DNA shuffling of monooxygenase genes for production of industrial chemicals

Abstract

This invention provides improved monoxygenases, dehydrogenases, and transferases that are useful for the biocatalytic synthesis of compounds such as α-hydroxycarboxylic acids, and aryl- and alkyl-hydroxy compounds. The polypeptides provided herein are improved in properties such as regioselectivity, enzymatic activity, stereospecificity, and the like. Methods for obtaining recombinant polynucleotides that encode these improved polypeptides are also provided, as are organisms that express the polypeptides and are thus useful for carrying out said biocatalytic syntheses. Also provided by the invention are methods for increasing said solvent resistance of organisms that are used in the synthetic methods.

What is claimed is:  
     
         1 . A method for obtaining a polynucleotide that encodes an improved polypeptide comprising monooxygenase activity, wherein said improved polypeptide has at least one property improved over a naturally occurring monooxygenase polypeptide. said method comprising: 
 (a) creating a library of recombinant polynucleotides encoding a recombinant monooxygenase polypeptide; and    (b) screening said library to identify a recombinant polynucleotide that encodes an improved recombinant monooxygenase polypeptide that has at least one property improved over said naturally occurring polypeptide.    
     
     
         2 . The method according to  claim 1 , wherein said creating a library comprises: 
 shuffling a plurality of parental polynucleotides to produce one or more recombinant monooxygenase polynucleotide encoding said improved property.    
     
     
         3 . The method according to  claim 1 , wherein said monooxygenase activity is a member selected from alkene epoxidation, alkane hydroxylation, aromatic hydroxylation, N-dealkylation of alkylamines, S-dealkylation of reduced thio-organics, O-dealkylation of alkyl ethers, oxidation of aryloxy phenols, conversion of aldehydes to acids, dehydrogenation, decarbonylation, oxidative dehalogenation of haloaromatics and halohydrocarbons, Baeyer-Villiger monoxygenation, modification of cyclosporins, hydroxylation of mevastatin, oxygenation of sulfonylureas and combinations thereof.  
     
     
         4 . The method of  claim 2 , wherein at least one of said parental polynucleotides encode at least one monooxygenase activity.  
     
     
         5 . The method of  claim 2 , wherein said parental polynucleotides are homologous.  
     
     
         6 . The method of  claim 2 , wherein at least one of said parental polynucleotides does not encode a monooxygenase activity.  
     
     
         7 . The method of  claim 2 , wherein said parental monooxygenase polynucleotide encodes a polypeptide or polypeptide subsequence selected from a P450 oxygenase, a heme-dependent peroxidase, an iron sulfur monooxygenase, a quinone-dependent monooxygenase and combinations thereof.  
     
     
         8 . The method of  claim 2 , wherein a member selected from said parental polynucleotides, said one or more recombinant monooxygenase polynucleotide, said identified recombinant monooxygenase polynucleotide and combinations thereof is cloned into an expression vector.  
     
     
         9 . The method of  claim 1 , wherein said identified recombinant monooxygenase polynucleotide has an ability to catalyze an enzymatic reaction using a redox partner other than NADPH.  
     
     
         10 . The method of  claim 2 , further comprising: 
 creating a library of recombinant peroxide production activity polynucleotides encoding a recombinant hydrogen peroxide production activity;    screening said library to identify a recombinant polynucleotide that encodes an improved hydrogen peroxide production activity; and    co-expressing one or more of said identified hydrogen peroxide production activity polynucleotides and said identified recombinant monooxygenase polynucleotide in a cell.    
     
     
         11 . The method of  claim 2 , further comprising: 
 creating a library of recombinant epoxide hydrolase activity polynucleotides encoding a recombinant epoxide hydrolase activity;    screening said library to identify a recombinant polynucleotide that encodes an improved epoxide hydrolase activity; and    co-expressing one or more of said identified recombinant epoxide hydrolase activity polynucleotides and said identified recombinant monooxygenase polynucleotide in a cell.    
     
     
         12 . The method of  claim 2 , further comprising: 
 creating a library of recombinant dehydrogenase activity polynucleotides encoding a recombinant dehydrogenase activity;    screening said library to identify a recombinant polynucleotide that encodes an improved dehydrogenase activity; and    co-expressing one or more of said identified recombinant dehydrogenase activity polynucleotides and said identified recombinant monooxygenase polynucleotide in a cell.    
     
