSTRITPYIFLKPSRIPNSINI

[0924] Coding sequence of WP_002793167.1 – SEQ ID NO:157

[0925]

[0926] Amino acid sequence of WP_002793167.1 – SEQ ID NO:158

[0927] MIPSLPQNDADSIKRQELLQRQKQVYIYDYVSGITLVKDLPAQENFSISYQLMLRKGLSALIANGVATRIENVFDPFDKLEDYEQLFPILPKPTSIKTWQSNTGFAYQRLAGTNPMVIRGISSLPDNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLNNLTLGSYQRGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTLPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLSHTHLVAEAFVLATATELALNHPLAILLKPHFQFTLAINTLAENELISPGGFVDRLLAGTLEASIEIIKTSYRQRLDNFADYTLPKQLAFRQVDDTSLLPDYPYRDDALLLWEATETYVKDYLSLYYTSDADVNEDTELQAWVRKLMSPEGGGIKKLVSDGKLDTLAKLIEVVTQIIFIAGPQHAAVNYSQYDYLAFCANIPLAGYQSPPKAAEEVDMDYILRLLPPQAQATYQLEIMHTLTAFQFNRFGYPSRNDFPDQRTYPILAVFQAKLKAIENEIDRRNSTRITPYIFLKPSRIPNSINI

[0928] Coding sequence of WP_061431977.1 – SEQ ID NO:159

[0929]

[0930] Amino acid sequence of WP_061431977.1 – SEQ ID NO:160

[0931] MMIPSLPKNDPDPVKRQDLLRRQKQVYIYDSVNGITLVKDLPTHENFSISYQVMRGKGFSALIANGVATRVENIFDPFDKLEDYEQLFPILPQPTSIKTWQSNTSFAYQRLAGANPMVIRGISSLPNNFPVSDAIFQKAMGPDKTIASEAAKGNLFLADYAPLHHLTLGSYQRGMKTVTAPLVLFCWRARGLRGQGGLVPVAIQLYQDPTLPNQRIYTPDDGLNWLMAKIFVQIADGNHHELVSHLTHTHLVAEAFVLATATELALNHPLAILLRPHFQFTLAINSLAESELINPGGFVDRLLAGTLEASIELIKSSYRQRLDNFADYTLPKELELRQVQDTSLLPDYPYRDDALLLWQATETYVKDYLSLYYTSDADVNEDTELQAWVRTLMSPEGGGIKKLVSDGELDTLAKLVEVVTQIIFVAGPQHAAVNYPQYDYLAFSPNIPLAGYQSPPKAAEEVDIDYILRLLPPQAQAAYQLEIMQTLTAFQFNRFGYPSRSAFPDQRAYPILAVFQAKLKAIENEIDRRNLTRFTPYIFLKPSRIPNSINI

[0932] Coding sequence of OUS02327.1 – SEQ ID NO:161

[0933]

[0934] Amino acid sequence of OUS02327.1 – SEQ ID NO:162

[0935] MVGHDGPKYAHESNQPSLPQNDTPAEQEARRTALGLTQEKYHLSNDNDLGLPLLKEVPAEEAFSNIYEAGRAIDTFPLLENHDKVMSQLTNPYGPFTGLADYESMFIDIPKPAVTKNWLTDESFGEQRLSGVNPVMIERVKNAKDLASKFNVSQLKDVLDSDINLDELIKDELLYITDLSPYLKDIPEGKVPSPGGYIPKYLPKPIGLFYWHKDGAKLKDPSLKSGRLLPLAIQVDLEGDQVKILTPKSPELLWTIAKMCFSIADVNVHEMSTHLGRAHFAQESFGAITPCQLAPKHPLAILLKPHLRFLVANNQAGIEKLVNTGGPVDMLLASTLQGSLDISTTAAKSWSVTETFPESIQARNVASEESLPHYPYRDDGILIWDAVVGYVNEYVNIYYKNEEDVVKDYELQAWAKNLADTGVHGGNIKDMPSQIESIKQLSQLLSVIIFHNSAGHSSINYPQYPCIGFCPNMPLAGYSNYREFLAKEKTTQEEQLTFLLSFAPPQALALGQIDITNSLSIYHYDTLGDYAKELTDPLAKHALYCFTQKLTAIEQQIEVRNSQRAEPYKYMLPSEILNSASI

[0936] Coding sequence of WP_106300061.1 – SEQ ID NO:163

[0937]

[0938] Amino acid sequence of WP_106300061.1 – SEQ ID NO:164

[0939] MLQPSLPQDDTLDRQQQRNQAIAQQREDYQYSQTAGILLIKELPQSEMFSLKYLLERDAGLVSLIANTLASSIENVFDPFDKLEDYQEMFPLLPKPSVWETFRNDAVFARQRIAGANPMVIERVIDKLPDNFPVTDAIFQKIMLTKKTLAEAIAEGRIFLTNYQGLDGLKPGGYQYERDGQQVKVTKTIAAPLVLYCWKPTGYGDYRGNLAPIAIQINQQPDPIANPIYTPRDGRHWLMAKIFAQMADGNYHEAISHLGRTHLVLEPFVLATANELAPNHPLSVLLKPHFQFTLAINELAREQLISPGGYADDLLAGTLEASIGVIKAAIKEYLENFTEFAIPKELTRRGVGETDVDGSGENFLPDYPYRDDALLLWNAIKVYVSDYLNLYYTSSAKIIGDPELQNWAKKLISPEGGNVTGLVPNGQLTTLEQLVEIVTQLIFVSGPQHGAVNYPQYDYMAFVPNIPLATYGNPPSRDVEINEETILNILPPQKLAAKQLELMRTLSVFRANRLGYPDREFVDVRARGVLQKFQARLQEIEQEISVRNETRLEPYLFLLPSNVPNSLNI

[0940] Coding sequence of WP_099065794.1 – SEQ ID NO:165

[0941]

[0942] Amino acid sequence of WP_099065794.1 – SEQ ID NO:166

[0943] MTQPSLPQDDSPEQQLQRKQEIARQREDYQYSETAGILLIKELPQSEMFSFKYLLERDKSLISLIANTLATNIDNVFDPFDSLEDYQQMFPLLPKPSTLQTFRNDGVFARQRIAGANPMVIERVVGKLPDNFAVTDAIFQKIMLTQKTLAQAIAEGRIFITNYQGLDGLTPGTYEQGTKTIAAPLVLYCWKPVGYGDYRGSLTPIAIQLNQQPHPENNPIYTPMDGMHWFMAKIYAQMADGNYHEAISHLGRTHLVLEPFVLATANELAPNHPLSVLLKPHFQFTLAINELAREQLISPGGYADTLLAGTLEASISVIKAAIKEYLENFSDFALPKELTRRGVGETDVDGQGENFLPDYPYRDDGLLLWKAIEAYVSNYLDLYYTSPVQIIKDTELQNWVQKLISPEGGGVKGLVPNGQLQTVEQLVAIATQLIFISGPQHGAVNYPQYDYLAFVPNMPLATYAPPPSRDREINEATILKILPPQKLAAKQLELMRTLTVFQPNRLGYPDKNFVDVRAQNVLRQFQAKLQEVEQVINQRNQTRLEPYTFLLPSNVPNSLNI

[0944] Coding sequence of WP_012596348.1 – SEQ ID NO:167

[0945]

[0946] Amino acid sequence of WP_012596348.1 – SEQ ID NO:168

[0947] MVQPSLPQDDTPDQQEQRNRAIAQQREAYQYSETAGILLIKTLPQSEMFSLKYLIERDKGLVSLIANTLASNIENIFDPFDKLEDFEEMFPLLPKPLVMNTFRNDRVFARQRIAGPNPMVIERVVDKLPDNFPVTDAMFQKIMFTKKTLAEAIAQGKLFITNYKGLAELSPGRYEYQKNGTLVQKTKTIAAPLVLYAWKPEGFGDYRGSLAPIAIQINQQPDPITNPIYTPRDGKHWFIAKIFAQMADGNCHEAISHLARTHLILEPFVLATANELAPNHPLSVLLKPHFQFTLAINELAREQLISAGGYADDLLAGTLEASIAVIKAAIKEYMDNFTEFALPRELARRGVGIGDVDQRGENFLPDYPYRDDAMLLWNAIEVYVRDYLSLYYQSPVQIRQDTELQNWVRRLVSPEGGRVTGLVSNGELNTIEALVAIATQVIFVSGPQHAAVNYPQYDYMAFIPNMPLATYATPPNKESNISEATILNILPPQKLAARQLELMRTLCVFYPNRLGYPDTEFVDVRAQQVLHQFQERLQEIEQRIVLCNEKRLEPYTYLLPSNVPNSTSI

[0948] Coding sequence of WP_036533591.1 – SEQ ID NO:169

[0949]

[0950] Amino acid sequence of WP_036533591.1 – SEQ ID NO:170

[0951] MLPPSLPQDDTPDQQLQRNQAIAQQREDYQYSQTAGILLIKTLPQSEMFSFKYLLERDKGLVSLIVNTLASKIENIFDPFEKLEDYQEMFPLLPKPSVLETFRHDAVFARQRIAGANPMVIERVISKLPDNFPVTDAMFQKIMSTKKTLAEAIAEGRLFLTNYKGLDGLTPGHYERGTKTIAAPLVLYCWKPTGYGDYRGNLAPIAIQINQKPDPIINPIYTPRDGMHWFMAKIFAQMADGNYHEAISHLGRTHLVLEPFVLATANELAPNHPLSILLKPHFQFTLAINELAREQLISKGGYADTLLAGTLEASISVIKAAIQEYFENFTEFAVPKELTRRGIGETDLDAQGENFLPDYPYRDDALLLWDAIKNYVRDYLNLYYTSQDKILKDTELKNWVSKLISPEGGNVKGLVPNGELTTLDQLVEIATQLIFVSGPQHAAVNYPQYDYMAFVPNMPLATYAPPSSDPTIDETTILKILPPQKLAAKQLELMKTLSVFRANRLGYPDNEFVDVRAQNVLIKFQGNLKKVEDKITARNETRLEPYVFLLPSNVPNSTNI

[0952] Coding sequence of WP_015784471.1 – SEQ ID NO:171

[0953]

[0954] Amino acid sequence of WP_015784471.1 – SEQ ID NO:172

[0955] MVQPSLPQDDTPDQQEQRNRAIAQQREAYQYSETAGILLIKTLPQSEMFSLKYLIERDKGLVSLIANTLASNIENIFDPFDKLEDFEEMFPLLPKPLVMNTFRNDRVFARQRIAGPNPMVIERVVDKLPDNFPVMDAMFQKIMFTKKTLAEAIAQGKLFITNYKGLAELSPGRYEYQKNGTLVQKTKTIAAPLVLYAWKPEGFGDYRGSLAPIAIQINQQPDPITNPIYTPRDGKHWFIAKIFAQMADGNCHEAISHLARTHLILEPFVLAMANELAPNHPLSVLLKPHFQFTLAINELAREQLISAGGYADALLAGTLEASIAVIKAAIKEYMDNFTEFALPRELARRGVGVADVDQTGENFLPDYPYRDDAMLLWNAIEVYVRDYLSLYYQSPVQIRQDTELQNWVRRLVSPEGGSVTGLVPNGELNTIEQLVAIATQVIFVSGPQHAAVNYPQYDYMAFIPNMPLATYATPPHKDSNISEATILNILPPQKLAARQLELMRTLCVFYPNRLGYPDTEFVDVRAQRVLHQFQERLQEIEQRIVLCNEKRLEPYTYLLPSNVPNSTSI

[0956] Coding sequence of WP_094531790.1 – SEQ ID NO:173

[0957]

[0958] Amino acid sequence of WP_094531790.1 – SEQ ID NO:174

[0959] MIFSLLSGVARILNFVAAKLVDLADWISRRSPSSKYPLLPQNDPEINQRQAFLNNARQLYQYNYTYIDSLPMVETVPTIERFSLSWGLLVGKAVVTVLLNERANLSLEKDKLASQAKQREFSKRLLEAGMSHSDTAILDLLDELPTVLETPPSDLEGVNIEEYNNLFWVIPLPTISQNYISNTEFARLRVAGFNPLVIQRVKALDARFPLTEEQFQTVLPNDSLALAGAEGRLYLADYAELEAIAGGTFPTGEQKYVNAPLALFAIPQGERSLTPIAIQLGQDPNINPIFLRRVGDEPNWLIAKTVVQIADANHHQLISHLGRTHLFVEPFVIATNRQLASNHPLYILLKPHFQGTLAINDAAQSNLVSVGGGVDSLLAGTIASSRAVSVHGVKSYQFEDALLPNALKKRGVDDPSLLPDYPYRDDALLIWEAIATWVKSYLSIYYFNDDAVVRDTELQAWAKEIIANDGGRVTSFGENGQIRTLSYLADALTAVIFTGSAQHAAVNFPQGDLIVYTPAIPLAGYTPAPTQTTGAEEADFFAMLPPIEQAKGQLKLTYILGSVYYTTLGDYGTDYFSDDRIQQPLRDFQDLLKEIESTIKSRNEQRVADYNYLRPSRIPQSINI

