P450 enzymes, nucleic acids encoding them and methods of making and using them

Abstract

The invention is directed to polypeptides having P450 activity, polynucleotides encoding the polypeptides, antibodies that bind to these polypeptides, and methods for making and using these polynucleotides and polypeptides. The P450 enzymes can be used to catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols.

1 . An isolated, synthetic or recombinant nucleic acid comprising 
 (a) a nucleic acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, or SEQ ID NO:53 over a region of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or more residues, a nucleic acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) sequence identity to SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:43, or SEQ ID NO:55 over a region of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or more residues,    a nucleic acid sequence having at least 70% 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) sequence identity to SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:35 over a region of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or more residues,    a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) sequence identity to SEQ ID NO:27 over a region of at least about 100 residues, or    a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) sequence identity to SEQ ID NO:19 over a region of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or more residues,    wherein the nucleic acid encodes at least one polypeptide having a P450 activity or encodes a polypeptide or peptide capable of generating an antibody that specifically binds to a polypeptide having the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 or SEQ ID NO:56 (a polypeptide or peptide that acts as an epitope or immunogen), and optionally the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection, and optionally the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d “nr pataa”-F F, and all other options are set to default; or,    (b) a nucleic acid encoding at least one polypeptide having a P450 activity, wherein the nucleic acid comprises a sequence that hybridizes under stringent conditions to a nucleic acid comprising the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 or SEQ ID NO:51,    (c) a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 or SEQ ID NO:56, or P450 enzymatically active fragments thereof;    (d) (i) the nucleic acid (polynucleotide) of (a), (b) or (c) and encoding a polypeptide having at least one conservative amino acid substitution and retaining its P450 activity; or, (ii) the nucleic acid of (d)(i), wherein the at least one conservative amino acid substitution comprises substituting an amino acid with another amino acid of like characteristics; or, a conservative substitution comprises: replacement of an aliphatic amino acid with another aliphatic amino acid; replacement of a Serine with a Threonine or vice versa; replacement of an acidic residue with another acidic residue; replacement of a residue bearing an amide group with another residue bearing an amide group; exchange of a basic residue with another basic residue; or replacement of an aromatic residue with another aromatic residue;    (e) the nucleic acid (polynucleotide) of (a), (b), (c) or (d) encoding a polypeptide having a P450 activity but lacking a signal sequence or proprotein sequence;    (f) the nucleic acid (polynucleotide) of (a), (b), (c), (d) or (e) encoding a polypeptide having a P450 activity further comprising a heterologous sequence;    (g) the nucleic acid (polynucleotide) of (f), wherein the heterologous sequence comprises, or consists of a sequence encoding a heterologous (leader) signal sequence, or a tag or an epitope;    (h) the nucleic acid (polynucleotide) of (a), (b), (c), (d), (e), (f) or (g) wherein the P450 activity comprises: a monooxygenation; catalysis of incorporation of oxygen into a substrate; catalysis of incorporation of oxygen into a substrate and hydroxylation of aliphatic or aromatic carbons; catalysis of incorporation of oxygen into a substrate and epoxidation; catalysis of incorporation of oxygen into a substrate and N-, O-, or S-dealkylation; catalysis of incorporation of oxygen into a substrate and dehalogenation; catalysis of incorporation of oxygen into a substrate and oxidative deamination; catalysis of incorporation of oxygen into a substrate and N-oxidation; catalysis of incorporation of oxygen into a substrate and N-hydroxylation; catalysis of incorporation of oxygen into a substrate and sulphoxide formation; catalysis of incorporation of oxygen into a substrate and an epoxidase activity comprising an alkene substrate; catalysis of incorporation of oxygen into a substrate and an epoxidase activity comprising production of a chiral product; or, catalysis of incorporation of oxygen into a substrate and an enantioselective epoxidase activity;    (i) the nucleic acid (polynucleotide) of (a), (b), (c), (d), (e), (f), (g) or (h) wherein the P450 activity thermostable or thermotolerant; or    (j) a nucleic acid sequence (polynucleotide) fully (completely) complementary to (a), (b) (c), (d), (e), (f), (g), (h) or (i).    
     