     
         13 . The method of  claim 1 , further comprising: 
 creating a library of recombinant transferase activity polynucleotides encoding a recombinant transferase activity;    screening said library to identify a recombinant polynucleotide that encodes an improved transferase activity; and    co-expressing one or more of said identified recombinant transferase activity polynucleotides and said identified recombinant monooxygenase polynucleotide in a cell.    
     
     
         14 . The method according to  claim 13 , wherein said transferase polynucleotide is a member selected from acyltransferases, glycosyltransferases, methyl transferases and combinations thereof.  
     
     
         15 . The method of  claim 2 , wherein said plurality of parental polynucleotides are shuffled to produce a library of recombinant polynucleotides comprising one or more library member polynucleotide encoding one or more monooxygenase activity, which library is selected for one or more monooxygenase activity selected from alkene epoxidation, alkane hydroxylation, aromatic hydroxylation, N-dealkylation of alkylanines, S-dealkylation of reduced thio-organics, O-dealkylation of alkyl ethers, oxidation of aryloxy phenols, conversion of aldehydes to acids, dehydrogenation, decarbonylation, oxidative dehalogenation of haloaromatics and halohydrocarbons, Baeyer-Villiger monoxygenation, modification of cyclosporins, hydroxylation of mevastatin, conversion of cholesterol to pregnenolone, and oxygenation of sulfonylureas.  
     
     
         16 . A library of recombinant polynucleotides comprising one or more monooxygenase activity made by said method of  claim 1 .  
     
     
         17 . The library of  claim 16 , wherein said library is a phage display library.  
     
     
         18 . An improved monooxygenase encoding nucleic acid prepared by the method according to  claim 1 .  
     
     
         19 . The method of  claim 2 , wherein said parental polynucleotides are shuffled in a plurality of cells, which cells are prokaryotes or eukaryotes.  
     
     
         20 . The method of  claim 2 , wherein said parental polynucleotides are shuffled in a plurality of cells, which cells are yeast, bacteria, or fungi.  
     
     
         21 . The method of  claim 2 , wherein said parental polynucleotides are shuffled in a plurality of cells; said method optionally further comprises one or more members selected from 
 (a) recombining DNA from said plurality of cells that display monooxygenase activity with a library of DNA fragments, at least one of which undergoes recombination with a segment in a cellular DNA present in said cells to produce recombined cells, or recombining DNA between said plurality of cells that display monooxygenase activity to produce cells with modified monooxygenase activity;    (b) recombining and screening said recombined or modified cells to produce further recombined cells that have evolved additionally modified monooxygenase activity; and    (c) repeating (a) or (b) until said further recombined cells. have acquired a desired monooxygenase activity.    
     
     
         22 . The method of  claim 2 , wherein said method further comprises: 
 (a) recombining at least one distinct or improved recombinant polynucleotide with a further monooxygenase activity polynucleotide, which further polynucleotide is identical to or different from one or more of said plurality of parental polynucleotides to produce a library of recombinant monooxygenase polynucleotides;    (b) screening said library to identify at least one further distinct or improved recombinant monooxygenase polynucleotide that exhibits a further improvement or distinct property compared to said plurality of parental polynucleotides; and, optionally,    (c) repeating (a) and (b) until said resulting further distinct or improved recombinant polynucleotide shows an additionally distinct or improved monooxygenase property.    
     
     
         23 . The method of  claim 2 , wherein said recombinant monooxygenase polynucleotide is present in one or more bacterial, yeast, or fungal cells and said method comprises: 
 pooling multiple separate monooxygenase polynucleotides;    screening said resulting pooled monooxygenase polynucleotides to identify an improved recombinant monooxygenase polynucleotides that exhibits an improved monooxygenase activity compared to a non-recombinant monooxygenase activity polynucleotide; and    cloning said improved recombinant nucleic acid.    
     
     
         24 . The method of  claim 23 , further comprising transducing said distinct or improved nucleic acid into a prokaryote or eukaryote.  
     
     
         25 . The method of  claim 2 , wherein said shuffling of a plurality of parental polynucleotides comprises family gene shuffling.  
     
     
         26 . The method of  claim 2 , wherein said shuffling of a plurality of parental nucleic acids comprises individual gene shuffling.  
     
     
         27 . A selected shuffled monooxygenase nucleic acid made by said method of  claim 2 .  
     
