HYDROCARBON-FORMING OXIDATIVE DECARBONYLASE ENZYME, HYDROCARBONS PRODUCED THEREBY, AND METHOD OF USE

Abstract

The present disclosure relates to oxidative decarbonylase enzymes, methods of making hydrocarbons with such enzymes, hydrocarbons produced therefrom and uses thereof. More particularly, the present disclosure relates to isolated polypeptide sequences that are cytochrome P450 enzymes with oxidative decarbonylase activity and methods of their use to generate hydrocarbon products, such as biofuels.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 12/620,328, filed Nov. 17, 2009, which in turn claims the benefit of U.S. Provisional Application No. 61/115,382 filed Nov. 17, 2008, both of which are herein incorporated by reference in their entirety.

FIELD

[0003] The present disclosure generally relates to a hydrocarbon-forming oxidative decarbonylase enzyme, hydrocarbons produced thereby and uses thereof.

BACKGROUND

[0004] Hydrocarbons are ubiquitous compounds in nature. The surface waxes of plants and insects contain very-long chain, non-isoprenoid hydrocarbons of 21 to over 50 carbons. Plant cuticular hydrocarbons are generally straight-chain, n-alkanes whereas insect cuticular hydrocarbons also often contain methyl-branched and unsaturated components. Long-chain hydrocarbons are also present in algae, uropygial glands of water birds, and in small amounts in many other organisms. Long-chain hydrocarbons of insects play central roles in waterproofing the insect cuticle and function extensively in chemical communication where relatively non-volatile chemicals are required. The recognition of the roles that hydrocarbons serve as sex pheromones, kairomones, species and gender recognition cues, nestmate recognition, dominance and fertility cues, chemical mimicry, primer pheromones, task specific cues and even as cues for maternal care of offspring has resulted in an explosion of new information in this area.

[0005] The ability of insects to withstand desiccation was recognized in the 1930s to be due to the epicuticular wax layer on the cuticle. The development and application of combined gas-liquid chromatography and mass spectrometry allowed rapid and efficient analyses of insect hydrocarbons. In the late 1960s and during the next few decades, it was recognized that for many insect species, very complex mixtures of normal (straight-chain), methyl-branched and unsaturated components existed, with chain lengths ranging from 21 to 50+ carbons. It was also recognized that the variety of chain lengths, the number and positions of the methyl branches and double bonds provided insects with the chemical equivalent of the visually variable colored plumage of birds.

[0006] Insects synthesize hydrocarbons by elongating fatty acyl-CoAs to produce the very long-chain fatty acids that are then converted to hydrocarbons by loss of the carboxyl group. Methyl-branched hydrocarbons (with the exception of 2-methylalkanes) arise from the incorporation of a propionyl-CoA group (as methylmalonyl-CoA derived from valine, isoleucine or methionine) in place of an acetyl-CoA group at specific points during chain elongation. 2-Methylalkanes arise from the elongation of the carbon skeleton of either valine (even number of carbons in the chain) or isoleucine (odd number of carbons in the chain). Insect hydrocarbon biosynthesis occurs in oenocytes (large secretory cells found in clusters underlying the epidermis of larval abdominal segments).

[0007] Although it is now clear that fatty acyl-CoAs are reduced to aldehydes and then converted to hydrocarbons by the loss of the carbonyl carbon, the mechanism by which the latter step occurs remains to be identified.

SUMMARY

[0008] Disclosed is the surprising identification of the mechanism by which fatty aldehydes are converted into hydrocarbons. Isolated polypeptides that are cytochrome P450 enzymes that have hydrocarbon-forming oxidative decarbonylase activity are disclosed. Cells, for instance fungal or bacterial cells, transformed with one or more nucleic acid sequences that encode one or more of the disclosed polypeptides, can be used as a source for hydrocarbons. As such, this discovery provides methods of producing hydrocarbons that can be used for the production of a wide range of products, such as hydrocarbon sex-pheromone components for Musca domestica control, biofuels, lubricants, or solvents.

[0009] One embodiment of the disclosure is an isolated polypeptide with an amino acid sequence set forth by SEQ ID NO: 1 or 2 or a sequence having at least 95% sequence identity, such as 99% sequence identity with SEQ ID NO: 1 or 2. Another embodiment is a polynucleotide that encodes an isolated polypeptide with an amino acid sequence set forth by SEQ ID NO: 1 or 2 or a sequence having at least 95% sequence identity, such as 99% sequence identify with SEQ ID NO: 1 or 2.

[0010] Another embodiment is a method of producing a hydrocarbon. In one example, the method includes transforming a cell with a recombinant construct containing a promoter operably linked to a nucleic acid sequence, wherein the nucleic acid sequence encodes a protein comprising SEQ ID NO: 1 or 2 or a sequence having at least 95% sequence identity with SEQ ID NO: 1 or 2 and culturing the cell under conditions wherein the cell expresses the protein, thereby producing the hydrocarbon.

[0011] In another example, the method of producing a hydrocarbon includes methods of making hydrocarbons in vitro, or partially in vitro. For example, one or more of the peptides described herein can be isolated and then allowed to react with a substrate in vitro to make an intermediate. That intermediate can then be added to a cell culture wherein the cells convert the intermediate to the desired product. In instances where the desired product is made entirely in vitro all of the necessary enzymes are reacted in vitro. However, the enzymes can be added sequentially or simultaneously and at various stages in the reaction, for example after intermediate purification or partial purification.

[0012] Also disclosed are embodiments of a method for using the disclosed enzymes and hydrocarbons produced therefrom. One use of the disclosed enzymes is the production of synthetic hydrocarbon sex-pheromone components for Musca domestica control. Another use is the production of hydrocarbons as biofuels, either in vitro, or by inserting the isolated disclosed sequences, such as SEQ ID NO: 1 or 2 (or related sequences, see Table A) into an organism (e.g., plant, bacteria, algae, etc.) in order to alter the hydrocarbon content, such as increasing the content, for production of fuel, lubricant, solvent, etc. For example, biofuel produced by a provided method is disclosed.

[0013] The foregoing and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures and sequence listing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1A is a schematic diagram illustrating an exemplary pathway of hydrocarbon biosynthesis in accordance with the present disclosure in which the oxidative decarbonylation pathway uses cytochrome P450 and produces carbon dioxide while the decarbonylation pathway produces carbon monoxide.

[0015] FIG. 1B is a schematic diagram illustrating the function of a hydrocarbon-forming oxidative decarbonylase according to the present disclosure, which converts fatty aldehydes to linear hydrocarbons.

[0016] FIG. 2A is a bar graph and a pair of tracings illustrating that in housefly microsomes, incubation of (Z)-15-[1-¹⁴C]- and (Z)-15-[15,16-³H₂]tetracosenoyl-CoA and the corresponding aldehydes in the presence of NADPH gave equal amounts of ¹⁴CO₂ and [³H]-(Z)-9-tricosene.

[0017] FIG. 2B is a bar graph demonstrating that NADPH and oxygen are required for hydrocarbon formation from 24:1-CoA.

[0018] FIG. 3 is a bar graph illustrating the amount of hydrocarbon production for control and knocked down (1405Cyp and M485Cyp) flies.

[0019] FIG. 4 is an image of a northern blot of housefly RNA isolated from male and female integuments and fat bodies hybridized with labeled CYP4G2 and actin (housekeeping control gene) cDNAs.

[0020] FIG. 5 is a representative gas chromatography (GC) profile of male 485 line D. melanogaster that have either knocked down CYP4G1 (lower line) or wildtype activity (upper line). Carbon lengths of various hydrocarbons are noted above their corresponding peaks.

[0021] FIG. 6 is a representative GC profile of male 1405 line D. melanogaster that have either knocked down CYP4G1 (lower line) or wildtype activity (upper line). Carbon lengths of various hydrocarbons are noted above their corresponding peaks.

[0022] FIG. 7 is a plot of hydrocarbon content (HC) vs cis-vaccinyl acetate content for male and female 485 and 1405 line D. melanogaster. Control flies have normal CYP4G1 activity, while the "Cyp" samples have CYP4G1 activity removed by RNAi. Each point represents an individual fly.

[0023] FIG. 8 is a ClustalW 2.0 multiple sequence alignment of Homo sapiens cytochrome CYP3A4 (SEQ ID NO: 48, GenBank Accession No. P08684, PDB 1TQN, top), Musca domestica CYP4G2 (SEQ ID NO: 1, middle), and Mycobacterium tuberculosis P450 51 (SEQ ID NO: 49, GenBank Accession No. P08512, PDB 2CIB, bottom). Invariant glycines and prolines (designated by a "G" and "P", respectively) that are conserved in all three sequences are boxed; conserved cysteines are double-underlined and a C; and the highly conserved region is overlined and the less conserved region is in not.

[0024] FIG. 9 is a digital image of a model of Homo sapiens microsomal cytochrome P450 3A4 (PDB 1TQN) illustrating the variant regions.

[0025] FIG. 10 is a digital image of a model of Homo sapiens microsomal cytochrome P450 3A4 (PDB 1TQN) exemplifying both the conserved and less conserved regions are intermixed unlike the alignment (FIG. 8) where there are distinct regions.

[0026] FIG. 11 is a tracing illustrating Drosophila melanogaster CYP4G1 expressed in yeast. The full-length CYP4G1 sequence recoded for optimal yeast codon usage was cloned into the pYeDP60 vector and expressed in a modified WR yeast strain after induction by galactose. The CO, reduced difference spectrum of yeast microsomes shows approx. 50 pmol CYP4G1/mg protein.

SEQUENCE LISTING

[0027] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0028] SEQ ID NO: 1 is the amino acid sequence of cytochrome P450 4g2 (CYP4G2).

[0029] SEQ ID NO: 2 is the amino acid sequence of cytochrome P450 4g1 (CYP4G1).

[0030] SEQ ID NO: 3 is the amino acid sequence of GG12761 from Drosophila erecta.

[0031] SEQ ID NO: 4 is the amino acid sequence of GD24639 from Drosophila simulans.

[0032] SEQ ID NO: 5 is the amino acid sequence of GE16587 from Drosophila yakuba.

[0033] SEQ ID NO: 6 is the amino acid sequence of GA17813 from Drosophila pseudoobscura.

[0034] SEQ ID NO: 7 is the amino acid sequence of GI11123 from Drosophila mojavensis.

[0035] SEQ ID NO: 8 is the amino acid sequence of GJ15981 from Drosophila virilis.

[0036] SEQ ID NO: 9 is the amino acid sequence of GF20812 from Drosophila ananassae.

[0037] SEQ ID NO: 10 is the amino acid sequence of GH24346 from Drosophila grimshawi.

[0038] SEQ ID NO: 11 is the amino acid sequence of GK25658 from Drosophila willistoni.

[0039] SEQ ID NO: 12 is the amino acid sequence of cytochrome P450 4g15 from Culex quinquefasciatus.

[0040] SEQ ID NO: 13 is an amino acid sequence of cytochrome P450 from Aedes aegypti.

[0041] SEQ ID NO: 14 is the amino acid sequence of cytochrome P450 from Aedes aegypti.

[0042] SEQ ID NO: 15 is an amino acid sequence similar to cytochrome P450 from Nasonia vitripennis.

[0043] SEQ ID NO: 16 is an amino acid sequence similar to cytochrome P450 monooxygenase from Nasonia vitripennis.

[0044] SEQ ID NO: 17 is the amino acid sequence of cytochrome P450 family 4 from Chironomus tentans.

[0045] SEQ ID NO: 18 is the amino acid sequence of AGAP000877-PA from Anopheles gambiae str. PEST.

[0046] SEQ ID NO: 19 is the amino acid sequence of AGAP001076-PA from Anopheles gambiae str. PEST.

[0047] SEQ ID NO: 20 is the amino acid sequence of cytochrome P450 monooxygenase CYP4G7 from Tribolium castaneum.

[0048] SEQ ID NO: 21 is the amino acid sequence of cytochrome P450 monooxygenase Tribolium castaneum.

[0049] SEQ ID NO: 22 is the amino acid sequence of cytochrome P450 from Bombyx mori.

[0050] SEQ ID NO: 23 is the amino acid sequence of cytochrome P450 4G25 (CYP4G25) Bombyx mori.

[0051] SEQ ID NO: 24 is the amino acid sequence of cytochrome P450 from Leptinotarsa decemlineata.

[0052] SEQ ID NO: 25 is the amino acid sequence of GM13084 from Drosophila sechellia.

[0053] SEQ ID NO: 26 is the amino acid sequence of cytochrome P450 monooxygenase from Apis mellifera.

[0054] SEQ ID NO: 27 is the amino acid sequence of cytochrome P450 4G19 monooxygenase (CYP4G19) from Blattella germanica.

[0055] SEQ ID NO: 28 is the amino acid sequence of CYP4G27 from Ips paraconfusus.

[0056] SEQ ID NO: 29 is the amino acid sequence of cytochrome P450 4G25 (CYP4G25) from Antheraea yamamai.

[0057] SEQ ID NO: 30 is the amino acid sequence of antennal cytochrome P450 4 (CYP4) from Mamestra brassicae.

[0058] SEQ ID NO: 31 is an amino acid sequence similar to cytochrome P450 from Acyrthosiphon pisum.

[0059] SEQ ID NO: 32 is the amino acid sequence of GL20168 from Drosophila persimilis.

[0060] SEQ ID NO: 33 is the amino acid sequence of cytochrome P450 4C1 (CYPIVC1) from Blaberus discoidalis (roaches).

[0061] SEQ ID NO: 34 is the amino acid sequence of cytochrome P450 4C39 (CYP4C39) from green crab, common shore crab.

[0062] SEQ ID NO: 35 is the amino acid sequence of hypothetical protein BRAFLDRAFT_--57954 from Branchiostoma floridae.

[0063] SEQ ID NO: 36 is the amino acid sequence of cytochrome 4V6 from Balaenoptera acutorostrata.

[0064] SEQ ID NO: 37 is the amino acid sequence of cytochrome P450 4M6 monooxygenase (CYP4M6) from Helicoverpa zea.

[0065] SEQ ID NO: 38 is the amino acid sequence of cytochrome P450 4 family from Daphnia magna.

[0066] SEQ ID NO: 39 is the amino acid sequence of cytochrome P450 from Nilaparvata lugens.

[0067] SEQ ID NO: 40 is the amino acid sequence of hypothetical protein L00562008 Danio rerio.

[0068] SEQ ID NO: 41 is the amino acid sequence of cytochrome P450, family 735, subfamily A, polypeptide 1 (CYP735A1) oxygen binding protein from Arabidopsis thaliana.

[0069] SEQ ID NO: 42 is the amino acid sequence of cytochrome P450 like protein from Arabidopsis thaliana.

[0070] SEQ ID NO: 43 is the amino acid sequence of hypothetical protein OsI_--028301 from Oryza sativa (indica cultivar-group).

[0071] SEQ ID NO: 44 is the amino acid sequence of hypothetical protein OsI_--003357 from Oryza sativa (indica cultivar-group).

[0072] SEQ ID NO: 45 is the amino acid sequence of hypothetical protein OsI_--005901 from Oryza sativa (indica cultivar-group).

[0073] SEQ ID NO: 46 is a nucleic acid sequence of cytochrome P450 4g2 (CYP4G2).

[0074] SEQ ID NO: 47 is the nucleic acid sequence of cytochrome P450 4g2 (CYP4G2).

[0075] SEQ ID NO: 48 is the nucleic acid sequence of cytochrome P450 4g1 (CYP4G1).

[0076] SEQ ID NO: 49 is the amino acid sequence of cytochrome CYP3A4 from Homo sapiens.

[0077] SEQ ID NO: 50 is the amino acid sequence of cytochrome P450 51 from Mycobacterium tuberculosis.

[0078] SEQ ID NO: 51 is a nucleic acid sequence of cytochrome P450 chimera 9T2/4G2.

[0079] SEQ ID NO: 52 is an amino acid sequence of cytochrome P450 chimera 9T2/4G2.

DETAILED DESCRIPTION

I. Overview of Several Embodiments

[0080] Fatty acyl-CoAs are reduced to aldehydes (FIG. 1A) and then converted to hydrocarbons by the loss of the carbonyl carbon. However, prior to the present disclosure the mechanism of the last step in this process, the conversion of aldehyde to hydrocarbon, was unclear. Previously it had been suggested that in plants, algae, vertebrates and insects, the aldehyde is decarbonylated to hydrocarbon and carbon monoxide (FIG. 1A) in a process that does not require cofactors. In contrast to these previous findings, it disclosed herein that the conversion of the aldehyde to hydrocarbon and carbon dioxide involves a cytochrome P450 enzyme with hydrocarbon-forming oxidative decarbonylase activity, molecular oxygen and NADPH (FIG. 1B).

[0081] For example, in housefly microsomes, incubation of (Z)-15-[1-¹⁴C]- and (Z)-15-[15,16-³H₂]tetracosenoyl-CoA and the corresponding aldehydes in the presence of NADPH gave equal amounts of ¹⁴CO₂ and [³H]-(Z)-9-tricosene (FIG. 2A). The formation of labeled carbon dioxide and not carbon monoxide was verified by both radio-GLC (FIG. 2B) and trapping agents.

[0082] The demonstration of a requirement for NADPH and O₂ and inhibition by CO and antibody to cytochrome P450 reductase implicated a cytochrome P450 in the reaction. However, to resolve the controversy of whether hydrocarbon formation involved decarboxylation or decarbonylation, the enzyme(s) involved in such process needed to not only be identified, but characterized both molecularly and biologically.

[0083] Herein, the inventors have identified several integument enriched cytochrome P450 cDNAs in the housefly, Musca domestica. One of these, CPY4G2 was found to have 71.7% amino acid identity and 81.8% similarity to its ortholog, CYP4G1, in Drosophila melanogaster. Two transgenic D. melanogaster lines (3972-R1 and 3972-R2) bearing CYP4G1 hairpin sequences under control of the yeast UAS promoter were crossed individually with a transgenic line carrying the Ga14 transcription factor gene under control of an oenocyte-specific promoter. Offspring from these crosses expressed CYP4G1 hairpin RNAs specifically in their oenocytes, thus triggering RNAi-mediated post-transcriptional gene silencing of CYP4G1 in oenocytes. The amount of hydrocarbon produced by these flies was less than 100 ng/fly, as compared to about 1500 ng/fly in parental insects (FIG. 3). The amount of cis-valeryl acetate was constant in test samples and control samples, indicating that fatty acid synthesis was not affected (FIG. 3).

[0084] These studies demonstrate that CYP4G2 and CYP4G1 are cytochrome P450 enzymes with oxidative decarbonylase activity involved in hydrocarbon biosynthesis and can be utilized to produce hydrocarbons, such as those used for biofuel production. For example, cells, for instance fungal, plant, or bacterial cells, that have been transformed with one or more of genes that encode these enzymes can be used as a source for hydrocarbons, such as a source for hydrocarbons that can be used as fuel in place of limited, non-renewable hydrocarbon resources. By controlling the host organism and/or the reaction substrates (for instance, controlling for chain length, branching and saturation and/or location of double bonds), microorganisms can be created that produce a wide range of hydrocarbons, including those having particular branches or unsaturated points.

[0085] One aspect of the disclosure provides isolated polypeptides that have oxidative decarbonylase activity. In one particular aspect, isolated recombinant nucleic acid sequences that encode proteins having oxidative decarbonylase activity are provided, such as isolated recombinant nucleic acid sequences that encode proteins having oxidative decarbonylase activity and share at least 95% sequence identity with amino sequence set forth by SEQ ID NO: 1 or 2. In one example, an isolated recombinant nucleic acid includes a promoter operably linked to a nucleic acid sequence encoding: (a) SEQ ID NO: 1 or 2 or (b) a sequence having at least 95% sequence identity with SEQ ID NO: 1 or 2. In some examples, the isolated recombinant nucleic acid includes a vector, and in certain examples, the vector is a plasmid, for instance pET-21b(+), pCOLADuet-1, pcDNA3.1(+), pCMV SPORT6.1, pCDFDuet-1, pENTR4 (Invitrogen), pBluescript SK- (Staratgene), pOT2 (Berkely Drosophila Resource Center), pMT-DEST48 (Invitrogen) or the vector is a virus, for instance BaculoDirect (Invitrogen).

[0086] In some examples, the isolated recombinant nucleic acid also includes at least one additional sequence, such as one or more of (a) a regulatory sequence operatively coupled to the nucleic acid; (b) a selection marker operatively coupled to the nucleic acid; (c) a purification moiety operatively coupled to the nucleic acid; (d) a secretion sequence operatively coupled to the nucleic acid; and (e) a targeting sequence operatively coupled to the nucleic acid. In certain examples, the selection marker is ampicillin/carbenicillin resistance, kanamycin resistance, chloramphenicol resistance, tetracycline resistance or bancyclovir resistance.

[0087] Also provided are cells transformed with any of the isolated recombinant nucleic acid sequences described herein, for example a bacterial cell, a yeast cell, a fungal cell, an animal cell, or a plant cell. In specific examples, the cell is an Escherichia coli cell, an Stenotrohomonas. maltophilia cell, a Kineococcus radiotolerans cell, a cell from an organism belonging to the Rhodococcus genus, a cell from an organism belonging to the Clostridium genus, a cell from an organism belonging to the Zymomonas genus, a cell from an organism belonging to the Klebsiella genus, a cell from an organism belonging to the Acinetobacter genus, a cell from an organism belonging to the Corynebacterium genus, a cell from an organism belonging to the Geobacillus genus, a cell from an organism belonging to the Proteus genus, a cell from an organism belonging to the Rhodobacter genus, a cell from an organism belonging to the Streptomyces genus, a Saccharomyces cerevisiae cell, an Aspergillus cell, a Trichoderma cell, a Neurospora cell, a Fusarium cell, a Chrysosporium cell, a Pichia cell, a Yarrowia cell, a Kluyveromyces cell, a Hansenula cell, a Schizosaccharomyces cell, or a Debaromyces cell.

[0088] Also disclosed is a bacterial cell that includes a recombinant nucleic acid encoding one or more of (a) SEQ ID NO: 1 or 2 or (b) a sequence having at least 95% sequence identity with SEQ ID NO: 1 or 2. In certain examples, the cell expresses the protein sequence of: (a) SEQ ID NO: 1 or 2 or (b) a sequence having at least 95% sequence identity with SEQ ID NO: 1 or 2. In particular examples, the expressed protein is secreted by the cell, and in even more particular examples the expressed protein has oxidative decarbonylase activity.

[0089] Other embodiments of the disclosure include a method for producing a hydrocarbon. In one example, a method for producing a hydrocarbon includes culturing a transformed cell described herein under conditions permitting expression of a protein having oxidative decarbonylase activity. In some examples of the method, the protein having oxidative decarbonylase activity includes: (a) an amino acid sequence as set forth in SEQ ID NO: 1 or 2 or (b) a sequence having at least 95% sequence identity with SEQ ID NO: 1 or 2; or (c) a combination thereof. For instance, in certain examples, the cell expresses: SEQ ID NO: 1 and SEQ ID NO: 2. In certain examples, the method also includes isolating the hydrocarbon from the cell or from the medium in which the cell is cultured, and in other examples, the method includes culturing the cell in the presence of at least one substrate of oxidative decarbonylase activity, for instance in the presence of a fatty acid, a fatty acylCoA, NADPH, NADP and/or O₂.

[0090] In some examples, a method of producing a hydrocarbon includes culturing a cell that expresses a recombinant construct containing a promoter operably linked to a nucleic acid sequence, wherein the nucleic acid sequence encodes a protein comprising SEQ ID NO: 1 or 2 or a sequence having at least 95% sequence identity with SEQ ID NO: 1 or 2; under conditions wherein the cell expresses the protein, thereby producing the hydrocarbon. In particular examples, the protein has oxidative decarbonylase activity. In some examples, the promoter is a constitutive promoter or an inducible promoter, for instance an oenocyte-specific promoter or a T7 promoter. In some examples, the cell is a bacterial cell, for instance an E. coli cell, an S. maltophilia cell, a K. radiotolerans cell, a cell from an organism belonging to the Rhodococcus genus, a Saccharomyces cerevisiae cell, an Aspergillus cell, a Trichoderma cell, a Neurospora cell, a Fusarium cell, or a Chrysosporium cell. In still other examples, the method also includes isolating the hydrocarbon from the cell or from the medium in which the cell is cultured.

[0091] In another example, the method of producing a hydrocarbon includes methods of making hydrocarbons in vitro, or partially in vitro. For example, one or more of the peptides described herein can be isolated and then allowed to react with a substrate in vitro to make an intermediate that intermediate can then be added to a cell culture wherein the cells convert the intermediate to the desired product. In instances where the desired product is made entirely in vitro all of the necessary enzymes are reacted in vitro. However, the enzymes can be added sequentially or simultaneously and at various stages in the reaction, for example after intermediate purification or partial purification.

[0092] The present disclosure also provides for the use of the disclosed enzymes and hydrocarbons produced therefrom. One use of the disclosed enzymes is the production of synthetic hydrocarbon sex-pheromone components for Musca domestica control. Another use is the production of hydrocarbons as biofuels, either in vitro, or by inserting the isolated disclosed sequences, such as CYP4G2 (or related sequences, see Table A) into an organism (e.g., plant, bacteria, algae, etc.) in order to alter the hydrocarbon content, such as increasing the content, for production of fuel, lubricant, solvent, etc.

II. Abbreviations and Terms

[0093] CO: carbon monoxide

[0094] CYP4G1: cytochrome P450 4G1

[0095] CYP4G2: cytochrome P450 4G2

[0096] GC: gas chromatography

[0097] HC: hydrocarbon content

[0098] MS: mass spectrometry

[0099] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0100] Aldehyde: An organic compound containing a terminal carbonyl group including a carbon atom bonded to a hydrogen atom and double-bonded to an oxygen atom (chemical formula O═CH--).

[0101] Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0102] The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (V_L) and "variable heavy chain" (V_H) refer, respectively, to these light and heavy chains.

[0103] As used herein, the term "antibody" includes intact immunoglobulins as well as a number of well-characterized fragments. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')₂, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

[0104] Antibodies for use in the methods and compositions of this disclosure can be monoclonal or polyclonal. These antibodies can be prepared by methods known to those of skill in the art, including as described herein (see Example Section below). Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495-97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.

[0105] Bacteria: As used herein, both Archaea and Eubacteria are encompassed by the term "bacteria." The term "Eubacteria" refers to prokaryotic organisms that are distinguishable from Archaea. Similarly, "Archaea" refers to prokaryotes that are distinguishable from Eubacteria. Eubacteria and Archaea can be distinguished by a number morphological and biochemical criteria known in the art. For example, differences in ribosomal RNA sequences, RNA polymerase structure, the presence or absence of introns, antibiotic sensitivity, the presence or absence of cell wall peptidoglycans and other cell wall components, the branched versus unbranched structures of membrane lipids, and the presence/absence of histones and histone-like proteins are used to assign an organism to Eubacteria or Archaea.

[0106] Examples of Eubacteria include, but are not limited to, members of the phyla Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Chlamydiae, Chlorobi, Chloroflexi Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus, Thermus, Dictyoglomi, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Tenericutes, Thermodesulfobacteria, Thermomicrobia, Thermotogae, and Verrucomicrobia. Specific, non-limiting examples of Eubacteria include Escherichia coli, Thermus thermophilus, Stenotrophomonas maltophilia, Kineococcus radiotolerans and Bacillus stearothermophilus. Example of Archaea include Methanococcus jannaschii, Methanosarcina mazei, Methanobacterium thermoautotrophicum, Methanococcus maripaludis, Methanopyrus kandleri, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-i, Archaeoglobus fulgidus, Pyrococcus fit riosus, Pyrococcus horikoshii, Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Aeuropyrum pernix, Thermoplasma acidophilum, and Thermoplasma volcanium. Other specific examples of Eubacteria can be found at world wide web address bacterio.cict.fr/classifphyla.html (last accessed on Nov. 17, 2009).

[0107] Biodiesel fuels: A diesel-equivalent processed fuel derived from biological sources which can be used in unmodified diesel-engine vehicles. Biodiesels are attractive for fuels, and some other uses, because they have a low vapor pressure, are non-toxic and are stable, as per HMIS regulation, and do not deteriorate or detonate upon mild heating. Chemically, biodiesels are generally defined as the mono alkyl esters of long chain fatty acids derived from renewable lipid sources.

[0108] Biofuel: Any fuel that derives from biomass--recently living organisms or their metabolic byproducts, such as manure from cows. A biofuel may be further defined as a fuel derived from a metabolic product of a living organism. It is a renewable energy source, unlike other natural resources such as petroleum, coal and nuclear fuels.

[0109] Conditions that permit production: Any fermentation or culturing conditions that allow a microorganism to produce a desired product, such as a hydrocarbon or hydrocarbon intermediate. Such conditions usually include temperature ranges, levels of aeration, and media selection that, when combined, allow the microorganism to grow. Exemplary mediums include broths or gels. Generally, the medium includes a carbon source (such as glucose, fructose, cellulose, or the like) that can be metabolized by the microorganism directly, or enzymes can be used in the medium to facilitate metabolizing the carbon source. To determine if culture conditions permit product production, the microorganism can be cultured for 2, 4, 6, 8, 12, 24, 36, 48 or 72 hours and a sample can be obtained and analyzed. For example, the cells in the sample or the medium in which the cells were grown can be tested for the presence of the desired product. When testing for the presence of a product, assays can be used, such as those provided herein, including those presented in the Examples below.

[0110] Contacting: Placement in direct physical association; includes both in solid and liquid form. Contacting includes contact between one molecule and another molecule. Contacting can occur in vitro with isolated cells or tissue or in vivo by administering it to an organism.

[0111] Cytochrome P450: A very large and diverse superfamily of hemoproteins found in all domains of life. Cytochromes P450 use a plethora of both exogenous and endogenous compounds as substrates in enzymatic reactions. Usually they form part of multi-component electron transfer chains, called P450-containing systems. The most common reaction catalysed by cytochrome P450 is a monooxygenase reaction, e.g. insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water: RH+O2+2H++2e-→ROH+H2O.

[0112] Cytochrome P450 enzymes have been identified from all lineages of life, including mammals, birds, fish, insects, worms, sea squirts, sea urchins, plants, fungi, slime molds, bacteria and archaea. More than 8100 distinct cytochrome P450 sequences are known. Exemplary cytochrome P450s are described herein and provided in the attached Sequence listings. In particular examples, exemplary cytochrome P450s include CYP4G1 (SEQ ID NO: 2) and CYP4G2 (SEQ ID NO: 1).

[0113] Decarbonylase: An enzyme that catalyses the decarboxylation of aldehydes to form carbon monoxide and hydrocarbons. Differs from an oxidative decarbonylase in that an oxidative decarbonylase catalyses the conversion of aldehydes to carbon dioxide and hydrocarbons.

[0114] Decarboxylase: An enzyme that hydrolyzes a carboxyl radical.

[0115] Deoxyribonucleic acid (DNA): A long chain polymer that includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid, RNA). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides, referred to as codons, in DNA molecules code for amino acid in a peptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

[0116] Encode: As used herein, the term "encode" refers to any process whereby the information in a polymeric macromolecule or sequence is used to direct the production of a second molecule or sequence that is different from the first molecule or sequence. As used herein, the term is construed broadly, and can have a variety of applications. In some aspects, the term "encode" describes the process of semi-conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase.

[0117] In another aspect, the term "encode" refers to any process whereby the information in one molecule is used to direct the production of a second molecule that has a different chemical nature from the first molecule. For example, a DNA molecule can encode an RNA molecule (for instance, by the process of transcription incorporating a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a peptide, as in the process of translation. When used to describe the process of translation, the term "encode" also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, for instance, by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a peptide, where it is understood that "encode" as used in that case incorporates both the processes of transcription and translation.

[0118] Endogenous: As used herein with reference to a nucleic acid molecule and a particular cell or microorganism, the term endogenous refers to a nucleic acid sequence or peptide that is in the cell and was not introduced into the cell using recombinant engineering techniques. For example, a gene that was present in the cell when the cell was originally isolated from nature.

[0119] Exogenous: As used herein with reference to a nucleic acid molecule and a particular cell, the term exogenous refers to any nucleic acid molecule that does not originate from that particular cell as found in nature. Thus, a non-naturally-occurring nucleic acid molecule is considered to be exogenous to a cell once introduced into the cell. A nucleic acid molecule that is naturally-occurring also can be exogenous to a particular cell.

[0120] Fermentation Broth: Any medium that supports microorganism life (for instance, a microorganism that is actively metabolizing carbon). A fermentation medium usually contains a carbon source. The carbon source can be anything that can be utilized, with or without additional enzymes, by the microorganism for energy.

[0121] Fungi: A kingdom of eukaryotic organisms. They are heterotrophic and digest their food externally, absorbing nutrient molecules into their cells. Yeasts, molds, and mushrooms are examples of fungi. The major phyla of fungi include Chytridiomycota, Zygomycota, Glomeromycota, Ascomycota, and Basidiomycota.

[0122] The Chytridiomycota are commonly known as chytrids. These fungi produce zoospores that are capable of moving on their own through liquid menstrua by simple flagella. The Zygomycota are known as zygomycetes and reproduce sexually with meiospores called zygospores and asexually with sporangiospores. Rhizopus stolonifer, Pilobolus, Mucor, Rhizomucor, and Rhizopus are Zygomycota.

[0123] Specific, non-limiting examples of fungi that are useful in the disclosed methods include Saccharomyces cerevisiae, Aspergillus, Trichoderma, Neurospora, Fusarium, and Chrysosporium.

[0124] Gene expression: The process by which the coded information of a nucleic acid transcriptional unit (including, for example, genomic DNA or cDNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for instance, exposure of a cell, tissue or subject to an agent that increases or decreases gene expression. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for instance, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level and by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).

[0125] Hydrocarbon: A chemical compound that contains the elements carbon (C) and hydrogen (H). All hydrocarbons have a carbon backbone and hydrogen atoms attached to that backbone. Sometimes, the term is used as a shortened form of the term "aliphatic hydrocarbon." There are essentially three types of hydrocarbons: (1) aromatic hydrocarbons, which have at least one aromatic ring; (2) saturated hydrocarbons, also known as alkanes, which lack double, triple or aromatic bonds; and (3) unsaturated hydrocarbons, which have one or more double or triple bonds between carbon atoms, are divided into: alkenes, alkynes, and dienes. Liquid geologically-extracted hydrocarbons are referred to as petroleum (literally "rock oil") or mineral oil, while gaseous geologic hydrocarbons are referred to as natural gas. All are significant sources of fuel and raw materials as a feedstock for the production of organic chemicals and are commonly found in the earth's subsurface using the tools of petroleum geology. Oil reserves in sedimentary rocks are the principal source of hydrocarbons for the energy and chemicals industries. Hydrocarbons are of interest because they encompass the constituents of the major fossil fuels (coal, petroleum, natural gas, for instance, and biofuels, as well as plastics, waxes, solvents and oils).

[0126] Hydrocarbon-forming oxidative decarbonylase activity: The activity of one or more peptides that causes the conversion of an aldehyde to a hydrocarbon, with the release of CO₂. Examples of enzymes having oxidative decarbonylase activity include those with an amino acid sequence provided by SEQ ID NO: 1 or 2 or having at least 70% sequence identity to SEQ ID NO: 1 or 2, such as at least 80%, at least 90%, at least 95% sequence identity to SEQ ID NO: 1 or 2. Other examples of enzymes with oxidative decarbonylase activities are provided in Table A or peptides known to have cytochrome P450 activity. The term "oxidative decarbonylase activity" is used interchangeably herein with the term "hydrocarbon-forming oxidative-decarbonylase activity." Oxidative decarbonylase activity can be tested methods known to those of ordinary skill in the art including, but not limited to those, provided in the Examples below.

[0127] Isolated: An "isolated" biological component (such as a nucleic acid molecule, peptide, or cell) has been substantially purified away from other biological components in a mixed sample (such as a cell extract). For example, an "isolated" peptide or nucleic acid molecule is a peptide or nucleic acid molecule that has been separated from the other components of a cell in which the peptide or nucleic acid molecule was present (such as an expression host cell for a recombinant peptide or nucleic acid molecule). The term "isolated nucleic acid" thus encompasses nucleic acids purified by standard nucleic acid purification methods. The term also embraces nucleic acids prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0128] Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In some examples, a disclosed polypeptide is labeled with a detectable label.

[0129] Microorganism: A member of the prokaryotic or eukaryotic microbial species from the domains Archaea, Bacteria, and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

[0130] Nucleic acid molecule: A polymeric form of nucleotides, which can include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. A "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

[0131] Nucleic acid molecules can be modified chemically or biochemically or can contain non-natural or derivatized nucleotide bases, as will be readily appreciated. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendent moieties (for example, peptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular and padlocked conformations.

[0132] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. Configurations of separate genes that are transcribed in tandem as a single messenger RNA are denoted as operons. Thus, placing genes in close proximity, for example in a plasmid vector, under the transcriptional regulation of a single promoter, constitutes a synthetic operon.

[0133] Optional or optionally: A term to describe a subsequently described event or circumstance can but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0134] Oxidative decarbonylation: A process that involves the removal of a carbonyl carbon of a fatty aldehyde as CO₂. In one example, this process is catalyzed by a hydrocarbon-forming oxidative decarbonylase enzyme (referred to as oxidative decarbonylase), such as any of those disclosed herein.

[0135] Peptide: Any compound composed of amino acids, amino acid analogs, chemically bound together. Peptide as used herein includes oligomers of amino acids, amino acid analog, or small and large peptides, including polypeptides or proteins. Any chain of amino acids, regardless of length or post-translational modification (such as glycosylation or phosphorylation). In one example, a peptide is two or more amino acids joined by a peptide bond.

[0136] "Peptide" applies to amino acid polymers to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example a artificial chemical mimetic of a corresponding naturally occurring amino acid.

[0137] A "polypeptide" is a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0138] As used herein, the term "polypeptide fragment" refers to a portion of a polypeptide which exhibits at least one useful epitope or functional domain. Polypeptide fragments contemplated herein include all fragments of a polypeptide that retain a particular desired activity of the polypeptide. Biologically functional fragments can vary in size and will depend on the polypeptide of interest.

[0139] The term "soluble" refers to a form of a polypeptide that is not inserted into a cell membrane.

[0140] Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Examples of conservative substitutions are shown below.

TABLE-US-00001 Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0141] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0142] The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

[0143] Plant: any living stage or form of any member of the plant kingdom including, but not limited to, eukaryotic algae, mosses, club mosses, ferns, angiosperms, gymnosperms, and lichens (which contain algae) including any parts (for instance, pollen, seeds, cells, tubers, stems) thereof.

[0144] Plasmid: A DNA molecule separate from chromosomal DNA and capable of autonomous replication. It is typically circular and double-stranded, and usually occurs in bacteria, and sometimes in eukaryotic organisms (for instance, the 2-micrometre-ring in Saccharomyces cerevisiae). The size of plasmids can vary from 1 to over 400 kilobase pairs. Plasmids often contain genes or gene cassettes that confer a selective advantage to the bacterium (or other cell) harboring them, such as the ability to make the bacterium (or other cell) antibiotic resistant.

[0145] Plasmids contain at least one DNA sequence that serves as an origin of replication, which enables the plasmid DNA to be duplicated independently from the chromosomal DNA. The chromosomes of most bacteria are circular, but linear plasmids are also known.

[0146] Plasmids used in genetic engineering are referred to as vectors. They can be used to transfer genes from one organism to another, and typically contain a genetic marker conferring a phenotype that can be selected for or against. Most also contain a polylinker or multiple cloning site, which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Specific, non-limiting examples of plasmids include pOT2 (Berkeley Drosophila Resource Center, Berkeley, Calif.), pMT-DEST48 (Invitrogen, Carlsbad, Calif.).

[0147] Primers: Short nucleic acids, for example DNA oligonucleotides 10 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for instance using the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.

[0148] Probes and primers as used herein typically include, for example, at least 12 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers also can be employed, such as probes and primers that include at least 15, 20, 30, 40, 50, or more consecutive nucleotides of the disclosed nucleic acid sequences.

[0149] Methods for preparing and using probes and primers are described, for example Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor, N.Y., 2000; Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, 1987; Innis et al., PCR Protocols, A Guide to Methods and Applications, 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).

[0150] Probe: An isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, fluorophores, and enzymes.

[0151] Promoter: A region of DNA that generally is located upstream (within the 5' flanking region of a gene) that is needed for transcription. Promoters permit the proper activation or repression of the gene which they control. A promoter contains specific sequences that are recognized by transcription factors. These factors bind to the promoter DNA sequences and result in the recruitment of RNA polymerase, the enzyme that synthesizes the RNA from the coding region of the gene.

[0152] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified hydrocarbon preparation is one in which the product is more concentrated than the product is in its environment within a cell. For example, a purified hydrocarbon is one that is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and peptides) that can accompany it. In another example, a purified hydrocarbon preparation is one in which the hydrocarbon is substantially-free from contaminants, such as those that might be present following fermentation.

[0153] In one example, a hydrocarbon is purified when at least about 50% by weight of a sample is composed of the hydrocarbon, for example when at least about 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more of a sample is composed of the hydrocarbon.