     
         2 - 45 . (canceled)  
     
     
         46 . A nucleic acid probe for identifying a nucleic acid encoding a polypeptide with a P450 activity, wherein the probe comprises at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive bases of a sequence of  claim 1 , 
 wherein the probe identifies the nucleic acid by binding or hybridization, wherein the probe comprises an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 base pair residues.    
     
     
         47 - 52 . (canceled)  
     
     
         53 . An amplification primer pair for amplifying a nucleic acid encoding a polypeptide having a P450 activity, wherein the primer pair is capable of amplifying a nucleic acid comprising a sequence of  claim 1 .  
     
     
         54 . (canceled)  
     
     
         55 . A method of amplifying a nucleic acid encoding a polypeptide having a P450 activity comprising amplification of a template nucleic acid with an amplification primer pair as set forth in  claim 53 .  
     
     
         56 . An expression cassette or vector comprising a nucleic acid of  claim 1 .  
     
     
         57 . (canceled)  
     
     
         58 . A cloning vehicle comprising a vector or an expression cassette as set forth in  claim 56 , wherein the cloning vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome, 
 wherein optionally the viral vector comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector, or the cloning vehicle comprises a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).    
     
     
         59 - 60 . (canceled)  
     
     
         61 . A transformed cell comprising a vector or an expression cassette of  claim 56 , 
 or a nucleic acid of  claim 1 , or a cloning vehicle of  claim 58 ,    wherein optionally the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.    
     
     
         62 - 63 . (canceled)  
     
     
         64 . A transgenic non-human animal, or a transgenic plant, or a transgenic seed, comprising 
 a nucleic acid of  claim 1 , or a vector or an expression cassette of  claim 56 , or a cloning vehicle of  claim 58 .    
     
     
         65 - 69 . (canceled)  
     
     
         70 . An antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid sequence of  claim 1 .  
     
     
         71 - 72 . (canceled)  
     
     
         73 . An isolated, synthetic or recombinant polypeptide having a P450 activity or capable of generating an antibody that specifically binds to a polypeptide having the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 or SEQ ID NO:56 (a polypeptide or peptide that acts as an epitope or immunogen), comprising 
 (a) a polypeptide comprising    an amino acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) identity to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, or SEQ ID NO:54, over a region of at least about 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 or more residues,    an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) identity to SEQ ID NO:2, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:44, or SEQ ID NO:56 over a region of at least about 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 or more residues,    an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) identity to SEQ ID NO:28 over a region of at least about 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 or more residues, or    an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) identity to SEQ ID NO:20 over a region of at least about 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 or more residues,    wherein optionally the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection, and optionally the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d “nr pataa”-F F, and all other options are set to default, or    (b) a polypeptide encoded by the nucleic acid of  claim 1     (c) (i) the polypeptide of (a) or (b) having at least one conservative amino acid substitution and retaining its P450 activity; or, (ii) the polypeptide of (c)(i), wherein the at least one conservative amino acid substitution comprises substituting an amino acid with another amino acid of like characteristics; or, a conservative substitution comprises: replacement of an aliphatic amino acid with another aliphatic amino acid; replacement of a Serine with a Threonine or vice versa; replacement of an acidic residue with another acidic residue; replacement of a residue bearing an amide group with another residue bearing an amide group; exchange of a basic residue with another basic residue; or replacement of an aromatic residue with another aromatic residue;    (d) the polypeptide of (a), (b) or (c) lacking a signal sequence or proprotein sequence;    (e) the polypeptide of (a), (b), (c) or (d) further comprising a heterologous sequence;    (f) the polypeptide of (e), wherein the heterologous sequence comprises, or consists of a sequence encoding a heterologous (leader) signal sequence, or a tag or an epitope;    (g) the polypeptide of (a), (b), (c), (d), (e) or (f) wherein the P450 activity comprises: a monooxygenation; catalysis of incorporation of oxygen into a substrate; catalysis of incorporation of oxygen into a substrate and hydroxylation of aliphatic or aromatic carbons; catalysis of incorporation of oxygen into a substrate and epoxidation; catalysis of incorporation of oxygen into a substrate and N-, O-, or S-dealkylation; catalysis of incorporation of oxygen into a substrate and dehalogenation; catalysis of incorporation of oxygen into a substrate and oxidative deamination; catalysis of incorporation of oxygen into a substrate and N-oxidation; catalysis of incorporation of oxygen into a substrate and N-hydroxylation; catalysis of incorporation of oxygen into a substrate and sulphoxide formation; catalysis of incorporation of oxygen into a substrate and an epoxidase activity comprising an alkene substrate; catalysis of incorporation of oxygen into a substrate and an epoxidase activity comprising production of a chiral product; or, catalysis of incorporation of oxygen into a substrate and an enantioselective epoxidase activity;    (h) the polypeptide of (a), (b), (c), (d), (e), (f) or (g) wherein the P450 activity is thermostable or thermotolerant; or    (i) the polypeptide of (a), (b), (c), (d), (e), (f), (g) or (h) wherein the polypeptide comprises at least one glycosylation site, and optionally the glycosylation is an N-linked glycosylation;    (j) the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h) or (i) wherein the polypeptide retains a P450 activity under conditions comprising about pH 4.5 or pH 5, or polypeptide retains a P450 activity under conditions comprising about pH 9.0, pH 9.5, or pH 10.    
     