     
         28 . A DNA shuffling mixture, comprising: at least three homologous DNAs, each of which is derived from a polynucleotide encoding a member selected from a polypeptide encoding monooxygenase activity, a polypeptide fragment encoding monooxygenase activity and combinations thereof.  
     
     
         29 . The DNA shuffling mixture of  claim 28 , wherein said at least three homologous DNAs are present in cell culture or in vitro.  
     
     
         30 . A method for increasing monooxygenase activity in a cell, comprising: performing whole genome shuffling of a plurality of genomic polynucleotides in said cell and selecting for one or more monooxygenase activity.  
     
     
         31 . The method of  claim 30 , wherein said genomic nucleic acids are from a species or strain different from said cell.  
     
     
         32 . The method of  claim 30 , wherein said cell is of prokaryotic or eukaryotic origin.  
     
     
         33 . The method of  claim 30 , wherein said monooxygenase activity to be selected is alkene epoxidation, alkane hydroxylation, aromatic hydroxylation, N-dealkylation of alkylamines, S-dealkylation of reduced thio-organics, O-Dealkylation of alkyl ethers, oxidation of aryloxy phenols, conversion of aldehydes to acids, dehydrogenation, decarbonylation, oxidative dehalogenation of haloaromatics and halohydrocarbons, Baeyer-Villiger monoxygenation, modification of cyclosporins, hydroxylation of mevastatin, conversion of cholesterol to pregnenolone, oxygenation of sulfonylureas and combinations thereof.  
     
     
         34 . A method for obtaining a polynucleotide encoding an improved polypeptide acting on a substrate comprising a target group selected from an olefin. a terminal methyl group, a methylene group, an aryl groupland combinations thereof, wherein said improved polypeptide exhibits one or more improved properties compared to a naturally occurring polypeptide acting on said substrate, said method comprising: 
 creating a library of recombinant polynucleotides that encoding a monooxygenase polypeptide acting on said substrate; and    screening said library to identify a recombinant polynucleotide encoding an improved polypeptide that exhibits one or more improved properties compared to a naturally occurring monooxygenase polypeptide.    
     
     
         35 . The method according to  claim 34 , wherein said library of recombinant polynucleotides is created by recombining at least a first form and a second form of a nucleic acid, at least one form encoding said naturally occurring polypeptide or a fragment thereof, wherein said first form and said second form differ from each other in two or more nucleotides.  
     
     
         36 . The method according to  claim 35 , wherein said first and second forms of said nucleic acid are homologous.  
     
     
         37 . The method according to  claim 35 , wherein at least one of said first and second forms of said nucleic acid does not encode a polypeptide having monooxygenase activity.  
     
     
         38 . A polypeptide encoded by a polynucleotide according to  claim 34 .  
     
     
         39 . The polypeptide according to  claim 38  wherein said polypeptide has an activity comprising, converting an olefin to an epoxide.  
     
     
         40 . The polypeptide according to  claim 38 , wherein said polypeptide has an activity comprising, converting said terminal methyl group to a hydroxymethyl group.  
     
     
         41 . The polypeptide according to  claim 38 , wherein said polypeptide has an activity comprising, converting a methylene group to a hydroxmethylene group.  
     
     
         42 . The polypeptide according to  claim 38 , wherein said polypeptide has an activity comprising, converting an aryl group to a hydroxyaryl group.  
     
     
         43 . The polypeptide according to  claim 38 , wherein said improved property is selected from: 
 improved regiospecificity of said acting on a substrate, wherein said substrate comprises at least two target groups;    enhanced production of a desired enantiomeric form of a reaction product;    enhanced expression of said polypeptide by a host cell that comprises said recombinant polynucleotide; and    enhanced stability of said polypeptide in said presence of an organic solvent.    
     
     
         44 . A method of oxidizing a substrate comprising a target group selected from an olefin, a terminal methyl group, a methylene group, an aryl group and combinations thereof, said method comprising contacting said substrate with a polypeptide according to  claim 38   
     
     
         45 . The method according to  claim 44 , wherein said absolute configuration of a product of said monooxygenase is R, S, or a mixture thereof.  
     
     
         46 . A method for preparing an epoxide group, said method comprising contacting a substrate comprising a carbon-carbon double bond with a polypeptide according to  claim 39 .  
     
