Genes encoding baeyer-villiger monooxygenases

Abstract

Genes have been isolated from a variety of bacteria encoding Baeyer-Villiger monooxygenase activity. The genes and their products are useful for the conversion of ketones to the corresponding esters. A series of motifs, common to all genes, has been identified as diagnostic for genes encoding proteins of this activity.

What is claimed is:  
     
         1 . An isolated nucleic acid fragment selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding a Baeyer-Villiger monooxygenase polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46;    (b) an isolated nucleic acid molecule encoding a Baeyer-Villiger monooxygenase polypeptide that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or    an isolated nucleic acid fragment that is complementary to (a) or (b).    
     
     
         2 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 542 amino acids that has at least 55% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:8 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         3 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 541 amino acids that has at least 53% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:10 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         4 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 439 amino acids that has at least 37% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:22 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         5 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 518 amino acids that has at least 44% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:24 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         6 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 541 amino acids that has at least 64% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:26 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         7 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 462 amino acids that has at least 65% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:28 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         8 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 523 amino acids that has at least 45% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:30 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         9 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 493 amino acids that has at least 55% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:32 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         10 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 539 amino acids that has at least 51% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:34 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         11 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 649 amino acids that has at least 39% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:36 or a second nucleotide sequence comprising-the complement of the first nucleotide sequence.  
     
     
         12 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 494 amino acids that has at least 43% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:38 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         13 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 499 amino acids that has at least 53% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:40 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         14 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 493 amino acids that has at least 44% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:42 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         15 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 541 amino acids that has at least 54% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:44 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         16 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 545 amino acids that has at least 42% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:46 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         17 . The isolated nucleic acid fragment of  claim 1  selected from the group consisting of SEQ ID NOs:7, 9, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45.  
     
     
         18 . An isolated nucleic acid fragment of  claim 1  isolated from  Rhodococcus.    
     
     
         19 . A polypeptide encoded by the isolated nucleic acid fragment of  claim 1 .  
     
     
         20 . The polypeptide of  claim 19  selected from the group consisting of SEQ ID NOs:8, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46.  
     
     
         21 . An isolated nucleic acid fragment selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding a Baeyer-Villiger monooxygenase polypeptide having an amino acid sequence as set forth in SEQ ID NO:12;    (b) an isolated nucleic acid molecule encoding a Baeyer-Villiger monooxygenase polypeptide that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or    an isolated nucleic acid fragment that is complementary to (a), or (b).    
     
     
         22 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 532 amino acids that has at least 57% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:11 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         23 . An isolated nucleic acid fragment of  claim 21  isolated from  Arthrobacter.    
     
     
         24 . A polypeptide encoded by the isolated nucleic acid fragment of  claim 21 .  
     
     
         25 . The polypeptide of  claim 24  as set forth in SEQ ID NO:12.  
     
     
         26 . An isolated nucleic acid fragment selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding a Baeyer-Villiger monooxygenase polypeptide having an amino acid sequence as set forth in SEQ ID NO:18;    (b) an isolated nucleic acid molecule encoding a Baeyer-Villiger monooxygenase polypeptide that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or    an isolated nucleic acid fragment that is complementary to (a), or (b).    
     
     
         27 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 538 amino acids that has at least 57% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:17 or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         28 . An isolated nucleic acid fragment of  claim 26  isolated from  Acidovorax.    
     
     
         29 . A polypeptide encoded by the isolated nucleic acid fragment of  claim 26 .  
     
     
         30 . The polypeptide of  claim 29  selected from the group consisting of SEQ ID NO:18.  
     
     
         31 . A chimeric gene comprising the isolated nucleic acid fragment of any one of claims  1 ,  19 ,  25 ,  30 , or  35  operably linked to suitable regulatory sequences.  
     
     
         32 . A transformed host cell comprising a host cell and the chimeric gene of  claim 31 .  
     
     
         33 . The transformed host cell of  claim 32  wherein the host cell is selected from the group consisting of bacteria, yeast, filamentous fungi, and green plants.  
     