     
         74 - 116 . (canceled)  
     
     
         117 . A protein preparation comprising the polypeptide of  claim 73 , wherein the protein preparation comprises a liquid, a solid or a gel.  
     
     
         118 . A heterodimer comprising the polypeptide of  claim 73  and a second domain, wherein optionally the second domain is a polypeptide and the heterodimer is a fusion protein, and optionally the second domain is an epitope.  
     
     
         119 - 121 . (canceled)  
     
     
         122 . An immobilized polypeptide having a P450 activity, wherein the polypeptide comprises the sequence of  claim 73 , and optionally the polypeptide is immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.  
     
     
         123 . (canceled)  
     
     
         124 . An array comprising the immobilized polypeptide of  claim 73  or the nucleic acid of  claim 1 .  
     
     
         125 . (canceled)  
     
     
         126 . An isolated or recombinant antibody that specifically binds to the polypeptide of in  claim 73 , wherein optionally the antibody is a monoclonal or a polyclonal antibody.  
     
     
         127 . (canceled)  
     
     
         128 . A hybridoma comprising an antibody that specifically binds to the polypeptide of  claim 73 .  
     
     
         129 . A method of isolating or identifying a polypeptide with a P450 activity comprising the steps of: 
 (a) providing the antibody of  claim 126;     (b) providing a sample comprising polypeptides; and    (c) contacting the sample of step (b) with the antibody of step (a) under conditions wherein the antibody can specifically bind to the polypeptide, thereby isolating or identifying a polypeptide having a P450 activity.    
     
     
         130 . (canceled)  
     
     
         131 . A method of producing a recombinant polypeptide comprising the steps of: 
 (a) providing a nucleic acid operably linked to a promoter; wherein the nucleic acid comprises the sequence of  claim 1;  and    (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide,    wherein optionally the method further comprises transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.    
     
     
         132 . (canceled)  
     
     
         133 . A method for identifying a polypeptide having a P450 activity comprising the following steps: 
 (a) providing the polypeptide of  claim 73;     (b) providing a P450 substrate; and    (c) contacting the polypeptide or a fragment or variant thereof of step (a) with the substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of a reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having a P450 activity,    wherein optionally the substrate is an alkene.    
     
     
         134 . (canceled)  
     
     
         135 . A method for identifying a P450 substrate comprising the following steps: 
 (a) providing the polypeptide of  claim 73;     (b) providing a test substrate; and    (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as a P450 substrate.    
     