[0154] Recombinant nucleic acid: A nucleic acid sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence, or that is placed next to a non-native DNA sequence, for example a nucleic acid sequence that is integrated into another host's chromosome. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for instance by genetic engineering techniques such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor, N.Y., 2000. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid can include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid can be part of a vector, used to transform a cell.

[0155] Reporter: An agent that can be used to identify and/or select target components of a system of interest. For example, a reporter can include a protein, for instance, an enzyme, that confers antibiotic resistance or sensitivity (for instance, 3-lactamase, chloramphenicol acetyltransferase (CAT), and the like), a fluorescent screening marker (for instance, green fluorescent protein (GFP), YFP, EGFP, RFP, etc.), a luminescent marker (for instance, a firefly luciferase protein), an affinity based screening marker, or positive or negative selectable marker genes such as lacZ, 3-gal/lacZ (13-galactosidase), ADH (alcohol dehydrogenase), his3, ura3, leu2, lys2, or the like.

[0156] A reporter gene is a nucleic acid sequence that encodes an easily assayed product (for instance firefly luciferase, CAT, and β-galactosidase), whose presence can be assayed. A reporter gene can be operably linked to a regulatory control sequence and transduced into cells. If the regulatory control sequence is transcriptionally active in a particular cell type, the reporter gene product normally will be expressed in such cells and its activity can be measured using techniques known in the art. The activity of a reporter gene product can be used, for example, to assess the transcriptional activity of an operably linked regulatory control sequence. In addition, the ability to produce hydrocarbons can be assayed for in a small scale experiment in which disclosed oxidative decarbonylase genes can be used themselves as reporters of their own activity.

[0157] Sequence identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.

[0158] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene 73:237-244, 1988; Higgins & Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Huang, et al., CABIOS 8:155-165, 1992; and Pearson et al., Methods in Molecular Biology 24:307-331, 1994. Altschul et al., J. Mol. Biol. 215:403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0159] The NCBI Basic Local Alignment Search Tool (BLAST®; Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NBCI, Bethesda, Md.), for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. BLAST® can be accessed on the internet at the NBCI website. As used herein, sequence identity is commonly determined with the BLAST® software set to default parameters. For instance, blastn (version 2.0) software can be used to determine sequence identity between two nucleic acid sequences using default parameters (expect=10, matrix=BLOSUM62, filter=DUST (Tatusov and Lipmann, in preparation as of Dec. 1, 1999; and Hancock and Armstrong, Comput. Appl. Biosci. 10:67-70, 1994), gap existence cost=11, per residue gap cost=1, and lambda ratio=0.85). For comparison of two polypeptides, blastp (version 2.0) software can be used with default parameters (expect 10, filter=SEG (Wootton and Federhen, Computers in Chemistry 17:149-163, 1993), matrix=BLOSUM62, gap existence cost=11, per residue gap cost=1, lambda=0.85).

[0160] For comparisons of amino acid sequences of greater than about 30 amino acids, the "Blast 2 sequences" function of the BLAST® program is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 35%, at least 45%, at least 50%, at least 60%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or 2.

[0161] For comparisons of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST® (Blastn) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default=11]; cost to extend a gap [default=1]; expectation value (E) [default=10.0]; word size [default=11]; number of one-line descriptions (V) [default=100]; number of alignments to show (B) [default=100]).

[0162] Substrate: As used herein, a substrate is a compound suitable to be used as the starting chemical in an enzymatic reaction. Typically the chemical formed by the enzymatic reaction is termed a product (products and substrates can also be termed intermediates). Specific, non-limiting examples of substrates that can be used with the disclosed methods include fatty acids, acyl CoAs, acyl ACPs, acyl AMP, and hydrocarbon intermediates.

[0163] Transduction: The process by which genetic material, for instance, DNA or another nucleic acid molecule, is inserted into a cell. Common transduction techniques include the use of viral vectors (including bacteriophages), electroporation, and chemical reagents that increase cell permeability. Transfection and transformation are other terms for transduction, although these sometimes imply expression of the genetic material as well. The term transformed refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. The term encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transformation with plasmid vectors (for example, by electroporation, conjugation, transduction, or natural transformation), transfection with viral vectors, and introduction of naked DNA by electroporation, natural transformation, lipofection, and particle gun acceleration.

[0164] Vector: A nucleic acid molecule capable of transporting a non-vector nucleic acid sequence that has been introduced into the vector. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which non-plasmid DNA segments can be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments can be ligated into all or part of the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for example, vectors having a bacterial origin of replication replicate in bacteria hosts). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell and are replicated along with the host genome. Some vectors contain expression control sequences (such as promoters) and are capable of directing the transcription of an expressible nucleic acid sequence that has been introduced into the vector. Such vectors are referred to as "expression vectors." A vector can also include one or more selectable marker genes and/or genetic elements known in the art.

[0165] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including explanations of terms, will control. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" means "including;" hence, "comprising A or B" means including A or B, as well as A and B together. All numerical ranges given herein include all values, including end points (unless specifically excluded) and any and all intermediate ranges between the endpoints.

[0166] Suitable methods and materials for the practice and testing of the disclosure are described below. However, the provided materials, methods, and examples are illustrative only and are not intended to be limiting. Accordingly, except as otherwise noted, the methods and techniques of the present disclosure can be performed according to methods and materials similar or equivalent to those described and/or according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification (see, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, 2000; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999).

III. Oxidative Decarbonylases and Methods of Their Use to Produce Hydrocarbons

[0167] Disclosed is the surprising identification of cytochrome P450 enzymes that function as aldehyde oxidative decarbonylases and are involved in the biosynthesis of hydrocarbons. Microorganisms (for instance fungal or bacterial cells) transformed with one or more of the genes that encodes one or more of these enzymes can be used as a source for hydrocarbons. Such a novel, renewable source of hydrocarbons is desirable because it provides a supplement to the existing limited resources of non-renewable hydrocarbons. As such, this discovery provides methods of producing hydrocarbons that can be used for the production of a wide range of products, such as hydrocarbon sex-pheromone components for Musca domestica control, biofuels, lubricants, or solvents. In use, a cell (such as a bacterial cell or a fungal cell) is transformed with one or more of these genes or their homologs, and the cell is then cultured under conditions that permit the generation of specific hydrocarbon species.

[0168] A. Isolated Polypeptides and Polynucleotides

[0169] 1. Structure

[0170] Polynucleotides and polypeptides were isolated from Musca domestica and identified to be involved in the biosynthesis of hydrocarbons. For example, such polypeptides were determined to be cytochrome P450 enzymes that possess oxidative decarbonylase activity. In one particular example, isolated polypeptide sequences with oxidative decarbonylase activity are provided, such as isolated polypeptide sequences with an amino acid sequence as set forth in SEQ ID NO: 1 or 2. In another particular example, isolated polynucleotides that encode sequences with oxidative decarbonylase activity are provided, such as encode a polypeptide with an amino acid sequence set forth in SEQ ID NO: 1 or 2. These polypeptide and nucleic acid sequences are listed in the accompanying Sequence Listing. One of ordinary skill in the art will appreciate that by using the information provided herein relating to the structure and function of the M. domestica sequences, other sequences having similar activity to the CYP4G2 and CYP4G1 can be obtained. For example, the present disclosure also relates to sequences similar to CYP4G2. For example, CYP4G2v1 is curated in GenBank as accession No. EF615002 (incorporated by reference in its entirety as available on GenBank on Nov. 17, 2009; Zhu et al., 2009, BMC Physiol. 8:18). Notably, Zhu et al. do not identify the CYP4G2v1 sequence as an oxidative decarbonylase. Rather, Zhu et al. imply that CYP4G2v1 is involved in permethrin metabolism. Additional related sequences include sequence 46640 (GenBank Accession No. AAU65695, incorporated by reference in its entirety as available on GenBank on Nov. 17, 2009) from U.S. Pat. No. 6,703,491, which has an E value of 6e-82 with CYP4G2, with an E value of 6e-82. Sequence 46640 encodes a portion of CYP4G15 from Drosophila melanogaster.

[0171] Oxidative decarbonylase activity can reasonably be expected to be found in all insects. Similar activities likely also exist in some bacteria, plants, algae, and birds that synthesize hydrocarbons. BLASTP searches comparing CYP4G2 and CYP4G1 against known sequences in GenBank returned highly significant hits to uncharacterized P450s in many insects, invertebrates, vertebrates, and plants (as provided in Table A below).

[0172] Definitively predicting oxidative decarbonylase activity for other P450s can be difficult. In some cases, minor amino acid changes can affect P450 activity. Also, P450 enzymes can show some sequence divergence. It is suggested that the more closely related the insects are taxonomically, the more common motifs they should have.

[0173] In the case of oxidative decarbonylase, sequences with the strongest "E-value" scores from BLASTP surveys of GenBank are expected to show the greatest degree of similarity. The E-value represents the probability that an observed alignment resulted by random chance; thus, the smaller the E-value, the more confident one can be that the alignment represents related sequences. The best possible E-value is 0.0. These sequences have the highest probability of encoding oxidative decarbonylase enzymes; indeed, CYP9T1 falls into this category.

[0174] In Table A, the BLASTP hits with 0.0 E-values are sequences from Drosophila spp. An alignment shows these sequences have 48.6% a.a. identity, and 98.6% a.a. similarity. The weakest insect hit is 8E-82 (8×10^-82). This is still very high, particularly for P450 sequences. When all the insect sequences are aligned, they have 23.7% identity and 77% similarity, with identical positions pretty much scattered throughout the sequences. If plant P450s are included (by restricting a blastp search to just plants) the best hits are significantly weaker than the insect sequences, and when all insect and plant hits are aligned, identity falls to 0.6%, and similarity is 58.6%.

[0175] Assuming that all or most of the sequences in Table A represent hydrocarbon decarbonylases, the portions of each protein responsible for activity; i.e. which amino acids determine the substrate binding site(s), can be determined. For example, a molecular model of CYP4G13 can be used to locate residues in channels. Similar models can be constructed for CYP4G2. Similarly, some of the weaker blastp hits in Table A can be reasonably assumed not to be hydrocarbon decarbonylases because of their expression pattern, etc.

TABLE-US-00002 TABLE A Additional examples of oxidative decarbonylase enzymes. Score E Value Musca domestica [flies] taxid 7370 gb|ABV48808.1|cytochrome P450 CYP4G2v1 [Musca domestica] 1148 0.0 gb|AAK40120.1|cytochrome P450 CYP4G13v2 [Musca domestica] 709 0.0 Drosophila erecta [flies] taxid 7220 ref|XP_001982391.1|GG12761 [Drosophila erecta] 825 0.0 gb|EDV45360.1|GG12761 [Drosophila erecta] 825 0.0 Drosophila melanogaster [flies] taxid 7227 ref|NP_525031.1|Cytochrome P450-4g1 CG3972-PA [Drosophila . . . 825 0.0 sp|Q9V3S0|CP4G1_DROME Cytochrome P450 4g1 (CYPIVG1) 825 0.0 emb|CAA15672.1|EG: 165H7.1 [Drosophila melanogaster] 825 0.0 gb|AAF45503.1|CG3972-PA [Drosophila melanogaster] 825 0.0 gb|ABY20430.1|GH01123p [Drosophila melanogaster] 825 0.0 Drosophila simulans [flies] taxid 7240 ref|XP_002076787.1|GD24639 [Drosophila simulans] 824 0.0 gb|EDX16353.1|GD24639 [Drosophila simulans] 824 0.0 Drosophila yakuba [flies] taxid 7245 ref|XP_002099674.1|GE16587 [Drosophila yakuba] 824 0.0 gb|EDX00782.1|GE16587 [Drosophila yakuba] 824 0.0 Drosophila pseudoobscura pseudoobscura [flies] taxid 46245 ref|XP_001354787.1|GA17813 [Drosophila pseudoobscura pseu . . . 823 0.0 gb|EAL31842.1|GA17813 [Drosophila pseudoobscura pseudoobs . . . 823 0.0 Drosophila mojavensis [flies] taxid 7230 ref|XP_002011612.1|GI11123 [Drosophila mojavensis] 818 0.0 gb|EDW05602.1|GI11123 [Drosophila mojavensis] 818 0.0 Drosophila virilis [flies] taxid 7244 ref|XP_002058244.1|GJ15981 [Drosophila virilis] 816 0.0 gb|EDW66352.1|GJ15981 [Drosophila virilis] 816 0.0 Drosophila ananassae [flies] taxid 7217 ref|XP_001964253.1|GF20812 [Drosophila ananassae] 813 0.0 gb|EDV34702.1|GF20812 [Drosophila ananassae] 813 0.0 Drosophila grimshawi [flies] taxid 7222 ref|XP_001992189.1|GH24346 [Drosophila grimshawi] 798 0.0 gb|EDV91896.1|GH24346 [Drosophila grimshawi] 798 0.0 Drosophila willistoni [flies] taxid 7260 ref|XP_002071182.1|GK25658 [Drosophila willistoni] 769 0.0 gb|EDW82168.1|GK25658 [Drosophila willistoni] 769 0.0 Culex quinquefasciatus [flies] taxid 7176 ref|XP_001869039.1|cytochrome P450 4g15 [Culex quinquefas . . . 585 2e-165 gb|EDS28283.1|cytochrome P450 4g15 [Culex quinquefasciatus] 585 2e-165 ref|XP_001851084.1|cytochrome P450 4g15 [Culex quinquefas . . . 532 3e-149 gb|EDS33030.1|cytochrome P450 4g15 [Culex quinquefasciatus] 532 3e-149 Aedes aegypti [flies] taxid 7159 ref|XP_001658068.1|cytochrome P450 [Aedes aegypti] 585 3e-165 ref|XP_001659149.1|cytochrome P450 [Aedes aegypti] 585 5e-165 ref|XP_001648376.1|cytochrome P450 [Aedes aegypti] 523 1e-146 Nasonia vitripennis [wasps &c.] taxid 7425 ref|XP_001600301.1|PREDICTED: similar to cytochrome P450 . . . 574 8e-162 ref|XP_001606417.1|PREDICTED: similar to cytochrome P450 . . . 528 6e-148 Chironomus tentans [flies] taxid 7153 gb|AAW78325.1|cytochrome P450 family 4 [Chironomus tentans] 568 4e-160 Anopheles gambiae str. PEST [flies] taxid 180454 ref|XP_555875.3|AGAP000877-PA [Anopheles gambiae str. PEST] 561 7e-158 gb|EAL39767.3|AGAP000877-PA [Anopheles gambiae str. PEST] 561 7e-158 ref|XP_558699.5|AGAP001076-PA [Anopheles gambiae str. PEST] 524 8e-147 gb|EAL40625.3|AGAP001076-PA [Anopheles gambiae str. PEST] 524 8e-147 Tribolium castaneum (rust-red flour beetle) [beetles] taxid 7070 ref|NP_001107860.1|cytochrome P450 monooxigenase CYP4G7 [ . . . 551 5e-155 ref|NP_001107791.1|cytochrome P450 monooxygenase [Triboli . . . 531 7e-149 Bombyx mori (silk moth, . . . ) [moths] taxid 7091 ref|NP_001106221.1|cytochrome P450 [Bombyx mori] 550 1e-154 gb|ABF51451.1|cytochrome P450 [Bombyx mori] 550 1e-154 ref|NP_001106223.1|cytochrome P450 CYP4G25 [Bombyx mori] 506 2e-141 gb|ABF51415.1|cytochrome P450 CYP4G25 [Bombyx mori] 506 2e-141 Leptinotarsa decemlineata [beetles] taxid 7539 gb|AAZ94273.1|cytochrome P450 [Leptinotarsa decemlineata] 543 1e-152 Drosophila sechellia [flies] taxid 7238 ref|XP_002044080.1|GM13084 [Drosophila sechellia] 529 2e-148 gb|EDW51392.1|GM13084 [Drosophila sechellia] 529 2e-148 ref|XP_002040228.1|GM19042 [Drosophila sechellia] 427 1e-117 gb|EDW43699.1|GM19042 [Drosophila sechellia] 427 1e-117 Apis mellifera (bee, . . . ) [bees] taxid 7460 ref|NP_001035323.1|cytochrome P450 monooxygenase [Apis me . . . 528 4e-148 gb|ABB36785.1|cytochrome P450 monooxygenase [Apis mellifera] 528 4e-148 Blattella germanica [roaches] taxid 6973 gb|AAO20251.1|cytochrome P450 monooxygenase CYP4G19 [Blat . . . 521 9e-146 Ips paraconfusus [beetles] taxid 89938 gb|ABF06553.1|CYP4G27 [Ips paraconfusus] 517 1e-144 Antheraea yamamai (oak silkmoth, . . . ) [moths] taxid 7121 dbj|BAD81026.1|cytochrome P450 CYP4G25 [Antheraea yamamai] 507 1e-141 Mamestra brassicae [moths] taxid 55057 gb|AAR26517.1|antennal cytochrome P450 CYP4 [Mamestra bra . . . 502 3e-140 Acyrthosiphon pisum [aphids] taxid 7029 ref|XP_001944205.1|PREDICTED: similar to cytochrome P450 . . . 484 6e-135 Drosophila persimilis [flies] taxid 7234 ref|XP_002023479.1|GL20168 [Drosophila persimilis] 429 4e-118 gb|EDW27627.1|GL20168 [Drosophila persimilis] 429 4e-118 Blaberus discoidalis [roaches] taxid 6981 sp|P29981|CP4C1_BLADI Cytochrome P450 4C1 (CYPIVC1) 352 5e-95 gb|AAA27819.1|cytochrome P450 352 5e-95 Carcinus maenas (common shore crab) [crustaceans] taxid 6759 pir||JC8026 cytochrome P450 enzyme, CYP4C39 enzyme - green . . . 339 4e-91 gb|AAQ93010.1|cytochrome P450 CYP4C39 [Carcinus maenas] 339 4e-91 Branchiostoma floridae [lancelets] taxid 7739 gb|EEA69963.1|hypothetical protein BRAFLDRAFT_57954 [Bran . . . 328 5e-88 gb|EEA70036.1|hypothetical protein BRAFLDRAFT_210358 [Bra . . . 322 7e-86 Balaenoptera acutorostrata (lesser rorqual) [whales & dolphins] taxid 9767 dbj|BAF64512.1|cytochrome 4V6 [Balaenoptera acutorostrata] 322 5e-86 Helicoverpa zea (tomato fruitworm, . . . ) [moths] taxid 7113 gb|AAM54722.1|cytochrome P450 monooxygenase CYP4M6 [Helic . . . 322 6e-86 Daphnia magna [crustaceans] taxid 35525 dbj|BAF35771.1|cytochrome P450 4 family [Daphnia magna] 321 1e-85 Nilaparvata lugens [bugs] taxid 108931 emb|CAQ57675.1|cytochrome P450 [Nilaparvata lugens] 313 2e-83 emb|CAQ57674.1|cytochrome P450 [Nilaparvata lugens] 308 8e-82 Danio rerio (leopard danio, . . . ) [bony fishes] taxid 7955 ref|NP_001073465.1|hypothetical protein LOC562008 [Danio . . . 313 2e-83 gb|AAI25941.1|Zgc: 154042 [Danio rerio] 313 2e-83 Arabidopsis thaliana (thale-cress, . . . ) [eudicots] taxid 3702 ref|NP_198661.1|CYP735A1 (cytochrome P450, family 735, su . . . 144 1e-33 dbj|BAB09357.1|cytochrome P450-like protein [Arabidopsis . . . 144 1e-33 emb|CAB10290.1|cytochrome P450 like protein [Arabidopsis . . . 131 1e-29 emb|CAB78553.1|cytochrome P450 like protein [Arabidopsis . . . 131 1e-29 Oryza sativa Indica Group (Indian rice) [monocots] taxid 39946 gb|EAZ07069.1|hypothetical protein OsI_028301 [Oryza sati . . . 144 1e-33 gb|EAY75510.1|hypothetical protein OsI_003357 [Oryza sati . . . 137 2e-33 gb|EAY75509.1|hypothetical protein OsI_003356 [Oryza sati . . . 137 2e-31 gb|EAY84668.1|hypothetical protein OsI_005901 [Oryza sati . . . 136 4e-31

[0176] Given these teachings, one of ordinary skill in the art will appreciate that sequences similar to CYP4G1 and CYP4G2 can readily be cloned and used to make hydrocarbons and hydrocarbon intermediates. Therefore, throughout this description reference to CYP4G1 and CYP4G2 should be understood to mean all proteins displaying the respective activity (as well as all polynucleotides encoding such proteins), including, for example those in Table A, the Examples as well as others that can be identified or engineered through various molecular techniques such as antibody binding, nucleic acid hybridization, PCR and the like.

[0177] Although particular embodiments of hydrocarbon and hydrocarbon intermediate forming sequences are disclosed, it will be understood that sequences that have similar structural characteristics can be isolated from other microorganisms. These newly isolated sequences can be assayed for oxidative decarbonylase activity (see Table A for list of specific, non-limiting examples of related sequences) by methods known to those of skill in the art including those disclosed herein (such as in the Examples). In addition, it will be understood that other functionally equivalent forms of the sequences disclosed herein can be readily identified and/or generated using conventional molecular biological techniques, including for instance site-directed mutagenesis or M13 primer mutagenesis. Details of these techniques are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor, N.Y., 2000, Ch. 15. Thus, in addition to structurally related sequences and homologous sequences, the disclosure also encompasses amino acid sequences that have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NOs: 1 and 2, for instance at least 95%, 96%, 97%, 98%, or 99% sequence identity. Moreover, the disclosure also encompasses nucleic acid sequences that encode polypeptides that have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NOs: 1 and 2, for instance at least 95%, 96%, 97%, 98%, or 99% sequence identity.

[0178] Sequences retaining structural and functional similarity to CYP4G1 and CYP4G2 can be identified by any of a number of known methods. One such method involves the screening of genomic sequences for sequence alignment with the known sequence(s). Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene 73:237-244, 1988; Higgins & Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Huang, et al., CABIOS 8:155-165, 1992; and Pearson et al., Methods in Molecular Biology 24:307-331, 1994. Altschul et al., J. Mol. Biol. 215:403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0179] When a genomic sequence is not available for a particular species of interest, related sequences can be amplified from total RNA using RT-PCR. Briefly, total RNA is extracted from the cells of interest by any one of a variety of well known methods. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor, N.Y., 2000, and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992) provide descriptions of methods for RNA isolation. Generally, any microorganism can be used as a source of such RNA. The extracted RNA is then used as a template for performing reverse transcription-polymerase chain reaction (RT-PCR) amplification of cDNA. Methods and conditions for RT-PCR are described in Kawasaki et al., (In PCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., San Diego, Calif., 1990).

[0180] The selection of amplification primers will be made according to the particular cDNA that is to be amplified. Variations in amplification conditions can be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed for instance in Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990).

[0181] Sequencing of PCR products obtained by these amplification procedures can be used to facilitate confirmation of the amplified sequence and provide information about natural variation of this sequence in different species. Oligonucleotides derived from the provided CYP4G1 and CYP4G2 sequences can be used in such sequencing methods. Closely related orthologous CYP4G1 and CYP4G2 molecules can share at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 98% sequence identity with the disclosed CYP4G1 and CYP4G2 sequences (see, the sequence listing).

[0182] 2. Function

[0183] Hydrocarbons and intermediates thereof can be formed by expressing one or more of the disclosed polypeptides in a host cell, such as E. coli. Therefore, E. coli, or other organisms that naturally or are engineered to make hydrocarbons such as S. maltophilia, C. aggregans or X. axonopodis, can be used to determine the oxidative decarbonylase activity of a specific protein. Briefly, the protein to be tested, for example a sequence similar to CYP4G2, is expressed in a host that is known to display oxidative decarbonylase activity. The CYP4G2-like sequence is deemed to be active (i.e. have oxidative decarbonylase activity) if the host produces or increases production of hydrocarbons, such as an increase of at least 10%, at least 20%, at least 50%, or at least 90%.

[0184] Production hosts can be engineered using the peptides disclosed herein to produce hydrocarbons and hydrocarbon intermediates having defined structural characteristics (degrees of branching, saturation, and length). One method of making hydrocarbon intermediates involves expressing, increasing the expression of, or expressing more active forms of, one or more enzymes having oxidative decarbonylase activity. Exemplary enzymes that can be manipulated to increase hydrocarbon production include CYP4G1 and CYP4G2, as well as other enzymes that increase or modify fatty acid production. One of ordinary skill in the art will appreciate that the products produced from such enzymes vary with the acyl chain of the substrate.

[0185] There are several methods of identifying peptides having hydrogen decarboxylase activity. Product (hydrocarbon) formation using one or more of these methods indicates that the peptide has dehydrogenase activity. For example, the peptide can be expressed from an exogenous nucleic acid sequence in a cell and then a cell lysate can be prepared. Various substrates such as NADPH and/or NADH can be added to the lysate and products can be detected using the GC/MS methods described herein (see, Examples below). In another example, the peptide can be purified and incubated with cell lysate from a cell that is not expressing the peptide (herein after wild-type lysate). The purified peptide, wild-type lysate and various substrates can be incubated and the resulting products can be characterized using the methods described herein. Peptides having oxidative decarbonylase activity are identified as those that produce hydrocarbons. One of ordinary skill in the art will appreciate that when a cell lysate is used that already contains hydrocarbon products, peptides having decarboxylase activity will be recognized by an increase in hydrocarbon production compared to the lysate without the addition of substrate.

[0186] B. Recombinant Nucleic Acid Constructs

[0187] Also disclosed herein are recombinant nucleic acid constructs that include one or more nucleic acid sequences encoding CYP4G1 or CYP4G2; homologs of SEQ ID NO: 1 or 2; conservative variants of SEQ ID NO: 1 or 2 (including those provided in the Examples below); and/or sequences having at least 50% sequence identity, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% (such as about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99%) with SEQ ID NO: 1 or 2 or those provided in Table A. Exemplary recombinant nucleic acid constructs of use include cloning vectors, expression vectors or synthetic operons. A cloning vector is a self-replicating DNA molecule that serves to transfer a DNA segment into a host cell. Three common types of cloning vectors are bacterial plasmids, phages, and other viruses. An expression vector is a cloning vector designed so that a coding sequence inserted at a particular site will be transcribed and translated into a protein. A synthetic operon is a fragment of DNA encoding the gene of interest flanked by promoter regions and regions that will allow integration into a hetrologous host.

[0188] Both cloning and expression vectors contain nucleotide sequences that allow the vectors to replicate in one or more suitable host cells. In cloning vectors, this sequence is generally one that enables the vector to replicate independently of the host cell chromosomes, and also includes either origins of replication or autonomously replicating sequences. Various bacterial and viral origins of replication are well known and include, but are not limited to, the pBR322 plasmid origin and the SV40, polyoma, adenovirus, VSV and BPV viral origins.

[0189] The nucleic acid sequences disclosed herein can be used to produce proteins by the use of recombinant expression vectors containing the sequence(s). A great variety of expression vectors can be used, for instance chromosomal, episomal and virus-derived vectors, including vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papoviruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses; pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host cell can be used for expression in this regard. Therefore, any other vector that is replicable and viable in the host cell can be used.

[0190] The appropriate DNA sequence is inserted into the vector by any of a variety of well-known and routine techniques. In general, a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4-DNA ligase. Procedures for restriction and ligation are well known. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known, are set forth in great detail in Sambrook et al. (2000); Ausubel et al. (1995).

[0191] In an expression vector, the sequence of interest is operably linked to a suitable regulatory sequence, expression control sequence or promoter recognized by the host cell to direct mRNA synthesis. Promoters are untranslated sequences located generally within 100 to 1000 base pairs upstream from the start codon of a structural gene that regulate the transcription and translation of nucleic acid sequences under their control. Promoters are generally either inducible or constitutive.

[0192] Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in the environment, for instance the presence or absence of a nutrient or a change in temperature. Constitutive promoters, in contrast, maintain a relatively constant level of transcription. In addition, useful promoters can also confer appropriate cellular and temporal specificity. Such promoters include those that are developmentally-regulated and/or cell-specific.

[0193] A nucleic acid sequence is operably linked to antoher nucleic acid sequence when it is placed into a functional relationship with the other nucleic acid sequence. For example, DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked sequences are contiguous and, in the case of a secretory leader, contiguous and in reading frame.

[0194] Linking is achieved by conventional techniques such as SOE PCR, DNA synthesis, blunt end ligation, or ligation at restriction enzyme sites. If suitable restriction sites are not available, then synthetic oligonucleotide adapters or linkers can be used (Sambrook et al., 2000; Ausubel et al., 1995).

[0195] It will be recognized that numerous promoters are functional in bacterial cells, and have been described in the literature, including constitutive, inducible, developmentally regulated, and environmentally regulated promoters. Of particular interest is the use of promoters (also referred to as transcriptional initiation regions) functional in the appropriate microbial host cell. For example if E. coli is used as a host cell then exemplary promoters that can be used include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, promoters of retroviral LTRs, the CaMV 35S promoter, coconut foliar decay virus (CFDV) DNA (U.S. Pat. No. 6,303,345), and the endogenous promoters of P. citrorellolis. If Saccharomyces cerevisiae is the host then the sequences of interest are under the control of yeast promoters. A specific, non-limiting example of a useful yeast promoter includes the GAL/CYC promoter. It will be understood that numerous promoters that are not mentioned are suitable for use and are well known, and can be readily employed in the manner illustrated herein. Other promoters known to control the expression of genes in prokaryotic or eukaryotic cells can be used. Expression vectors can also contain a ribosome binding site for translation initiation, and a transcription terminator. The vector can also contain sequences useful for the amplification of gene expression.

[0196] Expression and cloning vectors can and usually do contain a structural gene or selection marker having the necessary regulatory regions for expression in a host cell and providing for selection of transformant cells. The gene can provide for resistance to a cytotoxic agent, for instance an antibiotic, heavy metal, or toxin, complementation providing prototrophy to an auxotrophic host, viral immunity or the like. Depending upon the number of different host species into which the expression construct or components thereof are introduced, one or more markers can be employed, where different conditions for selection are used for the different hosts.

[0197] Specific, non-limiting examples of suitable selection markers include genes that confer resistance to bleomycin, gentamycin, glyphosate, hygromycin, kanamycin, methotrexate, nalidixic acid, phleomycin, phosphinotricin, spectinomycin, streptomycin, sulfonamide, sulfonylureas, ampicillin/carbenicillin, chloramphenicol, or streptomycin/spectinomycin, and tetracycline. Specific, non-limiting examples of markers include, but are not limited to, alkaline phosphatase (AP), myc, hemagglutinin (HA), 13 glucuronidase (GUS), luciferase, and green fluorescent protein (GFP).

[0198] In addition, expression vectors also can contain marker sequences operatively linked to a nucleotide sequence for a protein that encodes an additional protein used as a marker. The result is a hybrid or fusion protein comprising two linked and different proteins. The marker protein can provide, for example, an immunological or enzymatic marker for the recombinant protein produced by the expression vector. Additionally, the end of the polynucleotide can be modified by the addition of a sequence encoding an amino acid sequence useful for purification of the protein produced by affinity chromatography. Various methods have been devised for the addition of such affinity purification moieties to proteins. Representative examples can be found in U.S. Pat. Nos. 4,703,004, 4,782,137, 4,845,341, 5,935,824, and 5,594,115. Any method known in the art for the addition of nucleotide sequences encoding purification moieties can be used, for example those contained in Innis et al. (1990) and Sambrook et al. (2000).

[0199] More particularly, the present disclosure includes recombinant constructs that include one or more isolated nucleic acid sequences that encode CYP4G1 (SEQ ID NO: 2) or CYP4G2 (SEQ ID NO: 1) or variants and homologs of these sequences. The constructs can include a vector, such as a plasmid or viral vector, into which the sequence has been inserted, either in the forward or reverse orientation. The recombinant construct can further include a regulatory sequence, including for example, a promoter operatively linked to the sequence. Large numbers of suitable vectors and promoters are known and are commercially available. In one embodiment, the pET-21b(+), pCOLADuet-1, pCDFDuet-1, pcDNA3.1(+), and/or pCMV SPORT6.1 (Invitrogen) vectors are used. It will be understood however, that other plasmids or vectors can be used as long as they are replicable and viable or capable of expressing the encoded protein in the host. It will also be understood that recombinant DNA technology resulting in the integration of the respective DNA sequences encoding for CYP4G1 (SEQ ID NO: 2) or CYP4G2 (SEQ ID NO: 1) and/or variants and homologs of these sequences into the chromosome of any living organism can result in expression and production of the proteins.

[0200] The polynucleotide sequence also can be part of an expression cassette that at a minimum includes, operably linked in the 5' to 3' direction, a promoter, one or more nucleic acids of the present disclosure, and a transcriptional termination signal sequence functional in a host cell. The promoter can be of any of the types discussed herein, for example, an inducible promoter or constitutive promoter, and the expression cassette can further include an operably linked targeting sequence, or transit or secretion peptide coding region capable of directing transport of the protein produced. The expression cassette can also further include a nucleotide sequence encoding a selectable marker and/or a purification moiety.

[0201] C. Host Cells

[0202] Host cells (for instance, bacterial, fungal eukaryotic, plant, or algae cells) are provided that are genetically engineered (for instance, transformed, transduced or transfected) with one or more nucleic acid molecules encoding one or more of CYP4G1 (SEQ ID NO: 2) or CYP4G2 (SEQ ID NO: 1) or a variant or homolog of one or more of these sequences. These sequences can be expressed from vector constructs or directly from the chromosome after gene integration or from extrachromosomal arrays. For example, an CYP4G1 (SEQ ID NO: 2) or CYP4G2 (SEQ ID NO: 1) protein is operably linked to gene expression control elements that are functional in the desired host cell, for instance a T7 promoter in E. coli.

[0203] Methods of expressing proteins in heterologous expression systems are well known in the art. Typically a bacterial or yeast host cell is transformed by natural transformation, electroporation, conjugation of transduction to contain the expression construct either extrachromosomally as with a plasmid on integrated into the chromosome after recombination. In eukaryotic cells, typically, a host cell is transfected with (or infected with a virus containing) an expression vector using any method suitable for the particular host cell. Such transfection methods are also well known in the art and non limiting exemplary methods are described herein. The transfected (also called, transformed) host cell is capable of expressing the protein encoded by the corresponding nucleic acid sequence in the expression cassette. Transient or stable transfection of the host cell with one or more expression vectors is contemplated by the present disclosure.

[0204] Many different types of cells can be used to express heterologous proteins provided herein, such as bacteria, yeasts, fungi, algae, insects, vertebrate cells (such as mammalian cells), and plant cells, including (as appropriate) primary cells and immortal cell lines. Numerous representatives of each cell type are commonly used and are available from a wide variety of commercial sources, including, for example, ATCC, Pharmacia, and Invitrogen.

[0205] Various yeast strains and yeast derived vectors are used commonly for the expression of heterologous proteins. For instance, specific, non-limiting examples of suitable yeast cells include Saccharomyces cerevisiae cells, Aspergillus cells, Trichoderma cells, Neurospora cells, Fusarium cells, or a Chrysosporium cells. In one specific, non-limiting example, Pichia pastoris expression systems, obtained from Invitrogen (Carlsbad, Calif.), can be used to express a CYP4G1 (SEQ ID NO: 2) or CYP4G2 (SEQ ID NO: 1) peptide. Such systems include suitable Pichia pastoris strains, vectors, reagents, transformants, sequencing primers, and media. Available strains include KM71H (a prototrophic strain), SMD1168H (a prototrophic strain), and SMD1168 (a pep4 mutant strain) (Invitrogen, Carlsbad, Calif.).

[0206] Saccharomyces cerevisiae is another commonly used yeast. The plasmid YRp7 (Stinchcomb et al., Nature, 282:39, 1979; Kingsman et al., Gene, 7:141, 1979; Tschemper et al., Gene, 10:157, 1980) is commonly used as an expression vector in Saccharomyces. This plasmid contains the trp1 gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1 (Jones, Genetics, 85:12, 1977). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0207] Yeast host cells can be transformed using the polyethylene glycol method, as described by Hinnen (Proc. Natl. Acad. Sci. USA, 75:1929, 1978). Additional yeast transformation protocols are set forth in Gietz et al. (Nucl. Acids Res., 20(17):1425, 1992) and Reeves et al. (FEMS, 99(2-3):193-197, 1992).

[0208] In the construction of suitable expression vectors, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Any plasmid vector containing a yeast-compatible promoter capable of transcribing a nucleic acid sequence encoding a prokaryotic tRNA, an origin of replication, and a termination sequence, is suitable.

[0209] Other suitable host cells are bacterial cells. Specific, non-limiting examples of suitable bacterial phyla include Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Chlamydiae, Chlorobi, Chloroflexi Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus, Thermus, Dictyoglomi, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Tenericutes, Thermodesulfobacteria, Thermomicrobia, Thermotogae, and Verrucomicrobia. Specific, non-limiting examples bacterial species of use include Escherichia coli, Thermus thermophilus, Stenotrophomonas maltophilia, Kineococcus radiotolerans Bacillus stearothermophilus, Methanococcus jannaschii, Methanosarcina mazei, Methanobacterium thermoautotrophicum, Methanococcus maripaludis, Methanopyrus kandleri, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-i, Archaeoglobus fulgidus, Pyrococcus fitriosus, Pyrococcus horikoshii, Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Aeuropyrum pernix, Thermoplasma acidophilum, and Thermoplasma volcanium. In one specific, non-limiting embodiment, the host cell is an E. coli cell, a S. maltophilia cell, a Pseudomonas species cell, a Bacillus sp. cell or an actinomycetes cell or cells belonging to the genus Rhodococcus genus. Introduction of the construct into the host cell can be accomplished by a variety of methods including calcium phosphate transfection, DEAE-dextran mediated transfection, polybrene mediated transfection, protoplast fusion, liposome mediated transfection, conjugation, natural transformation, electroporation, and other methods known in the art.

[0210] Still other suitable host cells are plant cells, including, but not limited to species of eukaryotic algae, mosses, club mosses, ferns, angiosperms, gymnosperms, and lichens. Any known method can be employed for plant cell transformation, culture, and regeneration can be employed. Methods for introduction of foreign DNA into plant cells include, but are not limited to: transfer involving the use of Agrobacterium tumefaciens and appropriate Ti vectors, including binary vectors; chemically induced transfer (for instance, with polyethylene glycol); biolistics; and microinjection. See, for instance, An et al., Plant Molecular Biology Manual A3:1-19, 1988. Various promoters suitable for expression of heterologous genes in plant cells are known in the art, including constitutive promoters, for instance, the cauliflower mosaic virus (CaMV) 35S promoter, which is expressed in many plant tissues, organ- or tissue-specific promoters, and promoters that are inducible by chemicals such as methyl jasminate, salicylic acid, or safeners, for example.

[0211] Host cells are grown under appropriate conditions to a suitable cell density. If the sequence of interest is operably linked to an inducible promoter, the appropriate environmental alteration is made to induce expression. If the product (for instance the hydrocarbon) accumulates in the host cell, the cells are harvested by, for example, centrifugation or filtration. Whole cell extractions can be performed to purify the hydrocarbon products from the whole cells. If the host cells secrete the product into the medium, the cells and medium are separated and the medium retained for purification of the desired product.

[0212] D. Product Production and Uses Thereafter

[0213] The disclosure provides methods of making hydrocarbons. Various production hosts are provided that can be used to produce products having engineered carbon chain lengths, saturation sites, and branch points. Methods of making such products are also provided as well as methods of further modifying the products, such as through cracking, to create high quality biofuels and specialty chemicals. For example, the present disclosure also provides for the use of the disclosed enzymes and hydrocarbons produced therefrom. One use is the production of hydrocarbons as biofuels, either in vitro, or by inserting the isolated disclosed sequences, such as CYP4G2 (or related sequences, see Table A) into an organism (e.g., plant, bacteria, algae, etc.) as described in detail herein in order to alter the hydrocarbon content, such as increasing the content, for production of fuel, lubricant, solvent, etc.

[0214] Another use of the disclosed enzymes is the production of synthetic hydrocarbon sex-pheromone components for Musca domestica control. For example, lures or traps may be baited with synthetic hydrocarbon sex-pheromones to attract Musca domestica

[0215] 1. Carbon Chain Characteristics

[0216] The hydrocarbons can be engineered to have specific carbon chain characteristics by expressing various enzymes or attenuating the expression of various enzymes in the production host. For example, carbon chain length can be controlled by expressing various thioesterases in the production host while attenuating the expression of endogenous thioesterases. Similarly, various branch points can be introduced into the carbon chain by expressing various bkd genes, and the degree of saturation can also be controlled by expressing various genes for example by over-expressing fabB.

[0217] 2. Methods of Making Hydrocarbons

[0218] One of ordinary skill in the art will appreciate that hydrocarbons can be produced using in vitro reactions, including chemical or enzymatic conversions as well as through in vivo reactions. Additionally, a combination of in vivo and in vitro conversions can be used. Moreover, specific hydrocarbons can be produced by selectively providing selected fatty acids, acyl-ACP, acyl-CoA, or aliphatic ketones (in the instance where the product desired is a specific hydrocarbon).

