# Patent

## Title
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.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 61/115,382 filed Nov. 17, 2008, which is herein incorporated by reference in its entirety.


ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with government support under IBN-9630916 awarded by the National Science Foundation and United States Department of Agriculture-National Research Initiative, Grant/Contract Nos. 94-37302-0612 and 96-35302-3416. The United States Government has certain rights in the invention.


FIELD
The present disclosure generally relates to a hydrocarbon-forming oxidative decarbonylase enzyme, hydrocarbons produced thereby and uses thereof.
BACKGROUND
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.
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.
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).
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
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.
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.
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.
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.
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.
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
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.
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.
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.
FIG. 2B is a bar graph demonstrating that NADPH and oxygen are required for hydrocarbon formation from 24:1-CoA.
FIG. 3 is a bar graph illustrating the amount of hydrocarbon production for control and knocked down (1405Cyp and M485Cyp) flies.
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.
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.
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.
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.
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.
FIG. 9 is a digital image of a model of Homo sapiens microsomal cytochrome P450 3A4 (PDB 1TQN) illustrating the variant regions.
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.
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 μmol CYP4G1/mg protein.



SEQUENCE LISTING
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:
SEQ ID NO: 1 is the amino acid sequence of cytochrome P450 4g2 (CYP4G2).
SEQ ID NO: 2 is the amino acid sequence of cytochrome P450 4g1 (CYP4G1).
SEQ ID NO: 3 is the amino acid sequence of GG12761 from Drosophila erecta. 
SEQ ID NO: 4 is the amino acid sequence of GD24639 from Drosophila simulans. 
SEQ ID NO: 5 is the amino acid sequence of GE16587 from Drosophila yakuba. 
SEQ ID NO: 6 is the amino acid sequence of GA17813 from Drosophila pseudoobscura. 
SEQ ID NO: 7 is the amino acid sequence of GI11123 from Drosophila mojavensis. 
SEQ ID NO: 8 is the amino acid sequence of GJ15981 from Drosophila virilis. 
SEQ ID NO: 9 is the amino acid sequence of GF20812 from Drosophila ananassae. 
SEQ ID NO: 10 is the amino acid sequence of GH24346 from Drosophila grimshawi. 
SEQ ID NO: 11 is the amino acid sequence of GK25658 from Drosophila willistoni. 
SEQ ID NO: 12 is the amino acid sequence of cytochrome P450 4g15 from Culex quinquefasciatus. 
SEQ ID NO: 13 is an amino acid sequence of cytochrome P450 from Aedes aegypti. 
SEQ ID NO: 14 is the amino acid sequence of cytochrome P450 from Aedes aegypti. 
SEQ ID NO: 15 is an amino acid sequence similar to cytochrome P450 from Nasonia vitripennis. 
SEQ ID NO: 16 is an amino acid sequence similar to cytochrome P450 monooxygenase from Nasonia vitripennis. 
SEQ ID NO: 17 is the amino acid sequence of cytochrome P450 family 4 from Chironomus tentans. 
SEQ ID NO: 18 is the amino acid sequence of AGAP000877-PA from Anopheles gambiae str. PEST.
SEQ ID NO: 19 is the amino acid sequence of AGAP001076-PA from Anopheles gambiae str. PEST.
SEQ ID NO: 20 is the amino acid sequence of cytochrome P450 monooxygenase CYP4G7 from Tribolium castaneum. 
SEQ ID NO: 21 is the amino acid sequence of cytochrome P450 monooxygenase Tribolium castaneum. 
SEQ ID NO: 22 is the amino acid sequence of cytochrome P450 from Bombyx mori. 
SEQ ID NO: 23 is the amino acid sequence of cytochrome P450 4G25 (CYP4G25) Bombyx mori. 
SEQ ID NO: 24 is the amino acid sequence of cytochrome P450 from Leptinotarsa decemlineata. 
SEQ ID NO: 25 is the amino acid sequence of GM13084 from Drosophila sechellia. 
SEQ ID NO: 26 is the amino acid sequence of cytochrome P450 monooxygenase from Apis mellifera. 
SEQ ID NO: 27 is the amino acid sequence of cytochrome P450 4G19 monooxygenase (CYP4G19) from Blattella germanica. 
SEQ ID NO: 28 is the amino acid sequence of CYP4G27 from Ips paraconfusus.
SEQ ID NO: 29 is the amino acid sequence of cytochrome P450 4G25 (CYP4G25) from Antheraea yamamai. 
SEQ ID NO: 30 is the amino acid sequence of antennal cytochrome P450 4 (CYP4) from Mamestra brassicae. 
SEQ ID NO: 31 is an amino acid sequence similar to cytochrome P450 from Acyrthosiphon pisum . 
SEQ ID NO: 32 is the amino acid sequence of GL20168 from Drosophila persimilis. 