     
         47 . A method for preparing a hydroxymethyl group, said method comprising contacting a substrate comprising a terminal methyl group with a polypeptide according to  claim 40 .  
     
     
         48 . A method for preparing a hydroxymethylene group, said method comprising contacting a substrate comprising a methylene group with a polypeptide according to  claim 41 .  
     
     
         49 . A method for preparing a hydroxyaryl group, said method comprising contacting a substrate comprising an aryl group with a polypeptide according to  claim 42 .  
     
     
         50 . An organism comprising a recombinant monooxygenase polynucleotide encoding an improved polypeptide that catalyzes a reaction selected from epoxidation of an olefin, hydroxylation of a terminal methyl group, hydroxylation of a methylene group, hydroxylation of an aryl group and combinations thereof wherein said polypeptide exhibits one property improved relative to a corresponding property of a naturally occurring monooxygenase polypeptide.  
     
     
         51 . The organism according to  claim 50 , further comprising an improved transferase polypeptide that exhibits one or more improved properties improved relative to a corresponding property of a naturally occurring transferase polypeptide.  
     
     
         52 . The organism according to  claim 51 , wherein said transferase is selected from S-adenosylmethionine dependent O-methyltransferase, acyl-CoA transferase and combinations thereof.  
     
     
         53 . The organism according to  claim 50 , further comprising an improved ligase peptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring ligase polypeptide.  
     
     
         54 . The organism according to  claim 53 , wherein said ligase is an acyl CoA ligase.  
     
     
         55 . The organism according to  claim 50 , further comprising an improved racemase polypeptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring racemase polypeptide.  
     
     
         56 . The organism according to  claim 55 , wherein said racemase s mandelate racemase.  
     
     
         57 . The organism according to  claim 50 , further comprising a dehydrogenase polypeptide that exhibits one or more properties improved relative to a corresponding property in a naturally occurring dehydrogenase polypeptide.  
     
     
         58 . The organism according to  claim 57 , said organism dehydrogenating a hydroxyalkyl group of a substrate having the structure:  
       
         
           
           
               
               
           
         
         wherein 
 R 11 , R 12 , R 13  and R 14  are independently selected from H and OH and at least one of R 111 , R 12 , R 13  and R 14  is OH;  
 n and s are independently selected from the numbers 0 to 16; and  
 p and t are independently selected from 0 to 6, wherein at least one of p and t must be at least one and p+t≦6,  
 said hydroxyalkyl group being dehydrogenated to a member selected from a carboxylic acid, a ketone carbonyl and an aldehyde carbonyl.  
 
       
     
     
         59 . The organism according to  claim 50 , further comprising an improved solvent resistance polypeptide that confers upon said organism a resistance to an organic solvent that is improved relative to that conferred by a naturally occurring solvent resistance-conferring polypeptide.  
     
     
         60 . The organism according to  claim 59 , wherein said improved solvent resistance polypeptide imparts to the organism a resistance to one or more organic compounds selected from olefins, α-hydroxycarboxylic acids, diols, aldehydes, ketones, halogenated hydrocarbons, perfluorocarbons, esters, aryl compounds, carboxylic acids, alcohols, ethers and combinations thereof.  
     
     
         61 . The organism of  claim 59 , wherein said improved solvent resistance polypeptide imparts to the organism a resistance to said solvent, wherein the solvent is present in a medium at hypersaturating concentrations.  
     
     
         62 . The organism according to  claim 50 , wherein said organism firher comprises an epoxide hydrolase polypeptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring epoxide hydrolase polypeptide.  
     
     
         63 . The organism according to  claim 50 , wherein said microorganism further comprises an epoxide isomerase polypeptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring epoxide isomerase polypeptide.  
     
     
         64 . The organism of  claim 50 , wherein said organism further comprises two or more recombinant polynucleotides selected from the group consisting of an improved transferase polypeptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring transferase polypeptide; 
 an improved epoxide hydrolase peptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring epoxide hydrolase polypeptide;    an improved ligase peptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring ligase polypeptide;    an improved racemase polypeptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring racemase polypeptide;    an improved dehydrogenase polypeptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring dehydrogenase polypeptide;    an improved epoxide isomerase polypeptide that exhibits one or more properties improved relative to a corresponding property of a naturally occurring epoxide isomerase polypeptide; and    an improved solvent resistance polypeptide that confers upon said organism a resistance to an organic solvent that is improved relative to that conferred by a naturally occurring solvent resistance-conferring polypeptide.    
     