     
         136 . A method of determining whether a test compound specifically binds to a polypeptide comprising the following steps: 
 (a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid has the sequence of  claim 1 , or, providing the polypeptide of  claim 73;     (b) providing a test compound;    (c) contacting the polypeptide with the test compound; and    (d) determining whether the test compound of step (b) specifically binds to the polypeptide.    
     
     
         137 . A method for identifying a modulator of a P450 activity comprising the following steps: 
 (a) providing the polypeptide of  claim 73;     (b) providing a test compound;    (c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the P450, wherein a change in the P450 activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the P450 activity.    
     
     
         138 - 140 . (canceled)  
     
     
         141 . A computer system comprising a processor and a data storage device wherein said data storage device has stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises the sequence of  claim 73 , and the nucleic acid comprises the sequence of  claim 1 .  
     
     
         142 - 144 . (canceled)  
     
     
         145 . A computer readable medium having stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises the sequence of in  claim 73 , and the nucleic acid comprises the sequence of  claim 1 .  
     
     
         146 . A method for identifying a feature in a sequence comprising the steps of: 
 (a) reading the sequence using a computer program which identifies one or more features in a sequence, wherein the sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises the sequence of  claim 73 , and the nucleic acid comprises the sequence of  claim 1;  and    (b) identifying one or more features in the sequence with the computer program.    
     
     
         147 - 150 . (canceled)  
     
     
         151 . A method for isolating or recovering a nucleic acid encoding a polypeptide with a P450 activity from an environmental sample comprising the steps of: 
 (a) providing an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide with a P450 activity, wherein the primer pair is capable of amplifying SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, or a subsequence thereof;    (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to the amplification primer pair; and,    (c) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the environmental sample, thereby isolating or recovering a nucleic acid encoding a polypeptide with a P450 activity from an environmental sample.    
     
     
         152 . (canceled)  
     
     
         153 . A method for isolating or recovering a nucleic acid encoding a polypeptide with a P450 activity from an environmental sample comprising the steps of: 
 (a) providing a polynucleotide probe comprising the sequence of  claim 1;     (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a);    (c) combining the isolated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (a); and    (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide with an epoxide hydrolase activity from an environmental sample.    
     
     
         154 - 155 . (canceled)  
     
     
         156 . A method of generating a variant of a nucleic acid encoding a polypeptide with a P450 activity comprising the steps of: 
 (a) providing a template nucleic acid comprising the sequence of  claim 1;  and    (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid.    
     
     
         157 - 201 . (canceled)  
     
     
         202 . A method for hydrolyzing an alkene comprising the following steps: 
 (a) providing a polypeptide having a P450 activity, wherein the polypeptide comprises the amino acid sequence of  claim 73;     (b) providing a composition comprising an alkene; and    (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the polypeptide oxygenases the alkene.    
     
     
         203 . A method for producing a compound of a desired chirality comprising the following steps: 
 (a) providing a polypeptide having a enantioselective P450 activity, wherein the polypeptide comprises the amino acid sequence of  claim 73;     (b) providing a composition comprising a P450 substrate;    (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the polypeptide catalyzes the conversion of the P450 substrate to the chiral epoxide; and    (d) converting the chiral epoxide to a chiral product,    wherein optionally the chiral product comprises a diol, an amino alcohol, a halohydrin or a branched-chain alkyl moiety, or optionally the chiral product is an antibiotic.    
     
     
         204 - 208 . (canceled)  
     
     
         209 . A growth-based method for selecting a cell comprising a nucleic acid encoding an epoxidase comprising the following steps: 
 (a) providing a plurality of cells, wherein the cells lack a composition essential for growth;    (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by a P450 to a composition essential for growth of the cell and the essential composition must comprise an epoxide to induce growth of the cell;    (c) growing the cells in a medium lacking the epoxide, and adding the precursor or substrate of step (b); and    (d) screening the cells for growth, wherein the cells in the growth stimulated clone are identified as comprising a nucleic acid encoding a P450 capable of converting the precursor or substrate to the composition comprising the epoxide, thereby selecting a cell comprising a nucleic acid encoding a P450.    
     