[0219] The term "convert" refers to the use of either chemical means or polypeptides in a reaction which changes a first intermediate to a second intermediate. The term "chemical conversion" refers to reactions that are not actively facilitated by polypeptides. The term "biological conversion" refers to reactions that are actively facilitated by peptides. Conversions can take place in vivo or in vitro. When biological conversions are used the peptides and/or cells can be immobilized on supports such as by chemical attachment on polymer supports. The conversion can be accomplished using any reactor known to one of ordinary skill in the art, for example in a batch or a continuous reactor.

[0220] a. In vitro

[0221] Given the disclosure provided herein, large scale enzyme production of the peptides CYP4G1 (SEQ ID NO: 2) or CYP4G2 (SEQ ID NO: 1) and homologues, variants thereof is now possible. Briefly, the coding sequences from anyone of these peptides or homologues of these peptides can be cloned into a high expression plasmid such as pET-21B(+) or pCOLADuet-1 (EMD Chemicals, Inc., Germany) and the plasmid can be induced. The resulting peptides can then be purified and used in batch production.

[0222] When in vitro methods are used, the peptides supplied to the reaction will depend upon the starting material. For example, when a hydrocarbon is desired and the starting material is acyl-ACP, a thioesterase and appropriate co-reactants can be added in conjunction with CYP4G1 (SEQ ID NO: 2) or CYP4G2 (SEQ ID NO: 1) peptides.

[0223] Additionally, a combination of chemical conversions and biological conversions can be used to produce a desired product. For example, one of ordinary skill in the art will appreciate that two fatty acids can be condensed to make an aliphatic ketone via chemical conversion and the resulting aliphatic ketone can then be converted a hydrocarbon using biological conversions.

[0224] b. In Vivo

[0225] Given the disclosure provided herein, hydrocarbons can be produced in a recombinant cell. The recombinant cell can produce one or more CYP4G1 (SEQ ID NO: 2) or CYP4G2 (SEQ ID NO: 1) and related sequences thereof (see Table A). One of ordinary skill in the art will appreciate that the choice of peptides to express in the recombinant cell will depend upon the desired product and the starting material provided to the cells. The in vivo methods described herein can also be used in combination with chemical conversions and in vitro biological conversions. The disclosure allows for the large scale production of hydrocarbons that have defined carbon chain lengths, saturation levels, and branch points. The production of such engineered molecules provides a diversity of products that can be used a fuels, and specialty chemicals.

[0226] 3. Post Production Processing

[0227] The generated hydrocarbons can be subjected to cracking to convert the high molecular weight carbon chains (for example, about C₂₂ to about C₃₆) to lower molecular weight hydrocarbons (for example, about C₁ to about C₁₈). In particular, the cracking can selectively target the double bond positions for cleavage in the feedstock. For example, a C₂₆ hydrocarbon with a single internal double bond can be cleaved to make two products, such as a C₁₂ alkane and a C₁₄ alkane. In some examples, any unsaturated hydrocarbon, especially a C₁₄ to C₂0 hydrocarbon is cracked to make an octane, nonane, etc. These are especially useful for producing high value products for jet fuel (for instance, C₁₄ to C₁₈), diesel (for instance, C₈ to C₁₄), and gasoline (for instance, C₅ to C₁0). Any of the methods of thermal cracking, hydrocracking, and catalytic cracking known to those of skill in the art can be used to further modify the products produced.

[0228] The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1

CYP4G1 is a Oxidative Decarbonylase

[0229] This example shows that CYP4G1 is a cytochrome P450 that functions as a oxidative decarbonylase.

[0230] CYP4G1 (Flybase I.D. CG3972; GenBank Accession Nos. NM080292 and AAF45503 each of which is herein incorporated by reference in its entirety) is a fruitfly (Drosophila melanogaster) cytochrome P450 that is most similar (71.7% identity and 81.8% similarity at the amino acid level) to CYP4G2 of any known sequence. To date, CYP4G1 mRNA had been found exclusively in oenocytes in D. melanogaster.

[0231] Fruitfly lines were generated based on an RNAi-fly stock that specifically knocks down CYP4G1 mRNA levels in oenocytes. These flies were essentially missing, or have significantly reduced, CYP4G1 activity. Hydrocarbons (HC) and cis-vaccinyl acetate (cVA) from these flies and from control (normal) flies were extracted and compared. FIG. 3 summarizes two RNAi-based lines (1405 and 485) used to knockdown CYP4G1 gene expression in Drosophila melanogaster. Quantitative analyses indicated the CYP4G1-knockdown flies had between 10- to 30-fold lower hydrocarbon levels than wildtype flies (FIGS. 3 and 7). Cis-vaccinyl acetate, a fatty acid-derived component specific to males, appeared at similar amounts in both strains, suggesting that fatty acid production was unaffected. Gas chromatograph traces (FIGS. 5 and 6) clearly show reduced hydrocarbon levels in the CYP4G1 knockdown flies. These studies demonstrate that CYP4G1 functions to produce hydrocarbons, i.e. it is a oxidative decarbonylase. Additionally, FIG. 11 provides a tracing illustrating Drosophila melanogaster CYP4G1 expressed in yeast. The full-length CYP4G1 sequence recoded for optimal yeast codon usage was cloned into the pYeDP60 vector and expressed in a modified WR yeast strain after induction by galactose. The CO-reduced difference spectrum of yeast microsomes showed approximately 50 pmol CYP4G1/mg protein. These studies are contrary to the prior studies in which Drosophila melanogaster CYP4G1 was predicted to be an omega hydroxylase.

Example 2

Expression and Characterization of CYP4G2

[0232] This example describes the expression and characterization of CYP4G2.

[0233] Housefly RNA was isolated from male and female integruments and fat bodies by methods known to those of skill in the art. Samples were subjected to northern blot analysis in which housefly RNA isolated from male and female integuments and fat bodies was hybridized with labeled CYP4G2 and actin (housekeeping control gene) cDNAs. As illustrated in FIG. 4, CYP4G2 expression was localized to the integrument in both sexes, but not in the fat body. This supports that CYP4G2 mRNA is localized to oenocytes.

[0234] The results from the RNAi silencing of CYP4G1 (an ortholog of the housefly CYP4G2) provided strong evidence that both these genes encoded the cytochrome P450s that convert aldehydes to hydrocarbons in insects. To verify that they are, CYP4G2 from the housefly and CYP4G1 from the fruitfly were expressed and assayed. The CYP4G2 cDNA was amplified by PCR and directionally cloned into the BamHI and XhoI sites of pENTR4 (Invitrogen) (modified to remove the NcoI site in the poly-linker, Sandstrom et al., 2006, Insect Biochem. Molec. Biol. 36(11):835-845) by standard methods and transformed into DH5α cells.

[0235] The BaculoDirect expression system (Invitrogen, Carlsbad, Calif.) was first used to express CYP4G2 as this system was previously used to express CYP9T2, CYP9T1, and other CYP6 and CYP9 P450s. For example, various I. pini and D. ponderosae P450 cDNAs in baculoviral vectors were expressed in Sf9 cells with (9T2, 6BW1) or without (9T1, 9Z18) housefly P450 reductase and all preparations showed the characteristic 450 nm peak. However, using this system with CYP4G2 resulted in no detectable 450 nm peak in the CO difference spectrum of recombinant microsomes. It was hypothesized that CYP4G2 may not be folding correctly in the heterologous system. The CYP4G2 membrane anchor is not well defined compared to other P450s, and has three contiguous valines close to the catalytic portion of the enzyme.

[0236] To address this problem, the second valine of the three contiguous valines in CYP4G2 (amino acid 38 of SEQ ID NO: 1) was mutated to alanine with a site-directed mutagenesis kit (Stratagene), producing CYP4G2.sub.V38A. In addition, a chimera containing the signal sequence of CYP9T2 followed by the CYP4G2 catalytic domain (CYP9T2/4G2) was constructed (SEQ ID NO: 51 provides the nucleic acid sequence and SEQ ID NO: 52 provides the amino acid sequence of which amino acids 1-25 form 9T2). KpnI sites were created by mutagenesis (Stratagene) at the C-terminal of the signal sequence in CYP9T2, and at the N-terminal end of the CYP4G2 catalytic domain. The regions were amplified by PCR, digested with KpnI, purified, and ligated together. The ligation product was amplified by PCR using a CYP9T2-specific forward primer and a CYP4G2-specific reverse primer, directionally cloned into the SalI and XhoI sites of pENTR4, and transformed into Top 10 cells. Either or both of these strategies were hypothesized to relieve the problem of misfolding CYP4G2 in Sf9 cells.

[0237] In additional characterization studies, recombinant baculoviral CYP4G2.sub.V38A and CYP9T2/4G2 clones are produced by LR recombinase reaction between each pENTR4 recombinant clone and BaculoDirect Linear DNA. The recombinant baculoviral virus is transfected into Sf9 cells, and the cells are grown in the presence of ganciclovir to select for recombinant virus. The titers of viral stocks are determined by a plaque assay.

[0238] Recombinant CYP9T2/4G2 and CYP4G2.sub.V38A constructs are co-expressed with housefly P450 reductase for functional assays essentially as described previously (Sandstrom et al., 2006, Insect Biochem. Molec. Biol. 36(11):835-845; Sandstrom et al., 2008, J. Chem. Ecol. 34(12):1584-1592. Sf9 cells are infected with recombinant CYP9T2/4G2 or CYP4G2.sub.V38A baculovirus and housefly reductase baculovirus at multiplicities of infection (MOIs, pfu/cell) ranging from 0.2 to 2. A fixed, optimized MOI ratio for recombinant CYP9T2/4G2 or CYP4G2.sub.V38A: housefly P450 reductase is used in all infections. Hemin (final concentration 0.06 mM) is added 24 hours after transfection. Sf9 are harvested 96 hours post-infection and microsomes are prepared by differential centrifugation according to Sandstrom et al. (Insect Biochem. Molec. Biol. 36(11):835-845, 2006). Briefly, cells are collected by centrifugation at 3000×g at 4° C. for 10 minutes and then lysed in ice cold lysis buffer (100 mM sodium phosphate pH 7.8, containing 1.1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF, 1/1000 volume of Sigma protease inhibitor cocktail, and 20% glycerol) by sonication for 30 seconds on ice using a Branson Sonifier 450. The lysate is centrifuged at 10,000×g for 20 minutes at 4° C. The supernatant is collected and centrifuged at 120,000×g for 1 hour to pellet the microsomes. The microsomes are resuspended in the same buffer and used immediately. Alternatively, because CYP4G2 activity is stable in frozen housefly microsome preparations for at least a month, recombinant Sf9 microsomes can be flash-frozen in liquid nitrogen and stored at -80° C. for later analysis. Protein concentrations are assayed by a Bradford assay, utilizing BSA as a standard. The amount of P450 expressed in Sf9 microsomes are measured by adsorption at 450 nm according to the carbon monoxide (CO)-difference spectrum analysis method.

[0239] Hydrocarbon product is extracted, isolated and assayed by standard procedures, including those disclosed herein. Alternatively, a Drosophila cell expression system can be used to express either CYP4G2, CYP9T2/4G2, or CYP4G2.sub.V38A and CYP4G1. Thus, these studies will characterize the disclosed polypeptides and assess their enzymatic activity.

Example 3

Purification of CYP9T2/4G2 or CYP4G2.sub.V38A

[0240] This example describes methods for purifying exemplary insect P450 enzymes and in particular, their oxidative decarbonylase activity.

[0241] Compared to plants and mammals, much less is known about functions of the different insect P450 enzymes. The biochemical characteristics of insect P450 enzymes, such as CYP9T2/4G2 or CYP4G2.sub.V38A, can be determined through assays of purified, recombinant protein. CYP9T2/4G2 or CYP4G2.sub.V38A is purified using an immune affinity column.

[0242] Antibody production: In order to identify a suitable antigenic region for antibody production, an alignment of CYP4G2 to CYP4G13 was compared, which revealed a 62% amino acid identity to CYP4G2. One of several loop regions identified in the model had very high identity with CYP4G2 and revealed a 15 amino acid peptide in this region with very high antigenic potential.

[0243] To generate antisera against CYP4G2, the 15 residue peptide corresponding to a predicted antigenic portion of the sequence (WQHHRKMIAPTFHQS, amino acids 157-170 of SEQ ID NO: 1) is synthesized and purified by HPLC purification by the Nevada Proteomic Center (Reno, Nev., USA). The synthesized peptide is conjugated to a keyhole limpet hemocyanin carrier via an added C-terminal cysteine, and is used to immunize rabbits at Cocalico Biologicals (Reamstown, Pa., USA). ELISA assays of collected antisera are performed to confirm immunoreactivity. The positive samples are affinity purified using a SulfoLink column (Pierce, Rockford, Ill., USA) coupled with the peptide. The immunoreactivity of the affinity-purified rabbit anti-CYP4G2 antiserum is further confirmed by western blots of bacterially-expressed recombinant CYP4G2.

[0244] In addition or alternatively, an approach relying on intact CYP4G2 as an antigen can be used. The ORF for CYP4G2 (truncated to remove the membrane anchor) is inserted into pENT4 so that the vector-encoded His tag is fused to the C-terminal. This truncated CYP4G2 ("CYP4G2_cat") is transferred to the BaculoDirect expression vector by recombination, and high-titre virus stocks is prepared as described herein. Recombinant CYP4G2_cat-His fusion protein is subsequently purified on a Ni²+-column and incubated with enterokinase to remove the His tag. Purification of CYP4G2_cat-His is confirmed by SDS-PAGE and peptide sequencing.

[0245] Protein purification: Recombinant CYP9T2/4G2 or CYP4G2.sub.V38A is purified with an immune affinity column. Microsomal fractions of the cells are prepared according to methods described herein and solubilized with detergent. The solubilized enzyme is applied to a CYP4G2 affinity column prepared by fixing purified rabbit anti-CYP4G2 antibody to a protein A column (Pierce Protein Research Products, Rockford, Ill.). The column is washed to remove unbound protein and recombinant CYP4G2 is eluted with high salt buffer.

[0246] Alternatively, if this procedure reduces CYP4G2 catalytic activity, CYP4G2 is purified by standard ion exchange chromatography. The microsomal fraction is resuspended in 10 mM potassium phosphate buffer, ph 7.5, containing 20% glycerol, 0.1 mM EDA, 0.1 mM DTT and 0.1 mM BHT (Buffer A). A mixture of 1.7% (v/v) Lubrol PX and 4.25% (w/v) cholate is added to the microsomal suspension under stifling to give a final concentration of 0.2% Lubrol PX and 0.5% cholate. The suspension is stirred gently for 40 minutes at 4° C., and then centrifuged at 105,000×g for 60 minutes. Calcium phosphate gel is added to the supernatant, and the supernatant is collected after centrifugation at 2000×g for 5 min. The precipitated gel is washed with 7 ml of 10 mM potassium phosphate buffer pH 7.5, containing 20% glycerol, 0.1 mM EDTA, 0.2% Lubro PX and 0.5% cholate. After centrifugation at 2000×g for 5 minutes, the supernatant is collected and mixed with the first supernatant to form the enzyme fraction. The supernatant is applied to a HiTrap DEAE FF column connected to an AKTApurifier (Amersham Biosciences). The column is washed with one bed-volume of buffer A and the with two bed-volume of 50 mM NaCl. Finally, the enzyme is eluted with a linear gradient of 50-200 mM NaCl. The amount of heme-containing CYP9T2/4G2 or CYP4G2.sub.V38A in the elution is monitored by measuring the absorption at 450 nm. The fractions containing the CYP4G2 activity is concentrated with Centriprep concentrators (Amicon, Billerica, Mass.) and the buffer is exchanged 10 mM potassium phosphate buffer, pH 7.5, containing 20% glycerol. The sample is concentrated to a small volume and applied to a hydroxyapatite column. The enzyme in the pass through fraction is collected, measured by carbon monoxide (CO)-difference spectrum analysis method and stored at -80° C. until use. Purification is confirmed by SDS-PAGE, native gel electrophoresis and isoelectric focusing. Amino acid sequencing and MALDI-MS analyses are also performed to verify the identity and integrity of the isolated proteins. The CO-reduced difference spectra are measured to observe the peak at 450 which confirms the preparation of active P450

Example 4

Characterization of CYP9T2/4G2 and CYP4G2.sub.V38A

[0247] This example provides methods for characterizing disclosed cytochrome P450 enzymes.

[0248] Isolated P450s require lipid for activity. The approach used to mimic the structural arrangement of lipids and enzymes within the endoplasmic reticulum is to physically incorporate the cytochromes P450 in a vesicle bilayer of phospholipids. To obtain optimal activity, phospholipid is added to the assay mixture. The procedure is as follows: dilaurylphosphateidylcholine (DLPC), is suspended at a concentration of 5 mM in a solution of 50 mM potassium phosphate (pH7.25), 20% glycerol, 0.1 M NaCl, and 5 mM EDTA. The suspension lipid is sonicated in a glass tube in a water bath until it turns completely clear. Once the lipid is clear, purified CYP9T2/4G2 or CYP4G2.sub.V38A or other disclosed purified polypeptides and lipid is mixed in microfuge tubes. The above protein and lipid is incubated at room temperature for 2 hours. After the incubation, the mixture is aliquoted into assay tubes, and buffer and reaction components are added directly to the mixture. The assay solutions are incubated at 30° C. in the presence of substrate for 5 minutes, then reactions are started by adding NADPH. The standard experimental procedure in our lab will performed as described in objective-1. The lipid is added at varying ratios (100:1 to 500:1) to find the ideal ratio for CYP9T2/4G2 or CYP4G2.sub.V38A by assaying the activity. If adding lipid is difficult, protein can be added back from housefly integument tissue to see if a hydrocarbon binding protein is present that would bind hydrocarbon as it is produced and increase activity or time of linearity of reaction.

[0249] Chain length specificity: Long chain hydrocarbons of insects play central roles in the waterproofing of the insect cuticle and function extensively in chemical communication. Thus, deuterium- or tritium-labeled aldehydes of 14, 16, 18, 20, 22, 24 and 28 carbons are prepared by the method used by Reed et al. (Proc. Natl. Acad. Sci. U.S.A. 91(21): 10000-10004, 1994). Each aldehyde is assayed individually and the rate of conversion of aldehyde to hydrocarbon is monitored by GC-MS (deuterium labeled) or liquid scintillation counting (tritium labeled). In addition, groups of aldehydes are assayed together to determine the chain length preference.

[0250] Selectivity for the cofactor: Reed et al. (Biochemistry 34: 26221-26227, 1995) found when NADH replaced NADPH as a reductant, both males and females produced much less hydrocarbon. In contrast to the results using NADPH, males produced more hydrocarbon than females over the entire range of NADH concentrations tested. Purified P450 is assayed in the presence of varying amounts of NADPH, NADH or a combination of NADPH and NADH to determine the specificity of the reductant. If applicable, the Michaelis-Menton equation is used to evaluate K_M, k_cat, and K_cat/K_M for the various substrates. These studies will allow the disclosed polypeptides to be characterized.

Example 5

Molecular Modeling of CYP4G2

[0251] This example provides techniques to gain insight into the structure of CYP4G2. Molecular modeling has become an increasingly useful tool in understanding the mechanisms underlying biological phenomena. Since proteins are long, linear molecules even with limited flexibility the number of possible conformations is enormous: thus construction of models based simply from first principles is impractical at this time. Historically molecular models of proteins rely on constraints generated from experimental data (e.g., X-ray diffraction of single crystals). As the Protein Data Bank has accumulated a large number of experimentally constrained models of protein structure, it became evident that proteins form a limited number of folds (perhaps 1000-2000) thus making it possible to use this knowledge of protein folds to constrain model building. The combination of knowledge and energy based methods (constraining the protein so that it is at the lowest energy conformation) has had many successful predictions of protein structure. Success depends on correctly identifying a homologous protein whose structure has been determined experimentally. This so-called template is then used to constrain model building of the unknown protein (the target). Since protein sequence determines the secondary, tertiary, and quaternary structure of protein, sequence homology is commonly used to identify template structures.

[0252] Cytochromes P450 are quite varied in function and have a broad substrate specificity thus making the molecular modeling of P450s challenging. A ClustalW 2.0 alignment (FIG. 8) of Musca domestica cytochrome CYP4G2 with other P450s from the PDB, specifically Homo sapiens cytochrome CYP3A4 (pdb 1TQN) and Mycobacterium tuberculosis CYP51 (pdb 2CIB), show an area that contains many gaps, areas of varying homology, and few invariant residues as exemplified in the model and alignment. While there are differences within this class of enzymes, there are also many similarities. One important region of similarity includes the heme binding at the active center (FIG. 9), thus placing constraints on the protein fold around the binding/active site. Invariant glycines and prolines (FIGS. 9 and 10) are of interest because these residues have unique effects on Ramachandran space and thus on backbone geometry. The tiny side chain of glycine also facilitates tertiary interactions such as alpha-helix crossings, as seen in FIG. 9. Prolines strongly stiffen the peptide chain, while glycines cause the protein to have greater flexibility, thus they are often found where turns are located as exemplified in FIG. 10. The alignment in FIG. 8 shows the characteristic invariant cysteine, which binds to the iron center of the heme in the active site forming the 6^th ligand of the heme group (FIG. 9), as is seen with cytochromes P450. Upon further examination of this template (1TQN) a pocket where a substrate has the potential to fit was observed in an area on the opposite side of the heme from the cysteine. The model of 1TQN shows that the more conserved and the less conserved regions make up approximately equal halves of the protein, the more conserved areas being more closely associated with the heme binding center (FIG. 10). Note the two halves of the primary structure are interdigitated in the tertiary structure. Thus conserved and non-conserved regions strongly interact. CYP4G2 is longer than the other enzymes examined in the above alignment, thus the fly enzyme will contain multiple insertions. In this example, the amino acid sequence is used to predict putative solution conformations of the protein. Complementarity of the binding site to possible substrates (e.g., aldehydes 18 carbons, 24 carbons long, etc) are evaluated using molecular dynamics and visualized using programs such as MOLCAD (Heiden et al., 1993, J. Comput. Aided Mol. Des. 7(5): 503-14).

[0253] The methods utilized are calibrated against solved structures of known P450 proteins with ligands bound in the binding/active site. The energy of interaction between substrate and enzyme using free energy perturbation are calculated and compared with empirical methods using the AMBER suite of computational programs (Case et al., 2005, J. Comput. Chem. 26(16):1668-1688). When this model is experimentally confirmed, then it is applied to predict substrate specificity. In addition, upon determination that the model is realistic, it can be used to find ways to engineer the P450 to function as needed. Models are built by a combination of sequence homology (FUGUE) (Williams et al., 2001, Proteins 5:92-97) and threading, which uses recognition of protein folds of known structure, rather than homology, to base a structure prediction on using programs like Matchmaker, or GeneFold. Ligands are docked (e.g., Morris et al., 1996, J. Comput. Aided Mol. Des. 10(4): 293-304) and energies of interaction are determined by using free energy perturbation (examining energies between two states of a protein) and integration methods (calculates free energy for consecutive points in time and is calculated from an average over all points).

[0254] Other computational tools are investigated and incorporated if they are found to be beneficial. In addition to measurement of catalytic properties (substrate specificity, product analysis, inhibition), conformational verification can be done through optical spectroscopy (such as CD, UV-vis, fluorescence perturbation, fluorescence quenching, depolarization of fluorescence), hydrodynamics (e.g., gel permeation chromatography, analytical ultracentrifugation, inelastic light scattering), chemical modification (number and rates of reaction), site-directed mutagenesis and kinetics of proteolytic digestion.

[0255] It is to be understood that the above discussion provides a detailed description of various embodiments. The above descriptions will enable those of ordinary skill in the art to make and use the disclosed embodiments, and to make departures from the particular examples described above to provide embodiments of the methods and apparatuses constructed in accordance with the present disclosure. The embodiments are illustrative, and not intended to limit the scope of the present disclosure. The scope of the present disclosure is rather to be determined by the scope of the claims as issued and equivalents thereto.

Sequence CWU 1

1

521559PRTMusca domestica 1Met Thr Ala Asp Thr Leu Val Leu Glu Thr Met Asp Ser Ala Lys Asn 1 5 10 15 Ser Thr Ala Gly Pro Ala Thr Val Leu Asn Pro Ile Trp Thr Ala Leu 20 25 30 Leu Gly Ile Ala Val Val Val Ser Leu Tyr Glu Ile Trp Leu Arg Asn 35 40 45 Thr Arg Lys Tyr Lys Leu Thr Ala Asn Met Pro Asn Pro Pro Met Leu 50 55 60 Pro Leu Ile Gly Asn Gly His Leu Val Ala His Leu Thr Asn Ala Glu 65 70 75 80 Ile Leu Ala Arg Gly Ile Gly Tyr Met Gln Thr Tyr Gly Gly Ala Met 85 90 95 Arg Gly Phe Leu Gly Pro Met Leu Val Val Phe Leu Trp Asn Ala Pro 100 105 110 Asp Ile Glu Leu Ile Leu Ser Thr His Thr His Leu Glu Lys Ser Ile 115 120 125 Glu Tyr Arg Phe Phe Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile Ser 130 135 140 Asn Gly His His Trp Gln His His Arg Lys Met Ile Ala Pro Thr Phe 145 150 155 160 His Gln Ser Ile Leu Lys Ser Phe Val Pro Ala Phe Val Gln His Ser 165 170 175 Lys Lys Val Val Glu Arg Met Ala Lys Glu Leu Gly Lys Glu Phe Asp 180 185 190 Val His Asp Tyr Met Ser Gln Thr Thr Val Glu Ile Leu Leu Ser Thr 195 200 205 Ala Met Gly Val Lys Lys Val Pro Glu Asp Asn Lys Ser Leu Glu Tyr 210 215 220 Ala Lys Ala Val Val Asp Met Cys Asp Ile Ile His Lys Arg Gln Leu 225 230 235 240 Lys Phe Phe Tyr Arg Met Asp Ala Leu Tyr Asn Leu Ser Ser Met Ser 245 250 255 Glu Lys Gly Lys Lys Met Met Asp Ile Ile Leu Gly Met Thr Arg Lys 260 265 270 Val Val Thr Glu Arg Gln Gln Asn Phe Asn Ala Glu Ser Arg Ala Ile 275 280 285 Val Glu Glu Asp Asp Glu Ile Ser Lys Gln Lys Gln Gln Ala Lys Lys 290 295 300 Lys Glu Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp Glu Asn Asp Val 305 310 315 320 Gly Ala Lys Lys Arg Leu Ala Leu Leu Asp Ala Met Met Ala Met Ser 325 330 335 Lys Asn Pro Asp Val Glu Trp Thr Asp Lys Asp Val Met Asp Glu Val 340 345 350 Asn Thr Ile Met Phe Glu Gly His Asp Thr Thr Ser Ala Gly Ser Ser 355 360 365 Phe Val Leu Cys Met Leu Gly Ile Tyr Lys Asp Ile Gln Glu Lys Val 370 375 380 Leu Ala Glu Gln Lys Ala Ile Phe Gly Asp Asn Phe Leu Arg Asp Cys 385 390 395 400 Thr Phe Ala Asp Thr Met Glu Met Lys Tyr Leu Glu Arg Val Ile Met 405 410 415 Glu Thr Leu Arg Leu Tyr Pro Pro Val Pro Leu Ile Ala Arg Arg Ala 420 425 430 Glu Phe Asp Val Lys Leu Ala Ser Gly Pro Tyr Thr Ile Pro Lys Gly 435 440 445 Thr Thr Val Val Ile Ala Gln Phe Ala Val His Arg Asn Pro Gln Tyr 450 455 460 Phe Pro Asn Pro Glu Lys Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg 465 470 475 480 Met Ala Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser Ala Gly Pro 485 490 495 Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Val Leu 500 505 510 Leu Ser Thr Ile Ile Arg Asn Tyr Ser Val Gln Ser Asn Gln Gln Glu 515 520 525 Lys Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Ile Glu Asn Gly 530 535 540 Phe Asn Ile Met Leu Asn Arg Arg Pro Glu Ala Met Lys Ala Met 545 550 555 2486PRTMusca domestica 2Val Ala Ala Gly Leu Ser Asn Ala Glu Ile Leu Ala Val Gly Leu Gly 1 5 10 15 Tyr Leu Asn Lys Tyr Gly Glu Thr Met Lys Ala Trp Leu Gly Asn Val 20 25 30 Leu Leu Val Phe Leu Thr Asn Pro Ser Asp Ile Glu Leu Ile Leu Ser 35 40 45 Gly His Gln His Leu Thr Lys Ala Glu Glu Tyr Arg Tyr Phe Lys Pro 50 55 60 Trp Phe Gly Asp Gly Leu Leu Ile Ser Asn Gly His His Trp Arg His 65 70 75 80 His Arg Lys Met Ile Ala Pro Thr Phe His Gln Ser Ile Leu Lys Ser 85 90 95 Phe Val Pro Thr Phe Val Asp His Ser Lys Ala Val Val Ala Arg Met 100 105 110 Gly Leu Glu Ala Gly Lys Ser Phe Asp Val His Asp Tyr Met Ser Gln 115 120 125 Thr Thr Val Asp Ile Leu Leu Ser Thr Ala Met Gly Val Lys Lys Leu 130 135 140 Pro Glu Gly Asn Lys Ser Phe Glu Tyr Ala Gln Ala Val Val Asp Met 145 150 155 160 Cys Asp Ile Ile His Lys Arg Gln Val Lys Leu Leu Tyr Arg Leu Asp 165 170 175 Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu Lys Gly Asp Arg Met Met 180 185 190 Asn Ile Ile Leu Gly Met Thr Ser Lys Val Val Lys Asp Arg Lys Glu 195 200 205 Asn Phe Gln Glu Glu Ser Arg Ala Ile Val Glu Glu Ile Ser Thr Pro 210 215 220 Val Ala Ser Thr Pro Ala Ser Lys Lys Glu Gly Leu Arg Asp Asp Leu 225 230 235 240 Asp Asp Ile Asp Glu Asn Asp Val Gly Ala Lys Arg Arg Leu Ala Leu 245 250 255 Leu Asp Ala Met Val Glu Met Ala Lys Asn Pro Asp Ile Glu Trp Asn 260 265 270 Glu Lys Asp Ile Met Asp Glu Val Asn Thr Ile Met Phe Glu Gly His 275 280 285 Asp Thr Thr Ser Ala Gly Ser Ser Phe Ala Leu Cys Met Met Gly Ile 290 295 300 His Lys Asp Ile Gln Ala Lys Val Phe Ala Glu Gln Lys Ala Ile Phe 305 310 315 320 Gly Asp Asn Met Leu Arg Asp Cys Thr Phe Ala Asp Thr Met Glu Met 325 330 335 Lys Tyr Leu Glu Arg Val Ile Leu Glu Thr Leu Arg Leu Tyr Pro Pro 340 345 350 Val Pro Leu Ile Ala Arg Arg Leu Asp Tyr Asp Leu Lys Leu Ala Ser 355 360 365 Gly Pro Tyr Thr Val Pro Lys Gly Thr Thr Val Ile Val Leu Gln Tyr 370 375 380 Cys Val His Arg Arg Pro Asp Ile Tyr Pro Asn Pro Thr Lys Phe Asp 385 390 395 400 Pro Asp Asn Phe Leu Pro Glu Arg Met Ala Asn Arg His Tyr Tyr Ser 405 410 415 Phe Ile Pro Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr 420 425 430 Ala Met Leu Lys Leu Lys Val Leu Leu Ser Thr Ile Val Arg Asn Tyr 435 440 445 Ile Val His Ser Thr Asp Thr Glu Ala Asp Phe Lys Leu Gln Ala Asp 450 455 460 Ile Ile Leu Lys Leu Glu Asn Gly Phe Asn Val Ser Leu Glu Lys Arg 465 470 475 480 Gln Tyr Ala Thr Val Ala 485 3556PRTDrosophila erecta 3Met Ala Val Glu Val Val Gln Glu Thr Leu Gln Gln Ala Ala Ala Ser 1 5 10 15 Ser Ser Thr Thr Val Leu Gly Phe Ser Pro Met Phe Thr Thr Leu Val 20 25 30 Gly Thr Leu Val Ala Met Ala Leu Tyr Glu Tyr Trp Arg Arg Asn Ser 35 40 45 Arg Glu Tyr Arg Met Val Ala Asn Ile Pro Ser Pro Pro Glu Leu Pro 50 55 60 Ile Leu Gly Gln Ala His Val Ala Ala Gly Leu Ser Asn Ala Glu Ile 65 70 75 80 Leu Ala Val Gly Leu Gly Tyr Leu Asn Lys Tyr Gly Glu Thr Met Lys 85 90 95 Ala Trp Leu Gly Asn Val Leu Leu Val Phe Leu Thr Asn Pro Ser Asp 100 105 110 Ile Glu Leu Ile Leu Ser Gly His Gln His Leu Thr Lys Ala Glu Glu 115 120 125 Tyr Arg Tyr Phe Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile Ser Asn 130 135 140 Gly His His Trp Arg His His Arg Lys Met Ile Ala Pro Thr Phe His 145 150 155 160 Gln Ser Ile Leu Lys Ser Phe Val Pro Thr Phe Val Asp His Ser Lys 165 170 175 Ala Val Val Ala Arg Met Gly Leu Glu Ala Gly Lys Ser Phe Asp Val 180 185 190 His Asp Tyr Met Ser Gln Thr Thr Val Asp Ile Leu Leu Ser Thr Ala 195 200 205 Met Gly Val Lys Lys Leu Pro Glu Gly Asn Lys Ser Phe Glu Tyr Ala 210 215 220 Gln Ala Val Val Asp Met Cys Asp Ile Ile His Lys Arg Gln Val Lys 225 230 235 240 Leu Leu Tyr Arg Leu Asp Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu 245 250 255 Lys Gly Asp Arg Met Met Asn Ile Ile Leu Gly Met Thr Ser Lys Val 260 265 270 Val Lys Asp Arg Lys Glu Asn Phe Gln Glu Glu Ser Arg Ala Ile Val 275 280 285 Glu Glu Ile Ser Thr Pro Ala Ala Ser Thr Pro Ala Ser Lys Lys Glu 290 295 300 Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp Glu Asn Asp Val Gly Ala 305 310 315 320 Lys Arg Arg Leu Ala Leu Leu Asp Ala Met Val Glu Met Ala Lys Asn 325 330 335 Pro Asp Ile Glu Trp Asn Glu Lys Asp Ile Met Asp Glu Val Asn Thr 340 345 350 Ile Met Phe Glu Gly His Asp Thr Thr Ser Ala Gly Ser Ser Phe Ala 355 360 365 Leu Cys Met Met Gly Ile His Lys Asp Ile Gln Ala Lys Val Phe Ala 370 375 380 Glu Gln Lys Ala Ile Phe Gly Asp Asn Met Leu Arg Asp Cys Thr Phe 385 390 395 400 Ala Asp Thr Met Glu Met Lys Tyr Leu Glu Arg Val Ile Leu Glu Thr 405 410 415 Leu Arg Leu Tyr Pro Pro Val Pro Leu Ile Ala Arg Arg Leu Asp Tyr 420 425 430 Asp Leu Lys Leu Ala Ser Gly Pro Tyr Thr Val Pro Lys Gly Thr Thr 435 440 445 Val Ile Val Leu Gln Tyr Cys Val His Arg Arg Pro Asp Ile Tyr Pro 450 455 460 Asn Pro Thr Lys Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg Met Ala 465 470 475 480 Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser Ala Gly Pro Arg Ser 485 490 495 Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Val Leu Leu Ser 500 505 510 Thr Ile Val Arg Asn Tyr Ile Val His Ser Thr Asp Thr Glu Ala Asp 515 520 525 Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Leu Glu Asn Gly Phe Asn 530 535 540 Val Ser Leu Glu Lys Arg Gln Tyr Ala Thr Val Ala 545 550 555 4556PRTDrosophila simulans 4Met Ala Val Glu Val Val Gln Glu Thr Leu Gln Gln Ala Ala Ala Ser 1 5 10 15 Ser Ser Thr Thr Val Leu Gly Phe Ser Pro Met Leu Thr Thr Leu Val 20 25 30 Gly Thr Leu Val Ala Met Ala Leu Tyr Glu Tyr Trp Arg Arg Asn Ser 35 40 45 Arg Glu Tyr Arg Met Val Ala Asn Ile Pro Ser Pro Pro Glu Leu Pro 50 55 60 Ile Leu Gly Gln Ala His Val Ala Ala Gly Leu Ser Asn Ala Glu Ile 65 70 75 80 Leu Ala Val Gly Leu Gly Tyr Leu Asn Lys Tyr Gly Glu Thr Met Lys 85 90 95 Ala Trp Leu Gly Asn Val Leu Leu Val Phe Leu Thr Asn Pro Ser Asp 100 105 110 Ile Glu Leu Ile Leu Ser Gly His Gln His Leu Thr Lys Ala Glu Glu 115 120 125 Tyr Arg Tyr Phe Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile Ser Asn 130 135 140 Gly His His Trp Arg His His Arg Lys Met Ile Ala Pro Thr Phe His 145 150 155 160 Gln Ser Ile Leu Lys Ser Phe Val Pro Thr Phe Val Asp His Ser Lys 165 170 175 Ala Val Val Ala Arg Met Gly Leu Glu Ala Gly Lys Ser Phe Asp Val 180 185 190 His Asp Tyr Met Ser Gln Thr Thr Val Asp Ile Leu Leu Ser Thr Ala 195 200 205 Met Gly Val Lys Lys Leu Pro Glu Gly Asn Lys Ser Phe Glu Tyr Ala 210 215 220 Gln Ala Val Val Asp Met Cys Asp Ile Ile His Lys Arg Gln Val Lys 225 230 235 240 Leu Leu Tyr Arg Leu Asp Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu 245 250 255 Lys Gly Asp Arg Met Met Asn Ile Ile Leu Gly Met Thr Ser Lys Val 260 265 270 Val Lys Asp Arg Lys Glu Asn Phe Gln Glu Glu Ser Arg Ala Ile Val 275 280 285 Glu Glu Ile Ser Thr Pro Val Ala Ser Thr Pro Ala Ser Lys Lys Glu 290 295 300 Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp Glu Asn Asp Val Gly Ala 305 310 315 320 Lys Arg Arg Leu Ala Leu Leu Asp Ala Met Val Glu Met Ala Lys Asn 325 330 335 Pro Asp Ile Glu Trp Asn Glu Lys Asp Ile Met Asp Glu Val Asn Thr 340 345 350 Ile Met Phe Glu Gly His Asp Thr Thr Ser Ala Gly Ser Ser Phe Ala 355 360 365 Leu Cys Met Met Gly Ile His Lys Asp Ile Gln Ala Lys Val Phe Ala 370 375 380 Glu Gln Lys Ala Ile Phe Gly Asp Asn Met Leu Arg Asp Cys Thr Phe 385 390 395 400 Ala Asp Thr Met Glu Met Lys Tyr Leu Glu Arg Val Ile Leu Glu Thr 405 410 415 Leu Arg Leu Tyr Pro Pro Val Pro Leu Ile Ala Arg Arg Leu Asp Tyr 420 425 430 Asp Leu Lys Leu Ala Ser Gly Pro Tyr Thr Val Pro Lys Gly Thr Thr 435 440 445 Val Ile Val Leu Gln Tyr Cys Val His Arg Arg Pro Asp Ile Tyr Pro 450 455 460 Asn Pro Thr Lys Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg Met Ala 465 470 475 480 Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser Ala Gly Pro Arg Ser 485 490 495 Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Val Leu Leu Ser 500 505 510 Thr Ile Val Arg Asn Tyr Ile Val His Ser Thr Asp Thr Glu Ala Asp 515 520 525 Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Leu Glu Asn Gly Phe Asn 530 535 540 Val Ser Leu Glu Lys Arg Gln Tyr Ala Thr Val Ala 545 550 555 5556PRTDrosophila yakuba 5Met Ala Val Glu Val Val Gln Glu Thr Leu Gln Gln Ala Ala Ala Ser 1 5 10 15 Ser Ser Thr Thr Val Leu Gly Phe Ser Pro Met Phe Thr Thr Leu Val 20 25 30 Gly Thr Leu Val Ala Met Ala Leu Tyr Glu Tyr Trp Arg Arg Asn Ser 35 40 45 Arg Glu Tyr Arg Met Val Ala Asn Ile Pro Ser Pro Pro Glu Leu Pro 50 55 60 Ile Leu Gly Gln Ala His Val Ala Ala Gly Leu Ser Asn Ala Glu Ile 65 70 75 80 Leu Ala Val Gly Leu Gly Tyr Leu Asn Lys Tyr Gly Glu Thr Met Lys 85 90 95 Ala Trp Leu Gly Asn Val Leu Leu Val Phe Leu Thr Asn Pro Ser Asp 100 105 110 Ile Glu Leu Ile Leu Ser Gly His Gln His Leu Thr Lys Ala Glu Glu 115 120 125 Tyr Arg Tyr Phe Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile

Ser Asn 130 135 140 Gly His His Trp Arg His His Arg Lys Met Ile Ala Pro Thr Phe His 145 150 155 160 Gln Ser Ile Leu Lys Ser Phe Val Pro Thr Phe Val Asp His Ser Lys 165 170 175 Ala Val Val Ala Arg Met Gly Leu Glu Ser Gly Lys Ser Phe Asp Val 180 185 190 His Asp Tyr Met Ser Gln Thr Thr Val Asp Ile Leu Leu Ser Thr Ala 195 200 205 Met Gly Val Lys Lys Leu Pro Glu Gly Asn Lys Ser Phe Glu Tyr Ala 210 215 220 Gln Ala Val Val Asp Met Cys Asp Ile Ile His Lys Arg Gln Val Lys 225 230 235 240 Leu Leu Tyr Arg Leu Asp Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu 245 250 255 Lys Gly Asp Arg Met Met Asn Ile Ile Leu Gly Met Thr Ser Lys Val 260 265 270 Val Lys Asp Arg Lys Glu Asn Phe Gln Glu Glu Ser Arg Ala Ile Val 275 280 285 Glu Glu Ile Ala Thr Pro Val Ala Ser Thr Pro Ala Ser Lys Lys Glu 290 295 300 Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp Glu Asn Asp Val Gly Ala 305 310 315 320 Lys Arg Arg Leu Ala Leu Leu Asp Ala Met Val Glu Met Ala Lys Asn 325 330 335 Pro Asp Ile Glu Trp Asn Glu Lys Asp Ile Met Asp Glu Val Asn Thr 340 345 350 Ile Met Phe Glu Gly His Asp Thr Thr Ser Ala Gly Ser Ser Phe Ala 355 360 365 Leu Cys Met Met Gly Ile His Lys Asp Ile Gln Ala Lys Val Phe Ala 370 375 380 Glu Gln Lys Ala Ile Phe Gly Asp Asn Met Leu Arg Asp Cys Thr Phe 385 390 395 400 Ala Asp Thr Met Glu Met Lys Tyr Leu Glu Arg Val Ile Leu Glu Thr 405 410 415 Leu Arg Leu Tyr Pro Pro Val Pro Leu Ile Ala Arg Arg Leu Asp Tyr 420 425 430 Asp Leu Lys Leu Ala Ser Gly Pro Tyr Thr Val Pro Lys Gly Thr Thr 435 440 445 Val Ile Val Leu Gln Tyr Cys Val His Arg Arg Pro Asp Ile Tyr Pro 450 455 460 Asn Pro Thr Lys Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg Met Ala 465 470 475 480 Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser Ala Gly Pro Arg Ser 485 490 495 Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Val Leu Leu Ser 500 505 510 Thr Ile Val Arg Asn Tyr Ile Val His Ser Thr Asp Thr Glu Ala Asp 515 520 525 Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Leu Glu Asn Gly Phe Asn 530 535 540 Val Ser Leu Glu Lys Arg Gln Tyr Ala Thr Val Ala 545 550 555 6552PRTDrosophila pseudoobscura 6Met Thr Val Asp Thr Val Gln Glu Thr Leu Gln His Ala Ala Thr Ser 1 5 10 15 Thr Ser Gly Leu Gly Phe Ser Pro Met Leu Thr Thr Leu Val Gly Thr 20 25 30 Ile Val Ala Leu Gly Leu Tyr Glu Tyr Trp Arg Arg Asn Thr Arg Glu 35 40 45 Tyr Arg Met Val Ala Asn Ile Pro Ser Pro Pro Gly Leu Pro Leu Leu 50 55 60 Gly Gln Ala His Met Val Ala Gly Leu Ser Asn Ala Glu Ile Leu Asn 65 70 75 80 Val Gly Leu Gly Tyr Leu Asn Lys Tyr Gly Glu Thr Met Lys Ala Trp 85 90 95 Leu Gly Asn Val Leu Leu Val Phe Leu Thr Asn Pro Asn Asp Ile Glu 100 105 110 Leu Ile Leu Ser Gly His Gln His Leu Thr Lys Ala Glu Glu Tyr Arg 115 120 125 Tyr Phe Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile Ser Asn Gly His 130 135 140 His Trp Arg His His Arg Lys Met Ile Ala Pro Thr Phe His Gln Ser 145 150 155 160 Ile Leu Lys Ser Phe Val Pro Thr Phe Val Asp His Ser Lys Ser Val 165 170 175 Val Gly Arg Met Gly Leu Glu Thr Gly Lys Ser Phe Asp Val His Asp 180 185 190 Tyr Met Ser Thr Thr Thr Val Asp Ile Leu Leu Ser Thr Ala Met Gly 195 200 205 Val Lys Lys Leu Pro Glu Gly Asn Lys Ser Phe Glu Tyr Ala Gln Ala 210 215 220 Val Val Asp Met Cys Asp Ile Ile His Lys Arg Gln Val Lys Leu Leu 225 230 235 240 Tyr Arg Leu Asp Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu Lys Gly 245 250 255 Asp Arg Met Met Asn Ile Ile Leu Gly Met Thr Ser Lys Val Val Lys 260 265 270 Asp Arg Lys Gln Asn Phe Gln Glu Glu Ser Arg Ala Ile Val Asp Glu 275 280 285 Val Gln Ala Val Ser Thr Pro Ala Thr Lys Lys Glu Gly Leu Arg Asp 290 295 300 Asp Leu Asp Asp Ile Asp Glu Asn Asp Val Gly Ala Lys Arg Arg Leu 305 310 315 320 Ala Leu Leu Asp Ala Met Val Glu Met Ala Lys Asn Pro Asp Ile Glu 325 330 335 Trp Asn Glu Lys Asp Ile Ile Asp Glu Val Asn Thr Ile Met Phe Glu 340 345 350 Gly His Asp Thr Thr Ser Ala Gly Ser Ser Phe Ala Leu Cys Met Met 355 360 365 Gly Ile His Lys Asp Ile Gln Glu Lys Val Phe Ala Glu Gln Lys Ala 370 375 380 Ile Phe Gly Asp Asn Met Leu Arg Asp Cys Thr Phe Ala Asp Thr Asn 385 390 395 400 Glu Met Lys Tyr Leu Glu Arg Val Ile Leu Glu Thr Leu Arg Leu Tyr 405 410 415 Pro Pro Val Pro Leu Ile Ala Arg Arg Leu Asp Tyr Asp Leu Lys Leu 420 425 430 Ala Ser Gly Pro Tyr Thr Val Pro Lys Gly Thr Thr Val Ile Val Leu 435 440 445 Gln Tyr Cys Val His Arg Arg Ala Asp Ile Tyr Pro Asn Pro Thr Lys 450 455 460 Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg Met Ala Asn Arg His Tyr 465 470 475 480 Tyr Ser Phe Ile Pro Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg 485 490 495 Lys Tyr Ala Met Leu Lys Leu Lys Val Leu Leu Ser Thr Ile Val Arg 500 505 510 Asn Tyr Ile Val His Ser Thr Asp Thr Glu Ala Asp Phe Lys Leu Gln 515 520 525 Ala Asp Ile Ile Leu Lys Leu Glu Asn Gly Phe Asn Ile Ser Leu Glu 530 535 540 Lys Arg Lys Tyr Ala Thr Val Ala 545 550 7552PRTDrosophila mojavensis 7Met Ser Val Glu Thr Val Gln Glu Thr Leu Gln Gln Ala Ala Lys Ser 1 5 10 15 Ser Gly Gly Phe Ser Pro Ile Leu Thr Gly Leu Leu Gly Thr Ile Ile 20 25 30 Val Met Ala Leu Tyr Glu Tyr Trp His Arg Asn Thr Arg Glu Tyr Arg 35 40 45 Met Val Ala Asn Ile Pro Ser Pro Pro Ser Leu Pro Leu Ile Gly Met 50 55 60 Ala His Leu Ala Ala Gly Leu Ser Asn Ala Glu Ile Leu Ser Val Gly 65 70 75 80 Leu Gly Tyr Leu Asn Lys Tyr Gly Glu Thr Met Lys Gly Trp Leu Gly 85 90 95 Asn Val Leu Leu Val Phe Leu Thr Asn Pro Asn Asp Ile Glu Leu Ile 100 105 110 Leu Ser Gly His Gln His Leu Thr Lys Ala Glu Glu Tyr Arg Tyr Phe 115 120 125 Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile Ser Asn Gly His His Trp 130 135 140 Arg His His Arg Lys Met Ile Ala Pro Thr Phe His Gln Ser Ile Leu 145 150 155 160 Lys Ser Phe Val Pro Thr Phe Val Ala His Ser Lys Ala Val Ser Ala 165 170 175 Arg Met Ala Lys Glu Ala Gly Lys Ser Phe Asp Val His Asp Tyr Met 180 185 190 Ser Gln Thr Thr Val Asp Ile Leu Leu Thr Thr Ala Met Gly Val Lys 195 200 205 Lys Leu Pro Glu Gly Asn Lys Ser Phe Glu Tyr Ala Gln Ala Val Val 210 215 220 Asp Met Cys Asp Ile Ile His Thr Arg Gln Val Lys Leu Leu Tyr Arg 225 230 235 240 Leu Asp Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu Lys Gly Asp Arg 245 250 255 Met Met Asn Ile Ile Leu Gly Met Thr Arg Lys Val Val Lys Asp Arg 260 265 270 Asn Glu Asn Tyr Ser Pro Glu Ser Arg Ala Ile Ile Glu Asp Val Ala 275 280 285 Glu Pro Thr Pro Ala Lys Gln Ala Thr Lys Thr Glu Gly Leu Arg Asp 290 295 300 Asp Leu Asp Asp Ile Asp Glu Asn Asp Val Gly Ala Lys Arg Arg Leu 305 310 315 320 Ala Leu Leu Asp Ala Met Val Glu Met Ala Lys Asn Pro Asp Ile Glu 325 330 335 Trp Asn Glu Lys Asp Ile Met Asp Glu Val Asn Thr Ile Met Phe Glu 340 345 350 Gly His Asp Thr Thr Ser Ala Gly Ser Ser Phe Ala Leu Cys Met Met 355 360 365 Gly Ile His Lys Asp Val Gln Glu Arg Val Phe Ala Glu Gln Lys Ala 370 375 380 Ile Phe Gly Asp Asn Met Leu Arg Asp Cys Thr Phe Ala Asp Thr Met 385 390 395 400 Glu Met Lys Tyr Leu Glu Arg Val Ile Leu Glu Thr Leu Arg Met Tyr 405 410 415 Pro Pro Val Pro Leu Ile Ala Arg Arg Leu Asp His Asp Val Lys Leu 420 425 430 Ala Ser Gly Pro Tyr Thr Val Pro Lys Gly Thr Thr Cys Val Val Leu 435 440 445 Gln Tyr Cys Val His Arg Arg Pro Asp Ile Tyr Glu Asn Pro Thr Lys 450 455 460 Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg Ala Ala Lys Arg His Tyr 465 470 475 480 Tyr Ser Phe Ile Pro Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg 485 490 495 Lys Tyr Ala Met Leu Lys Leu Lys Val Leu Leu Ser Thr Ile Val Arg 500 505 510 Asn Phe Ile Ile His Ser Thr Asp Thr Glu Ala Asp Phe Lys Leu Gln 515 520 525 Ala Asp Ile Ile Leu Lys Leu Glu Asn Gly Phe Asn Ile Ser Leu Glu 530 535 540 Pro Arg Gln Tyr Pro Thr Ala Ala 545 550 8558PRTDrosophila virilis 8Met Ser Val Glu Thr Val Gln Glu Thr Leu Gln Gln Ala Thr Gly Ser 1 5 10 15 Thr Gly Ser Phe Met Leu Ser Pro Leu Leu Thr Gly Leu Val Gly Thr 20 25 30 Met Leu Ile Met Ala Leu Tyr Glu Tyr Trp His Arg Asn Thr Arg Glu 35 40 45 Tyr Arg Met Val Ala Asn Ile Pro Ser Pro Pro Ser Leu Pro Ile Ile 50 55 60 Gly Met Ala His Leu Ala Ala Gly Leu Ser Asn Ala Glu Ile Leu Ala 65 70 75 80 Val Gly Leu Gly Tyr Leu Asn Lys Tyr Gly Glu Thr Met Lys Ala Trp 85 90 95 Leu Gly Asn Val Leu Ile Val Phe Leu Thr Asn Pro Ser Asp Ile Glu 100 105 110 Leu Ile Leu Ser Gly His Gln His Leu Thr Lys Ala Glu Glu Tyr Arg 115 120 125 Tyr Phe Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile Ser Asn Gly His 130 135 140 His Trp Arg His His Arg Lys Met Ile Ala Pro Thr Phe His Gln Ser 145 150 155 160 Ile Leu Lys Ser Phe Val Pro Thr Phe Val Asp His Ser Lys Ala Val 165 170 175 Ser Ala Arg Met Ala Lys Glu Ala Gly Lys Ser Phe Asp Val His Asp 180 185 190 Tyr Met Ser Gln Thr Thr Val Asp Ile Leu Leu Ser Thr Ala Met Gly 195 200 205 Val Lys Lys Leu Pro Glu Gly Asn Lys Ser Phe Glu Tyr Ala Gln Ala 210 215 220 Val Val Asp Met Cys Asp Ile Ile His Lys Arg Gln Val Lys Leu Leu 225 230 235 240 Tyr Arg Leu Asp Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu Lys Gly 245 250 255 Asp Arg Met Met Asn Ile Ile Leu Gly Met Thr Arg Lys Val Val Lys 260 265 270 Asp Arg Lys Asp Asn Phe Gln Asn Glu Thr His Ala Ile Ile Glu Glu 275 280 285 Val Glu Glu Thr Pro Val Lys Gln Ser Arg Val Thr Ser Ala Thr Lys 290 295 300 Lys Glu Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp Glu Asn Asp Val 305 310 315 320 Gly Ala Lys Arg Arg Leu Ala Leu Leu Asp Ala Met Val Glu Met Ala 325 330 335 Lys Asn Pro Asp Ile Glu Trp Asn Glu Lys Asp Ile Met Asp Glu Val 340 345 350 Asn Thr Ile Met Phe Glu Gly His Asp Thr Thr Ser Ala Gly Ser Ser 355 360 365 Phe Ala Leu Cys Met Leu Gly Ile His Lys His Ile Gln Glu Arg Val 370 375 380 Phe Ala Glu Gln Lys Ser Ile Phe Gly Asp Asn Met Gln Arg Asp Cys 385 390 395 400 Thr Phe Ala Asp Thr Met Glu Met Lys Tyr Leu Glu Arg Val Ile Leu 405 410 415 Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Leu Ile Ala Arg Arg Leu 420 425 430 Asp His Asp Val Lys Leu Val Ser Gly Pro Tyr Thr Val Pro Lys Gly 435 440 445 Thr Thr Val Val Leu Leu Gln Tyr Cys Val His Arg Arg Pro Asp Ile 450 455 460 Tyr Pro Asn Pro Thr Glu Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg 465 470 475 480 Ala Ala Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser Ala Gly Pro 485 490 495 Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Val Leu 500 505 510 Leu Ser Thr Ile Val Arg Asn Phe Ile Val His Ser Thr Asp Thr Glu 515 520 525 Ala Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Leu Glu Asn Gly 530 535 540 Phe Asn Ile Ser Leu Glu Pro Arg Lys Tyr Gln Thr Val Ala 545 550 555 9540PRTDrosophila ananassae 9Met Thr Val Glu Ala Ala Ala Thr Ser Thr Ser Leu Leu Gly Tyr Ser 1 5 10 15 Pro Thr Leu Thr Thr Leu Val Ala Thr Met Val Ala Leu Gly Leu Tyr 20 25 30 Glu Tyr Trp Arg Arg Asn Thr Arg Glu Tyr Arg Met Val Ala Asn Ile 35 40 45 Pro Ser Pro Pro Glu Leu Pro Leu Leu Gly Gln Ala His Leu Ala Ala 50 55 60 Gly Leu Ser Asn Ala Glu Ile Met Asn Val Gly Leu Gly Tyr Leu Ser 65 70 75 80 Lys Tyr Gly Glu Thr Leu Lys Ala Trp Leu Gly Ser Val Leu Leu Val 85 90 95 Phe Ile Thr Asn Pro Asn Asp Ile Glu Leu Ile Leu Ser Gly His Gln 100 105 110 His Leu Thr Lys Ala Glu Glu Tyr Arg Tyr Phe Lys Pro Trp Phe Gly 115 120 125 Asp Gly Leu Leu Ile Ser Asn Gly His His Trp Arg His His Arg Lys 130 135 140 Met Ile Ala Pro Thr Phe His Gln Ser Ile Leu Lys Ser Phe Val Pro 145 150 155 160 Thr Phe Val Asn His Ser Lys Ala Val Val Asp Arg Met Gly Leu Glu 165 170 175 Ala Gly Lys Ser Phe Asp Val His Asp Tyr Met Ser Gln Thr Thr Val 180 185 190 Asp Ile Leu Leu Ser Thr Ala Met Gly Val Lys Lys Leu Pro Glu Gly 195 200 205 Asn Lys Ser Phe Glu Tyr Ala

Gln Ala Val Val Asp Met Cys Asp Ile 210 215 220 Ile His Lys Arg Gln Ile Lys Leu Leu Tyr Arg Leu Asp Ser Ile Tyr 225 230 235 240 Lys Phe Thr Lys Leu Arg Glu Lys Gly Asp Arg Met Met Asn Ile Ile 245 250 255 Leu Gly Met Thr Ser Lys Val Val Lys Asp Arg Lys Glu Asn Phe Gln 260 265 270 Glu Glu Ser Arg Ala Ile Val Glu Glu Ile Thr Thr Pro Ala Thr Pro 275 280 285 Ala Ala Lys Lys Glu Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp Glu 290 295 300 Asn Asp Val Gly Ala Lys Arg Arg Leu Ala Leu Leu Asp Ala Met Val 305 310 315 320 Glu Met Ala Lys Asn Pro Asp Ile Glu Trp Asn Glu Lys Asp Ile Ile 325 330 335 Asp Glu Val Asn Thr Ile Met Phe Glu Gly His Asp Thr Thr Ser Ala 340 345 350 Gly Ser Ser Phe Ala Leu Cys Met Met Gly Ile His Lys Asp Ile Gln 355 360 365 Glu Lys Val Phe Ala Glu Gln Lys Ala Ile Phe Gly Asp Asn Met Leu 370 375 380 Arg Asp Cys Thr Phe Ala Asp Thr Met Glu Met Lys Tyr Leu Glu Arg 385 390 395 400 Val Ile Leu Glu Thr Leu Arg Leu Tyr Pro Pro Val Pro Leu Ile Ala 405 410 415 Arg Arg Val Asp Tyr Asp Leu Lys Leu Ala Ser Gly Pro Tyr Thr Val 420 425 430 Pro Lys Gly Thr Thr Val Ile Val Leu Gln Tyr Cys Val His Arg Arg 435 440 445 Pro Asp Ile Tyr Pro Asn Pro Thr Lys Phe Asp Pro Asp Asn Phe Leu 450 455 460 Pro Glu Arg Met Ala Asn Arg His Tyr Tyr Ala Phe Ile Pro Phe Ser 465 470 475 480 Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu 485 490 495 Lys Val Leu Leu Ser Thr Ile Val Arg Asn Tyr Ile Val His Ser Thr 500 505 510 Asp Thr Glu Ala Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Leu 515 520 525 Glu Asn Gly Phe Asn Ile Ser Leu Glu Lys His Met 530 535 540 10562PRTDrosophila grimshawi 10Met Ser Val Glu Thr Val Gln Glu Thr Leu Gln Gln Ala Ala Ser Gly 1 5 10 15 Gly Gly Gly Tyr Ile Leu Ser Pro Leu Leu Thr Gly Val Leu Gly Thr 20 25 30 Ile Leu Ile Met Ala Leu Tyr Glu Tyr Trp His Arg Asn Ser Arg Glu 35 40 45 Tyr Arg Met Val Glu Asn Ile Pro Ser Pro Pro Thr Leu Pro Leu Val 50 55 60 Gly Met Ala His Leu Val Val Gly Leu Ser Asn Ala Glu Ile Leu Ser 65 70 75 80 Val Gly Leu Gly Tyr Leu Asn Lys Tyr Gly Glu Thr Met Lys Ala Trp 85 90 95 Leu Gly Asn Val Leu Val Val Phe Leu Thr Asn Pro Ser Asp Ile Glu 100 105 110 Leu Ile Leu Ser Gly His Gln His Leu Thr Lys Ala Glu Glu Tyr Arg 115 120 125 Tyr Phe Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile Ser Asn Gly His 130 135 140 His Trp Arg His His Arg Lys Met Ile Ala Pro Thr Phe His Gln Ser 145 150 155 160 Ile Leu Lys Ser Phe Val Pro Thr Phe Val Asp His Ser Lys Ala Val 165 170 175 Ser Ala Arg Met Gly Lys Glu Ser Gly Lys Pro Phe Asp Val His Asp 180 185 190 Tyr Met Ser Gln Thr Thr Val Asp Ile Leu Leu Ser Thr Ala Met Gly 195 200 205 Val Lys Lys Leu Pro Glu Gly Asn Thr Ser Phe Glu Tyr Ala Gln Ala 210 215 220 Val Val Asp Met Cys Asp Ile Ile His Lys Arg Gln Val Lys Leu Leu 225 230 235 240 Tyr Arg Leu Asp Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu Lys Gly 245 250 255 Asp Arg Met Met Asn Ile Ile Leu Gly Met Thr Arg Lys Val Val Lys 260 265 270 Asp Arg Lys Asp Asn Phe Ile Thr Glu Ser Arg Pro Ile Ile Asp Glu 275 280 285 Val Glu Glu Thr Pro Asn Pro Lys Leu Ser Arg Ala Thr Pro Pro Ala 290 295 300 Ala Ala Ala Lys Lys Glu Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp 305 310 315 320 Glu Asn Asp Val Gly Ala Lys Arg Arg Leu Ala Leu Leu Asp Ala Met 325 330 335 Val Glu Met Ala Lys Asn Pro Glu Ile Glu Trp Asn Glu Lys Asp Ile 340 345 350 Met Asp Glu Val Asn Thr Ile Met Phe Glu Gly His Asp Thr Thr Ser 355 360 365 Ala Gly Ser Ser Phe Ala Leu Cys Met Leu Gly Ile His Lys Asp Ile 370 375 380 Gln Glu Arg Val Phe Ala Glu Gln Lys Ala Ile Phe Gly Asp Lys Met 385 390 395 400 Gln Arg Asp Cys Thr Phe Ala Asp Thr Met Glu Met Lys Tyr Leu Glu 405 410 415 Arg Val Ile Leu Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Leu Ile 420 425 430 Ala Arg Arg Leu Asp His Asp Val Lys Leu Thr Ser Gly Pro Tyr Thr 435 440 445 Val Pro Lys Gly Thr Thr Val Val Val Leu Gln Tyr Cys Val His Arg 450 455 460 Arg Ala Asp Ile Tyr Pro Asn Pro Thr Lys Phe Asp Pro Asp Asn Phe 465 470 475 480 Leu Pro Glu Arg Ala Ala Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe 485 490 495 Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys 500 505 510 Leu Lys Val Leu Leu Ser Thr Ile Val Arg Asn Tyr Ile Val His Ser 515 520 525 Thr Asp Thr Glu Ala Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys 530 535 540 Leu Glu Asn Gly Phe Asn Ile Ser Leu Glu Pro Arg Lys Tyr Gln Thr 545 550 555 560 Val Ala 11505PRTDrosophila willistoni 11Met Val Ser Asn Ile Pro Ser Pro Pro Gly Leu Pro Leu Leu Gly Gln 1 5 10 15 Ala His Leu Ala Ala Gly Leu Ser Asn Ala Glu Ile Met Ser Val Gly 20 25 30 Met Gly Tyr Leu Asn Lys Tyr Gly Glu Thr Val Lys Ala Trp Leu Gly 35 40 45 His Val Leu Leu Val Phe Leu Thr Asn Pro Asn Asp Ile Glu Leu Ile 50 55 60 Leu Ser Gly His Gln His Leu Thr Lys Ala Glu Glu Tyr Arg Tyr Phe 65 70 75 80 Lys Pro Trp Phe Gly Asp Gly Leu Leu Ile Ser Asn Gly His His Trp 85 90 95 Arg His His Arg Lys Met Ile Ala Pro Thr Phe His Gln Ser Ile Leu 100 105 110 Lys Ser Phe Val Pro Thr Phe Val Asp His Ser Lys Asn Val Val Ala 115 120 125 Arg Met Asp Thr Glu Ala Gly Lys Ser Phe Asp Val His Asp Tyr Met 130 135 140 Ser Gln Thr Thr Val Asp Ile Leu Leu Ser Thr Ala Met Gly Val Lys 145 150 155 160 Lys Leu Pro Glu Gly Asn Lys Ser Phe Glu Tyr Ala Gln Ala Val Val 165 170 175 Asp Met Cys Asp Ile Ile His Lys Arg Gln Ile Lys Leu Leu Tyr Arg 180 185 190 Leu Asp Ser Ile Tyr Lys Phe Thr Lys Leu Arg Glu Lys Gly Asp Lys 195 200 205 Met Met Asn Ile Ile Leu Gly Met Thr Ser Lys Val Val Lys Asp Arg 210 215 220 Lys Glu Asn Phe Gln Ala Asp Thr Arg Ala Ile Ile Glu Glu Glu Leu 225 230 235 240 Thr Lys Pro Ala Ala Thr Ser Pro Ser Ala Lys Lys Glu Gly Leu Arg 245 250 255 Asp Asp Leu Asp Asp Ile Asp Glu Asn Asp Val Gly Ala Lys Arg Arg 260 265 270 Leu Ala Leu Leu Asp Ala Met Val Glu Met Ala Lys Asn Pro Asp Ile 275 280 285 Glu Trp Asn Glu Lys Asp Ile Ile Asp Glu Val Asn Thr Ile Met Phe 290 295 300 Glu Gly His Asp Thr Thr Ser Ala Gly Ser Ser Phe Ala Leu Cys Met 305 310 315 320 Met Gly Ile His Lys His Ile Gln Glu Arg Val Phe Ala Glu Gln Lys 325 330 335 Ala Ile Phe Gly Asp Asn Met Gln Arg Asp Cys Thr Phe Ala Asp Ala 340 345 350 Met Glu Met Lys Tyr Leu Glu Arg Val Ile Leu Glu Thr Leu Arg Leu 355 360 365 Tyr Pro Pro Val Pro Leu Ile Ala Arg Arg Leu Asp His Asp Leu Lys 370 375 380 Leu Ala Ser Gly Pro Tyr Thr Val Pro Lys Gly Thr Thr Val Ile Val 385 390 395 400 Leu Gln Tyr Cys Val His Arg Arg Pro Asp Ile Tyr Pro Asn Pro Thr 405 410 415 Thr Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg Met Ala Asn Arg His 420 425 430 Tyr Tyr Ala Phe Ile Pro Phe Ser Ala Gly Pro Arg Ser Cys Val Gly 435 440 445 Arg Lys Tyr Ala Met Leu Lys Leu Lys Val Leu Leu Ser Thr Ile Val 450 455 460 Arg Asn Tyr Ile Ile His Ser Thr Asp Thr Glu Ala Asp Phe Lys Leu 465 470 475 480 Gln Ala Asp Ile Ile Leu Lys Leu Glu Asn Gly Phe Asn Ile Ser Leu 485 490 495 Glu Lys Arg Lys Tyr Pro Thr Val Ala 500 505 12557PRTCulex quinquefasciatus 12Met Asn Val Glu Phe Val His Glu Arg Ser Ser Leu Ala Ala Leu Ala 1 5 10 15 Met Pro Thr Val Ile Val Met Thr Leu Val Leu Val Val Ser Val Leu 20 25 30 Phe His Met Trp Met Leu Ser Arg Arg Tyr Val Lys Leu Gly Asn Met 35 40 45 Ile Pro Gly Pro Arg Ala Tyr Pro Leu Ile Gly Asn Ala Asn Met Leu 50 55 60 Leu Gly Lys Ser His Asp Glu Ile Met Lys Arg Ala Ile Glu Leu Ser 65 70 75 80 Phe Val Tyr Gly Ser Val Ala Arg Gly Trp Leu Gly Tyr His Leu Val 85 90 95 Val Phe Leu Thr Glu Pro Ala Asp Ile Glu Leu Ile Leu Asn Ser Tyr 100 105 110 Val His Leu Thr Lys Ser Asn Glu Tyr Arg Phe Phe Lys Pro Trp Leu 115 120 125 Gly Asp Gly Leu Leu Ile Ser Ser Gly Asp Lys Trp Lys Ser His Arg 130 135 140 Lys Leu Ile Ala Pro Ala Phe His Gln Asn Val Leu Lys Thr Phe Ile 145 150 155 160 Asp Val Phe Asn Asp Asn Ser Leu Ala Val Val Glu Arg Met Arg Lys 165 170 175 Glu Val Gly Lys Val Phe Asp Val His Asp Tyr Met Ser Glu Val Thr 180 185 190 Val Asp Ile Leu Leu Glu Thr Ala Met Gly Ser Asn Arg Thr Gly Glu 195 200 205 Asn Lys Glu Gly Phe Asp Tyr Ala Met Ala Val Met Lys Met Cys Asp 210 215 220 Ile Leu His Ser Arg Gln Ile Lys Ile His Leu Arg Met Asp Pro Ile 225 230 235 240 Phe Asn Met Thr Lys Thr Lys Lys Glu Gln Glu Arg Leu Leu Gly Ile 245 250 255 Ile His Gly Leu Thr Arg Lys Val Val Lys Gln Lys Lys Glu Leu Phe 260 265 270 Glu Lys Asn Leu Ala Glu Gly Lys Leu Pro Ser Pro Ser Leu Ser Glu 275 280 285 Ile Ile Gly Lys Glu Glu Glu Ser Ser Gly Thr Thr Lys Val Glu Glu 290 295 300 Pro Ala Val Ile Ser Gln Gly Ser Met Leu Arg Asp Asp Leu Asp Ala 305 310 315 320 Ile Asp Glu Asn Asp Ile Gly Glu Lys Arg Arg Leu Ala Phe Leu Asp 325 330 335 Leu Met Ile Glu Thr Ala Lys Thr Gly Ala Asp Leu Ser Asp Glu Glu 340 345 350 Ile Lys Glu Glu Val Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr 355 360 365 Ala Ala Gly Ser Ser Phe Val Leu Cys Leu Leu Gly Ile His Gln Asp 370 375 380 Ile Gln Asp Arg Val Tyr Lys Glu Ile Lys Gln Ile Phe Gly Asp Ser 385 390 395 400 Lys Arg Lys Ala Thr Phe Asn Asp Thr Met Glu Met Lys Tyr Leu Glu 405 410 415 Arg Val Ile Phe Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Ala Ile 420 425 430 Ala Arg Lys Leu Thr Gln Glu Val Arg Leu Ala Ser His Asp Tyr Val 435 440 445 Val Pro Ser Gly Thr Thr Val Val Ile Gly Thr Tyr Lys Leu His Arg 450 455 460 Arg Glu Asp Ile Tyr Pro Asn Pro Asp Val Phe Asn Pro Asp Asn Phe 465 470 475 480 Leu Pro Glu Arg Thr Ser Asn Arg His Tyr Tyr Ser Tyr Ile Pro Phe 485 490 495 Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys 500 505 510 Leu Lys Val Leu Leu Thr Thr Ile Leu Arg Asn Tyr Arg Val Val Ser 515 520 525 Asn Leu Lys Glu Ser Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys 530 535 540 Arg Thr Asp Gly Phe Arg Ile Gln Leu Glu Pro Arg Val 545 550 555 13560PRTAedes aegypti 13Val Ser Gly Val Ile Tyr Lys Met Ser Ala Glu Ile Val Ala Glu Arg 1 5 10 15 Gly Ser Ser Leu Val Ser Leu Ala Val Pro Met Val Ile Phe Met Thr 20 25 30 Leu Val Leu Val Ala Ser Ala Leu Phe His Phe Trp Met Ile Ser Arg 35 40 45 Arg Tyr Val Gln Leu Gly Asn Lys Ile Pro Gly Pro Arg Ala Tyr Pro 50 55 60 Phe Ile Gly Asn Ala Asn Met Leu Leu Gly Met Asn His Asn Glu Ile 65 70 75 80 Met Glu Arg Ala Met Gln Leu Ser Tyr Ile Tyr Gly Ser Val Ala Arg 85 90 95 Gly Trp Leu Gly Tyr His Leu Val Val Phe Leu Thr Glu Pro Ala Asp 100 105 110 Ile Glu Ile Ile Leu Asn Ser Tyr Val His Leu Thr Lys Ser Ser Glu 115 120 125 Tyr Arg Phe Phe Lys Pro Trp Leu Gly Asp Gly Leu Leu Ile Ser Ser 130 135 140 Gly Glu Lys Trp Arg Ser His Arg Lys Leu Ile Ala Pro Ala Phe His 145 150 155 160 Met Asn Val Leu Lys Thr Phe Val Asp Val Phe Asn Asp Asn Ser Leu 165 170 175 Ala Val Val Glu Arg Met Arg Lys Glu Val Gly Lys Glu Phe Asp Val 180 185 190 His Asp Tyr Met Ser Glu Val Thr Val Asp Ile Leu Leu Glu Thr Ala 195 200 205 Met Gly Ser Gln Arg Thr Ser Glu Ser Lys Glu Gly Phe Asp Tyr Ala 210 215 220 Met Ala Val Met Lys Met Cys Asp Ile Leu His Ser Arg Gln Leu Lys 225 230 235 240 Phe His Leu Arg Met Asp Ser Val Phe Asn Phe Thr Lys Ile Lys Gln 245 250 255 Glu Gln Glu Arg Leu Leu Gly Ile Ile His Gly Leu Thr Arg Lys Val 260 265 270 Val Lys Gln Lys Lys Glu Leu Phe Glu Lys Asn Phe Ala Asp Gly Lys 275 280 285 Leu Pro Ser Pro Ser Leu Ser Glu Ile Ile Ala Lys Glu Glu Ser Glu 290 295 300 Ser Lys Glu Ser Leu Pro Val Ile Ser Gln Gly Ser Leu Leu Arg Asp 305 310 315 320 Asp Leu Asp Phe Asn Asp Glu Asn Asp Ile Gly Glu Lys Arg Arg Leu 325 330 335 Ala Phe Leu Asp Leu Met Ile Glu Thr Ala