[0960] Coding sequence of PZO42668.1 – SEQ ID NO:175

[0961]

[0962] Amino acid sequence of PZO42668.1 – SEQ ID NO:176

[0963] MVFSLLSGVAKTLNFVASKLKDLADWISRRSPSSKYPLLPQNDPEIKQRQSFLDNARQLYQYNYTYIDSLPLVETVPTNERFSLSWGLLVGKAAIKVLLNERANPLLLEAGKQTSKAKQQDFSKRLLEASVAQSESALLELLEDLPTVLETPPSELEGVNIEEYNNLFWVIPLPSISQNYTSNKEFARLRVAGFNPLVIQRITALDARFPLTEAQFQKVLPNDSLAVAGAEGRLYLADYAELEAIVGGTFPTGEQKYINAPLALFAIPQGEKSLTPIAIQLGQDPNTHPIFLHQVGDEPNWLIAKTVVQIADANHHQLISHLGRTHLFVEPFVIATNRQLASNHPLYILLKPHFQGTLAINDAAQSKLVSAGGGVDSLLAGTIESARAVSVHGVKTYKFEDALLPKALKKRGVDDPNLLPDYPYRDDALLVWEAIATWVKNYLSIYYFNDEDVIRDTELQAWAKEIIANDGGRATSFGENGQIRTLSYLADALTAVIFTGSAQHAAVNFPQGDLIVYTPAIPLAGYTPAPTQTTGATEADFFSLLPPIEQAKGQLKLTYILGSVYYTTLGEYGDGYFTDDRIEKPLRDFQDNLKAIESEIKSRNEKRVADYNYLKPSRIPQSINI

[0964] Coding sequence of WP_106893977.1 – SEQ ID NO:177

[0965]

[0966] Amino acid sequence of WP_106893977.1 – SEQ ID NO:178

[0967] MSLFSRVRPTLPQNDSPAAQQQRQEALLDEQSKYVWKDDFETLPGIPLAASVPRDDRPTITWLLEVADVGIDIVANQILAQTGRGDSLKSQTAAAAIRPHLDSMRQTIATIRSEQKATPDSPLRIVDHVAGTLLSLHRSRLDNELKTLQNMIAATYLGKLENPSLEQYRKLFVTLPLPAIADTFMDDATFARMRVAGPNSVLIAGLSAWPLKFGLSEAQYQSVMGTNDSLASALTEQRLYWLDYEELSTLKTGTTGGKPKFLCAPLALFAIPKGGGALTPVAIQLGQSPADGLFLRVSDQNSPDWWSWQMAKTFVQAAEGNYHELFVHLARTHLVIEAFAVATHRRLAPEHPLNVLLLPHFEGTLFINNSAAGSLIAEGGPIDHIFAGQITSTQTLAGSDRLAFDVTAHMLPNDLASRRVADVAALPDYPYRDDALLVWQAIQDWVRQYVSVYYLNDANVAGDTELQGWRDELLGLGKIKGLPELKDRETLISVVTMVIFTASAQHAAVNFPQKDLMSFAPAISGAAWAPVPKPDQPQSEAAWLKLLPPIKEAQEQLNVLWLLGSVHYRPLGDYRVNHWPYLPWFQDPRITGKNGPLARFKLALKAVEMEIDNRNAEREVPYPYLQPSLIPTSINI

[0968] Coding sequence of BBC22503.1 – SEQ ID NO:179

[0969]

[0970] Amino acid sequence of BBC22503.1 – SEQ ID NO:180

[0971] MIFSILSGVARILNFLSDKLANLANLISKPSKSSNYPLLPQNDPEISQRQALLNKSRQLYQYNYTYIDSLPMVEKVPTSERFSLSWGLLVGKVVVKVLLNDRANPAAFIDKEKSKAKQLEFSKKLLEASMAKSDTALVELLSNLPAILEDDPIDVAGSNIQEYNELFWIIPLPTISQSLFSNTEFARLRVAGFNPLMIQRVTSLDARFPVTEAQFQSVLADDSLAAAGAEGRLYLADYAELEALTGGTFPKGKQKYINAPLALFAVPKGKKSLTPIAIQLGQDPNTHPIFVSQHGDEPNWLIAKTVVQIADANYHQLISHLGRTHLFIEPFAIATNRQLANNHPLYILLKPHFQGTLAINDAAQSGLVSAGGTVDSLLAGTIDTARALSVHGVKTYNFDEAMLPVALKKRGVDDPKLLPEYPYRDDALLVWEAIATWVKNYLSVYYENDNDVARDSELQAWVKEITANDGGRVTSFGQNGQIRTLSYLVDAVTLLIFTSSAQHAAVNFPQGDLMDYAPAVPLAGYTPAPTSTTGATIDNFWSMIPAIDQAKSQLTMTYILGSVYYTTLGDYGNAYFTDDRIEQPLRDFQDNLKAIESTIKSRNEQRNVDYSYLRPSRIPQSINI

[0972] Coding sequence of WP_055077131.1 – SEQ ID NO:181

[0973]

[0974] Amino acid sequence of WP_055077131.1 – SEQ ID NO:182

[0975] MISSILRGIAQILNFLATKLSDLANLILRRSPSSKYPLLPQNDPEIDRRQALLNQSRQLYQYNYTYVAPLPMVEKVPTGEQFSLSWGLLVGKAVIEILLNDIANPFLLSEKGKNASKARQQDFSKRLLEAGVAQSNSAIIGLLSEIPTLLETEPTNVEGSNIKEYNDLFWIISLPKISQNFTTNSEFARLRVAGFNPVTIQRIKTLDAKFPLTEDQFQTVLAGDSLAEAGAQGRLYLADYAELTAIAGGTFPKGAQKYINAPLALFAVPKGQQSLTPIAIQLGQDPSAYPIFVCQADDEPNWLLAKTVVQIADANYHELISHLGRTHLFIEPFAIATNRQLASNHPLYILLKPHFQGTLAINDAAQSGLISAGGTVDSLLAGTIASSRTLSAQSVENYNFNEAMLPVALKKRGVDDVNMLPDYPYRDDALLVWGAIATWVKNYLSIYYFSDTDVMRDVELQAWAKEITSIDGGRVKSFGQNGQIQTFDYLVDAVTLLIFTSSAQHAAVNFPQGDLMDYTPAIPLAGYTPAPTATTGATEADFFAMLPPIDQAKSQLTMTYILGSVYYTTLGDYGSDYFNDDRLQQPLRDFQDGLKAIESTIKSRNETRAADYNYLKPSRIPQSINI

[0976] Coding sequence of WP_009629598.1 – SEQ ID NO:183

[0977]

[0978] Amino acid sequence of WP_009629598.1 – SEQ ID NO:184

[0979] MISSILRGIAQILNFLATKLSDLASLILRRSPSSKYPLLPQNDPEIDQRQALLNQSRQLYQYNYTYVAPLPMVEKVPTSEQFSLSWGLLVGKAAIEVLLNDIANPFLLSEKGKNASKAREQDFSKRLLEAGIAQSNSAIIGLLSEIPSLLETEPTNVEGSNIKEYNDLFWIISLPTISQSFTTNSEFARLRVAGFNPVTIQRIKTLDAKFPLTEDQFQTVLAGDSLAEAGAQGRLYLADYVDLTAIAGGTFPKGAQKYINAPLALFAVPKGQQSLTPIAIQLGQDPSAYPIFVCQADDEPNWLLAKTVVQIADANYHELISHLGRTHLFIEPFAIATNRQLASNHPLYILLKPHFQGTLAINNAAQSGLISAGGTVDSLLAGTIASSRTLSVQSVKNYNFNEAMLPVALKKRGVDDVNMLPDYPYRDDALLVWGAIATWVKNYLSIYYFSDTDVLRDSELQAWAKEITSVDGGRVTSFGQDGQIQTFDYLVDAVTLLIFTSSAQHAAVNFPQGDLMDYTPAIPLAGYTPAPKSTTGATEADFFAMLPPIDQAKSQLTMTYILGSVYYTTLGDYGSDYFNDDRLQQPLRDFQDGLKAIESTIKSRNETRVADYNYLKPSRIPQSINI

[0980] Coding sequence of WP_015133151.1 – SEQ ID NO:185

[0981]

[0982] Amino acid sequence of WP_015133151.1 – SEQ ID NO:186

[0983] MTATSPSSSQNLSDKQEKYQYNYRYMPPLAMVDSLPEEEQWSTSWKMTVGKVGFQLLVNKIILNYGDQGEAGAADDVRAFLISTFKQTLAEQKGFSKVGILLQGAKFLPRLIWGKITTQIVDVEDLMKEMIESMSRKFLEDFAANVMQKLTEDAPKGRFSSIKEFETLFTEIDLPDIAYTYQEDETFAYMRVAGPNAVMLQKITEPDPRFPVTEAHYQAVMGEEDSLAAARSEGRLYLCDYAILDGAIEGDFPVAQKYLYAPLALFAVPKADAVKRNLMPVAIQLGQVPKQNPILTPKSNKYAWLCAKTAVQIADANFHEAVTHLARTHLFMGPFAIATHRQLPESHPLFKLLKPHFFGMLAINDSAQAKLIAKGGGVNKILSATIDNARLFAILGVQTYGFNSAMLRKQLAARGVDDTEGLPIYPYRDDALLIWDAINNWVQSYLKTYYANDAAVRRDQAIQAWVKELISEDGGRVVEFGEDGGIQTLEYLIEAVTLIIFTVSAQHAAVNFPQKNLMSFAPGMPTAGYSPLDNLGEHTTEQDYLDLLPPMSQAQEQLKLCHLLGSAHFTELGQYDAKHFTDFKIQGALKQFQARLKEIEGIIHKRNRDRPEYEYLLPSLIPQSINI

[0984] Coding sequence of WP_063872765.1 – SEQ ID NO:187

[0985]

[0986] Amino acid sequence of WP_063872765.1 – SEQ ID NO:188

[0987] MTTSSPDNSRSLPITQNLELARQEYQYNYTHIPPIPMVNQLPNQENFTTRWTFLLAQQLREIFINTLITNRGDRSSKSVRDQVKRFILEALFKGAIPAKVSVIARLFQIIPQFLIQGISKDFHELDDLFFSLFKTNGLLIFRDSLNRITALLDKGHPTGHVNSLKDYQKLFTTIELPAIAKTFDQDQVFAYMQVAGYNPLVIKRVNSPGANFPVEETHYQAVMGSDDSLAAAGQEGRLYLADYQILDGAINGTYLNYQKYAYAPLALFAIPKNSDPNRLLRPIAIQCGQTPGADYPIITPNSGKYAWLFAKTIVHIADGNFHEAVSHLARTHLFVGVFVIATHRQLSPSHPLSLLLRPHFEGTLAINNAAQEVLIAPGGGVDRLLSSTIDNSRILAVRGLQSYSFNEAMLPNQLKQRGVDDPELLPVYPYRDDALLIWNAIHQWVSDYLSLYYPTDKDIQNDTALQAWAAEAKAENGGRVPDFGENGGIQTLDYLVDAATLIIFTASAQHAAVNFPQKDLMSYAPAFPLAGYVSASIKGEVSEQDYLNLLPPLEQAQQQFNLLTLLGSIYYNQLGEYPKSHFANPKVQTLLQKFQSQLQQIEITINQRNLHRPTYEYLLPSKIPQSINI

[0988] Coding sequence of WP_096687527.1 – SEQ ID NO:189

[0989]

[0990] Amino acid sequence of WP_096687527.1 – SEQ ID NO:190

[0991] MRSPTPKQRRQELIEQYVLSRRTMMALMAFACTPGLETLLVGDNKSSKPKQLDNPNGCTPGLETLLSNDNKPSKPKPPNNPSIPSLPQNDTKATQQERLTQLGKTREEYQLGLRLPNSARVKTLPATELFSEGYEKNRVILSQKIGANQQAFLQNPKPFQSFDDYSALFPVLPLPDIAKTFRNDSVFARQRLSGCNPMELKNVLALDYNLRSKLAITDEIFQAVLNATRTRERINKTLNSAIREGSLFVTDYAILDSIQPKEKQFVCAPIALYYAQRIRGDFQLIPIAIQLGQAPGSSLLCTPNDGVDWTLAKLITQMADFYVNQLYRHLGQTHLVMEPIALATARELAAKHPVNVLLKPHFEFTMAINSLGDEVLINPGGAVDIILPGTLESSLKLTDTGVADFFNNFSSFALPTNLRQRGVDNPYTLPDFPYRDDGLLVWNALEDYVSKYIGIYYKSNRDIREDFELQNWFQVLRKPKSEGGFGIVSLPANLTNRDQLIDILTIIIFTAGPQHSAIAWTQYQYMAFIPNMPGAIYQPIPTTKGKFADENSLTSFLPGIKPSLTQVQFMSLVGTKRDPKAFTDFGVNSFQDPQAIRVLRDFQNRLESIEKRIEAQNQRREECYPAFLPSRMSNSVSG