     
         65 . A method for preparing an epoxide group, said method comprising contacting a substrate comprising a carbon-carbon double bond with an organism according to  claim 50 , thereby forming said epoxide group.  
     
     
         66 . The method according to  claim 65 , wherein said substrate is selected from styrene, styrene substituted on the phenyl group, divinylbenzene, divinylbenzene substituted on the phenyl group, isoprene, butadiene, diallyl ether, allyl phenyl ether, allyl phenyl ether substituted on the phenyl group, allyl alkyl ether, allyl aralkyl ether, vinylcyclohexene, vinylnorbornene, and acrolein.  
     
     
         67 . A method for converting an olefin into a vicinal diol, said method comprising: 
 (a) contacting said olefin with an organism according to  claim 50  to form an epoxide; and    (b) contacting said epoxide with an organism comprising an epoxide hydrolase polypeptide, thereby forming said vicinal diol.    
     
     
         68 . The method according to  claim 67 , wherein said epoxide hydrolase polypeptide exhibits one or more properties improved relative to corresponding properties of a naturally occurring epoxide hydrolase polypeptide.  
     
     
         69 . The method according to  claim 67 , wherein said polypeptide of (a) and said polypeptide of (b) are expressed in the same host cell.  
     
     
         70 . The method according to  claim 67 , further comprising, 
 (c) contacting said vicinal diol with an organism comprising a polypeptide selected from a ligase polypeptide and a transferase polypeptide, thereby forming a vicinal diol adduct.    
     
     
         71 . The method according to  claim 70 , wherein said polypeptide of (c) is a polypeptide exhibiting one or more properties improved over a corresponding property of an analogous naturally occurring polypeptide.  
     
     
         72 . The method according to  claim 70 , wherein said polypeptide of (a), said polypeptide of (b) and said polypeptide of (c) are expressed in the same host cell.  
     
     
         73 . The method according to  claim 67 , wherein said vicinal diol has the structure:  
       
         
           
           
               
               
           
         
         wherein 
 R 1  is selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, —NR 2 R 3 , —OR 2 , —CN, C(R 4 )NR 2 R 3  and C(R 4 )OR 2  groups,  
 R 2  and R 3  are members independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl groups;  
 R 4  is selected from ═O and ═S, and n is a number between 0 and 10, inclusive.  
 
       
     
     
         74 . The method according to  claim 73 , wherein 
 R 1  is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl —NR 2 R 3 , —OCR 2 , —CN, C(R 4 )NR 2 R 3  and C(R 4 )OR 2  groups,    R 2  and R 3  are members independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl groups; and    R 4  is selected from ═O and ═S.    
     
     
         75 . A method for converting an olefin into an α-hydroxycarboxylic acid, said method comprising: 
 (a) contacting said olefin with an organism according to  claim 50  to form an epoxide;  
 (b) contacting said epoxide with an organism comprising an epoxide hydrolase polypeptide to form a vicinal diol; and  
 (c) contacting said vicinal diol with an organism comprising a dehydrogenase polypeptide to form said α-hydroxycarboxylic acid.  
 
     
     
         76 . The method according to  claim 75 , wherein at least one of said hydrolase polypeptide and said dehydrogenase polypeptide exhibits at least one property improved relative to a corresponding property in an analogous naturally occurring polypeptide.  
     
     
         77 . The method according to  claim 78 , wherein said polypeptide of (a), of (b) and of (c) are expressed in the same host cell.  
     
     
         78 . A method for converting an olefin into an α-hydroxycarboxylic acid, said method comprising, contacting said olefin with an organism according to  claim 64 , wherein said two or more recombinant polynucleotides are an improved epoxide hydrolase and an improved dehydrogenase.  
     
     
         79 . The method according to  claim 78 , further comprising: 
 (d) contacting said α-hydroxycarboxylic acid with an organism comprising an improved polypeptide having an activity selected from ligase, transferase and combinations thereof, thereby forming a α-hydroxycarboxylic acid adduct.    
     
     
         80 . The method according to  claim 79 , wherein at least two of said polypeptide of (a), (b), (c), (d) are expressed in the same host cell.  
     
     
         81 . The method according to  claim 79 , wherein at least one of said polypeptide selected from ligase, transferase and combinations thereof is an improved polypeptide.  
     