     
         210 . A growth-based method for selecting a nucleic acid encoding a P450 comprising the following steps: 
 (a) providing a nucleic acid encoding a polypeptide;    (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by a P450 to a composition essential for growth of the cell and the composition must comprise an epoxide to induce growth of the cell;    (c) providing a plurality of cells, wherein the cells cannot make the composition of step (b);    (d) inserting the nucleic acid into the cells and growing the cells under conditions wherein the nucleic acid is expressed and its encoded polypeptide is translated, and the cells are grown in a medium lacking the composition comprising the epoxide essential for growth, and adding the precursor or substrate of step (b); and    (e) screening the cells for growth, wherein the nucleic acid in the growth stimulated clone is identified as encoding a P450 capable of converting the precursor or substrate to the composition comprising the epoxide essential for growth, thereby selecting a nucleic acid encoding a P450.    
     
     
         211 . A method for identifying a nucleic acid encoding a P450 comprising the following steps: 
 (a) providing a nucleic acid library;    (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by a P450 to a composition essential for growth and the essential composition must comprise an epoxide to induce growth of the cell;    (c) providing a plurality of cells, wherein the cells cannot make the composition of step (b);    (d) inserting in a cell a member of the gene library and culturing the cells in a medium lacking the composition comprising the epoxide essential for growth;    (e) adding the precursor or substrate of step (b) to the culture;    (f) selecting a growing cell and identifying the inserted library member of step (d), wherein the cell is capable of growth by enzymatic conversion of the precursor to the composition comprising the epoxide essential for growth, and the enzyme is encoded by the library member, thereby identifying a nucleic acid encoding a P450,    wherein optionally the precursor or substrate comprises an alkene.    
     
     
         212 . A growth-based method for selecting a cell comprising a nucleic acid encoding a P450 comprising the following steps: 
 (a) providing a plurality of cells comprising an epoxide hydrolase, wherein the cells lack a composition essential for growth;    (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by a P450 to an epoxide, wherein the epoxide is capable of being converted by an epoxide hydrolase to a composition essential for growth of the cell;    (c) growing the cells in a medium lacking the epoxide and composition essential for growth, and adding the precursor or substrate of step (b); and,    (d) screening the cells for growth, wherein the cells in the growth stimulated clone are identified as comprising the nucleic acid encoding a P450 enzyme capable of converting the precursor or substrate to the epoxide, thereby selecting a cell comprising a nucleic acid encoding a P450 enzyme having an epoxidase activity,    wherein optionally the precursor or substrate comprises an alkene.    
     
     
         213 . A growth-based method for selecting a nucleic acid encoding a P450 comprising the following steps: 
 (a) providing a nucleic acid encoding a polypeptide;    (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by a P450 to an epoxide, wherein the epoxide is capable of being converted by an epoxide hydrolase to a composition essential for growth of the cell;    (c) providing a plurality of cells comprising an epoxide hydrolase, wherein the cells cannot make the composition of step (b);    (d) inserting the nucleic acid into the cells and growing the cells under conditions wherein the nucleic acid is expressed and its encoded polypeptide is translated, and the cells are grown in a medium lacking the composition comprising the epoxide essential for growth, and adding the substrate of step (c); and,    (e) screening the cells for growth, wherein the nucleic acid in the growth stimulated clone is identified as encoding a P450 capable of converting the precursor or substrate to the composition comprising the epoxide, thereby selecting a nucleic acid encoding a P450 having an epoxidase activity,    wherein optionally the precursor or substrate comprises an alkene, and optionally the precursor or substrate comprises allyl alcohol, 3,3-dimethyl acrylate or trans-3-methyl-2-pentenoate.    
     
     
         214 - 217 . (canceled)