[0992] Coding sequence of WP_015138267.1 – SEQ ID NO:191

[0993]

[0994] Amino acid sequence of WP_015138267.1 – SEQ ID NO:192

[0995] MNVASADNSRSSPSNHNLDIARQQYQYNYTHIPPLAMVNQLPPAEEFTTRWYCLLAKELRLIFINTLIVNRGNRGFKSVKDDVIAFLLEALIKGAIPFRLGVIARLLQILPQFLLRSVSKDLRELDDLFLSLLKEIGLSIFTDSLNRITKLLFEKQPKGRVTSLKDYEKLLPVLGLPKIASTYQEDEVFAYMQVAGYNPLMIKRVTSPGDRFPVTDEHYQAVMGSDDSLAAAGEDGRLYLADYGILDGAINGTHPKLQKYVYAPLALFAVPKGADAHRLLRPVAIQCGQTPDADHPIITPNSGKYAWLFAKTIVLIADANFHEAVSHLARTHLFVGVFVMATHRQLPSNHPLSLLLRPHFEGTLAINNAAQENLIARDGGVDLLLSSTIDNSRILAVRGLQSYNFNAAMLPKQLKQRGVDDPNLLPVYPYRDDALLIWDAIRDWVSDYLKLYYPTDADVEKDAALQAWATEAQAYEGGRITGFGEDGGIKTREYLIDAVTLIIFTASVQHAAVNFPQKDIMGYAPVVPLAGYMPASTLKGEVTEQDYLNLLPPLEQAQGQYNLLYLLGSVYYNKLGQYPQPHFTDPQVTSLLQSFQDKLQLIEDTINQRNLNRPAYEYLLPSKIPQSINI

[0996] Coding sequence of WP_094347473.1 – SEQ ID NO:193

[0997]

[0998] Amino acid sequence of WP_094347473.1 – SEQ ID NO:194

[0999] MTASSPENSISLSSTHTLDIARQEYQYNYTHIPSIAMLDRLSIAEEFATNWYFLLAQQLRVVFINTLIVNRGNQGSKSIRDDVERFILEAFLKGAVPVKITILARILQIIPQFLLNGISKDVRELDDLFYSILKENGLVILRDALNRIINLLYEGQPTGHATSLKDYENLFPVIGVPGIAKTYQEDEVFAYMRVAGYNPVTIARVTTPGDRFPVIDEHYQGVMGTDDSLAAAGLEGRLYLADYKILDGAVNGTFPHEQKYLYAPLALFALPKGSDPTRLLRPIAIQCGQTPDPDYPIVTPNSGKYSWLFAKTVVQIADANYHEAVTHLARTHLFVGVFAIATARQLPLTHPLRILLHPHFDSTLAINDAAQRILIAPGGGVDRLLSSSIDNSRVLVVLGLQSYGFNSAILPKQFQQRGVDDPNLLPVYPYRDDALLVWDAIHQWVADYLNLYYTTDEDIQKDTALQAWAAEISAYDGGRIPDFGEDGGIKTRNYLIDATTLIIFTASAQHAAVNFPQKDFMSYAAAIPMAGYLPASTLKREVTEQDYLNLLPPLDQAQRQYNLLSLLGSVYYNKLGDYQQGYFTDQKVKPLLQAFQSNLQQVEDTIKQRNLHRPPYEYLLPSKIPQSINI

[1000] Coding sequence of WP_012164252.1 – SEQ ID NO:195

[1001]

[1002] Amino acid sequence of WP_012164252.1 – SEQ ID NO:196

[1003] MTPQYEYRYDALKDVSPELKYPMAKEVFPADQSLTKWPWTRDLVSVVLRIIANQAMQDISVRRGSACRLITFIRLYRILENPLYQSGLERVFNAINNLVRGLSNIFGNRAQSQNIKHDVKDEQHPEKVSARISAIAKDIQETAESREAREQTSLADYRDLFQIIYLPDISNHFLEDRAFAAQRVAGANPLVINRISELPDHFQVTDQQFKAVMGDSESLQAALNDGRVYLADYQILEEIDAGTVEVKDREIPKYRYAPLALFAIASGNCPGRLLQPIAIQCHQEAGSPIFTPPSLEADKEERLAWRMAKTVVQIADGNYHELISHLGRTHLWIEPIALGTYRRLGTEHPLGKLLLPHFEGTLFINNAAANSLIAPGGTVDKILFGTLKSSVQLSVKGAKGYPFSFNDSMLPQTFASRGVDDLQKLPDYPYRDDALLIWHAIHDWVEAYLQIYYKDDDAVLKDDILQDWLAELRAEDGGQMTEIGESTPEEPEPKIRTLDYLINATTLIIFTCSAQHASVNFPQASLMTFVPNMPLAGFNEGPTAEKASEADYFSLLPPLSLAEQQLDLGYTLGSVYYTQLGYYKANDVDLDDINDHTYFKDLQVKQALRDFQQRLEEIELIIQDRNETRPTYYDILLPSKIPQSTNI

[1004] Coding sequence of WP_015121985.1 – SEQ ID NO:197

[1005]

[1006] Amino acid sequence of WP_015121985.1 – SEQ ID NO:198

[1007] MTDLSENNQNNLSPVDKLKLARQEYQYNYSHIPPIAMVDQLPSNENFSTGWLRLLAKELKVVFINTLIANRGNRGSESVRDDVRLFLIEVLAKGALPFNLTVSARILQIIPNLLLTGISKDYSEIDELFFSILRESGLSIFQDSLSRVKSLLYEKRPRGHAKSLNDYHKLFPEMGIPKIAENFSTDEQFAYMRVAGYNPVMIEQVNKLGDRFPVTEAQYREVMGDDSLAAAGEEGRLYLADYGILKGAVNGTFPSQQKYIYAPLALFAIPKNSNSNKPTLMRPVAIQCGQNPQDNPIITPKSDKYAWLFAKTIVQIADANYHEAVTHLGRTHLLVGPFVVATHRQLPDSHPLNILLSPHFEGTLAINDAAQRRLIAAGGGVDKLLASTIDNSRVLAAVGLQSYGFNEAMLPKQLEKRGVNDTQKLPVYPYRDDALLVWNTIHQWVGDYLNIYYKSDADVKNDTKLQNWAIEAGAFDGGRVPDFGQQHGLIQTLDYLIDAITLIIFTASAQHAAVNFPQGDMMNYAPAVPLAGYQPASILEGKVTEENYLNLLPPLEQAQEQLNLVHLLGSIYYQTLGDYPENYFKDTLVKPALQQFRNNLIEVEATIHQRNQNRPTYEYLLPSKIPQSINI

[1008] Coding sequence of WP_038083060.1 – SEQ ID NO:199

[1009]

[1010] Amino acid sequence of WP_038083060.1 – SEQ ID NO:200

[1011] MTASSQDNSINVPNADNLDIARQEYQYSYTHIPPLAMVDRLPPAEDFASAWYFLLAQQVRGLFVNTLITNRGNRGSESIRDDVRLFILEVLLKGAIPFQTNIIVKVLQIVPQILAQGISRDYRELDDLLFSILKDSGITILKDSLNKVIELLYEGQPTGRPTSLNDYEKLFPVLGVPAIATTFQDDEVFAYMRVAGYNPVIIERVSSPGDRFPVTEEHYQVVMGTDDSLAAAGEEGRLYLTDYGILEGTIGGTFPYYQKYLYAPLALFALPKGSDPNRLLRPIAIQCGQTPGPDYPIVTPNSGKYAWLFAKTVVQIADANVHEAVTHLARTHLFVGAFVLATHRQLLRTHPLSVLLRPHFEGTLAINDAAQRALIAPGGGVDRLLSATIDNSRVLAVYGLQSYSFNNAILPKQFKQRGVEDPNLLPVYPYRDDALLVWNAIHQWVSSYVNLYYSTNEDIQKDAALQAWVAEARSYDGGRVFDFGEDGGIKTREYLADALTLIIFTASAQHAAVNFPQKSLMGYAAAVPLAGYAPASTLTKEVSEEDYLKLLAPLDQAQRQYNLLALLSAVYYNKLGEYPQGHFTNPQVQPLLQEFQSNLKQVEATINQRNLKRPIYNYLLPSKIPQSINI

[1012] Coding sequence of WP_006516541.1 – SEQ ID NO:201

[1013]

[1014] Amino acid sequence of WP_006516541.1 – SEQ ID NO:202

[1015] MTASYKNQNLQEKKQQYQYNYTHIPPVAMVDKLSEEEGFSPGWRLLVAKVGFELLVNTIIANRGDQGKSGAADDVKIFLIETVKETLVDYKGFSRLKILWQGAKYTPRLLFGRLSINVEEIEDLITDIIKSVSADFLRDFAANVQQKLILDSPKGKGDDLKDFQELFQTIDLPAIAYTYEEDEVFASMRVAGPNPVMLQRLTEPEARLPITEAQYQAVMGATDSLTEAYAEGRVYLTDYAILEGAINGSFPADQKYLYAPLALFAVPKADVGDRRLRPVAIQCGQNPNDFPIHTPKSNPYAWLCAKTIVQVADANFHEAVTHLARTHLFIGPFAIATHRQLPDNHPLSLLLRPHFQGMLAINNEAQAKLIAAGGGVNKILSATIDTSRVFAVLGVQTYGFNSAMFPKQLQQRGVDDTNSLPIYPYRDDGSLIWDAIHNWVEDYLKLYYADDAAVQQDANLQAWAQELIAYDGGRVIEFGETDEQLQTLLQTLTYLIDAITLIIFTASAQHAAVNFPQKDIMSFTPAMPTAGYDELPDLGDQTTKEDYLSLLPPLNQAQEQLKLLHLLGSVHFTELGQYEKGHFQDSQVQAPL

[1016] QRFQNRLEEITDVIYQRNRNRPAYEYLLPKNIPQSINI

[1017] Coding sequence of WP_099100980.1 – SEQ ID NO:203

[1018]

[1019] Amino acid sequence of WP_099100980.1 – SEQ ID NO:204

[1020] MTASSPENSISSSSTHTLDIARQEYQYNYTHIPSIAMLDRLSIAEEFATNWYFLLAQQLRVVFINTLIVNRGNQGSKSIRDDVERFILEAFLKGAVPAKISILARILQIIPQFLLKSISKDVRELDDLFYSILKENGLVILRDALNRIINLLYEGQPTGHATSLKDYENLFPVIGMPAIAKTYQEDEVFAYMRVAGYNPVTIARVTTPGDRFPVTDEHYQAVMGTDDSLAAAGLEGRLYLADYKILDGAVNGTFPHEQKYLYAPLALFALPKGSDPTRLLRPIAIQCGQTPGPDYPIVTPNSGKYSWLFAKTVVQIADANYHEAVTHLARTHLLVGVFAIATARQLPLTHPLRILLHPHFDSTLAINDAAQRILIAPGGGVDRLLSSSIDNSRVLAVLGLQSYGFNSAILPKQFQQRGVDDPNLLPVYPYRDDALLVWDAIHQWVSDYLNLYYTTDEDIQKDTALQAWAVEISAYDGGRIRDFGEDGSIKTRNYLIDATTLIIFTASAQHAAVNFPQKDFMGYAAAIPLAGYLPASTLKREVTEQDYLNLLPPLDQAQRQYNLLSLLGSVYYNKLGDYQQGYFTDQKVKPLLQAFQSNLQQVEDTIKQRNLHRPPYEYLLPSKIPQSINI

[1021] Coding sequence of WP_096578311.1 – SEQ ID NO:205

[1022]