     
         82 . The method according to  claim 78 , wherein said α-hydroxycarboxylic acid has the structure:  
       
         
           
           
               
               
           
         
         wherein 
 R 1  is selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, R 2 R 3 , —OR 2 , —CN, C(R 4 )NR 2 R 3  and C(R 4 )OR 2  groups,  
 R 2  and R 3  are members independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl groups;  
 R 4  is selected from ═O and ═S, and  
 n is a number between 0 and 10, inclusive.  
 
       
     
     
         83 . The method according to  claim 82  wherein 
 R 1  is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl —NR 2 R 3 , —OR 2 , —CN, C(R 4 )NR 2 R 3  and C(R 4 )OR 2  groups,  
 R 2  and R 3  are members independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl groups; and  
 R 4  is selected from ═O and ═S.  
 
     
     
         84 . The method according to  claim 79 , wherein said transferase activity is selected from glycosyl transferase activity and methyltransferase activity.  
     
     
         85 . The method according to  claim 84 , wherein said methyl transferase is a S-adenosylmethionine dependent O-methyltransferase.  
     
     
         86 . The method according to  claim 79 , wherein said α-hydroxycarboxylic acid adduct has the structure:  
       
         
           
           
               
               
           
         
         wherein 
 R 1  is selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, —NR 2 R 3 (R 4 ) m , —OR 2 , —CN, C(R 5 )NR 2 R 3  and C(R 5 )OR 2  groups,  
 R 2 , R 3  and R 4  are members independently selected from said group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl groups;  
 R 5  is selected from ═O and ═S;  
 R 6  is selected from H, alkyl and substituted alkyl groups;  
 R 7  is C(O)R 8 , wherein R 8  is selected from H alkyl and substituted alkyl groups and R 7  and R 8  are not both H;  
 m is 0 or 1, such that when m is 1, an ammonium salt is provided; and  
 n is a number between 0 and 10, inclusive.  
 
       
     
     
         87 . The method according to  claim 86  wherein 
 R 1  is selected from phenyl, substituted phenyl, pyridyl, substituted pyridyl —NR 2 R 3 , —OR 2 , —CN, C(R 5 )NR 2 R 3  and C(R 5 )OR 2 ,groups  
 R 2  and R 3  are members independently selected from said group consisting of H, C 1 -C 6  alkyl and allyl; and  
 R 5  is ═O.  
 
     
     
         88 . A method for preparing a hydroxy group, said method comprising: 
 (a) contacting a substrate comprising a terminal methyl group with a microorganism according to  claim 50 , thereby forming a hydroxymethyl group.    
     
     
         89 . The method according to  claim 88 , wherein said substrate comprises an alkyl-terminal methyl group as a component of a substrate selected from arylalkyl groups, substituted arylalkyl groups, heteroarylalkyl groups, and substituted heteroarylalkyl groups.  
     
     
         90 . The method according to  claim 88 , wherein said substrate has the structure  
       
         
           
           
               
               
           
         
         wherein, 
 each of said n R groups is a member selected from the group consisting of H, alkyl groups and substituted alkyl groups;  
 m is a number from 0 to 10, inclusive; and  
 n is a number from 0 to 5, inclusive.  
 
       
     
     
         91 . The method according to  claim 90 , wherein said substrate comprises benzene substituted with a member selected from the group of straight-chain alkyl groups branched-chain alkyl groups and combinations thereof.  
     
     
         92 . The method according to  claim 91 , wherein said substrate comprises benzene substituted with a member selected from C 1 -C 6  straight-chain, C 1 -C 6  branched-chain alkyl and combinations thereof.  
     
     
         93 . The method according to  claim 92 , wherein said alkyl group is selected from ethyl, n-propyl, i-propyl, t-butyl and combinations thereof.  
     
     
         94 . The method according to  claim 92 , wherein said substrate is  
       
         
           
           
               
               
           
         
         wherein n is a number between 0 and 9, inclusive.  
       
     
     
         95 . The method according to  claim 92 , wherein said substrate has the structure:  
       
         
           
           
               
               
           
         
         wherein n is a number between 1 and 6, inclusive.  
       
     
     
         96 . The method according to  claim 88 , wherein said hydroxy group is a component of a member selected from benzyl alcohol, substituted benzyl alcohol, 2-phenylethanol, substituted 2-phenylethanol, 3-phenylpropanol and substituted 3-phenylpropanol.  
     