Lys Ser Gly Ala Asp Leu 340 345 350 Thr Asp Glu Glu Ile Lys Glu Glu Val Asp Thr Ile Met Phe Glu Gly 355 360 365 His Asp Thr Thr Ala Ala Gly Ser Ser Phe Val Leu Cys Leu Leu Gly 370 375 380 Ile His Gln Asp Val Gln Asp Arg Val Tyr Lys Glu Ile Tyr Gln Ile 385 390 395 400 Phe Gly Asn Ser Lys Arg Lys Ala Thr Phe Asn Asp Thr Leu Glu Met 405 410 415 Lys Tyr Leu Glu Arg Val Ile Phe Glu Thr Leu Arg Met Tyr Pro Pro 420 425 430 Val Pro Val Ile Ala Arg Lys Val Thr Gln Asp Val Arg Leu Ala Ser 435 440 445 His Asp Tyr Val Val Pro Ala Gly Thr Thr Val Val Ile Gly Thr Tyr 450 455 460 Lys Val His Arg Arg Ala Asp Ile Tyr Pro Asn Pro Asp Val Phe Asn 465 470 475 480 Pro Asp Asn Phe Leu Pro Glu Arg Thr Gln Asn Arg His Tyr Tyr Ser 485 490 495 Tyr Ile Pro Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr 500 505 510 Ala Met Leu Lys Leu Lys Val Leu Leu Ser Thr Ile Leu Arg Asn Tyr 515 520 525 Arg Val Val Ser Asn Leu Lys Glu Ser Asp Phe Lys Leu Gln Gly Asp 530 535 540 Ile Ile Leu Lys Arg Thr Asp Gly Phe Arg Ile Gln Leu Glu Pro Arg 545 550 555 560 14566PRTAedes aegypti 14Met Ser Ala Thr Val Ala Pro Ala Asp Pro Val Met Ala Asn Ala Asn 1 5 10 15 Ile Ala Ser Pro Met Asn Val Phe Tyr Phe Leu Leu Ala Pro Ala Leu 20 25 30 Leu Leu Trp Phe Ile Tyr Trp Arg Ile Ser Arg Gln His Met Leu Lys 35 40 45 Leu Ala Glu Lys Ile Pro Gly Pro Pro Gly Leu Pro Leu Leu Gly Asn 50 55 60 Ala Leu Glu Leu Ile Gly Thr Ser His Ser Val Phe Arg Asn Val Ile 65 70 75 80 Glu Lys Gly Lys Asp Phe Asn Gln Val Ile Lys Ile Trp Ile Gly Pro 85 90 95 Lys Leu Ile Val Phe Leu Val Asp Pro Arg Asp Val Glu Leu Leu Leu 100 105 110 Ser Ser His Val Tyr Ile Asp Lys Ser Pro Glu Tyr Arg Phe Phe Lys 115 120 125 Pro Trp Leu Gly Asn Gly Leu Leu Ile Ser Thr Gly His Lys Trp Arg 130 135 140 Gln His Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys 145 150 155 160 Ser Phe Ile Asp Leu Phe Asn Glu Asn Ser Arg Leu Val Val Glu Lys 165 170 175 Met His Lys Glu Ala Gly Lys Thr Phe Asp Cys His Asp Tyr Met Ser 180 185 190 Glu Cys Thr Val Glu Ile Leu Leu Glu Thr Ala Met Gly Val Ser Lys 195 200 205 Lys Thr Gln Asp Gln Ser Gly Phe Asp Tyr Ala Met Ala Val Met Lys 210 215 220 Met Cys Asp Ile Leu His Leu Arg His Arg Lys Met Trp Leu Tyr Pro 225 230 235 240 Asp Leu Phe Phe Asn Met Ser Gln Tyr Ala Lys Arg Gln Val Lys Leu 245 250 255 Leu Asp Thr Ile His Ser Leu Thr Arg Lys Val Ile Arg Asn Lys Lys 260 265 270 Ala Ala Phe Ala Thr Gly Thr Arg Gly Ser Leu Ala Thr Thr Ser Ile 275 280 285 Lys Thr Ala Glu Phe Glu Lys Pro Lys Ser Asn Ile Asn Thr Asn Ser 290 295 300 Val Glu Gly Leu Ser Phe Gly Gln Ser Ala Asn Leu Lys Asp Asp Leu 305 310 315 320 Asp Val Asp Glu Asn Asp Val Gly Glu Lys Lys Arg Leu Ala Phe Leu 325 330 335 Asp Leu Leu Leu Glu Ser Ala Glu Asn Gly Ala Leu Ile Ser Asp Glu 340 345 350 Glu Ile Lys Asn Gln Val Asp Thr Ile Met Phe Glu Gly His Asp Thr 355 360 365 Thr Ala Ala Gly Ser Ser Phe Phe Leu Ser Met Met Gly Ile His Gln 370 375 380 His Ile Gln Asp Lys Val Ile Gln Glu Leu Asp Asp Ile Phe Gly Asp 385 390 395 400 Ser Asp Arg Pro Ala Thr Phe Gln Asp Thr Leu Glu Met Lys Tyr Leu 405 410 415 Glu Arg Cys Leu Met Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Ile 420 425 430 Ile Ala Arg Ser Leu Lys Gln Asp Leu Lys Leu Ala Ser Ser Asp Leu 435 440 445 Val Val Pro Ser Gly Ala Thr Ile Val Val Ala Thr Tyr Lys Leu His 450 455 460 Arg Leu Glu Thr Ile Tyr Pro Asn Pro Asn Val Phe Asp Pro Asp Asn 465 470 475 480 Phe Leu Pro Glu Arg Gln Ala Asn Arg His Tyr Tyr Ala Phe Val Pro 485 490 495 Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu 500 505 510 Lys Leu Lys Val Ile Leu Ser Thr Ile Leu Arg Asn Phe Arg Val Ile 515 520 525 Ser Asp Leu Lys Glu Glu Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu 530 535 540 Lys Arg Glu Glu Gly Phe Gln Ile Arg Leu Glu Pro Arg Gln Arg Lys 545 550 555 560 Pro Lys Ala Ala Lys Ala 565 15552PRTNasonia vitripennis 15Met Asp Ala Val Pro Met Ser Thr Ser Tyr Leu Pro Ala Thr Leu Phe 1 5 10 15 Trp Pro Leu Val Leu Ile Ala Ala Ala Leu Ala Ala Val His Tyr Tyr 20 25 30 Ile Glu Thr Ser Arg Ile Val Arg Leu Gly Asn Lys Leu Pro Gly Pro 35 40 45 Lys Thr Val Pro Phe Phe Gly Asn Ala Leu Met Ala Leu Gly Val Gln 50 55 60 Pro Lys Asp Val Leu Thr Glu Val Met Lys Tyr Asp Ile Tyr Gly Asn 65 70 75 80 Val Ala Arg Ala Phe Leu Gly Pro Lys Leu Val Val Phe Leu Val Asp 85 90 95 Pro Arg Asp Val Glu Ile Ile Leu Gly Ser His Val His Ile Asp Lys 100 105 110 Ser Pro Glu Tyr Arg Tyr Phe Ala Pro Trp Leu Gly Glu Gly Leu Leu 115 120 125 Ile Ser Thr Gly Glu Lys Trp Arg Ser His Arg Lys Ile Ile Ala Pro 130 135 140 Thr Phe His Leu Asn Val Leu Lys Ser Phe Val Pro Leu Phe Tyr Glu 145 150 155 160 Asn Ser Ile Asp Leu Val Lys Arg Leu Lys Ser Glu Val Gly Lys Glu 165 170 175 Phe Asp Cys His Asp Tyr Met Ser Gly Ile Thr Val Asp Ile Leu Leu 180 185 190 Glu Thr Ala Met Gly Val Arg Gly Thr Gln Lys Glu Lys Ser Ser Tyr 195 200 205 Asp Tyr Ala Met Ala Val Met Lys Met Cys Asn Ile Ile His Gln Arg 210 215 220 Gln Tyr Asn Phe Met Leu Arg Leu Asp Thr Phe Phe Gln Phe Thr Ser 225 230 235 240 Phe Ala Lys Gln Gln Thr Lys Phe Leu Asp Ile Ile His Gly Leu Thr 245 250 255 Lys Arg Val Ile Lys Lys Arg Asn Val Glu Phe Lys Asp Lys Met Asp 260 265 270 Ser Pro Met Met Asn Ser Ile Met Lys Glu Leu Lys Lys Asp Ser Thr 275 280 285 Glu Ile Val Asp Glu Lys Gln Pro Glu Glu Gln Lys Met Arg Tyr Val 290 295 300 Arg Asp Asp Leu Asp Glu Ile Asp Glu Asn Asp Val Gly Glu Lys Arg 305 310 315 320 Arg Leu Ala Phe Leu Asp Leu Met Leu Glu Met Arg Lys Asn Gly Glu 325 330 335 Gln Leu Thr Asp Glu Glu Ile Lys Glu Glu Val Asp Thr Ile Met Phe 340 345 350 Glu Gly His Asp Thr Thr Ala Ala Gly Ser Ser Phe Val Leu Cys Val 355 360 365 Leu Gly Ile His Gln Asp Val Gln Asp Arg Val Ile Glu Glu Leu Asn 370 375 380 Glu Ile Phe Lys Gly Ser Asp Arg Pro Cys Thr Phe Gln Asp Thr Leu 385 390 395 400 Glu Met Lys Tyr Leu Glu Arg Val Ile Leu Glu Thr Leu Arg Leu Phe 405 410 415 Pro Pro Val Pro Ala Ile Ala Arg Gln Leu Asn Gln Asp Val Lys Leu 420 425 430 Ala Ser Gly Asp Tyr Ile Leu Pro Ser Gly Cys Thr Val Val Ile Pro 435 440 445 Gln Phe Lys Ile His Arg Leu Lys Glu Tyr Tyr Pro Asn Pro Asp Val 450 455 460 Phe Asp Pro Asp Asn Phe Leu Pro Asp Lys Thr Gln Asp Arg His Tyr 465 470 475 480 Tyr Ala Tyr Ile Pro Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg 485 490 495 Lys Tyr Ala Met Leu Lys Leu Lys Val Leu Leu Ser Thr Ile Leu Arg 500 505 510 Asn Tyr Lys Ile Asn Ser Asp Leu Thr Glu Glu Asp Phe Lys Leu Gln 515 520 525 Val Asp Ile Ile Leu Lys Arg Ser Asp Gly Phe Arg Ile Gln Ile Glu 530 535 540 Pro Arg Asn Gln Ala Val Ile Val 545 550 16561PRTNasonia vitripennis 16Met Ser Ala Ala Gly Pro Glu Val Val Ala Gly Ser Val Ala Ala Ala 1 5 10 15 Ala Ala Ser Gly Phe Ser Ala Thr Ser Val Phe Phe Thr Leu Leu Val 20 25 30 Pro Ala Ile Leu Leu Tyr Tyr Val Tyr Phe Arg Ile Ser Arg Arg His 35 40 45 Met Ile Glu Leu Ser Asp Lys Ile Pro Gly Pro Lys Gly Leu Pro Leu 50 55 60 Leu Gly Asn Ala Leu Glu Leu Ile Gly Ser Ser Asp Thr Ile Phe Arg 65 70 75 80 Asn Val Tyr Lys Arg Ser Phe Glu Phe Asp Gln Val Ile Lys Leu Trp 85 90 95 Val Gly Pro Lys Leu Val Ile Phe Leu Ile Asp Pro Arg Asp Val Glu 100 105 110 Val Ile Leu Ser Ser His Val Tyr Ile Asp Lys Ser Pro Glu Tyr Arg 115 120 125 Phe Phe Gln Pro Trp Leu Gly Asn Gly Leu Leu Ile Ser Thr Gly Gln 130 135 140 Lys Trp Arg Ala His Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn 145 150 155 160 Val Leu Lys Ser Phe Ile Asp Leu Phe Asn Ala Asn Ser Arg Ala Val 165 170 175 Val Gln Lys Met Arg Lys Glu Asp Glu Arg Glu Phe Asp Ile His Asp 180 185 190 Tyr Met Ser Glu Thr Thr Val Glu Ile Leu Leu Glu Thr Ala Met Gly 195 200 205 Val Ser Lys Ser Thr Gln Asp Lys Ser Gly Phe Glu Tyr Ala Met Ala 210 215 220 Val Met Lys Met Cys Asp Ile Leu His Leu Arg His Thr Arg Val Trp 225 230 235 240 Leu Arg Pro Asp Trp Leu Phe Asn Leu Thr Lys Tyr Gly Lys Glu Gln 245 250 255 Val His Leu Leu Asp Ile Ile His Gly Leu Thr Lys Lys Val Ile Ala 260 265 270 Arg Lys Lys Glu Asp Tyr Lys Ser Gly Lys Arg Asn Phe Val Asp Thr 275 280 285 Ser Ala Ala Ala Lys Asp Asn Lys Ser Thr Thr Val Val Glu Gly Leu 290 295 300 Ser Phe Gly Gln Ser Ala Gly Leu Lys Asp Asp Leu Asp Val Asp Asp 305 310 315 320 Asn Asp Val Gly Glu Lys Lys Arg Gln Ala Phe Leu Asp Leu Leu Val 325 330 335 Glu Ala Ser Gln Asn Gly Val Val Leu Thr Asp Glu Glu Val Lys Glu 340 345 350 Gln Val Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala Gly 355 360 365 Ser Ser Phe Phe Leu Ser Met Met Gly Cys His Pro Asp Ile Gln Glu 370 375 380 Lys Val Ile Gln Glu Leu Asp Glu Ile Phe Gly Asp Ser Asp Arg Pro 385 390 395 400 Ala Thr Phe Gln Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg Cys Leu 405 410 415 Met Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Ile Ile Ala Arg Glu 420 425 430 Val Lys Thr Asp Leu Lys Leu Ala Ser Gly Asp Tyr Thr Ile Pro Ala 435 440 445 Gly Cys Thr Val Val Val Ala Thr Phe Lys Leu His Arg Gln Pro His 450 455 460 Ile Tyr Pro Asn Pro Asp Val Phe Asn Pro Asp Asn Phe Leu Pro Glu 465 470 475 480 Lys Thr Ala Asn Arg His Tyr Tyr Ala Phe Val Pro Phe Ser Ala Gly 485 490 495 Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Ile 500 505 510 Leu Leu Ser Thr Ile Leu Arg Asn Phe Arg Val Arg Ser Thr Val Lys 515 520 525 Glu Glu Asp Phe Arg Leu Gln Ala Asp Ile Ile Leu Lys Arg Ala Glu 530 535 540 Gly Phe Lys Val Lys Leu Glu Pro Arg Lys Arg Ala Ala Gly Leu Lys 545 550 555 560 Ala 17559PRTChironomus tentans 17Met Ala Val Glu Gln Ile Ile Gln Ser Ser Val Phe Ser Ser Pro Leu 1 5 10 15 Leu Met Pro Leu Leu Ala Ile Val Phe Val Leu Ala Ala Val His Phe 20 25 30 Trp Gln Met Ser Arg Arg Glu Arg Lys Ile Gly Asp Leu Leu Pro Gly 35 40 45 Pro Pro Thr Val Pro Ile Ile Gly Asn Ala Tyr Leu Phe Met Asn Gly 50 55 60 Thr Asn His Glu Met Phe Lys Lys Ala Val Asp Leu Val Asn Cys Tyr 65 70 75 80 Gly Ser Val Val Arg Gly Trp Val Gly His Lys Leu Leu Val Gly Leu 85 90 95 Ser Asp Pro Arg Asp Val Glu Ile Ile Leu Gly Ser Gln Val His Ile 100 105 110 Asp Lys Ser Asp Glu Tyr Arg Phe Phe Arg Pro Trp Leu Gly Asn Gly 115 120 125 Leu Leu Ile Ser Ser Gly Asp Lys Trp Arg Thr His Arg Lys Leu Ile 130 135 140 Ala Pro Ala Phe His Met Asn Val Leu Lys Ser Phe Met Ala Thr Phe 145 150 155 160 Asn Asp Asn Ser Arg Phe Val Ile Lys Lys Leu Met Lys Glu Ala Gly 165 170 175 Lys Glu Phe Asp Cys His Asp Tyr Met Ser Glu Ala Thr Val Asp Ile 180 185 190 Leu Leu Glu Thr Ala Met Gly Ser Lys Arg Thr Ser Glu Ser Glu Glu 195 200 205 Gly Phe Lys Tyr Ala Met Ala Val Met Lys Met Cys Asp Ile Leu His 210 215 220 Arg Arg Gln Phe Lys Ile Phe Ser Arg Phe Glu Pro Phe Phe Thr Leu 225 230 235 240 Thr Gly Met Lys Glu Gln Gln Lys Lys Leu Leu Gly Ile Ile His Gly 245 250 255 Met Thr Gln Arg Val Leu Asn Glu Lys Lys Ala Ile Phe Asp Lys Asn 260 265 270 Leu Ser Glu Gly Asn Leu Pro Ser Pro Ser Leu Gln Glu Ile Ile Lys 275 280 285 Thr Asp Ala Ser Val Asp Gln Ala Ile Lys Lys Ala Lys Ala Lys Ala 290 295 300 Gln Asn Ile Asp Ala Gly Leu His Asp Asp Leu Asp Asp Ile Asp Glu 305 310 315 320 Asn Asp Val Gly Glu Lys Arg Arg Leu Ala Phe Leu Asp Leu Met Ile 325 330 335 Glu Thr Ser His Tyr Asn Pro Gln Gln Leu Ser Gln Glu Glu Ile Lys 340 345 350 Gln Gln Val Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala 355 360 365 Gly Ser Ser Phe Thr Leu Cys Met Leu Gly Cys His Pro Asp Ile Gln 370 375 380 Glu Lys Val Tyr Gln Glu Gln Lys Ala Ile Phe Gly Asp Ser Asp Arg 385 390

395 400 Asp Cys Thr Phe Ala Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg Val 405 410 415 Ile Phe Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Leu Ile Ala Arg 420 425 430 Lys Ile Asn Lys Asp Ile Arg Leu Ala Ser Cys Asp Gln Val Val Pro 435 440 445 Ala Gly Thr Thr Ile Ile Ile Ala Thr Val Lys Ile His Arg Arg Pro 450 455 460 Asp Ile Tyr Pro Asn Pro Asp Lys Phe Asp Pro Asp Asn Phe Leu Pro 465 470 475 480 Glu Arg Thr Ser Asn Arg His Tyr Tyr Gly Phe Ile Pro Phe Ser Ala 485 490 495 Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys 500 505 510 Val Leu Leu Ser Thr Ile Val Arg Asn Phe Tyr Val Lys Ser Thr Val 515 520 525 Pro Glu Lys Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Arg Thr 530 535 540 Asp Gly Phe Arg Ile Lys Leu Glu Pro Arg Lys Thr Lys Ala Asn 545 550 555 18566PRTAnopheles gambiae 18Val Ser Glu Ile Met Gly Ile Glu Thr Ile Pro Glu Arg Met Ala Met 1 5 10 15 Asp Glu Thr Val Pro Gly Ser Trp Val Leu Ser Val Thr Val Ala Thr 20 25 30 Val Leu Leu Leu Val Ala Gly Thr Leu Phe His Leu Trp Met Gln Thr 35 40 45 Arg Arg Tyr Val Gln Leu Gly Asn Leu Ile Pro Gly Pro Val Ala Tyr 50 55 60 Pro Leu Ile Gly Asn Ala Asn Met Leu Leu Gly Lys Thr His Asn Gln 65 70 75 80 Ile Met Glu Lys Ala Met Glu Leu Ser Tyr Ile Tyr Gly Thr Val Ala 85 90 95 Arg Gly Trp Leu Gly Tyr His Leu Val Val Phe Leu Thr Glu Pro Ala 100 105 110 Asp Val Glu Ile Ile Leu Asn Ser Tyr Val His Leu Glu Lys Ser Ser 115 120 125 Glu Tyr Arg Phe Phe Lys Pro Trp Leu Gly Asp Gly Leu Leu Ile Ser 130 135 140 Ser Gly Asp Lys Trp Lys Ser His Arg Lys Leu Ile Ala Pro Ala Phe 145 150 155 160 His Gln Asn Val Leu Lys Thr Phe Ile Asp Val Phe Asn Asp Asn Ser 165 170 175 Leu Ala Val Val Lys Arg Met Ser Lys Glu Val Gly His Val Phe Asp 180 185 190 Cys His Asp Tyr Met Ser Glu Val Thr Val Asp Ile Leu Leu Glu Thr 195 200 205 Ala Met Gly Ser Thr Arg Thr Gly Glu Asn Lys Glu Gly Phe Glu Tyr 210 215 220 Ala Met Ala Val Met Lys Met Cys Asp Ile Leu His Lys Arg Gln Leu 225 230 235 240 Lys Ile His Leu Arg Leu Asp Pro Leu Phe Asn Leu Thr Gly Val Lys 245 250 255 Lys Glu Gln Glu Arg Leu Leu Gln Ile Ile His Gly Leu Thr Arg Lys 260 265 270 Val Val Arg Glu Lys Lys Gln Leu Tyr Glu Arg Gln Met Ala Glu Gly 275 280 285 Lys Met Pro Ser Pro Ser Leu Thr Glu Ile Ile Gly Lys Glu Glu Lys 290 295 300 Pro Gly Glu Gly Gln Leu Gly Gly Ser Pro Ala Phe Ile Ser Gln Gly 305 310 315 320 Ser Met Leu Arg Asp Asp Leu Asp Asp Asn Asp Glu Asn Asp Ile Gly 325 330 335 Glu Lys Arg Arg Leu Ala Phe Leu Asp Leu Met Ile Glu Thr Ala Asn 340 345 350 Asn Gly Ala Asn Ile Ser Asp Glu Glu Ile Lys Glu Glu Val Asp Thr 355 360 365 Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala Gly Ser Ser Phe Val 370 375 380 Leu Cys Leu Leu Gly Ile His Gln His Val Gln Glu Gln Val Tyr Ala 385 390 395 400 Glu Leu Arg Gln Ile Phe Gly Asp Ser Lys Arg Lys Ala Thr Phe Gly 405 410 415 Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg Val Ile Phe Glu Thr Leu 420 425 430 Arg Met Phe Pro Pro Val Pro Met Ile Ala Arg Lys Ile Asn Glu Asp 435 440 445 Val Gln Leu Ala Ser Lys Asn Tyr Thr Ile Pro Ala Gly Thr Thr Val 450 455 460 Val Ile Gly Thr Tyr Lys Ile His Arg Arg Glu Asp Leu Tyr Pro His 465 470 475 480 Pro Glu Thr Phe Asn Pro Asp Asn Phe Leu Pro Glu Arg Thr Gln Asn 485 490 495 Arg His Tyr Tyr Ser Tyr Ile Pro Phe Ser Ala Gly Pro Arg Ser Cys 500 505 510 Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Val Leu Leu Ser Thr 515 520 525 Val Leu Arg His Tyr Arg Val Val Ser Asn Leu Thr Glu Lys Asp Phe 530 535 540 Lys Leu Gln Ala Asp Ile Ile Leu Lys Arg Thr Asp Gly Phe Gln Ile 545 550 555 560 Gln Leu Glu Pro Arg Ala 565 19565PRTAnopheles gambiae 19Met Ser Ala Thr Ile Ala His Thr Asp Gly Leu Asn Ser Ser Ala Asn 1 5 10 15 Ile Ile Ser Pro Ile Asn Met Phe Tyr Phe Leu Leu Thr Pro Ala Leu 20 25 30 Leu Leu Trp Phe Phe Tyr Trp Arg Leu Ser Arg Arg His Met Leu Glu 35 40 45 Leu Ala Glu Arg Ile Pro Gly Pro Lys Gly Leu Pro Leu Ile Gly Asn 50 55 60 Ala Leu Asp Leu Val Gly Ser Ser His Ser Val Phe Arg Thr Ile Ile 65 70 75 80 Glu Lys Gly Lys Glu Tyr Asn Glu Val Ile Lys Ile Trp Ile Gly Pro 85 90 95 Lys Leu Ile Val Phe Leu Val Asp Pro Arg Asp Ile Glu Leu Leu Leu 100 105 110 Ser Ser His Val Tyr Ile Asp Lys Ser Pro Glu Tyr Arg Phe Phe Lys 115 120 125 Pro Trp Leu Gly Asn Gly Leu Leu Ile Ser Thr Gly His Lys Trp Arg 130 135 140 Gln His Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys 145 150 155 160 Ser Phe Ile Asp Leu Phe Asn Glu Asn Ser Arg Leu Val Val Lys Lys 165 170 175 Met Gln Lys Glu Asn Gly Lys Val Phe Asp Cys His Asp Tyr Met Ser 180 185 190 Glu Cys Thr Val Glu Ile Leu Leu Glu Thr Ala Met Gly Val Ser Lys 195 200 205 Lys Thr Gln Asp Gln Ser Gly Tyr Asp Tyr Ala Met Ala Val Met Lys 210 215 220 Met Cys Asp Ile Leu His Leu Arg His Arg Lys Met Trp Leu Tyr Pro 225 230 235 240 Asp Leu Phe Phe Asn Leu Thr Gln Tyr Ala Lys Lys Gln Val Lys Leu 245 250 255 Leu Asn Thr Ile His Ser Leu Thr Lys Lys Val Ile Arg Asn Lys Lys 260 265 270 Ala Ala Phe Asp Thr Gly Thr Arg Gly Ser Leu Ala Thr Thr Ser Ile 275 280 285 Asn Thr Val Asn Ile Glu Lys Ser Lys Ser Asp Ser Thr Lys Thr Asn 290 295 300 Thr Val Glu Gly Leu Ser Phe Gly Gln Ser Ser Asn Leu Lys Asp Asp 305 310 315 320 Leu Asp Val Glu Glu Asn Asp Val Gly Glu Lys Lys Arg Leu Ala Phe 325 330 335 Leu Asp Leu Leu Leu Glu Ser Ala Glu Asn Gly Ala Leu Ile Ser Asp 340 345 350 Glu Glu Ile Lys Asn Gln Val Asp Thr Ile Met Phe Glu Gly His Asp 355 360 365 Thr Thr Ala Ala Gly Ser Ser Phe Phe Leu Ser Met Met Gly Val His 370 375 380 Gln Gln Ile Gln Asp Lys Val Ile Gln Glu Leu Asp Glu Ile Phe Gly 385 390 395 400 Glu Ser Asp Arg Pro Ala Thr Phe Gln Asp Thr Leu Glu Met Lys Tyr 405 410 415 Leu Glu Arg Cys Leu Met Glu Thr Leu Arg Met Tyr Pro Pro Val Pro 420 425 430 Ile Ile Ala Arg Ser Leu Lys Gln Asp Leu Lys Leu Ala Ser Ser Asp 435 440 445 Ile Val Val Pro Ala Gly Ala Thr Ile Thr Val Ala Thr Phe Lys Leu 450 455 460 His Arg Leu Glu Ser Ile Tyr Pro Asn Pro Asp Val Phe Asn Pro Asp 465 470 475 480 Asn Phe Leu Pro Glu Lys Gln Ala Asn Arg His Tyr Tyr Ala Phe Val 485 490 495 Pro Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met 500 505 510 Leu Lys Leu Lys Ile Ile Leu Ser Thr Ile Leu Arg Asn Phe Arg Val 515 520 525 Tyr Ser Asp Leu Lys Glu Glu Glu Phe Lys Leu Gln Ala Asp Ile Ile 530 535 540 Leu Lys Arg Glu Glu Gly Phe Gln Ile Arg Leu Glu Pro Arg Gln Arg 545 550 555 560 Lys Ser Lys Thr Leu 565 20553PRTTribolium castaneum 20Met Val Val Val Glu Glu Ser Leu Asn His Ser Leu Asn Leu Gly Asn 1 5 10 15 Ser Val Leu Ile Ser Leu Gly Val Val Ala Val Ile Leu Ala Val Tyr 20 25 30 His Phe Trp Leu Gln Ser Leu Arg Tyr Thr Lys Leu Gly Asn Lys Ile 35 40 45 Pro Gly Tyr Asp Pro Leu Pro Ile Ile Gly Asn Ala His Met Val Met 50 55 60 Asn Lys Asn Pro Thr Gln Val Met Glu Leu Ala Leu Arg Val Ala Ser 65 70 75 80 Glu Lys Gly Ser Val Val Arg Phe Trp Phe Gly Ser Lys Leu Gly Val 85 90 95 Ala Leu Leu Asp Pro Arg Asp Ile Glu Leu Ile Leu Gly Ser Asn Val 100 105 110 His Leu Glu Lys Ser Ser Glu Tyr Arg Phe Phe Glu Pro Trp Leu Gly 115 120 125 Asp Gly Leu Leu Ile Ser Lys Gly Asp Lys Trp Arg Ser His Arg Lys 130 135 140 Met Ile Ala Pro Thr Phe His Gln Ser Ile Leu Lys Thr Phe Val Pro 145 150 155 160 Val Phe Asn Lys Asn Ala Met Asp Leu Val Glu Gln Leu Arg Asn Glu 165 170 175 Ala Leu Asp Gln Ile Cys Asp Val His Asp Tyr Leu Ser Gly Ala Thr 180 185 190 Val Asp Val Leu Leu Glu Thr Val Met Gly Val Lys Lys Thr Lys Glu 195 200 205 Ala Arg Thr Ser Tyr Lys Tyr Ala Lys Ala Val Met Asp Met Cys Thr 210 215 220 Ile Leu His Phe Arg His Val Lys Leu Trp Leu Arg Ser Asp Trp Ile 225 230 235 240 Phe Ser Phe Thr Lys Leu Phe Lys Glu Gln Thr Ser Leu Leu Arg Ile 245 250 255 Ile His Asn Leu Thr Asp Arg Val Ile Lys Gln Lys Lys Lys Ala Tyr 260 265 270 Phe Glu Arg Val Lys Asp Gly Asp Val Ser Leu Tyr Asn Asn Ala Val 275 280 285 Lys Glu Thr Glu Glu Glu Asn Leu Lys Ile Lys Asn Glu Gln Thr Phe 290 295 300 Asn Phe Gly Ser Gly Leu Arg Asp Asp Leu Asp Glu Asn Asp Glu Asn 305 310 315 320 Leu Gly Glu Lys Lys Arg Leu Ala Phe Leu Asp Phe Met Val Glu Ala 325 330 335 Ser Gln Thr Glu Gly Asn Lys Leu Asn Asp Glu Glu Ile Arg Glu Glu 340 345 350 Val Asn Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala Ala Ser 355 360 365 Ser Phe Phe Ile Cys Ile Leu Gly Val Tyr Pro Glu Ile Gln Glu Lys 370 375 380 Val Tyr Gln Glu Leu Arg Asp Ile Phe Gln Asp Ser Asp Arg Pro Ile 385 390 395 400 Thr Phe Asn Asp Thr Leu Gln Met Lys Tyr Leu Glu Arg Val Leu Leu 405 410 415 Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Ile Ile Thr Arg Val Ile 420 425 430 Asn Glu Glu Val Lys Leu Ala Ser Gly Asp Tyr Thr Leu Pro Val Gly 435 440 445 Thr Thr Val Gly Ile Gly Gln Phe Leu Val His Arg Asn Pro Lys Tyr 450 455 460 Phe Pro Asn Pro Asp Lys Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg 465 470 475 480 Cys Gln Gln Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser Ala Gly Pro 485 490 495 Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Ile Leu 500 505 510 Leu Ala Ser Ile Val Arg Asn Phe Lys Ile Lys Ser Val Val Lys Glu 515 520 525 Lys Asp Phe Gln Leu Gln Ala Asp Ile Ile Leu Lys Arg Ala Asp Gly 530 535 540 Phe Arg Val Ile Leu Thr Ser Arg Thr 545 550 21560PRTTribolium castaneum 21Met Ser Thr Thr Val Pro Thr Pro Asp Ile Ser Thr Pro Ser Gly Ile 1 5 10 15 Leu Ser Ala Ser Asn Val Phe Tyr Phe Leu Leu Ile Pro Ala Leu Leu 20 25 30 Leu Trp Tyr Ala Tyr Trp Arg Ile Ser Lys Arg His Met Leu Glu Leu 35 40 45 Ala Ala Lys Ile Pro Gly Pro Pro Gly Leu Pro Ile Leu Gly Asn Ala 50 55 60 Leu Asp Leu Val Gly Lys Pro His Gln Val Phe Ser His Val Tyr Gln 65 70 75 80 Lys Ser Phe Glu Tyr Lys Lys Val Val Lys Met Trp Ala Gly Pro Lys 85 90 95 Leu Leu Val Phe Leu Thr Asp Pro Ser Asp Ile Glu Leu Ile Leu Ser 100 105 110 Ser Tyr Val His Ile Asp Lys Ser Ser Glu Tyr Arg Phe Phe Lys Pro 115 120 125 Trp Leu Gly Asp Gly Leu Leu Ile Ser Thr Gly Gln Lys Trp Lys Ala 130 135 140 His Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys Ser 145 150 155 160 Phe Ile Asp Leu Phe Asn Ala Asn Ser Arg Asp Val Ile Arg Lys Leu 165 170 175 Gln Lys Glu Ile Gly Lys Glu Phe Asp Cys His Asp Tyr Met Ser Glu 180 185 190 Ala Thr Val Glu Met Leu Leu Glu Thr Ala Met Gly Val Ser Lys Lys 195 200 205 Thr Gln Asp Gln Ser Gly Tyr Asp Tyr Ala Met Ala Val Met Lys Met 210 215 220 Cys Asp Ile Leu His Leu Arg His Thr Lys Phe Trp Leu Arg Pro Asp 225 230 235 240 Ile Ile Phe Asn Gln Thr Lys Tyr Ala Glu Tyr Gln Lys Ser Leu Ile 245 250 255 Asn Thr Ile His Ser Leu Thr Arg Lys Val Ile Lys Arg Lys Arg Ala 260 265 270 Asp Phe Asp Lys Gly Ile Arg Gly Ser Thr Ala Glu Val Pro Pro Glu 275 280 285 Leu Gln Thr Lys Asn Tyr Asp Lys Thr Glu Ser Lys Thr Val Val Glu 290 295 300 Gly Leu Ser Tyr Gly Gln Ser Ala Gly Leu Lys Asp Asp Leu Asp Val 305 310 315 320 Asp Asp Asn Asp Ile Gly Glu Lys Lys Arg Met Ala Phe Leu Asp Leu 325 330 335 Met Ile Glu Ala Ser Gln Asn Gly Val Val Ile Asn Asp Glu Glu Ile 340 345 350 Lys Glu Gln Val Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala 355 360 365 Ala Gly Ser Ser Phe Phe Leu Ser Met Met Gly Val His Gln Asp Ile 370 375 380 Gln Asp Lys Val Val Gln Glu Leu Tyr Asp Ile Phe Gly Asp Ser Asp 385 390 395 400 Arg Pro Ala Thr Phe Ala Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg 405 410 415 Cys Leu Met Glu Thr Leu Arg Met Phe Pro Pro Val Pro Ile Ile Ala 420 425 430 Arg Gln Leu Asn Gln Asp Leu Lys Leu Ala Ser Gly Asp Tyr Thr Val 435 440

445 Pro Ala Gly Cys Thr Val Val Ile Gly Thr Phe Lys Val His Arg Leu 450 455 460 Glu Glu Tyr Tyr Pro Asn Pro Asp Lys Phe Asp Pro Asp Asn Phe Leu 465 470 475 480 Pro Glu Arg Thr Ala Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser 485 490 495 Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu 500 505 510 Lys Ile Leu Leu Ser Thr Ile Leu Arg Asn Tyr Arg Ile Tyr Ser Asp 515 520 525 Leu Lys Glu Lys Asp Phe Gln Leu Gln Gly Asp Ile Ile Leu Lys Arg 530 535 540 Ala Glu Gly Phe Lys Val Arg Leu Glu Pro Arg Lys Met Ala Lys Ala 545 550 555 560 22562PRTBombyx mori 22Met Thr Ser Leu Val Asp Glu Thr Glu Gly Tyr His Val Asn Ser Arg 1 5 10 15 Val Ile Phe Tyr Pro Leu Leu Gly Leu Thr Thr Ala Ile Trp Ile Leu 20 25 30 Tyr Arg Trp Gln Gln Asn Ser His Met His Lys Leu Ala Glu Leu Ile 35 40 45 Pro Gly Pro Ala Pro Ile Pro Ile Phe Gly Asn Ala Leu Thr Leu Met 50 55 60 Arg Lys Asp Pro His Glu Leu Val Asn Leu Ala Leu Gly Tyr Ala Gln 65 70 75 80 Thr Phe Gly Asn Val Val Arg Val Trp Leu Gly Ser Lys Leu Ile Val 85 90 95 Phe Leu Ala Asp Ala Asp Asp Ile Glu Ile Ile Leu Asn Ser His Val 100 105 110 His Ile Asp Lys Ala Thr Glu Tyr Lys Phe Phe Lys Pro Trp Leu Gly 115 120 125 Glu Gly Leu Leu Ile Ser Ser Gly Pro Lys Trp Arg Ser His Arg Lys 130 135 140 Met Ile Ala Pro Thr Phe His Ile Asn Ile Leu Lys Ser Phe Val Gly 145 150 155 160 Ile Phe Asn Gln Asn Ser Asn Asn Val Val Glu Lys Leu Lys Ser Glu 165 170 175 Val Gly Lys Thr Phe Asp Val His Asp Tyr Met Ser Gly Thr Thr Val 180 185 190 Asp Ile Leu Leu Glu Thr Ala Met Gly Ile Ser Arg Lys Thr Gln Asp 195 200 205 Glu Ser Gly Phe Asp Tyr Ala Met Ala Val Met Lys Met Cys Asp Ile 210 215 220 Ile His Gln Arg His Tyr Lys Phe Trp Met Arg Ser Glu Ile Val Phe 225 230 235 240 Lys Leu Thr Ser Phe Phe Lys Gln Gln Thr Lys Leu Trp Gly Ile Ile 245 250 255 His Gly Leu Thr Asn Lys Val Ile Lys Asn Lys Lys Glu Thr Tyr Leu 260 265 270 Glu Asn Lys Ala Lys Gly Ile Ile Pro Pro Thr Leu Glu Glu Trp Thr 275 280 285 His His Ser Gly Glu Ile Leu Ala Asn Asn Ala Lys Thr Leu Ser Asp 290 295 300 Thr Val Phe Lys Gly Tyr Arg Asp Asp Leu Asp Phe Asn Asp Glu Asn 305 310 315 320 Asp Val Gly Glu Lys Lys Arg Arg Ala Phe Trp Asp Leu Met Ile Glu 325 330 335 Ser Ser Gln Asn Gly Thr Asn Lys Ile Ser Asp His Glu Ile Lys Glu 340 345 350 Glu Val Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala Gly 355 360 365 Ser Ser Phe Val Leu Cys Leu Leu Gly Ile His Gln Asp Val Gln Ala 370 375 380 Arg Val Tyr Asp Glu Leu Tyr Gln Ile Leu Gly Asp Ser Asp Arg Pro 385 390 395 400 Ala Thr Phe Ala Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg Val Ile 405 410 415 Leu Glu Ser Leu Arg Leu Tyr Pro Pro Val Pro Val Ile Ala Arg Lys 420 425 430 Leu Asn Arg Asp Val Thr Ile Ser Thr Lys Asn Tyr Val Ile Pro Ala 435 440 445 Gly Thr Thr Val Val Ile Gly Thr Phe Met Leu His Arg Gln Pro Lys 450 455 460 Tyr His Lys Asp Pro Glu Val Phe Asn Pro Asp Asn Phe Leu Pro Glu 465 470 475 480 Asn Thr Gln Asn Arg His Tyr Tyr Ser Tyr Ile Pro Phe Ser Ala Gly 485 490 495 Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Leu Leu Lys Leu Lys Ile 500 505 510 Leu Leu Ser Thr Ile Leu Arg Asn Phe Arg Thr Ile Ser Glu Ile Pro 515 520 525 Glu Lys Glu Phe Lys Leu Gln Gly Asp Ile Ile Leu Lys Arg Ala Glu 530 535 540 Gly Phe Gln Met Lys Val Glu Pro Arg Lys Arg Val Pro Thr Asn Val 545 550 555 560 Ala Arg 23556PRTBombyx mori 23Met Ser Tyr Thr Asn Ala Glu Asn Val Val Pro Thr Ser Thr Phe Ser 1 5 10 15 Ala Ile Asn Leu Phe Tyr Val Leu Leu Val Pro Ala Val Ile Leu Trp 20 25 30 Tyr Ala Tyr Trp Arg Met Ser Arg Arg Arg Leu Tyr Glu Leu Ala Asp 35 40 45 Lys Leu Asn Gly Pro Pro Gly Leu Pro Leu Leu Gly Asn Ala Leu Glu 50 55 60 Phe Val Gly Gly Ser Ala Asp Ile Phe Arg Asn Ile Val Gln Lys Ser 65 70 75 80 Ala Asp Tyr Asp His Glu Ser Val Val Lys Ile Trp Ile Gly Pro Arg 85 90 95 Leu Leu Val Phe Leu Tyr Asp Pro Arg Asp Val Glu Val Ile Leu Ser 100 105 110 Ser His Val Tyr Ile Asp Lys Ala Glu Glu Tyr Arg Phe Phe Lys Pro 115 120 125 Trp Leu Gly Asn Gly Leu Leu Ile Ser Thr Gly Gln Lys Trp Arg Ser 130 135 140 His Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys Ser 145 150 155 160 Phe Ile Asp Leu Phe Asn Ala Asn Ser Arg Ala Val Val Asp Lys Leu 165 170 175 Lys Lys Glu Ala Ser Asn Phe Asp Cys His Asp Tyr Met Ser Glu Cys 180 185 190 Thr Val Glu Ile Leu Leu Glu Thr Ala Met Gly Val Ser Lys Ser Thr 195 200 205 Gln Asp Gln Ser Gly Phe Glu Tyr Ala Met Ala Val Met Lys Met Cys 210 215 220 Asp Ile Leu His Leu Arg His Thr Lys Ile Trp Leu Arg Pro Asp Leu 225 230 235 240 Leu Phe Lys Phe Thr Asp Tyr Ala Lys Asn Gln Thr Lys Leu Leu Asp 245 250 255 Ile Ile His Gly Leu Thr Lys Lys Val Ile Lys Arg Lys Lys Glu Glu 260 265 270 Phe Ala Ser Gly Lys Lys Pro Ser Asn Leu Asn Glu Thr Ala Thr Thr 275 280 285 Ser Glu Pro Ser Thr Gly Lys Leu Thr Ser Val Glu Gly Leu Ser Phe 290 295 300 Gly Gln Ser Ser Gly Leu Lys Asp Asp Leu Asp Val Asp Asp Asp Val 305 310 315 320 Gly Gln Lys Lys Arg Leu Ala Phe Leu Asp Leu Leu Leu Glu Ser Ser 325 330 335 Gln Ser Gly Val Ala Ile Ser Asp Glu Glu Ile Lys Glu Gln Val Asp 340 345 350 Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala Gly Ser Ser Phe 355 360 365 Phe Leu Ser Met Met Gly Ile His Gln Asp Ile Gln Asp Lys Val Ile 370 375 380 Glu Glu Leu Asp Gln Ile Phe Gly Asp Ser Asp Arg Pro Val Thr Phe 385 390 395 400 Gln Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg Cys Leu Met Glu Thr 405 410 415 Leu Arg Leu Tyr Pro Pro Val Pro Ile Ile Ala Arg Gln Val Asn Gln 420 425 430 Glu Ile Thr Leu Pro Ser Asn Gly Lys Lys Ile Pro Ala Gly Thr Thr 435 440 445 Leu Val Ile Ala Thr Tyr Lys Leu His Arg Arg Pro Asp Val Tyr Pro 450 455 460 Asn Pro Asn Lys Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg Ser Ala 465 470 475 480 Asn Arg His Tyr Tyr Ala Phe Val Pro Phe Ser Ala Gly Pro Arg Ser 485 490 495 Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Val Ile Leu Ser 500 505 510 Thr Ile Leu Arg Asn Phe Arg Val Ile Ser Val Leu Lys Glu Ser Asp 515 520 525 Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Arg Ala Glu Gly Phe Gln 530 535 540 Val Arg Leu Gln Pro Arg Lys Arg Met Ala Lys Ala 545 550 555 24561PRTLeptinotarsa decemlineata 24Met Ser Ala Ala Thr Ala Ser Val Asp Leu Glu Asn Pro Thr Thr Leu 1 5 10 15 Leu Thr Pro Lys Asn Ile Phe Tyr Phe Leu Leu Ile Pro Ala Leu Val 20 25 30 Leu Trp Tyr Ala Tyr Trp Lys Ile Ser Arg Arg His Met Val Glu Leu 35 40 45 Ala Ser Lys Ile Pro Gly Pro Glu Gly Leu Pro Leu Leu Gly Ser Ala 50 55 60 Leu Glu Phe Val Gly Thr Ser Ala Asp Ile Phe Lys Arg Met Tyr Ala 65 70 75 80 Lys Ser Phe Glu Tyr Gly Asn Thr Val Lys Val Trp Ile Gly Pro Lys 85 90 95 Leu Leu Ile Phe Leu Val Asp Pro Arg Asp Val Glu Ile Ile Leu Ser 100 105 110 Ser His Val His Ile Asp Lys Ala Ser Glu Tyr Arg Phe Phe Gln Pro 115 120 125 Trp Leu Gly Asp Gly Leu Leu Ile Ser Thr Gly Gln Lys Trp Arg Ala 130 135 140 His Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys Ser 145 150 155 160 Phe Ile Asp Leu Phe Asn Ala Asn Ser Arg Glu Val Val Gln Lys Leu 165 170 175 Lys Lys Glu Val Gly Lys Glu Phe Asp Cys His Asp Tyr Met Ser Glu 180 185 190 Ala Thr Val Glu Ile Leu Leu Glu Thr Ala Met Gly Val Ser Lys Lys 195 200 205 Thr Gln Asp Gln Ser Gly Tyr Asp Tyr Ala Met Ala Val Met Lys Met 210 215 220 Cys Asp Ile Leu His Leu Arg His Thr Lys Val Trp Leu Arg Pro Asp 225 230 235 240 Phe Ile Phe Asn Leu Thr Asn Tyr Ala Lys Lys Gln Glu Gly Leu Ile 245 250 255 Gly Ile Ile His Ser Leu Thr Arg Lys Val Ile Lys Arg Lys Arg Ala 260 265 270 Asp Phe Glu Lys Gly Ile Arg Gly Ser Thr Ala Glu Val Pro Glu Glu 275 280 285 Leu Lys Thr Lys Asn Phe Asp Lys Asn Val Ser Ser Lys Thr Val Val 290 295 300 Glu Gly Leu Ser Tyr Gly Gln Ala Ala Gly Leu Lys Asp Asp Leu Asp 305 310 315 320 Val Asp Asp Asp Val Gly Glu Lys Lys Arg Met Ala Phe Leu Asp Leu 325 330 335 Met Ile Glu Ala Ser Gln Asn Gly Val Val Ile Asn Asp Glu Glu Ile 340 345 350 Lys Glu Gln Val Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala 355 360 365 Ala Gly Ser Ser Phe Phe Leu Ser Met Met Gly Val His Gln Asp Ile 370 375 380 Gln Asp Lys Val Val Gln Glu Ile Asp Glu Ile Phe Gly Asp Ser Asp 385 390 395 400 Arg Pro Ala Thr Phe Ala Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg 405 410 415 Cys Leu Met Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Ile Ile Ala 420 425 430 Arg Gln Leu Arg Gln Asp Val Lys Leu Ala Ser Gly Asp Tyr Thr Leu 435 440 445 Pro Ala Gly Ala Thr Ile Val Ile Gly Thr Phe Lys Ile His Arg Gln 450 455 460 Glu Asp Val Tyr Pro Asn Pro Asp Lys Phe Asp Pro Asp Asn Phe Leu 465 470 475 480 Pro Glu Arg Ser Ala Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser 485 490 495 Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu 500 505 510 Lys Ile Leu Leu Ser Thr Ile Leu Arg Asn Tyr Arg Ile Tyr Ser Thr 515 520 525 Val Glu Glu Lys Asp Phe Gln Leu Gln Gly Asp Ile Ile Leu Lys Arg 530 535 540 Ala Asp Gly Phe Arg Ile Lys Leu Glu Pro Arg Lys Arg Val Leu Lys 545 550 555 560 Ala 25577PRTDrosophila sechellia 25Met Glu Val Leu Lys Lys Asp Ala Ala Leu Gly Ser Pro Ser Asn Val 1 5 10 15 Phe Tyr Phe Leu Leu Leu Pro Thr Leu Val Leu Trp Tyr Ile Tyr Trp 20 25 30 Arg Leu Ser Arg Ala His Leu Tyr Arg Leu Ala Gly Arg Leu Pro Gly 35 40 45 Pro Arg Gly Leu Pro Ile Val Gly His Leu Phe Asp Val Ile Gly Pro 50 55 60 Ala Ser Ser Val Phe Arg Thr Val Ile Arg Lys Ser Ala Pro Phe Glu 65 70 75 80 His Ile Ala Lys Met Trp Ile Gly Pro Lys Leu Val Val Phe Ile Tyr 85 90 95 Asp Pro Arg Asp Val Glu Leu Leu Leu Ser Ser His Val Tyr Ile Asp 100 105 110 Lys Ala Ser Glu Tyr Lys Phe Phe Lys Pro Trp Leu Gly Asp Gly Leu 115 120 125 Leu Ile Ser Thr Gly Gln Lys Trp Arg Ser His Arg Lys Leu Ile Ala 130 135 140 Pro Thr Phe His Leu Asn Val Leu Lys Ser Phe Ile Glu Leu Phe Asn 145 150 155 160 Glu Asn Ser Arg Asn Val Val Arg Lys Leu Arg Ala Glu Asp Gly Arg 165 170 175 Thr Phe Asp Cys His Asp Tyr Met Ser Glu Ala Thr Val Glu Ile Leu 180 185 190 Leu Glu Thr Ala Met Gly Val Ser Lys Lys Thr Gln Asp Lys Ser Gly 195 200 205 Phe Glu Tyr Ala Met Ala Val Met Arg Met Cys Asp Ile Leu His Ala 210 215 220 Arg His Arg Ser Ile Phe Leu Arg Asn Glu Phe Val Phe Thr Leu Thr 225 230 235 240 Arg Tyr Tyr Lys Glu Gln Gly Arg Leu Leu Asn Ile Ile His Gly Leu 245 250 255 Thr Thr Lys Val Ile Arg Ser Lys Lys Ala Ala Phe Glu Gln Gly Thr 260 265 270 Arg Gly Ser Leu Ala Gln Cys Glu Leu Lys Ala Ala Ala Leu Glu Arg 275 280 285 Glu Arg Glu Gln Asp Gly Gly Val Gly Gly Gly Asp Gln Thr Ala Ser 290 295 300 Thr Ala Gly Ser Glu Glu Lys Asp Arg Glu Lys Asp Lys Glu Lys Ala 305 310 315 320 Ser Pro Val Ala Gly Leu Ser Tyr Gly Gln Ser Ala Gly Leu Lys Asp 325 330 335 Asp Leu Asp Val Glu Asp Asn Asp Ile Gly Glu Lys Lys Arg Leu Ala 340 345 350 Phe Leu Asp Leu Met Leu Glu Ser Ala Gln Asn Gly Ala Leu Ile Thr 355 360 365 Asp Thr Glu Ile Lys Glu Gln Val Asp Thr Ile Met Phe Glu Gly His 370 375 380 Asp Thr Thr Ala Ala Gly Ser Ser Phe Phe Leu Ser Leu Met Gly Ile 385 390 395 400 His Gln Asp Ile Gln Asp Arg Val Leu Ala Glu Leu Asp Ser Ile Phe 405 410 415 Gly Asp Ser Gln Arg Pro Ala Thr Phe Gln Asp Thr Leu Glu Met Lys 420 425 430 Tyr Leu Glu Arg Cys Leu Met Glu Thr Leu Arg Met Tyr Pro Pro Val 435 440 445 Pro Leu Ile Ala Arg Glu Leu Gln Glu Asp Leu Lys Leu Asn Ser Gly 450 455 460 Asn Tyr Val Ile Pro Arg Gly Ala Thr Val Thr Val Ala Thr Val Leu 465 470 475 480 Leu His Arg Asn Pro Lys Val Tyr Ala Asn Pro Asn Val Phe Asp Pro 485 490 495 Asp Asn Phe Leu Pro Glu Arg Gln Ala Asn