[1023] Amino acid sequence of WP_096578311.1 – SEQ ID NO:206

[1024] MLPTLPQNDPNPSVRQAQLARSRYIYKFTHKYQGCPGNSPLPNGIALAEHVPPDQEFTPDYLLRVTQVNATLLANHAAIDLEYLTGGNAGSSFSLSDWLGLTRAVGNKHLLFSTPLKVTSRIDSSFPINLDAYDAMFALIQKPEIVYKLKQGRDVCDRAFAWQRLAGANPMVLQGITHLPPTFQLTNQQYQAAIRDENDTLEAAGKEGRLYVADYSLLSGLPHGTWSDGVLGVPRNKYIFDPIALFAWKKETPLELGGLLPVAIQCQQTQDSISWCRSVAPIFTPNDGIFWEMAKAIVQSADGNIQEMVYHLGHTHFVMEAVIVAAERNLAAVHPIHVLLKPHFEFTLSLNDYAYKHLIAPGGAVDSVMGSTLEGSLTLMLRGMKNYAFNQALPPLDFKNRGVDNLDGLPEYPYRDDGLLVWTAIRKFVSKYLRLYYTNDIDVKTDTELQNWVKSIGNSQEGNIQGVEEIQTLEKLIDMVALIIFTASAQHGSLNYAQFPMMGYVPNVSGAIYAEAPTNTTPQNQDNYLMLLAPVQQALIQFTTLYQLSNVRYGKLGHYPCLYFQDSRVLPLVKEFQQNLAVVESEILDRDQTRFMSYPFLLPSQIGNSIFI

[1025] Coding sequence of RCJ33284.1 – SEQ ID NO:207

[1026]

[1027] Amino acid sequence of RCJ33284.1 – SEQ ID NO:208

[1028] MTASSPENSISSSSTHTLDIARQEYQYNYTHIPSIAMLDRLSIAEEFATNWYFLLAQQLRVVFINTLIVNRGNQGSKSIRDDVERFILEAFLKGAVPVKISILARILQIIPQFLLKSISQDVRELDDLFYSILKENGLVILRDALNRIINLLYEGQPTGHATSLKDYENLFPVIGVPAIAKTYQEDEVFAYMRVAGYNPVTIARVTTPGDRFPVTDEHYQGVMGTDDSLAAAGLEGRLYLADYKILDGAVNGTFPHEQKYLYAPLALFALPKGSDPTRLLRPIAIQCGQTPDPDYPIVTPNCSKYSWLFAKTVVQIADANYHEAVTHLARTHLFVGVFAIATARQLPLTHPLRILLHPHFDSTLAINDAAQRILIAPGGGVDRLLSSSIDNSRVLAVLGLQSYGFNSAILPKQFQQRGVDDPNLLPVYPYRDDALLVWDAIHQWVSDYLNLYYTTDEDIQKDRALQAWAAEIPAYDGGRIPDFGEDGGIKTRNYLIDATTLIIFTASAQHAAVNFPQKDFMGYAAAIPMAGYLPASTLKREVTEQDYLNLLPPLDQAQRQYNLLSLLGSVYYNKLGDYQQGYFTDQKVKPLLQAFQSNLQQVEDTIKQRNLRRPSYEYLLPSKIPQSINI

[1029] Coding sequence of WP_052555973.1 – SEQ ID NO:209

[1030]

[1031] Amino acid sequence of WP_052555973.1 – SEQ ID NO:210

[1032] MARTARYRFGPELPGARPDAQVVHPMSAFLPAFDPDPETRAAGRAAKRGEYTYNHEYVSPLAFVGEVPSRDRFPIDFTTLVLGKIMTNVANQADADSALRRRLRAMDVPIADMVLAGSTAVRAVGAAVGAVIGAAADARRLQTIDDYNALFHVIGLPPIAKDFEFDSTFAELRLAGPNPVMIHRVDKPDDRFPVTDAHFQVALPGDTLAAAGAEGRLFLVDYQRLDGVETGVSPCGLPKYLYAPLALFAVNKDTRKLVPVAIQCKQRPGPENPIFTPDDGYNWRIAKTIVEIADGNYHEAITHLGRTHLTVEPFVVAAHRQFGPNHPLNVLLQPHFGGTLAINHLARLKLISPDGVVDRLLGAKISAALELSAWGVQGHAFMDLLPPASFRRRGVDNTATLPSYSYRDDALLHWEAVREWVATYLRCFYRSDAEVAADVEVAAWLTEASAKTGGRINGIEPARTFAELVDVTALVIFTASAQHAAVNFPQYDIMSYAPAMPLAGYAPAPTSKTGATEADYMAMLPPRDQAALQMNTGFMLGTAHYTRLGHYEPGYFGEPRINELAARFAAKMDEIEATITERNRHRRPYPFMLPSGVPQSINI

[1033] Coding sequence of WP_103667398.1 – SEQ ID NO:211

[1034]

[1035] Amino acid sequence of WP_103667398.1 – SEQ ID NO:212

[1036] MIFSLLSGVARVLNFVSAKLTDLANLISRRSQSSKYPLLPQNDPATTQRQASLNQSRQLYQYNYTYIESLPMVEKVPKNERFSLSWGLLVGKVVVKVLLNDRANPSAFIDKEKSKAQQLDFSKRLLEASMSQSENALIELLSELPTILEDEPIDLEGSNIQEYNNLFWIIPLPAISQNFKSNSEFARLRVAGFNPLVIQKVKALDAKFPLTEAQFQKVLAGDSLAAAGAEGRLYLADYVELTAIAGGTFPKSEQKYINAPLALFAIPKGKKSLTPIAIQLGQDPNTNPIFVCQAGDEPNWMLAKTVVQIADANYHELISHLGRTHLFIEPFAIATNRQLASNHPLYVLLKPHFQGTLAINDAAQSGLINAGGTIDSLLAGTITSSRALSVQGVKTYNFDEAILPVALKKRGVDDPNLLPDYPYRDDALLVWDAISTWVKSYLSIYYFNDNDVIRDSELQAWAQEIISDNGGRVTSFGQSGQIRTFDYLVNAVTLLIFTGSAQHAAVNFPQGDLMVYAPAFPLAGYTPAPTSTTGASEADFFAMLPPIDQAKSQLTMTYILGSVYYTTLGEYGPSYFNDDRIKQPLLDFQDQLKAIESTIKSRNEKRVTDYNYLRPSRIPQSINI

[1037] Coding sequence of WP_023071825.1 – SEQ ID NO:213

[1038]

[1039] Amino acid sequence of WP_023071825.1 – SEQ ID NO:214

[1040] MTASYSNPDQHKKRLEYQYNYTHIPPIAMVDKLSEEEQFSSRWRLMVAKVGFEILVNTIIVNRGDQGKSGAADDVKAFLIETFQETLADYSVRSRLKILWQGAKFIPRILFTRLSLKAEELENLIKEIIQSVNGDFLRDFAANVQQKLKLDAPVGRGQDIKDFQALFQTIDLPDIAYTYETDEVFASMQVAGPNPVMIKRLSTPDARLPITETLYKGGMGETDSLADAYAEGRLYLADYGILDGAINGSFPEAQKYLYAPLALFAVAKTGDRRLRPVAIQCGQNPEEFPLYTPQSNPYAWLCAKTMVQIADANFHEAVTHLARTHLLIGPFAIATHRQLSDDHPLSLLLRPHFQGMLAINNEAQAKLIAPGGGVNKILSATIDTSRVFAVIGVQTYGFNSAMLPKQLQQRGVDDTDSLPIYPYRDDSILIWDAIHDWAENYLSLYYANDAAVQQDNALQAWAQELSAHNGGRVQEFGEAEGQLQTLAYLIDAITLIIFTASAQHAAVNFPQKEIMSYAPAMPTAGYAALENLGEHTTQANYLSLLPPIDQAQEQLKLLHLLGSVHFTQLGQYEKNHFQDANIKIPLEQFQNRLEEITDIIHERNRDRSPYEYLLPKNIPQSINI

[1041] Coding sequence of WP_096618242.1 – SEQ ID NO:215

[1042]

[1043] Amino acid sequence of WP_096618242.1 – SEQ ID NO:216

[1044] MRSPTPKQRRQELIDTYILSRRSMMMLMAVAATPGIEMLLFGGNKSSQASATGNFENCNPGLETLLSNENQPSKPKPPNNPNIPTLPHKDTKATQQERLLQLGKAREEYQTGLRLPNSAKVKTLPAQEAFSERYNNNRVILSEKIAANQQAFLSNPQPFQSFDDYAALFPVLPLPGIAKTFRNDDVFARQRLSGCNPMELKNVLKLGYSLRDKMGITDEIFQAVLGATRGRKPIHNNQTLNSAIREGSLFVTDYAVLDSVTPKETQYLCAPIALYYAARIRGDFHLIPIAIQLGQVPGESLLCTPLDGVDWTLAKLITQMADFSINQLYRHLGQTHLVMEPIALATVRELAARHPVNVLLKPHVEFTMAINSLGDQVLINPGGAVDVILPGTLESSLKLTERGVSDFCNNFSNFALPTNLRQRGVDNSSILQDFPYRDDGLLIWNALEEYVSQYIGIYYKSNRDIREDFELQKWFQALRKPVSEGGFGIVSLPASLTNRNQLIDILTIIIFTAGPQHSAIAWTQYQYMAFIPNMPGALYQPIPTTKGKFANENSLTSFLPGVKPSLTQVQFMSLVGTKRDPKAFTDFGTNSFQDPRAIRVLRDLQNRLESVEKRIKILNKRRQECYPAFLPSRMSNSVSG

[1045] Coding sequence of WP_107806740.1 – SEQ ID NO:217

[1046]

[1047] Amino acid sequence of WP_107806740.1 – SEQ ID NO:218

[1048] MTTSSPDNSRSLPITQNLELVRQEYQYNYTHIPPIPMVNQLPNQENFTTRWTFLLAQQLREIFINTLITNRGDRSSKSVRDQVKRFILEALFKGAIPAKVSVIARLFQIIPQFLIQGISKDFHELDDLFFSLFKTNGLLIFRDSLNRITALLDKGHPTGHVNSLKDYQKLFTTIELPAIAKTFDQDQVFAYMQVAGYNPLVIKRVKSPGANFPVEDTHYQAVMGSDDSLAAAGQEGRLYLADYQILDGAINGIYLNYQKYAYAPLALFAIPKNSDPNRLLRPIAIQCGQTPGADYPIITPNSGKYAWLFAKTIVHIADGNFHEAVSHLARTHLFVGVFVIATHRQLSPSHPLSLLLRPHFEGTLAINNAAQEVLIAPGGGVDILLSSTIDNSRILAVRGLQSYSFNEAMLPNQLKQRGVDDPELLPVYPYRDDALLIWNAIHQWVSDYLSLYYPTDKDIQNDTALQAWAAEAKADNGGRVPDFGENGGIQTLDYLVDAATLIIFTASAQHAAVNFPQKDLMSYAPAFPLAGYVSASINGEVSEQDYLNLLPPLEQAQQQFNLLTLLGSIYYNQLGEYPKSHFANPKVQILLQKFQSRLQQIEITINQRNLHRPTYEYLLPSKIPQSINI

[1049] Coding sequence of WP_017804222.1 – SEQ ID NO:219

[1050]

[1051] Amino acid sequence of WP_017804222.1 – SEQ ID NO:220

[1052] MTTSSPDNSRSLPITQNLELVRQEYQYNYTHIPPIPMVNQLPNQENFTTRWTFLLAQQLREIFINTLITNRGDRSSKSVRDQVKRFILEALFKGAIPAKVSVIARLFQIIPQFLIQGISKDFHELDDLFFSLFKTNGLLIFRDSLNRITALLDKGHPTGHVNSLKDYQKLFTTIELPAIAKTFDQDQVFAYMQVAGYNPLVIKRVKSPGANFPVEDTHYQAVMGSDDSLAAAGQEGRLYLADYQILDGAINGIYLNYQKYAYAPLALFAIPKNSDPNRLLRPIAIQCGQTPGADYPIITPNSGKYAWLFAKTIVHIADGNFHEAVSHLARTHLFVGVFVIATHRQLSPSHPLSLLLRPHFEGTLAINNAAQEVLIAPGGGVDILLSSTIDNSRILAVRGLQSYSFNEAMLPNQLKQRGVDDPELLPVYPYRDDALLIWNAIHQWVSDYLSLYYPTDKDIQNDTALQAWAAEAKADNGGRVPDFGENGGIQTLDYLVDAATLIIFTASAQHAAVNFPQKDLMSYAPAFPLAGYVSASINGEVSEQDYLNLLPPLEQAQQQFNLLSLLGSIYYNQLGEYPKSHFANPKVQILLQKFQSRLQQIEITINQRNLHRPTYEYLLPSKIPQSINI

[1053] Coding sequence of WP_010472182.1 – SEQ ID NO:221

[1054]