SEQ ID NO: 33 is the amino acid sequence of cytochrome P450 4C1 (CYPIVC1) from Blaberus discoidalis (roaches).
SEQ ID NO: 34 is the amino acid sequence of cytochrome P450 4C39 (CYP4C39) from green crab, common shore crab.
SEQ ID NO: 35 is the amino acid sequence of hypothetical protein BRAFLDRAFT—57954 from Branchiostoma floridae. 
SEQ ID NO: 36 is the amino acid sequence of cytochrome 4V6 from Balaenoptera acutorostrata. 
SEQ ID NO: 37 is the amino acid sequence of cytochrome P450 4M6 monooxygenase (CYP4M6) from Helicoverpa zea. 
SEQ ID NO: 38 is the amino acid sequence of cytochrome P450 4 family from Daphnia magna. 
SEQ ID NO: 39 is the amino acid sequence of cytochrome P450 from Nilaparvata lugens. 
SEQ ID NO: 40 is the amino acid sequence of hypothetical protein L00562008 Danio rerio. 
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. 
SEQ ID NO: 42 is the amino acid sequence of cytochrome P450 like protein from Arabidopsis thaliana. 
SEQ ID NO: 43 is the amino acid sequence of hypothetical protein OsI—028301 from Oryza sativa (indica cultivar-group).
SEQ ID NO: 44 is the amino acid sequence of hypothetical protein OsI—003357 from Oryza sativa (indica cultivar-group).
SEQ ID NO: 45 is the amino acid sequence of hypothetical protein OsI—005901 from Oryza sativa (indica cultivar-group).
SEQ ID NO: 46 is a nucleic acid sequence of cytochrome P450 4g2 (CYP4G2).
SEQ ID NO: 47 is the nucleic acid sequence of cytochrome P450 4g2 (CYP4G2).
SEQ ID NO: 48 is the nucleic acid sequence of cytochrome P450 4g1 (CYP4G1).
SEQ ID NO: 49 is the amino acid sequence of cytochrome CYP3A4 from Homo sapiens. 
SEQ ID NO: 50 is the amino acid sequence of cytochrome P450 51 from Mycobacterium tuberculosis. 
SEQ ID NO: 51 is a nucleic acid sequence of cytochrome P450 chimera 9T2/4G2.
SEQ ID NO: 52 is an amino acid sequence of cytochrome P450 chimera 9T2/4G2.
DETAILED DESCRIPTION
I. Overview of Several Embodiments
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).
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.
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.
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).
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.
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).
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.
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.
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.
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.
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.
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.
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
CO: carbon monoxide
CYP4G1: cytochrome P450 4G1
CYP4G2: cytochrome P450 4G2
GC: gas chromatography
HC: hydrocarbon content
MS: mass spectrometry
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:
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—).
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.
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” (VL) and “variable heavy chain” (VH) refer, respectively, to these light and heavy chains.
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).
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.
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.
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 Methanococcusjannaschii, Methanosarcina mazei, Methanobacterium thermoautotrophicum, Methanococcus maripaludis, Methanopyrus kandleri, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-i, Archaeoglobusfulgidus, 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).
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.
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.
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.
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.
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.
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).
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.
Decarboxylase: An enzyme that hydrolyzes a carboxyl radical.
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.
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.
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.
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.
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.
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.
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.
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.
Specific, non-limiting examples of fungi that are useful in the disclosed methods include Saccharomyces cerevisiae, Aspergillus, Trichoderma, Neurospora, Fusarium, and Chrysosporium. 
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).
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
“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.
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.
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.
The term “soluble” refers to a form of a polypeptide that is not inserted into a cell membrane.
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.