Arg His Tyr Tyr Ala Phe 500 505 510 Val Pro Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala 515 520 525 Met Leu Lys Leu Lys Ile Leu Leu Ser Thr Ile Leu Arg Asn Tyr Arg 530 535 540 Val Tyr Ser Asp Leu Thr Glu Ser Asp Phe Lys Leu Gln Ala Asp Ile 545 550 555 560 Ile Leu Lys Arg Glu Glu Gly Phe Arg Val Arg Leu Gln Pro Arg Thr 565 570 575 Arg 26548PRTApis mellifera 26Met Ala Ala Ala Ser Ala Thr Gly Phe Ser Ala Ser Ser Val Phe Leu 1 5 10 15 Ser Leu Leu Ile Pro Ala Leu Ile Leu Tyr Phe Ile Tyr Phe Arg Ile 20 25 30 Ser Arg Arg His Leu Leu Glu Leu Ala Glu Lys Ile Pro Gly Pro Pro 35 40 45 Ala Leu Pro Leu Ile Gly Asn Ala Leu Asp Leu Phe Gly Ser Pro Asp 50 55 60 Ala Met Phe Ser Gln Val Leu Lys Lys Ala Glu Asn Phe Lys Asp Val 65 70 75 80 Val Lys Ile Trp Val Gly Pro Lys Leu Val Ile Cys Leu Ile Asp Pro 85 90 95 Arg Asp Val Glu Ile Ile Leu Ser Ser Asn Val Tyr Ile Asp Lys Ser 100 105 110 Thr Glu Tyr Arg Phe Phe Lys Pro Trp Leu Gly Asp Gly Leu Leu Ile 115 120 125 Ser Thr Gly Gln Lys Trp Arg Asn His Arg Lys Leu Ile Ala Pro Thr 130 135 140 Phe His Leu Asn Val Leu Lys Ser Phe Ile Asp Leu Phe Asn Ala Asn 145 150 155 160 Ala Arg Ser Val Val Glu Lys Met Arg Lys Glu Asn Gly Lys Glu Phe 165 170 175 Asp Cys His Asn Tyr Met Ser Glu Leu Thr Val Asp Ile Leu Leu Glu 180 185 190 Thr Ala Met Gly Val Ser Lys Pro Thr Arg Asp His Asn Ala Phe Glu 195 200 205 Tyr Ala Met Ala Val Met Lys Met Cys Asp Ile Leu His Leu Arg His 210 215 220 Thr Lys Ile Trp Leu Arg Pro Asp Trp Leu Phe Asn Leu Thr Lys Tyr 225 230 235 240 Gly Lys Asn Gln Ile Lys Leu Leu Glu Ile Ile His Gly Leu Thr Lys 245 250 255 Lys Val Ile Gln Leu Lys Lys Glu Glu Tyr Lys Ser Gly Lys Arg Asn 260 265 270 Ile Ile Asp Asn Ser Ala Gln Lys Thr Glu Ser Lys Thr Asn Asn Ile 275 280 285 Val Val Glu Gly Val Ser Phe Gly Gln Ser Val Gly Leu Lys Asp Asp 290 295 300 Leu Asp Ile Asp Asp Asp Val Gly Glu Lys Lys Arg Gln Ala Phe Leu 305 310 315 320 Asp Leu Leu Ile Glu Ala Gly Gln Asn Gly Val Leu Leu Thr Asp Lys 325 330 335 Glu Val Lys Glu Gln Val Asp Thr Ile Met Phe Glu Gly His Asp Thr 340 345 350 Thr Ala Ser Gly Ser Ser Phe Phe Leu Ala Val Met Gly Cys His Pro 355 360 365 Asp Ile Gln Glu Lys Val Ile Gln Glu Leu Asp Glu Ile Phe Gly Asp 370 375 380 Ser Asp Arg Pro Ala Thr Phe Gln Asp Thr Leu Glu Met Lys Tyr Leu 385 390 395 400 Glu Arg Cys Leu Leu Glu Thr Leu Arg Met Tyr Pro Pro Val Pro Leu 405 410 415 Ile Ala Arg Glu Ile Lys Thr Asp Leu Lys Leu Ala Ser Gly Asp Tyr 420 425 430 Thr Ile Pro Ala Gly Cys Thr Val Val Ile Gly Thr Phe Lys Leu His 435 440 445 Arg Gln Pro His Ile Tyr Pro Asn Pro Asp Val Phe Asp Pro Asp Asn 450 455 460 Phe Leu Pro Glu Lys Thr Ala Asn Arg His Tyr Tyr Ala Phe Val Pro 465 470 475 480 Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu 485 490 495 Lys Leu Lys Ile Val Leu Ser Thr Ile Leu Arg Asn Phe Arg Val Arg 500 505 510 Ser Asp Val Lys Glu Ser Glu Phe Arg Leu Gln Ala Asp Ile Ile Leu 515 520 525 Lys Arg Ala Asp Gly Phe Lys Ile Arg Leu Glu Pro Arg Lys Gln Val 530 535 540 Ala Ser Thr Ala 545 27546PRTBlattella germanica 27Met Ser Val Thr Val Glu Thr Ala Thr Val Glu Ser Pro Ala Ser Ser 1 5 10 15 Ile Ser Thr Phe Met Val Leu Leu Val Ser Ala Val Ala Leu Phe Phe 20 25 30 Ala Tyr Trp Lys Ile Ser Arg Arg Arg Phe Leu Gln Leu Ala Glu Lys 35 40 45 Ile Pro Gly Pro Lys Gly Tyr Pro Ile Ile Gly Asn Ala Leu Asp Phe 50 55 60 Leu Gly Ser Ser Ser Gln Val Thr Asp Arg Met Ile Gln Ile Gly Phe 65 70 75 80 Gln Phe Thr Thr Ile Ala Lys Val Trp Ile Leu His Lys Leu Val Val 85 90 95 Phe Ile Ala Asp Pro Arg Asp Ile Glu Leu Ile Leu Gly Asn Ser Thr 100 105 110 His Leu Glu Lys Ser Glu Glu Tyr Arg Phe Phe Lys Pro Trp Leu Gly 115 120 125 Asp Gly Leu Leu Ile Ser Ser Gly Gln Lys Trp Lys Ser His Arg Lys 130 135 140 Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys Ser Phe Val Asp 145 150 155 160 Leu Phe Asn Ala Asn Ser Arg Ala Val Cys Asp Lys Met Ala Lys Glu 165 170 175 Asn Gly Arg Thr Phe Asp Cys His Asp Tyr Met Ser Glu Cys Thr Val 180 185 190 Glu Val Leu Leu Glu Thr Ala Met Gly Val Ser Lys Lys Thr Gln Asn 195 200 205 Lys Ser Gly Phe Glu Tyr Ala Met Ala Val Met Lys Met Cys Asn Ile 210 215 220 Leu His Leu Arg His Ser Lys Val Trp Leu Arg Pro Asp Trp Leu Phe 225 230 235 240 Asn Leu Thr Lys Tyr Gly Lys Glu Gln Val Asp Leu Leu Asp Val Ile 245 250 255 His Gly Leu Thr Lys Lys Val Ile Lys Asn Lys Lys Glu Ile Ile Ser 260 265 270 Ser Gly Thr Lys Lys Tyr Ile Glu Glu Ser Val Thr Gln Glu Glu Lys 275 280 285 Ala Ile Ala Ser Thr Pro Val Lys Gly Leu Arg Asp Asp Leu Asp Glu 290 295 300 Gln Asp Glu Asn Asp Val Gly Gln Lys Lys Arg Leu Ala Phe Leu Asp 305 310 315 320 Leu Met Ile Glu Ser Ala Gln Asn Gly Val Val Leu Thr Asp Glu Glu 325 330 335 Ile Lys Glu Glu Val Gly Thr Ile Met Phe Glu Gly His Asp Thr Thr 340 345 350 Ala Ala Gly Ser Ser Phe Phe Leu Cys Leu Met Gly Ile His Gln Lys 355 360 365 Tyr Gln Asp Met Cys Val Gln Glu Leu Asn Gln Ile Phe Gly Asp Ser 370 375 380 Asp Arg Pro Ala Thr Phe Ala Asp Thr Leu Glu Met Lys Phe Leu Glu 385 390 395 400 Arg Cys Leu Leu Glu Ala Leu Arg Met Tyr Pro Pro Val Pro Val Ile 405 410 415 Ala Arg Lys Leu Ala Glu Asp Leu Thr Leu Ala Ser Thr Gly Val Val 420 425 430 Ile Pro Gln Gly Thr Thr Ile Val Val Ser Thr Val Lys Thr His Arg 435 440 445 Leu Glu Glu His Trp Pro Asn Pro Asp Val Tyr Asp Pro Asp Asn His 450 455 460 Leu Pro Glu Lys Ala Ala Glu Arg His Tyr Tyr Ser Phe Val Pro Phe 465 470 475 480 Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Leu Leu Lys 485 490 495 Leu Lys Ile Ile Leu Ser Thr Ile Leu Arg Asn Phe Lys Val His Ser 500 505 510 Asp Ile Ser Glu Asp Glu Phe Lys Leu Gln Gly Asp Ile Ile Leu Lys 515 520 525 Arg Ala Asp Gly Phe Met Ile Arg Leu Glu Pro Arg Lys Lys Thr Val 530 535 540 Ala Ala 545 28564PRTIps paraconfusus 28Met Ser Thr Ala Thr Leu Ser Ser Ala Ala Ala Pro Gly Leu Leu Thr 1 5 10 15 Ser Thr Asn Leu Phe Leu Phe Leu Leu Ala Pro Ala Leu Ala Leu Leu 20 25 30 Tyr Val Tyr Trp Lys Val Ser Arg Lys His Met Val Glu Leu Ala Glu 35 40 45 Arg Ile Pro Gly Pro Ser Gly Leu Pro Ile Leu Gly Asn Ala Leu Glu 50 55 60 Phe Ile Gly Thr Pro Asn Gln Ile Phe Asn Thr Ile Tyr Gln Lys Ser 65 70 75 80 Phe Glu Phe Gly Arg Thr Ile Lys Val Trp Val Gly Pro Arg Leu Leu 85 90 95 Ile Phe Leu Thr Asp Pro Arg Asp Val Glu Ile Ile Leu Ser Ser His 100 105 110 Val His Ile Asp Lys Ser Pro Glu Tyr Arg Phe Phe Lys Pro Trp Leu 115 120 125 Gly Asp Gly Leu Leu Ile Ser Thr Gly Gln Lys Trp Arg Ala His Arg 130 135 140 Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys Ser Phe Ile 145 150 155 160 Asp Leu Phe Asn Lys Asn Ser Ile Glu Thr Val Asn Lys Leu Glu Lys 165 170 175 Glu Leu Gly Lys Glu Phe Asp Cys His Asp Tyr Met Ser Glu Ala Thr 180 185 190 Val Glu Ile Leu Leu Glu Thr Ala Met Gly Val Ser Lys Lys Thr Gln 195 200 205 Asp Gln Ser Gly Tyr Asp Tyr Ala Met Ala Val Met Lys Leu Cys Asp 210 215 220 Ile Leu His Leu Arg His Thr Lys Leu Trp Phe Arg Pro Asp Ile Ile 225 230 235 240 Phe Asn Leu Thr Ser Thr Ala Lys Tyr Gln Glu Lys Leu Ile Asn Val 245 250 255 Ile His Ser Leu Thr Arg Lys Val Ile Gln Lys Lys Lys Ala Asp Phe 260 265 270 Glu Lys Gly Ile Arg Gly Ser Thr Ala Glu Val Pro Glu Glu Leu Lys 275 280 285 Thr Gln Lys Tyr Glu Thr Ala Val Pro Thr Lys Thr Thr Thr Leu Glu 290 295 300 Gly Thr Ser Tyr Gly Gln Ser Val Gly Leu Lys Asp Asp Leu Asp Val 305 310 315 320 Asp Asp Asp Ile Gly Glu Lys Lys Arg Met Ala Phe Leu Asp Leu Met 325 330 335 Ile Glu Ala Ser Gln Asn Gly Val Val Ile Asn Asp Glu Glu Ile Lys 340 345 350 Glu Gln Val Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala 355 360 365 Gly Ser Ser Phe Phe Leu Cys Gln Met Ala Ala His Pro Glu Ile Gln 370 375 380 Glu Lys Val Leu Gln Glu Ile Asp Glu Ile Phe Gln Gly Ser Asp Arg 385 390 395 400 Pro Ala Thr Phe Ala Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg Cys 405 410 415 Leu Leu Glu Thr Leu Arg Leu Phe Pro Pro Val Pro Ile Ile Ala Arg 420 425 430 Gln Leu Gln Gln Asp Val Lys Leu Ala Ser Asn Pro Ser Tyr Val Leu 435 440 445 Pro Ser Gly Ala Thr Ile Ile Ile Gly Thr Phe Lys Val His Arg Leu 450 455 460 Glu Glu Ile Tyr Gly Pro Asn Ala Asp Lys Phe Asp Pro Asp Asn Phe 465 470 475 480 Leu Pro Glu Arg Ala Ala Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe 485 490 495 Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys 500 505 510 Leu Lys Ile Leu Leu Ser Thr Ile Leu Arg Asn Tyr Lys Ile Lys Ser 515 520 525 Asn Leu Lys Glu Ser Asp Tyr Lys Leu Gln Gly Asp Ile Ile Leu Lys 530 535 540 Arg Ala Asp Gly Phe Lys Ile Met Leu Glu Lys Arg Lys Pro Ile Val 545 550 555 560 Ser Val Lys Ala 29557PRTAntheraea yamamai 29Met Ser Tyr Thr Thr Ala Glu Asn Val Val Pro Ser Ser Thr Phe Ser 1 5 10 15 Ala Ile Asn Leu Phe Tyr Val Leu Leu Val Pro Ala Ile Ile Leu Trp 20 25 30 Tyr Thr Tyr Trp Arg Met Ser Arg Arg Arg Leu Tyr Glu Leu Ala Glu 35 40 45 Lys Leu Ser Gly Pro Glu Pro Leu Pro Ile Ile Gly Asn Ala Leu Glu 50 55 60 Phe Val Gly Gly Ser Asn Asp Ile Phe Asn Asn Ile Ile Ala Lys Ser 65 70 75 80 Leu Pro Phe Asp Asp Glu Ala Val Val Arg Leu Trp Ile Gly Pro Arg 85 90 95 Leu Leu Val Phe Ile Tyr Asp Pro Arg Asp Val Glu Val Ile Leu Ser 100 105 110 Ser His Val His Ile Asp Lys Ala Asp Glu Tyr Arg Phe Phe Lys Pro 115 120 125 Trp Leu Gly Asn Gly Leu Leu Ile Ser Thr Gly Gln Lys Trp Arg Ser 130 135 140 His Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys Ser 145 150 155 160 Phe Ile Asp Leu Phe Asn Ala Asn Ser Arg Ala Val Val Asp Lys Leu 165 170 175 Lys Lys Glu Ser Gly Thr Phe Asp Cys His Asp Tyr Met Ser Glu Cys 180 185 190 Thr Val Glu Ile Leu Leu Glu Thr Ala Met Gly Val Ser Lys Ser Thr 195 200 205 Gln Asp Gln Ser Gly Phe Glu Tyr Ala Met Ala Val Met Lys Met Cys 210 215 220 Asp Ile Leu His Leu Arg His Thr Lys Ile Trp Leu Arg Pro Asp Leu 225 230 235 240 Leu Phe Lys Leu Thr Asp Tyr Ala Lys Asn Gln Thr Arg Leu Leu Asp 245 250 255 Val Ile His Gly Leu Thr Lys Lys Val Ile Lys Arg Lys Lys Glu Glu 260 265 270 Phe Gln Ser Gly Lys Lys Ala Thr Ile Met Pro Glu Gly Asn Asp Val 275 280 285 Thr Asn Glu Val Pro Ser Ser Lys Leu Thr Ser Val Glu Gly Leu Ser 290 295 300 Phe Gly Gln Ser Ser Gly Leu Lys Asp Asp Leu Asp Val Asp Asp Asp 305 310 315 320 Val Gly Gln Lys Lys Arg Leu Ala Phe Leu Asp Leu Leu Leu Glu Ser 325 330 335 Ser Gln Ser Gly Val Val Ile Thr Asp Glu Glu Ile Lys Glu Gln Val 340 345 350 Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala Gly Ser Ser 355 360 365 Phe Phe Leu Ser Met Met Gly Ile His Gln His Ile Gln Asp Lys Val 370 375 380 Ile Glu Glu Leu Asp His Ile Phe Gly Asp Ser Asp Arg Pro Ala Thr 385 390 395 400 Phe Gln Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg Cys Leu Met Glu 405 410 415 Thr Leu Arg Leu Tyr Pro Pro Val Pro Ile Ile Ala Arg His Leu Lys 420 425 430 Glu Glu Ile Thr Leu Pro Ser Asn Gly Lys Lys Val Pro Ile Gly Thr 435 440 445 Thr Leu Ile Val Gly Thr Tyr Lys Leu His Arg Arg Pro Asp Val Tyr 450 455 460 Pro Asn Pro His Lys Phe Asp Pro Asp Asn Phe Leu Pro Glu Arg Ser 465 470 475 480 Ala Asn Arg His Tyr Tyr Ala Phe Val Pro Phe Ser Ala Gly Pro Arg 485 490 495 Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Ile Ile Leu 500 505 510 Ser Thr Ile Leu Arg Asn Phe Arg Val Tyr Ser Asp Leu Asn Glu Ser 515 520 525 Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Arg Ala Glu Gly Phe 530 535 540 Lys Val Arg Leu Gln Pro Arg Lys Lys Gln Ala Lys Val 545 550 555 30557PRTMamestra brassicae 30Met Ser

Tyr Ala Ala Ala Glu Ser Val Val Pro Thr Ser Thr Trp Ala 1 5 10 15 Ala Thr Ser Leu Phe Tyr Val Leu Leu Val Pro Ala Leu Ile Leu Trp 20 25 30 Tyr Ala Tyr Trp Arg Met Ser Arg Arg His Met Tyr Glu Leu Ala Ala 35 40 45 Lys Leu His Gly Pro Pro Gly Leu Pro Leu Leu Gly Asn Ala Leu Glu 50 55 60 Phe Thr Gly Gly Ser His Asp Ile Phe Arg Asn Val Ile Glu Lys Ser 65 70 75 80 Ile Pro Tyr Asp Gly Glu Ser Val Val Lys Ile Trp Ile Gly Pro Arg 85 90 95 Phe Trp Cys Ser Cys Thr Ile Leu Val Thr Trp Ser Leu Ile Leu Ser 100 105 110 Ser His Thr His Ile Asp Lys Ala Asp Glu Tyr Arg Phe Phe Lys Pro 115 120 125 Trp Leu Gly Asp Gly Leu Leu Ile Ser Thr Gly Gln Lys Trp Arg Ser 130 135 140 His Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys Ser 145 150 155 160 Phe Ile Asp Leu Phe Asn Ala Asn Ser Arg Ala Val Val Ser Lys Leu 165 170 175 Lys Lys Glu Ala Gly Glu Phe Asp Cys His Asp Tyr Met Ser Glu Cys 180 185 190 Thr Val Glu Ile Leu Leu Glu Thr Ala Met Gly Val Ser Lys Ser Thr 195 200 205 Gln Asp Gln Ser Gly Phe Glu Tyr Ala Met Ala Val Met Lys Met Cys 210 215 220 Asp Ile Leu His Leu Arg His Thr Lys Ile Trp Leu Arg Pro Asn Leu 225 230 235 240 Leu Phe Lys Leu Thr Asp Tyr Ala Lys Lys Gln Thr Lys Leu Leu Asp 245 250 255 Val Ile His Gly Leu Thr Lys Lys Val Ile Arg Arg Lys Lys Glu Glu 260 265 270 Phe Asn Ser Gly Lys Arg Pro Thr Ile Leu Gln Asp Cys Thr Thr Thr 275 280 285 Thr Thr Glu Glu Ala Asn Lys Thr Thr Ser Val Glu Gly Leu Ser Phe 290 295 300 Gly Gln Ser Ala Gly Leu Lys Asp Asp Leu Asp Val Asp Asp Ala Asp 305 310 315 320 Val Gly Gln Lys Lys Arg Leu Ala Phe Leu Asp Leu Leu Leu Glu Ser 325 330 335 Ser Gln Ser Gly Val Val Ile Ser Asp Glu Glu Ile Lys Glu Gln Val 340 345 350 Asp Thr Ile Met Phe Glu Gly His Asp Thr Thr Ala Ala Gly Ser Ser 355 360 365 Phe Phe Leu Ser Met Met Gly Ile His Gln Asp Ile Gln Asp Lys Val 370 375 380 Ile Asp Glu Leu Asp Lys Ile Phe Gly Asp Ser Asp Arg Pro Ala Thr 385 390 395 400 Phe Gln Asp Thr Leu Glu Met Lys Tyr Leu Glu Arg Cys Leu Met Glu 405 410 415 Thr Leu Arg Met Phe Pro Pro Val Pro Ile Ile Ala Arg His Leu Lys 420 425 430 Gln Asp Ile Thr Leu Pro Ser Cys Gly Lys Gln Val Pro Ala Gly Thr 435 440 445 Thr Val Val Val Ala Thr Tyr Lys Leu His Arg Arg Pro Asp Val Tyr 450 455 460 Pro Asn Pro Thr Glu Phe Asp Pro Asp Asn Phe Leu Pro Glu Lys Ser 465 470 475 480 Ala Asn Arg His Tyr Tyr Ala Phe Val Pro Phe Ser Ala Gly Pro Arg 485 490 495 Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys Ile Ile Leu 500 505 510 Ser Thr Ile Leu Arg Ser Phe Arg Val His Ser Asp Leu Lys Glu Ser 515 520 525 Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Arg Ala Glu Gly Phe 530 535 540 Lys Val Arg Leu Glu Pro Arg Lys Thr Thr Lys Ala Tyr 545 550 555 31565PRTAcyrthosiphon pisum 31Met Val Thr Asn Val Gln Gly Val Asn Pro Leu Phe Ala Leu Ser Ala 1 5 10 15 Phe Asn Leu Phe Phe Tyr Leu Leu Thr Pro Ala Ile Val Leu Trp Tyr 20 25 30 Ile Tyr Phe Arg Met Ser Arg Lys Gln Leu Tyr Asp Leu Ala Ser Lys 35 40 45 Ile Pro Gly Ser Glu Gly Leu Pro Leu Leu Gly Asn Ala Leu Asp Phe 50 55 60 Met Gln Asp Pro His Thr Ile Phe Glu Lys Ile Tyr Glu Arg Ser Phe 65 70 75 80 Glu Phe Glu Lys Asn Ser Pro Ile Lys Met Trp Ile Gly Pro Arg Leu 85 90 95 Leu Val Phe Leu Thr Asp Pro Arg Asp Val Glu Val Ile Leu Ser Ser 100 105 110 Asn Val Tyr Ile Asp Lys Ser Pro Glu Tyr Arg Leu Phe Glu Pro Trp 115 120 125 Leu Gly Asn Gly Leu Leu Ile Ser Thr Gly Asp Lys Trp Arg Ala His 130 135 140 Arg Lys Leu Ile Ala Pro Thr Phe His Leu Asn Val Leu Lys Ser Phe 145 150 155 160 Val Thr Leu Phe Asn Val Asn Ser Arg Asp Thr Val Ser Lys Leu Arg 165 170 175 Lys Met Gly Ser Ser Thr Phe Asp Ile His Asp Phe Met Ser Glu Cys 180 185 190 Thr Val Glu Ile Leu Leu Glu Thr Ala Met Gly Val Ser Lys Lys Thr 195 200 205 Gln Lys Lys Ser Gly Phe Glu Tyr Ala Ala Ala Val Met Lys Met Cys 210 215 220 Asp Ile Leu His Met Arg His Thr Asn Leu Trp Leu Lys Pro Asp Phe 225 230 235 240 Ile Phe Asn Phe Thr Lys Tyr Ala Lys Glu Gln Val Gly Leu Leu Asp 245 250 255 Leu Ile His Gly Leu Thr Asn Asn Val Leu Ala Lys Lys Lys Glu Glu 260 265 270 Phe Leu Lys Lys Lys Ser Leu Met Lys Glu Val Ser Asp Ile Pro Ala 275 280 285 Ala Ser Glu Glu Ile Val Glu Thr Ser Ser Thr Leu Glu Val Glu Glu 290 295 300 Val Pro Tyr Gly Asn Ser Phe Gly Gln Ser Ala Gly Leu Lys Asp Asp 305 310 315 320 Leu Asp Val Glu Asp Asp Gly Ile Gly Glu Lys Lys Arg Val Ala Phe 325 330 335 Leu Asp Leu Leu Ile Glu Cys Ser Glu Asn Gly Val Val Leu Ser Asp 340 345 350 Glu Glu Val Arg Glu Gln Val Asp Thr Ile Met Phe Glu Gly His Asp 355 360 365 Thr Thr Ala Ala Gly Ser Ser Phe Phe Leu Cys Leu Met Gly Ala His 370 375 380 Gln Asp Val Gln Gln Lys Val Val Asp Glu Leu Tyr Ser Ile Phe Gly 385 390 395 400 Asp Ser Asp Arg Pro Val Thr Phe Gln Asp Thr Leu Gln Met Lys Tyr 405 410 415 Met Glu Arg Cys Ile Met Glu Thr Leu Arg Met Tyr Pro Pro Val Pro 420 425 430 Ile Ile Ser Arg Gln Ile Lys Glu Lys Val Lys Leu Gly Glu Asp Ile 435 440 445 Thr Leu Pro Val Gly Ala Thr Ile Val Ile Ala Thr Phe Lys Ile His 450 455 460 Arg Asn Glu Asp Val Phe Pro Asn Pro Glu Val Phe Asn Pro Asp Asn 465 470 475 480 Phe Leu Pro Glu Lys Ser Ala Ser Arg His Tyr Tyr Ala Tyr Val Pro 485 490 495 Phe Ser Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu 500 505 510 Lys Leu Lys Ile Ile Leu Ser Thr Ile Leu Arg Asn Phe Lys Ile Asn 515 520 525 Ser Asn Leu Thr Glu Lys Asp Trp Lys Leu Gln Ala Asp Ile Ile Leu 530 535 540 Lys Arg Thr Asp Gly Phe Lys Leu Ser Leu Glu Pro Arg Lys Ser Leu 545 550 555 560 Ala Lys Thr Ala Ala 565 32817PRTDrosophila persimilis 32Met Ser Thr Val Glu Val Leu Leu His Glu Phe Tyr Gln Pro Thr Thr 1 5 10 15 Ser Asn Ala Arg Lys Arg Glu Ile Glu Thr Asn Leu Leu Ala Phe Lys 20 25 30 Ser Gln Pro Glu Ala Trp Gln Leu Cys Leu Arg Val Ala Thr Ala Gly 35 40 45 Asn Ser Phe Thr Asp Asn Gln Phe Leu Trp Phe Phe Ser Thr Ser Thr 50 55 60 Leu Glu His Thr Ile Thr Arg Arg Trp Ala Gln Leu Thr Pro Ser Asp 65 70 75 80 Arg Ala Gln Leu Arg Glu Thr Leu Trp Asn Thr Tyr Ala Gln Leu Gly 85 90 95 Met Leu Asn Gly Ala Arg Arg His Arg Asp Thr Leu Ala Gln Leu Ile 100 105 110 Ala Leu Met Gly Lys Arg Glu Phe Pro Glu Gln Asp Pro Asn Tyr Met 115 120 125 Gln His Cys Met Glu Leu Thr Lys Thr Arg Phe Ala Leu Gly Ile Asn 130 135 140 Leu Leu Arg Val Thr Ser Glu Glu Val Val Ser Asn Arg Gly Asp Leu 145 150 155 160 Thr Thr Glu Trp Lys Gln Tyr Phe Tyr Ser Cys Ile Ser Met Cys Ile 165 170 175 Pro Asp Val Met Asp Leu Val Thr Lys Tyr Leu Leu Ile Ala Val Cys 180 185 190 His Ile Asn Gly Lys Asp Ile Gln Ser Thr Ile Pro Asn Thr Leu Met 195 200 205 Asp Phe Ser Leu Thr Ser Ala Leu Pro Asn Asp Asn Gln Leu Ser Ser 210 215 220 Ser Ile Leu Glu Leu Leu Gly Cys Val Gln His Leu Val Ser Trp Ile 225 230 235 240 Arg Thr Glu Leu Ile Ser Glu Tyr Phe Leu Met Ser Ile Leu Asp Leu 245 250 255 Ser Gln Trp Arg Pro Ala Asn Glu Pro Ile Ser Leu Ala Ala Leu Ser 260 265 270 Val Leu Asn Glu Leu Leu Tyr Leu Gln Lys Pro Leu Pro Tyr Ala Gly 275 280 285 Thr Leu Met Gly Gly Val Ser Ser Leu Leu Glu Gln His Asn Val Asn 290 295 300 Lys Gln Gln Ser Glu Met Tyr Ser Asp Lys Leu Arg Glu Leu Leu Arg 305 310 315 320 Leu Tyr Thr Thr Lys Tyr Ala Gly Lys Leu Met Gln Glu Pro Glu Val 325 330 335 Leu Glu Thr Phe Leu Asn Gln Leu Tyr Gly Cys Thr Thr Glu Leu His 340 345 350 Gly Ala Leu Asp Phe Thr Glu Lys Leu Asp Ile Trp Ser Pro Ile Ile 355 360 365 Lys Ala Ile Ala Gln Gln Pro Ala Lys Ile Thr Arg Phe Asn Gln Val 370 375 380 Phe Thr Gln Leu Val Asp Glu Ile Met Arg Arg Thr Gln Phe Glu Ala 385 390 395 400 Asn Lys Pro Glu Leu Glu Val Leu Asp Asn Glu Leu Met Glu Asp Asp 405 410 415 Thr Pro Thr Thr Glu Trp Gln Gln Phe Leu Asp Gln Cys Phe Glu Cys 420 425 430 Leu Ala Leu Leu Ala Ser Thr Arg Gly Ala His Ile Val Phe Ala Gln 435 440 445 Val Phe Ala His Trp Ser Arg Pro Gln Met Tyr Leu Met Ser Leu Glu 450 455 460 His Ala Leu Asp His Gly Ser Ser Arg Ser Tyr Glu Ala Ala Arg Lys 465 470 475 480 Leu Lys His Ala Asn Val Gly Glu Ile Leu Arg Asp Phe Ala Thr Val 485 490 495 Cys Gln Ala Val Val Arg Leu Ala Pro Leu Met Asp Thr Ser Thr Ala 500 505 510 Ala Pro Gly Val Ala Asp Glu Met Glu Ala Gln Leu Gln Met Leu Ser 515 520 525 Asp Ser Leu Cys Arg Arg Cys Ser Phe Trp Pro Ala Ile Ala Ser Glu 530 535 540 Glu Gln Ile Trp Thr Arg Pro Pro Phe Arg Leu Ile Trp Ile Thr Ser 545 550 555 560 Ala Thr Lys Lys Glu Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp Glu 565 570 575 Asn Asp Val Gly Ala Lys Arg Arg Leu Ala Leu Leu Asp Ala Met Val 580 585 590 Glu Met Ala Lys Asn Pro Asp Ile Glu Trp Asn Glu Lys Asp Ile Ile 595 600 605 Asp Glu Val Asn Thr Ile Met Phe Glu Gly His Asp Thr Thr Ser Ala 610 615 620 Gly Ser Ser Phe Ala Leu Cys Met Met Gly Ile His Lys Asp Ile Gln 625 630 635 640 Glu Lys Val Phe Ala Glu Gln Lys Ala Ile Phe Gly Asp Asn Met Leu 645 650 655 Arg Asp Cys Thr Phe Ala Asp Thr Asn Glu Met Lys Tyr Leu Glu Arg 660 665 670 Val Ile Leu Glu Thr Leu Arg Leu Tyr Pro Pro Val Pro Leu Ile Ala 675 680 685 Arg Arg Leu Asp Tyr Asp Leu Lys Leu Ala Ser Gly Pro Tyr Thr Val 690 695 700 Pro Lys Gly Thr Thr Val Ile Val Leu Gln Tyr Cys Val His Arg Arg 705 710 715 720 Ala Asp Ile Tyr Pro Asn Pro Thr Lys Phe Asp Pro Asp Asn Phe Leu 725 730 735 Pro Glu Arg Met Ala Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser 740 745 750 Ala Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu 755 760 765 Lys Val Leu Leu Ser Thr Ile Val Arg Asn Tyr Ile Val His Ser Thr 770 775 780 Asp Thr Glu Ala Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Leu 785 790 795 800 Glu Asn Gly Phe Asn Ile Ser Leu Glu Lys Arg Lys Tyr Ala Thr Val 805 810 815 Ala 33511PRTBlaberus discoidalis 33Met Glu Phe Ile Thr Ile Leu Leu Ser Thr Ala Leu Phe Ile Val Thr 1 5 10 15 Phe Leu Phe Leu Phe Arg Gln Gly Ala Lys Arg Ala Arg Phe Val Tyr 20 25 30 Leu Val Asn Lys Leu Pro Gly Pro Thr Ala Tyr Pro Val Val Gly Asn 35 40 45 Ala Ile Glu Ala Ile Val Pro Arg Asn Lys Leu Phe Gln Val Phe Asp 50 55 60 Arg Arg Ala Lys Leu Tyr Gly Pro Leu Tyr Arg Ile Trp Ala Gly Pro 65 70 75 80 Ile Ala Gln Val Gly Leu Thr Arg Pro Glu His Val Glu Leu Ile Leu 85 90 95 Arg Asp Thr Lys His Ile Asp Lys Ser Leu Val Tyr Ser Phe Ile Arg 100 105 110 Pro Trp Leu Gly Glu Gly Leu Leu Thr Gly Thr Gly Ala Lys Trp His 115 120 125 Ser His Arg Lys Met Ile Thr Pro Thr Phe His Phe Lys Ile Leu Asp 130 135 140 Ile Phe Val Asp Val Phe Val Glu Lys Ser Glu Ile Leu Val Lys Lys 145 150 155 160 Leu Gln Ser Lys Val Gly Gly Lys Asp Phe Asp Ile Tyr Pro Phe Ile 165 170 175 Thr His Cys Ala Leu Asp Ile Ile Cys Glu Thr Ala Met Gly Ile Gln 180 185 190 Met Asn Ala Gln Glu Glu Ser Glu Ser Glu Tyr Val Lys Ala Val Tyr 195 200 205 Glu Ile Ser Glu Leu Thr Met Gln Arg Ser Val Arg Pro Trp Leu His 210 215 220 Pro Lys Val Ile Phe Asp Leu Thr Thr Met Gly Lys Arg Tyr Ala Glu 225 230 235 240 Cys Leu Arg Ile Leu His Gly Phe Thr Asn Lys Val Ile Gln Glu Arg 245 250 255 Lys Ser Leu Arg Gln Met Thr Gly Met Lys Pro Thr Ile Ser Asn Glu 260 265 270 Glu Asp Glu Leu Leu Gly Lys Lys Lys Arg Leu Ala Phe Leu Asp Leu 275 280 285 Leu Leu Glu Ala Ser Glu Asn Gly Thr Lys Met Ser Asp Thr Asp Ile 290 295 300 Arg Glu Glu Val Asp Thr Phe Met Phe Glu Gly His Asp Thr Thr Ser 305 310 315 320 Ala Gly Ile Cys Trp Ala Leu Phe Leu Leu Gly Ser His Pro Glu Ile 325 330 335 Gln Asp Lys Val Tyr Glu Glu Leu Asp His Ile Phe Gln Gly Ser Asp 340 345 350 Arg Ser Thr Thr Met Arg Asp Leu