[1055] Amino acid sequence of WP_010472182.1 – SEQ ID NO:222

[1056] MTPQYEYRYDALKDVSPELKYPMAKEVFPADQSLTKWPWTRDLVSVVLRIIANQAMQDISVRRGSACRLITFIRLYRILENPLYQSGLERLFNAVNNLVRGLSNIFGNRAQSQNIKHDVKEEQHPDKVSARISAMVKDIQETAESREAKEQPSLADYRDLFQIIYLPDISNHFLEDRAFAAQRVAGANPLVINRISELPDHFQVTDQQFKSVMGDSESLQAALNDGRVYLVDYQILEEIDAGTVEVKDREILKYRYAPLALFAIASGNCPGRLLQPIAIQCHQEAGSPIFTPPSLEADKEERLAWRMAKTVVQIADGNYHELISHLGRTHLWIEPIALGTYRRLGTEHPLGKLLLPHFEGTLFINNAAANSLIAPGGTVDKILFGTLKSSVQLSVKGAKGYPFSFNDSMLPQTFASRGVDDLQKLPDYPYRDDALLIWHAIHDWVEAYLQIYYKDDDAVLKDETLQDWLTELRAEDGGQMTEIGESTPEEPEPKIRTLDYLVNATTLIIFTCSAQHASVNFPQASLMTFVPNMPLAGFNEGPTAEKASEADYFSLLPPLSLAEQQLDLGYTLGSVYYTQLGYYKANDVDLGDINNHTYFNDLQVKQALLSFQQRLEEIELIIQDRNETRPTYYDILLPSKIPQSTNI

[1057] Coding sequence of WP_103139451.1 – SEQ ID NO:223

[1058]

[1059] Amino acid sequence of WP_103139451.1 – SEQ ID NO:224

[1060] MTNSLTSATTNSNLESAREQYKYNYSYIPPIAMVDELPDGEDFSRQWLLLLAKELKVIFVNILITNRGNRGSQKIRDDVRNFILEVILKGAIPANISVIARFMQIVPQLLIRGFSTDFHELDDLLFSLIKESGLLILSDSFQRITKLLDKGKPTGHVSSLADYQKLFPVIPPPKIAKTFQNDAEFAYMRVAGYNPVMIQRVSELDERFPVTDAQYQAVMGSDDSLALAGQEGRLYLADYGIFNGGLNGSCPSYQKYLYAPLALFAVPPGSNPNRLLQPVAIQCGQNPKENPIITPKSSEYAWLIAKAIVQIADANFHEPITHLARTHLLAGIFAIATHRQLPNSHPLYVLLTPHFEGTLAINDAAQRALIAPLGGVDILLSSTIDNSRVLTVLGLQSYGFNHAMLPKQFQQRGVDDANLLPVYPYRDDGLLLWDAIHQWVADYIQIYYHTDQEIQADAYIQAWAKEVQAYDGGRLTEFGEDGKIQTREYLIDAVTLIIFTASVQHAAVNFPQKDVMGYTPAVPLAGYLPASILQGEVTEKDYLNFLPPLDQAQQQYNLLALLGSVYYNRLGEYPPQHFADPKVEPLLRSFQKNLQEIETIIQKRNSDRPPYEYLLPSKIPQSINI

[1061] Coding sequence of WP_075890025.1 – SEQ ID NO:225

[1062]

[1063] Amino acid sequence of WP_075890025.1 – SEQ ID NO:226

[1064] MTATSGSQNLGLIEKQEKYKYNYSHIPPVAMVDTLPESEKWSIPWKLMVAKVGYQLLVNKIIVTYGDQGKAGAANDVRAFLIARLKETFGEQKGLSKVRVLLQGARFLPRIIWGEITTDVVDVEEVMRDAIKTVSRDFLEDFAANVMEQLTVDGKDGRCLSSTDFERLFATIDLPEIAYEYQTDESFAYMRVAGPNAVMLEKITEPDPRFPVTEAHYQAVMGEGDSLAAARAEGRLFLCDYEILDGAVNGSFPTDQKYLYAPLALFAVPKADAGKRDLRPVAIQLGQKPKEYPILTPKSNRYAWLCAKTAVQVADANFHEAVTHLGRTHLFMGPFVIATHRQLPENHPLFKLLTPHFLGMLAINDSAQAKLIYKGGGVDKILATTIDNARLFAVLGVQTYGFNRAMLPDQLAARGVDDTEALPVYPYRDDALLIWEAIYNWVKAYLKTYYPGDSAVQRDQALQAWAKELISYKGGRVVDFGEDGDIKTLSYLIDAVTLIIFTVSAQHAAVNFPQKGLMSFAPGMPTAGYAPLDNLGDQTAEQDYLDLLPPISQAQEQLKLCHLLGSVHFTQLGQYDKKHLGDPKIQKPLRQFQGRLEEIEMIIHKRNGDRPTYEYLLPSLIPQSINI

[1065] Coding sequence of WP_050046589.1 – SEQ ID NO:227

[1066]

[1067] Amino acid sequence of WP_050046589.1 – SEQ ID NO:228

[1068] MRSRSGCFAASPNRQQRHQQLIEQYVFSRRTMLALLGFICAPGLEHFIVSDTQPREPTLPANPQIPTLPQKNSLASQKERQQQLEIARSKYQLTPRLPNSVRVSTLPIEEAFDGGYSSNRASITRKITENQQAFFQNPKPFLALEDYTNVFQVLPVPDIAKTFRKDAIFAGQRLWGPNPMELTNVLALNYDLQEKLGITNEIFQTVLGAARGTAYVSETLESATKNGGLFVTDYAILATDGITSKTKRYLIAPIALYYADRDRGNWRLIPIAIQLGQVPQESLLCTPLDGVDWTLAKLIAQMADFSVHELVRHLGQTHLALEPIALATVRELPALHPVHVLLKPHFEFTMAINAFGDRVLINPGGYVDVILGGTLESSLNLVNLGVSEMFDNFSNFALPNNLQRRGVGDRSLLKDFPYRDDGVLVWDALSEYVSRYVGIYYRSSKDIREDFELQNWLKALRTPVSDGGFGVTSLPSYLKDRDQLIDLLTQIIFTAGPQHSAIAWTQYQYMSFVPNMPGAIYQPVPITKGTIEDEKSLTSFLPGIEPTFAQVNVISGIGVKLDVKAFTDFGVNSFQDPRAIAVLKGLQNRLEVVEKQIEQRNKRREECYPGFLPSRMANSTSG

[1069] Coding sequence of WP_012163949.1 – SEQ ID NO:229

[1070]

[1071] Amino acid sequence of WP_012163949.1 – SEQ ID NO:230

[1072] MTHQYSLTGLPTQITPVEIQQDKHQPTLAPTRPNPTQPEPIPAALKAARRKYQYNYSHIAPVAMVDRLPKEELPSRAWWSKLIRTMFKILSNAIVGAHNHHHEHEAEQHASRLIRKTLVDILRQRPEVRWRLIWHLLKTAPTTLLNGLRLSFSDAESLLHSLAAHLEHDLLRILHLNLKEHLAHECGQDRPTSIADFNQQFATIPLPECAEYFQEDEFFAYLRVAGPNPVLLQQVRHLSGDILCSHFPVTNQHYQTVMGEDDSLQIAITEGRLYIADYAILAGAINGNYPDQQKYISAPIALFAVPSADAPCRNLQPIAIQCRQSPGPETPILTPPTDQNPDQKQAWDMAKTCVQVADSNYHEAVTHLGRTHLFISPFVIATHRQLLPSHPVSVLLRPHFEGTLSINNGAQSMLMAPEGGVDTVLAATIDCARVLAVKGVQSYSFNQAMLPQQLRQLGLDNAEALPIHPYRDDALLIWQAIETWVTDYVSLYYPTDDSVQTDAALQAWAQELQAEEGGRVPDFGEDGQLRTQAYLIQALTLIIFTASAQHAAVNFPQGDIMVYTPGMPLAGYQPAPNSTAMSSQDRLNQLPSLHQALNQLELTYLLGQIYHTQLGQYEKSWFSDQRVQAPLHRFQANLLDIETAIAERNRHRPYPYRYLQPSNIPQSINI

[1073] Coding sequence of WP_050046033.1 – SEQ ID NO:231

[1074]

[1075] Amino acid sequence of WP_050046033.1 – SEQ ID NO:232

[1076] MRSRSGCFAASPNRQQRHQQLIEQYVFSRRTMLALLGFVCAPGLEHFIVGDTQPREPKLPANPQIPTLPQKNSLASQKERQQQLEIARSEYQLTSRLPNSVRVSTLPIKEAFDGGYSNNRASITQKITENQQAFFQNPKPFLALEDYTNVFQVLPVPDIAKTFRKDVIFAGQRLWGPNPMELTNVLALNYDLQEKLGITNEIFQTVLGAARGTAYVSETLESATKNGGLFVTDYAILATDGITSKTNRYLIAPIALYYADRNRGNWRLIPIAIQLGQVPQESLLCTPLDGVDWTLAKLIAQMADFSVHELVRHLGQTHLALEPIALATVRELPALHPVNVLLKPHFEFTMAINAFGDRVLINPGGYVDVILGGTLESSLKLTNLGVSEMFDNFSNFALPNNLQRRGVGDRSLLKDFPYRDDGVLVWDALSEYVSRYVGIYYKSSKDIREDFELQNWLKALRTPVSDGGFGVTSLPSYLQDRDQLIDLLTQIIFTAGPQHSAIAWTQYQYMSFVPNMPGAIYQPVPITKGTIEDEKSLTSFLPGIEPTFAQVNVISGIGVKLDIKAFTDFGVNSFQDPRAIAVLKGLQNRLDVVEKQIEQRNKRREECYPGFLPSRMANSTSG

[1077] Coding sequence of WP_096660823.1 – SEQ ID NO:233

[1078]

[1079] Amino acid sequence of WP_096660823.1 – SEQ ID NO:234

[1080] MTDLSQNNSTSVDKLKLARQEYQYSYIHIPPIAMVDKLPSNENFSTGWLRLLARELKVVFINTLIANRGNRGSENVRDDVRLFFLEVLAKGALPFNLGVTARVLQIIPNLLLKGTSKDFSEIDDLFFSILKESGLSIFQDSLSRVKSLLYEKRPTGHVSSLNDYQKLFPEMEIPKIADNFSTDEQFAYMRVAGYNPVMIERVNKLGDRFPVTEAQYQEVMGDDSLTAAGEEGRLYLADYGILEGAVNGTFPSQQKYIYAPLALFAIPKNSENDESSLMRPVAIQCGQNPQNNPICTPKSDKYAWLFAKTIVQIADANYHEAVTHLGRTHLLVGPFVVATHRQLPDSHPLNILLRPHFEGTLAINNAAQSSLIAAGGGVDKLLASTIDNSRVLAAVGLQSYGFNEAMLPKQLEKRGVNDTQKLPIYPYRDDALLIWNAIHTWVADYLSIYYKDDTSIQNDTYLQNWAIEAGAYDGGRVPDFGQENGLIQTLDYLIDATTLIIFTASAQHAAVNFPQGDMMIYAAAVPLAGYQPASILEGKVTQEDYLNLLPPLEQAQEQLNLVYLLGSIYYKTLGDYSDNYFKDALVKPALQEFRNNLLEAEATIHQRNQNRPTYEYLLPSKIPQSINI

[1081] Coding sequence of WP_110989156.1 – SEQ ID NO:235

[1082]

[1083] Amino acid sequence of WP_110989156.1 – SEQ ID NO:236

[1084] MTDSNTAQEAQSQQYEYRYDAFKNISPKLIYPMAVKVLPADQSFTKWKWTKNVVSLVLRLVANQAMQNVSLRKGSACRLITFIRLYRILEDPKNSSYIERLFDFIISIARALTNRFKRRPKSQDIEQDVKQNQKPDQVQARVEAMVDDIQQQSKTKDPVKHLSFEDYRNLFQIIYLPDISNHFLEDRSFAAQRVAGANPLVIMQVSELPEYFKVTEEHYTKVMGKDDSLQAALDEGRIYLADYKILDEIDPGTVEVGVNGSIKETIEKFGYAPLALFAIASGDCPGRLLTPVAIQCSQDAGSLIFTPPSIAAVDEERWAWRMAKTVVQVADGNYHELISHLGRTHLWIEPIALGTYRRLAKHKLGKLLLPHFEGTFFINNAAAGSLIAKGGVVESILSGTLLSSVTLSVKAAKGYPFAFNDSMLPKTFAARGVDDPQKLPDYPYRDDALLIWDAIHKWVKSYLEVYYSSDDEVLSDAVLQAWLAELVAEDGGQMTEIGEVIPEDRRPKIRTLDYLIDATTLIIFTCSVQHAAVNFTQASLMSFAPNMPLAGFNAAPTTLKVSEADYFSMLPSLSLAEQQMNFGYTLGSVYYTQIGQYKANEVELEEMNQHDYFGDSRISHHLEIFQNKLKEIELTIQQRNETRPTFYDILLPSKIPQSTNI

[1085] Coding sequence of WP_010473598.1 – SEQ ID NO:237

[1086]