     
         97 . The method according to  claim 88 , further comprising: 
 (b) contacting said hydroxymethyl group with an organism comprising an acyltransferase, thereby forming an acylated hydroxy adduct.    
     
     
         98 . The method according to  claim 97 , wherein said acyltransferase exhibits one or more properties improved relative to a corresponding property of a naturally occurring acyltransferase.  
     
     
         99 . The method according to  claim 97 , wherein said polypeptide of (a) and said polypeptide of (b) are expressed by the same host cell.  
     
     
         100 . The method according to  claim 88 , further comprising: 
 (b) contacting said hydroxymethyl group with a microorganism comprising an improved glycosyltransferase, thereby forming a glycosylated hydroxy adduct.    
     
     
         101  The method according to  claim 100 , wherein said glycosyltransferase exhibits one or more properties improved relative to a corresponding property of a naturally occurring glycosyltransferase.  
     
     
         102 . The method according to  claim 100 , wherein said polypeptide of (a) and said polypeptide of (b) are expressed by the same host cell.  
     
     
         103 . The method according to  claim 88 , further comprising: 
 (b) contacting said hydroxy group with a microorganism comprising a dehydrogenase, thereby forming a carboxylic acid.    
     
     
         104 . The method according to  claim 103 , wherein said dehydrogenase exhibits one or more properties improved relative to a corresponding property of a naturally occurring dehydrogenase.  
     
     
         105 . The method according to  claim 103 , wherein said polypeptide of (a) and said polypeptide of (b) are expressed by the same host cell.  
     
     
         106 . The method according to  claim 110 , further comprising, contacting said carboxylic acid with a microorganism comprising an improved transferase, thereby forming a carboxylic acid ester.  
     
     
         107 . A method for preparing a hydroxymethylene group, said method comprising contacting a substrate comprising a methylene group with a microorganism according to  claim 50 .  
     
     
         108 . The method according to  claim 107 , wherein said substrate comprises a member selected from 3,4-dihydrocoumarin and 3,4-dihydrocoumarin residues.  
     
     
         109 . The method according to  claim 107 , wherein said substrate is 3,4-dihydrocoumarin and said polypeptide converts said substrate to 4-hydroxy-,4-dihydrocoumarin.  
     
     
         110 . A method for preparing a hydroxyaryl group, said method comprising: 
 (a) contacting a substrate comprising an aryl group with a microorganism according to  claim 50 .    
     
     
         111 . The method according to  claim 110 , wherein said substrate comprises a group selected from aryl groups, substituted aryl groups, heteroaryl groups and substituted heteroaryl groups.  
     
     
         112 . The method according to  claim 110 , further comprising: 
 (b) contacting said hydroxyaryl group with an organism comprising an acyltransferase, thereby forming an acylated hydroxyaryl adduct.    
     
     
         113 . The method according to  claim 112 , wherein said acyltransferase exhibits one or more properties improved relative to a corresponding property of a naturally occurring acyltransferase.  
     
     
         114 . The method according to  claim 112 , wherein said polypeptide of (a) and said polypeptide of (b) are expressed by the same host cell.  
     
     
         115 . The method according to  claim 112 , further comprising: 
 (b) contacting said hydroxyaryl group with a microorganism comprising a glycosyltransferase, thereby forming a glycosylated hydroxyaryl adduct.    
     
     
         116  The method according to  claim 115 , wherein said glycosyltransferase exhibits one or more properties improved relative to a corresponding property of a naturally occurring glycosyltransferase.  
     
     
         117 . The method according to  claim 115 , wherein said polypeptide of (a) and said polypeptide of (b) are expressed by the same host cell.  
     
     
         118 . A screening process comprising: 
 (a) introducing the library of recombinant polynucleotides into a population of test microorganisms such that the recombinant polynucleotides are expressed;    (b) placing the organisms in a medium comprising at least one substrate; and    (c) and identifying those organisms exhibiting an improved propertv compared to microorganisms without the recombinant polynucleotide.    
     
     
         119 . A bioreactor comprising: 
 (a) an improved monooxygenase polypeptide;    (b) a redox partner;    (c) oxygen;    (d) an oxidizable substrate.    
     