     
         34 . The transformed host cell of  claim 33  wherein the host cell is selected from the group consisting of proteobacteria and actinomycetes.  
     
     
         35 . The transformed host cell of  claim 34  wherein the host cell is selected from the group consisting of  Burkholderia, Alcaligenes, Pseudomonas, Sphingomonas, Pandoraea, Delftia  and  Comamonas.    
     
     
         36 . The transformed host cell of  claim 33  wherein the host cell is selected from the group consisting of  Rhodococcus, Acinetobacter, Mycobacteria, Nocardia, Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Streptomyces, Escherichia, Salmonella, Pseudomonas, Aspergillus, Saccharomyces, Pichia, Candida, Corynebacterium , and  Hansenula.    
     
     
         37 . The transformed host cell of  claim 33  wherein the host cell is selected from the group consisting of soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, Arabidopsis, cruciferous vegetables, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses  
     
     
         38 . A method of obtaining a nucleic acid fragment encoding a Baeyer-Villiger monooxygenase polypeptide comprising: 
 (a) probing a genomic library with the nucleic acid fragment of any one of claims  1 ,  21 , or  26 ;    (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of any one of claims  1 ,  21 , or  26 ;    (c) sequencing the genomic fragment that comprises the clone identified in step (b);    wherein the sequenced genomic fragment encodes a Baeyer-Villiger monooxygenase polypeptide.    
     
     
         39 . A method of obtaining a nucleic acid fragment encoding a Baeyer-Villiger monooxygenase polypeptide comprising: 
 (a) synthesizing at least one oligonucleotide primer corresponding to a portion of the isolated nucleic acid sequence of any one of claims  1 ,  21 , or  26 ; and    (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a);    wherein the amplified insert encodes a Baeyer-Villiger monooxygenase polypeptide.    
     
     
         40 . A method for the identification of a polypeptide having monooxygenase activity comprising: 
 (a) obtaining the amino acid sequence of a polypeptide suspected of having monooxygenase activity; and    (b) aligning the amino acid sequence of step (a) with the amino acid sequence of a Baeyer-Villiger monooxygenase consensus sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49;    wherein where at least 80% of the amino acid residues at positions p1-p74 of SEQ ID NO:47, or at least 80% of the amino acid residues at p1-p76 of SEQ ID NO:48 or at least 80% of the amino acid residues of p1-p41 of SEQ ID NO:49 are completely conserved, the polypeptide of (a) is identified as having monooxygenase activity.    
     
     
         41 . A method according to  claim 40  wherein least 100% of the amino acid residues at positions p1-p74 of SEQ ID NO:47, or at least 100% of the amino acid residues at p1-p76 of SEQ ID NO:48 or at least 100% of the amino acid residues of p1-p41 of SEQ ID NO:49 are completely conserved.  
     
     
         42 . A method for identifying a gene encoding a Baeyer-Villiger monooxygenase polypeptide comprising: 
 (a) probing a genomic library with a nucleic acid fragment encoding a polypeptide wherein where at least 80% of the amino acid residues at positions p1-p74 of SEQ ID NO:47, or at least 80% of the amino acid residues at p1-p76 of SEQ ID NO:48 or at least 80% of the amino acid residues of p1-p41 of SEQ ID NO:49 are completely conserved;    (b) identifying a DNA clone that hybridizes with a nucleic acid fragment of step (a);    (c) sequencing the genomic fragment that comprises the clone identified in step (b);    wherein the sequenced genomic fragment encodes a Baeyer-Villiger monooxygenase polypeptide.    
     
     
         43 . A method according to  claim 42  wherein least 100% of the amino acid residues at positions p1-p74 of SEQ ID NO:47, or at least 100% of the amino acid residues at p1-p76 of SEQ ID NO:48 or at least 100% of the amino acid residues of p1-p41 of SEQ ID NO:49 are completely conserved.  
     
     
         44 . The product of either of claims  40  or  42 .  
     
     
         45 . A method for the biotransformation of a ketone substrate to the corresponding ester, comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of ketone substrate whereby the corresponding ester is produced, said transformed host cell comprising a nucleic acid fragment encoding an isolated nucleic acid fragment of any of claims  1 ,  21 ,  26  or  44 ; under the control of suitable regulatory sequences.  
     