[1087] Amino acid sequence of WP_010473598.1 – SEQ ID NO:238

[1088] MTHQYSLTGLPTQITPVEIQQDKHQPTLTSTRPNPTQPEPIPAALKAARRKYQYNYSHIAPVAMVDRLPQEELPSRTWWSKLFRTMFKILSNAIVGAHNHHHEHEAEQHISRLIRKTLVNILTQRPEVRWRLIWHLLKTAPTTLINGLRLSFADSESLLHSLAAHLEHDLLRILHLNLKEHLAHECRQDRPTSIADFNQQFATIPLPECAEYFQEDEFFAYLRVAGPNPVLLQQVRHLSGDTLCSHFPVTNQHYQAVMGADDSLQTAVTEGRLYIADYAILAGAINGNYPDQQKYISAPIALFAVPSADAPCRNLQPIAIQCRQSPGPETPILTPPTDQNPDQKQAWDMAKTCVQVADSNYHEAVTHLGRTHLFISPFVIATHRQLLPSHPVSVLLRPHFEGTLSINNGAQSMLMAPEGGVDTVLAATIDCARVLAVKGLQSYSFNQAMLPQQLQQLGLDNAAALPIHPYRDDALLIWQAIETWVTDYVSLYYPTDDSVQKDAALQAWAQELQAEEGGRVPDFGEDGQLRTQAYLIQALTLIIFTASAQHAAVNFPQGDIMVYTPGMPLAGYQPAPNTTAMSSQDRLNQLPPLHQALNQLELTYLLGQIYHTQLGQYEKSWFSDQRVLAPLHRFQANLLDIETAIAERNRHRPYPYRYLQPSNIPQSINI

[1089] Amino acid sequence of 5MEE_A – SEQ ID NO:239

[1090] MVQPSLPQDDTPDQQEQRNRAIAQQREAYQYSETAGILLIKTLPQSEMFSLKYLIERDKGLVSLIANTLASNIENIFDPFDKLEDFEEMFPLLPKPLVMNTFRNDRVFARQRIAGPNPMVIERVVDKLPDNFPVTDAMFQKIMFTKKTLAEAIAQGKLFITNYKGLAELSPGRYEYQKNGTLVQKTKTIAAPLVLYAWKPEGFGDYRGSLAPIAIQINQQPDPITNPIYTPRDGKHWFIAKIFAQMADGNCHEAISHLARTHLILEPFVLATANELAPNHPLSVLLKPHFQFTLAINELAREQVISAGGYADDLLAGTLEASIAVIKAAIKEYMDNFTEFALPRELARRGVGIGDVDQRGENFLPDYPYRDDAMLLWNAIEVYVRDYLSLYYQSPVQIRQDTELQNWVRRLVSPEGGRVTGLVSNGELNTIEALVAIATQVIFVSGPQHAAVNYPQYDYMAFIPNMPLATYATPPNKESNISEATILNILPPQKLAARQLELMRTLCVFYPNRLGYPDTEFVDVRAQQVLHQFQERLQEIEQRIVLCNEKRLEPYTYLLPSNVPNSTSI

[1091] 8. Consensus sequence motif

[1092] AKxxxxxADxxxxxxxxHxxxxHxxxxPxA (SEQ ID NO:240),

[1093] VxGxxxxxxxxxxLxxxxxxxxxxxxxxHxxxNxxQxxYxxxxxN (SEQ ID NO:241),

[1094] LxxxxxxIxxxNxxxxxxYxxxxPxxxxxSI (SEQ ID NO:242);

[1095] LxxxxxYxxxxxX 1 xxxxxxX 2 GxxxxxxxKxLPxPxxxFxWxxxX 3 xxxPxxI (SEQ ID NO:243)

[1096] WxxAKxCxQxADxxHxExxxHxxxxHxxMxPxA(SEQ ID NO:244);

[1097] GxVxGxxxxxxxxxxLxxxxxxxxxxCxPxHxxxNxxQxxYxxxxxNMPxAxY(SEQ ID NO:245),

[1098] QxxxxxxLxxxxxDxxGxYxxxX 4 F(SEQ ID NO:246),

[1099] QxxLxxxxxxIxxxNxxRxxxYxxxxxxxxxNSI(SEQ ID NO:247),

[1100] LxxxxxYxxxxxX 1 xxxxxxX 2 GGxxxxxxKxLPxPxAxFxWxxxX 3 xxxPxxI(SEQ ID NO:248),

[1101] WxxAKxCxQxADxNHxExxxHxxxTHxVMxPxAxxT(SEQ ID NO:249),

[1102] GxVxGxxxxxxxxxxLxxxxxxxxxxCxPxHxxxNxxQxxYxxxxxNMPxAxY(SEQ ID NO:250),

[1103] QxxxxxxLxxxxYDxLGxYxxx X 4 F(SEQ ID NO:251),

[1104] FQxxLxxxxxxIxxxNxxRxxxYxxxxPxxxxNSI(SEQ ID NO:252)

[1105] 9. LOX mutant

[1106] Codon-optimized coding sequence of WP_002738122.1mut – SEQ ID NO:253

[1107]

[1108] Amino acid sequence of WP_002738122.1mut – SEQ ID NO:254

[1109] MVNTPPPTPCLPQNEPDANRRADSLNLQRQAYRYDYQYLPPLVLMESVPAAENFSFQYITERLAATAELPANMLAVKVKSFLDPLDELQDYEDFFAIIPLPKIAKVYQTNDAFAEQRLSGANPLVLHLLKPGDARAQVLNQIPSSKTDFEPLFQVNQELAAGNIYICDYTGTDINYLGPSLIQGGTHAKGRKYLPKPRAFFWWRKSGIRDRGKLVPIAIQFGENAEKLYTPFEKNPLAWLFAKICVQVADSNHHEMNSHLCRTHFVMEPIAICTARQLAENHPLSLLLKPHLRFMLTNNSLGQERLINPGGPVDELLAGTLGESMALVKDAYANWNLRDFAFPKEISNRGMDDTERLPHYPYRDDGMLVWQSINQFVSDYLHYFYPNPQDITNDQELQAWAGECSNSAADQGGNVKGMPANFTDVEDLIEVVTTIIFICGPLHSAVNYGQYDYMTFAANMPLAAYCDLPEAIKDTTGSIIGDARGSITEKDILQLLPPYKKAADQLQSLFTLSDYRYDQLGYYDKAFRELYGRKFEEVFAEGDQATITGFLRQFQQNLNMNEQEIDANNQKRIVPYTYLKPSLILNSISI

[1110] Codon-optimized coding sequence of WP_002738122.1mut2 – SEQ ID NO:255

[1111]

[1112] Amino acid sequence of WP_002738122.1 mut2 – SEQ ID NO:256

[1113] MVNTPPPTPCLPQNEPDANRRADSLNLQRQAYRYDYQYLPPLVLMESVPAAENFSFQYITERLAATAELPANMLAVKVKSFLDPLDELQDYEDFFAIIPLPKIAKVYQTNDAFAEQRLSGANPLVLHLLKPGDARAQVLNQIPSSKTDFEPLFQVEQELAAGNIYICDYTGTDINYLGPCMIQGGTHAKGRKYLPKPRAFFWWRKSGIRDRGKLVPIAIQFGENAEKLYTPFEKNPLAWLFAKICVQVADSNHHEMNSHLCRTHFVMEPIAICTARQLAENHPLSLLLKPHLRFMLTNNHLGQERLINPGGPVDELLAGTLGESMALVKDAYANWNLRDFAFPKEISNRGMDDTERLPHYPYRDDGMLVWQSINQFVSDYLHYFYPNPQDITNDQELQAWAGECSNSAADQGGNVKGMPANFTDVEDLIEVVTTIIFICGPLHSAVNYGQYDYMTFAANMPLAAYCDLPEAIKDTTGSIIGDARGSITEKDILQLLPPYKKAADQLQSLFTLSDYRYDQLGYYDKAFRELYGRKFEEVFAEGDQATITGFLRQFQQNLNMNEQEIDANNQKRIVPYTYLKPSLILNSISI

[1114] Codon-optimized coding sequence of WP_015204462.1 mut – SEQ ID NO:257

[1115]

[1116] Amino acid sequence of WP_015204462.1mut – SEQ ID NO:258

[1117] MPQPCLPQNEPNPEKRNNDLSDQQQAYEYDYKYLPPLVLLKKIPAFENFSAQYIAERVVATSELVPNMLAAKARSFLDPLDDIKDYEDLFTLLPLPEVAKVYQTNNSFAEQRLSGANPFVIRLLDEDDARSQVLEQIPSFKDDFEPLFDVRKELAAGNIYICDYTGTDEYYRGPSMVQGGTYEKGRKYLPKPLAFFWWQRTGISDRGKLVPIAIQLDASKNSKVYTPTNSKVYTPFEQNPLDWLFAKLCVQIADGNHHEMSSHLCRTHFVMEPIAICTAHQLAENHPLSLLLRPHFLFMLTNNSLGQQRLINPGGPVDELLAGTLPESMELVKDAYEGWNIKEFAFPTEIKNRGMDNTERLPHYPYRDDGMLVWKAIHTFVSDYVNHFYPTPEDITGDTELQAWAKECSDQSAQTNGGKVKGMPTSFTTVQELIEIVTTIIFICGPQHSAVNYAQDGYMTFAANMPLAAYRDIPKQSHKPQDQPTATPSVAVQTTAEQTTAEQTKAVEITADKATLDQNTVLQKRAVQTTTVEIPEDQITEEQILKLLPPYKRTADQLQSLFVLSAYQYDRLGYYEKAFQQLYNDKFEDVFKDDNNQAIIAIVRQFQQNLNMVEQEIDANNKKRVVPYLYLKPSLILNSISI

[1118] Codon-optimized coding sequence of WP_015204462.1mut2 – SEQ ID NO:259

[1119]

[1120] Amino acid sequence of WP_015204462.1 mut2 – SEQ ID NO:260

[1121] MPQPCLPQNEPNPEKRNNDLSDQQQAYEYDYKYLPPLVLLKKIPAFENFSAQYIAERVVATSELVPNMLAAKARSFLDPLDDIKDYEDLFTLLPLPEVAKVYQTNNSFAEQRLSGANPFVIRLLDEDDPRSQVLEQIPSFKDDFEPLFDVRKELAAGNIYICDYTGTDEYYRGPSMVQGGTYEKGRKYLPKPLAFFWWQRTGISDRGKLVPIAIQLDASKNSKVYTPTNSKVYTPFEQNPLDWLFAKLCVQIADSNHHEMSSHLCRTHFVMEPIAICTAHQLAENHPLSLLLRPHFLFMLTNNSLGQQRLINPGGPVDELLAGTLPESMELVKDAYEGWNIKEFAFPTEIKNRGMDNTERLPHYPYRDDGMLVWKAIHTFVSDYVNHFYPTPEDITGDTELQAWAKECSDQSAQTNGGKVKGMPTSFTTVQELIEIVTTIIFICGPQHSAVNYAQDGYMTFAANMPLAAYRDIPKQSHKPQDQPTATPSVAVQTTAEQTTAEQTKAVEITADKATLDQNTVLQKRAVQTTTVEIPEDQITEEQILKLLPPYKRTADQLQSLFVLSAYQYDRLGYYEKAFQQLYGDKFEDVFKDDNNQAIIAIVRQFQQNLNMVEQEIDANNKKRVVPYLYLKPSLILNSISI

[1122] Codon-optimized coding sequence of WP_015204462.1 mut3 – SEQ ID NO:261

[1123]

[1124] Amino acid sequence of WP_015204462.1 mut3 – SEQ ID NO:262

[1125] MPQPCLPQNEPNPEKRNNDLSDQQQAYEYDYKYLPPLVLLKKIPAFENFSAQYIAERVVATSELVPNMLAAKARSFLDPLDDIKDYEDLFTLLPLPEVAKVYQTNNSFAEQRLSGANPFVIRLLDEDDARSQVLEQIPSFKDDFEPLFDVEKELAAGNIYICDYTGTDEYYRGPSMVQGGTYEKGRKYLPKPLAFFWWQRTGISDRGQLVPIAIQLDPSKNSKVYTPTNSKVYTPFEQNPLDWLFAKLCVQIADANHHEMSSHLCRTHFVMEPIAICTAHQLAENHPLSLLLRPHFLFMLTNNSLGQQRLINPGGPVDELLAGTLPESMELVKDAYEGWNIKEFAFPTEIKNRGMDNTERLPHYPYRDDGMLVWKAIHTFVSDYVNHFYPTPEDITGDTELQAWAKECSDQSAQTNGGKVKGMPTSFTTVQELIEIVTTIIFICGPQHSAVNYAQDGYMTFAANMPLAAYRDIPKQSHKPQDQPTATPSVAVQTTAEQTTAEQTKAVEITADKATLDQNTVLQKRAVQTTTVEIPEDQITEEQILKLLPPYKRTADQLQSLFVLSAYQYDRLGYYEKAFQQLYNDKFEDVFKDDNNQAIIAIVRQFQQNLNMVEQEIDANNKKRVVPYLYLKPSLILNSISI