Ala Asp Met Lys Tyr Leu Glu Arg 355 360 365 Val Ile Lys Glu Ser Leu Arg Leu Phe Pro Ser Val Pro Phe Ile Gly 370 375 380 Arg Val Leu Lys Glu Asp Thr Lys Ile Gly Asp Tyr Leu Val Pro Ala 385 390 395 400 Gly Cys Met Met Asn Leu Gln Ile Tyr His Val His Arg Asn Gln Asp 405 410 415 Gln Tyr Pro Asn Pro Glu Ala Phe Asn Pro Asp Asn Phe Leu Pro Glu 420 425 430 Arg Val Ala Lys Arg His Pro Tyr Ala Tyr Val Pro Phe Ser Ala Gly 435 440 445 Pro Arg Asn Cys Ile Gly Gln Lys Phe Ala Thr Leu Glu Glu Lys Thr 450 455 460 Val Leu Ser Ser Ile Leu Arg Asn Phe Lys Val Arg Ser Ile Glu Lys 465 470 475 480 Arg Glu Asp Leu Thr Leu Met Asn Glu Leu Ile Leu Arg Pro Glu Ser 485 490 495 Gly Ile Lys Val Glu Leu Ile Pro Arg Leu Pro Ala Asp Ala Cys 500 505 510 34515PRTCarcinus maenas 34Met Ala Leu Leu Leu Gly Arg Glu Phe Val Trp Trp Ser Ser Val Ala 1 5 10 15 Ser Tyr Ser Leu Gly Thr Ala Cys Leu Ala Leu Leu Leu Thr Trp Phe 20 25 30 Ile Arg Arg Gln Gln Thr Val Trp Leu Ile Glu Lys Leu Pro Gly Pro 35 40 45 Arg Ser Leu Pro Ile Leu Gly Asn Ala Leu Asp Val Asn Val Ala Pro 50 55 60 Arg Glu Leu Phe Leu Lys Ile Met Glu Phe Cys Glu Tyr Gly Asn Thr 65 70 75 80 Val Lys Ile Trp Leu Gly Met Tyr Pro Tyr Cys Leu Val Ser Glu Ala 85 90 95 Lys Ser Ala Glu Val Leu Leu Ser Ser Asn Lys His Leu Asp Lys Ser 100 105 110 Arg Asp Tyr Asn Phe Leu His Pro Trp Leu Gly Thr Gly Leu Leu Thr 115 120 125 Ser Thr Gly Lys Lys Trp His Ser Arg Arg Lys Ile Leu Thr Pro Ala 130 135 140 Phe His Phe Lys Ile Leu Glu Asp Phe Val Glu Val Phe Asn Ser Gln 145 150 155 160 Ser Asn Lys Met Leu Asp Lys Leu Thr Pro Lys Ala Asp Gly Lys Ala 165 170 175 Phe Asp Ile Phe Pro Tyr Ile Thr Leu Cys Thr Leu Asp Ile Ile Cys 180 185 190 Glu Thr Ala Met Gly Ile Asn Ile Asn Ala Gln Gly Asn Ser Asn Ser 195 200 205 Glu Tyr Val Asn Ala Val Tyr Arg Ile Gly Ala Leu Val Gln His Arg 210 215 220 Gln Thr Arg Pro Trp Ile Gln Pro Asp Phe Leu Phe Arg Leu Phe Gly 225 230 235 240 Tyr Ala Lys Leu His Asp Glu Tyr Leu Arg Val Leu His His Phe Ser 245 250 255 Asn Ser Ala Ile Glu Asn Arg Arg Lys Glu Tyr Gln Leu Glu Lys Leu 260 265 270 Asn Ala Lys Glu Asn Ile Asp Asp Asp Val Ile Gly Lys Lys Arg Arg 275 280 285 Leu Ala Phe Leu Asp Leu Leu Leu Asn Tyr Ser Glu Thr Gln Met Pro 290 295 300 Leu Ser Asn Glu Asp Ile Arg Glu Glu Val Asp Thr Phe Met Phe Glu 305 310 315 320 Gly His Asp Thr Thr Ala Ala Ala Leu Asn Trp Ser Val Tyr Leu Leu 325 330 335 Gly Cys His Pro Glu Ile Gln Ala Lys Val His Glu Glu Leu Asp Ala 340 345 350 Leu Phe Gly Asp Ser Asp Arg Pro Val Thr Met Ala Asp Leu Arg Glu 355 360 365 Met Lys Tyr Thr Glu Asn Cys Ile Lys Glu Ala Leu Arg Leu Phe Pro 370 375 380 Ser Val Pro Phe Leu Ala Arg Glu Leu Arg Glu Glu Ala Val Ile Asn 385 390 395 400 Asn Tyr Arg Ile Pro Val Gly Thr Thr Val Met Val Ile Thr Tyr Arg 405 410 415 Leu His Arg Asp Pro Glu Gln Phe Pro Asn Pro Glu Thr Phe Asp Pro 420 425 430 Asp Arg Phe Leu Pro Glu Asn Val Ala Lys Arg His Pro Tyr Ser Tyr 435 440 445 Val Pro Phe Ser Ala Gly Pro Arg Asn Cys Ile Gly Gln Lys Phe Ala 450 455 460 Ile Met Glu Glu Lys Ile Val Leu Ser Ser Ile Met Arg Arg Phe Arg 465 470 475 480 Val Glu Ser Thr Thr Arg Arg Glu Glu Leu Lys Leu Leu Gly Glu Leu 485 490 495 Ile Leu Arg Pro Glu Asn Gly Asn Thr Val Lys Leu Ile Pro Arg Thr 500 505 510 Pro Lys Val 515 35516PRTBranchiostoma floridae 35Met Ala Ala Val Leu Trp Thr Ala Ala Val Ala Val Ala Val Ser Val 1 5 10 15 Thr Ile Trp Ala Ile Phe Ala Phe Ala Arg Trp Trp Lys Leu Trp Lys 20 25 30 Thr Ile Asn Lys Ile Pro Gly Pro Pro Ala Tyr Pro Leu Val Gly Asn 35 40 45 Ala Leu Glu Phe Lys Pro Gly Ala Val Glu Phe Phe Ala Gln Leu Phe 50 55 60 Gly Trp Gly Lys Ala Tyr Ala Ser Ala Pro Val Leu Arg Trp Trp Ile 65 70 75 80 Gly Pro His Pro Met Val Ala Leu His His Pro Glu Met Leu Gln Val 85 90 95 Leu Phe Ser Ser Ser Lys His Ile Glu Lys Ser Phe Val Tyr Asp Phe 100 105 110 Leu His Pro Trp Leu Gly Thr Gly Leu Leu Thr Ser Ala Gly Asp Lys 115 120 125 Trp Lys Thr Arg Arg Arg Leu Ile Thr Pro Thr Phe His Phe Lys Ile 130 135 140 Leu Gly Asp Phe Leu His Glu Phe Asn Asp Gln Ser Glu Ile Met Val 145 150 155 160 Arg Lys Leu Glu Glu Met Ala Gly Thr Gly Glu Glu Phe Asp Val Phe 165 170 175 Pro Phe Ile Thr Leu Cys Ala Leu Asp Ile Ile Cys Gly Thr Ala Met 180 185 190 Gly Gln Ser Leu Asn Ala Gln Glu Asn Thr Asp Ser Asp Tyr Val Arg 195 200 205 Ala Ile Tyr Arg Ile Ser Asp Leu Ile Gln Val Arg Gln Lys Ser Pro 210 215 220 Trp Tyr Trp Ser Asp Pro Ile Tyr Lys Gly Phe Gly Pro Gly Arg Glu 225 230 235 240 Phe Glu Glu Thr Leu Arg Ile Leu His Asp Phe Thr Arg Ser Val Ile 245 250 255 Lys Glu Arg Ser Glu Gln Phe Gln Lys Gln Leu Glu Ser Gln Ser Gln 260 265 270 Asp Ala Phe Asp Ile Val Glu Asp Pro Asp Lys Pro Ile Ala Ile Gly 275 280 285 Gly Arg Lys Arg Leu Ala Phe Leu Asp Met Leu Leu Tyr Ala Ser Val 290 295 300 Gly Glu Thr Lys Leu Thr Asn Glu Asp Ile Gln Glu Glu Val Asp Thr 305 310 315 320 Phe Met Phe Glu Gly His Asp Thr Thr Ala Ala Ala Ala Asn Trp Ala 325 330 335 Ile Phe Leu Ile Gly Ser His Pro Asp Val Gln Arg Lys Val His Glu 340 345 350 Glu Met Asp Arg Val Met Ser Asp Pro Asp Glu Lys Pro Thr Met Asp 355 360 365 Asp Leu Arg Glu Met Lys Tyr Leu Glu Cys Cys Ile Lys Glu Ala Leu 370 375 380 Arg Leu Tyr Pro Ser Val Pro Phe Phe Ala Arg Thr Leu Ser Glu Asp 385 390 395 400 Cys Val Ile Gly Gly Tyr Glu Val Pro Lys Gly Val Thr Ala Ile Val 405 410 415 Pro Thr Tyr Asn Val His Arg Asp Pro Asn His Trp Pro Asp Ala Glu 420 425 430 Lys Phe Asp Pro Glu Arg Phe Phe Pro Glu Asn Cys Ala Gly Arg His 435 440 445 Pro Tyr Ala Tyr Ile Pro Phe Ser Ala Gly Ser Arg Asn Cys Ile Gly 450 455 460 Gln Arg Phe Ala Leu Met Glu Glu Lys Ala Ile Leu Ser Ser Ile Phe 465 470 475 480 Arg Arg Phe Arg Ile Glu Thr Met Gln Asn Arg Glu Asp Leu Lys Pro 485 490 495 Leu Gly Glu Leu Ile Leu Arg Pro Glu Ser Gly Val Arg Ile Lys Leu 500 505 510 Phe Arg Arg Glu 515 36525PRTBalaenoptera acutorostrata 36Met Leu Ala Leu Trp Leu Leu Ser Val Gly Gln Lys Leu Leu Leu Trp 1 5 10 15 Gly Gly Leu Cys Ala Val Ser Leu Ala Gly Ala Ile Leu Thr Leu Asn 20 25 30 Leu Leu Arg Met Ala Ala Ser Tyr Ala Trp Thr Trp Gln Arg Met Arg 35 40 45 Ala Val Pro Thr Leu Glu Gly Ala Tyr Pro Phe Leu Gly His Ala Leu 50 55 60 Leu Leu Lys Pro Asp Ala Arg Asp Phe Phe Gln Gln Met Ile Gln Tyr 65 70 75 80 Thr Glu Glu His Arg His Leu Pro Leu Leu Lys Leu Trp Leu Gly Pro 85 90 95 Ile Pro Val Val Phe Leu Tyr Asn Ala Glu Asn Val Glu Val Ile Leu 100 105 110 Thr Ser Ser Lys His Ile Asp Lys Ser Tyr Met Tyr Lys Phe Leu Glu 115 120 125 Pro Trp Leu Gly Leu Gly Leu Leu Thr Ser Thr Gly Asn Lys Trp Arg 130 135 140 Ser Arg Arg Lys Met Leu Thr Pro Thr Phe His Phe Thr Ile Leu Glu 145 150 155 160 Asp Phe Leu Asp Val Met Asn Glu Gln Ala Asn Ile Leu Val Asn Lys 165 170 175 Leu Glu Lys Tyr Val Asn Gln Glu Ala Phe Asn Cys Phe Ser Tyr Ile 180 185 190 Thr Leu Cys Ala Leu Asp Ile Ile Cys Glu Thr Ala Met Gly Lys Asn 195 200 205 Ile Gly Ala Gln Ser Asn Asn Asp Ser Glu Tyr Val Gln Ala Val Tyr 210 215 220 Arg Met Ser Asp Ser Ile His Gln Arg Met Lys Met Pro Trp Leu Trp 225 230 235 240 Leu Asp Leu Leu Phe Phe Ile Phe Lys Asp Gly Trp Glu His Lys Arg 245 250 255 Ser Leu Lys Ile Leu His Asn Phe Thr Lys Asn Val Ile Thr Glu Arg 260 265 270 Ala Asn Glu Met Lys Arg His Glu Glu Gly Arg Ser Asn Asp Lys Asp 275 280 285 Phe Pro Pro His Asn Asn Lys Arg Arg Gly Phe Leu Asp Leu Leu Leu 290 295 300 Asn Val Thr Asp Asp Gln Gly Asn Lys Leu Ser Tyr Glu Glu Ile Arg 305 310 315 320 Glu Glu Val Asp Thr Phe Met Phe Glu Gly His Asp Thr Thr Ala Ala 325 330 335 Ala Ile Asn Leu Ser Leu Tyr Leu Leu Gly Ser Tyr Pro Glu Val Gln 340 345 350 Gln Lys Val Asp Asn Glu Leu Glu Glu Val Phe Gly Arg Ser Asp Arg 355 360 365 Pro Ala Thr Leu Asp Asp Leu Lys Lys Leu Lys Tyr Leu Glu Cys Val 370 375 380 Val Lys Glu Ser Leu Arg Leu Phe Pro Ser Val Pro Phe Phe Ala Arg 385 390 395 400 Asn Leu Asn Glu Asp Cys Glu Val Ala Gly Tyr Lys Ile Val Lys Gly 405 410 415 Ser Gln Val Ile Ile Met Pro Tyr Ala Leu His Arg Asp Gln Arg Tyr 420 425 430 Phe Pro Asn Pro Glu Glu Phe Lys Pro Glu Arg Phe Phe Pro Glu Asn 435 440 445 Ser Lys Gly Arg His Ser Tyr Ala Tyr Val Pro Phe Ser Ala Gly Pro 450 455 460 Arg Asn Cys Ile Gly Gln Lys Phe Ala Met Met Glu Glu Lys Thr Ile 465 470 475 480 Leu Ser Cys Ile Leu Arg His Phe Trp Val Glu Ser Asn Gln Lys Arg 485 490 495 Glu Glu Leu Gly Leu Ala Gly Glu Leu Ile Leu Arg Pro Ser Asn Gly 500 505 510 Ile Trp Ile Lys Leu Lys Arg Arg Asn Thr Asn Glu Ser 515 520 525 37501PRTHelicoverpa zea 37Met Phe Trp Phe Leu Leu Phe Phe Val Gly Phe Leu Cys Leu Leu His 1 5 10 15 Leu Leu Leu Asn Tyr Asn Glu Arg Ala Arg Leu Ile Arg Lys Leu Pro 20 25 30 Gly Pro Glu Asp Ser Phe Ile Leu Gly Asn Gly Pro Ala Val Met Leu 35 40 45 Ser Ser Val Glu Val Met Lys Leu Ala Arg Lys Leu Ala Gln Glu Asn 50 55 60 Ser Gly Ile Tyr Arg Leu Trp Met Tyr Pro Val Ala Ala Val Ser Ile 65 70 75 80 Tyr Asn Pro Glu Asp Ile Glu Thr Ile Val Ser Ser Met Lys Tyr Asn 85 90 95 Glu Lys Ser Gln Val Tyr Arg Phe Leu Lys Pro Trp Leu Gly Asp Gly 100 105 110 Leu Leu Leu Ser Lys Gly Gln Lys Trp Gln Gln Arg Arg Lys Ile Leu 115 120 125 Thr Pro Thr Phe His Phe Asn Ile Leu Lys Gln Phe Cys Glu Val Ile 130 135 140 Ser Glu Asn Thr Gln Arg Phe Val Glu Asn Leu Lys Glu Val Ser Gly 145 150 155 160 Arg Pro Ile Asp Val Val Pro Val Ile Ser Glu Phe Thr Leu Asn Ser 165 170 175 Ile Cys Glu Thr Ala Met Gly Thr Asn Leu Thr Glu Tyr Asp Lys Thr 180 185 190 Ala Ala Ser Ala Tyr Lys Glu Ala Ile His Asn Leu Gly Tyr Ile Phe 195 200 205 Tyr Gln Arg Phe Ile Lys Val Tyr Tyr Phe Phe Asp Phe Ile Phe Asn 210 215 220 Leu Ser Ser Leu Ser Lys Lys Gln Asp Gly Tyr Leu Lys Thr Val His 225 230 235 240 Ser Phe Thr Lys Lys Val Ile Asp Glu Arg Ser Ala Tyr Ile Glu Lys 245 250 255 His Gly Ile Lys Ile Pro Asp Glu Asn Asp Asp Asp Asp Thr Tyr Val 260 265 270 Tyr Lys Ser Lys Lys Lys Thr Ala Met Leu Asp Val Leu Ile Ser Ala 275 280 285 Arg Lys Glu Gly His Ile Ser Asp Thr Gly Val Gln Glu Glu Val Asp 290 295 300 Thr Phe Met Phe Glu Gly His Asp Thr Thr Ala Gly Gly Leu Thr Tyr 305 310 315 320 Cys Phe Met Leu Leu Ala Asn His Lys Glu Ala Gln Asp Lys Ile Leu 325 330 335 Glu Glu Leu Lys Glu Ile Leu Gly Asp Asp Lys Arg Pro Ile Thr Met 340 345 350 Glu Asp Leu Pro Lys Met Lys Tyr Leu Glu Arg Cys Ile Lys Glu Ser 355 360 365 Leu Arg Leu Phe Pro Pro Val His Phe Ile Ser Arg Ser Leu Asn Glu 370 375 380 Thr Val Thr Leu Ser Asn Tyr Lys Ile Pro Ala Gly Thr Leu Cys His 385 390 395 400 Ile Gln Ile Tyr Asp Leu His Arg Arg Ala Asp Leu Phe Lys Asn Pro 405 410 415 Thr Ser Phe Asp Pro Asp Arg Phe Leu Pro Glu Asn Ser Val Gly Arg 420 425 430 His Pro Tyr Ala Tyr Ile Pro Phe Ser Ala Gly Pro Arg Asn Cys Ile 435 440 445 Gly Gln Lys Phe Ala Met Met Glu Met Lys Ile Ala Val Ala Glu Val 450 455 460 Leu Arg Glu Phe Glu Leu Gln Pro Val Thr Arg Pro Ser Asp Ile Arg 465 470 475 480 Met Ile Ala Asp Ala Val Phe Arg Asn Asp Gly Pro Val Glu Val Thr 485 490 495 Phe Val Lys Arg Gln 500 38526PRTDaphnia magna 38Met Asp Val Thr Ser Gly Gly Glu Ser Ser Val Trp Ile Ser Ser Phe 1 5 10 15 Ser Val Tyr Thr Val Thr Thr Ile Leu Val Thr Leu Val Val Leu Ala 20 25 30 Val Val Lys Arg Tyr Asn Phe Met Gln Arg Cys Asn Lys Val Leu Gly 35 40 45 Ser Pro Thr Asp Ile Pro Leu Phe Gly Gly Gly Ser Leu Ile Phe Val 50 55 60 Pro Pro Glu Glu Ile Met Asn Leu Leu Leu Leu Leu His Val Val Phe 65 70

75 80 Gly Arg Leu Ser Pro Ser Gly Ile Ile Arg Ala Trp Ile Gly Pro Leu 85 90 95 Pro Met Phe Phe Ala Thr Thr Ala Glu Ala Val Glu Ala Val Leu Ser 100 105 110 Ser Asn Lys Ile Ile Thr Lys Ser Arg Glu Tyr Asp Phe Leu His Pro 115 120 125 Trp Leu Asn Thr Gly Leu Leu Thr Ser Thr Gly Ser Lys Trp Gln Thr 130 135 140 Arg Arg Lys Leu Leu Thr Pro Ala Phe His Phe Lys Ile Leu Glu Asp 145 150 155 160 Phe Val His Val Phe Asn Glu Gln Ser Leu Ile Leu Val Asn Lys Leu 165 170 175 Asn Gln Ala Val Ala Lys Asp Lys Asp Leu Asn Ile Phe Pro Phe Val 180 185 190 Thr Leu Cys Thr Leu Asp Ile Ile Cys Glu Thr Ala Met Gly Arg Asn 195 200 205 Val Glu Ala Gln Ser Lys Thr Asp Ser Ala Tyr Val Gln Ala Val Tyr 210 215 220 Asn Met Ser Gln Leu Ile Gln His Arg Gln Val Arg Phe Tyr Leu Trp 225 230 235 240 Leu Asp Trp Met Phe Lys Leu Ser Ser His Trp Pro Glu Gln Arg Lys 245 250 255 Thr Leu Gly Ile Leu His Gly Phe Thr Asn Lys Val Ile Gln Glu Arg 260 265 270 Lys Ala Glu His Gln Gln Arg Ser Ser Asp Ile Ala Glu Pro Ser Lys 275 280 285 Asp Val Thr Glu Asp Ala Val Phe Ser Lys Arg Arg Leu Ala Phe Leu 290 295 300 Asp Leu Leu Ile Glu Phe Ser Gln Gly Gly Thr Val Leu Ser Ala Ser 305 310 315 320 Asp Ile Arg Glu Glu Val Asp Thr Phe Met Phe Glu Gly His Asp Thr 325 330 335 Thr Ser Ala Ala Ile Thr Trp Ser Ile Phe Leu Ile Gly Ser His Pro 340 345 350 Glu Val Gln Glu Met Val Asn Glu Glu Leu Asp Arg Val Phe Gly Asp 355 360 365 Ser Asp Arg Pro Ala Thr Met Ala Asp Leu Ser Glu Leu Lys Tyr Leu 370 375 380 Glu Cys Cys Val Lys Glu Ala Leu Arg Leu Tyr Pro Ser Val Pro Ile 385 390 395 400 Ile Ser Arg Thr Cys Val Glu Asp Thr Val Ile Gly Gly Asp Glu Ile 405 410 415 Pro Ala Gly Thr Ser Val Ser Ile Cys Ser Tyr Tyr Leu His Arg Asp 420 425 430 Pro Lys Tyr Phe Pro Asp Pro Glu Leu Tyr Gln Pro Lys Arg Phe Leu 435 440 445 Ala Glu His Ala Glu Arg Arg His Pro Tyr Ser Tyr Val Pro Phe Ser 450 455 460 Ala Gly Pro Arg Asn Cys Ile Gly Gln Arg Phe Ala Leu Met Glu Glu 465 470 475 480 Lys Ala Val Leu Ser Ala Ile Leu Arg Asn Phe His Val Gln Ser Leu 485 490 495 Asp Lys Arg Glu Glu Ile Ile Leu Leu Ala Glu Leu Ile Leu Arg Pro 500 505 510 Arg Asp Gly Ile Arg Val Arg Leu Glu Pro Lys Lys Lys Gln 515 520 525 39520PRTNilaparvata lugens 39Met Ala Lys Thr Ala Asn Gly Ser Ile Lys Met Asp Tyr Thr Thr Thr 1 5 10 15 Ile Leu Ser Leu Val Leu Phe Ile Phe Ser Ala Leu Tyr Leu Leu Arg 20 25 30 Gln Ala Phe Arg Arg Ile Lys Ile Ile Asn Met Val Asp Gln Leu Pro 35 40 45 Gly Pro Arg Ala Tyr Pro Ile Ile Gly Asn Ala Leu Asp Phe Met Val 50 55 60 Pro Arg Ser Glu Leu Met Asn Val Phe Asp Ser Arg Thr Lys Lys Tyr 65 70 75 80 Gly Pro Leu Phe Arg Thr Trp Ala Gly Pro Val Pro Gln Ile His Ile 85 90 95 Thr Arg Pro Glu His Met Glu Ile Val Met Ser Ser Leu Lys His Ile 100 105 110 Asp Lys Ser Lys Ala Tyr Thr Phe Leu Gln Pro Gly Leu Gly Thr Gly 115 120 125 Leu Leu Thr Gly Thr Gly Ala Lys Trp His Ser His Arg Lys Met Ile 130 135 140 Thr Pro Thr Leu His Phe Lys Ile Leu Asp Val Phe Val Glu Val Phe 145 150 155 160 Gly Glu Lys Cys Gln Thr Leu Ile Glu Asn Leu Leu Lys Lys Ala Asp 165 170 175 Gly Gln Glu Phe Asp Ile Tyr Pro Phe Ile Thr His Cys Ala Leu Asp 180 185 190 Ile Ile Cys Glu Thr Ala Met Gly Thr Gln Ile Asn Ala Gln Asn Glu 195 200 205 Ser Asp Ser Asp Tyr Val Arg Ala Ile Tyr Asp Ile Ser Glu Leu Thr 210 215 220 Thr Glu Arg Thr Thr Lys Pro Trp Leu His Ser Asp Leu Ile Trp Lys 225 230 235 240 Ser Ser Lys Arg Gly Ala Arg Tyr Ala His Asp Leu Ser Ile Leu His 245 250 255 Gly Phe Thr Asn Arg Val Ile Ser Glu Arg Lys Val Ala Arg Leu Ala 260 265 270 Asp Lys Glu Arg Ile Lys Asn His Glu Asp Asp Asp Glu Phe Leu Gly 275 280 285 Lys Lys Lys Arg Met Ala Phe Leu Asp Leu Leu Leu Glu Ala Ser Glu 290 295 300 Leu Gly Gln Lys Leu Thr Asp Asp Glu Ile Arg Glu Glu Val Asp Thr 305 310 315 320 Phe Met Phe Glu Gly His Asp Thr Thr Thr Ala Gly Ile Cys Trp Ser 325 330 335 Leu Phe Met Leu Gly Asn His Pro Glu Tyr Gln Asp Gln Val Ala Gln 340 345 350 Glu Leu Asp Gln Ile Phe Gly Asp Ser Asn Leu Pro Pro Thr Met Lys 355 360 365 Asp Leu Asn Glu Met Lys Tyr Leu Glu Arg Val Ile Lys Glu Ser Leu 370 375 380 Arg Leu Phe Pro Ser Val Pro Phe Ile Gly Arg Tyr Leu Gly Glu Asp 385 390 395 400 Thr Lys Phe Asp Asn Tyr Ile Val Pro Ala Gly Cys Val Met Asn Leu 405 410 415 Gln Ile Phe His Val His Arg Cys Pro Asp Gln Phe Pro Asp Pro Glu 420 425 430 Lys Phe Asn Pro Asp Asn Phe Leu Pro Glu Arg Thr Gln Gly Arg His 435 440 445 Pro Tyr Ala Tyr Ile Pro Phe Ser Ala Gly Pro Arg Asn Cys Ile Gly 450 455 460 Gln Lys Phe Ala Val Leu Glu Glu Lys Thr Val Leu Ser Ser Ile Leu 465 470 475 480 Arg Asn Tyr Arg Val Glu Ser Val Glu Lys Leu Glu Asp Leu Asn Leu 485 490 495 Met Asn Glu Leu Ile Leu Arg Pro Glu Ser Gly Ile Arg Met Arg Ile 500 505 510 Tyr Pro Arg Lys Lys Thr Gln Ser 515 520 40510PRTDanio rerio 40Met Gly Ile Leu Phe Gly Leu Tyr Ile Leu Gly Ile Leu Phe Thr Ala 1 5 10 15 Val Leu Leu Leu Leu Leu Ala Ser Thr Ala Tyr Asn Pro Leu Lys Asn 20 25 30 Tyr Ile Gly Lys Trp Asn Glu Met Arg Pro Ile Pro Gly Met Ala Gly 35 40 45 Ala Tyr Pro Ile Ile Gly Asn Ala Leu Gln Phe Lys Thr Asn Ala Gly 50 55 60 Asp Phe Phe Asn Gln Ile Ile Glu Gly Thr Asn Glu Asn Arg His Leu 65 70 75 80 Pro Leu Ala Lys Val Trp Val Gly Pro Val Pro Phe Leu Ile Leu Tyr 85 90 95 His Ala Glu Asn Ile Glu Val Val Leu Ser Asn Ser Arg His Leu Asp 100 105 110 Lys Ser Tyr Ser Tyr Arg Phe Leu His Pro Trp Leu Gly Thr Gly Leu 115 120 125 Leu Thr Ser Thr Gly Glu Lys Trp Arg Asn Arg Arg Lys Met Leu Thr 130 135 140 Pro Thr Phe His Phe Ser Ile Leu Ser Asp Phe Leu Glu Val Met Asn 145 150 155 160 Glu Gln Thr Asp Ile Leu Ile Gln Lys Met Gln Lys Leu Glu Asp Gly 165 170 175 Glu Pro Phe Asn Cys Phe Asn Phe Ile Thr Leu Cys Ala Leu Asp Ile 180 185 190 Ile Cys Glu Thr Ala Met Gly Lys Lys Ile Tyr Ala Gln Ser Asn Ala 195 200 205 Asp Ser Glu Tyr Val Gln Ser Val Tyr Lys Met Ser Asp Ile Ile Thr 210 215 220 Lys Arg Gln Arg Ala Pro Trp Leu Trp Pro Asp Trp Ile Tyr Asn Lys 225 230 235 240 Leu Lys Glu Gly Lys Glu His Ala Lys Arg Leu Lys Ile Leu His Ser 245 250 255 Phe Thr Ala Asn Val Ile Arg Glu Arg Ala Glu Phe Met Ser Ser Glu 260 265 270 Pro Asp Ser Asp Ser Asp Gln Gly Glu Arg Lys Arg Gln Ala Phe Leu 275 280 285 Asp Met Leu Leu Lys Thr Thr Tyr Glu Asn Gly Gln Lys Leu Ser His 290 295 300 Glu Asp Ile Gln Glu Glu Val Asp Thr Phe Met Phe Glu Gly His Asp 305 310 315 320 Thr Thr Ala Ala Ser Met Asn Trp Ala Leu His Leu Ile Gly Ser His 325 330 335 Pro Glu Val Gln Lys Ala Val Gln Ala Glu Leu Gln Glu Val Phe Gly 340 345 350 Ser Ser Glu Arg His Val Gly Val Glu Asp Leu Lys Lys Leu Arg Tyr 355 360 365 Leu Glu Cys Val Ile Lys Glu Ser Leu Arg Ile Phe Pro Ser Val Pro 370 375 380 Leu Phe Ala Arg Ser Ile Cys Glu Ala Cys His Ile Asn Gly Phe Lys 385 390 395 400 Val Pro Lys Gly Val Asn Ala Val Ile Ile Pro Tyr Ala Leu His Arg 405 410 415 Asp Pro Arg Tyr Phe Pro Glu Pro Glu Glu Phe Gln Pro Glu Arg Phe 420 425 430 Met Pro Glu Asn Ser Lys Gly Arg His Pro Tyr Ala Tyr Ile Pro Phe 435 440 445 Ser Ala Gly Pro Arg Asn Cys Ile Gly Gln Arg Phe Ala Met Met Glu 450 455 460 Glu Lys Val Val Leu Ala Thr Ile Leu Arg His Phe Asp Val Glu Ala 465 470 475 480 Cys Gln Ser Arg Glu Glu Leu Arg Pro Leu Gly Glu Leu Ile Leu Arg 485 490 495 Pro Glu Lys Gly Ile Trp Ile Lys Leu Gln Arg Arg Ser Lys 500 505 510 41518PRTArabidopsis thaliana 41Met Leu Leu Thr Ile Leu Lys Ser Leu Leu Val Ile Phe Val Thr Thr 1 5 10 15 Ile Leu Arg Val Leu Tyr Asp Thr Ile Ser Cys Tyr Trp Leu Thr Pro 20 25 30 Arg Arg Ile Lys Lys Ile Met Glu Gln Gln Gly Val Thr Gly Pro Lys 35 40 45 Pro Arg Pro Leu Thr Gly Asn Ile Leu Glu Ile Ser Ala Met Val Ser 50 55 60 Gln Ser Ala Ser Lys Asp Cys Asp Ser Ile His His Asp Ile Val Gly 65 70 75 80 Arg Leu Leu Pro His Tyr Val Ala Trp Ser Lys Gln Tyr Gly Lys Arg 85 90 95 Phe Ile Val Trp Asn Gly Thr Asp Pro Arg Leu Cys Leu Thr Glu Thr 100 105 110 Glu Leu Ile Lys Glu Leu Leu Met Lys His Asn Gly Val Ser Gly Arg 115 120 125 Ser Trp Leu Gln Gln Gln Gly Thr Lys Asn Phe Ile Gly Arg Gly Leu 130 135 140 Leu Met Ala Asn Gly Gln Asp Trp His His Gln Arg His Leu Ala Ala 145 150 155 160 Pro Ala Phe Thr Gly Glu Arg Leu Lys Gly Tyr Ala Arg His Met Val 165 170 175 Glu Cys Thr Ser Lys Leu Val Glu Arg Leu Arg Lys Glu Val Gly Glu 180 185 190 Gly Ala Asn Glu Val Glu Ile Gly Glu Glu Met His Lys Leu Thr Ala 195 200 205 Asp Ile Ile Ser Arg Thr Lys Phe Gly Ser Ser Phe Glu Lys Gly Lys 210 215 220 Glu Leu Phe Asn His Leu Thr Val Leu Gln Arg Arg Cys Ala Gln Ala 225 230 235 240 Thr Arg His Leu Cys Phe Pro Gly Ser Arg Phe Leu Pro Ser Lys Tyr 245 250 255 Asn Arg Glu Ile Lys Ser Leu Lys Lys Glu Val Glu Arg Leu Leu Ile 260 265 270 Glu Ile Ile Gln Ser Arg Arg Asp Cys Ala Glu Met Gly Arg Ser Ser 275 280 285 Thr His Gly Asp Asp Leu Leu Gly Leu Leu Leu Asn Glu Met Asp Ile 290 295 300 Asp Lys Asn Asn Asn Asn Asn Asn Asn Asn Leu Gln Leu Ile Met Asp 305 310 315 320 Glu Cys Lys Thr Phe Phe Phe Ala Gly His Glu Thr Thr Ala Leu Leu 325 330 335 Leu Thr Trp Thr Thr Met Leu Leu Ala Asp Asn Pro Thr Trp Gln Glu 340 345 350 Lys Val Arg Glu Glu Val Arg Glu Val Phe Gly Arg Asn Gly Leu Pro 355 360 365 Ser Val Asp Gln Leu Ser Lys Leu Thr Ser Leu Ser Lys Val Ile Asn 370 375 380 Glu Ser Leu Arg Leu Tyr Pro Pro Ala Thr Leu Leu Pro Arg Met Ala 385 390 395 400 Phe Glu Asp Leu Lys Leu Gly Asp Leu Thr Ile Pro Lys Gly Leu Ser 405 410 415 Ile Trp Ile Pro Val Leu Ala Ile His His Ser Glu Glu Leu Trp Gly 420 425 430 Lys Asp Ala Asn Gln Phe Asn Pro Glu Arg Phe Gly Gly Arg Pro Phe 435 440 445 Ala Ser Gly Arg His Phe Ile Pro Phe Ala Ala Gly Pro Arg Asn Cys 450 455 460 Ile Gly Gln Gln Phe Ala Leu Met Glu Ala Lys Ile Ile Leu Ala Thr 465 470 475 480 Leu Ile Ser Lys Phe Asn Phe Thr Ile Ser Lys Asn Tyr Arg His Ala 485 490 495 Pro Ile Val Val Leu Thr Ile Lys Pro Lys Tyr Gly Val Gln Val Ile 500 505 510 Leu Lys Pro Leu Val Ser 515 42576PRTArabidopsis thaliana 42Met Ala Phe Pro Ala Ala Ala Thr Tyr Pro Thr His Phe Gln Gly Gly 1 5 10 15 Ala Leu His Leu Gly Arg Thr Asp His Cys Leu Phe Gly Phe Tyr Pro 20 25 30 Gln Thr Ile Ser Ser Val Asn Ser Arg Arg Ala Ser Val Ser Ile Lys 35 40 45 Cys Gln Ser Thr Glu Pro Lys Thr Asn Gly Asn Ile Leu Asp Asn Ala 50 55 60 Ser Asn Leu Leu Thr Asn Phe Leu Ser Gly Gly Ser Leu Gly Ser Met 65 70 75 80 Pro Thr Ala Glu Gly Ser Val Ser Asp Leu Phe Gly Lys Pro Leu Phe 85 90 95 Leu Ser Leu Tyr Asp Trp Phe Leu Glu His Gly Gly Ile Tyr Lys Leu 100 105 110 Ala Phe Gly Pro Lys Ala Phe Val Val Ile Ser Asp Pro Ile Ile Ala 115 120 125 Arg His Val Leu Arg Glu Asn Ala Phe Ser Tyr Asp Lys Gly Val Leu 130 135 140 Ala Glu Ile Leu Glu Pro Ile Met Gly Lys Gly Leu Ile Pro Ala Asp 145 150 155 160 Leu Asp Thr Trp Lys Leu Arg Arg Arg Ala Ile Thr Pro Ala Phe His 165 170 175 Lys Leu Tyr Leu Glu Ala Met Val Lys Val Phe Ser Asp Cys Ser Glu 180 185 190 Lys Met Ile Leu Lys Ser Glu Lys Leu Ile Arg Glu Lys Glu Thr Ser 195 200 205 Ser Gly Glu Asp Thr Ile Glu Leu Asp Leu Glu Ala Glu Phe Ser Ser 210 215 220 Leu Ala Leu Asp Ile Ile Gly Leu Ser Val Phe Asn Tyr Asp Phe Gly 225 230 235 240 Ser Val Thr Lys Glu Ser Pro Val Ile Lys Ala Val Tyr Gly Thr Leu 245 250 255 Phe Glu Ala Glu His Arg Ser Thr Phe Tyr Phe Pro Tyr Trp Asn Phe 260 265 270 Pro Pro Ala Arg Trp Ile Val Pro Arg Gln Arg Lys Phe Gln Ser Asp 275 280 285 Leu Lys Ile Ile Asn Asp Cys Leu Asp Gly Leu Ile Gln