     
         46 . The method of  claim 45  wherein the ketone substrate is selected from the group consisting of cyclic ketones and ketoterpenes having the general formula:  
       
         
           
           
               
               
           
         
       
       wherein R and R 1  are independently selected from substituted or unsubstituted phenyl, substituted or unsubstituted alkyl, or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene.  
     
     
         47 . The method of  claim 46  wherein the ketone substrate is selected from the group consisting of Norcamphor, Cyclobutanone, Cyclopentanone, 2-methyl-cyclopentanone, Cyclohexanone, 2-methyl-cyclohexanone, Cyclohex-2-ene-1-one, 1,2-cyclohexanedione, 1,3-cyclohexanedione, 1,4-cyclohexanedione, Cycloheptanone, Cyclooctanone, Cyclodecanone, Cycloundecanone, Cyclododecanone, Cyclotridecanone, Cyclopenta-decanone, 2-tridecanone, dihexyl ketone, 2-phenyl-cyclohexanone, Oxindole, Levoglucosenone, dimethyl sulfoxide, dimethy-2-piperidone, Phenylboronic acid, and beta-ionone.  
     
     
         48 . A method for the in vitro transformation of a ketone substrate to the corresponding ester, comprising: contacting a ketone substrate under suitable reaction conditions with an effective amount of a Baeyer-Villiger monooxygenase enzyme, the enzyme having an amino acid seqeunce selected from the group consisting of SEQ ID NOs:8, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46.  
     
     
         49 . A method according to  claim 49  wherein the ketone substrate is selected from the group consisting of cyclic ketones and ketoterpenes having the general formula:  
       
         
           
           
               
               
           
         
         wherein R and R 1  are independently selected from substituted or unsubstituted phenyl, substituted or unsubstituted alkyl, or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene.  
       
     
     
         50 . A method according to  claim 48  wherein the ketone substrate is selected from the group consisting of Norcamphor, Cyclobutanone, Cyclopentanone, 2-methyl-cyclopentanone, Cyclohexanone, 2-methyl-cyclohexanone, Cyclohex-2-ene-1-one, 1,2-cyclohexanedione, 1,3-cyclohexanedione, 1,4-cyclohexanedione, Cycloheptanone, Cyclooctanone, Cyclodecanone, Cycloundecanone, Cyclododecanone, Cyclotridecanone, Cyclopenta-decanone, 2-tridecanone, dihexyl ketone, 2-phenyl-cyclohexanone, Oxindole, Levoglucosenone, dimethyl sulfoxide, dimethy-2-piperidone, Phenylboronic acid, and beta-ionone.  
     
     
         51 . A mutated microbial gene encoding a protein having an altered biological activity produced by a method comprising the steps of 
 (i) digesting a mixture of nucleotide sequences with restriction endonucleases wherein said mixture comprises: 
 a) a native microbial gene selected from the group consisting of SEQ ID NOs:7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45;  
 b) a first population of nucleotide fragments which will hybridize to said native microbial sequence;  
 c) a second population of nucleotide fragments which will not hybridize to said native microbial sequence;  
 wherein a mixture of restriction fragments are produced;  
   (ii) denaturing said mixture of restriction fragments;    (iii) incubating the denatured said mixture of restriction fragments of step (ii) with a polymerase;    (iv) repeating steps (ii) and (iii) wherein a mutated microbial gene is produced encoding a protein having an altered biological activity.    
     
     
         52 . An  Acidovorax  sp. comprising the 16s rDNA sequence as set forth in SEQ ID NO:5  
     
     
         53 . An  Arthrobacter  sp. comprising the 16s rDNA sequence as set forth in SEQ ID NO:1  
     
     
         54 . A  Rhodococcus  sp. comprising the 16s rDNA sequence as set forth in SEQ ID NO:6  
     
     
         55 . An isolated nucleic acid useful for the identification of a BV monooxygenase selected from the group consisting of SEQ ID 70-113.