[1126] Codon-optimized coding sequence of WP_006635899.1 mut – SEQ ID NO:263

[1127]

[1128] Amino acid sequence of WP_006635899.1mut – SEQ ID NO:264

[1129] MVDNMKPCLPQDDPNPEQRHDSLNRQQQAYQFDYESLSPLALLKDVPAVENFSSKYLAERILATSELPANMLAADSRTFLDPLDELQDYEDFFTWLPLPGVAKIYQTDRSFAEQRLSGANPMVLRLLHQEDARAETLAQLCCLQPLFDLRKELQDKNIYICDYTGTDEHYRGPAKVAGGTYEKGRKYLPKPRAFFAWRWTGIRDRGEMTPIAIQLDPKPGSHLYTPFDPPIDWLYAKLCVQVADANHHEMSSHLGRTHLVMEPIAICTARQLAKNHPLSLLLKPHFRFMLTNNSLARSHLIAPGGPVDELLGGTLAETMELTREACSTWSLDEFALPAELKNRGMDDPNQLPHYPYRDDGLLLWDAIETFVSGYLKFFYPTNEGIVQDVELQTWAKECASDDGGKVKGMPHHIDTVEQLIAIVTTVIFTCGPQHSAVNFPQYDYMSFAANMPLAAYRDIPGITASGHLEVITENDILRLLPPYKRAADQLQILFILSAYRYDRLGYYDKSFRELYRMSFDEVFAGTPIQLLARQFQQNLNMAEQKIDANNQKRVIPYFALKPSLVLNSISM

[1130] Codon-optimized coding sequence of WP_015178512.1mut – SEQ ID NO:265

[1131]

[1132] Amino acid sequence of WP_015178512.1mut – SEQ ID NO:266

[1133] MVDNMKPCLPQDDPNQEQRKDSLNRQQQAYQFDYESLSPLALLKNVPAVENFSSKYIGERILATSELPANMLAADSRTFLDPLDELQDYEDFFTLLPLPAVAKIYQTDRSFAEQRLSGANPMVLRLLDAGDARAQTLAQISSFHPLFDLGQELQQKNIYVCDYTGTDEHYRAPSKIGGGSYEKGRKFLPKPRAFFAWRWTGIRDRGEMTPIAIQLDPTPDSHVYTPFDPPVDWLFAKLCVQVADANHHEMSSHLGRTHLVMEPIAICTARQLAQNHPLSLLLKPHFRFMLTNNSLARSYLIAPGGPVDELLGGTLPETMEIAREACSTWSLDEFALPAELKNRGMDDTNQLPHYPYRDDGLLLWDAIETFVSGYLKFFYPTEIAIVQDVELQTWAQECASDRGGKVKGMPPRINTVEQLIKIVTTIIFTCGPQHSAVNFPQYEYMSFAANMPLAAYRDIPKITASGNLEVITEKDILRLLPPYKRAADQLKILFTLSAYRYDRLGYYDKSFRELYRMSFDEVFAGTPIQLLARQFQQNLNMAEQKIDANNQKRVIPYIALKPSLVINSISM

[1134] Codon-optimized coding sequence of WP_028091425.1mut – SEQ ID NO:267

[1135]

[1136] Amino acid sequence of WP_028091425.1mut – SEQ ID NO:268

[1137] MQPCLPQNDPNPSQRQSSLEKGRKEYQFMYDFLPPMAMIKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMDARFAFTIEELQDKFGSSINLIERLATGNLYVCDYRSLAFIQGGTYAKGKKYLPAPLAFFCWRTSGFQDRGQLVPVAIQINPKAGKASPLLTPFDDPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVCTPRQLAENHPLRILLKPHFRFMLANNSLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVNDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYQSSADLKADAELQAWARECVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQPIQQKGDIKDRQALIDFLPPAKPTSTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELNMVQRKIELNNKRRLVNYKYLQPRLILNSISI

[1138] Codon-optimized coding sequence of OBQ01436.1mut – SEQ ID NO:269

[1139]

[1140] Amino acid sequence of OBQ01436.1mut – SEQ ID NO:270

[1141] MQPCLPQNDPNPAQRQSCLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPILQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMDARFAFTIEELQAKFGNSINLIERLATGNLYVCDYRSLAFIQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGKASPLLTPFDDPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVCTPRQLAENHPLRILLRPHFRFMLANNSLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARECVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQEIQQNGDIEDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELSVVQRKIELNNKGRLVNYEYLQPGLILNSISI

[1142] Codon-optimized coding sequence of OBQ25779.1mut – SEQ ID NO:271

[1143]

[1144] Amino acid sequence of OBQ25779.1mut – SEQ ID NO:272

[1145] MINIMQPCLPQNDPNPGQRQSSLEKGRKEYQFMYDFLPPMAMLKSVPPAENFSTKYIAERTLEAAELPLNMMAVKTHAMWDPLDELQDYEDFFPVLQKPNVMKTYETDDSFAEQRLCGVNPMVLRQIKQMDARFAFTIEELQAKFGNSINLIERLATGNLYVCDYRSLAFIQGGTYAKGKKYLPAPLAFFCWRSSGFQDRGQLVPVAIQINPKAGQASPLLTPFDKPLTWFYAKSCVQIADANHHEMSSHLCRTHLVMEPFAVCTPRQLAENHPLRILLKPHFRFMLANNSLARKRLVSRGGFVDELLAGTLQESLQIVVDAYKSWSLDQFALPRELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLQLYYKSPADLKADGELQAWARECVAQDGGRVKGMSDRIDTLEQLVEIVTTIIYICGPQHSAVNFSQYEYMGFIPNMPLAAYQAIQQKGDIKDRQALIDFLPPAKPTNTQLSTVYILSDYRYDRLGYYEEEEFTDPNADQVVNKFQQELNVVQRKIELNNKGRLVNYEYLQPRLILNSISI

[1146] Codon-optimized coding sequence of WP_039200563.1mut – SEQ ID NO:273

[1147]

[1148] Amino acid sequence of WP_039200563.1mut – SEQ ID NO:274

[1149] MKPCLPQNDPNPTQRQSSLEKGRKEYEFRYDFLPPMAMLKNVPPSENFSTKYIAERTIETAELPSNMMAVKAHAMWDPLDELQDYEDFFPVLQKPNVMKNYETDDSFAEQRLCGVNPVVLCQIKQMDARFAFTIEELQAKFGNSIDLRERLATGNLYVCDYRPLAFIRGGTFAKGKKYLPAPLAFFCWRSSGFQDRGQLVPIAIQINPKEGKASPLLTPFDDSSTWFYAKSCVQIADANHHEMSSHLCRTHFVMEPFAVCTPRQLAQNHPLRILLKPHFRFMLANNSLGRQRLVNRGGPVDELLAGTLQESLQIVVDAYTDWRLDQFALPTELKNRGVDDVKNLPHYPYRDDGILLWNAINKFVFNYLELYYKSPADLTADVELQAWARECVAQDGGRVKGMSDRIDTLKQLVEIVTTIIYTCGPLHSAVNFPQYEYMGFIPNMPLAAYQPIKKEGVCTRKELIDFLPAAKPTSSQLTTLFTLSAYRYDRLGYYEEEEFEDPNADDVVNKFQQELNVVQRKIELSNKGRLVNYEYLQPRLILNSISI

[1150] Codon-optimized coding sequence of WP_012407347.1mut – SEQ ID NO:275

[1151]

[1152] Amino acid sequence of WP_012407347.1mut – SEQ ID NO:276

[1153] MKPCLPQNDPDPTKRQILLERNQGEYEFDYDFLVPMAMLKNVPSIENFSTKYIAERTLETAELPINMLAVKTRSLWDPLDELQDYEDYFPVLPKPNIIKTYQSDDSFCEQRLCGANPFVLRRIEQMDARFAFTILELQEKFGDSINLVEKLANGNLYVCDYRALAFVKGGSYERGKKFLPTPIAFFCWRSSGFSDRGQLVPIVIQINPADGKQSQLITPFDDPLTWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAICTARQLAENHPLSLLLKPHFRFMLANNSLARKRLISRGGPVDELLAGTLQESLQIVVNAYTEWSLDQFSLPTELKNRGMDDPDNLPHYPYRDDGLLLWNAIKKFVSEYLQIYYKTPQDLAEDLELQSWVQECVSQSGGRVKGISDRINTLDQLVDIATAVIFTCGPQHAAVNYSQYEYMTFMPNMPLAAYKQMTSEGTIPDRKSLLSFLPPSKQTADQLSILFILSAYRYDRLGYYDDKFLDPEAQDVLAKFQQELNEAEREIELNNKSRLINYNYLKPRLVTNSISV

[1154] Codon-optimized coding sequence of WP_027843955.1mut – SEQ ID NO:277

[1155]

[1156] Amino acid sequence of WP_027843955.1mut – SEQ ID NO:278

[1157] MKPCLPQNDPNPEKRKDWLNKNREEYQFNFNYLSPLPLIDDVPNNEAFSPKYLAERLPLTFGKLSANTLGIRLRSFWDPFDEFQDYEDFFPVLPTPELLKTYQNDEYFAEQRLSGVNPMVIRSIKELDARFAFSIRDLQAEFGTSLNLEQELNNGNLYICDYTSLSFVRGGSYLRGRKSLPAPIALFCWRNSGYCDRGELTPIAIQLVPELGTGSRILTPFDSHLNWLYAKICMQIADANHHEMSSHLCHTHLVMEPFAVCTARQLAENHPLGLLLRPHFRFMLHNNSLARKNLINQGGYVDNLLGGTLRESLQIVRDAYFKNAEEFWSLDEFALPKEIANRGLDDTDRLPHYPYRDDGMLLWNAIEKFVSNYLSIYYPNPGDIKDDRELQAWAAECVAADGGRVKGVPSQFENLQQLIDVVTGIIFTCGPQHSAVNYPQYEYMAFVPNMPLAGYQAVDSNPNMDLKSLMAFLPPPNQTADQLQIIYGLSAYRYDRLGYYDREFSDPHAEEVVRLFQQDLNQVERKIELRNKNRLVEYNFLKPSLVLNSISI

[1158] Codon-optimized coding sequence of WP_073641301.1mut – SEQ ID NO:279

[1159]

[1160] Amino acid sequence of WP_073641301.1mut – SEQ ID NO:280

[1161] MKPCLPQNDPDPIKRKYSLEHKKEEYEFDHDFLSPMAMLKDVPAVENFSTRYIAERTVETAELPINMLAVKTRALWDPLDELQDYEDYFPVLPKPNVIKTYQTDDSFCEQRLCGANPMALQQIKEMDARFEFTIEELQEKFGESINLVEKLADGNLYVCDYRPLSFVKGGTYERGKKYLPTPLAFFCWRSSGFSDRGQLVPIAIQLNPAVGRQSQLITPFDDPLTWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAICTARQLADNHPLNLLLKPHFRFMLANNSLGRKRLVNRGGPVDELLAGTLQESLQIVVNAYKEWSLDEFALPTEIKNRGMDDKLKLPHYPYRDDGMLLWNAIKKFVSEYLKLYYKTPQDLTADLELQAWAQECVSESGGRVKGVPSRIEKLEQLVDIATAVIFTCGPQHAAVNYSQYEYMTFMPNMPLAAYKQMTAEGTIADRKSLLSFLPPSKQTADQLSILFILSAYRYDRLGYYDDKFADPEAQDILVTFQQDLNEVERKIELNNKSRLIKYNYLKPRLVTNSISV

[1162] Codon-optimized coding sequence of WP_096647440.1mut – SEQ ID NO:281

[1163]

[1164] Amino acid sequence of WP_096647440.1mut – SEQ ID NO:282

[1165] MKPCLPQNDPEPTQRKNFLERKQGEYEFDHKFLKPMAMLKNVPSIENFSTKYIAERTVETAELPLNMLAVKTRSLWDPLDELQDYEDYFPVLPKPNVIKTYQTDNSFCEQRLCGANPLVLRQIQQMDARFAFTISELQEKFGDSIDLEERLKTGNLYVCDYRALAFVKGGTYERGKKYLPTPIAFFCWRSSGFSDRGQLVPIAIQINPTDGKQSQLITPFDEPLVWFHAKLCVQIADANHHEMSSHLCRTHFVMEPFAICTARQLADNHPLNLLLKPHFRFMLANNSLGRQRLVNRGGPVDELLAGTLQESLQIVVNAYKEWSLDQFSLPTELKNRGMDNSDKLPHYPYRDDGLLLWNAIKKFVSEYLKLYYKTPQDLTADFELQSWAQECVSQSGGRVKGVSDRITTLDQLIDIATAVIFTCGPQHAAVNYSQYEYMTFIPNMPLAAYKQITSEGNIPDRKSLLSFLPPSKQTADQLSILFILSAYRYDRLGYYDDKFLDPEAQEILVTFQQELNEAERQIELNNKSRLINYDYLKPRLVTNSISV