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









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.
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.
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.
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.
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.
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.).
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.
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.
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.).
Probe: An isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, fluorophores, and enzymes.
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.
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.
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.
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.
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.
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.
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.
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.
The NCBI Basic Local Alignment Search Tool (BLASTTM; 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. BLASTTM can be accessed on the internet at the NBCI website. As used herein, sequence identity is commonly determined with the BLASTTM 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).
For comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function of the BLASTTM 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.
For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLASTTM (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]).
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.
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.
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.
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.
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
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.
A. Isolated Polypeptides and Polynucleotides
1. Structure
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. 
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).
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.
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.
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%.
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 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









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.
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.
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.
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).
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).
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).
2. Function
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%.
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.
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.
B. Recombinant Nucleic Acid Constructs
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 heterologous host.
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.
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.
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).
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.
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.
A nucleic acid sequence is operably linked to another 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.
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).
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.
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.
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).
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).
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.
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.
C. Host Cells
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. 
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.
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.
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.).
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.
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).
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.
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, Archaeoglobusfulgidus, Pyrococcus fit riosus, 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.
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.
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.
D. Product Production and Uses Thereafter
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.
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 
1. Carbon Chain Characteristics
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.
2. Methods of Making Hydrocarbons
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).
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.
a. In Vitro
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.
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.
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.
b. In Vivo
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.
3. Post Production Processing
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 C1-4 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.
The disclosure is illustrated by the following non-limiting Examples.
EXAMPLES
Example 1
CYP4G1 is a Oxidative Decarbonylase
This example shows that CYP4G1 is a cytochrome P450 that functions as a oxidative decarbonylase.
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. 
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 μmol 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
This example describes the expression and characterization of CYP4G2.
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.
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.
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.
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 CYP4G2V38A. 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.
In additional characterization studies, recombinant baculoviral CYP4G2V38A 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.
Recombinant CYP9T2/4G2 and CYP4G2V38A 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 CYP4G2V38A 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 CYP4G2V38A: 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.
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 CYP4G2V38A and CYP4G1. Thus, these studies will characterize the disclosed polypeptides and assess their enzymatic activity.
Example 3
Purification of CYP9T2/4G2 or CYP4G2V38A 
This example describes methods for purifying exemplary insect P450 enzymes and in particular, their oxidative decarbonylase activity.
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 CYP4G2V38A, can be determined through assays of purified, recombinant protein. CYP9T2/4G2 or CYP4G2V38A is purified using an immune affinity column.
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.
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.
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 (“CYP4G2cat”) is transferred to the BaculoDirect expression vector by recombination, and high-titre virus stocks is prepared as described herein. Recombinant CYP4G2cat-His fusion protein is subsequently purified on a Ni2+-column and incubated with enterokinase to remove the His tag. Purification of CYP4G2cat-His is confirmed by SDS-PAGE and peptide sequencing.
Protein purification: Recombinant CYP9T2/4G2 or CYP4G2V38A 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.
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 stiffing 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 CYP4G2V38A 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 CYP4G2V38A 
This example provides methods for characterizing disclosed cytochrome P450 enzymes.
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 CYP4G2V38A 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 CYP4G2V38A 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.
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.
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 KM, kcat, and Kcat/KM for the various substrates. These studies will allow the disclosed polypeptides to be characterized.
Example 5
Molecular Modeling of CYP4G2
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.
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 6th 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).)
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).
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.
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.

## Claims

1. An isolated polypeptide sequence having an amino acid sequence at least 99% identity to the amino acid sequence of cytochrome P450 CYP4G2 (SEQ ID NO: 1) and having cytochrome P450 monooxygenase activity.

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

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

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