     
         120 . The bioreactor according to  claim 119 , wherein said polypeptide is immobilized.  
     
     
         121 . The bioreactor according to  claim 119 , wherein said polypeptide is a chimeric polypeptide.  
     
     
         122 . The bioreactor according to  claim 119 , wherein said polypeptide is a P-450 polypeptide.  
     
     
         123 . The bioreactor according to  claim 122 , wherein said P-450 is a peroxide-stable P-450.  
     
     
         124 . A kit comprising: 
 (a) at least one improved monooxygenase polypeptide; and    (b) directions for using said polypeptide to carry out a chemical reaction.    
     
     
         125 . The kit according to  claim 124 , wherein said at least one improved monooxygenase polypeptide is a constituent of a library of improved polypeptides.  
     
     
         126 . A recombinant P450 polypeptide comprising a backbone domain and an active site domain, wherein at least one of said domains comprises at least two contiguous amino acids that are not contiguous in a naturally occurring P450 enzyme.  
     
     
         127 . The recombinant P450 polypeptide according to  claim 126 , wherein the junction between the active site domain and the backbone domain is at a location selected from an end of the I helix and within the G-H loop.  
     
     
         128 . The recombinant P450 polypeptide according to  claim 126 , wherein the F and G helices are transferred into the backbone P450.  
     
     
         129 . A polynucleotide that encodes a recombinant P450 polypeptide according to  claim 126 .  
     
     
         130 . A method of obtaining a polynucleotide that encodes a recombinant P450 polypeptide comprising a backbone domain and an active site domain, said method comprising: 
 (a) recombining at least first and second forms of a nucleic acid that encodes a P450 active site domain, wherein the first and second forms differ from each other in two or more nucleotides to produce a library of recombinant active site domain encoding polynucleotides; and    (b) linking the recombinant active site domain-encoding polynucleotide to a backbone-encoding polynucleotide so that the active site-encoding domain and the backbone-encoding domain are in-frame.    
     
     
         131 . The method according to  claim 130 , wherein said backbone is derived from P450 BMP .  
     
     
         132 . The method according to  claim 130 , wherein said backbone domain and said recombinant active-site domain are joined at a member selected from an end of the I helix and within the G-H loop.  
     
     
         133 . The method according to  claim 130 , wherein the F and G helices are transferred into the backbone P450.  
     
     
         134 . A method of obtaining a polynucleotide that encodes a recombinant P450 polypeptide comprising a backbone domain and an active site domain, said method comprising: 
 (a) recombining at least first and second forms of a nucleic acid that encodes a P450 backbone domain, wherein the first and second forms differ from each other in two or more nucleotides to produce a library of recombinant backbone domain encoding polynucleotides; and    (b) linking the recombinant backbone domain-encoding polynucleotide to a active site-encoding polynucleotide so that the backbone-encoding domain and the active site-encoding domain are in-frame.    
     
     
         135 . The method according to  claim 134 , wherein said backbone is derived from P450 BMP .  
     
     
         136 . The method according to  claim 134 , wherein said backbone domain and said recombinant active-site domain are joined at a member selected from an end of the I helix and within the G-H loop.  
     
     
         137 . The method according to  claim 134 , wherein the F and G helices are transferred into the backbone P450.  
     
     
         138 . A method of obtaining a polynucleotide that encodes a recombinant P450 polypeptide comprising a backbone domain and an active site domain, said method comprising: 
 (a) recombining at least first and second forms of a nucleic acid that encodes a P450 active site domain, wherein the first and second forms differ from each other in two or more nucleotides to produce a library of recombinant active site domain encoding polynucleotides;    (b) recombining at least first and second forms of a nucleic acid that encodes a P450 backbone domain, wherein the first and second forms differ from each other in two or more nucleotides to produce a library of recombinant backbone domain encoding polynucleotides; and    (c) linking the recombinant active site domain-encoding polynucleotide to the recombinant backbone-encoding polynucleotide so that the recombinant active site-encoding domain and the recombinant backbone-encoding domain are in-frame.    
     
     
         139 . The method according to  claim 138 , wherein said backbone is derived from P450 BMP .  
     
     
         140 . The method according to  claim 138 , wherein said backbone domain and said recombinant active-site domain are joined at a member selected from an end of the I helix and within the G-H loop.  
     
     
         141 . The method according to  claim 138 , wherein the F and G helices are transferred into the backbone P450.