[1166] Codon-optimized coding sequence of WP_099099431.1mut – SEQ ID NO:283

[1167]

[1168] Amino acid sequence of WP_099099431.1mut – SEQ ID NO:284

[1169] MKPCLPQKDPDVKVRINWLDKNREEYKFNYDYLAPLPVIDKVPHKEIFSAEYTTKRLASMASLAPNMLAAKARNFLDPLDELEEYEELLSLLPKPDVIKNYKTDSCFAEQRLSGANPLAIQKIDVLDARFAVTDAHFQKVAGTEFTLEKALKEGKLYFCDYPLLSDIKGGVYNNVKKYLPKPQALFYWQSNDSPNGGSLVPVAIQINHDSGGKSVIYTPDDPHLDWFLAKTCVQIADGNHQELGSHFAYTHAVMAPFAICTARQLAENHPIALLLKPHFRFMLFDNSLGRTQFLQPGGPVDEFMAGSLAESLGFVAKVYEEWSVEKFTFPRLIKSRRTDDPEILPHFPFRDDGMLIWNAVEKFVYEYLQLYYKTSQDLIDDYELQNWARECVAQDGGRVKGMPAKIETLEQLIEIISVVVFTCAPLHSALNFSQYEYMAFVPNMPYAAYHPIPETKGVDLETIMKILPPFKQAADQVMWTEILTSYHYDKLGFYDEEFADPLAQEIVVQFQQNLHEIERQIDIRNQTRPIPYNYFKPSQIINSINT

[1170] Codon-optimized coding sequence of WP_052672367.1mut – SEQ ID NO:285

[1171]

[1172] Amino acid sequence of WP_052672367.1mut – SEQ ID NO:286

[1173] MKPCLPQHEPDAIARQNRLIKNRADYVLDYNYLPPIPLQTPVPQQERFSAEYTARRLASFANLVPNMLMARARNAFDPLDTLEEYADLLPVLPKPNVIKNYQADWCFAEQRLSGINPPAIRRIDALDARLPISNSSFQHSVGAEHNLEQALKEGKLYCCDYPLLSGIGGGNYQNLPKYLPKPQALFYWRSDNSKIGGSLVPVAIKILNELGGKNLVYTPNDAPLDWFLAKTCVQMADANHQELGTHFAKTHAVMAPIAACTARELGENHPLTLLLKPHFRFMLFDNSLGRTQFLQPTGPTEELLAGTLEESVQLVVQAYEEWSIDTTFPLELQQRQMHDPEILPHYPFRDDGILVWNAIHQFVTEYLQIYYHTPQDISADYEVQNWARECVDSGRVKGMPESIDTLAQLIDIIAVVIFTCAPLHSCLNLAQYEYMTFVPNMPYAAYHPIPTTKGVDMATIVKIMPPFQRAIDQILWTDILSAFQYDKLGFYEEDFADPKAQEVLQRFQDNLQQVEEKIEMHNQIRPIPYNYLKPSRIMNSINT

[1174] Codon-optimized coding sequence of WP_073631249.1mut – SEQ ID NO:287

[1175]

[1176] Amino acid sequence of WP_073631249.1mut – SEQ ID NO:288

[1177] MKPCLPQHDPNPEARRNWLEQNREDYKFDHNYLAPIPILDKVPHKELFSPQYTAKRLASMADLVPNMLAAKARNFFDPLDELEEYEALLSILPKPSVIKNYKTDSCFAEQRLSGANPMAMHRIDELDARFPVTNDHFQKAVGAEHNLEAALKEGKLYLCDYPLLFDIKGGTYQNIKKYLPKPQALFYWQSNGNKNSGSLVPIAIQIHNDTGGDSLIYTPDDPHLDWFLAKTCVQIADANHQELGSHFARTHAVMAPFAICTARQLGENHPLALLLKPHFRFMLYDNSLGRTHFLQAGGPVDEFMAGTLQESLGFVAKAYEEWSLDNAVFPTEVKNRKMDDPDILPHYPFRDDGMLLWDAVKKFVTEYLQLYYKTPQDLSEDYELQNWARECAAQDGGCVKGMPEKIETIEQLIHVVTVVVFTCAPLHSALNFSQY

[1178] EYMAFVPNMPYAAYYPVPETKGVDMQTIMKMLPPFKQAADQVMWSDILTSFHYDKLGHYDEEFANPMAQ

[1179] AILLQFQQNLHEVERQIEIKNQSRPIPYNYLKPSEIINSINT

[1180]

[1181] Amino acid sequence of WP_013220336.1mut – SEQ ID NO:290

[1182] MNTCLPQNDSDPQGRKDRLERRRALYVFNYDYVPPIPMIDKVPHEEYFSPKYTAERLASMAKLAPNMLAAKTKRLFDPLDELNEYDEMFIFLDKPGIVRGYRTDESFGEQRLSGVNPMSIRRLDKLDARFPIMDEYLEQSLGSPHTLAQALQEGRLYFCDYPQLAHVKEGGLYRGRKKYLPKPRALFCWDGNHLQPVAIQISGQPGGRLFIPRDSDLDWFVAKLCVQIADANHQELGTHFARTHVVMAPFAVCTHRQLAENHPLHILLRPHFRFMLYDNSLGRTRFIQPDGPVEHMMAGTLEESIGISAAFYKEWRLDEAAFPIEIARRKMDDPEVLPHYPFRDDGMLLWDGIQKFVKEYLALYYQSPEDLVQDQELRNWARECTANDGGRVAGMPGRIETVDQLTSILSTVIYTCAPLHSALNFAQYEYIGYVPNMPYAAYHPIPEEGGVDMETLMKILPPYEQAALQLKWTEILTSYHYDRLGHYDEKFEDPQAQAVVEQFQQELAAVEQEIDQRNQDRPLAYTYLKPSEIINSINT。

Claims

1. A method for preparing at least one mono- or poly-unsaturated aliphatic aldehyde, the method comprising: (1) contacting at least one polyunsaturated fatty acid (PUFA) substrate with a polypeptide, The polypeptide contains lipoxygenase enzymatic activity and: a) the amino acid sequence of the polypeptide is SEQ ID NO: 18, or b) the polypeptide is a mutant of SEQ ID NO: 18, wherein 1 to 10 amino acid mutations are made at sequence positions different from potential critical positions selected from C7, D134, R136, C161, A219, S256, C278, S305, C409 and G526 of SEQ ID NO: 18, and the mutant retains the enzymatic activity of lipoxygenase; or c) the polypeptide is a mutant retaining the enzymatic activity of lipoxygenase, and its amino acid sequence is selected from SEQ ID NO: 254, 256, 258, 260, 262, 262, 264, 266 and 284, thereby converting the at least one PUFA compound into a reaction product comprising at least one mono- or polyunsaturated aliphatic aldehyde; and (2) Optionally, the at least one mono- or poly-unsaturated aliphatic aldehyde obtained in step (1) is isolated.

2. The method of claim 1, wherein the polypeptide comprises the enzymatic activity of a bifunctional lipoxygenase.

3. The method according to claim 2, wherein the enzymatic activity of the bifunctional lipoxygenase is a combination of lipoxygenase and hydroperoxide lyase activities.

4. The method of claim 1, wherein the polypeptide has the ability to convert at least one PUFA into at least one mono- or poly-unsaturated aliphatic aldehyde.

5. The method according to claim 4, wherein the PUFA is selected from omega-3 and omega-6 PUFA.

6. The method according to claim 4, wherein the at least one mono- or polyunsaturated aliphatic aldehyde is at least one polyunsaturated aliphatic C 10 -aldehyde.

7. The method according to claim 6, wherein the at least one polyunsaturated aliphatic C 10 - the aldehyde is selected from decadienal and decatrienal.

8. The method according to claim 7, wherein the decadienal is selected from 2E,4E-decadienal and 2E,4Z-decadienal and mixtures thereof; and wherein the decatrienal is selected from 2E,4E,7Z-decatrienal and 2E,4Z,7Z-decatrienal and mixtures thereof.

9. The method according to claim 1, wherein the PUFA is selected from C 16 -C 22 -PUFA.

10. The method according to claim 9, wherein the PUFA is selected from omega-3C 16 -C 22 -PUFA and omega-6C 16 -C 22 -PUFA.

11. The method according to claim 9, wherein the PUFA is selected from omega-3C 18 -C 20 -PUFA and omega-6C 18 -C 20 -PUFA.

12. The method of claim 9, wherein the PUFA is selected from the group consisting of (4Z,7Z,10Z,13Z)-hexadecane-4,7,10,13-tetraenoic acid, SDA, EPA, DHA, GLA and ARA.

13. The method of claim 1, wherein step (1) is performed in vivo in cell culture in the presence of oxygen, or in vitro in a liquid reaction medium in the presence of oxygen.

14. The method of claim 1, wherein step (1) is performed by culturing a non-human host organism or cell expressing at least one polypeptide having the enzymatic activity of a lipoxygenase in the presence of a PUFA substrate under conditions that favor peroxidation and subsequent cleavage of at least one PUFA.

15. The method of claim 1, wherein the PUFA substrate is an isolated PUFA compound or a natural or synthetic composition comprising at least one PUFA convertible by the lipoxygenase; and wherein the natural PUFA composition is selected from: a) Borage oil, b) arachidonic acid oil, c) corresponding oil hydrolysates of fish oil, d) a mixture of LA and ALA; and e) A mixture comprising at least two of a) to d).

16. The method according to claim 1, further comprising chemical or enzymatic isomerization of the obtained mono- or poly-unsaturated aliphatic aldehyde; or chemical or enzymatic conversion of the obtained mono- or poly-unsaturated aliphatic aldehyde to the corresponding alcohol or hydrocarbyl ester.

17. A polypeptide comprising the enzymatic activity of lipoxygenase, wherein: a) the amino acid sequence of the polypeptide is SEQ ID NO: 18, or b) the polypeptide is a mutant of SEQ ID NO: 18, wherein 1 to 10 amino acid mutations are made at sequence positions different from potential critical positions selected from C7, D134, R136, C161, A219, S256, C278, S305, C409 and G526 of SEQ ID NO: 18, and the mutant retains the enzymatic activity of lipoxygenase; or c) the polypeptide is a mutant retaining the enzymatic activity of lipoxygenase, and its amino acid sequence is selected from SEQ ID NO: 254, 256, 258, 260, 262, 262, 264, 266 and 284.

18. A nucleic acid encoding the polypeptide of claim 17 or a complementary sequence thereof.

19. The nucleic acid according to claim 18, wherein the encoding nucleotide is: a) SEQ ID NO: 16 or 17; b) a nucleotide sequence having at least 98% sequence identity with at least one of the sequences in a) and encoding a polypeptide having the enzymatic activity of lipoxygenase; c) a nucleotide sequence selected from the group consisting of SEQ ID NO: 253, 255, 257, 259, 261, 263, 265 and 283; or d) the complement of any one of sequences a), b), or c).

20. An expression vector comprising the encoding nucleic acid according to any one of claims 18 and 19.

21. A recombinant non-human host organism or cell carrying at least one nucleic acid according to any one of claims 18 and 19, or carrying at least one expression vector according to claim 20.

22. A method for producing at least one polypeptide according to claim 17, the method comprising: a) cultivating a non-human host organism or a cell carrying at least one nucleic acid according to any of claims 18 and 19 and expressing or overexpressing at least one polypeptide according to claim 17; b) Optionally, isolating the polypeptide from the non-human host organism or cell cultured in step a).

23. A method for preparing a mutant polypeptide capable of converting at least one polyunsaturated fatty acid selected from omega-3 or omega-6 polyunsaturated fatty acids into at least one mono- or polyunsaturated aliphatic aldehyde, the method comprising the steps of: a) selecting a nucleic acid according to any one of claims 18 and 19; b) modifying the selected nucleic acid to obtain at least one mutant nucleic acid; c) providing the mutant nucleic acid sequence to a host cell or a unicellular organism to express a polypeptide encoded by the mutant nucleic acid sequence; d) screening for at least one mutant polypeptide having the activity of converting at least one polyunsaturated fatty acid selected from omega-3 or omega-6 polyunsaturated fatty acids into at least one mono- or polyunsaturated aliphatic aldehyde; e) optionally, if the mutant polypeptide does not have the desired activity, repeating the process of steps a) to d) until a polypeptide having the desired activity is obtained; and, f) Alternatively, if a mutant polypeptide having the desired activity is identified in step d) or e), the corresponding mutant nucleic acid is isolated.

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