Asn Ala Lys 290 295 300 Glu Thr Arg Gln Glu Thr Asp Val Glu Lys Leu Gln Glu Arg Asp Tyr 305 310 315 320 Thr Asn Leu Lys Asp Ala Ser Leu Leu Arg Phe Leu Val Asp Met Arg 325 330 335 Gly Val Asp Ile Asp Asp Arg Gln Leu Arg Asp Asp Leu Met Thr Met 340 345 350 Leu Ile Ala Gly His Glu Thr Thr Ala Ala Val Leu Thr Trp Ala Val 355 360 365 Phe Leu Leu Ser Gln Asn Pro Glu Lys Ile Arg Lys Ala Gln Ala Glu 370 375 380 Ile Asp Ala Val Leu Gly Gln Gly Pro Pro Thr Tyr Glu Ser Met Lys 385 390 395 400 Lys Leu Glu Tyr Ile Arg Leu Ile Val Val Glu Val Leu Arg Leu Phe 405 410 415 Pro Gln Pro Pro Leu Leu Ile Arg Arg Thr Leu Lys Pro Glu Thr Leu 420 425 430 Pro Gly Gly His Lys Gly Glu Lys Glu Gly His Lys Val Pro Lys Gly 435 440 445 Thr Asp Ile Phe Ile Ser Val Tyr Asn Leu His Arg Ser Pro Tyr Phe 450 455 460 Trp Asp Asn Pro His Asp Phe Glu Pro Glu Arg Phe Leu Arg Thr Lys 465 470 475 480 Glu Ser Asn Gly Ile Glu Gly Trp Ala Gly Phe Asp Pro Ser Arg Ser 485 490 495 Pro Gly Ala Leu Tyr Pro Asn Glu Ile Ile Ala Asp Phe Ala Phe Leu 500 505 510 Pro Phe Gly Gly Gly Pro Arg Lys Cys Ile Gly Asp Gln Phe Ala Leu 515 520 525 Met Glu Ser Thr Val Ala Leu Ala Met Leu Phe Gln Lys Phe Asp Val 530 535 540 Glu Leu Arg Gly Thr Pro Glu Ser Val Glu Leu Val Ser Gly Ala Thr 545 550 555 560 Ile His Ala Lys Asn Gly Met Trp Cys Lys Leu Lys Arg Arg Ser Lys 565 570 575 43534PRTOryza sativa 43Met Ala Ala Ala Val Leu Val Ala Ile Ala Leu Pro Val Ser Leu Ala 1 5 10 15 Leu Leu Leu Val Ala Lys Ala Val Trp Val Thr Val Ser Cys Tyr Tyr 20 25 30 Leu Thr Pro Ala Arg Ile Arg Arg Val Leu Ala Ser Gln Gly Val Arg 35 40 45 Gly Pro Pro Pro Arg Pro Leu Val Gly Asn Leu Arg Asp Val Ser Ala 50 55 60 Leu Val Ala Glu Ser Thr Ala Ala Asp Met Ala Ser Leu Ser His Asp 65 70 75 80 Ile Val Ala Arg Leu Leu Pro His Tyr Val Leu Trp Ser Asn Thr Tyr 85 90 95 Gly Arg Arg Phe Val Tyr Trp Tyr Gly Ser Glu Pro Arg Val Cys Val 100 105 110 Thr Glu Ala Gly Met Val Arg Glu Leu Leu Ser Ser Arg His Ala His 115 120 125 Val Thr Gly Lys Ser Trp Leu Gln Arg Gln Gly Ala Lys His Phe Ile 130 135 140 Gly Arg Gly Leu Leu Met Ala Asn Gly Ala Thr Trp Ser His Gln Arg 145 150 155 160 His Val Val Ala Pro Ala Phe Met Ala Asp Arg Leu Lys Gly Arg Val 165 170 175 Gly His Met Val Glu Cys Thr Arg Gln Thr Val Arg Ala Leu Arg Glu 180 185 190 Ala Val Ala Arg Ser Gly Asn Glu Val Glu Ile Gly Ala His Met Ala 195 200 205 Arg Leu Ala Gly Asp Val Ile Ala Arg Thr Glu Phe Asp Thr Ser Tyr 210 215 220 Glu Thr Gly Lys Arg Ile Phe Leu Leu Ile Glu Glu Leu Gln Arg Leu 225 230 235 240 Thr Ala Arg Ser Ser Arg Tyr Leu Trp Val Pro Gly Ser Gln Tyr Phe 245 250 255 Pro Ser Lys Tyr Arg Arg Glu Ile Lys Arg Leu Asn Gly Glu Leu Glu 260 265 270 Arg Leu Leu Lys Glu Ser Ile Asp Arg Ser Arg Glu Ile Ala Asp Glu 275 280 285 Gly Arg Thr Pro Ser Ala Ser Pro Cys Gly Arg Gly Leu Leu Gly Met 290 295 300 Leu Leu Ala Glu Met Glu Lys Lys Glu Ala Gly Gly Asn Gly Gly Gly 305 310 315 320 Glu Val Gly Tyr Asp Ala Gln Met Met Ile Asp Glu Cys Lys Thr Phe 325 330 335 Phe Phe Ala Gly His Glu Thr Ser Ala Leu Leu Leu Thr Trp Ala Ile 340 345 350 Met Leu Leu Ala Thr His Pro Ala Trp Gln Asp Lys Ala Arg Ala Glu 355 360 365 Val Ala Ala Val Cys Gly Gly Gly Ala Pro Ser Pro Asp Ser Leu Pro 370 375 380 Lys Leu Ala Val Leu Gln Met Val Ile Asn Glu Thr Leu Arg Leu Tyr 385 390 395 400 Pro Pro Ala Thr Leu Leu Pro Arg Met Ala Phe Glu Asp Ile Glu Leu 405 410 415 Gly Gly Gly Ala Leu Arg Val Pro Ser Gly Ala Ser Val Trp Ile Pro 420 425 430 Val Leu Ala Ile His His Asp Glu Gly Ala Trp Gly Arg Asp Ala His 435 440 445 Glu Phe Arg Pro Asp Arg Phe Ala Pro Gly Arg Pro Arg Pro Pro Ala 450 455 460 Gly Ala Phe Leu Pro Phe Ala Ala Gly Pro Arg Asn Cys Val Gly Gln 465 470 475 480 Ala Tyr Ala Met Val Glu Ala Lys Val Ala Leu Ala Met Leu Leu Ser 485 490 495 Ser Phe Arg Phe Ala Ile Ser Asp Glu Tyr Arg His Ala Pro Val Asn 500 505 510 Val Leu Thr Leu Arg Pro Arg His Gly Val Pro Val Arg Leu Leu Pro 515 520 525 Leu Pro Pro Pro Arg Pro 530 44519PRTOryza sativa 44Met Ala Val Gly Leu Leu Val Val Ala Tyr Leu Tyr Glu Pro Tyr Arg 1 5 10 15 Lys Val Trp His Val Pro Gly Pro Val Pro Val Pro Leu Ile Gly His 20 25 30 Leu His Leu Leu Ala Met His Gly Pro Asp Val Phe Ser Val Leu Ala 35 40 45 Arg Lys His Gly Pro Val Phe Arg Phe His Met Gly Arg Gln Pro Leu 50 55 60 Ile Ile Val Ala Asp Ala Glu Leu Cys Lys Glu Val Gly Val Lys Lys 65 70 75 80 Phe Lys Ser Ile Pro Asn Arg Ser Met Pro Ser Pro Ile Ala Asn Ser 85 90 95 Pro Ile His Lys Lys Gly Leu Phe Phe Ile Arg Gly Pro Arg Trp Thr 100 105 110 Ser Met Arg Asn Met Ile Ile Ser Ile Tyr Gln Pro Ser His Leu Ala 115 120 125 Ser Leu Ile Pro Thr Met Glu Ser Cys Ile Gln Arg Ala Ser Lys Asn 130 135 140 Leu Asp Gly Gln Lys Glu Ile Thr Phe Ser Asp Leu Ser Leu Ser Leu 145 150 155 160 Ala Thr Asp Val Ile Gly Leu Ala Ala Phe Gly Thr Asp Phe Gly Leu 165 170 175 Ser Lys Val Pro Val Thr Pro Asp Asp Ser Asn Ile Asp Lys Ile Ala 180 185 190 Ala Asp Thr Ser Val Glu Ala Lys Ala Ser Ser Glu Phe Ile Lys Met 195 200 205 His Met His Ala Thr Thr Ser Leu Lys Met Asp Leu Ser Gly Ser Leu 210 215 220 Ser Ile Leu Val Gly Met Leu Leu Pro Phe Leu Gln Glu Pro Phe Arg 225 230 235 240 Gln Val Leu Lys Arg Ile Pro Gly Met Gly Asp Tyr Lys Ile Asp Arg 245 250 255 Val Asn Arg Ala Leu Lys Thr His Met Asp Ser Ile Val Ala Glu Arg 260 265 270 Glu Ala Ala Met Glu His Asp Leu Ala Ala Ser Gln Gln Arg Lys Asp 275 280 285 Phe Leu Ser Val Val Leu Thr Ala Arg Glu Ser Asn Lys Ser Ser Arg 290 295 300 Glu Leu Leu Thr Pro Asp Tyr Ile Ser Ala Leu Thr Tyr Glu His Leu 305 310 315 320 Leu Ala Gly Ser Thr Thr Thr Ala Phe Thr Leu Ser Thr Val Leu Tyr 325 330 335 Leu Val Ala Lys His Pro Glu Val Glu Glu Lys Leu Leu Lys Glu Ile 340 345 350 Asp Ala Phe Gly Pro Arg Asp Arg Val Pro Met Ala Asp Asp Leu Gln 355 360 365 Thr Lys Phe Pro Tyr Leu Asp Gln Val Val Lys Glu Ser Met Arg Phe 370 375 380 Tyr Met Met Ser Pro Leu Leu Ala Arg Glu Thr Leu Glu Gln Val Glu 385 390 395 400 Ile Gly Gly Tyr Val Leu Pro Lys Gly Thr Trp Val Trp Leu Ala Pro 405 410 415 Gly Val Leu Ala Lys Asp Pro Lys Asn Phe Pro Glu Pro Glu Ile Phe 420 425 430 Arg Pro Glu Arg Phe Asp Pro Asn Gly Glu Glu Glu Arg Arg Arg His 435 440 445 Leu Tyr Ala Phe Ile Pro Phe Gly Ile Gly Pro Arg Val Cys Ile Gly 450 455 460 Gln Lys Phe Ser Ile Gln Glu Ile Lys Leu Ser Val Ile His Leu Tyr 465 470 475 480 Arg His Tyr Val Phe Arg His Ser Pro Ser Met Glu Ser Pro Leu Glu 485 490 495 Phe Gln Phe Ala Ile Ile Cys Asp Phe Lys Tyr Gly Val Lys Leu Gln 500 505 510 Ala Ile Lys Arg His His Ala 515 45571PRTOryza sativa 45Met Ala Ile Thr Ala Ala Thr Ala Ala Ala Ala Ala Thr Pro His Pro 1 5 10 15 Trp Gln Ala Asp Ala Ser Pro Arg Arg His Ala Ala Cys Pro Ala Leu 20 25 30 Arg Gly Arg Arg Arg Leu Pro Val Val Arg Cys Gln Ser Ser Ser Val 35 40 45 Asp Asp Lys Pro Lys Ser Lys Arg Gly Leu Leu Asp Asn Ala Ser Asn 50 55 60 Leu Leu Thr Asn Leu Leu Ser Gly Gly Ser Leu Gly Ala Met Pro Val 65 70 75 80 Ala Glu Gly Ala Val Thr Asp Leu Phe Gly Arg Pro Leu Phe Phe Ser 85 90 95 Leu Tyr Asp Trp Phe Leu Glu His Gly Ser Val Tyr Lys Leu Ala Phe 100 105 110 Gly Pro Lys Ala Phe Val Val Val Ser Asp Pro Ile Val Ala Arg His 115 120 125 Ile Leu Arg Glu Asn Ala Phe Cys Tyr Asp Lys Gly Val Leu Ala Glu 130 135 140 Ile Leu Lys Pro Ile Met Gly Lys Gly Leu Ile Pro Ala Asp Leu Asp 145 150 155 160 Thr Trp Lys Gln Arg Arg Lys Val Ile Thr Pro Gly Phe His Ala Leu 165 170 175 Phe Ile Glu Ala Met Val Gly Val Phe Thr Lys Cys Ser Glu Arg Thr 180 185 190 Ile Phe Lys Leu Glu Glu Leu Ile Glu Arg Gly Glu His Gly Glu Lys 195 200 205 Tyr Thr Ile Val Asp Leu Glu Ala Glu Phe Ser Asn Leu Ala Leu Asp 210 215 220 Ile Ile Gly Leu Gly Val Phe Asn Phe Asp Phe Asp Ser Val Thr Lys 225 230 235 240 Glu Ser Pro Val Ile Lys Ala Val Tyr Gly Thr Leu Phe Glu Ala Glu 245 250 255 His Arg Ser Thr Phe Tyr Ile Pro Tyr Trp Asn Leu Pro Leu Thr Arg 260 265 270 Trp Ile Val Pro Arg Gln Arg Lys Phe His Ser Asp Leu Lys Val Ile 275 280 285 Asn Asp Cys Leu Asp Ser Leu Ile Lys Asn Ala Lys Glu Thr Arg Gln 290 295 300 Glu Ala Asp Val Glu Lys Leu Gln Gln Arg Asp Tyr Ser Ser Leu Lys 305 310 315 320 Asp Ala Ser Leu Leu Arg Phe Leu Val Asp Met Arg Gly Ala Asp Val 325 330 335 Asp Asp Arg Gln Leu Arg Asp Asp Leu Met Thr Met Leu Ile Ala Gly 340 345 350 His Glu Thr Thr Ala Ala Val Leu Thr Trp Ser Val Phe Leu Leu Ala 355 360 365 Gln Asn Pro Ser Lys Met Arg Lys Ala Gln Ala Glu Val Asp Ser Val 370 375 380 Leu Ser Asn Glu Thr Ile Asn Val Asp Gln Leu Lys Lys Leu Glu Tyr 385 390 395 400 Ile Arg Leu Ile Ile Val Glu Ala Leu Arg Leu Tyr Pro Gln Pro Pro 405 410 415 Leu Leu Ile Arg Arg Ala Leu Arg Pro Asp Lys Leu Pro Gly Gly Tyr 420 425 430 Asn Gly Ala Lys Glu Gly Tyr Glu Ile Pro Ala Gly Thr Asp Ile Phe 435 440 445 Leu Ser Ile Tyr Asn Leu His Arg Ser Pro Tyr Phe Trp Asp Arg Pro 450 455 460 Asp Glu Phe Glu Pro Glu Arg Phe Ser Val Pro Lys Lys Asp Glu Ser 465 470 475 480 Ile Glu Gly Trp Ala Gly Phe Asp Pro Asp Arg Ser Pro Gly Ala Met 485 490 495 Tyr Pro Asn Glu Ile Leu Ala Asp Phe Ala Phe Leu Pro Phe Gly Gly 500 505 510 Gly Pro Arg Lys Cys Val Gly Asp Gln Phe Ala Leu Leu Glu Ser Thr 515 520 525 Val Ala Leu Ala Leu Leu Leu Gln Lys Phe Asp Val Glu Leu Arg Gly 530 535 540 Ser Pro Asp Glu Val Glu Met Val Thr Gly Ala Thr Ile His Thr Lys 545 550 555 560 Ser Gly Leu Trp Cys Arg Val Arg Arg Arg Thr 565 570 462108DNADrosophila melanogaster 46cttcggataa gctgttcacg cgattgagag gatccgttcg gtgaaaaaaa caaaaattta 60aacaaagcaa atcaaatcga aaagaattat aaattaaatt aaagaaaaaa aaataataga 120ataaaaatga cagcggatac attggtgctg gaaacaatgg atagtgcaaa aaattcaact 180gcgggacccg caaccgtatt gaacccaatt tggactgctc ttttgggtat tgcagtggtt 240gtgagtttgt acgaaatatg gctaaggaat accaggaaat ataaattaac ggcaaatatg 300ccaaacccac ctatgctgcc actcatcgga aatggccatt tggtggccca tttgacaaat 360gccgaaatcc ttgcccgtgg cattggttac atgcaaacct atggtggtgc catgcgtggc 420tttttgggtc ccatgttggt tgtgttcctc tggaacgctc ccgatattga attgattctc 480agtacccaca cccatttgga gaagtcaatt gaatatcgtt tcttcaaacc ctggtttggt 540gatggtctac ttatttcgaa tggtcaccat tggcaacatc atcgcaaaat gattgctcca 600acattccatc aaagcatttt gaagagtttc gtgccagcct ttgtgcaaca ctccaagaag 660gtggtggaac gtatggccaa ggaattgggc aaggaattcg atgtccatga ctatatgtca 720cagaccactg tggaaatttt gctctccact gccatgggtg ttaagaaggt gcccgaggac 780aacaagagtt tggaatatgc caaggctgtg gtggacatgt gcgacatcat tcacaagcgc 840caattgaaat tcttctatcg catggatgcc ctctacaatt tgagcagcat gagtgagaag 900ggcaagaaga tgatggacat tattttgggc atgacccgta aagtggtgac ggaacgtcaa 960cagaatttca atgccgaatc gcgtgccatt gtcgaggagg atgatgaaat tagcaagcag 1020aagcaacagg ccaagaagaa ggagggtttg cgtgatgatt tggatgacat tgatgaaaat 1080gatgtgggtg ccaagaaacg tttggctctg ctggatgcca tgatggccat gtccaagaac 1140cccgatgttg agtggaccga caaggatgtc atggatgaag tcaacaccat tatgtttgag 1200ggccacgata ccacctcagc tggttccagt tttgtcctct gcatgttggg catctacaag 1260gatatccaag agaaagtcct ggccgaacaa aaggccatct ttggtgacaa cttcctgcgc 1320gactgtacct ttgccgatac catggaaatg aaatacctgg aacgtgtgat tatggagact 1380ttgcgtttgt atccaccagt accccttatt gcccgtcgtg ctgagttcga tgtaaaattg 1440gcttccggtc cctacacaat tcccaagggc acaacagtgg tgattgccca atttgctgtg 1500catcgcaatc cccaatactt ccccaatccc gagaaattcg atcctgacaa tttcctaccc 1560gaacgtatgg ccaatcgtca ttactacagt ttcattccct tcagtgccgg ccccagaagt 1620tgcgttggcc gcaaatatgc catgctgaaa ttaaaggtcc tgctctccac cattattcgt 1680aactattcgg tgcagagcaa ccaacaggag aaggacttta agttgcaggc cgatattatc 1740ctaaagattg aaaatggttt caatatcatg ttgaaccgac gacccgaagc catgaaagct 1800atgtaaagta aagtagagat gagtgtgtaa agaagttttt tgtcccaact atcctaagaa 1860tgacccgtat ccttgtatgt ggtttccatt ttattgataa gtaaaagaag aaagaaacta 1920accccccgca ttataccaaa taataagaaa taacgaaact actaaccgac gtgagagtgt 1980taagttttat cctattgtta ttgactaaaa tgtatgttaa gatttttttt ttataaattt 2040tattgttttt gaagaaaaca caaaaatttt aaaaaaaaat aataaagagc atgccagttt 2100aaaactgt 2108471680DNAMusca domestica 47atgacagcgg atacattggt gctggaaaca atggatagtg caaaaaattc aactgcggga 60cccgcaaccg tattgaaccc aatttggact gctcttttgg gtattgcagt ggttgtgagt 120ttgtacgaaa tatggctaag gaataccagg aaatataaat taacggcaaa tatgccaaac 180ccacctatgc tgccactcat cggaaatggc catttggtgg cccatttgac aaatgccgaa 240atccttgccc gtggcattgg ttacatgcaa acctatggtg gtgccatgcg tggctttttg 300ggtcccatgt tggttgtgtt cctctggaac gctcccgata ttgaattgat tctcagtacc

360cacacccatt tggagaagtc aattgaatat cgtttcttca aaccctggtt tggtgatggt 420ctacttattt cgaatggtca ccattggcaa catcatcgca aaatgattgc tccaacattc 480catcaaagca ttttgaagag tttcgtgcca gcctttgtgc aacactccaa gaaggtggtg 540gaacgtatgg ccaaggaatt gggcaaggaa ttcgatgtcc atgactatat gtcacagacc 600actgtggaaa ttttgctctc cactgccatg ggtgttaaga aggtgcccga ggacaacaag 660agtttggaat atgccaaggc tgtggtggac atgtgcgaca tcattcacaa gcgccaattg 720aaattcttct atcgcatgga tgccctctac aatttgagca gcatgagtga gaagggcaag 780aagatgatgg acattatttt gggcatgacc cgtaaagtgg tgacggaacg tcaacagaat 840ttcaatgccg aatcgcgtgc cattgtcgag gaggatgatg aaattagcaa gcagaagcaa 900caggccaaga agaaggaggg tttgcgtgat gatttggatg acattgatga aaatgatgtg 960ggtgccaaga aacgtttggc tctgctggat gccatgatgg ccatgtccaa gaaccccgat 1020gttgagtgga ccgacaagga tgtcatggat gaagtcaaca ccattatgtt tgagggccac 1080gataccacct cagctggttc cagttttgtc ctctgcatgt tgggcatcta caaggatatc 1140caagagaaag tcctggccga acaaaaggcc atctttggtg acaacttcct gcgcgactgt 1200acctttgccg ataccatgga aatgaaatac ctggaacgtg tgattatgga gactttgcgt 1260ttgtatccac cagtacccct tattgcccgt cgtgctgagt tcgatgtaaa attggcttcc 1320ggtccctaca caattcccaa gggcacaaca gtggtgattg cccaatttgc tgtgcatcgc 1380aatccccaat acttccccaa tcccgagaaa ttcgatcctg acaatttcct acccgaacgt 1440atggccaatc gtcattacta cagtttcatt cccttcagtg ccggccccag aagttgcgtt 1500ggccgcaaat atgccatgct gaaattaaag gtcctgctct ccaccattat tcgtaactat 1560tcggtgcaga gcaaccaaca ggagaaggac tttaagttgc aggccgatat tatcctaaag 1620attgaaaatg gtttcaatat catgttgaac cgacgacccg aagccatgaa agctatgtaa 1680482285DNAMusca domestica 48acagttgagc actggcggct gatatagcaa cagtgccatc ttcagaagac aaaaaggatt 60tgcaccagag gaccagggat caggagcaaa gaagcaacag caaccatggc agtggaagta 120gttcaggaga cgctgcaaca agcggcgtcc agttcgtcga cgacggtcct gggattcagt 180cctatgttaa ccaccttagt gggcaccctg gtggccatgg cattgtacga gtattggcgc 240aggaatagcc gggaataccg catggttgcc aatataccat ccccaccgga gttgcctatt 300ttgggacagg ctcatgtggc cgccggcttg agcaatgccg agatcctggc cgttggcttg 360ggttacctca acaagtacgg agaaaccatg aaggcctggt tgggcaacgt cctgttggtg 420tttctaacca atcccagtga catcgagttg atcctgagtg ggcaccagca cttgaccaag 480gcggaggagt atcgctactt caagccctgg ttcggtgatg gtctactgat cagcaatgga 540catcattggc gtcatcatcg taagatgatt gcccccacct tccaccagag catcttgaag 600agcttcgtgc ctacatttgt ggatcactca aaggcggtag ttgccaggat gggcttagaa 660gcgggcaaat cctttgatgt tcatgactat atgtcgcaga ccacggttga catcctgttg 720tctaccgcca tgggtgtgaa gaagcttccg gagggtaaca agagtttcga atacgcccaa 780gccgtcgtcg acatgtgtga tatcatacat aagaggcagg ttaaattact gtaccgcctg 840gattccatct acaagtttac taagcttcgc gagaagggcg atcgcatgat gaacatcatc 900ttgggtatga ccagcaaggt ggtcaaggat cgtaaggaga acttccaaga ggagtcacgt 960gcgattgttg aggagatttc tacacctgtt gccagcactc ccgcttccaa gaaggagggt 1020cttcgcgatg atctggatga tatcgatgaa aatgatgtgg gcgccaagag gcgattggct 1080cttctagatg ccatggtgga aatggctaag aaccccgata tcgagtggaa cgagaaggac 1140atcatggatg aggtgaatac aattatgttt gagggccacg ataccacctc ggcgggatct 1200agtttcgccc tctgcatgat gggaatccac aaggacatcc aggctaaagt cttcgccgaa 1260cagaaggcca tcttcgggga taatatgctg agggattgca cctttgccga taccatggag 1320atgaaatatt tggagcgcgt aattttagag actttgaggt tgtacccacc agtaccactt 1380atcgccaggc gtctggacta cgacctgaag ttggccagtg gtccgtacac ggttcccaag 1440ggcactacgg tcatcgtgct gcagtactgc gtgcacagac gtccagacat ctaccccaat 1500cccaccaaat tcgatccgga caacttccta cccgagagga tggccaacag gcattactac 1560tccttcattc cctttagcgc tggacccaga agctgtgtgg gccgcaagta cgccatgctg 1620aagctaaagg tcctgctatc caccatcgtg aggaactata ttgtccactc caccgacacg 1680gaggcagatt tcaagctgca ggctgacatc atcctaaagc ttgagaatgg attcaatgtc 1740tcgttggaga agcgtcagta cgccacggtg gcctagaatc cagaaatcta ggaccccgac 1800tacacacacg caaccccgaa cccgaaaccg gaatccagcc ctgtatatag atgatgaata 1860ccgatgaata tcccaaaccg aaaacttgat gacgaactta taaatctaaa acaccgaata 1920agaacccaac gcacaagcca gccagagagt caattaattt ttctttcgtt ttttaactcg 1980ttacttttat atttgattaa tacctttttg tttgttggtc tttagcgagt ggtgccccta 2040tataatgtat acgtatatac tatatatcct tttaaccaac tattcaacgc aactgtttgt 2100gctcttcacc tttttagtac tcctactttt accactatct atactttttt ttcgtagcca 2160tgtagtgtga ttttttttct ttattctagt atttattaag tcaaatggtt taaacgaaac 2220ccaaaaaata tgaaaaatac acgtatgcga ggcacgtagc cgatagagct gcaaaacaat 2280tgtaa 228549503PRTHomo sapiens 49Met Ala Leu Ile Pro Asp Leu Ala Met Glu Thr Trp Leu Leu Leu Ala 1 5 10 15 Val Ser Leu Val Leu Leu Tyr Leu Tyr Gly Thr His Ser His Gly Leu 20 25 30 Phe Lys Lys Leu Gly Ile Pro Gly Pro Thr Pro Leu Pro Phe Leu Gly 35 40 45 Asn Ile Leu Ser Tyr His Lys Gly Phe Cys Met Phe Asp Met Glu Cys 50 55 60 His Lys Lys Tyr Gly Lys Val Trp Gly Phe Tyr Asp Gly Gln Gln Pro 65 70 75 80 Val Leu Ala Ile Thr Asp Pro Asp Met Ile Lys Thr Val Leu Val Lys 85 90 95 Glu Cys Tyr Ser Val Phe Thr Asn Arg Arg Pro Phe Gly Pro Val Gly 100 105 110 Phe Met Lys Ser Ala Ile Ser Ile Ala Glu Asp Glu Glu Trp Lys Arg 115 120 125 Leu Arg Ser Leu Leu Ser Pro Thr Phe Thr Ser Gly Lys Leu Lys Glu 130 135 140 Met Val Pro Ile Ile Ala Gln Tyr Gly Asp Val Leu Val Arg Asn Leu 145 150 155 160 Arg Arg Glu Ala Glu Thr Gly Lys Pro Val Thr Leu Lys Asp Val Phe 165 170 175 Gly Ala Tyr Ser Met Asp Val Ile Thr Ser Thr Ser Phe Gly Val Asn 180 185 190 Ile Asp Ser Leu Asn Asn Pro Gln Asp Pro Phe Val Glu Asn Thr Lys 195 200 205 Lys Leu Leu Arg Phe Asp Phe Leu Asp Pro Phe Phe Leu Ser Ile Thr 210 215 220 Val Phe Pro Phe Leu Ile Pro Ile Leu Glu Val Leu Asn Ile Cys Val 225 230 235 240 Phe Pro Arg Glu Val Thr Asn Phe Leu Arg Lys Ser Val Lys Arg Met 245 250 255 Lys Glu Ser Arg Leu Glu Asp Thr Gln Lys His Arg Val Asp Phe Leu 260 265 270 Gln Leu Met Ile Asp Ser Gln Asn Ser Lys Glu Thr Glu Ser His Lys 275 280 285 Ala Leu Ser Asp Leu Glu Leu Val Ala Gln Ser Ile Ile Phe Ile Phe 290 295 300 Ala Gly Tyr Glu Thr Thr Ser Ser Val Leu Ser Phe Ile Met Tyr Glu 305 310 315 320 Leu Ala Thr His Pro Asp Val Gln Gln Lys Leu Gln Glu Glu Ile Asp 325 330 335 Ala Val Leu Pro Asn Lys Ala Pro Pro Thr Tyr Asp Thr Val Leu Gln 340 345 350 Met Glu Tyr Leu Asp Met Val Val Asn Glu Thr Leu Arg Leu Phe Pro 355 360 365 Ile Ala Met Arg Leu Glu Arg Val Cys Lys Lys Asp Val Glu Ile Asn 370 375 380 Gly Met Phe Ile Pro Lys Gly Val Val Val Met Ile Pro Ser Tyr Ala 385 390 395 400 Leu His Arg Asp Pro Lys Tyr Trp Thr Glu Pro Glu Lys Phe Leu Pro 405 410 415 Glu Arg Phe Ser Lys Lys Asn Lys Asp Asn Ile Asp Pro Tyr Ile Tyr 420 425 430 Thr Pro Phe Gly Ser Gly Pro Arg Asn Cys Ile Gly Met Arg Phe Ala 435 440 445 Leu Met Asn Met Lys Leu Ala Leu Ile Arg Val Leu Gln Asn Phe Ser 450 455 460 Phe Lys Pro Cys Lys Glu Thr Gln Ile Pro Leu Lys Leu Ser Leu Gly 465 470 475 480 Gly Leu Leu Gln Pro Glu Lys Pro Val Val Leu Lys Val Glu Ser Arg 485 490 495 Asp Gly Thr Val Ser Gly Ala 500 50451PRTMycobacterium tuberculosis 50Met Ser Ala Val Ala Leu Pro Arg Val Ser Gly Gly His Asp Glu His 1 5 10 15 Gly His Leu Glu Glu Phe Arg Thr Asp Pro Ile Gly Leu Met Gln Arg 20 25 30 Val Arg Asp Glu Cys Gly Asp Val Gly Thr Phe Gln Leu Ala Gly Lys 35 40 45 Gln Val Val Leu Leu Ser Gly Ser His Ala Asn Glu Phe Phe Phe Arg 50 55 60 Ala Gly Asp Asp Asp Leu Asp Gln Ala Lys Ala Tyr Pro Phe Met Thr 65 70 75 80 Pro Ile Phe Gly Glu Gly Val Val Phe Asp Ala Ser Pro Glu Arg Arg 85 90 95 Lys Glu Met Leu His Asn Ala Ala Leu Arg Gly Glu Gln Met Lys Gly 100 105 110 His Ala Ala Thr Ile Glu Asp Gln Val Arg Arg Met Ile Ala Asp Trp 115 120 125 Gly Glu Ala Gly Glu Ile Asp Leu Leu Asp Phe Phe Ala Glu Leu Thr 130 135 140 Ile Tyr Thr Ser Ser Ala Cys Leu Ile Gly Lys Lys Phe Arg Asp Gln 145 150 155 160 Leu Asp Gly Arg Phe Ala Lys Leu Tyr His Glu Leu Glu Arg Gly Thr 165 170 175 Asp Pro Leu Ala Tyr Val Asp Pro Tyr Leu Pro Ile Glu Ser Phe Arg 180 185 190 Arg Arg Asp Glu Ala Arg Asn Gly Leu Val Ala Leu Val Ala Asp Ile 195 200 205 Met Asn Gly Arg Ile Ala Asn Pro Pro Thr Asp Lys Ser Asp Arg Asp 210 215 220 Met Leu Asp Val Leu Ile Ala Val Lys Ala Glu Thr Gly Thr Pro Arg 225 230 235 240 Phe Ser Ala Asp Glu Ile Thr Gly Met Phe Ile Ser Met Met Phe Ala 245 250 255 Gly His His Thr Ser Ser Gly Thr Ala Ser Trp Thr Leu Ile Glu Leu 260 265 270 Met Arg His Arg Asp Ala Tyr Ala Ala Val Ile Asp Glu Leu Asp Glu 275 280 285 Leu Tyr Gly Asp Gly Arg Ser Val Ser Phe His Ala Leu Arg Gln Ile 290 295 300 Pro Gln Leu Glu Asn Val Leu Lys Glu Thr Leu Arg Leu His Pro Pro 305 310 315 320 Leu Ile Ile Leu Met Arg Val Ala Lys Gly Glu Phe Glu Val Gln Gly 325 330 335 His Arg Ile His Glu Gly Asp Leu Val Ala Ala Ser Pro Ala Ile Ser 340 345 350 Asn Arg Ile Pro Glu Asp Phe Pro Asp Pro His Asp Phe Val Pro Ala 355 360 365 Arg Tyr Glu Gln Pro Arg Gln Glu Asp Leu Leu Asn Arg Trp Thr Trp 370 375 380 Ile Pro Phe Gly Ala Gly Arg His Arg Cys Val Gly Ala Ala Phe Ala 385 390 395 400 Ile Met Gln Ile Lys Ala Ile Phe Ser Val Leu Leu Arg Glu Tyr Glu 405 410 415 Phe Glu Met Ala Gln Pro Pro Glu Ser Tyr Arg Asn Asp His Ser Lys 420 425 430 Met Val Val Gln Leu Ala Gln Pro Ala Cys Val Arg Tyr Arg Arg Arg 435 440 445 Thr Gly Val 450 511948DNAArtificial Sequencenucleic acid sequence of recombinant variant of cytochrome p450 (chimera 9T2/4G2) 51atgttggtcg agttggtatt ggtcgccatt ttggcgttgt tgttttacta ccagttcgtg 60agaccgctag gtaccagttt gtacgaaata tggctaagga ataccaggaa atataaatta 120acggcaaata tgccaaaccc acctatgctg ccactcatcg gaaatggcca tttggtggcc 180catttgacaa atgccgaaat ccttgcccgt ggcattggtt acatgcaaac ctatggtggt 240gccatgcgtg gctttttggg tcccatgttg gttgtgttcc tctggaacgc tcccgatatt 300gaattgattc tcagtaccca cacccatttg gagaagtcaa ttgaatatcg tttcttcaaa 360ccctggtttg gtgatggtct acttatttcg aatggtcacc attggcaaca tcatcgcaaa 420atgattgctc caacattcca tcaaagcatt ttgaagagtt tcgtgccagc ctttgtgcaa 480cactccaaga aggtggtgga acgtatggcc aaggaattgg gcaaggaatt cgatgtccat 540gactatatgt cacagaccac tgtggaaatt ttgctctcca ctgccatggg tgttaagaag 600gtgcccgagg acaacaagag tttggaatat gccaaggctg tggtggacat gtgcgacatc 660attcacaagc gccaattgaa attcttctat cgcatggatg ccctctacaa tttgagcagc 720atgagtgaga agggcaagaa gatgatggac attattttgg gcatgacccg taaagtggtg 780acggaacgtc aacagaattt caatgccgaa tcgcgtgcca ttgtcgagga ggatgatgaa 840attagcaagc agaagcaaca ggccaagaag aaggagggtt tgcgtgatga tttggatgac 900attgatgaaa atgatgtggg tgccaagaaa cgtttggctc tgctggatgc catgatggcc 960atgtccaaga accccgatgt tgagtggacc gacaaggatg tcatggatga agtcaacacc 1020attatgtttg agggccacga taccacctca gctggttcca gttttgtcct ctgcatgttg 1080ggcatctaca aggatatcca agagaaagtc ctggccgaac aaaaggccat ctttggtgac 1140aacttcctgc gcgactgtac ctttgccgat accatggaaa tgaaatacct ggaacgtgtg 1200attatggaga ctttgcgttt gtatccacca gtacccctta ttgcccgtcg tgctgagttc 1260gatgtaaaat tggcttccgg tccctacaca attcccaagg gcacaacagt ggtgattgcc 1320caatttgctg tgcatcgcaa tccccaatac ttccccaatc ccgagaaatt cgatcctgac 1380aatttcctac ccgaacgtat ggccaatcgt cattactaca gtttcattcc cttcagtgcc 1440ggccccagaa gttgcgttgg ccgcaaatat gccatgctga aattaaaggt cctgctctcc 1500accattattc gtaactattc ggtgcagagc aaccaacagg agaaggactt taagttgcag 1560gccgatatta tcctaaagat tgaaaatggt ttcaatatca tgttgaaccg acgacccgaa 1620gccatgaaag ctatgtaaag taaagtagag atgagtgtgt aaagaagttt tttgtcccaa 1680ctatcctaag aatgacccgt atccttgtat gtggtttcca ttttattgat aagtaaaaga 1740agaaagaaac taaccccccg cattatacca aataataaga aataacgaaa ctactaaccg 1800acgtgagagt gttaagtttt atcctattgt tattgactaa aatgtatgtt aagatttttt 1860ttttataaat tttattgttt ttgaagaaaa cacaaaaatt ttaaaaaaaa ataataaaga 1920gcatgccagt ttaaaactgt aaaaaaaa 194852545PRTArtificial Sequenceamino acid sequence of recombinant variant of cytochrome p450 (chimera 9T2/4G2) 52Met Leu Val Glu Leu Val Leu Val Ala Ile Leu Ala Leu Leu Phe Tyr 1 5 10 15 Tyr Gln Phe Val Arg Pro Leu Gly Thr Ser Leu Tyr Glu Ile Trp Leu 20 25 30 Arg Asn Thr Arg Lys Tyr Lys Leu Thr Ala Asn Met Pro Asn Pro Pro 35 40 45 Met Leu Pro Leu Ile Gly Asn Gly His Leu Val Ala His Leu Thr Asn 50 55 60 Ala Glu Ile Leu Ala Arg Gly Ile Gly Tyr Met Gln Thr Tyr Gly Gly 65 70 75 80 Ala Met Arg Gly Phe Leu Gly Pro Met Leu Val Val Phe Leu Trp Asn 85 90 95 Ala Pro Asp Ile Glu Leu Ile Leu Ser Thr His Thr His Leu Glu Lys 100 105 110 Ser Ile Glu Tyr Arg Phe Phe Lys Pro Trp Phe Gly Asp Gly Leu Leu 115 120 125 Ile Ser Asn Gly His His Trp Gln His His Arg Lys Met Ile Ala Pro 130 135 140 Thr Phe His Gln Ser Ile Leu Lys Ser Phe Val Pro Ala Phe Val Gln 145 150 155 160 His Ser Lys Lys Val Val Glu Arg Met Ala Lys Glu Leu Gly Lys Glu 165 170 175 Phe Asp Val His Asp Tyr Met Ser Gln Thr Thr Val Glu Ile Leu Leu 180 185 190 Ser Thr Ala Met Gly Val Lys Lys Val Pro Glu Asp Asn Lys Ser Leu 195 200 205 Glu Tyr Ala Lys Ala Val Val Asp Met Cys Asp Ile Ile His Lys Arg 210 215 220 Gln Leu Lys Phe Phe Tyr Arg Met Asp Ala Leu Tyr Asn Leu Ser Ser 225 230 235 240 Met Ser Glu Lys Gly Lys Lys Met Met Asp Ile Ile Leu Gly Met Thr 245 250 255 Arg Lys Val Val Thr Glu Arg Gln Gln Asn Phe Asn Ala Glu Ser Arg 260 265 270 Ala Ile Val Glu Glu Asp Asp Glu Ile Ser Lys Gln Lys Gln Gln Ala 275 280 285 Lys Lys Lys Glu Gly Leu Arg Asp Asp Leu Asp Asp Ile Asp Glu Asn 290 295 300 Asp Val Gly Ala Lys Lys Arg Leu Ala Leu Leu Asp Ala Met Met Ala 305 310 315 320 Met Ser Lys Asn Pro Asp Val Glu Trp Thr Asp Lys Asp Val Met Asp 325 330 335 Glu Val Asn Thr Ile Met Phe Glu Gly His Asp Thr Thr Ser Ala Gly 340 345 350 Ser Ser Phe Val Leu Cys Met Leu Gly Ile Tyr Lys Asp Ile Gln Glu 355 360 365 Lys Val Leu Ala Glu Gln Lys Ala Ile Phe Gly Asp Asn Phe Leu Arg 370 375 380 Asp Cys Thr Phe Ala Asp Thr Met Glu Met Lys Tyr Leu Glu Arg Val 385 390 395 400 Ile Met Glu Thr Leu Arg Leu Tyr Pro Pro Val Pro Leu Ile Ala Arg 405 410 415 Arg Ala Glu Phe Asp Val Lys Leu Ala Ser Gly Pro Tyr Thr Ile Pro 420 425 430 Lys Gly Thr Thr Val Val Ile Ala Gln Phe Ala Val His Arg Asn Pro 435 440

445 Gln Tyr Phe Pro Asn Pro Glu Lys Phe Asp Pro Asp Asn Phe Leu Pro 450 455 460 Glu Arg Met Ala Asn Arg His Tyr Tyr Ser Phe Ile Pro Phe Ser Ala 465 470 475 480 Gly Pro Arg Ser Cys Val Gly Arg Lys Tyr Ala Met Leu Lys Leu Lys 485 490 495 Val Leu Leu Ser Thr Ile Ile Arg Asn Tyr Ser Val Gln Ser Asn Gln 500 505 510 Gln Glu Lys Asp Phe Lys Leu Gln Ala Asp Ile Ile Leu Lys Ile Glu 515 520 525 Asn Gly Phe Asn Ile Met Leu Asn Arg Arg Pro Glu Ala Met Lys Ala 530 535 540 Met 545

Claims

1. An isolated polypeptide sequence having an amino acid sequence with at least 95% identity to the amino acid sequence of cytochrome P450 CYP4G1 (SEQ ID NO: 2) and having cytochrome P450 monooxygenase activity.

2. The isolated polypeptide sequence of claim 1, wherein the amino acid sequence has at least 99% identity to the amino acid sequence of SEQ ID NO: 2.

3. The isolated polypeptide sequence of claim 1, wherein the polypeptide sequence is as set forth by SEQ ID NO: 2.

4. An isolated polynucleotide sequence that encodes the polypeptide sequence of claim 1 or a polypeptide sequence having an amino acid sequence with at least 95% identity to the amino acid sequence of cytochrome P450 CYP4G2 (SEQ ID NO: 1) and having cytochrome P450 monooxygenase activity SEQ ID NO: 1.

5. A method of producing a polypeptide having oxidative decarbonylase activity comprising: introducing the polynucleotide of claim 4 into an isolated host cell; culturing the host cell; and expressing from the host cell a polypeptide having oxidative decarbonylase activity.

6. The method of claim 5, further comprising isolating the polypeptide with oxidative decarbonylase activity following expressing the polypeptide with oxidative decarbonylase activity.

7. The method of claim 5, further comprising measuring oxidative decarbonylase activity of the expressed polypeptide.

8. A method of catalyzing hydrocarbon formation comprising contacting a sample comprising a fatty aldehyde with the polypeptide of claim 1.

9. A method of catalyzing hydrocarbon formation comprising contacting a sample comprising a fatty aldehyde with the polypeptide of claim 3.

10. A transgenic host cell produced by inserting the polynucleotide of claim 4 into the host cell.

11. The transgenic host cell of claim 10, wherein the transgenic host cell is a yeast cell, an algae cell, a plant cell, an animal cell or a bacterial cell.

12. A method for producing a hydrocarbon comprising culturing the cell of claim 10 under conditions sufficient to produce hydrocarbons.

13. The method of claim 12, further comprising isolating a hydrocarbon from the cell or from medium in which the cell is cultured.

14. The method claim 12, wherein the cell is cultured in the presence of at least one substrate of oxidative decarbonylase activity.

15. The method of claim 14, wherein the substrate is a fatty aldehyde.

16. A biofuel comprising hydrocarbons produced by the method of claim 12.

17. A biofuel comprising hydrocarbons produced by the method of claim 8.

18. A recombinant nucleic acid sequence comprising a promoter operably linked to a nucleic acid sequence encoding a peptide selected from: a) a sequence as set forth by SEQ ID NO: 1 or 2; or b) a sequence sharing 95% sequence identity with SEQ ID NO: 1 or 2.

19. A cell comprising the recombinant nucleic acid sequence of claim 18.

20. A method of making a hydrocarbon comprising culturing the cell of claim 19 under conditions sufficient to produce a hydrocarbon.

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